Force dependent effects of chronic overuse on fibrosis-related genes and proteins in skeletal muscles.

AIM: To examine the chronic effect of force on mRNA and protein expression levels of fibrosis-related genes in flexor digitorum muscles in a rat model of repetitive overuse injury that induces muscle fibrosis at high force levels. MATERIALS AND METHODS: Two groups of rats were trained to perform a voluntary repetitive lever-pulling task at either a high (HFHR) or a low force (LFHR) for 18 weeks, while a control group (FRC) performed no task. RNA and protein were prepared from forelimb flexor digitorum muscles. Fibrosis-related gene RNA transcripts were evaluated using quantitative PCR (qPCR) and analyzed using the geometric mean of three housekeeping genes or the mean of each individually as reference. Protein levels were quantified using ELISA, western blot, or immunohistofluorescence. RESULTS: Of eight fibrosis-related mRNAs examined, only FGF2 demonstrated a consistent significant increase in the HFHR group, compared to the FRC group. However, protein amounts of collagen type 1, collagen type 3, and TGFß1 were significantly higher in the HFHR, compared to the FRC and LFHR groups, while CCN2 and FGF2 were higher in both HFHR and LFHR, compared to the FRC group. CONCLUSIONS: Our results suggest that there is steady-state transcription of fibrogenic genes in muscles with established fibrosis, implying that post-transcriptional processes are responsible for the increased protein levels of fibrotic factors during muscle overuse conditions. We hypothesize that targeting such pathways represents a valid approach to treat overuse injury. Alternatively, FGF2 gene expression may represent a valid target for therapy.

Genetic variation and exercise-induced muscle damage: implications for athletic performance, injury and ageing.

Prolonged unaccustomed exercise involving muscle lengthening (eccentric) actions can result in ultrastructural muscle disruption, impaired excitation-contraction coupling, inflammation and muscle protein degradation. This process is associated with delayed onset muscle soreness and is referred to as exercise-induced muscle damage. Although a certain amount of muscle damage may be necessary for adaptation to occur, excessive damage or inadequate recovery from exercise-induced muscle damage can increase injury risk, particularly in older individuals, who experience more damage and require longer to recover from muscle damaging exercise than younger adults. Furthermore, it is apparent that inter-individual variation exists in the response to exercise-induced muscle damage, and there is evidence that genetic variability may play a key role. Although this area of research is in its infancy, certain gene variations, or polymorphisms have been associated with exercise-induced muscle damage (i.e. individuals with certain genotypes experience greater muscle damage, and require longer recovery, following strenuous exercise). These polymorphisms include ACTN3 (R577X, rs1815739), TNF (-308 G>A, rs1800629), IL6 (-174 G>C, rs1800795), and IGF2 (ApaI, 17200 G>A, rs680). Knowing how someone is likely to respond to a particular type of exercise could help coaches/practitioners individualise the exercise training of their athletes/patients, thus maximising recovery and adaptation, while reducing overload-associated injury risk. The purpose of this review is to provide a critical analysis of the literature concerning gene polymorphisms associated with exercise-induced muscle damage, both in young and older individuals, and to highlight the potential mechanisms underpinning these associations, thus providing a better understanding of exercise-induced muscle damage.

Astroglial glutamate transporter deficiency increases synaptic excitability and leads to pathological repetitive behaviors in mice.

An increase in the ratio of cellular excitation to inhibition (E/I ratio) has been proposed to underlie the pathogenesis of neuropsychiatric disorders, such as autism spectrum disorders (ASD), obsessive-compulsive disorder (OCD), and Tourette's syndrome (TS). A proper E/I ratio is achieved via factors expressed in neuron and glia. In astrocytes, the glutamate transporter GLT1 is critical for regulating an E/I ratio. However, the role of GLT1 dysfunction in the pathogenesis of neuropsychiatric disorders remains unknown because mice with a complete deficiency of GLT1 exhibited seizures and premature death. Here, we show that astrocyte-specific GLT1 inducible knockout (GLAST(CreERT2/+)/GLT1(flox/flox), iKO) mice exhibit pathological repetitive behaviors including excessive and injurious levels of self-grooming and tic-like head shakes. Electrophysiological studies reveal that excitatory transmission at corticostriatal synapse is normal in a basal state but is increased after repetitive stimulation. Furthermore, treatment with an N-methyl-D-aspartate (NMDA) receptor antagonist memantine ameliorated the pathological repetitive behaviors in iKO mice. These results suggest that astroglial GLT1 has a critical role in controlling the synaptic efficacy at corticostriatal synapses and its dysfunction causes pathological repetitive behaviors.

Atypical presentation of GNE myopathy with asymmetric hand weakness.

GNE myopathy is a rare autosomal recessive muscle disease caused by mutations in GNE, the gene encoding the rate-limiting enzyme in sialic acid biosynthesis. GNE myopathy usually manifests in early adulthood with distal myopathy that progresses slowly and symmetrically, first involving distal muscles of the lower extremities, followed by proximal muscles with relative sparing of the quadriceps. Upper extremities are typically affected later in the disease. We report a patient with GNE myopathy who presented with asymmetric hand weakness. He had considerably decreased left grip strength, atrophy of the left anterior forearm and fibro-fatty tissue replacement of left forearm flexor muscles on T1-weighted magnetic resonance imaging. The patient was an endoscopist and thus the asymmetric hand involvement may be associated with left hand overuse in daily repetitive pinching and gripping movements, highlighting the possible impact of environmental factors on the progression of genetic muscle conditions.

Upregulation of BDNF and NGF in cervical intervertebral discs exposed to painful whole-body vibration.

STUDY DESIGN: In vivo study defining expression of the neurotrophins, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), in cervical intervertebral discs after painful whole-body vibration (WBV). OBJECTIVE: The goal of this study is to determine if BDNF and NGF are expressed in cervical discs after painful WBV in a rat model. SUMMARY OF BACKGROUND DATA: WBV is a possible source of neck pain and has been implicated as increasing the risk for disc disorders. Typically, aneural regions of painful human lumbar discs exhibit hyperinnervation, suggesting nerve ingrowth as potentially contributing to disc degeneration and pain. BDNF and NGF are upregulated in painfully degenerate lumbar discs and hypothesized to contribute to this pathology. METHODS: Male Holtzman rats underwent 7 days of repeated WBV (15 Hz, 30 min/d) or sham exposures, followed by 7 days of rest. Cervical discs were collected for analysis of BDNF and NGF expression through RT-qPCR and Western blot analysis. Immunohistochemistry also evaluated their regional expression in the disc. RESULTS: Vibration significantly increases BDNF messenger ribonucleic acid (mRNA) levels (P=0.036), as well as total-NGF mRNA (P=0.035). Protein expression of both BDNF (P=0.006) and the 75-kDa NGF (P=0.045) increase by nearly 4- and 10-fold, respectively. Both BDNF mRNA (R=0.396; P=0.012) and protein (R=0.280; P=0.035) levels are significantly correlated with the degree of behavioral sensitivity (i.e., pain) at day 14. Total-NGF mRNA is also significantly correlated with the extent of behavioral sensitivity (R=0.276; P=0.044). Both neurotrophins are most increased in the inner annulus fibrosus and nucleus pulposus. CONCLUSION: The increases in BDNF and NGF in the cervical discs after painful vibration are observed in typically aneural regions of the disc, consistent with reports of its hyperinnervation. Yet, the induction of nerve ingrowth into the disc was not explicitly investigated. Neurotrophin expression also correlates with behavioral sensitivity, suggesting a role for both neurotrophins in the development of disc pain. LEVEL OF EVIDENCE: N/A.

Increased mast cell numbers in a calcaneal tendon overuse model.

Tendinopathy is often discovered late because the initial development of tendon pathology is asymptomatic. The aim of this study was to examine the potential role of mast cell involvement in early tendinopathy using a high-intensity uphill running (HIUR) exercise model. Twenty-four male Wistar rats were divided in two groups: running group (n = 12); sedentary control group (n = 12). The running-group was exposed to the HIUR exercise protocol for 7 weeks. The calcaneal tendons of both hind limbs were dissected. The right tendon was used for histologic analysis using Bonar score, immunohistochemistry, and second harmonic generation microscopy (SHGM). The left tendon was used for quantitative polymerase chain reaction (qPCR) analysis. An increased tendon cell density in the runners were observed compared to the controls (P = 0.05). Further, the intensity of immunostaining of protein kinase B, P = 0.03; 2.75 ± 0.54 vs 1.17 ± 0.53, was increased in the runners. The Bonar score (P = 0.05), and the number of mast cells (P = 0.02) were significantly higher in the runners compared to the controls. Furthermore, SHGM showed focal collagen disorganization in the runners, and reduced collagen density (P = 0.03). IL-3 mRNA levels were correlated with mast cell number in sedentary animals. The qPCR analysis showed no significant differences between the groups in the other analyzed targets. The current study demonstrates that 7-week HIUR causes structural changes in the calcaneal tendon, and further that these changes are associated with an increased mast cell density.

Epidemiology, genetics and biological factors of rotator cuff tears.

Rotator cuff disease is among the most common musculoskeletal disorders with high direct and indirect costs in industrialized countries. Not all rotator cuff tears are symptomatic. Genetics has recently been investigated as a factor involved in the pathogenesis of rotator cuff pathology. Genetic factors seem to be involved in symptom presentation and tear progression. As rotator cuff disease is multifactorial, no single gene is directly involved in the pathology. Phenotypic expression of genetic susceptibility manifests at the level of ultrastructure of the tendon. Predisposing genes may also operate through apoptosis and regenerative capacity. Studies on cellular and molecular biology are more numerous, but still incomplete, and recently have focussed on the role of apoptosis in tendinopathy, analyzing its key mediators and cellular changes. Oxidative stress is responsible for reduction of collagen synthesis. Biological investigations have identified recently new risk factors. Preliminary reports introduced the possible role of glucose as a risk factor for rotator cuff tear. Further studies are required to fully clarify the genetic and biological factors involved in rotator cuff tears.

Genetic variant of HTR2A associates with risk of impulse control and repetitive behaviors in Parkinson's disease.

OBJECTIVES: To determine the role of a serotonin 2A receptor gene (=HTR2A) variant in the development of impulse control and repetitive behaviors in Parkinson's disease. METHODS: We performed a genetic association analysis to a cohort of 404 Korean patients with Parkinson's disease who had been enrolled in a previous study. Presence of impulse control and repetitive behaviors was screened using modified version of Minnesota Impulsive Disorders Interview and genotyping for HTR2A c.102T > C was performed using the TaqMan assay. RESULTS: The T allele, which is presumably linked to higher receptor expression, was marginally associated with impulse control and repetitive behaviors in Parkinson's disease; the genetic influence was significantly enhanced in the lower levodopa-equivalent-dose group, increasing the risk by 2.8 and 6.9 times in CT and TT carriers, respectively (p-trend = 0.011). There was no significant interaction between the c.102T > C variant and clinical risk variables such as sex, age at onset, total daily levodopa-equivalent-dose, use of dopamine agonist and daily dose of dopamine agonist. CONCLUSION: Our data support a possible contribution of genetic variation in the HTR2A to the susceptibility to impulse control and repetitive behaviors in Parkinson's disease.

A pathway-based approach investigating the genes encoding interleukin-1ß, interleukin-6 and the interleukin-1 receptor antagonist provides new insight into the genetic susceptibility of Achilles tendinopathy.

OBJECTIVES: Achilles tendinopathy (AT) is a multifactorial condition for which genetic risk factors have been identified. A pathway-based approach was used to investigate genes within the inflammatory pathway. METHODS: Functional polymorphisms within IL-1ß (-31T→C and -511C→T), IL-1RN (variable number tandem repeat) and IL-6 (-172G→C) were investigated for associations with AT in a South African (SA) and Australian (AUS) case-control studies. A total of 369 (161 SA and 208 AUS) asymptomatic control participants (CON) and 175 (90 SA and 85 AUS) participants with AT (TEN) were genotyped. Allele combinations were constructed using the above polymorphisms in combination with the COL5A1 BstUI RFLP. RESULTS: Independently, no associations were observed between any of the polymorphisms tested and risk of TEN. The allele combinations of five polymorphisms were, however, found to have a highly significant relationship with AT (p=0.005), after adjusting for gender and country (SA or AUS). CONCLUSIONS: Variations within the interleukin genes and the COL5A1 BstUI CC genotype are collectively significantly associated with risk of AT. This research emphasises that a pathway-based genetic association study may be a more effective approach to capture and understand the genetic risk factors underlying the multifactorial conditions, such as AT.

CCL2 and CCR2 polymorphisms are associated with markers of exercise-induced skeletal muscle damage.

Novel eccentric (lengthening contraction) exercise typically results in muscle damage, which manifests as prolonged muscle dysfunction, delayed onset muscle soreness, and leakage of muscle proteins into circulation. There is a large degree of variability in the damage response of individuals to eccentric exercise, with higher responders at risk for potentially fatal rhabdomyolysis. We hypothesized that single nucleotide polymorphisms (SNPs) in chemokine ligand 2 (CCL2) and its receptor chemokine receptor 2 (CCR2) associate with the high degrees of variability in the muscle damage response. We based this hypothesis on CCL2's roles in macrophage and satellite cell signaling in injured muscle. DNA was obtained from 157 untrained men and women following maximal eccentric exercise. Strength loss, soreness, serum creatine kinase (CK), and myoglobin levels before and during recovery from a single exercise bout were tested for association with 16 SNPs in CCL2 and CCR2. The rare alleles for rs768539 and rs3918358 (CCR2) were significantly (P<0.05) associated with lower preexercise strength in men, whereas CCL2 SNPs (rs13900, rs1024611, and rs1860189) and CCR2 (rs1799865) were associated with altered preexercise CK levels in women. During recovery, the rs3917878 genotype (CCL2) was associated with attenuated strength recovery in men and an elevated CK response in women. CCR2 variants were associated with slower strength recovery in women (rs3918358) and elevated soreness (rs1799865) across all subjects. In summary, we found that SNPs in CCL2 and CCR2 are associated with exercise-induced muscle damage and that the presence of certain variants may result in an exaggerated damage response to strenuous exercise.

Extensive mononuclear infiltration and myogenesis characterize recovery of dysferlin-null skeletal muscle from contraction-induced injuries.

We studied the response of dysferlin-null and control skeletal muscle to large- and small-strain injuries to the ankle dorsiflexors in mice. We measured contractile torque and counted fibers retaining 10-kDa fluorescein dextran, necrotic fibers, macrophages, and fibers with central nuclei and expressing developmental myosin heavy chain to assess contractile function, membrane resealing, necrosis, inflammation, and myogenesis. We also studied recovery after blunting myogenesis with X-irradiation. We report that dysferlin-null myofibers retain 10-kDa dextran for 3 days after large-strain injury but are lost thereafter, following necrosis and inflammation. Recovery of dysferlin-null muscle requires myogenesis, which delays the return of contractile function compared with controls, which recover from large-strain injury by repairing damaged myofibers without significant inflammation, necrosis, or myogenesis. Recovery of control and dysferlin-null muscles from small-strain injury involved inflammation and necrosis followed by myogenesis, all of which were more pronounced in the dysferlin-null muscles, which recovered more slowly. Both control and dysferlin-null muscles also retained 10-kDa dextran for 3 days after small-strain injury. We conclude that dysferlin-null myofibers can survive contraction-induced injury for at least 3 days but are subsequently eliminated by necrosis and inflammation. Myogenesis to replace lost fibers does not appear to be significantly compromised in dysferlin-null mice.

Cytokines and apoptosis in supraspinatus tendinopathy.

The role of inflammatory cells and their products in tendinopathy is not completely understood. Pro-inflammatory cytokines are upregulated after oxidative and other forms of stress. Based on observations that increased cytokine expression has been demonstrated in cyclically-loaded tendon cells we hypothesised that because of their role in oxidative stress and apoptosis, pro-inflammatory cytokines may be present in rodent and human models of tendinopathy. A rat supraspinatus tendinopathy model produced by running overuse was investigated at the genetic level by custom micro-arrays. Additionally, samples of torn supraspinatus tendon and matched intact subscapularis tendon were collected from patients undergoing arthroscopic shoulder surgery for rotator-cuff tears and control samples of subscapularis tendon from ten patients with normal rotator cuffs undergoing arthroscopic stabilisation of the shoulder were also obtained. These were all evaluated using semiquantitative reverse transcription polymerase chain-reaction and immunohistochemistry. We identified significant upregulation of pro-inflammatory cytokines and apoptotic genes in the rodent model (p = 0.005). We further confirmed significantly increased levels of cytokine and apoptotic genes in human supraspinatus and subscapularis tendon harvested from patients with rotator cuff tears (p = 0.0008). These findings suggest that pro-inflammatory cytokines may play a role in tendinopathy and may provide a target for preventing tendinopathies.

COL2A1 gene polymorphisms and susceptibility to osteoarthritis of the hand in Finnish women.

OBJECTIVES: To study the role of two COL2A1 single nucleotide polymorphisms (rs3737548 and rs2276455) and their haplotypes in individual susceptibility to osteoarthritis (OA) of the hand in Finnish women. METHODS: Bilateral hand radiographs of 543 Finnish female dentists and teachers aged 45-63 years were examined and classified for the presence of OA by using reference images. The COL2A1 genotypes were determined by PCR-based methods. Data regarding other risk factors were collected by questionnaire. The haplotypes were statistically reconstructed from the genotype data by the PHASE program. Associations between the genotypes/diplotypes and hand OA were studied by logistic regression. RESULTS: Allowing for age and occupation, the carriage of at least one COL2A1 intron 33 minor allele was associated with an increased risk of hand OA (odds ratio (OR) 1.58, 95% CI 1.05 to 2.36) and the number of affected joints. When stratified by occupation, the increased risk associated with the intron 33 minor allele carriage appeared to be mainly attributable to the dentists (OR 2.18, 95% CI 1.18 to 4.06). The 2-1 haplotype (exon 5B minor allele-intron 33 major allele) posed a significantly higher risk of hand OA (OR 3.21, 95% CI 1.08 to 9.55) compared with non-carriers. Moreover, an interaction was observed between intron 33 minor allele carriage and low task variation history in dental work (OR 2.87, 95% CI 1.05 to 7.89 for their joint effect). CONCLUSIONS: The results suggest that the studied COL2A1 gene polymorphisms may play a role in the aetiology of hand OA and that this effect may be enhanced by repetitive loading work tasks.

Coordinate regulation of IL-1beta and MMP-13 in rat tendons following subrupture fatigue damage.

Mechanical overloading is a major causative factor of tendinopathy; however, its underlying mechanisms are unclear. We hypothesized mechanical overloading would damage tendons and alter genes associated with tendinopathy in a load-dependent manner. To test this hypothesis, we fatigue loaded rat patellar tendons in vivo and measured expression of the matrix-degrading enzyme MMP-13 and the inflammatory cytokine IL-1beta. We also examined these responses in cultured tenocytes exposed to intermittent hydrostatic pressure in vitro. Additionally, we hypothesized load-induced changes in tenocyte MMP-13 expression would be dependent on expression of IL-1beta. In vivo fatigue loading at 1.7% strain caused overt microstructural damage and upregulated expression of MMP-13 and IL-1beta, while 0.6% strain produced only minor changes in matrix microstructure and downregulated expression of both MMP-13 and IL-1beta. Loading of cultured tenocytes at 2.5 and 7.5 MPa produced comparable changes in expression to those of in vivo tendon loading. Blocking IL-1beta expression with siRNA suppressed load-induced both MMP-13 mRNA expression and activity. The data suggest fatigue loading alters expression of MMP-13 and IL-1beta in tendons in vivo and tenocytes in vitro in a load-dependent manner. The data also suggest MMP-13 is regulated by both IL-1beta-dependent and IL-1beta-independent pathways.

Gene expression in rat supraspinatus tendon recovers from overuse with rest.

Rest is a common treatment for overuse injuries, but its effectiveness on gene expression has not been systematically evaluated under controlled experimental conditions. We asked whether genes regulated in the supraspinatus tendon as a result of overuse would return to normal levels after 2 or 4 weeks of rest. We used a rat model of tendon overuse that generates reproducible changes in the histology, geometry, gene expression, and mechanical properties consistent with an overuse injury. Animals were subjected to the overuse protocol for 2 or 4 weeks followed by either 2 or 4 weeks of rest. Microarray analysis was used to measure global changes in gene expression after the overuse plus rest protocol. Genes upregulated as a result of the overuse returned to near normal levels after rest in most animals. The biochemical composition of the tendon was similar to normal after the imposed rest period, except for slightly lower collagen content. These results suggest as little as 2 weeks of rest is often sufficient to recover from the molecular and biochemical effects of 2 and 4 weeks of overuse in this rat model.

Heat shock protein and apoptosis in supraspinatus tendinopathy.

Heat shock proteins (HSPs) are often upregulated following oxidative and other forms of stress. Based on reports of excessive apoptosis in torn supraspinatus tendon and mechanically loaded tendon cells, we hypothesized heat shock proteins may be present in rodent and human models of tendinopathy due to their central role in caspase dependent apoptotic cell signaling. We used a running rat supraspinatus tendinopathy overuse model with custom microarrays to investigate the process at a genetic level. Additionally torn supraspinatus tendon and matched intact subscapularis tendon samples were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from 10 patients undergoing arthroscopic stabilization surgery and evaluated using semiquantative RT-PCR and immunohistochemistry. We identified substantial upregulation of heat shock proteins and apoptotic genes in the rodent model. We further confirmed increased levels of heat shock protein and apoptotic regulatory genes in human supraspinatus and subscapularis tendon. This finding suggests heat shock proteins play a role in the cascade of stress-activated programmed cell death and degeneration in tendinopathy and may provide a novel target in preventing tendinopathies.

Rat supraspinatus tendon expresses cartilage markers with overuse.

The goals of this study were to investigate the response of the rat supraspinatus tendon to overuse at the molecular level using transcriptional profiling, and to identify potential markers of tendinopathy. Adult rats were subjected to an overuse protocol that consists of downhill running (10% grade) at 17 m/min for 1 h/day, 5 days/week, for a total of either 1, 2, or 4 weeks. Another group of rats served as nonrunning time 0 controls. Transcriptional profiling was performed on the supraspinatus and patellar tendons using an Affymetrix rat genome array. A gene was considered to be differentially expressed if the p value from an ANOVA test was less than 0.01 and the difference between runners and controls was at least twofold at any time point. The supraspinatus tendon had increased expression of well-known cartilage genes such as col2a1, aggrecan, and sox9. These genes were not regulated in the patellar tendon, an internal comparator. Few genes associated with inflammation, or angiogenesis, were differentially expressed, and no significant change in the regulation of matrix metalloproteinases was detected. The results of this study suggest that by expressing more cartilage genes, the tendon is converting toward a fibrocartilage phenotype as a result of the repetitive loading and repeated compression of the tendon as it passes through the acromial arch.

Pathophysiology of writer's cramp.

Writer's cramp is a task-specific focal hand dystonia. The abnormality of task specificity is a curious one and indicates that we need to learn more about the coupling of motor programs and their effectors. Writer's cramp appears to be triggered by spending much time writing by an individual with a fertile physiological substrate for producing the disorder. The fertile background, which is likely genetic, may be a decrease of inhibition, an increase of plasticity or an impairment in sensory function. Recent pathophysiological findings have implications for new therapies.

Gender and neurogenic variables in tendon biology and repetitive motion disorders.

The incidence of repetitive motion disorders is increasing. Numerous studies have indicated that the incidence in females exceeds that in males. Some of the evidence regarding gender related factors in tendon biology is discussed and new data related to the regulation of gene expression in an animal model of tendon overuse, the determination of sex hormone receptors in tendons, and the influence of pregnancy associated factors on gene expression in four different tendons is provided. Furthermore, because neurogenic mechanisms may contribute to inflammatory conditions, new evidence is provided that supports the concept that neurotransmitters can influence expression of genes that could participate in such inflammation. By increasing our understanding of the regulation of tendon cellular and molecular biology, new approaches to preventing disease development and treatment of existing disease may evolve.