CB1 cannabinoid receptors promote oxidative stress and cell death in murine models of doxorubicin-induced cardiomyopathy and in human cardiomyocytes

Abstract

Aims: Here we investigated the mechanisms by which cardiovascular CB1 cannabinoid receptors may modulate the cardiac dysfunction, oxidative stress, and interrelated cell death pathways associated with acute/chronic cardiomyopathy induced by the widely used anti-tumour compound doxorubicin (DOX).

Methods and results: Both load-dependent and -independent indices of left-ventricular function were measured by the Millar pressure-volume conductance system. Mitogen-activated protein kinase (MAPK) activation, cell-death markers, and oxidative/nitrosative stress were measured by molecular biology/biochemical methods and flow cytometry. DOX induced left-ventricular dysfunction, oxidative/nitrosative stress coupled with impaired antioxidant defense, activation of MAPK (p38 and JNK), and cell death and/or fibrosis in hearts of wide-type mice (CB1(+/+)), and these effects were markedly attenuated in CB1 knockouts (CB1(-/-)). In human primary cardiomyocytes expressing CB1 receptors (demonstrated by RT-PCR, western immunoblot, and flow cytometry) DOX, likewise the CB1 receptor agonist HU210 and the endocannabinoid anandamide (AEA), induced MAPK activation and cell death. The DOX-induced MAPK activation and cell death were significantly enhanced when DOX was co-administered with CB1 agonists AEA or HU210. Remarkably, cell death and MAPK activation induced by AEA, HU210, and DOX +/- AEA/HU210 were largely attenuated by either CB1 antagonists (rimonabant and AM281) or by inhibitors of p38 and JNK MAPKs. Furthermore, AEA or HU210 in primary human cardiomyocytes triggered increased reactive oxygen species generation.

Conclusion: CB1 activation in cardiomyocytes may amplify the reactive oxygen/nitrogen species-MAPK activation-cell death pathway in pathological conditions when the endocannabinoid synthetic or metabolic pathways are dysregulated by excessive inflammation and/or oxidative/nitrosative stress, which may contribute to the pathophysiology of various cardiovascular diseases.

Figure 1

Improved DOX-induced acute cardiac dysfunction in CB₁ knockout mice. (A) Representative baseline pressure–volume (P–V) loops and derived parameters. Panels at right show volume (red), pressure (blue), and ±dP/dt (green) derived from the pressure signal. DOX-induced profound cardiac dysfunction in CB₁^+/+ mice evidenced by rightward shift of P–V relations, decrease in left-ventricular systolic pressure (LVSP), maximum first derivative of ventricular pressure with respect to time (+dP/dt), stroke work, ejection fraction, cardiac output, and an increase in left-ventricular end-diastolic pressure (LVEDP), and prolongation of relaxation time constants (τ Weiss and Glantz) 5 days following the administration of 20 mg/kg intra-peritoneally. These changes were largely attenuated in CB₁^−/− mice. (B) Representative pressure–volume (P–V) loops at different preloads after vena cava occlusion, showing differences in the end-systolic P–V relation (ESPVR) in CB₁^+/+ and CB₁^−/− mice treated with vehicle or DOX. The less steep ESPVR in DOX-treated mice indicates decreased contractile function, which was less pronounced in CB₁^−/− mice treated with DOX compared with CB₁^+/+ mice treated with the drug. DOX markedly decreased load-independent indexes of contractility [preload-recruitable stroke work (PRSW), dP/dt–end-diastolic volume relation (dP/dt–EDV), and end-systolic pressure–volume relation (Emax), respectively] in CB₁^+/+ mice, which was attenuated in knockouts. Results are mean ± SEM of 8–11 experiments/group. *P < 0.05 vs. vehicle; ^#P < 0.05 CB₁^+/+ + DOX.

Figure 2

Acute DOX-induced myocardial oxidative/nitrosative stress, impaired antioxidant defense is attenuated in CB₁^−/− mice. (A) Shows the lipid peroxides (4hydroxynonenal—4HNE) and nitrotyrosine (NT) accumulation in the groups as indicated. (B) Depicts myocardial glutathione peroxidase, superoxide dismutase (SOD) activities, and glutathione content. (C) Myocardial endocannabinoid metabolizing enzyme (FAAH) activity as indicated. *P < 0.05 vs. vehicle; ^#P < 0.05 vs. CB₁^+/+ + DOX (n = 6–10/group).

Figure 3

Acute DOX-induced myocardial MAPKs activation and cytochrome c release is attenuated in CB₁^−/− mice. (A) Representative blots depicting the MAPK activation in CB₁^+/+ or CB₁^−/− mice treated with DOX. (B) Representative western immunoblot depicting the cytochrome c release in the cytosol, 5 days after DOX administration. *P < 0.05 vs. vehicle; ^#P < 0.05 vs. CB₁^+/+ + DOX (n = 6/group).

Figure 4

Acute DOX-induced myocardial apoptosis/necrosis is attenuated in CB₁^−/− mice. (A) Cleaved caspase 3 activation by western blot analysis and (B) caspase 3/7 and (C) PARP activity in myocardial tissues from CB₁^+/+ or CB₁^−/− mice treated with vehicle or DOX 5 days following the exposure. *P < 0.05 vs. vehicle; ^#P < 0.05 vs. CB₁^+/+ + DOX (n = 6/group). (D) Representative TUNEL staining (green), showing that the DOX-induced apoptosis was present both in cardiomyocytes and endothelial cells, which was attenuated in CB₁^−/− mice, irrespective of the cell type. Red colour represents cardiomyocyte specific α-actinin staining, blue nuclei of all cells, green or light green/white TUNEL positive cells (the latter in overlaid images). There were no TUNEL positive cells in vehicle treated mice (data not shown).

Figure 5

Improved DOX-induced chronic cardiac dysfunction and decreased myocardial fibrosis in CB₁ knockout mice. (A) Treatment of wide type CB₁^+/+ mice with (cDOX), 5 mg/kg intra-peritoneally at days 1, 7, 14, and 21 induced a significantly decreased 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 (PRSW, dP/dt–EDV, Emax), and an increase in left-ventricular end-diastolic pressure (LVEDP) and prolongation of relaxation time constants (τ Weiss and Glantz) 35 days following its administration, which was attenuated in CB₁^−/− mice. (B) Genetic deletion of CB₁ attenuates chronic DOX(cDOX)-induced myocardial fibrosis Left: Sirius red staining of paraffin sections of heart tissues from respective groups and treatments as shown. Right: quantitative real-time PCR for the mRNA expression of fibrosis markers in the myocardium as indicated. Results are mean ± SEM of 10–14 experiments in each group for haemodynamics measurements, and 6/group for gene expressions. *P < 0.05 vs. vehicle in CB₁^+/+/CB₁^−/− mice; ^#P < 0.05 vs. CB₁^+/+ + DOX.

Figure 6

cDOX-induced myocardial oxidative/nitrosative stress and cell death is attenuated in CB₁^−/− mice. Myocardial (A) HNE and nitrotyrosine accumulation, (B) apoptosis markers and mRNA expressions of (C) NADPH oxidase isoforms (gp91phox, NOX4, p67, and p47phox) and (D) metalloproteinases 2 and 9 (MMP2/9). *P < 0.05 vs. vehicle; ^#P < 0.05 vs. CB₁^+/+ + DOX (n = 6–10/group).

Figure 7

CB₁ receptor activation with synthetic/endogenous agonists or DOX induces CB₁-dependent MAPK activation in human cardiomyocytes (HCM). Concentration- and time-dependent effects of HU210 (A) and AEA (B) on activation of p38 and JNK MAPKs. (C) DOX-, AEA-, HU210, or DOX ± AEA/HU210-induced MAPKs activation is attenuated by CB₁ receptor antagonist SR141716(SR1). HCM were treated with HU210, AEA, DOX, or SR1 alone at indicated concentrations for 40 min or first treated with SR1 for 1 h, followed by continued incubation with DOX alone or in combination with AEA/HU210 for 40 min, and MAPK activation was determined by western blot. Shows MAPK activation upon respective treatments. Adjacent panels describe the quantification (represented as fold change). *P < 0.05 vs. vehicle; ^#P < 0.05 vs. cells treated without SR1 with indicated treatments (n = 4).

Figure 8

HU210/AEA/DOX or their combination-induced cell death is attenuated by CB₁ antagonists in human cardiomyocytes (HCM); CB₁ activation increases ROS generation in HCM. (A and B) Cells were treated as indicated and cell death was determined by flow cytometry as described in the methods (A) *P < 0.05 vs. vehicle; ^#P < 0.05 vs. HU210 (n = 4) (B) *P < 0.05 vs. vehicle; ^†P < 0.05 vs. AEA/HU210 alone; ^#P < 0.05 vs. AEA ± HU210 ± DOX treated cells (n = 4). (C) Shows the quantification of ROS generation by flow cytometry as per the treatments indicated. *P < 0.05 vs. vehicle; #P < 0.05 vs. AEA/HU210 (n = 4). (see also Supplementary material online, Figure S4).