Hyperglycemia is an independent risk factor for diabetic cardiac injury.
21-24 To determine whether HG affected mitophagy, we infected NRVCs with Ad-mt-Rosella and compared mitophagy events (red fluorescent dots or puncta on the merged confocal images) in cells cultured under HG (30 mM) and normal glucose (NG; 5.5 mM) conditions. As shown in
Figure 2, HG increased the number of red dots compared with NG, suggesting that HG might have enhanced mitophagy. However, mitophagy is a dynamic process, and its functional status cannot be determined simply by a snapshot of the number of mitochondrial fragments (red dots) present in the lysosome. Instead, mitophagy flux must be determined in the absence and presence of lysosomal degradation inhibitors to reveal the true dynamic changes of the mitophagic events or red dots during the whole degradation process. Thus, we treated cells with pepstatin A (PepA) and E64d, 2 lysosomal protease inhibitors that block lysosomal degradation, the last step of mitophagy. If a treatment enhances mitophagy, then a blockage of lysosomal degradation would cause further accumulation of the red dots on the merged confocal images. As shown in
Figure 2, compared with DMSO control, PepA/E64d treatment led to a significant increase in the number of red dots in cells cultured in 5.5 mM of glucose, indicating an active mitophagy flux under an NG condition. However, PepA and E64d only resulted in a small increase in the number of red dots in cells cultured in 30 mM of glucose (HG, mean [SD], 9.2 [7.7] vs NG, 57.6 [20.3];
P<.01), suggesting that HG reduced the mitochondria degradation rate in the lysosome. In other words, HG inhibited mitophagy flux. This effect was not caused by increased osmolarity, since 24.5 mM of mannitol was added to the culture media containing 5.5 mM of glucose.