Effects of increased systolic Ca(2+) and β-adrenergic stimulation on Ca(2+) transient decline in NOS1 knockout cardiac myocytes.

We have previously shown that the main factor responsible for the faster [Ca(2+)](i) decline rate with β-adrenergic (β-AR) stimulation is the phosphorylation of phospholamban (PLB) rather than the increase in systolic Ca(2+) levels. The purpose of this study was to correlate the extent of augmentation of PLB Serine(16) phosphorylation to the rate of [Ca(2+)](i) decline. Thus, ventricular myocytes were isolated from neuronal nitric oxide synthase knockout (NOS1(-/-)) mice, which we observed had lower basal PLB Serine(16) phosphorylation levels, but equal levels during β-AR stimulation. Ca(2+) transients (Fluo-4) were measured in myocytes superfused with 3mM extracellular Ca(2+) ([Ca(2+)](o)) and a non-specific β-AR agonist isoproterenol (ISO, 1μM) with 1mM [Ca(2+)](o). This allowed us to get matched Ca(2+) transient amplitudes in the same myocyte. Similar to our previous work, Ca(2+) transient decline was significantly faster with ISO compared to 3mM [Ca(2+)](o), even with matched Ca(2+) transient amplitudes. Interestingly, when we compared the effects of ISO on Ca(2+) transient decline between NOS1(-/-) and WT myocytes, ISO had a larger effect in NOS1(-/-) myocytes, which resulted in a greater percent decrease in the Ca(2+) transient RT(50). 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 Serine(16) phosphorylation are the major determinants for the Ca(2+) decline rate. Furthermore, our data suggest that the molecular mechanisms of Ca(2+) transient decline is normal in NOS1(-/-) myocytes and that the slow basal Ca(2+) transient decline is predominantly due to decreased PLB phosphorylation.