Aldosterone stimulates the cardiac Na(+)/H(+) exchanger via transactivation of the epidermal growth factor receptor

The use of antagonists of the mineralocorticoid receptor in the treatment of myocardial hypertrophy and heart failure has gained increasing importance in the last years. The cardiac Na(+)/H(+) exchanger (NHE-1) upregulation induced by aldosterone could account for the genesis of these pathologies. We tested whether aldosterone-induced NHE-1 stimulation involves the transactivation of the epidermal growth factor receptor (EGFR). Rat ventricular myocytes were used to measure intracellular pH with epifluorescence. Aldosterone enhanced the NHE-1 activity. This effect was canceled by spironolactone or eplerenone (mineralocorticoid receptor antagonists), but not by mifepristone (glucocorticoid receptor antagonist) or cycloheximide (protein synthesis inhibitor), indicating that the mechanism is mediated by the mineralocorticoid receptor triggering nongenomic pathways. Aldosterone-induced NHE-1 stimulation was abolished by the EGFR kinase inhibitor AG1478, suggesting that is mediated by transactivation of EGFR. The increase in the phosphorylation level of the kinase p90(RSK) and NHE-1 serine703 induced by aldosterone was also blocked by AG1478. Exogenous epidermal growth factor mimicked the effects of aldosterone on NHE-1 activity. Epidermal growth factor was also able to increase reactive oxygen species production, and the epidermal growth factor-induced activation of the NHE-1 was abrogated by the reactive oxygen species scavenger N-2-mercaptopropionyl glycine, indicating that reactive oxygen species are participating as signaling molecules in this mechanism. Aldosterone enhances the NHE-1 activity via transactivation of the EGFR, formation of reactive oxygen species, and phosphorylation of the exchanger. These results call attention to the consideration of the EGFR as a new potential therapeutic target of the cardiovascular pathologies involving the participation of aldosterone.     

NHE-1-dependent intracellular sodium overload in hypertrophic hereditary cardiomyopathy: prevention by NHE-1 inhibitor

The purpose of this study was to verify whether myocardial intracellular Na(+) overload may take place in the hereditary cardiomyopathic hamster (CMH), as a result of an increased activity of the Na(+)-H(+) exchanger isoform-1 (NHE-1). Our results showed that simultaneous intracellular Na(+) and Ca(2+) overloads as well as an increase of NHE-1 protein level took place during the development of necrosis and hypertrophy in the CMH. Treatment of 30-day-old CMHs during the development of necrosis and in the absence of hypertrophy with the specific NHE-1 inhibitor EMD87580 (EMD) for 50 days significantly prevented the increase of NHE-1 protein level and intracellular Na(+) and Ca(2+) overloads as well as necrosis. Treatment of CMHs during the development of hypertrophy with EMD for 198 days prevented the development of both necrosis and hypertrophy. In conclusion, our results suggest that NHE-1 overexpression as well as Na(+) and Ca(2+) overloads do take place during the development of necrosis and hypertrophy in hereditary CMHs. Moreover, our results suggest that the blockade of NHE-1 by EMD87580 prevents these diseases by preventing the increase of Na(+) influx through the NHE-1.     

Mineralocorticoid and angiotensin receptor antagonism during hyperaldosteronemia

Elevated aldosterone levels induce a spironolactone-inhibitable decrease in cardiac sarcolemmal Na+-K+ pump function. Because pump inhibition has been shown to contribute to myocyte hypertrophy, restoration of Na+-K+ pump function may represent a possible mechanism for the cardioprotective action of mineralocorticoid receptor (MR) blockade. The present study examines whether treatment with the angiotensin type 1 receptor antagonist losartan, with either spironolactone or eplerenone, has additive effects on sarcolemmal Na+-K+ pump activity in hyperaldosteronemia. New Zealand White rabbits were divided into 7 different groups: controls, aldosterone alone, aldosterone plus spironolactone, aldosterone plus eplerenone, aldosterone plus losartan, aldosterone plus losartan and spironolactone, and aldosterone plus losartan and eplerenone. After 7 days, myocytes were isolated by enzymatic digestion. Electrogenic Na+-K+ pump current (I(p)), arising from the 3:2 Na+:K+ exchange ratio, was measured by the whole-cell patch clamp technique. Elevated aldosterone levels lowered I(p); treatment with losartan reversed aldosterone-induced reduced pump function, as did MR blockade. Coadministration of spironolactone or eplerenone with losartan enhanced the losartan effect on pump function to a level similar to that measured in rabbits given losartan alone in the absence of hyperaldosteronemia. In conclusion, hyperaldosteronemia induces a decrease in I(p) at near physiological levels of intracellular Na+ concentration. Treatment with losartan reverses this aldosterone-induced decrease in pump function, and coadministration with MR antagonists produces an additive effect on pump function, consistent with a beneficial effect of MR blockade in patients with hypertension and congestive heart failure treated with angiotensin type 1 receptor antagonists.     

Overexpression of csk inhibits acid-induced activation of NHE-3.

Opossum kidney OKP cells express an apical membrane Na+/H+ antiporter that is encoded by NHE-3 (for Na+/H+ exchanger 3) and is similar in many respects to the renal proximal tubule apical membrane Na+/H+ antiporter. Chronic incubation of OKP cells in acid medium for 24 hr increases Na+/H(+)-antiporter activity and NHE-3 mRNA abundance. The increase in Na+/H(+)-antiporter activity was not prevented by H7, a protein kinase C/protein kinase A inhibitor, but was prevented by herbimycin A, a tyrosine kinase inhibitor. Incubation of cells in acid medium increased c-src activity, and this was inhibited by herbimycin A. To determine the role of the src family of nonreceptor protein-tyrosine kinases, Csk (for carboxyl-terminal src kinase), a physiologic inhibitor of these kinases, was overexpressed in OKP cells. In three clones overexpressing csk, acid-induced increases in Na+/H(+)-antiporter activity and NHE-3 mRNA abundance were inhibited. In these clones, inhibition of acid activation of Na+/H(+)-antiporter activity paralleled inhibition of acid activation of c-src. Neither herbimycin A nor overexpression of csk inhibited dexamethasone-induced increases in Na+/H(+)-antiporter activity. These studies show that decreases in pH activate c-src and that the src family nonreceptor protein-tyrosine kinases play a key role in acid activation of NHE-3.     

Estrogen exerts concentration-dependent pro-and anti-hypertrophic effects on adult cultured ventricular myocytes. Role of NHE-1 in estrogen-induced hypertrophy

Estrogen has been shown to protect the heart and attenuate myocardial hypertrophy and left ventricular remodelling through as yet to be defined mechanisms. In the present study we examined concentration-dependent effects of estrogen on hypertrophy of adult rat cardiomyocytes, potential underlying mechanisms related to intracellular pH (pH_i) and possible sex-dependent responses. Cardiomyocytes were isolated from adult male and female Sprague-Dawley rats and used immediately for pH_i determinations or cultured and subsequently treated for 24 h with 17β-estradiol to assess hypertrophic responses. Fluorometric measurements with the pH_i-sensitive dye BCECF demonstrated that at 1 pM 17β-estradiol increased pH_i (+ 0.05 pH units in females and + 0.12 pH units in males, P < 0.05) by a rapid non-genomic mechanism that was blocked by the sodium-hydrogen exchange isoform 1 (NHE-1) specific inhibitor AVE-4890 (AVE, 5 μM). Treatment with 1 pM 17β-estradiol for 24 h increased cell size (females: 20%, P < 0.05; males: 29%, P < 0.05) and ANP expression (females: 414%, P < 0.05; males: 497%, P < 0.05) in a NHE-1-, and ERK1/2 MAPK-dependent manner. At 1 nM, 17β-estradiol decreased pH_i (females: − 0.24 pH units, P < 0.05; males: − 0.07 pH units, P < 0.05) which was also prevented by AVE, although at this concentration the hormone had no direct hypertrophic effect but instead prevented hypertrophy induced by phenylephrine. Our results show that low levels of estrogen produce cardiomyocyte hypertrophy through ERK/NHE-1 activation and intracellular alkalinization whereas an antihypertrophic effect is seen at high concentrations. These effects may further our understanding of the role of estrogen in heart disease particularly associated with hypertrophy.     

Mechanisms for aldosterone and spironolactone-induced positive inotropic actions in the rat heart

Previously, we reported that aldosterone and spironolactone have inotropic effects in the isolated perfused heart. To address the mechanisms underlying these inotropic effects, we examined the effects of aldosterone and spironolactone on isolated cardiac myocyte shortening, intracellular calcium ([Ca+2]i), pHi, and calcium-dependent actinomyosin ATPase activity. Aldosterone significantly increased shortening in cardiac myocytes (8.0+/-1.0 versus 16.0+/-1.3%, P<0.01) but neither diastolic [Ca+2]i (61.0+/-1.1 versus 66.0+/-4.4 nmol/L) nor peak systolic [Ca+2]i (302+/-11 versus 304+/-17 nmol/L) was affected. Spironolactone-increased shortening was also not coupled with changes in peak systolic calcium; however, diastolic calcium was significantly increased by spironolactone. Aldosterone, but not spironolactone, increased pHi from 7.23+/-0.03 to 7.59+/-0.02 (P<0.01); this was completely blocked by coadministration of 100 micromol/L of ethyl-isopropyl amiloride (EIPA), an inhibitor of the Na+/H+ exchanger (P<0.01). Consistent with this finding, aldosterone increased cytosolic sodium concentration ([Na+]i) from 9.2+/-0.15 to 11.4+/-0.2 mmol/L and produced a leftward shift in the pCa ATPase curve (pCa=5.82+/-0.02 versus 6.35+/-0.02, P<0.01) without affecting maximal myosin ATPase activity. Conversely, spironolactone, but not aldosterone, significantly increases maximal actomyosin ATPase activity (837+/-59 versus 355+/-52 nmol inorganic phosphate (P(i)) x min(-1) x g tissue(-1)). Collectively, these data strongly suggest that the inotropic actions of aldosterone and spironolactone are caused by different mechanisms of action. Aldosterone appeared to increase inotropy primarily through increased cytosolic pH, whereas spironolactone increased myosin ATPase calcium sensitivity and diastolic calcium concentration.     

Hypertrophic responsiveness of cardiomyocytes to alpha- or beta-adrenoceptor stimulation requires sodium-proton-exchanger-1 (NHE-1) activation but not cellular alkalization

The influence of the sodium-proton-exchanger-1 (NHE-1) inhibitor HOE694 on alpha- or beta-adrenoceptor mediated stimulation of protein synthesis was investigated in cultured ventricular cardiomyocytes from adult rat pre-treated with fetal calf serum to induce hypertrophic responsiveness to beta-adrenoceptor stimulation. Stimulation of alpha-adrenoceptors with phenylephrine (10 microM) in bicarbonate-free medium caused cellular alkalization (Delta pH(i): + 0.17 +/- 0.02, n = 5, P < 0.05). HOE694, an NHE-1 inhibitor, completely abolished this effect. [14C]phenylalanine incorporation into cellular protein mass increased in the presence of phenylephrine by 23+/-8%, and this effect was also abolished in the presence of HOE694. HOE694 (1 mciroM) neither influenced basal protein synthesis nor interfered with alpha-adrenoceptor mediated activation of ERK2. Phorbol myristate acetate, a direct stimulator of protein kinase C, mimicked the effect of alpha-adrenoceptor stimulation in regard to protein synthesis, but did not lead to cellular alkalization. Protein synthesis increased in the presence of isoprenaline, a beta-adrenoceptor agonist also. Again, HOE694 attenuated the stimulation of protein synthesis although isoprenaline did not cause cellular alkalization. In conclusion, the growth response to different hypertrophic stimuli, namely alpha- or beta-adrenoceptor stimulation, is attenuated in the presence of the NHE-1 inhibitor HOE694 and this inhibition is independent from cellular alkalization.     

Aldosterone stimulates matrix metalloproteinases and reactive oxygen species in adult rat ventricular cardiomyocytes

Matrix metalloproteinases (MMPs), aldosterone, and reactive oxygen species (ROS) are implicated in myocardial remodeling. Although ROS, cytokines, and neurohormones regulate MMP in cardiac fibroblasts, it is unknown whether aldosterone regulates MMP in cardiomyocytes. Therefore, we tested the hypothesis that aldosterone regulates MMP in cultured adult rat ventricular myocytes (ARVMs). ARVMs were treated with aldosterone for 24 hours, and MMP-2 and MMP-9 activities were measured by zymography. Aldosterone (50 nmol/L) increased MMP-2 (43+/-5%) and MMP-9 (55+/-15%; P<0.001 for both) activities. Pretreatment with spironolactone (100 nmol/L) abolished the aldosterone-induced increase in MMP activities. Aldosterone (50 nmol/L; 30 minutes) increased mitogen/extracellular signal-regulated kinase (MEK) (31+/-3%) and extracellular signal-regulated kinase 1/2 (ERK1/2; 41+/-7%; P<0.001 for both) phosphorylation. U0126 (10 micromol/L), an MEK1/2 inhibitor, abolished the aldosterone-induced increase in MMP activities. Aldosterone increased intracellular ROS as assessed by dichlorofluorescein diacetate (27+/-4%; P<0.05). This increase was inhibited by apocynin, an NADPH oxidase inhibitor. Apocynin likewise inhibited aldosterone-induced ERK1/2 phosphorylation and the increase in MMP activities. Furthermore, the antioxidants MnTMPyP and N-acetylcysteine inhibited the aldosterone-induced increase in ERK1/2 phosphorylation and MMP activities, respectively. Protein kinase C (PKC) is implicated in the nongenomic effects of aldosterone. To test the role of PKC, ARVMs were pretreated with chelerythrine, a PKC inhibitor. Chelerythrine prevented the aldosterone-induced increase in ERK1/2 phosphorylation and MMP activities. Thus, aldosterone induces MMP activity in ARVM via activation of the mineralocorticoid receptor, PKC, and ROS-dependent activation of the MEK/ERK pathway. NADPH oxidase is a likely source of ROS in this system.     

The obesity-associated peptide leptin induces hypertrophy in neonatal rat ventricular myocytes

One of the major manifestations of obesity is increased production of the adipocyte-derived 16-kDa peptide leptin, which is also elevated in heart disease, including congestive heart failure. However, whether leptin can directly alter the cardiac phenotype is not known. We therefore studied the effect of leptin as a potential hypertrophic factor in cultured myocytes from 1- to 4-day-old neonatal rat heart ventricles. Using RT-PCR, we demonstrate that these cells express the short-form (OB-Ra) leptin receptor. Twenty-four hours of exposure to leptin (0.31 to 31.3 nmol/L) produces a significantly increased cell surface area that peaked at 0.63 nmol/L. Subsequent experiments were done with 3.1 nmol/L leptin, which significantly increased cell area by 42%, protein synthesis by 32%, and alpha-skeletal actin and myosin light chain-2 expression by 250% and 300%, respectively. These events occurred in the absence of any increased cell death. Hypertrophy was preceded by rapid activation of the mitogen-activated protein kinase system including p38 and p44/42 as early as 5 minutes after leptin addition, whereas hypertrophy was inhibited by the p38 inhibitor SB203580 but not by the p44/42 inhibitor PD98059. Our results demonstrate a direct hypertrophic effect of leptin and may offer a biological link between hypertrophy and hyperleptinemic conditions such as obesity.     

Cardioprotective mechanisms of spironolactone associated with the angiotensin-converting enzyme/epidermal growth factor receptor/extracellular signal-regulated kinases, NAD(P)H oxidase/lectin-like oxidized low-density lipoprotein receptor-1, and Rho-kinase pathways in aldosterone/salt-induced hypertensive rats.

Studies were performed to test the hypothesis that the angiotensin-converting enzyme (ACE)/epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinases (ERK) pathway, nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase/lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) pathway, and Rho-kinase pathway contribute to the pathogenesis of aldosterone/salt-induced hypertensive rats. Wistar rats were given 1% NaCl to drink and treated with one of the following combinations for 6 weeks: vehicle; aldosterone (0.75 microg/h); aldosterone plus a mineralocorticoid receptor antagonist, spironolactone (20 mg/kg/day); aldosterone plus an ACE inhibitor, imidapril (1 mg/kg/day); aldosterone plus an NAD(P)H oxidase inhibitor, apocynin (0.5 mmol/l); and aldosterone plus an Rho-kinase inhibitor, Y-27632 (3 mg/kg/day). Upregulated expression of ACE and EGFR and p44/p42ERK phosphorylation were suppressed by spironolactone or imidapril. Upregulated NAD(P)H oxidase subunits and LOX-1 expression were inhibited by spironolactone or apocynin. Increased expression of RhoA and Rho-kinase and myosin light chain phosphorylation were decreased by spironolactone or Y-27632. Moreover, these drugs effectively inhibited the vascular lesion formation, as measured by the medial thickness and level of perivascular fibrosis, and suppressed the expression of transforming growth factor-beta1, type I and III collagen, and monocyte chemoattractant protein-1 mRNA. Spironolactone may be useful as a cardioprotective agent to prevent cardiovascular remodeling via the ACE/EGFR/ERK, NAD(P)H oxidase/LOX-1, and Rho-kinase pathways.     

Aldosterone increases plasminogen activator inhibitor-1 synthesis in rat cardiomyocytes

Plasminogen activator inhibitor-1 (PAI-1) is an anti-thrombolytic factor that also promotes tissue fibrosis. Under certain conditions, exposure to aldosterone can result in cardiac fibrosis by an unknown mechanism. In the current study, we tested the hypothesis that PAI-1 is a mediator of aldosterone's fibrotic effects. Aldosterone increased levels of PAI-1 mRNA and protein in the H9c2 rat cardiac cell line, responses that could be blocked by the mineralocorticoid receptor (MR) antagonist spironolactone. Confocal microscopy confirmed an effect of aldosterone to increase PAI-1 expression with reversal by spironolactone. Aldosterone also increased PAI-1 expression in neonatal rat cardiomyocytes, which was again blocked by spironolactone. In the neonatal cardiomyocytes (but not the H9c2 cells), anti-transforming growth factor-beta1 antibody inhibited the PAI-1 response to aldosterone. In summary, aldosterone directly increased PAI-1 expression in two different cardiac muscle cell types, an effect that was dependent on MR. In the neonatal cells, there appeared to be a requirement for transforming growth factor-beta1.     

NHE-1 inhibition improves impaired mitochondrial permeability transition and respiratory function during postinfarction remodelling in the rat

The mechanism by which Na+-H+ exchange (NHE) inhibition results in attenuation and reversal of postinfarction remodelling and heart failure remains controversial. In this study, we investigated the possible contribution of mitochondrial involvement by determining the effect of the NHE-1-specific inhibitor EMD-87580 (EMD) on mitochondrial permeability transition (MPT) and respiratory function during the postinfarction remodelling process. Male Sprague-Dawley rats were subjected to either 12 or 18 weeks of coronary artery ligation (CAL) or sham procedure. EMD was provided in the diet immediately after ligation. MPT pore opening was determined by perfusing hearts with 2-deoxy-[3H]-glucose ([3H]-DOG) and measurement of mitochondrial [3H]-DOG entrapment. The respiratory function of isolated mitochondria was measured using Clark-type oxygen electrode. Remodelling was associated with significant hypertrophy and there was an increase in MPT pore opening in hearts both 12 and 18 weeks following CAL. Mitochondrial respiratory function, especially state 2 and state 3 rates were significantly decreased in hearts subjected to CAL. EMD suppressed MPT pore opening by 40% (P <0.01) and 35% (P <0.01) 12 and 18 weeks after ligation, respectively. Mitochondria isolated from EMD treated hearts exhibited increased respiratory chain activity for oxidation of substrates at complex I and II. These beneficial effects of EMD were associated with decreased mitochondrial vulnerability to exogenous Ca2+. We conclude that NHE-1 inhibition has a protective effect on mitochondrial function, attenuating MPT pore opening and improving the respiratory function, which may contribute to the salutary effect of NHE-1 inhibitors in heart failure.     

Distinct roles of p42/p44ERK and p38 MAPK in oxidant-induced AP-1 activation and cardiomyocyte hypertrophy

Cardiac hypertrophy is an adaptive response to a number of heart diseases including myocardial infarction. Although it can be compensatory at first, sustained hypertrophy is often a transition to heart failure. We have found that cardiomyocytes in culture can survive mild doses of H₂O₂ but develop hypertrophy involving activation of p70 S6 kinase 1 (p70S6K1). Here, the role of p42/p44^ERK and p38 MAPK in oxidant-induced hypertrophy is tested. H₂O₂-induced phosphorylation (activation) of p42/p44^ERK and p38 within 10 min of 200 μM H₂O₂ exposure. Although p42/p44^ERK remained highly phosphorylated from 60 to 120 min, the level of p38 phosphorylation reached highest at 60 min and started to decline at 90 min. Inhibiting ERKs with PD98059 attenuated H₂O₂-induced AP-1 activation but did not affect H₂O₂-induced p70S6k1 activation or cardiomyocyte enlargement as measured by increases in cell volume and protein content. In contrast, the p38 inhibitor SB202190 has not inhibitory effect on AP-1 activation but partially prevented H₂O₂ from inducing p70S6K1 activation and cell enlargement. These data suggest that while p42/p44^ERK participates in gene expression associated with hypertrophy, p38 may regulate cell size increase by p70S6K1 activation.     

Hyperaldosteronemia in rabbits inhibits the cardiac sarcolemmal Na(+)-K(+) pump

Aldosterone upregulates the Na(+)-K(+) pump in kidney and colon, classical target organs for the hormone. An effect on pump function in the heart is not firmly established. Because the myocardium contains mineralocorticoid receptors, we examined whether aldosterone has an effect on Na(+)-K(+) pump function in cardiac myocytes. Myocytes were isolated from rabbits given aldosterone via osmotic minipumps and from controls. Electrogenic Na(+)-K(+) pump current, arising from the 3:2 Na(+):K(+) exchange ratio, was measured in single myocytes using the whole-cell patch clamp technique. Treatment with aldosterone induced a decrease in pump current measured when myocytes were dialyzed with patch pipette solution containing Na(+) in a concentration of 10 mmol/L, whereas there was no effect measured when the solution contained 80 mmol/L Na(+). Aldosterone had no effect on myocardial Na(+)-K(+) pump concentration evaluated by vanadate-facilitated [(3)H]ouabain binding or by K(+)-dependent paranitrophenylphosphatase activity in crude homogenates. Aldosterone induced an increase in intracellular Na(+) activity. The aldosterone-induced decrease in pump current and increased intracellular Na(+) were prevented by cotreatment with the mineralocorticoid receptor antagonist spironolactone. Our results indicate that hyperaldosteronemia decreases the apparent Na(+) affinity of the Na(+)-K(+) pump, whereas it has no effect on maximal pump capacity.     

Aldosterone activates vascular p38MAP kinase and NADPH oxidase via c-Src

Increasing evidence indicates that aldosterone elicits vascular effects through nongenomic signaling pathways. We tested the hypothesis that aldosterone induces activation of vascular mitogen-activated protein (MAP) kinases and NADPH oxidase via c-Src-dependent mechanisms in vascular smooth muscle cells (VSMCs). Aldosterone effects on activation of c-Src, p38MAP kinase, and NADPH oxidase, and incorporation of [3H]proline, an index of collagen synthesis, were assessed in cultured rat VSMCs. Studies were performed in the absence and presence of eplerenone, a selective mineralocorticoid receptor blocker, PP2, a selective Src inhibitor, and SB212190, a selective p38MAPK inhibitor. Phosphorylation of c-Src was dose-dependently increased by aldosterone, with maximal responses obtained at 10(-7) mol/L. Aldosterone increased p38MAP kinase phosphorylation, NAD(P)H oxidase activation, and [3H]proline incorporation. These responses were abrogated by eplerenone and almost abolished by PP2. Aldosterone-stimulated incorporation of [3H]proline was significantly reduced by SB212190, indicating that p38MAP kinase plays a role in profibrotic actions of aldosterone. To unambiguously demonstrate the importance of aldosterone in c-Src signaling, VSMCs from c-Src+/+ and c-Src+/- mice were also studied. Aldosterone increased phosphorylation of c-Src, p38MAP kinase, and cortactin, a Src-specific substrate, in c-Src+/+ VSMCs, but not in c-Src-deficient cells. Taken together, our findings demonstrate that nongenomic signaling by aldosterone occurs through c-Src-dependent pathways. These processes may play an important role in profibrotic actions of aldosterone.     

Effects of adding spironolactone to an angiotensin-converting enzyme inhibitor in chronic congestive heart failure secondary to coronary artery disease

In chronic heart failure, a diuretic plus an angiotensin-converting enzyme (ACE) inhibitor only partially suppresses aldosterone despite the fact that aldosterone has many harmful effects independent of angiotensin II. These possible harmful effects of aldosterone are magnesium loss, increased cardiac sympathetic activity, and increased ventricular arrhythmias. We have therefore assessed whether adding the aldosterone antagonist, spironolactone, to a loop diuretic and ACE inhibitor reverses any of these potentially harmful effects of residual aldosterone. In a preliminary animal study, we found that exogenous aldosterone reduced myocardial norepinephrine uptake by 24% in anesthetized rats in vivo. In our main study, 42 patients with New York Heart Association II to III congestive heart failure were randomized to spironolactone (50 to 100 mg/ day, titrated to blood pressure and plasma potassium) or placebo in a double-blind fashion. Our principal finding is that cardiac norepinephrine uptake as assessed by ¹²³I-metaiodobenzylguanidine scintigraphy increased with spironolactone (p < 0.01). Spironolactone also elevated plasma magnesium (p < 0.05), reduced urinary magnesium excretion (p < 0.05), and caused a reduction in ventricular arrhythmias on 24-hour ambulatory electrocardiography (p < 0.05). Spironolactone increased plasma renin activity, plasma aldosterone (p < 0.01), 24-hour urinary sodium excretion (p < 0.05), and urinary sodium/potassium ratio (p < 0.01). Echocardiographic-determined measurements of left ventricular systolic and diastolic function were unaltered by spironolactone. Therefore, spironolactone has potentially beneficial effects on cardiac adrenergic tone, divalent cation balance, and ventricular arrhythmias in patients with chronic heart failure who are already receiving standard doses of ACE inhibitors. All of these factors could be significant in the prevention of sudden cardiac death in this population.