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Aim Arrhythmogenic cardiomyopathy (ACM) is a primary myocardial disease that typically manifests with cardiac arrhythmias, progressive heart failure and sudden cardiac death (SCD). ACM is mainly caused by mutations in genes encoding desmosome proteins. Desmosomes are cell-cell adhesion structures and hubs for mechanosensing and mechanotransduction. The objective was to identify the dysregulated molecular and biological pathways in human ACM in the absence of overt heart failure. Methods and results Transcriptomes in the right ventricular endomyocardial biopsy samples from three independent individuals carrying truncating mutations in the DSP gene and 5 control samples were analyzed by RNA-Seq (discovery group). These cases presented with cardiac arrhythmias and had a normal right ventricular function. The RNA-Seq analysis identified ∼5,000 differentially expressed genes (DEGs), which predicted suppression of the Hippo and canonical WNT pathways, among others.Dysregulated genes and pathways, identified by RNA-Seq, were tested for validation in the right and left ventricular tissues from 5 independent autopsy-confirmed ACM cases with defined mutations (validation group), who were victims of SCD and had no history of heart failure. Protein levels and nuclear localization of the cWNT and Hippo pathway transcriptional regulators were reduced in the right and left ventricular validation samples. In contrast, levels of acetyltransferase EP300, known to suppress the Hippo and canonical WNT pathways, were increased and its bona fide target TP53 was acetylated. RNA-Seq data identified apical junction, reflective of cell-cell attachment, as the most disrupted biological pathway, which was corroborated by disrupted desmosomes and intermediate filament structures. Moreover, the DEGs also predicted dysregulation of over a dozen canonical signal transduction pathways, including the Tec kinase and integrin signaling pathways. The changes were associated with increased apoptosis and fibro-adipogenesis in the ACM hearts. Conclusion Altered apical junction structures is associated with activation of the EP300-TP53 and suppression of the Hippo/cWNT pathways in human ACM caused by defined mutations in the absence of an overt heart failure. The findings implicate altered mechanotransduction in the pathogenesis of ACM. Translational perspective The findings suggest that altered mechanosensing at the cell-cell junction instigates a cascade of molecular events through the activation of acetyltransferase EP300/TP53 and suppression of gene expression through the Hippo/canonical WNT pathways in human arrhythmogenic cardiomyopathy (ACM) caused by defined mutations. These molecular changes occur early and in the absence of overt heart failure. Consequently, one may envision cell type-specific interventions to target the dysregulated transcriptional, mechanosensing, and mechanotransduction pathways to prevent the evolving phenotype in human ACM.

Background: Arrhythmogenic cardiomyopathy (ACM) manifests with sudden death, arrhythmias, heart failure, apoptosis, and myocardial fibro-adipogenesis. The phenotype typically starts at the epicardium and advances transmurally. Mutations in genes encoding desmosome proteins, including DSP (desmoplakin), are major causes of ACM. Methods: To delineate contributions of the epicardium to the pathogenesis of ACM, the Dsp allele was conditionally deleted in the epicardial cells in mice upon expression of tamoxifen-inducible Cre from the Wt1 locus. Wild type (WT) and Wt1-Cre ERT2 :Dsp W/F were crossed to Rosa26 mT/mG (R26 mT/mG ) dual reporter mice to tag the epicardial-derived cells with the EGFP (enhanced green fluorescent protein) reporter protein. Tagged epicardial-derived cells from adult Wt1-Cre ERT2 :R26 mT/mG and Wt1-Cre ERT2 : R26 mT/mG :Dsp W/F mouse hearts were isolated by fluorescence-activated cell staining and sequenced by single-cell RNA sequencing. Results: WT1 (Wilms tumor 1) expression was progressively restricted postnatally and was exclusive to the epicardium by postnatal day 21. Expression of Dsp was reduced in the epicardial cells but not in cardiac myocytes in the Wt1-Cre ERT2 :Dsp W/F mice. The Wt1-Cre ERT2 :Dsp W/F mice exhibited premature death, cardiac dysfunction, arrhythmias, myocardial fibro-adipogenesis, and apoptosis. Single-cell RNA sequencing of ≈18 000 EGFP-tagged epicardial-derived cells identified genotype-independent clusters of endothelial cells, fibroblasts, epithelial cells, and a very small cluster of cardiac myocytes, which were confirmed on coimmunofluorescence staining of the myocardial sections. Differentially expressed genes between the paired clusters in the 2 genotypes predicted activation of the inflammatory and mitotic pathways—including the TGFβ1 (transforming growth factor β1) and fibroblast growth factors—in the epicardial-derived fibroblast and epithelial clusters, but predicted their suppression in the endothelial cell cluster. The findings were corroborated by analysis of gene expression in the pooled RNA-sequencing data, which identified predominant dysregulation of genes involved in epithelial–mesenchymal transition, and dysregulation of 146 genes encoding the secreted proteins (secretome), including genes in the TGFβ1 pathway. Activation of the TGFβ1 and its colocalization with fibrosis in the Wt1-Cre ERT2 :R26 mT/mG :Dsp W/F mouse heart was validated by complementary methods. Conclusions: Epicardial-derived cardiac fibroblasts and epithelial cells express paracrine factors, including TGFβ1 and fibroblast growth factors, which mediate epithelial–mesenchymal transition, and contribute to the pathogenesis of myocardial fibrosis, apoptosis, arrhythmias, and cardiac dysfunction in a mouse model of ACM. The findings uncover contributions of the epicardial-derived cells to the pathogenesis of ACM.

Arrhythmogenic cardiomyopathy (ACM) encompasses a group of inherited cardiomyopathies including arrhythmogenic right ventricular cardiomyopathy (ARVC) whose molecular disease mechanism is associated with dysregulation of the canonical WNT signalling pathway. Recent evidence indicates that ARVC and ACM caused by pathogenic variants in the FLNC gene encoding filamin C, a major cardiac structural protein, may have different molecular mechanisms of pathogenesis. We sought to identify dysregulated biological pathways in FLNC-associated ACM. RNA was extracted from seven paraffin-embedded left ventricular tissue samples from deceased ACM patients carrying FLNC variants and sequenced. Transcript levels of 623 genes were upregulated and 486 genes were reduced in ACM in comparison to control samples. The cell adhesion pathway and ILK signalling were among the prominent dysregulated pathways in ACM. Consistent with these findings, transcript levels of cell adhesion genes JAM2, NEO1, VCAM1 and PTPRC were upregulated in ACM samples. Moreover, several actin-associated genes, including FLNC, VCL, PARVB and MYL7, were suppressed, suggesting dysregulation of the actin cytoskeleton. Analysis of the transcriptome for dysregulated biological pathways predicted activation of inflammation and apoptosis and suppression of oxidative phosphorylation and MTORC1 signalling in ACM. Our data suggests dysregulated cell adhesion and ILK signalling as novel putative pathogenic mechanisms of ACM caused by FLNC variants which are distinct from the postulated disease mechanism of classic ARVC caused by desmosomal gene mutations. This knowledge could help in the design of future gene therapy strategies which would target specific components of these pathways and potentially lead to novel treatments for ACM.

Aims Arrhythmogenic cardiomyopathy (ACM) is a myocardial disease caused mainly by mutations in genes encoding desmosome proteins ACM patients present with ventricular arrhythmias, cardiac dysfunction, sudden cardiac death, and a subset with fibro-fatty infiltration of the right ventricle predominantly. Endurance exercise is thought to exacerbate cardiac dysfunction and arrhythmias in ACM. The objective was to determine the effects of treadmill exercise on cardiac phenotype, including myocyte gene expression in myocyte-specific desmoplakin (Dsp) haplo-insufficient (Myh6-Cre:DspW/F) mice. Methods and results Three months old sex-matched wild-type (WT) and Myh6-Cre:DspW/F mice with normal cardiac function, as assessed by echocardiography, were randomized to regular activity or 60 min of daily treadmill exercise (5.5 kJ work per run). Cardiac myocyte gene expression, cardiac function, arrhythmias, and myocardial histology, including apoptosis, were analysed prior to and after 3 months of routine activity or treadmill exercise. Fifty-seven and 781 genes were differentially expressed in 3- and 6-month-old Myh6-Cre:DspW/F cardiac myocytes, compared to the corresponding WT myocytes, respectively. Genes encoding secreted proteins (secretome), including inhibitors of the canonical WNT pathway, were among the most up-regulated genes. The differentially expressed genes (DEGs) predicted activation of epithelial–mesenchymal transition (EMT) and inflammation, and suppression of oxidative phosphorylation pathways in the Myh6-Cre:DspW/F myocytes. Treadmill exercise restored transcript levels of two-third (492/781) of the DEGs and the corresponding dysregulated transcriptional and biological pathways, including EMT, inflammation, and secreted inhibitors of the canonical WNT. The changes were associated with reduced myocardial apoptosis and eccentric cardiac hypertrophy without changes in cardiac function. Conclusion Treadmill exercise restored transcript levels of the majority of dysregulated genes in cardiac myocytes, reduced myocardial apoptosis, and induced eccentric cardiac hypertrophy without affecting cardiac dysfunction in a mouse model of ACM. The findings suggest that treadmill exercise has potential beneficial effects in a subset of cardiac phenotypes in ACM.

PURPOSE OF REVIEW: Human genome is pervasively transcribed, producing coding and noncoding RNAs. Recent studies have revealed the roles of a class of noncoding RNAs, the long noncoding RNAs (lncRNAs), in heart failure and other cardiovascular diseases. This review provides a brief summary of recent findings on lncRNA function. RECENT FINDINGS: Recent studies have documented the roles of lncRNAs in cardiac regeneration, conduction, hypertrophy/dysfunction, and endothelial function. LncRNAs perform these functions through acting as competing RNA (by binding and sequestering miRNAs) or acting as guides to protein targeting. A few lncRNAs also encode small peptides (for example; Dworf RNA) and in the context of heart regulate cardiac calcium homeostasis. SUMMARY: Noncoding RNA provides a versatile mechanism of gene regulation and thereby present as novel targets for intervention in various cardiovascular disease. Future studies aimed at defining the context-dependent lncRNA mechanisms will be required to advance our understanding and relish the goal of RNA therapeutics.

Rationale: LMNA (Lamin A/C), a nuclear membrane protein, interacts with genome through lamin-associated domains (LADs) and regulates gene expression. Mutations in the LMNA gene cause a diverse array of diseases, including dilated cardiomyopathy (DCM). DCM is the leading cause of death in laminopathies. Objective: To identify LADs and characterize their associations with CpG methylation and gene expression in human cardiac myocytes in DCM. Methods and Results: LMNA chromatin immunoprecipitation-sequencing, reduced representative bisulfite sequencing, and RNA-sequencing were performed in 5 control and 5 LMNA-associated DCM hearts. LADs were identified using enriched domain detector program. Genome-wide 331±77 LADs with an average size of 2.1±1.5 Mbp were identified in control human cardiac myocytes. LADs encompassed ≈20% of the genome and were predominantly located in the heterochromatin and less so in the promoter and actively transcribed regions. LADs were redistributed in DCM as evidenced by a gain of 520 and loss of 149 genomic regions. Approximately, 4500 coding genes and 800 long noncoding RNAs, whose levels correlated with the transcript levels of coding genes in cis, were differentially expressed in DCM. TP53 (tumor protein 53) was the most prominent among the dysregulated pathways. CpG sites were predominantly hypomethylated genome-wide in controls and DCM hearts, but overall CpG methylation was increased in DCM. LADs were associated with increased CpG methylation and suppressed gene expression. Integrated analysis identified genes whose expressions were regulated by LADs or CpG methylation, or by both, the latter pertained to genes involved in cell death, cell cycle, and metabolic regulation. Conclusions: LADs encompass ≈20% of the genome in human cardiac myocytes comprised several hundred coding and noncoding genes. LADs are redistributed in LMNA-associated DCM in association with markedly altered CpG methylation and gene expression. Thus, LADs through genomic alterations contribute to the pathogenesis of DCM in laminopathies.

Rationale: Mutations in the LMNA gene, encoding nuclear inner membrane protein lamin A/C, cause distinct phenotypes, collectively referred to as laminopathies. Heart failure, conduction defects, and arrhythmias are the common causes of death in laminopathies. Objective: The objective of this study was to identify and therapeutically target the responsible mechanism(s) for cardiac phenotype in laminopathies. Methods and Results: Whole-heart RNA sequencing was performed before the onset of cardiac dysfunction in the Lmna −/− and matched control mice. Differentially expressed transcripts and their upstream regulators were identified, validated, and targeted by adeno-associated virus serotype 9-short hairpin RNA constructs. A total of 576 transcripts were upregulated and 233 were downregulated in the Lmna −/− mouse hearts ( q Lmna −/− cardiac myocytes and in the myocardium of human heart failure patients. Nuclear localization of FOXO1 and 3 was increased, whereas phosphorylated (inactive) FOXO1 and 3 levels were reduced in the Lmna −/− hearts. Gene set enrichment analysis and gene ontology showed activation of apoptosis and inflammation and suppression of cell cycle, adipogenesis, and oxidative phosphorylation in the Lmna −/− hearts. Adeno-associated virus serotype 9-short hairpin RNA–mediated suppression of FOXO TFs rescued selected molecular signatures, improved apoptosis, and prolonged survival by ≈2-fold. Conclusions: FOXO TFs are activated and contribute to the pathogenesis of cardiac phenotype in laminopathies. Suppression of the FOXO TFs in cardiac myocytes partially rescues the phenotype and prolongs survival. The findings identify FOXO TFs as potential therapeutic targets for cardiac phenotype in laminopathies.

Lamin A/C (LMNA) is a major component of the nuclear membrane, involved in regulation of gene expression. Mutations in the LMNA gene cause a heterogeneous group of diseases, collectively referred to as laminopathies. Cardiac involvement is one of the most prominent feature of the laminopathies, presenting with dilated cardiomyopathy (DCM), conduction defects, and sudden death. Molecular mechanisms involved in the pathogenesis of cardiac phenotypes in laminopathies are not well known. To gain insights into molecular pathogenesis of DCM caused by LMNA deficiency, Lmna gene was deleted in cardiac myocytes ( Myh6-Cre:Lmna Flox ). Homozygous ( Myh6-Cre:Lmna F/F ) had a normal cardiac function and histology at 2 weeks of age. However, the mice showed progressive cardiac dilatation and dysfunction, interstitial fibrosis, increased apoptosis, and died by 4 weeks of age. To identify transcriptomic changes that precede cardiac dysfunction, cardiomyocytes were isolated from 2-week old wild type (WT) and Myh6-Cre:Lmna F/F mice and analyzed by RNA-Seq. A total of 1,420 genes were differentially expressed between WT and Myh6-Cre:Lmna F/F . Pathways analysis of differentially expressed genes (DEGs) showed enrichment of inflammatory, epithelial to mesenchymal transition, and cell death pathways, whereas mitochondrial oxidative function, adipogenesis, and fatty acid metabolism pathways were depressed in Myh6-Cre:Lmna F/F . Consistently, DEGs were enriched for the binding motif of RELA, the main transcription factor of the NFκB1 pathway. Activation of the NFκB1 pathway was validated by western blot showing increased nuclear localization of RELA. To determine therapeutic benefits of targeting the NFκB1 pathway, mice are being treated a specific inhibitor of NFκB (IKK-16). Effects on survival, cardiac function, apoptosis, and fibrosis are being analyzed. In conclusion, LMNA deficiency results in dysregulation of a large number genes, including NFκB1 pathway target genes that precede the onset of cardiac dysfunction and fibrosis. The findings point to the prominent role of inflammation in the development of cardiac dysfunction in myocyte-specific LMNA deficiency and identify the NFκB1 pathway as a therapeutic target in DCM caused by LMNA deficiency.

Rationale: Lamin A/C (LMNA) is a nuclear inner membrane protein that interacts with genome through Lamin-Associated Domains (LADs) and regulates gene expression. Mutations in the LMNA gene causes a diverse array of diseases that are collectively referred to as laminopathies. Dilated cardiomyopathy (DCM) is a major component of laminopathies and the leading cause of premature death. Objective: To identify and characterize LADs in cardiac myocytes, cardiac DNA methylation status, and transcriptomes and integrate the findings to find the pathogenic pathways in DCM Methods: We performed ChIP-Seq, reduced representative bisulfite sequencing (RRBS), and RNA-Seq in 5 control and 5 DCM hearts, the later with defined pathogenic variants. ChIP-Seq was performed using an anti-LMNA antibody on protein extracts from FACS-isolated myocyte nuclei, identified by expression of PCM1. RRBS was performed on whole heart DNA extracts to identify methylation state of ~ 500,000 CpG sites. RNA-Seq was performed on whole heart ribosome-depleted RNA extracts. LADs were identified using Enriched Domain Detector (EDD). Results: We identified 370±50 LADs with an average size of ~ 1Mbp (range: 0.5 to 10 Mb), collectively covering about 12,000 genes and 12% of the genomic regions in the control myocytes. The number and size of the LADs were unchanged in DCM. However, genomic locations of LADs had shifted, leading to loss of 3,302 genes from the LADs. Integration of LADs, CpG methylation, and transcript levels showed CpG hypermethylation and suppressed transcript levels in the LADs both in control and in DCM hearts. However, the effects were differential and LADs were associated with increased hypermethylation and further suppression of gene expression in DCM hearts. Pathway analysis of integrated omic datasets identified TGFβ1 and cell cycle pathways as the most activated and MPAK and glycolytic pathways as the most repressed pathways in DCM hearts. Conclusions: LADs, CpG methylation and transcriptomes are dysregulated in cardiac myocytes in patients with DCM caused by LMNA pathogenic variants. The dysregulated biological pathways provide potential targets for interventions in DCM caused by LMNA mutations.

Noncoding RNAs regulate many aspects of cardiovascular biology and are potential therapy targets. In this review, we summarize and highlight current discoveries in the field of microRNAs, a class of noncoding RNAs.miRNAs regulate posttranscriptional gene expression and have been shown to control cardiac development, hypertrophy, fibrosis, and regeneration. Of note are the miRNAs that regulate cardiac contractility (for example, miR-25 and miR-22), cardiac regeneration (like miR-302-367 and miR99/100 families), and fibrosis (as miR-125b). Consistently with these roles of miRNAs, pharmacological intervention using anti-miRNA oligonucleotides (antagomirs or LNA-anti-miRs) has been shown to improve cardiac contractility and mitigate fibrosis, alleviating cardiac dysfunction in the setting of heart failure.miRNAs are crucial regulators of cardiac phenotype and have enthused both basic scientists and clinicians alike. With advancement of technology and better understanding of mechanisms governing miRNA deregulation, we are at the crossroads for deciphering miRNA function and modulating it for therapeutics.

Rationale: PKP2 , encoding plakophilin 2 (PKP2), is the most common causal gene for arrhythmogenic cardiomyopathy. Objective: To characterize miRNA expression profile in PKP2-deficient cells. Methods and Results: Control and PKP2-knockdown HL-1 (HL-1 Pkp2-shRNA ) cells were screened for 750 miRNAs using low-density microfluidic panels. Fifty-nine miRNAs were differentially expressed. MiR-184 was the most downregulated miRNA. Expression of miR-184 in the heart and cardiac myocyte was developmentally downregulated and was low in mature myocytes. MicroRNA-184 was predominantly expressed in cardiac mesenchymal progenitor cells. Knockdown of Pkp2 in cardiac mesenchymal progenitor cells also reduced miR-184 levels. Expression of miR-184 was transcriptionally regulated by the E2F1 pathway, which was suppressed in PKP2-deficient cells. Activation of E2F1, on overexpression of its activator CCND1 (cyclin D1) or knockdown of its inhibitor retinoblastoma 1, partially rescued miR-184 levels. In addition, DNA methyltransferase-1 was recruited to the promoter region of miR-184, and the CpG sites at the upstream region of miR-184 were hypermethylated. Treatment with 5-aza-2′-deoxycytidine, a demethylation agent, and knockdown of DNA methyltransferase-1 partially rescued miR-184 level. Pathway analysis of paired miR-184:mRNA targets identified cell proliferation, differentiation, and death as the main affected biological processes. Knockdown of miR-184 in HL-1 cells and mesenchymal progenitor cells induced and, conversely, its overexpression attenuated adipogenesis. Conclusions: PKP2 deficiency leads to suppression of the E2F1 pathway and hypermethylation of the CpG sites at miR-184 promoter, resulting in downregulation of miR-184 levels. Suppression of miR-184 enhances and its activation attenuates adipogenesis in vitro. Thus, miR-184 contributes to the pathogenesis of adipogenesis in PKP2-deficient cells.

Rationale: Mutations in desmosome proteins cause arrhythmogenic cardiomyopathy (AC), a disease characterized by excess myocardial fibroadipocytes. Cellular origin(s) of fibroadipocytes in AC is unknown. Objective: To identify the cellular origin of adipocytes in AC. Methods and Results: Human and mouse cardiac cells were depleted from myocytes and flow sorted to isolate cells expressing platelet-derived growth factor receptor-α and exclude those expressing other lineage and fibroblast markers (CD32, CD11B, CD45, Lys76, Ly −6c and Ly 6c , thymocyte differentiation antigen 1, and discoidin domain receptor 2). The PDGFRA pos :Lin neg :THY1 neg :DDR2 neg cells were bipotential as the majority expressed collagen 1 α-1, a fibroblast marker, and a subset CCAAT/enhancer-binding protein α, a major adipogenic transcription factor, and therefore, they were referred to as fibroadipocyte progenitors (FAPs). FAPs expressed desmosome proteins, including desmoplakin, predominantly in the adipogenic but not fibrogenic subsets. Conditional heterozygous deletion of Dsp in mice using Pdgfra-Cre deleter led to increased fibroadipogenesis in the heart and mild cardiac dysfunction. Genetic fate mapping tagged 41.4±4.1% of the cardiac adipocytes in the Pdgfra-Cre : Eyfp:Dsp W /F mice, indicating an origin from FAPs. FAPs isolated from the Pdgfra-Cre : Eyfp:Dsp W /F mouse hearts showed enhanced differentiation to adipocytes. Mechanistically, deletion of Dsp was associated with suppressed canonical Wnt signaling and enhanced adipogenesis. In contrast, activation of the canonical Wnt signaling rescued adipogenesis in a dose-dependent manner. Conclusions: A subset of cardiac FAPs, identified by the PDGFRA pos :Lin neg :THY1 neg :DDR2 neg signature, expresses desmosome proteins and differentiates to adipocytes in AC through a Wnt-dependent mechanism. The findings expand the cellular spectrum of AC, commonly recognized as a disease of cardiac myocytes, to include nonmyocyte cells in the heart.

Arrhythmogenic cardiomyopathy (AC) is a genetic disease caused by mutations in genes encoding the intercalated disk (ID) proteins. ID is a multiprotein complex providing cell-cell junction and connecting the nucleus to the extracellular matrix, and playing a pivotal role for maintaining the structural integrity of the heart tissue. Mutations in PKP2 encoding plakophilin 2 (PKP2) are the most common causes of AC. The objective of this study was to investigate the biomechanical properties of the PKP2-knock down HL-1 mouse cardiac myocytes (PKP2-KD). PKP2 protein levels were reduced by 66 to 75% upon shRNA-targeting of the Pkp2 mRNA. Nuclear stiffness (Young’s modulus) and the force required to deform the nucleus were assessed by atomic force microscopy (AFM). Nuclear stiffness was decreased significantly (5 times) in PKP2-KD HL-1 myocytes as compared to wild-type (WT) myocytes, indicating an increased elasticity and/or susceptibility of the nucleus to be deformed (2 times). Interestingly, cell adhesion was also reduced (10 times) in the PKP2-KD myocytes, indicating a pivotal role of ID proteins in maintaining cell adhesion and mechanical integrity in cardiomyocytes. In the stress-relaxation test, while the decay time value was comparable between WT and PKP2-KD cells, relaxation force and cell deformation values were decreased 3 and 8 times, respectively, in PKP2-KD HL-1 myocytes, confirming the finding in the Young’s modulus. Moreover, the relaxation test revealed a reduced viscosity, highlighting a compromised cytoskeletal stability induced by the PKP2 deficiency. RNA-Sequencing and Ingenuity Canonical Pathway analysis identified integrin, tight junction, and the canonical Wnt signaling, among the most disturbed pathways in the PKP2-KD HL-1 myocytes. The findings indicate that the deficiency of a component of the ID protein markedly affects the whole-cellular biomechanics, which is expected to affect tissue stability, leading to activation of the mechano-transduction pathways.

Introduction: A phenotypic characteristic of arrhythmogenic cardiomyopathy (AC) is a gradual replacement of cardiac myocytes by fibro-adipocytes, which leads to cardiac arrhythmias, dysfunction and sudden death. Hypothesis: Differentially expressed miRNAs contribute to the pathogenesis of AC Results: Control and plakophilin-2 (PKP2)-suppressed HL-1 myocytes (HL-1Pkp2-shRNA) were screened for the expression of 750 miRNA using Taqman low-density microfluidic panels. Thirty-one miRNAs were up- and 28 were down-regulated in the HL-1Pkp2-shRNA myocytes. MiR-184 was the most down-regulated miRNA (~10-fold). It was also down-regulated in the heart of mouse models of AC. MiR-184 was developmentally regulated in the mouse heart as its levels were the highest in cardiac myocytes islated from the newborn mice, the lowest in adult myocytes and intermediate in myocytes isolated from 3-week old mice. Ingenuity pathway analysis of paired miR-184 and mRNA sequencing data identified cell proliferation, differentiation and death as the major affected functions. Knock down of miR-184 by shRNA reduced cellular proliferation/viability, increased apoptosis, and enhanced adipogenesis. Levels of over a dozen regulators of lipid synthesis were increased along with fat droplets. Over-expression of miR-184 had reciprocal effects. Bisulfite sequencing identified differential hypermethylation of the CpG sites at the upstream region of miR-184. Treatment with 5-aza-2’-deoxycytidine, a demethylation agent, partially rescued suppressed miR-184 levels. However, activation or suppression of the Hippo and the canonical Wnt signaling pathways, implicated in the pathogenesis of AC, did not affect miR-184 levels. Likewise, miR-184 over-expression or suppression did not affect the Hippo and Wnt signaling. Conclusions: miR-184 is developmentally regulated with the highest levels of expression in the newborn myocytes. It levels are suppressed in the AC models, partially because of hypermethylation of the CpG sites at its genomic regions. MiR-184 by regulating cellular proliferation and differentiation contributes to the pathogenesis of AC.

Arrhythmogenic cardiomyopathy (AC) is clinically characterized by arrhythmias, heart failure and sudden death and pathologically by replacement of cardiomyocytes by fibro-adipocytes. Mutations in desmosome genes are the main causes of AC. Desmosome proteins are known to be expressed only in cardiac myocytes. Cardiac progenitor cells (CPCs) expressing mutant desmosome proteins could differentiate to adipocytes. However, genetic fate-mapping identify CPCs as the source of a fraction of adipocytes in AC. We posit that cardiac cells other than CPCs express desmosome proteins and differentiate to adipocytes in AC. We systematically determined expression of the desmosome proteins in different cardiac cells. Cardiac fibro-adipocyte progenitors (FAPs), marked by expression of platelet-derived growth factor receptor-α (PDGFRA), expressed mRNAs and proteins of major desmosome genes. FAPs isolated from the heart of Nkx2.5Cre:Dsp W/F mice, an established model of AC, showed enhanced adipogenesis associated with suppression of canonical Wnt signaling (reduced Ccnd1, Ctgf and cMyc mRNA levels by 10-, 5- and 3-fold, respectively, N=5, all pPdgfra-Cre deleter, and conditionally deleted one copy of desmoplakin ( Dsp ) while expressing enhanced yellow fluorescence protein (EYFP) in the FAPs. Recombination efficiency (% of PDGFRA+ cells expressing EYFP) was ~75%. Six to 12 months old lineage tracer mice ( Pdgfra-Cre:Dsp W/F ) showed mild cardiac dysfunction (fractional shortening: 57±9%, N=14 vs. 66±7%, N=10 in controls, p< 0.005) and increased adipocytes in the heart (14±9, N=5 vs. 1±1, N=3 in controls, p=0.03). Co-expression of EYFP and C/EBPA adipogenic marker was detected in 54% of adipocytes in the lineage tracer mice, indicating an origin from FAPs. Dsp +/- FAPs showed enhanced differentiation to adipocytes as compared to control FAPs. The Hippo, canonical Wnt and NOTCH1 signaling pathways in mediating differentiation of Dsp +/- FAPs to adipocytes are being delineated. The findings show cardiac FAPs express desmosome proteins and when haplo-insufficient for Dsp differentiate to adipocytes. Thus, FAPs are a source of adipocytes in AC.

The genome continues to fascinate the enthusiasts, and the captivation seems unabating because of the continuous stream of new discoveries. What was once considered a junk DNA has now emerged to contain a large number of important regulatory elements.1 As an example of such discoveries is the recent finding that the genome contains ≈6200 enhancer elements that are operational in the human fetal and adult hearts.2 Genes occupy only ≈1.5% and coding exons only ≈1% of the genome, which make up only ≈60 million nucleotides of the 6.4 billion nucleotides (3.2 billion base pairs) in the genome.3 Yet, ≈5% of the human genome has undergone purifying selection and hence are likely functional.4 It is intriguing that about two third of the evolutionary constrained genomic elements are located in introns and the intergenic regions, suggestive of their regulatory roles in the genome.4 These conserved regions are enriched in loci that have been found to be associated with clinical phenotypes in the genome-wide association studies.4 The initial findings of the ENCODE (Encyclopedia of DNA Elements) project, although preliminary, illustrate the presence of numerous regulatory elements in the genome, including the enhancers.1 The discoveries largely made possible by the recent advances in the high-throughput RNA sequencing point to enormous RNA splicing diversity and the plethora of alternative splicing variants. Approximately 95% of the multiexon genes undergo alternative splicing, resulting in ≈100 000 abundant splice variants in various tissues.5 Moreover, it seems that almost all genomic regions in 1 form or shape are transcribed, which seems perplexing as only ≈1% of the genome codes for proteins, as has been understood to date. Only recently we have started to appreciate the diverse biological functions of these nonprotein coding transcripts, which are referred to as noncoding RNAs (ncRNAs). …

Background— Delineating the role of microRNAs (miRNAs) in the posttranscriptional gene regulation offers new insights into how the heart adapts to pathological stress. We developed a knockout of miR-22 in mice and investigated its function in the heart. Methods and Results— Here, we show that miR-22 –deficient mice are impaired in inotropic and lusitropic response to acute stress by dobutamine. Furthermore, the absence of miR-22 sensitized mice to cardiac decompensation and left ventricular dilation after long-term stimulation by pressure overload. Calcium transient analysis revealed reduced sarcoplasmic reticulum Ca 2+ load in association with repressed sarcoplasmic reticulum Ca 2+ ATPase activity in mutant myocytes. Genetic ablation of miR-22 also led to a decrease in cardiac expression levels for Serca2a and muscle-restricted genes encoding proteins in the vicinity of the cardiac Z disk/titin cytoskeleton. These phenotypes were attributed in part to inappropriate repression of serum response factor activity in stressed hearts. Global analysis revealed increased expression of the transcriptional/translational repressor purine-rich element binding protein B, a highly conserved miR-22 target implicated in the negative control of muscle expression. Conclusion— These data indicate that miR-22 functions as an integrator of Ca 2+ homeostasis and myofibrillar protein content during stress in the heart and shed light on the mechanisms that enhance propensity toward heart failure.

Recent evidence suggests that miRNAs play an important role in cardiac morphogenesis and pathophyiology of heart failure. To explore the role of miR-22 in the mouse heart physiology, we generated miR-22 null (KO) mice. Although, miR-22 KO mice showed normal cardiac structure and function at baseline, these mice are sensitized to maladaptive remodeling (cardiac dilation) and decompensation in response to pressure overload by transverse aortic constrictions (TAC) stimulation. Genome-wide molecular analysis of KO hearts revealed attenuated expression of numerous CarG-dependent genes encoding proteins that reside at the sarcomeric Z-disc (including Myh7, Acta1, Mlp, Melusin, MyoZ2) indicating that miR-22 is required for optimum muscle gene expression. Alterations in sarcomeric gene expression is especially interesting as this suggests a primary role of miR-22 in controlling cardiac contractility and adaptation to stress. Targetomics analysis revealed that mechanistically this effect could be modulated in part by miR-22 target PURB (Purine Rich element binding protein B), a transcriptional/translational repressor. In conclusion we define a critical role of miR-22 in cardiac adaptation to hemodynamic stress. Furthermore, these data provides a previously unseen essential molecular mechanism that underlies homeostatic control of sarcomeric protein expression in the heart.