Original article
Arginylation regulates myofibrils to maintain heart function and prevent dilated cardiomyopathy

https://doi.org/10.1016/j.yjmcc.2012.05.007Get rights and content

Abstract

Protein arginylation mediated by arginyltransferase (ATE1) is essential for heart formation during embryogenesis, however its cell-autonomous role in cardiomyocytes and the differentiated heart muscle has never been investigated. To address this question, we generated cardiac muscle-specific Ate1 knockout mice, in which Ate1 deletion was driven by α-myosin heavy chain promoter (αMHC-Ate1 mouse). These mice were initially viable, but developed severe cardiac contractility defects, dilated cardiomyopathy, and thrombosis over time, resulting in high rates of lethality after 6 months of age. These symptoms were accompanied by severe ultrastructural defects in cardiac myofibrils, seen in the newborns and far preceding the onset of cardiomyopathy, suggesting that these defects were primary and likely underlay the development of the future heart defects. Several major sarcomeric proteins were arginylated in vivo. Moreover, Ate1 deletion in the hearts resulted in a significant reduction of active and passive myofibril forces, suggesting that arginylation is critical for both myofibril structural integrity and contractility. Thus, arginylation is essential for maintaining the heart function by regulation of the major myofibril proteins and myofibril forces, and its absence in the heart muscle leads to progressive heart failure through cardiomyocyte-specific defects.

Highlights

► New role of posttranslational arginylation in the heart muscle. ► New regulatory mechanism of myofibril maintenance and contractility. ► New insights into posttranslational regulation of the sarcomeric proteins. ► New correlation between cardiac myofibril forces, heart defects, and arginylation.

Introduction

Protein arginylation is a poorly understood posttranslational modification mediated by arginyltransferase ATE1 [1], which transfers arginine (Arg) from arginyl-tRNA onto proteins [2], [3], [4], [5], [6]. While Ate1 preferentially transfers Arg to amino acid residues with acidic side chains (Wang et al., 2011), a large number of proteins arginylated in vivo on different sites have been identified [7], [8]. The chemistry of the arginylation reaction, as well as its downstream effects on the majority of protein targets, are not well understood.

In yeast, Ate1 gene is not essential for cell viability, but Ate1 knockout in mice results in embryonic lethality between embryonic days E12.5 and E17.5 and severe defects in cardiovascular development and angiogenesis [9]. Among these defects, especially prominent are severe abnormalities in cardiac morphogenesis, including thin myocardium, underdeveloped septa, and non-separation of the aorta and pulmonary artery (persistent truncus arteriosus, PTA) [9]. Analysis of the heart muscle and cardiomyocytes isolated from Ate1 knockout embryos at E12.5–E14.5 reveals defects in cardiac contractility, heart integrity, and myofibril development [10], including myofibril disorganization, sarcomere collapse, and disintegration of the intercalated disks that become more prominent at later developmental stages (E14.5 and on). However it is not clear whether any of these defects are cell-autonomous or whether they arise through impairments in tissue signaling and/or are secondary to the onset of the embryonic lethality, leading to the heart muscle disintegration as the animals die.

To address these questions and test the specific role of arginylation in cardiomyocytes during heart development and postnatal function, we generated cardiac muscle-specific Ate1 knockout mice, in which Ate1 deletion via Cre-mediated recombination is driven by cardiomyocyte-specific alpha myosin heavy chain (αMHC) promoter [11]. These mice, termed αMHC-Ate1, survived to adulthood, but developed age-related dilated cardiomyopathy and thrombosis, accompanied by early myofibril defects and resulting in high rates of lethality after 6 month of age. Our results demonstrate a cell-autonomous role of arginylation in cardiomyocytes, where it is essential for the maintenance of the normal heart function and prevention of cardiomyopathy and heart failure. Our study also provides a mouse model of age-related heart failure with symptoms reminiscent of human heart disease, which has a potentially broad outreach in developing novel heart disease therapeutics.

Section snippets

Transgenic mice

To obtain αMHC-Ate1 mice, Ate1-floxed mice [12], [13] were crossed with the mouse strain expressing Cre recombinase under αMHC promoter [11]. For the reporter analysis shown in Fig. S1, αMHC-Ate1 mice were crossed to R26R ROSA reporter strain and fixed embryos were stained with X-gal as described in [12].

Anatomical and histological analysis

For anatomical examination, mice found dead or euthanized were dissected to assess the morphology of the chest cavity (for fluid accumulation) and the hearts. To determine heart:body weight

Generation of αMHC-Ate1 mice

To develop a mouse model with cardiomyocyte specific Ate1 knockout, we used our previously developed ‘Ate1-floxed’ mouse line [12], [13] and crossed it with αMHC-Cre mice [11], in which Cre recombinase is expressed under cardiomyocyte-specific α-myosin heavy chain promoter that activates upon differentiation, resulting in Ate1 deletion in the heart muscle. These mice, termed αMHC-Ate1 mice, were used in the present study. To confirm the efficiency and specificity of the Cre transgene

Discussion

Our results demonstrate a cell-autonomous role of arginylation in the development and function of the heart muscle, and identify arginylation as a novel mechanism that maintains cardiac health and prevents the development of dilated cardiomyopathy in mice (Fig. 5). It has been previously shown that arginylation is essential for cardiac morphogenesis and the development of heart muscle in embryogenesis [9], [10], however only the use of the current αMHC-Ate1 mouse model made it possible to

Disclosure statement

There are no conflicts of interest to disclose.

The following are the supplementary related to this article.

Supplementary Figures.

Acknowledgments

We thank Dr. Catherine Wong for help with manual validation of arginylated protein mass spectra and Dr. Susan Shultz (University of Pennsylvania Small Animal Imaging Facility) for performing mouse echocardiography. This work was supported by NIH R01 HL084419, W.W. Smith Charitable Trust, and Philip Morris Research Management Group awards to A.K. and NIH P41 RR011823 and N01-HV-00243 awards to J.R.Y.

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    1

    Present address: University of Hiroshima, Hiroshima, Japan.

    2

    Present address: Tezpur University, Napaam, India.

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