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

Roof SR, Shannon TR, Janssen PM, Ziolo MT. Effects of increased systolic Ca2+ and phospholamban phosphorylation during beta-adrenergic stimulation on Ca2+ transient kinetics in cardiac myocytes. Am J Physiol Heart Circ Physiol 301: H1570-H1578, 2011. First published July 15, 2011; doi:10.1152/ajpheart.00402.2011.Previous studies demonstrated higher systolic intracellular Ca2+ concentration ([Ca2+](i)) amplitudes result in faster [Ca2+](i) decline rates, as does beta-adrenergic (beta-AR) stimulation. The purpose of this study is to determine the major factor responsible for the faster [Ca2+](i) decline rate with beta-AR stimulation, the increased systolic Ca2+ concentration levels, or phosphorylation of phospholamban. Mouse myocytes were perfused under basal conditions [1 mM extracellular Ca2+ concentration ([Ca2+](o))], followed by high extracellular Ca2+ (3 mM [Ca2+](o)), washout with 1 mM [Ca2+](o), followed by 1 mu M isoproterenol (ISO) with 1 mM [Ca-2+](o). ISO increased Ser(16) phosphorylation compared with 3 mM [Ca2+](o), whereas Thr(17) phosphorylation was similar. Ca2+ transient (CaT) (fluo 4) data were obtained from matched CaT amplitudes with 3 mM [Ca2+](o) and ISO. [Ca2+](i) decline was significantly faster with ISO compared with 3 mM [Ca2+](o). 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 [Ca2+](o). A Ca2+/calmodulin-dependent protein kinase (CAMKII) inhibitor (KN-93) did not affect the CaT decline rates with 3 mM [Ca2+](o) or ISO but normalized ISO's time to peak with 3 mM [Ca2+](o). Thus, during beta-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.