Cardiac troponin C (cTnC) is the primary calcium binding protein of the sarcomere, which contains four calcium binding sites. Unlike in skeletal muscle, cTnC site I does not bind calcium, while site II binds calcium and serves as a critical point of regulation. Mutations in cTnC can result in a variety of hereditary cardiomyopathies, with unique clinical phenotypes. Here, we investigate two mutations in site I: Gly34Ser (G34R) and Gly34Arg (G34R) that are associated with dilated cardiomyopathy and left ventricular noncompaction respectively. We first exchanged recombinant G34R and G34S containing troponin complexes into permeabilized porcine tissue and found that the G34R reduced maximum tension, while the G34S variant increased it. We next investigated elastic modulus, quantified as the slope of the tension-stiffness relationship, and found that G34S was stiffer compared with WT while G34R was more compliant. Moreover, there was no significant difference in calcium sensitivity with G34R, which is atypical of DCM-causing mutations in cTnC. To investigate troponin function, we titrated cTnI with INABD labeled cTnC and measured the subsequent fold change in fluorescence. G34S had a significantly higher affinity for cTnI vs WT for both the apo (1.93 ± 0.09 vs. 1.74 ± 0.04, p = 0.01) and calcium saturated states (2.46 ± 0.38 vs. 2.40 ± 0.11, p = 0.01). This was not true for G34R, suggesting that differences do not occur at the level of the isolated troponin. Measures for calcium binding affinity when G34R was incorporated into thin filaments also did not differ for G34R vs. WT. In summary, we find that, despite being at the same residue, G34R and G34S have widely disparate biochemical and functional effects, providing some insight into their different clinical presentation.
Jani et al. (Sun,) studied this question.