Abstract

Skeletal muscle plays an important role in systemic metabolism and overall health. Within skeletal muscle, there is a decline in mitochondrial function with age. Under normal healthy conditions, mitochondria remodel via two dynamic processes, mitochondrial protein turnover and mitochondrial dynamics, which includes mitochondrial fission and fusion events. While capturing the dynamic nature of mitochondrial protein turnover in skeletal muscle is now possible, assessing mitochondrial fission and fusion relies on snapshots of protein markers from muscle homogenate at distinct timepoints. Recent technical advancements allow for measurement of mitochondrial dynamics in skeletal muscle in vitro; however, rates of mitochondrial dynamics in other tissues are significantly slower in vivo compared to in vitro conditions. Therefore, the purpose of this study was to develop novel imaging methods to assess mitochondrial dynamics in vivo. Our hypothesis was that we could capture and assess in vivo mitochondrial fission/fusion events using a novel imaging approach. Using multiphoton microscopy, we activated a mitochondrial‐targeted photo‐activated GFP that was electroporated into the tibialis anterior (TA) muscle of 5 adult C57Bl6 mice to assess the rates of mitochondrial fission and fusion events. In vivo mitochondrial fission and fusion rates were about 50 times slower in comparison to previously reported rates in in vitro models. Due to the slower than expected rates of mitochondrial fission/fusion events in skeletal muscle in vivo, we used an additional imaging approach to explore changes mitochondrial morphology over more prolonged periods of time. To do so, we imaged control and denervated TA muscles of 5 adult C57Bl6 mice using an electroporated mitochondrial‐targeted GFP (mitochondrial matrix) and TOM20_tdTomato (outer mitochondrial matrix). We determined that two weeks of denervation decreased the cross‐sectional area that was co‐stained in the denervated limb compared to a control limb, suggesting a decrease in cristae structure. In addition, two weeks of denervation decreased the cross‐sectional area of the population of subsarcolemmal mitochondria in the denervated limb compared to a control limb. Together, our data emphasizes the importance of assessing mitochondrial dynamics in vivo. Additionally, changes to mitochondrial morphology in skeletal muscle fibers are regional, which stresses the importance of assessing mitochondrial subpopulations.