Figure 2 | Scientific Reports

Figure 2

From: Cardiac inflammatory CD11b/c cells exert a protective role in hypertrophied cardiomyocyte by promoting TNFR2- and Orai3- dependent signaling

Figure 2

TNFα activates a store- and voltage-independent Ca2+ influx in hypertrophied cardiomyocytes further identified as Orai3 dependent. Hypertrophied cardiomyocytes were loaded with Fura2 before measurement of voltage- and store-independent Ca2+ influx. (A) Two successive applications of the same TNFα perfusion medium on iso-treated hypertrophied cardiomyocytes gave rise to similar rates of Ca2+ entry. The first TNFα-induced Ca2+ influx was arbitrarily set to 2.2 to allow comparisons with the control conditions (see Fig. 1B). Preincubation with Orai inhibitors, YM58483 or Synta66, prior the second application of TNFα perfusion medium blunted activation of Ca2+ entry by TNFα. (B) Hypertrophied cardiomyocytes isolated from hearts injected with Cy3-tagged scramble or Orai3 siRNAs three days before isolation and iso-treatment. Typical images show Cy3-fluorescence in cardiomyocytes. TNFα activates Ca2+ influx in scramble siRNA-transfected cells but not in siOrai3-transfected cardiomyocytes. Number of cells analyzed and number of cell isolations (rats) as indicated, mean ± SEM of cells, Wilcoxon matched-paired tests to examine if the mean of the 2nd rate differs from the 1st one, arbitrarily set as 2.2 (A) or 1 (B), **p < 0.01, ***p < 0.001, ****p < 0.0001. (C) Efficient knockdown of Orai3 mRNA and protein in cardiomyocytes isolated from Wistar rats at day 3 following injection with Cy3-tagged scramble or Orai3 siRNAs. Histograms representing relative transcript levels normalized to the RPL32 mRNA or relative protein levels normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. Mean ± SEM of cardiomyocytes from 2–6 rats/group, Mann-Whitney U test, *p < 0.05. Full length blots were included in SI. (D) Hypertrophied cardiomyocytes isolated from rats after fourteen days of chronic iso-infusion injected with Cy3-tagged scramble or Orai3 siRNAs three days before isolation. TNFα activates Ca2+ influx in hypertrophied cardiomyocytes isolated from hearts injected or not with Cy3-tagged scramble siRNA, but not in hypertrophied cardiomyocytes isolated from hearts injected with Cy3-tagged Orai3 siRNA, three days before isolation. Number of cells analyzed and number of cell isolations (rats) as indicated, mean ± SEM of cells, Wilcoxon matched-paired tests to examine if the mean of the 2nd rate differs from the 1st one, arbitrarily set as 1, **p < 0.01, ****p < 0.0001.

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