Development of doxorubicin-induced chronic cardiotoxicity in the B6C3F₁ mouse model

Abstract

Serum levels of cardiac troponins serve as biomarkers of myocardial injury. However, troponins are released into the serum only after damage to cardiac tissue has occurred. Here, we report development of a mouse model of doxorubicin (DOX)-induced chronic cardiotoxicity to aid in the identification of predictive biomarkers of early events of cardiac tissue injury. Male B6C3F₁ mice were administered intravenous DOX at 3 mg/kg body weight, or an equivalent volume of saline, once a week for 4, 6, 8, 10, 12, and 14 weeks, resulting in cumulative DOX doses of 12, 18, 24, 30, 36, and 42 mg/kg, respectively. Mice were sacrificed a week following the last dose. A significant reduction in body weight gain was observed in mice following exposure to a weekly DOX dose for 1 week and longer compared to saline-treated controls. DOX treatment also resulted in declines in red blood cell count, hemoglobin level, and hematocrit compared to saline-treated controls after the 2nd weekly dose until the 8th and 9th doses, followed by a modest recovery. All DOX-treated mice had significant elevations in cardiac troponin T concentrations in plasma compared to saline-treated controls, indicating cardiac tissue injury. Also, a dose-related increase in the severity of cardiac lesions was seen in mice exposed to 24 mg/kg DOX and higher cumulative doses. Mice treated with cumulative DOX doses of 30 mg/kg and higher showed a significant decline in heart rate, suggesting drug-induced cardiac dysfunction. Altogether, these findings demonstrate the development of DOX-induced chronic cardiotoxicity in B6C3F₁ mice.

Introduction

Doxorubicin (DOX) is one of the most potent anthracycline antibiotics widely used to treat several types of solid tumors and hematologic malignancies (Ahmad et al., 2012, Batty et al., 2012, Carr, 2004, Honda et al., 2010, Masci et al., 2004, Stavridi and Palmieri, 2008). However, one of the serious complications associated with the clinical use of the drug is the risk of developing dose-related cumulative and irreversible cardiomyopathy, which can lead to life-threatening congestive heart failure (reviewed in Wouters et al., 2005). Among several biochemical markers of cardiac tissue damage, troponins are routinely utilized as the most sensitive and specific clinical biomarkers of myocardial injury (Lipshultz et al., 2004, Lipshultz et al., 2011). Several preclinical studies also have demonstrated the usefulness of serum levels of cardiac troponin T in determining the extent of myocardial damage caused by DOX (Bertinchant et al., 2003, Herman et al., 1998, Herman et al., 1999, Herman et al., 2001). However, these proteins are released in serum only after cardiac tissue damage has occurred. To control or prevent damage to the heart tissue during DOX treatment, it is crucial to identify biomarkers that would predict early stages of cardiac events before irreversible damage has occurred.

Doxorubicin has been extensively investigated for toxic effects of cumulative doses in the heart in various animal models. These include the rabbit (Herman and Ferrans, 1983, Jaenke, 1976, Solcia et al., 1981, Van Vleet and Ferrans, 1980), dog (Astra et al., 2003, Herman and Ferrans, 1983, Solcia et al., 1981, Tomlinson et al., 1986), pig (Van Vleet et al., 1979), monkey (Denine and Schmidt, 1975, Sieber et al., 1980), and the rat (Berthiaume and Wallace, 2007, Herman et al., 1985, Lebrecht et al., 2003, Solcia et al., 1981, Thompson et al., 2010, Zhou et al., 2001). Several studies have utilized mouse models in the evaluation of DOX-induced cardiac toxicity. However, these studies have primarily examined molecular events of cardiac toxicity at acute high doses instead of the chronic doses of DOX used in the clinical situation (Bjelogrlic et al., 2007, Doroshow et al., 1981, Matsumura et al., 1994, Olson et al., 2003, Papadopoulou et al., 1999, Rosenoff et al., 1975, Shuai et al., 2011, Tan et al., 2011). In addition, DOX was administered to the mice intraperitoneally, whereas DOX is administered intravenously in cancer patients. As a result, information concerning cardiotoxicity caused by chronic DOX exposure in mice is inadequate. One of the limitations of using the mouse in chronic toxicity studies is the difficulty of administering multiple intravenous injections of DOX over an extended period of time (Herman and Ferrans, 1998). This may, in part, be due to the ability of the drug to cause localized tissue damage.

Development of a mouse model of DOX-induced cardiotoxicity using the clinically relevant treatment strategy of smaller doses over a longer period of time will provide important insights into the early molecular events during drug exposure. This further will allow identification of early molecular markers of DOX-induced cardiac tissue injury that leads to a release of troponins. Genomic and genetic tools, such as a well-annotated complete genome sequence and transgenic models, are uniquely available for the mouse system. Application of these powerful genetic methods in the mouse can help validate the identified biomarkers for drug-induced cardiac tissue damage. In addition, comparison of functional and toxicological data from extensively investigated chronically exposed animal models to the findings in this newly developed chronic cardiotoxicity mouse model could advance our mechanistic understanding of DOX-mediated cardiotoxicity across the species, which may lead to the design of new, improved predictive biomarkers of cardiotoxicity. Translation of such biomarkers to the clinic may help in the prediction or early diagnosis of adverse cardiac events and also in designing novel treatment strategies to prevent or alleviate unwanted adverse events during DOX therapy.