Differential regulation of Krüppel-like factor family transcription factor expression in neonatal rat cardiac myocytes: effects of endothelin-1, oxidative stress and cytokines

Krüppel-like transcription factors (Klfs) modulate fundamental cell processes. Cardiac myocytes are terminally-differentiated, but hypertrophy in response to stimuli such as endothelin-1. H2O2 or cytokines promote myocyte apoptosis. Microarray studies of neonatal rat myocytes identified several Klfs as endothelin-1-responsive genes. We used quantitative PCR for further analysis of Klf expression in neonatal rat myocytes. In response to endothelin-1, Klf2 mRNA expression was rapidly increased ( approximately 9-fold; 15-30 min) with later increases in expression of Klf4 and Klf6 ( approximately 5-fold; 30-60 min). All were regulated as immediate early genes (cycloheximide did not inhibit the increases in expression). Klf5 expression was increased at 1-2 h ( approximately 13-fold) as a second phase response (cycloheximide inhibited the increase). These increases were transient and attenuated by U0126. H2O2 increased expression of Klf2, Klf4 and Klf6, but interleukin-1beta or tumor necrosis factor alpha downregulated Klf2 expression with no effect on Klf4 or Klf6. Of the Klfs which repress transcription, endothelin-1 rapidly downregulated expression of Klf3, Klf11 and Klf15. The dynamic regulation of expression of multiple Klf family members in cardiac myocytes suggests that, as a family, they are actively involved in regulating phenotypic responses (hypertrophy and apoptosis) to extracellular stimuli

Regulation and Function of MEF2 in Cardiomyocytes

Regular formation of the mammalian heart needs precise spatial and temporal transcriptional regulation of gene programs in ardiomyocytes. Cardiac transcription factors are defined, in this context, as essential transcriptional activators that are expressed predominantly in the myocardium which regulate the expression of the cardiac genes encoding structural proteins of cardiomyocytes. Unsurprisingly, disruptions in this elaborate transcriptional machinery can lead to severe cardiac abnormalities including hypertension, cardiomyopathy, and congenital heart disease. In this field, Myocyte Enhancer Factor 2 (MEF2) transcription factor is considered one of only a few core cardiac transcription factors that play important roles in cardiac development, survival, contractility, and in postnatal adaption to a wide array of physiological and pathological signals. MEF2 functions as a transcriptional switch by potently activating or repressing transcription through interaction with a variety of co-factors which serve as positive and negative regulators of transcription. The interaction of MEF2 with its co-factors is controlled by a multitude of signaling pathways that result in post-translational modification of MEF2 and in the subsequent MEF2-dependent repression or activation of target gene transcription. Our project studied regulation and function of MEF2A in cardiomyocytes. We hypothesized that the combinatorial interactions between transcription factors and promoter elements that are required for the regulation of cardiac gene expression may operate in pathological cardiac remodeling and hypertrophy. Therefore, studying and characterizing the regulation of proteins which bind to MEF2A in cardiomyocytes may unravel the underlying dysregulation of the cardiac transcriptome in the pathogenesis of cardiovascular disease and heart failure. In this project, HL-1 cardiomyocytes have been chosen as a model of study. An agonist (Isoproterenol) was used to mimic cardiomyocytes hypertrophy in HL-1 cells. Isoproterenol activates adrenergic signaling which can trigger many mechanisms in the heart contributing to the hypertrophic phenotype. We developed two different methods to capture MEF2A interacting partners (interactome), including immunoprecipitation (IP) of endogenous MEF2A and IP of Flag-MEF2A proteins in normal and hypertrophy conditions. Our optimization will allow characterization of MEF2A interactome partners through state of the art quantitative proteomics approaches. In previous research, transcriptome analysis (RNA-seq) from left ventricular RNA samples and MEF2A depleted cardiomyocytes identified some genes, including kf2, junb, alas2 and rarres2 which may have implications for cardiac hypertrophy. Our ChIP-qPCR data indicated that MEF2A is recruited to the rarres2 promoter in primary cardiomyocytes. Thus, rarres2 is a novel MEF2A target gene and further, it will be interesting to uncover functions of MEF2A interactome partners on rarres2 gene regulation in cardiac diseases. A study has indicated that klf2, junB, alas2, and rarres2 may have a role in promoting cardiomyocyte hypertrophy in cultured HL-1 cells and primary neonatal rat cardiomyocytes. Taken together, this project developed the methods to study the characterization of MEF2A interactome in cardiomyocytes. Additionally, we showed the capacity of some MEF2 target genes, including rarres2 to promote cardiomyocyte hypertrophy

Role of Transcription Factor MEF2A in Development of Coronary Artery Disease (CAD) and Myocardial Infarction (MI)

Coronary artery disease (CAD) and myocardial infarction (MI) are the leading cause of death in developed countries. Genetic factors play an important role in the pathogenesis of CAD and MI. The revolutionary advances in molecular genetics have provided new insights into the genetic pathways involved in this disease. Recently, our laboratory used linkage analysis to map the first disease-causing gene for CAD and MI to chromosome 15q26 in a large family and subsequently mutation analysis identified a 7-amio acid deletion in MEF2A, a gene encoding a transcription factor. Later we identified three novel mutations N263S, P279L, G283D in four other patients or families, which has validated our initial finding of MEF2A as a gene for CAD and MI. We recently screened another set of 200 CAD and MI patients using the single strand conformation polymorphism (SSCP) method and found another variant, T215A, in the sixth coding exon of MEF2FA in one CAD patient. The T215A mutation is clustered close to the major transcriptional activation domain of MEF2A.We then performed functional studies using the luciferase assay and found that the mutation significantly reduced the transcriptional activity of MEF2A and acted by a loss of function mechanism as the earlier mutations. To further investigate the role of MEF2A in atherogenesis, we studied knockout mice targeted to the mouse Mef2a gene and created a novel Mef2a+/- Apoe-/- knockout mouse strain. We then analyzed diet-induced atherosclerosis and spontaneous lesion formation in the Mef2a and Mef2a+/- Apoe-/- mice. A total of 39 Mef2a knockout mice, including 20 males (8 wild type Mef2a+/+ and 12 heterozygous Mef2a+/-) and 19 females (9 wild type Mef2a+/+ and 10 heterozygous Mef2a+/-) were fed on atherogenic/paigen diet for 38 weeks. A total of 47 Mef2a +/+, Mef2a+/- mice on Apoe-/- knockout background, including 26 males (15 wild type and 11 heterozygous) and 21 females (9 wild type and 12 heterozygous) were maintained on a Western-type diet for 12 weeks. The mice were analyze

Extracellular signal-regulated kinase 5 (ERK5) is required for the Yes-associated protein (YAP) co-transcriptional activity

Yes-associated protein (YAP) is a transcriptional cofactor involved in the regulation of various physio-pathological cellular processes. YAP is the main downstream effector of the tumor-suppressive Hippo pathway. When Hippo signaling is activated, it induces YAP phosphorylation and cytoplasmic sequestration, thus inhibiting its co-transcriptional activity. On the contrary, when Hippo signaling is inhibited, YAP translocates into the nucleus where it drives several transcriptional programs in a cell- and context-dependent manner. However, recent observations indicate that YAP activity can be also modulated by Hippo independent/integrating pathways, still largely unexplored. In this study, we demonstrated the role of the extracellular signal-regulated kinase 5 (ERK5)/mitogen activated protein kinase in the regulation of YAP activity. By means of ERK5 inhibition/silencing and overexpression experiments, and by using as model liver stem cells, hepatocytes, and hepatocellular carcinoma (HCC) cell lines, we provided evidence that ERK5 is required for YAP transcriptional activity. Mechanistically, ERK5 is required for the recruitment of YAP on the promoters of target genes and for its physical interaction with the transcriptional partner TEAD4. Moreover, we observed that ERK5 modulates YAP activation in cell adhesion dynamics, TGFβ-induced EMT of liver cells and cell migration. Furthermore, we demonstrated that ERK5 modulates the activity also of a YAP mutant non-phosphorylatable by LATS1/2, thus providing evidence of its, at least in part, independence from the Hippo pathway. Finally, preliminary results obtained in mice overexpressing YAP in the liver confirmed the relevance of ERK5/YAP axis in vivo and suggested the ERK5 involvement in the YAP-induced liver fibrosis. Therefore, our observations identify ERK5 as a novel upstream regulator of YAP activity, thus unveiling a new target for therapeutic approaches aimed at interfering with its function

The Immediate Early Response of Proliferating Myoblasts to One Bout of Electrical Stimulation

The maintenance of muscle mass is important across the lifespan. The activation of satellite cells, followed by their proliferation and subsequent differentiation is important in this maintenance. Cell cycle arrest must precede differentiation, and preservation of the molecular networks involved within the regenerative process are necessary. Electrical stimulation is a common method of altering activity within a cell, and is known to alter the phenotype of myogenic cells. This thesis looks at the immediate effects of electrical stimulation on proliferating C2C12 myoblasts, in order to determine what induces the long term reductions in cell number associated with electrical stimulation. The results indicate that stimulation alters intracellular processes within these cells, promoting cell cycle arrest and autophagy-mediated cellular remodelling, explaining the long term reduction in cell number associated with stimulation. The research conducted is important in our understanding of muscle regeneration and muscle health

Regulation of Cardiac Gene Expression by B-adrenergic Signaling and Heart Failure

Cardiac diseases such as coronary artery disease are the major causes of death around the world. Regulation of -adrenergic receptors by catecholamines is an important facet of understanding cardiac function in health and disease. Acute sympathetic activation of ARs results in an increase in cardiac output; however, sustained stimulation of the ARs is cytotoxic, leading to myocyte death and cardiac remodeling. -adrenergic blockers are a seminal class of drugs that play an important role in improving mortality and symptom control in various cardiac diseases. An important transcriptional regulatory protein target of -adrenergic signaling is MEF2, which plays a crucial role in cardiac gene expression during pathologic and physiological adaptation of the heart. In our experiments, we have observed a robust effect of -blockers on MEF2 transcriptional activity. Myocardial MEF2 responses to -blocker treatment indicates an important physiological linkage between -adrenergic signaling and MEF2 activity in the heart, which underpins changes in cardiac gene expression in response to -adrenergic blockade. In the first set of experiments, the link between MEF2 and cardiac survival pathways in the heart was examined. Collectively, data indicate a mechanism of the beneficial effects of acute 1AR blocker treatment through up-regulation of MEF2 activity, leading to cardiomyocyte survival. In the second set of experiments, changes in MEF2 activity and global gene transcription networks during heart failure and in response to chronic 1-blockade was studied. Together, data demonstrate that chronic 1-blockade inhibits myocardial MEF2 activity while also minimizing dynamic changes in heart failure associated transcriptome dynamics. Communally, both studies indicate molecular events resulting from 1-adrenergic blockade that result in positive effects on heart pathology. These studies define novel molecules and pathways involved in heart pathology that may represent new genetic or pharmacologic targets for heart failure therapies