Journal of the American Society of Hypertension 9(2) (2015) 77–85
Research Article
Effects of high and low salt intake on left ventricular remodeling after myocardial infarction in normotensive rats Ludimila Forechi, MSca, Marcelo Perim Baldo, PhDa, Isabela Binotti de Araujo, MScb, Breno Valentim Nogueira, PhDb, and Jose Geraldo Mill, MD, PhDa,* a
Department of Physiological Science, Federal University of Espirito Santo, Vit oria, Brazil; and b Department of Morphology, Federal University of Espirito Santo, Vit oria, Brazil Manuscript received September 7, 2014 and accepted November 30, 2014
Abstract The dietary–sodium restriction is a standard approach following an acute myocardial infarction (MI). We examined the hypothesis in which the use of a high or low–sodium diet would worsen post–infarction left ventricular remodeling in rats and facilitate the development of heart failure. Left coronary artery ligation or sham–operated (SO) was produced in male Wistar rats (250–290 g). After surgery, animals were assigned to one of the three diets: standard amount of sodium (0.3% NaCl, SO and MI groups), a high–sodium diet (0.6% NaCl, SO–High and MI–High groups), or a low–sodium diet (0.03% NaCl, SO– Low and MI–Low groups). Diets were provided for 8 weeks post–surgery. Mortality rate was elevated in high–salt group (MI–Low, 21.4%; MI, 35.3%; MI–High, 47.6%). Contractility parameter was seen to be impaired in MI–Low animals (3195 211 mm Hg/s) compared with MI (3751 200 mm Hg/s). Low–salt diet did not prevent myocardial collagen deposition (MI–Low, 5.2 0.5%; MI, 5.0 0.4%) nor myocyte hypertrophy (MI–Low, 608 41m2; MI, 712 53 mm2) in left ventricle after MI. High–salt intake increases collagen volume fraction (SO, 3.3 0.4%; SO–High, 4.7 0.4%) in animals sham, but no major changes after MI. Our results show that ventricular remodeling was not altered by immediate introduction of low sodium after MI, and it may be a safe strategy as a therapeutic intervention to avoid volume retention. However, high sodium can be harmful, accelerating the post–infaction ventricular remodeling. J Am Soc Hypertens 2015;9(2):77–85. Ó 2015 American Society of Hypertension. All rights reserved. Keywords: Cardiac fibrosis; diet; heart failure; sodium consumption.
Introduction Excessive salt intake has been extensively linked to cardiovascular morbidity and mortality. Over the last decades, clinical and experimental studies have shown that increased salt intake is associated with deleterious effects, including endothelial dysfunction, hypertension, stroke, heart failure, and kidney disease.1–3 Thus, high salt intake has been
This work was supported by grants from Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico–CNPq [470748/2013-3; 400484/2013-7]. The authors declare that they have no conflicts of interest. *Corresponding author: Jose Geraldo Mill, MD, PhD, Department of Physiological Sciences, Federal University of Espırito Santo, Av. Marechal Campos 1468, Maruıpe, 29042-755 – Vit oria, ES, Brazil. Tel: þ55 27-33357335; Fax: þ55 27-33357330. E-mail: [email protected]
considered a powerful risk factor for cardiovascular disease (CVD). Most population–based studies have found that salt consumption currently exceeds acceptable levels established by regulatory agencies.4 In fact, current recommendations advocate a reduction in salt intake to reduce the incidence of CVD, the most frequent cause of death worldwide. However, some authors have raised a number of issues about the current recommendations for a significant reduction in salt intake in the general population.5,6 It has been argued that low–salt diets may activate the sympathetic nervous system and the systemic renin–angiotensin–aldosterone system (RAAS), two effects that should be avoided during the post–infarct period because of their deleterious effects on left ventricular remodeling. Thus, low–salt diets could potentially be harmful after infarction because cardiac hypertrophy and fibrosis may facilitate the development of heart failure.
1933-1711/$ - see front matter Ó 2015 American Society of Hypertension. All rights reserved. http://dx.doi.org/10.1016/j.jash.2014.11.006
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Acute myocardial infarction (MI) is an extreme situation that is associated with complex changes in cardiac architecture and function that result in a high mortality rate. This process of change in the infarcted heart is known as myocardial remodeling, and it progressively leads to heart failure.7 It is noteworthy that, aside from standard pharmacologic approaches, a reduction in sodium intake is one of the first therapeutic actions after acute myocardial infarction. Based on the well–known effects of high salt consumption, it is expected that reducing salt intake after MI would attenuate left ventricular remodeling, thus preventing heart failure. However, this hypothesis has not been confirmed. For instance, Klein et al8 and Paterna et al9 did not observe beneficial effects after lowering sodium intake in heart failure patients. Instead, they observed increased hospitalization times for cardiovascular causes, increased mortality rates,8 and detrimental renal and neurohormonal effects.9 Thus, we sought to determine whether strict salt restriction or increased consumption of salt would affect the mortality rate and ventricular remodeling after acute MI in rats.
Materials and Methods Animals Male Wistar rats (260–290 g) from the animal core facility of the Federal University of Espirito Santo were used in the experiments. The animals were housed in appropriate cages in a room with controlled temperature and with a 12 hour light:12 hour dark cycle. Animals had free access to rat chow and tap water. All procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication N. 85–23, revised 1996) and approved by the institutional Committee for Ethics in Animal Research (CEUA–UFES n. 068/2012).
Coronary Artery Occlusion and Groups MI was induced as described previously.10 Briefly, the animals were anesthetized with ketamine (50 mg/kg intraperitoneal [ip]; Agener Uni~ao) and xylazine (Bayer; 10 mg/kg, ip), and left thoracotomy was performed in the fourth intercostal space. The heart was exposed rapidly, and the left coronary artery was permanently occluded using 6–0 monofilament nylon sutures. The thorax was closed, and the animals recovered normal respiratory movements. Sham–operated (SO) rats that underwent the same surgical procedures except coronary ligation were used as non–infarcted control animals. Immediately after surgery, the animals were randomly assigned into six groups to receive a diet containing the standard amount of sodium (0.3% NaCl, SO and MI groups), a high–sodium diet (0.6% NaCl, SO–High and MI–High groups), or a low–sodium diet (0.03% NaCl, SO–Low and MI–Low groups).
Diets were specifically prepared for this project, and all components of the rats’ diets were held constant except the sodium content. The animals were maintained on one of the three experimental diets for 8 weeks.
Noninvasive Blood Pressure Measurement Systolic blood pressure was recorded by noninvasive tail cuff plethysmography before surgery and 30 and 60 days after surgery. Briefly, the rat was placed in a pre–warmed restraining chamber, and occluding cuffs and pneumatic pulse transducers were positioned on the rat’s tail. The cuff was inflated and deflated automatically, and the signal was automatically collected in an IITC apparatus (IITC Inc).11 Five blood pressure measurements were made for each rat, and the mean of these measurements was recorded.
Physiological Parameters Each rat’s body weight was measured before surgery and then once per week. To determine the food and water intake and urinary parameters for each rat, the animals were placed into individual metabolic cages (Tecniplast 304) for 2 consecutive days 60 days after surgery. The first day was used as an adaptation period; 24–hour food and water consumption, as well as urine production, were measured during the second day. The urine production was used to measure the urinary excretion of sodium (MH-LAB ISE Electrolyte Analyzer, Diamond Diagnostics Inc).
Hemodynamic Measurements At the end of the 8–week follow–up period, the animals were anesthetized with ketamine (50 mg/kg ip) and xylazine (10 mg/kg ip). The right common carotid artery was catheterized with a fluid–filled polyethylene catheter (P50) connected to a pressure transducer (TRI 21, Letica Scientific Instruments) and to a digital system (Powerlab/ 4SP ML750, ADInstrument) to record the pulsatile blood pressure. The catheter was then advanced into the left ventricular cavity to record intracavitary pressure and its first temporal derivative. Left ventricular peak systolic pressure (LVSP) and end–diastolic pressure (LVEDP) and the maximum positive and negative values of dP/dt (þdP/dt max and dP/dt max) were recorded under regular rhythm with 1–kHz filtering.12 After hemodynamic recordings, the naso–anal length of the animal was measured, and the animal was euthanized with an overdose of anesthetic medication. The heart was rapidly removed, flushed in cold saline solution, dried with filter paper, weighed, and fixed in a buffered solution of paraformaldehyde (4%, pH 7.4) for subsequent histologic analysis. The lungs, kidney, liver, and the gastrocnemius muscle were also removed and weighed.
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Histologic Analysis
Statistical Analyses
After the atrial appendages were removed, the ventricles were transversely divided into three slices of approximately 3–mm thickness, embedded in paraffin blocks, and processed for conventional optical microscopy. At the level of the upper boundary of the anterior left papillary muscle, four 6 mm-thick sections were obtained and stained with picrosirius red stain to determine the collagen volume fraction and infarct size. Slices were also stained with hematoxylin–eosin to determine the myocyte cross– sectional area. Images were captured with a video camera (AxioCam ERc 5s, Carl Zeiss) coupled to an optical microscope (Olympus AX70, Olympus Corporation) under 400 magnification. Fifteen areas of high–power fields were chosen in the non–infarcted area of each heart, and the percentage of the area that was stained by picrosirius red was determined. In each rat, we measured the cross–sectional area of 30–50 myocytes positioned perpendicularly to the plane of the section with a clearly visible nucleus occupying the central region of the cell.12 The infarct size was calculated as (endocardial þ epicardial circumference of infarcted tissue)/(endocardial þ epicardial circumference of the left ventricle) and was expressed as a percentage. All histologic analysis was performed using ImageJ software (v. 1.43u, National Institutes of Health). Because infarct size is a critical factor influencing left ventricular remodeling, only animals with an infarct size greater than 35% were included in the present analysis.
The data are presented as means standard errors of the mean (SEM). The unpaired Student’s t–test was used to compare two means. Means in the six groups were compared via a two–way analysis of variance (ANOVA) followed by the Fisher post post–hoc test when necessary. The mortality rates in the infarcted groups are presented via the Kaplan–Meier method and compared using the log–rank test. Statistical significance was set at P < .05.
Results General Characteristics No mortality was observed in the SO groups. The mortality in the follow–up period (8 weeks) for each group was as follows: six out of 21 animals in the MI–Low group, three out of 23 animals in the MI group, and 10 out of 23 animals in the MI–High group. The Kaplan–Meier curves shown in Figure 1A suggest that mortality occurred earlier in MI when followed by a low–salt diet. However, the high–salt diet increased the mortality rate significantly (P ¼ .037). Among the surviving animals, four out of 15 rats in the MI–low group, two out of 20 rats in the MI group, and two out 13 rats in the MI–High group were excluded from the analysis because the infarct size was smaller than 35%. This exclusion resulted in three groups of rats with similar infarct sizes; the infarct sizes were 43.0% 3.1% in animals fed a standard diet,
Figure 1. Panel shows the (A) mortality rate after 60 days of follow–up, (B) mean infarct size, (C) body weight, and (D) systolic blood pressure. Standard diet is shown in black, low–sodium diet (Low) in gray, and high–sodium diet (High) in light gray. The values are presented as the mean standard error of the mean (SEM). P < .05 aSO vs. SO– Low; bSO vs. SO–High; cSO–Low vs. SO–High; dMI vs. MI–Low; eMI vs. MI–High; fMI–Low vs. MI–High; gSO– Low vs. MI–Low; hSO vs. MI; iSO– High vs. MI–High. MI, Myocardial infarction; SO, sham operation.
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Table 1 Morphologic parameters after 60 days of follow–up Parameters BW (g) Naso–anal length (cm) Tibia length (mm) Ventricles (mg) Lungs (mg) Liver (g) Kidney (mg) Gastrocnemius (mg)
SO–Low 306 22.1 41 752 1602 10.5 1009 1716
SO ,z
9* 0.3*,z 0.4z 28*,y 65z 0.4*,z 38*,z 88*,z
415 23.6 42 979 1823 14.0 1400 2112
SO–High
9 0.2 0.5 22 62 0.6 29 57
438 24.8 43 1270 1991 12.7 1596 2146
12 0.3 0.4 38y 64 0.4 57y 222
MI–Low 329 22.9 42 1039 2094 11.7 1081 1663
MI x,{
11 0.4 0.5 41x,{,# 132{,# 0.5x,{ 50x,{ 53x,{
391 23.3 43 1253 2215 13.2 1231 2121
MI–High
10 0.2 0.3 69** 207** 0.5 59** 85
407 23.5 43 1351 2810 15.6 1451 2062
12 0.2 0.6 51 212k,yy 0.6k 48k,yy 67
BW: Body weight; High, high–sodium diet; Low, low–sodium diet; MI, myocardial infarction; SO, sham operation. The values are shown as mean SEM. P < .05. * SO vs. SO–Low. y SO vs. SO–High. z SO-Low vs. SO–High. x MI vs. MI–Low. k MI vs. MI–High. { MI–Low vs. MI–High. # SO–Low vs. MI–Low. ** SO vs. MI. yy SO–High vs. MI–High.
48.0% 3.9% in those fed a low–sodium diet, and 40.1% 2.7% in those fed a high–sodium diet (Figure 1B). Body weight on the seventh day after surgery was smaller in the animals subjected to coronary ligation (Figure 1C). During the follow–up period, only animals fed the standard or high–sodium diet recovered their body weight. From the third week on, SO and MI animals fed a low–sodium diet exhibited reduced body weight gain compared with the animals on the standard diet or a high–salt diet (Figure 1C). At the end of the follow–up period, no difference in body weight was found between the SO and infarcted groups fed a low–sodium diet.
Morphologic and Physiologic Parameters Table 1 shows the organ weights for the six experimental groups. The weights of the gastrocnemius muscle, liver, left kidney, and ventricles were lower in SO rats fed a low–sodium diet compared with those fed a standard diet. In addition, the naso–anal length was smaller in the SO–low group. Interestingly, the low–sodium diet did not affect the compensatory gain of ventricular mass after MI. In contrast, the high–salt diet significantly increased the weights of the ventricles and kidneys in SO rats. However, the extent of hypertrophic growth in the ventricles secondary to infarction was unaffected by increasing the sodium content in the diet (Table 1). As expected, the lung weight was greater in all infracted groups compared with their corresponding SO groups. Physiologic data observed 8 weeks after MI or SO are depicted in Table 2. As expected, groups consuming high–salt diets exhibited greater water intake and,
consequently, greater urine production as well. Low salt content in the diet was associated with lower absolute food intake. However, in relative terms, the rats fed a low–salt diet ingested amounts of chow similar to those ingested by rats fed normal or high–salt diets. The urinary excretion of sodium in sham–operated rats was proportional to the consumption (0.07 0.04 mmol/24h in SO– Low group, 1.76 0.45 mmol/24h in SO group, and 11.68 1.49 mmol/24h in SO–High group), being significantly higher in the high–salt group (P < .05). Similar results were found in animals subjected to coronary ligation (0.04 0.04 mmol/24h, 0.24 0.05 mmol/24h and 17.23 1.69 mmol/24h in MI–Low, MI, and MI–High groups, respectively). The urinary excretion of sodium was elevated even further after MI in those animals fed a high–salt diet (11.68 1.49 mmol/24h in SO–High group and 17.23 1.69 mmol/24h in MI–High group; P < .05).
Blood Pressure and Left Ventricular Function To assess the effect of sodium content in the diet on blood pressure, we noninvasively measured animals’ systolic blood pressure (Figure 1D). A small and non–significant increase in the systolic blood pressure was found 4 weeks after surgery in SO animals consuming either low– sodium or standard diets. Dietary salt excess, however, caused SO animals’ blood pressure to increase and remain elevated throughout the follow–up period. This effect was not observed in infarcted rats fed a high–salt diet. Systolic blood pressure measurements were stable in all infarcted groups and did not vary significantly with the salt content of animals’ diets.
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Table 2 Metabolic parameters at 60 days after surgery Parameters Water intake (mL/day) Food intake (g/day) Urinary output (mL/day)
SO–Low
SO z
36.0 2.6 15.1 0.7*,z 19.2 1.7z
35.5 1.9 19.7 0.8 17.0 1.2
SO–High
MI–Low y
80.2 3.9 19.5 1.0 57.9 3.6y
{
36.8 3.9 17.9 0.6# 23.1 3.7x,{
MI
MI–High
41.0 2.9 20.3 1.2 13.6 1.5
88.4 5.5k 19.6 1.2 72.1 4.1k,yy
High, High–sodium diet; Low, low–sodium diet; MI, myocardial infarction; SO, sham operation. The values are shown as mean SEM. P < .05 * SO vs. SO–Low. y SO vs. SO–High. z SO–Low vs. SO–High. x MI vs. MI–Low. k MI vs. MI–High. { MI–Low vs. MI–High. # SO–Low vs. MI–Low. yy SO–High vs. MI–High.
Dietary sodium restriction per se reduced peak LVSP in anesthetized animals compared with rats fed a control diet and did not significantly affect other hemodynamic parameters (Figure 2A). Left ventricular (LV) dysfunction in infarcted animals was confirmed by a finding of increased LVEDP (Figure 2B), along with impaired LV contractile function and relaxation (Figure 2C, D, respectively). The frequency of contraction was only slightly impaired in MI–Low animals compared with MI animals.
Cardiac Histology To further evaluate the influence of different levels of sodium intake on post–infarct remodeling, we investigated whether changes in diet could alter myocardial collagen deposition and the myocyte cross–sectional area in non–
infarcted areas of the left ventricle. In SO rats fed a high– salt diet, the myocardial area occupied by collagen was significantly increased (Figure 3A). Although all groups subjected to MI exhibited increased fibrosis, the extent of fibrosis was not influenced by different levels of sodium intake. A similar pattern was observed for the myocyte cross–sectional area; the myocyte cross–sectional area increased slightly, but not significantly, with increasing salt in the diet. MI was associated with a significant increase in the myocyte cross–sectional area. However, this increase was unaffected by the salt content in the diet (Figure 3B).
Discussion The World Health Organization recommends reducing daily sodium intake to prevent cardiovascular disease.
Figure 2. Cardiac function assessed by intraventricular pressure measurements. Graphs show (A) left ventricular systolic pressure, (B) left ventricular end–diastolic pressure, and the maximum rate of pressure rise and fall (C and D), respectively. P < .05 aSO vs. SO–Low; b SO vs. SO–High; cSO–Low vs. SO– High; dMI vs. MI–Low; eMI vs. MI– High; fMI–Low vs. MI–High; gSO–Low vs. MI–Low; hSO vs. MI; iSO–High vs. MI–High. High, High–sodium diet; Low, low–sodium diet; MI, myocardial infarction; SO, sham operation.
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Figure 3. Photomicrographs of cardiac sections stained with Sirius red to visualize collagen deposition and stained with hematoxylin–eosin to determine the myocyte cross–sectional area. (A) The quantitation of collagen deposition in the non–infarcted interventricular septum area; (B) Myocyte cross–section area. P < .05 bSO vs. SO–High; cSO–Low vs. SO–High; gSO–Low vs. MI–Low; h SO vs. MI; iSO–High vs. MI–High. High, High–sodium diet; Low, low–sodium diet; MI, myocardial infarction; SO, sham operation.
However, there is some clinical and experimental evidence that lowering sodium intake increases the risk of cardiovascular disease, as has been observed with high salt intake. Several mechanisms may account for the association between low salt intake and cardiovascular diseases, for example, activation of the renin–angiotensin system,13–15 sympathetic overactivity,16 endothelial dysfunction and oxidative stress,17 insulin resistance, higher plasma triacylglycerol levels,18 and increased atherogenesis.14,19 Considering that dietary sodium restriction is a standard approach after acute MI in patients aiming to reduce fluid retention, the present study investigated whether changes in dietary sodium content affect post–infarction mortality. Moreover, we have investigated whether long–term diets with differing sodium content result in different courses of post–infarction remodeling in normotensive rats. In order to perform this study, we used a high–sodium diet (2–fold higher as compared with the standard diet) and a low–sodium diet (10–fold lower as compared with the standard diet). Our choice for the amount of sodium used in in the high–sodium diet was based in the American consumption of about 3400 milligrams of sodium a day, twice as high as the 1500 milligrams recommended by the American Heart Association.5 Also, the low–sodium diet was created to mimic the strict reduction in the sodium intake observed in patients that suffered an acute coronary event. Our results demonstrate that after 8 weeks of follow–up, a low–sodium diet did not significantly alter the mortality after infarction, while an increased mortality rate was observed in infarcted rats fed a high–salt diet. As previously reported, mortality after permanent coronary ligation has two leading causes. In the acute phase, ventricular arrhythmias are associated with high mortality; subsequently,
left ventricular dysfunction and heart failure are associated with high mortality.7 Examining the survival curve, we observed that rats fed a high–sodium diet exhibited higher mortality, while in the group fed a low–sodium diet, mortality was lower but occurred earlier compared with animals given a normal or high–sodium diet. Sympathetic activation after infarction increases mortality because it facilitates the development of life–threatening ventricular arrhythmias.7 Therefore, we suggest that arrhythmias may be a putative mechanism involved in the earlier mortality in the MI– low–salt group. In those animals subjected to high salt intake, however, mortality may be caused by an increased susceptibility to ventricular arrhythmias early after infarction and by an acceleration of the development of heart failure. Both hypotheses have already been demonstrated.20,21 The loss of contractile tissue after MI initiates a progressive process in infarcted and non–infarcted areas of the heart, to preserve cardiac function. One of the key markers of cardiac failure after infarction is LV enlargement, and the extent of LV enlargement is primarily determined by the infarct size. In our study, changes in dietary sodium introduced immediately after coronary ligature appears to have no effect on infarct size. Similar results have been reported by Dikow et al,22 who reported that infarct size was similar in nephrectomized animals fed a low–salt diet, and by Resende and Mill,23 who reported a similar infarct size in rats fed a larger quantity of sodium. However, our data do not allow us to determine whether dietary manipulations before coronary ligature are able to influence the extent of cardiac damage produced during coronary occlusion. Further studies are necessary to answer this question. Our data also suggest that manipulation of sodium intake after infarction has little influence on left ventricular
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performance. In SO rats, salt restriction decreased LVSP, while high salt intake increased LVSP; analogous effects were observed for dP/dt. Changes occurred in the same direction in infarcted animals. However, the differences were smaller and non–significant. These results are consistent with the results of an in vitro study in which a low–sodium diet (0.05% NaCl) did not affect left ventricular function in isolated hearts after MI.13 These results are also consistent with an in vivo study that demonstrated that MI rats had significantly lower LV maximum þ dP/dt compared with the control group under salt overload.23 Moreover, chronic low–sodium (0.015% NaCl) and high–sodium (0.9% NaCl) intake had no effect on ejection fraction or fractional shortening after MI.24 However, studies should be compared cautiously because the dietary sodium contents and the treatment periods were not uniform. Increased collagen deposition and myocyte hypertrophy are two changes that contribute to the development of heart failure in the post–infarct period. The increase in collagen levels in noninfarcted tissue is a critical step that contributes to cardiac contractile dysfunction and structural remodeling after infarction.7,25 Numerous studies have observed an activation of the cardiac renin–angiotensin– aldosterone system (RAAS) after MI. Increased local production of aldosterone and angiotensin II stimulates collagen deposition along the cardiac myocyte strands. The involvement of aldosterone in this process has been confirmed by the observation that aldosterone antagonists reduce the extent of fibrosis.26 The relationships between sodium intake and local RAAS are still poorly understood. Urabe et al24 showed that a low–salt diet positively contributes to cardioprotection and that this effect may be related to reduced collagen deposition, despite high levels of plasma aldosterone. These data, however, were not confirmed in our study because increased collagen deposition after infarction remained stable even in the infarcted group given a low–salt diet. Because the myocardial deposition of collagen is strongly regulated by the local RAAS, our data suggest that the cardiac RAAS is not regulated by sodium, in contrast with the endocrine RAAS, which is strongly activated by reduced sodium intake. Alternatively, our data suggest that the local RAAS may be fully activated in infarcted rats. Thus, additional manipulations of the systemic RAAS did not exert additional influences in the heart. As has been observed for collagen deposition, the development of cardiac hypertrophy after MI is also stimulated by several factors, including hemodynamic overload and neurohumoral activation. Cardiac hypertrophy processes occur asymmetrically to ensure functional adaptation to the hemodynamic overload. Our data support this view because infarcted animals exhibited increased cardiac mass and increased myocyte cross–sectional areas compared with SO rats. Dietary salt excess has repeatedly been demonstrated to increase the weight of the heart chambers by increasing
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the amount of extracellular matrix and cardiomyocyte size, an effect that is partially modulated by the local RAAS and known to be independent of blood pressure (1). Here, we also observed a small but non–significant effect in SO rats and no effect in rats with MI. Therefore, our data suggest that hypertrophic growth appears to be fully activated by infarction, with no additional activation that can be attributed to other factors. It has been reported that low sodium intake suppresses post–MI cardiac hypertrophy24 and potentiates the antihypertrophic effect of some drugs after MI13 or in spontaneously hypertensive rats.27 However, the mechanism underlying this phenomenon is not completely understood. In our study, a low–sodium diet did not affect either the cardiac mass or the myocyte cross–sectional area. Our data showed that strict salt restriction depresses body growth in infarcted and non–infarcted rats. Growth suppression seems not to be due to a lower caloric intake because diets were isocaloric and relative food intake was relatively stable among groups consuming different amounts of sodium. Very low sodium intake, however, may change hormonal profiles. In our experiment, the SO rats fed a low–sodium diet exhibited either lower body weight or smaller body size compared with those animals fed standard or high–sodium diets. In a study by Coelho et al,28 rats were given a low–sodium diet from weaning until 12 weeks of age. Low sodium intake increased weight gain but did not affect the plasma levels of T3 or T4 or the exploratory activity of animals. However, it decreased the levels of TSH, thermogenin, and leptin.28 The results of studies examining different animal species and different degrees of salt restriction have been inconsistent, with studies showing either weight gain18 or no change in weight.13–15,17,29–31 In addition, we cannot rule out the possibility that reduced sodium intake may alter the absorption of essential nutrients,32,33 even though the only difference between the standard chow and the low–sodium chow was the amount of sodium in the chow. In summary, low salt intake after a MI did not prevent increased collagen deposition and ventricular hypertrophy in rats. Moreover, lowering sodium intake had no effect on the mortality rate, infarct size, or ventricular function. Thus, the immediate introduction of a low–sodium diet after MI did not worsen the progression of cardiac remodeling and left ventricular dysfunction caused by MI in rats. High salt intake increased the collagen volume and the weight of the ventricles and kidneys in sham–treated animals. In addition, high salt intake increased the mortality rate, but no major changes in post–infarction cardiac remodeling were observed in rats fed a high–salt diet. Acknowledgment The authors are grateful to the Laboratory of Cellular Ultrastructure Carlos Alberto Redins (LUCCAR) for technical assistance and use of the microscope.
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References 1. Ferreira DN, Katayama IA, Oliveira IB, Rosa KT, Furukawa LN, Coelho MS, et al. Salt-induced cardiac hypertrophy and interstitial fibrosis are due to a blood pressure-independent mechanism in Wistar rats. J Nutr 2010;140:1742–51. 2. Cordaillat M, Rugale C, Casellas D, Mimran A, Jover B. Cardiorenal abnormalities associated with high sodium intake: correction by spironolactone in rats. Am J Physiol Regul Integr Comp Physiol 2005; 289:R1137–43. 3. Drenjancevic-Peric I, Jelakovic B, Lombard JH, Kunert MP, Kibel A, Gros M. High-salt diet and hypertension: focus on the renin-angiotensin system. Kidney Blood Press Res 2011;34:1–11. 4. He FJ, MacGregor GA. Reducing population salt intake worldwide: from evidence to implementation. Prog Cardiovasc Dis 2010;52:363–82. 5. Whelton PK, Appel LJ, Sacco RL, Anderson CA, Antman EM, Campbell N, et al. Sodium, blood pressure, and cardiovascular disease: further evidence supporting the American Heart Association sodium reduction recommendations. Circulation 2012;126: 2880–9. 6. Taylor RS, Ashton KE, Moxham T, Hooper L, Ebrahim S. Reduced dietary salt for the prevention of cardiovascular disease: a meta-analysis of randomized controlled trials (Cochrane review). Am J Hypertens 2011;24:843–53. 7. Mill JG, Stefanon I, dos Santos L, Baldo MP. Remodeling in the ischemic heart: the stepwise progression for heart failure. Braz J Med Biol Res 2011;44:890–8. 8. Klein L, O’Connor CM, Leimberger JD, GattisStough W, Pina IL, Felker GM, et al. Lower serum sodium is associated with increased short-term mortality in hospitalized patients with worsening heart failure: results from the Outcomes of a Prospective Trial of Intravenous Milrinone for Exacerbations of Chronic Heart Failure (OPTIME-CHF) study. Circulation 2005;111:2454–60. 9. Paterna S, Gaspare P, Fasullo S, Sarullo FM, Di Pasquale P. Normal-sodium diet compared with lowsodium diet in compensated congestive heart failure: is sodium an old enemy or a new friend? Clin Sci 2008;114:221–30. 10. Baldo MP, Davel AP, Nicoletti-Carvalho JE, Bordin S, Rossoni LV, Mill JG. Granulocyte colony-stimulating factor reduces mortality by suppressing ventricular arrhythmias in acute phase of myocardial infarction in rats. J Cardiovasc Pharmacol 2008;52:375–80. 11. Baldo MP, Forechi L, Morra EA, Zaniqueli D, Machado RC, Lunz W, et al. Long-term use of lowdose spironolactone in spontaneously hypertensive
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
rats: effects on left ventricular hypertrophy and stiffness. Pharmacol Rep 2011;63:975–82. Forechi L, Baldo MP, Meyerfreund D, Mill JG. Granulocyte colony-stimulating factor improves early remodeling in isoproterenol-induced cardiac injury in rats. Pharmacol Rep 2012;64:643–9. Westendorp B, Schoemaker RG, Buikema H, de Zeeuw D, van Veldhuisen DJ, van Gilst WH. Dietary sodium restriction specifically potentiates left ventricular ACE inhibition by zofenopril, and is associated with attenuated hypertrophic response in rats with myocardial infarction. J Renin Angiotensin Aldosterone Syst 2004;5:27–32. Tikellis C, Pickering RJ, Tsorotes D, Huet O, ChinDusting J, Cooper ME, et al. Activation of the reninangiotensin system mediates the effects of dietary salt intake on atherogenesis in the apolipoprotein E knockout mouse. Hypertension 2012;60:98–105. Mori T, Kurumazuka D, Matsumoto C, Shirakawa H, Kimura S, Kitada K, et al. Dietary salt restriction activates mineralocorticoid receptor signaling in volumeoverloaded heart failure. Eur J Pharmacol 2009;623: 84–8. Campese VM, Romoff MS, Levitan D, Saglikes Y, Friedler RM, Massry SG. Abnormal relationship between sodium intake and sympathetic nervous system activity in salt-sensitive patients with essential hypertension. Kidney Int 1982;21:371–8. Suematsu N, Ojaimi C, Recchia FA, Wang Z, Skayian Y, Xu X, et al. Potential mechanisms of lowsodium diet-induced cardiac disease: superoxide-NO in the heart. Circ Res 2010;106:593–600. Ruivo GF, Leandro SM, do Nascimento CA, Catanozi S, Rocha JC, Furukawa LN, et al. Insulin resistance due to chronic salt restriction is corrected by alpha and beta blockade and by L-arginine. Physiol Behav 2006;88:364–70. Raz-Pasteur A, Gamliel-Lazarovich A, Gantman A, Coleman R, Keidar S. Mineralocorticoid receptor blockade inhibits accelerated atherosclerosis induced by a low sodium diet in apolipoprotein E-deficient mice. J Renin Angiotensin Aldosterone Syst 2012;15: 228–35. Baldo MP, Teixeira AK, Rodrigues SL, Mill JG. Acute arrhythmogenesis after myocardial infarction in normotensive rats: influence of high salt intake. Food Chem Toxicol 2012;50:473–7. Katayama IA, Pereira RC, Dopona EP, Shimizu MH, Furukawa LN, Oliveira IB, et al. High-salt intake induces cardiomyocyte hypertrophy in rats in response to local angiotensin II type 1 receptor activation. J Nutr 2014;144:1571–8. Dikow R, Kihm LP, Zeier M, Kapitza J, Tornig J, Amann K, et al. Increased infarct size in uremic
L. Forechi et al. / Journal of the American Society of Hypertension 9(2) (2015) 77–85
23.
24.
25.
26.
27.
28.
rats: reduced ischemia tolerance? J Am Soc Nephrol 2004;15:1530–6. de Resende MM, Mill JG. Effect of high salt intake on local renin-angiotensin system and ventricular dysfunction following myocardial infarction in rats. Clin Exp Pharmacol Physiol 2007;34:274–9. Urabe A, Izumi T, Abe Y, Taniguchi I, Mochizuki S. Effects of eplerenone and salt intake on left ventricular remodeling after myocardial infarction in rats. Hypertens Res 2006;29:627–34. Bujak M, Frangogiannis NG. The role of TGF-beta signaling in myocardial infarction and cardiac remodeling. Cardiovasc Res 2007;74:184–95. Fraccarollo D, Galuppo P, Hildemann S, Christ M, Ertl G, Bauersachs J. Additive improvement of left ventricular remodeling and neurohormonal activation by aldosterone receptor blockade with eplerenone and ACE inhibition in rats with myocardial infarction. J Am Coll Cardiol 2003;42:1666–73. Griffiths CD, Morgan TO, Delbridge LM. Effects of combined administration of ACE inhibitor and angiotensin II antagonist are prevented by a high NaCl intake. J Hypertens 2001;19:2087–95. Coelho MS, Passadore MD, Gasparetti AL, Bibancos T, Prada PO, Furukawa LL, et al. High- or low-salt diet
29.
30.
31.
32.
33.
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from weaning to adulthood: effect on body weight, food intake and energy balance in rats. Nutr Metab Cardiovasc Dis 2006;16:148–55. Trongtorsak P, Morgan TO, Delbridge LM. Combined renin-angiotensin system blockade and dietary sodium restriction impairs cardiomyocyte contractility. J Renin Angiotensin Aldosterone Syst 2003;4:213–9. Lima NK, Lima FB, dos Santos EA, Okamoto MM, Sumida DH, Hell NS, et al. Effect of lifelong highor low-salt intake on blood pressure, left ventricular mass and plasma insulin in Wistar rats. Am J Med Sci 2006;331:309–14. Shao W, Seth DM, Prieto MC, Kobori H, Navar LG. Activation of the renin-angiotensin system by a lowsalt diet does not augment intratubular angiotensinogen and angiotensin II in rats. Am J Physiol Renal Physiol 2013;304:F505–14. Garriga C, Moreto M, Planas JM. Hexose transport in the apical and basolateral membranes of enterocytes in chickens adapted to high and low NaCl intakes. J Physiol 1999;514:189–99. Garriga C, Planas JM, Moreto M. Aldosterone mediates the changes in hexose transport induced by low sodium intake in chicken distal intestine. J Physiol 2001;535: 197–205.
Research Article
Effects of high and low salt intake on left ventricular remodeling after myocardial infarction in normotensive rats Ludimila Forechi, MSca, Marcelo Perim Baldo, PhDa, Isabela Binotti de Araujo, MScb, Breno Valentim Nogueira, PhDb, and Jose Geraldo Mill, MD, PhDa,* a
Department of Physiological Science, Federal University of Espirito Santo, Vit oria, Brazil; and b Department of Morphology, Federal University of Espirito Santo, Vit oria, Brazil Manuscript received September 7, 2014 and accepted November 30, 2014
Abstract The dietary–sodium restriction is a standard approach following an acute myocardial infarction (MI). We examined the hypothesis in which the use of a high or low–sodium diet would worsen post–infarction left ventricular remodeling in rats and facilitate the development of heart failure. Left coronary artery ligation or sham–operated (SO) was produced in male Wistar rats (250–290 g). After surgery, animals were assigned to one of the three diets: standard amount of sodium (0.3% NaCl, SO and MI groups), a high–sodium diet (0.6% NaCl, SO–High and MI–High groups), or a low–sodium diet (0.03% NaCl, SO– Low and MI–Low groups). Diets were provided for 8 weeks post–surgery. Mortality rate was elevated in high–salt group (MI–Low, 21.4%; MI, 35.3%; MI–High, 47.6%). Contractility parameter was seen to be impaired in MI–Low animals (3195 211 mm Hg/s) compared with MI (3751 200 mm Hg/s). Low–salt diet did not prevent myocardial collagen deposition (MI–Low, 5.2 0.5%; MI, 5.0 0.4%) nor myocyte hypertrophy (MI–Low, 608 41m2; MI, 712 53 mm2) in left ventricle after MI. High–salt intake increases collagen volume fraction (SO, 3.3 0.4%; SO–High, 4.7 0.4%) in animals sham, but no major changes after MI. Our results show that ventricular remodeling was not altered by immediate introduction of low sodium after MI, and it may be a safe strategy as a therapeutic intervention to avoid volume retention. However, high sodium can be harmful, accelerating the post–infaction ventricular remodeling. J Am Soc Hypertens 2015;9(2):77–85. Ó 2015 American Society of Hypertension. All rights reserved. Keywords: Cardiac fibrosis; diet; heart failure; sodium consumption.
Introduction Excessive salt intake has been extensively linked to cardiovascular morbidity and mortality. Over the last decades, clinical and experimental studies have shown that increased salt intake is associated with deleterious effects, including endothelial dysfunction, hypertension, stroke, heart failure, and kidney disease.1–3 Thus, high salt intake has been
This work was supported by grants from Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico–CNPq [470748/2013-3; 400484/2013-7]. The authors declare that they have no conflicts of interest. *Corresponding author: Jose Geraldo Mill, MD, PhD, Department of Physiological Sciences, Federal University of Espırito Santo, Av. Marechal Campos 1468, Maruıpe, 29042-755 – Vit oria, ES, Brazil. Tel: þ55 27-33357335; Fax: þ55 27-33357330. E-mail: [email protected]
considered a powerful risk factor for cardiovascular disease (CVD). Most population–based studies have found that salt consumption currently exceeds acceptable levels established by regulatory agencies.4 In fact, current recommendations advocate a reduction in salt intake to reduce the incidence of CVD, the most frequent cause of death worldwide. However, some authors have raised a number of issues about the current recommendations for a significant reduction in salt intake in the general population.5,6 It has been argued that low–salt diets may activate the sympathetic nervous system and the systemic renin–angiotensin–aldosterone system (RAAS), two effects that should be avoided during the post–infarct period because of their deleterious effects on left ventricular remodeling. Thus, low–salt diets could potentially be harmful after infarction because cardiac hypertrophy and fibrosis may facilitate the development of heart failure.
1933-1711/$ - see front matter Ó 2015 American Society of Hypertension. All rights reserved. http://dx.doi.org/10.1016/j.jash.2014.11.006
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Acute myocardial infarction (MI) is an extreme situation that is associated with complex changes in cardiac architecture and function that result in a high mortality rate. This process of change in the infarcted heart is known as myocardial remodeling, and it progressively leads to heart failure.7 It is noteworthy that, aside from standard pharmacologic approaches, a reduction in sodium intake is one of the first therapeutic actions after acute myocardial infarction. Based on the well–known effects of high salt consumption, it is expected that reducing salt intake after MI would attenuate left ventricular remodeling, thus preventing heart failure. However, this hypothesis has not been confirmed. For instance, Klein et al8 and Paterna et al9 did not observe beneficial effects after lowering sodium intake in heart failure patients. Instead, they observed increased hospitalization times for cardiovascular causes, increased mortality rates,8 and detrimental renal and neurohormonal effects.9 Thus, we sought to determine whether strict salt restriction or increased consumption of salt would affect the mortality rate and ventricular remodeling after acute MI in rats.
Materials and Methods Animals Male Wistar rats (260–290 g) from the animal core facility of the Federal University of Espirito Santo were used in the experiments. The animals were housed in appropriate cages in a room with controlled temperature and with a 12 hour light:12 hour dark cycle. Animals had free access to rat chow and tap water. All procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication N. 85–23, revised 1996) and approved by the institutional Committee for Ethics in Animal Research (CEUA–UFES n. 068/2012).
Coronary Artery Occlusion and Groups MI was induced as described previously.10 Briefly, the animals were anesthetized with ketamine (50 mg/kg intraperitoneal [ip]; Agener Uni~ao) and xylazine (Bayer; 10 mg/kg, ip), and left thoracotomy was performed in the fourth intercostal space. The heart was exposed rapidly, and the left coronary artery was permanently occluded using 6–0 monofilament nylon sutures. The thorax was closed, and the animals recovered normal respiratory movements. Sham–operated (SO) rats that underwent the same surgical procedures except coronary ligation were used as non–infarcted control animals. Immediately after surgery, the animals were randomly assigned into six groups to receive a diet containing the standard amount of sodium (0.3% NaCl, SO and MI groups), a high–sodium diet (0.6% NaCl, SO–High and MI–High groups), or a low–sodium diet (0.03% NaCl, SO–Low and MI–Low groups).
Diets were specifically prepared for this project, and all components of the rats’ diets were held constant except the sodium content. The animals were maintained on one of the three experimental diets for 8 weeks.
Noninvasive Blood Pressure Measurement Systolic blood pressure was recorded by noninvasive tail cuff plethysmography before surgery and 30 and 60 days after surgery. Briefly, the rat was placed in a pre–warmed restraining chamber, and occluding cuffs and pneumatic pulse transducers were positioned on the rat’s tail. The cuff was inflated and deflated automatically, and the signal was automatically collected in an IITC apparatus (IITC Inc).11 Five blood pressure measurements were made for each rat, and the mean of these measurements was recorded.
Physiological Parameters Each rat’s body weight was measured before surgery and then once per week. To determine the food and water intake and urinary parameters for each rat, the animals were placed into individual metabolic cages (Tecniplast 304) for 2 consecutive days 60 days after surgery. The first day was used as an adaptation period; 24–hour food and water consumption, as well as urine production, were measured during the second day. The urine production was used to measure the urinary excretion of sodium (MH-LAB ISE Electrolyte Analyzer, Diamond Diagnostics Inc).
Hemodynamic Measurements At the end of the 8–week follow–up period, the animals were anesthetized with ketamine (50 mg/kg ip) and xylazine (10 mg/kg ip). The right common carotid artery was catheterized with a fluid–filled polyethylene catheter (P50) connected to a pressure transducer (TRI 21, Letica Scientific Instruments) and to a digital system (Powerlab/ 4SP ML750, ADInstrument) to record the pulsatile blood pressure. The catheter was then advanced into the left ventricular cavity to record intracavitary pressure and its first temporal derivative. Left ventricular peak systolic pressure (LVSP) and end–diastolic pressure (LVEDP) and the maximum positive and negative values of dP/dt (þdP/dt max and dP/dt max) were recorded under regular rhythm with 1–kHz filtering.12 After hemodynamic recordings, the naso–anal length of the animal was measured, and the animal was euthanized with an overdose of anesthetic medication. The heart was rapidly removed, flushed in cold saline solution, dried with filter paper, weighed, and fixed in a buffered solution of paraformaldehyde (4%, pH 7.4) for subsequent histologic analysis. The lungs, kidney, liver, and the gastrocnemius muscle were also removed and weighed.
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Histologic Analysis
Statistical Analyses
After the atrial appendages were removed, the ventricles were transversely divided into three slices of approximately 3–mm thickness, embedded in paraffin blocks, and processed for conventional optical microscopy. At the level of the upper boundary of the anterior left papillary muscle, four 6 mm-thick sections were obtained and stained with picrosirius red stain to determine the collagen volume fraction and infarct size. Slices were also stained with hematoxylin–eosin to determine the myocyte cross– sectional area. Images were captured with a video camera (AxioCam ERc 5s, Carl Zeiss) coupled to an optical microscope (Olympus AX70, Olympus Corporation) under 400 magnification. Fifteen areas of high–power fields were chosen in the non–infarcted area of each heart, and the percentage of the area that was stained by picrosirius red was determined. In each rat, we measured the cross–sectional area of 30–50 myocytes positioned perpendicularly to the plane of the section with a clearly visible nucleus occupying the central region of the cell.12 The infarct size was calculated as (endocardial þ epicardial circumference of infarcted tissue)/(endocardial þ epicardial circumference of the left ventricle) and was expressed as a percentage. All histologic analysis was performed using ImageJ software (v. 1.43u, National Institutes of Health). Because infarct size is a critical factor influencing left ventricular remodeling, only animals with an infarct size greater than 35% were included in the present analysis.
The data are presented as means standard errors of the mean (SEM). The unpaired Student’s t–test was used to compare two means. Means in the six groups were compared via a two–way analysis of variance (ANOVA) followed by the Fisher post post–hoc test when necessary. The mortality rates in the infarcted groups are presented via the Kaplan–Meier method and compared using the log–rank test. Statistical significance was set at P < .05.
Results General Characteristics No mortality was observed in the SO groups. The mortality in the follow–up period (8 weeks) for each group was as follows: six out of 21 animals in the MI–Low group, three out of 23 animals in the MI group, and 10 out of 23 animals in the MI–High group. The Kaplan–Meier curves shown in Figure 1A suggest that mortality occurred earlier in MI when followed by a low–salt diet. However, the high–salt diet increased the mortality rate significantly (P ¼ .037). Among the surviving animals, four out of 15 rats in the MI–low group, two out of 20 rats in the MI group, and two out 13 rats in the MI–High group were excluded from the analysis because the infarct size was smaller than 35%. This exclusion resulted in three groups of rats with similar infarct sizes; the infarct sizes were 43.0% 3.1% in animals fed a standard diet,
Figure 1. Panel shows the (A) mortality rate after 60 days of follow–up, (B) mean infarct size, (C) body weight, and (D) systolic blood pressure. Standard diet is shown in black, low–sodium diet (Low) in gray, and high–sodium diet (High) in light gray. The values are presented as the mean standard error of the mean (SEM). P < .05 aSO vs. SO– Low; bSO vs. SO–High; cSO–Low vs. SO–High; dMI vs. MI–Low; eMI vs. MI–High; fMI–Low vs. MI–High; gSO– Low vs. MI–Low; hSO vs. MI; iSO– High vs. MI–High. MI, Myocardial infarction; SO, sham operation.
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Table 1 Morphologic parameters after 60 days of follow–up Parameters BW (g) Naso–anal length (cm) Tibia length (mm) Ventricles (mg) Lungs (mg) Liver (g) Kidney (mg) Gastrocnemius (mg)
SO–Low 306 22.1 41 752 1602 10.5 1009 1716
SO ,z
9* 0.3*,z 0.4z 28*,y 65z 0.4*,z 38*,z 88*,z
415 23.6 42 979 1823 14.0 1400 2112
SO–High
9 0.2 0.5 22 62 0.6 29 57
438 24.8 43 1270 1991 12.7 1596 2146
12 0.3 0.4 38y 64 0.4 57y 222
MI–Low 329 22.9 42 1039 2094 11.7 1081 1663
MI x,{
11 0.4 0.5 41x,{,# 132{,# 0.5x,{ 50x,{ 53x,{
391 23.3 43 1253 2215 13.2 1231 2121
MI–High
10 0.2 0.3 69** 207** 0.5 59** 85
407 23.5 43 1351 2810 15.6 1451 2062
12 0.2 0.6 51 212k,yy 0.6k 48k,yy 67
BW: Body weight; High, high–sodium diet; Low, low–sodium diet; MI, myocardial infarction; SO, sham operation. The values are shown as mean SEM. P < .05. * SO vs. SO–Low. y SO vs. SO–High. z SO-Low vs. SO–High. x MI vs. MI–Low. k MI vs. MI–High. { MI–Low vs. MI–High. # SO–Low vs. MI–Low. ** SO vs. MI. yy SO–High vs. MI–High.
48.0% 3.9% in those fed a low–sodium diet, and 40.1% 2.7% in those fed a high–sodium diet (Figure 1B). Body weight on the seventh day after surgery was smaller in the animals subjected to coronary ligation (Figure 1C). During the follow–up period, only animals fed the standard or high–sodium diet recovered their body weight. From the third week on, SO and MI animals fed a low–sodium diet exhibited reduced body weight gain compared with the animals on the standard diet or a high–salt diet (Figure 1C). At the end of the follow–up period, no difference in body weight was found between the SO and infarcted groups fed a low–sodium diet.
Morphologic and Physiologic Parameters Table 1 shows the organ weights for the six experimental groups. The weights of the gastrocnemius muscle, liver, left kidney, and ventricles were lower in SO rats fed a low–sodium diet compared with those fed a standard diet. In addition, the naso–anal length was smaller in the SO–low group. Interestingly, the low–sodium diet did not affect the compensatory gain of ventricular mass after MI. In contrast, the high–salt diet significantly increased the weights of the ventricles and kidneys in SO rats. However, the extent of hypertrophic growth in the ventricles secondary to infarction was unaffected by increasing the sodium content in the diet (Table 1). As expected, the lung weight was greater in all infracted groups compared with their corresponding SO groups. Physiologic data observed 8 weeks after MI or SO are depicted in Table 2. As expected, groups consuming high–salt diets exhibited greater water intake and,
consequently, greater urine production as well. Low salt content in the diet was associated with lower absolute food intake. However, in relative terms, the rats fed a low–salt diet ingested amounts of chow similar to those ingested by rats fed normal or high–salt diets. The urinary excretion of sodium in sham–operated rats was proportional to the consumption (0.07 0.04 mmol/24h in SO– Low group, 1.76 0.45 mmol/24h in SO group, and 11.68 1.49 mmol/24h in SO–High group), being significantly higher in the high–salt group (P < .05). Similar results were found in animals subjected to coronary ligation (0.04 0.04 mmol/24h, 0.24 0.05 mmol/24h and 17.23 1.69 mmol/24h in MI–Low, MI, and MI–High groups, respectively). The urinary excretion of sodium was elevated even further after MI in those animals fed a high–salt diet (11.68 1.49 mmol/24h in SO–High group and 17.23 1.69 mmol/24h in MI–High group; P < .05).
Blood Pressure and Left Ventricular Function To assess the effect of sodium content in the diet on blood pressure, we noninvasively measured animals’ systolic blood pressure (Figure 1D). A small and non–significant increase in the systolic blood pressure was found 4 weeks after surgery in SO animals consuming either low– sodium or standard diets. Dietary salt excess, however, caused SO animals’ blood pressure to increase and remain elevated throughout the follow–up period. This effect was not observed in infarcted rats fed a high–salt diet. Systolic blood pressure measurements were stable in all infarcted groups and did not vary significantly with the salt content of animals’ diets.
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Table 2 Metabolic parameters at 60 days after surgery Parameters Water intake (mL/day) Food intake (g/day) Urinary output (mL/day)
SO–Low
SO z
36.0 2.6 15.1 0.7*,z 19.2 1.7z
35.5 1.9 19.7 0.8 17.0 1.2
SO–High
MI–Low y
80.2 3.9 19.5 1.0 57.9 3.6y
{
36.8 3.9 17.9 0.6# 23.1 3.7x,{
MI
MI–High
41.0 2.9 20.3 1.2 13.6 1.5
88.4 5.5k 19.6 1.2 72.1 4.1k,yy
High, High–sodium diet; Low, low–sodium diet; MI, myocardial infarction; SO, sham operation. The values are shown as mean SEM. P < .05 * SO vs. SO–Low. y SO vs. SO–High. z SO–Low vs. SO–High. x MI vs. MI–Low. k MI vs. MI–High. { MI–Low vs. MI–High. # SO–Low vs. MI–Low. yy SO–High vs. MI–High.
Dietary sodium restriction per se reduced peak LVSP in anesthetized animals compared with rats fed a control diet and did not significantly affect other hemodynamic parameters (Figure 2A). Left ventricular (LV) dysfunction in infarcted animals was confirmed by a finding of increased LVEDP (Figure 2B), along with impaired LV contractile function and relaxation (Figure 2C, D, respectively). The frequency of contraction was only slightly impaired in MI–Low animals compared with MI animals.
Cardiac Histology To further evaluate the influence of different levels of sodium intake on post–infarct remodeling, we investigated whether changes in diet could alter myocardial collagen deposition and the myocyte cross–sectional area in non–
infarcted areas of the left ventricle. In SO rats fed a high– salt diet, the myocardial area occupied by collagen was significantly increased (Figure 3A). Although all groups subjected to MI exhibited increased fibrosis, the extent of fibrosis was not influenced by different levels of sodium intake. A similar pattern was observed for the myocyte cross–sectional area; the myocyte cross–sectional area increased slightly, but not significantly, with increasing salt in the diet. MI was associated with a significant increase in the myocyte cross–sectional area. However, this increase was unaffected by the salt content in the diet (Figure 3B).
Discussion The World Health Organization recommends reducing daily sodium intake to prevent cardiovascular disease.
Figure 2. Cardiac function assessed by intraventricular pressure measurements. Graphs show (A) left ventricular systolic pressure, (B) left ventricular end–diastolic pressure, and the maximum rate of pressure rise and fall (C and D), respectively. P < .05 aSO vs. SO–Low; b SO vs. SO–High; cSO–Low vs. SO– High; dMI vs. MI–Low; eMI vs. MI– High; fMI–Low vs. MI–High; gSO–Low vs. MI–Low; hSO vs. MI; iSO–High vs. MI–High. High, High–sodium diet; Low, low–sodium diet; MI, myocardial infarction; SO, sham operation.
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Figure 3. Photomicrographs of cardiac sections stained with Sirius red to visualize collagen deposition and stained with hematoxylin–eosin to determine the myocyte cross–sectional area. (A) The quantitation of collagen deposition in the non–infarcted interventricular septum area; (B) Myocyte cross–section area. P < .05 bSO vs. SO–High; cSO–Low vs. SO–High; gSO–Low vs. MI–Low; h SO vs. MI; iSO–High vs. MI–High. High, High–sodium diet; Low, low–sodium diet; MI, myocardial infarction; SO, sham operation.
However, there is some clinical and experimental evidence that lowering sodium intake increases the risk of cardiovascular disease, as has been observed with high salt intake. Several mechanisms may account for the association between low salt intake and cardiovascular diseases, for example, activation of the renin–angiotensin system,13–15 sympathetic overactivity,16 endothelial dysfunction and oxidative stress,17 insulin resistance, higher plasma triacylglycerol levels,18 and increased atherogenesis.14,19 Considering that dietary sodium restriction is a standard approach after acute MI in patients aiming to reduce fluid retention, the present study investigated whether changes in dietary sodium content affect post–infarction mortality. Moreover, we have investigated whether long–term diets with differing sodium content result in different courses of post–infarction remodeling in normotensive rats. In order to perform this study, we used a high–sodium diet (2–fold higher as compared with the standard diet) and a low–sodium diet (10–fold lower as compared with the standard diet). Our choice for the amount of sodium used in in the high–sodium diet was based in the American consumption of about 3400 milligrams of sodium a day, twice as high as the 1500 milligrams recommended by the American Heart Association.5 Also, the low–sodium diet was created to mimic the strict reduction in the sodium intake observed in patients that suffered an acute coronary event. Our results demonstrate that after 8 weeks of follow–up, a low–sodium diet did not significantly alter the mortality after infarction, while an increased mortality rate was observed in infarcted rats fed a high–salt diet. As previously reported, mortality after permanent coronary ligation has two leading causes. In the acute phase, ventricular arrhythmias are associated with high mortality; subsequently,
left ventricular dysfunction and heart failure are associated with high mortality.7 Examining the survival curve, we observed that rats fed a high–sodium diet exhibited higher mortality, while in the group fed a low–sodium diet, mortality was lower but occurred earlier compared with animals given a normal or high–sodium diet. Sympathetic activation after infarction increases mortality because it facilitates the development of life–threatening ventricular arrhythmias.7 Therefore, we suggest that arrhythmias may be a putative mechanism involved in the earlier mortality in the MI– low–salt group. In those animals subjected to high salt intake, however, mortality may be caused by an increased susceptibility to ventricular arrhythmias early after infarction and by an acceleration of the development of heart failure. Both hypotheses have already been demonstrated.20,21 The loss of contractile tissue after MI initiates a progressive process in infarcted and non–infarcted areas of the heart, to preserve cardiac function. One of the key markers of cardiac failure after infarction is LV enlargement, and the extent of LV enlargement is primarily determined by the infarct size. In our study, changes in dietary sodium introduced immediately after coronary ligature appears to have no effect on infarct size. Similar results have been reported by Dikow et al,22 who reported that infarct size was similar in nephrectomized animals fed a low–salt diet, and by Resende and Mill,23 who reported a similar infarct size in rats fed a larger quantity of sodium. However, our data do not allow us to determine whether dietary manipulations before coronary ligature are able to influence the extent of cardiac damage produced during coronary occlusion. Further studies are necessary to answer this question. Our data also suggest that manipulation of sodium intake after infarction has little influence on left ventricular
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performance. In SO rats, salt restriction decreased LVSP, while high salt intake increased LVSP; analogous effects were observed for dP/dt. Changes occurred in the same direction in infarcted animals. However, the differences were smaller and non–significant. These results are consistent with the results of an in vitro study in which a low–sodium diet (0.05% NaCl) did not affect left ventricular function in isolated hearts after MI.13 These results are also consistent with an in vivo study that demonstrated that MI rats had significantly lower LV maximum þ dP/dt compared with the control group under salt overload.23 Moreover, chronic low–sodium (0.015% NaCl) and high–sodium (0.9% NaCl) intake had no effect on ejection fraction or fractional shortening after MI.24 However, studies should be compared cautiously because the dietary sodium contents and the treatment periods were not uniform. Increased collagen deposition and myocyte hypertrophy are two changes that contribute to the development of heart failure in the post–infarct period. The increase in collagen levels in noninfarcted tissue is a critical step that contributes to cardiac contractile dysfunction and structural remodeling after infarction.7,25 Numerous studies have observed an activation of the cardiac renin–angiotensin– aldosterone system (RAAS) after MI. Increased local production of aldosterone and angiotensin II stimulates collagen deposition along the cardiac myocyte strands. The involvement of aldosterone in this process has been confirmed by the observation that aldosterone antagonists reduce the extent of fibrosis.26 The relationships between sodium intake and local RAAS are still poorly understood. Urabe et al24 showed that a low–salt diet positively contributes to cardioprotection and that this effect may be related to reduced collagen deposition, despite high levels of plasma aldosterone. These data, however, were not confirmed in our study because increased collagen deposition after infarction remained stable even in the infarcted group given a low–salt diet. Because the myocardial deposition of collagen is strongly regulated by the local RAAS, our data suggest that the cardiac RAAS is not regulated by sodium, in contrast with the endocrine RAAS, which is strongly activated by reduced sodium intake. Alternatively, our data suggest that the local RAAS may be fully activated in infarcted rats. Thus, additional manipulations of the systemic RAAS did not exert additional influences in the heart. As has been observed for collagen deposition, the development of cardiac hypertrophy after MI is also stimulated by several factors, including hemodynamic overload and neurohumoral activation. Cardiac hypertrophy processes occur asymmetrically to ensure functional adaptation to the hemodynamic overload. Our data support this view because infarcted animals exhibited increased cardiac mass and increased myocyte cross–sectional areas compared with SO rats. Dietary salt excess has repeatedly been demonstrated to increase the weight of the heart chambers by increasing
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the amount of extracellular matrix and cardiomyocyte size, an effect that is partially modulated by the local RAAS and known to be independent of blood pressure (1). Here, we also observed a small but non–significant effect in SO rats and no effect in rats with MI. Therefore, our data suggest that hypertrophic growth appears to be fully activated by infarction, with no additional activation that can be attributed to other factors. It has been reported that low sodium intake suppresses post–MI cardiac hypertrophy24 and potentiates the antihypertrophic effect of some drugs after MI13 or in spontaneously hypertensive rats.27 However, the mechanism underlying this phenomenon is not completely understood. In our study, a low–sodium diet did not affect either the cardiac mass or the myocyte cross–sectional area. Our data showed that strict salt restriction depresses body growth in infarcted and non–infarcted rats. Growth suppression seems not to be due to a lower caloric intake because diets were isocaloric and relative food intake was relatively stable among groups consuming different amounts of sodium. Very low sodium intake, however, may change hormonal profiles. In our experiment, the SO rats fed a low–sodium diet exhibited either lower body weight or smaller body size compared with those animals fed standard or high–sodium diets. In a study by Coelho et al,28 rats were given a low–sodium diet from weaning until 12 weeks of age. Low sodium intake increased weight gain but did not affect the plasma levels of T3 or T4 or the exploratory activity of animals. However, it decreased the levels of TSH, thermogenin, and leptin.28 The results of studies examining different animal species and different degrees of salt restriction have been inconsistent, with studies showing either weight gain18 or no change in weight.13–15,17,29–31 In addition, we cannot rule out the possibility that reduced sodium intake may alter the absorption of essential nutrients,32,33 even though the only difference between the standard chow and the low–sodium chow was the amount of sodium in the chow. In summary, low salt intake after a MI did not prevent increased collagen deposition and ventricular hypertrophy in rats. Moreover, lowering sodium intake had no effect on the mortality rate, infarct size, or ventricular function. Thus, the immediate introduction of a low–sodium diet after MI did not worsen the progression of cardiac remodeling and left ventricular dysfunction caused by MI in rats. High salt intake increased the collagen volume and the weight of the ventricles and kidneys in sham–treated animals. In addition, high salt intake increased the mortality rate, but no major changes in post–infarction cardiac remodeling were observed in rats fed a high–salt diet. Acknowledgment The authors are grateful to the Laboratory of Cellular Ultrastructure Carlos Alberto Redins (LUCCAR) for technical assistance and use of the microscope.
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References 1. Ferreira DN, Katayama IA, Oliveira IB, Rosa KT, Furukawa LN, Coelho MS, et al. Salt-induced cardiac hypertrophy and interstitial fibrosis are due to a blood pressure-independent mechanism in Wistar rats. J Nutr 2010;140:1742–51. 2. Cordaillat M, Rugale C, Casellas D, Mimran A, Jover B. Cardiorenal abnormalities associated with high sodium intake: correction by spironolactone in rats. Am J Physiol Regul Integr Comp Physiol 2005; 289:R1137–43. 3. Drenjancevic-Peric I, Jelakovic B, Lombard JH, Kunert MP, Kibel A, Gros M. High-salt diet and hypertension: focus on the renin-angiotensin system. Kidney Blood Press Res 2011;34:1–11. 4. He FJ, MacGregor GA. Reducing population salt intake worldwide: from evidence to implementation. Prog Cardiovasc Dis 2010;52:363–82. 5. Whelton PK, Appel LJ, Sacco RL, Anderson CA, Antman EM, Campbell N, et al. Sodium, blood pressure, and cardiovascular disease: further evidence supporting the American Heart Association sodium reduction recommendations. Circulation 2012;126: 2880–9. 6. Taylor RS, Ashton KE, Moxham T, Hooper L, Ebrahim S. Reduced dietary salt for the prevention of cardiovascular disease: a meta-analysis of randomized controlled trials (Cochrane review). Am J Hypertens 2011;24:843–53. 7. Mill JG, Stefanon I, dos Santos L, Baldo MP. Remodeling in the ischemic heart: the stepwise progression for heart failure. Braz J Med Biol Res 2011;44:890–8. 8. Klein L, O’Connor CM, Leimberger JD, GattisStough W, Pina IL, Felker GM, et al. Lower serum sodium is associated with increased short-term mortality in hospitalized patients with worsening heart failure: results from the Outcomes of a Prospective Trial of Intravenous Milrinone for Exacerbations of Chronic Heart Failure (OPTIME-CHF) study. Circulation 2005;111:2454–60. 9. Paterna S, Gaspare P, Fasullo S, Sarullo FM, Di Pasquale P. Normal-sodium diet compared with lowsodium diet in compensated congestive heart failure: is sodium an old enemy or a new friend? Clin Sci 2008;114:221–30. 10. Baldo MP, Davel AP, Nicoletti-Carvalho JE, Bordin S, Rossoni LV, Mill JG. Granulocyte colony-stimulating factor reduces mortality by suppressing ventricular arrhythmias in acute phase of myocardial infarction in rats. J Cardiovasc Pharmacol 2008;52:375–80. 11. Baldo MP, Forechi L, Morra EA, Zaniqueli D, Machado RC, Lunz W, et al. Long-term use of lowdose spironolactone in spontaneously hypertensive
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
rats: effects on left ventricular hypertrophy and stiffness. Pharmacol Rep 2011;63:975–82. Forechi L, Baldo MP, Meyerfreund D, Mill JG. Granulocyte colony-stimulating factor improves early remodeling in isoproterenol-induced cardiac injury in rats. Pharmacol Rep 2012;64:643–9. Westendorp B, Schoemaker RG, Buikema H, de Zeeuw D, van Veldhuisen DJ, van Gilst WH. Dietary sodium restriction specifically potentiates left ventricular ACE inhibition by zofenopril, and is associated with attenuated hypertrophic response in rats with myocardial infarction. J Renin Angiotensin Aldosterone Syst 2004;5:27–32. Tikellis C, Pickering RJ, Tsorotes D, Huet O, ChinDusting J, Cooper ME, et al. Activation of the reninangiotensin system mediates the effects of dietary salt intake on atherogenesis in the apolipoprotein E knockout mouse. Hypertension 2012;60:98–105. Mori T, Kurumazuka D, Matsumoto C, Shirakawa H, Kimura S, Kitada K, et al. Dietary salt restriction activates mineralocorticoid receptor signaling in volumeoverloaded heart failure. Eur J Pharmacol 2009;623: 84–8. Campese VM, Romoff MS, Levitan D, Saglikes Y, Friedler RM, Massry SG. Abnormal relationship between sodium intake and sympathetic nervous system activity in salt-sensitive patients with essential hypertension. Kidney Int 1982;21:371–8. Suematsu N, Ojaimi C, Recchia FA, Wang Z, Skayian Y, Xu X, et al. Potential mechanisms of lowsodium diet-induced cardiac disease: superoxide-NO in the heart. Circ Res 2010;106:593–600. Ruivo GF, Leandro SM, do Nascimento CA, Catanozi S, Rocha JC, Furukawa LN, et al. Insulin resistance due to chronic salt restriction is corrected by alpha and beta blockade and by L-arginine. Physiol Behav 2006;88:364–70. Raz-Pasteur A, Gamliel-Lazarovich A, Gantman A, Coleman R, Keidar S. Mineralocorticoid receptor blockade inhibits accelerated atherosclerosis induced by a low sodium diet in apolipoprotein E-deficient mice. J Renin Angiotensin Aldosterone Syst 2012;15: 228–35. Baldo MP, Teixeira AK, Rodrigues SL, Mill JG. Acute arrhythmogenesis after myocardial infarction in normotensive rats: influence of high salt intake. Food Chem Toxicol 2012;50:473–7. Katayama IA, Pereira RC, Dopona EP, Shimizu MH, Furukawa LN, Oliveira IB, et al. High-salt intake induces cardiomyocyte hypertrophy in rats in response to local angiotensin II type 1 receptor activation. J Nutr 2014;144:1571–8. Dikow R, Kihm LP, Zeier M, Kapitza J, Tornig J, Amann K, et al. Increased infarct size in uremic
L. Forechi et al. / Journal of the American Society of Hypertension 9(2) (2015) 77–85
23.
24.
25.
26.
27.
28.
rats: reduced ischemia tolerance? J Am Soc Nephrol 2004;15:1530–6. de Resende MM, Mill JG. Effect of high salt intake on local renin-angiotensin system and ventricular dysfunction following myocardial infarction in rats. Clin Exp Pharmacol Physiol 2007;34:274–9. Urabe A, Izumi T, Abe Y, Taniguchi I, Mochizuki S. Effects of eplerenone and salt intake on left ventricular remodeling after myocardial infarction in rats. Hypertens Res 2006;29:627–34. Bujak M, Frangogiannis NG. The role of TGF-beta signaling in myocardial infarction and cardiac remodeling. Cardiovasc Res 2007;74:184–95. Fraccarollo D, Galuppo P, Hildemann S, Christ M, Ertl G, Bauersachs J. Additive improvement of left ventricular remodeling and neurohormonal activation by aldosterone receptor blockade with eplerenone and ACE inhibition in rats with myocardial infarction. J Am Coll Cardiol 2003;42:1666–73. Griffiths CD, Morgan TO, Delbridge LM. Effects of combined administration of ACE inhibitor and angiotensin II antagonist are prevented by a high NaCl intake. J Hypertens 2001;19:2087–95. Coelho MS, Passadore MD, Gasparetti AL, Bibancos T, Prada PO, Furukawa LL, et al. High- or low-salt diet
29.
30.
31.
32.
33.
85
from weaning to adulthood: effect on body weight, food intake and energy balance in rats. Nutr Metab Cardiovasc Dis 2006;16:148–55. Trongtorsak P, Morgan TO, Delbridge LM. Combined renin-angiotensin system blockade and dietary sodium restriction impairs cardiomyocyte contractility. J Renin Angiotensin Aldosterone Syst 2003;4:213–9. Lima NK, Lima FB, dos Santos EA, Okamoto MM, Sumida DH, Hell NS, et al. Effect of lifelong highor low-salt intake on blood pressure, left ventricular mass and plasma insulin in Wistar rats. Am J Med Sci 2006;331:309–14. Shao W, Seth DM, Prieto MC, Kobori H, Navar LG. Activation of the renin-angiotensin system by a lowsalt diet does not augment intratubular angiotensinogen and angiotensin II in rats. Am J Physiol Renal Physiol 2013;304:F505–14. Garriga C, Moreto M, Planas JM. Hexose transport in the apical and basolateral membranes of enterocytes in chickens adapted to high and low NaCl intakes. J Physiol 1999;514:189–99. Garriga C, Planas JM, Moreto M. Aldosterone mediates the changes in hexose transport induced by low sodium intake in chicken distal intestine. J Physiol 2001;535: 197–205.
