Uncoupling of myofilament Ca2+-sensitivity from troponin I phosphorylation by mutations can be reversed by Epigallocatechin-3-Gallate.

AIMS: Heart muscle contraction is regulated via the β-adrenergic response that leads to phosphorylation of Troponin I (TnI) at Ser22/23, which changes the Ca(2+)-sensitivity of the cardiac myofilament. Mutations in thin filament proteins that cause Dilated Cardiomyopathy (DCM) and some mutations that cause Hypertrophic Cardiomyopathy (HCM) abolish the relationship between TnI phosphorylation and Ca(2+)-sensitivity (uncoupling). Small molecule Ca(2+)-sensitisers and Ca(2+)-desensitisers that act upon troponin alter the Ca(2+)-sensitivity of the thin filament but their relationship with TnI phosphorylation has never been studied before. METHODS AND RESULTS: Quantitative in vitro motility assay showed that 30 μM EMD57033 and 100 μM Bepridil increase Ca(2+)-sensitivity of phosphorylated cardiac thin filaments by 3.1 and 2.8-fold respectively. Additionally they uncoupled Ca(2+)-sensitivity from TnI phosphorylation, mimicking the effect of HCM mutations. EGCG decreased Ca(2+)-sensitivity of phosphorylated and unphosphorylated wild-type thin filaments equally (by 2.15±0.45 and 2.80±0.48-fold respectively), retaining the coupling. Moreover, EGCG also reduced Ca(2+)-sensitivity of phosphorylated but not unphosphorylated thin filaments containing DCM and HCM-causing mutations, thus the dependence of Ca(2+)-sensitivity upon TnI phosphorylation of uncoupled mutant thin filaments was restored in every case. In single mouse heart myofibrils, EGCG reduced Ca(2+)-sensitivity of force and k(ACT) and also preserved coupling. Myofibrils from the ACTC E361G (DCM) mouse were uncoupled; EGCG reduced Ca(2+)-sensitivity more for phosphorylated than unphosphorylated myofibrils, thus restoring coupling. CONCLUSION: We conclude that it is possible to both mimic and reverse the pathological defects in troponin caused by cardiomyopathy mutations pharmacologically. Re-coupling by EGCG may be of potential therapeutic significance for treating cardiomyopathies

Instrumentation to study myofibril mechanics from static to artificial simulations of cardiac cycle

Copyright © 2016 The Authors. Many causes of heart muscle diseases and skeletal muscle diseases are inherited and caused by mutations in genes of sarcomere proteins which play either a structural or contractile role in the muscle cell. Tissue samples from human hearts with mutations can be obtained but often samples are only a few milligrams and it is necessary to freeze them for storage and transportation. Myofibrils are the fundamental contractile components of the muscle cell and retain all structural elements and contractile proteins performing in contractile event; moreover viable myofibrils can be obtained from frozen tissue. • We are describing a versatile technique for measuring the contractility and its Ca2+ regulation in single myofibrils. The control of myofibril length, incubation medium and data acquisition is carried out using a digital acquisition board via computer software. Using computer control it is possible not only to measure contractile and mechanical parameters but also simulate complex protocols such as a cardiac cycle to vary length and medium independently. • This single myofibril force assay is well suited for physiological measurements. The system can be adapted to measure tension amplitude, rates of contraction and relaxation, Ca2+ dependence of these parameters in dose-response measurements, length-dependent activation, stretch response, myofibril elasticity and response to simulated cardiac cycle length changes. Our approach provides an all-round quantitative way to measure myofibrils performance and to observe the effect of mutations or posttranslational modifications. The technique has been demonstrated by the study of contraction in heart with hypertrophic or dilated cardiomyopathy mutations in sarcomere proteins.British Heart Foundation (RG/11/20/29266)

The dilated cardiomyopathy-causing mutation ACTC E361G in cardiac muscle myofibrils specifically abolishes modulation of Ca2+ regulation by phosphorylation of Troponin I

Phosphorylation of troponin I by protein kinase A (PKA) reduces Ca2þ sensitivity and increases the rate of Ca2þ release from troponin C and the rate of relaxation in cardiac muscle. In vitro experiments indicate that mutations that cause dilated cardiomyopathy (DCM) uncouple this modulation, but this has not been demonstrated in an intact contractile system. Using a Ca2þ-jump protocol, we measured the effect of the DCM-causing mutation ACTC E361G on the equilibrium and kinetic parameters of Ca2þ regulation of contractility in single transgenic mouse heart myofibrils. We used propranolol treatment of mice to reduce the level of troponin I and myosin binding protein C (MyBP-C) phosphorylation in their hearts before isolating the myo- fibrils. In nontransgenic mouse myofibrils, the Ca2þ sensitivity of force was increased, the fast relaxation phase rate constant, kREL, was reduced, and the length of the slow linear phase, tLIN, was increased when the troponin I phosphorylation level was reduced from 1.02 to 0.3 molPi/TnI (EC50 P/unp ¼ 1.8 5 0.2, p < 0.001). Native myofibrils from ACTC E361G transgenic mice had a 2.4-fold higher Ca2þ sensitivity than nontransgenic mouse myofibrils. Strikingly, the Ca2þ sensitivity and relaxation parameters of ACTC E361G myofibrils did not depend on the troponin I phosphorylation level (EC50 P/unp ¼ 0.88 5 0.17, p ¼ 0.39). Nevertheless, modulation of the Ca2þ sensitivity of ACTC E361G myofibrils by sarcomere length or EMD57033 was indistinguishable from that of nontransgenic myofibrils. Overall, EC50 measured in different conditions varied over a 7-fold range. The time course of relaxation, as defined by tLIN and kREL, was correlated with EC50 but varied by just 2.7- and 3.3-fold, respectively. Our results confirm that troponin I phosphorylation specifically alters the Ca2þ sensitivity of isometric tension and the time course of relaxation in cardiac muscle myofibrils. Moreover, the DCM-causing mutation ACTC E361G blunts this phosphorylation-dependent response without affecting other parameters of contraction, including length-dependent activation and the response to EMD57033

Author Correction: Abnormal contractility in human heart myofibrils from patients with dilated cardiomyopathy due to mutations in TTN and contractile protein genes

Correction to: Scientific Reportshttps://doi.org/10.1038/s41598-017-13675-8, published online 01 November 201

Distinct hypertrophic cardiomyopathy genotypes result in convergent sarcomeric proteoform profiles revealed by top-down proteomics

Hypertrophic cardiomyopathy (HCM) is the most common heritable heart disease. Although the genetic cause of HCM has been linked to mutations in genes encoding sarcomeric proteins, the ability to predict clinical outcomes based on specific mutations in HCM patients is limited. Moreover, how mutations in different sarcomeric proteins can result in highly similar clinical phenotypes remains unknown. Posttranslational modifications (PTMs) and alternative splicing regulate the function of sarcomeric proteins; hence, it is critical to study HCM at the level of proteoforms to gain insights into the mechanisms underlying HCM. Herein, we employed high-resolution mass spectrometry–based top-down proteomics to comprehensively characterize sarcomeric proteoforms in septal myectomy tissues from HCM patients exhibiting severe outflow track obstruction (n = 16) compared to nonfailing donor hearts (n = 16). We observed a complex landscape of sarcomeric proteoforms arising from combinatorial PTMs, alternative splicing, and genetic variation in HCM. A coordinated decrease of phosphorylation in important myofilament and Z-disk proteins with a linear correlation suggests PTM cross-talk in the sarcomere and dysregulation of protein kinase A pathways in HCM. Strikingly, we discovered that the sarcomeric proteoform alterations in the myocardium of HCM patients undergoing septal myectomy were remarkably consistent, regardless of the underlying HCM-causing mutations. This study suggests that the manifestation of severe HCM coalesces at the proteoform level despite distinct genotype, which underscores the importance of molecular characterization of HCM phenotype and presents an opportunity to identify broad-spectrum treatments to mitigate the most severe manifestations of this genetically heterogenous disease