Abstract 12912: The Plasma Membrane Calcium Pump (PMCA4) Modulates Cardiac Contractility by Tethering Neuronal Nitric Oxide Synthase to the Plasma Membrane Compartment
Calcium is involved in the regulation of two essential processes in the myocardium: myofilament contraction and signal transduction. We have previously shown in animals overexpressing the plasma membrane calcium ATPase isoform 4 (PMCA4) that this calcium pump regulates nitric oxide (NO) signalling through cardiac nNOS (neuronal NO synthase), but its role in contraction/relaxation remained unclear. We here addressed the mechanism by which PMCA4 modulates nNOS activity and the role of PMCA4 in cardiac physiology using mice carrying a genetic deletion of PMCA4 (PMCA4 KO). In vivo contractility was enhanced in PMCA4 KO (dP/dtmax 8049 ± 628 vs 6604 ± 296 mmHg/s in wild type, WT; p<0.05, n=10) with no change in the relaxation rate. Ca2+ transients in cardiomyocytes from PMCA4 KO showed an increase in amplitude (492±28nM vs. 299±24nM in WT; p<0.05, n=8) with no change in the rate of decay. Sarcoplasmic reticulum (SR) Ca2+ load as well as expression of all other calcium transporters was unchanged. Cardiomyocytes from PMCA4 KO showed a similar Ca2+-decay pattern to WT cells when perfused with Na+/Ca2+ free solution (to inhibit the Na+/Ca2+ exchanger) in the presence of caffeine (to empty the SR). These findings were incompatible with a direct role for PMCA4 in relaxation. Interestingly, nNOS inhibition with vinyl-L-NIO mimicked the effect of PMCA4 gene deletion in WT animals, as it increased contraction and Ca2+-amplitude, but had no lusitropic effect. Vinyl-L-NIO was ineffective in PMCA4 KO. There was no change in total nNOS protein expression in KOs, but over 50% of sarcolemmal nNOS protein and activity was delocalised to the cytosol, resulting in a significant decrease in cellular cGMP. This reduction in cGMP led to a significant elevation in cAMP levels as well as RYR receptor phosphorylation in PMCA4 KO compared to WT, both via modification of phosphodiesterase 2 activity. In conclusion, PMCA4 has no direct role in determining diastolic calcium. It plays a key structural role in tethering nNOS to the membrane and in maintaining its physiological activity. Binding to PMCA4 defines a functionally distinct and spatially confined set of nNOS molecules in the myocardium. These results place the PMCA4 at the heart of a novel Ca2+/nNOS signalling complex in the cardiac sarcolemma.
- © 2010 by American Heart Association, Inc.