UNIVERSIDADE ESTADUAL DE CAMPINAS SISTEMA DE BIBLIOTECAS DA UNICAMP REPOSITÓRIO DA PRODUÇÃO CIENTIFICA E INTELECTUAL DA UNICAMP Versão do arquivo anexado / Version of attached file: Versão do Editor / Published Version Mais informações no site da editora / Further information on publisher's website: https://physoc.onlinelibrary.wiley.com/doi/full/10.14814/phy2.12409 DOI: 10.14814/phy2.12409 Direitos autorais / Publisher's copyright statement: ©2015 by John Wiley & Sons. All rights reserved. DIRETORIA DE TRATAMENTO DA INFORMAÇÃO Cidade Universitária Zeferino Vaz Barão Geraldo CEP 13083-970 – Campinas SP Fone: (19) 3521-6493 http://www.repositorio.unicamp.br Physiological Reports ISSN 2051-817X ORIGINAL RESEARCH Expression of calcium-buffering proteins in rat intrinsic laryngeal muscles Renato Ferretti1, Maria Julia Marques2, Tejvir S. Khurana3 & Humberto Santo Neto2 1 Departamento de Anatomia, Instituto de Biociencias de Botucatu, Universidade Estadual Paulista, Botucatu, Sao~ Paulo, Brazil 2 Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, Sao~ Paulo, Brazil 3 Department of Physiology, Perelman School of Medicine and Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, Pennsylvania Keywords Abstract Calcium, laryngeal muscles, Ncx, Pmca1, Serca, store-operated calcium entry. Intrinsic laryngeal muscles (ILM) are highly specialized muscles involved in phonation and airway protection, with unique properties that allow them to Correspondence perform extremely rapid contractions and to escape from damage in muscle Humberto Santo Neto, Departamento de dystrophy. Due to that, they may differ from limb muscles in several physio- Biologia Estrutural e Funcional, Instituto de logical aspects. Because a better ability to handle intracellular calcium has been Biologia, Universidade Estadual de Campinas suggested to explain ILM unique properties, we hypothesized that the profile (UNICAMP). Campinas, SP - 13083-970, of the proteins that regulate calcium levels in ILM is different from that in a Brazil. Tel: +55-19-3521-6395 limb muscle. Calcium-related proteins were analyzed in the ILM, cricothyroid Fax: 55-19-3521-6185 (CT), and tibialis anterior (TA) muscles from male Sprague–Dawley rats E-mail: [email protected] (8 weeks of age) using quantitative PCR and western blotting. Higher expres- sion of key Ca2+ regulatory proteins was detected in ILM compared to TA, Funding Information such as the sarcoplasmic reticulum (SR) Ca2+-reuptake proteins (Sercas 1 and This work was supported by CAPES/PDEE + 2+ 2+ ß~ 2), the Na /Ca exchanger, phospholamban, and the Ca -binding protein (grant #2037/10-6) and Fundacao de 2+ Amparo a Pesquisa do Estado de Sao~ Paulo calsequestrin. Parvalbumin, calmodulin and the ATPase, Ca -transporting, (FAPESP, grants 04/15526-9, 08/58491-1 and and plasma membrane 1 were also expressed at higher levels in ILM compared 13/10633-0). H.S.N. and M.J.M. are to TA. The store-operated calcium entry channel molecule was decreased in recipients of fellowships from Conselho ILM compared to the limb muscle and the voltage-dependent L-type and Nacional de Pesquisas (CNPq; 302831/2013- ryanodine receptor were expressed at similar levels in ILM and TA. These 4; 303320/2013-3; 474708/06-3). R.F. was results show that ILM have a calcium regulation system profile suggestive of a recipient of a fellowship from CAPES/PDEE better ability to handle calcium changes in comparison to limb muscles, and (2037/10-6), PROPe UNESP (2052/002/14) and CNPq (143170/08-2). this may provide a mechanistic insight for their unique pathophysiological properties. Received: 20 February 2015; Revised: 27 April 2015; Accepted: 29 April 2015 doi: 10.14814/phy2.12409 Physiol Rep, 3 (6), 2015, e12409, doi: 10.14814/phy2.12409 Introduction Dulhunty 1982; DelGaudio et al. 1995; Hoh 2005). These functions are possible due to ILM unique features that The intrinsic laryngeal muscles (ILM) are highly special- include specific fiber-type composition, high speed con- ized muscles involved in vital functions that include respi- traction, and resistance to fatigue (Hinrichsen and Dulh- ration, phonation, and airway protection (Hinrichsen and unty 1982; Hoh 2005). Their ability to precisely control ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of 2015 | Vol. 3 | Iss. 6 | e12409 the American Physiological Society and The Physiological Society. Page 1 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Calcium Proteins in Laryngeal Muscles R. Ferretti et al. airway caliber during phonation requires specific charac- pumped back into the SR by sarcoplasmic reticulum Ca2+ teristics reflected in their mechanical properties, innerva- ATPases (SERCA), extruded from the cell by the Na+/ tion pattern (Maranillo et al. 2005; Shinners et al. 2006; Ca2+ exchanger (NCX) (Berridge et al. 2003) and buffered Xu et al. 2009), regenerative capacity, response to age by the low-affinity, high capacity Ca2+-binding protein (Kersing and Jennekens 2004; Goding et al. 2005; calsequestrin (CASQ) (Beard et al. 2004). Other proteins McLoon et al. 2007), and disease (Marques et al. 2007; that also play a role in Ca2+ regulation include the T-sys- Thomas et al. 2008; Smythe 2009). Within the ILM, the tem plasma membrane Ca2+-ATPase type I (PMCA1) and posterior cricoarytenoid (PCA) muscle abducts the vocal the store-operated calcium entry (SOCE), which uses the fold, whereas the thyroarytenoid and cricothyroid (CT) SR Ca2+ sensor Orai1 Ca2+ channel (Lang et al. 2012). In adducts and tenses the vocal fold to aid in phonation and addition, cytosolic Ca2+-binding proteins, such as parval- airway protection during swallowing (Hinrichsen and bumin (PVALB), calmodulin (CaM), and regucalcin Dulhunty 1982; Hoh 2005). An interesting property of (RGN), can modify Ca2+ transients to ensure Ca2+ the ILM, in comparison to limb muscles, is their differen- homeostasis (Gailly 2002; Berridge et al. 2003; Beard et al. tial involvement in neuromuscular disorders. While ILM 2004; Yamaguchi 2005; Lang et al. 2012). are involved early in myasthenia gravis, amyotrophic lat- In this study, we evaluated the profile of calcium regu- eral sclerosis with bulbar involvement, and mitochondrial lation systems in ILM compared to limb muscle (tibialis myopathy (Debain et al. 1968; Mao et al. 2001), they are anterior, TA), in adult rats, using qPCR and Western histologically spared in Duchenne muscle dystrophy blotting. We were interested to see whether ILM shows a (DMD), except for the CT muscle (Marques et al. 2007; constitutive calcium-buffering proteins profile that could Thomas et al. 2008; Smythe 2009). predict their better capacity to handle calcium changes in The ILM play important roles in initiation of and comparison to limb muscle and EOM. We found that recovery from acute respiratory distress syndrome Ca2+ reuptake-related proteins of the sarcoplasmic reticu- (ARDS) and critical illnesses, with both swallowing and lum (Serca1, Serca2, Pln, Casq1, and Casq2) were phonation disability (Nixon et al. 2010). Additionally, expressed at higher levels in ILM in comparison to the vocal muscle paralysis is an important and frequent con- limb muscle. ILM showed higher levels of Orai1 in rela- dition seen in otolaryngology clinics, most frequently tion to TA and CaM was 17-fold increased in ILM com- caused by lesions to the recurrent laryngeal nerve (Li pared to TA. A similar pattern of gene expression of et al. 2014), although idiopathic etiologies remain com- Sercas 1 and 2 and calsequestrins was observed in the mon causes of unilateral vocal fold immobility (Rosenthal dystrophin-deficient mdx mice compared to the rat. These et al. 2007). Possible mechanisms underlying this differ- results provide a mechanistic insight into ILM physiologi- ential response of ILM to muscle diseases, compared to cal properties and response to pathophysiological condi- limb muscles, may be related to differences regarding tions. their neuromuscular junction structure or satellite cell regenerative potential (Feng et al. 2012). In the mdx Methods mouse model of DMD (Bulfield et al. 1984), we have sug- gested that ILM differ from the affected limb muscles in Animals terms of their ability to handle changes in calcium due to differential levels of calcium-buffering proteins (Ferretti All animal experiments were performed in accordance et al. 2009), similarly to what is observed in the extraocu- with the ARVO Statement for the Use of Animals in lar muscles, which are also protected in dystrophy (Khur- Ophthalmic and Vision Research, using protocols ana et al. 1995; Porter et al. 1998). approved by the Institutional Animal Care and Use Com- In skeletal muscle fibers, intracellular calcium (Ca2+) mittee of the University of Pennsylvania School of Medi- plays an important role in regulating muscle force pro- cine (protocol #A3079-01) and by the Committee of the duction, metabolism, and muscle gene expression (Berch- University of Campinas (protocol #1463-1). Male told et al. 2000; Chin 2010). The sarcoplasmic reticulum Sprague–Dawley rats (n = 20; 8 weeks of age), control (SR) and T-tubule membrane proteins play central roles C57BL/10ScSnJ (Ctrl, n = 10; 8 weeks of age), and mdx in calcium regulation. In excitation contraction coupling, mice (C57BL/10ScSn-Dmdmdx/J; n = 10; 8 weeks of age)