Titin-truncating mutations associated with dilated cardiomyopathy alter length-dependent activation and its modulation via phosphorylation.

Petr G Vikhorev,Amy Li,Cristobal G Dos Remedios,Maya Guglin,Kenneth S Campbell,Natalia N Vikhoreva,Steven B Marston,Waichun Yeung,Pieter De Tombe,Magdi H Yacoub,Sean Lal,Cheavar A Blair

doi:10.1093/cvr/cvaa316

Abstract

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.

Highlights

Heart failure is a serious, life-threatening condition.[1]
Titin-truncating mutations lead to decreased length-dependent activation and increased elasticity of myofibrils
Phosphorylation levels of troponin I (TnI) and myosin binding protein-C (MyBP-C) seen in the left ventricles are essential for the length-dependent changes in Ca2þ sensitivity in healthy donors, but they are reduced in Dilated cardiomyopathy (DCM) patients with TTN-truncating variants

Summary

Introduction

Heart failure is a serious, life-threatening condition.[1]. Studies have indicated that it affects 1.4% of the total population in the UK2,3 and 2.2% of adults aged 20 and older in the USA.[4]. Heart failure has two main causes[5,6,7]: coronary artery disease leading to infarction, and remodelling of the heart tissue, which is initially compensatory but leads to deterioration of function and failure; and non-ischaemic heart failure (40.8% of cases in the USA7) with mechanisms linked to mutations in genes encoding cardiac contractile proteins. Non-ischaemic heart failure is frequent in younger patients.[8]. Dilated cardiomyopathy (DCM) is a major cause of non-ischaemic heart failure.[7,9] It is characterized by enlarged left ventricular enddiastolic dimension, decreased left ventricular ejection fraction, and reduced fractional shortening. In about half of cases, DCM is familial and is associated with more than 50 genes mostly encoding sarcomeric and cytoskeletal proteins.[11,12]

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