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Objective The transcription factor nuclear factor-kappaB (NF-kappaB) has been implicated in cardiomyocyte hypertrophy in vitro as well as in vivo; however, it is unknown if activation of NF-kappaB plays a mandatory role in the hypertrophic process. Here we characterize the importance of NF-kappaB signaling in moderate and severe left ventricular (LV) hypertrophy in rats with chronic pressure overload induced by angiotensin II (Ang II) infusion. Methods and results Electrophoretic mobility shift assay analysis revealed that Ang II infusion (2.5 microg/kg per min) for 6 days increased LV NF-kappaB/DNA-binding activity in a biphasic manner in Sprague-Dawley rats. Pyrrolidine dithiocarbamate (PDTC) (100 mg/kg per day), an NF-kappaB inhibitor, abolished Ang II-induced NF-kappaB activation and concomitant increase in tumor necrosis factor-alpha gene expression, while activator protein-1/DNA binding was not affected. Inhibition of NF-kappaB signaling for 6 days significantly attenuated Ang II-induced increases in LV/body weight ratio, LV mean wall thickness and cardiomyocyte cross-sectional area, without compromising LV systolic function. Moreover, PDTC abolished Ang II-induced cardiomyocyte apoptosis and interstitial fibrosis, and attenuated the gene expression of type I collagen. In contrast, a moderate LV hypertrophy induced by Ang II at a lower dose (0.5 microg/kg per min) was not associated with a significant activation of NF-kappaB, and PDTC treatment had no effect on the hypertrophic indices. Conclusion Our in-vivo data indicate a critical role of NF-kappaB signaling in the advanced stage of the remodeling process, whereas development of moderate LV hypertrophy is not dependent on NF-kappaB activation.

The transcription factors involved in the activation of cardiac gene expression by angiotensin II (Ang II) in vivo are not well understood. Here we studied the contribution of transcriptional elements to the activation of the cardiac B-type natriuretic peptide (BNP) gene promoter by Ang II in conscious rats and in angiotensin II type 1 receptor (AT1R) transgenic mice. Rat BNP luciferase reporter gene constructs were injected into the left ventricular wall. The mean luciferase activity was 1.8-fold higher (P

The Na(+)/K(+)-ATPase inhibitor ouabain has been shown to trigger hypertrophic growth of cultured cardiomyocytes; however, the significance of endogenous ouabain-like compound (OLC) in the hypertrophic process in vivo is unknown. Here we characterized the involvement of OLC in left ventricular (LV) hypertrophy induced by norepinephrine (NE) and angiotensin II (Ang II) infusions in rats. Administration of NE (300 microg/kg/h) via subcutanously implanted osmotic minipumps for 72 h resulted in a significant increase in left ventricular weight to body weight (LVW/BW) ratio (P0.001) and a substantial up-regulation of atrial natriuretic peptide (ANP) gene expression (13.2-fold, P0.001). NE infusion induced a transient increase in plasma OLC levels at 12 h (P0.05), which returned to control levels by 72 h. Adrenalectomy markedly reduced both basal and NE-induced increase in plasma OLC levels. LVW/BW ratio was not modulated by adrenalectomy; however, ANP gene expression was blunted by 44% (P0.01) and 47% (P0.05) at 12 and 72 h, respectively. In agreement, adrenalectomy reduced up-regulation of ANP without affecting LV mass in rats infused with Ang II (33 microg/kg/h). Administration of exogenous ouabain (1 nM to 100 microM) for 24 h had no effect on ANP gene expression in cultured neonatal rat ventricular myocytes. However, the up-regulation of ANP mRNA levels induced by the alpha-adrenergic agonist phenylephrine (1 microM) was markedly enhanced by ouabain (100 microM) (5.6-fold vs. 9.6-fold, P0.01). These data show that OLC as an adrenal-derived factor may be required for the induction LV ANP gene expression during the hypertrophic process.

Long-term dietary fatty acid intake alters the development of left ventricular hypertrophy, but the linking signaling pathways are unclear. We studied the role and underlying signaling mechanisms of dietary fat intake in the early phase of the hypertrophic process. Rats assigned for 4 weeks of high-oil, high-fat, or standard diet were subjected to angiotensin II (Ang II; 33 microg/kg/h, subcutaneous) or vehicle infusion for 24 h. The Ang II-induced increase in left ventricular mRNA levels of hypertrophy-associated genes was higher in rats fed the high-oil diet compared with the standard diet. Western blotting revealed that, in parallel with changes in gene expression, the high-oil diet increased c-Jun N-terminal kinase phosphorylation (P < 0.001). Ang II increased p38 mitogen-activated protein kinase (MAPK) phosphorylation in rats fed the high-fat diet (3-fold; P < 0.01). The increase in transcription factor activator protein-1 (AP-1) DNA binding activity in response to Ang II was higher in rats fed the high-oil diet compared with those fed the standard diet (P < 0.001). Ang II downregulated inducible nitric oxide synthase mRNA levels in fatty acid-supplemented groups compared with the standard diet group. These results show that dietary fat type modulates the early activation of hypertrophic genes in pressure-overloaded myocardium involving the distinct activation of AP-1 and MAPK signal transduction pathways.

To gain insight into the cardiac adaptive mechanisms in diabetes, we studied whether angiotensin II (Ang II) alters expression of the atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and adrenomedullin (AM) genes in the left ventricle of the diabetic rat heart.Diabetes was induced by streptozotocin (STZ; 60 mg/kg body weight intravenously). During the last 24 h of 2.5 or 7 weeks of treatment of male Wistar rats with STZ or vehicle, Ang II (33 microg/kg per h) was administered via osmotic minipumps.Diabetes was associated with an increased left ventricular weight to body weight (LV/BW) ratio, an index of left ventricular hypertrophy, at week 7 but not at week 2.5, and with increased ANP mRNA content at 2.5 weeks, but not with altered expression of the AM and BNP genes. Mean arterial pressure and LV/BW ratio were increased by Ang II in all groups except in the 7-week diabetic group. Levels of ANP mRNA were increased fourfold (P0.001) and threefold (P0.05) by Ang II at 2.5 and 7 weeks in control animals, respectively, and 11-fold (P0.001) and sevenfold (P0.001) at 2.5 and 7 weeks in diabetic animals, respectively. Ang II increased ventricular concentrations of BNP mRNA in control and diabetic animals at 2.5 weeks (1.3-fold, P0.001; and 1.6-fold, P0.001) and at 7 weeks (1.3-fold, P0.05; and 1.8-fold, P0.001), respectively. Left ventricular levels of adrenomedullin mRNA were increased by treatment with Ang II for 24 h in 2.5-week diabetic animals.Ang II markedly increased the levels of natriuretic peptide mRNAs in the left ventricle of normal and diabetic rat hearts, whereas it increased adrenomedullin mRNA levels only in 2.5-week diabetic rats and failed to cause hypertension in 7-week diabetic rats. Left ventricular levels of ANP and BNP mRNA were increased by Ang II in diabetic animals more than the additive effects of diabetes and Ang II alone, showing that Ang II induced an amplified response with respect to cardiac concentrations of ANP and BNP in diabetes.

Background— The precise function of angiotensin II type 2 receptor (AT 2 -R) in the mammalian heart in vivo is unknown. Here, we investigated the role of AT 2 -R in cardiac pressure overload. Methods and Results— Rats were infused with vehicle, angiotensin II (Ang II), PD123319 (an AT 2 -R antagonist), or the combination of Ang II and PD123319 via subcutaneously implanted osmotic minipumps for 12 or 72 hours. Ang II–induced increases in mean arterial pressure, left ventricular weight/body weight ratio, and elevation of skeletal α-actin and β-myosin heavy chain mRNA levels were not altered by PD123319. In contrast, AT 2 -R blockade resulted in a marked increase in the gene expression of c-fos, endothelin-1, and insulin-like growth factor-1 in Ang II–induced hypertension. In parallel, Ang II–stimulated mRNA and protein expression of atrial natriuretic peptide were significantly augmented by AT 2 -R blockade. Moreover, PD123319 markedly increased the synthesis of B-type natriuretic peptide. Furthermore, the expression of vascular endothelial growth factor and fibroblast growth factor-1 was downregulated by Ang II only in the presence of AT 2 -R blockade. Conclusions— Our results provide evidence that AT 2 -R plays a functional role in the cardiac hypertrophic process in vivo by selectively regulating the expression of growth-promoting and growth-inhibiting factors.

The orphan receptor APJ and its recently identified endogenous ligand, apelin, are expressed in the heart. However, their importance in the human cardiovascular system is not known. This study shows that apelin-like immunoreactivity is abundantly present in healthy human heart and plasma. Gel filtration HPLC analysis revealed that atrial and plasma levels of high molecular weight apelin, possibly proapelin, were markedly higher than those of mature apelin-36 itself. As assessed by quantitative RT-PCR analysis, left ventricular apelin mRNA levels were increased 4.7-fold in chronic heart failure (CHF) due to coronary heart disease (p

Background— Extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38-MAPK) have been shown to regulate various cellular processes, including cell growth, proliferation, and apoptosis in the heart. However, the function of these signaling pathways in the control of cardiac contractility is unclear. Here, we characterized the contribution of ERK1/2 and p38-MAPK to the inotropic effect of endothelin-1 (ET-1). Methods and Results— In isolated perfused rat hearts, infusion of ET-1 (1 nmol/L) for 10 minutes increased contractility and phosphorylation of ERK1/2 and their downstream target p90 ribosomal S6 kinase (p90RSK). Suppression of ERK1/2 activation prevented p90RSK phosphorylation and attenuated the inotropic effect of ET-1. Pharmacological inhibition of epidermal growth factor receptor kinase activity abolished ET-1–induced epidermal growth factor receptor transactivation and ERK1/2 and p90RSK phosphorylation and reduced ET-1–mediated inotropic response. Moreover, inhibition of the p90RSK target Na + -H + exchanger 1 attenuated the inotropic effect of ET-1. In contrast to ERK1/2 signaling, suppression of p38-MAPK activity further augmented ET-1–enhanced contractility, which was accompanied by increased phosphorylation of phospholamban at Ser-16. Conclusions— MAPKs play opposing roles in the regulation of cardiac contractility in that the ERK1/2-mediated positive inotropic response to ET-1 is counterbalanced by simultaneous activation of p38-MAPK. Hence, selective activation of ERK1/2 signaling and inhibition of p38-MAPK signaling may represent novel means to support cardiac function in disease.

The zinc finger transcription factor GATA-4 has been implicated as a critical regulator of inducible cardiac gene expression and as a potential mediator of the hypertrophic program. However, the precise intracellular mechanisms that regulate the DNA-binding activity of GATA-4 are not fully understood. The aim of the present study was to examine the role of mitogen-activated protein kinases (p38 kinase, extracellular signal-regulated protein kinase, and c-Jun N-terminal protein kinase) in the left ventricular wall stress-induced activation of GATA-4 DNA binding in adult heart. Isolated perfused rat hearts were subjected to increased left ventricular wall stress by inflating a balloon in the ventricle. Gel mobility shift assays were used to analyze the transacting factors that interact with the GATA motifs of the B-type natriuretic peptide promoter. The left ventricular wall stress rapidly activated GATA-4 DNA binding and significantly increased the levels of phosphorylated p38 kinase, extracellular signal-regulated protein kinase, and c-Jun N-terminal protein kinase. The wall stress-induced increase in the DNA-binding activity of GATA-4 was abolished both in the presence of the p38 inhibitor SB239063 and MEK1/2 inhibitor U0126. In contrast, the inhibition of c-Jun N-terminal protein kinase by CEP11004 had no effect on the baseline or stretch-induced GATA-4 DNA binding. Moreover, GATA-4 DNA binding was up-regulated by mechanical stretch in the isolated rat atria via p38 and extracellular signal-regulated protein kinase. In conclusion, the present study demonstrates that both p38 and extracellular signal-regulated protein kinase are required for the stretch-induced GATA-4 binding in intact heart.