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.