Phospholamban phosphorylation sites enhance the recovery of intracellular Ca2+ after perfusion arrest in isolated, perfused mouse heart

Objective: To investigate the importance of the phosphorylation of Ser(16) and Thr(17) sites of phospholamban (PLN) on intracellular Ca2+ (Ca-i(2)+) handling and contractile recovery of the stunned myocardium. Methods: Ca-i(2)+ (Rhod-2, pulsed local-field fluorescence microscopy) and contractility (isovolumic left ventricular developed pressure, LVDP) were simultaneously measured in Langendorff perfused hearts from transgenic mice expressing either intact PLN (PLN-WT) or PLN with both phosphorylation sites mutated to Ala (PLN-DM), subjected to 12 min of global ischemia followed by a reperfusion period of 30 min. Results: Pre-ischemic values of Ca-i(2)+ and LvDP were similar in both groups. In PLN-WT, a transient increase in Thr(17) phosphorylation at early reperfusion preceded a recovery of Ca2+ transient amplitude, virtually completed by the end of reperfusion. LVDP at 30 min reperfusion was 67.9 +/- 7.6% of pre-ischemic values, n =14. In contrast, in PLN-DM, there was a poor recovery of Ca-i(2)+ transient amplitude and LVDP was significantly lower (28.3 +/- 6.7%, n = 11, 30 min reperfusion) than in PLN-WT hearts. Although myofilament Ca2+ responsiveness and troponin I (TnI) degradation did not differ between groups, the episodes of mechanical alternans, typical of Ca-i(2)+ overload, were significantly prolonged in PLN-DM vs. PLN-WT hearts. Conclusions: PLN phosphorylation appears to be crucial for the mechanical and Ca-i(2)+ recovery during stunning and protective against the mechanical abnormalities typical of Ca-i(2)+ overload. The importance of PLN phosphorylation would primarily reside in the Thr(17) residue, which is phosphorylated during the critical early phase of reperfusion. Our results emphasize that, although ablation of PLN phosphorylation does not affect basal contractility, it does alter Ca2+ handling and mechanical performance under stress situations. (c) 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.