Doxorubicin-induced cardiomyopathy associated with inhibition of autophagic degradation process and defects in mitochondrial respiration

Abstract

Doxorubicin (Dox) is a highly effective anticancer drug but cause acute ventricular dysfunction, and also induce late-onset cardiomyopathy and heart failure. Despite extensive studies, the pathogenic sequelae leading to the progression of Dox-associated cardiomyopathy remains unknown. We assessed temporal changes in autophagy, mitochondrial dynamics, and bioenergetics in mouse models of acute and chronic Dox-cardiomyopathy. Time course study of acute Dox-treatment showed accumulation of LC3B II in heart lysates. Autophagy flux assays confirmed that the Dox-induced accumulation of autophagosomes occurs due to blockage of the lysosomal degradation process. Dox-induced autophagosomes and autolysosome accumulation were confirmed in vivo by using GFP-LC3 and mRFP-GFP-LC3 transgenic (Tg) mice. Mitochondria isolated from acute Dox-treated hearts showed significant suppression of oxygen consumption rate (OCR). Chronic Dox-cardiotoxicity also exhibited time-dependent accumulation of LC3B II levels and increased accumulation of green puncta in GFP-LC3 Tg hearts. Mitochondria isolated from chronic Dox-treated hearts also showed significant suppression of mitochondrial OCR. The in vivo impairment of autophagic degradation process and mitochondrial dysfunction data were confirmed in vitro using cultured neonatal cardiomyocytes. Both acute and chronic Dox-associated cardiomyopathy involves a multifocal disease process resulting from autophagosomes and autolysosomes accumulation, altered expression of mitochondrial dynamics and oxidative phosphorylation regulatory proteins, and mitochondrial respiratory dysfunction.

Figure 1

Cardiac function and survival in acute Dox cardiomyopathy mice. (A) Schematic of acute Dox administration protocol. FVB/N mice of 8 to 10-weeks of age were treated with a single dose of Dox (20 mg/kg) and vehicle by i.p. injections. (B) Kaplan Meier survival curve showing significant mortality in mice after acute Dox (n = 22) treatment compared to Vehicle (n = 10) treated mice. M mode echocardiography was used to examine cardiac function before as well as 3 and 5 days after Dox- and vehicle-injection. (C) LV systolic internal dimension (LVID; s). (D) LV diastolic internal dimension (LVID; d). (E) Percentage fractional shortening (%FS). (F) LV systolic volume (LV Vol; s). (G) LV diastolic volume (LV Vol; d). (H) Percentage ejection fraction (%EF). (I) LV diastolic posterior wall thickness (LVPW; d). (J) LV diastolic interventricular septum thickness (IVS; d). (K) LV mass. Data represent mean ± SEM. n = 9 mice for Dox-treatment group and n = 5 mice for vehicle treatment group. P value versus vehicle-treated mice by Tukey’s post hoc test. NS, not significant.

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.

Figure 3

Inhibition of autophagic degradation process in the hearts of acute Dox-cardiomyopathy mice. (A) Representative fluorescence images of heart tissue sections from tf-LC3 Tg mice 5 days after acute Dox as well as saline administered heart treated with chloroquine (CQ) or vehicle. Autophagic flux assay showed an accumulation of autophagosomes (yellow puncta), autolysosomes (red puncta) and defective autophagosome/autolysosome fusion (red and yellow pucta coming together) in acute Dox-treated hearts (indicated by white arrow). (B) Quantification of puncta numbers in the heart showing accumulation of autophagosomes and autolysosome in the acute Dox-cardiomyopathy heart at 5 days after Dox-treatment with or without CQ treatment. n = 4–5 hearts per group with 10 microscopic fields (2.2 × 10⁵ μm²) per heart section analyzed. Scale bar, 10 μm. Bars represent mean ± SEM. P value versus vehicle-treated mice by Tukey’s post hoc test. NS = not significant.

Figure 4

Expression of mitochondrial dynamics and OXPHOS regulatory protein in the hearts of acute Dox-cardiomyopathy mice. (A) Representative Western blot of the whole cell fraction showing expression of mitochondrial dynamic regulatory proteins in the acute Dox-treated hearts: Drp1, OPA1, and MFN2. Ponceau S protein staining of the transfer membrane confirmed approximately equal loading across the gel. (B) Representative Western blot showing expression of Complex I, Complex II, Complex III, Complex V and PDH complex protein derived from the whole cell fraction isolated from acute Dox-treated hearts at different time points. Ponceau S protein stain of the transfer membrane was used to confirm approximately equal loading. (C) Densitometric quantification of the temporal changes in Drp1, OPA1, and MFN2 protein expression in Dox-treated hearts. (D) Densitometric quantification of OXPHOS complex and PDH complex protein. Bars represent mean ± SEM. n = 4 mice per group at each time point. P values were determined by Tukey’s post-hoc test. NS = not significant.

Figure 5

Suppression of mitochondrial respiration in the hearts of acute Dox-cardiomyopathy mice. (A) Mitochondrial oxygen consumption rate (OCR) profiles in isolated mitochondria from 3 days after acute Dox-treated hearts. Arrow indicates the sequential addition of oligomycin (1 µM), FCCP (4 µM), and rotenone (0.5 µM) plus antimycin A (0.5 µM). OCR profile is expressed as pMolesO₂/min/µg of protein. Graph showing OCR under (B) baseline as well as with the addition of (C) oligomycin, (D) FCCP, and (E) rotenone plus antimycin A. Key parameters of mitochondrial function, including (F) ATP turnover, (G) maximal respiration, (H) state apparent and (I) coupling efficiency were significantly decreased in Dox mice. Bars represent mean ± SEM. n = 4–6 mice per group. P values were determined by Tukey’s post-hoc test.

Figure 6

Cardiac function and survival in chronic Dox-associated cardiomyopathy mice. (A) Schematic of chronic Dox administration protocol. FVB/N mice of 8 to 10-weeks of age were subjected to four serial Dox (5 mg/kg) injections weekly by i.p. (B) Kaplan Meier survival curve showing mortality in mice after chronic Dox (n = 13) treatment compared to Vehicle (n = 10) treated mice. M mode echocardiography was used to examine cardiac functions before as well as 4, 8 and 12 weeks after Dox and vehicle-injection. (C) LV systolic internal dimension (LVID; s). (D) LV diastolic internal dimension (LVID; d). (E) Percentage fractional shortening (%FS). (F) LV systolic volume (LV Vol; s). (G) LV diastolic volume (LV Vol; d). (H) Percentage ejection fraction (%EF). (I) LV diastolic posterior wall thickness (LVPW; d). (J) LV diastolic interventricular septum thickness (IVS; d). (K) LV mass. n = 15 mice for Dox-treatment group and n = 5 mice for vehicle treatment group. Bars represent mean ± SEM. P value versus vehicle-treated mice by Tukey’s post hoc test. NS, not significant.

Figure 7

Accumulation of autophagosomes and autolysosomes in the hearts of chronic Dox-cardiomyopathy mice. (A) Representative Western blot and densitometric quantification showing temporal changes in LC3B, Cathepsin D and Lamp 2a protein levels in the heart of 4, 6, 8 and 12 weeks after Dox- and vehicle-treatment. Ponceau S protein stain of the transfer membrane was used to confirm approximately equal loading. n = 4 hearts per group on each time point. Bars represent mean ± SEM. (B) Representative fluorescence images of heart tissue sections from GFP-LC3 Tg mice 1, 4, 8, and 12 weeks after 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 chronic Dox-cardiomyopathy heart 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.

Figure 8

Altered expression of mitochondrial dynamics and OXPHOS regulatory protein in the hearts of chronic Dox-cardiomyopathy mice. (A) Representative Western blot of the whole cell fraction showing expression of mitochondrial dynamic regulatory proteins in the acute Dox-treated hearts: Drp1, OPA1, and MFN2. Ponceau S protein staining of the transfer membrane confirmed approximately equal loading across the gel. (B) Representative Western blot showing expression of Complex I, Complex II, Complex III, Complex V and PDH complex protein derived from the whole cell fraction isolated from acute Dox-treated hearts at different time points. Ponceau S protein stain of the transfer membrane was used to confirm approximately equal loading. (C) Densitometric quantification of the temporal changes in Drp1, OPA1, and MFN2 protein expression in Dox-treated hearts. (D) Densitometric quantification of OXPHOS complex and PDH complex protein. Bars represent mean ± SEM. n = 4 mice per group at each time point. P values were determined by Tukey’s post-hoc test. NS = not significant.

Figure 9

Suppression of mitochondrial respiration in the hearts of chronic Dox-cardiomyopathy mice. (A) Mitochondrial oxygen consumption rate (OCR) profiles in isolated mitochondria from 12 weeks after Dox- and vehicle-treated hearts. Arrow indicates the sequential addition of oligomycin (1 µM), FCCP (4 µM), and rotenone (0.5 µM) plus antimycin A (0.5 µM). OCR profile are expressed as pMolesO₂/min/µg of protein. Graph showing OCR under (B) baseline as well as with the addition of (C) oligomycin, (D) FCCP, and (E) rotenone plus antimycin A. Key parameters of mitochondrial function, including (F) ATP turnover, (G) state apparent, (H) respiratory control ratio and, (I) coupling efficiency were significantly decreased in Dox mice. Bars represent mean ± SEM. n = 6 mice per group. P values were determined by Tukey’s post-hoc test.

Figure 10

Inhibition of autophagic flux and suppression of mitochondrial respiration by Dox-treatment in cultured cardiomyocytes. (A) Representative Western blot and densitometric quantification showing LC3B II expression at different doses of Dox (1–25 μM, 24 hours) in NRCs. GAPDH was used as a loading control. n = 6 independent experiments. (B) Inhibition of autophagic flux by 10 μM Dox (24 hours) in NRC examined by immunoblotting of LC3B II. Bafilomycin A1 (50 μg/mL) was added for 4 hours to block lysosomal degradation, and GAPDH was used as a loading control. n = 6 independent experiments. Bars represent mean ± SEM. (C) Dox treatment (1–10 μM Dox, 24 hours) dose-dependently suppress mitochondrial oxygen consumption rate (OCR) profiles in NRCs. Arrow indicates the sequential addition of oligomycin (1 µM), FCCP (4 µM), and rotenone (0.5 µM) plus antimycin A (0.5 µM). OCR profile are expressed as pMolesO₂/min/µg of protein. Graph showing OCR under baseline as well as with the addition of oligomycin, FCCP, and rotenone plus antimycin A. Key parameters of the mitochondrial function, including basal and maximal respiration were dose-dependently decreased in Dox-treated NRCs. Bars represent mean ± SEM. n = 5 wells per group. P values were determined by Tukey’s post-hoc test.

Figure 11

Altered mitochondrial morphology by Dox-treatment in cultured cardiomyocytes. (A) Representative images showing MitoTracker Green staining of mitochondria in NRCs treated with 1 μM and 10 μM Dox for 24 hours. Scale bar, 10 μm. Quantitative data for (B) mitochondrial length, (C) mitochondrial size distribution frequency and (D) mitochondrial aspect ratio. Bars represent mean ± SEM. P values were determined by Tukey’s post-hoc test.