Clinical trials delivering high doses of adeno-associated viruses (AAVs) expressing truncated dystrophin molecules (micro-dystrophins) are underway for individuals with Duchenne muscular dystrophy (DMD). We examined the efficiency and efficacy of this strategy with four micro-dystrophin constructs (three in clinical trials and a variant of the largest clinical construct), in a severe mouse model of DMD, using doses of AAV comparable to those used in the clinical trials. We achieved high levels of micro-dystrophin expression in striated muscle with cardiac expression ~10 fold higher than that observed in skeletal muscle. Significant, albeit incomplete, correction of the skeletal muscle disease was observed. Surprisingly, a lethal acceleration of cardiac disease progression occurred with two of the micro-dystrophins. The detrimental impact on the heart appears to be caused by the high levels of micro-dystrophin resulting in variable competition (dependent on the design of the micro-dystrophin) between micro-dystrophin and utrophin at the cardiomyocyte membrane. There may also be a contribution from an overloading of protein degradation. The significance of these observations for patients currently being treated with AAV-micro-dystrophin therapies is unclear since the levels of expression being achieved in the DMD hearts are unknown. However, it suggests that micro-dystrophin treatments need to avoid excessively high levels of expression in the heart and cardiac function should be carefully monitored in these patients.
Cora C. Hart, Young il Lee, Jun Xie, Guangping Gao, Brian L. Lin, David W. Hammers, H. Lee Sweeney
Chronic kidney disease (CKD) causes an accumulation of uremic metabolites that negatively impact skeletal muscle function. Tryptophan-derived uremic metabolites are agonists of the aryl hydrocarbon receptor (AHR) which has been shown to be activated in the blood of CKD patients. This study investigated the role of the AHR in skeletal muscle pathology of CKD. Compared to control participants with normal kidney function, AHR-dependent gene expression (CYP1A1 and CYP1B1) was significantly upregulated in skeletal muscle of patients with CKD (P=0.032) and the magnitude of AHR activation was inversely correlated with mitochondrial respiration (P<0.001). In mice with CKD, muscle mitochondrial oxidative phosphorylation (OXPHOS) was significantly impaired and strongly correlated with both the serum level of tryptophan-derived uremic metabolites and AHR activation. Muscle-specific deletion of the AHR significantly improved mitochondrial OXPHOS in male mice with the greatest uremic toxicity (CKD+probenecid) and abolished the relationship between uremic metabolites and OXPHOS. The uremic metabolite-AHR-mitochondrial axis in skeletal muscle was further confirmed using muscle-specific AHR knockdown in C57BL6J that harbour a high-affinity AHR allele, as well as ectopic viral expression of constitutively active mutant AHR in mice with normal renal function. Notably, OXPHOS changes in AHRmKO mice were only present when mitochondria were fueled by carbohydrates. Further analyses revealed that AHR activation in mice led to significant increases in Pdk4 expression (P<0.05) and phosphorylation of pyruvate dehydrogenase enzyme (P<0.05). These findings establish a uremic metabolite-AHR-Pdk4 axis in skeletal muscle that governs mitochondrial deficits in carbohydrate oxidation during CKD.
Trace Thome, Nicholas A. Vugman, Lauren E. Stone, Keon Wimberly, Salvatore T. Scali, Terence E. Ryan
Skeletal muscle wasting results from numerous pathological conditions impacting both the musculoskeletal and nervous systems. A unifying feature of these pathologies is the upregulation of members of the E3 ubiquitin ligase family, resulting in increased proteolytic degradation of target proteins. Despite the critical role E3 ubiquitin ligases in regulating muscle mass, the specific proteins they target for degradation and the mechanisms by which they regulate skeletal muscle homeostasis remain ill-defined. Here, using zebrafish loss of function models combined with in vivo cell biology and proteomic approaches, we reveal a role of atrogin-1 in regulating the levels of the endoplasmic reticulum chaperone BiP. Loss of atrogin-1 results in an accumulation of BiP, leading to impaired mitochondrial dynamics and a subsequent loss in muscle fibre integrity. We further implicate a disruption in atrogin-1 mediated BiP regulation in the pathogenesis of Duchenne muscular dystrophy. We reveal that BiP is not only upregulated in Duchenne muscular dystrophy, but its inhibition using pharmacological strategies, or by upregulating atrogin-1, significantly ameliorates pathology in a zebrafish model of Duchenne muscular dystrophy. Collectively, our data implicates atrogin-1 and BiP in the pathogenesis of Duchenne muscular dystrophy, and highlights atrogin-1’s essential role in maintaining muscle homeostasis.
Avnika A. Ruparelia, Margo Montandon, Jo Merriner, Cheng Huang, Siew Fen Lisa Wong, Carmen Sonntag, Justin P. Hardee, Gordon S. Lynch, Lee B. Miles, Ashley Siegel, Thomas E. Hall, Ralf B. Schittenhelm, Peter D. Currie
Tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK) are clinically overlapping disorders characterized by childhood-onset muscle weakness and a variable occurrence of multisystemic signs, including short stature, thrombocytopenia, and hyposplenism. TAM/STRMK is caused by gain-of-function mutations in the Ca2+ sensor STIM1 or the Ca2+ channel ORAI1, both of which regulate Ca2+ homeostasis through the ubiquitous store-operated Ca2+ entry (SOCE) mechanism. Functional experiments in cells have demonstrated that the TAM/STRMK mutations induce SOCE overactivation, resulting in excessive influx of extracellular Ca2+. There is currently no treatment for TAM/STRMK, but SOCE is amenable to manipulation. Here, we crossed Stim1R304W/+ mice harboring the most common TAM/STRMK mutation with Orai1R93W/+ mice carrying an ORAI1 mutation partially obstructing Ca2+ influx. Compared with Stim1R304W/+ littermates, Stim1R304W/+Orai1R93W/+ offspring showed a normalization of bone architecture, spleen histology, and muscle morphology; an increase of thrombocytes; and improved muscle contraction and relaxation kinetics. Accordingly, comparative RNA-Seq detected more than 1,200 dysregulated genes in Stim1R304W/+ muscle and revealed a major restoration of gene expression in Stim1R304W/+Orai1R93W/+ mice. Altogether, we provide physiological, morphological, functional, and molecular data highlighting the therapeutic potential of ORAI1 inhibition to rescue the multisystemic TAM/STRMK signs, and we identified myostatin as a promising biomarker for TAM/STRMK in humans and mice.
Roberto Silva-Rojas, Laura Pérez-Guàrdia, Alix Simon, Sarah Djeddi, Susan Treves, Agnès Ribes, Lorenzo Silva-Hernández, Céline Tard, Jocelyn Laporte, Johann Böhm
The polymerization of myosin molecules into thick filaments in muscle sarcomeres is essential for cardiac contractility, with the attenuation of interactions between the heads of myosin molecules within the filaments being proposed to result in hypercontractility, as observed in hypertrophic cardiomyopathy (HCM). However, experimental evidence demonstrates the structure of these giant macromolecular complexes is highly dynamic, with molecules exchanging between the filaments and a pool of soluble molecules on the minute timescale. Therefore, we sought to test the hypothesis that the enhancement of interactions between the heads of myosin molecules within thick filaments limits the mobility of myosin by taking advantage of mavacamten, a small molecule approved for the treatment of HCM. Myosin molecules were labeled in vivo with a green fluorescent protein (GFP) and imaged in intact hearts using multiphoton microscopy. Treatment of the intact hearts with mavacamten resulted in an unexpected >5-fold enhancement in GFP-myosin mobility within the sarcomere. In vitro biochemical assays suggested that mavacamten enhanced the mobility of GFP-myosin by increasing the solubility of myosin molecules, through the stabilization of a compact/folded conformation of the molecules, once disassociated from the thick filaments. These findings provide alternative insight into the mechanisms by which molecules exchange into and out of thick filaments and have implications for how mavacamten may impact cardiac contractility.
Colleen M. Kelly, Jody L. Martin, Michael J. Previs
BACKGROUND While the benefits of statin therapy on atherosclerotic cardiovascular disease are clear, patients often experience mild to moderate skeletal myopathic symptoms, the mechanism for which is unknown. This study investigated the potential effect of high-dose atorvastatin therapy on skeletal muscle mitochondrial function and whole-body aerobic capacity in humans.METHODS Eight overweight (BMI, 31.9 ± 2.0) but otherwise healthy sedentary adults (4 females, 4 males) were studied before (day 0) and 14, 28, and 56 days after initiating atorvastatin (80 mg/d) therapy.RESULTS Maximal ADP-stimulated respiration, measured in permeabilized fiber bundles from muscle biopsies taken at each time point, declined gradually over the course of atorvastatin treatment, resulting in > 30% loss of skeletal muscle mitochondrial oxidative phosphorylation capacity by day 56. Indices of in vivo muscle oxidative capacity (via near-infrared spectroscopy) decreased by 23% to 45%. In whole muscle homogenates from day 0 biopsies, atorvastatin inhibited complex III activity at midmicromolar concentrations, whereas complex IV activity was inhibited at low nanomolar concentrations.CONCLUSION These findings demonstrate that high-dose atorvastatin treatment elicits a striking progressive decline in skeletal muscle mitochondrial respiratory capacity, highlighting the need for longer-term dose-response studies in different patient populations to thoroughly define the effect of statin therapy on skeletal muscle health.FUNDING NIH R01 AR071263.
Terence E. Ryan, Maria J. Torres, Chien-Te Lin, Angela H. Clark, Patricia M. Brophy, Cheryl A. Smith, Cody D. Smith, E. Matthew Morris, John P. Thyfault, P. Darrell Neufer
The Murphy Roths Large (MRL) mouse strain has “super-healing” properties that enhance recovery from injury. In mice, the DBA/2J strain intensifies many aspects of muscular dystrophy so we evaluated the ability of the MRL strain to suppress muscular dystrophy in the Sgcg null mouse model of limb girdle muscular dystrophy. A comparative analysis of Sgcg null mice in the DBA/2J versus MRL strains showed greater myofiber regeneration with reduced structural degradation of muscle in the MRL strain. Transcriptomic profiling of dystrophic muscle indicated strain-dependent expression of the extracellular matrix (ECM) and TGF-β signaling genes. To investigate the MRL ECM, cellular components were removed from dystrophic muscle sections to generate decellularized myoscaffolds. Decellularized myoscaffolds from dystrophic mice in the protective MRL strain had significantly less deposition of collagen and matrix-bound TGF-β1 and TGF-β3 throughout the matrix. Dystrophic myoscaffolds from the MRL background, but not the DBA/2J background, were enriched in myokines like IGF-1 and IL-6. C2C12 myoblasts seeded onto decellularized matrices from Sgcg–/– MRL and Sgcg–/– DBA/2J muscles showed the MRL background induced greater myoblast differentiation compared to dystrophic DBA/2J myoscaffolds. Thus, the MRL background imparts its effect through a highly regenerative ECM, which is active even in muscular dystrophy.
Joseph G. O'Brien, Alexander B. Willis, Ashlee M. Long, Jason M. Kwon, GaHyun Lee, Frank W. Li, Patrick G.T. Page, Andy H. Vo, Michele Hadhazy, Melissa J. Spencer, Rachelle H. Crosbie, Alexis R. Demonbreun, Elizabeth M. McNally
Cachexia is a debilitating skeletal muscle wasting condition for which we currently lack effective treatments. In the context of cancer, certain chemotherapeutics cause DNA damage and cellular senescence. Senescent cells exhibit chronic activation of the transcription factor nuclear factor (NF)-κB, a known mediator of the pro-inflammatory senescence-associated secretory phenotype (SASP) and skeletal muscle atrophy. Thus, targeting NF-κB represents a logical therapeutic strategy to alleviate unintended consequences of genotoxic drugs. Herein, we show that treatment with the IKK/NF-κB inhibitor SR12343 during a course of chemotherapy reduces markers of cellular senescence and the SASP in liver, skeletal muscle, and circulation and, correspondingly, attenuates features of skeletal muscle pathology. Lastly, we demonstrate SR12343 mitigates chemotherapy-induced reductions in body weight, lean mass, fat mass, and muscle strength. These findings support senescent cells as a promising druggable target to counteract the SASP and skeletal muscle wasting in the context of chemotherapy.
Davis A. Englund, Alyssa M. Jolliffe, Gabriel J. Hanson, Zaira Aversa, Xu Zhang, Xinyi Jiang, Thomas A. White, Lei Zhang, David G. Monroe, Paul D. Robbins, Laura J. Niedernhofer, Theodore M. Kamenecka, Sundeep Khosla, Nathan K. LeBrasseur
Hyperuricemia is implicated in numerous pathologies but the mechanisms underlying uric acid production are poorly understood. Using a combination of mouse studies, cultured cell studies, and human serum samples, we sought to determine the cellular source of uric acid. In mice, fasting and glucocorticoid treatment increased serum uric acid and uric acid release from ex vivo incubated skeletal muscle. In vitro, glucocorticoids and the transcription factor FoxO3 increased purine nucleotide degradation and purine release from differentiated muscle cells, which coincided with the transcriptional upregulation of AMP deaminase 3, a rate-limiting enzyme in adenine nucleotide degradation. Heavy isotope tracing during co-culture experiments revealed that oxidation of muscle purines to uric acid required their transfer from muscle cells to a cell type that expresses xanthine oxidoreductase, such as endothelial cells. Lastly, in healthy women, matched for age and body composition, serum uric acid was greater in individuals scoring below average on standard physical function assessments. Together, these studies reveal skeletal muscle purine degradation is an underlying driver of uric acid production, with the final step of uric acid production occurring primarily in a non-muscle cell type. This suggests that skeletal muscle fiber purine degradation may represent a therapeutic target to reduce serum uric acid and treat numerous pathologies.
Spencer G. Miller, Catalina Matias, Paul S. Hafen, Andrew S. Law, Carol A. Witczak, Jeffrey J. Brault
BACKGROUND. Although 25-hydroxyvitamin D (25(OH)D) concentrations ≥30ng/mL are known to reduce injury risk and boost strength, the influence on anterior cruciate ligament reconstruction (ACLR) outcomes remains unexamined. This study aimed to define the vitamin D signaling response to ACLR, assess the relationship between vitamin D status and muscle fiber cross-sectional area (CSA) and bone density outcomes, and discover vitamin D receptor (VDR) targets post-ACLR. METHODS. 21 young, healthy, physically active participants with recent ACL tears were enrolled (17.8 ± 3.2 yr, BMI: 26.0 ± 3.5 kg/m2). Data were collected through blood samples, vastus lateralis biopsies, DXA bone density measurements, and isokinetic dynamometer measures at baseline, 1 week, 4 months, and 6 months post-ACLR. The biopsies facilitated CSA, western blot, RNA-seq, and VDR ChIP-seq analyses. RESULTS. ACLR surgery led to decreased circulating bioactive vitamin D and increased VDR and activating enzyme expression in skeletal muscle one week post-operation. Participants with < 30 ng/mL 25(OH)D levels (n = 13) displayed more significant quadriceps fiber CSA loss one week and 4 months post-ACLR than those with ≥30 ng/mL (n = 8; P < 0.01 for post-hoc comparisons; P = 0.041 for time x vitamin D status interaction). RNA-seq and ChIP-seq data integration revealed genes associated with energy metabolism and skeletal muscle recovery, potentially mediating the impact of vitamin D status on ACLR recovery. No difference in bone mineral density (BMD) losses between groups was observed. CONCLUSION. Correcting vitamin D status prior to ACLR may aid in preserving skeletal muscle during recovery.
Yuan Wen, Christine M. Latham, Angelique N. Moore, Nicholas T. Thomas, Brooke D. Lancaster, Kelsey A. Reeves, Alexander R. Keeble, Christopher S. Fry, Darren L. Johnson, Katherine L. Thompson, Brian Noehren, Jean L. Fry
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