New Findings: Hindlimb Unloading Causes Nucleocytoplasmic Ca²⁺ Overload and DNA Damage in Skeletal Muscle

Disuse atrophy of skeletal muscle is associated with a severe imbalance in cellular Ca²⁺ homeostasis and marked increase in nuclear apoptosis. Nuclear Ca²⁺ is involved in the regulation of cellular Ca²⁺ homeostasis. However, it remains unclear whether nuclear Ca²⁺ levels change under skeletal muscle disuse conditions, and whether changes in nuclear Ca²⁺ levels are associated with nuclear apoptosis. In this study, changes in Ca²⁺ levels, Ca²⁺ transporters, and regulatory factors in the nucleus of hindlimb unloaded rat soleus muscle were examined to investigate the effects of disuse on nuclear Ca²⁺ homeostasis and apoptosis. Results showed that, after hindlimb unloading, the nuclear envelope Ca²⁺ levels ([Ca²⁺]_NE) and nucleocytoplasmic Ca²⁺ levels ([Ca²⁺]_NC) increased by 78% (p < 0.01) and 106% (p < 0.01), respectively. The levels of Ca²⁺-ATPase type 2 (Ca²⁺-ATPase2), Ryanodine receptor 1 (RyR1), Inositol 1,4,5-tetrakisphosphate receptor 1 (IP₃R1), Cyclic ADP ribose hydrolase (CD38) and Inositol 1,4,5-tetrakisphosphate (IP₃) increased by 470% (p < 0.001), 94% (p < 0.05), 170% (p < 0.001), 640% (p < 0.001) and 12% (p < 0.05), respectively, and the levels of Na⁺/Ca²⁺ exchanger 3 (NCX3), Ca²⁺/calmodulin dependent protein kinase II (CaMK II) and Protein kinase A (PKA) decreased by 54% (p < 0.001), 33% (p < 0.05) and 5% (p > 0.05), respectively. In addition, DNase X is mainly localized in the myonucleus and its activity is elevated after hindlimb unloading. Overall, our results suggest that enhanced Ca²⁺ uptake from cytoplasm is involved in the increase in [Ca²⁺]_NE after hindlimb unloading. Moreover, the increase in [Ca²⁺]_NC is attributed to increased Ca²⁺ release into nucleocytoplasm and weakened Ca²⁺ uptake from nucleocytoplasm. DNase X is activated due to elevated [Ca²⁺]_NC, leading to DNA fragmentation in myonucleus, ultimately initiating myonuclear apoptosis. Nucleocytoplasmic Ca²⁺ overload may contribute to the increased incidence of myonuclear apoptosis in disused skeletal muscle.