Resveratrol attenuates azidothymidine-induced cardiotoxicity by decreasing mitochondrial reactive oxygen species generation in human cardiomyocytes

Abstract

Nucleotide reverse transcriptase inhibitors, such as zidovudine (azidothymidine, AZT) and stavudine, represent a class of approved antiretroviral agents for highly active antiretroviral therapy, which prolongs the life expectancy of patients infected with human-immunodeficiency virus. Unfortunately, the use of these drugs is associated with known toxicities in the liver, skeletal muscle, heart and other organs, which may involve increased reactive oxygen species (ROS) generation, among other mechanisms. Resveratrol is a polyphenolic plant-derived antioxidant abundantly found in certain grapes, roots, berries, peanuts and red wine. This study, using primary human cardiomyocytes, evaluated the effects of AZT and pre-treatment with resveratrol on mitochondrial ROS generation and the cell death pathways. AZT induced concentration-dependent cell death, involving both caspase-3 and -7 and poly(ADP-ribose) polymerase activation, coupled with increased mitochondrial ROS generation in human cardiomyocytes. These effects of AZT on mitochondrial ROS generation and cell death may be attenuated by resveratrol pre-treatment. The results demonstrate that mitochondrial ROS generation plays a pivotal role in the cardiotoxicity of AZT in human cardiomyocytes, and resveratrol may provide a potential strategy to attenuate these pathological alterations, which are associated with widely used antiretroviral therapy.

Figure 1

Resveratrol attenuates AZT-induced cell death in human cardiomyocytes. Human cardiomyocytes were pre-treated with or without various concentrations of resveratrol for 2 h, followed by treatment with various doses of AZT with or without resveratrol. After 48 h, the cells were harvested for flow cytometric analysis with Annexin V for apoptosis and Sytox Green for necrosis. Left panel, representative flow cytometry charts demonstrating the effects of AZT with or without resveratrol pre-treatment on cell death (apoptotic and necrotic) in human primary cardiomyocytes; right panel, the quantification of apoptotic and necrotic cell death from the left panel. Values represent the means ± SEM (n=4). *P<0.05, vehicle vs. AZT; ^#P<0.05, AZT with or without resveratrol, respectively.

Figure 2

Resveratrol attenuates the AZT induction of caspase-3 and -7 activity in human cardiomyocytes. Human cardiomyocytes were treated with resveratrol and AZT, as described in Fig. 1. The cells were harvested and caspase-3 and -7 activity was measured. Left panel, the concentration-dependent effect of AZT on caspase-3 and -7 activity in human cardiomyocytes; right panel, the effects of AZT with or without resveratrol pre-treatment. Values represent the means ± SEM (n=3-6). *P<0.05, vehicle vs. AZT; ^#P<0.05, AZT with or without resveratrol, respectively.

Figure 3

Resveratrol attenuates the AZT induction of PARP activity in human cardiomyocytes. Human cardiomyocytes were treated as described in Fig. 1. The cells were harvested and PARP activity was measured. Left panel, the concentration-dependent effect of AZT on PARP activity in human cardiomyocytes; right panel, the effects of AZT with or without resveratrol pre-treatment. Values represent the means ± SEM (n=8). *P<0.05, vehicle vs. AZT; ^#P<0.05, AZT with or without resveratrol, respectively.

Figure 4

Resveratrol attenuates AZT-induced mitochondrial ROS generation in human cardiomyocytes. Human cardiomyocytes were treated as described in Fig. 1. The cells were harvested and analyzed by flow cytometric analysis using MitoSOX Red dye. Left panel, representative flow cytometry charts demonstrating the effects of AZT with or without resveratrol pre-treatment on mitochondrial ROS generation in human primary cardiomyocytes; right panel, the quantification of MitoSOX Red mean intensity from the left panel (3 experiments). Values represent the means ± SEM (n=4). *P<0.05, vehicle vs. AZT; ^#P<0.05, AZT with or without resveratrol, respectively.