Abstract
The mechanism by which mutations of the cardiac troponin I (cTnI) gene evoke familial hypertrophic cardiomyopathy (fHCM) is unknown. In this investigation the potential effects of three fHCM-related cTnI mutations on Calpain-1-induced cTnI degradation were tested, and a study was made of whether additional conformational changes due to troponin complex formation and protein kinase A-induced phosphorylation affect the intensity of cTnI proteolysis. Purified recombinant wild-type cTnI and three of its fHCM-related missense mutants (R145G, G203S and K206Q), alone or in the troponin complex (i.e. together with troponin C and troponin T), in the non-phosphorylated or protein kinase A-bisphosphorylated forms were proteolyzed in vitro in the presence of Calpain-1 (0.05–2.5U) at 30°C. Following incubation with Calpain-1 for 0.5, 30, 60 or 120 min, the extent of protein degradation was evaluated through the use of Western immunoblotting and densitometry. The results indicated that both the wild-type and the mutant cTnI molecules were susceptible to Calpain-1. However, the degradation of the cTnI molecules in the troponin complex was less intense than that of the non-complexed forms. Moreover, phosphorylation by protein kinase A conferred effective protection against cTnI proteolysis. The data suggested that mutations in the central inhibitory domain (R145G) and in the C-terminal region (G203S and K206Q) of cTnI do not affect its Calpain-1-mediated degradation, or the phosphorylation-induced protection against proteolysis.
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Kimura A, Harada H, Park JE, Nishi H, Satoh M, Takahashi M, Hiroi S, Sasaoka T, Ohbuchi N, Nakamura T, Koyanagi T, Hwang TH, Choo JA, Chung KS, Hasegawa A, Nagai R, Okazaki O, Nakamura H, Matsuzaki M, Sakamoto T, Toshima H, Koga Y, Imaizumi T, Sasazuki T: Mutations in the cardiac troponin I gene associated with hypertrophic cardiomyopathy. Nat Genet 16: 379-382, 1997
Solaro RJ, Rarick HM: Troponin and tropomyosin: Proteins that switch on and tune in the activity of cardiac myofilaments. Circ Res 83: 471-480, 1998
Solaro RJ: Troponin I, stunning, hypertrophy, and failure of the heart. Circ Res 84: 122-124, 1999
James J, Zhang Y, Osinska H, Sanbe A, Klevitsky R, Hewett TE, Robbins J: Transgenic modeling of a cardiac troponin I mutation linked to familial hypertrophic cardiomyopathy. Circ Res 87: 805-811, 2000
Takahashi-Yanaga F, Morimoto S, Harada K, Minakami R, Shiraishi F, Ohta M, Lu QW, Sasaguri T, Ohtsuki I: Functional consequences of the mutations in human cardiac troponin I gene found in familial hypertrophic cardiomyopathy. J Mol Cell Cardiol 33: 2095-2107, 2001
Lang R, Gomes AV, Zhao J, Housmans PR, Miller T, Potter JD: Functional analysis of a troponin I (Arg145Gly) mutation associated with familial hypertrophic cardiomyopathy. J Biol Chem 277: 11670-11678, 2002
Elliott K, Watkins H, Redwood CS: Altered regulatory properties of human cardiac troponin I mutants that cause hypertrophic cardiomyopathy. J Biol Chem 275: 22069-22074, 2000
Gao WD, Atar D, Liu Y, Perez NG, Murphy AM, Marban E: Role of troponin I proteolysis in the pathogenesis of stunned myocardium. Circ Res 80: 393-399, 1997
McDonough JL, Arrell DK, Van Eyk JE: Troponin I degradation and covalent complex formation accompanies myocardial ischemia/reperfusion injury. Circ Res 84: 9-20, 1999
Papp Z, van der Velden J, Stienen GJM: Calpain-I induced alterations in the cytoskeletal structure and impaired mechanical properties of single myocytes of rat heart. Cardiovasc Res 45: 981-993, 2000
Di Lisa F, De Tullio R, Salamino F, Barbato R, Melloni E, Siliprandi N, Schiaffino S, Pontremoli S: Specific degradation of troponin T and I by μ-calpain and its modulation by substrate phosphorylation. Biochem J 308: 57-61, 1995
Murphy AM, Kögler H, Georgakopoulos D, McDonough JL, Kass DA, Van Eyk JE, Marbán E: Transgenic mouse model of stunned myocardium. Science 287: 488-491, 2000
Goll DE, Thompson VF, Taylor RG, Christiansen JA: Role of the calpain system in muscle growth. Biochimie 74: 225-237, 1992
Richard I, Broux O, Allamand V, Fougerousse F, Chiannilkulchai N, Bourg N, Brenguier L, Devaud C, Pasturaud P, Roudaut C, Hillaire D, Passos-Bueno MR, Zatz M, Tischfield JA, Fardeau M, Jackson CE, Cohen D, Beckmann JS: Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A. Cell 81: 27-40, 1995
Deng Y, Schmidtmann A, Redlich A, Westerdorf B, Jaquet K, Thieleczek R: Effects of phosphorylation and mutation R145G on human cardiac troponin I function. Biochemistry 40: 14593-14602, 2001
Reiffert SU, Jaquet K, Heilmeyer LMG, Herberg FW: Stepwise subunit interaction changes by mono-and bisphosphorylation of cardiac troponin I. Biochemistry 37: 13516-13525, 1998
Reiffert SU, Maytum R, Geeves M, Lohmann K, Greis T, Blüggel M, Meyer HE, Heilmeyer LMG, Jaquet K: Characterization of the cardiac holotroponin complex reconstituted from native cardiac troponin T and recombinant I and C. Eur J Biochem 261: 40-47, 1999
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Barta, J., Tóth, A., Jaquet, K. et al. Calpain-1-dependent degradation of troponin I mutants found in familial hypertrophic cardiomyopathy. Mol Cell Biochem 251, 83–88 (2003). https://doi.org/10.1023/A:1025485916872
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DOI: https://doi.org/10.1023/A:1025485916872