Effects of aging on sarcoplasmic reticulum Ca2+-cycling proteins and theirphosphorylation in rat myocardium

Diminished Ca2+-sequestering activity of the sarcoplasmic reticulum (SR) is implicated in the age-associated slowing of cardiac muscle relaxation. In attempting to further define the underlying mechanisms, the present study i nvestigated the impact of aging on the contents of major SR Ca2+-cycling pr oteins and SR protein phosphorylation by endogenous Ca2+/calmodulin-depende nt protein kinase (CaM kinase). The studies were performed using homogenate s and SR vesicles derived from the ventricular myocardium of adult (6-8 mo old) and aged (26-28 mo old) Fischer 344 rats. Western immunoblotting analy sis showed no significant age-related difference: in the relative amounts o f ryanodine receptor-Ca2+-release channel (RyR-CRC), the Ca2+-storage prote in calsequestrin, Ca2+-pumping ATPase (Ca2+-ATPase), and Ca2+-ATPase-regula tory protein phospholamban (PLB) in SR or homogenate. On the other hand, th e relative amount of immunoreactive CaM kinase II (delta-isoform) was simil ar to 50% lower in the aged heart. CaM kinase-mediated phosphorylation of R yR-CRC, Ca2+-ATPase, and PLB was reduced significantly (similar to 25-40%) in the aged compared with adult rat. ATP-dependent Ca2+-uptake activity of SR and the stimulatory effect of calmodulin on Ca2+ uptake were also reduce d significantly with aging. Treatment of SR vesicles with anti-PLB antibody (PLBab) invoked relatively less stimulation of Ca2+ uptake in the aged (le ss than or equal to 26%) compared with the adult (less than or equal to 65% ) rat. Ca2+-ATPase but not PLB underwent phosphorylation by CaM kinase in P LBab-treated SR with resultant stimulation of Ca2+ uptake. The rates of Ca2 + uptake by PLBab-treated SR were significantly lower (45-55%) in the aged compared with adult rat in the absence and presence of calmodulin. These fi ndings imply that changes in the intrinsic functional properties of SR Ca2-cycling proteins and/or their phosphorylation-dependent regulation contrib ute to impaired SR function in the aging heart.