Neuronal nitric oxide synthase signaling within cardiac myocytes targets phospholamban

Studies have shown that neuronal nitric oxide synthase (nNOS, NOS1) knockout mice (NOS1(-/-)) have increased or decreased contractility, but consistently have found a slowed rate of intracellular Ca2+ ([Ca2+](i)) decline and relengthening. Contraction and [Ca2+](i) decline are determined by many factors, one of which is phospholamban (PLB). The purpose of this study is to determine the involvement of PLB in the NOS1-mediated effects. Force-frequency experiments were performed in trabeculae isolated from NOS1(-/-) and wild-type (WT) mice. We also simultaneously measured Ca2+ transients (Fluo-4) and cell shortening (edge detection) in myocytes isolated from WT, NOS1(-/-), and PLB-/- mice. NOS1(-/-) trabeculae had a blunted force-frequency response and prolonged relaxation. We observed similar effects in myocytes with NOS1 knockout or specific NOS1 inhibition with S-methyl-L-thiocitrulline (SMLT) in WT myocytes (i. e., decreased Ca2+ transient and cell shortening amplitudes and prolonged decline of [Ca2+](i)). Alternatively, NOS1 inhibition with SMLT in PLB-/- myocytes had no effect. Acute inhibition of NOS1 with SMLT in WT myocytes also decreased basal PLB serine16 phosphorylation. Furthermore, there was a decreased SR Ca2+ load with NOS1 knockout or inhibition, which is consistent with the negative contractile effects. Perfusion with FeTPPS (peroxynitrite decomposition catalyst) mimicked the effects of NOS1 knockout or inhibition. beta-Adrenergic stimulation restored the slowed [Ca2+](i) decline in NOS1(-/-) myocytes, but a blunted contraction remained, suggesting additional protein target(s). In summary, NOS1 inhibition or knockout leads to decreased contraction and slowed [Ca2+](i) decline, and this effect is absent in PLB-/- myocytes. Thus NOS1 signaling modulates PLB serine16 phosphorylation, in part, via peroxynitrite.