Eduardo Mascareno

JAK/STAT Signaling Is Associated With Cardiac Dysfunction During Ischemia and Reperfusion

Background—Activation of the heart renin-angiotensin system (RAS) under pathophysiological conditions has been correlated with the development of ischemic injury. The binding of angiotensin II to its receptors triggers induction of several, perhaps multifunctional, intracellular signaling pathways, notable among them the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. In this study, we investigated whether the JAK/STAT signaling is involved in the ischemia/reperfusion injury in adult rat myocardium. Methods and Results—We report here that 2 components of the JAK/STAT signaling pathway, namely STAT 5A and STAT 6, are selectively activated in the rat heart subjected to ischemia/reperfusion. The activated STATs bind to a conserved nucleotide sequence (St domain) in the promoter of the angiotensinogen (ANG) gene and consequently upregulate the level of ANG mRNA. Treatment of the hearts with losartan (4.5 &mgr;mol/L), an AT1 blocker, or with tyrphostin AG490 (5 &mgr;mol/L), an inhibitor of JAK 2 phosphorylation, results in loss of the STAT/ANG promoter binding activity and an upregulated level of ANG mRNA. Hearts treated with the JAK 2 inhibitor tyrphostin AG490 showed a reduction in myocardial infarct size and in number of cardiomyocytes undergoing apoptosis. The treated hearts also showed a recovery in functional hemodynamics of the myocardium. Conclusions—These findings suggest that activation of the JAK/STAT signaling pathway is a significant contributing factor to the pathogenesis of myocardial ischemia and that interference in activation of the pathway potentiates recovery in cardiac function.

The functional role of the JAK–STAT pathway in post-infarction remodeling

OBJECTIVES Recently, the Janus kinase/signal transducer and activator of transcription (JAK-STAT) signaling pathway was found to be prominently associated with activation of the autocrine loop of the heart tissue-localized renin angiotensin system (RAS). We investigated if the JAK-STAT pathway is activated in the post-myocardial infarction (MI) non-ischemic myocardium (NIM), destined to undergo remodeling and whether blockade of the pathway in vivo can modify early post-MI remodeling. METHODS We investigated the time course of tyrosine phosphorylation of JAK-STAT and gp130 proteins in the NIM of post-MI rat heart as well as the binding activity of STAT proteins to the St-domain of the angiotensinogen gene promoter. We further compared the effects of in vivo blockade of RAS by the AT(1) receptor (AT(1)R) blocker losartan with the in vivo blockade of JAK-STAT pathway by the specific JAK2 blocker tyrphostin AG490 on certain aspects of early post-MI remodeling. RESULTS We showed that JAK2, STATs 1, 3, 5a and 6 and gp130 proteins are tyrosine phosphorylated as early as 5-30 min post-MI and that STATs 1, 3, and 5a remain activated up to 7 days post-MI. Gel mobility shift assay showed a strong binding activity of STAT proteins to the St-domain of angiotensinogen gene promoter in 1-day post-MI NIM. The binding was significantly reduced in rat hearts previously treated with losartan or tyrphostin AG490. Supershift experiments identified STATs 3 and 5a as specifically interacting with the St-domain. Both AT(1)R and JAK2 blockade resulted in significant amelioration of the increase of protein phosphatase 1 activity and decrease in basal level of p16-phospholamban that may underlie early diastolic dysfunction, as well as partial amelioration of early downregulation of Kv4.2 gene expression that may underlie increased arrhythmogenicity of 3-day post-MI heart. On the other hand, while blockade of AT(1)R significantly ameliorated apoptotic changes in 1-day post-MI border zone, blockade of JAK2 increased apoptosis. CONCLUSIONS The study provides compelling evidence in favor of the linkage of the JAK-STAT pathway with the angiotensin II autocrine loop and uncovers a mechanism by which selective activation of a set of STAT proteins underlies mobilization of the gene activation program intrinsic to post-MI remodeling. It also suggests that drugs that inhibit JAK-STAT phosphorylation may provide a new approach to modify post-MI remodeling. This needs to be confirmed in long term in vivo studies in the post-MI heart.

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.

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.

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.

Cardiac Lineage Protein-1 (CLP-1) Regulates Cardiac Remodeling via Transcriptional Modulation of Diverse Hypertrophic and Fibrotic Responses and Angiotensin II-transforming Growth Factor β (TGF-β1) Signaling Axis

Background: CLP-1 heterozygous mice exhibit enhanced susceptibility to cardiac stress. Results: Angiotensin-II-induced left ventricular hypertrophy and fibrosis were enhanced, and the Smad3 and Stat3 signaling was stimulated in CLP-1+/− mice. Conclusion: CLP-1 controls the hypertrophy and fibrotic response during cardiac remodeling. Significance: Our results offer the potential of targeting CLP-1 for therapeutic intervention in cardiac disease. It is well known that the renin-angiotensin system contributes to left ventricular hypertrophy and fibrosis, a major determinant of myocardial stiffness. TGF-β1 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-β-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.

Signal transduction and transcriptional adaptation in embryonic heart development and during myocardial hypertrophy.

In comparing the pathological state of cardiac hypertrophy with early embryonic growth and development of the primitive heart, important and informative aspects of mechanisms that underlie activation of the gene expression pattern become apparent. Interestingly, in both cases the muscle phenotypes share the expression of a ‘fetal’ gene expression program, raising the question whether the same genetic mechanism is being called upon by signals associated with the onsets of cardiogenesis and myocardial hypertrophy. A cell specific transcription factor, CLP-1, was recently identified in our laboratory that is likely to play a crucial role, in conjunction with other known regulatory factors, in early cardiac events leading to cardiogenic cell specification and differentiation. We have also identified a novel mechanism that involves activation of the Jak/Stat signaling pathway that is linked to the autocrine angiotensin-II loop associated with the hypertrophic response in cardiomyocytes. Since early cardiac cell development and the hypertrophic state involve the expression of the same battery of genes, one may speculate that common transcription factors may account for assembling a competent apparatus responsible for transcribing the genes. Our present studies are designed to investigate the potential role of these factors in control of both processes.

Cardiac Hypertrophy: Signal Transduction, Transcriptional Adaptation, and Altered Growth Control

Abstract: Cardiac hypertrophy results from the enlargement of cardiac muscle and fibroblast cells. This abnormal pattern of growth can be elicited by a number of hypertrophic agents, such as cytokines and hormones that participate in normal cell‐cell signaling events during development. Under conditions yet to be defined, these same signaling molecules can cause hypertrophy of the heart. Intracellular signal transduction pathways appear to be the prime means by which the hypertrophic signal is transduced in cardiomyocytes. There is no evidence that the signal transduction pathways in hypertrophic cardiomyocytes differ from those of normal cardiomyocytes. Perhaps the signal itself is aberrant, mistimed, misplaced, or occurring at non‐physiological concentrations. Alternatively, as a quiescent cell, the cardiomyocyte may not be able to respond completely to a growth signal by turning on its proliferative machinery. Three avenues of research are described: (1) the study of the upregulation of the cardiac MLC‐2 gene, (2) STAT proteins and activation of angiotensin II, and (3) hypertrophy as a perturbation of cell cycle controls.

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.

Hexim‐1 modulates androgen receptor and the TGF‐β signaling during the progression of prostate cancer

Androgen and TGF‐β 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.