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

First published online 15 December 2004
doi: 10.1242/jcs.01553

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 HighWire Citing Articles via Google Scholar Google Scholar Articles by Araya, R. Articles by Sáez, J. C. Search for Related Content PubMed PubMed Citation Articles by Araya, R. Articles by Sáez, J. C.

Expression of connexins during differentiation and regeneration of skeletal muscle: functional relevance of connexin43

Roberto Araya^1,*,, Dominik Eckardt², Stephan Maxeiner², Olaf Krüger², Martin Theis^2,, Klaus Willecke² and Juan C. Sáez¹

¹ Departamento de Fisiología, Pontificia Universidad Católica de Chile, Santiago, Chile
² Institut für Genetik, Universität Bonn, Bonn, Germany

View larger version (48K): [in a new window]   Fig. 2. Cx45 and Cx43 are expressed in tibialis anterior muscles (TA) and up-regulated during skeletal muscle regeneration. (A) Western blot analysis of Cx45 present in homogenate samples (100 µg protein) of TAs at different days post BaCl2 injection (PI) (3, 5, 7, 10 and 14) and in HeLa Cx45 transfectants (H) and control (C) muscles. The different patterns of reactive bands in HeLa and C2C12 cells are due to the differential posttranscriptional modifications of Cx45, which will be described elsewhere. (B) Western blot analysis of Cx43 in aliquots of heart homogenates (H, used as positive control) and of TAs (100 µg) under control conditions and at days 3, 5, 7 or 10 post BaCl2 injection (PI). The non-phosphorylated (NP) and phosphorylated (P) forms of Cx43 are indicated. Tub: -tubulin levels measured in stripped blots.

View larger version (98K): [in a new window]   Fig. 1. Time course of BaCl2-induced regeneration of tibialis anterior muscle (TA). TAs from C57BL/6 mice under control conditions or after 3, 5 and 7 days of BaCl2-induced injury were dissected, counterstained and examined histologically (A) or analyzed for myogenin (Myo) levels (B). (A) The Hematoxylin and Eosin stained sections show that in the control most stained nuclei were located in the cell periphery. At 3 and 5 days post BaCl2 injection (PI), fibers probably undergoing necrosis (arrows) and small mononucleated cells (<15 µm; arrowheads) were abundant in the muscle core. At 7 days PI, nearly 90% of the muscle section area was occupied by centrally nucleated myotubes. Scale bar: 60 µm. (B) Myogenin (Myo) was measured by western blot analysis in 100 µg protein aliquots of homogenates from control TA (C) and from TA at different days PI (3, 5, 7, 10 or 14). Differentiated C2C12 myoblasts were used as positive control.

View larger version (87K): [in a new window]   Fig. 3. Cx45 gene expression in regenerating tibialis anterior muscles (TA). (A) Eosin stained TA cross sections of control Cx45+/– mice showed X-gal reactivity in microvessels (empty arrowhead). At day 3, numerous cells showed cytoplasmic X-gal positive dots (open arrowheads). At 7 days PI X-gal positive dots were found within (arrows) and between (open arrowheads) fibers. (B-D) Co-localization of X-gal staining with specific cell markers in regenerating TAs from Cx45+/– mice. Arrows indicate sites of co-localization; both arrow and arrowheads indicate positive X-gal staining. These panels show co-localization of von Willebrand factor (VWF) with X-gal staining under control conditions and at D3 PI (B), with CD14 at D3 PI (C) and with myogenin (Myo) at D5 PI (D). Scale bars: In A, 100 µm (left column) and 50 µm (right column); in B, 45 µm (upper panels), 30 µm (middle panels) and 50 µm (bottom panels); (C,D) 20 µm.

View larger version (78K): [in a new window]   Fig. 4. Cx43 gene expression in regenerating TA. (A) Cross sections of TAs from Cx43del/+ mice under control conditions and at different days PI (3 or 7 PI) show X-gal-stained cells. (a,b) Under control conditions, cells of the epymisum (a, arrow), myofibers (b.1, arrows) and cells closely associated with myofibers (b, open arrowhead) were X-gal positive. Inset in b shows co-localization of M-cadherin (fluorescence, left panel) and X-gal staining (phase contrast, right panel). (c,d) At 3 days PI (D3 PI) cells adhered (d, arrows) or close to necrotic fibers showed X-gal stained nucleus. (e,f) At day 7 (D7 PI), numerous myofibers (f, arrows) and cells located between myofibers (f, open arrowheads) showed X-gal reactivity. Scale bar: 160 µm (a); 85 µm (b); 100 µm (b.1); 270 µm (c); 70 µm (d); 90 µm (e); 50 µm (f and inset in b). (B) Co-localization of specific cell markers and X-gal staining in normal and regenerating TA of Cx43del/+ mice. (Left panels) Co-localization of von Willebrand factor (VWF) in control and at 3 days PI (D3 PI); (top right panels) CD14 reactive cells with X-gal staining at D3 PI; (bottom right panels) co-localization of myogenin (Myo) reactive cells with X-gal staining at 5 days PI (D5 PI). Arrows indicate sites of co-localization. Scale bar in left pair of panels: 50 µm (top), 60 µm (bottom). Scale bar in right pair of panels: 30 µm (top), 20 µm (bottom).

View larger version (36K): [in a new window]   Fig. 5. Primary cultures of myoblasts express Cx43. (A) Primary cultures of satellite cell-derived myoblasts maintained for 48 hours in differentiation medium were fixed with ethanol and Cx43 was detected by immunofluorescence. Scale bar: 20 µm. (B) Western blot analysis of Cx43 in aliquots (100 µg of protein) of myoblast homogenates obtained at 0, 24 and 48 hours of differentiation. The non-phosphorylated (NP) and phosphorylated (P) forms of Cx43 are indicated.

View larger version (34K): [in a new window]   Fig. 6. Induced ablation of Cx43 expression inhibits myogenesis in primary cultures of myoblasts. (A) Western blot analysis of Cx43, myogenin (Myo), MyoD and -tubulin (Tub) in homogenates (100 µg) of Cx43fl/fl or Cx43Cre-ER(T)/fl satellite cell-derived myoblasts treated with 4-OH-tamoxifen every 24 hours for 5 days followed by 24 hours of differentiation. (B) At 24 hours of differentiation, positive Cx43 immunolabeling was detected in control Cx43fl/fl myoblasts (left) but not in 4-OH-tamoxifen-treated cells of Cx43Cre-ER(T)/fl mice (right; upper panel shows phase contrast view). Scale bar: 40 µm. (C) Bar chart of the incidence of coupling (%) of 4-OH-tamoxifen-treated myoblasts of Cx43Cre-ER(T)/fl or Cx43fl/fl mice after 24 hours of differentiation. (D) Bar chart showing the percentage of the average number of nuclei present in myotubes at day 4 of differentiation in 4-OH-tamoxifen-treated myoblasts of Cx43Cre-ER(T)/fl mice as compared to 4-OH-tamoxifen-treated myoblasts of Cx43fl/fl mice (**P<0.01 vertical bars indicate the standard deviation (s.d.), n=4 independent experiments). Upper images are phase-contrast views at day 4 of differentiation (D4) of 4-OH-tamoxifen-treated Cx43fl/fl or Cx43Cre-ER(T)/fl myoblasts. Scale bar: 40 µm. (E) Bar chart showing the relative creatine kinase activity of cultured myoblasts at day 4 of differentiation. 4-OH-tamoxifen-treated cells were from Cx43Cre-ER(T)/fl or Cx43fl/fl mice. (*P<0.05 vertical bars indicate s.d., n=3).

View larger version (82K): [in a new window]   Fig. 7. Cell-specific deletion of Cx43 delays TA regeneration. (A) Levels of Cx43 were measured by western blot analysis in TA homogenates (100 µg of protein) obtained at day (D) 3, 7, 10 or 14 of regeneration after BaCl2 treatment. Muscles were from pI-pC-treated Cx43fl/fl, Mx-cre mice (fl/fl, Mx-cre) and pI-pC-treated Cx43fl/fl mice (fl/fl). The non-phosphorylated (NP) and phosphorylated (P) forms of Cx43 are indicated. (B) Hematoxylin and Eosin-stained cross sections of TAs from pI-pC-treated Cx43fl/fl and pI-pC-treated Cx43fl/fl, Mx-cre mice after 7, 10 or 14 days post BaCl2 administration (D7 PI, D10 PI, or D14 PI, respectively). A small group of damaged cells is present in the 14 days PI (D14 PI) sample, (indicated by a dashed rectangle). Scale bar: 100 µm. (C) At 7 days PI (D7 PI), X-gal stained nuclei were observed in sections of TAs from pI-pC-treated Cx43fl/fl, Mx-cre mice but not in TAs from pI-pC-treated Cx43fl/fl mice. Scale bar: 100 µm. (D) Co-localization of specific cell markers and X-gal staining in regenerating TAs from pI-pC-treated Cx43fl/fl, Mx-cre mice. These panels showed co-localization of X-gal staining with myogenin (Myo) at D5 PI and desmin, CD14 or von Willebrand factor (VWF) at D3 PI. Arrows indicate co-localization sites. Scale bar: 12 µm for Myo, Desmin and CD14 panels and 20 µm for VWF panel. (E) Relative levels of myogenin (Myo) were measured by western blot analysis in samples (100 µg of protein) of TA homogeneates obtained from pI-pC-treated Cx43fl/fl or pI-pC-treated Cx43fl/fl, Mx-cre mice at different days (3, 5, 7, 10, and 14) PI.