Cardiac Peroxynitrite Formation and Left Ventricular Dysfunction following Doxorubicin Treatment in Mice

Abstract

Selective cardiotoxicity of doxorubicin remains a significant and dose-limiting clinical problem. The mechanisms involved have not been fully defined but may involve the production of reactive oxygen species and/or alteration of cardiac energetics. Here, we tested the hypotheses that doxorubicin causes left ventricular dysfunction in mice and is associated with dysregulation of nitric oxide in cardiac tissue, leading to the accumulation of 3-nitrotyrosine (a biomarker of peroxynitrite formation). Animals were dosed with doxorubicin (20 mg/kg i.p.), and left ventricular performance was assessed in vivo using M-mode and Doppler echocardiography. Five days after doxorubicin administration, left ventricular fractional shortening, cardiac output, and stroke volume parameters were significantly reduced relative to control values (30.0 +/- 3.6 versus 46.1 +/- 1. 6%, 8.9 +/- 0.9 versus 11.5 +/- 0.6 ml/min, and 21.2 +/- 0.1 versus 29.5 +/- 0.1 microl for doxorubicin versus control, P <.05). Statistically significant (P <.05) increases in the immunoprevalence of myocardial inducible nitric oxide synthase (33 +/- 18 versus 9 +/- 2%, via quantitative image analysis) and 3-nitrotyrosine formation (56 +/- 24 versus 0.3 +/- 0.4%) were also observed after doxorubicin. Correlation analyses revealed a highly significant inverse relationship between left ventricular fractional shortening and cardiac 3-nitrotyrosine immunoprevalence (P <.01). No such relationship was observed for inducible nitric oxide synthase. Western blot analyses of cardiac myofibrillar fractions revealed extensive nitration of an abundant 40-kDa protein, shown to be the myofibrillar isoform of creatine kinase. These data demonstrate that alteration of cardiac nitric oxide control and attendant peroxynitrite formation may be an important contributor to doxorubicin-induced cardiac dysfunction. Furthermore, nitration of key myofibrillar proteins and alteration of myocyte energetics are implicated.