Biochemical and Biophysical Research Communications
Drastic Ca2+ sensitization of myofilament associated with a small structural change in troponin I in inherited restrictive cardiomyopathy
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.
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