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Molecular Mechanisms of Cardiotoxicity
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Molecular Mechanisms of Cardiotoxicity

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Toxicology".

Deadline for manuscript submissions: closed (31 January 2025) | Viewed by 2882

Special Issue Editor

Dr. Antonio Lax

E-Mail Website
Guest Editor
Department of Medicine, University of Murcia, 30120 Murcia, Spain
Interests: myocardial infarction; adverse remodeling; heart failure; cardiotoxicity; cardiac dysfunction; ventricular remodeling; cardioprotection; ischemic heart disease; cardiomyopathy; cardiac regeneration
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Cardiotoxicity is heart damage that arises from certain cancer treatments or drugs. It can develop years after cancer treatment. Although the molecular mechanism related to chemotherapy-induced cardiotoxicity is associated with activation of oxidative stress in oncologic patients, studies with a more standardized design and better characterized populations are necessary to evaluate novel molecular axes. Further, the development of new technologies allows the analysis of a large volume of data, which may lead to enabling a more precise description of the molecular processes related to cardiotoxicity after chemotherapy.

Here, I propose an ambitious special issue to change the conceptual framework used for the management of cardiotoxicity. The aim will be to detail novel molecular mechanisms related to cardiotoxicity, with novel pharmacological targets described to design pioneering therapies to treat or prevent cardiotoxicity related to chemotherapeutic treatments.

Dr. Antonio Lax
Guest Editor

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Keywords

  • cardiac dysfunction
  • cardiotoxicity
  • atrophy
  • antagomiR
  • AAV
  • gene therapy
  • heart failure
  • oxidative stress
  • apoptosis
  • myocardial remodeling

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27 pages, 20721 KiB  
Article
Doxorubicin-Induced Cardiotoxicity Through SIRT1 Loss Potentiates Overproduction of Exosomes in Cardiomyocytes
by Shuai Zhang, Yu Yang, Xinchen Lv, Xue Zhou, Wangqian Zhao, Linfeng Meng, Hongfei Xu, Shaohua Zhu and Ying Wang
Int. J. Mol. Sci. 2024, 25(22), 12376; https://doi.org/10.3390/ijms252212376 - 18 Nov 2024
Viewed by 1449
Abstract
Mutual interaction between doxorubicin (DOX) and cardiomyocytes is crucial for cardiotoxicity progression. Cardiomyocyte injury is an important pathological feature of DOX-induced cardiomyopathy, and its molecular pathogenesis is multifaceted. In addition to the direct toxic effects of DOX on cardiomyocytes, DOX-induced exosomes in the [...] Read more.
Mutual interaction between doxorubicin (DOX) and cardiomyocytes is crucial for cardiotoxicity progression. Cardiomyocyte injury is an important pathological feature of DOX-induced cardiomyopathy, and its molecular pathogenesis is multifaceted. In addition to the direct toxic effects of DOX on cardiomyocytes, DOX-induced exosomes in the extracellular microenvironment also regulate the pathophysiological states of cardiomyocytes. However, the mechanisms by which DOX regulates exosome secretion and subsequent pathogenesis remain incompletely understood. Here, we found that DOX significantly increased exosome secretion from cardiomyocytes, and inhibiting this release could alleviate cardiomyocyte injury. DOX promoted exosome secretion by reducing cardiomyocyte silencing information regulator 1 (SIRT1) expression, exacerbating cardiotoxicity. DOX impaired lysosomal acidification in cardiomyocytes, reducing the degradation of intracellular multivesicular bodies (MVBs), resulting in an increase in MVB volume before fusing with the plasma membrane to release their contents. Mechanistically, SIRT1 loss inhibited lysosomal acidification by reducing the expression of the ATP6V1A subunit of the lysosomal vacuolar-type H+ ATPase (V-ATPase) proton pump. Overexpressing SIRT1 increased ATP6V1A expression, improved lysosomal acidification, inhibited exosome secretion, and thereby alleviated DOX-induced cardiotoxicity. Interestingly, DOX also induced mitochondrial-derived vesicle formation in cardiomyocytes, which may further increase the abundance of MVBs and promote exosome release. Collectively, this study identified SIRT1-mediated impairment of lysosomal acidification as a key mechanism underlying the increased exosome secretion from cardiomyocytes induced by DOX, providing new insights into DOX-induced cardiotoxicity pathogenesis. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Cardiotoxicity)
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Figure 1

Figure 1
<p>DOX−induced cardiotoxicity evaluated in vivo and in vitro. (<b>A</b>) Representative M−mode images of transthoracic echocardiography. (<b>B</b>,<b>C</b>) Quantification of (<b>B</b>) left ventricular ejection fraction (LVEF) and (<b>C</b>) left ventricular fractional shortening (LVFS) (<span class="html-italic">n</span> = 6 animals per group). (<b>D</b>) Representative H&amp;E staining images of heart sections (scale bar: 50 μm). (<b>E</b>) Representative Masson’s trichrome staining images of heart sections (<span class="html-italic">n</span> = 6 animals per group) (scale bar: 50 μm). (<b>F</b>) Quantification of fibrotic area. (<b>G</b>) Representative images of wheat germ agglutinin (WGA) staining in heart sections (<span class="html-italic">n</span> = 6 animals per group) (scale bar: 50 μm). Nuclei stained with 4′,6−diamidino−2−phenylindole (DAPI, blue) and WGA represented cardiomyocyte borders (green). (<b>H</b>) Quantification of cardiomyocyte hypertrophy. (<b>I</b>) Representative images of TdT−mediated dUTP nick−end labeling (TUNEL) staining (green) of heart sections (<span class="html-italic">n</span> = 6 animals per group) (scale bar: 50 μm). Apoptotic cell nuclei appear in green fluorescence, and normal nuclei appear in blue fluorescence (DAPI). (<b>J</b>–<b>M</b>) Cardiac reactive oxygen species (ROS), malondialdehyde (MDA), serum lactate dehydrogenase (LDH), and cardiac superoxide dismutase (SOD) levels (<span class="html-italic">n</span> = 6 animals per group). (N, O) H9c2 cell viability following (<b>N</b>) gradient DOX concentrations and (<b>O</b>) treatment durations (<span class="html-italic">n</span> = 3 independent cell culture experiments). (<b>P</b>–<b>S</b>) ROS, MDA, supernatant LDH, and SOD levels in H9c2 cells (<span class="html-italic">n</span> = 3 independent cell culture experiments). Data are presented as mean ± SD. *** <span class="html-italic">p</span> &lt; 0.001 vs. control group.</p> Full article ">Figure 2
<p>DOX−induced exosome secretion from cardiomyocytes. (<b>A</b>) Transmission electron micrographs of exosomes derived from H9c2 cells in control and DOX−treated groups (scale bar: 1 μm and 200 nm). (<b>B</b>) Nanoparticle tracking analysis of H9c2 cell−derived exosome size distribution. (<b>C</b>) Western blot analysis of exosome−specific markers CD9, CD63, and TSG101 and the non-exosomal marker GM130 in H9c2 cell−derived exosomes. Whole−cell lysates were used as controls. (<b>D</b>) Exosome concentration measured using acetylcholine esterase (AChE) activity (<span class="html-italic">n</span> = 3 independent cell culture experiments). (<b>E</b>) Immunofluorescence staining for CD63 and TSG101 (red) in heart sections from control and DOX−treated groups (scale bar: 50 μm). Sarcomeric alpha−actinin labeled cardiomyocytes (green). Nuclei stained with DAPI (blue). (<b>F</b>) Uptake of 3,3′−dioctadecyloxacarbocyanine perchlorate (DiO, green)−labeled exosomes by H9c2 cells (scale bar: 50 μm). (<b>G</b>–<b>J</b>) ROS, MDA, supernatant LDH, and SOD levels in H9c2 cells after exposure to exosomes from control and DOX−treated groups (<span class="html-italic">n</span> = 3 independent cell culture experiments). Data are presented as mean ± SD. * <span class="html-italic">p</span> &lt; 0.05 vs. control group.</p> Full article ">Figure 3
<p>GW4869 attenuated DOX−induced cardiotoxicity. (<b>A</b>) LDH levels in H9c2 cells showed 0.005% DMSO and GW4869 at 10 μM and 20 μM were not cytotoxic compared to control (<span class="html-italic">n</span> = 3 independent cell culture experiments). (<b>B</b>–<b>E</b>) ROS, MDA, supernatant LDH, and SOD levels in H9c2 cells (<span class="html-italic">n</span> = 3 independent cell culture experiments). (<b>F</b>–<b>I</b>) ROS, MDA, supernatant LDH, and SOD levels in H9c2 cells after treatment with culture supernatants from the CON, GW4869, DOX, and GW4869 + DOX groups (<span class="html-italic">n</span> = 3 independent cell culture experiments). (<b>J</b>) Representative M−mode images of transthoracic echocardiography. (<b>K</b>,<b>L</b>) Quantification of (<b>K</b>) LVEF and (<b>L</b>) LVFS (<span class="html-italic">n</span> = 6 animals per group). (<b>M</b>) H&amp;E staining of heart sections (scale bar: 50 μm). (<b>N</b>) Representative Masson’s trichrome staining images of heart sections (<span class="html-italic">n</span> = 6 animals per group) (scale bar: 50 μm). (<b>O</b>) Quantification of fibrotic area. (<b>P</b>) Representative WGA staining images of heart sections (<span class="html-italic">n</span> = 6 animals per group) (scale bar: 50 μm). Nuclei stained with DAPI (blue) and WGA represented cardiomyocyte borders (green). (<b>Q</b>) Quantification of cardiomyocyte hypertrophy. (<b>R</b>) Representative TUNEL staining images of heart sections (<span class="html-italic">n</span> = 6 animals per group) (scale bar: 50 μm). Apoptotic cell nuclei appear in green fluorescence, and normal nuclei appear in blue fluorescence (DAPI). (<b>S</b>–<b>V</b>) Cardiac ROS, MDA, serum LDH, and cardiac SOD levels (<span class="html-italic">n</span> = 6 animals per group). Data are presented as mean ± SD. NS, not significant (<span class="html-italic">p</span> &gt; 0.05), * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001 vs. control group.</p> Full article ">Figure 3 Cont.
<p>GW4869 attenuated DOX−induced cardiotoxicity. (<b>A</b>) LDH levels in H9c2 cells showed 0.005% DMSO and GW4869 at 10 μM and 20 μM were not cytotoxic compared to control (<span class="html-italic">n</span> = 3 independent cell culture experiments). (<b>B</b>–<b>E</b>) ROS, MDA, supernatant LDH, and SOD levels in H9c2 cells (<span class="html-italic">n</span> = 3 independent cell culture experiments). (<b>F</b>–<b>I</b>) ROS, MDA, supernatant LDH, and SOD levels in H9c2 cells after treatment with culture supernatants from the CON, GW4869, DOX, and GW4869 + DOX groups (<span class="html-italic">n</span> = 3 independent cell culture experiments). (<b>J</b>) Representative M−mode images of transthoracic echocardiography. (<b>K</b>,<b>L</b>) Quantification of (<b>K</b>) LVEF and (<b>L</b>) LVFS (<span class="html-italic">n</span> = 6 animals per group). (<b>M</b>) H&amp;E staining of heart sections (scale bar: 50 μm). (<b>N</b>) Representative Masson’s trichrome staining images of heart sections (<span class="html-italic">n</span> = 6 animals per group) (scale bar: 50 μm). (<b>O</b>) Quantification of fibrotic area. (<b>P</b>) Representative WGA staining images of heart sections (<span class="html-italic">n</span> = 6 animals per group) (scale bar: 50 μm). Nuclei stained with DAPI (blue) and WGA represented cardiomyocyte borders (green). (<b>Q</b>) Quantification of cardiomyocyte hypertrophy. (<b>R</b>) Representative TUNEL staining images of heart sections (<span class="html-italic">n</span> = 6 animals per group) (scale bar: 50 μm). Apoptotic cell nuclei appear in green fluorescence, and normal nuclei appear in blue fluorescence (DAPI). (<b>S</b>–<b>V</b>) Cardiac ROS, MDA, serum LDH, and cardiac SOD levels (<span class="html-italic">n</span> = 6 animals per group). Data are presented as mean ± SD. NS, not significant (<span class="html-italic">p</span> &gt; 0.05), * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001 vs. control group.</p> Full article ">Figure 4
<p>DOX affected exosome secretion through SIRT1 in cardiomyocytes. (<b>A</b>) SIRT1 protein expression levels in H9c2 cells as measured using western blot. (<b>B</b>) Immunofluorescence staining of SIRT1 (green) in H9c2 cells (<span class="html-italic">n</span> = 3 independent cell culture experiments) (scale bar: 50 μm). (<b>C</b>) SIRT1 overexpression in H9c2 cells as measured using western blot. (<b>D</b>) Immunofluorescence staining of SIRT1 (green) in H9c2 cells (<span class="html-italic">n</span> = 3 independent cell culture experiments) (scale bar: 50 μm). (<b>E</b>) Western blot analysis of exosomal markers CD63 and TSG101 in H9c2 cell−derived exosomes. (<b>F</b>) Exosome concentration measured using AChE activity (<span class="html-italic">n</span> = 3 independent cell culture experiments). (<b>G</b>–<b>J</b>) ROS, MDA, supernatant LDH, and SOD levels in H9c2 cells (<span class="html-italic">n</span> = 3 independent cell culture experiments). Data are presented as mean ± SD. * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01 vs. control group.</p> Full article ">Figure 5
<p>Overexpression of SIRT1 attenuated DOX−induced cardiotoxicity. (<b>A</b>) SIRT1 protein expression levels in mouse hearts as measured using western blot. (<b>B</b>) Immunohistochemistry staining of SIRT1 (brown) in heart sections (scale bar: 50 μm). (<b>C</b>) SIRT1 overexpression in mouse hearts as measured using western blot. (<b>D</b>) Immunohistochemistry staining of SIRT1 overexpression (brown) in heart sections (scale bar: 50 μm). (<b>E</b>,<b>F</b>) Immunofluorescence staining of exosome markers (<b>E</b>) CD63 and (<b>F</b>) TSG101 (red) in heart sections after SIRT1 overexpression (scale bar: 25 μm). Sarcomeric alpha−actinin labeled cardiomyocytes (green). Nuclei stained with DAPI (blue). (<b>G</b>) Representative M−mode images of transthoracic echocardiography for each group 2 weeks after AAV9−SIRT1 treatment. (<b>H</b>,<b>I</b>) Quantification of (<b>H</b>) LVEF and (<b>I</b>) LVFS (<span class="html-italic">n</span> = 6 animals per group). (<b>J</b>) Representative images of H&amp;E staining in heart sections (scale bar: 50 μm). (<b>K</b>) Representative images of Masson’s trichrome staining in heart sections (<span class="html-italic">n</span> = 6 animals per group) (scale bar: 50 μm). (<b>L</b>) Quantification of fibrotic area. (<b>M</b>) Representative images of WGA staining in heart sections (<span class="html-italic">n</span> = 6 animals per group) (scale bar: 50 μm). Nuclei stained with DAPI (blue) and WGA represented cardiomyocyte borders (green). (<b>N</b>) Quantification of cardiomyocyte hypertrophy. (<b>O</b>) Representative images of TUNEL staining of heart sections (<span class="html-italic">n</span> = 6 animals per group) (scale bar: 50 μm). Apoptotic cell nuclei appear in green fluorescence, and normal nuclei appear in blue fluorescence (DAPI). (<b>P</b>–<b>S</b>) Cardiac ROS, MDA, serum LDH, and cardiac SOD levels. Data are presented as mean ± SD. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001 vs. control group.</p> Full article ">Figure 5 Cont.
<p>Overexpression of SIRT1 attenuated DOX−induced cardiotoxicity. (<b>A</b>) SIRT1 protein expression levels in mouse hearts as measured using western blot. (<b>B</b>) Immunohistochemistry staining of SIRT1 (brown) in heart sections (scale bar: 50 μm). (<b>C</b>) SIRT1 overexpression in mouse hearts as measured using western blot. (<b>D</b>) Immunohistochemistry staining of SIRT1 overexpression (brown) in heart sections (scale bar: 50 μm). (<b>E</b>,<b>F</b>) Immunofluorescence staining of exosome markers (<b>E</b>) CD63 and (<b>F</b>) TSG101 (red) in heart sections after SIRT1 overexpression (scale bar: 25 μm). Sarcomeric alpha−actinin labeled cardiomyocytes (green). Nuclei stained with DAPI (blue). (<b>G</b>) Representative M−mode images of transthoracic echocardiography for each group 2 weeks after AAV9−SIRT1 treatment. (<b>H</b>,<b>I</b>) Quantification of (<b>H</b>) LVEF and (<b>I</b>) LVFS (<span class="html-italic">n</span> = 6 animals per group). (<b>J</b>) Representative images of H&amp;E staining in heart sections (scale bar: 50 μm). (<b>K</b>) Representative images of Masson’s trichrome staining in heart sections (<span class="html-italic">n</span> = 6 animals per group) (scale bar: 50 μm). (<b>L</b>) Quantification of fibrotic area. (<b>M</b>) Representative images of WGA staining in heart sections (<span class="html-italic">n</span> = 6 animals per group) (scale bar: 50 μm). Nuclei stained with DAPI (blue) and WGA represented cardiomyocyte borders (green). (<b>N</b>) Quantification of cardiomyocyte hypertrophy. (<b>O</b>) Representative images of TUNEL staining of heart sections (<span class="html-italic">n</span> = 6 animals per group) (scale bar: 50 μm). Apoptotic cell nuclei appear in green fluorescence, and normal nuclei appear in blue fluorescence (DAPI). (<b>P</b>–<b>S</b>) Cardiac ROS, MDA, serum LDH, and cardiac SOD levels. Data are presented as mean ± SD. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001 vs. control group.</p> Full article ">Figure 6
<p>DOX inhibited lysosomal acidification by regulating ATP6V1A expression through SIRT1 in cardiomyocytes. (<b>A</b>) Western blot analysis of ATP6V1A protein levels in mouse hearts. (<b>B</b>) Representative immunohistochemistry staining of ATP6V1A (brown) staining in heart sections (scale bar: 50 μm). (<b>C</b>) Western blot of ATP6V1A expression in H9c2 cells. (<b>D</b>) Representative immunofluorescence staining of ATP6V1A (green) in H9c2 cells (scale bar: 50 μm). (<b>E</b>) DOX treatment for 24 h reduced Lysotracker Red−positive puncta (Cat#C1046, Beyotime Biotechnology, Haimen, China) in H9c2 cell cytoplasm (scale bar: 50 μm). (<b>F</b>) DOX increased lysosomal pH as measured using Dextran, Oregon Green 514 (<span class="html-italic">n</span> = 3 independent cell culture experiments). (<b>G</b>,<b>H</b>) Representative double immunofluorescence images of VPS16 (green) and LAMP−1 (red) co−localization in (<b>G</b>) H9c2 cells and (<b>H</b>) mouse hearts, indicating lysosome−MVBs interaction (scale bar: 10 μm and 25 μm). Sarcomeric alpha−actinin labeled cardiomyocytes (gray, central panels). Nuclei stained with DAPI (blue). (<b>I</b>,<b>J</b>) Transmission electron micrographs showing MVBs in (<b>I</b>) H9c2 cells and (<b>J</b>) mouse hearts (scale bar: 500 nm). The white arrows pointed to the MVBs. (<b>K</b>–<b>N</b>) SIRT1 overexpression restored ATP6V1A expression in mouse hearts and H9c2 cells as measured using western blot and immunofluorescence staining. (<b>O</b>,<b>P</b>) SIRT1 increased (<b>O</b>) Lysotracker Red−positive puncta (scale bar: 50 μm) and decreased (<b>P</b>) lysosomal pH (<span class="html-italic">n</span> = 3 independent cell culture experiments). (<b>Q</b>,<b>R</b>) SIRT1 enhanced VPS16 (green) and LAMP−1 (red) co−localization in (<b>Q</b>) H9c2 cells (scale bar: 10 μm) and (R) mouse hearts (scale bar: 25 μm). Sarcomeric alpha−actinin labeled cardiomyocytes (gray, central panels). Nuclei stained with DAPI (blue). (<b>S</b>,<b>T</b>) Transmission electron micrographs showing MVBs in (<b>S</b>) H9c2 cells and (<b>T</b>) mouse hearts (scale bar: 500 nm). The white arrows pointed to the MVBs. Data are presented as mean ± SD. ** <span class="html-italic">p</span> &lt; 0.01 vs. control group.</p> Full article ">Figure 6 Cont.
<p>DOX inhibited lysosomal acidification by regulating ATP6V1A expression through SIRT1 in cardiomyocytes. (<b>A</b>) Western blot analysis of ATP6V1A protein levels in mouse hearts. (<b>B</b>) Representative immunohistochemistry staining of ATP6V1A (brown) staining in heart sections (scale bar: 50 μm). (<b>C</b>) Western blot of ATP6V1A expression in H9c2 cells. (<b>D</b>) Representative immunofluorescence staining of ATP6V1A (green) in H9c2 cells (scale bar: 50 μm). (<b>E</b>) DOX treatment for 24 h reduced Lysotracker Red−positive puncta (Cat#C1046, Beyotime Biotechnology, Haimen, China) in H9c2 cell cytoplasm (scale bar: 50 μm). (<b>F</b>) DOX increased lysosomal pH as measured using Dextran, Oregon Green 514 (<span class="html-italic">n</span> = 3 independent cell culture experiments). (<b>G</b>,<b>H</b>) Representative double immunofluorescence images of VPS16 (green) and LAMP−1 (red) co−localization in (<b>G</b>) H9c2 cells and (<b>H</b>) mouse hearts, indicating lysosome−MVBs interaction (scale bar: 10 μm and 25 μm). Sarcomeric alpha−actinin labeled cardiomyocytes (gray, central panels). Nuclei stained with DAPI (blue). (<b>I</b>,<b>J</b>) Transmission electron micrographs showing MVBs in (<b>I</b>) H9c2 cells and (<b>J</b>) mouse hearts (scale bar: 500 nm). The white arrows pointed to the MVBs. (<b>K</b>–<b>N</b>) SIRT1 overexpression restored ATP6V1A expression in mouse hearts and H9c2 cells as measured using western blot and immunofluorescence staining. (<b>O</b>,<b>P</b>) SIRT1 increased (<b>O</b>) Lysotracker Red−positive puncta (scale bar: 50 μm) and decreased (<b>P</b>) lysosomal pH (<span class="html-italic">n</span> = 3 independent cell culture experiments). (<b>Q</b>,<b>R</b>) SIRT1 enhanced VPS16 (green) and LAMP−1 (red) co−localization in (<b>Q</b>) H9c2 cells (scale bar: 10 μm) and (R) mouse hearts (scale bar: 25 μm). Sarcomeric alpha−actinin labeled cardiomyocytes (gray, central panels). Nuclei stained with DAPI (blue). (<b>S</b>,<b>T</b>) Transmission electron micrographs showing MVBs in (<b>S</b>) H9c2 cells and (<b>T</b>) mouse hearts (scale bar: 500 nm). The white arrows pointed to the MVBs. Data are presented as mean ± SD. ** <span class="html-italic">p</span> &lt; 0.01 vs. control group.</p> Full article ">Figure 7
<p>Overexpression of ATP6V1A inhibited exosome secretion. (<b>A</b>) Western blot analysis of ATP6V1A protein levels in H9c2 cells. (<b>B</b>) Representative immunofluorescence staining of ATP6V1A (green) in H9c2 cells (scale bar: 50 μm). (<b>C</b>) ATP6V1A overexpression increased Lysotracker Red−positive puncta in H9c2 cell cytoplasm (<span class="html-italic">n</span> = 3 independent cell culture experiments) (scale bar: 50 μm). (<b>D</b>) ATP6V1A decreased lysosomal pH as measured using Dextran, Oregon Green 514 (<span class="html-italic">n</span> = 3 independent cell culture experiments). (<b>E</b>) Representative double immunofluorescence images of VPS16 (green) and LAMP−1 (red) in H9c2 cells, indicating lysosome−MVBs interaction (scale bar: 10 μm). (<b>F</b>) Transmission electron micrographs of H9c2 cells from different treatment groups, with representative images showing MVBs (scale bar: 500 nm). The white arrows pointed to the MVBs. (<b>G</b>) Western blot analysis of exosome markers CD63 and TSG101 in H9c2 cell−derived exosomes. Whole−cell lysates were used as controls. (<b>H</b>) Exosome concentration measured using AChE activity (<span class="html-italic">n</span> = 3 independent cell culture experiments). Data are presented as mean ± SD. * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01 vs. control group.</p> Full article ">Figure 8
<p>DOX induced mitochondrial−derived vesicle formation. (<b>A</b>) Representative transmission electron micrographs of heart tissues. Low magnification images showed general cardiac ultrastructure (scale bar: 5 μm). Higher magnification images revealed mitochondrial morphology and vesicles (scale bar: 1 μm). The white boxes indicated the zoomed−in area shown in the figure on the right. The white triangles pointed to the MDVs. (<b>B</b>) Representative immunofluorescence staining showing PDH and TOM20 (red) expression in mouse hearts; both were markers of MDVs (scale bar: 50 μm). Sarcomeric alpha−actinin labeled cardiomyocytes (green). Nuclei stained with DAPI (blue). (<b>C</b>) Representative transmission electron micrographs of H9c2 cells depicting mitochondrial morphology and vesicles (scale bar: 500 nm). The white triangles pointed to the MDVs. (<b>D</b>) Representative double immunofluorescence images for PDH or TOM20 (green, both were markers of MDVs) with LAMP1\2 (red) in H9c2 cells, indicating interaction between lysosome−TOM20+ or PDH+ MDVs (scale bar:10 μm).</p> Full article ">Figure 9
<p>DOX promoted the transport of mitochondria−derived vesicles to MVBs. (<b>A</b>) OPA1 and SIRT1 protein expression levels in H9c2 cells measured using western blot. (<b>B</b>) Representative transmission electron micrographs of H9c2 cells depicting mitochondrial morphology and vesicles (scale bar: 500 nm). The white triangles pointed to the MDVs. (<b>C</b>) Representative double immunofluorescence images of H9c2 cells showing PDH, TOM20, or VPS16 (green) with LAMP1\2 (red), indicating lysosome−TOM20+\PDH+ MDVs or MVBs interaction (scale bar:10 μm). (<b>D</b>) Transmission electron micrographs of H9c2 cells from different treatment groups, with representative images showing MVBs (scale bar: 500 nm). The white arrows pointed to the MVBs. (<b>E</b>) Western blot analysis of exosomal markers CD63 and TSG101 in H9c2 cell−derived exosomes. Whole−cell lysates were used as controls. (<b>F</b>) Exosome concentration measured using AChE activity (<span class="html-italic">n</span> = 3 independent cell culture experiments). (<b>G</b>–<b>J</b>) ROS, MDA, supernatant LDH, and SOD levels in H9c2 cells (<span class="html-italic">n</span> = 3 independent cell culture experiments). Data are presented as mean ± SD. * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01 vs. control group.</p> Full article ">

Review

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21 pages, 2045 KiB  
Review
Evolution of Theories on Doxorubicin-Induced Late Cardiotoxicity-Role of Topoisomerase
by Jaroslaw Szponar, Erwin Ciechanski, Magda Ciechanska, Jaroslaw Dudka and Sławomir Mandziuk
Int. J. Mol. Sci. 2024, 25(24), 13567; https://doi.org/10.3390/ijms252413567 - 18 Dec 2024
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Abstract
Doxorubicin (DOX) has been widely used as a cytotoxic chemotherapeutic. However, DOX has a number of side effects, such as myelotoxicity or gonadotoxicity, the most dangerous of which is cardiotoxicity. Cardiotoxicity can manifest as cardiac arrhythmias, myocarditis, and pericarditis; life-threatening late cardiotoxicity can [...] Read more.
Doxorubicin (DOX) has been widely used as a cytotoxic chemotherapeutic. However, DOX has a number of side effects, such as myelotoxicity or gonadotoxicity, the most dangerous of which is cardiotoxicity. Cardiotoxicity can manifest as cardiac arrhythmias, myocarditis, and pericarditis; life-threatening late cardiotoxicity can result in heart failure months or years after the completion of chemotherapy. The development of late cardiomyopathy is not yet fully understood. The most important question is how DOX reprograms the cardiomyocyte, after which DOX is excreted from the body, initially without symptoms. However, clinically overt cardiomyopathy develops over the following months and years. Since the 1980s, DOX-induced disorders in cardiomyocytes have been thought to be related to oxidative stress and dependent on the Fe/reactive oxygen species (ROS) mechanism. That line of evidence was supported by dexrazoxane (DEX) protection, the only Food and Drug Administration (FDA)-approved drug for preventing DOX-induced cardiomyopathy, which complexes iron. Thus, the hypothesis related to Fe/ROS provides a plausible explanation for the induction of the development of late cardiomyopathy via DOX. However, in subsequent studies, DEX was used to identify another important mechanism in DOX-induced cardiomyopathy that is related to topoisomerase 2β (Top2β). Does the Top2β hypothesis explain the mechanisms of the development of DOX-dependent late heart failure? Several of these mechanisms have been identified to date, proving the involvement of Top2β in the regulation of the redox balance, including oxidative stress. Thus, the development of late cardiomyopathy can be explained based on mechanisms related to Top2β. In this review, we highlight free radical theory, iron imbalance, calcium overload, and finally, a theory based on Top2β. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Cardiotoxicity)
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<p>Late cardiotoxic effects of doxorubicin, involving dilated cardiomyopathy caused by cardiac remodeling, ventricle dilatation, progression of fibrosis, and finally, left ventricular ejection fraction reduction; ↑—increase, ↓—decrease.</p> Full article ">Figure 2
<p>The cytotoxic effect of doxorubicin is directly related to reactive oxygen species (ROS). Briefly, DOX easily obtains electrons from NADH and NADPH in the presence of iron in reactions catalyzed by NADPH, cytochrome P-450 reductase, iNOS, and others. After obtaining an electron, DOX forms a semiquinone radical and transfers an electron to molecular oxygen, forming O<sub>2</sub><sup>•−</sup>. The presence of O<sub>2</sub><sup>•−</sup> favors the generation of other ROS, which damage lipids, proteins, and mitochondrial DNA (mtDNA). NADH is largely consumed in the DOX redox cycle once DOX is attached to cardiolipin in the inner mitochondrial membrane, through which adenosine triphosphate (ATP) synthesis decreases, leading to mitochondrial electron transport chain dysfunction. The four-electron reduction of oxygen to water decreases in favor of one-, two-, and three-electron reduction, which triggers oxidative stress and mtDNA damage. These lead to a positive feedback effect, finally manifesting as heart failure. DEX and the active Top2β cluster together and inhibit DOX–Top2β complex formation, stopping the cycle at an early stage; ↑—increase, ↓—decrease; A—adriamycin (doxorubicin); ADP—adenosine diphosphate; D—dexrazoxane; ETC—electron transport chain.</p> Full article ">Figure 3
<p>Iron imbalance. Transferrin-bound Fe<sup>3+</sup> enters the cell through transferrin receptor 1 (TfR1), whereas Fe<sup>2+</sup> predominantly enters via divalent metal transporter 1 (DMT1), as well as through L- and T-type calcium channels (LTCC and TTCC). Ferric iron is reduced inside the cell’s endosome to ferrous iron by the six-transmembrane epithelial antigen of prostate 3 (STEAP3). The formation of the DOX semiquinone radical concurrently occurs with the release of iron from ferritin. The increased concentrations of H<sub>2</sub>O<sub>2</sub>, O<sub>2</sub><sup>•−</sup> and ONOO<sub>−</sub> induced by DOX trigger the release of an iron atom from [4Fe-4S] aconitase, converting it to the [3Fe-4S] conformation. Consequently, the transcriptional activity of ferritin is reduced, leading to ferritinophagy and decreasing the expression of the iron regulatory gene, lowering the human homeostatic iron regulator protein (HFE) synthesis, in the nucleus. These processes result in the accumulation of a labile iron pool in Fe<sup>2+</sup> form, which promotes the Fenton reaction, causing lipid peroxidation and triggering ferroptosis, thereby exacerbating cardiotoxicity; ↑—increase, ↓—decrease, DOX—doxorubicin, A—adriamycin (doxorubicin).</p> Full article ">Figure 4
<p>Role of Top2β in the cytotoxic effect of DOX. Once the DOX–Top2β complex is created, DSBs and DNA transcription changes lead to defective mitochondrial biogenesis. Mitochondrial dysfunction is secondary to the suppression of PPARGC1A and PPARGC1B transcription, which regulate genes involved in the electron transport chain, the tricarboxylic acid cycle, and the β-oxidation of fatty acids via ESR1 and NRF1/NRF2. Once the PPARGC1A and PPARGC1B expression is suppressed, the superoxide dismutase (SOD), peroxiredoxin, and thioredoxin expression is also suppressed, promoting ROS production. Thus, disturbances in the mitochondrial electron transport chain lead to the one-, two-, and three-electron reduction of oxygen, triggering oxidative stress and a snowball effect in the mitochondria and leading to heart failure; ↑—increase; ↓—decrease; A—adriamycin (doxorubicin); ADP—adenosine diphosphate; ATP—adenosine triphosphate; DSBs—double-strand breaks; ETC—electron transport chain.</p> Full article ">
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