β-Adrenergic Receptor–Stimulated Apoptosis in Cardiac Myocytes Is Mediated by Reactive Oxygen Species/c-Jun NH2-Terminal Kinase–Dependent Activation of the Mitochondrial Pathway
Stimulation of β-adrenergic receptors (βARs) causes apoptosis in adult rat ventricular myocytes (ARVMs). The role of reactive oxygen species (ROS) in mediating βAR-stimulated apoptosis is not known. Stimulation of βARs with norepinephrine (10 μmol/L) in the presence of prazosin (100 nmol/L) for 24 hours increased the number of apoptotic myocytes as determined by TUNEL staining by 3.6- fold. The superoxide dismutase/catalase mimetics Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP; 10 μmol/L) and Euk-134 decreased βAR-stimulated apoptosis by 89±6% and 76±10%, respectively. Infection with an adenovirus expressing catalase decreased βAR-stimulated apoptosis by 82±15%. The mitochondrial permeability transition pore inhibitor bongkrekic acid (50 μmol/L) decreased βAR-stimulated apoptosis by 76±8%, and the caspase inhibitor zVAD-fmk (25 μmol/L) decreased βAR-stimulated apoptosis by 62±11%. βAR-stimulated cytochrome c release was inhibited by MnTMPyP. βAR stimulation caused c-Jun NH2-terminal kinase (JNK) activation, which was abolished by MnTMPyP. Transfection with an adenovirus expressing dominant-negative JNK inhibited βAR-stimulated apoptosis by 81±12%, and the JNK inhibitor SP600125 inhibited both βAR-stimulated apoptosis and cytochrome c release. Thus, βAR-stimulated apoptosis in ARVMs involves ROS/JNK-dependent activation of the mitochondrial death pathway.
Beta-adrenergic receptor (βAR) stimulation in adult rat ventricular myocytes (ARVMs) causes apoptosis,1–5 which involves caspase activation.4 However, little is known about the signals proximal to caspase. Reactive oxygen species (ROS) can cause myocyte apoptosis,6–8 and ROS may mediate myocyte apoptosis in response to remodeling stimuli.9,10 ROS can induce myocyte apoptosis through c-Jun NH2-terminal kinase (JNK)-dependent activation of a mitochondrial pathway.11 We therefore tested the roles of ROS, JNK, and mitochondria in mediating βAR-stimulated cardiac myocyte apoptosis.
Materials and Methods
Isolation of ARVMs
ARVMs prepared as previously described1 were plated 30 to 50 cells/mm2 on 100-mm plastic culture dishes for Western blotting or 40×22 mm2 glass coverslips precoated with laminin (1 μg/cm2, Becton-Dickinson) for TUNEL staining.
l-Norepinephrine (NE; 10 μmol/L; Sigma) was added for 24 hours (TUNEL), 6 hours (cytochrome c), or 15 minutes (JNK). Prazosin (PZ; 0.1 μmol/L; Sigma) was added 30 minutes before NE. All plates were supplemented with ascorbic acid (100 μmol/L). Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP; 10 μmol/L; Calbiochem), Euk-13412 (Euk; 50 μmol/L; Eukarion), bongkrekic acid (BA; 50 μmol/L; Calbiochem), zVAD-fmk (zVAD; 25 μmol/L, Calbiochem), and SP600125 (SP; 2 μmol/L; Calbiochem) were added 30 minutes before NE.
Western Blotting for Cytochrome c and Phospho-JNK
ARVMs were infected with adenovirus expressing β-galactosidase (10 multiplicity of infection [MOI]), catalase (50 MOI; ATCC), or dominant-negative JNK (10 MOI; the T183A, Y185F JNK1 mutant,14 courtesy of R. Davis, University of Massachusetts, Worcester, Mass) 48 hours before addition of NE.
All data are mean±SEM. Differences across multiple conditions were tested by one-way ANOVA for repeated measures. Comparisons between conditions were tested by Student’s unpaired t test using the Bonferroni correction for multiple comparisons.
βAR-Stimulated Apoptosis Is ROS-Dependent and Involves the Mitochondrial Permeability Transition Pore and Caspase Activation
Under control conditions, 4% to 7% of cells were apoptotic as assessed by TUNEL staining. βAR stimulation with NE in the presence of PZ (NE/PZ) for 24 hours increased the number of apoptotic myocytes by 3.6±0.2-fold (Figure 1A). The superoxide dismutase (SOD)/catalase mimetics MnTMPyP and Euk decreased the magnitude of βAR-stimulated apoptosis by 89±6% and 76±10%, respectively (Figure 1A). Likewise, infection with an adenovirus expressing catalase decreased βAR-stimulated apoptosis by 82±15% (Figure 1B). Bongkrekic acid, an inhibitor of the mitochondrial permeability transition pore,15 decreased βAR-stimulated apoptosis by 76±8% (Figure 1A). The caspase inhibitor zVAD-fmk decreased βAR-stimulated apoptosis by 62±11% (Figure 1A).
βAR Stimulation Causes ROS-Dependent Mitochondrial Cytochrome c Release
βAR-Stimulated Apoptosis and Cytochrome c Release Are JNK-Dependent
βAR stimulation (15 minutes) caused a 2.3±0.4-fold increase in JNK activity. MnTMPyP decreased βAR-stimulated JNK activation to 1.0±0.3-fold (Figure 2A). The JNK inhibitor SP60012516 decreased βAR-stimulated apoptosis to 1.3±0.1-fold (Figure 2B) and decreased βAR-stimulated cytochrome c release to 1.6±0.4-fold (P<0.05; n=6). Likewise, infection with an adenovirus expressing dominant-negative JNK, which inhibited JNK activation by ≈70% (data not shown), decreased βAR-stimulated apoptosis by 81±12% (Figure 2C).
We1,2 and others3–5 have shown that βAR stimulation causes apoptosis in ARVMs. In the present study, βAR-stimulated apoptosis was inhibited by SOD/catalase mimetics or overexpression of catalase, thereby demonstrating that ROS play a necessary role in βAR-stimulated apoptosis and suggesting that the responsible ROS is H2O2 or a derivative.
We found that βAR stimulation caused mitochondrial cytochrome c release, which was ROS-dependent, and that the mitochondrial permeability transition pore inhibitor bongkrekic acid15 decreased βAR-stimulated apoptosis, thus implicating the mitochondrial pathway. ROS are known to cause mitochondrial cytochrome c release in cardiac myocytes.8,11
βAR stimulation in ARVMs causes activation of JNK, p38, and to a lesser extent, extracellular signal-regulated kinase (ERK).13 In the present study, βAR-stimulated JNK activation was inhibited by MnTMPyP, thus indicating that it is ROS-dependent. Further, infection with an adenovirus expressing dominant-negative JNK14 inhibited βAR-stimulated apoptosis, and the JNK inhibitor SP6001216 prevented both βAR-stimulated apoptosis and cytochrome c release. Thus, βAR activation of the mitochondrial death pathway requires the ROS-dependent activation of JNK. JNK can associate with mitochondria and mediate activation of the mitochondrial death pathway in cardiac myocytes in response to H2O2.11 In contrast, JNK may exert antiapoptotic effects in neonatal rat cardiac myocytes exposed to nitric oxide,17 suggesting that the role of JNK is cell-type and/or context-dependent. We previously found that βAR-stimulated apoptosis was increased by inhibition of p38 and not affected by inhibition of ERK.13
It is now recognized that ROS play an important role in signal transduction.18 We previously found that α-adrenergic receptor (αAR) stimulation causes ROS-dependent activation of the ras-raf-MEK-ERK1/2 cascade leading to hypertrophy.19,20 In contrast to βAR stimulation, αAR stimulation does not cause myocyte apoptosis.19 We have also found that ROS can mediate either hypertrophy or apoptosis in cardiac myocytes in response to mechanical strain, depending on the magnitude of strain.10 That αAR versus βAR and different amplitudes of mechanical strain link via ROS to distinct myocyte phenotypes suggests that qualitative and/or quantitative differences in ROS are critical determinants of ROS signaling in cardiac myocytes. The source of ROS that links βAR stimulation to apoptosis remains to be determined but may involve mitochondria, xanthine oxidase, NADPH oxidase, nitric oxide synthase, and/or cyclooxygenase.
These findings indicate that ROS play a central role in the regulation of myocyte apoptosis via JNK-dependent activation of the mitochondrial death pathway. ROS are thus a potential new target for the prevention of myocardial failure.
This work was supported by NIH grants HL61639 and HL20612 (to W.S.C.), HL057947 (to K.S.), HL03878 (to D.B.S.), and HL07224 (to S.K.); a grant from the Swiss National Science Foundation (to A.R.); and a Merit grant from the Department of Veterans Affairs (to K.S.).
Original received October 29, 2002; resubmission received December 17, 2002; accepted December 17, 2002.
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