Loss of mitochondria long-chain fatty acid oxidation impairs skeletal muscle contractility by disrupting myofibril structure and calcium homeostasis

Abstract

Objective: Abnormal lipid metabolism in mammalian tissues can be highly deleterious, leading to organ failure. Carnitine Palmitoyltransferase 2 (CPT2) deficiency is an inherited metabolic disorder affecting the liver, heart, and skeletal muscle due to impaired mitochondrial oxidation of long-chain fatty acids (mLCFAO) for energy production.

Methods: However, the basis of tissue damage in mLCFAO disorders is not fully understood. Mice lacking CPT2 in skeletal muscle (Cpt2^Sk-/-) were generated to investigate the nexus between mFAO deficiency and myopathy.

Results: Compared to controls, ex-vivo contractile force was reduced by 70% in Cpt2^Sk-/- oxidative soleus muscle despite the preserved capacity to couple ATP synthesis to mitochondrial respiration on alternative substrates to long-chain fatty acids. Increased mitochondrial biogenesis, lipid accumulation, and the downregulation of 80% of dystrophin-related and contraction-related proteins severely compromised the structure and function of Cpt2^Sk-/- soleus. CPT2 deficiency affected oxidative muscles more than glycolytic ones. Exposing isolated sarcoplasmic reticulum to long-chain acylcarnitines (LCACs) inhibited calcium uptake. In agreement, Cpt2^Sk-/- soleus had decreased calcium uptake and significant accumulation of palmitoyl-carnitine, suggesting that LCACs and calcium dyshomeostasis are linked in skeletal muscle.

Conclusions: Our data demonstrate that loss of CPT2 and mLCFAO compromise muscle structure and function due to excessive mitochondrial biogenesis, downregulation of the contractile proteome, and disruption of calcium homeostasis.

Keywords: CPT2; Calcium; Fatty acid oxidation; Muscle contraction; Palmitoyl-carnitine.

Figure 1

Impaired long-chain fatty acid oxidation compromises contraction in a muscle-type-dependent manner. (A) Treadmill running performance in Cpt2^Skf/f and Cpt2^Sk−/− mice. (B) Inverted screen test performance. (C) Muscle wet mass in grams (g) for EDL and Soleus from Cpt2^Skf/f and Cpt2^Sk−/− mice. (D) Ex-vivo Extensor Digitorum Longus (EDL) muscle contraction. (E) Ex-vivo EDL muscle contraction normalized to muscle size. (F) Percentage change of ex-vivo contraction in Cpt2^Sk−/− EDL muscle compared to control. (G) Ex-vivo soleus (SOL) muscle contraction. (H) Ex-vivo Soleus contraction normalized to muscle size. (I) Percentage change of ex-vivo contraction in Cpt2^Sk−/− Soleus muscle compared to control. All data were generated in adult male mice and presented as mean ± SEM. N = 4–6. ∗P ≤ 0.05 by T-Test.

Figure 2

Bioenergetic capacity of CPT2 deficient skeletal muscle mitochondria. (A–D) Representative traces of oxygen consumption rates (JO2) in isolated mitochondria from Cpt2^Skf/f and Cpt2^Sk−/− red gastrocnemius muscle (RGa) energized with pyruvate (Pyr), octanoylcarnitine (OctC), palmitoylcarnitine (PC), or succinate (S) with rotenone (Rot) and malate (M) across energy demands control by phosphocreatine (PCr) and uncoupling induced by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Substrate-supported oxygen consumption (JO2) under low (E) or high (F) energy demands and the fold-change from low to high energy (G) in isolated mitochondria from RGa. Comparison of mitochondrial oxygen consumption rates at low (H) or high (I) energy demands between genotypes for RGa muscle presented as Cpt2^Sk−/− percentage change from control. (J) Blood glucose immediately after a high-intensity treadmill challenge. (K) Rate of ATP production (JATP) and (L) ATP production per oxygen molecule (P/O ratio) on different energetic substrates. Data were generated in adult male mice and presented as mean ± SEM. N = 5–6. ∗P ≤ 0.05 by T-Test.

Figure 3

In oxidative muscles, loss of CPT2 deregulates the myofibril-sarcolemma-ECM anchorage system and disrupts the contractile machinery. (A–C) Expression abundance heatmaps of proteins involved in the ECM-sarcolemma-myofibril anchorage system in Cpt2^Skf/f and Cpt2^Sk−/− soleus muscle; asterisk denotes significant differences (q < 0.1) between genotypes. (D) TEM images of Cpt2^Skf/f and Cpt2^Sk−/− Soleus muscle. Data was generated in adult male mice and presented as Log2 abundance. ∗adjusted p-value (q < 0.1) calculated by Benjamini Hochberg FDR correction. N = 6 for A-C. For (C) magnification 20× and scale bar = 200 μm.

Figure 4

Upon loss of CPT2, oxidative myofibers accumulate polar lipids. (A) Lipid visualization by Oil-Red-O (ORO) (upper 4 images), Sudan Black (middle 4 images), and Nile Red (lower 4 images) in soleus and EDL muscle. (B) Relative abundance of triacylglycerides (TAGs) in Soleus. (C–I) Relative abundance of phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and ceramides in Soleus muscles. Data was generated in adult male mice and presented as mean ± SEM. N = 3–4. ∗P ≤ 0.05 by T-Test. For (A) magnification 20× and scale bar = 200 μm.

Figure 5

The loss of CPT2 disrupts SR structure and intracellular calcium homeostasis. (A) Expression abundance heatmaps of SR tethering proteins in Cpt2^Skf/f and Cpt2^Sk−/− soleus muscle; asterisk denotes significant differences (q < 0.1) between genotypes. (B) Transverse TEM images of Cpt2^Skf/f and Cpt2^Sk−/− Soleus, red arrows indicate dilated SR. (C) Expression abundance heatmaps of calcium handling proteins in Soleus muscle, asterisk denotes significant differences (q < 0.1) between genotypes. (D) Representative traces of muscle force production over time and at peak rate of relaxation in control and Cpt2^Sk−/− Soleus muscle stimulated with 100 Hz. (E, F, G) Relative calcium uptake over time, area under the curve (AUC), and time to half-life (1/2 Life) in homogenate from Cpt2^Skf/f and Cpt2^Sk−/− Soleus muscle. N = 5–6. For (A) data are presented as Log2 abundance. ∗adjusted p-value (q < 0.1) calculated by Benjamini Hochberg FDR correction. For (D–G), data are presented as Mean ± SEM and ∗P ≤ 0.05 by T-Test.

Figure 6

Long-chain acylcarnitines interfere with SR-mediated calcium dynamics in skeletal muscle. (A) Immunohistochemistry of myosin heavy chain type 2A (green), type 2B (red), type 2X (black), and dystrophin (yellow) in TA muscle of Cpt2^Sk−/− mice. Palmitoyl-carnitine (C16:0) and oleoyl-carnitine (C18:1) detection by mass-spectroscopy-based lipid scanning in TA muscle of Cpt2^Sk−/− mice. (B) Relative abundance of LCACs in TA and soleus (SOL) muscle of Cpt2^Skf/f and Cpt2^Sk−/− mice. (C) Abundance of palmitoyl-carnitine (C16:0) and oleoyl-carnitine (C18:1) relative to internal standards (IS) in Cpt2^Sk−/− white quadriceps (WQuad) and soleus. (D) Representative traces of calcium uptake and release in isolated sarcoplasmic reticulum (SR) from control muscle in the absence of fatty acids, and in the presence of palmitoyl-carnitine (PC) or octanoyl-carnitine (OctC). (E) Quantitation of the calcium uptake and release slope over time relative to protein concentration. Data was generated in adult male mice. Data are presented as mean ± SEM and ∗P ≤ 0.05 by T-Test or 1-way ANOVA. N = 3–6.