Phospholamban phosphorylation in ischemia-reperfused heart. Effect of pacing during ischemia and response to a beta-adrenergic challenge

Abstract

The status of phospholamban (PLB) phosphorylation in the ischemia-reperfused hearts remains controversial. Although a decrease in the phosphorylation of both PLB residues (Ser16, PKA site, and Thr17, CaMKII site) was previously reported, experiments from our laboratory failed to detect this decrease. In an attempt to elucidate the cause for this discrepancy, experiments were performed in Langendorff-perfused rat hearts with two main goals: (1) To determine whether keeping pacing during ischemia, a protocol followed in other ischemia-reperfusion models, decreases the phosphorylation of PLB residues, below pre-ischemic values; (2) To investigate whether a maximal beta-adrenergic challenge allows to detect a decrease in the ability of PLB to be phosphorylated in ischemia-reperfused hearts. Hearts were submitted to a global ischemia/reperfusion protocol (20/30 min) with (P) or without (NP) pacing during ischemia, and phosphorylation of PLB residues was assessed by immunodetection. The recovery of contractility upon reperfusion was lower in P vs. NP hearts. Ser16 of PLB, was phosphorylated at the end of ischemia in NP hearts. This increase appeared earlier in P hearts and was significantly diminished by catecholamine depletion and beta-blockade. Thr17 site was phosphorylated at the beginning of ischemia and the onset of reperfusion. The ischemia-induced phosphorylation of Thr17 was higher and more sustained in P vs. NP hearts, and inhibited by the calcium channel blocker, nifedipine, whereas the reperfusion-induced increase in Thr17 phosphorylation was similar in P and NP hearts and was significantly diminished by the Na+/Ca2+ exchanger inhibitor KB-R7943. Phosphorylation of PLB residues did not decrease below basal levels at any time during ischemia and reperfusion. However, the phosphorylation, inotropic and lusitropic response to beta-adrenergic stimulation was significantly decreased both in P and NP hearts.