Figure 3. MCUb induction with 48 h of fasting regulates metabolic dynamics in muscle.
(A) Schematic identifying PDK4 as the only antithetically regulated metabolic gene from global RNA microarray analysis in muscle-specific Mcu versus Mcub-deleted mice.
(B) RNA expression of PDK4 from quadriceps at 6 months of age from the indicated groups of mice under baseline and post 48-h fasting conditions. Data are presented as mean ± SEM. ***p < 0.001 vs. the Mcubfl/fl fasting group, using two-way ANOVA and Bonferroni’s multiple-comparisons test. #p < 0.05 versus the Mcubfl/fl fed group, using Student’s t test.
(C) Representative western blot of PDK4 from quadriceps muscle from the indicated groups of mice at 6 months of age. The arrow shows the correct size to identify PDK4 from muscle. Gapdh and the voltage-dependent anion channel (VDAC) were used as controls.
(D) Quantification of PDK4 as shown in (C). n = 3 per group. Data are presented as mean ± SEM. Two-way ANOVA and Bonferroni’s multiple comparison test were used for statistical analysis. *p < 0.05.
(E) Scheme of introducing MyoAAV-PDK4 or MyoAAV-luciferase as a control at 2 months of age, followed by harvest at 4 months.
(F) Western blot of the indicated proteins from quadriceps in the indicated groups of mice. Gapdh was used as a control.
(G and H) Glucose dependency with UK5099 and fatty acid dependency with etomoxir in isolated FDB myofibers from the indicated groups, presented as the percentage of the basal OCR (detailed in STAR Methods). n = 5 in the Mcubfl/fl MyoAAV-luciferase group, n = 6 in the Mcubfl/fl-Myod-Cre MyoAAV-luciferase group, n = 7 in the Mcubfl/fl MyoAAV-PDK4 group, n = 7 in the Mcubfl/fl-Myod-Cre MyoAAV-PDK4 group. two-way ANOVA and Bonferroni’s multiple comparison test were used for statistical analysis. **p < 0.01, ***p < 0.001. #p < 0.05 vs. the Mcubfl/fl MyoAAV-PDK4-treated group using Student’s t test.