WO2023183371A2

WO2023183371A2 - Engineered heart tissue model of restrictive cardiomyopathy for drug discovery

Info

Publication number: WO2023183371A2
Application number: PCT/US2023/015878
Authority: WO
Inventors: Barry FINE; Xiaokan ZHANG; Bryan Wang; Trevor Ray NASH; Gordana Vunjak-Novakovic
Original assignee: The Trustees Of Columbia University In The City Of New York
Priority date: 2022-03-22
Filing date: 2023-03-22
Publication date: 2023-09-28
Also published as: WO2023183371A8; WO2023183371A3

Abstract

Described herein is a novel mutation in Filamin C (FLNC) in a patient with early onset restrictive cardiomyopathy and utilization of induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) derived from this patient to generate a cell-based model of impaired cardiomyocyte relaxation. In certain aspects, the provided herein are methods of identifying a compound that modulates relaxation velocity of a contractile cell or a beating cardiomyocyte, methods of observing a dynamic physical property of a beating cardiomyocyte, methods of identifying compounds that modulate a dynamic physical property of an engineered cardiac tissue, methods of treating a cardiomyopathy, cell lines, and engineered cardiac tissue.

Description

ENGINEERED HEART TISSUE MODEL OF RESTRICTIVE CARDIOMYOPATHY FOR DRUG DISCOVERY

[0001] This International Patent Application claims the benefit of and priority to U.S. Application No. 63/322,599, filed March 22, 2022, and U.S. Application No. 63/327,639, filed April 5, 2022, the contents of each of which are hereby incorporated by reference in their entireties.

[0002] All patents, patent applications and publications, and other literature references cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

GOVERNMENT SUPPORT

[0004] This invention was made with government support under Grant EB027062 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0005] Restrictive cardiomyopathy (RCM) is characterized by increased cardiac filling pressures in the setting of diastolic dysfunction and normal left ventricular wall thickness. Because of its functional definition, RCM has been difficult to recapitulate in vitro and there are currently no cell models that capture the clinical phenotype. Described herein is a novel mutation in Filamin C (FLNC) in a patient with early onset RCM and utilization of induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) derived from this patient to generate a cell-based model of impaired cardiomyocyte relaxation. SUMMARY OF THE INVENTION

[0006] In certain aspects, described herein is a method of identifying a compound that modulates relaxation velocity of a contractile cell, the method comprising: (a) culturing contractile cells in two-dimensional culture, wherein the contractile cells are derived from an induced pluripotent stem cell (iPSC); (b) contacting the contractile cells with a calciumsensitive indicator; (c) measuring a signal of calcium flux generated from the calcium sensitive indicator; (d) contacting the contractile cells with a test compound; (e) measuring a signal of calcium flux generated from the calcium sensitive indicator; (f) determining a time constant of relaxation value using the calcium flux signal measured in step (e); and (g) identifying the test compound as a compound that modulates relaxation velocity of a contractile cell if the time constant of relaxation value determined in step (f) is significantly higher or lower than either: (i) a time constant of relaxation value for the contractile cell determined using the calcium flux signal measured in step (c); or (ii) a time constant of relaxation value for a control contractile cell.

[0007] In certain aspects, described herein is a method of identifying a compound that modulates relaxation velocity of a beating cardiomyocyte, the method comprising: (a) culturing beating cardiomyocytes in two-dimensional culture, wherein the cardiomyocytes are derived from an induced pluripotent stem cell (iPSC); (b) contacting the beating cardiomyocytes with a calcium-sensitive indicator; (c) measuring a signal of calcium flux generated from the calcium sensitive indicator; (d) contacting the beating cardiomyocytes with a test compound; (e) measuring a signal of calcium flux generated from the calcium sensitive indicator; (f) determining a time constant of relaxation value using the calcium flux signal measured in step (e); and (g) identifying the test compound as a compound that modulates relaxation velocity of a beating cardiomyocyte if the time constant of relaxation value determined in step (f) is significantly higher or lower than either: (i) a time constant of relaxation value for the beating cardiomyocyte determined using the calcium flux signal measured in step (c); or (ii) a time constant of relaxation value for a control beating cardiomyocyte.

[0008] In some embodiments, the determining of step (f) in the method of identifying a compound that modulates relaxation velocity of a contractile cell or the method of identifying a compound that modulates relaxation velocity of a beating cardiomyocyte comprises: normalizing the signal of calcium flux measured in step (e) to the signal of calcium flux measured in step (c); identifying calcium flux signal peaks in the normalized calcium flux signal; for each peak, segmenting the portion of the normalized calcium flux signal from a maximum amplitude to a minimum value; performing a linear regression on the segmented normalized calcium flux signal to obtain a value of tau (T) corresponding to a time constant of relaxation value; averaging the value of tau across the number of signal peaks. In some embodiments, the linear regression is performed using the following equation:

wherein y is the normalized calcium flux signal and t is time.

[0009] In some embodiments, the test compound is identified as a compound that modulates relaxation velocity of a beating cardiomyocyte if the time constant of relaxation value determined in step (f) is significantly higher or lower than a time constant of relaxation value for the beating cardiomyocyte determined using the calcium flux signal measured in step (c). In some embodiments, the test compound is identified as a compound that modulates relaxation velocity of a beating cardiomyocyte if the time constant of relaxation value determined in step (f) is significantly higher or lower than a time constant of relaxation value for a control beating cardiomyocyte.

[0010] In some embodiments, the control beating cardiomyocyte is a control by virtue of being contacted with DMSO instead of a test compound. In some embodiments, the time constant of relaxation value for the control beating cardiomyocyte is determined comprising: culturing beating cardiomyocytes in two-dimensional culture, wherein the cardiomyocytes are derived from an induced pluripotent stem cell (iPSC); contacting the beating cardiomyocytes with a calcium-sensitive indicator; measuring a signal of calcium flux generated from the calcium sensitive indicator; contacting the beating cardiomyocytes with DMSO; measuring a signal of calcium flux generated from the calcium sensitive indicator; and determining a time constant of relaxation value using the calcium flux signal measured after contacting the beating cardiomyocytes with DMSO. In some embodiments, determining the time constant of relaxation for the beating cardiomyocytes with DMSO comprises normalizing the signal of calcium flux measured after contacting the beating cardiomyocytes with DMSO to the signal of calcium flux measured prior to contacting the beating cardiomyocytes with DMSO; identifying calcium flux signal peaks in the normalized calcium flux signal; for each peak, segmenting the portion of the normalized calcium flux signal from a maximum amplitude to a minimum value; performing a linear regression on the segmented normalized calcium flux signal to obtain a value of tau (T) corresponding to a time constant of relaxation value; and averaging the value of tau across the number of signal peaks. In some embodiments, the linear regression is performed using the following equation:

wherein y is the normalized calcium flux signal and t is time.

[0011] In some embodiments, the control beating cardiomyocyte and the cardiomyocyte contacted with the test compound are derived from the same iPSC culture.

[0012] In some embodiments, culturing beating cardiomyocytes in two-dimensional culture comprises: isolating peripheral mononuclear blood cells from a blood sample from a subject; reprograming the isolated peripheral mononuclear blood cells to generate induced pluripotent stem cells (iPSCs); culturing the iPSCs in a culture medium comprising a cardiac differentiation media (CDM) containing RPMI1640, albumin and ascorbic acid to generate beating cardiomyocytes; culturing the beating cardiomyocytes in a culture medium comprising RPMI1640 with B27 supplement; culturing the beating cardiomyocytes at least one passage in a culture medium comprising RPMI1640 with B27 supplement and CHIR99021; and culturing the beating cardiomyocytes in a culture medium comprising RPMI160 with B27 supplement. In some embodiments, the beating cardiomyocytes are maintained in a culture medium comprising RPMI160 with B27 supplement. In some embodiments, the contractile cells are human. In some embodiments, the beating cardiomyocytes are human. In some embodiments, the peripheral mononuclear blood cells are isolated by immunomagnetic cell separation. In some embodiments, the immunomagnetic cell separation uses an antibody against Epithelial Cell Adhesion Molecule (EpCAM). In some embodiments, the methods further comprises: serially passaging the beating cardiomyocytes.

[0013] In some embodiments, the beating cardiomyocytes are cultured in a multi-well plate and each well of the multi-well plate is used to test one or more different test compounds or different concentrations of one or more test compounds. In some embodiments, the control beating cardiomyocyte is cultured in a well of the same multi-well plate as the beating cardiomyocytes contacted with test compound. In some embodiments, a second control beating cardiomyocyte is cultured in a well of the same multi-well plate as the beating cardiomyocytes contacted with test compound, wherein the second control is contacted with a compound that is known to modulate the relaxation velocity of a beating cardiomyocyte. In some embodiments, the multi -well plate is a 384 well plate.

[0014] In some embodiments, the method of identifying a compound that modulates relaxation velocity of a beating cardiomyocyte is for identifying a test compound that increases the relaxation velocity of a beating cardiomyocyte, wherein if the time constant of relaxation value determined in step (f) is significantly lower than either condition (i) or (ii) described above, the test compound is identified as a compound that increases the relaxation velocity of a beating cardiomyocyte. In some embodiments, the method of identifying a compound that modulates relaxation velocity of a beating cardiomyocyte is for identifying a test compound that decreases the relaxation velocity of a beating cardiomyocyte, wherein if the time constant of relaxation value determined in step f) is significantly higher than either condition (i) or (ii) described above the test compound is identified as a compound that decreases the relaxation velocity of a beating cardiomyocyte. In some embodiments, culture medium of the culturing of step (a) is replaced with fresh culture medium before contacting the beating cardiomyocytes with the calcium-sensitive indicator.

[0015] In some embodiments, the calcium sensitive indicator is a chemical sensitive dye, a genetically encoded calcium indicator, or a combination thereof. In some embodiments, the chemical indicator is a calcium sensitive dye. In some embodiments, the genetically encoded calcium indicator is GCaMP. In some embodiments, the calcium-sensitive indicator is Calcium 6.

[0016] In some embodiments, the beating cardiomyocytes are contacted with the test compound for 1 hour. In some embodiments, the beating cardiomyocytes are contacted with the test compound for 3 hours. In some embodiments, the beating cardiomyocytes are contacted with the test compound for 1 day. In some embodiments, the beating cardiomyocytes are contacted with the test compound for 1 week.

[0017] In some embodiments, the beating cardiomyocytes comprise filamin C (FLNC) with a mutation, a deletion or an addition of at least one nucleic acid residue or amino acid. In some embodiments, the FLNC deletion comprises a deletion of the nucleic acid residues from position 7416 to 7418 as described herein. In some embodiments, the beating cardiomyocytes comprise a FLNC mutation, deletion, or addition that has been corrected to wild-type.

[0018] In some embodiments, the beating cardiomyocytes display a phenotype of cardiomyopathy. In some embodiments, the beating cardiomyocytes express a genetically encoded calcium indicator. In some embodiments, the genetically encoded calcium indicator is GcaMP.

[0019] In certain aspects, described herein is a method of observing a dynamic physical property of a beating cardiomyocyte, the method comprising: culturing beating cardiomyocytes in two-dimensional culture, wherein the cardiomyocytes are derived from an induced pluripotent stem cell (iPSC); observing the beating cardiomyocytes with brightfield video microscopy; determining the contractile amplitude, contraction velocity, relaxation velocity, or a combination thereof. In some embodiments, the determining is performed using pixel intensity subtraction. In some embodiments, the video microscopy is performed at about 100 frames per second. In some embodiments, the beating cardiomyocytes are cultured at about 200,000 cells per cm². In some embodiments, the beating cardiomyocytes are cultured in a multi-well plate. In some embodiments, the methods further comprise contacting the cells with a test compound and determining the contractile amplitude, contraction velocity, relaxation velocity, or a combination thereof in the presence of the test compound.

[0020] In certain aspects, described herein is a method of identifying a compound that modulates a dynamic physical property of an engineered cardiac tissue, the method comprising: (a) culturing an engineered cardiac tissue (ECT) comprising cardiomyocytes derived from iPSCs and human primary cardiac fibroblasts in a bioreactor, wherein the cardiomyocytes and fibroblasts are encapsulated in a fibrin gel and grow around two horizontal PDMS pillars of the bioreactor, wherein the bioreactor further comprises electrodes for electrical stimulation of the ECT; (b) stimulating the ECT with an electrical stimulation; (c) maintaining the ECT with a lower electric stimulation than the electric stimulation used in step (b); (d) determining a dynamic physical property of the ECT comprising (i) stimulating the ECT with electrical stimulation and capturing video of ECT contraction; (ii) analyzing pillar deflection.

[0021] In some embodiments, analyzing the pillar deflection comprises: tracking pillar head movement and calculating displacement values. In some embodiments, the method further comprises using the displacement values to calculate force. In some embodiments, the force is calculated using the known force needed to deflect a pillar of the bioreactor. In some embodiments, the force calculated is the force that displaces pillars during ECT contraction (active force). In some embodiments, the force calculated is the force causing pillar deflection during maximum relaxation of the ECT (residual or passive force). In some embodiments, the force calculated is the sum of the active force that displaces pillars during ECT contraction and the residual force causing pillar deflection during maximum relaxation of the ECT (total force). In some embodiments, the method further comprises determining a relaxation velocity measurement, a contraction velocity measurement, or both, using the derivative of the displacement values.

[0022] In some embodiments, the method further comprises contacting the ECT with a test compound and determining whether the test compound modulates a dynamic physical property of the ECT comprising: stimulating the ECT in the presence of the test compound with electrical stimulation and capturing video of ECT contraction and analyzing pillar deflection. In some embodiments, analyzing the pillar deflection in the presence of the test compound comprises: tracking pillar head movement and calculating displacement values in the presence of the test compound. In some embodiments, the method further comprises using the displacement values in the presence of the test compound to calculate force in the presence of the test compound. In some embodiments, the force in the presence of the test compound is calculated using the known force needed to deflect a pillar of the bioreactor. In some embodiments, the force calculated is the force that displaces pillars during ECT contraction in the presence of the test compound (active force). In some embodiments, the force calculated is force causing pillar deflection during maximum relaxation of the ECT in the presence of the test compound (residual or passive force). In some embodiments, the force calculated is the sum of the active force that displaces pillars during ECT contraction in the presence of the test compound and the residual force causing pillar deflection during maximum relaxation of the ECT in the presence of the test compound (total force). In some embodiments, the method further comprises determining a relaxation velocity measurement in the presence of the test compound, a contraction velocity measurement in the presence of the test compound, or both, using the derivative of the displacement values in the presence of the test compound. [0023] In some embodiments, in the method of identifying a compound that modulates a dynamic physical property of an engineered cardiac tissue the culturing an ECT of step (a) comprises: dissociating cardiomyocytes and primary cardiac fibroblasts; suspending the dissociated cells in a fibrinogen solution; mixing the suspended cells with thrombin and placing in contact with the PDMS pillars to generate an ECT; maintaining the ECT in a culture medium comprising B27 media and 6-aminocaproic acid; and maintaining the tissue in a culture medium comprising RPMI-based metabolic maturation media, AlbuMAX, higher calcium content and lower glucose content than the culture medium of the prior step. In some embodiments, the dissociating further comprising dissociating the cardiomyocytes and the primary cardiac fibroblasts with TrypLE™ or trypsin.

[0024] In some embodiments, in the method of identifying a compound that modulates a dynamic physical property of an engineered cardiac tissue the test compound is identified as a compound that modulates a dynamic physical property of the ECT tissue generated if in the presence of the test compound the ECT has significantly higher or lower active force, passive tension, total force, contraction velocity, relaxation velocity, or a combination thereof compared to the ECT cultured without the test compound or to a ECT comprising an isogenic control tissue or a wild-type tissue. In some embodiments, the test compound is identified as a compound that modulates a dynamic physical property of the ECT tissue generated if in the presence of the test compound the ECT has significantly higher or lower active force, passive tension, total force, contraction velocity, relaxation velocity, or a combination thereof compared to the ECT cultured without the test compound. In some embodiments, the test compound is identified as a compound that modulates a dynamic physical property of the ECT tissue generated if in the presence of the test compound the ECT has significantly higher or lower active force, passive tension, total force, contraction velocity, relaxation velocity, or a combination thereof compared to a ECT comprising an isogenic control tissue or a wildtype tissue.

[0025] In some embodiments, the cardiomyocytes are human. In some embodiments, the cardiomyocytes comprise filamin C (FLNC) with a mutation, a deletion or an addition of at least one nucleic acid residue or amino acid. In some embodiments, the FLNC deletion comprises a deletion of the nucleic acid residues from position 7416 to 7418. In some embodiments, the beating cardiomyocytes comprise a FLNC mutation, deletion, or addition that has been corrected to wild-type. [0026] In some embodiments, the culture medium of any of the methods described herein further comprises Glutamax. In some embodiments, the culture medium of any of the methods described herein further comprises EGF. In some embodiments, the culture medium of any of the methods described herein comprises 10 ng/ml of EGF. In some embodiments, the culture medium of any of the methods described herein further comprises antibiotic- antimycotic. In some embodiments, the culture medium of any of the methods described herein comprises 5% Matrigel. In some embodiments, the culture medium of any of the methods described herein comprises 5% heat-inactivated charcoal-stripped FBS.

[0027] In some embodiments, the test compound that increases the relaxation velocity of a beating cardiomyocyte is identified as a treatment for cardiomyopathy. In some embodiments, the test compound that increases passive tension of the ECT, increases the relaxation velocity of the ECT, or a combination thereof is identified as a treatment for cardiomyopathy.

[0028] In some embodiments, the test compound is a small molecule. In some embodiments, the test compound is an antibody. In some embodiments, the test compound is an antisense oligonucleotide. In some embodiments, the test compound is a phosphodiesterase 3 (PDE3) inhibitor.

[0029] In certain aspect, described herein is a method of treating a cardiomyopathy in a subject in need thereof, comprising: (a) culturing beating cardiomyocytes in two-dimensional culture, wherein the cardiomyocytes are derived from an induced pluripotent stem cell (iPSC); (b) contacting the beating cardiomyocytes with a test compound; (c) observing the beating cardiomyocytes with brightfield video microscopy; and (d) determining a contractile amplitude, a contraction velocity, a relaxation velocity or a combination thereof, wherein the test compound is administered to the subject if a contractile amplitude, a contractile velocity, a relaxation velocity or a combination thereof of the beating cardiomyocytes is increased as compared to a contractile amplitude, a contractile velocity, a relaxation velocity or a combination thereof of the beating cardiomyocyte in the absence of the test compound.

[0030] In certain aspect, described herein is a method of treating a cardiomyopathy in a subject in need thereof, comprising: (a) culturing beating cardiomyocytes in two-dimensional culture, wherein the cardiomyocytes are derived from an induced pluripotent stem cell (iPSC); (b) contacting the beating cardiomyocytes with a calcium-sensitive indicator; (c) measuring a signal of calcium flux generated from the calcium sensitive indicator; (d) contacting the beating cardiomyocytes with a test compound; (e) measuring a signal of calcium flux generated from the calcium sensitive indicator; and (f) determining a time constant of relaxation value using the calcium flux signal measured in step (e); and (g) identifying the test compound as a compound that modulates relaxation velocity of a beating cardiomyocyte if the time constant of relaxation value determined in step (f) is significantly higher or lower than either: i) a time constant of relaxation value for the beating cardiomyocyte determined using the calcium flux signal measured in step (c); or ii) a time constant of relaxation value for a control beating cardiomyocyte, wherein, the test compound is administered to the subject if relaxation velocity of a beating cardiomyocyte is higher in the presence of the test compound as compared to relaxation velocity of a beat cardiomyocytes in the absence of a test compound.

[0031] In certain aspect, described herein is a method of treating a cardiomyopathy in a subject in need thereof, comprising: (a) culturing an engineered cardiac tissue (ECT) comprising cardiomyocytes derived from iPSCs and human primary cardiac fibroblasts in a bioreactor, wherein the cardiomyocytes and fibroblasts are encapsulated in a fibrin gel and molded around two horizontal PDMS pillars and wherein the bioreactor further comprises electrodes for electrical stimulation of the ECT; (b) stimulating the ECT with an electrical stimulation; (c) maintaining the ECT with a lower electric stimulation than the electric stimulation used in step (b); (d) contacting the ECT with a test compound; and (e) determining a dynamic physical property of the ECT comprising (i) stimulating the ECT with electrical stimulation and capturing video of ECT contraction; (ii) analyzing pillar deflection by tracking pillar head movement and calculating displacement values, wherein the displacement values are used to calculate an active force, a residual force, a total force or a combination thereof, wherein the test compound is administered to the subject if the active force, the residual force, the total force or a combination thereof in the ECT is decreased in the presence of the test compound, as compared to the forces in the ECT in the absence of the test compound.

[0032] In some embodiments, the test compound is a small molecule. In some embodiments, the test compound is an antibody. In some embodiments, the test compound is an antisense oligonucleotide. In some embodiments, the test compound is a phosphodiesterase 3 (PDE3) inhibitor. In some embodiments, the phosphodiesterase inhibitor is trequinsin. In some embodiments, the test compound comprises at least one of the candidates in Fig. 4C. In some embodiments, the test compound is 17oc-hydroxyprogesterone. In some embodiments, the test compound is denbuphyilline. In some embodiments, the test compound is E4031.

[0033] In certain aspects, described herein is an in vitro cell line comprising a beating cardiomyocyte derived from an induced pluripotent stem cell (iPSC), wherein the beating cardiomyocyte comprises filamin C (FLNC) with a mutation, a deletion or an addition of at least one nucleic acid residue or amino acid.

[0034] In certain aspects, described herein is an in vitro cell line comprising a beating cardiomyocyte that is derived from an induced pluripotent stem cell (iPSC), wherein the beating cardiomyocyte comprises filamin C (FLNC) with a mutation, a deletion or an addition of at least one nucleic acid residue or amino acid; wherein the beating cardiomyocyte is cultured on a two-dimensional surface.

[0035] In some embodiments, the FLNC deletion comprises a deletion of the nucleic acid residues from position 7416 to 7418 as described herein. In some embodiments, the beating cardiomyocytes comprise a FLNC mutation, deletion, or addition that has been corrected to wild-type.

[0036] In certain aspects, described herein is an engineered cardiac tissue that comprises cardiomyocytes derived from an induced pluripotent stem cell (iPSC) and human primary cardiac fibroblasts, wherein the engineered cardiac tissue comprises filamin C (FLNC) with a mutation, a deletion or an addition of at least one nucleic acid residue or amino acid. In some embodiments, the FLNC deletion comprises a deletion of the nucleic acid residues from position 7416 to 7418 as described herein. In some embodiments, the beating cardiomyocytes comprise a FLNC mutation, deletion, or addition that has been corrected to wild-type.

[0037] In certain aspects, described herein is a cDNA library that comprises filamin C (FLNC) with a mutation, a deletion or an addition of at least one nucleic acid residue or amino acid. In some embodiments, the FLNC deletion comprises a deletion of the nucleic acid residues is from position 7416 to 7418 as described herein. BRIEF DESCRIPTION OF THE FIGURES

[0038] The patent or application file contains at least one drawing executed in color. To conform to the requirements for PCT patent applications, many of the figures presented herein are black and white representations of images originally created in color.

[0039] FIG. 1 shows a schematic of overall strategy of modeling restrictive cardiomyopathy and high throughput compound screening.

[0040] FIGS. 2A-2D show patient-derived iPSC-cardiomyocytes. FIG. 2A: Echocardiogram of proband with restrictive cardiomyopathy. Top left: apical four chamber image with enlarged left atria with normal LV thickness; top right', pulse doppler of mitral inflow with restrictive pattern; bottom left: tissue doppler imaging with low velocity of the mitral annulus ; bottom right: family pedigree. FIG. 2B: Immunofluorescence staining of iPSC-CM show colocalization of cardiac actinin and filamin C. FIG. 2C: Brightfield contraction analysis of FLNC^AGAA and FLNC^vWT iPSC-CM. (n = 24 wells, representative of 3 independent differentiations). FIG. 2D: Analysis of calcium decay by Tau in iPSC-CM (n = 384 wells, representative of 2 independent differentiations) *** p <0.001; **** p < 0.0001 by two-tailed student’s t-test. Error bars represent standard deviation. DAPI, 4',6-diamidino-2- phenylindole.

[0041] FIGS. 3A-3F show FLNC^AGAA impairs ECT function and relaxation. FIG. 3A: Representative brightfield microscopy of FLNC^AGAA and FLNC^vWT tissues at max relaxation. FIG. 3B: Immunofluorescence of ECT revealed significant sarcomere disarray in FLNC^AGAA compared o FLNC^vWT. FIG. 3C: Quantification of sarcomere fiber angle relative to tissue axis (n = 30 measurements of 3 tissues per group). FIG. 3D: Time-course of contractile force of cardiac tissues over three weeks of electromechanical stimulation, n = 3-6 tissues per group, representative of 3 independent experiments. FIG. 3E: Measurements of contractile force of cardiac tissues at 3 weeks after tissue formation, n = 3-6 tissues per group, representative of 3 independent experiments. FIG. 3F: Measurements of contraction and relaxation velocities at 3 weeks after tissue formation, n = 3-6 tissues per group, representative of 3 independent experiments. * p<0.05, *** <0.001, ****<0.0001 using Welch’s t-test or two-way ANOVA with Bonferroni correction. Error bars represent standard deviation. [0042] FIGS. 4A-4D show high throughput compound screening reveals phosphodiesterase inhibition as potential therapy. FIG. 4A: Calcium transients in FLNC^AGAA knock-in GcaMP6 iPSC-CM, showing lengthened tau (n = 96-127 per group, representative of 15 independent plates). FIG. 4B: Schematic of screen landscape. FIG. 4C: Top drugs altering tau at both 1 and 3 hour time point. FIG. 4D: Effect of trequinsin-HCl on the calcium tau in proband and corrected patient-derived iPSC-CM (n=24 wells, representative of 3 independent experiments). **** p <0.0001 with two-tailed student’s t-test or two-way ANOVA with Bonferonni correction. Error bars represent standard deviation. FC, fold change.

[0043] FIGS. 5A-5E show PDE3 inhibition ameliorates restrictive cardiomyopathy phenotype in ECT. Contraction analysis of tissues treated with lOpM trequinsin at 1 and 3 hour time points measuring FIG. 5A: total force generation FIG. 5B: active force FIG. 5C: passive tension FIG. 5D: contraction velocity, and FIG. 5E: relaxation velocity. N = 11-12 tissues per genotype, representative of 3 independent experiments. **** p<0.0001, *** p <0.001, ** p<0.01, repeated measures ANOVA with Dunnett’s multiple comparison’s test. Error bars represent standard deviation.

[0044] FIG. 6 shows CRISPR/Cas9 and homology directed repair of proband iPSC. Figure 6 discloses SEQ ID NOs: 1 and 2 in order of appearance.

[0045] FIGS. 7A-7C show characterization of iPSC-CM. FIG. 7A: Quantification of iPSC-CM cell size via confocal microscopy. FIG. 7B: Sarcomere length of iPSC-CM measured by ACTN2 staining. FIG. 7C: Western blot of soluble and insoluble fractions of iPSC-CM. Mean +/- SD, significance tested by two-tailed student’s t-test.

[0046] FIGS. 8A-8B show tissue formation protocol. FIG. 8A: Schematic of tissue formation protocol. FIG. 8B: Photo of tissue bioreactor casted from PDMS.

[0047] FIG. 9 shows sanger sequences of the WTC-11 Gcamp6f line with knocked-in FLNC^AGAA mutation. Figure 9 discloses SEQ ID NOs: 3 and 4 in order of appearance.

[0048] FIG. 10 shows cardiac tissue PKA activity measured after 1 hour treatment with trequinsin compared to an untreated DMSO control. N = per group, **** p< 0.0001 with two-tailed student’s t-test between groups within genotypes. [0049] FIGS. 11A-D show patient-derived iPSC cardiomyocytes. FIG. 11 A: Representative traces of bright-field contraction analysis of FLNCAGAA and FLNCyWT iPSC-CMs. FIG. 11B: Representative traces of calcium flux of FLNCAGAA and FLNCyWT iPSC-CMs. FIG. 11C: Amplitude measured using high-throughput fluorescence imaging (n = 384 wells, representative of 2 independent differentiations, captured using high- throughput fluorimetry). FIG. 11D: Time to peak measured using high-throughput fluorescence imaging (n = 384 wells, representative of 2 independent differentiations, captured using high-throughput fluorimetry).

[0050] FIGS. 12A-F show PDE3 inhibition ameliorates restrictive cardiomyopathy phenotype in ECT. FIG. 12A: Representative traces of ECT stimulated at 1 Hz before and after 1 h of trequisin treatment. FIG. 12B: Rescue of passive tension with trequinsin. FIG. 12C: Response of relaxation velocity in response to trequinsin treatment. FIG. 12D: Response of contraction velocity in response to trequinsin treatment. FIG. 12E: Time to 50% relaxation. FIG. 12F: Time to 50% contraction, (n = 11-12 tissues per genotype, representative of 3 independent experiments).

[0051] FIG. 13 shows characterization of iPSC-CM. Colocalization and Pearson correlation of FLNC and a-actinin from FIG. IB. n =3 independent images at 20x magnification per genotype.

[0052] FIGS. 14A-B show engineered heart tissues from milliPillar. FIG. 14A: Comparison of maturation media (MM) versus B27 media on engineered cardiac tissues after 4 weeks of culture and electromechanical stimulation. FIG. 14B: Maturation media causes a decrease in tissue width and an increase in tissue stress generation. N = 9-12 per group; normalized to B27 average. *p<0.05 **** P<0.0001 by two-tailed student’s t-test.

[0053] FIGS. 15A-E show validation in a knock-in cell line. FIG. 15A: Representative traces of GCAMP6AGAA and GCAMP6 ECTs. FIGS. 15B and 15C: Force and velocities from GCAMP6AGAA and isogenic control cardiac tissues. N = 9 per genotype. FIGS. 15D and 15E: Representative gating and flow cytometry analysis of cardiomyocyte size, dissociated from cardiac tissues. N = 3 **p <0.01, **** p<0.0001 by two-tailed student’s t- test.

[0054] FIGS. 16A-F show PDE inhibition in by trequinsin. FIG. 16A: Representative calcium decay in proband and pseudowild-type iPSC-CM from Figure 3D. FIG. 16B: 17a- hydroxyprogesterone and FIG. 16C: denbuphyilline measured over three hours. N = 3-5 tissues per group, * p<0.05 by two-way ANOVA and Sidak’s multiple comparison test. FIG. 16D: Representative traces of single cells paced at 0.5Hz under baseline or drug treatment. FIG. 16E: Quantification of average beat-to-beat frequency under baseline or drug treatment. N = 8-15 randomly selected cells per group. *p<0.05, **** p<0.0001 by two-way ANOVA with Dunnett’s multiple comparisons test. FIG. 16F: Lactate dehydrogenase cytotoxicity assay reveals no toxicity in response to trequinsin. N = 3 tissues per group, significance tested with two way ANOVA with Sidak’s multiple comparison test.

DETAILED DESCRIPTION

Definitions and Abbreviations

[0055] The term “FLNC” designates Filamin C.

[0056] The term “RCM” designates restrictive cardiomyopathy.

[0057] The term “HCM” designates hypertrophic cardiomyopathy.

[0058] The term “DCM” designates dilated cardiomyopathy.

[0059] The term “iPSC” designates induced pluripotent stem cell.

[0060] The term “iPSC-CM” designates induced pluripotent stem cell derived cardiomyocytes.

[0061] The term “PDE3” designates phosphodiesterase 3.

[0062] The term “2-D” designates 2-dimensional.

[0063] The term “3-D” designates 3 -dimensional.

[0064] The term “PBMC” designates peripheral mononuclear blood cells.

[0065] The term “ECT” designates engineered cardiac tissue.

[0066] The term “PDMS” designates polydimethylsiloxane.

[0067] The term “FITS” designates high throughput screening. [0068] The term “PKA” designates protein kinase A.

[0069] The term “TNNI3” designates troponin I.

[0070] The term “PLN” designates phospholamban.

[0071] The term “SERCA” designates sarcoplasmic reticulum Ca2+ ATPase.

[0072] The term “ACTN2” designates a-actinin 2.

[0073] The term “DAPI” designates 4',6-diamidino-2-phenylindole.

[0074] The singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0075] Described herein are stem cell derived cardiomyocytes from a patient with restrictive cardiomyopathy and a mutation in FLNC that display deficits in relaxation. Engineered cardiac tissues generated with these cells demonstrate increased passive tension and decreased relaxation velocity. High throughput calcium imaging of stem cell derived cardiomyocytes is able to identify compounds which alter relaxation. Engineered cardiac tissues can be used to validate compounds which improve cardiomyocyte relaxation.

[0076] Described herein are induced pluripotent stem cell derived cardiomyocytes (iPSC- CM) used to model restrictive cardiomyopathy (RCM) and to identify potential therapeutic candidates to improve cardiomyocyte relaxation.

[0077] Whole exome sequencing identified the novel mutation in FLNC (c.7416_7418delGAA, p.Glu2472_Asn2473delinAsp) in a patient with early onset restrictive cardiomyopathy. Pluripotent stem cells were generated from the patient (FLNC^AGAA) and an isogenic control line reverting the mutation to the wild type allele (/7,M'^{uW I}) was engineered using CRISPR-Cas9. Cardiomyocytes were differentiated and contractile properties were assessed using brightfield microscopy and calcium imaging. Engineered cardiac tissues (ECT) were constructed in a bioreactor and mechanical function was measured using video microscopy. High throughput compound screening was performed on cardiomyocytes in two- dimensional culture to identify compounds which alter calcium decay. Compound validation was then performed on engineered cardiac tissues. In some embodiments, aspects of the invention use a filamin C (FLNC) with a mutation, a deletion or an addition of at least one nucleic acid residue or amino acid. In some embodiments, the FLNC deletion comprises a deletion of the nucleic acid residues from position 7416 to 7418, wherein position 1 corresponds to the start codon of FLNC mRNA transcript variant 1 (e.g. Genbank Sequence ID NM 001458.5, incorporated herein by refernence). Deletion of “GAA” at position 7416- 7418 of the nucleic acid sequence encoding FLNC results in deletion of the last nucleic acid residue of the codon GAG encoding glutamic acid at position 2472 and the first two nucleic acid residues of codon AAC encoding asparagine at position 2473 and generates a codon GAC encoding aspartic acid.

[0078] FLNC^AGAA cardiomyocytes exhibited slower relaxation and prolonged calcium decay when grown in 2-dimensional culture. FLNC^AGAA CC displayed increased passive tension and slower relaxation velocities when compared to FLNC^vWT. High throughput screening and subsequent validation in ECT show that trequinsin, a phosphodiesterase inhibitor, improves measurements of passive tension and increases relaxation velocity in mutant cardiac tissues.

[0079] Restrictive cardiomyopathy can be modeled for drug discovery in both two and three-dimensional culture using cardiomyocytes derived from pluripotent stem cells.

Methods of Identifying Compounds

[0080] In certain aspects, described herein is a method of identifying a compound that modulates relaxation velocity of a contractile cell, the method comprising: (a) culturing contractile cells in two-dimensional culture, wherein the contractile cells are derived from an induced pluripotent stem cell (iPSC); (b) contacting the contractile cells with a calciumsensitive indicator; (c) measuring a signal of calcium flux generated from the calcium sensitive indicator; (d) contacting the contractile cells with a test compound; (e) measuring a signal of calcium flux generated from the calcium sensitive indicator; and (f) determining a time constant of relaxation value using the calcium flux signal measured in step (e); and (g) identifying the test compound as a compound that modulates relaxation velocity of a contractile cell if the time constant of relaxation value determined in step (f) is significantly higher or lower than either: (i) a time constant of relaxation value for the contractile cell determined using the calcium flux signal measured in step (c); or (ii) a time constant of relaxation value for a control contractile cell. [0081] In some embodiments, the contractile cell is a striated muscle cell. In some embodiments, the contractile cell is a smooth muscle cell. In some embodiments, the contractile cell is a skeletal muscle cell. In some embodiments, the contractile cell is cardiac cell.

[0082] In certain aspects, described herein is a method of identifying a compound that modulates relaxation velocity of a beating cardiomyocyte, the method comprising: (a) culturing beating cardiomyocytes in two-dimensional culture, wherein the cardiomyocytes are derived from an induced pluripotent stem cell (iPSC); (b) contacting the beating cardiomyocytes with a calcium-sensitive indicator; (c) measuring a signal of calcium flux generated from the calcium sensitive indicator; (d) contacting the beating cardiomyocytes with a test compound; (e) measuring a signal of calcium flux generated from the calcium sensitive indicator; and (f) determining a time constant of relaxation value using the calcium flux signal measured in step (e); and (g) identifying the test compound as a compound that modulates relaxation velocity of a beating cardiomyocyte if the time constant of relaxation value determined in step (f) is significantly higher or lower than either: (i) a time constant of relaxation value for the beating cardiomyocyte determined using the calcium flux signal measured in step (c); or (ii) a time constant of relaxation value for a control beating cardiomyocyte.

[0083] In some embodiments, the determining of step (f) in the method of identifying a compound that modulates relaxation velocity of a contractile cell or the method of identifying a compound that modulates relaxation velocity of a beating cardiomyocyte comprises: normalizing the signal of calcium flux measured in step (e) to the signal of calcium flux measured in step (c); identifying calcium flux signal peaks in the normalized calcium flux signal; for each peak, segmenting the portion of the normalized calcium flux signal from a maximum amplitude to a minimum value; performing a linear regression on the segmented normalized calcium flux signal to obtain a value of tau (T) corresponding to a time constant of relaxation value; averaging the value of tau across the number of signal peaks. In some embodiments the linear regression is performed using the following equation:

wherein y is the normalized calcium flux signal and t is time. [0084] In some embodiments, the test compound is identified as a compound that modulates relaxation velocity of a beating cardiomyocyte if the time constant of relaxation value determined in step (f) is significantly higher or lower than a time constant of relaxation value for the beating cardiomyocyte determined using the calcium flux signal measured in step (c). In some embodiments, the test compound is identified as a compound that modulates relaxation velocity of a beating cardiomyocyte if the time constant of relaxation value determined in step (f) is significantly higher or lower than a time constant of relaxation value for a control beating cardiomyocyte.

[0085] In some embodiments, the control beating cardiomyocyte is a control by virtue of being contacted with DMSO instead of a test compound. DMSO is an organosulfur compound that can be used as a vehicle for a test compound. Other known vehicles for test compounds can be used. Accordingly, in some embodiments, a control is contacted with the vehicle used to deliver the test compounds of interest.

[0086] In some embodiments, the time constant of relaxation value for the control beating cardiomyocyte is determined comprising: culturing beating cardiomyocytes in two- dimensional culture, wherein the cardiomyocytes are derived from an induced pluripotent stem cell (iPSC); contacting the beating cardiomyocytes with a calcium-sensitive indicator; measuring a signal of calcium flux generated from the calcium sensitive indicator; contacting the beating cardiomyocytes with DMSO; measuring a signal of calcium flux generated from the calcium sensitive indicator; and determining a time constant of relaxation value using the calcium flux signal measured after contacting the beating cardiomyocytes with DMSO. In some embodiments, determining the time constant of relaxation for the beating cardiomyocytes with DMSO comprises normalizing the signal of calcium flux measured after contacting the beating cardiomyocytes with DMSO to the signal of calcium flux measured prior to contacting the beating cardiomyocytes with DMSO; identifying calcium flux signal peaks in the normalized calcium flux signal; for each peak, segmenting the portion of the normalized calcium flux signal from a maximum amplitude to a minimum value; performing a linear regression on the segmented normalized calcium flux signal to obtain a value of tau (T) corresponding to a time constant of relaxation value; averaging the value of tau across the number of signal peaks. In some embodiments, the linear regression is performed using the following equation:

wherein y is the normalized calcium flux signal and t is time.

[0087] In some embodiments, the control beating cardiomyocyte and the cardiomyocyte contacted with the test compound are derived from the same iPSC culture. In some embodiments, culturing beating cardiomyocytes in two-dimensional culture comprises: isolating peripheral mononuclear blood cells from a blood sample from a subject; reprograming the isolated peripheral mononuclear blood cells to generate induced pluripotent stem cells (iPSCs); culturing the iPSCs in a culture medium comprising a cardiac differentiation media (CDM) containing RPMI1640, albumin and ascorbic acid to generate beating cardiomyocytes; culturing the beating cardiomyocytes in a culture medium comprising RPMI1640 with B27 supplement; culturing the beating cardiomyocytes at least one passage in a culture medium comprising RPMI1640 with B27 supplement and CHIR99021; and culturing the beating cardiomyocytes in a culture medium comprising RPMI160 with B27 supplement. In some embodiments, the beating cardiomyocytes are maintained in a culture medium comprising RPMI160 with B27 supplement. In some embodiments, the contractile cells are human. In some embodiments, beating cardiomyocytes are human. In some embodiments, the peripheral mononuclear blood cells are isolated by immunomagnetic cell separation. In some embodiments, the immunomagnetic cell separation uses an antibody against Epithelial Cell Adhesion Molecule (EpCAM). In some embodiments, the method further comprises: serially passaging the beating cardiomyocytes.

[0088] In some embodiments, the beating cardiomyocytes are cultured in a multi-well plate and each well of the multi-well plate is used to test one or more different test compounds or different concentrations of one or more test compounds. In some embodiments, the control beating cardiomyocyte is cultured in a well of the same multi-well plate as the beating cardiomyocytes contacted with test compound. In some embodiments, a second control beating cardiomyocyte is cultured in a well of the same multi-well plate as the beating cardiomyocytes contacted with test compound, wherein the second control is contacted with a compound that is known to modulate the relaxation velocity of a beating cardiomyocyte. In some embodiments, the multi -well plate is a 384 well plate. In some embodiments, the multi-well plate is a 6 well plate, a 12 well plate, a 24 well plate, a 48 well plate, a 96 well plate, a 384 well plate, or a 1536 well plate. [0089] In some embodiments, the method of identifying a compound that modulates relaxation velocity of a beating cardiomyocyte is for identifying a test compound that increases the relaxation velocity of a beating cardiomyocyte, wherein if the time constant of relaxation value determined in step (f) is significantly lower than either condition (i) or (ii) described above, the test compound is identified as a compound that increases the relaxation velocity of a beating cardiomyocyte. In some embodiments, the method of identifying a compound that modulates relaxation velocity of a beating cardiomyocyte is for identifying a test compound that decreases the relaxation velocity of a beating cardiomyocyte, wherein if the time constant of relaxation value determined in step (f) is significantly higher than either condition (i) or (ii) described above the test compound is identified as a compound that decreases the relaxation velocity of a beating cardiomyocyte. In some embodiments, culture medium of the culturing of step (a) is replaced with fresh culture medium before contacting the beating cardiomyocytes with the calcium-sensitive indicator.

[0090] In some embodiments, the calcium sensitive indicator is a chemical sensitive dye, a genetically encoded calcium indicator, or a combination thereof. In some embodiments, the chemical indicator is a calcium sensitive dye. In some embodiments, the genetically encoded calcium indicator is GCaMP. In some embodiments, the calcium-sensitive indicator is Calcium 6. Calcium sensitive dyes are known in the art and can be utilized for the optical assessment of Ca2+ fluctuations. Calcium sensitive dyes are typically fluorescent molecules that respond to the binding of Ca2+ ions by fluorescence properties. Genetically encodable calcium indicators are proteins whose sequence is encoded by one or more nucleic acids that can be introduced into cells using known techniques.

[0091] In some embodiments, the beating cardiomyocytes are contacted with the test compound for 1 hour. In some embodiments, the beating cardiomyocytes are contacted with the test compound for 3 hours. In some embodiments, the beating cardiomyocytes are contacted with the test compound for 1 day. In some embodiments, the beating cardiomyocytes are contacted with the test compound for 1 week. In some embodiments, the beating cardiomyocytes are contacted with the test compound for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours. In some embodiments, the beating cardiomyocytes are contacted with the test compound for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days. In some embodiments, the beating cardiomyocytes are contacted with the test compound for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or longer.

[0092] In some embodiments, the beating cardiomyocytes comprise filamin C (FLNC) with a mutation, a deletion or an addition of at least one nucleic acid residue or amino acid. In some embodiments, the FLNC deletion comprises a deletion of the nucleic acid residues from position 7416 to 7418, as described herein. In some embodiments, the beating cardiomyocytes comprise a FLNC mutation, deletion, or addition that has been corrected to wild-type. FLMN C as described herein has GenBank Gene ID: 2318. The nucleotide and amino acid sequences can be readily obtained by one of ordinary skill in the art using the listed accession numbers and are hereby incorporated by reference in their entireties.

[0093] In some embodiments, the beating cardiomyocytes display a phenotype of cardiomyopathy. In some embodiments, the beating cardiomyocytes express a genetically encoded calcium indicator. In some embodiments, the genetically encoded calcium indicator is GcaMP.

[0094] In certain aspects, described herein is a method of observing a dynamic physical property of a beating cardiomyocyte, the method comprising: culturing beating cardiomyocytes in two-dimensional culture, wherein the cardiomyocytes are derived from an induced pluripotent stem cell (iPSC); observing the beating cardiomyocytes with brightfield video microscopy; and determining the contractile amplitude, contraction velocity, relaxation velocity, or a combination thereof. In some embodiments, the determining is performed using pixel intensity subtraction. In some embodiments, the video microscopy is performed at about 100 frames per second. In some embodiments, the beating cardiomyocytes are cultured at about 200,000 cells per cm². In some embodiments, the beating cardiomyocytes are cultured in a multi-well plate. In some embodiments, the method further comprises contacting the cells with a test compound and determining the contractile amplitude, contraction velocity, relaxation velocity, or a combination thereof in the presence of the test compound. [0095] Pixel intensity subtraction can be performed as described in Sala L, van Meer B J, Tertoolen LGJ, Bakkers J, Beilin M, Davis RP, Denning C, Dieben MAE, Eschenhagen T, Giacomelli E, Grandela C, Hansen A, Holman ER, Jongbloed MRM, Kamel SM, Koopman CD, Lachaud Q, Mannhardt I, Mol MPH, Mosqueira D, Orlova VV, Passier R, Ribeiro MC, Saleem U, Smith GL, Burton FL, Mummery CL. MUSCLEMOTION: A Versatile Open Software Tool to Quantify Cardiomyocyte and Cardiac Muscle Contraction In Vitro and In Vivo. Circ Res. 2018 Feb 2;122(3):e5-el6, the content of which is hereby incorporated by reference in its entirety. In some embodiments, the method of observing a dynamic physical property of a beating cardiomyocyte is performed before or after performing the method of identifying a compound that modulates relaxation velocity of a beating cardiomyocyte as described herein. For example, the method of observing a dynamic physical property of a beating cardiomyocyte could be used to validate a compound identified as modulating a relaxation velocity of a beating cardiomyocyte as described herein or vice versa.

[0096] In certain aspects, described herein is a method of identifying a compound that modulates a dynamic physical property of an engineered cardiac tissue, the method comprising: (a) culturing an engineered cardiac tissue (ECT) comprising cardiomyocytes derived from iPSCs and human primary cardiac fibroblasts in a bioreactor, wherein the cardiomyocytes and fibroblasts are encapsulated in a fibrin gel and grow around two horizontal PDMS pillars of the bioreactor, wherein the bioreactor further comprises electrodes for electrical stimulation of the ECT; (b) stimulating the ECT with an electrical stimulation; (c) maintaining the ECT with a lower electric stimulation than the electric stimulation used in step (b); (d) determining a dynamic physical property of the ECT comprising (i) stimulating the ECT with electrical stimulation and capturing video of ECT contraction; (ii) analyzing pillar deflection.

[0097] As described here, a software pipeline can be used to automatically track the movement of the pillars across frames in a recording of the ECT contracting followed by calculating the velocity from the displacement. See Tamargo MA, Nash TR, Fleischer S, Kim Y, Vila OF, Yeager K, Summers M, Zhao Y, Lock R, Chavez M, Costa T, Vunjak- Novakovic G. milliPillar: A Platform for the Generation and Real-Time Assessment of Human Engineered Cardiac Tissues. ACS Biomater Sci Eng. 2021 Nov 8;7(11):5215-5229, the content of which is hereby incorporated by reference in its entirety. Described herein is the use of this technique to measure altered relaxation velocity recapitulating a disease phenotype.

[0098] In certain aspects, electrical stimulation used to stimulate the ECT is a 2-week ramped electrical stimulation regimen which lasted from 2Hz and increased 0.33Hz every 24 hours until 6Hz. After this two-week regimen, tissues were maintained electrically paced at 1 Hz.

[0099] In some embodiments, analyzing the pillar deflection comprises: tracking pillar head movement and calculating displacement values. In some embodiments, the method further comprises using the displacement values to calculate force. In some embodiments, the force is calculated using the known force needed to deflect a pillar of the bioreactor. In some embodiments, the force calculated is the force that displaces pillars during ECT contraction (active force). In some embodiments, the force calculated is the force causing pillar deflection during maximum relaxation of the ECT (residual or passive force). In some embodiments, the force calculated is the sum of the active force that displaces pillars during ECT contraction and the residual force causing pillar deflection during maximum relaxation of the ECT (total force). In some embodiments, the method further comprises determining a relaxation velocity measurement, a contraction velocity measurement, or both, using the derivative of the displacement values.

[00100] In some embodiments, the method further comprises contacting the ECT with a test compound and determining whether the test compound modulates a dynamic physical property of the ECT comprising: stimulating the ECT in the presence of the test compound with electrical stimulation and capturing video of ECT contraction and analyzing pillar deflection. In some embodiments, analyzing the pillar deflection in the presence of the test compound comprises: tracking pillar head movement and calculating displacement values in the presence of the test compound. In some embodiments, the method further comprises using the displacement values in the presence of the test compound to calculate force in the presence of the test compound. In some embodiments, the force in the presence of the test compound is calculated using the known force needed to deflect a pillar of the bioreactor. In some embodiments, the force calculated is the force that displaces pillars during ECT contraction in the presence of the test compound (active force). In some embodiments, the force calculated is force causing pillar deflection during maximum relaxation of the ECT in the presence of the test compound (residual or passive force). In some embodiments, the force calculated is the sum of the active force that displaces pillars during ECT contraction in the presence of the test compound and the residual force causing pillar deflection during maximum relaxation of the ECT in the presence of the test compound (total force). In some embodiments, the method further comprises determining a relaxation velocity measurement in the presence of the test compound, a contraction velocity measurement in the presence of the test compound, or both, using the derivative of the displacement values in the presence of the test compound.

[00101] In some embodiments, in the method of identifying a compound that modulates a dynamic physical property of an engineered cardiac tissue the culturing an ECT of step (a) comprises: dissociating cardiomyocytes and primary cardiac fibroblasts; suspending the dissociated cells in a fibrinogen solution; mixing the suspended cells with thrombin and placing in contact with the PDMS pillars to generate an ECT; maintaining the ECT in a culture medium comprising B27 media and 6-aminocaproic acid; and maintaining the tissue in a culture medium comprising RPMI-based metabolic maturation media, AlbuMAX, higher calcium content and lower glucose content than the culture medium of the prior step. In some embodiments, the dissociating further comprising dissociating the cardiomyocytes and the primary cardiac fibroblasts with TrypLE™ or trypsin.

[00102] In some embodiments, in the method of identifying a compound that modulates a dynamic physical property of an engineered cardiac tissue the test compound is identified as a compound that modulates a dynamic physical property of the ECT tissue generated if in the presence of the test compound the ECT has significantly higher or lower active force, passive tension, total force, contraction velocity, relaxation velocity, or a combination thereof compared to the ECT cultured without the test compound or to a ECT comprising an isogenic control tissue or a wild-type tissue. In some embodiments, the test compound is identified as a compound that modulates a dynamic physical property of the ECT tissue generated if in the presence of the test compound the ECT has significantly higher or lower active force, passive tension, total force, contraction velocity, relaxation velocity, or a combination thereof compared to the ECT cultured without the test compound. In some embodiments, the test compound is identified as a compound that modulates a dynamic physical property of the ECT tissue generated if in the presence of the test compound the ECT has significantly higher or lower active force, passive tension, total force, contraction velocity, relaxation velocity, or a combination thereof compared to a ECT comprising an isogenic control tissue or a wildtype tissue.

[00103] In some embodiments, the cardiomyocytes are human. In some embodiments, the cardiomyocytes comprise filamin C (FLNC) with a mutation, a deletion or an addition of at least one nucleic acid residue or amino acid. In some embodiments, the FLNC deletion comprises a deletion of the nucleic acid residues from position 7416 to 7418 as described herein. In some embodiments, the beating cardiomyocytes comprise a FLNC mutation, deletion, or addition that has been corrected to wild-type.

[00104] In some embodiments, the culture medium of any of the methods described herein further comprises Glutamax. In some embodiments, the culture medium of any of the methods described herein further comprises EGF. In some embodiments, the culture medium of any of the methods described herein comprises 10 ng/ml of EGF. In some embodiments, the culture medium of any of the methods described herein further comprises antibiotic- antimycotic. In some embodiments, the culture medium of any of the methods described herein comprises 5% Matrigel. In some embodiments, the culture medium of any of the methods described herein comprises 5% heat-inactivated charcoal-stripped FBS.

[00105] In some embodiments, the test compound is a small molecule. In some embodiments, the test compound is an antibody. In some embodiments, the test compound is an antisense oligonucleotide. In some embodiments, the test compound is a phosphodiesterase 3 (PDE3) inhibitor. In some embodiments, the phosphodiesterase inhibitor is trequinsin. In some embodiments, the test compound comprises at least one of the candidates in Fig. 4C. In some embodiments, the test compound is 17oc-hydroxyprogesterone. In some embodiments, the test compound is denbuphyilline. In some embodiments, the test compound is E4031. In certain embodiments, a test compound according to the methods described herein can be added alone, or in combination with other drugs therapies, small molecules, biologically active or inert compounds, or other additive intended to enhance the delivery, efficacy, tolerability, or function of the test compound.

[00106] Test compounds can be screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available, or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means.

[00107] Methods for preparing libraries of molecules are well known in the art and many libraries are commercially available. Libraries of interest include peptide libraries, randomized oligonucleotide libraries, synthetic organic combinatorial libraries, and the like. Degenerate peptide libraries can be readily prepared in solution, in immobilized form as bacterial flagella peptide display libraries or as phage display libraries. Peptide ligands can be selected from combinatorial libraries of peptides containing at least one amino acid. Libraries can be synthesized of peptoids and non-peptide synthetic moieties. Such libraries can further be synthesized which contain non-peptide synthetic moieties, which are less subject to enzymatic degradation compared to their naturally-occurring counterparts. Libraries are also meant to include for example but are not limited to peptide-on-plasmid libraries, polysome libraries, aptamer libraries, synthetic peptide libraries, synthetic small molecule libraries, neurotransmitter libraries, and chemical libraries. The libraries can also comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the functional groups.

[00108] Small molecule combinatorial libraries can also be generated and screened. A combinatorial library of small organic compounds is a collection of closely related analogs that differ from each other in one or more points of diversity and are synthesized by organic techniques using multi-step processes. Combinatorial libraries include a vast number of small organic compounds. One type of combinatorial library is prepared by means of parallel synthesis methods to produce a compound array. A compound array can be a collection of compounds identifiable by their spatial addresses in Cartesian coordinates and arranged such that each compound has a common molecular core and one or more variable structural diversity elements. The compounds in such a compound array are produced in parallel in separate reaction vessels, with each compound identified and tracked by its spatial address. Examples of parallel synthesis mixtures and parallel synthesis methods are known in the art. Numerous examples of chemically synthesized libraries are described in the art. Methods of Treatment

[00109] In certain aspects, described herein is a method of treating a cardiomyopathy in a subject in need thereof, comprising: (a) culturing beating cardiomyocytes in two-dimensional culture, wherein the cardiomyocytes are derived from an induced pluripotent stem cell (iPSC); (b) contacting the beating cardiomyocytes with a test compound; (c) observing the beating cardiomyocytes with brightfield video microscopy; and (d) determining a contractile amplitude, a contraction velocity, a relaxation velocity or a combination thereof, wherein the test compound is administered to the subject if a contractile amplitude, a contractile velocity, a relaxation velocity or a combination thereof of the beating cardiomyocytes is increased as compared to a contractile amplitude, a contractile velocity, a relaxation velocity or a combination thereof of the beating cardiomyocyte in the absence of the test compound.

[00110] In certain aspects, described herein is a method of treating a cardiomyopathy in a subject in need thereof, comprising: (a) culturing beating cardiomyocytes in two-dimensional culture, wherein the cardiomyocytes are derived from an induced pluripotent stem cell (iPSC); (b) contacting the beating cardiomyocytes with a calcium-sensitive indicator; (c) measuring a signal of calcium flux generated from the calcium sensitive indicator; (d) contacting the beating cardiomyocytes with a test compound; (e) measuring a signal of calcium flux generated from the calcium sensitive indicator; and (f) determining a time constant of relaxation value using the calcium flux signal measured in step (e); and (g) identifying the test compound as a compound that modulates relaxation velocity of a beating cardiomyocyte if the time constant of relaxation value determined in step (f) is significantly higher or lower than either:!) a time constant of relaxation value for the beating cardiomyocyte determined using the calcium flux signal measured in step (c); or ii) a time constant of relaxation value for a control beating cardiomyocyte, wherein the test compound is administered to the subject if relaxation velocity of a beating cardiomyocyte is higher in the presence of the test compound as compared to relaxation velocity of a beat cardiomyocytes in the absence of a test compound.

[00111] In certain aspects, described herein is a method of treating a cardiomyopathy in a subject in need thereof, comprising: (a) culturing an engineered cardiac tissue (ECT) comprising cardiomyocytes derived from iPSCs and human primary cardiac fibroblasts in a bioreactor, wherein the cardiomyocytes and fibroblasts are encapsulated in a fibrin gel and molded around two horizontal PDMS pillars and wherein the bioreactor further comprises electrodes for electrical stimulation of the ECT; (b) stimulating the ECT with an electrical stimulation; (c) maintaining the ECT with a lower electric stimulation than the electric stimulation used in step (b); (d) contacting the ECT with a test compound; and (e) determining a dynamic physical property of the ECT comprising (i) stimulating the ECT with electrical stimulation and capturing video of ECT contraction; (ii) analyzing pillar deflection by tracking pillar head movement and calculating displacement values, wherein the displacement values are used to calculate an active force, a residual force, a total force or a combination thereof; wherein the test compound is administered to the subject if the active force, the residual force, the total force or a combination thereof in the ECT is decreased in the presence of the test compound, as compared to the forces in the ECT in the absence of the test compound.

[00112] In some embodiments, the test compound is a small molecule. In some embodiments, the test compound is an antibody. In some embodiments, the test compound is an antisense oligonucleotide. In some embodiments, the test compound is a phosphodiesterase 3 (PDE3) inhibitor. In some embodiments, the phosphodiesterase inhibitor is trequinsin. In some embodiments, the test compound comprises at least one of the candidates in Fig. 4C. In some embodiments, the test compound is 17oc-hydroxyprogesterone. In some embodiments, the test compound is denbuphyilline. In some embodiments, the test compound is E4031. In certain embodiments, a test compound to be administered according to the methods described herein can be administered alone, or in combination with other drugs therapies, small molecules, biologically active or inert compounds, or other additive intended to enhance the delivery, efficacy, tolerability, or function of the test compound.

Cell Lines and Nucleic Acids

[00113] In certain aspects, described herein is an in vitro cell line comprising a beating cardiomyocyte derived from an induced pluripotent stem cell (iPSC), wherein the beating cardiomyocyte comprises filamin C (FLNC) with a mutation, a deletion or an addition of at least one nucleic acid residue or amino acid.

[00114] In certain aspects, described herein is an in vitro cell line comprising a beating cardiomyocyte that is derived from an induced pluripotent stem cell (iPSC), wherein the beating cardiomyocyte comprises filamin C (FLNC) with a mutation, a deletion or an addition of at least one nucleic acid residue or amino acid; wherein the beating cardiomyocyte is cultured on a two-dimensional surface. [00115] In some embodiments, the cell line is derived from a stem cell, a progenitor cell, or a cell obtained by directed differentiation of the stem cell or the progenitor cell. In some embodiments, a stem cell is an embryonic stem cell, an induced pluripotent stem cell, or a totipotent stem cell. In another embodiment, the cell line is a cell obtained by in vitro differentiation of a stem cell or a progenitor cell wherein the stem cell or progenitor cell is genetically modified and then differentiated in vitro.

[00116] In some embodiments, a cell is genetically modified by expressing an exogenous nucleic acid. In some embodiment, the exogenous expression nucleic acid is an expression vector, the expression vector is a retroviral vector or a recombinant viral vector. In some embodiments, where the exogenous nucleic acid is an expression vector, the expression vector is a targeting vector.

[00117] In some embodiments, the FLNC deletion comprises a deletion of the nucleic acid residues from position 7416 to 7418 as described herein. In some embodiments, the beating cardiomyocytes comprise a FLNC mutation, deletion, or addition that has been corrected to wild-type.

[00118] In certain aspects, described herein is an engineered cardiac tissue that comprises cardiomyocytes derived from an induced pluripotent stem cell (iPSC) and human primary cardiac fibroblasts, wherein the engineered cardiac tissue comprises filamin C (FLNC) with a mutation, a deletion or an addition of at least one nucleic acid residue or amino acid.

[00119] In some embodiments, the FLNC deletion comprises a deletion of the nucleic acid residues from position 7416 to 7418 as described herein. In some embodiments, the beating cardiomyocytes comprise a FLNC mutation, deletion, or addition that has been corrected to wild-type.

[00120] In certain aspects, described herein is a cDNA library that comprises filamin C (FLNC) with a mutation, a deletion or an addition of at least one nucleic acid residue or amino acid.

[00121] In some embodiments, the FLNC deletion comprises a deletion of the nucleic acid residues from position 7416 to 7418 as described herein. Methods of Administering

[00122] Indications, dosage and methods of administration of the drugs of the present invention are known to one of skill in the art. In some embodiments, a drug of the present invention can be supplied in the form of a pharmaceutical composition, comprising an isotonic excipient prepared under sufficiently sterile conditions for human administration. Choice of the excipient and any accompanying elements of the composition will be adapted in accordance with the route and device used for administration. In some embodiments, a composition comprising a drug of the present invention can also comprise, or be accompanied with, one or more other ingredients that facilitate the delivery or functional mobilization of the drugs of the present invention.

[00123] These methods described herein are by no means all-inclusive, and further methods to suit the specific application is understood by the ordinary skilled artisan. Moreover, the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect.

[00124] According to the invention, a pharmaceutically acceptable carrier can comprise any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is compatible with the active compound can be used. Supplementary active compounds can also be incorporated into the compositions.

[00125] Pharmaceutical compositions for use in accordance with the invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. The therapeutic compositions of the invention can be formulated for a variety of routes of administration, including systemic and topical or localized administration. Techniques and formulations generally can be found in Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa (20^th ed., 2000), the entire disclosure of which is herein incorporated by reference.

[00126] Any of the therapeutic applications described herein can be applied to any subject in need of such therapy, including, for example, a mammal such as a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human. In certain embodiments, the therapeutic applications described herein can be applied to a human. [00127] Administration of a drug of the present invention is not restricted to a single route, but may encompass administration by multiple routes. Multiple administrations may be sequential or concurrent. Other modes of application by multiple routes will be apparent to one of skill in the art.

***

[00128] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[00129] All publications and other references mentioned herein are incorporated by reference in their entirety, as if each individual publication or reference were specifically and individually indicated to be incorporated by reference. Publications and references cited herein are not admitted to be prior art.

EXAMPLES

EXAMPLE 1 - Engineered heart tissue model of restrictive cardiomyopathy for drug discovery

[00130] Restrictive cardiomyopathy is defined as increased myocardial stiffness and impaired diastolic relaxation leading to elevated ventricular filling pressures. As opposed to the morphological phenotypes in hypertrophic and dilated cardiomyopathy, restrictive cardiomyopathy is functionally defined and thus has been difficult to model in two- dimensional culture. Human mutations in FLNC have been linked to a variety of cardiomyopathies and described herein is a novel mutation (c.7416_7418delGAA, p.Glu2472_Asn2473delinAsp) in a patient with RCM. iPSC-CM with this mutation display impaired contraction and reduced calcium kinetics in two-dimensional culture when compared to a CRISPR-Cas9 corrected isogenic control line. In order to recapitulate a restrictive phenotype, three-dimensional engineered heart tissue was generated and demonstrated that with both mechanical and metabolic maturation, patient derived engineered cardiac tissues displayed increased passive tension and impaired relaxation velocity compared to an isogenic control. High throughput small molecule screening utilizing calcium fluorescence in iPSC-CM identified phosphodiesterase 3 (PDE3) inhibition by trequinsin as a potential therapy to improve cardiomyocyte relaxation. This was validated in engineered heart tissue as trequinsin ameliorated the restrictive phenotype, improving both passive tension and relaxation velocity. Together, these data demonstrate the first engineered heart tissue model of RCM and establish the therapeutic potential of this system to identify therapeutics targeting myocardial relaxation.

Introduction

[00131] Restrictive cardiomyopathy is defined as increased myocardial stiffness and impaired relaxation leading to pulmonary hypertension and heart failure (Muchtar E et. al. Circ Res. 121 (7): 819-837, 2017). Though less common than hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM), RCM prognosis is one of the poorest owing to a lack of therapies (Felker GM et. al. N Engl J Med 342(15): 1077-1084, 2000). The phenotype of RCM arises from several etiologies including infiltrative processes, storage diseases, endomyocardial processes, radiation, drug exposure and mutations to the sarcomeric apparatus (Muchtar E et. al. Circ Res. 121 (7) :819-837, 2017). There are no approved therapies that directly target RCM and treatment centers on careful volume management and identifying reversible causes.

[00132] Pluripotent stem cells have been increasingly used to study patient-specific mutations as in-vitro models for disease modeling and therapeutic screening. There are currently no published models of restrictive cardiomyopathy using iPSC-CM. RCM is inherently more difficult to model, as unlike DCM and HCM which rely on morphological criteria, RCM is defined functionally as restricted ventricular filling. The hallmarks of RCM are an increase in myocardial wall tension and failure to relax during diastole (Mogensen J & Arbustini E, Curr Opinion Cardiol, 24(3)214-220, 2009), parameters which are difficult to measure in cells attached to plastic substrate. Recent advances in induced pluripotent stem cell (iPSC)-based cardiac tissue engineering have enabled the ability to capture more clinically relevant measures of cardiac phenotypes, for which classical 2-dimensional (2D) cell culture is lacking (Ronaldson-Bouchard K et. al Nature, 2018). Uniquely, cardiac tissues provide measurements of contractile force, and promote the maturation of iPSC-CM in a 3- dimensional microenvironment that mimics native tissue (Mosqueira D et. al. Eur Heart J. 39(43)2879-3892, 2018; Wang G et. al. Nat Med. 20(6):616-623, 2014). Recently, a platform was reported for the generation and real-time assessment of engineered cardiac tissues in a medium-throughput manner, enabling the quantification of clinical-like parameters throughout the cardiac contraction cycle such as contractile force and muscle tension, and relaxation velocity (Tamargo MA et. al. ACS Biomater Sci Eng. 2021).

[00133] Filamin C (FLNC) is an actin cross-linking protein with a known role in sarcomeric protein organization (Van der Flier A * Sonnenberg A, Biochim Biophys Acta - Mol Cell Res. 1538(2-3) :99-l 17, 2001). Deletion of Fine in mice causes early death with a severe cardiac phenotype (Dalkilic I et. al. Mol Cell Biol 26(17):6522-6534, 2006; Zhou Y et. al. Circulation 869-871, 2020). In the last decade, pathogenic mutations in FLNC have been identified in myofibrillar myopathy (Furst DO et. al. Acta Neuropathol 125(1): 33 -46, 2013; Ruparelia AA et. al. Hum Mol Genet. 25(11):2131-2142, 2016), DCM (Janin A et. al. Clin Genet. 92(6):616-623, 2017), HCM (Valdes-Mas R et. al. Nat Commun. 5, 2014; Gomez J et. al. Circ Cardiovasc Genet.10(2), 2017), and RCM (Brodehl A et. al. Hum Mutat. 37(3):269- 279, 2016; Roldan-Sevilla A et. al. Circ Genomic Precis Med. 12(3), 2020; Schubert J et. al. Hum Mutat. 39(12):2083-2096, 2018; Tucker NR et. al. Circ Cardiovasc Genet. 10(6), 2017), underscoring its importance in myocardial integrity. Described herein is a novel heterozygous in frame deletion in FLNC (c.7416_7418delGAA, p.Glu2472_Asn2473delinAsp) in a young patient with RCM. iPSC-CM’s were generated and studied successively using a 2D high-throughput drug screening approach and 3D engineered cardiac tissues (FIG. 1). Also described herein is the use of engineered tissues to recapitulate the phenotype of restrictive cardiomyopathy displaying decreased relaxation velocity and increased passive tension. Further described herein is the identification of PDE3 inhibition as a potential therapeutic target for FLNC RCM.

Results

An inframe deletion in FLNC leads to restrictive cardiomyopathy and defects in cardiomyocyte relaxation.

[00134] Echocardiography of a 3 year old male patient showed normal left ventricular function and thickness with a dilated left atrium, right ventricular hypertrophy, restrictive filling pattern, reduced tissue doppler velocity and elevated estimated pulmonary pressures, all consistent with a restrictive cardiomyopathy (FIG. 2A). The patient and his parents underwent whole exome sequencing revealing a de novo in-frame mutation in FLNC (c.7416_7418delGAA p.Glu2472_Asn2473delinsAsp) which was further confirmed by exon specific PCR. This mutation is located in exon 44, in the 22^nd immunoglobulin-like domain repeat (R22) of FLNC. Both Glu2472 and Asn2473 are strictly conserved across 100 species and the mutation was predicted to be deleterious by PROVEAN. The patient’s family provided consent for generation of an iPSC line under a protocol approved by the Columbia University Institutional Review Board.

[00135] Patient-specific iPSC’s (FLNC^AGAA) were reprogrammed PBMC’s isolated from the proband, and an isogenic cell line with the mutation corrected to pseudo-wildtype ( NC'^JWG) was engineered using CRISPR-Cas9 (FIG. 6). FLNC^AGAA and FLNC^ iPSC were differentiated into cardiomyocytes. Immunofluorescence of iPSC-CM showed colocalization of FLNC and sarcomeric u-actinin (FIG. 2B). No significant differences in either cell size, or sarcomere length between FLNC^AGAA and FLNC^vWT cardiomyocytes were observed (FIGS. 7A and 7B). There were also no differences observed in the solubility FLNC^AGAA and FLNC^vWT as had been previously reported for other mutations in FLNC (FIG. 7C) (Lowe T et. al. Hum Mol Genet 16(11): 1351-1358, 2007; Agarwal R et. al. Circ Res. 751-766, 2021). Video analysis of beating iPSC-CM revealed decreased contractile amplitude, contraction velocity, and relaxation velocity in FLNC^AGAA cardiomyocytes (FIG. 2C). Since calcium flux is critical for sarcomeric relaxation, the time constant of relaxation, tau(r), was evaluated using a calcium dye on beating cardiomyocytes. FLNC^AGAA cardiomyocytes displayed significantly longer tau compared to FLNC^vWT cardiomyocytes, paralleling the deficit in relaxation observed in contraction analysis (FIG. 2D). Together, these data demonstrate the FLNC^AGAA mutation causes deficiencies in cardiomyocyte relaxation velocity and calcium flux decay.

FLNC^AGAA engineered cardiac tissues model restrictive cardiomyopathy.

[00136] Recent advances have highlighted the relative immaturity of iPSC-CM cultured on 2-dimensional (2D) surfaces, and suggest that engineering of 3 -dimensional cardiac tissues enables maturation of iPSC-CM in a native-like microenvironment (Ronaldson-Bouchard K et. al Nature, 2018; Campostrini G et. al. Circ Res. 128:775-801, 2021). Without being bound by theory, the use of engineered cardiac tissue (ECT) and both metabolic and physical maturation could be used to further model the relaxation deficit in FLNC^AGAA iPSC-CM. ECT’s were created from FLNC^AGAA and FLNC^vWT iPSC-CM using the previously published method, in which cells encapsulated in a fibrin gel are molded around two horizontal pillars cast from polydimethylsiloxane (PDMS) (FIG. 3A and FIGS. 8A-8B) (Tamargo MA et. al. ACS Biomater Sci Eng. 2021). A two-staged method was used to encourage tissue maturation. First, a culture medium high in fatty acids was used to favor fatty acid oxidation, which improves physiological function (Feyen DAM et. al. Cell Rep. 32(3), 2020). One week after formation, tissues were then subjected to two weeks of electromechanical stimulation which was shown previously to improve iPSC-CM maturation and gene expression(Ronaldson-Bouchard K et. al Nature, 2018).

[00137] Whole-mount immunofluorescence imaging at 3 weeks was used to assess sarcomeric structure in situ. Compared to FLNC^vWT ECTs, FLNC^AGAA ECTs exhibited sarcomeric disorganization with significant deviation of actinin fibers from the tissue longitudinal axis (FIGS. 3B and C). ECT function was assessed using video microscopy to track pillar deflection. Based on pillar displacement and the previously performed empirical measurements (Tamargo MA et. al. ACS Biomater Sci Eng. 2021), force generation is able to be calculated with three specific metrics: (1) an active force, which is the force that displaces the pillars during tissue contraction; (2) passive tension, which denotes the residual force causing pillar deflection during maximum relaxation; (3) total force, which is the sum of both passive and active forces. Overall force generated by FLNC^AGAA and FLNC^vWT tissues when paced at 1Hz increased a similar amount over the maturation period. However, the increase in passive tension, and its contribution to overall force was significantly higher for FLNC^AGAA tissues when compared to FLNC^vWT while active force remained significantly lower in FLNC^AGAA compared to FLNC^vWT (FIGS. 3D and 3E). Furthermore, FLNC^AGAA tissues exhibited slower contraction and relaxation velocities when compared to FLNC^vWT tissues (FIG. 3F). These data indicate ECTs capture clinically relevant elements of restrictive cardiomyopathy, illustrated through the measurement of tissue-specific parameters not available to 2D culture, such as passive tension and force generation.

High Throughput (HTS) drug screening to identify potential therapy for RCM.

[00138] Compared to video analysis of beating, calcium flux can be measured quickly and efficiently using high throughput fluorimetry. Given that calcium flux differences between FLNC^AGAA and FLNC^vWT iPSC-CM paralleled the decreased relaxation velocity in two and three dimensional models, tau was used as a surrogate output for relaxation. To facilitate the HTS, the 7416_7418delGAA in frame deletion was knocked-in into an allele in the iPSC calcium reporter line GCAMP6 (obtained by MTA from the Gladstone Institute) (FIG. 9) (Huebsch N et. al. Tissue Eng - Part C Methods. 21(5):467-479, 2015). The introduction of this mutation resulted in a broad prolongation in tau (FIG. 4A). Cells were plated onto 384- well plates and three compound libraries consisting of 2,185 total compounds were screened (FIG. 4B). Calcium fluorescence were recorded prior to compound treatment and at 1 and 3 hours post treatment with each well serving as its own control for drug response. Compounds were selected for further testing by their consistent effect on tau at both time points. As shown in Figure 4C, trequinsin, a phosphodiesterase 3 (PDE3) inhibitor, was identified as a top candidate with approximately 50 percent reduction in tau. This finding was validated in the patient cell line, and demonstrated that trequinsin causes significant reductions in tau as predicted in both FLNC^AGAA and FLNC^vWT iPSC-CM (FIG. 4D).

PDE inhibition ameliorates restrictive cardiomyopathy phenotype in ECT.

[00139] In order to make a direct assessment of myocardial relaxation, ECT were treated with trequinsin and the acute mechanical response of tissues were measured. Trequinsin treatment significantly reduced overall contractile force generated in FLNC^AGAA while it had no effect in FLNC^vWT (FIG. 5A). This reduction was due to a decrease in passive tension in the FLNC^AGAA tissues while active force remained unchanged (FIGS. 5B and 5C).

Significant increases in contraction and relaxation velocities were measured for both FLNC^AGAA and FLNC^vWT tissues (FIGS. 5D and 5E). These changes were sustained 3 hours after treatment. As inhibition of PDE3 in the heart leads to accumulation of cAMP and activation of the cAMP dependent kinase PKA, PKA activity in tissue lysates was assessed and significant increases in PKA kinase activity in both genotypes were observed (FIG. 10). Taken together, these data demonstrate the potential therapeutic efficacy of modulating phosphodiesterase activity for improving myocardial relaxation in RCM.

[00140] Described herein is a previously unidentified autosomal dominant in-frame deletion in FLNC which causes early onset RCM. This mutation is located in exon 44, in R22 of FLNC and is the first identified mutation in this region to cause RCM, though nearby missense mutations in R21 and R23 causing RCM have been identified (Schuber J et. al.

Hum Mutat 39(12):2083-2096, 2018; Mao Z & Nakamura F, Int J Mol Sci 21(8), 2020; Eden M & Frey N J Clin Med 10(4):577, 2021). Using patient specific iPSC-CMs, and a genetically corrected isogenic cell line, described herein demonstrates that this mutation causes impairment of cardiomyocyte contractility, and alteration of calcium flux during the cardiac contraction cycle. There are significant differences in contractility, relaxation velocity, and tissue passive tension caused by this mutation when these cells are incorporated into engineered cardiac tissues. Utilizing high-throughput compound screening targeted at modulating calcium flux, described here is PDE3 inhibition as a potential therapeutic target for RCM and demonstrated amelioration of passive tension in this tissue system with a PDE3 inhibitor trequinsin.

[00141] The results described herein adds to a small but growing body of work using iPSC models to study cardiac filaminopathies (Chen SN et. al. Sci Adv 38(8):52, 2022). Recently, in a two-dimensional model of DCM, homozygous loss of the C terminus of FLNC in iPSC- CM resulted in aberrant sarcomeric structure and function while heterozygous loss and an in frame deletion (not patient based) were mechanically and structurally similar to isogenic controls (Agarwal R et. al. Circ Res 751-766, 2021). That study and others have also reported aberrant lysosomal accumulation and autophagic flux leading to protein aggregation as a significant contributor to muscle dysfunction (Ruparelia AA et. al. Hum Mol Genet 25(11):2131-2142, 2016; Lowe T et. al. Hum Mol Genet 16(11): 1351-1358, 2007). As described herein, deficits in contractile properties and calcium flux were demonstrated in two-dimensional culture without evidence of FLNC accumulation or changes in solubility. This finding may represent a divergence in cellular phenotypes between FLNC variants that generate DCM or HCM versus those that lead to RCM. Genotype phenotype relationship may also be contributory since a dominant and highly penetrant mutation that manifested early in life while other models are based on mutations that result in cardiomyopathy in adulthood or are not based on human mutation data.

[00142] A significant advancement described herein is the first 3-dimensional ECT model of an FLNC mutation and restrictive cardiomyopathy. Demonstrated herein is a tissue engineered model that is able to capture clinically relevant properties of RCM including passive tension and relaxation velocity. These results underscore the importance of a nativelike microenvironment and cardiac tissue maturation as a necessary facet of cardiac disease modeling. The phenotypic differences between FLNC^AGAA and FLNC^vWT tissues relevant to RCM increased after electrical and metabolic maturation. This is a critical step in disease modeling as iPSC-CM are immature and physiologically not representative of adult myocardium. The previous publication has shown dedicated maturation protocol improves tissue function, structure and mature gene expression and described herein is the power of such an approach in eliciting genotype-phenotype relationships in vitro (Ronaldson-Bouchard K et. al Nature, 2018; Tamargo MA et. al. ACS Biomater Sci Eng. 2021). [00143] While the tissues are relatively medium-throughput (24-96 tissues/experiment), high throughput compound testing with this technology is not feasible without excessive personnel or time. Measurements of calcium transients paralleled contractile deficits in two dimensional FLNC^AGAA cardiomyocytes and thus without being bound by theory it is thought that the rate of calcium fluorescence decay could be used as a high throughput surrogate for relaxation. What is observed herein are consistent with previous studies in mouse models of RCM with troponin I (TNNI3) mutations, which show lengthened calcium transient decay (Davis J et. al. Cir Res 100(10): 1494-1502, 2007; Wen Y et. al. J Mol Biol 392(5): 1158- 1167, 2009; Li Y et. al. J Mol Cell Cardiol 49(3):402-411, 2010). The identification of a PDE3 inhibitor as a positive regulator of relaxation in FLNC^AGAA is not unexpected. One of the primary mechanism of PDE3i is activation of PKA which then phosphorylates a number of targets involved in calcium flux and mechanical contraction, including phospholamban (PLN) and ryanodine receptor 2 (RYR2) (Materson LR et. al. J Mol Biol 412(2): 155-164, 2011; Marks AR J Clin Invest 123(l):46-52, 2013). PKA also phosphorylates TNNI3, which increases the rate of muscle relaxation (Zhang et. al. Circ Res 76(6): 1028-1035, 1995; Kentish JC et. al. Cir Res 88(10): 1059-1065, 2001). Testing of PDE3 inhibition in ECT confirmed the validity of our calcium-based screen and showed its effectiveness in increasing tissue relaxation velocity and contractile dynamics.

[00144] Clinically, PDE3 inhibitors are in use for the treatment of acute heart failure. However, a number of randomized controlled trials have shown their ineffectiveness in treating chronic heart failure due to toxicity (Packer M et. al. N Engl J Med 325(21): 1468- 1475, 1991; Cuffe MS et. al. J Am Med Assoc 287(12): 1541-1547, 2002; Metra et. al. Eur Heart J 30(24):3015-3026, 2009; Dibianco et. al. N Engl J Med 320(11):677-683, 1989). Nevertheless, the more recent development of specific small molecule PDE inhibitors targeting different isoforms to reduce toxicity (Kim GE & Kass DA Hanbook of Experimental Pharmacology 243:249-269, 2017) as well as the paucity of druggable targets in RCM means that PDEi as a potential therapy warrants further investigation in RCM. It should be noted that in those trials, the main focus of PDE3 inhibition was to increase cardiac contractility for systolic dysfunction, and that the dosing a PDE3 inhibitor with the goal of improving cardiac relaxation may differ significantly. Indeed, in the ECTs, the addition of trequinsin does not increase the force or active force generated, but does improve relaxation dynamics. [00145] The integration of 2D and 3D culture methods yielded a potential druggable pathway in FLNC RCM. However, the study described herein may be limited in that it only models cardiomyocyte-specific effects of FLNC^AGAA, as commercial primary cardiac fibroblasts isolated from healthy human hearts were used in the ECTs. The contribution of non-myocyte cell types to cardiac disease is becoming increasingly recognized. FLNC^AGAA may cause cell-type specific changes which affect signaling in these other types. Fibroblasts in particular modulate cardiac extracellular matrix which contributes to ventricular compliance, and may accelerate the development of RCM. These factors were not part of the model. As fibroblasts comprise about 15% of the healthy human heart, and perhaps more in disease, this may affect the utility of PDE3i in clinical disease. Furthermore, though identified calcium dysregulation was identified as a key indicator of RCM, exactly how FLNC^AGAA alters calcium decay is unknown and requires further study. FLNC is a scaffolding protein with numerous binding partners, including membrane receptors, ion channels, and sarcomeric proteins such as titin (Mao Z & Nakamura F Int J Mol Sci 21(8), 2020). Many of these bind at R22, and without being bound by theory, impaired scaffolding with its partners may contribute to the phenotype.

[00146] In conclusion, FLNC^AGAA causes an autosomal dominant form of restrictive cardiomyopathy, which can be modeled in-vitro utilizing 2D and 3D iPSC-CM models. Complementary approaches in 2D and 3D identified PDE inhibition as a potential target to ameliorate RCM phenotypes. This work strengthens the application of iPSC-CM and cardiac tissues to model genetic cardiac diseases. Whether the pathomechanism of the FLNC^AGAA mutation and PDE3i treatment are generalizable to other forms of primary RCM remains to be seen in future studies. If so, this model could be further applied to identify and translate much needed novel therapies for RCM.

[00147] While restrictive cardiomyopathies is considered rare, it has been increasingly diagnosed due to improved detection of infiltrative and genetic causes. Here, the findings describe a previously unknown mutation in Filamin C and establish a genotype phenotype relationship using engineered cardiac tissues. The model outlined here will aid future research and treatment discovery pipelines for restrictive cardiomyopathies.

[00148] Engineered cardiac tissues has the ability to capture the hallmarks of increased muscle tension and reduced relaxation in restrictive cardiomyopathy. Studies using this platform may provide mechanistic insights regarding the function of Filamin C in the cardiomyocyte and refine preclinical drug testing of agents which improve myocardial relaxation. These studies may have broad implications for patients who have RCM, which presently has no effective mortality reducing treatments.

Methods

[00149] Method of generating patient-specific and CRISPR/Cas9 iPSC: Blood samples were collected from the affected patient and parents. Whole exome sequencing of the trio was performed by GeneDx (Gaithersburg, MD) as part of the patient’s clinical care. Isolated PBMCs were reprogrammed to iPSC’s using Sendai virus (Yang W StemBook, 2014). See method 2.2 CRISPR-Cas9 was used to revert the mutation back to the wild type allele for an isogenic control cell line and to generate the mutation in the GCAMP6 iPSC reporter line. See Method 2.3. Table 1 shows sequences used in CRISPR-Cas9 editing.

Consent for the study was obtained from the patient’s parents. Human subjects protocol was approved by the Columbia University Institutional Review Board (IRB AAAR1017).

Table 1 - Sequences used in CRISPR-Cas9 editing

[00150] Method of generating induced pluripotent stem cell: PBMC (2xl0⁶) were cultured in 12-well plates in a serum-free media that supports hematopoietic stem/progenitor cells, in the presence of cytokines that help the expansion of the erythroblast population (Hossain MM et. al. Analyst 135(7): 1624-1630, 2010). 9 to 12 days after collection, the expanded erythroblast population were reprogrammed using a Sendai virus-based approach (Cytotune iPS 2.0 Sendai Reprogramming kit, Life Technologies) containing the four recombinant viral vectors (Oct4, Sox2, KLF4, c-myc). One week after infection, cells were transferred onto irradiated MEF feeder cells in hESC culture media supplemented with 20% KO-SR (Life Technologies) and 4ng/ml bFGF (R&D Systems). After about 25-30 days, cells with iPSC characteristic morphology were isolated and further grown for expansion, freezing and characterization. Clones were tested to determine their sternness by staining for pluripotency markers Oct4 and Nanog (Cell Signaling Technology) and Tra-1-60 and SSEA4 (BD Biosciences) by flow cytometry. G-band karyotyping (Cell Line Genetics or NYP Clinical Cytogenetics Laboratory) was used to assess chromosomal stability on at least twenty metaphase cells at 450-500 band resolution. In vitro differentiation into the three germ layers was assessed by using the Human Pluripotent Stem Cell Functional Identification kit (R&D Systems). Absence of mycoplasma contamination was confirmed by PCR (e-Myco™ plus Mycoplasma PCR Detection Kit, Bulldog Bio).

[00151] Method of CRISPR-Cas9 modification of iPS cells: CRISPR/Cas9 technology was used to correct the FLNC frameshift mutation c.7416_7418delGAA in the patient- derived iPSC line as well as knockin the mutation into the GCAMP6 reporter iPS line, ribonucleoprotein delivery (RNPs) with a purified Cas9 protein (from IDT) were used and synthetic guide RNAs (sgRNA, from Synthego) were validated. The online tool from Synthego/benchling was used to choose the sgRNAs sequences with highest specificity and efficiency to target this region. Three synthetic sgRNAs (CRISPR evolution sgRNA EZ Kit, Synthego) were used for initial screening to choose the best sgRNA sequence. Cells were electroporated with three separate Ribonucleoprotein (RNPs)-sgRNA complex mix consisting in lOug of purified Cas9 protein (Alt-R® S.p. HiFi Cas9 Nuclease V3, IDT) and 5ug of each sgRNA, delivered in 2X10^A5 cells/reaction via electroporation with the Amaxa Nucleofector 4D (program CA-137) and P3 Primary Cell 4D-Nucleofector X Kit L (Lonza, cat. no. V4XP- 3012). Cells were allowed to recover for 2-3 days, then DNA was isolated for Sanger sequencing to assess the efficiency of cleavage by the Cas9. The genomic DNA was used in a PCR reaction to amplify the region of interest. The PCR products from electroporated samples and control (DNA from non -electroporated cells) were processed for Sanger Sequencing. To determine cleavage efficiency, the resulting electropherograms for both the electroporated and non-electroporated cells were applied to inference of CRISPR editing (ICE) analysis using the ICE online tool from Synthego (www.synthego.com/products/bioinformatics/crispr-analysis). Based on ICE results, electroporation was repeated using IxlO⁶ cells using the selected reagents (20ug Cas9, 15ug sgRNA and 15ug single stranded modified donor DNA (IDT). Electroporated cells were allowed to grow for 48hrs and then seeded at low density (3xlO^b cells in a 10cm Matrigel-coated plate) in mTeSR Plus (Cat #100-0276, Stem Cell Technologies) and CloneR (cat# 05888 Stem Cell Technologies) to grow for 7-10 days before picking isolated iPSC colonies. Colonies were expanded and further analyzed by genotyping. The sequences of the corrected clones were confirmed by genotyping, then clones were further expanded, and final confirmation was obtained by Sanger sequencing. Clones were subsequently karyotyped and only those with normal karyotyping were used in this study.

[00152] Method of iPSC-CMs differentiation: Two days before differentiation iPSCs were replated in 6 well plates at a density of 2 million cells per well. iPSCs were differentiated into cardiomyocytes as previously described, using a cardiac differentiation media (CDM) containing RPMI1640, albumin, and ascorbic acid (Burridge PW et. al. Nat Methods 11(8):855-860, 2014). At day 10 post differentiation, beating cardiomyocytes were switched to RPMI 1640+B27 Supplement (ThermoFisher), and were expanded one passage with the addition of 2pM CHIR99021 (Tocris), per a recently published method (Buikema JW et. al. Cell Stem Cell 27(l):50-63, 2020). Cardiomyocytes were maintained in RPMI1640 + B27 for experiments in 2D or until incorporation into tissues.

[00153] Primary cardiac fibroblast culture: Human primary cardiac fibroblasts (Lonza CC-2904) were thawed and expanded in Fibroblast Growth Medium 3 (Promocell C-23025) for two passages before freezing. A singular lot of cardiac fibroblasts was used for all experiments in the examples.

[00154] Method of 2D immunofluorescence and sarcomere length measurement: Cell were seeded onto glass coverslips coated with matrigel. After cells were recovered in media for 3-5 days, iPSC-CM were fixed in 4% PFA for 15 minutes followed by permeabilization with 0.1% Triton-X in PBS. Primary antibodies were incubated overnight followed by secondary antibody at room temperature for 1 hour. Antibodies used are in Table 2. Sarcomere length was measured by taking pixel intensity along a line parallel to the sarcomere using a-actinin staining and calculating the distance between intensity peaks using ImageJ.

[00155] 2D contractility analysis: iPSC-CM were replated in 24 well plates at a density of 200,000 per cm². Brightfield videos were taken at a frame rate of 100 frames per second and analyzed with custom written Python code using the principle of pixel intensity subtraction (Sala L et. al. Circ Res. 122(3):e5-el6, 2018; Hossain MM et. al. Analyst. 135(7): 1624-1630, 2010).

[00156] High-throughput compound screen: Cells were plated in 384-well black with clear bottom plates (Greiner 781096). On the day of the assay, medium was aspirated from the assay plates and replaced with 10 pL of new media. After 30 minutes incubation for cells to acclimate to the media, 10 pL of FLIPR Calcium 6 dye (Molecular Devices) was added to the assay plates followed by centrifugation at 46 g for 10 sec. Assay plates were incubated for 2 hours at 37°C in a humidified 5% CO2 incubator to load the dye. At the end of the incubation, a baseline reading was taken on the FLIPR Tetra (Molecular Devices). The exposure time was 0.05 sec and 388 reads were collected at a read time interval of 0.125 sec. Screening compounds (20 nl of 10 mM stock in DMSO) was added to the assay plates using a Labcyte Echo 550 acoustic dispenser (Beckman Coulter), resulting in a final concentration of 10 pM compound and 0.1% DMSO. Negative (DMSO vehicle) and positive control wells (300 pM nisoldipine and 10 pM NKH477) were included on every plate. Assay plates were centrifuged at 46 g for 10 seconds and incubated for 1 hour at 37°C in a humidified 5% CO2 incubator before reading on the FLIPR. One minute of fluorescent flux were captured for each well. Libraries screened included FDA-approved drug library (Enzo Life Sciences), Pharmakon (MicroSource Discovery Systems), and Tested-In-Humans collection (Yale Center for Molecular Discovery). Calcium traces for each well obtained from FLIPR Tetra were analyzed using a custom R script. Briefly, each trace was normalized to baseline fluorescence. A peak finding algorithm was implemented using the ‘findpeaks’ function in the R package ‘pracma’ . For each peak, the portion of the trace ranging from maximum amplitude to the mininum of the curve obtained from findpeaks was segmented. Linear regression using the equation:

where y is the fluorescence value and t is time, was used to obtain the value of tau. The value tau is reported as the average of all beats in the well.

[00157] Compound Validation: iPSC-CM were replated in 24 well plates at a density of 200,000 per cm² and allowed 3 days to recover. Cells were stained with Fluo-4 (ThermoFisher F14201) calcium dye using a 1 :4 dilutions in culture media for fifteen minutes. Then media was replaced with fresh media containing a dilution of 1 : 10 Fluo-4 dye. Trequinsin or DMSO was used to treat iPSC-CM at 5 M for 30 minutes. Fluorescent videos were taken of cardiomyocyte beating 30 minutes post treatment. Videos were processed to extract a curve of calcium fluorescence, and subsequent analysis of calcium transients was analyzed using a Python script.

[00158] Generation of 3D ECT: ECTs were generated and analyzed in our recently reported pipeline(Tamargo MA et. al. ACS Biomater Sci Eng. 2021). Briefly, bioreactors were cast from PDMS in custom-milled molds containing electrodes for electrical stimulation. Cardiomyocytes and human primary cardiac fibroblasts (Promocell) were dissociated using 10X TrypLE (ThermoFisher) for 15-20 minutes. Cell were resuspended at a concentration of 500,000 cells per tissue in a ratio of 75% cardiomyocytes and 25% cardiac fibroblasts, in a solution of 5mg/ml fibrinogen. 12pl of cell suspension was mixed with 3 pl of thrombin (5U/ml) in each well to cast one tissue. For three days after tissue formation, tissues were maintained in fresh B27 media containing 5mg/mL 6-aminocaproic acid (Sigma- Aldrich A7824). Seven days after tissue formation, media was changed to a RPMI-based metabolic maturation media containing AlbuMAX (ThermoFisher 11020021) higher calcium content, and lower glucose content to promote fatty acid oxidation, as detailed previously (Feyen DAM et. al. Cell Rep. 32(3), 2020). Also at day 7, tissues began a 2-week ramped electrical stimulation regimen which lasted from 2Hz and increased 0.33Hz every 24 hours until 6Hz. After this two-week regimen, tissues were maintained electrically paced at 1 Hz.

[00159] Force Analysis of ECT: Analysis of tissue function and force generation was performed by capturing video of tissue contraction while stimulated at 1Hz and analyzing pillar deflection using a custom-written Python code, previously outlined in detail (Tamargo MA et. al. ACS Biomater Sci Eng. 2021). Briefly, a computer vision package containing an object-tracking algorithm was adapted to track pillar head movement and calculate displacement from videos of beating tissues. Displacement measurements were then used to calculate force based on previously determined empirical measurements, which determined the force needed to deflect the pillar using a microscale mechanical tester (Microtester MT- LT, CellScale). Relaxation and contraction velocity measurements were calculated using the derivative of displacement.

[00160] Drug treatments on human cardiac microtissue: Matured cardiac tissue following 4 weeks of culture were treated with lOpM trequinsin-HCl. Contractile analysis was performed prior and post compound treatment at 1 and 3 hours. [00161] Whole-mount staining of ECT : Tissues were fixed and permeabilized with 100% methanol for 15min at room temperature. After Ih blocking with 5% BSA in PBS, cells or tissues were incubated with antibodies against a-actinin 2 (MACS 130-119-766), Vimentin (Abeam ab202504) and Cardiac Troponin T (BD Biosciences 565744) for Ih at room temperature in the dark. For whole mount immunofluorescence staining, cardiac microtissues were embedded in ProLong Glass Antifade Mountant with NucBlue Stain (Invitrogen P36981) in CoverWell incubation Chambers (Grace Bio-Labs 645501), ECTs were imaged on a Nikon Al confocal microscope.

[00162] Western Blot: iPSC-CM were washed with cold PBS and lysed using Pierce IP Lysis Buffer (Thermofisher 87787) containing protease and phosphatase inhibitors (ThermoFisher 78442) Lysate was transferred to ice for 30 minutes, then centrifuged at 16,000g for 10 minutes. The supernatant was removed to a clean tube as the soluble portion. The pellet was then resuspended in lysis buffer containing 4% SDS. Lysates were quantified with Pierce BCA kit and 20ug total protein was loaded in 4-20% Tris-glycine gels (Thermo, XP04205BOX). Antibodies used are in Table 2.

Table 2 - List of antibodies used for IF and western blot

[00163] PKA activity assay: Cells were washed with PBS and then lysed in ice-cold IP Lysis Buffer (ThermoFisher 87787) containing protease and phosphatase inhibitors (ThermoFisher 78442) for 30 minutes. Protein concentration was quantified using BCA, then 5 pg of protein was used in a protein kinase A (PKA) activity colorimetric kit (ThermoFisher EIAPKA) per manufacturer’s instructions.

[00164] Fractionation of soluble and insoluble fractions: Cells were washed with PBS and then lysed in ice-cold IP Lysis Buffer (ThermoFisher 87787) containing protease and phosphatase inhibitors (ThermoFisher 78442) for 30 minutes. The cells were centrifuged at 16,000g for 15 minutes and supernatants were collected, generating the soluble fraction. The insoluble pellets were dissolved in lysis buffer containing 200U/ml DNAse I (Roche) and 2% sodium dodecyl sulfate (SDS), and sonicated, generating the insoluble fraction. Insoluble fractions were then used for Western blotting. Example 2 - Engineered Cardiac tissue model of restrictive cardiomyopathy for drug discovery

[00165] In-frame deletion in FLNC leads to RCM and defects in cardiomyocyte relaxation.

[00166] A 3 -year-old boy presented to our pediatric heart failure and transplant clinic for evaluation of heart failure, developmental delay, and arthrogryposis. Echocardiography revealed a systolic ejection fraction of 55%, normal left ventricle (LV) wall thickness, a dilated left atrium, right ventricular hypertrophy, restrictive filling Doppler of the mitral valve, reduced tissue Doppler velocity, and elevated estimated pulmonary pressures, all consistent with an RCM (Figure 2A). The patient and his parents underwent exome sequencing, revealing a rare de novo in-frame mutation in FLNC (c.7416_7418delGAA p.Glu2472_Asn2473delinsAsp), which was further confirmed by Sanger sequencing. This variant is located in exon 44, in the 22nd immunoglobulin-like domain repeat (R22) of the ROD2 domain of FLNC. Both Glu2472 and Asn2473 are strictly conserved across species, and the mutation was classified as a “pathogenic variant” by GeneDx.

[00167] Patient-specific iPSCs (FLNCAGAA) were reprogrammed from peripheral blood mononuclear cells (PBMCs) isolated from the proband, and an isogenic cell line with the mutation corrected to pseudo-wild type (FLNCyWT) was engineered using CRISPR-Cas9 (Figure 6A). FLNCAGAA and FLNCyWT iPSCs were differentiated into cardiomyocytes. Immunofluorescence of iPSC-CMs showed colocalization of FLNC and sarcomeric a-actinin (Figure 2B). No significant differences in either cell size, sarcomere length, or FLNC/actin colocalization were observed between FLNCAGAA and FLNCyWT cardiomyocytes (Figures 7A-7C, 13). There were also no differences observed in FLNC protein solubility between FLNCAGAA and FLNCyWT genotypes, as had been previously reported for other mutations in FLNC (Figure 7C). (Lowe, T., Kley, R.A., van der Ven, P.F.M., Himmel, M., Huebner, A., Vorgerd, M., and F€urst, D.O. (2007). The pathomechanism of filaminopathy: altered biochemical properties explain the cellular phenotype of a protein aggregation myopathy. Hum. Mol. Genet. 16, 1351-1358.; Agarwal, R., Paulo, J. A., Toepfer, C.N., Ewoldt, J.K., Sundaram, S., Chopra, A., Zhang, Q., Gorham, J., DePalma, S.R., Chen, C.S., et al. (2021). Filamin C cardiomyopathy variants cause protein and lysosome accumulation. Circ. Res. 129, 751-766). [00168] The spontaneous beating function of iPSC-CMs was analyzed using video analysis. FLNCAGAA cardiomyocytes had significantly diminished beating amplitudes associated with a decrease in both peak contraction and relaxation velocities (Figures 2C, 11 A). Because calcium mediates excitation-contraction coupling, calcium flux was measured in spontaneously beating cardiomyocytes and observed paradoxically increased amplitudes and decreased time to peak in FLNCAGAA cardiomyocytes (Figures 11B-D). This suggests that calcium flux and contraction may be decoupled in FLNC mutant cardiomyocytes, as previously reported. (Powers, J.D., Kirkland, N.J., Liu, C., Razu, S.S., Fang, X., Engler, A. J., Chen, J., and McCulloch, A.D. (2022). Subcellular remodeling in filamin C deficient mouse hearts impairs myocyte tension development during progression of dilated cardiomyopathy. Int. J. Mol. Sci. 23, 871). However, as opposed to calcium flux during contraction, the time constant of decay tau (t), a measurement of calcium reuptake efficiency during relaxation was significantly longer in FLNCAGAA compared with FLNC\|/WT cardiomyocytes (Figure 2D). Together, these data demonstrate that the FLNCAGAA mutation causes deficiencies in contractile function and aberrant calcium flux in cultured cardiomyocytes.

/ /.A CAGAA engineered cardiac tissues model RCM

[00169] Recent advances have highlighted the relative immaturity of iPSC-CMs cultured on 2D surfaces and suggest that engineering of 3D cardiac tissues enables maturation of iPSC-CMs in a native-like microenvironment. (Ronaldson-Bouchard, K., Ma, S.P., Yeager, K., Chen, T., Song, L., Sirabella, D., Morikawa, K., Teles, D., Yazawa, M., and Vunjak- Novakovic, G. (2018). Advanced maturation of human cardiac tissue grown from pluripotent stem cells. Nature 556, 239-243; Campostrini, G., Windt, L.M., van Meer, B.J., Beilin, M., and Mummery, C.L. (2021). Cardiac tissues from stem cells: new routes to maturation and cardiac regeneration. Circ. Res. 128, 775-801). The use of engineered cardiac tissue (ECT) and both metabolic and physical maturation could be used to further model the relaxation deficit in FLNCAGAA iPSC-CMs. ECTs were created from FLNCAGAA and FLNC\|/WT iPSC-CMs using other published method, in which cells encapsulated in a fibrin gel are molded around two horizontal pillars cast from polydimethylsiloxane (PDMS) (Figures 3 A and 8A-8B). (Tamargo, M.A., Nash, T.R., Fleischer, S., Kim, Y., Vila, O.F., Yeager, K., Summers, M., Zhao, Y., Lock, R., Chavez, M., et al. (2021). milliPillar: a platform for the generation and real-time assessment of human engineered cardiac tissues. ACS Biomater. Sci. Eng. 7, 5215-5229). Two methods were combined to encourage tissue maturation. First, tissues were subjected to 2 weeks of electromechanical stimulation, which was shown previously to improve iPSC-CM maturation and gene expression. (Ronaldson-Bouchard, K., Ma, S.P., Yeager, K., Chen, T., Song, L., Sirabella, D., Morikawa, K., Teles, D., Yazawa, M., and Vunjak-Novakovic, G. (2018). Advanced maturation of human cardiac tissue grown from pluripotent stem cells. Nature 556, 239-243). A culture medium high in fatty acids was used to favor fatty acid oxidation, which has been shown to improve physiological function. (Feyen, D.A.M., McKeithan, W.L., Bruyneel, A.A.N., Spiering, S., Hormann, L., Ulmer, B., Zhang, H., Briganti, F., Schweizer, M., Hegyi, B., et al. (2020). Metabolic maturation media improve physiological function of human iPSC-derived cardiomyocytes. Cell Rep. 32, 107925) The addition of the maturation media resulted in more compacted tissues and higher stress, or force per unit area, generated when compared with the standard RPMI-based media (FIGS. 14A-14B).

[00170] Whole-mount immunofluorescence imaging at 3 weeks was used to assess sarcomeric structure in situ. Compared with FLNC\|/WT ECTs, FLNCAGAA ECTs exhibited sarcomeric disorganization with significant deviation of actinin fibers from the tissue longitudinal axis (Figures 3B and 3C). ECT function was assessed using video microscopy to track pillar deflection. Based on pillar displacement and the previously performed empirical measurements (Tamargo, M.A., Nash, T.R., Fleischer, S., Kim, Y., Vila, O.F., Yeager, K., Summers, M., Zhao, Y., Lock, R., Chavez, M., et al. (2021). milliPillar: a platform for the generation and real-time assessment of human engineered cardiac tissues. ACS Biomater. Sci. Eng. 7, 5215-5229), force generation is able to be calculated with three specific metrics: (1) active force, which is the force that displaces the pillars during tissue contraction; (2) passive tension, which denotes the residual force causing pillar deflection during maximum relaxation; and (3) total force, which is the sum of both passive and active forces. Total force generated by FLNCAGAA and FLNC\|/WT tissues when paced at 1 Hz increased a similar amount over the maturation period. However, the increase in passive tension, and its contribution to total force, was significantly higher for FLNCAGAA tissues when compared with FLNC\|/WT, while active force remained significantly lower in FLNCAGAA compared with FLNC\|/WT (Figures 3D and 3E). Furthermore, FLNCAGAA tissues exhibited slower contraction and relaxation velocities when compared with FLNC\|/WT tissues (Figure 3F).

[00171] These findings were validated by generating a 7416_7418delGAA knockin using the WT iPSC calcium reporter line GCAMP6 using CRISPR-Cas9 (Figure 9). As observed in the patient-derived tissues, the knockin mutation GCAMP6AGAA resulted in decreased active force and increased passive tension along with overall decreased velocities when compared with the isogenic WT GCAMP6 tissues (Figuresl5A-C). There was no significant difference in cell size from this knockin mutation as measured by flow cytometry (Figures 15D-15E). Together, these data indicate ECTs capture clinically relevant elements of RCM, illustrated through the measurement of tissue-specific parameters such as passive tension and relaxation velocity.

High-throughput drug screening to identify potential therapy for RCM

[00172] Compared with video analysis of contractility, calcium flux can be measured quickly and efficiently using high-throughput fluorimetry. Given that deficits in calcium relaxation were observed (Figure 2D) correlated with impaired mechanical relaxation in both 2D (Figure 11 A) and 3D (Figures 3E and 3F) cardiomyocytes, tau was utilized as a surrogate endpoint for relaxation kinetics. GCAMP6AGAA iPSC-CMs similarly displayed a significantly increased tau compared with isogenic WT controls (Figure 4A). These cells were plated onto 384-well plates, and three compound libraries consisting of 2,185 total compounds were screened (Figure 4B). Calcium fluorescence was recorded prior to compound treatment and at 1 and 3 h post treatment, with each well serving as its own control for drug response. Because the initial screen was single pass, compounds were selected by their consistent effect on tau at both time points with the goal of improving the specificity of the results. As shown in Figure 4C, trequinsin, a PDE3 inhibitor, was identified as a top candidate with approximately 50% reduction in tau. This finding was cross-validated in the proband cell line and demonstrated that trequinsin causes significant reductions in tau as predicted in both FLNCAGAA and FLNC\|/WT iPSC-CMs (Figures 4D and 16A).

PDE inhibition ameliorates the RCM phenotype ECT

[00173] In order to directly assess myocardial relaxation, FLNCAGAA and FLNC\|/WT ECTs were treated with trequinsin, and the acute functional responses of tissues were measured with pacing at 1 Hz (Figure 12A). After 1 h of trequinsin treatment, passive tension was reduced in FLNCAGAA tissues (Figure 12B). Trequinsin caused increases in relaxation and contraction velocities in both FLNCAGAA and FLNC\|/WT tissues (Figures 12C and 12D). Importantly, trequinsin treatment also rescued the time-dependent metrics of contractile dynamics, including time to 50% relaxation and contraction (Figures 4E and 4F).

[00174] As inhibition of PDE3 in the heart leads to accumulation of cAMP and activation of the cAMP-dependent kinase PKA, PKA activity was measured in tissue lysates and observed significant increases in PKA kinase activity in both genotypes (Figure 10). Given these data, two other compounds were selected, 17a-hydroxyprogesterone and denbuphylline (a non-selective PDE3/4 inhibitor), for testing in our engineered tissues. 17a- hydroxyprogesterone was ineffective at increasing the relaxation velocity of tissues when compared with the baseline. However, a genotype-specific response of FLNCAGAA tissues to denbuphylline was observed, resulting in about 1.3-fold increase in relaxation velocity after 3 h (Figures 16B, 16D).

[00175] PDE inhibition is known to predispose to arrhythmia. (Cuffe, M.S., Califf, R.M., Adams, K.F., Jr., Benza, R., Bourge, R., Colucci, W.S., Massie, B.M., O’Connor, C.M., Pina,

I., Quigg, R., et al. (2002). JAMA 287, 1541-1547; DiBianco, R., Shabetai, R., Kostuk, W., Moran, J., Schlant, R.C., and Wright, R. (1989). N. Engl. J. Med. 320, 677-683; Metra, M., Eichhorn, E., Abraham, W.T., Linseman, J., Bohm, M., Corbalan, R., DeMets, D., De Marco, T., Elkayam, U., Gerber, M., et al. (2009). Eur. Heart J. 30, 3015-3026; Packer, M., Carver,

J.R., Rodeheffer, R.J., Ivanhoe, R.J., DiBianco, R., Zeldis, S.M., Hendrix, G.H., Bommer, W.J., Elkayam, U., Kukin, M.L., et al. (1991) N. Engl. J. Med. 325, 1468-1475). The arrhythmogenicity of trequinsin was tested in monolayer-cultured GCAMP6AGAA and GCAMP6 iPSC-CMs using the hERG channel blocker E-4031 as a positive control. (Harris,

K., Aylott, M., Cui, Y., Louttit, J.B., McMahon, N.C., and Sridhar, A. (2013). Toxicol. Sci. 134, 412-426). Under 0.5 Hz stimulation, E-4031 induced arrhythmic events (as measured by calcium flux), while trequinsin did not, in both GCAMP6AGAA and GCAMP6 iPSC-CMs (Figure 16D). Quantification of the average beat-to-beat frequency demonstrated significant dispersion in cells treated with E-4031, while trequinsin did not alter capture at 0.5 Hz (Figure 16E). This suggests that at the concentration tested, trequinsin does not increase afterdepolarizations. Finally, to quantify the cytotoxicity of trequinsin, GCAMP6AGAA and GCAMP6 ECTs were treated with trequinsin for 3 h and subsequently measured the release of lactate dehydrogenase (LDH) into the supernatant. No significant increase in LDH was observed when compared with DMSO treatment (Figure 16F). Taken together, these data demonstrate the potential therapeutic efficacy of modulating PDE activity to improve myocardial relaxation in RCM caused by the FLNCAGAA mutation. Methods

Table 3 - Reagents or Resources

Experimental model and subject details

Generation of patient-specific and CRISPR/Cas9 iPSC

[00176] Blood samples were collected from the affected patient and parents. Exome sequencing of the trio was performed by GeneDx (Gaithersburg, MD) as part of the patient’s clinical care. Isolated PBMCs were reprogrammed to iPSCs using Sendai virus (described below). (Yang, W., Mills, J.A., Sullivan, S., Liu, Y., French, D.L., and Gadue, P (2014) In StemBook). CRISPR-Cas9 was used to revert the mutation back to the wild type allele for an isogenic control cell line and to generate the mutation in the GCAMP6 iPSC reporter line. Consent for the study was obtained from the patient’s parents. The protocol was approved by the Columbia University Institutional Review Board (IRB AAAR1017). iPSC culture

[00177] iPSCs were cultured in MTESR Plus media (Stem Cell Technologies) on Matrigel (Corning 354,230) coated plates until 70% confluence, then passaged using 0.5mM EDTA every 4-6 days. iPSC-CMs differentiation

[00178] Two days before differentiation iPSCs were replated in 6 well plates at a density of 2 million cells per well. iPSCs were differentiated into cardiomyocytes as previously described, using a cardiac differentiation media (CDM) containing RPMI1640, albumin, and ascorbic acid.53 At day 10 post differentiation, beating cardiomyocytes were switched to RPMI 1640 + B27 Supplement (ThermoFisher), and were expanded one passage with the addition of 2pM CHIR99021 (Tocris), per a recently published method.54 Cardiomyocytes were maintained in RPMI 1640 + B27 for experiments in 2D or until incorporation into tissues.

Primary cardiac fibroblast culture

[00179] Human primary cardiac fibroblasts (Lonza CC-2904)were thawed and expanded in Fibroblast Growth Medium 3 (Promocell C-23025) for two passages before freezing. A singular lot of cardiac fibroblasts was used for all experiments in this study.

Experimental design

[00180] Experiments were replicated as described in figure legends. Generally, key experiments involved replication with at least n = 3 independent cardiomyocyte differentiations. The sample size for each experiment was selected based on anticipated means, a = 0.05, and 80% power, in addition to experimental workflow (for example, each cardiac tissue reactor contains 6 tissues). Regarding tissue experiments, experimental groups were randomized and labeled by letter. The operator obtaining video data was blinded and collection and analysis of videos were fully automated; videos were re-labeled after analysis. Prior to analysis, all tissues were subject to individual quality inspection. Those which exhibited 1) complete mechanical disruption due to operator manipulation, or 2) spontaneous detachment from a pillar, were excluded from analysis, as these conditions precluded data analysis from the pillar-tracking algorithm (which requires a continuous single tissue to be in contact with both pillars). All other tissues were included in the analysis. In general, these events occurred at a rate of 1-2 times per batch of 12 tissues.

Induced pluripotent stem cell generation

[00181] PBMC (2 x 106) were cultured in 12-well plates in a serum-free media that supports hematopoietic stem/progenitor cells, in the presence of cytokines that help the expansion of the erythroblast population. (Sala, L., van Meer, B.J., Tertoolen, L.G.J., Bakkers, J., Beilin, M., Davis, R.P., Denning, C., Dieben, M.A.E., Eschenhagen, T., Giacomelli, E., et al. (2018) Circ. Res. 122, e5-el6). 9 to 12 days after collection, the expanded erythroblast population were reprogrammed using a Sendai virus-based approach (Cytotune iPS 2.0 Sendai Reprogramming kit, Life Technologies) containing the four recombinant viral vectors (Oct4, Sox2, KLF4, c-myc). One week after infection, cells were transferred onto irradiated MEF feeder cells in hESC culture media supplemented with 20% KO-SR (Life Technologies) and 4 ng/mL bFGF (R&D Systems). After about 25-30 days, cells with iPSC characteristic morphology (high ratio of nucleus to cytoplasm, prominent nucleoli, well-defined borders) were isolated and further grown for expansion, freezing and characterization. Clones were tested to determine their sternness by staining for pluripotency markers Oct4 and Nanog (Cell Signaling Technology) and Tra-1-60 and SSEA4 (BD Biosciences) by flow cytometry. G-band karyotyping (Cell Line Genetics or NYP Clinical Cytogenetics Laboratory) was used to assess chromosomal stability on at least twenty metaphase cells at 450-500 band resolution. In vitro differentiation into the three germ layers was assessed by using the Human Pluripotent Stem Cell Functional Identification kit (R&D Systems). Absence of mycoplasma contamination was confirmed by PCR (e-Myco plus Mycoplasma PCR Detection Kit, Bulldog Bio).

CRISPR-Cas9 modification of iPS cells

[00182] CRISPR/Cas9 technology was used to correct the FLNC frameshift mutation c.7416_7418delGAA in the patient-derived iPSC line as well as knockin the mutation into the GCAMP6 reporter iPS line, ribonucleoprotein delivery (RNPs) was used with a purified Cas9 protein (from IDT) and validated synthetic guide RNAs (sgRNA, from Synthego). The online tool from Synthego/benchling was used to choose the sgRNAs sequences with highest specificity and efficiency to target this region. Three synthetic sgRNAs (CRISPR evolution sgRNA EZ Kit, Synthego) were used for initial screening to choose the best sgRNA sequence. Cells were electroporated with three separate Ribonucleoprotein (RNPs)-sgRNA complex mix consisting in lOug of purified Cas9 protein (Alt-R S.p. HiFi Cas9 Nuclease V3, IDT) and 5ug of each sgRNA, delivered in 2 * 10'5 cells/reaction via electroporation with the Amaxa Nucleofector 4D (program CA-137) and P3 Primary Cell 4D-Nucleofector X Kit L (Lonza, cat. no. V4XP-3012). Cells were allowed to recover for 2-3 days, then DNA was isolated for Sanger sequencing to assess the efficiency of cleavage by the Cas9. The genomic DNA was used in a PCR reaction to amplify the region of interest. The PCR products from electroporated samples and control (DNA from non -electroporated cells) were processed for Sanger Sequencing. To determine cleavage efficiency, the resulting electropherograms for both the electroporated and non-electroporated cells were applied to inference of CRISPR editing (ICE) analysis using the ICE online tool from Synthego (www.synthego.com/products/bioinformatics/crispr-analysis). Based on ICE results, electroporation was repeated using 1 * 106 cells using the selected reagents (20pg Cas9, 15ug sgRNA and 15ug single stranded modified donor DNA (IDT). Electroporated cells were allowed to grow for 48hrs and then seeded at low density (3 x 106 cells in a 10cm Matrigel - coated plate) in mTeSR Plus (Cat #100-0276, Stem Cell Technologies) and CloneR (cat# 05888 Stem Cell Technologies) to grow for 7-10 days before picking isolated iPSC colonies. Colonies were expanded and further analyzed by genotyping. The sequences of the corrected clones were confirmed by genotyping, then clones were further expanded, and final confirmation was obtained by Sanger sequencing. Clones were subsequently karyotyped and only those with normal karyotyping were used in this study.

2D immunofluorescence and sarcomere length measurement

[00183] Cell were seeded onto glass coverslips coated with Matrigel. After cells were recovered in media for 3-5 days, iPSC-CM were fixed in 4% PF A for 15 min followed by permeabilization with 0.1% Triton X- in PBS at room temperature. Primary antibodies were incubated overnight followed by secondary antibody at room temperature for 1 h. Antibodies used are in Table 3. Sarcomere length was measured by taking pixel intensity along a line parallel to the sarcomere using a-actinin staining and calculating the distance between intensity peaks using ImageJ.

Colocalization analysis

[00184] In each fluorescent image, a 256 by 256 pixel region of interest was selected. Colocalization analysis was run in ImageJ using the Coloc2 plugin, which provides pixel intensity correlation of space using the Pearson method.

2D contractility analysis

[00185] iPSC-CM were replated in Matrigel coated 24 well plates at a density of 200,000 per cm2. Brightfield videos of spontaneously beating cells were taken at a frame rate of 20 frames per second and analyzed with custom written Python code using the principle of pixel intensity subtraction. (Giacomelli, E., Meraviglia, V., Campostrini, G., Cochrane, A., Cao, X., van Helden, R.W.J., Krotenberg Garcia, A., Mircea, M., Kostidis, S., Davis, R.P., et al. (2020). Cell Stem Cell 26, 862-879. el l.; Sala, L., van Meer, B.J., Tertoolen, L.G.J., Bakkers, J., Beilin, M., Davis R.P., Denning, C., Dieben, M.A.E., Eschenhagen, T., Giacomelli, E., et al (2018) Circ. Res. 122, e5-el6). All code is available at github.com/GVNLab.

High-throughput compound screen [00186] Cells were plated in 384-well black with clear bottom plates coated with Matrigel (Greiner 781,096). On the day of the assay, medium was aspirated from the assay plates and replaced with 10 pL of fresh media. After 30 min incubation for cells to acclimate to the media, 10 pL of FLIPR Calcium 6 dye (Molecular Devices) was added to the assay plates followed by centrifugation at 46g for 10 s. Assay plates were incubated for 2 h at 37°C in a humidified 5% CO2 incubator to load the dye. At the end of the incubation, a baseline reading was taken on the FLIPR Tetra (Molecular Devices). The exposure time was 0.05 s and 388 reads were collected at a read time interval of 0.125 s. Screening compounds (20 nL of 10 mM stock in DMSO) was added to the assay plates using a Labcyte Echo 550 acoustic dispenser (Beckman Coulter), resulting in a final concentration of 10 pM compound and 0.1% DMSO. Negative (DMSO vehicle) and positive control wells (300 pM nisoldipine and 10 pM NKH477) were included on every plate. Assay plates were centrifuged at 46 g for 10 s and incubated for 1 h at 37°C in a humidified 5% CO2 incubator before reading on the FLIPR. One minute of fluorescent flux of spontaneously beating cells was captured for each well (excitation/emission 470/530). An additional read was also taken 3 h post-drug treatment. Libraries screened included FDA-approved drug library (Enzo Life Sciences), Pharmakon (Micro-Source Discovery Systems), and Tested-In-Humans collection (Yale Center for Molecular Discovery). Calcium traces for each well obtained from FLIPR Tetra were analyzed using a custom R script. Briefly, each trace was normalized to baseline fluorescence. A peak finding algorithm was implemented using the ‘findpeaks’ function in the R package ‘pracma’. For each peak, the portion of the trace ranging from maximum amplitude to the minimum of the curve obtained from findpeaks was segmented. Linear regression using the equation:

where y is the fluorescence value and t is time, was used to obtain the value of tau. The value tau is reported as the average of all beats in the well. Compounds were rank ordered by their effect on tau at each time point, and the intersection of the top 100 compounds from each list were used to determine candidate therapies to validate.

Compound validation

[00187] iPSC-CM were replated in Matrigel coated 24 well plates at a density of 200,000 per cm2 and allowed 3 days to recover. Cells were stained with Fluo-4 (ThermoFisher F14201) calcium dye using a 1 :4 dilution in culture media for 15 min. Then media was replaced with fresh media containing a dilution of 1 : 10 Fluo-4 dye. Trequinsin or DMSO was used to treat iPSC-CM at 5pM for 30 min. Fluorescent videos were taken of cardiomyocytes beating 30 min post treatment. Videos were processed to extract a curve of calcium fluorescence, and subsequent analysis of calcium transients was analyzed using a Python script.

Generation of ECT

[00188] ECTs were generated and analyzed in our recently reported pipeline. (Tamargo, M.A., Nash, T.R., Fleischer, S., Kim, Y., Vila, O.F., Yeager, K., Summers, M., Zhao, Y., Lock, R., Chavez, M., et al. (2021). ACS Biomater. Sci. Eng. 7, 5215-5229.) Briefly, bioreactors were cast from PDMS in custom-milled molds containing electrodes for electrical stimulation. Cardiomyocytes and human primary cardiac fibroblasts were dissociated using 10X TrypLE (ThermoFisher) for 15-20 min. Cells were resuspended at a concentration of 500,000 cells per tissue in a ratio of 75% cardiomyocytes and 25% cardiac fibroblasts, in a solution of 5 mg/mL fibrinogen. 12pL of cell suspension was mixed with 3pL of thrombin (12.5U/mL) in each well to cast one tissue. For three days after tissue formation, tissues were maintained in fresh B27 media containing 5 mg/mL 6-aminocaproic acid (Sigma-Aldrich A7824). Seven days after tissue formation, media was changed to an RPMLbased metabolic maturation media containing AlbuMAX (ThermoFisher 11,020,021) higher calcium content, and lower glucose content to promote fatty acid oxidation, as detailed previously.25 Also at day 7, tissues began a 2-week ramped electrical stimulation regimen which lasted from 2Hz and increased 0.33Hz every 24 h until 6Hz. After this two-week regimen, tissues were electrically paced at 1 Hz.

Force analysis of ECT

[00189] Analysis of tissue function and force generation was performed by capturing video of tissue contraction while stimulated at 1Hz and analyzing pillar deflection using a custom-written Python code, previously outlined in detail. (Tamargo, M.A., Nash, T.R., Fleischer, S., Kim, Y., Vila, O.F., Yeager, K., Summers, M., Zhao, Y., Lock, R., Chavez, M., et al. (2021). ACS Biomater. Sci. Eng. 7, 5215-5229.) Briefly, a computer vision package containing an object-tracking algorithm was adapted to track pillar head movement and calculate displacement from videos of beating tissues. Displacement measurements were then used to calculate force based on previously determined empirical measurements, which determined the force needed to deflect the pillar using a microscale mechanical tester (Microtester MT-LT, CellScale). Relaxation and contraction velocity measurements were calculated using the derivative of displacement.

Drug treatments of human cardiac microtissue

[00190] Matured cardiac tissue following 4 weeks of culture were treated with lOpM trequinsin-HCl. Contractile analysis was performed prior and post compound treatment at 1 and 3 h, using the method described above.

Arrhythmia testing on iPSC-CM

[00191] GCAMP6AGAA and GCAMP6 iPSC-CMs were seeded on coverslips and treated with Trequinsin (lOpM), E-4031 (50pM) or forskolin (lOpM) for Ih before imaging. The coverslip with cells was placed in a bath of Tyrode buffer and were stimulated with a frequency of 0.5 Hz for 1 min. Emission was detected at 510 nm through a Nikon Fluor x 10 objective by a Prime BSI-Express sCMOS (scientific Complementary Metal-Oxide- S emi conductor) camera (Teledyne Photometries) and Nikon Elements (Version 5.21) as previously described. (Papa, A., Zakharov, S.I., Katchman, A.N., Kushner, J.S., Chen, B.X., Yang, L., Liu, G., Jimenez, A.S., Eisert, R.J., Bradshaw, G.A., et al. (2022). Nat. Cardiovasc. Res. 1, 1022-1038). Cells were randomly selected for rhythmicity analysis. Calcium fluorescence was analyzed using a written Python code available on Github.

Whole-mount staining of ECT

[00192] Tissues were fixed and permeabilized with 100% methanol for 15 min at room temperature. After Ih blocking with 5% BSA in PBS, cells or tissues were incubated with antibodies against a-actinin 2 (MACS 130-119-766), Vimentin (Abeam ab202504) and Cardiac Troponin T (BD Biosciences 565744) for Ih at room temperature in the dark. For whole mount immunofluorescence staining, cardiac microtissues were embedded in Pro-Long Glass Antifade Mountant with NucBlue Stain (Invitrogen P36981) in CoverWell incubation Chambers (Grace Bio-Labs 645501), ECTs were imaged on a Nikon Al confocal microscope.

Relative cardiomyocyte size assessment by flow cytometry

[00193] Per replicate, 3 cardiac tissues were pooled into a 1.5mL Eppendorf containing a solution of 200U/mL Type II Collagenase (Worthington) in Hank’s Buffered Saline Solution (HBSS). Tissues were dissociated for 1-2 h at 37°C with constant agitation. The solution was spun at 10g for 5 min to remove large debris, and the supernatant collected. Supernatant was then pelleted again with a spin at 300g for 5 min, and washed 3X with PBS. Pellet was then stained with anti-SIRPA and CD90 antibodies (Biolegend 323806, Thermofisher 11-0909-42) in PBS at a concentration of 1 :500. Cells were incubated at room temperature in the dark for 30 min. Following another 3 PBS washes, cells were suspended in PBS containing 1 : 1000 Sytox Blue (Thermofisher S34857) and 1 : 1000 Vibrant DyeCycle Ruby (Thermofisher V10309). Samples were run on a Bio-Rad ZE5 cell analyzer. Cells were discriminated from debris by being Sytox Blue negative and DyeCycle Ruby positive. Cardiomyocytes were selected on the basis of SIRPA+ but CD90- 58.

Lactate dehydrogenase cytotoxicity assay

[00194] After ECTs were treated with trequinsin for 3 h, the supernatant from each tissue was collected. LDH in the supernatant was quantified using the LDH-Glo Cytotoxicity Assay following manufacturer’s instructions.

Western Blot

[00195] iPSC-CM were washed with cold PBS and lysed using Pierce IP Lysis Buffer (Thermofisher 87787) containing protease and phosphatase inhibitors (ThermoFisher 78442) Lysate was transferred to ice for 30 min, then centrifuged at 16,000g for 10 min. The supernatant was removed to a clean tube as the soluble portion. The pellet was then resuspended in lysis buffer containing lOmM Tris-HCl and 4% SDS. Lysates were quantified with Pierce BCA kit, mixed 1 : 1 with 2X Laemmli buffer (Biorad) and 20ug total protein was loaded in 4-20% Tris-glycine gels (Thermo, XP04205BOX).

PKA activity assay

[00196] Cells were washed with PBS and then lysed in ice-cold IP Lysis Buffer (ThermoFisher 87787) containing protease and phosphatase inhibitors (ThermoFisher 78442) for 30 min. Protein concentration was quantified using BCA, then 5pg of protein was used in a PKA activity colorimetric kit (ThermoFisher EIAPKA) per manufacturer’s instructions.

Fractionation of soluble and insoluble fractions

[00197] Cells were washed with PBS and then lysed in ice-cold IP Lysis Buffer (ThermoFisher 87787) containing protease and phosphatase inhibitors (ThermoFisher 78442) for 30 min. The cells were centrifuged at 16,000g for 15 min and supernatants were collected, generating the soluble fraction. The insoluble pellets were dissolved in lysis buffer containing 200U/mL DNAse I (Roche) and 2% sodium dodecyl sulfate (SDS), and sonicated, generating the insoluble fractions that were used for Western blotting.

Quantification and statistical analysis

[00198] Graphpad Prism Version 9 was used for all statistical analysis. The details of statistical analysis including n, test used, and significance are reported in the figure legends. Assumptions of normality were analyzed using qq-plots.

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Claims

What is claimed:

1. A method of identifying a compound that modulates relaxation velocity of a contractile cell, the method comprising: a) culturing contractile cells in two-dimensional culture, wherein the contractile cells are derived from an induced pluripotent stem cell (iPSC); b) contacting the contractile cells with a calcium-sensitive indicator; c) measuring a signal of calcium flux generated from the calcium sensitive indicator; d) contacting the contractile cells with a test compound; e) measuring a signal of calcium flux generated from the calcium sensitive indicator; and f) determining a time constant of relaxation value using the calcium flux signal measured in step e); and g) identifying the test compound as a compound that modulates relaxation velocity of a contractile cell if the time constant of relaxation value determined in step f) is significantly higher or lower than either: i. a time constant of relaxation value for the contractile cell determined using the calcium flux signal measured in step c); or ii. a time constant of relaxation value for a control contractile cell.

2. A method of identifying a compound that modulates relaxation velocity of a beating cardiomyocyte, the method comprising: a) culturing beating cardiomyocytes in two-dimensional culture, wherein the cardiomyocytes are derived from an induced pluripotent stem cell (iPSC); b) contacting the beating cardiomyocytes with a calcium-sensitive indicator; c) measuring a signal of calcium flux generated from the calcium sensitive indicator; d) contacting the beating cardiomyocytes with a test compound; e) measuring a signal of calcium flux generated from the calcium sensitive indicator; and f) determining a time constant of relaxation value using the calcium flux signal measured in step e); and g) identifying the test compound as a compound that modulates relaxation velocity of a beating cardiomyocyte if the time constant of relaxation value determined in step f) is significantly higher or lower than either: i. a time constant of relaxation value for the beating cardiomyocyte determined using the calcium flux signal measured in step c); or ii. a time constant of relaxation value for a control beating cardiomyocyte. The method of claim 1 or 2, wherein the determining of step f) comprises: normalizing the signal of calcium flux measured in e) to the signal of calcium flux measured in c); identifying calcium flux signal peaks in the normalized calcium flux signal; for each peak, segmenting the portion of the normalized calcium flux signal from a maximum amplitude to a minimum value; performing a linear regression on the segmented normalized calcium flux signal to obtain a value of tau (T) corresponding to a time constant of relaxation value; averaging the value of tau across the number of signal peaks. The method of claim 3, wherein the linear regression is performed using the following equation:

wherein y is the normalized calcium flux signal and t is time. The method of claims 2-4, wherein the test compound is identified as a compound that modulates relaxation velocity of a beating cardiomyocyte if the time constant of relaxation value determined in step f) is significantly higher or lower than a time constant of relaxation value for the beating cardiomyocyte determined using the calcium flux signal measured in step c). The method of claims 2-4, wherein the test compound is identified as a compound that modulates relaxation velocity of a beating cardiomyocyte if the time constant of relaxation value determined in step f) is significantly higher or lower than a time constant of relaxation value for a control beating cardiomyocyte. The method of claims 2 or 6, wherein the control beating cardiomyocyte is a control by virtue of being contacted with DMSO instead of a test compound. The method of claims 2 or 6, wherein the time constant of relaxation value for the control beating cardiomyocyte is determined comprising: culturing beating cardiomyocytes in two-dimensional culture, wherein the cardiomyocytes are derived from an induced pluripotent stem cell (iPSC); contacting the beating cardiomyocytes with a calcium-sensitive indicator; measuring a signal of calcium flux generated from the calcium sensitive indicator; contacting the beating cardiomyocytes with DMSO; measuring a signal of calcium flux generated from the calcium sensitive indicator; and determining a time constant of relaxation value using the calcium flux signal measured after contacting the beating cardiomyocytes with DMSO. The method of claim 8, wherein determining the time constant of relaxation for the beating cardiomyocytes with DMSO comprises normalizing the signal of calcium flux measured after contacting the beating cardiomyocytes with DMSO to the signal of calcium flux measured prior to after contacting the beating cardiomyocytes with DMSO; identifying calcium flux signal peaks in the normalized calcium flux signal; for each peak, segmenting the portion of the normalized calcium flux signal from a maximum amplitude to a minimum value; performing a linear regression on the segmented normalized calcium flux signal to obtain a value of tau (T) corresponding to a time constant of relaxation value; averaging the value of tau across the number of signal peaks. The method of claim 9, wherein the linear regression is performed using the following equation:

wherein y is the normalized calcium flux signal and t is time. The method of claims 8-10, wherein the control beating cardiomyocyte and the cardiomyocyte contacted with the test compound are derived from the same iPSC. The method of claim 2, wherein culturing beating cardiomyocytes in two-dimensional culture comprises: isolating peripheral mononuclear blood cells from a blood sample from a subject; reprograming the isolated peripheral mononuclear blood cells to generate induced pluripotent stem cells (iPSCs); culturing the iPSCs in a culture medium comprising a cardiac differentiation media (CDM) containing RPMI1640, albumin and ascorbic acid to generate beating cardiomyocytes; culturing the beating cardiomyocytes in a culture medium comprising RPMI1640 with B27 supplement; culturing the beating cardiomyocytes at least one passage in a culture medium comprising RPMI1640 with B27 supplement and CHIR99021; culturing the beating cardiomyocytes in a culture medium comprising RPMI160 with B27 supplement. The method of claim 1, wherein the contractile cells are human. The method of claim 2, wherein the beating cardiomyocytes are human. The method of claims 12, wherein the isolating peripheral mononuclear blood cells is by immunomagnetic cell separation. The method of claim 15, wherein the immunomagnetic cell separation uses an antibody against Epithelial Cell Adhesion Molecule (EpCAM). The method of claim 12, further comprising: serially passaging the beating cardiomyocytes. The method of claim 2, wherein the beating cardiomyocytes are cultured in a multiwell plate and each well of the multi-well plate is used to test one or more different test compounds or different concentrations of one or more test compounds. The method of claim 18, wherein the control beating cardiomyocyte is cultured in a well of the same multi-well plate as the beating cardiomyocytes contacted with test compound. The method of claims 18 or 19, wherein a second control beating cardiomyocyte is cultured in a well of the same multi-well plate as the beating cardiomyocytes contacted with test compound, wherein the second control is contacted with a compound that is known to modulate the relaxation velocity of a beating cardiomyocyte. The method of claims 18-20, wherein the multi -well plate is a 384 well plate. The method of claim 2, wherein the method is for identifying a test compound that increases the relaxation velocity of a beating cardiomyocyte, wherein if the time constant of relaxation value determined in step f) is significantly lower than either i) or ii) the test compound is identified as a compound that increases the relaxation velocity of a beating cardiomyocyte. The method of claim 2, wherein the method is for identifying a test compound that decreases the relaxation velocity of a beating cardiomyocyte, wherein if the time constant of relaxation value determined in step f) is significantly higher than either i) or ii) the test compound is identified as a compound that decreases the relaxation velocity of a beating cardiomyocyte. The method of claim 2, wherein culture medium of the culturing of step a) is replaced with fresh culture medium before contacting the beating cardiomyocytes with the calcium-sensitive indicator. The method of claim 24, wherein the calcium sensitive indicator is a chemical sensitive dye, a genetically encoded calcium indicator, or a combination thereof. The method of claim 25, wherein the chemical indicator is a calcium sensitive dye. The method of claim 25, wherein the genetically encoded calcium indicator is GCaMP. The method of claim 2, wherein the calcium-sensitive indicator is Calcium 6. The method of claim 2, wherein the beating cardiomyocytes are contacted with the test compound for 1 hour. The method of claim 2, wherein the beating cardiomyocytes are contacted with the test compound for 3 hours. The method of claim 2, wherein the beating cardiomyocytes are contacted with the test compound for 1 day. The method of claim 2, wherein the beating cardiomyocytes are contacted with the test compound for 1 week. The method of claim 2, wherein the beating cardiomyocytes comprise filamin C (FLNC) with a mutation, a deletion or an addition of at least one nucleic acid or amino acid. The method of claim 33, wherein the deletion of the nucleic acid is from position 7416 to 7418. The method of claim 33, wherein the beating cardiomyocytes comprise a FLNC mutation, deletion, or addition that has been corrected to wild-type. The method of claims 2, wherein the beating cardiomyocytes display a phenotype of cardiomyopathy. The method of claims 2 or 33, wherein the beating cardiomyocytes express a genetically encoded calcium indicator. A method of observing a dynamic physical property of a beating cardiomyocyte, the method comprising: a) culturing beating cardiomyocytes in two-dimensional culture, wherein the cardiomyocytes are derived from an induced pluripotent stem cell (iPSC); b) observing the beating cardiomyocytes with brightfield video microscopy; c) determining the contractile amplitude, contraction velocity, relaxation velocity, or a combination thereof. The method of claim 38, wherein the determining is performed using pixel intensity subtraction. The method of claim 38, wherein the video microscopy is performed at about 100 frames per second. The method of claim 38, wherein the beating cardiomyocytes are cultured at about 200,000 cells per cm². The method of claim 38, wherein the beating cardiomyocytes are cultured in a multiwell plate. The method of claims 38-42, further comprising contacting the cells with a test compound and determining the contractile amplitude, contraction velocity, relaxation velocity, or a combination thereof in the presence of the test compound. A method of identifying a compound that modulates a dynamic physical property of an engineered cardiac tissue, the method comprising: a) culturing an engineered cardiac tissue (ECT) comprising cardiomyocytes derived from iPSCs and human primary cardiac fibroblasts in a bioreactor, wherein the cardiomyocytes and fibroblasts are encapsulated in a fibrin gel and grow around two horizontal PDMS pillars of the bioreactor, wherein the bioreactor further comprises electrodes for electrical stimulation of the ECT; b) stimulating the ECT with an electrical stimulation; c) maintaining the ECT with a lower electric stimulation than the electric stimulation used in step (b); d) determining a dynamic physical property of the ECT comprising

1. stimulating the ECT with electrical stimulation and capturing video of ECT contraction; ii. analyzing pillar deflection. The method of claim 44, wherein analyzing the pillar deflection comprises: tracking pillar head movement; and calculating displacement values. The method of claim 45, further comprising using the displacement values to calculate force. The method of claim 46, wherein the force is calculated using the known force needed to deflect a pillar of the bioreactor. The method of claim 47, wherein the force calculated is force that displaces pillars during ECT contraction (active force). The method of claim 47, wherein the force calculated is force causing pillar deflection during maximum relaxation of the ECT (residual force). The method of claim 47, wherein the force calculated is the sum of the active force that displaces pillars during ECT contraction and the residual force causing pillar deflection during maximum relaxation of the ECT (total force). The method of claims 45-50, further comprising determining a relaxation velocity measurement, a contraction velocity measurement, or both, using the derivative of the displacement values. The method of any of claims 44 to 51, further comprising contacting the ECT with a test compound and determining whether the test compound modulates a dynamic physical property of the ECT comprising: stimulating the ECT in the presence of the test compound with electrical stimulation and capturing video of ECT contraction; and analyzing pillar deflection. The method of claim 52 wherein analyzing the pillar deflection in the presence of the test compound comprises: tracking pillar head movement; and calculating displacement values in the presence of the test compound. The method of claim 53, further comprising using the displacement values in the presence of the test compound to calculate force in the presence of the test compound. The method of claim 54, wherein the force in the presence of the test compound is calculated using the known force needed to deflect a pillar of the bioreactor. The method of claim 55, wherein the force calculated is force that displaces pillars during ECT contraction in the presence of the test compound (active force). The method of claim 55, wherein the force calculated is force causing pillar deflection during maximum relaxation of the ECT in the presence of the test compound (residual force). The method of claim 55, wherein the force calculated is the sum of the active force that displaces pillars during ECT contraction in the presence of the test compound and the residual force causing pillar deflection during maximum relaxation of the ECT in the presence of the test compound (total force). The method of claims 53-58, further comprising determining a relaxation velocity measurement in the presence of the test compound, a contraction velocity measurement in the presence of the test compound, or both, using the derivative of the displacement values in the presence of the test compound. The method of claim 44, wherein the culturing an ECT of step (a) comprises: dissociating cardiomyocytes and primary cardiac fibroblasts; suspending the dissociated cells in a fibrinogen solution; mixing the suspended cells with thrombin and placing in contact with the PDMS pillars to generate an ECT; maintaining the ECT in a culture medium comprising B27 media and 6- aminocaproic acid; and maintaining the tissue in a culture medium comprising RPMI-based metabolic maturation media, AlbuMAX, higher calcium content and lower glucose content than the culture medium of the prior step. The method of claim 60, wherein the dissociating further comprising dissociating the cardiomyocytes and the primary cardiac fibroblasts with TrypLE™ or trypsin. The method of claims 53-58, wherein the test compound is identified as a compound that modulates a dynamic physical property of the ECT tissue generated if in the presence of the test compound the ECT has significantly higher or lower active force, passive tension, total force, contraction velocity, relaxation velocity, or a combination thereof compared to the ECT cultured without the test compound or to a ECT comprising an isogenic control tissue or a wild-type tissue. The method of claims 53-58, wherein the test compound is identified as a compound that modulates a dynamic physical property of the ECT tissue generated if in the presence of the test compound the ECT has significantly higher or lower active force, passive tension, total force, contraction velocity, relaxation velocity, or a combination thereof compared to the ECT cultured without the test compound. The method of claims 53-58, wherein the test compound is identified as a compound that modulates a dynamic physical property of the ECT tissue generated if in the presence of the test compound the ECT has significantly higher or lower active force, passive tension, total force, contraction velocity, relaxation velocity, or a combination thereof compared to a ECT comprising an isogenic control tissue or a wild-type tissue. The method of claim 44, wherein the cardiomyocytes are human. The method of claim 44, wherein the cardiomyocytes comprise filamin C (FLNC) with a mutation, a deletion or an addition of at least one nucleic acid or amino acid. The method of claim 66, wherein the deletion of the nucleic acid is from position 7416 to 7418. The method of claim 66, wherein the beating cardiomyocytes comprise a FLNC mutation, deletion, or addition that has been corrected to wild-type. The method of any one of claims 12, 24 or 60, wherein the culture medium further comprises Glutamax. The method of claims 12, 24 or 60, wherein the culture medium further comprises EGF. The method of claim 70, wherein the culture medium comprises 10 ng/ml of EGF. The method of claims 12, 24 or 60, wherein the culture medium further comprises antibiotic-antimycotic. The method of claims 12, 24 or 60, wherein the culture medium comprises 5% Matrigel. The method of claims 12, 24 or 60, wherein the culture medium comprises 5% heat- inactivated charcoal-stripped FBS. The method of claim 22, wherein the test compound that increases the relaxation velocity of a beating cardiomyocyte is identified as a treatment for cardiomyopathy. The method of claim 62, wherein the test compound that increases passive tension of the ECT, increases the relaxation velocity of the ECT, or a combination thereof is identified as a treatment for cardiomyopathy. The method of any one of claims 1-37, 43, 52-59, 62-64, 75 or 76, wherein the test compound is a small molecule. The method of any one of claim 1-37, 43, 52-59, 62-64, 75 or 76, wherein the test compound is an antibody. The method of any one of claim 1-37, 43, 52-59, 62-64, 75 or 76, wherein the test compound is an antisense oligonucleotide. The method of any one of claims 1-37, 43, 52-59, 62-64, 75 or 76, wherein the test compound is a phosphodiesterase 3 (PDE3) inhibitor. A method of treating a cardiomyopathy in a subject in need thereof, comprising: a) culturing beating cardiomyocytes in two-dimensional culture, wherein the cardiomyocytes are derived from an induced pluripotent stem cell (iPSC); b) contacting the beating cardiomyocytes with a test compound; c) observing the beating cardiomyocytes with brightfield video microscopy; and d) determining a contractile amplitude, a contraction velocity, a relaxation velocity or a combination thereof wherein the test compound is administered to the subject if a contractile amplitude, a contractile velocity, a relaxation velocity or a combination thereof of the beating cardiomyocytes is increased as compared to a contractile amplitude, a contractile velocity, a relaxation velocity or a combination thereof of the beating cardiomyocyte in the absence of the test compound. A method of treating a cardiomyopathy in a subject in need thereof, comprising: a) culturing beating cardiomyocytes in two-dimensional culture, wherein the cardiomyocytes are derived from an induced pluripotent stem cell (iPSC); b) contacting the beating cardiomyocytes with a calcium-sensitive indicator; c) measuring a signal of calcium flux generated from the calcium sensitive indicator; d) contacting the beating cardiomyocytes with a test compound; e) measuring a signal of calcium flux generated from the calcium sensitive indicator; and f) determining a time constant of relaxation value using the calcium flux signal measured in step e); and g) identifying the test compound as a compound that modulates relaxation velocity of a beating cardiomyocyte if the time constant of relaxation value determined in step f) is significantly higher or lower than either: i. a time constant of relaxation value for the beating cardiomyocyte determined using the calcium flux signal measured in step c); or ii. a time constant of relaxation value for a control beating cardiomyocyte wherein, the test compound is administered to the subject if relaxation velocity of a beating cardiomyocyte is higher in the presence of the test compound as compared to relaxation velocity of a beat cardiomyocytes in the absence of a test compound. ethod of treating a cardiomyopathy in a subject in need thereof, comprising: a) culturing an engineered cardiac tissue (ECT) comprising cardiomyocytes derived from iPSCs and human primary cardiac fibroblasts in a bioreactor, wherein the cardiomyocytes and fibroblasts are encapsulated in a fibrin gel and molded around two horizontal PDMS pillars and wherein the bioreactor further comprises electrodes for electrical stimulation of the ECT; b) stimulating the ECT with an electrical stimulation; c) maintaining the ECT with a lower electric stimulation than the electric stimulation used in (b); d) contacting the ECT with a test compound; and e) determining a dynamic physical property of the ECT comprising i. stimulating the ECT with electrical stimulation and capturing video of ECT contraction; ii. analyzing pillar deflection by tracking pillar head movement and calculating displacement values, wherein the displacement values are used to calculate an active force, a residual force, a total force or a combination thereof; wherein the test compound is administered to the subject if the active force, the residual force, the total force or a combination thereof in the ECT is decreased in the presence of the test compound, as compared to the forces in the ECT in the absence of the test compound. The method of any one of claims 81-83, wherein the test compound is a small molecule. The method of any one of claims 81-83, wherein the test compound is an antibody. The method of any one of claims 81-83, wherein the test compound is an antisense oligonucleotide. The method of any one of claims 81-83, wherein the test compound is a phosphodiesterase 3 (PDE3) inhibitor. The method of claim 87, wherein the phosphodiesterase inhibitor is trequinsin. An in vitro cell line comprising a beating cardiomyocyte derived from an induced pluripotent stem cell (iPSC), wherein the beating cardiomyocyte comprises filamin C (FLNC) with a mutation, a deletion or an addition of at least one nucleic acid or amino acid. An in vitro cell line comprising a beating cardiomyocyte that is derived from an induced pluripotent stem cell (iPSC), wherein the beating cardiomyocyte comprises filamin C (FLNC) with a mutation, a deletion or an addition of at least one nucleic acid or amino acid; wherein the beating cardiomyocyte is cultured on a two-dimensional surface. The beating cardiomyocyte of claim 89 or 90, wherein the deletion of the nucleic acid is from position 7416 to 7418. The beating cardiomyocyte of claim 89 or 90, wherein the beating cardiomyocytes comprise a FLNC mutation, deletion, or addition that has been corrected to wild-type. An engineered cardiac tissue that comprises cardiomyocytes derived from an induced pluripotent stem cell (iPSC) and human primary cardiac fibroblasts wherein the engineered cardiac tissue comprises filamin C (FLNC) with a mutation, a deletion or an addition of at least one nucleic acid or amino acid. The engineered cardiac tissue of claim 93, wherein the deletion of the nucleic acid is from position 7416 to 7418. The engineered cardiac tissue of claim 93, wherein the beating cardiomyocytes comprise a FLNC mutation, deletion, or addition that has been corrected to wild-type. A cDNA library that comprises filamin C (FLNC) with a mutation, a deletion or an addition of at least one nucleic acid or amino acid. The cDNA library of claim 96, wherein the deletion of the nucleic acid is from position 7416 to 7418.