Reactive oxygen species mediate amplitude-dependent hypertrophic and apoptotic responses to mechanical stretch in cardiac myocytes.

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)H-leucine 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 approximately 3-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.

Norepinephrine stimulates apoptosis in adult rat ventricular myocytes by activation of the beta-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.