Search

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inherited disorder of mitochondrial fatty acid β-oxidation (FAO) in humans. Patients exhibit clinical episodes often associated with fasting. Symptoms include hypoketotic hypoglycemia and Reye-like episodes. With limited treatment options, we explored the use of human MCAD (hMCAD) mRNA in fibroblasts from patients with MCAD deficiency to provide functional MCAD protein and reverse the metabolic block. Transfection of hMCAD mRNA into MCAD- deficient patient cells resulted in an increased MCAD protein that localized to mitochondria, concomitant with increased enzyme activity in cell extracts. The therapeutic hMCAD mRNA-lipid nanoparticle (LNP) formulation was also tested in vivo in Acadm−/− mice. Administration of multiple intravenous doses of the hMCAD mRNA-LNP complex (LNP-MCAD) into Acadm−/− mice produced a significant level of MCAD protein with increased enzyme activity in liver, heart and skeletal muscle homogenates. Treated Acadm−/− mice were more resistant to cold stress and had decreased plasma levels of medium-chain acylcarnitines compared to untreated animals. Furthermore, hepatic steatosis in the liver from treated Acadm−/− mice was reduced compared to untreated ones. Results from this study support the potential therapeutic value of hMCAD mRNA-LNP complex treatment for MCAD deficiency.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is an inborn error of long chain fatty acid β-oxidation (FAO) with limited treatment options. Patients present with heterogeneous clinical phenotypes affecting predominantly heart, liver, and skeletal muscle. While VLCAD deficiency is a systemic disease, restoration of liver FAO has the potential to improve symptoms more broadly due to increased total body ATP production and reduced accumulation of potentially toxic metabolites. We explored the use of synthetic human VLCAD (hVLCAD) mRNA and lipid nanoparticle encapsulated hVLCAD mRNA (LNP-VLCAD) to generate functional VLCAD enzyme in patient fibroblasts derived from VLCAD deficient patients, mouse embryonic fibroblasts, hepatocytes isolated from VLCAD knockout (Acadvl

Objective To define the transcriptomic changes responsible for the histologic alterations in skeletal muscle and their progression in collagen VI‐related muscular dystrophy (COL6‐RD). Methods COL6‐RD patient muscle biopsies were stratified into three groups based on the overall level of pathologic severity considering degrees of fibrosis, muscle fiber atrophy, and fatty replacement of muscle tissue. Using microarray and RNA‐Seq, we then performed global gene expression profiling on the same muscle biopsies and compared their transcriptome with age‐ and sex‐matched controls. Results COL6‐RD muscle biopsy transcriptomes as a group revealed prominent upregulation of muscle extracellular matrix component genes and the downregulation of skeletal muscle and mitochondrion‐specific genes. Upregulation of the TGFβ pathway was the most conspicuous change across all biopsies and was fully evident even in the mildest/earliest histological group. There was no difference in the overall transcriptional signature between the different histologic groups but polyserial analysis identified relative changes along with COL6‐RD histological severity. Interpretation Overall, our study establishes the prominent dysregulation of extracellular matrix genes, TGFβ signaling, and its downstream cellular pathways at the transcriptomic level in COL6‐RD muscle.

Striated muscle needs to maintain cellular homeostasis in adaptation to increases in physiological and metabolic demands. Failure to do so can result in rhabdomyolysis. The identification of novel genetic conditions associated with rhabdomyolysis helps to shed light on hitherto unrecognized homeostatic mechanisms. Here we report seven individuals in six families from different ethnic backgrounds with biallelic variants in MLIP, which encodes the muscular lamin A/C-interacting protein, MLIP. Patients presented with a consistent phenotype characterized by mild muscle weakness, exercise-induced muscle pain, variable susceptibility to episodes of rhabdomyolysis, and persistent basal elevated serum creatine kinase levels. The biallelic truncating variants were predicted to result in disruption of the nuclear localizing signal of MLIP. Additionally, reduced overall RNA expression levels of the predominant MLIP isoform were observed in patients’ skeletal muscle. Collectively, our data increase the understanding of the genetic landscape of rhabdomyolysis to now include MLIP as a novel disease gene in humans and solidifies MLIP’s role in normal and diseased skeletal muscle homeostasis.

Skeletal muscle regeneration following injury is a complex multi-stage process involving the recruitment of inflammatory cells, the activation of muscle resident fibroblasts, and the differentiation of activated myoblasts into myocytes. Dysregulation of these cellular processes is associated with ineffective myofiber repair and excessive deposition of extracellular matrix proteins leading to fibrosis. PI3K/Akt signaling is a critical integrator of intra- and intercellular signals connecting nutrient availability to cell survival and growth. Activation of the PI3K/Akt pathway in skeletal muscle leads to hypertrophic growth and a reversal of the changes in body composition associated with obesity and advanced age. Though the molecular mechanisms mediating these effects are incompletely understood, changes in paracrine signaling are thought to play a key role. Here, we utilized modified RNA to study the biological role of the transient translocation of Akt to the myonuclei of maturing myotubes. Using a conditioned medium model system, we show that ectopic myonuclear Akt suppresses fibrogenic paracrine signaling in response to oxidative stress, and that interventions that increase or restore myonuclear Akt may impair fibrosis.

Glycogen Storage Disease 1a (GSD1a) is a rare, inherited metabolic disorder caused by deficiency of glucose 6-phosphatase (G6Pase-α). G6Pase-α is critical for maintaining interprandial euglycemia. GSD1a patients exhibit life-threatening hypoglycemia and long-term liver complications including hepatocellular adenomas (HCAs) and carcinomas (HCCs). There is no treatment for GSD1a and the current standard-of-care for managing hypoglycemia (Glycosade®/modified cornstarch) fails to prevent HCA/HCC risk. Therapeutic modalities such as enzyme replacement therapy and gene therapy are not ideal options for patients due to challenges in drug-delivery, efficacy, and safety. To develop a new treatment for GSD1a capable of addressing both the life-threatening hypoglycemia and HCA/HCC risk, we encapsulated engineered mRNAs encoding human G6Pase-α in lipid nanoparticles. We demonstrate the efficacy and safety of our approach in a preclinical murine model that phenotypically resembles the human condition, thus presenting a potential therapy that could have a significant therapeutic impact on the treatment of GSD1a., Glycogen Storage Disease 1a (Gsd1a) is an inherited disorder caused by glucose 6-phosphatase (G6Pase-α) deficiency and characterized by hypoglycaemia and high risk of liver cancer. Here the authors develop a mRNA-based G6Pase-α delivery therapy that is efficacious and safe in a mouse model of GSD1a.

Alpha 1-antitrypsin (AAT) deficiency arises from an inherited mutation in the SERPINA1 gene. The disease causes damage in the liver where the majority of the AAT protein is produced. Lack of functioning circulating AAT protein also causes uninhibited elastolytic activity in the lungs leading to AAT deficiency-related emphysema. The only therapy apart from liver transplantation is augmentation with human AAT protein pooled from sera, which is only reserved for patients with advanced lung disease caused by severe AAT deficiency. We tested modified mRNA encoding human AAT in primary human hepatocytes in culture, including hepatocytes from AAT deficient patients. Both expression and functional activity were investigated. Secreted AAT protein increased from 1,14 to 3,43 µg/ml in media from primary human hepatocytes following mRNA treatment as investigated by ELISA and western blot. The translated protein showed activity and protease inhibitory function as measured by elastase activity assay. Also, mRNA formulation in lipid nanoparticles was assessed for systemic delivery in both wild type mice and the NSG-PiZ transgenic mouse model of AAT deficiency. Systemic intravenous delivery of modified mRNA led to hepatic uptake and translation into a functioning protein in mice. These data support the use of systemic mRNA therapy as a potential treatment for AAT deficiency.

Signal transducer and activator of transcription 3 (STAT3) signaling plays critical roles in regulating skeletal muscle mass, repair, and diseases. In this review, we discuss the upstream activators of STAT3 in skeletal muscles, with a focus on interleukin 6 (IL6) and transforming growth factor beta 1 (TGF-β1). We will also discuss the double-edged effect of STAT3 activation in the muscles, including the role of STAT3 signaling in muscle hypertrophy induced by exercise training or muscle wasting in cachectic diseases and muscular dystrophies. STAT3 is a critical regulator of satellite cell self-renewal after muscle injury. STAT3 knock out affects satellite cell myogenic progression by impairing proliferation and inducing premature differentiation. Recent studies in STAT3 signaling demonstrated its direct role in controlling myogenic capacity of myoblasts and satellite cells, as well as the potential benefit in using STAT3 inhibitors to treat muscle diseases. However, prolonged STAT3 activation in muscles has been shown to be responsible for muscle wasting by activating protein degradation pathways. It is important to balance the extent of STAT3 activation and the duration and location (cell types) of the STAT3 signaling when developing therapeutic interventions. STAT3 signaling in other tissues and organs that can directly or indirectly affects skeletal muscle health are also discussed.

UNLABELLED: Profiling of protein species is important because gene polymorphisms, splice variations and post-translational modifications may combine and give rise to multiple protein species that have different effects on cellular function. Two-dimensional gel electrophoresis is one of the most robust methods for differential analysis of protein species, but bioinformatic interrogation is challenging because the consequences of changes in the abundance of individual protein species on cell function are unknown and cannot be predicted. We conducted DIGE of soleus muscle from male and female rats artificially selected as either high- or low-capacity runners (HCR and LCR, respectively). In total 696 protein species were resolved and LC-MS/MS identified proteins in 337 spots. Forty protein species were differentially (P

Transforming growth factor beta 1 (TGFβ1) is a key player in skeletal muscle degenerative and regenerative processes. We previously showed that conditionally overexpressing TGFβ1 in skeletal muscles caused myofiber atrophy and endomysial fibrosis in mice. However, the disease severity varied significantly among individual mice. While 40% of mice developed severe muscle pathology and lost body weight within 2 weeks of TGFβ1 transgene induction in muscles, the rest showed milder or no phenotype. This study aims at determining whether signal transducer and activator of transcription 3 (STAT3) plays a role in the phenotypic difference and whether it can be activated by TGFβ1 directly in muscle cells. Our results show that while total STAT3 was not differentially expressed between the two groups of mice, there was significantly higher pSTAT3 (Tyr705) in the muscles of the mice with severe phenotype. Immunohistochemistry showed that pSTAT3 (Tyr705) was localized in approximately 50% of the nuclei of the muscles. We further showed that TGFβ1 induced Tyr705 phosphorylation of STAT3 in C2C12 cells within 30 minutes of treatment while total STAT3 was not affected. Our findings suggest that TGFβ1 alone can induce Tyr705 phosphorylation of STAT3 in skeletal muscle cells and contribute to disease severity in transgenic TGFβ1 mice.

Platelet-derived growth factors (PDGFs) belong to a family of polypeptide growth factors that signal through cell surface tyrosine kinase receptors to stimulate growth, proliferation and differentiation. Platelet-derived growth factor receptors (PDGFRs) are also considered important targets for specific kinase inhibitors in the treatment of several human tumours. The aim of this study was to investigate the role of PDGF-A, PDGFB, PDGFR-alpha and PDGFR-beta in canine lymphoma by determining gene and protein expression in lymph nodes of dogs with diffuse large B-cell lymphoma (DLBCL), peripheral T-cell lymphoma (PTCL), T-Iymphoblastic lymphoma (T-LBL) and in healthy control dogs. One lymph node was also studied at the end of therapy in a subset of dogs in remission for DLBCL. In controls, PDGF-A, PDGFR-alpha and PDGFR-beta mRNA levels were significantly higher than in DLBCLs, PTCLs and T-LBLs. However, PDGFR-alpha and PDGFR-beta were minimally expressed by lymphocytes and plasma cells in normal lymph nodes as determined by immunohistochemistry, while neoplastic B and T cells showed the highest score (P

The most promising techniques for detecting minimal residual disease (MRD) in canine lymphoma are flow cytometry (FC) and polymerase chain reaction amplification of antigen receptor genes (PARR). However, the agreement between these methods has not been established. MRD was monitored by FC and PARR following treatment of dogs affected with diffuse large B-cell lymphoma (DLBCL), comparing results in lymph node (LN), peripheral blood (PB) and bone marrow (BM) samples. The prognostic impact of MRD on time to relapse (TTR) and lymphoma-specific survival (LSS) was also assessed.Fourteen dogs with previously untreated DLBCL were enrolled into the study; 10 dogs eventually relapsed, while four dogs with undetectable MRD were still in remission at the end of the study. At diagnosis, the concordance rate between FC and PARR was 100%, 78.6%, and 643% for LN, PB and BM, respectively. At the end of treatment, the agreement rates were 35.7%, 50%, and 57.1% for LN, PB and BM, respectively. At least one of the follow-up samples from dogs experiencing relapse was PARR.; conversely, FC was not able to detect MRD in seven of the dogs that relapsed. PARR was more sensitive than FC in predicting TTR, whereas the combination of PARR and FC was more sensitive than either technique alone in predicting LSS using PB samples. The results suggest that immunological and molecular techniques should be used in combination when monitoring for MRD in canine DLBCL. (C) 2014 Elsevier Ltd. All rights reserved.

Mitochondria are crucial organelles in the production of energy and in the control of signalling cascades. A machinery of pro-fusion and fission proteins regulates their morphology and subcellular localization. In muscle this results in an orderly pattern of intermyofibrillar and subsarcolemmal mitochondria. Muscular atrophy is a genetically controlled process involving the activation of the autophagy-lysosome and the ubiquitin-proteasome systems. Whether and how the mitochondria are involved in muscular atrophy is unknown. Here, we show that the mitochondria are removed through autophagy system and that changes in mitochondrial network occur in atrophying muscles. Expression of the fission machinery is per se sufficient to cause muscle wasting in adult animals, by triggering organelle dysfunction and AMPK activation. Conversely, inhibition of the mitochondrial fission inhibits muscle loss during fasting and after FoxO3 overexpression. Mitochondrial-dependent muscle atrophy requires AMPK activation as inhibition of AMPK restores muscle size in myofibres with altered mitochondria. Thus, disruption of the mitochondrial network is an essential amplificatory loop of the muscular atrophy programme.