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. 2008 Dec;135(23):3881-9.
doi: 10.1242/dev.022723. Epub 2008 Oct 23.

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

Reena Rai  1 , Catherine C L Wong, Tao Xu, N Adrian Leu, Dawei W Dong, Caiying Guo, K John McLaughlin, John R Yates 3rd, Anna Kashina
Affiliations

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

Reena Rai et al. Development. 2008 Dec.

Erratum in

  • Development. 2008 Dec;135(23):3971

Abstract

Post-translational arginylation mediated by arginyltransferase (Ate1) is essential for cardiovascular development and angiogenesis in mammals and directly affects myocardium structure in the developing heart. We recently showed that arginylation exerts a number of intracellular effects by modifying proteins involved in the functioning of the actin cytoskeleton and in cell motility. Here, we investigated the role of arginylation in the development and function of cardiac myocytes and their actin-containing structures during embryogenesis. Biochemical and mass spectrometry analyses showed that alpha cardiac actin undergoes arginylation at four sites during development. Ultrastructural analysis of the myofibrils in wild-type and Ate1 knockout mouse hearts showed that the absence of arginylation results in defects in myofibril structure that delay their development and affect the continuity of myofibrils throughout the heart, predicting defects in cardiac contractility. Comparison of cardiac myocytes derived from wild-type and Ate1 knockout mouse embryos revealed that the absence of arginylation results in abnormal beating patterns. Our results demonstrate cell-autonomous cardiac myocyte defects in arginylation knockout mice that lead to severe congenital abnormalities similar to those observed in human disease, and outline a new function of arginylation in the regulation of the actin cytoskeleton in cardiac myocytes.

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Figures

Figure 1
Figure 1. Alpha cardiac actin is arginylated in vivo
Left, areas of a Coomassie stained 2D gel obtained by fractionation of the whole E12.5 mouse hearts from wild type (+/+, top) and knockout (−/−, bottom) embryos under a shallow pH gradient (pH 4–8) to enable separation of individual actin isoforms. pH increases left to right. Arrowheads indicate the position of spots, which were used for the horizontal alignment of the two gels to enable observation of gel shifts of individual actin spots. Arrows indicate the position of the 43 kDa marker at the actin molecular weight. ‘Alpha’ (α) symbol denotes the position of alpha cardiac actin, as identified by mass spectrometry. Right, 3D structure of an alpha cardiac actin monomer (PDB identifier 1J6Z) with posttranslationally arginylated sites indicated in pink within the blue actin backbone anddenoted with capital R. Pale pink R indicates a site, for which the mass spectrum is not shown and will be described elsewhere.
Figure 2
Figure 2. Ate1 knockout results in delayed development and disorganization of cardiac myofibrils
Electron microscopic images of sections of wild type (left) and knockout (right) E14.5 hearts at 2500× magnification. Copies of the original images with myofibrils highlighted in green are shown on the bottom. While in wild type at E14.5 myofibrils are prominent and oriented along the axis of the heart muscle, in knockout at the same stage myofibrils are much less abundant and difficult to trace over continuous distances, suggesting a defect in overall myofibril organization and cardiac contractility. Bars, 10 μm.
Figure 3
Figure 3. Ate1 knockout results in defects in the sarcomeric structure of cardiac myofibrils
Left top, wild type sarcomere with measured parameters indicated. Left middle, frequency distribution of sarcomere length and Z-band thickness in wild type (WT) and knockout (KO) hearts at embryonic days E12.5 (E12) and E14.5 (E14). While in wild type and E12.5 hearts both sarcomere length and Z-band thickness are relatively constant with small variations due to differences in the contractile state of individual myocytes, in knockout hearts at later stages (E14.5) the frequency distribution of both parameters becomes wider, suggesting disorganization of the sarcomeres. Left bottom and right top panels illustrate the defects in Z-band thickness and sarcomere length, respectively. Average sarcomere lengths were 1389 +/− 141 nm (WT E12, n=27), 1490 +/− 101 nm (KO E12, n=39), 1682 +/− 116 (WT E14, n=116) and 1179 +/− 199 (KO E14, n=124). Average Z-band thicknesses were 86 +/− 19 nm (WT E12, n=27), 87 +/− 23 nm (KO E12, n=39), 120 +/− 23 (WT E14, n=116) and 142 +/− 36 (KO E14, n=124) (errors represent SD). Bottom right, examples of other defects in myofibril structure seen in Ate1 knockout hearts, including myofibril branching at Z-bands and asymmetric sarcomeres, in which the density of the filaments on the two sides is markedly different. Bars, 500 nm.
Figure 4
Figure 4. Ate1 knockout results in defects in myofibril continuity at intercataled disks
Left top, an illustration of the parameters measured. Left middle, frequency distribution of myofibril angle average at each intercalated disk (top diagram) and deviation of this angle from 90° (bottom diagram). Left bottom, cell-cell distance averaged for all wild type hearts together (light gray bars) and for individual knockout hearts (dark gray bars), sorted by embryo age. As the severity of the knockout phenotype progresses, average cell-cell distance as well as the standard deviation between distances in a single heart increase, as seen at E14 for the three KO hearts shown. Right column, examples of intercalated disks in wild type (top) and knockout (bottom four panels). Abnormalities at intercalated disks included disorganization of the myofibrils, resulting in angle deviations for incoming filaments from 90° (top two images), myofibril asymmetry on two sides of intercalated disk (middle images), and disruption of the cell-cell contacts at intercalated disks (bottom image). Bars, 500 nm for top images and 2 μm for the bottom right image.
Figure 5
Figure 5. Ate1 knockout results in defects in cardiac myocyte beating patterns
Left top, comparison of mean beats per minute (bpm), percentage of cells beating with high frequency, and percentage of cells with visible irregularities in the beating patterns between wild type (WT) and knockout (KO) cultured myocytes derived from E12.5 embryonic hearts. Calculations were made for 164 WT and 233 KO cells/islands for the left set of bars at low sampling rate (LS, 2 frames per second), 28 WT and 17 KO cells/islands for the next two sets of bars (phase and calcium, sampled at 4 frames per second for the same cell/islands in phase contrast and fluorescence (calcium) channel), and 194 WT and 250 KO cells for the two right-hand sets of bars. See the images of individual beating curves in Supplemental Figures 2 and 3. Left bottom, examples of beating frequency curves, which were considered as regular, irregular, and high frequency during manual calculation of curves shown in Supplemental Figures 2, 3, and 4 to derive the percentages shown above. Right top, illustration of the beat and calcium wave measurements as total gray level in a square region taken in the area of the beating cell with most visible changes (usually, the center). Right bottom, correlation plot between the physical beats observed in phase contrast (x axis) and calcium changes over time in the same cells (y axis) shows that for the most part beats are correlated with calcium waves in both cultures. See Supplemental Figure 4 for beating curves obtained by imaging of the same cells by phase contrast and Fluo4 Ca fluorescence.
Figure 6
Figure 6. Model of the regulation of myofibril assembly and function by arginylation
In wild type (WT) arginylated actin assembles into stable filaments and eventually into normal myofibrils. In Ate1 knockout, unarginylated actin forms destabilized filaments, resulting in delayed myofibril development and profound structural defects.
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