Using cultured canine cardiac slices to model the autophagic flux with doxorubicin

Figures

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  Fig 1. Canine cardiac slices are viable for >7 days in culture. A. No significant reduction in adult canine cardiac slice viability through 14 days of culturing (arrow) by MTT staining was noted. N = 7 hearts. B. H&E staining demonstrated minimal myofibrillar damage during Day 1 (Billingham grade 0), 3 (Billingham grade 0), 5 (Billingham grade 1), and 7 (Billingham grade 1) of culture, and TUNEL staining showed minimal TUNEL+ apoptotic cells. Red arrows: TUNEL+ nuclei. C. Quantification shows a low baseline of TUNEL+ nuclei which did not increase during 7 days in culture (n = 3 per condition). D. Adult mouse cardiac slices had much lower viability when cultured in the same canine media (CM) compared to the Claycomb media (MTT purple staining indicates viable tissue), E. in which tissues showed no significant decrease in viability within the 7 days of culture by MTT staining. F. However, TUNEL staining showed significant increase in apoptotic cells by day 3 of culture. N = 4, ANOVA with Dunnett’s post-test. *p<0.05, **p<0.01, ns = no significant change.

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  Fig 2. Basal autophagy is preserved in cultured canine cardiac slices. A-D. Representative immunoblots for cardiac slices cultured for 1–7 days. Quantification showed no change in expression of autophagy proteins LC3-II and p62. E-H. Autophagy was induced by 0.1 μM rapamycin (Rap) or blocked by 20 μM chloroquine (Cq) for 24 hours. Rapamycin significantly increased LC3-II expression but not p62 compared to vehicle (Veh) control. Chloroquine led to the accumulation of LC3-II and p62. Protein quantifications were normalized to Actin. N = 3, ***p<0.001, ****p<0.0001, ns = no significant change, ANOVA with Tukey post-test.

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  Fig 3. Doxorubicin induces cardiac tissue damage, apoptotic cell death, and impairs autophagy. Canine cardiac slices were exposed to increasing concentrations of doxorubicin (Dox, 10–50 μM) for 24 hours. A. Representative H&E and TUNEL stained sections exposed to 0–50 μM of doxorubicin. 0 μM: Bellingham grade 0; 10 μM: Bellingham grade 2; 30 and 50 μM: Bellingham grade 3. Blue arrows: disorganized myocyte fibers. Red arrows: TUNEL+ nuclei. B. Quantification of doxorubicin retention in cardiac slices by direct fluorescence tissue imaging. SNR = signal-to-noise ratio. N = 4. C. Quantification of TUNEL+ nuclei in cardiac slices exposed to 0, 10, 30, or 50 μM of doxorubicin for 24 hours (n = 4). D-G. Representative western blots and quantification of autophagy proteins (LC3-II, p62) in slices treated with increasing concentrations of doxorubicin for 24 hours. N = 3. H. Immunostaining of LC3 demonstrated robustly increased LC3 accumulation (green = LC3, blue = DAPI) in cardiomyocytes of slices exposed to doxorubicin. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to untreated controls unless otherwise noted, ANOVA with Tukey post-test.

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  Fig 4. Autophagy induction by rapamycin significantly attenuated cardiomyocyte apoptotic cell death. Cardiac slices were exposed to 50 μM doxorubicin with or without rapamycin (Rap) co-treatment. A-D. Representative immunoblots and quantification of LC3-II and p62. E. Expression increases of LC3-II and p62 with doxorubicin were mitigated with rapamycin co-treatment for 24 hours, leading to cardiomyocyte apoptosis reduction. N = 3. F. The effects of doxorubicin, rapamycin, and chloroquine treatment on LC3-II and p62 expression, and the overall effect on autophagic flux and apoptotic cell death are summarized. **p<0.01, ***p<0.001, ****p<0.0001, ANOVA with Tukey post-test.