Figure 4

DMB fails to limit inflammation, fibrosis and induce mitochondrial turnover in PKO mice. Age-matched PKO mice underwent permanent coronary artery ligation and were given vehicle (50 µL DMSO) or DMB (10 pmoles/25 g) i.p. 2 hours after surgery and again 2 and 5 days later. Mice were sacrificed 3 days after PCAL (n = 3–4 mice/group) for H&E staining and MitoTimer ratiometric shifts, and 7 days after PCAL for Masson Trichrome staining. A second cohort of mice received vehicle (50 µL DMSO) or DMB (10 pmoles/25 g) i.p. One hour later, mice received chloroquine (CQ, 10 mg/kg i.p.) and were sacrificed 16 hours later (n = 3/group) for western blot analysis of autophagy and mitophagy markers. (a) Representative 60X and 20X magnification of heart sections stained with H&E showing immune cell infiltration in PKO mice 3 days post-MI; (b) Representative 20X magnification of heart sections with Masson Trichrome 7 days after PCAL showing fibrosis in PKO vs. wild type in DMB-treated mice. (c) Western blot analysis and quantification of autophagy markers p62 and LC3 in heart whole lysates of PKO mice; (d) Western blot analysis and quantification of mitophagy markers PINK1, Optineurin (OPTN) and BNIP3 in the mitochondrial-enriched fraction of PKO mice; (e) Cryopreserved sections of the heart were examined for MitoTimer ratiometric shifts (green/red ratio = new/old mitochondria) as an indication of mitochondrial biogenesis (n = 4 mice/group). All values are presented as mean ± standard deviation. Scale bars as indicated. Standard Student’s t-test was used to compare the groups. WB figures were cropped and all densitometry was performed using NIH Image J software v 1.51 (https://imagej.nih.gov/ij/download.html). Full-length blots/gels are presented in Supplementary Figure 3.