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

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 varyi...

Troponin structure and function: a view of recent progress

“…In wildtype, but 1.0 for the HCM and DCM mutations shown on the x-axis, indicating uncoupling. From (Sheehan et al 2018) switch by phosphorylation and mutations. These do not produce detectable changes in the steady state but in both cases significant changes in troponin dynamics, exemplified by the A/B helix angle, can be demonstrated by NMR and molecular dynamics simulations.…”

Epigallocatechin-3 gallate prevents pressure overload-induced heart failure by up-regulating SERCA2a via histone acetylation modification in mice

“…EGCG has been reported to exert lipid-lowering effects, in addition to effects on angiogenesis and osteogenesis; it also affects many abnormal pathophysiological changes, as it has been demonstrated to have anti-cancer, anti-inflammatory, anti-collagenase, autoxidative, and anti-fibrotic effects [ 35 – 44 ], and possess therapeutic activity for a number of diseases such as cancer, oral disease, diabetes, and especially cardiovascular diseases [ 19 , 20 ]. Previously, several mechanisms were suggested for EGCG with respect to heart failure, such as alleviating cardiac fibrosis [ 45 ], reducing desensitization of the β1 adrenoceptor [ 46 ], modulating the Nrf2/ARE antioxidant system [ 47 ], and altering myofilament Ca2+-sensitivity [ 26 , 48 ]. Recently, studies have demonstrated that EGCG exerts a tremendous effect on events associated with epigenetic regulation, including histone acetylation, methylation, and DNA methylation [ 22 – 24 ].…”

Approaches to High-Throughput Analysis of Cardiomyocyte Contractility

“…As shown above, this dramatically improves the statistical power of measurements. Figure 7 shows an example of a simple drug screening protocol using a single rat myocyte preparation with several established β-agonists and antagonists, recently developed drugs that act on myocyte contractility and novel compounds that are proposed to have “recoupling” activity ( Tadano et al, 2010 ; Papadaki et al, 2015 ; Sheehan et al, 2018 ). The 13 compounds were tested in about 5 h. Myocytes are kept at 4°C and plated onto laminin-coated dishes in three batches at approximately 2-h intervals to avoid run down in older preparations of cardiomyocytes.…”

“…None of these compounds showed a reduction of% shortening or reduction of TTB90 indicative of enhanced relaxation in our study. Most of the compounds proposed to affect the dobutamine response of mutant myocytes ("recouplers": Silybin A and B, dehydrosilybin A and B, resveratrol, and novobiocin) had little effect on the baseline response in wild type as predicted (Papadaki et al, 2015;Wilkinson et al, 2015;Sheehan et al, 2018).…”