Pore-forming toxins (PFTs) are used by both the immune system and by pathogens to disrupt cell membranes. Cells attempt to repair this disruption in various ways, but the exact mechanism(s) that cells use are not fully understood, nor agreed upon. Current models for membrane repair include (1) patch formation (e.g., fusion of internal vesicles with plasma membrane defects), (2) endocytosis of the pores, and (3) shedding of the pores by blebbing from the cell membrane. In this study, we sought to determine the specific mechanism(s) that cells use to resist three different cholesterol-dependent PFTs: Streptolysin O, Perfringolysin O, and Intermedilysin. We found that all three toxins were shed from cells by blebbing from the cell membrane on extracellular microvesicles (MVs). Unique among the cells studied, we found that macrophages were 10 times more resistant to the toxins, yet they shed significantly smaller vesicles than the other cells. To examine the mechanism of shedding, we tested whether toxins with engineered defects in pore formation or oligomerization were shed. We found that oligomerization was necessary and sufficient for membrane shedding, suggesting that calcium influx and patch formation were not required for shedding. However, pore formation enhanced shedding, suggesting that calcium influx and patch formation enhance repair. In contrast, monomeric toxins were endocytosed. These data indicate that cells use two interrelated mechanisms of membrane repair: lipid-dependent MV shedding, which we term 'intrinsic repair', and patch formation by intracellular organelles. Endocytosis may act after membrane repair is complete by removing inactivated and monomeric toxins from the cell surface.

We sought to identify biomarkers which delineated individual hypertrophic responses to resistance training. Untrained, college-aged males engaged in full-body resistance training (3 d/wk) for 12 weeks. Body composition via dual x-ray absorptiometry (DXA), vastus lateralis (VL) thickness via ultrasound, blood, VL muscle biopsies, and three-repetition maximum (3-RM) squat strength were obtained prior to (PRE) and following (POST) 12 weeks of training. K-means cluster analysis based on VL thickness changes identified LOW [n = 17; change (mean±SD) = +0.11±0.14 cm], modest (MOD; n = 29, +0.40±0.06 cm), and high (HI; n = 21, +0.69±0.14 cm) responders. Biomarkers related to histology, ribosome biogenesis, proteolysis, inflammation, and androgen signaling were analyzed between clusters. There were main effects of time (POST>PRE, p<0.05) but no cluster×time interactions for increases in DXA lean body mass, type I and II muscle fiber cross sectional area and myonuclear number, satellite cell number, and macronutrients consumed. Interestingly, PRE VL thickness was ~12% greater in LOW versus HI (p = 0.021), despite POST values being ~12% greater in HI versus LOW (p = 0.006). However there was only a weak correlation between PRE VL thickness scores and change in VL thickness (r2 = 0.114, p = 0.005). Forced post hoc analysis indicated that muscle total RNA levels (i.e., ribosome density) did not significantly increase in the LOW cluster (351±70 ng/mg to 380±62, p = 0.253), but increased in the MOD (369±115 to 429±92, p = 0.009) and HI clusters (356±77 to 470±134, p<0.001; POST HI>POST LOW, p = 0.013). Nonetheless, there was only a weak association between change in muscle total RNA and VL thickness (r2 = 0.079, p = 0.026). IL-1β mRNA levels decreased in the MOD and HI clusters following training (p<0.05), although associations between this marker and VL thickness changes were not significant (r2 = 0.0002, p = 0.919). In conclusion, individuals with lower pre-training VL thickness values and greater increases muscle total RNA levels following 12 weeks of resistance training experienced greater VL muscle growth, although these biomarkers individually explained only ~8–11% of the variance in hypertrophy.

We sought to determine the effects of L-leucine (LEU) or different protein supplements standardized to LEU (~3.0 g/serving) on changes in body composition, strength, and histological attributes in skeletal muscle and adipose tissue. Seventy-five untrained, college-aged males (mean ± standard error of the mean (SE); age = 21 ± 1 years, body mass = 79.2 ± 0.3 kg) were randomly assigned to an isocaloric, lipid-, and organoleptically-matched maltodextrin placebo (PLA, n = 15), LEU (n = 14), whey protein concentrate (WPC, n = 17), whey protein hydrolysate (WPH, n = 14), or soy protein concentrate (SPC, n = 15) group. Participants performed whole-body resistance training three days per week for 12 weeks while consuming supplements twice daily. Skeletal muscle and subcutaneous (SQ) fat biopsies were obtained at baseline (T1) and ~72 h following the last day of training (T39). Tissue samples were analyzed for changes in type I and II fiber cross sectional area (CSA), non-fiber specific satellite cell count, and SQ adipocyte CSA. On average, all supplement groups including PLA exhibited similar training volumes and experienced statistically similar increases in total body skeletal muscle mass determined by dual X-ray absorptiometry (+2.2 kg; time p = 0.024) and type I and II fiber CSA increases (+394 μm2 and +927 μm2; time p < 0.001 and 0.024, respectively). Notably, all groups reported increasing Calorie intakes ~600–800 kcal/day from T1 to T39 (time p < 0.001), and all groups consumed at least 1.1 g/kg/day of protein at T1 and 1.3 g/kg/day at T39. There was a training, but no supplementation, effect regarding the reduction in SQ adipocyte CSA (−210 μm2; time p = 0.001). Interestingly, satellite cell counts within the WPC (p < 0.05) and WPH (p < 0.05) groups were greater at T39 relative to T1. In summary, LEU or protein supplementation (standardized to LEU content) does not provide added benefit in increasing whole-body skeletal muscle mass or strength above PLA following 3 months of training in previously untrained college-aged males that increase Calorie intakes with resistance training and consume above the recommended daily intake of protein throughout training. However, whey protein supplementation increases skeletal muscle satellite cell number in this population, and this phenomena may promote more favorable training adaptations over more prolonged periods.

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 μm 2 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.

Adopting low carbohydrate, ketogenic diets remains a controversial issue for individuals who resistance train given that this form of dieting has been speculated to reduce skeletal muscle glycogen levels and stifle muscle anabolism. We sought to characterize the effects of a 12-week ketogenic diet (KD) on body composition, metabolic, and performance parameters in participants who trained recreationally at a local CrossFit facility. Twelve participants (nine males and three females, 31 ± 2 years of age, 80.3 ± 5.1 kg body mass, 22.9 ± 2.3% body fat, 1.37 back squat: body mass ratio) were divided into a control group (CTL; n = 5) and a KD group (n = 7). KD participants were given dietary guidelines to follow over 12 weeks while CTL participants were instructed to continue their normal diet throughout the study, and all participants continued their CrossFit training routine for 12 weeks. Pre, 2.5-week, and 12-week anaerobic performance tests were conducted, and pre- and 12-week tests were performed for body composition using dual X-ray absorptiometry (DXA) and ultrasound, resting energy expenditure (REE), blood-serum health markers, and aerobic capacity. Additionally, blood beta hydroxybutyrate (BHB) levels were measured weekly. Blood BHB levels were 2.8- to 9.5-fold higher in KD versus CTL throughout confirming a state of nutritional ketosis. DXA fat mass decreased by 12.4% in KD (p = 0.053). DXA total lean body mass changes were not different between groups, although DXA dual-leg lean mass decreased in the KD group by 1.4% (p = 0.068), and vastus lateralis thickness values decreased in the KD group by ~8% (p = 0.065). Changes in fasting glucose, HDL cholesterol, and triglycerides were similar between groups, although LDL cholesterol increased ~35% in KD (p = 0.048). Between-group changes in REE, one-repetition maximum (1-RM) back squat, 400 m run times, and VO2peak were similar between groups. While our n-sizes were limited, these preliminary data suggest that adopting a ketogenic diet causes marked reductions in whole-body adiposity while not impacting performance measures in recreationally-trained CrossFit trainees. Whether decrements in dual-leg muscle mass and vastus lateralis thickness in KD participants were due to fluid shifts remain unresolved, and increased LDL-C in these individuals warrants further investigation.