The dose–response effects of arachidonic acid on primary human skeletal myoblasts and myotubes

Figures & data

Table 1. Materials and reagents information.

Material/Reagent	Company	Catalog #
Human Skeletal Myoblasts	Lonza Technologies (Portsmouth, NH, USA)	2580
Skeletal Muscle Growth Media and Bullet Kit		3245
Trypsin and Trypsin Neutralizing Solution Reagent Pack		5034
MTT Proliferation Assay	ATCC (Manassas, VA, USA)	30-1010K
Human Skeletal Myoblasts	Thermo Fisher Scientific (Waltham, MA, USA)	A11440 & A12555
Low-Glucose Dulbecco’s Modified Eagle’s Medium		11885092
Horse Serum		26050070
ptgs1		Hs00377726_m1
ptgs2		Hs00153133_m1
elf1		Hs00152844_m1
myh1		Hs00428600_m1
myh2		Hs00430042
myh3		Hs01074230
myh7		Hs01110632
myh8		Hs00267293
Vinculin	Cell Signaling Technologies Antibodies (Danvers, MA, USA)	13901
p-p70 T389		9234
Total p70		2708
p-AKT S473		9271
Total AKT		9272
p-S6 S235/236		4858
p-S6 S240/244		2215
Total S6		2217
COX1		9896
COX2		12282
p-TSC1 S1387		23402
Total TSC2		4308

Figure 1. AA stimulates prostaglandin production in a dose-dependent manner. Prostaglandin hormones PGE₂ and PGF_2α were measured using ELISAs. (A) PGE₂ and (B) PGF_2α concentrations are shown for proliferating myoblasts treated with AA for 72 hours. (C) PGE₂ and (D) PGF_2α concentrations for differentiating myotubes treated with AA for 48 hours. Data are expressed as mean ± SEM (***p < 0.001; **p < 0.01; *p < 0.05; all compared to respective control).

Figure 2. AA reduces myoblast viability at high and moderate doses. Skeletal muscle myoblasts were proliferated for 48 hours with various concentrations of AA. (A) Cell viability was measured using 3-(4, 5-Dimethylthiazol-2-Yl)-2, 5-Diphenyltetrazolium Bromide assay. Viability was affected at concentrations of 50, 25, 12.5, 6.25, 3.15, and 1.56 µM AA. (B) Cell counts were measured using Trypan Blue (0.4%) and an automated counter. Data are expressed as mean ± SEM (***p < 0.001; **p < 0.01; *p < 0.05; all compared to control). n = 3 independent experiments.

Figure 3. AA reduces myotube formation in a dose-dependent manner. Differentiating skeletal muscle myoblasts were treated with decreasing concentrations of AA for 72 hours and then fixed and prepared for fluorescent confocal microscopy and gene using an antibody that recognizes embryonic myosin heavy chain. (A) Representative images were generated using a Zeiss LSM 700 using Zen software. AA concentrations are provided for each image. Analyses of images from two independent experiments for (B) myotube area, (C) myotube fusion percentage, and (D) total DAPI. Myotubes were also analyzed for myosin heavy chain mRNA expression of (E) MYH1, MYH2, MYH3, MYH7, and MYH8. Data are expressed as mean ± SEM (***p < 0.001; **p < 0.01; *p < 0.05; all compared to control). n = 3 independent experiments.

Figure 4. High doses of AA impair the protein kinase B/AKT pathway in proliferating human skeletal myoblasts. Western immunoblotting was performed using lysates from human skeletal myoblasts that were treated for 48 hours with AA. (A) Representative images are shown. Densiometric quantification was used and proteins were normalized to their respective totals for (B) p-TSC2 S138, (C) p-AKT S473, (D) p-S6 235, and (E) p-4EBP1 S65. Data are expressed as mean ± SEM and represent four independent experiments (***p < 0.001; **p < 0.01; *p < 0.05; all compared to control). n = 4 independent experiments.

Figure 5. Effect of AA on COX inflammatory pathway in proliferating human skeletal myoblasts. (A) PTGS1 and PTGS2 mRNA levels were examined in cells treated with various concentrations of AA for 48 hours. COX protein levels were examined in cells treated with various concentrations of AA or equivalent diluent (ethanol) for 48 hours. (B) Representative western blots are shown; purified COX proteins were run to ensure accuracy of band identification. (C) Densiometric quantifications of Cox-1 and Cox-2. Data are expressed as mean ± SEM and represent three independent experiments (***p < 0.001; **p < 0.01 *p < 0.05; all compared to control).

Figure 6. AA does not impair the protein kinase B/AKT pathway in differentiating human skeletal myotubes. Various concentrations of AA were used to treat human skeletal myotubes for 72 hours. (A) Representative western blots are shown. Densitometric quantification was used and proteins were normalized to their respective totals for (B) p-AKT, (C) p-S6 235, (D) p-S6 240, and (E) p-p70 T389. Data are expressed as mean ± SEM and represent four independent experiments (***p < 0.001; **p < 0.01 *p < 0.05).

Figure 7. Effect of AA on COX inflammatory pathway in differentiating human skeletal myotubes. (A) PTGS1 and PTGS2 mRNA levels were examined in cells treated with various concentrations of AA for 48 hours. COX protein levels were examined in cells treated with various concentrations of AA or equivalent diluent (ethanol) for 48 hours. (B) Representative western blots are shown; purified COX proteins were run to ensure accuracy of band identification. (C) Cox1 and Cox2 protein levels in cells treated with various concentrations of AA for 48 hours. Data are expressed as mean ± SEM and represent three independent experiments (***p < 0.001; **p < 0.01 *p < 0.05; compared to control).

Figure 8. Celecoxib does not attenuate the effect of AA at high doses on myotubes. Differentiating skeletal muscle myoblasts were treated with decreasing concentrations of AA and Celecoxib for 72 hours and then fixed and prepared for fluorescent confocal microscopy and gene using an antibody that recognizes embryonic myosin heavy chain. Analyses of images experiments for (A) myotube area, (B) myotube fusion percentage, and (C) total DAPI. Myotubes were also analyzed for myosin heavy chain mRNA expression of (D) MYH1, MYH2, MYH3, MYH7, and MYH8. Data are expressed as mean ± SEM (***p < 0.001; **p < 0.01; *p < 0.05; all compared to control). n = 3 independent experiments.