Angiotensin II-induced cardiomyocyte hypertrophy in vitro is TAK1-dependent and Smad2/3-independent

Cardiac hypertrophy occurs as an adaptation to hypertension but a sustained hypertrophic response can ultimately lead to heart failure. Angiotensin-II (Ang II) is released following hemodynamic overload and stimulates a cardiac hypertrophic response. AngII also increases expression of the regulatory cytokine, transforming growth factor-β1 (TGFβ1), which is also implicated in the cardiac hypertrophic response and can stimulate activation of Smad2/3 as well as TGFβ-activated kinase 1 (TAK1) signaling mediators. To better understand the downstream signaling events in cardiac hypertrophy, we therefore investigated activation of Smad2/3 and TAK1 signaling pathways in response to Ang II and TGFβ1 using primary neonatal rat cardiomyocytes to model cardiac hypertrophic responses. Small interfering RNA (siRNA) knockdown of Smad 2/3 or TAK1 protein or addition of the TGFβ type I receptor inhibitor, SB431542, were used to investigate the role of downstream mediators of TGFβ signaling in the hypertrophic response. Our data revealed that TGFβ1 stimulation leads to cardiomyocyte hypertrophic phenotypes that were indistinguishable from those occurring in response to Ang II. In addition, inhibition of the TGFβ1 type receptor abolished Ang II-induced hypertrophic changes. Furthermore, the hypertrophic response was also prevented following siRNA knockdown of TAK1 protein, but was unaffected by knockdown of Smad2/3 proteins. We conclude that Ang II-induced cardiomyocyte hypertrophy in vitro occurs in a TAK1-dependent, but Smad-independent, manner.     

The TIR/BB-loop mimetic AS-1 prevents cardiac hypertrophy by inhibiting IL-1R-mediated MyD88-dependent signaling

Activation of NF-κB contributes to cardiac hypertrophy and the interleukin-1 receptor (IL-1R)-mediated MyD88-dependent signaling pathway predominately activates NF-κB. Recent studies have shown that the TIR/BB-Loop mimetic (AS-1) disrupted the interaction of MyD88 with the IL-1R, resulting in blunting of NF-κB activation. We have examined the effects of AS-1 on the IL-1β-induced hypertrophic response using cultured neonatal cardiac myocytes in vitro and transverse aortic constriction (TAC) pressure overload-induced cardiac hypertrophy in vivo. Neonatal cardiac myocytes were treated with AS-1 15?min prior to IL-1β stimulation for 24?h. AS-1 treatment significantly attenuated IL-1β-induced hypertrophic responses of cardiac myocytes. In vivo experiments showed that AS-1 administration prevented cardiac hypertrophy and dysfunction induced by pressure overload. AS-1 administration disrupted the interaction of IL-1R with MyD88 in the pressure overloaded hearts and prevented activation of NF-κB. In addition, AS-1 prevented increases in activation of the MAPK pathway (p38 and p-ERK) in TAC-induced hypertrophic hearts. Our data suggest that the IL-1R-mediated MyD88-dependent signaling pathway plays a role in the development of cardiac hypertrophy and AS-1 attenuation of cardiac hypertrophy is mediated by blocking the interaction between IL-1R and MyD88, resulting in decreased NF-κB binding activity and decreased MAPK activation.     

PARP inhibition prevents postinfarction myocardial remodeling and heart failure via the protein kinase C/glycogen synthase kinase-3beta pathway

The inhibition of glycogen synthase kinase-3beta (GSK-3beta) via phosphorylation by Akt or protein kinase C (PKC), or the activation of mitogen-activated protein kinase (MAPK) cascades can play a pivotal role in left ventricular remodeling following myocardial infarction. Our previous data showed that MAPK and phosphatidylinositol-3-kinase/Akt pathways could be modulated by poly(ADP-ribose)polymerase (PARP) inhibition raising the possibility that cardiac hypertrophic signaling responses may be favorably influenced by PARP inhibitors. A novel PARP inhibitor (L-2286) was tested in a rat model of chronic heart failure following isoproterenol-induced myocardial infarction. Subsequently, cardiac hypertrophy and interstitial collagen deposition were assessed; additionally, mitochondrial enzyme activity and the phosphorylation state of GSK-3beta, Akt, PKC and MAPK cascades were monitored. PARP inhibitor (L-2286) treatment significantly reduced the progression of postinfarction heart failure attenuating cardiac hypertrophy and interstitial fibrosis, and preserving the integrity of respiratory complexes. More importantly, L-2286 repressed the hypertrophy-associated increased phosphorylation of panPKC, PKC alpha/betaII, PKC delta and PKC epsilon, which could be responsible for the activation of the antihypertrophic GSK-3beta. This work provides the first evidence that PARP inhibition beneficially modulates the PKC/GSK-3beta intracellular signaling pathway in a rat model of chronic heart failure identifying a novel drug target to treat heart failure.     

Atrial natriuretic peptide inhibits transforming growth factor beta-induced Smad signaling and myofibroblast transformation in mouse cardiac fibroblasts

This study tested the hypothesis that activation of atrial natriuretic peptide (ANP)/cGMP/protein kinase G signaling inhibits transforming growth factor (TGF)-beta1-induced extracellular matrix expression in cardiac fibroblasts and defined the specific site(s) at which this molecular merging of signaling pathways occurs. Left ventricular hypertrophy and fibrosis, collagen deposition, and myofibroblast transformation of cardiac fibroblasts in response to pressure overload by transverse aortic constriction were exaggerated in ANP-null mice compared with wild-type controls. ANP and cGMP inhibited TGF-beta1-induced myofibroblast transformation, proliferation, collagen synthesis, and plasminogen activator inhibitor-1 expression in cardiac fibroblasts isolated from wild-type mice. Following pretreatment with cGMP, TGF-beta1 induced phosphorylation of Smad3, but the resultant pSmad3 could not be translocated to the nucleus. pSmad3 that had been phosphorylated with recombinant protein kinase G-1alpha was analyzed by use of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ion trap tandem mass spectrometry. The analysis revealed phosphorylation of Ser309 and Thr388 residues, sites distinct from the C-terminal Ser423/425 residues that are phosphorylated by TGF-beta receptor kinase and are critical for the nuclear translocation and down-stream signaling of pSmad3. These results suggest that phosphorylation of Smad3 by protein kinase G is a potential molecular mechanism by which activation of ANP/cGMP/protein kinase G signaling disrupts TGF-beta1-induced nuclear translocation of pSmad3 and downstream events, including myofibroblast transformation, proliferation, and expression of extracellular matrix molecules in cardiac fibroblasts. We postulate that this process contributes to the antifibrogenic effects of the natriuretic peptide in heart.     

Important role of endogenous norepinephrine and epinephrine in the development of in vivo pressure-overload cardiac hypertrophy

OBJECTIVES: We sought to define the role of norepinephrine and epinephrine in the development of cardiac hypertrophy and to determine whether the absence of circulating catecholamines alters the activation of downstream myocardial signaling pathways. BACKGROUND: Cardiac hypertrophy is associated with elevated plasma catecholamine levels and an increase in cardiac morbidity and mortality. Although considerable evidence suggests that G-protein-coupled receptors are involved in the hypertrophic response, it remains controversial whether catecholamines are required for the development of in vivo cardiac hypertrophy. METHODS: We performed transverse aortic constriction (TAC) in dopamine beta-hydroxylase knockout mice (Dbh(-/-), genetically altered mice that are completely devoid of endogenous norepinephrine and epinephrine) and littermate control mice. After induction of cardiac hypertrophy, the mitogen-activated protein kinase (MAPK) signaling pathways were measured in pressure-overloaded/wild-type and Dbh(-/-) hearts. RESULTS: Compared with the control animals, cardiac hypertrophy was significantly blunted in Dbh(-/-) mice, which was not associated with altered cardiac function, as assessed by transthoracic echocardiography in conscious mice. The extracellularly regulated kinase (ERK 1/2), c-jun-NH(2)-terminal kinase (JNK) and p38 MAPK pathways were all activated by two- to threefold after TAC in the control animals. In contrast, induction of the three pathways (ERK 1/2, JNK and p38) was completely abolished in Dbh(-/-) mice. CONCLUSIONS: These data demonstrate a nearly complete requirement of endogenous norepinephrine and epinephrine for the induction of in vivo pressure-overload cardiac hypertrophy and for the activation of hypertrophic signaling pathways.     

Na(+)influx via Na(+)/H(+)exchange activates protein kinase C isozymes delta and epsilon in cultured neonatal rat cardiac myocytes.

Protein kinase C (PKC) is one of the important signaling molecules in the development of the cardiac hypertrophic response, and activation of Na(+)/H(+)exchange is caused by PKC in myocytes. In this study we examined the contribution of Na(+)/H(+)exchange in cardiac hypertrophy induced by the activation of PKC and its mechanism using cultured neonatal rat cardiac myocytes. Phenylephrine (PE), endothelin-1 (ET-1) and phorbol 12-myristate 13-acetate (PMA) increased cytoplasmic pH in myocytes, and this effect was strongly inhibited by treatment with HOE694, an inhibitor of Na(+)/H(+)exchange. These substances increased the [(3)H]phenylalanine incorporation, total protein content and beta -myosin heavy chain protein content in myocytes. These hypertrophic responses were also attenuated by HOE694. To clarify the role of Na(+)influx through activation of Na(+)/H(+)exchange in cardiac hypertrophy, we next examined the hypertrophic responses to veratridine and ouabain, which increase the intracellular Na(+)content. Veratridine and ouabain increased the [(3)H]phenylalanine incorporation. Staurosporine, a PKC inhibitor, completely abolished veratridine-induced hypertrophic response, but did not affect increment of intracellular Na(+)concentration by veratridine. PMA caused increases of alpha -, delta -and epsilon -PKC in the particulate fraction, but PE, ET-1 and veratridine affected only those of delta - and epsilon -PKC. HOE694 significantly inhibited only increases of delta - and epsilon -PKC caused by PE, ET-1 or PMA, but not those by veratridine. These results demonstrate that Na(+)influx via activation of Na(+)/H(+)exchange reactivates PKC in myocytes. delta - and epsilon -PKC appear to be involved in the signal mechanism of the hypertrophic response induced by Na(+)influx through Na(+)/H(+)exchange in myocytes.     

The A-kinase anchoring protein (AKAP)-Lbc-signaling complex mediates alpha1 adrenergic receptor-induced cardiomyocyte hypertrophy

In response to various pathological stresses, the heart undergoes a pathological remodeling process that is associated with cardiomyocyte hypertrophy. Because cardiac hypertrophy can progress to heart failure, a major cause of lethality worldwide, the intracellular signaling pathways that control cardiomyocyte growth have been the subject of intensive investigation. It has been known for more than a decade that the small molecular weight GTPase RhoA is involved in the signaling pathways leading to cardiomyocyte hypertrophy. Although some of the hypertrophic pathways activated by RhoA have now been identified, the identity of the exchange factors that modulate its activity in cardiomyocytes is currently unknown. In this study, we show that AKAP-Lbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor activity, is critical for activating RhoA and transducing hypertrophic signals downstream of alpha1-adrenergic receptors (ARs). In particular, our results indicate that suppression of AKAP-Lbc expression by infecting rat neonatal ventricular cardiomyocytes with lentiviruses encoding AKAP-Lbc-specific short hairpin RNAs strongly reduces both alpha1-AR-mediated RhoA activation and hypertrophic responses. Interestingly, alpha1-ARs promote AKAP-Lbc activation via a pathway that requires the alpha subunit of the heterotrimeric G protein G12. These findings identify AKAP-Lbc as the first Rho-guanine nucleotide exchange factor (GEF) involved in the signaling pathways leading to cardiomyocytes hypertrophy.     

TGF-beta1 and angiotensin networking in cardiac remodeling

The renin-angiotensin system (RAS) and transforming growth factor-beta1 (TGF-beta1) play a pivotal role in the development of cardiac hypertrophy and heart failure. Recent studies indicate that angiotensin II (Ang II) and TGF-beta1 do not act independently from one another but rather act as part of a signalling network in order to promote cardiac remodeling, which is a key determinant of clinical outcome in heart disease. This review focuses on recent advances in the understanding, how Ang II and TGF-beta1 are connected in the pathogenesis of cardiac hypertrophy and dysfunction. Increasing evidence suggests that at least some of the Ang II-induced effects on cardiac structure are mediated via indirect actions. Ang II upregulates TGF-beta1 expression via activation of the angiotensin type 1 (AT1) receptor in cardiac myocytes and fibroblasts, and induction of this cytokine is absolutely required for Ang II-induced cardiac hypertrophy in vivo. TGF-beta induces the proliferation of cardiac fibroblasts and their phenotypic conversion to myofibroblasts, the deposition of extracellular matrix (ECM) proteins such as collagen, fibronectin, and proteoglycans, and hypertrophic growth of cardiomyocytes, and thereby mediates Ang II-induced structural remodeling of the ventricular wall in an auto-/paracrine manner. Downstream mediators of cardiac Ang II/TGF-beta1 networking include Smad proteins, TGFbeta-activated kinase-1 (TAK1), and induction of hypertrophic responsiveness to beta-adrenergic stimulation in cardiac myocytes.     

PICOT inhibits cardiac hypertrophy and enhances ventricular function and cardiomyocyte contractility

Multiple signaling pathways involving protein kinase C (PKC) have been implicated in the development of cardiac hypertrophy. We observed that a putative PKC inhibitor, PICOT (PKC-Interacting Cousin Of Thioredoxin) was upregulated in response to hypertrophic stimuli both in vitro and in vivo. This suggested that PICOT may act as an endogenous negative feedback regulator of cardiac hypertrophy through its ability to inhibit PKC activity, which is elevated during cardiac hypertrophy. Adenovirus-mediated gene transfer of PICOT completely blocked the hypertrophic response of neonatal rat cardiomyocytes to enthothelin-1 and phenylephrine, as demonstrated by cell size, sarcomere rearrangement, atrial natriuretic factor expression, and rates of protein synthesis. Transgenic mice with cardiac-specific overexpression of PICOT showed that PICOT is a potent inhibitor of cardiac hypertrophy induced by pressure overload. In addition, PICOT overexpression dramatically increased the ventricular function and cardiomyocyte contractility as measured by ejection fraction and end-systolic pressure of transgenic hearts and peak shortening of isolated cardiomyocytes, respectively. Intracellular Ca(2+) handing analysis revealed that increases in myofilament Ca(2+) responsiveness, together with increased rate of sarcoplasmic reticulum Ca(2+) reuptake, are associated with the enhanced contractility in PICOT-overexpressing cardiomyocytes. The inhibition of cardiac remodeling by of PICOT with a concomitant increase in ventricular function and cardiomyocyte contractility suggests that PICOT may provide an efficient modality for treatment of cardiac hypertrophy and heart failure.     

Upregulation of TRPC1 in the development of cardiac hypertrophy

The importance of Ca(2+) entry in the cardiac hypertrophic response is well documented, but the actual Ca(2+) entry channels remain unknown. Transient receptor potential (TRP) proteins are thought to form either homo- or heteromeric Ca(2+) entry channels that are involved in the proliferation and differentiation of various cells. The purpose of this study was to explore the potential involvement of TRP channels in the development of cardiac hypertrophy. The mRNA and protein expression of several TRP channel subunits were evaluated using hearts from abdominal aortic-banded (AAB) rats. Although TRPs C1, C3, C5, and C6 were constitutively expressed, only TRPC1 expression was significantly increased in the hearts of AAB rats compared to sham-operated rats. Using primary cultures of neonatal rat cardiomyocytes, we detected increases in the expression of TRPC1, brain natriuretic peptide (BNP), and atrial natriuretic factor (ANF), as well as increases in store-operated Ca(2+) entry (SOCE) and cell surface area, following endothelin-1 (ET-1) treatment. Silencing of the TRPC1 gene via small interfering RNA (siRNA) attenuated SOCE and prevented ET-1-, angiotensin-II (AT II)-, and phenylephrine (PE)-induced cardiac hypertrophy. In HEK 293T cells, overexpression of TRPC1 augmented SOCE, leading to an increase in nuclear factor of activated T cells (NFAT) promoter activity, while co-transfection with dominant-negative forms of TRPC1 suppressed it. In conclusion, TRPC1 functions in Ca(2+) influx, and its upregulation is involved in the development of cardiac hypertrophy; moreover, it plays an important role in the regulation of the signaling pathways that govern cardiac hypertrophy. These findings establish TRPC1 as a functionally important regulator of cardiac hypertrophy.     

Asiatic acid inhibits cardiac hypertrophy by blocking interleukin-1β-activated nuclear factor-κB signaling in vitro and in vivo

Background

Activated interleukin (IL)-1β signaling pathway is closely associated with pathological cardiac hypertrophy. This study investigated whether asiatic acid (AA) could inhibit IL-1β-related hypertrophic signaling, and thus suppressing the development of cardiac hypertrophy.

Methods

Transverse aortic constriction (TAC) induced cardiac hypertrophy in C57BL/6 mice and cultured neonatal cardiac myocytes stimulated with IL-1β were used to evaluate the role of AA in cardiac hypertrophy. The expression of atrial natriuretic peptide (ANP) was evaluated by quantitative polymerase chain reaction (qPCR) and the nuclear factor (NF)-κB binding activity was measured by electrophoretic mobility shift assays (EMSA).

Results

AA pretreatment significantly attenuated the IL-1β-induced hypertrophic response of cardiomyocytes as reflected by reduction in the cardiomyocyte surface area and the inhibition of ANP mRNA expression. The protective effect of AA on IL-1β-stimulated cardiomyocytes was associated with the reduction of NF-κB binding activity. In addition, AA prevented TAC-induced cardiac hypertrophy in vivo. It was found that AA markedly reduced the excessive expression of IL-1β and ANP, and inhibited the activation of NF-κB in the hypertrophic myocardium.

Conclusions

Our data suggest that AA may be a novel therapeutic agent for cardiac hypertrophy. The inhibition of IL-1β-activated NF-κB signaling may be the mechanism through which AA prevents cardiac hypertrophy.     

Signaling Pathways Involved in Cardiac Hypertrophy

Cardiac hypertrophy is the heart's response to a variety of extrinsic and intrinsic stimuli that impose increased biomechanical stress. Traditionally, it has been considered a beneficial mechanism; however, sustained hypertrophy has been associated with a significant increase in the risk of cardiovascular disease and mortality. Delineating intracellular signaling pathways involved in the different aspects of cardiac hypertrophy will permit future improvements in potential targets for therapeutic intervention. Generally, there are two types of cardiac hypertrophies, adaptive hypertrophy, including eutrophy (normal growth) and physiological hypertrophy (growth induced by physical conditioning), and maladaptive hypertrophy, including pathologic or reactive hypertrophy (growth induced by pathologic stimuli) and hypertrophic growth caused by genetic mutations affecting sarcomeric or cytoskeletal proteins. Accumulating observations from animal models and human patients have identified a number of intracellular signaling pathways that characterized as important transducers of the hypertrophic response, including calcineurin/nuclear factor of activated T- cells, phosphoinositide 3-kinases/Akt (PI3Ks/Akt) , G protein-coupled receptors, small G proteins, MAPK, PKCs, Gpl30/STAT3, Na /H exchanger, peroxisome proliferator-activated receptors, myocyte enhancer factor 2/histone deacetylases, and many others. Furthermore, recent evidence suggests that adaptive cardiac hypertrophy is regulated in large part by the growth hormone/insulin-like growth factors axis via signaling through the PI3K/Akt pathway. In contrast, pathological or reactive hypertrophy is triggered by autocrine and paracrine neurohormonal factors released during biomechanical stress that signal through the Gq/phosphorlipase C pathway, leading to an increase in cytosolic calcium and activation of PKC.     

Natriuretic Peptide Signaling via Guanylyl Cyclase (GC)-A: An Endogenous Protective Mechanism of the Heart

Atrial and brain natriuretic peptides (ANP and BNP, respectively) are cardiac hormones, secretions of which are markedly upregulated during cardiac failure, making their plasma levels clinically useful diagnostic markers. ANP and BNP exert potent diuretic, natriuretic and vasorelaxant effects, which are mediated via their common receptor, guanylyl cyclase (GC)-A (also called natriuretic peptide receptor (NPR)-A). Mice deficient for GC-A are mildly hypertensive and show marked cardiac hypertrophy and fibrosis that is disproportionately severe, given their modestly higher blood pressure. Indeed, the cardiac hypertrophy seen in these mice is enhanced in a blood pressure-independent manner and is suppressed by cardiomyocyte-specific overexpression of GC-A. These results suggest that the actions of a local cardiac ANP/BNP-GC-A system are essential for maintenance of normal cardiac architecture. In addition, GC-A was shown to exert its cardioprotective effects by inhibiting angiotensin II-induced hypertrophic signaling, and recent evidence suggests that regulator of G protein signaling (RGS) subtype 4 is involved in the GC-A-mediated inhibition of Gαq-coupled hypertrophic signal transduction. Furthermore, several different groups have reported that functional mutations in the promoter region of the human GC-A gene are associated with essential hypertension and ventricular hypertrophy. These findings suggest that endogenous GC-A protects the heart from pathological hypertrophic stimuli, and that humans who express only low levels of GC-A are genetically predisposed to cardiac remodeling and hypertension.     

Identification of intracellular pathways through which TGF-beta1 upregulates PAI-1 expression in endothelial cells

Upregulation of plasminogen activator inhibitor type 1 (PAI-1) expression is a critical mechanism through which transforming growth factor-beta1 (TGF-beta1) accelerates intimal growth. The aim of this study was to identify signaling pathways through which TGF-beta1 upregulates PAI-1 expression in endothelial cells (EC) and test interventions for blocking these pathways. We transduced cultured bovine EC with an adenoviral vector containing the PAI-1 promoter fused to a beta-galactosidase reporter gene. We used these cells, along with vectors expressing potential modifiers of TGF-beta1 signaling and pharmacologic antagonists of mitogen-activated protein kinase (MAPK) pathways to identify key mediators of basal and TGF-beta1-regulated PAI-1 expression. Basal activity of the PAI-1 promoter was directly correlated with Ras activation and was blocked by a dominant negative (DN) type I TGF-beta receptor. TGF-beta1-stimulated activity of the PAI-1 promoter did not require Ras activation, and was lessened or eliminated by expression of either DN type I or type II TGF-beta receptors and by inhibition of either of two MAPKs: MEK and p38. Our results suggest unanticipated pathways of TGF-beta1 signaling in EC and point to new strategies to limit TGF-beta1-induced vascular disease.     

Role of FAK signaling in chagasic cardiac hypertrophy

Cardiac hypertrophy and dysfunction are a significant complication of chronic Chagas disease, with heart failure, stroke, and sudden death related to disease progression. Thus, understanding the signaling pathways involved in the chagasic cardiac hypertrophy may provide potential targets for pharmacological therapy. Herein, we investigated the implication of focal adhesion kinase (FAK) signaling pathway in triggering hypertrophic phenotype during acute and chronic T. cruzi infection. C57BL/6 mice infected with T. cruzi (Brazil strain) were evaluated for electrocardiographic (ECG) changes, plasma levels of endothelin-1 (ET-1) and activation of signaling pathways involved in cardiac hypertrophy, including FAK and ERK1/2, as well as expression of hypertrophy marker and components of the extracellular matrix in the different stages of T. cruzi infection (60–210 dpi). Heart dysfunction, evidenced by prolonged PR interval and decrease in heart rates in ECG tracing, was associated with high plasma ET-1 level, extracellular matrix remodeling and FAK signaling activation. Upregulation of both FAK tyrosine 397 (FAK-Y397) and serine 910 (FAK-S910) residues phosphorylation as well as ERK1/2 activation, lead to an enhancement of atrial natriuretic peptide gene expression in chronic infection. Our findings highlight FAK-ERK1/2 signaling as a regulator of cardiac hypertrophy in Trypanosoma cruzi infection. Both mechanical stress, induced by cardiac extracellular matrix (ECM) augment and cardiac overload, and ET-1 stimuli orchestrated FAK signaling activation with subsequent activation of the fetal cardiac gene program in the chronic phase of infection, highlighting FAK as an attractive target for Chagas disease therapy.