No abstract

Neuregulin (NRG)-1␤ has a prosurvival effect on cardiac myocytes via the phosphatidylinositol-3-kinase/Akt pathway, but the physiological regulators of this system in the intact heart are unknown. In this study, we tested the hypothesis that reactive oxygen species regulate NRG/erbB signaling. We used isolated adult rat ventricular myocytes (ARVMs) or cardiac microvascular endothelial cells (CMECs) in monoculture, or together in coculture. H 2 O 2 induced NRG-1␤ release from CMECs in a concentration-dependent manner, and conditioned medium from H 2 O 2 -treated CMEC activated ARVM erbB4. NRG-1␤ release occurred via proteolytic cleavage of 115-kDa transmembrane NRG-1␤ and was inhibited by the metalloproteinase inhibitor 1,10-phenanthroline. In myocyte monoculture, H 2 O 2 induced erbB4-dependent, but NRG-independent, activation of Akt. To elucidate the bioactivity of CMEC-derived NRG-1␤ on ARVMs, we examined H 2 O 2 -induced myocyte apoptosis in co-culture using an antibody to NRG-1␤. The percentages of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells were significantly higher in the anti-NRG-1␤ group than in the control group. The change in apoptosis induced by anti-NRG-1␤ in co-culture was similar in magnitude to the protection of myocytes by addition of recombinant NRG-1␤ to ARVM monocultures. Activation of NRG/erbB paracrine signaling was also seen in the intact heart subjected to oxidative stress by ischemia-reperfusion injury. Isolated perfused mouse hearts subjected to 15 min of ischemia, followed by 30 min of reperfusion, showed complete proteolytic cleavage of 115-kDa NRG-1␤, with concomitant erbB4 phosphorylation. These results demonstrate that reactive oxygen species activate NRG-1␤/erbB4 paracrine signaling in the heart and suggest that this system is involved in cardiac adaptation to oxidative stress. Neuregulin-1 (NRG)1 /erbB signaling is essential for cardiac development (1-5). Moreover, cardiotoxicity of trastuzumab (Herceptin), an erbB2-targeted antibody used in treatment of erbB2-positive cancer, suggests that this pathway also plays a role in the regulation of cardiac structure and function in the postnatal heart (6). Cardiac microvascular endothelial cells (CMECs) express NRG (7), and recombinant NRG-1␤ protects myocytes against anthracycline-and ␤-adrenergic receptor-induced cell injury and death (8 -10). This prosurvival effect of NRG-1␤ occurs through the activation of erbB2 and erbB4 receptor tyrosine kinases in myocytes with downstream signaling in the phosphatidylinositol-3-kinase/Akt pathway. The in vivo role of erbB signaling in cardioprotection is further supported by the finding that mice with a cardiac-specific erbB2 conditional knock-out survive to birth but have accelerated heart failure after injury by pressure-overload or Adriamycin (11, 12). These observations suggest that the NRG/erbB pathway acts as a stress-responsive signal between CMECs and myocytes to maintain cell survival and cardiac function.We have cloned eight isoforms of NRG-1␤ fr...