Carmen C. Sucharov

PGC-1α and ERRα target gene downregulation is a signature of the failing human heart

Heart failure is a cause of significant morbidity and mortality in developed nations, and results from a complex interplay between genetic and environmental factors. To discover gene regulatory networks underlying heart failure, we analyzed DNA microarray data based on left ventricular free-wall myocardium from 59 failing (32 ischemic cardiomyopathy, 27 idiopathic dilated cardiomyopathy) and 33 non-failing explanted human hearts from the Cardiogenomics Consortium. In particular, we sought to investigate cardiac gene expression changes at the level of individual genes, as well as biological pathways which contain groups of functionally related genes. Utilizing a combination of computational techniques, including Comparative Marker Selection and Gene Set Enrichment Analysis, we identified a subset of downstream gene targets of the master mitochondrial transcriptional regulator, peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha), whose expression is collectively decreased in failing human hearts. We also observed decreased expression of the key PGC-1alpha regulatory partner, estrogen-related receptor alpha (ERRalpha), as well as ERRalpha target genes which may participate in the downregulation of mitochondrial metabolic capacity. Gene expression of the antiapoptotic Raf-1/extracellular signal-regulated kinase (ERK) pathway was decreased in failing hearts. Alterations in PGC-1alpha and ERRalpha target gene sets were significantly correlated with an important clinical parameter of disease severity - left ventricular ejection fraction, and were predictive of failing vs. non-failing phenotypes. Overall, our results implicate PGC-1alpha and ERRalpha in the pathophysiology of human heart failure, and define dynamic target gene sets sharing known interrelated regulatory mechanisms capable of contributing to the mitochondrial dysfunction characteristic of this disease process.

Prevalence of Desmin Mutations in Dilated Cardiomyopathy

Background— Desmin-related myofibrillar myopathy (DRM) is a cardiac and skeletal muscle disease caused by mutations in the desmin (DES) gene. Mutations in the central 2B domain of DES cause skeletal muscle disease that typically precedes cardiac involvement. However, the prevalence of DES mutations in dilated cardiomyopathy (DCM) without skeletal muscle disease is not known. Methods and Results— Denaturing high-performance liquid chromatography was used to screen DES for mutations in 116 DCM families from the Familial Dilated Cardiomyopathy Registry and in 309 subjects with DCM from the Beta-Blocker Evaluation of Survival Trial (BEST). DES mutations were transfected into SW13 and human smooth muscle cells and neonatal rat cardiac myocytes, and the effects on cytoskeletal desmin network architecture were analyzed with confocal microscopy. Five novel missense DES mutations, including the first localized to the highly conserved 1A domain, were detected in 6 subjects (1.4%). Transfection of DES mutations in the 2B domain severely disrupted the fine intracytoplasmic staining of desmin, causing clumping of the desmin protein. A tail domain mutation (Val459Ile) showed milder effects on desmin cytoplasmic network formation and appears to be a low-penetrant mutation restricted to black subjects. Conclusions— The prevalence of DES mutations in DCM is between 1% and 2%, and mutations in the 1A helical domain, as well as the 2B rod domain, are capable of causing a DCM phenotype. The lack of severe disruption of cytoskeletal desmin network formation seen with mutations in the 1A and tail domains suggests that dysfunction of seemingly intact desmin networks is sufficient to cause DCM.

MicroRNA-124 Controls the Proliferative, Migratory, and Inflammatory Phenotype of Pulmonary Vascular Fibroblasts

Rationale: Pulmonary hypertensive remodeling is characterized by excessive proliferation, migration, and proinflammatory activation of adventitial fibroblasts. In culture, fibroblasts maintain a similar activated phenotype. The mechanisms responsible for generation/maintenance of this phenotype remain unknown. Objective: We hypothesized that aberrant expression of microRNA-124 (miR-124) regulates this activated fibroblast phenotype and sought to determine the signaling pathways through which miR-124 exerts effects. Methods and Results: We detected significant decreases in miR-124 expression in fibroblasts isolated from calves and humans with severe pulmonary hypertension. Overexpression of miR-124 by mimic transfection significantly attenuated proliferation, migration, and monocyte chemotactic protein-1 expression of hypertensive fibroblasts, whereas anti–miR-124 treatment of control fibroblasts resulted in their increased proliferation, migration, and monocyte chemotactic protein-1 expression. Furthermore, the alternative splicing factor, polypyrimidine tract–binding protein 1, was shown to be a direct target of miR-124 and to be upregulated both in vivo and in vitro in bovine and human pulmonary hypertensive fibroblasts. The effects of miR-124 on fibroblast proliferation were mediated via direct binding to the 3′ untranslated region of polypyrimidine tract–binding protein 1 and subsequent regulation of Notch1/phosphatase and tensin homolog/FOXO3/p21Cip1 and p27Kip1 signaling. We showed that miR-124 directly regulates monocyte chemotactic protein-1 expression in pulmonary hypertension/idiopathic pulmonary arterial hypertension fibroblasts. Furthermore, we demonstrated that miR-124 expression is suppressed by histone deacetylases and that treatment of hypertensive fibroblasts with histone deacetylase inhibitors increased miR-124 expression and decreased proliferation and monocyte chemotactic protein-1 production. Conclusions: Stable decreases in miR-124 expression contribute to an epigenetically reprogrammed, highly proliferative, migratory, and inflammatory phenotype of hypertensive pulmonary adventitial fibroblasts. Thus, therapies directed at restoring miR-124 function, including histone deacetylase inhibitors, should be investigated.

Beta-adrenergic adaptation in paediatric idiopathic dilated cardiomyopathy

BACKGROUND Although the pathophysiology and treatment of adult heart failure (HF) are well studied, HF in children remains poorly understood. In adults, adrenergic receptor (AR)-mediated adaptation plays a central role in cardiac abnormalities in HF, and these patients respond well to β-blocker (BB) therapy. However, in children with HF, there is a growing body of literature suggesting a lack of efficacy of adult HF therapies. Due to these unanticipated differences in response to therapy and the paucity of data regarding the molecular adaptation of the paediatric heart, we investigated the molecular characteristics of HF in children. METHODS AND RESULTS Explanted hearts from adults and children with idiopathic dilated cardiomyopathy and non-failing controls were used in the study. Our results show that the molecular characteristics of paediatric HF are strikingly different from their adult counterparts. These differences include: (i) down-regulation of β1- and β2-AR in children, whereas β2-AR expression is maintained in adults; (ii) up-regulation of connexin43 in children, whereas down-regulation is observed in adults; (iii) no differences in phosphatase expression, whereas up-regulation is observed in adults; (iv) no decrease in the phosphorylation of phospholamban at the Ser16 or Thr17 sites in children, which are known characteristics of adult HF. CONCLUSION There is a different adaptation of β-AR and adrenergic signalling pathways in children with HF compared with adults. Our results begin to address the disparities in cardiovascular research specific to children and suggest that age-related differences in adaptation could influence the response to therapy. These findings could lead to a paradigm shift in the contemporary management of children with HF.

YY1 Protects Cardiac Myocytes from Pathologic Hypertrophy by Interacting with HDAC5

YY1 is a transcription factor that can repress or activate the transcription of a variety of genes. Here, we show that the function of YY1 as a repressor in cardiac myocytes is tightly dependent on its ability to interact with histone deacetylase 5 (HDAC5). YY1 interacts with HDAC5, and overexpression of YY1 prevents HDAC5 nuclear export in response to hypertrophic stimuli and the increase in cell size and re-expression of fetal genes that accompany pathological cardiac hypertrophy. Knockdown of YY1 results in up-regulation of all genes present during fetal development and increases the cell size of neonatal cardiac myocytes. Moreover, overexpression of a YY1 deletion construct that does not interact with HDAC5 results in transcription activation, suggesting that HDAC5 is necessary for YY1 function as a transcription repressor. In support of this relationship, we show that knockdown of HDAC5 results in transcription activation by YY1. Finally, we show that YY1 interaction with HDAC5 is dependent on the HDAC5 phosphorylation domain and that overexpression of YY1 reduces HDAC5 phosphorylation in response to hypertrophic stimuli. Our results strongly suggest that YY1 functions as an antihypertrophic factor by preventing HDAC5 nuclear export and that up-regulation of YY1 in human heart failure may be a protective mechanism against pathological hypertrophy.

Role of microRNAs in cardiovascular disease: therapeutic challenges and potentials.

MicroRNAs (miRNAs, miRs) are short approximately 22-nucleotide noncoding RNAs that bind to messenger RNA transcripts and in doing so modulate cognate gene expression. In eukaryotes, miRNAs act primarily by causing translational repression although they may also act to destabilize RNA transcripts. During the past few years, a number of studies have demonstrated that miR expression changes as a result of cardiac hypertrophy or heart failure. Additionally, cell-based and transgenic mouse studies have demonstrated that individual miRs can affect a number of aspects of cardiac biology including developmental processes, stem cell differentiation, progression of hypertrophy and failure, ion channel function, as well as angiogenesis, rates of apoptosis, and fibroblast proliferation. In this review, we will summarize several of the miRs known to change in expression in association with heart failure and outline details of what is known about their putative targets. In addition, we will review several aspects of regulation of miR expression that have not been addressed in a cardiovascular context. Finally, as is common to all new and rapidly moving fields, we will highlight some of the gaps and inconsistencies related to miR expression and cardiac phenotypes, particularly those associated with heart failure.

The Ku Protein Complex Interacts with YY1, Is Up-Regulated in Human Heart Failure, and Represses α Myosin Heavy-Chain Gene Expression

ABSTRACT Human heart failure is accompanied by repression of genes such as α myosin heavy chain (αMyHC) and SERCA2A and the induction of fetal genes such as βMyHC and atrial natriuretic factor. It seems likely that changes in MyHC isoforms contribute to the poor contractility seen in heart failure, because small changes in isoform composition can have a major effect on the contractility of cardiac myocytes and the heart. Our laboratory has recently shown that YY1 protein levels are increased in human heart failure and that YY1 represses the activity of the human αMyHC promoter. We have now identified a region of the αMyHC promoter that binds a factor whose expression is increased sixfold in failing human hearts. Through peptide mass spectrometry, we identified this binding activity to be a heterodimer of Ku70 and Ku80. Expression of Ku represses the human αMyHC promoter in neonatal rat ventricular myocytes. Moreover, overexpression of Ku70/80 decreases αMyHC mRNA expression and increases skeletal α-actin. Interestingly, YY1 interacts with Ku70 and Ku80 in HeLa cells. Together, YY1, Ku70, and Ku80 repress the αMyHC promoter to an extent that is greater than that with YY1 or Ku70/80 alone. Our results suggest that Ku is an important factor in the repression of the human αMyHC promoter during heart failure.

Increased Phosphorylation‐Dependent Nuclear Export of Class II Histone Deacetylases in Failing Human Heart

In the failing human heart (FHH) the induction of a fetal contractile protein gene program is directly and selectively associated with the dilated cardiomyopathy (DCM) phenotype and involves multiple signaling pathways. In response to cardiac stress signals, class II HDACs are subject to phosphorylation dependent nuclear export, which allows for activation of fetal cardiac genes via the transcription factor MEF2. The current study tests the hypothesis that MEF2 activation produced by class II HDAC de‐repression is present in the FHH. In this study, human left ventricular tissue from nonfailing and failing adult hearts was analyzed for the presence of MEF2, HDACs 4 and 5. CaMK and HDAC kinase activities were measured in tissue homogenates. In nuclear fractions from failing ventricles, HDAC4 and HDAC5 protein was decreased versus nonfailing controls. MEF2 was not reduced in failing nuclear fractions. CaMK and HDAC kinase activities were increased in failing versus nonfailing hearts. PKCμ (PKD1) activity was increased in nuclear fractions from failing human LVs. These data provide support for decreased nuclear compartment class II HDACs in the FHH, associated with increased activities of kinases known to phosphorylate class II HDACs.

Temporal expression of miRNAs and mRNAs in a mouse model of myocardial infarction

Analysis of changes in gene expression is an important means to define molecular differences associated with the phenotypic changes observed in response to myocardial infarction (MI). Several studies in humans or animal models have reported differential miRNA expression in response to MI acutely (animal) or chronically (human). To determine the relative contribution of microRNA (miRNA) and mRNAs to acute and chronic temporal changes in response to MI, mRNA and miRNA expression profiles were performed in three time points post-MI. Changes in mRNA and miRNA expression was analyzed by arrays and confirmed by RT-PCR. Bioinformatic analysis demonstrated that several genes and miRNAs in various pathways are regulated in a temporal or phenotype-specific manner. Furthermore miRNA analyses indicated that miRNAs can target expression of several genes involved in multiple cardiomyopathy-related pathways. Our results suggest that: 1) Differentially regulated miRNAs are predicted to target expression of several genes in multiple biological processes involved in the response to MI; 2) antithetical and compensatory changes in miRNA expression are observed at later disease stages, including antithetical regulation of miR-29, which correlates with the expression of collagen genes, and upregulation of apoptosis-related miRNAs at early stages and antiapoptotic/growth promoting miRNAs at later stages; 3) temporally dependent changes in miRNA and mRNA expression post-MI are generally characterized by dramatic changes acutely postinjury and are normalized as disease progresses; 4) A combinatorial analysis of mRNA and miRNA expression may aid in determining factors involved in compensatory and decompensated responses to cardiac injury.

β-adrenergic pathways in human heart failure

β-adrenergic receptor activation plays an important role in the progression of human heart failure and the treatment of patients with β-blockers has greatly improved the outcome of the disease. However, heart failure still is one of the leading causes of death in various countries and there is an imperative need for additional targets for the treatment of the disease. Recent studies by various groups have analyzed the downstream signaling pathways activated in response to β-adrenergic stimulation that have the potential to become important targets for future treatments of heart failure. This review focuses on the significance of these pathways in the pathophysiology of heart failure in response to β-adrenergic stimulation. More specifically the roles of PDE3, phosphorylation of phospholamban, and CaMKII activation are extensively discussed.