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(Circulation. 1997;96:3436-3442.)
© 1997 American Heart Association, Inc.

Articles

Left Ventricular Contractile Effects of Inducible Nitric Oxide Synthase in the Human Allograft

Walter J. Paulus, MD, PhD; Stefanie Kästner, BS; Pénélope Pujadas, MD; Ajay M. Shah, MD, MRCP; Helmut Drexler, MD; ; Marc Vanderheyden, MD

From the Cardiovascular Center, OLV Ziekenhuis, Aalst, Belgium (W.J.P., P.P., M.V.); the Department of Cardiology, University of Wales College of Medicine, Cardiff, UK (A.M.S); and Abteilung Kardiologie, Medizinische Hochschule, Hannover, Germany (S.K., H.D.).

Correspondence to Dr Walter J. Paulus, MD, PhD, Cardiovascular Center, OLV Ziekenhuis, Moorselbaan 164, B 9300 Aalst, Belgium. E-mail Walter.Paulus{at}ping.be

	Abstract

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Background Myocardial expression of inducible (i) nitric oxide (NO) synthase (iNOS) gene has been reported in transplant recipients and in dilated cardiomyopathy. NO derived from NO donor or from coronary endothelium has previously been shown in the human heart to reduce end-systolic left ventricular (LV) pressure, especially during ß-adrenoreceptor stimulation, because of earlier onset of LV relaxation. The present study investigated in transplant recipients whether a similar cardiodepressant effect could be attributed to NO derived from iNOS.

Methods and Results In 16 transplant recipients who were free of rejection or graft vasculopathy, microtip LV pressure recordings, LV angiograms, and endomyocardial biopsies were obtained at annual coronary angiography. In 8 transplant recipients, microtip LV pressure recordings were obtained during intravenous dobutamine(5 µg · kg^-1 · min^-1). Competitive reverse transcription–polymerase chain reaction of iNOS mRNA was performed on the endomyocardial biopsies, and the intensity of iNOS mRNA expression was quantified on a scale ranging from 0 to 5+. All measures of baseline LV function were comparable in transplant recipients with low (2+) or high myocardial iNOS mRNA. During intravenous dobutamine infusion, there was a significant correlation between the abbreviation of LV electromechanical systole time (LVEST is the time from onset of QRS to dP/dt_min) and the rise of LV dP/dt_max (r=.79; P<.02). By use of a multiple regression analysis, addition of the intensity of iNOS mRNA expression as an independent variable significantly (P<.005) improved the correlation between LVEST and LV dP/dt_max (P<.001; r=.97), implying a larger abbreviation of LV contraction for a similar rise in LV dP/dt_max, when myocardial iNOS mRNA was higher. The larger abbreviation of LV contraction in-patients with high iNOS mRNA was associated with a decrease in LV end-systolic pressure (-31±16 mm Hg).

Conclusions Myocardial iNOS gene expression in the human allograft influences the LV contractile response to ß-adrenergic stimulation through earlier onset of LV relaxation and reduction of LV end-systolic pressure. These effects are similar to the LV contractile effects of NO derived from NO donor or from coronary endothelium.

Key Words: transplantation • receptors, adrenergic, beta • endothelium-derived factors • myocardial contraction

	Introduction

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In the human heart, the LV contractile effects of myocardial iNOS gene expression remain unclear.¹ Expression of iNOS gene has first been reported in ventricular myocardium of patients with dilated cardiomyopathy after myocarditis,² subsequently in transplant recipients at the time of surveillance endomyocardial biopsy³ or during episodes of clinically and biopsy-proven rejection,⁴ and finally in myocardium of failing human hearts regardless of the cause.⁵ In transplant recipients, with the use of Doppler echocardiographic measures of LV performance, the presence of iNOS mRNA was associated with systolic, diastolic, or combined LV dysfunction.³ In patients with dilated cardiomyopathy, the functional significance for myocardial contractile performance of iNOS gene expression was investigated in isolated muscle strips obtained from the explanted hearts at the time of cardiac transplantation.⁶ In this study, iNOS gene expression induced an abbreviation of the twitch contraction of the isolated muscle strip. This abbreviation significantly reduced peak active tension after ß-adrenergic stimulation. Similar contractile effects had previously been observed in normal control subjects, in transplant recipients, and in dilated cardiomyopathy patients after intracoronary infusion of NO donor substances or of substance P, which releases NO from the coronary endothelium.^7-9 These studies also documented an NO-induced abbreviation of LV electromechanical systole, which was accompanied by a slight reduction in peak and end-systolic LV pressures. In cardiac allografts and in cardiomyopathic hearts, this NO-induced abbreviation of LV contraction was more pronounced after pretreatment with intravenous dobutamine and resulted in considerable decreases in LV peak and end-systolic pressures.⁹

Because of the absence of invasive data on the in vivo functional significance for the human heart of myocardial iNOS gene expression, high-fidelity tip-micromanometer LV pressure recordings and simultaneous LV angiograms were obtained in transplant recipients at the time of annual coronary angiography, and measures of LV function were correlated with the intensity of myocardial iNOS gene expression in simultaneously procured endomyocardial biopsies. Moreover, because of the aforementioned interaction between NO and ß-agonists on LV function, hemodynamic data were also obtained after intravenous infusion of dobutamine. Finally, in patients with low iNOS gene expression, LV hemodynamics were recorded during combined intravenous infusion of dobutamine and intracoronary infusion of substance P, which releases NO from the coronary endothelium, and these data were compared with LV hemodynamics observed in patients with high iNOS gene expression during a single intravenous infusion of dobutamine.

	Methods

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Patients
Sixteen transplant recipients were studied at the time of annual coronary angiography and cardiac catheterization after orthotopic heart transplantation. The study group consisted of 2 women and 14 men (mean age, 55 years; range, 26 to 65 years). Two patients were studied 1 year after heart transplantation; 3 patients, 2 years after heart transplantation; 5 patients, 3 years after heart transplantation; 3 patients, 4 years after heart transplantation; 1 patient, 5 years after heart transplantation and 1 patient, 2 and 3 years after heart transplantation. All patients were on immunosuppressive therapy, which consisted of various combinations of cyclosporine, prednisone, and azathioprine. For ethical reasons, immunosuppressive and antihypertensive therapies, [ACE inhibitors (n=3), and calcium channel blockers (n=3)] were maintained at the time of study. Coronary angiography, which preceded the study protocol, revealed angiographically normal coronary arteries without evidence of accelerated graft atherosclerosis.¹⁰ No patient had biopsy evidence requiring adjustment of immunosuppressive therapy at the time of study. Informed consent was obtained from all patients, the study protocol was approved by the local ethical committee, and there were no complications related to procedure or study protocol.

Study Protocol
As described previously,¹¹ left and right heart catheterization was performed from the right femoral artery and vein with a high-fidelity tip-micromanometer catheter to measure LV pressure. The high-fidelity-tip micromanometer catheter was calibrated externally against a mercury reference and matched against luminal pressure. Fast-paper-speed recordings (250 mm/s), covering several respiratory cycles of LV pressure and LV dP/dt were obtained on a Gould ES 1000 multichannel recorder. In 8 transplant recipients (Table 1, patients 4, 8, 9, and 12 through 16), recordings were obtained at baseline and during intravenous infusion of dobutamine, which was progressively titrated upward to a dose of 5 µg · kg^-1 · min^-1 and then maintained for a 5-minute period, at the end of which additional fast-paper-speed recordings were obtained. Subsequently, while the intravenous dobutamine infusion was maintained, in 4 patients (Table 1, patients 4, 8, 9, and 12) a 5-minute intracoronary infusion of substance P (20 pmol/min)^8,9 was performed by use of a 4F left coronary catheter inserted from the left femoral artery and positioned in the left coronary ostium. In these patients, additional fast-paper-speed recordings were obtained at 1-minute intervals during the intracoronary substance P infusion. LV angiography was performed under baseline conditions in all patients. Right ventricular endomyocardial biopsies were obtained at the end of the study in all patients. Additional biopsy samples were immediately frozen in liquid nitrogen and stored at -80°C for subsequent detection of iNOS mRNA by reverse transcription–PCR.

View this table: [in this window] [in a new window]   Table 1. LV Function at Rest and Myocardial iNOS Gene Expression in the Human Allograft

Competitive Reverse Transcription–PCR for Quantification of iNOS mRNA
Quantification of iNOS mRNA was performed by reverse transcription followed by PCR^6,12 in the presence of a defined concentration of a shortened iNOS competitor RNA that served as an internal standard as previously described for human ACE and chymase.¹³

Reverse Transcription
For first-strand cDNA synthesis, equal amounts of total RNA (2 µg) were mixed with increasing quantities of iNOS competitor RNA(12.5 to 6.25x10⁴ molecules) in 1x reverse transcription buffer (50 mmol/L Tris-HCl, pH 8.3, 75 mmol/L KCl, 3 mmol/L MgCl₂) completed with 0.5 mmol/L dNTPs (Pharmacia Biosystems Ltd) and 250 pmol of random hexanucleotide primers. These mixtures were heated to 72°C for 3 minutes. Then, dithiothreitol (10 mmol/L), RNase inhibitor (2 U/100 ng total RNA, Amersham Buchler, Ltd), and Moloney murine leukemia virus reverse transcriptase (10 U/100 ng total RNA, Life Technologies Ltd) were added to the reverse transcription reaction to a total volume of 25 µL and incubated at 42°C for 60 minutes, followed by a denaturation at 95°C for 5 minutes.

PCR Amplification
Duplicate samples of PCR were performed in a total volume of 50 µL, respectively, each containing 10 µL of reverse transcription reaction, 35 µL of a PCR master mix (16 mmol/L Tris-HCl, pH 8.3, 40 mmol/L KCl, 0.4 mmol/L MgCl₂, 20 pmol of sense and antisense primer), and 2.5 U of TAQ-DNA polymerase (Pharmacia Biosystems, Ltd). The mixture was overlaid with mineral oil (Sigma Ltd) and then subjected to 36 cycles of PCR amplification by use of a DNA thermal cycler (Perkin Elmer Ltd). The cycle profile included denaturation for 1 minute at 94°C, annealing for 2 minutes at 62°C, and extension for 3 minutes at 72°C. As a negative control, no amplification product occurred if reverse transcriptase or total RNA was omitted in the first-strand cDNA reaction. The PCR products of iNOS mRNA were found to be of the expected size as shown by gel electrophoresis. In addition, the specificity of the amplified sequences was confirmed by restriction enzyme analysis and by hybridization with specific internal oligonucleotide probes.

Quantitative Analysis
The amplification products of 10 µL of each PCR reaction were separated by electrophoresis on a 1.5% agarose gel, stained with ethidium bromide, visualized by UV irradiation, and photographed (Polaroid 665 negative film, Polaroid Ltd). The negative film was used to evaluate the band densities by use of a laser densitometer. To account for differences in molecular weight between target and competitor DNA, the ratio of target to competitor DNA was calculated. Consequently, to correct for less incorporation of ethidium bromide, the band densities of the iNOS competitor were multiplied by 1.33 (419 bp:314 bp). The mean value of duplicate samples (variation between duplicate samples <5%) was plotted as logarithm of the ratio of competitor to gene target PCR products versus the logarithm of the known number of competitor molecules (r>.94, P<.0005). At the competition equivalence point (log ratio=0), the original number of target mRNAs corresponds to the initial number of competitor RNA molecules used. In control experiments, the optimal amount of total RNA and PCR cycle profile was determined.

Selection and Synthesis of the PCR Primers
Appropriate sense and antisense primer oligonucleotides were selected from the human cDNA sequences of iNOS (sense primer: 1614 to 1633, 5'-GGGAGCATCACCCCCGTGTT-3'; antisense primer: 2012 to 2033, 5'-GAGCGATTTCTTCAGTTTCTCT-3') by computer analysis with the Oligo program (National Biosciences Inc). To ensure that no genomic DNA contamination was present in the RNA solution, the chosen primer oligonucleotides spanned splice junctions.¹⁴

Construction and In Vivo Transcription of the Competitor Templates
For construction of internal standard competitor RNAs, shortened fragments of the human cDNAs of iNOS were made and transcribed into RNA. The iNOS competitor template was obtained from a 419-bp cDNA fragment that had been amplified with the sense and the antisense primer as described above with the human iNOS cDNA¹⁴ used as template in the PCR reaction. The amplified cDNA fragment was phosphorylated, blunted, and ligated into a blunt-ended, dephosphorylated Bluescript SK(+) vector (Stratagene Ltd). For in vitro transcription, the shortened iNOS cDNA clone was first linearized with the restriction enzyme Xba I. Then, 1 µg of the digested cDNA template was transcribed into RNA by use of a T3-/T7-RNA polymerase in vitro transcription kit according to the supplier's recommendation (Stratagene Ltd). Subsequently, the DNA template was removed by addition of 1 U of Rnase-free Dnase (Life Technologies Ltd) and incubation for 30 minutes at 37°C. The competitor RNA template was purified by phenol extraction, precipitated, and quantified by absorption at 260 nm and stored at -20°C until use. No remaining DNA was detectable when each competitor RNA was subjected to RNA-PCR omitting reverse transcriptase in the first-strand cDNA reaction.

Data Analysis
LV end-diastolic volume and ejection fraction were derived from single-plane right anterior oblique LV angiograms with the area-length method and a regression equation.¹⁵ The time constant of LV pressure decay (Table 1) was calculated from the digitized pressure data points of isovolumic LV relaxation with the use of an exponential curve fit with zero asymptote.¹⁶ The duration of LVEST (Table 1), which indicates the time to onset of LV relaxation, was measured as the interval from the Q wave on the ECG to the moment of LV dP/dt_min. Intensity of iNOS mRNA expression was converted to a scale ranging from 0 to 5+.

All data are expressed as mean±SD. Comparison of LV hemodynamics in low (2+) and high iNOS gene expression patients (Table 2) was performed using Student's t test. Statistical significance was set at a two-tailed level of P<.05. Multiple regression analysis was performed by use of SAS software (SAS Institute).

View this table: [in this window] [in a new window]   Table 2. LV Function at Rest in the Human Allograft: Low Versus High iNOS Gene Expression

	Results

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iNOS Gene Expression and LV Function at Rest
Table 1 summarizes hemodynamic data and intensity of iNOS mRNA expression observed at rest in 16 transplant recipients. There was no significant correlation between any hemodynamic measure of LV function at rest and myocardial iNOS gene expression. When patients with low (2+) myocardial iNOS gene expression were compared with patients with high (>2+) myocardial iNOS gene expression, every measure of LV function at rest yielded comparable values, as evident from Table 2.

iNOS Gene Expression and LV Function During Dobutamine Infusion
Intravenous infusion of dobutamine (n=8; Table 1, patients 4, 8, 9, and 12 through 16) caused significant increases in heart rate (25 ± 10 bpm) and in LV dP/dt_max (720±280 mm Hg/s) and significant decreases in LV minimum diastolic pressure (-4±4 mm Hg), in LVEDP (-15±6 mm Hg), in LVEST (-84±33 ms), and in the time constant of LV relaxation (-9±4 ms). For the group as a whole, there was no significant change in LV peak systolic pressure (-9±22 mm Hg) or in LVESP (-11±18 mm Hg).

Fig 1 shows representative examples of LV pressure recordings obtained under baseline conditions and during intravenous infusion of dobutamine in a patient with high iNOS gene expression (Table 1, patient 16) and in a patient with low iNOS gene expression (Table 1, patient 14). During intravenous dobutamine, despite a larger increase in LV dP/dt_max, the patient with low iNOS gene expression had less abbreviation of LV contraction and higher LVESP. Fig. 2 shows the reverse transcription–PCR amplification product of iNOS mRNA derived from endomyocardial biopsies obtained in 6 patients, including the 2 patients whose LV pressure recordings are shown in Fig 1. The patient with high iNOS gene expression (Fig 1, left) is shown in lane 4 of Fig 2, and the patient with low iNOS gene expression (Fig 1, right) is shown in lane 5 of Fig 2.

View larger version (57K): [in this window] [in a new window]   Figure 1. Microtip LV pressure (LVP) recordings obtained under baseline conditions and during intravenous infusion of dobutamine in a patient with high iNOS gene expression (left) and in a patient with low iNOS gene expression (right). In the patient with high iNOS gene expression, intravenous dobutamine resulted in lower LVESP and shorter LV contraction (interval from Q wave to LV dP/dtmin) despite the smaller increase in LV dP/dtmax.

View larger version (53K): [in this window] [in a new window]   Figure 2. Reverse transcription–PCR amplification products of iNOS mRNA derived from endomyocardial biopsies obtained in 6 patients, including the patients whose LV pressure recordings are shown in Fig 1 (lane 4, left panels of Fig 1; lane 5, right panels of Fig 1).

During intravenous dobutamine infusion, there was a significant correlation between LVEST and LV dP/dt_max (r=.79; P<.02). Using a multiple regression analysis, addition of iNOS mRNA as an independent variable significantly (P<.005) improved the correlation between LVEST and LV dP/dt_max (r=.97; P<.001). When the data are expressed as a ratio of LVEST divided by LV dP/dt_max, a significant correlation (r= 0.89; P<.005) was observed between this ratio and iNOS mRNA. This last correlation, which is shown in Fig 3, is also consistent with a larger dobutamine-induced abbreviation of LV contraction for a similar dobutamine-induced rise in LV dP/dt_max when myocardial iNOS gene expression is higher.

View larger version (25K): [in this window] [in a new window]   Figure 3. Linear regression analysis between the ratio of the dobutamine-induced decrease in LVEST divided by the dobutamine-induced increase in LV dP/dtmax and the intensity of myocardial iNOS mRNA expression quantified on a scale ranging from 0 to 5+. A larger dobutamine-induced abbreviation of LV contraction for a similar dobutamine-induced increase in LV dP/dtmax is observed at higher myocardial iNOS mRNA.

Intravenous dobutamine infusion was associated in patients with high myocardial iNOS gene expression with a larger abbreviation of LVEST (low -66±30 ms versus high, -113±8 ms; P<.05) and a fall in LVESP (low, -2±6 mm Hg versus high, -31±16 mm Hg; P<.01), despite a comparable increase in LV dP/dt_max (low, 680±271 mm Hg/s versus high, 787±160 mm Hg/s; P=NS). In 4 patients with low iNOS gene expression (Table 1, patients 4, 8, 9, and 12), substance P, which releases NO from the coronary endothelium, was infused at a dose of 20 pmol/min IC during the intravenous infusion of dobutamine. This induced a further abbreviation of LVEST from -66±30 to -98±32 ms (P<.05) and a decrease in LVESP from -2±6 mm Hg to -34±8 mm Hg (P<.05). These values of LVEST and LVESP during combined intravenous dobutamine and intracoronary substance P in patients with low iNOS gene expression were comparable to the values observed in patients with high iNOS gene expression during intravenous dobutamine.

	Discussion

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Myocardial Contractile Effects of iNOS: Experimental Evidence
Following the demonstration of calcium-independent NO production in cardiomyocytes isolated from rats treated with endotoxin,¹⁷ evidence has accumulated over the last couple of years that myocardial activation of iNOS could contribute to the cardiodepression frequently observed in immune-mediated conditions.^1,18-24 The mechanism underlying the NO-induced myocardial contractile depression, however, remains unresolved. NO-induced myocardial contractile depression has been variably explained by cGMP-mediated desensitization of cardiac myofilaments,²⁵ by cGMP-mediated stimulation of phosphodiesterase with a consequent decrease in cAMP,²² by a reduction of the calcium transient²³ or of the c-AMP–stimulated sarcolemmal calcium influx,²⁶ or by direct actions of NO on excitation-contraction coupling²⁷ or on energetic pathways.²⁸

The functional significance of iNOS gene expression for global LV performance was recently assessed in myosin-immunized rats.²⁹ In this model, the selective iNOS inhibitor aminoguanidine exerted a favorable hemodynamic effect, which could have resulted from a direct inhibition of contractile effects of NO or from prevention of peroxynitrite production, which inhibits sarcoplasmic reticulum Ca²⁺-ATPase through formation of hydroxyl radicals³⁰ and induces irreversible suppression of mitochondrial respiration.³¹ In the same myocarditis model, another group of investigators³² indeed observed in aminoguanidine-treated animals reduced or absent superoxide, peroxynitrite, and nitrotyrosine production and no histopathological evidence of myocardial destruction. Reduced myocardial destruction was also observed in a murine myocarditis model³³ after administration of the NOS inhibitor N^G-monomethyl-L-arginine and of amlodipine, which decreased myocardial iNOS expression. In other myocarditis models,^34,35 however, infected animals fed NOS inhibitors and infected iNOS "knockout" mice³⁶ had higher mortality. The beneficial actions of NO in these latter models probably resulted from a temporally and spatially restricted activation of iNOS³⁷ as part of an appropriate inflammatory response.

Immune-associated myocardial contractile depression was also investigated in conscious dogs.^38,39 Both studies observed a delayed LV contractile depression after injection of recombinant human tumor necrosis factor-, consistent with cytokine-induced synthesis of iNOS and subsequent NO generation. In dogs with pacing-induced heart failure, myocardial NOS activity was significantly increased compared with control dogs, and administration of an NOS inhibitor had no effect on basal myocardial contractility but augmented the inotropic response to isoproterenol.⁴⁰

Myocardial Contractile Effects of iNOS: Clinical Evidence
In normal subjects, transplant recipients, and congestive cardiomyopathy patients,^7-9,12 bicoronary infusion of the NO donor sodium nitroprusside or of substance P induced a significant abbreviation of LV contraction with a concomitant reduction in peak and end-systolic pressures and an increase in LV diastolic distensibility as evident from larger diastolic LV volumes at lower diastolic LV pressures. The present study failed to detect a baseline abbreviation of LV contraction or a baseline increase in LV diastolic distensibility in transplant recipients with high myocardial iNOS gene expression. This probably resulted from the small magnitude under baseline conditions of NO-induced myocardial contractile effects^7,8 and the absence of a paired design. In isolated cardiac muscle strips, the NO-induced drop in peak isometric tension is small (12%)⁴¹ because of counterbalancing positive and negative inotropic effects of NO with predominant positive inotropic effects in the low dose range and predominant negative inotropic effects in the high dose range.^41,42 Previous studies^7,8 with bicoronary infusions of NO donor or of substance P compared in the same patient baseline values to data obtained during bicoronary infusion. Such a paired design was absent in the present study. The use of sequential data in the same patient comparing LV function at a time of low myocardial iNOS gene expression to LV function at a time of high myocardial iNOS gene expression could possibly result in detection of changes in baseline systolic or diastolic LV performance^3,43 as a function of iNOS gene expression.

In isolated cat papillary muscle strips, ß-adrenergic agonists⁴¹ potentiated the negative inotropic effect of NO derived from NO donors. A potentiating effect of ß-adrenergic agonists on the cardiodepressant action of NO was recently confirmed in transplant recipients and in congestive cardiomyopathy patients.⁹ These observations prompted the use in the present study of intravenous dobutamine to facilitate recognition of iNOS-induced LV contractile effects. In these patients, who had high iNOS gene expression, intravenous dobutamine infusion resulted in a larger decrease in LVESP and in LVEST than in patients with low iNOS gene expression. These findings were also consistent with data obtained in isolated muscle strips from explanted human cardiomyopathic hearts.⁶ In this study, muscle strips derived from hearts with high iNOS gene expression had a larger decrease in peak active tension and a larger abbreviation of twitch contraction during ß-adrenergic stimulation.

In the present study, multiple regression analysis revealed a significant correlation between the dobutamine-induced abbreviation of LV contraction as a dependent variable and both the dobutamine-induced increase in LV dP/dt_max and the intensity of myocardial iNOS gene expression as independent variables. A close correlation was also observed between a ratio consisting of the dobutamine-induced abbreviation of LVEST divided by the dobutamine-induced increase in LV dP/dt_max and the intensity of myocardial iNOS gene expression. Both correlations imply a larger dobutamine-induced abbreviation of LV contraction for a similar dobutamine-induced increase in LV dP/dt_max in transplant recipients with higher iNOS gene expression and probably result from a potentiating interaction between NO and ß-adrenergic stimulation on the onset of LV relaxation because of additive effects on myofilamentary calcium sensitivity of an NO-induced increase in cGMP and a ß-agonist–induced increase in cAMP.⁹ The dobutamine-induced change in LV dP/dt_max failed to be significantly correlated with myocardial iNOS gene expression, in contrast to a previous study in which patients with LV dysfunction⁴⁴ experienced an increase in LV dP/dt_max after NOS inhibition during dobutamine infusion. This study, however, titrated the dose of dobutamine to achieve a preset increase in LV dP/dt_max whereas the present study used a fixed dose of dobutamine. The latter resulted in a variable response of LV dP/dt_max to the dobutamine infusion because of individual variations in number of ß-adrenergic receptors and in ß-adrenergic signal transduction pathway.

Study Limitations
The presence of iNOS in the myocardium of the transplant recipients was established in the present study by demonstration of iNOS mRNA by reverse transcription–PCR and not by direct demonstration in the myocardium of iNOS protein or of elevated cGMP, which would provide definite proof of myocardial presence of iNOS because of posttranscriptional modification of iNOS protein translation. In a previous study,³ however, iNOS protein immunostaining was detected in 80% of all biopsies that showed iNOS mRNA expression by reverse transcription–PCR, and myocardial cGMP was significantly increased in biopsies with iNOS mRNA expression. In this study, the observed elevation of cGMP validated the role of NO as mediator of LV contractile effects in patients with iNOS mRNA expression. In the present study, the role of NO as mediator of the altered LV hemodynamics during dobutamine infusion in patients with high iNOS gene expression was validated by comparison of the LV response to combined intravenous dobutamine and intracoronary substance P in patients with low iNOS gene expression to the LV response to intravenous dobutamine in patients with high iNOS gene expression. During combined intravenous dobutamine and intracoronary substance P infusion in patients with low iNOS mRNA, the decrease in LVEST and in LVESP was comparable to the decrease observed during intravenous dobutamine in patients with high iNOS mRNA. Intracoronary infusion of highly iNOS-specific antagonists, which was not performed in the present study, could provide definite and direct evidence for NO as mediator of the observed effects.

In the present study, intensity of iNOS gene expression was derived from a single additional snap-frozen biopsy. This limited procurement of additional biopsies failed to account for the frequently observed spatial heterogeneity of myocardial iNOS gene expression. Patients with high iNOS gene expression had no clinical or histopathological evidence of rejection. A similar dissociation in routine surveillance endomyocardial biopsies between histopathological grades of rejection and iNOS gene expression had previously been reported by Lewis et al³ and confirms the poor correlation between biopsy histology and cytotoxic T-cell activation or cytokine gene expression.

Conclusions and Clinical Relevance
The present study provides the first evidence for a well-defined LV contractile effect of myocardial iNOS gene expression in the human heart, namely an enhanced abbreviation of LV contraction with respect to the increase in LV dP/dt_max after intravenous administration of dobutamine. In transplant recipients, these measurements could be of relevance to detect forms of allograft rejection related to immune components such as cytokines, which result in elevation of myocardial iNOS⁴⁵ and not necessarily in a concurrent change in histology. Because of the potential role of iNOS gene expression in the development of graft vasculopathy,⁴⁶ these hemodynamic measures could help to detect allograft recipients at risk for graft vasculopathy. Repeating these studies in patients with congestive cardiomyopathy could lead to the development of a similar LV hemodynamic index for myocardial iNOS gene expression in congestive cardiomyopathy. Such an index could guide prognosis because it reflects myocardial exposure to cytokines, which have plasma levels that have previously been shown to be inversely related to survival,⁴⁷ and could perhaps help to select patients who will react favorably to ß-blockers⁴⁸ or to calcium channel blockers⁴⁹ because of modulation of myocardial iNOS gene expression by ß-adrenergic stimulation⁵⁰ and by amlodipine.³³

	Selected Abbreviations and Acronyms

 

EST = electromechanical systole time ESP = end-systolic pressure iNOS = inducible nitric oxide synthase LV = left ventricular

Received May 5, 1997; revision received July 10, 1997; accepted August 1, 1997.

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