Priyatansh Gurha

Targeted Deletion of MicroRNA-22 Promotes Stress-Induced Cardiac Dilation and Contractile Dysfunction

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 Ca2+ load in association with repressed sarcoplasmic reticulum Ca2+ 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 Ca2+ homeostasis and myofibrillar protein content during stress in the heart and shed light on the mechanisms that enhance propensity toward heart failure.

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:DspW/F and Myh6:Jup mouse models of AC, and in the PKP2 knockdown HL-1 myocytes (HL-1PKP2:shRNA). Level of active protein kinase C-&agr; 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-&agr; 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 &bgr;-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 &bgr;-catenin and adipogenesis in the HL-1PKP2: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.

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.

The Mef2 transcription network is disrupted in myotonic dystrophy heart tissue, dramatically altering miRNA and mRNA expression

Cardiac dysfunction is the second leading cause of death in myotonic dystrophy type 1 (DM1), primarily because of arrhythmias and cardiac conduction defects. A screen of more than 500 microRNAs (miRNAs) in a DM1 mouse model identified 54 miRNAs that were differentially expressed in heart. More than 80% exhibited downregulation toward the embryonic expression pattern and showed a DM1-specific response. A total of 20 of 22 miRNAs tested were also significantly downregulated in human DM1 heart tissue. We demonstrate that many of these miRNAs are direct MEF2 transcriptional targets, including miRNAs for which depletion is associated with arrhythmias or fibrosis. MEF2 protein is significantly reduced in both DM1 and mouse model heart samples, and exogenous MEF2C restores normal levels of MEF2 target miRNAs and mRNAs in a DM1 cardiac cell culture model. We conclude that loss of MEF2 in DM1 heart causes pathogenic features through aberrant expression of both miRNA and mRNA targets.

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 Ca2+ transient, Ca2+ 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.

Archaeal Pus10 proteins can produce both pseudouridine 54 and 55 in tRNA

Pus10, a recently identified pseudouridine (Psi) synthase, does not belong to any of the five commonly identified families of Psi synthases. Pyrococcus furiosus Pus10 has been shown to produce Psi55 in tRNAs. However, in vitro studies have identified another mechanism for tRNA Psi55 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 Psi54 in the tRNAs of Archaea and higher eukaryotes and its absence in yeast and bacteria. No tRNA Psi54 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 Psi54 and Psi55. The two modifications seem to occur independently. Salt concentration dependent variations in these activities of both proteins are observed. The Psi54 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 x A58 reverse Hoogstein base pair and pyrimidine at position 56 to convert tRNA U55 to Psi55. The homology of eukaryal Pus10 with archaeal Pus10 suggests that the former may also have a tRNA Psi54 synthase activity.

MicroRNAs in cardiovascular disease.

Purpose of review 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. Recent findings 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. Summary 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.

Cardiac Fibro-Adipocyte Progenitors Express Desmosome Proteins and Preferentially Differentiate to Adipocytes Upon Deletion of the Desmoplakin Gene

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.

Role of forefinger and thumb loops in production of Ψ54 and Ψ55 in tRNAs by archaeal Pus10.

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 Crohns 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.

Noncoding RNAs in Cardiovascular Biology and Disease

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). …