Mini invasive skeletal muscle biopsy technique with a tri-axial end cut needle.

OBJECTIVE: Skeletal Muscle Biopsy is a minor surgical procedure for the diagnosis of different neuromuscular pathological conditions and has recently gained popularity also in the research field of age-related muscular modifications and sarcopenia. Few studies focused on the application of mini-invasive muscular biopsy in both normal and pathological conditions. The aim of our study was to describe a mini invasive ultrasound-guided skeletal muscular biopsy technique in complete spinal cord injured (SCI) patients and healthy controls with a tri-axial end-cut needle. PATIENTS AND METHODS: Skeletal muscle biopsies were collected from 6 chronic SCI patients and 3 healthy controls vastus lateralis muscle with a tri-axial end cut needle (Biopince© - Angiotech). Muscle samples were stained for ATPase to determine fibers composition, moreover, gene expression of cyclooxygenase-1 (COX-1) and prostaglandin E2 receptor has been analyzed by Real Time RT-PCR. RESULTS: All the procedures were perfomed easily without failures and complications. Control tissue was macroscopically thicker than SCI one. Control specimen displayed an equal distribution of type I and type II fibers, while SCI sample displayed a prevalence of type II fibers SCI specimen displayed a significant reduction in COX-1 gene expression. This mini-invasive approach was easy, accurate and with low complication rate in performing skeletal muscle biopsy in both SCI patients and controls. CONCLUSIONS: This technique could be useful in conditions in which the overall quantity of specimen required is small like for molecular biology analysis. For histological diagnostic purposes and/or conditions in which the original tissue is already pathologically modified, this technique should be integrated with more invasive techniques.

Serum activity of creatine kinase and aminotransferase aspartate enzymes of horses submitted to muscle biopsy and incremental jump test

The objective was to evaluate serum activity of the enzymes creatine kinase (CK) and aspartate aminotransferase (AST), which are leakage enzymes responsive to muscle injury, of athletic horses that underwent muscle biopsy and incremental jump test (IJT) involving incremental jumps. The animals were grouped as follows: the first group, horses with history of superior performance (SP); the second, with a history of inferior performance (IP); and lastly, a control group (CG). All groups underwent biopsy of the gluteus medius muscle, while groups SP and IP were also submitted to the incremental jump test (IJT) 24 hours after biopsy. The IJT consisted of three stages with 40 jumps each, where jump height increased progressively, from 40 to 60 and last, 80cm. Blood samples were drawn before biopsy, and 6 and 24 hours after the exercise as well. The levels of CK serum activity increased 6 hours after exercise and decreased 24 hours later in all groups, including CG. AST activity did not increase after biopsy and exercise. There was no increase of both enzyme activities that could be attributed to the exercise, possibly due to exercise short duration and/or low intensity. We conclude that the muscle biopsy was able to show that there was enough stimulus to cause CK enzyme leakage into the plasma, and consequent detection of increased serum activity, while the incremental jump test did not.

Determination of creatine and phosphocreatine in muscle biopsy samples by capillary electrophoresis with contactless conductivity detection

A capillary electrophoresis method with contactless conductivity detection was evaluated as a new approach for quantification of creatine and phosphocreatine in human quadriceps femoris biopsy samples. The running buffers employed consisted of 1 M acetic acid at a pH of 2.3 for the determination of creatine and 50 mM 3-(N-morpholino)propanesulfonic acid/30 mM histidine at a pH of 6.4 for the determination of phosphocreatine in the centrifuged muscle extracts. The limits of detection for creatine and phosphocreatine were found to be 2.5 and 1.0 μM, respectively. Creatine and phosphocreatine were determined in six human muscle biopsy samples and the results were found comparable to those of a standard enzymatic assay. The procedures developed for creatine and phosphocreatine also allow the determination of creatinine as well as adenosine diphosphate and adenosine triphosphate.

Acute resistance exercise increases the expression of chemotactic factors within skeletal muscle

Intense resistance exercise causes mechanical loading of skeletal muscle, followed by muscle adaptation. Chemotactic factors likely play an important role in these processes. Purpose We investigated the time course of changes in the expression and tissue localization of several key chemotactic factors in skeletal muscle during the early phase of recovery following resistance exercise. Methods Muscle biopsy samples were obtained from vastus lateralis of eight untrained men (22+-0.5 yrs) before and 2, 4 and 24 h after three sets of leg press, squat and leg extension at 80% 1 RM. Results Monocyte chemotactic protein-1 (95×), interleukin-8 (2,300×), IL-6 (317×), urokinase-type plasminogen activator (15×), vascular endothelial growth factor (2×) and fractalkine (2.5×) mRNA was significantly elevated 2 h post-exercise. Interleukin-8 (38×) and interleukin-6 (58×) protein was also significantly elevated 2 h post-exercise, while monocyte chemotactic protein-1 protein was significantly elevated at 2 h (22×) and 4 h (21×) post-exercise. Monocyte chemotactic protein-1 and interleukin-8 were expressed by cells residing in the interstitial space between muscle fibers and, in some cases, were co-localized with CD68+ macrophages, PAX7+ satellite cells and blood vessels. However, the patterns of staining were inconclusive and not consistent. Conclusion In conclusion, resistance exercise stimulated a marked increase in the mRNA and protein expression of various chemotactic factors in skeletal muscle. Myofibers were not the dominant source of these factors. These findings suggest that chemotactic factors regulate remodeling/adaptation of skeletal muscle during the early phase of recovery following resistance exercise.

Ibuprofen supplementation and its effects on NF-KB activation in skeletal muscle following resistance exercise

Resistance exercise triggers a subclinical inflammatory response that plays a pivotal role in skeletal muscle regeneration. Nuclear factor‐κB (NF‐κB) is a stress signalling transcription factor that regulates acute and chronic states of inflammation. The classical NF‐κB pathway regulates the early activation of post‐exercise inflammation; however there remains scope for this complex transcription factor to play a more detailed role in post‐exercise muscle recovery. Sixteen volunteers completed a bout of lower body resistance exercise with the ingestion of three 400 mg doses of ibuprofen or a placebo control. Muscle biopsy samples were obtained prior to exercise and at 0, 3 and 24 h post‐exercise and analysed for key markers of NF‐κB activity. Phosphorylated p65 protein expression and p65 inflammatory target genes were elevated immediately post‐exercise independent of the two treatments. These changes did not translate to an increase in p65 DNA binding activity. NF‐κB p50 protein expression and NF‐κB p50 binding activity were lower than pre‐exercise at 0 and 3 h post‐exercise, but were elevated at 24 h post‐exercise. These findings provide novel evidence that two distinct NF‐κB pathways are active in skeletal muscle after resistance exercise. The initial wave of activity involving p65 resembles the classical pathway and is associated with the onset of an acute inflammatory response. The second wave of NF‐κB activity comprises the p50 subunit, which has been previously shown to resolve an acute inflammatory program. The current study showed no effect of the ibuprofen treatment on markers of the NF‐κB pathway, however examination of the within group effects of the exercise protocol suggests that this pathway warrants further research.

Expression of toll-like receptors by human muscle cells in vitro and in vivo:TLR3 is highly expressed in inflammatory and HIV myopathies, mediates IL-8 release and up-regulation of NKG2D-ligands

The particular microenvironment of the skeletal muscle can be the site of complex immune reactions. Toll-like receptors (TLRs) mediate inflammatory stimuli from pathogens and endogenous danger signals and link the innate and adaptive immune system. We investigated innate immune responses in human muscle. Analyzing TLR1-9 mRNA in cultured myoblasts and rhabdomyosarcoma cells, we found constitutive expression of TLR3. The TLR3 ligand Poly (I:C), a synthetic analog of dsRNA, and IFN-gamma increased TLR3 levels. TLR3 was mainly localized intracellularly and regulated at the protein level. Poly (I:C) challenge 1) activated nuclear factor-kappaB (NF-kappaB), 2) increased IL-8 release, and 3) up-regulated NKG2D ligands and NK-cell-mediated lysis of muscle cells. We examined muscle biopsy specimens of 6 HIV patients with inclusion body myositis/polymyositis (IBM/PM), 7 cases of sporadic IBM and 9 nonmyopathic controls for TLR3 expression. TLR3 mRNA levels were elevated in biopsy specimens from patients with IBM and HIV-myopathies. Muscle fibers in inflammatory myopathies expressed TLR3 in close proximity of infiltrating mononuclear cells. Taken together, our study suggests an important role of TLR3 in the immunobiology of muscle, and has substantial implications for the understanding of the pathogenesis of inflammatory myopathies or therapeutic interventions like vaccinations or gene transfer.

Dominant and Recessive RYR1 Mutations in Adults with Core Lesions and Mild Muscle Symptoms

INTRODUCTION: Ryanodine receptor gene (RYR1) mutations have been associated with central core disease (CCD), multiminicore/minicore/multicore disease (MmD), and susceptibility to malignant hyperthermia (MH). METHODS: Patients with muscle symptoms in adulthood, who had features compatible with CCD/MmD, underwent clinical, histological, and genetic (RYR1 and SEPN1 genes) evaluations. Published cases of CCD and MmD with adult onset were also reviewed. RESULTS: Eight patients fulfilled the criteria for further analysis. Five RYR1 mutations, 4 of them unreported, were detected in 3 patients. Compound heterozygosity was proven in 1 case. CONCLUSIONS: To our knowledge, this is the only report of adult onset associated with recessive RYR1 mutations and central core/multiminicores on muscle biopsy. Although adult patients with CCD, MmD, and minimally symptomatic MH with abnormal muscle biopsy findings usually have a mild clinical course, differential diagnosis and carrier screening is crucial for prevention of potentially life-threatening reactions to general anesthesia.

Effects of endurance training status and sex difference on Na+, K+-pump mRNA expression, content and maximal activity in human skeletal muscle

Aim: This study investigated the effects of endurance training status and sex differences on skeletal muscle Na⁺,K⁺-pump mRNA expression, content and activity. Methods: Forty-five endurance-trained males (ETM), 11 recreationally active males (RAM), and nine recreationally active females (RAF) underwent a vastus lateralis muscle biopsy. Muscle was analysed for Na⁺,K⁺-pump α1, α2, α3, β1, β2 and β3 isoform mRNA expression (real-time reverse transcription-polymerase chain reaction), content ([³H]-ouabain-binding site) and maximal activity (3-O-methylfluorescein phosphatase, 3-O-MFPase). Results: ETM demonstrated lower α1, α3, β2 and β3 mRNA expression by 74%, 62%, 70% and 82%, respectively, than RAM (P < 0.04). In contrast, [³H]-ouabain binding and 3-O-MFPase activity were each higher in ETM than in RAM, by 16% (P < 0.03). RAM demonstrated a 230% and 364% higher α3 and b3 mRNA expression than RAF, respectively (P < 0.05), but no significant sex differences were found for α1, α2, β1 or β2 mRNA, [³H]-ouabain binding  or 3-O-MFPase activity. No significant correlation was found between years of endurance training and either [³H]-ouabain binding or 3-O-MFPase activity. Significant but weak correlations were found between the number of training hours per week and 3-O-MFPase activity (r = 0.31, P < 0.02) and between incremental exercise V O_2(peak) and both   [³H]-ouabain binding (r = 0.33, P < 0.01) and 3-O-MFPase activity (r = 0.28, P < 0.03). Conclusions: Isoform-specific differences in Na⁺,K⁺-pump mRNA expression were found with both training status and sex differences, but only training status influenced Na⁺,K⁺-pump content and maximal activity in human skeletal muscle.

STAT3 signaling is activated in human skeletal muscle following acute resistance exercise

The transcription factor signal transducer and activator of transcription 3 (STAT3) has been identified as a mediator of cytokine signaling and implicated in hypertrophy; however, the importance of this pathway following resistance exercise in human skeletal muscle has not been investigated. In the present study, the phosphorylation and nuclear localization of STAT3, together with STAT3-regulated genes, were measured in the early recovery period following intense resistance exercise. Muscle biopsy samples from healthy subjects (7 males, 23.0 + 0.9 yr) were harvested before and again at 2, 4, and 24 h into recovery following a single bout of maximal leg extension exercise (3 sets, 12 repetitions). Rapid and transient activation of phosphorylated (tyrosine 705) STAT3 was observed at 2 h postexercise. STAT3 phosphorylation paralleled the transient localization of STAT3 to the nucleus, which also peaked at 2 h postexercise. Downstream transcriptional events regulated by STAT3 activation peaked at 2 h postexercise, including early responsive genes c-FOS (800-fold), JUNB (38-fold), and c-MYC (140-fold) at 2 h postexercise. A delayed peak in VEGF (4-fold) was measured 4 h postexercise. Finally, genes associated with modulating STAT3 signaling were also increased following exercise, including the negative regulator SOCS3 (60-fold). Thus, following a single bout of intense resistance exercise, a rapid phosphorylation and nuclear translocation of STAT3 are evident in human skeletal muscle. These data suggest that STAT3 signaling is an important common element and may contribute to the remodeling and adaptation of skeletal muscle following resistance exercise.

Live high: train low increases muscle buffer capacity and submaximal cycling efficiency

This study investigated whether hypoxic exposure increased muscle buffer capacity (βm) and mechanical efficiency during exercise in male athletes. A control (CON, n=7) and a live high:train low group (LHTL, n=6) trained at near sea level (600 m), with the LHTL group sleeping for 23 nights in simulated moderate altitude (3000 m). Whole body oxygen consumption (V˙O₂) was measured under normoxia before, during and after 23 nights of sleeping in hypoxia, during cycle ergometry comprising 4×4-min submaximal stages, 2-min at 5.6 ± 0.4 W kg^–1, and 2-min 'all-out' to determine total work and V˙O_2peak. A vastus lateralis muscle biopsy was taken at rest and after a standardized 2-min 5.6 ± 0.4 W kg^–1 bout, before and after LHTL, and analysed for βm and metabolites. After LHTL, βm was increased (18%, P < 0.05). Although work was maintained, V˙O_2peak fell after LHTL (7%, P < 0.05). Submaximal V˙O₂ was reduced (4.4%, P < 0.05) and efficiency improved (0.8%, P < 0.05) after LHTL probably because of a shift in fuel utilization. This is the first study to show that hypoxic exposure, per se, increases muscle buffer capacity. Further, reduced V˙O₂ during normoxic exercise after LHTL suggests that improved exercise efficiency is a fundamental adaptation to LHTL.

Intense exercise up-regulates Na+, K+ -ATPase isoform mRNA, but not protein expression in human skeletal muscle

Characterization of expression of, and consequently also the acute exercise effects on, Na+,K+-ATPase isoforms in human skeletal muscle remains incomplete and was therefore investigated. Fifteen healthy subjects (eight males, seven females) performed fatiguing, knee extensor exercise at 40% of their maximal work output per contraction. A vastus lateralis muscle biopsy was taken at rest, fatigue and 3 and 24 h postexercise, and analysed for Na+,K+-ATPase 1, 2, 3, ß1, ß2 and ß3 mRNA and crude homogenate protein expression, using Real-Time RT-PCR and immunoblotting, respectively. Each individual expressed gene transcripts and protein bands for each Na+,K+-ATPase isoform. Each isoform was also expressed in a primary human skeletal muscle cell culture. Intense exercise (352 ± 69 s; mean ±S.E.M.) immediately increased 3 and ß2 mRNA by 2.4- and 1.7-fold, respectively (P < 0.05), whilst 1 and 2 mRNA were increased by 2.5- and 3.5-fold at 24 h and 3 h postexercise, respectively (P < 0.05). No significant change occurred for ß1 and ß3 mRNA, reflecting variable time-dependent responses. When the average postexercise value was contrasted to rest, mRNA increased for 1, 2, 3, ß1, ß2 and ß3 isoforms, by 1.4-, 2.2-, 1.4-, 1.1-, 1.0- and 1.0-fold, respectively (P < 0.05). However, exercise did not alter the protein abundance of the 1–3 and ß1–ß3 isoforms. Thus, human skeletal muscle expresses each of the Na+,K+-ATPase 1, 2, 3, ß1, ß2 and ß3 isoforms, evidenced at both transcription and protein levels. Whilst brief exercise increased Na+,K+-ATPase isoform mRNA expression, there was no effect on isoform protein expression, suggesting that the exercise challenge was insufficient for muscle Na+,K+-ATPase up-regulation.

Real time RT-PCR analysis of housekeeping genes in human skeletal muscle following acute exercise

Studies examining gene expression with RT-PCR typically normalize their mRNA data to a constitutively expressed housekeeping gene. The validity of a particular housekeeping gene must be determined for each experimental intervention. We examined the expression of various housekeeping genes following an acute bout of endurance (END) or resistance (RES) exercise. Twenty-four healthy subjects performed either a interval-type cycle ergometry workout to exhaustion (~75 min; END) or 300 single-leg eccentric contractions (RES). Muscle biopsies were taken before exercise and 3 h and 48 h following exercise. Real-time RT-PCR was performed on ß-actin, cyclophilin (CYC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ß2-microglobulin (ß2M). In a second study, 10 healthy subjects performed 90 min of cycle ergometry at ~65% of O2 max, and we examined a fifth housekeeping gene, 28S rRNA, and reexamined ß2M, from muscle biopsy samples taken immediately postexercise. We showed that CYC increased 48 h following both END and RES exercise (3- and 5-fold, respectively; P < 0.01), and 28S rRNA increased immediately following END exercise (2-fold; P = 0.02). ß-Actin trended toward an increase following END exercise (1.85-fold collapsed across time; P = 0.13), and GAPDH trended toward a small yet robust increase at 3 h following RES exercise (1.4-fold; P = 0.067). In contrast, ß2M was not altered at any time point postexercise. We conclude that ß2M and ß-actin are the most stably expressed housekeeping genes in skeletal muscle following RES exercise, whereas ß2M and GAPDH are the most stably expressed following END exercise.

Depressed Na+-K+-ATPase activity in skeletal muscle at fatigue is correlated with increased Na+-K+-ATPase mRNA expression following intense exercise

We investigated whether depressed muscle Na⁺-K⁺-ATPase activity with exercise reflected a loss of Na⁺-K⁺-ATPase units, the time course of its recovery postexercise, and whether this depressed activity was related to increased Na⁺-K⁺-ATPase isoform gene expression. Fifteen subjects performed fatiguing, knee extensor exercise at ~40% maximal work output per contraction. A vastus lateralis muscle biopsy was taken at rest, fatigue, 3 h, and 24 h postexercise and analyzed for maximal Na⁺-K⁺-ATPase activity via 3-O-methylfluorescein phosphatase (3-O-MFPase) activity, Na⁺-K⁺-ATPase content via [3H]ouabain binding sites, and Na⁺-K⁺-ATPase α1-, α2-, α3-, ß1-, ß2- and ß3-isoform mRNA expression by real-time RT-PCR. Exercise [352 (SD 267) s] did not affect [³H]ouabain binding sites but decreased 3-O-MFPase activity by 10.7 (SD 8)% (P < 0.05), which had recovered by 3 h postexercise, without further change at 24 h. Exercise elevated α1-isoform mRNA by 1.5-fold at fatigue (P < 0.05). This increase was inversely correlated with the percent change in 3-O-MFPase activity from rest to fatigue (%Δ3-O-MFPaserest-fatigue) (r = –0.60, P < 0.05). The average postexercise (fatigue, 3 h, 24 h) {alpha}1-isoform mRNA was increased 1.4-fold (P < 0.05) and approached a significant inverse correlation with %Δ3-O-MFPaserest-fatigue (r = –0.56, P = 0.08). Exercise elevated α2-isoform mRNA at fatigue 2.5-fold (P < 0.05), which was inversely correlated with %Δ3-O-MFPaserest-fatigue (r = –0.60, P = 0.05). The average postexercise α2-isoform mRNA was increased 2.2-fold (P < 0.05) and was inversely correlated with the %Δ3-O-MFPaserest-fatigue (r = –0.68, P < 0.05). Nonsignificant correlations were found between %Δ3-O-MFPaserest-fatigue and other isoforms. Thus acute exercise transiently decreased Na^+-K⁺-ATPase activity, which was correlated with increased Na⁺-K⁺-ATPase gene expression. This suggests a possible signal-transduction role for depressed muscle Na⁺-K⁺-ATPase activity with exercise.

Chronic intermittent hypoxia and incremental cycling exercise independently depress muscle in vitro maximal Na+-K+-ATPase activity in well-trained athletes

Athletes commonly attempt to enhance performance by training in normoxia but sleeping in hypoxia [live high and train low (LHTL)]. However, chronic hypoxia reduces muscle Na⁺-K⁺-ATPase content, whereas fatiguing contractions reduce Na⁺-K⁺-ATPase activity, which each may impair performance. We examined whether LHTL and intense exercise would decrease muscle Na⁺-K⁺-ATPase activity and whether these effects would be additive and sufficient to impair performance or plasma K⁺ regulation. Thirteen subjects were randomly assigned to two fitness-matched groups, LHTL (n = 6) or control (Con, n = 7). LHTL slept at simulated moderate altitude (3,000 m, inspired O₂ fraction = 15.48%) for 23 nights and lived and trained by day under normoxic conditions in Canberra (altitude ~600 m). Con lived, trained, and slept in normoxia. A standardized incremental exercise test was conducted before and after LHTL. A vastus lateralis muscle biopsy was taken at rest and after exercise, before and after LHTL or Con, and analyzed for maximal Na⁺-K⁺-ATPase activity [K⁺-stimulated 3-O-methylfluorescein phosphatase (3-O-MFPase)] and Na⁺-K⁺-ATPase content ([³H]ouabain binding sites). 3-O-MFPase activity was decreased by –2.9 ± 2.6% in LHTL (P < 0.05) and was depressed immediately after exercise (P < 0.05) similarly in Con and LHTL (–13.0 ± 3.2 and –11.8 ± 1.5%, respectively). Plasma K⁺ concentration during exercise was unchanged by LHTL; [3H]ouabain binding was unchanged with LHTL or exercise. Peak oxygen consumption was reduced in LHTL (P < 0.05) but not in Con, whereas exercise work was unchanged in either group. Thus LHTL had a minor effect on, and incremental exercise reduced, Na⁺-K⁺-ATPase activity. However, the small LHTL-induced depression of 3-O-MFPase activity was insufficient to adversely affect either K⁺ regulation or total work performed.