Figure 4. ATPIF1 overexpression inhibited complex V activity and slowed electron flow through the ETC.
(A) Measurement of ADP-dependent OCR in isolated mitochondria from cardiomyocytes by Seahorse analyzer using pyruvate and malate as substrates (n = 5). (B) Rate of ATP production in cardiomyocytes (n = 4). (C) Measurements of OCR driven by FCCP to assess complex I–IV activities in isolated mitochondria from cardiomyocytes using Seahorse analyzer (n = 4). Rotenone was added to inhibit complex I activity, and succinate was used to activate complex II–mediated respiration. Antimycin A was added to inhibit complex III activity, and complex IV was stimulated by the addition of TMPD and ascorbate. (D) Quantification of NAD+ and NADH levels in the cardiomyocyte lysates (n = 6). Data are means ± SEM of the values. Comparisons between ATPIF1 OE and its respective controls were made using unpaired 2-tailed Student’s t test; **P < 0.01, ****P < 0.0001.