Neuregulin-1β1 rapidly modulates nitric oxide synthesis and calcium handling in rat cardiomyocytes

The ErbB-neuregulin-1 beta 1 (Nrg1 beta 1) pathway is required for cardiac development and exerts chronic effects on the postnatal adult heart. Long-term application of Nrg1 beta 1 results in hypertrophy and protection against oxidative stress and cytotoxic agents. We performed experiments with acute Nrg1 beta 1 treatment to find evidence for a further protective role due to rapid modulation of adult cardiomyocyte function. In confocal fluorimetric measurements, Nrg1 beta 1 induced a calcium-independent increase in nitric oxide (NO) production in isolated adult rat ventricular myocytes (ARVCMs) that was blocked by the phosphoinositide-3-kinase (PI3K) inhibitor Wortmannin. Western blot analysis showed enhancement of endothelial nitric oxide synthase phosphorylation in Nrg1 beta 1-treated ARVCMs, which was attenuated by Wortmannin. Nrg1 beta 1 induced a significant increase in calcium transient amplitude (indo-1 ratiometric measurement) and accelerated the recovery of cytosolic calcium in the sarcoplasmic reticulum without affecting whole-cell L-type calcium current. Wortmannin or the protein kinase G inhibiting peptide (DT-2) abolished the increase in calcium transient amplitude and the acceleration of calcium recovery induced by Nrg1 beta 1 treatment. Immunofluorescence analysis revealed that Nrg1 beta 1 treatment increased phospholamban phosphorylation, and the effect was blocked by PI3K and protein kinase G inhibition. Caffeine-releasable sarcoplasmic reticulum calcium content was also higher during Nrg1 beta 1 administration. Rapid activation of PI3K, endothelial nitric oxide synthase and protein kinase G and a consequent improvement in diastolic calcium can be added to established Nrg1 protective roles.