Research
Abstract
X-linked myotubular myopathy (XLMTM) due to MTM1 mutations is a rare and often lethal congenital myopathy. Its downstream molecular and cellular mechanisms are currently incompletely understood. The most abundant protein in muscle, myosin, has been implicated in the pathophysiology of other congenital myopathies. Hence, in the present study, we aimed to define whether myosin is also dysfunctional in XLMTM and whether it thus may constitute a potential drug target. To this end, we used skeletal muscle tissue from human patients and canine/mouse models; we performed Mant-ATP chase experiments coupled with X-ray diffraction analyses and LC/MS-based proteomics studies. In XLMTM humans, we found that myosin molecules are structurally disordered and preferably adopt their ATP-consuming biochemical state. This phosphorylation-related (mal)adaptation was mirrored by a striking remodelling of the myofibre energetic proteome in XLMTM dogs. In line with these, we confirmed an accrued myosin ATP consumption in mice lacking MTM1. Hence, we treated these, with a myosin ATPase inhibitor, mavacamten. After a four-week treatment period, we observed a partial restoration of the myofibre proteome, especially proteins involved in cytoskeletal, sarcomeric and energetic pathways. Altogether, our study highlights myosin inhibition as a new potential drug mechanism for the complex XLMTM muscle phenotype.
Authors
Elise Gerlach Melhedegaard, Fanny Rostedt, Charlotte Gineste, Robert A.E. Seaborne, Hannah F. Dugdale, Vladimir Belhac, Edmar Zanoteli, Michael W. Lawlor, David L. Mack, Carina Wallgren-Pettersson, Anthony L. Hessel, Heinz Jungbluth, Jocelyn Laporte, Yoshihiko Saito, Ichizo Nishino, Julien Ochala, Jenni Laitila
Abstract
Chronic liver injury results in activation of quiescent Hepatic Stellate Cells (qHSCs) into Collagen Type I-producing activated HSCs that make liver fibrotic. We identified ETS1/2 (E26 transformation-specific transcription factors 1/2) as lineage-specific transcription factors regulating HSC phenotypes. Here we investigated the role of ETS1/2 in HSCs in liver fibrosis using toxic liver injury models and 3D human liver spheroids. Liver fibrosis was induced in wild-type and HSC-specific Ets1 (Ets1ΔHSC) and Ets2 (Ets2ΔHSC) knockout mice by administration of carbon tetrachloride for 6 weeks, following cessation of liver injury for 2 weeks. Liver fibrosis was more severe in Ets1ΔHSC, and to lesser extent in Ets2ΔHSC, compared to wild-type mice. Regression of liver fibrosis was suppressed only in Ets1ΔHSC, indicating Ets1 as the predominant isoform maintaining quiescent-like phenotype in HSCs. Similar results were obtained in a MASH model using 3D human liver spheroids. Knockdown of ETS1 in human HSCs caused upregulation of fibrogenic genes in MASH human liver spheroids and prevented fibrosis regression. ETS1 regulated the qHSC phenotype via CRTC2/PGC1α/PPARγ pathway. Knockdown of CRTC2 (cAMP response element-binding protein (CREB)-regulated transcription co-activator 2) abrogated PPARγ responses and facilitated HSC activation. These findings suggest that ETS1 may represent a therapeutic target for anti-fibrotic therapy.
Authors
Wonseok Lee, Xiao Liu, Sara Brin Rosenthal, Charlene Miciano, Sadatsugu Sakane, Kanani Hokutan, Debanjan Dhar, Hyun Young Kim, David A. Brenner, Tatiana Kisseleva
Abstract
Impaired cardiac lipid metabolism has been reported to cause heart failure. Lipin1, a multifunctional protein, is a phosphatidate phosphatase that generates diacylglycerol from phosphatidic acid and a transcriptional cofactor that regulates lipid metabolism-related gene expression. Here, we investigated the roles of lipin1 in cardiac remodeling after myocardial infarction (MI). The expression levels of lipin1 significantly decreased in cardiomyocytes of the human failing heart and murine ischemic myocardium. Cardiomyocyte-specific Lpin1 knockout (cKO) mice showed left ventricle enlargement and reduced fractional shortening after MI, compared to control mice. This was accompanied by elevated cardiac fibrosis, accumulation of reactive oxygen species, and increased expression of inflammatory cytokines. In contrast, cardiomyocyte-specific Lpin1 overexpression (cOE) mice showed reduced fibrosis and inflammation and improved cardiac function compared to control mice. Cardiac lipid droplets (LDs) were reduced after MI in wild-type (WT) mice hearts and were further downregulated in the hearts of cKO mice with a decrease in triglyceride and free fatty acid content, while cOE mice hearts exhibited increased LDs and lipid content. Expression levels of genes involved in fatty acid oxidation, such as Ppargc1a (PGC1A) and Acaa2, were decreased and increased in the MI hearts of cKO mice and cOE mice, respectively. These results suggest the protective role of lipin1 against ischemic injury by maintaining lipid metabolism in ischemic cardiomyocytes.
Authors
Jiaxi Guo, Kohei Karasaki, Kazutaka Ueda, Manami Katoh, Masaki Hashimoto, Toshiyuki Ko, Masato Ishizuka, Satoshi Bujo, Chunxia Zhao, Risa Kishikawa, Haruka Yanagisawa-Murakami, Hiroyuki Sowa, Bowen Zhai, Mutsuo Harada, Seitaro Nomura, Norihiko Takeda, Brian N. Finck, Haruhiro Toko, Issei Komuro
Abstract
Clear cell renal cell carcinomas (ccRCC) are largely driven by HIF2α and are avid consumers of glutamine. However, inhibitors of glutaminase1 (GLS1), the first step in glutaminolysis, have not shown benefit in phase III trials, and HIF2α inhibition, recently FDA-approved for treatment of ccRCC, shows significant but incomplete benefits. This highlights the need to better understand the interplay between glutamine metabolism and HIF2α in ccRCC. Here, we report that glutamine deprivation rapidly redistributes GLS1 into isolated clusters within mitochondria in diverse cell types, but not in ccRCC. GLS1 clustering occurs rapidly within 1 to 3 hours, is reversible, is specifically triggered by reduced intracellular glutamate, and is dependent on mitochondrial fission. Clustered GLS1 markedly enhances glutaminase activity and promotes cell death under glutamine-deprived conditions. HIF2α prevents GLS1 clustering, independently of its transcriptional activity, thereby maintaining low GLS activity and protecting ccRCC cells from glutamine deprivation-induced cell death. Forced clustering of GLS1, using constitutively clustering mutants, restores high GLS activity, promotes apoptosis, and suppresses ccRCC tumor growth in vivo. These findings reveal multiple insights into cellular glutamine handling, including a previously unrecognized process by which HIF2α promotes ccRCC: by suppressing GLS1 clustering and maintaining low GLS activity. This mechanism provides a potential explanation for the lack of clinical efficacy of GLS inhibitors in ccRCC and suggests a therapeutic avenue to combine HIF2α inhibition with strategies that restore GLS1 clustering.
Authors
Wencao Zhao, Sara M. Demczyszyn, Nathan J. Coffey, Yanqing Jiang, Boyoung Kim, Schuyler Bowers, Caitlyn E. Bowman, Michael C. Noji, Cholsoon Jang, M. Celeste Simon, Zoltan Arany, Boa Kim
Abstract
The lung’s mechanosensitive immune response to alveolar overdistension impedes ventilation therapy for hypoxemic respiratory failure. Though mechanistically unclear, the prevailing hypothesis is that the immune response results when alveolar overdistension stretches alveolar macrophages (AMs). Since this hypothesis is untested in live lungs, we optically imaged live mouse alveoli to detect alveolus-adherent, sessile AMs that communicate with the alveolar epithelium through connexin43 (Cx43)-containing gap junctions. Alveolar hyperinflation did not stretch the AMs, but it increased AM Ca2+. AM-specific Cx43 deletion blocked the Ca2+ response, as well lung injury due to mechanical ventilation at high tidal volume (HTV). HTV was also inhibited by AM-targeted delivery of liposomes containing the inhibitor of endosomal Ca2+ release, Xestospongin C. We conclude, Cx43- and Ca2+-dependent AM-epithelial interactions determine the lung’s mechanosensitive immunity, providing a basis for therapy for ventilator-induced lung injury.
Authors
Liberty Mthunzi, Mohammad Islam, Galina A Gusarova, Brian Karolewski, Sunita Bhattacharya, Jahar Bhattacharya
Abstract
Elite controllers (ECs) maintain undetectable levels of plasma viremia in the absence of treatment, but small reservoirs of replication-competent proviruses persist in the vast majority of these persons. We longitudinally studied paired blood and inguinal lymph node samples (LNMC) from two ECs to better characterize distinguishing features of viral reservoir cell dynamics in ECs. In both participants, we observed a 7- to 10-fold lower frequency of intact proviruses in LNMC samples relative to reservoir cells circulating in blood. The landscape of intact proviruses in both tissue compartments was dominated by shared large clones that were frequently integrated in non-coding DNA regions, but the frequency and diversity of intact proviruses was more limited in LNMCs. Of note, over 9-10 years of longitudinal follow-up, a 3- to 18-fold reduction of intact proviruses was observed. Together, these data support the hypothesis that viral reservoirs in ECs’ blood and lymphoid tissues are under strong, likely immune-mediated selection pressure.
Authors
Samantha K. Marzi, Chloé M. Naasz, Leah Carrere, Carmen Gasca-Capote, Isabelle C. Roseto, Ce Gao, Matthias Cavassini, Andrea Mastrangelo, Mathias Lichterfeld, Matthieu Perreau, Xu G. Yu
Abstract
Angiopoietin-like 3 (ANGPTL3) is a major regulator of lipoprotein metabolism. ANGPTL3 deficiency results in lower levels of triglycerides, LDL-cholesterol (LDL-C), and HDL-cholesterol (HDL-C), and may protect from cardiovascular disease. ANGPTL3 oligomerizes with ANGPTL8 to inhibit lipoprotein lipase (LPL), the enzyme responsible for plasma triglyceride hydrolysis. Independent of ANGPTL8, oligomers of ANGPTL3 can inhibit endothelial lipase (EL), which regulates circulating HDL-C and LDL-C levels through the hydrolysis of lipoprotein phospholipids. The N-terminal region of ANGPTL3 is necessary for both oligomerization and lipase inhibition. However, our understanding of the specific residues that contribute to these functions is incomplete. In this study, we performed mutagenesis of the N-terminal region to identify residues important for EL inhibition and oligomerization. We also assessed the presence of different ANGPTL3 species in human plasma. We identified a motif important for lipase inhibition, and protein structure prediction suggested that this region interacted directly with EL. We also found that recombinant ANGPTL3 formed a homotrimer and was unable to inhibit EL activity when trimerization was disrupted. Surprisingly, we observed that human plasma contained more monomeric ANGPTL3 than trimeric ANGPTL3. An important implication of these findings is that previous correlations between circulating ANGPTL3 and circulating triglyceride-rich lipoproteins need to be revisited.
Authors
Sydney G. Walker, Yan Q. Chen, Kelli L. Sylvers-Davie, Alex Dou, Eugene Y. Zhen, Yuewei Qian, Yi Wen, Mariam E. Ehsani, Sydney A. Smith, Rakshya Thapa, Maxwell J. Mercer, Lucy Langmack, Bharat Raj Bhattarai, Michael Ploug, Robert J. Konrad, Brandon S.J. Davies
Abstract
Mammalian skin wounds typically heal with a scar, characterized by fibrotic tissue that disrupts original tissue architecture and function. Therapies that limit fibrosis and promote regenerative healing remain a major unmet clinical need. Rosemary extract, particularly in the form of topical oils and creams, has gained widespread public attention for its purported wound-healing properties. However, its efficacy and mechanism of action remain poorly understood. We show in adult wound healing mouse models that an ethanol-based rosemary extract accelerates the speed of wound healing and mitigates fibrosis. Mechanistically, we identify that carnosic acid, a major bioactive component of rosemary leaves, activates the TRPA1 nociceptor on cutaneous sensory neurons to enhance tissue regeneration. Mice lacking TRPA1 in sensory neurons do not exhibit these pro-regenerative responses, confirming its role as a critical mediator. Together, these findings suggest that topical rosemary extract may represent an effective and accessible therapeutic approach to improve skin repair outcomes.
Authors
Emmanuel Rapp, Jiayi Pang, Borna Saeednia, Stephen Marsh Prouty, Christopher A. Reilly, Thomas H. Leung
Abstract
Anthracycline chemotherapy, widely used in cancer treatment, poses a significant risk of cardiotoxicity that results in functional decline. Current diagnostic methods poorly predict cardiotoxicity because they do not detect early damage that precedes dysfunction. Positron emission tomography (PET) is well-suited to address this need when coupled with suitable imaging biomarkers. We used PET to evaluate cardiac molecular changes in male C57BL/6J mice exposed to doxorubicin (DOX). These mice initially developed cardiac atrophy, experienced functional deficits within 10 weeks of treatment, and developed cardiac fibrosis by 16 weeks. Elevated cardiac uptake of [68Ga]Ga-FAPI-04, a PET tracer targeting fibroblast activation protein alpha (FAP), was evident by 2 weeks and preceded the onset of functional deficits. Cardiac PET signal correlated with FAP expression and activity as well as other canonical indicators of cardiac remodeling. By contrast, cardiac uptake of [18F]DPA-714 and [18F]MFBG, which target translocator protein 18-kDa (TSPO) and the norepinephrine transporter (NET), respectively, did not differ between the DOX animals and their controls. These findings identify FAP as an early imaging biomarker for DOX-induced cardiac remodeling in males and support the use of FAP PET imaging to detect some cancer patients at risk for treatment-related myocardial damage before cardiac function declines.
Authors
Chul-Hee Lee, Onorina L. Manzo, Luisa Rubinelli, Sebastian E. Carrasco, Sungyun Cho, Thomas M. Jeitner, John W. Babich, Annarita Di Lorenzo, James M. Kelly
Abstract
Laminin-α2-related Congenital Muscular Dystrophy (LAMA2-CMD) is a severe neuromuscular disorder caused by mutations in the LAMA2 gene, leading to loss of heterotrimers laminin-211/221, key components of the skeletal muscle extracellular matrix. Their absence disrupts adhesion between the cytoskeleton and extracellular matrix, resulting in progressive muscle wasting. Laminin-211/221 interacts with adhesion complexes such as the dystrophin/Utrophin glycoprotein complex and the α7β1-integrin. However, the regulatory mechanisms of these laminin-binding complexes and the broader role of laminin’s influence on the formation of the macromolecular network in skeletal muscle remain unclear. We previously demonstrated that mouse laminin-111 delivered in the dyW⁻/⁻ mouse model of LAMA2-CMD prevented disease progression, improved strength, and extended survival. We hypothesize that laminin-111, the embryonic laminin isoform, restores key adhesion-signaling networks. Using spatial-proteomics on patient and mouse muscle, we identified loss of essential signaling components: heat shock proteins 27 and 70, c-Jun N-terminal kinase, and glucose transporter 1 in laminin-α2 deficient muscle. Treatment with recombinant human laminin-111 (rhLAM-111) restored protein localization, reduced ROS, and promoted glycolytic, pro-survival signaling. These findings highlight laminin’s role in maintaining muscle homeostasis and metabolism and support the therapeutic potential of rhLAM-111 for treating LAMA2-CMD by restoring adhesion and intracellular signaling in dystrophic muscle.
Authors
Hailey J. Hermann, Ryan D. Wuebbles, Marisela Dagda, Axel Munoz, Lauren L. Parker, Paula C. C Guzman, Lola T. Byrne, Steven A. Moore, Dean J. Burkin
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