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Updated: Apr 23, 2026
A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish
Published on: June 7, 2018
Mitochondrial apoptosis-inducing factor is involved in doxorubicin-induced toxicity on H9c2 cardiomyoblasts
Ana C Moreira1, Ana F Branco1, Susana F Sampaio1
1CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; Department of Life Sciences, University of Coimbra, Portugal.
Abstract:
The cardiotoxicity induced by the anti-cancer doxorubicin involves increased oxidative stress, disruption of calcium homeostasis and activation of cardiomyocyte death. Nevertheless, antioxidants and caspase inhibitors often show little efficacy in preventing cell death. We hypothesize that a caspase-independent cell death mechanism with the release of the apoptosis-inducing factor from mitochondria is involved in doxorubicin toxicity. To test the hypothesis, H9c2 cardiomyoblasts were used as model for cardiac cells. Our results demonstrate that z-VAD-fmk, a pan-caspase inhibitor, does not prevent doxorubicin toxicity in this cell line. Doxorubicin treatment results in AIF translocation to the nuclei, as confirmed by Western Blotting of cell fractions and confocal microscopy. Also, doxorubicin treatment of H9c2 cardiomyoblasts resulted in the appearance of 50kbp DNA fragments, a hallmark of apoptosis-inducing factor nuclear effects. Apoptosis-inducing factor knockdown using a small-interfering RNA approach in H9c2 cells resulted in a reduction of doxorubicin toxicity, including decreased p53 activation and poly-ADP-ribose-polymerase cleavage. Among the proteases that could be responsible for apoptosis-inducing factor cleavage, doxorubicin decreased calpain activity but increased cathepsin B activation, with inhibition of the latter partly decreasing doxorubicin toxicity. Altogether, the results support that apoptosis-inducing factor release is involved in doxorubicin-induced H9c2 cell death, which explains the limited ability of caspase inhibitors to prevent toxicity.
Insights
Doxorubicin cardiotoxicity involves apoptosis-inducing factor (AIF) release, not just caspase activation. Inhibiting AIF reduces doxorubicin-induced cardiac cell death, explaining why caspase inhibitors are often ineffective.
Area of Science:
- Cardiovascular Pharmacology
- Cell Death Mechanisms
- Mitochondrial Biology
Background:
- Doxorubicin (DOX) induces cardiotoxicity via oxidative stress and cardiomyocyte death.
- Standard treatments targeting oxidative stress and caspase activation show limited efficacy.
- A caspase-independent cell death pathway involving apoptosis-inducing factor (AIF) is hypothesized.
Purpose of the Study:
- To investigate the role of AIF in doxorubicin-induced cardiotoxicity.
- To determine if AIF translocation and nuclear effects are involved in DOX toxicity.
- To explore the potential of targeting AIF for mitigating DOX cardiotoxicity.
Main Methods:
- H9c2 cardiomyoblasts were used as a model system.
- Assessed doxorubicin toxicity with and without pan-caspase inhibitor z-VAD-fmk.
- Confirmed AIF translocation via Western Blotting and confocal microscopy.
- Analyzed DNA fragmentation and employed small-interfering RNA (siRNA) for AIF knockdown.
- Investigated the role of calpain and cathepsin B in AIF cleavage.
Main Results:
- z-VAD-fmk did not prevent doxorubicin-induced H9c2 cell death.
- Doxorubicin treatment led to AIF nuclear translocation and DNA fragmentation.
- AIF knockdown significantly reduced doxorubicin toxicity and associated p53 activation/PARP cleavage.
- Cathepsin B activity increased with doxorubicin treatment, and its inhibition partially reduced toxicity.
Conclusions:
- AIF release from mitochondria is a key mechanism in doxorubicin-induced cardiotoxicity.
- This caspase-independent pathway explains the limited efficacy of caspase inhibitors.
- Targeting AIF or cathepsin B may offer novel therapeutic strategies against doxorubicin cardiotoxicity.
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