Search

Introduction: Adrenergic activation of protein kinase A (PKA) in cardiac muscle targets the sarcolemma, sarcoplasmic reticulum, and contractile apparatus to increase contractile force and heart rate. In the thin filaments of the contractile apparatus, cardiac troponin I (cTnI) Ser22 and Ser23 in the cardiac-specific N-terminal peptide (NcTnI: residues 1 to 32) are the targets for PKA phosphorylation. Phosphorylation causes 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). Cardiomyopathy-linked mutations primarily affect Ca2+ regulation or the PKA-dependent modulatory system, such that Ca2+-sensitivity becomes independent of phosphorylation level (uncoupling) and this could be sufficient to induce cardiomyopathy. A drug that could restore the phosphorylation-dependent modulation of Ca2+-sensitivity could have potential for treatment of these pathologies. We have found that a number of small molecules, including silybin B, resveratrol and EGCG, can restore coupling in single filament assays. Methods: We did molecular dynamics simulations (5x1500ns for each condition) of the unphosphorylated and phosphorylated cardiac troponin core with the G159D DCM mutation in the presence of the 5 ligands and analysed the effects on several dynamic parameters. We also studied the effect of the ligands on the contractility of cardiac muscle myocytes with ACTC E99K and TNNT2 R92Q mutations in response to dobutamine. Results: Silybin B, EGCG and resveratrol restored the phosphorylation-induced change in molecular dynamics to wild-type values, whilst silybin A, an inactive isomer of silybin B, and Epicatechin gallate, an EGCG analogue that does not recouple, did not. We analysed the atomic-level changes induced by ligand binding to explain recoupling. Mutations ACTC E99K and TNNT2 R92Q blunt the increased relaxation speed response to β1 adrenergic stimulation of cardiac myocytes and we found that resveratrol, EGCG and silybin B could restore the β1 adrenergic response, whereas silybin A did not. Discussion: The uncoupling phenomenon caused by cardiomyopathy-related mutations and the ability of small molecules to restore coupling in vitro and lusitropy in myocytes is observed at the cellular, molecular and atomistic levels therefore, restoring lusitropy is a suitable target for treatment. Further research on compounds that restore lusitropy is thus indicated as treatments for genetic cardiomyopathies. Further molecular dynamics simulations could define the specific properties needed for recoupling and allow for the prediction and design of potential new drugs. [ABSTRACT FROM AUTHOR]

Aims  Dilated cardiomyopathy (DCM) is associated with mutations in many genes encoding sarcomere proteins. Truncating mutations in the titin gene TTN are the most frequent. Proteomic and functional characterizations are required to elucidate the origin of the disease and the pathogenic mechanisms of TTN- truncating variants. Methods and results  We isolated myofibrils from DCM hearts carrying truncating TTN mutations and measured the Ca2+ sensitivity of force and its length dependence. Simultaneous measurement of force and adenosine triphosphate (ATP) consumption in skinned cardiomyocytes was also performed. Phosphorylation levels of troponin I (TnI) and myosin binding protein-C (MyBP-C) were manipulated using protein kinase A and λ phosphatase. mRNA sequencing was employed to overview gene expression profiles. We found that Ca2+ sensitivity of myofibrils carrying TTN mutations was significantly higher than in myofibrils from donor hearts. The length dependence of the Ca2+ sensitivity was absent in DCM myofibrils with TTN -truncating variants. No significant difference was found in the expression level of TTN mRNA between the DCM and donor groups. TTN exon usage and splicing were also similar. However, we identified down-regulation of genes encoding Z-disk proteins, while the atrial-specific regulatory myosin light chain gene, MYL7, was up-regulated in DCM patients with TTN -truncating variants. Conclusion  Titin - truncating mutations lead to decreased length-dependent activation and increased elasticity of myofibrils. Phosphorylation levels of TnI and MyBP-C seen in the left ventricles are essential for the length-dependent changes in Ca2+ sensitivity in healthy donors, but they are reduced in DCM patients with TTN -truncating variants. A decrease in expression of Z-disk proteins may explain the observed decrease in myofibril passive stiffness and length-dependent activation. [ABSTRACT FROM AUTHOR]