Omega-3 Polyunsaturated Fatty Acids Mitigate Palmitate-Induced Impairments in Skeletal Muscle Cell Viability and Differentiation

Abstract

Accumulation of excess saturated free fatty acids such as palmitate (PAL) in skeletal muscle leads to reductions in mitochondrial integrity, cell viability and differentiation. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) counteract PAL-induced lipid accumulation. EPA and DHA, as well as the n-3 PUFA docosapentaenoic acid (DPA), may therefore mitigate PAL-induced lipotoxicity to promote skeletal muscle cell survival and differentiation. C2C12 myoblasts were treated with 50 μM EPA, DPA, or DHA in the absence or presence of 500 μM PAL for 16 h either prior to myoblast analysis or induction of differentiation. Myoblast viability and markers of apoptosis, endoplasmic reticulum (ER) stress and myotube differentiation capacity were investigated using fluorescence microscopy and immunoblotting. High-resolution respirometry was used to assess mitochondrial function and membrane integrity. PAL induced cell death via apoptosis and increased protein content of ER stress markers BiP and CHOP. EPA, DPA, and DHA co-treatment maintained cell viability, prevented PAL-induced apoptosis and attenuated PAL-induced increases in BiP, whereas only DPA prevented increases in CHOP. PAL subsequently reduced protein content of the differentiation marker myogenin and inhibited myotube formation, and all n-3 PUFAs promoted myotube formation in the presence of PAL. Furthermore, DPA prevented PAL-induced release of cytochrome c and maintained mitochondrial integrity. These findings demonstrate the n-3 PUFAs EPA, DPA and DHA elicit similar protective effects against PAL-induced impairments in muscle cell viability and differentiation. Mechanistically, the protective effects of DPA against PAL lipotoxicity are attributable in part to its ability to maintain mitochondrial respiratory capacity via mitigating PAL-induced loss of mitochondrial membrane integrity.

Keywords: DHA; DPA; EPA; mitochondria; myoblast; myotube; n-3 PUFA; saturated fatty acid.

FIGURE 1

EPA, DPA, and DHA protect against PAL-induced reductions in cell viability. Trypan blue exclusion test dose response experiment in myoblasts following 16 h of palmitate (PAL) treatment to assess cell viability (A). Trypan blue exclusion test of cell viability following treatment with 500 μM PAL and/or 50 μM EPA, DPA, or DHA (B). Representative images of nuclear DAPI staining following treatments (C). Average number of nuclei/field (D), brightness/nuclei (E), nuclear area (F), and nuclear perimeter (μm) (G). Treatments that do not share letters are considered statistically significant (p < 0.05; myoblasts were cultured and harvested independently to generate a total sample size of 3–4 biological replicates). Scale bar represents 125 μm. Values are presented as mean ± SEM.

FIGURE 2

EPA, DPA, and DHA attenuate PAL-induced increases in DNA fragmentation characteristic of apoptosis. Representative images of myoblast TUNEL staining 16 h following treatment with 500 μM PAL and/or 50 μM EPA, DPA or DHA (A). Percentage of TUNEL positive cells relative to total number of cells in the field (B). Treatments that do not share letters are considered statistically significant (p < 0.05; myoblasts were cultured and harvested independently to generate a total sample size of 3 biological replicates). Scale bar represents 125 μm. Values are presented as mean ± SEM.

FIGURE 3

EPA, DPA, and DHA attenuate PAL-induced increases in the ER stress markers BiP and CHOP. Representative images of ER stress proteins BiP and CHOP following 16 h treatment with 500 μM PAL and/or 50 μM EPA, DPA or DHA (A) and representative image of a stain free blot used for normalization shown with molecular weight markers indicated. Quantified protein content of BiP (B) and CHOP (C). In the absence of PAL, little to no CHOP protein was detectable. All densitometry values are expressed relative to protein content determined by stain free imaging and presented in arbitrary units relative to CON. Treatments that do not share letters are considered statistically significant (p < 0.05; myoblasts were cultured and harvested independently to generate a total sample size of 5 biological replicates). Values are presented as mean ± SEM.

FIGURE 4

EPA, DPA, and DHA protect against the inhibitory effects of PAL on myotube differentiation. Protein expression of the myogenic protein myogenin 120 h following treatment with 500 μM PAL and/or 50 μM EPA, DPA, or DHA, with a representative image of a stain free blot used for normalization shown with molecular weight markers indicated (A). Quantified protein expression of the myogenic protein myogenin (B). All densitometry values are expressed relative to protein content determined by stain free imaging and presented in arbitrary units relative to CON. Treatments that do not share letters are considered statistically significant (p < 0.05; n = 6 biological replicates). Average number of myotubes/field (C) and fusion index (number of nuclei in tubes relative to the total number of nuclei expressed as a percentage) (D). Representative images of each treatment group at 120 h (n = 3 biological replicates) (E). Scale bar represents 275 μm. Values are presented as mean ± SEM.

FIGURE 5

Treatment with DPA and/or PAL does not alter mitochondrial respiration, but DPA maintains mitochondrial membrane integrity in response to PAL. In (A), JO₂ represents mitochondrial O₂ flux. Cytochrome c retention test response expressed as (PMDGS-CytoC/CytoC) × 100 (B). Treatments that do not share letters are considered statistically significant (p < 0.05). Myoblasts were cultured and harvested independently to generate a total sample size of 4 biological replicates. Values are presented as mean ± SEM.

FIGURE 6

Treatment with DPA and/or PAL does not alter myoblast mitochondrial content. Protein content of mitochondrial OXPHOS complexes following 16 h treatment with 500 μM PAL and/or 50 μM EPA, DPA, or DHA and representative images (A–F). All densitometry values are expressed relative to protein content determined by stain free imaging (A; representative image shown with molecular weight markers indicated) and presented in arbitrary units relative to CON. Myoblasts were cultured and harvested independently to generate a total sample size of 6 biological replicates. Values are presented as mean ± SEM.