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Atrophy and fat accumulation are debilitating aspects of muscle diseases and are rarely prevented. Using a vertical approach combining anatomic techniques with omics methodology in a tenotomy-induced sheep model of rotator cuff disease, we tested whether mitochondrial dysfunction is implicated in muscle wasting and perturbed lipid metabolism, speculating that both can be prevented by the stimulation of β-oxidation with l-carnitine. The infraspinatus muscle lost 22% of its volume over the first 6 weeks after tenotomy before the area-percentage of lipid increased from 8% to 18% at week 16. Atrophy was associated with the down-regulation of mitochondrial transcripts and protein and a slow-to-fast shift in muscle composition. Correspondingly, amino acid levels were increased 2 weeks after tendon release, when the levels of high-energy phosphates and glycerophospholipids were lowered. l-Carnitine administration (0.9 g/kg per day) prevented atrophy over the first 2 weeks, and mitigated alterations of glutamate, glycerophospholipids, and carnitine levels in released muscle, but did not prevent the level decrease in high-energy phosphates or protein constituents of mitochondrial respiration, promoting the accumulation of longer lipids with an increasing saturation. We conclude that the early phase of infraspinatus muscle degeneration after tendon release involves the elimination of oxidative characteristics associated with an aberrant accumulation of lipid species but is largely unrelated to the prevention of atrophy with oral l-carnitine administration.

Background:Muscle atrophy and fatty infiltration are limiting factors for successful rotator cuff (RC) repair. Quantitative data regarding these hallmarks of degenerative muscle changes after RC repair in humans are scarce. By utilizing a new application of the 6-point Dixon magnetic resonance imaging technology, 3-dimensional volume and fat fraction analysis of the whole RC muscle have become possible.Purpose:Quantitative analysis of atrophy and fatty infiltration of the supraspinatus muscle after healed and failed RC tendon-to-bone repair.Study Design:Cohort study; Level of evidence, 3.Methods:Muscle volume and fat fraction were measured preoperatively and at 3 and 12 months postoperatively in 19 failed and 21 healed arthroscopic supraspinatus tendon repairs, with full muscle volume segmentation and magnetic resonance Dixon sequences.Results:In both groups, the muscle volume initially decreased 3 months after RC repair by –3% in intact ( P = .140) and –10% in failed repair ( P = .004) but recovered between 3 and 12 months to 103% ( P = .274) in intact and 92% ( P = .040) in failed repairs when compared with the preoperative volume (difference of change between groups, preoperative to 12 month: P = .013). The supraspinatus muscle’s fat fraction did not significantly change after successful repair (6.5% preoperative, 6.6% after 3 months, and 6.7% after 12 months; all nonsignificant). There was, however, a significant increase from 7.8% to 10.8% at 3 months ( P = .014) and 11.4% at 12 months ( P = .020) after failed repair (difference between groups at 3- and 12-month follow-up: P = .018 and P = .001, respectively).Conclusion:After successful arthroscopic repair, RC tendon tear–induced fatty infiltration can be almost stopped, and muscle atrophy can even be slightly reversed. In case of a failed repair, however, these changes are further pronounced during the first 3 postoperative months but seem to stabilize thereafter.

INTRODUCTION We evaluated the contribution of denervation-related molecular processes to rotator cuff muscle degeneration after tendon release. METHODS We assessed the levels of myogenic (myogenin and myogenic differentiation factor [myoD]) and pro-adipogenic (peroxisome proliferator-activated receptor gamma) transcription factors; the denervation-associated proteins tenascin-C, laminin-2, and calcium/calmodulin dependent kinase II (CaMKII); and cellular alterations in sheep after infraspinatus tenotomy (TEN), suprascapular neurectomy (NEU), or both (TEN-NEU). RESULTS Extracellular ground substance increased at the expense of contractile tissue 16 weeks after surgery, correlating with CaMKII isoform levels. Sheep undergoing NEU and TEN-NEU had exaggerated infraspinatus atrophy and increased fast fibers compared with TEN sheep. The βMCaMKII isoform levels increased with tenotomy, and myoD levels tripled after denervation and were associated with slow fibers. DISCUSSION In sheep, denervation did not affect muscle-to-fat conversion after tenotomy of the infraspinatus. Furthermore, concurrent neurectomy mitigated the loss of fast fibers after tenotomy by inducing a fast-contractile phenotype. This article is protected by copyright. All rights reserved.

Background Borderline ovarian tumors (BOT) are uncommon but not rare epithelial ovarian neoplasms, intermediate between benign and malignant categories. Emerging knowledge supports the notion that subtypes of borderline ovarian tumors comprise distinct biologic, pathogenetic, and molecular entities, precluding a single unifying concept for BOT. Therefore, the identification of valuable markers for the diagnosis and classification of these tumors is in need. Among the molecular candidates, the Retinoblastoma (Rb) family members Rb/p105 and Rb2/p130 seem to play a pivotal role in ovarian cancer. In particular, Rb/p105, when in the unphosphorylated form, acts as a growth suppressor and plays a pivotal role in the negative control of the cell cycle and in tumor progression; whereas, the phosphorylated form (p-pRB) activates genic transcription and cellular proliferation. While Rb/p105 is ubiquitously confined to the nuclei of cycling and quiescent cells, Rb2/p130 activity is also regulated by intracellular localization. According to this premise, Rb family members could represent a novel marker in diagnosis and classification risk for patients with borderline ovarian tumors (BOT). Aims In this study, we evaluated the immunohistochemical expression and subcellular localization of proteins of the retinoblastoma (Rb) gene family: Rb/p105 and Rb2/p130 in 65 ovarian borderline tumors (26 serous, 19 sero-mucinous and 20 mucinous subtypes). Results Statistically significant differences were found in nuclear and cytoplasmic expression of Rb/p105 and Rb2/p130 according to different examined histotypes. In detail, the nuclear expression of Rb/p105 and Rb2/p130 was more frequently detected in serous (84.6%) than sero-mucinous (42.1%) and mucinous (50%) types. Conversely, the cytoplasmic expression of Rb2/p130 was not detected in serous tumors and frequently observed in mucinous subtypes (80%). Conclusions Our findings suggest that Rb proteins do not play a key role in the tumor progression of serous borderline tumors since they are always located in the nucleus and no cases showed a cytoplasmic localization. By contrast, the observed higher cytoplasmic expression of Rb2/p130 in mucinous BOTs (intestinal) types, is suggestive of Rb proteins involvement in the cancerogenesis pathway of mucinous ovarian tumors. Our results also suggest that mucinous BOTs of intestinal type, exhibiting low nuclear and high cytoplasmic levels of Rb2/p130 might potentially be considered a high risk category of malignant evolution. Further studies on larger series are needed in order to clarify how BOTs could be stratified in different prognostic groups according to their Rb proteins immunohistochemical profile.

Background:The effect of an additional neurological injury (suprascapular nerve traction injury) to a chronically retracted rotator cuff muscle is incompletely understood and warrants clarification.Purpose:To investigate the microscopic and macroscopic muscle degeneration patterns caused by tendon release and/or muscle denervation in a sheep rotator cuff model.Study Design:Controlled laboratory study.Methods:Infraspinatus muscle biopsy specimens (for histological analysis) were obtained from 18 Swiss alpine sheep before and 16 weeks after release of the infraspinatus tendon (tenotomy [T] group; n = 6), transection of the suprascapular nerve (neurectomy [N] group; n = 6), or tendon release plus nerve transection (tenotomy + neurectomy [T&N] group; n = 6). Magnetic resonance imaging (MRI) and computed tomography (CT) were used to assess retraction (CT), muscle density (CT), volume (MRI T2), and fat fraction (MRI Dixon). Stiffness of the infraspinatus was measured with a spring scale.Results:At 16 weeks postoperatively, the mean infraspinatus muscle volume had decreased significantly more after neurectomy (to 47% ± 7% of the original volume; P = .001) and tenotomy plus neurectomy (48% ± 13%; P = .005) than after tenotomy alone (78% ± 11%). Conversely, the mean amount of intramuscular fat (CT/MRI Dixon) was not significantly different in the 3 groups (T group: 50% ± 9%; N group: 40% ± 11%; T&N group: 46% ± 10%) after 16 weeks. The mean myotendinous retraction (CT) was not significantly different in the T and T&N groups (5.8 ± 1.0 cm and 6.4 ± 0.4 cm, respectively; P = .26). Stiffness was, however, most increased after additional neurectomy. In contrast to muscle changes after tendon release, denervation of the muscle led to a decrease in the pennation angle of lengthened muscle fibers, with a reduced mean cross-sectional area of pooled muscle fibers, a slow- to fast-type transformation, and an increase in the area percentage of hybrid fibers, leading to overall significantly greater atrophy of the corresponding muscle.Conclusion:Although it is unclear which experimental group (T or T&N) most accurately reflects the clinical scenario in a given case, these findings provide baseline information for clinical differentiation between muscle changes caused by denervation or rotator cuff tendon lesions.Clinical Relevance:The findings of this study help to understand how and to which extent a neurological lesion of the supplying suprascapular nerve could influence the pattern of anatomic-physiological muscular changes after rotator cuff tendon tears.

Skeletal muscle responds to endurance exercise with an improvement of biochemical pathways that support substrate supply and oxygen-dependent metabolism. This is reflected by enhanced expression of associated factors after exercise and is specifically modulated by tissue perfusion and oxygenation. We hypothesized that transcript expression of pro-angiogenic factors (VEGF, tenascin-C, Angpt1, Angpt1R) and oxygen metabolism (COX4I1, COX4I2, HIF-1α) in human muscle after an endurance stimulus depends on vasoconstriction, and would be modulated through angiotensin-converting enzyme inhibition by intake of lisinopril. Fourteen non-specifically trained, male Caucasians subjects, carried out a single bout of standardized one-legged bicycle exercise. Seven of the participants consumed lisinopril in the 3 days before exercise. Biopsies were collected pre- and 3 h post-exercise from the m. vastus lateralis. COX4I1 (P = 0.03), COX4I2 (P = 0.04) mRNA and HIF-1α (P = 0.05) mRNA and protein levels (P = 0.01) showed an exercise-induced increase in the group not consuming the ACE inhibitor. Conversely, there was a specific exercise-induced increase in VEGF transcript (P = 0.04) and protein levels (P = 0.03) and a trend for increased tenascin-c transcript levels (P = 0.09) for subjects consuming lisinopril. The observations indicate that exercise-induced expression of transcripts involved in angiogenesis and mitochondrial energy metabolism are to some extent regulated via a hypoxia-related ACE-dependent mechanism.

Introduction Gene polymorphisms are associated with athletic phenotypes relying on maximal or continued power production and affect the specialization of skeletal muscle composition with endurance or strength training of untrained subjects. We tested whether prominent polymorphisms in genes for angiotensin converting enzyme (ACE), tenascin-C (TNC), and actinin-3 (ACTN3) are associated with the differentiation of cellular hallmarks of muscle metabolism and contraction in high level athletes. Methods Muscle biopsies were collected from m. vastus lateralis of three distinct phenotypes; endurance athletes (n = 29), power athletes (n = 17), and untrained non-athletes (n = 63). Metabolism-, and contraction-related cellular parameters (such as capillary-to-fiber ratio, capillary length density, volume densities of mitochondria and intramyocellular lipid, fiber mean cross sectional area (MCSA) and volume densities of myofibrils) and the volume densities of sarcoplasma were analyzed by quantitative electron microscopy of the biopsies. Gene polymorphisms of ACE (I/D (insertion/deletion), rs1799752), TNC (A/T, rs2104772), and ACTN3 (C/T, rs1815739) were determined using high-resolution melting polymerase chain reaction (HRM-PCR). Genotype distribution was assessed using Chi$^{2}$ tests. Genotype and phenotype effects were analyzed by univariate or multivariate analysis of variance and post hoc test of Fisher. P-values below 0.05 were considered statistically significant. Results The athletes demonstrated the specialization of metabolism- and contraction-related cellular parameters. Differences in cellular parameters could be identified for genotypes rs1799752 and rs2104772, and localized post hoc when taking the interaction with the phenotype into account. Between endurance and power athletes these concerned effects on capillary length density for rs1799752 and rs2104772, fiber type distribution and volume densities of myofibrils (rs1799752), and MSCA (rs2104772). Endurance athletes carrying the I-allele of rs1799752 demonstrated 50%-higher volume densities of mitochondria and sarcoplasma, when power athletes that carried only the D-allele showed the highest fiber MCSAs and a lower percentage of slow type muscle fibers. Discussion ACE and tenascin-C gene polymorphisms are associated with differences in cellular aspects of muscle metabolism and contraction in specifically-trained high level athletes. Quantitative differences in muscle fiber type distribution and composition, and capillarization in knee extensor muscle explain, in part, identified associations of the insertion/deletion genotypes of ACE (rs1799752) with endurance- and power-type Sports.

We investigated molecular and cellular parameters which set metabolic and mechanical functioning of knee extensor muscles in the operated and contralateral control leg of 9 patients with a chronically insufficient anterior cruciate ligament (ACL; 26.6 ± 8.3 years, 8 males, 1 female) after open reconstructive surgery (week 0), after ambulant physiotherapy under cast immobilization (week 9), succeeding rehabilitation training (up to week 26), and subsequent voluntary physical activity (week 260). Clinical indices of knee function in the operated leg were improved at 52 weeks and remained at a comparable level at week 260. CSA of the quadriceps (-18%), MCSA of muscle fibers (-24%), and capillary-to-fiber ratio (-24%) in m. vastus lateralis from the ACL insufficient leg were lower at week 0 than reference values in the contralateral leg at week 260. Slow type fiber percentage (-35%) and mitochondrial volume density (-39%) were reduced in m. vastus lateralis from the operated leg at weeks 9 and 26. Composition alterations in the operated leg exceeded those in the contralateral leg and, with the exception of the volume density of subsarcolemmal mitochondria, returned to the reference levels at week 260. Leg-specific deterioration of metabolic characteristics in the vasti from the operated leg was reflected by the down-regulation of mitochondrial respiration complex I-III markers (-41–57%) at week 9. After rehabilitation training at week 26, the specific Y397 phosphorylation of focal adhesion kinase (FAK), which is a proxy for mechano-regulation, was elevated by 71% in the operated leg but not in the contralateral leg, which had performed strengthening type exercise during ambulant physiotherapy. Total FAK protein and Y397 phosphorylation levels were lowered in both legs at week 26 resulting in positive correlations with mitochondrial volume densities and mitochondrial protein levels. The findings emphasize that a loss of mechanical and metabolic characteristics in knee extensor muscle remains detectable years after untreated ACL rupture, which may be aggravated in the post-operative phase by the deterioration of slow-oxidative characteristics after reconstruction due to insufficient load-bearing muscle activity. The reestablishment of muscle composition subsequent to years of voluntary physical activity reinforces that slow-to-fast fiber transformation is reversible in humans., Frontiers in Physiology, 9, ISSN:1664-042X

Non-specific chronic low back pain (nsCLBP) is a multifactorial condition of unknown etiology and pathogenesis. Physical and genetic factors may influence the predisposition of individuals to CLBP, which in many instances share a musculoskeletal origin. A reduced pain level in low back pain patients that participate in exercise therapy highlights that disuse-related muscle deconditioning may predispose individuals to nsCLBP. In this context, musculoskeletal pain may be the consequence of capillary rarefaction in inactive muscle as this would lower local tissue drainage and washing out of toxic waste. Muscle activity is translated into an angio-adaptative process, which implicates angiogenic-gene expression and individual response differences due to heritable modifications of such genes (gene polymorphisms). The pathophysiologic mechanism underlying nsCLBP is still largely unaddressed. We hypothesize that capillary rarefaction due to a deconditioning of dorsal muscle groups exacerbates nsCLBP by increasing noxious sensation, reducing muscle strength and fatigue resistance by initiating a downward spiral of local deconditioning of back muscles which diminishes their load-bearing capacity. We address the idea that specific factors such as angiotensin-converting enzyme and Tenascin-C might play an important role in altering susceptibility to nsCLBP via their effects on microvascular perfusion and vascular remodeling of skeletal muscle, inflammation, and pain sensation. The genetic profile may help to explain the individual predisposition to nsCLBP, thus identifying subgroups of patients, which could benefit from ad hoc treatment types. Future therapeutic approaches aimed at relieving the pain associated with nsCLBP should be based on the verification of mechanistic processes of activity-induced angio-adaptation and muscle-perfusion.

The insertion/deletion polymorphism in the gene for the regulator of vascular tone, angiotensin-converting enzyme (ACE), is the prototype of a genetic influence on physical fitness and this involves an influence on capillary supply lines and dependent aerobic metabolism in skeletal muscle. The respective interaction of ACE-I/D genotype and training status on local metabolic and angiogenic reactions in exercised muscle is not known. Toward this end we characterized the metabolomic and angiogenic response in knee extensor muscle, m. vastus lateralis, in 18 untrained and 34 endurance-trained (physically active, V˙O2max > 50 mL min−1 kg−1) white British men to an exhaustive bout of one-legged cycling exercise. We hypothesized that training status and ACE-I/D genotype affect supply-related muscle characteristics of exercise performance in correspondence to ACE expression and angiotensin 2 levels. ACE-I/D genotype and training status developed an interaction effect on the cross-sectional area (CSA) of m. vastus lateralis and mean CSA of slow type fibers, which correlated with peak power output (r ≥ 0.44). Genotype × training interactions in muscle also resolved for exercise-induced alterations of 22 metabolites, 8 lipids, glycogen concentration (p = 0.016), ACE transcript levels (p = 0.037), and by trend for the pro-angiogenic factor tenascin-C post exercise (p = 0.064). Capillary density (p = 0.001), capillary-to-fiber ratio (p = 0.010), systolic blood pressure (p = 0.014), and exercise-induced alterations in the pro-angiogenic protein VEGF (p = 0.043) depended on the ACE-I/D genotype alone. Our observations indicate that variability in aerobic performance in the studied subjects was in part reflected by an ACE-I/D-genotype-modulated metabolic phenotype of a major locomotor muscle. Repeated endurance exercise appeared to override this genetic influence in skeletal muscle by altering the ACE-related metabolic response and molecular aspects of the angiogenic response to endurance exercise., Frontiers in Physiology, 8, ISSN:1664-042X

Inter-individual variation in running and cycling exercise economy (EE) remains unexplained although studied for more than a century. This study is the first to comprehensively evaluate the importance of biochemical, structural, physiological, anthropometric, and biomechanical influences on running and cycling EE within a single study. In 22 healthy males (VO2 max range 45.5 to 72.1 ml.min(-1) .kg(-1) ) no factor related to skeletal muscle structure (% slow twitch fibre content, number of capillaries per fibre), mitochondrial properties (volume density, oxidative capacity, or mitochondrial efficiency) or protein content (UCP3 and MFN2 expression) explained variation in cycling and running EE among subjects. In contrast, biomechanical variables related to vertical displacement correlated well with running EE, but were not significant when taking body weight into account. Thus, running EE and body weight were correlated (R(2) = 0.94; P < 0.001), but was lower for cycling EE (R(2) = 0.23; P < 0.023). To separate biomechanical determinants of running EE we contrasted individual running and cycling EE considering that during cycle ergometer exercise the biomechanical influence on EE would be small because of the fixed movement pattern. Differences in cycling and running exercise protocols, e.g., related to biomechanics, play however only a secondary role in determining EE. There was no evidence for an impact of structural or functional skeletal muscle variables on EE. Body weight was the main determinant of EE explaining 94% of variance in running EE, although more than 50% of the variability of cycling EE remains unexplained.

AIMS We assessed focal adhesion kinase (FAK) response to concentric (CON) versus eccentric (ECC) resistance training (RT) at two vastus lateralis (VL) sites, and the relationships between FAK, muscle protein synthesis (MPS), and morphological remodeling. METHODS Six young males trained both legs unilaterally 3 times/week for 8 weeks; one leg performed CON RT, the contralateral performed ECC RT. Muscle biopsies were collected after training from VL midbelly (MID) and distal (Distal) sites at 0, 4, 8 weeks. FAK content and activation were evaluated by immunoblotting. MPS was assessed by deuterium-oxide tracer; morphological adaptations were evaluated by ultrasound and DXA. RESULTS pY397-FAK 8 weeks levels were ~4-fold greater after ECC at the Distal site compared to CON (p

We hypothesised that load-sensitive expression of costameric proteins, which hold the sarcomere in place and position the mitochondria, contributes to the early adaptations of antigravity muscle to unloading and would depend on muscle fibre composition and chymotrypsin activity of the proteasome. Biopsies were obtained from vastus lateralis (VL) and soleus (SOL) muscles of eight men before and after 3 days of unilateral lower limb suspension (ULLS) and subjected to fibre typing and measures for costameric (FAK and FRNK), mitochondrial (NDUFA9, SDHA, UQCRC1, UCP3, and ATP5A1), and MHCI protein and RNA content. Mean cross-sectional area (MCSA) of types I and II muscle fibres in VL and type I fibres in SOL demonstrated a trend for a reduction after ULLS (0.05≤P<0.10). FAK phosphorylation at tyrosine 397 showed a 20% reduction in VL muscle (P=0.029). SOL muscle demonstrated a specific reduction in UCP3 content (-23%;P = 0.012). Muscle-specific effects of ULLS were identified for linear relationships between measured proteins, chymotrypsin activity and fibre MCSA. The molecular modifications in costamere turnover and energy homoeostasis identify that aspects of atrophy and fibre transformation are detectable at the protein level in weight-bearing muscles within 3 days of unloading.

PURPOSE We explored whether altered expression of factors tuning mitochondrial metabolism contributes to muscular adaptations with endurance training in the condition of lowered ambient oxygen concentration (hypoxia) and whether these adaptations relate to oxygen transfer as reflected by subsarcolemmal mitochondria and oxygen metabolism in muscle. METHODS Male volunteers completed 30 bicycle exercise sessions in normoxia or normobaric hypoxia (4,000 m above sea level) at 65% of the respective peak aerobic power output. Myoglobin content, basal oxygen consumption, and re-oxygenation rates upon reperfusion after 8 min of arterial occlusion were measured in vastus muscles by magnetic resonance spectroscopy. Biopsies from vastus lateralis muscle, collected pre and post a single exercise bout, and training, were assessed for levels of transcripts and proteins being associated with mitochondrial metabolism. RESULTS Hypoxia specifically lowered the training-induced expression of markers of respiratory complex II and IV (i.e. SDHA and isoform 1 of COX-4; COX4I1) and preserved fibre cross-sectional area. Concomitantly, trends (p < 0.10) were found for a hypoxia-specific reduction in the basal oxygen consumption rate, and improvements in oxygen repletion, and aerobic performance in hypoxia. Repeated exercise in hypoxia promoted the biogenesis of subsarcolemmal mitochondria and this was co-related to expression of isoform 2 of COX-4 with higher oxygen affinity after single exercise, de-oxygenation time and myoglobin content (r ≥ 0.75). Conversely, expression in COX4I1 with training correlated negatively with changes of subsarcolemmal mitochondria (r < -0.82). CONCLUSION Hypoxia-modulated adjustments of aerobic performance with repeated muscle work are reflected by expressional adaptations within the respiratory chain and modified muscle oxygen metabolism.