Abstract
Measuring mitochondrial respiration in cultured cells is a valuable tool to investigate the influence of physiological and disease-related factors on cellular metabolism; however, the details of the experimental workflow greatly influence the informative value of the results. Working with primary cells and cell types capable of differentiation can be particularly challenging. We present a streamlined workflow optimised for investigation of primary human skeletal muscle cells. We applied the workflow to differentiated and undifferentiated cells and we investigated the effect of TGFβ1 treatment. Differentiation of myoblasts to myotubes increased mitochondrial respiration and abundance of mitochondrial enzymes and mitochondrial marker proteins. Differentiation also induced qualitative changes in mitochondrial protein composition and respiration. TGFβ1 reduced complex IV protein MTCO1 abundance in both myoblasts and myotubes. In myoblasts, spare electron transport system (ETS) capacity was reduced due to a reduction in maximal oxygen consumption. In TGFβ1-treated myotubes, the reduction in spare ETS capacity is mainly a consequence of increased oxidative phosphorylation capacity and complex III protein UQCRC2. Taken together, our data shows that it is important to monitor muscle cell differentiation when mitochondrial function is studied. Our workflow is not only sensitive enough to detect physiological-sized differences, but also adequate to form mechanistic hypotheses.
Highlights
Skeletal muscle is by far the biggest organ of the human body, accounting for roughly 40% of body mass in non-obese individuals and is responsible for 22% of basic metabolic rate[1]
We describe the difference between the differentiated and undifferentiated cells and as an example we interrogate the influence of TGFβ1 on mitochondrial respiration and abundance of key mitochondrial enzymes in differentiated and undifferentiated cells
TGFβ1 treatment serves as an example to demonstrate that our workflow is suitable for uncovering small differences in mitochondrial respiration and enzyme abundance in human skeletal muscle cells and can be successfully employed to elucidate the underlying mechanisms
Summary
Skeletal muscle is by far the biggest organ of the human body, accounting for roughly 40% of body mass in non-obese individuals and is responsible for 22% of basic metabolic rate[1]. Peripheral insulin resistance in skeletal muscle leads to reduced postprandial glucose clearance and is considered the starting point for the development of type 2 diabetes[4,5]. It is accompanied by ectopic lipid accumulation and mitochondrial dysfunction[6,7]. We describe a workflow for assaying the influence of candidate factors on mitochondrial respiration and enzyme content in differentiated and undifferentiated primary human skeletal muscle cells. TGFβ1 treatment serves as an example to demonstrate that our workflow is suitable for uncovering small differences in mitochondrial respiration and enzyme abundance in human skeletal muscle cells and can be successfully employed to elucidate the underlying mechanisms
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