Combining in vivo 2-photon imaging with photoactivatable fluorescent labeling reveals strikingly low rates of mitochondrial dynamics in skeletal muscle.

Mitochondrial dynamics involve two distinct and opposing processes, fusion and fission. Traditionally we assess fusion and fission by snapshots of protein markers at distinct time points or in vitro models to infer outcomes in vivo. Recent technological advancements enable visualization of mitochondrial dynamics in vivo using fluorescent microscopy. Our study modified this technique to evaluate mitochondrial dynamics in skeletal muscle, comparing young (6mo) and old (24mo) mice in vivo and contrasting this to ex vivo and in vitro models. We hypothesized that in vitro and ex vivo models would have higher rates of dynamics than in vivo models and that young animals would have higher rates than old animals. We electroporated mitochondrial matrix-targeted photo-activatable GFP into the tibialis anterior (TA) of young and old C57Bl6 mice and imaged using multiphoton microscopy. We also measured rates of mitochondrial dynamics using single fibers isolated from the TA of the electroporated mice, as well as C2C12 myotubes transfected with the same plasmids. We found that the rates of dynamic events in vivo are slower than previously indicated, with the C2C12 myoblasts having the fastest rates of dynamic events across all models. We also observed that dynamic rates are slower in old animals compared to young animals. Finally, we found that rates of dynamic events were higher in old animals after an acute bout of exercise. Our data demonstrate it is possible to directly measure rates of mitochondrial dynamics in vivo. This technique provides a powerful tool to answer experimental questions about mitochondrial dynamics of skeletal muscle.