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Several published protocols exist for isolating contractile or myofibrillar (MF) proteins from skeletal muscle, however, achieving complete resuspension of the myofibril pellet can be technically challenging. We performed several previously published MF isolation methods with the intent of determining which method was most suitable for MF protein isolation and solubilization. Here, we provide an optimized protocol to isolate sarcoplasmic and solubilized MF protein fractions from mammalian skeletal muscle suitable for several downstream assays.

The anatomical area of the extrahepatic bile ducts exhibits plethora of anatomic variants. The detailed study and comprehension of anatomic variations of extrahepatic bile ducts is a prerequisite in order to avoid the intraoperative biliary or tract damages, but they are also necessary for the targeted treatment of any complications. Gallbladder agenesis is a rare congenital anomaly of the biliary tree with an estimated incidence of 0.007-0.027% in surgical series which is much lower compared to the incidence of other gallbladder anomalies. It may be asymptomatic, but sometimes is associated with symptoms such as upper quadrant abdominal pain, which may be mistaken for cholecystitis and can lead the patient to the operating room. We present a case of a 30-year-old male patient without any significant past medical history presented with a 2-year history of upper abdominal complaints, dyspepsia, epigastric abdominal pain and weight loss, normal laboratory workup and unclear radiological signs which led him to exploratory laparoscopy due to the patient’s chronic symptoms, in order to exclude the presence of another underlying pathologic process. In addition to our case presentation, a relative review of literature was conducted. As a conclusion, examinations, such as transabdominal ultrasonography, may be misleading and MCRP should be the principal method of investigation to establish a presumptive diagnosis. However, in cases with a strong suspicion for a different underlying pathology, further investigation with exploratory laparoscopy may be warranted.

The activity of the heart and vessels is permanently modulated in response to electrical, mechanical and chemical signals to maintain cardiovascular system homeostasis. Some effects are rapidly manifested (e.g. contraction after an electrical stimulus), while others are observed at long-term (e.g. hypertrophy resulting from gene expression modulation). In any case, an orchestrated set of events follows from receptor to intracellular messengers and effectors via complex signaling routes. These include neurohumoral signaling targeting G protein-coupled receptors (such as adrenaline, angiotensin II and endothelin-1 receptors), growth factor pathways initiated at tyrosine (including insulin, vascular endothelial growth factor and fibroblast growth factor) or serine/threonine kinase receptors (transforming growth factor-β) or even direct intracellular/nuclear pathways (triggered by calcium, nitric oxide or thyroid hormones). Herein, the signaling pathways taking place in cardiomyocytes, endothelial cells, vascular smooth muscle cells and fibroblasts, mainly involved in the regulation of cardiac contraction, vasorelaxation, mechanotransduction, cell survival and hypertrophy are described. Finally, the role of extracellular matrix in cardiac remodeling and fibrosis is reviewed.

Dietary carotenoids have been proposed to boost immune system and antioxidant functions in vertebrate animals, but studies aimed at testing these physiological functions of carotenoids have often failed to find support. Here we subject yellow canaries (Serinus canaria), which possess high levels of carotenoids in their tissue, and white recessive canaries, which possess a knockdown mutation that results in very low levels of tissue carotenoids, to oxidative and pathogen challenges. Across diverse measures of physiological performance, we detect no differences between carotenoid-rich yellow and carotenoid-deficient white canaries. These results add further challenge to the assumption that carotenoids are directly involved in supporting physiological function in vertebrate animals. While some dietary carotenoids provide indirect benefits as retinoid precursors, our observations suggest that carotenoids themselves may play little to no direct role in key physiological processes in birds., Dietary carotenoids have been proposed to have physiological benefits in addition to contributing to coloration. Here, Koch et al. compare immune and antioxidant functions in yellow, carotenoid-rich vs. white, carotenoid-deficient canaries and find no difference, suggesting a limited physiological role of carotenoids.

Mechanical ventilation (MV) results in the rapid development of ventilator-induced diaphragm dysfunction (VIDD). While the mechanisms responsible for VIDD are not fully understood, recent data reveal that prolonged MV activates autophagy in the diaphragm, which may occur as a result of increased cellular reactive oxygen species (ROS) production. Therefore, we tested the hypothesis that (1) accelerated autophagy is a key contributor to VIDD; and that (2) oxidative stress is required to increase the expression of autophagy genes in the diaphragm. Our findings reveal that targeted inhibition of autophagy in the rat diaphragm prevented MV-induced muscle atrophy and contractile dysfunction. Attenuation of VIDD in these animals occurred as a result of increased diaphragm concentration of the antioxidant catalase and reduced mitochondrial ROS emission, which corresponded to reductions in the activity of calpain and caspase-3. To determine if increased ROS production is required for the upregulation of autophagy biomarkers in the diaphragm, rats that were administered the mitochondrial-targeted peptide SS-31 during MV. Results from this study demonstrated that mitochondrial ROS production in the diaphragm during MV is required for the increased expression of key autophagy genes (i.e. LC3, Atg7, Atg12, Beclin1 and p62), as well as for increased activity of cathepsin L. Together, these data reveal that autophagy is required for VIDD, and that autophagy inhibition reduces MV-induced diaphragm ROS production and prevents a positive feedback loop whereby increased autophagy is stimulated by oxidative stress, resulting in further increases in ROS and autophagy.

Mitochondria are hypothesized to display a biphasic response to reactive oxygen species (ROS) exposure. In this study, we evaluated the time course changes in mitochondrial performance and oxidative stress in house mice following X-irradiation. Forty-eight mice were equally divided among six groups, including a nonirradiated control and five experimental groups that varied in time between X-ray exposure and euthanasia (1 h and 1, 4, 7, and 10 days after X-irradiation). We measured parameters associated with mitochondrial respiratory function and ROS emission from isolated liver and skeletal muscle mitochondria and levels of oxidative damage and antioxidants in liver, skeletal muscle, and heart tissues. Mitochondrial function dropped initially after X-irradiation but recovered quickly and was elevated 10 days after the exposure. Hydrogen peroxide production, lipid peroxidation, and protein carbonylation showed inverse U-shaped curves, with levels returning to control or lower than control, 10 days after X-irradiation. Enzymatic antioxidants and markers for mitochondrial biogenesis exhibited a tissue-specific response after irradiation. These data provide the first chronological description of the mitohormetic response after a mild dose of irradiation and highlight the protective response that cells display to ROS exposure. This study also provides valuable information and application for future mitochondrial and oxidative stress studies in numerous physiological settings.

Energy allocation theory predicts that a lactating female should alter the energetic demands of its organ systems in a manner that maximizes nutrient allocation to reproduction while reducing nutrient use for tasks that are not vital to immediate survival. We posit that organ-specific plasticity in the function of mitochondria plays a key role in mediating these energetic trade-offs. The goal of this project was to evaluate mitochondrial changes that occur in response to lactation in two of the most energetically demanding organs in the body of a rodent, the liver and skeletal muscle. This work was conducted in wild-derived house mice (Mus musculus) kept in seminatural enclosures that allow the mice to maintain a natural social structure and move within a home range size typical of wild mice. Tissues were collected from females at peak lactation and from age-matched nonreproductive females. Mitochondrial respiration, oxidative damage, antioxidant, PGC-1α, and uncoupling protein levels were compared between lactating and nonreproductive females. Our findings suggest that both liver and skeletal muscle downregulate specific antioxidant proteins during lactation. The liver, but not skeletal muscle, of lactating females displayed higher oxidative damage than nonreproductive females. The liver mass of lactating females increased, but the liver displayed no change in mitochondrial respiratory control ratio. Skeletal muscle mass and mitochondrial respiratory control ratio were not different between groups. However, the respiratory function of skeletal muscle did vary among lactating females as a function of stage of concurrent pregnancy, litter size, and mass of the mammary glands. The observed changes are predicted to increase the efficiency of skeletal muscle mitochondria, reducing the substrate demands of skeletal muscle during lactation. Differences between our results and prior studies highlight the role that an animals' social and physical environment could play in how it adapts to the energetic demands of reproduction.

We examined molecular mechanisms that were altered during rapid soleus (type I fiber-dominant) and plantaris (type II fiber-dominant) hypertrophy in rats. Twelve Wistar rats (3.5 mo old; 6 female, 6 male) were subjected to surgical right-leg soleus and plantaris dual overload [synergist ablation (SA)], and sham surgeries were performed on left legs (CTL). At 14 days after surgery, the muscles were dissected. Plantaris mass was 27% greater in the SA than CTL leg ( P < 0.001), soleus mass was 13% greater in the SA than CTL leg ( P < 0.001), and plantaris mass was higher than soleus mass in the SA leg ( P = 0.001). Plantaris total RNA concentrations and estimated total RNA levels (suggestive of ribosome density) were 19% and 47% greater in the SA than CTL leg ( P < 0.05), protein synthesis levels were 64% greater in the SA than CTL leg ( P = 0.038), and satellite cell number per fiber was 60% greater in the SA than CTL leg ( P = 0.003); no differences in these metrics were observed between soleus SA and CTL legs. Plantaris, as well as soleus, 20S proteasome activity was lower in the SA than CTL leg ( P < 0.05), although the degree of downregulation was greater in the plantaris than soleus muscle (−63% vs. −20%, P = 0.001). These data suggest that early-phase plantaris hypertrophy occurs more rapidly than soleus hypertrophy, which coincided with greater increases in ribosome biogenesis, protein synthesis, and satellite cell density, as well as greater decrements in 20S proteasome activity, in the plantaris muscle.

Impairments in oxygen delivery and consumption can lead to reduced muscle endurance and physical disability. Perfusion, a measure of microvascular blood flow, provides information on nutrient delivery. T2* provides information about relative tissue oxygenation. Changes in these parameters following stress, such as exercise, can yield important information about imbalance between delivery and consumption. In this study, we implemented novel golden angle radial MRI acquisition technique to simultaneously quantify muscle perfusion and T2* at 7T with improved temporal resolution, and demonstrated assessment of spatial and temporal changes in these parameters within calf muscles during recovery from plantar flexion exercise. Nine healthy subjects participated the studies. At rest, perfusion and T2* in gastrocnemius muscle group within calf muscle were 5 ± 2 mL/100 g/min and 21.1 ± 3 ms respectively. Then the subjects performed plantar flexion exercise producing a torque of ~8ft-lb. Immediately after the exercise, perfusion was elevated to 79.3 ± 9 mL/100 g/min and T2* was decreased by 6 ± 3%. The time constants for 50% perfusion and T2* recovery were 54.1 ± 10 s and 68.5 ± 7 s respectively. These results demonstrate successful simultaneous quantification of perfusion and T2* in skeletal muscle using the developed technique.

Resistance training generally increases skeletal muscle hypertrophy, whereas aging is associated with a loss in muscle mass. Interestingly, select studies suggest that aging, as well as resistance training, may lead to a reduction in the abundance of skeletal muscle myofibrillar (or contractile) protein (per mg tissue). Proteomic interrogations have also demonstrated that aging, as well as weeks to months of resistance training, lead to appreciable alterations in the muscle proteome. Given this evidence, the purpose of this small pilot study was to examine total myofibrillar as well as total sarcoplasmic protein concentrations (per mg wet muscle) from the vastus lateralis muscle of males who were younger and resistance-trained (denoted as YT, n = 6, 25 &plusmn, 4 years old, 10 &plusmn, 3 self-reported years of training), younger and untrained (denoted as YU, n = 6, 21 &plusmn, 1 years old), and older and untrained (denoted as OU, n = 6, 62 &plusmn, 8 years old). The relative abundances of actin and myosin heavy chain (per mg tissue) were also examined using SDS-PAGE and Coomassie staining, and shotgun proteomics was used to interrogate the abundances of individual sarcoplasmic and myofibrillar proteins between cohorts. Whole-body fat-free mass (YT &gt, YU = OU), VL thickness (YT &gt, YU = OU), and leg extensor peak torque (YT &gt, YU = OU) differed between groups (p &lt, 0.05). Total myofibrillar protein concentrations were greater in YT versus OU (p = 0.005), but were not different between YT versus YU (p = 0.325). The abundances of actin and myosin heavy chain were greater in YT versus YU (p &lt, 0.05) and OU (p &lt, 0.001). Total sarcoplasmic protein concentrations were not different between groups. While proteomics indicated that marginal differences existed for individual myofibrillar and sarcoplasmic proteins between YT versus other groups, age-related differences were more prominent for myofibrillar proteins (YT = YU &gt, OU, p &lt, 0.05: 7 proteins, OU &gt, YT = YU, p &lt, 0.05: 11 proteins) and sarcoplasmic proteins (YT = YU &gt, 0.05: 8 proteins, YT&amp, YU, p &lt, 0.05: 29 proteins). In summary, our data suggest that modest (~9%) myofibrillar protein packing (on a per mg muscle basis) was evident in the YT group. This study also provides further evidence to suggest that notable skeletal muscle proteome differences exist between younger and older humans. However, given that our n-sizes are low, these results only provide a preliminary phenotyping of the reported protein and proteomic variables.

The life-history patterns that animals display are a product of their ability to maximize reproductive performance while concurrently balancing numerous metabolic demands. For example, the energetic costs of reproduction may reduce an animal's ability to support self-maintenance and longevity. In this work, we evaluated the impact of parity on mitochondrial physiology in laboratory mice. The theory of mitohormesis suggests that modest exposure to reactive oxygen species can improve performance, while high levels of exposure are damaging. Following this theory, we hypothesized that females that experienced one bout of reproduction (primiparous) would display improved mitochondrial capacity and reduced oxidative damage relative to non-reproductive (nulliparous) mice, while females that had four reproductive events (multiparous) would have lower mitochondrial performance and greater oxidative damage than both nulliparous and primiparous females. We observed that multiple reproductive events enhanced the mitochondrial respiratory capacity of liver mitochondria in females with high body mass. Four-bout females showed a positive relationship between body mass and mitochondrial capacity. In contrast, non-reproductive females showed a negative relationship between body mass and mitochondrial capacity and primiparous females had a slope that did not differ from zero. Other measured variables, too, were highly dependent on body mass, suggesting that a female's body condition has strong impacts on mitochondrial physiology. We also evaluated the relationship between how much females allocated to reproduction (cumulative mass of all young weaned) and mitochondrial function and oxidative stress in the multiparous females. We found that females that allocated more to reproduction had lower basal respiration (state 4), lower mitochondrial density, and higher protein oxidation in liver mitochondria than females that allocated less. These results suggest that, at least through their first four reproductive events, female laboratory mice may experience bioenergetic benefits from reproduction but only those females that allocated the most to reproduction appear to experience a potential cost of reproduction.

We investigated the effect of ischemic preconditioning (IPC) on performance of a 3 min maximal effort arm ergometer test in young women. Twenty healthy women (23.1 (SD 3.3) years) performed a 3 min maximal effort arm cycling exercise, preceded by IPC on both arms or SHAM in a counterbalanced randomized crossover design. Both blood flow (via high resolution ultrasound; n = 17) and muscle oxygenation/deoxygenation (via near infrared spectroscopy; n = 5) were measured throughout the IPC/SHAM. Performance and perceptual/physiological (i.e., heart rate, blood lactate, rating of perceived exertion, and triceps brachialis oxygenation) parameters were recorded during the exercise test. Occlusion during IPC completely blocked brachial artery blood flow, decreased oxygenated hemoglobin/myoglobin (Δ[oxy(Hb + Mb)]), and increased deoxygenated Hb/Mb (Δ[deoxy(Hb + Mb)]). There were no differences (P > 0.797) in performance (peak, mean, and end power output) or in any perceptual/physiological variables during the 3 min all-out test between IPC/SHAM. During exercise, Δ[oxy(Hb + Mb)] initially decreased with no differences (P ≥ 0.296) between conditions and returned towards baseline by the completion of the test while Δ[deoxy(Hb + Mb)] increased with no differences between conditions and remained elevated until completion of the test (P ≥ 0.755). We verified the successful application of IPC via blood flow and NIRS measures but found no effects on performance of a 3 min maximal effort arm cranking test in young women.

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.

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

The aim of the study was to track changes of perceived stress and body composition across an entire calendar year in National Collegiate Athletic Association (NCAA) division I female volleyball players. We hypothesized that perceived stress and body composition would vary between the competitive season and off-season, with the largest changes occurring during time points prior to the onset and after the end of the competitive season. Eight female volleyball players participated in a longitudinal study. Body mass, body mass index (BMI), percent body fat, fat mass, and fat free mass were obtained during the early, mid, late, and off season and during the pre, early, mid, and late competitive season. The perceived stress scale-10 was used to appraise stress levels. BMI and body mass were significantly higher in pre-season compared to early-offseason. Changes in BMI between these points were due to increase in fat mass. Fat mass and percent body fat were significantly higher in pre-season compared to late off-season, mid-season, and late season. Perceived stress was significantly higher at the mid-season compared to early offseason. A significant positive correlation existed between BMI and body fat (p

Carotenoid-based coloration in birds is widely considered an honest signal of individual condition, but the mechanisms responsible for condition dependency in such ornaments remain debated. Currently, the most common explanation for how carotenoid coloration serves as a reliable signal of condition is the resource trade-off hypothesis, which proposes that use of carotenoids for ornaments reduces their availability for use by the immune system or for protection from oxidative damage. However, two main assumptions of the hypothesis remain in question: whether carotenoids boost the performance of internal processes such as immune and antioxidant defenses, and whether allocating carotenoids to ornaments imposes a trade-off with such benefits. In this study, we tested these two fundamental assumptions using types of domestic canary (Serinus canaria domestica) that enable experiments in which carotenoid availability and allocation can be tightly controlled. Specifically, we assessed metrics of immune and antioxidant performance in three genetic variants of the color-bred canary that differ only in carotenoid phenotype: ornamented, carotenoid-rich yellow canaries; unornamented, carotenoid-rich 'white dominant' canaries; and unornamented, carotenoid-deficient 'white recessive' canaries. The resource trade-off hypothesis predicts that carotenoid-rich individuals should outperform carotenoid-deficient individuals and that birds that allocate carotenoids to feathers should pay a cost in the form of reduced immune function or greater oxidative stress compared with unornamented birds. We found no evidence to support either prediction; all three canary types performed equally across measures. We suggest that testing alternative mechanisms for the honesty of carotenoid-based coloration should be a key focus of future studies of carotenoid-based signaling in birds.

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