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

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.