This renewal application describes ongoing studies on characterization of key proteins regulating Ca release from junctional sarcoplasmic reticulum (SR) in cardiac muscle. Four proteins will be studied, which are abundantly expressed in heart, co-localize to the junctional SR membrane, and form a complex. The four proteins investigated are junctin, triadin 1, calsequestrin, and the ryanodine receptor. The interactions stabilizing the complex of these four proteins at the junctional SR membrane will be resolved and the role of the complex in regulating Ca release elucidated. Junctin and triadin 1 are homologous, integral membrane proteins which bind to each other, to calsequestrin, and to the ryanodine receptor. They are responsible for anchoring calsequestrin to the ryanodine receptor at the lumenal face of the SR membrane. The sites of junctin and triadin 1 binding to each other, to calsequestrin, and to the ryanodine receptor will be localized, and the effects of junctin and triadin 1 on the channel activity of the ryanodine receptor will be defined, alone and in combination with calsequestrin. Junctin appears to promote junction formation with sarcolemmal membranes, and an attempt will be made to identify putative sarcolemmal docking protein(s) which may bind to the cytoplasmic domain of junctin. Calsequestrin is the major Ca-binding protein located in the lumen of the junctional SR of cardiac muscle, which stores the Ca required for Ca release. The sites of calsequestrin binding to junctin and to triadin 1 will be localized. Junctin and triadin 1 effects on Ca binding to calsequestrin will be characterized, and the effects of calsequestrin on the channel activity of the ryanodine receptor will be determined. Calsequestrin will be crystallized, and its 3-D structure determined in the presence and absence of Ca, complexed with and without the calsequestrin-binding domains of junctin and triadin 1. To assess the function(s) of junctin, triadin 1, and calsequestrin in intact myocytes and in live animals, the wild-type and mutated proteins will be overexpressed in transgenic mouse hearts. Effects of overexpression of the proteins on cardiac membrane biochemistry, ultrastructure, and intact cellular physiology will be investigated. Completion of these studies will increase our understanding of the role(s) of junctin, triadin 1, and calsequestrin in regulating the channel activity of the ryanodine receptor, in maintaining the molecular architecture at the junctional SR membrane, and in controlling the strength of the heartbeat. New animal models useful for investigation of cardiac hypertrophy and heart failure will result.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Pharmacology A Study Section (PHRA)
Program Officer
Przywara, Dennis
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Indiana University-Purdue University at Indianapolis
Internal Medicine/Medicine
Schools of Medicine
United States
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Chopra, Nagesh; Kannankeril, Prince J; Yang, Tao et al. (2007) Modest reductions of cardiac calsequestrin increase sarcoplasmic reticulum Ca2+ leak independent of luminal Ca2+ and trigger ventricular arrhythmias in mice. Circ Res 101:617-26
Knollmann, Bjorn C; Chopra, Nagesh; Hlaing, Thinn et al. (2006) Casq2 deletion causes sarcoplasmic reticulum volume increase, premature Ca2+ release, and catecholaminergic polymorphic ventricular tachycardia. J Clin Invest 116:2510-20
Kirchhefer, Uwe; Hanske, Gabriela; Jones, Larry R et al. (2006) Overexpression of junctin causes adaptive changes in cardiac myocyte Ca(2+) signaling. Cell Calcium 39:131-42
Kirchhefer, Uwe; Baba, Hideo A; Hanske, Gabriela et al. (2004) Age-dependent biochemical and contractile properties in atrium of transgenic mice overexpressing junctin. Am J Physiol Heart Circ Physiol 287:H2216-25
Gyorke, Inna; Hester, Nichole; Jones, Larry R et al. (2004) The role of calsequestrin, triadin, and junctin in conferring cardiac ryanodine receptor responsiveness to luminal calcium. Biophys J 86:2121-8
Kirchhefer, Uwe; Jones, Larry R; Begrow, Frank et al. (2004) Transgenic triadin 1 overexpression alters SR Ca2+ handling and leads to a blunted contractile response to beta-adrenergic agonists. Cardiovasc Res 62:122-34
Yang, Alexander; Sonin, Dimitry; Jones, Larry et al. (2004) A beneficial role of cardiac P2X4 receptors in heart failure: rescue of the calsequestrin overexpression model of cardiomyopathy. Am J Physiol Heart Circ Physiol 287:H1096-103
Tijskens, Pierre; Jones, Larry R; Franzini-Armstrong, Clara (2003) Junctin and calsequestrin overexpression in cardiac muscle: the role of junctin and the synthetic and delivery pathways for the two proteins. J Mol Cell Cardiol 35:961-74
Kirchhefer, Uwe; Neumann, Joachim; Bers, Donald M et al. (2003) Impaired relaxation in transgenic mice overexpressing junctin. Cardiovasc Res 59:369-79
Kirchhefer, Uwe; Baba, Hideo A; Kobayashi, Yvonne M et al. (2002) Altered function in atrium of transgenic mice overexpressing triadin 1. Am J Physiol Heart Circ Physiol 283:H1334-43

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