Understanding the Role of Mitochondrial Remodeling during Myogenesis, Postnatal Muscle Growth, and Disuse Atrophy

Abstract

Mitochondria are characterized as the chemical factory of cells. This organelle is fundamental to life and death, by generating chemical energy (i.e., ATP) and regulating cellular stress responses. Importantly, mitochondria have evolved elegant mechanisms to respond to numerous stressors/stimuli. These stressors/stimuli including metabolic and oxidative stress elicit differential responses at the mitochondria level, which is accompanied by a change in its structure and function. Collectively, the change in mitochondrial structure and function is termed mitochondrial remodeling. At the organelle level, the dynamic balance of mitochondrial morphology (i.e., fission/fusion balance) coupled with mitochondrial turnover (i.e., biogenesis and mitophagy) is required for appropriate mitochondrial remodeling. These remodeling processes must occur in a controlled manner to prevent excessive activation of downstream mitochondrial apoptotic signaling events. Although the primary function of mitochondria is to produce energy; their behaviour and response to stressors/stimuli can vary between different tissues. This is particularly relevant in tissues with high metabolic demand, such as skeletal muscle. Within skeletal muscle, there are phenotypically distinct myofibers (e.g., slow-twitch and fast-twitch), and within each myofiber, there are distinct pools of mitochondria (e.g., subsarcolemmal and intermyofibrillar). Given the uniqueness and complexity of skeletal muscle mitochondria, there are several unknowns with respect to mitochondrial remodeling in skeletal muscle. Therefore, the studies in this thesis were designed to better understand mitochondrial remodeling during three important stages: skeletal muscle formation (myogenesis), postnatal muscle growth, and disuse muscle atrophy.

Chapter 1 provides a literature review of mitochondria, mitochondrial quality control, skeletal muscle mitochondria and mitochondrial remodeling during myogenesis, postnatal muscle growth, and disuse atrophy.

Chapter 2 is focused on understanding the interaction between mitochondrial dynamics and turnover during myogenesis in vitro. Enhancing mitochondrial fission increased mitochondrial network fragmentation and mitophagic flux during myogenic differentiation of C2C12 cells, resulting in smaller myotubes without impairing the myogenesis. Despite these morphological changes, higher fission did not affect the levels of mitochondrial turnover proteins. In contrast, greater mitochondrial fusion reduced mitophagic flux, significantly impairing myogenesis and increasing mitochondrial apoptotic signaling. Cells with hyperfused mitochondria also display diminished mitochondrial biogenesis and mitophagy signaling. Enhancing mitophagy in fission-deficient cells reduced mitochondrial apoptotic signaling and biogenesis signaling without impacting myogenesis. Finally, upregulation of mitochondrial biogenesis worsened myogenic defects in fission-deficient cells, independent of changes in mitophagy or mitochondrial protein levels. These findings demonstrate that optimal mitochondrial fission is crucial for regulating both mitophagy and biogenesis during myogenesis.

Chapter 3 then explored the role of mitochondrial remodeling on postnatal skeletal muscle growth. RNA sequencing analyses identified several differentially expressed genes during postnatal development, including upregulation of metabolic genes and a downregulation of genes involved in cell growth and differentiation. In vivo experiments revealed significant increases in body mass, muscle mass, and myofiber cross-sectional area. Mitochondrial maturation during this period was evidenced by increased mitochondrial function, and elevated mitophagic flux, along with increased mitochondrial localization of autophagy and mitophagy proteins. Cellular signaling revealed an increase in anabolic signaling, which was accompanied by enhanced mitophagy and fusion signaling and a simultaneous decrease in mitochondrial biogenesis signaling. In skeletal muscle-specific autophagy-deficient mice, there were no changes in body or muscle mass, nor in mitochondrial function despite ablated mitophagic flux. These mice exhibited compensatory activation of alternative degradative enzymes, including mitochondrial apoptotic signaling and ubiquitin-proteasome signaling, suggesting a shift in degradative pathways to preserve muscle mass and function in young mice. These findings demonstrate that postnatal development is marked by increased mitochondrial activity and mitophagy. Furthermore, while constitutive autophagy deficiency abolishes mitophagic flux, it does not impair muscle growth in young mice.

Chapter 4 examined the role of mitochondrial remodeling with an emphasis on mitophagy during disuse atrophy of mature skeletal muscle. RNA sequencing analyses reveal an upregulation of genes associated with protein degradation, particularly those linked to the ubiquitin-proteasome system and apoptosis, while downregulating genes involved in muscle development and mitochondrial components. Immobilization-induced muscle atrophy affected the large muscles of the hindlimb, with partial recovery following remobilization. Immobilization increased mitophagic flux, which remained elevated following remobilization, alongside a reduction in mitochondrial function. Mitochondrial translocation of mitophagy receptors were identified in immobilization and remobilization muscles. Immobilization also enhanced mitochondrial apoptotic signaling, with increased mitophagy and suppressed mitochondrial biogenesis signaling. Antioxidant during immobilization suppressed mitophagy flux but exacerbated atrophy in fast/glycolytic myofibers without significantly altering markers of mitochondrial remodeling or the localization of autophagy/mitophagy-related proteins. Autophagy inhibition during immobilization also led to atrophy in fast/glycolytic myofibers, inhibiting mitophagic flux without affecting mitochondrial tagging with mitophagy or apoptosis-related molecules. Together, these findings suggest that mitophagy protects against excessive atrophy is muscle due to immobilization.

Finally, Chapter 5 integrates and summarizes the findings from all the studies and highlights the physiological implications. Overall, these insights suggest that targeted therapeutic strategies aimed at enhancing the coordination of mitochondrial remodeling processes could optimize skeletal muscle function. Such strategies would focus on stabilizing the balance between mitochondrial fission and fusion, ensuring efficient mitophagic clearance of damaged or dysfunctional mitochondria, and promoting mitochondrial biogenesis to maintain a healthy mitochondrial network in skeletal muscle cells and tissues.