Syed Haq

Differential activation of signal transduction pathways in human hearts with hypertrophy versus advanced heart failure.

Background —Left ventricular failure is commonly preceded by a period of hypertrophy. Intriguingly, many of the signaling pathways that have been implicated in the regulation of hypertrophy, including the 3 mitogen-activated protein kinases (MAPKs: extracellular signal-regulated kinase, stress-activated protein kinase, and p38), protein phosphatase, calcineurin, and the protein kinase Akt and its target glycogen synthase kinase-3 (GSK-3), also regulate the apoptotic response. Methods and Results —To understand the mechanisms that might regulate the progression of heart failure, we analyzed the activity of these signaling pathways in the hearts of patients with advanced heart failure, patients with compensated cardiac hypertrophy, and normal subjects. In patients with hypertrophy, neither the MAPK nor the Akt/GSK-3 pathways were activated, and the dominant signaling pathway was calcineurin. In failing hearts, calcineurin activity was increased but less so than in the hypertrophied hearts, and all 3 MAPKs and Akt were activated (and, accordingly, GSK-3&bgr; was inhibited), irrespective of whether the underlying diagnosis was ischemic or idiopathic cardiomyopathy. Conclusions —In the failing heart, there is a clear prohypertrophic activity profile, likely occurring in response to increased systolic wall stress and neurohormonal mediators. However, with the activation of these hypertrophic pathways, potent proapoptotic and antiapoptotic signals may also be generated. Therapies directed at altering the balance of activity of these signaling pathways could potentially alter the progression of heart failure.

17β-Estradiol Reduces Cardiomyocyte Apoptosis In Vivo and In Vitro via Activation of Phospho-Inositide-3 Kinase/Akt Signaling

Female gender and estrogen-replacement therapy in postmenopausal women are associated with improved heart failure survival, and physiological replacement of 17&bgr;-estradiol (E2) reduces infarct size and cardiomyocyte apoptosis in animal models of myocardial infarction (MI). Here, we characterize the molecular mechanisms of E2 effects on cardiomyocyte survival in vivo and in vitro. Ovariectomized female mice were treated with placebo or physiological E2 replacement, followed by coronary artery ligation (placebo-MI or E2-MI) or sham operation (sham) and hearts were harvested 6, 24, and 72 hours later. After MI, E2 replacement significantly increased activation of the prosurvival kinase, Akt, and decreased cardiomyocyte apoptosis assessed by terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) staining and caspase 3 activation. In vitro, E2 at 1 or 10 nmol/L caused a rapid 2.7-fold increase in Akt phosphorylation and a decrease in apoptosis as measured by TUNEL staining, caspase 3 activation, and DNA laddering in cultured neonatal rat cardiomyocytes. The E2-mediated reduction in apoptosis was reversed by an estrogen receptor (ER) antagonist, ICI 182,780, and by phospho-inositide-3 kinase inhibitors, LY294002 and Wortmannin. Overexpression of a dominant negative-Akt construct also blocked E2-mediated reduction in cardiomyocyte apoptosis. These data show that E2 reduces cardiomyocyte apoptosis in vivo and in vitro by ER- and phospho-inositide-3 kinase–Akt–dependent pathways and support the relevance of these pathways in the observed estrogen-mediated reduction in myocardial injury.

Stabilization of β-catenin by a Wnt-independent mechanism regulates cardiomyocyte growth

β-Catenin is a transcriptional activator that regulates embryonic development as part of the Wnt pathway and also plays a role in tumorigenesis. The mechanisms leading to Wnt-induced stabilization of β-catenin, which results in its translocation to the nucleus and activation of transcription, have been an area of intense interest. However, it is not clear whether stimuli other than Wnts can lead to important stabilization of β-catenin and, if so, what factors mediate that stabilization and what biologic processes might be regulated. Herein we report that β-catenin is stabilized in cardiomyocytes after these cells have been exposed to hypertrophic stimuli in culture or in vivo. The mechanism by which β-catenin is stabilized is distinctly different from that used by Wnt signaling. Although, as with Wnt signaling, inhibition of glycogen synthase kinase-3 remains central to hypertrophic stimulus-induced stabilization of β-catenin, the mechanism by which this occurs involves the recruitment of activated PKB to the β-catenin-degradation complex. PKB stabilizes the complex and phosphorylates glycogen synthase kinase-3 within the complex, inhibiting its activity directed at β-catenin. Finally, we demonstrate via adenoviral gene transfer that β-catenin is both sufficient to induce growth in cardiomyocytes in culture and in vivo and necessary for hypertrophic stimulus-induced growth. Thus, in these terminally differentiated cells, β-catenin is stabilized by hypertrophic stimuli acting via heterotrimeric G protein-coupled receptors. The stabilization occurs via a unique Wnt-independent mechanism and results in cellular growth.

Regulation of cardiac hypertrophy in vivo by the stress-activated protein kinases/c-Jun NH2-terminal kinases

Cardiac hypertrophy often presages the development of heart failure. Numerous cytosolic signaling pathways have been implicated in the hypertrophic response in cardiomyocytes in culture, but their roles in the hypertrophic response to physiologically relevant stimuli in vivo is unclear. We previously reported that adenovirus-mediated gene transfer of SEK-1(KR), a dominant inhibitory mutant of the immediate upstream activator of the stress-activated protein kinases (SAPKs), abrogates the hypertrophic response of neonatal rat cardiomyocytes to endothelin-1 in culture. We now report that gene transfer of SEK-1(KR) to the adult rat heart blocks SAPK activation by pressure overload, demonstrating that the activity of cytosolic signaling pathways can be inhibited by gene transfer of loss-of-function mutants in vivo. Furthermore, gene transfer of SEK-1(KR) inhibited pressure overload-induced cardiac hypertrophy, as determined by echocardiography and several postmortem measures including left ventricular (LV) wall thickness, the ratio of LV weight to body weight, cardiomyocyte diameter, and inhibition of atrial natriuretic factor expression. Our data suggest that the SAPKs are critical regulators of cardiac hypertrophy in vivo, and therefore may serve as novel drug targets in the treatment of hypertrophy and heart failure.

The β-Catenin/T-Cell Factor/Lymphocyte Enhancer Factor Signaling Pathway Is Required for Normal and Stress-Induced Cardiac Hypertrophy

ABSTRACT In cells capable of entering the cell cycle, including cancer cells, β-catenin has been termed a master switch, driving proliferation over differentiation. However, its role as a transcriptional activator in terminally differentiated cells is relatively unknown. Herein we utilize conditional, cardiac-specific deletion of the β-catenin gene and cardiac-specific expression of a dominant inhibitory mutant of Lef-1 (Lef-1Δ20), one of the members of the T-cell factor/lymphocyte enhancer factor (Tcf/Lef) family of transcription factors that functions as a coactivator with β-catenin, to demonstrate that β-catenin/Tcf/Lef-dependent gene expression regulates both physiologic and pathological growth (hypertrophy) of the heart. Indeed, the profound nature of the growth impairment of the heart in the Lef-1Δ20 mouse, which leads to very early development of heart failure and premature death, suggests β-catenin/Tcf/Lef targets are dominant regulators of cardiomyocyte growth. Thus, our studies, employing complementary models in vivo, implicate β-catenin/Tcf/Lef signaling as an essential growth-regulatory pathway in terminally differentiated cells.

Deletion of cytosolic phospholipase A2 promotes striated muscle growth.

Generation of arachidonic acid by the ubiquitously expressed cytosolic phospholipase A2 (PLA2) has a fundamental role in the regulation of cellular homeostasis, inflammation and tumorigenesis. Here we report that cytosolic PLA2 is a negative regulator of growth, specifically of striated muscle. We find that normal growth of skeletal muscle, as well as normal and pathologic stress-induced hypertrophic growth of the heart, are exaggerated in Pla2g4a−/− mice, which lack the gene encoding cytosolic PLA2. The mechanism underlying this phenotype is that cytosolic PLA2 negatively regulates insulin-like growth factor (IGF)-1 signaling. Absence of cytosolic PLA2 leads to sustained activation of the IGF-1 pathway, which results from the failure of 3-phosphoinositide-dependent protein kinase (PDK)-1 to recruit and phosphorylate protein kinase C (PKC)-ζ, a negative regulator of IGF-1 signaling. Arachidonic acid restores activation of PKC-ζ, correcting the exaggerated IGF-1 signaling. These results indicate that cytosolic PLA2 and arachidonic acid regulate striated muscle growth by modulating multiple growth-regulatory pathways.

Activation of β-catenin Signaling Pathways by Classical G-Protein-Coupled Receptors: Mechanisms and Consequences in Cycling and Non-cycling Cells

Wnt signaling pathways are some of the most intensely studies in all of biology. Recently, a number of classical heterotrimeric G protein coupled receptors (GPCRs) have been shown to activate the canonical Wnt pathway, culminating in the stabilization of β-catenin and induction of transcription of genes regulated by the Tcf/Lef family of transactivators. However, mechanisms by which these GPCRs accomplish this differ in key ways, and in some circumstances, the phenotypes produced are novel. Herein, we will examine mechanisms by which classical GPCRs interact with the canonical Wnt pathway, culminating in its activation, and describe the consequences of this activation, focusing on the heart. In the heart, the contractile cells, or cardiomyocytes, are terminally differentiated and virtually exclusively grow by increasing cell size (hypertrophy) rather than cell number, and we will describe how GPCR-mediated activation of the canonical pathway can drive this process.