Molecular mechanisms and structural features of cardiomyopathy-causing troponin T mutants in the tropomyosin overlap region

Abstract

Point mutations in genes encoding sarcomeric proteins are the leading cause of inherited primary cardiomyopathies. Among them are mutations in the gene that encodes cardiac troponin T (TnT). These mutations are clustered in the tropomyosin (Tm) binding region of TnT, TNT1 (residues 80-180). To understand the mechanistic changes caused by pathogenic mutations in the TNT1 region, six hypertrophic cardiomyopathy (HCM) and two dilated cardiomyopathy (DCM) mutants were studied by biochemical approaches. Binding assays in the absence and presence of actin revealed changes in the affinity of some, but not all, TnT mutants for Tm relative to WT TnT. HCM mutants were hypersensitive and DCM mutants were hyposensitive to Ca in regulated actomyosin ATPase activities. To gain better insight into the disease mechanism, we modeled the structure of TNT1 and its interactions with Tm. The stability predictions made by the model correlated well with the affinity changes observed in vitro of TnT mutants for Tm. The changes in Ca sensitivity showed a strong correlation with the changes in binding affinity. We suggest the primary reason by which these mutations between residues 92 and 144 cause cardiomyopathy is by changing the affinity of TnT for Tm within the TNT1 region.

Pathogenic troponin T mutants with opposing effects on myofilament Ca2+ sensitivity attenuate cardiomyopathy phenotypes in mice

“…Previous studies report on the TnT1 domain’s crucial flexibility and its role in regulating Tm positioning for contraction [28–30]. Further evidence for the importance of this domain in our study of TnT1 mutations lies in their ability to alter the affinity of troponin complex for Tm [31]. Therefore, we suggest that the most targeted approach to “rescuing” either HCM or DCM phenotype would be to have a mutation within that same domain to counteract manifestation of the disease, probably through TnT1’s flexibility and eventually altered contraction.…”

“…Therefore, we suggest that the most targeted approach to “rescuing” either HCM or DCM phenotype would be to have a mutation within that same domain to counteract manifestation of the disease, probably through TnT1’s flexibility and eventually altered contraction. The increased binding affinity between R141W troponin and Tm [31] possibly underlies the increased cooperativity of thin filament activation displayed by the HET mice within this study (Table 1). Interestingly, I79N/HET mice displayed a normalization of cooperativity of thin filament activation (Table 1).…”

Cardiomyopathy mutation (F88L) in troponin T abolishes length dependency of myofilament Ca2+ sensitivity

“…When cooperative stabilization between neighboring RUs is decreased by TnT F88L , it decreases the threshold for Ca 2+ to activate RUs, leading to increased Ca 2+ sensitivity of thin filaments. Such attenuation of RU–RU cooperativity may result from the weakening of TnT–Tm interactions ( Gangadharan et al, 2017 ) and/or an effect on Tm–Tm overlap junction; in theory, this would lower the effective stiffness of the Tm chain and decrease cooperative communication between RUs. This line of reasoning is consistent with previous findings that have suggested that one primary mechanism by which HCM-linked thin filament mutations enhance myofilament Ca 2+ sensitivity is through attenuation of RU–RU cooperativity ( Palm et al, 2001 ; Hinkle and Tobacman, 2003 ).…”

Do Actomyosin Single-Molecule Mechanics Data Predict Mechanics of Contracting Muscle?

“…Both may play roles [ 119 , 120 , 121 ] in controlling the degree to which myosin heads leave their sequestered (parked) super-relaxed state [ 40 , 41 ] on the thick filaments under different conditions, e.g., in case of stress or need for increased power. These properties are of increasing interest to study using in vitro systems with isolated proteins because of potentially important roles in effects of myosin active drugs [ 21 , 24 , 40 , 122 ] and/or in cardiomyopathies [ 30 , 31 , 38 , 123 , 124 , 125 , 126 , 127 ]. In these regards the degree of phosphorylation of the regulatory light chains of striated muscle myosin is also of interest to control as this property affects both the state of activation of the thick filaments and drug effects [ 38 , 128 , 129 , 130 , 131 , 132 , 133 ].…”