NRG-1 modulation of contractility and NO synthesis in isolated ventricular myocytes.

Objectives: Neuregulins (Nrg) are growth factors that mediate cellular interactions in different systems through the activation of tyrosine kinase receptors of the ErbB family. The ErbB-Nrg pathway is a mediator of cardiac development and different long-term effects on postnatal and adult cardiomyocytes have been demonstrated. As it is not clear whether Nrg1 has any rapid effects on cardiac activity, we investigated acute effects in isolated adult rat ventricular myocytes (ARVCMs). Methods: The effects induced by recombinant binding domain of Nrg1beta1 on nitric oxide (NO) production were studied in unstimulated ARVCMs using simultaneous Ca2+ and NO fluorimetric confocal imaging. Nrg1 effect on Ca2+ handling was investigated by Ca2+ transients and Ca2+ current (ICa2+) measurements. eNOS and phospholamban (PLN) phosphorylation state were evaluated by western blot analysis and immunofluorescence staining. Results: In isolated cardiomyocytes Nrg1 induced a Ca2+ independent increase in NO production, blocked by the PI3K inhibitor Wortmannin (Wm). Western blot analysis revealed an increase in eNOS phosphorylation in Nrg1 treated myocytes respect to control and phosphorylation was attenuated by Wm. Nrg1 treatment induced a significant increase in Ca2+ transients amplitude under basal condition while it was ineffective upon beta-adrenergic stimulation. Moreover Nrg1 treatment accelerated the recovery of cytosolic Ca2+ as indicated by the decrease of the constant of cytosolic Ca2+ decline, suggesting a role of Nrg1 in Ca2+ release and/or reuptake from SR. The increase in Ca2+ transients amplitude exerted by Nrg1 didn’t involve the regulation of L-Type Ca2+ channels. The role of PI3K and PKG were also investigated as potential signaling steps involved in Nrg1 mediated Ca2+ handling: the presence of Wm or PKG inhibitor DT2 abolished the increase in transient Ca2+ amplitude and the acceleration of Ca2+ recovery induced by Nrg1 treatment. Immunofluorescence analysis revealed that Nrg1 treatment increased PLN phosphorylation, blocked by inhibitors of PI3K, eNOS and PKG. Conclusions: In ARVCMs Nrg1 increases eNOS phosphorylation and NO production through PI3K-Akt pathway, without affecting basal Ca2+ and ICa2+. The stimulation of NO synthesis activates the cGMP-PKG dependent pathway, with consequent increase in PLN phosphorylation, acceleration of cytosolic Ca2+ recovery in the SR and increase in Ca2+ transients amplitude.