Impaired Skeletal Muscle Function Research Articles

Impact of Passaging Primary Skeletal Muscle Cell Isolates on the Engineering of Skeletal Muscle.

Volumetric muscle loss (VML) is a clinical state that results in impaired skeletal muscle function. Engineered skeletal muscle can serve as a treatment for VML. Currently, large biopsies are required to achieve the cells necessary for the fabrication of engineered muscle, leading to donor-site morbidity. Amplification of cell numbers using cell passaging may increase the usefulness of a single muscle biopsy for engineering muscle tissue. In this study, we evaluated the impact of passaging cells obtained from donor muscle tissue by analyzing characteristics of in vitro cellular growth and tissue-engineered skeletal muscle unit (SMU) structure and function. Human skeletal muscle cell isolates from three separate donors (P0-Control) were compared with cells passaged once (P1), twice (P2), or three times (P3) by monitoring SMU force production and determining muscle content and structure using immunohistochemistry. Data indicated that passaging decreased the number of satellite cells and increased the population doubling time. P1 SMUs had slightly greater contractile force and P2 SMUs showed statistically significant greater force production compared with P0 SMUs with no change in SMU muscle content. In conclusion, human skeletal muscle cells can be passaged twice without negatively impacting SMU muscle content or contractile function, providing the opportunity to potentially create larger SMUs from smaller biopsies, thereby producing clinically relevant sized grafts to aid in VML repair.

Estrogen deficiency reduces maximal running capacity and affects serotonin levels differently in the hippocampus and nucleus accumbens in response to acute exercise.

Estrogen deficiency is associated with unfavorable changes in body composition and metabolic health. While physical activity ameliorates several of the negative effects, loss of ovarian function is associated with decreased physical activity levels. It has been proposed that the changes in brain neurochemical levels and /or impaired skeletal muscle function may underlie this phenomenon. We studied the effect of estrogen deficiency induced via ovariectomy (OVX) in female Wistar rats (n = 64). Rats underwent either sham or OVX surgery and were allocated thereafter into four groups matched for body mass and maximal running capacity: sham/control, sham/max, OVX/control, and OVX/max, of which the max groups had maximal running test before euthanasia to induce acute response to exercise. Metabolism, spontaneous activity, and maximal running capacity were measured before (PRE) and after (POST) the surgeries. Three months following the surgery, rats were euthanized, and blood and tissue samples harvested. Proteins were analyzed from gastrocnemius muscle and retroperitoneal adipose tissue via Western blot. Brain neurochemical markers were measured from nucleus accumbens (NA) and hippocampus (HC) using ultra-high performance liquid chromatography. OVX had lower basal energy expenditure and higher body mass and retroperitoneal adipose tissue mass compared with sham group (p ≤ 0.005). OVX reduced maximal running capacity by 17% (p = 0.005) with no changes in muscle mass or phosphorylated form of regulatory light chain (pRLC) in gastrocnemius muscle. OVX was associated with lower serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) level in the NA compared with sham (p = 0.007). In response to acute exercise, OVX was associated with low serotonin level in the HC and high level in the NA (p ≤ 0.024). Our results highlight that OVX reduces maximal running capacity and affects the response of brain neurochemical levels to acute exercise in a brain region-specific manner. These results may offer mechanistic insight into why OVX reduces willingness to exercise.

Immunogenetic Aspects of Sarcopenic Obesity.

Sarcopenic obesity (SO) is a combination of obesity and sarcopenia, with diagnostic criteria defined as impaired skeletal muscle function and altered body composition (e.g., increased fat mass and reduced muscle mass). The mechanism of SO is not yet perfectly understood; however, the pathogenesis includes aging and its complications, chronic inflammation, insulin resistance (IR), and hormonal changes. Genetic background is apparent in the pathogenesis of isolated obesity, which is most often polygenic and is characterized by the additive effect of various genetic factors. The genetic etiology has not been strictly established in SO. Still, many data confirm the existence of pathogenic gene variants, e.g., Fat Mass and Obesity Associated Gene (FTO), beta-2-adrenergic receptor (ADRB2) gene, melanocortin-4 receptor (MC4R) and others with obesity. The literature on the role of these genes is scarce, and their role has not yet been thoroughly established. On the other hand, the involvement of systemic inflammation due to increased adipose tissue in SO plays a significant role in its pathophysiology through the synthesis of various cytokines such as monocyte chemoattractant protein-1 (MCP-1), IL-1Ra, IL-15, adiponectin or CRP. The lack of anti-inflammatory cytokine (e.g., IL-15) can increase SO risk, but further studies are needed to evaluate the exact mechanisms of implications of various cytokines in SO individuals. This manuscript analyses various immunogenetic and non-genetic factors and summarizes the recent findings on immunogenetics potentially impacting SO development.

Pharmacologic Inhibition of Myostatin With a Myostatin Antibody Improves the Skeletal Muscle and Bone Phenotype of Male Insulin-Deficient Diabetic Mice.

Type 1 diabetes (T1D) is associated with low bone and muscle mass, increased fracture risk, and impaired skeletal muscle function. Myostatin, a myokine that is systemically elevated in humans with T1D, negatively regulates muscle mass and bone formation. We investigated whether pharmacologic myostatin inhibition in a mouse model of insulin-deficient, streptozotocin (STZ)-induced diabetes is protective for bone and skeletal muscle. DBA/2J male mice were injected with low-dose STZ (diabetic) or vehicle (non-diabetic). Subsequently, insulin or palmitate Linbits were implanted and myostatin (REGN647-MyoAb) or control (REGN1945-ConAb) antibody was administered for 8 weeks. Body composition and contractile muscle function were assessed in vivo. Systemic myostatin, P1NP, CTX-I, and glycated hemoglobin (HbA1c) were quantified, and gastrocnemii were weighed and analyzed for muscle fiber composition and gene expression of selected genes. Cortical and trabecular parameters were analyzed (micro-computed tomography evaluations of femur) and cortical bone strength was assessed (three-point bending test of femur diaphysis). In diabetic mice, the combination of insulin/MyoAb treatment resulted in significantly higher lean mass and gastrocnemius weight compared with MyoAb or insulin treatment alone. Similarly, higher raw torque was observed in skeletal muscle of insulin/MyoAb-treated diabetic mice compared with MyoAb or insulin treatment. Additionally, muscle fiber cross-sectional area (CSA) was lower with diabetes and the combination treatment with insulin/MyoAb significantly improved CSA in type II fibers. Insulin, MyoAb, or insulin/MyoAb treatment improved several parameters of trabecular architecture (eg, bone volume fraction [BV/TV], trabecular connectivity density [Conn.D]) and cortical structure (eg, cortical bone area [Ct. Ar.], minimum moment of inertia [Imin]) in diabetic mice. Lastly, cortical bone biomechanical properties (stiffness and yield force) were also improved with insulin or MyoAb treatment. In conclusion, pharmacologic myostatin inhibition is beneficial for muscle mass, muscle function, and bone properties in this mouse model of T1D and its effects are both independent and additive to the positive effects of insulin. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

An evaluation of clinical presentation and genetic testing approaches for patients with neuromuscular disorders.

Inherited neuromuscular disorders (NMDs) are a large group of genetic conditions characterized by impaired peripheral nerve, motor neuron, neuromuscular junction, or skeletal muscle function. These conditions are also known to have clinical and genetic heterogeneity and variable ages of onset. Clinical evaluation for NMDs has increasingly incorporated molecular genetics. However, genetic testing is complicated by the variety of testing options and the ambiguity of NMD phenotypes. Examining test selection and yield may elucidate testing recommendations and improve the diagnostic journey for these patients. This retrospective chart review evaluated the clinical presentations, genetic testing approaches, and diagnostic outcomes of 155 patients with suspected NMDs at Cincinnati Children's Hospital Medical Center. A total of 262 individual tests were ordered, averaging 1.7 tests per patient. The clinic utilized 26 separate genetic tests, with test yields ranging from 0% to 66%. Overall, 21% of patients received a genetic diagnosis. Of all the clinical findings evaluated, elevated CPK levels with or without muscle weakness were the most informative symptoms correlated with a diagnostic result. This study highlights several genetic testing considerations for NMDs, including the variability of diagnostic outcomes. This knowledge is relevant to clinicians and patients, especially during the pretest counseling and consenting process.

Adiposity is associated with widespread transcriptional changes and downregulation of longevity pathways in aged skeletal muscle.

Amongst healthy older people, a number of correlates of impaired skeletal muscle mass and function have been defined. Although the prevalence of obesity is increasing markedly in this age group, information is sparse about the particular impacts of obesity on ageing skeletal muscle or the molecular mechanisms that underlie this and associated disease risk. Here, we examined genome-wide transcriptional changes using RNA sequencing in muscle biopsies from 40 older community-dwelling men from the Hertfordshire Sarcopenia Study with regard to obesity (body mass index [BMI] >30kg/m2 , n=7), overweight (BMI 25-30, n=19), normal weight (BMI<25, n=14), and per cent and total fat mass. In addition, we used EPIC DNA methylation array data to investigate correlations between DNA methylation and gene expression in aged skeletal muscle tissue and investigated the relationship between genes within altered regulatory pathways and muscle histological parameters. Individuals with obesity demonstrated a prominent modified transcriptional signature in muscle tissue, with a total of 542 differentially expressed genes associated with obesity (false discovery rate ≤0.05), of which 425 genes were upregulated when compared with normal weight. Upregulated genes were enriched in immune response (P=3.18×10-41 ) and inflammation (leucocyte activation, P=1.47×10-41 ; tumour necrosis factor, P=2.75×10-15 ) signalling pathways and downregulated genes enriched in longevity (P=1.5×10-3 ) and AMP-activated protein kinase (AMPK) (P=4.5×10-3 ) signalling pathways. Furthermore, differentially expressed genes in both longevity and AMPK signalling pathways were associated with a change in DNA methylation, with a total of 256 and 360 significant cytosine-phosphate-guanine-gene correlations identified, respectively. Similar changes in the muscle transcriptome were observed with respect to per cent fat mass and total fat mass. Obesity was further associated with a significant increase in type II fast-fibre area (P=0.026), of which key regulatory genes within both longevity and AMPK pathways were significantly associated. We provide for the first time a global transcriptomic profile of skeletal muscle in older people with and without obesity, demonstrating modulation of key genes and pathways implicated in the regulation of muscle function, changes in DNA methylation associated with such pathways and associations between genes within the modified pathways implicated in muscle regulation and changes in muscle fibre type.

ANTI-RANKL TREATMENT ENHANCES MUSCLE STRENGTH THROUGH MACROPHAGE MODULATION MECHANISM IN SARCOPENIC MICE

Sarcopenia is an age-related geriatric syndrome which is associated with subsequent disability and morbidity. Currently there is no promising therapy approved for the treatment of sarcopenia. The receptor activator of nuclear factor NF-κB ligand (RANKL) and its receptor (RANK) are expressed in bone and skeletal muscle. Activation of the NF-κB pathway mainly inhibits myogenic differentiation, which leads to skeletal muscle dysfunction and loss. LYVE1 and CD206 positive macrophage has been reported to be associated with progressive impairment of skeletal muscle function with aging. The study aims to investigate the effects of an anti-RANKL treatment on sarcopenic skeletal muscle and explore the related mechanisms on muscle inflammation and the polarization status of macrophages.Sarcopenic senescence-accelerated mouse P8 (SAMP8) mice at month 8 were treated intraperitoneally with 5mg/kg anti-RANKL (IK22/5) or isotype control (2A3; Bio X Cell) antibody every 4 weeks and harvested at month 10. Senescence accelerated mouse resistant-1 (SAMR1) were collected at month 10 as the age-matched non-sarcopenic group. Ex-vivo functional assessment, grip strength and immunostaining of C/EBPa, CD206, F4/80, LYVE1 and PAX7 were performed. Data analysis was done with one-way ANOVA, and the significant level was set at p≤0.05.At month 10, tetanic force/specific tetanic force, twitch force/specific twitch force in anti-RANKL group were significantly higher than control group (all p&lt;0.01). The mice in the anti-RANKL treatment group also showed significantly higher grip strength than Con group (p&lt;0.001). The SAMP8 mice at month 10 expressed significantly more C/EBPa, CD206 and LYVE1 positive area than in SAMR1, while anti-RANKL treatment significantly decreased C/EBPa, CD206 and LYVE1 positive area.The anti-RANKL treatment protected against skeletal muscle dysfunctions through suppressing muscle inflammation and modulating M2 macrophages, which may represent a novel therapeutic approach for sarcopenia.Acknowledgment: Collaborative Research Fund (CRF, Ref: C4032-21GF)

Melatonin and Exercise Counteract Sarcopenic Obesity through Preservation of Satellite Cell Function.

Sarcopenic obesity (SO) is characterized by atrophic skeletal muscle impairment (sarcopenia) and obesity, which is associated with adverse outcomes of morbidity and mortality in elderly people. We investigated the effects of melatonin and exercise training on SO in 32-week-old senescence-accelerated mouse-prone-8 (SAMP8) mice fed a normal diet or a high-fat diet for 16 weeks. Melatonin, exercise, or melatonin and exercise for 8 weeks displayed reductions in the SO-induced impairment of skeletal muscle function and atrophy. Specifically, a decrease in mitochondrial calcium retention capacity in skeletal muscles observed in the HFD-con group was attenuated in melatonin and/or exercise intervention groups. More importantly, HFD-con mice displayed a lower number of Pax7+ satellite cells (SCs) and higher expression of p16ink than P8ND mice, which were attenuated by melatonin and/or exercise interventions. The cellular senescence in SC-derived primary myoblasts from HFD-con mice was significantly attenuated in myoblasts from the melatonin and/or exercise groups, which was reproduced in a senescence model of H2O2-treated C2C12 myoblasts. Our results suggest that melatonin and exercise training attenuate SO-induced skeletal muscle dysfunction, at least in part, through preserving the SC pool by inhibiting cellular senescence and attenuating mitochondrial dysfunction.

Punicalagin protects against impaired skeletal muscle function in high-fat-diet-induced obese mice by regulating TET2.

The function of skeletal muscles can be markedly hampered by obesity. Ten-eleven translocation 2 (TET2) is an important therapeutic target for ameliorating skeletal muscle dysfunction. Our previous study revealed that punicalagin (PUN) regulated TET2 in obese mice; however, whether PUN can prevent obesity-induced skeletal muscle dysfunction by regulating TET2 remains unclear. In the present study, 40 male C57BL/6J mice were divided into four groups (n = 10 per group): the control (CON) group, the high-fat-diet (HFD, negative control) group, the resveratrol (positive control) group, and the PUN group. The ratio of gastrocnemius weight to body weight (0.0097 ± 0.0016 vs. 0.0080 ± 0.0011), the grip strength (120.04 g ± 11.10 vs. 98.89 g ± 2.79), and the muscle fiber count (314.56 per visual field ± 92.73 vs. 236.44 per visual field ± 50.58) in the PUN group were higher than those in the HFD group. Moreover, the levels of the TET2 protein, 5-hydroxymethylcytosine (5hmC), and 5-formylcytosine (5fC) in skeletal muscles were significantly lower in the HFD group than those in the CON group; these levels increased after PUN treatment. Compared with the HFD group, the phosphorylation level of AMP-activated protein kinase (AMPK) α in the PUN group was higher, which effectively enhanced the stability of the TET2 protein. Besides, the ratio of (succinic acid + fumaric acid)/α-ketoglutarate in the PUN group was lower than that in the HFD group (43.21 ± 12.42 vs. 99.19 ± 37.07), and a lower ratio led to a higher demethylase activity of TET2 in the PUN group than in the HFD group. This study highlights that PUN supplementation protects against obesity-induced impairment of the skeletal muscle function via regulating the protein stability of TET2 and the enzymatic activity of TET2 demethylation.

Bergamot (Citrus bergamia) leaf extract improves metabolic, antioxidant and anti-inflammatory activity in skeletal muscles in a metabolic syndrome experimental model

Metabolic Syndrome (MetS), inflammation and oxidative stress contribute to impairment of skeletal muscle function. Bergamot (Citrus bergamia) leaf extract (BLE) has shown protective effects against comorbidities associated with MetS through its anti-inflammatory and antioxidant effects. The aim of this work was to elucidate the antioxidant and anti-inflammatory activity of BLE in skeletal muscles in an experimental model of MetS. Once metabolic syndrome was diagnosed, animals were divided into groups receiving different treatments for 10 weeks, including control diet (n = 10), control + BLE (n = 10), High Sugar-fat diet (HSF) (n = 10), HSF + BLE (n = 10). Evaluation included nutritional, metabolic and hormonal analyses, along with measurements of inflammatory status and oxidative stress in soleus and extensor digitorum longus (EDL) muscles. BLE showed positive metabolic effects, with a reduction of plasma triglycerides and insulin resistance and an increase in high-density lipoprotein cholesterol, and protective activity against oxidative stress and inflammation in Soleus and EDL muscles in animals with MetS.

Abstract 11480: Telmisartan to Improve Walking Performance in People With Lower Extremity Peripheral Artery Disease: The Telex Randomized Clinical Trial

BACKGROUND. People with lower extremity peripheral artery disease (PAD) have reduced lower extremity perfusion, impaired lower extremity skeletal muscle function, and poor walking performance. Telmisartan, an angiotensin receptor blocker (ARB), has properties that reverse these abnormalities. This randomized clinical trial tested whether telmisartan improved six-minute walk distance, compared to placebo, in people with PAD at 6-month follow-up. METHODS. This multi-centered randomized clinical trial was designed as a 2 x 2 factorial design, comparing the effects of telmisartan plus supervised exercise therapy (SET) to telmisartan alone and SET alone, respectively and comparing telmisartan alone to placebo. PAD participants were randomized to one of four groups: telmisartan+SET (N=30), telmisartan + attention control (N=29), placebo + SET (N=28), or placebo+ attention control (N=27) for six months. Due to slower than anticipated enrollment, the primary comparison was changed to a comparison of the two telmisartan groups combined to the two placebo groups combined. The primary outcome was 6-month change in 6-minute walk distance. Secondary outcomes were maximal treadmill walking distance, the Walking Impairment Questionnaire distance, speed, and stair climbing scores, and the short-form 36 physical functioning score. Results adjusted for site, baseline six-minute walk distance, randomization to SET vs. attention control, and group differences in the prevalence of sex and heart failure at baseline. RESULTS. Of 114 randomized patients (mean [SD] age, 67.3 [9.9] years, 46 (40.4%) female, 81 (71.1%) Black), 105 (92%) completed 6-month follow-up. See the Table for results. CONCLUSIONS. These results do not support telmisartan for improving objectively measured walking performance or patient reported outcomes in patients with PAD.

Effect of Telmisartan on Walking Performance in Patients With Lower Extremity Peripheral Artery Disease

Patients with lower extremity peripheral artery disease (PAD) have reduced lower extremity perfusion, impaired lower extremity skeletal muscle function, and poor walking performance. Telmisartan (an angiotensin receptor blocker) has properties that reverse these abnormalities. To determine whether telmisartan improves 6-minute walk distance, compared with placebo, in patients with lower extremity PAD at 6-month follow-up. Double-blind, randomized clinical trial conducted at 2 US sites and involving 114 participants. Enrollment occurred between December 28, 2015, and November 9, 2021. Final follow-up occurred on May 6, 2022. The trial randomized patients using a 2 × 2 factorial design to compare the effects of telmisartan plus supervised exercise vs telmisartan alone and supervised exercise alone and to compare telmisartan alone vs placebo. Participants with PAD were randomized to 1 of 4 groups: telmisartan plus exercise (n = 30), telmisartan plus attention control (n = 29), placebo plus exercise (n = 28), or placebo plus attention control (n = 27) for 6 months. The originally planned sample size was 240 participants. Due to slower than anticipated enrollment, the primary comparison was changed to the 2 combined telmisartan groups vs the 2 combined placebo groups and the target sample size was changed to 112 participants. The primary outcome was the 6-month change in 6-minute walk distance (minimum clinically important difference, 8-20 m). The secondary outcomes were maximal treadmill walking distance; Walking Impairment Questionnaire scores for distance, speed, and stair climbing; and the 36-Item Short-Form Health Survey physical functioning score. The results were adjusted for study site, baseline 6-minute walk distance, randomization to exercise vs attention control, sex, and history of heart failure at baseline. Of the 114 randomized patients (mean age, 67.3 [SD, 9.9] years; 46 were women [40.4%]; and 81 were Black individuals [71.1%]), 105 (92%) completed 6-month follow-up. At 6-month follow-up, telmisartan did not significantly improve 6-minute walk distance (from a mean of 341.6 m to 343.0 m; within-group change: 1.32 m) compared with placebo (from a mean of 352.3 m to 364.8 m; within-group change: 12.5 m) and the adjusted between-group difference was -16.8 m (95% CI, -35.9 m to 2.2 m; P = .08). Compared with placebo, telmisartan did not significantly improve any of the 5 secondary outcomes. The most common serious adverse event was hospitalization for PAD (ie, lower extremity revascularization, amputation, or gangrene). Three participants (5.1%) in the telmisartan group and 2 participants (3.6%) in the placebo group were hospitalized for PAD. Among patients with PAD, telmisartan did not improve 6-minute walk distance at 6-month follow-up compared with placebo. These results do not support telmisartan for improving walking performance in patients with PAD. ClinicalTrials.gov Identifier: NCT02593110.

MicroRNA-29a Regulates ADAM12 Through Direct Interaction With ADAM12 mRNA and Modulates Postischemic Perfusion Recovery.

BackgroundPeripheral artery disease is caused by atherosclerotic occlusion of vessels outside the heart and most commonly affects vessels of the lower extremities. Angiogenesis is a part of the postischemic adaptation involved in restoring blood flow in peripheral artery disease. Previously, in a murine hind limb ischemia model of peripheral artery disease, we identified ADAM12 (a disintegrin and metalloproteinase gene 12) as a key genetic modifier of postischemic perfusion recovery. However, less is known about ADAM12 regulation in ischemia. MicroRNAs are a class of small, noncoding, single‐stranded RNAs that regulate gene expression primarily through transcriptional repression of messenger RNA (mRNA). We showed microRNA‐29a (miR‐29a) modulates ADAM12 expression in the setting of diabetes and ischemia. However, how miR‐29a modulates ADAM12 is not known. Moreover, the physiological effects of miR‐29a modulation in a nondiabetic setting is not known.Methods and ResultsWe overexpressed or inhibited miR‐29a in ischemic mouse gastrocnemius and tibialis anterior muscles, and quantified the effect on perfusion recovery, ADAM12 expression, angiogenesis, and skeletal muscle regeneration. In addition, using RNA immunoprecipitation–based anti‐miR competitive assay, we investigated the interaction of miR‐29a and ADAM12 mRNA in mouse microvascular endothelial cell, skeletal muscle, and human endothelial cell lysates. Ectopic expression of miR‐29a in ischemic mouse hind limbs decreased ADAM12 mRNA expression, increased skeletal muscle injury, decreased skeletal muscle function, and decreased angiogenesis and perfusion recovery, with no effect on skeletal muscle regeneration and myofiber cross‐sectional area following hind limb ischemia. RNA immunoprecipitation–based anti‐miR competitive assay studies showed miR‐29a antagomir displaced miR‐29a and ADAM12 mRNA from the AGO‐2 (Argonaut‐2) complex in a dose dependent manner.ConclusionsTaken together, the data show miR‐29a suppresses ADAM12 expression by directly binding to its mRNA, resulting in impaired skeletal muscle function, angiogenesis, and poor perfusion. Hence, elevated levels of miR‐29a, as seen in diabetes and aging, likely contribute to vascular pathology, and modulation of miR‐29a could be a therapeutic target.

The Functional Role of Long Non-Coding RNA in Myogenesis and Skeletal Muscle Atrophy.

Skeletal muscle is a pivotal organ in humans that maintains locomotion and homeostasis. Muscle atrophy caused by sarcopenia and cachexia, which results in reduced muscle mass and impaired skeletal muscle function, is a serious health condition that decreases life longevity in humans. Recent studies have revealed the molecular mechanisms by which long non-coding RNAs (lncRNAs) regulate skeletal muscle mass and function through transcriptional regulation, fiber-type switching, and skeletal muscle cell proliferation. In addition, lncRNAs function as natural inhibitors of microRNAs and induce muscle hypertrophy or atrophy. Intriguingly, muscle atrophy modifies the expression of thousands of lncRNAs. Therefore, although their exact functions have not yet been fully elucidated, various novel lncRNAs associated with muscle atrophy have been identified. Here, we comprehensively review recent knowledge on the regulatory roles of lncRNAs in skeletal muscle atrophy. In addition, we discuss the issues and possibilities of targeting lncRNAs as a treatment for skeletal muscle atrophy and muscle wasting disorders in humans.

CRISPR/Cas9-Mediated Constitutive Loss of VCP (Valosin-Containing Protein) Impairs Proteostasis and Leads to Defective Striated Muscle Structure and Function In Vivo.

Valosin-containing protein (VCP) acts as a key regulator of cellular protein homeostasis by coordinating protein turnover and quality control. Mutations in VCP lead to (cardio-)myopathy and neurodegenerative diseases such as inclusion body myopathy with Paget’s disease of the bone and frontotemporal dementia (IBMPFD) or amyotrophic lateral sclerosis (ALS). To date, due to embryonic lethality, no constitutive VCP knockout animal model exists. Here, we generated a constitutive CRISPR/Cas9-induced vcp knockout zebrafish model. Similar to the phenotype of vcp morphant knockdown zebrafish embryos, we found that vcp-null embryos displayed significantly impaired cardiac and skeletal muscle function. By ultrastructural analysis of skeletal muscle cells and cardiomyocytes, we observed severely disrupted myofibrillar organization and accumulation of inclusion bodies as well as mitochondrial degeneration. vcp knockout was associated with a significant accumulation of ubiquitinated proteins, suggesting impaired proteasomal function. Additionally, markers of unfolded protein response (UPR)/ER-stress and autophagy-related mTOR signaling were elevated in vcp-deficient embryos, demonstrating impaired proteostasis in VCP-null zebrafish. In conclusion, our findings demonstrate the successful generation of a stable constitutive vcp knockout zebrafish line that will enable characterization of the detailed mechanistic underpinnings of vcp loss, particularly the impact of disturbed protein homeostasis on organ development and function in vivo.

Predictors of Prolonged Cardiopulmonary Exercise Impairment After COVID-19 Infection: A Prospective Observational Study.

Objectives: Coronavirus disease 2019 (COVID-19) is a global pandemic affecting individuals to varying degrees. There is emerging evidence that even patients with mild symptoms will suffer from prolonged physical impairment.Methods: In this prospective observational study, lung function, and cardiopulmonary exercise testing have been performed in 100 patients for 3–6 months after COVID-19 diagnosis (post-CoVG). Depending on the severity of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, patients were divided into asymptomatic, or mild to moderate (mild post-CoVG), and severe post-CoVG [hospitalization with or without intensive care unit/non-invasive ventilation (ICU/NIV)]. Results have been compared with age, sex, and body mass index (BMI) matched control group (CG, N = 50).Results: Both lung function (resting) and exercise capacity (peak workload, Wpeak and peak oxygen uptake, VO2 peak - % predicted) were considerably affected in patients with severe post-CoV (81.7 ± 27.6 and 86.1 ± 20.6%), compared to the mild post-CoVG (104.8 ± 24.0%, p = 0.001 and 100.4 ± 24.8; p = 0.003). In addition, also the submaximal exercise performance was significantly reduced in the severe post-CoVG (predicted VT1/VO2 peak; p = 0.013 and VT2/VO2 peak; p = 0.001). Multiple linear regression analyses revealed that 74 % (adjusted R2) of the variance in relative VO2 peak of patients who had CoV could be explained by the following variables: lower age, male sex, lower BMI, higher DLCO, higher predicted heart rate (HR) peak, lower breathing reserve (BR), and lower SaO2 peak, which were related to higher relative VO2 peak values. Higher NT-proBNP and lower creatinine kinase (CK) values were seen in severe cases compared to patients who experienced mild CoV.Discussion: Maximal and submaximal exercise performance in patients recovering from severe COVID-19 remain negatively affected for 3–6 months after COVID-19 diagnosis. The presented findings reveal that impaired pulmonary, cardiac, and skeletal muscle function contributed to the limitation of VO2 peak in those patients, which may have important implications on rehabilitation programs.

Human skeletal muscle CD90+ fibro-adipogenic progenitors are associated with muscle degeneration in type 2 diabetic patients

Type 2 diabetes mellitus (T2DM) is associated with impaired skeletal muscle function and degeneration of the skeletal muscles. However, the mechanisms underlying the degeneration are not well described in human skeletal muscle. Here we show that skeletal muscle of T2DM patients exhibit degenerative remodeling of the extracellular matrix that is associated with a selective increase of a subpopulation of fibro-adipogenic progenitors (FAPs) marked by expression of THY1 (CD90)-the FAPCD90+. We identify platelet-derived growth factor (PDGF) as a key FAP regulator, as it promotes proliferation and collagen production at the expense of adipogenesis. FAPsCD90+ display a PDGF-mimetic phenotype, with high proliferative activity, clonogenicity, and production of extracellular matrix. FAPCD90+ proliferation was reduced by invitro treatment with metformin. Furthermore, metformin treatment reduced FAP content in T2DM patients. These data identify a PDGF-driven conversion of a subpopulation of FAPs as a key event in the fibrosis development in T2DM muscle.

SOD2 in skeletal muscle: New insights from an inducible deletion model

Metabolic conditions such as obesity, insulin resistance and glucose intolerance are frequently associated with impairments in skeletal muscle function and metabolism. This is often linked to dysregulation of homeostatic pathways including an increase in reactive oxygen species (ROS) and oxidative stress. One of the main sites of ROS production is the mitochondria, where the flux of substrates through the electron transport chain (ETC) can result in the generation of oxygen free radicals. Fortunately, several mechanisms exist to buffer bursts of intracellular ROS and peroxide production, including the enzymes Catalase, Glutathione Peroxidase and Superoxide Dismutase (SOD). Of the latter, there are two intracellular isoforms; SOD1 which is mostly cytoplasmic, and SOD2 which is found exclusively in the mitochondria. Developmental and chronic loss of these enzymes has been linked to disease in several studies, however the temporal effects of these disturbances remain largely unexplored. Here, we induced a post-developmental (8-week old mice) deletion of SOD2 in skeletal muscle (SOD2-iMKO) and demonstrate that 16 weeks of SOD2 deletion leads to no major impairment in whole body metabolism, despite these mice displaying alterations in aspects of mitochondrial abundance and voluntary ambulatory movement. This is likely partly explained by the suggestive data that a compensatory response may exist from other redox enzymes, including catalase and glutathione peroxidases. Nevertheless, we demonstrated that inducible SOD2 deletion impacts on specific aspects of muscle lipid metabolism, including the abundance of phospholipids and phosphatidic acid (PA), the latter being a key intermediate in several cellular signaling pathways. Thus, our findings suggest that post-developmental deletion of SOD2 induces a more subtle phenotype than previous embryonic models have shown, allowing us to highlight a previously unrecognized link between SOD2, mitochondrial function and bioactive lipid species including PA.

Hand grip strength and type 2 diabetes are mutually independent predictors of cardiovascular events and of mortality in patients with established cardiovascular disease

Background and Aims: Impaired skeletal muscle function is involved in the pathogenesis of type 2 diabetes (T2DM) as well as of cardiovascular disease (CVD). Hand grip strength (HGS) is a simple, inexpensive and non-invasive diagnostic parameter for muscle function. Its association with cardiovascular events and mortality in patients with established CVD is unclear and is addressed in the present study.

Gait disturbances and muscle dysfunction in fibroblast growth factor 2 knockout mice

Fibroblast growth factor 2 (FGF2) is important in musculoskeletal homeostasis, therefore the impact of reduction or Fgf2 knockout on skeletal muscle function and phenotype was determined. Gait analysis as well as muscle strength testing in young and old WT and Fgf2KO demonstrated age-related gait disturbances and reduction in muscle strength that were exacerbated in the KO condition. Fgf2 mRNA and protein were significantly decreased in skeletal muscle of old WT compared with young WT. Muscle fiber cross-sectional area was significantly reduced with increased fibrosis and inflammatory infiltrates in old WT and Fgf2KO vs. young WT. Inflammatory cells were further significantly increased in old Fgf2KO compared with old WT. Lipid-related genes and intramuscular fat was increased in old WT and old Fgf2KO with a further increase in fibro-adipocytes in old Fgf2KO compared with old WT. Impaired FGF signaling including Increased β-Klotho, Fgf21 mRNA, FGF21 protein, phosphorylated FGF receptors 1 and 3, was observed in old WT and old Fgf2KO. MAPK/ ERK1/2 was significantly increased in young and old Fgf2KO. We conclude that Fgf2KO, age-related decreased FGF2 in WT mice, and increased FGF21 in the setting of impaired Fgf2 expression likely contribute to impaired skeletal muscle function and sarcopenia in mice.