Mitochondrial PE potentiates respiratory enzymes to amplify skeletal muscle aerobic capacity

Abstract

Exercise capacity is a strong predictor of all-cause mortality. Skeletal muscle mitochondrial respiratory capacity, its biggest contributor, adapts robustly to changes in energy demands induced by contractile activity. While transcriptional regulation of mitochondrial enzymes has been extensively studied, there is limited information on how mitochondrial membrane lipids are regulated. Here, we show that exercise training or muscle disuse alters mitochondrial membrane phospholipids including phosphatidylethanolamine (PE). Addition of PE promoted, whereas removal of PE diminished, mitochondrial respiratory capacity. Unexpectedly, skeletal muscle-specific inhibition of mitochondria-autonomous synthesis of PE caused respiratory failure because of metabolic insults in the diaphragm muscle. While mitochondrial PE deficiency coincided with increased oxidative stress, neutralization of the latter did not rescue lethality. These findings highlight the previously underappreciated role of mitochondrial membrane phospholipids in dynamically controlling skeletal muscle energetics and function.

Fig. 1. Skeletal muscle mitochondrial PE promotes oxidative capacity.

(A to C) Untrained (UT, n = 6) or trained (T, n = 7 or 8) C57BL6/J mice. (A) Skeletal muscle mitochondrial phospholipidome. LPC, lyso-PC; PG, phosphatidylglycerol; n.s., not significant. (B) Mitochondrial phospholipids quantified by thin-layer chromatography (TLC). (C) Skeletal muscle PSD mRNA. (D to L) Studies on PSD-MKI mice (n = 3 to 9). (D) Generation of mice with conditional knock-in of PSD. 5′UTR, 5′ untranslated region. (E) Skeletal muscle PSD mRNA. (F) Muscle mitochondrial PE. (G and H) Rates for oxygen consumption or ATP production in permeabilized muscle fibers with Krebs cycle substrates. DQ, duroquinol; AA, antimycin A. (I) Protein abundance of respiratory complexes II to V. (J) Myosin heavy chain (MHC) fiber-type distribution. (K) Endurance running test. (L) Ex vivo twitch endurance test. Means ± SEM.

Fig. 2. Deficiency of mitochondrial PE promotes atrophy and respiratory failure.

(A to C) Control (Ctrl; n = 6 or 7) and hindlimb-unloaded (HU; n = 7) C57BL6/J mice. (A) Gastrocnemius weight. (B) Skeletal muscle mitochondrial phospholipidome. (C) Skeletal muscle PSD mRNA. (D to P) Studies on PSD-MKO mice. (D) Generation of PSD-MKO mice. (E) PSD mRNA levels in multiple tissues (n = 5 to 6). (F) TLC analysis of mitochondrial phospholipids. (G) Muscle mitochondrial PE (n = 3 to 4). (H) Body weights after tamoxifen injection (n = 9 to 22). (I) Kyphosis in PSD-MKO mice. (J) Kaplan-Meier survival curve. (K and L) Breathing rate and peripheral capillary oxygen saturation (SpO₂) 6 weeks after tamoxifen injection (n = 3). bpm, breaths per minute. (M to P) Diaphragm 4 weeks after tamoxifen injection. (M) Diaphragm weight (n = 4 to 6). (N) Distribution of fiber cross-sectional area (n = 9). (O) Fibrosis and fiber type. (P) Force-frequency curve (n = 4 to 6). Means ± SEM.

Fig. 3. PE deficiency in skeletal muscle mitochondria.

(A) Electron micrograph of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. (B and C) Rates of oxygen consumption and ATP production in permeabilized fibers with Krebs cycle substrates (n = 3 to 5). (D) Protein abundance of respiratory complexes I to V. (E) Activities of respiratory enzymes (n = 4 to 6). (F) Blue native gel of isolated mitochondria revealing supercomplexes (n = 4). High–molecular weight supercomplexes (HMW SCs). (G) Mitochondrial H₂O₂ production and emission with pyruvate normalized to O₂ consumption (n = 3). (H) 4-HNE. (I) MDA (n = 5). (J) Reduced glutathione (GSH) and oxidized glutathione (GSSG) (n = 3 to 5). Means ± SEM.

Fig. 4. Overexpression of mitochondrial catalase does not rescue PSD deficiency.

(A) PSD-MKO mice were crossed with mCAT transgenic mice to generate mCAT × PSD-MKO mice. (B) Mitochondrial H₂O₂ production and emission with pyruvate normalized to O₂ consumption (n = 5 to 7). (C) Kaplan-Meier survival curve. (D) Body weights after tamoxifen injection (n = 6 to 9). (E) Force-frequency curve of diaphragm muscles (n = 4 to 11). (F) Rates of oxygen consumption in permeabilized fibers with Krebs cycle substrates (n = 4 to 10). (G and H) Pathway analyses for differentially expressed genes between control, PSD-MKO, and mCAT × PSD-MKO diaphragms (n = 3 to 4). (G) Area-proportional Venn diagram of differentially activated pathways. (H) Normalized enrichment scores (NES) of differentially activated pathways. ECM, extracellular matrix. (I) Schematic illustration of the consequences of mitochondrial PE deficiency. ROS, reactive oxygen species. Means ± SEM.