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Updated: Dec 10, 2025
Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays
Published on: February 10, 2022
Skeletal muscle mitochondrial fragmentation and impaired bioenergetics from nutrient overload are prevented by carbon
Heath G Gasier1,2, Jacob Dohl2, Hagir B Suliman1,3
1Department of Anesthesiology, Duke University Medical Center, Durham, North Carolina.
Abstract:
Nutrient excess increases skeletal muscle oxidant production and mitochondrial fragmentation that may result in impaired mitochondrial function, a hallmark of skeletal muscle insulin resistance. This led us to explore whether an endogenous gas molecule, carbon monoxide (CO), which is thought to prevent weight gain and metabolic dysfunction in mice consuming high-fat diets, alters mitochondrial morphology and respiration in C2C12 myoblasts exposed to high glucose (15.6 mM) and high fat (250 µM BSA-palmitate) (HGHF). Also, skeletal muscle mitochondrial morphology, distribution, respiration, and energy expenditure were examined in obese resistant (OR) and obese prone (OP) rats that consumed a high-fat and high-sucrose diet for 10 wk with or without intermittent low-dose inhaled CO and/or exercise training. In cells exposed to HGHF, superoxide production, mitochondrial membrane potential (ΔΨm), mitochondrial fission regulatory protein dynamin-related protein 1 (Drp1) and mitochondrial fragmentation increased, while mitochondrial respiratory capacity was reduced. CO decreased HGHF-induced superoxide production, Drp1 protein levels and mitochondrial fragmentation, maintained ΔΨm, and increased mitochondrial respiratory capacity. In comparison with lean OR rats, OP rats had smaller skeletal muscle mitochondria that contained disorganized cristae, a normal mitochondrial distribution, but reduced citrate synthase protein expression, normal respiratory responses, and a lower energy expenditure. The combination of inhaled CO and exercise produced the greatest effect on mitochondrial morphology, increasing ADP-stimulated respiration in the presence of pyruvate, and preventing a decline in resting energy expenditure. These data support a therapeutic role for CO and exercise in preserving mitochondrial morphology and respiration during metabolic overload.
Insights
Carbon monoxide (CO) and exercise preserve mitochondrial function during metabolic overload. These interventions protect against nutrient excess-induced mitochondrial damage and improve energy expenditure, suggesting therapeutic potential.
Area of Science:
- Mitochondrial biology
- Metabolic disease research
- Cellular physiology
Background:
- Nutrient excess impairs skeletal muscle mitochondrial function, contributing to insulin resistance.
- Carbon monoxide (CO) may prevent metabolic dysfunction in high-fat diet models.
- Mitochondrial morphology and respiration are critical for metabolic health.
Purpose of the Study:
- To investigate CO's effects on mitochondrial morphology and respiration in C2C12 myoblasts under high-glucose, high-fat conditions (HGHF).
- To examine skeletal muscle mitochondrial morphology, respiration, and energy expenditure in obese prone (OP) versus obese resistant (OR) rats.
- To assess the combined effects of CO and exercise training on mitochondrial function and energy expenditure in rats.
Main Methods:
- C2C12 myoblasts were exposed to HGHF conditions with or without CO.
- Skeletal muscle mitochondria from OR and OP rats were analyzed for morphology, distribution, and respiration.
- Rats were subjected to high-fat, high-sucrose diets with intermittent low-dose inhaled CO and/or exercise training.
Main Results:
- HGHF increased superoxide production, mitochondrial fragmentation, and decreased respiratory capacity in myoblasts.
- CO treatment reduced oxidative stress, mitochondrial fragmentation, and enhanced mitochondrial respiration in HGHF-exposed myoblasts.
- OP rats exhibited smaller mitochondria with disorganized cristae and lower energy expenditure compared to OR rats.
- Combined CO and exercise treatment yielded the most significant improvements in mitochondrial respiration and energy expenditure.
Conclusions:
- Carbon monoxide (CO) preserves mitochondrial morphology and function under metabolic stress.
- Exercise training, especially combined with CO, enhances mitochondrial respiration and energy expenditure.
- CO and exercise represent potential therapeutic strategies for metabolic overload and insulin resistance.
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