Russian Journal of PhysiologyRussian Journal of PhysiologyРоссийский физиологический журнал им. И.М. Сеченова0869-81392658-655XThe Russian Academy of Sciences68090310.31857/S0869813925040053UEYBBIEXPERIMENTAL ARTICLESЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИResearch ArticleTime-course Expression of Mechanosensitive Ion Channels in Rat Postural Muscle under Hindlimb UnloadingДинамика экспрессии механочувствительных ионных каналов в постуральной мышце крысы в условиях функциональной разгрузкиVilchinskayaN. A.ВильчинскаяН. А.
Russian Federation
tmirzoev@yandex.ruShenkmanB. S.ШенкманБ. С.
Russian Federation
tmirzoev@yandex.ruMirzoevT. M.МирзоевТ. М.
Russian Federation
tmirzoev@yandex.ruInstitute of Biomedical Problems of the Russian Academy of SciencesИнститут медико-биологических проблем РАН15042025111462563628052025Copyright ©; 2025, Russian Academy of SciencesCopyright ©; 2025, Российская академия наук2025Russian Academy of SciencesРоссийская академия наукhttps://journals.eco-vector.com/0869-8139/article/view/680903

Atony and atrophy of mammalian postural muscles can occur due to mechanical unloading (weightlessness, hypokinesia). There is reason to believe that calcium-permeable mechanosensitive channels may contribute to the development of muscle atrophy caused by mechanical unloading. The aim of the study was to assess time-course changes in the expression of key mechanosensitive channels in rat soleus muscle under conditions of mechanical unloading. Male Wistar rats were subjected to hindlimb suspension (HS) for 1, 3, 7 and 14 days. Expression of Piezo1, TRPC1, TRPC3, TRPC6, TRPM3, TRPM7 and TMEM63B mRNA was determined using PCR. Piezo1 protein content was assessed using Western blotting. Piezo1 mRNA expression transiently increased after 24 h of HS, but did not differ from the control after 3, 7 and 14 days of unloading. A decrease in Piezo1 protein content was observed after 3, 7 and 14 days of HS relative to the control. At the early stages of HS, there was a significant increase in the mRNA expression of TRPC3, TRPM3, TRPM7 and TMEM63B, while TRPC6 expression was reduced. The level of TRPC1 mRNA expression was increased only after 3 days of HS. Seven-day unloading did not cause changes in the mRNA expression of TRPC1, TRPC3, TRPM3 and TMEM63B but led to increased TRPM7 expression. After two weeks of HS in the soleus muscle, a decrease in the mRNA expression of TRPC1, TRPC6, TRPM3 and TMEM63B was observed. Thus, at an early stage of mechanical unloading (1 and 3 days), a transient increase in the mRNA expression of Piezo1, TRPC1, TRPC3 and TMEM63B is observed, then at a later stage of unloading (14 days), reduced expression of TRPC1, TRPC6, TRPM3, TMEM63B at the mRNA level and Piezo1 at the protein level is observed.

При функциональной разгрузке (невесомость, гипокинезия) происходит атония и атрофия постуральных мышц млекопитающих. Есть основания полагать, что кальций-проницаемые механочувствительные каналы могут вносить вклад в развитие мышечной атрофии, вызванной функциональной разгрузкой. Цель исследования состояла в оценке динамики экспрессии ключевых механочувствительных каналов в камбаловидной мышце крысы в условиях функциональной разгрузки. Самцы крыс Вистар подвергались вывешиванию задних конечностей в течение 1, 3, 7 и 14 суток. Экспрессия мРНК Piezo1, TRPC1, TRPC3, TRPC6, TRPM3, TRPM7 и TMEM63B определялась с помощью ПЦР. Содержание белка Piezo1 оценивалось с помощью Вестерн-блоттинга. Экспрессия мРНК Piezo1 временно увеличилась спустя 24 ч разгрузки, но не отличалась от контроля после 3, 7 и 14 суток разгрузки. Снижение содержания белка Piezo1 относительно контроля наблюдалось после 3, 7 и 14 суток функциональной разгрузки. На ранних стадиях разгрузки наблюдалось значительное увеличение экспрессии мРНК TRPC3, TRPM3, TRPM7 и TMEM63B, при этом экспрессия TRPC6 была понижена. Уровень экспрессии мРНК TRPC1 был повышен только после трехсуточной разгрузки. Семисуточная разгрузка не вызвала изменений в экспрессии мРНК TRPC1, TRPC3, TRPM3 и TMEM63B, но привела к повышенной экспрессии TRPM7. После двухнедельной разгрузки в камбаловидной мышце наблюдалось снижение экспрессии мРНК TRPC1, TRPC6, TRPM3 и TMEM63B. Таким образом, на ранней стадии функциональной разгрузки (первые и третьии сутки) наблюдалось транзиторное увеличение экспрессии мРНК Piezo1, TRPC1, TRPC3 и TMEM63B, но на более поздней стадии разгрузки (14 суток) отмечалась пониженная экспрессия TRPC1, TRPC6, TRPM3, TMEM63B на уровне мРНК и Piezo1 на уровне белка.

soleus musclemechanical unloadingPiezo1TRPCTRPMTMEM63Bкамбаловидная мышцафункциональная разгрузкаPiezo1TRPCTRPMTMEM63BРоссийский научный фондRussian Science Foundation22-75-10046Fitts RH, Riley DR, Widrick JJ (2001) Functional and structural adaptations of skeletal muscle to microgravity. J Exp Biol 204 (Pt 18): 3201–3208. https://doi.org/ 10.1242/jeb.204.18.3201Deane CS, Piasecki M, Atherton PJ (2024) Skeletal muscle immobilisation-induced atrophy: Мechanistic insights from human studies. Clin Sci 138 (12): 741–756. https://doi.org/10.1042/CS20231198Bodine SC (2013) Disuse-induced muscle wasting. Int J Biochem Cell Biol 45 (10): 2200–2208. https://doi.org/10.1016/j.biocel.2013.06.011Mirzoev TM, Shenkman BS (2023) Mechanosensory Structures in the Mechanotransduction System of Muscle Fibers. J Evol Biochem Physiol 59 (4): 1341–1359. https://doi.org/10.1134/s0022093023040269Kefauver JM, Ward AB, Patapoutian A (2020) Discoveries in structure and physiology of mechanically activated ion channels. Nature 587 (7835): 567–576. https://doi.org/10.1038/s41586-020-2933-1Benavides Damm T, Egli M (2014) Calcium's role in mechanotransduction during muscle development. Cell Physiol Biochem 33(2): 249–272. https://doi.org/10.1159/000356667Michelucci A, Liang C, Protasi F, Dirksen RT (2021) Altered Ca(2+) Handling and Oxidative Stress Underlie Mitochondrial Damage and Skeletal Muscle Dysfunction in Aging and Disease. Metabolites 11(7): 424. https://doi.org/ 10.3390/metabo110704248.Valentim MA, Brahmbhatt AN, Tupling AR (2022) Skeletal and cardiac muscle calcium transport regulation in health and disease. Biosci Rep 42(12): BSR20211997. https://doi.org/ 10.1042/BSR20211997Shenkman BS, Nemirovskaya TL (2008) Calcium-dependent signaling mechanisms and soleus fiber remodeling under gravitational unloading. J Muscle Res Cell Motil 29(6–8): 221–230. https://doi.org/10.1007/s10974-008-9164-7Ito N, Ruegg UT, Kudo A, Miyagoe-Suzuki Y, Takeda S (2013) Activation of calcium signaling through Trpv1 by nNOS and peroxynitrite as a key trigger of skeletal muscle hypertrophy. Nat Med 19(1): 101–106. https://doi.org/10.1038/nm.3019Hyatt HW, Powers SK (2020) The Role of Calpains in Skeletal Muscle Remodeling with Exercise and Inactivity-induced Atrophy. Int J Sports Med 41(14): 994–1008. https://doi.org/ 10.1055/a-1199-7662Spangenburg EE, McBride TA (2006) Inhibition of stretch-activated channels during eccentric muscle contraction attenuates p70S6K activation. J Appl Physiol 100(1): 129–135. https://doi.org/10.1152/japplphysiol.00619.2005Mirzoev TM, Tyganov SA, Petrova IO, Shenkman BS (2019) Acute recovery from disuse atrophy: Тhe role of stretch-activated ion channels in the activation of anabolic signaling in skeletal muscle. Am J Physiol Endocrinol Metab 316(1): 86–95. https://doi.org/10.1152/ajpendo.00261.2018Tyganov S, Mirzoev T, Shenkman B (2019) An Anabolic Signaling Response of Rat Soleus Muscle to Eccentric Contractions Following Hindlimb Unloading: A Potential Role of Stretch-Activated Ion Channels. Int J Mol Sci 20(5): 1165. https://doi.org/ 10.3390/ijms20051165Butterfield TA, Best TM (2009) Stretch-activated ion channel blockade attenuates adaptations to eccentric exercise. Med Sci Sports Exerc 41(2): 351–356. https://doi.org/10.1249/MSS.0b013e318187cffaJuffer P, Bakker AD, Klein-Nulend J, Jaspers RT (2014) Mechanical loading by fluid shear stress of myotube glycocalyx stimulates growth factor expression and nitric oxide production. Cell Biochem Biophys 69(3): 411–419. https://doi.org/10.1007/s12013-013-9812-4Maroto R, Raso A, Wood TG, Kurosky A, Martinac B, Hamill OP (2005) TRPC1 forms the stretch-activated cation channel in vertebrate cells. Nat Cell Biol 7(2): 179–185. https://doi.org/10.1038/ncb1218Yamaguchi Y, Iribe G, Nishida M, Naruse K (2017) Role of TRPC3 and TRPC6 channels in the myocardial response to stretch: Linking physiology and pathophysiology. Prog Biophys Mol Biol 130(Pt B): 264–272. https://doi.org/10.1016/j.pbiomolbio.2017.06.010Grimm C, Kraft R, Sauerbruch S, Schultz G, Harteneck C (2003) Molecular and functional characterization of the melastatin-related cation channel TRPM3. J Biol Chem 278(24): 21493-21501. https://doi.org/10.1074/jbc.M300945200Numata T, Shimizu T, Okada Y (2007) Direct mechano-stress sensitivity of TRPM7 channel. Cell Physiol Biochem 19(1–4): 1–8. https://doi.org/10.1159/000099187Nikolaev YA, Cox CD, Ridone P, Rohde PR, Cordero-Morales JF, Vasquez V, Laver DR, Martinac B (2019) Mammalian TRP ion channels are insensitive to membrane stretch. J Cell Sci 132(23): jcs238360. https://doi.org/ 10.1242/jcs.238360Coste B, Mathur J, Schmidt M, Earley TJ, Ranade S, Petrus MJ, Dubin AE, Patapoutian A (2010) Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science 330(6000): 55–60. https://doi.org/10.1126/science.1193270Coste B, Xiao B, Santos JS, Syeda R, Grandl J, Spencer KS, Kim SE, Schmidt M, Mathur J, Dubin AE, Montal M, Patapoutian A (2012) Piezo proteins are pore-forming subunits of mechanically activated channels. Nature 483(7388): 176–181. https://doi.org/10.1038/nature10812Tsuchiya M, Hara Y, Okuda M, Itoh K, Nishioka R, Shiomi A, Nagao K, Mori M, Mori Y, Ikenouchi J, Suzuki R, Tanaka M, Ohwada T, Aoki J, Kanagawa M, Toda T, Nagata Y, Matsuda R, Takayama Y, Tominaga M, Umeda M (2018) Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation. Nat Commun 9(1): 2049. https://doi.org/10.1038/s41467-018-04436-wBosutti A, Giniatullin A, Odnoshivkina Y, Giudice L, Malm T, Sciancalepore M, Giniatullin R, D'Andrea P, Lorenzon P, Bernareggi A (2021) “Time window” effect of Yoda1-evoked Piezo1 channel activity during mouse skeletal muscle differentiation. Acta Physiol (Oxf) 233(4): e13702. https://doi.org/ 10.1111/apha.13702.26Ma N, Chen D, Lee JH, Kuri P, Hernandez EB, Kocan J, Mahmood H, Tichy ED, Rompolas P, Mourkioti F (2022) Piezo1 regulates the regenerative capacity of skeletal muscles via orchestration of stem cell morphological states. Sci Adv 8(11): eabn0485. https://doi.org/10.1126/sciadv.abn0485Hirano K, Tsuchiya M, Shiomi A, Takabayashi S, Suzuki M, Ishikawa Y, Kawano Y, Takabayashi Y, Nishikawa K, Nagao K, Umemoto E, Kitajima Y, Ono Y, Nonomura K, Shintaku H, Mori Y, Umeda M, Hara Y (2023) The mechanosensitive ion channel PIEZO1 promotes satellite cell function in muscle regeneration. Life Sci Alliance 6(2): e202201783. https://doi.org/ 10.26508/lsa.202201783Hirata Y, Nomura K, Kato D, Tachibana Y, Niikura T, Uchiyama K, Hosooka T, Fukui T, Oe K, Kuroda R, Hara Y, Adachi T, Shibasaki K, Wake H, Ogawa W (2022) A Piezo1/KLF15/IL-6 axis mediates immobilization-induced muscle atrophy. J Clin Invest 132(10): 1–13. https://doi.org/10.1172/JCI154611Chen X, Wang N, Liu JW, Zeng B, Chen GL (2023) TMEM63 mechanosensitive ion channels: Activation mechanisms, biological functions and human genetic disorders. Biochem Biophys Res Commun 683: 149111. https://doi.org/10.1016/j.bbrc.2023.10.043Zhao X, Yan X, Liu Y, Zhang P, Ni X (2016) Co-expression of mouse TMEM63A, TMEM63B and TMEM63C confers hyperosmolarity activated ion currents in HEK293 cells. Cell Biochem Funct 34(4): 238–241. https://doi.org/10.1002/cbf.3185Murthy SE, Dubin AE, Whitwam T, Jojoa-Cruz S, Cahalan SM, Mousavi SAR, Ward AB, Patapoutian A (2018) OSCA/TMEM63 are an Evolutionarily Conserved Family of Mechanically Activated Ion Channels. Elife 7: e41844. https://doi.org/ 10.7554/eLife.41844Zheng W, Rawson S, Shen Z, Tamilselvan E, Smith HE, Halford J, Shen C, Murthy SE, Ulbrich MH, Sotomayor M, Fu TM, Holt JR (2023) TMEM63 proteins function as monomeric high-threshold mechanosensitive ion channels. Neuron 111(20): 3195–3210. https://doi.org/ 10.1016/j.neuron.2023.07.006Novikov VE, Ilyin EA (1981) Age-related reactions of rat bones to their unloading. Aviat Space Environ Med 52(9): 551–553.Morey-Holton ER, Globus RK (2002) Hindlimb unloading rodent model: Тechnical aspects. J Appl Physiol 92(4): 1367–1377. https://doi.org/10.1152/japplphysiol.00969.2001Mirzoev TM, Tyganov SA, Shenkman BS (2017) Akt-dependent and Akt-independent pathways are involved in protein synthesis activation during reloading of disused soleus muscle. Muscle Nerve 55(3): 393–399. https://doi.org/10.1002/mus.25235Tyganov SA, Mochalova EP, Melnikov IY, Vikhlyantsev IM, Ulanova AD, Sharlo KA, Mirzoev TM, Shenkman BS (2021) NOS-dependent effects of plantar mechanical stimulation on mechanical characteristics and cytoskeletal proteins in rat soleus muscle during hindlimb suspension. FASEB J 35(10): e21905. https://doi.org/10.1096/fj.202100783RSander H, Wallace S, Plouse R, Tiwari S, Gomes AV (2019) Ponceau S waste: Ponceau S staining for total protein normalization. Anal Biochem 575: 44–53. https://doi.org/10.1016/j.ab.2019.03.010Ingalls CP, Warren GL, Armstrong RB (1999) Intracellular Ca2+ transients in mouse soleus muscle after hindlimb unloading and reloading. J Appl Physiol 87(1): 386–390. https://doi.org/10.1152/jappl.1999.87.1.386Ingalls CP, Wenke JC, Armstrong RB (2001) Time course changes in [Ca2+]i, force, and protein content in hindlimb-suspended mouse soleus muscles. Aviat Space Environ Med 72(5): 471–476.Mukhina AM, Altaeva EG, Nemirovskaya TL, Shenkman BS (2008) The role of L-type calcium channels in the accumulation of Ca2+ in soleus muscle fibers in the rat and changes in the ratio of myosin and serca isoforms in conditions of gravitational unloading. Neurosci Behav Physiol 38(2): 181–188. https://doi.org/10.1007/s11055-008-0027-xEnns DL, Belcastro AN (2006) Early activation and redistribution of calpain activity in skeletal muscle during hindlimb unweighting and reweighting. Can J Physiol Pharmacol 84(6): 601–609. https://doi.org/10.1139/y06-013Mirzoev TM, Shenkman BS, Ushakov IB, Ogneva IV (2012) Desmin and alpha-actinin-2 content in rat soleus muscle in the dynamics of gravitational unloading and subsequent reloading. Dokl Biochem Biophys 444: 144–146. https://doi.org/10.1134/S1607672912030052Melnikov IY, Tyganov SA, Sharlo KA, Ulanova AD, Vikhlyantsev IM, Mirzoev TM, Shenkman BS (2022) Calpain-dependent degradation of cytoskeletal proteins as a key mechanism for a reduction in intrinsic passive stiffness of unloaded rat postural muscle. Pflugers Arch 474(11): 1171–1183. https://doi.org/10.1007/s00424-022-02740-5Sergeeva KV, Tyganov SA, Zaripova KA, Bokov RO, Nikitina LV, Konstantinova TS, Kalamkarov GR, Shenkman BS (2024) Mechanical and signaling responses of unloaded rat soleus muscle to chronically elevated β-myosin activity. Arch Biochem Biophys 754: 109961. https://doi.org/ 10.1016/j.abb.2024.109961Sergeeva KV, Tyganov SA, Kalashnikov VE, Shenkman BS, Mirzoev TM (2023) Analysis of the Role of Piezo1 Channels in Mechano-Anabolic Coupling in Rat Soleus Muscle. Biol Membrany 40(5): 362–369. https://doi.org/10.31857/S0233475523050080Vilchinskaya NA, Sergeeva KV, Tyganov SА, Shenkman BS, Mirzoev ТМ (2024) Role of the Mechanically Activated Channels in the Regulation of Anabolic Markers in the Isolated Rat Postural Muscle in Response to Passive Stretching. Aviakosm Ekolog Med 58(4): 44–51. https://doi.org/10.21687/0233-528x-2024-58-4-44-51Zanou N, Schakman O, Louis P, Ruegg UT, Dietrich A, Birnbaumer L, Gailly P (2012) Trpc1 ion channel modulates phosphatidylinositol 3-kinase/Akt pathway during myoblast differentiation and muscle regeneration. J Biol Chem 287(18): 14524–14534. https://doi.org/10.1074/jbc.M112.341784Xia L, Cheung KK, Yeung SS, Yeung EW (2016) The involvement of transient receptor potential canonical type 1 in skeletal muscle regrowth after unloading-induced atrophy. J Physiol 594(11): 3111–3126. https://doi.org/10.1113/JP271705Damm TB, Richard S, Tanner S, Wyss F, Egli M, Franco‐Obregón A (2013) Calcium‐dependent deceleration of the cell cycle in muscle cells by simulated microgravity. The FASEB J 27(5): 2045–2054. https://doi.org/10.1096/fj.12-218693Zhang BT, Yeung SS, Cheung KK, Chai ZY, Yeung EW (2014) Adaptive responses of TRPC1 and TRPC3 during skeletal muscle atrophy and regrowth. Muscle Nerve 49(5): 691–699. https://doi.org/10.1002/mus.23952Singh BB, Liu X, Tang J, Zhu MX, Ambudkar IS (2002) Calmodulin Regulates Ca2+-Dependent Feedback Inhibition of Store-Operated Ca2+ Influx by Interaction with a Site in the C Terminus of TrpC1. Mol Cell 9(4): 739–750. https://doi.org/10.1016/s1097-2765(02)00506-3