Cardiac sarcoplasmic reticulum (SR) Ca(2+)
ATPase (SERCA2a) promotes Ca(2+) uptake in the SR. Dephosphorylated
phospholamban (PLB) inhibits SERCA2a activity. We found a distinct dephosphorylation of PLB at Thr(17) and Ser(16) after 20-30min of
ischemia produced by coronary artery occlusion in rats. The aim of the study was to investigate how PLB is dephosphorylated in
ischemia and to determine whether PLB dephosphorylation causes myocardial hypercontraction and
calpain activation through Ca(2+) overload in reperfusion.
Protein inhibitor-1 (I-1) specifically inhibits
protein phosphatase 1 (PP1), the predominant PLB
phosphatase in heart. A Ca(2+)-dependent
phosphatase calcineurin may also induce PLB dephosphorylation.
Ischemia for 30min induced PKC-α translocation, resulting in inactivation of I-1 through PKC-α-dependent phosphorylation at Ser(67). The PP1 activation following I-1 inactivation was thought to induce PLB dephosphorylation in
ischemia.
Ischemia for 30min activated
calcineurin, and pre-treatment with a
calcineurin inhibitor,
cyclosporine A (CsA), inhibited PKC-α translocation, I-1 phosphorylation at Ser(67), and PLB dephosphorylation in
ischemia. Reperfusion for 5min following 30min of
ischemia induced spreading of contraction bands (CBs) and proteolysis of
fodrin by
calpain. Both CsA and an anti-PLB antibody that inhibits binding of PLB to SERCA2a reduced the CB area and
fodrin breakdown after reperfusion. These results reveal a novel pathway via which
ischemia induces
calcineurin-dependent activation of PKC-α, inactivation of I-1 through PKC-α-dependent phosphorylation at Ser(67), and PP1-dependent PLB dephosphorylation. The pathway contributes to the spreading of CBs and
calpain activation through Ca(2+) overload in early reperfusion.