Robust intrinsic differences in mitochondrial respiration and H2O2 emission between L6 and C2C12 cells

Abstract

Rat L6 and mouse C2C12 cell lines are commonly used to investigate myocellular metabolism. Mitochondrial characteristics of these cell lines remain poorly understood despite mitochondria being implicated in the development of various metabolic diseases. To address this need, we performed high-resolution respirometry to determine rates of oxygen consumption and H₂O₂ emission in suspended myoblasts during multiple substrate-uncoupler-inhibitor titration protocols. The capacity for oxidative phosphorylation supported by glutamate and malate, with and without succinate, or supported by palmitoyl-l-carnitine was lower in L6 compared with C2C12 myoblasts (all P < 0.01 for L6 vs. C2C12). Conversely, H₂O₂ emission during oxidative phosphorylation was greater in L6 than C2C12 myoblasts (P < 0.01 for L6 vs. C2C12). Induction of noncoupled respiration revealed a significantly greater electron transfer capacity in C2C12 compared with L6 myoblasts, regardless of the substrate(s) provided. Mitochondrial metabolism was also investigated in differentiated L6 and C2C12 myotubes. Basal rates of oxygen consumption were not different between intact, adherent L6, and C2C12 myotubes; however, noncoupled respiration was significantly lower in L6 compared with C2C12 myotubes (P = 0.01). In summary, L6 myoblasts had lower respiration rates than C2C12 myoblasts, including lesser capacity for fatty acid oxidation and greater electron leak toward H₂O₂. L6 cells also retain a lower capacity for electron transfer compared with C2C12 following differentiation to form fused myotubes. Intrinsic differences in mitochondrial metabolism between these cell lines should be considered when modeling and investigating myocellular metabolism.

Keywords: electron transfer system; lipid oxidation; muscle cells; reactive oxygen species.

Fig. 1.

Glutamate, malate, and succinate (GMS)-supported respiration and H₂O₂ emission of L6 and C2C12 myoblasts. Glutamate and malate are N-linked (N) substrates, donating electrons to complex I. Succinate is S-linked (S), donating electrons to complex II. A and B: rates of oxygen consumption (J̇o₂) and H₂O₂ emission (J̇h₂o₂), respectively, for 0.5 × 10⁶ cells/ml. Inset: respiratory states and paths for electron donation. C: electron leak to H₂O₂ during simultaneous measures of oxygen consumption and H₂O₂ emission. D and E: relative contribution of substrate-linked electron flow to maximal oxidative phosphorylation (OxPhos) (D) and noncoupled electron transfer (E). F: relative contribution of respiratory state to maximal respiration. *P < 0.05 for L6 vs. C2C12. Within-cell line comparisons of interest are described in the main text. Data are presented as mean and standard deviation; n = 5 experiments were run for each cell line. Standard deviation is not presented in D–F. Full details regarding statistical analysis are provided in materials and methods (see Statistical analysis). P, oxidative phosphorylation; E, noncoupled electron transfer; L, leak respiration; ROX, residual oxygen consumption; GM, glutamate + malate; FCCP, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone.

Fig. 2.

Palmitoyl-l-carnitine and malate (PCM)-supported respiration and H₂O₂ emission L6 and C2C12 myoblasts. Palmitoyl-l-carnitine is F-linked (F) and donates electrons to electron-transferring flavoprotein complex and complex I. Malate is provided as a substrate for oxaloacetate production to alleviate build-up of acetyl-CoA, with minimal contribution to respiration. A and B: rates of oxygen consumption (J̇o₂) and H₂O₂ emission (J̇h₂o₂) for 0.5 × 10⁶ cells/ml, respectively. Inset: respiratory states and paths for electron donation. C: electron leak during simultaneous measures of oxygen consumption and H₂O₂ emission. D: relative contribution of respiratory state to maximal respiration. *P < 0.05 for L6 vs. C2C12. Within-cell line comparisons and main effects of interest are described in the main text. Data are presented as mean and standard deviation; n = 5 experiments were run for each cell line. Standard deviation is not presented in D. Full details regarding statistical analysis are provided in materials and methods (see Statistical analysis). P, oxidative phosphorylation; E, noncoupled electron transfer; L, leak respiration; ROX, residual oxygen consumption; FCCP, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone.

Fig. 3.

Reference protocol 2 (RP2) respiration for L6 and C2C12 myoblasts. Octanoyl-l-carnitine is F-linked (F), donating electrons to electron-transferring flavoprotein complex and complex I; malate, pyruvate, and glutamate are N-linked (N), donating electrons to complex I; succinate is S-linked (S), donating electrons to complex II; and glycerophosphate is Gp-linked (Gp), donating electrons to glycerophosphate dehydrogenase complex. A: rates of oxygen consumption (J̇o₂) for 1 × 10⁶ cells expressed relative to the respiration chamber volume. Inset: respiratory states and paths for electron donation. Relative contribution of substrate-linked electron flow to maximal oxidative phosphorylation (OxPhos) (B) and noncoupled electron transfer (C) is shown. D: relative contribution of respiratory state to maximal respiration. *P < 0.05 for L6 vs. C2C12. Statistical significance noted in B reflects differences in F(N) and S between cell lines. Within-cell line comparisons of interest are described in the main text. Data are presented as mean and standard deviation; n = 3 experiments were run for each cell line. Standard deviation is not presented in B–D. Full details regarding statistical analysis are provided in materials and methods (see Statistical analysis). R, routine respiration; P, oxidative phosphorylation; E, noncoupled electron transfer; ROX, residual oxygen consumption; FCCP, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone.

Fig. 4.

Western blot analysis of mitochondrial proteins for L6 and C2C12 myoblasts. A: abundance of mitochondrial subunits from complexes I-V (CI-CV), hydroxyacyl-coenzyme A dehydrogenase (HADH), and tubulin. B: complete Western blot images with molecular weight markers presented in kDa. Data are presented as mean relative to L6 and standard deviation for n = 6 samples for each cell line. Between-cell line differences in Western blot data were analyzed using unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. *P < 0.05 for L6 vs. C2C12.

Fig. 5.

Extracellular flux analysis of L6 and C2C12 myotubes. A: triplicate measures of oxygen consumption rate (OCR) before (basal) and after sequential addition of oligomycin (Oligo), to determine ATP-linked respiration and proton leak; carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), to assess the mitochondrial reserve capacity; and antimycin A (Anti-A) and rotenone (Rot), to assess nonmitochondrial respiration, with no statistical comparisons performed. B: averaged OCR values to perform statistical analysis. C: relative respiratory control after correction for nonmitochondrial rates of oxygen consumption. Data are presented as mean and standard deviation in A and B, with all technical replicates presented in A (n = 36 and n = 39 for L6 and C2C12, respectively) and n = 3 averaged experimental values for each cell line in B. Standard deviation is not presented in C. Differences in OCR and relative respiratory control between L6 and C2C12 myotubes were analyzed using unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. *P < 0.05 for L6 vs. C2C12.