Salt Excess Causes Left Ventricular Diastolic Dysfunction in Rats With Metabolic Disorder
Metabolic syndrome is a highly predisposing condition for cardiovascular disease and could be a cause of excess salt–induced organ damage. Recently, several investigators have demonstrated that salt loading causes left ventricular diastolic dysfunction associated with increased oxidative stress and mineralocorticoid receptor activation. We, therefore, investigated whether excess salt induces cardiac diastolic dysfunction in metabolic syndrome via increased oxidative stress and upregulation of mineralocorticoid receptor signals. Thirteen-week-old spontaneously hypertensive rats and SHR/NDmcr-cps, the genetic model of metabolic syndrome, were fed a normal salt (0.5% NaCl) or high-salt (8% NaCl) diet for 4 weeks. In SHR/NDmcr-cps, salt loading induced severe hypertension, abnormal left ventricular relaxation, and perivascular fibrosis. Salt-loaded SHR/NDmcr-cps also exhibited overproduction of reactive oxygen species and upregulation of mineralocorticoid receptor–dependent gene expression, such as Na+/H+ exchanger-1 and serum- and glucocorticoid-inducible kinase-1 in the cardiac tissue. However, in spontaneously hypertensive rats, salt loading did not cause these cardiac abnormalities despite a similar increase in blood pressure. An antioxidant, tempol, prevented salt-induced diastolic dysfunction, perivascular fibrosis, and upregulation of mineralocorticoid receptor signals in SHR/NDmcr-cps. Moreover, a selective mineralocorticoid receptor antagonist, eplerenone, prevented not only diastolic dysfunction but also overproduction of reactive oxygen species in salt-loaded SHR/NDmcr-cps. These results suggest that metabolic syndrome is a predisposed condition for salt-induced left ventricular diastolic dysfunction, possibly via increased oxidative stress and enhanced mineralocorticoid receptor signals.
Metabolic syndrome (MetS) is associated with a high rate of cardiovascular disease morbidity.1,2 Patients with MetS have been reported to exhibit left ventricular (LV) diastolic dysfunction,3,4 which eventually leads to diastolic heart failure with a poor prognosis.5,6 However, LV diastolic dysfunction has not always been seen in a MetS animal model. For example, in obesity-prone Sprague-Dawley rats, a moderate fat diet induced metabolic abnormalities but did not cause LV diastolic dysfunction.7 This discrepancy indicates a possibility that some exogenous factors deteriorate cardiac function in MetS. In patients with MetS, high salt intake increased blood pressure (BP) greater than in those without MetS.8 In an MetS rat model, evident renal injury was induced by salt loading.9 These findings suggest that the MetS model is highly susceptible to salt-induced organ damage. Salt loading induced LV hypertrophy or LV diastolic dysfunction in several hypertension models.10–14 Therefore, it is speculated that LV diastolic impairment could be accelerated with excess salt in an MetS model.
Reactive oxygen species (ROS) are important elements causing cardiac functional damage.10,15,16 MetS should be associated with an increase in oxidative stress,17–19 possibly through enhanced ROS-inducible adipocytokines.20–22 Also, salt loading increased oxidative stress in several salt-sensitive hypertension models.10,23–25 Given that those models with MetS are predisposed to salt-induced ROS overproduction, salt loading is expected to induce ROS augmentation and resultant LV diastolic dysfunction in MetS.
The beneficial effect of a mineralocorticoid receptor (MR) antagonist has been proven by clinical trials in patients with severe heart failure.26,27 MR antagonists showed a cardioprotective effect despite a low circulating aldosterone level in salt-loaded animal models.13,14,28,29 Furthermore, we reported recently that MR blockade prevented salt-induced renal damage in a rat MetS model.9 These results suggest that an MR signal is involved in the pathogenesis of salt-induced organ damage, including cardiac dysfunction in MetS.
In the present study, therefore, we investigated whether salt loading induces LV diastolic dysfunction in a MetS model, and, if so, whether ROS or MR signaling is involved in the mechanisms of salt-induced diastolic dysfunction. As a genetic rat model of MetS, we used the SHR/NDmcr-cp (SHR/cp), a derivative of the spontaneously hypertensive rat (SHR) with leptin receptor deficiency, characterized by obesity, hyperinsulinemia, dyslipidemia, and hypertriglyceridemia, all of which are consistent with MetS.30
All of the animals were handled in an accredited facility in accordance with the institutional animal care guidelines, and all of the research protocols conformed to the guiding principles for animal experimentation as outlined by the ethics committee on animal research of the University of Tokyo.
Thirteen-week-old male SHR/cps (SLC, Hamamatsu, Japan) were randomized into 4 groups: a normal salt (0.5% NaCl) diet group (NS-SHR/cps, n=10); high-salt (8% NaCl) diet group (HS-SHR/cps, n=12); high-salt diet with a superoxide dismutase mimetic, 4-hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl (tempol; 1 mmol/L in drinking water) group (HS+TE-SHR/cps, n=12); or high-salt diet with a selective MR antagonist (eplerenone; 1.25 g/kg of chow: 50 to 80 mg/kg per day) group (HS+EP-SHR/cps, n=12). The groups were treated for 4 weeks. SHRs (Eisai, Tokyo, Japan) with or without salt loading (HS-SHRs: n=4; NS-SHRs: n=6) were used as controls. The animals were maintained in a regulated environment at 26±2°C with a 12-hour:12-hour light-dark cycle.
Systolic BP (SBP) was monitored every week with the tail-cuff method (BP-98A, Softron). In each examination, we measured SBP 5 times at each time point for each rat and calculated the average.
Evaluation of Excretion of Urinary 8-Hydroxy-2′-Deoxyguanosine
The urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) concentration was evaluated using a commercially available 8-OHdG ELISA kit (Japanese Institute for the Control of Aging).
Transthoracic echocardiographic studies were performed as described previously.10 For details, see the data supplement (available online at http://hyper.ahajournals.org).
Measurement of Metabolic Parameters and Serum Aldosterone
For more information, please see the data supplement.
Hemodynamic parameters were recorded from a catheter placed in the cavity of the left ventricle, and maximal negative slope (−dp/dtmax) and time constant (T) at the isovolumic relaxation phase were calculated as reported previously.10 For details, see the data supplement.
ROS Production Induced by Addition of Nicotinamide-Adenine Dinucleotide Phosphate in Cardiac Tissue
ROS production in the cardiac tissue was evaluated by lucigenin chemiluminescence, as described previously.10 For details, see the data supplement.
For details see the data supplement.
Real-Time Quantitative PCR
The mRNA was extracted by the guanidinium method (Isogen, Nippon Gene). After the procedure of reverse transcription real-time quantitative PCR was performed using an ABI PRISM 7000 (Applied Biosystems).31 Commercially available, ready-made primers were used for the real-time quantitative-PCR (Applied Biosystems).
All of the values are expressed as means±SEMs. Comparisons were made with 1-way ANOVA followed by Scheffe’s method in all of the groups of rats. P values of <0.05 were considered to indicate significance. Furthermore, we analyzed the data using 2-way ANOVA to evaluate the independent and interactive influence of strains and salt loading on various parameters, including cardiac diastolic function and oxidative stress, in the 4 groups of rats (NS-SHR, HS-SHR, NS-SHR/cp, and HS-SHR/cp).
Body Weight, BP, and Cardiac Weight
Body weight was significantly higher in SHR/cps than in SHRs (Table 1). Body weight was not affected by salt loading, with or without tempol or eplerenone. Salt loading elevated SBP in both SHRs and SHR/cps, although this effect was slightly weak in SHR/cps at the early phase (Figure 1). Concomitantly, salt loading increased cardiac weight and LV wall thickness, as shown by the echocardiography (Tables 1 and 2⇓ and Figure S1). In HS-SHR/cps, tempol did not affect SBP. On the other hand, eplerenone decreased SBP slightly but not significantly in HS-SHR/cps. The cardiac weight was not affected by tempol in HS-SHR/cps, whereas eplerenone decreased it slightly but not significantly.
Metabolic parameters such as plasma leptin, serum insulin, and free fatty acids were significantly higher in SHR/cps than in SHRs, with or without salt loading (Table 3). The levels of serum triglyceride and fasting glucose were also higher in SHR/cps than in SHRs. These metabolic parameters in salt-loaded SHR/cps were not altered by treatment with tempol or eplerenone. Large SEs were detected in triglyceride and free fatty acid levels in SHR/cps.
LV Systolic and Diastolic Function
LV systolic function in echocardiography was preserved among all of the groups (Table 2 and Figure S1). NS-SHR/cps rats exhibited elongation in the deceleration time of early mitral inflow (EDcT) and in T in the hemodynamic study compared with NS-SHRs; however, these elongations were very small (Figures 2A through 2C, S2, and S3). Actually, −dp/dtmax of NS-SHR/cps was equivalent to those of NS-SHRs. On the other hand, salt loading caused evident LV abnormal relaxation in SHR/cps, which was confirmed by markedly elongated EDcT, blunted −dp/dtmax, and prolonged T (EDcT: 75.2±4.2 versus 58.4±2.1 ms, P<0.01; −dp/dtmax: 3.9±0.3 versus 5.5±0.3 mm Hg/ms, P<0.01; T: 15.4±2.2 versus 8.4±0.9 ms, P<0.01), whereas, in SHRs, salt loading did not cause LV diastolic dysfunction. There was no difference in the LV end-diastolic pressure among all of the groups (Table 4). These results suggest that SHR/cp, a rat model of MetS, is more predisposed to salt-induced LV diastolic dysfunction than SHR.
Coronary Perivascular Fibrosis in the SHR/cp Group
The histological study showed that salt loading caused evident perivascular fibrosis of the coronary artery in SHR/cps (2.8±0.2 versus 2.1±0.04; P<0.01) but not in SHRs (2.2±0.1 versus 2.1±0.1; Figures 3A and S4). The fibrotic change was restricted to the perivascular region of the heart. Concomitantly, salt loading significantly increased cardiac mRNA expression of connective tissue growth factor and collagen type III in SHR/cps but not in SHRs (Figure 3B and 3C).
Without salt loading, markers of ROS, such as urinary excretion of 8-OHdG, superoxide production induced by addition of NADPH in cardiac tissue, and mRNA expression of NADPH oxidase components p22phox, p47phox and gp91phox, were equivalent between SHRs and SHR/cps (Figure 4A through 4F). Salt loading significantly increased these ROS markers in SHR/cps but did not in SHRs.
Serum Aldosterone and MR Signals
Serum aldosterone level was significantly higher in NS-SHR/cps than in NS-SHRs (Figure 5A). Salt loading decreased the serum aldosterone level, but serum aldosterone was still slightly but not significantly higher in HS-SHR/cps as compared with HS-SHRs. In SHR/cps, salt loading enhanced cardiac mRNA expression of Na+/H+ exchanger-1 (NHE-1) and serum- and glucocorticoid-inducible kinase-1 (SgK-1), the markers of MR signals (Figure 5B and 5C). However, SHRs did not show any salt-induced upregulation of MR signals. Concomitantly, gene expression of angiotensin-converting enzyme (ACE) in the cardiac tissue was enhanced by salt loading in SHR/cps but not in SHRs (Figure 5D).
Tempol Inhibited LV Diastolic Dysfunction and MR Signal Activation in the Salt-Loaded SHR/cps Rats
In HS-SHR/cps, tempol not only inhibited ROS markers (Figure 4) but also prevented salt-induced LV diastolic function (EDcT: 54.5±0.8 ms, P<0.01; −dp/dtmax: 5.9±0.5 mm Hg/ms, P<0.01; T: 8.8±0.7 ms, P<0.01; Figures 2, S2, and S3). Also, tempol suppressed perivascular fibrosis (2.2±0.2; P<0.05) and overexpression of connective tissue growth factor and collagen type III (Figures 3 and S4). These cardioprotective effects were not accompanied by a reduction in BP (Figure 1). Furthermore, tempol prevented salt-induced upregulation of NHE-1, SgK-1, and ACE in SHR/cps without any change in serum aldosterone (Figure 5).
Eplerenone Inhibited LV Diastolic Dysfunction and Inhibited ROS Overproduction in Salt-Loaded SHR/cps Rats
In HS-SHR/cps, a selective MR antagonist, eplerenone, not only downregulated MR signals (Figure 5B and 5C) but also preserved LV diastolic function (EDcT: 53.6±1.2 ms, P<0.01; −dp/dtmax: 5.4±0.2 mm Hg/ms, P<0.01; T: 7.6±0.6 ms, P<0.01; Figures 2, S2, and S3). Eplerenone prevented perivascular fibrosis (2.0±0.1; P<0.01), associated with the inhibition of fibrotic markers (Figures 3 and S4). Furthermore, eplerenone inhibited a salt-induced increase in oxidative stress (Figure 4). In addition, eplerenone inhibited the overexpression of ACE in HS-SHR/cps (Figure 5D).
Influences of Strain and Salt Loading: Analysis by 2-Way ANOVA
Obesity and metabolic abnormalities were marked in SHR/cps as compared with SHRs, whereas SBP and cardiac weight were increased by salt loading (Table S1). However, there was no interaction between strains and salt loading from these data.
With regard to LV diastolic dysfunction, EDcT was significantly influenced by strains or salt loading, and the interaction between both was significant. Also, T was influenced by these 2 factors, and the interaction was marginally significant. Although −dp/dtmax was not significantly influenced by either of them, the interaction was marginally significant.
Cardiac ROS production induced by the addition of NADPH was significantly influenced by strain or salt loading, and the interaction was marginally significant. Urinary 8-OHdG showed a similar tendency but was not significant, suggesting that redox changes occur mainly in cardiac tissue of salt-loaded SHR/cps. Although the expression of NADPH oxidase components was not influenced by either of them because of large SEs, the huge increase in these parameters was compatible with the data of nicotinamide-adenine dinucleotide phosphate (NADPH)–induced cardiac ROS production. The changes in MR signal expression also showed similar tendencies to those in NADPH oxidase components. Thus, the data of LV diastolic function analyzed by 2-way ANOVA were compatible with the original results analyzed by 1-way ANOVA.
In the present study, SHR/cp, a rat model of MetS, showed salt-induced LV diastolic dysfunction, as indicated by either elongated EDcT by Doppler echocardiography or blunted −dp/dtmax and prolonged T in the hemodynamic study. This abnormal LV relaxation may be regarded as an early functional change without an increase in LV chamber stiffness, because LV systolic function was preserved, LV end-diastolic pressure was not elevated, and fibrotic change appeared only in the perivascular region.
Although patients with MetS are often associated with LV diastolic dysfunction,3,4 diastolic function was not markedly impaired without salt loading in SHR/cps. This is compatible with a previous study7 and suggests that, in addition to metabolic disorder, some exogenous factors may be involved in the early progression of diastolic dysfunction. Salt-induced BP hyperreactivity or renal injury has been reported in MetS patients or in MetS animal models.8,9 In the present study, LV diastolic function was apparently impaired when salt was loaded in SHR/cps. Therefore, excess salt may play a pivotal role in the progression of organ damage in MetS.
Increased oxidative stress may be involved in salt-induced cardiac damage in salt-sensitive hypertensive rats.10,24,25 Excess ROS may contribute to LV diastolic dysfunction by inhibition of the sarcoplasmic reticular Ca2+ uptake pump15 or by the disturbance of phospholamban.16 In addition, the beneficial effect of the MR antagonist has been shown in patients with severe heart failure26,27 and also in animal models of salt-induced cardiac damage.13,14,28,29 In fact, in SHR/cps, salt-induced LV diastolic dysfunction was accompanied by increased oxidative stress and by the upregulated expression of cardiac NHE-1 and SgK-1, both being downstream signals of MR.32,33 Moreover, antioxidant or selective MR antagonist prevented LV diastolic dysfunction. Thus, salt can be considered to be an important trigger of increased ROS and stimulated MR signals in MetS, both of which may play an important role in salt-induced progression of cardiac damage in MetS.
In other words, the degree of susceptibility to salt-related organ damage may be an important factor. In SHRs, LV diastolic dysfunction or the upregulation of MR signals was induced by long-term (6 to 12 weeks) salt loading.11–13,34 These previous findings are compatible with the present ones in that short-term (4 weeks) salt loading was not sufficient to induce LV diastolic dysfunction in SHRs. However, short-term salt loading caused diastolic dysfunction associated with ROS overproduction and MR promotion in SHR/cps. Similarly, in salt-sensitive rats, such as Dahl salt-sensitive rats and stroke-prone SHRs, cardiac damage or an increase in oxidative stress was induced by a relatively short period (4 to 6 weeks) of salt loading.10,14,24,25,35 Taken together, this shows, for the first time, that the SHR/cp is a more predisposed model to salt-induced organ abnormalities, such as LV diastolic dysfunction, oxidative stress, and MR promotion, compared with the SHR.
Salt-related ROS augmentation might be explained partly by increased ROS-inducible adipocytokines, such as tumor necrosis factor-α or nonesterified fatty acids in MetS.20–22 However, further investigation is required to evaluate the response of adipocytokines to salt loading. On the other hand, it is hypothesized that MR could be activated by excess salt in an aldosterone-independent manner in MetS. Even with a reduced level of circulating aldosterone, MR blockers were found to be protective against salt-induced cardiac dysfunction or proteinuria.9,28,31 Similarly, in SHR/cps, high salt intake promoted MR signals in the heart despite reduced circulating aldosterone, and eplerenone prevented LV diastolic dysfunction. It should be noted that NS-SHR/cps exhibited a significantly higher level of serum aldosterone than NS-SHRs, as reported previously.30 Although salt decreased circulating aldosterone, aldosterone was slightly higher in HS-SHR/cps than in HS-SHRs. Indeed, this higher level of aldosterone might affect salt-induced renal injury in SHR/cp rats,9 but this might not be the case in the heart. With the small increase in serum aldosterone in HS-SHR/cps, local aldosterone does not seem to overwhelm the huge amount of corticosterone in the heart. 11-β-Hydroxysteroid dehydrogenase type 2 was undetectable in the heart, and thus corticosterone can be prevented from being metabolized to cortisone.28 Therefore, MR should be occupied mostly by corticosterone in the cardiac tissue. Thus, MR might be stimulated by an aldosterone-independent mechanism in the heart of HS-SHR/cps.
It is questioned whether ROS augmentation and MR signal promotion emerged merely in parallel or whether they interacted with each other in salt-loaded SHR/cps. A causal linkage between MR activation and ROS has been proposed. First, administration of aldosterone augments ROS production via NADPH oxidase in vascular endothelial cells, which was abolished by an MR antagonist.36 Consistently, in our salt-loaded SHR/cps, eplerenone inhibited oxidative stress. Therefore, MR activation can induce ROS overproduction. Second, ROS could be speculated to be an upstream element in MR activation.9,37,38 This hypothesis is compatible with our finding that tempol inhibited salt-induced upregulation of NHE-1 and SgK-1. Thus, the involvement of ROS is one possible explanation for aldosterone-independent MR activation. However, the possibility of MR activation by ROS is still speculative and should be further evaluated.
The upregulation of ACE might be involved in the proposed association between ROS and MR signals. It has been reported that aldosterone increases the expression of ACE in cardiomyocytes.39 In SHR/cps, along with the increased expression of ROS and MR signals, ACE expression was upregulated by salt loading and, in turn, tempol or eplerenone inhibited the overexpression of ACE. These results suggest a feed-forward pathway from ROS overproduction or MR signal stimulation to ACE upregulation, which stimulates production of angiotensin II, resulting in further ROS overproduction or MR activation. However, this should also be evaluated by further experiments.
It is known that elevated BP accelerates LV diastolic dysfunction. In eplerenone-treated SHR/cps, a small reduction in BP might contribute to the preservation of LV function. However, LV relaxation was apparently impaired in HS-SHR/cps but not in HS-SHRs, even with a slightly lower BP elevation, and tempol prevented salt-induced LV abnormal relaxation with suppressed MR signals and without a reduction in BP. Therefore, a BP-independent mechanism may be at least partly involved in salt-induced diastolic dysfunction in MetS, as mentioned above. However, we could not clarify the extent to which BP contributes to eplerenone-induced amelioration of LV dysfunction because of the limitations involved in measuring BP.
As expected, metabolic parameters such as insulin and lipids were increased in SHR/cps compared with SHRs. Large SEs were detected in serum triglyceride and free fatty acid in SHR/cps but not in SHRs, although the blood specimens were collected after overnight fasting in both. Therefore, the fluctuation of lipids should be involved in the characteristics of SHR/cps. However, salt-induced diastolic dysfunction was not accompanied by additive exacerbation of metabolic parameters in SHR/cps. Consistently, tempol or eplerenone prevented salt-induced diastolic dysfunction without amelioration of these metabolic parameters. Thus, our results do not indicate that this metabolic disorder directly affects salt-induced LV diastolic dysfunction.
In conclusion, the present study demonstrates that SHR/cp, a model of MetS, is a highly predisposed condition for salt-induced LV diastolic dysfunction. It also suggests that increased oxidative stress and enhanced MR signals are involved in accelerating the pathogenesis of salt-induced diastolic dysfunction in MetS.
MetS is a highly predisposing condition for cardiovascular disease,1,2 and one of the early cardiac involvements is LV diastolic dysfunction. Thus, an appropriate interventional strategy should be prescribed to preserve LV function in MetS. We demonstrated that high salt intake may be one exacerbating factor in the progression of LV diastolic dysfunction in an MetS animal model. Renal damage is also accelerated with salt loading in the same model.9 Therefore, our series of results suggest that salt restriction should be prescribed as a first-line lifestyle modification to prevent obesity-related LV diastolic dysfunction and renal damage.9 Moreover, we demonstrated the possible involvement of oxidative stress and MR signals in the pathogenesis of salt-induced LV diastolic dysfunction in MetS. Therefore, it is also suggested that treatment with an antioxidant or a selective MR blocker could be highly effective in preventing cardiac dysfunction in patients with MetS.
Sources of Funding
This work was supported by grants from the Daiichi Sankyo Company, Ltd.
- Received February 10, 2008.
- Revision received March 3, 2008.
- Accepted June 12, 2008.
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