FIGURE 3.

Autophagic flux is increased in starved cardiomyocytes. Cardiomyocytes infected with Ad-GFP-LC3 adenovirus were analyzed for induction of autophagy in fed and starved conditions. Autophagic flux in cardiomyocytes was determined in the absence or presence of lysosomal inhibitors. A, fed cardiomyocytes show diffuse, uniform expression of GFP-LC3 with no detectable LC3 puncta. B, starvation results in increased GFP-LC3 puncta in cardiomyocytes. Increased GFP-LC3 puncta were observed in the presence of lysosomal inhibitors both fed (D) and starved (E) cardiomyocytes compared with the absence of inhibitors (A and B). C, number of AVs/cell was significantly increased in starved cardiomyocytes compared with fed cardiomyocytes and also in presence of inhibitors compared with their respective controls in both conditions. F, significant increase in autophagic flux was observed in starved compared with fed cardiomyocytes. GFP-LC3 puncta are indicated by white arrows in B, D, and E. G–I, cardiomyocytes were incubated in fed and starved media either in the presence or in the absence of the specific autophagy inhibitor 3MA (10 μm) for 24 h. Cardiomyocytes are immunostained with sarcomeric α-actinin (cardiac muscle actinin, green) and cell size was measured. Inhibition of autophagy with 3MA completely rescues the starvation-induced cell size reduction. Significance (*) was determined by one-way analysis of variance (ANOVA) (p < 0.05; n = 3).