The mitochondrial protein MAO-B has recently been shown to contribute to ischemia-reperfusion injury in cardiomyocyte-specific MAO-B knockout mice. Since in those mice MAO-B-dependent ROS production was decreased but not abolished, it can be assumed that, in addition to cardiomyocytes, another cell type, i.e. fibroblasts, contributes to MAO-B-dependent ROS production. We therefore investigated whether stimulation of MAO-B in cardiac fibroblasts induces ROS production and myofibroblast development, and whether these responses are absent in fibroblast-specific MAO-B knockout cells.
To investigate the impact of MAO-B in cardiac fibroblasts we have crossed Col1a2CreERT mice with MAO-Bfl/fl mice. MAO-B knockout (KO) was induced by tamoxifen injection. Then, fibroblasts of KO mice and MAO-Bfl/fl mice as controls (WT) were isolated from mice hearts via collagenase digestion and selective adhesion to the culture dishes. The purity of the cultures was determined by the detection of fibroblast-specific marker genes. While the cardiomyocyte marker gene MYH6 and the endothelial marker gene PECAM1 were not detected by real-time RT-PCR, the cultures showed an enrichment of COL1A2 and PDGFRA expression as fibroblast markers. Furthermore, reduction of MAO-B in fibroblasts from KO mice was confirmed in RT-PCR (0.3-fold mRNA expression vs. 1.1-fold in WT, p<0.05) and western blots (MAO-B levels reduced to 55 ± 28 %, p<0.05 vs. WT). In isolated mitochondria from the left ventricle of these mice, ROS production, measured with Amplex Red, was significantly reduced under stimulation with the specific MAO-B substrate β-phenylethylamine (PEA) compared to WTs. Also, mitochondrial ROS production in cells, detected by MitoSox Red staining, increased within 10 minutes under PEA (1000 µM) stimulation only in WT cells (584-fold, n=14, p<0.05 vs. unstimulated control and stimulated MAO-B KO). Furthermore, mRNA of the marker gene for myofibroblast differentiation, COL1A1 , increased 2-fold in WT but not in MAO-B KO fibroblasts 24 h after PEA stimulation (p<0.05 vs. unstimulated controls). To analyse, if MAO-B influences the behaviour of fibroblasts, migration assays were performed. Under PEA stimulation 57 ± 12 % WT-fibroblasts migrated into the scratch within 48 h, compared to about 39 % in MAO-B KO-cells or unstimulated controls (n=9, p<0.05).
In conclusion, stimulation of MAO-B in cardiac fibroblasts leads to the formation of mitochondrial ROS, enhancement of myofibroblast marker gene expression and migration of the cells. Enhanced migration indicates an increased invasiveness of fibroblasts, which is often associated with pro-fibrotic processes. Excessive fibrosis caused by elevated MAO-B activity in myocardial infarction can therefore contribute to cardiac dysfunction.
