Cytokine-mediated apoptosis in cardiac myocytes: the role of inducible nitric oxide synthase induction and peroxynitrite generation.

Increased production of nitric oxide (NO) after induction of the cytokine-inducible isoform of nitric oxide synthase (iNOS or NOS2) in cardiac myocytes and other parenchymal cells within the heart may in addition to contributing to myocyte contractile dysfunction also contribute to the induction of programmed cell death (apoptosis). To investigate the mechanism(s) by which increased NO production leads to apoptosis, we examined the role of NO in primary cultures of neonatal rat ventricular myocytes (NRVMs) after induction by the cytokines interleukin-1beta (IL-1beta) and interferon gamma (IFNgamma) or exposure to the exogenous NO donor S-nitroso-N-acetylcysteine (SNAC) or peroxynitrite (ONOO(-)). Both SNAC (1 mmol/L) and ONOO(-) (100 micromol/L), but not their respective controls (ie, N-acetylcysteine and pH-inactivated ONOO(-)), induced apoptosis in confluent, serum-starved NRVMs at 48 hours. Similarly, incubation of NRVMs with IL-1beta and IFNgamma for 48 hours resulted in an increase in iNOS expression, nitrite production, and programmed cell death. Both the cytokine-induced nitrite accumulation and myocyte apoptosis could be completely prevented by the nonselective NOS inhibitor L-nitroarginine (3 mmol/L) or the specific iNOS inhibitor 2-amino-5, 6-dihydro-6-methyl-4H-1,3-thiazine (AMT, 100 micromol/L). NO-mediated myocyte apoptosis was not attenuated by the inhibition of soluble guanylyl cyclase with ODQ, nor could apoptosis be induced by the incubation of NRVMs with 1 mmol/L 8-bromo-cGMP, a cell-permeant cGMP analogue. However, NO-mediated apoptosis was significantly attenuated by the superoxide dismutase mimetic and ONOO(-) scavenger Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP, 100 micromol/L). NO/ONOO(-)-mediated apoptosis was associated with increased expression of Bax with no change in Bcl-2 mRNA abundance. Furthermore, apoptotic cell death was also confirmed in adult rat ventricular myocytes (ARVMs) when grown in heteroculture with IL-1beta- and IFNgamma-treated rat cardiac microvascular endothelial cells. Therefore, cytokine-induced apoptosis in NRVMs and ARVMs is mediated by iNOS induction, ONOO(-), and associated with an increase in Bax levels.

Mice lacking inducible nitric oxide synthase have improved left ventricular contractile function and reduced apoptotic cell death late after myocardial infarction.

Nitric oxide produced by inducible nitric oxide synthase (NOS2) has been implicated in the pathophysiology of chronic myocardial remodeling and failure. We tested the role of NOS2 in left ventricular (LV) remodeling early (1 month) and late (4 months) after myocardial infarction (MI) in mice lacking NOS2. MI size measured 7 days, 1 month, and 4 months after MI was the same in NOS2 knockout (KO) and wild-type (WT) mice. The LV end-diastolic pressure-volume relationship measured by the isovolumic Langendorff technique showed a progressive rightward shift from 1 to 4 months after MI in WT mice. LV developed pressure measured over a range of LV volumes was reduced at 1 and 4 months after MI in WT mice (P<0.05 and P<0.01 versus shams, respectively). In KO mice, the rightward shift was similar to that in WT mice at 1 and 4 months after MI, as was peak LV developed pressure at 1 month after MI. In contrast, at 4 months after MI, peak LV developed pressure in KO mice was higher than in WT mice (P<0.05 versus WT) and similar to that in sham-operated mice. At 1 month after MI, the frequency of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive myocytes in the remote myocardium was increased to a similar extent in WT and KO mice. At 4 months after MI, the frequency of apoptotic myocytes was increased in WT mice but not in KO mice (P<0.05 versus WT). Improved contractile function and reduced apoptosis were associated with reduced mortality rate in KO mice at 4 months after MI. Thus, NOS2 does not play an important role in determining infarct size or early LV remodeling during the first month after MI. In contrast, during late (ie, 4 months after MI) remodeling, NOS2 in remote myocardium contributes to decreased contractile function, increased myocyte apoptosis in remote myocardium, and reduced survival.

Exaggerated left ventricular dilation and reduced collagen deposition after myocardial infarction in mice lacking osteopontin.

Osteopontin (OPN), an extracellular matrix protein, is expressed in the myocardium with hypertrophy and failure. We tested the hypothesis that OPN plays a role in left ventricular (LV) remodeling after myocardial infarction (MI). Accordingly, OPN expression and LV structural and functional remodeling were determined in wild-type (WT) and OPN knockout (KO) mice 4 weeks after MI. Northern analysis showed increased OPN expression in the infarcted region, peaking 3 days after MI and gradually decreasing over the next 28 days. In the remote LV, OPN expression was biphasic, with peaks at 3 and 28 days. In situ hybridization and immunohistochemical analyses showed increased OPN mRNA and protein primarily in the interstitium. Infarct size, heart weight, and survival were similar in KO and WT mice after MI (P=NS), whereas the lung wet weight/dry weight ratio was increased in the KO mice (P<0.005 versus sham-operated mice). Peak LV developed pressure was reduced to a similar degree after MI in the KO and WT mice. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive myocytes was similar in KO and WT mice after MI. In contrast, post-MI LV chamber dilation was approximately twice as great in KO versus WT mice (P<0.001). Myocyte length increased after MI in WT mice (P<0.001) but not in KO mice. Electron microscopy showed increased collagen content in WT mice after MI but not in KO mice after MI. Type I collagen content was increased approximately 3-fold and approximately 7-fold in remote and infarcted regions, respectively, of WT hearts after MI but not in KO hearts (P<0.01 versus WT hearts). Likewise, Northern analyses showed increased collagen I(alpha(1)) mRNA after MI in remote regions of WT hearts but not in KO hearts. Thus, increased OPN expression plays an important role in regulating post-MI LV remodeling, at least in part, by promoting collagen synthesis and accumulation.

Reactive oxygen species mediate amplitude-dependent hypertrophic and apoptotic responses to mechanical stretch in cardiac myocytes.

Oxidative stress stimulates both growth and apoptosis in cardiac myocytes in vitro. We investigated whether oxidative stress mediates hypertrophy and apoptosis in cyclically stretched ventricular myocytes. Neonatal rat ventricular myocytes cultured on laminin-coated silastic membranes were stretched cyclically (1 Hz) at low (nominal 5%) and high (nominal 25%) amplitudes for 24 hours. Stretch caused a graded increase in superoxide anion production as assessed by superoxide dismutase (SOD)-inhibitable cytochrome c reduction or electron paramagnetic resonance spectroscopy. The role of reactive oxygen species (ROS) was assessed using the cell-permeable SOD/catalase mimetics Mn(II/III)tetrakis(1-methyl-4-peridyl) (MnTMPyP) and EUK-8. Stretch-induced increases in protein synthesis ((3)H-leucine incorporation) and cellular protein content were completely inhibited by MnTMPyP (0.05 mmol/L) at both low and high amplitudes of stretch. In contrast, while MnTMPyP inhibited basal atrial natriuretic factor (ANF) mRNA expression, the stretch-induced increase in ANF mRNA expression was not inhibited by MnTMPyP. In contrast to hypertrophy, only high-amplitude stretch increased myocyte apoptosis, as reflected by increased DNA fragmentation on gel electrophoresis and an approximately 3-fold increase in the number of TUNEL-positive myocytes. Similarly, only high-amplitude stretch increased the expression of bax mRNA. Myocyte apoptosis and bax expression stimulated by high-amplitude stretch were inhibited by MnTMPyP. Both low- and high-amplitude stretch caused rapid phosphorylation of ERK1/2, while high-, but not low-, amplitude stretch caused phosphorylation of JNKs. Activation of both ERK1/2 and JNKs was ROS-dependent. Thus, cyclic strain causes an amplitude-related increase in ROS, associated with differential activation of kinases and induction of hypertrophic and apoptotic phenotypes.

Platelet-activating factor is a general membrane perturbant.

Platelet-activating factor (PAF) is, at physiological (nanomolar) concentrations, a potent mediator of inflammation and coagulation. At pharmacological (micromolar) concentrations, PAF induces a variety of effects in diverse tissues. Here we show that PAF at micromolar concentrations is a membrane perturbant. Micromolar PAF alters the properties of channels formed by gramicidin A, and at concentrations greater than or equal to 4 microM disrupts the barrier properties of the host lipid bilayer. PAF thus can act as a detergent and non-specifically alter the behavior of membranes and membrane proteins. This may provide an explanation for some of the effects of PAF seen at high concentrations in vitro.

Opposing effects of beta(1)- and beta(2)-adrenergic receptors on cardiac myocyte apoptosis : role of a pertussis toxin-sensitive G protein.

BACKGROUND: beta-Adrenergic receptor (beta-AR) stimulation increases apoptosis in adult rat cardiac (ventricular) myocytes (ARVMs) via activation of adenylyl cyclase. beta(2)-ARs may couple to a G(i)-mediated signaling pathway that can oppose the actions of adenylyl cyclase. METHODS AND RESULTS: In ARVMs, beta-AR stimulation for 24 hours increased the number of apoptotic cells as measured by flow cytometry. beta-AR-stimulated apoptosis was abolished by the beta(1)-AR-selective antagonist CGP 20712A (P<0.05 versus beta-AR stimulation alone) but was potentiated by the beta(2)-AR-selective antagonist ICI 118,551 (P<0.05 versus beta-AR stimulation alone). The muscarinic agonist carbachol also prevented beta-AR-stimulated apoptosis (P<0.05 versus beta-AR stimulation alone), whereas pertussis toxin potentiated the apoptotic action of beta-AR stimulation (P<0.05 versus beta-AR stimulation alone) and prevented the antiapoptotic action of carbachol. CONCLUSIONS: In ARVMs, stimulation of beta(1)-ARs increases apoptosis via a cAMP-dependent mechanism, whereas stimulation of beta(2)-ARs inhibits apoptosis via a G(i)-coupled pathway. These findings have implications for the pathophysiology and treatment of myocardial failure.

Cell therapy attenuates deleterious ventricular remodeling and improves cardiac performance after myocardial infarction.

BACKGROUND: Myocardial infarction (MI) promotes deleterious remodeling of the myocardium, resulting in ventricular dilation and pump dysfunction. We examined whether supplementing infarcted myocardium with skeletal myoblasts would (1) result in viable myoblast implants, (2) attenuate deleterious remodeling, and (3) enhance in vivo and ex vivo contractile performance. METHODS AND RESULTS: Experimental MI was induced by 1-hour coronary ligation followed by reperfusion in adult male Lewis rats. One week after MI, 10(6) myoblasts were injected directly into the infarct region. Three groups of animals were studied at 3 and 6 weeks after cell therapy: noninfarcted control (control), MI plus sham injection (MI), and MI plus cell injection (MI+cell). In vivo cardiac function was assessed by maximum exercise capacity testing and ex vivo function was determined by pressure-volume curves obtained from isolated, red cell-perfused, balloon-in-left ventricle (LV) hearts. MI and MI+cell hearts had indistinguishable infarct sizes of approximately 30% of the LV. At 3 and 6 weeks after cell therapy, 92% (13 of 14) of MI+cell hearts showed evidence of myoblast graft survival. MI+cell hearts exhibited attenuation of global ventricular dilation and reduced septum-to-free wall diameter compared with MI hearts not receiving cell therapy. Furthermore, cell therapy improved both post-MI in vivo exercise capacity and ex vivo LV systolic pressures. CONCLUSIONS: Implanted skeletal myoblasts form viable grafts in infarcted myocardium, resulting in enhanced post-MI exercise capacity and contractile function and attenuated ventricular dilation. These data illustrate that syngeneic myoblast implantation after MI improves both in vivo and ex vivo indexes of global ventricular dysfunction and deleterious remodeling and suggests that cellular implantation may be beneficial after MI.

Adrenergic regulation of myocardial apoptosis.

Increased sympathetic nerve activity to the myocardium is a central feature in patients with heart failure. Norepinephrine, the primary transmitter of the sympathetic nervous system, signals via binding to alpha- and beta-adrenergic receptors (AR) that are coupled to G-proteins. Pharmacologic studies of cardiac myocytes in vitro demonstrate that beta-AR can stimulate apoptosis. Likewise, in transgenic mice overexpression of beta 1-AR or G alpha s is associated with myocyte apoptosis and the development of dilated cardiomyopathy. Whereas beta 1-AR stimulate apoptosis in vitro and in vivo, beta 2-AR may either stimulate or inhibit apoptosis and myocardial failure depending on the level of expression. Receptors coupling to Gi and Gq may also be able to mediate or modulate apoptosis and the development of myocardial failure, suggesting the potential for interactions between the beta-AR system and numerous remodeling stimuli that act through Gi or Gq signaling pathways. It appears likely that the mitogen-activated protein kinase superfamily plays a key role in mediating the actions of adrenergic pathways on myocyte apoptosis. These observations suggest that the adrenergic nervous system plays an important role in the regulation of myocyte apoptosis, and may thus contribute to the development of myocardial failure.

Nitric oxide in the failing myocardium.

It is now apparent that NO is produced in the myocardium. There it plays a central role in normal myocardial physiology. In addition, NO has the ability to exert whether beneficial or deleterious effects on the structure and function of the myocardium. At low, "physiologic" concentrations, NO may protect from deleterious stimuli such as mechanical stress and norepinephrine. At higher, "pathologic" concentrations, NO may cause the loss of myocytes. The mechanisms by which NO exerts these contrasting effects may involve decreases and increases in oxidative stress, respectively. A better understanding of the role NO plays in the development and progression of myocardial failure may lead to new treatment strategies.

Mechanism of fluid secretion in isolated shark renal proximal tubules.

Renal proximal tubules from the glomerular Squalus acanthias were studied for evidence of fluid secretion by closing one end of isolated tubules and leaving the other end open so that secreted fluid could be collected. When tubules were bathed in shark Ringer, fluid secretion rate was 27.6 +/- 3.9 (SE) pl X min-1 X mm-1 (21 tubules). Dibutyryl cAMP stimulated fluid secretion 50% (P less than 0.02, n = 14), furosemide inhibited fluid secretion 50% (P less than 0.01, n = 6), and metabolic inhibitors blocked fluid secretion nearly 100%. Secreted fluid was slightly hyperosmotic to peritubular bath (P less than 0.01, n = 7) but Na, Cl, S, K, and Ca concentrations were not significantly different from bath concentrations (wavelength-dispersive spectroscopy, electron probe analysis). cAMP had no effect on secreted fluid composition, and in some tubules cAMP did not stimulate fluid secretion. In conjunction with previous data we propose that spontaneous fluid secretion is driven by secretion of NaCl. However, finding the mechanism of NaCl and fluid secretion in glomerular renal tubules offers new perspectives of some previously inexplicable phenomena in the renal physiology of fish.

Dibutyryl-cAMP increases basolateral sodium conductance of mosquito Malpighian tubules.

Dibutyryladenosine 3',5'-cyclic monophosphate (cAMP) stimulates fluid secretion in isolated Malpighian tubules of the mosquito Aedes aegypti. In the present study the effects of cAMP on the basolateral membrane were studied with conventional microelectrodes. Membrane conductances were evaluated from the changes of the basolateral membrane potential (Vbl) consequent to ion changes in the bath. Under control conditions, Vbl measured -65.2 +/- 1.5 mV [83 impalements, 67 tubules]. A fivefold decrease in the bath Na concentration hyperpolarized Vbl by 10.2 +/- 0.6 mV [7], whereas a 4.4-fold increase in the bath K concentration depolarized Vbl by 7.9 +/- 1.0 mV [9]. In the presence of cAMP (10(-4) M) Vbl depolarized to -24.8 +/- 2.7 mV [9]. Vbl now hyperpolarized by 22.7 +/- 1.5 mV [7] for the bath Na change and depolarized by only 3.8 +/- 1.1 mV [6] for the bath K change. Thus the dominant effect of cAMP is the increase of the basolateral membrane Na conductance. This increase is consistent with 1) the depolarization of Vbl and 2) the hyperpolarization of the transepithelial voltage, the decrease of the transepithelial resistance, and the increase of Na and fluid secretion observed previously. Spontaneous oscillations of Vbl were observed and could not be attributed to cyclical changes of the basolateral membrane Na conductance.

Gramicidin single-channel properties show no solvent-history dependence.

The structure of membrane-associated gramicidins can depend on the solvent in which they were dissolved prior to membrane incorporation (LoGrasso, P. V., F. Moll, and T. A. Cross 1988. Biophys. J. 54:259-267; Killian, J. A., K. U. Prasad, D. Hains, and D. W. Urry. 1988. Biochemistry. 27:4848-4855). The peptide's solvent history might thus affect the functional characteristics of gramicidin channels (op. cit.). We tested this proposal by examining the properties (conductance, conductance dispersity, and average duration) of channels formed by [Val1]gramicidin A that had been dissolved in eight different solvents. The peptide was incorporated into lipid bilayers either by addition to the aqueous phase (and subsequent adsorption to the membrane) or by cosolubilization with the membrane-forming phospholipid. When the peptide was cosolubilized with the phospholipid, the channel properties did not vary with the solvent used. When the peptide was dissolved in chloroform, benzene, or trifluoroethanol and added through the aqueous phase, the channel properties differed from those found when gramidicin was dissolved in methanol, ethanol, dioxane, dimethylsulfoxide, or ethylacetate. The changes observed with the former three solvents were reproduced by adding them to the aqueous phase, and are therefore due to the ability of these solvents to partition into the membrane and alter the channels' behavior.

Induction of conductance heterogeneity in gramicidin channels.

In previous work from our laboratory, 5-10% of the channels formed by [Val1]gramicidin A have conductances that fall outside the narrow range that conventionally has defined the standard gramicidin channel [e.g., see Russell et al. (1986) Biophys. J. 49, 673]. Reports from other laboratories, however, show that up to 50% of [Val1]gramicidin channels have conductances that fall outside the range for standard channels [e.g., see Prasad et al. (1986) Biochemistry 25, 456]. This laboratory-to-laboratory variation in the distribution of gramicidin single-channel conductances suggests that the conductance variants are induced by some environmental factor(s) [Busath et al. (1987) Biophys. J. 51, 79]. In order to test whether extrinsic agents can induce such conductance heterogeneity, we examined the effects of nonionic or zwitterionic detergents upon gramicidin channel behavior. In phospholipid bilayers, detergent addition induces many changes in gramicidin channel behavior: all detergents tested increase the channel appearance rate and average duration; most detergents decrease the conductance of the standard channel; and all but one of the detergents increase the conductance heterogeneity. These results show that the conductance heterogeneity can result from environmental perturbations, thus providing a possible explanation for the laboratory-to-laboratory variation in the heterogeneity of gramicidin channels. In addition, the differential detergent effects suggest possible mechanisms by which detergents can induce the conformational perturbations that result in gramicidin single-channel conductance variations.

Renal proximal tubule of flounder II. Transepithelial Mg secretion.

The kinetics of transepithelial Mg secretion were studied in isolated perfused proximal tubule II of the flounder, Pseudopleuronectes americanus. To detect measurable changes of luminal [Mg] as a function of bath [Mg], the lumen had to be perfused at subnanoliter rates. Transepithelial Mg secretion did not obey first-order reaction kinetics; hence values for half saturation of transport (0.22 mM) and transport maximum (1.5 pmol X min-1 X mm-1) are apparent and suggest a high-affinity low-capacity transport system. Because all experiments were done in the absence of bath SO4, the independence of Mg transport from SO4 transport is established. In the absence of perfusion, when tubules secrete fluid spontaneously, secreted fluid contained Cl (156 +/- 3 mM), Na (130 +/- 6 mM), and Mg (27 +/- 5 mM), all significantly different from the bath. Rates of Cl, Na, and Mg secretion were all positively correlated with fluid secretion, but Na and Mg concentrations in secreted fluid were inversely proportional. The results indicate that NaCl secretion provides basal rates of fluid secretion, and when MgCl2 is secreted in addition, fluid secretion increases with the effect of generating inverse relationships between luminal Na and Mg concentrations.

Adrenergic regulation of cardiac myocyte apoptosis.

The direct effects of catecholamines on cardiac myocytes may contribute to both normal physiologic adaptation and pathologic remodeling, and may be associated with cellular hypertrophy, apoptosis, and alterations in contractile function. Norepinephrine (NE) signals via alpha- and beta-adrenergic receptors (AR) that are coupled to G-proteins. Pharmacologic studies of cardiac myocytes in vitro demonstrate that stimulation of beta1-AR induces apoptosis which is cAMP-dependent and involves the voltage-dependent calcium influx channel. In contrast, stimulation of beta2-AR exerts an anti-apoptotic effect which appears to be mediated by a pertussis toxin-sensitive G protein. Stimulation of alpha1-AR causes myocyte hypertrophy and may exert an anti-apoptotic action. In transgenic mice, myocardial overexpression of either beta1-AR or G(alpha)s is associated with myocyte apoptosis and the development of dilated cardiomyopathy. Myocardial overexpression of beta2-AR at low levels results in improved cardiac function, whereas expression at high levels leads to dilated cardiomyopathy. Overexpression of wildtype alpha1B-AR does not result in apoptosis, whereas overexpression of G(alpha)q results in myocyte hypertrophy and/or apoptosis depending on the level of expression. Differential activation of the members of the mitogen-activated protein kinase (MAPK) superfamily and production of reactive oxygen species appear to play a key role in mediating the actions of adrenergic pathways on myocyte apoptosis and hypertrophy. This review summarizes current knowledge about the molecular and cellular mechanisms involved in the regulation of cardiac myocyte apoptosis via stimulation of adrenergic receptors and their coupled effector pathways.

Adrenergic overload and apoptosis in heart failure: implications for therapy.

Sympathetic nervous system activity to the myocardium is increased in patients with heart failure. It is now appreciated that norepinephrine (NE), the primary sympathetic neurotransmitter, can exert direct adverse effects on cardiac myocytes and might thereby contribute to pathological remodeling, a chronic process which leads to progressive left ventricular (LV) chamber dilation and loss of contractile function. The demonstration of apoptosis in failing human hearts has led to the thesis that continuing loss of viable myocytes is a mechanism for progressive myocardial failure. For many years it has been appreciated that chronic exposure to catecholamines can exert a toxic effect on the myocardium. In vitro studies in cultured cardiac myocytes show that tonic exposure to NE increases the number of apoptotic myocytes via stimulation of the beta-adrenergic receptor (beta-AR) pathway. Interestingly, a beta1-AR selective antagonist completely prevented NE-stimulated apoptosis, whereas a beta2-AR selective antagonist increased the amount of apoptosis, suggesting that beta1- versus beta2-AR may couple to different signaling pathways. In rats, isoproterenol infusion for as little as 12 hours increased the frequency of terminal deoxynucleotidyltransferase-mediated nick end-labeling (TUNEL)-positive myocytes. Likewise, mice that overexpress beta1-AR or G alpha s in the myocardium develop left ventricular dilation, contractile dysfunction and apoptosis. Although the link between apoptosis and myocardial failure remains to be proven, these in vitro and in vivo observations provide a rational mechanism by which beta-AR antagonists may help to prevent or slow LV remodeling and failure in patients.

High-throughput assessment of calcium sensitivity in skinned cardiac myocytes.

Isolated permeabilized cardiac myocytes have been used in the study of myofilament calcium sensitivity through measurement of the isometric force-pCa curve. Determining this force-pCa relationship in skinned myocytes is relatively expensive and carries a high degree of variability. We therefore attempted to establish an alternative high-throughput method to measure calcium sensitivity in cardiac myocytes. With the use of commercially available software that allows for precise measurement of sarcomere spacing, we measured sarcomere length changes in unloaded skinned cardiac myocytes over a range of calcium concentrations. With the use of this technique, we were able to accurately detect acute increases or decreases in myofilament calcium sensitivity after exposure to 10 mM caffeine or 5 mM 2,3-butanedione monoxime, respectively. This technique allows for the simple and rapid determination of myofilament calcium sensitivity in cardiac myocytes in a reproducible and inexpensive manner and could be used for high-throughput screening of pharmacological agents and/or transgenic mouse models for changes in myofilament calcium sensitivity.

Daunorubicin-induced apoptosis in rat cardiac myocytes is inhibited by dexrazoxane.

-The clinical efficacy of anthracycline antineoplastic agents is limited by a high incidence of severe and usually irreversible cardiac toxicity, the cause of which remains controversial. In primary cultures of neonatal and adult rat ventricular myocytes, we found that daunorubicin, at concentrations /=10 micromol/L induced necrotic cell death within 24 hours, with no changes characteristic of apoptosis. To determine whether reactive oxygen species play a role in daunorubicin-mediated apoptosis, we monitored the generation of hydrogen peroxide with dichlorofluorescein (DCF). However, daunorubicin (1 micromol/L) did not increase DCF fluorescence, nor were the antioxidants N-acetylcysteine or the combination of alpha-tocopherol and ascorbic acid able to prevent apoptosis. In contrast, dexrazoxane (10 micromol/L), known clinically to limit anthracycline cardiac toxicity, prevented daunorubicin-induced myocyte apoptosis, but not necrosis induced by higher anthracycline concentrations (>/=10 micromol/L). The antiapoptotic action of dexrazoxane was mimicked by the superoxide-dismutase mimetic porphyrin manganese(II/III)tetrakis(1-methyl-4-peridyl)porphyrin (50 micromol/L). The recognition that anthracycline-induced cardiac myocyte apoptosis, perhaps mediated by superoxide anion generation, occurs at concentrations well below those that result in myocyte necrosis, may aid in the design of new therapeutic strategies to limit the toxicity of these drugs.