Effects of increased systolic Ca²⁺ and phospholamban phosphorylation during β-adrenergic stimulation on Ca²⁺ transient kinetics in cardiac myocytes.

Previous studies demonstrated higher systolic intracellular Ca(2+) concentration (Ca(2+)) amplitudes result in faster Ca(2+) decline rates, as does β-adrenergic (β-AR) stimulation. The purpose of this study is to determine the major factor responsible for the faster Ca(2+) decline rate with β-AR stimulation, the increased systolic Ca(2+) concentration levels, or phosphorylation of phospholamban. Mouse myocytes were perfused under basal conditions [1 mM extracellular Ca(2+) concentration (Ca(2+))], followed by high extracellular Ca(2+) (3 mM Ca(2+)), washout with 1 mM Ca(2+), followed by 1 μM isoproterenol (ISO) with 1 mM Ca(2+). ISO increased Ser(16) phosphorylation compared with 3 mM Ca(2+), whereas Thr(17) phosphorylation was similar. Ca(2+) transient (CaT) (fluo 4) data were obtained from matched CaT amplitudes with 3 mM Ca(2+) and ISO. Ca(2+) decline was significantly faster with ISO compared with 3 mM Ca(2+). Interestingly, the faster decline with ISO was only seen during the first 50% of the decline. CaT time to peak was significantly faster with ISO compared with 3 mM Ca(2+). A Ca(2+)/calmodulin-dependent protein kinase (CAMKII) inhibitor (KN-93) did not affect the CaT decline rates with 3 mM Ca(2+) or ISO but normalized ISO's time to peak with 3 mM Ca(2+). Thus, during β-AR stimulation, the major factor for the faster CaT decline is due to Ser(16) phosphorylation, and faster time to peak is due to CAMKII activation.