Phosphorylation of MAP4 (p-MAP4) causes cardiac remodeling, with the cardiac microvascular endothelium being considered a vital mediator of this process. In the current study, we investigated the mechanism underlying p-MAP4 influences on cardiac microvascular density. We firstly confirmed elevated MAP4 phosphorylation in the myocardium of MAP4 knock-in (KI) mice. When compared with the corresponding control group, we detected the decreased expression of CD31, CD34, VEGFA, VEGFR2, ANG2, and TIE2 in the myocardium of MAP4 KI mice, accompanied by a reduced plasma concentration of VEGF. Moreover, we observed apoptosis and mitochondrial disruption in the cardiac microvascular endothelium of MAP4 KI animals. Consistently, we noted a decreased cardiac microvascular density, measured by CD31 and lectin staining, in MAP4 KI mice. To explore the underlying mechanism, we targeted the NLRP3-related pyroptosis and found increased expression of the corresponding proteins, including NLRP3, ASC, mature IL-1β, IL-18, and GSDMD-N in the myocardium of MAP4 KI mice.
Fig: Angiogenic signaling pathways are inhibited in the myocardium of MAP4 KI mice: A Representative bands of western blotting for detecting p-MAP4 in the myocardium; n = 5. B, C The statistical analysis of p-MAP4, and MAP4. ***p < 0.001 versus the Con group. N.S. represents no statistical difference between the Con and MAP4 KI groups; n = 5. D Representative immunoblotting bands of the levels of CD31, CD34, VEGFA, VEGFR2, ANG2, and TIE2 in the myocardium; n = 5. E–J The statistical analysis of CD31, CD34, VEGFA, VEGFR2, ANG2, and TIE2. ***p < 0.001 versus the Con-young group; ##p < 0.01 and ###p < 0.001 versus the Con-aged group; n = 5. K A scatter diagram showing the concentration of VEGF in the plasma. N.S. represents no statistical difference between the Con-young and MAP4 KI-young groups. ##p < 0.01 versus the Con-aged group; n = 8.L Representative TEM images of endothelial cells in the myocardium. Yellow arrows point to disrupted mitochondria. Scale bar, 0.5 µm; n = 3.
Furthermore, we utilized a MAP4 (Glu) adenovirus to mimic cellular p-MAP4. After incubating HUVECs with MAP4 (Glu) adenovirus, the angiogenic ability was inhibited, and NLRP3-related pyroptosis were significantly activated. Moreover, both cytotoxicity and PI signal were upregulated by the MAP4 (Glu) adenovirus. Finally, NLRP3 inflammasome blockage alleviated the inhibited angiogenic ability induced by MAP4 (Glu) adenovirus. These results demonstrated that p-MAP4 reduced cardiac microvascular density by activating NLRP3-related pyroptosis in both young and aged mice. We thus managed to provide clues explaining MAP4 phosphorylation-induced cardiac remodeling and enriched current knowledge regarding the role of MAP4.
Feng, Yh., Li, Lf., Zhang, Q. et al. Microtubule associated protein 4 (MAP4) phosphorylation reduces cardiac microvascular density through NLRP3-related pyroptosis. Cell Death Discov. 7, 213 (2021). https://doi.org/10.1038/s41420-021-00606-w