Abstract
To understand the molecular function of troponin T (TnT) in the Ca(2+) regulation of muscle contraction as well as the molecular pathogenesis of familial hypertrophic cardiomyopathy (FHC), eight FHC-linked TnT mutations, which are located in different functional regions of human cardiac TnT (HCTnT), were produced, and their structural and functional properties were examined. Circular dichroism spectroscopy demonstrated different secondary structures of these TnT mutants. Each of the recombinant HCTnTs was incorporated into porcine skinned fibers along with human cardiac troponin I (HCTnI) and troponin C (HCTnC), and the Ca(2+) dependent isometric force development of these troponin-replaced fibers was determined at pH 7.0 and 6.5. All eight mutants altered the contractile properties of skinned cardiac fibers. E244D potentiated the maximum force development without changing Ca(2+) sensitivity. In contrast, the other seven mutants increased the Ca(2+) sensitivity of force development but not the maximal force. R92L, R92W, and R94L also decreased the change in Ca(2+) sensitivity of force development observed on lowering the pH from 7 to 6.5, when compared with wild type TnT. The examination of additional mutants, H91Q and a double mutant H91Q/R92W, suggests that mutations in a region including residues 91-94 in HCTnT can perturb the proper response of cardiac contraction to changes in pH. These results suggest that different regions of TnT may contribute to the pathogenesis of TnT-linked FHC through different mechanisms.
Highlights
To understand the molecular function of troponin T (TnT) in the Ca2؉ regulation of muscle contraction as well as the molecular pathogenesis of familial hypertrophic cardiomyopathy (FHC), eight FHC-linked TnT mutations, which are located in different functional regions of human cardiac TnT (HCTnT), were produced, and their structural and functional properties were examined
We have previously demonstrated that endogenous porcine cardiac Tn can be replaced by recombinant HCTnT WT nearly completely under certain conditions, and HCTnT WT-incorporated fibers showed almost no Ca2ϩ regulation unless they were reconstituted with human cardiac troponin I (HCTnI) and troponin C (TnC) [18]
Because FHC that is caused by TnT mutations might be a consequence of an altered molecular function of TnT, that would lead to abnormal cardiac muscle contraction, the functional analyses of these disease-causing TnT mutations could help our understanding of the molecular function of TnT as well as the pathogeneses of FHC
Summary
To understand the molecular function of troponin T (TnT) in the Ca2؉ regulation of muscle contraction as well as the molecular pathogenesis of familial hypertrophic cardiomyopathy (FHC), eight FHC-linked TnT mutations, which are located in different functional regions of human cardiac TnT (HCTnT), were produced, and their structural and functional properties were examined. The examination of additional mutants, H91Q and a double mutant H91Q/R92W, suggests that mutations in a region including residues 91–94 in HCTnT can perturb the proper response of cardiac contraction to changes in pH These results suggest that different regions of TnT may contribute to the pathogenesis of TnTlinked FHC through different mechanisms. Many studies have been attempted to exploit the molecular pathogenesis of FHC caused by each TnT mutation from molecular to physiological levels and have shown that most FHC-TnT mutations alter the contractile properties of cardiac muscle, especially the Ca2ϩ sensitivity of force development and ATPase activity in vitro and in vivo [14, 16, 17]. Similar results have been seen at both the molecular and physiological levels, implying that altered Ca2ϩ regulation of muscle contraction by TnT mutations might be the primary mechanism for TnT-linked FHC
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