Signalling mechanisms underlying doxorubicin and Nox2 NADPH oxidase‐induced cardiomyopathy: involvement of mitofusin‐2

Abstract

Background and PurposeThe anthracycline doxorubicin (DOX), although successful as a first‐line cancer treatment, induces cardiotoxicity linked with increased production of myocardial ROS, with Nox2 NADPH oxidase‐derived superoxide reported to play a key role. The aim of this study was to identify novel mechanisms underlying development of cardiac remodelling/dysfunction further to DOX‐stimulated Nox2 activation.Experimental ApproachNox2−/− and wild‐type (WT) littermate mice were administered DOX (12 mg·kg−1 over 3 weeks) prior to study at 4 weeks. Detailed mechanisms were investigated in murine HL‐1 cardiomyocytes, employing a robust model of oxidative stress, gene silencing and pharmacological tools.Key ResultsDOX‐induced cardiac dysfunction, cardiomyocyte remodelling, superoxide production and apoptosis in WT mice were attenuated in Nox2−/− mice. Transcriptional analysis of left ventricular tissue identified 152 differentially regulated genes (using adjusted P < 0.1) in DOX‐treated Nox2−/− versus WT mice, and network analysis highlighted ‘Cell death and survival’ as the biological function most significant to the dataset. The mitochondrial membrane protein, mitofusin‐2 (Mfn2), appeared as a strong candidate, with increased expression (1.5‐fold), confirmed by qPCR (1.3‐fold), matching clear published evidence of promotion of cardiomyocyte cell death. In HL‐1 cardiomyocytes, targeted siRNA knockdown of Nox2 decreased Mfn2 protein expression, but not vice versa. While inhibition of Nox2 activity along with DOX treatment attenuated its apoptotic and cytotoxic effects, reduced apoptosis after Mfn2 silencing reflected a sustained cytotoxic response and reduced cell viability.Conclusions and ImplicationsDOX‐induced and Nox2‐mediated up‐regulation of Mfn2, rather than contributing to cardiomyocyte dysfunction through apoptotic pathways, appears to promote a protective mechanism.Linked ArticlesThis article is part of a themed section on New Insights into Cardiotoxicity Caused by Chemotherapeutic Agents. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.21/issuetoc

Protection against Doxorubicin-Induced Cytotoxicity by Geniposide Involves AMPKα Signaling Pathway

“…Cardiac tissue is especially vulnerable to free radicals induced by DOX because the hearts had a lot of mitochondria and cardiac antioxidant enzymes were relatively lower compared with other organs [10]. Previous studies have shown that increased levels of cardiac reactive oxygen species (ROS) were observed during the development of DOX-induced cardiomyopathy [11]. Excessive ROS accumulation caused lipid peroxidation, structural changes of biological macromolecules, and eventually result in cell apoptosis.…”

Doxorubicin-Induced Cardiotoxicity: From Mechanisms to Development of Efficient Therapy

“…DOX-induced cardiomyocyte apoptosis and atrophy, interstitial fibrosis, leukocyte infiltration, and cardiac dysfunction in wild-type (WT) mice were attenuated in Nox2 −/− mice [33,39]. DOX-induced superoxide production was also mitigated in this animal model [39].…”

“…An in vitro study using NADPH oxidase inhibitors (diphenyliodonium and apocynin) on H9c2 cells showed that DOX-induced apoptosis was mitigated, demonstrating NADPH oxidase is also involved in the development of cardiac toxicity induced by DOX [36]. Furthermore, there is accumulating evidence to support an important role for Nox2 NADPH oxidase (one of the seven different NADPH oxidase isoforms) in DIC, identified using Nox2-deficient (Nox2 −/− ) or gp91phox knock-out (gp91 −/− ) mice [33,[37][38][39]. DOX-induced cardiomyocyte apoptosis and atrophy, interstitial fibrosis, leukocyte infiltration, and cardiac dysfunction in wild-type (WT) mice were attenuated in Nox2 −/− mice [33,39].…”

“…Furthermore, there is accumulating evidence to support an important role for Nox2 NADPH oxidase (one of the seven different NADPH oxidase isoforms) in DIC, identified using Nox2-deficient (Nox2 −/− ) or gp91phox knock-out (gp91 −/− ) mice [33,[37][38][39]. DOX-induced cardiomyocyte apoptosis and atrophy, interstitial fibrosis, leukocyte infiltration, and cardiac dysfunction in wild-type (WT) mice were attenuated in Nox2 −/− mice [33,39]. DOX-induced superoxide production was also mitigated in this animal model [39].…”

New insights into cardiotoxicity caused by chemotherapeutic agents

“…The anthracycline agent, doxorubicin, is commonly used to treat a diverse range of cancers and is notorious for associated risk of cardiomyopathy, which may largely be mediated by oxidative stress. To specifically investigate the role of the NADPH oxidase NOX2, previously implicated in doxorubicin-induced cardiotoxicity (Zhao et al, 2010), McLaughlin et al (2017) performed microarray analysis of left ventricular tissue from doxorubicin-treated Nox2 À/À and wild-type mice, which highlighted the particular involvement of cell death and survival pathways. They specifically focussed on mitofusin-2 (Mfn2), a mitochondrialshaping protein that has been implicated in cardiomyocyte health and survival (Ong et al, 2017), and reported that activation of Mfn2 signalling appears to be protective against doxorubicin cardiotoxicity.…”