The role of Nox2 in chronic cocaine administration-induced cardiac oxidative stress and cardiotoxicity

Lampson Fan, Alexis Bailey, Ian Kitchen, Jian-Mei Li

University of Surrey, Guildford, UK

Cocaine is one of the most common illicitly used drugs in the world and causes the most frequent drug-related deaths in young adults (<40 years). Chronic cocaine consumption is associated with serious cardiovascular complications such as hypertension, cardiac hypertrophy, arrhythmia, infarction and sudden cardiac death1. However, our understanding of the underlying cocaine cardiotoxicity is far from complete and pharmacological management of such patients is problematic. The cardiac tissue expresses constitutively an NADPH oxidase, which generates reactive oxygen species (ROS) and is involved in the pathogenesis of cardiac disorders2. In this study, we investigated the hypothesis of ROS-mediated cocaine cardiotoxicity in a mouse model (C57BL/6J, male, 5 weeks old) of chronic escalating “binge” cocaine administration3. The control group was given intra-peritoneal (ip) injections of saline (10ml/kg), and the cocaine group was given cocaine dissolved in saline (10ml/kg) three times daily at 1 hourly interval for 14 days (mg/kg): d1-4 at 3x15mg; d5-8 at 3x20mg; d9-12 at 3x25mg and d13-14 at 3x30mg (n=12/per group). Compared to saline controls, cocaine increased significantly the NADPH-dependent O2.- production by 1.96±0.4 fold (p<0.01) in cardiac homogenates as detected by tiron (an O2.- scavenger)-inhibitable lucigenin-chemiluminescence and this was confirmed by DHE fluorescence on cardiac sections. Cocaine-induced O2.- production was associated with significant increases (>2-fold) in the protein levels of Nox2 (an isoform of the NADPH oxidase family), p22phox, p47phox, p67phox and Rac1 as detected by western blotting, and in p47phox serine phosphorylation (3.2±0.5 fold) notably in the cardiomyocytes as revealed by confocal microscopy. The increase in Nox2 activity was accompanied by a significant activation of ERK1/2, p38MAPK and JNK in the myocardium as detected by phospho-specific antibodies. The cocaine effect on myocardial oxidative stress was further examined in cultured cardiac myocytes (H9C2 cells). Cocaine at a concentration as low as 5 μM (for 24 h) had significantly increased (1.8±0.3 fold, P<0.01) the ROS production by cultures myocytes, and at a concentration of 25 μM or above, the ROS production by cocaine-stimulated myocytes was 3 fold of the control level and this was accompanied by severe cellular injury and cell death as revealed by TUNEL assay. In vitro knockout of Nox2 using siRNA completely inhibited cocaine-induced oxidative stress in cardiomyocytes. In conclusion, cocaine administration causes severe cardiac oxidative stress through the activation of Nox2. Increased ROS production contributes to MAPK activation and the subsequent cardiac myocyte injury and death. Inhibitors to Nox2 and antioxidants may have potential therapeutic value to the treatment of cocaine cardiotoxicity.

Afonso L, Mohammad T and Thatai D (2007) Crack whips the heart: A review of the cardiovascular toxicity of cocaine. Am J Cardiol 100:1040-1043.

Li J-M, Gall NP, Grieve DJ, Chen M and Shah AM (2002a) Activation of NADPH oxidase during progression of cardiac hypertrophy to failure. Hypertension 40:477-484

Bailey A, Yoo J-H, Racz I, Zimmer A and Kitchen I (2007) Preprodynorphin mediates locomotion and D2 dopamine and m- opioid receptor changes induced by chronic "binge" cocaine administration. J Neurochem 102:1817-1830..