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Increased utrophin expression is known to reduce pathology in dystrophin-deficient skeletal muscles. Transgenic over-expression of PGC-1α has been shown to increase levels of utrophin mRNA and improve the histology of mdx muscles. Other reports have shown that PGC-1α signaling can lead to increased oxidative capacity and a fast to slow fiber type shift. Given that it has been shown that slow fibers produce and maintain more utrophin than fast skeletal muscle fibers, we hypothesized that over-expression of PGC-1α in post-natal mdx mice would increase utrophin levels via a fiber type shift, resulting in more slow, oxidative fibers that are also more resistant to contraction-induced damage. To test this hypothesis, neonatal mdx mice were injected with recombinant adeno-associated virus (AAV) driving expression of PGC-1α. PGC-1α over-expression resulted in increased utrophin and type I myosin heavy chain expression as well as elevated mitochondrial protein expression. Muscles were shown to be more resistant to contraction-induced damage and more fatigue resistant. Sirt-1 was increased while p38 activation and NRF-1 were reduced in PGC-1α over-expressing muscle when compared to control. We also evaluated if the use a pharmacological PGC-1α pathway activator, resveratrol, could drive the same physiological changes. Resveratrol administration (100 mg/kg/day) resulted in improved fatigue resistance, but did not achieve significant increases in utrophin expression. These data suggest that the PGC-1α pathway is a potential target for therapeutic intervention in dystrophic skeletal muscle.

The study objectives were to test the hypothesis that heat stress (HS) during gestational development alters postnatal growth, body composition, and biological response to HS conditions in pigs. To investigate this, 14 first parity crossbred gilts were exposed to one of four environmental treatments (TNTN, TNHS, HSTN, or HSHS) during gestation. TNTN and HSHS dams were exposed to thermal neutral (TN, cyclical 18-22°C) or HS conditions (cyclical 28-34°C) during the entire gestation, respectively. Dams assigned to HSTN and TNHS treatments were heat-stressed for the first or second half of gestation, respectively. Postnatal offspring were exposed to one of two thermal environments for an acute (24 h) or chronic (five weeks) duration in either constant TN (21°C) or HS (35°C) environment. Exposure to chronic HS during their growth phase resulted in decreased longissimus dorsi cross-sectional area (LDA) in offspring from HSHS and HSTN treated dams whereas LDA was larger in offspring from dams in TNTN and TNHS conditions. Irrespective of HS during prepubertal postnatal growth, pigs from dams that experienced HS during the first half of gestation (HSHS and HSTN) had increased (13.9%) subcutaneous fat thickness compared to pigs from dams exposed to TN conditions during the first half of gestation. This metabolic repartitioning towards increased fat deposition in pigs from dams heat-stressed during the first half of gestation was accompanied by elevated blood insulin concentrations (33%; P = 0.01). Together, these results demonstrate HS during the first half of gestation altered metabolic and body composition parameters during future development and in biological responses to a subsequent HS challenge.

Abstract Heat-related complications continue to be a major health concern for humans and animals and lead to potentially life-threatening conditions. Heat stress (HS) alters metabolic parameters and may alter glucose metabolism and insulin signaling. Therefore, the purpose of this investigation was to determine the extent to which 12 h of HS-altered energetic metabolism in oxidative skeletal muscle. To address this, crossbred gilts (n = 8/group) were assigned to one of three environmental treatments for 12 h: thermoneutral (TN; 21 °C), HS (37 °C), or pair-fed to HS counterparts but housed in TN conditions (PFTN). Following treatment, animals were euthanized and the semitendinosus red (STR) was recovered. Despite increased relative protein abundance of the insulin receptor, insulin receptor substrate (IRS1) phosphorylation was increased (P = 0.0005) at S307, an inhibitory site, and phosphorylated protein kinase B (AKT) (S473) was decreased (P = 0.03) likely serving to impair insulin signaling following 12 h of HS. Further, HS increased phosphorylated protein kinase C (PKC) ζ/λ (P = 0.02) and phosphorylated PKCδ/θ protein abundance (P = 0.02), which are known to regulate inhibitory serine phosphorylation of IRS1 (S307). Sarcolemmal glucose transporter 4 (Glut4) was decreased (P = 0.04) in the membrane fraction of HS skeletal muscle suggesting diminished glucose uptake capacity. HS-mediated increases (P = 0.04) in mechanistic target of rapamycin (mTOR) were not accompanied by phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1). HS decreased (P = 0.0006) glycogen synthase (GS) and increased (P = 0.02) phosphorylated GS suggesting impaired glycogen synthesis. In addition, HS altered fatty acid metabolic signaling by increasing (P = 0.02) Acetyl-CoA carboxylase (ACC), decreasing (P = 0.005) phosphorylated ATP-citrate lyase (pATPCL) and fatty acid synthase (P = 0.01) (FAS). These data suggest that 12 h of HS blunted insulin signaling, decreased protein synthesis, and altered glycogen and fatty acid metabolism.

Prolonged exposure to inescapable heat and humidity can lead to environment‐induced heat stress (EIHS). The extent to which EIHS damages the heart is largely unknown, though our previous work indicated EIHS caused ventricle‐dependent changes. The purpose of this investigation was to determine the extent to which EIHS increased proteolysis and altered calcium homeostasis in the left (LV) and right ventricles (RV). We hypothesized that in the RV, EIHS would increase proteolysis, whereas in the LV, EIHS would cause calcium dysregulation. To test this hypothesis, 3‐month‐old female pigs were assigned to thermoneutral (TN; 20 ± 0.2°C; n = 8) or EIHS (37.4 ± 0.2°C; n = 8) conditions for 24 h and hearts were removed. In the RV, we discovered increased markers of proteolysis such that the relative protein abundance of calpain II, MuRF‐1, and MAFbx/Atrogin1 was increased, as was a marker of calpain activity. Conversely, in the LV, we discovered that EIHS increased the relative protein abundance of calcium regulatory proteins, including PMCA, SERCA2a, STIM1, calsequestrin, CaMKII, and VDAC. These data demonstrate EIHS caused ventricular‐dependent changes such that in the RV, the balance of proteostasis was shifted toward proteolysis and in the LV, calcium dysregulation may underlie, at least in part, our previous discovery of ventricular thickening.

Semen cryopreservation has transformed the livestock industry by enabling the long-term storage and global distribution of genetic material from high-indexing sires. However, despite its benefits, cryopreservation of bull semen is known to impair sperm quality, as seen by declines in motility, membrane viability, and fertilization potential. The molecular mechanisms behind these impairments remain poorly understood, prompting the need for proteomic profiling to uncover biomarkers associated with cryosurvival and reproductive function. This study employed a bottom-up proteomic approach to characterize proteomic changes in bovine sperm before and after cryopreservation. Semen samples from five sexually mature bulls with proven fertility were divided into fresh group and cryopreserved groups and analyzed by LC-MS/MS. A total of 1,373 proteins were identified across all samples after filtering was applied. Principal component and hierarchical clustering analyses revealed 84 proteins with significant differential abundance (greater than log2FC of 2 and p < 0.05); 59 were more abundant in fresh sperm and 25 more abundant in cryopreserved sperm. Functional enrichment analysis indicated that proteins increased after cryopreservation were primarily associated with metabolic processes and keratinization, while proteins more abundant before cryopreservation were primarily involved in reproductive processing and cytoskeletal organization. Image-based flow cytometry further demonstrated cryo-induced redistribution cytochrome c (CYCT), dolichyl-diphosphooligosaccharide-protein glycosyltransferase 48kDa subunit (DDOST), and 40s ribosomal protein S3a (RPS3A) across distinct sperm cell regions. These results implicate mitochondrial integrity, redox homeostatic, and cytoskeletal structure as pathways disrupted during semen cryopreservation, consistent with apoptosis-like and capacitation-like changes. This work establishes a framework for future evaluation of protein biomarkers involved in critical sperm functions associated with cryoinjury and detecting susceptible sperm populations.

Duchenne muscular dystrophy (DMD) results from a genetic lesion in the dystrophin gene and leads to progressive muscle damage. PGC-1α pathway activation improves muscle function and decreases histopathological injury. We hypothesized that mild disease found in the limb muscles of mdx mice may be responsive to quercetin-mediated protection of dystrophic muscle via PGC-1α pathway activation. To test this hypothesis muscle function was measured in the soleus and EDL from 14 month old C57, mdx, and mdx mice treated with quercetin (mdxQ; 0.2% dietary enrichment) for 12 months. Quercetin reversed 50% of disease-related losses in specific tension and partially preserved fatigue resistance in the soleus. Specific tension and resistance to contraction-induced injury in the EDL were not protected by quercetin. Given some functional gain in the soleus it was probed with histological and biochemical approaches, however, in dystrophic muscle histopathological outcomes were not improved by quercetin and suppressed PGC-1α pathway activation was not increased. Similar to results in the diaphragm from these mice, these data suggest that the benefits conferred to dystrophic muscle following 12 months of quercetin enrichment were underwhelming. Spontaneous activity at the end of the treatment period was greater in mdxQ compared to mdx indicating that quercetin fed mice were more active in addition to engaging in more vigorous activity. Hence, modest preservation of muscle function (specific tension) and elevated spontaneous physical activity largely in the absence of tissue damage in mdxQ suggests dietary quercetin may mediate protection.

Duchenne muscular dystrophy (DMD) is caused by the absence of functional dystrophin protein and results in progressive muscle wasting. Dystrophin deficiency leads to a host of dysfunctional cellular processes including impaired autophagy. Autophagic dysfunction appears to be due, at least in part, to decreased lysosomal abundance mediated by decreased nuclear localization of transcription factor EB (TFEB), a transcription factor responsible for lysosomal biogenesis. PGC‐1α overexpression decreased disease severity in dystrophin‐deficient skeletal muscle and increased PGC‐1α has been linked to TFEB activation in healthy muscle. The purpose of this study was to determine the extent to which PGC‐1α overexpression increased nuclear TFEB localization, increased lysosome abundance, and increased autophagosome degradation. We hypothesized that overexpression of PGC‐1α would drive TFEB nuclear translocation, increase lysosome biogenesis, and improve autophagosome degradation. To address this hypothesis, we delivered PGC‐1α via adeno‐associated virus (AAV) vector injected into the right limb of 3‐week‐old mdx mice and the contralateral limbs received a sham injection. At 6 weeks of age, this approach increased PGC‐1α transcript by 60‐fold and increased TFEB nuclear localization in gastrocnemii from PGC‐1α treated limbs by twofold compared to contralateral controls. Furthermore, lamp2, a marker of lysosome abundance, was significantly elevated in muscles from limbs overexpressing PGC‐1α. Lastly, increased LC3II and similar p62 in PGC‐1α overexpressing‐limbs compared to contralateral limbs are supportive of increased degradation of autophagosomes. These data provide mechanistic insight into PGC‐1α‐mediated benefits to dystrophin‐deficient muscle, such that increased TFEB nuclear localization in dystrophin‐deficient muscle leads to increased lysosome biogenesis and autophagy. Degradation of autophagosomes appears to be impaired in dystrophic skeletal muscle in part due to reduced lysosome abundance. We found that PGC‐1alpha mediated TFEB nuclear translocation, increased lysosome abundance, and degradation of autophagosomes.

Heat stress (HS) diminishes animal production, reducing muscle growth and increasing adiposity, especially in swine. Excess heat creates a metabolic phenotype with limited lipid oxidation that relies on aerobic and anaerobic glycolysis as a predominant means of energy production, potentially reducing metabolic rate. To evaluate the effects of HS on substrate utilization and energy expenditure, crossbred barrows (15.2 ± 2.4 kg) were acclimatized for 5 days (22 °C), then treated with 5 days of TN (thermal neutral, 22 °C, n = 8) or HS (35 °C, n = 8). Pigs were fed ad libitum and monitored for respiratory rate (RR) and rectal temperature. Daily energy expenditure (DEE) and respiratory exchange ratio (RER, CO2:O2) were evaluated fasted in an enclosed chamber through indirect calorimetry. Muscle biopsies were obtained from the longissimus dorsi pre/post. HS increased temperature (39.2 ± 0.1 vs. 39.6 ± 0.1 °C, p < 0.01) and RER (0.91 ± 0.02 vs. 1.02 ± 0.02 VCO2:VO2, p < 0.01), but decreased DEE/BW (68.8 ± 1.7 vs. 49.7 ± 4.8 kcal/day/kg, p < 0.01) relative to TN. Weight gain (p = 0.80) and feed intake (p = 0.84) did not differ between HS and TN groups. HS decreased muscle metabolic flexibility (~33%, p = 0.01), but increased leucine oxidation (~35%, p = 0.02) compared to baseline values. These data demonstrate that HS disrupts substrate regulation and energy expenditure in growing pigs.

Muscle stem cells (MuSCs) hold great potential as a regenerative therapeutic but have met numerous challenges in treating systemic muscle diseases. Muscle stem cell-derived extracellular vesicles (MuSC-EVs) may overcome these limitations. We assessed the number and size distribution of extracellular vesicles (EVs) released by MuSCs ex vivo, determined the extent to which MuSC-EVs deliver molecular cargo to myotubes in vitro, and quantified MuSC-EV-mediated restoration of mitochondrial function following oxidative injury. MuSCs released an abundance of EVs in culture. MuSC-EVs delivered protein cargo into myotubes within 2 h of incubation. Fluorescent labeling of intracellular mitochondria showed co-localization of delivered protein and mitochondria. Oxidatively injured myotubes demonstrated a significant decline in maximal oxygen consumption rate and spare respiratory capacity relative to untreated myotubes. Remarkably, subsequent treatment with MuSC-EVs significantly improved maximal oxygen consumption rate and spare respiratory capacity relative to the myotubes that were damaged but received no subsequent treatment. Surprisingly, MuSC-EVs did not affect mitochondrial function in undamaged myotubes, suggesting the cargo delivered is able to repair but does not expand the existing mitochondrial network. These data demonstrate that MuSC-EVs rapidly deliver proteins into myotubes, a portion of which co-localizes with mitochondria, and reverses mitochondria dysfunction in oxidatively-damaged myotubes.

Environment‐induced heat stress (EIHS) results from sustained body temperature elevation owing to prolonged exposure to heat and humidity. We hypothesized that EIHS would cause kidney injury and cellular dysfunction. To test this hypothesis, female C57 mice were exposed to EIHS ( n  = 14; 37.6°C, 42.0% relative humidity) or thermoneutral (TN) conditions ( n  = 12; 31.2°C, 35.0% relative humidity) for 24 h. EIHS increased rectal temperature by 2.1°C ( p  < 0.001), decreased body mass by 10% ( p  = 0.036), decreased absolute kidney mass by 10% ( p  = 0.026), increased renal water content by 19% ( p  < 0.007) and increased blood urea nitrogen by 102% ( p  < 0.001) compared with TN. Histological analysis revealed that EIHS caused proximal tubule vacuolation ( p  = 0.001) and alterations in glomerular structure, supported by markers of lipid accumulation, tubular injury and inflammatory signalling. EIHS increased relative protein abundance of heat shock proteins 70 (48%, p  = 0.002), 90 (37%, p  = 0.001) and 60 (36%, p  = 0.026), in addition to heat shock factor 1 (2‐fold, p  = 0.020) compared with TN. In addition, there was biochemical evidence of mitochondrial remodelling, increased autophagic flux and robust activation of endoplasmic reticulum stress in kidneys from EIHS mice compared with TN mice. Collectively, these data suggest that 24 h of EIHS is sufficient to cause histological injury and cellular dysfunction, which might undermine renal health and function. What is the central question of this study? Does environment‐induced heat stress impair kidney health? What is the main finding and its importance? One day of environment‐induced heat stress is sufficient to alter the architecture of the renal corpuscle and cause cellular injury, which might be reflective of renal thermic injury.