Distinct aging-induced intracellular calcium accumulation pattern in mouse fast-twitch skeletal muscle

BACKGROUND: Age-related skeletal muscle atrophy primarily manifests as a heterogeneous decline in the size and function of individual fibers. Intracellular calcium ion concentration (Ca 2 + i ) is a pivotal second messenger and regulates numerous cellular phenomena via its temporal and spatial dynamics. Dysregulation of Ca 2 + i homeostasis has been identified as a characteristic of old skeletal muscle, however, the spatiotemporal pattern of Ca 2 + i dynamics remains unknown. PURPOSE: To characterize the spatiotemporal dynamics and regulatory mechanisms of Ca 2 + i in aged skeletal muscle at rest and in response to twitch contractions. METHODS: Male young (Y; 6 months old, n = 6) and old (O; 26 months old, n = 4) C57BL/6J mice were subjected to in vivo Ca 2 + i imaging. Under anesthesia, the tibialis anterior (TA) muscle was gently exteriorized and loaded with ratiometric Ca 2 + indicator Fura 2-AM (40 μM). Ca 2 + i was measured from the fluorescence ratio (R: 340/380) at rest (30 min) and in response to twitch contractions (1 Hz, 4 ms pulse, -10V, 10 min). After imaging, the TA and white gastrocnemius muscles were sampled for biochemical and morphological analysis. RESULTS: Resting Ca 2 + i was significantly increased in old skeletal muscle compared to young (Y: 1.49 ± 0.02, O: 1.64 ± 0.05, p = 0.012). The spatial distribution of Ca 2 + i within muscle fibers in aged muscle exhibited a distinct localized accumulation along the longitudinal muscle axis. This spatial heterogeneity was quantitatively characterized by a significant increase in the coefficient of variation of Ca 2 + i in the fiber (Y: 4.46 ± 0.24%, O: 6.91 ± 0.51%, p = 0.0008). No statistical differences in the amplitude of temporal Ca 2 + i changes at rest were observed between young and aged muscle (Y: 0.18 ± 0.05%, O: 0.14 ± 0.08%, p = 0.254). Twitch contraction-induced Ca 2 + i elevation was observed in both young and old muscle (Y: 6.3 ± 1.9%, O: 8.5 ± 2.5%, main effect of contraction: p < 0.0001), while muscle torque was relatively decreased in aged muscle (main effect of age: p = 0.013). This result suggests that old muscle displayed higher Ca 2 + i per unit of relative muscle force during the twitch contractions (main effect of age: p = 0.032). Levels of major Ca 2 + -handling proteins located on sarcoplasmic reticulum (RyR: p = 0.565, SERCA1: p = 0.258, CSQ1: p = 0.466) and mitochondria (MCU: p = 0.957, MICU1: p = 0.798, MICU2: p = 0.953, COXIV: p = 0.125), and SR-mitochondrial tethering protein (Mfn1: p = 0.869, Mfn2: p = 0.785) were maintained with aging. The muscle section revealed an abnormal sarcoplasmic reticulum with tubular aggregates arranged longitudinally in 91.4 % of aged muscle fibers. CONCLUSION: Our findings reveal novel aspects of Ca 2 + i dysregulation in old skeletal muscle. We demonstrated that old muscle exhibited a chronically elevated basal Ca 2 + i with a unique spatially heterogeneous distribution within individual fibers. This distinct, longitudinally localized, Ca 2 + i pattern in concert with the emergence of tubular aggregates could potentially underlie the mechanisms promoting muscle atrophy in aging. Funding information: This study was supported in part by JSPS KAKENHI Grant (No.20H04074, 21J14283). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.