Effects of increased systolic Ca2+ and β-adrenergic stimulation on Ca2+ transient decline in NOS1 knockout cardiac myocytes

Abstract

We have previously shown that the main factor responsible for the faster [Ca2+]i decline rate with β-adrenergic (β-AR) stimulation is the phosphorylation of phospholamban (PLB) rather than the increase in systolic Ca2+ levels. The purpose of this study was to correlate the extent of augmentation of PLB Serine16 phosphorylation to the rate of [Ca2+]i decline. Thus, ventricular myocytes were isolated from neuronal nitric oxide synthase knockout (NOS1−/−) mice, which we observed had lower basal PLB Serine16 phosphorylation levels, but equal levels during β-AR stimulation. Ca2+ transients (Fluo-4) were measured in myocytes superfused with 3mM extracellular Ca2+ ([Ca2+]o) and a non-specific β-AR agonist isoproterenol (ISO, 1μM) with 1mM [Ca2+]o. This allowed us to get matched Ca2+ transient amplitudes in the same myocyte. Similar to our previous work, Ca2+ transient decline was significantly faster with ISO compared to 3mM [Ca2+]o, even with matched Ca2+ transient amplitudes. Interestingly, when we compared the effects of ISO on Ca2+ transient decline between NOS1−/− and WT myocytes, ISO had a larger effect in NOS1−/− myocytes, which resulted in a greater percent decrease in the Ca2+ transient RT50. We believe this is due to a greater augmentation of PLB Serine16 phosphorylation in these myocytes. Thus, our results suggest that not only the amount but the extent of augmentation of PLB Serine16 phosphorylation are the major determinants for the Ca2+ decline rate. Furthermore, our data suggest that the molecular mechanisms of Ca2+ transient decline is normal in NOS1−/− myocytes and that the slow basal Ca2+ transient decline is predominantly due to decreased PLB phosphorylation.