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Department of Molecular Physiology

Members

Director Osamu NAKAGAWA
Laboratory Chief Yuko IWATA, Tomoe Y. NAKAMURA-NISHITANI, Takashi HISAMITSU, Yusuke WATANABE
Research Fellow Yumi KIUGASA-KATAYAMA, Toshiharu FUKAYAMA, Daiki SEYA, Toru TANAKA
Visiting Researcher Teruhisa KAWAMURA, Yuichiro ARIMA
Trainee Masahide FUJITA, Kazuki OKUMURA
Research Assistant Haruka KAWAKAMI, Natsuko YOSHIDA, Masae SUZUKI, Yukihiro HARADA
Administrative Assistant Kazuyo ENDO

Research activities

The Department of Molecular Physiology has been engaged in studies of the molecular mechanisms of the ionic regulation of contraction and relaxation of cardiac and smooth muscles, which are the major constituents of the cardiovascular system. In particular, we focus on the molecular physiology and pharmacology of the sarcolemmal ion transporters. Another subject is a study of the cellular mechanism underlying the functional failure and death of myocytes associated with cardiomyopathy. This study is particularly concerned with the role of cytoskeletal proteins in the genesis of dilated cardiomyopathy using animal disease models.

Studies on cellular ionic mechanisms regulating the contractile state of myocytes

Ionic homeostasis in the cardiovascular system is regulated by multiple ion transporters and ion channels. Among them, the plasma membrane Na+/Ca2+ (NCX) and Na+/H+ (NHE) exchangers play crucial roles in ionic metabolism of intracellular Na+, Ca2+ and H+ in cardiovascular cells. These transporters participate not only in physiological regulation of contractile activity but also in pathological aspects of myocyte function such as cardiac ischemia/reperfusion injury, cardiac hypertrophy/failure, and hypertension. In this laboratory, we investigate various mechanistic, physiological, and pharmacological aspects of functions of these transporters, in particular, ion transport mechanisms, interaction with signaling molecules, and functions in cardiovascular tissues, using multiple methodologies including analysis of expressed mutant molecules, biochemical and cell biological approaches, and analysis using genetically modified mice.

We have developed the first selective inhibitor of NCX, KB-R7943, that has therapeutic potential for clinical use. This compound is being widely used as a useful tool for studies of NCX function at different levels of experimental systems. Recently, we analyzed the topology of the cardiac NCX molecule as well as its interaction with transport substrate Ca2+, KB-R7943, a nonselective inhibitor Ni2+, and an activator Li+. Based on detailed analysis by cysteine scanning and site-directed mutagenesis, we found that the highly conserved alpha-1 and alpha-2 repeat regions in NCX may be involved in the interactions with these inhibitors and ligands, suggesting that these regions participate in the formation of ion transport pathway in NCX. Our study have also obtained evidence that NCX consists of 9 transmembrane segments, with regions containing alpha-1 and alpha-2 repeats forming re-entrant membrane loops originating from the opposite sides of the membrane.

NHE is an excellent target for the study of cellular signal transduction leading to modulation of transport activity, because it is regulated by a variety of extracellular signals. We have recently analyzed the interaction of calmodulin (CaM) and the calcineurin-homologous protein CHP with various NHE isoforms, and found that CaM is involved in Ca2+-induced activation of NHE1, whereas CHP is an essential cofactor to support physiological activity of multiple NHE isoforms. We have also determined that a hallmark of NHE regulation"pH-sensor" is regulated via several cytoplasmic subdomains of NHE1. More recently, we analyzed membrane topology of NHE1, and found that NHE1 comprises 12 transmembrane segments with N- and C-termini in the cytosol. Our future goal is to understand the structure-function relationships in these ion transporters and its regulatory machinery at a higher resolution as well as to obtain insights into their definitive physiological roles in cardiovascular and other tissues.

Studies on roles of cytoskeletal proteins in the genesis of cardiomyopathy

Cardiomyopathy is caused by a variety of etiologies. Deficiency in the dystrophin gene causes a high incidence of dilated cardiomyopathy in Duchenne and Becker muscular dystrophy patients and also causes X-linked dilated cardiomyopathy. In animal models, dilated cardiomyopathies have been associated with genetic deficiencies of several cytoskeletal proteins, indicating that these proteins are essential for the maintenance of the normal contractile function of cardiac myocytes. However, the cellular mechanisms by which particular defects in these proteins generate cardiomyopathy are not known.

In striated muscles, the dystrophin complex consists of dystrophin and its associated proteins: alpha- and beta-dystroglycans, alpha-, beta-, gamma- and delta-sarcoglycans, sarcospan, syntrophins, and dystrobrevins. The complex confers a linkage between laminin in the extracellular matrix and F-actin cytoskeleton via the alpha- and beta-dystroglycan subcomplex and is thought to protect sarcolemma from damage induced by contractile activity. Sarcoglycans are intrinsic membrane proteins and form a subcomplex with sarcospan. Loss-of-function mutations in sarcoglycan genes cause skeletal muscle dystrophy and cardiomyopathy in patients, BIO14.6 hamsters and sarcoglycan KO mice.

In skeletal and cardiac muscles of BIO14.6 hamsters, we found that sarcoglycans are greatly reduced or lost and that the dystrophin complex are totally disrupted, although dystrophin and beta-dystroglycan are present at mildly reduced levels. Using this hamster model, we have been studying the functions of sarcoglycans and the cellular mechanism for pathogenesis of cardiac and skeletal myopathies. We have recently provided evidence that sarcoglycan deficiency in BIO14.6 hamster myocytes or in myocytes rendered sarcoglycans deficient using sarcoglycan antisense oligonucleotides are susceptible to applied mechanical stress leading to cell damage and that these cells exhibit an increased basal rate of Ca2+ influx. In BIO14.6 hamster myocytes, we have further shown that the resting activity of stretch-activated non-specific cation channels is markedly elevated. The latter finding suggests that increased activity of these channels may ultimately cause Ca2+ overload, which contributes to cell damage observed in the myopathic myocytes. We have also studied the bidirectional signaling between sarcoglycan subcomplex and integrin cell adhesion systems in cultured L6 myocytes and the interaction of syntrophin with actin cytoskeleton in skeletal and cardiac myocytes. Furthermore, we are engaged in search of genes that are possibly involved in pathogenesis of cardiac and skeletal myopathies.

Selected Publication

Osamu NAKAGAWA

  1. Mizuta K, Sakabe M, Hashimoto A, Ioka T, Sakai C, Okumura K, Hattammaru M, Fujita M, Araki M, Somekawa S, Saito Y, Nakagawa O. Impairment of endothelial-mesenchymal transformation during atrioventricular cushion formation in Tmem100 null embryos. Dev Dyn 2014, in press.
  2. Morioka T, Sakabe M, Ioka T, Iguchi T, Mizuta K, Hattammaru M, Sakai C, Itoh M, Sato GE, Hashimoto A, Fujita M, Okumura K, Araki M, Xin M, Pedersen RA, Utset MF, Kimura H, Nakagawa O. An important role of endothelial Hairy-related transcription factors in mouse vascular development. Genesis 52:897-906, 2014.
  3. Somekawa S, Imagawa K, Hayashi H, Sakabe M, Ioka T, Sato GE, Inada K, Iwamoto T, Mori T, Uemura S, Nakagawa O, Saito Y. Tmem100, an ALK1 signaling-dependent gene essential for arterial endothelium differentiation and vascular morphogenesis. Proc Nat Acad Sci USA 109:12064-9, 2012.
  4. Xin M, Small EM, van Rooij E, Qi X, Richardson JA, Nakagawa O, Olson EN. Essential roles of the bHLH transcription factor Hrt2 in repression of atrial gene expression and maintenance of postnatal cardiac function. Proc Nat Acad Sci USA 104:7975-80, 2007.
  5. Nakagawa O, Arnold M, Nakagawa M, Hamada H, Shelton JM, Kusano H, Harris TM, Childs G, Campbell KP, Richardson JA, Nishino I, Olson EN. Centronuclear myopathy in mice lacking a novel muscle-specific protein kinase transcriptionally regulated by MEF2. Genes Dev 19:2066-77, 2005.
  6. Murakami M, Nakagawa M, Olson EN, Nakagawa O. A WW domain protein TAZ is a critical co-activator for TBX5, a transcription factor implicated in Holt-Oram syndrome. Proc Nat Acad Sci USA 102:18034-9, 2005.
  7. Kathiriya IS, King IN, Murakami M, Nakagawa M, Astle JA, Gardner KA, Gerard RD, Olson EN, Srivastava D, Nakagawa O. Hairy-related transcription factors inhibit GATA-dependent cardiac gene expression through a signal-responsive mechanism. J Biol Chem 279:54937-43, 2004.
  8. Gottlieb PD, Pierce SA, Sims RJ III, Yamagishi H, Weihe EK, Harriss JV, Maika SD, Kuziel WA, King HL, Olson EN, Nakagawa O, Srivastava D. Bop encodes a muscle-restricted protein containing MYND and SET domains and is essential for cardiac differentiation and morphogenesis. Nature Genet 31:25-32, 2002.
  9. Nakagawa O, McFadden DG, Nakagawa M, Yanagisawa H, Hu T, Srivastava D, Olson EN. Members of the HRT family of bHLH proteins act as transcriptional repressors downstream of Notch signaling. Proc Nat Acad Sci USA 97:13655-60, 2000.
  10. Nakagawa O, Nakagawa M, Richardson JA, Olson EN, Srivastava D. HRT1, HRT2, HRT3: a new subclass of bHLH transcription factors marking specific cardiac, somitic and pharyngeal arch segments. Dev Biol 216:72-84, 1999.

Yuko IWATA

  1. Iwata Y, Ohtake H, Suzuki O, Matsuda J, Komamura K, Wakabayashi S. Blockade of sarcolemmal TRPV2 accumulation inhibits progression of dilated cardiomyopathy. Cardiovasc Res 99:760-8, 2013.
  2. Maekawa K*, Hirayama A*, Iwata Y*, Tajima Y, Nishimaki-Mogami T, Sugawara S, Ueno N, Abe H, Ishikawa M, Murayama M, Matsuzawa Y, Nakanishi H, Ikeda K, Arita M, Taguchi R, Minamino N, Wakabayashi S, Soga T, Saito Y (* Equal Contribution). Global metabolic analysis of heart tissue in a hamster model for dilated cardiomyopathy. J Mol Cell Cardiol 59:76-85, 2013.
  3. Iwata Y, Suzuki O, Wakabayashi S. Decreased surface sialic acid content is a sensitive indicator for muscle damage. Muscle Nerve 47:372-378, 2013.
  4. Iwata Y, Katanosaka Y, Arai Y, Shigekawa M, Wakabayashi S. Dominant-negative inhibition of Ca2+ influx via TRPV2 ameliorates muscular dystrophy in animal models. Hum Mol Genet 18:824-34, 2009.
  5. Zanou N, Iwata Y, Schakman O, Lebacq J, Wakabayashi S, Gailly P. Essential role of TRPV2 ion channel in the sensitivity of dystrophic muscle to eccentric contractions. FEBS Lett 583:3600-4, 2009.
  6. Nakamura TY*, Iwata Y*, Arai Y, Komamura K, Wakabayashi S (* Equal Contribution). Activation of Na+/H+ exchanger 1 is sufficient to generate Ca2+ signals that induce cardiac hypertrophy and heart failure. Circ Res 103:891-9, 2008.
  7. Iwata Y, Katanosaka Y, Hisamitsu T, Wakabayashi S. Enhanced Na+/H+ exchange activity contributes to the pathogenesis of muscular dystrophy via involvement of P2 receptors. Am J Pathol 171:1576-87, 2007.
  8. Iwata Y, Katanosaka Y, Shijun Z, Kobayashi Y, Hanada H, Shigekawa M, Wakabayashi S. Protective effects of Ca2+ handling drugs against abnormal Ca2+ homeostasis and cell damage in myopathic skeletal muscle cells. Biochem Pharmacol 70:740-51, 2005.
  9. Iwata Y, Katanosaka Y, Arai Y, Komamura K, Miyatake K, Shigekawa M. A novel mechanism of myocyte degeneraion involving the Ca2+-permeable growth factor-regulated channel. J Cell Biol 161:957-67, 2003.
  10. Muraki K, Iwata Y, Katanosaka Y, Ito T, Ohya S, Shigekawa M, Imaizumi Y. TRPV2 is a component of osmotically sensitive cation channels in murine aortic myocytes. Circ Res 93:829-38, 2003.

Tomoe Y. NAKAMURA

  1. Wakabayashi S, Hisamitsu T, Nakamura TY. Regulation of the cardiac Na+/H+ exchanger in health and disease. J Mol Cell Cardiol 61:68-76, 2013.
  2. Nakamura TY, Wakabayashi S. Role of neuronal calcium sensor-1 in the cardiovascular system. Trends Cardiovasc Med 22:12-7, 2012.
  3. Nakamura TY, Jeromin A, Mikoshiba K, Wakabayashi S. Neuronal calcium sensor-1 promotes immature heart function and hypertrophy by enhancing Ca2+ signals. Circ Res 109:512-23, 2011.
  4. Nakamura TY*, Iwata Y*, Arai Y, Komamura K, Wakabayashi S (* Equal Contribution). Activation of Na+/H+ exchanger 1 is sufficient to generate Ca2+ signals that induce cardiac hypertrophy and heart failure. Circ Res 103:891-9, 2008.
  5. Nakamura TY, Jeromin A, Smith G, Kurushima H, Koga H, Nakabeppu Y, Wakabayashi S, Nabekura J. Novel role of neuronal Ca2+ sensor-1 as a survival factor up-regulated in injured neurons. J Cell Biol 172:1081-91, 2006.
  6. Nakamura TY, Pountney DJ, Ozaita A, Nandi S, Ueda S, Rudy B, Coetzee WA. A role for frequenin, a Ca2+-binding protein, as a regulator of Kv4 K+-currents. Proc Natl Acad Sci USA 98:12808-13, 2001.
  7. Nakamura TY, Iwata Y, Sampaolesi M, Hanada H, Saito N, Artman M, Coetzee WA, Shigekawa M. Stretch-activated cation channels in skeletal muscle myotubes from sarcoglycan-deficient hamsters. Am J Physiol Cell Physiol 281:C690-9, 2001.
  8. Nakamura TY, Yamamoto I, Nishitani H, Matozaki T, Suzuki T, Wakabayashi S, Shigekawa M, Goshima K. Detachment of cultured cells from the substratum induced by the neutrophil-derived oxidant NH2Cl: Synergistic role of phosphotyrosine and intracellular Ca2+ concentration. J Cell Biol 131:509-24, 1995.
  9. Nakamura TY, Yamamoto I, Kanno Y, Shiba Y, Goshima K. Metabolic coupling of glutathione between mouse and quail cardiac myocytes and its protective role against oxidative stress. Circ Res 74:806-16, 1994.
  10. Nakamura TY, Goda K, Okamoto T, Kishi T, Nakamura T, Goshima K. Contractile and morphological impairment of cultured fetal mouse myocytes induced by oxygen radicals and oxidants. Correlation with intracellular Ca2+ concentration. Circ Res 73:758-70, 1993.

Takashi HISAMITSU

  1. Shimada-Shimizu N, Hisamitsu T, Nakamura TY, Hirayama N, Wakabayashi S. Na+/H+ exchanger 1 is regulated via its lipid-interacting domain which functions as a molecular switch: A pharmacological approach using indolocarbazole compounds. Mol Pharmacol 85:18-28, 2014.
  2. Wakabayashi S, Hisamitsu T, Nakamura TY. Regulation of the cardiac Na+/H+ exchanger in health and disease. J Mol Cell Cardiol 61:68-76, 2013.
  3. Shimada-Shimizu N, Hisamitsu T, Nakamura TY, Wakabayashi S. Evidence that Na+/H+ exchanger 1 is an ATP-binding protein. FEBS J 280:1430-42, 2013.
  4. Hisamitsu T, Nakamura TY, Wakabayashi S. Na+/H+ exchanger 1 directly binds to calcineurin A and activates downstream NFAT signaling, leading to cardiomyocyte hypertrophy. Mol Cell Biol 32:3265-80, 2012.
  5. Wakabayashi S, Nakamura TY, Kobayashi S, Hisamitsu T. Novel phorbol ester-binding motif mediates hormonal activation of Na+/H+ exchanger. J Biol Chem 285:26652-61, 2010.
  6. Iwata Y, Katanosaka Y, Hisamitsu T, Wakabayashi S. Enhanced Na+/H+ exchange activity contributes to the pathogenesis of muscular dystrophy via involvement of P2 receptors. Am J Pathol 171:1576-87, 2007.
  7. Hisamitsu T*, Yamada K*, Nakamura TY, Wakabayashi S (* Equal Contribution). Functional importance of charged residues within the putative intracellular loops in pH regulation by Na+/H+ exchanger NHE1. FEBS J 274:4326-35, 2007.
  8. Hisamitsu T, BenAmmar Y, Nakamura TY, Wakabayashi S. Dimerization is crucial for function of the Na+/H+ exchanger NHE1. Biochemistry 45:13346-55, 2006.
  9. BenAmmar Y, Takeda S, Hisamitsu T, Mori H, Wakabayashi S. Crystal structure of CHP2 complexed with NHE1-cytosolic region and an implication for pH regulation. EMBO J 25:2315-25, 2006.
  10. Hisamitsu T, Pang T, Shigekawa M, Wakabayashi S. Dimeric interaction between the cytoplasmic domains of the Na+/H+ exchanger NHE1 revealed by symmetrical intermolecular cross-linking and selective co-immunoprecipitation. Biochemistry 31:11135-43, 2004.

Soushi KOBAYASHI

  1. Wakabayashi S, Nakamura TY, Kobayashi S, Hisamitsu T. Novel phorbol ester-binding motif mediates hormonal activation of Na+/H+ exchanger. J Biol Chem 285:26652-61, 2010.
  2. Tsuneki H, Kobayashi S, Takagi K, Kagawa S, Tsunoda M, Murata M, Matsuoka T, Wada T, Kurachi M, Kimura I, Sasaoka T. Novel G423S mutation of human alpha7 nicotinic receptor promotes agonist-induced desensitization by a protein kinase C-dependent mechanism. Mol Pharmacol 71:777-86, 2007.
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