Background: Arrhythmogenic cardiomyopathy (ACM) is a heritable cardiac disease characterized by arrhythmias, progressive myocardial injury, and sudden death. Inflammation is central to disease progression. 2-deoxy-2-[18F]-Fluoroglucose positron emission tomography ([18F]FDG PET) is increasingly used to identify glycolytically active immune cells in inflammatory cardiomyopathies. However, whether [18F]FDG uptake in ACM reflects myocardial immune cell recruitment remains unclear. Methods and Results: We performed cardiac [18F]FDG PET imaging in wild-type and desmoglein-2 (DSG2) mutant mice, a well established model of ACM, followed by immune cell profiling using flow cytometry. DSG2 mutant mice exhibited significantly increased cardiac [18F]FDG uptake compared with wild type controls. Although both CCR2+ and CCR2- macrophage populations correlated with the [18F]FDG uptake, pharmacologic depletion of macrophages with liposomal clodronate did not reduce [18F]FDG uptake. High resolution confocal microscopy using the fluorescent glucose analog 2-NBDG demonstrated increased uptake localized predominantly to cardiomyocytes in DSG2 mutant hearts.Conclusions: Myocardial [18F]FDG uptake is not dependent on macrophage abundance and is predominantly localized to cardiomyocytes. These findings suggest that [18F]FDG uptake reflects cardiomyocyte metabolic remodeling driven by cell-intrinsic inflammatory signaling pathways, rather than serving as a direct marker of immune cell infiltration.

ABSTRACT Background Cardiomyocytes respond to stress by undergoing hypertrophic growth driven by dynamic changes in gene expression. Epigenetic mechanisms, including histone methylation, play critical roles in regulating these transcriptional programs, yet the enzymes controlling these modifications during cardiac disease remain largely unknown. The SMYD family of histone methyltransferases regulates gene expression in multiple biological contexts, but the function of SMYD5 in the mammalian heart has never been investigated. Methods SMYD5 expression was assessed in human heart failure samples and in a mouse model of cardiac hypertrophy. To define its functional role in vivo, we generated inducible cardiomyocyte-specific Smyd5 knockout mice and characterized their cardiac phenotype using molecular, histological, and functional analyses. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) was performed to examine histone H4 lysine 20 trimethylation (H4K20me3) at the Il-6 promoter. Results SMYD5 expression was altered in diseased human and mouse hearts. Under basal conditions, cardiomyocyte-specific deletion of Smyd5 resulted in baseline structural cardiac remodeling and transcriptional signatures characteristic of pathological stress. Smyd5 -deficient hearts exhibited marked inflammatory activation resembling a cytokine storm with immune cell infiltration and heart failure. Notably, Smyd5 knockout mice displayed a 100-fold increase in Il-6 expression, accompanied by a global reduction in H4K20me3. ChIP-qPCR analysis of the Il-6 promoter, together with loss- and gain-of-function analysis of SMYD5, supports a direct epigenetic role of SMYD5 in regulating Il-6 expression through H4K20me3 in cardiomyocytes. Conclusions SMYD5 is a previously unrecognized epigenetic regulator of cardiac homeostasis that restrains inflammatory signaling in cardiomyocytes under normal conditions. Loss of Smyd5 disrupts H4K20me3, leading to derepression of Il-6 in cardiomyocytes and a robust inflammatory response characterized by immune cell recruitment and fibrosis, accompanied by rapid progression of cardiac remodeling and heart failure. These findings identify SMYD5 as a critical regulator of intrinsic cardiomyocyte inflammatory signaling and reveal a novel chromatin-based mechanism contributing to inflammatory cardiomyopathies. NOVELTY AND SIGNIFICANCE What Is Known? Elevated levels of pro-inflammatory cytokines such as IL-6 are strongly associated with adverse cardiac remodeling and poor outcomes in heart failure. Histone methylation is a key epigenetic mechanism regulating gene expression in the heart, but the role of histone H4K20 trimethylation and its regulatory enzymes in cardiomyocytes remains poorly understood. The histone methyltransferase SMYD5 regulates gene expression and inflammatory pathways in several non-cardiac cell types, but its role in the mammalian heart has not been examined. What New Information Does This Article Contribute? This study provides the first in vivo characterization of SMYD5 in the heart and identifies it as a critical epigenetic regulator of cardiomyocyte homeostasis. Cardiomyocyte-specific loss of Smyd5 triggers pathological hypertrophy, elevated inflammatory signaling, immune cell infiltration, fibrosis, and heart failure. SMYD5 directly represses Il-6 expression in cardiomyocytes through histone H4K20 trimethylation at the Il-6 promoter, revealing a previously unrecognized epigenetic mechanism controlling intrinsic cardiomyocyte-driven inflammation. Epigenetic mechanisms that regulate inflammatory signaling in cardiomyocytes remain largely unknown. Here, we identify the histone methyltransferase SMYD5 as a critical regulator of cardiac homeostasis and intrinsic inflammatory signaling. Using an inducible cardiomyocyte-specific Smyd5 knockout mouse model, we demonstrate that loss of Smyd5 induces rapid progression to heart failure accompanied by robust inflammatory activation, including a ∼100-fold increase in Il-6 expression, inflammatory immune cell infiltration, fibrosis, and severe cardiac dysfunction. Mechanistically, SMYD5 directly regulates Il-6 expression by catalyzing histone H4 lysine 20 trimethylation at the Il-6 promoter, thereby restraining pro-inflammatory gene expression in cardiomyocytes. Ablation of Smyd5 markedly reduces global H4K20 trimethylation, and results in dramatic upregulation of Il-6 and downstream cytokine signaling pathways, producing a phenotype resembling cytokine storm-like inflammatory cardiomyopathy. These findings establish SMYD5 as the first epigenetic regulator shown to suppress intrinsic cardiomyocyte inflammatory signaling and uncover a novel chromatin-based mechanism controlling cytokine production in the heart. Targeting SMYD5-dependent pathways therefore represents a new strategy for limiting maladaptive inflammation in heart failure and inflammatory cardiomyopathies.

Histone H4K20 methylation is critical in regulating the cell cycle, DNA damage response, and gene repression in proliferating cells. However, its role in the heart remains poorly understood. Our previous work revealed that histone H4K20 tri-methylation is elevated in acute cell models of cardiomyocyte hypertrophy but is reduced in mouse models of cardiac hypertrophy and ischemia. Although these findings highlight the dynamic nature of this modification and its significance in regulating gene expression, the data on enzymes regulating H4K20 methylation is sparse. To build upon this work and investigate H4K20 di-methylation and the enzymes modulating this site in cardiac pathology, we quantified histone H4K20 di-methylation and 12 methyltransferases and demethylases across one cell model, two mouse models of cardiac dysfunction, and cardiac tissue from heart failure patients. While we observed no global changes in H4K20 di-methylation, we detected alterations in methyltransferases KMT5C and SMYD5 and demethylases RAD23A and KDM7C in humans and mice. These findings suggest changes in H4K20 di-methylation may occur on an individual gene basis but do not lead to global alterations in H4K20 di-methylation. Additionally, this work identified four enzymes differentially modulated in cardiac dysfunction to advance our understanding of epigenetic mechanisms involved in heart disease.

Amniotic products are potent immunomodulators used clinically to repair tissue injury. This study sought to determine the impact of acellular human amniotic fluid (hAF) on cardiac remodeling. Amniotic fluid was obtained from volunteer donors at the time of elective caesarean section, proprietarily processed into a sterile filtered acellular hAF, and administered to rats following coronary ligation. Compared to controls, hAF treated animals had a nearly sixfold decrease in both infarct size and fibrosis. Under hypoxic stress, hAF-treated H9C2 cells demonstrated higher cell viability and mitochondrial membrane stability and less apoptosis compared to saline-treated cells. Here we demonstrate that a single intravenous dose of acellular hAF provides functional cardioprotection that is mechanistically associated with cellular tolerance to hypoxic insult likely afforded by the plethora of naturally produced cytokines, chemokines, and immune-modulating proteins present in hAF. The ubiquitous availability of acellular hAF offers promising potential for its use as a cardioprotective adjunct.

GJA1-20k, the internally translated isoform of the gap junction protein Connexin-43 (Cx43) is required for trafficking of the full length Cx43 to the membrane. We recently found that GJA1-20k expression is reduced in patients and mice with arrhythmogenic cardiomyopathy (ACM), and that gene therapy-mediated introduction of GJA1-20k in a mouse model of ACM rescues Cx43 trafficking to the intercalated disc and reduces arrhythmia burden, indicating that loss of GJA1-20k contributes to arrhythmogenesis in ACM. Here, we examined cardiac muscle from patients with a confirmed diagnosis of ACM for GJA1-20k expression before and after left ventricular assist device (LVAD) support. The goal of this study was to determine if left ventricular (LV) unloading benefits endogenous expression of this critical trafficking protein. Cardiac tissue was collected from consecutive, prospectively enrolled patients undergoing LVAD implantation or heart transplantation in hospitals comprising the Utah Cardiac Recovery program and donor patients from hospitals throughout the Intermountain West. Samples were prepared for analysis using a standard lysis buffer and analyzed via Western Blotting using antibodies to GAPDH and Cx43-CT. Consistent with prior findings, cardiac muscle samples from patients with ACM had decreased GJA1-20k expression relative to muscle from healthy donor hearts not allocated for transplantation. However, tissue obtained from the same ACM patients post LVAD support (at the time of heart transplantation) revealed a large and significant increase in GJA1-20k expression compared to the pre LVAD timepoint (1 +/- 0.5 vs 10.2+/- 1, p=0.0013, n=3 ACM pre/post). In contrast to patients with ACM, patients with non-ischemic cardiomyopathy (NICM) that underwent LVAD implantation had no significant change in GJA1-20k expression after LV unloading (1+/- 0.5 vs 1.4+/- 0.7, p= 0.424, n=4 NICM pre/post). These data indicate that LV mechanical unloading increases GJA1-20k expression in hearts of patients with known ACM. Further work will focus on how mechanical unloading regulates GJA1-20k expression and whether mechanical unloading is sufficient to recover Cx43 trafficking and limit arrhythmias in ACM.

Arrhythmogenic cardiomyopathy (ACM) is an inherited disease caused by mutations in desmosome proteins. Patients with ACM are born with normal hearts but develop arrhythmias, fibrofatty infiltration, and sudden death. The desmoglein 2 (DSG2) mutant mouse is a well-established model of ACM that recapitulates the human phenotype of the disease. This mouse develops severe ventricular arrhythmias, systolic dysfunction, myocardial fibrosis and exercise induced sudden death. Here, we present evidence of early myocardial immune cell infiltration as well as an inverse relationship between cardiac function and adult myocardial cytokine expression in DSG2 mutant mice. We examined 20 week old adult DSG2 homozygote mice and wildtype littermate controls for systolic function using echocardiography, cytokine expression using rtPCR and fibrosis using masons trichrome staining. Consistent with previous studies we found ventricular fibrosis is significantly increased in mutant hearts (p=0.003). We demonstrate that osteopontin, an inflammatory cytokine released by macrophages, is highly expressed in these hearts (p=2.0x10 -4 ). We detected an inverse correlation between systolic function and the osteopontin expression levels in these mutant mice (p=0.0015, r=-0.8315, R 2 =0.6914). These results lead us to suspect that immune cell infiltration may contribute to the disease progression. Subsequently, we examined immune cell populations in 3 week old DSG2 hearts, a period of time before the development of fibrosis, systolic dysfunction or arrhythmias. We measured >40-fold higher number of CD45 + leukocytes (with increased Ly6G + neutrophils and Ly6C + monocytes) within the heart muscle of DSG2 homozygotes via flow cytometry (p=7.4x10 -5 ), consistent with myocarditis. These results suggest myocarditis precedes fibrosis in the DSG2 mutant mouse model. Our ongoing work is focused on further characterizing the immune response in early post-natal life in the DSG2 mouse heart and determining whether early myocarditis contributes to the phenotype in ACM. Our results highlight potentially novel therapeutic targets to prevent disease progression in ACM.

SMYD1, a striated muscle-specific lysine methyltransferase, was originally shown to play a key role in embryonic cardiac development but more recently we demonstrated that loss of Smyd1 in the murine adult heart leads to cardiac hypertrophy and failure. However, the effects of SMYD1 overexpression in the heart and its molecular function in the cardiomyocyte in response to ischemic stress are unknown. In this study, we show that inducible, cardiomyocyte-specific overexpression of SMYD1a in mice protects the heart from ischemic injury as seen by a > 50% reduction in infarct size and decreased myocyte cell death. We also demonstrate that attenuated pathological remodeling is a result of enhanced mitochondrial respiration efficiency, which is driven by increased mitochondrial cristae formation and stabilization of respiratory chain supercomplexes within the cristae. These morphological changes occur concomitant with increased OPA1 expression, a known driver of cristae morphology and supercomplex formation. Together, these analyses identify OPA1 as a novel downstream target of SMYD1a whereby cardiomyocytes upregulate energy efficiency to dynamically adapt to the energy demands of the cell. In addition, these findings highlight a new epigenetic mechanism by which SMYD1a regulates mitochondrial energetics and functions to protect the heart from ischemic injury.

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.). , 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. , 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. , 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. , 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. , 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. , 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. , 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. , 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.). 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. 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

SMYD1 is a lysine methyltransferase, which has been shown to methylate lysine 4 on histone H3, an established mark of gene activation. SMYD1 is only expressed in skeletal and cardiac muscle and was originally shown to play a significant role in regulating cardiac development. In the adult myocardium, using inducible, cardiomyocyte-specific Smyd1 knockout mice, loss of SMYD1 leads to massive downregulation of mitochondrial bioenergetics and overt heart failure. However, the effects of SMYD1 gain-of-function in the heart and its molecular function in the cardiomyocyte in response to ischemic stress remains unknown. Here we demonstrate that SMYD1a, the mouse ortholog of human SMYD1, positively regulates cardiac energetics and protects the heart from ischemic injury. To delineate how SMYD1a controls energy efficiency and metabolism in the cardiomyocyte, we generated a novel mouse model capable of inducible cardiomyocyte-specific SMYD1a overexpression. When subjected to ischemic injury these transgenic mice display reduced infarct size and cardiomyocyte death concomitant with enhanced mitochondrial respiratory efficiency. In addition, our molecular analysis revealed that the cardiac tissue in these animals is protected from ischemic injury through SMYD1a’s synergistic regulation of two key mitochondrial pathways. First, through its histone methyltransferase activity, SMYD1a maintains metabolic homeostasis by preserving basal expression of PGC-1α and its downstream targets including electron transport chain subunits. Second, SMYD1a regulates expression of OPA1, a key regulator of cristae morphology which drives the formation of electron transport chain supercomplexes to enhance mitochondrial respiration and ATP production. This work highlights SMYD1a as the only known epigenetic regulator of cristae morphology and identifies a novel molecular pathway by which cardiomyocytes dynamically regulate energy efficiency to protect from ischemic injury.

Heart disease is the leading cause of death in the developed world, and its comorbidities such as hypertension, diabetes, and heart failure are accompanied by major transcriptomic changes in the heart. During cardiac dysfunction, which leads to heart failure, there are global epigenetic alterations to chromatin that occur concomitantly with morphological changes in the heart in response to acute and chronic stress. These epigenetic alterations include the reversible methylation of lysine residues on histone proteins. Lysine methylations on histones H3K4 and H3K9 were among the first methylated lysine residues identified and have been linked to gene activation and silencing, respectively. However, much less is known regarding other methylated histone residues, including histone H4K20. Trimethylation of histone H4K20 has been shown to repress gene expression; however, this modification has never been examined in the heart. Here, we utilized immunoblotting and mass spectrometry to quantify histone H4K20 trimethylation in three models of cardiac dysfunction. Our results show that lysine methylation at this site is differentially regulated in the cardiomyocyte, leading to increased H4K20 trimethylation during acute hypertrophic stress in cell models and decreased H4K20 trimethylation during sustained ischemic injury and cardiac dysfunction in animal models. In addition, we examined publicly available data sets to analyze enzymes that regulate H4K20 methylation and identified two demethylases (KDM7B and KDM7C) and two methyltransferases (KMT5A and SMYD5) that were all differentially expressed in heart failure patients. This is the first study to examine histone H4K20 trimethylation in the heart and to determine how this post-translational modification is differentially regulated in multiple models of cardiac disease.

The histone lysine methyltransferase SMYD1 has been shown to be critical for embryonic cardiac development and in maintaining cardiomyocyte homeostasis in adult mice. Subsequently, we reported that loss of Smyd1 in the adult mouse myocardium leads to progressive cardiac hypertrophy and heart failure, which is accompanied with downregulation of mitochondrial proteins involved in oxidative phosphorylation, including Ppargc1a , and reduction of mitochondrial respiration capacity. To build upon these results and evaluate if SMYD1a can attenuate disease-induced remodeling in an animal model, we generated transgenic mice which inducibly express SMYD1a (the human ortholog) in cardiomyocytes and subjected them to permanent occlusion (PO) of the LAD. This lead to >50% reduction in infarct size and preserved cardiac function, as compared to littermate controls. Additionally, we demonstrated that under physiological conditions SMYD1a maintains metabolic homeostasis by regulating expression of Ppargc1a and its downstream targets, including components of the electron transport chain. Our molecular analysis shows that observed protection from ischemic injury results from enhanced mitochondrial respiration through Complex I and II as well as increased ATP production. This is associated with increased mitochondria cristae, and formation and stabilization of respiratory chain supercomplexes within the cristae. These changes in cristae structure occur concomitant with enhanced OPA1 expression, a major regulator of mitochondrial fusion and cristae morphology. Through this work we have established that OPA1 is a novel, functionally important downstream target of SMYD1a by which cardiomyocytes upregulate energy efficiency, protecting them from ischemic injury. These results also highlight SMYD1a as the only known epigenetic regulator of cristae morphology and provide broad implications for understanding the epigenetic mechanisms driving cardiac metabolism. Ultimately this work has identified a novel signaling pathway by which cardiomyocytes regulate energy efficiency, protecting them from ischemic injury.

The Connexin43 gap junction gene GJA1 has one coding exon, but its mRNA undergoes internal translation to generate N-terminal truncated isoforms of Connexin43 with the predominant isoform being only 20 kDa in size (GJA1-20k). Endogenous GJA1-20k protein is not membrane bound and has been found to increase in response to ischemic stress, localize to mitochondria, and mimic ischemic preconditioning protection in the heart. However, it is not known how GJA1-20k benefits mitochondria to provide this protection. Here, using human cells and mice, we identify that GJA1-20k polymerizes actin around mitochondria which induces focal constriction sites. Mitochondrial fission events occur within about 45 s of GJA1-20k recruitment of actin. Interestingly, GJA1-20k mediated fission is independent of canonical Dynamin-Related Protein 1 (DRP1). We find that GJA1-20k-induced smaller mitochondria have decreased reactive oxygen species (ROS) generation and, in hearts, provide potent protection against ischemia-reperfusion injury. The results indicate that stress responsive internally translated GJA1-20k stabilizes polymerized actin filaments to stimulate non-canonical mitochondrial fission which limits ischemic-reperfusion induced myocardial infarction.

SMYD1a, a myosin-specific histone lysine methyltransferase, plays a major role in regulating disease-induced remodeling in the adult heart. Previously, we demonstrated that the inducible loss of this chromatin-bound enzyme is sufficient to induce cardiac hypertrophy and failure in vivo , which is preceded by downregulation of mitochondrial proteins involved in oxidative phosphorylation (OXPHOS), and reduction of mitochondrial respiration capacity. However, our most recent data in transgenic mice (TG) displaying inducible, cardiomyocyte-specific overexpression of SMYD1a show that these mice are protected from ischemic injury after permanent occlusion (PO) of the LAD manifested by reduced infarct size and cardiac dysfunction compared to littermate controls (WT), suggesting that SMYD1 plays a protective role in the heart and mitigates disease-induced remodeling. Additionally, global proteomic evaluation of cardiac tissue from TG mice showed unique expression of metabolic enzymes, including proteins from the electron transport chain, and our high-resolution mitochondrial respirometry analysis showed that overexpression of SMYD1a leads to increased oxygen consumption rates through Complex I and II. To further asses OXPHOS efficiency in TG mice we subjected them to permanent occlusion of the LAD and evaluated ATP production rates in isolated mitochondria from TG and WT mice, by measuring the molar amount of ATP produced per mole of atomic oxygen consumed (known as ATP:O ratio). Interestingly, we observed a significant increase in ATP:O ratio in TG mice 24h after PO suggesting that they are much more efficient at producing ATP. Finally, we show that the global regulation of mitochondrial respiration in TG mice occurs through transcriptional control of Ppargc1α . Our results confirm that cardiac expression of Ppargc1α was significantly reduced in WT mice (48h after PO) but maintained at basal levels in TG mice, which also corroborated with our ChIP-qPCR data showing SMYD1a binding to the Ppargc1α promoter and regulating its expression. Overall, these results show that SMYD1a can mitigate ischemic injury and adverse remodeling in the adult myocardium, which occurs through Ppargc1α expression and regulation of cardiac energetics and metabolism.

Introduction: Arrhythmogenic cardiomyopathy (ACM) is an inherited condition caused by mutations of junctional proteins resulting in heart rhythm abnormalities, heart failure and sudden cardiac death. ACM subjects exhibit a loss of normal trafficking of the gap junction protein Connexin 43 (Cx43) to the intercalated disk. Our group has previously demonstrated that the Cx43 gene, GJA1, undergoes internal translation to form multiple truncations of the full-length protein. The 20 kilodalton isoform, (GJA1-20k) is required for trafficking of Cx43 gap junction channels, and exogenous expression of GJA1-20k increases trafficking of full length Cx43 to the membranes and the intercalated disk. Here, we hypothesize that GJA1-20K will reduce dysrhythmias by improving trafficking of Cx43 to the intercalated disc in a mouse model of ACM. Methods: Desmoglein 2 (DSG2) homozygous knock-out mice, producing a typical ACM phenotype, were injected with a viral vector containing either GJA1-20K-GFP or GFP alone at four weeks of age along with wild-type littermate controls. Echocardiography was performed every four weeks and Telemetry was recorded at ~18-19 weeks of age to record cardiac electrical activity over a continuous 12 hour period. At 20 weeks, the mice were sacrificed, and hearts were harvested for further analysis. Results: Compared to WT littermates DSG2-KO mice that received a GFP viral vector demonstrated over a 100 fold increase in mean number of PVCs observed over a 12 hour period (2 vs 371 respectively p=0.0005). Mice that received the GJA1-20k viral vector in contrast demonstrated significantly fewer PVCs than their GFP treated littermates 43 vs 371. (analysis of variance of Log(#PVC) p =0.015 N = 6-7 mice/group). Quantitative immunofluorescence analysis of hearts confirmed an increase in Cx43 localization at the intercalated disk (as measured as a percent of N-Cadherin-Cx43 colabeling) in mice treated with GJA1-20k relative to GFP (25% vs 6% respectively N= 7 images/mouse and 3 mice/ group) t-test p=0.0009). Conclusions: Our data indicate that GJA1-20K significantly reduces the number of PVCs and recovers Cx43 trafficking to the intercalated disk in a mouse model of ACM. Our results point to a therapeutic option for the treatment of ACM related arrhythmias.