—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-1β (IL-1β) and interferon γ (IFNγ) or exposure to the exogenous NO donorS-nitroso-N-acetylcysteine (SNAC) or peroxynitrite (ONOO⁻). Both SNAC (1 mmol/L) and ONOO⁻(100 μmol/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-1β and IFNγ 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 inhibitorl-nitroarginine (3 mmol/L) or the specific iNOS inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT, 100 μmol/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 μmol/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-1β– and IFNγ-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.

—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 ≤1 μmol/L, induced myocyte programmed cell death within 24 hours, as defined by several complementary techniques. In contrast, daunorubicin concentrations ≥10 μmol/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 μmol/L) did not increase DCF fluorescence, nor were the antioxidantsN-acetylcysteine or the combination of α-tocopherol and ascorbic acid able to prevent apoptosis. In contrast, dexrazoxane (10 μmol/L), known clinically to limit anthracycline cardiac toxicity, prevented daunorubicin-induced myocyte apoptosis, but not necrosis induced by higher anthracycline concentrations (≥10 μmol/L). The antiapoptotic action of dexrazoxane was mimicked by the superoxide-dismutase mimetic porphyrin manganese(II/III)tetrakis(1-methyl-4-peridyl)porphyrin (50 μmol/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.

Cardiac myocytes express both constitutive and cytokine-inducible nitric oxide syntheses (NOS). NO and its congeners have been implicated in the regulation of cardiac contractile function. To determine whether NO could affect myocardial energetics, 31P NMR spectroscopy was used to evaluate high-energy phosphate metabolism in isolated rat hearts perfused with the NO donor S-nitrosoacetylcysteine (SNAC). All hearts were exposed to an initial high Ca2+ (3.5 mM) challenge followed by a recovery period, and then, either in the presence or absence of SNAC, to a second high Ca2+ challenge. This protocol allowed us to monitor simultaneously the effect of SNAC infusion on both contractile reserve (i.e., baseline versus high workload contractile function) and high-energy phosphate metabolism. The initial high Ca2+ challenge caused the rate-pressure product to increase by 74 +/- 5% in all hearts. As expected, ATP was maintained as phosphocreatine (PCr) content briefly dropped and then returned to baseline during the subsequent recovery period. Control hearts responded similarLy to the second high Ca2+ challenge, but SNAC-treated hearts did not demonstrate the expected increase in rate-pressure product. In these hearts, ATP declined significantly during the second high Ca2+ challenge, whereas phosphocreatine did not differ from controls, suggesting that phosphoryl transfer by creatine kinase (CK) was inhibited. CK activity, measured biochemically, was decreased by 61 +/- 13% in SNAC-treated hearts compared to controls. Purified CK in solution was also inhibited by SNAC, and reversal could be accomplished with DTT, a sulfhydryl reducing agent. Thus, NO can regulate contractile reserve, possibly by reversible nitrosothiol modification of CK.

The endothelial isoform of nitric oxide synthase (eNOS) is dually acylated and thereby targeted to signal-transducing 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.

Background N -acetylcysteine (NAC) has been shown to potentiate the effects of nitroglycerin (NTG) and to have antioxidant activity. This is the first study to assess the safety and effect of NAC in the treatment of evolving acute myocardial infarction (AMI). Methods and Results Patients with AMI received either 15 g NAC infused over 24 hours (n=20) or no NAC (n=7), combined with intravenous NTG and streptokinase. Peripheral venous plasma malondialdehyde (MDA), reduced (GSH) and oxidized (GSSG) glutathione concentrations, and rate of reperfusion (using continuous ST-segment analysis) were measured. Cardiac catheterization was performed between days 2 and 5. No significant adverse events occurred. Less oxidative stress occurred in patients treated with NAC than in patients not receiving NAC (GSH to GSSG ratio 44±25 versus 19±13 at 4 hours, P <.05). NAC concentration (mean 172±79 μmol/L at 4 hours) was correlated to GSH concentration ( P =.006). MDA concentrations were lower ( P =.001) over the first 8 hours of treatment with NAC. There was a trend toward more rapid reperfusion (median 58 minutes, 95% confidence interval [CI] 48 to 98 minutes versus median 95 minutes, 95% CI 59 to 106 minutes; P =.17) and better preservation of left ventricular function (cardiac index 3.4±0.8 versus 2.6±0.27 L · min · m ² , P =.009) with NAC treatment. Conclusions NAC in combination with NTG and streptokinase appeared to be safe for the treatment of evolving AMI and was associated with significantly less oxidative stress, a trend toward more rapid reperfusion, and better preservation of left ventricular function.

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 voltage-sensitive 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 (CCh) on I _Ca-L and contractile amplitude in isoproterenol (ISO)-prestimulated ventricular myocytes isolated from adult mice, designated eNOS ^null mice, with targeted disruption of the eNOS gene. Although both eNOS ^null and wild-type (WT) ventricular myocytes exhibited similar increases in I _Ca-L in response to ISO, there was no measurable suppression of I _Ca-L by CCh in cells from eNOS ^null mice, in contrast to cells from WT mice. These results were reflected in the absence of an effect of CCh on the positive inotropic effect of ISO in eNOS ^null myocytes. Also, unlike myocytes from WT animals, eNOS ^null myocytes failed to exhibit an increase in cGMP content in response to CCh. Nevertheless, the pharmacologic nitric oxide donors 3-morpholino-sydnonimine and S-nitroso-acetyl-cystein increased cGMP generation and suppressed ISO-augmented I _Ca-L in eNOS ^null cells, suggesting that the signal transduction pathway(s) downstream of eNOS remained intact. Of importance, activation of the acetylcholine-activated K ⁺ channel by CCh was unaffected in atrial and ventricular eNOS ^null myocytes. These results confirm the obligatory role of eNOS in coupling muscarinic receptor activation to cGMP-dependent control of I _Ca-L in cardiac myocytes.