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First published online 23 October 2008
doi: 10.1242/dev.022723

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

¹ Department of Animal Biology and School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
² The Scripps Research Institute, La Jolla, CA 92037, USA.
³ Florida Atlantic University, Boca Raton, FL 33431, USA.
⁴ Janelia Farm, Ashburn, VA 20147, USA.
⁵ Mari-Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

View larger version (34K): [in this window] [in a new window]   Fig. 1. Alpha cardiac actin is arginylated in vivo. (A) Areas of a Coomassie-stained 2D gel of protein samples obtained by fractionation of whole E12.5 mouse hearts from wild-type (+/+, top) and Ate1 knockout (-/-, bottom) embryos under a shallow pH gradient (pH 4-8) to enable separation of individual actin isoforms. pH increases from left to right. Arrowheads indicate the position of spots that 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, equivalent to the molecular weight of intact alpha actin. , the position of alpha cardiac actin as identified by mass spectrometry. (B) Three-dimensional structure of an alpha cardiac actin monomer (PDB identifier 1J6Z) with post-translationally arginylated sites (R) indicated in pink within the blue actin backbone. The site indicated in pale pink, for which the mass spectrum is not shown, will be described elsewhere.

View larger version (136K): [in this window] [in a new window]   Fig. 2. Ate1 knockout results in the delayed development and the disorganization of cardiac myofibrils. Electron microscopy images of sections of wild-type (WT, left column) and knockout (KO, right column) E14.5 mouse hearts at 2500x magnification. The same images are shown in the bottom row, but with myofibrils highlighted in green. In the wild type at E14.5, myofibrils are prominent and are oriented along the axis of the heart muscle. In the 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. Scale bars: 10 µm.

View larger version (105K): [in this window] [in a new window]   Fig. 3. Ate1 knockout results in defects in the sarcomeric structure of cardiac myofibrils. (A) Wild-type sarcomere illustrating parameters measured. (B) Frequency distribution of sarcomere length and Z-band thickness in wild-type (WT) and knockout (KO) mouse hearts at E12.5 (E12) and E14.5 (E14). In wild-type and mutant 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. (C,D) Defects in Z-band thickness (C) and sarcomere length (D). Average sarcomere lengths (± s.d.) 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). (E) 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. Scale bars: 500 nm.

View larger version (106K): [in this window] [in a new window]   Fig. 4. Ate1 knockout results in defects in myofibril continuity at intercataled disks. (A) Illustration of the structural parameters measured. (B) Frequency distribution of average myofibril angle at each intercalated disk (above) and deviation of this angle from 90° (below). (C) Cell-cell distance averaged for all wild-type mouse 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, the 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. (D,E) Examples of intercalated disks in the wild type (D) and knockout (E). Abnormalities at intercalated disks included disorganization of the myofibrils resulting in angle deviations from 90° for incoming filaments (E, top two images), myofibril asymmetry on the two sides of the intercalated disk (E, the middle two images), and disruption of the cell-cell contacts at intercalated disks (E, bottom). Scale bars: 500 nm in D; 500 nm in E, except for bottom image at 2 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 5. Ate1 knockout results in defects in cardiac myocyte beating patterns. (A) Comparison of mean beats per minute (bpm), percentage of cells beating at high frequency, and percentage of cells with visible irregularities in the beating pattern between wild-type (WT) and knockout (KO) cultured myocytes derived from E12.5 embryonic mouse hearts. Calculations were made for 164 WT and 233 KO cells/islands for the left-most set of bars at low sampling rate (LS, two frames per second), 28 WT and 17 KO cells/islands for the next two sets of bars [calcium and phase, sampled at four frames per second for the same cell/islands in fluorescence (calcium) channel and phase-contrast], and 194 WT and 250 KO cells for the two right-hand sets of bars (irregular and high frequency). For images of individual beating curves, see Figs S4-6 in the supplementary material. (B) Examples of beating frequency curves, which were considered as regular, irregular or high frequency during manual calculation of the curves shown in Figs S4-S6 (see Figs S4-S6 in the supplementary material) to derive the percentages shown in A. (C) Illustration of how beat and calcium waves were measured from the total `gray level' in a region (square) of the beating cell that showed the most obvious changes (usually, the center). (D) Correlation plot between the physical beats observed in phase-contrast (x-axis) and calcium changes over time in the same cells (y-axis). For the most part, beats are correlated with calcium waves in both WT and KO cultures. For beating curves obtained by imaging of the same cells by phase-contrast and Fluo-4 calcium fluorescence, see Fig. S6 in the supplementary material.

View larger version (37K): [in this window] [in a new window]   Fig. 6. Model of the regulation of myofibril assembly and function by arginylation. In wild-type (WT) mice, arginylated actin assembles into stable filaments and eventually into normal myofibrils. In the Ate1 knockout, non-arginylated actin forms destabilized filaments, resulting in delayed myofibril development and profound structural defects.

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