Dataset Information

Acute Heat Exposure Alters Autophagy Signaling in C2C12 Myotubes.

ABSTRACT: Autophagy is a major intracellular degradation process that is essential for the clearance of unnecessary proteins/organelles and the maintenance of cellular homeostasis. The inhibition of autophagy results in cellular consequences associated with many skeletal muscle pathologies, and therapies designed to elevate autophagic activity may provide protection from such pathologies. Acute exposure to low levels of heat has therapeutic effects; however, the impact of heat on skeletal muscle autophagy remains unclear. In the present study, C2C12 myotubes were maintained at 37°C thermoneutral (TN) or heated at 40°C heat treatment (HT) for 1 h. Myotubes were harvested immediately after heating, or returned to 37°C for recovery of 2 or 24 h. HT resulted in an elevation in pAMPK (T172), Beclin-1, and LC3 II, a marker for autophagosome formation, but no change in p62. In the context of autophagy inhibition with Bafilomycin A1, HT resulted in lower LC3 II compared to TN. The applied heat load induced the heat shock response, as evidenced by immediate upregulation of HSF1 and Hsp70. Hsp70 continued to increase during recovery, whereas pHsp27 was downregulated acutely in response to HT, but retuned to TN levels by 2 h of recovery. HT also reduced the phosphorylation of the MAP-kinases p38 and JNK. These findings suggest that an acute, short bout of mild heat may be beneficial to skeletal muscle by increasing AMPK activity, markers of autophagasome formation, and the heat shock response.

SUBMITTER: Summers CM

PROVIDER: S-EPMC6960406 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Acute Heat Exposure Alters Autophagy Signaling in C2C12 Myotubes.

Summers Corey M CM Valentine Rudy J RJ

Frontiers in physiology 20200108

Autophagy is a major intracellular degradation process that is essential for the clearance of unnecessary proteins/organelles and the maintenance of cellular homeostasis. The inhibition of autophagy results in cellular consequences associated with many skeletal muscle pathologies, and therapies designed to elevate autophagic activity may provide protection from such pathologies. Acute exposure to low levels of heat has therapeutic effects; however, the impact of heat on skeletal muscle autophagy ...[more]

PMID: 31969827

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Project description: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-1? (+?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-1?, 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.

Project description:Muscle protein synthesis and proteolysis are tightly coupled processes. Given that muscle growth is promoted by increases in net protein balance, it stands to reason that bolstering protein synthesis through amino acids while reducing or inhibiting proteolysis could be a synergistic strategy in enhancing anabolism. However, there is contradictory evidence suggesting that the proper functioning of proteolytic systems in muscle is required for homeostasis. To add clarity to this issue, we sought to determine if inhibiting different proteolytic systems in C2C12 myotubes in conjunction with acute and chronic leucine treatments affected markers of anabolism. In Experiment 1, myotubes underwent 1-h, 6-h, and 24-h treatments with serum and leucine-free DMEM containing the following compounds (n = 6 wells per treatment): (i) DMSO vehicle (CTL), (ii) 2 mM leucine + vehicle (Leu-only), (iii) 2 mM leucine + 40 μM MG132 (20S proteasome inhibitor) (Leu + MG132), (iv) 2 mM leucine + 50 μM calpeptin (calpain inhibitor) (Leu + CALP), and (v) 2 mM leucine + 1 μM 3-methyladenine (autophagy inhibitor) (Leu + 3MA). Protein synthesis levels significantly increased (p < 0.05) in the Leu-only and Leu + 3MA 6-h treatments compared to CTL, and levels were significantly lower in Leu + MG132 and Leu + CALP versus Leu-only and CTL. With 24-h treatments, total protein yield was significantly lower in Leu + MG132 cells versus other treatments. Additionally, the intracellular essential amino acid (EAA) pool was significantly greater in 24-h Leu + MG132 treatments versus other treatments. In a follow-up experiment, myotubes were treated for 48 h with CTL, Leu-only, and Leu + MG132 for morphological assessments. Results indicated Leu + MG132 yielded significantly smaller myotubes compared to CTL and Leu-only. Our data are limited in scope due to the utilization of select proteolysis inhibitors. However, this is the first evidence to suggest proteasome and calpain inhibition with MG132 and CALP, respectively, abrogate leucine-induced protein synthesis in myotubes. Additionally, longer-term Leu + MG132 treatments translated to an atrophy phenotype. Whether or not proteasome inhibition in vivo reduces leucine- or EAA-induced anabolism remains to be determined.

Dataset Information

Acute Heat Exposure Alters Autophagy Signaling in C2C12 Myotubes.

Publications

Acute Heat Exposure Alters Autophagy Signaling in C2C12 Myotubes.

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