Loss of long-chain acyl-CoA synthetase isoform 1 impairs cardiac autophagy and mitochondrial structure through mechanistic target of rapamycin complex 1 activation

Abstract

Because hearts with a temporally induced knockout of acyl-CoA synthetase 1 (Acsl1(T-/-)) are virtually unable to oxidize fatty acids, glucose use increases 8-fold to compensate. This metabolic switch activates mechanistic target of rapamycin complex 1 (mTORC1), which initiates growth by increasing protein and RNA synthesis and fatty acid metabolism, while decreasing autophagy. Compared with controls, Acsl1(T-/-) hearts contained 3 times more mitochondria with abnormal structure and displayed a 35-43% lower respiratory function. To study the effects of mTORC1 activation on mitochondrial structure and function, mTORC1 was inhibited by treating Acsl1(T-/-) and littermate control mice with rapamycin or vehicle alone for 2 wk. Rapamycin treatment normalized mitochondrial structure, number, and the maximal respiration rate in Acsl1(T-/-) hearts, but did not improve ADP-stimulated oxygen consumption, which was likely caused by the 33-51% lower ATP synthase activity present in both vehicle- and rapamycin-treated Acsl1(T-/-) hearts. The turnover of microtubule associated protein light chain 3b in Acsl1(T-/-) hearts was 88% lower than controls, indicating a diminished rate of autophagy. Rapamycin treatment increased autophagy to a rate that was 3.1-fold higher than in controls, allowing the formation of autophagolysosomes and the clearance of damaged mitochondria. Thus, long-chain acyl-CoA synthetase isoform 1 (ACSL1) deficiency in the heart activated mTORC1, thereby inhibiting autophagy and increasing the number of damaged mitochondria.

Keywords: ATP synthase; glucose metabolism; heart metabolism; lipid metabolism; β-oxidation.

Figure 1.

Loss of cardiac ACSL1 decreased fatty acid use and increased the use of glucose. A) ACSL1 protein in ventricles 20 wk after tamoxifen-induced knockout of Acsl1. B) Gene expression in ventricles (n = 6). C) ACSL specific activity in ventricular membranes (n = 3). D) [1-¹⁴C]palmitate oxidation to CO₂ or acid-soluble metabolites (ASM) in isolated cardiac mitochondria (n = 4). E) TAG content in ventricles (n = 5). F) In vivo 2-Br-[1-¹⁴C]palmitate uptake in ventricles (n = 5). G) Phosphorylated AMPK relative to total AMPK in ventricles (n = 4–5). H) [2-¹⁴C]pyruvate oxidation to CO₂ in isolated cardiac mitochondria (n = 5). I) Tissues were collected from female mice 10 wk after tamoxifen at 7:00 am (fed), or at 11:00 am after 4 h of withholding food (unfed). Glucose from glycogen was measured after acid hydrolysis (n = 4). *P < 0.05.

Figure 2.

Two weeks of rapamycin treatment inhibited mTORC1 activation in Acsl1^T−/− hearts. Control and Acsl1^T−/− mice were treated with vehicle or 1 mg/kg rapamycin daily for 2 wk. A) Representative immunoblot of phosphorylation of mTORC1 targets, S6K and 4E-BP1, in ventricles. B) Heart weight normalized to body weight (n = 5–8). C) Gene expression in ventricles from mice treated with vehicle or rapamycin for 2 wk (n = 3–5). Rapa, rapamycin; Veh, vehicle. *P < 0.05 comparing genotype. ^#P < 0.05 comparing treatment.

Figure 3.

mTORC1 inhibition improved mitochondrial structure in Acsl1^T−/− hearts. A) Representative electron microscopy images of left ventricles for untreated mice and mice treated daily with rapamycin for 1 or 2 wk; ×5000 or ×20,000 magnification. White arrows indicate autophagic vesicles. B) Abnormal mitochondria in untreated mice or mice treated with rapamycin for 1 or 2 wk relative to total mitochondria (n = 3). Abnormal mitochondria were defined as those containing vacuoles, inclusions, or disrupted cristae. C) Autophagic mitochondria relative to total mitochondria after 1 or 2 wk of rapamycin treatment (n = 3). D) Mitochondrial area (n = 3). Mitochondrial area was quantified using ImageJ software for at least 1000 mitochondria per heart (4–7 images). Rapa, rapamycin. *P < 0.05 comparing genotype. ^#P < 0.05 comparing treatment.

Figure 4.

Rapamycin treatment normalized high mitochondrial number in Acsl1^T−/− hearts. Control and Acsl1^T−/− mice were treated with vehicle or rapamycin daily for 2 wk. A) Mitochondrial DNA normalized to the nuclear gene H19 (n = 8). B) Number of mitochondria per cell area (n = 3). C) Mitochondrial area per cellular area (n = 3). Mitochondrial number and area were quantified using ImageJ software for at least 1000 mitochondria per heart (4–7 images). D) mRNA expression of genes controlling mitochondrial biogenesis (Pgc1a, Erra) (n = 4). Rapa, rapamycin; Veh, vehicle. *P < 0.05 comparing genotype. ^#P < 0.05 comparing treatment.

Figure 5.

Inhibition of mTORC1 activated autophagy in Acsl1^T−/− hearts. A) Control and Acsl1^T−/− mice were treated with vehicle or rapamycin for 14 d and vehicle or 60 mg/kg body weight chloroquine, a lysosome inhibitor, for the last 7 d (n = 4). Representative immunoblot of LC3B-I and LC3B-II (n = 4). B) p62 protein in ventricles from mice treated with vehicle or rapamycin for 2 wk (n = 4). C) Parkin localization, normalized to vehicle control (n = 3). D) Mitochondrial fission and fusion gene expression in ventricles from mice treated with vehicle or rapamycin for 2 wk (n = 4–8). Rapa, rapamycin. *P < 0.05 comparing genotype. ^#P < 0.05 comparing treatment.

Figure 6.

Rapamycin treatment partially normalized mitochondrial function in Acsl1^T−/− hearts. Control and Acsl1^T−/− mice were treated with vehicle or rapamycin daily for 2 wk. A and B) Mitochondrial function was measured in isolated mitochondria using a Seahorse XF24 Analyzer, which sequentially injected ADP, oligomycin, FCCP, and antimycin A (n = 4–5). C) Mitochondrial complexes were separated by native electrophoresis and stained for either complex V (ATP synthase) activity or total complex V (n = 4). OCR, O₂ consumption rate; Rapa, rapamycin. *P < 0.05 comparing genotype. ^#P < 0.05 comparing treatment.