M.A.Q. Siddiqui

Mechanical Regulation of the Proangiogenic Factor CCN1/CYR61 Gene Requires the Combined Activities of MRTF-A and CREB-binding Protein Histone Acetyltransferase

Smooth muscle-rich tissues respond to mechanical overload by an adaptive hypertrophic growth combined with activation of angiogenesis, which potentiates their mechanical overload-bearing capabilities. Neovascularization is associated with mechanical strain-dependent induction of angiogenic factors such as CCN1, an immediate-early gene-encoded matricellular molecule critical for vascular development and repair. Here we have demonstrated that mechanical strain-dependent induction of the CCN1 gene involves signaling cascades through RhoA-mediated actin remodeling and the p38 stress-activated protein kinase (SAPK). Actin signaling controls serum response factor (SRF) activity via SRF interaction with the myocardin-related transcriptional activator (MRTF)-A and tethering to a single CArG box sequence within the CCN1 promoter. Such activity was abolished in mechanically stimulated mouse MRTF-A−/− cells or upon inhibition of CREB-binding protein (CBP) histone acetyltransferase (HAT) either pharmacologically or by siRNAs. Mechanical strain induced CBP-mediated acetylation of histones 3 and 4 at the SRF-binding site and within the CCN1 gene coding region. Inhibition of p38 SAPK reduced CBP HAT activity and its recruitment to the SRF·MRTF-A complex, whereas enforced induction of p38 by upstream activators (e.g. MKK3 and MKK6) enhanced both CBP HAT and CCN1 promoter activities. Similarly, mechanical overload-induced CCN1 gene expression in vivo was associated with nuclear localization of MRTF-A and enrichment of the CCN1 promoter with both MRTF-A and acetylated histone H3. Taken together, these data suggest that signal-controlled activation of SRF, MRTF-A, and CBP provides a novel connection between mechanical stimuli and angiogenic gene expression.

The role of Jak/STAT signaling in heart tissue renin-angiotensin system

The involvement of the Renin Angiotensin System (RAS) and the role of its primary effector, angiotensin II (Ang II), in etiology of myocardial hypertrophy and ischemia is well documented. In several animal models, the RAS is activated in cardiac cell types that express the receptor AT1, and/or AT2, through which the Ang II mediated effects are promoted. In this article, we briefly review recent experimental evidence on the critical role of a prominent signaling pathway, the Jak/Stat pathway in activation and maintenance of the local RAS in cardiac hypertrophy and ischemia. Recent studies in our laboratory document that the promoter of the prohormone angiotensinogen (Ang) gene serves as the target site for STAT proteins, thereby linking the Jak/Stat pathway to activation of heart tissue autocrine Ang II loop. Stat5A and Stat6, are selectively activated when the heart is subjected to ischemic injury, whereas activation of Stat3 and Stat5A is involved in myocardial hypertrophy. Blockage of RAS activation by treatment with specific inhibitor promotes a remarkable recovery in functional hemodynamics of the myocardium. Thus, activation of selective sets of Stat proteins constitutes the primary signaling event in the pathogenesis of myocardial hypertrophy and ischemia.

Enhanced hypertrophy in ob/ob mice due to an impairment in expression of atrial natriuretic peptide

RATIONALEnWe investigated the molecular mechanism(s) that play a role in leptin signaling during the development of left ventricular hypertrophy (LVH) due to pressure overload. To this end, ob/ob leptin deficient and C57BL/6J control mice were subjected transverse aortic constriction (TAC).nnnMETHODSnControl sham C57BL/6J and ob/ob mice, along with C57BL/6J and ob/ob leptin deficient mice were subjected transverse aortic constriction (TAC) for 15 days and then evaluated for morphological, physiological, and molecular changes associated with pressure overload hypertrophy.nnnRESULTSnEvaluation by echocardiography revealed a significant increase in left ventricular mass (LVmass) and wall thickness in ob/ob mice subjected to transverse aortic constriction (TAC) as compared to C57BL/6J. Analysis of the expression of molecular markers of LVH, such as atrial natriuretic peptide (ANP), revealed a blunted increase in the level of ANP in ob/ob mice as compared to C57BL/6J mice. We observed that leptin plays a role in modulating the transcriptional activity of the promoter of the ANP gene. Leptin acts by regulating NFATc4, a member of the nuclear factor activated T cell (NFAT) family of transcription factors in cardiomyocytes. Our in vivo studies revealed that ob/ob mice subjected to TAC failed to activate the NFATc4 in the heart, however, intraperitoneal injection of leptin in ob/ob mice restored the NFATc4 DNA-binding activity and induced expression of the ANP gene.nnnCONCLUSIONnThis study establishes the role of leptin as an anti-hypertrophic agent during pressure overload hypertrophy, and suggests that a key molecular event is the leptin mediated activation of NFATc4 that regulates the transcriptional activation of the ANP gene promoter.

Cross-talk between calcineurin/NFAT and Jak/STAT signalling induces cardioprotective αB-crystallin gene expression in response to hypertrophic stimuli

Among the stress proteins that are up‐regulated in the heart due to imposed biomechanical stress, αB‐crystallin (CryAB) is the most abundant and pivotal in rendering protection against stress‐induced cell damage. Cardiomyocyte‐specific expression of the CryAB gene was shown to be dependent upon an intact αBE4 cis‐element located in the CryAB enhancer. To date, there is no evidence on the identity of regulatory proteins and associated signalling molecules that control CryAB expression in cardiomyocytes. In this study, we define a mechanism by which the calcineurin/NFAT and Jak/STAT pathways regulate CryAB gene expression in response to a hypertrophic agonist endothelin‐1 (En‐1), in hypertrophic hearts of mice with pressure overload (TAC) and in heart‐targeted calcineurin over‐expressing mice (MHC‐CnA). We observed that in response to various hypertrophic stimuli the transcription factors NFAT, Nished and STAT3 form a dynamic ternary complex and interact with the αBE4 promoter element of the CryAB gene. Both dominant negative NFAT and AG490, an inhibitor of the Jak2 phosphorylation, inhibited CryAB gene transcription in transient transfection assays. AG490 was also effective in blocking the nuclear translocation of NFAT and STAT3 in cardiomyocytes treated with En‐1. We observed a marked increase in CryAB gene expression in MHC‐CnA mouse hearts accompanied with increased phosphorylation of STAT3. We conclude that hypertrophy‐dependent CryAB gene expression can be attributed to a functional linkage between the Jak/STAT and calcineurin/NFAT signalling pathways, each of which are otherwise known to be involved independently in the deleterious outcome in cardiac hypertrophy.

RNAi inhibition of Pax3/7 expression leads to markedly decreased expression of muscle determination genes

Skeletal muscle regeneration by cell transplantation for the treatment of muscle diseases requires the identification and isolation of well-defined, early skeletal muscle progenitor cells. It is known that myogenesis is governed by the sequential and compound activation of the muscle determination genes, the myogenic regulatory factors (MRFs). Recently it has been proposed that the transcription factors Pax3 and Pax7 trigger the expression of the MRFs and thereby specify a novel population of cells destined to enter the myogenic program. We directly tested this hypothesis using RNA interference methodology to reduce the levels of Pax3 and Pax7 RNA in mouse embryoid bodies developing inxa0vitro. We found that decreasing the levels of Pax3/Pax7 RNA leads to a marked and selective decrease in Myf5, MyoD and Desmin expression. Pax3 and Pax7 expressing cells from developing embryos may thus serve as the earliest known skeletal muscle progenitor cells potentially useful for cell transplantation studies.

Down‐regulation of cardiac lineage protein (CLP‐1) expression in CLP‐1 +/− mice affords cardioprotection against ischaemic stress

In order to understand the transcriptional mechanism that underlies cell protection to stress, we evaluated the role of CLP‐1, a known inhibitor of the transcription elongation complex (pTEFb), in CLP‐1 +/− mice hearts. Using the isolated heart model, we observed that the CLP‐1+/− hearts, when subjected to ischaemic stress and evaluated by haemodynamic measurements, exhibit significant cardioprotection. CLP‐1 remains associated with the pTEFb complex in the heterozygous hearts, where as it is released in the wild‐type hearts suggesting the involvement of pTEFb regulation in cell protection. There was a decrease in Cdk7 and Cdk9 kinase activity and consequently in phosphorylation of serine‐5 and serine‐2 of Pol II CTD in CLP‐1 +/− hearts. However, the levels of mitochondrial proteins, PGC‐1α and HIF‐1α, which enhance mitochondrial activity and are implicated in cell survival, were increased in CLP‐1+/− hearts subjected to ischaemic stress compared to that in wild‐type CLP‐1+/+ hearts treated identically. There was also an increase in the expression of pyruvate dehydrogenase kinase (PDK‐1), which facilitates cell adaptation to hypoxic stress. Taken together, our data suggest that regulation of the CLP‐1 levels is critical to cellular adaptation of the survival program that protects cardiomyocytes against stress due collectively to a decrease in RNA Pol II phosphorylation but an increase in expression of target proteins that regulate mitochondrial function and metabolic adaptation to stress.

Downregulation of cardiac lineage protein‐1 confers cardioprotection through the upregulation of redox effectors

CLP‐1, the mouse homologue of human Hexim1 protein, exerts inhibitory control on transcriptional elongation factor‐b of RNA transcript elongation. Previously, we have demonstrated that downregulation of cardiac lineage protein‐1 (CLP‐1) in CLP‐1+/− heterozygous mice affords cardioprotection against ischemia–reperfusion injury. Our current study results show that the improvement in cardiac function in CLP‐1+/− mice after ischemia–reperfusion injury is achieved through the potentiation of redox signaling and their molecular targets including redox effector factor‐1, nuclear factor erythroid 2‐related factor, and NADPH oxidase 4 and the active usage of thioredoxin‐1, thioredoxin‐2, glutaredoxin‐1 and glutaredoxin‐2. Our results suggest that drugs designed to down regulate CLP‐1 could confer cardioprotection through the potentiation of redox cycling.

Cardiac Remodeling in the Hypertrophic Heart: Signal-Dependent Regulation of the Fibrotic Gene Program by CLP-1

The response of the heart to hypertrophic stress stimuli is designed to normalize heart function under conditions of increased demand. To achieve this, the heart must mount a genomic stress response that is itself responsive to the signal transduction pathways used by heart cells to transmit stress signals. The ability to adaptively link stress signal to genomic stress response is critical to how the heart responds to stress. Our laboratory has been studying the molecular events involved in this adaptive linkage. Our studies have shown that CLP-1 (Cardiac Lineage Protein-1), the mouse homolog of the human HEXIM1, acts as the molecular “go-between” linking stress signal with genomic stress response. Critical to this linkage is HEXIM1/CLP-1’s control of cyclin-dependent kinase 9 (cdk9), the kinase responsible for activating RNA polymerase (pol) II to complete synthesis of nascent stress gene transcripts. Through its control of cdk9, HEXIM1/CLP-1 controls the transcriptional output of stress response genes by regulating the ability of RNA polymerase (pol) II, and as more recent data has shown, the activity of specific transcription factors such as those of the small mother against decapentaplegic(smad) family, to transcribe stress response genes. Together, these observations provide strong support for the idea that HEXIM1/CLP-1 plays a critical role in controlling the response of cardiac cells to hypertrophic stress stimuli.

CLP-1-Mediated Transcriptional Control of Hypertrophic Gene Programs Underlying Cardiac Hypertrophy

Cardiac hypertrophy is the heart’s response to increased work, pressure, or volume overload. It begins with a compensatory phase that allows the heart to meet imposed demand through rapid expression of stress response genes. A decompensatory phase follows marked by additional adaptive stress response gene expression that with prolonged stress progressively turns maladaptive, leading the heart into failure. The transition from compensatory to decompensatory hypertrophy is likely to reflect changes in the transcription factors and regulatory molecules that control these programs in response to changes in stress stimuli and the status of cardiomyocytes throughout the hypertrophic process. Our laboratory has been studying the role of one such transcriptional regulatory molecule, CLP-1 (cardiac lineage protein-1), in the cellular response to hypertrophic stimuli. CLP-1, the mouse homolog of the human HEXIM1 gene, is an inhibitor of P-TEFb (transcription elongation factor b), a component of the transcriptional apparatus that controls RNA polymerase II activity and gene transcription. Knockout of the CLP-1 gene results in a severe form of hypertrophy in fetal mice suggesting that in the absence of the CLP-1 inhibitor, uninhibited P-TEFb activity may lead to unregulated expression of stress response genes and decompensatory hypertrophy. Because of its critical role in regulating the stress gene response to hypertrophic stimuli, we review our laboratory’s work on CLP-1, its control of P-TEFb under various hypertrophic conditions, and how it may play an important role in a novel gene control mechanism, called promoter proximal pausing, that ensures the rapid expression of stress response genes in response to hypertrophic stimuli.

Repression of the cardiac myosin light chain-2 gene in skeletal muscle requires site-specific association of antithetic regulator, Nished, and HDACs.

The transcriptional activation mechanisms that regulate tissue‐specific expression of cardiac muscle genes have been extensively investigated, but little is known of the regulatory events involved in repression of cardiac‐specific genes in non‐cardiac cells. We have previously reported that Nished, a ubiquitous transcription factor, interacts with a positive sequence element, the Intron Regulatory Element (IRE) as well as a negatively acting element, the Cardiac‐Specific Sequence (CSS), in myosin light chain‐2 (MLC2v) gene to promote activation and repression of the gene in cardiac and skeletal muscle cells respectively. Here, we show that the negative regulation of cardiac MLC2v gene in skeletal muscle cells is mediated via the interaction of Nished with histone deacetylase (HDAC) co‐repressor. Treatment of cells with the HDAC inhibitor, Trichostatin A (TSA), alleviates the repressor activity of Nished in a dose‐dependent manner. Co‐transfection studies in primary muscle cells in culture and in Nished expressing stable skeletal muscle cell line demonstrate that Nished down‐regulates the cardiac MLC2 gene expression when its association is restricted to CSS alone. Chromatin immunoprecipitation data suggest that the CSS‐mediated repression of cardiac MLC2v gene in skeletal muscle cells excludes the participation of the positive element IRE despite the presence of an identical Nished binding site. Taken together, it appears that the negative control of MLC2v transcription is based on a dual mode of regulations, one that affords inaccessibility of IRE to Nished and second that promotes the formation of the transcription repression complex at the inhibitory CSS site to silence the cardiac gene in skeletal muscle cell.