Examination of Mitochondrial Morphology and Movement Within Skeletal Muscle

Mitochondria are vital organelles, critical for energy supply and cell survival. Such functional versatility is paralleled by the structural complexity of the organelle. Mitochondria are dynamic and can continuously alter their morphology. The movement of these organelles is controlled by their interaction with the cytoskeleton. To date, the morphology and movement of mitochondria in muscle has yet to be fully explored. Thus, the overall purpose of this Dissertation was to: 1) examine the effects of chronic muscle use and disuse on mitochondrial morphology and regularity protein expression, 2) investigate the cytoskeletal filaments responsible for governing mitochondrial movement within skeletal muscle cells, and 3) elucidate the effects of oxidative stress on organelle moment and morphology. The mitochondrial network within cells is mediated by opposing fission and fusion processes. In Manuscript #1, we investigated the expression of the proteins responsible for these events during conditions of altered oxidative capacity within rat skeletal muscle. The results demonstrate that chronic muscle use increases the ratio of fusion:fission proteins, leading to reticular mitochondria, whereas muscle disuse and aging result in a decrease in this ratio, culminating in a disruption of the mitochondrial network, and fragmented organelles. Manuscript #2 focuses on determining the cytoskeletal elements that are primarily responsible for mitochondrial movements in muscle cells, and asks whether calcium can influence organelle motility. Dynein and kinesins motor proteins use microtubules to transport cargo throughout the cell. The ability of mitochondria to respond to calcium has previously been found to be mediated by motor protein/adaptor complexes in neurons. Using live-cell imaging, we monitored mitochondrial motility and found that they move primarily along microtubules tracks. Moreover, displacement of mitochondria is regulated by calcium, which inhibits organelle movements through the microtubule motor protein adapter, Milton. By understanding the regulation of these movements we can elucidate the underlying basis for organelle interactions, leading to the formation of the mitochondrial reticulum, and the distribution of energy in muscle cells. The focus of Manuscript #3 was intended to assess whether mitochondrial dynamics are altered with an acute exposure to oxidative stress. In addition, we sought to elucidate the interplay between mitochondrial morphology and stress responses. Mitochondrial stress is speculated to result in the activation of at least three cellular responses, including mitophagy, and the mitochondrial- and ER-unfolded protein responses. In our study, we found that oxidative stress halts mitochondrial movement and induces mitochondrial fragmentation in myoblasts. The observed oxidative stress-induced mitochondrial fragmentation is mediated by Drp1. Moreover, we demonstrated that the unfolded protein responses are activated prior to the initiation of mitophagy during oxidative stress.
Collectively, our data illustrate that mitochondrial morphology is affected during conditions of chronic muscle use and disuse, and the movement of mitochondria in muscle cells is influenced by both oxidative stress and calcium signalling.