GJA1-20k Rescues Cx43 Localization and Arrhythmias in Arrhythmogenic Cardiomyopathy

HomeCirculation ResearchVol. 132, No. 6GJA1-20k Rescues Cx43 Localization and Arrhythmias in Arrhythmogenic Cardiomyopathy Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBGJA1-20k Rescues Cx43 Localization and Arrhythmias in Arrhythmogenic Cardiomyopathy Joseph A. Palatinus, Steven Valdez, Lindsey Taylor, Claire Whisenant, Craig H. Selzman, Stavros G Drakos, Ravi Ranjan, TingTing Hong, Jeffrey E. Saffitz and Robin M. Shaw Joseph A. PalatinusJoseph A. Palatinus https://orcid.org/0000-0002-6424-0105 Nora Eccles Harrison Cardiovascular Research and Training Institute (J.A.P., S.V., L.T., C.W., C.H.S., S.G.D., R.R., T.H., R.M.S.), University of Utah, Salt Lake City. Department of Medicine, Intermountain Medical Center, Murray, UT (J.A.P.). Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA (J.A.P., J.E.S.). Search for more papers by this author , Steven ValdezSteven Valdez Nora Eccles Harrison Cardiovascular Research and Training Institute (J.A.P., S.V., L.T., C.W., C.H.S., S.G.D., R.R., T.H., R.M.S.), University of Utah, Salt Lake City. Search for more papers by this author , Lindsey TaylorLindsey Taylor Nora Eccles Harrison Cardiovascular Research and Training Institute (J.A.P., S.V., L.T., C.W., C.H.S., S.G.D., R.R., T.H., R.M.S.), University of Utah, Salt Lake City. Search for more papers by this author , Claire WhisenantClaire Whisenant Nora Eccles Harrison Cardiovascular Research and Training Institute (J.A.P., S.V., L.T., C.W., C.H.S., S.G.D., R.R., T.H., R.M.S.), University of Utah, Salt Lake City. Search for more papers by this author , Craig H. SelzmanCraig H. Selzman https://orcid.org/0000-0001-9218-4764 Nora Eccles Harrison Cardiovascular Research and Training Institute (J.A.P., S.V., L.T., C.W., C.H.S., S.G.D., R.R., T.H., R.M.S.), University of Utah, Salt Lake City. Search for more papers by this author , Stavros G DrakosStavros G Drakos https://orcid.org/0000-0002-1223-327X Nora Eccles Harrison Cardiovascular Research and Training Institute (J.A.P., S.V., L.T., C.W., C.H.S., S.G.D., R.R., T.H., R.M.S.), University of Utah, Salt Lake City. Search for more papers by this author , Ravi RanjanRavi Ranjan Nora Eccles Harrison Cardiovascular Research and Training Institute (J.A.P., S.V., L.T., C.W., C.H.S., S.G.D., R.R., T.H., R.M.S.), University of Utah, Salt Lake City. Search for more papers by this author , TingTing HongTingTing Hong https://orcid.org/0000-0002-0243-5046 Nora Eccles Harrison Cardiovascular Research and Training Institute (J.A.P., S.V., L.T., C.W., C.H.S., S.G.D., R.R., T.H., R.M.S.), University of Utah, Salt Lake City. Department of Pharmacology and Toxicology, College of Pharmacy (T.H.), University of Utah, Salt Lake City. Search for more papers by this author , Jeffrey E. SaffitzJeffrey E. Saffitz https://orcid.org/0000-0001-8568-9457 Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA (J.A.P., J.E.S.). Search for more papers by this author and Robin M. ShawRobin M. Shaw Correspondence to: Robin Shaw, MD, PhD, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, 95 S 2000 E Room 207 D, Salt Lake City, UT 84112. Email E-mail Address: [email protected] https://orcid.org/0000-0001-7429-6092 Nora Eccles Harrison Cardiovascular Research and Training Institute (J.A.P., S.V., L.T., C.W., C.H.S., S.G.D., R.R., T.H., R.M.S.), University of Utah, Salt Lake City. Search for more papers by this author Originally published22 Feb 2023https://doi.org/10.1161/CIRCRESAHA.122.322294Circulation Research. 2023;132:744–746is related toMeet the First AuthorsOther version(s) of this articleYou are viewing the most recent version of this article. Previous versions: March 16, 2023: Previous Version of Record February 22, 2023: Ahead of Print Meet the First Author, see p 673Arrhythmogenic cardiomyopathy (ACM) is a heritable heart muscle disease associated with increased risk of sudden cardiac death. The only treatment proven to reduce this risk is an implantable defibrillator. Originally named arrhythmogenic right ventricular dysplasia after autopsy studies revealed fibro-fatty replacement of the right ventricle, nomenclature has been updated to reflect a genetic disease with a structurally normal heart at birth that can perturb myocytes in both ventricles and increase risk for sudden death. In other nonischemic cardiomyopathies, such as hypertrophic or dilated cardiomyopathy, the risk of sudden death is associated with progressive structural remodeling of the heart involving myocyte disarray, fibrosis, and changes in cardiac chamber dimensions. Although progressive myocardial injury and accumulation of fibro-fatty scar tissue contribute to development of arrhythmias in advanced ACM, recent evidence has highlighted the importance of early subclinical electrophysiological changes which contribute to the arrhythmogenic substrate in this disease.1 Importantly, arrhythmias are typically the first clinical manifestation of ACM and often precede histologic changes in heart muscle or onset of ventricular dysfunction2 supporting a primary mechanism independent of macroscopic or histological changes.A majority of cases of ACM can be attributed to variants in genes that encode desmosome proteins, such as the desmosomal cadherin, desmoglein 2 (Dsg2). How mutations in cell-cell adhesion components of intercalated discs promote arrhythmias prior to development of structural changes in the myocardium is not well established. However, reduced immunoreactive signal at intercalated discs for the major ventricular gap junction protein, Cx43 (connexin 43), is a prominent feature in ACM. Reduced Cx43 at intercalated discs has been linked to impaired forward trafficking of Cx43,3 and likely contributes to arrhythmogenesis. We previously demonstrated that normal cytoskeleton based forward trafficking of cardiac Cx43 to the intercalated disc is dependent on a truncated 20 kDa isoform of Cx43 that is generated by internal translation initiation of Gja1 mRNA (GJA1-20k).4 Here, we provide evidence that expression of this critical auxiliary Cx43 subunit is decreased in the hearts of patients with clinically diagnosed ACM and a documented history of ventricular arrhythmias and in a well-characterized mouse model of ACM involving homozygous knock-in of a variant in the desmosomal gene, Dsg2 resulting in a loss of Dsg 2 expression (Dsg2−/− mice). This mouse demonstrates arrhythmias, fibrosis, and increased sudden death with exercise consistent with patient phenotypes of ACM.3 In this study of sedentary mice, we observed arrhythmias and fibrosis. Furthermore, we found that gene therapy via AAV9 (adeno associated virus 9)-mediated myocardial expression of exogenous GJA1-20k improves trafficking of Cx43 to intercalated discs in Dsg2−/− mice and reduces their arrhythmogenic phenotype, independent of changes in left ventricular function.Cx43 and GJA1-20k levels were measured by Western blots in lysates of ventricular myocardium from nonfailing control hearts and hearts of patients with ACM who underwent heart transplant (n=7 ACM hearts, 2 with confirmed genotypes and 5 with diagnosis by task force criteria and n=7 nonfailing control donor hearts). The amount of total Cx43 was similar in control and ACM ventricular tissue but GJA1-20k expression was reduced in ACM (Figure [A]). This observation was unexpected as GJA1-20k expression is increased in patients and animal models with ischemic cardiomyopathy.5 Consistent with the patient samples, preserved total Cx43 but decreased GJA1-20k expression also occurred in Dsg2−/− mice (Figure [B]). To determine if augmentation of GJA1-20k expression can rescue the ACM disease phenotype, Dsg2−/− and wild-type mice underwent echocardiography at 4 weeks of age followed by retro-orbital introduction of AAV9-GJA1-20k-green fluorescent protein (GFP) or control AAV9-GFP (n=9, wild-type AAV9-GFP; n=10, AAV9-GFP Dsg2−/−; n=9, AAV9-GJA1-20k-GFP Dsg2−/−). Echocardiograms were repeated every 4 weeks, and at 16 weeks of age, telemetry devices were implanted for ECG monitoring. Mice were euthanized at 20 weeks of age, and hearts were analyzed by histology and biochemical analysis. At the time of AAV9 injection, Dsg2−/− mice (4 weeks of age) ejection fractions were no different than wild type. At 16 weeks postinjection, Dsg2−/− mice (20 weeks of age) ejection fractions were reduced independent of treatment with GJA1-20k (Figure [C]). Analysis of Masson trichrome stained sections showed increased myocardial fibrosis in Dsg2−/− mice relative to control, with no apparent difference with GJA-20k therapy (Figure [C]). High-resolution confocal microscopy imaging of frozen heart tissue sections revealed decreased immunoreactive Cx43 signal (relative to N-cadherin) at intercalated discs in Dsg2−/− mice (Figure [D] and [E]), consistent with published findings.3 However, GJA1-20k therapy normalized Cx43 signal at intercalated discs (Figure [E]). Furthermore, nocturnal telemetry recordings showed that GJA1-20k treated animals had significantly fewer ventricular arrhythmias as quantified by premature ventricular contraction (PVCs) per hour (Figure [F]).Download figureDownload PowerPointFigure. GJA1-20k normalizes Cx43 (connexin 43) immunoreactive signal at gap junctions and reduces arrhythmias in Dsg2−/− mice. A, Immunoblot (left) and quantification (right) of Cx43 GJA1-20k and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) from lysates of ventricular tissue obtained from patients with arrhythmogenic cardiomyopathy (ACM; n=7) at the time of heart transplantation. Control samples from nonfailing (NF) donor hearts (n=6) processed identically to the ACM samples. Middle, Table with patient with ACM diagnosis, number indicates lane on immunoblot. B, Left, Representative immunoblot (left) and quantification(right) of expression levels of Cx43, GJA1-20k and GAPDH in whole-heart lysates from wild-type (WT; n=9) or age/liter matched Dsg2−/− mice (n=9). Right, Quantification of ejection fraction from B-mode echocardiograms in WT (n=13) and Dsg2−/− mice treated with green fluorescent protein (GFP) (n=10) or GJA1-20k virus (n=12) at injection time (4 weeks of age) and 16 weeks postinjection. C, Trichrome staining of WT, Dsg2−/− GFP, or Dsg2−/− GJA1-20k injected hearts (left) showing increase fibrosis in Dsg2−/− GFP mice with no change in GJA1-20k-treatment, (right) quantification (% fibrosis of section area) in each group (WT, n=8; GFP, n=13; GJA1-20k, n=9). D, Illustration of cytoskeleton based trafficking of Cx43 by GJA1-20k (created with Biorender). E, Large: Immunofluorescent micrographs from WT and Dsg2−/− hearts treated with AAV-GFP (adeno associated virus 9-green fluorescent protein) or AAV-GJA1-20k (Dsg2−/− GJA1-20k). Cx43 is green; N-cadherin is red. Small, Zoomed images of representative intercalated discs showing decreased Cx43 relative to N-cadherin labeling in Dsg2−/− mice and increased signal in Dsg2−/− 20k treated mice. Relative intensity Cx43/N-cadherin signals in all groups was quantified in 3 images per mouse, (WT, n=9; GFP, n=8; GJA1-20k, n=9). F, ECGs from WT, Dsg2−/− AAV-GFP and Dsg2−/− GJA1-20k mice with plotted premature ventricular contraction (PVCs)/hour for each group (WT, n=10; GFP, n=10; GJA1-20k, n=8). All statistical tests performed in GraphPad Prism, pairwise comparisons were performed using a Mann-Whitney test, and multiple comparisons were performed using a Kruskal Wallis test with Dunn correction. All error bars in graphs represent median +/− interquartile range. DSP indicates desmoplakin; PLN, phospholambam; and TFC, task force criteria.In conclusion, we provide evidence that recovery of Cx43 cytoskeleton based forward trafficking after GJA1-20k gene therapy improves gap junction localization and reduces ventricular arrhythmias in a mouse model of ACM, independent of systolic dysfunction, or degree of fibrosis. These results suggest that defective trafficking of Cx43 to intercalated discs is a potential therapeutic strategy to reduce risk of lethal arrhythmias. Continuing work will focus on the ribosomal translational mechanisms that decrease GJA1-20k expression in patients with ACM.All procedures involving animals were approved by the institutional and national authorities. All animals were randomly assigned to experimental groups regardless of sex. Overall, 46% of animals were male, with no obvious sex differences in results. All data analysis was performed in a blinded manner. Successful viral transduction was defined as 30 cycles by rtPCR (reverse transtricption polymerase chain reaction). GJA1-20k overexpression, by rtPCR, was on average 2% of GJA1 mRNA expression.Article InformationAcknowledgmentsThe authors appreciate the assistance of Zane Zobell. The authors are grateful to the donor families for their generosity, and DonorConnect (https://www.donorconnect.life/), Salt Lake City, Utah, for facilitating the work of our research team members acquiring myocardial tissue in the operating rooms of several hospitals of the Mountain West.Sources of FundingThe authors acknowledge support from the National Institutes of Health (J.A. Palatinus, T. Hong, J.E. Saffitz, and R.M. Shaw), The Harold Geneen Charitable Trust (Dr Palatinus), and the Nora Eccles Treadwell Foundation (T. Hong and R.M. Shaw).Data AvailabilityThe data that support the findings are available from the corresponding author on request who had full access to all the data in the study and takes responsibility for its integrity and the data analysis.Nonstandard Abbreviations and AcronymsACMarrhythmogenic cardiomyopathyCx43connexin 43Disclosures R.M. Shaw had a prior sponsored research agreement with Renovacor. The other authors report no conflicts.FootnotesFor Sources of Funding and Disclosures, see page 746.Correspondence to: Robin Shaw, MD, PhD, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, 95 S 2000 E Room 207 D, Salt Lake City, UT 84112. Email robin.[email protected]utah.eduReferences1. van Opbergen CJM, Bagwan N, Maurya SR, Kim JC, Smith AN, Blackwell DJ, Johnston JN, Knollmann BC, Cerrone M, Lundby A, et al. Exercise causes arrhythmogenic remodeling of intracellular calcium dynamics in plakophilin-2-deficient hearts.Circulation. 2022; 145:1480–1496. doi: 10.1161/CIRCULATIONAHA.121.057757LinkGoogle Scholar2. Asimaki A, Kleber AG, MacRae CA, Saffitz JE. Arrhythmogenic cardiomyopathy - new insights into disease mechanisms and drug discovery.Prog Pediatr Cardiol. 2014; 37:3–7. doi: 10.1016/j.ppedcard.2014.10.001CrossrefMedlineGoogle Scholar3. Chelko SP, Asimaki A, Andersen P, Bedja D, Amat-Alarcon N, DeMazumder D, Jasti R, MacRae CA, Leber R, Kleber AG, et al. Central role for GSK3beta in the pathogenesis of arrhythmogenic cardiomyopathy.JCI Insight. 2016; 1:e85923. doi: 10.1172/jci.insight.85923CrossrefMedlineGoogle Scholar4. Xiao S, Shimura D, Baum R, Hernandez DM, Agvanian S, Nagaoka Y, Katsumata M, Lampe PD, Kleber AG, Hong T, et al. Auxiliary trafficking subunit GJA1-20k protects connexin-43 from degradation and limits ventricular arrhythmias.J Clin Invest. 2020; 130:4858–4870. doi: 10.1172/JCI134682CrossrefMedlineGoogle Scholar5. Basheer WA, Fu Y, Shimura D, Xiao S, Agvanian S, Hernandez DM, Hitzeman TC, Hong T, Shaw RM. Stress response protein GJA1-20k promotes mitochondrial biogenesis, metabolic quiescence, and cardioprotection against ischemia/reperfusion injury.JCI Insight. 2018; 3:e121900. doi: 10.1172/jci.insight.121900CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesMeet the First AuthorsCirculation Research. 2023;132:672-673 March 17, 2023Vol 132, Issue 6 Advertisement Article InformationMetrics © 2023 American Heart Association, Inc.https://doi.org/10.1161/CIRCRESAHA.122.322294PMID: 36927183 Originally publishedFebruary 22, 2023 Keywordsmyocardiumarrhythmias, cardiacmutationheart failureconnexinPDF download Advertisement SubjectsArrhythmiasGenetically Altered and Transgenic ModelsSudden Cardiac Death