Browsing by Author "Quadrilatero, Joe"

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Autophagy Regulates the NOTCH Signaling Pathway During Skeletal Muscle Cell Differentiation
(University of Waterloo, 2021-06-01) Pathmarajan, Rishiga; Quadrilatero, Joe
Research has indicated a crucial role for autophagy during skeletal muscle differentiation. More so, the inhibition of autophagy using 3MA (3-methyladenine), CQ (chloroquine), and shRNA against Atg7 has been shown to impair myocyte fusion and differentiation. Thus far, research in skeletal muscle literature has primarily focused on the degradative system of autophagy, highlighting cell survival, and thereby overlooking the direct effects on numerous signaling processes, including the regulation of NOTCH signaling. The NOTCH signaling developmental pathway is implicated in a broad range of developmental processes, including cell fate, proliferation, and differentiation. Although, there is growing evidence of crosstalk between autophagy and NOTCH signaling in hemopoiesis, cardiogenesis, and neurogenesis, limited studies have investigated the role of autophagy in regulating NOTCH signaling during skeletal muscle cell differentiation. Therefore, in order to examine the role of NOTCH signaling, our laboratory has characterized NOTCH signaling during C2C12 myoblast differentiation. We have inhibited γ-secretase with DAPT treatment that decreased the production of NOTCH1 receptor and NOTCH1ICD intracellular domain (ICD) levels to discern whether NOTCH signaling is required in myogenesis. Furthermore, autophagy was induced with rapamycin (RAPA), and inhibited with CQ to manipulate NOTCH signaling and assess whether autophagy is an important regulator of NOTCH signaling. Additionally, using a genetic approach, C2C12 cells were stably transfected with shRNA against Atg7 (shAtg7) to assess differences in NOTCH1ICD levels during myogenic differentiation. We first confirmed that the downregulation of NOTCH1 and NOTCH1ICD occurs alongside increased autophagic flux during C2C12 myoblast differentiation. Furthermore, we found that induction of autophagy with RAPA was associated with decreased NOTCH1ICD levels, while inhibition of autophagy with CQ was associated with increased NOTCH1ICD levels in proliferating myoblasts. Similarly, shAtg7 cells showed 0.6-fold increase in NOTCH1ICD levels during differentiation. However, inhibition of γ-secretase with DAPT in shAtg7 cells decreased NOTCH1ICD levels and was associated with rescued myogenic differentiation. Collectively, our results indicate that autophagy may be an important regulator of NOTCH signaling, thus playing a critical role in both skeletal muscle cell maintenance and myogenesis.
The Development of a Green Fluorescent Protein Reporter System for Apoptosis Repressor with Caspase Recruitment Domain Promoter Analysis
(University of Waterloo, 2017-01-31) Turner, Erin; Quadrilatero, Joe
Apoptosis is a form of controlled cell death that is important to tissue homeostasis. There are a variety of pro- and anti-apoptotic proteins that play an important role in the regulation of cell death. Apoptosis repressor with caspase recruitment domain (ARC) is a potent anti-apoptotic protein that has the ability to inhibit cell death through all the major apoptotic pathways. ARC is highly expressed in post-mitotic tissue such as cardiac muscle, neurons, and skeletal muscle, but is expressed at much lower levels in other tissues. However, ARC protein levels can increase dramatically when some tissues become cancerous. Research has shown that this increase in ARC protein content results in resistance to chemotherapeutic drugs. Additionally, decreasing ARC protein in cancer cell lines has been shown to be effective in increasing the sensitivity of these cancer cells to apoptosis-inducing agents. Therefore, ARC presents an attractive therapeutic target. The development of a reporter system that is driven by the ARC promoter region would allow for the monitoring of ARC expression during a high-throughput drug screen. Green fluorescent protein (GFP) is a valuable tool commonly used to analyse gene expression in cells and presents numerous advantages over a luciferase-based reporter system. First, GFP can be detected without the lysing the cells and without the addition of costly substrates. Second, fluorescence can be detected by a simple plate reader assay. Thus, a GFP reporter plasmid under the control of the ARC promoter region was developed. However, GFP production was limited in cells transfected with the reporter plasmid and significant changes in GFP fluorescence as ARC protein levels increased was not detected by the fluorescent plate reader assay. Ultimately, more research is needed to develop a reporter plasmid that responds to ARC promoter activation and can be detected by a plate reader assay.
Examining Autophagy and Mitophagy as Inducible Mechanisms of Cellular Remodelling
(University of Waterloo, 2017-11-20) Bloemberg, Darin; Quadrilatero, Joe
Despite an explosion of knowledge regarding the molecular regulation of autophagy since its initial characterization in yeast in the 1990s (including the 2016 Nobel Prize in Physiology or Medicine for Dr Yoshinori Ohsumi), essential questions concerning its biological relevance are unanswered. Importantly, given autophagy’s logical links to the beneficial health effects of relative caloric restriction and exercise, progress is being made towards developing autophagy-inducing drugs intended to generally benefit human health. Although many candidates appear to have such effects in model organisms and are well-tolerated by humans, it remains unclear whether these effects are due to autophagy specifically, as direct autophagy-inducing chemicals have not yet been publicly identified. This lack of precise autophagy-targeting chemicals amplifies and confounds the fact that the biological and physiological impacts of specific autophagy induction are relatively unexplored. Here, several basic cellular effects resulting from autophagy induction by amino acid starvation or rapamycin (mTOR inhibitor) as well as mitophagy induction by CCCP (depolarizes mitochondrial membranes) were examined. These effects were investigated in Atg7-knockdown C2C12 cells (considered to be autophagy-deficient) and those with Bnip3-knockout. First, previous research has examined the relationship between autophagy and senescence caused by various stimuli; results have shown that autophagy promotes and attenuates senescence, depending on the study. Although, whether autophagy induction itself causes senescence has not been examined. We demonstrate that repeated administration of C2C12 cells with low staurosporine (STS) doses causes senescence characterized by G1 cell cycle arrest, enlarged cell and nuclei size, increased senescence-associated heterochromatic foci (SAHF), increased senescence-associated acid b–galactosidase activity (SA-Bgal), and myogenic differentiation impairment. However, none of these cellular features occurred in cells repeatedly incubated in amino acid and serum free media (HBSS), which massively induced autophagy. Additionally, while senescent cells were protected from cell death caused by the DNA damaging agent cisplatin, HBSS-treated cells were not. When Atg7-deficient cells were intermittently given low dose STS, senescence did not occur, likely due to the vastly decreased ability to actually survive without functional autophagy. Therefore, Chapter II demonstrates that although autophagic activity is implicated in senescence development, massive sub-lethal autophagy induction itself does not cause senescence. Next, we wanted to further examine autophagy-induced stress resistance development, as some protection from STS-induced cell death was observed in HBSS-treated cells in Chapter II. To do this, normal and Atg7-deficient cells were intermittently incubated in amino acid free media or rapamycin to induce autophagy, and the sensitivity to cell death caused by STS, cisplatin, or hydrogen peroxide was examined. Results indicated that prior repeated amino acid withdrawal protected cells from STS-induced cell death, and this required Atg7. This effect was likely due to reduced mitochondrial-mediated caspase activation, as caspase-9 activity was significantly lower in amino acid starved cells and administering a chemical inhibitor of caspase-3 could mimic the protective effect. Surprisingly, not only were rapamycin-treated cells not similarly protected, but they displayed increased sensitivity to cell death induced by hydrogen peroxide and cisplatin in an Atg7-independent manner. These cells were additionally characterized by greatly enlarged cell size, altered cell cycle profiles, and completely prevented myogenic differentiation. Therefore, Chapter III demonstrates autophagy’s potential as a cellular remodelling mechanism that causes context-dependent stress resistance, and highlights the significant differences between metabolic stimulation of autophagy and that caused by mTOR inhibition. Lastly, to investigate the relevance of mitophagy and mitochondria-specific mechanisms in mediating this observed autophagy-induced stress resistant phenotype, similar experiments were performed to compare the effects caused by intermittently incubating cells in HBSS or CCCP. Although CCCP treatments did not protect from STS-induced cell death to the same extent as HBSS, both treatments attenuated calcium-induced mitochondrial membrane depolarization and permeability pore formation. In fact, this protection was abrogated in Atg7-deficient cells, demonstrating that autophagy is required for this adaptation. Further examination into mitochondrial function showed that previous intermittent amino acid starvation increased maximal ADP-stimulated cellular oxygen consumption when provided with complex-I and/or complex-II substrates. Additionally, not only was mitochondrial respiration significantly impaired in Atg7-deficient cells, but amino acid starvation did not increase oxygen consumption without Atg7. By generating Bnip3-deficient cells with CRISPR/Cas9, it was also shown that Bnip3 is dispensable for repeated amino acid starvation to cause resistance to STS-induced cell death and to increase maximal mitochondrial respiration. Therefore, Chapter IV demonstrates that specific amino acid starvation-induced autophagy causes mitochondrial remodelling resulting in increased stress resistance and function, and furthermore that Bnip3 may have a redundant role in this regard.
Examining the role of autophagy and mitophagy in regulating muscle differentiation
(University of Waterloo, 2019-05-22) Baechler, Brittany Lindsay; Quadrilatero, Joe
Autophagy is a degradative process that is used to eliminate intracellular organelles and protein aggregates. Further, a selective form of autophagy, termed mitophagy, is used to specifically degrade mitochondria. Autophagy/mitophagy is important for eliminating damaged/dysfunctional mitochondria to limit ROS levels and apoptosis, and is also required during erythrocyte and myoblast differentiation. Moreover, recent studies have demonstrated that mitophagy is required to initiate mitochondrial biogenesis during myogenic differentiation. Previous work in our lab has demonstrated that autophagy-deficient myoblasts fail to differentiate, have increased mitochondrial dysfunction, and have elevated levels of apoptotic signaling. Therefore, the purpose of this thesis was to determine the role of autophagy- and mitophagy-related proteins during myogenic differentiation. Chapter 2 demonstrated that canonical mitophagy is disrupted in ATG7-deficient cells, but that mitochondria can still be degraded using an alternative mitophagy pathway. However, we also determined that mitochondrial damage was increased in ATG7-deficient cells, suggesting that targeted degradation of damaged mitochondria specifically is impaired in ATG7-deficient cells. Moreover, we found that increasing the expression of the mitophagy receptor protein BNIP3 was able to partially recover myogenesis in ATG7-deficient cells. Chapter 3 then explored the requirement for the mitophagy-related proteins BNIP3L/NIX and BNIP3 during myogenic differentiation, and found that a deficiency in either of these proteins was disruptive to myogenesis. Further, we demonstrated that bnip3-/- cells showed elevated levels of mitochondria-mediated apoptotic signaling, suggesting impairment in the elimination of dysfunctional mitochondria. Moreover, bnip3-/- cells had increased autophagy-related protein expression. Interestingly, we found that overexpression of ATG7 or treatment with the autophagy inducer rapamycin can disrupt myogenic differentiation in C2C12 myoblasts, suggesting that elevated autophagy might inhibit myogenesis. Additionally, Chapter 2 and Chapter 3 demonstrated that mitochondrial signaling and mitochondrial protein expression is reduced in both shAtg7 and bnip3-/- cells, suggesting impairment in mitochondrial remodelling during differentiation. Therefore, Chapter 4 examined whether upregulating mitochondrial biogenesis can compensate for a potential reduction in autophagy/mitophagy during differentiation. Interestingly, we found that treating ATG7- and BNIP3-deficient cells with SNP, a mitochondrial biogenesis inducer, caused increased mitochondrial biogenesis- and mitochondria-related protein expression, as well as an increase in differentiation and myotube formation. Overall, this thesis demonstrated that autophagy and mitophagy are important during myogenic differentiation, and that these processes must be tightly regulated in order to ensure that cell death is limited and differentiation can progress properly.
Examining the role of caspase-2 in skeletal muscle cell differentiation
(University of Waterloo, 2016-09-26) Boonstra, Kristen; Quadrilatero, Joe
Prior research has indicated a crucial role for apoptotic signaling in skeletal muscle cell differentiation. Although a number of caspases (-3, -8, -9) have been implicated in this process, few prior investigations have identified a role for the most enigmatic member of the caspase family, caspase-2. Due to its unique nuclear localization as well as its purported roles in cell cycle regulation and DNA damage response; caspase-2 is a likely candidate for regulating differentiation. In order to examine the role of caspase-2 in myocyte differentiation, we assessed enzyme activity throughout the time course of C2C12 differentiation. Additionally, we stably transfected C2C12 cells with caspase-2 shRNA to assess the impact of a caspase-2 knockdown on myocyte differentiation. Finally, we identified the subcellular localization of caspase-2 and p21 throughout the early stages of differentiation. Enzyme activity of caspase-2 transiently increased more than two-fold within 24 hours of differentiation induction, with levels returning to normal by day 7, indicating that the enzyme likely plays a role in the differentiation process. Furthermore, knockdown of caspase-2 dramatically impaired myotube formation and induction of cell cycle inhibitor p21 and myogenic regulatory factor myogenin. Caspase-3 activity was also ablated in the caspase-2 knockdown C2C12 cells. Finally, subcellular fractionation of C2C12 cells at early time points in differentiation revealed a nuclear retention of both caspase-2 and p21 throughout the process. Given the nuclear localization of caspase-2 and p21 as well as the impairment in p21 induction in caspase-2 KD cells, we propose that the role of caspase-2 in myocyte differentiation is to regulate p21 induction at the onset of differentiation. Collectively, the results of this study highlight a novel function for caspase-2 in regulating myocyte differentiation.
Examining the Role of DNM1L-Dependent Mitochondrial Fission and Mitophagy Receptors During Skeletal Muscle Differentiation
(University of Waterloo, 2020-07-27) Ma, Andrew; Quadrilatero, Joe
Mitochondrial fission is necessary for the remodelling of existing mitochondria during skeletal muscle differentiation. When there is a need for muscle regeneration or repair, stem cell-like myoblasts exit the cell cycle, restructure their mitochondrial networks, and fuse together to form multinucleated myotubes. Studies investigating the cellular process of DNM1L (dynamin 1-like)-dependent mitochondrial fission during skeletal muscle differentiation are limited. Here, we demonstrated the effect of DNM1L inhibition with mdivi 1 (mitochondrial division inhibitor-1), a pharmacological inhibitor of DNM1L self-assembly, during murine C2C12 myoblast differentiation. Myoblasts treated with mdivi-1 exhibited a hyperfused mitochondrial network which consisted of increased branch lengths and inversely decreased the number of mitochondrial puncta, suggesting the reduction of DNM1L-driven mitochondrial fission events. Furthermore, inadequate mitochondrial fission suppressed MYH (myosin heavy chain) levels and concomitantly decreased myoblast cell fusion during myotube formation. To further support the notion that DNM1L is required for myoblast differentiation, we attempted to genetically knockdown Dnm1l expression with a short hairpin RNA which marginally decreased DNM1L protein content and dramatically abolished MYH levels. In contrast, DNM1L overexpression in differentiated myoblasts did not impair MYH levels. Moreover, we examined the effect of excess mitochondrial fission with CCCP (carbonyl cyanide 3 chlorophenylhydrazone), a chemical inducer of mitochondrial depolarization, which promoted MAP1LC3B (microtubule associated protein 1 light chain 3 beta) conversion indicating that CCCP stimulated autophagy, an intracellular degradation system for the removal of proteins and organelles. Previous studies demonstrated the importance for the degradation of mitochondria facilitated by the mitophagy receptors BNIP3 (BCL2 interacting protein 3) and PRKN (parkin RBR E3 ubiquitin protein ligase) but overlooked the relation of DNM1L during skeletal muscle differentiation. In this study, we examined the mitophagy-related proteins BNIP3 and PRKN in the context of DNM1L inhibition during myoblast differentiation. Importantly, mdivi-1 treated myoblasts overexpressed with BNIP3 ameliorated myoblast cell fusion and upregulated MYH, whereas PRKN overexpression ineffectively restored myotube formation. Collectively, our findings further support the integral role of DNM1L in the process of mitochondrial fission, as well as emphasize the interplay between DNM1L and mitophagy receptors during C2C12 myoblast differentiation.
Interplay between Autophagy and Apoptosis During Skeletal Muscle Differentiation
(University of Waterloo, 2020-07-07) Fatemeh, Keyvani; Quadrilatero, Joe
Our previous studies showed that autophagy regulates apoptosis and is required for proper skeletal muscle differentiation. Previously, we inhibited autophagy in C2C12 cells using 3MA (3-methyladenine) treatment or shRNA against ATG7, both of which resulted in elevated apoptotic signaling and impaired skeletal muscle differentiation. In the present study, we treated C2C12 cells with ad-DN-ATG5 (adenovirus expressing the dominant negative form of the autophagy related protein 5) to interrupt autophagosome formation and inhibit autophagy. The ad-DN-ATG5 treated C2C12 cells displayed elevated apoptosis (increased CASP3 (caspase3) activation) as well as lower MYH (myosin heavy chain) expression and impaired myoblast fusion and differentiation. The increased CASP3 activation in ad-DN-ATG5 treated C2C12 cells was accompanied by significantly reduced BECN1 (beclin 1) levels. Studies in other cell types and contexts have indicated that CASP3 could cleave BECN1 and inactivate BECN1 autophagic function. Therefore, we investigated whether STS (staurosporine)-induced CASP3 activation is also accompanied by lower BECN1 levels. STS treatment also resulted in reduced BECN1 levels in differentiating C2C12 cells. Next, we investigated the importance of BECN1 regulator; AMBRA1 (activating molecule in BECN1 regulated autophagy protein 1), during C2C12 differentiation. Silencing AMBRA1 expression in C2C12 cells using an AMBRA1 siRNA, lead to reduced levels of anti-apoptotic protein BCL2 (BCL2 apoptosis regulator) as well as significantly lower MYH expression. Collectively, the result of this study i) confirm the previously observed protective role of autophagy against apoptosis during skeletal muscle differentiation, ii) introduce BECN1 as a potential factor contributing in the interplay between autophagy and apoptosis, and iii) indicate an important role for AMBRA1 in regulating apoptotic signaling and skeletal muscle differentiation.
Investigating the Importance of RAB7 in C2C12 Myoblast Differentiation
(University of Waterloo, 2021-06-01) Thoms, James; Quadrilatero, Joe
Prior research has shown that autophagy and mitophagy are intimately linked to skeletal muscle differentiation and myogenesis. Recent studies show that the RAB7 cycle is crucial in multiple stages of autophagy and mitophagy; however, few studies examine the importance of RAB7 in muscle physiology. The objective of this study was to explore the significance of RAB7 in mammalian myoblast differentiation and myogenesis. The protein expression and localization patterns of 4 constituents of the RAB7 cycle (RAB5, CCZ1, RAB7, and RABGDI) were characterized over 5 days of differentiation. These experiments showed two major findings. Differentiation of C2C12 cells induced changes in protein content and localization of RAB5, CCZ1, RAB7, and RABGDI, meaning that differentiation and the RAB7 cycle are linked. Furthermore, RAB7 mostly localizes to mitochondrial-enriched fractions, suggesting that RAB7 is highly active and participates in mitochondria dynamics. Next, differentiating C2C12 cells were transfected with RAB7 siRNA or chronically treated with CID1067700. These experiments show three notable findings. RAB7 inhibition results in negligible changes to RAB5, CCZ1, and RABGDI content suggesting there is a compensatory RAB7-independent mechanism. Differentiation and myogenesis are affected by RAB7 inhibition as observed by dramatic decreases in MYH content and various morphological measures. These differentiation deficits were likely caused by defective autophagy and ubiquitin proteasome system (UPS), as given evidence by accumulating LC3-II, unstable SQSTM1, decreased proteasome activity, and potentially lessened autophagic flux. Overall, this is the first study to show that RAB7 is critical to mammalian myoblast differentiation and myogenesis, and that RAB7-mediated defects in differentiation are likely caused by faulty autophagy, the UPS, and the crosstalk between them.
Rapid determination of myosin heavy chain expression in rat, mouse, and human skeletal muscle using multicolor immunofluorescence analysis
(Public Library of Science (PLOS), 2012) Bloemberg, Darin; Quadrilatero, Joe
Skeletal muscle is a heterogeneous tissue comprised of fibers with different morphological, function, and metabolic properties. Different muscles contain varying proportions of fiber types; therefore, accurate identification is important. A number of histochemical methods are used to determine muscle fiber type; however, these techniques have several disadvantages. Immunofluorescence analysis is a sensitive method that allows for simultaneous evaluation of multiple MHC isoforms on a large number of fibers on a single cross-section, and offers a more precise means of identifying fiber types. In this investigation we characterized pure and hybrid fiber type distribution in 10 rat and 10 mouse skeletal muscles, as well as human vastus lateralis (VL) using multicolor immunofluorescence analysis. In addition, we determined fiber type-specific cross-sectional area (CSA), succinate dehydrogenase (SDH) activity, and a-glycerophosphate dehydrogenase (GPD) activity. Using this procedure we were able to easily identify pure and hybrid fiber populations in rat, mouse, and human muscle. Hybrid fibers were identified in all species and made up a significant portion of the total population in some rat and mouse muscles. For example, rat mixed gastrocnemius (MG) contained 12.2% hybrid fibers whereas mouse white tibialis anterior (WTA) contained 12.1% hybrid fibers. Collectively, we outline a simple and time-efficient method for determining MHC expression in skeletal muscle of multiple species. In addition, we provide a useful resource of the pure and hybrid fiber type distribution, fiber CSA, and relative fiber type-specific SDH and GPD activity in a number of rat and mouse muscles.
Understanding the Role of Mitochondrial Remodeling during Myogenesis, Postnatal Muscle Growth, and Disuse Atrophy
(University of Waterloo, 2024-09-23) Rahman, Fasih; Quadrilatero, Joe
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.