Vastus Lateralis Biopsy Samples Research Articles

Serum inflammatory cytokines as disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease, caused by mutations in the dystrophin gene, characterised by cycles of muscle degeneration, inflammation and regeneration. Recently, there has been renewed interest specifically in drugs that ameliorate muscle inflammation in DMD patients. The DE50-MD dog is a model of DMD that closely mimics the human DMD phenotype. We quantified inflammatory proteins in serum from wild-type (WT) and DE50-MD dogs aged 3-18 months to identify biomarkers for future pre-clinical trials. Significantly higher concentrations of C-C motif chemokine ligand 2 (CCL2), granulocyte-macrophage colony-stimulating factor (GM-CSF or CSF2), keratinocyte chemotactic-like (KC-like, homologous to mouse CXCL1), TNFα (or TNF), and interleukins IL2, IL6, IL7, IL8 (CXCL8), IL10, IL15 and IL18 were detected in DE50-MD serum compared to WT serum. Of these, CCL2 best differentiated the two genotypes. The relative level of CCL2 mRNA was greater in the vastus lateralis muscle of DE50-MD dogs than in that of WT dogs, and CCL2 was expressed both within and at the periphery of damaged myofibres. Serum CCL2 concentration was significantly associated with acid phosphatase staining in vastus lateralis biopsy samples in DE50-MD dogs. In conclusion, the serum cytokine profile suggests that inflammation is a feature of the DE50-MD phenotype. Quantification of serum CCL2 in particular is a useful non-invasive biomarker of the DE50-MD phenotype.

Muscle Adaptations to Heavy-Load and Blood Flow Restriction Resistance Training Methods.

Resistance-based blood flow restriction training (BFRT) improves skeletal muscle strength and size. Unlike heavy-load resistance training (HLRT), there is debate as to whether strength adaptations following BFRT interventions can be primarily attributed to concurrent muscle hypertrophy, as the magnitude of hypertrophy is often minor. The present study aimed to investigate the effect of 7 weeks of BFRT and HLRT on muscle strength and hypertrophy. The expression of protein growth markers from muscle biopsy samples was also measured. Male participants were allocated to moderately heavy-load training (HL; n = 9), low-load BFRT (LL + BFR; n = 8), or a control (CON; n = 9) group to control for the effect of time. HL and LL + BFR completed 21 training sessions (3 d.week−1) comprising bilateral knee extension and knee flexion exercises (HL = 70% one-repetition maximum (1-RM), LL + BFR = 20% 1-RM + blood flow restriction). Bilateral knee extension and flexion 1-RM strength were assessed, and leg muscle CSA was measured via peripheral quantitative computed tomography. Protein growth markers were measured in vastus lateralis biopsy samples taken pre- and post the first and last training sessions. Biopsy samples were also taken from CON at the same time intervals as HL and LL + BFR. Knee extension 1-RM strength increased in HL (19%) and LL + BFR (19%) but not CON (2%; p < 0.05). Knee flexion 1-RM strength increased similarly between all groups, as did muscle CSA (50% femur length; HL = 2.2%, LL + BFR = 3.0%, CON = 2.1%; TIME main effects). 4E-BP1 (Thr37/46) phosphorylation was lower in HL and LL + BFR immediately post-exercise compared with CON in both sessions (p < 0.05). Expression of other growth markers was similar between groups (p > 0.05). Overall, BFRT and HLRT improved muscle strength and size similarly, with comparable changes in intramuscular protein growth marker expression, both acutely and chronically, suggesting the activation of similar anabolic pathways. However, the low magnitude of muscle hypertrophy was not significantly different to the non-training control suggesting that strength adaptation following 7 weeks of BFRT is not driven by hypertrophy, but rather neurological adaptation.

Identifying the hub genes for Duchenne muscular dystrophy and Becker muscular dystrophy by weighted correlation network analysis

BackgroundThe goal of this study is to identify the hub genes for Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) via weighted correlation network analysis (WGCNA).MethodsThe gene expression profile of vastus lateralis biopsy samples obtained in 17 patients with DMD, 11 patients with BMD and 6 healthy individuals was downloaded from the Gene Expression Omnibus (GEO) database (GSE109178). After obtaining different expressed genes (DEGs) via GEO2R, WGCNA was conducted using R package, modules and genes that highly associated with DMD, BMD, and their age or pathology were screened. Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analysis and protein–protein interaction (PPI) network analysis were also conducted. Hub genes and highly correlated clustered genes were identified using Search Tool for the Retrieval of Interacting Genes (STRING) and Cystoscape software.ResultsOne thousand four hundred seventy DEGs were identified between DMD and control, with 1281 upregulated and 189 downregulated DEGs. Four hundred and twenty DEGs were found between BMD and control, with 157 upregulated and 263 upregulated DEGs. Fourteen modules with different colors were identified for DMD vs control, and 7 modules with different colors were identified for BMD vs control. Ten hub genes were summarized for DMD and BMD respectively, 5 hub genes were summarized for BMD age, 5 and 3 highly correlated clustered genes were summarized for DMD age and BMD pathology, respectively. In addition, 20 GO enrichments were found to be involved in DMD, 3 GO enrichments were found to be involved in BMD, 3 GO enrichments were found to be involved in BMD age.ConclusionIn DMD, several hub genes were identified: C3AR1, TLR7, IRF8, FYB and CD33(immune and inflammation associated genes), TYROBP, PLEK, AIF1(actin reorganization associated genes), LAPTM5 and NT5E(cell death and arterial calcification associated genes, respectively). In BMD, a number of hub genes were identified: LOX, ELN, PLEK, IKZF1, CTSK, THBS2, ADAMTS2, COL5A1(extracellular matrix associated genes), BCL2L1 and CDK2(cell cycle associated genes).

Differential Autophagy Response in Men and Women After Muscle Damage.

Following muscle damage, autophagy is crucial for muscle regeneration. Hormones (e.g., testosterone, cortisol) regulate this process and sex differences in autophagic flux exist in the basal state. However, to date, no study has examined the effect of a transient hormonal response following eccentric exercise-induced muscle damage (EE) between untrained young men and women. Untrained men (n = 8, 22 ± 3 years) and women (n = 8, 19 ± 1 year) completed two sessions of 80 unilateral maximal eccentric knee extensions followed by either upper body resistance exercise (RE; designed to induce a hormonal response; EE + RE) or a time-matched rest period (20 min; EE + REST). Vastus lateralis biopsy samples were collected before (BL), and 12 h, and 24 h after RE/REST. Gene and protein expression levels of selective markers for autophagic initiation signaling, phagophore initiation, and elongation/sequestration were determined. Basal markers of autophagy were not different between sexes. For EE + RE, although initiation signaling (FOXO3) and autophagy-promoting (BECN1) genes were greater (p < 0.0001; 12.4-fold, p = 0.0010; 10.5-fold, respectively) for women than men, autophagic flux (LC3-II/LC3-I protein ratio) did not change for women and was lower (p < 0.0001 3.0-fold) than men. Furthermore, regardless of hormonal changes, LC3-I and LC3-II protein content decreased (p = 0.0090; 0.547-fold, p = 0.0410; 0.307-fold, respectively) for men suggesting increased LC3-I lipidation and autophagosome degradation whereas LC3-I protein content increased (p = 0.0360; 1.485-fold) for women suggesting decreased LC3-I lipidation. Collectively, our findings demonstrated basal autophagy was not different between men and women, did not change after EE alone, and was promoted with the acute hormonal increase after RE only in men but not in women. Thus, the autophagy response to moderate muscle damage is promoted by RE-induced hormonal changes in men only.

MTORC1 Activity Occurs Predominantly in the Periphery of Human Skeletal Muscle Fibers Following Anabolic Stimuli

The mechanistic target of rapamycin complex 1 (mTORC1) is regarded as the master regulator of protein synthesis in skeletal muscle and is essential for optimal responses to acute anabolic stimuli. We have shown mTORC1 to translocate toward the cell periphery where it colocalizes with upstream activators, downstream effectors and the microvasculature after protein feeding and muscle contraction. These events occur at similar times to elevated whole muscle mTORC1 kinase activity and protein synthesis; however, whether mTORC1 activity occurs in a region-specific pattern is yet to be studied. Therefore, the aim of the current study was to develop an immunofluorescent staining (IF) protocol to measure mTORC1 activation in a region-specific manner following anabolic stimuli in human skeletal muscle. Fourteen young, healthy males (22±4yrs, 14±4% body fat, mean±SD) participated in the study and were randomized to complete trials where a protein-carbohydrate beverage was ingested at rest (FED, n=7) or following a whole-body resistance exercise session (EXFED, n=7). Vastus lateralis biopsy samples were obtained prior to feeding/exercise (basal) and 120 and 300min following anabolic stimuli. Phosphorylated ribosomal protein S6Ser240/244 (p-RPS6Ser240/244) was chosen as the marker of mTORC1 activation as this phosphorylation event is rapamycin-sensitive. We first optimized and validated an IF protocol for p-RPS6Ser240/244 through the omission of primary and secondary antibodies. Using this protocol, we then showed that RPS6Ser240/244 phosphorylation was elevated at 120min (p<0.04) with EXFED being greater than FED (p=0.02). p-RPS6Ser240/244 remained elevated above basal values at 300min in EXFED only (p=0.04). Importantly, a strong positive correlation was observed between RPS6Ser240/244 phosphorylation measured by immunoblotting and IF (r=0.85, p<0.001) showing our novel IF method is a valid readout of mTORC1 activity. In central regions of skeletal muscle fibers, p-RPS6Ser240/244 phosphorylation was only elevated in EXFED at 120min. In the outer 5.5µm of fibers (periphery), mTORC1 activity increased at 120min in both conditions (p<0.03) but to a greater extent in EXFED (p=0.002), remaining elevated above basal levels at 300min only in EXFED (p=0.02). Similarly, the peripheral-to-central ratio of RPS6Ser240/244 phosphorylation was elevated above basal values in both conditions at 120min and remained elevated only in EXFED at 300min, suggesting mTORC1 activity occurs to a greater extent in the periphery of skeletal muscle fibers. In conclusion, we have optimized and validated an IF technique to investigate mTORC1 activity in a region-specific manner in human skeletal muscle. We show that, following physiological anabolic stimuli, mTORC1 activity occurs to a greater extent in the periphery of fibers, regions where mTORC1 is commonly visualized in humans and protein synthesis is observed in animal models. These findings extend our knowledge regarding the spatial regulation of mTORC1 in human skeletal muscle and provide a novel method to investigate mTORC1 dynamics in various populations.

Transcriptional Regulation Of Substrate Metabolism Following Carbohydrate Ingestion During Exercise Initiated With Different Glycogen Concentrations

BACKGROUND: Starting aerobic exercise with low muscle glycogen content elicits greater fat and less carbohydrate utilization than exercise started with high muscle glycogen content, even when exogenous carbohydrate is ingested during exercise. The mechanisms contributing to greater fat utilization despite ingesting carbohydrate during exercise initiated with low glycogen are not delineated. PURPOSE: Characterize transcriptional regulation of substrate metabolism in response to carbohydrate ingestion during steady-state exercise initiated with low muscle glycogen. METHODS: In a randomized, crossover design, 12 men (mean ± SD, age, 21 ± 4 y; body mass, 83 ± 11 kg; VO2peak, 44 ± 3 mL/kg/min) completed two cycle ergometry glycogen depletion trials, followed by a 24 h period of either high fat (1.5 g/kg carbohydrate, 3.0 g/kg fat) or high carbohydrate (6.0 g/kg carbohydrate, 1.0 g/kg fat) isocaloric refeeding to elicit low (LOW) or adequate (AD) glycogen content the following morning before initiating 80-min of cycle ergometry (64 ± 3% VO2peak) while ingesting 146 g of carbohydrate. Transcriptional regulation of substrate metabolism was assessed using RT-qPCR in vastus lateralis biopsy samples obtained before (PRE) and after (POST) the 80-min exercise bout. RESULTS: PRE glycogen synthase kinase 3α expression was 40% lower (P<0.05; time-by-treatment interaction) in LOW than AD. GLUT4, hexokinase 2, phosphofructokinase, and pyruvate kinase expression were not different between LOW and AD. PRE fatty acid translocase was 40% higher (P<0.05; time-by-treatment interaction) in LOW than AD. Independent of time, fatty acid binding protein, carnitine palmitoyltransferase 1A, and hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA expression were each ~40% higher (P<0.05; time effect) in LOW than AD. In LOW, POST peroxisome proliferator-activated receptor δ was 177% higher (P<0.05 time-by-treatment interaction) than PRE, with no change in AD. CONCLUSION: Initiating aerobic exercise with low muscle glycogen content upregulates the transcriptional control of fat oxidation without modulating intramuscular regulation of glucose metabolism, even when exogenous glucose is ingested during exercise.

Exercise and β-alanine supplementation on carnosine-acrolein adduct in skeletal muscle

Previous studies have demonstrated that exercise results in reactive aldehyde production and that β-alanine supplementation increases carnosine content in skeletal muscle. However, little is known about the influence exercise and β-alanine supplementation have on the formation of carnosine-aldehydes. The goal of the present study was to monitor the formation of carnosine-aldehyde adducts, following high-intensity intermittent exercise, before and after β-alanine supplementation. Vastus lateralis biopsy samples were taken from 14 cyclists, before and after a 28 day β-alanine supplementation, following 4 bouts of a 30 s all-out cycling test, and carnosine and CAR-aldehyde adducts [carnosine-acrolein, CAR-ACR (m/z 303), carnosine-4-hydroxy-2-hexenal, CAR-HHE (m/z 341) and carnosine-4-hydroxy-2-nonenal, CAR-HNE (m/z 383)] were quantified by HPLC-MS/MS. β-alanine supplementation increased muscle carnosine content by ~50% (p = 0.0001 vs. Pre-Supplementation). Interestingly, there was a significant increase in post-exercise CAR-ACR content following β-alanine supplementation (p < 0.001 vs. post-exercise before supplementation), whereas neither exercise alone nor supplementation alone increased CAR-ACR formation. These results suggest that carnosine functions as an acrolein-scavenger in skeletal muscle. Such a role would be relevant to the detoxification of this aldehyde formed during exercise, and appears to be enhanced by β-alanine supplementation. These novel findings not only have the potential of directly benefiting athletes who engage in intensive training regimens, but will also allow researchers to explore the role of muscle carnosine in detoxifying reactive aldehydes in diseases characterized by abnormal oxidative stress.

Loss of maximal explosive power of lower limbs after 2weeks of disuse and incomplete recovery after retraining in older adults.

Disuse in older adults can critically decrease lower limb muscle power, leading to compromised mobility and overall quality of life. We studied how muscle power and its determinants (muscle mass, single muscle fibre properties and motor control) adapted to 2weeks of disuse and subsequent 2weeks of physical training in young and older people. Disuse decreased lower limb muscle power in both groups; however, different adaptations in single muscle fibre properties and co-contraction of leg muscles were observed between young and older individuals. Six physical training sessions performed after disuse promoted the recovery of muscle mass and power. However, they were not sufficient to restore muscle power to pre-disuse values in older individuals, suggesting that further countermeasures are required to counteract the disuse-induced loss of muscle power in older adults. Disuse-induced loss of muscle power can be detrimental in older individuals, seriously impairing functional capacity. In this study, we examined the changes in maximal explosive power (MEP) of lower limbs induced by a 14-day disuse (bed-rest, BR) and a subsequent 14-day retraining, to assess whether the impact of disuse was greater in older than in young men, and to analyse the causes of such adaptations. Sixteen older adults (Old: 55-65years) and seven Young (18-30years) individuals participated in this study. In a subgroup of eight Old subjects, countermeasures based on cognitive training and protein supplementation were applied. MEP was measured with an explosive ergometer, muscle mass was determined by magnetic resonance, motor control was studied by EMG, and single muscle fibres were analysed in vastus lateralis biopsy samples. MEP was ∼33% lower in Old than in Young individuals, and remained significantly lower (-19%) when normalized by muscle volume. BR significantly affected MEP in Old (-15%) but not in Young. Retraining tended to increase MEP; however, this intervention was not sufficient to restore pre-BR values in Old. Ankle co-contraction increased after BR in Old only, and remained elevated after retraining (+30%). Significant atrophy occurred in slow fibres in Old, and in fast fibres in Young. After retraining, the recovery of muscle fibre thickness was partial. The proposed countermeasures were not sufficient to affect muscle mass and power. The greater impact of disuse and smaller retraining-induced recovery observed in Old highlight the importance of designing suitable rehabilitation protocols for older individuals.

Use of high-throughput liquid chromatography mass spectrometry to measure association between vitamin D metabolites and body composition and muscle mass: a cross-sectional study

BackgroundSarcopenia contributes to functional decline and metabolic dysfunction in elderly people, and there is no accepted pharmacological treatment. Vitamin D deficiency is common, and can affect tissues such as muscle. Existing assays focus on one metabolite, 25-hydroxyvitamin D3 (25(OH)D3). We aimed to perform high-throughput analysis of vitamin D metabolites with novel liquid chromatography tandem mass spectrometry (LC-MS/MS) techniques to evaluate the association between vitamin D metabolites and body composition and muscle function. MethodsThis cross-sectional observational study was carried out at the National Institute for Health Research-Wellcome Trust Clinical Research Facility. Participants were healthy non-obese volunteers. We performed body composition analysis (DEXA), assessed grip strength, measured serum metabolites of vitamin D (LC-MS/MS) and glucocorticoids in 24 h urine collection (gas chromatography mass spectography), and determined gene expression in vastus lateralis biopsy samples. Non-parametric testing was completed. Research ethics committee approval was obtained. Findings116 volunteers took part in the study (79 women, 37 men; age range 20–74 years). Body fat correlated negatively with 25(OH)D3 and 3-epi-25(OH)D3 (Pearson r=−0·20 [p=0·04] and −0·21 [p=0·03], respectively). Total lean mass correlated positively with 3-epi-25(OH)D3 (r=0·24, p=0·02) but not with 25(OH)D3. Grip strength correlated positively with 3-epi-25(OH)D3 (r=0·22, p=0·04) but not with 25(OH)D3. In women, the tetrahydrocortisol plus 5α-tetrahydrocortisol to tetrahydrocortisone ratio (an 11β-HSD1 activity marker) in urine correlated negatively with serum 25(OH)D3 (r=−0·31, p=0·02) and 24,25(OH)2D3 (r=−0·26, p=0·04). Conversely urinary cortisol to cortisone ratio (an 11β-HSD2 activity marker) correlated positively with 25(OH)D3 (r=0·28, p=0·04), 24,25(OH)2D3 (r=0·29, p=0·03), and 25(OH)D2 (r=0·35, p=0·04). Serum 25(OH)D3 and 3-epi-25(OH)D3 correlated positively with genes with anabolic functions in muscle including myogenin, myosin chain components, and insulin and IGF1 receptors. InterpretationUsing a novel high-throughput approach, we found that vitamin D metabolites, including epimers, correlated with reduced fat mass and increased lean mass and grip strength. Data were also consistent with effects on 11β-HSD function, which we have previously shown to be important in determining muscle mass and strength. Large scale supplementation trials are required to examine the therapeutic potential in sarcopenia and obesity. FundingThe clinical study was funded by a European Research Council Advanced Grant awarded to PS. Vitamin D analyses and method development were funded by a National Institues of Health P50 grant awarded to MH and JA (AR063020-01 NIH/NIAMS).

A single bout of whole-leg, peristaltic pulse external pneumatic compression upregulates PGC-1α mRNA and endothelial nitric oxide sythase protein in human skeletal muscle tissue.

What is the central question of this study? Does 60 min of peristaltic pulse external pneumatic compression (EPC) alter gene and protein expression patterns related to metabolism, vascular biology, redox balance and inflammation in vastus lateralis biopsy samples? What is the main finding and its importance? A single bout of EPC transiently upregulates PGC-1α mRNA, while also upregulating endothelial nitric oxide synthase protein and nitric oxide metabolite concentrations in vastus lateralis biopsy samples. We investigated whether a single 60 min bout of whole-leg, lower pressure external pneumatic compression (EPC) altered select vascular, metabolic, antioxidant and inflammation-related mRNAs. Ten participants (eight male, two female; aged 22.0 ± 0.4 years) reported to the laboratory 4 h postprandial, and vastus lateralis muscle biopsies were obtained before (PRE) and 1 and 4 h after EPC treatment. Messenger RNA expression was analysed using real-time RT-PCR, and significant mRNA findings were investigated further by Western blot analysis of respective protein concentrations. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) mRNA increased by 77% 1 h following EPC compared with PRE levels (P = 0.005), but no change in protein concentration 1 or 4 h post-EPC was observed. Increases in endothelial nitric oxide sythase (eNOS) mRNA (+44%) and superoxide dismutase 2 (SOD2) mRNA (+57%) 1 h post-EPC as well as an increase in interleukin-10 mRNA (+132%) 4 h post-EPC compared with PRE levels were observed, but only approached significance (P = 0.076, 0.077 and 0.074, respectively). Interestingly, eNOS protein (+40%, P = 0.025) and nitrate and nitrite (NOx) concentrations (+69%, P = 0.025) increased 1-4 h post-EPC. Moreover, SOD2 protein tended to increase from PRE to 4 h post-EPC (+43%, P = 0.074), although no changes in tissue 4-hydroxnonenal levels was observed. An acute bout of EPC transiently upregulates PGC-1α mRNA, while also upregulating eNOS protein and NOx concentrations in vastus lateralis biopsy samples. Future research should characterize the origin of these responses (e.g. vascular or muscle fibre cells) and how the acute effects of EPC application on gene and protein expression observed herein are associated with functional improvements (e.g. metabolism, vascular function) in acute and chronic models.

Novel events in the molecular regulation of muscle mass in critically ill patients

Critically ill patients experience marked skeletal muscle atrophy, but the molecular mechanisms responsible for this are largely unresolved. Therefore, we investigated key genes and proteins, identified from cell and animal studies to control protein synthesis and breakdown, in vastus lateralis biopsy samples obtained from 10 patients and 10 age- and sex-matched healthy controls. Muscle cytokines IL-6 and TNF-α mRNA were higher in patients than in controls(6.5-fold; P < 0.001 and 2-fold; P < 0.01). From the perspective of muscle protein breakdown, muscle-specific E3-ligases (MAFbx and MuRF1) were higher in patients at mRNA (4.5-fold; P < 0.05 and 2.5-fold; P < 0.05) and protein (5-fold; P < 0.001 and 4.5-fold; P < 0.001) level. Furthermore, 20S proteasome mRNA and protein were higher in patients (5-fold; P < 0.001 and 2.5-fold; P < 0.01). Cathepsin-L mRNA was 2-fold higher (P < 0.01), whilst calpain-3 mRNA(2-fold; P < 0.01) and protein (4-fold; P < 0.01)were lower inpatients. Another novel observation was the 3-fold (P < 0.05) and 8.5-fold (P < 0.001) higher expression of myostatin mRNA and protein in patients. Widespread dephosphorylation (inactivation) of proteins regulating translation initiation factor activation and protein synthesis (Akt1, GSK3α,β, mTOR, p70S6K and 4E-BP1) was observed in patients, which was paralleled by increases in their mRNAs. Finally, PDK4 mRNA and protein was 2-fold (P < 0.05) and 2.6-fold (P < 0.01), respectively, higher inpatients. In conclusion, we showed comprehensive alterations in molecular events thought to reduce muscle mass and carbohydrate (CHO) oxidation in critically ill patients. Nevertheless,these catabolic events were matched by a cellular programme of anabolic restoration at the transcriptional level. This shows a high molecular plasticity in the muscle of patients, and strategies to preserve muscle mass and metabolic function should focus on maintaining Akt phosphorylation and inhibiting myostatin expression.C

Exercise training normalizes skeletal muscle vascular endothelial growth factor levels in patients with essential hypertension

Vascular endothelial growth factor (VEGF) protein and capillarization were determined in muscle vastus lateralis biopsy samples in individuals with essential hypertension (n = 10) and normotensive controls (n = 10). The hypertensive individuals performed exercise training for 16 weeks. Muscle samples as well as muscle microdialysis fluid samples were obtained at rest, during and after an acute exercise bout, performed prior to and after the training period, for the determination of muscle VEGF levels, VEGF release, endothelial cell proliferative effect and capillarization. Prior to training, the hypertensive individuals had 36% lower levels of VEGF protein and 22% lower capillary density in the muscle compared to controls. Training in the hypertensive group reduced (P < 0.01) mean arterial blood pressure by 7.1 +/- 0.8 mmHg, enhanced (P < 0.01) the capillary-to-fiber ratio by 17% and elevated (P < 0.05) muscle VEGF protein by 67%. Before training, acute exercise did not induce an increase in muscle interstitial VEGF levels above resting levels, but a five-fold increase (P < 0.05) was observed after the training period. Acute exercise induced an elevated (P < 0.05) endothelial cell proliferative effect of muscle dialysate after, but not before, training. In summary, exercise training markedly elevates VEGF protein levels in muscle tissue, increases exercise-induced VEGF release from muscle and the cell proliferative effect of muscle dialysate. These alterations are paralleled by a lowering of blood pressure and an increased capillary-per-fiber ratio, but unaltered capillary density.

Age, Obesity, and Sex Effects on Insulin Sensitivity and Skeletal Muscle Mitochondrial Function

OBJECTIVEReductions in insulin sensitivity in conjunction with muscle mitochondrial dysfunction have been reported to occur in many conditions including aging. The objective was to determine whether insulin resistance and mitochondrial dysfunction are directly related to chronological age or are related to age-related changes in body composition.RESEARCH DESIGN AND METHODSTwelve young lean, 12 young obese, 12 elderly lean, and 12 elderly obese sedentary adults were studied. Insulin sensitivity was measured by a hyperinsulinemic-euglycemic clamp, and skeletal muscle mitochondrial ATP production rates (MAPRs) were measured in freshly isolated mitochondria obtained from vastus lateralis biopsy samples using the luciferase reaction.RESULTSObese participants, independent of age, had reduced insulin sensitivity based on lower rates of glucose infusion during a hyperinsulinemic-euglycemic clamp. In contrast, age had no independent effect on insulin sensitivity. However, the elderly participants had lower muscle MAPRs than the young participants, independent of obesity. Elderly participants also had higher levels inflammatory cytokines and total adiponectin. In addition, higher muscle MAPRs were also noted in men than in women, whereas glucose infusion rates were higher in women.CONCLUSIONSThe results demonstrate that age-related reductions in insulin sensitivity are likely due to an age-related increase in adiposity rather than a consequence of advanced chronological age. The results also indicate that an age-related decrease in muscle mitochondrial function is neither related to adiposity nor insulin sensitivity. Of interest, a higher mitochondrial ATP production capacity was noted in the men, whereas the women were more insulin sensitive, demonstrating further dissociation between insulin sensitivity and muscle mitochondrial function.

Inadequate protein intake affects skeletal muscle transcript profiles in older humans

Background: Inadequate dietary protein intake causes adverse changes in the morphology and function of skeletal muscle. These changes may be reflected in early alterations in muscle messenger RNA levels.Objective: This study assessed whether inadequate protein intake differentially affects skeletal muscle transcript concentrations and expression profiles in older adults.Design: Twenty-one older men and women (aged 55–80 y) consumed controlled diets that provided 1.2 g protein · kg−1 · d−1 (adequate protein) for 1 wk and then were randomly assigned to consume either 0.5 g protein · kg−1 · d−1 [inadequate protein (IP) group; n = 11] or 1.2 g protein · kg−1 · d−1 (control group; n = 10) for a second week. RNA was isolated from fasting-state vastus lateralis biopsy samples obtained at the end of each period, and transcript levels in the IP group were measured by using microarray anlysis. Changes in selected transcript levels were confirmed by real-time polymerase chain reaction in both groups.Results: Analysis of variance showed 529 differentially expressed transcripts (P < 0.05) after inadequate protein intake. Using the false discovery rate (FDR) correction to adjust for multiple comparisons, we observed that 85 transcripts were differentially expressed: 54 were up-regulated and 31 were down-regulated. The differentially expressed transcripts were in functional classes for immune, inflammatory, and stress responses (predominantly up-regulated); contraction, movement, and development (up-regulated); extracellular connective tissue (up-regulated); energy metabolism (down-regulated); protein synthesis (down-regulated); and proliferation (down-regulated). Diet-related differences in the expression of 9 transcripts were cross-validated by using real-time polymerase chain reaction.Conclusion: The results document changes in skeletal muscle transcript levels induced by short-term inadequate protein intakes in older humans that might precede adverse metabolic, functional, and structural events, including muscle wasting.

The Catabolic Effects of Prolonged Inactivity and Acute Hypercortisolemia Are Offset by Dietary Supplementation

We compared the anabolic stimulus provided by an essential amino acid and carbohydrate (AA/CHO) supplement to a mixed clinical meal during bed rest (BR) and episodic hypercortisolemia ( approximately 24 microg.dl(-1)). In the experimental (EXP; n = 7) and control (CON; n = 6) groups, femoral arteriovenous blood samples and vastus lateralis biopsy samples were obtained during a primed constant infusion of l-[ring-(2)H(5)]phenylalanine and a 14-h infusion of hydrocortisone sodium succinate (60 microg.kg.h(-1)) before (pre-BR) and after (post-BR) 28 d of BR. Muscle protein kinetics were calculated during the postabsorptive state, for 2.5 h after ingestion of a meal and for 2.5 h after ingestion of an AA/CHO supplement (EXP) or placebo (CON). Postabsorptive net phenylalanine balance values were as follows: EXP, -35.14 +/- 2.93, and CON, -32.60 +/- 6.65 (pre-BR); and EXP, -32.91 +/- 5.67, and CON, -30.43 +/- 6.28 nmol phe.ml(-1).100 ml leg volume(-1) (post-BR). After AA/CHO supplementation, net phenylalanine balance improved to 33.51 +/- 8.06 (pre-BR) and 24.15 +/- 11.4 nmol phe.ml(-1).100 ml leg volume(-1) (post-BR), but remained negative after the meal. Cumulative 5.5-h mixed muscle fractional synthetic rate was greater in the EXP group pre-BR (EXP, 0.108 +/- 0.01, and CON, 0.073 +/- 0.04%.h(-1)) and post-BR (EXP, 0.111 +/- 0.015, and CON, 0.05 +/- 0.002%.h(-1)). Unlike a typical clinical meal, AA/CHO supplementation stimulated net muscle protein synthesis despite acute hypercortisolemia and prolonged inactivity.

Skeletal muscle myosin heavy-chain synthesis rate in healthy humans.

Mixed muscle protein synthetic rate has been measured in humans. These measurements represent the average of synthetic rates of all muscle proteins with variable rates. We determined to what extent the synthesis rate of mixed muscle protein in humans reflects that of myosin heavy chain (MHC), the main contractile protein responsible for the conversion of ATP to mechanical energy as muscle contraction. Fractional synthetic rates of MHC and mixed muscle protein were measured from the increment of [13C]leucine in these proteins in vastus lateralis biopsy samples taken at 5 and 10 h during a primed continuous infusion of L-[1-13C]leucine in 10 young healthy subjects. Calculations were done by use of plasma [13C]ketoisocaproate (KIC) and muscle tissue fluid [13C]leucine as surrogate measures of leucyl-tRNA. Fractional synthetic rate of MHC with plasma KIC (0.0299 +/- 0.0043%/h) and tissue fluid leucine (0.0443 +/- 0.0056%/h) were only 72 +/- 3% of that of mixed muscle protein (0.0408 +/- 0.0032 and 0.0603 +/- 0.0059%/h, respectively, with KIC and tissue fluid leucine). Contribution of MHC (7 +/- 1 mg.kg-1.h-1) to synthetic rates of whole body mixed muscle protein (36 +/- 5 mg.kg-1.h-1) and whole body protein (127 +/- 4 mg.kg-1.h-1) is only 18 +/- 1 and 5 +/- 1%, respectively. This relatively low contribution of MHC to whole body and mixed muscle protein synthesis warrants direct measurement of synthesis rate of MHC in conditions involving abnormalities of muscle contractile function.

Neuromuscular Differences Between Volleyball Players, Middle Distance Runners and Untrained Controls

Volleyball players, middle distance runners and non-athletes (n = 10/group) were tested to determine whether neuromuscular differences existed between groups and to clarify the roles of factors involved in maximal power production. The runners were leaner than controls, while the volleyball players were taller, heavier and had larger thigh volumes than the other groups. The volleyball players had higher absolute cycle ergometer power than both middle distance (26%) and control (15%) groups, but differences disappeared when expressed relative to body mass or thigh volume. Volleyball athletes were also stronger than both middle distance (51, 52%) and control subjects (33, 35%) for isokinetic leg extension and plantar flexion respectively (0-4.19 rad.s-1). In leg press they were stronger than middle distance (32%) and control subjects (36%) for only the isometric and 1.05 rad.s-1 contraction. The volleyball players also had higher rates of isometric torque development than the other groups, however nerve conduction velocity did not vary. Vastus lateralis biopsy samples revealed no differences in percent Type II muscle fibers, or fiber cross-sectional area between groups, yet volleyball athletes had larger Type II/I fiber area ratio than controls (15%). Both strength, rate of torque development and power were related to muscle and muscle fiber size variables, but not fiber distribution or nerve conduction velocity. The size of type II muscle fibers seemed to be especially important since this was the only variable related to power when adjusted for body size.