ORM1-like 3 (ORMDL3) has strong genetic linkage to childhood onset asthma. To determine whether ORMDL3 selective expression in airway smooth muscle (ASM) influences ASM function we used cre/lox techniques to generate transgenic mice (hORMDL3Myh11eGFP-cre) which express human ORMDL3 selectively in smooth muscle cells. In vitro studies of ASM cells isolated from the bronchi of hORMDL3Myh11eGFP-cre mice demonstrated that they developed hypertrophy (quantitated by FACS and image analysis), hyperplasia (assessed by BrdU incorporation), and expressed increased levels of tropomysin proteins TPM1 and TPM4. siRNA knockdown of TPM1 or TPM4 demonstrated their importance to ORMDL3 mediated ASM proliferation but not hypertrophy. In addition, ASM derived from hORMDL3Myh11eGFP-cre mice had increased contractility to histamine in vitro which was associated with increased levels of intracellular Ca2+, increased cell surface membrane Orai1 Ca2+ channels which mediate influx of Ca2+ into the cytoplasm, and increased expression of ASM contractile genes Serca2b and Sm22. In vivo studies of hORMDL3Myh11eGFP-cre mice demonstrated that they had a spontaneous increase in ASM and AHR. ORMDL3 expression in ASM thus induces changes in ASM (hypertrophy, hyperplasia, increased contractility) which may explain the contribution of ORMDL3 to the development of AHR in childhood onset asthma which is highly linked to ORMDL3 on chromosome 17q12-21.
Alexa K. Pham, Marina Miller, Peter Rosenthal, Sudipta Das, Ning Weng, Sunghoon Jang, Richard C. Kurten, Jana Badrani, Taylor A. Doherty, Brian G. Oliver, David H. Broide
During denervation induced muscle atrophy, the loss of neuro-muscular junction (NMJ) integrity and the consequent cessation of nerve signal transmission to muscle, lead to a decline in myofiber size mass and contractile activity. However, the identity of the cell types implicated in the muscle response to nerve injury has not been clearly defined. Here, we describe a subpopulation of muscle resident glial cells activated by loss of NMJ integrity. Gene expression analysis at bulk and single cell level revealed the existence of a population of Itga7-expressing cells, which are distinct from muscle satellite cells and are selectively activated upon nerve injury. Upon nerve lesion, these cells expanded and activated a neurotrophic gene program, including the expression of a prospective selection marker – Ngfr – and a number of neurotrophic genes as well as ECM components. Among them, we observed that Tenascin C (Tnc) was specifically produced by muscle glial cells activated by nerve injury and preferentially localized to NMJ. Activation of muscle-resident glial cells by nerve injury induced a neurotrophic phenotype, which was reversible upon recovery of NMJ integrity; by contrast, muscle-resident glial cells in skeletal muscles of a mouse model of Amyotrophic Lateral Sclerosis (ALS) steadily increased over the course of the disease and exhibited an impaired neurotrophic activity, suggesting that pathogenic activation of glial cells may be implicated in ALS progression.
Daisy Proietti, Lorenzo Giordani, Marco De Bardi, Chiara D'Ercole, Biliana Lozanoska-Ochser, Susanna Amadio, Cinzia Volontè, Sara Marinelli, Antoine Muchir, Marina Bouchè, Giovanna Borsellino, Alessandra Sacco, Pier Lorenzo Puri, Luca Madaro
Cantu Syndrome (CS) is caused by gain-of-function (GOF) mutations in pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) KATP channel subunits, the most common mutations being SUR2[R1154Q] and SUR2[R1154W], carried by ~30% of patients. We used CRISPR/Cas9 genome engineering to introduce the equivalent of human SUR2[R1154Q] mutation to the mouse ABCC9 gene. Along with minimal CS disease features, R1154Q cardiomyocytes and vascular smooth muscle showed much lower KATP current density and pinacidil activation than WT cells. Almost complete loss of SUR2-dependent protein and KATP in homozygous R1154Q ventricles revealed an underlying diazoxide-sensitive SUR1-dependent KATP channel activity. Surprisingly, sequencing of SUR2 cDNA revealed divergent transcripts, one encoding full length SUR2 protein, and the other with in-frame deletion of 93 bases (corresponding to 31 amino acids encoded by exon 28) that was present in ~40% and ~90% of transcripts from hetero- and homozygous R1154Q tissues, respectively. Recombinant expression of SUR2A protein lacking exon 28 resulted in non-functional channels. SUR2[R1154Q] CS patient tissue and iPSC-derived cardiomyocytes showed only full length SUR2 transcripts, although further studies will be required to fully test whether SUR2[R1154Q] or other CS mutations might result in aberrant splicing and variable expressivity of disease features in human CS.
Haixia Zhang, Alex M. Hanson, Tobias U. Scherf de Almeida, Christopher H. Emfinger, Conor McClenaghan, Theresa Harter, Zihan Yan, Paige E. Cooper, G. Schuyler Brown, Eric C. Arakel, Robert P. Mecham, Attila Kovacs, Carmen M. Halabi, Blanche Schwappach, Maria S. Remedi, Colin G. Nichols
Chronic kidney disease (CKD) results in a progressive skeletal myopathy involving atrophy, weakness, and fatigue. Mitochondria have been thought to contribute to skeletal myopathy, however, the molecular mechanisms underlying changes in muscle metabolism in CKD are unknown. This study employed a comprehensive mitochondrial phenotyping platform to elucidate the mechanisms of skeletal muscle mitochondrial impairment in mice with adenine-induced CKD. CKD mice displayed significant reductions in mitochondrial oxidative phosphorylation (OXPHOS), which was strongly correlated with glomerular filtration rate, suggesting a link between kidney function and muscle mitochondrial health. Biochemical assays uncovered that OXPHOS dysfunction was driven principally by reduced activity of matrix dehydrogenases. Untargeted metabolomics analyses in skeletal muscle revealed a distinct metabolite profile in CKD muscle including accumulation of uremic toxins that strongly associated with the degree of mitochondrial impairment. Additional muscle phenotyping found that CKD mice experienced muscle atrophy and increased muscle protein degradation, but only male CKD mice had lower maximal contractile force. CKD mice also had morphological changes indicative of destabilization in the neuromuscular junction. This study provides the first comprehensive evaluation of mitochondrial health in murine CKD muscle and uncovers several unknown uremic metabolites that are strongly associated with the degree of mitochondrial impairment.
Trace Thome, Ravi A. Kumar, Sarah K. Burke, Ram B. Khattri, Zachary R. Salyers, Rachel C. Kelley, Madeline D. Coleman, Demetra D. Christou, Russell T. Hepple, Salvatore T. Scali, Leonardo F. Ferreira, Terence E. Ryan
Cantύ Syndrome (CS), caused by gain-of-function (GOF) mutations in pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) ATP-sensitive potassium (KATP) channel subunit genes, is frequently accompanied by gastrointestinal (GI) dysmotility, and we describe one CS patient who required an implanted intestinal irrigation system for successful stooling. We used gene-modified mice to assess the underlying KATP channel subunits in gut smooth muscle, and to model the consequences of altered KATP channels in CS gut. We show that Kir6.1/SUR2 subunits underlie smooth muscle KATP channels throughout the small intestine and colon. Knock-in mice, carrying human KCNJ8 and ABCC9 CS mutations in the endogenous loci, exhibit reduced intrinsic contractility throughout the intestine, resulting in death when weaned onto solid food in the most severely affected animals. Death is avoided by weaning onto a liquid gel diet, implicating intestinal insufficiency and bowel impaction as the underlying cause, and GI transit is normalized by treatment with the KATP inhibitor glibenclamide. We thus define the molecular basis of intestinal KATP channel activity, the mechanism by which overactivity results in GI insufficiency, and a viable approach to therapy.
Nathaniel W. York, Helen Parker, Zili Xie, David Tyus, Maham A. Waheed, Zihan Yan, Dorothy K. Grange, Maria S. Remedi, Sarah K. England, Hongzhen Hu, Colin G. Nichols
Compromised muscle mitochondrial metabolism is a hallmark of peripheral arterial disease, especially in patients with the most severe clinical manifestation - critical limb ischemia (CLI). We asked whether inflexibility in metabolism is critical for the development of myopathy in ischemic limb muscles. Using Polg mtDNA mutator (D257A) mice, we reveal remarkable protection from hindlimb ischemia (HLI) due to a unique and beneficial adaptive enhancement of glycolytic metabolism and elevated ischemic muscle PFKFB3. Similar to the relationship between mitochondria from CLI and claudicating patient muscles, BALB/c muscle mitochondria are uniquely dysfunctional after HLI onset as compared to the BL6 parental strain. AAV-mediated over-expression of PFKFB3 in BALB/c limb muscles improved muscle contractile function and limb blood flow following HLI. Enrichment analysis of RNA sequencing data on muscle from CLI patients revealed a unique deficit in the Glucose Metabolism Reactome. Muscles from these patients express lower PFKFB3 protein and their muscle progenitor cells possess decreased glycolytic flux capacity in vitro. Here we show supplementary glycolytic flux as sufficient to protect against ischemic myopathy in instances where reduced blood flow related mitochondrial function is compromised pre-clinically. Additionally, our data reveal reduced glycolytic flux as a common characteristic of CLI patient limb skeletal muscle.
Terence E. Ryan, Cameron A. Schmidt, Michael D. Tarpey, Adam J. Amorese, Dean Yamaguchi, Emma Goldberg, Melissa R. Iñigo, Reema Karnekar, Allison R. O’Rourke, James M. Ervasti, Patricia Brophy, Thomas Green, P. Darrell Neufer, Kelsey H. Fisher-Wellman, Espen Spangenburg, Joseph McClung
Classical dynamins are large GTPases regulating membrane and cytoskeleton dynamics and are linked to different pathological conditions ranging from neuromuscular diseases to encephalopathy and cancer. Dominant DNM2 (dynamin 2) mutations lead to either mild adult onset or severe neonatal centronuclear myopathy (ADCNM). Our objectives were to better understand the pathomechanism of severe ADCNM and test a potential therapy. Here, we created the Dnm2SL/+ mouse line harboring the common S619L mutation found in patients with severe ADCNM and impairing the conformational switch regulating dynamin self-assembly and membrane remodeling. The Dnm2SL/+ mouse faithfully reproduces severe ADCNM hallmarks with early impaired muscle function and force together with myofibers hypotrophy. It revealed swollen mitochondria lacking cristae as the main ultrastructural defect and potential cause of the disease. Patient analysis confirmed this structural hallmark. In addition, DNM2 reduction with antisense oligonucleotides after disease onset efficiently reverted locomotor and force defects after only 3 weeks of treatment. Most histological defects including mitochondria alteration were partially or fully rescued. Overall, this study highlights an efficient approach to revert the severe form of dynamin-related centronuclear myopathy. These data also reveal that the dynamin conformational switch is key for muscle function and should be targeted for future therapeutic developments.
Xènia Massana Muñoz, Christine Kretz, Roberto Silva-Rojas, Julien Ochala, Alexia Menuet, Norma B. Romero, Belinda S. Cowling, Jocelyn Laporte
Specialized pro-resolving mediators (SPMs) actively limit inflammation and expedite its resolution by modulating leukocyte recruitment and function. Here we profiled intramuscular lipid mediators via LC-MS based metabolipidomics following myofiber injury and investigated the potential role of SPMs in skeletal muscle inflammation and repair. Both pro-inflammatory eicosanoids and SPMs increased following myofiber damage induced by either intramuscular injection of barium chloride or synergist ablation-induced functional muscle overload. Daily systemic administration of the SPM resolvin D1 (RvD1) as an immunoresolvent limited the degree and duration of inflammation, enhanced regenerating myofiber growth, and improved recovery of muscle strength. RvD1 suppressed inflammatory cytokine expression, enhanced polymorphonuclear cell clearance, modulated the local muscle stem cell response, and polarized intramuscular macrophages to a more pro-regenerative subset. RvD1 had minimal direct impact on in-vitro myogenesis but directly suppressed myokine production and stimulated macrophage phagocytosis, showing that SPMs can modulate both infiltrating myeloid and resident muscle cell populations. These data reveal the efficacy of immunoresolvents as a novel alternative to classical anti-inflammatory interventions in the management of muscle injuries to modulate inflammation while stimulating tissue repair.
James F. Markworth, Lemuel A. Brown, Eunice Lim, Carolyn Floyd, Jacqueline Larouche, Jesus A. Castor-Macias, Kristoffer B. Sugg, Dylan C. Sarver, Peter C. D. Macpherson, Carol S. Davis, Carlos A. Aguilar, Krishna Rao Maddipati, Susan V. Brooks
The severity of Duchenne muscular dystrophy (DMD), an incurable disease caused by the lack of dystrophin, might be modulated by different factors, including miRNAs. Among them, miR-378 is considered of high importance for muscle biology, but intriguingly, its role in DMD and its murine model (mdx mice) has not been thoroughly addressed so far. Here, we demonstrate that dystrophic mice additionally globally lacking miR-378 (double-KO [dKO] animals) exhibited better physical performance and improved absolute muscle force compared with mdx mice. Accordingly, markers of muscle damage in serum were significantly decreased in dKO mice, accompanied by diminished inflammation, fibrosis, and reduced abundance of regenerating fibers within muscles. The lack of miR-378 also normalized the aggravated fusion of dystrophin-deficient muscle satellite cells (mSCs). RNA sequencing of gastrocnemius muscle transcriptome revealed fibroblast growth factor 1 (Fgf1) as one of the most significantly downregulated genes in mice devoid of miR-378, indicating FGF1 as one of the mediators of changes driven by the lack of miR-378. In conclusion, we suggest that targeting miR-378 has the potential to ameliorate DMD pathology.
Paulina Podkalicka, Olga Mucha, Iwona Bronisz-Budzyńska, Magdalena Kozakowska, Katarzyna Pietraszek-Gremplewicz, Anna Cetnarowska, Urszula Głowniak-Kwitek, Karolina Bukowska-Strakova, Maciej Cieśla, Maria Kulecka, Jerzy Ostrowski, Michał Mikuła, Anna Potulska-Chromik, Anna Kostera-Pruszczyk, Alicja Józkowicz, Agnieszka Łoboda, Józef Dulak
Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy. When human induced pluripotent stem cells (hiPSCs) were differentiated into myoblasts, the myoblasts derived from DMD patients’ hiPSCs (DMD hiPSC-derived myoblasts) exhibited an identifiable DMD relevant phenotype: myogenic fusion deficiency. Based on this model, we developed a DMD hiPSC-derived myoblast screening platform employing a high-content imaging (BD pathway 855) approach to generate parameters describing morphological as well as myogenic marker protein expression. Following treatment of the cells with 1524 compounds from the Johns Hopkins Clinical Compound Library, compounds that enhanced myogenic fusion of DMD hiPSC-derived myoblasts were identified. The final hits were ginsenoside Rd and fenofibrate. Transcriptional profiling revealed that ginsenoside Rd is functionally related to FLT3 signaling, while fenofibrate is linked to TGF-β signaling. Preclinical tests in mdx mice showed that treatment with these two hit compounds can significantly ameliorate some of the skeletal muscle phenotypes caused by dystrophin deficiency, supporting their therapeutic potential. Further study with hiPSC-derived cardiomyocytes revealed that fenofibrate could inhibit mitochondria-induced apoptosis in the DMD hiPSC-derived cardiomyocytes. We have developed a platform based on DMD hiPSC-derived myoblasts for drug screening and identified two promising small molecules with in vivo efficacy.
Congshan Sun, In Young Choi, Yazmin I. Rovira Gonzalez, Peter Andersen, C. Conover Talbot Jr., Shama R. Iyer, Richard M. Lovering, Kathryn R. Wagner, Gabsang Lee
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