Fig 2: 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.

Fig 3: Increased mtROS level was necessary for ATPIF1-triggered HIF1a activation.(A) Real-time measurement of mitochondrial superoxide production in cardiomyocytes by MitoSOX (n = 4). (B) Immunoblot analysis of protein carbonyls in isolated mitochondria from cardiomyocytes (n = 3). (C) Quantification of glutathione (GSH) levels in the cardiomyocyte lysates (n = 6). (D–F) Representative immunoblot and group average of HIF1a protein level (D, n = 5), ECAR measurement (E, n = 5), and real-time PCR analysis of glycolytic gene expression (F, n = 6) of cardiomyocytes with coexpression of ATPIF1 and mitochondrial catalase (mCat). Gene expression values were normalized to 18S rRNA expression and are shown as relative change over the average of control sets (F). Data are means ± SEM of the values. P values were determined using unpaired 2-tailed Student’s t test (C and F) or 2-way ANOVA followed by Tukey’s multiple-comparison test (D and E); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 4: Cardiac-specific deletion of ATPIF1 partially restored respiratory function induced by pathological cardiac hypertrophy.(A) Immunoblot of ATPIF1 expression from liver or ventricular lysates (n = 4–5). (B) Analysis of isolated mouse heart mitochondrial OCR by Seahorse analyzer (n = 5). (C and D) Representative OCR tracing and tabulated data of isolated mouse heart mitochondrial OCR at 28 days after TAC (C), MI (D), or sham surgery. (E) Average OCR of TAC and MI groups during ADP or FCCP stimulation, normalized to the respective sham group (n = 6). Data are means ± SEM of the values. P values were determined using unpaired 2-tailed Student’s t test (E) or 1-way ANOVA followed by Dunnett’s multiple-comparison test (A); **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 5: GSEA profiles showing a significant enrichment of gene sets associated with liver cancer progression (A), and glycolysis (KEGG, (B)) in human HCC with high levels of ATPIF1 mRNA (GSE20238). See heat-map in Supplementary Fig. S1.