Hypotonic Swelling Method for the Isolation of Pure Mitochondria From Primary Human Skeletal Myoblasts for Proteomic Studies

Mitochondria play a fundamental role in energy metabolism, particularly in high-energy-demand tissues such as skeletal muscle. Understanding the proteomic composition of mitochondria in these cells is crucial for elucidating the mechanisms underlying muscle physiology and pathology. However, effective isolation of mitochondria from primary human skeletal muscle cells has been challenging due to the complex cellular architecture and the propensity for contamination with other organelles. Here, we compared four different methods to isolate mitochondria from primary human skeletal myoblasts regarding total protein yield, mitochondrial enrichment capacity and purity of the isolated fraction. We presented a modified method that combines differential centrifugation with a hypotonic swelling step and a subsequent purification process to minimise cellular contamination. We validated our method by demonstrating its ability to obtain highly pure mitochondrial fractions, as confirmed by Western Blot with mitochondrial, cytosolic and nuclear markers. We demonstrated that proteomic analysis can be performed with isolated mitochondria. Our approach provides a valuable tool for investigating mitochondrial dynamics, biogenesis and function in the context of skeletal muscle biology in health and disease. This methodological advancement opens new avenues for mitochondrial research and its implications in myopathies, sarcopenia, cachexia and metabolic disorders.