A deletion mutation ⌬K210 in cardiac troponin T (cTnT) was recently found to cause familial dilated cardiomyopathy (DCM). To explore the effect of this mutation on cardiac muscle contraction under physiological conditions, we determined the Ca 2؉ -activated force generation in permeabilized rabbit cardiac muscle fibers into which the mutant and wild-type cTnTs were incorporated by using our TnT exchange technique. The free Ca 2؉ concentrations required for the force generation were higher in the mutant cTnTexchanged fibers than in the wild-type cTnT-exchanged ones, with no statistically significant differences in maximal force-generating capability and cooperativity. Exchanging the mutant cTnT into isolated cardiac myofibrils also increased the free Ca 2؉ concentrations required for the activation of ATPase. In contrast, a deletion mutation ⌬E160 in cTnT that causes familial hypertrophic cardiomyopathy (HCM) decreased the free Ca 2؉ concentrations required for force generation, just as in the case of the other HCM-causing mutations in cTnT. The results indicate that cTnT mutations found in the two distinct forms of cardiomyopathy (i.e., HCM and DCM) change the Ca 2؉ sensitivity of cardiac muscle contraction in opposite directions. The present study strongly suggests that Ca 2؉ desensitization of force generation in sarcomere is a primary mechanism for the pathogenesis of DCM associated with the deletion mutation ⌬K210 in cTnT.C ontraction of the vertebrate-striated muscles (i.e., skeletal and cardiac muscles) is regulated by Ca 2ϩ through its binding to a specific regulatory protein complex, troponin (Tn), which is distributed at regular intervals along the entire thin filament (1, 2). Tn is a complex of three different proteins, troponin T (TnT; tropomyosin-binding component), troponin I (TnI; inhibitory component), and troponin C (TnC; Ca 2ϩ -binding component). On Ca 2ϩ binding to TnC, a Ca 2ϩ -induced interaction of TnC with TnI relieves the inhibitory action of TnI exerted on the thin filament and enables the myosin head to cyclically interact with actin in the thin filament and generate force. The Ca 2ϩ sensitivity of muscle contraction is determined by the Ca 2ϩ -binding affinity of TnC, which is dynamically altered through interaction with TnI and TnT in the myofilament lattice (3-8).Mutations in genes for cardiac troponin T (cTnT) and cardiac troponin I (cTnI) have been found to cause familial hypertrophic cardiomyopathy (HCM), an autosomal dominant heart disease characterized by asymmetrical ventricular hypertrophy with a high incidence of sudden death in young adults (9). We have already examined the effects of eight HCM-linked cTnT mutations (I79N, R92Q, ⌬E160, E244D, R278C, and two truncated mutants produced by a splice donor site mutation Int15G 1 3A) and six HCM-linked cTnI mutations (R145G, R145Q, R162W, ⌬K183, G203S, and K206Q) on the contractile functions of cardiac muscle by using a technique for exchanging the exogenous Tn complex into skinned muscle fibers and isolated myofibrils. We found t...

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The effects of Troponin T (TnT) mutants R141W and ⌬K210, the only two currently known mutations in TnT that cause dilated cardiomyopathy(DCM) independent of familial hypertrophic cardiomyopathy (FHC), and TnT-K273E, a mutation that leads to a progression from FHC to DCM, were investigated. Studies on the Ca 2؉ sensitivity of force development in porcine cardiac fibers demonstrated that TnT-⌬K210 caused a significant decrease in Ca 2؉ sensitivity, whereas the TnT-R141W did not result in any change in Ca 2؉ sensitivity when compared with human cardiac wild-type TnT (HCWTnT). TnT-⌬K210 also caused a decrease in maximal force when compared with HCWTnT and TnT-R141W. In addition, the TnT-⌬K210 mutant decreased maximal ATPase activity in the presence of Ca 2؉ . However, the TnT-K273E mutation caused a significant increase in Ca 2؉ sensitivity but behaved similarly to HCWTnT in actomyosin activation assays. Inhibition of ATPase activity in reconstituted actin-activated myosin ATPase assays was similar for all three TnT mutants and HCWTnT. Additionally, circular dichroism studies suggest that the secondary structure of all three TnT mutants was similar to that of the HCWTnT. These results suggest that a rightward shift in Ca 2؉ sensitivity is not the only determinant for the phenotype of DCM.

To study the functional consequences of various cardiomyopathic mutations in human cardiac ␣-tropomyosin (Tm), a method of depletion/reconstitution of native Tm and troponin (Tn) complex (Tm-Tn) in cardiac myofibril preparations has been developed. The endogenous Tm-Tn complex was selectively removed from myofibrils and replaced with recombinant wild-type or mutant proteins. Successful depletion and reconstitution steps were verified by SDS-gel electrophoresis and by the loss and regain of Ca 2؉ -dependent regulation of ATPase activity. Five Tm mutations were chosen for this study: the hypertrophic cardiomyopathy (HCM) mutations E62Q, E180G, and L185R and the dilated cardiomyopathy (DCM) mutations E40K and E54K. Through the use of this new depletion/ reconstitution method, the functional consequences of these mutations were determined utilizing myofibrillar ATPase measurements. The results of our studies showed that 1) depletion of >80% of Tm-Tn from myofibrils resulted in a complete loss of the Ca 2؉ -regulated ATPase activity and a significant loss in the maximal ATPase activity, 2) reconstitution of exogenous wild-type Tm-Tn resulted in complete regain in the calcium regulation and in the maximal ATPase activity, and 3) all HCM-associated Tm mutations increased the Ca 2؉ sensitivity of ATPase activity and all had decreased abilities to inhibit ATPase activity. In contrast, the DCMassociated mutations both decreased the Ca 2؉ sensitivity of ATPase activity and had no effect on the inhibition of ATPase activity. These findings have demonstrated that the mutations which cause HCM and DCM disrupt discrete mechanisms, which may culminate in the distinct cardiomyopathic phenotypes.

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 TnTlinked FHC through different mechanisms.