WO2019006512A1 - Cardiomyocyte regeneration - Google Patents

Cardiomyocyte regeneration Download PDF

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WO2019006512A1
WO2019006512A1 PCT/AU2018/050700 AU2018050700W WO2019006512A1 WO 2019006512 A1 WO2019006512 A1 WO 2019006512A1 AU 2018050700 W AU2018050700 W AU 2018050700W WO 2019006512 A1 WO2019006512 A1 WO 2019006512A1
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cardiomyocytes
catenin
cardiomyocyte
heart
yap
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PCT/AU2018/050700
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French (fr)
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James Hudson
Enzo PORRELLO
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The University Of Queensland
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Priority claimed from AU2017902668A external-priority patent/AU2017902668A0/en
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Publication of WO2019006512A1 publication Critical patent/WO2019006512A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • a central feature of the cardiac regenerative response in neonatal mice is the activation of cardiomyocyte proliferation following injury (Haubner et al., 2012; Porrello et al., 2011; Porrello et al., 2013).
  • Genetic studies in the mouse have identified cardiomyocyte proliferation as the primary source of regenerated cardiomyocytes (Porrello et al., 2011), which is similar to findings in adult zebrafish (Jopling et al., 2010; Kikuchi et al., 2010).
  • the developmental timing of regenerative arrest in rodents coincides with the postnatal window when most cardiomyocytes withdraw from the cell cycle and become terminally differentiated (Soonpaa et al., 1996).
  • the one or more genetic elements is, or includes YAP- 1 protein.
  • the invention provides a method of regenerating cardiomyocytes, said method including the step facilitating nuclear expression of a constitutively active ⁇ -catenin protein and, optionally, modulation of one or more genetic elements in one or more cardiomyocytes of the heart to thereby promote or enhance cardiomyocyte regeneration.
  • the invention provides a method of identifying, screening or producing a molecule capable of inducing or facilitating cardiomyocyte regeneration, said method including the step of identifying a molecule that mimics or facilitates ⁇ - catenin-mediated gene expression in a cardiomyocyte and thereby induces cardiomyocyte regeneration.
  • the molecule facilitates nuclear localization of an endogenous ⁇ -catenin protein in a cardiomyocyte.
  • Figure 19 Validation of cell purity and CHIR activity in RNA-seq samples.
  • IWR-1 significantly reduced the percentage of PH3+ cardiomyocytes in vivo.
  • FIG. 21 Cardiomyocyte proliferation and WNT-P-catenin signaling are repressed in hCOs cultured in MM.
  • mice Cardiomyocytes from AAV6-YAP1-S127A and AAV6-N90-BCAT i.v. injected mice had significantly reduced cross sectional area compared to AAV6-CMV-GFP injected controls.
  • AAV6-YAP1-S127A and AAV6-N90-BCAT co-administration resulted in elevated Birc5 expression in adult mice cardiomyocytes.
  • Adult mice (P56) were intracardially injected with AAVs (2xlO u viral particles per animal) and cardiomyocytes and nonmyocytes were isolated by enzymatic digestion 3 days later.
  • Control animals were injected with AAV6-CMV-GFP.
  • n 2 mice.
  • GSK3 -mediated phosphorylation of the bolded serine and threonine residues in ⁇ catenin shown in SEQ ID NO: l is required for binding by ubiquitin ligases such as E3 ubiquitin ligase TrCP-1 that cause resultant ubiquitination and proteolytic degradation of ⁇ catenin.
  • ubiquitin ligases such as E3 ubiquitin ligase TrCP-1 that cause resultant ubiquitination and proteolytic degradation of ⁇ catenin.
  • certain embodiments of a constitutively active ⁇ catenin may have some or all of the bolded serine and threonine residues in SEQ ID NO: l mutated (e.g. to alanine or other non- conservative substitution) or deleted, thereby preventing phosphorylation and resultant degradation of ⁇ catenin.
  • the constitutively active ⁇ catenin protein may comprise an N-terminal deletion of some or all of the N-terminal region comprising the serine and threonine residues that can be phosphorylated by GSK3. In some embodiments at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or all of the 89 amino acids underlined in SEQ ID NCv l may be deleted.
  • constitutively active ⁇ -catenin may be administered or expressed in combination with modulation of one or more genetic elements.
  • genetic elements may include molecules that may facilitate, enhance, potentiate or synergize with constitutively active ⁇ -catenin to promote cardiomyocyte regeneration.
  • the one or more genetic elements are, or act by way of, epigenetic factors (Sim et al, FASEB J, 2015; Quaife-Ryan et al, Sem. Cell. Dev. Biol, 2016).
  • YAP and/or TAZ When activated, YAP and/or TAZ can bind to several transcription factors including p73, API, TBX5, Runx2 and several TEA domain family members (TEADs).
  • Potential gene targets of YAP-1 -mediated transcriptional activation in cardiomyocytes are provided in the Examples.
  • Serine 127 is critical for Large Tumour Suppressor (LATS) kinase-mediated cytoplasmic sequestration of YAP by the Hippo pathway. Phosphorylation by LATS kinases promotes cytoplasmic sequestration of YAP in a manner that involves 14-3-3 proteins and a-catenin. LATS-mediated phosphorylation of YAP serine 127 also can promote YAP-1 ubiquitination and subsequent proteasomal degradation. Serine 127 is bolded and underlined in SEQ ID NO:2 below.
  • the NLS may be monopartite or bipartite, a bipartite NLS sequence comprising a linker of up to about 10-12 amino acids connecting each NLS sequence.
  • NLS sequence motifs include K(R/K)X(R/K) (SEQ ID NO:27), K(K/R)X(K/R) (SEQ ID NO:28), KR(R/X)K (SEQ ID NO:29), KRRR (SEQ ID NO:30), KR(K/R)R (SEQ ID NO:31) or K(K/R)RK(SEQ ID NO:32) for a monopartite NLS and (K/R)(K/R)Xio-i2(K/R) 3 / 5 (SEQ ID NO:33), KRX10-12KRRK (SEQ ID NO:34), KRXio-i 2 K(K/R)(K/R) (SEQ ID NO:35) or KRXio- i 2 K(K
  • the NLS is located at or near the N-terminus of the constitutively active ⁇ catenin protein and/or the constitutively active YAP-1 protein.
  • a particular aspect of the invention provides a genetic construct comprising: (i) a nucleotide sequence that encodes a constitutively active ⁇ -catenin protein operably linked to one or more regulatory nucleotide sequences which facilitate expression of the ⁇ -catenin protein in a cardiomyocyte nucleus; (ii) a nucleotide sequence of one or more genetic elements, such as encoding a constitutively active YAP-1 protein, operably linked to one or more regulatory nucleotide sequences which facilitate expression of the YAP-1 protein in a cardiomyocyte; or (iii) a nucleotide sequence that encodes a constitutively active ⁇ -catenin protein and one or more genetic elements, such as a constitutively active YAP-1 protein, operably linked to one or more regulatory nucleotide sequences which facilitate expression of the constitutively active ⁇ -catenin protein and the constitutively active YAP-1 protein in a cardiomyocyte nucleus.
  • nucleic acid includes and encompasses single and double-stranded DNA and RNA inclusive of genomic DNA, cDNA, RNA such as mRNA, cRNA, miRNA and tRNA and DNA:RNA hybrids.
  • Nucleic acids may comprise A, G, C, T and/or U nucleotides and/or nucleotides that comprise modified pyrimidine or purine bases such as pseudouridine, 5-methyluridine, 2-thiouridine, N 6 -methyladenosine and 5-methylcytidine dihydrouridine, inosine or 7- methylguanosine, although without limitation thereto.
  • nucleic acid forms may include oligonucleotides, restriction fragments, amplification products, primers and probes, although without limitation thereto.
  • Promoters are well known in the art and can include constitutive, inducible or repressible promoters and tissue-specific promoters.
  • a constitutive promoter that is active in many mammalian cell types, including cardiomyocytes is a CMV promoter.
  • the genetic construct may comprise cardiomyocyte-specific promoters or enhancers such as cTNT, a-MHC, NCX1, MLC2v.
  • Modified RNA molecules can be delivered to cardiomyocytes for transient expression, such as described in Kanji et al., 2013 Nat. Biotechnol. 31 898. Reference is also made to International Publication WO2016010119, European Publication EP2694660 and United States Patent 8278036 which also describe the production and delivery of modified RNA.
  • Carriers that facilitate delivery or administration of genetic constructs to cardiomyocytes in vitro may include carriers that facilitate transfection or transformation such as microparticles, CaCh, lipofectin, lipofectamine, DEAE Dextran, although without limitation thereto.
  • the cardiomyocytes are suitable for adoptive transfer or transplantation to a patient.
  • the cardiomyocytes may be autologous cardiomyocytes obtained from the patient or non-autologous cardiomyocytes at least antigenically or immunologically matched with the patient (e.g HLA-matched) to minimize the risk of immune rejection of the transferred cardiomyocytes.
  • the molecule may facilitate nuclear localization of an endogenous ⁇ -catenin protein.
  • the molecule is, or mimics the action of, one or more genetic elements selected from: include: DNA methyltransferases (D MTs), histone acetyl transferases (HATs), histone deacteylases (HDACs), HMTs, histone methyltransferases (HMTs), KDMs, histone lysine demethylases (KDMs), eRNAs, enhancer RNAs (eRNAs), IncRNAs, long non-coding RNAs (RNAs), microRNAs (miRNAs), polycomb repressive complexes (PRC), TETs, ten-eleven translocation (TETs) and/or TFs, transcription regulators (TRs).
  • D MTs DNA methyltransferases
  • HATs histone acetyl transferases
  • HDACs histone deacteylases
  • HMTs histone methyltransferases
  • KDMs histone lysine demethylases
  • the molecule is YAP-1 or activates or mimics
  • the molecule facilitates nuclear localization of an active ⁇ -catenin protein and/or said one or more genetic elements disclosed herein, such as a YAP-1 protein.
  • the method further includes the step of determining whether the molecule is capable of inducing cardiomyocyte regeneration.
  • the inventors decided to globally examine the transcriptional networks driving cardiac injury responses in multiple cell populations at different stages of development.
  • the inventors provide a comprehensive transcriptomic analysis of the major cell populations within the regenerative (PI) and non-regenerative (P56) mouse heart with and without injury (myocardial infarction).
  • PI regenerative
  • P56 non-regenerative
  • These analyses identify unique developmentally regulated and injury -induced transcriptional responses in cardiomyocytes, fibroblasts, endothelial cells and leukocytes that govern distinct cellular behaviours following cardiac injury.
  • These comparative analyses also identify a unique cardiomyocyte transcriptional signature associated with cell cycle shutdown and repression of a Wnt-P-catenin gene regulatory network in both rodent and human cardiomyocytes.
  • Neonatal and adult MI surgeries For neonatal surgeries, timed pregnant CDl female mice were housed under standard conditions with 12h light/dark cycles and ad libitum access to food and water. At postnatal day 1 mice (PI), neonatal MI surgeries were performed as previously described (Mahmoud et al., 2014). For adult male surgeries, the animals were housed under standard conditions with 12h light/dark cycles and ad libitum access to food and water. At 8 weeks of age, male CDl mice were anesthetized with 4% isoflurane (Bayer) and maintained with 2% isoflurane and 0.25 1/min oxygen during surgery.
  • the animals were intubated and ventilated (Minivent, Harvard Apparatus) with a tidal volume of 250 ⁇ and a respiration rate of 133 strokes/min.
  • a thoracotomy at the 3 rd and 4 th intercostal space was performed and the pericardial sac removed.
  • the left descending coronary artery was permanently ligated with 7-0 prolene suture (Ethicon). Following ligation, blanching of the myocardium was observed to determine if adequate ischaemia was achieved.
  • the chest wall was then sutured closed, the mouse removed from aneasthesia, supplied with s.c. injection of buprenorphine (0.05mg/kg) as required and allowed to recover from the surgery. Ethical approval for these neonatal and adult mouse experiments was obtained from The University of Queensland's Animal Ethics Committee (SBMS/101/13/NHMRC).
  • the aorta was then cannulated with a 21 gauge cannula, secured with 3-0 silk suture and perfused with 37°C, oxygenated perfusion buffer using a Langendorff apparatus (4ml/min).
  • digestion buffer 200 ⁇ g/ml Liberase DH (Roche) in perfusion buffer
  • atria and excess tissue was removed.
  • hearts were minced with fine scissors into small pieces and triturated to release cells.
  • Cell isolates were passed through a 100 ⁇ cell strainer and centrifuged at 30xg for 3 minutes at room temperature.
  • RNAseq experiment ribosomal RNA was depleted with Ribo Zero Gold and cDNA was generated with Superscript II Reverse Transcriptase (ThermoFisher). Libraries were created with TruSeq Stranded Total RNA kits (Illumina) and read with HiSeq SR Cluster v4 kit (Illumina) on a HiSeq 2500 sequencer. Each sample contained -45 million 50-bp single-end reads. For the CHIR experiment, Libraries were constructed with Nugen Ovation RNA-Seq system V2 (for SPIA amplifications and cDNA generation) coupled with the Ovation Ultralow System (NuGEN).
  • Nugen Ovation RNA-Seq system V2 for SPIA amplifications and cDNA generation
  • NuGEN Ovation Ultralow System
  • Hearts were fixed in 4% PFA in PBS, washed in PBS and then halved with a single transverse cut at the ligature mark. The hearts were dehydrated and then embedded in paraffin wax. Each sample was sectioned at 6 ⁇ . Sections were then rehydrated, blocked with 10% goat serum in PBS and stained with Anti-phospho-Histone H3 (SerlO) (Millipore, 06-570), ⁇ -catenin (PY489) (Developmental Studies Hybridoma Bank, ⁇ -catenin (PY489)), Tnnt2 (Thermo Scientific, MS-295-P0) diluted 1 : 100 in 2% goat serum/PBS overnight at 4°C.
  • Anti-phospho-Histone H3 SerlO
  • PY489 ⁇ -catenin
  • Tnnt2 Thermo Scientific, MS-295-P0
  • Sections were then stained with secondary antibodies goat anti -rabbit IgG Alexa 633 (A-21070), goat anti-mouse IgM Alexa- 488 (A-21042), and goat anti-mouse Alexa-555 (A-21422) diluted 1 :400 and hoerscht diluted 1 : 1000 in 2% goat serum/PBS for 1 hour at room temperature and mounted in FluoMount®. Each slide was then imaged using a Leica DMi8 confocal microscope
  • Cardiomyocytes were derived from PI Sprague-Dawley neonatal rats as previously described (Thomas et al 2002). Briefly, neonatal rats were sacrificed and hearts were excised, washed in ADS buffer (put in the protocol) and atria removed. Myocytes were then isolated using collagenase (get the Wharton number) and separated with PercollR® gradients. Percoll gradients were then constructed by layering 1 : 1.2 Percoll:ADS layer on a 1 :0.5 Percoll:ADS layer in a 15 ml Falcon tube. Isolated myocytes were plated in Cardiac Medium (see below in 2D experiments) without BRDU and allowed to recover overnight before experiments.
  • Cardiomyocytes were produced based on recently published protocols (Hudson et al., 2012; Titmarsh et al., 2016), which give both cardiomyocytes and stromal cells which are critical for tissue function (Hudson et al., 2011). Briefly, cardiomyocyte/stromal cell cultures were produced from HES3 human embryonic stems cells (hESCs, WiCell) which were maintained as TypLE passaged cultures using mTeSR-1 (Stem Cell Technologies)/Matrigel (Millipore).
  • hESCs were seeded at 2xl0 4 cells/cm 2 in Matrigel coated flasks and cultured for 4 days using mTeSR-1. Subsequently, the hESCs were firstly differentiated into cardiac mesoderm using RPMI B27- medium (RPMI1640 GlutaMAX+ 2% B27 supplement minus insulin, 200 ⁇ L-ascorbic acid 2 phosphate sesquimagnesium salt hydrate (Sigma) and 1% Penecillin/Streptomycin) containing 5 ng/ml BMP -4 (RnD Systems), 9 ng/ml Activin A (RnD Systems), 5 ng/ml FGF-2 (RnD Systems) and 1 ⁇ CHIR99021 (Stem Cell Technologies) with daily medium exchange for 3 days.
  • RPMI B27- medium RPMI1640 GlutaMAX+ 2% B27 supplement minus insulin, 200 ⁇ L-ascorbic acid 2 phosphate sesquimagnesium salt hydrate (S
  • RPMI B27- containing 5 ⁇ IWP-4 Stem Cell Technologies
  • RPMI B27+ RPMI 1640 GlutaMAX + 2% B27 supplement with insulin, 200 ⁇ L- ascorbic acid 2 phosphate sesquimagnesium salt hydrate (Sigma) and 1% Penecillin/Streptomycin
  • the differentiated cells (-75% cardiomyocytes, -25% CD90 + stromal cells) were then cultured in RPMI B27+ until digestion at 15 days using 0.2% collagenase type I (Sigma) in 20% fetal bovine serum (FBS) in PBS (with Ca 2+ and Mg 2+ ) for 45 min at 37°C, followed by 0.25% trypsin-EDTA for 10 min.
  • the cells were filtered using a 100 ⁇ mesh cell strainer (BD Biosciences), centrifuged at 300 x g for 3 min, and resuspended at the required density in Cardiac Medium: a-MEM GlutaMAX, 10% FBS, 200 ⁇ L- ascorbic acid 2 phosphate sesquimagnesium salt hydrate (Sigma) and 1% Penecillin/Streptomycin.
  • a-MEM GlutaMAX 10% FBS
  • 200 ⁇ L- ascorbic acid 2 phosphate sesquimagnesium salt hydrate Sigma
  • Penecillin/Streptomycin 1% Penecillin/Streptomycin.
  • the cells were transfected at 50 nM with scramble miR control (All Stars Negative Control, Qiagen), miR mimic hsa-miR-199a-3p (Qiagen) or miR mimic hsa-miR-590-3p (Qiagen).
  • scramble miR control All Stars Negative Control, Qiagen
  • miR mimic hsa-miR-199a-3p Qiagen
  • miR mimic hsa-miR-590-3p Qiagen
  • constitutively active YAP cells were infected in Cardiac Medium with an adenovirus containing a mutated version of murine YAP-1, CMV-YAP(S112A) at an MOI of 10.
  • the hCO were cultured in the circular molds for 5 days with a medium change after 2 days. Subsequently, the hCO were cultured on PDMS exercise poles (designed to create 20 % stretch at rest and the hCO to shorten by 0.33 ⁇ / ⁇ with contractions) with media changes every 2-3 days (2 ml/hCO). Small molecules were added to Cardiac Medium and given to the cells for 24 or 48 h: DMSO (Sigma), CHIR99021 (Stem Cell Technologies) and ICRT14 (Tocris).
  • the Heart-Dyno design facilitates the self-formation of tissues around in-built PDMS exercise poles (designed to deform 0.33 ⁇ / ⁇ ).
  • the medium was changed every 2-3 days (150 ⁇ /hCO) until treatment after 6 days.
  • Small molecules were added to Cardiac Medium and given to the cells for 48 h: DMSO (Sigma), CHIR99021 (Stem Cell Technologies), NRG-1 (RnD Systems) and ICRT14 (Tocris).
  • DMSO Stemethanol
  • CHIR99021 Stem Cell Technologies
  • NRG-1 RnD Systems
  • ICRT14 Tocris.
  • the cells were transfected for 4 h using Lipofectamine RNAiMax (3 ⁇ /hCO) in 150 ⁇ /hCO OptiMEM followed by a medium change into Cardiac Medium.
  • Echocardiography Mice were anesthetized using 2% isofluorane for the duration of echocardiographic recordings. Mice were positioned supine on a 37°C heating pad. Images of the left ventricular wall were taken in M-mode using a HD-15 ultrasound (Phillips) and a 14 Hz ultrasound probe (Phillips).
  • Cellular identity is governed by a core transcriptional program, which is epigenetically cemented during development. We postulated that core cell identity programs similarly exist in both neonatal and adult stages. To test this, we isolated genes enriched for a particular cell type relative to other cell types in all conditions including infarcted and sham-operated neonates and adults. In these "cellular identity datasets" many transcription factors, signalling pathways and gene ontologies classically associated with each cell type were highly enriched ( Figure 3). For example, both neonatal and adult myocytes were highly enriched for transcriptional networks controlled by cardiogenic transcription factors, including Nkx2-5, Gata4, Mef2c and Mef2a, as well as genes associated with cellular metabolic processes (Figure 3).
  • CHIR could induce proliferation in standard two-dimensional (2D) cultures of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) using KI67 at 24 h ( Figure 13).
  • hPSC-CMs human pluripotent stem cell-derived cardiomyocytes
  • Figure 5B 3D human cardiac organoid
  • CHIR was capable of activating ⁇ -catenin and driving it into the nucleus in hCO ( Figure 5D), whilst simultaneously maintaining localisation of ⁇ -catenin at cell-cell junctions (Figure 5C), which may be critical for maintaining cardiomyocyte coupling during proliferation (Swope et al., 2012).
  • YAP-1 For overexpression of constitutively active YAP-1, cells were infected in CTRL medium with an adenovirus containing a mutated version of murine YAP-1, CMV- YAP(S112A) at an MOI of 10. hCO were cultured in CTRL medium for 7 days before treatment. Small molecules were added and given to the cells for 48 h: DMSO, CHIR99021 and NRG-1. For transfection experiments, the cells were transfected for 4 h using Lipofectamine RNAiMax (3 ⁇ /hCO) in 150 ⁇ /hCO OptiMEM followed by a medium change into CTRL medium. The cells were transfected at 50 nM with scramble miR control, miR mimic hsa-miR-199a-3p or miR mimic hsa-miR-590-3p.
  • beta-Catenin-driven cancers require a YAP-1 transcriptional complex for survival and tumorigenesis. Cell 151, 1457-1473.
  • Polizzotti BD Ganapathy B, Walsh S, Choudhury S, Ammanamanchi N, Bennett DG, dos Remedios CG, Haubner BJ, Penninger JM, Kuhn B. Neuregulin stimulation of cardiomyocyte regeneration in mice and human myocardium reveals a therapeutic window. Science translational medicine. 2015;7:281ra245
  • Zanconato F Forcato M, Battilana G, Azzolin L, Quaranta E, Bodega B, Rosato A, Bicciato S, Cordenonsi M, Piccolo S. Genome-wide association between yap/taz/tead and ap-1 at enhancers drives oncogenic growth. Nature cell biology. 2015; 17: 1218- 1227
  • Pate KT Stringari C, Sprowl-Tanio S, Wang K, TeSlaa T, Hoverter NP, McQuade MM, Garner C, Digman MA, Teitell MA, Edwards RA, Gratton E, Waterman ML. Wnt signaling directs a metabolic program of glycolysis and angiogenesis in colon cancer. The EMBO Journal. 2014;33 : 1454-1473.

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Abstract

Activation of β-catenin in cardiomyocytes enhances or stimulates cardiomyocyte regeneration to thereby prevent or treat diseases or conditions of the heart such as ischaemic heart disease. One or more other genetic elements such as Yes-associated protein-1 protein may facilitate or assist the action of β-catenin in cardiomyocyte regeneration.

Description

TITLE
CARDIOMYOCYTE REGENERATION TECHNICAL FIELD THIS INVENTION relates to cardiomyocytes. More particular, this invention relates to inducing or promoting cardiomyocyte regeneration of damaged cardiac tissue.
BACKGROUND
Ischemic heart disease is the leading cause of death worldwide. Due to the adult heart's extremely limited capacity for regeneration, myocardial infarction (MI) causes irreparable damage to the heart and heart failure (Senyo et al., 2013). Recent evidence suggests that there is a developmental basis for this lack of regenerative capacity. In contrast to adults, neonates are able to mount a swift cardiac regenerative response following amputation or myocardial ischemia, but this regenerative capability is lost within the first postnatal week in mice (Porrello et al., 2011; Porrello et al., 2013). As such, there is substantial interest in defining the hallmarks of the cardiac injury response in neonates and identifying underlying mechanisms that distinguish the neonatal regenerative response from pathological repair processes in adulthood.
A central feature of the cardiac regenerative response in neonatal mice is the activation of cardiomyocyte proliferation following injury (Haubner et al., 2012; Porrello et al., 2011; Porrello et al., 2013). Genetic studies in the mouse have identified cardiomyocyte proliferation as the primary source of regenerated cardiomyocytes (Porrello et al., 2011), which is similar to findings in adult zebrafish (Jopling et al., 2010; Kikuchi et al., 2010). The developmental timing of regenerative arrest in rodents coincides with the postnatal window when most cardiomyocytes withdraw from the cell cycle and become terminally differentiated (Soonpaa et al., 1996). Accordingly, genetic mutants with defective cardiomyocyte proliferation fail to mount a full regenerative response following injury in the neonatal period (Mahmoud et al., 2013; Porrello et al., 2013; Tao et al., 2016; Xin et al., 2013a). Re- igniting proliferation in the adult heart has been pursued as a regenerative strategy (Pasumarthi and Field, 2002), with recent studies reinforcing the possibility of augmenting adult cardiomyocyte proliferation rates through manipulation of growth factor receptors (D'Uva et al., 2015), transcription factors (von Gise et al., 2014), microRNAs (Eulalio et al., 2012) and the cell cycle machinery (Shapiro et al., 2014). However, despite these recent advances and even with the most potent cardiac mitogens, cardiomyocyte proliferation rates remain low in the adult heart and are not sufficient to drive a full regenerative response (Quaife-Ryan et al., 2016). It is also unclear whether these factors are capable of driving human cardiomyocyte proliferation, which will be crucial for clinical translation.
Human cardiac organoids offer a model system to study human cardiac biology and regeneration (Tiburcy et al, 2017, Voges et al., 2017). We have recently developed conditions to promote maturation of these human cardiac organoids and can now use this model system to discover novel pro-regenerative strategies.
SUMMARY
The present invention is broadly directed to activation of β-catenin in cardiomyocytes to enhance or stimulate cardiomyocyte regeneration and/or proliferation. In some embodiments, activation of β-catenin in cardiomyocytes enhances or stimulates cardiomyocyte regeneration and/or proliferation to thereby prevent or treat diseases or conditions of the heart such as ischaemic heart disease. In a further aspect, one or more genetic elements such as Yes-associated protein- 1 (YAP-1) protein may facilitate or assist the action of β-catenin in cardiomyocytes.
In a first aspect, the invention provides a method of preventing or treating a disease or condition of the heart, said method including the step facilitating nuclear expression of a constitutively active β-catenin protein and, optionally, modulation of one or more genetic elements in one or more cardiomyocytes of the heart to thereby promote or enhance cardiomyocyte regeneration and thereby prevent or treat said disease or condition.
In some embodiments, the one or more genetic elements is, or includes YAP- 1 protein.
In a second aspect, the invention provides a method of regenerating cardiomyocytes, said method including the step facilitating nuclear expression of a constitutively active β-catenin protein and, optionally, modulation of one or more genetic elements in one or more cardiomyocytes of the heart to thereby promote or enhance cardiomyocyte regeneration.
In some embodiments, the one or more genetic elements is, or includes, a YAP-1 protein. In a third aspect, the invention provides a genetic construct comprising: (i) a nucleotide sequence that encodes a constitutively active β-catenin protein operably linked to one or more regulatory nucleotide sequences which facilitate expression of the β-catenin protein in a cardiomyocyte; optionally (ii) a nucleotide sequence of one or more genetic elements operably linked to one or more regulatory nucleotide sequences which facilitates expression of the genetic element in a cardiomyocyte; or (iii) a nucleotide sequence that encodes a constitutively active β-catenin protein and optionally one or more genetic elements operably linked to one or more regulatory nucleotide sequences which facilitates expression of the constitutively active β- catenin protein and the one or more genetic elements in a cardiomyocyte.
In some embodiments, the one or more genetic elements is, or includes YAP- 1 protein.
In a fourth aspect, the invention provides a cardiomyocyte comprising the genetic construct of the third aspect.
In some embodiments, the cardiomyocyte comprises a constitutively active β-catenin protein and one or more genetic elements such as a constitutively active YAP-1 protein located in the nucleus of the cardiomyocyte.
In a fifth aspect, the invention provides a composition comprising the genetic construct of the third aspect and/or one or more cardiomyocytes of the fourth aspect, together with a pharmaceutically acceptable carrier, diluent or excipient.
In a sixth aspect, the invention provides a method of identifying, screening or producing a molecule capable of inducing or facilitating cardiomyocyte regeneration, said method including the step of identifying a molecule that mimics or facilitates β- catenin-mediated gene expression in a cardiomyocyte and thereby induces cardiomyocyte regeneration.
In an embodiment, the molecule facilitates nuclear localization of an endogenous β-catenin protein in a cardiomyocyte.
In an embodiment, the molecule facilitates nuclear localization of an expressed, constitutively active β-catenin protein in a cardiomyocyte.
In an embodiment, the molecule facilitates targeting of a constitutively active β-catenin protein to a cardiomyocyte.
In some embodiments, the molecule is, or mimics the action of, one or more genetic elements selected from: DNA methyltransferases (DNMTs), histone acetyl transferases (HATs), histone deacteylases (HDACs), HMTs, histone methyltransferases (HMTs), KDMs, histone lysine demethylases (KDMs), eRNAs, enhancer RNAs (eRNAs), IncRNAs, long non-coding RNAs (RNAs), microRNAs (miRNAs), polycomb repressive complexes (PRC), TETs, ten-eleven translocation (TETs) and/or TFs, transcription regulators (TRs).
In a particular embodiment, the molecule is YAP-1 or activates or mimics
YAP-1.
A related aspect provides a molecule identified, screened or produced by the method of this aspect.
Suitably, the cardiomyocytes are human cardiomyocytes.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Isolation, sorting and RNA-seq analysis of multiple cardiac cell populations during development and regeneration.
(A) Schematic of cardiac cell sorting strategy and RNA-seq pipeline.
(B) Purity of isolated cardiac cell populations based on expression of known cardiac cell type-specific markers, n = 4 per group.
(C) Percentage of non-myocyte populations in each group measured by FACS.
(D) Differentially expressed genes for each cell type between sham-operated and infarcted mice at neonatal or adult stages.
Figure 2. Analysis of RNA-seq data from multiple cardiac cell populations during development, repair and regeneration.
(A) Principal coordinate analysis for each cell population. CM = cardiomyocytes, LK = leukocytes, FIB = fibroblasts, ETC = endothelial cells.
(B) Pearson correlations between RNA-seq samples for each cell type. Blue = sham surgery at PI (ShamPl), purple = MI at PI (MIP1), green = sham surgery at P56 (ShamP56) and red = MI at P56 (MIP56).
(C) Hierarchical clustering of significantly regulated genes. GO analysis of cell- specific clusters reveals enrichment of cell-specific GO terms. Statistical significance is represented by FDR.
Figure 3. Identification of transcription factors and signaling pathways governing cardiac cell identity.
(A) Over-connected transcriptional networks and signaling pathways for each cell type. (B) Enriched pathways and gene ontologies in each cell type.
Figure 4. Analysis of transcription factors and signaling networks associated with neonatal versus adult responses to MI in each cardiac cell population.
(A) Over-connected transcription factors and signaling molecules are shown for each cellular dataset. Over-connected genes in neonatal cell types are shown in red boxes, whereas over-connected adult genes are shown in blue boxes. # denotes genes that are specifically regulated following MI (MIP1 vs MIP56), whereas genes without # are also regulated during development (ShamPl vs ShamP56).
(B) Gene ontologies enriched in neonatal and adult cell types following MI. Red
= enriched in neonate, blue = enriched in adult.
(C) Gene networks and associated signaling pathways were assembled and ranked for each cell type.
Figure 5: Beta-catenin activation induces cardiomyocyte cell cycle activity in hCO.
(A) Activated β-catenin expression in the nucleus of cardiomyocytes decreases as cardiomyocytes mature and exit the cell cycle (P1-P14). Sections from postnatal mouse hearts (PI, P7, PI 4, P21) were stained for a-actinin (cardiomyocytes), pH3 (mitosis) and phosphorylated Y450 β-catenin (activated β-catenin). Scale bars = 20μπι. High magnification insets are also shown.
(B) Schematic for directed differentiation of human embryonic stem cells into cardiac cells (15 days), formation of 3D tissue molds (5 days), and exercising of 3D tissues (7 days) to form hCO for proliferation studies.
(C) Treatment of hCO with CHIR did not affect inactive β-catenin localization at cell-cell junctions.
(D) Treatment of hCO with CHIR resulted in active β-catenin in cardiomyocyte nuclei.
(E) Treatment of hCO with CHIR increases Ki67+ cardiomyocytes in hCO after 24h. Data pooled from 2 independent experiments. *P<0.05, 4784 cardiomyocytes.
(F) Treatment of hCO with CHIR increases pH3+ cardiomyocytes in hCO after 48h. Data pooled from 2 independent experiments. ***P<0.001, 2641 cardiomyocytes. (G) Schematic showing cardiomyocyte purification strategy from hCO using the KX2-5GFP/+ human embryonic stem cell line. hCO were treated with either CHIR or DMSO as a vehicle control and cardiomyocytes were purified via FACS for qPCR analysis.
(H) qPCR for the β-catenin transcriptional target AXIN2 confirms transcriptional activation in cardiomyocytes (GFP+ fraction) following CHIR treatment in hCO at 24h. n = 3-4 independent experiments. **P<0.01.
(I) Inhibition of β-catenin transcriptional activity using ICRT14 reduces AXIN2 expression in hCO treated with CHIR for 24h. Data pooled from 3 independent experiments. **P<0.01.
(J) Inhibition of β-catenin transcriptional activity using ICRT14 blunts cardiomyocyte proliferation in hCO treated with CHIR for 24 h. Data pooled from 3 independent experiments. *P<0.05, 6466 cardiomyocytes.
Figure 6: β-catenin transcriptionally regulates a regenerative network of genes in human and mouse cardiomyocytes.
(A) Schematic of cell sorting strategy for RNA-seq of 2D differentiated human cardiomyocytes and fibroblasts treated with CHIR or DMSO vehicle for 24h. Venn diagram depicts differentially expressed genes in CHIR-treated myocytes and fibroblasts.
(B) Volcano plot of gene expression changes induced by treatment with CHIR for 24 h in 2D differentiated human cardiomyocytes (CD90-).
(C) GO term enrichment for genes up-regulated by CHIR treatment in cardiomyocytes.
(D) Transcription factor binding site analysis for CHIR treated myocytes showing enrichment for the cell cycle-related transcription factor E2F 1.
(E) Venn diagram showing overlapping genes shared between the mouse reversion signature and CHIR-treated myocytes. Transcription factors predicted to regulate these networks are also shown.
(F) Heatmap of genes that revert to a neonatal-like transcriptional signature following MI. The highest ranked network in neonatal myocytes (p = 3.66E-
29) is driven by Wnt/ -catenin and 116 signalling and is enriched for cell cycle terms. Figure 7: Constitutively active β-catenin is sufficient to drive cardiomyocyte proliferation in hCO and partial regeneration of the adult mouse heart in vivo
(A) Schematic outlining 3D hCO culture in a 96-well cardiac microtissue screening platform (Heart-Dyno).
(B) AAV6 AN90PCAT activates nuclear β-catenin (β-catenin PY450 staining) in cardiomyocytes (a-actinin) versus AAV6 MCS control.
(C) AAV6 AN90pCAT is sufficient to activate mitosis (pH3 staining) of cardiomyocytes (a-actinin) versus AAV6 MCS control.
(D) AAV6 AN90pCAT activates mitosis of cardiomyocytes to a similar level as CHIR treatment at 48 h. Data pooled from 2 independent experiments.
*P<0.05, n=15653 cardiomyocytes.
(E) AAV6 AN90pCAT does not affect force of contraction in hCO, whereas CHIR has a large negative impact. Data pooled from 2 independent experiments. ***P<0.001.
(F) qPCR analysis of a subset of CHIR-induced β-catenin target genes in AAV6
AN90pCAT infected hCOs. n = 3-5 hCO, *P<0.05. The expression of each CHIR-induced β-catenin target gene was also assessed in mouse cardiomyocyte by RNAseq and confirmed by qPCR. n = 4, ***P<0.001, ****p<0.0001.
Figure 8. Neonatal mice regenerate following myocardial infarction.
(A) Echocardiography (ejection fraction and fractional shortening) at day 28 following MI or sham surgery in neonatal mice (PI).
(B) Quantification of fibrosis at day 3 and day 28 following neonatal MI.
(C) Transverse sections were stained with Masson's tri chrome. Representative images of infarcted noenatal hearts at day 3 and day 28 following MI are shown. Scale bar = 2 mm.
Figure 9. Percentage and quality of isolated adult mouse cardiomyocytes following Langendorff perfusion of sham-operated and infarcted hearts.
(A) Relative proportion of myocytes and non-myocytes isolated from sham- operated and infarcted adult hearts.
(B) Adult cardiac cells isolated by Langendorff retrograde perfusion using liberase DH. Top panel 4x magnification and bottom panels lOx magnification Figure 10. Differential gene expression of different cardiac cell types following neonatal or adult MI. Red dots denote significantly differentially expressed genes (FDR < 0.05, Log2(FC) < -1 or > 1). Myo = cardiomyocytes, Fibro - fibroblasts, Leuko = leukocytes, Endo = endothelial cells. ShamPl = sham surgery at PI, ShamP56 = sham surgery at P56, MIP1 = MI at PI, MIP56 = MI at P56.
Figure 11. Heat maps showing expression of common signaling pathways involved in cardiac development and regeneration.
Figure 12: CHIR induces proliferation of neonatal rat cardiomyocytes.
(A) Treatment of neonatal rat cardiomyocytes with CHIR increases Ki67+ cardiomyocytes (a-actinin) after 24 h. n = 3-4 independent experiments.
***P<0.001, 7823 cardiomyocytes.
(B) Treatment of neonatal rat cardiomyocytes with CHIR increases pH3+ cardiomyocytes (a-actinin) after 24 h. n = 3-4 independent experiments. *P<0.05, n = 7797 cardiomyocytes.
(C) Treatment of neonatal rat cardiomyocytes with CHIR increases Aurora B kinase-positive cardiomyocytes (a-actinin) after 24 h. Data from 3-4 independent experiments. n=3712 cardiomyocytes.
Scale bars = 20 μπι.
Figure 13 : CHIR induces proliferation of 2D human cardiomyocytes.
Treatment of 2D human cardiomyocytes with CHIR increases Ki67+ cardiomyocytes (a-actinin) after 24 h. Data from 4 independent experiments. *P<0.05, n=3160 cardiomyocytes. Scale bar = 20 μπι.
Figure 14: CHIR induces proliferation of cardiomyocytes in hCO cultured without serum.
(A) Schematic outlining protocol for direct formation of 3D hCO tissues from human embryonic stem cells cultured under serum-free conditions without any dissociation steps. After 13 days, hCO were removed from molds and cultured on exercise poles for the remainder of the experiments.
(B) CHIR increases Ki67+ cardiomyocytes in hCO formed and cultured under serum-free conditions. Results show induction of proliferation of cardiomyocytes (a-actinin) after 24 h using KI67 as a marker of general cell cycle induction, n = 4 hCO per group. *P<0.05, 4219 cardiomyocytes.
Figure 15: Analysis of cell purity in cardiomyocytes and non-myocytes sorted from KX2-5+/GFP hCO treated with CHIR. (A) Representative FACS plots showing separation of cell populations into GFP+ cardiomyocytes and GFP- non-cardiomyocytes in both DMSO and CHIR treated hCO at 24 h.
(B) qPCR of target genes confirms separation of cell populations and purification of cardiomyocytes in the GFP+ fraction in both DMSO and CHIR treated hCO at 24 h. n = 3-4 independent experiments. *P<0.05, **P<0.01, ***P<0.001.
Figure 16: Comparison of known cardiomyocyte proliferative agents in neonatal rat cardiomyocytes.
(A) Proliferation marked by pH3 was evident in cardiomyocytes (a-actinin) following all treatments after 48 h culture.
(B) Quantification of proliferation revealed that CHIR, miR-199a, miR-590 and Ad-YAP(S112A) were capable of inducing proliferation in neonatal rat cardiomyocytes. n = 4 replicates per group. 14339 cardiomyocytes
Bars = 20 μιη.
Figure 17: Effective transfection of microRNA in Heart-Dyno hCO.
(A) Whole-tissue imaging after transfection of a Cy3 labelled siRNA demonstrates efficient transfection of small RNAs throughout the hCO in the Heart-Dyno.
(B) qPCR demonstrates increased expression of miR-199a after transfection versus a scramble control, n = 5-6 from 2 independent experiments. **P<0.01.
(C) qPCR demonstrates increased expression of miR-590 after transfection versus a scramble control, n = 6 from 2 independent experiments. *P<0.05 Figure 18: CHIR and YAP(S127A) induce proliferation in hCO cardiomyocytes.
(A) Schematic outlining protocol for directed differentiation of human embryonic stem cells into cardiac cells (15 days) and formation and exercise of hCO in the Heart-Dyno (6 days). The tissues were then stimulated for 2 days before analysis.
(B) pH3 staining showing CHIR induced cell cycle activity in cardiomyocytes
(a-actinin) in the Heart-Dyno hCO. Bars = 20 μπι.
(C) Whole-tissue images showing that cardiomyocyte (a-actinin) pH3 staining is present throughout the Heart-Dyno hCO treated with CHIR. Scale bars = 200 μπι. (D)pH3 quantification in cardiomyocytes (a-actinin) reveals that CHIR and constitutively active YAP- 1 (SI 27 A) are the only conserved activators of cardiomyocyte proliferation in hCO in the Heart-Dyno. Data pooled from 2 independent experiments. **P<0.01, 33828 cardiomyocytes.
(E) Analysis of force of contraction demonstrates that CHIR decreases force and
ICRT14 cannot rescue this effect. Data pooled from 2 independent experiments. *P<0.05, **P<0.01
(F) qPCR analysis reveals that CHIR activates the β-catenin target gene AXIN2. n = 4-6 from 2 independent experiments. Data normalized to the relevant controls (0.15% DMSO- media additives, SCR - transfection and AAV6
MCS - viral infection). *P<0.05.
Figure 19: Validation of cell purity and CHIR activity in RNA-seq samples.
(A) Representative FACS plots showing separation of cell populations into CD90- cardiomyocytes and CD90+ non-cardiomyocytes in both DMSO and CHIR treated 2D cardiac cells at 24 h.
(B) qPCR of target genes confirms separation of cell populations and purification of cardiomyocytes in the CD90- fraction in both DMSO and CHIR treated human 2D cardiac cultures at 24 h. n = 3-4 independent experiments. *P<0.05, **P<0.01.
qPCR of the β-catenin transcriptional target AXIN2 confirms transcriptional activation in cardiomyocytes (CD90- fraction) in CHIR treated human 2D cardiac cultures at 24 h. n = 3-4 independent experiments. *P<0.05.
Figure 20: Modulation of in vivo β-catenin demonstrates β-catenin is critical for cardiomyocyte proliferation.
A) Birc5 expression was significantly repressed following IWR treatment.
Neonatal mice (P0) were subcutaneously injected with 20 mg/kg IWR each day and hearts were collected at P2 for qPCR. n = 6 mice.
B) IWR-1 significantly reduced the percentage of PH3+ cardiomyocytes in vivo.
Neonatal mice (P0) were injected subcutaneously with daily doses of IWR-1 (20 mg/kg) and hearts were collected and stained at P7. Control mice were injected with DMSO. n=6 mice.
C) Assessment of ejection fraction using echocardiography in adult mice after MI and delivery of AAV6-N90-BCAT or a AAV6-CMV-GFP control, n = 5- 7 mice. Data is mean ± s.e.m. * P <0.05, using t-test (a,b) or two-way ANOVA (c).
Figure 21 : Cardiomyocyte proliferation and WNT-P-catenin signaling are repressed in hCOs cultured in MM.
a) Culture of hCOs in MM (total 11 days) reduces cardiomyocyte (a-actinin) proliferation (Ki-67). n= 11-14 hCOs from 3-4 experiments. 4396 cardiomyocytes were manually counted. Scale bars = 20 μιη.
b) Culture of hCOs in MM (total 11 days) reduces cardiomyocyte (a-actinin) mitosis (pH3). n= 10 from 5 experiments. 7838 cardiomyocytes were manually counted.
c) KI67 intensity in hCOs cultured in different metabolic conditions after 48 h reveals that lack of insulin is responsible for the initial drop in cell cycle activity, n = 6-13 from 3 experiments. Scale bars = 20 μιη.
d) Activated β-catenin intensity in hCOs cultured in different metabolic conditions after 48 h reveals that lack of insulin is responsible for a decrease in activated β-catenin. n = 6-13 from 3 experiments.
e) Cell cycle activity (Ki-67) and activated β-catenin were highly correlated in hCOs cultured under different metabolic conditions, n = 146 from 3 experiments.
f) Re-introduction of insulin for 48 h after longer term culture (9 days in MM) could not restore proliferation (Ki-67) of cardiomyocytes (a-actinin).
g) Quantification of Ki-67 intensity, n = 10- 11 hCOs from 2 experiments. h) Quantification of proliferating (Ki-67) cardiomyocytes (a-actinin). n = 3-4 hCOs from 2 experiments. 2441 cardiomyocytes were manually counted.
Scale bars = 20 μπι. Data is mean ± s.e.m. **P<0.01, using t-test (a), p-values calculated using t-test (b,g,h), Pearson correlation (r) and p-value (e). PO.0001, INS- statistically different from INS+ using two-way ANOVA (c,d)
Figure 22: Ki-67 intensity in hCO derived from different lines and force of contraction under different metabolic conditions in support of the data presented in Figure 21.
a) Ki-67 intensity in H9-derived hCOs. n = 10.
b) Ki-67 intensity in hIPSC-derived hCOs. n = 4.
c) Force of contraction in F£ES3-derived hCOs cultured in different metabolic conditions after 48 h. n = 11-15 from 3 experiments.
Data is mean ± s.e.m. **P < 0.01, using t-test (a,b). Figure 23 : Restoration of β-catenin and YAP-1 activation restores proliferation in MM.
a) hCOs cultured in MM have a blunted proliferative response (KI67 intensity) to CHIR99021 (24 h treatment). n= 11-14 hCOs from 3-4 experiments.
b) Quantification of proliferating (Ki-67) cardiomyocytes (a-actinin) confirms hCOs cultured in MM have a blunted proliferative response to CHIR99021 (24 h treatment). n= 7-8 from 2 experiments. 10,609 cardiomyocytes were manually counted.
c) YAP/TAZ target genes are repressed in hCOs cultured in MM. Heat map data from significantly regulated targets in RNA-seq data, n = 4 experiments.
d) Delivery of constitutively active β-catenin or YAP-1 individually does not activate proliferation (Ki-67 intensity) in hCOs cultured in MM. n = 11-13 from 3 experiments.
e) Delivery of constitutively active β-catenin or YAP-1 individually do not activate proliferation (Ki-67) of cardiomyocytes (a-actinin) in hCOs cultured in MM. n= 10 from 3 experiments. 17,214 cardiomyocytes were manually counted.
f) Delivery of constitutively active β-catenin or YAP-1 individually do not activate mitosis (pH3) of cardiomyocytes (a-actinin) in hCOs cultured in MM. n= 6 from 2 experiments. 10,243 cardiomyocytes were manually counted.
g) Delivery of constitutively active β-catenin or YAP-1 individually do not activate BIRC5 in hCOs cultured in MM. n= 7-8 from 2 experiments
h) Delivery of constitutively active β-catenin and YAP-1 co-operate to activate proliferation (Ki-67 intensity) in hCOs cultured in MM. n= 11-14 from 3 experiments.
i) Delivery of constitutively active β-catenin and YAP-1 co-operate to activate proliferation (Ki-67) of cardiomyocytes (a-actinin) in hCOs cultured in MM. n= 9 from 3 experiments. 10,434 cardiomyocytes were manually counted. j) Delivery of constitutively active β-catenin and YAP-1 co-operate to induce of mitosis (pH3) in cardiomyocytes (a-actinin) in hCOs cultured in MM. n= 3-4. 3,770 cardiomyocytes were manually counted. k) β-catenin and YAP-1 co-operate in hCOs cultured in MM to induce expression of BIRC5. n = 6-7 from 2 experiments.
1) Compound 6.28 induces dose dependent increases in proliferation (Ki-67 intensity) in hCOs cultured in MM. n = 6-9 from 2 experiments.
m) Compound 6.28 induces dose dependent increases in proliferation (Ki-67) of cardiomyocytes (a-actinin) in hCOs cultured in MM. n = 6-9 from 2 experiments. 10,742 cardiomyocytes were manually counted,
n) Compound 6.28 induces mitosis (pH3) of cardiomyocytes (a-actinin) in hCOs cultured in MM. n = 7 from 2 experiments. 4,782 cardiomyocytes were manually counted.
o) Compound 6.28 induces expression of BIRC5 in hCOs cultured in MM. n =
5-6.
p) Compound 6.28 induces cell cycle activity (BrdU+) in adult mouse cardiomyocytes (MLC2v+) in vivo, n = 6 sections from 3 mice per group. Scale bars = 20 μιη. CM- cardiomyocyte, NM- non-myocyte.
q) Schematic illustrating the mechanism of cardiomyocyte maturation in hCOs cultured in MM. Insulin can only be removed in long-term culture with maintenance of contractile function if fatty acids replace metabolic substrates. Initially, the removal of insulin is responsible for a decrease in proliferation. However, in the longer term, repression of β-catenin and YAP-
1 results in cell cycle exit.
Data is mean ± s.e.m. * < 0.05, ** <0.01, *** P <0.001, **** P O.0001, using ANOVA with Tukey's post-test (a,b), Dunnet's post-test (l,m) or t-test (h-k,n,o,p). Figure 24: Regulated YAP/TAZ targets, force of contraction with viral treatment, and compound 6.28 data in support of the data presented in Figure 24.
a) Heatmap with gene names of YAP/TAZ targets down-regulated in hCO in MM, n = 4 experiments from RNA-seq data.
b) Force of contraction is not affected by overexpression of constitutively active β-catenin and YAP-1. n = 9-12.
c) Chemical structure of compound 6.28.
d) Dose-response curves for compound 6.28
Figure 25: β-catenin and YAPl can enhance post-natal cardiomyocyte proliferation. A) AAV6-YAP1-S127A and AAV6-N90-BCAT co-administration induces cardiomyocyte proliferation in vivo. Neonatal mice (PI) were injected (i.v.) with AAVs (2xlOu viral particles per animal) and hearts were collected and stained 28 days later. Control animals were injected with AAV6-CMV-GFP. Following viral injection, mice were injected (s.c.) with BrdU (100 mg/kg) every second day for 12 days, n = 6 mice.
B) Cardiomyocytes from AAV6-YAP1-S127A and AAV6-N90-BCAT i.v. injected mice had significantly reduced cross sectional area compared to AAV6-CMV-GFP injected controls. Neonatal mice (PI) were injected (i.v.) with viruses (2xlOu viral particles per animal) and the hearts were collected and stained 28 days later, n = 6 mice.
C) Heart size is not altered following delivery of AAV6-YAP1-S127A and AAV6-N90-BCAT i.v. injected mice as they have similar heart weight to tibia length. Neonatal mice (PI) were injected (i.v.) with viruses (2xlOu viral particles per animal) and the hearts were collected and stained 28 days later, n = 6 mice.
Data is mean ± s.e.m. ** P < 0.01, *** P < 0.001, using t-tests. Scale bars represent
Figure 26: AAV6-YAP1-S127A and AAV6-N90-BCAT co-administration results in therapeutic effects in adult mice following MI.
A) AAV6-YAP1-S127A and AAV6-N90-BCAT co-administration resulted in elevated Birc5 expression in adult mice cardiomyocytes. Adult mice (P56) were intracardially injected with AAVs (2xlOu viral particles per animal) and cardiomyocytes and nonmyocytes were isolated by enzymatic digestion 3 days later. Control animals were injected with AAV6-CMV-GFP. n = 2 mice.
B) Assessment of ejection fraction using echocardiography in adult mice after MI and delivery of AAV6-YAP1-S127A and AAV6-N90-BCAT or a AAV6- CMV-GFP control, n = 5-7 mice.
Figure 27: Delivery of constitutively active β-catenin or YAP-1 control restore heart function (ejection fraction, EF) following a heart attack in mice.
A) Experimental outline
B) Delivery of either β-catenin or YAP as a positive control improve heart function (ejection fraction, EF) following a heart attack in mice; C) Delivery of either β-catenin or YAP as a positive control improve heart function (fraction shortening, FS) following a heart attack in mice;
D) Sections of the heart showing muscle (red) and fibrosis (blue);
E) Quantification of the sections show less fibrosis in the beta catenin hearts; F) Delivery of β-catenin does not impact cardiomyocyte size;
G) Delivery of β-catenin does not impact cardiomyocyte proliferation (mitotsis with pH3);
H) Delivery of β-catenin does not impact cardiomyocyte proliferation (DNA synthesis with BrDU plus chase labelling any proliferating myocyte during the exp), YAP a pro-proliferative stimulus used as a positive control.
DETAILED DESCRIPTION
The present invention is at least partly predicated on data indicating that β-catenin and optionally one or more other genetic elements such as YAP-1 signalling becomes repressed in mature cardiomyocytes. We show that re-activation of both pathways works co-operatively and synergistically to enhance cardiomyocyte regeneration. Accordingly, the invention provides methods that utilize nuclear expression of a constitutively active β-catenin protein and YAP-1 protein in cardiomyocytes to stimulate cardiomyocyte regeneration to thereby prevent or treat diseases or conditions of the heart, such as myocardial infarction. It is proposed that the present invention is superior to promoting β catenin activity indirectly via 'upstream' targets (e.g GSK3, Wnt), as this latter alternative approach may have undesired side effects.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described.
As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps. It will be appreciated that the indefinite articles "a" and "an" are not to be read as singular indefinite articles or as otherwise excluding more than one or more than a single subject to which the indefinite article refers. For example, "a" cell includes one cell, one or more cells and a plurality of cells.
For the purposes of this invention, by "isolated" is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material {e.g., cells) may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. This also includes within its scope terms such as "enriched" and "purified'.
The term "protein" refers to an amino acid polymer comprising natural and/or non-natural amino acids inclusive of D- and L-amino acids. A peptide is a protein comprising no more than 50 contiguous amino acids. A polypeptide is a protein comprising more than 50 contiguous amino acids.
In an aspect, the invention provides a method of preventing or treating a disease or condition of the heart, said method including the step facilitating nuclear expression of a constitutively active β-catenin protein and, optionally, modulating one or more genetic elements such as a YAP-1 protein in one or more cardiomyocytes of the heart to thereby promote or enhance cardiomyocyte regeneration and prevent or treat said disease or condition. Suitably, the constitutively active β-catenin protein and the one or more genetic elements facilitate transcription of one or more genes associated with, or involved in, cardiac cell regeneration.
In a related aspect, the invention provides a method of regenerating cardiomyocytes, said method including the step facilitating nuclear expression of a constitutively active β-catenin protein and, optionally, modulating one or more genetic elements such as a YAP-1 protein in one or more cardiomyocytes of the heart to thereby promote or enhance cardiomyocyte regeneration. Suitably, the constitutively active β-catenin protein and the one or more genetic elements facilitate transcription of one or more genes associated with, or involved in, cardiac cell regeneration. As used herein "cardiomyocytes" are cardiac muscle cells also known as myocardiocytes or cardiac myocytes, that make up cardiac muscle such as found in the atria and ventricles of the heart. Each myocardial cell contains myofibrils, which are the fundamental contractile units of cardiac muscle cells. The majority of cardiomyocytes contain only one or two nuclei, although they may have as many as four and a relatively high mitochondrial density, facilitating production of adenosine triphosphate (ATP) for muscle contraction. Myocardial infarction causes the death of cardiomyocytes. In adults, the heart's limited capacity to regenerate these lost cardiomyocytes leads to compromised cardiac function and high morbidity and mortality.
As generally used herein "regeneration" broadly refers to at least partial restoration of a structure or function, such as of a cardiac cell, tissue or organ, or the at least partial reversal of damage to, or deterioration or degeneration of, that structure or function. In some embodiments, regeneration may include cell proliferation, although cell proliferation does not necessarily underlie, cause or be associated with regeneration. By way of example only, regeneration may occur at least partly as a result of modulating or reprogramming gene expression to at least partially restore structure or function, or at least partially reverse damage to, or deterioration or degeneration of, that structure or function.
Diseases and conditions of the heart include any that are characterized by or associated with damage, degeneration or dysfunction of cardiac tissue comprising cardiomyocytes. Non-limiting examples include acute coronary syndrome (ACS), hypertrophic cardiomyopathy, myocardial infarction inclusive of ST-segment elevation myocardial infarction (STEMI) non-ST-segment elevation myocardial infarction (NSTEMI), coronary artery disease, ischemic heart disease, heart failure, heart failure with preserved ejection fraction (HFPEF) heart failure with reduced injection fraction (HFREF) and/or congestive heart failure atherosclerosis, angina, ventricular arrhythmia and/or ventricular fibrillation.
As used herein, "treating" or "treat" or "treatment" refers to a therapeutic intervention that at least party eliminates or ameliorates one or more existing or previously identified pathologies or symptoms of a disease or condition.
As used herein, "preventing" or "prevent" or "prevention" refers to a prophylactic treatment initiated prior to the onset of pathologies or symptoms of a disease or condition, whereby the prophylactic treatment at least partly or temporarily prevents or suppresses the occurrence of the symptom.
As used herein, a "constitutively active β catenin" can be any mutant or otherwise modified form of a β catenin protein that can promote transcriptional activation, such as via TCF and LEF transcription factors, even in the presence of negative regulators of β catenin activity. Typically, β catenin activity is controlled or negatively regulated by intracellular proteolytic degradation. Accordingly, a preferred constitutively active β catenin is at least partly resistant to intracellular proteolytic degradation. Typically, GSK3 -mediated phosphorylation of the bolded serine and threonine residues in β catenin shown in SEQ ID NO: l is required for binding by ubiquitin ligases such as E3 ubiquitin ligase TrCP-1 that cause resultant ubiquitination and proteolytic degradation of β catenin. Accordingly, certain embodiments of a constitutively active β catenin may have some or all of the bolded serine and threonine residues in SEQ ID NO: l mutated (e.g. to alanine or other non- conservative substitution) or deleted, thereby preventing phosphorylation and resultant degradation of β catenin. In other particular embodiments, the constitutively active β catenin protein may comprise an N-terminal deletion of some or all of the N-terminal region comprising the serine and threonine residues that can be phosphorylated by GSK3. In some embodiments at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or all of the 89 amino acids underlined in SEQ ID NCv l may be deleted.
1 matqadlmel dmamepdrka avshwqqqsy ldsgihsgat ttapslsgkg npeeedvdts
61 qylyeweqgf sqsftqeqva didgqyamtr aqrvraamfp etldegmqip stqfdaahpt 121 nvqrlaepsq mlkhavvnli nyqddaelat raipeltkll ndedqvvvnk aavmvhqlsk 181 keasrhaimr spqmvsaivr tmqntndvet arctagtlhn lshhreglla ifksggipal 241 vkmlgspvds vlfyaittlh nlllhqegak mavrlagglq kmvallnktn vkflaittdc 301 lqilaygnqe skliilasgg pqalvnimrt ytyekllwtt srvlkvlsvc ssnkpaivea 361 ggmqalglhl tdpsqrlvqn clwtlrnlsd aatkqegmeg llgtlvqllg sddinvvtca 421 agilsnltcn nyknkmmvcq vggiealvrt vlragdredi tepaicalrh ltsrhqeaem 481 aqnavrlhyg lpvvvkllhp pshwplikat vglirnlalc panhaplreq gaiprlvqll 541 vrahqdtqrr tsmggtqqqf vegvrmeeiv egctgalhil ardvhnrivi rglntiplfv 601 qllyspieni qrvaagvlce laqdkeaaea ieaegatapl tellhsrneg vatyaaavlf 661 rmsedkpqdy kkrlsvelts slfrtepmaw netadlgldi gaqgeplgyr qddpsyrsfh 721 sggygqdalg mdpmmehemg ghhpgadypv dglpdlghaq dlmdglppgd snqlawfdtd 781 1 (SEQ ID NO: 1)
It will be understood from the foregoing that constitutively active β-catenin may be administered or expressed in combination with modulation of one or more genetic elements. As broadly used herein, "genetic elements" may include molecules that may facilitate, enhance, potentiate or synergize with constitutively active β-catenin to promote cardiomyocyte regeneration. Non-limiting examples of other genetic elements include DNA methyltransferases (DNMTs), histone acetyl transferases (HATs), histone deacteylases (HDACs), HMTs, histone methyltransferases (HMTs), KDMs, histone lysine demethylases (KDMs), eRNAs, , enhancer RNAs (eRNAs), IncRNAs, long non-coding RNAs (RNAs), microRNAs (miRNAs), polycomb repressive complexes (PRC), TETs, ten-eleven translocation (TETs) and/or TFs, transcription regulators (TRs).
In this context, by "modulating" the one or more genetic elements, the genetic elements may be at least partly activated or repressed in a manner that facilitates or assists β-catenin-mediated gene expression. This may be achieved genetically through expression of mutant or wild-type forms of the one or more genetic elements and/or molecules that control the activity of the one or more genetic elements, and/or or by chemical or pharmacological targeting of the one or more genetic elements, molecules that control the activity of the one or more genetic elements.
In some embodiments, there may be epigenetic mechanisms that blunt the reactivation of β-catenin target genes, as there have been observed epigenetic changes during postnatal development. It is known that β-catenin target genes are highly regulated by epigenetics and this may therefore play a key role. Thus, the one or more genetic elements are, or act by way of, epigenetic factors (Sim et al, FASEB J, 2015; Quaife-Ryan et al, Sem. Cell. Dev. Biol, 2016).
In some embodiments, the one or more genetic elements is, or includes, a transcriptional activator such as YAP-1. According to a preferred embodiment, YAPl is activated. Activation may be achieved by expression of a constitutively active YAPl protein, chemically or pharmacologically activating YAP-1 and/or by inhibiting protein kinases (or other signaling molecules upstream of YAP) such as Salvador, LATS or MST1 which normally negatively regulate YAP-1 activity. Non- limiting examples of chemical activators of YAP-1 include iysophosphatidic acid (LPA) and sphingosine 1-phosphophat.e (SIP; Fa-Xing Yu et al., 2012, Cell 150, 780). A non-limiting example of a pharmacological inhibitor of MST1 is 4-((5,10- dimethyl-6-oxo-6,10-dihydro-5H-pyrimido[5,4-b]thieno[3,2-e][l,4]diazepin-2- yl)amino) benzenesulfonamide (XMU-MP-1) such as described in Fan et al., 2016, Regen. Med. 352 108. As used herein, a "constitutively active YAP-V can be any mutant or otherwise modified form of a YAP-1 protein that can promote transcriptional activation. In mammals, YAP can work alone or together with the transcriptional coactivator with PDZ-binding motif (TAZ). When activated, YAP and/or TAZ can bind to several transcription factors including p73, API, TBX5, Runx2 and several TEA domain family members (TEADs). Potential gene targets of YAP-1 -mediated transcriptional activation in cardiomyocytes are provided in the Examples. Serine 127 is critical for Large Tumour Suppressor (LATS) kinase-mediated cytoplasmic sequestration of YAP by the Hippo pathway. Phosphorylation by LATS kinases promotes cytoplasmic sequestration of YAP in a manner that involves 14-3-3 proteins and a-catenin. LATS-mediated phosphorylation of YAP serine 127 also can promote YAP-1 ubiquitination and subsequent proteasomal degradation. Serine 127 is bolded and underlined in SEQ ID NO:2 below.
1 mdpgqqpppq PaPqgc[gc[PP Ξ<ΪΡΡ¾3¾3ΡΡ sgpgqpapaa tqaapqappa ghqivhvrgd 61 setdlealfn avmnpktanv pqtvpmrlrk lpdsffkppe pkshsrqast dagtagaltp 121 qhvrahsspa slqlgavspg tltptgvvsg paatptaqhl rqssfeipdd vplpagwema 181 ktssgqryfl nhidqtttwq dprkamlsqm nvtaptsppv qqnmmnsasg plpdgweqam 241 tqdgeiyyin hknkttswld prldprfamn qrisqsapvk qppplapqsp qggvmggsns 301 nqqqqmrlqq lqmekerlrl kqqellrqam rninpstans pkcqelalrs qlptleqdgg 361 tqnpvsspgm sqelrtmttn ssdpflnsgt yhsrdestds glsmssysvp rtpddflnsv 421 demdtgdtin qstlpsqqnr fpdyleaipg tnvdlgtleg dgmniegeel mpslqealss 481 dilndmesvl aatkldkesf ltwl (SEQ ID NO: 2)
It is proposed that a non-conservative substitution of serine 127 to another amino acid such as alanine results in constitutive activation of YAP-1 by locating YAP-1 in the nucleus rather than being sequestered in the cytoplasm. Other non- limiting examples of YAP-1 mutants potentially useful according to the present invention are disclosed in Ehmer et al., 2014, Cell Rep. 8 371.
Suitably, the constitutively active β catenin protein and the constitutively active YAP-1 protein are expressed in one or more cardiomyocytes, preferably targeted to the cardiomyocyte nucleus. Although it is expected that a constitutively active β catenin protein and constitutively active YAP-1 protein such as described above may translocate to the cardiomyocyte nucleus, optionally the constitutively active β catenin and/or the constitutively active YAP-1 protein further comprise a nuclear localization signal ( LS). Typically, LS sequences are about four (4) or five (5) residues in length and comprise one or more acidic residues such as Lys and Arg. The NLS may be monopartite or bipartite, a bipartite NLS sequence comprising a linker of up to about 10-12 amino acids connecting each NLS sequence. Non- limiting examples of NLS sequence motifs include K(R/K)X(R/K) (SEQ ID NO:27), K(K/R)X(K/R) (SEQ ID NO:28), KR(R/X)K (SEQ ID NO:29), KRRR (SEQ ID NO:30), KR(K/R)R (SEQ ID NO:31) or K(K/R)RK(SEQ ID NO:32) for a monopartite NLS and (K/R)(K/R)Xio-i2(K/R)3/5 (SEQ ID NO:33), KRX10-12KRRK (SEQ ID NO:34), KRXio-i2K(K/R)(K/R) (SEQ ID NO:35) or KRXio- i2K(K/R)X(K/R) (SEQ ID NO:36) for a bipartite NLS; wherein X is any amino acid.
Suitably, the NLS is located at or near the N-terminus of the constitutively active β catenin protein and/or the constitutively active YAP-1 protein.
A particular aspect of the invention provides a genetic construct comprising: (i) a nucleotide sequence that encodes a constitutively active β-catenin protein operably linked to one or more regulatory nucleotide sequences which facilitate expression of the β-catenin protein in a cardiomyocyte nucleus; (ii) a nucleotide sequence of one or more genetic elements, such as encoding a constitutively active YAP-1 protein, operably linked to one or more regulatory nucleotide sequences which facilitate expression of the YAP-1 protein in a cardiomyocyte; or (iii) a nucleotide sequence that encodes a constitutively active β-catenin protein and one or more genetic elements, such as a constitutively active YAP-1 protein, operably linked to one or more regulatory nucleotide sequences which facilitate expression of the constitutively active β-catenin protein and the constitutively active YAP-1 protein in a cardiomyocyte nucleus.
The genetic construct may therefore encode either or both of a constitutively active β-catenin protein and one or more genetic elements such as a constitutively active YAP-1 protein.
The genetic construct(s) is/are suitable for delivery to cardiomyocytes and expression of the constitutively active β catenin protein and the one or more genetic elements, such as a constitutively active YAP-1 protein, in cardiomyocytes.
As used herein the term "nucleic acid" includes and encompasses single and double-stranded DNA and RNA inclusive of genomic DNA, cDNA, RNA such as mRNA, cRNA, miRNA and tRNA and DNA:RNA hybrids. Nucleic acids may comprise A, G, C, T and/or U nucleotides and/or nucleotides that comprise modified pyrimidine or purine bases such as pseudouridine, 5-methyluridine, 2-thiouridine, N6-methyladenosine and 5-methylcytidine dihydrouridine, inosine or 7- methylguanosine, although without limitation thereto. By way of example only particular nucleic acid forms may include oligonucleotides, restriction fragments, amplification products, primers and probes, although without limitation thereto.
A "genetic construct" is any artificially constructed nucleic acid that comprises a nucleic acid encoding the constitutively active β catenin protein and/or the one or more genetic elements and, optionally one or more additional nucleotide sequences. The genetic construct may be suitable for stable or transient expression of the constitutively active β catenin protein and/or the one or more genetic elements. Suitably, the one or more additional nucleotide sequences may include regulatory nucleotide sequences such as promoters, enhancers, splice acceptor/donor sequences, polyadenylation sequences, selection marker sequences (e.g. to facilitate selection of stable transformants), translation start and/or termination sequences, although without limitation thereto. Promoters are well known in the art and can include constitutive, inducible or repressible promoters and tissue-specific promoters. One example of a constitutive promoter that is active in many mammalian cell types, including cardiomyocytes, is a CMV promoter. The genetic construct may comprise cardiomyocyte-specific promoters or enhancers such as cTNT, a-MHC, NCX1, MLC2v.
The genetic construct may be in the form of a DNA plasmid, a viral construct or an RNA construct, although without limitation thereto.
In one broad embodiment, the genetic construct may be suitable for delivery or administration to a patient in vivo, whereby the constitutively active β catenin protein is expressed in situ by cardiomyocytes in the patient heart.
In another broad embodiment, the genetic construct may be suitable for delivery to one or more cardiomyocytes in vitro, whereby the cardiomyocytes are administered to the heart of a patient in vivo, whereby the constitutively active β catenin protein is expressed in situ by the cardiomyocytes transferred to the patient heart.
In embodiments where the genetic construct is plasmid DNA, the construct may comprise a cardiac specific promoter (e.g aMHC promoter) to increase the specificity of expression and restrict expression to cardiomyocytes. In this embodiment, plasmid DNA(s) encoding the constitutively active β catenin protein and the one or more genetic elements, such as constitutively active YAP-1 protein, can be injected directly into the heart to facilitate transient expression of constitutively active β catenin protein and the one or more genetic elements, such as constitutively active YAP-1 protein, in the heart. Suitably, plasmid DNA is delivered to achieve transient expression of the constitutively active β catenin protein and the one or more genetic elements in cardiomyocytes.
In some embodiments the genetic construct is a viral construct, which may be particularly suited to stable or long-term expression of constitutively active β catenin protein and the one or more genetic elements in cardiomyocytes. In embodiments where the genetic construct is a viral construct, the genetic construct may be an adenovirus, adeno-associated virus, vaccinia or lentivirus construct or "vector", although without limitation thereto. In certain embodiments, the genetic construct is an adeno-associated viral vector such as an AAV6 or AAV9 viral vector. These viruses target striated muscle specifically to delivery constitutively active β-catenin protein and the constitutively active YAP-1 protein. Specificity can be further enhanced by incorporating a cardiomyocyte specific enhancer (e.g. a-MHC, NCX1, MLC2v) together with a constitutive, non tissue-specific promoter such as a CMV promoter.
In other embodiments, the constitutively active β-catenin and/or the one or more genetic elements may be delivered in the form of RNA, such as "modified RNA". Modified RNA is RNA in which non-natural or modified nucleosides have been incorporated to reduce the immunogenicity of the RNA molecule, particularly in relation to activation of TLR receptors. Non-limiting examples of non-natural or modified nucleosides include, for example pseudouridine, 5-methyluridine, 2- thiouridine, N6-methyladenosine and 5-methylcytidine, such as described in Kariko et al, 2008, Mol. Ther. 16 1833. Modified RNA molecules can be delivered to cardiomyocytes for transient expression, such as described in Kanji et al., 2013 Nat. Biotechnol. 31 898. Reference is also made to International Publication WO2016010119, European Publication EP2694660 and United States Patent 8278036 which also describe the production and delivery of modified RNA.
In some embodiments, targeted delivery of RNA to cardiomyocytes, such as modified RNA, may be achieved using miRNA "switches", such as described in Miki et al, 2015, Cell Stem Cell 4 699. More generally, an miRNA recognition sequence may be incorporated into the delivered RNA molecule to target the expression of a desired RNA sequence (e.g. encoding a constitutively active β- catenin protein and/or a constitutively active YAP-1 protein) to cardiomyocytes.
Also provided are compositions comprising the genetic construct disclosed herein. Suitably, the composition comprises a pharmaceutically acceptable carrier, diluent or excipient. Diluents and excipients may include substances such as water, salts, saline, minerals, vitamins, encapsulating substances, matrices, emulsifiers, thickeners, emollients, lubricants, gels, glidants, buffers, stabilizers, gums, resins and/or powders that facilitate formulating the composition so as to be deliverable or administrable to one or more cardiomyocytes in a patient in vivo or to one or more cardiomyocytes in vitro.
Carriers that may facilitate delivery or administration of genetic constructs in vivo may include liposomes (e.g polycationic liposomes), microbubbles, bacterial minicells, dendrimers, nanofilms (e.g layer-by-layer nanofilms), and particles inclusive of nanospheres, nanobeads and other nanoparticles such as metal nanoparticles (e.g gold or magnetite particles), polycationic polymer particles (e.g chitosan, polyethyleneimine polyamidoamine and poly(a-(4-aminobutyl)-L-glycolic acid), dendrimers and biodegradable nanoparticles (e.g. carbonite apatite), although without limitation thereto.
In some embodiments, microbubbles may be particularly useful for non- invasive, repeated delivery of transiently-expressed genetic constructs such as modified RNA. Typically, although not exclusively, microbubbles may be delivered in conjunction with ultrasound-mediated delivery such as described in Sun et al., 2013, Biomaterials 34 2107.
Carriers that facilitate delivery or administration of genetic constructs to cardiomyocytes in vitro may include carriers that facilitate transfection or transformation such as microparticles, CaCh, lipofectin, lipofectamine, DEAE Dextran, although without limitation thereto.
In yet another aspect, the invention provides one or more cardiomyocytes that comprise a genetic construct disclosed herein, suitably expressing a constitutively active β catenin protein in the cell nucleus.
In one particular embodiment, the cardiomyocytes are suitable for adoptive transfer or transplantation to a patient. The cardiomyocytes may be autologous cardiomyocytes obtained from the patient or non-autologous cardiomyocytes at least antigenically or immunologically matched with the patient (e.g HLA-matched) to minimize the risk of immune rejection of the transferred cardiomyocytes.
Suitably the one or more cardiomyocytes have been engineered in vitro to transiently or stably express the genetic construct by any transfection or transformation method known in the art. These include DEAE Dextran-mediated transfection, electroporation, CaCh precipitation, lipofection, impalefection, microprojectile bombardment and viral transduction, although without limitation thereto.
Also provided are compositions comprising cardiomyocytes that comprise the genetic construct disclosed herein, suitably expressing the constitutively active β- catenin protein in the cell nucleus. Suitably, the composition comprises a pharmaceutically acceptable carrier, diluent or excipient such as hereinbefore described. However, in some embodiments the pharmaceutically acceptable carrier, diluent or excipient may be selected to facilitate delivery of the composition comprising the cardiomyocytes to the heart of a patient in vivo. Suitable pharmaceutically acceptable carrier, diluent or excipients may include encapsulating substances, matrices such as collagen-based matrices, gels, hydrogels, patches, scaffolds including biodegradable polymer scaffolds and decellularized cardiac tissue, polyester fleece or other three-dimensional systems that assist the placement and retention of the cardiomyocytes in heart tissue (such as in damaged cardiac tissue following myocardial infarction) so that the cardiomyocytes can proliferate and regenerate in three dimensions to thereby repair the damaged cardiac tissue.
In some embodiments, the genetic construct encoding the constitutively active β-catenin protein, the one or more genetic elements or one or more cardiomyocytes comprising same, may be co-administered (i.e. in combination therapies)
A broad embodiment of the invention therefore provides a method where cardiomyocytes engineered in vitro to transiently or stably express a constitutively active β-catenin protein and one or more genetic elements, such as a constitutively active YAP-1 protein, are adoptively transferred to the heart of a patient to prevent or treat a disease or condition of the heart, such as myocardial infarction.
In one particular embodiment, the method may include the steps of: (i) delivering a genetic construct, or respective genetic constructs, encoding a constitutively active β catenin protein and optionally one or more genetic elements, such as a constitutively active YAP-1 protein, to one or more cardiomyocytes in vitro; and
(ii) transferring the one or more cardiomyocytes produced at step (i) to the heart of a patient.
In step (i), the genetic construct(s) may be used to transfect or transform the cardiomyocytes as hereinbefore described.
In some embodiments, the method may include further step intermediate steps (i) and (ii) where the cardiomyocytes are cultured under conditions suitable to facilitate proliferation or expansion of the cardiomyocytes before transfer to the heart of the patient.
In another broad embodiment, the method may include the step of delivering a single genetic construct, or respective genetic constructs, encoding a constitutively active β catenin protein and one or more genetic elements such as a constitutively active YAP-1 protein systemically so that the genetic construct locates to the heart of a patient, or directly to the heart of a patient. As will be described in more detail hereinafter, the invention contemplates the identification of one or more molecules that may facilitate targeted delivery of the genetic construct to cardiomyocytes.
It is proposed that the timing of delivery of the cardiomyocytes and/or genetic construct may be variable. By way of example, delivery may immediately follow a myocardial infarction (MI) or other disease or condition of the heart, for example using a drug-eluting stent to deliver the genetic construct, as well as later times (e.g. days, weeks, months or years) after the MI. Dosages may be single doses that provide long-term expression of the constitutively active β catenin protein and/or one or more genetic elements, such as a constitutively active YAP-1 protein, or multiple, repeated dosages that are administered regularly over a time course or on an "as-needed" basis such as to prevent or treat a recurrence of the disease or condition of the heart.
In a further aspect, the invention provides a method of identifying, screening or producing a molecule capable of inducing or facilitating cardiomyocyte regeneration, said method including the step of identifying a molecule that mimics or facilitates β-catenin-mediated gene expression in a cardiomyocyte and thereby induces cardiomyocyte regeneration.
As used herein by "mimics" or "mimics the action of is meant that a molecule has a biological activity similar to another molecule (e.g. activated β- catenin) or produces a biological response similar to that of another molecule (e.g. β- catenin-mediated gene expression in a cardiomyocyte).
Suitably, the method further includes the step of determining whether the molecule is capable of inducing cardiomyocyte regeneration.
According to this aspect, the molecule may facilitate or otherwise assist β- catenin in inducing cardiomyocyte gene expression and/or proliferation. In an embodiment, the molecule facilitates transcription of one or more genes associated with, or involved in, cardiac cell regeneration.
The molecule may facilitate nuclear localization of an endogenous β-catenin protein.
The molecule may facilitate nuclear localization of an expressed, constitutively active β-catenin protein in a cardiomyocyte.
The molecule may facilitate targeting of a constitutively active β-catenin protein to a cardiomyocyte.
It will be appreciated that the present invention is at least partly predicated on the discovery that constitutively active β-catenin protein and one or more genetic elements, such as constitutively active YAP-1 protein, are at least partly capable of inducing cardiomyocyte regeneration. Thus, it is proposed that molecules that induce, activate or promote β-catenin protein-mediated gene expression in cardiomyocytes may be candidates for facilitating cardiomyocyte regeneration. It may be particularly advantageous to identify molecules that selectively target the genetic construct disclosed herein to cardiomyocytes to avoid unwanted expression of the constitutively active β-catenin protein in other cells or tissues.
In some embodiments, the molecule is, or mimics the action of, one or more genetic elements selected from: include: DNA methyltransferases (D MTs), histone acetyl transferases (HATs), histone deacteylases (HDACs), HMTs, histone methyltransferases (HMTs), KDMs, histone lysine demethylases (KDMs), eRNAs, enhancer RNAs (eRNAs), IncRNAs, long non-coding RNAs (RNAs), microRNAs (miRNAs), polycomb repressive complexes (PRC), TETs, ten-eleven translocation (TETs) and/or TFs, transcription regulators (TRs).
In a particular embodiment, the molecule is YAP-1 or activates or mimics
YAP-1.
In another embodiment, the molecule facilitates nuclear localization of an active β-catenin protein and/or said one or more genetic elements disclosed herein, such as a YAP-1 protein.
Suitably, the active β-catenin protein and/or said one or more genetic elements disclosed herein, such as a YAP-1 protein facilitate transcription of one or more genes associated with, or involved in, cardiac cell regeneration.
It will be appreciated that that nuclear localization of an active β-catenin protein is at least one event that is necessary to induce cardiomyocyte regeneration. Thus, it is proposed that the identification of molecules that facilitate nuclear localization of an endogenous, active β-catenin protein may be candidates as inducers of cardiomyocyte regeneration. Additionally, nuclear localization of said one or more genetic elements disclosed herein, such as a YAP-1 protein may also facilitate induction of cardiomyocyte regeneration.
The method preferably includes the steps of contacting the molecule with one or more cardiomyocytes and determining whether the molecule facilitates nuclear localization in a cardiomyocyte. The activity and localization of β-catenin protein and/or other genetic elements such as YAP-1 may be measured by any method known in the art.
Suitably, the method further includes the step of determining whether the molecule is capable of inducing cardiomyocyte regeneration.
The method of this aspect may be adapted to a "high-throughput" screening system whereby cardiomyocytes are contacted with libraries of molecules to identify one or more molecules that facilitate nuclear localization of an active β-catenin protein in a cardiomyocyte.
The molecules may be or comprise a protein, nucleic acid, carbohydrate, lipid, small organic molecule, any other molecule or chemical moiety or combinations of these. The molecules may be present in synthetic chemical libraries, natural product libraries, combinatorial libraries, phage display libraries and inhibitory RNA libraries, although without limitation thereto. Methods for measuring cardiomyocyte regeneration are well known in the art and non-limiting examples of such methods are provided hereinafter.
The invention disclosed herein may pertain to any mammal, inclusive of humans and non-human mammals such as domestric pets, livestock and performance animals, although without limitation thereto.
So that the invention may be readily understood and put into practical effect, reference is made to the following non-limiting examples.
EXAMPLES
Introduction
Given the multitude of possible different regulators of cardiac regeneration, the inventors decided to globally examine the transcriptional networks driving cardiac injury responses in multiple cell populations at different stages of development. Here, the inventors provide a comprehensive transcriptomic analysis of the major cell populations within the regenerative (PI) and non-regenerative (P56) mouse heart with and without injury (myocardial infarction). These analyses identify unique developmentally regulated and injury -induced transcriptional responses in cardiomyocytes, fibroblasts, endothelial cells and leukocytes that govern distinct cellular behaviours following cardiac injury. These comparative analyses also identify a unique cardiomyocyte transcriptional signature associated with cell cycle shutdown and repression of a Wnt-P-catenin gene regulatory network in both rodent and human cardiomyocytes. The inventors demonstrate that re-activation of β- catenin in the adult mouse heart enhances cardiomyocyte proliferation and regeneration following myocardial infarction, thus demonstrating the utility of such comparative transcriptomic approaches for cardiac regeneration studies. The current study represents the first comparative transcriptomic analysis of different cell lineages during mammalian development, regeneration and repair, thus providing a key resource for ongoing studies in regenerative biology and medicine. Materials Methods
Reagents were purchased from Life Technologies unless otherwise indicated. For antibody manufacturer details please see Table 2.
Neonatal and adult MI surgeries For neonatal surgeries, timed pregnant CDl female mice were housed under standard conditions with 12h light/dark cycles and ad libitum access to food and water. At postnatal day 1 mice (PI), neonatal MI surgeries were performed as previously described (Mahmoud et al., 2014). For adult male surgeries, the animals were housed under standard conditions with 12h light/dark cycles and ad libitum access to food and water. At 8 weeks of age, male CDl mice were anesthetized with 4% isoflurane (Bayer) and maintained with 2% isoflurane and 0.25 1/min oxygen during surgery. The animals were intubated and ventilated (Minivent, Harvard Apparatus) with a tidal volume of 250 μΐ and a respiration rate of 133 strokes/min. A thoracotomy at the 3rd and 4th intercostal space was performed and the pericardial sac removed. The left descending coronary artery was permanently ligated with 7-0 prolene suture (Ethicon). Following ligation, blanching of the myocardium was observed to determine if adequate ischaemia was achieved. The chest wall was then sutured closed, the mouse removed from aneasthesia, supplied with s.c. injection of buprenorphine (0.05mg/kg) as required and allowed to recover from the surgery. Ethical approval for these neonatal and adult mouse experiments was obtained from The University of Queensland's Animal Ethics Committee (SBMS/101/13/NHMRC).
Neonatal heart cell isolation Neonatal CD-I (ICR) mice were decapitated 3 days after surgery and cardiac ventricles were dissected and washed in ADS buffer (116.3 mmol/1 NaCl, 20mmol/l HEPES, lmmol/1 NaH2P04, 5.5mmol/l glucose, 5.36 mmol/1 KC1, 0.83 mmol/1 MgS04,) and then cut into small pieces and pooled into groups of 6 ventricles per cell stirrer. Hearts were then digested at 37°C under constant agitation for 30-40 minutes (or until tissue was fully homogenised). The digestion buffer was made with 200 μg/ml Liberase DH (Roche) in ADS buffer. Myocytes and non-myocytes were then separated by Percoll® as described above. Cell isolates were then layered on percoll gradients and centrifuged at 2100xg for 30 minutes. The bottom layer (containing myocytes) and the top layer (containing enriched non-myocytes) were then collected separately and washed twice in ADS. The myocyte fraction was then lysed in Trizol. FACS was then performed on the non-myocyte fraction.
Adult mouse heart cell isolation Adult CD-I (ICR) mice were anaesthetised with an i.p injection of ketamine (200 mg/kg) 3 days post-surgery. Hearts were quickly dissected and washed in perfusion buffer (120.4 mmol/1 NaCl, 14.7 mmol/1 KC1, 0.6 mmol/1 KH2PO4, 0.6 mmol/1 Na2HP04, 1.2 mmol/1 MgS04'7H20, 4.6 mmol/1 NaHCC-3, 10 mmol/1 Na-HEPES, 30 mmol/1 taurine, 5.5 mmol/1 glucose and 10 mM 2,3-Butanedione 2-monoxime). The aorta was then cannulated with a 21 gauge cannula, secured with 3-0 silk suture and perfused with 37°C, oxygenated perfusion buffer using a Langendorff apparatus (4ml/min). Once all blood was washed from the heart, digestion buffer (200 μg/ml Liberase DH (Roche) in perfusion buffer) was passed through the heart for ~8 minutes (until the hearts appeared waxy in colour and flaccid to the touch). During perfusion, atria and excess tissue was removed. After enzymatic digestion, hearts were minced with fine scissors into small pieces and triturated to release cells. Cell isolates were passed through a 100 μιη cell strainer and centrifuged at 30xg for 3 minutes at room temperature. Supernatant (containing the non-myocyte cells) was collected and the myocyte pellet was washed with perfusion buffer and recentrifuged. The supernatant was again collected and the purified myocyte pellet was lysed with 1 ml of Trizol (ThermoFisher). The non-myocyte supernatant was centrifuged at 500xg for 5 minutes FACS was performed.
FACS Following cell extraction, the non-myocyte fraction was resuspended in lOOul 5 mg/ml bovine serum albumin in PBS solution. Cell isolates were then incubated at 4°C for 20 minutes CD90-APC (A14727, ThermoFisher), CD45-FITC (130-102- 778, Miltenyi Biotec), CD31-BV421 (102423, BioLegend) and Podoplanin-PE/Cy7 (127411, BioLegned). The stained cells were washed in PBS/BSA solution and sorted using a BD FACS ARIA cell sorter. Each purified cell population was centrifuged at lOOOxg for 5 minutes, cell media was aspirated and 1 ml Trizol was added to isolate RNA.
Isolation of RNA for RNA-seq
RNA was isolated with trizol according to manufacturer's instructions. Each sample was treated with DNase I (Roche) for 10 minutes and then RNA was further column purified with MinElute RNeasy kit (QIAGEN). RNA-seq
For mouse RNAseq experiment, ribosomal RNA was depleted with Ribo Zero Gold and cDNA was generated with Superscript II Reverse Transcriptase (ThermoFisher). Libraries were created with TruSeq Stranded Total RNA kits (Illumina) and read with HiSeq SR Cluster v4 kit (Illumina) on a HiSeq 2500 sequencer. Each sample contained -45 million 50-bp single-end reads. For the CHIR experiment, Libraries were constructed with Nugen Ovation RNA-Seq system V2 (for SPIA amplifications and cDNA generation) coupled with the Ovation Ultralow System (NuGEN). Libraries were read with 50bp SR HiSeq V4 Rapid Mode (Illumina) chemistry on a HiSeql500. Each sample produced -20 million 50-bp reads. The read quality of each sample was determined with FASTQC and poor quality sequence (<20 phred score) and adapter sequence was timed with Trimmomatic. Reads were mapped with STAR to either mm 10 or hg38. Count matrices were generated using htseq-count on union mode and differential expression analysis performed with EdgeR. Bioinformatics
Pathway and TF analysis was performed with Metacore™ and Ingenuity® Pathway Analysis. Gene Ontology analysis was performed with either Metaore™ or the DAVID webtool. Transcription factor binding site analysis was performed on in the CHIR RNA-seq dataset using Trawler, RSAT and TRANSFAC. Staining of mouse heart sections
Hearts were fixed in 4% PFA in PBS, washed in PBS and then halved with a single transverse cut at the ligature mark. The hearts were dehydrated and then embedded in paraffin wax. Each sample was sectioned at 6 μπι. Sections were then rehydrated, blocked with 10% goat serum in PBS and stained with Anti-phospho-Histone H3 (SerlO) (Millipore, 06-570), β-catenin (PY489) (Developmental Studies Hybridoma Bank, β-catenin (PY489)), Tnnt2 (Thermo Scientific, MS-295-P0) diluted 1 : 100 in 2% goat serum/PBS overnight at 4°C. Sections were then stained with secondary antibodies goat anti -rabbit IgG Alexa 633 (A-21070), goat anti-mouse IgM Alexa- 488 (A-21042), and goat anti-mouse Alexa-555 (A-21422) diluted 1 :400 and hoerscht diluted 1 : 1000 in 2% goat serum/PBS for 1 hour at room temperature and mounted in FluoMount®. Each slide was then imaged using a Leica DMi8 confocal microscope
Neonatal Rat Cardiomyocyte Culture
Cardiomyocytes were derived from PI Sprague-Dawley neonatal rats as previously described (Thomas et al 2002). Briefly, neonatal rats were sacrificed and hearts were excised, washed in ADS buffer (put in the protocol) and atria removed. Myocytes were then isolated using collagenase (get the Wharton number) and separated with PercollR® gradients. Percoll gradients were then constructed by layering 1 : 1.2 Percoll:ADS layer on a 1 :0.5 Percoll:ADS layer in a 15 ml Falcon tube. Isolated myocytes were plated in Cardiac Medium (see below in 2D experiments) without BRDU and allowed to recover overnight before experiments.
Cardiac Differentiation of Human Embryonic Stem Cells
Ethical approval for the use of human embryonic stem cells was obtained from The University of Queensland's Medical Research Ethics Committee (2014000801). Cardiomyocytes were produced based on recently published protocols (Hudson et al., 2012; Titmarsh et al., 2016), which give both cardiomyocytes and stromal cells which are critical for tissue function (Hudson et al., 2011). Briefly, cardiomyocyte/stromal cell cultures were produced from HES3 human embryonic stems cells (hESCs, WiCell) which were maintained as TypLE passaged cultures using mTeSR-1 (Stem Cell Technologies)/Matrigel (Millipore). hESCs were seeded at 2xl04 cells/cm2 in Matrigel coated flasks and cultured for 4 days using mTeSR-1. Subsequently, the hESCs were firstly differentiated into cardiac mesoderm using RPMI B27- medium (RPMI1640 GlutaMAX+ 2% B27 supplement minus insulin, 200 μΜ L-ascorbic acid 2 phosphate sesquimagnesium salt hydrate (Sigma) and 1% Penecillin/Streptomycin) containing 5 ng/ml BMP -4 (RnD Systems), 9 ng/ml Activin A (RnD Systems), 5 ng/ml FGF-2 (RnD Systems) and 1 μΜ CHIR99021 (Stem Cell Technologies) with daily medium exchange for 3 days. Subsequently, they were specified into a cardiomyocyte/stromal cell mixture using RPMI B27- containing 5 μΜ IWP-4 (Stem Cell Technologies) followed by another 7 days and of RPMI B27+ (RPMI 1640 GlutaMAX + 2% B27 supplement with insulin, 200 μΜ L- ascorbic acid 2 phosphate sesquimagnesium salt hydrate (Sigma) and 1% Penecillin/Streptomycin) with medium exchange every 2-3 days. The differentiated cells (-75% cardiomyocytes, -25% CD90+ stromal cells) were then cultured in RPMI B27+ until digestion at 15 days using 0.2% collagenase type I (Sigma) in 20% fetal bovine serum (FBS) in PBS (with Ca2+ and Mg2+) for 45 min at 37°C, followed by 0.25% trypsin-EDTA for 10 min. The cells were filtered using a 100 μπι mesh cell strainer (BD Biosciences), centrifuged at 300 x g for 3 min, and resuspended at the required density in Cardiac Medium: a-MEM GlutaMAX, 10% FBS, 200 μΜ L- ascorbic acid 2 phosphate sesquimagnesium salt hydrate (Sigma) and 1% Penecillin/Streptomycin.
2D Cultures and Treatments For both neonatal rat cardiomyocyte and human cardiomyocyte assays in 2D the cells were seeded in Cardiac Medium on gelatine coated glass cover-slips at 1 x 105 cells/cm2 for 24 h. Small molecules/growth factors were added to Cardiac Medium and given to the cells for 24 or 48 h: DMSO (Sigma), CHIR99021 (Stem Cell Technologies) and NRG-1 (RnD Systems). For transfection experiments the cells were transfected for 8 h using Lipofectamine RNAiMax (3 μ1/24 well) in 500 μ1/24 well OptiMEM followed by a medium change into Cardiac Medium. The cells were transfected at 50 nM with scramble miR control (All Stars Negative Control, Qiagen), miR mimic hsa-miR-199a-3p (Qiagen) or miR mimic hsa-miR-590-3p (Qiagen). For overexpression of constitutively active YAP cells were infected in Cardiac Medium with an adenovirus containing a mutated version of murine YAP-1, CMV-YAP(S112A) at an MOI of 10.
Large 3D hCO
For each hCO, 5xl05 cardiac cells in Cardiac Medium were mixed with collagen I to make a 150 μΐ final solution containing 1 mg/ml collagen I. The bovine acid- solubilised collagen I (Devro) was firstly salt balanced and pH neutralised using 10X DMEM and 0.1 M NaOH, respectively, prior to mixing with cells. The mixture was prepared on ice and pipetted into circular PDMS molds (Sylgard 184, Dow Corning) with an internal diameter of 3.9 mm and an external diameter of 8.1 mm. The collagen was then gelled at 37°C for 1 h prior to adding Cardiac Medium to cover the tissues (-3 ml/hCO). The hCO were cultured in the circular molds for 5 days with a medium change after 2 days. Subsequently, the hCO were cultured on PDMS exercise poles (designed to create 20 % stretch at rest and the hCO to shorten by 0.33 μιη/μΝ with contractions) with media changes every 2-3 days (2 ml/hCO). Small molecules were added to Cardiac Medium and given to the cells for 24 or 48 h: DMSO (Sigma), CHIR99021 (Stem Cell Technologies) and ICRT14 (Tocris).
Serum-free and digestion free 3D hCO Serum-free and digestion free 3D hCO were fabricated as previously described. Briefly, 5 x 105 hESCs were mixed with collagen I to make a 150 μΐ final solution containing 1 mg/ml collagen I. The bovine acid-solubilised collagen I (Devro) was firstly salt balanced and pH neutralised using 10X DMEM and 0.1 M NaOH, respectively, prior to mixing with cells. The mixture was prepared on ice and pipetted into circular PDMS molds (Sylgard 184, Dow Corning) with an internal diameter of 3.9 mm and an external diameter of 8.1 mm. The collagen was then gelled at 37°C for 1 h prior to adding mTeSR-1 (Stem Cell Technologies) to cover the tissues (~3 ml/hCO). After 24 h the cardiac differentiation protocol outlined in the Cardiac Differentiation of Human Embryonic Stem Cells Screening 3D hCO
For each hCO, 5 x 104 cardiac cells in Cardiac Medium were mixed with collagen I to make a 3.5 μΐ final solution containing 2.6 mg/ml collagen I and 9% Matrigel (Millipore). The bovine acid-solubilised collagen I (Devro) was firstly salt balanced and pH neutralised using 10X DMEM and 0.1 M NaOH, respectively, prior to mixing with Matrigel and cells. The mixture was prepared on ice and pipetted into a Heart-Dyno device which allows measurement of contractile force by movement of elastic poles of the device The mixture was then gelled at 37°C for 30 min prior to adding Cardiac Medium to cover the tissues (150 μΙ/hCO). The Heart-Dyno design facilitates the self-formation of tissues around in-built PDMS exercise poles (designed to deform 0.33 μτη/μΝ). The medium was changed every 2-3 days (150 μΙ/hCO) until treatment after 6 days. Small molecules were added to Cardiac Medium and given to the cells for 48 h: DMSO (Sigma), CHIR99021 (Stem Cell Technologies), NRG-1 (RnD Systems) and ICRT14 (Tocris). For transfection experiments the cells were transfected for 4 h using Lipofectamine RNAiMax (3 μΙ/hCO) in 150 μΙ/hCO OptiMEM followed by a medium change into Cardiac Medium. The cells were transfected at 50 nM with scramble miR control (All Stars Negative Control, Qiagen), miR mimic hsa-miR-199a-3p (Qiagen) or miR mimic hsa-miR-590-3p (Qiagen). For overexpression of constitutively active YAP, hCO were infected in Cardiac Medium with an AAV6 containing a mutated version of human YAP-1, CMV-YAP(S127A) (Vector Biolabs) at 2.5 x 1010 vg/cell an AAV6- MCS control was also used in these experiments. For overexpression of constitutively active β-catenin cells were infected in Cardiac Medium with an AAV6 containing a mutated version of human CTN B 1 without the amino acids 2-90, AAV6-AN90pCAT (Vector Biolabs), at 2.5 x 1010 vg/cell an AAV6-MCS control (a gift from Paul Gregorevic) was also used in these experiments.
Analysis of force of contraction Force of contraction was analysed by taking 10 second videos (at 50fps) of the elastic poles in the Heart-Dyno device using a customised Leica DMI8 Microscope and an in-house batch analysis pole-tracking program written in Matlab (Mathworks). The pole movement was converted to force, which we validated using force transducers. qPCR
RNA was extracted from cells or tissues using Trizol and treated with DNase I (Roche). The RNA was then either used as a template for either cDNA synthesis using Superscript III for SYBR-base qPCR or as a template for RNU6B, hsa-miR- 199a-3p or hsa-miR-590-3p Taqman assays. qPCR was performed on a Applied Biosystems Step One Plus machine and expression analysed using the 2"ΔΔα method. Primers used for S YBR qPCR are in Table 1.
Immunostaining of cultured cells and tissues
For 2D experiments cells were fixed on cover-slips for 10 min with 1% paraformaldehyde (Sigma) and washed 3X with PBS. The cells were then incubated with primary antibodies (see Table 2) in Blocking Buffer, 5% FBS and 0.025% Triton-X- 100 (Sigma) in PBS for 90 min at room temperature. Cells were then washed in Blocking Buffer 2X and subsequently incubated with secondary antibodies and Hoescht (1 : 1000) for 45 min at room temperature. Cells were then washed in Blocking Buffer 2X and then the cover-slips mounted on microscope slides using Fluoromount-G (Southern Biotech). The cells were then imaged using an Olympus BX51 fluorescence microscope where images of random fields of cardiomyocytes were taken using 10X (KI67 and pH3) or 20X (Aurora B) objectives for quantification analysis or at 40X for representative images.
For 3D experiments cells were fixed for 60 min with 1% para-formaldehyde (Sigma) at room temperature and washed 3X with PBS. The cells were then incubated with primary antibodies (see Table 2) in Blocking Buffer, 5% FBS and 0.025% Triton-X- 100 (Sigma) in PBS overnight at 4°C. Cells were then washed in Blocking Buffer 2X for 2 h and subsequently incubated with secondary antibodies and Hoescht (1 : 1000) overnight at 4°C. Cells were then washed in Blocking Buffer 2X for 2 h and then the cover-slips mounted on microscope slides using Fluoromount-G (Southern Biotech). The cells were then imaged using an Olympus 1X81 confocal microscope where images 3X random fields of cardiomyocytes were taken for quantification analysis per tissue.
FACS of Human Cardiac Cells
For the FACS from large 3D hCO for the experiment outlined in Figure 5, hCO treated with either DMSO or CHIR for 24 h were dissociated using 1.8 mg/ml collagen B and 2.4 mg/ml collagenase D in PBS for 1 h at 37°C. The digested cells were then passed through a 100 μπι mesh cell strainer (BD Biosciences), centrifuged at 300 x g for 3 min, and resuspended in PBS for FACS. Live cells were gated on forward- and side-scatter. For the FACS from 2D cardiac cells for the RNA-seq experiment outlined in Figure 6, cells were treated with either DMSO or CHIR for 24 h were dissociated using the protocol outlined in the Cardiac Differentiation of Human Embryonic Stem Cells section. The digested cells were then passed through a 100 μπι mesh cell strainer (BD Biosciences), centrifuged at 300 x g for 3 min, and stained with CD90 for FACS. Cells were incubated with CD90 for 15 min at 4°C in FACS buffer, 5% FBS in PBS. Subsequently the cells were washed in FACS buffer then incubated with Alexa Fluor® 488 Goat anti -Mouse IgG (H+L) in FACS buffer for 15 min at 4°C. The cells were then washed in FACS buffer and re-suspended in PBS for FACS. Live cells were gated on forward- and side-scatter, doublets were excluded based on forward- and side-scatter width and height.
Neonatal IWR-1 injections Neonatal CD-I mice (P0) were injected (s.c.) daily with 8 μΐ of IWR-1 or DMSO. IWR-1 was dissolved in DMSO and injected at a concentration of 20 mg/kg. All injections were carried out with a 30-gauge syringe. Hearts were isolated at P2 for qPCR and P7 for immunofluorescent staining. Adult AAV6-YAP1-S127A and AAV6-N90-BCAT injections
At 8 weeks of age, male CD1 mice were anesthetized with 4% isoflurane (Bayer) and maintained with 2% isoflurane and 0.25 1/min oxygen during surgery. The animals were intubated and ventilated (Minivent, Harvard Apparatus) with a tidal volume of 250 μΐ and a respiration rate of 133 strokes/min. A thoracotomy at the 3rd and 4th intercostal space was performed and the pericardial sac removed. 5 μΐ of either AAV6-YAP1-S127A and AAV6-N90-BCAT or AAV6-CMV-GFP were injected at 4 sites surrounding the left anterior descending artery using a 30-gauge needle and syringe (Hamilton). For the co-administered dose, lxlO11 viral particles of each virus was injected. The control mice were injected with 2xlOu viral particles AAV6-CMV-GFP. The chest wall was then sutured closed, the mouse removed from anaesthesia, supplied with s.c. injection of buprenorphine (0.05 mg/kg) as required and allowed to recover from the surgery. 72 hours later the mice were sacrificed with a single intraperitoneal dose of ketamine (200 mg/kg) and xylazil (16 mg/kg). Hearts were quickly dissected and washed in perfusion buffer (120.4 mmol/1 NaCl, 14.7 mmol/1 KC1, 0.6 mmol/1 KH2P04, 0.6 mmol/1 Na2HP04, 1.2 mmol/1 MgS04»7H20, 4.6 mmol/1 NaHC03, 10 mmol/1 Na-HEPES, 30 mmol/1 taurine, 5.5 mmol/1 glucose and 10 mM 2,3-Butanedione 2-monoxime). The aorta was then cannulated with a 21 -gauge cannula, secured with 3-0 silk suture and perfused with 37°C, oxygenated perfusion buffer using a Langendorff apparatus (4ml/min). Once all blood was washed from the heart, digestion buffer (200 μg/ml Liberase DH (Roche) in perfusion buffer) was passed through the heart for ~8 minutes (until the hearts appeared waxy in color and flaccid to the touch). During perfusion, atria and excess tissue were removed. After enzymatic digestion, hearts were minced with fine scissors into small pieces and triturated to release cells. Cell isolates were passed through a 100 μπι cell strainer and centrifuged at 30 xg for 3 minutes at room temperature. Supernatant (containing the non-myocyte cells) was collected and the myocyte pellet was washed with perfusion buffer and re-centrifuged. RNA from both cell populations were isolated with Trizol, using glycogen are a carrying agent. Ethical approval for neonatal and adult mouse procedures was obtained from The University of Queensland's Animal Ethics Committee (SBMS/101/13/NHMRC).
Echocardiography Mice were anesthetized using 2% isofluorane for the duration of echocardiographic recordings. Mice were positioned supine on a 37°C heating pad. Images of the left ventricular wall were taken in M-mode using a HD-15 ultrasound (Phillips) and a 14 Hz ultrasound probe (Phillips).
Statistical Analysis Statistics were analysed using Microsoft Excel (Microsoft) or GraphPAD Prism 6 (Graphpad Software Inc) using the appropriate statistical tests. All data is displayed as mean ± standard error of the mean unless otherwise indicated. For RNAseq differential expression analysis, false discovery rate comparisons were generated using the Benjamani-Hochberg method. Results
Isolation of purified cardiac cell populations from infarcted and non-infarcted neonatal and adult mouse hearts
Recent analyses of the cellular composition of the murine heart have revealed that fibroblasts, leukocytes and vascular endothelial cells comprise the majority of non- myocyte cell populations in the heart (Pinto et al., 2016). Of relevance to this study, each of these cell populations has been implicated in neonatal cardiac proliferative or regenerative processes (Aurora et al., 2014; Ieda et al., 2009). In order to perform transcriptional profiling of the different cardiac cell populations under regenerative versus reparative conditions, we devised a strategy to isolate cardiomyocytes, fibroblasts, leukocytes and vascular endothelial cells from neonatal (postnatal day 1; PI) or adult (postnatal day 56; P56) mice following MI or sham surgery (Figure 1A). All samples were obtained at day 3 post-surgery, which represents an early stage of the injury response when all of the cell types of interest are engaged and can be easily isolated. In order to obtain highly pure and viable cell populations, we used Liberase DH enzymatic digestion in combination with spinner flask dissociation for neonatal hearts or Langendorff retrograde perfusion for adult hearts (Figure 1A). Cardiomyocytes were immediately isolated for RNA extraction using differential density fractionation on a Percoll gradient for neonatal cardiomyocytes or low speed centrifugation for adult cardiomyocytes (see Figure 1 and methods). FACS was performed on the non-myocyte fraction to isolate leukocytes (CD45+), fibroblasts (CD90+/CD457CD31-) and vascular endothelial cells (CD31+/CD457Podo"). In concordance with recent findings (Pinto et al., 2016), the largest population of non- myocyte cells from non-infarcted adult hearts were endothelial cells (51.9%) followed by fibroblasts (26.5%) and leukocytes (19.9%) (Figure 1C). Furthermore, 96.7% of all CD31+/CD45" cells were vascular endothelial cells (CD31+/Podo"), while the remaining 3.3% were lymphatic endothelial cells (CD31+/Podo+), which is also consistent with a recent report (Pinto et al., 2016)(Figure 1C). It should be noted that in our isolation strategy the negative population (CD457CD907CD31") yielded little RNA and we concluded this population was primarily comprised of cellular debris. Therefore, the positively sorted populations represent the major cells types within the neonatal and adult heart and we subsequently excluded the negative population from further analysis.
As would be expected, infarcted neonatal and adult hearts had a proportional increase in the number of leukocytes compared to non-infarcted controls (Figure 1C). As development progressed from neonatal to adult stages, steady-state (non- infarcted) leukocyte populations within the heart also increased in number (Figure 1C). Conversely, the percentage of fibroblasts relative to other non-myocyte populations in the heart decreased during development and was markedly reduced after adult infarction (Figure 1C). The percentage of fibroblasts in non-infarcted adult mice in this study (26.5%) is similar to the previously reported percentage of CD90+/CD457CD31- fibroblasts (-30%) by Ali et al (Ali et al., 2014). Another study recently reported a similar fraction of fibroblasts (-27%), but a lower fraction of CD90+ cells (Pinto et al., 2016), which could be attributed to the use of different enzymatic dissociation protocols, different antibodies and different mouse strains. Our findings are highly concordant with other recent reports and suggest that endothelial cells are the most numerous non-myocyte population in the heart and that fibroblasts comprise -30% of non-myocytes in the adult mouse heart. Multi-cellular RNA-seq reveals distinct transcriptional programs in different cell types during cardiac regeneration or repair
To determine cell type-specific transcriptional programs deployed during cardiac development, repair and regeneration, we performed RNA-seq on cardiomyocytes, fibroblasts, leukocytes and vascular endothelial cells isolated from the neonatal and adult heart before or after MI. RNA-seq libraries were prepared using TruSeq Stranded Total RNA kits (Illumina) with 50 base-pair single-end reads. This process resulted in exceptionally high quality reads with a mean phred quality score of >35 and an average number of 46 million reads per sample. Analysis of cell type-specific transcripts confirmed that our purified cell populations were highly enriched for cell- specific gene markers (Figure IB). For example, myosin light chain 7 (Myl ) and myosin light chain 2 {MylT) were specifically expressed in the cardiomyocyte fraction and were highly enriched at neonatal and adult stages, respectively (Figure IB). In total, >9964 differentially expressed genes were identified in at least one comparison within a cell type during development (ShamPl vs ShamP56) or following MI (MIP1 vs MIP56; log2 expression > 1 or < -1, FDR < 0.05). Hundreds of differentially regulated genes were identified within each cell population following MI in neonatal versus adult hearts, with the vast majority of differences accounted for by the cardiomyocyte compartment (Figure ID). To visualize the transcriptional relatedness and reproducibility of each RNA-seq sample, we employed principal coordinate analysis (PCA) (Figure 2A). Each cell type within the RNA-seq data set grouped into a defined cluster within the PCA plot and RNA-seq samples within each experimental group were also tightly clustered together (Figure 2A). Major transcriptional differences were apparent between neonatal and adult samples for all cell populations (in the absence of injury) (Figure 2A). Pearson correlations and unsupervised hierarchical clustering of RNA-seq samples further confirmed the high reproducibility of transcriptional signatures within groups (Figure 2B). As a further validation of our cell sorting strategy, we performed unsupervised hierarchical clustering of RNA-seq samples followed by gene ontology (GO) analysis for each cluster. As would be expected, up-regulated genes within each specific cluster were highly enriched for GO terms associated with that specific cell type (Figure 2C). For example, cluster 1 1 contained genes that were highly expressed in leukocytes and this cluster was enriched for GO terms such as inflammatory response and T cell activation (Figure 2C). Similarly, genes highly expressed in adult cardiomyocytes were contained within cluster 2, which was highly enriched for terms associated with heart development, regulation of heart contraction and mitochondrial organization (Figure 2C). Clusters 8 and 9 contained genes that were highly enriched in fibroblast and endothelial cells, respectively (Figure 2C).
Defining the transcriptional programs governing cellular identity
Cellular identity is governed by a core transcriptional program, which is epigenetically cemented during development. We postulated that core cell identity programs similarly exist in both neonatal and adult stages. To test this, we isolated genes enriched for a particular cell type relative to other cell types in all conditions including infarcted and sham-operated neonates and adults. In these "cellular identity datasets" many transcription factors, signalling pathways and gene ontologies classically associated with each cell type were highly enriched (Figure 3). For example, both neonatal and adult myocytes were highly enriched for transcriptional networks controlled by cardiogenic transcription factors, including Nkx2-5, Gata4, Mef2c and Mef2a, as well as genes associated with cellular metabolic processes (Figure 3). Similarly, fibroblasts were enriched for extracellular matrix proteins (Figure 3). Although very little is known about the transcription factors that dictate cardiac fibroblast identity, our data set revealed three over-connected transcription factors within the fibroblast identity network; Nr2fl, Prdm5 and Zfp384, which are all associated with extracellular matrix deposition and tumor suppression (Burkitt Wright et al., 2011; Furuya et al., 2000; Sosa et al., 2015). Therefore, this dataset provides a rich and highly predictive resource for interrogation of transcriptional circuits controlling cardiac cell identity, which could be leveraged for cell reprogramming studies.
Defining the transcriptional processes governing multi-cellular responses to MI
We next sought to understand the cellular processes, signaling pathways and transcriptional networks that distinguish adult from neonatal cellular responses following MI. Consistent with enhanced cardiomyocyte proliferation in the neonatal heart following MI, regenerative neonatal cardiomyocytes were highly enriched for transcription factors and GO terms associated with cell cycle (Figure 4A-B). Of particular interest, the highest ranked transcriptional network associated with infarcted neonatal cardiomyocytes was associated with the Wnt signaling pathway (Figure 4C). Alternatively, adult myocytes and endothelial cells deployed distinct transcriptional responses to MI. Many pathways and transcription factors related to metabolism and mitochondrial organization were enriched in both adult myocytes, with autophagic (Tfeb, Amfr, Gabarap, Gabarapll) and oxidative stress response (Sodl) genes specifically upregulated in response to infarction (Figure 4A). Similar to neonatal myocytes, neonatal vascular endothelial cells were also enriched for GO terms and transcription factors associated with cell cycle (Figure 4A-B). However, in addition, several cell cycle transcription factors were specifically upregulated in neonatal endothelial cells following MI (Figure 4A). In contrast, adult endothelial cells appear to be refractory to cell cycle induction at 3 days post MI and instead deploy an inflammatory response characterize by C3 complement signaling (Figure 4A&B). Adult leukocytes and fibroblasts from the infarcted heart were also enriched for genes related to the immune response and inflammatory processes (Figure 4A-C), with infarcted adult leukocytes specifically characterized by expression of H2-Aa and H2-Abl (Figure 4A). Interestingly, H2-Aa and H2-Abl are both highly expressed in B cells, dendritic cells and monocytes (Heng et al., 2008), which display unique cell recruitment profiles following neonatal MI (Aurora et al., 2014). Thus, neonatal and adult cardiomyocytes and non-myocytes were characterised by unique transcriptional signatures following MI but it was unclear whether these differences arose during development or in response to injury.
Adult cardiomyocytes, endothelial cells and fibroblasts deploy an injury- induced transcriptional program that is distinct from the neonatal injury response. We next attempted to dissociate developmentally regulated transcripts from injury- induced gene expression changes within each cardiac cell population. Strikingly, we noted that neonatal cardiomyocytes, fibroblasts and vascular endothelial cells failed to mount a robust transcriptional response to MI (Figure 10A-D). In contrast, adult cardiomyocytes, fibroblasts and vascular endothelial cells activated a distinct injury- induced transcriptional program in response to MI (Figure 10E-H). On the other hand, leukocytes displayed injury -induced transcriptional shifts following MI, with distinct suites of transcripts activated in response to injury at neonatal and adult stages. For example, in response to MI, neonatal leukocytes were highly enriched for networks associated with retinoic acid signaling (Figure 4C), whereas the MyD88- dependent toll-like receptor signaling pathway was activated in adult leukocytes (p = 7.25e-l l). These data point to underlying differences in the inflammatory response to MI in neonatal and adult hearts, which could reflect differences in the recruitment and activation of specific leukocyte subsets, as well as the induction of distinct suites of inflammatory cytokines.
An important implication of this result is that neonatal cardiomyocytes, fibroblasts and endothelial cells are almost transcriptionally identical to their sham-operated controls following ischemic injury. This result implies that neonatal cardiomyocytes, fibroblasts and endothelial cells do not activate a specific transcriptional network following MI but rather already possess the transcriptional requirements for regeneration. Our analyses reveal enormous transcriptional differences between neonatal and adult cell types, with the vast majority of differences occurring during cardiomyocyte maturation (Figures 1, 2 and 10). The transcriptional profiles of neonatal and adult cardiomyocytes were so disparate that neonatal cardiomyocytes were transcriptionally more similar to endothelial cells and leukocytes than they were to adult cardiomyocytes (Figure 2C). Therefore, neonatal heart regeneration is predominantly linked with a permissive regenerative transcriptional state rather than induction of a specific injury-induced regenerative transcriptional program. Failure of adult cardiomyocytes to re-activate the neonatal gene program following MI is associated with aberrant Wnt/ -catenin signaling
Adult cardiac pathologies are commonly associated with reversion to a fetal/neonatal gene program. However, few studies have assessed this phenomenon using genome- wide methods with cellular resolution. We next sought to understand global transcriptional responses to MI at neonatal and adult stages. Following MI, adult fibroblasts and leukocytes underwent a transcriptional reversion to a "neonatal-like" state, with their transcriptomes clustering more closely with infarcted and non- infarcted neonatal cells than non-infarcted adult cells (Figure 2A). A similar reversion phenomenon did not occur in adult cardiomyocytes and vascular endothelial cells following MI. The post-infarction reversion of adult leukocytes and adult fibroblasts to a neonatal-like state was largely associated with the re-induction of cell cycle genes (Figure 2C, cluster 6; GO 0000278 :mitotic_cell_cycle, Leukocytes FDR= 2.334e-31; fibroblasts FDR = 5.166e-67). In contrast, cardiomyocytes (and to a lesser extent endothelial cells) failed to re-activate the same set of cell cycle genes following MI (Figure 3C, cluster 6). Further analysis of this reversion network revealed that all cardiac cell populations down-regulated a common set of 683 genes during development from neonatal to adult stages but only fibroblasts and leukocytes were able to re-activate this set of genes in adulthood following MI (Figure 4).
In order to identify the signaling networks controlling the expression of this reversion network, we assessed the signaling pathways driving cellular networks in each cell type during cardiac development and regeneration. We first surveyed several cell signaling pathways that are known to contribute to cardiac regeneration including the Hippo pathway (Xin et al., 2011), F-κΒ pathway, JAK-STAT pathway (Fang et al., 2013; Mahmoud et al., 2015), epidermal growth factor receptor signaling (Bersell et al., 2009), retinoic acid signaling (Kikuchi et al., 2011), and Notch signaling (Raya et al., 2003). Matrices of genes for each signaling pathway were constructed to visualize pathway usage in different cell types during development and following MI at neonatal or adult stages (Figure 11). However, we could not identify a consistent pattern of transcriptional regulation for these signaling pathways that could explain the cell cycle reversion phenomenon that was strongly associated with cardiac regenerative capacity.
We subsequently employed an unbiased approach to investigate which gene networks were dominant during development (ShamPl vs ShamP56) and regeneration (MIP1 vs MIP56) in each cell type (Figure 4C). The highest ranked signaling pathway associated with neonatal regenerative networks in cardiomyocytes and endothelial cells, which failed to revert following adult MI, was the Wnt/β- catenin signaling pathway (Figure 4C). Moreover, the expression profile of Wnt/β- catenin signaling components closely resembled the cell cycle gene expression profile within cardiomyocytes, with several Wnt activators becoming repressed in adult cardiomyocytes (Figure 11). These Wnt activators were not re-activated in adult cardiomyocytes following MI (Figure 11). Conversely, several Wnt inhibitors were activated during cardiomyocyte development from neonatal to adult stages (Figure 11). Similarly, these Wnt inhibitors failed to revert to their neonatal expression levels following MI in adulthood (Figure 11). Based upon these insights, we sought to determine whether Wnt/p-catenin signaling was a major driver of cardiomyocyte proliferative capacity and heart regeneration. β-catenin is regulated during cardiac development and Wnt/ -catenin signaling induces cardiomyocyte proliferation in immature rodent and human cardiomyocytes
We first sought to determine whether the downstream effector of the canonical Wnt pathway, β-catenin, was altered during postnatal heart development in the mouse. We used an antibody that was specific for the active, nuclear localized, form of β- catenin (PY489)(Sturzu et al., 2015). We found that there was a progressive loss of active β-catenin from cardiomyocyte nuclei from PI to P21, coinciding with the loss of cardiomyocyte proliferative capacity and regenerative potential during postnatal development (Porrello et al., 2011)(Figure 5A).
We next assessed whether activation of Wnt/p-catenin signaling was capable of inducing cardiomyocyte proliferation in rodent and human cardiomyocytes. For these experiments, we used the specific small molecule inhibitor of GSK3 (Kramer et al., 2012), CHIR99021 (CHIR) to stimulate the Wnt/p-catenin pathway. We firstly demonstrated that CHIR was capable of inducing cardiomyocyte proliferation in commonly used neonatal rat cardiomyocyte cultures, as demonstrated by increases in the general cell cycle marker KI67, the mitosis marker pH3 and the cytokinesis marker Aurora B after 24 h (Figure 12). Additionally, we demonstrated that CHIR could induce proliferation in standard two-dimensional (2D) cultures of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) using KI67 at 24 h (Figure 13). We next tested whether CHIR could also induce proliferation in more mature hPSC-CMs cultured in a 3D human cardiac organoid (hCO) format (Figure 5B). CHIR was capable of activating β-catenin and driving it into the nucleus in hCO (Figure 5D), whilst simultaneously maintaining localisation of β-catenin at cell-cell junctions (Figure 5C), which may be critical for maintaining cardiomyocyte coupling during proliferation (Swope et al., 2012). Additionally, treatment with CHIR led to increased cell cycle activity in hCO marked by KI67 at 24 h (Figure 5E), followed by increased mitosis marked by pH3+ cardiomyocytes with disassembled sarcomeres at 48 h (Figure 5F). β-catenin-dependent transcription is required for cardiomyocyte proliferation We next undertook a series of experiments to dissect the relative contributions of serum, mechanical stimulation and β-catenin transcription to the CHIR-induced proliferative response of human cardiomyocytes. We initially performed the CHIR stimulation experiments in the presence of FBS to ensure the tissues were healthy (see methods). FBS contains many factors that could have been potentially required for CHIR to induce cardiomyocyte proliferation. We therefore firstly sought to determine whether FBS was required for CHIR to induce a proliferative response. For these experiments, we used the recently developed serum-free direct formation of hCO from pluripotent stem cells, whereby the cells do not pass through a dissociation step and are cultured under serum-free conditions throughout the protocol (Hudson et al., 2015) (see methods). We found that CHIR was still capable of inducing cardiomyocyte cell cycle entry marked by KI67 at 24 h under these serum-free conditions (Figure 14).
We next sought to determine whether activation of a β-catenin transcriptional response was required to induce proliferation in the hCO. We firstly confirmed that CHIR was inducing a β-catenin transcriptional response in the hCO cardiomyocytes. For these studies we used a KX2-5+/GFP knock-in cardiomyocyte reporter line (Elliott et al., 2011) coupled with FACS sorting (Figure 5G). Using FACS from control (DMSO) or CHIR treated hCO, we were able to purify GFP+ cardiomyocytes (Figure 15 A), which we confirmed were depleted in the stromal cell marker COL1A1 and enriched in the cardiomyocyte-specific marker MYH6 using qPCR (Figure 16B), as these are the two predominant cell types present in our tissues (data not shown). We next assessed the expression of the widely used generic β-catenin target gene, AXIN2, and found that CHIR induced activation of β-catenin transcription in the purified cardiomyocytes (Figure 5H). We next wanted to confirm functionality of β- catenin transcription in the induction of cardiomyocyte cell cycle. However, for these experiments we could not simply knock-down or knock-out β-catenin, as it also plays a critical role in maintaining cardiomyocyte cell-cell junctions (Swope et al., 2012). In addition, we wanted to specifically target the transcriptional response, as inhibiting the Wnt pathway further upstream (eg. using IWP-4 or IWR-1 (Hudson and Zimmermann, 2011)) would not be sufficient to block β-catenin activation of target genes in response to CHIR. We therefore utilized ICRT14 to block β-catenin- TCF interaction and activation of target genes (Gonsalves et al., 2011). Treatment of hCO with ICRT14 suppressed the CHIR-induced activation of the β-catenin target gene AXIN2 (Figure 51) and also suppressed CHIR-induced proliferative responses (Figure 5 J), suggesting that these effects were dependent on β-catenin transcriptional activity. Wnt/ -catenin signaling is a highly conserved regulator of cardiomyocyte proliferation
As a number of inducers of adult cardiomyocyte proliferation have recently been identified in rodents, we wanted to benchmark the effects of CHIR against a panel of previously reported activators of adult cardiomyocyte proliferation. We initially confirmed that the proliferative activators were capable of inducing proliferation in commonly used neonatal rat cardiomyocyte cultures. We found that CHIR, miR- 199a (Eulalio et al., 2012), miR-590 (Eulalio et al., 2012) and overexpression of constitutively active murine YAP-1(S 1 12A) (Xin et al., 2013b) were all capable of inducing mitosis in neonatal rodent cardiomyocytes marked by pH3+ cardiomyocytes with disassembled sarcomeres (Figure 16). Neuregulin was unable to induce proliferation over baseline in neonatal rat cardiomyocyte cultures, although this could be due to the presence of serum in our cultures, which induces a similar proliferative rate as neuregulin (D'Uva et al., 2015; Zhao et al., 1998).
We further confirmed robust overexpression of miRs in hCO cultured in a 96-well screening platform following transfection (Figure 17) and then screened the pro- proliferative factors in a human cardiac microtissue device (Figure 18). Interestingly, we found only CHIR and constitutively active human YAP- 1 (S I 27 A) induced proliferation above baseline in hCO (Figure 18). No treatments other than CHIR were associated with activation of a β-catenin dependent transcriptional responses marked by AXIN2 (Figure 18). These results confirm that Wnt/p-catenin is a highly conserved regulator of cardiomyocyte proliferation in rodent and human cardiomyocytes.
Intersection of mouse and human cardiomyocyte transcriptomes identifies a β- catenin gene regulatory network driving cardiomyocyte proliferation We next wanted to define the β-catenin gene regulatory network in human cardiomyocytes to determine whether a conserved set of β-catenin transcriptional targets is associated with cardiac regenerative capacity. To identify β-catenin transcriptional targets we performed RNA-seq on hPSC-CM after 24h of CHIR treatment. To get enough high quality RNA from purified cardiomyocytes for RNA- seq we performed this experiment on 2D hPSC-CM, which were purified using CD90" FACS (Figure 6A). We confirmed the CD90" cells were depleted in the stromal cell marker COL1A1 and enriched in the cardiomyocyte-specific marker MYH6, and that AXIN2 was activated with CHIR treatment in the CD90- cardiomyocyte population using qPCR (Figure 20).
RNA-seq was performed on FACS sorted fibroblasts (CD90+) and myocytes (CD90) from 2D cultures treated with or without CHIR. Interestingly, stromal cells and cardiomyocytes underwent distinct transcriptional responses to CHIR treatment, with 235 genes regulated in myocytes and 52 genes regulated in fibroblasts and only 7 genes, including AXIN2, co-regulated in both cell types (Figure 6A&B). The highest regulated GO terms in cardiomyocytes treated with CHIR were associated with cell cycle, chromosome organization and DNA replication (Figure 6C). The CHIR- induced transcriptional network in human cardiomyocytes was highly enriched for transcription factor motifs and networks governed by common cell cycle transcription factors (E2F1, FOXMl, E2F8, MYBL2) and downstream effectors of the Wnt signaling pathway (TCF7L2 and LEFl)(Figure 6D&E). Therefore, CHIR induces the expression of a network of genes controlled by cell cycle and Wnt/β- catenin transcription factors in human cardiomyocytes.
In order to determine whether the CHIR-induced cardiomyocyte transcriptional network represents a core set of cell cycle genes implicated in the regulation of cardiac regenerative capacity, we intersected the human and mouse RNA-seq data sets. Of the 235 genes that were induced by CHIR in human cardiomyocytes, a large subset (65%) overlapped with a set of developmental^ regulated genes in mouse cardiomyocytes that failed to revert to a neonatal-like state following MI (Figure 6E&F). Moreover, the mouse reversion cluster and the CHIR-induced human myocyte network showed strong overlap with a common set of transcription factors that are predicted to control this transcriptional network (Figure 6E). Further analysis of the highest ranked network in neonatal versus adult cardiomyocytes similarly revealed strong enrichment for components of the Wnt/ -catenin signaling pathway including interactions with multiple cell cycle transcription factors (Figure 6G). Therefore, neonatal regenerative potential is linked with the transcriptional repression of a β-catenin gene regulatory network, which fails to re-activate in adult cardiomyocytes following MI. Re-activation of β-Catenin in the adult heart improves cardiac function and induces partial regeneration following MI
The aforementioned results pointed towards a central role for β-catenin in the activation of cell cycle genes. Therefore, we next tested whether direct activation of β-catenin was sufficient to drive proliferation in hCO cardiomyocytes (Figure 7A). For these experiments we over-expressed a constitutively active form of β-catenin using AAV6 (AN9(^CAT, see methods). We confirmed that AAV6 AN9(^CAT was capable of activating β-catenin in hCO cardiomyocytes (Figure 7B). Moreover, AAV6 AN9C^CAT induced cardiomyocyte mitosis (marked by pH3+ cardiomyocytes with disassembled sarcomeres) after 48 h (Figure 7C) to a similar level as CHTR-treated hCO cardiomyocytes (Figure 7D). Importantly, while treatment with CHIR resulted in a severe reduction in the contractile properties of hCO (significantly reduced force of contraction), AAV6 AN9C^CAT had no deleterious effects on contractile force (Figure 7E). We also confirmed that AAV6 AN9C^CAT was able to induce the expression of a core set of developmentally regulated genes in hCO, which were also induced by CHIR in 2D myocytes and which failed to revert to a neonatal-like state following MI in adult mouse cardiomyocytes (Figure 7F). Together, these results demonstrate that β-catenin is sufficient to drive human cardiomyocyte proliferation without causing any detrimental effects on cardiac contractility.
We next tested whether β-catenin plays a role in proliferation of cardiomyocytes in vivo. IWR-1 inhibits WNT signaling by blocking the WNT^-catenin pathway reporter response (IC50 value of 180 nM) (Chen et al., 2009, Nat. Chem. Biol. 5 100). Its effects are potentially through the stabilization of the destruction complex member AXIN2 (Chen et al., 2009, supra). We found that inhibition of β-catenin in neonates using IWR-1 reduced the β-catenin target gene Birc5 (Fig. 20A). Treatment of neonatal mouse hearts with IWR-1 reduced proliferation (Fig. 20B), indicating that β-catenin plays a key role in regulating proliferation in neonates in vivo. We next assessed whether delivery of β-catenin could induce therapeutic benefits in vivo in adult mouse hearts following MI. We found that delivery of β-catenin resulted in a significant improvement in ejection fraction following MI (Fig. 20C).
EXAMPLE 2 Materials & Methods
Mitogen screening
Cardiomyocytes were derived from PI Sprague-Dawley neonatal rats, as previously described (Thomas et al 2002). Briefly, neonatal rats were sacrificed and hearts were excised, washed in ADS buffer and atria removed. Myocytes were isolated using collagenase II and separated with Percoll gradients. Percoll gradients were constructed by layering 1 : 1.2 Percoll: ADS layer on a 1 :0.5 Percoll: ADS layer in a 15 ml Falcon tube. Isolated myocytes were plated in CTRL medium without BRDU on gelatine coated glass cover-slips at 1 x 105 cells/cim and allowed to recover overnight before experiments. Small molecules/growth factors were added to CTRL medium and given to the cells for 24 or 48 h: DMSO (Sigma), CHIR99021 and NRG-1 (RnD Systems). For transfection experiments, the cells were transfected for 8 h using Lipofectamine RNAiMax (3 μ1/24 well) in 500 μ1/24 well OptiMEM followed by a medium change into CTRL medium. The cells were transfected at 50 nM with scramble miR control (All Stars Negative Control, Qiagen), miR mimic hsa-miR-199a-3p (Qiagen) or miR mimic hsa-miR-590-3p (Qiagen). For overexpression of constitutively active YAP-1, cells were infected in CTRL medium with an adenovirus containing a mutated version of murine YAP-1, CMV- YAP(S112A) at an MOI of 10. hCO were cultured in CTRL medium for 7 days before treatment. Small molecules were added and given to the cells for 48 h: DMSO, CHIR99021 and NRG-1. For transfection experiments, the cells were transfected for 4 h using Lipofectamine RNAiMax (3 μΙ/hCO) in 150 μΙ/hCO OptiMEM followed by a medium change into CTRL medium. The cells were transfected at 50 nM with scramble miR control, miR mimic hsa-miR-199a-3p or miR mimic hsa-miR-590-3p.
Expression of YAP-1 and β catenin For overexpression of constitutively active YAP-1, hCOs were infected in
with an AAV6 containing a mutated version of human YAP-1, CMV-YAP(S127A) (Vector Biolabs) at 1.25-2.5 x 10io vg/hCO. For overexpression of constitutively active β-catenin hCO were infected with an AAV6 containing a mutated version of human CTN B 1 without the amino acids 2-90, AAV6-AN90pCAT (Vector Biolabs), at 1.25-2.5 x lOio vg/hCO. Control AAV6-MCS or AAV6-GFP (Vector Biolabs) controls were used at the same titres in these experiments.
Whole-mount immunostaining
hCOs were fixed for 60 min with 1% paraformaldehyde (Sigma) at room temperature and washed 3X with PBS, after which they were incubated with primary antibodies (Table 2) in Blocking Buffer, 5% FBS and 0.025% Triton-X-100 (Sigma) in PBS overnight at 4°C. Cells were then washed in Blocking Buffer 2X for 2 h and subsequently incubated with secondary antibodies (Table 2) and Hoescht (1 : 1000) overnight at 4°C. They were washed in Blocking Buffer 2X for 2 h and imaged in situ or mounted on microscope slides using Fluoromount-G (Southern Biotech). The cells were imaged using a Leica DMi8 high content imaging microscope for in situ imaging, or an Olympus 1X81 confocal microscope or a Nikon Diskovery Spinning Disk confocal microscope for mounted imaging.
In vivo Experiments
Ethical approval for adult mouse experiments was obtained from The University of Queensland's Animal Ethics Committee (SBMS/101/13/NHMRC). Adult Males CD-I mice were housed under standard conditions with 12h light/dark cycles and ad libitum access to food and water. At 8 weeks of age, mice were anesthetized with 4% isoflurane (Bayer) and maintained with 2% isoflurane and 0.25 1/min oxygen during surgery. The animals were intubated and ventilated (Minivent, Harvard Apparatus) with a tidal volume of 250 μΐ and a respiration rate of 133 strokes/min. A thoracotomy at the 3rd and 4th intercostal space was performed and the pericardial sac removed. 15 μΐ of DMSO or 15 μΐ of Compound 6.28 (1.7 mg/kg) was directly injected into the myocardium using a 30g needle (Hamilton). The chest wall was then closed using a 6-0 prolene suture (Ethicon), the mouse removed from anaesthesia, supplied with s.c. injection of buprenorphine (0.05mg/kg) and allowed to recover from the surgery. Animals were BrdU pulsed with 100 mg/kg i.p. injections at day 0, 2, 4 and 6 post-surgery. Hearts were collected at day 7 post- surgery for immunofluorescent quantification of myocyte proliferation.
For BrdU staining, mice were sacrificed by cervical dislocation and hearts collected, washed in PBS and fixed in 4% paraformaldehyde overnight. Each heart was washed in PBS, halved with a single transverse cut, dehydrated and embedded in paraffin wax. 6 μπι sections were mounted on SuperFrost Ultra Plus slides. Sections were then rehydrated and blocked with 10% goat serum in PBS. Sections were stained with BrdU and MLC2v antibodies, relevant secondary antibodies (Table 3) and Hoechst (1 : 1000) to quantify proliferating cardiomyocytes. Slides were mounted with FluoMount-G and imaging was performed using a Leica DMi8 confocal microscope and every BrdU positive cell on each section was analysed in high resolution to determine whether it was a proliferating cardiomyocyte.
Neonatal AAV6-YAP1-S127A and AAV6-N90-BCAT injections Neonatal CD-I mice (PI) were gently restrained and intravenously injected (temporal vein) with a cocktail of AAV6-YAP1-S127A and AAV6-N90-BCAT or AAV6-CMV-GFP. For the co-administered dose, lxlO11 viral particles of each virus was injected. The AAV6-CMV-GFP virus injected at a concentration of 2xlOu viral particles. Staring at PI, mice were subcutaneously injected with 100 mg/kg BrdU every second day for 12 days. All injections were carried out with a 30-gauge syringe. Hearts were isolated at P2 for qPCR and P7 for immunofluorescent staining.
Adult AAV6-YAP1-S127A and AAV6-N90-BCAT injections
At 8 weeks of age, male CD1 mice were anesthetized with 4% isoflurane (Bayer) and maintained with 2% isoflurane and 0.25 1/min oxygen during surgery. The animals were intubated and ventilated (Minivent, Harvard Apparatus) with a tidal volume of 250 μΐ and a respiration rate of 133 strokes/min. A thoracotomy at the 3rd and 4th intercostal space was performed and the pericardial sac removed. 5 μΐ of either AAV6-YAP1-S127A and AAV6-N90-BCAT or AAV6-CMV-GFP were injected at 4 sites surrounding the left anterior descending artery using a 30-gauge needle and syringe (Hamilton). For the co-administered dose, lxlO11 viral particles of each virus was injected. The control mice were injected with 2xlOu viral particles AAV6-CMV-GFP. The chest wall was then sutured closed, the mouse removed from anaesthesia, supplied with s.c. injection of buprenorphine (0.05 mg/kg) as required and allowed to recover from the surgery. 72 hours later the mice were sacrificed with a single intraperitoneal dose of ketamine (200 mg/kg) and xylazil (16 mg/kg). Hearts were quickly dissected and washed in perfusion buffer (120.4 mmol/1 NaCl, 14.7 mmol/1 KC1, 0.6 mmol/1 KH2P04, 0.6 mmol/1 Na2HP04, 1.2 mmol/1 MgS04»7H20, 4.6 mmol/1 NaHC03, 10 mmol/1 Na-HEPES, 30 mmol/1 taurine, 5.5 mmol/1 glucose and 10 mM 2,3-Butanedione 2-monoxime). The aorta was then cannulated with a 21 -gauge cannula, secured with 3-0 silk suture and perfused with 37°C, oxygenated perfusion buffer using a Langendorff apparatus (4ml/min). Once all blood was washed from the heart, digestion buffer (200 μg/ml Liberase DH (Roche) in perfusion buffer) was passed through the heart for ~8 minutes (until the hearts appeared waxy in color and flaccid to the touch). During perfusion, atria and excess tissue were removed. After enzymatic digestion, hearts were minced with fine scissors into small pieces and triturated to release cells. Cell isolates were passed through a 100 μπι cell strainer and centrifuged at 30 xg for 3 minutes at room temperature. Supernatant (containing the non-myocyte cells) was collected and the myocyte pellet was washed with perfusion buffer and re-centrifuged. RNA from both cell populations were isolated with Trizol, using glycogen are a carrying agent. Ethical approval for neonatal and adult mouse procedures was obtained from The University of Queensland's Animal Ethics Committee (SBMS/101/13/NHMRC).
Echocardiography
Mice were anesthetized using 2% isofluorane for the duration of echocardiographic recordings. Mice were positioned supine on a 37°C heating pad. Images of the left ventricular wall were taken in M-mode using a HD-15 ultrasound (Phillips) and a 14 Hz ultrasound probe (Phillips).
Statistical analysis
Statistics were analysed using Microsoft Excel (Microsoft) or GraphPAD Prism 6 (Graphpad Software Inc).
Results
In other, previous studies performed in this laboratory and described in Australian Provisional Patent Application 2016903404, it was established that culture in a maturation medium (MM) induced cell cycle repression and a switch from glycolysis to fatty acid oxidation. We therefore wanted to assess how metabolic substrates influenced the cardiac cell cycle. In these studies, we specifically examined the Wnt/p-catenin signaling pathway, as we have previously identified CHIR99021 (Titmarsh et al 2016) as one of the most potent activators of human cardiomyocyte proliferation and have also found this pathway to be transcriptionally repressed during postnatal maturation (Sim et al 2014).
We first confirmed that the general cell cycle marker, Ki-67 (Fig. 21a), and the mitosis specific marker, pH3 (Fig. 21b), were repressed in cardiomyocytes in hCOs. Cardiomyocyte proliferation was markedly reduced in hCOs cultured in MM, with very low overall rates of cardiomyocyte mitosis (-0.2% pH3+ cardiomyocytes), which is similar to the postnatal human heart (Polizotti et al 2015). Importantly, all conditions had a similar force of contraction during the first 48 h of culture (Fig. 22c, indicating that the hCOs have similar functional properties and viability at this early stage, even in the absence of glucose and palmitate (in contrast to longer term cultures, Fig. 21g). As a read-out of β-catenin activity in our screen, we performed quantification of activated β-catenin using an antibody that only binds to activated, nuclear-localized β-catenin (Sturzu et al., 2015). Activated β-catenin was highly dependent on insulin regardless of the presence of either glucose or palmitate (Fig. 21c), as was proliferation (Fig. 2 Id), and there was a highly significant correlation between activated β-catenin and Ki-67 intensity (Fig. 21e over all hCOs in all conditions, n = 164). Despite this dependence on insulin signaling for proliferation immediately after culture in MM, the addition of insulin after 11 days in MM was not sufficient to re-activate cardiomyocyte proliferation (Fig. 21g,h). This indicates that longer term culture in MM induces desensitization of cardiomyocytes to the proliferative actions of insulin.
Interestingly, only CHIR99021 treatment resulted in a reduction of force of contraction at 48h, indicating that it is the inhibition of GSK3, but not the activation of β-catenin or proliferation per se, that results in a reduction in function (Fig. 22 g). These results confirmed that Wnt^-catenin is a highly conserved and potent regulator of cardiomyocyte proliferation in rodent and human cardiomyocytes.
We next tested whether hCOs cultured in MM were refractory to activation of proliferation using CHTR99021, which in our screen activated proliferation throughout the hCO. While hCOs cultured in CTRL medium (for standard 16 days in culture) mounted a robust proliferative response to CHIR99021, mature hCOs cultured in MM had a blunted proliferative response (Fig. 23a,b). This finding suggested that mature cardiomyocytes in MM were resistant to proliferative stimuli.
We next hypothesized that the β-catenin and YAP pathways act in synergy to activate proliferation. Both β-catenin and YAP activated proliferation in immature hCOs (Fig. 23), have been shown to act co-operatively with the core cardiogenic transcription factor TBX5 to regulate the cell cycle (Rosenbluh et al. 2012), and are required for cardiomyocyte proliferation during embryonic development (Heallen et al., 2011; Xin et al., 2011) , 42). Given the lack of a proliferative response to CHTR99021, we therefore hypothesized that YAP/TAZ -mediated transcription may be also be repressed in hCOs cultured in MM. YAP/TAZ (or Hippo) signaling was not overrepresented in our RNA-seq or proteomics analysis (data not shown). However, this may be because YAP/TAZ mediates the transcription of the majority of its transcriptional targets through enhancers (Zanconato et al., 2015), for which the targets have only recently been determined by cross-referencing ChlP-seq data to the 3D chromatin interactome (Augeri et al., 2012). Therefore, to determine whether YAP/TAZ transcriptional targets were regulated, we cross-referenced downregulated genes in hCOs cultured in MM versus CTRL medium (FDR < 0.05, 2120 genes) to YAP/TAZ targets (Fig. 21c).
We found that 57 YAP/TAZ targets were repressed by hCOs cultured in MM
(Fig. 23C, only 8 targets were up-regulated (for a list of downregulated gene names see Fig. 24a). The YAP/TAZ targets repressed in hCOs cultured in MM included the well-characterized YAP/TAZ targets CTGF, CYR61, ANKRD1, and AXLA (Augeri et al., 2012).
The top GO term for the repressed YAP/TAZ targets was "regulation of cell cycle" (10 genes, p= 9.7 x 10-4), thereby indicating that loss of YAP/TAZ transcriptional activity could by partially responsible for the cell cycle arrest observed in hCO cultured in MM.
We also tested whether co-activation of β-catenin and YAP-1 was sufficient to drive cardiomyocyte cell cycle re-entry in MM-cultured hCOs. We found that overexpression of either constitutively active YAP-1 (SI 27 A) or constitutively active β-catenin (ΔΝ90) alone was insufficient to facilitate cell cycle re-entry in hCO culture in MM (Fig. 23d,e,f) and failed the activate BIRC5 transcription (Fig. 23g), which has been shown to be dependent on a β- catenin and YAP-1 complex40. However, overexpression of both β-catenin (ΔΝ90) and YAP-1 (SI 27 A) were synergistic in combination, together they augmented proliferation (Fig. 23h,i) and mitosis (Fig. 23j) of cardiomyocytes, and also activated BIRC5 transcription (Fig. 23k) in the hCOs cultured in MM. This proliferative response did not alter the force of contraction (Fig. 24b). In order to confirm these findings with an independent method, we co-activated both signaling pathways using a small molecule, compound 6.2845 (Fig. 24c). Compound 6.28 inhibits both GSK3A/B (IC50 13 and 16 nM, respectively) and MST1 (12 nM) (Fig. 24d), which are critical in inhibiting β-catenin (Clevers et al., 2006) and YAP (Xu et al., 2013), respectively.
Compound 6.28 induced concentration dependent increases in cardiomyocyte proliferation (Fig. 231, m) and also induced mitosis (Fig. 23n) and activation of BIRC5 (Fig. 23 o) in hCOs cultured in MM. Additionally, a single intramyocardial injection (1.7 mg/kg) of Compound 6.28 was sufficient to induce cell cycle re-entry of adult mouse cardiomyocytes in vivo (Fig. 24p). Using 5-bromo-2-deoxyuridine (BrdU) in a pulse-chase experiment, we detected BrdU positive cardiomyocytes within treated hearts up to 7 days post-injection, indicative of cardiomyocytes that had re-entered the cell cycle. Relative to DMSO control treated hearts, BrdU+ cardiomyocytes were 2.7-fold higher in Compound 6.28 treated hearts (Fig. 23p). Together these results demonstrate that both β-catenin and YAP-1 act synergistically to drive proliferation in mature cardiomyocytes, and that hCO can be used as an in vitro model system to predict in vivo biology.
We next assessed the direct role of β-catenin and YAPl in proliferation in vivo. Delivery of β-catenin and YAPl was also sufficient to induce proliferation in neonatal mouse hearts in vivo (Fig. 25a), resulting in hearts with smaller cardiomyocytes (Fig. 25b), but preserved heart size (Fig. 25c). Together indicating that β-catenin and YAPl drive proliferation in vivo.
Finally, we tested whether delivery of β-catenin and YAPl could have therapeutic effects when delivered to adult hearts. Delivery of β-catenin and YAPl resulted in increased expression of Birc5 in purified adult cardiomyocytes (Fig. 26a). This indicates that β-catenin and YAPl may control the expression of the key cell cycle genes in vivo. We therefore assessed whether β-catenin and YAPl could have therapeutic effects when delivered to an adult mouse MI model. Delivery of β- catenin and YAPl had a therapeutic effect when delivered in vivo at the time of MI and resulted in increased ejection fraction following MI (Fig. 26b). To determine whether these therapeutic effects were due to β-catenin or YAP1, we administered either β-catenin or YAPl individually via direct injection into the ischemic border zone at the time of MI (Fig. 27a). β-catenin overexpression mediated a significant improvement in cardiac function (ejection fraction and fractional shortening) following MI, which was evident as early as 6 days post-MI and persisted until day 28 (Fig. 27b&c). The degree of functional improvement seen with β-catenin over-expression was similar to that obtained by over-expression of YAPl alone (Fig. 27b&c). Importantly, β-catenin over-expression resulted in a significant reduction in cardiac fibrosis (Fig. 27d&e). β-catenin overexpression did not significantly affect cardiomyocyte size (Fig. 27f). In contrast to the modest increase in cardiomyocyte proliferation (DNA synthesis) induced by over-expression of YAPl, β-catenin over-expression alone was not sufficient to promote myocyte proliferation in the adult heart (Fig. 27g&h).
DISCUSSION
The key upstream drivers of cardiomyocyte maturation and cell cycle arrest are largely unknown.
Our findings show that switching cardiomyocyte metabolism to fatty acid oxidation and in vivo maturation of cardiomyocytes induces long-lasting changes in β-catenin and YAP signaling. Importantly, simultaneous activation of both of these signaling pathways was required to overcome the metabolism and maturation- induced proliferative barrier in mature hCO and in adult mice in vivo. Cooperativity between β-catenin and YAP signaling has also been reported in the embryonic heart where they interact to control cardiomyocyte proliferation during heart development (Heallen et al., 2011). Moreover, Hippo/Yap signaling has emerged as a central regulator of cardiac regenerative capacity in the neonatal period (Xin et al., 2013; Morikawa et al., 205; Wang et al., 2015). Our study suggests that postnatal alterations in cardiomyocyte metabolism could operate as a key switch leading to cardiomyocyte cell cycle shut down via repression of β-catenin and YAP signaling after birth. Similarly, alterations in metabolism are known to influence β-catenin and YAP activity and vice versa (Chocarro-Calvo et al., 2013; Pate et al., 2014) in other cell types. Therefore, these findings support a model whereby β-catenin, YAPl and metabolism are intimately linked and cooperate to regulate the cardiac cell cycle. Throughout the specification, the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Various changes and modifications may be made to the embodiments described and illustrated without departing from the present invention.
The disclosure of each patent and scientific document, computer program and algorithm referred to in this specification is incorporated by reference in its entirety.
Table 1: qPCR primers
Figure imgf000061_0001
Table 2: Antibodies
Antibody Species Samples Company Cat No Dilution
CD90-APC Rat lgG2c FACS mouse RNA-seq ThermoFisher A14727 1 M9
CD45-FITC Rat lgG2 FACS mouse RNA-seq Miltenyi Biotec 130-102- 1 M9
778
CD31-BV421 Rat lgG2a FACS mouse RNA-seq BioLegend 102423 1 μα
Podoplanin- Syrian FACS mouse RNA-seq BioLegned 127411 1 M9 PE/Cy7 Hamster IgG
a-actinin (clone Mouse IgG! Rodent and human Sigma A781 1 1 :1000 EA-53) immunostaining and imaging Immunostaining and imaging
Troponin T, Mouse IgG Rodent immunostaining and Thermo MS-295- 1 :100 Cardiac Isoform imaging Scientific P0 Immunostaining and Ab-1 imaging
Rabbit IgG Rodent and human 1 :400 immunostaining and imaging Cell Signaling Immunostaining and
Ki-67 (D3B5) 9129
Technology imaging
Anti-phospho- Rabbit Rodent and human Millipore 06-570 1 :200 Histone H3 Polyclonal immunostaining and imaging Immunostaining and (SeM O) imaging
Aurora B Rabbit Rodent and human Sigma A5102 1 :50 Immunostaining
Polyclonal immunostaining and imaging and imaging
Titin Mouse IgM Immunostaining Developmental 9D10 5 μg mL studies
hybridoma bank
BrdU Mouse IgG! Immunostaining Developmental G3G4 1 :100 studies
hybridoma bank
LC2v Rabbit IgG Immunostaining Protein Tech 10906-1- 1 :100
Group AP
β-catenin (total) Rabbit Rodent and human Cell Signaling 9562 1 :200
Polyclonal immunostaining and imaging Technology Immunostaining and imaging β-catenin (PY489) Mouse IgM Rodent and human Developmental PY489-B- 5 pg/ml immunostaining and imaging studies catenin
hybridoma bank
YAP Antibody Rabbit Human immunostaining and Cell Signaling 4912 1 :200
Polyclonal imaging Technology Immunostaining and imaging
Human Mouse lgG2a Human FACS RnD Systems MAB2067 1 pg/ml FACS CD90/Thy1 Ab
(Clone Thy-1A1)
Alexa Fluor® 488 NA NA Life A-11034 1 :400 goat anti-Rabbit Technologies Immunostaining and IgG (H+L) imaging
1 :400 Flow cytometry
Alexa Fluor® 488 NA NA Life A-11029 1 :400 Goat anti-Mouse Technologies Immunostaining and IgG (H+L) imaging
Alexa Fluor® 488 NA NA Life A-21042 1 :400 Goat Anti-Mouse Technologies Immunostaining and IgM (μ chain) imaging
Alexa Fluor® 555 NA NA Life A-21428 1 :400 goat anti-Rabbit Technologies Immunostaining and IgG (H+L) imaging
Alexa Fluor® 555 NA NA Life A-21422 1 :400 Goat Anti-Mouse Technologies Immunostaining and IgG (H+L) imaging
Alexa Fluor® 633 NA NA Life A-21070 1 :400
Figure imgf000063_0001
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Claims

1. A method of preventing or treating a disease or condition of the heart of a mammal, said method including the step facilitating nuclear expression of a constitutively active β-catenin protein and, optionally, modulating one or more genetic elements in one or more cardiomyocytes of the heart to thereby promote or enhance cardiomyocyte regeneration.
2. A method of regenerating cardiomyocytes, said method including the step facilitating nuclear expression of a constitutively active β-catenin protein and, optionally, modulating one or more genetic elements in one or more cardiomyocytes of the heart of a mammal to thereby promote or enhance cardiomyocyte regeneration.
3. The method of Claim 1 or Claim 2, which includes delivering one or more genetic constructs encoding the constitutively active β-catenin protein and, optionally, one or more genetic elements to one or more cardiomyocytes of the heart to thereby promote or enhance cardiomyocyte regeneration in the heart.
4. The method of Claim 1 or Claim 2, which includes delivering one or more cardiomyocytes expressing the constitutively active β-catenin protein and, optionally the one or more genetic elements to the heart to thereby promote or enhance regeneration of the cardiomyocytes in the mammalian heart.
5. The method of Claim 4, wherein the one or more cardiomyocytes are autologous or immunologically matched cardiomyocytes.
6. The method of Claim 4 or Claim 5, which includes the step of expanding the cardiomyocytes before delivery to the heart.
7. The method of any preceding claim, wherein regeneration includes cardiomyocyte proliferation.
8. The method of any preceding claim, wherein the mammal is a human.
9. A genetic construct for delivery to one or more cardiomyocytes, said genetic construct comprising: (i) a nucleotide sequence that encodes a constitutively active β-catenin protein; optionally (ii) a nucleotide sequence that encodes one or more genetic elements; or optionally (iii) a nucleotide sequence that encodes a constitutively active β-catenin protein and one or more genetic elements.
10. The method of any one of Claims 1-8 or the genetic construct of Claim 9, wherein the constitutively active β-catenin protein comprises a substitution or deletion of at least one of Ser33, Ser37 Thr4i and Ser45 in SEQ ID NO: l .
1 1 . The method of any one of Claims 1-8 or 10 or the genetic construct of Claim 9 or Claim 10, wherein the one or more genetic elements include: DNA methyltransferases (DNMTs), histone acetyl transferases (HATs), histone deacteylases (HDACs), HMTs, histone methyltransferases (HMTs), KDMs, histone lysine demethylases (KDMs), eRNAs, , enhancer RNAs (eRNAs), IncRNAs, long non-coding RNAs (RNAs), microRNAs (miRNAs), polycomb repressive complexes (PRC), TETs, ten-eleven translocation (TETs) and/or TFs, transcription regulators
(TRs).
12. The method or the genetic construct of Claim 1 1, wherein the one or more genetic elements include a YAP-1 protein.
13. The method of Claim 12, wherein YAP-1 protein is activated by pharmacologic inhibition of MST1.
14. The method or genetic construct of Claim 12, wherein the YAP-1 protein is constitutively active.
15. The method or the genetic construct of Claim 14, wherein the constitutively active YAP-1 protein comprises a substitution or deletion of Serine 127 in SEQ ID NO:2.
16. The genetic construct of any one of Claims 9-12, 14 or 15 which can be selectively targeted to cardiomyocytes.
17. A cardiomyocyte comprising the genetic construct of any one of Claims 9-12 or 14-16.
18. The cardiomyocyte of Claim 15, which comprises the constitutively active β-catenin protein and one or more genetic elements located in the nucleus of the cardiomyocyte.
19. A composition comprising the genetic construct of any one of Claims 9- 12 or 14-16, and/or one or more cardiomyocytes of Claim 17 or Claim 18, together with a pharmaceutically acceptable carrier, diluent or excipient.
20. A method of identifying, screening or producing a molecule capable of inducing or facilitating cardiomyocyte regeneration, said method including the step of identifying a molecule that mimics or facilitates β-catenin-mediated gene expression in a cardiomyocyte and thereby induces cardiomyocyte regeneration.
21. The method of Claim 20, wherein the molecule facilitates nuclear localization of an endogenous β-catenin protein in a cardiomyocyte.
22. The method of Claim 20 or Claim 21, wherein the molecule facilitates nuclear localization of an expressed, constitutively active β-catenin protein in a cardiomyocyte.
23. The method of Claim 20, wherein the molecule facilitates targeting of a constitutively active β-catenin protein to a cardiomyocyte.
24. The method of any one of Claims 20-23, wherein the molecule is, or mimics the action of, one or more genetic elements selected from: DNA methyltransferases (DNMTs), histone acetyl transferases (HATs), histone deacteylases (HDACs), HMTs, histone methyltransferases (HMTs), KDMs, histone lysine demethylases (KDMs), eRNAs, enhancer RNAs (eRNAs), IncRNAs, long non-coding RNAs (RNAs), microRNAs (miRNAs), polycomb repressive complexes
(PRC), TETs, ten-eleven translocation (TETs) and/or TFs, transcription regulators (TRs).
25. The method of Claim 24, wherein the molecule is YAP-1 or mimics the action of YAP-1.
26. A molecule identified, screened or produced by the method of any one of
Claims 20-25.
27. The molecule of Claim 26, for use in preventing or treating a disease or condition of the heart, by promoting or enhancing cardiomyocyte regeneration in a mammal, preferably a human.
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