A common cause for the development of cardiomyopathies (CM) are point mutations in the TNNI3 gene, encoding cardiac Troponin I (cTnI).  The newly detected cTnI-R170G/W amino acid replacements, identified in infants, leads to restrictive cardiomyopathy (RCM). These mutations are located in the regulatory C-Terminus of cTnI, which regulates its inhibitory region. Therefore, we wanted to determine the functional consequences of these mutations and the effects on interactions between thin and thick filament proteins, such as myosin binding protein C (cMyBPC).

Hereby we found a direct troponin/cMyBPC-C0C2 interaction by using microscale thermophoresis (MST). We identified cTnI and cTnT as binding partners for cMyBPC, but not cTnC. The binding affinities of the mutant subunits (cTnI R170G/W) alone towards C0C2 are unchanged compared to wildtype in MST. But regarding the whole troponin comlex (Tn), the affinity of cTn mutant cTnIR170G towards C0C2 was significantly increased compared to wildtype cTn. The binding affinity of cTnI-R170W was unchanged. 

In vivo, under endogenous protein expression levels, we could verify cTnI as binding partner of MyBPC with the Duolink PLA Technology. 

To study the effects of troponin mutations on its function we measured the Ca²⁺ dependent activation of reconstituted thin filaments by using pyrene maleimide labeled tropomyosin (PM-Tpm). cTnI R170G/W mutations show no effect on Ca²⁺ dependent activation on reconstituted filaments. Due to the involvement of other sarcomeric proteins, which bind to the filaments, we included the cMyBPC C0C2 fragment. In the presence of C0C2 the cooperativity of thin filament activation was increased with wildtype cTn, but not with R170G/W.

Electron microscopy (EM) reveal reduced stability of reconstituted thin filaments, such as fragmentation and waves in case of R170G/W.

Taken together we have shown that cMyBPC affects the mutant filaments (cTnI R170G/W) differently and have proven for the first time that cTnI is its binding partner on cTn.