The IgCAM CAR Regulates Gap Junction-Mediated Coupling on Embryonic Cardiomyocytes and Affects Their Beating Frequency

Figures

• 

  Figure 1. Increased frequency of beating and calcium transients in monolayer cultures of embryonic cardiomyocytes in the absence of the CAR. (A) Localization of the CAR in cultures from embryonic E10.5 hearts cultured 4 DIV in DMEM/FCS. Cardiomyocytes were identified by labeling with an anti-sarcomeric actinin antibody (green). Scale bar, 80 μm. (B) The beating frequencies of cardiomyocytes were counted manually at DIV 4 at 37 °C in a humidified atmosphere of 95% air and 5% CO2 in DMEM/FCS by using an inverted microscope. Cells were from 13 independent cultures and 27 wild type and 25 knockout cell clusters were counted. (C) Individual traces of calcium transients of cultured cardiomyocytes are shown. FU, relative fluorescence units. (D) Summary of the frequency of calcium transients from wild type, heterozygote, and CAR-deficient cardiomyocytes. Measurements were done from Fura-2-loaded cardiomyocytes in ACSF at room temperature. The numbers in the columns represent the number of cell clusters from independent cultures. (E) Decay time constants of calcium transients of wild type, heterozygote, and CAR-deficient cardiomyocytes. (F,G) Individual traces of caffeine-induced calcium transients…

• 

  Figure 2. Calcium extrusion is enhanced in CAR knockout cardiomyocytes, which correlates with the increased beating frequency. Calcium extrusion is enhanced in CAR knockout cardiomyocytes, which correlates with the increased beating frequency. (A) Intracellular calcium concentrations at diastolic and systolic phases of wild type, heterozygote, and CAR-deficient cultured cardiomyocytes are depicted. (B) Amplitudes of calcium transients after the application of 10 mM of caffeine, which might be considered a measure of calcium levels stored in the sarcoplasmic reticulum. No differences were observed between the different genotypes, suggesting that similar amounts of calcium are stored in the sarcoplasmic reticulum. (C) Spontaneous calcium events via the RyR, so-called calcium leaks, can induce spontaneous beating and fibrillation of the heart [42,52,53]. The Ca2+ leak can be quantified by measuring the changes of cytosolic Ca2+ in the presence of 1 mM of tetracaine (RyR inhibitor) in Na+- and Ca2+- free ACSF. Tetracaine completely blocked the RyR and, therefore, the SR Ca2+ leak, consequently, the cytosolic Ca2+ concentration decreased. Wild type and CAR knockout cardiomyocytes did not show…

• 

  Figure 3. The sizes of connexin43 spots and dye coupling are increased in the absence of the CAR. (A–D) Quantification of connexin43 spots at cell–cell contact sites of cultured E10.5 wild type and CAR-deficient cardiomyocytes. Cells were stained by rabbit anti-connexin43, mAb anti-sarcomeric actinin, and DAPI. The average sizes of connexin43 clusters increased; (B) data are from three independent cultures, Mann–Whitney test p = 0.0007. In total, 56 wild type and 66 knockout cardiomyocytes were analyzed. Roughly 20 spots per cell were counted and the average size per cell was calculated. (C) The size distribution of connexin43 plaques shows a shift toward a larger cluster while smaller ones are reduced (chi-square test p = 0.0182). (D) The total number of connexin43 plaques per cell–cell contact area was reduced. (Mann–Whitney U test p < 0.001). Scale bar in A, 50 μm. (E) Western blot of wild type and CAR-deficient embryonic hearts using antibodies to the phosphorylated Akt (pAkt) or total Akt (lower panel) are shown. (F,G) Individual cardiomyocytes from the E10.5 CAR wild type and CAR knockout cultures were injected with Lucifer Yellow and the spreading was examined after 5 min. The…

• 

  Figure 4. Decreased expressions of connexin43 and 45 in CAR-deficient embryonic hearts. (A,B) Expressions of connexin43, 45, 40, and 50 in wild type and CAR-deficient E10.5/E11.5 hearts shown by Western blots. For comparison, β-catenin and GAPDH are shown (from top to bottom). Band intensities from blots were quantified. Reductions were observed for connexin43 and 45 (t-test p = 0.0095 and p < 0.0001, respectively) but not for connexins 40 or 50 (t-test 0.5773 and 0.1179, respectively) in knockouts. Individual hearts were directly solubilized in the SDS-PAGE sample buffer. Molecular mass markers are indicated on the left of the panel. AU, arbitrary units. (C,D) Selected Affymetrix microarray data of mRNA expression levels of connexins and cell–cell contact proteins of E10.5 hearts of wild type and CAR-deficient mice did not reveal differences. For further details, see Table S1 and the database entry (GEO GSE138831). (E,F) On E10.5 cardiomyocytes, the CAR was not found to co-localize with connexin43. ZO-1 is shown for comparison. Scale bar, 10 μm. The Pearson correlation of co-localization was calculated using Fiji/Image J software (F); 48 and 43 images were analyzed for CAR-ZO-1 and…

• 

  Figure 5. Localization of the CAR in the developing and mature heart and the increased spontaneous beating frequency of heart organ cultures in the absence of the CAR or the presence of the fiber knob or antibodies to CAR. (A) Overview of the localization of the CAR in the heart at E11.5. Cryostat sections were stained by rabbit anti-mCAR generated against the extracellular domain of the CAR (Rb80) and by a monoclonal antibody against sarcomeric α-actinin. Sections from knockout hearts indicated the specificity of Rb80. Scale bar, 100 μm. (B) Localization of the CAR at different developmental stages and in the mature heart. Arrowheads in sections of P10 or the adult heart indicate intercalated discs. Scale bar, 50 μm. (C) In vitro culture of E10.5 heart in Matrigel to determine the beating rate. Scale bar, 200 μm. (D) Purified recombinantly expressed fiber knob Ad2 is shown in 12% SDS-PAGE stained by Coomassie blue. Specificity of the rabbit antibody to the extracellular region of mCAR is demonstrated by Western blotting using wild type and knockout embryonic hearts. Loading was monitored by a monoclonal antibody to clathrin. The specificity of Rb80 is also shown in the cryostat section of…