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. 2023 May 18;26(6):106895.
doi: 10.1016/j.isci.2023.106895. eCollection 2023 Jun 16.

Genetic deletion of skeletal muscle iPLA2γ results in mitochondrial dysfunction, muscle atrophy and alterations in whole-body energy metabolism

Sung Ho Moon  1 , Beverly Gibson Dilthey  1 , Shaoping Guan  1 , Harold F Sims  1 , Sara K Pittman  2 , Amy L Keith  2 , Christopher M Jenkins  1 , Conrad C Weihl  2 , Richard W Gross  1   3   4   5
Affiliations

Genetic deletion of skeletal muscle iPLA2γ results in mitochondrial dysfunction, muscle atrophy and alterations in whole-body energy metabolism

Sung Ho Moon et al. iScience. .

Abstract

Skeletal muscle is the major site of glucose utilization in mammals integrating serum glucose clearance with mitochondrial respiration. To mechanistically elucidate the roles of iPLA2γ in skeletal muscle mitochondria, we generated a skeletal muscle-specific calcium-independent phospholipase A2γ knockout (SKMiPLA2γKO) mouse. Genetic ablation of skeletal muscle iPLA2γ resulted in pronounced muscle weakness, muscle atrophy, and increased blood lactate resulting from defects in mitochondrial function impairing metabolic processing of pyruvate and resultant bioenergetic inefficiency. Mitochondria from SKMiPLA2γKO mice were dysmorphic displaying marked changes in size, shape, and interfibrillar juxtaposition. Mitochondrial respirometry demonstrated a marked impairment in respiratory efficiency with decreases in the mass and function of oxidative phosphorylation complexes and cytochrome c. Further, a pronounced decrease in mitochondrial membrane potential and remodeling of cardiolipin molecular species were prominent. Collectively, these alterations prevented body weight gain during high-fat feeding through enhanced glucose disposal without efficient capture of chemical energy thereby altering whole-body bioenergetics.

Keywords: Cell biology; Cellular physiology; Physiology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Skeletal muscle-specific deletion of iPLA2γ results in altered mitochondrial structure and impaired protein synthesis leading to skeletal muscle atrophy (A) iPLA2γ protein levels in multiple tissues including white adipose tissue, liver, and skeletal muscle from WT and SKMiPLA2γKO mice were analyzed by western blot analysis indicating skeletal muscle-specific ablation of iPLA2γ. The predominant isoforms of iPLA2γ (i.e., 85, 74, 63, and 52 kDa bands) in WT skeletal muscle are indicated by arrows. (B) Tibialis anterior (TA) and quadriceps (Quad) muscles were excised from both legs of WT and SKMiPLA2γKO mice and weighed. The numbers of mice used for each sample set in the bar graphs are indicated in parentheses. (C) Representative transmission electron microscopy (TEM) images of longitudinal sections of skeletal muscle TA fibers from WT and SKMiPLA2γKO mice (7 months old) demonstrating mitochondria with irregular cristae and electron dense inclusions present in the KO mouse resulting in disruption of normal striated muscle structure. See also Figure S1. (D) Mitochondria were isolated from WT and SKMiPLA2γKO mice (7 months old) by differential centrifugation and the total yield of mitochondrial protein was determined by a BCA protein assay (left panel). The mitochondrial weight to tissue mass ratio was calculated for each indicated condition (right panel). Values from multiple independent preparations (indicated in parentheses in the bar graphs) are the average ± SEM. N.S., not significant. (E) Copy number of mitochondrial DNA (ND1 gene) relative to nuclear DNA (HK2 gene) in TA from WT and SKMiPLA2γKO mice were determined using qPCR. (F) Immunoblot analyses of mitochondrial proteins including MFN1, MFN2, OPA1-l (long form), OPA1-s (short form), DRP1, ANT, citrate synthase (CS), and VDAC from WT and SKMiPLA2γKO mouse TA muscle homogenates. See also Figure S2. (G) Immunoblot analyses of proteins involved in protein synthesis and autophagy. ∗p < 0.05 and ∗∗∗p < 0.001.
Figure 2
Figure 2
Skeletal muscle weakness with concomitant muscle fiber degeneration is present in SKMiPLA2γKO mice (A–C) A forearm grip test (A), four-limb hanging test (B), and treadmill exercise test (C) were performed with WT and SKMiPLA2γKO male mice at 7–8 months of age. Grip force was measured as pond units (p = 0.00980665 N). See also Figure S3. (D and E) Blood glucose (D) and lactate levels (E) in mice fed either a normal-chow (NC) or high-fat (HF) diet at rest (R) before and after treadmill exercise (T) were measured from blood samples taken from the tail vein. Values are expressed as the mean of multiple measurements from the indicated mice (mouse numbers indicated in the parenthesis in the bar graphs) ± SEM. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. N.S., not significant. See also Figure S3. (F) Isometric maximal tetanic force measurements were performed on EDL muscles isolated from 4 WT and 4 SKMiPLA2γKO mice. The specific force after normalization to muscle cross-sectional area (PCSA) was determined. Individual values for PCSA, EDL muscle mass, and body weights of the mice that were utilized for force measurements are shown. ∗p < 0.05. (G) Relative fiber type distribution present in the tibialis anterior (TA) muscle of WT and SKMiPLA2γKO mice are compared by immunoblot analysis for fast-skeletal and slow-skeletal myosin. (H) Representative histological images of H & E staining of the TA muscles from WT and SKMiPLA2γKO are shown for comparison. TA from SKMiPLA2γKO mice displayed decreased muscle fiber size and an increased number of central nuclei. Scale bar: 50 μm. See also Figure S4. (I) Immunoblot analyses of MyoD, Myogenin, ATP citrate lyase (ACL), and phospho-ACL (Ser455) from WT and SKMiPLA2γKO mouse TA muscle (n = 4). ∗p < 0.05 and ∗∗p < 0.01. N.S., not significant.
Figure 3
Figure 3
Mitochondrial respiration in SKMiPLA2γKO skeletal muscle is significantly attenuated with dramatic decreases in the activities and expression levels of electron transfer chain complexes (A) High-resolution mitochondrial respirometry was performed by utilizing permeabilized tibialis anterior (TA) muscle tissue. 5 WT and 5 KO mice fed an NC diet, and 5 WT and 6 KO mice fed an HF diet were utilized for independent measurements. ∗p < 0.05 and ∗∗p < 0.01. See also Figure S5. (B) Protein expression levels of respiratory electron chain complexes including Complex I ∼ IV and cytochrome c in WT vs. SKMiPLA2γKO TA were determined by western blot analyses. ∗∗p < 0.01 and ∗∗∗p < 0.001. (C and D) Complex I and II catalytic activities were assayed by using membrane-disrupted mitochondria isolated from TA muscle of 5 WT and 5 SKMiPLA2γKO mice fed an NC diet, in the presence of substrates (i.e., NADH and succinate, respectively) with decylubiquinone and DCIP as a terminal electron acceptor. Complex I and II activities were confirmed by using their specific inhibitors, rotenone (Rot) and TTFA, respectively. Values are expressed as means ± SEM. (E) Membrane potential of mitochondria isolated from TA muscle was measured by using a TPP·Cl electrode. Independent measurements with 5 WT and 5 KO mice were utilized. Glutamate/pyruvate/malate (G P M) or succinate (Succ) were used as substrates for mitochondrial membrane potentiometry. ∗p < 0.05. (F) Mitochondria isolated from TA muscle of 6 WT and 6 KO mice were placed in respiration buffer. Mitochondria were incubated with either glutamate/pyruvate/malate (G P M) or succinate (Succ) substrate in the presence or absence of 1 μM rotenone (Rot). The amount of H2O2 in the buffer (indicative of superoxide production) was determined by Amplex Red. (G and H) Glucose (G) and lactate levels (H) in blood from 6 WT to 6 SKMiPLA2γKO female mice before (R) and after (T) intense treadmill exercise at a speed of 18 m/min for 4 min were measured. Note that 3 out of 6 KO mice were completely exhausted within 1–2.5 min and their glucose and lactate levels were measured immediately upon reaching exhaustion. ∗p < 0.05, ∗∗p < 0.01 and §p < 1.0 × 10−7.
Figure 4
Figure 4
Genetic ablation of iPLA2γ alters mitochondrial membrane cardiolipin content Male mice (WT and SKMiPLA2γKO) were fed either a normal-chow (NC) or high-fat (HF) diet for 3 months starting at 3 months of age. (A) Lipids from TA muscle were extracted and cardiolipin (CL) molecular species were identified. CL molecular species were quantified using tetra 14:0-CL as an internal standard (I.S.) through comparing peak intensities in the negative ion mode using the [M-2H+1]2- isotopologue peak of CL. 6 WT fed an NC diet, 5 WT fed an HF diet, 6 SKMiPLA2γKO fed an NC diet, and 5 SKMiPLA2γKO fed an HF diet were utilized. A mirror plot of CL molecular species with indicated acyl chain compositions from WT and KO mice fed an NC diet (top panel) or HF diet (lower panel) are presented with calculated differences (CLNC-CLHF) displayed between the two panels. Values are the means ± SEM. ∗p < 0.05 and §p < 0.005. (B and C) Total contents of TAG (B) and DAG (C) in TA tissues from 5 WT to 5 SKMiPLA2γKO mice fed HF diet were determined. ∗p < 0.05.
Figure 5
Figure 5
Deletion of skeletal muscle iPLA2γ results in improved glucose tolerance and the inability to gain weight during high-fat feeding Genetic ablation of skeletal muscle iPLA2γ improves glucose tolerance and reduces weight gain during HF feeding. (A) Body weights of 6 WT and 6 SKMiPLA2γKO male mice fed an HF diet were monitored over 11 weeks following initiation of HF feeding at 3 months of age. (B) Comparison of body weights of WT and SKMiPLA2γKO mice at the end of 11 weeks (3 months) of NC or HF feeding. (C–F) Tissue weights of liver (C), epididymal white adipose tissue (WAT) (D), quadriceps (Quad) (E), and heart (F) normalized to body weight were compared between WT and SKMiPLA2γKO mice. Mouse numbers are indicated in the parenthesis in the bar graphs. (G–J) glucose tolerance test (G and H) and insulin tolerance tests (I and J) for WT and SKMiPLA2γKO mice either on a normal-chow (NC) diet or a high-fat (HF) diet were performed. AUC, area under the curve in arbitrary units (A.U.). (K) Blood insulin levels from 5 WT to 6 SKMiPLA2γKO mice after HF feeding were determined by ELISA analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and §p < 0.0001.
Figure 6
Figure 6
Pathologic accumulation of neutral lipids is attenuated in the liver of the SKMiPLA2γKO mouse following high-fat feeding Skeletal muscle specific knockout of iPLA2γ alters energy metabolism preventing accumulation of neutral lipids in liver during HF feeding. (A) Hepatic steatosis was evaluated by histological microscopic imaging. H & E staining was performed with liver sections obtained from WT and SKMiPLA2γKO mice after HF feeding. Fat droplets appear as vacuoles in the images. Scale bar: 100 μm. (B–E) Triglyceride (B-C) and diglyceride (D-E) molecular species and amounts in WT (upper spectrum) and SKMiPLA2γKO (lower spectrum) hepatic tissue following HF feeding were determined by shotgun lipidomics utilizing mass spectrometry. Triacylglycerol (TAG) and diacylglycerol (DAG) levels were markedly increased in the livers of WT (n = 5) mice after HF feeding relative to SKMiPLA2γKO (n = 5) mice. Peak heights are normalized to tri-17:1-TAG and di-20:0-DAG internal standards (I.S.). ∗p < 0.05 and ∗∗p < 0.01. See also Figure S6.
Figure 7
Figure 7
Increased glycogen content and decreased fibrosis in liver from the SKMiPLA2γKO mouse (A) Glycogen present in hepatic tissue from WT and SKMiPLA2γKO mice fed a normal-chow diet was visualized by periodic acid-Shiff (PAS) staining with diastase digestion. The intensity of purple-magenta staining reflects the glycogen content of the hepatocytes in the representative histological images. Scale bar: 100 μm. (B) Expression and phosphorylation levels of proteins involved in insulin signaling in livers from 3 WT to 3 SKMiPLA2γKO male mice fed either an NC or HF diet as determined by western blot analyses. Values are the means ± SEM. ∗p < 0.05 and ∗∗p < 0.01. (C) Fibrosis in hepatic tissues from WT and SKMiPLA2γKO mice subjected to HF feeding was visualized by Masson’s trichrome histopathological staining. Collagen fibers appear as filamentous blue structures in the representative histologic images. Scale bar: 100 μm.
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