Nox2 NADPH Oxidase Promotes Pathologic Cardiac Remodeling Associated with Doxorubicin Chemotherapy

Abstract

Doxorubicin is a highly effective cancer treatment whose use is severely limited by dose-dependent cardiotoxicity. It is well established that doxorubicin increases reactive oxygen species (ROS) production. In this study, we investigated contributions to doxorubicin cardiotoxicity from Nox2 NADPH oxidase, an important ROS source in cardiac cells, which is known to modulate several key processes underlying the myocardial response to injury. Nox2-deficient mice (Nox2 −/− ) and wild-type (WT) controls were injected with doxorubicin (12 mg/kg) or vehicle and studied 8 weeks later. Echocardiography indicated that doxorubicin-induced contractile dysfunction was attenuated in Nox2 −/− versus WT mice (fractional shortening: 29.5 ± 1.4 versus 25.7 ± 1.0%; P < 0.05). Similarly, in vivo pressure-volume analysis revealed that systolic and diastolic function was preserved in doxorubicin-treated Nox2 −/− versus WT mice (ejection fraction: 52.6 ± 2.5 versus 28.5 ± 2.3%, LVdP/dt min : −8,379 ± 416 versus −5,198 ± 527 mmHg s −1 ; end-diastolic pressure-volume relation: 0.051 ± 0.009 versus 0.114 ± 0.012; P < 0.001). Furthermore, in response to doxorubicin, Nox2 −/− mice exhibited less myocardial atrophy, cardiomyocyte apoptosis, and interstitial fibrosis, together with reduced increases in profibrotic gene expression (procollagen IIIαI, transforming growth factor-β 3 , and connective tissue growth factor) and matrix metalloproteinase-9 activity, versus WT controls. These alterations were associated with beneficial changes in NADPH oxidase activity, oxidative/nitrosative stress, and inflammatory cell infiltration. We found that adverse effects of doxorubicin were attenuated by acute or chronic treatment with the AT1 receptor antagonist losartan, which is commonly used to reduce blood pressure. Our findings suggest that ROS specifically derived from Nox2 NADPH oxidase make a substantial contribution to several key processes underlying development of cardiac contractile dysfunction and remodeling associated with doxorubicin chemotherapy.

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

“…In samples diluted to a concentration of 1 mg protein·mL −1 , NADPH‐dependent superoxide production was measured by lucigenin (5 μM)‐enhanced chemiluminescence at 37°C for 30 min (Zhao et al, 2010). Potential sources of superoxide were assessed in experiments including the following: (a) tiron (20 mM), cell‐permeable superoxide scavenger; (b) diphenyleneiodonium (DPI, 10 μM), inhibitor of NADPH oxidase and other flavoproteins; (c) L‐N G ‐nitroarginine methyl ester (L‐NAME, 1 mM), inhibitor of superoxide production by dysfunctional NOS; (d) oxypurinol (100 μM), xanthine oxidase inhibitor and (e) rotenone (10 μM), which inhibits the mitochondrial electron transport chain.…”

“…LV protein was extracted by homogenization with ice‐cold RIPA buffer, as previously described (Zhao et al, 2010), and 20 μg loaded onto a 10% SDS‐PAGE gel before blotting on a PVDF membrane (Immobilon‐FL; Millipore). Membranes were incubated overnight at 4°C with a rabbit monoclonal antibody against Nox2 (1:1000, Abcam ab129068) using hypoxanthine phosphoribosyltransferase (HPRT) antibody (1:10 000, ab109021 Abcam) as a loading control.…”

“…In addition to NOS signalling, ROS generated through NADPH oxidase play an essential role in cardiac pathophysiology, regulating major elements of cardiac remodelling, such as fibrosis and apoptosis (Grieve et al, 2006; Gilleron et al, 2009). There is accumulating evidence to support an important role for Nox2 NADPH oxidase in DOX‐induced cardiotoxicity, identified using Nox2‐deficient (Nox2 −/− ) mice (Wojnowski et al, 2005; Deng et al, 2007; Zhao et al, 2010). Indeed, our group previously reported that DOX‐induced interstitial fibrosis, leukocyte infiltration, cardiomyocyte apoptosis and atrophy, and cardiac dysfunction were attenuated in Nox2 −/− mice (Zhao et al, 2010).…”

“…There is accumulating evidence to support an important role for Nox2 NADPH oxidase in DOX‐induced cardiotoxicity, identified using Nox2‐deficient (Nox2 −/− ) mice (Wojnowski et al, 2005; Deng et al, 2007; Zhao et al, 2010). Indeed, our group previously reported that DOX‐induced interstitial fibrosis, leukocyte infiltration, cardiomyocyte apoptosis and atrophy, and cardiac dysfunction were attenuated in Nox2 −/− mice (Zhao et al, 2010). …”

“…Use of mRNA microarray technology (Kuhn et al, 2004) and the Nox2 −/− mouse model (Zhao et al, 2010) was considered a suitable approach. Having identified the mitochondrial membrane protein, mitofusin‐2 (Mfn2), as a strong candidate, the hypothesis that up‐regulated Mfn2 contributes to cardiomyocyte death processes induced by DOX was tested.…”