Monoamine Oxidase B (MAO-B) as an Inducer of Mitochondrial Reactive Oxygen Species (ROS) Production and Myofibroblast Differentiation in Cardiac Fibroblasts of Mice

Figures

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  Figure 1. Characterization of isolated cells from collagenase-digested mouse hearts. After isolation and cultivation of cardiomyocytes (CMs) and fibroblasts (FBs) from control mice, purity of cultures was verified in (A) real-time RT-PCR using specific primers for Myh6 as a cardiomyocyte marker, Pecam1 as an endothelial marker, and Col1a2 and PdgfRA as fibroblast markers. Normalization of relative expression was done with bestkeeper using B2M, HPRT, GAPDH and 18SrRNA as housekeeping genes and expressed as percent of their expression in whole heart tissues (indicated by the dashed line). (n = 6 for whole hearts and n = 11–15 for CMs or FBs, * p ≤ 0.05 vs. whole heart expression.) (B) Immunofluorescence of fibroblast cultures (magnification 20×) was performed using antibodies against the fibroblast-specific marker protein DDR2 and TRITC-labeled secondary antibodies. DAPI was used for nuclear staining.

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  Figure 2. Reduction in MAO-B expression in fibroblasts after MAO-B knockdown. After induction of MAO-B KD via tamoxifen injections, fibroblasts were isolated. MAO-B expression was analyzed in real-time RT-PCR (7–11, # p ≤ 0.05 vs. C). MAO-B (n = 9–12, # p ≤ 0.05 vs. C) and MAO-A (n = 5–6) were analyzed in Western blots.

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  Figure 3. ROS production of isolated cardiac mitochondria of MAO-B knockdown mice. Mitochondria of control and MAO-B KD mice hearts were isolated and the ROS production was measured in the presence of complex I substrate and after the addition of PEA (250 µM) and the inhibitor selegiline (Sel, 1 µM) or clorgyline (Clg, 1 µM) (C, n = 7; KD, n = 9, * p ≤ 0.05).

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  Figure 4. Reduced mitochondrial ROS production in MAO-B KD fibroblasts. Fibroblasts were incubated with 1 µM MitoSOX™ Red, followed by PEA stimulation for 10 min. Then, fluorescence images were taken and quantified. (n = 11–16, * p ≤ 0.05 vs. unstimulated control, # p ≤ 0.05 vs. KD, 1000 µM PEA.).

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  Figure 5. Myofibroblast marker genes are not increased under PEA stimulation in MAO-B KD fibroblasts. Fibroblasts were stimulated with PEA. (A) After 24 h, RNA was isolated and real-time RT-PCR was performed for detection of Col1a1 and periostin mRNA expression. Normalization of relative expression was done with bestkeeper using actin, HPRT, GAPDH and 18SrRNA as housekeeping genes. (n = 7–12, * p ≤ 0.05 vs. unstimulated control, # p ≤ 0.05 vs. KD, 1000 µM PEA.) (B) After 48 h, fibroblasts were isolated and Col1a1 protein expression was analyzed in Western blots. GAPDH was used as a loading control (n = 6, p ≤ 0.05 vs. 0 PEA).

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  Figure 6. Reduced migration potential of MAO-B KD fibroblasts. In a confluent monolayer of fibroblasts, a scratch was made and first pictures were taken (0 h). Then, fibroblasts were stimulated with PEA (100 and 1000 µM). Pictures were taken at the same location before and 24 and 48 h after stimulation. The area of migrating cells into the scratch area was related to the initial scratch area. (n = 6–9, * p ≤ 0.05 vs. time point 0 h, # p ≤ 0.05 vs. related 48 h control value).