Aksu-Menges Evrim, Kumtepe Eray Taha, Akpinar Gurler, Balci-Hayta Burcu
Department of Medical Biology, Faculty of Medicine, Hacettepe University, Ankara, Turkey.
Department of Medical Biology, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey.
J Cell Mol Med. 2025 Jan;29(2):e70370. doi: 10.1111/jcmm.70370.
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.
线粒体在能量代谢中发挥着重要作用，尤其是在骨骼肌等对能量需求较高的组织中。了解这些细胞中线粒体的蛋白质组组成对于阐明肌肉生理和病理机制至关重要。然而，由于复杂的细胞结构以及容易被其他细胞器污染，从原代人骨骼肌细胞中有效分离线粒体一直具有挑战性。在此，我们比较了四种从原代人骨骼肌成肌细胞中分离线粒体的不同方法，涉及总蛋白产量、线粒体富集能力以及分离组分的纯度。我们提出了一种改进方法，该方法将差速离心与低渗肿胀步骤以及随后的纯化过程相结合，以尽量减少细胞污染。我们通过用线粒体、胞质和核标记物进行蛋白质免疫印迹证实其能够获得高度纯净的线粒体组分，从而验证了我们的方法。我们证明可以对分离出的线粒体进行蛋白质组分析。我们的方法为在健康和疾病状态下研究骨骼肌生物学背景下的线粒体动力学、生物发生和功能提供了一个有价值的工具。这一方法学进展为线粒体研究及其在肌病、肌肉减少症、恶病质和代谢紊乱中的应用开辟了新途径。
