Reduced phospholamban phosphorylation is associated with impaired relaxation in left ventricular myocytes from neuronal NO synthase-deficient mice

Stimulation of nitric oxide (NO) release from the coronary endothelium facilitates myocardial relaxation via a cGMP-dependent reduction in myofilament Ca2+ sensitivity. Recent evidence suggests that NO released by a neuronal NO synthase (nNOS) in the myocardium can also hasten left ventricular relaxation; however, the mechanism underlying these findings is uncertain. Here we show that both relaxation (TR50) and the rate of [Ca2+](i) transient decay (tau) are significantly prolonged in field-stimulated or voltage-clamped left ventricular myocytes from nNOS(-/-) mice and in wild-type myocytes (nNOS(+/+)) after acute nNOS inhibition. Disabling the sarcoplasmic reticulum abolished the differences in TR50 and tau, suggesting that impaired sarcoplasmic reticulum Ca2+ reuptake may account for the slower relaxation in nNOS(-/-) mice. In line with these findings, disruption of nNOS (but not of endothelial NOS) decreased phospholamban phosphorylation (P-Ser(16) PLN), whereas nNOS inhibition had no effect on TR50 or tau in PLN-/- myocytes. Inhibition of cGMP signaling had no effect on relaxation in either group whereas protein kinase A inhibition abolished the difference in relaxation and PLN phosphorylation by decreasing P-Ser(16) PLN and prolonging TR50 in nNOS(+/+) myocytes. Conversely, inhibition of type 1 or 2A protein phosphatases shortened TR50 and increased P-Ser(16) PLN in nNOS(-/-) but not in nNOS(-/-) myocytes, in agreement with data showing increased protein phosphatase activity in nNOS(-/-) hearts. Taken together, our findings identify a novel mechanism by which myocardial nNOS promotes left ventricular relaxation by regulating the protein kinase A-mediated phosphorylation of PLN and the rate of sarcoplasmic reticulum Ca2+ reuptake via a cGMP-independent effect on protein phosphatase activity.