WO2023230464A2

WO2023230464A2 - A murine model to test the effect of pkp2 mutations on living adult hearts and uses thereof

Info

Publication number: WO2023230464A2
Application number: PCT/US2023/067343
Authority: WO
Inventors: Mario Delmar; Marina CERRONE
Original assignee: New York University
Priority date: 2022-05-23
Filing date: 2023-05-23
Publication date: 2023-11-30
Also published as: WO2023230464A3

Abstract

The present disclosure provides a murine model for arrhythmogenic cardiomyopathy, such as arrhythmogenic right ventricular cardiomyopathy (ARVC) Further provided are related cells, nucleotides, vectors and kits. Methods for identifying an agent capable of treating or preventing an arrhythmogenic cardiomyopathy, such as arrhythmogenic right ventricular cardiomyopathy (ARVC) using the murine model are also described.

Description

A MURINE MODEL TO TEST THE EFFECT OF PKP2 MUTATIONS ON LIVING ADULT HEARTS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of Provisional U.S. Application No.63/344,776, filed May 23, 2022, the contents of which is herein incorporated by reference in its entirety for all purposes. SEQUENCE LISTING [0002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 19, 2023, is named 243735_000275_SL.xml and is 12,754 bytes in size. FIELD OF THE DISCLOSURE [0003] The present disclosure describes a murine model for arrhythmogenic cardiomyopathy, such as arrhythmogenic right ventricular cardiomyopathy (ARVC). Further provided are related cells, nucleotides, vectors and kits. Methods for identifying an agent capable of treating or preventing an arrhythmogenic cardiomyopathy such as ARVC using the murine model are also described. BACKGROUND [0004] Plakophilin-2 (PKP2) is a protein of the desmosome, an intercellular adhesion structure¹. Mutations in PKP2 are associated with gene-positive arrhythmogenic right ventricular cardiomyopathy (ARVC), a disease mainly electrical, structural or both depending on stage of disease progression². Catecholaminergic sudden cardiac arrest is common in the subclinical (“concealed”) phase^{1, 2}. Current ARVC therapy is limited by the lack of effective antiarrhythmic drugs³, and by lack of effective treatments to arrest/delay the progression of the structural disease. Progress in gene delivery methods offers a toolbox to study disease mechanisms and forward therapy. Early reports indicate that gene therapy in humans may arrest progression of otherwise-deadly cardiac disease such as Danon disease, an X-linked dominant mutisystemic disorder that affects cardiac muscle. Pre-clinical evidence in experimental animal models remains necessary prior to implementation of gene therapy for ARVC. SUMMARY OF THE DISCLOSURE [0005] As specified in the Background section above, there is a great need in the art for experimental animal models, for example, transgenic mice comprising cells, e.g., cardiomyocytes, harboring a mutated PKP2 in the absence of any native PKP2 expression, to inform successful design and implementation of gene therapy for ARVC. The present application addresses these and other needs. [0006] In one aspect, provided herein is a transgenic mouse comprising cells that (i) comprise an endogenous plakophilin-2 (PKP2) gene, or a functional portion thereof, flanked by a pair of loxP sequences; (ii) express a ligand-inducible CRE recombinase under control of a promoter; and (iii) comprise a nucleotide sequence encoding a mutant PKP2 gene. [0007] In some embodiments, exon 2 and exon 3 of the endogenous PKP2 gene is flanked by a pair of loxP sequences. [0008] In some embodiments, the ligand-inducible CRE recombinase is under control of a tissue-specific promoter. [0009] In some embodiments, the ligand-inducible CRE recombinase is under control of a cardiac-specific promoter. [0010] In some embodiments, the cardiac-specific promoter is an α myosin heavy chain promoter or a cardiac troponin T (cTNT) promoter. [0011] In some embodiments, the ligand-inducible CRE recombinase comprises a ligand binding domain of the human estrogen receptor. [0012] In some embodiments, the ligand-inducible CRE recombinase is inducible by tamoxifen. [0013] In some embodiments, induction by tamoxifen results in inactivation of the endogenous PKP2 gene. [0014] In some embodiments, the cells are cardiomyocytes. [0015] In some embodiments, the mutant PKP2 gene is associated with an arrhythmogenic cardiomyopathy. In certain embodiments, arrhythmogenic cardiomyopathy, may comprise, e.g., arrhythmogenic right ventricular cardiomyopathy (ARVC), arrhythmogenic right ventricular dysplasia (ARVD) and/or arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D). [0016] In some embodiments, the mutant PKP2 gene is a human PKP2 gene. [0017] In some embodiments, the mutant PKP2 gene is a truncation mutant. [0018] In some embodiments, the truncation mutant is Arg79X, Gln378X, Arg413X, Arg651X, or Arg735X. [0019] In some embodiments, the mutant PKP2 gene is operably linked to one or more regulatory sequences that can mediate expression of a mutant PKP2 protein in the cells. [0020] In some embodiments, the nucleotide sequence of the mutant PKP2 gene is present in a vector. [0021] In some embodiments, the vector is a viral vector. [0022] In some embodiments, the viral vector is an AAV vector. [0023] In some embodiments, the mutant PKP2 gene is integrated into a chromosome within the cell. [0024] In another aspect, provided herein is a cardiomyocyte isolated from the above- described transgenic mouse. [0025] In another aspect, provided herein is a recombinant vector comprising a mutant PKP2 gene. [0026] In some embodiments, the mutant PKP2 gene is a truncation mutant. [0027] In some embodiments, the truncation mutant is Arg79X, Gln378X, Arg413X, Arg651X, or Arg735X. [0028] In some embodiments, the vector is a viral vector. [0029] In some embodiments, the vector is an AAV vector. [0030] In some embodiments, the AAV vector has tropism for the heart. [0031] In some embodiments, the AAV is AAV9. [0032] In some embodiments, the mutant PKP2 gene is operably linked to one or more regulatory sequences which can mediate expression of a mutant PKP2 protein in a cell. [0033] In some embodiments, the cell is a cardiomyocyte. [0034] In another aspect, provided herein is a method of identifying an agent capable of treating or preventing an arrhythmogenic cardiomyopathy, comprising: a) administering to the above-described transgenic mouse an effective amount of tamoxifen to induce inactivation of the endogenous PKP2 gene; b) administering an agent to the transgenic mouse; c) measuring a cardiac function associated with the arrhythmogenic cardiomyopathy in the transgenic mouse after administration of the agent and comparing the cardiac function with that in a control transgenic mouse which has been administered tamoxifen to induce inactivation of the endogenous PKP2 gene but has not been administered the agent; and d) determining that i) the agent is capable of treating or preventing the arrhythmogenic cardiomyopathy if the cardiac function is improved as compared to the cardiac function in the control mouse; or ii) the agent is not capable of treating or preventing the arrhythmogenic cardiomyopathy if the cardiac function is the same or decreased as compared to the cardiac function in the control mouse. [0035] In some embodiments, step (b) is carried out after step (a). [0036] In some embodiments, step (b) is carried out between about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, or about 7 weeks or more, after step (a). In some embodiments, step (b) is carried out between about 1 week to about 9 weeks after step (a). In some embodiments, step (b) is carried out between about 2 week to about 8 weeks after step (a). In some embodiments, step (b) is carried out between about 3 week to about 7 weeks after step (a). In some embodiments, step (b) is carried out between about 4 week to about 6 weeks after step (a). [0037] In some embodiments, step (b) is carried out between about 3 weeks to about 7 weeks after step (a). [0038] In some embodiments, step (b) is carried out at the same time as step (a). [0039] In some embodiments, the cardiac function is measured by echocardiogram, electrocardiogram, determining arrhythmia burden, measuring fibrosis, determining intracellular calcium dynamics, or any combination thereof. [0040] In some embodiments, the arrhythmogenic cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy (ARVC). [0041] In some embodiments, the agent comprises a polynucleotide molecule encoding a full-length wild-type PKP2 protein. [0042] In some embodiments, the agent comprises a polynucleotide encoding a functional fragment of a wild-type PKP2 protein. [0043] In some embodiments, the functional fragment of the wild-type PKP2 protein comprises an N-terminal domain of PKP2, a C-terminal domain of PKP2, or both. [0044] In some embodiments, the functional fragment of the wild-type PKP2 protein does not comprise one or more armadillo repeats of wild-type PKP2. [0045] In some embodiments, the agent is a small molecule, an antibody, an siRNA, an shRNA, an antisense oligonucleotide, or a site-specific nuclease. [0046] In another aspect, provided herein is an agent capable of treating or preventing arrhythmogenic cardiomyopathy identified by any of the above-described methods. [0047] In another aspect, provided herein is a transgenic mouse expressing a functional fragment of the wild-type PKP2 protein identified by any of the above-described methods. [0048] In another aspect, provided herein is a cell line expressing a functional fragment of a wild-type PKP2 protein identified by any of the above-described methods. [0049] In another aspect, provided herein is a polynucleotide comprising or consisting of the nucleotide sequence encoding a functional fragment of a wild-type PKP2 protein identified by any of the above-described methods. [0050] In another aspect, provided herein is a recombinant vector expressing a functional fragment of a wild-type PKP2 protein identified by any of the above-described methods. [0051] In some embodiments, the vector is a viral vector. [0052] In some embodiments, the vector is an AAV vector. [0053] In some embodiments, the AAV has tropism for the heart. [0054] In some embodiments, the AAV is AAV9. [0055] In some embodiments, the nucleotide sequence encoding the functional fragment of the wild-type PKP2 protein is operably linked to one or more regulatory sequences for expression of said functional fragment of the wild-type PKP2 protein in a cell. [0056] In another aspect, provided herein is a kit comprising one or more of the above- described recombinant vectors, and optionally packaging and/or instructions for the same. [0057] In another aspect, provided herein is a method of treating an arrhythmogenic cardiomyopathy in a subject in need thereof, comprising administering to the subject the agent identified by the above-described methods, the above-described polynucleotide, and/or the above-described recombinant vector. [0058] In some embodiments, the subject has a mutant PKP2 gene. [0059] In some embodiments, the mutant PKP2 gene is a truncation mutant. [0060] In some embodiments, the truncation mutant is Arg79X, Gln378X, Arg413X, Arg651X, or Arg735X. [0061] In some embodiments, the arrhythmogenic cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy (ARVC). [0062] In some embodiments, the above-described methods may further comprise administering one or more additional agents. [0063] In some embodiments, the one or more additional agents are selected from an antiarrhythmic agent, an RyR stabilizer, a muscle relaxer, and an Cx43-Hs inhibitor. [0064] In some embodiments, the one or more additional agents are selected from flecainide, ent-verticilide, dantrolene, TAT-Gap19 or other GAP19 peptide derivative, RRNYRRNY peptide (SEQ ID NO: 1), RyRHCIp peptide (SEQ ID NO: 2), and analogs or derivatives thereof. [0065] In some embodiments, the one or more additional agents are one or more polynucleotides encoding the GAP19 peptide, the RRNYRRNY peptide (SEQ ID NO: 1), and/or the RyRHCIp peptide (SEQ ID NO: 2). [0066] In some embodiments, the one or more additional agents are one or more recombinant vectors encoding the GAP19 peptide, the RRNYRRNY peptide (SEQ ID NO: 1), and/or the RyRHCIp peptide (SEQ ID NO: 2). [0067] In some embodiments, the one or more additional agents are one or more AAV vectors encoding the GAP19 peptide, the RRNYRRNY peptide (SEQ ID NO: 1), and/or the RyRHCIp peptide (SEQ ID NO: 2). BRIEF DESCRIPTION OF THE DRAWINGS [0068] Figure 1 shows implementation of adeno-associated virus (AAV)-mediated gene delivery in Plakophilin-2 (PKP2) knockout (PKP2cKO) mice. Immunofluorescence image of right ventricle (RV) and septum of a mouse heart infected with AAV-vehicle as a control (left panel). RV and septum of a mouse heart 4 weeks after infection with AAV-green fluorescent protein (GFP) (middle panel). Magnification of the RV wall of the same heart as shown in the middle panel, depicting high levels of GFP localization obtained through AAV infection. Scale bars are present in each panel. [0069] Figures 2A-2C demonstrate the effect of ent-verticilide in the PKP2cKO mouse at 21 days post injection (dpi). Frequency of Ca²⁺ sparks at baseline and in response to isoproterenol (ISO) (Figure 2A). Leftmost two bars: control myocytes. Middle two bars: PKP2cKO myocytes in baseline and with ISO. Rightmost two bars: ent-verticilide with and without ISO in PKP2cKO myocytes. Significance was assessed by Two-way ANOVA followed by Tukey’s multiple comparison’s test (and corroborated by linear mixed-effects analysis. Ent-verticilide controls ISO-induced arrhythmias in anesthetized PKP2cKO mice (Figure 2B). Data reported as percent of animals studied (n=10). Number inside bars indicate mean ± standard deviation (SD) of ventricular extrasystoles. Cumulative data of ectopic beats in dimethyl sulfoxide (DMSO) and ent-verticilide treated mice (Figure 2C). Data presented as mean ± SD. [0070] Figures 3A-3B show the effect of dantrolene in the PKP2cKO mouse. Dantrolene controls ISO-induced arrhythmias in anesthetized PKP2cKO mice at 21 dpi. Data reported as percent of animals (n=8 and 7 mice) studied (Figure 3A). Number inside the bars indicate mean ± SD of ventricular extrasystoles. Cumulative data of ectopic beats in DMSO and dantrolene treated mice (Figure 3B). Data presented as mean ± SD. [0071] Figure 4 shows the effect of the connexin43 hemichannel (CX43Hs) blocker TAT- Gap19 on intracellular calcium (Ca²⁺i) in PKP2cKO myocytes 14 days post-Tamoxifen administration. Left panels: data without TAT-Gap19. Right panels: data with TAT-Gap19. Magnitude of Ca²⁺ transients (top row). Sarcoplasmic reticulum (SR) load (middle row). Diastolic Ca²⁺ (bottom row). LV: left ventricle myocytes. RV: right ventricle myocytes. N = number of cells tested, shown inside each column. Statistics: Two-way repeated measures analysis of variance (ANOVA)-Bonferroni for Ca²⁺ transients. *p<0.05. I p<0.05 vs. LV. From⁶. [0072] Figures 5A-5C show human PKP2 nucleotide sequence (Accession No. NM_001005242.3) (Figures 5A-5B) and human PKP2 amino acid sequence (Figure 5C). The armadillo repeat region, N-terminal domain, and C-terminal domain are indicated in the amino acid sequence. [0073] Figures 6A-6C show exemplary mutant human PKP2 sequences. Human PKP2 nucleotide sequence (SEQ ID NO: 5) with mutations marked within the sequence (indicated with boxes). The corresponding amino acid sequence (SEQ ID NO: 4) is also shown (Figures 6A-6B). Human PKP2 amino acid sequence (SEQ ID NO: 4) with positions of the mutations shown in boxed text. The positions of the corresponding nucleotides (Arg79x c 235 C>T; Gln378x c.1132C>T; Arg413x c.1237C>T; Arg651x c.1819 C>T; Arg735x c.2071C>T) are also indicated (Figure 6C). DETAILED DESCRIPTION [0074] Mutations in PKP2 are the primary cause of gene-positive ARVC, a disease with high incidence of adrenergically-mediated sudden death in the young². Fatal arrhythmias occur often in the preclinical (“concealed”) phase of the disease; in its natural history, ARVC progresses from a disease of right ventricular predominance into a biventricular cardiomyopathy leading to heart failure and often cardiac transplant^{1, 2, 10}, mostly because of the lack of effective therapies³ able to control arrhythmias and arrest ventricular damage. Patients with PKP2-ARVC carry most frequently heterozygous radical mutations leading to premature interruption of the coding sequence⁹. As of now, few experimental adult mammalian models of desmosomal dysfunction have been developed and none in which specific PKP2 variants are tested. A cardiac-selective, tamoxifen-activated model with complete loss of PKP2 (PKP2cKO) was developed and characterized⁴. This animal model allows use of AAV-mediated gene transfer to study the endophenotype of specific clinically-relevant PKP2 mutants in a background that lacks PKP2. Restoring full-length PKP2 in the knock-out background improves survival and cardiac function, and abolishes arrhythmias⁵. Whether this is the case for a heart expressing a PKP2 mutant is addressed in the present disclosure. Insights from these results provide highly relevant information for the potential of gene therapy in the setting of the clinical disease. [0075] The potential for gene replacement therapy prompts the need to identify the essential PKP2 domains for function. As disclosed herein, a partial construct including only selected domains is able to rescue the phenotype, thereby facilitating gene therapy approaches, i.e., by reducing to a minimum the size of the exogenous gene, and advancing knowledge on the structure-function relation of PKP2, which enables an improved adjudication of the effect of genetic variants. [0076] ARVC life-threatening arrhythmias may be associated with Ca²⁺ _i mishandling. Recent data show that in addition to cell-cell adhesion, PKP2 and its intercalated disc (ID) partners translate information initiated at the cell-cell contact into intracellular signals that modulate Ca²⁺i^{4, 6}. The relationship between adrenergic input and ARVC converges with studies showing that adrenergic stimulation leads to increased [Ca²⁺]_i and increased amplitude of Ca²⁺i transients and with experimental studies indicating that PKP2 deficiency increases basal [Ca²⁺]_i and the propensity of Ryanodine Receptor 2 (RyR2) channels to release Ca²⁺ into the intracellular space^{4, 6}. It was demonstrated that flecainide can abolish arrhythmias in the PKP2cKO mouse⁴. In certain aspects of the present disclosure, it will be desirable to measure intracellular calcium dynamics to investigate, as an example, calcium dysregulation, e.g., in isolated myocytes disclosed herein. Intracellular calcium dynamics may be measured in accordance with conventional methodology by those of skill in the art. The experiments disclosed herein demonstrate that agents with a more selective RyR2-blocking effect than flecainide and without sodium channel blocking properties, such as ent-verticilide, can be effective for ARVC treatment. Such experiments offer the opportunity of testing the effect of these potentially new drugs in the setting of a clinically relevant PKP2 mutant in an adult mammalian heart. [0077] Cx43-Hs provides a linkage in the PKP2-Ca²⁺i axis. Cx43-Hs may be the conduit by which Ca²⁺ moves into the intracellular space, acting as an initial event that leads to Ca²⁺i dysregulation, culminating in triggered activity and life-threatening ventricular arrhythmias. Cx43Hs do not share a pharmacological effect with gap junctions¹². Peptides that block Cx43-Hs do not block gap junction conductance. Also, cardiac Cx43-Hs are mostly in the closed state (with increased probability to open when Ca²⁺ _i is in the 250-500 nM range) and are more likely to open under pathological conditions ¹³. As such, blockers of Cx43Hs would not affect normal gap junction-mediated propagation, but would prevent the Ca²⁺i imbalance resulting from their increased activity in the pathological state^{12, 13}. Overall, understanding the relationship between PKP2 integrity and Ca²⁺i homeostasis at a time preceding the structural disease is paramount to understand the disease mechanisms and, in turn, preventing the occurrence of sudden death in ARVC patients. It is within this framework that the role of Cx43-Hs in the setting of PKP2 deficiency and of selected PKP2 mutants is disclosed herein. [0078] The present disclosure describes gene therapy approaches for patients with ARVC resulting from PKP2 mutations. The experiments of the present disclosure, in particular, take advantage of an experimental animal model developed and characterized by the inventors and modifies its phenotype by identifying, at the same time, the minimum gene therapeutic approach. Definitions [0079] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments. [0080] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. [0081] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. [0082] The following terms, unless otherwise indicated, shall be understood to have the following meanings: [0083] The terms “polypeptide” and “protein” used interchangeably herein encompass native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. A polypeptide or protein may be monomeric or polymeric. [0084] The term “fragment” or “portion” in regard to polypeptides refers to a polypeptide that has an amino-terminal, carboxy-terminal, and/or internal deletion, but where the remaining amino acid sequence is substantially identical to the corresponding positions in the full-length naturally-occurring sequence. Also, fragments according to the invention may be made by truncation, e.g., by removal of one or more amino acids from the amino- and/or carboxy- terminal ends of a polypeptide. Up to 10, up to 20, up to 30, up to 40 or more amino acids may be removed from the N and/or C terminal in this way. Fragments may also be generated by one or more internal deletions. In some embodiments, fragments are at least 5, 6, 8, or 10 amino acids long. In other embodiments, the fragments are at least 14, at least 20, at least 50, or at least 70, 80, 90, 100, 150, 200, or 400 amino acids long. [0085] In certain embodiments, amino acid substitutions of a protein or portion thereof are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, and/or (4) confer or modify other physicochemical or functional properties. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the normally- occurring sequence. [0086] A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence. Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al., Nature 354:105 (1991), which are each incorporated herein by reference. [0087] As used herein, the twenty naturally occurring amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2^nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. [0088] The term “polynucleotide” as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms. [0089] The term “oligonucleotide” as used herein includes naturally occurring, and modified nucleotides linked together by naturally occurring and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a polynucleotide subset generally comprising a length of 200 bases or fewer. Preferably oligonucleotides are 10 to 60 bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded, e.g. for primers and probes; although oligonucleotides may be double stranded, e.g. for use in the construction of a gene mutant. Oligonucleotides of the invention can be either sense or antisense oligonucleotides. [0090] “Operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term “expression control sequence” as used herein means polynucleotide sequences that are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such expression control sequences differs depending upon the host organism; in prokaryotes, such expression control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such expression control sequences include promoters and transcription termination sequence. The term “expression control sequences” is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. [0091] The term “vector”, as used herein, means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some embodiments, the vector is a plasmid, i.e., a circular double stranded DNA loop into which additional DNA segments may be ligated. In some embodiments, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In other embodiments, the vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). [0092] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. As used herein, the term “regulatory sequence” means a nucleic acid sequence which can regulate expression of a gene product operably linked to the regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter or regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner. [0093] A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell. [0094] An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell. [0095] A reference to a nucleotide sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. [0096] The term “substantial similarity” or “substantial sequence similarity,” when referring to a nucleic acid or fragment thereof, means that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 85%, preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well- known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above. [0097] As applied to polypeptides, the term “substantial identity” or “substantially identical” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, as supplied with the programs, share at least 70%, 75%, 80% or 85% sequence identity, preferably at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or 99% sequence identity. In certain embodiments, residue positions that are not identical differ by conservative amino acid substitutions. [0098] A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson, Methods Mol. Biol.243:307-31 (1994). Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Conservative amino acids substitution groups are: valine-leucine- isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. [0099] Alternatively, a conservative substitution or replacement, as the terms are used interchangeably herein, is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., Science 256:1443-45 (1992), herein incorporated by reference. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix. [00100] The phrase “effective amount” or “therapeutically effective amount” as used herein refers to an amount necessary (at dosages and for periods of time and for the means of administration) to achieve the desired therapeutic result. An effective amount is at least the minimal amount, but less than a toxic amount, of an active agent which is necessary to impart therapeutic benefit to a subject. [00101] The phrase “pharmaceutically acceptable”, as used in connection with compositions described herein, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human). Preferably, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. [00102] As used herein, “pharmaceutically acceptable carrier” or “pharmaceutical acceptable excipient” includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000). [00103] The term “treating”, as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, delaying the progression of, delaying the onset of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term “treatment”, as used herein, unless otherwise indicated, refers to the act of treating as “treating” is defined immediately above. The term “treating” also includes adjuvant and neo-adjuvant treatment of a subject. For the avoidance of doubt, reference herein to “treatment” includes reference to curative, palliative and prophylactic treatment. [00104] The terms “patient”, “individual”, “subject”, and “animal” are used interchangeably herein and refer to mammals, including, without limitation, human and veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models. In a preferred embodiment, the subject is a human. Transgenic Mice [00105] In one aspect, the present disclosure provides a transgenic mouse comprising cells that (i) comprise an endogenous plakophilin-2 (PKP2) gene, or a functional portion thereof, flanked by a pair of loxP sequences; (ii) express a ligand-inducible CRE recombinase under control of a promoter; and (iii) comprise a nucleotide sequence encoding a mutant PKP2 gene. In some embodiments, exon 2 and exon 3 of the endogenous PKP2 gene is flanked by a pair of loxP sequences. In some embodiments, the ligand-inducible CRE recombinase is under control of a tissue-specific promoter. [00106] In some embodiments, the transgenic mouse described herein may comprise cells that may comprise elements of a CRE-LoxP system. The CRE-loxP system is a site-directed recombination reaction system where, e.g., bacteriophage P1 CRE recombinase acts on a specific DNA sequence called a loxP sequence. CRE recombinase is a site-directed DNA recombination enzyme, which can be a member of a phage λ integrase family. A loxP sequence can include 34 bp, namely inverted repeats (CRE recombinase binding domain) of 13 bp on both ends and a region of 8 bp located therebetween. Concerning the mechanism of the Cre- loxP system, when the two loxP sequences are located extending in the same direction, CRE recombinase can recognize the loxP sequences, and the sequence located between the loxP sequences can be cut out, or excised. When the two loxP sequences are located extending in the opposite directions, CRE recombinase can recognize the loxP sequences, and the sequence located between the LoxP sequences can be inverted with respect to the sequences located outside the loxP sequences. That is, CRE recombinase is an enzyme that recognizes the loxP sequences, and when the two loxP sequences are located extending in the same direction, CRE recombinase can catalyze a reaction of cutting out of the sequence (i.e., excising the sequence) between the loxP sequences, while when the two loxP sequences are located extending in the opposite directions, CRE recombinase can catalyze a reaction of inverting the sequence between the loxP sequences. [00107] In some embodiments, the CRE recombinase described herein may be under the control of a promoter. In some embodiments, the promoter can be a tissue-specific promoter. As an example, the tissue-specific promoter may be a cardiac-specific promoter. In some embodiments, the cardiac-specific promoter is an α myosin heavy chain promoter (αMyHC) or a cardiac troponin T (cTNT) promoter. [00108] In some embodiments, the CRE recombinase described herein may by inducible and can be controlled by, for example, regulatory elements such as, but not limited to, promoters and enhancers. In some embodiments, the CRE recombinase of the present disclosure may be inducible such as by way of an exogenous inducer, e.g., tamoxifen or tetracycline. In some embodiments, a tamoxifen-inducible CRE recombinase may be achieved, e.g., by a CRE recombinase protein that can be fused with an estrogen receptor (ER) which may comprise a ligand binding domain (e.g., a mutated ligand binding domain). [00109] In some embodiments, the CRE recombinase described herein may be a ligand- inducible CRE recombinase comprising a ligand binding domain of an estrogen receptor (ER), e.g., a human estrogen receptor. In some embodiments, the ligand-inducible CRE recombinase is induced by tamoxifen. In some embodiments, the tamoxifen results in inactivation of the endogenous PKP2 gene. [00110] In some embodiments, CRE recombinase-mediated nucleotide (e.g., DNA) editing and promoter-specific CRE expression of the present disclosure may be used to generate a transgenic mouse described herein, e.g., a cardiac-specific, PKP2 knockout (KO) mouse. Without wishing to be bound by theory, for generation of the cardiac-specific, PKP2 knockout (KO) mouse, a C57BL/6 PKP2 fl/fl mouse line can be generated and crossed with the αMyHC- Cre-ER (T2) line. Two forward loxP sites can be designed and introduced into the construct flanking mouse PKP2 exons 2 and 3, with a downstream neomycin selection cassette. The linearized targeting construct can be electroporated into C57B/6 derived embryonic stem (ES) cells and the resultant ES cell clones can be identified. The confirmed positive ES cells can then be injected into isogenic blastocysts, and microinjected into foster mice. The neo cassette can be excised by crossing the F1 heterozygous mice with FRT mice. The PKP2 flox/flox mice were mated to αMyHC-Cre-ER(T2) mice to obtain flox/flox/Cre+ mice which contain the α myosin heavy chain promoter and the ligand binding domain of the human estrogen receptor (ER). The resulting mice (PKP2-cKO) can develop normally without functional or structural deficits. [00111] In some embodiments, the cells described herein are cardiomyocytes. In various embodiments, the present disclosure encompasses a cardiomyocyte which may be isolated from a transgenic mouse described herein. [00112] In some embodiments, the mutant PKP2 gene is associated with an arrhythmogenic cardiomyopathy. [00113] In some embodiments, the mutant PKP2 gene is a human PKP2 gene. [00114] In some embodiments, the mutant PKP2 gene is a truncation mutant. [00115] In some embodiments, the truncation mutant is, for example, without limitation, Arg79X, Gln378X, Arg413X, Arg651X, or Arg735X. [00116] In some embodiments, the mutant PKP2 gene is operably linked to one or more regulatory sequences that can mediate expression of a mutant PKP2 protein in the cells. Examples of regulatory sequences include transcription initiation, promoter, terminator, and enhancer sequences. In some embodiments, the mutant PKP2 gene is operably linked to a promoter. The promoter may be a constitutive promoter or inducible promoter. Vectors [00117] In one aspect, the present disclosure provides a recombinant vector comprising a mutant PKP2 gene. In some embodiments, a nucleotide sequence encoding a mutant PKP2 gene described herein may be present in a vector, e.g. a recombinant vector. In some embodiments, the mutant PKP2 gene can be integrated into a chromosome within a cell described herein. [00118] In some embodiments, the vector is a viral vector. [00119] Suitable viral vectors that can be used in the present disclosure include, but are not limited to, an adenoviral vector, a baculoviral vector, an adeno-associated viral (AAV) vector, a herpes viral vector, a retroviral vector, and/or an adeno-associated viral (AAV) vector. In some embodiments, the viral vector is an adenovirus. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector. In some embodiments the AAV vector is selected from, without limitation, AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5,AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11,AAV type hu11 (AAV hu11), AAV12, AAV13, AAVDJ, AAV retro (AAV retro), Anc80L65, AAVLK03, AAV type rh32.33 (AAVrh.32.33), AAV PHP.S, AAV PHP.B, AAV PHP.eB, AAVrh.64R1, AAVhu.37, AAVrh.8, and AAV2/8,AAV2G9. In some embodiments, the AAV vector is AAV8 vector. [00120] In some embodiments, the AAV vector is AAV9. [00121] In some embodiments, the AAV vector has tropism for the heart. [00122] In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a transposon such as, but are not limited to, a PiggyBac transposon and a sleeping beauty transposon. [00123] In some embodiments, the vector is a plasmid. [00124] In some embodiments, when a recombinant vector described herein comprises a mutant PKP2 gene, the mutant PKP2 gene is a truncation mutation. Non-limiting examples of truncation mutations are Arg79X, Gln378X, Arg413X, Arg651X, and Arg735X. In some embodiments, the mutant PKP2 gene can be operably linked to one or more regulatory sequences described herein which can mediate expression of a mutant PKP2 protein in a cell such as, but not limited to, a cardiomyocyte. Pharmaceutical Compositions [00125] Any of various agents, polynucleotides, and/ or recombinant vectors described herein can be present in a pharmaceutical composition (such as a formulation) that can include other agents, excipients, or stabilizers. In various embodiments, a pharmaceutical composition described herein may comprise (i) an agent described herein, (ii) a polynucleotide described herein, and/or (iii) a recombinant vector described herein, and a pharmaceutically acceptable carrier or adjuvant. [00126] It is understood that the compounds of the present disclosure can be present in one or more stereoisomers (e.g., diastereomers). The disclosure includes, within its scope, all of these stereoisomers, either isolated (e.g., in enantiomeric isolation) or in combination (including racemic and diastereomeric mixtures). The present disclosure uses amino acids independently selected from L and D forms (e.g., the peptide may contain two serine residues, each serine residue having the same or opposite absolute stereochemistry), etc., are intended for the use of both L- and D-form amino acids. [00127] Accordingly, the compounds of the present disclosure also include substantially pure stereoisomeric form of the specific compound with respect to the asymmetric center of the amino acid residue, for example about 90% de, such as greater than about 95% to 97% de, or 99% de. For larger compounds, as well as mixtures thereof (such as racemic mixtures). Such diastereomers may be prepared, for example, by asymmetric synthesis using chiral intermediates, or the mixture may be divided by conventional methods, such as chromatography or the use of dividing agents. [00128] If the compounds of the disclosure require purification, chromatographic techniques such as high-performance liquid chromatography (HPLC) and reverse phase HPLC can be used. Peptides may be characterized by mass spectrometry and/or other suitable methods. [00129] If the compound contains one or more functional groups that can be protonated or deprotonated (e.g., at physiological pH), the compound can be prepared and / or isolated as a pharmaceutically acceptable salt. It will be appreciated that the compound can be zwitterion at a given pH. As used herein, the expression "pharmaceutically acceptable salt" refers to a salt of a given compound, which salt is suitable for pharmaceutical administration. Such salts can be formed, for example, by reacting an acid or base with an amine or carboxylic acid group, respectively. [00130] Pharmaceutically acceptable acid addition salts can be prepared from inorganic and organic acids. Examples of inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like. Examples of organic acids include acetic acid, propionic acid, glycolic acid, pyruvate, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartrate acid, citrate, benzoic acid, cinnamic acid, mandelic acid, Examples thereof include methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid and salicylic acid. [00131] Pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. Corresponding counterions derived from inorganic bases include salts of sodium, potassium, lithium, ammonium, calcium and magnesium. Organic bases include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, prokine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, Substituted amines such as primary, secondary and tertiary amines such as N-alkylglucamine, theobromine, purines, piperazine, piperazine and N-ethylpiperidine, substituted amines such as natural substituted amines and cyclic amines can be mentioned. [00132] Acid/base addition salts tend to be more soluble in aqueous solvents than the corresponding free acid/base forms. [00133] In some embodiments, it is envisioned that two or more combinations of the compounds of the disclosure will be administered to the subject. It is believed that the compound (s) may also be administered in combination with one or more additional therapeutic agents. This combination can allow separate, continuous or simultaneous administration with the other active ingredients of the above compounds. This combination may be provided in the form of a pharmaceutical composition. [00134] As used herein, the term "combination" is used by the combination agents as defined above dependently or independently, or by the use of different fixed combinations with different amounts of combination agents, i.e. simultaneously or at different times. Refers to a kit of compositions or parts that can be administered. The combination agents can then be administered, for example, simultaneously or staggered in time (i.e., at different times and at equal or different time intervals for any part of the kit). The ratio of the total amount of combination agents administered in a combination can vary, e.g., to address the needs of a subpopulation of patients to be treated or the needs of a single patient, and different needs are the age of the patient, it can be due to gender, weight, etc. [00135] The route of administration and the type of pharmaceutically acceptable carrier will depend on the condition being treated and the type of mammal. Formulations containing the active compound may be prepared such that the activity of the compound is not disrupted during the process and the compound can reach its site of action without disruption. In some cases, it may be necessary to protect the compound by means known in the art, such as microencapsulation. Similarly, the route of dosing selected should be such that the compound reaches its site of action. [00136] In some embodiments, the composition further comprises a targeting agent or a carrier that promotes the delivery of the inhibitors of endocytosis to an area affected by the chronic pain. Exemplary carriers include liposomes, micelles, nanodisperse albumin and its modifications, polymer nanoparticles, dendrimers, inorganic nanoparticles of different compositions. [00137] The appropriate formulation for the compound of the disclosure can be adjusted for pH. Buffer systems are routinely used to provide pH values in the desired range and include carboxylic acid buffers such as acetates, citrates, lactates and succinates. In some embodiments, the composition is formulated to have a pH range of about 4.5 to about 9.0, including for example pH ranges of about any of 5.0 to about 8.0, about 6.5 to about 7.5, and about 6.5 to about 7.0. In some embodiments, the pH of the composition is formulated to no less than about 6, including for example no less than about any of 6.5, 7, or 8 (such as about 8). The composition can also be made to be isotonic with blood by the addition of a suitable tonicity modifier, such as glycerol. [00138] The formulation may also include suitable excipients, such as antioxidants. Examples of antioxidants include phenolic compounds such as BHT or Vitamin E, reducing agents such as methionine or sulfites, and metal chelating agents such as EDTA. [00139] The compounds or pharmaceutically acceptable salts thereof described herein can be prepared in parenteral dosage forms such as those suitable for, e.g., intravascular (intravenous or intraarterial), intraperitoneal, intratumoral, intraventricular, intrapleural or intramuscular administration delivery. Suitable pharmaceutical forms for injectable use include sterile injectable or dispersions and sterile powders for the immediate preparation of sterile injectable solutions. They must be stable under manufacturing and storage conditions and protected from reduction or oxidation and the contaminating effects of microorganisms such as bacteria or fungi. [00140] The solvent or dispersion medium for the injectable solution or dispersion may include either conventional solvents or carrier systems for the active compound, e.g., water, ethanol, polyols (e.g., glycerol, propylene glycol and). Liquid polyethylene glycol, etc., suitable mixtures thereof, and vegetable oils may be included. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, the maintenance of the required particle size in the case of dispersions, and the use of surfactants. Prevention of the action of microorganisms can be performed as needed by incorporating various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it may be preferable to include agents that regulate osmotic pressure, such as sugar or sodium chloride. Preferably, the injectable formulation is isotonic with blood. Sustained absorption of the injectable composition can be brought about by the use of agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition. Suitable pharmaceutical forms for injection can be delivered by any suitable route, including intravenous, intramuscular, intracerebral, intrathecal, epidural injection or infusion. [00141] Sterilized injectable solutions are prepared by adding the required amount of the compounds of the disclosure to a suitable solvent containing various other components, such as those listed above, as needed, followed by filtration sterilization. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and other required ingredients from those described above. For sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying or lyophilization of the pre-sterile filtered solution of the active ingredient plus any additional desired ingredients. [00142] Other pharmaceutical forms include the oral and enteral formulations, where the active compound can be formulated with an inert diluent or an assimilated edible carrier, or encapsulated in hard or softshell gelatin capsules. The formulations can also be tableted, or it can be incorporated directly into diet foods. For oral therapeutic administration, the active compound is taken up with excipients and used in the form of ingestible tablets, buccal or sublingual tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. The amount of active compound in such a therapeutically useful composition is such that an appropriate dose can be obtained. [00143] Tablets, lozenges, pills, capsules, etc. may also contain the ingredients listed below: binders such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; corn starch, Disintegrants such as potato starch, arginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose or saccharin, or flavors such as peppermint, winter green oil, or cherry flavor may be added. If the dosage unit form is a capsule, it may contain a liquid carrier in addition to the above types of materials. Various other materials may be present as a coating or in other ways to alter the physical form of the dosage unit. For example, tablets, pills, or capsules can be coated with shellac, sugar, or both. The syrup or elixir may contain active compounds, sucrose as a sweetener, methyl and propylparabens as preservatives, pigments and flavors such as cherry or orange flavors. Of course, any substance used to prepare the dosage unit form must be pharmaceutically pure and substantially non-toxic in the amount used. In addition, the compounds of the disclosure may be incorporated into sustained release formulations and formulations comprising those that specifically deliver the active peptide to a particular region of the intestine. [00144] Liquid formulations can also be administered enterally via the stomach or esophageal canal. The enteral preparation can be prepared in the form of a suppository by mixing with a suitable base such as an emulsifying base or a water-soluble base. It is possible, but not necessary, to administer the compound of the present disclosure topically, intranasally, intravaginally, intraocularly or the like. [00145] Pharmaceutically acceptable vehicles and / or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarders, and the like. The use of such vehicles and agents for pharmaceutically active substances is well known in the art. Its use in therapeutic compositions is intended unless any conventional vehicle or agent is incompatible with the active ingredient. Auxiliary active ingredients can also be incorporated into the composition. [00146] It is particularly advantageous to formulate the composition in unit dosage form for ease of administration and uniformity of dosage. As used herein, a dosage unit form means a physically distinct unit suitable as a unit dosage for a mammalian subject to be treated; each unit is a required pharmaceutically acceptable vehicle. A dosage unit form may contain a predetermined amount of active substance calculated to produce a desired therapeutic effect described herein. Details of the novel dosage unit forms of the disclosure include (a) the unique properties of the active substance and the particular therapeutic effect to be achieved, and (b) physical health as disclosed in detail herein. It is determined by and directly dependent on the technology-specific limitations of the active substances formulated for the treatment of the disease in living subjects with impaired disease states. [00147] As mentioned above, the main active ingredient may be formulated for convenient and effective administration in therapeutically effective amounts using a suitable pharmaceutically acceptable vehicle in the form of a dosage unit. The unit dosage form can contain, for example, the major active compound in an amount ranging from 0.25 μg to about 2000 mg. Expressed in proportion, the active compound may be present in a carrier of about 0.25 μg to about 2000 mg / mL. In the case of a composition containing an auxiliary active ingredient, the dose is determined with reference to the usual dosage and mode of administration of the ingredient. [00148] In some embodiments, the composition is suitable for administration to a human. In some embodiments, the composition is suitable for administration to a mammal such as, in the veterinary context, domestic pets and agricultural animals. There are a wide variety of suitable formulations of the composition comprising the inhibitor of endocytosis. The following formulations and methods are merely exemplary and are in no way limiting. Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice, (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules, (c) suspensions in an appropriate liquid, and (d) suitable emulsions. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art. [00149] Examples of suitable carriers, excipients, and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline solution, syrup, methylcellulose, methyl and propylhydroxybenzoates, talc, magnesium stearate, and mineral oil. In some embodiments, the composition comprising the inhibitor of endocytosis with a carrier as discussed herein is present in a dry formulation (such as lyophilized composition). The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents. [00150] Formulations suitable for parenteral administration include aqueous and non- aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation compatible with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Methods [00151] In one aspect, the present disclosure provides a method of identifying an agent capable of treating or preventing an arrhythmogenic cardiomyopathy, the method comprising a) administering to a transgenic mouse described herein an effective amount of tamoxifen to induce inactivation of the endogenous PKP2 gene; b) administering an agent to the transgenic mouse; c) measuring a cardiac function associated with the arrhythmogenic cardiomyopathy in the transgenic mouse after administration of the agent and comparing the cardiac function with that in a control transgenic mouse which has been administered tamoxifen to induce inactivation of the endogenous PKP2 gene but has not been administered the agent; and d) determining that i) the agent is capable of treating or preventing the arrhythmogenic cardiomyopathy if the cardiac function is improved as compared to the cardiac function in the control mouse; or ii) the agent is not capable of treating or preventing the arrhythmogenic cardiomyopathy if the cardiac function is the same or decreased as compared to the cardiac function in the control mouse. [00152] In some embodiments of the above-described method, step (b) is carried out after step (a). [00153] In some embodiments, step (b) is carried out between about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, or about 7 weeks or more, after step (a). In some embodiments, step (b) is carried out between about 1 week to about 9 weeks after step (a). In some embodiments, step (b) is carried out between about 2 week to about 8 weeks after step (a). In some embodiments, step (b) is carried out between about 3 week to about 7 weeks after step (a). In some embodiments, step (b) is carried out between about 4 week to about 6 weeks after step (a). [00154] In some embodiments, step (b) is carried out between about 3 weeks to about 7 weeks after step (a). In some embodiments, step (b) is carried out at about 3, 4, 5, 6, or 7 weeks, or more, after step (a). [00155] In some embodiments, step (b) is carried out at the same time as step (a). [00156] In some embodiments, the cardiac function is measured by, without limitation, echocardiogram, electrocardiogram, determining arrhythmia burden, measuring fibrosis, determining intracellular calcium dynamics, or any combination thereof. [00157] In some embodiments, the arrhythmogenic cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy (ARVC). [00158] In some embodiments, the agent comprises a polynucleotide molecule encoding a full-length wild-type PKP2 protein. [00159] In some embodiments, the agent comprises a polynucleotide encoding a functional fragment of a wild-type PKP2 protein. [00160] In some embodiments, the functional fragment of the wild-type PKP2 protein comprises an N-terminal domain of PKP2, a C-terminal domain of PKP2, or both. [00161] In some embodiments, the functional fragment of the wild-type PKP2 protein does not comprise one or more armadillo repeats of wild-type PKP2. [00162] In some embodiments, the agent is a small molecule, an antibody, an siRNA, an shRNA, an antisense oligonucleotide, or a site-specific nuclease. In some embodiments, the agent is a microRNA or a gapmer. In some embodiments, the small molecule is a synthetic compound or a plant derived compound. In some embodiments, the site-specific nuclease may comprise a gene editing nuclease such as, but not limited to, Cas protein, zinc finger nuclease (ZFN), and transcription activator-like effector nuclease (TALEN). [00163] In various embodiments, the present disclosure contemplates an agent capable of treating or preventing arrhythmogenic cardiomyopathy identified by any of various methods described herein. The present disclosure further contemplates a transgenic mouse expressing a functional fragment of the wild-type PKP2 protein identified by any of various methods described herein. The present disclosure still further contemplates a cell line expressing a functional fragment of a wild-type PKP2 protein identified by any of various methods described herein. [00164] In various embodiments, the present disclosure encompasses a polynucleotide comprising or consisting of a nucleotide sequence encoding a functional fragment of a wild- type PKP2 protein identified by any of various methods described herein. The present disclosure also encompasses a recombinant vector expressing a functional fragment of a wild- type PKP2 protein identified by any of various methods described herein. The vector can be any of various vectors described herein. In some embodiments, the nucleotide sequence encoding the functional fragment of the wild-type PKP2 protein can operably linked to one or more regulatory sequences described herein for expression of the functional fragment of the wild-type PKP2 protein in a cell. [00165] In various embodiments, the present disclosure provides a kit comprising one or more of the recombinant vectors described herein, and optionally packaging and/or instructions for the same. [00166] In one aspect, the present disclosure provides a method of treating an arrhythmogenic cardiomyopathy in a subject in need thereof, comprising administering to the subject the agent identified by any of various methods described herein, a polynucleotide described herein, and/or a recombinant vector described herein. [00167] In some embodiments, the subject has a mutant PKP2 gene. [00168] In some embodiments, the mutant PKP2 gene is a truncation mutant such as, but not limited to, Arg79X, Gln378X, Arg413X, Arg651X, or Arg735X. [00169] The method of any one of claims 51-54, wherein the arrhythmogenic cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy (ARVC). [00170] In some embodiments, the method of treating may comprise administering one or more additional agents. Non-limiting examples of additional agents are an antiarrhythmic agent, an RyR stabilizer, a muscle relaxer, and an Cx43-Hs inhibitor. [00171] In some embodiments, the one or more additional agents is flecainide, ent- verticilide, dantrolene, TAT-Gap19 or other GAP19 peptide derivative, RRNYRRNY peptide (SEQ ID NO: 1), RyRHCIp peptide (SEQ ID NO: 2), or an analog or derivatives thereof. [00172] In some embodiments, the one or more additional agents are one or more polynucleotides encoding the GAP19 peptide, the RRNYRRNY peptide (SEQ ID NO: 1), and/or the RyRHCIp peptide (SEQ ID NO: 2). [00173] In some embodiments, the one or more additional agents are one or more recombinant vectors encoding the GAP19 peptide, the RRNYRRNY peptide (SEQ ID NO: 1), and/or the RyRHCIp peptide (SEQ ID NO: 2). [00174] In some embodiments, the one or more additional agents are one or more AAV vectors encoding the GAP19 peptide, the RRNYRRNY peptide (SEQ ID NO: 1), and/or the RyRHCIp peptide (SEQ ID NO: 2) EXAMPLES [00175] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments. Example 1. Gene replacement therapy using adenovirus vectors in PKP2cKO mice [00176] A murine model of cardiac specific, Tamoxifen-activated PKP2 knockout (PKP2cKO) developed by the inventors of the present disclosure was used to examine gene replacement. In the context of complete loss of PKP2 expression, a single gene dose injection of an AAV (adeno-associated virus):PKP2 construct prolonged survival, prevented arrhythmias, and modified the course of disease progression in PKP2cKO mice ⁵. Figure 1 shows implementation of AAV-mediated gene delivery in PKP2cKO mice. The coding region of Green Fluorescent Protein (eGFP) was packaged into a commercially available AAV9 which uses CAG, a ubiquitous promoter known to drive strong expression in mammalian cells (Virovek Inc). AAVs were injected into adult mice via the tail vein. Specifically, Figure 1 shows an example of strong GFP expression in the cardiac tissue of the treated mice (n=5 total mice, and n=1 mouse control with empty vector). Expression is detected as early as two weeks after injection and the transgene can be manifest more than three months later. Example 2. Ent-verticilide limits isoproterenol (ISO)-induced premature ventricular complex (PVC) burden and reduces Ca²⁺ release events in isolated ISO-treated PKP2cKO cardiomyocytes [00177] Ca²⁺i dysregulation may be an effective target for arrhythmia therapy in ARVC. Flecainide, a Class IC antiarrhythmic, was tested in the PKP2-cKO mouse model. The results showed that flecainide has a strong antiarrhythmic effect on mice deficient in PKP2⁴. These results converged with anecdotal clinical observations indicating that flecainide can be effective as combination therapy in ARVC^{7, 8}. The cumulative data served as the bases for a clinical trial to examine the possible use of flecainide to treat arrhythmias in patients with ARVC. [00178] Whether ent-verticilide, a selective RyR2 blocker, affects arrhythmogenesis in PKP2-deficient mice was tested in the present Example. The results show that ent- verticilide limited isoproterenol (ISO)-induced PVCs burden and reduced Ca²⁺ release events in isolated ISO-treated PKP2cKO cardiomyocytes (Figure 2). Example 3. Effectiveness of dantrolene in controlling ISO-induced arrhythmias in PKP2-deficient hearts [00179] An alternative for a selective RyR stabilizer currently in clinical practice is dantrolene, which is used as emergency intervention for malignant hyperthermia. Data showing effectiveness of dantrolene in controlling ISO-induced arrhythmias in PKP2- deficient hearts are shown in Figure 3. Experiments were conducted 21 days post Tamoxifen (TAM) injection (21 days post-injection [dpi]). At this stage, this murine model mimics the concealed stage of the disease, with a manifest right ventricular (RV) dilation, preserved left ventricular (LV) structure and function, and increased susceptibility to ISO- induced arrhythmias⁴. Specifically, as shown in Figure 3, a single dose of dantrolene (30 mg/kg, intraperitoneal [i.p.]) delivered 30 minutes before an ISO challenge (3 mg/kg^{4, 19}) significantly dampened the ISO-induced PVC burden. The effect of dantrolene on arrhythmia burden, cardiac function and Ca²⁺-release events is further explored in PKP2cKO mice in Example 7. Example 4. Cx43-Hs inhibitor (TAT-Gap19) protects the cells from excess Ca²⁺ accumulation [00180] Cx43-Hs become functionally relevant to cardiac electrophysiology under a pathological state. Studies recently showed that loss of PKP2 expression leads to increased open probability of Cx43-Hs in cardiac myocytes, a shift in their Ca²⁺ _i dependence and/or phosphorylation state, and a consequent increase in the concentration of Ca²⁺ inside the cells, thus creating a pro-arrhythmic state⁶. The studies herein further demonstrated that a Cx43-Hs inhibitor (TAT-Gap19) protects the cells from excess Ca²⁺ accumulation (Figure 4). [00181] Whether inhibition of Cx43-H activity can reduce arrhythmia burden is further explored in Example 7. The experimental approach includes both pharmacological intervention, as well as AAV-mediated gene delivery. Example 5. Establish the endophenotype of disease-relevant PKP2 truncations [00182] The present Example tests whether a loss of the PKP2 carboxyl terminal (CT) domain leads to an ARVC phenotype comparable to that of the complete loss of the PKP2 gene. Previous experiments have shown that loss of PKP2 expression leads to an ARVC phenotype, which is prevented by exogenous expression of the full-length wild-type PKP2⁵. A limitation of the PKP2cKO model is the fact that therapeutic manipulations are conducted in the setting of a complete loss of PKP2, which is different from what occurs in real disease in humans, which is associated with PKP2 mutations, such as, e.g., PKP2 truncations^{2, 9, 23}. In the present Example, the inventors set out to create a model for clinically-relevant PKP2 mutations. The present new model allows for testing the efficacy of gene replacement and other therapies in the setting of a disease-causing mutation. [00183] The following truncation mutants, known to associate with ARVC in humans, are tested: Arg79X, Gln378X, Arg413X, Arg651X, Arg735X^{2, 9, 23}. Arg79X indicates a stop codon that replaces the arginine residue expected to occupy position 79. Gln378X is the replacement of the glutamine at position 378 for a stop codon, and the same is the case for Arginine 651 and for Arginine 735 (see, e.g., Figures 6A-6C). Constructs are HA-tagged, as in previous studies, to facilitate detection²⁴. Young adult mice (3 months of age) are injected with AAVs carrying transgenes encoding the specific mutant PKP2 proteins. In an initial set of experiments, echocardiograms are obtained at time of AAV injection (time zero) and eight weeks after to determine whether, in the setting of wild-type PKP2 expression, heart dysfunction is detected. These animals are then euthanized and the hearts assessed post-mortem to detect fibrosis (trichrome) as well as expression of the transgene (by detection of the HA epitope). Such post-mortem analysis of the heart, e.g., to measure the extent of fibrosis in the heart, may be used in the practice of the present disclosure. These experiments will allow inform whether excess of a mutant PKP2 leads to a disease phenotype even in the presence of endogenous PKP2 expression. [00184] In a second set of experiments, tamoxifen (TAM) is injected four weeks after expression of the PKP2 mutant transgene. Previous studies have shown that AAV- mediated expression of full-length wild-type PKP2 is sufficient to prevent the ARVC phenotype in PKP2cKO mice⁵. Here, the phenotype that associates with a given PKP2 mutation is tested. In the absence of a transgene, the ARVC phenotype may manifest, e.g., 21 days after TAM injection, reduction of left ventricular ejection fraction (LVEF) may be present, e.g., 28 days post-TAM, and survival may not exceed 50 days in various instances. PKP2 mutant-expressing animals are monitored by weekly echocardiography to determine the timing of functional loss. Clinically-relevant truncations may generate a non-functional protein and, as such, the phenotype observed in these animals may be similar to that of the PKP2cKO. The functional boundaries at which a shorter, CT- truncated PKP2 fails to provide normal function are determined by the experiments disclosed in the present Example. Guided by the time of significant LVEF reduction (using paired comparisons; each animal in a time series serving as its own control), mice are euthanized to determine the extent of fibrosis (trichrome) and the expression of the transgene (HA detection), as well as its subcellular localization. [00185] Cell culture experiments have suggested that loss of the CT end of PKP2 causes a mislocalization of the protein ^{25, 26}, which is no longer found at the junctional site. Instead, at least some of the signal can be detected in the cell nucleus. This observation, however, has not been confirmed in adult mammalian preparations. The experiments in the present Example test for this possibility. Finally, on selected mutations, Kaplan-Meier curves are generated to determine whether survival in PKP2 mutant-expressing animals is the same as in the full knockout (KO). Experiments in isolated cells oriented toward understanding arrhythmia mechanisms and antiarrhythmic therapy are further detailed in the below-described Examples. [00186] Together, the present Example tests whether the loss of PKP2 gene leads to an ARVC phenotype, preventable by exogenous expression of the gene. In contrast, disease- relevant PKP2 constructs may fail to rescue the PKP2cKO. A main focus of the experiments is on the C-terminal domain, given that PKP2 truncations lead to ARVC^{2, 9}. Whether the resulting phenotype is the same as that of complete loss of PKP2, and whether the mutants act in a dominant negative manner is determined. These experiments generate a model on which to test the efficacy of gene replacement therapy in the setting of a disease-causing mutation. Example 6. Design of transgenic mice with cells comprising mutant PKP2 gene(s), and establishment of the minimum PKP2 sequence(s) for the treatment and/or prevention ARVC [00187] The present Example describes the design of transgenic mice with cells harboring a nucleotide sequence that encodes a mutant PKP2 gene(s), which is specifically used to test the effect of PKP2 mutations relevant to arrhythmogenic cardiomyopathy, e.g., arrhythmogenic right ventricular cardiomyopathy (ARVC), on the heart. The mouse is then used to identify agents effective in the treatment and/or prevention arrhythmogenic cardiomyopathy. Importantly, such agents include the minimum necessary PKP2 sequence (e.g., functional fragment(s) of the wild-type PKP2 protein), the generation of which is described in detail below. The design and testing of various mutant PKP2 genes, as well as related recombinant vectors and cells, are also described. These experiments provide high translational value, as they may serve as pre-clinical evidence for use of gene therapy, for example, in ARVC patients. [00188] For generation of the transgenic mouse, the mouse is first designed to comprise cells that: (i) comprise an endogenous PKP2 gene, or a functional portion of an endogenous PKP2 gene that is flanked by a pair of loxP sequences; (ii) express a ligand-inducible CRE recombinase under the control of a promoter; and, (iii) harbor a nucleotide sequence which encodes a mutant PKP2 gene. In particular, exon 2 and exon 3 of the endogenous PKP2 gene is flanked by the pair of loxP sequences, and the ligand-inducible CRE recombinase is under the control of a tissue-specific promoter, e.g., a cardiac-specific promoter. The cardiac-specific promoters used include, but are not limited to, α myosin heavy chain promoter and cardiac troponin T (cTNT) promoter, however any such promoters known to one of skill in the art may be useful. The ligand-inducible CRE recombinase possesses a ligand binding domain of the human estrogen receptor that is inducible by tamoxifen (TAM) such that induction by tamoxifen results in complete inactivation of the endogenous PKP2 gene. The specific cells that are targeted in this instance are cardiomyocytes, however other cardiac-specific cells may be of interest and specific promoters for such alternate cell types may be selected accordingly. [00189] Exogenous full-length wild-type PKP2 has been shown to prevent the ARVC phenotype consequent to loss of native PKP2. The future gene therapy requires that the transgene is capable of rescuing function in the background of a disease-relevant PKP2 mutation(s), rather than total absence of the native (i.e., endogenous) protein. To this end, nucleotide sequences which encode mutant PKP2 genes are specifically designed for use in the above-described transgenic mice such that the transgenic mouse cells (e.g., cardiomyocytes) express the mutant PKP2 gene. In certain designs, the mutant PKP2 gene is a truncation mutant including, for example, Arg79X, Gln378X, Arg413X, Arg651X, or Arg735X (Figures 6A- 6C). As disease penetrance is low for cases of other mutations, e.g., missense mutations or insertions, a focal point of various experiments disclosed herein may be truncations, which may have the most penetrance and a clearer relation to disease. However, a skilled artisan will appreciate that the present invention also encompasses any disease-relevant PKP2 mutation (e.g., PKP2 mutation associated with arrhythmogenic cardiomyopathy). The mutant PKP2 gene may be operably linked to one or more regulatory sequences, e.g., cardiac-specific promoters, for expression of a corresponding mutant PKP2 protein, when desirable. In some cases, the nucleotide sequence encoding the mutant PKP2 gene is present on a vector such as a viral vector, e.g., an adeno-associated virus (AAV) vector such as, but not limited to, AAV9. As an alternative, the nucleotide sequence encoding the PKP2 gene is integrated into a chromosome. [00190] Cardiomyocytes are also isolated from the transgenic mouse for further experimental use and/or testing. Recombinant vectors comprising various nucleotide sequences including those encoding the mutant PKP2 genes are further developed for additional use. As discussed above, the mutant PKP2 genes encoded by the nucleotide sequences are, e.g., truncation mutations such as, without limitation, Arg79X, Gln378X, Arg413X, Arg651X, or Arg735X. In various embodiments, the recombinant vectors are viral vectors such as, without limitation, AAV9. The mutant PKP2 gene is also, in some cases, linked to a one or more regulatory sequences for expression of the mutant PKP2 protein. [00191] In addition to the above-described transgenic mouse, and related cardiomyocytes and recombinant vectors, the present Example encompasses method steps for identifying agent(s) capable of treating of preventing arrhythmogenic cardiomyopathy, e.g., arrhythmogenic right ventricular cardiomyopathy (ARVC). For these experiments, first, an effective amount of tamoxifen is administered to the transgenic mouse thereby inducing complete inactivation of the endogenous PKP2 gene. After a pre-determined, set period of time, e.g., between about 3 weeks to about 7 weeks, an agent is administered to the transgenic mouse. Following administration of the agent, various functional assays are performed to test cardiac function associated with arrhythmogenic cardiomyopathy in the transgenic mouse for comparison to the cardiac function of a control transgenic mouse which has not been administered the agent. For example, the cardiac function is assessed using: 1) Echocardiogram to determine heart function, using methods such as those described by Cerrone and colleagues (2017)⁴, the content of which is incorporated herein by reference in its entirety for all purposes. Briefly, mice are anesthetized, placed on a warm platform and conventional echocardiography procedures known to those of skill in the art are used to determine, e.g., the thickness of the left ventricular wall, the volumes during the cardiac cycle, left ventricular ejection fraction, fractional shortening, and right ventricular area. Through these parameters the mechanical function of the beating heart is captured.2) Survival (Kaplan-Meier curves). In this procedure, mice are kept under standard housing conditions, provided with food and water and observed for survival and/or clinically-apparent deterioration. Body weights are captured, e.g., every week or two weeks. End point can be death, or if the animals appear severely deteriorated, euthanasia. 3) Arrhythmia burden. For these experiments, animals are anesthetized, instrumented for recording of the electrocardiogram, and then given a bolus intraperitoneal (i.p.) injection of isoproterenol. Electrocardiograms are recorded for a total of 30 minutes, and the number of premature ventricular contractions (PVCs) are counted within the 30 minutes that follow the isoproterenol bolus injection.4) The extent of fibrosis in post-mortem analysis of the heart. In this case, after euthanasia, the hearts are extracted, fixed, and histological sections are obtained along the long axis of the heart (i.e., so-called 4-chamber view). The sections are stained with a method (trichrome) that allows to discern collagen (stained in blue) from muscle (stained in red). The samples are then observed under a microscope and the area of the ventricular walls that is occupied by muscle, or by collagen, is quantified. 5) Analysis of intracellular calcium dynamics in isolated (cardio)myocytes. For these experiments, hearts are excised via a thoracotomy under deep anesthesia, placed in a perfusion system, and exposed to enzymes that allow for the separation of the tissue into individual cells. These isolated cells are then exposed to fluorescent dyes that emit light, detectable by a microscope (e.g., a fluorescence microscope), as a function of the concentration of calcium in the intracellular space. The agent is capable of treating or preventing arrhythmogenic cardiomyopathy if such echocardiographic, electrocardiographic, structural and survival data show improved cardiac function in the transgenic mouse receiving the agent as compared to a control transgenic mouse that did not receive the agent. If the agent is not capable of treating or preventing arrhythmogenic cardiomyopathy, then the cardiac function as interrogated by assays such as those listed above, is the same or decreased. [00192] In some cases, the agent comprises a nucleotide sequence encoding a wild-type full- length PKP2 protein (see, e.g., Figures 5A-5C). However, the method steps may involve an agent comprising a nucleotide sequence that encodes a functional portion (e.g., a fragment) of the wild-type PKP2 protein, rather than its full-length version. Based on previous studies^{25, 26}, the C-terminal end is necessary for proper PKP2 localization and it is therefore essential. The N-terminal domain may be critical for expression and localization, as well. Whether all, or only a small fraction of the protein comprising the armadillo repeat domain(s) is necessary for PKP2 function remains as yet unknown. Hence, various iterations and combinations of protein domains (e.g., C-terminal domain, N-terminal domain, armadillo domain (s)) of the PKP2 gene are tested. By way of a non-limiting example, for some experiments, the functional portion of the PKP2 gene that is tested comprises the N- terminal domain of PKP2, the C-terminal domain of PKP2, or both. In further experiments, the functional portion of the PKP2 protein does not comprise one or more armadillo domains. For follow-up experiments to these, one armadillo domain may then be removed at a time, in sequence. Ultimately, the selection of domains is based, among other considerations, on clinical evidence^{9, 23} showing that most disease-relevant mutations occur in the C-terminus and in some armadillo repeats. The experiments to assess function, extent of fibrosis, transgene expression and survival are performed as described above. Functional studies of relevance to arrhythmia mechanisms disclosed herein are also conducted. [00193] Exogenous full-length wild-type PKP2 prevents the ARVC phenotype consequent to loss of native PKP2. Experiments of the present Example further define and test the “minimum PKP2” that exerts the same effect by testing any number of nucleotide sequence that encode a functional portion of the PKP2 protein, using approaches such as those described above. The identified “PKP2min” is tested in the background of a disease- relevant PKP2 truncation mutant. These experiments provide evidence of high translational value, as they may serve as pre-clinical evidence for future attempts to use gene therapy in ARVC-patients. Indeed, previous studies show that exogenous full-length wild-type PKP2 prevents the ARVC phenotype consequent to loss of native PKP2 in mice⁵. Thus the agent used in the methods disclosed herein also comprises a nucleotide sequence encoding a functional portion of the PKP2 protein that is the minimum portion of the protein needed to exert potentially beneficial effects. [00194] The agent, e.g., agents comprising, for example, the nucleotide sequence encoding a full-length PKP2 protein and/or the nucleotide sequence encoding a functional fragment of the PKP2 transgene, may be delivered by way of a recombinant vector, e.g., a viral vector such as, but not limited to an AAV. In some cases, this “secondary” AAV is injected at the time of tamoxifen injection. These “rescue” constructs are tagged using a V5 tag, so that they can be localized separate from the mutant constructs, which, at times, express HA. [00195] Various other agents for the treatment and/or prevention of arrhythmogenic cardiomyopathy, e.g., ARVC, may also be tested and used. Such agents include, but are not limited to a small molecule, an antibody, an siRNA, an shRNA, an antisense oligonucleotide, and/or a site-specific nuclease. [00196] The present Example aims to demonstrate that the N-terminal and C-terminal domains of PKP2 are essential for PKP2 function. Expression of the N-terminal and C- terminal domains of PKP2, even in the absence of the armadillo domain(s), may prevent an ARVC phenotype associated with a disease-relevant PKP2 truncation or with loss of PKP2 expression. Exogenous full-length PKP2 prevents the ARVC phenotype consequent to loss of native PKP2. This Example aims to define the “minimum PKP2” that exerts the same effect. The identified “PKP2min” is tested in the background of a disease-relevant PKP2 truncation mutant. These experiments provide evidence of high translational value, and may serve as pre-clinical evidence for future use of gene therapy in ARVC-afflicted patients. Example 7. Determination of the ability of Cx43-H blockers and/or RyR2 blockers to prevent the ARVC phenotype associated with a clinically-relevant PKP2 mutation [00197] The present Example tests whether Cx43-Hs and/or RyR2 channels are actionable targets for antiarrhythmic therapy. Reducing the eagerness of RyR2 to release Ca²⁺ and/or blocking Cx43-Hs may limit Ca²⁺ _i accumulation and triggered activity in PKP2-deficient myocytes. Gene delivery methods as well as drug repurposing approaches to interfere with disease progression in a murine model of ARVC are tested. [00198] Previous data show that flecainide prevents ISO-induced arrhythmias in PKP2cKO mice⁴. Additional studies show the same for ent-verticilide²⁰, and a single dose of dantrolene may be effective as well. Preventing RyR2 mediated Ca²⁺ release, in the setting of PKP2 loss of expression may have beneficial effects on arrhythmia burden. The present Example evaluates whether a similar result can be observed when a disease- relevant ARVC mutation is expressed. The first set of experiments assesses these treatments in the setting of the PKP2 mutants as described above. Arrhythmia burden is determined similar to or the same as described in Figure 2 and Figure 3. A loss of function may be apparent in truncation mutants. As the size of the transgene is reduced, a separate (non-concatenated) coding region for GFP is inserted. The latter allows for identification of single cells expressing the transgene, as well as single cell experiments test whether the mutants may reproduce the behavior of PKP2cKO myocytes in terms of intracellular calcium dysregulation^{4, 6}. [00199] A single administration of dantrolene may acutely dampen ISO-induced premature ventricular contraction (PVC) burden, likely by dampening excess release of Ca²⁺ through the RyR2 channels. As an extension of these experiments, the experiments of the present Example assess, in the setting of an ARVC-relevant PKP2 mutant, whether chronic use of dantrolene in the PKP2cKO mouse can control arrhythmic burden and can delay/arrest the cardiomyopathy progression. Mice are treated daily with intraperitoneal (i.p.) dantrolene injections for 14 days, starting 7 days after TAM injection and ventricular function is assessed (e.g., via echocardiography). ISO-dependent arrhythmias at, e.g., 21 dpi and 28 dpi (or timing adjusted if the progression of the cardiomyopathy in the background of a PKP2 mutant is different from that of the PKP2cKO), are measured. Serial doses of dantrolene (e.g., 30, 20 and 10 mg/kg) are tested to identify the lowest effective pharmacological dose. Dantrolene controls arrhythmias, e.g., at 21 and/or 28 dpi, and/or delays or arrests the decline in ventricular function and dimensions. These experiments are complemented with studies in isolated cardiomyocytes to determine the extent of the effect of dantrolene on the various components of calcium dysregulation previously identified in PKP2cKO myocytes⁶. [00200] It is hypothesized that AAV-mediated delivery of a Cx43-Hs inhibitor may delay or even arrest the development of the structural dysfunction in PKP2cKO hearts. AAV- mediated gene transfer similar approach is used to introduce either GAP19 or RRNYRRNY (SEQ ID NO: 1) which is fused to a fluorescent protein (e.g., GFP) and expressed with a cardiac selective promoter. The latter is a peptide originally described to prevent chemically-mediated gap junction closure²⁸. Recently it was shown that the peptide has also powerful blocking effects on Cx43-Hs¹³. Importantly both GAP19 and RRNYRRNY (SEQ ID NO: 1) are genetically-encoded linear peptides, exert their action from the intracellular space and maintain gap junctions open thus avoiding a negative effect on action potential propagation. As an alternative, the RyRHCIp peptide KNRRNPRLVPY (SEQ ID NO: 2) such as that described by Lissoni and colleagues (2021)²⁹ which is incorporated herein by reference in its entirety, is used, which has been shown to impact Cx43-Hs function²⁹. These are rather small constructs and as such, packing them together with a truncated PKP2 mutant does not represent an insurmountable hurdle. The AAV-peptide construct is delivered, e.g., 4 weeks before TAM injection. The first set of experiments uses the PKP2cKO mice. Delivery of the AAV-peptide construct may improve survival, contractility and arrhythmia burden in the living animals, arresting or delaying the development of the disease. At the cellular level, AAV-mediated peptide delivery may impact the number of Cx43-H-mediated events which is tested such as by patch clamp, as well as limit of the accumulation of Ca²⁺i. After validation in the complete loss of function model (i.e., PKP2cKO), experiments are conducted in the background of a PKP2 mutant, as described above. [00201] In an alternative approach, the AAV-peptide construct is delivered, e.g., 7 days after TAM injection, to investigate the potential of this approach to arrest the disease after initial loss of PKP2 is already ongoing. This latter model provides insights into the potential of Cx43-Hs blockade in the setting of the clinical disease, where genetically mediated loss of PKP2 occurs since birth and before the diagnosis of ARVC. [00202] Together, the present Example aims investigate reducing the eagerness of RyR2 to release Ca²⁺ and/or blocking Cx43-Hs limits Ca²⁺i accumulation and its effect on activity in PKP2-deficient cardiac myocytes. Evidence from experimental models⁶, and anecdotal clinical evidence^{7, 8}, indicate that PKP2-dependent arrhythmias involve Cx43-H dysfunction, and excess RyR2 activity. Here, gene delivery methods as well as drug repurposing approaches are used to interfere with disease progression in a murine model of ARVC. [00203] In conclusion, the experimental paradigms disclosed herein seek to advance the pre-clinical phase of gene therapy for patients with ARVC resulting from PKP2 mutations. The above-described experiments take advantage of an experimental animal model developed and characterized by the inventors of the present disclosure and modifies its phenotype by identifying, at the same time, the minimum gene therapeutic approach. Data from experiments disclosed herein represent a transformational step to guide experimental gene therapy into clinic. [00204] Below are the methods used in the Examples described above. [00205] Generation of cardiac-specific, PKP2 KO mice is described in Cerrone et al⁴, which is incorporated herein by reference in its entirety. Briefly, a C57BL/6 PKP2 fl/fl mice line was generated and crossed with the αMyHC-Cre-ER(T2) line, as previously published³⁰. Two forward loxP sites were designed and introduced into the construct flanking mouse PKP2 exons 2 and 3, with a downstream neomycin selection cassette. The linearized targeting construct was electroporated into C57B/6 derived embryonic stem (ES) cells and the resultant ES cell clones were identified. The confirmed positive ES cells were injected into isogenic blastocysts, and microinjected into the foster mice. The neo cassette was excised by crossing the F1 heterozygous mice with FRT mice. The PKP2 flox/flox mice were mated to ^MyHC-Cre- ER(T2) mice to obtain flox/flox/Cre+ mice which contains the ^ myosin heavy chain promoter and the ligand binding domain of the human estrogen receptor. The resulting mice (PKP2- cKO) developed normally without functional or structural deficits. Mice were injected 4 consecutive days with tamoxifen (3 mg dissolved in sterile peanut oil with 10% ethanol; mice weight hovered ∼28–30 g, giving an approximate tamoxifen dose of 0.1 mg of tamoxifen per gram of body weight). Binding of tamoxifen to the estrogen receptor induced the cardiomyocyte specific Cre-mediated deletion of the Pkp2 gene. All experiments were performed in PKP2-cKO mice and CRE-negative, tamoxifen treated, littermates were used as controls. Both genders were included. All animals were between 3 and 4 months of age. [00206] All experiments in cardiac PKP2-cKO mice (3-6 months old) proposed are controlled with fl/fl, Cre-negative mice (either littermates or gender and age-matched) injected with tamoxifen. Equal numbers are used for both genders. In the absence of gender-based differences, data are combined. Numbers of experiments are based on power analysis. A difference of 10% between means, with 5% SD in the largest mean, at >0.9 power and alpha of 0.05 requires a minimum sample size of 6. As in⁶, n values will be above 10. For experiments in isolated myocytes obtained from more than one mouse, statistical significance is first assessed by hierarchical analysis based on the open resource of Sikkel et al¹⁶, which detects adequacy of hierarchical analysis versus other methods, primarily two-way ANOVA of repeated measures and Bonferroni test. Non-parametric tests, e.g., Mann-Whitney U, Wilcoxon signed rank and/or Kruskal-Wallis tests. are performed as appropriate. References 1. Delmar M and McKenna WJ. The cardiac desmosome and arrhythmogenic cardiomyopathies: from gene to disease. Circ Res.2010;107:700-14. 2. 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Lissoni A, Hulpiau P, Martins-Marques T, Wang N, Bultynck G, Schulz R, Witschas K, Girao H, De Smet M and Leybaert L. RyR2 regulates Cx43 hemichannel intracellular Ca2+- dependent activation in cardiomyocytes. Cardiovasc Res.2021;117(1): 123-136. 30. Lubkemeier I, Requardt RP, Lin X, Sasse P, Andrie R, Schrickel JW, Chkourko H, Bukauskas FF, Kim JS, Frank M, Malan D, Zhang J, Wirth A, Dobrowolski R, Mohler PJ, Offermanns S, Fleischmann BK, Delmar M and Willecke K. Deletion of the last five C- terminal amino acid residues of connexin43 leads to lethal ventricular arrhythmias in mice without affecting coupling via gap junction channels. Basic Res Cardiol.108, 348 (2013) * * * [00207]The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. [00208]All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.

Listing of Sequences

Claims

Claims 1. A transgenic mouse comprising cells that (i) comprise an endogenous plakophilin-2 (PKP2) gene, or a functional portion thereof, flanked by a pair of loxP sequences; (ii) express a ligand-inducible CRE recombinase under control of a promoter; and (iii) comprise a nucleotide sequence encoding a mutant PKP2 gene.

2. The transgenic mouse of claim 1, wherein exon 2 and exon 3 of the endogenous PKP2 gene is flanked by a pair of loxP sequences.

3. The transgenic mouse of claim 1 or 2, wherein the ligand-inducible CRE recombinase is under control of a tissue-specific promoter.

4. The transgenic mouse of claim 3, wherein the ligand-inducible CRE recombinase is under control of a cardiac-specific promoter.

5. The transgenic mouse of claim 4, wherein the cardiac-specific promoter is an α myosin heavy chain promoter or a cardiac troponin T (cTNT) promoter.

6. The transgenic mouse of any one of claims 1-5, wherein the ligand-inducible CRE recombinase comprises a ligand binding domain of the human estrogen receptor.

7. The transgenic mouse of claim 6, wherein the ligand-inducible CRE recombinase is inducible by tamoxifen.

8. The transgenic mouse of claim 7, wherein induction by tamoxifen results in inactivation of the endogenous PKP2 gene.

9. The transgenic mouse of any one of claims 1-8, wherein the cells are cardiomyocytes.

10. The transgenic mouse of any one of claims 1-9, wherein the mutant PKP2 gene is associated with an arrhythmogenic cardiomyopathy.

11. The transgenic mouse of any one of claims 1-10, wherein the mutant PKP2 gene is a human PKP2 gene.

12. The transgenic mouse of any one of claims 1-11, wherein the mutant PKP2 gene is a truncation mutant.

13. The transgenic mouse of claim 12, wherein the truncation mutant is Arg79X, Gln378X, Arg413X, Arg651X, or Arg735X.

14. The transgenic mouse of any one of claims 1-13, wherein the mutant PKP2 gene is operably linked to one or more regulatory sequences that can mediate expression of a mutant PKP2 protein in the cells.

15. The transgenic mouse of any one of claims 1-14, wherein the nucleotide sequence of the mutant PKP2 gene is present in a vector.

16. The transgenic mouse of claim 15, wherein the vector is a viral vector.

17. The transgenic mouse of claim 16, wherein the viral vector is an AAV vector.

18. The transgenic mouse of any one of claims 1-17, wherein the mutant PKP2 gene is integrated into a chromosome within the cell.

19. A cardiomyocyte isolated from the transgenic mouse of any one of claims 1-18.

20. A recombinant vector comprising a mutant PKP2 gene.

21. The recombinant vector of claim 20, wherein the mutant PKP2 gene is a truncation mutant.

22. The recombinant vector of claim 21, wherein the truncation mutant is Arg79X, Gln378X, Arg413X, Arg651X, or Arg735X.

23. The recombinant vector of any one of claims 20-22, wherein the vector is a viral vector.

24. The recombinant vector of claim 23, wherein the vector is an AAV vector.

25. The recombinant vector of claim 24, wherein the AAV vector has tropism for the heart.

26. The recombinant vector of claim 24, wherein the AAV is AAV9.

27. The recombinant vector of any one of claims 20-26, wherein the mutant PKP2 gene is operably linked to one or more regulatory sequences which can mediate expression of a mutant PKP2 protein in a cell.

28. The recombinant vector of claim 27, wherein the cell is a cardiomyocyte.

29. A method of identifying an agent capable of treating or preventing an arrhythmogenic cardiomyopathy, comprising: e) administering to the transgenic mouse of any one of claims 1-18 an effective amount of tamoxifen to induce inactivation of the endogenous PKP2 gene; f) administering an agent to the transgenic mouse; g) measuring a cardiac function associated with the arrhythmogenic cardiomyopathy in the transgenic mouse after administration of the agent and comparing the cardiac function with that in a control transgenic mouse which has been administered tamoxifen to induce inactivation of the endogenous PKP2 gene but has not been administered the agent; and h) determining that i) the agent is capable of treating or preventing the arrhythmogenic cardiomyopathy if the cardiac function is improved as compared to the cardiac function in the control mouse; or ii) the agent is not capable of treating or preventing the arrhythmogenic cardiomyopathy if the cardiac function is the same or decreased as compared to the cardiac function in the control mouse.

30. The method of claim 29, wherein step (b) is carried out after step (a).

31. The method of claim 30, wherein step (b) is carried out between about 3 weeks to about 7 weeks after step (a).

32. The method of claim 29, wherein step (b) is carried out at the same time as step (a).

33. The method of any one of claims 29-32, wherein the cardiac function is measured by echocardiogram, electrocardiogram, determining arrhythmia burden, measuring fibrosis, determining intracellular calcium dynamics, or any combination thereof.

34. The method of any one of claims 29-33, wherein the arrhythmogenic cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy (ARVC).

35. The method of any one of claims 29-34, wherein the agent comprises a polynucleotide molecule encoding a full-length wild-type PKP2 protein.

36. The method of any one of claims 29-34, wherein the agent comprises a polynucleotide encoding a functional fragment of a wild-type PKP2 protein.

37. The method of claim 36, wherein the functional fragment of the wild-type PKP2 protein comprises an N-terminal domain of PKP2, a C-terminal domain of PKP2, or both.

38. The method of claim 36 or claim 37, wherein the functional fragment of the wild-type PKP2 protein does not comprise one or more armadillo repeats of wild-type PKP2.

39. The method of any one of claims 29-34, wherein the agent is a small molecule, an antibody, an siRNA, an shRNA, an antisense oligonucleotide, or a site-specific nuclease.

40. An agent capable of treating or preventing arrhythmogenic cardiomyopathy identified by the method of any one of claims 29-39.

41. A transgenic mouse expressing a functional fragment of the wild-type PKP2 protein identified by the method of any one of claims 29-39.

42. A cell line expressing a functional fragment of a wild-type PKP2 protein identified by the method of claims any one of claims 29-39.

43. A polynucleotide comprising or consisting of the nucleotide sequence encoding a functional fragment of a wild-type PKP2 protein identified by the method of any one of claims 29-38.

44. A recombinant vector expressing a functional fragment of a wild-type PKP2 protein identified by the method of any one of claims 29-38.

45. The recombinant vector of claim 44, wherein the vector is a viral vector.

46. The recombinant vector of claim 45, wherein the vector is an AAV vector.

47. The recombinant vector of claim 46, wherein the AAV has tropism for the heart.

48. The recombinant vector of claim 46 or claim 47, wherein the AAV is AAV9.

49. The recombinant vector of any one of claims 44-49, wherein the nucleotide sequence encoding the functional fragment of the wild-type PKP2 protein is operably linked to one or more regulatory sequences for expression of said functional fragment of the wild-type PKP2 protein in a cell.

50. A kit comprising one or more of the recombinant vectors of claims 20-28 or 44-49, and optionally packaging and/or instructions for the same.

51. A method of treating an arrhythmogenic cardiomyopathy in a subject in need thereof, comprising administering to the subject the agent identified by the method of any one of claims 29-39, the polynucleotide of claim 43, and/or the recombinant vector of any one of claims 44-49.

52. The method of claim 51, wherein the subject has a mutant PKP2 gene.

53. The method of claim 52, wherein the mutant PKP2 gene is a truncation mutant.

54. The method of claim 53, wherein the truncation mutant is Arg79X, Gln378X, Arg413X, Arg651X, or Arg735X.

55. The method of any one of claims 51-54, wherein the arrhythmogenic cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy (ARVC).

56. The method of any one of claims 51-55, further comprising administering one or more additional agents.

57. The method of claim 56, wherein the one or more additional agents are selected from an antiarrhythmic agent, an RyR stabilizer, a muscle relaxer, and an Cx43-Hs inhibitor.

58. The method of claim 56, wherein the one or more additional agents are selected from flecainide, ent-verticilide, dantrolene, TAT-Gap19 or other GAP19 peptide derivative, RRNYRRNY peptide (SEQ ID NO: 1), RyRHCIp peptide (SEQ ID NO: 2), and analogs or derivatives thereof.

59. The method of claim 56, wherein the one or more additional agents are one or more polynucleotides encoding the GAP19 peptide, the RRNYRRNY peptide (SEQ ID NO: 1), and/or the RyRHCIp peptide (SEQ ID NO: 2).

60. The method of claim 56, wherein the one or more additional agents are one or more recombinant vectors encoding the GAP19 peptide, the RRNYRRNY peptide (SEQ ID NO: 1), and/or the RyRHCIp peptide (SEQ ID NO: 2).

61. The method of claim 56, wherein the one or more additional agents are one or more AAV vectors encoding the GAP19 peptide, the RRNYRRNY peptide (SEQ ID NO: 1), and/or the RyRHCIp peptide (SEQ ID NO: 2).