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(Circulation Research. 1995;77:486-493.)
© 1995 American Heart Association, Inc.

Articles

Contractile Responsiveness of Ventricular Myocytes to Isoproterenol Is Regulated by Induction of Nitric Oxide Synthase Activityin Cardiac Microvascular EndothelialCells in Heterotypic Primary Culture

Dan Ungureanu-Longrois, Jean-Luc Balligand, Ikutaro Okada, William W. Simmons, Lester Kobzik, Charles J. Lowenstein, Steven L. Kunkel, Thomas Michel, Ralph A. Kelly, Thomas W. Smith

From the Cardiovascular Division, Department of Medicine, and Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, and the Respiratory Physiology Program, Harvard School of Public Health (L.K.), Boston, Mass; the Department of Medicine, Johns Hopkins University, Baltimore, Md (C.J.L.); and the Department of Pathology, University of Michigan Medical School, Ann Arbor (S.L.K.).

Correspondence to Ralph A. Kelly, MD, Cardiovascular Division, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115.

	Abstract

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Abstract Unlike large-vessel endothelial cells in cell culture, cardiac microvascular endothelial cells (CMEC) isolated from adult rat ventricular muscle exhibit little detectable constitutive nitric oxide (NO) synthase activity after isolation in vitro but respond to specific combinations of inflammatory mediators with an increase in inducible NO synthase (iNOS; type 2 NO synthase) activity. CMEC iNOS is induced by soluble inflammatory mediators in lipopolysaccharide-activated rat alveolar macrophage–conditioned medium at 24 hours, and this induction can be partially prevented by either interleukin-1 (IL-1) receptor antagonist or a polyclonal anti–rat tumor necrosis factor- (TNF-) antiserum. Interferon- (IFN-), which by itself does not induce iNOS in CMEC, potentiates and accelerates iNOS induction by IL-1ß. Transforming growth factor-ß (TGF-ß) decreases iNOS activity, protein content, and mRNA abundance in IL-1ß– and IFN-–pretreated CMEC. To determine whether NO released by CMEC would affect myocyte contractile function in vitro, freshly isolated ARVM were allowed to settle onto confluent, serum-starved CMEC that had been pretreated for 24 hours with IL-1ß, a cytokine that alone does not affect myocyte contractile function in vitro. Baseline contractile amplitude, at 2 Hz and 37°C, of myocytes in heterotypic culture with IL-1ß–pretreated CMEC was not different from that of myocytes in control, homotypic myocyte cultures. However, cocultured myocytes exhibited decreased contractile responsiveness to 2 nmol/L isoproterenol compared with control cells, and this could be reversed by the addition of 1 mmol/L N^G-monomethyl-L-arginine, an inhibitor of NOS. Thus, activation of endothelial cell iNOS by specific cytokines affects the contractile responsiveness to isoproterenol of adjacent cardiac myocytes in vitro. Reciprocal cell-cell signaling leading to TGF-ß release and activation could act to limit the extent of endothelial cell iNOS induction in vivo.

Key Words: interleukin-1 • interferon- • transforming growth factor-ß • interleukin-1 receptor antagonist

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
There is increasing evidence from a number of laboratories that both the endocardial endothelium and coronary microvascular endothelium may contribute to the regulation of cardiac muscle contractile function by releasing cardioactive agents that act on subjacent or downstream cardiac myocytes, analogous to the regulation of vascular tone by endothelial cells.^{1 2} However, the use of endothelial cells derived from large conduit vessels or derived from the microvasculature of tissues other than cardiac muscle to study the regulation of release of cytokines or other locally acting autacoids that could affect cardiac myocyte function may be inappropriate. This is due to the marked phenotypic differences between endothelial cells derived from large vessels and from the microvasculature and to the phenotypic heterogeneity exhibited by microvascular endothelium from different tissues and organs.^{3 4 5 6} Within the heart, the repetitive changes in ventricular wall stress, microvascular blood flow, and mechanical stretch with cycles of diastolic filling and systolic contraction, as well as the unique tissue microenvironment of a highly vascular organ richly innervated by the autonomic nervous system, all may act to determine the phenotype of endothelial cells in the coronary microvasculature. In contrast to aortic endothelial cells, for example, confluent low-passage homotypic primary cultures of CMEC do not constitutively express mRNA for endothelin-precursor peptides, although preproendothelin transcripts can easily be detected in CMEC in coculture with cardiac myocytes.⁷

In a recent report from this laboratory, we determined that microvascular endothelial cells isolated from adult rat ventricular tissue demonstrate no significant levels of constitutive NO release in culture but exhibit a robust increase in NOS activity after a 24-hour exposure to soluble inflammatory mediators in medium conditioned by LPS-activated rat alveolar macrophages.⁸ In another report, we determined that the NOS isoform in these microvascular endothelial cells responsible for the NO release is identical to the iNOS (type 2 NOS) isoform originally cloned and sequenced from activated murine macrophages.⁹ This is in contrast to large-vessel endothelial cells in vitro (such as human umbilical vein or BAEC), in which the expression of iNOS, induced by LPS or by inflammatory cytokines, has not been documented.

In this report we examine the cytokines and cellular mechanisms responsible for iNOS expression in CMEC. Since increased NOS activity in cardiac myocytes can regulate contractile responsiveness to ß-adrenergic agonists,^{8 10} we also examined whether microvascular endothelial cells, after activation by specific inflammatory mediators that alone do not affect cardiac myocyte contractile function, could alter the inotropic response to isoproterenol of adjacent ventricular myocytes in heterotypic culture.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolation and Preparation of CMEC and ARVM
The methods for the isolation and characterization of CMEC from adult rat ventricular tissue have been described in detail elsewhere.¹¹ After isolation, dissociated cells were washed and resuspended in DMEM (GIBCO-BRL) with 20% FCS (GIBCO) and penicillin/streptomycin and plated on laminin-coated (1 µg/cm²; GIBCO) culture dishes at a density of 2500 cells/cm². The medium was removed, and the cells were washed once at 1 hour to remove loosely adherent cells. These primary isolates have been documented to contain >90% endothelial cells, with a phenotype at low passage number consistent with their microvascular origin as previously described.¹¹

Calcium-tolerant ARVM were isolated from adult male Sprague-Dawley rats (250 to 300 g) by the approach originally described by Claycomb and Palazzo¹² with modifications to minimize the number of contaminating nonmyocyte cells as previously described.¹³ Myocytes were cultured in a defined medium that is a modification of that originally described by Volz et al¹⁴ and consisted of medium 199 with Earle's balanced salts, including 25 mmol/L HEPES and NaHCO₃ (pH 7.4 at 37°C) without L-glutamine (Sigma), supplemented with 2 mg/mL BSA, 2 mmol/L L-carnitine, 5 mmol/L creatine, 5 mmol/L taurine, with 100 IU/mL penicillin and 100 µg/mL streptomycin (GIBCO-BRL) as described previously (ie, "ACCT" in Reference 15¹⁵ ), which is referred to as "defined medium" throughout the text.

Heterotypic ARVM-CMEC Primary Cultures
Short-term (3 to 7 hours) heterotypic primary culture of CMEC and adult rat cardiac myocytes was the coculture format used for the experiments reported in this article. Freshly isolated ARVM were plated directly on confluent, serum-starved low-passage CMEC primary cultures. Approximately 5000 myocytes were plated either directly on laminin-coated plastic coverslips (13 mm in diameter; Thermonox, Nunc, Inc) or seeded at the same density onto confluent, serum-starved homotypic CMEC cultures that had been plated on laminin-coated coverslips. Both control and heterotypic CMEC-myocyte cultures were routinely cultured in defined medium containing 0.1 µmol/L insulin and 0.1 nmol/L 3,5,3'-triiodothyronine unless otherwise stated. Two hours after myocyte plating, cultures were routinely washed to remove nonadherent cells.

Since previous studies from this laboratory have documented changes in myocyte morphology and function in heterotypic primary culture with CMEC,^{7 11} these parameters were examined under the experimental conditions used for assay of myocyte contractile function in this report. The percentage of cells retaining a rod-shaped morphology was examined by randomly selecting microscopic fields (at x100 magnification) in homotypic and heterotypic myocyte cultures at 3, 6, 18, and 24 hours. There was no significant difference in the percentage of rod-shaped cells at 3 and 6 hours after myocyte plating in homotypic and heterotypic culture. However, the percentage of rod-shaped cells declined significantly at 18 and 24 hours (to 33% and 15%, respectively, of the total number of cells examined) in heterotypic primary culture compared with 70% in myocyte monocultures at 24 hours. All myocyte contractility assays in heterotypic primary cultures were completed within 7 hours of plating in the experiments reported here.

After isolation and plating, coverslips containing homotypic and heterotypic myocyte-CMEC cultures were transferred to a superfusion chamber, and measurements of the amplitude and velocity of unloaded ventricular myocyte shortening and relengthening were made on the stage of an inverted phase-contrast microscope (Diavert; E. Leitz Inc) with an optical-video system with on-line digital storage and analysis of the optical signal, as previously described.¹³ Unless otherwise stated, cells were stimulated with a platinum electrode at 2.0 Hz (1 ms duration at 60 V) while being superfused with KHB buffer at 37°C at 0.8 mL/min with supplements as noted. The amplitude of myocyte shortening was examined 2 to 7 hours after plating during simulation at 2.0 Hz for at least 2 minutes before recording of baseline contractility. Myocytes chosen were rod-shaped, did not exhibit spontaneous contractions, and had a baseline contractile amplitude between 2 and 4 µm under basal conditions when driven at 2 Hz. After a baseline recording had been obtained for each cell, the superfusion buffer was changed to KHB buffer containing 2 nmol/L isoproterenol, approximately the EC₅₀ for the positive inotropic effect of this ß-adrenergic agonist in these isolated cells, and 1 mmol/L ascorbate. Cell motion was recorded in real time (3 to 5 minutes) until the increase in contractile amplitude had stabilized. Only one cell per coverslip in each experimental group was examined.

Isolation and Preparation of Rat Alveolar Macrophage-Conditioned Medium
Alveolar macrophages were obtained by tracheal lavage of sodium pentobarbital–anesthetized male Sprague-Dawley rats (250 to 275 g), as previously described.⁸ The first cell pellet after washes and perfusion was resuspended at a concentration of 0.5x10⁶ cells/mL in endotoxin-free DMEM containing 0.1% BSA with 100 IU/mL penicillin and 100 µg/mL streptomycin and was cultured at a density of 2.5x10⁶ cells per 60-mm culture dish in a 95% O₂/5% CO₂ atmosphere at 37°C. Each dish was washed three times in DMEM to remove nonadherent cells. Alveolar macrophages were then exposed to either endotoxin (the LPS component of Salmonella typhimurium [Sigma, lot 87F402]) at a concentration of 10 µg/mL or endotoxin-free DMEM for 24 hours. Macrophage-conditioned medium was harvested, centrifuged at 1500g for 10 minutes to remove cell debris, and then stored at -70°C for further use.

Measurement of NOS Activity
Conversion of [³H]L-Arginine to [³H]L-Citrulline
CMEC homogenates were prepared by suspending approximately 10⁶ cells in warm HBSS (without MgCl₂, CaCl₂, or MgSO₄; GIBCO-BRL) containing 0.25% trypsin and 1 mmol/L EDTA, centrifuging at 100g at 4°C, washing in ice-cold PBS, and resuspending the final pellet in 0.5 mL of lysis buffer containing 20 mmol/L Tris-HCl (pH 7.4 at 4°C), 0.5 mmol/L EDTA, 0.5 mmol/L EGTA, 1 mmol/L DTT, 1 µmol/L tetrahydrobiopterin (Dr B. Schircks Laboratories), 1 µmol/L leupeptin, and 0.2 mmol/L PMSF (Sigma). The cells were sonicated on ice for 30 seconds with a Branson Sonifier 450, three times for 10 seconds, and the cell homogenates were centrifuged at 1500g for 15 minutes at 4°C.

To determine NOS activity, 25 µL of total homogenate was incubated at 37°C for 2 hours in the presence of 50 mmol/L HEPES (pH 7.4 at 37°C), 1.25 mmol/L CaCl₂, 1 mmol/L EDTA, 0.5 mmol/L NADPH, 10 µmol/L FAD, 10 µmol/L FMN, 10 µmol/L tetrahydrobiopterin, 10 µg/mL calmodulin, and 0.3 nmol/L [³H]L-arginine (Amersham) for a final total volume of 150 µL. The reactions were stopped by the addition of 2 mL ice-cold 20 mmol/L HEPES (pH 5.5) and 2 mmol/L EDTA, and the total volume was applied to a Dowex-50W x8 column preequilibrated with 20 mmol/L HEPES (pH 5.5). [³H]L-citrulline was eluted with 2 mL of deionized water, and radioactivity was determined by scintillation counting. The protein content of the cell homogenate was determined by the Bradford technique with a BioRad kit. The data from this NOS activity assay are reported as counts per minute per milligram protein per hour.

Nitrite Release in CMEC-Conditioned Medium
The nitrite content of endothelial cell–conditioned medium was determined by standard techniques. Microvascular endothelial cells were cultured in 12-well tissue culture plates (Costar) in DMEM with 20% FCS until confluent and then for 24 hours in 400 µL of DMEM modified to use phenol red–free DMEM. After a 24-hour incubation, the medium was collected and centrifuged once at 1500g for 15 minutes at 4°C to remove cellular debris, and 150 µL of this supernatant was added to a 1:1 (vol/vol) mixture of Griess reagent (0.75% sulfanilamide [final concentration] in 0.5N HCl/0.075% naphthylethylenediamine; Sigma), and absorbance was determined at 543 nm spectrophotometrically. A standard curve was constructed by use of known concentrations of sodium nitrite.

Cell Respiration Assay
To verify endothelial cell viability after exposure to specific recombinant cytokines or to LPS-activated macrophage-con-ditioned medium, CMEC were plated in 12-well plates (Costar) in DMEM plus 20% FCS and allowed to reach confluency before transfer into defined medium with and without recombinant cytokines or other reagents or a 1:1 (vol/vol) dilution of LPS-activated macrophage-conditioned medium. After a 24-hour incubation, the medium was supplemented with 0.2 mg/mL MTT (Sigma). At successive time intervals, the culture medium was removed, and endothelial cells were solubilized in 1 mL of dimethylsulfoxide. The extent of reduction of MTT to formazan within cells, a measure of cellular respiration,¹⁶ was quantified by measurement of the ratio of absorbance at 550 and 630 nm. The results are expressed as a percentage of the ratio 550/630 of cells incubated for the same time duration in defined medium alone.

Western Blot of iNOS Protein
Endothelial cell extracts were prepared as described above, and total protein content was determined by the Bradford technique. Protein extracts were reconstituted in sample buffer containing 0.062 mol/L Tris-HCl, 2% SDS, 10% glycerol, and 5% (vol/vol) ß-mercaptoethanol, and the mixture was boiled for 5 minutes. Equal amounts (60 µg) of the denatured proteins were loaded per lane, separated on a 12% SDS polyacrylamide gel (Mini Protean II, BioRad), and transferred to a nitrocellulose membrane (HATF 20200, Millipore) in 25 mmol/L CAPSO buffer (Sigma; pH 10) with 20% methanol overnight at 4°C. The membrane was blocked with 1% BSA in TBST (Sigma). Membranes were incubated with rabbit polyclonal anti-mouse iNOS primary antibody that had undergone affinity purification on a synthetic peptide composed of a unique sequence (residues 1 through 20 of the N-terminus of the murine macrophage iNOS¹⁷ ) for 2 hours in TBST with 1% BSA. After three washes (10 minutes each), the membranes were incubated for 1 hour at room temperature with [¹²⁵I]-labeled goat anti-rabbit secondary antibody (NEN Dupont) in TBST with 1% BSA. The membranes were dried and autoradiographed by exposure to Kodak XAR film for 24 hours at -80°C.

Northern Blots of CMEC iNOS mRNA
Northern blot hybridizations were performed with the 217-bp cDNA probe representing a portion of the CMEC iNOS mRNA sequence identified by this laboratory by reverse transcriptase–polymerase chain reaction techniques on mRNA isolated from cytokine-pretreated CMEC, as reported elsewhere.⁹ Hybridizations were performed by electrophoresing 15 µg of total RNA, isolated by the method of Chomczynski and Sacchi,¹⁸ through a 1.5% formaldehyde-agarose gel and blotting onto a nylon membrane overnight by capillary transfer. cDNA probes were radiolabeled with [³²P]dCTP by random primer labeling. After 4 hours of prehybridization at 42°C, the blots were hybridized overnight at 42°C, then washed with 2x SSC/0.1% SDS (SSC is 0.15 mol/L NaCl and 0.015 mol/L sodium citrate) for 30 minutes at room temperature, followed by 1x SSC/0.1% SDS at 37°C and 0.2x SSC/0.1% SDS at 65°C. The blots were prepared for autoradiography at -70°C for at least 6 hours.

Note on Usage
Whenever a general statement is made regarding the effects of a cytokine, the standard abbreviation is used throughout the text (eg, "IFN-"). However, when the specific recombinant peptide reagent used to generate the data in this article is mentioned in the text, the prefix "r" and the species from which the sequence was derived (human [h] or murine [m]) are noted as well (eg, "rmIFN-").

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Role of IL-1 in the Induction of iNOS in CMEC by MacLPS(+) Medium
Since MacLPS(+) medium contains significant amounts of IL-1 as well as TNF- bioactivity,⁸ concentrations of rhIL-RA and dilutions of an anti-rat TNF- antiserum were determined that completely inhibited the IL-1 and TNF- bioactivity present in MacLPS(+) medium. As shown in Fig 1A, rhIL-1RA at 100 ng/mL diminished but did not abolish the induction of iNOS activity by MacLPS(+) medium, as judged by the accumulation of nitrite in the endothelial cell cultures after removal of the MacLPS(+) medium. The polyclonal anti-rat TNF antiserum had a similar effect on nitrite release by CMEC at dilutions that were sufficient (ie, 1:1000 dilution) to completely inhibit endogenous TNF- activity in MacLPS(+) medium in a cytokine-selective bioassay (data not shown). CMEC incubated in a 1:1 dilution of MacLPS(+) medium in defined medium for up to 48 hours showed no evidence of decreased viability, as judged by a cell respiration assay.

View larger version (46K): [in this window] [in a new window]   Figure 1. Effect of IL-1 receptor antagonist on iNOS induction in CMEC by MacLPS(+) medium. Confluent, serum-starved low-passage CMEC were incubated for 24 hours in a defined medium alone (control), or in a 1:1 dilution with MacLPS(+) medium alone, or with graded concentrations of rhIL-1 receptor antagonist (IL-1 RA). The medium was aspirated and nitrite content analyzed by the Griess reaction. The data shown are mean±SEM of duplicate samples from three independent experiments [*P<.05 vs nitrite accumulation in CMEC exposed to MacLPS(+) medium alone].

As expected from these data, CMEC preincubated for 24 hours in medium containing rhIL-1ß do exhibit induction of iNOS activity, as measured by accumulation of nitrite in myocyte-conditioned medium (Fig 2A and 2B) and by the conversion of [³H]L-arginine to [³H]L-citrulline in endothelial cell homogenates (data not shown). At 2 ng/mL of IL-1ß, the induction of iNOS activity was maximal at 18 hours of preincubation and sustained for up to 48 hours, as measured by the conversion of [³H]L-arginine to [³H]L-citrulline. Nitrite accumulation in the CMEC-conditioned medium did not increase above baseline until 18 hours and continued to increase at 48 hours of incubation in IL-1ß–containing medium (Fig 2B). Note that the absolute concentration of nitrite detected is a function of the extent of induction of iNOS activity in these primary cultures and could vary somewhat from preparation to preparation (Fig 2A and 2B, for example).

View larger version (22K): [in this window] [in a new window]   Figure 2. Effect of IL-1ß and IFN- on iNOS induction in CMEC. The rhIL-1ß concentration-effect relation (A) and time course of induction with 2 ng/mL of rhIL-1ß (B) are illustrated on iNOS induction in confluent, serum-starved CMEC primary cultures, as detected by the release and accumulation of nitrite into endothelial cell–conditioned medium. C, Addition of rmIFN- (up to 1000 U/mL [100 ng/mL]) potentiates both the extent and the rapidity of iNOS induction to 2 ng/mL rhIL-1ß, as detected by nitrite release into the medium. All data represent duplicate samples from three independent experiments, expressed as mean±SEM.

Addition of rmIFN- resulted in a potentiation of the extent and rapidity of induction of iNOS by IL-1ß, as detected by nitrite accumulation (note the difference in scale of Fig 2C compared with Fig 2B). The accumulation of nitrite in medium conditioned by IL-1ß/IFN-–treated CMEC could be completely inhibited by DAHP (Sigma), an inhibitor of GTP cyclohydrolase I, with an IC₅₀ between 0.1 and 0.5 mmol/L. This indicates the essential role of de novo synthesis of tetrahydrobiopterin in the activity of NOS in these cells (data not shown).

Effect of TGF-ß on CMEC iNOS Induction
We investigated the effects of TGF-ß, a peptide signaling factor known to be produced by cellular constituents of cardiac muscle in vivo and in vitro.^{7 19 20} TGF-ß has been shown in cardiac myocytes, as well as other tissues and cell types, to modulate NOS activity.^{21 22 23 24 25} rhTGF-ß2 decreases the iNOS response in these cells exposed to rhIL-1ß and rmIFN-, as assayed by nitrite accumulation, by 50%, with an EC₅₀ between 0.1 and 1 ng/mL (data not shown). As shown in Fig 3A, TGF-ß2 also reduced iNOS protein content, detected by Western analysis with a polyclonal antiserum directed against the murine iNOS protein.¹⁷ This was paralleled by a decrease in iNOS mRNA abundance at 24 hours after TGF-ß exposure when added to IL-1ß– and IFN-–containing medium, as shown in Fig 3B.

View larger version (27K): [in this window] [in a new window]   Figure 3. Autoradiographs showing TGF-ß attenuation of the induction of iNOS in CMEC. TGF-ß caused a decline at 24 hours after IL-1ß and IFN- both in iNOS protein levels (A), as detected by Western analysis with an antibody against the murine macrophage iNOS isoform, and in iNOS mRNA abundance (B), as detected by Northern analysis with the 217-bp iNOS cDNA probe isolated by reverse transcriptase–polymerase chain reaction techniques on mRNA from cytokine-treated CMEC.9 The Western and the Northern blot data with TGF-ß were repeated three times with similar results.

Myocyte Contractile Function in Short-term Heterotypic Cardiac Myocyte–CMEC Cultures: Effect of LPS and IL-1ß
To determine whether microvascular endothelial cells, after exposure to a specific antigenic or immunologic stimulus, could generate autacoids that would affect the function of adjacent cardiac myocytes, a coculture system was designed in which myocyte contractile function was used as a bioassay (see "Methods"). Low-passage CMEC homotypic primary cultures were grown to confluency on coverslips, then switched from serum-containing medium to defined medium for 24 hours. Freshly isolated ARVM were plated directly on these serum-starved CMEC cultures. The amplitude of shortening was studied in myocytes 3 to 7 hours after plating before and after superfusion with Krebs-Henseleit bicarbonate buffer containing 2 nmol/L isoproterenol. Myocytes attached to endothelial cells could be paced over a range of stimulation frequencies from 0.5 to 5 Hz with amplitudes of contraction at driving rates above 1 Hz that were stable but consistently 20% to 30% below that of myocytes in homotypic cultures (Fig 4). Nevertheless, cocultured myocytes exhibited a positive rate-staircase phenomenon apparent at 3 to 5 Hz, analogous to that previously observed in myocytes attached directly to laminin-coated plastic substrate.²⁶

View larger version (22K): [in this window] [in a new window]   Figure 4. Contractile function of ARVM in heterotypic coculture with cardiac microvascular endothelial cells. Freshly isolated ventricular myocytes were plated directly on confluent, serum-starved primary cultures of CMEC. The coverslips were transferred within 2 to 7 hours to a superfusion chamber for determination of myocyte contractile function and responsiveness to isoproterenol. The figure represents the contractile amplitude of control rat ventricular myocytes (open bars) and cocultured myocytes (stippled bars) over a range of frequencies from 0.5 to 5 Hz, normalized to the response at 0.5 Hz, demonstrating that myocytes cocultured with endothelial cells exhibited a characteristic positive rate staircase (ie, at 3 Hz; *P<.05 and **P<.01 vs amplitude ratio at 1 Hz).

The baseline contractile amplitude of myocytes paced at 2 Hz was not different in myocytes plated on CMEC that had been pretreated for 24 hours with 10 µg/mL LPS compared with myocytes plated on CMEC exposed only to control medium (2.3±0.2 versus 2.2±0.2 µm, respectively, mean±SEM; n=11 cells in both groups). When myocytes were then superfused with 2 nmol/L isoproterenol, there was again no significant difference in the increased amplitude of contraction in myocytes plated on LPS-pretreated endothelial cells compared with myocytes plated on microvascular endothelial cells exposed to control medium (2.5±0.4 versus 2.3±0.2, mean±SEM, expressed as the ratio of the amplitude of contraction after isoproterenol divided by the amplitude of contraction at baseline; n=11 cells in both groups).

IL-1ß alone does not have any effect on baseline or on isoproterenol-stimulated contractile function in cardiac myocytes after a 24-hour exposure to the cytokine.²⁷ To determine whether IL-1ß could alter the content or function of myocytes plated on cytokine-pretreated CMEC cultures, microvascular endothelial cells were exposed to defined medium containing 2 ng/mL rhIL-1ß. After 24 hours, myocytes were added and allowed to attach for 2 hours, and baseline contractility was recorded. There was no difference in the baseline contractile amplitude between control and IL-1ß–pretreated myocytes (data not shown) or between myocytes plated on IL-1ß–pretreated CMEC compared with myocytes plated on control endothelial cell cultures (Fig 5A). However, cardiac myocytes plated on IL-1ß–pretreated endothelial cells had a significantly reduced inotropic response to isoproterenol compared with myocytes plated on control endothelial cell cultures (Fig 5B).

View larger version (54K): [in this window] [in a new window]   Figure 5. iNOS induction in microvascular endothelial cells decreases contractile responsiveness of ARVM in heterotypic coculture. Primary isolates of ARVM were plated on confluent, serum-starved primary microvascular endothelial cell cultures that had been pretreated for 24 hours with rhIL-1ß (2 ng/mL) or control, defined medium alone. After a minimum of 2 hours (range, 2 to 7 hours), myocytes were stimulated at 2 Hz at 37°C, and their amplitude of contraction was recorded at baseline (A) and in response to 2 nmol/L isoproterenol (B; expressed as the ratio of the increase in amplitude of contraction with isoproterenol compared with the basal amplitude). Myocytes cocultured with IL-1ß–pretreated CMEC (black bars) but not with CMEC exposed to control medium alone (stippled bars) exhibited a decreased contractile responsiveness to isoproterenol. This could be reversed by adding 1 mmol/L L-NMMA to the incubation and superfusion medium (diagonal bars) (*P<.01 vs myocytes in control cocultures; mean±SEM). Contractility assays were performed on 8 to 18 cells per experimental condition to obtain each data point.

To determine whether NO was responsible for the decreased contractile responsiveness to isoproterenol in ARVM plated on IL-1ß–pretreated endothelial cells, 1 mmol/L L-NMMA was added to the CMEC incubation medium at the time of addition of rhIL-1ß. There was no effect of L-NMMA on baseline contractile amplitude of cocultured myocytes from control or IL-1ß–treated groups (Fig 5A). However, L-NMMA restored the responsiveness to isoproterenol of myocytes plated on IL-1ß–pretreated endothelial cells (Fig 5B).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The observations of Brutsaert and Andries² that the endocardial endothelium modifies the contractile function of subjacent myocardium, presumably by release of locally acting autacoids, has led a number of laboratories to investigate the role of both the endocardial endothelium and coronary microvascular endothelium in regulating heart muscle function. Relatively selective removal of the coronary vascular endothelium by intracoronary infusion of detergent in intact hearts resulted in changes in papillary muscle twitch duration and maximal twitch tension from those hearts that were qualitatively similar to those observed by removal of the endocardial endothelium.²⁸ Evidence has also been presented by Ramaciotti et al^{29 30} and McClellan et al³¹ that endocardial endothelium and coronary microvascular endothelium release both positively inotropic and negatively inotropic autacoids, possibly in response to changes in arteriolar oxygen tension and coronary flow rate, respectively.

In heterotypic primary cultures of cardiac myocytes and microvascular endothelial cells separately isolated from adult rat ventricular muscle, we have documented reciprocal cell-cell signaling between these two cell types.⁷ This includes the release of endothelial cell mitogens by cardiac myocytes¹¹ and the induction, synthesis, and activation of TGF-ß by microvascular endothelial cells in coculture, which then acts as an autocrine secretagogue to induce the expression of precursors of endothelin, a peptide with well-described inotropic and hypertrophic effects on cardiac myocytes.⁷

Both endocardial endothelial cells and CMEC also express iNOS (ie, type II NOS) activity, in contrast to most large-vessel endothelial cells, in which detection of iNOS mRNA or enzymatic activity in response to inflammatory cytokines remains controversial. We and others have shown that iNOS is also induced in cardiac myocytes in response to specific cytokines^{8 21} and that NO plays an obligatory intermediate role in diminishing the inotropic response of cardiac myocytes to ß-adrenergic agonists. In response to the species-specific combination of soluble inflammatory mediators contained in LPS-activated rat alveolar macrophage–conditioned medium, both ARVM⁸ and CMEC (Fig 1) markedly increase iNOS activity at 24 hours. Both IL-1RA and an anti–rat TNF- antiserum were effective in reducing nitrite release by CMEC exposed to MacLPS(+) medium, indicating that IL-1ß and TNF- activities in the activated macrophage-conditioned medium were contributing to iNOS activation in these cells. This is in contrast to the myocyte response to these two cytokine antagonists, since only IL-1RA and not anti–TNF- antiserum was effective in blocking iNOS induction in response to MacLPS(+) medium.²⁷ Although IL-1ß induces iNOS activity in both cell types in vitro and the combination of IL-1ß and IFN- results in a synergistic induction of iNOS in CMEC (Fig 2C), these microvascular endothelial cells do not express iNOS in response to IFN- alone, in contrast to cardiac myocytes and confluent aortic smooth muscle cells in vitro.⁹ The molecular signaling mechanisms underlying the differential regulation of iNOS induction by specific cytokines in these cellular components of ventricular muscle are currently under study.

The inhibition of iNOS induction in CMEC with inflammatory cytokines by TGF-ß has also been observed in activated macrophages²² and in neonatal²³ and adult rat ventricular myocytes.²¹ TGF-ß₂ was the isoform used for the experimental protocols described in this article, on the basis of previous evidence from this laboratory that this isoform was expressed by CMEC in heterotypic primary culture with cardiac myocytes.⁷ In IFN-–treated murine peritoneal macrophages, Vodovotz et al²² demonstrated that TGF-ß, when given concurrently with IFN-, did not affect iNOS transcription but did increase the rate of iNOS mRNA degradation and iNOS protein degradation. We cannot comment on whether the site of TGF-ß regulation of iNOS induction by IL-1ß and IFN- was transcriptional or posttranscriptional, since our data are consistent with either mechanism. The decline in iNOS activity and protein content in TGF-ß–treated cells did parallel the decline in iNOS mRNA abundance, suggesting that the predominant effect was either transcriptional or on iNOS mRNA stability.

IL-1 and Myocyte Contractile Responsiveness to Isoproterenol in CMEC–Cardiac Myocyte Primary Cultures
The purpose of the short-term CMEC–cardiac myocyte coculture experiments was to determine whether these microvascular endothelial cells could transduce and/or amplify inflammatory signals, leading to functional changes in adjacent cardiac myocytes. The experimental model was to allow a suspension of freshly isolated ARVM to settle directly on established, confluent, serum-starved homotypic CMEC primary cultures that had been pretreated for 24 hours with soluble inflammatory mediators. The myocytes remained on the endothelial cell layer for 2 to 3 hours, sufficient time to allow most cells to become stably attached but not for significant iNOS induction to occur in the myocytes before initiation of contractility studies. Myocytes plated on both control and cytokine-pretreated CMEC demonstrated a reduced baseline amplitude of contraction compared with myocytes plated on laminin, presumably due to altered integrin-mediated cell attachment. Nevertheless, myocytes under these experimental conditions exhibited an expected positive force-frequency relation (Fig 4) and an inotropic response to isoproterenol in control cocultured cells that was comparable to that observed in myocytes in monoculture. It should be emphasized that all the observations reported here were made on cells that undergo shortening against an internal load, in contrast to contraction against an external load that more closely represents conditions in vivo. However, Kent et al³² documented that the rate and extent of shortening of isolated adult feline ventricular myocytes is analogous to the force-velocity relation of isolated cardiac muscle preparations. Nevertheless, the isolated myocyte preparation can serve only as an approximation of cardiac muscle cell function in situ.

Brady et al³³ used a similar short-term myocyte coculture protocol to study the effects of NO on contractile function of isolated guinea pig ventricular myocytes that had been plated for several minutes on confluent BAEC cultures. They observed a small decline in baseline contractile amplitude in myocytes plated on BAEC exposed to bradykinin that was prevented by an L-arginine analogue NOS antagonist. This was presumably due to activation of a constitutive (cNOS) activity by bradykinin in BAEC. It remains unclear whether the cellular mechanism responsible for decreased contractile responsiveness to isoproterenol differs whether activated by NO from these large-vessel endothelial cells or generated by an endogenous cNOS within cardiac myocytes themselves¹⁰ compared with the potentially much higher levels of NO that would be expected to accumulate after induction of either a cardiac myocyte or microvascular endothelial cell iNOS.

Neither IL-1ß nor LPS alone had any effect on baseline or isoproterenol-stimulated myocyte contractile amplitude in homotypic control cardiac myocyte primary cultures. IL-1ß does induce iNOS activity in cardiac myocytes,²⁷ but only after 12 to 24 hours. In contrast, myocytes plated on IL-1ß–pretreated endothelial cells exhibited an L-NMMA–reversible decline in isoproterenol-stimulated contractile amplitude but not in baseline contractile function. This is similar to what we have observed in adult cardiac myocytes exposed for 24 hours to a mixture of inflammatory mediators contained in LPS-activated rat alveolar macrophage–conditioned media⁸ or to a combination of inflammatory cytokines, including rhIL-1ß and rmIFN-.²⁷ Although it is possible that IL-1ß could have altered integrin expression in microvascular endothelial cells that might have affected myocyte attachment and loading during shortening, this is unlikely to have been a dominant effect, since baseline shortening was unaffected and L-NMMA rapidly restored myocyte contractile responsiveness to isoproterenol.

It is likely that release of NO or related bioactive congeners from CMEC adjacent to cardiac myocytes in situ in cardiac muscle, as well as other cytokines and autacoids released by activated endothelial cells, will affect the function and potentially modify the phenotype of cardiac muscle cells in certain pathophysiological conditions. Malinski et al,^{34 35} using a porphyrin/Nafion–coated NO-selective microsensor technology, have verified that NO concentrations as high as 10 nmol/L can be detected in vascular smooth muscle in situ in rabbit aortic ring preparations that are 100 µm from intimal endothelium within 7 seconds after administration of bradykinin. NO, or biologically active adducts,^{36 37} may diffuse up to 200 to 400 µm in some tissues.³⁸ In the heart, in which the ratio of microvessels to myofibrils is approximately 1:1 and the distance between capillaries, even in hypertrophied cardiac muscle, is <40 to 50 µm,³⁹ physiologically significant concentrations of NO derived from increased iNOS activity in the microvascular endothelium would be expected to accumulate in close proximity to adjacent cardiac myocytes. As we have demonstrated in longer-term heterotypic primary cultures of CMEC and cardiac myocytes in serum-containing medium,⁷ it is likely that increased expression and activation of TGF-ß, as well as other locally acting cytokines and autacoids, will act to modify the extent of iNOS activation in vivo as well.

In summary, specific inflammatory cytokines, including TNF-, IL-1ß, and IFN-, induce iNOS activation within cellular constituents of cardiac muscle, including microvascular endothelial cells. Although increased iNOS activity in response to viral or bacterial infection may be beneficial, inappropriate or excessive activation of this enzyme could contribute to regional or to global myocardial dysfunction, such as that observed in the systemic inflammatory response syndrome.⁴⁰

	Selected Abbreviations and Acronyms

 

ARVM = adult rat ventricular myocytes BAEC = bovine aortic endothelial cells CMEC = cardiac microvascular endothelial cells cNOS = constitutive nitric oxide synthase FCS = fetal calf serum IFN- = interferon- IL-1 = interleukin-1 iNOS = inducible nitric oxide synthase KHB = Krebs-Henseleit bicarbonate L-NMMA = NG-monomethyl-L-arginine LPS = lipopolysaccharide MacLPS(+) medium = LPS-activated rat alveolar macrophage–conditioned medium NOS = nitric oxide synthase RA = receptor antagonist TBST = Tris-buffered saline with 0.05% (vol/vol) Tween 20 TGF-ß = transforming growth factor-ß TNF- = tumor necrosis factor-

	Acknowledgments

 
This work was supported by grants R37-HL-36141 (to Dr Smith) and R29-HL-46457 (to Dr Michel) from the National Institutes of Health. Dr Michel is the recipient of an Established Investigator Award from the American Heart Association. Drs Balligand and Ungureanu-Longrois were supported by Fellowship Awards from the Massachusetts Affiliate of the American Heart Association. Dr Simmons is supported by a fellowship award from the Medical Research Council of Canada. Dr Okada was the recipient of a fellowship award from the Japanese Heart Foundation. rhTGF-ß₂ was a gift of Celtrix Pharmaceuticals, Santa Clara, Calif, and rhIL-1RA was a gift of Synergen, Boulder, Colo.

	Footnotes

 
This manuscript was sent to Dr Leslie A. Leinwand, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received March 14, 1994; accepted April 20, 1995.

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