Restrictive cardiomyopathy mutations demonstrate functions of the C-terminal end-segment of troponin I

•The C-terminal end-segment of TnI is a highly conserved regulatory structure.•Restrictive cardiomyopathy mutations R193H and R205H affect the I–T interface.•R193H and R205H mutation increase the binding affinity of TnI for TnT and TnC.•R193H mutation increases the binding affinity of TnI for tropomyosin at pCa 9.

The C-terminal end-segment of Troponin I (TnI) corresponding to the last 27–33 amino acids is the most conserved structure of TnI and interacts with tropomyosin in a Ca2+-regulated manner, suggesting a role in muscle relaxation. Mutations in the C-terminal end-segment of cardiac TnI cause restrictive cardiomyopathy. Here we demonstrate that mouse cardiac TnI containing R193H or R205H mutation have significantly conformational changes in the region interfacing with troponin T (TnT) and increased binding affinity for TnT. These restrictive cardiomyopathy mutations also exhibit increased binding affinity for troponin C at pCa 4. The effects of R193H mutation were more profound than that of R205H. Tertiary troponin complex was reconstituted using the TnI mutants and a mini TnT lacking tropomyosin-binding sites to examine the interaction between the C-terminal end-segment of TnI and tropomyosin. The results showed that, R193H, but not R205H, caused a moderate but statistically significant increase in the binding affinity for tropomyosin at pCa 9. Similar trend was observed at pCa 5.5 but not pCa 4. These results provide novel evidence for the function of the C-terminal end-segment of TnI, where mutations with conformational effects alter TnI’s interaction with other troponin subunits and tropomyosin to cause diastolic dysfunction.

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