Abstract 1391: Specific Role of nNOS when Tethered to the Plasma Membrane Calcium Pump in the Myocardium
The role of cardiac nNOS (neuronal nitric oxide synthase) as a modulator of cardiac contractility has been clearly demonstrated using nNOS knockout models. Cardiac nNOS however is localized to specific cellular compartments including the plasma membrane, sarcoplasmic reticulum and mitochondria. We have previously described that the sarcolemmal calcium pump (hPMCA4b) binds to nNOS in the heart via its PDZ domain and this complex regulates β-adrenergic signal transmission in vivo (Oceandy et al 2007). We here investigated whether the plasma membrane complex (nNOS/PMCA4b) serves as a specific signaling modulator in the heart. In transgenic mice overexpressing hPMCA4b in the heart, and in neonatal rat cardiomyocytes (NRCM) with adenoviral overexpression of hPMCA4b, a significant 3 fold increase in phosphorylation of phospholamban (PLB) at Serine-16, as well as troponin-I was observed prior to β-adrenergic stimulation (n=5, p<0.05). However, phosphorylation of PLB and troponin-I following isoproterenol treatment was severely reduced, which explains the blunted physiological response to β-adrenergic stimulation that we have observed in vivo. This effect was emulated by treatment with the specific nNOS inhibitor S-methyl-L-thiocitrulline. hPMCA4b overexpression in both models reduced nNOS activity, as measured by the ability to convert tritium-labelled L-arginine to L-citrulline, and led to a reduction in the subsequent production of NO by 21.4±5.11% and cGMP by 24±5.09% (n=6, p<0.05). cAMP was significantly increased by 32±5.5% (n=8, p<0.05) leading to stimulation of protein kinase A activity and ultimately phospholamban phosphorylation. Regulation of cardiac phosphodiesterase activities determined the balance between cGMP and cAMP following PMCA4b overexpression. These results showed that the downstream effector of the plasma membrane nNOS/PMCA4 complex is soluble guanylate cyclase rather than niytrosylation of NO-target proteins. In addition, this complex regulates contractility via cAMP and PLB as well as troponin-I phosphorylation. Overall, this is the first demonstration that specific binding partners of nNOS, such as the strictly plasma membrane bound PMCA4 confer specificity of action to nNOS in the membrane compartment.