The Role of Electrostatics in the Desensitization of Cardiac Muscle Contraction

Abstract

Heart muscle contraction is regulated by Ca2+ binding to cardiac troponin C. This induces troponin I (cTnI) switch region binding to the regulatory domain of troponin C (cNTnC), pulling the cTnI inhibitory region off actin, and triggering muscle contraction. Small molecules targeting this cNTnC-cTnI interface have potential for treating heart disease. The calmodulin antagonist, W7, is a calcium desensitizer. W7 binds to the interface of cNTnC and cTnI switch region, and weakens cTnI binding, possibly by electrostatic repulsion between a positively charged amino group on W7 and the positively charged side chain of arginine 147 on cTnI. To test the role of electrostatics, we synthesized A7, replacing the amino group of W7 with a carboxyl group. We determined the high-resolution solution NMR structure of A7 bound to cNTnC-cTnI chimera. The structure shows that A7 does not change the overall structure of the cNTnC-cTnI interface, and that the naphthalene ring of A7 sits in the same hydrophobic pocket as W7. We measured the affinities of A7 for cNTnC, the cNTnC-cTnI complex and a cNTnC-cTnI chimera, as well as that of cTnI for the cNTnC-A7 complex. A7 decreases the binding affinity of cTnI much less than W7. We compared the effect of W7 and A7 on the calcium sensitivity of force of demembranated rat ventricular trabeculae, and found that A7 has a much weaker desensitization effect than W7. We synthesized A6 and A4, which have one and three less methylene groups on the hydrocarbon chain than A7, respectively. A6 and A4 did not affect binding of cTnI switch peptide, nor change the calcium sensitivity of trabeculae. These results suggest that the negative inotropic effect of W7 may result from a combination of electrostatic repulsion and steric hindrance with cTnI.