TRPV4 inhibition to treat arrhythmic cardiomyopathy

Abstract

Genome sequencing identified a new gene associated with a cardiac arrhythmic disease, TAXI -binding protein 3. From cell line models and a conditional knock-out mouse models of the gene deletion, it was found that a specific molecule, transient receptor potential vanilloid 4 (TRPV4), is up-regulated and is associated with calcium mediated arrhythmic depolarizations.

Description

Atty Docket No.: 47381-00073TRPV4 Inhibition to Treat Arrhythmic Cardiomyopathy Inventors: Andrew Landstrom Robin Perelli TECHNICAL FIELD

[0001] A new gene associated with arrhythmogenic cardiomyopathy, TAX1-binding protein 3 was identified. Further it was found that a specific gene, transient receptor potential vanilloid 4 (TRPV4), is up-regulated in the cardiac myocytes from cell line models and is associated with calcium-mediated arrhythmic depolarizations leading to the identification of new treatments for cardiac arrhythmic disorders that have the characteristic of calcium leak or waves. BACKGROUND

[0002] Arrhythmogenic cardiomyopathy (ACM) is one of the leading causes of sudden cardiac death in children and is characterized by the fibrofatty replacement of the myocardium, predominantly of the right ventricle. Sixty percent of patients with ACM have a known genetic cause, but for the remainder, the etiology is unknown. This lack of mechanistic understanding has also slowed development of new therapies, and children with ACM have a high degree of morbidity and mortality. As such, there is an ongoing need for improved understanding and treatment of ACM and similar conditions. While 60% of individuals with ACM host variants in known disease-associated genes, for 40% of patients, the cause is unknown. There are currently no treatments to reverse or halt disease progression. Thus, there is a critical need for improved mechanistic understanding of ACM pathogenesis to facilitate the development of novel therapies.

[0003] Life-threatening arrhythmias are often the first presenting symptom in ACM. A well- established mechanism of arrhythmogenesis is intracellular Ca2+leak. Tight regulation of Ca2+inside the cardiomyocyte is critical for the coordinated contraction of the cell. When the cardiomyocyte depolarizes, Ca2+enters the cell through the L-type Ca2+channel and binds to the ryanodine receptor (RyR2) on the sarcoplasmic reticulum (SR). This channel then opens, allowing the intracellular store of Ca2+to be released and then bind to troponin C which causes contraction of the myofilaments. Aberrant Ca2+can leak out of RyR2 can lead to spontaneous depolarizations which, at the tissue level, can leaded to triggered arrhythmias. SUMMARY

[0004] The Summary is provided to introduce a selection of concepts that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of theAtty Docket No.: 47381-00073claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter.

[0005] One aspect of the present disclosure provides a method treating and / or preventing cardiac disease in a subject, the method comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of a transient receptor potential vanilloid 4 (TRPV4) inhibitor such that the cardiac disease in the subject is treated and / or prevented.

[0006] In one embodiment, the TRPV4 inhibitor is selected from the group consisting of HC-067047, GSK2193874, GSK2798745, RN-1734, RN-1747, RN 9893 hydrochloride, Piperidine-Benzimidazole, PF-05214030, amino bithiazlole antagonists, and the like.

[0007] In one embodiment, the cardiac disease comprises ischemia. In another embodiment, the cardiac disease comprises arrhythmic cardiomyopathy (ACM). Another aspect of the present disclosure provides all that is described and illustrated herein.

[0008] The accompanying figures and Examples are provided by way of illustration and not by way of limitation. The foregoing aspects and other features of the disclosure are explained in the following description, taken in connection with the accompanying example figures (also “FIG.”) relating to one or more embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0009] FIG 1A-1G: Pedigree of TAX1BP3M78T / delKindred. FIG 1A). Delayed enhancement cardiac magnetic resonance images (CMRI) of healthy mother (I.2, p.M78T / WT) and sister (II.3,WT / WT) demonstrating healthy myocardium (black signal of the right ventricle (RV) free wall and youngest brother (II.2, p.M78T / del) and oldest brother (II.4, p.M78T / del) with fibrofatty hyperenhancement (bright signal) of the RV free wall. Yellow arrows show location RV free wall. Scale bar = 2cm. FIG 1B). Pedigree of TAX1BP3 variants co-segregating in kindred with ACM. Subjects are labeled by position on phylogenetic tree. Roman numerals indicate generation and numbers label kindred member. Black fill = SCD or diagnosis of ACM. Slash = deceased. FIG 1C) Sanger sequencing chromatograms of wild type, heterozygous variant p.M78T, and compound heterozygous p.M78T / del. FIG 1D) Genomic location of the TAX1BP3 locus on chromosome 17 in purple, with location of deletion in red. FIG 1E) TAX1BP3 locus with location of variant on exon 3. FIG 1F) Linear topology of TAX1BP3 with a predicted acetylation site (Ac), phosphorylation site (P), PDZ domain, and p.M78T variant location noted. FIG 1G) Amino acid conservation analysis demonstrating the percent conservation across 195 species at each TAX1BP3 position relative to the human sequence.Atty Docket No.: 47381-00073

[0010] FIG 2A-2C: ECG Evaluation. FIG 2A) Representative 12-lead ECG from individual II.4 showing normal sinus rhythm with T wave inversions in septal leads (V1 and V2). FIG 2B) 12-led ECG from individual II.2 showing normal sinus rhythm with premature ventricular contractions (PVCs) and FIG 2C) 12-led ECG from individual II.4 showing wide complex tachycardia consistent with ventricular tachycardia.

[0011] FIG 3: CMA findings demonstrating loss of TAX1BP3 heterozygosity. CMA results for individuals II.1, II.2, and II.4 showing TAX1BP3 deletion (17p13.2). The deletion is paternally inherited and encompasses the entire TAX1BP3 gene and is a total of 242 kb (chr17:3,392,348-3,634,096).

[0012] FIG 4 is a Table of Clinical and genetic information for the family.

[0013] FIG 5 is a Table of in silico variant prediction aggregate analysis of the TAXBP3-M78T variant.

[0014] FIG 6A-6B is Validation of iPSCs. FIG 6A and FIG 6B) Immunofluorescence for iPSC marker OCT4 (green) or NANOG (green) and DAPI (blue) on the three iPSC lines. Scale bar = 50μm.

[0015] FIG 7A-7B Validation of iPSC-CMs FIG 7A) Sanger sequencing of iPSC-CMs to confirm T>C missense variant in II.2 and II.4. FIG 7B) Immunofluorescence examples of DAPI (blue) and TNNT2 (green) positive iPSC-CMs. Scale bar = 20μm. Quantification of TNNT2 and ACTC2 cells per DAPI positive cell.

[0016] FIG 8A-8E: TAX1BP3 loss-of-function results in increased adipogenesis. FIG 8A) Western blot of iPSC-CMs showing decrease of TAX1BP3 protein in iPSC-CMM78T / del. FIG 8B) Quantification of A (Mean ± s.e.m. n = 3;4, unpaired t-test: p = 0.0149. FIG 8C) Confocal images of iPSC-CMWT / WTand iPSC-CMM78T / dellines showing immunofluorescence TNNT2 (cyan) and Nile Red (red). Scale bar = 500μm. FIG 8D) Quantification of the percentage of Nile Red fluorescence relative to TNNT2 fluorescence. (Mean ± s.e.m. n = 4; 3; 4, one-way ANOVA with multiple comparisons: p = 0.0018,WT / WTvs.M78T / del– II.2: p = 0.0014,WT / WTvs.M78T / del– II.4: p = 0.0065) FIG 8E) RT-qPCR for PPARG and CEBPa of iPSC-CMs following vehicle or 3F treatment.3F treatments for both iPSC-CMWT / WTand iPSC- CMM78T / dellines were normalized to the vehicle treatment of each line (Mean ± s.e.m. n = 5; 6; 4; 6, multiple unpaired t-tests, not significant). FIG 9A‐9E iPSC-CMM78T / delhave differences in Ca2+handling. FIG 9A) Example Ca2+transient traces of iPSC-CMs taken from line scans while cells were paced at 0.5Hz. FIG 9B) Quantifications of Ca2+transient amplitude (Mean ± s.e.m, n differentiations = 5; 4; 5, n cells = 31; 34; 37, WT / WT vs. M78T / del – II.2: p = 0.057, WT / WT vs. M78T / del – II.4: p < 0.01), time to peak (Mean ± s.e.m, nAtty Docket No.: 47381-00073differentiations = 5; 4; 5, n cells = 31; 34; 37, not significant), tau (Mean ± s.e.m, n differentiations = 5; 4; 5, n cells = 31; 34; 37, p < 0.001, WT / WT vs. M78T / del – II.2: p < 0.01, WT / WT vs. M78T / del – II.4: not significant). FIG 9C) Example Ca2+traces of iPSC-CMs following treatment with 10mM caffeine to trigger SR store Ca2+release. FIG 9D) Quantifications of SR store Ca2+release amplitude (Mean ± s.e.m, n differentiations = 5; 5; 6, n cells = 34; 28; 31, WT / WT vs. M78T / del – II.2: p < 0.05, WT / WT vs.

[0001] M78T / del – II.4: p = 0.0815) and tau (Mean ± s.e.m, n differentiations = 5; 5; 6, n cells = 34; 28; 31, not significant). E) Quantification of 340 / 380 ratio of Fura-2 fluorescence measuring intracellular Ca2+levels (Mean ± s.e.m, n = 158; 132; 98, not significant. p < 0.01.* p < 0.05, ** p < 0.01, *** p <0.001

[0002] FIG 10A-10C iPSC-CMM78T / delhave greater Ca2+leak. FIG 10A) Representative examples of confocal line scans with insets showing example regions of interest showing Ca2+sparks. FIG 10B) Quantifications of spark frequency (Mean ± s.e.m. n differentiations = 5; 4; 5, n cells = 41; 34; 53, p < 0.001, WT / WT vs M78T / del—II.2: not significant, WT / WT vs M78T / del—II.4: p < 0.05) FIG 10C) Quantification of Ca2+leak normalized to SR store (average amplitude per cell line of caffeine induced Ca2+release) (Mean ± s.e.m, n differentiations = 5; 4; 5, n cells = 41; 34; 53, WT / WT vs. M78T / del— II.2: p < 0.05, WT / WT vs. M78T / del—II.4: p < 0.01). * p < 0.05, ** p < 0.01, *** p <0.001)

[0003] FIG 11A-11C: iPSC-CMM78T / delhave spontaneous Ca2+release and inducible sustained cellular arrhythmias. FIG 11A) Example Ca2+fluorescent traces of iPSC-CMs during pacing. Asterisks denote spontaneous Ca2+release. FIG 11B) Quantification of the percentage of cells with spontaneous Ca2+release (Mean ± 95% confidence interval, n = 32; 28; 34, Fisher’s Exact Test, not significant). FIG 11C) An example trace from iPSC-CMM78T / delII.4 iPSC-CMs with a triggered sustained arrhythmia.

[0004] FIG 12 is a table of Canonical Wnt signaling gene expression changes in iPSC-CM RNA- sequencing.FIG 13A‐13F: TRPV4 localization changes in iPSC-CMM78T / del. FIG^13A) Volcano plots of RNA-sequencing of iPSC-CMs showing genes with an adjusted p value < 0.05 in II.2 vs WT (II.3, left)and II.4 vs. WT (right). FIG 13B,C) Representative traces of ITRPV4 in iPSC-CMWT / WT vs. iPSC-CMM78T / delcells . D,E) ) Group mean values of ITRPV4 currents of iPSC-CMWTvs.iPSC- CMM78T / delcells measured at -80mV (Mean ± s.e.m, n = 8; 9, p < 0.05) and +80mV Mean ± s.e.m, n = 8; 9, p <0.05) FIG 13F) Representative confocal images of immunofluorescence of DSP (cyan) and TRPV4(red) in iPSC-CMs with additional DAPI stainingAtty Docket No.: 47381-00073(white). White arrows denote TRPV4 and nucleus co-localization in iPSC-CMWT / WT and DSP andTRPV4 signal overlap at the cell membrane in iPSC-CMM78T / del, respectively. Scale bar = 20μm. FIG^13G) Representative confocal images of immunofluorescence of RyR2 (cyan) and Trpv4 (red) onisolated mouse ventricular myocytes with additional Hoechst staining (white) from Tax1bp3+ / +and Tax1bp3‐ / ‐mice, respectively. H) Graphs of the representative fluorescence plot profiles ofprimary myocytes from Tax1bp3+ / + and Tax1bp3‐ / ‐ mice, respectively, showing RyR2 (cyan) andTrpv4 (red) localization patterns. * p < 0.05, ** p < 0.01, *** p <0.001

[0005] FIG 13E-13F)FIG 14A‐14H: TRPV4 inhibition with HC-067047 rescues Ca2+ leak. FIG^14A) Example confocalimages of regions of interest for Ca2+ sparks in and II.4 iPSC-CMs treated with DMSO or 1µM HC-067047. FIG 14B) Quantification of spark frequency (mean ± s.e.m, hierarchical test, M78T / delII.4: n differentiations = 3;3, n cells = 29; 31, p = 0.0034) and Ca2+leak (mean ± s.e.m, hierarchical test, M78T / del II.4: n differentiations = 3;3, n cells = 29; 31, p < 0.0001.). FIG 14C-and 14D) Examples tracings of action potentials recordings from iPSC-CMWT / WTand iPSC-CMM78T / del, respectively. Black arrows denote delayed afterdepolarizations (DADs) FIG 14E) iPSC-CMM78T / del-II.4action potential example trace with 10µM HC067047 pre-treatment. FIG 14F) Bar graph demonstrating the quantification of the percentage of cells which demonstrated DADs (mean, n = 13; 11; 12, WT / WT vs. M78T / del, P < 0.0001; M78T / del vs. M78T / del + HC-067047, P < 0.01). * p < 0.05, ** p < 0.01, *** p <0.001 FIG 15 Proposed mechanism of TAX1BP3-mediated arrhythmia development. 1) Loss of Tax1bp3 results in Trpv4 translocation to the t-tubules. 2) This leads towards development of ITRPV4. 3) The increased Ca2+triggers Ca2+leak from the sarcoplasmic reticulum (SR) via RyR2 that 4) drives development of delayed after depolarizations (DADs). 5) This leads to triggered arrhythmogenesis of the myocardium. FIG 16A‐16G:^Tax1bp3‐ / ‐^mice have increased Ca2+leak and inducible ventricular arrhythmias. FIG 16A) Schematic showing inducible, cardiac specific Tax1bp3‐ / ‐^mouse breeding strategy. FIG16B) Example Western blot for Gapdh and Tax1bp3 on mouse right ventricle (RV), left ventricle(LV) and liver tissue samples FIG 16C) Representative ROI images showing Ca2+sparks fromconfocal line scans in Tax1bp3+ / +^and Tax1bp3‐ / ‐^mice. White arrows denote examples of sparks.FIG 16D) Quantification of spark frequency after 0.5Hz pacing (mean ± s.e.m, n mice = 3; 4, n cells = 35; 37, p < 0.01) and Ca2+leak (mean ± s.e.m, n mice = 3; 4, n cells = 34; 34, p < 0.001). FIG 16E)Atty Docket No.: 47381-00073Representative surface ECG trace of sinus rhythm follow ventricular pacing in Tax1bp3+ / +mice and polymorphic and bidirectional ventricular tachycardia (VT) surface ECG traces in Tax1bp3- / -mice. FIG 16F) Quantification of percentage of mice with sustained VT (Mean ± 95% confidence interval, n = 17; 21, p < 0.05) FIG 16G) Quantification of the number of sustained VT events per mouse (mean ± s.e.m, n = 17; 21, p < 0.01). * p < 0.05, ** p < 0.01, *** p <0.001

[0006] FIG 17A-17C Electrocardiograms in Tax1bp3- / -mice demonstrate no differences compared to controls. FIG 17A) Example surface ECG traces in Tax1bp3+ / + and Tax1bp3- / -mice, respectively. FIG 17B) Graphs demonstrating the quantification of PR, RR, QRS and QTc intervals. Open circles are female mice and closed circles are male mice (mean ± s.e.m., n = 17; 22,). FIG 17C) Quantification of sinus node recovery time (SNRT) (mean ± s.e.m., n = 17; 22) and AV node effective refractory period (AVERP) (Mean ± s.e.m., n= 11; 17). Open circles are female mice and closed circles are male mice.

[0007] FIG 18A-18B:Tax1bp3- / -mouse transthoracic echocardiograms demonstrate no differences compared to controls. FIG 18A) Example long axis M-mode echocardiograms from Tax1bp3+ / + and Tax1bp3- / -mice, respectively. FIG 18B) Graphs with quantification of echocardiogram measurements of the left ventricular (LV) wall thickness, interventricular septum width (IVSW), fractional shortening, ejection fraction, systolic right ventricle (RV) area, and diastolic RV area (mean ± s.e.m., n= 9; 22; 6).

[0008] FIG 19 TRPV4 localization changes in iPSC-CMM78T / del. Representative confocal images of immunofluorescence of Hoechst (white), RyR2 (cyan), and TRPV4 (red) on isolated mouse ventricular myocytes with corresponding fluorescence plot profiles (n = 4 Tax1bp3+ / +, n = 3 Tax1bp3- / -). Scale bars = 25μm, inset scale bars = 5μm.

[0009] FIG 20A-20F TRPV4 inhibition rescues Ca2+ leak in Tax1bp3- / -mice and PKP2-fs iPSC- CMs. FIG 20A). Representative line scans from live cell Ca2+ confocal imaging after 0.5Hz pacing from Tax1bp3- / -mice with either DMSO or 1µM HC-067047 (white arrows denote Ca2+ sparks). FIG 20B) Graph of the quantification of Ca2+spark frequency (mean ± s.e.m, n mice = 3; 3, n cells = 33; 35, not significant) and Ca2+ leak (mean ± s.e.m, n mice = 3; 3, n cells = 33; 35, p < 0.05). FIG 20C) Representative confocal line scans with 5Hz pacing with either DMSO or 1µM HC-067047 (black arrows denote spontaneous release events, SREs). FIG 20D) Graph quantifying the number of SREs per ventricular cell (mean ± s.e.m, n mice = 3; 3, n cells = 35; 35, p < 0.001). FIG 20E) Representative images from live cell, Ca2+imaging confocal line scans after 0.5Hz pacing from iPSC-CMPKP2-fsand iPSC- CMPKP2-WTafter either DMSO (red circles) or 1µM HC-067047 (cyan triangles) exposure. FIG 20F) Quantification of Ca2+spark frequency (mean ± s.e.m, n differentiations = 3; 3; 3; 3, n cells = 34; 26; 32;Atty Docket No.: 47381-0007337, iPSC-CMPKP2-WT+DMSO vs iPSC-CMPKP2-fs+ HC-067047, p <0.05) and Ca2+leak (mean ± s.e.m, n differentiations = 3; 3; 3; 3, n cells = 34; 26; 32; 37, iPSC- CMPKP2-WT+ DMSO vs iPSC-CMPKP2-WT+ HC-067047, not significant, iPSC-CMPKP2-WT+ DMSO vs iPSC-CMPKP2-fs+ DMSO, not significant (p = 0.07), iPSC-CMPKP2-WT+ HC-067047 vs iPSC- CMPKP2-fs+ DMSO, p < 0.05, iPSC-CMPKP2-fs+DMSO vs iPSC-CMPKP2-fs+ HC-067047, p < 0.01.* p < 0.05, ** p < 0.01, *** p <0.001.

[0010] FIG 21A-21D FIG 21A) Representative regions of interest from line scans of TAX1BP3 iPSC-CMM78T / delwith DMSO or 4nM GSK2798745. FIG 21B) Quantification of spark frequency (mean ± s.e.m, n cells = 9; 10, M78T / del – DMSO vs. M78T / del – GSK2798745, p < 0.01) and calcium leak (mean ± s.e.m, n cells = 8; 10, M78T / del – DMSO vs. M78T / del – GSK2798745, p < 0.01). FIG 21C) Representative regions of interest from line scans of PKP2-fs with DMSO or 4nM GSK2798745. FIG 21D) Quantification of spark frequency (mean ± s.e.m, n cells = 5; 6, PKP2-fs – DMSO vs. PKP2-fs – GSK2798745, p < 0.05) and calcium leak (mean ± s.e.m, n cells = 5; 6, PKP2-fs – DMSO vs. PKP2-fs – GSK2798745, p = 0.0519), p < 0.01.* p < 0.05, ** p < 0.01, *** p <0.001. DETAILED DESCRIPTION

[0011] The inventors were presented with a family with early onset ACM that was genetically unexplained. Genome sequencing was used to identify a new gene associated with the disease and created cell line models (induced pluripotent stem cell derived cardiac myocytes) from the affected and unaffected family members and a conditional KO mouse model of the gene - TAX1BP3. It was found that a specific gene, transient receptor potential vanilloid 4 (TRPV4), is up-regulated in the cardiac myocytes from the iPSC-CMs and is associated with calcium mediated arrhythmic depolarizations from the myocytes (delated after depolarizations, DADs). The present disclosure proves that inhibiting TRPV4 suppresses the calcium leak, which drives the arrhythmias and suppresses all of the DADs seen in the cells.

[0012] In one aspect of the invention, any TRPV4 inhibitor, including for example HC-067047, GSK2193874, GSK2798745, RN-1734, RN-1747, RN 9893 hydrochloride, Piperidine-Benzimidazole, PF-05214030, amino bithiazlole antagonists, or any combination thereof serves as an inhibitor of calcium sparks in cardiac tissue, and thereby an inhibitor of calcium leaks, thereby an inhibitor of calcium waves, and thereby an inhibitor of unregulated twitching and thereby an inhibitor of arrhythmia.

[0013] In one aspect of the invention, any TRPV4 inhibitor as described above is a prophylactic compound that may prevent calcium leak in cardiac cells and thereby prevent the cascade of events leading to an arrhythmia.Atty Docket No.: 47381-00073

[0014] In one aspect of the invention, any TRPV4 inhibitor as described above, in the form of a pharmaceutical formulation may be administered to a patient diagnosed with arrhythmogenic cardiomyopathy (ACM) or another cardiac arrhythmia syndrome in order to prevent calcium leak in cardiac cells and thereby prevent the cascade of events leading to an arrhythmia

[0015] In one aspect of the invention, this prophylactic pharmaceutical composition may be administered to a subject diagnosed with a cardiac arrhythmia syndrome, the family member of a subject that has been diagnosed with a cardiac arrhythmia syndrome, or a subject suspected to have a cardiac arrhythmia syndrome.

[0016] In one aspect of the invention, a screening method for identifying a subject at risk for a cardiac arrhythmia syndrome is described. The screening method searching for a deletion or mutation of the tax1- binding protein 3 (TAX1BP3) gene of a subject or a deletion or mutation causing chronic upregulation of the TRPV4 gene.

[0017] In one aspect of the invention, a kit or method for identification of the TAX1BP3 gene is provided. The kit may include a labeled complementary sequence to the TAX1BP3 gene for identification of the presence or notable absence of the gene in standard gene testing protocols.

[0018] In one aspect of the invention, the TAX1BP3 gene is provided. In one aspect of the invention, TAX1BP3 as part of a panel for cardiac testing is provided.

[0019] In one aspect of the invention, an in vivo mouse model of arrhythmogenic cardiomyopathy is provided so as to, for example, evaluate new therapeutics for the presence or prevention of calcium leak, calcium sparks, calcium waves, unregulated twitching, arrhythmia or tachycardia or atrial fibrillation. Definitions

[0020] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.

[0021] Non-standard Abbreviations and Acronyms: Arrhythmogenic cardiomyopathy (ACM), tax1- binding protein 3 (TAX1BP3), transient receptor potential vanilloid type 4 (TRPV4), ryanodine receptor 2 (RyR2), undiagnosed diseases network (UDN), electrocardiogram (ECG), cardiac magnetic resonance imaging (CMRI), chromosomal microarray analysis (CMA), copy number variation (CNV), peripheral blood mononuclear cells (PBMCs), induced pluripotent stem cells (iPSCs), induced pluripotent stem cell-Atty Docket No.: 47381-00073derived cardiomyocytes (iPSC-CMs), phosphate buffered solution (PBS), immunofluorescence (IF), paraformaldehyde (PFA), sarcoplasmic reticulum (SR), desmoplakin (DSP), sodium calcium exchanger (NCX).

[0022] Articles “a” and “an” are used herein to refer to one or to more than one (i.e. at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0023] “About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result. The term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration, or percentage is meant to encompass variations of ±1% from the specified amount. The term “about X wt. %” can be taken to means that the wt. % may vary within 1 wt. % of the X reference numeral unless otherwise specified. That is if X is 5 wt. %, and thus about 5 wt. % may encompass between 4 wt. % and 6 wt % unless otherwise described as encompassing a different range.

[0024] The use herein of the terms "including," "comprising," or "having," and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

[0025] As used herein, the transitional phrase "consisting essentially of" (and grammatical variants) is to be interpreted as encompassing the recited materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. Thus, the term "consisting essentially of" as used herein should not be interpreted as equivalent to "comprising."

[0026] Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0027] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and includingAtty Docket No.: 47381-00073the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0028] As used herein, "treatment,” “therapy” and / or “therapy regimen” refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and / or the remission of the disease, disorder or condition.

[0029] As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disease, disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder or condition.

[0030] The term "effective amount" or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results.

[0031] As used herein, the term "administering" an agent, such as a therapeutic entity to an animal or cell, is intended to refer to dispensing, delivering or applying the substance to the intended target. In terms of the therapeutic agent, the term "administering" is intended to refer to contacting or dispensing, delivering or applying the therapeutic agent to a subject by any suitable route for delivery of the therapeutic agent to the desired location in the animal, including delivery by either the parenteral or oral route, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, intrathecal administration, buccal administration, transdermal delivery, topical administration, and administration by the intranasal or respiratory tract route.

[0032] The term “disease” as used herein includes, but is not limited to, any abnormal condition and / or disorder of a structure or a function that affects a part of an organism. It may be caused by an external factor, such as an infectious disease, injury, etc. or by internal dysfunctions, such as cancer, cancer metastasis, and the like. In some embodiments, the disease comprises cardiac disease. As used herein, the term “cardiac disease” includes those conditions, illnesses, and the like that effect the heart and / or cardiovascular system. Examples of cardiac disease include, but are not limited to, unstable angina, ischemia (e.g., resulting from a heart attack / myocardial infarction, heart failure, etc.), arrhythemia (including arrhythmic cardiomyopathy (ACM)), valve disease, high blood pressure, congenital heart conditions, inherited (e.g., genetic) heart conditions, and the like.

[0033] As used herein, the term "subject" and "patient" are used interchangeably herein and refer to both human and nonhuman animals. The term "nonhuman animals" of the disclosure includes allAtty Docket No.: 47381-00073vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. The methods and compositions disclosed herein can be used on a sample either in vitro (for example, on isolated cells or tissues) or in vivo in a subject (i.e. living organism, such as a patient). In some embodiments, the subject comprises a human who is undergoing treatment as prescribed herein.

[0034] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0035] EXAMPLES

[0036] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred aspects and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.

[0037] Example 1: TAX1BP3 is associated with autosomal recessive pediatric-onset ACM

[0038] Disclosed herein is identification of the cause of genetically elusive ACM in kindred presenting with pediatric ACM. The kindred pedigree and selected cardiac MRI (CMRI) images are shown in FIG 1A-1B. The proband was a White male (II.1) with no significant past medical history who died suddenly at 17 years of age while playing baseball. The autopsy showed that he had undiagnosed ACM. The proband’s two brothers (II.2 and II.4) were subsequently diagnosed with ACM. Electrocardiograms (ECGs) are included in FIG 2. The proband’s sister (II.3) and biological parents (I.1 and I.2) were found to have no evidence of ACM after extensive testing including CMRI, ECG, and Holter monitoring. A next generation sequencing panel for variants in 13 known ACM-associated genes did not identify a diagnostic genetic variant. The two surviving brothers were subsequently evaluated through the Undiagnosed Diseases Network (UDN). Exome sequencing (ES) through the UDN identified a paternally inherited deletion of approximately 238 kb encompassing the entire TAX1BP3 (MIM# 616484) gene locus and a maternally inherited hemizygous missense variant in TAX1BP3. (NM_014604.4:c.233T>C, p.M78T) in both brothers (II.2 and II.4). The missense variant was confirmed by Sanger sequencing (FIG 1C-1E). The 17p13.2 deletion was confirmed by chromosomal microarray analysis (CMA) and reported as 242 kb (chr17:3,392,348-3,634,096, FIG 3). Due to the previous report of TAX1BP3 biallelic variants in a sibling pair with dilated cardiomyopathy and septo-optic dysplasia, ophthalmologic exams were obtained in both affected brothers and were normal. Both brothers had largely normal evaluations asideAtty Docket No.: 47381-00073from the cardiac phenotype (FIG 4). The deceased proband (II.1) was confirmed to have both the 242 kb deletion and the p.M78T missense variant through CMA and Sanger sequencing on blood that had been stored by the state laboratory following the autopsy. The unaffected sister (II.3) was found to not have either of the two variants. Therefore, the two TAX1BP3 variants co-segregated with disease in an autosomal recessive manner.

[0039] TAX1BP3 is ubiquitously expressed, including in cardiac myocytes (v.19.3.proteinatlas.org). It is a small protein of 124 amino acids (13.74 kDa) and consists of a single PDZ domain. These domains are common protein interaction sites, and TAX1BP3 has been shown to physically interact with other proteins with this domain such as ß-catenin and rhotekin. The p.M78T variant localizes within this PDZ domain in an area with high sequencing conservation across species (86.7%), and a GERP++RS score of 5.31 (FIG 1F-1G).

[0040] TAX1BP3 is a tolerant gene with an RVIS score of 45 and pLI of 0.005. The p.M78T variant was not identified in ~13,000 internal controls, gnomAD, or ClinVar databases. In silico modeling also suggests that the p.M78T variant would be deleterious to protein function (FIG 5). Taken together, these results suggest that biallelic variants in TAX1BP3 are associated with autosomal recessive ACM.

[0041] Example 2: iPSC-CMM78T / deldemonstrate myocardial remodeling with adipogenesis

[0042] To study the role TAX1BP3 in the heart, we generated induced pluripotent stem cells (iPSCs) from the patient peripheral blood mononuclear cell (PBMC) lines from the two surviving affected brothers (II.2 and II.4, p.M78T / del) and the sister (II.3,WT / WT) to use as the control. We validated that the iPSC lines expressed iPSC markers OCT4 and NANOG (FIG 6A-6B). We then differentiated the iPSCs into cardiomyocytes (iPSC-CMs) and confirmed the TAX1BP3 M78T variant status in the iPSC-CMs by Sanger sequencing (FIG 7A). Reduction of TAX1BP3 protein was confirmed in the iPSC-CMs with Western blot (FIG 8A-8B) Differentiation was validated by immunofluorescence of cardiomyocyte markers TNNT2 and ACTC2 and robust beating of cells in the dish (FIG 7B). Because ACM is associated with adipogenesis, we hypothesized that the affected iPSC-CMs would be susceptible to fat accumulation. We applied the 3 factor (3F) reagent protocol to stimulate adipogenesis and found that this media resulted in fat aggregates in both the iPSC-CMWT / WTand iPSC-CMM78T / dellines, but the iPSC-CMM78T / delline had more than double the quantity of fat accumulation (FIG 8C-8D). The increase in fat in the iPSC- CMM78T / dellines suggests that genetic loss of TAX1BP3 predisposes iPSC-CMs to undergo adipogenic remodeling of the cardiac myocytes, reflective of a distinguishing property of ACM. We applied the 3 factor (3F) reagent protocol to stimulate lipogenesis and found that this media resulted in lipid droplets inAtty Docket No.: 47381-00073both the iPSC-CMWT / WTand iPSC-CMM78T / dellines, but the iPSC-CMM78T / dellines had more than double the quantity of fat accumulation (FIG 8C). To validate that the protocol was indeed inducing a lipogenesis- state, we performed RT-qPCR for lipogenesis genes PPARG and CEBPA and found that both were upregulated following treatment with the 3F media (FIG 8E).

[0043] Example 3: iPSC-CMM78T / deldemonstrates pro-arrhythmogenic Ca2+leak from the sarcoplasmic reticulum

[0044] Given the marked arrhythmogenic phenotype of the genotype-positive kindred, we hypothesized that the iPSC-CMsM78T / dellines would have a Ca2+-overload state marked by pathologic Ca2+leak from the SR store – a known mechanism of arrhythmogenesis in other ACM models. To assess Ca2+dynamics, we examined calcium transients using live cell calcium imaging finding that iPSC-CMM78T / delpaced at 0.5Hz demonstrated increase Ca2+transient amplitude, decreased tau of decay, and trend towards increased NCX activity compared to iPSC- CMWT / WT(FIG 9A-9B). In addition, SR store Ca2+was smaller in iPSC-CMM78T / delcompared to iPSC-CMWT / WT(FIG 9C-9D). We also found that iPSC- CMM78T / dellines also had increased diastolic Ca2+as measured by Fura-2 AM 340 / 380 ratios suggesting an overall Ca2+-overload state of the cardiac myocyte (FIG 9E). Together, these results suggest that the variants in TAX1BP3 result in shifting of the intracellular Ca2+out of the SR store and into the cytosol. We next attempted to identify the cause of this shift, hypothesizing that SR store Ca2+-leak would be increased in the mutant cells. Indeed, we found that the iPSC-CMM78T / dellines have greater spark frequency and Ca2+leak from the SR via RyR2(FIG 10A-10B). This difference was more pronounced when this leak was normalized to the relatively low SR store to account for the total amount of Ca2+available (FIG 10C). The iPSC-CMM78T / dellines also trended towards a greater percentage of cells with spontaneous Ca2+release during pacing. Specifically, iPSC-CMM78T / delells exhibited an increased propensity for delayed-after depolarization-like spontaneous Ca2+release events (FIG 11A-11B), a known cellular etiology for triggered arrhythmogenesis of the myocardium. Moreover, some iPSC-CMM78T / delcells also demonstrated significant runs of spontaneous Ca2+release events (FIG 11C). These findings suggest that the mechanism of reduced SR store and the arrhythmogenesis in the affected kindred from TAX1BP3 loss-of-function is due to Ca2+leak from RyR2.

[0045] Example 4: TRPV4 current is activated in iPSC-CMM78T / del

[0046] Given that the function of TAX1BP3 in the heart is unknown, we took a nonbiased approach to begin investigating how TAX1BP3 may be modulating intracellular Ca2+leak. We performed bulk differential expression RNA-sequencing on iPSC-CMs from the two iPSC- CMM78T / dellines and the iPSC-Atty Docket No.: 47381-00073CMWT / WTline. Due to the known role of Wnt signaling in ACM and that TAX1BP3 has been shown to bind to ß-catenin in other tissues, we first looked at canonical Wnt signaling transcripts. There were no significant differences in canonical Wnt signaling mRNA expression between the iPSC-CMM78T / dellines and the iPSC-CMWT / WTline (FIG 12). Following subsequent manual curation of the differentially expressed genes that were consistent between both affected lines, we identified TRPV4 as being increased in iPSC- CMM78T / dellines compared to iPSC-CMWT / WT(FIG 13A). TRPV4 is a nonselective cation channel permeable to Ca2+. This finding was of particular interest as increased TRPV4 current activity has been linked with increased Ca2+ spark frequency in cerebral arteries and aged mouse ventricular myocytes28, and antagonism of TRPV4 suppressed atrial fibrillation in rats which has been linked with SR Ca2+ leak23. To test for TRVP4 functionally, we performed whole cell patch clamp. We found that iPSC- CMWT / WThad minimal TRPV4 current (ITRPV4) at basal conditions as well as following application of GSK1016790A, a potent and specific TRPV4 agonist. Conversely, iPSC- CMM78T / deldemonstrated a large increase in ITRPV4 following application of GSK1016790A (FIG 13B-13E). Taken together with our previous findings, this suggests that TAX1BP3 loss results in induction of functionally active TRPV4 expression..

[0047] Example 5: TRPV4 activity is increased in iPSC-CMM78T / del

[0048] To test if this change in expression and localization affect TRVP4 functionally, we performed whole cell patch clamp. We found that iPSC-CMWT / WThad minimal TRPV4 current (ITRPV4) at basal conditions as well as following application of GSK1016790A, a potent and specific TRPV4 agonist. Conversely, iPSC-CMM78T / deldemonstrated a large increase in ITRPV4 following application of GSK1016790A (FIG 13C-13F). Taken together with our previous findings, this suggests that TAX1BP3 loss-of-function variants results in induction of functionally active TRPV4 expression.

[0049] Example 6: Ca2+leak can be suppressed by TRPV4 inhibition

[0050] We now demonstrate that TRPV4 inhibition would rescue the increased Ca2+leak in iPSC- CMM78T / dellines. To test this, we added a TRPV4-specific inhibitor small molecule HC- 067047 to the iPSC-CMs and found a reduction of Ca2+spark frequency and leak in the iPSC- CMM78T / delcells. This reduction was equivalent to untreated iPSC-CMWT / WTCa2+leak levels (FIG 14A-14D). We next evaluated whether application of HC-067047 would suppress spontaneous Ca2+release events in iPSC- CMM78T / dellines. Indeed, we found that ITRPV4 inhibition also suppressed spontaneous Ca2+release events similar to the levels of untreated iPSC-CMWT / WTcells (FIG 14E-14H). These findings suggest that loss of TAX1BP3 leads to increased Ca2+leak, and this can be prevented by TRPV4 antagonism. WhenAtty Docket No.: 47381-00073combined with our previous findings, these results suggest that loss of function mutations in TAX1BP3 result in increased TRPV4 expression, increased ITRPV4, and increased store Ca2+leak which allows for DAD-mediated ventricular arrhythmias (FIG 15). Furthermore, iPSC-CMM78T / delcells exhibited an increased propensity for delayed- afterdepolarizations, a known cellular etiology for triggered arrhythmogenesis of the myocardium, which was also ameliorated by TRPV4 inhibition (FIG 14C-14F). These findings indicate that loss of TAX1BP3 leads to increased Ca2+leak, and this can be prevented by TRPV4 antagonism.

[0051] Example 7: Inducible, cardiac-specific Tax1bp3 knockout mice have increased Ca2+ leak and ventricular arrhythmias

[0052] To determine the effect of TAX1BP3 loss in an in vivo model, we generated an inducible, cardiomyocyte-specific Tax1bp3 knockout mouse (Tax1bp3- / -) by crossing mice harboring Tax1bp3 alleles with loxP sites flanking exons 2-4 with the alpha-MHC MerCreMer (MCM) mouse (FIG 16A). Cre induction resulted in a reduction of Tax1bp3 protein from ventricle myocardium while not altering Tax1bp3 expression levels in non-cardiac organs (FIG 16B). To test for evidence of cardiomyopathic remodeling, we performed transthoracic echocardiograms of conscious mice and processed Tax1bp3- / -hearts for myocardial histology analysis to look for evidence of cardiac remodeling. There were no differences in the LV dimensions or systolic function by echocardiogram (FIG 17). We found no significant changes in the LV dimensions, ventricular wall thickness, or other structural abnormalities by H&E staining of the whole hearts. Further, we found no evidence of myocardial fibrosis, and no evidence of adipose deposition / remodeling, as assessed by Masson’s Trichrome and Oil Red-O staining, respectively (FIG 18). Given the evidence of Ca2+-mediated arrhythmogenesis in the iPSC-CM models, we next assessed whether reduced Tax1bp3 expression causes increased Ca2+leak from the SR. To do this, we Langendorff-perfused hearts from Tax1bp3- / -and Tax1bp3+ / +mice to isolate ventricular myocytes and then performed live-cell Ca2+imaging following Cal520 dye loading. We found that primary ventricular myocytes from Tax1bp3- / -mice had greater Ca2+spark frequency and Ca2+leak compared with Tax1bp3+ / +mice (FIG 16C and 16D). To evaluate arrhythmia inducibility in vivo, we next performed both ECG and intracardiac electrophysiology studies with programmed electrical stimulation. While there were no differences in surface ECG measurements (FIG 17), 30% of Tax1bp3- / -mice had sustained ventricular tachycardia immediately following programmed electrical stimulation of the ventricular myocardium, while Tax1bp3+ / +mice demonstrated no arrhythmias (FIG 16E-16G). Taken together, these data indicateAtty Docket No.: 47381-00073that loss of Tax1bp3 does not result in heart failure or cardiomyopathic remodeling yet increases ventricular arrhythmia predisposition and intracellular Ca2+leak, in vivo at 6 months.

[0053] Example 8: TRPV4 localizes to the dyad in Tax1bp3- / -mice

[0054] Given our findings in iPSC-CMs that ITRPV4 is induced with loss of TAX1BP3 expression, we hypothesized that this is due to re-localization of TRPV4 to the sarcolemma where ITRPV4 was detected. To test this, we performed immunofluorescence staining for TRPV4 which showed that the TRPV4 expression localization is primarily perinuclear and nuclear in control iPSC-CMWT / WT. Conversely, in iPSC-CMM78T / del, TRPV4 localized to the sarcolemma and co-localizes with desmoplakin (DSP), a cardiac desmosomal protein (FIG 13F). Similar IF staining for Trpv4 in Tax1bp3- / -ventricular myocytes revealed that Trpv4 was localized to the t-tubular sarcolemma in close proximity with RyR2. This was in stark contrast to Trpv4 staining in Tax1bp3+ / +mice, which showed Trpv4-positive fluorescence in the nucleus and perinuclear area without clear colocalization with RyR2 (FIG 13 G and FIG 19). Overall, these findings suggest that TRPV4 changes subcellular localization from predominantly nuclear / perinuclear to the T-tubular sarcolemma in iPSC-CMM78T / deland Tax1bp3 KO, compared to respective controls.

[0055] Example 9:TRPV4 inhibition rescues Ca2+ leak in Tax1bp3- / -mice

[0056] Based on the observation that TRPV4 inhibition normalized Ca2+-mediated DADs and SR store leak, we hypothesized that it would also reduce aberrant Ca2+leak and spontaneous Ca2+release events (SREs) in Tax1bp3- / -myocytes. To test this, we applied HC-067047 to isolated primary ventricular myocytes derived from Tax1bp3- / -mice. We found that application of HC- 067047 normalized the elevated Ca2+leak in the isolated ventricular myocytes from Tax1bp3- / -mice to approximately WT levels (FIG 20A and 20B). Furthermore, we observed a marked reduction in SREs from Tax1bp3- / -ventricular myocytes, again to WT levels (FIG 20C and 20D). Overall, these findings suggesting that TRPV4 inhibition decreases the aberrant Ca2+leak and SREs which is the cellular substrate for triggered ventricular arrhythmias in these mice.

[0057] Example 10:TRPV4 inhibition rescues Ca2+ leak in iPSC-CMs derived from a PKP2- positive ACM donor

[0058] To explore if TRPV4-mediated Ca2+leak could be an underlying phenomenon in other patients with ACM, we moved to an iPSC-CM model derived from an ACM patient with a heterozygous PKP2- His773AlafsX8 (c.2197_2202delCACACCinsG) variant in PKP2 (iPSC-CMPKP2-fs). The donor has early onset ventricular tachycardia requiring ICD placement. The loss-of- function variant in PKP2 represents the most commonly observed genetic subtype of ACM and is diagnostic by current ACMG criteria.30Atty Docket No.: 47381-00073Importantly, ACM-associated variants in PKP2 have been previously linked with Ca2+leak from RyR2.21To determine whether these cardiac myocytes demonstrated increased Ca2+leak, we first conducted live cell Ca2+imagining and found that iPSC-CMPKP2-fshad greater Ca2+leak from the SR, driven largely by increased spark frequency, when compared to a sex-matched, healthy control iPSC-CM line (iPSC- CMPKP2-WT). Strikingly, exposure of these cells to HC-067047, and resultant TRPV4 inhibition suppressed normalized it to that of WT control levels (FIG 20E and 20F). When combined with ourprevious findings, these results suggest that loss of TAX1BP3 results in increased TRPV4 expression and translocation to the t-tubules, increased ITRPV4, and increased store Ca2+leak which allows for DAD- mediated ventricular arrhythmias (FIG 15). Finally, given the ability of TRPV4 inhibition to suppress Ca2+leak in both the iPSC-CMM78T / deland Tax1bp3- / -mouse model, as well as a separate ACM iPSC-CM patient cell line derived from a donor with PKP2-mediated disease, this strategy could be a novel therapeutic target for treating arrhythmias associated with ACM more broadly

[0059] Example 11: Tax1bp3- / -are susceptible to inducible ventricular tachycardia

[0060] The results of an intracardiac electrophysiology study are shown in FIG 16E-16G. Briefly, after measurement of surface electrocardiograms (ECGs), the attire and ventricle are paced and arrhythmias are observed in response to caffeine and isoproterenol. To complete the study, ECGs are remeasured and atrial and ventricular pacing repeated. Tax1bp3- / -are susceptible to inducible ventricular tachycardia whereas Tax1bp3+ / -and Tax1bp3+ / +are not.

[0061] Example 12: TRPV4 inhibitors demonstrate reduction in calcium leak in multiple cell strains

[0062] As shown in FIG 21A-21D, the effect of reducing calcium sparks and reducing calcium leak in iPSCs is not specific to the genetic mutation used to derive the cardiac arrhythmia.

[0063] Common Materials and Methods

[0064] Clinical evaluation

[0065] The siblingship was evaluated at the UDN at the Duke / Columbia (https: / / undiagnosed.hms.harvard.edu / ) site. This study was approved by the central institutional review board at the National Human Genome Research Institute (NHGRI, 15-HG-0130) and locally by the Duke University IRB (Pro00056651). Informed consent was obtained from each participant or participant’s parent / legal guardian in accordance with the Declaration of Helsinki. Prior to UDN participation, the participants had been diagnosed by their local providers with ACM based on the modified Task Force criteria.12Specifically, pertinent demographics, personal and family history, physical examination wereAtty Docket No.: 47381-00073performed by both board certified pediatric medical geneticists and pediatric cardiologists. Standard 12- lead ECG analysis, echocardiographic testing, and CMRI were obtained. Pre-UDN ACM gene-panel analysis included the following genes: CTNNA3, DES, DSC2, DSG2, DSP, FLNC, JUP, LDB3, LMNA, PKP2, PLN, RYR2, SCN5A, TGFB3, TMEM43, and TTN.

[0066] Exome sequencing

[0067] Exome sequencing (ES) was performed at the UDN sequencing core laboratory in the Baylor College of Medicine, with methods previously published. On the younger affected brothers and both parents, the ES underwent a secondary analysis through a phenotype-agnostic research pipeline at the Duke / Columbia UDN clinical site, with methods described previously. In addition to the 242 kb deletion described above (chr17:3,392,348-3,634,096), the youngest brother (II.4) was found to have two additional deletions on CMA: a 592 kb deletion of unknown significance at 3p26.3 (chr3:858,547- 1,450,741) and a likely pathogenic deletion at 14q23.3 (chr14:67,198,080-67,285-061), which could be related to his non-cardiac findings of learning disabilities. To confirm the absence of the identified missense variant in ostensibly healthy individuals, the publicly-available Genome Aggregation Database (gnomAD) was used as a control cohort which is comprised of a total of 15,708 genomes and 125,748 exomes from 141,456 individuals. The variant was cross-referenced in ClinVar (National Center for Biotechnology Information). Sanger sequencing confirmation of the TAX1BP3 locus was performed on all kindred. For this, genomic DNA was extracted according to the ReliaPrep™ Blood gDNA Miniprep System kit (A5082, Promega). Primers are available on reasonable request. Polymerase chain reaction (PCR) was conducted using the 2.0X Taq Red Master Mix kit (42 -138, Genesee) and the subsequent product was sequenced using direct Sanger sequencing with BigDye Terminator v1.1 (43-374-50, Applied Biosystems) according to the manufacturer's protocol and electrophoresed on an 3730xl DNA Analyzer (3730XL, Applied Biosystems).

[0068] Chromosomal microarray analysis

[0069] Chromosomal microarray analysis (CMA) was performed using the Affymetrix Cytoscan HD array (901835) at the clinical cytogenetics laboratory at Duke University. This array includes about 2.7 million genetic markers incorporating 743,304 single nucleotide polymorphism probes as well as 1,953,246 non-polymorphic copy number variation (CNV) probes with a median spacing of 0.88 kb. Genomic linear positions are given relative to NCBI build 37 (hg19).

[0070] Sequence homology and domain mappingAtty Docket No.: 47381-00073

[0071] TAX1BP3 consensus sequence (NP_055419.1) was obtained from the Ensembl browser. Primary sequence conservation among 194 independent TAX1BP3 orthologues was compared to determine degree of conservation across species using the National Center for Biotechnology Information (NCBI). Percent identity was calculated as the proportion of species with predicted TAX1BP3 orthologues containing an identical amino acid at a given position relative to human.

[0072] In silico variant pathogenicity modeling

[0073] In silico variant pathogenicity prediction was performed using PolyPhen-2, SIFT, PANTHER, Provean, and MUpro, and Revel prediction tools.

[0074] Peripheral blood mononuclear cell proliferation

[0075] Peripheral blood samples were diluted in an equal volume of 1X Phosphate buffered solution (PBS) with 2% FBS and added to SepMate tubes (85450, Stemcell Technologies) loaded with Lympoprep (07851, Stemcell Technologies). Following manufacturer’s protocol, isolated peripheral blood mononuclear cells (PBMCs) were cultured in Erythroid Expansion Media: Stemspan SFEM II (09655, Stemcell Technologies) with Erythroid Expansion Supplement (02692, Stemcell Technologies) and 100μg / ml Primocin (ant-pm-1, InvivoGen). The PBMCs were grown for 24 hours before being re-plated, the media was changed every 2 days, and cells were cultured for a total of 6 days before being cryopreserved.

[0076] Generation of iPSCs

[0077] Human induced pluripotent stem cells (iPSCs) were generated from the expanded PBMC lines through the Human Stem Cell Core at Baylor College of Medicine. To initiate reprogramming, approximately 1x10^5 erythroid progenitors were spinfected at 1,040 x g for 90 minutes with non- integrating Sendai viruses expressing OCT4, SOX2, KLF4, and C-MYC by CytoTune-iPS 2.0 (A16517, Invitrogen). Clonal human iPSC colonies were derived under feeder- free conditions using hESC-qualified Matrigel (354230, Corning) and ReproTeSR medium (05926, Stemcell Technologies). Individual colonies were manually picked and expanded in TeSR-E8 medium (05990, Stemcell Technologies).24Lines of iPSCs were validated by colony morphology, g-banding karyotyping (Baylor Medical Genetics Lab) to confirm normal diploid chromosomes and were tested for mycoplasma. Two clones per iPSC line were used for downstream experiments to control for interline variability.

[0078] iPSC culture and differentiation of cardiomyocytes

[0079] Cells were plated in mTeSR media (85857, StemCell Technologies) with 10% CloneR (StemCell Technologies) on a 2% Matrigel-coated (354230, Corning) 6 well plate (353036, Corning).Atty Docket No.: 47381-00073After 24 hours, mTeSR without CloneR was used and changed daily. Cells were passaged by release from the Matrigel coating with ReLeSR (05873, StemCell Technologies). iPSCs were seeded to Matrigel-coated wells. iPSCs were differentiated into iPSC-CMs utilizing a method adapted from Burridge et al, 2014.25Day 0 of differentiation began when iPSCs were 80-90% confluent with the addition of 60ng / μl Activin A (78001.1, StemCell Technologies), 12uM CHIR 99021 (13122, Cayman Chemical), 50ug / ml Ascorbic Acid (Tocris Bioscience, Bristol, UK) in RPMI 1640 with 2% B27 without insulin (A1895602, Life Technologies) (RPMI / B27 -). On day 1 and day 2, the cells were given RPMI / B27- with 5μM IWR1 (StemCell Technologies) and 50μg / ml Ascorbic Acid. On day 4, the media was changed to RPMI / B27- with 5μM IWR1. From day 6 onwards the cells are given RPMI 1640 (11875199, Thermofisher Scientific) with 2% B27 with insulin (17504044, Life Technologies) every other day and observed for beating. iPSC-CMs were used for downstream experiments after day 30 of differentiation.

[0080] Adipogenesis in iPSC-CMs

[0081] To drive fatty acid oxidation from glycolysis, we supplemented insulin, dexamethasone, and 3-isobutyl-1- methylxanthine (IBMX, 3F media) to iPSC-CMs.18This media was applied when the iPSC- CMs were day 30 of differentiation and treated for 30 days every other day. To visualize fat accumulation, the iPSC-CMs were stained with Nile Red and co-stained with TNNT2. Cells were imaged using a Zeiss LSM 510 confocal microscope: 4.5mm x 4.5mm areas were imaged by taking a 5 x 5 tile scan at 10X magnification.

[0082] Western blot

[0083] iPSC-CMs were lysed with RIPA buffer and 30μg of lysate loaded onto a polyacrylamide gel and run for 35min at 200V at 4C. Proteins were transferred to a nitrocellulose membrane. The membrane was blocked for 1 hour at room temperature with 5% milk. The membrane was blotted overnight at 4C with mouse anti-GAPDH (1:2000, AKR-001, Cell Biolabs) and rabbit anti- TAX1BP3 (1:1000, ab155992, Abcam) antibodies. The next day, the membrane was blotted with goat anti-mouse and anti- rabbit HRP secondaries (1:2000) for 1 hour at room temperature. ECL substrate was added before imaging (32106, Thermofisher Scientific).

[0084] Ca2+imaging and analysis

[0085] To conduct live cell Ca2+imaging, iPSC-CMs were washed once with 1X Ca2+-free Tyrode solution and then were stained with 10µM CAL-520 (ab171868, Abcam) for 1 hour. iPSC-CMs were then incubated with a 1:1 solution of RPMI / B27+ and 1X Tyrode with 1.8mM CaCl2 for 30 minutes priorAtty Docket No.: 47381-00073to imaging. Line scans of iPSC- CMs were acquired on a Zeiss Laser Scanning Confocal 510 Meta Microscope (Carl Zeiss AG) at 0.1μm per pixel. iPSC-CMs were paced at 0.5Hz with an IonOptix MyoPacer field stimulator (IonOptix) for ten seconds. Fiji ImageJ v.1.53c (National Institutes of Health) was used to analyze Ca2+transients. Fiji was used in conjunction with the 2007 SparkMaster plug-in to quantify sparks.19SparkMaster calculated and displayed a set threshold for quantifying sparks and their features at 3.8 times the standard deviation of surroundings. Features include spark duration, width, time to peak, amplitude and tau. Regions of interest were generated 0.22 seconds from the last paced beat. The average amplitude, width, and duration at half the maximum of all the sparks for a cell were multiplied with the spark frequency to generate a total leak value per cell. For the calcium TRPV4 inhibition experiments, DMSO or 1μM HC-067047 (4100, Tocris) was added in 1:1 solution of RPMI / B27+ and 1X Tyrode with 1.8mM CaCl2. For caffeine analysis, cells were imaged with a Zeiss spinning disk Axio Observer.Z1 at 215ms intervals at 40X.10mM caffeine was added to the iPSC-CMs after a 10 second pause following 10 seconds of 1Hz pacing. For Fura-2 imaging, iPSC-CMs were loaded with 10µM Fura-2;AM (AS-84017, Anaspec) in calcium-free 1X Tyrode containing 0.1% Pluronic F- 127 (P2443, Sigma-Aldrich) for 30 minutes and then incubated in 1:1 solution of RPMI / B27+ and 1X Tyrode with 1.8mM CaCl2for 30 minutes prior to imaging. Experiments were performed at room temperature. Cells were imaged with a Nikon TE2000-E with 340 / 380 dual excitation filter. The iPSC- CMs were paced for ten seconds at 0.5Hz first before data collection. The MetaFluor software (Molecular Devices) was used to generate 340 / 380 ratios. NCX activity was calculated as 1 / taucaffeine.

[0086] Immunofluorescence (IF)

[0087] To fix the iPSC-CMs, 4% paraformaldehyde (PFA) was applied for 15 minutes. 0.5% TritonX-100 (v / v, in PBS) was added for 15 minutes to the slide for permeabilization. A blocking solution of 3% (w / v) bovine serum albumin in PBS was added for 30 minutes. The primary antibody solution of 1:300 mouse anti-TNNT2 (TNNT2, ab209813, Abcam), 1:1000 anti-Nanog (sc-374001, Santa Cruz Biotechnology), or 1:1000 anti-Oct3 / 4 (sc-5279, Santa Cruz Biotechnology) into 1% BSA in PBS and incubated at 4°C overnight. The secondary antibody solution of Alexa Fluor 488-conjugated AffiniPure donkey anti-mouse IgG (715-545-150, Jackson Immunoresearch) was diluted 1:1000 in PBS and incubated with the cells at room temperature for an hour. Nile Red Stain (ab228553, Abcam) was also added 1:4 to the entire secondary solution if required. The cells were covered in VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (H-1700-10, Vector Laboratories) and stored at 4°C. For Nile Red image analysis, 5x510x tilescans with z-stacks to include all cells were obtained per dish.Atty Docket No.: 47381-00073Fluorescence of TNNT2 signal (green) and Nile Red (red) was thresholded and quantified using FIJI, and the percentage of Nile Red was normalized to the percentage of TNNT2 to account for variability in cellnumber

[0088] Patch clamp

[0089] Whole cell voltage-clamp experiments were performed to record membrane current using aMultiClamp-700A amplifier with Digidata 1322A interface and Clampex software (Axon Instruments) as described previously.20The cover-glass with iPSC-CM cells was continuously perfused (at 2ml / min, room temperature) with external solution containing (in mM): 150 NaCl, 6 CsCl, 5 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, (pH 7.4 with NaOH). The patch pipettes had a resistance of 3~6 MΩ when filled with pipette solution contained (in mM) 100 Cs-aspartate, 20 CsCl, 1 MgCl2, 4 Na2-ATP, 10 HEPES, 10 EGTA, and 0.08 CaCl2 (pH 7.2 with CsOH). To block LTCC,

[0090] Nifedipine (10μM) was added in external solution. Cells were held at a potential of 0 mV, and ramps from +100 mV to -100 mV with a duration of 400 ms were applied at a frequency of 0.1 Hz. Data were acquired at sample rate of 10 kHz (filtered at 1 kHz). After stabilization, a TRPV4 specific agonist GSK 1016790A (1μM, Sigma- Aldrich) was used to stimulate TRPV4 current (ITRPV4). Membrane currents were normalized by cell membrane capacitance(pA / pF) and leak subtracted. The differences (at + / - 80mV) before and after GSK 1016790A administration were compared in WT and mutant iPSC-CM cells.21Action potentials were recorded in TAX1BP3WT / WTand TAX1BP3M78T / deliPSC-CMs. Patch-clamp experiments were performed in whole cell current- clamp mode using a MultiClamp-700A amplifier with Digidata 1322A interface and Clampex software (Axon Instruments). Cells on cover-glass were continuously perfused (at 2ml / min) with Tyrode’s solution containing (in mM): 140 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, pH 7.4.). The patch pipettes had a resistance of 6~10 MΩ when filled with pipette solution contained (in mM) 130 K-aspartate, 6 NaCl, 3 Mg-ATP, 0.4GTP,10 HEPES, 5 EGTA, and pH 7.2 (with KOH). Experiments were performed at 35.0 + / - 0.5 Cº. Data were acquired at sample rate of 10 kHz (filtered at 1 kHz) and analyzed with pCLAMP 10 software (Axon Instruments).

[0091] RNA-sequencing

[0092] Bulk RNA-sequencing was conducted on RNA from iPSC-CMs which included 3 iPSC experimental replicates (three different passages) per clone for each of the 2 clones per line. RNA was extracted using Direct-Zol RNA Purification Kit (R2052, Zymo Research). The Sequencing and Genomics Technologies Core Facility at Duke University performed RNA- sequencing. mRNA wasAtty Docket No.: 47381-00073enriched from total RNA and reversed transcribed into cDNA to build sequencing libraries using the Kapa mRNA HyperPrep Kit from Roche (Code: KK8581). Libraries were pooled to equimolar concentration and sequenced on the NovaSeq 6000 S1 flow cell to produce 50 bp paired-end reads.

[0093] RNA-seq data was processed using the fastp toolkit to trim low-quality bases and sequencing adapters from the 3’ end of reads, then mapped to GRCh38 (downloaded from Ensembl, version 106) using the STAR RNA-seq alignment tool, and reads aligning to a single genomic location were summarized across genes. For genes having an overlap of at least 10 reads, gene counts were normalized and differential expression was carried out using the DESeq2 Bioconductor package implemented for the R programming environment (www.r- project.org). Consistent with the recommendation of the DESeq authors, independent filtering was utilized prior to calculating adjusted p-values and moderated log2 fold-changes were derived using the ashr package.

[0094] Generation of Tax1bp3 knockout mouse

[0095] Mice with the Tm1c allele with loxP sites flanking Tax1bp3 exons 2-4 were obtained by the European Mouse Mutant Archive (EMMA). These mice were crossed with alpha-MHC-MerCreMer mice to create an inducible, cardiac-specific Tax1bp3 knockout line. Pups injected with 100μl of 10mg / ml tamoxifen (T5648, Sigma-Aldrich) at 1.5 months once a day for 3 days for induction of Cre. Experiments were conducted when mice were 5.5-6 months of age.

[0096] Mouse experimental design

[0097] Mice were kept in standard house conditions with standard light cycles with standard ad lib food and water. All mice were treated in accordance with the Duke University School of Medicine Institutional Animal Care and Use Committees and reflected proposed guidelines for rigor and reproducibility in preclinical animal studies.30,31 Exclusion criteria were 1) non-experimental mice used for mating and 2) mice that were homozygous for Cre or WT for Cre for both alleles. All experimental protocols were prospectively written. All studies were conducted blinded to mouse genotype unless otherwise noted. No randomization was performed. Littermate Tax1bp3+ / + mice that were heterozygous for MCM allele and treated with tamoxifen were used for controls for all experiments. Both males and females were utilized in equal proportions. Unless otherwise noted, for all studies, mice were anesthetized in an induction chamber with 2% isoflurane mixed with 1 L / min 100% O2 for 10-15 minutes until the withdrawal reflex was absent (R583S Vaporizer, RWD Life Science, San Diego, CA).

[0098] Ventricular myocyte isolationAtty Docket No.: 47381-00073

[0099] Langendorff perfusion protocol was adapted from O’Connell et al.32 Briefly, mouse hearts were rapidly excised and aorta canulated and perfused with Tyrode solution (130 mM NaCl, 5.4 mM KCl, 2 mM NaHCO3, 1.2 mM MgSO4, 10 mM HEPES, and 15 mM Glucose, pH 7.35) for 5 minutes before being transferred to a Langendorff apparatus for perfusion with 2.4mg / ml Collagenase II in Tyrode for 7- 10 minutes. The heart was then removed and placed in stopping buffer (10% FBS in Tyrode) and manually triturated. Large debris was separated and the single cell suspension supernatant was then used for either Ca2+imaging or immunofluorescence experiments.

[0100] Immunofluorescence

[0101] Isolated ventricular myocytes were plated onto 200μg / ml laminin coated coverslips for one hour before fixing in 4% paraformaldehyde (PFA) was applied for 10 minutes.0.5% Triton X-100 (v / v, in PBS) was added for 10 minutes to the slide for permeabilization. A blocking solution of 3% (w / v) bovine serum albumin in PBS was added for 30 minutes. Rabbit anti-TRPV4 (ACC-034, Alomone Labs) was used at 1:500 and mouse-anti RyR2 (CBL173, EMD Millipore) was used at 1:100 and incubated overnight at 4 degrees Celsius. Secondaries were used at 1:1000 and incubated for 1 hour at room temperature (Alexa Fluor 594-AffiniPure Goat Anti-Rabbit IgG and Alexa Fluor 488-AffiniPure Goat Anti-mouse IgG, Jackson Immunoresearch, 111-585-003 and 715-545-150). 1:10000 Hoechst 33258 (Thermofisher Scientific, H21491) was incubated for 5 minutes prior to mounting with Permount Mounting Medium (Thermofisher Scientific, SP15100). Z-stacks were obtained of the entire cell at 63X with a Zeiss Laser Scanning Confocal 510 Meta Microscope (Carl Zeiss AG).

[0102] Echocardiography

[0103] Conscious mice were briefly restrained by hand or placed on chamber to remove chest and abdominal hair by either a clipper or depilatory cream and perform echocardiographic imaging. Ultrasonic gel and / or 70% ETOH was applied on the imaging surface to enhance image quality. Two-dimensional (2D) guided B-mode and M- mode echocardiography was performed using an echocardiograph machine (Vevo 2100 High Resolution Imaging System, VisualSonics, Toronto, ON, Canada). All imaging and subsequent echo-based measurements were conducted blinded to genotype.

[0104] Histology

[0105] Rapidly excised mouse hearts were perfused with 5 mL of cold 1X PBS followed by 5 mL 4% PFA (158127, Sigma-Aldrich) until the heart stiffens. Hearts were then put in 4% PFA, incubated at 4°C for overnight, washed 2X in cold PBS, and covered in 100 mM glycine (VWR) in 1 mM EDTA (T9650, Sigma-Aldrich). Following incubation at 4 °C, the heart was washed in ice cold PBS then submerged inAtty Docket No.: 47381-0007330% sucrose (MP Biomedicals) in PBS and incubated at 4°C until tissue sank. Hearts were placed in casts (Polysciences, Warrington, PA), filled with O.C.T. solution (4583, Sakura Finetek) for embedding and put on the dry ice until covered and frozen.10 µm frozen sections were prepared on a Leica microtome (Leica Biosystems, Nussloch, DE). To determine the morphology of the heart, the hematoxylin and eosin staining was performed on sections following the manufacturer’s protocol (ab24880, Abcam). To determine the collagenous connective tissue fibers of the heart, the Masson's Trichrome staining was performed on sections following the manufacturer's protocol (ab1500686, Abcam). For visualizing fat accumulation, Oil Red-O was performed on sections following the manufacturer’s protocol (ab150678, Abcam). Image acquisition was performed on a Zeiss Axio Imager Z2 Upright Microscope (Carl Zeiss, Oslo, NO) and analyzed ZEN 2 pro software (Carl Zeiss, Oslo, NO).

[0106] Mouse intracardiac electrophysiology

[0107] Mice were anesthetized using a volatile anesthetic system with induction chamber (R5835, RWD Life Science, Dover, Delaware) with 2% isofluorane mixed with 2L / min 100% O2. Subdermal leads were placed for surface electrocardiographic (ECG) analysis which includes two total leads: I and II. Baseline ECGs were recorded at 1000 samples / second and measured when mice reached internal temperature of 37 degrees Celsius. Jugular venous cutdown was performed using a dissection microscope (A60, Leica, Buffalo Grove, Illinois) and an 8- lead 1.1F, 8E, 1.0mm octapolar catheter electrophysiology catheter (iWire-BIO8, ADInstruments, Colorado Springs, Colorado) was placed in the internal jugular vein and positioned in the right ventricle and right atrium.33 ECG parameters, including RR, PR, QRS, QT, and corrected QT were measured as well as sinus node recovery time (SNRT) and AV node effective refractory period (AVERP) obtained. Electrical pacing was performed utilizing customized electrical stimulator to elicit ectopy and arrhythmia generation with progressive electrical challenge.34 Induction of atrial and ventricular arrhythmias was tested using this electrical stimulation. Rhythm detection was captured by an iWorx-RA-834 Eight Channel 16bit Data Acquisition System (iWorx, Dover New Hampshire, United States). Data was viewed using a custom-built ECG Analysis Module software program for LabScribe4. Adrenergic stimulation and ryanodine receptor sensitization was achieved with 200μg / g caffeine and 1μg / g isoproterenol IP and repeat ECG measurements made after two minutes. Atrial and ventricular pacing was then repeated. Sustained VT was defined as greater than 10. All studies were performed blinded to genotype.

[0108] Statistical analysisAtty Docket No.: 47381-00073

[0109] To determine statistical significance among groups, a paired Student’s t-test was performed for data with a normal distribution and two groups, and a one-way ANOVA with multiple comparisons was used for 3 groups. For non-parametric data, a Mann-Whitney test was used when comparing two groups and a Kruskall-Wallace test with Dunn post hoc tests were used when comparing 3 groups. Fisher’s exact tests were used for categorical data, plotted with 95% confidence intervals. For nested data, we performed the hierarchical analysis described in Sikkel et al. 2017 using the R code provided using R studio.35 With non-parametric data, the data was first Log10-transformed before applying the R code. Data are presented as the mean ± SEM. P<0.05 were considered statistically significant.

[0110] One skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The disclosures described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the present disclosure. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the present disclosure as defined by the scope of the claims.

[0111] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

Claims

Atty Docket No.: 47381-00073CLAIMS:

1. A method of inhibiting transient receptor potential vanilloid type 4 (TRPV4) in cardiac tissue of a subject comprising administering to the cardiac tissue of a subject an inhibitor of TRPV4.

2. The method of claim 1, wherein the inhibitor of TRPV4 is selected from the group consisting of HC-067047, GSK2193874, GSK2798745, RN-1734, RN-1747, RN 9893 hydrochloride, Piperidine-Benzimidazole, PF-05214030, or amino bithiazlole antagonists.

3. The method of claim 1, wherein the inhibitor of TRPV4 is combined with a second compound prior to administration to the cardiac tissue.

4. The method of claim 1, wherein, prior to the administering step, a step of determining if the subject has a cardiac disorder that is arrhythmic cardiomyopathy (ACM), heart failure, dilated cardiomyopathy, hypertrophic cardiomyopathy (HCM), ventricular arrhythmia, ischemia, ventricular tachycardia, a cardiac arrhythmia syndrome, or an arrhythmia disposition.

5. The method of claim 1, wherein the subject is a family member of a subject diagnosed with arrhythmic cardiomyopathy (ACM).

6. The method of claim 1, wherein the cardiac tissue of a subject is a cell or cell line derived from a subject, or the subject is an animal model of arrhythmic cardiomyopathy (ACM) derived from a knock-out of the TAX1BP3 gene.

7. A method of inhibiting small Calcium release event, also known as sparks, in cardiac tissue of a subject, the method comprising administering to the cardiac tissue of a subject an inhibitor of TRPV4, wherein the inhibition of TRPV4 is effective to prevent small Calcium release events.

8. The method of claim 7, wherein the inhibitor of TRPV4 is selected from the group consisting of HC-067047, GSK2193874, GSK2798745, RN-1734, RN-1747, RN 9893 hydrochloride,Atty Docket No.: 47381-00073Piperidine-Benzimidazole, PF-05214030, or amino bithiazlole antagonists in a pharmaceutically acceptable composition.

9. The method of claim 8, wherein the inhibitor of TRPV4 is combined with a second compound in a pharmaceutically acceptable composition.

10. The method of claim 7, wherein, prior to step of administering a TRPV4 inhibitor, a subject undergoes genetic screening to identify the presence of genetic loss or mutation of tax1-binding protein 3 (TAX1BP3) gene such that a genetic loss or mutation of tax1-binding protein 3 gene of the subject renders the subject prone to a cardiac disorder.

11. The method of claim 7, wherein the cardiac disorder is arrhythmic cardiomyopathy (ACM), heart failure, dilated cardiomyopathy, hypertrophic cardiomyopathy (HCM), ventricular arrhythmia, ischemia, ventricular tachycardia, a cardiac arrhythmia syndrome, or an arrhythmia disposition.

12. The method of claim 7, wherein, prior to step of administering a TRPV4 inhibitor, a subject has been identified as exhibiting chronic upregulation of the TRPV4 gene such that any condition that chronic upregulation of the TRPV4 gene renders the subject prone to a cardiac disorder.

13. The method of claim 7, wherein the step of administering to the cardiac tissue of a subject an inhibitor of TRPV4 is continuous, daily, and a prophylactic.

14. The method of claim 7, wherein inhibition small Calcium release events prevents the release of calcium sparks, leaks, and waves, thereby preventing unregulated twitching or contraction and arrhythmia.

15. The method of claim 7, wherein the cardiac tissue of a subject is a cell or cell line derived from cardiac tissue, or the subject is an animal model of arrhythmic cardiomyopathy (ACM) derived from a knock-out of the TAX1BP3 gene.Atty Docket No.: 47381-0007316. A method of screening for the presence of a cardiac disorder, or conditions favorable to product early events occurring in a cardiac disorder in a subject, the method comprising: obtaining a genomic sample from the subject, analyzing the sample for genetic loss or mutation of tax1-binding protein 3 (TAX1BP3) gene or chronic upregulation of the TRPV4 gene, wherein deletion or mutation of the TAX1BP3 gene in the genome of the subject or chronic upregulation of the TRPV4 gene indicates the presence of a cardiac disorder in the subject.

17. The method of 17, wherein the cardiac disorder is arrhythmogenic cardiomyopathy.

18. The method of 17, wherein the cardiac disorder is heart failure, dilated cardiomyopathy, hypertrophic cardiomyopathy (HCM), ventricular arrhythmia, ischemia, ventricular tachycardia, a cardiac arrhythmia syndrome, or an arrhythmia disposition.

Images