Fiber Atrophy Research Articles

CHANNELOPATHIES AND RELATED DISORDERS: EP.225 Autosomal dominant early onset vacuolar myopathy with granular material, without periodic paralysis, associated with c.1583C>A CACNA1S gene mutation

Dominant mutations in CACNA1S gene mainly causes hypokalemic periodic paralysis (PP)(hypoPP). We report a patient with progressive early onset myopathy associated with heterozygous CACNA1S mutation and aim to extend the description of phenotypical spectrum of CACNA1S-related muscle diseases. A 68-year-old man developed progressive proximal weakness from the age of 35 years. He had four siblings including an affected brother. His mother had PP and became wheelchair bound at 55 years. CK level was elevated (<1.5 upper normal value). Electroneuromyography showed axonal sensorimotor polyneuropathy and myogenic potentials. Thigh magnetic resonance imaging showed extensive muscle fatty replacement and muscle biopsy showed atrophic fibers with vacuoles containing granular material. Clinical exome identified the heterozygous c.1583G>A well-known pathogenic variant in CACNA1S. The patient's brother had PP from the age of 27. Neurological examination was normal at the age of 39. Muscle biopsy showed a vacuolar myopathy with granular material. No further medical information was available. The patient died at the age of 72 yrs. Molecular characterisation was not achieved. CACNA1S-HypoPP evolving to fixed myopathy in late adulthood is well described but isolated progressive myopathy was only exceptionally reported and never with an onset <50 years. We hereby report the earliest “myopathy onset” ever described, enlarging the phenotypical spectrum of CACNA1S-related myopathies and suggest considering it even in young adult-onset myopathies, especially when granular vacuoles are present.

NEUROMUSCULAR JUNCTION RELATED DISORDERS: EP.295 Brachio-cervical inflammatory myopathy associated with myasthenia gravis

Among idiopathic inflammatory myopathies (IIM), brachio-cervical inflammatory myopathy (BCIM) constitutes a rare entity basically characterized by progressive weakness in the proximal regions of the arms and neck, the frequent association with other autoimmune diseases, and peculiar histopathological features at muscle biopsy. A 57-year-old woman presented with a 1-yr history of weight loss (-10kg), cervicalgia, bulbar signs and weakness in upper limbs. Clinical examination showed drop head with cervical muscle atrophy, severe proximal muscle weakness in upper limbs and nasal voice with swallowing disorders. No sign of scleroderma. Biological assessment disclosed increased CK serum levels and anti-AChR and anti-Scl autoantibodies. EMG showed myogenic changes in upper limbs but no decrement. Deltoid muscle biopsy revealed type-II fiber atrophy, randomly distributed active myonecrosis, and nodular inflammatory infiltrates, mainly perivascular. Immunohistochemical techniques showed ubiquitous myofiber expression of major histocompatibility complex (MHC)-class I and multifocal MHC-II-expressing myofibers. B-cells (CD20) were majority in infiltrates. C5b-9 immunostaining showed deposition in endomysium and capillaries walls. Significant clinical improvement was observed under medical treatment (anticholinesterase, corticosteroid, immunoglobulin and rituximab), ventilatory and nutritional support. Present case enlightened the specificities of BCIM, including (i) the remarkable distribution of muscle deficit, (ii) the frequent association with myasthenia gravis, and (iii) the highly recognizable histopathological pattern with active myopathy, C5b-9 staining of endomysium, focal perivascular and perimysial inflammation often with a prominent B cell component. Treatment is usually modeled on that of IIM, but considering histopathological features and the association with myasthenia, BCIM could constitute a privileged indication for anti-CD20 biotherapy.

Sex-dependent effects of amyloid precursor-like protein 2 in the SOD1-G37R transgenic mouse model of MND

Motor neurone disease (MND) is a neurodegenerative disorder characterised by progressive destruction of motor neurons, muscle paralysis and death. The amyloid precursor protein (APP) is highly expressed in the central nervous system and has been shown to modulate disease outcomes in MND. APP is part of a gene family that includes the amyloid precursor-like protein 1 (APLP1) and 2 (APLP2) genes. In the present study, we investigated the role of APLP2 in MND through the examination of human spinal cord tissue and by crossing APLP2 knockout mice with the superoxide dismutase 1 (SOD1-G37R) transgenic mouse model of MND. We found the expression of APLP2 is elevated in the spinal cord from human cases of MND and that this feature of the human disease is reproduced in SOD1-G37R mice at the End-stage of their MND-like phenotype progression. APLP2 deletion in SOD1-G37R mice significantly delayed disease progression and increased the survival of female SOD1-G37R mice. Molecular and biochemical analysis showed female SOD1-G37R:APLP2−/− mice displayed improved innervation of the neuromuscular junction, ameliorated atrophy of muscle fibres with increased APP protein expression levels in the gastrocnemius muscle. These results indicate a sex-dependent role for APLP2 in mutant SOD1-mediated MND and further support the APP family as a potential target for further investigation into the cause and regulation of MND.

Long-term wheel-running prevents reduction of grip strength in type 2 diabetic rats.

Diabetic skeletal muscles show reduced contractile force and increased fatigability. Hands are a target for several diabetes‐induced complications. Therefore, reduced handgrip strength often occurs as a consequence of diabetes. The aim of this study was to examine whether long‐term exercise can prevent reduction of grip strength in type 2 diabetes mellitus (T2DM) model OLETF rats, and to explore the mechanisms underlying diabetes‐induced grip strength reduction. Ten 5‐week‐old OLETF rats were used as experimental animals, and five non‐diabetic LETO rats as controls of OLETF rats. Half OLETF rats performed daily voluntary wheel‐running for 17 months (OLETF + EXE), and the rest of OLETF and LETO rats were sedentary. Grip strength was higher in OLETF + EXE and LETO groups than in OLETF group. OLETF group with hyperglycemia showed an increase in HbA1c, serum TNF‐α, and muscle SERCA activity, but a decrease in circulating insulin. Each fiber area, total fiber area, and % total fiber area in type IIb fibers of extensor digitorum longus muscles were larger in OLETF + EXE and LETO groups than in OLETF group. There was a positive correlation between grip strength and the above three parameters concerning type IIb fiber area. Therefore, type IIb fiber atrophy may be the major direct cause of grip strength reduction in OLETF group, although there seems multiple etiological mechanisms. Long‐term wheel‐running may have blocked the diabetes‐induced reduction of grip strength by preventing type IIb fiber atrophy. Regular exercise may be a potent modality for preventing not only the progression of diabetes but muscle dysfunction in T2DM patients.

Ultrasonographic examination of skeletal muscles in traumatic ischemia (experimental study)

Post-traumatic muscles ischemia of the extremities is the result of severe trauma and leads to permanent dysfunction. The tactics of management and treatment of patients are decisive at all stages, so the latest methods of treatment using cellular technologies are being developed to prevent the development of complications and improve long-term results. Aim: to determine the correlation between histological and ultrasonographic parameters (qualitative and quantitative) in the muscles after traumatic ischemia and to compare the results. Material and methods. The study was performed on rabbits breed Chinchilla. The animals were placed on a tourniquet on the lower limb from the middle third of the thigh to the ankle joint, after 6 hours it was removed. Developed software to determine the texture of the sonographic image (δ-entropy) allowed to assess the qualitative and quantitative changes in the damaged muscles. Correlation analysis was performed between sonographic and histological changes in the muscles. Results. The relative amount of structurally altered tissue was morphometrically estimated and compared with entropy. A high correlation between muscle damage and vertical δ-entropy has been found, indicating that high-probability sonography can detect areas of necrosis and, to a lesser extent, fibrosis in the development of ischemic limb contracture. Conclusions. The obtained high positive correlation between the histological index of muscle tissue damage (especially atrophy of muscle fibers and fibrosis) and the results of sonographic data processing, namely vertical δ-entropy, have clinical importance in terms of establishing the severity of necrotic lesions segments of the extremities and the prediction of fibrous changes, formation of limb contracture. Key words: traumatic ischemia, necrosis, histological changes in the muscles.

Expanding the clinical and genetic spectrum of pathogenic variants in STIM1.

Stromal interaction molecule 1 (STIM1) is a reticular Ca2+ sensor composed of a luminal and a cytosolic domain. Autosomal dominant mutations in STIM1 cause tubular aggregate myopathy and Stormorken syndrome or its variant York platelet syndrome. In this study we aimed to expand the features related to new variants in STIM1. We performed a cross-sectional study of individuals harboring monoallelic STIM1 variants recruited at five tertiary centers involved in a study of inherited myopathies analyzed with a multigene-targeted panel. We identified seven individuals (age range, 26-57 years) harboring variants in STIM1, including five novel changes: three located in the EF-hand domain, one in the sterile α motif (SAM) domain, and one in the cytoplasmatic region of the protein. Functional evaluation of the pathogenic variants using a heterologous expression system and measuring store-operated calcium entry demonstrated their causative role and suggested a link of new variants with the clinical phenotype. Muscle contractures, found in three individuals, showed variability in body distribution and in the number of joints involved. Three patients showed cardiac and respiratory involvement. Short stature, hyposplenism, sensorineural hearing loss, hypothyroidism, and Gilbert syndrome were variably observed among the patients. Laboratory tests revealed hyperCKemia in six patients, thrombocytopenia in two patients, and hypocalcemia in one patient. Muscle biopsy showed the presence of tubular aggregates in three patients, type I fiber atrophy in one patient, and nonspecific myopathic changes in two patients. Our clinical, histological, and molecular data expand the genetic and clinical spectrum of STIM1-related diseases.

Histopathological, Ultrastructural, and Immunohistochemical Findings in Radial Longitudinal Deficiency: A Prospective, Observational Study

To report the histopathological, electron microscopic, and immunohistochemical findings of tissue samples obtained from patients with radial longitudinal deficiency (RLD) and investigate the contribution of abnormality in soft tissues as a secondary driver of deformity in RLD. Specimens from radial-sided muscles and tendons were obtained at the time of surgery (either radialization or centralization) from 14 patients with 16 limbs affected with Bayne type 3 and type 4 RLD. The specimens were evaluated using light microscopy, electron microscopy, and immunohistochemical examination. Among the 16 frozen muscle specimens, 6 (37%) showed normal muscle, while 10 (63%) showed the presence of atrophic fibers. The 6 cases with normal muscle showed no abnormality in fiber type distribution. Six patients showed predominance of type 1 muscle fibers. None of the specimens had myofibroblasts; 4 of 16 specimens had mast cells, and 9 of 16 specimens showed the presence of platelet derived growth factor-positive cells. Features of myofibroblasts (the presence of basal lamina, intercellular junctions, or pinocytic vesicles) were not identified in any specimen on electron microscopy. The histopathological, electron microscopic, and immunohistochemical findings, in particular the absence of myofibroblasts, in tissue samples obtained from patients with RLD, do not support the assumption of abnormality in soft tissues as a secondary driver of deformity in RLD. This study provides a preliminary insight into a possible role of soft tissues in the development of the deformity in RLD.

Effects of long-term immobilisation on endomysium of the soleus muscle in humans.

What is the central question of this study? While muscle fibre atrophy in response to immobilisation has been extensively examined, intramuscular connective tissue, particularly endomysium, has been largely neglected: does endomysium content of the soleus muscle increase during bed rest? What is the main finding and its importance? Absolute endomysium content did not change, and previous studies reporting an increase are explicable by muscle fibre atrophy. It must be expected that even a relative connective tissue accumulation will lead to an increase in muscle stiffness. Muscle fibres atrophy during conditions of disuse. Whilst animal data suggest an increase in endomysium content with disuse, that information is not available for humans. We hypothesised that endomysium content increases during immobilisation. To test this hypothesis, biopsy samples of the soleus muscle obtained from 21 volunteers who underwent 60days of bed rest were analysed using immunofluorescence-labelled laminin γ-1 to delineate individual muscle fibres as well as the endomysium space. The endomysium-to-fibre-area ratio (EFAr, as a percentage) was assessed as a measure related to stiffness, and the endomysium-to-fibre-number ratio (EFNr) was calculated to determine whether any increase in EFAr was absolute, or could be attributed to muscle fibre shrinkage. As expected, we found muscle fibre atrophy (P=0.0031) that amounted to shrinkage by 16.6% (SD 28.2%) on day 55 of bed rest. ENAr increased on day 55 of bed rest (P<0.001). However, when analysing EFNr, no effect of bed rest was found (P=0.62). These results demonstrate that an increase in EFAr is likely to be a direct effect of muscle fibre atrophy. Based on the assumption that the total number of muscle fibres remains unchanged during 55days of bed rest, this implies that the absolute amount of connective tissue in the soleus muscle remained unchanged. The increased relative endomysium content, however, could be functionally related to an increase in muscle stiffness.

TMEM182 interacts with integrin beta 1 and regulates myoblast differentiation and muscle regeneration.

BackgroundTransmembrane proteins are vital for intercellular signalling and play important roles in the control of cell fate. However, their physiological functions and mechanisms of action in myogenesis and muscle disorders remain largely unexplored. It has been found that transmembrane protein 182 (TMEM182) is dramatically up‐regulated during myogenesis, but its detailed functions remain unclear. This study aimed to analyse the function of TMEM182 during myogenesis and muscle regeneration.MethodsRNA sequencing, quantitative real‐time polymerase chain reaction, and immunofluorescence approaches were used to analyse TMEM182 expression during myoblast differentiation. A dual‐luciferase reporter assay was used to identify the promoter region of the TMEM182 gene, and a chromatin immunoprecipitation assay was used to investigate the regulation TMEM182 transcription by MyoD. We used chickens and TMEM182‐knockout mice as in vivo models to examine the function of TMEM182 in muscle growth and muscle regeneration. Chickens and mouse primary myoblasts were used to extend the findings to in vitro effects on myoblast differentiation and fusion. Co‐immunoprecipitation and mass spectrometry were used to identify the interaction between TMEM182 and integrin beta 1 (ITGB1). The molecular mechanism by which TMEM182 regulates myogenesis and muscle regeneration was examined by Transwell migration, cell wound healing, adhesion, glutathione‐S‐transferse pull down, protein purification, and RNA immunoprecipitation assays.Results TMEM182 was specifically expressed in skeletal muscle and adipose tissue and was regulated at the transcriptional level by the myogenic regulatory factor MyoD1. Functionally, TMEM182 inhibited myoblast differentiation and fusion. The in vivo studies indicated that TMEM182 induced muscle fibre atrophy and delayed muscle regeneration. TMEM182 knockout in mice led to significant increases in body weight, muscle mass, muscle fibre number, and muscle fibre diameter. Skeletal muscle regeneration was accelerated in TMEM182‐knockout mice. Furthermore, we revealed that the inhibitory roles of TMEM182 in skeletal muscle depend on ITGB1, an essential membrane receptor involved in cell adhesion and muscle formation. TMEM182 directly interacted with ITGB1, and this interaction required an extracellular hybrid domain of ITGB1 (aa 387–470) and a conserved region (aa 52–62) within the large extracellular loop of TMEM182. Mechanistically, TMEM182 modulated ITGB1 activation by coordinating the association between ITGB1 and laminin and regulating the intracellular signalling of ITGB1. Myogenic deletion of TMEM182 increased the binding activity of ITGB1 to laminin and induced the activation of the FAK‐ERK and FAK‐Akt signalling axes during myogenesis.ConclusionsOur data reveal that TMEM182 is a novel negative regulator of myogenic differentiation and muscle regeneration.

Sensitivity of subcellular components of neuromuscular junctions to decreased neuromuscular activity

Muscle unloading imparts subtotal disuse on the neuromuscular system resulting in reduced performance capacity. This loss of function, at least in part, can be attributed to disruptions at the neuromuscular junction (NMJ). However, research has failed to document morphological remodeling of the NMJ with short term muscle unloading. Here, rather than quantifying cellular components of the NMJ, we examined subcellular active zone responses to 2 weeks of unloading in male Wistar rats. It was revealed that in the plantaris, but not the soleus muscles, unloading elicited significant (P ≤ 0.05) decrements in active zone staining as measured by Bassoon, and calcium channel expression. It was also discovered that unloading decreased the area of calcium channels staining relative to active zone areas of staining suggesting potential interference in the ability of calcium influx to trigger the release of vesicles docked at the active zone. Post-synaptic adaptations of the motor endplate were not evident. This presynaptic subcellular size reduction was not associated with atrophy of the underlying plantaris muscle fibers, although atrophy of the weight-bearing soleus fibers, where no subcellular remodeling was evident, was noted. These results suggest that the active zone is highly sensitive to alterations in neuromuscular activity, and that morphological adaptation of excitatory and contractile components of the NMJ can occur independently of each other.

Sarcopenia versus cancer cachexia: the muscle wasting continuum in healthy and diseased aging.

Muscle wasting is one of the major health problems in older adults and is traditionally associated to sarcopenia. Nonetheless, muscle loss may also occur in older adults in the presence of cancer, and in this case, it is associated to cancer cachexia. The clinical management of these conditions is a challenge due to, at least in part, the difficulties in their differential diagnosis. Thus, efforts have been made to better comprehend the pathogenesis of sarcopenia and cancer cachexia, envisioning the improvement of their clinical discrimination and treatment. To add insights on this topic, this review discusses the current knowledge on key molecular players underlying sarcopenia and cancer cachexia in a comparative perspective. Data retrieved from this analysis highlight that while sarcopenia is characterized by the atrophy of fast-twitch muscle fibers, in cancer cachexia an increase inthe proportion of fast-twitch fibers appears to happen. The molecular drivers for these specificmuscleremodeling patterns are still unknown; however, among the predominant contributors to sarcopenia is the age-induced neuromuscular denervation, and in cancer cachexia, the muscle disuse experienced by cancer patients seems to play an important role. Moreover, inflammation appears to be more severe in cancer cachexia. Impairment of nutrition-related mediators may also contribute to sarcopenia and cancer cachexia, being distinctly modulated in each condition.

Macular ganglion cell complex injury in different stages of anterior ischemic optic neuropathy.

BACKGROUNDAnterior ischemic optic neuropathy (AION) is a group of ophthalmic diseases in which the optic nerve is injured causing blindness. However, the pathogenesis, clinical manifestations, and clinical treatments of AION are yet elusive. Only a few related experimental or clinical reports are available on the disease. In this study, spectral domain optical coherence tomography (SD-OCT) was used to examine the morphology of thickness swelling and atrophic changes of macular ganglion cell complex (mGCC) in the different stages of AION that were then compared with the visual fields. Thus, the clinical value of mGCC examination was alleged to be similar to that of the visual field.AIMTo explore the mGCC injury at different stages in AION and the clinical significance.METHODSCases with AION were analyzed in a retrospective study. SD-OCT was used to analyze the correlation between mGCC and peripapillary retinal nerve fiber layer thicknesses at different stages of AION and the changes in the corresponding stages of visual fields.RESULTSA total of 21 cases (28 eyes) presented AION. The onset time of AION was defined as early stage (within 3 wk of onset), middle stage (from 3 wk to 2 mo), and late stage (disease span > 2 mo). In the early stage, the mGCC thickness of SD-OCT was within the normal high limit, and the perioptic nerve fibers thickness was more than the normal. The changes in the visual field in early stage were not consistent with the swelling changes in mGCC and peri-disc nerve fibers. In addition, atrophy and thinning appeared in mGCC, and the perioptic nerve fibers were swollen. However, the thickness was lower in the middle period than that in the early stage. The change in visual field was consistent with that of mGCC in this period. In the late stage, mGCC shrank and thinned, and the thickness of the nerve fibers around the optic disc in the corresponding region shrank and thinned.CONCLUSIONThe changes in mGCC thickness in patients with AION showed early, middle, and late stages of development by SD-OCT. Although the early stage visual field changes of AION were not consistent with the swelling changes of mGCC, the horizontal delimitation or annular atrophy of mGCC was consistent with that in the middle and late stage of the disease. The atrophy of peripheral nerve fibers was later than that of the mGCC atrophy.

Shear Wave Elastography, a New Tool for Diaphragmatic Qualitative Assessment: A Translational Study.

Rationale: Prolonged mechanical ventilation is often associated with either a decrease (known atrophy) or an increase (supposed injury) in diaphragmatic thickness. Shear wave elastography is a noninvasive technique that measures shear modulus, a surrogate of tissue stiffness and mechanical properties. Objectives: To describe changes in shear modulus (SM) during the ICU stay and the relationship with alterations in muscle thickness. To perform a comprehensive ultrasound-based characterization of histological and force production changes occurring in the diaphragm. Methods: Translational study using critically ill patients and mechanically ventilated piglets. Serial ultrasound examination of the diaphragm collecting thickness and SM was performed in both patients and piglets. Transdiaphragmatic pressure and diaphragmatic biopsies were collected in piglets. Measurements and Main Results: We enrolled 102 patients, 88 of whom were invasively mechanically ventilated. At baseline, SM was 14.3 ± 4.3 kPa and diaphragm end-expiratory thickness was 2.0 ± 0.5 mm. Decrease or increase by more than 10% from baseline was reported in 86% of the patients for thickness and in 92% of the patients for SM. An increase in diaphragmatic thickness during the stay was associated with a decrease in SM (β = -9.34 ± 4.41; P = 0.03) after multivariable analysis. In the piglet sample, a decrease in SM over 3 days of mechanical ventilation was associated with loss of force production, slow and fast fiber atrophy, and increased lipid droplets accumulation. Conclusions: Increases in diaphragm thickness during critical illness is associated with decreased tissue stiffness as demonstrated by shear wave ultrasound elastography, consistent with the development of muscle injury and weakness. Clinical trial registered with www.clinicaltrials.gov (NCT03550222).

Hypothyroidism-associated rhabdomyolysis

Background: Hypothyroidism alone or in combination with other factors has the risk of triggering rhabdomyolysis. In this article, we aim to describe hypothyroidism-associated rhabdomyolysis and its outcomes. Methods: We reported a new case of hypothyroidism-associated rhabdomyolysis and reviewed similar reported cases from the literature for in-depth knowledge. Results: Eighty-one cases, including the one reported in this article, met the inclusion criteria for this review. The mean age of the patients was 45.6 ± 15.8 years. Out of these, 57 (70.4%) patients were males and 24 (29.6%) were females. The precipitating factor was absent in 45 (55.6%) cases. A total of 8 (9.9%) cases had chronic renal failure at time of presentation, while 25 (30.9%) had hypertension, 11 (13.6%) had diabetes mellitus, and 32 (39.5%) had dyslipidemia. The median creatine kinase (CK) level was 5885 U/L (Interquartile range: 3280.5–11550.5 U/L). Electromyography was performed in 12 patients with 10 (12.3%) cases showing myopathic changes including polyphasic potential and fiber necrosis. Muscle biopsy was performed in 7 (8.4%) cases, with Type II fiber atrophy observed in 4 (4.9%) biopsies. Sixty-two cases developed acute kidney injury, of which 14 (17.3%) required hemodialysis. All patients were treated with levothyroxine and most patients (67, 82.7%) were treated by hydration. All the reported patients made good recovery. A statistically nonsignificant correlation was found between CK and thyroid-stimulating hormone (r = 0.218; P = 0.052). Conclusions: Rhabdomyolysis is a recognized complication of hypothyroidism even in the absence of additional risk factors. Clinicians should be aware of the impact of rhabdomyolysis and hypothyroidism on renal function and promptly initiate hormone replacement therapy and vigorous hydration to preserve the renal function.

Skeletal muscle cellular contractile dysfunction after anterior cruciate ligament reconstruction contributes to quadriceps weakness at 6-month follow-up.

Muscle dysfunction following anterior cruciate ligament reconstruction (ACLR) may evolve from alterations in muscle contractility at the myofilament protein level. Using a prospective, within-subject case-control design, we evaluated cellular-level contractility, cross-sectional area (CSA), and myosin heavy chain (MHC) isoform expression on single muscle fibers 3 weeks post ACLR, and evaluated their relationship to whole muscle strength and patient-oriented outcomes 6 months post operation. Biopsies of the vastus lateralis were performed 3 weeks post ACLR in 11 subjects (5 females, mean age ± SD = 24.7 ± 6.5 years, height = 172.7 ± 8.2 cm, mass = 75.7 ± 12.5 kg) following first-time ACL rupture and whole muscle strength and self-reported pain, function, and quality of life assessed 6 months post ACLR. At 3 weeks post ACLR, force production was reduced (p < 0.01) in MHC I (-36%) and IIA (-48%) fibers compared with the non-injured leg. When force production was expressed relative to CSA to account for fiber atrophy, reductions remained in MHC IIA fibers (-40%; p < 0.001), but MHC I fibers showed only a trend toward being lower (-13%; p = 0.09). Finally, skeletal muscle fiber functional deficits at 3 weeks post ACLR were associated with whole muscle weakness and less favorable patient-reported outcomes at 6-month follow-up. Thus, ACLR promotes early cellular contractile dysfunction that may contribute to decreased whole muscle strength and patient function, and increased patient-reported symptoms, at 6-month follow-up.

Hydrogen sulfide donor protects against mechanical ventilation-induced atrophy and contractile dysfunction in the rat diaphragm.

Mechanical ventilation (MV) is a clinical tool providing adequate alveolar ventilation in patients that require respiratory support. Although a life‐saving intervention for critically ill patients, prolonged MV results in the rapid development of inspiratory muscle weakness due to both diaphragmatic atrophy and contractile dysfunction; collectively known as “ventilator‐induced diaphragm dysfunction” (VIDD). VIDD is a severe clinical problem because diaphragmatic weakness is a risk factor for difficulties in weaning patients from MV. Currently, no standard treatment to prevent VIDD exists. Nonetheless, growing evidence reveals that hydrogen sulfide (H2S) possesses cytoprotective properties capable of protecting skeletal muscles against several hallmarks of VIDD, including oxidative damage, accelerated proteolysis, and mitochondrial damage. Therefore, we used an established animal model of MV to test the hypothesis that treatment with sodium sulfide (H2S donor) will defend against VIDD. Our results confirm that sodium sulfide was sufficient to protect the diaphragm against both MV‐induced fiber atrophy and contractile dysfunction. H2S prevents MV‐induced damage to diaphragmatic mitochondria as evidenced by protection against mitochondrial uncoupling. Moreover, treatment with sodium sulfide prevented the MV‐induced activation of the proteases, calpain, and caspase‐3 in the diaphragm. Taken together, these results support the hypothesis that treatment with a H2S donor protects the diaphragm against VIDD. These outcomes provide the first evidence that H2S has therapeutic potential to protect against MV‐induced diaphragm weakness and to reduce difficulties in weaning patients from the ventilator. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Mechanical ventilation (MV) results in diaphragm atrophy and contractile dysfunction, known as ventilator‐induced diaphragm dysfunction (VIDD). VIDD is important because diaphragm weakness is a risk factor for problems in weaning patients from MV. Currently, no accepted treatment exists to protect against VIDD. Growing evidence reveals that hydrogen sulfide (H2S) donors protect skeletal muscle against ischemia‐reperfusion‐induced injury. Nonetheless, it is unknown if treatment with a H2S donor can protect against VIDD. WHAT QUESTION DID THIS STUDY ADDRESS? Can treatment with an H2S donor protect against VIDD? WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? This study provides the first evidence that treatment with a H2S donor protects against VIDD. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? These new findings provide the basis for further exploration of H2S donors as a therapy to prevent VIDD and reduce the risk of problems in weaning patients from MV.

FGF23, a novel muscle biomarker detected in the early stages of ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive muscle weakness. Skeletal muscle is a prime source for biomarker discovery since it is one of the earliest sites to manifest disease pathology. From a prior RNA sequencing project, we identified FGF23 as a potential muscle biomarker in ALS. Here, we validate this finding with a large collection of ALS muscle samples and found a 13-fold increase over normal controls. FGF23 was also increased in the SOD1G93A mouse, beginning at a very early stage and well before the onset of clinical symptoms. FGF23 levels progressively increased through end-stage in the mouse. Immunohistochemistry of ALS muscle showed prominent FGF23 immunoreactivity in the endomysial connective tissue and along the muscle membrane and was significantly higher around grouped atrophic fibers compared to non-atrophic fibers. ELISA of plasma samples from the SOD1G93A mouse showed an increase in FGF23 at end-stage whereas no increase was detected in a large cohort of ALS patients. In conclusion, FGF23 is a novel muscle biomarker in ALS and joins a molecular signature that emerges in very early preclinical stages. The early appearance of FGF23 and its progressive increase with disease progression offers a new direction for exploring the molecular basis and response to the underlying pathology of ALS.

Temporal changes in the markers of ribosome biogenesis in rat soleus muscle under simulated microgravity

Skeletal muscle inactivity due to real or simulated microgravity results in a significant atrophy of muscle fibers. One of the key causes of disuse atrophy is a decrease in the rate of muscle protein synthesis (MPS). MPS is dependent not only on translational efficiency but also on the number of ribosomes available for translation (translational capacity), however a time course for biomarkers of ribosome biogenesis during muscle disuse remains understudied. The aim of the study was to analyze temporal changes in the regulators/markers of ribosome biogenesis in rat soleus muscle during the course of disuse. Rats were subjected to hindlimb unloading (HU) for 1, 3, 7 and 14 days. 18S + 28S rRNA content was assessed by gel-electrophoresis; expression levels of 45S pre-rRNA and c-Myc were determined by RT-PCR and the rate of MPS was measured using SUnSET method. The content of 18S + 28S rRNA and total RNA as well as 45S pre-rRNA expression significantly decreased after 1-day HU and remained diminished for the entire unloading period. c-Myc mRNA expression was downregulated following 1, 3 and 7 days of HU, but did not significantly differ from control after 14-day HU. MPS showed a progressive decrease from the 3rd to 14th day of HU. Thus, a decrease in MPS in rat soleus during 3-, 7- and 14-day HU was accompanied by downregulation of the key markers of translational capacity.

Alterations in Autophagy and Mammalian Target of Rapamycin (mTOR) Pathways Mediate Sarcopenia in Patients with Cirrhosis

The pathophysiology of sarcopenia in cirrhosis is poorly understood. We aimed to evaluate the histological alterations in the muscle tissue of patients with cirrhosis and sarcopenia, and identify the regulators of muscle homeostasis. Computed tomography images at third lumbar vertebral level were used to assess skeletal muscle index (SMI) in 180 patients. Sarcopenia was diagnosed based on the SMI cut-offs from a population of similar ethnicity. Muscle biopsy was obtained from the vastus lateralis in 10 sarcopenic patients with cirrhosis, and the external oblique in five controls (voluntary kidney donors during nephrectomy). Histological changes were assessed by hematoxylin and eosin staining and immunohistochemistry for phospho-FOXO3, phospho-AKT, phospho-mTOR, and apoptosis markers (annexin V and caspase 3). The messenger ribonucleic acid (mRNA) expressions for MSTN, FoxO3, markers of ubiquitin-proteasome pathway (FBXO32, TRIM63), and markers of autophagy (Beclin-1 and LC3) were also quantified. The prevalence of sarcopenia was 14.4%. Muscle histology in sarcopenics showed atrophic angulated fibers (P= 0.002) compared to controls. Immunohistochemistry showed a significant loss of expression of phospho-mTOR (P= 0.026) and an unaltered phospho-AKT (P= 0.089) in sarcopenic patients. There were no differences in the immunostaining for annexin-V, caspase-3, and phospho-FoxO3 between the two groups. The mRNA expressions of MSTN and Beclin-1 were higher in sarcopenics (P= 0.04 and P= 0.04, respectively). The two groups did not differ in the mRNA levels for TRIM63, FBXO32, and LC3. Significant muscle atrophy, increase in autophagy, MSTN gene expression, and an impaired mTOR signaling were seen in patients with sarcopenia and cirrhosis.

Muscle-specific deletion of the vitamin D receptor in mice is associated with diaphragm muscle weakness.

Diseases or conditions where diaphragm muscle (DIAm) function is impaired, including chronic obstructive pulmonary disease, cachexia, asthma, and aging, are associated with an increased risk of pulmonary symptoms, longer duration of hospitalizations, and increasing requirements for mechanical ventilation. Vitamin D deficiency is associated with proximal muscle weakness that resolves following therapy with vitamin D3. Skeletal muscle expresses the vitamin D receptor (VDR), which responds to the active form of vitamin D, 1,25-dihydroxyvitamin D3 by altering gene expression in target cells. In knockout mice without skeletal muscle VDRs, there is marked atrophy of muscle fibers and a change in skeletal muscle biochemistry. We used a tamoxifen-inducible skeletal muscle Cre recombinase in Vdrfl/fl mice (Vdrfl/fl actin.iCre+) to assess the role of muscle-specific VDR signaling on DIAm-specific force, fatigability, and fiber type-dependent morphology. Vdrfl/fl actin.iCre+ mice treated with vehicle and Vdrfl/fl mice treated with tamoxifen served as controls. Seven days following the final treatment, mice were euthanized, the DIAm was removed, and isometric force and fatigue were assessed in DIAm strips using direct muscle stimulation. The proportion and cross-sectional areas of DIAm fiber types were evaluated by immunolabeling with myosin heavy chain antibodies differentiating type I, IIa and IIx, and/or IIb fibers. We show that in mice with skeletal muscle-specific VDR deletion, maximum specific force and residual force following fatigue are impaired, along with a selective atrophy of type IIx and/or IIb fibers. These results show that the VDR has a significant biological effect on DIAm function independent of systemic effects on mineral metabolism.NEW & NOTEWORTHY Vitamin D deficiency and vitamin D receptor (VDR) polymorphisms are associated with adverse pulmonary and diaphragm muscle (DIAm)-associated respiratory outcomes. We used a skeletal muscle-specific tamoxifen-inducible VDR knockout to investigate DIAm dysfunction following reduced VDR signaling. Marked DIAm weakness and atrophy of type IIx and/or IIb fibers are present in muscle-specific tamoxifen-induced VDR knockout mice compared with controls. These results show that the VDR has a significant biological effect on DIAm function independent of systemic effects on mineral metabolism.