Arginyltransferase regulates alpha cardiac actin function, myofibril formation and contractility during heart development

INTRODUCTION Protein arginylation is a poorly understood post-translational modification mediated by the arginyltransferase Ate1 (Balzi et al., 1990), which transfers arginine (Arg) from tRNA onto proteins (Kaji et al., 1963; Soffer, 1968; Takao and Samejima, 1999). Ate1 is encoded by a single gene that is highly conserved in evolution from yeast to human, and is expressed as one or more isoforms produced by alternative splicing in different mouse tissues (Kwon et al., 1999; Rai and Kashina, 2005; Rai et al., 2006). No other arginyl transferases have been identified in eukaryotic species to date. In yeast, the Ate1 gene is not essential for cell viability, whereas Ate1 knockout in mice results in embryonic lethality and severe defects in cardiovascular development and angiogenesis (Kwon et al., 2002). Multiple proteins are arginylated in vivo (Bongiovanni et al., 1999; Decca et al., 2006a; Decca et al., 2006b; Eriste et al., 2005; Fissolo et al., 2000; Hallak and Bongiovanni, 1997; Kopitz et al., 1990; Lee et al., 2005), including 43 different proteins isolated from various mouse cells and tissues (Wong et al., 2007). Ate1 knockout mice die between E12.5 and E17, with large hemorrhages, impairment of embryonic angiogenesis, and severe heart defects that include underdeveloped myocardium, septation defects [ventricular and atrial septal defects (VSD and ASD)], and non-separation of the aorta and pulmonary artery [persistent truncus arteriosus (PTA)] (Kwon et al., 2002). The underlying molecular mechanisms and cell lineage(s) responsible for these defects are unknown. It has been hypothesized that some, or all, of these defects might be due to impaired embryonic cell migration during heart formation. VSD, ASD and PTA defects are often observed in mice with knockout of genes that affect the migration of cells of the neural crest lineage (Gitler et al., 2002) and cell adhesion (Conti et al., 2004; George et al., 1997; George et al., 1993; Tullio et al., 1997). Cells derived from Ate1 knockout embryos display severe defects in lamella formation that result in impairment of directional migration along the substrate, linked to arginylation of beta actin, a ubiquitously expressed non-muscle actin isoform that plays a crucial role in lamella formation and non-muscle cell locomotion (Karakozova et al., 2006). However, whether the heart defects seen in Ate1 knockout embryos are linked to impairments in cell migration during development, or whether additional cellautonomous changes in the cells composing the heart contribute to the Ate1 knockout phenotype, remains to be addressed. It has recently been found that a number of proteins in embryonic and adult mouse tissues are arginylated (Wong et al., 2007). Among these, a prominent arginylation substrate is alpha cardiac actin (also known as cardiac alpha actin, Actc1), the major component of the myofibrils in the cardiac muscle, the arginylation of which is likely to affect myofibril development and function. It is likely that impairment of this arginylation in the Ate1 knockout mouse would lead to defects in myofibril development that would be evident upon comparison of the hearts of wild-type and Ate1 knockout embryos, shedding light on the molecular role of arginylation in heart development and myofibril function. To address the question of whether Ate1 knockout results in cellautonomous changes in cardiac myocytes and whether arginylation plays a role in myofibril development and function, we characterized embryonic actin by gel fractionation and mass spectrometry and found that during development, alpha cardiac actin exists in a highly arginylated state and contains a total of four arginylated sites, Arginyltransferase regulates alpha cardiac actin function, myofibril formation and contractility during heart development