This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cao, L. Articles by Gardner, D. G. Search for Related Content PubMed PubMed Citation Articles by Cao, L. Articles by Gardner, D. G.

(Hypertension. 1995;25:227-234.)
© 1995 American Heart Association, Inc.

Articles

Natriuretic Peptides Inhibit DNA Synthesis in Cardiac Fibroblasts

Li Cao; David G. Gardner

From the Metabolic Research Unit and Department of Medicine, University of California at San Francisco.

Correspondence to David G. Gardner, Metabolic Research Unit and Department of Medicine, Box 0540, 1142 HSW, University of California at San Francisco, San Francisco, CA 94143.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract We have examined the effects of the natriuretic peptides on DNA synthesis in primary cultures of neonatal rat cardiac fibroblasts. Binding analysis using ¹²⁵I-labeled atrial natriuretic peptide identified a single class of high-affinity binding sites (K_d=0.03±0.01 nmol/L) in these cells. Of these sites, 80% appear to be of the natriuretic peptide C receptor subtype, with the remainder being A and B receptor subtypes. Northern blot analysis confirmed the presence of all three natriuretic peptide receptors in these cells. Atrial natriuretic peptide (10^-7 mol/L) effected a modest but consistent reduction in both agonist- and stretch-stimulated [³H]thymidine incorporation (17% to 41%). Moreover, brain natriuretic peptide (10^-7 mol/L), C-type natriuretic peptide (10^-7 mol/L), and des-[Gln¹⁸,Ser¹⁹,Gly²⁰,Leu²¹,Gly²²]-ANF 4-23-NH₂ (10^-7 to 10^-6 mol/L) all proved capable of antagonizing growth factor–dependent [³H]thymidine incorporation (the inhibition ranged from 14% to 28%) and cell proliferation, suggesting that all three natriuretic peptide receptor subtypes are involved in the regulation of mitogenesis in these cultures. The inhibition by atrial natriuretic peptide was amplified by cotreatment with phosphodiesterase inhibitors. Similar reduction in [³H]thymidine incorporation was seen after treatment with 8-bromo-cGMP (10^-4 to 10^-3 mol/L) or nitroprusside (10^-4 to 10^-3 mol/L). These results suggest an important paracrine role for the natriuretic peptides in regulating fibroblast growth during cardiac hypertrophy.

Key Words: natriuretic peptide • fibroblast growth factor • fibroblasts • nucleotides, cyclic

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Hemodynamic overload evokes a number of acute and chronic responses in the heart that are directed toward neutralizing the stresses it engenders. The acute responses are focused on making the heart more mechanically efficient and hence more capable of dealing with the increased workload.¹ The chronic changes are typified by a growth response within the cellular elements that make up the heart.^{2 3} This is characterized by hypertrophy of nondividing myocardial cells, which serves to increase wall thickness and thereby (through Laplace's law) reduce wall stress, and hyperplasia of the mesenchymal elements (predominantly fibroblasts) that make up the cardiac interstitium. These latter cells are responsible for generating the collagenous framework that supports the myocardium. The increased density of this framework that is effected during cardiac hypertrophy serves to abate the increase in wall tension that accompanies hemodynamic overload.

We now have a reasonable understanding of the events that lead to and result from cardiac hypertrophy. A number of biochemical^{4 5} and mechanical^{6 7} surrogates of the hypertrophic stimulus have been shown to trigger a series of predictable responses in myocardial cells in vitro, culminating in an increase in cell size and sarcomeric organization. The responses occur in a specific temporal sequence, with triggering of the immediate early gene cascade (ie, c-jun, c-fos, c-myc, and egr-1) preceding activation of the so-called embryonic repertoire (ie, atrial natriuretic peptide [ANP], -skeletal actin, and ß-myosin heavy chain), which in turn is followed by increased expression of a host of constitutively expressed cardiac structural genes. The ANP gene, as a representative of the embryonic repertoire, is of particular interest in that reactivation of its expression in the stimulated ventricular myocardium has come to represent one of the most sensitive markers of hypertrophy in this tissue.⁸ Its role in contributing to or antagonizing the hypertrophic process has yet to be defined, although its hemodynamic properties (ie, improvement in the loading conditions of the ventricular myocardium) suggest potential efficacy in at least temporarily forestalling the progression to ventricular failure. However, it is noteworthy that this peptide has also been shown to have important growth-suppressing properties in a variety of tissues, including the renal mesangium,^{9 10} neural glia,¹¹ and vascular endothelial¹² and smooth muscle^{13 14 15} cells. These effects have been related to occupancy of both the guanylate cyclase–linked natriuretic peptide type A receptor (NPR-A) and type B receptor (NPR-B) as well as the unlinked type C, or clearance, receptor (NPR-C).

As noted above, expansion of the interstitial fibroblast compartment and enhanced collagen deposition are well-documented components of the growth response seen in cardiac hypertrophy. Given the close proximity of the activated myocardial cell, with its proclivity for ANP gene expression, to the interstitial compartment, we chose to examine the possibility that ANP might regulate the growth response (assessed by [³H]thymidine incorporation) of cultured neonatal cardiac fibroblasts to exogenous stimuli. Our findings suggest that each of the natriuretic peptides possesses such growth-suppressing activity and raise the intriguing possibility that they may function in a paracrine fashion to modulate growth in the interstitial compartment during cardiac hypertrophy.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
ANP (rANF, 28 amino acids), brain natriuretic peptide (porcine, BNP_1-32), C-type natriuretic peptide (porcine, CNP_1-22), and cANF (rat, des-[Gln¹⁸,Ser¹⁹,Gly²⁰,Leu²¹,Gly²²]-ANF 4-23-NH₂) were purchased from Peninsula Laboratories, Inc. Human recombinant basic fibroblast growth factor (bFGF), acidic fibroblast growth factor, insulin-like growth factor I, and platelet-derived growth factor-AA were purchased from Upstate Biotechnology Inc. Angiotensin II, endothelin, 3-isobutyl-1-methylxanthine (IBMX), nitroprusside, 8-bromo-cAMP, 8-bromo-cGMP, and reagents for cGMP and cAMP radioimmunoassay (cGMP and cAMP standards and antibodies) were purchased from Sigma Chemical Co. Guanosine 3',5'-cyclic phosphoric acid, 2-O-succinyl [¹²⁵I]iodotyrosine methyl ester (¹²⁵I-cGMP), adenosine 2-O-succinyl [¹²⁵I]iodotyrosine methyl ester (¹²⁵I-cAMP), [³²P]-dCTP, and [³H]thymidine were purchased from New England Nuclear Corp. Enriched calf serum was purchased from Gemini Bioproducts, Inc. Other chemicals and cell culture reagents were purchased from standard commercial suppliers. The bovine NPR-C cDNA and the rat NPR-A and NPR-B cDNAs were generously provided by Drs Gordon Porter and David Garbers, respectively.

Cell Culture
Ventricular mesenchymal cells (fibroblasts) from 1-day-old Sprague-Dawley rat pups were used in the experiments. The rat pups were killed, intact hearts were removed, and the lower 70% of the heart was dispersed to generate the individual cells. Cells were separated using alternating cycles of trypsin (0.1%) digestion and mechanical trituration through a 10-mL wide-bore pipette. After removal of cell debris with a Cellector filter (30 mesh, Belco), the cells were plated for 30 minutes at 37°C to allow fibroblasts to attach to the cell culture plates. Myocardial cells were decanted from the plates. The ventricular fibroblasts were cultured in Dulbecco's modified Eagle's medium H-21 supplemented with 10% enriched calf serum, 2 mmol/L glutamine, and penicillin/streptomycin (100 IU/mL and 100 mg/mL, respectively) at 37°C in a 95% air/5% CO₂ humidified atmosphere for 4 to 6 days to allow them to reach confluence. Cells were then split to 24-well cell culture plates or six-well flexible-bottomed dishes (see below) at a density of 5x10⁴ cells per well and grown to confluence in serum-containing medium. To achieve quiescence, the cells were then transferred to serum-free medium containing 10% serum substitute,¹⁶ 2 mmol/L glutamine, 100 IU/mL penicillin, 100 mg/mL streptomycin, 5 mg/mL insulin, 5 mg/mL transferrin, and 1 mg/mL vitamin B₁₂ for 48 hours. At that point, fresh medium containing different growth factors or peptides was added, and individual experiments were carried out as described below.

Measurement of DNA Synthesis and Cell Number
DNA synthesis in the ventricular fibroblasts was assayed by measuring [³H]thymidine incorporation into the acid-insoluble fraction of the cells.¹⁴ In brief, after growth factor treatment or mechanical stimulation, cells were pulsed with [³H]thymidine (2 mCi/mL) for 4 hours in thymidine-free Eagle's minimal essential medium with Earle's balanced salt solution (Cell Culture Facility, University of California at San Francisco). Cells were then washed twice with cold phosphate-buffered saline (PBS) and once with 10% cold trichloroacetic acid and incubated with 10% trichloroacetic acid at 4°C for 30 minutes. Cell residues were rinsed in ethanol (95%), solubilized in 0.25N NaOH at 4°C for 2 hours, and neutralized with 2.5 mol/L HCl and 1 mol/L Tris-HCl (pH 7.5), and the radioactivity was determined by liquid scintillation spectrophotometry.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, Sigma) was used to estimate cell number in the individual cultures. MTT is a tetrazolium salt that is selectively taken up and converted to a dark blue formazan product by living but not by dead cells. This product, which is linearly related to cell density in the cultures,¹⁷ can be measured colorimetrically by its absorbance at 570 nm. Ventricular fibroblasts were split to 96-well plates at a density of 12 000 cells per well and cultured in serum-containing medium in the presence or absence of bFGF (50 ng/mL) and natriuretic peptides (10^-7 mol/L) for the intervals indicated. Twenty microliters of MTT (2.5 mg/mL) per 100 µL of culture media was then added to each well, and the incubation was continued for 4 hours at 37°C. At that point, 150 µL of 0.04N HCl and 0.1% NP-40 in 2-propanol was added to each well, and absorbance at 570 nm was read on a multiwell scanning spectrophotometer (Microplate, EL310, Bio-Tek Instruments).

Measurement of ANP Binding
ANP was labeled with Na¹²⁵I using the chloramine T method and purified as described previously.¹⁸ The specific activity of the ¹²⁵I-ANP was 300 to 700 mCi/mg (919 to 2142 Ci/mmol). The binding properties of this material, after intermittent repurification, remained constant for at least 4 weeks. Binding assays were performed in 24-well plates on confluent cell monolayers, as described by Leitman et al,¹⁹ with slight modification. Briefly, cells were washed twice with 1 mL PBS and incubated with 200 mL Dulbecco's modified Eagle's medium containing 10 mmol/L HEPES, 0.2% bovine serum albumin, and increasing concentrations of ¹²⁵I-ANP. After 25 minutes of incubation at 37°C, the cells were washed four times with cold PBS containing 0.2% bovine serum albumin and solubilized with 1 mL of 1N NaOH, and 900-mL aliquots were assayed for the total bound radioactivity. Nonspecific binding was determined by incubating parallel cultures with ¹²⁵I-ANP in the presence of 10^-6 mol/L unlabeled ANP. Specific binding was computed as the difference between total and nonspecific binding. The dissociation constant (K_d) and maximum binding (B_max) were calculated using a computer program described previously.²⁰ Unlabeled cANF (10^-6 mol/L), a ligand that binds selectively to NPR-C, was used in an analogous fashion to determine specific binding to that receptor.

Measurement of Cyclic Nucleotide Levels
Cells were grown to confluence in 24-well plates. After treatment with growth factors and/or other agonists as indicated in the individual experiments, the cells were washed twice with cold PBS and incubated in 0.5 mL of cold 10% trichloroacetic acid at 4°C for 30 minutes. The cell extracts were collected and centrifuged at 12 000 rpm for 3 minutes. The supernatant was then extracted four times with water-saturated ether. cAMP and cGMP were measured directly by radioimmunoassay using commercially available antibodies and ¹²⁵I-labeled cAMP and cGMP.

RNA Isolation and Northern Blot Analysis
Ventricular fibroblasts were grown to confluence in 10-cm plates, and total RNA was extracted according to the method of Chirgwin et al.²¹ Thirty milligrams of total RNA was electrophoresed on a 1% agarose gel containing 2.2 mol/L formaldehyde, transferred by capillary action to a nitrocellulose membrane (Genescreen Plus, New England Nuclear Corp) in 10x standard saline citrate (SSC, 1.5 mol/L sodium chloride and 0.15 mol/L sodium citrate) for 8 to 16 hours, and fixed to the membrane by UV irradiation (DNA transfer lamp, Fotodyne Inc). The blots were hybridized to a full-length (2.1-kb) bovine NPR-C cDNA, isolated as a HindIII-EcoRI fragment,²² a 1.2-kb EcoRI fragment from the 5' end of the rat NPR-A cDNA,²³ or a 1.3-kb EcoRI-BamHI fragment from the rat NPR-B cDNA,²⁴ all of which were labeled with [³²P]-dCTP (3000 Ci/mmol) using the random primer technique.²⁵ Hybridizations were performed overnight at 42°C using conventional techniques.²⁵ Blots were washed with 2x SSC and 0.1x SSC in the presence of 0.1% sodium dodecyl sulfate at room temperature and 42°C, respectively, then exposed to x-ray film from 5 to 10 days.

Mechanical Stretch of Ventricular Fibroblasts
The Flexcell Strain Unit (Flexcell Corp)²⁶ was used in these experiments. Cells were cultured on specially manufactured six-well plates containing a flexible collagen-coated silicone rubber membrane at the bottom of each well. Cells were plated at a density of 5x10⁴ cells per well and allowed to grow to confluence over the ensuing 48 to 72 hours. Cultures were then placed in serum-free medium for another 24 hours before being stretched. Cells destined for incubation in the dynamic (ie, stretched) environment were then placed in an airtight vacuum manifold (Flexcell Corp) and subjected to constant strain (25 kPa of applied vacuum) at fixed cycle lengths (0.5-second strain for each 1-second cycle). Maximal percent elongation of the culture surface (ie, that present 3 to 5 mm internal to the edge of the well) has been calculated to be 30% at -25 kPa.²⁷ These calculations represent approximations of the elongation of the culture surface itself; elongation of attached cells is likely to be less than this.²⁸ The serum-free medium was changed every 24 hours throughout the experiment.

Statistical Analysis
Data were subjected to one-way ANOVA and the Newman-Keuls test for significance.

	Results

Top Abstract Introduction Methods Results Discussion References

 
As a prelude to examining natriuretic peptide activity in the cardiac fibroblast cultures, we sought to identify and characterize receptors for the peptides on these cells. As shown in Fig 1, top, Scatchard analysis of ¹²⁵I-ANP binding (using unlabeled ANP to assess nonspecific binding) identified a single class of high-affinity binding sites (K_d=0.03±0.01 nmol/L). In these particular cultures, total receptor concentration was approximated at 43.1 fmol/mg protein. When cANF rather than ANP was used as the unlabeled binding competitor, a similar binding affinity was obtained (K_d=0.025±0.008 nmol/L), but total receptor concentration was reduced to 34.2 fmol/mg protein. Since cANF binds selectively to the clearance receptor, or NPR-C, at the concentrations used here, the measured affinity and binding capacity should reflect primarily binding to that receptor. Studies carried out with unlabeled ANP, on the other hand, would be expected to identify binding to any of the three receptor subtypes (ie, NPR-A, NPR-C, and to a lesser extent NPR-B). Based on this analogy, we can infer that approximately 80% of the total receptor concentration in these cells are of the NPR-C subtype and the remaining 20% are of the combined NPR-A+NPR-B class. Since the latter is difficult to dissect with conventional binding analysis, we examined fibroblast RNA for the presence of transcripts encoding each of the receptor subtypes. As shown in Fig 1, bottom, Northern blot hybridization analysis suggests that transcripts for each of the receptors are present in these cultured cells. Both the NPR-A and NPR-B transcripts migrate as approximately 4.0-kb bands on the gel, a size compatible with that reported for these transcripts in other systems.²⁹ Independent experiments confirmed that both ANP and CNP increase cGMP in these cell cultures, implying that these transcripts are effectively translated to functionally active products (L.C., unpublished observations, 1994). The major portion of NPR-C transcript activity migrates as a broad band extending from 4.8 to 2.3 kb. Larger species, migrating at 7 kb, represent minor components of the total transcript population. A similar pattern, albeit with different ratios of larger-to-smaller transcript levels, has been demonstrated in vascular smooth muscle and endothelial cells (L.C., unpublished observation, 1994).

View larger version (23K): [in this window] [in a new window]   Figure 1. Line graph and Northern blot show natriuretic peptide receptor subtypes in primary ventricular fibroblast cultures. Top, Scatchard analyses were carried out on confluent cells. Increasing concentrations of 125I-labeled atrial natriuretic peptide (ANP) in the absence or presence of 10-6 mol/L unlabeled ANP were used to measure total and nonspecific binding, respectively. Unlabeled cANF (10-6 mol/L) was used to assess specific binding to natriuretic peptide receptor type C (NPR-C). Residual binding in the presence of the latter presumably reflects a combination of nonspecific binding and specific binding to the type A and type B receptors. indicates with ANP competitor (Kd=0.03±0.01 nmol/L, Bmax=43.11 fmol/mg protein); , with cANF competitor (Kd=0.025±0.008 nmol/L, Bmax=34.2 fmol/mg protein). Similar findings were obtained in three independent experiments. Bottom, Total RNA was collected as described in "Methods." Thirty micrograms was loaded in each of three lanes on a 2.2 mol/L formaldehyde/1% agarose gel and subjected to electrophoresis. RNA was then transferred to a nitrocellulose filter and hybridized separately with [32P]-dCTP–labeled cDNA probes for NPR-A (lane 1), NPR-B (lane 2), or NPR-C (lane 3). Data shown are representative of two separate experiments.

Next, we sought to determine whether the natriuretic peptides possessed the capacity to regulate [³H]thymidine incorporation, as an indirect measure of DNA synthesis, in these cultures. As shown in Fig 2A, ANP (10^-7 mol/L), the prototypic ligand for NPR-A, effected a modest but consistent reduction in both basal and agonist-stimulated thymidine incorporation. Inhibition ranged from 17% with bFGF-dependent to 41% with basal thymidine incorporation. Of interest, the vasoactive peptides angiotensin II and endothelin each effected a modest but significant increase in [³H]thymidine incorporation, supporting a potentially important role for these peptides in shaping the interstitial compartment of the heart. Of equal importance, ANP antagonized the effects of these peptides, much as it has been shown to antagonize their activity in a variety of other systems.¹⁴ The inhibitory properties of ANP were shared by other members of the natriuretic peptide family (Fig 2B). Both BNP and CNP proved to be as effective as ANP in reducing bFGF-dependent [³H]thymidine incorporation (25% inhibition by BNP and 21% inhibition by CNP at 10^-7 mol/L). This inhibition, by CNP in particular, suggests that the suppression may arise from occupancy of either NPR-A or NPR-B.

View larger version (19K): [in this window] [in a new window]   Figure 2. A, Bar graph shows atrial natriuretic peptide (ANP) effect on vasoactive peptide- or growth factor–dependent [3H]thymidine incorporation in cultured ventricular fibroblasts. Confluent cells were treated with angiotensin II (AII, 10-7 mol/L), endothelin (ET, 10-7 mol/L), acidic fibroblast growth factor (aFGF, 50 ng/mL), insulin-like growth factor I (IGF, 50 ng/mL), and basic fibroblast growth factor (bFGF, 50 ng/mL) for 24 hours in serum-free medium, and ANP (10-7 mol/L) was added to the culture for the last 6 hours. Cells were then pulsed with [3H]thymidine in thymidine-free Eagle's minimal essential medium with Earle's balanced salt solution for another 4 hours. Open bars indicate without ANP; closed bars, with ANP. Data are mean±SD; n=5-8. *P<.01 compared with untreated control cells; **P<.01 compared with agonist-treated cells without ANP. B, Bar graph shows effect of brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) on bFGF-dependent [3H]thymidine incorporation. Cells were treated with bFGF for 24 hours and with ANP, BNP, or CNP (each at 10-7 mol/L) for the final 6 hours. [3H]Thymidine incorporation was measured as described above. Data are mean±SD; n=6-12. *P<.01 compared with untreated cells; **P<.01 compared with bFGF-treated cells. All values are normalized to the levels found in untreated cells.

To take this analysis one step farther, we decided to examine the effect of ANP on a nonpharmacological stimulus of [³H]thymidine incorporation. Mechanical stretch has been implicated as an activator of collagen gene expression in cardiac fibroblasts cultured in vitro.³⁰ We cultured cells on collagen-coated flexible silicone rubber supports and then distorted these supports (thereby applying mechanical strain to the adherent cell monolayer) using a commercially available apparatus designed for this purpose. As shown in Fig 3A, application of cyclical strain (ie, 60 cycles per minute in which the strain stimulus is applied for 0.5 second of each cycle) effected a doubling of [³H]thymidine incorporation at 24 hours. The effect persisted as the stimulus was maintained for an additional 24 hours. Importantly, ANP proved capable of reversing this stimulation (Fig 3B). In this case, the smaller stretch-dependent increment in [³H]thymidine incorporation was completely reversed by the peptide. These data suggest that both biochemical (ie, growth factor) and mechanical activators of [³H]thymidine incorporation can be reversed by the natriuretic peptides in these cells.

View larger version (14K): [in this window] [in a new window]   Figure 3. Bar graphs show atrial natriuretic peptide (ANP) effects on mechanical stretch–stimulated [3H]thymidine incorporation in cultured ventricular fibroblasts. A, Confluent cells were either cultured in a static environment (open bars) or subjected to mechanical distension (closed bars) for 24 to 48 hours. Cells were pulsed with [3H]thymidine during the last 4 hours of incubation. All values are normalized to levels found in static cells and presented as mean±SD; n=6. Similar findings were obtained from four independent experiments. *P<.01 compared with static group at each time point. B, Cells were either cultured in static environment (open bars) or subjected to mechanical distension (closed bars) for 24 hours, again in the presence or absence of 10-7 mol/L ANP for the final 6 hours of the experiment. Cells were then pulsed with [3H]thymidine. Values are normalized to those of the static, untreated group. Data are mean±SD; n=6. *P<.01 compared with static cells; **P<.01 compared with stretched cells not treated with ANP.

Next, we asked whether occupancy of NPR-C might contribute in any way to the inhibitory activity of the natriuretic peptides noted above. NPR-C occupancy has in fact been implicated in the suppression of mitogenesis in renal mesangial cells and rat glial cells in culture.^{11 12 15} To address this issue, we used the C receptor–selective ligand cANF_4-23. As shown in Fig 4, this agonist did prove capable of suppressing platelet-derived growth factor–dependent [³H]thymidine incorporation (14% inhibition at 10^-7 mol/L and 28% inhibition at 10^-6 mol/L), although in general it was considerably less efficacious than ANP in promoting this effect (a finding that has been borne out with several additional experiments).

View larger version (18K): [in this window] [in a new window]   Figure 4. Bar graph shows that cANF inhibits platelet-derived growth factor (PDGF)–stimulated [3H]thymidine incorporation in cultured ventricular fibroblasts. Cells were treated with PDGF (2 ng/mL) for 24 hours, and atrial natriuretic peptide (open bars) or cANF (closed bars) was added to the culture in varying concentrations for the final 6 hours. Values are mean±SD (n=5-8) and compared with levels found in cells treated with PDGF alone. *P<.01 vs PDGF-treated samples.

We next turned our attention to the investigation of the mechanism or mechanisms underlying the effects seen with the various natriuretic peptides. Both NPR-A and NPR-B harbor particulate guanylate cyclase as an intrinsic part of their protein structure. Most if not all of the activity signaled through these receptors appears to involve activation of this cyclase and the subsequent accumulation of intracellular cGMP. If such a signaling modality were involved here, one might predict that cotreatment with a phosphodiesterase inhibitor, which prevents the degradation of the cyclic nucleotide, would serve to amplify the natriuretic peptide effect. As shown in Fig 5, treatment with either the nonselective phosphodiesterase inhibitor IBMX or the cGMP-selective phosphodiesterase (type V) inhibitor M&B 22,948 led to a moderate reduction in either basal or bFGF-stimulated [³H]thymidine incorporation. When added together with ANP, these phosphodiesterase inhibitors effected a further reduction in [³H]thymidine incorporation, implying amplification of a cGMP-dependent effect. Interestingly, their effect on the cANF-mediated inhibition was considerably more modest, suggesting that this latter ligand operates through a pathway that does not directly involve cGMP.

View larger version (27K): [in this window] [in a new window]   Figure 5. Bar graph shows effect of phosphodiesterase inhibitors on growth factor–dependent [3H]thymidine incorporation. Confluent cells were treated with basic fibroblast growth factor (bFGF, 50 ng/mL) for 24 hours, and 10-7 mol/L atrial natriuretic peptide (ANP) or 10-7 mol/L cANF was added to the cultures in the presence or absence of 3-isobutyl-1-methylxanthine (IBMX, 0.3 mmol/L) or M&B 22,948 (0.3 mmol/L) for the final 6 hours. [3H]Thymidine incorporation was measured as described in Fig 2. Open bars indicate without IBMX or M&B 22,948; hatched bars, with M&B 22,948; closed bars, with IBMX. Data (mean±SD, n=4) are normalized to levels in untreated cells. P<.01 compared with cells without treatment; *P<.01 compared with bFGF alone; **P<.05 compared with cells not exposed to phosphodiesterase inhibitors in the same group.

To explore the cGMP dependence of the natriuretic peptide effect more fully, we used the permeable derivate 8-bromo-cGMP in an attempt to promote the inhibitory effect at a locus distal to the natriuretic peptide receptor interaction. As shown in Fig 6, 8-bromo-cGMP effected a dose-dependent reduction in [³H]thymidine incorporation that reached a peak of 28% at 10^-3 mol/L. 8-Bromo-cAMP was without effect over the same dose range. Nitroprusside, a reducing agent that activates the soluble guanylate cyclase and hence raises intracellular cGMP levels in a variety of cell types, effected a modest reduction in [³H]thymidine incorporation similar to that seen with the cGMP analogue.

View larger version (14K): [in this window] [in a new window]   Figure 6. Line graph shows nitroprusside, 8-bromo-cGMP, and 8-bromo-cAMP effects on basic fibroblast growth factor (bFGF)–stimulated [3H]thymidine incorporation in cultured ventricular fibroblasts. Cells were treated with bFGF (50 ng/mL) for 24 hours in the presence of varying concentrations of nitroprusside (), 8-bromo-cGMP (), or 8-bromo-cAMP () for the final 6 hours and then pulsed with [3H]thymidine for another 4 hours. Values are normalized to levels found in the group treated with bFGF alone. Data are mean±SD; n=3. *P<.01 compared with untreated cells.

These studies left an obvious question as to the mechanism underlying the cANF-dependent activity. We questioned whether this ligand might exert its effects through a traditional guanylate cyclase–linked pathway, either through direct interaction with NPR-A or NPR-B or by impairing the clearance of an endogenous natriuretic peptide product present in these cultures in limiting amounts. To address this question, we examined the capacity of cANF in these cultures to increase cellular cGMP accumulation. As shown in Fig 7A, cANF had no effect on cGMP levels, whereas ANP promoted the expected increments in cGMP at doses associated with suppression of [³H]thymidine incorporation. In parallel studies, neither ANP nor cANF had a significant effect on cellular cAMP levels (Fig 7B).

View larger version (10K): [in this window] [in a new window]   Figure 7. Line graphs show cGMP (A) and cAMP (B) responses to atrial natriuretic peptide (ANP) or cANF treatment in cultured ventricular fibroblasts. Cells were treated with basic fibroblast growth factor (50 ng/mL) and ANP (, 10-7 mol/L) or cANF (, 10-7 mol/L). After 24 hours, intracellular cGMP or cAMP was collected and measured as described in "Methods." Data are mean±SD; n=3.

Finally, to relate the natriuretic peptide effects on [³H]thymidine incorporation to growth-suppressant activity, we examined the effect of prolonged exposure to these peptides on the number of viable fibroblasts present in our cultures. As shown in the Table, each of the natriuretic peptides proved capable of reducing the bFGF-dependent increment in fibroblast number. This effect was more apparent with CNP and cANF at the earlier time points but was found with all of the natriuretic peptides after 6 days of incubation. Importantly, cANF proved to be as effective as or more effective than the guanylate cyclase–activating ligands in decreasing cell number, supporting a role for NPR-C as a mediator of natriuretic peptide–dependent growth-suppressant activity.

View this table: [in this window] [in a new window]   Table 1. Natriuretic Peptide Effect on Ventricular Fibroblast Density

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Mesenchymal cells make up more than two thirds of the cell population in the heart despite contributing less than one third of the total heart mass.³¹ Hemodynamic overload activates both a hypertrophic response within the nondividing cardiac myocyte population and a hyperplastic response among the fibroblasts of the cardiac interstitium. This latter response is accompanied by an increase in collagen synthesis and deposition in the extracellular space, resulting in amplification of the collagen network that provides the skeletal support for the myocardium.³² Increased collagen synthesis would be predicted to assist the heart in withstanding the increased intracardiac pressures associated with overload. On the other hand, overproduction of collagen could also lead to a reduction in diastolic compliance with reduced ventricular filling, a phenomenon that could seriously compromise pump function in a situation in which reserve is at a minimum. In addition, an increase in interstitial collagen might lead to conduction abnormalities,³³ which predispose to arrhythmias. Thus, understanding the regulation of the hyperplastic response in the interstitial compartment may provide important insight into the changes that accompany hemodynamic overload and potentially contribute to the progression to congestive heart failure when it occurs.

A number of factors have been linked to fibroblast proliferation in the heart. In addition to the in vivo models of cardiac hypertrophy^{2 8} mentioned above, a number of growth factors and vasoactive peptides have been linked to mitogenic responses in these cells. Angiotensin II in particular has received a good deal of attention as a promoter of fibroblast proliferation and a potentially important factor in cardiac hypertrophy^{4 34} and remodeling.³⁵ Our own data support a role for angiotensin II in promoting cardiac fibroblast growth and extend this to endothelin as well as a variety of more traditional growth factors. Of the group, the growth factors clearly appeared to be more efficacious than the vasoactive peptides in stimulating thymidine incorporation, but the relative contributions of the individual agonists in generating the fibroblast growth response attendant to hypertrophy in vivo remain to be defined.

Each of the natriuretic peptides examined, including ANP, BNP, and CNP, proved capable of reducing agonist-stimulated [³H]thymidine incorporation in these cell cultures. By and large, the effects were modest, averaging 25% to 30% inhibition, although this level of inhibition is well in line with that reported in other systems^{11 12 13 14} in which reductions of 20% to 50% were noted. Our data suggest that at least a portion of the inhibition traffics through the guanylate cyclase–linked NPR-A and NPR-B. Both 8-bromo-cGMP and nitroprusside proved capable of mimicking the natriuretic peptide effect, and the inclusion of a phosphodiesterase inhibitor amplified the inhibitory properties of ANP (see Fig 5). The fact that both ANP and CNP were effective in promoting the inhibition implies that both NPR-A and NPR-B are present on these cells (confirmed by RNA analysis; see Fig 1, bottom) and that both are capable of signaling the antimitogenic activity. One needs to recall, however, that these are cultured cells and may not reflect the in vivo receptor phenotype with fidelity.³⁶ Thus, the relative importance of NPR-A versus NPR-B as antimitogenic effectors in vivo depends on their relative abundance on the surface of fibroblasts in the normal and hypertrophic heart and the availability of their respective cognate ligands (ie, ANP and CNP) in the microenvironment of the cardiac interstitium.

Of interest, we found that the NPR-C–specific ligand cANF_4-23 also produced an inhibition of [³H]thymidine incorporation, as well as a reduction in cell density, in these fibroblast cultures. Similar NPR-C–dependent antimitogenic activity has been described in a number of other systems.^{11 12 15} As in those reports, the effect in our fibroblast cultures did not appear to operate either directly or indirectly through a guanylate cyclase–dependent pathway. The inhibition by cANF_4-23 was not amplified by the phosphodiesterase inhibitor IBMX. Furthermore, this ligand failed to increase cGMP levels at concentrations at which it possessed inhibitory activity. The precise signaling mechanism underlying the NPR-C effect remains undefined. Several studies have suggested that this receptor can inhibit adenylate cyclase activity in a pertussis toxin–sensitive manner³⁷ ; however, simultaneous measurement of cellular cAMP levels revealed no differences between the control and cANF_4-23–treated cells. This would suggest that this receptor possesses the ability to signal through an as yet unknown pathway in these cells.

The possibility of a role for the natriuretic peptide receptors in controlling fibroblast growth in the cardiac interstitium is intriguing. As noted above, although limited expansion of this compartment with increased collagen deposition may be beneficial, overgrowth of the interstitium may have detrimental effects that lead to aberrant hemodynamics, electrical conduction defects, and cardiac decompensation. ANP, as a component of the embryonic repertoire, is avidly expressed early in the hypertrophied ventricular myocardium.⁸ Thus, at a time when the stimulus to mitogenesis in the interstitium is being activated, the antiproliferative regulatory peptide increases in parallel, providing a convenient intracardiac paracrine mechanism to contain fibroblast growth at an appropriate level. Breakdown or inactivation of this counterregulatory mechanism could accelerate the development of fibrosis in hypertrophied hearts and contribute to the progressive decline in hemodynamic function as failure develops.

	Acknowledgments

 
This work was supported by grants HL-35753 and HL-45637 from the National Institutes of Health, Bethesda, Md. The authors are grateful to Karl Narkmura for assistance with preparation of the cells and Hui Yuan for help with the MTT assay.

Received April 18, 1994; first decision May 26, 1994; accepted October 3, 1994.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Katz AM. Cardiomyopathy of overload. N Engl J Med. 1990;322:100-110. [Medline] [Order article via Infotrieve]
2. Parker TG, Schneider M. Growth factors, proto-oncogenes, and plasticity of the cardiac phenotype. Annu Rev Physiol. 1991;53:179-200. [Medline] [Order article via Infotrieve]
3. Weber KT, Clark WA, Janicki JS, Shroff SG. Physiologic versus pathologic hypertrophy and the pressure-overloaded myocardium. J Cardiovasc Pharmacol. 1987;10(suppl 6):S37-S49.
4. Sadoshima J, Izumo S. Molecular characterization of angiotensin II-induced hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts: critical role of the AT1 receptor subtype. Circ Res. 1993;73:413-423. [Abstract/Free Full Text]
5. Shubeita HE, McDonough PM, Harris AN, Knowlton KU, Glembotski CC, Brown JH, Chien KR. Endothelin induction of inositol phospholipid hydrolysis, sarcomere assembly, and cardiac gene expression in ventricular myocytes. J Biol Chem. 1990;265:20555-20562. [Abstract/Free Full Text]
6. Komuro I, Katoh Y, Kaida T, Shibazaki Y, Kurabayashi M, Hoh E, Takaku F, Yazaki Y. Mechanical loading stimulates cell hypertrophy and specific gene expression in cultured rat cardiac myocytes. J Biol Chem. 1991;266:1265-1268. [Abstract/Free Full Text]
7. Sadoshima J, Takahashi T, Jahn L, Izumo S. Role of mechano-sensitive ion channels, cytoskeleton, and contractile activity in stretch-induced immediate-early gene expression and hypertrophy of cardiac myocytes. Proc Natl Acad Sci U S A. 1992;89:9905-9909. [Abstract/Free Full Text]
8. Day ML, Schwartz D, Wiegand RC, Stockman PT, Brunnert SR, Tolunay HE, Currie MG, Standaert DG, Needleman P. Ventricular atriopeptin: unmasking of messenger RNA and peptide synthesis by hypertrophy or dexamethasone. Hypertension. 1987;9:485-491. [Abstract/Free Full Text]
9. Kohno M, Ikeda M, Johchi M, Horio T, Yasunari K, Kurihara N, Takeda T. Interaction of PDGF and natriuretic peptides on mesangial cell proliferation and endothelin secretion. Am J Physiol. 1993;265:E673-E679. [Abstract/Free Full Text]
10. Haneda M, Kikkawa R, Koya D, Sakamoto K, Nakanishi S, Matsuda Y, Shigeta Y. Biological receptors mediate anti-proliferative action of atrial natriuretic peptide in cultured mesangial cells. Biochem Biophys Res Commun. 1993;192:642-648. [Medline] [Order article via Infotrieve]
11. Levin ER, Frank HJL. Natriuretic peptides inhibit rat astroglial proliferation: mediation by C receptor. Am J Physiol. 1991;261:R453-R457. [Abstract/Free Full Text]
12. Itoh H, Pratt RE, Ohno M, Dzau VJ. Atrial natriuretic polypeptide as a novel antigrowth factor of endothelial cells. Hypertension. 1992;19:758-761. [Abstract/Free Full Text]
13. Abell TJ, Richards AM, Ikram H, Espiner EA, Yandle T. Atrial natriuretic factor inhibits proliferation of vascular smooth muscle cells stimulated by platelet-derived growth factor. Biochem Biophys Res Commun. 1989;160:1392-1396. [Medline] [Order article via Infotrieve]
14. Itoh H, Pratt RE, Dzau VJ. Atrial natriuretic polypeptide inhibits hypertrophy of vascular smooth muscle cells. J Clin Invest. 1990;86:1690-1697.
15. Cahill PA, Hassid A. Clearance receptor-binding atrial natriuretic peptides inhibit mitogenesis and proliferation of rat aortic smooth muscle cells. Biochem Biophys Res Commun. 1991;179:1606-1613. [Medline] [Order article via Infotrieve]
16. Bauer RF, Arthur LO, Fine DL. Propagation of mouse tumor virus in serum-free medium. In Vitro. 1976;12:558-563. [Medline] [Order article via Infotrieve]
17. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55-63. [Medline] [Order article via Infotrieve]
18. Gardner DG, Deschepper CF, Ganong WF, Hane S, Fiddes J, Baxter JD, Lewicki J. Extra-atrial expression of the gene for atrial natriuretic factor. Proc Natl Acad Sci U S A. 1986;83:6697-6701. [Abstract/Free Full Text]
19. Leitman DC, Andresen JW, Catalano RM, Waldman SA, Tuan JJ, Murad F. Atrial natriuretic peptide binding, cross-linking, and stimulation of cyclic GMP accumulation and particulate guanylate cyclase activity in cultured cells. J Biol Chem. 1988;263:3720-3728. [Abstract/Free Full Text]
20. Murlas C, Nadel JA, Roberts JM. The muscarinic receptors of airway smooth muscle: their characterization in vitro. J Appl Physiol. 1982;52:1084-1091. [Abstract/Free Full Text]
21. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979;18:5294-5299. [Medline] [Order article via Infotrieve]
22. Fuller F, Porter JG, Arfsten AE, Miller J, Schilling JW, Scarborough RM, Lewicki JA, Schenk DB. Atrial natriuretic peptide clearance receptor. J Biol Chem. 1988;263:9395-9401. [Abstract/Free Full Text]
23. Chinkers M, Garbers DL, Chang MS, Lowe DG, Chin H, Goeddel DV, Schulz S. A membrane form of guanylate cyclase is an atrial natriuretic peptide receptor. Nature. 1989;338:78-83. [Medline] [Order article via Infotrieve]
24. Schulz S, Singh S, Bellet RA, Singh G, Tubb DJ, Chin H, Garbers DL. The primary structure of a plasma membrane guanylate cyclase demonstrates diversity within this new receptor family. Cell. 1989;58:1155-1162. [Medline] [Order article via Infotrieve]
25. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.
26. Banes AJ, Gilbert J, Taylor D, Monbureau O. A new vacuum-operated stress-providing instrument that applies static or variable duration cycle tension or compression to cells in vitro. J Cell Sci. 1985;75:35-42. [Abstract]
27. Buckley MJ, Banes AJ, Levin LG, Sumpio BE, Sato M, Jordan R, Gilbert J, Link GW, Tay RTS. Osteoblasts increase the rate of division and align in response to cyclical, mechanical tension in vitro. Bone Miner. 1988;4:225-236. [Medline] [Order article via Infotrieve]
28. Wirtz HRW, Dobbs LG. Calcium mobilization and exocytosis after one mechanical stretch of lung epithelial cells. Science. 1990;250:1266-1269. [Abstract/Free Full Text]
29. Suga S, Nakao K, Hosoda K, Mukoyama M, Ogawa Y, Shirakami G, Arai H, Saito Y, Kambayashi Y, Inouye K, et al. Receptor selectivity of natriuretic peptide family, atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide. Endocrinology. 1992;130:229-239. [Abstract]
30. Carver W, Nagpal ML, Nachtigal M, Borg TK, Terracio L. Collagen expression in mechanically stimulated cardiac fibroblasts. Circ Res. 1991;69:116-122. [Abstract/Free Full Text]
31. Zak R. Cell proliferation during cardiac growth. Am J Cardiol. 1973;31:211-219. [Medline] [Order article via Infotrieve]
32. Weber KT. Cardiac interstitium in health and disease: the fibrillar collagen network. J Am Coll Cardiol. 1989;13:1637-1652. [Abstract]
33. Spach MS, Miller WT, Dolber PC, Kootsey JM, Sommer JR, Mosher CE. The functional role of structured complexities in the propagation of depolarization in the atrium of the dog: cardiac conduction disturbances due to discontinuities of effective axial resistivity. Circ Res. 1982;50:175-191. [Free Full Text]
34. Schorb W, Booz GW, Dostal DE, Conrad KM, Chang KC, Baker KM. Angiotensin II is mitogenic in neonatal rat cardiac fibroblasts. Circ Res. 1993;72:1245-1254. [Abstract/Free Full Text]
35. Brilla CG, Maisch B, Weber KT. Myocardial collagen matrix remodeling in arterial hypertension. Eur Heart J. 1992;13(suppl D):24-32.
36. Suga S, Nakao K, Kishimoto I, Hosoda K, Mukoyama M, Arai H, Shirakami G, Ogawa Y, Komatsu Y, Nakagawa O, et al. Phenotype-related alteration in expression of natriuretic peptide receptors in aortic smooth muscle cells. Circ Res. 1992;71:34-39. [Abstract/Free Full Text]
37. Anand-Srivastava MB, Sairam MR, Cantin M. Ring deleted analogs of atrial natriuretic factor inhibit adenylate cyclase/cAMP system. J Biol Chem. 1990;265:8556-8572.

This article has been cited by other articles:

				H. M. Nef, H. Mollmann, S. Kostin, C. Troidl, S. Voss, M. Weber, T. Dill, A. Rolf, R. Brandt, C. W. Hamm, et al. Tako-Tsubo cardiomyopathy: intraindividual structural analysis in the acute phase and after functional recovery Eur. Heart J., October 2, 2007; 28(20): 2456 - 2464. [Abstract] [Full Text] [PDF]

				J. E. Rame, M. H. Drazner, W. Post, R. Peshock, J. Lima, R. S. Cooper, and D. L. Dries Corin I555(P568) Allele Is Associated With Enhanced Cardiac Hypertrophic Response to Increased Systemic Afterload Hypertension, April 1, 2007; 49(4): 857 - 864. [Abstract] [Full Text] [PDF]

				L. J Ellmers, N. J A Scott, J. Piuhola, N. Maeda, O. Smithies, C. M Frampton, A M. Richards, and V. A Cameron Npr1-regulated gene pathways contributing to cardiac hypertrophy and fibrosis J. Mol. Endocrinol., February 1, 2007; 38(2): 245 - 257. [Abstract] [Full Text] [PDF]

				P. De Paolis, V. Nobili, A. Lombardi, D. Tarasi, D. Barbato, S. Marchitti, U. Ganten, E. Brunetti, M. Volpe, and S. Rubattu Role of a Molecular Variant of Rat Atrial Natriuretic Peptide Gene in Vascular Remodeling Ann. Clin. Lab. Sci., January 1, 2007; 37(2): 135 - 140. [Abstract] [Full Text] [PDF]

				H. Masuyama, T. Tsuruda, J. Kato, T. Imamura, Y. Asada, J.-P. Stasch, K. Kitamura, and T. Eto Soluble Guanylate Cyclase Stimulation on Cardiovascular Remodeling in Angiotensin II-Induced Hypertensive Rats Hypertension, November 1, 2006; 48(5): 972 - 978. [Abstract] [Full Text] [PDF]

				M. D. Jarvis, M. T. Rademaker, L. J. Ellmers, M. J. Currie, J. L. McKenzie, B. R. Palmer, C. M. Frampton, A. M. Richards, and V. A. Cameron Comparison of infarct-derived and control ovine cardiac myofibroblasts in culture: response to cytokines and natriuretic peptide receptor expression profiles. Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1952 - H1958. [Abstract] [Full Text] [PDF]

				A. Koch, S. Zink, and H. Singer B-type natriuretic peptide in paediatric patients with congenital heart disease Eur. Heart J., April 1, 2006; 27(7): 861 - 866. [Abstract] [Full Text] [PDF]

				T. E. H. Christoffersen, M. Aplin, C. C. Strom, S. P. Sheikh, O. Skott, P. K. Busk, S. Haunso, and L. B. Nielsen Increased natriuretic peptide receptor A and C gene expression in rats with pressure-overload cardiac hypertrophy Am J Physiol Heart Circ Physiol, April 1, 2006; 290(4): H1635 - H1641. [Abstract] [Full Text] [PDF]

				L. R. Potter, S. Abbey-Hosch, and D. M. Dickey Natriuretic Peptides, Their Receptors, and Cyclic Guanosine Monophosphate-Dependent Signaling Functions Endocr. Rev., February 1, 2006; 27(1): 47 - 72. [Abstract] [Full Text] [PDF]

				T. Nishikimi, N. Maeda, and H. Matsuoka The role of natriuretic peptides in cardioprotection Cardiovasc Res, February 1, 2006; 69(2): 318 - 328. [Abstract] [Full Text] [PDF]

				E. Vellaichamy, M. L. Khurana, J. Fink, and K. N. Pandey Involvement of the NF-{kappa}B/Matrix Metalloproteinase Pathway in Cardiac Fibrosis of Mice Lacking Guanylyl Cyclase/Natriuretic Peptide Receptor A J. Biol. Chem., May 13, 2005; 280(19): 19230 - 19242. [Abstract] [Full Text] [PDF]

				J. C. Burnett Nesiritide: new hope for acute heart failure syndromes? Eur. Heart J. Suppl., April 1, 2005; 7(suppl_B): B25 - B30. [Abstract] [Full Text] [PDF]

				M Jankowski, D Wang, S Mukaddam-Daher, and J Gutkowska Pregnancy alters nitric oxide synthase and natriuretic peptide systems in the rat left ventricle J. Endocrinol., January 1, 2005; 184(1): 209 - 217. [Abstract] [Full Text] [PDF]

				V. Franco, Y.-F. Chen, S. Oparil, J. A. Feng, D. Wang, F. Hage, and G. Perry Atrial Natriuretic Peptide Dose-Dependently Inhibits Pressure Overload-Induced Cardiac Remodeling Hypertension, November 1, 2004; 44(5): 746 - 750. [Abstract] [Full Text] [PDF]

				K. L. Tran, X. Lu, M. Lei, Q. Feng, and Q. Wu Upregulation of corin gene expression in hypertrophic cardiomyocytes and failing myocardium Am J Physiol Heart Circ Physiol, October 1, 2004; 287(4): H1625 - H1631. [Abstract] [Full Text] [PDF]

				D. Rahmutula, J. Cui, S. Chen, and D. G. Gardner Transcriptional Regulation of Type B Human Natriuretic Peptide Receptor Gene Promoter: Dependence on Sp1 Hypertension, September 1, 2004; 44(3): 283 - 288. [Abstract] [Full Text] [PDF]

J. Magga, M. Puhakka, S. Hietakorpi, K. Punnonen, P. Uusimaa, J. Risteli, O. Vuolteenaho, H. Ruskoaho, and K. Peuhkurinen Atrial natriuretic peptide, B-type natriuretic peptide, and serum collagen markers after acute myocardial infarction J Appl Physiol, April 1, 2004; 96(4): 1306 - 1311.