Natriuretic peptide infusion reduces myocardial injury during acute ischemia/reperfusion

Kousholt, Birgitte S.a,b; Rolighed Larsen, Jens K.a; Bisgaard, Lineb; Burnett, John C. Jrc; Hasenkam, John Michaela; Goetze, Jens P.b

Cardiovascular Endocrinology:
doi: 10.1097/XCE.0b013e328356c67b
Original article

Aim: The aim of this study was to determine whether a natriuretic peptide infusion during reperfusion can reduce cardiomyocyte ischemia–reperfusion damage.

Materials and methods: The effect of B-type natriuretic peptide (BNP) activity was assessed in vitro and in vivo: the cellular effect was determined by assessment of intracellular caspase activity and troponin T release from cultured HL-1 cells subjected to short-term hypoxia–reperfusion. Cardiac effects were further examined in pigs (n=25) that had been subjected to 1 h of regional cardiac ischemia, followed by 3 h of reperfusion.

Results: HL-1 cardiomyocytes responded to exogenous BNP with increased cGMP activity (∼3-fold, P=0.0037) and hypoxia–reperfusion with increased vascular endothelial growth factor and BNPmRNA contents (2.3- and 2.5-fold, respectively, P<0.0001) and caspase activity (2.9-fold, P=0.03), but without a decrease in apoptotic changes in the BNP-stimulated cells. Pigs tolerated the BNP and CD-NP (a CNP analogue) infusion well, with a decrease in systemic blood pressure (∼15 mmHg) and increased diuresis compared with the controls. Left ventricular pressure decreased in the pigs that received BNP infusion compared with controls (P=0.02). A similar trend was observed in the pigs that received CD-NP infusion, although this was not significant (P=0.3). BNP and CD-NP infusion in pigs reduced total cardiac troponin T release by 46 and 40%, respectively (P=0.0015 and 0.0019), and were associated with improved RNA integrity in the ischemic left ventricular region (P<0.05).

Conclusion: We report that natriuretic peptide infusion in vivo reduces cardiomyocyte injury in acute ischemia–reperfusion, possibly through indirect mechanisms (e.g. increased diuresis and vasodilation). The results suggest a role for natriuretic peptide therapy in human cardiac ischemia.

Author Information

aDepartment of Cardiothoracic and Vascular Surgery, University of Aarhus, Aarhus

bDepartment of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark

cCardiorenal Research Laboratory, Mayo Clinic, Rochester, Minnesota, USA

Correspondence to Jens P. Goetze, Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, 2100 Copenhagen, Denmark Tel: +45 354 52202; fax: +45 354 52524; e-mail:

Received April 19, 2012

Accepted June 8, 2012

Article Outline
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Ischemic heart disease is a major health problem in the western world 1. Modern treatment strategies aim at reperfusing the ischemic area as early as possible 2. However, patients are still at risk of heart failure and death. Thus, there is a need for adjunctive interventions to further prevent cardiomyocyte dysfunction and injury 3.

The natriuretic peptides [atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP)] are released from the cardiomyocytes in response to increased workload 4 and acute ischemia 5. ANP and BNP induce an increase in renal sodium excretion and vascular permeability, and elicit vasodilation 6,7. Interestingly, genetically modified mice devoid of the BNP gene or its receptor [natriuretic peptide receptor type-A (NPR-A)] develop cardiac hypertrophy and fibrosis 8,9. These findings suggest that natriuretic peptides may be cardioprotective during myocardial stress and disease 10. A few reports have confirmed this suggestion 11–14. Beneficial effects, such as reduced release of myocardial creatinine kinase isoform MB, have also been suggested in a clinical placebo-controlled trial on ANP infusion during myocardial infarction 15.

The underlying mechanism for reducing ischemia–reperfusion injury may be mediated in several ways, for example through cardiac antiapoptosis 16,17 and altered coronary blood flow 18, or indirectly by reducing systemic blood pressure and cardiac workload 19. The cardiac effect may also be associated with other neurohumoral systems, such as the renin–angiotensin–aldosterone axis. In this study, we examined whether BNP stimulation reduced ischemia–reperfusion injury in cultured murine cardiomyocytes and in anesthetized pigs subjected to regional cardiac ischemia, followed by reperfusion. Our results show that natriuretic peptides reduce cardiomyocyte injury, possibly through indirect mechanisms.

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Materials and methods

The experimental surgery and protocols described below were submitted to and approved by the Animal Experiments Expectorate in Denmark.

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Murine HL-1 cells (a generous gift from Dr Claycomb) were cultured as described previously 20. The cell concentrations and viability were measured using a cell counter (Countess Automated Cell Counter; Invitrogen, Taastrup, Denmark).

An experimental setup of simulated ischemia–reperfusion (SIR) was established. HL-1 cells were grown at a density of 3×105 cells/well (six-well plates). After 48 h, the cell medium was substituted with a hypoxia medium as described previously 21. The plates were transferred to a hypoxia box containing an AnaeroGen sachet (Oxoid Ltd, Roakilde, Denmark) and incubated at 37°C. Anoxia was achieved after approximately 2½ h, determined by an Oxoid Anaerobic Indicator (Oxoid Ltd). After 4 h of anoxia, cells were removed from the hypoxia box and their medium was changed to supplemented Claycomb media without noradrenaline (NA). Cells were then stimulated with murine BNP-45 (10−6 or 10−7 mol/l; Phoenix Pharmaceuticals, Mannheim, Germany) and incubated under normal O2 conditions for 4 h. Control cells were incubated with supplemented medium without NA from the beginning of the experiment.

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Pigs subjected to 1 h of regional ischemia followed for 48 h

Four Danish-bred pigs, all 3–4-month-old females weighing ∼25 kg, were used. They were premedicated with midazolam (0.5 mg/kg; Dormicum, Hameln, Germany). A mixture of S-ketamine (5 mg/kg; Pfizer, Ballerup, Denmark) and midazolam (0.5 mg/kg) was then administered intramuscularly to maintain sedation. Anesthesia was administered by an intravenous injection of propofol (4–6 mg/kg; B Braun Melsungen AG, Melsungen, Germany). The pigs were intubated and ventilated with 50% oxygen (Datex Bikeroed, Ohmeda 5/s Avance; GE Health Care, Ballerup, Denmark). Anesthesia was maintained using sevoflurane (100%; Abbott Scandinavia AB, Solna, Sweden) (Mac value 1.2–1.8%). Analgesia was induced by two boluses of 100 µg fentanyl intravenously (Haldid; Janssen-Cilag, Binkeroed, Denmark) administered at a 10-min interval and continued by an infusion of 100–200 µg/min throughout the anesthesia period. Potassium infusion (10–15 mmol/h) and saline infusion (0.9%, 10–15 ml/kg/h) were maintained throughout the anesthesia period to prevent fluid loss and to maintain serum potassium concentrations between 4 and 5 mmol/l.

A central venous catheter was placed transcutaneously into the right external jugular vein (0.032 inch, CVC with a blue flex tip; Arrow International, Diegem, Belgium). A 6-Fr sheath was surgically inserted into the left carotid artery. Heparin was administered (2500, i.e., followed by 1500, i.e., each hour during anesthesia) (Heparin; SAD, Copenhagen, Denmark) to prevent thrombus formation. Amiodarone (1 mg/kg, Cordarone; Sanofi-Aventis, Copenhagen, Denmark) was administered intravenously to avoid arrhythmia. A coronary balloon dilatation catheter (Sprinter RX; Medtronic Inc., Minneapolis, Minnesota, USA) was inserted through the carotid artery sheath using a 4 JL-type launcher (Medtronic Inc.) 22. The left anterior descending coronary artery (LAD) was occluded distal to the second diagonal branch. The balloon (2.5×6 mm) was left inflated for 1 h. Contrast fluoroscopy verified a zero-flow occlusion. Anesthesia was maintained for 1½ h after reperfusion was established. Peroperatively, the pigs were injected intramuscularly with flunixin (2.8 mg/kg, Finadyne; Intervet/Schering-Plough, Ballerup, Denmark), and treated again 24 h later (2.8 mg/kg) to sustain analgesia. Blood samples were collected every hour during the first 4 h and every fourth hour during the remaining 44 h. After 48 h, animals were sedated using midazolam intravenously (0.5 mg/kg). Anesthesia was induced by propofol (4–6 mg/kg) intravenously. The pigs were intubated and ventilated with 50% oxygen. Anesthesia was maintained by sevoflurane (Mac value 1.2–1.8%). Analgesia was induced by two doses of 100 µg fentanyl intravenously administered at a 10-min interval and a continuous infusion of 200 µg/min. A sternotomy was performed. A ligation band was placed around the LAD at the occlusion site according to the contrast fluoroscopy performed 48 h before. The LAD was reoccluded and 8 ml Evans blue dye (10%; AppliChem Scandinavia, Kongens Lyngby, Denmark, Denmark) was injected into the left auricle to delineate the area at risk (AAR). Animals were then sacrificed by evisceration. The myocardium was trimmed and sliced into 8 mm thick slices. Planimetry was performed using computer-imaging software (ImageJ 1.34j; National Institutes of Health, Bethesda, Washington, USA) to determine infarct size (IS) and AAR. Sizes were calculated by multiplying the area by the thickness of the slice 23.

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Pigs subjected to myocardial ischemia–reperfusion and peptide infusion

Twenty-five Danish-bred pigs (as described above) were allocated to one of three groups: a BNP-32 infusion, a C-type natriuretic peptide (CD-NP) infusion, and a control group. BNP-32 and CD-NP groups received synthetic porcine BNP-32 (Phoenix Peptides, Karlsruhe, Germany) or CD-NP (consisting of human CNP-22 C-terminally extended with 15 amino acid C-terminus of the dendroaspis natriuretic peptides. CD-NP was a generous gift from Nile Therapeutics (San Mateo, California, USA). Both peptides were infused at a dose of 0.05 µg/kg/min. This dose was chosen on the basis of earlier reports in humans 6,13,19 and pilot studies in our group. The control group received an equal volume of an isotonic saline infusion.

The pigs were premedicated as described above. Anesthesia was initiated by intravenous pentobarbital (3–5 mg/kg, Mebumal, SAD, Copenhagen, Denmark). The pigs were ventilated with 50% oxygen. Analgesia was induced using two doses of 50 µg fentanyl intravenously at 10-min intervals. The anesthesia was maintained by an intravenous administration of pentobarbital (10 mg/kg/min) and fentanyl (50 µg/h). A potassium infusion and a saline infusion, as mentioned above, were maintained throughout the experiment.

A sheath was surgically inserted into the right and the left external jugular vein and carotid artery. A Swan-Ganz catheter was positioned in the pulmonary artery. A Millar Mikro-Tip pressure catheter (Vingmed A/S, Taastrup, Denmark) was positioned in the left ventricle. Heparin (2500, i.e., followed by 1500, i.e., each hour) and amiodarone (1 mg/kg) were administered. The left anterior descending (LAD) was occluded as described above and by Larsen et al. 22. An infusion of BNP-32, CD-NP, or placebo was initiated 5 min before reperfusion. The ischemic area was reperfused for 3 h (Fig. 1). The animals were maintained at a steady body temperature throughout the procedure.

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Plasma sampling

Blood was collected before the induction of ischemia and every hour in Na2-EDTA tubes. The blood samples were left on ice for 30 min, centrifuged at 2500 rpm at 4°C, and the plasma was stored at −80°C.

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Hemodynamic assessments

Left ventricular pressure (LVPmax) was monitored using a Millar Mikro-Tip pressure catheter (Vingmed A/S). All data were converted digitally using NI Lab-VIEW 2009 (Texas Instruments, Horsholm, Denmark).

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Myocardial tissue

A sternotomy was performed at the end of reperfusion. A ligation band was placed around the LAD corresponding to the occlusion site. The snare was tightened and reoccluded the LAD. Eight milliliters of Evans blue dye (10%) was injected directly into the left auricle to delineate the ischemic area. The animal was sacrificed by evisceration. Transmural tissue biopsies were collected from the appendage of the left and the right atria, from the right ventricle, and from the ischemic and nonischemic areas of the left ventricle. The biopsies (250–500 mg) were frozen immediately in liquid nitrogen and stored at −80°C.

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Estimation of myocardial IS and AAR

The heart was sliced into 5–7 mm transverse sections and incubated in 1% triphenyltetrazolium chloride (TTC) in phosphate buffer (pH 7.4) at 37°C for 10 min. Planimetry was performed using computer-imaging software as described above to estimate IS and AAR. Sizes were calculated by multiplying the area by the weight of the slice 23. The precision was tested by calculating the variation coefficient from duplicate measurements.

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cTnT measurement

Cardiac troponin T (cTnT) was measured in cell media and blood plasma using an automated assay (Modular P; Roche Diagnostics, Mannheim, Germany). This assay utilizes monoclonal antibodies directed against conserved epitopes in pig, mouse, and human cTnT. The interassay coefficient of variation is less than 3%.

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Intracellular transduction activity mediated by BNP stimulation

Intracellular cGMP was measured using an ELISA kit (Sigma-Aldrich, Copenhagen, Denmark). Briefly, HL-1 cells were stimulated with murine BNP-45 for 15 min and subsequently lysed in 0.1 mmol/l HCl at 37°C for 20 min. The supernatant was centrifuged for 20 min at 600g at room temperature, and the cGMP content was quantified according to the kit insert.

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Apoptosis assay

Caspase activity was assessed using a Caspase-Glo 3/7 Assay (Promega, Nacka, Sweden). This luminescent assay measures caspase-3 and caspase-7 activities in cultures of adherent or suspension cells. HL-1 cells were harvested from the plates and the cell concentrations were determined. The caspase activity in 1000 cells were then determined according to the manufacturer’s protocol.

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Natriuretic peptide measurement

The plasma concentrations of the infused peptides were quantified by radioimmunoassays for porcine BNP-32 and CNP-22, respectively (Phoenix Peptides). Endogenous porcine proANP was measured using a processing-independent immunoassay developed recently in our department 24; the analytical principle has been reviewed elsewhere 25. The interassay CV was less than 15%.

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mRNA analyses

Total RNA was isolated with TRIzol (Invitrogen). The RNA integrity was evaluated on an Agilent Bioanalyzer 2100 (Agilent Technologies, Waldbronn, Germany). The integrity was expressed as RNA integrity number (RIN) values on the basis of the ratio of 28S to 18S of ribosomal RNA 26. The mRNA was thereafter evaluated by real-time PCR. The contribution of contaminating DNA was earlier found to be negligible. cDNA and subsequent RNA amplification was performed as described previously for BNP, vascular endothelial growth factor (VEGF), β-actin, and glyceraldehyde 3-phosphate-dehydrogenase (GAPDH) 5. For ANP mRNA amplification, we used the following primers: 5′-ACGACGCCAGCATGAGCTCCTTC-3′ and 5-GCTGTTATCTTCAGTACCGGAA-3′. All data were normalized to β-actin and GAPDH before data analyses. RNA extraction and analysis of mRNA from HL-1 cells were performed as described above. The primers used for PCR were as follows: VEGF, 5′-AGTCCCATGAAGTGATCAAGTTCA-3′ and 5′-ATCCGCATGATCTGCATGG-3′; 18S, 5′-CGCGGTTCTATTTTGTTGGT-3′ and 5′-AGTCGGCATCGTTTATGGTC-3′; ANP, 5′-CCTGTGTACAGTGCGGTGTC-3′ and 5′-AGATCTATCGGAGGGGTCCCA-3′; and BNP, 5′-CTGAAGGTGCTGTCCCAGAT-3′ and 5′-GTTCTTTTGTGAGGCCTTGG-3′. All data were normalized to 18S before data analyses.

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Statistical analyses

Data are expressed as mean±SEM unless otherwise stated. LVPmax was analyzed by two-way analysis of variance with repeated measurements. Otherwise, for comparison between groups, we used one-way analysis of variance with Tukey’s multiple comparison tests. For comparison between two groups, we used Student’s t-test with Welch’s correction. P-values less than 0.05 were considered statistically significant; the Prism 5.0 software was used for all calculations (Graphpad Software Inc., La Jolla, California, USA).

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BNP stimulation dose-dependently increases intracellular cGMP

To determine an intracellular effect of BNP-45 in HL-1 cells, we assessed the cGMP response in a dose-dependent manner. BNP stimulation at 100 nmol/l increased cGMP by ∼three-fold (P=0.0037; Fig. 2a), which corroborates the functional presence of NPRs on HL-1 cells.

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SIR increases VEGF and BNP mRNA contents

VEGF mRNA contents in HL-1 cells increased 2.3-fold in the SIR setup compared with the control cells (P<0.0001; Fig. 2b). Moreover, we observed a 2.5-fold increase (P<0.0001) in the BNP mRNA content in the ischemia–reperfusion setup cells (data not shown). Taken together, this confirms hypoxia-induced transcriptional activation in HL-1 cells 5,27.

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No effect of BNP stimulation on apoptosis and cellular damage

Apoptosis was evaluated after SIR with (10−6 or 10−7 mol/l) and without postconditional BNP stimulation in HL-1 cells (Fig. 2c). Caspase activity increased 2.9-fold [12 807±2703 relative light units (RLU)] compared with the control cells (4457±612 RLU, P=0.03). However, no effect on caspase activity was observed after BNP stimulation (BNP 10−6 mol/l=9178±973 RLU, P=0.25; BNP 10−7 mol/l=10 688±1736 RLU, P=0.53) compared with the controls (12 807±2703 RLU). cTnT in cell media increased after hypoxia/reperfusion (100±12 ng/l, Fig. 2d) compared with the controls (25±2 ng/l, P=0.002). No cTnT changes were observed in the intervention groups compared with the controls (BNP 10−6 mol/l=91±7 ng/l, P=0.29; BNP 10−7 mol/l=83±10 ng/l, P=0.50). Taken together, the in-vitro results do not support a local effect of BNP stimulation on ischemia–reperfusion damage. We therefore tested BNP and CD-NP infusion in a porcine cardiac ischemia–reperfusion model.

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Single-phase cTnT release in pigs subjected to 1 h of regional ischemia followed for 48 h

One pig was excluded because of failure to induce sufficient ischemia. In the remaining animals, the IS, defined as IS/AAR, was 30.3±2% after 48 h, which parallels earlier experiments using porcine models. Furthermore, the cTnT release (Fig. 3a) was single phased after the ischemic period and not followed by later washout or release. The cTnT release was therefore considered to be a valid measure of myocardial damage in a 4-h model.

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Physiological concentrations of infused natriuretic peptides in pigs subjected to acute ischemia–reperfusion

In the peptide infusion experiment, the animals tolerated the infusion regimens well. Three pigs developed cardiac arrest before peptide infusion. Three animals (one in the BNP-32 group and two in the CD-NP group) were excluded after the experiments because of unsuccessful peptide infusion. The study thus included 19 consecutively included animals. The peak natriuretic peptide plasma concentrations were similar in the BNP-32 and the CD-NP groups (1003±212.5 vs. 982±45 pmol/l) 2 h after the initiation of reperfusion.

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Natriuretic peptide infusion lowers blood pressure and stimulates diuresis

There was no difference in the baseline hemodynamic measures and body core temperature at baseline (Table 1). A decrease in the mean arterial pressure in the BNP-32 group was observed during the last hour of reperfusion compared with the controls (∼15 mmHg, P<0.0001), and a similar decrease was observed in the CD-NP group (∼12 mmHg, P=0.0052). A reduction in the mean pulmonary artery pressure was also found in both the groups. The BNP-treated and CD-NP-treated animals showed increased diuresis throughout the experiment (data not shown). No differences were found in cardiac output, pH, temperature, bicarbonate, lactate, nor glucose between the three groups.

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Effects of natriuretic peptides on left ventricular function

We found no difference in left ventricular function between the three groups during stabilization. During reperfusion, there was a decrease in the LVPmax in the BNP-32 infusion group compared with the control group (P=0.02, Fig. 4a). In the CD-NP group, this trend was not significant (P=0.3; Fig. 4b).

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Reduced myocardial damage in natriuretic peptide-infused animals

The total cTnT release was assessed as the area under the curve during the peptide infusion period, that is second, third, and fourth hour (Fig. 3b). A 46% decrease was found in the BNP-32 infusion group compared with the controls (P=0.0015, Fig. 3c). In parallel, a 40% decrease was found in the CD-NP group (P=0.0194, Fig. 3d), with no significant difference between the BNP-32 and the CD-NP groups.

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Estimation of IS evaluated by TTC staining did not indicate significant differences between the peptide-infused animals and the control group (35–44%, n=14). However, the quality of staining varied for the AAR and could only be defined clearly in 14 animals (BNP group: 36±7, n=4; CD-NP group: 44±10, n=5; control 38±10, n=5). Also, the analytical variation (CV) for this method was found to be greater than 20%.

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Preservation of mRNA integrity in natriuretic peptide-infused animals

In the nonischemic left ventricle, the mRNA contents were evaluated by RT-PCR. The ANP mRNA content was marginally reduced in the BNP-infused animals compared with the controls (Supplementary data, Fig. 5a). In contrast, there was no effect on the BNP mRNA contents in the normoxic myocardium (Supplementary data, Fig. 5b). Before gene-specific mRNA analyses, the total RNA integrity was tested. This integrity analysis yields a more robust overview of the ribosomal RNA status within the cells 26. Interestingly, the RIN was better preserved in the infusion groups, with an average 1.2–1.4 higher RIN values compared with the control animals (Supplementary data, Fig. 5c). The RIN in all the samples generally indicated ongoing RNA degradation reflected by relatively reduced RIN values. Specific mRNA analyses of the ANP and BNP transcripts in the ischemic myocardium indicated no difference in the ANP mRNA contents (Supplementary data, Fig. 5d), whereas the BNP mRNA contents were higher in the BNP infusion group compared with the controls (Supplementary data, Fig. 5e). The VEGF mRNA showed 7.6–18-fold higher contents in the infusion groups (Supplementary data, Fig. 5f). Hence, our in-vivo data parallel the findings in HL-1 cells on hypoxia-induced VEGF and BNP mRNA stimulation.

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BNP infusion selectively reduces endogenous proANP in plasma

An expected increase in plasma proANP after induction of ischemia was observed in the porcine model (Supplementary data, Fig. 6a). Surprisingly, BNP infusion resulted in an inverse plasma profile with decreased proANP concentrations after ischemia and throughout the reperfusion period (Supplementary data, Fig. 6b). This effect was not observed in the CD-NP group (Supplementary data, Fig. 6c).

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We report that the infusion of BNP-32 and a chemical homologue to CD-NP during reperfusion reduces cardiomyocyte injury in an acute porcine model of ischemia and reperfusion damage, possibly through indirect mechanisms (e.g. increased diuresis and vasodilation).

First, we assessed whether a potential cardioprotective role for BNP stimulation could be established in-vitro using cultured cardiomyocytes. BNP stimulation increased intracellular cGMP in a concentration-dependent manner, which corresponds well to earlier reports by Kato et al. 16. In contrast, we did not observe a reduction in cardiomyocyte injury as assessed by intracellular caspase activity. This difference may be because of differences in the choice of cell culture, induction of simulated ischemia, and natriuretic peptide stimulation (concentration) setup. Although the number of NPR receptors expressed on HL-1 cells was not quantified, we found that the model is responsive to BNP stimulation, and that, at least at the concentrations used, no apparent effect was observed when estimating apoptosis by caspase activity.

As no effect of BNP stimulation on cardiomyocyte injury in vitro was observed, we tested the peptide infusion in a whole animal model using pigs. Moreover, we tested two different natriuretic peptides: BNP-32, which preferentially binds to the NPR-A receptor, and chimeric CD-NP, which possibly binds to both the NPR-A and the NPR-B receptor. Notably, CD-NP has been reported not to affect blood pressure in humans 28. Surprisingly, BNP-32 and CD-NP showed almost similar peripheral effects with a decrease in the mean arterial pressure and a cumulative increase in diuresis. These findings indicate that the peripheral effects are mainly mediated through the NPR-A receptor, which is the principal peripheral receptor for ANP-mediate and BNP-mediated vascular and renal actions. Moreover, the similar decreases in blood pressure using both peptides also suggest that this decrease may contribute to the reduction in myocardial injury and preserved RNA integrity. Future experiments should ideally directly compare peptide infusion against a drug that only lowers blood pressure and has a limited effect on the nitric oxide system. This could be disclosed by using a porcine specific-blocker of NRP-A in combination with a blood pressure lowering compound. Unfortunately, the authors do not know of any porcine specific NPR-A blocker. The receptor affinity for CD-NP seems more complex, where studies in humans have shown that this chimeric peptide has an affinity for both the NPR-A receptor as well as the CNP-specific NPR-B receptor 29. Whether these receptor affinities can be extrapolated to porcine receptors is unclear. Natriuretic peptide infusion in canine heart failure suggests that both BNP and CD-NP may be beneficial, although the peripheral effects of BNP may be problematic. Different peptide doses would probably also elicit different hemodynamic responses. To this end, CD-NP has earlier been found to cause less hypotension than BNP – at least in canine models and humans 28,30.

Our porcine model of acute ischemia and reperfusion showed a marked reduction in cTnT release. The ∼45% reduction in total cTnT during the experiment strongly suggests reduced cellular necrosis. Our 48-h cTnT measurement in an identical model of regional cardiac ischemia showed a single-phase cTnT release during the first few hours after the onset of reperfusion. Thus, we believe that the cTnT measures are representative of the total cellular injury and membrane lysis – also in the 4-h experiments. Unfortunately, our histological evaluation of IS/AAR was unsuccessful. Several factors may contribute to this: the histological technique was not optimal in our hands, with an imprecision profile greater than 20%, probably because of the indecisive delineation of the AAR. Second, the short period of ischemia and reperfusion is not likely to represent the final infarct and can only be used as an indicator of the final anatomical lesion. Finally, species differences exist, where the final size of the myocardial infarct is more rapidly accomplished in rodents than in dogs and pigs (reviewed in 31). All possible measures of myocardial damage should consequently be considered when interpreting the end myocardial effects in whole animal models 10. In this context, it is noteworthy that the total RNA integrity measured as RIN in the ischemic area showed a clear reduction in RNA degradation in the BNP-infused and CD-NP-infused animals. Although it is difficult to evaluate total RNA degradation from singular mRNAs (because of, for instance, the transcripts used for normalization), the total integrity analysis provides a more robust overview of the ribosomal RNA status within the cells 26. We speculate that cytochemical measures of ischemia other than TTC and Evans Blue staining may be informative in the early period of ischemia–reperfusion damage, for example, staining to complement C4D deposition 32.

The selective response on endogenous proANP in plasma is surprising. Although this has been suggested in an earlier report 13, this is the first demonstration of BNP-32 mediated feedback on cardiac peptide secretion. As the effect was observed during the short duration of the experiments, we suspect that a lack of acute secretion primarily represents effects on atrial myocytes, as ventricular myocytes do not contain granules for peptide storage 5. An effect on gene transcription would first be expected after 2–3 h of the start of infusion. As the effect was BNP specific, the NPR-A receptor seems to be the main receptor mediating this effect. Interestingly, the expected increase in plasma proANP during ischemia 33 may also have pathophysiological relevance. First, endogenous peptides may serve as a molecular ‘guard’ against acute ischemic damage, where the observed inhibition of endogenous release could be associated with increased long-term cardiac damage. In addition, the use of proANP-derived peptides as prognostic biomarkers during AMI 34 could be complicated by this negative feedback effect during BNP-32 infusion. Survival experiments must therefore determine whether the inhibition in itself will affect the long-term infarct size, fibrotic infiltration, and cardiac function.

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We show that an infusion of natriuretic peptides reduces cardiomyocyte injury and maintains mRNA integrity in the acute phase of ischemia and reperfusion damage, possibly mediated through indirect mechanisms (vasodilation and increase in diuresis leading to a decrease in preload and afterload). Future studies should determine which peptide will be most beneficial in long-term models of cardiac damage and survival.

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The study was supported by the Danish Heart Foundation, Copenhagen, Denmark, the Lundbeck Foundation, Copenhagen, Denmark, the Familien Hede Nielsens Foundation, Horsens, Denmark, Raimond and Dagmar Ringgaard Bohn’s Foundation, Copenhagen, Denmark (to B.S. Kousholt), and the Messerschmidt Foundation, Copenhagen, Denmark, and a grant from the Research Council at Rigshospitalet (to J.P. Goetze).

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Conflicts of interest

There are no conflicts of interest.

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BNP; CD-NP; ischemia–reperfusion injury; myocardial infarction; porcine

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