Histone demethylase KDM5 regulates cardiomyocyte maturation by promoting fatty acid oxidation, oxidative phosphorylation, and myofibrillar organization.

AIMS: Human pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a platform to identify and characterize factors that regulate the maturation of CMs. The transition from an immature foetal to an adult CM state entails coordinated regulation of the expression of genes involved in myofibril formation and oxidative phosphorylation (OXPHOS) among others. Lysine demethylase 5 (KDM5) specifically demethylates H3K4me1/2/3 and has emerged as potential regulators of expression of genes involved in cardiac development and mitochondrial function. The purpose of this study is to determine the role of KDM5 in iPSC-CM maturation. METHODS AND RESULTS: KDM5A, B, and C proteins were mainly expressed in the early post-natal stages, and their expressions were progressively downregulated in the post-natal CMs and were absent in adult hearts and CMs. In contrast, KDM5 proteins were persistently expressed in the iPSC-CMs up to 60 days after the induction of myogenic differentiation, consistent with the immaturity of these cells. Inhibition of KDM5 by KDM5-C70 -a pan-KDM5 inhibitor, induced differential expression of 2372 genes, including upregulation of genes involved in fatty acid oxidation (FAO), OXPHOS, and myogenesis in the iPSC-CMs. Likewise, genome-wide profiling of H3K4me3 binding sites by the cleavage under targets and release using nuclease assay showed enriched of the H3K4me3 peaks at the promoter regions of genes encoding FAO, OXPHOS, and sarcomere proteins. Consistent with the chromatin and gene expression data, KDM5 inhibition increased the expression of multiple sarcomere proteins and enhanced myofibrillar organization. Furthermore, inhibition of KDM5 increased H3K4me3 deposits at the promoter region of the ESRRA gene and increased its RNA and protein levels. Knockdown of ESRRA in KDM5-C70-treated iPSC-CM suppressed expression of a subset of the KDM5 targets. In conjunction with changes in gene expression, KDM5 inhibition increased oxygen consumption rate and contractility in iPSC-CMs. CONCLUSION: KDM5 inhibition enhances maturation of iPSC-CMs by epigenetically upregulating the expressions of OXPHOS, FAO, and sarcomere genes and enhancing myofibril organization and mitochondrial function.

Genetic inactivation of ß-catenin is salubrious, whereas its activation is deleterious in desmoplakin cardiomyopathy.

AIMS: Mutations in the DSP gene encoding desmoplakin, a constituent of the desmosomes at the intercalated discs (IDs), cause a phenotype that spans arrhythmogenic cardiomyopathy (ACM) and dilated cardiomyopathy. It is typically characterized by biventricular enlargement and dysfunction, myocardial fibrosis, cell death, and arrhythmias. The canonical wingless-related integration (cWNT)/ß-catenin pathway is implicated in the pathogenesis of ACM. The ß-catenin is an indispensable co-transcriptional regulator of the cWNT pathway and a member of the IDs. We genetically inactivated or activated ß-catenin to determine its role in the pathogenesis of desmoplakin cardiomyopathy. METHODS AND RESULTS: The Dsp gene was conditionally deleted in the 2-week-old post-natal cardiac myocytes using tamoxifen-inducible MerCreMer mice (Myh6-McmTam:DspF/F). The cWNT/ß-catenin pathway was markedly dysregulated in the Myh6-McmTam:DspF/F cardiac myocytes, as indicated by a concomitant increase in the expression of cWNT/ß-catenin target genes, isoforms of its key co-effectors, and the inhibitors of the pathway. The ß-catenin was inactivated or activated upon inducible deletion of its transcriptional or degron domain, respectively, in the Myh6-McmTam:DspF/F cardiac myocytes. Genetic inactivation of ß-catenin in the Myh6-McmTam:DspF/F mice prolonged survival, improved cardiac function, reduced cardiac arrhythmias, and attenuated myocardial fibrosis, and cell death caused by apoptosis, necroptosis, and pyroptosis, i.e. PANoptosis. In contrast, activation of ß-catenin had the opposite effects. The deleterious and the salubrious effects were independent of changes in the expression levels of the cWNT target genes and were associated with changes in several molecular and biological pathways, including cell death programmes. CONCLUSION: The cWNT/ß-catenin was markedly dysregulated in the cardiac myocytes in a mouse model of desmoplakin cardiomyopathy. Inactivation of ß-catenin attenuated, whereas its activation aggravated the phenotype, through multiple molecular pathways, independent of the cWNT transcriptional activity. Thus, suppression but not activation of ß-catenin might be beneficial in desmoplakin cardiomyopathy.

Effects of tamoxifen inducible MerCreMer on gene expression in cardiac myocytes in mice.

The Cre-LoxP technology, including the tamoxifen (TAM) inducible MerCreMer (MCM), is increasingly used to delineate gene function, understand the disease mechanisms, and test therapeutic interventions. We set to determine the effects of TAM-MCM on cardiac myocyte transcriptome. Expression of the MCM was induced specifically in cardiac myocytes upon injection of TAM to myosin heavy chain 6-MCM (Myh6-Mcm) mice for 5 consecutive days. Cardiac function, myocardial histology, and gene expression (RNA-sequencing) were analyzed 2 weeks after TAM injection. A total of 346 protein coding genes (168 up- and 178 down-regulated) were differentially expressed. Transcript levels of 85 genes, analyzed by a reverse transcription-polymerase chain reaction in independent samples, correlated with changes in the RNA-sequencing data. The differentially expressed genes were modestly enriched for genes involved in the interferon response and the tumor protein 53 (TP53) pathways. The changes in gene expression were relatively small and mostly transient and had no discernible effects on cardiac function, myocardial fibrosis, and apoptosis or induction of double-stranded DNA breaks. Thus, TAM-inducible activation of MCM alters cardiac myocytes gene expression, provoking modest and transient interferon and DNA damage responses without exerting other discernible phenotypic effects. Thus, the effects of TAM-MCM on gene expression should be considered in discerning the bona fide changes that result from the targeting of the gene of interest.

The EP300/TP53 pathway, a suppressor of the Hippo and canonical WNT pathways, is activated in human hearts with arrhythmogenic cardiomyopathy in the absence of overt heart failure.

AIMS: 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 five control samples were analysed by RNA-Seq (discovery group). These cases presented with cardiac arrhythmias and had a normal right ventricular function. The RNA-Seq analysis identified ∼5000 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 five 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 were 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 signalling pathways. The changes were associated with increased apoptosis and fibro-adipogenesis in the ACM hearts. CONCLUSION: Altered apical junction structures are 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.

Single-Cell RNA Sequencing Uncovers Paracrine Functions of the Epicardial-Derived Cells in Arrhythmogenic Cardiomyopathy.

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-CreERT2:DspW/F were crossed to Rosa26mT/mG (R26mT/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-CreERT2:R26mT/mG and Wt1-CreERT2: R26mT/mG:DspW/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-CreERT2:DspW/F mice. The Wt1-CreERT2:DspW/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 TGFß1 (transforming growth factor ß1) 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 TGFß1 pathway. Activation of the TGFß1 and its colocalization with fibrosis in the Wt1-CreERT2:R26mT/mG:DspW/F mouse heart was validated by complementary methods. CONCLUSIONS: Epicardial-derived cardiac fibroblasts and epithelial cells express paracrine factors, including TGFß1 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.

Haploinsufficiency of Tmem43 in cardiac myocytes activates the DNA damage response pathway leading to a late-onset senescence-associated pro-fibrotic cardiomyopathy.

AIMS: Arrhythmogenic cardiomyopathy (ACM) encompasses a genetically heterogeneous group of myocardial diseases whose manifestations are sudden cardiac death, cardiac arrhythmias, heart failure, and in a subset fibro-adipogenic infiltration of the myocardium. Mutations in the TMEM43 gene, encoding transmembrane protein 43 (TMEM43) are known to cause ACM. The purpose of the study was to gain insights into the molecular pathogenesis of ACM caused by TMEM43 haploinsufficiency. METHODS AND RESULTS: The Tmem43 gene was specifically deleted in cardiac myocytes by crossing the Myh6-Cre and floxed Tmem43 mice. Myh6-Cre:Tmem43W/F mice showed an age-dependent phenotype characterized by an increased mortality, cardiac dilatation and dysfunction, myocardial fibrosis, adipogenesis, and apoptosis. Sequencing of cardiac myocyte transcripts prior to and after the onset of cardiac phenotype predicted early activation of the TP53 pathway. Increased TP53 activity was associated with increased levels of markers of DNA damage response (DDR), and a subset of senescence-associated secretary phenotype (SASP). Activation of DDR, TP53, SASP, and their selected downstream effectors, including phospho-SMAD2 and phospho-SMAD3 were validated by alternative methods, including immunoblotting. Expression of SASP was associated with epithelial-mesenchymal transition and age-dependent expression of myocardial fibrosis and apoptosis in the Myh6-Cre:Tmem43W/F mice. CONCLUSION: TMEM43 haploinsufficiency is associated with activation of the DDR and the TP53 pathways, which lead to increased expression of SASP and an age-dependent expression of a pro-fibrotic cardiomyopathy. Given that TMEM43 is a nuclear envelope protein and our previous data showing deficiency of another nuclear envelope protein, namely lamin A/C, activates the DDR/TP53 pathway, we surmise that DNA damage is a shared mechanism in the pathogenesis of cardiomyopathies caused by mutations involving nuclear envelope proteins.

Identification of Genes and Pathways Regulated by Lamin A in Heart.

Background Mutations in the LMNA gene, encoding LMNA (lamin A/C), causes distinct disorders, including dilated cardiomyopathies, collectively referred to as laminopathies. The genes (coding and noncoding) and regulatory pathways controlled by LMNA in the heart are not completely defined. Methods and Results We analyzed cardiac transcriptome from wild-type, loss-of-function (Lmna-/-), and gain-of-function (Lmna-/- injected with adeno-associated virus serotype 9 expressing LMNA) mice with normal cardiac function. Deletion of Lmna (Lmna-/-) led to differential expression of 2193 coding and 629 long noncoding RNA genes in the heart (q<0.05). Re-expression of LMNA in the Lmna-/- mouse heart, completely rescued 501 coding and 208 non-coding and partially rescued 1862 coding and 607 lncRNA genes. Pathway analysis of differentially expressed genes predicted activation of transcriptional regulators lysine-specific demethylase 5A, lysine-specific demethylase 5B, tumor protein 53, and suppression of retinoblastoma 1, paired-like homeodomain 2, and melanocyte-inducing transcription factor, which were completely or partially rescued upon reexpression of LMNA. Furthermore, lysine-specific demethylase 5A and 5B protein levels were increased in the Lmna-/- hearts and were partially rescued upon LMNA reexpression. Analysis of biological function for rescued genes identified activation of tumor necrosis factor-α, epithelial to mesenchymal transition, and suppression of the oxidative phosphorylation pathway upon Lmna deletion and their restoration upon LMNA reintroduction in the heart. Restoration of the gene expression and transcriptional regulators in the heart was associated with improved cardiac function and increased survival of the Lmna-/- mice. Conclusions The findings identify LMNA-regulated cardiac genes and their upstream transcriptional regulators in the heart and implicate lysine-specific demethylase 5A and B as epigenetic regulators of a subset of the dysregulated genes in laminopathies.

RNA sequencing-based transcriptome profiling of cardiac tissue implicates novel putative disease mechanisms in FLNC-associated arrhythmogenic cardiomyopathy.

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.

Knock Down of Plakophillin 2 Dysregulates Adhesion Pathway through Upregulation of miR200b and Alters the Mechanical Properties in Cardiac Cells.

Background: Mutations in genes encoding intercalated disk/desmosome proteins, such as plakophilin 2 (PKP2), cause arrhythmogenic cardiomyopathy (ACM). Desmosomes are responsible for myocyte-myocyte attachment and maintaining mechanical integrity of the myocardium. Methods: We knocked down Pkp2 in HL-1 mouse atrial cardiomyocytes (HL-1Pkp2-shRNA) and characterized their biomechanical properties. Gene expression was analyzed by RNA-Sequencing, microarray, and qPCR. Immunofluorescence was used to detect changes in cytoskeleton and focal adhesion. Antagomirs were used to knock down expression of selected microRNA (miR) in the rescue experiments. Results: Knockdown of Pkp2 was associated with decreased cardiomyocyte stiffness and work of detachment, and increased plasticity index. Altered mechanical properties were associated with impaired actin cytoskeleton in HL-1Pkp2-shRNA cells. Analysis of differentially expressed genes identified focal adhesion and actin cytoskeleton amongst the most dysregulated pathways, and miR200 family (a, b, and 429) as the most upregulated miRs in HL-1Pkp2-shRNA cells. Knockdown of miR-200b but not miR-200a, miR-429, by sequence-specific shRNAs partially rescued integrin-α1 (Itga1) levels, actin organization, cell adhesion (on collagen), and stiffness. Conclusions: PKP2 deficiency alters cardiomyocytes adhesion through a mechanism that involves upregulation of miR-200b and suppression of Itga1 expression. These findings provide new insights into the molecular basis of altered mechanosensing in ACM.

Genomic Reorganization of Lamin-Associated Domains in Cardiac Myocytes Is Associated With Differential Gene Expression and DNA Methylation in Human Dilated Cardiomyopathy.

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.

DNA Damage Response/TP53 Pathway Is Activated and Contributes to the Pathogenesis of Dilated Cardiomyopathy Associated With LMNA (Lamin A/C) Mutations.

RATIONALE: Mutations in the LMNA gene, encoding LMNA (lamin A/C), are responsible for laminopathies. Dilated cardiomyopathy (DCM) is a major cause of mortality and morbidity in laminopathies. OBJECTIVE: To gain insights into the molecular pathogenesis of DCM in laminopathies. METHODS AND RESULTS: We generated a tet-off bigenic mice expressing either a WT (wild type) or a mutant LMNA (D300N) protein in cardiac myocytes. LMNAD300N mutation is associated with DCM in progeroid syndromes. Expression of LMNAD300N led to severe myocardial fibrosis, apoptosis, cardiac dysfunction, and premature death. Administration of doxycycline suppressed LMNAD300N expression and prevented the phenotype. Whole-heart RNA sequencing in 2-week-old WT and LMNAD300N mice led to identification of ≈6000 differentially expressed genes. Gene Set Enrichment and Hallmark Pathway analyses predicted activation of E2F (E2F transcription factor), DNA damage response, TP53 (tumor protein 53), NFκB (nuclear factor κB), and TGFß (transforming growth factor-ß) pathways, which were validated by Western blotting, quantitative polymerase chain reaction of selected targets, and immunofluorescence staining. Differentially expressed genes involved cell death, cell cycle regulation, inflammation, and epithelial-mesenchymal differentiation. RNA sequencing of human hearts with DCM associated with defined LMNA pathogenic variants corroborated activation of the DNA damage response/TP53 pathway in the heart. Increased expression of CDKN2A (cyclin-dependent kinase inhibitor 2A)-a downstream target of E2F pathway and an activator of TP53-provided a plausible mechanism for activation of the TP53 pathway. To determine pathogenic role of TP53 pathway in DCM, Tp53 gene was conditionally deleted in cardiac myocytes in mice expressing the LMNAD300N protein. Deletion of Tp53 partially rescued myocardial fibrosis, apoptosis, proliferation of nonmyocyte cells, left ventricular dilatation and dysfunction, and slightly improved survival. CONCLUSIONS: Cardiac myocyte-specific expression of LMNAD300N, associated with DCM, led to pathogenic activation of the E2F/DNA damage response/TP53 pathway in the heart and induction of myocardial fibrosis, apoptosis, cardiac dysfunction, and premature death. The findings denote the E2F/DNA damage response/TP53 axis as a responsible mechanism for DCM in laminopathies and as a potential intervention target.

A Calsequestrin Cis-Regulatory Motif Coupled to a Cardiac Troponin T Promoter Improves Cardiac Adeno-Associated Virus Serotype 9 Transduction Specificity.

Adeno-associated virus serotype 9 (AAV9) is an efficient vector for gene transfer to the myocardium. However, the use of ubiquitous promoters, such as the cytomegalovirus (CMV) promoter, can result in expression of the transgene in organs other than the heart. This study tested if the efficiency and specificity of cardiac transcription from a chicken cardiac troponin T (TnT) promoter could be further increased by incorporating a cardiomyocyte-specific transcriptional cis-regulatory motif from human calsequestrin 2 (CS-CRM4) into the expression cassette (Enh.TnT). The efficiency of luciferase expression from the TnT and Enh.TnT constructs was compared to expression of luciferase under the control of the CMV promoter in both adult and neonatal mice. Overall, expression levels of luciferase in the heart were similar in mice injected with AAV9.TnT.Luc, AAV9.Enh.TnT.Luc and AAV9.CMV.Luc. In contrast, expression levels of luciferase activity in nontarget organs, including the liver and muscle, was lower in mice injected with the AAV9.TnT.Luc compared to AAV9.CMV.Luc and was negligible with AAV9.Enh.TnT. In neonates, in organs other than the heart, luciferase expression levels were too low to be quantified for all constructs. Taken together, the data show that the AAV9 Enh.TnT constructs drives high levels of expression of the transgene in the myocardium, with insignificant expression in other organs. These properties reduce the risks associated with the AAV9-mediated expression of the therapeutic protein of interest in nontarget organs. The excellent cardiac specificity should allow for the use of higher vector doses than are currently used, which might be essential to achieve the levels of transgene expression necessary for therapeutic benefits. Taken together, the findings suggest that the Enh.TnT transcription unit is a potentially attractive tool for clinical cardiac gene therapy in adults.

Suppression of Activated FOXO Transcription Factors in the Heart Prolongs Survival in a Mouse Model of 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<0.05). Forkhead box O (FOXO) transcription factors (TFs) were the most activated while E2 factors were the most suppressed transcriptional regulators. Transcript levels of FOXO targets were also upregulated in the isolated 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.

Knockdown of Plakophilin 2 Downregulates miR-184 Through CpG Hypermethylation and Suppression of the E2F1 Pathway and Leads to Enhanced Adipogenesis In Vitro.

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.

The hippo pathway is activated and is a causal mechanism for adipogenesis in arrhythmogenic cardiomyopathy.

RATIONALE: Mutations in the intercalated disc proteins, such as plakophilin 2 (PKP2), cause arrhythmogenic cardiomyopathy (AC). AC is characterized by the replacement of cardiac myocytes by fibro-adipocytes, cardiac dysfunction, arrhythmias, and sudden death. OBJECTIVE: To delineate the molecular pathogenesis of AC. METHODS AND RESULTS: Localization and levels of selected intercalated disc proteins, including signaling molecules, were markedly reduced in human hearts with AC. Altered protein constituents of intercalated discs were associated with activation of the upstream Hippo molecules in the human hearts, in Nkx2.5-Cre:Dsp(W/F) and Myh6:Jup mouse models of AC, and in the PKP2 knockdown HL-1 myocytes (HL-1(PKP2:shRNA)). Level of active protein kinase C-α isoform, which requires PKP2 for activity, was reduced. In contrast, neurofibromin 2 (or Merlin), a molecule upstream of the Hippo pathway and that is inactivated by protein kinase C-α isoform, was activated. Consequently, the downstream Hippo molecules mammalian STE20-like protein kinases 1/2 (MST1/2), large tumor suppressor kinases 1/2 (LATS1/2), and Yes-associated protein (YAP) (the latter is the effector of the pathway) were phosphorylated. Coimmunoprecipitation detected binding of phosphorylated YAP, phosphorylated ß-catenin, and junction protein plakoglobin (the latter translocated from the junction). RNA sequencing, transcript quantitative polymerase chain reaction, and reporter assays showed suppressed activity of SV40 transcriptional enhancer factor domain (TEAD) and transcription factor 7-like 2 (TCF7L2), which are transcription factors of the Hippo and the canonical Wnt signaling, respectively. In contrast, adipogenesis was enhanced. Simultaneous knockdown of Lats1/2, molecules upstream to YAP, rescued inactivation of YAP and ß-catenin and adipogenesis in the HL-1(PKP2:shRNA) myocytes. CONCLUSIONS: Molecular remodeling of the intercalated discs leads to pathogenic activation of the Hippo pathway, suppression of the canonical Wnt signaling, and enhanced adipogenesis in AC. The findings offer novel mechanisms for the pathogenesis of AC.

microRNA-22 promotes heart failure through coordinate suppression of PPAR/ERR-nuclear hormone receptor transcription.

Increasing evidence suggests that microRNAs are intimately involved in the pathophysiology of heart failure. MicroRNA-22 (miR-22) is a muscle-enriched miRNA required for optimum cardiac gene transcription and adaptation to hemodynamic stress by pressure overload in mice. Recent evidence also suggests that miR-22 induces hypertrophic growth and it is oftentimes upregulated in end stage heart failure. However the scope of mRNA targets and networks of miR-22 in the heart failure remained unclear. We analyzed transgenic mice with enhanced levels of miR-22 expression in adult cardiomyocytes to identify important pathophysiologic targets of miR-22. Our data shows that forced expression of miR-22 induces a pro-hypertrophic gene expression program, and it elicits contractile dysfunction leading to cardiac dilation and heart failure. Increased expression of miR-22 impairs the Ca(2+) transient, Ca(2+) loading into the sarcoplasmic reticulum plus it interferes with transcription of estrogen related receptor (ERR) and PPAR downstream genes. Mechanistically, miR-22 postranscriptionally inhibits peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), PPARα and sirtuin 1 (SIRT1) expression via a synergistic circuit, which may account for deleterious actions of unchecked miR-22 expression on the heart.

Small noncoding differentially methylated copy-number variants, including lncRNA genes, cause a lethal lung developmental disorder.

An unanticipated and tremendous amount of the noncoding sequence of the human genome is transcribed. Long noncoding RNAs (lncRNAs) constitute a significant fraction of non-protein-coding transcripts; however, their functions remain enigmatic. We demonstrate that deletions of a small noncoding differentially methylated region at 16q24.1, including lncRNA genes, cause a lethal lung developmental disorder, alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV), with parent-of-origin effects. We identify overlapping deletions 250 kb upstream of FOXF1 in nine patients with ACD/MPV that arose de novo specifically on the maternally inherited chromosome and delete lung-specific lncRNA genes. These deletions define a distant cis-regulatory region that harbors, besides lncRNA genes, also a differentially methylated CpG island, binds GLI2 depending on the methylation status of this CpG island, and physically interacts with and up-regulates the FOXF1 promoter. We suggest that lung-transcribed 16q24.1 lncRNAs may contribute to long-range regulation of FOXF1 by GLI2 and other transcription factors. Perturbation of lncRNA-mediated chromatin interactions may, in general, be responsible for position effect phenomena and potentially cause many disorders of human development.