Development of skeletal muscle fibrosis in a rodent model of cancer cachexia.

Cachexia is characterized by losses in lean body mass and its progression results in worsened quality of life and exacerbated outcomes in cancer patients. However, the role and impact of fibrosis during the early stages and development of cachexia in under-investigated. The purpose of this study was to determine if fibrosis occurs during cachexia development, and to evaluate this in both sexes. Female and male C57BL6/J mice were injected with phosphate-buffered saline or Lewis Lung Carcinoma (LLC) at 8-week of age, and tumors were allowed to develop for 1, 2, 3, or 4 weeks. 3wk and 4wk female tumor-bearing mice displayed a dichotomy in tumor growth and were reassigned to high tumor (HT) and low tumor (LT) groups. In vitro analyses were also performed on cocultured C2C12 and 3T3 cells exposed to LLC conditioned media. Immunohistochemistry and quantitative polymerase chain reaction (qPCR) analysis were used to investigate fibrosis and fibrosis-related signaling in skeletal muscle. Collagen deposition in skeletal muscle was increased in the 1wk, LT, and HT groups in female mice. However, collagen deposition was only increased in the 4wk group in male mice. In general, female mice displayed earlier alterations in extracellular matrix (ECM)-related genes beginning at 1wk post-LLC injection. Whereas this was not seen in males. While overall tumor burden is tightly correlated to cachexia development in both sexes, fibrotic development is not. Male mice did not exhibit early-stage alterations in ECM-related genes contrary to what was noted in female mice.

Transcriptomic analysis of the development of skeletal muscle atrophy in cancer-cachexia in tumor-bearing mice.

Cancer-cachexia (CC) is a wasting condition directly responsible for 20-40% of cancer-related deaths. The mechanisms controlling development of CC-induced muscle wasting are not fully elucidated. Most investigations focus on the postcachectic state and do not examine progression of the condition. We recently demonstrated mitochondrial degenerations precede muscle wasting in time course progression of CC. However, the extent of muscle perturbations before wasting in CC is unknown. Therefore, we performed global gene expression analysis in CC-induced muscle wasting to enhance understanding of intramuscular perturbations across the development of CC. Lewis lung carcinoma (LLC) was injected into the hind-flank of C57BL6/J mice at 8 wk of age with tumor allowed to develop for 1, 2, 3, or 4 wk and compared with PBS-injected control. Muscle wasting was evident at 4 wk LLC. RNA sequencing of gastrocnemius muscle samples showed widespread alterations in LLC compared with PBS animals with largest differences seen in 4 wk LLC, suggesting extensive transcriptomic alterations concurrent to muscle wasting. Commonly altered pathways included: mitochondrial dysfunction and protein ubiquitination, along with other less studied processes in this condition regulating transcription/translation and cytoskeletal structure. Current findings present novel evidence of transcriptomic shifts and altered cellular pathways in CC-induced muscle wasting.

Cancer cachexia-induced muscle atrophy: evidence for alterations in microRNAs important for muscle size.

Muscle atrophy is a hallmark of cancer cachexia resulting in impaired function and quality of life and cachexia is the immediate cause of death for 20-40% of cancer patients. Multiple microRNAs (miRNAs) have been identified as being involved in muscle development and atrophy; however, less is known specifically on miRNAs in cancer cachexia. The purpose of this investigation was to examine the miRNA profile of skeletal muscle atrophy induced by cancer cachexia to uncover potential miRNAs involved with this catabolic condition. Phosphate-buffered saline (PBS) or Lewis lung carcinoma cells (LLC) were injected into C57BL/6J mice at 8 wk of age. LLC animals were allowed to develop tumors for 4 wk to induce cachexia. Tibialis anterior muscles were extracted and processed to isolate small RNAs, which were used for miRNA sequencing. Sequencing results were assembled with mature miRNAs, and functions of miRNAs were analyzed by Ingenuity Pathway Analysis. LLC animals developed tumors that contributed to significantly smaller tibialis anterior muscles (18.5%) and muscle cross-sectional area (40%) compared with PBS. We found 371 miRNAs to be present in the muscle above background levels. Of these, nine miRNAs were found to be differentially expressed. Significantly altered groups of miRNAs were categorized into primary functionalities including cancer, cell-to-cell signaling, and cellular development among others. Gene network analysis predicted specific alterations of factors contributing to muscle size including Akt, FOXO3, and others. These results create a foundation for future research into the sufficiency of targeting these genes to attenuate muscle loss in cancer cachexia.

Autophagy activation, not peroxisome proliferator-activated receptor γ coactivator 1α, may mediate exercise-induced improvements in glucose handling during diet-induced obesity.

NEW FINDINGS: What is the central question of this study? What are the individual and combined effects of muscle-specific peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) overexpression and physical activity during high-fat feeding on glucose and exercise tolerance? What is the main finding and its importance? Our main finding is that muscle-specific PGC-1α overexpression provides no protection against lipid-overload pathologies nor does it enhance exercise adaptations. Instead, physical activity, regardless of PGC-1α content, protects against high-fat diet-induced detriments. Activation of muscle autophagy was correlated with exercise protection, suggesting that autophagy might be a mediating factor for exercise-induced protection from lipid overload. The prevalence of glucose intolerance is alarmingly high. Efforts to promote mitochondrial biogenesis through peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) to mitigate glucose intolerance have been controversial. However, physical activity remains a primary means to alleviate the condition. The aim of this study was to determine the combined effects of muscle-specific overexpression of PGC-1α and physical activity on glucose handling during diet-induced obesity. Wild-type (WT, ∼20) and PGC-1α muscle transgenic (MCK-PGC-1α, ∼20) mice were given a Western diet (WD) at 8 weeks age and allowed to consume food ab libitum throughout the study. At 12 weeks of age, all animals were divided into sedentary (SED) or voluntary wheel running (VWR) interventions. At 7, 11 and 15 weeks of age, animals underwent glucose tolerance tests (GTT) and graded exercise tests (GXT). At 16 weeks of age, tissues were collected. At 11 weeks, the MCK-PGC-1α animals had 50% greater glucose tolerance integrated area under the curve compared with WT. However, at 15 weeks, SED animals also had greater GTT integrated area under the curve compared with VWR, regardless of genotype; furthermore, SED animals demonstrated reduced exercise capacity compared with earlier time points, which was not seen in VWR animals. Voluntary distance run per day was correlated with GTT in VWR-WT, but not VWR-MCK-PGC-1α mice. Voluntary wheel running and genotype independently resulted in a greater LC3II/LC3I ratio, suggesting enhanced autophagosome formation, which was correlated with exercise-induced improvements in GTT. In conclusion, artificially increasing mitochondrial content does not protect from lipid-induced pathologies nor does it augment exercise adaptations. Physical activity ameliorates the effects of lipid overload-induced glucose intolerance, an effect that appears to be related to enhanced activation of autophagy.

microRNA-16 Is Downregulated During Insulin Resistance and Controls Skeletal Muscle Protein Accretion.

Insulin resistant diabetes, currently at epidemic levels in developed countries, begins in the skeletal muscle and is linked to altered protein turnover. microRNAs downregulate targeted mRNA translation decreasing the amount of translated protein, thereby regulating many cellular processes. Regulation of miRNAs and their function in skeletal muscle insulin resistance is largely unexplored. The purpose of this study was to identify the effects of insulin resistance on contents of skeletal muscle miRNAs with potential functions in protein turnover. We examined miRs -1, -16, -23, -27, -133a, -133b, and -206 in muscles of Zucker rats. miR-1 was 5- to 10-fold greater in obesity, whereas miRs-16 and -133b were repressed ∼50% in obese compared to lean rats, with no other alterations in miRNA contents. miR-16 correlated to protein synthesis in lean, but not obese rats. miR-16 reduction by lipid overload was verified in-vivo by diet-induced obesity and in-vitro using a diacylglycerol analog. A role for miR-16 in protein turnover of skeletal myocytes was established using transient overexpression and anti-miR inhibition. miR-16 overexpression resulted in lower protein synthesis (puromycin incorporation, ∼25-50%), mTOR (∼25%), and p70S6K1 (∼40%) in starved and insulin stimulated myoblasts. Conversely, anti-miR-16 increased basal protein synthesis (puromycin incorporation, ∼75%), mTOR (∼100%), and p70S6K1 (∼100%). Autophagy was enhanced by miR-16 overexpression (∼50% less BCL-2, ∼100% greater LC3II/I, ∼50% less p62) and impaired with miR-16 inhibition (∼45% greater BCL-2, ∼25% less total LC3, ∼50% greater p62). This study demonstrates reduced miR-16 during insulin resistance and establishes miR-16 control of protein accretion in skeletal muscle. J. Cell. Biochem. 117: 1775-1787, 2016. © 2015 Wiley Periodicals, Inc.

No Change in 24-Hour Hydration Status Following a Moderate Increase in Fluid Consumption.

PURPOSE: To investigate changes in 24-hour hydration status when increasing fluid intake. METHODS: Thirty-five healthy males (age 23.8 ± 4.7 years; mass 74.0 ± 9.4 kg) were divided into 4 treatment groups for 2 weeks of testing. Volumes of 24-hour fluid ingestion (including water from food) for weeks 1 and 2 was 35 and 40 ml/kg body mass, respectively. Each treatment group was given the same proportion of beverages in each week of testing: water only (n = 10), water + caloric cola (n = 7), water + noncaloric cola (n = 10), or water + caloric cola + noncaloric cola + orange juice (n = 8). Serum osmolality (Sosm), total body water (TBW) via bioelectrical impedance, 24-hour urine osmolality (Uosm), and volume (Uvol) were analyzed at the end of each 24-hour intervention. RESULTS: Independent of treatment, total beverage consumption increased 22% from week 1 to 2 (1685 ± 320 to 2054 ± 363 ml; p < 0.001). Independent of beverage assignment, the increase in fluid consumption between weeks 1 and 2 did not change TBW (43.4 ± 5.2 vs 43.0 ± 4.8 kg), Sosm (292 ± 5 vs 292 ± 5 mOsm/kg), 24-hour Uosm (600 ± 224 vs 571 ± 212 mOsm/kg), or 24-hour Uvol (1569 ± 607 vs 1580 ± 554 ml; all p > 0.05). CONCLUSIONS: Regardless of fluid volume or beverage type consumed, measures of 24-hour hydration status did not differ, suggesting that standard measures of hydration status are not sensitive enough to detect a 22% increase in beverage consumption.

Hydration Status over 24-H Is Not Affected by Ingested Beverage Composition.

OBJECTIVE: To investigate the 24-h hydration status of healthy, free-living, adult males when given various combinations of different beverage types. METHODS: Thirty-four healthy adult males participated in a randomized, repeated-measures design in which they consumed: water only (treatment A), water+cola (treatment B), water+diet cola (treatment C), or water+cola+diet cola+orange juice (treatment D) over a sedentary 24-h period across four weeks of testing. Volumes of fluid were split evenly between beverages within each treatment, and when accounting for food moisture content and metabolic water production, total fluid intake from all sources was equal to 35 ± 1 ml/kg body mass. Urine was collected over the 24-h intervention period and analyzed for osmolality (Uosm), volume (Uvol) and specific gravity (USG). Serum osmolality (Sosm) and total body water (TBW) via bioelectrical impedance were measured after the 24-h intervention. RESULTS: 24-h hydration status was not different between treatments A, B, C, and D when assessed via Uosm (590 ± 179; 616 ± 242; 559 ± 196; 633 ± 222 mOsm/kg, respectively) and Uvol (1549 ± 594; 1443 ± 576; 1690 ± 668; 1440 ± 566 ml) (all p > 0.05). A -difference in 24-h USG was observed between treatments A vs. D (1.016 ± 0.005 vs. 1.018 ± 0.007; p = 0.049). There were no differences between treatments at the end of the 24-h with regard to Sosm (291 ± 4; 293 ± 5; 292 ± 5; 293 ± 5 mOsm/kg, respectively) and TBW (43.9 ± 5.9; 43.8 ± 6.0; 43.7 ± 6.1; 43.8 ± 6.0 kg) (all p > 0.05). CONCLUSIONS: Regardless of the beverage combination consumed, there were no differences in providing adequate hydration over a 24-h period in free-living, healthy adult males. This confirms that beverages of varying composition are equally effective in hydrating the body.

Impaired exercise-induced mitochondrial biogenesis in the obese Zucker rat, despite PGC-1α induction, is due to compromised mitochondrial translation elongation.

Previously, we demonstrated that high-volume resistance exercise stimulates mitochondrial protein synthesis (a measure of mitochondrial biogenesis) in lean but not obese Zucker rats. Here, we examined factors involved in regulating mitochondrial biogenesis in the same animals. PGC-1α was 45% higher following exercise in obese but not lean animals compared with sedentary counterparts. Interestingly, exercised animals demonstrated greater PPARδ protein in both lean (47%) and obese (>200%) animals. AMPK phosphorylation (300%) and CPT-I protein (30%) were elevated by exercise in lean animals only, indicating improved substrate availability/flux. These findings suggest that, despite PGC-1α induction, obese animals were resistant to exercise-induced synthesis of new mitochondrial and oxidative protein. Previously, we reported that most anabolic processes are upregulated in these same obese animals regardless of exercise, so the purpose of this study was to assess specific factors associated with the mitochondrial genome as possible culprits for impaired mitochondrial biogenesis. Exercise resulted in higher mRNA contents of mitochondrial transcription factor A (∼50% in each phenotype) and mitochondrial translation initiation factor 2 (31 and 47% in lean and obese, respectively). However, mitochondrial translation elongation factor-Tu mRNA was higher following exercise in lean animals only (40%), suggesting aberrant regulation of mitochondrial translation elongation as a possible culprit in impaired mitochondrial biogenesis following exercise with obesity.

Effects of PGC-1α overexpression on the myogenic response during skeletal muscle regeneration.

The ability of skeletal muscle to regenerate from injury is crucial for locomotion, metabolic health, and quality of life. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1A) is a transcriptional coactivator required for mitochondrial biogenesis. Increased mitochondrial biogenesis is associated with improved muscle cell differentiation, however PGC1A's role in skeletal muscle regeneration following damage requires further investigation. The purpose of this study was to investigate the role of skeletal muscle-specific PGC1A overexpression during regeneration following damage. 22 C57BL/6J (WT) and 26 PGC1A muscle transgenic (A1) mice were injected with either phosphate-buffered saline (PBS, uninjured control) or Bupivacaine (MAR, injured) into their tibialis anterior (TA) muscle to induce skeletal muscle damage. TA muscles were extracted 3- or 28-days post-injury and analyzed for markers of regenerative myogenesis and protein turnover. Pgc1a mRNA was ∼10-20 fold greater in A1 mice. Markers of protein synthesis, AKT and 4EBP1, displayed decreases in A1 mice compared to WT at both timepoints indicating a decreased protein synthetic response. Myod mRNA was ∼75% lower compared to WT 3 days post-injection. WT mice exhibited decreased cross-sectional area of the TA muscle at 28 days post-injection with bupivacaine compared to all other groups. PGC1A overexpression modifies the myogenic response during regeneration.

The effect of diet-induced obesity on extracellular matrix remodeling during skeletal muscle regeneration.

Diet-induced obesity has previously been shown to occur with the concomitant rise in the expression of proinflammatory cytokines and increases in collagen deposition. While it has been known that the regenerative process of skeletal muscle is altered in obese mice following an acute muscle injury, we sought to examine differences in the expression of various markers of extracellular matrix remodeling and repair. Our laboratory has previously reported an impaired inflammatory and protein synthetic signaling in these mice that may contribute negatively to the muscle regenerative process. To expand upon this previous investigation, tissues from these animals underwent further analysis to determine the extent of changes to the regenerative response within the extracellular matrix, including transcriptional changes in Collagen I, Collagen III, and Fibronectin. Here, we show that the expression of Collagen III:I is significantly increased at 3-days post-injury in obese injured animals compared to lean injured animals (p â€‹= â€‹0.0338), and by 28-days the obese injured animals exhibit a significantly lower Collagen III:I than their lean injured counterparts (p â€‹= â€‹0.0035). We demonstrate an impaired response to an acute muscle injury in obese mice when compared with lean counterparts. However, further studies are required to elucidate translational consequences of these changes, as well as to determine any causative mechanisms that may be driving this effect.

Metabolipidomic profiling reveals an age-related deficiency of skeletal muscle pro-resolving mediators that contributes to maladaptive tissue remodeling.

Specialized pro-resolving mediators actively limit inflammation and support tissue regeneration, but their role in age-related muscle dysfunction has not been explored. We profiled the mediator lipidome of aging muscle via liquid chromatography-tandem mass spectrometry and tested whether treatment with the pro-resolving mediator resolvin D1 (RvD1) could rejuvenate the regenerative ability of aged muscle. Aged mice displayed chronic muscle inflammation and this was associated with a basal deficiency of pro-resolving mediators 8-oxo-RvD1, resolvin E3, and maresin 1, as well as many anti-inflammatory cytochrome P450-derived lipid epoxides. Following muscle injury, young and aged mice produced similar amounts of most pro-inflammatory eicosanoid metabolites of cyclooxygenase (e.g., prostaglandin E2 ) and 12-lipoxygenase (e.g., 12-hydroxy-eicosatetraenoic acid), but aged mice produced fewer markers of pro-resolving mediators including the lipoxins (15-hydroxy-eicosatetraenoic acid), D-resolvins/protectins (17-hydroxy-docosahexaenoic acid), E-resolvins (18-hydroxy-eicosapentaenoic acid), and maresins (14-hydroxy-docosahexaenoic acid). Similar absences of downstream pro-resolving mediators including lipoxin A4 , resolvin D6, protectin D1/DX, and maresin 1 in aged muscle were associated with greater inflammation, impaired myofiber regeneration, and delayed recovery of strength. Daily intraperitoneal injection of RvD1 had minimal impact on intramuscular leukocyte infiltration and myofiber regeneration but suppressed inflammatory cytokine expression, limited fibrosis, and improved recovery of muscle function. We conclude that aging results in deficient local biosynthesis of specialized pro-resolving mediators in muscle and that immunoresolvents may be attractive novel therapeutics for the treatment of muscular injuries and associated pain in the elderly, due to positive effects on recovery of muscle function without the negative side effects on tissue regeneration of non-steroidal anti-inflammatory drugs.

Sestrins regulate muscle stem cell metabolic homeostasis.

The health and homeostasis of skeletal muscle are preserved by a population of tissue-resident muscle stem cells (MuSCs) that maintain a state of mitotic and metabolic quiescence in adult tissues. The capacity of MuSCs to preserve the quiescent state declines with aging and metabolic insults, promoting premature activation and stem cell exhaustion. Sestrins are a class of stress-inducible proteins that act as antioxidants and inhibit the activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling complex. Despite these pivotal roles, the role of Sestrins has not been explored in adult stem cells. We show that SESTRIN1,2 loss results in hyperactivation of the mTORC1 complex, increased propensity to enter the cell cycle, and shifts in metabolic flux. Aged SESTRIN1,2 knockout mice exhibited loss of MuSCs and a reduced ability to regenerate injured muscle. These findings demonstrate that Sestrins help maintain metabolic pathways in MuSCs that protect quiescence against aging.

Cancer-induced Cardiac Atrophy Adversely Affects Myocardial Redox State and Mitochondrial Oxidative Characteristics.

Cachexia presents in 80% of advanced cancer patients; however, cardiac atrophy in cachectic patients receives little attention. This cardiomyopathy contributes to increased occurrence of adverse cardiac events compared to age-matched population norms. Research on cardiac atrophy has focused on remodeling; however, alterations in metabolic properties may be a primary contributor. PURPOSE: Determine how cancer-induced cardiac atrophy alters mitochondrial turnover, mitochondrial mRNA translation machinery and in-vitro oxidative characteristics. METHODS: Lewis lung carcinoma (LLC) tumors were implanted in C57BL6/J mice and grown for 28days to induce cardiac atrophy. Endogenous metabolic species, and markers of mitochondrial function were assessed. H9c2 cardiomyocytes were cultured in LLC-conditioned media with(out) the antioxidant MitoTempo. Cells were analyzed for ROS, oxidative capacity, and hypoxic resistance. RESULTS: LLC heart weights were ~10% lower than controls. LLC hearts demonstrated ~15% lower optical redox ratio (FAD/FAD+NADH) compared to PBS controls. When compared to PBS, LLC hearts showed ~50% greater COX-IV and VDAC, attributed to ~50% lower mitophagy markers. mt-mRNA translation machinery was elevated similarly to markers of mitochondrial content. mitochondrial DNA-encoded Cytb was ~30% lower in LLC hearts. ROS scavengers GPx-3 and GPx-7 were ~50% lower in LLC hearts. Treatment of cardiomyocytes with LLC-conditioned media resulted in higher ROS (25%), lower oxygen consumption rates (10% at basal, 75% at maximal), and greater susceptibility to hypoxia (~25%) -- which was reversed by MitoTempo. CONCLUSION: These results substantiate metabolic cardiotoxic effects attributable to tumor-associated factors and provide insight into interactions between mitochondrial mRNA translation, ROS mitigation, oxidative capacity and hypoxia resistance.

Moderators of skeletal muscle maintenance are compromised in sarcopenic obese mice.

The purpose of this study was to determine whether sarcopenic obesity accelerates impairments in muscle maintenance through the investigation of cell cycle progression and myogenic, inflammatory, catabolic and protein synthetic signaling in mouse gastrocnemius muscles. At 4 weeks old, 24 male C57BL/6 mice were fed either a high fat diet (HFD, 60 % fat) or normal chow (NC, 17 % fat) for either 8-12 weeks or 21-23 months. At 3-4 months or 22-24 months the gastrocnemius muscles were excised. In addition, plasma was taken for C2C12 differentiation experiments. Mean cross-sectional area (CSA) was reduced by 29 % in aged HFD fed mice compared to the aged NC mice. MyoD was roughly 50 % greater in the aged mice compared to young mice, whereas TNF-α and IGF-1 gene expression in aged HFD fed mice were reduced by 52 % and 65 % in comparison to aged NC fed mice, respectively. Myotubes pretreated with plasma from aged NC fed mice had 14 % smaller myotube diameter than their aged HFD counterparts. Aged obese mice had greater impairments to mediators of muscle maintenance as evident by reductions in muscle mass, CSA, along with alterations in cell cycle regulation and inflammatory and insulin signaling.

Murine muscle stem cell response to perturbations of the neuromuscular junction are attenuated with aging.

During aging and neuromuscular diseases, there is a progressive loss of skeletal muscle volume and function impacting mobility and quality of life. Muscle loss is often associated with denervation and a loss of resident muscle stem cells (satellite cells or MuSCs); however, the relationship between MuSCs and innervation has not been established. Herein, we administered severe neuromuscular trauma to a transgenic murine model that permits MuSC lineage tracing. We show that a subset of MuSCs specifically engraft in a position proximal to the neuromuscular junction (NMJ), the synapse between myofibers and motor neurons, in healthy young adult muscles. In aging and in a mouse model of neuromuscular degeneration (Cu/Zn superoxide dismutase knockout - Sod1-/-), this localized engraftment behavior was reduced. Genetic rescue of motor neurons in Sod1-/- mice reestablished integrity of the NMJ in a manner akin to young muscle and partially restored MuSC ability to engraft into positions proximal to the NMJ. Using single cell RNA-sequencing of MuSCs isolated from aged muscle, we demonstrate that a subset of MuSCs are molecularly distinguishable from MuSCs responding to myofiber injury and share similarity to synaptic myonuclei. Collectively, these data reveal unique features of MuSCs that respond to synaptic perturbations caused by aging and other stressors.

Emerging molecular mediators and targets for age-related skeletal muscle atrophy.

The age-associated decline in muscle mass has become synonymous with physical frailty among the elderly due to its major contribution in reduced muscle function. Alterations in protein and redox homeostasis along with chronic inflammation, denervation, and hormonal dysregulation are all hallmarks of muscle wasting and lead to clinical sarcopenia in older adults. Reduction in skeletal muscle mass has been observed and reported in the scientific literature for nearly 2 centuries; however, identification and careful examination of molecular mediators of age-related muscle atrophy have only been possible for roughly 3 decades. Here we review molecular targets of recent interest in age-related muscle atrophy and briefly discuss emerging small molecule therapeutic treatments for muscle wasting in sarcopenic susceptible populations.

Dissecting Murine Muscle Stem Cell Aging through Regeneration Using Integrative Genomic Analysis.

During aging, there is a progressive loss of volume and function in skeletal muscle that impacts mobility and quality of life. The repair of skeletal muscle is regulated by tissue-resident stem cells called satellite cells (or muscle stem cells [MuSCs]), but in aging, MuSCs decrease in numbers and regenerative capacity. The transcriptional networks and epigenetic changes that confer diminished regenerative function in MuSCs as a result of natural aging are only partially understood. Herein, we use an integrative genomics approach to profile MuSCs from young and aged animals before and after injury. Integration of these datasets reveals aging impacts multiple regulatory changes through significant differences in gene expression, metabolic flux, chromatin accessibility, and patterns of transcription factor (TF) binding activities. Collectively, these datasets facilitate a deeper understanding of the regulation tissue-resident stem cells use during aging and healing.

Resolvin D1 supports skeletal myofiber regeneration via actions on myeloid and muscle stem cells.

Specialized proresolving mediators (SPMs) actively limit inflammation and expedite its resolution by modulating leukocyte recruitment and function. Here we profiled intramuscular lipid mediators via liquid chromatography-tandem mass spectrometry-based metabolipidomics following myofiber injury and investigated the potential role of SPMs in skeletal muscle inflammation and repair. Both proinflammatory eicosanoids and SPMs increased following myofiber damage induced by either intramuscular injection of barium chloride or synergist ablation-induced functional muscle overload. Daily systemic administration of the SPM resolvin D1 (RvD1) as an immunoresolvent limited the degree and duration of inflammation, enhanced regenerating myofiber growth, and improved recovery of muscle strength. RvD1 suppressed inflammatory cytokine expression, enhanced polymorphonuclear cell clearance, modulated the local muscle stem cell response, and polarized intramuscular macrophages to a more proregenerative subset. RvD1 had minimal direct impact on in vitro myogenesis but directly suppressed myokine production and stimulated macrophage phagocytosis, showing that SPMs can modulate both infiltrating myeloid and resident muscle cell populations. These data reveal the efficacy of immunoresolvents as a novel alternative to classical antiinflammatory interventions in the management of muscle injuries to modulate inflammation while stimulating tissue repair.

Regulation of mitochondrial quality following repeated bouts of hindlimb unloading.

Muscle disuse impairs muscle quality and is associated with increased mortality. Little is known regarding additive effects of multiple bouts of disuse, which is a common occurrence in patients experiencing multiple surgeries. Mitochondrial quality is vital to muscle health and quality; however, to date mitochondrial quality control has not been investigated following multiple bouts of disuse. Therefore, the purpose of this study was to investigate mitochondrial quality controllers during multiple bouts of disuse by hindlimb unloading. Male rats (n ∼ 8/group) were assigned to the following groups: hindlimb unloading for 28 days, hindlimb unloading with 56 days of reloading, 2 bouts of hindlimb unloading separated by a recovery phase of 56 days of reloading, 2 bouts of hindlimb unloading and recovery after each disuse, or control animals with no unloading. At designated time points, tissues were collected for messenger RNA and protein analysis of mitochondrial quality. Measures of mitochondrial biogenesis, such as proliferator-activated receptor gamma coactivator 1 alpha, decreased 30%-40% with unloading with no differences noted between unloading conditions. Measures of mitochondrial translation were 40%-50% lower in unloading conditions, with no differences noted between bouts of unloading. Measures of mitophagy were 40%-50% lower with reloading, with no differences noted between reloading conditions. In conclusion, disuse causes alterations in measures of mitochondrial quality; however, multiple bouts of disuse does not appear to have additive effects. Novelty Disuse atrophy causes multiple alterations to mitochondrial quality control. With sufficient recovery most detriments to mitochondrial quality control are fixed. In general, multiple bouts of disuse do not produce additive effects.

Late life maintenance and enhancement of functional exercise capacity in low and high responding rats after low intensity treadmill training.

Intrinsic exercise capacity is predictive of both lifespan and healthspan but whether adaptive exercise capacity influences the benefits achieved from aerobic training implemented later in life is not known. AIM: To determine if exercise late in life provides any functional improvements or underlying beneficial biochemical adaptations in rats bred to have a high response to training (HRT rats) or little to no response to training (LRT rats). METHODS: Adult (11 months) and old (22 months) female LRT and HRT rats either remained sedentary (SED) or were exercised (EXER) on a treadmill 2-3 times/week at 60% of their initial maximum running speed and distance for 4 months. At 26 months of age, exercise capacity was re-evaluated and extensor digitorum longus, gastrocnemius (GTN), and tibialis anterior (TA) muscles were excised for histological and biochemical analysis. RESULTS: Both SED-HRT and SED-LRT rats showed decreased exercise capacity from 22 to 26 months, but with 4 months of treadmill training, EXER-HRT rats displayed a 50% improvement in exercise capacity while EXER-LRT rats maintained pre-training levels. Protein levels of antioxidant enzymes PRDX3, CuZnSOD, and PRXV were 6-fold greater in TA muscles of aged HRT rats compared to LRT rats. PGC-1α protein levels were ~2-fold greater in GTN and TA muscles of aged HRT than in LRT rats and TFAM protein was similarly elevated in GTN muscles of aged HRT rats compared with LRT rats. BNIP3 protein levels were 5-fold greater in TA muscles of aged HRT than in LRT rats while PINK1 protein content was reduced by 78% in GTN muscles of aged HRT rats compared with LRT rats. CONCLUSION: HRT rats retained the ability to improve exercise capacity into late life and that ability was associated with inherent and adaptive changes in antioxidant enzyme levels and markers of and mitochondrial quality related to healthspan benefits in aging. Moreover, low intensity exercise prevented the age-associated decline in functional exercise capacity in LRT rats.