Nitric oxide (NO) has long been known to regulate protein phosphorylation in discreet myocardial subcellular domains via a cyclic GMP-mediated regulation of PKG and phosphodiesterase activity. More recently, nNOS-derived NO has been shown to hasten basal myocardial relaxation by enhancing PKA- phosphorylation of phospholamban via a cGMP-independent inhibition of protein phosphatase activity. However, the mechanism underlying the effect of myocardial nNOS-derived NO on inotropy and the sarcolemmal Ca2+ current (ICa) remains to be explored.Methods and Results
PKA inhibition (PKI, 1 μmol/L) did not affect basal contraction in voltage-clamped LV myocytes from WT mice but caused a significant reduction in shortening in nNOS-/- myocytes (from 9.15 ± 1.17% to 6.27 ± 0.8% with PKI, n=9-16 cells, P < 0.05). In agreement with these findings, the phosphorylated fraction α1.2 subunit of the L-type Ca channel was significantly larger in nNOS-/- myocytes (0.93 ± 0.15 v. 0.68 ± 0.17 in WT, n=12, P < 0.05) and in WT myocytes after nNOS inhibition with SMTC (n = 3, P < 0.05).Methods and Results
Total protein phosphatase activity in α1.2 immunoprecipitates did not differ between genotypes (RFU: 2.75 ± 0.02 in WT v. 2.81 ± 0.02 in nNOS-/-; n=15, P=NS) but the PP2A contribution, i.e., the okadaic acid (OA, 10 nmol/L)-inhibitable fraction of phosphatase activity, was significantly reduced in nNOS-/- α1.2 immunoprecipitates (12 ± 1.2% v. 21 ± 1.0% in WT, n=8 hearts per genotype, P=0.03) in the absence of changes in PP2A protein. Consistent with this result, PP2A inhibition by intracellular perfusion of OA significantly increased cell shortening and ICa in WT mice but not in nNOS-/- (pA/pF: 5.85 ± 0.6 v. 7.97 ± 0.58 after OA in WT, n=9-10, P=0.03 and 7.92 ± 1.05 v. 7.54 ± 0.49 in nNOS-/-, n=9-10, P=NS) whereas PP1 inhibition had no effect in either group. Similarly, OA enhanced α1.2 phosphorylation in WT but not nNOS-/- myocytes. Soluble guanylate cyclase inhibition with ODQ (10 μM) did not affect the difference α1.2 phosphorylation or PP2A activity between genotypes.Conclusions
Myocardial NO production by nNOS targets phosphorylation of proteins involved in EC coupling and regulates myocardial function via differential cGMP-independent effects on protein phosphatase activity in discreet subcellular domains.