Fig. 4.

Enhanced mitochondrial bioenergetics and SC assembly in the heart of Ndufab1 transgenic mice (TG). TG and WT littermates of 2–4 months old were used. a Western blots for NDUFAB1 expression in WT and TG hearts. ATPB served as the loading control. b Decreased mitochondrial ROS level in TG cardiomyocytes. The intensity of mitoSOX fluorescence of TG was normalized to that of WT (n = 34–41 cells from 3 male mice; **p < 0.01 versus WT). c Increased ΔΨ_m in TG cardiomyocytes. The intensity of FCCP-sensitive TMRM fluorescence of TG was normalized to that of WT (n = 12–15 cells from three male mice per group; *p < 0.05 versus WT). d, e Whole-cell OCR measured with Seahorse using glucose/pyruvate (d) or palmitate (e) as the substrate. Arrows indicate the time of adding palmitate (PAL, 200 µM), oligomycin (Oligo, 1 µM), FCCP (0.5 µM), and rotenone/antimycin A (Rot/AA, 1 µM for each). f, g Statistical analysis of maximal OCR in d and e (mean ± s.e.m.; n = 10–13 wells from 3 male mice per group; *p < 0.05 versus WT). h Changes of respiratory control ratio (RCR) with different substrates in isolated mitochondria (mean ± s.e.m.; n = 3–8 male mice per group; *p < 0.05, **p < 0.01 versus WT). i In-gel activity of SCs and complex I. j Statistical analysis of i. The activity of TG was normalized to WT (mean ± s.e.m.; n = 5 male mice per group; *p < 0.05, **p < 0.01 versus WT). k BN-PAGE immunoblots of SCs and individual ETC complexes as in Fig. 3c. l Statistical analysis of k. The expression level of TG complexes and supercomplexes was normalized to that of WT (mean ± s.e.m.; n = 4–8 male mice per group; *p < 0.05 versus WT)