Kumar Pandya

MicroRNA-208a is a regulator of cardiac hypertrophy and conduction in mice

MicroRNAs (miRNAs) are a class of small noncoding RNAs that have gained status as important regulators of gene expression. Here, we investigated the function and molecular mechanisms of the miR-208 family of miRNAs in adult mouse heart physiology. We found that miR-208a, which is encoded within an intron of alpha-cardiac muscle myosin heavy chain gene (Myh6), was actually a member of a miRNA family that also included miR-208b, which was determined to be encoded within an intron of beta-cardiac muscle myosin heavy chain gene (Myh7). These miRNAs were differentially expressed in the mouse heart, paralleling the expression of their host genes. Transgenic overexpression of miR-208a in the heart was sufficient to induce hypertrophic growth in mice, which resulted in pronounced repression of the miR-208 regulatory targets thyroid hormone-associated protein 1 and myostatin, 2 negative regulators of muscle growth and hypertrophy. Studies of the miR-208a Tg mice indicated that miR-208a expression was sufficient to induce arrhythmias. Furthermore, analysis of mice lacking miR-208a indicated that miR-208a was required for proper cardiac conduction and expression of the cardiac transcription factors homeodomain-only protein and GATA4 and the gap junction protein connexin 40. Together, our studies uncover what we believe are novel miRNA-dependent mechanisms that modulate cardiac hypertrophy and electrical conduction.

MicroRNA-Mediated In Vitro and In Vivo Direct Reprogramming of Cardiac Fibroblasts to Cardiomyocytes

Rationale: Repopulation of the injured heart with new, functional cardiomyocytes remains a daunting challenge for cardiac regenerative medicine. An ideal therapeutic approach would involve an effective method at achieving direct conversion of injured areas to functional tissue in situ. Objective: The aim of this study was to develop a strategy that identified and evaluated the potential of specific micro (mi)RNAs capable of inducing reprogramming of cardiac fibroblasts directly to cardiomyocytes in vitro and in vivo. Methods and Results: Using a combinatorial strategy, we identified a combination of miRNAs 1, 133, 208, and 499 capable of inducing direct cellular reprogramming of fibroblasts to cardiomyocyte-like cells in vitro. Detailed studies of the reprogrammed cells demonstrated that a single transient transfection of the miRNAs can direct a switch in cell fate as documented by expression of mature cardiomyocyte markers, sarcomeric organization, and exhibition of spontaneous calcium flux characteristic of a cardiomyocyte-like phenotype. Interestingly, we also found that miRNA-mediated reprogramming was enhanced 10-fold on JAK inhibitor I treatment. Importantly, administration of miRNAs into ischemic mouse myocardium resulted in evidence of direct conversion of cardiac fibroblasts to cardiomyocytes in situ. Genetic tracing analysis using Fsp1Cre-traced fibroblasts from both cardiac and noncardiac cell sources strongly suggests that induced cells are most likely of fibroblastic origin. Conclusions: The findings from this study provide proof-of-concept that miRNAs have the capability of directly converting fibroblasts to a cardiomyocyte-like phenotype in vitro. Also of significance is that this is the first report of direct cardiac reprogramming in vivo. Our approach may have broad and important implications for therapeutic tissue regeneration in general.

Fibrosis, not cell size, delineates β-myosin heavy chain reexpression during cardiac hypertrophy and normal aging in vivo

Reexpression of the fetally expressed β-myosin heavy chain (β-MHC) gene is a well documented marker of pathological cardiac hypertrophy and normal aging in many experimental models. To gain insights into factors affecting this reexpression of β-MHC within the complex anatomical structure of the heart, we investigated the spatial pattern of its expression at the level of single cells during aging and hypertrophy. We generated mice that express yellow fluorescent protein fused to the N terminus of the β-MHC and examined its expression pattern during normal aging and in mice with hypertrophy induced by constitutive expression of a renin transgene. The localization of fibrosis within the hearts also was determined by using a fluorescent lectin. The results show that reexpression of β-MHC occurs in discrete subsets of myocytes within the subendocardium rather than uniformly throughout the heart, that β-MHC induction is not an obligatory consequence of cellular hypertrophy, and that β-MHC-expressing cells in the normal aging heart and the hypertrophic heart are distributed predominantly in clusters within and surrounding foci of fibrosis. We conclude that β-MHC gene expression in the normal aging adult and hypertrophic mouse heart is a marker of fibrosis rather than of cellular hypertrophy.

Three-dimensional diffusion tensor microscopy of fixed mouse hearts

The relative utility of 3D, microscopic resolution assessments of fixed mouse myocardial structure via diffusion tensor imaging is demonstrated in this study. Isotropic 100‐μm resolution fiber orientation mapping within 5.5° accuracy was achieved in 9.1 hr scan time. Preliminary characterization of the diffusion tensor primary eigenvector reveals a smooth and largely linear angular rotation across the left ventricular wall. Moreover, a higher level of structural hierarchy is evident from the organized secondary and tertiary eigenvector fields. These findings are consistent with the known myocardial fiber and laminar structures reported in the literature and suggest an essential role of diffusion tensor microscopy in developing quantitative atlases for studying the structure–function relationships of mouse hearts. Magn Reson Med 52:453–460, 2004.

Heterogeneous myocyte enhancer factor-2 (Mef2) activation in myocytes predicts focal scarring in hypertrophic cardiomyopathy

Unknown molecular responses to sarcomere protein gene mutations account for pathologic remodeling in hypertrophic cardiomyopathy (HCM), producing myocyte growth and increased cardiac fibrosis. To determine if hypertrophic signals activated myocyte enhancer factor-2 (Mef2), we studied mice carrying the HCM mutation, myosin heavy-chain Arg403Gln, (MHC403/+) and an Mef2-dependent β-galactosidase reporter transgene. In young, prehypertrophic MHC403/+ mice the reporter was not activated. In hypertrophic hearts, activation of the Mef2-dependent reporter was remarkably heterogeneous and was observed consistently in myocytes that bordered fibrotic foci with necrotic cells, MHC403/+ myocytes with Mef2-dependent reporter activation reexpressed the fetal myosin isoform (βMHC), a molecular marker of hypertrophy, although MHC403/+ myocytes with or without βMHC expression were comparably enlarged over WT myocytes. To consider Mef2 roles in severe HCM, we studied homozygous MHC403/403 mice, which have accelerated remodeling, widespread myocyte necrosis, and neonatal lethality. Levels of phosphorylated class II histone deacetylases that activate Mef2 were substantially increased in MHC403/403 hearts, but Mef2-dependent reporter activation was patchy. Sequential analyses showed myocytes increased Mef2-dependent reporter activity before death. Our data dissociate myocyte hypertrophy, a consistent response in HCM, from heterogeneous Mef2 activation and reexpression of a fetal gene program. The temporal and spatial relationship of Mef2-dependent gene activation with myocyte necrosis and fibrosis in MHC403/+ and MHC403/403 hearts defines Mef2 activation as a molecular signature of stressed HCM myocytes that are poised to die.

β-MyHC and Cardiac Hypertrophy Size Does Matter

See related article, pages 629–638 In response to stress signals, the mammalian heart responds by an increase in its size.1 This is largely accomplished by an increase in the size of myocytes (hypertrophy) rather than by increasing their numbers (hyperplasia). At the molecular level, pathological stresses induce multiple changes, including genetic reprogramming—the reexpression of a battery of fetal genes and the downregulation of multiple adult genes.2 Together, the changes in gene expression result in substantial phenotypic changes, including changes in size, contractility, metabolic state, and electric conductance. Indeed, the reexpression of fetal genes, including β-myosin heavy chain (β-MyHC), atrial natriuretic factor (ANF), and alpha-skeletal actin, has for many years been looked on as an important molecular indicator of pathological hypertrophy. In spite of this use of β-MyHC reexpression as a marker of pathological hypertrophy, until recently there have been surprisingly few reports on β-MyHC reexpression at the level of individual myocytes within the hypertrophic heart. Nevertheless, previous work using in situ hybridization against β-MyHC mRNA has shown that reexpression of β-MyHC does not take place in all areas of the hypertrophic heart; rather, it is distributed in distinct regions.3 More recently, it has been possible to study individual cells from the heart using genetically altered mice in which a fluorescent protein (yellow fluorescent protein [YFP]) is tagged onto the native β-MyHC gene. This procedure confirmed that β-MyHC reexpression takes place in cells located in distinct regions, including regions adjacent to areas of …

Reversible epigenetic modifications of the two cardiac Myosin Heavy Chain genes during changes in expression

The two genes of the cardiac myosin heavy chain (MHC) locus-alpha-MHC (aMHC) and beta-MHC (bMHC)--are reciprocally regulated in the mouse ventricle during development and in adult conditions such as hypothyroidism and pathological cardiac hypertrophy. Their expressions are under the control of thyroid hormone T3 levels. To gain insights into the epigenetic mechanisms that underlie this inducible and reversible switching of the aMHC and bMHC isoforms, we have investigated the histone modification patterns that occur over the two cardiac MHC promoters during T3-mediated reversible switching of gene expression. Mice fed a diet of propylthiouracil (PTU, an inhibitor of T3 synthesis) for 2 weeks dramatically reduce aMHC mRNA expression and increase bMHC mRNA levels to high levels, while a subsequent withdrawal of PTU diet for 2 weeks completely reverses the T3-mediated changes in MHC expression. Using hearts from mice treated in this way, we carried out chromatin immunoprecipitation-qPCR assays with antibodies against acetylated histone H3 (H3ac) and trimethylated histone (H3K4me3)-two well-documented markers of activation. Our results show that the reexpression of bMHC is associated at the bMHC promoter with increased H3ac but not H3K4me3. In contrast, the silencing of aMHC is associated at its promoter with decreased H3K4me3, but not decreased H3ac. The epigenetic changes at the two MHC promoters are completely reversed when the gene expression returns to initial levels. These data indicate that during reciprocal and inducible gene expression H3ac parallels bMHC isoform expression while H3K4me3 parallels expression of the tightly linked aMHC isoform.

Discordant on/off switching of gene expression in myocytes during cardiac hypertrophy in vivo

To determine whether the expression of cardiac genes changes in a graded manner or by on/off switching when cardiac myocytes change genetic programs in living animals, we have studied two indicator genes that change their expression oppositely in mouse binucleate ventricular cardiomyocytes during development and in response to cardiac hypertrophy. One is a single-copy transgene controlled by an α-myosin heavy chain (aMHC) promoter and coding for CFP. The other is the endogenous β-myosin heavy chain (bMHC) gene modified to code for a YFP–bMHC fusion protein. Using high-resolution confocal microscopy, we determined the expression of the two indicator genes in individual cardiomyocytes perinatally and after inducing cardiac hypertrophy by transverse aortic constriction. Our results provide strong evidence that the cardiac genes respond by switching their expression in an on/off rather than graded manner, and that responding genes within a single cell and within the two nuclei of cardiomyocytes do not necessarily switch concordantly.

Diuretics Prevent Thiazolidinedione-Induced Cardiac Hypertrophy without Compromising Insulin-Sensitizing Effects in Mice

Much concern has arisen regarding critical adverse effects of thiazolidinediones (TZDs), including rosiglitazone and pioglitazone, on cardiac tissue. Although TZD-induced cardiac hypertrophy (CH) has been attributed to an increase in plasma volume or a change in cardiac nutrient preference, causative roles have not been established. To test the hypothesis that volume expansion directly mediates rosiglitazone-induced CH, mice were fed a high-fat diet with rosiglitazone, and cardiac and metabolic consequences were examined. Rosiglitazone treatment induced volume expansion and CH in wild-type and PPARγ heterozygous knockout (Pparg(+/-)) mice, but not in mice defective for ligand binding (Pparg(P465L/+)). Cotreatment with the diuretic furosemide in wild-type mice attenuated rosiglitazone-induced CH, hypertrophic gene reprogramming, cardiomyocyte apoptosis, hypertrophy-related signal activation, and left ventricular dysfunction. Similar changes were observed in mice treated with pioglitazone. The diuretics spironolactone and trichlormethiazide, but not amiloride, attenuated rosiglitazone effects on volume expansion and CH. Interestingly, expression of glucose and lipid metabolism genes in the heart was altered by rosiglitazone, but these changes were not attenuated by furosemide cotreatment. Importantly, rosiglitazone-mediated whole-body metabolic improvements were not affected by furosemide cotreatment. We conclude that releasing plasma volume reduces adverse effects of TZD-induced volume expansion and cardiac events without compromising TZD actions in metabolic switch in the heart and whole-body insulin sensitivity.

Decreased beta-adrenergic responsiveness following hypertrophy occurs only in cardiomyocytes that also re-express beta-myosin heavy chain

Cardiac hypertrophy is associated with a reduction in the contractile response to beta‐adrenergic stimulation, and with re‐expression of foetal genes such as beta‐myosin heavy chain (MHC). However, whether these two markers of pathology develop concordantly in the same individual cells or independently in different cells is not known.