Altered Regulatory Properties of Human Cardiac Troponin I Mutants That Cause Hypertrophic Cardiomyopathy*

Cardiac troponin I (cTnI) is the inhibitory component of the troponin complex and is involved in the calcium control of heart muscle contraction. Recently, specific missense mutations of the cTnI gene (TNNI3) have been shown to cause familial hypertrophic cardiomyopathy (HCM). We have analyzed the functional effects of two HCM mutations (R145G and R162W) using purified recombinant cTnI. Both mutations gave reduced inhibition of actin-tropomyosin-activated myosin ATPase, both in experiments using cTnI alone and in those using reconstituted human cardiac troponin under relaxing conditions. Both mutant troponin complexes also conferred increased calcium sensitivity of ATPase regulation. Studies on wild type/R145G mutant mixtures showed that the wild type phenotype was dominant in that the inhibition and the calcium sensitivity conferred by a 50:50 mixture was more similar to wild type than expected. Surface plasmon resonance-based assays showed that R162W mutant had an increased affinity for troponin C in the presence of calcium. This observation may contribute to the increased calcium sensitivity found with this mutant and also corroborates recent structural data. The observed decreased inhibition and increased calcium sensitivity suggest that these mutations may cause HCM via impaired relaxation rather than the impaired contraction seen with some other classes of HCM mutants.

[1]  H. Taussky,et al.  A microcolorimetric method for the determination of inorganic phosphorus. , 1953, The Journal of biological chemistry.

[2]  J. Seidman,et al.  A molecular basis for familial hypertrophic cardiomyopathy: A β cardiac myosin heavy chain gene missense mutation , 1990, Cell.

[3]  H. Cheung,et al.  Coupling of calcium to the interaction of troponin I with troponin C from cardiac muscle. , 1994, Biochemistry.

[4]  M. Way,et al.  Expression of human plasma gelsolin in Escherichia coli and dissection of actin binding sites by segmental deletion mutagenesis , 1989, The Journal of cell biology.

[5]  C. Ramos,et al.  Structural and regulatory functions of the NH2- and COOH-terminal regions of skeletal muscle troponin I. , 1994, The Journal of biological chemistry.

[6]  R. Hodges,et al.  A synthetic peptide mimics troponin I function in the calcium‐dependent regulation of muscle contraction , 1993, FEBS letters.

[7]  H. Watkins,et al.  Functional analyses of troponin T mutations that cause hypertrophic cardiomyopathy: insights into disease pathogenesis and troponin function. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[8]  E. Eisenberg,et al.  Troponin-tropomyosin complex. Column chromatographic separation and activity of the three, active troponin components with and without tropomyosin present. , 1974, The Journal of biological chemistry.

[9]  F. Studier,et al.  Use of T7 RNA polymerase to direct expression of cloned genes. , 1990, Methods in enzymology.

[10]  M. James,et al.  Refined crystal structure of troponin C from turkey skeletal muscle at 2.0 A resolution. , 1988, Journal of molecular biology.

[11]  J. D. Pardee,et al.  [18] Purification of muscle actin , 1982 .

[12]  B. Maron,et al.  A mutant tropomyosin that causes hypertrophic cardiomyopathy is expressed in vivo and associated with an increased calcium sensitivity. , 1998, Circulation research.

[13]  D. Szczesna,et al.  Altered Regulation of Cardiac Muscle Contraction by Troponin T Mutations That Cause Familial Hypertrophic Cardiomyopathy* , 2000, The Journal of Biological Chemistry.

[14]  B. Hainque,et al.  Familial hypertrophic cardiomyopathy: from mutations to functional defects. , 1998, Circulation research.

[15]  D. K. Arrell,et al.  Troponin I degradation and covalent complex formation accompanies myocardial ischemia/reperfusion injury. , 1999, Circulation research.

[16]  G Butler-Browne,et al.  Molecular cloning of human cardiac troponin I using polymerase chain reaction , 1990, FEBS letters.

[17]  L. Leinwand,et al.  Heterologous expression of a cardiomyopathic myosin that is defective in its actin interaction. , 1994, The Journal of biological chemistry.

[18]  D. Kass,et al.  Transgenic mouse model of stunned myocardium. , 2000, Science.

[19]  J. Stull,et al.  Structural and functional responses of mammalian thick filaments to alterations in myosin regulatory light chains. , 1998, Journal of structural biology.

[20]  J. Trewhella,et al.  A model structure of the muscle protein complex 4Ca2+.troponin C.troponin I derived from small-angle scattering data: implications for regulation. , 1994, Biochemistry.

[21]  R. Hodges,et al.  Mapping of a second actin-tropomyosin and a second troponin C binding site within the C terminus of troponin I, and their importance in the Ca2+-dependent regulation of muscle contraction. , 1997, Journal of molecular biology.

[22]  M. Villain,et al.  Conformation of the Regulatory Domain of Cardiac Muscle Troponin C in Its Complex with Cardiac Troponin I* , 1999, The Journal of Biological Chemistry.

[23]  S. Lowey,et al.  [7] Preparation of myosin and its subfragments from rabbit skeletal muscle , 1982 .

[24]  H. Watkins,et al.  Properties of mutant contractile proteins that cause hypertrophic cardiomyopathy. , 1999, Cardiovascular Research.

[25]  E. Homsher,et al.  Functional Consequences of Troponin T Mutations Found in Hypertrophic Cardiomyopathy* , 1999, The Journal of Biological Chemistry.

[26]  L. Tobacman,et al.  Thin filament-mediated regulation of cardiac contraction. , 1996, Annual review of physiology.

[27]  H. Watkins,et al.  Investigation of a truncated cardiac troponin T that causes familial hypertrophic cardiomyopathy: Ca(2+) regulatory properties of reconstituted thin filaments depend on the ratio of mutant to wild-type protein. , 2000, Circulation research.

[28]  S. Minchin,et al.  Overexpression of human cardiac troponin-I and troponin-C in Escherichia coli and their purification and characterisation. Two point mutations allow high-level expression of troponin-I. , 1994, European journal of biochemistry.

[29]  R. Hodges,et al.  Distinct Regions of Troponin I Regulate Ca2+-dependent Activation and Ca2+ Sensitivity of the Acto-S1-TM ATPase Activity of the Thin Filament* , 1997, The Journal of Biological Chemistry.

[30]  B. Sykes,et al.  Structure of Cardiac Muscle Troponin C Unexpectedly Reveals a Closed Regulatory Domain* , 1997, The Journal of Biological Chemistry.

[31]  L. Heilmeyer,et al.  Stepwise subunit interaction changes by mono- and bisphosphorylation of cardiac troponin I. , 1998, Biochemistry.

[32]  M. Matsuzaki,et al.  Mutations in the cardiac troponin I gene associated with hypertrophic cardiomyopathy , 1997, Nature Genetics.

[33]  R. Hodges,et al.  The biological importance of each amino acid residue of the troponin I inhibitory sequence 104-115 in the interaction with troponin C and tropomyosin-actin. , 1988, The Journal of biological chemistry.

[34]  R. Hodges,et al.  Calmodulin and troponin C: a comparative study of the interaction of mastoparan and troponin I inhibitory peptide [104-115]. , 1986, Biochemistry.

[35]  R. Solaro,et al.  The C Terminus of Cardiac Troponin I Is Essential for Full Inhibitory Activity and Ca2+ Sensitivity of Rat Myofibrils* , 1997, The Journal of Biological Chemistry.

[36]  P. Powers,et al.  Cardiac troponin I gene knockout: a mouse model of myocardial troponin I deficiency. , 1999, Circulation research.

[37]  B. Sykes,et al.  Binding of cardiac troponin-I147-163 induces a structural opening in human cardiac troponin-C. , 1999, Biochemistry.

[38]  J. Potter,et al.  The effect of troponin I phosphorylation on the Ca2+-binding properties of the Ca2+-regulatory site of bovine cardiac troponin. , 1982, The Journal of biological chemistry.

[39]  R. Solaro,et al.  Troponin and tropomyosin: proteins that switch on and tune in the activity of cardiac myofilaments. , 1998, Circulation research.