Venkatesh Rajapurohitam

The Obesity-Associated Peptide Leptin Induces Hypertrophy in Neonatal Rat Ventricular Myocytes

One of the major manifestations of obesity is increased production of the adipocyte-derived 16-kDa peptide leptin, which is also elevated in heart disease, including congestive heart failure. However, whether leptin can directly alter the cardiac phenotype is not known. We therefore studied the effect of leptin as a potential hypertrophic factor in cultured myocytes from 1- to 4-day-old neonatal rat heart ventricles. Using RT-PCR, we demonstrate that these cells express the short-form (OB-Ra) leptin receptor. Twenty-four hours of exposure to leptin (0.31 to 31.3 nmol/L) produces a significantly increased cell surface area that peaked at 0.63 nmol/L. Subsequent experiments were done with 3.1 nmol/L leptin, which significantly increased cell area by 42%, protein synthesis by 32%, and &agr;-skeletal actin and myosin light chain-2 expression by 250% and 300%, respectively. These events occurred in the absence of any increased cell death. Hypertrophy was preceded by rapid activation of the mitogen-activated protein kinase system including p38 and p44/42 as early as 5 minutes after leptin addition, whereas hypertrophy was inhibited by the p38 inhibitor SB203580 but not by the p44/42 inhibitor PD98059. Our results demonstrate a direct hypertrophic effect of leptin and may offer a biological link between hypertrophy and hyperleptinemic conditions such as obesity.

Signalling mechanisms underlying the metabolic and other effects of adipokines on the heart

Adipokines represent a family of proteins released by adipocytes that affect various biological processes including metabolism, satiety, inflammation, and cardiovascular function. The first adipokine to be identified is leptin, a product of the obesity gene whose primary function is to act as a satiety factor. However, it is now recognized that leptin and many of the newly discovered adipokines produce effects on numerous organ systems including the heart. Indeed, various adipokines including leptin, adiponectin, and apelin exert potent and diverse cardiovascular effects which are mediated by their specific receptors and involve complex and multifaceted cell-signalling pathways. Among these are effects on the heart as well as blood pressure where leptin has been proposed to potentially contribute to obesity-related hypertension. In this review, we focus primarily on the diverse effects of adipokines on the heart and discuss the potential cell-signalling mechanisms underlying their actions. The potential role of adipokines in the regulation of cardiac metabolism and function is discussed. Discussion is also presented on the emerging role, both deleterious and salutary, of various adipokines in heart disease with an examination of the possible underlying mechanisms which contribute to these effects.

Antihypertrophic Effect of Na+/H+ Exchanger Isoform 1 Inhibition Is Mediated by Reduced Mitogen-Activated Protein Kinase Activation Secondary to Improved Mitochondrial Integrity and Decreased Generation of Mitochondrial-Derived Reactive Oxygen Species

Although inhibition of Na+/H+ exchanger isoform 1 (NHE-1) reduces cardiomyocyte hypertrophy, the mechanisms underlying this effect are not known. Recent evidence suggests that this may be associated with improved mitochondrial function. To understand the mechanistic bases for mitochondrial involvement in the antihypertrophic effect of NHE-1 inhibition, we examined the effect of the NHE-1-specific inhibitor N-[2-methyl-4,5-bis(methylsulphonyl)-benzoyl]-guanidine, hydrochloride (EMD, EMD87580; 5 μM) on the hypertrophic phenotype, mitogen-activated protein kinase (MAPK) activity, mitochondrial membrane potential (Δψm), permeability transition (MPT) pore opening, and superoxide generation in phenylephrine (PE)-treated neonatal rat cardiomyocytes. EMD significantly suppressed markers of cell hypertrophy, including cell surface area and gene expression of atrial natriuretic peptide and α-skeletal actin. EMD inhibited the PE-induced MPT pore opening, prevented the loss in Δψm, and attenuated superoxide generation induced by PE. Moreover, the activation of p38 MAPK (p38) and extracellular signal-regulated kinase (ERK) 1/2 MAPKs induced by PE was significantly attenuated in the presence of EMD as well as the antioxidant catalase. To examine the role of MPT and mitochondrial Ca2+ uniport in parallel with EMD, the effects of cyclosporin A (0.2 μM) and ruthenium red (10 μM) were evaluated. Both agents significantly attenuated PE-induced hypertrophy and inhibited both mitochondrial dysfunction and p38 and ERK1/2 MAPK activation. Our results suggest a novel mechanism for attenuation of the hypertrophic phenotype by NHE-1 inhibition that is mediated by a reduction in PE-induced MAPK activation and superoxide production secondary to improved mitochondrial integrity.

Ginseng Inhibits Cardiomyocyte Hypertrophy and Heart Failure via NHE-1 Inhibition and Attenuation of Calcineurin Activation

Background—Ginseng is a medicinal plant used widely in Asia that has gained popularity in the West during the past decade. Increasing evidence suggests a therapeutic role for ginseng in the cardiovascular system. The pharmacological properties of ginseng are mainly attributed to ginsenosides, the principal bioactive constituents in ginseng. The present study was carried out to determine whether ginseng exerts a direct antihypertrophic effect in cultured cardiomyocytes and whether it modifies the heart failure process in vivo. Moreover, we determined the potential underlying mechanisms for these actions. Methods and Results—Experiments were performed on cultured neonatal rat ventricular myocytes as well as adult rats subjected to coronary artery ligation (CAL). Treatment of cardiomyocytes with the &agr;1 adrenoceptor agonist phenylephrine (PE) for 24 hours produced a marked hypertrophic effect as evidenced by significantly increased cell surface area and ANP gene expression. These effects were attenuated by ginseng in a concentration-dependent manner with a complete inhibition of hypertrophy at a concentration of 10 &mgr;g/mL. Phenylephrine-induced hypertrophy was associated with increased gene and protein expression of the Na+-H+ exchanger 1 (NHE-1), increased NHE-1 activity, increased intracellular concentrations of Na+ and Ca2+, enhanced calcineurin activity, increased translocation of NFAT3 into nuclei, and GATA-4 activation, all of which were significantly inhibited by ginseng. Upregulation of these systems was also evident in rats subjected to 4 weeks of CAL. However, animals treated with ginseng demonstrated markedly reduced hemodynamic and hypertrophic responses, which were accompanied by attenuation of upregulation of NHE-1 and calcineurin activity. Conclusions—Taken together, our results demonstrate a robust antihypertrophic and antiremodeling effect of ginseng, which is mediated by inhibition of NHE-1–dependent calcineurin activation.

Probiotic Administration Attenuates Myocardial Hypertrophy and Heart Failure After Myocardial Infarction in the Rat

Background—Probiotics are extensively used to promote gastrointestinal health, and emerging evidence suggests that their beneficial properties can extend beyond the local environment of the gut. Here, we determined whether oral probiotic administration can alter the progression of postinfarction heart failure. Methods and Results—Rats were subjected to 6 weeks of sustained coronary artery occlusion and administered the probiotic Lactobacillus rhamnosus GR-1 or placebo in the drinking water ad libitum. Culture and 16s rRNA sequencing showed no evidence of GR-1 colonization or a significant shift in the composition of the cecal microbiome. However, animals administered GR-1 exhibited a significant attenuation of left ventricular hypertrophy based on tissue weight assessment and gene expression of atrial natriuretic peptide. Moreover, these animals demonstrated improved hemodynamic parameters reflecting both improved systolic and diastolic left ventricular function. Serial echocardiography revealed significantly improved left ventricular parameters throughout the 6-week follow-up period including a marked preservation of left ventricular ejection fraction and fractional shortening. Beneficial effects of GR-1 were still evident in those animals in which GR-1 was withdrawn at 4 weeks, suggesting persistence of the GR-1 effects after cessation of therapy. Investigation of mechanisms showed a significant increase in the leptin:adiponectin plasma concentration ratio in rats subjected to coronary ligation, which was abrogated by GR-1. Metabonomic analysis showed differences between sham control and coronary artery ligated hearts particularly with respect to preservation of myocardial taurine levels. Conclusions—The study suggests that probiotics offer promise as a potential therapy for the attenuation of heart failure.

A neutralizing leptin receptor antibody mitigates hypertrophy and hemodynamic dysfunction in the postinfarcted rat heart

The 16 kDa adipokine leptin has been shown to exert direct hypertrophic effects on cultured cardiomyocytes although its role as an endogenous contributor to postinfarction remodeling and heart failure has not been determined. We therefore investigated the effect of leptin receptor blockade in vivo on hemodynamic function and cardiac hypertrophy following coronary artery ligation (CAL). Cardiac function and biochemical parameters were measured in rats subjected to 7 or 28 days of left main CAL in the presence and absence of a leptin receptor antibody. Animals subjected to an identical treatment in which the artery was not tied served as sham-operated controls. CAL produced myocardial hypertrophy, which was most pronounced 28 days postinfarction as demonstrated by increases in both left ventricular weight-to-body weight ratio and atrial natriuretic peptide gene expression, both of which were abrogated by leptin receptor antagonism. Leptin receptor blockade also significantly improved left ventricular systolic function, attenuated the increased left ventricular end-diastolic pressure, and reduced the expression of genes associated with extracellular matrix remodeling 28 days following CAL. In conclusion, the ability of a leptin receptor-neutralizing antibody to improve cardiac function offers evidence that endogenous leptin contributes to cardiac hypertrophy following CAL. The possibility exists that targeting the myocardial leptin receptor represents a viable and novel approach toward attenuating postinfarction remodeling.

Anti-hypertrophic effect of NHE-1 inhibition involves GSK-3β-dependent attenuation of mitochondrial dysfunction

Although Na(+)-H(+) exchanger 1 (NHE-1) inhibition has been demonstrated to have anti-hypertrophic effect indirectly through mitochondria, the detailed cellular mechanisms mediating this effect remain elusive. In this study we sought to determine whether NHE-1 inhibition exerts an anti-hypertrophic effect by modulating the mitochondrial permeability transition pore (mPTP) opening through the AMP-activated protein kinase (AMPK)/glycogen synthase kinase 3beta (GSK-3beta) pathway during hypertrophy in cardiomyocytes. An in vivo model of hypertrophy was induced in male Sprague-Dawley rats by subjecting them to 3, 7 or 28 days of coronary artery ligation (CAL). To induce hypertrophy in vitro, cardiomyocytes isolated from hearts of neonatal (1-3 days) Sprague-Dawley rats were exposed to endothelin-1 (ET-1, 10 nM) in the presence or absence of various treatments. The results demonstrate that CAL affected both AMPKalpha and GSK-3beta phosphorylation in a time-dependent manner. In cultured cardiomyocytes, ET-1 increased phosphorylation of AMPKalpha(1)/alpha(2)(Ser485/Ser491) and GSK-3beta(Ser9) by 80% (P<0.05) and 225% (P<0.05) respectively, both of which were significantly blunted by the NHE-1 inhibitor AVE-4890 (5 microM). ET-1-induced phosphorylation of GSK-3beta(Ser9) was attenuated by inhibitors of phosphatidylinositol 3-kinase (LY294002), Akt (Akt inhibitor VIII), ERK1/2 (PD98059) and by the AMPK agonist 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). Prevention of GSK-3beta(Ser9) phosphorylation was also accompanied by suppression of ET-1-induced increases in cell surface area, ANP and alpha-skeletal actin gene expression. Co-immunoprecipitation studies revealed that GSK-3beta interacts with components of the mPTP, voltage-dependent anion channel (VDAC) and adenine nucleotide translocase. Furthermore, ET-1 reduced phosphorylation of VDAC, which was associated with both mPTP opening and mitochondrial membrane depolarization. These effects were mimicked by the GSK-3beta inhibitor SB216763, thus showing that modulation of mPTP formation is GSK-3beta-dependent. In conclusion, anti-hypertrophic effect of NHE-1 inhibition can be mediated through activation of GSK-3beta which in turn induces inhibition of mPTP opening due to VDAC phosphorylation.

Actin Cytoskeleton Dynamics Promotes Leptin-Induced Vascular Smooth Muscle Hypertrophy via RhoA/ROCK- and Phosphatidylinositol 3-Kinase/Protein Kinase B-Dependent Pathways

Obesity is associated with increased leptin production that may contribute to cardiovascular pathology through a multiplicity of effects. Leptin has been shown to contribute to vascular remodeling through various mechanisms, including production of vascular smooth muscle (VSMC) hypertrophy; however, the mechanisms underlying the vascular hypertrophic effect of leptin remain unknown. In the present study, we investigated the contributions of the RhoA/Rho kinase (ROCK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathways, actin dynamics, and the expression of serum-response factor (SRF) in the hypertrophic effects of leptin on vascular tissue. Strips of rat portal vein (RPV) were cultured with or without leptin at 3.1 nM for 1 to 3 days. Leptin significantly increased RhoA activity by 163 ± 20%, whereas phosphorylation of downstream factors, including LIM kinase 1 and cofilin-2, was increased by 160 ± 25 and 290 ± 25%, respectively. Leptin also significantly phosphorylated Akt by 130 ± 30%, which was inhibited by the PI3K inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). RhoA/ROCK and PI3K/Akt activation was associated with a significant increase in RPV wet weight (11 ± 1%), protein synthesis (45 ± 7%), SRF expression (136 ± 11%), and polymerization of actin, as reflected by an increase in the F-/G-actin ratio, effects that were significantly attenuated by a leptin receptor (leptin obese receptor) antibody, the ROCK inhibitor (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) (Y-27632) as well as the PI3K inhibitor LY294002. Our results indicate that the activation of RhoA/ROCK and PI3K/Akt plays a pivotal role in leptin signaling, leading to the development of VSMC hypertrophy through a mechanism involving altered actin dynamics.

Nitric oxide inhibits endothelin-1-induced neonatal cardiomyocyte hypertrophy via a RhoA-ROCK-dependent pathway

Although nitric oxide (NO) has received extensive attention as an anti-hypertrophic agent the mechanisms underlying its regulation of endothelin-1 (ET-1) have not been fully elucidated. Since RhoA has been identified as an important mediator of cardiac hypertrophy and is inhibited by NO in vascular tissue, we sought to determine whether the anti-ET-1 effects of NO in cardiomyocytes were mediated via inhibition of the RhoA-ROCK cascade in the context of cardiac hypertrophy. Neonatal rat ventricular myocytes were cultured in the presence of ET-1 (10 nM) with or without pre-treatment with the NO donor S-nitroso-n-acetylpenicillamine (SNAP; 100 microM), 8-Br-cGMP (cGMP; 100 microM), the RhoA inhibitor C3 exoenzyme (C3; 30 ng/ml), or the ROCK inhibitor Y-27632 (10 microM). ET-1-induced cardiomyocyte hypertrophy was prevented by pre-treatment with SNAP, cGMP, C3, or Y-27632. The hypertrophic response to ET-1 was associated with significantly increased gene and protein expression of both NOS2 and NOS1 although NOS3 was unaffected. ET-1 treatment for 15 min increased membrane-bound RhoA 2.6-fold (p<0.05), which was prevented by both SNAP and cGMP (p<0.05). These effects were associated with a complete abrogation of ET-1-induced phosphorylation of the downstream target of RhoA, cofilin-2, that was mimicked by direct inhibition of RhoA and ROCK. In addition, confocal microscopy and Western blotting revealed that 24 h ET-1 treatment reduced the G- to F-actin ratio 67% (p<0.05) which was prevented by SNAP, cGMP, C3 and Y (p<0.05). Taken together, these results suggest that the anti-hypertrophic effects of NO are due, in part, to cGMP-dependent inhibition of the RhoA-ROCK-cofilin signalling pathway. These findings may be important in understanding the mechanisms of anti-ET-1 and anti-hypertrophic effects of NO as well as in the development of novel RhoA-targeted therapeutic interventions for treating cardiac hypertrophy.

A novel chimeric natriuretic peptide reduces cardiomyocyte hypertrophy through the NHE-1-calcineurin pathway

AIMS Natriuretic peptides (NPs) inhibit cardiomyocyte hypertrophy through a cyclic GMP (cGMP)-dependent process, although these effects are associated with substantial vasodilatation. In this study, we used CU-NP, a non-vasodilatating novel NP synthesized from the ring structure of human C-type NP (CNP) and both C- and N-termini of urodilatin, and investigated whether it can directly modulate cardiomyocyte hypertrophy. METHODS AND RESULTS Experiments were carried out in cultured neonatal rat ventricular myocytes exposed to phenylephrine, angiotensin II, or endothelin-1 in the absence or presence of CU-NP. CU-NP produced a concentration- and time-dependent increase in intracellular cGMP levels. The hypertrophic responses to all agonists were abrogated by 10 nM CU-NP. CU-NP treatment also prevented increased activity, gene and protein expression of sodium-hydrogen exchanger-1 (NHE-1) as well as elevations in intracellular Na(+) concentrations caused by hypertrophic agents. In addition, these effects were associated with a more than two-fold increase in activity of the Ca(2+)-dependent protein phosphatase calcineurin that peaked 6 h after addition of hypertrophic stimuli. Early (1-3 h) calcineurin activation was unaffected by CU-NP, although activation at 6 and 24 h was prevented by CU-NP as was the resultant translocation of the transcriptional factor NFAT into nuclei. CONCLUSION Our study demonstrates a direct anti-hypertrophic effect of the chimeric peptide CU-NP via NHE-1 inhibition, thereby preventing calcineurin activation and NFAT nuclear import. Thus, CU-NP represents a novel fusion peptide of CNP and urodilatin that has the potential to be developed into a therapeutic agent to treat cardiac hypertrophy and heart failure.