207 Temporal heat stress impact on gene regulation of skeletal muscle hypertrophy in bovine myocytes

Abstract Skeletal muscle can be susceptible to environmental stress stimuli, causing shifts in molecular responses. This study elucidated the degree of molecular response in bovine myocytes by measuring exome-wide transcriptome abundance following temporal heat stress. Bovine satellite cells (BSCs) were extracted from Holstein calves (n=3, BW: 77.10 ± 2.02 kg). Following myogenic differentiation, confluent myocytes were exposed to one of three treatment groups for 3 h duration: 38 °C (control; CON; n=3), 39.5 °C (moderate heat stress; MHS; n=3), and 41 °C (extreme heat stress; EHS; n=3). Post temporal heat stress exposure, RNA was extracted for total RNA sequencing (RNASeq). RNA sequencing was performed using 1×100 bp format, with raw reads aligned to the bovine ARS-UCD1.2 reference genome. Myocytes underwent mRNA extraction for gene expression and were analyzed via RT-qPCR. Protein differentiations were analyzed using western blot (WB), and myocytes were stained for immunofluorescence microscopy to evaluate differentiation index, myotube diameter, and protein synthesis rate. Differentially expressed genes (DEGs) were assessed for gene ontology enrichment using GOrilla and selected using a significance threshold of P < 0.05 after a Benjamini and Hochberg multiple testing correction, regardless of Log2 fold change. Gene expression, protein levels, differentiation index, myotube diameter, and protein synthesis were analyzed using ANOVA with Tukey’s HSD post hoc test with a significance threshold of P < 0.05. Samples submitted for RNASeq detected 888 DEGs for MHS vs. CON and 2,590 DEGs for EHS vs. CON, with 590 DEGs shared between MHS and EHS treatments compared to CON. Elevated expression by EHS of FOXO6 and a 3-fold enrichment for genes associated with heat shock protein isoforms (HSP) binding (q < 0.001) was detected under RNASeq analysis. In addition, exposure to EHS conditions, resulted in the upregulation of HSPA6, HSPA1A, HSPH1, HSPA8, and HSP90AA1 detected in RNASeq (P < 0.05), and HSP20, HSP27, HSP70, and HSP90 confirmed by RT-qPCR (P < 0.001). Gene expression of MyoD1 and protein abundance increased under MHS and EHS conditions (P < 0.05). Myogenin gene expression in EHS was upregulated (P < 0.01) in gene expression and protein abundance. Myocytes exposed to EHS upregulated IGF-1 gene expression and led to subsequent altering of Akt/mTOR/p70S6 pathway (P < 0.05), associated with protein synthesis. Calculated differentiation index and subsequent myotube diameter increased in MHS and EHS vs. CON cells, coinciding with elevated expression levels in myosin heavy chain isoforms (MHC) I, IIA, and IIX (P < 0.05). Alteration of key anabolic pathways associated with protein synthesis, modulation of myogenic regulatory factors, and the large number of DEGs detected in HS exposed myocytes demonstrates the potential of phenotypic alterations to myotube size and protein synthesis.