Molecular Defects in Cardiac Myofilament Ca2+-Regulation Due to Cardiomyopathy-Linked Mutations Can Be Reversed by Small Molecules Binding to Troponin.

Alice Sheehan,Vladimír Kren,David Biedermann,Andrew E Messer,Maria Papadaki,Steven B Marston,Anuj Khandelwal,Brian Blagg,Afnan Choudhry

doi:10.3389/fphys.2018.00243

Abstract

The inherited cardiomyopathies, hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are relatively common, potentially life-threatening and currently untreatable. Mutations are often in the contractile proteins of cardiac muscle and cause abnormal Ca2+ regulation via troponin. HCM is usually linked to higher myofilament Ca2+-sensitivity whilst in both HCM and DCM mutant tissue there is often an uncoupling of the relationship between troponin I (TnI) phosphorylation by PKA and modulation of myofilament Ca2+-sensitivity, essential for normal responses to adrenaline. The adrenergic response is blunted, and this may predispose the heart to failure under stress. At present there are no compounds or interventions that can prevent or treat sarcomere cardiomyopathies. There is a need for novel therapies that act at a more fundamental level to affect the disease process. We demonstrated that epigallocatechin-3 gallate (EGCG) was found to be capable of restoring the coupled relationship between Ca2+-sensitivity and TnI phosphorylation in mutant thin filaments to normal in vitro, independent of the mutation (15 mutations tested). We have labeled this property “re-coupling.” The action of EGCG in vitro to reverse the abnormality caused by myopathic mutations would appear to be an ideal pharmaceutical profile for treatment of inherited HCM and DCM but EGCG is known to be promiscuous in vivo and is thus unsuitable as a therapeutic drug. We therefore investigated whether other structurally related compounds can re-couple myofilaments without these off-target effects. We used the quantitative in vitro motility assay to screen 40 compounds, related to C-terminal Hsp90 inhibitors, and found 23 that can re-couple mutant myofilaments. There is no correlation between re-couplers and Hsp90 inhibitors. The Ca2+-sensitivity shift due to TnI phosphorylation was restored to 2.2 ± 0.01-fold (n = 19) compared to 2.0 ± 0.24-fold (n = 7) in wild-type thin filaments. Many of these compounds were either pure re-couplers or pure desensitizers, indicating these properties are independent; moreover, re-coupling ability could be lost with small changes of compound structure, indicating the possibility of specificity. Small molecules that can re-couple may have therapeutic potential.HIGHLIGHTS - Inherited cardiomyopathies are common diseases that are currently untreatable at a fundamental level and therefore finding a small molecule treatment is highly desirable.- We have identified a molecular level dysfunction common to nearly all mutations: uncoupling of the relationship between troponin I phosphorylation and modulation of myofilament Ca2+-sensitivity, essential for normal responses to adrenaline.- We have identified a new class of drugs that are capable of both reducing Ca2+-sensitivity and/or recouping the relationship between troponin I phosphorylation and Ca2+-sensitivity.- The re-coupling phenomenon can be explained on the basis of a single mechanism that is testable.- Measurements with a wide range of small molecules of varying structures can indicate the critical molecular features required for recoupling and allows the prediction of other potential re-couplers.

Highlights

The contractile apparatus of cardiac muscle is controlled by the sarcoplasmic Ca2+-level acting upon the Ca2+ sensor, troponin
In our studies of the uncoupling effects of mutations we demonstrated that epigallocatechin-3 gallate (EGCG), originally studied as a Ca2+ desensitizer (Tadano et al, 2010; Friedrich et al, 2016), was found to be capable of restoring the coupled relationship between Ca2+-sensitivity and TnI phosphorylation in mutant thin filaments to normal in vitro, a property we refer to as “recoupling” (Papadaki et al, 2015; Messer et al, 2016, 2017)
We measured the Ca2+ activation of contractility using an in vitro motility assay that measures the movement of reconstituted thin filaments over a bed of immobilized myosin heads

Summary

INTRODUCTION

The contractile apparatus of cardiac muscle is controlled by the sarcoplasmic Ca2+-level acting upon the Ca2+ sensor, troponin. Whilst the structural mechanism of the troponin Ca2+ switch is well-documented (Takeda et al, 2003), the structural basis of lusitropy is poorly understood since both the phosphorylated N-terminal peptide and the regulatory “switch peptide” of TnI are intrinsically disordered, recent studies using molecular dynamics simulations have begun to explain this phenomenon (Papadaki and Marston, 2016) The study of this phosphorylation dependent regulation has been recently stimulated by the discovery that a primary effect of many mutations in thin filament proteins associated with cardiomyopathy is the uncoupling of this relationship (Bayliss et al, 2012; Memo et al, 2013; Messer and Marston, 2014). Consideration of the molecular structures of re-coupling molecules (structure-activity relationships, SAR), compared with similar inactive molecules can provide considerable insight into the mechanism of recoupling and may lead to the discovery of more potent recoupling (lead) compounds with therapeutic potential

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