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Physiol. Genomics 27: 231-236, 2006. First published August 22, 2006; doi:10.1152/physiolgenomics.00022.2006
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Received 11 February 2006; accepted in final form 19 July 2006.
Physiological Genomics 27:231-236 (2006)
1094-8341/06 $8.00 © 2006 American Physiological Society

Small gene effect and exercise training-induced cardiac hypertrophy in mice: an Ace gene dosage study

Fabiana Sant'Anna Evangelista and José E. Krieger

Heart Institute (InCor), Department of Medicine-LIM13, University of São Paulo Medical School, São Paulo, Brazil


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Small gene effects influence complex phenotypes in a context dependent manner. Here we evaluated whether increasing dosage of the angiotensin I converting enzyme (Ace) gene influence exercise-induced cardiac hypertrophy. Mice harboring one, two, three, and four copies of the Ace gene were assigned to sedentary (S1–4) and swimming exercise-trained (T1–4) groups (1.5 h twice daily, 5 days/wk, 4 wk). Exercising resulted in comparable bradycardia and elevated skeletal muscle citrate synthase activity, while blood pressure remained unchanged. Left ventricle mass index and cardiomyocyte diameter were similar among sedentary mice and the magnitude of their increase associated to exercising was not influenced by the Ace genotype (T1: 12.6 and 17.9%, T2: 15.2 and 13.8%, T3: 16.9 and 20%, T4: 17 and 19%, respectively). Plasma renin activity (PRA) levels were higher in one vs. three or four copies mice (4.89 ± 0.5 vs. 2.43 ± 0.6 and 2.12 ± 01.1 ng/ml Ang I, P < 0.05), while cardiac ACE activity was higher in three vs. two or one copy mice (5,946 ± 590.8 vs. 2,951.5 ± 328.3 and 3,504.1 ± 258.9 µF·min–1·ml–1, P < 0.05). With exercise, PRA remained unchanged in each group, while cardiac immunostaining for Ang II reached comparable levels. In summary: 1) exercise training led to similar aerobic adaptation regardless of the Ace genotype, and 2) higher number of Ace gene copies per se, which alters cardiac ACE activity, did not influence basal cardiac mass or, most importantly, the magnitude of swimming-induced cardiac hypertrophy. Collectively, these data indicate that small isolated genetic disturbances in ACE cardiac levels can be well compensated under physiological perturbations.

angiotensin I converting enzyme; renin-angiotensin system; transgenic mice; swimming training


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
THE RENIN-ANGIOTENSIN SYSTEM (RAS) plays an important role in the regulation of myocardial remodeling (8, 32, 33, 40). Angiotensin I converting enzyme (ACE) is responsible for the breakdown of several substrates including vasodilator kinins, while promoting formation of the vasoconstrictor angiotensin II (Ang II). Both clinical and experimental data have established the association of Ang II and cardiovascular diseases such as heart failure (7) and cardiac hypertrophy (28, 35).

A combination of genetic, physiological, and environmental factors contributes to different forms of cardiac hypertrophy. Physical training has long been used as a model for inducing volume overload to study different aspects of physiological cardiac remodeling (1, 45); it appears that structural and functional characteristics explain differences observed between physiological and pathological patterns of cardiac hypertrophy (15).

The molecular mechanisms underlying both exercise-induced (physiological) and pathological hypertrophy are poorly understood. Moreover, the role of Ang II in the development of physiological cardiac hypertrophy remains unclear. In trained rats, cardiac hypertrophy appears to be unrelated to Ang II levels (15); conversely, the deletion polymorphism of the Ace gene, which is coupled with higher levels of tissue and serum ACE activity, is associated with cardiac hypertrophy in human athletes (2, 14, 23).

Despite such findings, establishing the individual contribution of a single gene to a complex phenotype such as cardiac hypertrophy remains a challenge since this is a context dependent response. In the present study we evaluate the influence of increasing the dosage of the Ace gene on the magnitude of exercise induced cardiac hypertrophy. More importantly, we used genetic engineered mice carrying one, two, three, or four copies of the Ace gene at its endogenous locus resulting in lower and above normal ACE levels comparable in magnitude to the variation in plasma or tissue ACE levels observed with naturally occurring human ACE insertion-deletion polymorphism (20).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals.
Male genetically engineered mice harboring either an inactivation or a duplication of the Ace gene on chromosome 11, which resulted in them having one, two, three, or four functional copies of the gene (19, 20) were used. These mice have similar background since they have been maintained by backcross breeding to C57BL/6J mice for several generations and differ only at the Ace locus. They represent a controlled genetic model in which the role of this locus can be quantitatively assessed under different environmental conditions (e.g., physical training). Identification of genetically modified offspring was determined at 21 days of age by PCR amplification of DNA isolated from ear biopsies, as described previously (19).

The animals were randomly assigned to four sedentary (S1, n = 16; S2, n = 19; S3, n = 15; S4, n = 9) and four exercise-trained (T1, n = 17; T2, n = 17; T3, n = 17; T4, n = 10) groups. The mice were housed in a light (12-h light cycle)- and temperature (22°C)-controlled environment and fed a pellet rodent diet ad libitum, with free access to water. The training sessions were performed during the mice's dark cycle. Experiments were approved by the Ethics Committee of the University of São Paulo Medical School, and all procedures followed Institutional guidelines.

Training protocol.
Physical training associated to the development of ~20% cardiac hypertrophy, as described in detail by Evangelista et al. (11), consisted of two 1.5-h daily sessions of swimming training, 5 days/wk, for 4 wk. The swimming sessions started at 20 min and were progressively increased by 10 min per day until reaching 1.5 h. To minimize the influence of the water stress, sedentary mice were placed in the swimming apparatus for 5 min twice/weekly during the experimental protocol.

The swimming apparatus consists of two 200-liter, coupled, glass water tanks of different dimensions. The outer tank measures 60 cm in diameter, 100 cm in width, and 50 cm in height; the inner tank is divided into 14 lanes with a surface area of 15 cm2 per lane and a sufficient depth (35 cm) to allow individual training. To prevent animals from floating and ceasing to exercise, water bubbling was induced by connecting tubes to an air pump system. A heating system maintained the water temperature between 30–32°C (16).

Resting blood pressure and heart rate measurements.
Tail-cuff blood pressure (BP) and heart rate (HR), estimated from the pulse rate, were determined during the 4-wk study period via use of a computerized tail-cuff system (BP 2000 Visitech Systems). BP values for each animal were determined by averaging BP measurements obtained on two different days of the week during the mice's dark cycle.

Skeletal muscle oxidative enzyme activity.
Muscle samples were taken from the left and right soleus at the time of killing and frozen in liquid nitrogen for future processing. Citrate synthase activity, used as an index of physical training, was determined spectrophotometrically in whole muscle homogenates, according to the method of Srere (38). The assay is based on the reaction of a colorimetric reagent, DTNB (acetyl-CoA + oxaloacetate + water {leftrightarrow} citrate + CoA-SH + DTNB -> mercaptide ion). The rate of change in color was monitored at a wavelength of 412 nm. The solubilized protein extracts from the homogenates were quantified in duplicate by the Bradford method (6) using bovine albumin standards. The citrate synthase activity was then normalized for the total protein content and reported in nanomoles per milligram of protein per minute.

Cardiac structural analysis.
Twenty-four hours after the end of the study session, sedentary and exercise-trained mice were killed and tissues harvested. Whole heart weight and dissected chambers' weight [atria, right ventricle, and left ventricle (LV)] were measured. The weight of each cardiac chamber was normalized for the total body weight of the animal. For the morphometric analysis, LV samples were then fixed by immersion in 4% buffered formalin and embedded in paraffin for routine histological processing. Sections (4 µm) were stained with hematoxylin and eosin for examination with a light microscope. Cardiomyocyte width in the LV free wall was measured with a computer-assisted morphometric system (Leica Quantimet 500, Cambridge, UK). A single transverse measurement of width, passing through the nucleus, was made in 10 cardiomyocytes and averaged for each animal.

Plasma renin activity assay.
Plasma renin activity (PRA) was determined by radioimmunoassay, using commercially available kits and conducted according to the manufacture's instructions (PRA: REN-CIS).

ACE activity assay.
ACE activity was determined via fluorometric assay of samples of cardiac tissue, frozen in liquid nitrogen, and stored at –70°C (3). Briefly, the supernatant from homogenized LV tissue (15 µl) was incubated with a substrate containing 15 µM Abz-FRK(Dnp)P-OH (Abz = o-aminobenzoic acid; Dnp = dinitrophenyl) in 0.1 M Tris·HCl buffer, 50 mM NaCl, and 10 µM ZnCl2, pH 7.0 (200 µl final volume). Enzymatic activity of the fluorogenic peptide was continuously assessed by using a fluorometer to measure fluorescence at {lambda}em = 420 nm and {lambda}ex = 320 nm. The slope was converted into micromoles of substrate hydrolyzed per minute, based on the calibration curve obtained from complete hydrolysis of each peptide.

Cardiac immunohistochemistry for Ang II.
Tissue samples for Ang II immunohistochemistry were obtained from animals with one and three copies of the Ace gene, which were anesthetized with pentobarbital sodium (120 mg/kg; Cristalia, Sao Paulo, Brazil) before tissue perfusion. The presence of Ang II was confirmed in paraffin sections of LV tissue using anti-Ang II rabbit antiserum (1:400; Peninsula, Belmont, CA) (13). Optimal working dilutions of primary antibody were previously determined by titration experiments. The antigen was marked by fast red dye, and the specificity of the secondary antibody was established in positive and negative controls. Images were obtained with a computer-assisted morphometric system (Leica Quantimet 500, Cambridge, UK). The number of positive signals in cardiomyocytes was evaluated and expressed as cells of Ang II/mm2.

Data analysis.
All values are expressed as means ± SE. Data from exercise-trained groups were compared with data from sedentary groups by two and three-way ANOVA followed by a Tukey post hoc test. Statistical significance was considered for P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Resting BP and HR during the exercise-training period.
After training, baseline BP remained unchanged among all groups (Fig. 1A). In contrast, HR decreased significantly by the second week of training in all trained mice compared with both the preexercise period and each sedentary littermate (Fig. 1B). Interestingly, the magnitude of resting bradycardia remained comparable among the trained groups with different copies of the Ace gene. The development of comparable resting bradycardia in the exercising mice indicates that aerobic conditioning was achieved and the genetic variation in Ace did not modulate that response to physical training.


Figure 1
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  		Fig. 1. Resting blood pressure (A) and heart rate (B) during experimental period. BPT, before physical training. Data are presented as means ± SE (3-way ANOVA test). *P < 0.05 for comparisons with sedentary groups (S1–4); #P < 0.05 for comparisons with trained groups (T1–4) before and after physical training. As shown, resting blood pressure did not change after physical training among the groups; however, heart rate decreased in all trained groups after 2 wk of physical training. The magnitude of bradycardia was similar among mice with 1–4 copies of the angiotensin I converting enzyme (Ace) gene.

 
Skeletal muscle oxidative enzyme activity.
Maximal citrate synthase activity in the soleus muscle was significantly higher in exercise-trained mice from T1 (41.8%), T2 (34.4%), T3 (51.8%), and T4 (26.2%) compared with the respective sedentary groups S1, S2, S3, and S4. A small but significant difference in the magnitude of enzymatic increase was observed between T1 and T4 groups (234 ± 23 and 191 ± 23 µmol·min –1·mg –1, respectively; P < 0.05) (Fig. 2).


Figure 2
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  		Fig. 2. Skeletal muscle maximal citrate synthase activity following physical training. Data are presented as means ± SE (2-way ANOVA test). *P < 0.05 for comparisons with sedentary groups; #P < 0.05 for comparisons with trained groups (1 vs. 4 copies). Compared with the sedentary groups, citrate synthase activity was increased in the trained groups. Among the trained groups, a statistically significant difference in levels of citrate synthase activity was only found between groups with 1 or 4 copies of the Ace gene.

 
Cardiac structure analysis.
The body weights of the trained mice (T1: 24.32 ± 0.47; T2: 23.91 ± 0.56; T3: 23.33 ± 0.46; T4: 23.17 ± 0.59) were not significantly different from those of the sedentary mice (S1: 23.86 ± 0.61; S2: 24.17 ± 0.57; S3: 23.16 ± 0.66; S4: 24.10 ± 0.94). As shown in Table 1 and Fig. 3A, LV mass index increased in all trained groups (T1, T2, T3, and T4) compared with the sedentary controls. The increased heart weight-to-body weight ratio was due to an increase in the weight of all cardiac chambers. The measurements of cardiomyocyte width paralleled the changes observed in LV-to-body weight ratios (LV mass index). Compared with the sedentary littermates, a significant increase in cardiomyocyte width was observed in all exercise-trained groups (Fig. 3B). However, both the heart weight-to-body weight ratio and cardiomyocyte width did not significantly differ among the trained groups, indicating that genetic variation in the Ace gene did not influence cardiac structural adaptation associated with physical training.


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  		Table 1. Cardiac morphometric analysis following physical training

 

Figure 3
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  		Fig. 3. Left ventricular (LV) mass index (A) and cardiomyocyte dimensions (B) following physical training. Data are presented as means ± SE (2-way ANOVA test). *P < 0.05 for comparisons with sedentary groups. Trained mice with different numbers of copies of the Ace gene showed similar increases in LV mass index and myocyte dimensions compared with sedentary controls.

 
RAS adaptations to physical training.
Figure 4A summarizes the responses of the RAS to exercise training. The sedentary group carrying one copy of the Ace gene showed 50.3 and 56.6% higher PRA (P < 0.05) than the S3 and S4 groups, respectively. Upon exercise training, the same pattern was maintained i.e., the one-copy group showed 39.1 and 35.4% higher PRA (P < 0.05) than the three- and four-copy groups, respectively. There was no significant difference in PRA between sedentary and trained mice within each ACE genotype.


Figure 4
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  		Fig. 4. Renin-angiotensin system (RAS) response to physical training: plasma renin activity (PRA, A), cardiac ACE activity (B), and immunostaining for Ang II (angiotensin II, C). Data are presented as means ± SE (2-way ANOVA test).*P < 0.05 for comparisons among groups S1, S3, and S4 (A); S1, S2, and S3 (B); S1 and T1 (C). #P < 0.05 for comparisons among groups T1, T3, and T4 (A).

 
As expected, cardiac ACE activity was higher in S3 compared with S1 and S2 (5,946.3 ± 590.8, 3,504.1 ± 258.9, and 2,951.5 ± 328.3 µF·min –1·mg–1, respectively; P < 0.05) (Fig. 4B). Exercise training had no effect on cardiac ACE activity in all Ace genotype groups.

Interestingly, immunostaining for Ang II, revealed by red-brown staining in LV, showed similar levels of signal among sedentary or trained mice harboring one or three copies of the Ace gene (S1 vs. S3, 0.0025 ± 0.0004 and 0.0046 ± 0.0009 and T1 vs. T3, 0.0072 ± 0.0015 and 0.0069 ± 0.0010 cells/mm2, respectively; P > 0.05) (Fig. 4C).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our data indicate that small genetic perturbations in a component of the RAS, Ace, are well tolerated under a physiological stimulus associated with the development of cardiac hypertrophy. Interestingly, the magnitude of cardiac hypertrophy achieved in all groups was similar regardless of the Ace genotype, which was directly related to cardiac ACE activity but inversely related to PRA.

BP and HR responses.
Exercise training had no influence on tail-cuff blood pressure regardless of the Ace genotype. These results are consistent with previous observations from our group (21, 31) and others (9) that have shown that arterial pressure remains unchanged in exercise-trained normotensive animals and humans.

Most likely, the systemic activation of ACE is counteracted by decreased PRA; the opposite occurs in animals with lower levels of ACE, indicating well-balanced compensation with regard to BP regulation under physiological conditions (20). Also, evidence from experimental and modeling data show that modest increases in ACE activity may affect the steady-state plasma concentration of its substrate (e.g., lowers Ang I and bradykinin) but not of its products (e.g., Ang II) (39) consistent with the lack of correlation between cardiac mass and Ace genotypes observed either under basal or physiological stressor conditions.

Resting bradycardia is an useful and reliable indicator of endurance conditioning (22). Our assessment of the effects of physical training on resting HR and Ace genotype was determined indirectly by tail-cuff system. Despite the potential confounding effects of restraint and heating associated with this technique, baseline HR values were similar in our study to those reported by authors using telemetry (18). After 2 wk of training, all exercise-trained groups showed a lower resting HR compared with the sedentary control mice (P < 0.05).

To determine whether genetic changes in Ace affect HR values, we compared trained animals with different complements of the Ace gene. Although Krege et al. (20) showed that HR was modestly but significantly inversely proportional to the number of Ace gene copies, the magnitude of the resting HR in trained groups was not influenced by the Ace genotype in the present study. Moreover, we have observed an increase in basal HR only in double Ace knockout mice in our laboratory (data not shown).

Skeletal muscle oxidative capacity.
An increase in muscle oxidative activity concomitant with an improvement in aerobic work capacity is one of the hallmarks of skeletal muscle adaptation to aerobic conditioning (43). The maximal activity of citrate synthase in soleus muscle was significantly increased in all exercise-trained mice, suggesting that the swimming training was sufficient to increase oxidative capacity similarly in all groups. Elevations in citrate synthase activity between 37 and 45% have been well documented in previous studies involving swimming training (11, 29).

Lower ACE levels have been associated with higher efficiency of skeletal muscle metabolism after physical training (24, 44). The small but higher oxidative response shown by one-copy mice is consistent with previous evidence correlating the functional ACE I gene variant and exercise performance in humans. Furthermore, almost all studies performed with resistance athletes have shown improved muscular capacity with aerobic physical training associated with the ACE II genotype and low ACE activity (12, 24, 26). However, Sonna et al. (37) and Nazarov et al. (30) did not observe variability in sports performance among those with different ACE genotypes. These discrepancies could be, at least in part, due to the inherent difficulties in studying heterogeneous cohorts of mixed athletic abilities and disciplines.

To minimize the differences among physiological variables in humans, Bahi et al. (4) produced an animal model with chronic ACE inhibition. Their data show that decreased ACE activity produced by chronic ACE inhibition had no marked effect on endurance time in sedentary animals and did not alter oxidative capacity, as indicated by citrate synthase activity. Yet one can argue that the level of ACE inhibition induced by pharmacological means can vary and may reflect properties of specific compounds, even within the same pharmacological class. This is not the case with genetic manipulation of animals, as shown in the present study. In addition, the Bahi et al. (4) study did not include exercise-trained animals.

Ventricular weights and LV cardiomyocyte dimensions.
Physiological myocardial hypertrophy is a morphological adaptive response to chronic volume overload imposed on the heart during aerobic physical training (11). In the present study, heart weight-to-body weight ratio increase was consistently observed in all trained groups compared with their sedentary littermates (T1: 15.7%; T2: 16.4%; T3: 17.5%; T4: 20.2%). These percentages (16–20%) are within the same range observed by us and other groups previously (11, 16). Other groups using different physical training protocols, such as voluntary wheel running (1) and treadmill running (17), reported smaller degrees of cardiac hypertrophy (10 and 12.3%, respectively). Thus, the magnitude of differences in cardiac hypertrophy may reflect the intensity and specificity of each training modality (5).

The effect of exercise was observed in all cardiac chambers; this is demonstrated by the significant increase in normalized values for all chambers in all groups upon comparing sedentary animals with trained, regardless of the Ace genotype. Consistent with this observation, the hypertrophic response was accompanied by an increase in cardiomyocyte width in all trained groups.

Cardiac hypertrophy and RAS.
Exercise training has been shown to significantly reduce PRA levels to a given workload in wrestlers following vigorous swimming (41). In the present study, comparisons between the sedentary and trained groups with the same Ace genotype revealed similar PRA values 24 h following the exercise. So it is possible that methodological issues, choice of models or modalities of exercise may explain some of the discrepancies reported.

We found an inverse relationship between the PRA and cardiac ACE activity, as determined by the number of Ace gene copies. Krege et al. (20) reported a similar inverse relationship between Ace genotype (activity) and renin gene transcription. These findings argue in favor of compensatory adaptations in the RAS directed at maintaining BP homeostasis in genetically manipulated animals harboring different copies of the Ace gene.

In sedentary animals, ACE activity tended to increase in proportion to the number of copies of the Ace gene (from 1 to 3), as previously demonstrated by Wei et al. (42). Upon exercise training, the same trend was observed, but there was no significant difference in ACE activity levels between groups. This response may reflect compensatory changes that took place during the exercising training period, since the estimated cardiac immunoreactive Ang II levels were similar between sedentary as well as trained one- or three-copy mice; this finding is also consistent with the fact that cardiac mass was similar among sedentary animals and increased by the same magnitude after exercise, regardless of the Ace genotype. The local production of Ang II to promote cardiac hypertrophy seems to be relevant. Danser and Schalekamp (8) have estimated that the levels of Ang II are higher in cardiac tissue than in serum, suggesting that local production is likely. Our results show a close relationship between estimated immunoreactive Ang II and cardiac hypertrophy, regardless of the Ace genotype. In contrast, using a pathologic stimulus, ACE genotype and cardiac Ang II levels are directly associated to the magnitude of pressure overload-induced cardiac hypertrophy in these mice (36).

Activation of the RAS has been associated with pathological cardiac remodeling (33, 34). Similarly, patients with asymptomatic LV dysfunction after myocardial infarction have shown improved survival and reduced morbidity and mortality with long-term administration of an ACE inhibitor (32). However, it is important to point out that the contribution of ACE to the development of cardiac hypertrophy in athletes remains controversial. Some evidence supports an association between the deletion homozygous ACE gene polymorphism, in which plasma and tissue ACE levels are expected to be higher than for the II genotype, and the degree of cardiac hypertrophy (2, 23, 25, 27). However, others have failed to reproduce these findings (10).

The exercise training-induced cardiac hypertrophy reported in this study emphasizes potential discrepancies that may be expected when dissecting complex modulation of important homeostatic systems under physiological or pathological conditions. Thus, only the excess production of Ang II may indeed influence cardiac trophic state underlying the difference between physiological and pathological states (28, 46).

We demonstrated that the magnitude of myocardial hypertrophy was not associated with Ace gene copy number in this physiological, swimming-induced cardiac hypertrophy model. This finding is consistent with the notion that under physiological condition increases or decreases in cardiac ACE can be counteracted by reversal changes in other components of the RAS, resulting in comparable cardiac Ang II levels in both sedentary and trained animals.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
F. S. Evangelista holds a doctoral fellowship from Fundação de Amparo a Pesquisa do Estado de São Paulo, Brasil (FAPESP 01/08592-7). The study was supported by grants from Fundacao Amparo Pesquisa do Estado de São Paulo (FAPESP 01/00009-0) and from Conselho Nacional de Pesquisa e Desenvolvimento, Brasil (CNPq 471219/01-0).


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the technical assistance of Dr. Luis Fernando Rosa (citrate synthase activity), Dr. Edilamar M. Oliveira for renin assay, Dr. Irene Noronha, and Rita Cavaglieri for Ang II immunoreactivity assays and Dr. Marcelo Nobrega for critical reading of the manuscript.


   FOOTNOTES
 
Address for reprint requests and other correspondence: J. E. Krieger, Instituto do Coração (InCor), Laboratório de Genética e Cardiologia Molecular, Av. Dr. Enéas de Carvalho Aguiar, 44 Bloco B, 10° andar, São Paulo - SP, CEP 05403-000-Brazil (e-mail: krieger{at}incor.usp.br)

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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