Abstract-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 Յ1 mol/L, induced myocyte programmed cell death within 24 hours, as defined by several complementary techniques. In contrast, daunorubicin concentrations Ն10 mol/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 mol/L) did not increase DCF fluorescence, nor were the antioxidants N-acetylcysteine or the combination of ␣-tocopherol and ascorbic acid able to prevent apoptosis. In contrast, dexrazoxane (10 mol/L), known clinically to limit anthracycline cardiac toxicity, prevented daunorubicin-induced myocyte apoptosis, but not necrosis induced by higher anthracycline concentrations (Ն10 mol/L). The antiapoptotic action of dexrazoxane was mimicked by the superoxide-dismutase mimetic porphyrin manganese(II/III)tetrakis(1-methyl-4-peridyl)porphyrin (50 mol/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.(Circ Res. 1999;84:257-265.)

Cardiac myocytes have been shown to express constitutively endothelial nitric oxide synthase (eNOS) (nitric oxide synthase 3), the activation of which has been implicated in the regulation of myocyte L-type voltagesensitive calcium channel current (I Ca-L ) and myocyte contractile responsiveness to parasympathetic nervous system signaling, although this implication remains controversial. Therefore, we examined the effect of the muscarinic cholinergic agonist carbachol ( The regulation of the beating rate and force of contraction of the heart by the autonomic nervous system has been one of the most intensively studied aspects of cardiovascular pharmacology. Recently, a number of studies have implicated an important role for nitric oxide (NO) generation in mediating some aspects of muscarinic cholinergic and adrenergic signaling, both in intact hearts and in isolated myocytes (1-12). NO, whether released by pharmacologic NO donors or generated by activation of endogenous NO synthases (NOS), has been shown to suppress the activation of voltage-sensitive Ca 2ϩ current (I Ca-L ) by ␤-adrenergic agonists (7-12). Conversely, inhibition of NO generation within cardiac myocytes, including specialized pacemaker and conduction system cells, has been shown to blunt the negative inotropic and chronotropic effects of parasympathetic nerve stimulation in the mammalian heart in situ when infused with a ␤-adrenergic agonist (3-6).The enzyme responsible for the generation of NO within cardiac myocytes is the NOS originally described in endothelial cells (i.e., eNOS or NOS3). eNOS is a Ca 2ϩ -calmodulinactivated enzyme that is localized in both myocytes and endothelial cells to specialized plasmalemmal microdomains termed caveolae (15, 16). A principal target of eNOSgenerated NO is the soluble guanylyl cyclase (17-21). Indeed, agents that prevent cGMP generation have been shown to attenuate significantly the inhibitory effect of NO on I Ca-L (8)(9)(10)17).Nevertheless, the importance of endogenous NO generation in the regulation of cardiac I Ca-L , particularly in response to muscarinic cholinergic agonists, remains controversial (see refs. 16, 21, and 22 for reviews). In frog ventricular myocytes, for example, NOS inhibitors failed to modify cholinergic inhibition of I Ca-L (23). Inhibition of a cAMP-stimulated chloride current by cholinergic agonists in guinea pig ventricular myocytes also was unaffected by NOS inhibitors (24). This controversy prompted us to examine the control of I Ca-L by muscarinic cholinergic signaling in atrial and ventricular myocytes of mice with targeted disruption of eNOS (eNOS null ). We found that muscarinic cholinergic agonists significantly increased intracellular cGMP levels in wild-type (WT) but not eNOS null myocytes and that cholinergic inhibition of ␤-adrenergic agonist-stimulated I Ca-L and myocyte contractile amplitude both were impaired in myocytes lacking functional eNOS. METHODSCell Isolation. Calcium-tolerant ventricular and atrial myocytes were isolated from mice as described (2...

The endothelial isoform of nitric oxide synthase (eNOS) is dually acylated and thereby targeted to signaltransducing microdomains termed caveolae. In endothelial cells, eNOS interacts with caveolin-1, which represses eNOS enzyme activity. In cardiac myocytes, eNOS associates with the muscle-specific caveolin-3 isoform, but whether this interaction affects NO production and regulates myocyte function is unknown. We isolated neonatal cardiac myocytes from mutant mice with targeted disruption of the eNOS gene and transfected them with wild-type (WT) eNOS or myristoylation-deficient (myr ؊ ) eNOS mutant cDNA. In myocytes expressing WT eNOS, the muscarinic cholinergic agonist carbachol completely abrogated the spontaneous beating rate and induced a 4-fold elevation of the cGMP level. By contrast, in the myr ؊ eNOS myocytes, carbachol failed to exert its negative chronotropic effect and to increase cGMP levels. We then used a reversible permeabilization protocol to load intact neonatal rat myocytes with an oligopeptide corresponding to the caveolin-3 scaffolding domain. This peptide completely and specifically inhibited the carbachol-induced negative chronotropic effect and the accompanying cGMP elevation. Thus, our results suggest that acylated eNOS may couple muscarinic receptor activation to heart rate control and indicate a key role for eNOS/caveolin interactions in the cholinergic modulation of cardiac myocyte function.The endothelial isoform of nitric-oxide synthase (eNOS) 1 , originally identified in large vessel endothelium, is now known to be expressed in numerous cell types, including cardiac myocytes. The eNOS enzyme is dually acylated (for review, see Refs. 1 and 2) and is thereby specifically targeted to plasmalemmal signal-transducing microdomains termed caveolae (3, 4). In endothelial cells and cardiac myocytes, eNOS is quantitatively associated with caveolin (4), the structural protein of caveolae, and a stable protein-protein interaction takes place at consensus sequences present within both proteins and leads to the inhibition of the eNOS activity (5-8). More recently, we have also documented that, in endothelial cells, when intracellular Ca 2ϩ concentration is increased by an agonist, a regulatory cycle is initiated (2, 9), wherein (i) Ca 2ϩ /calmodulin activates eNOS by disrupting the heteromeric complex formed between eNOS and caveolin; (ii) activated, caveolin-free eNOS is translocated from caveolae, probably associated with enzyme desensitization; (iii) when Ca 2ϩ returns to basal levels, eNOS reassociates with caveolin; and (iv) the inhibitory complex is restored to caveolae, a process facilitated by eNOS palmitoylation. The role (if any) of this eNOS-caveolin regulatory cycle in cardiac myocytes, however, remains to be established.The location of eNOS in plasmalemmal caveolae and its interaction with caveolin may find several biological justifications. First, the compartmentation of eNOS with other signaling proteins may facilitate, or improve the efficacy of, the coupling between agonist stim...