PSIII-5 Effects of heat stress and citrulline and arginine supplementation on muscle-related gene expression and myotube formation in bovine myocytes.

Abstract This study examined the effects of heat stress (HS) (41°C) and amino acid treatments, including citrulline (CIT) and arginine (ARG), on muscle-related gene expression in bovine myocytes. We hypothesized that CIT would increase ARG availability, which increases protein synthesis and myotube formation under HS conditions. Bovine satellite cells were isolated from a female fetus (23.1 kg) and plated at a density of 4 × 10⁴ cells in DMEM containing 10% FBS. The cells were incubated at 38°C with 5% CO₂ until they reached 80% confluency. The culture medium was replaced with differentiation medium (control media) consisting of 2% HS SILAC DMEM Flex Media (DMEM without ARG and CIT). The treatment groups were as follows: T1 (control media), T2 (control media with 120 µM ARG), and T3 (control media with 120 µM CIT). The cells were exposed to 38°C or 41°C, and harvested at 48 hours after temperature exposure. Real-time q-PCR was used to determine the gene responses related to HS and muscle growth for all treatment groups. The cells were stained to determine the differentiation fusion index and muscle fiber size. Data was analyzed using two-way ANOVA to evaluate the effects of treatments, temperature, and their interaction on gene expression, differentiation fusion index, and muscle fiber size. Tukey’s test was used for post-hoc comparisons. Results are presented as significant at P < 0.05. Analyses were conducted using GraphPad Prism 9.4.1 to assess treatment effects under normal and HS conditions. Gene expression of IGF-I was affected by treatment (P = 0.013) and its interaction with temperature (P = 0.003), with the highest expression in the CIT group under HS. Myf5 decreased with heat and ARG treatment (P < 0.001, P = 0.006). MyoD increased at 41°C (P = 0.015), with the highest levels in control. Myogenin was upregulated under HS, especially with CIT (P < 0.001, P = 0.004). Expression of MHC I was elevated with ARG at 41°C (P < 0.001). MHC ⅡA was affected by the interaction (P = 0.018), while MHC ⅡX expression was influenced by treatment (P = 0.01) and temperature (P= 0.015). Heat stress altered gene expression, with CIT enhancing IGF-1 and myogenin, while ARG increased MHC I. At 38°C, the treatments were statistically different (P=0.004) from one another with CIT (34.0%) being the highest. There were no differences between treatments in myotube length (P=0.820) or width (P=0.34) at 38°C. At 41°C, CIT and ARG treatments were both significantly different (P< 0.001) than control and both caused a greater myotube length (P=0.007). However, there was no difference in myotube width (P = 0.760). Therefore, CIT and ARG can increase fusion index and myotube length during times of HS while not affecting myotube width.