Effects of aging on sarcoplasmic reticulum Ca2+-cycling proteins and their phosphorylation 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 investigated the impact of aging on the contents of major SR Ca2+-cycling proteins and SR protein phosphorylation by endogenous Ca2+/calmodulin-dependent protein kinase (CaM kinase). The studies were performed using homogenates 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 analysis showed no significant age-related difference in the relative amounts of ryanodine receptor-Ca2+-release channel (RyR-CRC), the Ca2+-storage protein calsequestrin, Ca2+-pumping ATPase (Ca2+-ATPase), and Ca2+-ATPase-regulatory protein phospholamban (PLB) in SR or homogenate. On the other hand, the relative amount of immunoreactive CaM kinase II (delta-isoform) was approximately 50% lower in the aged heart. CaM kinase-mediated phosphorylation of RyR-CRC, Ca2+-ATPase, and PLB was reduced significantly ( approximately 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 reduced significantly with aging. Treatment of SR vesicles with anti-PLB antibody (PLBab) invoked relatively less stimulation of Ca2+ uptake in the aged (