Skeletal Muscle Deficits Research Articles

PS-B10-11: PATHOPHYSIOLOGICAL STUDY OF COGNITIVE FRAILTY USING A MOUSE MODEL OF ALZHEIMER'S DISEASE

Objective: Hypertension is known to increase the risk of developing dementia including Alzheimer's disease (AD). The concept of cognitive frailty or motoric cognitive risk syndrome, representing a coexistence of physical and cognitive decline, has been attracting attention in the global aging society. However, the molecular mechanisms underlying the pathological interplay between these conditions remain largely unknown. Methods: We investigated the effects of AD pathogenesis on skeletal muscle by using APP23 mice, a mouse model of AD. Three-month-old male APP23 mice and wild-type mice from the same litter were evaluated for skeletal muscle function by grip strength, for cognitive function by passive avoidance and open field tests, and for glucose tolerance and 24-hour voluntary locomotion using a running wheel as factors related to both skeletal muscle function and cognitive function. After dissection, various organs, mainly skeletal muscle, were harvested and wet weight was evaluated. Results: Grip strength and some skeletal muscle weights of APP23 mice were lower than those of wild-type mice. The ipGTT was used to assess glucose tolerance, and the passive avoidance test and open field test were used to assess cognitive function, but there were no significant differences between genotypes. In the running wheel test, which measures voluntary locomotion, APP23 mice were significantly more active than wild-type mice suggesting that the muscle weakness in APP23 mice was not due to disuse. Conclusions: This study demonstrates that APP23 mice exhibit skeletal muscle deficits, including muscle weakness, along with cognitive dysfunction. In addition, APP23 mice showed behavioral abnormalities such as increased voluntary locomotion, which should contribute to increased skeletal muscle function, but skeletal muscle function was conversely decreased, suggesting that the effect of AD on skeletal muscle function is mediated by factors other than behavioral abnormalities due to cognitive dysfunction.

Skeletal muscle deficits are associated with worse exercise performance in pediatric pulmonary hypertension.

BackgroundSkeletal muscle deficits are associated with worse exercise performance in adults with pulmonary hypertension (PH) but the impact is poorly understood in pediatric PH.ObjectiveTo study muscle deficits, physical inactivity, and performance on cardiopulmonary exercise test (CPET) and exercise cardiac magnetic resonance (eCMR) in pediatric PH.MethodsYouth 8–18 years participated in a prospective, cross-sectional study including densitometry (DXA) for measurement of leg lean mass Z-score (LLMZ), handheld dynamometer with generation of dominant and non-dominant handgrip Z-scores, Physical Activity Questionnaire (PAQ), CPET, and optional eCMR. CPET parameters were expressed relative to published reference values. CMR protocol included ventricular volumes and indexed systemic flow at rest and just after supine ergometer exercise. Relationships between LLMZ, PAQ score, and exercise performance were assessed by Pearson correlation and multiple linear regression.ResultsThere were 25 participants (13.7 ± 2.8 years, 56% female, 64% PH Group 1, 60% functional class I); 12 (48%) performed both CPET and eCMR. Mean LLMZ (–0.96 ± 1.14) was associated with PAQ score (r = 50, p = 0.01) and with peak oxygen consumption (VO2) (r = 0.74, p = < 0.001), VO2 at anaerobic threshold (r = 0.65, p < 0.001), and peak work rate (r = 0.64, p < 0.01). Higher handgrip Z-scores were associated with better CPET and eCMR performance. On regression analysis, LLMZ and PAQ score were positively associated with peak VO2, while handgrip Z-score and PAQ score were positively associated with peak work rate.ConclusionMuscle mass and strength are positively associated with exercise performance in pediatric PH. Future studies should determine the effect of rehabilitation programs on muscle properties and exercise performance.

How Well Do Low Population-Specific Values for Muscle Parameters Associate with Indices of Poor Physical Health? Cross-Sectional Data from the Geelong Osteoporosis Study.

We aimed to examine associations between skeletal muscle deficits and indices of poor health. Cut-points for skeletal muscle deficits were derived using data from the Geelong Osteoporosis Study and definitions from the revised European Consensus on Definition and Diagnosis and the Foundation for the National Institutes of Health. Participants (n = 665; 323 women) aged 60–96 year had handgrip strength measured by dynamometry and appendicular lean mass by whole-body dual-energy X-ray absorptiometry. Physical performance was assessed using the Timed Up and Go test. Sex-specific cut-points were equivalent to two standard deviations below the mean young reference range from the Geelong Osteoporosis Study. Indices of poor health included fractures, falls, and hospitalisations. Low trauma fractures since age 50 year (excluding skull, face, digits) were self-reported and confirmed using radiological reports. Falls (≥1 in the past 12 months) and hospitalisations (past month) were self-reported. Logistic regression models (age- and sex-adjusted) were used to examine associations. Receiver Operating Characteristic curves were applied to determine optimal cut-points for handgrip strength, Timed Up and Go, appendicular lean mass/height2, and appendicular lean mass/body mass index that discriminated poor health outcomes. There were 48 participants (6.9%) with hospitalisations, 94 (13.4%) with fractures, and 177 (25.3%) with at least one fall (≥1). For all cut-points, low handgrip strength was consistently associated with falls. There was little evidence to support an association between low appendicular lean mass, using any cut-point, and indices of poor health. Optimal cut-offs for predicting falls (≥1) were: handgrip strength 17.5 kg for women and 33.5 kg for men; Timed Up and Go 8.6 s for women and 9.9 s for men; appendicular lean mass/height2 6.2 kg/m2 for women and 7.46 kg/m2 for men; and appendicular lean mass/body mass index 0.6 m2 for women and 0.9 m2 for men. In conclusion, muscle strength and function performed better than lean mass to indicate poor health. These findings add to the growing evidence base to inform decisions regarding the selection of skeletal muscle parameters and their optimal cut-points for identifying sarcopenia.

Targeting the Ubiquitin-Proteasome System in Limb-Girdle Muscular Dystrophy With CAPN3 Mutations.

LGMDR1 is caused by mutations in the CAPN3 gene that encodes calpain 3 (CAPN3), a non-lysosomal cysteine protease necessary for proper muscle function. Our previous findings show that CAPN3 deficiency leads to reduced SERCA levels through increased protein degradation. This work investigates the potential contribution of the ubiquitin-proteasome pathway to increased SERCA degradation in LGMDR1. Consistent with our previous results, we observed that CAPN3-deficient human myotubes exhibit reduced SERCA protein levels and high cytosolic calcium concentration. Treatment with the proteasome inhibitor bortezomib (Velcade) increased SERCA2 protein levels and normalized intracellular calcium levels in CAPN3-deficient myotubes. Moreover, bortezomib was able to recover mutated CAPN3 protein in a patient carrying R289W and R546L missense mutations. We found that CAPN3 knockout mice (C3KO) presented SERCA deficits in skeletal muscle in the early stages of the disease, prior to the manifestation of muscle deficits. However, treatment with bortezomib (0.8 mg/kg every 72 h) for 3 weeks did not rescue SERCA levels. No change in muscle proteasome activity was observed in bortezomib-treated animals, suggesting that higher bortezomib doses are needed to rescue SERCA levels in this model. Overall, our results lay the foundation for exploring inhibition of the ubiquitin-proteasome as a new therapeutic target to treat LGMDR1 patients. Moreover, patients carrying missense mutations in CAPN3 and presumably other genes may benefit from proteasome inhibition by rescuing mutant protein levels. Further studies in suitable models will be necessary to demonstrate the therapeutic efficacy of proteasome inhibition for different missense mutations.

Development of a Rat Model of Fasciotomy Treatment for Compartment Syndrome.

Skeletal muscle injuries are a major cause of disability for military and civilian populations. Compartment syndrome (CS) in skeletal muscle results from an edema-induced increase in intracompartmental pressure (ICP) after primary injury. Untreated ICP will occlude the tissue vasculature, tissue necrosis, and potential loss of limb. The current standard of care for CS is surgical fasciotomy, an incision through the muscle fascia to relieve ICP. Early fasciotomy will preserve the limb, but often leaves patients with long-term scarring and reduced muscle function. Our group previously developed and characterized a rat model of CS to explore the pathophysiology of CS and test new therapies. We present an expansion of this CS model, including the fasciotomy, to better simulate clinical treatment. CS was induced on the hind limb of adult male Lewis rats and fasciotomy was performed 24 h later. Less than 20% of the rats that underwent fasciotomy showed detectable force 4 days after injury, compared with the 75% of rats that underwent CS induction without fasciotomy. Muscles undergoing fasciotomy showed a significant increase in fibrosis and an increased number of macrophages, Pax7+ satellite cells, and α-smooth muscle actin+ myofibroblasts at 7 days postinjury. These data indicate that the use of fasciotomy in a rat model of CS resulted in injury sequelae that reflect the severity of human clinical disease presentation along with current standard of care. Impact Statement Current animal models of skeletal muscle injury struggle to accurately reflect the injury sequelae seen in humans, particularly in rats and mice. These animals also recover faster than humans do. More accurate recapitulation of the injury is needed to better study the injury progression, as well as screen for novel therapies. This research combines an existing model of compartment syndrome with its clinical standard of care (fasciotomy), creating a more accurate rat model of injury, and providing for a better treatment screening tool. These results show how our model leads to a sustained skeletal muscle deficit with increased inflammation.

AUF1 gene transfer increases exercise performance and improves skeletal muscle deficit in adult mice

Muscle function and mass begin declining in adults long before evidence of sarcopenia and include reduced mitochondrial function, although much remains to be characterized. We found that mRNA decay factor AU-rich mRNA binding factor 1 (AUF1), which stimulates myogenesis, is strongly reduced in skeletal muscle of adult and older mice in the absence of evidence of sarcopenia. Muscle-specific adeno-associated virus (AAV)8-AUF1 gene therapy increased expression of AUF1, muscle function, and mass. AAV8 AUF1 muscle gene transfer in 12-month-old mice increased the levels of activated muscle stem (satellite) cells, increased muscle mass, reduced markers of muscle atrophy, increased markers of mitochondrial content and muscle fiber oxidative capacity, and enhanced exercise performance to levels of 3-month-old mice. With wild-type and AUF1 knockout mice and cultured myoblasts, AUF1 supplementation of muscle fibers was found to increase expression of Peroxisome Proliferator-activated Receptor Gamma Co-activator 1-alpha (PGC1α), a major effector of skeletal muscle mitochondrial oxidative metabolism. AUF1 stabilized and increased translation of the pgc1α mRNA, which is strongly reduced in adult muscle in the absence of AUF1 supplementation. Skeletal muscle-specific gene transfer of AUF1 therefore restores muscle mass, increases exercise endurance, and may provide a therapeutic strategy for age-related muscle loss.

Muscle Function is Associated With Presence, New-onset, or Worsening of Hepatic Steatosis Assessed by Biochemical Parameters, Independent of Muscle Mass

Non-alcoholic fatty liver disease (NAFLD) becomes a major health problem leading to metabolic complications and end-stage liver disease, lacking effective therapeutic interventions. Skeletal muscle deficit, or sarcopenia, is related to accelerated accumulation of ectopic fat at liver. Skeletal muscle can be a novel intervention target of NAFLD. However, previous studies focused on the association between muscle mass and NAFLD, whereas the association of muscle function with NAFLD, a more clinically relevant assessment regarding skeletal muscle, remains unclear. Among participants enrolled between 2013 to 2014 in cardiovascular and Metabolic Diseases Etiology Research Center (CMERC) study (n=807), a cohort of community-dwelling Korean adults to study cardiovascular risk factors, a total of 500 individuals were recruited for 5-year follow-up. Hepatic steatosis was defined by hepatic steatosis index 36 or higher (HSI=8X(AST/ALT)+BMI+2 [if diabetes]+2 [if female]). New-onset of hepatic steatosis was defined as newly developed hepatic steatosis at current follow-up compared to baseline and worsening of HSI was defined as the highest quartile of HSI changes (2 or higher HSI increase). Muscle function was assessed by peak countermovement jump power relative weight (W/kg), 5 times chair rise test (CRT; in seconds), and grip strength (GS; in kg). Appendicular lean mass (ALM) was measured using bioimpedance analysis (InBody770, Biospace, Seoul, Korea). A total of 439 subjects (women 74%; mean age 57 ± 8 year) were analyzed after excluding those with excessive alcohol intake (n=51) and any missing values (n=10). Hepatic steatosis was present in 40% of subjects, which was increased from baseline period (33%, p<0.001). Low peak jump power (adjusted odds ratio [aOR] 1.14 per 1 W/kg decrease), GS (aOR 1.08 per 1kg decrease), and CRT performance (aOR 1.09 per 1 second increase; p<0.05 for all) were all associated with elevated odds of hepatic steatosis, after adjustment for age, gender, height, ALM, and metabolic syndrome components. Compared to those without hepatic steatosis at baseline and follow-up, those with persistent hepatic steatosis had significantly lower jump power in both men and women (33 vs. 40 W/kg in men, p=0.027; 26 vs. 29 W/kg in women, p<0.001). Jump power remained as robust predictor for new-onset hepatic steatosis or worsening of HSI (aOR 1.05 per 1W/kg decrease, p=0.044), whereas GS, CRT, and ALM were not. Muscle function measured by jump power was associated with presence or worsening of hepatic steatosis assessed by biochemical parameters, independent of muscle mass. Acknowledgement: We thank CMERC participants and all research staffs for this work. Funding: This study was supported by research grants from Hanmi Pharmaceutical Co.,Ltd. (4-2018-0845).

Marked skeletal muscle deficits are associated with 6-minute walk distance in paediatric pulmonary hypertension.

Poor growth is common in children with pulmonary hypertension; however, skeletal muscle deficits have not been described and the association between muscle deficits and functional status is unknown. Patients aged 8-18 years with pulmonary hypertension (diagnostic Groups 1, 2, or 3) and World Health Organization functional class I or II underwent dual-energy absorptiometry to measure leg lean mass Z-score (a surrogate for skeletal muscle). Muscle strength was assessed using dynamometry. Physical activity questionnaires were administered. Clinical data, including 6-minute walk distance, were reviewed. Relationships between skeletal muscle, physical activity score, and 6-minute walk distance were assessed by correlations and linear regression. Sixteen patients (12.1 ± 3.2 years, 50% female, 56% Group 1, 56% functional class II) were enrolled. Leg lean mass Z-score was significantly less than reference data (-1.40 ± 1.12 versus 0.0 ± 0.9, p < 0.001) and worse in those with functional class II versus I (-2.10 ± 0.83 versus -0.50 ± 0.73, p < 0.01). Leg lean mass Z-score was positively associated with right ventricular systolic function by tricuspid annular plane systolic Z-score (r = 0.54, p = 0.03) and negatively associated with indexed pulmonary vascular resistance (r = -0.78, p < 0.001). Leg lean mass Z-score and forearm strength were positively associated with physical activity score. When physical activity score was held constant, leg lean mass Z-score independently predicted 6-minute walk distance (R2 = 0.39, p = 0.03). Youth with pulmonary hypertension demonstrate marked skeletal muscle deficits in association with exercise intolerance. Future studies should investigate whether low leg lean mass is a marker of disease severity or an independent target that can be improved.

Model of radiation-induced ambulatory dysfunction

Approximately 50% of oncology patients receive radiotherapy, with the dose of radiation often being limited to mitigate normal tissue damage of proximal critical/sensitive structures. Currently, no preclinical models exist to study a putative drug's efficacy in terms of functional recovery of tissues adversely effected by radiation exposure. Such limitations inhibit the development of targeted therapeutics. To this end, we developed a model of radiation-induced soft-tissue damage with quantitative outcomes of tissue function. Mice received a single administration of radiation focused on the posterior limb while under anesthesia. A blinded observer weighed each mouse and performed motors skills assessments weekly for the duration of the study. At the end of the study, mice were euthanized, assessed for hindlimb range of motion and histological analysis was performed, evaluating tissue morphology and fibrotic scarring. Mice presented numerous significant ambulatory deficits that positively correlated with the dose of radiation administered. Irradiated mice presented with reduced range of motion and increased fibrotic scarring upon histological assessment across all dose groups as compared to controls. The current study is the first to describe a preclinical model of functional skeletal muscle deficits following focal radiation injury of a posterior limb, which will be useful for the assessment of novel drug candidates to treat radiotherapy-induced soft-tissue damage.

Motor axonopathies in a mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease caused by deleterious mutations in the DMD gene which encodes the dystrophin protein. Skeletal muscle weakness and eventual muscle degradation due to loss of dystrophin are well-documented pathological hallmarks of DMD. In contrast, the neuropathology of this disease remains understudied despite the emerging evidence of neurological abnormalities induced by dystrophin loss. Using quantitative morphological analysis of nerve sections, we characterize axonopathies in the phrenic and hypoglossal (XII) nerves of mdx mice. We observe dysfunction in these nerves – which innervate the diaphragm and genioglossus respectively – that we propose contributes to respiratory failure, the most common cause of death in DMD. These observations highlight the importance in the further characterization of the neuropathology of DMD. Additionally, these observations underscore the necessity in correcting both the nervous system pathology in addition to skeletal muscle deficits to ameliorate this disease.

Preferential deficit of fat-free soft tissue in the appendicular region of children with cerebral palsy and proposed statistical models to capture the deficit

Cerebral palsy (CP) is a neurological disorder characterized by a profound skeletal muscle deficit. However, whether there is a regional-specific skeletal muscle deficit in children with CP is unknown. The purpose of this study was to determine whether fat-free soft tissue mass (FFST), a commonly used surrogate for skeletal muscle mass, is more compromised in the limbs than in the trunk in children with CP. A second purpose was to determine whether physical characteristics can be used to accurately estimate appendicular FFST (AFFST) in children with CP. Forty-two children with CP (4-13y) and 42 typically developing children matched to children with CP for sex, age and race were studied. Whole body FFST (FFSTwhole), FFST in the upper limbs (FFSTupper), FFST in the lower limbs (FFSTlower), the ratio of AFFST to height (AFFST/ht), the ratio of AFFST to height2 (AFFST/ht2) and non-appendicular FFST were estimated from dual-energy X-ray absorptiometry. Statistical models were developed to estimate AFFST, AFFST/ht and AFFST/ht2 in both groups of children, and the leave-one-out method was used to validate the models. Children with CP had 21% lower FFSTwhole, 30% lower AFFST, 34% lower FFSTlower, 14% lower non-appendicular FFST, 23% lower AFFST/ht, 19% lower AFFST/ht2 and 9% lower AFFST/FFSTwhole (all p<0.05). Statistical models developed using data from typically developing children overestimated AFFST, AFFST/ht and AFFST/ht2 by 35%, 30% and 21% (all p<0.05), respectively, in children with CP. Separate models developed using data from children with CP yielded better accuracy, with the estimated results highly correlated (r2=0.78, 0.66 and 0.50, respectively; all p<0.001) and not different from calculated AFFST, AFFST/ht and AFFST/ht2 (all p>0.99). However, when the difference in estimated values and measured values of AFFST, AFFST/ht and AFFST/ht2 were plotted against measured values, there was an inverse relationship (r=-0.38,-0.47 and-0.61, respectively, all p<0.05). Children with CP have a remarkable deficit in FFST that is more pronounced in the appendicular than in the non-appendicular region and more pronounced in the lower than in the upper limbs. Preliminary models developed using data from children with CP can provide reasonable estimates of AFFST and indexes of AFFST relative to height, but further development of the models may be needed.

Dysregulation of NRAP degradation by KLHL41 contributes to pathophysiology in nemaline myopathy.

Nemaline myopathy (NM) is the most common form of congenital myopathy that results in hypotonia and muscle weakness. This disease is clinically and genetically heterogeneous, but three recently discovered genes in NM encode for members of the Kelch family of proteins. Kelch proteins act as substrate-specific adaptors for Cullin 3 (CUL3) E3 ubiquitin ligase to regulate protein turnover through the ubiquitin-proteasome machinery. Defects in thin filament formation and/or stability are key molecular processes that underlie the disease pathology in NM; however, the role of Kelch proteins in these processes in normal and diseases conditions remains elusive. Here, we describe a role of NM causing Kelch protein, KLHL41, in premyofibil-myofibil transition during skeletal muscle development through a regulation of the thin filament chaperone, nebulin-related anchoring protein (NRAP). KLHL41 binds to the thin filament chaperone NRAP and promotes ubiquitination and subsequent degradation of NRAP, a process that is critical for the formation of mature myofibrils. KLHL41 deficiency results in abnormal accumulation of NRAP in muscle cells. NRAP overexpression in transgenic zebrafish resulted in a severe myopathic phenotype and absence of mature myofibrils demonstrating a role in disease pathology. Reducing Nrap levels in KLHL41 deficient zebrafish rescues the structural and function defects associated with disease pathology. We conclude that defects in KLHL41-mediated ubiquitination of sarcomeric proteins contribute to structural and functional deficits in skeletal muscle. These findings further our understanding of how the sarcomere assembly is regulated by disease-causing factors in vivo, which will be imperative for developing mechanism-based specific therapeutic interventions.

Patients with inflammatory bowel disease have higher abdominal adiposity and less skeletal mass than healthy controls.

BackgroundAbdominal fat type and distribution have been associated with complicated Crohn’s disease and adverse postoperative outcomes. Few studies have assessed the abdominal distribution of fat and lean stores in patients with inflammatory bowel disease (IBD) and compared this with healthy controls. This retrospective study aimed to compare the abdominal body composition in IBD patients who failed medical treatment and who underwent computed tomography (CT) imaging prior to gastrointestinal surgery with healthy controls. Associations between preoperative abdominal body composition and postoperative outcomes within a year of surgery were explored.MethodsAbdominal body composition was evaluated in 22 presurgical patients with medically refractory IBD (18 with Crohn’s disease) and 22 healthy controls, using routinely acquired CT. Total fat, subcutaneous fat, visceral fat, and skeletal muscle cross-sectional area were measured.ResultsAn independent disease effect was observed, explaining a fat deposition excess of 38 cm2 and a skeletal muscle deficit of 15 cm2 in IBD. Abdominal skeletal muscle correlated with visceral fat for the control (rho=0.51, P=0.015), but not for the IBD group (rho=-0.13, P=0.553). A positive correlation observed between subcutaneous fat with skeletal muscle in the controls (rho=0.47, P=0.026) was inverted in the IBD group (rho=-0.43, P=0.045). Preoperative abdominal body composition was not predictive of postoperative outcomes.ConclusionsA higher degree of abdominal adiposity, a lower skeletal mass and a larger body size for the same anthropometry can be expected in IBD patients. Preoperative abdominal body composition is not associated with surgical outcomes.

Evaluation of Skeletal Muscle Function in Lung Transplant Candidates.

Lung transplantation (LTx) is offered to older and more complex patients who may be at higher risk of skeletal muscle dysfunction, but the clinical implications of this remain uncertain. The study aims were to characterize deficits in skeletal muscle mass, strength and physical performance, and examine the associations of these deficits with clinical outcomes. Fifty LTx candidates (58% men; age, 59 ± 9 years) were prospectively evaluated for skeletal muscle deficits: muscle mass using bioelectrical impedance, quadriceps, respiratory muscle and handgrip strength, and physical performance with the Short Physical Performance Battery. Comparisons between number of muscle deficits (low muscle mass, quadriceps strength and physical performance) and 6-minute walk distance (6MWD), London Chest Activity of Daily Living Questionnaire, and quality of life were assessed using one-way analysis of variance. Associations with pretransplant and posttransplant delisting/mortality, hospital duration, and 3-month posttransplant 6MWD were evaluated using Fisher exact test and Spearman correlation. Deficits in quadriceps strength (n = 27) and physical performance (n = 24) were more common than muscle mass (n = 8). LTx candidates with 2 or 3 muscle deficits (42%) compared with those without any deficits (26%) had worse 6MWD = -109 m (95% confidence interval [CI], -175 to -43), London Chest Activity of Daily Living Questionnaire = 18 (95% CI, 7-30), and St. George's Activity Domain = 12 (95% CI, 2-21). Number of muscle deficits was associated with posttransplant hospital stay (r = 0.34, P = 0.04), but not with delisting/mortality or posttransplant 6MWD. Deficits in quadriceps muscle strength and physical performance are common in LTx candidates and further research is needed to assess whether modifying muscle function pretransplant can lead to improved clinical outcomes.

Maternal creatine supplementation during pregnancy prevents acute and long-term deficits in skeletal muscle after birth asphyxia: a study of structure and function of hind limb muscle in the spiny mouse.

Maternal antenatal creatine supplementation protects the brain, kidney, and diaphragm against the effects of birth asphyxia in the spiny mouse. In this study, we examined creatine's potential to prevent damage to axial skeletal muscles. Pregnant spiny mice were fed a control or creatine-supplemented diet from mid-pregnancy, and 1 d before term (39 d), fetuses were delivered by c-section with or without 7.5 min of birth asphyxia. At 24 h or 33 ± 2 d after birth, gastrocnemius muscles were obtained for ex-vivo study of twitch-tension, muscle fatigue, and structural and histochemical analysis. Birth asphyxia significantly reduced cross-sectional area of all muscle fiber types (P < 0.05), and increased fatigue caused by repeated tetanic contractions at 24 h of age (P < 0.05). There were fewer (P < 0.05) Type I and IIa fibers and more (P < 0.05) Type IIb fibers in male gastrocnemius at 33 d of age. Muscle oxidative capacity was reduced (P < 0.05) in males at 24 h and 33 d and in females at 24 h only. Maternal creatine treatment prevented all asphyxia-induced changes in the gastrocnemius, improved motor performance. This study demonstrates that creatine loading before birth protects the muscle from asphyxia-induced damage at birth.

Role of Endolysosomes in Skeletal Muscle Pathology Observed in a Cholesterol-Fed Rabbit Model of Alzheimer’s Disease

Deficits in skeletal muscles contribute not only to the functional decline in people living with Alzheimer’s disease (AD), but also to AD pathogenesis. We have shown that endolysosome dysfunction plays an important role in the development of AD pathological features in a cholesterol-fed rabbit model of AD. Interestingly we observed in skeletal muscle from the rabbit AD model increased deposition of Aβ, phosphorylated tau, and ubiquitin. Here, we tested the hypothesis that endolysosome dysfunction commonly occurs in skeletal muscle and brain in this rabbit model of AD. In skeletal muscle of rabbits fed a 2% cholesterol-enriched diet for 12 weeks we observed the presence of abnormally enlarged endolysosomes, in which were increased accumulations of free cholesterol and multiple AD marker proteins subject to misfolding and aggregation including Aβ, phosphorylated tau, and ubiquitin. Moreover, in skeletal muscle of rabbits fed the cholesterol-enriched diet we observed decreased specific activities of three different lysosome enzymes. Our results suggest that elevated levels of plasma cholesterol can disturb endolysosome structure and function as well as promote the development of AD-like pathological features in skeletal muscle and that these organellar changes might contribute to the development of skeletal muscle deficits in AD.

622. Systemic β-Sarcoglycan Gene Therapy for Treatment of Cardiac and Skeletal Muscle Deficits in LGMD2E

Limb-girdle muscular dystrophy type 2E (LGMD2E) results from mutations in the beta-sarcoglycan (SGCB) gene causing loss of a sarcolemmal structural protein component of the dystrophin-associated protein complex (DAPC). This leads to a progressive dystrophy with numerous histopathological features, resulting in deteriorating muscle function. This occurs not only in limb muscle but also in the diaphragm and the heart. Consequences include respiratory failure and cardiomyopathy in 50% or more of LGMD2E patients. SGCB knockout mice share many of the phenotypic deficiencies of LGMD2E patients. In this mouse model we quantified dystrophic histopathology, fibrosis, and functional outcomes in lower limb, upper limb, and torso skeletal muscles, as well as the diaphragm and cardiac muscles. Diaphragms of SGCB-/- mice demonstrated reduced specific force output (116.24 mN/mm2) compared to wild-type (WT) mice (236.67 mN/mm2), and hearts from SGCB-/- mice had lower ejection fraction (58.19%) compared to WT mice (75.04%) as determined by MRI. Additionally, radiographic imaging defined the degree of kyphoscoliosis in SGCB-/- mice. Laser monitoring of open-field cage activity showed a reduction of ~55% in both total ambulation as well as hindlimb vertical rearing in SGCB-/- mice compared to WT. For treatment, we designed a self-complementary AAVrh74 vector containing a codon optimized human SGCB transgene driven by a muscle specific promoter. We next demonstrated efficacy of vector delivery by intramuscular (IM) injection to the tibialis anterior muscle, as well as isolated-limb perfusion (ILP) to the lower limb muscles of SGCB-/- mice. Along with restoration of SGCB expression in treated muscle, we saw histological and functional improvements and a reduction in fibrosis as indicated by reduced collagen deposition. These regional gene delivery studies were followed by systemic delivery of scAAV.hSGCB through the tail vein of SGCB-/- mice to provide a potential rationale for gene delivery in clinical trial that would lead to clinically meaningful results. Tail vein injection of scAAV.hSGCB resulted in nearly 100% transgene expression in numerous muscles throughout the hindlimbs, forelimbs, torso, and the heart, that was accompanied by improvements in histopathology including reduction in central nucleation and increased fiber diameter. Kyphoscoliosis of the spine was also improved, and total ambulation increased in scAAV.hSGCB treated mice by ~22% while hindlimb vertical rearing increased by ~77% in treated mice compared to KO. We also saw complete restoration of diaphragm function following treatment with specific force output improved to 226.07 mN/mm2. In this well-defined mode of LGMD2E, we have demonstrated that systemic delivery of scAAV.hSGCB normalizes histologic and functional outcome measures in limb, diaphragm, and heart. These findings have established a path for AAV mediated gene therapy for LGMD2E that we are currently pursuing.

Creatine supplementation in the aging population: effects on skeletal muscle, bone and brain.

This narrative review aims to summarize the recent findings on the adjuvant application of creatine supplementation in the management of age-related deficits in skeletal muscle, bone and brain metabolism in older individuals. Most studies suggest that creatine supplementation can improve lean mass and muscle function in older populations. Importantly, creatine in conjunction with resistance training can result in greater adaptations in skeletal muscle than training alone. The beneficial effect of creatine upon lean mass and muscle function appears to be applicable to older individuals regardless of sex, fitness or health status, although studies with very old (>90years old) and severely frail individuals remain scarce. Furthermore, there is evidence that creatine may affect the bone remodeling process; however, the effects of creatine on bone accretion are inconsistent. Additional human clinical trials are needed using larger sample sizes, longer durations of resistance training (>52weeks), and further evaluation of bone mineral, bone geometry and microarchitecture properties. Finally, a number of studies suggest that creatine supplementation improves cognitive processing under resting and various stressed conditions. However, few data are available on older adults, and the findings are discordant. Future studies should focus on older adults and possibly frail elders or those who have already experienced an age-associated cognitive decline.

Combined Strategies for Maintaining Skeletal Muscle Mass and Function in Aging: Myostatin Inactivation and AICAR-Associated Oxidative Metabolism Induction.

Myostatin (mstn) blockade, resulting in muscle hypertrophy, is a promising therapy to counteract age-related muscle loss. However, oxidative and mitochondrial deficit observed in young mice with myostatin inhibition could be detrimental with aging. The aim of this study was (a) to bring original data on metabolic and mitochondrial consequences of mstn inhibition in old mice, and (b) to examine whether 4-weeks of AICAR treatment, a pharmacological compound known to upregulate oxidative metabolism, may be useful to improve exercise capacity and mitochondrial deficit of 20-months mstn KO versus wild-type (WT) mice. Our results show that despite the enlarged muscle mass, the oxidative and mitochondrial deficit associated with reduced endurance running capacity is maintained in old mstn KO mice but not worsened by aging. Importantly, AICAR treatment induced a significant beneficial effect on running limit time only in old mstn KO mice, with a marked increase in PGC-1α expression and slight beneficial effects on mitochondrial function. We showed that AICAR effects were autophagy-independent. This study underlines the relevance of aged muscle remodelling by complementary approaches that impact both muscle mass and function, and suggest that mstn inhibition and aerobic metabolism activators should be co-developed for delaying age-related deficits in skeletal muscle.

Lean mass deficits, vitamin D status and exercise capacity in children and young adults after Fontan palliation

ObjectiveWe sought to evaluate body composition in children and young adults with Fontan physiology. Leg lean mass (LM) deficits correlate with diminished exercise capacity in other populations and may contribute...