C2C12 myoblasts (ATCC) were seeded onto non-coated 6-well plates and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Welgene, Korea) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin (Welgene, Korea) in a humidified atmosphere of 95% air and 5% CO2 at 37°C. For proliferation test, 5 mM L-carnitine or 300 uM oleic acid or a mixture (5 mM L-carnitine and 300 μM oleic acid) were added to the growth medium (10% FBS), based on previous studies¹⁵,¹⁷. When myoblasts were confluent (95%), the growth medium was changed to a differentiation medium (DM) supplemented with 2% horse serum (HS), 100 U/mL penicillin, and 100 mg/mL streptomycin (Welgene, Korea). DM was changed every 24 h. For the differentiation test and fusion index assay, 5 mM L-carnitine or 300 μM oleic acid or a mixture were added to DM supplemented with 2% HS and incubated for 96 h. The detailed incubation method for each experiment is described in each figure legend. Cell culture was performed a minimum of six independent times. As the fusion index is a myological indicator showing the degree of myogenic differentiation from myoblasts to myotubes, the index is calculated as the number of nuclei in myotubes divided by the total number of nuclei counted, which is expressed as a percentage. A schematic diagram of the cell culture protocol is presented in Figure 1.

Cell counting and viability assay were conducted using the trypan blue exclusion assay with minor modification¹⁸. C2C12 myoblasts (5 × 104/mL) were plated onto non-coated 60-mm culture dishes and grown in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin in a humidified atmosphere of 95% air and 5% CO² at 37°C for 48 h. DMEM was changed on a daily basis. Following incubation, the cells were trypsinized and stained with 10 μL trypan blue on a patch of parafilm. Non-stained viable cells and stained cells were counted on a hemocytometer under a phase-contrast microscope and expressed as % NT.

As described¹⁵, myotubes were fixed and permeabilized by incubation in ice-cold methanol (Sigma-Aldrich) for 10 min. After rehydration in DPBS three times, the myotubes were incubated in blocking solution including 2% BSA for 30 min at room temperature, followed by incubation in 2% BSA solution containing anti-sarcomeric myosin antibody MF-20 conjugated with Alexa Fluor 488 (eBioscience). After myonuclei were stained with DAPI (Molecular Probes), micrographs were acquired under a fluorescence microscope (Carl Zeiss, Axio Observer). The cells with a minimum number of three myonuclei were counted as differentiated myotubes. A fusion index is a myological indicator showing the degree of myogenic differentiation. The index is calculated as the number of nuclei in myotubes divided by the total number of nuclei counted, which is expressed as a percentage.

Differentiated myotubes were washed with cold DPBS and lysed with TRIzol reagent. Chloroform was added for the separation of RNA from DNA and protein fractions. RNA fraction was precipitated by the addition of isopropanol, followed by centrifugation at 12,000 g for 8 min at 4°C. The RNA pellet was washed with 75% ethanol and centrifuged at 12,000 g for 5 min at 4°C. After removal of ethanol, RNA pellets were air-dried before resuspending with RNAse free water. The RNA concentration was quantified using a NanoDrop spectrophotometer (ND-2000, Thermo Fisher Scientific, USA). Reverse transcription was performed with a cDNA synthesis kit according to the manufacturer’s instruction (Bioneer, Korea). Following complementary DNA synthesis, qRT-PCR was performed in 7500 Real-Time PCR system (Applied Biosystems, USA) using a SYBR Green Master Mix (Bioline, Korea), and the primer pair sets are described in Table 1. Cycle threshold (Ct) values were normalized to the housekeeping gene (HPRT1-F, 5'-GACTTGCTCGAGATGTCATG-3'; HPRT1-R, 5'-TACAGTCATAGGAATGGACC-3').

Immunoblotting was performed as described previously¹⁹. Briefly, differentiated myotubes were lysed in RIPA buffer (10 mM Tris-HCl, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholate, pH 7.5) with cell scraper. Following centrifugation (16,000 g for 15 min at 4°C), supernatants were collected for Bradford assay to quantify total protein concentrations. Following SDS-PAGE, samples were transferred to the PVDF membrane. Subsequently, the membranes were incubated in TBST containing 5% nonfat dry milk for 30 min prior to overnight incubation with respective primary antibodies (rabbit polyclonal anti-VDAC1/porin [Abcam] and mouse monoclonal α-tubulin [Sigma-Aldrich]). After washing twice in TBST, the membranes were further incubated with HRP-conjugated secondary antibodies for 40 min, followed by washing thrice with TBST. Then, membranes were subjected to standard enhanced chemiluminescence (Thermo Fisher Scientific) method for visualization. The resulting band intensities were quantified by ImageJ software (NIH).

The results were presented as mean + SEM for a minimum of six independent experiments. One-way ANOVA was followed by Tukey’s post hoc test to present statistical difference among groups. The statistical significance was set at P-value < 0.05.

To investigate whether L-carnitine and oleate have effects on cell proliferation and viability, myoblasts were incubated at 37°C for 48 h until the assay. Although no effect on cell proliferation was observed with L-carnitine, the cell number was significantly higher in myoblasts conditioned with oleate (24%) or a mixture (27%) than that in non-treated myoblasts (P < 0.05, Figure 2A). Cell viability was not different among all tested groups (Figure 2B).

To investigate the effects of L-carnitine and oleate on myogenic differentiation, the growth medium without L-carnitine and oleate was used for incubation until 95% confluency. After proliferation, the growth medium was changed to the respective DM. The morphological assay indicated that fusion index and myotube width were significantly higher in myotubes conditioned with L-carnitine or oleate than those of non-treated myotubes (P < 0.05). Interestingly, the values of the fusion index and myotube width were significantly higher in myotubes conditioned with a mixture (P < 0.05) in contrast to those with L-carnitine or oleate alone (Figure 3A-C).

As the mitochondrial biogenic process is coupled with initiation of myogenic differentiation, the related gene expressions were measured following differentiation for 96 h. The expressions of deacetylation of peroxisome proliferator- activated receptor-γ coactivator 1-alpha (Pgc1-α), nuclear respiratory factor (Nrf1), and mitochondrial transcription factor A (Tfam) were increased in myotubes conditioned with L-carnitine and oleate, individually, in contrast to those with no treatment, with a greater effect observed in myotubes conditioned with the mixture (P < 0.05, Figure 4). The expression of Ncor1, a corepressor and reciprocal regulator of Pgc1-α, was not changed by individual treatment of L-carnitine and oleate, whereas this gene expression was decreased significantly in myotubes conditioned with a mixture of L-carnitine and oleate than that in non-treated myotubes (P < 0.05, Figure 4).

The protein level of porin was measured in differentiated myotubes as a surrogate marker of mitochondrial mass. The protein expression of porin was significantly increased by individual treatment of L-carnitine and oleate than that in non-treated myotubes (P < 0.05, Figure 5). The protein levels of porin were significantly higher in myotubes conditioned with a mixture in contrast to those with L-carnitine or oleate alone (P < 0.05, Figure 5)