Redox regulation of MAPK pathways and cardiac hypertrophy in adult rat cardiac myocyte

OBJECTIVES: We analyzed the regulatory function of reactive oxygen species (ROS) on the hypertrophic signaling in adult rat cardiac myocytes: BACKGROUND: The ROS regulate mitogenic signal transduction in various cell types. In neonatal rat cardiac myocyte, antioxidants have been shown to inhibit cardiac hypertrophy, and ROS are suggested to modulate the hypertrophic signaling. However, the conclusion may not reflect the situation of mature heart, because of the different natures between neonatal and adult cardiac myocytes. METHODS: Cultured adult rat cardiac myocytes were stimulated with endothelin-1 (ET-1) or phenylephrine (PE), and intracellular ROS levels, the activities of mitogen-activated protein kinases (MAPKs; ERK, p38, and JNK), and 3H-phenylalanine incorporation were examined. We also examined the effects of antioxidant pretreatment of myocytes on MAPK activities and cardiac hypertrophy to analyze the modulatory function of redox state on MAPK-mediated hypertrophic signaling. RESULTS: The ROS levels in ET-1- or PE-stimulated myocytes were maximally increased at 5 min after stimulation. The origin of ROS appears to be from NADH/NADPH oxidase, because the increase in ROS was suppressed by pretreatment of myocytes with NADH/NADPH oxidase inhibitor diphenyleneiodonium. Extracellular signal-regulated kinase (ERK) activity was increased by the stimulation of ET-1 or PE. In contrast, p38 and c-Jun-N-terminal protein kinase (JNK) activities did not change after these stimulations. Antioxidant treatment of myocytes suppressed the increase in ROS and blocked ERK activation and the subsequent cardiac hypertrophy induced by these stimuli. CONCLUSIONS: These data demonstrate that ROS mediate signal transduction of cardiac hypertrophy induced by ET-1 or PE in adult rat cardiac myocytes.     

Aldosterone stimulates matrix metalloproteinases and reactive oxygen species in adult rat ventricular cardiomyocytes

Matrix metalloproteinases (MMPs), aldosterone, and reactive oxygen species (ROS) are implicated in myocardial remodeling. Although ROS, cytokines, and neurohormones regulate MMP in cardiac fibroblasts, it is unknown whether aldosterone regulates MMP in cardiomyocytes. Therefore, we tested the hypothesis that aldosterone regulates MMP in cultured adult rat ventricular myocytes (ARVMs). ARVMs were treated with aldosterone for 24 hours, and MMP-2 and MMP-9 activities were measured by zymography. Aldosterone (50 nmol/L) increased MMP-2 (43+/-5%) and MMP-9 (55+/-15%; P<0.001 for both) activities. Pretreatment with spironolactone (100 nmol/L) abolished the aldosterone-induced increase in MMP activities. Aldosterone (50 nmol/L; 30 minutes) increased mitogen/extracellular signal-regulated kinase (MEK) (31+/-3%) and extracellular signal-regulated kinase 1/2 (ERK1/2; 41+/-7%; P<0.001 for both) phosphorylation. U0126 (10 micromol/L), an MEK1/2 inhibitor, abolished the aldosterone-induced increase in MMP activities. Aldosterone increased intracellular ROS as assessed by dichlorofluorescein diacetate (27+/-4%; P<0.05). This increase was inhibited by apocynin, an NADPH oxidase inhibitor. Apocynin likewise inhibited aldosterone-induced ERK1/2 phosphorylation and the increase in MMP activities. Furthermore, the antioxidants MnTMPyP and N-acetylcysteine inhibited the aldosterone-induced increase in ERK1/2 phosphorylation and MMP activities, respectively. Protein kinase C (PKC) is implicated in the nongenomic effects of aldosterone. To test the role of PKC, ARVMs were pretreated with chelerythrine, a PKC inhibitor. Chelerythrine prevented the aldosterone-induced increase in ERK1/2 phosphorylation and MMP activities. Thus, aldosterone induces MMP activity in ARVM via activation of the mineralocorticoid receptor, PKC, and ROS-dependent activation of the MEK/ERK pathway. NADPH oxidase is a likely source of ROS in this system.     

Aldosterone stimulates the cardiac Na(+)/H(+) exchanger via transactivation of the epidermal growth factor receptor

The use of antagonists of the mineralocorticoid receptor in the treatment of myocardial hypertrophy and heart failure has gained increasing importance in the last years. The cardiac Na(+)/H(+) exchanger (NHE-1) upregulation induced by aldosterone could account for the genesis of these pathologies. We tested whether aldosterone-induced NHE-1 stimulation involves the transactivation of the epidermal growth factor receptor (EGFR). Rat ventricular myocytes were used to measure intracellular pH with epifluorescence. Aldosterone enhanced the NHE-1 activity. This effect was canceled by spironolactone or eplerenone (mineralocorticoid receptor antagonists), but not by mifepristone (glucocorticoid receptor antagonist) or cycloheximide (protein synthesis inhibitor), indicating that the mechanism is mediated by the mineralocorticoid receptor triggering nongenomic pathways. Aldosterone-induced NHE-1 stimulation was abolished by the EGFR kinase inhibitor AG1478, suggesting that is mediated by transactivation of EGFR. The increase in the phosphorylation level of the kinase p90(RSK) and NHE-1 serine703 induced by aldosterone was also blocked by AG1478. Exogenous epidermal growth factor mimicked the effects of aldosterone on NHE-1 activity. Epidermal growth factor was also able to increase reactive oxygen species production, and the epidermal growth factor-induced activation of the NHE-1 was abrogated by the reactive oxygen species scavenger N-2-mercaptopropionyl glycine, indicating that reactive oxygen species are participating as signaling molecules in this mechanism. Aldosterone enhances the NHE-1 activity via transactivation of the EGFR, formation of reactive oxygen species, and phosphorylation of the exchanger. These results call attention to the consideration of the EGFR as a new potential therapeutic target of the cardiovascular pathologies involving the participation of aldosterone.     

Alpha1-adrenergic stimulation induced hypertrophy in protein kinase C down-regulated cultured cardiac myocytes

To examine whether protein kinase C (PKC) activation is essential for the induction of cardiac myocyte hypertrophy caused by alpha1-adrenergic stimulation, we investigated the hypertrophic effect of phenylephrine in PKC down-regulated and non-treated cultured cardiac myocytes obtained from neonatal Sprague-Dawley rat ventricles. The treatment with 10 nmol/L 12-tetra decanoylphorbol-13-acetate (TPA) for more than 2 hours decreased PKC activity by approximately 80% without marked hypertrophy. Phenylephrine increased [14C] phenylalanine (Phe) incorporation in both TPA non-treated and treated cells, 1.54- and 1.71-fold as large as control, respectively. The cell surface area also enlarged in both groups, 1.67- and 1.74-fold, respectively. Thus, phenylephrine induced the similar grade hypertrophy in cultured cardiac myocytes even when PKC was down-regulated. These results suggest that conventional PKC activation may not be essential for mediating myocyte hypertrophy by alpha1-adrenergic stimulation.     

Raloxifene prevents cardiac hypertrophy and dysfunction in pressure-overloaded mice

17beta-estradiol reduces myocardial hypertrophy and left ventricular mass, suggesting that the selective estrogen receptor modulator raloxifene may have similar effects. However, it is not clear whether raloxifene inhibits both cardiac hypertrophy and dysfunction. We used transverse aortic-banded mice to produce pressure-overload cardiac hypertrophy and used neonatal rat ventricular cardiomyocytes to investigate the cellular mechanisms of raloxifene on cardiac hypertrophy. Left ventricular mass and fractional shortening of mice hearts were measured by transthoracic echocardiography. Protein synthesis of cardiomyocytes was evaluated by incorporation of [3H]leucine into cardiomyocytes exposed to angiotensin II. Phosphorylation of mitogen-activated protein (MAP) kinase was also observed in cardiomyocytes. Raloxifene prevented increases in left ventricular mass and decreases of fractional shortening at 4 weeks after aortic banding. Pretreatment with raloxifene before angiotensin II stimulation inhibited the increase in [3H]leucine incorporation into neonatal rat cardiomyocytes in a concentration-dependent manner. This inhibition was partially but not significantly attenuated by N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, and completely abolished by ICI182780, an estrogen receptor antagonist. Although the phosphorylation of p38 MAP kinase, c-Jun N-terminal kinase (JNK), or extracellular signal-regulated protein kinase (ERK) in cardiomyocytes was significantly increased by angiotensin II stimulation as compared with the control, pretreatment with raloxifene attenuated p38 MAP kinase phosphorylation, but neither JNK nor ERK phosphorylation. We conclude that raloxifene inhibits cardiac hypertrophy and dysfunction and that the inhibition of p38 MAP kinase phosphorylation after the stimulation of estrogen receptors may be involved in the cellular mechanisms of this agent.     

H(2)O(2) regulates cardiac myocyte phenotype via concentration-dependent activation of distinct kinase pathways

Reactive oxygen species (ROS) can act as signaling molecules to stimulate either hypertrophy or apoptosis in cardiac myocytes. We tested the hypothesis that the phenotypic effects of ROS are due to differential, concentration-dependent activation of specific kinase signaling pathways. Adult rat ventricular myocytes were exposed to H(2)O(2) over a broad concentration range (10-1000 microM). Low concentrations of H(2)O(2) (10-30 microM) increased protein synthesis without affecting survival. Higher concentrations of H(2)O(2) (100-200 microM) increased apoptosis (assessed by TUNEL). Still higher concentrations of H(2)O(2) (300-1000 microM) caused both apoptosis and necrosis. A hypertrophic concentration of H(2)O(2) (10 microM) increased the activity of ERK1/2, but not that of JNK, p38 kinase or Akt. An apoptotic concentration of H(2)O(2) (100 microM) activated JNK, p38 kinase and Akt, and further activated ERK1/2. The MEK1/2 inhibitor U0126 prevented the hypertrophic effect of 10 microM H(2)O(2). The apoptotic effect of 100 microM H(2)O(2) was inhibited bya dominant-negative JNK adenovirus, and was potentiated by U0126 or an Akt inhibitor. Thus, the concentration-dependent effects of ROS on myocyte hypertrophy and growth are due, at least in part, to the differential activation of specific kinase signaling pathways that regulate hypertrophy and apoptosis.     

α1-Adrenergic Stimulation Induced Hypertrophy in Protein Kinase C Down-Regulated Cultured Cardiac Myocytes

To examine whether protein kinase C (PKC) activation is essential for the induction of cardiac myocyte hypertrophy caused by α₁-adrenergic stimulation, we investigated the hypertrophic effect of phenylephrine in PKC down-regulated and non-treated cultured cardiac myocytes obtained from neonatal Sprague-Dawley rat ventricles. The treatment with 10 nmol/L 12-tetra decanoylphorbol-13-acetate (TPA) for more than 2 hours decreased PKC activity by approximately 80% without marked hypertrophy. Phenylephrine increased [¹⁴C] phenylalanine (Phe) incorporation in both TPA non-treated and treated cells, 1.54- and 1.71-fold as large as control, respectively. The cell surface area also enlarged in both groups, 1.67- and 1.74-fold, respectively. Thus, phenylephrine induced the similar grade hypertrophy in cultured cardiac myocytes even when     

Nongenomic effects of aldosterone in the human heart: interaction with angiotensin II

Aldosterone exerts rapid "nongenomic" effects in various nonrenal tissues. Here, we investigated whether such effects occur in the human heart. Trabeculae and coronary arteries obtained from 57 heart valve donors (25 males; 32 females; 17 to 66 years of age) were mounted in organ baths. Aldosterone decreased contractility in atrial and ventricular trabeculae by maximally 34+/-3% and 15+/-4%, respectively, within 5 to 15 minutes after its application. The protein kinase C (PKC) inhibitor chelerythrine chloride, but not the mineralocorticoid receptor antagonists spironolactone and eplerenone, blocked this effect. Aldosterone also relaxed trabeculae that were prestimulated with angiotensin II (Ang II), and its negative inotropic effects were mimicked by hydrocortisone (at 10-fold lower potency) but not 17beta-estradiol. Aldosterone concentrations required to reduce inotropy were present in failing but not in normal human hearts. Previous exposure of coronary arteries to 1 micromol/L aldosterone or 17beta-estradiol (but not hydrocortisone) doubled the maximum contractile response (Emax) to Ang II. DeltaEmax correlated with extracellular signal-regulated kinase (ERK) 1/2 phosphorylation (P<0.01). Spironolactone and eplerenone did not block the potentiating effect of aldosterone. Studies in porcine renal arteries showed that potentiation also occurred at pmol/L aldosterone levels but not at 17beta-estradiol levels <1 micromol/L. Aldosterone did not potentiate the alpha1-adrenoceptor agonist phenylephrine. In conclusion, aldosterone induces a negative inotropic response in human trabeculae (thereby antagonizing the positive inotropic actions of Ang II) and potentiates the vasoconstrictor effect of Ang II in coronary arteries. These effects are specific and involve PKC and ERK 1/2, respectively. Furthermore, they occur in a nongenomic manner, and require pathological aldosterone concentrations.     

Autonomous and growth factor-induced hypertrophy in cultured neonatal mouse cardiac myocytes. Comparison with rat

Cultured neonatal rat cardiac myocytes have been used extensively to study cellular and molecular mechanisms of cardiac hypertrophy. However, there are only a few studies in cultured mouse myocytes despite the increasing use of genetically engineered mouse models of cardiac hypertrophy. Therefore, we characterized hypertrophic responses in low-density, serum-free cultures of neonatal mouse cardiac myocytes and compared them with rat myocytes. In mouse myocyte cultures, triiodothyronine (T3), norepinephrine (NE) through a beta-adrenergic receptor, and leukemia inhibitory factor induced hypertrophy by a 20% to 30% increase in [(3)H]phenylalanine-labeled protein content. T3 and NE also increased alpha-myosin heavy chain (MyHC) mRNA and reduced beta-MyHC. In contrast, hypertrophic stimuli in rat myocytes, including alpha(1)-adrenergic agonists, endothelin-1, prostaglandin F(2alpha), interleukin 1beta, and phorbol 12-myristate 13-acetate (PMA), had no effect on mouse myocyte protein content. In further contrast with the rat, none of these agents increased atrial natriuretic factor or beta-MyHC mRNAs. Acute PMA signaling was intact by extracellular signal-regulated kinase (ERK1/2) and immediate-early gene (fos/jun) activation. Remarkably, mouse but not rat myocytes had hypertrophy in the absence of added growth factors, with increases in cell area, protein content, and the mRNAs for atrial natriuretic factor and beta-MyHC. We conclude that mouse myocytes have a unique autonomous hypertrophy. On this background, T3, NE, and leukemia inhibitory factor activate hypertrophy with different mRNA phenotypes, but certain Gq- and protein kinase C-coupled agonists do not.     

Aldosterone stimulates collagen gene expression and synthesis via activation of ERK1/2 in rat renal fibroblasts

Recently, we demonstrated that in rats treated chronically with aldosterone and salt, severe tubulointerstitial fibrosis is associated with the activation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERK1/2). Here, we investigated whether aldosterone stimulates collagen synthesis via ERK1/2-dependent pathways in cultured rat renal fibroblasts. Gene expression of mineralocorticoid receptor (MR) and types I, II, III, and IV collagen was measured by real-time polymerase chain reaction (PCR). MR protein expression and ERK1/2 activity were evaluated by Western blotting analysis with anti-MR and anti-phospho-ERK1/2 antibodies, respectively. Collagen synthesis was determined by [3H]-proline incorporation. Significant levels of MR mRNA and protein expression were observed in rat renal fibroblasts. Treatment with aldosterone (0.1 to 10 nmol/L) increased ERK1/2 phosphorylation in a concentration-dependent manner with a peak at 5 minutes. Aldosterone (10 nmol/L) also increased the mRNA levels of types I, III, and IV collagen at 36 hours but had no effect on the type II collagen mRNA level. [3H]-proline incorporation was significantly increased by aldosterone in both the medium and cell layer at 48 hours. Aldosterone-induced ERK1/2 phosphorylation was markedly attenuated by pretreatment with eplerenone (10 micromol/L), a selective MR antagonist, or PD98059 (10 micromol/L), a specific inhibitor of MAPK kinase/ERK kinase, which is the upstream activator of ERK1/2. In addition, both eplerenone and PD98059 prevented the aldosterone-induced increases in types I, III, and IV collagen mRNA and [3H]-proline incorporation. These results suggest that aldosterone stimulates collagen gene expression and synthesis via MR-mediated ERK1/2 activation in renal fibroblasts, which may contribute to the progression of aldosterone-induced tubulointerstitial fibrosis.     

c-Src-dependent nongenomic signaling responses to aldosterone are increased in vascular myocytes from spontaneously hypertensive rats

Aldosterone plays an important role in the pathogenesis of hypertension. We previously demonstrated that nongenomic signaling by aldosterone in vascular smooth muscle cells occurs through c-Src-dependent pathways. Here we tested the hypothesis that upregulation of c-Src by aldosterone plays a role in increased mitogen-activated protein (MAP) kinase activation, [3H]-proline incorporation, and NADPH-driven generation of reactive oxygen species, thereby inducing cell growth, collagen production, and inflammation, respectively, in vascular smooth muscle cells from spontaneously hypertensive rats. The time course of c-Src phosphorylation by aldosterone was shifted to the left in vascular myocytes from hypertensive animals. Aldosterone rapidly increased phosphorylation of p38 MAP kinase and extracellular signal-regulated kinase with significantly greater effects in cells from spontaneously hypertensive rats versus control cells (P<0.05). Aldosterone increased NADPH oxidase activity with significantly greater responses in vascular smooth muscle cells from hypertensive animals (P<0.05). These events were associated with enhanced [3H]proline incorporation (index of collagen synthesis) in cells from spontaneously hypertensive rats (P<0.05). The NADPH oxidase activity increase, collagen synthesis, c-Src, and MAP kinase phosphorylation induced by aldosterone were significantly reduced by eplerenone (selective mineralocorticoid receptor blocker) and PP2 (selective c-Src inhibitor). In conclusion, nongenomic signaling by exogenous aldosterone, mediated through c-Src, is increased in vascular smooth muscle cells from spontaneously hypertensive rats. Upregulation of c-Src signaling may be important in the profibrotic and proinflammatory actions of aldosterone in this genetic model of hypertension.     

Aldosterone activates vascular p38MAP kinase and NADPH oxidase via c-Src

Increasing evidence indicates that aldosterone elicits vascular effects through nongenomic signaling pathways. We tested the hypothesis that aldosterone induces activation of vascular mitogen-activated protein (MAP) kinases and NADPH oxidase via c-Src-dependent mechanisms in vascular smooth muscle cells (VSMCs). Aldosterone effects on activation of c-Src, p38MAP kinase, and NADPH oxidase, and incorporation of [3H]proline, an index of collagen synthesis, were assessed in cultured rat VSMCs. Studies were performed in the absence and presence of eplerenone, a selective mineralocorticoid receptor blocker, PP2, a selective Src inhibitor, and SB212190, a selective p38MAPK inhibitor. Phosphorylation of c-Src was dose-dependently increased by aldosterone, with maximal responses obtained at 10(-7) mol/L. Aldosterone increased p38MAP kinase phosphorylation, NAD(P)H oxidase activation, and [3H]proline incorporation. These responses were abrogated by eplerenone and almost abolished by PP2. Aldosterone-stimulated incorporation of [3H]proline was significantly reduced by SB212190, indicating that p38MAP kinase plays a role in profibrotic actions of aldosterone. To unambiguously demonstrate the importance of aldosterone in c-Src signaling, VSMCs from c-Src+/+ and c-Src+/- mice were also studied. Aldosterone increased phosphorylation of c-Src, p38MAP kinase, and cortactin, a Src-specific substrate, in c-Src+/+ VSMCs, but not in c-Src-deficient cells. Taken together, our findings demonstrate that nongenomic signaling by aldosterone occurs through c-Src-dependent pathways. These processes may play an important role in profibrotic actions of aldosterone.     

Expression and activity of protein kinase D/protein kinase C mu in myocardium: evidence for alpha1-adrenergic receptor- and protein kinase C-mediated regulation

Protein kinase D (PKD), which is also known as protein kinase C (PKC) mu, is a novel serine/threonine kinase that can be activated in parallel with or downstream of PKC in various cell types, but its expression and regulation in myocardium have not been characterized. In the present study, two proteins of 110 and 115 kDa were detected in rat ventricular myocardium using antibodies directed at the extreme N- or C-terminus of PKD. Both proteins were highly expressed in the fetal heart but showed a developmental decline in abundance. Fractionation studies showed that PKD was distributed between myocyte and non-myocyte fractions in the neonatal heart, but was found predominantly in the non-myocyte fraction in the adult heart. In cultured neonatal rat ventricular myocytes, an in vitro kinase assay revealed increased autophosphorylation of PKD (EC50 2.8 nM) in response to phorbol-12-myristate-13-acetate (PMA). Exposure to norepinephrine also induced a dose-dependent increase in PKD autophosphorylation (EC50 0.6 microM). Pretreatment with the alpha1-adrenergic receptor (AR) antagonist prazosin blocked norepinephrine-induced PKD autophosphorylation, while the beta1-AR antagonist atenolol had no effect, indicating that activation of PKD by norepinephrine occurred via the alpha1-AR. Involvement of the alpha1-AR was confirmed by exposure of myocytes to the alpha1-AR agonist phenylephrine, which induced a similar profile of PKD autophosphorylation to norepinephrine (EC50 0.6 microM). The effects of both alpha1-AR stimulation and PMA on PKD autophosphorylation were mediated by PKC, since these effects could be attenuated by pretreatment of myocytes with the PKC inhibitor bisindolylmaleimide. These data show that PKD is expressed in rat ventricular myocardium, where its expression is subject to developmental control, and that PKD activity in ventricular myocytes is regulated through alpha1-AR- and PKC-mediated pathways.     

Aldosterone increases NHE-1 expression and induces NHE-1-dependent hypertrophy in neonatal rat ventricular myocytes

We determined the effect of 24-hour aldosterone (100 nmol/L) treatment on hypertrophic responses in rat neonatal ventricular myocytes and the possible role of Na+-H+ exchange isoform 1 (NHE-1). Aldosterone significantly increased cell size by 61% and expression of atrial natriuretic peptide by 2-fold. NHE-1 mRNA expression and protein abundance were significantly increased, and intracellular Na+ levels were elevated. Both hypertrophy and elevated Na+ levels were prevented by the NHE-1-specific inhibitor EMD87580 as well as the aldosterone antagonist spironolactone, although the increased NHE-1 levels were prevented only by spironolactone. Aldosterone transiently (within 5 minutes) stimulated p44/42 phosphorylation, which decreased thereafter for the remaining 24 hours, whereas p38 phosphorylation was reduced. Neither a p38 nor a p44/42 inhibitor had any effect on aldosterone-induced hypertrophy or NHE-1 regulation. Our results therefore demonstrate a direct hypertrophic effect of aldosterone on cultured myocytes, which is dependent on NHE-1 activity.     

MEK1/2-ERK1/2 mediates alpha1-adrenergic receptor-stimulated hypertrophy in adult rat ventricular myocytes

We examined the relative roles of the mitogen-activated protein kinases (MAPK) in mediating the alpha1-adrenergic receptor (alpha1-AR) stimulated hypertrophic phenotype in adult rat ventricular myocytes (ARVM). Norepinephrine (NE; 1 microM) in the presence of the beta -AR antagonist propranolol (Pro; 2 microM) caused activation of Ras (>six-fold), MAPK/ERK kinase 1 and 2 (MEK1/2, >10-fold) and extracellular signal-regulated kinases 1 and 2 (ERK1/2, approximately 30-fold) within 5 min, as determined by kinase activity assays and Western blots using phospho-specific antibodies. Conversely, p38 and c-Jun amino-terminal kinases (JNK) were not activated by NE/Pro. Activated MEK1/2 signals remained detectable at 2 h, and activated ERK1/2 remained detectable at 48 h. The alpha1-AR selective inhibitor prazosin (100 nM) completely inhibited the NE/Pro-stimulated activation of Ras, MEK1/2 and ERK1/2. The MEK inhibitor PD98059 caused a concentration-dependent inhibition of NE/Pro-stimulated protein synthesis (as assessed by [3H]leucine incorporation and cellular protein accumulation) and ERK1/2 activation, with approximately 50% inhibition at a concentration between 10 and 50 microM, which is consistent with the known IC50 values of PD98059 for MEK1 (4 microM) and MEK2 (50 microM). Thus, these data show that alpha1-AR stimulated hypertrophy in ARVM is dependent on the MEK1/2-ERK1/2 signaling pathway.