Hypoxia impairs autophagy of cardiomyocytes via p38/MAPK/MAP4 pathway

Abstract

Background: Myocardial hypoxia occurs in severe burns and may cause severe cardiac dysfunction, in which the blockage of the autophagy flux plays an important role. Previous studies indicates that the p38/MAPK pathway is involved in regulating the microtubule structure by regulating MAP4 phosphorylation, and the microtubule structure affects the autophagy. However, as a complex degradation process, how autophagy is specifically affected by microtubules remains unknown. An in-depth understanding of hypoxia-related autophagy disorders is critical for the treatment of myocardial injury.

Methods: Cardiomyocytes (CMs) were isolated from the ventricles of neonatal Sprague-Dawley rats and cultured in an incubator filled with 1 % O₂, 5 % CO₂, and 94 % N₂. SB203580 and MKK6 (Glu) recombinant adenovirus were used to specifically inhibit and activate the p38/MAPK pathway, respectively. The adeno-associated viruses (AAVs) encoding MAP4 gene and MAP4 siRNA were used to up-regulate and down-regulate the expression of MAP4, respectively. After infection of cells with AAV encoding GFP-LC3 fusion proteins, the number of green spots under fluorescence microscopy shows the quantity of autophagosomes. Western blots access the expression of LC3-II, LC3-I and p62. The ratio of LC3-II to LC3-I (LC3-II/I) tells the quantity of autophagosomes, and the expression of p62 indicates the extent of autophagosome degradation. Cell Counting Kit 8 was used to detect cell viability. Rapamycin was used to recover the autophagy.

Results: Hypoxia reduced the viability of cardiomyocytes, in which the quantity of autophagosomes is increased, while the degradation is reduced, and the p38/MAPK pathway is activated. Activation of the p38/MAPK pathway could block the autophagy pathway. The phosphorylation of MAP4 did not affect the quantity of autophagosomes, but hindered its degradation. The p38/MAPK pathway could regulate the phosphorylation of MAP4. Finally, when the autophagy pathway was restored, cell viability has partially recovered.

Conclusions: Hypoxia regulates the phosphorylation of MAP4 through the p38/MAPK pathway, thereby hindering the degradation of autophagosomes, rather than the quantity, blocking autophagic flux and ultimately affecting cell viability.

Keywords: Autophagy; Cardiomyocytes; Hypoxia; Microtubule-associated protein.