Direct Effects of Aldosterone on Cardiomyocytes in the Presence of Normal and Elevated Extracellular Sodium
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《内分泌学杂志》
Department of Cardiovascular Medicine (Me.Y., Mi.Y., M.N., K.A., M.S., S.S., T.S., H.O.), Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan
First Department of Internal Medicine (Y.S.), Nara Medical University, Nara 634-8521, Japan
Department of Medicine and Clinical Science (K.N.), Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan
Division of Cardiology (H.Y.), Kumamoto Aging Research Institute, Kumamoto 860-8518, Japan
Abstract
It is now recognized that aldosterone is potentially cardiotoxic, although its local effects in the heart are not well understood. We examined the effects of aldosterone on cultured neonatal rat cardiomyocytes in the presence of normal and elevated extracellular Na+ ([Na+]o). We evaluated the intracellular volume of cardiomyocytes in the presence of normal (141 mEq/liter) and elevated (146 mEq/liter) [Na+]o by measuring cell size. Intracellular Na+ was measured using sodium-binding-benzofuran-isophthalate as a fluorescent sodium indicator, and cardiac hypertrophy was assessed using B-type natriuretic peptide transcription and 3H-leucine incorporation. Cardiomyocytes shrank in the presence of 146 mEq/liter Na+ due to the increased extracellular osmolarity at early phase. Aldosterone (10–7 mol/liter) mitigated the shrinkage by stimulating Na+ uptake by the cells. This effect of aldosterone was blocked by SM 20220, a Na+/H+ exchanger 1 (NHE1) inhibitor, but not by eplerenone, a mineralocorticoid receptor blocker. Seventy-two hours of exposure to aldosterone in the presence of 146 mEq/liter Na+ led to increases in cardiomyocyte size, 3H-leucine incorporation, and B-type natriuretic peptide and NHE1 transcription that were significantly greater than were seen in the presence of 141 mEq/liter Na+. All but the last were blocked by either eplerenone or SM 20220; the increase in NHE1 transcription was blocked only by eplerenone. Aldosterone exerts a beneficial effect via NHE1 to block cardiomyocyte shrinkage in the presence of elevated [Na+]o at early phase, but long-time exposure to aldosterone in the presence of elevated [Na+]o leads to cardiomyocyte hypertrophy via genomic effects mediated by the mineralocorticoid receptor.
Introduction
THE BODY RESPONDS to a salty meal by decreasing the secretion of aldosterone from the adrenal cortex, increasing the secretion of antidiuretic hormone from the posterior pituitary, and activating drinking behavior, thereby stabilizing levels of circulating Na+ (1, 2). However, if one’s diet is continuously high in Na+ or if a large amount of Na+ is acutely taken in, the circulating Na+ level will tend to rise. When that happens, somatic cells are subject to fluid loss due to the increase in the extracellular osmolarity, which causes them to shrink. Oxidative stress is also increased, and a life-threatening crisis can occur in severe cases (3, 4). But despite its vital importance, the regulatory system that strictly governs the levels of circulating Na+ is not well understood.
Aldosterone has traditionally been seen as a key regulator of fluid and electrolyte balance, acting via the mineralocorticoid receptor (MR) in the epithelium of the kidney (distal nephron), colon, and salivary glands (5). However, we recently showed that aldosterone is also synthesized in the hearts of patients with heart failure or hypertension (6, 7, 8), and that in neonatal rat cardiomyocytes it induces expression of angiotensin-converting enzyme, creating a vicious circular cascade involving the renin-angiotensin-aldosterone-system (9). In addition, others have shown that aldosterone induces vascular inflammation and apoptosis within the cardiovascular system (10, 11). The molecular mechanisms by which aldosterone exerts its local effects are not fully characterized, however.
The Na+/H+ exchanger 1 (NHE1) is a ubiquitously expressed housekeeping transporter that catalyzes the electroneutral countertransport of extracellular Na+ and intracellular protons (12, 13). In addition to mediating the transcellular absorption of Na+, NHE1 plays a major role in the regulation of intracellular pH, cell volume and, possibly, cell proliferation (13). Aldosterone regulates Na+ homeostasis and, consequently, extracellular volume in large part by controlling NHE1 activity in the kidney (12, 13, 14). There are also reports that aldosterone up-regulates NHE1 activity by both genomic or nongenomic means (15, 16); that NHE1 serves as a critical downstream regulator contributing to cardiac remodeling in response to various hypertrophic factors (16, 17); and that inhibition of NHE1 suppresses progression of cardiac hypertrophy (18).
With that as background, we hypothesized that, in the face of an acute increase in the extracellular Na+ concentration ([Na+]o), aldosterone would act nongenomically on NHE1 to promote cellular Na+ uptake and fluid retention to attenuate cell shrinkage. If those conditions persisted, however, aldosterone would act genomically, causing cardiomyocyte hypertrophy by way of NHE1. To test this hypothesis, we investigated the direct actions of aldosterone—i.e. those independent of hemodynamic overload—in cultured neonatal rat cardiomyocytes.
Materials and Methods
In this in vitro study, [Na+]o was either 141 mEq/liter or 146 mEq/liter, which is within the physiological range. Cellular fluid changes were evaluated by measuring cell size (19) and changes in the intracellular Na+ concentration ([Na+]i) were measured using sodium-binding benzofuran isophthalate (SBFI), a fluorescent Na+ indicator (20). To examine NHE1 activity, intracellular H+ concentration ([H+]i) was measured using LysoSensor Green DND-153 (21). Cardiac hypertrophy was assessed using 3H-leucine incorporation (22) and B-type natriuretic peptide (BNP) gene expression (9, 19, 23), two sensitive makers of cardiac hypertrophy, as indices. In addition, we also tested the effects of eplerenone, a specific MR blocker (24), and SM 20220, a NHE1 inhibitor (25), on the genomic and nongenomic actions of aldosterone.
Agents used
Aldosterone was purchased from Steraloid Co. (Wilton, NH). SBFI-acetoxymethyl ester (SBFI-AM) was purchased from Sigma Chemical Co. (St. Louis, MO) (20). LysoSensor Green DND-153 as a pH indicator was purchased from Molecular Probes (Eugene, OR) (21). Eplerenone was provided by Pfizer Co., Ltd. (New York, NY) (24). SM 20220, which is a specific inhibitor of NHE1 in cultured neurons and glial cells with an IC50 of 5 and 20 nM, respectively, was provided by Sumitomo Pharmaceuticals Co., Ltd., Research Division (Osaka, Japan) (25, 26).
Preparation of cardiomyocytes
All animal procedures conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Research Committee at Kumamoto University. Cardiomyocytes were obtained from 1- to 2-d-old Wistar rats. Ventricular cells were dispersed in a balanced Na+ solution containing 0.04% collagenase II (Sigma) and 0.06% pancreatin (Sigma) (9, 19, 22, 23). The cardiomyocytes were isolated on a discontinuous Percoll gradient using 40.5% and 58.5% Percoll (Sigma) prepared in balanced Na+ solution. Ventricular cells were initially suspended in the 58.5% Percoll layer (19, 22, 23). After centrifugation at 3000 rpm for 30 min at 15 C, the cardiomyocytes had migrated to the interface between the layers.
Cell culture
Purified cardiomyocytes were plated at a density of 3.0x104 cells/cm2 in six-well plates (2.9 x 105 cells/well) in DMEM (GIBCO, Carlsbad, CA) supplemented with 10% fetal bovine serum (Moregate Bio Tech, Bulimba, Australia) and antibiotics (100 U/ml penicillin G and 100 μg/ml streptomycin; GIBCO). The cells were allowed to attach for 30 h, after which the medium was replaced with serum-free DMEM, and the cells were incubated for an additional 12 h. After this preconditioning period, the cells were incubated in serum-free DMEM containing 1 mg/ml BSA (Sigma) with the indicated test substances (9, 19, 22, 23). Medium containing 146 mEq/liter Na+ was made by simply adding NaCl (Wako, Osaka, Japan) to the normal 141 mEq/liter Na+ medium (27). pH was 7.3 ± 0.2 in both media.
Measurement of cell size
Surface areas of cardiomyocytes were measured using Lumina Vision (Mitani Co., Fukui, Japan). Cardiomyocytes in the culture wells were chosen at random for measuring cell sizes from five to eight preparations and two different people blindly measured cell sizes.
3H-leucine incorporation
Cardiomyocytes were plated at 3.0x104 cells/cm2 in 96-well dishes and treated as described above. 3H-leucine (3 μCi/ml, 2.15 x 10–8 mol/liter; PerkinElmer, Yokohama, Japan) was then added to each well just after the last treatment, as previously described (22). After incubating 72 h, the cells were harvested using an Omnifilter-96 Harvester (PerkinElmer) (28), and 3H-leucine incorporation was measured using a MicroBeta TriLux (PerkinElmer) (28).
Quantitative real-time RT-PCR
For real-time RT-PCR, total RNA was extracted from cardiomyocytes cultured in six-well plates using an RNeasy Mini Kit (QIAGEN, Bulimba, Germany) (9, 23) and treated with deoxyribonuclease I (QIAGEN) to eliminate any contaminating genomic DNA (9, 23). The oligonucleotide primers and TaqMan probes used to analyze expression of rat BNP mRNA were designed from GenBank databases (M25297) using Primer Express version 1.0 (Applied Biosystems, Foster City, CA) as previously described (9, 23). The forward primer was 5'-181-CAG AAG CTG CTG GAG CTG ATA AG-203-3'; the reverse primer was 5'-258-TGT AGG GCC TTG GTC CTT TG-239-3'; and the TaqMan probe was 5'-207-AAA GTC AGA GGA AAT GGC TCA GAG ACA GCT C-237-3'. Primers and the TaqMan probe set for rat NHE1 (No. 185248084) were purchased from Assays-on-Demand Gene Expression Products (Applied Biosystems), and those for rat glyceraldehyde-3-phosphate dehydrogenase were from PerkinElmer Applied Biosystems. Two-step real time RT-PCR was carried out using TaqMan Reverse Transcription Reagents (Applied Biosystems) and a TaqMan Universal Master Mix kit (Applied Biosystems) (29) with an ABI Prism 7900 sequence detection system (Applied Biosystems) (29).
Measurement of BNP and NHE1 protein levels
We measured BNP levels in these culture mediums after 72 h using a BNP ELISA Kit (Peninsula Laboratories Inc., San Carlos, CA). NHE1 protein levels were measured by Western blotting using a polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) after 72 h culture as described (16).
Measurement of intracellular Na+
After incubating cardiomyocytes for 2 h in medium containing 141 or 146 mEq/liter Na+, [Na+]i was determined as previously described (20). Briefly, cardiomyocytes grown on glass-bottomed dishes were incubated with 10 μmol/liter SBFI-AM for 90 min at room temperature in presence of the nonionic surfactant Pluronic F-127 (0.05% wt/vol; Sigma). After washing out the external dye, we allowed the intracellular SBFI-AM to be deesterified to active SBFI for 20 min before proceeding with [Na+]i measurements. The cells were incubated in DMEM containing 141 and 146 mEq/liter Na+, and [Na+]i levels at a single cell were measured as a function of SBFI fluorescence using an Ion Optix dual-wavelength ratiometric photon counting system (11). The cells were pretreated with SM 20220 or eplerenone just before this measuring. After measuring for 60 sec, 10–7 mol/liter aldosterone was added to the medium.
Measurement of intracellular H+
After incubating cardiomyocytes for 2 h in a medium containing 141 or 146 mEq/liter Na+, intracellular H+ levels ([H+]i) were examined (21). Cardiomyocytes grown on glass-bottomed dishes were incubated with 1 μmol/liter LysoSensor Green DND-153 for 30 min. After washing out the external dye, the cells were incubated in DMEM containing 141 and 146 mEq/liter Na+, and [H+]i levels in a single cell with 10–7 mol/liter aldosterone were measured using LysoSensor Green DND-153 fluorescence with Lumina Vision (Mitani Co., Fukui, Japan). The cells were pretreated with SM 20220 or eplerenone just before this measurement.
Statistical analysis
Data are expressed as means ± SEM. Statistical analysis was performed using one-way ANOVA followed by multiple comparisons using Fisher’s protected least-significant difference and unpaired Student’s t tests, as appropriate. Values of P < 0.05 were considered significant.
Results
In the present study, we evaluate the actions of aldosterone in time course divided into early phase (0–6 h) and late phase (72 h).
Effect of aldosterone on cardiomyocyte size in the presence of normal and elevated [Na+]o at early phase
Figure 1A shows the morphology of neonatal rat cardiomyocytes after 2 h in the presence of normal (141 mEq/liter) or elevated (146 mEq/liter) [Na+]o with and without 10–7 mol/liter aldosterone. There was no change in the size of the cells in the presence of 141 mEq/liter Na+ with or without aldosterone. In the presence of 146 mEq/liter Na+ without aldosterone, however, the cells became substantially smaller, and this shrinkage was blocked by the addition of aldosterone.
Figure 1B shows the time-dependent changes in the size of cardiomyocytes over a period of 24 h. In the absence of aldosterone, the cells shrank significantly in the presence of 146 mEq/liter Na+ over the course of 6 h (P < 0.0001; vs. 141 mEq/liter Na+ without aldosterone at 1, 2, 3, and 6 h); again, this shrinkage was blocked by addition of 10–7 mol/liter aldosterone. The ability of aldosterone to block cell shrinkage in the presence of elevated [Na+]o apparently reflects its ability to stimulate Na+ uptake by the cells and thus increase [Na+]i. Figure 1C showed the effects of eplerenone and SM 20220 on the cell sizes of cardiomyocytes with 10–7 mol/liter aldosterone in the presence of 146 mEq/liter Na+ at 2 h. SM 20220, but not eplerenone, blocked cell recovery induced by aldosterone in the presence of 146 mEq/liter Na+.
Effect of aldosterone on the level of intracellular Na+ and intracellular H+ in the presence of normal and elevated [Na+]o at early phase
In Fig. 2, 10–7 mol/liter aldosterone did not elevate intracellular sodium concentrations in SBFI-loaded cardiomyocytes with 141 mEq/liter Na+ (Fig. 2A). There was an increase in [Na+]i after adding 10–7 mol/liter aldosterone to cardiomyocytes in the presence of 141 and 146 mEq/liter Na+ (Fig. 2B). It appears that the aldosterone-induced Na+ uptake was mediated by the NHE1, as the effect was significantly inhibited by the NHE1 antagonist SM 20220 (10–7 mol/liter) (Fig. 2C). In contrast, the MR antagonist eplerenone (10–5 mol/liter) had no effect (Fig. 2D).
In Fig. 3, we measured the level of intracellular H+ ([H+]i) in the cardiomyocytes using LysoSensor Green DND-153 with 10–7 mol/liter aldosterone for examination of the activity of NHE1. Levels of [H+]i in the cardiomyocytes with 10–7 mol/liter aldosterone in the presence of 146 mEq/liter Na+ were significantly lower than those in the presence of 141 mEq/liter Na+. SM 20220 blocked aldosterone-induced H+ discharge, but eplerenone did not block aldosterone-induced H+ discharge in the presence of 146 mEq/liter Na+.
Effect of aldosterone on cardiomyocytes size in the presence of normal and elevated [Na+]o at late phase
Seventy-two hours of exposure to 10–7 mol/liter aldosterone significantly increased cardiomyocyte size in the presence of both 141 mEq/liter and 146 mEq/liter Na+ (P < 0.005, P < 0.0005, respectively), although the increase was significantly greater the presence of the latter (P < 0.0005 vs. normal Na+) (Figs. 4A and 5A). The effect of aldosterone on cell size was significantly attenuated by either eplerenone or SM 20220 in the presence of 146 mEq/liter [Na+]o (both P < 0.0005) (Figs. 4B and 5A). In the absence of aldosterone, neither eplerenone nor SM 20220 affected the cell size in the presence of 141 mEq/liter Na+ at 72 h. In Figs. 1A and 4, they were different cells before and after aldosterone stimulation.
Effect of aldosterone on 3H-leucine incorporation by cardiomyocytes at late phase
Indicative of induction of cell hypertrophy, 10–7 mol/liter aldosterone significantly increased 3H-leucine incorporation by cardiomyocytes in the presence of either 141 mEq/liter or 146 mEq/liter Na+ at late phase (P < 0.05, P < 0.05, respectively) and, as with cell size, the effect was more pronounced in the presence of the latter (Fig. 5B). This effect in the presence of 146 mEq/liter Na+ was significantly attenuated by inhibiting NHE1 using SM 20220 (P < 0.0005). In the absence of aldosterone, neither eplerenone nor SM 20220 affected 3H-leucine incorporation in the presence of 141 mEq/liter Na+ at 72 h.
Effect of aldosterone on BNP gene expression and BNP expression by cardiomyocytes at late phase
We found that long-time exposure to 10–7 mol/liter aldosterone in the presence of 146 mEq/liter Na+ induced a significant increase in BNP gene expression by cardiomyocytes (P < 0.0005) that was accompanied by a substantial increase in cell size (Fig. 5C). Moreover, the effect of aldosterone on BNP gene expression was significantly greater in the presence of 146 mEq/liter Na+ than in the presence of 141 mEq/liter Na+ (P < 0.05). Both the BNP gene expression and the hypertrophy were attenuated by either eplerenone or SM 20220 (P < 0.005, P < 0.0005, respectively, for BNP gene expression). Eplerenone and SM 20220, without aldosterone, did not affect BNP gene expression levels in the presence of 141 mEq/liter Na+ at 72 h. SM 20220 or eplerenone with 10–7 mol/liter aldosterone did not inhibit the effect of aldosterone by these parameters in the presence of 141 mEq/liter Na+ at 72 h.
In the supplemental figure published on The Endo- crine Society’s Journals Online web site at http://endo.endojournals.org, 10–7 mol/liter aldosterone significantly increased BNP levels in cardiomyocytes in the presence of either 141 mEq/liter or 146 mEq/liter Na+ at the late phase (P < 0.05, P < 0.05, respectively) and, as with BNP gene expression, the effect was more pronounced in the presence of the latter. This effect in the presence of 146 mEq/liter Na+ was significantly attenuated by inhibiting NHE1 using SM 20220 (P < 0.05). In the absence of aldosterone, eplerenone and SM 20220 did not affect BNP levels in the presence of 141 mEq/liter Na+ at 72 h.
Effects of aldosterone on NHE1 gene and protein expression by cardiomyocytes at late phase
NHE1 gene expression was also significantly increased by 10–7 mol/liter aldosterone (P < 0.05) in the presence of Na+141 and 146 mEq/liter (Fig. 6A). Aldosterone also increased NHE1 protein levels in the presence of Na+ 141 and 146 mEq/liter (Fig. 6B). The effect of aldosterone on NHE1 gene expression was significantly attenuated by eplerenone (P < 0.05); in contrast, SM 20220 had no effect (Fig. 6A).
Discussion
We found that acute exposure to elevated [Na+]o caused cardiomyocytes to rapidly shrink as a result of fluid loss to the outside driven by the increase in extracellular osmolarity. Aldosterone strongly suppressed this loss of fluid by inducing Na+ uptake, as indicated by the observed increase in SBFI fluorescence, which diminished the osmotic pressure gradient across the cell membrane. We believe these findings show that aldosterone exerts a protective effect against cardiomyocyte dehydration in the presence of elevated [Na+]o at early phase. It appears that this effect is mediated by the NHE1 because it was blocked by the NHE1 antagonist SM 20220. At early phase, we found no evidence that aldosterone acts acutely via the MR, as indicated by eplerenone’s lack of effect, or that it acutely induces cellular hypertrophy, as indicated by the absence of up-regulated BNP transcription or 3H-leucine incorporation (data not shown). These findings also suggest that aldosterone may contribute to the rapid regulation and maintenance of circulating Na+ levels by regulating the movement of Na+ into cardiomyocytes like principal cells of the kidney distal tubules (30). It has been reported that the nongenomic effects of aldosterone on NHE were blocked by eplerenone in mesenteric resistance vessels (31). We cannot positively deny the effects of aldosterone on NHE1 via the classical MR in cardiomyocytes (31, 32).
In one recent study, aldosterone was shown to induce Na+ influx into human umbilical vein endothelial cells, leading to cell swelling, even when the Na+ concentration in the culture medium was unchanged (33). It was suggested that this effect was mediated via the epithelial Na+ channel (ENaC). At one time, ENaCs were thought to exist only in the kidney; that was until the ENaC -subunit was identified in human heart (34). The function and significance of the ENaC in heart is not yet known, but it is possible that, along with NHE1, it contributes to mediating the acute effects of aldosterone in response to a rise in [Na+]o.
Na+/K+ adenosine triphosphatase (Na+/K+ ATPase), which mediates the active transport of Na+ out and K+ into the cells, is present in cardiomyocytes (35). It is notable that Na+/K+ ATPase has been reported to be reversely suppressed by aldosterone in the myocardium (36). Therefore, the inhibitory action of aldosterone on Na+/K+ ATPase in the myocardium would not conflict with the possible effect of aldosterone on the intracellular influx of sodium by NHE1 because both actions take sodium into cells in the myocardium.
Secretion of aldosterone from the adrenal gland is diminished by a high Na+ diet, reducing circulating levels of the hormone (2). On the other hand, it was recently reported that, in rats, the concentration of aldosterone in cardiac and vascular tissues are increased by high Na+ intake (37). In view of our present findings, we suggest that cell shrinkage caused by the increase in extracellular osmolarity associated with high Na+ intake would stimulate local synthesis of aldosterone, which in turn stimulates uptake of extracellular Na+ and water into the cells, thereby stabilizing the electrolyte and fluid balance across the cell membrane. Naturally, we understand that there are many reports disputing the theory about cardiac aldosterone synthesis (38, 39). It is still unclear, however, which of sodium, potassium, chloride, angiotensin II, ACTH, or others is actually the key regulator for cardiac aldosterone synthesis in vivo (40, 41). There are many issues that we should study in the future.
In sharp contrast to the effects of aldosterone at early phase, we found that long-time aldosterone exposure induces cardiomyocyte hypertrophy, as indicated by increased cell size, increased incorporation of 3H-leucine, and increased BNP transcription. That these effects could be suppressed by either eplerenone or SM 20220 means that the effects of aldosterone are mediated via both the MR and NHE1 at late phase. In addition, we also found that aldosterone induces NHE1 gene expression via the MR (eplerenone sensitive) at late phase, which is consistent with earlier reports (15, 16).
The effects of aldosterone at late phase observed in the present study are in agreement with those of Karmazyn et al. (16) and complement them by adding the observation that aldosterone-induced myocardial hypertrophy is dependent on [Na+]o as well as on the NHE1 activity. Elevation of [Na+]i via NHE1 likely leads to Ca2+ overload via the Na+/Ca2+ exchanger, which would in turn stimulate hypertrophic signaling (42). Also, it is possible that the increase in cell pH directly resulting from NHE1 activation might be the signal for the induction of the hypertrophic response. Furthermore, inflammatory cytokines may be involved in this system of aldosterone-induced myocardial hypertrophy depending on the [Na+]o level. In any event, eplerenone would be useful for treating and/or preventing myocardial hypertrophy. The results of both the RALES trial and the EPHESUS trial support this idea for the MR antagonist (43, 44). Also, as suggested by Young and Funder (45), the NHE1 antagonist might also be good for reducing cardiac fibrosis, although this agent has not been used in a clinical stage yet.
We used aldosterone at a concentration of 10–7 mol/liter, which is a close approximation to the circulating levels seen in vivo, particularly under hyperaldosteronemic conditions. This actually may be somewhat conservative, however, because aldosterone concentrations are reportedly an order of magnitude higher in cardiac tissues than in the peripheral circulation (40).
The effect of aldosterone in the heart should be discussed because cardiomyocyte MR is normally occupied by endogenous glucocorticoid in physiological status because 11-hydroxysteroid dehydrogenase type 2 is not normally expressed in the cardiomyocytes and there are high levels of circulating cortisol (46); however, in pathophysiological states, such as hypertension, heart failure, or neonatal stage, it has been hypothesized that mineralocorticoids can access cardiac MR and thereby produce cardiac damage (46).
We previously reported that aldosterone synthesis is activated in both the adrenal gland and hearts of patients with heart failure or hypertension (6, 7, 8). Moreover, as mentioned above, aldosterone levels are higher in the myocardium than in the circulation (40). Taken together, these findings suggest that a continuous intake of excess salt stimulates cardiac hypertrophy together with local production of aldosterone in the heart, irrespective of circulating aldosterone levels. Consistent with that idea, we observed that long-time elevated [Na+]o induced a small increase in cardiac hypertrophy even in the absence of added aldosterone. This may be explained by the endogenous production of aldosterone by the cells. We have to regardless the fact that there are many reports disputing the theory about cardiac aldosterone synthesis (38, 39) and also that the detrimental effects of aldosterone on the heart may be due to adrenal derived aldosterone (47).
a clinical viewpoint, the results of the present study highlight the benefit of reducing salt in the diet and are consistent with earlier reports emphasizing the importance maintaining a low-salt diet to prevent cardiac hypertrophy and subsequent heart failure (27). In that regard, our findings indicate that by maintaining a low-salt diet and thus reducing local cardiovascular levels of aldosterone, one mitigates the chronic effects of this potent mediator of cardiac hypertrophy. Observational studies of the Yanomamo Indians, a no-salt culture, reinforce this view (48, 49).
In conclusion, aldosterone acutely induces Na+ uptake via NHE1 in the presence of elevated [Na+]o. This rapid, nongenomic protective effect against cellular fluid loss is a positive and physiological attribute. In the face of elevated [Na+]o, however, long-time exposure to aldosterone induces pathological genomic effects via MR that lead to cardiomyocyte hypertrophy. The MR antagonist eplerenone could thus be useful for suppressing cardiac hypertrophy, without affecting the beneficial effects of aldosterone at early phase.
Footnotes
This study was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology, Tokyo [B (2 )-15390248 and B (2 )-15390249], the Ministry of Health, Labor and Welfare, Tokyo [14C-4, 14A-1, 17A-1 and 17C-2], the Smoking Research Foundation, Tokyo and the Takeda Science Foundation (Tokyo, Japan).
First Published Online December 22, 2005
Abbreviations: BNP, B-type natriuretic peptide; ENaC, epithelial Na+ channel; MR, mineralocorticoid receptor; Na+/K+ ATPase, Na+/K+ adenosine triphosphatase; NHE1, Na+/H+ exchanger 1; SBFI, sodium-binding-benzofuran-isophthalate; SBFI-AM, SBFI-acetoxymethyl ester.
Accepted for publication December 9, 2005.
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First Department of Internal Medicine (Y.S.), Nara Medical University, Nara 634-8521, Japan
Department of Medicine and Clinical Science (K.N.), Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan
Division of Cardiology (H.Y.), Kumamoto Aging Research Institute, Kumamoto 860-8518, Japan
Abstract
It is now recognized that aldosterone is potentially cardiotoxic, although its local effects in the heart are not well understood. We examined the effects of aldosterone on cultured neonatal rat cardiomyocytes in the presence of normal and elevated extracellular Na+ ([Na+]o). We evaluated the intracellular volume of cardiomyocytes in the presence of normal (141 mEq/liter) and elevated (146 mEq/liter) [Na+]o by measuring cell size. Intracellular Na+ was measured using sodium-binding-benzofuran-isophthalate as a fluorescent sodium indicator, and cardiac hypertrophy was assessed using B-type natriuretic peptide transcription and 3H-leucine incorporation. Cardiomyocytes shrank in the presence of 146 mEq/liter Na+ due to the increased extracellular osmolarity at early phase. Aldosterone (10–7 mol/liter) mitigated the shrinkage by stimulating Na+ uptake by the cells. This effect of aldosterone was blocked by SM 20220, a Na+/H+ exchanger 1 (NHE1) inhibitor, but not by eplerenone, a mineralocorticoid receptor blocker. Seventy-two hours of exposure to aldosterone in the presence of 146 mEq/liter Na+ led to increases in cardiomyocyte size, 3H-leucine incorporation, and B-type natriuretic peptide and NHE1 transcription that were significantly greater than were seen in the presence of 141 mEq/liter Na+. All but the last were blocked by either eplerenone or SM 20220; the increase in NHE1 transcription was blocked only by eplerenone. Aldosterone exerts a beneficial effect via NHE1 to block cardiomyocyte shrinkage in the presence of elevated [Na+]o at early phase, but long-time exposure to aldosterone in the presence of elevated [Na+]o leads to cardiomyocyte hypertrophy via genomic effects mediated by the mineralocorticoid receptor.
Introduction
THE BODY RESPONDS to a salty meal by decreasing the secretion of aldosterone from the adrenal cortex, increasing the secretion of antidiuretic hormone from the posterior pituitary, and activating drinking behavior, thereby stabilizing levels of circulating Na+ (1, 2). However, if one’s diet is continuously high in Na+ or if a large amount of Na+ is acutely taken in, the circulating Na+ level will tend to rise. When that happens, somatic cells are subject to fluid loss due to the increase in the extracellular osmolarity, which causes them to shrink. Oxidative stress is also increased, and a life-threatening crisis can occur in severe cases (3, 4). But despite its vital importance, the regulatory system that strictly governs the levels of circulating Na+ is not well understood.
Aldosterone has traditionally been seen as a key regulator of fluid and electrolyte balance, acting via the mineralocorticoid receptor (MR) in the epithelium of the kidney (distal nephron), colon, and salivary glands (5). However, we recently showed that aldosterone is also synthesized in the hearts of patients with heart failure or hypertension (6, 7, 8), and that in neonatal rat cardiomyocytes it induces expression of angiotensin-converting enzyme, creating a vicious circular cascade involving the renin-angiotensin-aldosterone-system (9). In addition, others have shown that aldosterone induces vascular inflammation and apoptosis within the cardiovascular system (10, 11). The molecular mechanisms by which aldosterone exerts its local effects are not fully characterized, however.
The Na+/H+ exchanger 1 (NHE1) is a ubiquitously expressed housekeeping transporter that catalyzes the electroneutral countertransport of extracellular Na+ and intracellular protons (12, 13). In addition to mediating the transcellular absorption of Na+, NHE1 plays a major role in the regulation of intracellular pH, cell volume and, possibly, cell proliferation (13). Aldosterone regulates Na+ homeostasis and, consequently, extracellular volume in large part by controlling NHE1 activity in the kidney (12, 13, 14). There are also reports that aldosterone up-regulates NHE1 activity by both genomic or nongenomic means (15, 16); that NHE1 serves as a critical downstream regulator contributing to cardiac remodeling in response to various hypertrophic factors (16, 17); and that inhibition of NHE1 suppresses progression of cardiac hypertrophy (18).
With that as background, we hypothesized that, in the face of an acute increase in the extracellular Na+ concentration ([Na+]o), aldosterone would act nongenomically on NHE1 to promote cellular Na+ uptake and fluid retention to attenuate cell shrinkage. If those conditions persisted, however, aldosterone would act genomically, causing cardiomyocyte hypertrophy by way of NHE1. To test this hypothesis, we investigated the direct actions of aldosterone—i.e. those independent of hemodynamic overload—in cultured neonatal rat cardiomyocytes.
Materials and Methods
In this in vitro study, [Na+]o was either 141 mEq/liter or 146 mEq/liter, which is within the physiological range. Cellular fluid changes were evaluated by measuring cell size (19) and changes in the intracellular Na+ concentration ([Na+]i) were measured using sodium-binding benzofuran isophthalate (SBFI), a fluorescent Na+ indicator (20). To examine NHE1 activity, intracellular H+ concentration ([H+]i) was measured using LysoSensor Green DND-153 (21). Cardiac hypertrophy was assessed using 3H-leucine incorporation (22) and B-type natriuretic peptide (BNP) gene expression (9, 19, 23), two sensitive makers of cardiac hypertrophy, as indices. In addition, we also tested the effects of eplerenone, a specific MR blocker (24), and SM 20220, a NHE1 inhibitor (25), on the genomic and nongenomic actions of aldosterone.
Agents used
Aldosterone was purchased from Steraloid Co. (Wilton, NH). SBFI-acetoxymethyl ester (SBFI-AM) was purchased from Sigma Chemical Co. (St. Louis, MO) (20). LysoSensor Green DND-153 as a pH indicator was purchased from Molecular Probes (Eugene, OR) (21). Eplerenone was provided by Pfizer Co., Ltd. (New York, NY) (24). SM 20220, which is a specific inhibitor of NHE1 in cultured neurons and glial cells with an IC50 of 5 and 20 nM, respectively, was provided by Sumitomo Pharmaceuticals Co., Ltd., Research Division (Osaka, Japan) (25, 26).
Preparation of cardiomyocytes
All animal procedures conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Research Committee at Kumamoto University. Cardiomyocytes were obtained from 1- to 2-d-old Wistar rats. Ventricular cells were dispersed in a balanced Na+ solution containing 0.04% collagenase II (Sigma) and 0.06% pancreatin (Sigma) (9, 19, 22, 23). The cardiomyocytes were isolated on a discontinuous Percoll gradient using 40.5% and 58.5% Percoll (Sigma) prepared in balanced Na+ solution. Ventricular cells were initially suspended in the 58.5% Percoll layer (19, 22, 23). After centrifugation at 3000 rpm for 30 min at 15 C, the cardiomyocytes had migrated to the interface between the layers.
Cell culture
Purified cardiomyocytes were plated at a density of 3.0x104 cells/cm2 in six-well plates (2.9 x 105 cells/well) in DMEM (GIBCO, Carlsbad, CA) supplemented with 10% fetal bovine serum (Moregate Bio Tech, Bulimba, Australia) and antibiotics (100 U/ml penicillin G and 100 μg/ml streptomycin; GIBCO). The cells were allowed to attach for 30 h, after which the medium was replaced with serum-free DMEM, and the cells were incubated for an additional 12 h. After this preconditioning period, the cells were incubated in serum-free DMEM containing 1 mg/ml BSA (Sigma) with the indicated test substances (9, 19, 22, 23). Medium containing 146 mEq/liter Na+ was made by simply adding NaCl (Wako, Osaka, Japan) to the normal 141 mEq/liter Na+ medium (27). pH was 7.3 ± 0.2 in both media.
Measurement of cell size
Surface areas of cardiomyocytes were measured using Lumina Vision (Mitani Co., Fukui, Japan). Cardiomyocytes in the culture wells were chosen at random for measuring cell sizes from five to eight preparations and two different people blindly measured cell sizes.
3H-leucine incorporation
Cardiomyocytes were plated at 3.0x104 cells/cm2 in 96-well dishes and treated as described above. 3H-leucine (3 μCi/ml, 2.15 x 10–8 mol/liter; PerkinElmer, Yokohama, Japan) was then added to each well just after the last treatment, as previously described (22). After incubating 72 h, the cells were harvested using an Omnifilter-96 Harvester (PerkinElmer) (28), and 3H-leucine incorporation was measured using a MicroBeta TriLux (PerkinElmer) (28).
Quantitative real-time RT-PCR
For real-time RT-PCR, total RNA was extracted from cardiomyocytes cultured in six-well plates using an RNeasy Mini Kit (QIAGEN, Bulimba, Germany) (9, 23) and treated with deoxyribonuclease I (QIAGEN) to eliminate any contaminating genomic DNA (9, 23). The oligonucleotide primers and TaqMan probes used to analyze expression of rat BNP mRNA were designed from GenBank databases (M25297) using Primer Express version 1.0 (Applied Biosystems, Foster City, CA) as previously described (9, 23). The forward primer was 5'-181-CAG AAG CTG CTG GAG CTG ATA AG-203-3'; the reverse primer was 5'-258-TGT AGG GCC TTG GTC CTT TG-239-3'; and the TaqMan probe was 5'-207-AAA GTC AGA GGA AAT GGC TCA GAG ACA GCT C-237-3'. Primers and the TaqMan probe set for rat NHE1 (No. 185248084) were purchased from Assays-on-Demand Gene Expression Products (Applied Biosystems), and those for rat glyceraldehyde-3-phosphate dehydrogenase were from PerkinElmer Applied Biosystems. Two-step real time RT-PCR was carried out using TaqMan Reverse Transcription Reagents (Applied Biosystems) and a TaqMan Universal Master Mix kit (Applied Biosystems) (29) with an ABI Prism 7900 sequence detection system (Applied Biosystems) (29).
Measurement of BNP and NHE1 protein levels
We measured BNP levels in these culture mediums after 72 h using a BNP ELISA Kit (Peninsula Laboratories Inc., San Carlos, CA). NHE1 protein levels were measured by Western blotting using a polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) after 72 h culture as described (16).
Measurement of intracellular Na+
After incubating cardiomyocytes for 2 h in medium containing 141 or 146 mEq/liter Na+, [Na+]i was determined as previously described (20). Briefly, cardiomyocytes grown on glass-bottomed dishes were incubated with 10 μmol/liter SBFI-AM for 90 min at room temperature in presence of the nonionic surfactant Pluronic F-127 (0.05% wt/vol; Sigma). After washing out the external dye, we allowed the intracellular SBFI-AM to be deesterified to active SBFI for 20 min before proceeding with [Na+]i measurements. The cells were incubated in DMEM containing 141 and 146 mEq/liter Na+, and [Na+]i levels at a single cell were measured as a function of SBFI fluorescence using an Ion Optix dual-wavelength ratiometric photon counting system (11). The cells were pretreated with SM 20220 or eplerenone just before this measuring. After measuring for 60 sec, 10–7 mol/liter aldosterone was added to the medium.
Measurement of intracellular H+
After incubating cardiomyocytes for 2 h in a medium containing 141 or 146 mEq/liter Na+, intracellular H+ levels ([H+]i) were examined (21). Cardiomyocytes grown on glass-bottomed dishes were incubated with 1 μmol/liter LysoSensor Green DND-153 for 30 min. After washing out the external dye, the cells were incubated in DMEM containing 141 and 146 mEq/liter Na+, and [H+]i levels in a single cell with 10–7 mol/liter aldosterone were measured using LysoSensor Green DND-153 fluorescence with Lumina Vision (Mitani Co., Fukui, Japan). The cells were pretreated with SM 20220 or eplerenone just before this measurement.
Statistical analysis
Data are expressed as means ± SEM. Statistical analysis was performed using one-way ANOVA followed by multiple comparisons using Fisher’s protected least-significant difference and unpaired Student’s t tests, as appropriate. Values of P < 0.05 were considered significant.
Results
In the present study, we evaluate the actions of aldosterone in time course divided into early phase (0–6 h) and late phase (72 h).
Effect of aldosterone on cardiomyocyte size in the presence of normal and elevated [Na+]o at early phase
Figure 1A shows the morphology of neonatal rat cardiomyocytes after 2 h in the presence of normal (141 mEq/liter) or elevated (146 mEq/liter) [Na+]o with and without 10–7 mol/liter aldosterone. There was no change in the size of the cells in the presence of 141 mEq/liter Na+ with or without aldosterone. In the presence of 146 mEq/liter Na+ without aldosterone, however, the cells became substantially smaller, and this shrinkage was blocked by the addition of aldosterone.
Figure 1B shows the time-dependent changes in the size of cardiomyocytes over a period of 24 h. In the absence of aldosterone, the cells shrank significantly in the presence of 146 mEq/liter Na+ over the course of 6 h (P < 0.0001; vs. 141 mEq/liter Na+ without aldosterone at 1, 2, 3, and 6 h); again, this shrinkage was blocked by addition of 10–7 mol/liter aldosterone. The ability of aldosterone to block cell shrinkage in the presence of elevated [Na+]o apparently reflects its ability to stimulate Na+ uptake by the cells and thus increase [Na+]i. Figure 1C showed the effects of eplerenone and SM 20220 on the cell sizes of cardiomyocytes with 10–7 mol/liter aldosterone in the presence of 146 mEq/liter Na+ at 2 h. SM 20220, but not eplerenone, blocked cell recovery induced by aldosterone in the presence of 146 mEq/liter Na+.
Effect of aldosterone on the level of intracellular Na+ and intracellular H+ in the presence of normal and elevated [Na+]o at early phase
In Fig. 2, 10–7 mol/liter aldosterone did not elevate intracellular sodium concentrations in SBFI-loaded cardiomyocytes with 141 mEq/liter Na+ (Fig. 2A). There was an increase in [Na+]i after adding 10–7 mol/liter aldosterone to cardiomyocytes in the presence of 141 and 146 mEq/liter Na+ (Fig. 2B). It appears that the aldosterone-induced Na+ uptake was mediated by the NHE1, as the effect was significantly inhibited by the NHE1 antagonist SM 20220 (10–7 mol/liter) (Fig. 2C). In contrast, the MR antagonist eplerenone (10–5 mol/liter) had no effect (Fig. 2D).
In Fig. 3, we measured the level of intracellular H+ ([H+]i) in the cardiomyocytes using LysoSensor Green DND-153 with 10–7 mol/liter aldosterone for examination of the activity of NHE1. Levels of [H+]i in the cardiomyocytes with 10–7 mol/liter aldosterone in the presence of 146 mEq/liter Na+ were significantly lower than those in the presence of 141 mEq/liter Na+. SM 20220 blocked aldosterone-induced H+ discharge, but eplerenone did not block aldosterone-induced H+ discharge in the presence of 146 mEq/liter Na+.
Effect of aldosterone on cardiomyocytes size in the presence of normal and elevated [Na+]o at late phase
Seventy-two hours of exposure to 10–7 mol/liter aldosterone significantly increased cardiomyocyte size in the presence of both 141 mEq/liter and 146 mEq/liter Na+ (P < 0.005, P < 0.0005, respectively), although the increase was significantly greater the presence of the latter (P < 0.0005 vs. normal Na+) (Figs. 4A and 5A). The effect of aldosterone on cell size was significantly attenuated by either eplerenone or SM 20220 in the presence of 146 mEq/liter [Na+]o (both P < 0.0005) (Figs. 4B and 5A). In the absence of aldosterone, neither eplerenone nor SM 20220 affected the cell size in the presence of 141 mEq/liter Na+ at 72 h. In Figs. 1A and 4, they were different cells before and after aldosterone stimulation.
Effect of aldosterone on 3H-leucine incorporation by cardiomyocytes at late phase
Indicative of induction of cell hypertrophy, 10–7 mol/liter aldosterone significantly increased 3H-leucine incorporation by cardiomyocytes in the presence of either 141 mEq/liter or 146 mEq/liter Na+ at late phase (P < 0.05, P < 0.05, respectively) and, as with cell size, the effect was more pronounced in the presence of the latter (Fig. 5B). This effect in the presence of 146 mEq/liter Na+ was significantly attenuated by inhibiting NHE1 using SM 20220 (P < 0.0005). In the absence of aldosterone, neither eplerenone nor SM 20220 affected 3H-leucine incorporation in the presence of 141 mEq/liter Na+ at 72 h.
Effect of aldosterone on BNP gene expression and BNP expression by cardiomyocytes at late phase
We found that long-time exposure to 10–7 mol/liter aldosterone in the presence of 146 mEq/liter Na+ induced a significant increase in BNP gene expression by cardiomyocytes (P < 0.0005) that was accompanied by a substantial increase in cell size (Fig. 5C). Moreover, the effect of aldosterone on BNP gene expression was significantly greater in the presence of 146 mEq/liter Na+ than in the presence of 141 mEq/liter Na+ (P < 0.05). Both the BNP gene expression and the hypertrophy were attenuated by either eplerenone or SM 20220 (P < 0.005, P < 0.0005, respectively, for BNP gene expression). Eplerenone and SM 20220, without aldosterone, did not affect BNP gene expression levels in the presence of 141 mEq/liter Na+ at 72 h. SM 20220 or eplerenone with 10–7 mol/liter aldosterone did not inhibit the effect of aldosterone by these parameters in the presence of 141 mEq/liter Na+ at 72 h.
In the supplemental figure published on The Endo- crine Society’s Journals Online web site at http://endo.endojournals.org, 10–7 mol/liter aldosterone significantly increased BNP levels in cardiomyocytes in the presence of either 141 mEq/liter or 146 mEq/liter Na+ at the late phase (P < 0.05, P < 0.05, respectively) and, as with BNP gene expression, the effect was more pronounced in the presence of the latter. This effect in the presence of 146 mEq/liter Na+ was significantly attenuated by inhibiting NHE1 using SM 20220 (P < 0.05). In the absence of aldosterone, eplerenone and SM 20220 did not affect BNP levels in the presence of 141 mEq/liter Na+ at 72 h.
Effects of aldosterone on NHE1 gene and protein expression by cardiomyocytes at late phase
NHE1 gene expression was also significantly increased by 10–7 mol/liter aldosterone (P < 0.05) in the presence of Na+141 and 146 mEq/liter (Fig. 6A). Aldosterone also increased NHE1 protein levels in the presence of Na+ 141 and 146 mEq/liter (Fig. 6B). The effect of aldosterone on NHE1 gene expression was significantly attenuated by eplerenone (P < 0.05); in contrast, SM 20220 had no effect (Fig. 6A).
Discussion
We found that acute exposure to elevated [Na+]o caused cardiomyocytes to rapidly shrink as a result of fluid loss to the outside driven by the increase in extracellular osmolarity. Aldosterone strongly suppressed this loss of fluid by inducing Na+ uptake, as indicated by the observed increase in SBFI fluorescence, which diminished the osmotic pressure gradient across the cell membrane. We believe these findings show that aldosterone exerts a protective effect against cardiomyocyte dehydration in the presence of elevated [Na+]o at early phase. It appears that this effect is mediated by the NHE1 because it was blocked by the NHE1 antagonist SM 20220. At early phase, we found no evidence that aldosterone acts acutely via the MR, as indicated by eplerenone’s lack of effect, or that it acutely induces cellular hypertrophy, as indicated by the absence of up-regulated BNP transcription or 3H-leucine incorporation (data not shown). These findings also suggest that aldosterone may contribute to the rapid regulation and maintenance of circulating Na+ levels by regulating the movement of Na+ into cardiomyocytes like principal cells of the kidney distal tubules (30). It has been reported that the nongenomic effects of aldosterone on NHE were blocked by eplerenone in mesenteric resistance vessels (31). We cannot positively deny the effects of aldosterone on NHE1 via the classical MR in cardiomyocytes (31, 32).
In one recent study, aldosterone was shown to induce Na+ influx into human umbilical vein endothelial cells, leading to cell swelling, even when the Na+ concentration in the culture medium was unchanged (33). It was suggested that this effect was mediated via the epithelial Na+ channel (ENaC). At one time, ENaCs were thought to exist only in the kidney; that was until the ENaC -subunit was identified in human heart (34). The function and significance of the ENaC in heart is not yet known, but it is possible that, along with NHE1, it contributes to mediating the acute effects of aldosterone in response to a rise in [Na+]o.
Na+/K+ adenosine triphosphatase (Na+/K+ ATPase), which mediates the active transport of Na+ out and K+ into the cells, is present in cardiomyocytes (35). It is notable that Na+/K+ ATPase has been reported to be reversely suppressed by aldosterone in the myocardium (36). Therefore, the inhibitory action of aldosterone on Na+/K+ ATPase in the myocardium would not conflict with the possible effect of aldosterone on the intracellular influx of sodium by NHE1 because both actions take sodium into cells in the myocardium.
Secretion of aldosterone from the adrenal gland is diminished by a high Na+ diet, reducing circulating levels of the hormone (2). On the other hand, it was recently reported that, in rats, the concentration of aldosterone in cardiac and vascular tissues are increased by high Na+ intake (37). In view of our present findings, we suggest that cell shrinkage caused by the increase in extracellular osmolarity associated with high Na+ intake would stimulate local synthesis of aldosterone, which in turn stimulates uptake of extracellular Na+ and water into the cells, thereby stabilizing the electrolyte and fluid balance across the cell membrane. Naturally, we understand that there are many reports disputing the theory about cardiac aldosterone synthesis (38, 39). It is still unclear, however, which of sodium, potassium, chloride, angiotensin II, ACTH, or others is actually the key regulator for cardiac aldosterone synthesis in vivo (40, 41). There are many issues that we should study in the future.
In sharp contrast to the effects of aldosterone at early phase, we found that long-time aldosterone exposure induces cardiomyocyte hypertrophy, as indicated by increased cell size, increased incorporation of 3H-leucine, and increased BNP transcription. That these effects could be suppressed by either eplerenone or SM 20220 means that the effects of aldosterone are mediated via both the MR and NHE1 at late phase. In addition, we also found that aldosterone induces NHE1 gene expression via the MR (eplerenone sensitive) at late phase, which is consistent with earlier reports (15, 16).
The effects of aldosterone at late phase observed in the present study are in agreement with those of Karmazyn et al. (16) and complement them by adding the observation that aldosterone-induced myocardial hypertrophy is dependent on [Na+]o as well as on the NHE1 activity. Elevation of [Na+]i via NHE1 likely leads to Ca2+ overload via the Na+/Ca2+ exchanger, which would in turn stimulate hypertrophic signaling (42). Also, it is possible that the increase in cell pH directly resulting from NHE1 activation might be the signal for the induction of the hypertrophic response. Furthermore, inflammatory cytokines may be involved in this system of aldosterone-induced myocardial hypertrophy depending on the [Na+]o level. In any event, eplerenone would be useful for treating and/or preventing myocardial hypertrophy. The results of both the RALES trial and the EPHESUS trial support this idea for the MR antagonist (43, 44). Also, as suggested by Young and Funder (45), the NHE1 antagonist might also be good for reducing cardiac fibrosis, although this agent has not been used in a clinical stage yet.
We used aldosterone at a concentration of 10–7 mol/liter, which is a close approximation to the circulating levels seen in vivo, particularly under hyperaldosteronemic conditions. This actually may be somewhat conservative, however, because aldosterone concentrations are reportedly an order of magnitude higher in cardiac tissues than in the peripheral circulation (40).
The effect of aldosterone in the heart should be discussed because cardiomyocyte MR is normally occupied by endogenous glucocorticoid in physiological status because 11-hydroxysteroid dehydrogenase type 2 is not normally expressed in the cardiomyocytes and there are high levels of circulating cortisol (46); however, in pathophysiological states, such as hypertension, heart failure, or neonatal stage, it has been hypothesized that mineralocorticoids can access cardiac MR and thereby produce cardiac damage (46).
We previously reported that aldosterone synthesis is activated in both the adrenal gland and hearts of patients with heart failure or hypertension (6, 7, 8). Moreover, as mentioned above, aldosterone levels are higher in the myocardium than in the circulation (40). Taken together, these findings suggest that a continuous intake of excess salt stimulates cardiac hypertrophy together with local production of aldosterone in the heart, irrespective of circulating aldosterone levels. Consistent with that idea, we observed that long-time elevated [Na+]o induced a small increase in cardiac hypertrophy even in the absence of added aldosterone. This may be explained by the endogenous production of aldosterone by the cells. We have to regardless the fact that there are many reports disputing the theory about cardiac aldosterone synthesis (38, 39) and also that the detrimental effects of aldosterone on the heart may be due to adrenal derived aldosterone (47).
a clinical viewpoint, the results of the present study highlight the benefit of reducing salt in the diet and are consistent with earlier reports emphasizing the importance maintaining a low-salt diet to prevent cardiac hypertrophy and subsequent heart failure (27). In that regard, our findings indicate that by maintaining a low-salt diet and thus reducing local cardiovascular levels of aldosterone, one mitigates the chronic effects of this potent mediator of cardiac hypertrophy. Observational studies of the Yanomamo Indians, a no-salt culture, reinforce this view (48, 49).
In conclusion, aldosterone acutely induces Na+ uptake via NHE1 in the presence of elevated [Na+]o. This rapid, nongenomic protective effect against cellular fluid loss is a positive and physiological attribute. In the face of elevated [Na+]o, however, long-time exposure to aldosterone induces pathological genomic effects via MR that lead to cardiomyocyte hypertrophy. The MR antagonist eplerenone could thus be useful for suppressing cardiac hypertrophy, without affecting the beneficial effects of aldosterone at early phase.
Footnotes
This study was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology, Tokyo [B (2 )-15390248 and B (2 )-15390249], the Ministry of Health, Labor and Welfare, Tokyo [14C-4, 14A-1, 17A-1 and 17C-2], the Smoking Research Foundation, Tokyo and the Takeda Science Foundation (Tokyo, Japan).
First Published Online December 22, 2005
Abbreviations: BNP, B-type natriuretic peptide; ENaC, epithelial Na+ channel; MR, mineralocorticoid receptor; Na+/K+ ATPase, Na+/K+ adenosine triphosphatase; NHE1, Na+/H+ exchanger 1; SBFI, sodium-binding-benzofuran-isophthalate; SBFI-AM, SBFI-acetoxymethyl ester.
Accepted for publication December 9, 2005.
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