Unrestrained fatty acid oxidation triggers heart failure in mice via cardiolipin loss and mitochondrial dysfunction.

Figure 1. Genetic deletion of ACC1 and ACC2 in mouse cardiomyocytes. (A and B) Total RNA was isolated from hearts of 10-week-old WT, ACC1 HKO, ACC2 HKO, and ACC dHKO male mice, and RT-qPCR was performed to measure Acc1 and Acc2 mRNA expression (n = 6 WT, 5 ACC1 HKO, 2 ACC2 HKO, and 5 ACC dHKO for A; n = 6 WT, 5 ACC1 HKO, 2 ACC2 HKO, and 5 ACC dHKO for B). ***P < 0.001, ****P < 0.0001 by 1-way ANOVA. (C) Hearts from 20-week-old WT and ACC dHKO female mice were harvested and freeze-clamped to measure malonyl-CoA and acetyl-CoA levels, as described in Methods (n = 6 WT and 7 ACC dHKO). ****P < 0.0001 by unpaired 2-tailed Student’s t test. (D–F) ECHO assessment of cardiac function in male ACC1 HKO, ACC2 HKO, and ACC dHKO mice at 2, 4, 6, and 9 months of age (n = 10 WT, 5 ACC1 HKO, 7 ACC2 HKO, and 5 ACC dHKO at 2, 4, and 6 months; n = 10 WT, 5 ACC1 HKO, 7 ACC2 HKO, and 4 ACC dHKO at 9 months). Data are presented as the mean ± SEM.

Figure 2. Decreased heart function in ACC dHKO mice. (A) Transverse/2-chamber views of hearts from 11-month-old WT and ACC dHKO male mice. (B) Representative M-mode ECHO images from 10-month-old male WT and ACC dHKO mice. LVPW, left ventricular posterior wall. (C) ECHO assessment of cardiac function in 10-month-old WT and ACC dHKO male mice (n = 15 WT and 9 ACC dHKO). ****P < 0.0001 by unpaired 2-tailed Student’s t test. Data are presented as the mean ± SEM. (D) Total RNA was isolated from hearts of 10-week-old WT and ACC dHKO male mice, and RT-qPCR was performed to measure expression of hypertrophy-associated genes (Myh7, Nppa, and Nppb) (n = 5 WT and 5 ACC dHKO). *P < 0.05, ***P < 0.001 by unpaired 2-tailed Student’s t test. (E) Representative TC-stained sections from hearts of 11-month-old WT and ACC dHKO male mice are shown at ×10 magnification. Collagen area was quantified from TC-positive regions using multiple microscopy images from each group (11 images from 2 WT mice and 15 images from 3 ACC dHKO mice) with the split-red-channel method, as described in Methods. ****P < 0.0001 by unpaired 2-tailed Student’s t test.

Figure 3. Increased FAO in ACC dHKO hearts. (A) Fatty acylcarnitines were measured in hearts isolated from 24-week-old WT and ACC dHKO female mice, as described in Methods (n = 7 WT and 4 ACC dHKO). (B) Cardiac FA and glucose oxidation rates were measured with Langendorff working heart perfusion in live hearts isolated from 7-week-old WT or ACC dHKO mice (n = 4 per group), as described in Methods. LCFA, long-chain fatty acid. Data are presented as the mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001 by unpaired 2-tailed Student’s t test.

Figure 4. Impaired respiratory electron transfer function in mitochondria isolated from ACC dHKO hearts. (A) Mitochondria were isolated from WT and ACC dHKO male mice aged 2–3 months (18 total mice; Seahorse experiments performed in 5 independent runs, 3–4 mice per run with age-matched WT and ACC dHKO in each run). Electron transfer between respiratory complexes was assessed by measuring oxygen consumption rate (OCR) during sequential additions of pyruvate/malate/FCCP, rotenone, succinate, antimycin A, and TMPD/ascorbate, as described in Methods. (B–D) Hearts from 20-week-old female WT and ACC dHKO mice were used (n = 6 WT and 7 ACC dHKO). (B) Free CoA and CoA thioesters (propionyl-CoA and succinyl-CoA) were quantified by LC-MS as described for malonyl-CoA and acetyl-CoA. (C) NAD, NADH, and the NAD+/NADH ratio were measured by LC-MS in freeze-clamped hearts. (D) Total adenine nucleotides levels were quantified by LC-MS. Cellular energy charge was calculated as ([ATP] + 0.5 × [ADP])/([ATP] + [ADP] + [AMP]). Data are presented as the mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001 by unpaired 2-tailed Student’s t test.

Figure 5. Reduced linoleoyl phospholipids and tetralinoleoyl cardiolipin in ACC dHKO hearts. (A–E) Cardiac phospholipids from 8-week-old male WT and ACC dHKO mice were analyzed by LC-MS/MS, as described in Methods. Total phospholipids, each PL species, and their specific FA compositions were quantified (n = 5 WT and 5 ACC dHKO for A–C and E; n = 4 WT and 4 ACC dHKO for D). PE (A), PG (B), PC (C), PI (D), and linoleoyl cardiolipin (72:8, E). IS, internal standard. (F–I) Incorporation of labeled FAs into phospholipids and cardiolipins in WT and ACC dHKO hearts. Mice received intraperitoneal injections of LA-d4 (18:2) or OA-d17 (18:1). Enrichment of labeled species was determined 1 h after injection (n = 5 WT and 5 ACC dHKO). MS analysis quantified PC34:2 (16:0,18:2; D4-LA) (F), PC34:1 (16:0,18:1; D17-OA) (G), CL72:6 containing 1 18:2 chain (D4-LA) (H), and CL72:6 containing 1 18:1 chain (D17-OA) (I). Enrichment was calculated as labeled/(labeled + unlabeled) from isotopologue distributions, as described in Methods. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by unpaired 2-tailed Student’s t test.

Figure 6. Inhibition of FAO by etomoxir normalizes the amount of linoleic acid in ACC dHKO hearts. (A) Hearts from 4-week-old WT and ACC dHKO male mice fed chow or chow supplemented with etomoxir (20 mg/kg/day) for 1 month were harvested, and FA composition was measured by gas chromatography, as described in Methods (n = 4 WT-NT, 4 ACC dHKO-NT, 5 WT-etomoxir, and 7 ACC dHKO-etomoxir). (B–G) LC-MS/MS was used to quantify total phospholipids (B, PC; C, PE), linoleoyl-containing phospholipids (D, PC 18:2; E, PE 18:2), total cardiolipins (F), and linoleoyl cardiolipins (G, CL 72:8), in hearts from A, as described in Methods (n = 4 WT-NT, 4 ACC dHKO-NT, 5 WT-etomoxir, and 6 ACC dHKO-etomoxir). Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by unpaired 2-tailed Student’s t test.

Figure 7. Inhibition of FAO by etomoxir prevents the development of heart failure in ACC dHKO mice. (A) Eight-week-old WT and ACC dHKO male mice were fed either chow or chow containing etomoxir (20 mg/kg/day) for 2 weeks. Heart mitochondria were isolated, and oxygen consumption rates (OCRs) were measured during sequential additions of pyruvate/malate/FCCP, rotenone, succinate, antimycin A, and TMPD/ascorbate, as described in Methods (n = 10 WT and 10 ACC dHKO). Data are presented as the mean ± SEM; comparisons were made by unpaired 2-tailed Student’s t test. (B–E) Four-week-old WT and ACC dHKO male mice were fed chow or chow supplemented with etomoxir (20 mg/kg/day) for 1 month. ECHO was performed to quantify EF (B), FS (C), LVIDs (D), and LV Vols (E) (n = 11 WT-NT, 9 ACC dHKO-NT, 12 WT-etomoxir, and 13 ACC dHKO-etomoxir for B and C; n = 10 WT-NT, 9 ACC dHKO-NT, 12 WT-etomoxir, and 13 ACC dHKO-etomoxir for D and E). Data are presented as the mean ± SEM. **P < 0.01, ***P < 0.001 by 2-way ANOVA (genotype × treatment) with Šidák’s multiple-comparison test.