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Cardiovascular Research 2000 45(4):866-873; doi:10.1016/S0008-6363(99)00402-2
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Copyright © 2000, European Society of Cardiology

Gene expression of Na+/Ca2+ exchanger during development in human heart

Yongxia Qu, Ashwini Ghatpande, Nabil El-Sherif and Mohamed Boutjdir*

Cardiology Division, Department of Medicine, V.A. Medical Center and SUNY Health Science Center, Brooklyn, NY 11209, USA

* Corresponding author. Tel.: +1-718-630-3645; fax: +1-718-630-3796 mohamed.boutjdir{at}med.va.gov

Received 29 March 1999; accepted 25 October 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 
Objective: In immature animal hearts, lower activity of sarcoplasmic reticulum and lower densities of Ca2+ channels highlight the potentially vital role of the Na+/Ca2+ exchanger (NCX) to excitation–contraction coupling. To date, studies on NCX expression have been restricted to late developmental stages. The distribution and gene expression of NCX during early ontogeny is not known, especially in humans. In the present report, we systematically characterized changes in NCX gene expression in human heart during development, with particular emphasis in early ontogeny. Methods: Human hearts during early gestation (9- to 20-week gestation), neonatal (1 to 2 days after birth) and adulthood (18–40 years old) were used. NCX mRNA levels were studied using RNase Protection Assay (RPA) and NCX protein levels were assessed by Western blot. Wet weight was also used as the tissue base. Immunolocalization studies using confocal microscopy were performed in isolated fetal cardiac myocytes. Results: Normalization of NCX mRNA derived from ventricles against an early gestational age (10-week gestation) shows that NCX mRNA levels nominally increased from 1 to 1.13 at 19-week gestation then decreased to 0.74 (P<0.05) at neonate and further decreased to 0.23 (P<0.05) at adult stages. NCX protein levels increased from 1 at 9-week gestation to 3 (P<0.05) at 20-week gestation and then decreased to 1.8 (P<0.05) at neonate and to 1.87 (P<0.05) at adult stages. Confocal imaging of fetal cardiac myocytes revealed intense homogeneous membrane staining and abundance of NCX protein at this stage. Conclusions: The data demonstrate changes in NCX transcript and NCX protein levels as well as total RNA and proteins during human heart development. Per wet weight, NCX mRNA was 4.5 times greater at early fetal than adult stages and NCX protein was 2 times greater at adult than the early fetal stage indicating considerable post-transcriptional regulation. These findings provide new insights into the understanding of temporal changes in NCX in the developing heart at the gene level. The functional significance remains to be determined.

KEYWORDS Developmental biology; Gene expression; Myocytes; Na/Ca-exchanger


   1 Introduction
Top
 Abstract
 1 Introduction
2 Materials and methods
 3 Results
 4 Discussion
 References
 
During excitation–contraction coupling of mature cardiac myocytes, transsarcolemmal Ca2+ influx through Ca2+ channels triggers Ca2+ induced Ca2+ release from the sarcoplasmic reticulum (SR) to initiate contraction [1–4]. Striking morphological and functional differences in the SR of developing and mature mammalian hearts have been reported [5–11]. Both SR Ca2+ release and uptake are significantly underdeveloped in the neonate compared to the adult [12,13]. Lower SR function has been demonstrated based on ryanodine binding studies showing lower SR Ca2+ release channel number in fetal sheep ventricle than the adult [8]. While a negative inotropic response was elicited in adult hearts by ryanodine, there was no change in contraction in fetal and newborn hearts [8,12]. Recent co-immunolabeling studies suggest that a spatial disconnect exists between the sarcolemma and the interior of the SR of the newborn rabbit myocytes [13]. Altogether, these findings indicate that sarcolemmal Ca2+ influx through the Na+/Ca2+ exchanger (NCX) and/or Ca2+ channels likely play an important role in Ca2+ homeostasis in fetal and neonatal hearts.

Canine cardiac NCX (NCX1) was the first to be cloned [14] and the cDNA was shown to encode for a protein of 970 amino acids. Hydropathy analysis predicted a topological model consisting of 11 transmembrane segments, with the amino terminus localized in the extracellular region and an intracellular carboxyl terminus. Sequence analysis of a full-length clone of the human NCX revealed similar overall structure of transmembrane regions and amino acid identity compared to the canine NCX [14,15].

A number of studies have been carried out to characterize developmental changes in NCX but these were limited to late fetal stages [16–18]. A common finding is that NCX amounts increase from late fetal to reach a maximum at perinatal, then decrease at adult stages [16–19]. Conversely, the only available study of human fetal hearts reported a lesser amount of NCX in fetal (18- and 19-week gestation) compared to adult heart [20].

To date, the localization and gene expression of sarcolemmal NCX during development in humans, especially during early ontogeny, is not known. Therefore, we systematically characterized mRNA and protein levels of NCX at different gestational ages (9- to 20-week) using RNase protection assay and Western blots, respectively. We also examined the localization of the NCX in isolated cardiac myocytes using confocal microscopy.


   2 Materials and methods
Top
 Abstract
 1 Introduction
 2 Materials and methods
3 Results
 4 Discussion
 References
 
2.1 Hearts
Fetal hearts (ages 9-, 10-, 11-, 12-, 14-, 15-, 17-, 19- and 20-week gestation) were obtained after elective termination of normal pregnancy. All studies were performed in accordance with the guidelines of the Institutional Review Board and after obtaining informed consent from the mothers. The present study conforms to the principles outlined in the Declaration of Helsinki [21]. Fresh fetal hearts, transported on iced PBS, were used for cell isolation and immunofluorescence studies and snap frozen (in liquid nitrogen) fetal hearts were used for mRNA and protein levels experiments. Normal adult (18 and 40 years old) and neonate (1 and 2 days after birth) heart tissues were obtained frozen from the tissue bank in Baltimore, MD.

2.2 RNA preparation
Total cellular RNA was isolated from frozen ventricles using a RNAzolTM B kit (TEL-TEST, Inc.) based on the method developed by Chomczynski and Sacchi [22]. RNA was quantified by spectrophotometry at 260 nm, and the ratio of absorbance at 260 nm to that of 280 nm was >1.8 for all samples. Degradation of RNA samples was monitored by the observation of appropriate 28S to 18S ribosomal RNA ratios as determined by ethidium bromide staining of the agarose gels. Aliquots of RNA were stored in RNase free water at –80°C.

2.3 Protein preparation
Preparation of proteins for NCX was performed as described elsewhere [23]. Briefly, 100 mg of frozen heart tissue trimmed of atria and fat were homogenized in a seven-fold volume of buffer (in mmol/l: HEPES 20, EGTA 4, and dithiotreitol 1, pH 7.5) complete with protease inhibitors (in mmol/l: Leupeptin 0.1 and phenylmethylsulfony fluoride 0.3; Sigma Ltd.) and stirred for 45 min at 4°C. A first spin of 1000 g at 4°C was performed for 10 min to remove cell debris and nuclei. The supernatant was collected and subjected to a second centrifugation at 100 000 g for 60 min at 4°C. The supernatant was then removed, and the pellet containing the particulate fraction was re-suspended in a seven-fold volume of the same buffer. For determination of total protein concentration, tissue samples were homogenized in 20 volumes of ice-cold sterile bi-distilled water using a polytron homogenizer [24]. The protein concentration was determined in triplicate by a Bio-Rad DC protein assay Kit at absorbance of 750 nm using Bovine Serum Albumin as a standard according to Lowry [25]. Aliquots were stored at –80°C until used.

2.4 Western blot analysis
Samples of 50 µg protein were denatured by heating to 95°C in Tris-Glycine SDS sample buffer (NOVEX) and subjected to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to a PVDF membrane (NOVEX) by E19001-Xcell IITM at 100 mA for 1.5 h. The transfer was checked by staining the PVDF membrane with Ponceau S. The blot was blocked 2 h in 5% nonfat milk and 0.3% Tween-20TM and washed three times, once for 15 min and twice for five min in 1x PBS. For immmunoreaction, the blot was incubated with a 1:1000 diluted C2C12 monoclonal mouse anti-NCX antibody (Affinity Bioreagent, Inc.) for 2 h, washed three times for 15 min and then twice for five min. Immunodetection of the primary antibody against NCX was carried out with a 1:2000 diluted Peroxidase-conjugated anti-mouse IgG for 60 min, the blot was washed again as earlier, then detected with enhanced chemiluminescence (ECL).

2.5 RNase protection assays
RPAs were performed through concomitant measurement of cyclophilin mRNA (internal standard) as previously described [26,27]. DNA templates of NCX were prepared by ligating a cDNA fragment (251 bp) to the T7 promoter using Lig’nScribe kit from Ambion Inc. The cDNA fragment was prepared by reverse transcription and PCR amplification of total cellular RNA isolated from normal human hearts. The constructs were confirmed by sequencing and were used to prepare 32P-UTP radiolabeled antisense cRNA probes (MAXIscriptTM, Ambion). All cRNA probes were gel purified prior to using a 5% denatured polyacrylamide gel. Concomitant hybridization of the two probes using 10 µg total RNA from each age group was carried out at 48°C for 18 h followed by digestion with RNAses A and T1 at 37°C for 30 min. The reaction was terminated by addition of SDS and proteinase K, followed by Phenol-Chloroform extraction and ethanol precipitation. The protected fragments were visualized by autoradiography after electrophoresis on a 5% denatured polyacrylamide gel. Ten µg of yeast RNA was used as a negative control to test for the presence of probe self-protection bands. Quantitative evaluation was carried out using scanning densitometric analysis of the protected fragments.

2.6 Isolation of cardiac myocytes
Cardiac myocytes were obtained from hearts perfused by the Langendorff technique as previously described [28]. Hearts were perfused at 37°C with a HEPES buffered solution containing (in mmol/l): NaCl 117, KCl 5.7, NaHCO3 4.4, NaH2PO4 1.5, MgCl2 1.7, HEPES 20, glucose 11, creatine 10, taurine 20, and 21 milliunits/ml insulin and gassed with 100% O2. The pH was adjusted to 7.4 with NaOH. After 5 min of wash to eliminate the remaining blood, the heart was then perfused with fresh buffer mixed with 1 mg/ml collagenase type A or B (Boehringer Mannheim Corporation) and 20 µM CaCl2 for 5 to 10 min. Both atria were removed and the ventricles were then cut off and gently dissociated with forceps in the same solution without collagenase. Cells were suspended in Petri dishes containing HEPES buffer with 1 mmol/l CaCl2 and 0.5% bovine serum albumin (pH=7.4).

2.7 Indirect immunofluorescent labeling
Isolated myocytes were first fixed with 2% buffered formaldehyde for 15 min. The fixed cells were treated with 0.1% triton X-100 for 15 min. All cells were kept in blocking solution (3% bovine serum albumin and 5% donkey serum) for 1 h, followed by incubation with NCX monoclonal antibody C2C12 (Affinity Bioreagents, Inc.) (1:200) for 90 min [29]. After washing, the cells were incubated with TRITC labeled goat anti-mouse secondary antibody (1:50) for 45 min. Finally, the cells were washed several times in 1x PBS, and in distilled water and then mounted on slides with mounting medium. Control experiments were performed using secondary antibody alone. Serial thin sections of immunofluorescent images were carried out with a Leica confocal microscope.

2.8 Data analysis
For densitometric scans, significant differences between the groups were determined by ANOVA. When the F ratio exceeded the critical value (P<0.05), Bonferronic P-values were determined for identifying significant group to group differences. The values of NCX proteins and mRNA levels of the early gestational age are set to 1 for easy comparison with other developmental stages.


   3 Results
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
4 Discussion
 References
 
3.1 Determination of total RNA and protein
To investigate possible changes in the amount of RNA and protein during cardiac development and to allow for appropriate analysis of developmental changes of NCX mRNA and proteins [24,30], total ventricle RNA and protein was quantified at different stages. At the earliest phase of gestational hearts, total ventricular RNA was 2.9 times greater than the adult, but total protein concentration was 2.6 times greater in the adult than in the earliest phase of gestational hearts. Table 1 provides the average data for each age group (Table 1).


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  		Table 1 Developmental profile of the protein, RNA of the human heart (µg/mg wet weight)a

 
3.2 NCX transcript levels
To examine the developmental changes in NCX gene expression, the abundance of the NCX mRNA was determined by RNase protection assay. Fig. 1 illustrates RNase protection experiments using total RNA isolated from ventricles aged 10-, 12-, 14-, 17-, and 19-week gestation; 2 days after birth, and adult (40 years old). Panel A shows that the protected bands of NCX increased during fetal development and declined at neonatal and adult stages.


Figure 1
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  		Fig. 1 Developmental expression of NCX mRNA in human myocardium assessed by RNase Protection Assay (RPA). In panel A, total cellular RNA was isolated from 10-w (week), 12-w, 14-w, 17-w, 19-w, 2-day-old and adult human hearts. The bands represent the protected fragments corresponding to NCX and cyclophilin mRNA. Bar graph demonstrating quantitative analysis of NCX mRNA levels in hearts with different ages is shown in panel B. Results were normalized to signals of cyclophilin mRNA. Values are mean±S.E. and n=4 for each age. * Indicates P<0.05 when NCX mRNA levels are compared to that of the 10-week gestational age. # Indicates P<0.05 when NCX mRNA levels are compared to that of 19-week gestational age. & Indicates P<0.05 when NCX mRNA level at neonatal stage is compared to that of the adult stage.

 
Densitometric analysis was used for measuring the abundance of NCX transcript expressed in arbitrary units relative to cyclophilin levels for each age (Fig. 1B). As shown in Fig. 1A, no significant developmental changes in cyclophilin levels were found. Averaged data from four hearts at each stage showed an increase in the NCX transcript to 19-week gestation, and a subsequent decline at neonatal, with the lowest level reached at the adult stage. Normalization of NCX mRNA against an early gestational age (10-week, its NCX mRNA value was set to 1 for easy comparison) showed that NCX mRNA levels increased to 1.75 at 19 weeks (P<0.05) and then decreased to 1.3 at neonate (P<0.05 compared to 19-week) and further decreased to 0.68 at the adult stage (P<0.05 compared to 19 weeks). In addition, ventricular wet weight was used as tissue base [30] to re-evaluate the above developmental changes of NCX mRNA (Table 2). The findings show that NCX mRNA levels nominally increased to 1.13 at 19-week (P=NS), then decreased to 0.74 at neonatal (P<0.05 compared to 19-week heart) and further decreased to 0.23 at the adult stage (P<0.05 compared to 19-week and to neonatal). Compared to the early fetal stage (10-week gestation) at 19-week gestation, NCX mRNA increased to 1.75 per cyclophilin, but was not significantly different at 1.13 per wet weight. At the adult stage compared to the early fetal stage, NCX mRNA decreased to 0.68 per cyclophilin vs. 0.23 per wet weight. Both methods revealed a change in NCX mRNA expression during development, but the magnitude of the change was different (i.e., normalized to 10-week gestation, NCX mRNA in the adult was 0.68 per cyclophilin vs. 0.23 per wet weight (P<0.05)).


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  		Table 2 Developmental changes in NCX mRNA and NCX protein using ventricular wet weight (ww), cyclophilin mRNA and the same amount of total protein as tissue base. The values of the earliest gestational age of 9- and/or 10-week were set to 1 for easy comparison

 
3.3 Western blot analysis
To examine whether the observed transcript alterations are accompanied by changes at the protein level, the immunoreactive NCX protein was quantified by Western blot analysis using a specific monoclonal mouse anti-NCX antibody. Fig. 2A illustrates a representative Western blot experiment using hearts aged 9-, 11-, 12-, 14- and 20-week gestation; 1 day after birth, and adult (18 years old). The mouse monoclonal anti-NCX antibody cross-reacted with the particulate fractions of fetal, neonatal and adult human hearts. A single band of 120 kD was found in the immunoblots and a minor 70 kD band was also seen in approximately 10% of the gels. This 70 kD band who was suggested to be the proteolytic fragment of 120 kD band associated with NCX [14,29], was equally present in fetal, neonatal, and adult hearts. The protein levels of NCX increased with development to 20-week gestation then declined in neonates and further in adults. Densitometric analysis of average data from four hearts of each stage is shown in Fig. 2B. Normalization of NCX proteins against the earliest gestational age (9-week) showed that NCX protein increased to 2.1 at 20-week (P<0.05), then decreased to 1.2 at neonate (P<0.05 compared to 20-week) and further decreased to 0.83 at the adult stage (P<0.05 compared to 20-week). Similar to NCX mRNA, we also re-evaluated NCX protein levels relative to wet weight (Table 2). Normalization of NCX proteins against the earliest gestational age (9-week) showed that protein levels increased to 3 at 20-week (P<0.05), then decreased to 1.8 at neonatal (P<0.05 compared to 20-week) and to 1.87 at the adult stage (P<0.05 compared to 20-week). Again, the overall trend of NCX protein, that is, increase during fetal development followed by a decrease at neonatal and adult stages, remained the same. The NCX protein levels per wet weight in the adult (1.87) were not significantly different from neonatal heart (1.80).


Figure 2
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  		Fig. 2 Developmental expression of NCX proteins in human myocardium assessed by Western blot. In panel A, a 120 kD band was consistently present in all hearts at all ages. In panel B, a bar graph demonstrating quantitative analysis of Westerns for NCX protein levels from human heart at different stages is shown. Protein amount was quantified by scanning densitometry. Results in the Y-axis are shown in arbitrary units. Values are mean±S.E. and n=4 for each age. * Indicates P<0.05 when NCX protein levels are compared to that of the 9-week gestational age. # Indicates P<0.05 when NCX protein levels are compared to that of 20-week gestational age. & Indicates P<0.05 when NCX protein level at neonatal stage is compared to that of the adult stage.

 
3.4 Immunostaining
The localization and distribution of NCX was investigated in single human fetal ventricular myocytes using confocal microscopy in myocytes from 4 hearts (50 cells per heart, 19- to 20-week gestation). Exposure of myocytes to NCX monoclonal antibody followed by TRITC secondary antibody showed that the exchanger is homogeneously distributed in the cell surface with intense labeling in the periphery of the cell (Fig. 3). No such staining was observed in cells incubated with secondary antibody alone (data not shown).


Figure 3
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  		Fig. 3 Localization of NCX protein in ventricular myocytes from a 20-week old human fetal heart. Confocal thin sections were taken every 150 nm from the top to the bottom of the cell. Panel A shows a phase contrast image. Panels B and C are selected sections at the 8th (S8) and the 12th (S12) sections. Arrowheads indicate cell surface NCX staining.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
References
 
The present study demonstrates the abundance of NCX gene expression levels in human fetal hearts as early as 9-week gestation. The overall NCX mRNA and protein levels increases with fetal development then decreases at neonatal and at the adult stages. These data suggest that NCX may play a greater role in Ca2+ handling in the human fetal heart during early fetal stages where SR function is relatively immature [5–13]. This is further supported by the data showing that L-type Ca2+ current density is significantly lower in fetal rat [31] and neonatal rabbit hearts [32] than adult hearts. Recent mathematical computer modeling demonstrated that even when SR Ca2+ release or L-type ICa are omitted from the modeling equations, transsarcolemmal Ca2+ fluxes via the NCX1 are sufficient to account for the Ca2+ transient observed experimentally in newborn rabbit myocytes [13]. The implication is that fetal and neonatal hearts will encounter an increased dependency on transsarcolemmal Ca2+ flux in the setting of low L-type Ca2+ current density [31,32]. This indicates the existence of an alternative source of sarcolemmal Ca2+ transport mechanism. Therefore, it is tempting to speculate that NCX may provide an important source of Ca2+ flux in the developing myocardium.

4.1 Transcript and protein levels
In this study, Western blot analysis of human fetal, neonatal and adult heart preparations showed a consistent 120 kD band at all age groups, similar to the size shown to correspond to human cardiac NCX in previous reports [23,33,34]. Occasionally another 70 kD band was also detected; this has been suggested to be the proteolytic fragment of 120 kD NCX [14,29]. In each individual Western blot, there was a trend of an increase in NCX protein from early ontogeny to midterm gestation then a decrease at neonatal followed by a further decrease to the lowest levels in the adult heart. Similarly, mRNA levels paralleled the changes in the levels of immunoreactive NCX proteins when cyclophilin was used as an internal control.

Total protein is usually used as tissue base when assessing the levels of a specific protein, and housekeeping genes like cyclophilin and GAPDH-mRNA, as internal controls, when assessing specific mRNA levels [30]. This approach assumes that total protein and total RNA do not change significantly during the conditions studied [24,30]. The consequences of this assumption were elegantly elucidated in an editorial by Van den Hoff and Moorman [30]. Indeed, under our experimental conditions, total protein and total RNA change with development (Table 1). At the earliest phase of fetal development (9-week), total ventricular RNA was 2.9 times greater than in the adult, but total protein concentration was 2.6 times greater in the adult than in the earliest phase of fetal development (9-week). These data are reasonably comparable to previous data obtained using rat heart [24]. Therefore, we have re-evaluated the above Western blot data using wet weight as tissue base. The average data is shown in Table 2. Using ventricular wet weight as tissue base to assess both NCX protein and NCX mRNA revealed similar patterns of changes during development, but there were some differences in the magnitude of change for each age group. For example, the NCX protein per ventricular wet weight decreased from 3 at 20-week gestation to 1.87 at the adult stage, and NCX mRNA per ventricular wet weight decreased from 1.13 at 19-week gestation to 0.23 at the adult stage. These values are clearly different from the conventional method where NCX proteins decreased from 2.1 at 20-week gestation to 0.83 at the adult stage, and NCX mRNA decreased from 1.75 at 19-week gestation to 0.68 at the adult stage. While NCX mRNA per ventricular wet weight decreased at the neonate stage and continued to decrease at the adult stage, NCX proteins per ventricular wet weight was not significantly different between neonate and adult stages, suggesting a post-transcriptional gene regulation.

The overall NCX gene expression results are consistent with those reported for NCX protein and mRNA content from other species during development [16,17,19] but several differences were found. In this study NCX gene expression for protein and mRNA continued to increase to 19- to 20-week of gestation but decreased at neonatal stages where the NCX peak was previously reported for most of the published reports in animals [16–19,35]. This discrepancy may be due to species differences or to the fact that no studies have yet extended to early developmental stages as demonstrated in the present study. Alternatively, because the youngest and oldest gestational ages available to us were 9- and 20-week respectively, NCX gene expression beyond these ages remains to be determined. It is noteworthy that the only available study on humans by Komuro et al. [20] reported less NCX mRNA in 17- and 19-week-old human fetal hearts than in adult hearts. These findings are inconsistent with all other published reports [16,17,19] and with the present study. While we do not know the exact reason for this discrepancy, the paucity of data in human fetal heart justifies the need for further work on NCX gene expression, especially during early ontogeny.

4.2 Immunolocalization
The distribution of NCX is species and age dependent and somewhat controversial ([36], review). While Frank et al. [29] suggested that the exchanger is located in abundance predominantly in the T-tubules of adult heart myocytes, Kieval et al. [37] reported that the exchanger is uniformly distributed in the plasma membrane of cardiac myocytes. The immunolocalization data showed homogenous staining of the myocyte surface with intense labeling of the cell periphery supporting our findings concerning the presence of NCX proteins in these fetal myocytes.

4.3 Physiological and pathological significance
To date, most of the knowledge available about the potential role of NCX in cardiac Ca2+ handling comes from studies using late fetal, newborn and adult stages [16–18,36]. The present study provides additional and new insights on the gene expression of human cardiac NCX during early ontogeny. The correlation of our findings to functional significance remains to be determined. However, it is tempting to suggest that NCX may provide an alternative route for sarcolemmal Ca2+ handling during fetal and neonate stages. This is supported by studies showing that at birth, excitation–contraction coupling can occur independently of SR Ca2+ release by utilizing Ca2+ entry through outward NCX to directly activate the contractile proteins [13,38,39]. A critical role of NCX has been shown in disease states such as heart failure where an increase in NCX function is likely to compensate for the depressed SR function [23,34]. Characterizing human NCX gene expression and its comparison with SR and L-type Ca2+ channel expression during development is essential to understanding the relative contribution of one to the other and into Ca2+ homeostasis in both physiological and pathological settings.

Time for primary review 23 days.


   Acknowledgements
 
This work has been supported by National Heart, Lung and Blood Institute Grant #HL55401 (to M.B) and the V.A. Merit Grant Award (to M.B). We thank Drs. Mark Restivo, James Godde and June Stapleton for reviewing and editing this manuscript. We also thank Jonathan Feig, Hayf Al-Mussawir and Dr. Hazem Zufari for their technical assistance and collection of hearts. This paper is dedicated in memory of Professor Edward Coraboeuf.


   References
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 

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