Silybin B, resveratrol and epigallocatechin-3 gallate (EGCG) bind to troponin to restore the loss of lusitropy caused by cardiomyopathy mutations in vitro, in vivo, and in silico

Adrenergic activation of protein kinase A (PKA) targets the sarcolemma, sarcoplasmic reticulum, and contractile apparatus to increase contractile force and heart rate. In the thin filaments of the contractile apparatus, cTroponinI Ser22 and Ser23 are the targets for PKA phosphorylation. The effect of phosphorylation is a 2-3 fold decrease of affinity of cTn for Ca2+, associated with a higher rate of Ca2+ dissociation from cTnC leading to a faster relaxation rate of the cardiac muscle (lusitropy). This modulation of Ca2+-sensitivity is often suppressed by mutations that cause cardiomyopathy (uncoupling) and this could be sufficient to induce cardiomyopathy. Therefore, a drug that could restore the phosphorylation-dependent modulation of Ca2+-sensitivity could have potential for treatment of these pathologies. We have found that in single filament assays that a number of small molecules including silybin B, resveratrol and EGCG can restore coupling. We performed molecular dynamics simulations of the unphosphorylated and phosphorylated cardiac Troponin core with the TNNC1 G159D DCM mutation. We found that silybin B, EGCG and resveratrol restored the phosphoryation-induced change of most metrics to wild-type values, whilst silybin A, an inactive isomer of silybin B, and ECG did not. We analysed the atomic-level changes induced by ligand binding to explain recoupling. In parallel, we have extended our studies to intact TNNT2 R92Q-transfected cardiomyocytes. The mutation blunts the increased relaxation speed response to β1 adrenergic stimulation and we found that resveratrol, EGCG and silybin B could restore the β1 adrenergic response whilst silybin A did not. ### Competing Interest Statement The authors have declared no competing interest.