Peroxiredoxin Ⅲ safeguards cardiac function against doxorubicin by regulating mitochondrial quality control via H2O2 detoxification

Figures

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  Fig. 1. Doxorubicin increases mitochondrial H2O2 levels and causes cell death in rat cardiomyocytes. (A) H9c2 cells were treated with the indicated concentrations of DOX for 24 h. Cell death was assessed by Annexin V-FITC staining and analyzed using flow cytometer. Representative histograms of three independent experiments are shown. (B) H9c2 cells were treated with 1 μM DOX for the indicated times. Cell viability was measured using WST-1 reagent. (C-D) H9c2 cells were treated with 1 μM DOX for the indicated times and then stained with 5 μM CM-H2DCFDA (C) or 5 μM MitoPY-1 (D) for 15 min. Relative fluorescence intensity (RFI) was measured by flow cytometer. All data are presented as mean ± S.D. (n = 3). Statistical significance was determined by one-way ANOVA followed by Dunnett's post hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to untreated control.

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  Fig. 2. PrxⅢ regulates mitochondrial H2O2 accumulation and oxidative damage in DOX-treated cardiomyocytes. (A) H9c2 cells stably expressing either control (pSUPER) or PrxⅢ-targeting siRNA (pSUPER-siPrxⅢ) were transduced with Ad-PrxⅢ or Ad-Stuffer for 24 h and then exposed to 1 μM DOX for 9 h. Mitochondrial H2O2 levels were assessed using the mitochondria-targeted fluorescent probe MitoPY-1 and visualized by green fluorescence. Scale bar, 75 μm. Five randomly selected microscopic fields per sample were analyzed and combined to represent one biological replicate. (B) Cells were transduced with Ad-PrxⅢ or Ad-Stuffer for 24 h and then exposed to 1 μM DOX for 18 h. FOXO3a localization was assessed by immunofluorescence using anti-FOXO3a (green) antibody and DAPI (blue). Scale bar, 25 μm Five randomly selected microscopic fields per sample were analyzed and combined to represent one biological replicate. All data are expressed as mean ± S.D. Statistical significance was determined using two-way ANOVA followed by Bonferroni's post hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and ns, p > 0.05.

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  Fig. 3. PrxⅢ regulates oxidative damage and mitochondrial bioenergetics in DOX-treated cardiomyocytes. (A) Cells were transduced with Ad-PrxⅢ or Ad-Stuffer for 24 h and then exposed to 1 μM DOX for 12 h. Cells were labeled with 5 μM 10-NAO and relative fluorescence intensity (RFI) was analyzed by flow cytometer. Representative histograms and quantification are shown. (B) Cells were treated with 1 μM DOX for 12 h and labeled with 10 μM Rho-123 to assess mitochondrial membrane potential (ΔΨm). The percentage of cells with low ΔΨm was quantified by flow cytometry. Representative histograms and quantification are shown. (C) Cells were transduced with Ad-PrxⅢ or Ad-Stuffer for 24 h and then exposed to 1 μM DOX for 12 h. Representative bar graph showing relative ATP concentration normalized to protein contents. (D-H) Cells were transduced with Ad-PrxⅢ or Ad-Stuffer for 24 h and then exposed to 1 μM DOX for 12 h. (D) Representative tracing of the oxygen consumption rate (OCR). Arrows indicate time points when cells were treated with oligomycin, FCCP, and rotenone plus antimycin A (Rot/AA), respectively. The OCR is calculated as (E) basal respiration, (F) maximal respiration, (G) spare…

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  Fig. 4. PrxⅢ regulates mitochondrial biogenesis in DOX-treated cardiomyocytes. (A) Cells were transduced with Ad-PrxⅢ or Ad-Stuffer for 24 h and then exposed to 1 μM DOX for 6 h. Total RNA was isolated and the mRNA levels of PGC-1α, Nrf1, and mtTFA were determined by qRT-PCR. Representative bar graphs showing relative expression normalized to GAPDH are presented. (B) Relative mRNA expression of the mitochondrial DNA-encoded cytochrome c oxidase (COX) mRNA was measured as an indicator of mtDNA copy-dependent transcription. Representative bar graphs showing relative expression normalized to GAPDH are presented. (C) Cells were transduced with Ad-PrxⅢ or Ad-Stuffer for 24 h and then exposed to 1 μM DOX for 18 h. Representative immunoblots and quantitative analyses of PGC-1α, Nrf1, and mtTFA proteins. GAPDH and α-tubulin were used as loading controls. Densitometric quantification of protein expression was performed from three independent biological replicates. All data are presented as mean ± S.D. (n = 3). Statistical significance was determined using two-way ANOVA followed by Bonferroni's post hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

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  Fig. 5. PrxⅢ modulates mitochondrial dynamics and prevents mitochondrial imbalance under DOX-induced stress. (A) Cells were transduced with Ad-PrxⅢ or Ad-Stuffer for 24 h and then exposed to 1 μM DOX for 12 h. Mitochondria were stained with MitoTracker CMX Red and imaged using confocal microscopy. Mitochondrial morphology was quantified using MiNA plugin Fiji software and expressed as mitochondrial aspect ratio. Enlarged images (labeled “ZOOM”) represent magnified views of the boxed regions. Scale bars: 2.5 μm (upper), 0.4 μm (lower). Five randomly selected microscopic fields per sample were analyzed and combined to represent one biological replicate. (B) Cells were treated as described in (A). Western blotting was performed to assess the expression of mitochondrial fusion (Mfn1, Mfn2, and OPA1) and fission (Drp1, Drp1-phospho-Ser616) proteins. β-actin was used as a loading control. Phospho-Drp1 was normalized to total Drp1. Densitometric quantification was performed from three independent biological replicates. All data are presented as mean ± S.D. Statistical significance was determined using two-way ANOVA followed by Bonferroni's post hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and…

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  Fig. 6. PrxⅢ restores impaired autophagic flux and mitophagy under DOX-induced stress. (A) Cells were transduced with Ad-PrxⅢ or Ad-Stuffer for 24 h and then exposed to DOX (1 μM, 12 h). Immunoblotting was performed to assess LC3 conversion (LC3-I to LC3-II) and SQSTM1/p62 expression. Β-actin was used as a loading control. Densitometric quantification of protein expression was performed from three independent biological replicates. (B) Cells were treated with DOX (1 μM, 6 h) and additionally treated with DMSO or BafA1 (10 nM) for 4 h. Cell lysates were subjected to immunoblot analysis regarding LC3B. β-actin was used as a loading control. Densitometric quantification of protein expression was performed from three independent biological replicates (C-D) Cells were transfected with Ad-PrxⅢ or Ad-Stuffer in the presence of GFP-LC3 (C) or mCherry-GFP-LC3 (D) adenovirus for 24 h and then exposed to DOX (1 μM, 12 h). Representative confocal images are shown. The upper right image (scale bars, 5 μm) is a magnified image of the white box in each corresponding image (scale bars, 20 μm). Quantitative data of GFP-LC3 puncta (C) or autophagosomes (yellow puncta) and autolysosomes (red puncta) (D)…

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  Fig. 7. PrxⅢ rescues lysosomal dysfunction and promotes autophagosome–lysosome fusion under DOX-induced stress. Cells were transduced with Ad-PrxⅢ or Ad-Stuffer for 24 h and then exposed to 1 μM DOX for 6 h. (A) Lysosomal protease activity was analyzed using Magic Red (red), and nuclei were counterstained with Hoechst (blue). Scale bar, 25 μm. (B, C) Lysosomal acidity was analyzed by (B) DQ-BSA Red (red; scale bar, 10 μm) and (C) LysoSensor Green DND-189 (green; scale bar, 25 μm). Nuclei were stained with DAPI (blue). (D) Cells were transduced with Ad-PrxⅢ or Ad-Stuffer in the presence of GFP-LC3 (green) adenovirus for 24 h and treated as above. Fixed cells were immunostained with anti-LAMP1 antibody (red), and nuclei were stained with DAPI (blue). Colocalization of GFP-LC3 and LAMP1 was plotted as yellow dots in graph. Scale bar, 15 μm. Five randomly selected microscopic fields per sample were analyzed and combined to represent one biological replicate. All data are presented as mean ± S.D. Statistical significance was determined using two-way ANOVA followed by Bonferroni's post hoc test. ∗∗p < 0.01 and ∗∗∗p < 0.001.

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  Fig. 8. PrxⅢ protects cardiomyocytes against DOX-induced apoptotic cell death. Cells were transduced with Ad-PrxⅢ or Ad-Stuffer for 24 h and then exposed to 1 μM DOX for 18 h. (A) Cell lysates were subjected to immunoblot analyses for cleaved Caspase-3 and cleaved PARP-1 with normalization to β-actin. Densitometric quantification of protein expression was performed from three independent biological replicates. (B) Apoptotic cell death was assessed by Annexin V-FITC and 7-AAD double staining followed by flow cytometric analysis. Quantification of Annexin V and/or 7-AAD-positive cells is shown. All data are presented as mean ± S.D. Statistical significance was determined using two-way ANOVA followed by Bonferroni's post hoc test. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.

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  Fig. 9. PrxⅢ deficiency aggravates DOX-induced cardiac dysfunction and mitochondrial damage in vivo. (A) Longitudinal echocardiographic assessment of cardiac function in wild-type (WT) and PrxⅢ knockout (KO) mice before and after DOX administration (10 mg/kg, intraperitoneally, 2 weeks). Representative M-mode echocardiographic images from PrxⅢ WT and KO mice post-DOX are shown (top). Baseline (pre-DOX) and endpoint (post-DOX) measurements of ejection fraction (EF), fractional shortening (FS), heart rate (HR), cardiac output (CO), stroke volume (SV), and left ventricular end-systolic volume (LVESV) are presented as paired before-after plots, with dashed lines connecting individual animals across time points. Within-genotype comparisons (pre-DOX vs. post-DOX) were analyzed by paired Student's t-test, and between-genotype comparisons were analyzed by unpaired Student's t-test (N = 5/group). (B) Heart weight/body weight ratio between PrxⅢ WT and KO mice (N = 5/group) post DOX treatment. (C) Representative transmission electron microscopy (TEM) images of cardiac tissue from WT and KO mice post-DOX administration (scale bar, 5 μm for low-magnification images and 2 μm for high-magnification…

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  Fig. 10. Bioinformatic analysis of public transcriptome datasets confirms DOX-induced dysregulation of MQC pathways. (A) Volcano plot of differentially expressed genes (DEGs) in DOX-treated versus sham mouse hearts from publicly available RNA-seq data (GSE233644, n = 4/group). Black diamonds indicate target genes representing MQC pathways examined in this study. Dashed lines indicate significance thresholds (|log2FC| > 1 and adjusted p-value <0.05). (B) Heatmap showing Z-score normalized expression of 27 target genes across individual replicates (Sham #1–4 and DOX #1–4) from GSE233644. Genes are grouped by functional pathway category. Bold gene names indicate statistical significance (adjusted p < 0.05). Log2FC values and significance levels are shown on the right. (C) Cross-dataset comparison of log2FC values for target genes across three independent public DOX cardiotoxicity transcriptome datasets (GSE233644, mouse heart, n = 4; GSE236165, mouse heart, n = 2; GSE224157, mouse heart, n = 3).

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  Fig. 11. SS-31 rescues DOX-induced cardiac dysfunction and mitochondrial injury exacerbated by PrxIII deficiency in vivo. (A) Analysis of cardiac function using echocardiography in wild-type (WT) and PrxⅢ knockout (KO) mice. Mice were treated with PBS (Control), DOX (5 mg/kg/week, intraperitoneally, 4 weeks) alone, or DOX combined with daily administration of the mitochondria-targeted peptide SS-31 (3 mg/kg/day, intraperitoneally) in a complete 3 × 2 factorial design (N = 3/group). Representative M-mode echocardiographic images (top) and quantification of ejection fraction (EF), fractional shortening (FS), heart rate (HR), cardiac output (CO), stroke volume (SV), and left ventricular end-systolic volume (LVESV) are shown. (B) Serum cTnⅠ levels across all six groups. (C, D) Cardiac tissue lysates were subjected to immunoblot analyses for OPA1 and Tomm20 (C) and Bax and cleaved Caspase-3 (D), with β-actin as a loading control. For OPA1, the L-OPA1/S-OPA1 ratio was quantified as a functional indicator of mitochondrial inner membrane integrity and fusion competence; TOMM20, Bax, and cleaved caspase-3 were each normalized to β-actin. Densitometric quantification was performed from three…