Xu A, Narayanan N
Department of Physiology, The University of Western Ontario, London, Ontario, Canada N6A 5C1.
Am J Physiol. 1998 Dec;275(6):H2087-94. doi: 10.1152/ajpheart.1998.275.6.H2087.
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 (</=26%) compared with the adult (</=65%) rat. Ca2+-ATPase but not PLB underwent phosphorylation by CaM kinase in PLBab-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 findings imply that changes in the intrinsic functional properties of SR Ca2+-cycling proteins and/or their phosphorylation-dependent regulation contribute to impaired SR function in the aging heart.
肌浆网（SR）钙摄取活性降低与年龄相关的心肌舒张减慢有关。为了进一步明确其潜在机制，本研究调查了衰老对主要SR钙循环蛋白含量以及内源性钙/钙调蛋白依赖性蛋白激酶（CaM激酶）介导的SR蛋白磷酸化的影响。研究使用了来自成年（6 - 8月龄）和老年（26 - 28月龄）Fischer 344大鼠心室肌的匀浆和SR囊泡。蛋白质免疫印迹分析显示，SR或匀浆中兰尼碱受体 - 钙释放通道（RyR - CRC）、钙储存蛋白肌集钙蛋白、钙泵ATP酶（Ca2 + - ATPase）和Ca2 + - ATPase调节蛋白受磷蛋白（PLB）的相对含量在年龄相关方面无显著差异。另一方面，老年心脏中免疫反应性CaM激酶II（δ亚型）的相对含量约低50%。与成年大鼠相比，老年大鼠中CaM激酶介导的RyR - CRC、Ca2 + - ATPase和PLB的磷酸化显著降低（约25 - 40%）。随着衰老，SR的ATP依赖性钙摄取活性以及钙调蛋白对钙摄取的刺激作用也显著降低。与成年大鼠（≤65%）相比，用抗PLB抗体（PLBab）处理SR囊泡后，老年大鼠（≤26%）的钙摄取刺激相对较小。在PLBab处理的SR中，CaM激酶使Ca2 + - ATPase而非PLB发生磷酸化，从而刺激了钙摄取。在有无钙调蛋白的情况下，PLBab处理的SR的钙摄取速率在老年大鼠中均显著低于成年大鼠（45 - 55%）。这些发现表明，SR钙循环蛋白的内在功能特性及其磷酸化依赖性调节的变化导致了衰老心脏中SR功能受损。
