Heart failure is a severe pathology, which has displayed a dramatic increase in the occurrence of patients with chronic heart disease in developed countries, as a result of increases in the population's average age and in survival time. This pathology is associated with severe malnutrition, which worsens the prognosis. Although the cachexia associated with chronic heart failure is a well-known complication, there is no reference animal model of malnutrition related to heart failure. This study was designed to evaluate the nutritional status of rats in a model of loss of cardiac function obtained by ascending aortic banding. Cardiac overload led to the development of cardiac hypertrophy, which decompensates to heart failure, with increased brain natriuretic peptide levels. The rats displayed hepatic dysfunction and an associated renal hypotrophy and renal failure, evidenced by the alteration in renal function markers such as citrullinemia, creatininemia, and uremia. Malnutrition has been evidenced by the alteration of protein and amino acid metabolism. A muscular atrophy with decreased protein content and increased amino acid concentrations in both plasma and muscle was observed. These rats with heart failure displayed a multiorgan failure and malnutrition, which reflected the clinical situation of human chronic heart failure.
- multi-organ failure
- amino acids
the increasing average age of the population and the increased survival time of patients with chronic heart disease has led to the growing prevalence of congestive heart failure (CHF) in developed countries. As a common sequel of many forms of cardiovascular disease, CHF, described as the 21st century endemic disease, is becoming a major health problem and the first single cause of hospitalization in patients over 60 yr of age (23). Heart failure, mainly a consequence of hypertension and/or ischemia (50), is described as a chronic left ventricular dysfunction, which severely decreases effort capacity and life quality. Patients suffering from heart failure also display symptoms that can alter food intake, such as weakness when strained, breathing difficulties, and gastrointestinal complications, including nausea symptoms, loss of appetite, and ascites (33, 46, 47). The disease is associated with a poor prognosis, that is, repeated hospitalizations and shortened survival rate. Several studies have shown a correlation between the length of hospital stay and the nutritional status, with a prolonged stay linked to cases of malnutrition. Malnutrition is rarely the direct cause of death except in elderly dialyzed patients. However, it may contribute to a poor prognosis by aggravating the preexisting heart and renal failure and increasing the susceptibility to infection (6). Weight loss and cachexia are well-known complications of heart failure (4); the prognosis of patients with CHF and cachexia is worse than that of patients with similar degree of left ventricular dysfunction but without cachexia (5, 17). Basal metabolic rate is increased in heart failure, but the decreased intake of macro- and micronutrients may contribute to the development of cachexia (52). The CHF-associated cachexia is a well-known complication that has been little investigated, mainly due to the lack of an appropriate and well-described animal model. Cachexia is characterized by a loss in fat mass, a muscle weight loss, and a decrease in the muscle protein content. In the human disease, studies have reported controversial data, such as leptin levels in patients with cachexia (21, 26, 37, 42) or the resistance to growth hormone (2, 3). This discrepancy in the observations demonstrates the need for a reference animal model of malnutrition associated with heart failure. Moreover, there is a need to know more about the CHF-induced alterations at the tissue level to establish appropriate nutritional therapy.
With the aim of defining an animal model, in this work, we evaluated the nutritional component of heart failure in a rat model obtained by ascending aortic banding. Although the impairment of cardiac functions is well known, no information is available on the relationship between the degree of cachexia and the severity of the disease in this model. Our model rats developed a severe cardiac hypertrophy, which decompensates in CHF, as well as multiorgan failure and malnutrition, thus reflecting the clinical situation.
MATERIALS AND METHODS
The investigations were carried out in agreement with the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication no. 85-23, revised 1996). The authors are entitled to legal experimentation by authorizations no. 92–181 and no. 3273.
Animals and experimental design.
Male Wistar rats, weighing 60–70 g (Charles River Laboratories, L'Arbresle, France), were anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg body wt). The aortic stenosis was induced via a left thoracic incision by banding the ascending aorta with titanium clips (Weck Atrauclip, 0.6-mm internal diameter, Rüsch Pilling, France) (20, 24). Eleven rats were initially submitted to aortic banding, but two died after 4 wk (AB group, n = 9). The Sham group consisted of sham-operated rats prepared by a similar surgical treatment without placement of the clip (n = 11). The rats were housed for 8 wk with free access to tap water and a semipurified jellied diet (composition as described in Ref. 45) in a controlled environment (21°C and a 12:12-h light-dark cycle). Two days before euthanasia, the rats were individually housed in metabolic cages for a period of 48 h to evaluate food intake and to collect urine samples (diuresis was recorded daily). At the end of the 8th wk, the rats were euthanized with pentobarbital sodium. Rats from the Sham group received 60 mg/kg body wt. Rats from the AB group received only 30 mg/kg because of the increased sensitivity of these rats to anesthetic drugs (48). Plasma and tissues were sampled as described below.
Blood was collected in heparinized tubes and rapidly centrifuged (4,000 g for 15 min at +4°C). The plasma was collected to determine glucose, creatinine, and urea concentrations and aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), and creatine kinase activities using an Olympus AU 600 analyzer (7). For amino acid analyses, plasma was deproteinized with a sulfosalicylic acid solution (30% wt/vol). The supernatants were stored at −80°C until amino acid analysis by ion exchange chromatography, which was performed with an amino acid autoanalyzer (Aminotac, Jeol, Tokyo, Japan). Results of our participation in the European Quality Control Scheme (ERNDIM) indicate the accuracy of amino acid determinations. A part of the blood was collected on EDTA, centrifuged (1,000 g for 15 min at +4°C), and stored at −20°C for determination of plasma brain natriuretic peptide (BNP), a marker of heart failure severity (32). BNP-32 was extracted from 2 ml of plasma with 1-ml Vycor glass suspension (60 mg of activated glass powder/ml deionized water) (Corning Glassware). The absorbed BNP-32 was eluted with 2.5 ml of acetone-water (60:40) containing HCl (0.2%), and the elution fraction was evaporated to dryness. The resulting pellet was reconstituted in 0.5 ml of RIA buffer (0.1 M potassium phosphate, pH 7.4, containing 0.05 M NaCl, 0.1% BSA, 0.1% Triton X-100, and 0.01% sodium azide), and the BNP-32 concentration was determined by RIA. The BNP-32 antiserum (Peninsula Laboratories) showed no cross-reactivity with α-atrial natriuretic peptide (ANP) (1–28), endothelin-1, and ANG II. An aliquot (0.1 ml) of the extracted fraction was added to 0.1 ml of antiserum and 0.1 ml of RIA buffer. The mixture was incubated at 4°C for 24 h, and 125I-labeled BNP-32 (Amersham) at 6,000 counts/min was added for another 24-h incubation period. The separation of free tracer from antibody-bound tracer was obtained by batch addition of dextran-charcoal and centrifugation at 1,200 g for 15 min. The radioactivity of supernatant was counted with a gamma counter.
A macroscopic necropsy was done for each animal. Heart failure was defined as described by Desjardins et al. (19). The heart was withdrawn and separated into ventricles, atria, and septum. Atria was removed, and each of the other parts was weighed. The liver and kidneys were also withdrawn and weighed; a piece of the liver was frozen in liquid nitrogen and stored at −80°C to determine hydratation levels. The hindlimb muscles [soleus, extensor digitorum longus (EDL), and gastrocnemius] were rapidly removed, weighed, frozen in liquid nitrogen, and then stored at −80°C for amino acid analysis. These three muscles were selected because they differ largely in their functions, fiber types, and metabolic responses (30, 40). The right limb muscles were used to determine the hydratation levels, whereas the left limb muscles were used to determine proteins and amino acid concentrations. They were homogenized and deproteinized with 10% (wt/vol) TCA. The supernatant was stored at −80°C until analyses of amino acids as described above. For total muscle protein determination, the frozen tissue was pulverized and homogenized in ice-cold 10% TCA (1 ml TCA/100 mg tissue) using an Ultra-Turrax T25 tissue disrupter (Ika Labortechnik, Staufen, Germany). After delipidation with ethanol-ether (1:1, vol/vol), the precipitate was dissolved in 1 N NaOH (4 ml/100 mg tissue) for 12 h at 40°C. The total protein content was then determined according to the method of Gornall (27).
Water content was determined by using the wet weight-dry weight technique. The fresh tissue samples (liver, EDL, soleus, and gastrocnemius) were immediately weighed (wet weight) and placed in an incubator at 100°C for 24 h (liver) or 48 h (gastrocnemius). The samples were weighed once again to determine dry weight. Water content was calculated as follows: water content = [(wet weight − dry weight)/wet weight] × 100.
Urinary and plasmatic creatinine levels were measured by the Jaffé reaction on an Olympus AU 600 analyzer (7). The creatinine clearance was calculated as follows: C = U × V/P, where C is creatinine clearance (l/24 h), U is creatininuria (mmol/l), V is diuresis (liters), and P is creatininemia (mmol/l).
Data are expressed as means ± SE and submitted to a Student's t-test (16) using the NCSS program. Differences at P < 0.05 were considered significant.
The necropsic observations showed that, as expected, the Sham rats presented no pathological characteristics, whereas the clinical and anatomic signs of heart failure were obvious in the AB rats. These rats displayed a multifocal failure, as evidenced by congestive liver (7 of 9) (Fig. 1, A and B), renal hypotrophy (6 of 9), and hydrothorax and/or ascites (8 of 9). Moreover, the AB rats showed an elevated sensitivity to anesthesia, and two of them displayed accelerated respiration and hyperventilation. These observations are consistent with a transition from compensated heart hypertrophy to CHF and the onset of a multiorgan failure syndrome.
Body weight was clearly affected in AB rats, which showed a significantly reduced growth (−30%) and a reduced (−22%) food intake (32 g/day vs. 41 g/day; P < 0.001). The morphometric data are shown in Table 1. At the end of the experiment, the rats of the AB group developed cardiac hypertrophy (Fig. 1, C and D) and displayed an average heart weight of 1.74 ± 0.06 g vs. 1.05 ± 0.02 g for the Sham rat hearts. This cardiac hypertrophy reflects a significant left ventricle hypertrophy (+70%), but the right ventricle and atria were also affected (P < 0.001). The heart hypertrophy is represented in Table 1 as heart weight-to-body weight ratio for control (2.39 mg/g) and CHF (5.91 mg/g) rats (P < 0.001).
BNP and creatine kinase.
The plasma concentration of BNP was 10 times higher in AB rats than in control rats (14 ± 2.5 vs. 135 ± 11; P < 0.001). We noticed a large variability in BNP plasma levels among the rats of the AB group, which varied from 780% to 1,360% of the mean basal value of the Sham group. The plasma creatine kinase levels were significantly (P < 0.05) increased in AB rats compared with Sham rats (69 ± 15 IU/l vs. 49 ± 4 IU/l).
In addition to heart failure, the AB rats displayed a significant renal hypotrophy (−40%) compared with Sham rats (Table 1). This difference was still significant (P < 0.001) when the renal mass-to-body mass ratio was reported (0.62 ± 0.01 vs. 0.56 ± 0.02). Moreover, the creatinine clearance was reduced in AB rats compared with their controls. Other parameters reflecting renal function, including plasma citrulline, creatinine, and urea levels, were also abnormal in the AB rats (Table 2).
The liver mass was also affected by the ascending aortic banding. The livers of AB rats were significantly smaller than those of the Sham rats. However, when the liver mass was reported to body mass, the difference between AB and Sham rats was not significant (Table 1). Moreover, the aortic banding affected the circulating levels of ASAT (72.3 ± 1.5 IU/l for AB vs. 51.4 ± 2.1 IU/l for Sham rats; P < 0.001) reflecting the hepatic dysfunction. Conversely, the ALAT plasma levels (28.1 ± 2.8 IU/l for AB vs. 26.9 ± 2.6 IU/l for Sham rats) were not significantly affected by aortic banding in these animals.
The ascending aortic banding significantly reduced the muscle mass (−30%, P < 0.001) in all the muscle types tested (EDL, soleus, and gastrocnemius) as shown in Table 3. Muscle protein contents were significantly lower in AB rats (−30%, P < 0.001). Whichever tissue was considered (liver, EDL, gastrocnemius, or soleus), no significant variation in hydratation level could be detected between AB and Sham rats (Table 4).
Amino acid concentrations.
The plasma concentrations of amino acids are shown in Table 5. Several plasma amino acids were significantly affected by aortic banding. Most of them were significantly increased compared with the Sham group (from 18% for proline to 54% for tyrosine). Only alanine was significantly decreased (−16%, P < 0.001). In parallel, the muscle amino acid contents were evaluated in the gastrocnemius samples. As for plasma concentrations, most of the muscle amino acid concentrations were significantly increased in AB vs. Sham rats (Table 6) with the noticeable exception of alanine, which was decreased.
Plasma glucose concentrations.
The plasma glucose concentration was significantly reduced in AB vs. Sham rats (9.4 vs. 11 mmol/l, respectively; P < 0.01).
This study was designed to evaluate the nutritional status of rats with ascending aortic banding and to determine whether this model of chronic heart failure induced by chronic pressure overload could be a good model to investigate CHF-associated cachexia. This ascending aortic stenosis induced physiological and morphological alterations characteristic of heart failure, including weakness, breathing acceleration, hydrothorax and ascites, liver congestion, kidney hypotrophy, and decreased renal function. AB rats showed a clear decrease in activity, with prolonged rest times much longer than Sham rats of the same age. However, our study did not include exact measurements of their activity. In response to cardiac load increase, the rats developed a compensative heart hypertrophy due to the overdevelopment of the left ventricle but also of the atria and the right ventricle, as already described by Feldman et al. (24). In CHF patients, the natriuretic peptide system is activated to its highest degree, and the natriuretic peptides were considered as the most powerful predictor of morbidity and mortality. In the present study, the average plasma BNP levels in the CHF rats raised up to 10-fold over the levels of the control rats. These results are in the same range as those previously reported in the same model (48) and confirm the validity of this model to investigate heart failure. In this study, the ejection fraction in AB rats was not demonstrated by echocardiography, but the plasma BNP levels and the occurrence of hydrothorax strongly suggest a loss of cardiac function and a decrease in ejection fraction. This experimental heart failure rat model was used in a study reporting weekly echocardiographic follow-up until 11 wk and histopathological analysis. Zaha et al. (53) showed that these rat hearts were characteristic of chronic heart failure. Momken et al. (41) showed by the echocardiographic study that heart failure develops relatively early during the disease. Thus, according to our results and to the literature, we are confident that this model of aortic banding in rats is a good model of chronic heart failure. The plasma levels of creatine kinase increased in AB rats. Even if this marker is not the best biochemical marker for myocardial injury because of its weak specificity (11), the rise of creatine kinase concentrations in serum reveals a myocardial disturbance.
Aortic banding imposed on rats for 2 mo resulted in cardiac failure and was associated with circulation impairments that induce, in turn, renal and hepatic failure. The necropsy investigations revealed renal hypotrophy, suggesting that aortic banding resulted in renal function impairment. The evaluation of markers of renal function revealed a decreased creatinine clearance and increased citrullinemia, creatininemia, and uremia. These data confirm the disturbances in renal metabolism and a diminished renal elimination. These parameters are known to be representative of renal damage and related to chronic renal failure. Levillain et al. (35, 36) reported that, in renal failure, citrulline accumulates in the plasma; they also showed a correlation between citrullinemia and the severity of renal failure. Moreover, they considered citrullinemia as a more sensitive marker of renal damage than plasma urea and creatinine concentrations. In the present study, citrullinemia was increased by more than 30% in the AB rats, suggesting that these rats had undergone a severe renal failure. In humans, the dysfunction of renal parenchyma was reported to result in an alteration of other amino acid metabolic pathways, leading to a serine and tyrosine decrease that may, in turn, contribute to increased plasma concentrations of citrulline and glutamine (10). In this study, however, we observed an increased tyrosine plasma concentration in rats that could be related to hepatic dysfunction (38). As a matter of fact, in the AB rats, the livers were congestive (as evidenced by the dark zones in parts or in the whole liver; Fig. 1, A and B). In addition, this study evidenced an increase in ASAT levels in the AB group; conversely, there was no significant modification in the ALAT levels. Michielsen et al. (38) showed that ASAT levels are more useful than ALAT to assess the severity of liver disease, based on the fact that 1) this mitochondrial enzyme is present in higher quantities in the liver than in the cytosolic ALAT and 2) the amount released is related to the severity of tissue damage. This dissociation between ASAT and ALAT could be related to a mitochondrial dysfunction, although this was not investigated in this study. All of these results confirmed that this model of aortic banding in rats causes cardiac failure and that it may be relevant to investigate the malnutrition associated with heart failure.
Malnutrition is usually associated with a decrease in body weight and an alteration of protein and amino acid metabolism. The decrease in body weight may be caused by either anorexia or by an increased caloric expenditure or a combination of both. In this study, we clearly showed a decrease in food intake, but the caloric expenditure was not measured. Because there are multiple origins for decreases in body weight in humans, we did not include pair-fed rats; from a mechanistic point of view, the lack of a pair-fed group is a limit of our study. Further studies that include pair-fed rats may determine the implication of such a reduction in food intake on the evolution of heart failure. Several clinical trials (1, 3, 12) have reported an increase in energy expenditure in heart failure patients, related to the activation of sympathetic nervous system or elevated cytokine levels. Further studies are required to characterize the energy expenditure and oxygen consumption in this model and to determine whether signaling pathways are involved. In particular, cytokine activation is a potential causal mechanism for the development of cachexia, and plasma concentrations of TNF-α have been closely related to muscle wasting.
Malnutrition is often associated with CHF. Anker et al. (3) reported a catabolic/anabolic imbalance in CHF, which may play a key role in the development of cachexia. However, there are no available data in the literature on the alteration of protein metabolism in CHF. The present study describes for the first time the plasma and muscle amino acids profiles observed in a model of loss of cardiac function. Note that other models of CHF are available (52), and comparison of information provided by different models could be of interest in the future. In the present study, we observed that CHF is associated with a muscular atrophy and a decrease in protein content. This may be a direct effect of CHF or may be linked to the decrease in food intake. In previous studies, our group demonstrated that the muscle weight-to-body weight ratio is constant throughout life (39) in normal conditions and that food restriction programs in rats are responsible for a decrease in soleus and EDL weight, although only the protein content of EDL was affected (25, 51). We also observed important modifications in amino acid patterns in both plasma and muscle. Plasma amino acids reflect a balance in amino acid fluxes between muscle and visceral organs (15). Moreover, the degree of hypermetabolism and the fate of specific tissue alterations make the pattern of plasma and muscle amino acids characteristic of each pathological situation (8, 9, 14, 22, 43). In the present study, aortic banding led to renal and hepatic alterations, which have a major influence on amino acid metabolism. Amino acid levels in renal failure have been reported (9). However, the amino acid pattern reported here are quite different from those associated with pure renal failure, probably because of the multi-organ failure situation, including liver failure. The increase in plasma citrulline concentration can be attributed to renal failure (15). The inability of the kidney to synthesize arginine from citrulline accounts for the high level of citrulline observed during organic renal failure (28). Renal failure is also responsible for the increase in glutamine concentrations in plasma and tissues: the metabolic acidosis observed during renal failure leads to a decrease in hepatic ureagenesis due to a reduced glutaminase activity in periportal hepatocytes (15). Hence, the nonincorporated ammonium ions are trapped by the perivenous hepatocytes for glutamine synthesis. Glutamine is then deaminated by the kidney for ammonium excretion. However, the origin of the variations in plasma and muscular amino acid levels remains unclear.
In this study, all of the amino acid levels except alanine were increased in CHF rats; alanine was the single amino acid that decreased in both plasma and muscle. This profile cannot be related to amino acid modifications reported in acidosis or in liver or renal failure (13, 31, 44). Muscular alanine production is correlated with the use of this amino acid for gluconeogenesis in the liver. It is mainly formed from glucose carbon skeleton via pyruvate and from ammonia as the result of branched-chain amino acid (BCAA) catabolism (34). However, we observed that BCAA levels were increased in muscle. Both liver and renal failure may thus be associated with a decrease in gluconeogenesis capacity. We also observed that glycemia was decreased. Because alanine is mainly produced from glucose, the decrease in glucose availability could result in a decreased alanine synthesis, which would in turn lower the BCAA transamination in muscle. This hypothesis could explain the typical profile of plasma and muscle BCAA and alanine observed in AB rats. However, further studies are required to confirm this hypothesis and to explain these alterations of BCAA catabolism and glucose-alanine cycle during CHF (34). A subsequent question arises regarding the capacity of alanine supplementation to improve the nutritional status in these rats. To the best of our knowledge, there are no data about alanine supplementation in heart failure. Moreover, the few studies assessing the effects of alanine supplementation failed to indicate any beneficial effects in preterm infants or in the severely ill (29, 49).
It was reported recently that the prognosis of CHF patients with a high body mass index is better than that of patients displaying cachexia with a similar degree of left ventricular dysfunction (5), highlighting the importance of body mass on the prognosis of heart failure. Much research effort has been expended on drug treatments in CHF, but little attention has been paid to nonpharmacological management (52). Moreover, the low attention to nutrition is influenced directly by the intensity of critical care provided, potentially reflecting physicians' insufficient interest in appropriately feeding the most severely ill patients. Nutritional support should be considered as an integral part of basic care, like hemodynamic or respiratory support (18). This study highlights the importance of the nutritional status of rats with loss of cardiac function. This topic needs to be investigated in humans; it would be of utmost interest to determine whether a preventive approach through management of the nutritional status may improve the outcome in CHF. In our study, we clearly showed that this model reproduces some aspects of the clinical features of CHF patients and may be used to evaluate nutritional therapy. Further studies are required to define the optimal nutritional program that could improve the outcome of heart failure patients. Moreover, other studies regarding the time course of hypertrophy to heart failure and the evolution of the nutritional status during the disease should help the nutritional therapy. Alanine was the only amino acid to show a lower level in rat CHF, and the specific amino acids may be regarded as potentially predictive markers for development of cachexia in CHF patients.
In conclusion, ascending aortic banding in rats induces a chronic pressure overload, leading to chronic heart failure. In this model, CHF is associated with multiorgan dysfunction, including severe renal failure and hepatic dysfunction. The nutritional status was evaluated in this model and showed that these rats presented severe metabolic alterations that were mainly caused by multiple organ failure. This rat model could be considered a good model of cachexia associated with heart failure, as long as it reflects the clinical situation, and appears qualitatively different from the renal failure cachexia. This model can be used to test the efficacy of various nutrition strategies.
The authors are grateful to Dr A. Carayon (Hôpital de la Pitié, Paris) for plasma BNP determinations, to Stéphanie Ovide-Bordeaux and Carine Genthon for helpful assistance, and to Jallal Oussar for critical reading of the manuscript. The authors thank Cacao Barry for the generous gift of pure cocoa butter.
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