Aging is Associated with Reduced Calcium Release and a Transformation of the Fast Fiber Population in Mouse Lumbrical Muscles

Abstract

Impairments in sarcoplasmic reticulum (SR) calcium (Ca2+) release and re‐uptake have been implicated in age‐associated declines in skeletal muscle contractile function. The goal of this study was to determine the effects of aging on intracellular Ca2+ dynamics in mammalian skeletal muscle. Ca2+ release was monitored by recording the large, brief intracellular Ca2+ transient (ICT) that results from a single depolarization event and produces a twitch contraction. Re‐sequestration of Ca2+ was monitored by recording the slow restoration of resting levels following a long tetanic contraction. Because the characteristics of the ICT are influenced by fiber type, we also analyzed the fiber type composition of the muscles. Finally, levels of proteins associated with Ca2+ release and uptake were examined. Experiments were performed on lumbrical muscles isolated from adult (8 mo) or old (28 mo) C57BL/6 mice. The ratiometric, low‐affinity Ca2+‐sensitive fluorescent dye mag‐fura‐2 was used to provide a linear, kinetically accurate representation of the ICT, while the return to resting level was reported by the high‐affinity Ca2+‐sensitive fluorescent dye fluo‐4. The time required for the fluo‐4 response to decline by 50% beginning 1.5 s after a 5 s tetanic contraction was taken as the measure of Ca2+‐uptake performance. Fiber type composition was determined by immunofluorescence using antibodies for specific myosin heavy chains and Ca2+ handling proteins were examined by western blot. The peak level reached during the ICT in muscles from old mice was reduced by 36% (p<0.001) compared with that measured for muscles of adult mice (old: 0.033±0.004 ratio‐units; adult: 0.052±0.005 ratio‐units, n=9), and ICT full‐width at half‐maximum (an indication of duration) was 42% longer (p=0.002) in old compared with adult muscles (old: 4.2±1.0 ms, adult: 3.0±0.3 ms, n=9). In contrast, the time required for the Ca2+ level to decline by 50% following a 5 s tetanus was unchanged (p=0.23) with aging (old: 7.3±0.8 s, adult: 6.9±0.6 s, n=9). Analysis of fiber types showed a 5.7‐fold increase with age in the cross‐sectional area occupied by type 2a fibers (old: 64.3±14.1%; adult: 11.3±6.6%, n=6) with a corresponding decrease in total type 2b fiber area (old: 14.7±10.2%; adult: 68.2±16.1%). Western blot analyses (n=5–7) indicated a 26% decline (p=0.043) in expression of the dihydropyridine receptor in muscles from old compared with adult mice and a trend (p=0.06) for a decrease in the SR Ca2+‐ATPase (SERCA1). No change was observed in the levels of the ryanodine receptor or parvalbumin. We conclude that the Ca2+ release is diminished in lumbrical muscles from old mice, while the net effectiveness of post‐tetanic Ca2+ removal mechanisms is unchanged. Previous reports detailing differences between fiber types in the properties of the ICT suggest that the changes observed with aging in the present study are largely explained by the large shift from 2b to 2a fibers in lumbrical muscles of old compared with adult mice, rather than intrinsic impairments in the calcium handling capabilities.Support or Funding InformationSupported by AG051442