Flexibility underlies differences in mitochondrial respiratory performance between migratory and non-migratory White-crowned Sparrows (Zonotrichia leucophrys).

Migration is one of the most energy-demanding behaviors observed in birds. Mitochondria are the primary source of energy used to support these long-distance movements, yet how mitochondria meet the energetic demands of migration is scarcely studied. We quantified changes in mitochondrial respiratory performance in the White-crowned Sparrow (Zonotrichia leucophrys), which has a migratory and non-migratory subspecies. We hypothesized that the long-distance migratory Gambel's subspecies (Z. l. gambelii) would show higher mitochondrial respiratory performance compared to the non-migratory Nuttall's subspecies (Z. l. nuttalli). We sampled Gambel's individuals during spring pre-migration, active fall migration, and a period with no migration or breeding (winter). We sampled Nuttall's individuals during periods coinciding with fall migration and the winter period of Gambel's annual cycle. Overall, Gambel's individuals had higher citrate synthase, a proxy for mitochondrial volume, than Nuttall's individuals. This was most pronounced prior to and during migration. We found that both OXPHOS capacity (state 3) and basal respiration (state 4) of mitochondria exhibit high seasonal flexibility within Gambel's individuals, with values highest during active migration. These values in Nuttall's individuals were most similar to Gambel's individuals in winter. Our observations indicate that seasonal changes in mitochondrial respiration play a vital role in migration energetics.

Impact of capillary and sarcolemmal proximity on mitochondrial structure and energetic function in skeletal muscle.

Mitochondria within skeletal muscle cells are located either between the muscle contractile apparatus (interfibrillar mitochondria, IFM) or beneath the cell membrane (subsarcolemmal mitochondria, SSM), with several structural and functional differences reported between IFM and SSM. However, recent 3D imaging studies demonstrate that mitochondria are particularly concentrated in the proximity of capillaries embedded in sarcolemmal grooves rather than in proximity to the sarcolemma itself (paravascular mitochondria, PVM). To evaluate the impact of capillary vs. sarcolemmal proximity, we compared the structure and function of skeletal muscle mitochondria located either lateral to embedded capillaries (PVM), adjacent to the sarcolemma but not in PVM pools (SSM) or interspersed between sarcomeres (IFM). Mitochondrial morphology and interactions were assessed by 3D electron microscopy coupled with machine learning segmentation, whereas mitochondrial energy conversion was assessed by two-photon microscopy of mitochondrial membrane potential, content, calcium, NADH redox and flux in live, intact cells. Structurally, although PVM and SSM were similarly larger than IFM, PVM were larger, rounder and had more physical connections to neighbouring mitochondria compared to both IFM and SSM. Functionally, PVM had similar or greater basal NADH flux compared to SSM and IFM, respectively, despite a more oxidized NADH pool and a greater membrane potential, signifying a greater activation of the electron transport chain in PVM. Together, these data indicate that proximity to capillaries has a greater impact on resting mitochondrial energy conversion and distribution in skeletal muscle than the sarcolemma alone. KEY POINTS: Capillaries have a greater impact on mitochondrial energy conversion in skeletal muscle than the sarcolemma. Paravascular mitochondria are larger, and the outer mitochondrial membrane is more connected with neighbouring mitochondria. Interfibrillar mitochondria are longer and have greater contact sites with other organelles (i.e. sarcoplasmic reticulum and lipid droplets). Paravascular mitochondria have greater activation of oxidative phosphorylation than interfibrillar mitochondria at rest, although this is not regulated by calcium.

Reorganization of mitochondria-organelle interactions during postnatal development in skeletal muscle.

Skeletal muscle cellular development requires the integrated assembly of mitochondria and other organelles adjacent to the sarcomere in support of muscle contractile performance. However, it remains unclear how interactions among organelles and with the sarcomere relates to the development of muscle cell function. Here, we combine 3D volume electron microscopy, proteomic analyses, and live cell functional imaging to investigate the postnatal reorganization of mitochondria-organelle interactions in skeletal muscle. We show that while mitochondrial networks are disorganized and loosely associated with the contractile apparatus at birth, contact sites among mitochondria, lipid droplets and the sarcoplasmic reticulum are highly abundant in neonatal muscles. The maturation process is characterized by a transition to highly organized mitochondrial networks wrapped tightly around the muscle sarcomere but also to less frequent interactions with both lipid droplets and the sarcoplasmic reticulum. Concomitantly, expression of proteins involved in mitochondria-organelle membrane contact sites decreases during postnatal development in tandem with a decrease in abundance of proteins associated with sarcomere assembly despite an overall increase in contractile protein abundance. Functionally, parallel measures of mitochondrial membrane potential, NADH redox status, and NADH flux within intact cells revealed that mitochondria in adult skeletal muscle fibres maintain a more activated electron transport chain compared with neonatal muscle mitochondria. These data demonstrate a developmental redesign reflecting a shift from muscle cell assembly and frequent inter-organelle communication toward a muscle fibre with mitochondrial structure, interactions, composition and function specialized to support contractile function. KEY POINTS: Mitochondrial network organization is remodelled during skeletal muscle postnatal development. The mitochondrial outer membrane is in frequent contact with other organelles at birth and transitions to more close associations with the contractile apparatus in mature muscles. Mitochondrial energy metabolism becomes more activated during postnatal development. Understanding the developmental redesign process within skeletal muscle cells may help pinpoint specific areas of deficit in muscles with developmental disorders.

How does density of the inner mitochondrial membrane influence mitochondrial performance?

Our current understanding of variation in mitochondrial performance is incomplete. The production of ATP via oxidative phosphorylation is dependent, in part, on the structure of the inner mitochondrial membrane. Morphology of the inner membrane is crucial for the formation of the proton gradient across the inner membrane and, therefore, ATP synthesis. The inner mitochondrial membrane is dynamic, changing shape and surface area. These changes alter density (amount per volume) of the inner mitochondrial membrane within the confined space of the mitochondrion. Because the number of electron transport system proteins within the inner mitochondrial membrane changes with inner mitochondrial membrane area, a change in the amount of inner membrane alters the capacity for ATP production within the organelle. This review outlines the evidence that the association between ATP synthases, inner mitochondrial membrane density, and mitochondrial density (number of mitochondria per cell) impacts ATP production by mitochondria. Furthermore, we consider possible constraints on the capacity of mitochondria to produce ATP by increasing inner mitochondrial membrane density.

Mitochondrial network configuration influences sarcomere and myosin filament structure in striated muscles.

Sustained muscle contraction occurs through interactions between actin and myosin filaments within sarcomeres and requires a constant supply of adenosine triphosphate (ATP) from nearby mitochondria. However, it remains unclear how different physical configurations between sarcomeres and mitochondria alter the energetic support for contractile function. Here, we show that sarcomere cross-sectional area (CSA) varies along its length in a cell type-dependent manner where the reduction in Z-disk CSA relative to the sarcomere center is closely coordinated with mitochondrial network configuration in flies, mice, and humans. Further, we find myosin filaments near the sarcomere periphery are curved relative to interior filaments with greater curvature for filaments near mitochondria compared to sarcoplasmic reticulum. Finally, we demonstrate variable myosin filament lattice spacing between filament ends and filament centers in a cell type-dependent manner. These data suggest both sarcomere structure and myofilament interactions are influenced by the location and orientation of mitochondria within muscle cells.

Flying on empty: reduced mitochondrial function and flight capacity in food-deprived monarch butterflies.

Mitochondrial function is fundamental to organismal performance, health and fitness - especially during energetically challenging events, such as migration. With this investigation, we evaluated mitochondrial sensitivity to ecologically relevant stressors. We focused on an iconic migrant, the North American monarch butterfly (Danaus plexippus), and examined the effects of two stressors: 7 days of food deprivation and infection by the protozoan parasite Ophryocystis elektroscirrha (known to reduce survival and flight performance). We measured whole-animal resting metabolic rate (RMR) and peak flight metabolic rate, and mitochondrial respiration of isolated mitochondria from the flight muscles. Food deprivation reduced mass-independent RMR and peak flight metabolic rate, whereas infection did not. Fed monarchs used mainly lipids in flight (respiratory quotient 0.73), but the respiratory quotient dropped in food-deprived individuals, possibly indicating switching to alternative energy sources, such as ketone bodies. Food deprivation decreased mitochondrial maximum oxygen consumption but not basal respiration, resulting in lower respiratory control ratio (RCR). Furthermore, food deprivation decreased mitochondrial complex III activity, but increased complex IV activity. Infection did not result in any changes in these mitochondrial variables. Mitochondrial maximum respiration rate correlated positively with mass-independent RMR and flight metabolic rate, suggesting a link between mitochondria and whole-animal performance. In conclusion, low food availability negatively affects mitochondrial function and flight performance, with potential implications for migration, fitness and population dynamics. Although previous studies have reported poor flight performance in infected monarchs, we found no differences in physiological performance, suggesting that reduced flight capacity may be due to structural differences or low energy stores.

Reduced Mitochondrial Respiration in Hybrid Asexual Lizards.

AbstractThe scarcity of asexual reproduction in vertebrates alludes to an inherent cost. Several groups of asexual vertebrates exhibit lower endurance capacity (a trait predominantly sourced by mitochondrial respiration) compared with congeneric sexual species. Here we measure endurance capacity in five species of Aspidoscelis lizards and examine mitochondrial respiration between sexual and asexual species using mitochondrial respirometry. Our results show reduced endurance capacity, reduced mitochondrial respiration, and reduced phenotypic variability in asexual species compared with parental sexual species, along with a positive relationship between endurance capacity and mitochondrial respiration. Results of lower endurance capacity and lower mitochondrial respiration in asexual Aspidoscelis are consistent with hypotheses involving mitonuclear incompatibility.

Short and long-term effect of reproduction on mitochondrial dynamics and autophagy in rats.

We evaluated mitochondrial dynamics and autophagy by investigating the acute and long-term changes in the liver and skeletal muscle of rats in multiple reproductive stages. A total of 48 rats were used. Rats were randomly assigned to three groups (n = 16 per group): nonreproductive females; females that became pregnant, gave birth, but had their pups removed at birth, and thus, did not lactate; and females that experienced pregnancy, gave birth, and were allowed to lactate. Each group was further divided into two-time subgroups (n = 8 per subgroup) and data were collected at a time-point corresponding to 1) peak lactation (day 14 of lactation) in the lactating animals (4 months of age) and 2) 15 weeks after parturition (12 weeks post-weaning in lactating animals; 7 months of age). Levels of several proteins involved in mitochondrial dynamics and the autophagy system were measured in the liver and skeletal muscle. Beclin1 protein levels in the liver were higher in non-lactating rats two weeks after parturition, while Beclin1 protein levels were highest in 7-month-old animals that had previously experienced a standard reproductive event that included pregnancy and a full 3 week of lactation. These animals also exhibited higher protein levels of the mitochondrial fusion marker Mfn2 in the liver. In skeletal muscle, we also observed increased protein levels of the mitochondrial fission marker DRP1 in non-lactating animals compared to animals that lactated. In summary, our data provide insightful information on the mechanisms that influence liver and skeletal muscle remodeling in response to the metabolic challenges of reproduction, and lactation in particular. Autophagy remodeling and mitochondrial fusion seem to coincide with liver mass size during the lactation stage of reproduction. Our findings highlight the complex changes that occur in the liver and skeletal muscle during reproduction, and highlights the remarkable plasticity required during this demanding metabolic feat.

Development of a Mobile Mitochondrial Physiology Laboratory for Measuring Mitochondrial Energetics in the Field.

Mitochondrial energetics is a central theme in animal biochemistry and physiology, with researchers using mitochondrial respiration as a metric to investigate metabolic capability. To obtain the measures of mitochondrial respiration, fresh biological samples must be used, and the entire laboratory procedure must be completed within approximately 2 h. Furthermore, multiple pieces of specialized equipment are required to perform these laboratory assays. This creates a challenge for measuring mitochondrial respiration in the tissues of wild animals living far from physiology laboratories as live tissue cannot be preserved for very long after collection in the field. Moreover, transporting live animals over long distances induces stress, which can alter mitochondrial energetics. This manuscript introduces the Auburn University (AU) MitoMobile, a mobile mitochondrial physiology laboratory that can be taken into the field and used on-site to measure mitochondrial metabolism in tissues collected from wild animals. The basic features of the mobile laboratory and the step-by-step methods for measuring isolated mitochondrial respiration rates are presented. Additionally, the data presented validate the success of outfitting the mobile mitochondrial physiology laboratory and making mitochondrial respiration measurements. The novelty of the mobile laboratory lies in the ability to drive to the field and perform mitochondrial measurements on the tissues of animals captured on site.

Mitochondrial Bioenergetics of Extramammary Tissues in Lactating Dairy Cattle.

Lactation is physiologically demanding, requiring increased nutrient and energy use. Mammary and extramammary tissues undergo metabolic changes for lactation. Although it has long been recognized that mitochondria play a critical role in lactation, the mitochondrial adaptations for milk synthesis in supporting tissues, such as liver and skeletal muscle are relatively understudied. In this study, we assessed the mitochondrial function in these tissues across lactation in dairy cattle. Tissue biopsies were taken at 8 ± 2 d (early, n = 11), 75 ± 4 d (peak, n = 11) and 199 ± 6 d (late, n = 11) in milk. Early lactation biopsies were harvested from one group of cows and the peak and late biopsies from a second cohort. Milk yield (MY) was recorded at each milking and milk samples were collected for composition analysis. Mitochondrial efficiency was quantified as the respiratory control ratio (RCR), comparing maximal to resting respiration rates. Liver complex II RCR was positively associated with MY. Liver ROS emission increased across lactation whereas liver antioxidant activity was similar across lactation. No change was detected in skeletal muscle RCR or ROS emission, but muscle GPx activity decreased across lactation and muscle SOD was negatively associated with MY. Muscle oxidative damage was elevated at early and late lactation. Across lactation, genes involved in mitochondrial biogenesis were upregulated in the liver. Our results indicate that during lactation, liver mitochondrial biogenesis and efficiency are increased, which is associated with greater milk yield. In contrast, the mitochondrial efficiency in skeletal muscle remains consistent across lactation, but undergoes oxidative damage, which is associated with reduced antioxidant activity.

Energy transfer between the mitochondrial network and lipid droplets in insulin resistant skeletal muscle.

Mitochondria and lipid droplets in the insulin resistant skeletal muscle of type 2 diabetic individuals have both been heavily investigated independently and are characterized by more fragmented, dysfunctional mitochondrial networks and larger lipid droplets compared to skeletal muscle of healthy individuals. Specialized contacts between mitochondrial and lipid droplet membranes are known to decrease in diabetic muscle, though it remains unclear how energy transfer at the remaining mitochondria-lipid droplet contact sites may be altered by type 2 diabetes. The purpose of this review is to highlight recent data on mitochondrial structure and function and lipid droplet dynamics in type 2 diabetic skeletal muscle and to underscore the need for more detailed investigations into the functional nature of mitochondria-lipid droplet interactions in type 2 diabetes.

Acute and chronic effects of resistance training on skeletal muscle markers of mitochondrial remodeling in older adults.

We investigated the acute and chronic effects of resistance training (RT) on skeletal muscle markers of mitochondrial content and remodeling in older, untrained adults. Sixteen participants (n = 6 males, n = 10 females; age = 59 ± 4 years) completed 10 weeks of full-body RT (2 day/week). Muscle biopsies from the vastus lateralis were obtained prior to RT (Pre), 24 hr following the first training session (Acute), and 72 hr following the last training session (Chronic). Protein levels of mitochondrial electron transport chain complexes I-V (+39 to +180%, p ≤ .020) and markers of mitochondrial fusion Mfn1 (+90%, p = .003), Mfn2 (+110%, p < .001), and Opa1 (+261%, p = .004) increased following chronic RT. Drp1 protein levels also increased (+134%, p = .038), while Fis1 protein levels did not significantly change (-5%, p = .584) following chronic RT. Interestingly, protein markers of mitochondrial biogenesis (i.e., PGC-1α, TFAM, and NRF1) or mitophagy (i.e., Pink1 and Parkin) were not significantly altered (p > .050) after 10 weeks of RT. In summary, chronic RT promoted increases in content of electron transport chain proteins (i.e., increased protein levels of all five OXPHOS complexes) and increase in the levels of proteins related to mitochondrial dynamics (i.e., increase in fusion protein markers) in skeletal muscle of older adults. These results suggest that chronic RT could be a useful strategy to increase mitochondrial protein content in older individuals.

Human Skeletal Muscle Mitochondrial Adaptations Following Resistance Exercise Training.

It is universally accepted that resistance training promotes increases in muscle strength and hypertrophy in younger and older populations. Although less investigated, studies largely suggest resistance training results in lower skeletal muscle mitochondrial volume; a phenomenon which has been described as a "dilution of the mitochondrial volume" via resistance training. While this phenomenon is poorly understood, it is likely a result of muscle fiber hypertrophy outpacing mitochondrial biogenesis. Critically, there is no evidence to suggest resistance training promotes a net loss in mitochondria. Further, given the numerous reports suggesting resistance training does not decrease and may even increase VO2max in previously untrained individuals, it is plausible certain aspects of mitochondrial function may be enhanced with resistance training, and this area warrants further research consideration. Finally, there are emerging data suggesting resistance training may affect mitochondrial dynamics. The current review will provide an in-depth discussion of these topics and posit future research directions which can further our understanding of how resistance training may affect skeletal muscle mitochondrial physiology.

Uncarboxylated osteocalcin decreases insulin-stimulated glucose uptake without affecting insulin signaling and regulators of mitochondrial biogenesis in myotubes.

Uncarboxylated osteocalcin (uOC) is a circulating bone matrix protein, which has previously been shown to regulate glucose uptake and systemic metabolism. However, the cellular mechanism by which uOC acts has yet to be elucidated. C2C12 mouse myotubes were treated for 72 h with uOC (1-100 ng/mL). Cellular metabolism was analyzed using oxygen consumption and extracellular acidification rate. Metabolic gene and protein expression were measured via quantitative real-time polymerase chain reaction and Western blot, respectively. Additionally, C2C12 myotubes were treated with 10 ng/mL uOC to examine glucose uptake and activation of insulin signaling with or without insulin resistance. Finally, cellular lipid content was measured via Oil Red O and Nile Red staining. uOC treatment resulted in dose-dependent alterations of oxygen consumption with little effect on regulators of mitochondrial metabolism. Basal expression of regulators of glucose uptake were unaffected by uOC treatment. However, insulin-stimulated glucose uptake was blunted by uOC treatment with no concurrent alterations in insulin signaling. While chronic insulin treatment resulted in suppressed activation of Akt, concurrent uOC treatment was unable to prevent these detrimental effects on insulin signaling. uOC treatment had no effect on markers of lipogenesis and cellular lipid content. These findings suggest that 72-h uOC treatment may alter oxygen consumption without effect on regulators of mitochondrial biogenesis. Additionally, uOC treatment suppressed insulin-stimulated glucose uptake in cultured myotubes but had little effect on insulin signaling or regulators of cellular metabolism and was unable to mitigate insulin resistance.

MODIFYING MIDSOLE STIFFNESS of BASKETBALL FOOTWEAR AFFECTS FOOT and ANKLE BIOMECHANICS.

BACKGROUND: There is a growing incidence of foot injuries in basketball, which may be from the sport's repetitive, forceful multi-directional demands. Modifying midsole stiffness of the basketball shoe has been reported to alter ankle motion and plantar forces to reduce the risk of injury; however, the effects on anatomical, in-shoe foot (metatarsal), motion is not well understood. PURPOSE: The purpose of this study was to identify differences in foot and ankle biomechanics between basketball shoes with differing midsole stiffness values during single-leg jump landings. It was hypothesized that a stiffer midsole would elicit lower 1st metatarsophalangeal joint (MTPJ) dorsiflexion angles, higher ankle dorsiflexion angles, and higher plantar forces and relative loading in the distal foot. STUDY DESIGN: Experimental cross-sectional study. METHODS: Twenty high school and collegiate-aged basketball players performed a single-leg side drop jump and a single-leg cross drop jump in a pair of standard basketball shoes and a pair of shoes modified with a fiberglass plate to increase midsole stiffness. Three-dimensional motion analysis and flexible insoles quantified foot and ankle kinematics and plantar force distribution, respectively. Separate 2 (footwear) × 2 (task) repeated measures ANOVA models were used to analyze differences in 1) ankle kinematics, 2) 1st metatarsophalangeal kinematics, 3) maximal regional plantar forces, and 4) relative load. RESULTS: The stiffer shoe elicited decreased peak ankle plantarflexion (mean difference = 5.8 °, p = 0.01) and eversion (mean difference = 6.6 °, p = 0.03) and increased peak ankle dorsiflexion angles (mean difference = 5.0 °, p = 0.008) but no differences were observed in 1st MTPJ motion (p > 0.05). The stiffer shoe also resulted in lower peak plantar forces (mean difference = 24.2N, p = 0.004) and relative load (mean difference = 1.9%, p = 0.001) under the lesser toes. CONCLUSIONS: Altering the midsole stiffness in basketball shoes did not reduce motion at the MTPJ, indicating that added stiffness may reduce shoe motion, but does not reduce in-shoe anatomical motion. Instead, a stiffer midsole elicits other changes, including additional ankle joint motion and a reduction in plantar forces under the lesser toes. Collectively, this indicates that clinicians need to account for unintended compensations that can occur throughout the kinetic chain when altering a shoe property to alleviate a musculoskeletal injury. LEVEL OF EVIDENCE: 2b.

Five months of voluntary wheel running downregulates skeletal muscle LINE-1 gene expression in rats.

Transposable elements (TEs) are mobile DNA and constitute approximately half of the human genome. LINE-1 (L1) is the only active autonomous TE in the mammalian genome and has been implicated in a number of diseases as well as aging. We have previously reported that skeletal muscle L1 expression is lower following acute and chronic exercise training in humans. Herein, we used a rodent model of voluntary wheel running to determine whether long-term exercise training affects markers of skeletal muscle L1 regulation. Selectively bred high-running female Wistar rats (n = 11 per group) were either given access to a running wheel (EX) or not (SED) at 5 wk of age, and these conditions were maintained until 27 wk of age. Thereafter, mixed gastrocnemius tissue was harvested and analyzed for L1 mRNA expression and DNA content along with other L1 regulation markers. We observed significantly (P < 0.05) lower L1 mRNA expression, higher L1 DNA methylation, and less L1 DNA in accessible chromatin regions in EX versus SED rats. We followed these experiments with 3-h in vitro drug treatments in L6 myotubes to mimic transient exercise-specific signaling events. The AMP-activated protein kinase (AMPK) agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR; 4 mM) significantly decreased L1 mRNA expression in L6 myotubes. However, this effect was not facilitated through increased L1 DNA methylation. Collectively, these data suggest that long-term voluntary wheel running downregulates skeletal muscle L1 mRNA, and this may occur through chromatin modifications. Enhanced AMPK signaling with repetitive exercise bouts may also decrease L1 mRNA expression, although the mechanism of action remains unknown.

Plumage redness signals mitochondrial function in the house finch.

Carotenoid coloration is widely recognized as a signal of individual condition in various animals, but despite decades of study, the mechanisms that link carotenoid coloration to condition remain unresolved. Most birds with red feathers convert yellow dietary carotenoids to red carotenoids in an oxidation process requiring the gene encoding the putative cytochrome P450 enzyme CYP2J19. Here, we tested the hypothesis that the process of carotenoid oxidation and feather pigmentation is functionally linked to mitochondrial performance. Consistent with this hypothesis, we observed high levels of red ketolated carotenoids associated with the hepatic mitochondria of moulting wild house finches (Haemorhous mexicanus), and upon fractionation, we found the highest concentration of ketolated carotenoids in the inner mitochondrial membrane. We further found that the redness of growing feathers was positively related to the performance of liver mitochondria. Structural modelling of CYP2J19 supports a direct role of this protein in carotenoid ketolation that may be functionally linked to cellular respiration. These observations suggest that feather coloration serves as a signal of core functionality through inexorable links to cellular respiration in the mitochondria.

Muscle fiber hypertrophy in response to 6 weeks of high-volume resistance training in trained young men is largely attributed to sarcoplasmic hypertrophy.

Cellular adaptations that occur during skeletal muscle hypertrophy in response to high-volume resistance training are not well-characterized. Therefore, we sought to explore how actin, myosin, sarcoplasmic protein, mitochondrial, and glycogen concentrations were altered in individuals that exhibited mean skeletal muscle fiber cross-sectional area (fCSA) hypertrophy following 6 weeks of high-volume resistance training. Thirty previously resistance-trained, college-aged males (mean ± standard deviation: 21±2 years, 5±3 training years) had vastus lateralis (VL) muscle biopsies obtained prior to training (PRE), at week 3 (W3), and at week 6 (W6). Muscle tissue from 15 subjects exhibiting PRE to W6 VL mean fCSA increases ranging from 320-1600 µm2 was further interrogated using various biochemical and histological assays as well as proteomic analysis. Seven of these individuals donated a VL biopsy after refraining from training 8 days following the last training session (W7) to determine how deloading affected biomarkers. The 15 fCSA hypertrophic responders experienced a +23% increase in mean fCSA from PRE to W6 (p<0.001) and, while muscle glycogen concentrations remained unaltered, citrate synthase activity levels decreased by 24% (p<0.001) suggesting mitochondrial volume decreased. Interestingly, repeated measures ANOVAs indicated that p-values approached statistical significance for both myosin and actin (p = 0.052 and p = 0.055, respectively), and forced post hoc tests indicated concentrations for both proteins decreased ~30% from PRE to W6 (p<0.05 for each target). Phalloidin-actin staining similarly revealed actin concentrations per fiber decreased from PRE to W6. Proteomic analysis of the sarcoplasmic fraction from PRE to W6 indicated 40 proteins were up-regulated (p<0.05), KEGG analysis indicated that the glycolysis/gluconeogenesis pathway was upregulated (FDR sig. <0.001), and DAVID indicated that the following functionally-annotated pathways were upregulated (FDR value <0.05): a) glycolysis (8 proteins), b) acetylation (23 proteins), c) gluconeogenesis (5 proteins) and d) cytoplasm (20 proteins). At W7, sarcoplasmic protein concentrations remained higher than PRE (+66%, p<0.05), and both actin and myosin concentrations remained lower than PRE (~-50%, p<0.05). These data suggest that short-term high-volume resistance training may: a) reduce muscle fiber actin and myosin protein concentrations in spite of increasing fCSA, and b) promote sarcoplasmic expansion coincident with a coordinated up-regulation of sarcoplasmic proteins involved in glycolysis and other metabolic processes related to ATP generation. Interestingly, these effects seem to persist up to 8 days following training.

Bovine Milk Extracellular Vesicles (EVs) Modification Elicits Skeletal Muscle Growth in Rats.

The current study investigated how bovine milk extracellular vesicles (EVs) affected rotarod performance and biomarkers of skeletal muscle physiology in young, growing rats. Twenty-eight-day Fisher 344 rats were provided an AIN-93G-based diet for 4 weeks that either remained unadulterated [EVs and RNA-sufficient (ERS; n = 12)] or was sonicated [EVs and RNA-depleted (ERD; n = 12)]. Prior to (PRE) and on the last day of the intervention (POST), animals were tested for maximal rotarod performance. Following the feeding period, the gastrocnemius muscle was analyzed at the histological, biochemical, and molecular levels and was also used to measure mitochondrial function and reactive oxygen species (ROS) emission. A main effect of time was observed for rotarod time (PRE > POST, p = 0.001). Terminal gastrocnemius mass was unaffected by diet, although gastrocnemius muscle fiber cross sectional area was 11% greater (p = 0.018) and total RNA (a surrogate of ribosome density) was 24% greater (p = 0.001) in ERD. Transcriptomic analysis of the gastrocnemius indicated that 22 mRNAs were significantly greater in ERS versus ERD (p < 0.01), whereas 55 mRNAs were greater in ERD versus ERS (p < 0.01). There were no differences in gastrocnemius citrate synthase activity or mitochondrial coupling (respiratory control ratio), although mitochondrial ROS production was lower in ERD gastrocnemius (p = 0.016), which may be explained by an increase in glutathione peroxidase protein levels (p = 0.020) in ERD gastrocnemius. Dietary EVs profiling confirmed that sonication in the ERD diet reduced EVs content by ∼60%. Our findings demonstrate that bovine milk EVs depletion through sonication elicits anabolic and transcriptomic effects in the gastrocnemius muscle of rapidly maturing rats. While this did not translate into a functional outcome between diets (i.e., rotarod performance), longer feeding periods may be needed to observe such functional effects.

Pre-training Skeletal Muscle Fiber Size and Predominant Fiber Type Best Predict Hypertrophic Responses to 6 Weeks of Resistance Training in Previously Trained Young Men.

Limited evidence exists regarding differentially expressed biomarkers between previously-trained low versus high hypertrophic responders in response to resistance training. Herein, 30 college-aged males (training age 5 ± 3 years; mean ± SD) partook in 6 weeks of high-volume resistance training. Body composition, right leg vastus lateralis (VL) biopsies, and blood were obtained prior to training (PRE) and at the 3-week (W3) and 6-week time points (W6). The 10 lowest (LOW) and 10 highest (HIGH) hypertrophic responders were clustered based upon a composite hypertrophy score of PRE-to-W6 changes in right leg VL mean muscle fiber cross-sectional area (fCSA), VL thickness assessed via ultrasound, upper right leg lean soft tissue mass assessed via dual x-ray absorptiometry (DXA), and mid-thigh circumference. Two-way ANOVAs were used to compare biomarker differences between the LOW and HIGH clusters over time, and stepwise linear regression was performed to elucidate biomarkers that explained significant variation in the composite hypertrophy score from PRE to W3, W3 to W6, and PRE to W6 in all 30 participants. PRE-to-W6 HIGH and LOW responders exhibited a composite hypertrophy change of +10.7 ± 3.2 and -2.1 ± 1.6%, respectively (p < 0.001). Compared to HIGH responders, LOW responders exhibited greater PRE type II fCSA (+18%, p = 0.022). Time effects (p < 0.05) existed for total RNA/mg muscle (W6 > W3 > PRE), phospho (p)-4EBP1 (PRE > W3&W6), pan-mTOR (PRE > W3 < W6), p-mTOR (PRE > W3 < W6), pan-AMPKα (PRE > W3 < W6), pan-p70s6k (PRE > W3), muscle ubiquitin-labeled proteins (PRE > W6), mechano growth factor mRNA (W6 > W3&PRE), 45S rRNA (PRE > W6), and muscle citrate synthase activity (PRE > W3&W6). No interactions existed for the aforementioned biomarkers and/or other assayed targets (muscle 20S proteasome activity, serum total testosterone, muscle androgen receptor protein levels, muscle glycogen, or serum creatine kinase). Regression analysis indicated PRE type II fiber percentage (R 2 = 0.152, ß = 0.390, p = 0.033) and PRE type II fCSA (R 2 = 0.207, ß = -0.455, p = 0.019) best predicted the PRE-to-W6 change in the composite hypertrophy score. While our sample size is limited, these data suggest: (a) HIGH responders may exhibit more growth potential given that they possessed lower PRE type II fCSA values and (b) possessing a greater type II fiber percentage as a trained individual may be advantageous for hypertrophy in response to resistance training.