Characterizing mitochondrial phenotypes and MERCS in aged human skeletal muscle myoblasts.

Myoblasts from older donors exhibited senescence features including elevated SA-β-gal activity and reduced Lamin B1 expression.

• Primary skeletal muscle myoblasts from young and elderly donors were compared.
• SA-β-gal (senescence-associated beta-galactosidase) activity was elevated in aged myoblasts.
• Lamin B1 expression, a marker whose reduction is associated with cellular senescence, was decreased in older donor cells.
• These features confirmed the senescent phenotype of aged myoblasts used in the study.

Aged myoblasts displayed increased mitochondrial oxidative stress compared to young myoblasts.

• Mitochondrial reactive oxygen species (mtROS) levels were elevated in myoblasts from older donors.
• This increased oxidative stress was associated with other aging-related phenotypes observed in the cells.
• mtROS scavenging was used as an experimental intervention to assess the role of oxidative stress in MERCS formation.

Aged myoblasts exhibited marked mitochondrial hyperfusion and increased mitochondrial DNA content.

• Mitochondrial morphology was assessed and showed hyperfusion (elongated, interconnected mitochondrial networks) in old compared to young myoblasts.
• Mitochondrial DNA (mtDNA) content was significantly increased in aged myoblasts.
• Mitochondrial hyperfusion is described as a notable phenotype accompanying cellular aging in human myoblasts.

Mitochondrial oxygen consumption rate and membrane potential per mitochondrial area were comparable between young and old myoblasts despite aging-associated changes.

• Mitochondrial oxygen consumption rate (OCR) was measured and found to be similar between young and elderly donor myoblasts when normalized per mitochondrial area.
• Mitochondrial membrane potential per mitochondrial area was also not significantly different between the two groups.
• These findings suggest that mitochondrial functional capacity per unit area is maintained in aged myoblasts despite structural and oxidative changes.

MERCS were significantly elevated in aged myoblasts and were reduced by scavenging mitochondrial reactive oxygen species.

• Mitochondria-endoplasmic reticulum contact sites (MERCS) were quantified and found to be significantly elevated in myoblasts from older donors compared to young donors.
• Treatment with a mitochondrial ROS scavenger reduced MERCS in aged myoblasts, indicating an association between mtROS and MERCS formation.
• This finding links elevated oxidative stress to increased MERCS formation during cellular aging.
• MERCS elevation is proposed to accompany mitochondrial hyperfusion as part of an adaptive response to maintain mitochondrial function under elevated oxidative stress.