Myocyte contractile activity modulates norepinephrine cytotoxicity and survival effects of neuregulin-1β

Abstract

The purpose of this study is to test the hypothesis that mechanical and electrical activity in adult rat ventricular myocytes (ARVM) alters responses to proapoptotic and prosurvival ligands. The effects of electrical stimulation on myocyte survival, stress signaling, response to beta-adrenergic receptor (beta-AR)-stimulated apoptosis, and neuregulin-1beta (NRG) were examined. Electrical stimulation (6.6 V/cm; 0, 2, and 5 Hz; 2-ms duration; alternating polarity) of ARVM resulted in more than 70% capture. Although ARVM paced for 48 h showed higher mitochondrial uptake of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (P < 0.05, 0 vs. 2 and 5 Hz), electrical stimulation had little effect on cell survival assessed by trypan blue uptake, CPK release, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. Electrical stimulation for 24 h did not induce stress response (heat shock protein 70, 90) nor stress kinase (Erk, JNK, p38) activation. NRG stimulation of Erk and Akt was similar between paced and quiescent cells. Pacing sensitized myocytes to beta-AR-stimulated JNK phosphorylation and cell death with 0.1 microM norepinephrine (NE) in paced myocytes causing equivalent cytotoxicity to 10 microM NE in quiescent cells. NRG suppressed beta-AR-induced apoptosis through a phosphatidylinositol-3-kinase-dependent pathway in both paced and quiescent cells, although it is overwhelmed by high-NE concentration in paced cells. Thus myocyte contractility modulates both NE cytotoxicity as well as the cytoprotective effect of NRG. These results demonstrate the feasibility and importance of using electrically paced cardiomyocytes in primary culture when examining the signaling pathways of cell survival.

Neuregulin activates erbB2-dependent src/FAK signaling and cytoskeletal remodeling in isolated adult rat cardiac myocytes

“…To date, the explanation for the absolute requirement of NRG-1, and the erbB2/ erbB4 receptors in cardiac development [1][2][3] focused on the ability of this system to regulate myocyte growth and survival [4,28]. Our findings that NRG-1β/erbB2 signaling regulates FAK activation, FAC formation and directional spreading of myocytes demonstrate a novel function of NRG-1β/erbB signaling and suggest that NRG-1β regulates myocyte-myocyte and/or myocyte-matrix interactions.…”

“…NRG-1β binding and activation of erbB4/erbB2 receptor tyrosine kinases in myocytes induce activation of a MEK/Erk pathway, as well as a PI3-kinase/Akt pathway, leading to increased protein synthesis [10] and cell survival in the presence of cytotoxic stress [11,28], respectively. The NRG-1β/erbB2-dependent activation of Src/FAK observed here appears to be distinct from those pathways, as it was selectively suppressed by low concentrations of Src inhibitor or chronic treatment with an anti-erbB2 antibody.…”

Anthracyclines Induce Calpain-dependent Titin Proteolysis and Necrosis in Cardiomyocytes

“…Myocytes from the same experiments were subsequently stained with 0.4% trypan blue as described previously (35). Creatine kinase activity in the media samples was determined from a diagnostic kit according to manufacturer's instructions (CK-10, Sigma).…”

Cardiac Endothelial Cells Regulate Reactive Oxygen Species-induced Cardiomyocyte Apoptosis through Neuregulin-1β/erbB4 Signaling

“…The pellet was resuspended in modified RIPA buffer and designated as the membrane fraction, whereas the supernatant was designated as the cytosol fraction. Activation of erbB4, erbB2, Akt, Erk1/2, JNK, and p38 were determined using total cell lysate of ARVMs by immunoprecipitation or immunoblot as described previously (7)(8)(9).…”