Do skeletal muscle composition and gene expression as well as acute exercise-induced serum adaptations in older adults depend on fitness status?

Background Inactive physical behavior among the elderly is one risk factor for cardiovascular disease, immobility and increased all-cause mortality. We aimed to answer the question whether or not circulating and skeletal muscle biomarkers are differentially expressed depending on fitness status in a group of elderly individuals. Methods Twenty-eight elderly individuals (73.36 ± 5.46 years) participated in this exploratory study after participating as part of the multinational SITLESS-clinical trial (implementation of self-management and exercise programs over 16 weeks). A cardiopulmonary exercise test (CPX) and resting skeletal muscle biopsy were performed to determine individual physiological performance capacity. Participants were categorized into a high physical fitness group (HPF) and a low physical fitness group (LPF) depending on peak oxygen uptake (VO2peak). Serum blood samples were taken before (pre) and after (post) CPX and were examined regarding serum BDNF, HSP70, Kynurenine, Irisin and Il-6 concentrations. Skeletal muscle tissue was analyzed by silver staining to determine the myosin heavy chain (MyHC) composition and selected genes by qRT-PCR. Results HPF showed lower body weight and body fat, while skeletal muscle mass and oxygen uptake at the first ventilatory threshold (VO2T1) did not differ between groups. There were positive associations between VO2peak and VO2VT1 in HPF and LPF. MyHC isoform quantification revealed no differences between groups. qRT-PCR showed higher expression of BDNF and BRCA1 in LPF skeletal muscle while there were no differences in other examined genes regarding energy metabolism. Basal serum concentrations of Irisin were higher in HPF compared to LPF with a trend towards higher values in BDNF and HSP70 in HPF. Increases in Il-6 in both groups were observed post. Conclusions Although no association between muscle composition/VO2peak with fitness status in older people was detected, higher basal Irisin serum levels in HPF revealed slightly beneficial molecular serum and muscle adaptations. Trial registration ClinicalTrials.gov, NCT02629666. Registered 19 November 2015.

Dermatomyositis- Related Intestinal Dysmotility

Dermatomyositis (DM) is an inflammatory myopathy characterized by proximal muscle weakness and pathognomonic skin lesions. A 69-year-old woman with a recent diagnosis of DM one month prior, treated with corticosteroids and immunomodulators, presented to our inpatient rehabilitation with worsening dysphagia and constipation. At the time of our evaluation, physical examination was notable for erythematous papules over the MCPs, PIPs, elbows, and knees as well as a violaceous rash on the face. Muscle strength was diminished bilaterally with proximal distribution being affected greater than distal. Laboratory studies were notable for CK level 31 IU/ L, ANA by immunofluorescence of 1:80, and aldolase 4 u/L. The eleven-antibody myositis panel was negative. Skeletal muscle biopsy of the left thigh showed partially treated acquired inflammatory myopathy with perifascicular atrophy. During hospitalization, she was found to have pulmonary embolism. She received enoxaparin 1 mg/kg subcutaneous BID. Soon after, she developed rectal bleeding. Colonoscopy showed a stercoral ulcer caused by chronic constipation. While dysphagia is common, being present in 25-50% of patients with DM, lower gastrointestinal problems involving the small and large intestine are rare and typically present as a late manifestation of the disease. Decreased peristalsis in the large colon can lead to constipation, impaction, and subsequent mucosal ulceration, and pressure necrosis induced by fecaloma formation. Although rare, our case highlights the importance of recognizing gastrointestinal complications that dermatomyositis can cause and the effects that those complications have on morbidity and mortality.

Hindlimb Muscle Dissection, Permeabilization, and Respirometry v1

The use of permeabilized muscle fibers (PMF) has emerged as a gold standard for assessing skeletal muscle mitochondrial function. PMF provide an intermediate approach between in vivo strategies and isolated mitochondria that allows the mitochondria to be maintained in close to their native morphology in the myofiber while allowing greater control of substrate and inhibitor concentrations. However, like mitochondrial isolation, the primary drawback to PMF is disruption of the cellular environment during the muscle biopsy and preparation. Despite all the benefits of permeabilized muscle fibers in evaluating mitochondrial respiration and dynamics one of the major drawbacks is increased variability introduced during a muscle biopsy as well as intrinsic variation that exists due to sex and age. This study was designed to evaluate how age, sex, and biopsy preparations affect mitochondrial respiration in extensor digitorum longus, soleus, and gastrocnemius muscle of mice. Here we detail a modified approach to skeletal muscle biopsy of the gastrocnemius muscle of mice focused on maintenance of intact fibers that results in greater overall respiration compared to cut fibers. The improved respiration of intact fibers is sex specific as are some of the changes in mitochondrial respiration with age. This study shows the need for standard practices when measuring mitochondrial respiration in permeabilized muscle and provides a protocol to control for variation introduced during a typical mouse muscle biopsy.

The Use of Autologous Skeletal Muscle Derived Cells as a Sling in the Treatment of Induced Stress Urinary Incontinence, An Experimental Study in Dogs

Introduction:This is an experimental pre-clinical study, testing the applicability of autologous skeletal muscle derived cells as a treatment of SUI in canine modelMethods:10 Mongrel dogs included. Skeletal muscle biopsy was harvested in 4. 1 month later, incontinence was induced in 8 dogs through urethrolysis. Muscle biopsy was incubated and expanded for 8 weeks. Muscle derived cells were collected and covered a Polyglycolic acid (PGA) scaffold immersed in culture medium and coated with matrigel to be used as a sling. Placed suburethral in 8 dogs; 4 had cell- seeded and 4 had scaffold only. Urethral pressure (UP) measurement was done at baseline 2 &6 weeks after sling insertion. The urethra was harvested 4 weeks after sling insertion for histopathology.Results:UP shows increase of maximum urethral pressure during static measurement in all dogs with a scaffold inserted. The increase ranged from 5-40 cmH20 Histopathology shows significant periurethral proliferation of skeletal muscles in 4 dogs with cell-seeded scaffold. This was maximum in dogs # 1& 2. This was not the case in the 4 dogs that had sling only.Conclusion: Use of skeletal muscle –seeded PGA scaffold is a practical technique with preserved integrity of histological differentiation in canine model.

Cardarine (GW501516) Effects on Improving Metabolic Syndrome

The present study hypothesized that treatment with GW501516 (a selective PPAR-δ agonist) lowers lipids by increasing fatty acid oxidation without adverse effects on oxidative stress. Caucasian men (age 18-50 years, n=18) were randomly assigned to treatment with GW501516, GW590735, or placebo for two weeks while residing in a clinical research facility. A meal tolerance test, skeletal muscle biopsy, and blood/breath sampling were conducted. The study reported that treatment with GW501516 ameliorated multiple metabolic abnormalities associated with metabolic syndrome including oxidative stress, obesity, dyslipidemia, and insulin resistance, all while increasing fatty acid oxidation. Notably, no adverse effects were reported. However, the restricted living conditions and/or diets that the participants were subjected to likely do not resemble their normal lifestyle. Therefore, the beneficial effects of GW501516 on metabolic health observed in the study should further be investigated in a real-life setting. During participant recruitment, the use of dietary supplements were minimally considered, thereby increasing the risk for confounding effects on the metabolic parameters assessed in the study. Also, recruiting a larger and more diverse population would allow for a more detailed analysis that may benefit a broader range of people (i.e., examining the effects of GW501516 in certain ethnic groups or with/without exercise programs). Additional research on GW501516 and other PPAR-δ agonists is encouraged since it appears that this class of drugs can ameliorate multiple metabolic syndrome features. Future studies should consider additional metrics relevant to metabolic syndrome such as C-reactive protein, cortisol, and homocysteine.

The use of autologous skeletal muscle-derived cells as a sling in the treatment of induced stress urinary incontinence, an experimental study

Introduction & hypothesis: This is an experimental pre-clinical study testing for the applicability of autologous skeletal muscle derived cells as a seeded sling for the treatment of Stress urinary incontinence in canine model. Methods: 10 Mongrel dogs: In 4, skeletal muscle biopsy was harvested from Biceps Femoris. 1 month later, incontinence was induced in 8 dogs through surgical disruption of the pubourethral ligaments. Muscle biopsy was incubated in medium and after expansion for 8 weeks, Muscle derived cells were collected. Polyglycolic acid scaffold was immersed in culture medium, coated with matrigel and cells were seeded. The sling was placed suburethral in 8 dogs; 2 of which were cell-seeded and 4 had the scaffold only. Urethral pressure measurement was done at baseline and 2 weeks after insertion of the sling. The urethra with its surrounding was harvested 4 weeks after sling insertion for histopathology. 2 dogs were considered as control, in which no urethrolysis or insertion of slings was carried out. Results: Urethral pressure shows increase of maximum urethral pressure during static measurement in all dogs with a scaffold inserted. The increase ranged from 5-40 centimeter water (Median: 23 cmH20). Histopathology shows significant periurethral proliferation of skeletal muscles in 4 dogs with cell-seeded scaffold, as demonstrated by Desmin. This was maximum in dogs numbers 1and 2. This was not the case in the 4 dogs that had Polyglycolic acid sling only. Conclusion: The use of skeletal muscle-seeded scaffold is a practical technique with preserved integrity of histological differentiation in canine model at short term.

Case Report: Two Chinese Infants of Sengers Syndrome Caused by Mutations in AGK Gene

Sengers syndrome (OMIM #212350) is a rare autosomal recessive disorder due to mutations in acylglycerol kinase (AGK) gene. We report two cases that were diagnosed clinically and confirmed genetically. Both infants had typical clinical features characterized by hypertrophic cardiomyopathy, bilateral cataracts, myopathy, and lactic acidosis, and heart failure was the most severe manifestation. Genetic testing of a boy revealed a homozygous pathogenic variant for Sengers syndrome in AGK (c.1131+2T>C) which was classified as likely pathogenic according to the ACMG guideline; besides, his skeletal muscle biopsy and transmission electron microscope presented obvious abnormity. One girl had compound heterozygous (c.409C>T and c.390G>A) variants of AGK gene that was identified in the proband and further Sanger sequencing indicated that the parents carried a single heterozygous mutation each. After the administration of “cocktail” therapy including coenzyme Q10, carnitine, and vitamin B complex, as well as ACEI, heart failure and myopathy of the boy were significantly improved and the condition was stable after 1-year follow-up, while the cardiomyopathy of the girl is not progressive but the plasma lactate acid increased significantly. We present the first report of two infants with Sengers syndrome diagnosed via exome sequencing in China.

Insulin Infusion is Linked to Increased NPPC Expression in Muscle and Plasma C-type Natriuretic Peptide in Male Dogs

The purpose of this study was to assess insulin-stimulated gene expression in canine skeletal muscle with a particular focus on NPPC, the gene that encodes C-type natriuretic peptide, a key hormonal regulator of cardiometabolic function. Four conscious canines underwent hyperinsulinemic, euglycemic clamp studies. Skeletal muscle biopsy and arterial plasma samples were collected under basal and insulin-stimulated conditions. Bulk RNA sequencing of muscle tissue was performed to identify differentially expressed genes between these two steady-state conditions. Our results showed that NPPC was the most highly expressed gene in skeletal muscle in response to insulin infusion, rising fourfold between basal and insulin-stimulated conditions. In support of our RNA-sequencing data, we found that raising the plasma insulin concentration 15-fold above basal elicited a 2-fold (p = 0.0001) increase in arterial plasma concentrations of N-terminal prohormone C-type natriuretic peptide. Our data suggest insulin may play a role in stimulating secretion of C-type natriuretic peptide by skeletal muscle. In this context, C-type natriuretic peptide may act in a paracrine manner to facilitate muscle-vascular bed crosstalk and potentiate insulin-mediated vasodilation. This could serve to enhance insulin and glucose delivery, particularly in the postprandial absorptive state.

When multiple caveolins make the difference: Cav1 partly compensates Cav3 alterations and rescues ion channels expression

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Fondazione Cariplo Caveolae are small-membrane invagination that contribute both to buffering excessive contraction-dependent membrane strain and to initiation of membrane repair. Moreover, they constitute micro-domains where receptors and ion channels are clustered, favouring their functional interaction. Caveolin-3 (Cav3) is the key structural component of muscular caveolae. Mutations in Cav3 gene are associated with alterations of the skeletal muscle architecture leading to some rare forms of hereditary skeletal myopathies and/or cardiomyopathies called caveolinopathies. Notably, skeletal muscle dysfunctions usually precede cardiac dysfunctions, even though the mutated Cav3 is expressed in both cell types. An important difference between skeletal fibers and cardiomyocytes is that in the latter, caveolin-1 (Cav1) participates with Cav3 to form caveolae; skeletal myotubes instead do not express Cav1.The delay or lack of onset of cardiac alterations in caveolinopathies may depend on a preserved micro-domains organization in the heart compared to skeletal muscle, due to Cav1 expression. We decided to focus on a specific mutation T78K found in heterozygous in a patient with Ripple muscle disease and hyperCKemia. We have characterized human cardiomyocytes (CM) differentiated from induced pluripotent stem cells (iPSC) derived from this patient and one healthy control. In particular, we have investigated which caveolin isoforms are expressed at day 30 of differentiation, finding both Cav1 and Cav3, with a significant decrease of Cav3 isoform in T78K-CM. Their different expressions significantly increase T78K membrane resistance (3.27 ± 0.6 GΩ versus 1.64 ± 0.4 GΩ in the CTRL-CM), and consequently membrane excitability. The T78K_CM showed an increase spontaneous beating rate compared to CTRL (1,75± 0,08 Hz and 0,89 ± 0,4 Hz, respectively). Previous laboratory analysis conducted in caveolin-free MEF cells, co-transfected with WT and T78K Cav3 mutation, revealed that the T78K mutant is dominant, inducing the retention of WT Cav3 in the perinuclear areas and causes significant reduction in current density of three ion channels (HCN4, Kv1.5 and Kir2.1) known to interact with caveolins. The dominant decreased in cav3 expression is in line with previous data in skeletal muscle biopsy, however, electrophysiological data would be likely incompatible with life. For this reason, we decided to compare the impact of this mutation in CHO cells that exhibit high levels of Cav1, and in cav-1 expressing MEF line. In these systems, the membrane localization of Cav3 T78K is rescued both in heterozygous and homozygous conditions. In line with caveolin membrane expression, HCN4, Kv1.5 and Kir2.1 density is also rescued to normal levels. These results constitute the first evidence of a possible role of Cav1 in compensating membrane disorganization and disfunction due to Cav3 mutations in the heart, making this organ less susceptible to caveolinopathies.

Quantitative Muscle-MRI Correlates with Histopathology in Skeletal Muscle Biopsies

Background: Skeletal muscle biopsy is one of the gold standards in the diagnostic workup of muscle disorders. By histopathologic analysis, characteristic features like inflammatory cellular infiltrations, fat and collagen replacement of muscle tissue or structural defects of the myofibers can be detected. In the past years, novel quantitative MRI (qMRI) techniques have been developed to quantify tissue parameters, thus providing a non-invasive diagnostic tool in several myopathies. Objective: This proof-of-principle study was performed to validate the qMRI-techniques to skeletal muscle biopsy results. Methods: Ten patients who underwent skeletal muscle biopsy for diagnostic purposes were examined by qMRI. Fat fraction, water T2-time and diffusion parameters were measured in the muscle from which the biopsy was taken. The proportion of fat tissue, the severity of degenerative and inflammatory parameters and the amount of type 1- and type 2- muscle fibers were determined in all biopsy samples. The qMRI-data were then correlated to the histopathological findings. Results: The amount of fat tissue in skeletal muscle biopsy correlated significantly with the fat fraction derived from the Dixon sequence. The water T2-time, a parameter for tissue edema, correlated with the amount of vacuolar changes of myofibers and endomysial macrophages in the histopathologic analysis. No significant correlations were found for diffusion parameters. Conclusion: In this proof-of-principle study, qMRI techniques were related to characteristic histopathologic features in neuromuscular disorders. The study provides the basis for further development of qMRI methods in the follow-up of patients with neuromuscular disorders, especially in the context of emerging treatment strategies.