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 quantitative RT-qPCR was performed to measure Acc1 and Acc2 mRNA expression. ***P < 0.001, ****P < 0.0001 by one-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. ****P < 0.0001 by unpaired two-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.

Figure 2. Decreased heart function in ACC dHKO mice. (A) Transverse/two-chamber views of hearts from 11-month-old WT and ACC dHKO male mice. (B) Representative M-mode echocardiographic images from 10-month-old male WT and ACC dHKO mice. (C) Echocardiographic assessment of cardiac function in 10-month-old WT and ACC dHKO male mice. ****P < 0.0001 by unpaired two-tailed Student’s t test. (D) Total RNA was isolated from hearts of 10-week-old WT and ACC dHKO male mice, and quantitative RT-qPCR was performed to measure expression of hypertrophy-associated genes (Myh7, Nppa, Nppb). *P < 0.05, ***P < 0.001 by unpaired two-tailed Student’s t test. (E) Representative trichrome-stained sections from hearts of 11-month-old WT and ACC dHKO male mice are shown at ×10 magnification. Collagen area was quantified from trichrome-positive regions with the split-red-channel method as described in Methods. ****P < 0.0001 by unpaired two-tailed Student’s t test.

Figure 3.. Increased FA oxidation 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. (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. Results are shown as the mean ± SEM. *p<0.05, ***p < 0.001, ****p < 0.0001 assessed by 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 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. (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 shown as mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001 by unpaired two-tailed Student’s t test.

Figure 5.. Reduced linoleoyl phospholipids and tetra-linoleoyl 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. PE (A), PC (B), PI (C), PG (D), and linoleoyl cardiolipin (72:8, E). (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 one hour post-injection. Mass spectrometry analysis quantified *PC34:2 (16:0,18:2; D4-LA) (F), *PC34:1 (16:0,18:1; D17-OA) (G), *CL72:6 containing one 18:2 chain (D4-LA) (H), and *CL72:6 containing one 18:1 chain (D17-OA) (I). Enrichment was calculated as labeled/(labeled + unlabeled) from isotopologue distributions, as described in Methods. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Student’s t test.

Figure 6.. Inhibition of FA oxidation 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−1 day−1) for one month were harvested, and FA composition was measured by gas chromatography as described in Methods. (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. Results are shown as the mean ± SEM. *p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 assessed by Student’s t test.

Figure 7.. Inhibition of FA oxidation 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−1·day−1) for two weeks. Heart mitochondria were isolated, and oxygen consumption rates (OCR) were measured during sequential additions of pyruvate/malate/FCCP, rotenone, succinate, antimycin A, and TMPD/ascorbate, as described in Methods. Data are mean ± SEM; comparisons were made by 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−1·day−1) for one month. ECHO was performed to quantify EF; B), FS; C), LVIDₛ; D), and LV Volₛ; E). Data shown are mean ± SEM. **p < 0.01, ***p < 0.001 by two-way ANOVA (genotype × treatment) with Sidak’s multiple comparisons test.