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.

No abstract

Abstract-Osteopontin (OPN), an extracellular matrix protein, is expressed in the myocardium with hypertrophy and failure. We tested the hypothesis that OPN plays a role in left ventricular (LV) remodeling after myocardial infarction (MI). Accordingly, OPN expression and LV structural and functional remodeling were determined in wild-type (WT) and OPN knockout (KO) mice 4 weeks after MI. Northern analysis showed increased OPN expression in the infarcted region, peaking 3 days after MI and gradually decreasing over the next 28 days. In the remote LV, OPN expression was biphasic, with peaks at 3 and 28 days. In situ hybridization and immunohistochemical analyses showed increased OPN mRNA and protein primarily in the interstitium. Infarct size, heart weight, and survival were similar in KO and WT mice after MI (PϭNS), whereas the lung wet weight/dry weight ratio was increased in the KO mice (PϽ0.005 versus sham-operated mice). Peak LV developed pressure was reduced to a similar degree after MI in the KO and WT mice. Key Words: extracellular matrix proteins Ⅲ osteopontin Ⅲ collagen Ⅲ myocyte slippage Ⅲ myocyte elongation T he dynamic synthesis and breakdown of extracellular matrix (ECM) proteins may play an important role in myocardial remodeling. 1,2 Recently, using spontaneously hypertensive and aortic-banded rats, we showed increased expression of osteopontin (OPN), an ECM protein, coincident with the development of heart failure. 3 Although first isolated from mineralized bone matrix, OPN has since been shown to be synthesized by several cell types, including cardiac myocytes, microvascular endothelial cells, and fibroblasts. 4 -6 OPN, an adhesive glycophosphoprotein with an arginineglycine-aspartic acid (RGD) sequence, has been shown to interact with integrins (␣ V ␤ 3 , ␣ V ␤ 1 , and ␣ V ␤ 5 ) and the CD44 receptor in an RGD-dependent manner. 4,7 OPN appears capable of mediating diverse biological functions, including cell adhesion, chemotaxis, and signaling. 4,8 OPN has also been shown to interact with fibronectin and collagen, suggesting its possible role in matrix organization and/or stability. 9 -11 Recently, using a mammary cell line, we observed that suppression of OPN synthesis leads to increased activity of matrix metalloproteinase (MMP)-2. 12 In fact, there is increased expression of OPN in several tissues in response to injury, suggesting a role in wound healing. Using a skin incision model, Liaw et al 13 observed disorganization of the matrix and alteration of collagen fibrillogenesis, leading to collagen fibrils with smaller diameters in OPN knockout (KO) mice. Similarly, OPN has been shown to play a critical role in the generation of interstitial fibrosis in the kidney after obstructive nephropathy. 14 Remodeling after myocardial infarction (MI) is associated with left ventricular (LV) dilation, decreased cardiac function, and increased mortality. 15 Early dilation of the LV is likely due to scar expansion in the infarcted region, 16 -18 followed later by progressive remodeling 19 in the noninf...

In ARVMs, stimulation of beta(1)-ARs increases apoptosis via a cAMP-dependent mechanism, whereas stimulation of beta(2)-ARs inhibits apoptosis via a G(i)-coupled pathway. These findings have implications for the pathophysiology and treatment of myocardial failure.

We recently reported that alpha(1)-adrenoceptor (alpha(1)-AR) stimulation induces hypertrophy via activation of the mitogen/extracellular signal-regulated kinase (MEK) 1/2-extracellular signal-regulated kinase (ERK) 1/2 pathway and generates reactive oxygen species (ROS) in adult rat ventricular myocytes (ARVM). Here we investigate the intracellular source of ROS in ARVM and the mechanism by which ROS activate hypertrophic signaling after alpha(1)-AR stimulation. Pretreatment of ARVM with the ROS scavenger Mn(III)terakis(1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP) completely inhibited the alpha(1)-AR-stimulated activation of Ras-MEK1/2-ERK1/2. Direct addition of H(2)O(2) or the superoxide generator menadione activated ERK1/2, which is also prevented by MnTMPyP pretreatment. We found that ARVM express gp91(phox), p22(phox), p67(phox), and p47(phox), four major components of NAD(P)H oxidase, and that alpha(1)-AR-stimulated ERK1/2 activation was blocked by four structurally unrelated inhibitors of NAD(P)H oxidase [diphenyleneiodonium, phenylarsine oxide, 4-(2-aminoethyl)benzenesulfonyl fluoride, and cadmium]. Conversely, inhibitors for other potential ROS-producing systems, including mitochondrial electron transport chain, nitric oxide synthase, xanthine oxidase, and cyclooxygenase, had no effect on alpha(1)-AR-stimulated ERK1/2 activation. Taken together, our results show that ventricular myocytes express components of an NAD(P)H oxidase that appear to be involved in alpha(1)-AR-stimulated hypertrophic signaling via ROS-mediated activation of Ras-MEK1/2-ERK1/2.

Abstract-Oxidative stress stimulates both growth and apoptosis in cardiac myocytes in vitro. We investigated whether oxidative stress mediates hypertrophy and apoptosis in cyclically stretched ventricular myocytes. Neonatal rat ventricular myocytes cultured on laminin-coated silastic membranes were stretched cyclically (1 Hz) at low (nominal 5%) and high (nominal 25%) amplitudes for 24 hours. Stretch caused a graded increase in superoxide anion production as assessed by superoxide dismutase (SOD)-inhibitable cytochrome c reduction or electron paramagnetic resonance spectroscopy. The role of reactive oxygen species (ROS) was assessed using the cell-permeable SOD/catalase mimetics Mn(II/III)tetrakis(1-methyl-4-peridyl) (MnTMPyP) and EUK-8. Stretch-induced increases in protein synthesis ( 3 Hleucine incorporation) and cellular protein content were completely inhibited by MnTMPyP (0.05 mmol/L) at both low and high amplitudes of stretch. In contrast, while MnTMPyP inhibited basal atrial natriuretic factor (ANF) mRNA expression, the stretch-induced increase in ANF mRNA expression was not inhibited by MnTMPyP. In contrast to hypertrophy, only high-amplitude stretch increased myocyte apoptosis, as reflected by increased DNA fragmentation on gel electrophoresis and an Ϸ3-fold increase in the number of TUNEL-positive myocytes. Similarly, only high-amplitude stretch increased the expression of bax mRNA. Myocyte apoptosis and bax expression stimulated by high-amplitude stretch were inhibited by MnTMPyP. Both low-and high-amplitude stretch caused rapid phosphorylation of ERK1/2, while high-, but not low-, amplitude stretch caused phosphorylation of JNKs. Activation of both ERK1/2 and JNKs was ROS-dependent. Thus, cyclic strain causes an amplitude-related increase in ROS, associated with differential activation of kinases and induction of hypertrophic and apoptotic phenotypes.

Stimulation of beta-adrenergic receptors (betaARs) causes apoptosis in adult rat ventricular myocytes (ARVMs). The role of reactive oxygen species (ROS) in mediating betaAR-stimulated apoptosis is not known. Stimulation of betaARs with norepinephrine (10 micromol/L) in the presence of prazosin (100 nmol/L) for 24 hours increased the number of apoptotic myocytes as determined by TUNEL staining by 3.6- fold. The superoxide dismutase/catalase mimetics Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP; 10 micromol/L) and Euk-134 decreased betaAR-stimulated apoptosis by 89+/-6% and 76+/-10%, respectively. Infection with an adenovirus expressing catalase decreased betaAR-stimulated apoptosis by 82+/-15%. The mitochondrial permeability transition pore inhibitor bongkrekic acid (50 micromol/L) decreased betaAR-stimulated apoptosis by 76+/-8%, and the caspase inhibitor zVAD-fmk (25 micromol/L) decreased betaAR-stimulated apoptosis by 62+/-11%. betaAR-stimulated cytochrome c release was inhibited by MnTMPyP. betaAR stimulation caused c-Jun NH2-terminal kinase (JNK) activation, which was abolished by MnTMPyP. Transfection with an adenovirus expressing dominant-negative JNK inhibited betaAR-stimulated apoptosis by 81+/-12%, and the JNK inhibitor SP600125 inhibited both betaAR-stimulated apoptosis and cytochrome c release. Thus, betaAR-stimulated apoptosis in ARVMs involves ROS/JNK-dependent activation of the mitochondrial death pathway.

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.