Figure 2
Accumulation of autophagosomes and autolysosomes in the hearts of acute Dox-cardiomyopathy mice. (A) Representative Western blot and densitometric quantification showing temporal changes in LC3B II protein levels in the heart after acute Dox (20 mg/kg, i.p.) and vehicle-treatment. β-Actin was used as a loading control. n = 4 mice per group on each time point. (B) Autophagic flux assay showed accumulation of autophagosomes resulting from impaired autophagic degradation in acute Dox hearts. GRP75 was used as a loading control. n = 4–6 mice per group. Bars represent mean ± SEM. P value versus vehicle-treated mice by Tukey’s post hoc test. NS = not significant. (C) Representative fluorescence images of heart tissue sections from GFP-LC3 Tg mice 1 and 5 days after acute Dox- and vehicle treatment. Quantification of GFP-LC3 puncta/microscopic field in hearts from male GFP-LC3 Tg mouse showing accumulation of autophagosomes in the acute Dox-cardiomyopathy heart at 5 days after Dox-treatment. n = 4–5 hearts per group with 10 microscopic fields (2.2 × 105 μm2) per heart section analyzed. Scale bar, 20 μm. (D) Representative fluorescence images of heart tissue sections from tf-LC3 Tg mice 1 and 5 days after acute Dox- and vehicle treatment. Quantification of puncta numbers in the heart showing accumulation of autophagosomes (yellow puncta) and autolysosome (red puncta) in the acute Dox-cardiomyopathy heart at 5 days after Dox-treatment. n = 4–5 hearts per group with 10 microscopic fields (2.2 × 105 μm2) per heart section analyzed. Scale bar, 20 μm. Bars represent mean ± SEM. P value versus vehicle-treated mice by Tukey’s post hoc test. NS = not significant.
