Reduced ATP turnover during hibernation in relaxed skeletal muscle

Reduced ATP turnover during hibernation in relaxed skeletal muscle | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Reduced ATP turnover during hibernation in relaxed skeletal muscle Bert Blaauw, Cosimo de Napoli, Luisa Schmidt, Mauro Montesel, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4271626/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jan, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract In mammals, loss of food intake and reduced mechanical loading/activity of skeletal muscles leads to a very rapid loss in mass and function. However, during hibernation in bears, despite spending months without feeding and with very modest muscle activity, only moderate muscle wasting is observed. Part of this tissue sparing is due to a highly reduced metabolic activity in almost all tissues, including skeletal muscle. Myosin, one of the most abundant proteins in skeletal muscle, has different metabolic activities in resting muscle. To evaluate the ATPase activity of myosin in hibernating bears, we performed an analysis on a single muscle fiber level. Individual fibers were taken from biopsies of the same bears either during hibernation or during the active phase in the summer. We confirm that muscle fibers from hibernating bears show no loss of fiber size and a mild reduction in force generating capacity. Interestingly, we find a significant reduction in ATPase activity of single muscle fibers taken from hibernating bears, which is caused by a reduced myosin ATP turnover. Single fiber proteomics analysis shows a major remodeling of their proteome, which is similar between different fiber types. Both type 2A and type 1/2A mixed fibers show a marked reduction in mitochondrial proteins during hibernation, with a decrease in proteins linked to the TCA cycle and mitochondrial translation. Western blotting, electron microscope and immunohistochemical analyses confirm mitochondrial alterations in winter muscles. Using bioinformatical approaches based on the significant proteome changes, we found a decrease in Myosin Light Chain Kinase (MYLK2) targets in winter muscles compared to summer samples. This outcome was confirmed by western blotting analyses of both phosphorylated myosin light chain and MYLK2, which is a known stimulator of basal myosin ATPase activity. These results suggest that reduced myosin ATPase activity is one of the evolutionary adaptations adopted by resting skeletal muscle during hibernation to minimize energy expenditure. Interestingly, this suggests modulation of myosin ATPase activity as a new possible target to combat muscle wasting diseases, particularly those linked to altered metabolism. Health sciences/Medical research/Translational research Biological sciences/Zoology/Animal physiology Biological sciences/Physiology/Metabolism Full Text Additional Declarations There is NO Competing Interest. Cite Share Download PDF Status: Published Journal Publication published 02 Jan, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4271626","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":295452606,"identity":"b8ff8dc7-3b03-4793-98d0-4d0eb1170393","order_by":0,"name":"Bert Blaauw","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYBACAxDB2GABph4wMEgwMBxgbCCkhbGBsUECxGY2gGppxKsHWQsbmGQ4wIDfGnP23ucPGHdIyPPPyDGr5t1hkcd3gLn9AT4tlj3HDRsYz0gYzriRY3ab94xEsSRBh91IAzqsTYKx4Ubuttu8bRKJGwhquf8MrMV+PlBLMXFabrCBtSRuAGphJkqLZU8a44zEMxLJG8+8/yw5tw3ol8OMjTPwaTFnP8bw4eMOG9t5x9MSP7xtq8vjO97+4AM+LWCQgMJmJqgej/ZRMApGwSgYBWAAADbuT/FGzLTTAAAAAElFTkSuQmCC","orcid":"","institution":"University of Padua","correspondingAuthor":true,"prefix":"","firstName":"Bert","middleName":"","lastName":"Blaauw","suffix":""},{"id":295452607,"identity":"0b8635ab-49d4-4990-a050-d795e9eb5b31","order_by":1,"name":"Cosimo de Napoli","email":"","orcid":"","institution":"University of Padova, Venetian Institute of Molecular Medicine","correspondingAuthor":false,"prefix":"","firstName":"Cosimo","middleName":"","lastName":"de Napoli","suffix":""},{"id":295452608,"identity":"acdf6b38-609f-4108-8688-4616430e37eb","order_by":2,"name":"Luisa Schmidt","email":"","orcid":"","institution":"Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging‐Associated Diseases (CECAD), University of Cologne","correspondingAuthor":false,"prefix":"","firstName":"Luisa","middleName":"","lastName":"Schmidt","suffix":""},{"id":295452609,"identity":"e08c3a60-da73-4298-b31b-18c74b49ae8d","order_by":3,"name":"Mauro Montesel","email":"","orcid":"","institution":"University of Padova, Venetian Institute of Molecular Medicine","correspondingAuthor":false,"prefix":"","firstName":"Mauro","middleName":"","lastName":"Montesel","suffix":""},{"id":295452610,"identity":"439826d8-726a-46a4-80ab-7ae8dfdf15a5","order_by":4,"name":"Laura Cussonneau","email":"","orcid":"","institution":"University of Padova, Venetian Institute of Molecular Medicine","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Cussonneau","suffix":""},{"id":295452611,"identity":"15c3a672-23ae-493e-96a1-56b9b41efa0d","order_by":5,"name":"Samuele Sanniti","email":"","orcid":"","institution":"University of Padova, Venetian Institute of Molecular Medicine","correspondingAuthor":false,"prefix":"","firstName":"Samuele","middleName":"","lastName":"Sanniti","suffix":""},{"id":295452612,"identity":"a11e8e9b-4a6d-4b5c-99c6-7aa57caf4767","order_by":6,"name":"Lorenzo Marcucci","email":"","orcid":"https://orcid.org/0000-0002-9542-4417","institution":"University of Padua","correspondingAuthor":false,"prefix":"","firstName":"Lorenzo","middleName":"","lastName":"Marcucci","suffix":""},{"id":295452613,"identity":"6e811b4a-4fa6-4ad9-8afc-c90f1b28143b","order_by":7,"name":"Elena Germinario","email":"","orcid":"","institution":"University of Padova","correspondingAuthor":false,"prefix":"","firstName":"Elena","middleName":"","lastName":"Germinario","suffix":""},{"id":295452614,"identity":"e9077e39-9d62-49dd-975e-2796ae30aec8","order_by":8,"name":"Jonas Kindberg","email":"","orcid":"","institution":"Norwegian Institute for Nature Research, Trondheim; 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However, during hibernation in bears, despite spending months without feeding and with very modest muscle activity, only moderate muscle wasting is observed. Part of this tissue sparing is due to a highly reduced metabolic activity in almost all tissues, including skeletal muscle. Myosin, one of the most abundant proteins in skeletal muscle, has different metabolic activities in resting muscle. To evaluate the ATPase activity of myosin in hibernating bears, we performed an analysis on a single muscle fiber level. Individual fibers were taken from biopsies of the same bears either during hibernation or during the active phase in the summer. We confirm that muscle fibers from hibernating bears show no loss of fiber size and a mild reduction in force generating capacity. Interestingly, we find a significant reduction in ATPase activity of single muscle fibers taken from hibernating bears, which is caused by a reduced myosin ATP turnover. Single fiber proteomics analysis shows a major remodeling of their proteome, which is similar between different fiber types. Both type 2A and type 1/2A mixed fibers show a marked reduction in mitochondrial proteins during hibernation, with a decrease in proteins linked to the TCA cycle and mitochondrial translation. Western blotting, electron microscope and immunohistochemical analyses confirm mitochondrial alterations in winter muscles.\r\nUsing bioinformatical approaches based on the significant proteome changes, we found a decrease in Myosin Light Chain Kinase (MYLK2) targets in winter muscles compared to summer samples. This outcome was confirmed by western blotting analyses of both phosphorylated myosin light chain and MYLK2, which is a known stimulator of basal myosin ATPase activity. These results suggest that reduced myosin ATPase activity is one of the evolutionary adaptations adopted by resting skeletal muscle during hibernation to minimize energy expenditure. 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