Chronic Contractile Activity Research Articles

Intersection of chronic contractile activity and lysosomal defects in mediating lysosomal adaptations in skeletal muscle cells

Mitophagy is the cellular process that serves to degrade mitochondria when these organelles lose their potential for ATP production, generate excessive reactive oxygen species, and exhibit a reduced membrane potential. The terminal step of mitophagy is mediated by the lysosome, the organelle that degrades defective cellular cargos. Electron microscopy evidence shows that lysosomes become defective in aging muscle, as well as in lysosomal storage diseases, ultimately leading to cellular pathology. Recent research has shown that chronic contractile activity can induce rapid increases in lysosomal gene and protein expression in muscle. Our objective is to investigate whether this adaptation leads to greater lysosomal content and function, thus enhancing the capacity to degrade defective mitochondria. This result would suggest that reduced lysosomal function in muscle could be rescued by exercise. However, the underlying mechanisms mediating lysosomal turnover and function are not clear. To study this, we simulated lysosomal dysfunction in C2C12 myotubes using short-interfering RNA targeting the lysosomal calcium channel mucolipin-1 (MCOLN1) or the autophagosome-lysosome fusion protein, lysosomal-associated membrane protein 2 (LAMP2). We achieved a 60-70% knockdown, as well as a 40-50% knockdown, after a 24-hour transfection, in each lysosomal marker, respectively. The cells were then subjected to four days of electrical stimulation-induced chronic contractile activity. Changes in gene expression were measured via qPCR, while protein content was measured via western blotting techniques, in addition to lysosomal content and functional assessments via confocal microscopy and flow cytometry. Silencing of MCOLN1 or LAMP2 both resulted in a compensatory increase in the transcript and protein levels of lysosomal genes, including the lysosomal transcription factors TFEB, TFE3, as well as lysosomal cathepsins B and D. Interestingly, contractile activity superimposed on the MCOLN1 or LAMP2 knockdown did not further enhance these compensatory increases. This suggests that contractile activity and the cellular signaling that arises from lysosomal defects, operate similarly to initiate the biogenesis of lysosomes as a negative feedback mechanism to enhance lysosomal degradation capacity. The nature of these signaling pathways, potentially involving changes in lysosome-derived intracellular calcium driving organelle synthesis, recycling, or degradation represent novel avenues for upcoming research investigating skeletal muscle phenotypic adaptations. This work is supported by NSERC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Skeletal Muscle Extracellular Vesicles Enhance Mitochondrial Movement and have a Dose-dependent Effect on Increasing Mitochondrial Respiration

Chronic exercise leads to systemic health benefits across tissues, including an increase in mitochondrial function in skeletal muscle. One potential mechanism is via the release of extracellular vesicles (EVs), which can transfer functional cargo to recipient cells. Previously, we have shown that using an in vitro model of exercise, chronic contractile activity (CCA)-derived EVs (CCA-EV) increased mitochondrial biogenesis in healthy myoblasts. Here, we hypothesize that CCA-EVs will have a concentration-dependent effect on mitochondrial respiration, and also regulate mitochondrial dynamics in myoblasts. C2C12 myoblasts were differentiated into myotubes, and electrically paced (3hrs/day, 4 days, 14V, C-PACE EM, IonOptix). EVs from control and CCA-stimulated myotubes were isolated from conditioned media using differential ultracentrifugation and biophysically characterized by size and concentration using TRPS (Izon). C2C12 myoblasts were treated daily for 4 days using three different concentrations: 1.0E4; 2.0E4 and 4.0E4 EVs/cell (N=4-5). Basal and maximal oxygen consumption rates (OCR) were measured using the Seahorse XFe24 (Agilent). We used live-cell fluorescence microscopy and Mitometer software to access mitochondrial movement: area/volume, displacement, distance, length, perimeter, speed and velocity. Average size was unchanged between control-EVs (123±10.8 nm) and CCA-EVs (123±11.9 nm) (p=0.9137, N=3), and 2.3-fold more CCA-EVs were released vs. control-EVs (p=0.2072, N=3). Lowest EV concentration (1.0E+4 EVs/cell, N=5) had no effect on basal (p=0.4226) and maximal (p=0.1835) OCR. Intermediate dosage of CCA-EVs (2.0E+4 EVs/cell, N=5) increased basal OCR by 20% (p=0.0037) and maximal OCR by 18% (p=0.0095), while the highest concentration (4.0E+4 EVs/cell, N=4) decreased basal OCR by 9% (p=0.0001) and maximal OCR by 29% (p=0.0009), compared to control-EVs. Mitochondrial motility (N=484-976) in myoblasts treated daily after each day of CCA showed enhanced mitochondrial dynamics with CCA-EVs: increased area/volume (p<0.0001), displacement (p<0.0001), distance (p<0.0001), mean intensity (p<0.0001), perimeter/surface area (p=0.0137) and speed (p<0.0001), compared to control-EVs. In summary, CCA-EVs induced concentration-dependent improvements in mitochondrial respiration in healthy myoblasts, concomitant with enhanced mitochondrial movement. These results open exciting avenues for the therapeutic potential of CCA-EVs in rescuing mitochondrial dysfunction. TFGS is funded by a Research Manitoba Postdoctoral Fellowship. Grants from CHRIM, CHF, DREAM and NSERC to AS funded the research. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Activation of the Nrf-2 pathway improves the mitochondrial phenotype in skeletal muscle cells

Aging muscle displays a reduction in mitochondrial content, along with elevated reactive oxygen species (ROS) emission and reduced levels of antioxidant enzymes. These characteristics enhance tissue susceptibility to oxidative damage, facilitating sub-optimal cellular metabolism and subsequent muscle atrophy. While these perturbations to metabolic homeostasis are a natural course of the aging process, there has been ongoing research investigating how to implement interventions to reduce this decline in muscle health. Regular exercise is one possible treatment modality, however this may be unsuitable for some populations who are unable to participate due to their health status. The objective of this study was to examine the activation of the Nrf-2 pathway using the nutraceutical Sulforaphane (SFN) and evaluate its role as a potential exercise mimetic. We hypothesized that SFN would act through similar pathways activated with chronic exercise to improve mitochondrial content, respiration, and ROS emission. To address our objective, C2C12 myotubes were treated with SFN for various timepoints basally, and in the presence of the Complex-I inhibitor rotenone to induce oxidative stress. Western blotting revealed an 8-fold increase in Nrf-2 nuclear localization following 24hrs of SFN treatment, which subsequently led to the upregulation of antioxidant enzymes NQO1, Heme Oxygenase-1, catalase, glutathione reductase and Glucose-6-Phosphate Dehydrogenase by 2-to 3-fold. These protein changes, were accompanied by dramatic reductions in both total cellular and mitochondrial ROS emission with SFN, detected with CellROX Green and MitoSOX Red respectively. Interestingly, this led to an increase in mitochondrial membrane potential, detected via MitoTracker Red (MTR), despite rotenone-induced oxidative stress. Western blotting revealed TFAM upregulation at 24hrs, suggesting an increase in mitochondrial DNA (mtDNA) replication and transcription. This was corroborated by an increase in COX subunit I protein, a mitochondrially-encoded subunit of cytochrome c oxidase, as well as MitoTracker Green and MTR staining by 48hrs. Mitochondrial respiration was evaluated using Seahorse analysis, demonstrating a marked increase in ATP production as well as basal and maximal respiration. Together these data suggest that activation of the Nrf-2 pathway via SFN may lead to adaptations similar to chronic exercise. Additionally, SFN may have applications in disorders characterized by mitochondrial loss and high ROS emission. To fully elucidate the precise mechanisms underlying this agent, SFN will be combined with chronic contractile activity to determine if these two interventions operate through independent pathways. This work was supported by funds from the Natural Science and Engineering Research Council (NSERC). Sabrina Champsi is the recipient of the NSERC Alexander Graham Bell Canada Graduate Scholarship-Master’s (CGS-M). David A. Hood is the holder of a Canadian Research Chair in Cell Physiology. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Activating transcription factor 4 regulates mitochondrial content, morphology, and function in differentiating skeletal muscle myotubes.

Mitochondrial function is widely recognized as a major determinant of health, emphasizing the importance of understanding the mechanisms promoting mitochondrial quality in various tissues. Recently, the mitochondrial unfolded protein response (UPRmt) has come into focus as a modulator of mitochondrial homeostasis, particularly in stress conditions. In muscle, the necessity for activating transcription factor 4 (ATF4) and its role in regulating mitochondrial quality control (MQC) have yet to be determined. We overexpressed (OE) and knocked down ATF4 in C2C12 myoblasts, differentiated them to myotubes for 5 days, and subjected them to acute (ACA) or chronic (CCA) contractile activity. ATF4 mediated myotube formation through the regulated expression of myogenic factors, mainly Myc and myoblast determination protein 1 (MyoD), and suppressed mitochondrial biogenesis basally through peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1α). However, our data also show that ATF4 expression levels are directly related to mitochondrial fusion and dynamics, UPRmt activation, as well as lysosomal biogenesis and autophagy. Thus, ATF4 promoted enhanced mitochondrial networking, protein handling, and the capacity for clearance of dysfunctional organelles under stress conditions, despite lower levels of mitophagy flux with OE. Indeed, we found that ATF4 promoted the formation of a smaller pool of high-functioning mitochondria that are more responsive to contractile activity and have higher oxygen consumption rates and lower reactive oxygen species levels. These data provide evidence that ATF4 is both necessary and sufficient for mitochondrial quality control and adaptation during both differentiation and contractile activity, thus advancing the current understanding of ATF4 beyond its canonical functions to include the regulation of mitochondrial morphology, lysosomal biogenesis, and mitophagy in muscle cells.

Role of TFEB and TFE3 in mediating lysosomal and mitochondrial adaptations to contractile activity in skeletal muscle myotubes

Exercise is potent stimulus for mitochondrial adaptations, serving to activate mitochondrial biogenesis as well as mitochondrial turnover. Through the process of mitophagy, dysfunctional mitochondria are selectively targeted and recycled via the lysosomes, which is activated following a single bout of exercise. The microphthalamia (MiT) family of transcription factors, including TFEB and TFE3, are widely recognized as the master regulators of lysosomal biogenesis, as they homo- and hetero-dimerize to transcriptionally regulate lysosomal and macroautophagy-related genes. It is currently unknown to what extent TFEB and TFE3 regulate mitophagy, and whether these transcription factors mediate mitochondrial adaptations to contractile activity (CA). Here we show that following an acute bout of contractile activity in cultured C2C12 murine skeletal muscle myotubes, LC3-II mitophagy flux is induced and the absence of TFEB or TFE3 impairs this acute mitophagic response. However, the loss of either transcription factor alone does not mitigate the improvements in oxygen consumption seen following chronic contractile activity (CCA). Chronic contractile activity also elicited functional improvements in lysosomes including a reduction in size and increased proteolytic activity, evidenced by increased digestion and unquenching of DQ-BSA fluorophore, thereby illustrating a level of redundancy between the two transcription factors in mediating chronic contractile activity-induced adaptations. However, in the absence of both TFEB and TFE3, lysosomal adaptations were not observed following chronic contractile activity and subsequent mitochondrial adaptations were attenuated. These findings underscore the importance of the lysosomes, and of TFEB and TFE3, in mediating mitochondrial adaptations to chronic contractile activity.

Characterizing Extracellular Vesicles and Particles Derived from Skeletal Muscle Myoblasts and Myotubes and the Effect of Acute Contractile Activity.

Extracellular vesicles (EVs), released from all cells, are essential to cellular communication and contain biomolecular cargo that can affect recipient cell function. Studies on the effects of contractile activity (exercise) on EVs usually rely on plasma/serum-based assessments, which contain EVs from many different cells. To specifically characterize skeletal muscle–derived vesicles and the effect of acute contractile activity, we used an in vitro model where C2C12 mouse myoblasts were differentiated to form myotubes. EVs were isolated from conditioned media from muscle cells at pre-differentiation (myoblasts) and post-differentiation (myotubes) and also from acutely stimulated myotubes (1 h @ 14 V, C-Pace EM, IonOptix, Westwood, MA, USA) using total exosome isolation reagent (TEI, ThermoFisher (Waltham, MA, USA), referred to as extracellular particles [EPs]) and differential ultracentrifugation (dUC; EVs). Myotube-EPs (~98 nm) were 41% smaller than myoblast-EPs (~167 nm, p < 0.001, n = 8–10). Two-way ANOVA showed a significant main effect for the size distribution of myotube vs. myoblast-EPs (p < 0.01, n = 10–13). In comparison, myoblast-EPs displayed a bimodal size distribution profile with peaks at <200 nm and 400–600, whereas myotube-Eps were largely 50–300 nm in size. Total protein yield from myotube-EPs was nearly 15-fold higher than from the myoblast-EPs, (p < 0.001 n = 6–9). Similar biophysical characteristics were observed when EVs were isolated using dUC: myotube-EVs (~195 nm) remained 41% smaller in average size than myoblast-EVs (~330 nm, p = 0.07, n = 4–6) and had comparable size distribution profiles to EPs isolated via TEI. Myotube-EVs also had 4.7-fold higher protein yield vs. myoblast EVs (p < 0.05, n = 4–6). Myotube-EPs exhibited significantly decreased expression of exosomal marker proteins TSG101, CD63, ALIX and CD81 compared with myoblast-EPs (p < 0.05, n = 7–12). Conversely, microvesicle marker ARF6 and lipoprotein marker APO-A1 were only found in the myotube-EPs (p < 0.05, n = 4–12). There was no effect of acute stimulation on myotube-EP biophysical characteristics (n = 7) or on the expression of TSG101, ARF6 or CD81 (n = 5–6). Myoblasts treated with control or acute stimulation–derived EPs (13 µg/well) for 48 h and 72 h showed no changes in mitochondrial mass (MitoTracker Red, ThermoFisher, Waltham, MA, USA), cell viability or cell count (n = 3–4). Myoblasts treated with EP-depleted media (72 h) exhibited ~90% lower cell counts (p < 0.01, n = 3). Our data show that EVs differed in size, distribution, protein yield and expression of subtype markers pre vs. post skeletal muscle–differentiation into myotubes. There was no effect of acute stimulation on biophysical profile or protein markers in EPs. Acute stimulation–derived EPs did not alter mitochondrial mass or cell count/viability. Further investigation into the effects of chronic contractile activity on the biophysical characteristics and cargo of skeletal muscle–specific EVs are warranted.

Lysosomal Alterations in Skeletal Muscle Plasticity – An Investigation of Age, Exercise and Disuse

Skeletal muscle displays a high degree of plasticity in response to a variety of contractile activity stimuli. This is exemplified in response to exercise training, in which muscle mitochondrial content is enhanced to drive an increase in metabolic capacity. The opposite is true with chronic muscle disuse and advancing age, in which the muscle loses its oxidative capacity. This plasticity is regulated by alterations in the synthesis and degradation of mitochondria. A major intracellular degradation system is the autophagy‐lysosome pathway. This includes the targeting of damaged intracellular components by autophagosomes, followed by delivery of these to the lysosomes for degradation. However, there is little understanding of the regulation of the lysosomes in response to physiological stimuli. Thus, our objective is to understand how muscle lysosomes are regulated in response to physiological stimuli, including exercise, disuse and age. Thus, we utilized unilateral chronic contractile activity (CCA;7 days) to elicit endurance exercise adaptations, and denervation of the peroneal nerve (7 days) to elicit disuse adaptations. These opposing activity paradigms resulted in parallel increases (50%) and decreases (50%) in mitochondrial content within muscle, respectively. To assess the effects of age, 36‐month‐old rats and 24‐month‐old mice were used. CCA elicited increases in lysosomal proteins Lamp1, Lamp2 and Cathepsin D by 2.9‐,1.8‐ and 1.3‐fold respectively, an effect that was blunted in aged rats. Both denervated and aged muscle exhibited elevated levels of these proteins, such as 3.0‐ and 1.6‐fold increases in Lamp1 and Lamp2 in denervated muscle, and 5‐fold increases in these proteins with age. To understand how these opposing processes (i.e. CCA vs age/disuse) elicit directionally similar changes in these proteins, we measured TFEB, a transcription factor that drives the formation of lysosomes. CCA elicited modest, 50% elevations in TFEB protein, whereas denervation and aging increased TFEB by 70% and 180%, respectively. However, TFEB localization was elevated differentially, by 10%, 25% and 100% in CCA, denervation and aging, respectively. Importantly, electron micrographs of denervated and aged muscle exhibited evidence of lipofuscin accumulation, suggesting impairments in lysosomal function with these wasting conditions, and this could account for an accumulation of dysfunctional lysosomal proteins. To evaluate the transcriptional control of lysosomal biogenesis, young and aged mice were injected with the TFEB‐promoter luciferase reporter. Acute exercise elevated TFEB promoter activity by 1.6‐fold in young muscle, and 2.4‐fold in aged muscle, whereas nuclear TFEB was enhanced to a greater extent in young muscle. This could explain the blunted CCA‐induced lysosomal adaptations in aged muscle. Our data suggest increases in lysosomal proteins are evident with both enhanced (i.e. CCA) and reduced (i.e denervation and age) contractile stimuli. Future work will evaluate whether the changes in lysosomal proteins represent functional lysosomes or dysfunctional accumulation of organelles.Support or Funding InformationNSERC and CIHR

Endoplasmic reticulum stress and unfolded protein response in diaphragm muscle dysfunction of patients with stable chronic obstructive pulmonary disease.

Respiratory muscle dysfunction is common in patients with chronic obstructive pulmonary disease (COPD). Chronic contractile activity induces endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in animals (animals and humans). We hypothesized that the respiratory muscle dysfunction associated with COPD may upregulate ER stress and UPR expression in diaphragm of stable patients with different degrees of airway obstruction and normal body composition. In diaphragm muscle specimens of patients with mild and moderate-to-severe COPD with preserved body composition and non-COPD controls (thoracotomy because of lung localized neoplasms), expression of protein misfolding (ER stress) and UPR markers, proteolysis and apoptosis (qRT-PCR and immunoblotting), and protein aggregates (lipofuscin, histology) were quantified. All patients and non-COPD controls were also clinically evaluated: lung and muscle functions and exercise capacity. Compared with non-COPD controls, patients exhibited mild and moderate-to-severe airflow limitation and diffusion capacity and impaired exercise tolerance and diaphragm strength. Moreover, compared with the controls, in the diaphragm of the COPD patients, slow-twitch fiber proportions increased, gene expression but not protein levels of protein disulfide isomerase family A member 3 and phosphatidylinositol 3-kinase catalytic subunit type 3 were upregulated, and no significant differences were found in markers of UPR transmembrane receptor pathways (activating transcription factor-6, inositol-requiring enzyme-1α, and protein kinase-like ER kinase), lipofuscin aggregates, proteolysis, or apoptosis. In stable COPD patients with a wide range of disease severity, reduced diaphragm force of contraction, and normal body composition, ER stress and UPR signaling were not induced in the main respiratory muscle. These findings imply that ER stress and UPR are probably not involved in the documented diaphragm muscle dysfunction (reduced strength) observed in all the study patients, even in those with severe airflow limitation. Hence, in stable COPD patients with normal body composition, therapeutic strategies targeted to treat diaphragm muscle dysfunction should not include UPR modulators, even in those with a more advanced disease. NEW & NOTEWORTHY In stable chronic obstructive pulmonary disease patients with a wide range of disease severity, diaphragm muscle weakness, and normal body composition, endoplasmic reticulum stress and unfolded protein response (UPR) signaling were not induced in the main respiratory muscle. These findings imply that endoplasmic reticulum stress and UPR are not involved in the documented diaphragm muscle dysfunction observed in the study patients, even in those with severe airflow limitation. In stable chronic obstructive pulmonary disease patients with normal body composition, therapeutic strategies should not include UPR modulators.

Importance of TFEB and TFE3 in Mediating Lysosomal and Mitochondrial Adaptations

Autophagy is the process through which damaged cellular components are degraded by the lysosome, and it is essential for the overall maintenance of the cell. Mitochondria are recycled through a selective form of autophagy, known as mitophagy, in which dysfunctional mitochondria are poly‐ubiquitinated, engulfed in an autophagosomal membrane and transported to the lysosome for degradation. Chronic contractile activity (CCA) alters mitophagy flux in muscle, and this coincides with the activation of lysosomal biogenesis, which precedes changes in mitochondrial content. Thus, it is proposed that the clearance of dysfunctional mitochondria through the lysosomes may play an important role in mediating mitochondrial adaptations to CCA. We sought to understand the transcriptional regulation of lysosomal biogenesis brought about by CCA, by investigating the transcriptional regulators TFEB and TFE3, and their relationship to mitochondrial content. TFEB and TFE3 were silenced using siRNA in C2C12 myotubes, and subjected to 3 days of stimulation (3hr/day) to induce CA. Following a single bout, TFEB and TFE3 were elevated in both the nuclear and cytosolic fractions, which coincided with increases in Cathepsin D, preceding the increases in VDAC seen following 3 days of stimulation. The loss of TFEB or TFE3 individually had no impact on lysosomal adaptations, since LAMP1 and Cathepsin D remained responsive to CCA, demonstrating redundancy in role of TFEB and TFE3 in mediating lysosomal biogenesis. However, in the absence of both transcription factors, CCA‐induced lysosomal adaptations were reduced, illustrating their collective importance in mediating lysosomal biogenesis. Interestingly, the absence of TFEB had no effect on mitochondrial adaptations, measured by COX I and IV protein content, however this adaptation to CCA was blunted in the absence of TFE3. These results suggest that while TFEB and TFE3 may exhibit compensatory roles in mediating lysosomal biogenesis, TFE3 alone appears to be important for the mitochondrial response to CCA. Since, lysosomal adaptations proceed in the absence of TFE3, these data suggest a more direct role of TFE3 in the regulation of mitochondria in response to chronic contractile activity.Support or Funding InformationANO is funded by an Ontario Graduate Scholarship (OGS). JMM is funded by Natural Science and Engineering Research (NSERC) scholarship. DAH is the holder of a Canada Research Chair in Cell Physiology with funding from NSERC and CIHR.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Effect of Chronic Contractile Activity and Retinoic Acid on Mitochondrial Turnover in C2C12 Myotubes

Mitochondrial turnover is necessary for the optimization and maintenance of a healthy pool of functional organelles, able to meet the metabolic needs of the muscle cell. Exercise is known to improve mitochondrial turnover by activating both mitochondrial biogenesis and mitochondrial‐specific autophagy, known as mitophagy. Moreover, research in adipocytes and liver tissue has shown the potential of the dietary supplement retinoic acid to activate mitochondrial biogenesis as well. Retinoids have also been shown to be effective in activating autophagy in cancer cells. However, the combined effect of retinoic acid isomers and exercise in facilitating mitochondrial turnover has not been described. The purpose of this project was to evaluate the effect of retinoic acid (RA) isomers combined with exercise on muscle mitochondrial turnover. Mouse C2C12 myoblasts were plated on 6‐well plates and allowed to differentiate into myotubes. Myotubes were then treated with either vehicle (DMSO), or the retinoic acid (RA) isomers 9‐cis RA or All‐trans RA (ATRA) in the absence or presence of electrical stimulation to elicit Chronic Contractile Activity (CCA; 4days, 3hrs/day). Sample collection took place 21h after the fourth day. Mitochondrial biogenesis was assessed by examining the protein content of PGC‐1α, as well as nuclear and mitochondrially‐encoded mitochondrial proteins. CCA led to a 3‐fold increase in COX subunit IV expression, as well as a 2‐fold increase in PGC‐1α. The mtDNA‐encoded COX subunit I was modestly changed by CCA, but increased 2‐fold in response to 9cis‐RA or ATRA treatment. These RA isomers also increased PGC‐1α by 3‐fold, but had no significant impact on COX IV expression. When RA isomers were combined with CCA, no additive or synergistic effects were observed. To evaluate mitochondrial degradation, we examined several lysosomal and mitophagy proteins. While autophagy proteins Beclin and p62 showed no changes with CCA or RA treatment, the LC3II to LC3I ratio was reduced 2‐fold with CCA, suggesting an enhanced clearance of autophagosomes. RA isomers had no effect on this ratio. The lysosomal fusion protein Lamp1 was increased 1.8‐fold by ATRA, but not further by CCA. Our data suggest that while RA isomers and CCA both promote mitochondrial biogenesis, RA appears to target mitochondrially‐encoded proteins, while CCA appears to have greater effects on the expression of target nuclear‐encoded proteins. CCA also may serve as a stimulus for the clearance of autophagosomes containing damaged cargo. The lack of synergy among these pathways suggests that they operate independently to stimulate mitochondrial turnover.Support or Funding InformationSupported by NSERC, CanadaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation of autophagic and mitophagic flux during chronic contractile activity-induced muscle adaptations.

Autophagy and mitophagy are important for training-inducible muscle adaptations, yet it remains unclear how these systems are regulated throughout the adaptation process. Here, we studied autophagic and mitophagic flux in the skeletal muscles of Sprague-Dawley rats (300-500g) exposed to chronic contractile activity (CCA; 3h/day, 9V, 10Hz continuous, 0.1ms pulse duration) for 1, 2, 5, and 7days (N = 6-8/group). In order to determine the flux rates, colchicine (COL; 0.4mg/ml/kg) was injected 48h before tissue collection, and we evaluated differences of autophagosomal protein abundances (LC3-II and p62) between colchicine- and saline-injected animals. We confirmed that CCA resulted in mitochondrial adaptations, including improved state 3 respiration as early as day 1 in permeabilized muscle fibers, as well significant increases in mitochondrial respiratory capacity and marker proteins in IMF mitochondria by day 7. Mitophagic and autophagic flux (LC3-II and p62) were significantly decreased in skeletal muscle following 7days of CCA. Notably, the mitophagic system seemed to be downregulated prior (day 3-5) to changes in autophagic flux (day 7), suggesting enhanced sensitivity of mitophagy compared to autophagy with chronic muscle contraction. Although we detected no significant change in the nuclear translocation of TFEB, a regulator of lysosomal biogenesis, CCA increased total TFEB protein, as well as LAMP1, in skeletal muscle. Thus, chronic muscle activity reduces mitophagy in parallel with improved mitochondrial function, and this is supported by enhanced lysosomal degradation capacity.

Contractile activity attenuates autophagy suppression and reverses mitochondrial defects in skeletal muscle cells

ABSTRACT Macroautophagy/autophagy is a survival mechanism that facilitates protein turnover in post-mitotic cells in a lysosomal-dependent process. Mitophagy is a selective form of autophagy, which arbitrates the selective recognition and targeting of aberrant mitochondria for degradation. Mitochondrial content in cells is the net balance of mitochondrial catabolism via mitophagy, and organelle biogenesis. Although the latter process has been well described, mitophagy in skeletal muscle is less understood, and it is currently unknown how these two opposing mechanisms converge during contractile activity. Here we show that chronic contractile activity (CCA) in muscle cells induced mitochondrial biogenesis and coordinately enhanced the expression of TFEB (transcription factor EB) and PPARGC1A/PGC-1α, master regulators of lysosome and mitochondrial biogenesis, respectively. CCA also enhanced the expression of PINK1 and the lysosomal protease CTSD (cathepsin D). Autophagy blockade with bafilomycin A1 (BafA) reduced mitochondrial state 3 and 4 respiration, increased ROS production and enhanced the accumulation of MAP1LC3B-II/LC3-II and SQSTM1/p62. CCA ameliorated this mitochondrial dysfunction during defective autophagy, increased PPARGC1A, normalized LC3-II levels and reversed mitochondrially-localized SQSTM1 toward control levels. NAC emulated the LC3-II reductions induced by contractile activity, signifying that a decrease in oxidative stress could represent a mechanism of autophagy normalization brought about by CCA. CCA enhances mitochondrial biogenesis and lysosomal activity, and normalizes autophagy flux during autophagy suppression, partly via ROS-dependent mechanisms. Thus, contractile activity represents a potential therapeutic intervention for diseases in which autophagy is inhibited, such as vacuolar myopathies in skeletal muscle, by establishing a healthy equilibrium of anabolic and catabolic pathways. Abbreviations: AMPK: AMP-activated protein kinase; BafA: bafilomycin A1; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; CCA: chronic contractile activity; COX4I1: cytochrome c oxidase subunit 4I1; DMEM: Dulbecco’s modified Eagle’s medium; GFP: green fluorescent protein; LSD: lysosomal storage diseases; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NAC: N-acetylcysteine; PPARGC1A: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; PINK1: PTEN induced putative kinase 1; ROS: reactive oxygen species; SOD2: superoxide dismutase 2, mitochondrial; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB

Mitophagy flux in skeletal muscle during chronic contractile activity and ageing.

Mitophagy flux in skeletal muscle during chronic contractile activity and ageing.

Autophagy and mitophagy flux in skeletal muscle during chronic‐contractile activity

Autophagy and mitophagy are important cellular recycling mechanisms, however the regulation of autophagy and mitophagy flux in training‐induced muscle adaptations remains to be elucidated. Using the microtubule destabilizer colchicine (0.04 mg/kg), this study assessed LC3II and p62 flux in skeletal muscles of Sprague‐Dawley rats (501 ± 9 g) subjected to chronic contractile activity (CCA; 3 hours/day, 9V, 10 Hz continuous, 0.1 ms pulse duration) for 1, 2, 3, 5, and 7 days (N=6–8/group). Colchicine was given via an intraperitoneal injection 48 hours prior to tissue collection, whereas control animals were administered saline as a vehicle treatment. Following 7 days of CCA, there were significant increases in maximal state 3 respiration, accompanied by a decrease in ROS production in IMF mitochondria (P &lt; 0.05), indicating that mitochondrial adaptations were elicited in the stimulated muscle. Notably, whole muscle autophagy flux, as measured by LC3II and p62, was downregulated in response to 3 days of CCA, and further by 7 days of CCA (P &lt; 0.05). In addition, muscle mitochondrial LC3II flux was decreased by 3 days of CCA, indicative of reduced mitophagy. In contrast, the lysosomal system, measured by TFEB and LAMP1 protein levels, was gradually upregulated by CCA, culminating at day 7. Interestingly, the translocation of TFEB into the nucleus in response to CCA was increased at day 3 (P &lt; 0.05). However, this nuclear TFEB was not sustained and subsequently declined back to basal levels. In conclusion, we suggest that chronic exercise can lead to muscle adaptations through a coordinated mechanism, involving a decrease in autophagy/mitophagy flux, combined with an increase in the capacity of the lysosomal system.Support or Funding InformationThis work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations.

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

Regulation of the autophagy system during chronic contractile activity-induced muscle adaptations.

Skeletal muscle is adaptable to exercise stimuli via the upregulation of mitochondrial biogenesis, and recent studies have suggested that autophagy also plays a role in exercise‐induced muscle adaptations. However, it is still obscure how muscle regulates autophagy over the time course of training adaptations. This study examined the expression of autophagic proteins in skeletal muscle of rats exposed to chronic contractile activity (CCA; 6 h/day, 9V, 10 Hz continuous, 0.1 msec pulse duration) for 1, 3, and 7 days (n = 8/group). CCA‐induced mitochondrial adaptations were observed by day 7, as shown by the increase in mitochondrial proteins (PGC‐1α, COX I, and COX IV), as well as COX activity. Notably, the ratio of LC3 II/LC3 I, an indicator of autophagy, decreased by day 7 largely due to a significant increase in LC3 I. The autophagic induction marker p62 was elevated on day 3 and returned to basal levels by day 7, suggesting a time‐dependent increase in autophagic flux. The lysosomal system was upregulated early, prior to changes in mitochondrial proteins, as represented by increases in lysosomal system markers LAMP1, LAMP2A, and MCOLN1 as early as by day 1, as well as TFEB, a primary regulator of lysosomal biogenesis and autophagy flux. Our findings suggest that, in response to chronic exercise, autophagy is upregulated concomitant with mitochondrial adaptations. Notably, our data reveal the surprising adaptive plasticity of the lysosome in response to chronic contractile activity which enhances muscle health by providing cells with a greater capacity for macromolecular and organelle turnover.

The unfolded protein response in relation to mitochondrial biogenesis in skeletal muscle cells

Mitochondria comprise both nuclear and mitochondrially encoded proteins requiring precise stoichiometry for their integration into functional complexes. The augmented protein synthesis associated with mitochondrial biogenesis results in the accumulation of unfolded proteins, thus triggering cellular stress. As such, the unfolded protein responses emanating from the endoplasmic reticulum (UPRER) or the mitochondrion (UPRMT) are triggered to ensure correct protein handling. Whether this response is necessary for mitochondrial adaptations is unknown. Two models of mitochondrial biogenesis were used: muscle differentiation and chronic contractile activity (CCA) in murine muscle cells. After 4 days of differentiation, our findings depict selective activation of the UPRMT in which chaperones decreased; however, Sirt3 and UPRER markers were elevated. To delineate the role of ER stress in mitochondrial adaptations, the ER stress inhibitor TUDCA was administered. Surprisingly, mitochondrial markers COX-I, COX-IV, and PGC-1α protein levels were augmented up to 1.5-fold above that of vehicle-treated cells. Similar results were obtained in myotubes undergoing CCA, in which biogenesis was enhanced by ~2-3-fold, along with elevated UPRMT markers Sirt3 and CPN10. To verify whether the findings were attributable to the terminal UPRER branch directed by the transcription factor CHOP, cells were transfected with CHOP siRNA. Basally, COX-I levels increased (~20%) and COX-IV decreased (~30%), suggesting that CHOP influences mitochondrial composition. This effect was fully restored by CCA. Therefore, our results suggest that mitochondrial biogenesis is independent of the terminal UPRER Under basal conditions, CHOP is required for the maintenance of mitochondrial composition, but not for differentiation- or CCA-induced mitochondrial biogenesis.

Chronology of UPR activation in skeletal muscle adaptations to chronic contractile activity.

The mitochondrial and endoplasmic reticulum unfolded protein responses (UPR(mt) and UPR(ER)) are important for cellular homeostasis during stimulus-induced increases in protein synthesis. Exercise triggers the synthesis of mitochondrial proteins, regulated in part by peroxisome proliferator activator receptor-γ coactivator 1α (PGC-1α). To investigate the role of the UPR in exercise-induced adaptations, we subjected rats to 3 h of chronic contractile activity (CCA) for 1, 2, 3, 5, or 7 days followed by 3 h of recovery. Mitochondrial biogenesis signaling, through PGC-1α mRNA, increased 14-fold after 1 day of CCA. This resulted in 10-32% increases in cytochrome c oxidase activity, indicative of mitochondrial content, between days 3 and 7, as well as increases in the autophagic degradation of p62 and microtubule-associated proteins 1A/1B light chain 3A (LC3)-II protein. Before these adaptations, the UPR(ER) transcripts activating transcription factor-4, spliced X-box-binding protein 1, and binding immunoglobulin protein were elevated (1.3- to 3.8-fold) at days 1-3, while CCAAT/enhancer-binding protein homologous protein (CHOP) and chaperones binding immunoglobulin protein and heat shock protein (HSP) 70 were elevated at mRNA and protein levels (1.5- to 3.9-fold) at days 1-7 of CCA. The mitochondrial chaperones 10-kDa chaperonin, HSP60, and 75-kDa mitochondrial HSP, the protease ATP-dependent Clp protease proteolytic subunit, and the regulatory protein sirtuin-3 of the UPR(mt) were concurrently induced 10-80% between days 1 and 7 To test the role of the UPR in CCA-induced remodeling, we treated animals with the endoplasmic reticulum stress suppressor tauroursodeoxycholic acid and subjected them to 2 or 7 days of CCA. Tauroursodeoxycholic acid attenuated CHOP and HSP70 protein induction; however, this failed to impact mitochondrial remodeling. Our data indicate that signaling to the UPR is rapidly activated following acute contractile activity, that this is attenuated with repeated bouts, and that the UPR is involved in chronic adaptations to CCA; however, this appears to be independent of CHOP signaling.

Expression of mitochondrial fission and fusion regulatory proteins in skeletal muscle during chronic use and disuse

The mitochondrial network within cells is mediated by fission and fusion processes. We investigated the expression of the proteins responsible for these events during conditions of altered oxidative capacity. With chronic contractile activity, the mitochondrial reticulum increased in size, along with concomitant increases in the fusion proteins Opa1 and Mfn2 (by 36% and 53%; P < 0.05). When we induced muscle disuse through denervation for 7 days, fragmented mitochondria were observed, along with significant decreases in the expression of Mfn2 and Opa1 (by 84% and 70%). To assess the effects of aging on mitochondrial morphology, young (5 month) and aged (35 month) Fisher 344 Brown Norway rats were used. Aged animals also possessed smaller mitochondria and displayed increased levels of fission proteins. 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 fragmented organelles.

Mammalian target of rapamycin pathway is up-regulated by both acute endurance exercise and chronic muscle contraction in rat skeletal muscle

This study examined changes in the expression of translation initiation regulatory proteins and mRNA following both an acute bout of endurance exercise and chronic muscle contractile activity. Female Sprague Dawley rats ran for 2 h at 15 m·min(-1) followed by an increase in speed of 5 m·min(-1) every 5 min until volitional fatigue. The red gastrocnemius muscle was harvested from nonexercised animals (control; n = 6) and from animals that exercised either immediately after exercise (n = 6) or following 3 h of recovery from exercise (n = 6). Compared with control, ribosomal protein S6 (rpS6) mRNA was elevated (p < 0.05) at both 0 h (+32%) and 3 h (+47%). Both a catalytic subunit of eukaryotic initiation factor 2B (eIF2Bε) (+127%) and mammalian target of rapamycin (mTOR) mRNA (+44%) were increased at 3 h, compared with control. Phosphorylation of mTOR (+40%) and S6 kinase 1 (S6K1) (+266%) were increased immediately after exercise (p < 0.05). Female Sprague Dawley rats also underwent chronic stimulation of the peroneal nerve continuously for 7 days. The red gastrocnemius muscle was removed 24 h after cessation of the stimulation. Chronic muscle stimulation increased (p < 0.05) mTOR protein (+74%), rpS6 (+31%), and eukaryotic initiation factor 2α (+44%, p = 0.069), and this was accompanied by an increase in cytochrome c (+31%). Increased resting phosphorylation was observed for rpS6 (+51%) (p < 0.05) but not for mTOR or eukaryotic initiation factor 4E binding protein 1. These experiments demonstrate that both acute and chronic contractile activity up-regulate the mTOR pathway and mitochondrial content in murine skeletal muscle. This up-regulation of the mTOR pathway may increase translation efficiency and may also represent an important control point in exercise-mediated mitochondrial biogenesis.