Krishna Singh

Norepinephrine Stimulates Apoptosis in Adult Rat Ventricular Myocytes by Activation of the β-Adrenergic Pathway

BACKGROUND Myocardial sympathetic activity is increased in heart failure. We tested the hypothesis that norepinephrine (NE) stimulates apoptosis in adult rat ventricular myocytes in vitro. METHODS AND RESULTS Myocytes were exposed to NE alone (10 micromol/L), NE+propranolol (2 micromol/L), NE+prazosin (0.1 micromol/L), or isoproterenol (ISO, 10 micromol/L) for 24 hours. NE and ISO decreased the number of viable myocytes by approximately 35%. This effect was completely blocked by the beta-adrenergic antagonist propranolol but was not affected by the alpha1-adrenergic antagonist prazosin. NE increased DNA laddering on agarose gel electrophoresis and increased the percentage of cells that were stained by terminal deoxynucleotidyl transferase-mediated nick end-labeling from 5.8+/-1. 0% to 21.0+/-2.3% (P<0.01; n=4). NE likewise increased the percentage of apoptotic cells with hypodiploid DNA content as assessed by flow cytometry from 7.8+/-0.7% to 16.7+/-2.2% (P<0.01; n=6), and this effect was abolished by propranolol but not prazosin. ISO and forskolin (10 micromol/L) mimicked the effect of NE, increasing the percentage of apoptotic cells to 14.7+/-1.9% and 14. 4+/-2.2%, respectively. NE-stimulated apoptosis was abolished by the protein kinase A inhibitor H-89 (20 micromol/L) or the voltage-dependent calcium channel blockers diltiazem and nifedipine. CONCLUSIONS NE, acting via the ss-adrenergic pathway, stimulates apoptosis in adult rat cardiac myocytes in vitro. This effect is mediated by protein kinase A and requires calcium entry via voltage-dependent calcium channels. NE-stimulated apoptosis of cardiac myocytes may contribute to the progression of myocardial failure.

Opposing Effects of β1- and β2-Adrenergic Receptors on Cardiac Myocyte Apoptosis Role of a Pertussis Toxin–Sensitive G Protein

Background—β-Adrenergic receptor (β-AR) stimulation increases apoptosis in adult rat cardiac (ventricular) myocytes (ARVMs) via activation of adenylyl cyclase. β2-ARs may couple to a Gi-mediated signaling pathway that can oppose the actions of adenylyl cyclase. Methods and Results—In ARVMs, β-AR stimulation for 24 hours increased the number of apoptotic cells as measured by flow cytometry. β-AR–stimulated apoptosis was abolished by the β1-AR–selective antagonist CGP 20712A (P<0.05 versus β-AR stimulation alone) but was potentiated by the β2-AR–selective antagonist ICI 118,551 (P<0.05 versus β-AR stimulation alone). The muscarinic agonist carbachol also prevented β-AR–stimulated apoptosis (P<0.05 versus β-AR stimulation alone), whereas pertussis toxin potentiated the apoptotic action of β-AR stimulation (P<0.05 versus β-AR stimulation alone) and prevented the antiapoptotic action of carbachol. Conclusions—In ARVMs, stimulation of β1-ARs increases apoptosis via a cAMP-dependent mechanism, whereas stimulatio...

Inhibition of Copper-Zinc Superoxide Dismutase Induces Cell Growth, Hypertrophic Phenotype, and Apoptosis in Neonatal Rat Cardiac Myocytes In Vitro

Oxidative stress has been implicated in the pathophysiology of myocardial failure. We tested the hypothesis that inhibition of endogenous antioxidant enzymes can regulate the phenotype of cardiac myocytes. Neonatal rat ventricular myocytes in vitro were exposed to diethyldithiocarbamic acid (DDC), an inhibitor of cytosolic (Cu, Zn) and extracellular superoxide dismutase (SOD). DDC inhibited SOD activity and increased intracellular superoxide in a concentration-dependent manner. A low concentration (1 micromol/L) of DDC stimulated myocyte growth, as demonstrated by increases in protein synthesis, cellular protein, prepro-atrial natriuretic peptide, and c-fos mRNAs and decreased sarcoplasmic reticulum Ca(2+)ATPase mRNA. These actions were all inhibited by the superoxide scavenger Tiron (4,5-dihydroxy-1,3-benzene disulfonic acid). Higher concentrations of DDC (100 micromol/L) stimulated myocyte apoptosis, as evidenced by DNA laddering, characteristic nuclear morphology, in situ terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL), and increased bax mRNA expression. DDC-stimulated apoptosis was inhibited by the SOD/catalase mimetic EUK-8. The growth and apoptotic effects of DDC were mimicked by superoxide generation with xanthine plus xanthine oxidase. Thus, increased intracellular superoxide resulting from inhibition of SOD causes activation of a growth program and apoptosis in cardiac myocytes. These findings support a role for oxidative stress in the pathogenesis of myocardial remodeling and failure.

Adrenergic regulation of cardiac myocyte apoptosis.

The direct effects of catecholamines on cardiac myocytes may contribute to both normal physiologic adaptation and pathologic remodeling, and may be associated with cellular hypertrophy, apoptosis, and alterations in contractile function. Norepinephrine (NE) signals via α‐ and β‐adrenergic receptors (AR) that are coupled to G‐proteins. Pharmacologic studies of cardiac myocytes in vitro demonstrate that stimulation of β1‐AR induces apoptosis which is cAMP‐dependent and involves the voltage‐dependent calcium influx channel. In contrast, stimulation of β2‐AR exerts an anti‐apoptotic effect which appears to be mediated by a pertussis toxin‐sensitive G protein. Stimulation of α1‐AR causes myocyte hypertrophy and may exert an anti‐apoptotic action. In transgenic mice, myocardial overexpression of either β1‐AR or Gαs is associated with myocyte apoptosis and the development of dilated cardiomyopathy. Myocardial overexpression of β2‐AR at low levels results in improved cardiac function, whereas expression at high levels leads to dilated cardiomyopathy. Overexpression of wildtype α1B‐AR does not result in apoptosis, whereas overexpression of Gαq results in myocyte hypertrophy and/or apoptosis depending on the level of expression. Differential activation of the members of the mitogen‐activated protein kinase (MAPK) superfamily and production of reactive oxygen species appear to play a key role in mediating the actions of adrenergic pathways on myocyte apoptosis and hypertrophy. This review summarizes current knowledge about the molecular and cellular mechanisms involved in the regulation of cardiac myocyte apoptosis via stimulation of adrenergic receptors and their coupled effector pathways.

Adrenergic regulation of myocardial apoptosis

Increased sympathetic nerve activity to the myocardium is a central feature in patients with heart failure. Norepinephrine, the primary transmitter of the sympathetic nervous system, signals via binding to alpha- and beta-adrenergic receptors (AR) that are coupled to G-proteins. Pharmacologic studies of cardiac myocytes in vitro demonstrate that beta-AR can stimulate apoptosis. Likewise, in transgenic mice overexpression of beta 1-AR or G alpha s is associated with myocyte apoptosis and the development of dilated cardiomyopathy. Whereas beta 1-AR stimulate apoptosis in vitro and in vivo, beta 2-AR may either stimulate or inhibit apoptosis and myocardial failure depending on the level of expression. Receptors coupling to Gi and Gq may also be able to mediate or modulate apoptosis and the development of myocardial failure, suggesting the potential for interactions between the beta-AR system and numerous remodeling stimuli that act through Gi or Gq signaling pathways. It appears likely that the mitogen-activated protein kinase superfamily plays a key role in mediating the actions of adrenergic pathways on myocyte apoptosis. These observations suggest that the adrenergic nervous system plays an important role in the regulation of myocyte apoptosis, and may thus contribute to the development of myocardial failure.

Regulation of angiotensin II-stimulated osteopontin expression in cardiac microvascular endothelial cells: role of p42/44 mitogen-activated protein kinase and reactive oxygen species.

Using spontaneously hypertensive and aortic banded rats, we have shown that expression of myocardial osteopontin, an extracellular matrix protein, coincides with the development of heart failure and is inhibited by captopril, suggesting a role for angiotensin II (ANG II). This study tested whether ANG II induces osteopontin expression in adult rat ventricular myocytes and cardiac microvascular endothelial cells (CMEC), and if so, whether induction is mediated via activation of mitogen‐activated protein kinases (p42/44 MAPK) and involves reactive oxygen species (ROS). ANG II (1 μM, 16 h) increased osteopontin expression (fold increase 3.3±0.34, n = 12, P < 0.01) in CMEC as measured by northern analysis, but not in ARVM. ANG II stimulated osteopontin expression in CMEC in a time‐ (within 4 h) and concentration‐dependent manner, which was prevented by the AT1 receptor antagonist, losartan. ANG II elicited robust phosphorylation of p42/44 MAPK as measured using phospho‐specific antibodies, and increased superoxide production as measured by cytochrome c reduction and lucigenin chemiluminescence assays. These effects were blocked by diphenylene iodonium (DPI), an inhibitor of the flavoprotein component of NAD(P)H oxidase. PD98059, an inhibitor of p42/44 MAPK pathway, and DPI each inhibited ANG II‐stimulated osteopontin expression. Northern blot analysis showed basal expression of p22phox, a critical component of NADH/NADPH oxidase system, which was increased 40–60% by exposure to ANG II. These results suggest that p42/44 MAPK is a critical component of the ROS‐sensitive signaling pathways activated by ANG II in CMEC and plays a key role in the regulation of osteopontin gene expression. Published 2001 Wiley‐Liss, Inc.

Inhibition of protein phosphatase 1 induces apoptosis in neonatal rat cardiac myocytes: role of adrenergic receptor stimulation.

Abstract The mechanisms that regulate cardiac myocyte apoptosis are not well understood. To study the role of protein phosphatase 1 (PP1) and 2A (PP2A) in apoptosis, we exposed cultured neonatal rat cardiac myocytes to the phosphatase inhibitor okadaic acid (OA). Exposure (18 h) to 100 nM OA (a concentration which inhibits both PP1 and PP2A) decreased the number of adherent cells, caused genomic DNA fragmentation, and increased the percentage of apoptotic cells. These effects did not occur at a lower concentration of OA (1 nM) which is relatively specific for PP2A. Stimulation of α1- or β-adrenergic receptors with norepinephrine (NE) in the presence of propranolol or prazosin partially blocked OA-induced apoptosis as measured by flow cytometry. Likewise, stimulation of adenylyl cyclase with forskolin reduced OA-induced apoptosis. Conversely, inhibition of protein kinase A with H89 or protein kinase C with chelerethrine potentiated OA-induced apoptosis. OA increased caspase-3 activity, and this effect was reduced by NE. Thus, inhibition of PP1 stimulates apoptosis in NRVM and stimulation of adrenergic receptors protects against OA-induced apoptosis.