Increasing the calcium sensitivity of muscle using trifluoperazine-induced manipulations in silico, in vitro and in vivo systems.

Many therapeutics for cardiomyopathy treat the symptoms of the disease rather than the underlying mechanism. The mechanism of cardiomyopathy onset is believed to include two means: calcium sensitivity changes and myosin activity alteration. Trifluoperazine is a compound that binds troponin, and other components of the calcium pathway, which impacts calcium regulation of contraction. Here, the ability of TFP to shift calcium sensitivity was examined in vitro with purified proteins and the impact of TFP on heart function was assessed in vivo using embryonic zebrafish. The binding of TFP to troponin was modeled in silico and a model of zebrafish troponin was generated. TFP increased regulated cardiac actomyosin activity in vitro and elevated embryonic zebrafish heart rates at effective drug concentrations. Troponin structural changes predicted in silico suggest altered protein interactions within thin filaments that would affect the regulation of heart function.

The Dark Side of Actin: Cardiac actin variants highlight the role of allostery in disease development.

Mutations in the α-cardiac actin ACTC1 gene cause dilated or hypertrophic cardiomyopathy. These diseases are the result of changes in protein interactions between ACTC protein and force-generating ß-myosin or the calcium-dependent cardiac-tropomyosin (cTm) and cardiac troponin (cTn) regulatory complex, altering the overall contractile force. The T126I and S271F ACTC variants possess amino acid substitutions on the other side of actin relative to the myosin or regulatory protein binding sites on what we call the "dark side" of actin. The T126I change results in hyposensitivity to calcium, in accordance with the calcium sensitivity pathway of cardiomyopathy development while the S271F change alters the maximum in vitro motility sliding speed, reflecting a change in maximum force. These results demonstrate the role of actin allostery in the cardiac disease development.

M-class hypertrophic cardiomyopathy cardiac actin mutations increase calcium sensitivity of regulated thin filaments.

Hypertrophic cardiomyopathy is a commonly occurring cardiovascular disease resulting primarily from changes in proteins participating in muscle contraction in the heart, including the cardiac actin protein. Changes in cardiac actin located exclusively in the myosin binding site are called M-class variants and include the H88Y, R95C, and E99K substitutions and F90Δ deletion. The prevailing hypothesis for these mutations is that hypertrophic cardiomyopathy is the result of increased calcium sensitivity of contraction in the myocardium. To test this hypothesis, we studied the activity of myosin at varying calcium concentrations in the presence of regulated thin filaments containing M-class cardiac actin variants. We found that all M-class cardiac actin variants exhibit increased calcium sensitivity, with the R95C variant also displaying significant decreases in maximal myosin activity. This work represents the first characterization of all M-class variant proteins and suggests that drugs targeting contraction specifically to treat hypertrophic cardiomyopathy must consider the impact on both calcium sensitivity and maximal myosin activity on overall heart health.