(A) Strategy for the generation of the Mcu^{fl/fl-Ska-MCM} mice. Skel, skeletal muscle. (B) Tamoxifen dosing regimen to induce Mcu deletion. Mice were fed tamoxifen chow for 4 weeks (400 mg/kg) and examined at 28 weeks of age. (C) Western blot analysis of MCU expression from isolated muscle mitochondria prepared at 28 weeks of age from the indicated groups. The VDAC was used as the protein loading control. (D) MW/TL from the indicated groups. Muscles analyzed are shown, and heart weight is also normalized to tibia length. n = 8 (Ska-MCM), n = 5 (Mcu^fl/fl), and n = 11 (Mcu^{fl/fl-Ska-MCM}). One-way ANOVA with Dunnett’s multiple comparisons test was used for statistical analysis. (E) Calcium Green-5N assay for mitochondrial Ca²⁺ uptake in isolated muscle mitochondria. Mitochondria were challenged with 10 μM CaCl₂ pulses (red arrows). Rel, relative. (F) Representative transverse H&E-stained TA histological muscle sections at ×100 magnification. (G) Schematic for experiments using direct AAV9 TA muscle injections. Mcu^fl/fl animals were injected with virus at 6 weeks of age and analyzed at 14 weeks of age. (H) Western blot analysis of MCU expression from total TA protein lysates of AAV9-Cre– or AAV9-GFP–transduced Mcu^fl/fl animals. GAPDH was used as a protein loading control. (I) TA MW/TL from the indicated AAV9 experimental groups; n = 8 per group. Student’s 2-tailed t-test was used to analyze groups for statistical significance. (J) Representative H&E-stained TA muscle sections 8 weeks after AAV9 viral transduction. Scatter plots show individual values and mean ± SEM.