Nrf2 Deficiency Exaggerates Doxorubicin-Induced Cardiotoxicity And Cardiac Dysfunction

BackgroundThe anticancer drug doxorubicin (Dox) (also referred to as adriamycin) is highly effective in the treatment of a broad range of cancers; however, it is associated with dose-dependent acute and chronic cardiotoxicity, which significantly limits its chemotherapeutic dosage. The cause of Dox-induced cardiotoxicity is multifactorial and includes free radical-induced mitochondrial damage, DNA damage, inhibition of DNA and protein synthesis, and myofiber degeneration, which cumulatively leads to myocardial apoptotic and/or necrotic cell death. Nevertheless, the precise pathophysiology of Dox-induced cardiotoxicity is not fully understood.That oxidative stress is the primary cause of Dox-induced cardiomyopathy has been the prevailing hypothesis. However, antioxidant approaches of nonselective reactive oxygen species (ROS) scavenging for the treatment of Dox-induced cardiomyopathy have been shown to be ineffective. Therefore efficacious therapy may require more specific targeting of either the source of oxidative stress or the endogenous antioxidant defense system. However, such specific targets remain to be identified.Nuclear factor erythroid-2 related factor 2 (Nrf2) is a master transcription factor in controlling the basal and inducible expression of a battery of antioxidant genes and other cytoprotective phase II detoxifying enzymes. We have demonstrated that Nrf2 is a negative regulator of cardiac pathological remodeling and dysfunction via suppressing oxidative stress in diverse pathological settings. While it has been documented that Nrf2 plays a mediator role in hydrogen sulfide-mediated suppression of oxidative stress-induced cardiac dysfunction, we and others have demonstrated that Nrf2 might be a drug target for the treatment of cardiomyocyte injury and cardiac dysfunction. Of interest, a recent report has revealed that Nrf2 is able to enhance autophagic clearance of toxic ubiquitinated protein aggregates secondary to ROS formation, suggesting a novel mediator role of Nrf2 for sufficient activation of autophagy. Considering the causative role of oxidative stress and the protective effect of sufficient autophagy activation in Dox-induced cardiotoxicity as well as the Nrf2-mediated antioxidant defense and sufficient activation of autophagy, it is conceivable that Nrf2 is a negative regulator of Dox-induced cardiomyopathy.Therefore, in the present study, we explored the role of Nrf2 in the regulation of Dox-induced cardiomyopathy with a focus on oxidative stress and autophagic activity in the heart. We demonstrate that loss of Nrf2 function exaggerates Dox-induced oxidative stress, insufficient autophagic activities, and cardiomyocyte necrosis, as well as cardiac dysfunction. These results indicate that Nrf2 acts as a critical negative regulator of Dox-induced cardiomyopathy, thereby identifying a potential, novel target for the treatment of Dox-induced cardiomyopathy. Objectives1. We explored the role of Nrf2 in the regulation of Dox-induced cardiomyopathy with a focus on oxidative stress and autophagic activity in the heart.2. Nrf2 acts as a critical negative regulator of Dox-induced cardiomyopathy, thereby identifying a potential, novel target for the treatment of Dox-induced cardiomyopathy.Materials and methods1. Generate Nrf2 knockout (NrET-/-) mice.2. Set up doxorubicin-Induced Cardiomyopathy model.3. Echocardiography for Dox model.4. Measurement of Autophagic Flux In Vivo.5. Histology and Immunochemistry for oxidative stress and Nrf2 expression.6. Evans Blue Labelling for necrosis.7. Virus Preparation.8. Cell Culture and Adenovirus Infection.9. Protein expression detected by Western blot.10. Gene expression detected by Q-PCR.11. Statistical differences were analyzed by one-way ANOVA followed by Bonferroni test for multiple comparisons using GraphPad Prism software.Results1. Dox-Induced Cardiac Oxidative Stress, Toxicity, and Dysfunction.Following a single intraperitoneal injection of Dox (25 mg/kg), there was a time dependent onset of oxidative stress in the heart, which appeared at day 1 and was sustained by day 4 after injection. There was also substantial cardiomyocyte necrosis in the heart as evidenced by cytoplasmic vacuolization and sporadic Evans blue dye uptake in cardiomyocytes.2. Dox-Induced Impairment of Autophagy and Accumulation of Ubiquitinated Proteins in the Heart.Treatment of chloroquine (CQ), an inhibitor of autophagosome fusion with lysosome, led to increased myocardial protein levels of LC3-II and p62 (autophagic flux) in control vehicle treated mice. Although Dox treatment resulted in increased myocardial protein levels of LC3-II and p62, CQ treatment had no impact on the myocardial protein expression of LC3-II and p62 in the mice treated with Dox. In addition, Dox treatment resulted in time-dependent accumulation of ubiquitinated proteins in the heart.3. Dox-Induced Nrf2 Activation in the Heart.Dox treatment enhanced myocardial Nrf2 mRNA levels in a time dependent manner. Dox treatment also enhanced myocardial Nrf2 protein levels and triggered Nrf2 nuclear translocation in cardiomyocytes 4 days after the treatment. In addition, Dox treatment led to increases in myocardial mRNA levels of NQO1 and heme oxygenase-1HO-1, the downstream antioxidant genes of Nrf2.4. Nrf2 Deficiency Exaggerates Dox-Induced Myocardial Oxidative Stress and Impaired Autophagy as well as Cardiac Toxicity and Dysfunction.Loss of Nrf2 enhanced Dox-induced upregulation of LC3-II and p62 proteins in both soluble and insoluble fractions. Nrf2 deficiency did not affect Dox-induced accumulation of ubiquitinated proteins in soluble fractions, while enhancing the accumulation of ubiquitinated proteins in insoluble fractions.5. Nrf2 Activation Ameliorates Dox-Induced Autophagy Impairment and Cell Death in Cardiomyocytes via Facilitating Autophagic Clearance of Ubiquitinated Protein Aggregates.Dox at toxic dose of 1 μM increased the steady level of LC3-II; however, treatment of bafilomycin A1 (BafA1), an inhibitor of autophagosome fusion with lysosome, did not further accumulate the Dox-induced upregulation of LC3. In addition, the Dox (1 μM) treatment led to accumulation of ubiquitinated proteins in insoluble cellular fractions.Overexpression of Nrf2 upregulated whereas Nrf2 knockdown downregulated the steady levels of LC3-II. Dox treatment accumulated the steady levels of LC3-II in the Ad-Gfp control but not in Ad-Nrf2 infected cells. In contrast, Dox accumulated the steady levels of LC3-II in both Ad-scramble control and Ad-miNrf2 infected cells with a similar magnitude.Nrf2 overexpression dramatically inhibited Dox-induced accumulation of ubiquitinated protein aggregates; however, Nrf2 knockdown enhanced basal and Dox-induced accumulation of ubiquitinated protein aggregates.Conclusions1. Nrf2 is likely an endogenous inhibitor of Dox-induced cardiomyopathy. Mechanistically, Nrf2 is capable of suppressing Dox-induced oxidative stress and impaired autophagy.2. Further investigation of molecular mechanisms by which Nrf2 regulates the interplay between oxidative stress and insufficient autophagy in Dox-induced cardiotoxicity will provide valuable insights into the feasibility of targeting Nrf2 as a novel strategy for preventing or mitigating Dox-induced cardiotoxicity and cardiac dysfunction.