Objective To investigate influence of nicotinic acid adenine dinucleotide phosphate (NAADP) on autophagy in hypoxic cardiomyocytes of rats and its mechanism.
Methods Five neonatal Sprague-Dawley rats were collected and sacrificed to harvest the hearts, and primary cardiomyocytes were separated for the following experiments. (1) Primary cardiomyocytes were collected and divided into normoxia group, hypoxia 9 h group, and hypoxia 9 h+ NAADP group according to random number table, with 5 wells in each group. Cells in normoxia group were cultured routinely in the constant temperature incubator at 37 ℃ for 9 hours. Cells in hypoxia 9 h group and hypoxia 9 h+ NAADP group were cultured in hypoxic incubator with volume fraction 94% nitrogen, 5% carbon dioxide, and 1% oxygen for 9 hours. Before hypoxia, cells in hypoxia 9 h+ NAADP group were dealt with final amount-of-substance concentration 10 μmol/L NAADP. Cell counting kit 8 was used to measure cell viability. (2) Primary cardiomyocytes were collected and divided into normoxia group, hypoxia 9 h group, hypoxia 9 h+ NAADP group, hypoxia 9 h+ tran-Ned-19 group, and hypoxia 9 h+ trans-Ned-19+ NAADP group according to the random number table, with 2 wells in each group. Cells in normoxia group were cultured routinely in the constant temperature incubator at 37 ℃ for 9 hours. And cells in the other 4 groups were cultured in hypoxic incubator as that in experiment (1) Before hypoxia, cells in hypoxia 9 h+ NAADP group were dealt with amount-of-substance concentration 10 μmol/L NAADP, cells in hypoxia 9 h+ tran-Ned-19 group were dealt with amount-of-substance concentration 1 μmol/L trans-Ned-19, and cells in hypoxia 9 h+ trans-Ned-19 + NAADP group were dealt with amount-of-substance concentration 10 μmol/L NAADP and 1 μmol/L trans-Ned-19. Protein expressions of microtubule associated protein 1 light chain 3-Ⅱ and P62 were detected by Western blotting. (3) Primary cardiomyocytes were collected and grouped as those in experiment (1). The lysosomal acidity was determined by immunofluorescence method. Data were processed with one-way analysis of variance and least-significant difference test.
Results (1) The cell viability in normoxia group was 1.114±0.024, which was significantly higher than 0.685±0.079 of cells in hypoxia 9 h group (
P<0.01). The cell viability of hypoxia 9 h+ NAADP group was 0.886±0.061, which was obviously higher than that of cells in hypoxia 9 h group (
P<0.05). (2) Expressions of microtubule-associated protein 1 light chain 3-Ⅱ and P62 of cells in hypoxia 9 h group were significantly higher than those of cells in normoxia group (
P<0.01). Compared with those in hypoxia 9 h group, expression of P62 in hypoxia 9 h+ NAADP group was significantly decreased (
P<0.01), while expression of microtubule-associated protein 1 light chain 3-Ⅱ did not change significantly (
P>0.05). There were no significantly statistical difference in expressions of microtubule-associated protein 1 light chain 3-Ⅱ and P62 between hypoxia 9 h group and hypoxia 9 h+ trans-Ned-19 group (
P>0.05). Compared with those of cells in hypoxia 9 h+ NAADP group, expression of P62 of cells in hypoxia 9 h+ trans-Ned-19+ NAADP group was obviously increased (
P<0.01), while expression of microtubule-associated protein 1 light chain 3-Ⅱ did not change significantly (
P>0.05). (3) The intensity of green fluorescence of cells in normoxia group was strong and co-localized well with red fluorescence, and internal environment of lysosome was with stronger acidity. The intensity of green fluorescence in cells of hypoxia 9 h group was significantly lower than that of cells in normoxia group, and acidity of internal environment of lysosome was weakened. The intensity of green fluorescence and acidity of internal environment of lysosome in hypoxia 9 h+ NAADP were significantly stronger than those of cells in hypoxia 9 h group, but significantly lower than those of cells in normoxia group.
Conclusions NAADP can improve myocardial cell viability through acidifying internal environment of lysosome of cardiomyocyte after hypoxia, promoting degradation of autophagosomes, reducing autophagic lysosomal accumulation, and repairing damaged autophagic flow.
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