Pharmacological inhibition of CB1 cannabinoid receptor protects against doxorubicin-induced cardiotoxicity

Abstract

Objectives: We aimed to explore the effects of pharmacologic inhibition of cannabinoid-1 (CB1) receptor in in vivo and in vitro models of doxorubicin (DOX)-induced cardiotoxicity.

Background: Doxorubicin is one of the most potent antitumor agents available; however, its clinical use is limited because of the risk of severe cardiotoxicity. Endocannabinoids mediate cardiodepressive effects through CB1 receptors in various pathophysiological conditions, and these effects can be reversed by CB1 antagonists.

Methods: Left ventricular function was measured by Millar pressure-volume system. Apoptosis markers, CB1/CB2 receptor expression, and endocannabinoid levels were determined by immunohistochemistry, Western blot, reverse transcription-polymerase chain reaction, real-time polymerase chain reaction, flow cytometry, fluorescent microscopy, and liquid chromatography/in-line mass spectrometry techniques.

Results: Five days after the administration of a single dose of DOX (20 mg/kg intraperitoneally) to mice, left ventricular systolic pressure, maximum first derivative of ventricular pressure with respect to time (+dP/dt), stroke work, ejection fraction, cardiac output, and load-independent indexes of contractility (end-systolic pressure-volume relation, preload-recruitable stroke work, dP/dt-end-diastolic volume relation) were significantly depressed, and the myocardial level of the endocannabinoid anandamide (but not CB1/CB2 receptor expression) was elevated compared with vehicle-treated control mice. Treatment with the CB1 antagonists rimonabant or AM281 markedly improved cardiac dysfunction and reduced DOX-induced apoptosis in the myocardium. Doxorubicin also decreased cell viability and induced apoptosis in the H9c2 myocardial cell line measured by flow cytometry and fluorescent microscopy, which were prevented by the preincubation of the cells with either CB1 antagonist, but not with CB1 and CB2 agonists and CB2 antagonists.

Conclusions: These data suggest that CB1 antagonists may represent a new cardioprotective strategy against DOX-induced cardiotoxicity.

Figure 1. Effects of CB₁ Antagonists on DOX-Induced Cardiac Dysfunction

Effect of doxorubicin (DOX) on left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), LV maximum first derivative of ventricular pressure with respect to time (+dP/dt), LV −dP/dt, heart rate, stroke work, ejection fraction, and cardiac output and tau (Weiss and Glantz) in mice. Mice were pretreated either with vehicle, rimonabant, or AM281, and treated with either vehicle or DOX, as indicated in the Methods section. Hemodynamic parameters were measured 5 days after DOX administration. Results are mean ± standard error of the mean of 7 to 13 experiments in each group. *p < 0.05 versus vehicle; #p < 0.05 versus DOX. CB₁ = cannabinoid-1.

Figure 2. Effect of CB₁ Antagonists on DOX-Induced Depression of Load-Independent Indexes of Cardiac Contractility

(A) Representative pressure–volume (P–V) loops obtained with a P–V conductance catheter system at different preloads after vena cava occlusion, showing differences in the end-systolic P–V relation (ESPVR) and between mice pretreated with vehicle, rimonabant, or AM281 and treated with vehicle or DOX. The less steep ESPVR in DOX-treated mice indicates decreased contractile function, which was improved by CB₁ antagonists. (B) Effects of CB₁ antagonists on DOX-induced depression of load-independent indexes of cardiac contractility. Results are mean ± standard error of the mean of 9 to 18 experiments in each group. *p < 0.05 versus vehicle; #p < 0.05 versus DOX. EDV = end-diastolic volume relation; ESPVR or Emax = end-systolic pressure–volume relation; PRSW = preload-recruitable stroke work; other abbreviations as in Figure 1.

Figure 3. Effects of CB₁ Antagonists on DOX-Induced Cell Death and Apoptosis In Vitro

(A) Effects of CB₁ antagonists on cell viability measured by XTT assays. AM281 and rimonabant (1 μmol/l) prevent cell death induced by 1 or 5 μmol/l of DOX. *p < 0.05 versus control group; #p < 0.05 versus DOX (n = 4). (B) Effects of CB₁ antagonists on the early apoptosis marker fluorescent annexin V conjugate (Annexin V-FITC) and cell death detection dye propidium iodide (PI) measured by flow cytometric analysis of H9c2. Representative data from 3 experiments analyzed. (C) Effects of CB₁ antagonists on active caspase expression (green) and nuclear staining pattern by Hoechst 33342 dye (blue). Please also note that some of the nuclei are fragmented in DOX-treated cells (indicative of late apoptosis), but not in CB₁ antagonist-treated or control cells. Representative data from at least 15 experiments analyzed. Abbreviations as in Figure 1.

Figure 4. Effects of CB₁ Agonist, CB₂ Antagonists, and Agonists on DOX-Induced Apoptosis in H9c2 In Vitro

Effects of CB₁ agonist, CB₂ antagonists, and agonists on the early apoptosis marker Annexin V-FITC and cell death detection dye propidium iodide (PI) measured by flow cytometric analysis of H9c2. Representative data from 3 experiments analyzed. Abbreviations as in Figures 1 and 3.

Figure 5. Effects of CB₁ Antagonists on DOX-Induced Apoptosis In Vivo

(A) Effects of CB₁ antagonists on DOX-induced caspase-3 activation analyzed by Western blot from heart tissue homogenates. Treatment with rimonabant or AM281 reduced myocardial caspase-3 activation in DOX-treated mice. The blot was also probed for beta-actin level for loading control. Representative blot from at least 5 experiments. (B) Effects of CB₁ antagonists on DOX-induced caspase-3 activity analyzed by colorimetric method from heart tissue homogenates. Treatment with rimonabant or AM281 reduced caspase-3 activity in DOX-treated mice. *p < 0.05 versus vehicle; #p < 0.05 versus DOX (n = 4 per group). (C) Effects of CB₁ antagonists on DOX-induced caspase-3 and caspase-9 gene expression. Data were analyzed with 2 housekeeping genes, and data presented here were normalized with beta-actin. Treatment with rimonabant or AM281 reduced myocardial caspase-3 and caspase-9 gene expression in DOX-treated mice. *p < 0.05 versus vehicle; #p < 0.05 versus DOX (n = 6 to 15 per group). Abbreviations as in Figure 1.

Figure 6. Effects of Rimonabant and AM281 on DOX-Induced Myocardial Apoptosis In Vivo Determined by TUNEL and DNA Fragmentation Assay

(A) Note the increased myocardial terminal deoxynucleotidyltransferase-mediated nick-end labeling (TUNEL) staining from doxorubicin (DOX)-treated mice (brown). Treatment with cannabinoid-1 antagonist rimonabant and AM281 reduced myocardial TUNEL staining in DOX-treated mice. Similar immunohistochemical profiles were seen in n = 3 hearts per group. Quantitative measurements were carried out in 20 fields per group. *p < 0.05 versus vehicle (Veh); #p < 0.05 versus DOX. (B) Effects of rimonabant and AM281 on DOX-induced myocardial apoptosis in vivo by TUNEL assay. *p < 0.05 versus vehicle; #p < 0.05 versus DOX (n = 4 per group). (C) Effects of rimonabant and AM281 on DOX-induced deoxyribonucleic acid (DNA) fragmentation in vivo. Changes are expressed in % of myocardial DNA fragmentation in vehicle-treated mice (100%). *p < 0.05 versus vehicle; #p < 0.05 versus DOX (n = 4 per group). Eu = Europium.

Figure 7. Effects of DOX on Myocardial Endocannabinoid Content and CB _1/2 Receptor Expression

(A) Evidence of CB₁ receptor protein expression in total lysate of DOX-treated mouse heart tissue homogenate by Western blot analysis. (B and D) Evidence of CB₁ and CB₂ receptor gene expression by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) from complementary deoxyribonucleic acid of both untreated and DOX-treated mouse heart samples (B) and cardiomyocytes (D). (C) Evidence of CB₁ receptor and CB₂ receptor gene expression in heart tissue by quantitative real-time PCR after normalization to beta-actin (n = 6 to 15). (E) Effect of DOX on anandamide (AEA) and 2-arachidonylglycerol (2-AG) production in vivo by liquid chromatography mass spectrometry (LCMS) analysis of heart tissue samples. *p < 0.05 versus vehicle (n = 6). (F) Effect of DOX (1 μM for 2 h) on AEA and 2-AG production in vitro by LCMS analysis of H9c2 cells. Increased AEA levels were observed in doxorubicin treated samples. *p < 0.05 versus vehicle (n = 3). KO = knockout; other abbreviations as in Figure 1.