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Cardiovascular Research 1997 36(2):184-194; doi:10.1016/S0008-6363(97)00149-1
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

Nitric oxide induced contractile dysfunction is related to a reduction in myocardial energy generation

Malte Kelma,*, Stefan Schäfera, Rüdiger Dahmanna, Bahar Doluc, Stefan Peringsa, Ulrich K.M Deckingb, Jürgen Schraderb and Bodo E Strauera

aDepartment of Medicine, Division of Cardiology, Pulmonary Diseases, and Angiology, Heinrich Heine University, Moorenstr. 5, 40225 Düsseldorf, Germany
bDepartment of Physiology, Heinrich Heine University, Moorenstr. 5, 40225 Düsseldorf, Germany
cDepartment of Pharmacology, Ege University, 53100 Bornova, Izmir, Turkey

* Corresponding author. Tel.: +49-211-81-18323; fax: +49-211-3179737; email: sschaefe@uni-duesseldorf.de

Received 18 February 1997; accepted 23 May 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: It has been suggested that nitric oxide (NO) is involved in the regulation of myocardial function in a variety of diseases such as dilated cardiomyopathy, myocarditis, heart transplant rejection, and septic shock. However, the underlying mechanism of NO mediated reduction of cardiac contractility has not been clearly established so far. Therefore, we studied the effects of authentic NO on left ventricular function and myocardial energy status in the isolated heart. Methods: In 43 isolated perfused guinea pig hearts quantitative and kinetic changes in coronary flow (CF), left ventricular developed pressure (LVDP), the cardiac release of adenosine, lactate, cyclic GMP, and norepinephrine were measured during infusion of authentic NO. In parallel, myocardial phosphocreatine (PCr), ATP and the free energy change of ATP-hydrolysis ({Delta}GATP) were measured using 31P nuclear magnetic resonance spectroscopy. Results: At low concentrations (0.01 to 1.0 µmol/L) NO increased CF only; at higher concentrations (1 to 100 µmol/L) CF remained elevated and LVDP was significantly reduced. Onset and offset of changes in LVDP occurred always within 2 to 5 s after start and cessation of NO infusion. Contractile dysfunction was significantly correlated to a pronounced increase in adenosine formation (>70-fold), a significant decrease in myocardial PCr (–78%), ATP (–25%) and a decrease in {Delta}GATP from –61.76 kJ/mol to –50.75 kJ/mol. This was paralleled by a significant decrease in myocardial oxygen consumption (–65%) and a tenfold increase in lactate production. Coronary vasodilation (NO: 0.001 to 1.0 µmol/L) significantly correlated with the increase in cGMP release, whereas at negative inotropic concentrations (NO: 10 to 100 µmol/L) a clear quantitative and kinetic dissociation between NO-induced changes in cGMP and LVDP was observed. Contractile dysfunction was not related to cardiac release of norepinephrine. Conclusions: In the isolated heart NO can potently depress myocardial energy generation thus being an effective modulator of cardiac contractility. This effect of NO may be of pathophysiological significance in cardiac muscle disorders in vivo.

KEYWORDS Endothelium-derived factors (nitric oxide); Free radicals; Myocardial contraction; Guinea pig


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Nitric oxide (NO) has been demonstrated to modulate systolic and diastolic left ventricular function both in experimental [1–8]and clinical settings [9–12]. Recently, it has been hypothesized that NO accounts, at least in part, for the myocardial dysfunction associated with a variety of heart muscle disorders such as dilated cardiomyopathy, myocarditis, heart transplant rejection, and cardiac dysfunction in septic shock (for review see [13–15]). However, the underlying mechanism for the negative inotropic effect of NO is still under debate, and various factors have been implicated to mediate NO induced contractile dysfunction. From experiments with isolated cardiomyocytes the second messenger cyclic guanosine monophosphate (cGMP) has been suggested to mediate the NO-related negative inotropic effects but this hypothesis has been challenged in later experiments [8, 16–19]. In electrically stimulated adult rat ventricular myocytes NO has been demonstrated to blunt the inotropic effect of the β-adrenergic agonist isoproterenol suggesting that NO might regulate contractile function via an anti-adrenergic effect [20]. Recently, it has been postulated that NO inhibits myocardial creatine kinase [21]. Thus, up to now it is not clear whether NO induced contractile disfunction is mediated exclusively by cGMP or if other targets within the cardiomyocytes are affected by NO. Due to its radical nature and extraordinarily high affinity to hemoproteins it is also tempting to speculate that NO affects key enzymes of the mitochondrial electron transport chain thereby directly modulating oxidative phosphorylation and myocardial contraction.

In order to test this hypothesis, we characterized both kinetically and quantitatively the NO-induced changes of cardiac function and of myocardial energy status in the isolated heart.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Preparation of isolated hearts
A total of 43 guinea pigs (body mass 250–350 g) were stunned by a blow on the neck and hearts were quickly excised and perfused at 37°C with constant pressure (60 cmH2O or, 45 mmHg) according to the Langendorff technique. The perfusion medium consisted of a modified Krebs–Henseleit buffer containing (in mmol/L): NaCl 116, KCl 4.7, MgSO4 1.1, KH2PO4 1.17, NaHCO3 24.9, CaCl2 2.52, glucose 8.3, and pyruvate 2.0. The perfusion medium was equilibrated with a gas mixture composed of O2: CO2 (0.95: 0.05, v: v). The right ventricle was vented via the pulmonary artery with a polyethylene catheter (1.2 mm outer diameter, 7 cm length). The tip of the catheter was placed approximately 2 cm below the apex of the heart to permit an optimal drainage of the right ventricle. The mitral valve was cut to prevent fluid accumulation in the left ventricle. After completion of the experimental preparation, the heart was placed in a water jacketed chamber at 37°C and allowed to equilibrate for 30 min.

2.2 Cardiac and coronary dynamics
Coronary flow (CF) was measured two centimeters above the orifice of the coronary vessels using an electromagnetic flow probe (SP2202, Statham, Oxnard, CA, USA). For isovolumetric measurement of left ventricular function, a latex balloon (size 4, Hugo Sachs Elektronik KG, March, Germany) was introduced into the left ventricle via the cut mitral valve, which was fixed directly on a stainless steel cannula connected to a pressure transducer (Gould P23, Cleveland, OH, USA). The hearts were paced via the right ventricle at a constant rate 15% above the spontaneous sinus rhythm. Left ventricular pressure (LVP) and (by electronic differentiation) maximal rise of LVP (dP/dtmax) were continuously monitored on an ink recorder (Gould, Brush 480, Cleveland, OH, USA). Left ventricular end diastole was determined as the point when left ventricular dP/dt started its rapid upstroke after crossing the zero line. At the beginning of each experiment, left ventricular end diastolic pressure (LVEDP) was set to approximately 5 mmHg by adjusting the filling of the latex balloon; left ventricular developed pressure (LVDP) was calculated as maximal minus end diastolic LVP. Coronary resistance (CR, in mmHgxminxmL–1) was calculated as (45-LVEDP)/CF. Hearts were used for further study only when the following criteria were met: (i) basal coronary flow with no balloon inside was less than 7 mL/min, (ii) maximal coronary flow during reactive hyperemia after a transient 20 s coronary occlusion was at least 200% of the initial value, and (iii) LVDP at baseline was greater than 50 mmHg. About 25% of hearts were rejected from further study because not all of these criteria were fulfilled.

After termination of the experiments, the hearts were weighed after blotting the endo- and epicardial surface with a paper towel.

2.3 Preparation of nitric oxide standards
A concentration response curve for the cardiac dynamic effects of NO was derived by intracoronary infusion of standards of authentic NO. Standards of aqueous NO solution were prepared as previously described [22]. Briefly, NO was dissolved in deoxygenated purified water kept under argon atmosphere. Concentration of NO solutions were routinely determined using the oxyhemoglobin-assay [23, 24]. NO-solutions could be prepared reliably and reproducibly as assessed by low variability of determined concentrations (coefficient of variance 3.4%; n = 12). NO solutions were kept in gas-tight syringes mounted in a precision infusion pump (Infors AG, Basel, Switzerland) and connected to a high performance liquid chromatography (HPLC) steel cannula, which was placed in the aortic inflow line directly in front of the ostium of the coronary arteries in order to limit destruction of NO during its transport to the tissue (stability of NO solution up to 6 h).

2.4 Measurement of adenosine
Samples of coronary venous effluent were desalted via C18-Sep-Pak columns (Waters, Eschborn, Germany). Adsorbed purines were eluted with 2 mL of methanol/H2O (2:1 v:v) and evaporated to dryness (Vortex evaporator, Buchler, Lenexa, KA, USA), and thereafter redissolved in 200 µL H2O of which 20 µL were used for HPLC-analysis [25]. The chromatography system used (Sykam S1100 and S8110, Sykam, Gilching, Germany, and Axxiom 727 software package, Axxiom Inc., Moorpark, CA, USA) was programmed for gradient elution of samples injected onto a C18-reversed phase column (150x3.9 mm, particle size 10 µm, Waters, Eschborn, Germany). A linear gradient at a flow of 1 mL/min was used starting at 95% buffer A (ammonium acetate, 0.026 mol/L, pH 5.0) and 5% buffer B (70:30 v:v methanol:water) reaching 75% buffer A/25% buffer B after 8 min. Absorbance of column eluate was continuously recorded at {lambda} = 254 nm (Linear Instruments UVis 200, Reno, NE, USA).

2.5 Measurement of lactate, creatine kinase, coronary venous pH, and partial pressure of oxygen
For determination of coronary venous pH, partial pressure of oxygen, aliqouts from the coronary venous effluent perfusate were sampled anaerobically. Partial oxygen pressure, p(O2) and pH of the perfusate were measured immediately with a standard Astrup analyzer (ABL 510, Radiometer, Copenhagen, Denmark). Myocardial oxygen extraction (MVO2) was calculated from arteriovenous difference and coronary flow. Aliqouts of coronary venous effluent perfusate were sampled and frozen until determination of L-lactate and creatine kinase (CK) with commercially available enzyme assays (Boehringer Mannheim, Germany).

2.6 31P-NMR spectroscopy
31Phosphorus nuclear magnetic resonance spectroscopy (31P-NMR) spectra were acquired on an AMX400 WB pulsed Fourier transform NMR spectrometer (Bruker) coupled to a 9.4 Tesla superconducting magnet (Spectrospin) and equipped with a sensitive 20 mm probe-head (Fraunhofer Institute, St. Ingbert, Germany) as described previously [26]. In brief, partially saturated 31P spectra were accumulated using a radiofrequency pulse width of 58 µs, resulting in a 70° tilt angle, 2k data points in the time domain and a pulse interval of 3 s. Spectra were a result of 128 scans except in the kinetic studies where the number of scans was reduced to 16 scans. Spectra were processed using software (NMR1) by Tripos (St. Louis, USA). Zero filling to 4k and exponential multiplication of the data (5 Hz line broadening) was followed by Fourier transformation, automatic phasing and baseline correction. Manual interactive integration was employed for phosphocreatine (PCr) and adenosine triphosphate (ATP) and the PCr/ATP-ratio was calculated. A curve-fitting routine was used for the integration of the cytosolic Pi peak, which partially overlapped with the extracellular Pi resonance. Under basal conditions, [ATP], [PCr] and total [Cr] were taken to be 7.08, 13.3 and 22.2 mmol/L, respectively [27]. Intracellular pH, cytosolic free concentration of adenosine diphosphate (ADP) and the free energy change of ATP-hydrolysis ({Delta}GATP) were calculated as previously described [26], using the following equations.

Intracellular pH was determined from the chemical shift difference of PCr and intracellular Pi ({delta}, ppm):


Formula

Cytosolic free concentration of ADP was calculated as


Formula

and the {Delta}GATP was calculated as


Formula

where change in standard free energy ({Delta}G°Obs)=–30.5 kJ/mol at 37°C, [H+]=10–7.1 mol/L, and the equilibrium constant of the creatine kinase reaction (KCK)=109/mol; R is the gas constant, T is temperature.

2.7 Measurement of cGMP
Measurement of cGMP was performed according to a standard procedure from our laboratory, as previously described [22]. Samples of coronary effluent perfusate were passed over Sep-Pak C18 cartridges (Waters, Eschborn, Germany) that had been pretreated with 3 mL methanol, 2 mL isopropanol (40% v:v) and 3x2 mL distilled water, followed by 2 mL KH2PO4 (0.01 mol/L). Absorbed cGMP was eluted with 2 mL isopropanol and evaporated to dryness. Capacity and reproducibility of the separation procedure was tested with cGMP-standards yielding more than 80% recovery of cGMP. Data of cGMP release were not corrected for losses occurring during the sample purification. Cyclic GMP was determined with a commercially available radioimmunoassay (RPA 525, Amersham, Braunschweig, Germany). Radioactivity of the tracer (cGMP-2'-O-succinyl-3-[125]iodotyrosine methyl ester) was measured by a gamma scintillation counter (Model 05301, Hewlett Packard, Boston, MA, USA) and compared to a standard calibration curve.

2.8 Measurement of norepinephrine
Coronary effluent perfusate (10 mL) was sampled into ice-cold tubes containing Na-EGTA (ethylene glycol-bis(b-aminoethyl ether) N,N,N',N'-tetraacetic acid; final concentration: 180 mg/L) and glutathione (140 mg/L, Chromsystems, Munich, Germany). Norepinephrine (NE) was preconcentrated via solid phase extraction in 2 mL fractions given on cartridges containing aluminium oxide (Al2O3) together with 500 µL of alkaline TRIS buffer (pH 8.6) and 50 µL of the internal standard dihydroxybenzylamine (DHBA, 10 ng/mL, Chromsystems, Munich, Germany) and thereafter desorbed with 120 µL of perchloric acid containing buffer (pH 3.0) with a recovery ranging from 70% to 80%. Quantification of NE was accomplished as described previously [28]. Briefly, an electrochemical detector was connected to a reversed-phase high performance liquid chromatography system (R4A Chromatopac and system 6-A, Shimadzu Europe, Duisburg, Germany; Nucleosil-C18 column, Waters, Eschborn, Germany) using isocratic elution (1 mL/min, 38°C) with a mobile phase composed of 0.8 g octansulfonic acid (Fluka, Buchs, Switzerland), 1.94 mL dibutylamine (reagent D4, Waters, Eschborn, Germany), 8.2 g sodium acetate, 8.42 g carbomonohydrate, 0.074 g sodium-EDTA, 100 mL methanol (all Merck, Darmstadt, Germany), all added to a final volume of 2000 mL (pH=4.5). Using this setup, a detection limit of 11 pmol/L and a coefficient of variance of less than 5% was achieved.

2.9 Statistics
Results are expressed as means± standard error of the mean (SEM). Statistical significance between means was tested using a one-way analysis of variance, followed by a least significant difference post-hoc test where appropriate. A p<0.05 was considered significant. Linear regressions were calculated using the method of least squares. Data processing was performed using the SPSS® software package (release 5.0.1, SPSS Inc., Chicago, IL, USA).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effects of nitric oxide on cardiac function
The cardiac and coronary dynamic parameters at baseline and at the different concentrations of NO are given. NO infused into the coronary circulation significantly increased CF and decreased coronary resistance in a concentration-dependent manner (Fig. 1). In the range of 10 nmol/L to 1 µmol/L, there was no effect on LVDP or dP/dtmax. However, a further increase in the intracoronary NO concentration above the maximal vasodilatory concentration (1 µmol/L) significantly reduced LVDP and dP/dtmax in a concentration-dependent manner (n = 7–11; Fig. 1). At a maximal concentration of NO, LVDP was reduced by 66%, whereas CR remained decreased (see Fig. 1). No significant correlation between NO-induced changes in LVDP and CF or CR was observed. In a separate set of experiments the kinetics of NO-induced changes in cardiac function were documented. After start of NO infusion (100 µmol/L) NO-induced coronary vasodilation began after 2.3±0.3 s and reduction in LVDP after 2.9±0.3 s (n = 4). The reduction in LVDP was as rapidly reversed: LVDP began to increase again at 3.8±0.8 s after cessation of NO infusion and control values were reached always within less than 10 s (see Fig. 2). The observed changes in CF and LVDP were not mediated by the stable end products of NO decomposition, as neither nitrite (NO2–) nor nitrate (NO3–) affected CF, LVDP or dP/dtmax (n = 6, see Fig. 3). The wet weight of the hearts at the end of the experiments was 1.1±0.1 g (n = 35 out of 43).


Figure 1
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  		Fig. 1 Effect of nitric oxide (NO) on left ventricular developed pressure (LVDP) and coronary resistance (CR). Aqueous standards of authentic NO (10–9 to 10–4 mol/L) were infused into the coronary circulation of isolated constant-pressure perfused guinea pig hearts. *p<0.05 versus control, # p<0.05 versus 1 µmol/L NO (n = 7–11).

 

Figure 2
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  		Fig. 2 Original tracing that depicts the rapid kinetics in left ventricular pressure after onset (on) and cessation (off) of NO infusion (100 µmol/L). LVP, left ventricular pressure; LV dP/dt, time change in left ventricular pressure.

 

Figure 3
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  		Fig. 3 Effect of nitrite (NO2–, closed symbols) and nitrate (NO3–, open symbols) on left ventricular developed pressure (LVDP) and coronary flow (CF) in isolated constant-pressure perfused guinea pig hearts (n = 6).

 
3.2 Effects of nitric oxide on myocardial energy status
At baseline the release of adenosine into the coronary circulation amounted to 129±39 pmol/min (n = 6). At concentrations of 1 nmol/L to 1 µmol/L NO did not significantly affect adenosine release from isolated hearts. Only at the negative inotropic concentrations NO increased concentration-dependently the release of adenosine from isolated hearts, which at the maximum was 70-fold above control (see Fig. 4). The coronary venous concentration of adenosine increased from 21±7 to 742±89 nmol/L. A significant correlation was found between NO-induced (1 to 100 µmol/L) increase in adenosine release and decrease in LVDP (adenosine concentration: r = 0.79, adenosine release rate: r = 0.75, both p<0.0001). In control experiments infusion of adenosine (10 µmol/L) significantly increased CF (17±2 mL/min), but did not affect LVDP (68±4 mmHg, n = 4), excluding the possibility that the NO-induced decrease in LVDP is mediated by adenosine itself (see inset Fig. 4). In order to further assess the effects of NO on myocardial energy status, we quantified cardiac release of lactate into the coronary venous effluent. Under baseline conditions lactate levels amounted to 14±3 µmol/L. During infusion of NO at the highest concentration (100 µmol/L, n = 4) lactate significantly increased to 112±5 µmol/L.


Figure 4
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  		Fig. 4 Effect of nitric oxide (NO) on the release of adenosine into the coronary circulation of isolated constant-pressure perfused guinea pig hearts (n = 6). Inset: In control experiments adenosine was infused at a final concentration of 10 µmol/L, which is 10-fold beyond that from endogenously formed adenosine during NO infusion (n = 4). CF denotes coronary flow and LVDP, left ventricular developed pressure at control conditions (open bars) and infusion of adenosine (10 µmol/L, solid bars). *p<0.05 versus control.

 
In addition to cardiac release of adenosine the parameters of myocardial energy status PCr, ATP, PCr/ATP-ratio, ADP, pHi and {Delta}GATP (free energy change of ATP-hydrolysis) were determined by means of 31P NMR spectroscopy in a separate set of experiments (n = 4) at control and during infusion of NO at the concentrations which elicited maximal coronary vasodilation (1 µmol/L) and maximal depression of LVDP (100 µmol/L). These results are summarized in Fig. 5. No considerable changes in parameters of myocardial energy status were detected at 1 µmol/L NO as compared to control conditions. However, at 100 µmol/L of NO there was a decrease in LVDP accompanied by a 60% decrease in MVO2, a substantial increase in adenosine release and a pronounced decrease in PCr (–78%) and {Delta}GATP (–61.76 kJ/mol to –50.75 kJ/mol), all p<0.05 versus control (Fig. 5). ATP, however, only decreased by 26%. Thus PCr/ATP ratio changed from 2.20±0.26 to 0.64±0.07 at 100 µmol/L. Before NO infusion ADP was 36.57±0.68 µmol/L, and Pi was 1.07±0.33 mmol/L. At 100 µmol/L NO ADP rose to 261.79±31.20 µmol/L and Pi to 7.75±0.92 mmol/L. Intracellular pH was 7.13±0.01 at baseline conditions and 7.10±0.01 at 100 µmol/L NO, p = n.s.


Figure 5
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  		Fig. 5 Changes in left ventricular developed pressure (LVDP), myocardial oxygen extraction (MVO2), phosphocreatine (PCr), ATP, and free energy change of ATP hydrolysis ({Delta}GATP) before (control 1, open bars), during infusion of 1 (hatched bars) and 100 µmol/L (solid bars) NO and after cessation of NO infusion (cross hatched bars). *p<0.05 versus control 1 (n = 4).

 
Changes in LVDP and {Delta}GATP at onset and offset of NO infusion ran in parallel resulting in a significant correlation of both parameters (r = 0.69, p<0.05). PCr and {Delta}GATP reached control values within one minute after cessation of NO infusion.

3.3 Effects of nitric oxide on cyclic GMP
Basal release of cGMP into the coronary circulation of isolated hearts amounted to 360±76 fmol/min (n = 7). At concentrations of 1 nmol/L to 1 µmol/L NO significantly increased cGMP release from isolated guinea pig hearts (see Fig. 6). This increase does not merely reflect increases in CF, as cGMP concentration in the coronary venous effluent significantly increased from 65±8 pmol/L (control) to 323±73 pmol/L (1 µmol/L NO). A significant correlation was observed between NO-induced changes in CF (NO: 0.01–1.0 µmol/L) and either the concentration (r = 0.41) or release (r = 0.65) of cGMP into the coronary circulation (both p<0.05). A further almost 10-fold increase in intracoronary cGMP concentration was observed, when NO concentration was increased up to 10 µmol/l: 0.32±0.07 to 2.73±0.7 nmol/L cGMP. In contrast, NO did not further enhance cGMP concentration within its negative inotropic concentration range (at 100 µmol/L NO: 2.83±0.6 pmol/L cGMP) and consequently cardiac cGMP release remained unaffected at high concentrations of NO (see Fig. 6). Thus, we failed to demonstrate a significant correlation between NO-induced changes in LVDP (NO: 1–100 µmol/L) and changes in either concentration or release of cGMP (r<0.3, p>0.3). Furthermore, a clear dissociation in the kinetic responses between NO-mediated changes in LVDP and cGMP release was observed as determined in a separate set of experiments. LVDP declined from 71±4 to 18±2 mmHg during infusion of 100 µmol/L NO. Following cessation of NO infusion LVDP reached control values within 8 s: 20±1 (1 s), 21±1 (2 s), 25±3 (3 s), 33±3 (4 s), 44±6 (5 s), 53±6 (6 s), 62±6 (7 s), 69±6 mmHg (8 s after cessation of NO application). In contrast, cGMP release remained significantly elevated for more than one minute (31±5 pmol/min) and reached control values only after three minutes. In a further set of experiments the stable analogue 8-bromo-cGMP infused at increasing concentrations (10 µmol/L, 100 to 1000 µmol/L, each for 30 min) significantly increased CF in a concentration dependent manner to a maximum of 12±2 mL/min, whereas baseline LVDP and dP/dtmax remained unaffected, 68±4 mmHg and 1144±210 mmHg/s, respectively (n = 4).


Figure 6
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  		Fig. 6 Effect of nitric oxide (NO) on the rate of cyclic guanosine monophosphate (cGMP) release from isolated constant-pressure perfused guinea pig hearts (n = 7). *p<0.05 versus control, and # p<0.05 versus 1 µmol/L NO.

 
3.4 Effect of nitric oxide on coronary venous pH and release of norepinephrine and CK
At baseline norepinephrine was released into the coronary circulation at a rate of 24.5±8.6 pmol/min (n = 5). Infusion of NO (0.001 to 100 µmol/L) did not alter norepinephrine release from isolated hearts. When desipramine (0.5 µmol/L) was infused to deplete endogenous stores via inhibition of released norepinephrine, a small but not significant decrease in CF from 7.1 to 5.3 mL/min, in LVDP from 78 to 64 mmHg, and in dP/dtmax from 1243 to 900 mmHg/s (n = 2) was seen. Infusion of NO (1 to 100 µmol/L) following 60 min after start of desipramine application increased CF and decreased LVDP to a similar extent as seen without desipramine, excluding a significant interaction of NO-induced changes in LVDP and cardiac norepinephrine.

To assess the cellular integrity of cardiomyocytes and other cardiac cells during NO infusions the release rate of CK into the coronary venous effluent perfusate was measured. At baseline, CK activity was 39±9 mU/min and did not significantly change over the entire NO concentration range infused (100 µmol/L NO: 29±3 mU/min CK).At baseline, coronary venous pH was 7.32±0.05 and during NO infusion (100 µmol/L) 7.31±0.05 (p = n.s.).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The most important finding of the present study is that in the isolated heart authentic NO can potently depress myocardial energy generation and thus induce contractile dysfunction. NO affects cardiac contractility with extremely rapid kinetics suggesting that this NO-mediated mechanism may play an important role in the regulation of myocardial function under pathophysiological conditions in vivo.

4.1 NO and myocardial energy status
The myocardium depends almost exclusively on immediate energy supply by the oxidative mitochondrial metabolism [29]. Thus, the matching of energy supply to demand is of fundamental importance for cardiac function. In the present study, NO induced a reversible reduction in both cardiac oxygen consumption and myocardial function. Adenosine is considered to be a sensitive marker for any imbalance between ATP supply and demand, most likely due to the close coupling of adenosine formation to free cytosolic AMP and thus the cardiac energy status [25, 30]. Accordingly, the NO induced reduction of cardiac contractility was paralleled by a massive, concentration-dependent release of adenosine in our study. Notably, exogenous adenosine infused at comparable concentrations selectively dilated coronary arteries without affecting LVDP, suggesting that the NO induced contractile dysfunction is not mediated by this nucleoside.

Using 31P-NMR determination of cytosolic concentrations of PCr, ATP, Pi and the intracellular pH allows to calculate both the free cytosolic ADP concentration and the free energy change of ATP hydrolysis. This parameter reflects precisely the energy set free during the hydrolysis of ATP to ADP and thus is an index of the myocardial energy supply-to-demand ratio [29]. In principle, mitochondrial oxidative energy generation may be impaired by insufficient oxygen supply, or a lack of reducing equivalents, ADP and Pi. Blockade of the electron transport chain would have a similar effect. Given the more than 100% increase in coronary flow during infusion of NO, a decrease in cardiac oxygen supply as a cause for the myocardial dysfunction can be excluded in the present study. A lack of ADP or Pi is equally unlikely, given the increase of these parameters during 100 µmol/L NO.

Another possible explanation for the observed reduction of cardiac contractility could be opening of intracoronary shunts at higher concentrations of NO similar to the effect seen at high doses of other directly relaxing substances such as papaverine or calcium channel openers. Relevant opening of coronary shunt vessels should result in a considerable decrease in coronary resistance in parallel to a decrease in cardiac contractility. In the present study, coronary resistance decreased over a concentration range of NO (1 mmol/L to 1 µmol/L) that had no effect on myocardial performance. The dose dependent decrease in LVDP at higher concentrations of NO was, however, not accompanied by a further decrease in coronary resistance. Therefore, it appears highly unlikely that at least under our experimental conditions NO reduces myocardial contractility via shunting of coronary perfusate.

In a previous study, a selective decrease in intracellular ATP and a non selective decrease in phosphocreatine were observed in response to an inotropic challenge with intracoronary calcium in isolated rat hearts exposed to the NO donor S-nitrosoacetylcysteine [21]. The authors concluded that NO impaired myocardial creatine kinase thereby limiting energy transfer from phosphocreatine to ATP which could eventually lead to a decrease in cardiac contractility [21]. It is, however, also conceivable that these results may be explained by inhibition of mitochondrial oxidative phosphorylation, e.g. due to blockade of the electron transport chain.

In the present study, a decrease in both intracellular ATP and PCr and a massive release of adenosine was observed in parallel to the reduction of cardiac oxygen consumption and contractility. These data imply that myocardial energy generation is impaired at the mitochondrial level. While mitochondria are an important cellular source of oxygen derived radicals, they also represent a preferred target of free radical attack [31, 32]. There is an increasing experimental evidence that NO or its related compounds, such as peroxynitrite or nitrosothiols, interfere with the heme enzymes of the mitochondrial electron transport chain leading to insufficient myocardial energy generation [18, 31]. Moreover, the effects of NO on oxidative capacity in isolated mitochondria appear to depend critically not only on the amount of NO, but also on the concentration of oxygen, superoxide anions, peroxynitrite, on the substrate used (e.g., malate, succinate, or ascorbate) [33–37], and on the presence or absence of ATP [36]. In those experiments on isolated mitochondria, the kinetics in onset and offset of the inhibition of mitochondrial respiration were rapid, i.e., below or within minutes depending on the experimental conditions. Thus, both in the isolated mitochondria as well as in our preparation, the NO related effects on energy generation are rapid and reversible. In line with the present experimental data it is of interest that endothelium derived NO has been shown to modulate left ventricular systolic and diastolic function in healthy humans [12]. If these preliminary findings are confirmed, the NO mediated reduction in myocardial energy generation as demonstrated in the present study may well be of clinical relevance.

4.2 NO and cGMP
Many of the biological effects of NO, in particular smooth muscle relaxation, are mediated by the second messenger cGMP (reviewed by [38–40]). In accordance with previous work, the NO induced coronary vasodilation is most likely explained by an activation of soluble guanylyl cyclase within the smooth muscle, as indicated by the parallel increase in coronary flow and coronary venous cGMP concentration at the vasodilatory effective NO concentrations, i.e., 10 nmol/L to 1 µmol/L [22, 41]. It has been assumed that cGMP modulates myocardial contractility via inhibition of the L-type calcium channel current [42]. In line with this concept, the NO related reduction in contraction of isolated cardiomyocytes was blunted in the presence of the guanylyl cyclase inhibitor methylene blue [43], and in solubilized tissue of left ventricular wall from rat hearts cGMP content increased after induction of myocardial NO synthase [1]. In carbachol-stimulated cardiomyocytes isolated from guinea pigs [17], in isoproterenol-stimulated rat hearts [44], and in isolated electrically stimulated ferret papillary muscle [16]no direct correlation between changes in cardiac contractility and cGMP formation were observed. By contrast, in rat ventricular myocytes and in isolated human papillary muscles a probably cGMP mediated light increase in contractile response to NO-donors has been described [19, 45]. This item has become even more complicated by the very recent finding of a bi-directional response of contractility in isolated cat papillary muscles exposed to different types of NO donors, which at least in part appeared to be cGMP-mediated [8]. Based on these contradictory findings, the role of cGMP as a second messenger for the NO induced changes in myocardial contractility has been questioned recently (for review see [15]). However, up to now quantitative and kinetic data on NO-induced release of cardiac cGMP and their correlation to changes in cardiac function in isolated hearts have been missing. In the present study, NO did not further alter cardiac cGMP release within its negative inotropic concentration range. Consequently, no correlation between NO-induced changes in cGMP and LVDP was found. This effect might be caused by a generalized down regulation of cardiac energy generation during NO infusion (100 µmol/L), which would have prevented a further increase of guanylyl cyclase activity. Nevertheless, a clear dissociation between the kinetics of reversal of changes in cGMP release and LVDP after cessation of NO infusion was observed, indicating that LV function was already restored although cGMP was still elevated. Moreover, high doses of the stable analogue 8-bromo-cGMP failed to mimic NO induced depression of cardiac contractility. Taken together, these findings suggest that NO induced contractile dysfunction cannot be exclusively explained by an enhanced stimulation of guanylyl cyclase in our experimental model.

4.3 NO and norepinephrine
In electrically stimulated adult rat ventricular myocytes NO has been demonstrated to blunt the inotropic effect of the β-adrenergic agonist isoproterenol [20]suggesting that NO might modulate catecholaminergic myocardial effects. In the present study NO did not alter cardiac NE release. Moreover, NO induced changes of cardiac function were almost identical in the absence and presence of desipramine, which depleted neuronal NE stores via blockage of NE reuptake, as indicated by a decrease of CF in the respective control experiments. Thus, the NO-induced reduction of contractility at least in isolated guinea pig hearts appears not to be mediated by an altered release or reuptake of cardiac NE.

4.4 Critique of methods and study limitations
In the present study, aqueous solutions of authentic NO standards were chosen to investigate the effects of NO on cardiac function in an isolated heart preparation over a wide concentration range. These standards were prepared with a reliable reproducibility and offer several advantages compared to NO generated from pharmacological NO donors: (1) side effects were avoided, which could be related to the decomposition product of drugs following NO generation, such as organic nitrates or sodium nitroprusside [46]. (2) Because of the rapid inactivation of NO within the heart [22], the present protocol allows to measure exactly the kinetics of the NO induced changes in cardiac function without being hampered by slow on and off responses, due to bioactivation of NO donors, diffusion problems, or washout phenomena of these drugs. (3) In the present study the effects of NO on cardiac function were elicited not by its stable metabolites nitrite and nitrate, as indicated by our control experiments. This does not necessarily mean that the effects observed following intracoronary infusion of authentic NO are exclusively mediated by NO itself. Intramyocardial conversion along its route of diffusion to cardiomyocytes resulting in NO-related compounds such as nitrosothiols or peroxynitrite may at least in part contribute to the NO-induced contractile dysfunction.

A possible drawback of our experimental approach is that we cannot exactly determine the proportion of bioactive NO at the site of the cardiomyocytes. Kinetic modeling predicts that a major part of the NO in the vasculature is scavanged along its route to the interstitium [47]. In our study, even at the highest intracoronary concentrations of NO no long term toxic effects were present as evidenced by (i) the rapid recovery after cessation of NO infusion and (ii) the lack of cell damage measured as release of CK into the coronary effluent perfusate. However, the amount of bioactive NO necessary to modulate myocardial energy generation at the intracellular level in vivo remains to be determined. In this context, the very recent immunohistochemical localization of a mitochondrial NO synthase in rat hearts [48]is of particular interest since the NO produced within the mitochondria may control myocardial energy generation directly at its site of production.

4.5 Implications for future studies
The direct impact of NO on myocardial energy generation may be an important feature of NO under in vivo conditions, as this suggests that cardiac endothelial and endocardial cells as well as smooth muscle cells and cardiomyocytes may effectively modulate cardiac contractility via the generation and release of NO. Further in vivo studies devoted to the hypothesis that NO from different sources may influence mitochondrial energy generation and thus LV function in vivo are warranted, as this mechanism may offer a new therapeutic target in a variety of heart muscle disorders.

Time for primary review 28 days.


   Acknowledgements
 
We wish to thank Ms. Claudia Ferfers for expert technical assistance. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 242). B.D. is a Research Training Fellow sponsored by TUBITAK, Turkish Scientific and Technical Research Council of Turkey. M.K. is a Recipient of a Gerhard-Hess grant from the Deutsche Forschungsgemeinschaft.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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