Abstract 16735: Attenuated Subsarcolemmal Protein Phosphatase 2A Activity is Responsible for the Enhanced L-Type Ca Channel Phosphorylation and Contraction in nNOS−/− Mice
Background: Reversible phosphorylation of proteins involved in EC coupling plays an important role in the regulation of cardiac function. Nitric oxide (NO) is known to target protein phosphorylation to discreet myocardial subcellular domains via a cyclic GMP-mediated regulation of phosphodiesterase activity. In addition, myocardial nNOS-derived NO has been shown to hasten relaxation by enhancing PKA- phosphorylation of phospholamban via a previously unrecognized cGMP-independent inhibition of protein phosphatases (PP1 and PP2A). However, the mechanism underlying the effects of myocardial nNOS-derived NO on inotropy and the sarcolemmal Ca2+ current (I Ca) remains to be explored.
Methods and Results: Intracellular dialysis of the PKA inhibitor, PKI (1 μmol/L) had no effect in in voltage-clamped LV myocytes from WT mice but caused a significant reduction in cell shortening in nNOS−/− myocytes (9.15±1.17 v. 6.27±0.8% with PKI, n=9–16, P<0.05). In agreement with these findings, the phosphorylated fraction of the α1.2 subunit of cardiac L-type Ca channel was significantly larger in nNOS−/− mice (0.93±0.15 v. 0.68±0.17 in WT littermates, n=12, P=0.03). Protein phosphatase activity in α1.2 immunoprecipitates did not differ between genotypes (RFU: 2.75±0.02 v. 2.81±0.02 in nNOS−/−; n=15, P=0.07) but the okadaic acid (OA, 10 nmol/L)-inhibitable (PP2A-dependent) fraction was significantly reduced in nNOS−/− mice (9.4±1.3 in WT v. 1.8±0.7% in nNOS−/−, n=15, P<0.0001). This difference was maintained after inhibition of sGC with ODQ (10 μmol/L, n=25). PP2A inhibition significantly increased cell shortening and I Ca 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 after OA in nNOS−/−, n=9–10) whereas PP1 inhibition had no effect in either group.
Conclusions: The negative inotropic effect of nNOS-derived NO is due to a cGMP-independent reduction in the PKA phosphorylation of L-type Ca2+ channels secondary to a localized increase in PP2A activity at the sarcolemmal membrane. These data suggest that myocardial NO production by nNOS can target phosphorylation of protein involved in EC coupling by differentially modulating protein phosphatase activity in discreet subcellular domains.
- © 2010 by American Heart Association, Inc.