Search

RationaleHuman pluripotent stem cell-derived CMs (iPSC-CMs) are a valuable tool for disease modeling, cell therapy and to reconstruct the CM maturation process and identify, characterize factors that regulate maturation. The transition from immature fetal to adult CM entails coordinated regulation of the mature gene programming, which is characterized by the induction of myofilament and OXPHOS gene expression among others. Recent studies inDrosophila,C. elegans,and C2C12 myoblast cell lines have implicated the histone H3K4me3 demethylase KDM5 and its homologs, as a potential regulator of developmental gene program and mitochondrial function. We speculated that KDM5 may potentiate the maturation of iPSC-CMs by targeting a conserved epigenetic program that encompass mitochondrial OXPHOS and other CM specific maturation genes.ObjectivesThe purpose of this study is to determine the role of KDM5 in iPSC-CM maturation.Methods and ResultsImmunoblot analysis revealed that KDM5A, B, and C expression was progressively downregulated in postnatal cardiomyocytes and absent in adult hearts and CMs. Additionally, KDM5 proteins were found to be persistently expressed in iPSC-CMs up to 60 days after the onset of myogenic differentiation, consistent with the immaturity of these cells. Inhibition of KDM5 by KDM5-C70 -a pan-KDM5 inhibitor-resulted in differential regulation of 2,372 genes including upregulation of Fatty acid oxidation (FAO), OXPHOS, and myogenic gene programs in iPSC-CMs. Likewise, genome-wide profiling of H3K4me3 binding sites by the CUT&RUN assay revealed enriched H3K4me3 peaks at the promoter regions of FAO, OXPHOS, and sarcomere genes. Consistent with the chromatin and gene expression data, KDM5 inhibition led to increased expression of multiple sarcomere proteins, enhanced myofibrillar organization and improved calcium handling. Furthermore, inhibition of KDM5 increased H3K4me3 deposits at the promoter region of theESRRAgene, which is known to regulate OXPHOS and cardiomyocyte maturation, and resulted in its increased RNA and protein levels. Finally, KDM5 inhibition increased baseline, peak, and spare oxygen consumption rates in iPSC-CMs.ConclusionsKDM5 regulates the maturation of iPSC-CMs by epigenetically regulating the expression of ESRRA, OXPHOS, FAO, and sarcomere genes and enhancing myofibril organization and mitochondrial function.

AimMutations in theDSPgene encoding desmoplakin, a constituent of the desmosomes at the intercalated discs (IDs), cause a phenotype that spans arrhythmogenic cardiomyopathy (ACM) and dilated cardiomyopathy (DCM). It is typically characterized by biventricular enlargement and dysfunction, severe myocardial fibrosis, cell death, and arrhythmias.The canonical WNT (cWNT)/β-catenin signaling pathway is implicated in the pathogenesis of ACM. Given that β-catenin, an indispensable co-transcriptional regulator of the cWNT pathway, is also a member of the IDs, we genetically inactivated or activated β-catenin to determine its role in the pathogenesis of the desmoplakin cardiomyopathy.Methods and ResultsTheDspgene was conditionally deleted in cardiac myocytes concomitant with the genetic inactivation or activation of β-catenin using the tamoxifen-inducible MerCreMer mice. Inactivation and activation of β-catenin were achieved upon deletion of its transcriptional domain and degrons, respectively. Analysis of cardiac myocytes transcripts and proteins showed marked dysregulation of the cWNT/β-catenin pathway in the DSP-deficient mouse cardiac myocytes (Myh6-McmTam:DspF/F), as indicated by increased expression of cWNT/β-catenin targets along with its inhibitors and isoforms of its key co-effectors. Genetic inactivation of β-catenin in theMyh6-McmTam:DspF/Fmice prolonged survival, improved cardiac function, reduced cardiac arrhythmias, and attenuated myocardial fibrosis, and cell death caused by apoptosis, necroptosis, pyroptosis, i.e., PANoptosis, whereas its activation had the opposite effects. Inactivation of β-catenin was associated with partial restoration of the suppressed genes involved in OXPHOS, whereas its activation has the opposite effect. The beneficial effects were independent of the changes in the transcript levels of the cWNT target genes.ConclusionThe cWNT/β-catenin was markedly dysregulated in the cardiac myocytes from a mouse model of DC. Inactivation of β-catenin attenuated the phenotype partly through the recovery of OXPHOS genes whereas its activation had deleterious effects. The findings suggest suppression of β-catenin might be beneficial in desmoplakin-cardiomyopathy.SummaryGenetic inactivation of β-catenin improved desmoplakin cardiomyopathy, in part through the restoration of expression of genes involved in oxidative phosphorylation, whereas its activation was deleterious.

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.

Mutations in theThe purpose of the study was to determine the contribution of the fibroblasts to DCM caused by LMNA deficiency.TheDeletion of the

Mutation in the LMNA gene, encoding lamin A/C, causes a diverse group of diseases called laminopathies. Cardiac involvement is the major cause of death and manifests as dilated cardiomyopathy, heart failure, arrhythmias, and sudden death. There is no specific therapy for LMNA-associated cardiomyopathy. We report that deletion of Lmna in cardiomyocytes in mice leads to severe cardiac dysfunction, conduction defect, ventricular arrhythmias, fibrosis, apoptosis, and premature death within 4 weeks. The phenotype is similar to LMNA-associated cardiomyopathy in humans. RNA sequencing, performed before the onset of cardiac dysfunction, led to identification of 2338 differentially expressed genes (DEGs) in Lmna-deleted cardiomyocytes. DEGs predicted activation of bromodomain-containing protein 4 (BRD4), a regulator of chromatin-associated proteins and transcription factors, which was confirmed by complementary approaches, including chromatin immunoprecipitation sequencing. Daily injection of JQ1, a specific BET bromodomain inhibitor, partially reversed the DEGs, including those encoding secretome; improved cardiac function; abrogated cardiac arrhythmias, fibrosis, and apoptosis; and prolonged the median survival time 2-fold in the myocyte-specific Lmna-deleted mice. The findings highlight the important role of LMNA in cardiomyocytes and identify BET bromodomain inhibition as a potential therapeutic target in LMNA-associated cardiomyopathy, for which there is no specific effective therapy.

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., Graphical Abstract

The human genome is pervasively transcribed and yet only a small fraction of these RNAs (less than 2%) are known to code for proteins. The vast majority of the RNAs are classified as noncoding RNAs (ncRNAs) and are further subgrouped as small (shorter than 200 bases) and long noncoding RNAs. The ncRNAs have been identified in all three domains of life and regulate diverse cellular processes through transcriptional and posttranscriptional gene regulation. Most of these RNAs work in conjunction with proteins forming a wide array of base pairing interactions. The determinants of these base pairing interactions are now becoming more evident and show striking similarities among the diverse group of ncRNAs. Here we present a mechanistic overview of pairing between RNA-RNA or RNA-DNA that dictates the function of ncRNAs; we provide examples to illustrate that ncRNAs work through shared evolutionary mechanisms that encompasses a guide-target interaction, involving not only classical Watson-Crick but also noncanonical Wobble and Hoogsteen base pairing. We also highlight the similarities in target selection, proofreading, and the ruler mechanism of ncRNA-protein complexes that confers target specificity and target site selection. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA-Based Catalysis > RNA-Mediated Cleavage RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution.

Introduction: Arrhythmogenic cardiomyopathy (ACM) is hereditary cardiomyopathy caused by pathogenic variants (mutations) in genes encoding the intercalated disc (ID), particularly desmosome proteins. ACM caused by mutations in the DSP gene encoding desmoplakin (DSP) is characterized by the prominence of cell death, myocardial fibrosis, and inflammation, and is referred to as desmoplakin cardiomyopathy. Aim: The aim of this article was to gain insight into the pathogenesis of DSP cardiomyopathy. Methods and Results: The Dsp gene was exclusively deleted in cardiac myocytes using tamoxifen-inducible MerCreMer (Myh6-McmTam) and floxed Dsp (DspF/F) mice (Myh6-McmTam:DspF/F). Recombination was induced upon subcutaneous injection of tamoxifen (30 mg/kg/d) for 5 days starting post-natal day 14. Survival was analyzed by Kaplan-Meier plots, cardiac function by echocardiography, arrhythmias by rhythm monitoring, and gene expression by RNA-Seq, immunoblotting, and immunofluorescence techniques. Cell death was analyzed by the TUNEL assay and the expression levels of specific markers were by RT-PCR and immunoblotting. Myocardial fibrosis was assessed by picrosirius red staining of the myocardial sections, RT-PCR, and immunoblotting. The Myh6-McmTam:DspF/F mice showed extensive molecular remodeling of the IDs and the differential expression of ~10,000 genes, which predicted activation of KDM5A, IRFs, and NFκB and suppression of PPARGC1A and RB1, among others in the DSP-deficient myocytes. Gene set enrichment analysis predicted activation of the TNFα/NFκB pathway, inflammation, cell death programs, and fibrosis. Analysis of cell death markers indicated PANoptosis, comprised of apoptosis (increased CASP3, CASP8, BAD and reduced BCL2), necroptosis (increased RIPK1, RIPK3, and MLKL), and pyroptosis (increased GSDMD and ASC or PYCARD) in the DSP-deficient myocytes. Transcript levels of the pro-inflammatory and pro-fibrotic genes were increased and myocardial fibrosis comprised ~25% of the myocardium in the DSP-deficient hearts. The Myh6-McmTam:DspF/F mice showed severe cardiac systolic dysfunction and ventricular arrhythmias, and died prematurely with a median survival rate of ~2 months. Conclusion: The findings identify PANoptosis as a prominent phenotypic feature of DSP cardiomyopathy and set the stage for delineating the specific molecular mechanisms involved in its pathogenesis. The model also provides the opportunity to test the effects of pharmacological and genetic interventions on myocardial fibrosis and cell death. One sentence summary: Post-natal homozygous deletion of the Dsp gene leads to fulminant PANoptosis and severe myocardial fibrosis, cardiac dysfunction, arrhythmias, and premature death in mice.

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.

Our understanding of gene regulation is constantly evolving. It is now clear that the majority of cellular transcripts are non-coding RNAs. The spectrum of non-coding RNAs is diverse and includes short (200 nt) and long non-coding RNAs (lncRNAs) (200 nt). LncRNAs regulate gene expression through diverse mechanisms. In this review, we describe the emerging roles of lncRNA mediated plant gene regulation. We discuss the current classification of lncRNAs and their role in genome organization and gene regulation. We also highlight a subset of lncRNAs that are epigenetic regulators of plant gene expression. Lastly, we provide an overview of emerging techniques and databases that are employed for the identification and characterization of plant lncRNAs.

Background: Mutations in genes encoding intercalated disk/desmosome proteins, such as plakophilin 2 (PKP2), cause arrhythmogenic cardiomyopathy (ACM). Desmosomes are responsible for myocyte&ndash, 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-&alpha, 1 (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.

Deleting the box C/D RNA-containing intron in the Haloferax volcanii tRNA Trp gene abolishes RNA-guided 2′-O methylations of C34 and U39 residues of tRNA Trp . However, this deletion does not affect growth under standard conditions.

Pus10, a recently identified pseudouridine (Ψ) synthase, does not belong to any of the five commonly identified families of Ψ synthases. Pyrococcus furiosus Pus10 has been shown to produce Ψ55 in tRNAs. However, in vitro studies have identified another mechanism for tRNA Ψ55 production in Archaea, which uses Cbf5 and other core proteins of the H/ACA ribonucleoprotein complex, in a guide RNA-independent manner. Pus10 homologs have been observed in nearly all sequenced archaeal genomes and in some higher eukaryotes, but not in yeast and bacteria. This coincides with the presence of Ψ54 in the tRNAs of Archaea and higher eukaryotes and its absence in yeast and bacteria. No tRNA Ψ54 synthase has been reported so far. Here, using recombinant Methanocaldococcus jannaschii and P. furiosus Pus10, we show that these proteins can function as synthase for both tRNA Ψ54 and Ψ55. The two modifications seem to occur independently. Salt concentration dependent variations in these activities of both proteins are observed. The Ψ54 synthase activity of M. jannaschii protein is robust, while the same activity of P. furiosus protein is weak. Probable reasons for these differences are discussed. Furthermore, unlike bacterial TruB and yeast Pus4, archaeal Pus10 does not require a U54•A58 reverse Hoogstein base pair and pyrimidine at position 56 to convert tRNA U55 to Ψ55. The homology of eukaryal Pus10 with archaeal Pus10 suggests that the former may also have a tRNA Ψ54 synthase activity.

Assembly and guide–target interaction of an archaeal box C/D-guide sRNP was investigated under various conditions by analyzing the lead (II)-induced cleavage of the guide RNA. Guide and target RNAs derived from Haloferax volcanii pre-tRNATrp were used with recombinant Methanocaldococcus jannaschii core proteins in the reactions. Core protein L7Ae binds differentially to C/D and C′/D′ motifs of the guide RNA, and interchanging the two motifs relative to the termini of the guide RNA did not affect L7Ae binding or sRNA function. L7Ae binding to the guide RNA exposes its D′-guide sequence first followed by the D guide. These exposures are reduced when aNop5p and aFib proteins are added. The exposed guide sequences did not pair with the target sequences in the presence of L7Ae alone. The D-guide sequence could pair with the target in the presence of L7Ae and aNop5p, suggesting a role of aNop5p in target recruitment and rearrangement of sRNA structure. aFib binding further stabilizes this pairing. After box C/D-guided modification, target–guide pairing at the D-guide sequence is disrupted, suggesting that each round of methylation may require some conformational change or reassembly of the RNP. Asymmetric RNPs containing only one L7Ae at either of the two box motifs can be assembled, but a functional RNP requires L7Ae at the box C/D motif. This arrangement resembles the asymmetric eukaryal snoRNP. Observations of initial D-guide–target pairing and the functional requirement for L7Ae at the box C/D motif are consistent with our previous report of the sequential 2′-O-methylations of the target RNA.

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.

Archaeal RNA splicing involves at least two protein enzymes, a specific endonuclease and a specific ligase. The endonuclease recognizes and cleaves within a characteristic bulge-helix-bulge (BHB) structure formed by pairing of the regions near the two exon–intron junctions, producing 2‘,3′-cyclic phosphate and 5′-hydroxyl termini. The ligase joins the exons and converts the cyclic phosphate into junction phosphate. The ligated product contains a seven-base hairpin loop, in which the splice junction is in between the two 3′ terminal residues of the loop. Archaeal splicing endonucleases are also involved in rRNA processing, cutting within the BHB structures formed by pairing of the 5′ and 3′ flanking regions of the rRNAs. Large free introns derived from pre-rRNAs have been observed as stable and abundant circular RNAs in certain Crenarchaeota, a kingdom in the domain Archaea. In the present study, we show that the cells of Haloferax volcanii, a Euryarchaeote, contain circular RNAs formed by 3′,5′-phosphodiester linkage between the two termini of the introns derived from their pre-tRNAs. H. volcanii ligase, in vitro, can also circularize both endonuclease-cleaved introns, and non-endonuclease-produced substrates. Exon joining and intron circularization are mechanistically similar ligation reactions that can occur independently. The size of the ligated hairpin loop and position of the splice junction within this loop can be changed in in vitro ligation reactions. Overall, archaeal RNA splicing seems to involve two sets of two symmetric transesterification reactions each.

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). …

Pseudouridines (Ψ) are found in structurally and functionally important regions of RNAs. Six families of Ψ synthases, TruA, TruB, TruD, RsuA, RluA, and Pus10 have been identified. Pus10 is present in Archaea and Eukarya. While most archaeal Pus10 produce both tRNA Ψ54 and Ψ55, some produce only Ψ55. Interestingly, human PUS10 has been implicated in apoptosis and Crohn’s and Celiac diseases. Homology models of archaeal Pus10 proteins based on the crystal structure of human PUS10 reveal that there are subtle structural differences in all of these Pus10 proteins. These observations suggest that structural changes in homologous proteins may lead to loss, gain, or change of their functions, warranting the need to study the structure-function relationship of these proteins. Using comparison of structural models and a series of mutations, we identified forefinger loop (reminiscent of that of RluA) and an Arg and a Tyr residue of archaeal Pus10 as critical determinants for its Ψ54, but not for its Ψ55 activity. We also found that a Leu residue, in addition to the catalytic Asp, is essential for both activities. Since forefinger loop is needed for both rRNA and tRNA Ψ synthase activities of RluA, but only for tRNA Ψ54 activity of Pus10, archaeal Pus10 proteins must use a different mechanism of recognition for Ψ55 activity. We propose that archaeal Pus10 uses two distinct mechanisms for substrate uridine recognition and binding. However, since we did not observe any mutation that affected only Ψ55 activity, both mechanisms for archaeal Pus10 activities must share some common features.

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.

RNA-guided pseudouridine (Psi) synthesis in Archaea and Eukarya requires a four-protein one-RNA containing box H/ACA ribonucleoprotein (RNP) complex. The proteins in the archaeal RNP are aCbf5, aNop10, aGar1 and L7Ae. Pyrococcus aCbf5-aNop10 is suggested to be the minimal catalytic core in this synthesis and the activity is enhanced by L7Ae and aGar1. The protein aCbf5 is homologous to eukaryal Cbf5 (dyskerin, NAP57) as well as to bacterial TruB and eukaryal Pus4; the last two produce YPsi55 in tRNAs in a guide RNA-independent manner. Here, using recombinant Methanocaldococcus jannaschii proteins, we report that aCbf5 and aGar1 together can function as a tRNA Psi55 synthase in a guide RNA-independent manner. This activity is enhanced by aNop10, but not by L7Ae. The aCbf5 alone can also produce Psi55 in tRNAs that contain the canonical 3'-CCA sequence and this activity is stimulated by aGar1. These results suggest that the roles of accessory proteins are different in guide RNA-dependent and independent Psi synthesis by aCbf5. The presence of conserved C (or U) and A at tRNA positions 56 and 58, respectively, which are required for TruB/Pus4 activity, is not essential for aCbf5-mediated Psi55 formation. Conserved A58 in tRNA normally forms a tertiary reverse Hoogstein base pair with an equally conserved U54. This base pair is recognized by TruB. Apparently aCbf5 does not require this base pair to recognize U55 for conversion to Psi55.

Haloferax volcanii pre-tRNA(Trp) processing requires box C/D ribonucleoprotein (RNP)-guided 2'-O-methylation of nucleotides C34 and U39 followed by intron excision. Positioning of the box C/D guide RNA within the intron of this pre-tRNA led to the assumption that nucleotide methylation is guided by the cis-positioned box C/D RNPs. We have now investigated the mechanism of 2'-O-methylation for the H. volcanii pre-tRNA(Trp) in vitro by assembling methylation-competent box C/D RNPs on both the pre-tRNA and the excised intron (both linear and circular forms) using Methanocaldococcus jannaschii box C/D RNP core proteins. With both kinetic studies and single nucleotide substitutions of target and guide nucleotides, we now demonstrate that pre-tRNA methylation is guided in trans by the intron-encoded box C/D RNPs positioned in either another pre-tRNA(Trp) or in the excised intron. Methylation by in vitro assembled RNPs prefers but does not absolutely require Watson-Crick pairing between the guide and target nucleotides. We also demonstrate for the first time that methylation of two nucleotides guided by a single box C/D RNA is sequential, that is, box C'/D' RNP-guided U39 methylation first requires box C/D RNP-guided methylation of C34. Methylation of the two nucleotides of exogenous pre-tRNA(Trp) added to an H. volcanii cell extract also occurs sequentially and is also accomplished in trans using RNPs that pre-exist in the extract. Thus, this trans mechanism is analogous to eukaryal pre-rRNA 2'-O-methylation guided by intron-encoded but trans-acting box C/D small nucleolar RNPs. This trans mechanism could explain the observed accumulation of the excised H. volcanii pre-tRNA(Trp) intron in vivo. A trans mechanism would also eliminate the obligatory refolding of the pre-tRNA that would be required to carry out two cis-methylation reactions before pre-tRNA splicing.

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.