Figure 3.

Mitochondrial Outer Membrane Permeabilization–Mediated mtDNA Release Stimulates the cGAS-STING Pathway in Cardiomyocytes in a CKD Milieu

(A) Gene set enrichment analysis of gene sets associated with cytosolic DNA sensing pathway in the myocardia. (B) Relative enrichment of DNA fragments in NRCMs incubated with normal or CKD serum. (C) Representative images of cGAS and DNA (left), cGAS and 8-OHdG (middle), or cardiac troponin T (cTNT) and p-TBK1 (right) colocalization in the myocardia. The region marked by the box is magnified in the right panel. Colocalization of cGAS with DNA (left), colocalization of cGAS with 8-OHdG (middle), and colocalization of cTNT with p-TBK1 (right) are marked by the white arrow. Scale bar, 20 μm in the left panels and 4 μm in the right panels. (D) Representative images of cGAS and DNA (left), cGAS and 8-OHdG (middle), or cTNT and p-TBK1 (right) colocalization in NRCMs incubated with normal or CKD serum. The region marked by the box is magnified in the right panel. Colocalization of cGAS with DNA (left) and colocalization of cGAS with 8-OHdG (middle) with p-TBK1 (right) are marked by the white arrow. Scale bar, 20 μm in left panel and 4 μm in the right panel. (E) Representative western blot analysis of p-TBK1 in the myocardia from sham or CKD mice with or without VBIT-4 treatment. Data represent mean ± SD and were analyzed using 2-tailed Student’s t-test (B) or 1-way analysis of variance (E). n = 3 biologically independent experiments. ∗∗P < 0.01 and ∗∗∗P < 0.001. DAPI = 4′,6-diamidino-2-phenylindole; FDR = false discovery rate; mtDNA = mitochondrial DNA; n.d., not determined; nDNA = nuclear DNA; NES = normalized enrichment score; other abbreviations as in Figure 1.