Therapeutic Chemical Screen Identifies Phosphatase Inhibitors to Reconstitute PKB Phosphorylation and Cardiac Contractility in ILK-Deficient Zebrafish.

Alexander Pott,Steffen Just,Karolina Weinmann,Doreen Köhler,Wolfgang Rottbauer,Maryam Shahid,Christian Pylatiuk

doi:10.3390/biom8040153

Abstract

Patients with inherited dilated cardiomyopathy (DCM) often suffer from severe heart failure based on impaired cardiac contractility leading to increased morbidity and mortality. Integrin-linked kinase (ILK) as a part of the cardiac mechanical stretch sensor was found to be an essential genetic regulator of cardiac contractility. Integrin-linked kinase localizes to z-disks and costameres in vertebrate hearts and regulates the activity of the signaling molecule protein kinase B (PKB/Akt) by controlling its phosphorylation. Despite identification of several potential drug targets in the ILK signaling pathway, pharmacological treatment strategies to restore contractile function in ILK-dependent cardiomyopathies have not been established yet. In recent years, the zebrafish has emerged as a valuable experimental system to model human cardiomyopathies as well as a powerful tool for the straightforward high-throughput in vivo small compound screening of therapeutically active substances. Using the ILK deficient zebrafish heart failure mutant main squeeze (msq), which shows reduced PKB phosphorylation and thereby impaired cardiac contractile force, we identified here, in an automated small compound screen, the protein phosphatase inhibitors calyculin A and okadaic acid significantly restoring myocardial contractile function by reconstituting PKB phosphorylation in msq ILK-deficient zebrafish embryos.

Highlights

Dilated cardiomyopathy (DCM) is a life-threatening heart disease significantly contributing to systolic heart failure and sudden cardiac death based on reduced cardiac contractility [1,2,3,4]
We found that mean ventricular fractional shortening (FS) in wt treated with 0.1% DMSO was 53.2 ± 3.4% (n = 51)
Treated msq embryos displayed high phosphorylation status of PKB (pPKB) levels comparable to wild-type zebrafish (Figure 4B). These findings demonstrate that calyculin A treatment of integrin-linked kinase (ILK)-deficient msq embryos effectively inhibits further protein kinase B (PKB) dephosphorylation, thereby reconstituting phospho-PKB levels and rescuing cardiac contractility in msq heart failure mutants

Summary

Introduction

Dilated cardiomyopathy (DCM) is a life-threatening heart disease significantly contributing to systolic heart failure and sudden cardiac death based on reduced cardiac contractility [1,2,3,4]. Molecular and genetic studies have identified more than 30 different DCM disease genes, mainly coding for proteins of the sarcomere, the cardiac Z-disc and the cytoskeleton [5]. The detailed genetic and molecular underpinnings of this complex autoregulatory mechanism are not fully understood yet, but of high clinical importance, since impaired adaption of cardiac contractility is considered to cause a sizeable proportion of DCM-related heart failure cases in humans [2,6,8]. Genetic studies of cardiac stretch sensor components in zebrafish, mice and humans identified the integrin-linked kinase (ILK) as an essential regulator of cardiac contractility adaption on changing circulatory demands [7,9]

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