Mitochondrial dysfunction and consequences in calpain-3-deficient muscle

Abstract

Background: Nonsense or loss-of-function mutations in the non-lysosomal cysteine protease calpain-3 result in limb-girdle muscular dystrophy type 2A (LGMD2A). While calpain-3 is implicated in muscle cell differentiation, sarcomere formation, and muscle cytoskeletal remodeling, the physiological basis for LGMD2A has remained elusive.

Methods: Cell growth, gene expression profiling, and mitochondrial content and function were analyzed using muscle and muscle cell cultures established from healthy and calpain-3-deficient mice. Calpain-3-deficient mice were also treated with PPAR-delta agonist (GW501516) to assess mitochondrial function and membrane repair. The unpaired t test was used to assess the significance of the differences observed between the two groups or treatments. ANOVAs were used to assess significance over time.

Results: We find that calpain-3 deficiency causes mitochondrial dysfunction in the muscles and myoblasts. Calpain-3-deficient myoblasts showed increased proliferation, and their gene expression profile showed aberrant mitochondrial biogenesis. Myotube gene expression analysis further revealed altered lipid metabolism in calpain-3-deficient muscle. Mitochondrial defects were validated in vitro and in vivo. We used GW501516 to improve mitochondrial biogenesis in vivo in 7-month-old calpain-3-deficient mice. This treatment improved satellite cell activity as indicated by increased MyoD and Pax7 mRNA expression. It also decreased muscle fatigability and reduced serum creatine kinase levels. The decreased mitochondrial function also impaired sarcolemmal repair in the calpain-3-deficient skeletal muscle. Improving mitochondrial activity by acute pyruvate treatment improved sarcolemmal repair.

Conclusion: Our results provide evidence that calpain-3 deficiency in the skeletal muscle is associated with poor mitochondrial biogenesis and function resulting in poor sarcolemmal repair. Addressing this deficit by drugs that improve mitochondrial activity offers new therapeutic avenues for LGMD2A.

Keywords: Calpain-3 deficiency; LGMD2A; Mitochondria; Muscle membrane repair.

Fig. 1

Capn3-deficient and Capn3-sufficient immortalized primary muscle cells. Genotyping: agarose gel showing the WT (Capn3^+/+) Capn3 gene band at 500 bp and the Capn3-deficient (Capn3^−/−) gene band at 300 bp. The large T gene is shown at 700 bp (a). Differentiation test performed on Capn3^+/+ and Capn3^−/− primary immortalized cell cultures. Hoechst 33342 was used to stain the nuclei and show multinucleated myotubes formation after 2 days in a low-serum medium (b). Western blots and quantification of muscle-specific proteins in myoblasts and myotubes. Myogenin and dysferlin were used to verify the myogenicity of cells. The expression of myogenin and dysferlin was normalized to vinculin loading control. Since myogenin and dysferlin expression varied between calpain-deficient and calpain-sufficient myoblasts, we have expressed increased expression of these differentiation markers in myotubes as a percentage of expression seen in myoblasts (c). Analyses were performed on n = 4 different cell culture flasks for proliferation and n = 3 for differentiation. **Significantly different with p < 0.01; *significantly different with p < 0.05

Fig. 2

Gene expression profiling of Capn3-deficient vs. Capn3-sufficient myoblasts with an Illumina BeadChip array. Dendrogram results attesting the good clustering of Capn3-deficient (Capn3^−/−) myoblast samples when compared to corresponding WT (Capn3^+/+) muscle cells using Partek Genomics Suite, a statistical analysis and interactive visualization software (a). Pathway analysis was done using the Ingenuity Pathways Analysis software that allows functional integration of molecular pathways. Ingenuity pathway analysis demonstrating changes in mitochondrial biogenesis, lipid metabolism, and protein transport in myoblasts. Pink indicates an upregulation and green indicates a downregulation of the specific gene in Capn3^−/− myoblasts compared to Capn3^+/+ myoblasts (b). Three different samples were used per group. cRNA was synthesized from 250 ng of total RNA for each sample. Gene pathways were prepared by ingenuity pathways analysis according to a gene list based on the interaction of a gene candidate with a p value of 0.001 and a fold increase ≥ 4

Fig. 3

Capn3 deficiency induces a deficiency in mitochondrial biogenesis in vitro. Analysis of the mitochondrial DNA-to-nuclear DNA ratio in WT (Capn3^+/+) and Capn3-deficient (Capn3^−/−) myoblasts. The RT-qPCR analysis was performed on n = 2 flasks for Capn3^+/+ and n = 6 for Capn3^−/− (a). Flow cytometry analysis of 10-nonyl acridine orange fluorescence (NAO) (cardiolipin content) in WT and Capn3^−/− myoblasts (b, c). The analysis was done on n = 6 flasks per group. ***Significantly different with p < 0.001; *significantly different with p < 0.05

Fig. 4

Capn3 deficiency induces a mitochondrial metabolism deficiency in vitro. Live WT (Capn3^+/+) and Capn3-deficient (Capn3^−/−) myoblasts stained with DiOC6 to show their mitochondrial inner membrane potentials. Pictures were taken with the same focus, laser intensity, and exposure time in both panels (a). Flow cytometric analysis of 3,3′-dihexyloxacarbocyanine iodide (DiOC6) (mitochondrial inner membrane potential) in Capn3^+/+ and Capn3^−/− myoblasts (b). Histogram representing Capn3^−/− (pink), Capn3^+/+ (green), and Capn3^+/+ and CCCP (uncoupler, purple) DiOC6 fluorescence (c). The DiOC6/NAO fluorescence ratio in Capn3^+/+ and Capn3^−/− myoblasts (d). The analysis was done on n = 6 flasks per group. ***Significantly different with p < 0.001

Fig. 5

Capn3 deficiency induces mitochondrial deficiency in vivo. Western blot of vinculin (control), TFAM, and PDK1 in the TA muscle lysates from WT (Capn3^+/+) and Capn3-deficient (Capn3^−/−) mice (a). Western blot quantification of TFAM expression in Capn3^+/+ and Capn3^−/− muscle, normalized to vinculin expression (n = 3) (b). Western blot quantification of PDK1 expression in the Capn3^+/+ and Capn3^−/− muscle, normalized to vinculin expression (n = 3) (c). Creatine kinase (CK) activity in the Capn3^+/+ and Capn3^−/− TA muscle, normalized to protein concentration (n = 8) (d). LDH activity measured in the Capn3^+/+ and Capn3^−/− TA muscle, normalized to protein concentration (n = 8) (e). ***Significantly different with p < 0.001; **significantly different with p < 0.01; *significantly different with p < 0.05

Fig. 6

Myoblast proliferation behavior and effect of mitochondrial activity improvement on satellite cell markers. Proliferation kinetics of WT (Capn3^+/+) and Capn3-deficient (Capn3^−/−) myoblasts analyzed through flow cytometry by either CFSE fluorescence decrease (n = 4) (a) or manual counting (n = 5) (b). Expression of Pax7 mRNA (c) and MyoD mRNA (d) after 4 weeks of GW501516 treatment in the quadriceps muscle from 7-month-old Capn3^−/− mice (n = 2). ***Significantly different with p < 0.001; *significantly different with p < 0.05

Fig. 7

The effect of enhanced mitochondrial activity on muscle phenotype in 9-month-old Capn3-deficient mice. EDL and soleus muscle mass (a) specific force in Capn3-deficient (Capn3^−/−) mice treated with GW501516 (GW) or vehicle (b). Twitch-to-tetanus ratio of EDL and SOL in Capn3^−/− mice treated with GW or vehicle (c). Half-relaxation time (HRT) (s) (d), time to peak twitch tension (TTP) (s), of EDL muscle (e) of GW- or vehicle-treated mice. Maximal force relative to baseline for the EDL (f) and soleus (g) muscles in Capn3^−/− mice treated with GW or vehicle. The force is expressed relative to the maximal force at the beginning of the experiment. Significance indicated difference at 3 min

Fig. 8

Biochemical and behavioral assessments after GW501516 treatment. Creatine kinase (CK) activity in the blood serum extracted by heart puncture immediately after euthanasia from Capn3^−/− mice treated with GW or vehicle (a). Time on the Rotarod in seconds before and after treatment of Capn3^−/− mice with GW or vehicle (b). Behavioral activity measurements such as horizontal distance (c), total distance (d), movement number (e), and movement number (f) were used to assess the overall behavioral activity. Experiments were done with n = 6 mice per group. **Significantly different with p < 0.01; *significantly different with p < 0.05

Fig. 9

Membrane repair deficits in Capn3-deficient muscle after laser injury. Response to focal laser injury in isolated EDL fibers from WT ((Capn3^+/+) top panels at 0, 60, and 240 s) or Capn3-deficient ((Capn3^−/−) bottom panels at 0, 60, and 240 s) at 3 months of age. Overlay of the FM 1-43 dye fluorescence (green) on the bright-field (greyscale) image is shown

Fig. 10

Mitochondrial dependence of membrane repair efficiency after laser injury. Response to laser injury in Capn3-deficient (Capn3^−/−) (blue) or WT (black) 3-month-old mice: biceps brachii (n = 10) (a), soleus (n = 10) (b), and EDL (n = 10) muscles (c) and EDL isolated fibers (n = 10) (d). Effect of using CCCP to uncouple the mitochondrial inner potential in response to laser injury in WT EDL muscle (red) and untreated EDL muscle (black) (n = 4 Capn3^+/+, n = 5 Capn3^+/+ + CCCP) (e). Response to laser injury in Capn3^−/− EDL-isolated fibers after activation of mitochondrial activity with pyruvate (100 mM) (green) (n = 11), as compared to untreated Capn3^−/− EDL isolated fibers (blue) (n = 3) (f). Inner membrane potential assessed with DiOC6 dye fluorescence in Capn3^−/− EDL-isolated fibers after pyruvate treatment (n = 10), as compared to untreated Capn3^−/− EDL muscle (n = 10) (g)