The gene for rat atrial natriuretic factor

Atrial natriuretic factor (ANF), a peptide hormone recently isolated from heart atria, appears to play an important role in the regulation of extracellular fluid volume and blood pressure. Indeed, natural and synthetic ANF rapidly and markedly stimulate natriuresis and diuresis and produce smooth muscle relaxation. Consistent with the hypothesis that ANF is a novel hormone, it was recently shown that ANF is present in circulation, and high affinity membrane receptors specific for ANF have been described in renal, vascular, and adrenal tissues. These important biological activities suggest that conditions like hypertension could be associated with defective ANF gene expression. We and others have shown by cDNA cloning that ANF is part of a larger precursor, pro-natriodilatin (PND). We now describe the isolation and structural analysis of the rat PND gene. Southern blot analysis of rat genomic DNA suggests the presence of a single PND gene per haploid genome. The PND coding sequences are interrupted by two short introns. A long alternating purine-pyrimidine tract (GT)9GATG(GT)27 is found 111 base pairs downstream of the polyadenylation site; such sequences could adopt Z-DNA configuration and they have been associated with sequences that appear very active in intergenic recombination. Comparison of the rat and human PND genomic sequences shows highest homology in 5'-flanking as well as in coding sequences. The rat PND gene will be a useful model to study the physiology and pathology of this important regulator of the cardiovascular system.     

Age-dependent differences in the effect of atrial natriuretic factor

Replacement of drinking water by 1.8% NaCl solution produces a severe hypernatremia in prepubertal rats and only a moderate elevation of the plasma sodium concentration (pNa) in adults. Hypernatremia in prepubertal rats was reduced by infusion of blood from adult rats, whereas blood from young rats was without this effect. The aim of this study was to find out if the natriuretic factor containing atrial extracts from prepubertal (AEP) and adult (AEA) rats also differ in their ability to reduce hypernatremia in prepubertal rats. A decrease of pNa was observed 10 min after the administration of AEA (delta = -5.43 mmol/l). AEP failed to show this effect (delta = +0.92 mmol/l). No significant effect of AEA on pNa was observed in adult rats with moderate hypernatremia (delta = -1.88 mmol/l). Natriuretic and diuretic effects of AEP were also significantly lower than those of AEA if they were tested in prepubertal recipients, whereas no difference in activities of AEP and AEA was found in adult recipients. Possible mechanisms of the hypernatremia suppressing activity of AEA, causes of the differences in activities between AEA and AEP, and consequences of the low AEP activity for prepubertal rats are discussed.     

The physiology of atrial natriuretic factor

Following the discovery of the natriuretic effect of atrial extract, our laboratory attempted to dissect the possible physiological role of atrial natriuretic factor. Initial micropuncture experiments demonstrated that the reduction of tubular sodium reabsorption was localized in the medullary collecting duct, a nephron site in which sodium transport was known to be inhibited after acute hypervolemia. Partial removal of the endogenous source of atrial natriuretic factor was associated with a reduced renal response to hypervolemia, confirming that the factor is causally involved in acute sodium balance. In vitro incubation of atrial tissue was used to investigate mechanisms of release of atrial natriuretic factor. It was found that agonists known to activate the intracellular polyphosphoinositide system in other tissues were effective in releasing natriuretic activity from the atria into the incubation medium. To determine whether atrial natriuretic factor might play a role in hypertension, atrial natriuretic content was measured in spontaneously hypertensive rats and their normotensive controls. Hypertension was associated with increased content. Since the renal response to exogenous factor was not impaired in these animals, we suggested that the increased content might play a compensatory role. Our early studies thus indicated that atrial natriuretic factor was a previously unrecognized hormone involved in cardiovascular regulation.     

The relationship between contractile force and intracellular [Ca2+] in intact rat cardiac trabeculae

The control of force by [Ca2+] was investigated in rat cardiac trabeculae loaded with fura-2 salt. At sarcomere lengths of 2.1-2.3 microns, the steady state force-[Ca2+]i relationship during tetanization in the presence of ryanodine was half maximally activated at a [Ca2+]i of 0.65 +/- 0.19 microM with a Hill coefficient of 5.2 +/- 1.2 (mean +/- SD, n = 9), and the maximal stress produced at saturating [Ca2+]i equalled 121 +/- 35 mN/mm2 (n = 9). The dependence of steady state force on [Ca2+]i was identical in muscles tetanized in the presence of the Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA). The force-[Ca2+]i relationship during the relaxation of twitches in the presence of CPA coincided exactly to that measured at steady state during tetani, suggesting that CPA slows the decay rate of [Ca2+]i sufficiently to allow the force to come into a steady state with the [Ca2+]i. In contrast, the relationship of force to [Ca2+]i during the relaxation phase of control twitches was shifted leftward relative to the steady state relationship, establishing that relaxation is limited by the contractile system itself, not by Ca2+ removal from the cytosol. Under control conditions the force-[Ca2+]i relationship, quantified at the time of peak twitch force (i.e., dF/dt = 0), coincided fairly well with steady state measurements in some trabeculae (i.e., three of seven). However, the force-[Ca2+]i relationship at peak force did not correspond to the steady state measurements after the application of 5 mM 2,3-butanedione monoxime (BDM) (to accelerate cross-bridge kinetics) or 100 microM CPA (to slow the relaxation of the [Ca2+]i transient). Therefore, we conclude that the relationship of force to [Ca2+]i during physiological twitch contractions cannot be used to predict the steady state relationship.     

Relaxing effect of atrial natriuretic factor on endothelin-precontracted vascular strips

Endothelin (ET), a peptide recently isolated from the supernatant of cultured porcine aortic endothelial cells, is a potent vasoconstrictor. On the other hand, atrial natriuretic factor (ANF) is a powerful vasorelaxant found in cardiocytes. Its effect was investigated in ET-precontracted rabbit vascular strips. ANF-induced a dose-dependent relaxation of maximally-precontracted mesenteric, renal and aortic strips. Mesenteric artery strips were more sensitive to ANF than either renal or aortic strips. The relaxant effect of ANF on ET-precontracted arteries was more potent than that of other vasorelaxant agents, such as isoproterenol and sodium nitroprusside. Renal and aortic arteries were more sensitive to the vasoconstrictor effect of ET than mesenteric strips. From these results, we conclude that ANF may play a role as a physiological antagonist of ET. The different sensitivity of vascular segments to ET could be due to varying vascular ET receptor densities.     

Effects of atrial appendectomy on circulating atrial natriuretic factor during volume expansion in the rat

This study examined the changes in the circulating level of endogenous atrial natriuretic factor during diuresis and natriuresis produced by acute volume expansion in anesthetized rats with either bilateral atrial appendectomy (n = 9) or sham operation (n = 9). Following control measurements in the sham-operated rats, 1% body weight volume expansion with isotonic saline produced an increment in urinary sodium excretion of over 4 mueq/min (P less than 0.05) while urine volume increased by more than 20 microliter/min (P less than 0.05). These responses were associated with a significant increase in immunoreactive plasma atrial natriuretic factor from a baseline value of 82 +/- 10 pg/ml to a level of 120 +/- 14 pg/ml (P less than 0.05). In contrast, in the group of rats with bilateral atrial appendectomy an identical degree of volume expansion increased urinary sodium excretion and urine volume by only 0.61 mueq/min (P less than 0.05) and 3.07 microliter/min (P less than 0.05), respectively. In this group, immunoreactive plasma atrial natriuretic factor remained statistically unchanged from a control value of 70 +/- 12 pg/ml to a level of 82 +/- 16 pg/ml (P greater than 0.05). Comparison of the two groups indicates that the natriuresis, diuresis, and plasma atrial natriuretic factor levels during volume expansion were significantly reduced in the rats with bilateral atrial appendectomy. No differences in mean arterial pressure and heart rate were observed between the two groups. These data demonstrate that removal of both atrial appendages in the rat attenuated the release of atrial natriuretic factor during volume expansion; and this effect, in turn, was associated with a reduction in the natriuretic and diuretic responses.     

Lack of effect of rat atrial natriuretic factor (rANF) on the renal function in frogs

1. The aim of the present experiments was to examine the question whether the rat atrial natriuretic factor (rANF 1-28) could alter the fractional excretion of sodium (FENa) and other solutes in the frog (Rana esculenta). 2. Although experiments were performed throughout the year possible seasonal changes in the animals were considered in particular. 3. In all frogs, a hypotonic diuresis was induced. 4. Under these conditions in winter frogs, the control FENa was 8.8 +/- 5.8% (15) [means +/- SD (n)], and during rANF administration 7.7 +/- 6.6% (13) (NS). 5. In summer frogs, the control and experimental FENa was 5.2 +/- 2.8% (5) and 6.0 +/- 2.5% (5), respectively (NS). 6. These results show that there was no significant effect of this polypeptide on the fractional excretion of sodium in the frog.     

Tissue-specific determinants of human atrial natriuretic factor gene expression in cardiac tissue

Elements controlling tissue-specific expression of the human atrial natriuretic factor gene have been examined in primary cultures of neonatal rat cardiocytes. When a 68-base pair fragment from human atrial natriuretic factor (hANF) 5'-flanking sequence (positions -400 to -333) was placed upstream from the herpes simplex thymidine kinase promoter linked to a bacterial reporter gene (chloramphenicol acetyltransferase), a tissue-specific positive regulatory effect was observed in atrial as well as ventricular cardiocytes but not in nonmyocardial cells. The cis-acting element in this fragment was orientation- and position-dependent. Examination of nuclear protein extracts for the presence of factors capable of interacting with the 5'-flanking sequence of the hANF gene revealed a cardiocyte-specific factor which bound to the 68-base pair fragment. This association was both tissue- and sequence-specific. These findings indicate that a cis-acting element present in the proximal 5'-flanking sequence confers tissue-specific expression upon the hANF gene, possibly through association with a cardiac-specific nuclear protein.     

The effect of angiotensin II and ADH on the secretion of atrial natriuretic factor

Studies in intact animals have suggested that angiotensin II (AII) and antidiuretic hormone (ADH) increase the plasma concentration of atrial natriuretic factor (ANF). The purpose of these studies was to examine the effects of AII and ADH on ANF secretion in a rat heart-lung preparation under conditions where aortic pressure could be regulated and other indirect effects of these hormones eliminated. ANF secretion was estimated as the total amount of ANF present in a perfusion reservoir at the end of each 30-min period. A pump was used to deliver a fluorocarbon perfusate to the right atrium at rates of either 2 or 5 ml/min. In a time control series where venous return was maintained at 2 ml/min for three 30-min periods ANF secretion was 672 +/- 114, 794 +/- 91, and 793 +/- 125 pg/min (n = 6, P greater than 0.05). When venous return was increased from 2 to 5 ml/min ANF secretion increased from 669 +/- 81 to 1089 +/- 127 pg/min (P less than 0.01). The addition of AII to the perfusate in concentrations of 50, 100, or 200 pg/ml (n = 6 in each group) had no significant effect on basal ANF secretion or the ANF response to increasing venous return. Similarly, the addition of ADH to the perfusate in concentrations of 5, 25, or 100 pg/ml had no significant effect on ANF release from the heart. These results suggest that the ability of AII and ADH to increase plasma ANF concentration in vivo may be due to the effects of these hormones on right or left atrial pressure.     

Distribution of atrial natriuretic factor in fetal rat atria and ventricles

Summary An immunohistochemical study of rat fetal hearts at 20 days of gestation revealed the presence of immunoreactive atrial natriuretic factor (ANF) in cardiocytes of the left and right atria as well as in certain cells is the left and right ventricles. In the atria, cells of the adluminal pectinate muscles appear more densely labeled than the more peripheral mural cells. In the ventricles, immunoreactive cells were found only in adluminal cardiocytes of the presumptive trabeculae and papillary muscles. The results indicate that ANF is synthesized in the perinatal heart, and that the presence of this hormone in the ventricular cardiocytes may be of only temporary nature during certain stages of pre- and postnatal development.Supported by Miami Valley Chapter of American Heart Association MVH-86-019 and MVH-86-010     

心房钠泵因子对颈动脉窦压力感受器反射的易化作用

Effects of atriopeptin II (APII) on the carotid sinus baroreflex in anesthetized rats and on the sinus nerve afferent activity in the anesthetized rabbits were investigated. The results were as follows: (1) By perfusing the isolated left carotid sinus with APII (1 microgram/ml) in anesthetized rats (n = 10), the threshold pressure (TP) of the carotid baroreflex did not show any change, while the equilibrium pressure (EP), the saturation pressure (SP) and the operating range (OR) were decreased from 101 +/- 2.8 to 95 +/- 2.0 mmHg (P less than 0.05), 202 +/- 5.2 to 168 +/- 6.1 mmHg (P less than 0.001) and 128 +/- 5.5 to 93 +/- 6.3 mmHg (P less than 0.001), respectively. The function curve of the baroreflex was shifted to the left and downward with a peak slope (PS) increased during perfusing with APII. In contrast, by perfusing the carotid sinus with sodium nitroprusside (NP, 0.5 micrograms/ml), TP and EP remained unchanged, whereas SP and OR were increased from 188 +/- 6.4 to 218 +/- 6.0 mmHg (n = 6, P less than 0.01) and from 107 +/- 6.9 to 132 +/- 7.6 mmHg (P less than 0.05), respectively. The function curve of the baroreflex and its PS were not affected by NP. The sinus nerve afferent activity was quite stable with the perfusion of carotid sinus at constant intrasinus pressure (ISP) in the rabbits (n = 6) and increased during the elevation of ISP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of atrial natriuretic factor on norepinephrine release in the rat hypothalamus.

The effects of atrial natriuretic factor on the mechanisms involved in norepinephrine release were studied 'in vitro' in slices of Wistar rat hypothalamus. Atrial natriuretic factor (10, 50 and 100 nM) decreased spontaneous [3H]norepinephrine secretion in a concentration dependent way. In addition, the peptide (10 nM) also reduced acetylcholine induced output of norepinephrine. The atrial factor (10 nM) was unable to alter the amine secretion when the incubation medium was deprived of calcium or when a calcium channel blocker such as diltiazem (100 microM) was added. In conclusion, atrial natriuretic factor reduced both spontaneous and acetylcholine evoked [3H]norepinephrine release in the rat hypothalamus. These findings suggest that the atrial natriuretic factor may alter catecholamine secretion by modifying the calcium available for the exocytotic process of catecholamine output.     

Spontaneous and propagated contractions in rat cardiac trabeculae

Sarcomere length measurement by microscopic and laser diffraction techniques in trabeculae of rat heart, superfused with Krebs-Henseleit solution at 21 degrees C, showed spontaneous local sarcomere shortening after electrically stimulated twitches. The contractions originated in a region of several hundred micrometers throughout the width of the muscle close to the end of the preparation that was damaged by dissection. The contractions propagated at a constant velocity along the trabeculae. The velocity of propagation increased from 0 to 10 mm/s in proportion to the number of stimuli (3-30) in a train of electrically evoked twitches at 2 Hz and at an external calcium ion concentration ([Ca++]o) of 1.5 mM. At a constant number of stimuli (n), the velocity of propagation increased from 0 to 15 mm/s with [Ca++]o increasing from 1 to 7 mM. In addition, increase of n and [Ca++]o led to an increase of the extent of local sarcomere shortening during the spontaneous contractions, and the occurrence of multiple contractions. Spontaneous contractions with much internal shortening and a high velocity of propagation frequently induced spontaneous synchronized contractions and eventually arrhythmias. Propagation of spontaneous contractions at low and variable velocity is consistent with the hypothesis that calcium leakage into damaged cells causes spontaneous calcium release from the overloaded sarcoplasmic reticulum in the damaged cells. This process propagates as a result of diffusion of calcium into adjacent cells, which triggers calcium release from their sarcoplasmic reticulum. We postulate that the propagation velocity depends on the intracellular calcium ion concentration, with increases with n and [Ca++]o.     

The effect of isoproterenol on right and left atrial dynamics and plasma atrial natriuretic factor in anesthetized rabbits.

The effect of isoproterenol on mean right and left atrial pressures (RAP, LAP) and dimensions (RAD, LAD), and plasma immunoreactive atrial natriuretic factor (IR-ANF) was investigated in anesthetized rabbits. Infusion of isoproterenol (10 micrograms.kg-1.min-1 for 10 min) significantly increased plasma IR-ANF and heart rate. There were no significant changes in mean RAP or LAP following isoproterenol. Neither mean RAD, systolic RAD and diastolic RAD nor mean LAD, systolic LAD or diastolic LAD changed significantly. Systolic right and left atrial wall stress and diastolic right and left atrial wall stress did not change significantly during the infusion of isoproterenol. Since atrial dimensions did not increase, it is unlikely that the release of IR-ANF in response to isoproterenol is mediated by atrial stretch. These results suggest that the release of IR-ANF in response to this dose of isoproterenol is mediated by factors other than stretch or changes in atrial dynamics.     

The post-translational processing of rat pro-atrial natriuretic factor by primary atrial myocyte cultures

Primary cultures of neonatal rat atrial myocytes were maintained in two different serum-free media for up to 25 days. Reversed-phase high performance liquid chromatography coupled with atrial natriuretic factor (ANF)-specific radioimmunoassay demonstrated that the cultures maintained in our previously described serum-free medium (Glembotski, C.C., and Gibson, T. R. (1985) Biochem. Biophys. Res. Commun. 132, 1008-1017) secreted primarily ANF-(1-126)-like material, whereas those cultures maintained in a different formulation of medium secreted mostly ANF-(99-126)-like material. Cultures that secreted ANF(99-126)-like material were biosynthetically labeled with [35S]cysteine followed by immunoprecipitation of secreted ANF and analysis by reversed-phase, size exclusion, and ion-exchange high performance liquid chromatography. The labeled ANF-(99-126)-like peptide was shown to be chromatographically indistinguishable from other synthetic peptides related to ANF-(99-126). Labeled ANF purified from extracts of the cultured cells was chromatographically indistinguishable from authentic ANF-(1-126), and could be cleaved specifically by thrombin into labeled ANF-(99-126)-like material. These results indicate that primary atrial myocytes maintained under certain serum-free conditions are capable of secreting ANF-related material that is chromatographically indistinguishable from ANF-(99-126), the known circulating form of the hormone. Additional preliminary studies suggest that the presence of glucocorticoids in the culture medium may confer ANF processing ability on cultured myocytes.     

Alterations in atrial and plasma atrial natriuretic factor (ANF) content during development of hypoxia-induced pulmonary hypertension in the rat

Distension of the atrial wall has been proposed as a signal for the increased release of atrial natriuretic factor (ANF) from atrial myocytes in response to perceived volume overload. To determine whether pressure changes resulting from hypertension in the pulmonary circulation may stimulate release of ANF, rats were exposed to chronic hypobaric hypoxia for 3 or 21 days and the ANF concentration in the atria and plasma were determined by specific radioimmunoassay. Exposure to chronic hypoxia resulted in significant increases in hematocrit at both 3 (p less than 0.025) and 21 days (p less than 0.005) and in the development of right ventricular hypertrophy (RVH) expressed as the ratio of the weight of the right ventricle to the weight of the left ventricle and septum (RV/LV+S) at both 3 (RV/LV+S = 0.278 +/- 0.005) and 21 days (RV/LV+S = 0.536 +/- 0.021). After 21 days, left atrial (LA) ANF content was significantly increased in hypoxic rats compared to controls (508 +/- 70 ng/mg tissue vs 302 +/- 37 ng/mg), while right atrial (RA) ANF content was significantly reduced (440 +/- 45 vs 601 +/- 58 ng/mg). At this time, plasma ANF concentration was significantly elevated compared to controls (238 +/- 107 pg/ml vs 101 +/- 10 pg/ml). These results suggest that the development of pulmonary hypertension following chronic hypobaric exposure induces altered atrial ANF content and increased plasma ANF concentration as a result of altered distension of the atrial wall.     

The actions of atrial natriuretic factor on the vascular wall

The actions of atrial natriuretic factor (ANF) on the vascular wall are diverse and show a profound regional heterogeneity. ANF is a potent relaxant of aortic smooth muscle, a response which is associated with activation of particulate guanylate cyclase and elevation in tissue levels of cyclic GMP. However, many large and small muscular arteries and most veins are unresponsive to the peptide. The regional vascular heterogeneity may be due to an altered distribution of high affinity receptors and (or) alterations in the coupling of receptor activation to elevations in cyclic 3',5'-guanosine monophosphate (cGMP). Species differences exist in the structural requirements for receptor activation as well as the effects of infused ANF on peripheral resistance. Although the relaxation to ANF in vitro does not require an intact endothelium, endothelial cells contain multiple receptor subtypes for ANF. Differences amongst tissues and (or) species in the receptor profile for ANF may, in part, explain some of heterogeneity in responsiveness to ANF.     

Amiloride enhances atrial natriuretic factor stimulation of cGMP accumulation in rat glomeruli

The effects of amiloride on ANF binding and ANF stimulation of cGMP were evaluated in rat glomeruli. Amiloride increased ANF binding to whole glomeruli and to glomerular membrane preparations. In contrast, amiloride enhanced ANF-stimulated cGMP accumulation only at 37 degrees C in whole glomeruli, but not at 4 degrees C in whole glomeruli or at 37 degrees C in membrane preparations. These data suggest that under physiological conditions amiloride augments ANF-stimulated intracellular cGMP accumulation. The discrepancies between amiloride augmentation of ANF binding and failure to increase ANF-stimulated cGMP accumulation may result from ANF receptor heterogeneity. This is the first report of an amiloride augmentation of ANF-stimulated cGMP accumulation in renal tissue.     

Identification and characterization of atrial natriuretic factor receptors in the rat retina

The characteristics of atrial natriuretic factor (ANF) receptors where studied in rat retinal particulate preparations. Specific 125I-ANF binding to retinal particulate preparations was greater than 90% of total binding and saturable at a density (Bmax) of 40 +/- 8 fmol/mg protein with an apparent dissociation constant (Kd) of 6.0 +/- 2.0 pM (n = 3). Apparent equilibrium conditions were established within 30 min. The Kd value of 125I-ANF binding calculated by kinetic analysis was 4.0 pM. The Bmax of 60 +/- 10 fmol/mg protein and the Kd of 5 +/- 2 pM, calculated by competition analysis, were in close agreement with the values obtained from Scatchard plots or kinetic analysis. The 125I-ANF binding to retinal particulate preparations was not inhibited by 1 microM concentration of somatostatin, vasopressin, vasoactive intestinal peptide, adrenocorticotropin, thyrotropin releasing hormone, or leu-enkephalin. The rank order of potency of the unlabelled atrial natriuretic peptides for competing with specific 125I-ANF (101-126) binding sites was rANF (92-126) greater than rANF (101-126) greater than rANF (99-126) greater than rANF (103-126) greater than Tyro-Atriopeptin I greater than hANF (105-126) greater than rANF (1-126). Similar results have been obtained in peripheral tissues and mammalian brain, indicating that central and peripheral ANF-binding sites have somewhat similar structural requirements. Affinity cross-linking of 125I-ANF to retinal particulate preparations resulted in the labelling of two sites of molecular weight 140 and 66 kDa, respectively. This demonstration of specific high-affinity ANF receptors suggests that the peptide may act as a neurotransmitter or neuromodulator in the retina.     

Atrial natriuretic factor in the impulse-conduction system of rat cardiac ventricles

Summary A complex network of atrial natriuretic factor-producing cells has been delineated by biochemical and morphological techniques in the rat ventricular myocardium. The chordae tendineae spuriae (CTS; false tendons) contain ANF mRNA and the ANF propeptide (Asn 1-Tyr 126) as assessed by Northern blot analysis, high-pressure liquid chromatography and immunohisto- and -cytochemistry, using three different affinity-purified antibodies: monoclonal and polyclonal antibodies against C-terminal ANF (Arg 101-Tyr 126) and polyclonal antibodies against N-terminal ANF (Asp 11-Ala 37). Two types of cells harboring ANF-containing secretory granules constitute the CTS: the majority (Purkinje type I) have ultrastructural similarities with both atrial and classical Purkinje fibers. Purkinje type-II fibers resemble working ventricular cardiocytes. Both cell types harbor a large paranuclear Golgi complex. The subendocardial Purkinje network is also made up of these two cell types. In this location, Purkinje type-I fibers form cable-like structures while Purkinje type-II fibers are either located beneath the former or abut directly on the endocardium. The latter are not separated from adjacent working ventricular cardiocytes by connective tissue septa. Coronary arteries and arterioles, as in birds, are surrounded by a cushion of Purkinje type-II fibers which blend with the surrounding myocardium. These results indicate that, in the rat, the entire intraventricular conduction system is constituted of endocrine cells producing ANF.Supported by a Medical Research Council of Canada Group Grant to the Multidisciplinary Research Group on Hypertension, by the National Research Council of Canada, the Pfizer Company (England), Bio-Méga Inc. and the Canadian Heart Foundation