WO1999055317A2 - Treatment of advanced decompensated heart failure with nitric oxide donors - Google Patents

Abstract

Nitric oxide donor compounds which are positive inotropes can be used to treat severe advanced decompensated heart failure in patients, by nitrosylating the L-type calcium channel or the ryanodine receptor so as to increase myocardial contractility. Measuring nitrosylation of either the L-type calcium channel or the ryanodine receptor by a test compound can also be used to screen for candidate positive inotropic drugs.

Description

TREATMENT OF ADVANCED DECOMPENSATED HEART FAILURE WITH NITRIC OXIDE DONORS

This work was supported by NIH grant K08 HL03228. The government therefore has certain rights in the invention. This application claims the benefit of provisional application Serial No. 60/083,618 filed April 30, 1998, which is incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

This invention is related to treatment of heart failure. In particular, this invention is related to treatment of advanced decompensated heart failure with drugs which are capable of nitrosylating proteins.

BACKGROUND OF THE INVENTION

Congestive heart failure is a prevalent and serious condition. In the United States, 4.7 million people have congestive heart failure. Once the condition develops, the six-year mortality rate approaches 80% in men and 65% in women. (Kannel,

Cardiol Clin. 7, 1-9, 1989).

Patients with New York Heart Association (NYHA) class IV failure are especially severely affected. Typically, such patients cannot carry on any physical activity without discomfort, and symptoms of heart failure or anginal syndrome may be present even at rest. If these patients undertake any physical activity, their discomfort is increased.

Present guidelines for treating NYHA class IV heart failure include, for example, the use of angiotensin-converting enzyme (ACE) inhibitor therapy unless the inhibitors are contraindicated or not tolerated. (American Heart Association Guidelines for the Evaluation and Management of Heart Failure, available at <http://v/ww.americanheart.org>). Contraindications to treatment with ACE inhibitors include shock, angioneurotic edema, and significant hyperkalemia. Patients may be unable to tolerate ACE inhibitors because of symptomatic hypotension, azotemia, hyperkalemia, cough, rash, or angioneurotic edema.

Beneficial effects have also been seen in NYHA class IV patients in clinical trials of β-adrenergic blocking agents. (See, e.g.., Metra et al., J. Am. coll. Cardiol 24, 1678-87, 1994; Olsen et al, J. Am. Coll. Cardiol. 25, 1225-31, 1995; Krum et al., Circulation 92, 1499 -1506, 1995). However, initiation of β-blockade can exacerbate heart failure in some patients. (Krum et al., 1995; CIBIS Investigators and

Committees, Circulation 90, 1765-73, 1994).

Diuretic treatment is usually recommended for class IV heart failure patients; however, a variety of complications such as hypotension, vascular collapse, hypokalemia, and diuretic drug resistance can develop. While vasodilators can be used to treat NYHA class I-III heart failure, they are often detrimental to class IV patients.

Despite advances in the treatment of severe heart failure, there is still a need in the art for drugs which can be used alone or in combination with other therapies to treat advanced decompensated heart failure.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method of treating advanced decompensated heart failure.

It is another object of the present invention to provide methods for screening to identify candidate positive inotropic drugs for treating advanced heart failure.

These and other objects of the invention are achieved by one or more of the embodiments described below.

One embodiment of the invention provides a method of treating advanced decompensated heart failure. A nitric oxide donor which is a positive inotrope is administered to a patient with advanced decompensated heart failure in an amount sufficient to increase myocardial contractility. Another embodiment of the invention provides a method for screening to identify candidate positive inotropic drugs for treating advanced heart failure. A test substance is contacted with an L-type calcium channel. S-nitrosylation of the L-type calcium channel is determined. A test substance which donates a nitric oxide radical to the L-type calcium channel is identified as a candidate positive inotropic drug.

Still another embodiment of the invention provides a method for screening to identify candidate positive inotropic drugs for treating advanced heart failure. A test substance is contacted with a ryanodine receptor. S-nitrosylation of the ryanodine receptor is determined. A test substance which donates a nitric oxide radical to the ryanodine receptor is identified as a candidate positive inotropic drug.

Thus, the invention provides novel means for treating advanced decompensated heart failure by increasing myocardial contractility using drugs which nitrosylate the L-type calcium channel or the ryanodine receptor. The invention also provides novel means of screening for candidate positive inotropic drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Representative pressure- volume data generated with transient occlusion of the inferior vena cava. Shown are pressure- volume loops before (solid loops) and after SIN-1 infusion (dotted loops, 160 μg/Kg/min). The solid line indicates slope of the end-systolic pressure volume relationship (Ees) obtained by non-linear regression of end-systolic pressure volume points. In this example, the end-systolic pressure volume relationship shifted to the left, resulting in near-complete emptying of the ventricle at end-systole. This positive inotropic effect dramatically increased stroke volume.

Figure 2. Effects of SIN-1 on preload recruitable stroke work in the absence (diamonds, n=l 8) and presence (squares, n=6) of 30 U superoxide dismutase (SOD).

SIN-1 was infused via the internal jugular vein and pressure- volume data were obtained during transient IVC occlusion at each infusion rate. SIN-1 administration caused a biphasic positive inotropic response which was converted to a negative inotropic effect by pretreatment with SOD. *P<0.05 vs. control. Figure 3. Effect of SIN-1 on loading conditions in the absence (grey bars) and presence of SOD (black bars). Preload was indexed by LV end-diastolic pressure (LVEDP) and afterload by effective arterial elastance (Ea). *P<0.05 vs. control.

DETAILED DESCRIPTION

The present invention provides a method of treating patients with advanced decompensated heart failure. These are preferably patients who are classified as New

York Heart Association (NYHA) class IV patients. These patients are hemodynamically decompensated, have low blood pressure, and deteriorating kidney function and mental status. Typically, these patients cannot tolerate vasodilator drugs or angiotensin-converting enzyme inhibitors, and some patients cannot tolerate β- adrenergic blocking agents. Many of these patients are not ambulatory and/or are refractory to outpatient therapy.

The method of the invention for treating such patients involves administering a nitric oxide donor which is a positive inotrope to a patient with advanced decompensated heart failure in an amount sufficient to increase myocardial contractility. Nitric oxide donors activate the L-type Ca²⁺ channel and the ryanodine receptor by nitrosylating the thiol moieties of these proteins. NONOates are one class of nitric oxide donors which can nitrosylate proteins. Sydnonimes are another class of nitric oxide donors. For example, 3-Morpholinosydnonimine (SIN-1) releases equimolar NO^* and -O₂- that react to form peroxynitrite, an effective nitrosating agent. Nitrosothiols, such as S-nitrosocysteamine, are a third class of nitric oxide donors.

Nitric oxide donors which can be used to treat patients with advanced decompensated heart failure include, but are not limited to, members of the classes above, such as SIN-1, ((Z)-l-N-[3-aminopropyl]-N-[4-(3- aminopropylammonio)butyl]-aminodiazen- 1 -ium- 1 ,2-diolate (spermine NONOate), diethylamine NONOate, S,S'-Dinitrosodithio (±)-S-Nitroso-N-acetylpenicillinamine

(SNAP), Glyco-SNAP-1, Glyco-SNAP-2, NOC-9, NOR-3, sodium dinitroprusside dihydrate, S-Nitrosoglutathione (SNOG), S-Nitrosoglutathione monomethyl ester, 2- (dimethylamino)-ethylputreanine/NO (DMAEP/NO), ((Z)- 1 -N-Methyl-N-[6-(N- methylammoniohexyl)amino] diazen-l)-ium-l,2-diolate (MAHMA NONOate), S- nitrosocysteine, S-nitroso-N-acetylcysteine, S-nitrosocysteamine, S- nitrosohomocysteine, S-nitrosodithiothreitol, S-nitrosoalbumin, CAS 1609 (4- hydroxymethyl-furoxan-3-carboxamide), and diazeniumdiolate NONOate. Nitric oxide donors for use in the method of the invention can be purchased commercially from sources such as Sigma or can be synthesized in the laboratory. Means of synthesizing nitric oxide donors are known in the art and are taught, for example, in

Stamler & Feelisch, eds., METHODS IN NITRIC OXIDE RESEARCH, at pages 521-39 (1996).

Nitric oxide donors can be administered either alone or in combination with other therapies. Preferably, the patients are not treated with or are not concomitantly treated with other positive inotropic agents, such as digoxin or dobutamine. The nitric oxide donor is preferably administered by intravenous infusion. Dosages of the nitric oxide donors can be determined, for example, by carrying out routine testing in animal models of heart failures, which are known and widely used in the art. For SIN-1 , the dosage effective to increase myocardial contractility is between 10 and 200 μg/kg/min. or between 50 and 150 μg/kg/min. The total amount administered is between 100 and 2000 μg/kg or 500 and 1500 μg/kg. Ideally, the amount of the nitric oxide donor is sufficient to stabilize the hemodynamics of the patient. Hemodynamic stabilization can be assessed by means of common hemodynamic measurements, such as cardiac output, stroke output, stroke work, stroke power, ventricular end-diastolic pressure, ejection fraction, and ventricular end-diastolic volume, as is known in the art.

The method of the invention is especially effective for treatment of severely ill heart failure patients, such as those with a pulmonary wedge pressure of less than 18 or more than 28 mm Hg and/or a cardiac index of < 2 L/min/m². Pulmonary wedge pressure can be measured by methods known in the art, such as Doppler echocardiography or right heart catheterization. Cardiac index can be measured using the Fick method or other techniques known in the art. (See Hurst et al., eds., THE HEART, 4th ed., pp. 491-501, 1978).

The invention also provides a method for screening drugs to identify candidate positive inotropic drugs for treating advanced heart failure. In the screening methods, a test substance is contacted with an L-type calcium channel, and S-nitrosylation of the L-type calcium channel is determined. Alternatively, the test substance can be contacted with a ryanodine receptor, and S-nitrosylation of t e ryanodine receptor is determined. A test substance which donates a nitric oxide radical to the L-type calcium channel or the ryanodine receptor is identified as a candidate positive inotropic drug.

The test substance can be a pharmacologic agent already known in the art as a nitric oxide donor or can be a compound previously unknown to have any nitrosylating activity. The test substance can be naturally occurring or designed in the laboratory. It can be isolated from microorganisms, animals, or plants, or can be produced recombinantly or synthesized by chemical methods known in the art.

S-nitrosylation of the L-type calcium channel or ryanodine receptor can be determined, for example in isolated cardiac muscle strips, isolated cardiac myocytes, or non-cardiac muscle cells transfected with expression vectors encoding either of the two proteins. Alternatively, S-nitrosylation can be measured in in vitro systems comprising purified or recombinantly expressed L-type calcium channels or ryanodine receptors. Purification of these proteins has been described. (See Tauna & Murphy, Can. J. Physiol Pharmacol 68, 1482-88, 1990; Xu et al., Science 279:234-237 (1998). Either protein can be expressed using recombinant DNA techniques well known in the art. (See Hu et al, Cir. Res. 81, 742-52, 1997; Perez-Reyes et al, FEBS Zett. 342, 119-23, 1994). S-nitrosylation assays are taught, for example, in Xu et al.

(1998) and in Stamler & Feelisch (1996), above.

Optionally, the candidate positive inotropic drug is administered to a mammalian heart, such as a rabbit, ferret, mouse, rat, guinea pig, hamster, dog, cat, cow, sheep, pig, or goat heart, and myocardial contractility is measured. The heart can be an isolated, perfused heart or the heart can be in an intact animal. Myocardial contractility can be measured by any means known in the art. Methods of measuring myocardial contractility are taught, for example, in Kojda et al., Cir. Res. 78, 91-101 (1996); Mohan et al., Circulation 93, 1223-29 (1996); Preckel et al., Circulation 96, 2675-82 (1997); Keaney et al., Am. J. Physiol. 271, H2646-H2652 (1996); Hare et al., J. Clin. Invest. 101, 1424-31 (1998), and in Examples 1 and 8, below. A candidate drug which increases myocardial contractility in the heart is identified as a positive inotrope.

The above disclosure generally describes the present invention. All references cited in this disclosure are expressly incorporated herein by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.

EXAMPLE 1

Methods

Male C57BL/6 mice (n=25; 12-18 weeks old; 20-35 g body weight; Jackson Laboratories) were used in the studies described in the following examples. Animals were housed under diurnal lighting conditions and allowed food and tap water ad libitum. Animal treatment and care was provided in accordance with institutional guidelines, and the protocol was approved by the Animal Care and Use Committee of the Johns Hopkins University. The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of

Health (NIH Publication NO. 85-23, revised 1996).

Anesthesia was induced with inhalation of methoxyfluorane (Metofane^®), followed by intraperitoneal injection of urethane (500 mg/kg) and etomidate (20 mg/kg) dissolved in 0.25 ml of normal saline. Supplemental i.p. anesthesia (1/5* dose) was provided to maintain animals unresponsive to tail pinch by forceps, as assessed by changes in heart rate and blood pressure. Animals were placed on a heating pad set to 38 °C. Endotracheal intubation was achieved with a short PE20 tube via tracheotomy, and ventilation initiated using a custom-made constant pressure ventilator with 100% oxygen at 120 breaths/min, and a tidal volume of 200 μl. Using a dissecting microscope for visualization, a limited substernal abdominal incision was performed, and the diaphragm cut at the anterior chest wall. Through a small apical stab, a combined micromanometer-conductance catheter (20) (SPR-719, Millar Instruments, Houston, TX) was then advanced retrograde into the left ventricle along the cardiac longitudinal axis with the distal tip in the aortic root and proximal electrode just within the endocardial wall of the LV apex. The pericardium was left as intact as possible. For drug and hypertonic saline infusions, the right jugular vein was cannulated with a 30G needle.

The recorded volume signal from the conductance catheter requires calibration for absolute volume (offset) and stroke volume (gain). Calibration was performed in 7 mice. Absolute volume was derived using a saline wash-in technique (21). This saline wash-in method allows determination of the "parallel-conductance," a measure of the conductance signal which derives from structures outside the left ventricular cavity (i.e., muscle, bone, blood in the right ventricle, etc.).

Stroke volume calibration was derived from cardiac output obtained from direct measurement of aortic flow. With the catheter fixed in place, the mice were turned to the left-lateral position. Special care was taken to maintain catheter position as evidenced by the shape and position of the pressure-volume loops visualized on-line. A limited lateral thoracotomy was then performed and the descending aorta dissected free from the spinal column just above the level of the diaphragm. A flow probe (AT01RB, Transonic Systems, Ithaca, NY) was placed around the aorta, and flow per minute was recorded (ATI 06, Transonic Systems). Due to the observed negative inotropic effects of SIN-1 following SOD and the consequent deterioration of hemodynamic status of the animals, calibration of the volume signal could not be obtained in these experiments. Pressure, volume and flow signals were digitized at 1 kHz, stored to disk and analyzed with custom software.

EXAMPLE 2

Data Analysis

The analysis of pressure- volume relationships and the arterial pressure response allowed the evaluation of variables related to myocardial systolic and diastolic performance. Averaged data from 10-20 consecutive beats were used to derive steady-state parameters, and data measured during unloading of the heart by transient occlusion of the inferior vena cava (IVC) was used to assess pressure- volume relations. This yielded 10-20 successive cardiac cycles over the following 2 seconds from which the cardiac performance parameters was derived. Preload was indexed as the left ventricular end-diastolic volume (LVEDV) and pressure (LVEDP). Afterload was evaluated as effective arterial elastance (Ea, ratio of LV systolic pressure to stroke volume) (23, 24). Ea has been validated to closely approximate aortic impedance, which incorporates systemic vascular resistance and the reflected wave properties of the vasculature. Contractility was indexed using the load-independent parameters end-systolic elastance (Ees) and preload-recruitable stroke work (PRSW; slope of stroke work/end-diastolic volume relation) (23). Diastolic performance was measured by the time constant of isovolumic relaxation (tau) and the time to peak filling rate (ttpf) (25).

EXAMPLE 3 Experimental Protocol and Drugs

With a precision infusion pump (Model 22, Harvard Instruments, Waltham, MA), 100 μL of 10% dextran 40 in normal saline (Abbot Laboratories, North Chicago, 111.) was given as volume support over 2 min, 15 min before infusions of SIN-1 or SOD. SIN-1 (Sigma, St. Louis, MO) was dissolved in normal saline and given in a graded intravenous infusion at the doses 80, 160, 320 g/kg/min at a rate of

1.25 to 5 μL/min. The effect of each dose was observed for 10 min before the next and higher dose was started. Control experiments demonstrated that the vehicle had no effect on cardiac or systemic hemodynamics at the infusion rates used in the experiments. SIN-1 was prepared freshly for each experiment, protected from light and kept at 4 °C. In additional experiments (n=6), 30 U SOD (Sigma) was dissolved in 10 μL normal saline and injected through the left internal jugular vein line 1 min before SIN-1 was administered.

EXAMPLE 4

Statistics All results are reported as mean±SEM. An unpaired Student's t-test was used to compare pre-infusion hemodynamic variables in the SIN-1 alone and SOD experimental groups. For statistical analysis of hemodynamic concentration-effect relationships, a two-way ANOVA with a term for individual experiment was used (26). Differences were considered significant at P-values <0.05. All statistical analyses were performed using SYSTAT or SAS software.

EXAMPLE 5

Effects of SIN-1 on left ventricular performance

Table 1 summarizes baseline hemodynamic variables in all C57BL/6 mice studied. Heart rate was 699±17 bpm, SBP 98±3 mm Hg, Ees 18.7±4.2 mm Hg/μl and

PRSW 92.5±4.3 mm Hg. No significant differences were observed between mice that were and were not given SOD.

Graded infusion of SIN-1 (80, 160, 320 μg/kg/min; n=18) caused a positive inotropic response. Ventricular elastance, the slope of the end-systolic pressure- volume relation exhibited marked increases in slope (Figure 1, p<.05), providing qualitative evidence for increased myocardial contractility. Moreover,

PRSW (Figure 2) increased by 27±13% (p<0.05) from baseline at the peak response which occurred at the dose 160 μg/kg/min. At higher infusion rates of SIN-1 (320 μg/kg/min) this effect was partially reversed. In contrast, SIN-1 did not affect myocardial relaxation as indexed by tau and ttpf.

EXAMPLE 6

Effects of SIN-1 on loading conditions and cardiac performance

The effects of SIN-1 on cardiac loading conditions were assessed. Preload, measured by LVEDP or LVEDV, was not significantly affected by SIN-1 (Figure 2). On the other hand, SIN-1 exerted a vasodilator action. Ea (afterload) decreased from the control value of 14.4±6.4 mm Hg/μl by 23±7% (ρ=0.02; Figure 3). SIN-1 did not affect heart rate. Thus, at these infusion rates, SIN-1 had both a positive inotropic and vasodilator effect in mice. These changes were not accompanied by a lusitropic or chronotropic effect. To gauge the overall impact of these inotropic and vasodilator actions, we derived cardiac output from calibrated studies (n=7). Baseline CO (5.7 ± 1.2 ml/min) rose to 7.1(1.1 mL/min (P<.05) with SIN-1 160 μg/kg/min. EXAMPLE 7

Effects of SIN-1 in the presence of SOD

To assess whether SIN-1 acted by a nitrosylation-based mechanism, we pretreated mice with SOD to prevent peroxynitrite formation. In the presence of 30 U SOD (n=6), the positive inotropic effect of SLN-1 was abolished. End-systolic pressure volume relationship (Ees) did not change significantly in response to SIN-1. PRSW (Figure 2) decreased by 42±10% from baseline at the dose 160 μg/kg/min. At this infusion-rate, SIN-1 also decreased heart rate by 12±8% (p=0.04). After SOD, SIN-1 had no effect on diastolic performance (tau and ttpf), and did not change preload (LVEDP) or afterload (Ea) (Figure 3).

EXAMPLE 8

Measurement of myocardial contractility in isolated perfused hearts

Rat Langendorff preparations can be used to measure myocardial contractility. Hearts are excised from male Wistar rats and retrogradely perfused with oxygenated perfusion buffer at 37 °C . A polyvinyl chloride balloon attached to PE-190 tubing balloon is placed through the left atrium and mitral valve into the left ventricle. The balloon is filled with saline to achieve a maximum isovolumic developed pressure, which typically occurs at an end-diastolic pressure of 10-15 mm Hg.

Hearts are perfused at a constant flow with a peristaltic pump titrated to a coronary perfusion pressure of 80 mm Hg. Constant flow is used to avoid confounding alterations in contractility due to the Gregg effect . The perfusate contains (mmol/L) sodium 144, potassium 5, calcium 1.5, bicarbonate 17.5, magnesium 1.2, and chloride 134, along with 5 μg/ml lidocaine. This is equilibrated with a gas mixture of 95% O₂/5% CO₂, resulting in a perfusate pH of 7.4. The hearts are placed in a heated bath to maintain the temperature at " ° C and paced at 300 beats per minute with an electrode placed in the bath. The left ventricular pressure, the rate of change of left ventricular pressure (dP/dt), and the mean coronary perfusion pressure are measured continuously on a physiograph (Gould Electronics Corporation) and simultaneously digitized at 1000 Hz. After a 15 min. stabilization period, drugs can be infused at 1% of coronary flow using a Harvard 11 pump.

DISCUSSION

The examples presented above demonstrate in vivo that SIN-1, an effective nitrosating species, has a positive inotropic effect that can be blocked by superoxide dismutase (SOD). The purpose of this study was to test the hypothesis that nitrosating Nitric oxide donors would have positive inotropic effects in vivo. The demonstration that SOD blocked SIN-1 positive inotropic effects supports a nitrosylation-dependent mechanism of action. The physiologic roles of nitrosylation-based reactions are being increasingly appreciated. Thiol nitrosylation (S-NO) reactions have been shown to confer NO-like vasodilator activity to albumin (5) and other low molecular-weight thiols (31), as well as to enhance tissue plasminogen activator function (6). More recently, S-NO reactions have shown to be important in the ability of hemoglobin to regulate blood vessel tone (32). The present findings extend in vitro observations that the L-type channel and the ryanodine receptor undergo S-nitrosylation leading to increased calcium cycling. The nitrosylation has now been shown to increase the level of myocardial contractility. Thus, this pathway represents a mechanism by which NO may contribute to the maintenance of resting myocardial contractility and illustrates a novel regulatory mechanism involved in myocardial function.

Whether and how NO affects resting myocardial contractility has been controversial. Studies have shown inhibitory, neutral, and cardiostimulatory effects attributable to NO (reviewed in 4). A potential, albeit partial, explanation for these divergent observations is that NO influences myocardial contractility in a biphasic concentration-dependent manner, being stimulatory at low and inhibitory at high concentration (9). Biphasic effects have been largely recognized in vitro, whereas in vivo studies using infusions of organic nitrates and Nitric oxide donors have shown that weak stimulatory effects predominate, suggesting increased physiologic relevance of the stimulatory limb (12). The second messenger most often attributed to be responsible for the cardiovascular actions of the NO signaling pathway is cGMP, produced by NO activation of soluble gaunylyl cyclase. Studies of cGMP actions on contractility also suggest a biphasic response (33). A role for NO in myocardial contractility is more apparent in the setting of β-adrenergic (18, 34) or heart rate (force-frequency response) (35, 36) stimulated myocardial contractility. Here NO appears to offset these increases in contractile state, through a mechanism also likely involving cGMP. Why this response is more readily recognized is likely because both heart rate and β-adrenergic stimulation of the heart also activate NOS and the subsequent elevation in NO production is sufficient to have a negative inotropic effect. Increased cardiac

NO-production due to β-adrenergic stimulation has been directly measured with a porphyrinic microsensor (37) and by our laboratory using electron-paramagnetic resonance. The present study indicates that a non-cGMP mechanism, protein nitrosylation, also contributes to NO regulation of myocardial contractility. Certain features of our observations are notable. First, the magnitude of SIN- 1 positive inotropy was substantial. SIN-1 increased ventricular elastance dramatically, leading to very high slopes (often approaching vertical) or leftward shifts (toward near total chamber emptying) in the relationship (Figure 1). Because of this, the magnitude of the positive inotropic effect was quantitated with PRSW. This index is also relatively load-independent but is derived from the entire area of the PV loop, rather than a single point as is the case for Ees. Second, a biphasic response was also noted. At low infusion rates, SIN-1 had a positive inotropic effect which was not sustained at high concentrations. Peroxynitrite may also lead to cellular toxicity due to its potent oxidizing properties (27), shown both in isolated hearts (38) and intact animal studies (39), and to inhibition of mitochondrial respiration (40). The latter two actions could contribute to reversal of positive inotropic effects. A similar dual response has been noted with regard to peroxynitrite effects on vascular tone, acting as a vasodilator on the one hand and a cause of endothelial dysfunction on the other (41-43).

In this study, a novel miniaturized conductance catheter was used to assess hemodynamics in anesthetized mice. Using this conductance catheter and systemic infusions of SIN-1, the effects of SIN-1 on contractility, preload, and afterload (20, 23) were separated. In addition, anesthesia and mechanical ventilation were carefully selected to study mice with physiologic hemodynamic parameters (44). . At the infusion rates used in the study, SIN-1 had both a positive inotropic effect and reduced afterload, but did not significantly influence preload or heart rate. In summary, this study provides proof of the principle that an agent effective at nitrosylation can cause positive inotropic effects. These effects are seen at low concentrations of SIN-1 and are lost at high concentrations, indicating a biphasic response. Importantly, SOD pretreatment blocks SIN-1 positive inotropy, providing strong evidence that the effect is based upon peroxynitrite formations and subsequent nitro sy lation of proteins .

Table 1. Baseline hemodynamic conditions (n=24)

HR (bpm) 699(17

SBP (mmHg) 98(3

SV(μl) 9.4(1.9

CO (mL/min) 5.1 (1.2

Ees (mmHg/ μl) 18.7(4.2

PRSW (mmHg) 92.5(4.3

Tau (s) 4.9(0.3

ttpf (ms) 26.3(1.0

LVEDP (mmHg) 7.6 ( 0.6

LVEDV (μl) 15.2(2.9

Ea (mmHg/ μl) 14.4(6.4

Abbreviations: Heart rate, HR; Systolic blood pressure, SBP; Stroke

volume, SV; Cardiac output, CO; Ventricular elastance, Ees; Preload

recruitable stroke work, PRSW; Time to peak filling, ttpf; Left-ventricular end-diastolic pressure, LVEDP; Left-ventricular end-diastolic volume,

LVEDV; Arterial elastance, Ea. Reference List

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Claims

1. A method of treating advanced decompensated heart failure comprising: administering a nitric oxide donor which is a positive inotrope to a patient with advanced decompensated heart failure in an amount sufficient to increase myocardial contractility.

2. The method of claim 1 wherein the step of administering is by intravenous infusion.

3. The method of claim 1 wherein the patient has New York Heart Association (NYHA) class IV heart failure.

4. The method of claim 1 wherein the patient cannot tolerate angiotensin- con verting enzyme inhibitors.

5. The method of claim 1 wherein the patient cannot tolerate vasodilator drugs.

6. The method of claim 1 wherein the patient is not concomitantly treated with a positive inotropic drug which is not a nitric oxide donor.

7. The method of claim 1 wherein no positive inotropic drug which is not a nitric oxide donor is administered to the patient.

8. The method of claim 1 wherein an amount of the nitric oxide donor is administered which is sufficient to stabilize hemodynamics of the patient.

9. The method of claim 1 wherein the patient is not ambulatory.

10. The method of claim 1 wherein the nitric oxide donor is a NONOate.

11. The method of claim 1 wherein the nitric oxide donor is a nitrosothiol.

12. The method of claim 1 wherein the nitric oxide donor is a sydnonime.

13. The method of claim 1 wherein the sydnonime is SIN-1.

14. The method of claim 13 wherein the dosage is between 10 and 200 ╬╝g/kg/min.

15. The method of claim 13 wherein the dosage is between 50 and 150 ╬╝g/kg/min.

16. The method of claim 13 wherein the total amount administered is between 100 and 2000 ╬╝g/kg.

17. The method of claim 13 wherein the total amount administered is between 500 and 1500 ╬╝g/kg.

18. The method of claim 13 wherein the patient has a cardiac index of < 2 L/min/m².

19. The method of claim 13 wherein the patient has a pulmonary wedge pressure of less than 18 or more than 28 mm Hg.

20. A method for screening to identify candidate positive inotropic drugs for treating advanced heart failure, comprising: contacting a test substance with an L-type calcium channel; and determining S-nitrosylation of the L-type calcium channel, wherein a test substance which donates a nitric oxide radical to the L-type calcium channel is identified as a candidate positive inotropic drug.

21. The method of claim 20 wherein the candidate positive inotropic drug is administered to a mammalian heart and myocardial contractility is measured, wherein a candidate drug which increases myocardial contractility in the heart is identified as a positive inotrope.

22. The method of claim 21 wherein the heart is an isolated, perfused heart.

23. The method of claim 21 wherein the heart is in an intact animal.

24. A method for screening to identify candidate positive inotropic drugs for treating advanced heart failure, comprising: contacting a test substance with a ryanodine receptor; and determining S-nitrosylation of the ryanodine receptor, wherein a test substance which donates a nitric oxide radical to the ryanodine receptor is identified as a candidate positive inotropic drug.

25. The method of claim 24 wherein the candidate positive inotropic drug is administered to a mammalian heart, and myocardial contractility is measured, wherein a candidate drug which increases myocardial contractility in the heart is identified as a positive inotrope.

26. The method of claim 25 wherein the heart is an isolated, perfused heart.

27. The method of claim 25 wherein the heart is in an intact animal.