Blockade of the Adenosine 2A Receptor Mitigates the Cardiomyopathy Induced by Loss of Plakophilin-2 Expression

FIGURE 1. (A) Western blot confirming knock-down of expected proteins in CX43Δ, PKP2Δ, and PKP2Δ/ CX43Δ HL-1 cell lines. (B) ATP release measured in HL1 cells (black bar), PKP2-deficient HL1 cells (PKP2KD, gray bar) and PKP2 and connexin43-deficient HL1 cells (PKP2/CX43 KD, white bar). ATP levels are higher in PKP2KD cells when compared to controls. Concomitant loss of Cx43 blunts partially this effect. ∗p < 0.05; ∗∗∗∗p < 0.0001.

FIGURE 2. RNAseq data from PKP2cKO (n = 5) and littermate control (n = 4) mice showing that lack of PKP2 expression is linked to overexpression of genes involved in fibrosis and regulated by adenosine expression (Perez-Aso et al., 2014; Ferrari et al., 2016). In the abscissae, “log2FoldChange” refers to the magnitude of the differential (expressed as logarithm base 2) of the abundance of a given transcript in control when compared to that present in PKP2cKO mice hearts. The ordinates indicate the negative logarithm base 10 of the confidence (p-value) calculated from the difference between abundance in control versus abundance in the PKP2cKO.

FIGURE 3. (A) Kaplan Meyer survival curve showing that PKP2cKO mice treated with ISTRA (black line, n = 10) have increased survival when compared to untreated ones (red line, n = 22). (B) Left Ventricular ejection fraction (LVEF) in PKP2cKO mice 35 days after tamoxifen injection. ISTRA-treated mice (black bar, n = 10) showed improved values when compared to untreated mice (red bars, n = 22). ∗∗∗p < 0.001.

FIGURE 4. (A) Masson Trichrome staining of untreated (left) and ISTRA-treated (right) PKP2cKO mice hearts 35 days post-tamoxifen injection. (B) Collagen percentage in the right (left bars) and left (right bars) ventricles of PKP2cKO mice treated (black, n = 10) and untreated (red, n = 8) with ISTRA. ∗p < 0.05. (C,D) Picrosirius red staining in areas of the right (top) and left (bottom) ventricles in PKP2cKO untreated and ISTRA-treated hearts. Left panels show BW contrast and polarized light microscopy images.

FIGURE 5. (A) Examples of vimentin staining on sections of PKP2cKO ventricles at 35 days post tamoxifen untreated (left) and after ISTRA treatment (right), showing decreased fibroblast abundance. Note the vessel in the right bottom panel (LV) as positive control for appropriate staining. (B) Quantification of vimentin-positive cells in untreated (red bar) and ISTRA treated hearts (black bars).

FIGURE 6. (A) Left ventricular ejection fraction is worsened by PSB115 treatment in PKP2cKO mice 35 days after tamoxifen injection. (B) Treatment with PSB115 does not reduce fibrosis in the right (RV) or left ventricles (LV) in PKP2cKO mice and shows a trend toward increased fibrosis. (C) Masson trichrome stained examples of untreated (left) and PSB115-treated (right) hearts of PKP2cKO mice at 35 days post tamoxifen injection. Red bars: untreated animals. Gray bars: treated with PSB115. ∗p < 0.05.