Therapeutic effects of mitoquinol during an acute heat stress challenge in growing barrows.

Study objectives were to determine the effects of mitoquinol (MitoQ, a mitochondrial-targeted antioxidant) on biomarkers of metabolism and inflammation during acute heat stress (HS). Crossbred barrows [n = 32; 59.0 ±â€…5.6 kg body weight (BW)] were blocked by BW and randomly assigned to 1 of 4 environmental-therapeutic treatments: 1) thermoneutral (TN) control (n = 8; TNCon), 2) TN and MitoQ (n = 8; TNMitoQ), 3) HS control (n = 8; HSCon), or 4) HS and MitoQ (n = 8; HSMitoQ). Pigs were acclimated for 6 d to individual pens before study initiation. The trial consisted of two experimental periods (P). During P1 (2 d), pigs were fed ad libitum and housed in TN conditions (20.6 ±â€…0.8 °C). During P2 (24 h), HSCon and HSMitoQ pigs were exposed to continuous HS (35.2 ±â€…0.2 °C), while TNCon and TNMitoQ remained in TN conditions. MitoQ (40 mg/d) was orally administered twice daily (0700 and 1800 hours) during P1 and P2. Pigs exposed to HS had increased rectal temperature, skin temperature, and respiration rate (+1.5 °C, +6.8 °C, and +101 breaths per minute, respectively; P < 0.01) compared to their TN counterparts. Acute HS markedly decreased feed intake (FI; 67%; P < 0.01); however, FI tended to be increased in HSMitoQ relative to HSCon pigs (1.5 kg vs. 0.9 kg, respectively; P = 0.08). Heat-stressed pigs lost BW compared to their TN counterparts (-4.7 kg vs. +1.6 kg, respectively; P < 0.01); however, the reduction in BW was attenuated in HSMitoQ compared to HSCon pigs (-3.9 kg vs. -5.5 kg, respectively; P < 0.01). Total gastrointestinal tract weight (empty tissue and luminal contents) was decreased in HS pigs relative to their TN counterparts (6.2 kg vs. 8.6 kg, respectively; P < 0.01). Blood glucose increased in HSMitoQ relative to HSCon pigs (15%; P = 0.04). Circulating non-esterified fatty acids (NEFA) increased in HS compared to TN pigs (P < 0.01), although this difference was disproportionately influenced by elevated NEFA in HSCon relative to HSMitoQ pigs (251 µEq/L vs. 142 µEq/L; P < 0.01). Heat-stressed pigs had decreased circulating insulin relative to their TN counterparts (47%; P = 0.04); however, the insulin:FI ratio tended to increase in HS relative to TN pigs (P = 0.09). Overall, circulating leukocytes were similar across treatments (P > 0.10). Plasma C-reactive protein remained similar among treatments; however, haptoglobin increased in HS relative to TN pigs (48%; P = 0.03). In conclusion, acute HS exposure negatively altered animal performance, inflammation, and metabolism, which were partially ameliorated by MitoQ.

Heat stress (HS) compromises animal health and productivity, and this causes major economic losses in almost every livestock sector. The negative consequences of HS are thought to originate from intestinal barrier dysfunction and subsequent immune activation. The underlying causes of lost intestinal integrity during HS are likely multifactorial; however, intestinal ischemia, increased accumulation of reactive oxygen species, and the ensuing epithelial oxidative damage might be potential causes. Mitochondria-targeted antioxidants, such as mitoquinol (MitoQ), are probably more effective than traditional dietary antioxidants (i.e., selenium, vitamin E) at alleviating oxidative stress, as they localize and accumulate within the mitochondria, potentiating their antioxidant activity. Thus, the present study aimed to investigate MitoQ's role during a thermal event in growing pigs. Herein, HS increased all body temperature indices, decreased feed intake (FI), and induced substantial body weight (BW) loss. Interestingly, the reduction in FI and BW was less dramatic in pigs receiving MitoQ. Changes in circulating metabolism and the acute phase response were observed due to the HS challenge; however, contrary to our expectations, these changes were not offset by MitoQ administration. Although our results suggest a positive MitoQ effect on growth performance, future studies are needed to corroborate the replicability of this response during HS.

Biological sex impacts oxidative stress in skeletal muscle in a porcine heat stress model.

Oxidative stress contributes to heat stress (HS)-mediated alterations in skeletal muscle; however, the extent to which biological sex mediates oxidative stress during HS remains unknown. We hypothesized muscle from males would be more resistant to oxidative stress caused by HS than muscle from females. To address this, male and female pigs were housed in thermoneutral conditions (TN; 20.8 ± 1.6°C; 62.0 ± 4.7% relative humidity; n = 8/sex) or subjected to HS (39.4 ± 0.6°C; 33.7 ± 6.3% relative humidity) for 1 (HS1; n = 8/sex) or 7 days (HS7; n = 8/sex) followed by collection of the oxidative portion of the semitendinosus. Although HS increased muscle temperature, by 7 days, muscle from heat-stressed females was cooler than muscle from heat-stressed males (0.3°C; P < 0.05). Relative protein abundance of 4-hydroxynonenal (4-HNE)-modified proteins increased in HS1 females compared with TN (P = 0.05). Furthermore, malondialdehyde (MDA)-modified proteins and 8-hydroxy-2'-deoxyguanosine (8-OHdG) concentration, a DNA damage marker, was increased in HS7 females compared with TN females (P = 0.05). Enzymatic activities of catalase and superoxide dismutase (SOD) remained similar between groups; however, glutathione peroxidase (GPX) activity decreased in HS7 females compared with TN and HS1 females (P ≤ 0.03) and HS7 males (P = 0.02). Notably, HS increased skeletal muscle Ca2+ deposition (P = 0.05) and was greater in HS1 females compared with TN females (P < 0.05). Heat stress increased sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA)2a protein abundance (P < 0.01); however, Ca2+ ATPase activity remained similar between groups. Overall, despite having lower muscle temperature, muscle from heat-stressed females had increased markers of oxidative stress and calcium deposition than muscle from males following identical environmental exposure.NEW & NOTEWORTHY Heat stress is a global threat to human health and agricultural production. We demonstrated that following 7 days of heat stress, skeletal muscle from females was more susceptible to oxidative stress than muscle from males in a porcine model, despite cooler muscle temperatures. The vulnerability to heat stress-induced oxidative stress in females may be driven, at least in part, by decreased antioxidant capacity and calcium dysregulation.

Heat stress alters hematological parameters in barrows and gilts.

The purpose of this investigation was to establish the role biological sex plays in circulating factors following heat stress (HS). Barrows and gilts (36.8 ±â€…3.7 kg body weight) were kept in either thermoneutral (TN; 20.8 ±â€…1.6 °C; 62.0% ±â€…4.7% relative humidity; n = 8/sex) conditions or exposed to HS (39.4 ±â€…0.6 °C; 33.7% ±â€…6.3% relative humidity) for either 1 (HS1; n = 8/sex) or 7 (HS7; n = 8/sex) d. Circulating glucose decreased as a main effect of the environment (P = 0.03). Circulating non-esterified fatty acid (NEFA) had an environment × sex interaction (P < 0.01) as HS1 barrows had increased NEFA compared to HS1 gilts (P = 0.01) and NEFA from HS7 gilts increased compared to HS1 gilts (P = 0.02) and HS7 barrows (P = 0.04). Cortisol, insulin, glucagon, T3, and T4 were reduced as a main effect of environment (P ≤ 0.01). Creatinine was increased in HS1 and HS7 animals compared to TN (P ≤ 0.01), indicative of decreased glomerular filtration rate. White blood cell populations exhibited differential patterns based on sex and time. Neutrophils and lymphocytes had an environment × sex interaction (P ≤ 0.05) as circulating neutrophils were increased in HS1 barrows compared to TN and HS7 barrows, and HS1 gilts (P ≤ 0.01) and HS7 barrows had less neutrophils compared to TN barrows (P = 0.01), whereas they remained similar in gilts. In contrast, barrow lymphocyte numbers were similar between groups, but in HS7 gilts they were decreased compared to TN and HS1 gilts (P ≤ 0.04). In total, these data demonstrate that HS alters a host of circulating factors and that biological sex mediates, at least in part, the physiological response to HS.

Heat stress (HS) negatively impacts efficient pork production; however, the role of biological sex is largely unknown. The objective of this study was to determine the extent to which HS differentially impacted hematological parameters in barrows and gilts. To address this, 3-mo-old barrows and gilts were exposed to ambient temperature (TN) or constant HS for 1 or 7 d. Following the experimental period, blood was collected for analysis of hormones, metabolites, immune cells, and markers of organ damage. Overall, cortisol, insulin, glucagon, T3, and T4 were reduced following HS. Furthermore, 7 d of HS decreased circulating glucose, albeit slightly. Circulating fatty acids had a sex-specific response as HS1 barrows and HS7 gilts were increased compared to their environmental counterparts, though, these changes are minor compared to those expected with a similar feed restriction. HS caused immune system activation in barrows and gilts; however, circulating levels of specific white blood cells were time- and sex-dependent. Barrows appeared more resistant to HS-mediated kidney injury acutely; however, with continued heating, markers of kidney injury were similar between barrows and gilts. In total, these data suggest biological sex regulates some, but not all, aspects of HS-mediated biological changes in pigs.

The Interaction of Duchenne Muscular Dystrophy and Insulin Resistance.

Duchenne muscular dystrophy (DMD), caused by deficiency of functional dystrophin protein, is a fatal, progressive muscle disease that frequently includes metabolic dysregulation. Herein, we explore the physiologic consequences of dystrophin deficiency within the context of obesity and insulin resistance. We hypothesized that dystrophin deficiency increases the frequency of insulin resistance, and insulin resistance potentiates muscle pathology caused by dystrophin deficiency.

A fat- and sucrose-enriched diet causes metabolic alterations in mdx mice.

Duchenne muscular dystrophy (DMD), a progressive muscle disease caused by the absence of functional dystrophin protein, is associated with multiple cellular, physiological, and metabolic dysfunctions. As an added complication to the primary insult, obesity/insulin resistance (O/IR) is frequently reported in patients with DMD; however, how IR impacts disease severity is unknown. We hypothesized a high-fat, high-sucrose diet (HFHSD) would induce O/IR, exacerbate disease severity, and cause metabolic alterations in dystrophic mice. To test this hypothesis, we treated 7-wk-old mdx (disease model) and C57 mice with a control diet (CD) or an HFHSD for 15 wk. The HFHSD induced insulin resistance, glucose intolerance, and hyperglycemia in C57 and mdx mice. Of note, mdx mice on CD were also insulin resistant. In addition, visceral adipose tissue weights were increased with HFHSD in C57 and mdx mice though differed by genotype. Serum creatine kinase activity and histopathological analyses using Masson's trichrome staining in the diaphragm indicated muscle damage was driven by dystrophin deficiency but was not augmented by diet. In addition, markers of inflammatory signaling, mitochondrial abundance, and autophagy were impacted by disease but not diet. Despite this, in addition to disease signatures in CD-fed mice, metabolomic and lipidomic analyses demonstrated a HFHSD caused some common changes in C57 and mdx mice and some unique signatures of O/IR within the context of dystrophin deficiency. In total, these data revealed that in mdx mice, 15 wk of HFHSD did not overtly exacerbate muscle injury but further impaired the metabolic status of dystrophic muscle.

Indicators of increased ER stress and UPR in aged D2-mdx and human dystrophic skeletal muscles.

Duchenne muscular dystrophy (DMD) is a progressive muscle disease that results in muscle wasting, wheelchair dependence, and eventual death due to cardiac and respiratory complications. In addition to muscle fragility, dystrophin deficiency also results in multiple secondary dysfunctions, which may lead to the accumulation of unfolded proteins causing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). The purpose of this investigation was to understand how ER stress and the UPR are modified in muscle from D2-mdx mice, an emerging DMD model, and from humans with DMD. We hypothesized that markers of ER stress and the UPR are upregulated in D2-mdx and human dystrophic muscles compared to their healthy counterparts. Immunoblotting in diaphragms from 11-month-old D2-mdx and DBA mice indicated increased ER stress and UPR in dystrophic diaphragms compared to healthy, including increased relative abundance of ER stress chaperone CHOP, canonical ER stress transducers ATF6 and pIRE1α S724, and transcription factors that regulate the UPR such as ATF4, XBP1s, and peIF2α S51. The publicly available Affymetrix dataset (GSE38417) was used to analyze the expression of ER stress and UPR-related transcripts and processes. Fifty-eight upregulated genes related to ER stress and the UPR in human dystrophic muscles suggest pathway activation. Further, based on analyses using iRegulon, putative transcription factors that regulate this upregulation profile were identified, including ATF6, XBP1, ATF4, CREB3L2, and EIF2AK3. This study adds to and extends the emerging knowledge of ER stress and the UPR in dystrophin deficiency and identifies transcriptional regulators that may be responsible for these changes and be of therapeutic interest.

Environment-induced heat stress causes structural and biochemical changes in the heart.

Prolonged exposure to heat can lead to environment-induced heat stress (EIHS), which may jeopardize human health, but the extent to which EIHS affects cardiac architecture and myocardial cell health are unknown. We hypothesized EIHS would alter cardiac structure and cause cellular dysfunction. To test this hypothesis, 3-mo old female pigs were exposed to thermoneutral (TN; 20.6 ± 0.2 °C; n = 8) or EIHS (37.4 ± 0.2 °C; n = 8) conditions for 24 h, hearts were removed and dimensions measured, and portions of the left ventricle (LV) and right ventricle (RV) were collected. Environment-induced heat stress increased rectal temperature 1.3 °C (P < 0.01), skin temperature 11 °C (P < 0.01) and respiratory rate 72 breaths per minute (P < 0.01). Heart weight and length (apex to base) were decreased by 7.6% (P = 0.04) and 8.5% (P = 0.01), respectively, by EIHS, but heart width was similar between groups. Left ventricle wall thickness was increased (22%; P = 0.02) and water content was decreased (8.6%; P < 0.01) whereas in RV, wall thickness was decreased (26%; P = 0.04) and water content was similar in EIHS compared to TN. We also discovered ventricle-specific biochemical changes such that in RV EIHS increased heat shock proteins, decreased AMPK and AKT signaling, decreased activation of mTOR (35%; P < 0.05), and increased expression of proteins that participate in autophagy. In LV, heat shock proteins, AMPK and AKT signaling, activation of mTOR, and autophagy-related proteins were largely similar between groups. Biomarkers suggest EIHS-mediated reductions in kidney function. These data demonstrate EIHS causes ventricular-dependent changes and may undermine cardiac health, energy homeostasis, and function.

Indices of Defective Autophagy in Whole Muscle and Lysosome Enriched Fractions From Aged D2-mdx Mice.

Duchenne muscular dystrophy (DMD) is a fatal, progressive muscle disease caused by the absence of functional dystrophin protein. Previous studies in mdx mice, a common DMD model, identified impaired autophagy with lysosomal insufficiency and impaired autophagosomal degradation as consequences of dystrophin deficiency. Thus, we hypothesized that lysosomal abundance would be decreased and degradation of autophagosomes would be impaired in muscles of D2-mdx mice. To test this hypothesis, diaphragm and gastrocnemius muscles from 11 month-old D2-mdx and DBA/2J (healthy) mice were collected. Whole muscle protein from diaphragm and gastrocnemius muscles, and protein from a cytosolic fraction (CF) and a lysosome-enriched fraction (LEF) from gastrocnemius muscles, were isolated and used for western blotting. Initiation of autophagy was not robustly activated in whole muscle protein from diaphragm and gastrocnemius, however, autophagosome formation markers were elevated in dystrophic muscles. Autophagosome degradation was impaired in D2-mdx diaphragms but appeared to be maintained in gastrocnemius muscles. To better understand this muscle-specific distinction, we investigated autophagic signaling in CFs and LEFs from gastrocnemius muscles. Within the LEF we discovered that the degradation of autophagosomes was similar between groups. Further, our data suggest an expanded, though impaired, lysosomal pool in dystrophic muscle. Notably, these data indicate a degree of muscle specificity as well as model specificity with regard to autophagic dysfunction in dystrophic muscles. Stimulation of autophagy in dystrophic muscles may hold promise for DMD patients as a potential therapeutic, however, it will be critical to choose the appropriate model and muscles that most closely recapitulate findings from human patients to further develop these therapeutics.

Rapamycin administration during an acute heat stress challenge in growing pigs.

Study objectives were to determine the effects of rapamycin (Rapa) on biomarkers of metabolism and inflammation during acute heat stress (HS) in growing pigs. Crossbred barrows (n = 32; 63.5 ± 7.2 kg body weight [BW]) were blocked by initial BW and randomly assigned to 1 of 4 environmental-therapeutic treatments: 1) thermoneutral (TN) control (n = 8; TNCon), 2) TN and Rapa (n = 8; TNRapa), 3) HS control (n = 8; HSCon), or 4) HS and Rapa (n = 8; HSRapa). Following 6 d of acclimation to individual pens, pigs were enrolled in two experimental periods (P). During P1 (10 d), pigs were fed ad libitum and housed in TN conditions (21.3 ± 0.2°C). During P2 (24 h), HSCon and HSRapa pigs were exposed to constant HS (35.5 ± 0.4°C), while TNCon and TNRapa pigs remained in TN conditions. Rapamycin (0.15 mg/kg BW) was orally administered twice daily (0700 and 1800 hours) during both P1 and P2. HS increased rectal temperature and respiration rate compared to TN treatments (1.3°C and 87 breaths/min, respectively; P < 0.01). Feed intake (FI) markedly decreased in HS relative to TN treatments (64%; P < 0.01). Additionally, pigs exposed to HS lost BW (4 kg; P < 0.01), while TN pigs gained BW (0.7 kg; P < 0.01). Despite marked changes in phenotypic parameters caused by HS, circulating glucose and blood urea nitrogen did not differ among treatments (P > 0.10). However, the insulin:FI increased in HS relative to TN treatments (P = 0.04). Plasma nonesterified fatty acids (NEFA) increased in HS relative to TN treatments; although this difference was driven by increased NEFA in HSCon compared to TN and HSRapa pigs (P < 0.01). Overall, circulating white blood cells, lymphocytes, and monocytes decreased in HS compared to TN pigs (19%, 23%, and 33%, respectively; P ≤ 0.05). However, circulating neutrophils were similar across treatments (P > 0.31). The neutrophil-to-lymphocyte ratio (NLR) was increased in HS relative to TN pigs (P = 0.02); however, a tendency for reduced NLR was observed in HSRapa compared to HSCon pigs (21%; P = 0.06). Plasma C-reactive protein tended to differ across treatments (P = 0.06) and was increased in HSRapa relative to HSCon pigs (46%; P = 0.03). Circulating haptoglobin was similar between groups. In summary, pigs exposed to HS had altered phenotypic, metabolic, and leukocyte responses; however, Rapa administration had limited impact on outcomes measured herein.

The effect of Mitoquinol (MitoQ) on heat stressed skeletal muscle from pigs, and a potential confounding effect of biological sex.

Heat stress (HS) poses a major threat to human health and agricultural production. Oxidative stress and mitochondrial dysfunction appear to play key roles in muscle injury caused by HS. We hypothesized that mitoquinol (MitoQ), would alleviate oxidative stress and cellular dysfunction in skeletal muscle during HS. To address this, crossbred barrows (male pigs) were treated with placebo or MitoQ (40 mg/d) and were then exposed to thermoneutral (TN; 20 °C) or HS (35 °C) conditions for 24 h. Pigs were euthanized following the environmental challenge and the red portion of the semitendinosus (STR) was collected for analysis. Unexpectedly, malondialdehyde concentration, an oxidative stress marker, was similar between environmental and supplement treatments. Heat stress decreased LC3A/B-I (p < 0.05) and increased the ratio of LC3A/B-II/I (p < 0.05), while p62 was similar among groups suggesting increased degradation of autophagosomes during HS. These outcomes were in disagreement with our previous results in muscle from gilts (female pigs). To probe the impact of biological sex on HS-mediated injury in skeletal muscle, we compared STR from these barrows to archived STR from gilts subjected to a similar environmental intervention. We confirmed our previous findings of HS-mediated dysfunction in muscle from gilts but not barrows. These data also raise the possibility that muscle from gilts is more susceptible to environment-induced hyperthermia than muscle from barrows.

Heat Stress Reduces Metabolic Rate While Increasing Respiratory Exchange Ratio in Growing Pigs.

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 Cell-Derived Extracellular Vesicles Reverse Hydrogen Peroxide-Induced Mitochondrial Dysfunction in Mouse Myotubes.

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.

PGC-1α overexpression increases transcription factor EB nuclear localization and lysosome abundance in dystrophin-deficient skeletal muscle.

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.

Nutraceutical and pharmaceutical cocktails did not improve muscle function or reduce histological damage in D2-mdx mice.

Progressive muscle injury and weakness are hallmarks of Duchenne muscular dystrophy. We showed previously that quercetin (Q) partially protected dystrophic limb muscles from disease-related injury. As quercetin activates PGC-1α through Sirtuin-1, an NAD+-dependent deacetylase, the depleted NAD+ in dystrophic skeletal muscle may limit quercetin efficacy; hence, supplementation with the NAD+ donor, nicotinamide riboside (NR), may facilitate quercetin efficacy. Lisinopril (Lis) protects skeletal muscle and improves cardiac function in dystrophin-deficient mice; therefore, it was included in this study to evaluate the effects of lisinopril used with quercetin and NR. Our purpose was to determine the extent to which Q, NR, and Lis decreased dystrophic injury. We hypothesized that Q, NR, or Lis alone would improve muscle function and decrease histological injury and when used in combination would have additive effects. Muscle function of 11-mo-old DBA (healthy), D2-mdx (dystrophin-deficient), and D2-mdx mice was assessed after treatment with Q, NR, and/or Lis for 7 mo. To mimic typical pharmacology of patients with Duchenne muscular dystrophy, a group was treated with prednisolone (Pred) in combination with Q, NR, and Lis. At 11 mo of age, dystrophin deficiency decreased specific tension and tetanic force in the soleus and extensor digitorum longus muscles and was not corrected by any treatment. Dystrophic muscle was more sensitive to contraction-induced injury, which was partially offset in the QNRLisPred group, whereas fatigue was similar between all groups. Treatments did not decrease histological damage. These data suggest that treatment with Q, NR, Lis, and Pred failed to adequately maintain dystrophic limb muscle function or decrease histological damage.NEW & NOTEWORTHY Despite a compelling rationale and previous evidence to the contrary in short-term investigations, quercetin, nicotinamide riboside, or Lisinopril, alone or in combination, failed to restore muscle function or decrease histological injury in dystrophic limb muscle from D2-mdx mice after long-term administration. Importantly, we also found that in the D2-mdx model, an emerging and relatively understudied model of Duchenne muscular dystrophy dystrophin deficiency caused profound muscle dysfunction and histopathology in skeletal muscle.

The effect of recovery from heat stress on circulating bioenergetics and inflammatory biomarkers.

Heat stress (HS) jeopardizes animal productivity and health. The intestinal barrier is sensitive to HS and heat-induced hyperpermeability plays a key role in its pathophysiology. However, the biology of recovery following HS is less understood. Thus, study objectives were to determine the temporal pattern of metabolic, inflammatory, and intestinal histological parameters during HS recovery. Female pigs (n = 32; 19.5 ± 0.5 kg BW) were sacrificed following exposure to 1 of 4 environmental treatments: 1) constant thermoneutral (TN) conditions (TNC; 24.2 ± 0.5°C), 2) no TN recovery post HS (0D), 3) 3 d of TN recovery post HS (3D), and 4) 7 d of TN recovery post HS (7D). The HS protocol was cyclical (33.6 ± 1.8 to 37.4 ± 2.1°C) and lasted for 3 d for all HS treatments. During the 3 d of HS, rectal temperature, skin temperature, and respiration rates were increased (1.3°C, 4.8°C, and 77 breaths/min, respectively; P < 0.01) and ADFI was decreased (27%; P < 0.01) compared to TNC pigs. Skin temperature tended to be decreased 0.6°C in 3D pigs during days 1-3 of recovery (P = 0.06) and was decreased 1.6 and 0.7°C during days 1-3 and 4-7 of recovery, respectively, in 7D pigs (P ≤ 0.03) compared to TNC. Relative to TNC pigs, ADFI remained 14% decreased during days 1-3 of recovery in both 3D and 7D pigs, and 17% decreased during days 4-7 in 7D pigs (P ≤ 0.01). Plasma glucose was decreased (10%; P = 0.03) for 0D and 3D relative to TNC pigs. Circulating lipopolysaccharide-binding protein was increased in 3D and 7D vs. TNC pigs (110 and 147%, respectively; P = 0.01) and tended to increase linearly with increasing recovery time (P = 0.08). Circulating tumor necrosis factor alpha was decreased (15%) in 0D pigs and increased linearly with advancing recovery time (P < 0.01). Jejunum and ileum villus height were reduced 17 and 11% in 0D vs. TNC pigs and increased linearly with progressive recovery time (P < 0.01). Jejunum and ileum mucosal surface areas were reduced 17 and 9% in 0D pigs and remained decreased in the jejunum while the ileum recovered to TNC levels by day 3 of recovery. Relative to TNC pigs, goblet cell area was similar in jejunum and colon of 0D pigs but was reduced in the ileum of 0D pigs and in jejunum, ileum, and colon of 3D and 7D relative to TNC pigs (P < 0.01). In summary, HS has deleterious effects on intestinal morphology that seem to improve with recovery time. In contrast, feed consumption remained suppressed and inflammatory biomarkers indicative of leaky gut increased following the heat load.

Prolonged environment-induced hyperthermia alters autophagy in oxidative skeletal muscle in Sus scrofa.

Prolonged heat stress represents a continuing threat to human health and agricultural production. Despite the broad, negative impact of prolonged hyperthermia little is known about underlying pathological mechanisms leading to negative health outcomes, which has limited the development of etiological interventions and left clinicians and producers with only cooling and rehydration strategies. The purpose of this investigation was to determine the extent to which prolonged environment-induced hyperthermia altered autophagy in oxidative skeletal muscle in a large animal model, serving the dual purpose of accurately modeling human physiology as well as agricultural production. We hypothesized that prolonged hyperthermia would induce autophagy in skeletal muscle, independent of the accompanying caloric restriction. To test this hypothesis pigs were treated as follows: thermoneutral (20 °C), heat stress (35 °C), or were held under thermoneutral conditions but pair-fed to the heat stress group for seven days. Upon euthanasia the red portion of the semitendinosus was collected. We found that prolonged hyperthermic exposure increased oxidative stress without a corresponding change in antioxidant enzyme activities. Hyperthermia prevented initiation of autophagy despite increased markers of nucleation, elongation and autophagosome formation. However, p62 relative protein abundance, which is inversely correlated with autophagic degradation, was strongly increased suggesting suppressed degradation of autophagosomes. Markers of mitophagy and mitochondrial abundance were largely similar between groups. These data indicate that faulty autophagy plays a key role in hyperthermic muscle dysfunction.

Is Exercise the Right Medicine for Dystrophic Muscle?

INTRODUCTION: Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by a dystrophin protein deficiency. Dystrophin functions to stabilize and protect the muscle fiber during muscle contraction; thus, the absence of functional dystrophin protein leads to muscle injury. DMD patients experience progressive muscle necrosis, loss of function, and ultimately succumb to respiratory failure or cardiomyopathy. Exercise is known to improve muscle health and strength in healthy individuals as well as positively affect other systems. Because of this, exercise has been investigated as a potential therapeutic approach for DMD. METHODS: This review aims to provide a concise presentation of the exercise literature with a focus on dystrophin-deficient muscle. Our intent was to identify trends and gaps in knowledge with an appreciation of exercise modality. RESULTS: After compiling data from mouse and human studies, it became apparent that endurance exercises such as a swimming and voluntary wheel running have therapeutic potential in limb muscles of mice and respiratory training was beneficial in humans. However, in the comparatively few long-term investigations, the effect of low-intensity training on cardiac and respiratory muscles was contradictory. In addition, the effect of exercise on other systems is largely unknown. CONCLUSIONS: To safely prescribe exercise as a therapy to DMD patients, multisystemic investigations are needed including the evaluation of respiratory and cardiac muscle.

Short-term heat stress results in increased apoptotic signaling and autophagy in oxidative skeletal muscle in Sus scrofa.

Prolonged environment-induced hyperthermia causes morbidities and mortality in humans and animals and appears to cause organ-specific injury and dysfunction. We have previously determined autophagic dysfunction and apoptotic signaling in oxidative skeletal muscle following prolonged hyperthermia. The aim of this investigation was to extend our knowledge regarding the early chronology of heat stress-mediated apoptotic and autophagic signaling in oxidative skeletal muscle. We hypothesized that 2, 4, and 6 h of hyperthermia would increase apoptosis and autophagy in oxidative skeletal muscle compared to thermoneutral (TN) conditions. Pigs were assigned to four groups (n = 8/group) and exposed to environmental heat stress (37 °C) for 0, 2, 4, or 6 h. Immediately following environmental exposure animals were euthanized and the red portion of the semitendinosus was collected. Markers of apoptotic signaling were increased following 2 h of heating but returned to baseline thereafter, while caspase 3 activity remained elevated 2-3 fold (p < .05) throughout the hyperthermic period. Heat stress increased (p < .05) markers of autophagic activation, and nucleation as well as autophagosome formation and degradation linearly throughout the heating intervention. In addition, 6 h of hyperthermia increased (p < .05) markers of mitophagy. These data suggest that apoptotic signaling precedes increased autophagy during acute heat stress in oxidative skeletal muscle.

Short-term heat stress altered metabolism and insulin signaling in skeletal muscle.

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.

Heat stress induces autophagy in pig ovaries during follicular development.

Hyperthermia or heat stress (HS) occurs when heat dissipation mechanisms are overwhelmed by external and internal heat production. Hyperthermia negatively affects reproduction and potentially compromises oocyte integrity and reduces developmental competence of ensuing embryos. Autophagy is the process by which cells recycle energy through the reutilization of cellular components and is activated by a variety of stressors. Study objectives were to characterize autophagy-related proteins in the ovary following cyclical HS during the follicular phase. Twelve gilts were synchronized and subjected to cyclical HS (n = 6) or thermal neutral (n = 6) conditions for 5 days during the follicular phase. Ovarian protein abundance of Beclin 1 and microtubule associated protein light chain 3 beta II were each elevated as a result of HS (P = 0.001 and 0.003, respectively). The abundance of the autophagy related (ATG)12-ATG5 complex was decreased as a result of HS (P = 0.002). Regulation of autophagy and apoptosis occurs in tight coordination, and B-cell lymphoma (BCL)2 and BCL2L1 are involved in regulating both processes. BCL2L1 protein abundance, as detected via immunofluorescence, was increased in both the oocyte (∼1.6-fold; P < 0.01) and granulosa cells of primary follicles (∼1.4-fold P < 0.05) of HS ovaries. These results suggest that ovarian autophagy induction occurs in response to HS during the follicular phase, and that HS increases anti-apoptotic signaling in oocytes and early follicles. These data contribute to the biological understanding of how HS acts as an environmental stress to affect follicular development and negatively impact reproduction.