Drastic Ca2+ sensitization of myofilament associated with a small structural change in troponin I in inherited restrictive cardiomyopathy

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Abstract

Six missense mutations in human cardiac troponin I (cTnI) were recently found to cause restrictive cardiomyopathy (RCM). We have bacterially expressed and purified these human cTnI mutants and examined their functional and structural consequences. Inserting the human cTnI into skinned cardiac muscle fibers showed that these mutations had much greater Ca2+-sensitizing effects on force generation than the cTnI mutations in hypertrophic cardiomyopathy (HCM). The mutation K178E in the second actin-tropomyosin (Tm) binding region showed a particularly potent Ca2+-sensitizing effect among the six RCM-causing mutations. Circular dichroism and nuclear magnetic resonance spectroscopy revealed that this mutation does not extensively affect the structure of the whole cTnI molecule, but induces an unexpectedly subtle change in the structure of a region around the mutated residue. The results indicate that the K178E mutation has a localized effect on a structure that is critical to the regulatory function of the second actin-Tm binding region of cTnI. The present study also suggests that both HCM and RCM involving cTnI mutations share a common feature of increased Ca2+ sensitivity of cardiac myofilament, but more severe change in Ca2+ sensitivity is associated with the clinical phenotype of RCM.

Section snippets

Experimental procedures

Mutagenesis of human cTnI complementary DNAs. Mutagenesis of human cTnI complementary DNA (cDNA) was carried out by polymerase chain reaction (PCR) (Quickchange, Stratagene) to generate the RCM-causing mutations in full-length cTnI and its C-terminal peptide cTnI129–210 (Fig. 1). The results of the mutagenesis in cDNA were confirmed by DNA sequencing.

Expression and purification of recombinant human cardiac troponin components. The human wild-type cTnC, cTnT, cTnI, and the six cTnI mutants were

Results

All the six RCM-causing mutations were generated in full-length human cTnI by a PCR-based mutagenesis of cDNA and exchanged into skinned cardiac muscle fibers. Fig. 2A compares the force–pCa relationships in the skinned cardiac muscle fibers exchanged with RCM-causing mutant cTnIs with that in the fibers exchanged with wild-type cTnI. All six RCM-causing mutations shifted the force–pCa relationship to lower Ca2+ concentrations, indicating that RCM-causing mutations, like HCM-causing mutations,

Discussion

In 2003, six missense mutations were found in patients with idiopathic RCM, which is the first case in which a specific gene has ever been shown to be responsible for RCM [13]; a missense mutation D190G was found in 12 affected patients in the same family, with two mutations, K178E and R192H, being de novo, and three others, L144Q, R145W, and A171T, being identified only in the probands. Interestingly, patients with the D190G mutation in a large family showed clinical phenotypes with a mixed

Acknowledgments

We are grateful to Kayoko Yanatake for her help with rabbit Myosin B preparation. This work was supported in part by Grants-in-Aid, Special Coordination Funds, and the National Project on Protein Structural and Functional Analyses for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

References (43)

  • M. Hatakenaka et al.

    Replacement of three troponin components with cardiac troponin components within single glycerinated skeletal muscle fibers

    Biochem. Biophys. Res. Commun.

    (1991)
  • J.K. Heinonen et al.

    A new colorimetric determination of inorganic phosphate and its application to the assay of inorganic pyrophosphatase

    Anal. Biochem.

    (1981)
  • A.V. Gomes et al.

    Mutations in human cardiac troponin I that are associated with restrictive cardiomyopathy affect basal ATPase activity and the calcium sensitivity of force development

    J. Biol. Chem.

    (2005)
  • P. Robinson et al.

    Alterations in thin filament regulation induced by a human cardiac troponin T mutant that causes dilated cardiomyopathy are distinct from those induced by troponin T mutants that cause hypertrophic cardiomyopathy

    J. Biol. Chem.

    (2002)
  • I. Ohtsuki et al.

    Regulatory and cytoskeletal proteins of vertebrate skeletal muscle

    Adv. Protein Chem.

    (1986)
  • B. Tripet 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

    J. Mol. Biol.

    (1997)
  • K. Murakami et al.

    Structural basis for Ca2+-regulated muscle relaxation at interaction sites of troponin with actin and tropomyosin

    J. Mol. Biol.

    (2005)
  • R. Koradi et al.

    MOLMOL: a program for display and analysis of macromolecular structures

    J. Mol. Graph.

    (1996)
  • G.W. Dec et al.

    Idiopathic dilated cardiomyopathy

    N. Engl. J. Med.

    (1994)
  • B.J. Maron

    Hypertrophic cardiomyopathy: a systematic review

    JAMA

    (2002)
  • S.S. Kushwaha et al.

    Restrictive cardiomyopathy

    N. Engl. J. Med.

    (1997)
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