IntroductionDespite rigorous rehabilitation aimed at restoring muscle health, anterior cruciate ligament (ACL) injury is often hallmarked by significant long-term quadriceps muscle weakness. Derangements in mitochondrial function are a common feature of various atrophying conditions, yet it is unclear to what extent mitochondria are involved in the detrimental sequela of quadriceps dysfunction after ACL injury. Using a preclinical, non-invasive ACL injury rodent model, our objective was to explore the direct effect of an isolated ACL injury on mitochondrial function, muscle atrophy, and muscle phenotypic transitions.MethodsA total of 40 male and female, Long Evans rats (16-week-old) were exposed to non-invasive ACL injury, while 8 additional rats served as controls. Rats were euthanized at 3, 7, 14, 28, and 56 days after ACL injury, and vastus lateralis muscles were extracted to measure the mitochondrial respiratory control ratio (RCR; state 3 respiration/state 4 respiration), mitochondrial reactive oxygen species (ROS) production, fiber cross sectional area (CSA), and fiber phenotyping. Alterations in mitochondrial function and ROS production were detected using two-way (sex:group) analyses of variance. To determine if mitochondrial characteristics were related to fiber atrophy, individual linear mixed effect models were run by sex.ResultsMitochondria-derived ROS increased from days 7 to 56 after ACL injury (30–100%, P < 0.05), concomitant with a twofold reduction in RCR (P < 0.05). Post-injury, male rats displayed decreases in fiber CSA (days 7, 14, 56; P < 0.05), loss of IIa fibers (day 7; P < 0.05), and an increase in IIb fibers (day 7; P < 0.05), while females displayed no changes in CSA or phenotyping (P > 0.05). Males displayed a positive relationship between state 3 respiration and CSA at days 14 and 56 (P < 0.05), while females only displayed a similar trend at day 14 (P = 0.05).ConclusionLong-lasting impairments in quadriceps mitochondrial health are present after ACL injury and play a key role in the dysregulation of quadriceps muscle size and composition. Our preclinical data indicate that using mitoprotective therapies may be a potential therapeutic strategy to mitigate alterations in muscle size and characteristic after ACL injury.

Oxidative status is impacted by fitness and dietary nutrients, such as antioxidants. We hypothesized that astaxanthin (ASTX) supplementation would improve oxidative status in the circulation and muscle in response to exercise following deconditioning and reconditioning in horses. Twelve conditioned polo ponies (14.8 ± 1.7 yr) were assigned to control (CON; n = 6) or astaxanthin supplemented (ASTX; n = 6; 75 mg ASTX daily) groups. Horses performed 26 min submaximal exercise tests (SET) followed by 30 min of recovery while in condition (SET1), after 16 wk of deconditioning (SET2), and after 16 wk of reconditioning (SET3). Blood samples were collected 30 min before and 0, 15, 30, and 60 min after each SET. Semitendinosus muscle biopsies were collected 2 wk before and 2 hr after each SET. Using commercially available kits, plasma and muscle superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities and plasma malondialdehyde (MDA) concentrations were determined. At SET2, ASTX had 116% greater plasma SOD activities than CON (P=0.001). Both treatment groups increased from SET2 to SET3 (P< 0.001), but there were no treatment effects at SET3 (P=0.788). At SET2, plasma GPX activities were 49.3% greater in ASTX than CON (P=0.012), and ASTX tended to be 21.2% greater than CON at SET3 (P=0.096). Plasma MDA concentrations were 22.2% greater in CON than ASTX at SET2 (P=0.034), but not at SET1 or SET3 (P≥0.449). There were no detectable differences in muscle SOD or GPX activities 2 wk before or 2 hr after any SET (P≥0.309). In conclusion, ASTX supplementation maintained circulating antioxidant capacity and minimized oxidant activities during deconditioning, reducing oxidative stress in response to the SET in the circulation but not in the skeletal muscle. This may enable horses to adjust to strenuous exercise more efficiently, improving athletic performance, especially when they are re-introduced to exercise after deconditioning.

BACKGROUND Many patients that suffer from anterior cruciate ligament (ACL) injury have persistent quadriceps atrophy even after considerable rehabilitation. Our previous finding has revealed that mitochondrial dysfunction and redox disturbances are causal events in the initiation of muscle atrophy and central to maintenance of a healthy mitochondria is the removal of damaged mitochondria through mitophagy. However, the extent to which mitophagy play a key role in quadriceps muscle atrophy after ACL injury has yet to be explored. If mitophagy is found to play a central role in directing muscle atrophy after ACL injury than it may be an attractive therapeutic target. PURPOSE Using a pre‐clinical non‐invasive ACL injury model, our objective was to use a time course study to investigate the potential role of mitophagy in quadriceps muscle atrophy after ACL injury. METHODS 48 Long Evans rats (n=8 per group; 4m/4f) underwent non‐invasive rupture of the right ACL and were euthanized at 7, 14, 28, 56 days post‐injury. 8 rats (4m/4f) served as healthy controls (HC). Mitophagy‐related cellular components of the vastus lateralis were analyzed by Western Blot analysis. One‐way ANOVAs with LSD post‐hoc were used to determine differences between groups (P < 0.05). RESULTS Dynamin‐related protein 1 (DRP1), a protein regulating mitochondrial fission, was increased significantly at 56 days post‐injury [HC: n=8; 1.294 ± 0.364, 56D: n=7; 2.093 ± 1.519 (A.U), P<0.05]. Lysosomes are the terminal step in mitophagy and one of the lysosomal markers, Lysosomal‐associated membrane protein 1 (LAMP‐1) expression was significantly elevated at 56 days post‐injury [HC: n=8; 1.270 ± 0.840, 56D: n=8; 2.266 ± 1.137 (A.U), P<0.05]. Upstream autophagy marker, Beclin‐1 expression was also significantly increased at 56 days post‐injury (HC: n=8; 1.117 ± 0.339, 56D: n=6; 2.158 ± 1.274, P<0.05). CONCLUSION Collectively, these results imply that long‐term ACL injury dysregulates mitochondrial quality control including key mitophagy markers, which contributes to ACL injury‐induced quadriceps atrophy. Therefore, targeting mitophagy may be the one of the potential therapeutic interventions to prevent muscle atrophy in patients with an ACL injury.

BACKGROUND The accumulation of senescent cells is a hallmark of aging in the skeletal muscle which causes reactive oxygen species (ROS) and mitochondrial dysfunction. p21 is one of the major regulators and most recognized cellular markers for senescent cells. Recent evidence demonstrated that clearance of p21high cells enhances muscle function including grip strength, hanging endurance, and maximal walking speed in mice. However, it is still unclear how the muscle performance enhances through the clearance of p21high cells. One of the potential mechanisms is mitochondrial function and/or mitochondria‐derived ROS. Therefore, this study investigated the linkage between mitochondria and p21high cells in aging or high fat diet‐induced muscle dysfunction. METHODS Five p21‐Cre/+; +/+ (P) and p21‐Cre/+; DTA/+ (PD) obese mice fed by high fat diet were administrated with tamoxifen for twice. Five P lean mice fed by normal chow were adopted as normal control. We previously demonstrated that p21high cells accumulate in obese P mice, and can be eliminated in obese PD mice by tamoxifen treatment. Mitochondrial respiration was measured, by high‐resolution respirometry, in permeabilized muscle fibers from the soleus muscle. Mitochondria‐derive ROS production was determined using Amplex Red assays. One‐way ANOVAs with Bonferroni post‐hoc were used to determine differences between groups (P < 0.05). RESULTS Reductions in complex I + II state 3 respiration were observed in p21‐Cre mice with high fat diet but the clearance of p21high cells using tamoxifen enhanced complex I + II state 3 respiration (Lean P: 35.2 ± 3.13 pmol·s‐1·mg‐1; Obese P: 17.45 ± 2.85 pmol·s‐1·mg‐1; Obese PD: 30.04 ± 3.19 pmol·s‐1·mg‐1; P < 0.05). State 4 respiration did not differ between groups (P > 0.05). Respiratory Control Ratio (RCR), defined as respiration in state 3 divided by respiration in state 4, significantly decreased in high fat diet group but clearing p21high cells restored respiratory function (Lean P: 4.20 ± 1.18; Obese P: 2.21 ± 1.11; Obese PD: 3.85 ± 1.60; P<0.05). Also, high fat diet increased mitochondrial ROS production, but tamoxifen treatment attenuated ROS production (Lean P: 10.16 ± 0.41 pmol·s‐1·mg‐1; Obese P: 26.83 ± 0.54 pmol·s‐1·mg‐1; Obese PD: 12.23 ± 0.54 pmol·s‐1·mg‐1; P<0.05). CONCLUSION These results demonstrated that p21high cells may play a causal role in mitochondrial dysfunction and ROS emission in the skeletal muscle with obesity and other chronic diseases. Therefore, targeting p21high cells may enhance muscle performance through decreasing mitochondria‐derived ROS and enhancing mitochondrial function.