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 Ca 2+ 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 (TR 50 ) and the rate of [Ca 2+ ] 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 TR 50 and tau, suggesting that impaired sarcoplasmic reticulum Ca 2+ 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 TR 50 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 TR 50 in nNOS +/+ myocytes. Conversely, inhibition of type 1 or 2A protein phosphatases shortened TR 50 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 Ca 2+ reuptake via a cGMP-independent effect on protein phosphatase activity.