Serine phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase in the intact beating rabbit heart

Recent studies have demonstrated that Ca2+/calmodulin-dependent protein kin ase phosphorylates the Ca2+-pumping ATPase of cardiac sarcoplasmic reticulu m (SR) in vitro. Also, evidence from in vitro studies suggested that this p hosphorylation, occurring at Ser(38), results in stimulation of Ca2+ transp ort. In the present study, we investigated whether serine phosphorylation o f the SR Ca2+-ATPase occurs in the intact functioning heart. Hearts removed from anesthetized rabbits were subjected to retrograde aortic perfusion of the coronary arteries with oxygenated mammalian Ringer solution containing P-32(i) and contractions were monitored by recording systolic left ventric ular pressure development. Following 45-50 min of P-32 perfusion, the heart s were freeze-clamped, SR isolated, and analyzed for protein phosphorylatio n. SDS-polyacrylamide gel electrophoresis and autoradiography showed phosph orylation of several peptides including the Ca2+-ATPase and Ca2+ release ch annel (ryanodine receptor). The identity of Ca2+-ATPase as a phosphorylated substrate was confirmed by Western immunoblotting as well as immunoprecipi tation using a cardiac SR Ca2+-ATPase-specific monoclonal antibody. The Ca2 +-ATPase showed immunoreactivity with a phosphoserine monoclonal antibody i ndicating that the in situ phosphorylation occurred at the serine residue. Quantification of Ca2+-ATPase phosphorylation in situ yielded a value of 20 8 +/- 12 pmol P-32/mg SR protein which corresponded to the phosphorylation of similar to 20% of the Ca2+ pump units in the SR membrane. Since this pho sphorylation occurred under basal conditions (i.e., in the absence of any i notropic intervention), a considerable steady-state pool of serine-phosphor ylated Ca2+-ATPase likely exists in the normally beating heart. These findi ngs demonstrate that serine phosphorylation of the Ca2+-ATPase is a physiol ogical event which may be important in the regulation of SR function. (C) 1 999 Academic Press.