QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by van den Eijnde, S. M. Articles by Ramaekers, F. C. S. Search for Related Content PubMed PubMed Citation Articles by van den Eijnde, S. M. Articles by Ramaekers, F. C. S.

Transient expression of phosphatidylserine at cell-cell contact areas is required for myotube formation

¹ Department of Molecular Cell Biology, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, PO Box 616, 6200 MD Maastricht, The Netherlands
² Department of Anatomy & Embryology, Molecular and Experimental Cardiology Group, Academic Medical Center University of Amsterdam, 1105 AZ Amsterdam, The Netherlands
³ Department of Biochemistry, CARIM, University of Maastricht, PO Box 616, 6200 MD Maastricht, The Netherlands
⁴ Department of Plastic and Reconstructive Surgery, Erasmus University Medical School, PO Box 1738, 3000 DR Rotterdam, The Netherlands

View larger version (81K): [in a new window]   Fig. 1. Transient PS exposure by differentiating myoblasts in mouse embryos. Primary myotubes expose PS transiently at E13 (black arrows) in the cervical area (boxed area A1,A2), in-between the developing ribs (boxed area B1,B2) and the lumbar region (boxed area C1,C2). Frequently, annexin V-positive rounded cells were found attached to myotubes (D1,D2, arrowheads). Also in these sections, indications were found that annexin V-positive extensions arise from these developing myotubes (D1,D2, open arrowheads). Mitotic cells were negative for annexin V (B2, white arrowhead). At E14, annexin V staining of myotubes is virtually absent: compare the labeling for muscle using an anti--smooth muscle actin antibody (E1) with the labeling of surface-exposed PS (E2) in the same muscle (white arrows) in an adjacent section. For comparison, F shows annexin V-labeled apoptotic cells in the fusing E11 branchial arches, both in the mesodermal compartment (arrow) and in the ectoderm (arrowhead). Bars, 25 µm (C2,D1,D2,F); 40 µm (B2); 200 µm (A2,B1,C1); 500 µm (A1,E1,E2). Abbreviations: b, brain; l, limb; r, rib.

View larger version (69K): [in a new window]   Fig. 2. Double labeling with annexin V-fluo (green) and for DNA (DAPI, blue) of C2C12 (A) and H9C2 (B) muscle cells shows that differentiating myoblasts are labeled with annexin V at cell-cell contact areas (A1,A2,B1,B2, arrows), while mitotic myoblasts (A3,B3, arrowheads) and multinucleated myotubes (A4,B4, arrowheads) are negative for annexin V. Immunocytochemistry with an anti-human annexin V antibody (red) shows co-localization of these two markers resulting in yellow areas in the overlay (A2,B2). Bars, 20 µm (A1-3,B1-3); 60 µm (A4,B4).

View larger version (106K): [in a new window]   Fig. 3. Triple labeling with annexin V-fluo (green), for titin (red) and DNA (DAPI, blue) of C2C12 (A1,A2) and H9C2 (B1,B2) myoblasts shows that annexin V labels mainly early differentiating muscle cells with a punctate titin expression pattern (A1,B1, arrows). At later stages of differentiation, when the titin-expression pattern becomes filamentous, myoblasts were almost completely negative for annexin V-fluo (A2,B2). Bar, 20 µm.

View larger version (48K): [in a new window]   Fig. 4. Differentiating annexin V-binding C2C12 (A1,A2) and H9C2 (B1-3) myoblasts have an intact plasma membrane as detected by PI exclusion. In all cultures low numbers of solitary annexin V (green) and PI (red) labeled cells were found (A1,B1,B3, arrows). These postapoptotic/necrotic cells can clearly be discriminated from annexin V-positive differentiating myoblasts where annexin V labeling is preferentially located at cell-cell contact areas (A2, nuclei stained with DAPI; B2, asterices indicate location of nuclei), and which are PI impermeable (A2,B2,B3, asterisks indicate location of nuclei). In-between the cells shown in B2, an annexin V- and PI-positive cell fragment, most probably resulting from apoptotic cell membrane blebbing, is present (arrowhead). Bars, 25 µm (A1,A2,B1,B2); 200 µm (B3).

View larger version (54K): [in a new window]   Fig. 5. Recombinant human annexin V (AnxV) inhibits myotube formation in differentiating C2C12 and H9C2 muscle cell cultures. C2C12 cells were induced to differentiate in DM when the cultures were at 25% (not shown) and 50% confluency (A1,A2), and H9C2 cells at 95% confluency (B1,B2). In samples cultured in the presence of M1234 (not shown) or in the absence of annexin V (ctrl) myotube formation could clearly be observed in C2C12 cells at day 5 (DM d5, A3, arrows) and H9C2 cells at day 11 (DM d11, B3, arrows), and to a lesser extent in cells cultured in DM containing 100 µg/ml of recombinant human annexin V (A4,B4, arrows). Note that cells cultured in GM differentiate spontaneously and form myotubes when reaching 100% confluency (e.g. B1,B2, arrows). To assess the effect of annexin V on myotube formation quantitatively, multinucleated cells and their nuclei were counted in C2C12 cultures at DM d5 (A5) and H9C2 cultures at DM d11 (B5), after staining of DNA with DAPI (blue), and with titin (green) to detect differentiating cells. The quantitative results are shown in Fig. 6 and Table 1. Bar, 100 µm (A1-4,B1-4); 25 µm (A5,B5).

View larger version (23K): [in a new window]   Fig. 6. (A) Annexin V inhibits myotube formation in differentiating C2C12 and H9C2 myoblast cultures via an interaction with PS. C2C12 and H9C2 cells were induced to differentiate in DM (control), DM with PS binding-deficient M1234, or DM with 100 µg/ml annexin V (AnxV). The graphs depict myotube distribution as a function of the number of nuclei they contained on DMd5 in cultures of C2C12 cells that were induced to differentiate at 50% confluency, and on DMd11 in cultures of H9C2 cells that were induced to differentiate at 95% confluency. Data from 6 wells per group were combined. Statistical analysis of this data is shown in Table 1. (B) Annexin V inhibits myotube formation dose dependently. C2C12 cells were induced to differentiate in DM with indicated concentrations of annexin V and the number of myotubes were counted in each sample (n=6 per dose). The graph depicts the average number of myotubes per well as a percentage of control on DMd5 in myoblast cultures that were induced to differentiate at 50% confluency. A significant decrease in the number of myotubes formed was observed at a concentration of 40 µg/ml (**p=0.001) and 100 µg/ml (* p=0.002).

View larger version (53K): [in a new window]   Fig. 7. Annexin V-binding by differentiating C2C12 (A) and H9C2 (B) myoblasts does not require loss of mitochondrial inner membrane potential or caspase activity. Apoptotic myoblast are positive for annexin V-fluo (green) and show a diffuse labeling for CMXRos (A1,B1, arrowhead, red) and immunoreactivity for the anti-active caspase 3 antibody (A3,B3,B4, arrowhead, red). By contrast, differentiating C2C12 and H9C2 cells bind annexin V-fluo while having a granular labeling for CMXRos (A2,B2, arrow) and show no active caspase 3 protein (A4,B4, arrow). In line with the latter results, culturing cells in DM containing the caspase inhibitor zVAD-(OMe)-fmk at a dose of 100 µM blocks neither myotube formation (A5,B5, arrow) nor annexin V-fluo binding (A6,B6, arrow). These two figures, in which out-of-focus information has been removed by image restoration using deconvolution software, further illustrate the intracellular granular labeling of myoblasts with annexin V-fluo after longer incubation periods. Bars, 25 µm.

View larger version (101K): [in a new window]   Fig. 8. PS is transiently exposed by differentiating cardiomyocytes of E12 mouse embryos. Adjacent transverse sections were stained for -smooth muscle actin to detect cardiac muscle cells (A) and annexin V to label surface-exposed PS (B). Boxed areas in A1 and B1 are depicted at a higher magnification in A2 and B2 (upper box), and A3 and B3 (lower box). A strong annexin V-biotin labeling was observed in the smooth-walled atrial myocardium (A2,B2, arrow), the subepicardial ventricular myocardium (A2,A3,B2,B3, in-between arrowheads) and the epicardium (A2,A3,B2,B3, open arrowhead). Also, at this stage of development, some pyknotic cardiomyocytes were observed in the trabecular myocardium (A3,B3, open arrows). Bars, 25 µm (A2,B2); 50 µm (A3,B3); 500 µm (A1,B1). Abbreviations: n, neural tube; l, limb; p, pericardial cavity.