HEXIM1 controls satellite cell expansion after injury to regulate skeletal muscle regeneration.

The native capacity of adult skeletal muscles to regenerate is vital to the recovery from physical injuries and dystrophic diseases. Currently, the development of therapeutic interventions has been hindered by the complex regulatory network underlying the process of muscle regeneration. Using a mouse model of skeletal muscle regeneration after injury, we identified hexamethylene bisacetamide inducible 1 (HEXIM1, also referred to as CLP-1), the inhibitory component of the positive transcription elongation factor b (P-TEFb) complex, as a pivotal regulator of skeletal muscle regeneration. Hexim1-haplodeficient muscles exhibited greater mass and preserved function compared with those of WT muscles after injury, as a result of enhanced expansion of satellite cells. Transplanted Hexim1-haplodeficient satellite cells expanded and improved muscle regeneration more effectively than WT satellite cells. Conversely, HEXIM1 overexpression restrained satellite cell proliferation and impeded muscle regeneration. Mechanistically, dissociation of HEXIM1 from P-TEFb and subsequent activation of P-TEFb are required for satellite cell proliferation and the prevention of early myogenic differentiation. These findings suggest a crucial role for the HEXIM1/P-TEFb pathway in the regulation of satellite cell­mediated muscle regeneration and identify HEXIM1 as a potential therapeutic target for degenerative muscular diseases.

Cardiac lineage protein-1 (CLP-1) regulates cardiac remodeling via transcriptional modulation of diverse hypertrophic and fibrotic responses and angiotensin II-transforming growth factor ß (TGF-ß1) signaling axis.

It is well known that the renin-angiotensin system contributes to left ventricular hypertrophy and fibrosis, a major determinant of myocardial stiffness. TGF-ß1 and renin-angiotensin system signaling alters the fibroblast phenotype by promoting its differentiation into morphologically distinct pathological myofibroblasts, which potentiates collagen synthesis and fibrosis and causes enhanced extracellular matrix deposition. However, the atrial natriuretic peptide, which is induced during left ventricular hypertrophy, plays an anti-fibrogenic and anti-hypertrophic role by blocking, among others, the TGF-ß-induced nuclear localization of Smads. It is not clear how the hypertrophic and fibrotic responses are transcriptionally regulated. CLP-1, the mouse homolog of human hexamethylene bis-acetamide inducible-1 (HEXIM-1), regulates the pTEFb activity via direct association with pTEFb causing inhibition of the Cdk9-mediated serine 2 phosphorylation in the carboxyl-terminal domain of RNA polymerase II. It was recently reported that the serine kinase activity of Cdk9 not only targets RNA polymerase II but also the conserved serine residues of the polylinker region in Smad3, suggesting that CLP-1-mediated changes in pTEFb activity may trigger Cdk9-dependent Smad3 signaling that can modulate collagen expression and fibrosis. In this study, we evaluated the role of CLP-1 in vivo in induction of left ventricular hypertrophy in angiotensinogen-overexpressing transgenic mice harboring CLP-1 heterozygosity. We observed that introduction of CLP-1 haplodeficiency in the transgenic α-myosin heavy chain-angiotensinogen mice causes prominent changes in hypertrophic and fibrotic responses accompanied by augmentation of Smad3/Stat3 signaling. Together, our findings underscore the critical role of CLP-1 in remodeling of the genetic response during hypertrophy and fibrosis.

Hexim-1 modulates androgen receptor and the TGF-ß signaling during the progression of prostate cancer.

BACKGROUND: Androgen and TGF-ß signaling are important components during the progression of prostate cancer. However, whether common molecular events participate in the activation of these signaling pathways are less understood. METHOD: Hexim 1 expression was detected by immunohistochemistry of human tissue microarrays and TRAMP mouse models. The in vivo significance of Hexim-1 was established by crossing the TRAMP mouse model of prostate cancer with Hexim-1 heterozygous mice. TRAMP C2 cell line was also modified to delete one copy of Hexim-1 gene to generate TRAMP-C2-Hexim-1+/- cell lines. RESULTS: In this report, we observed that Hexim-1 protein expression is absent in normal prostate but highly expressed in adenocarcinoma of the prostate and a characteristic sub-cellular distribution among normal, benign hyperplasia, and adenocarcinoma of the prostate. Heterozygosity of the Hexim-1 gene in the prostate cancer mice model and the TRAMP-C2 cell line, leads to increased Cdk9-dependent serine phosphorylation on protein targets such as the androgen receptor (AR) and the TGF-ß-dependent downstream transcription factors, such as the SMAD proteins. CONCLUSION: Our results suggest that changes in the Hexim-1 protein expression and cellular distribution significantly influences the AR activation and the TGF-ß signaling. Thus, Hexim-1 is likely to play a significant role in prostate cancer progression.

The JAK-STAT pathway in hypertrophic stress signaling and genomic stress response.

The JAK-STAT signaling pathway plays a central role in transducing stress and growth signals in the hypertrophic heart. Unlike most signal transducers, JAKs and STATs signal in a number of different ways, both within the JAK-STAT pathway and in collaboration with other signaling pathways. In this review, we discuss how IL-6 activates cells lacking IL-6 receptors through trans-signaling and examine JAK-STAT pathway interaction with GPCR-linked pathways both within and between cells. Finally, we discuss recent studies showing how the JAK-STAT pathway can intersect with a general transcriptional regulatory mechanism to effect transcription of STAT-dependent stress response genes.

CLP-1 associates with MyoD and HDAC to restore skeletal muscle cell regeneration.

Emerging evidence suggests that eukaryotic gene transcription is regulated primarily at the elongation stage by association and dissociation of the inhibitory protein cardiac lineage protein 1 (CLP-1/HEXIM1) from the positive transcription elongation factor b (P-TEFb) complex. It was reported recently that P-TEFb interacts with skeletal muscle-specific regulatory factor, MyoD, suggesting a linkage between CLP-1-mediated control of transcription and skeletal myogenesis. To examine this, we produced CLP-1 knockdown skeletal muscle C2C12 cells by homologous recombination, and demonstrated that the C2C12 CLP-1 +/- cells failed to differentiate when challenged by low serum in the medium. We also showed that CLP-1 interacts with both MyoD and histone deacetylases (HDACs) maximally at the early stage of differentiation of C2C12 cells. This led us to hypothesize that the association might be crucial to inhibition of MyoD-target proliferative genes. Chromatin immunoprecipitation analysis revealed that the CLP-1/MyoD/HDAC complex binds to the promoter of the cyclin D1 gene, which is downregulated in differentiated muscle cells. These findings suggest a novel transcriptional paradigm whereby CLP-1, in conjunction with MyoD and HDAC, acts to inhibit growth-related gene expression, a requirement for myoblasts to exit the cell cycle and transit to myotubes.

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.

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

Among the stress proteins that are up-regulated in the heart due to imposed biomechanical stress, alphaB-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 alphaBE4 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 alphaBE4 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.

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.

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

RATIONALE: We 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). METHODS: Control 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. RESULTS: Evaluation 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. CONCLUSION: This 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.

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.

Positive transcription elongation factor b activity in compensatory myocardial hypertrophy is regulated by cardiac lineage protein-1.

Emerging evidence illustrates the importance of the positive transcription elongation factor (P-TEF)b in control of global RNA synthesis, which constitutes a major feature of the compensatory response to diverse hypertrophic stimuli in cardiomyocytes. P-TEFb complex, composed of cyclin T and cdk9, is critical for elongation of nascent RNA chains via phosphorylation of the carboxyl-terminal domain of RNA polymerase (Pol) II. We and others have shown that the activity of P-TEFb is inhibited by its association with cardiac lineage protein (CLP)-1, the mouse homolog of human HEXIM1, in various physiological and pathological conditions. To investigate the mechanism of control of P-TEFb activity by CLP-1 in cardiac hypertrophy, we used a transgenic mouse model of hypertrophy caused by overexpression of calcineurin in the heart. We observed that the level of CLP-1 associated with P-TEFb was reduced markedly in hypertrophic hearts. We also generated bigenic mice (MHC-cyclin T1/CLP-1(+/-)) by crossing MHC-cyclin T1 transgenic mice with CLP-1 heterozygote. The bigenic mice exhibit enhanced susceptibility to hypertrophy that is accompanied with an increase in cdk9 activity via an increase in serine 2 phosphorylation of carboxyl-terminal domain and an increase in GLUT1/GLUT4 ratio. These mice have compensated systolic function without evidence of fibrosis and reduced lifespan. These data suggest that the reduced level of CLP-1 introduced in the background of elevated levels of cyclin T1 elevates derepression of P-TEFb activity and emphasizes the importance of the role of CLP-1 in the mechanism governing compensatory hypertrophy in cardiomyocytes.

Down-regulation of cardiac lineage protein (CLP-1) expression in CLP-1 +/- mice affords.

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-1alpha and HIF-1alpha, 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.

Signal transduction in early heart development (I): cardiogenic induction and heart tube formation.

Heart development begins with the induction of cardiogenic cells from the embryonic mesoderm, followed by the coalescing of these cells into a linear heart tube. Subsequent looping of the heart tube brings the rudimentary atria and ventricles into alignment for further development into the four-chambered heart. Underlying these morphologic events is a complex program of signaling between cells and tissues that orchestrates their participation in heart development. Among these signals are bone morphogenetic proteins, fibroblast growth factors, Wnts, Hedgehog, and members of the transforming growth factor-beta family of signaling molecules. We review here the various properties of these signaling molecules and their signal transduction pathways in hopes of providing a greater appreciation of the molecular events driving heart development.

Signal transduction in early heart development (II): ventricular chamber specification, trabeculation, and heart valve formation.

The formation of a four-chambered heart with ventricular chambers aligned in a left-right orientation begins with the rightward looping of the linear heart tube in accordance with the left-right embryonic axis. The functional specification of the ventricular chambers in the looped heart occurs with the formation of a trabeculated myocardium along the outer curvature of the realigned heart tube. Two major signal transduction pathways are involved in this process, the retinoic acid and neuregulin signaling pathways, with the retinoic acid pathway also participating in rightward heart tube looping. With the establishment of the atrial and ventricular chambers, maintenance of a unidirectional flow of blood between the two chambers must be ensured. To achieve this, heart valves develop at the atrioventricular juncture. This process begins with formation of endocardial cushions, the primordia of heart valves, and ends with formation of heart valve leaflets. Underlying this process is a complex network of signal transduction pathways that mediate communication between the endocardial and myocardial cell layers to form the endocardial cushions and nascent heart valve. Some of the signaling molecules involved are vascular endothelial growth factor, Wnts, bone morphogenetic proteins, epidermal growth factor, hyaluronic acid, neurofibromin, and calcium.

Pivotal role of cardiac lineage protein-1 (CLP-1) in transcriptional elongation factor P-TEFb complex formation in cardiac hypertrophy.

OBJECTIVE: Our aim was to determine if the expression pattern of CLP-1 in developing heart is consistent with its role in controlling RNA transcript elongation by transcriptional elongation factor b (P-TEFb) and if the inhibitory control exerted over P-TEFb by CLP-1 is released under hypertrophic conditions. METHODS: We performed immunoblot and immunofluorescence analysis of CLP-1 and the P-TEFb components cdk9 and cyclin T in fetal mouse heart and 2 day post-natal mouse cardiomyocytes to determine if they are co-localized. We induced hypertrophy in rat cardiomyocytes either by mechanical stretch or treatment with hypertrophic agents such as endothelin-1 and phenylephrine to determine if CLP-1 is released from P-TEFb in response to hypertrophic stimuli. The involvement of the Jak/STAT signal transduction pathway in this process was studied by blocking this pathway with the Jak2 kinase inhibitor, AG490, and assessing the association of CLP-1 with P-TEFb complexes. RESULTS: We found that CLP-1 is expressed along with P-TEFb components in developing heart during the period in which knockout mice lacking the CLP-1 gene develop cardiac hypertrophy and die. Under conditions of hypertrophy induced by mechanical stretch or agonist treatment, CLP-1 dissociates from the P-TEFb complex, a finding consistent with the de-repression of P-TEFb kinase activity seen in hypertrophic cardiomyocytes. Blockage of Jak/STAT signaling by AG490 prevented release of CLP-1 from P-TEFb despite the ongoing presence of hypertrophic stimulation by mechanical stretch. CONCLUSIONS: CLP-1 expression in developing heart and isolated post-natal cardiomyocytes colocalizes with P-TEFb expression and therefore has the potential to regulate RNA transcript elongation by controlling P-TEFb cdk9 kinase activity in heart. We further conclude that the dissociation of CLP-1 from P-TEFb is responsive to hypertrophic stimuli transduced by cellular signal transduction pathways. This process may be part of the genomic stress response resulting in increased RNA transcript synthesis in hypertrophic cardiomyocytes.

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 in vitro. 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.

Inhibition of Jak2 phosphorylation attenuates pressure overload cardiac hypertrophy.

RATIONALE: We examined the role of Jak2 kinase phosphorylation in the development of pressure overload hypertrophy in mice subjected to transverse aortic constriction (TAC) and treated with tyrphostin AG490, a pharmacological inhibitor of Jak2. METHODS: Control mice (sham), subjected to TAC for 15 days (TAC) or to TAC and treated with 48 microg/kg/day i.p. of tyrphostin AG490 (TAC+AG490) were evaluated for morphological, physiological, and molecular changes associated with pressure overload hypertrophy. RESULTS: Mice subjected to TAC alone developed concentric hypertrophy that accompanied activation of the components of the Jak/STAT signaling pathway manifested by an increase in phosphorylation of Jak2 and STAT3. We also observed increased phosphorylation of MAPK p44/p42, p38 MAPK and JNK in the TAC group, as well as, an increase in expression of MKP-1 phosphatase which negatively regulates MAPK kinases. Treatment of aortic constricted mice with tyrphostin AG490 failed to develop hypertrophy and showed a marked reduction in phosphorylation of Jak2 and STAT3. There was, however, in TAC and AG490 treated mice, a notable increase in the phosphorylation state of the MAPK p44/42, whereas MKP-1 phosphatase was downregulated. CONCLUSION: These findings suggest that Jak2 kinase plays an important role in left ventricular remodeling during pressure overload hypertrophy. Pharmacological inhibition of Jak2 kinase during pressure overload blocks the development of concentric hypertrophy.

Janus kinase-2 signaling mediates apoptosis in rat cardiomyocytes.

We tested the hypothesis that activation Jak2, which is prominently involved in the up-regulation of the renin-angiotensin system (RAS), constitutes a focal point in relaying signals triggered by a Angiotensin II (Ang II) and hypoxia/reoxygenation separately to cause an enhanced susceptibility of cardiac myocyte to apoptotic cell death. Ang II-treated adult cardiomyocytes in culture exhibited an increased level of apoptosis that accompanied activation of pro-apoptotic as well as anti-apoptotic signaling pathways. We observed increased phosphorylation of Jak2 kinase, Stat1, JNK, with increased expression of Bax protein, followed by an increase in caspase-1 and caspase-3 activity. Activation of these pro-apoptotic pathways was blocked by the Jak2 pharmacological inhibitor, Tyrphostin AG490. We also observed an increase in phosphorylation of cardioprotective pathway components, namely S6 ribosomal protein, and heat shock protein 27 (HSP27). Likewise, the oxidative stress, via the hypoxia/reoxygenation treatment of rat adult cardiomyocytes, produced apoptosis that was dependent upon activation of Jak2. The apoptotic response was not only reduced by Losartan, an inverse agonist of the AT1, receptor, but by treatment with AG490 as well. Taken together, these observations provide clear evidence in favor of Jak2 signaling as mediator of the apoptotic response in cardiomyocytes. However, there was a concomitant induction of cytoprotective signaling that presumably provides a negative feed-back to the deleterious effects of the agonist.

Vitamin C inhibits hypoxia-induced damage and apoptotic signaling pathways in cardiomyocytes and ischemic hearts.

Reactive oxygen species play a central role in myocardial ischemic injury and are a target for therapeutic intervention. Vitamin C is an essential antioxidant yet difficult to deliver in pharmacologic concentration to the myocardium. We found that adult rat cardiomyocytes accumulate vitamin C by transporting dehydroascorbic acid (DHA), the oxidized form of vitamin C, but do not transport ascorbic acid. Loading cells with vitamin C by DHA treatment resulted in resistance to hypoxia- and hypoxia/reoxygenation-induced cell death associated with the quenching of reactive oxygen species. When rats were injected with DHA before coronary occlusion, the ascorbic acid content in the heart was six to eight times higher than in untreated controls and myocardial infarction was reduced by 62%. DHA also provided significant protection when administered intravenously 2 h after coronary occlusion. In cardiomyocytes subjected to hypoxia/reoxygenation, DHA treatment resulted in decreased apoptosis associated with inhibition of Bax expression, caspase-3 activation, and cytochrome c translocation into the cytoplasm. DHA treatment also inhibited Jak2, STAT1, and STAT5 phosphorylation, and increased STAT3 phosphorylation, in hypoxic cardiomyocytes and ischemic myocardial tissue. Our findings suggest that DHA may be useful as a cardioprotectant in ischemic heart disease.

A ternary complex of transcription factors, Nishéd and NFATc4, and co-activator p300 bound to an intronic sequence, intronic regulatory element, is pivotal for the up-regulation of myosin light chain-2v gene in cardiac hypertrophy.

Transcriptional up-regulation of the myosin light chain-2 (MLC-2v) gene is an established marker for hypertrophic response in cardiomyocytes. Despite the documentation on the role of several cis-elements in the MLC-2v gene and their cognate proteins in transcription, the mechanism that dictates the preferential increase in MLC-2v gene expression during myocardial hypertrophy has not been delineated. Here we describe the properties of a cardiac specific intronic activator element (IRE) that shares sequence homology with the repressor element, the cardiac specific sequence, in the chicken MLC-2v gene. The transcription factor, Nishéd, that recognizes both IRE and the cardiac specific sequence potentiates the transcription of the MLC-2v gene via interaction with another transcription factor, nuclear factor of activated T cells, and the co-activator p300 at the IRE site. Angiotensin II (Ang II), a potent agonist of hypertrophy, causes induction of the MLC-2v gene transcription, which correlates well with the enhanced binding of Nishéd-nuclear factor of the activated T cells-p300 complex to IRE in the gel mobility shift assay. Losartan, an antagonist of Ang II receptor (AT1), abolishes the agonist-dependent stimulation of IRE/protein interaction and the consequent increase in MLC-2v gene transcription. These results together have thus established a transcriptional role of IRE as a direct target sequence of Ang II-mediated signaling that appears to be pivotal in the mechanism underlying the up-regulation of the MLC-2v gene during cardiac hypertrophy.