Ethanol‐Impaired Myogenic Differentiation is Associated With Decreased Myoblast Glycolytic Function

Abstract

Background: Myopathy affects nearly half of individuals with alcohol use disorder (AUD), and impaired skeletal muscle regenerative potential is a probable contributing factor. Previous findings from our laboratory indicate that chronic in vivo and in vitro ethanol (EtOH) treatment decreases myogenic potential of skeletal muscle myoblasts. Myogenesis, a highly coordinated process, requires shifts in cellular metabolic state allowing for myoblasts to proliferate and differentiate into mature myotubes. The objective of this study was to determine whether alcohol interferes with myoblast mitochondrial and glycolytic metabolism and impairs myogenic differentiation. Methods: Myoblasts were isolated from vastus lateralis muscle excised from alcohol-na€ ıve adult male (n = 5) and female (n = 5) rhesus macaques. Myoblasts were proliferated for 3 days (day 0 differentiation; D0) and differentiated for 5 days (D5) with or without 50 mM EtOH. Metabolism was assessed using a mitochondrial stress test to measure oxygen consumption (OCR) and extracellular acidification (ECAR) rates at D0. Differentiation was examined at D5. Expression of mitochondrial and glycolytic genes and mitochondrial DNA (mtDNA) was measured at D0 and D5. Results: Ethanol significantly (p < 0.05) increased myoblast maximal OCR and decreased ECAR at D0, and decreased fusion index, myotubes per field, and total nuclei at D5. The EtOH-induced decrease in ECAR was associated with the EtOH-mediated decreases in fusion index and myotubes per field. EtOH did not alter the decrease in glycolytic gene expression and increase in mtDNA from D0 to D5. Conclusion: During myoblast proliferation, EtOH decreased glycolytic metabolism and increased maximal OCR, suggesting that myoblast metabolic phenotype was dysregulated with EtOH. The EtOH-induced decrease in ECAR was associated with decreased differentiation. These findings suggest that EtOH-mediated shifts in metabolic phenotype may underlie impaired differentiation, which has important clinical implications for myogenesis in those affected by alcoholic myopathy.

Serum-free cultures of C2C12 cells show different muscle phenotypes which can be estimated by metabolic profiling

“…4 A,B). Previously, significantly downregulated mRNA expression of hexokinase ( HK ), aldolase ( ALDO ), pyruvate kinase ( PK ) in myotubes versus myoblasts were reported 30 . Consistent results previously reported that glycolysis occurs in proliferating C2C12 myoblasts, which later is followed by a differentiation process accompanied by autophagy and mitophagy, resulting in increasing oxidative phosphorylation activity 31 , 32 .…”

Alcohol Impairs Immunometabolism and Promotes Naïve T Cell Differentiation to Pro-Inflammatory Th1 CD4+ T Cells

“…Naïve CD4 + T cells that were not activated and differentiated were plated (density of 2x10 6 /ml per well) in growth media without anti-CD3 and anti-CD28. Water pans containing 75 mM ethanol were changed daily to maintain the 50 mM ethanol concentration in the incubator atmosphere as previously described ( 26 ). Both 25 mM and 50 mM ethanol were used in preliminary studies, but 25 mM had no significant effect on CD4 + T cell differentiation (data not shown), hence all subsequent experiments were performed with 50 mM.…”

“…This impairment of glycolysis within the liver occurs, in part, due to an increase in the NADH/NAD + ratio, which depletes free NAD + cofactor availability for glycolytic enzymes, such as glyceraldehyde 3-phosphate dehydrogenase ( 25 ). Ethanol also decreases myoblast (muscle stem cells) glycolytic function and leads to decreased differentiation ( 26 ). Further, ethanol increases reactive oxygen species (ROS) formation in mitochondria, which can damage mitochondrial DNA and ETC proteins, leading to impaired mitochondrial function and cell death ( 24 , 27 ).…”

A review of alcohol–pathogen interactions: New insights into combined disease pathomechanisms

“…Parallel in vitro studies using naïve myoblasts exposed to 50 mM alcohol for 3 days show that direct alcohol exposure results in a contrasting shift in metabolic phenotype. This may be associated with the impaired myoblast differentiation as shown by the close correlation between ECAR and fusion index and the number of myotubes per field (Levitt et al, 2020).…”