Fig. 7: Endothelial RNF20 regulates cardiomyocyte contractility and rhythmicity. | Nature Communications

Fig. 7: Endothelial RNF20 regulates cardiomyocyte contractility and rhythmicity.

From: Endothelial RNF20 suppresses endothelial-to-mesenchymal transition and safeguards physiological angiocrine signaling to prevent congenital heart disease

Fig. 7

a Schematic representation of the transwell co-culture system: mouse ECs isolated from control and Rnf20iEC-KO hearts were treated with 10 µM 4-Hydroxytamoxifen for 72 h and subsequently plated on the top compartment of a transwell plate. 12 h beforehand rat CMs were plated in the bottom compartment and co-cultured with ECs for 2 days. b Plot of contraction amplitude and speed of beating rat CMs co-cultured with control orRnf20iEC-KO ECs extracted from video sequences using MYOCYTER. c Poincaré plot derived from image sequences of beating rat CMs co-cultured with control or Rnf20iEC-KO ECs. d Frequency and amplitude of image sequences recorded on rCMs after co-culture with ECs isolated from control (n = 6) or Rnf20iEC-KO (n = 6) hearts. e Percentage of proliferating EdU-positive rat CMs co-cultured with ECs isolated from control (n = 6) or Rnf20iEC-KO (n = 6) hearts. f Schematic representation of the transwell co-culture system: HUVECs were either transfected with control siRNA or siRNA against RNF20 or infected with control or RNF20 overexpressing (OE) lentivirus for 48 h, following a co-culture with rat CMs for 2 days. g Immunostaining for pH3S10 and MF20 (left panels) and percentage of mitotic pH3S10-positive rat CMs (right panel) after co-culture with HUVECs transfected with control siRNA (n = 5) or siRNA against RNF20 (n = 5) or infected with control (n = 5) of RNF20 OE lentivirus (n = 5). Scale bars, 50 µm. h Plot of contraction amplitude and speed of beating of rat CMs co-cultured with control, siRNF20 silenced or RNF20 OE HUVECs extracted from video sequences using MYOCYTER. i Poincaré plot derived from image sequences of beating rat CMs co-cultured with control, siRNF20 silenced or RNF20 OE HUVECs. j Frequency and amplitude of image sequences recorded on rCMs after co-culture with control (n = 3), siRNF20 silenced (n = 3) or RNF20 OE HUVECs (n = 3). Transwell co-culture system schematics in (a, f) created in BioRender.com. Dobreva, G. (2025) https://BioRender.com/yr6530z. Statistical analysis between two groups in (d, e, g, j), was performed using an unpaired two-tailed Student’s t test. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

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