Distinct myofibre domains of the human myotendinous junction revealed by single-nucleus RNA sequencing.

The myotendinous junction (MTJ) is a specialized domain of the multinucleated myofibre that is faced with the challenge of maintaining robust cell-matrix contact with the tendon under high mechanical stress and strain. Here, we profiled 24,124 nuclei in semitendinosus muscle-tendon samples from three healthy males by using single-nucleus RNA sequencing (snRNA-seq), alongside spatial transcriptomics, to gain insight into the genes characterizing this specialization in humans. We identified a cluster of MTJ myonuclei represented by 47 enriched transcripts, of which the presence of ABI3BP, ABLIM1, ADAMTSL1, BICD1, CPM, FHOD3, FRAS1 and FREM2 was confirmed at the MTJ at the protein level in immunofluorescence assays. Four distinct subclusters of MTJ myonuclei were apparent, comprising two COL22A1-expressing subclusters and two subclusters lacking COL22A1 expression but with differing fibre type profiles characterized by expression of either MYH7 or MYH1 and/or MYH2. Our findings reveal distinct myonuclei profiles of the human MTJ, which represents a weak link in the musculoskeletal system that is selectively affected in pathological conditions ranging from muscle strains to muscular dystrophies.

Mechanical properties, collagen and glycosaminoglycan content of equine superficial digital flexor tendons are not affected by training.

Physical activity can activate extracellular matrix (ECM) protein synthesis and influence the size and mechanical properties of tendon. In this study, we aimed to investigate whether different training histories of horses would influence the synthesis of collagen and other matrix proteins and alter the mechanical properties of tendon. Samples from superficial digital flexor tendon (SDFT) from horses that were either (a) currently race trained (n = 5), (b) previously race trained (n = 5) or (c) untrained (n = 4) were analysed for matrix protein abundance (mass spectrometry), collagen and glycosaminoglycan (GAG) content, ECM gene expression and mechanical properties. It was found that ECM synthesis by tendon fibroblasts in vitro varied depending upon the previous training history. In contrast, fascicle morphology, collagen and GAG content, mechanical properties and ECM gene expression of the tendon did not reveal any significant differences between groups. In conclusion, although we could not identify any direct impact of the physical training history on the mechanical properties or major ECM components of the tendon, it is evident that horse tendon cells are responsive to loading in vivo, and the training background may lead to a modification in the composition of newly synthesised matrix.

Insulin-like growth factor-1 infusion in preterm piglets does not affect growth parameters of skeletal muscle or tendon tissue.

Prematurity has physical consequences, such as lower birth weight, decreased muscle mass and increased risk of adult-onset metabolic disease. Insulin-like growth factor 1 (IGF-1) has therapeutic potential to improve the growth and quality of muscle and tendon in premature births, and thus attenuate some of these sequalae. We investigated the effect of IGF-1 on extensor carpi radialis muscle and biceps brachii tendon of preterm piglets. The preterm group consisted of 19-day-old preterm (10 days early) piglets, treated with either IGF-1 or vehicle. Term controls consisted of groups of 9-day-old piglets (D9) and 19-day-old piglets (D19). Muscle samples were analysed by immunofluorescence to determine the cross-sectional area (CSA) of muscle fibres, fibre type composition, satellite cell content and central nuclei-containing fibres in the muscle. Tendon samples were analysed for CSA, collagen content and maturation, and vascularization. Gene expression of the tendon was measured by RT-qPCR. Across all endpoints, we found no significant effect of IGF-1 treatment on preterm piglets. Preterm piglets had smaller muscle fibre CSA compared to D9 and D19 control group. Satellite cell content was similar across all groups. For tendon, we found an effect of age on tendon CSA, and mRNA levels of COL1A1, tenomodulin and scleraxis. Immunoreactivity for elastin and CD31, and several markers of tendon maturation, were increased in D9 compared to the preterm piglets. Collagen content was similar across groups. IGF-1 treatment of preterm-born piglets does not influence the growth and maturation of skeletal muscle and tendon.

Disruption of day-to-night changes in circadian gene expression with chronic tendinopathy.

Overuse injury in tendon tissue (tendinopathy) is a frequent and costly musculoskeletal disorder and represents a major clinical problem with unsolved pathogenesis. Studies in mice have demonstrated that circadian clock-controlled genes are vital for protein homeostasis and important in the development of tendinopathy. We performed RNA sequencing, collagen content and ultrastructural analyses on human tendon biopsies obtained 12 h apart in healthy individuals to establish whether human tendon is a peripheral clock tissue and we performed RNA sequencing on patients with chronic tendinopathy to examine the expression of circadian clock genes in tendinopathic tissues. We found time-dependent expression of 280 RNAs including 11 conserved circadian clock genes in healthy tendons and markedly fewer (23) differential RNAs with chronic tendinopathy. Further, the expression of COL1A1 and COL1A2 was reduced at night but was not circadian rhythmic in synchronised human tenocyte cultures. In conclusion, day-to-night changes in gene expression in healthy human patellar tendons indicate a conserved circadian clock as well as the existence of a night reduction in collagen I expression. KEY POINTS: Tendinopathy is a major clinical problem with unsolved pathogenesis. Previous work in mice has shown that a robust circadian rhythm is required for collagen homeostasis in tendons. The use of circadian medicine in the diagnosis and treatment of tendinopathy has been stifled by the lack of studies on human tissue. Here, we establish that the expression of circadian clock genes in human tendons is time dependent, and now we have data to corroborate that circadian output is reduced in diseased tendon tissues. We consider our findings to be of significance in advancing the use of the tendon circadian clock as a therapeutic target or preclinical biomarker for tendinopathy.

Human derived tendon cells contribute to myotube formation in vitro.

Skeletal muscle possesses remarkable adaptability to mechanical loading and regenerative potential following muscle injury primarily due to satellite cell activity. Although the roles of several types of interstitial cells in skeletal muscle have been documented, the signaling interplay between the skeletal muscle and the adjacent tendon tissue has not been elucidated. Here, we tested whether human tendon derived cells (tenocytes) could induce human myogenic cells (myoblasts) proliferation and differentiation in vitro using co-culture experiments that allowed us to investigate the effect of tenocytes secretion upon myogenic progression. This was done in vitro by introducing insert wells with either myoblasts, tenocytes, or no cells (control) into a myoblast containing well (co-culture). Immunofluorescence analysis revealed a higher fusion index (≥5 nuclei within one Desmin + myotube) and a higher myotube diameter in co-cultures with tenocytes compared to myoblasts condition. Correspondingly, MHC-IIX gene expression was up-regulated when co-cultured with tenocytes. However, the proliferation of myoblasts (either Ki67 or BrdU + cells) was not enhanced under the presence of tenocytes. These findings show that tenocytes influence myotube formation upon human primary cells in vitro and contribute to understanding the role of tendon derived cells in skeletal muscle during development and regeneration.

Human skeletal muscle acetylcholine receptor gene expression in elderly males performing heavy resistance exercise.

Muscle fiber denervation is a major contributor to the decline in muscle mass and function during aging. Heavy resistance exercise is an effective tool for increasing muscle mass and strength, but whether it can rescue denervated muscle fibers remains unclear. Therefore, the purpose of this study was to investigate the potential of heavy resistance exercise to modify indices of denervation in healthy elderly individuals. Thirty-eight healthy elderly men (72 ± 5 yr) underwent 16 wk of heavy resistance exercise, whereas 20 healthy elderly men (72 ± 6 yr) served as nonexercising sedentary controls. Muscle biopsies were obtained pre and post training, and midway at 8 wk. Biopsies were analyzed by immunofluorescence for the prevalence of myofibers expressing embryonic myosin [embryonic myosin heavy chain (MyHCe)], neonatal myosin [neonatal myosin heavy chain (MyHCn)], nestin, and neural cell adhesion molecule (NCAM), and by RT-qPCR for gene expression levels of acetylcholine receptor (AChR) subunits, MyHCn, MyHCe, p16, and Ki67. In addition to increases in strength and type II fiber hypertrophy, heavy resistance exercise training led to a decrease in AChR α1 and ε subunit messenger RNA (mRNA; at 8 wk). Changes in gene expression levels of the α1 and ε AChR subunits with 8 wk of heavy resistance exercise supports the role of this type of exercise in targeting stability of the neuromuscular junction. The number of fibers positive for NCAM, nestin, and MyHCn was not affected, suggesting that a longer timeframe is needed for adaptations to manifest at the protein level.

Preserved stem cell content and innervation profile of elderly human skeletal muscle with lifelong recreational exercise.

Muscle fibre denervation and declining numbers of muscle stem (satellite) cells are defining characteristics of ageing skeletal muscle. The aim of this study was to investigate the potential for lifelong recreational exercise to offset muscle fibre denervation and compromised satellite cell content and function, both at rest and under challenged conditions. Sixteen elderly lifelong recreational exercisers (LLEX) were studied alongside groups of age-matched sedentary (SED) and young subjects. Lean body mass and maximal voluntary contraction were assessed, and a strength training bout was performed. From muscle biopsies, tissue and primary myogenic cell cultures were analysed by immunofluorescence and RT-qPCR to assess myofibre denervation and satellite cell quantity and function. LLEX demonstrated superior muscle function under challenged conditions. When compared with SED, the muscle of LLEX was found to contain a greater content of satellite cells associated with type II myofibres specifically, along with higher mRNA levels of the beta and gamma acetylcholine receptors (AChR). No difference was observed between LLEX and SED for the proportion of denervated fibres or satellite cell function, as assessed in vitro by myogenic cell differentiation and fusion index assays. When compared with inactive counterparts, the skeletal muscle of lifelong exercisers is characterised by greater fatigue resistance under challenged conditions in vivo, together with a more youthful tissue satellite cell and AChR profile. Our data suggest a little recreational level exercise goes a long way in protecting against the emergence of classic phenotypic traits associated with the aged muscle. KEY POINTS: The detrimental effects of ageing can be partially offset by lifelong self-organized recreational exercise, as evidence by preserved type II myofibre-associated satellite cells, a beneficial muscle innervation status and greater fatigue resistance under challenged conditions. Satellite cell function (in vitro), muscle fibre size and muscle fibre denervation determined by immunofluorescence were not affected by recreational exercise. Individuals that are recreationally active are far more abundant than master athletes, which sharply increases the translational perspective of the present study. Future studies should further investigate recreational activity in relation to muscle health, while also including female participants.

Nestin and osteocrin mRNA increases in human semitendinosus myotendinous junction 7 days after a single bout of eccentric exercise.

The myotendinous junction (MTJ), a specialized interface for force transmission between muscle and tendon, has a unique transcriptional activity and is highly susceptible to muscle strain injury. Eccentric exercise training is known to reduce this risk of injury, but knowledge of the influence of exercise on the MTJ at the molecular and cellular levels is limited. In this study, 30 subjects were randomized to a single bout of eccentric exercise 1 week prior to tissue sampling (exercised) or no exercise (control). Samples were collected from the semitendinosus as part of reconstruction of the anterior cruciate ligament and divided into fractions containing muscle, MTJ and tendon, respectively. The concentrations of macrophages and satellite cells were counted, and the expression of genes previously known to be active at the MTJ were analyzed by real-time-quantitative PCR. An effect of the single bout of exercise was found on the expression of nestin (NES) and osteocrin (OSTN) mRNA in the MTJ and tendon fractions. Genes earlier identified at the MTJ (COL22A1, POSTN, ADAMTS8, MNS1, NCAM1) were confirmed to be expressed at a significantly higher level in the MTJ compared to muscle and tendon but were unaffected by exercise. In the exercise group a higher concentration of macrophages, but not of satellite cells, was seen in muscle close to the MTJ. The expression of NES and OSTN was higher in human semitendinosus MTJ 1 week after a single session of heavy eccentric exercise. Based on these results, NES and OSTN could have a part in explaining how the MTJ adapts to eccentric exercise.

Vitamin D controls T cell antigen receptor signaling and activation of human T cells.

Phospholipase C (PLC) isozymes are key signaling proteins downstream of many extracellular stimuli. Here we show that naive human T cells had very low expression of PLC-gamma1 and that this correlated with low T cell antigen receptor (TCR) responsiveness in naive T cells. However, TCR triggering led to an upregulation of approximately 75-fold in PLC-gamma1 expression, which correlated with greater TCR responsiveness. Induction of PLC-gamma1 was dependent on vitamin D and expression of the vitamin D receptor (VDR). Naive T cells did not express VDR, but VDR expression was induced by TCR signaling via the alternative mitogen-activated protein kinase p38 pathway. Thus, initial TCR signaling via p38 leads to successive induction of VDR and PLC-gamma1, which are required for subsequent classical TCR signaling and T cell activation.

Gene Expression Linked to Reepithelialization of Human Skin Wounds.

Our understanding of the regulatory processes of reepithelialization during wound healing is incomplete. In an attempt to map the genes involved in epidermal regeneration and differentiation, we measured gene expression in formalin-fixed, paraffin-embedded standardized epidermal wounds induced by the suction-blister technique with associated nonwounded skin using NanoString technology. The transcripts of 139 selected genes involved in clotting, immune response to tissue injury, signaling pathways, cell adhesion and proliferation, extracellular matrix remodeling, zinc transport and keratinocyte differentiation were evaluated. We identified 22 upregulated differentially expressed genes (DEGs) in descending order of fold change (MMP1, MMP3, IL6, CXCL8, SERPINE1, IL1B, PTGS2, HBEGF, CXCL5, CXCL2, TIMP1, CYR61, CXCL1, MMP12, MMP9, HGF, CTGF, ITGB3, MT2A, FGF7, COL4A1 and PLAUR). The expression of the most upregulated gene, MMP1, correlated strongly with MMP3 followed by IL6 and IL1B. rhIL-1ß, but not rhIL-6, exposure of cultured normal human epidermal keratinocytes and normal human dermal fibroblasts increased both MMP1 mRNA and MMP-1 protein levels, as well as TIMP1 mRNA levels. The increased TIMP1 in wounds was validated by immunohistochemistry. The six downregulated DEGs (COL7A1, MMP28, SLC39A2, FLG1, KRT10 and FLG2) were associated with epidermal maturation. KLK8 showed the strongest correlation with MKI67 mRNA levels and is a potential biomarker for keratinocyte proliferation. The observed gene expression changes correlate well with the current knowledge of physiological reepithelialization. Thus, the gene expression panel described in this paper could be used in patients with impaired healing to identify possible therapeutic targets.

RNA sequencing and immunofluorescence of the myotendinous junction of mature horses and humans.

The myotendinous junction (MTJ) is a specialized interface for transmitting high forces between the muscle and tendon and yet the MTJ is a common site of strain injury with a high recurrence rate. The aim of this study was to identify previously unknown MTJ components in mature animals and humans. Samples were obtained from the superficial digital flexor (SDF) muscle-tendon interface of 20 horses, and the tissue was separated through a sequential cryosectioning approach into muscle, MTJ (muscle tissue enriched in myofiber tips attached to the tendon), and tendon fractions. RT-PCR was performed for genes known to be expressed in the three tissue fractions and t-distributed stochastic neighbor embedding (t-SNE) plots were used to select the muscle, MTJ, and tendon samples from five horses for RNA sequencing. The expression of previously known and unknown genes identified through RNA sequencing was studied by immunofluorescence on human hamstring MTJ tissue. The main finding was that RNA sequencing identified the expression of a panel of 61 genes enriched at the MTJ. Of these, 48 genes were novel for the MTJ and 13 genes had been reported to be associated with the MTJ in earlier studies. The expression of known [COL22A1 (collagen XXII), NCAM (neural cell adhesion molecule), POSTN (periostin), NES (nestin), OSTN (musclin/osteocrin)] and previously undescribed [MNS1 (meiosis-specific nuclear structural protein 1), and LCT (lactase)] MTJ genes was confirmed at the protein level by immunofluorescence on tissue sections of human MTJ. In conclusion, in muscle-tendon interface tissue enriched with myofiber tips, we identified the expression of previously unknown MTJ genes representing diverse biological processes, which may be important in the maintenance of the specialized MTJ.

AXIN1 knockout does not alter AMPK/mTORC1 regulation and glucose metabolism in mouse skeletal muscle.

KEY POINTS: Tamoxifen-inducible skeletal muscle-specific AXIN1 knockout (AXIN1 imKO) in mouse does not affect whole-body energy substrate metabolism. AXIN1 imKO does not affect AICAR or insulin-stimulated glucose uptake in adult skeletal muscle. AXIN1 imKO does not affect adult skeletal muscle AMPK or mTORC1 signalling during AICAR/insulin/amino acid incubation, contraction and exercise. During exercise, α2/ß2/γ3AMPK and AMP/ATP ratio show greater increases in AXIN1 imKO than wild-type in gastrocnemius muscle. ABSTRACT: AXIN1 is a scaffold protein known to interact with >20 proteins in signal transduction pathways regulating cellular development and function. Recently, AXIN1 was proposed to assemble a protein complex essential to catabolic-anabolic transition by coordinating AMPK activation and inactivation of mTORC1 and to regulate glucose uptake-stimulation by both AMPK and insulin. To investigate whether AXIN1 is permissive for adult skeletal muscle function, a phenotypic in vivo and ex vivo characterization of tamoxifen-inducible skeletal muscle-specific AXIN1 knockout (AXIN1 imKO) mice was conducted. AXIN1 imKO did not influence AMPK/mTORC1 signalling or glucose uptake stimulation at rest or in response to different exercise/contraction protocols, pharmacological AMPK activation, insulin or amino acids stimulation. The only genotypic difference observed was in exercising gastrocnemius muscle, where AXIN1 imKO displayed elevated α2/ß2/γ3 AMPK activity and AMP/ATP ratio compared to wild-type mice. Our work shows that AXIN1 imKO generally does not affect skeletal muscle AMPK/mTORC1 signalling and glucose metabolism, probably due to functional redundancy of its homologue AXIN2.

Collagens in primary frozen shoulder: expression of collagen mRNA isoforms in the different phases of the disease.

OBJECTIVES: Primary frozen shoulder (pFS) has three phases that differ in clinical presentation. It is characterized by contracture of the joint capsule. We hypothesized that there is a general upregulation of collagens in pFS, and that this is highest in the first phase of the disease. The aims of this study were to investigate the expression of various collagens and degradation of collagens in patients with primary pFS and relate this to the three phases of the condition. METHODS: From twenty-six patients with pFS and eight control patients with subacromial impingement, biopsies were obtained during shoulder arthroscopy from the middle glenohumeral ligament and the anterior capsule, and mRNA levels for collagens, MMP-2 and -14 and TGF-ß1, - ß2 and -ß3 in the tissue were analysed using real-time PCR. RESULTS: Genes for collagens type I, III, IV, V, VI and XIV, were activated in pFS, and the total mRNA for all collagens was increased (P < 0.05). This upregulation was independent of disease phases in pFS. In addition, MMP-2, MMP-14, TGF-ß1 and TGF-ß3 were upregulated in all phases of the disease. CONCLUSION: There is a general upregulation and an increased degradation of collagens in pFS in all three phases of the disease. This indicates a constantly increased turnover of the fibrotic tissue in the capsule from pFS. The difference in clinical presentation of pFS observed in the three phases of the disease is not primarily a result of variations in collagen production.

Preserved capacity for satellite cell proliferation, regeneration, and hypertrophy in the skeletal muscle of healthy elderly men.

Blunted muscle hypertrophy and impaired regeneration with aging have been partly attributed to satellite cell (SC) dysfunction. However, true muscle regeneration has not yet been studied in elderly individuals. To investigate this, muscle injury was induced by 200 electrically stimulated (ES) eccentric contractions of the vastus lateralis (VL) of one leg in seven young (20-31 years) and 19 elderly men (60-73 years). This was followed by 13 weeks of resistance training (RT) for both legs to investigate the capacity for hypertrophy. Muscle biopsies were collected Pre- and Post-RT, and 9 days after ES, for immunohistochemistry and RT-PCR. Hypertrophy was assessed by MRI, DEXA, and immunohistochemistry. Overall, surprisingly comparable responses were observed between the young and elderly. Nine days after ES, Pax7+ SC number had doubled (P < .05), alongside necrosis and substantial changes in expression of genes related to matrix, myogenesis, and innervation (P < .05). Post-RT, VL cross-sectional area had increased in both legs (~15%, P < .05) and SCs/type II fiber had increased ~2-4 times more with ES+RT vs RT alone (P < .001). Together these novel findings demonstrate "youthful" regeneration and hypertrophy responses in human elderly muscle. Furthermore, boosting SC availability in healthy elderly men does not enhance the subsequent muscle hypertrophy response to RT.

Thyroid hormone receptor α in skeletal muscle is essential for T3-mediated increase in energy expenditure.

Thyroid hormones are important for homeostatic control of energy metabolism and body temperature. Although skeletal muscle is considered a key site for thyroid action, the contribution of thyroid hormone receptor signaling in muscle to whole-body energy metabolism and body temperature has not been resolved. Here, we show that T3-induced increase in energy expenditure requires thyroid hormone receptor alpha 1 (TRα1 ) in skeletal muscle, but that T3-mediated elevation in body temperature is achieved in the absence of muscle-TRα1 . In slow-twitch soleus muscle, loss-of-function of TRα1 (TRαHSACre ) alters the fiber-type composition toward a more oxidative phenotype. The change in fiber-type composition, however, does not influence the running capacity or motivation to run. RNA-sequencing of soleus muscle from WT mice and TRαHSACre mice revealed differentiated transcriptional regulation of genes associated with muscle thermogenesis, such as sarcolipin and UCP3, providing molecular clues pertaining to the mechanistic underpinnings of TRα1 -linked control of whole-body metabolic rate. Together, this work establishes a fundamental role for skeletal muscle in T3-stimulated increase in whole-body energy expenditure.

Early development of tendinopathy in humans: Sequence of pathological changes in structure and tissue turnover signaling.

Overloading of tendon tissue with resulting chronic pain (tendinopathy) is a common disorder in occupational-, leisure- and sports-activity, but its pathogenesis remains poorly understood. To investigate the very early phase of tendinopathy, Achilles and patellar tendons were investigated in 200 physically active patients and 50 healthy control persons. Patients were divided into three groups: symptoms for 0-1 months (T1), 1-2 months (T2) or 2-3 months (T3). Tendinopathic Achilles tendon cross-sectional area determined by ultrasonography (US) was ~25% larger than in healthy control persons. Both Achilles and patellar anterior-posterior diameter were elevated in tendinopathy, and only later in Achilles was the width increased. Increased tendon size was accompanied by an increase in hypervascularization (US Doppler flow) without any change in mRNA for angiogenic factors. From patellar biopsies taken bilaterally, mRNA for most growth factors and tendon components remained unchanged (except for TGF-beta1 and substance-P) in early tendinopathy. Tendon stiffness remained unaltered over the first three months of tendinopathy and was similar to the asymptomatic contra-lateral tendon. In conclusion, this suggests that tendinopathy pathogenesis represents a disturbed tissue homeostasis with fluid accumulation. The disturbance is likely induced by repeated mechanical overloading rather than a partial rupture of the tendon.

Postprandial muscle protein synthesis rate is unaffected by 20-day habituation to a high protein intake: a randomized controlled, crossover trial.

PURPOSE: During the last decade more researchers have argued in favor of an increased protein intake for older adults. However, there is a lack of knowledge on the long-term effects of conforming to such a high protein intake with regards to the basal and postprandial muscle protein turnover. The purpose of this study was to compare the postprandial synthesis response in muscle proteins, and the abundance of directly incorporated food-derived amino acids following habituation to high vs. recommended level of protein intake. METHODS: In a double blinded crossover intervention 11 older male participants (66.6 ± 1.7 years of age) were habituated for 20 days to a recommended protein (RP) intake (1.1 g protein/kg lean body mass (LBM)/day) and a high protein (HP) intake (> 2.1 g protein/kg LBM/day). Following each habituation period, intrinsically labelled proteins were ingested as part of a mixed meal to determine the incorporation of meal protein-derived amino acids into myofibrillar proteins. Furthermore, the myofibrillar fractional synthesis rate (FSR) and amino acid kinetics across the leg were determined using gold standard stable isotope tracer methodologies. RT qPCR was used to assess the expression of markers related to muscle proteinsynthesis and breakdown. RESULTS: No impact of habituation was observed on skeletal muscle amino acid or protein kinetics. However, the shunting of amino acids directly from artery to vein was on average 2.9 [Formula: see text]mol/min higher following habituation to HP compared to RP. CONCLUSIONS: In older males, habituation to a higher than the currently recommended protein intake did not demonstrate any adaptions in the muscle protein turnover or markers hereof when subjected to an intake of an identical mixed meal. CLINICAL TRIAL REGISTRY: Journal number NCT02587156, Clinicaltrials.org. Date of registration: October 27th, 2015.

A Human Cellular Model for Colorectal Anastomotic Repair: The Effect of Localization and Transforming Growth Factor-ß1 Treatment on Collagen Deposition and Biomarkers.

Anastomotic leakage (AL) is a devastating complication after colorectal surgery, possibly due to the loss of stabilizing collagen fibers in the submucosa. Our aim was to assess the formation of collagen in the colon versus the rectum with or without transforming growth factor (TGF)-ß1 exposure in a human cellular model of colorectal repair. Primary fibroblasts were isolated by an explant procedure from clinically resected tissue rings during anastomosis construction in 19 consecutive colorectal patients who underwent laparoscopy. The cells, identified as fibroblasts by morphologic characteristics and flow cytometry analysis (CD90+), were cultured for 8 days and in 12 patients in the presence of 1 ng/mL TGF-ß1. Total collagen deposition was measured colorimetrically after Sirius red staining of fixed cell layers, and type I, III, and VI collagen biosynthesis and degradation were specifically determined by the biomarkers PINP, PRO-C3, PRO-C6, and C3M in conditioned media by competitive enzyme-linked immunosorbent assays. Total collagen deposition by fibroblasts from the colon and rectum did not significantly differ. TGF-ß1 treatment increased PINP, PRO-C6, and total collagen deposition. Mechanistically, TGF-ß1 treatment increased COL1A1 and ACTA2 (encoding α-smooth muscle actin), and decreased COL6A1 and MMP2 mRNA levels in colorectal fibroblasts. In conclusion, we found no effect of anatomic localization on collagen production by fibroblasts derived from the large intestine. TGF-ß1 represents a potential therapeutic agent for the prevention of AL by increasing type I collagen synthesis and collagen deposition.

Insulin-stimulated glucose uptake partly relies on p21-activated kinase (PAK)2, but not PAK1, in mouse skeletal muscle.

KEY POINTS: Muscle-specific genetic ablation of p21-activated kinase (PAK)2, but not whole-body PAK1 knockout, impairs glucose tolerance in mice. Insulin-stimulated glucose uptake partly relies on PAK2 in glycolytic extensor digitorum longus muscle By contrast to previous reports, PAK1 is dispensable for insulin-stimulated glucose uptake in mouse muscle. ABSTRACT: The group I p21-activated kinase (PAK) isoforms PAK1 and PAK2 are activated in response to insulin in skeletal muscle and PAK1/2 signalling is impaired in insulin-resistant mouse and human skeletal muscle. Interestingly, PAK1 has been suggested to be required for insulin-stimulated glucose transporter 4 translocation in mouse skeletal muscle. Therefore, the present study aimed to examine the role of PAK1 in insulin-stimulated muscle glucose uptake. The pharmacological inhibitor of group I PAKs, IPA-3 partially reduced (-20%) insulin-stimulated glucose uptake in isolated mouse soleus muscle (P < 0.001). However, because there was no phenotype with genetic ablation of PAK1 alone, consequently, the relative requirement for PAK1 and PAK2 in whole-body glucose homeostasis and insulin-stimulated muscle glucose uptake was investigated. Whole-body respiratory exchange ratio was largely unaffected in whole-body PAK1 knockout (KO), muscle-specific PAK2 KO and in mice with combined whole-body PAK1 KO and muscle-specific PAK2 KO. By contrast, glucose tolerance was mildly impaired in mice lacking PAK2 specifically in muscle, but not PAK1 KO mice. Moreover, while PAK1 KO muscles displayed normal insulin-stimulated glucose uptake in vivo and in isolated muscle, insulin-stimulated glucose uptake was slightly reduced in isolated glycolytic extensor digitorum longus muscle lacking PAK2 alone (-18%) or in combination with PAK1 KO (-12%) (P < 0.05). In conclusion, glucose tolerance and insulin-stimulated glucose uptake partly rely on PAK2 in glycolytic mouse muscle, whereas PAK1 is dispensable for whole-body glucose homeostasis and insulin-stimulated muscle glucose uptake.

Influence of the integrin alpha-1 subunit and its relationship with high-fat diet upon extracellular matrix synthesis in skeletal muscle and tendon.

Integrins are important for mechanosensation in tissue and play, together with nutrition, a role in regulating extracellular matrix (ECM) in skeletal muscle and tendon. Integrin receptors are dimers that consist of an α and ß subunit and bridge extracellular and intracellular signals. The present study investigates whether the deletion of the integrin receptor α1 subunit influences collagen and other matrix proteins in the musculotendinous tissue and whether it causes any compensatory changes in other integrin subunits in C57BL/6J mice. In addition, we study whether a high-fat diet (HFD) influences these responses in muscle or tendon. Mice on a HFD had a higher number of non-enzymatic cross-links in skeletal muscle ECM and increased gene expression of collagen and other extracellular matrix proteins. In contrast to gene expression, total collagen protein content was decreased by HFD in the muscle with no change in tendon. Integrin α1 subunit knockout resulted in a decrease of collagen type I and III, TGF-ß1 and IGF-1 gene expression in muscle of HFD mice but did not affect total collagen protein compared with wild-type (WT) littermates in either muscle or tendon. There was no compensatory increase in the genes that express other integrin subunits. In conclusion, HFD induced a significant increase in expression of ECM genes in muscle. On the protein level, HFD resulted in a lower collagen content in muscle. Tendons were unaffected by the diet. Deletion of the integrin α1 subunit did not affect collagen protein or gene expression in muscle or tendon.