Heat acclimation increases mitochondrial respiration capacity of C2C12 myotubes and protects against LPS-mediated energy deficit

Abstract

This work investigated the effect of a 6-day heat acclimation (HA) protocol on myotube metabolic responses at baseline and in response to a subsequent lipopolysaccharide (LPS) challenge. C2C12 myotubes were incubated for 2 h/day at 40 °C for 6 days (HA) or maintained at 37 °C (C). Following 24-h recovery, myotubes were challenged with 500 ng/ml LPS for 2 h, then collected for analysis of protein markers of mitochondrial biogenesis and macronutrient storage. Functional significance of these changes was confirmed with mitochondrial respiration and glycolytic measurements on a Seahorse XF-96 analyzer. HA stimulated mitochondrial biogenesis and increased indicators of mitochondrial content [SIRT1 (+ 62%); PGC-1α (+ 57%); NRF-1 (+ 40%); TFAM (+ 141%); CS (+ 25%); CytC (+ 38%); all p < 0.05]. Altered lipid biosynthesis enzymes [p-ACCa:ACC (+ 59%; p = 0.04) and FAS (- 86%; p < 0.01)] suggest fatty acid generation may have been downregulated, whereas increased GLUT4 (+ 69%; p < 0.01) and LDH-B (+ 366%; p < 0.01) suggest aerobic glycolytic capacity may have been improved. Mitochondrial biogenesis signaling in HA myotubes was suppressed by 500 ng/ml LPS (PGC-1α, NRF-1, TFAM; all p > 0.05) but increased LDH-B (+ 30%; p = 0.02) and CPT-1 (+ 55%; p < 0.01) suggesting improved catabolic function. Basal respiration was increased in HA myotubes (+ 8%; p < 0.01) and HA myotubes maintained elevated basal respiration during LPS challenge (+ 8%; p < 0.01). LPS reduced peak respiration in C myotubes (- 6%; p < 0.01) but did not impair peak respiration in HA myotubes (p > 0.05). Oxidative reliance was elevated in HA over that in control (+ 25%; p < 0.01) and in HA + LPS over C + LPS (+ 30%; p < 0.01). In summary, HA stimulated mitochondrial biogenesis in C2C12 myotubes. HA myotubes exhibited (1) elevated basal/peak mitochondrial respiration capacities; (2) greater oxidative reliance; and (3) protection against LPS-mediated respiration impairment. Collectively, these data suggest HA may improve aerobic metabolism in skeletal muscle and protect against LPS-mediated energy deficit.

Heat acclimation increases inflammatory and apoptotic responses to subsequent LPS challenge in C2C12 myotubes

“…These findings are important because LPS compromises aerobic metabolism in skeletal muscle cells (Frisard et al 2010;Park and Jeoung 2016). We referred to the metabolic improvements in that study as Bmetabolic cross-tolerance^ (Patton et al 2018) and noted that our findings in skeletal muscle cells paralleled the Bheat-acclimation mediated cross-tolerance^(HACT) that had been reported by another group in cardiomyocytes (Assayag et al 2012).…”

“…These changes promoted mitochondrial biogenesis that led to improved functional metabolic outcomes when myotubes that received hyperthermia treatment (HA) were subsequently challenged with LPS. Specifically, we noted that HA myotubes exhibited a reduced suppression of peak respiratory capacity in response to LPS challenge and an overall lower reliance on anaerobic glycolysis to maintain energy status (Patton et al 2018). These findings are important because LPS compromises aerobic metabolism in skeletal muscle cells (Frisard et al 2010;Park and Jeoung 2016).…”

“…For that reason, each mild heat stress exposure was maintained for 127 min; afterwards, myotubes were returned to the control incubator for a minimum of 24 h recovery prior to harvest or any subsequent experiment being performed. This repeated mild heat stress protocol (2 h/day of 40°C exposure over 6 consecutive days) was utilized because it is known to mimic the intramuscular conditions that exist during a standard heat acclimation protocol (Watkins et al 2008) and matches the protocol that was utilized in our prior report (Patton et al 2018). For reference, two other research groups have utilized repeated thermal exposure to stimulate acquisition of the heat acclimation (HA) phenotype in C2C12 myotubes.…”

“…Protein expression was normalized to a representative β-actin, and protein activation/inactivation was normalized to either total protein or representative β-actin. Data presented in this manuscript are meant to accompany a recent report by our group in this journal which described the effect of HA on PPAR signaling, mitochondrial biogenesis, and regulators of macromolecule synthesis (both with and without LPS stimulation) (Patton et al 2018). To maintain consistency between the two studies, the same sets of cell lysates were analyzed and reported on in both manuscripts.…”

“…Myotubes that received the continuous hyperthermia protocol also exhibited elevated myosin heavy chain (MHC) type I and decreased MHC type II, suggesting a fast to slow shift in myotube Bfiber-type^that the authors attributed to activation of PGC-1α (Yamaguchi et al 2010). A recent study by our group indicates that 6 days of 2 h/day hyperthermia exposure (40°C) increases AMPK, SIRT-1, and PGC-1α levels in differentiated C2C12 myotubes (Patton et al 2018). These changes promoted mitochondrial biogenesis that led to improved functional metabolic outcomes when myotubes that received hyperthermia treatment (HA) were subsequently challenged with LPS.…”