Introduction

Despite effective medical therapies, heart failure (HF) remains a major cause of morbidity and mortality worldwide.1, 2, 3 Importantly, ischemic heart disease is a major cause of HF.4 Significant clinical efforts, therefore, are directed at preventing acute myocardial infarction (AMI) via coronary vasodilation and, in individuals experiencing AMI, reducing the size of the infarct and ischemia/reperfusion injury.5 The endogenous protein hormone atrial natriuretic peptide (ANP) is mainly released from atrial tissue, and carperitide—recombinant human ANP—is widely used in patients with HF.6, 7, 8 The beneficial effects of carperitide have been attributed to various cardiovascular-protective activities, including diuresis, natriuresis, vasodilatation and reduced activity in the sympathetic nervous system and renin–angiotensin–aldosterone system.9 Recently, we showed that carperitide limits infarct size and improves cardiac function in patients with AMI (J-WIND study).10 Among the many cardioprotective effects of carperitide, the most prominent in ischemic heart disease is coronary vasodilation. Because carperitide shares a signaling pathway with nitric oxide (NO),9, 11 and the two molecules are known to interact,12, 13, 14 we hypothesized that carperitide increases coronary blood flow (CBF) in ischemic hearts and that inhibiting endogenous NO signaling would blunt the observed coronary vasodilation.

To test these hypotheses, we determined whether carperitide mediates vasodilation and attenuates the severity of metabolic and contractile dysfunction in ischemic canine hearts. We also examined the role of endogenous NO in these effects. Finally, we investigated carperitide-mediated limitations of the infarct size and whether NO contributes to this cardioprotective activity.

Methods

Instrumentation

Female beagle dogs weighing 10–14 kg were anesthetized with intravenous pentobarbital sodium (30 mg kg−1). Preparative methods are detailed in a previous study.15 After the chest was opened, coronary perfusion pressure (CPP) and CBF to the perfused myocardium were measured. A pair of ultrasonic crystals was inserted ∼1 cm apart in the inner one-third of the myocardium to measure the myocardial segment length with an ultrasonic dimension gauge.

All procedures complied with the Guide for the Care and Use of Laboratory Animals (NIH publication no. 85–23, 1996 revision) and were approved by the National Cerebral and Cardiovascular Center Committee for Laboratory Animal Use.

Experimental protocols

Protocol I: effects of carperitide on coronary vasodilation in nonischemic hearts

Five dogs were used in this protocol. Coronary arterial and venous blood was sampled for blood gas analysis. Myocardial oxygen consumption (ml per 100 g per min) is calculated by CBF (ml per 100 g per min) × the oxygen difference between coronary arterial and venous blood (ml dl−1). We measured CPP and CBF after dogs were randomly administered carperitide at a dose of 0.025, 0.05, 0.1 or 0.2 μg kg−1 min−1 (Daiichi-Sankyo KK, Tokyo, Japan) into the left anterior descending coronary artery (LAD). A preliminary study showed that CBF stabilized 5–10 min after a change in the carperitide dose.

Protocol II: effects of carperitide on myocardial ischemia produced by coronary hypoperfusion with or without an ANP receptor antagonist

Twelve dogs were used in this protocol. Coronary arterial and venous blood was sampled for analysis of blood gas and the levels of lactate and plasma NO metabolites (nitrate and nitrite). The following hemodynamic parameters were measured: left ventricular pressure, dP/dt and the segmental length of the perfused myocardium. After hemodynamic parameters were stabilized, we infused either saline (n=7) or the ANP receptor antagonist HS-142-1 (40 μg kg−1 min−1, n=5). At 5 min after infusion onset, an occluder attached to the extracorporeal bypass tube was used to reduce CPP such that CBF decreased to one-third of the control value. Thereafter, the occluder was adjusted to maintain CPP at this level. We confirmed that 10 min was required to obtain a stable state in the hypoperfused myocardium. After 10 min, carperitide (0.1 μg kg−1 min−1) was infused into the LAD and all hemodynamic and metabolic parameters were measured again after 20 min. After acquiring the data, carperitide infusion was discontinued and all hemodynamic and metabolic parameters were measured after 20 min.

We injected microspheres before (10 min after the onset of coronary hypoperfusion), during (30 min) and after (50 min) carperitide infusion.

Protocol III: effects of carperitide on myocardial ischemia produced by coronary hypoperfusion with or without a NO synthase inhibitor

Twelve dogs were used in this protocol. We measured the same hemodynamic and metabolic parameters described in protocol II. We infused either saline (n=7) or Lω-nitroarginine methyl ester (L-NAME), an inhibitor of NO synthase (NOS; 10 μg kg−1 min−1; n=5). At 5 min after infusion onset, CPP was reduced such that CBF decreased to one-third of the control value. Thereafter, the occluder was adjusted to maintain CPP at this level. After 10 min, carperitide (0.1 μg kg−1 min−1) was infused into the LAD and all hemodynamic and metabolic parameters were measured again after 20 min. After acquiring the data, carperitide infusion was discontinued and all hemodynamic and metabolic parameters were measured after 20 min.

Protocol IV: effects of carperitide on cyclic GMP levels in epicardial coronary arteries of ischemic hearts

We determined whether carperitide increases coronary arterial cyclic guanosine monophosphate (GMP) levels in ischemic myocardium. An occluder attached at the extracorporeal bypass tube was used to reduce CPP such that CBF decreased to one-third of control CBF. Thereafter, the occluder was adjusted to maintain CPP at the low level. After 10 min, carperitide (0.1 μg kg−1 min−1) was infused into the LAD for 10 min and the epicardial LAD (ischemic region) and left circumflex (nonischemic control region) coronary arteries were rapidly removed in the presence (n=5) or absence (n=7) of L-NAME using precooled stainless steel scissors and tongs. Samples were quickly stored in liquid nitrogen.

Protocol V: effects of carperitide on myocardial infarct size following coronary occlusion and reperfusion

In 36 dogs, the bypass tube to the LAD was occluded for 90 min, followed by reperfusion for 6 h as saline, carperitide (0.1 μg kg−1 min−1), L-NAME (10 μg kg−1 min−1) with carperitide (0.1 μg kg−1 min−1) and L-NAME (10 μg kg−1 min−1) were administered (n=9 for each group) from 10 min before occlusion until 1 h after reperfusion onset, except at the time of coronary occlusion. In all groups, infarct size was assessed 6 h after reperfusion onset.

The area affected by myocardial necrosis and the area at risk were measured in the dogs after protocol completion by an individual who had no knowledge of the specific treatment given to each animal. Infarct size is expressed as a percentage of the area at risk. Regional myocardial blood flow was determined as described previously.16 Microspheres were administered 80 min after the onset of coronary occlusion.

Assays

Lactate was assessed using an enzymatic assay, and the lactate extraction ratio was obtained as the coronary arteriovenous difference in the lactate concentration multiplied by 100 and divided by the arterial lactate concentration. Levels of plasma NO metabolites (nitrate and nitrite) were analyzed using an automated procedure based on the Griess reaction.17 Nitrate and nitrite concentration differences between coronary venous and arterial blood were used to quantify cardiac NO levels.

The method used to measure cyclic GMP levels has been described previously.18 After removing adventitial connective tissues from the coronary arteries (20–40 mg), frozen tissue was powdered, homogenized at 4 °C in 1 ml of ice-cold 6% trichloroacetic acid and centrifuged at 2500 × g for 20 min. The supernatant was removed, extracted three times with 3 ml of diethyl ether saturated with water and stored at −80 °C. Cyclic GMP concentrations in the supernatant were measured within 7 days using a radioimmunoassay. Briefly, 100 μl of dioxane–triethylamine mixture containing succinic acid anhydride was used to succinylate cyclic GMP in 100 μl of supernatant. After a 10-min incubation, the reaction mixture was combined with 800 μl of 0.3 M imidazole buffer (pH 6.5). Then, 100 μl of succinyl cyclic GMP tyrosine methyl ester iodinated with 125I (15 000–20 000 counts per min) was added to the assay mixture containing 100 μl of the supernatant and 100 μl of diluted anti-sera in the presence of chloramines. The mixture was kept at 4 °C for 24 h. A cold solution of dextran-coated charcoal (500 μl) was added to the mixture in an ice-cold water bath. The charcoal was spun down, and 0.5 ml of the supernatant was assessed for radioactivity in a gamma spectrometer. Cyclic GMP levels were normalized based on protein content in the coronary artery that was assayed using the Lowry method.19

Measurements of regional CBF

Regional myocardial blood flow was determined using a microsphere-based technique as previously reported.20 Nonradioactive microspheres (Sekisui Plastic, Tokyo, Japan) are made of inert plastic labeled with different stable heavy elements as described in detail in previous studies.16, 20 Microspheres were suspended in isotonic saline with 0.01% Tween-80 to prevent aggregation. Microspheres were ultrasonicated for 5 min followed by 5 min of vortexing immediately before injection. Approximately 1 ml of the microsphere suspension (2–4 × 105 spheres per ml) was injected into the left atrium followed by several warm (37 °C) saline flushes (5 ml).

X-ray fluorescence of stable heavy elements was measured using a wavelength dispersive spectrometer (PW 1480; Phillips Co. Ltd., Almelo, Netherlands). This X-ray fluorescence spectrometer has been previously described in detail. In brief, when microspheres are irradiated by the primary X-ray beam, electrons fall back to a lower orbit and emit measurable energy as characteristic fluorescence depending on the element. Therefore, X-ray fluorescence from several differently labeled microspheres in the mixture can be assessed. In protocol II, myocardial blood flow in the endocardium versus that in the epicardium (End/Epi flow ratio) was calculated and normalized based on the wet weight of the sampled myocardium. In protocol V, regional myocardial blood flow was calculated according to the following formula: time flow=tissue count × reference flow/reference count. The results are expressed in ml min−1 per g of wet sample.

Statistical analysis

Statistical analysis was performed using two-way analysis of variance21, 22 to compare data among the groups. When analysis of variance reached significance, paired data were compared using Bonferroni’s test. Changes in the hemodynamic and metabolic parameters over time were compared by analysis of variance for repeated measures. Analysis of covariance, by endocardial collateral blood flow in the inner half of left ventricle wall as the covariate, was used to account for the effect of endocardial collateral blood flow on infarct size. All results are expressed as mean±s.e.m., and P<0.05 was considered significant.

Results

Mean blood pressure (103±2 mm Hg) and heart rate (139±2 beats per min) did not differ significantly among the groups. These systemic hemodynamic parameters did not change significantly before, during or after coronary hypoperfusion or complete coronary occlusion with or without administration of a pharmacologic agent.

Effects of carperitide on CBF in nonischemic hearts

Figure 1 shows CBF during infusions of carperitide. CBF increased based on the dose of carperitide, with saturation of coronary vasodilation observed at a dose of 0.1 μg kg−1 min−1 (Figure 1a) despite no changes in CPP (102±2 mm Hg) or myocardial oxygen consumption (Figure 1b).

Figure 1
figure 1

Effects of carperitide on coronary blood flow (CBF) and myocardial oxygen consumption (MVO2) in nonischemic hearts. CBF increased as the carperitide dose increased (a) despite no changes in myocardial oxygen consumption (b). *P<0.05, **P<0.01 vs. the control.

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Effects of carperitide on CBF and the severity of myocardial ischemia

Figure 2 shows CBF (Figure 2b) and fractional shortening (FS) (Figure 2c) whereas CPP was reduced (Figure 2a) with or without the denoted pharmacologic agents. Carperitide increased both CBF and FS whereas CPP was held constant, effects that were blunted by the ANP receptor antagonist HS-142-1. The myocardial End/Epi flow ratio was also augmented by carperitide (Figure 3). Carperitide increased both lactate extraction ratio (Figure 4a) and pH levels (Figure 4b) in coronary venous blood from the ischemic area; HS-142-1 inhibited these effects without increasing cardiac NOx levels (differences in nitrate and nitrite levels between coronary venous and arterial blood; Figure 4c).

Figure 2
figure 2

Effects of carperitide on coronary blood flow (CBF) and fractional shortening (FS) in ischemic hearts with or without HS-142-1. Carperitide increased both CBF (b) and FS (c) whereas coronary perfusion pressure (CPP; a) was held constant, effects that were blunted by the atrial natriuretic peptide (ANP) receptor antagonist HS-142-1. *P<0.05, **P<0.01 vs. the values at 10 minutes.

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Figure 3
figure 3

The ratio of epicardial flow to endocardial flow in the myocardium during ischemia. Carperitide predominantly increased endocardial blood flow relative to epicardial blood flow. This effect was attenuated by HS-142-1. *P<0.05 vs. ischemia.

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Figure 4
figure 4

Effects of carperitide on metabolic function in ischemic hearts with or without HS-142-1. Carperitide increased both (a) lactate extraction ratio (LER) and (b) pH levels in coronary venous blood from the ischemic area; HS-142-1 inhibited these effects without increasing cardiac nitric oxide (NO) levels (c). *P<0.01 vs. the control.

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Figure 5 shows CBF (Figure 5b) and FS (Figure 5c) whereas CPP was reduced (Figure 5a) with or without L-NAME. Carperitide increased both CBF and FS without changes in CPP, the effects that were inhibited by an inhibitor of NOS (L-NAME). Carperitide increased both lactate extraction ratio (Figure 6a) and pH levels (Figure 6b) in coronary venous blood from the ischemic area; L-NAME blunted these effects without increasing cardiac NOx levels (Figure 6c).

Figure 5
figure 5

Effects of carperitide on coronary blood flow (CBF) and fractional shortening (FS) in ischemic hearts with or without Lω-nitroarginine methyl ester (L-NAME). Carperitide increased both CBF (b) and FS (c) without changes in coronary perfusion pressure (CPP; a), effects that were inhibited by an inhibitor of nitric oxide (NO) synthase L-NAME. *P<0.05, **P<0.01 vs. the values at 10 minutes.

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Figure 6
figure 6

Effects of carperitide on metabolic function in ischemic hearts with or without Lω-nitroarginine methyl ester (L-NAME). Carperitide increased both (a) lactate extraction ratio LER and (b) pH levels in coronary venous blood from the ischemic area; L-NAME blunted these effects without increasing cardiac nitric oxide (NO) levels (c). *P<0.01 vs. the control.

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We also investigated cyclic GMP levels during carperitide-induced coronary vasodilation. Without carperitide treatment, myocardial ischemia (CPP: 105±4 to 42±2 mm Hg; CBF: 82±3 to 27±2 ml per 100 g per min) increased cyclic GMP concentrations in the coronary artery from 44±17 to 121±22 fmol per mg protein (Figure 7; P<0.01). Moreover, carperitide provided during myocardial ischemia further increased cyclic GMP levels in the involved coronary artery (P<0.05); L-NAME attenuated this effect. Furthermore, carperitide increased myocardial cyclic GMP levels (168±30 to 263±30 pmol per mg protein, n=3 each); L-NAME attenuated this effect (184±24 pmol per mg protein, n=3).

Figure 7
figure 7

Cyclic guanosine monophosphate (GMP) levels in the coronary arteries of ischemic hearts. Ischemia per se increased cyclic GMP levels that were further elevated by carperitide. The increases in cyclic GMP levels were attenuated by Lω-nitroarginine methyl ester (L-NAME). *P<0.05 vs. the control.

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Effects of carperitide on infarct size following ischemia and reperfusion

Of the 36 dogs, 6 were excluded from the analysis because their subendocardial collateral flow was >15 ml per 100 g per min, and hence 30 dogs completed the protocol satisfactorily. Of these 30 dogs, 6 developed ventricular fibrillation, and hence these animals were also excluded from analysis. The number of dogs that were excluded from the analysis were 2, 0, 2 and 2 in the saline, carperitide, carperitide and L-NAME or L-NAME groups, respectively.

Heart rate and aortic blood pressure were similar among the four groups throughout this protocol. Neither the area at risk nor endocardial collateral blood flow in the LAD region during myocardial ischemia differed among the groups receiving saline, carperitide, carperitide and L-NAME or L-NAME (Table 1). Carperitide, however, decreased the infarct size compared with results from the group treated with saline (18.1±3.6% vs. 39.8±5.1% of the area at risk, respectively; P<0.05); this effect was blunted by L-NAME (18.1±3.6% vs. 41.6±2.2% of the area at risk in the groups treated with carperitide or carperitide and L-NAME, respectively; P<0.05; Figure 8a). Figure 8b shows the regression plots of the area at risk and endocardial collateral blood flow during ischemia. Carperitide mediated the substantial cardioprotection irrespective of collateral flow that was again blunted by L-NAME.

Table 1 Risk area and endocardial collateral blood flow during myocardial ischemia in each group
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Figure 8
figure 8

Effects of carperitide on myocardial ischemia and reperfusion. (a) Infarcts were smaller in the carperitide group compared with the control group, a difference that was abolished by Lω-nitroarginine methyl ester (L-NAME). Infarct size expressed as the plot of infarct size because of 90 min of ischemia and regional collateral flow during ischemia (b). There are inverse relations between normalized infarct area and collateral flow, and a significant difference (P<0.05) is seen in the carperitide group compared with the control group. ANP, atrial natriuretic peptide. *P<0.05 vs. the control group.

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Discussion

The present study showed that carperitide causes coronary vasodilation and promotes myocardial contractility and metabolism in ischemic hearts, the effects that are mediated by accumulation of cyclic GMP in the coronary artery and myocardium. In addition, carperitide potently decreases the infarct size following sustained ischemia and reperfusion. We also showed that inhibiting NO production attenuates carperitide-induced coronary vasodilation and reductions in the infarct size.

Role of NO in carperitide-mediated coronary vasodilation in ischemic hearts

The present study showed that carperitide increases CBF in a manner dependent on cyclic GMP. Because cyclic GMP induces vasorelaxation in smooth muscle cells, the carperitide-induced coronary vasodilation was likely a result of increased cyclic GMP levels. Several other effects of carperitide, however, should be considered. First of all, because carperitide attenuates catecholamine-induced cellular responses,23 the reduction of α-adrenoceptor activation by carperitide may cause coronary vasodilation. Indeed, we could not exclude the possibility that the carperitide-induced coronary vasodilation is mediated by coronary α2-adrenoceptor blockade. On the other hand, myocardial effects of carperitide may also be involved in the present observation. If this myocardial effect of carperitide was the case, carperitide should have also reduced norepinephrine-induced myocardial hypercontraction, leading to lower myocardial contractility and oxygen consumption. However, the present study revealed that carperitide did not alter myocardial oxygen consumption (Figure 1). It is well known that an increase in coronary perfusion increases myocardial oxygen consumption, termed the Gregg phenomenon. In turn, carperitide has a potency to decrease myocardial oxygen consumption via increased myocardial cyclic GMP levels that may blunt the Gregg phenomenon. We may have observed the mixed effects of carperitide that may culminate in no changes in myocardial oxygen consumption. Second, because (1) NO increases CBF24 and (2) L-NAME attenuates carperitide-induced coronary vasodilation, carperitide may enhance NO production. Because the levels of the end-product of cardiac NO (Figures 4c and 6c) did not increase in response to carperitide, this possibility appears unlikely. Third, carperitide may inhibit leukocyte activation and adhesion that occur in ischemic hearts.25 Because leukocyte adhesion decreases CBF by plugging small coronary arteries, reduced leukocyte adhesion may restore CBF. However, this would not be likely. A significant amount of binding between leukocytes and coronary endothelial cells would be required to reduce CBF, and this degree of adhesion would not likely be attenuated quickly following carperitide infusion. The effects of carperitide, however, were reversible as shown in Figures 2 and 3. Fourth, carperitide may open collateral flow that may increase CBF. In the present study, carperitide increased CBF in the perfused region that was measured with an electromagnetic flow probe attached to the bypass tube in the coronary hypoperfusion model (Figure 2b), indicating that carperitide seemed to increase coronary forward flow independent of the collateral flow. Furthermore, the myocardial End/Epi flow ratio was augmented by carperitide (Figure 3), suggesting that carperitide-induced increases in coronary flow is not luxury flow but effective flow to the ischemic myocardium. On the other hand, the present study also showed that carperitide did not increase the myocardial collateral blood flow during myocardial ischemia in the total coronary occlusion model (Table 1), suggesting that carperitide may not increase collateral flow. Further study is needed to study the effects of carperitide on collateral flow in the animal model with less collateral flow in the future. Fifth, carperitide attenuates oxidative stress26, 27 that may contribute to coronary vasodilation in ischemic hearts because oxygen-derived free radicals reduce coronary vasodilation by reducing NO bioavailability.28, 29 However, cardiac NO levels decreased in response to carperitide, arguing against this possibility.

How does carperitide affect NO in ischemic hearts? Our results demonstrate that carperitide does not increase NO production in ischemic hearts. Alternatively, the inhibition of NOS may deactivate downstream ANP receptors because L-NAME attenuates increases in cyclic GMP levels following ANP receptor activation. Several lines of evidence support the idea that NO modulates carperitide activity:11, 12, 13, 14 both factors signal via cyclic GMP and the soluble and particulate guanylate cyclase pathways. It is reported that ANP increases cardiac NOS activity and thus cardiac NO synthesis;30 the same authors reported that ANP increased NOS activity, but the activation was lower in spontaneously hypertensive rats than Wistar-Kyoto rats.31 Intriguingly, carperitide significantly decreased NO production in ischemic hearts (Figures 4c and 6c) in the present study. These data seem to be contradictory, but this is not the case. One possible explanation is that cardiac NOS activity may be already saturated in the coronary hypoperfusion model of the present study, and carperitide-induced NO-independent coronary vasodilation may become a major determinant of coronary vascular tone. Another possibility is that these differences may be attributable to the variations in experimental models (normotensive vs. hypertensive hearts and nonischemic vs. ischemic hearts). Whatever the mechanisms are, this study is the first to reveal a relationship between carperitide and endogenous NO in ischemic hearts. At present, however, no available evidence details how the sensitivity of ANP receptors or signal transduction following ANP receptor activation is reduced by inhibiting NOS.

Furthermore, it is reported that ANP may ameliorate endothelial dysfunction by upregulating endothelial NOS (coded by NOS3 gene) and downregulating inducible NOS (coded by NOS2 gene).32 However, as we used L-NAME, a nonselective NOS inhibitor, to examine the involvement of NOS in the carperitide-induced cardioprotection, we could not clarify what type of NOS is activated in hypoperfused and/or ischemic canine hearts by treatment with carperitide. This would be the next target to elucidate the relationship between carperitide and NO.

Role of NO in carperitide-mediated cardiac function in ischemic hearts

It is reported that NO prevents cellular damages by induction of the rapid recovery to normal pH after ischemia/reperfusion via a guanylyl cyclase/cyclic GMP/protein kinase G (PKG) signaling cascade, and thus inhibits mitochondrial permeability transition (MPT).33 Yang et al.34 reported that ANP infusion decreased infarct size of the risk area, and this effect was mimicked by a cyclic GMP analog that directly activates PKG and likely by opening of mitochondrial KATP channel and stimulation of downstream kinases. Furthermore, Cohen et al.35 reported that postconditioning prevents mitochondrial permeability transition pore (MPTP) formation by maintaining acidosis during the first minutes of reperfusion. Recently, soluble and particulate guanylate cyclase activator exerts cardioprotective effects via cyclic GMP/PKG signaling cascade and activates phospholamban phosphorylation.36 Therefore, these myocardioprotective mechanistics of carperitide in addition to the coronary vasodilation may be involved in the cardioprotective effects of carperitide in ischemic heart.

Carperitide limits the infarct size

We showed that carperitide reduces infarct size, and this was also revealed in a previous study.37 Myocardial infarction is caused by many factors, including free radial generation, platelet aggregation, myocardial calcium ion overload, leukocyte activation and excess catecholamines, each of which is reportedly attenuated by carperitide. Carperitide exerts protective effect of cyclic GMP/PKG signaling pathway during reperfusion in isolated myocytes or isolated hearts on top of the vasodilatory effects of carperitide to reduce cardiac preload and afterload, all of which may mediate cardioprotection. However, even if collateral flow is increased by carperitide, the collateral flow-independent infarct size limitation caused by carperitide largely exists because the infarct size-limiting effect of carperitide is observed even after normalization by the collateral flow (Figure 8b). More importantly, clinical observations have shown that carperitide is cardioprotective against ischemia and reperfusion injury.10, 38

Clinical implications

Carperitide improves the pathophysiology of acute decompensated HF,8, 9 whereas a recent large clinical trial (ASCEND study) suggested that nesiritide, the recombinant human brain natriuretic peptide (BNP), does not reduce mortality and morbidity in patients with acute decompensated HF.39 Our group and others have reported that carperitide limits infarct size in humans,10, 38 and the present study showed this cardioprotection may require NO. It is to be noted that nitrate is usually administered to patients with AMI that may enhance the effects of carperitide. If this is the case, it would be important to maintain NO at levels that are sufficient to strengthen the effects of carperitide.