Scandinavian Journal of Clinical & Laboratory Investigation, 2014; 74: 27–36
ORIGINAL ARTICLE
Confined ischemia may improve remote myocardial outcome after rat cardiac arrest
ZANXIN WANG1,2, HONGYAN LI1, VILMA VUOHELAINEN1, JYRKI TENHUNEN3, MARI HÄMÄLÄINEN4, TIMO RINNE5, EEVA MOILANEN4, TIMO PAAVONEN6, MATTI TARKKA1 & ARI MENNANDER1 1Heart
Center, Cardiac Research, Tampere University Hospital and Tampere University, Tampere, Finland, of Cardiovascular Surgery, Tianjin Medical University General Hospital, Tianjin, P. R. China, 3Department of Surgical Sciences/Anesthesiology and Intensive Care Medicine, University of Uppsala, Sweden and Critical Care Medicine Research Group in the Department of Intensive Care Medicine, Tampere University Hospital, Tampere, Finland, 4The Immunopharmacology Research Group, University of Tampere School of Medicine and Tampere University Hospital, Tampere, Finland, 5Department of Anesthesiology, Tampere University Hospital, Tampere, Finland, and 6Department of Pathology, Fimlab and Tampere University, Tampere, Finland 2Department
Abstract Background. Confined ongoing ischemia after ischemia-reperfusion injury (IRI) may alter myocardial recovery. We evaluated in a rat cardiac transplantation model whether distal persistent myocardial ischemia (dMI) and remote preconditioning (RPreC) have a remote myocardial impact after IRI. Material and methods. Syngeneic heterotopic cardiac transplantation was performed on 29 Fischer344 rats to induce IRI, including nine rats which underwent distal ligation of the left anterior coronary artery (LAD) to yield distal MI (IRI dMI). RPreC was applied by occluding the left renal artery 5 min prior to reperfusion in six rats with IRI (IRI RPreC) as well as in seven with distal MI (IRI dMI RPreC). Microdialysis, histology and qRT-PCR for inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were performed after graft harvesting. Results. In contrast to IRI dMI RPreC (39 7 μmol), glutamate decreased in IRI RPreC and IRI dMI as compared with IRI (26 3 and 31 8 vs 91 20, μmol respectively, p 0.007). The relative number of vacuolated intramyocardial artery nuclei decreased in IRI dMI as compared with IRI (0.02 0.01, range 0–12 vs. 0.42 0.31, range 0–3.25 PSU respectively, p 0.04). iNOS expression decreased in IRI RPreC as compared with IRI (p 0.04), and eNOS expression decreased in IRI dMI RPreC as compared with IRI dMI (p 0.006) along with increased glycerol release. Conclusions. dMI after IRI has a potentially beneficial myocardial impact after cardiac arrest, which is hampered by RPreC. Key Words: Cardiac arrest, ischemia-reperfusion, rat, remote preconditioning, distal myocardial infarction
Introduction Complete reperfusion is the gold standard of cardiac surgery after ischemia-reperfusion injury (IRI) [1], though it is often anticipated that after coronary artery bypass grafting a small ischemic myocardial area causes minimal, if any, remote myocardial changes. However, the effects of a persistent local cardiac ischemic area on remote myocardial microvasculature remain poorly understood after cardiac arrest causing IRI [2]. A distal myocardial ischemia
(dMI) may have an impact on the remote myocardium after IRI, even though the confined ischemic area nourished by the non-revascularized coronary artery is significantly small, or a relatively short duration of cardiac arrest necessary during surgery provides quick early functional recovery [1,3,4]. We have previously shown that myocardial infarct has a remote myocardial impact on increasing small intramyocardial artery edema [5,6]; we accomplished myocardial infarct by occluding the proximal part of
Correspondence: Ari A. Mennander, Cardiac Research, Heart Center, Tampere University Hospital, SDSKIR, Teiskontie 35, PL 2000 Tampere, Finland. Tel: 358 3 31164945. Fax: 358 3 31165756. E-mail: [email protected] (Received 15 February 2013 ; accepted 12 October 2013) ISSN 0036-5513 print/ISSN 1502-7686 online © 2014 Informa Healthcare DOI: 10.3109/00365513.2013.855944
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the left anterior descending coronary artery (LAD), in order to obtain a prominent myocardial ischemic area. In this study, we investigated whether dMI may alter the remote myocardium during IRI [4]. It has been shown earlier that abrupt reperfusion flow may cause ischemia reperfusion injury and remote preconditioning exerts a beneficial impact [7]. However, it is not clear whether a relatively short duration of remote preconditioning would afford sustainable cardioprotection after cold and warm ischemia. Using the rat cardiac transplantation model, we also evaluated whether remote preconditioning of the kidney (RPreC) may have a comparable effect, as well as whether or not RPreC, together with dMI, may have an additional remote myocardial impact during IRI. Ideally, decreased excessive energy consumption of the heart during cardiac surgery would enhance recovery after IRI. This principle is applied during heart surgery, while the heart is arrested and cardioprotection induced with cold fluid to decrease energy consumption. Nevertheless, an especially indispensable warm ischemic period during suturing phase of surgery increases the release of metabolic byproducts, such as increased lactate and glycerol after reperfusion of the heart. The metabolic changes have an effect on the development of edema and fluid retention [8], soon observed histologically, but later affecting functional recovery and, at least theoretically, enhancing the development of chronic myocardial arteriopathy and fibrosis [9]; the early histological changes caused by IRI are specifically found at the endothelial layer and inner intramyocardial artery wall structure, the primal targets after reperfusion [9,10]. To explore the remote myocardial outcome, we performed microdialysis to evaluate metabolic parameters associated with energy consumption, such as lactate and pyruvate after reperfusion. Together with energy consumption, functional recovery after IRI may also be predicted by evaluating the release of interstitial glutamate and glycerol that determine histopathological changes associated with edema [8]. We also investigated the expressions of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS), and studied intramyocardial artery histology. Nitric oxide synthases mirror the molecular cascade activation related to recovery from IRI [11], and these parameters were chosen to confirm the effect of IRI on the remote myocardium by seeking for delicate changes in gene expressions influencing the vascular endothelium. The aim of this study was to interfere with complete and abrupt reperfusion flow after 15 min of cold ischemia and 45 min of warm ischemia, both necessary for heart surgery. We hypothesized that dMI simulates RPreC, but dMI may offer a more sustainable increased remote myocardial protection after IRI as compared with RPreC. Reperfusion
injury may be decreased, while a small persistent and confined area of the heart is left without abrupt reperfusion flow. Methods Ethics The study was approved by the Finnish State Provincial Office. A total of 58 inbred Fischer 344 rats (F344/ NHsd, Harlan Laboratories, The Netherlands) weighing 200–270 g, served as donors (n 18) and recipients (n 29). The rats were kept in Tampere University vivarium and received humane care in compliance with the ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research and the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication No. 86-23, revised 1996). Surgical procedure Rats were anesthetized with sevoflurane (Baxter, USA) for inhalation, and a mixture of ketamine (Ketalar®; Orion Pharma Oy, Espoo, Finland; 7.5 mg/100g) and medetomidine (Dormitor®; Pfizer Oy Animal Health, Espoo, Finland; 0.05 mg/100 g) intraperitoneally. A modified heterotopic transplantation of the heart was performed as previously described [12]. Both donor and recipient rats breathed room air freely before and during surgery. The anesthetized donor rats were sacrificed after harvesting the heart graft. Likewise, the recipient rats were deeply anesthetized but breathed spontaneously during the whole experiment, and were sacrificed only after procurement of the heart. Briefly, before harvesting, cold infusion with cardioplegia fluid (4oC, Custodiol®; Bretschneider HTK solution for cardioplegia and miltiorgan protection, Germany) was infused into the donor aorta in order to arrest the heart and maximize myocardial protection. After harvesting, the cardiac graft was immersed into cold (4oC) temperature physiologic saline fluid with heparin (Heparin Leo®; Vianex S.A., Greece; 100 U). All 18 grafts underwent heterotopic cardiac transplantation intra-abdominally by joining the graft aorta to the aorta and the graft pulmonary artery to the inferior vena cava of the recipient, as previously described [5,7]. From the recipient aorta, the transplanted heart received oxygenated blood that was introduced into the coronary arteries of the graft. Via the coronary sinus, this blood circulated into the right atrium and eventually the right ventricle, from where deoxygenated blood recirculated to the recipient rat throughout the pulmonary artery. The nutritional flow of the myocardium consisted of oxygenated blood, and the transplanted heart was not ischemic
Confined ischemia and rat cardiac arrest after reperfusion upon transplantation. Since the aortic valve was competent, oxygenated blood was not allowed to fill the left ventricle, and therefore the transplanted heart simulated a non-working resting state of the left side of the graft. This heterotopic transplantation model allowed us to study ischemiareperfusion in-vivo without interferences of myocardial stress factors. Simulating the clinical setup during cardiac surgery, the heterotopic cardiac transplantation utilized cold 4°C cardioplegia to arrest the heart and cold 4°C liquid storage for cardiac protection before suturing during a warm 37°C ischemic period of 45 min. The model thus simulated the clinical concept of acute cardiac arrest resuscitated with initiation of cardiopulmonary bypass and left ventricle assist device [12]. After the procedure, carprofen (Rimadyl®; Pfizer Oy Animal Health, Helsinki, Finland) 0.1~0.15 ml was given subcutaneously for pain relief. Experimental groups The rats were randomized into four groups. Seven grafts underwent heart transplantation only to serve as controls with ischemia-reperfusion injury (IRI). In six transplanted cardiac grafts, the left renal artery of the recipient was occluded for 5 min immediately before reperfusion of the graft upon completion of surgery (IRI RPreC); the renal artery of the recipient was occluded under direct visualization immediately before onset of reperfusion of the heart graft. The occlusion of the left renal artery was kept for 5 min in accordance with the experimental protocol, and the occlusion was released thereafter again under direct visualization and ensured by checking the evident change of color of the left kidney from dark brown to light brown upon reperfusion of the kidney. In nine grafts, the LAD was ligated permanently but at its very distal part with a single 7-0 suture yielding a confined local and distal myocardial ischemia (IRI dMI); the ligation knot for LAD obstruction was placed immediately before the bifurcation of the 3rd diagonal artery branch of the LAD, after the onset of the 2nd diagonal branch before onset of reperfusion while the heart remained still. A confined blue distal ischemic area after reperfusion at the apex was ensured to confirm the desired anatomic area size of the completely ischemic myocardium. All hearts were used for surgery, and we approximated the confined ischemic area to 2 2 mm. In seven additional transplanted cardiac grafts together with the distal LAD occlusion, the left renal artery of the recipient was occluded 5 min immediately before reperfusion of the graft upon completion of surgery (IRI dMI RPreC). The baseline body weight was comparable among the rats (IRI: 339 66 g, IRI RPreC: 282 17 g, IRI dMI: 293 8 g, IRI dMI RPreC: 237 10 g, p 0.07). The overall cross-sectional length of the
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hearts was 0.77 0.1 cm. A detailed protocol of the experimental setting is provided on Figure 1. Microdialysis Glutamate, pyruvate, lactate and glycerol were investigated. Immediately after surgery, a single microdialysis probe (CMA 70; 20,000 Dalton cutoffs, 0.6 mm diameter; CMA Microdialysis AB) was implanted in the left ventricular wall of the graft tangentially to the LAD, as previously described [5]. Care was taken to place the microdialysis catheter away from the distal ischemic area, in the basal remote myocardial part of the heart. The microdialysis probe was connected to a CMA 107 microinfusion pump at a flow rate of 0.2 μl/min. Immediately after onset of reperfusion upon release of the flow of the abdominal vessels of the recipient, a waste microdialysis sample was discarded, as required for the technique utilizing microdialysis; this ensures that the microtrauma of the inserted microdialysis catheter interferes minimally with the collected parameters. After the 10-min tissue stabilization period, samples were collected without interruption for 10 min and for three cycles in 30 min. They were subsequently immediately stored at 20°C. Analysis was performed by a CMA 600 Microdialysis Analyzer (CMA Microdialysis AB).
Tissue samples After the procedure, the recipient rats were sacrificed after 40 min of reperfusion. The basal part of the cardiac graft was snap frozen in liquid nitrogen and stored at 70°C for further analysis. The middle half of the graft was embedded in paraffin and 5 μm sections were cut and stained with hematoxylin-eosin. Histology Evaluation of histology was performed blinded to the study protocol. The following variables were evaluated: presence of cardiomyocyte edema, hemorrhage and inflammation. These were graded according to an arbitrary scale from 0–2 and expressed as point score units (PSU): 0, no cardiomyocyte edema, hemorrhage or inflammation; 1, presence of occasional edematous cardiomyocytes, spots of hemorrhage or inflammatory cells; 2, groups of edematous cardiomyocytes, hemorrhage, inflammatory or proliferating cells. Vacuolated nuclei of the media layer of intramyocardial arteries reflected edema, and were counted in a representative cross-sectional intramyocardial artery chosen randomly from the left anterior ventricular wall. The inspected remote intramyocardial arteries were evenly distributed. One section and one artery from every individual hearts were included for an absolute measurement value.
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Figure 1. Schema of the experimental setting of the study. (A) IRI hearts undergoing transplantation with ischemia-reperfusion injury only. (B) IRI RPreC transplanted cardiac grafts with remote preconditioning. (C) IRI dMI transplanted grafts with distal LAD ligation. (D) IRI dMI RPreC transplanted cardiac grafts with distal LAD occlusion and remote preconditioning. The sequence of the experimental protocol was as follows: Infusion of cold (4oC) temperature cardioplegia (2 ml) into the aortic root of the surgically prepared donor rat heart (1) inducing immediate cardiac arrest (2) and onset of a 15 min cold ischemic time, during which the heart was surgically dissected and procured (3) and immersed into cold (4oC) temperature physiologic saline fluid (4), and the anesthetized recipient rat was surgically prepared to reveal the abdominal cavity with its aorta and vena cava (5). These vessels were clamped (6), and heterotopic heart transplantation was initiated (7), which defined the onset of a 45 min warm (37oC) ischemic time. Depending on the study group, the distal LAD was first ligated (IRI dMI and IRI dMI RPreC), after which the left kidney artery was clamped for 5 min (IRI RPreC and IRI dMI RPreC) before the application of the microdialysis catheter in all hearts and reperfusion. Warm ischemic time was terminated by releasing of the recipient abdominal aorta and vena cava clamps to initiate the onset of reperfusion. Immediately after the onset of reperfusion, a waste microdialysis sample was discarded during a 10-min stabilization period, as required for the technique utilizing microdialysis. The stabilization period ensures that the microtrauma of the inserted microdialysis catheter had a minimal effect to interfere with the collected parameters. Thereafter the three phases of the subsequent 10-min microdialysis samples were collected. Finally, after a 40-min reperfusion time, the heart was transected and the basal part was snap frozen in liquid nitrogen for RNA analysis, and the middle part of the heart was procured for histology.
Histology did not encompass the additional ischemic area in grafts with dMI, since distal occlusion of LAD was distal to the chosen cross-sectional area that represented remote myocardium. Round and smooth-edged blue nuclei of the media cells were defined as normal. The number of vacuolated nuclei was divided by the number of round smooth-edged media cell nuclei to obtain the relative number of vacuolated nuclei.
Quantitative RT-PCR analysis The frozen tissue of the base of the heart of six grafts randomly chosen from each group was homogenized and RNA extraction was carried out with GenElute™ Mammalian Total RNA Miniprep kit (Sigma– Aldrich, St. Louis, MO, USA) with proteinase K treatment. Total RNA was then reverse-transcribed to cDNA using TaqMan® Reverse Transcription reagents and random hexamers (Applied Biosystems,
Confined ischemia and rat cardiac arrest
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Figure 1. (Continued)
Foster City, CA, USA). The cDNA obtained from the RT reaction (amount corresponding to approximately 1 ng of total RNA) was subjected to quantitative PCR using QuantiTect® Primer Assays (Qiagen, Valencia, CA, USA) for eNOS, iNOS and GAPDH, Maxima® SYBR Green/ROX qPCR Master Mix (Thermo Scientific, Waltham, MA, USA) and ABI PRISM 7000 Sequence detection system (Applied Biosystems, Foster City, CA, USA). PCR reaction parameters for SYBR® Green detection were as follows: incubation at 50°C for 2 min, incubation at 95°C for 10 min, and thereafter 40 cycles of denaturation at 95°C for 15 sec and annealing and extension at 60°C for one minute. Each sample was determined in duplicate. Ct values were determined, and the relative quantification was calculated using the 2ΔΔCt method [13]. The values of six control samples were used as a calibrator, and the expression levels of eNOS and iNOS were normalized against GAPDH.
Statistical analysis Statistical analysis was performed using SPSS for Windows (version 19.0). The data is presented as mean standard error of the mean (SEM) in the text and the median is shown in figures. The data was analyzed by means of Kruskal-Wallis test and Mann-Whitney U test where appropriate for all groups and parameters. The p-values 0.05 were considered significant. For clarity, all statistically significant comparisons are shown on the figures.
Results Microdialysis (Figure 2) A total of 10 min was allowed for the heart to recover from the microtrauma associated with the insertion of the microdialysis catheter. At 10 min after this stabilization period, while the heart had
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Figure 2. Interstitial glutamate, pyruvate, lactate and glycerol in the IRI (), IRI RPreC (□), IRI dMI (䉬) and IRI dMI RPreC (䉫) groups, respectively. The median is shown with a horizontal line in each group. (A) Glutamate, (B) pyruvate, (C) lactate, and (D) glycerol.
been reperfused for 20 min, glutamate decreased in IRI RPreC and IRI dMI as compared with the IRI group (39 9 and 83 18 vs. 160 27, μmol, p 0.002 and p 0.01, respectively). The differences in glutamate prevailed throughout the study; at 20 min after stabilization, glutamate decreased in IRI RPreC and IRI dMI as compared with the IRI group (26 4 and 36 7 vs. 153 34, μmol, p 0.001 and p 0.0001, respectively). At 30 min after stabilization, glutamate continued to decrease in IRI RPreC and IRI dMI as compared with the IRI group (26 3 and 31 8 vs. 91 20, μmol, p 0.007 and p 0.0001, respectively). Instead, glutamate increased in IRI dMI RPreC as compared with the IRI dMI group at 20 min after stabilization (50 6 vs. 36 7, μmol, p 0.04). Concomitantly, at 20 min after stabilization, glycerol decreased in only IRI dMI as compared
with the IRI group (59 8 vs. 132 39, μmol, p 0.04). In contrast, glycerol increased in IRI dMI RPreC as compared with IRI dMI at every time point (at 10 min: 204 18 vs. 112 23, μmol, p 0.002; at 20 min: 159 26 vs. 59 8, μmol, p 0.001; at 30 min: 148 32 vs. 53 8, μmol, p 0.001). Statistically, the same pattern was observed with lactate, that decreased in only IRI dMI as compared with the IRI group at every time point after the stabilization period (at 10 min: 0.4 0.1 vs 1.7 0.5, mmol, p 0.002; at 20 min: 0.4 0.1 vs. 1.7 0.5, mmol, p 0.002; at 30 min: 0.3 0.1 vs. 1.0 0.3, mmol, p 0.005). In contrast, lactate increased in IRI dMI RPreC as compared with IRI dMI (at 10 min: 1.3 0.1 vs. 0.4 0.1, mmol, p 0.002; at 20 min: 1.3 0.1 vs. 0.4 0.1, mmol, p 0.001; at 30 min: 1.2 0.2 vs. 0.3 0.1, mmol, p 0.001).
Confined ischemia and rat cardiac arrest Similarily, pyruvate decreased in only IRI dMI as compared with the IRI group at every time point after stabilization period (at 10 min: 5 1 vs. 40 10, μmol, p 0.006; at 20 min: 5 0.1 vs. 34 12, μmol, p 0.005; at 30 min: 4 0.1 vs. 39 13, μmol, p 0.05); whereas pyruvate increased in IRI dMI RPreC as compared with IRI dMI (at 10 min: 25 7 vs. 5 1, μmol, p 0.005; at 20 min: 29 6 vs. 5 0.1, μmol, p 0.01; at 30 min: 23 6 vs. 4 0.1, μmol, p 0.005). Statistically, all other comparisons between groups remained non-significant in regard to microdialysis. Histology Cardiomyocyte edema was comparable among all grafts; 0.50 0.17 PSU in grafts with IRI, 0.67 0.21 PSU with IRI RPreC, 0.32 0.11 PSU with IRI dMI and 0.14 0.14 PSU with IRI dMI RPreC. Myocardial inflammation and hemorrhage were not present in the grafts (0 PSU). Instead, vacuolated non-clear nuclei of intra-myocardial artery cells of the left anterior ventricular wall were observed in grafts with IRI, whereas the majority of the nuclei of
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grafts with IRI RPreC, IRI dMI and IRI dMI RPreC remained round-shaped representing non-ischemic intramyocardial arteries (Figure 3). The relative number of vacuolated non-clear nuclei in intra-myocardial artery wall was comparable in grafts with IRI RPreC (0.01 0.01 PSU, range 0–0.03), IRI dMI (0.02 0.01 PSU, range 0–0.12) and IRI dMI RPreC (0.02 0.03 PSU, range 0–0.08). However, according to Mann-Whitney test, the relative number of vacuolated non-clear nuclei in intra-myocardial artery wall was higher in grafts with IRI (0.42 0.31 PSU, range 0–3.25) as compared with IRI dMI (0.02 0.01 PSU, range 0–0.12; p 0.04) (Figure 4). eNOS and iNOS expressions (Figure 5) After 40 min of reperfusion, eNOS expression in the grafts with IRI dMI RPreC decreased as compared to IRI dMI (0.63 0.2 vs. 1.71 0.1, fold change, respectively p 0.006); whereas grafts with IRI RPreC (0.46 0.1, fold change) remained comparable to the control IRI group. Concomitantly, iNOS expression was decreased in the grafts
Figure 3. Representative histology of the heart showing a left ventricular intramyocardial artery in cardiac grafts after 40 min of reperfusion in the IRI (A), IRI dMI (B) and IRI dMI RPreC groups (C). Hematoxylin-eosin staining 40. Arrows show vacuolated nuclei. No histological differences were observed in IRI RPreC as compared with IRI dMI.
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Figure 4. The relative number of the vacuolated nuclei versus normal clear nuclei in the intra-myocardial artery wall in the IRI (), IRI RPreC (□), IRI dMI (䉬) and IRI dMI RPreC (䉫) groups, respectively. The horizontal line indicates the median in each group. The value of 3.25 PSU in IRI is beyond the vertical axis and not shown.
with IRI RPreC (0.44 0, fold change, p 0.04) as compared to the control IRI group, and comparable in the grafts with IRI dMI and IRI dMI RPreC (1.79 0.7 vs. 1.06 0.2, fold change, respectively). Discussion We have demonstrated in this study that, during IRI, a confined distal ischemic area has an effect on remote intramyocardial arteries after cardiac arrest while reducing excessive myocardial metabolic activity. An additional remote ischemia established prior to IRI and IRI dMI decreased the cardiac iNOS and eNOS expressions respectively, but did not add to cardioprotection as compared with either IRI alone and IRI dMI. Instead, both IRI RPreC
and IRI dMI RPreC showed increased metabolic activity in terms of excessive release of pyruvate and lactate. Moreover, glycerol increased in the IRI dMI RPreC group, indicating poorer cardioprotection as compared with the IRI dMI grafts. The clinical setting of cardiac arrest implicates that even a distal and small ischemic area does not represent an inert tissue, but has an important role in controlling remote myocardium. It is anticipated that reperfusion ameliorates the fate of the whole myocardium after prolonged ischemia. However, microvascular endothelial dysfunction may contribute to the no-reflow phenomenon seen during IRI [14]. IRI may provoke myocardial capillary no-reflow and persistent tissue hypoxia [15]. Sensitive myocardial microvessels have an important impact on the progression of advance lesions observed after IRI [16,17]. The early intramyocardial injury associated with IRI is histologically observed in our study as an increased number of vacuolized nuclei. Nuclear vacuolization of the arterial wall is a primal indication of endothelial and smooth muscle cell injury after IRI [10]. The number of nuclear vacuolization may even predict the development of intramyocardial arteriopathy associated with arterial wall thickening and vacuoles of smooth muscle cells [9]. We and others have previously shown that a complete and proximal LAD obstruction leading to local myocardial infarction after IRI had an additional detrimental impact on increasing intramyocardial artery vacuolization [5,6,10]. In this study, we applied a distal LAD occlusion at the very apex of the graft that resulted in a small, approximately 2 2 mm confined ischemic area devoid of early reperfusion. Interestingly, even such a small local area without initial reperfusion injury induced a remote intramyocardial artery effect during IRI. It is tempting to speculate that an abrupt total IRI after ischemia causes remote intramyocardial changes that are
Figure 5. Endothelial nitric oxide synthase (eNOS, A) and inducible nitric oxide synthase (iNOS, B) mRNA expression of rat heart grafts with IRI (), IRI RPreC (□), IRI dMI (䉬) and IRI dMI RPreC (䉫) groups, respectively. Expression shown as fold change compared with the mean of IRI. Horizontal bars indicate the medians.
Confined ischemia and rat cardiac arrest susceptible to respond to early additional intervention regulating reperfusion flow. The outcome of the heart with dMI after cardiac arrest and IRI is not to be compared with the heart model devoid of IRI encompassing LAD obstruction of the thoracic heart without cardiac arrest; our experimental approach using the heterotopic transplantation model enables to simulate the clinical setting including the possibility to perform microdialysis of the heart. Microdialysis provided important information for early detection of myocardial metabolism that coincided with histological outcome during early IRI; in overall, increased levels of glutamate, glycerol, pyruvate and lactate were observed during IRI indicating increased metabolic activity of the heart after ischemia. As previously shown, decreased myocardial glutamate was associated with prevailing beneficial remote intramyocardial artery integrity [5]. Since glutamate is a crucial substrate in myocardial ischemia [8], decreased glutamate may reflect preservation of metabolism involved during cardioprotection in the IRI dMI group. In contrast, IRI dMI RPreC showed a tendency for increased metabolism, while increasing glutamate and therefore suggesting the relative loss of cardioprotection. However, though glutamate also decreased in IRI RPreC, other parameters of metabolic activity such as pyruvate, lactate and glycerol remained relatively identical with the IRI group alone. Similarily, in IRI dMI RPreC, pyruvate, lactate and glycerol were clearly increased, strongly confirming the detrimental metabolic milieu of these hearts as compared with the IRI dMI group. The opposite metabolic changes were consistently observed in IRI dMI as, not only glutamate but pyruvate, lactate and to a lesser extent glycerol were all decreased – reflecting cardioprotection and subsequent decreased nuclear vacuolization of remote intramyocardial arteries. We may therefore speculate that though both RPreC and dMI have metabolic effects after IRI, RPreC does not add to cardioprotection offered by dMI alone. It seems that the liberation of renal artery occlusion affords no additional myocardial protection after IRI [7,18]. Taken together, microdialysis confirmed that a confined myocardial ischemic area after IRI suffices to interfere with the remote myocardial metabolism. As an additional means to study remote myocardium, we evaluated eNOS and iNOS expression changes. Nitric oxide synthases mirror the cardioprotective state of the heart after IRI [11], and these parameters were chosen to confirm the effect of dMI and RPreC on the remote myocardium after the experiment setting. After 15 min of cold ischemia and 45 min of warm ischemia, the metabolic changes of the remote myocardium are demonstrated in hearts with IRI dMI RPreC as compared with IRI dMI (decrease of eNOS, Figure 5), as well as in hearts with IRI RPreC as compared with IRI alone (decrease of iNOS, Figure 5). On the other
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hand – despite not being statistically significant – a slight increase of eNOS was observed in grafts with IRI dMI as compared with IRI alone. We therefore confirm that RPreC has an impact on the expression of nitric oxide synthases at the remote myocardium after IRI, but again RPreC did not add to the histological or metabolic protection after IRI dMI. Ongoing local ischemia induces myocardial iNOS expression after IRI [15]. In the brain, the association of glutamate metabolism and iNOS expression has been described [20]. In this study, there were no major differences in iNOS expression in hearts between the treatment groups, apart from the decrease observed in grafts with IRI RPrec. On the other hand, as also shown in the experimental brain study [20], decreased eNOS expression may predict worse outcome in grafts with IRI dMI RPreC. This is in line with the gradual increase of glycerol in these grafts, as discussed above. However, in a microdialysis-based analysis of interstitial NO, it was found that NOS-independent pathways are also involved in NO synthesis during myocardial ischemia [21]. We may cautiously state that the role of iNOS and eNOS remain speculative in terms of molecular mechanism, though NOS expression reflects early changes on remote intramyocardial arteries after IRI. Taken together, we were able to confirm that though RPreC has an impact after IRI, hearts with IRI dMI RPreC show no additional protection as compared with IRI dMI. Indeed, IRI dMI afforded decreased remote histological damage as evaluated by estimating vacuolization of intramyocardial arteries as well as metabolic parameters such as glutamate, pyruvate, lactate and glycerol after reperfusion. It is tempting to speculate that a confined local and persistent small ischemic area not only has a remote effect on the heart after IRI, but may even afford sustained early cardioprotection missing with RPreC alone due to the relatively short period of applying temporarily left renal artery occlusion. Interestingly, hemorrhage and myocardial edema did not show differences among the groups, and only the changes of intramyocardial arteries were distinctive histologically. This is in accord with previous studies, showing the impact of early ischemia on capillary and arteriole endothelium; the vascular endothelial layer being the primal target after IRI [9–11]. Limitations of the study are to be mentioned. Oxygen and saturation values were not included in the experimental protocol. In this acute setting, we did not measure left ventricle ejection fraction to describe cardiac function. IRI includes a dispersed range of histological findings necessitating careful statistical interpretation of the results. In this study, we wanted to simulate the clinical concept of IRI associated with cardiac arrest, and investigated the impact of a very distal local ischemia of the cardiac apex on the remote intramyocardial arteries during
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RPreC. Our results confirm the remote impact of confined ischemia after IRI on remote myocardium [22]; in a clinical setting with IRI, additional RPreC may not enhance the protection of remote myocardium changes caused by dMI. Regulating reperfusion flow during IRI may thus alter the remote myocardium outcome after cardiac arrest – a most important message for the clinician aiming to achieve complete reperfusion during coronary bypass grafting. The fundamental need in surgery with both cold and warm ischemic period may require the careful adjustment and timing of the reperfusion flow, and maneuvers relatively short in duration such as RPreC may not be sufficient to permanently abolish the remote myocardial effects of IRI. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This study was supported by research funding from The Competitive Research Funding of Pirkanmaa Hospital District, Finland, Tuberculosis Foundation, The Finnish Heart Association and The Finnish Cultural Foundation. Dr A. Mennander is the recipient of the Ingegeerd and Viking O. Bjork award for Scandinavian Cardiovascular Research. References [1] Mohammadi S, Kalavrouziotis D, Dagenais F, Voisine P, Charbonneau E. Completeness of revascularization and survival among octogenerians with triple-vessel disease. Ann Thorac Surg 2012;93:1432–8. [2] Bauer B, Simkhovich BZ, Kloner RA, Przyklenk K. Does preconditioning protect the coronary vasculature from subsequent ischemia/reperfusion injury? Circulation 1993; 88:659–72. [3] Harjai KJ, Boura J, Grines L, Goldstein J, Stone GW, Brodie B, Cox D, O’Neill WW, Grines C. Comparison of effectiveness of primary angioplasty for proximal versus distal right coronary artery culprit lesion during acute myocardial infarction. Am J Cardiol 2002;90:1193–7. [4] Przyklenk K, Bauer B, Ovize M, Kloner RA, Whittaker P. Regional ischemic ‘preconditioning’ protects remote virgin myocardium from subsequent sustained coronary occlusion. Circulation 1993;87:893–9. [5] Liu Z,Vuohelainen V, Tarkka M, Tenhunen J, Lappalainen RS, Narkilahti S, Paavonen T, Oksala N, Wu Z, Mennander A. Glutamate release predicts ongoing myocardial ischemia of rat hearts. Scand J Clin Lab Invest 2010;70:217–24. [6] Vuohelainen V, Raitoharju E, Levula M, Lehtimaki T, Pelto-Huikko M, Honkanen T, Huovila A, Paasonen T, Tarkka M, Mennander A. Myocardial infarction induces early increased remote ADAM8 expression of rat hearts after cadiac arrest. Sand J Clin Lab Invest 2011;71:553–62. [7] Bjornsson B, Windbladh A, Bojmar L, Trulsson LM, Olsson H, Sundquist T, Gulstrand P, Sandstrom P. Remote or conventional ischemic preconditioning – local liver metabolism in rats studied with microdialysis. JSR 2012;176:55–62. [8] Lofgren B, Povlsen JA, Rasmussen LE, Stottrup NB, Solskov L, Krarup P-M, Kristiansen SB, Botker HE,
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Nielsen TT. Amino acid transamination is crucial for ischaemic cardioprotection in normal and preconditioned isolated rat hearts – focus on l-glutamate. Exp Physiol 2010;95:140–52. Shyu J-J, Cheng C-H, Erlandson RA, Lin J-H, Liu S-K. Ultrastructure of intramural coronary arteries in pigs with hypertrophic cardiomyopathy. Cardiovasc Path 2002;11:104–11. Lin PJ, Chang C-H, Lee Y-S, Chou Y-Y, Chu J-J, Chang J-P, Hsieh M-J. Acute endothelial reperfusion injury after coronary artery bypass grafting. Ann Thorac Surg 1994; 58:782–8. Yang C, Talukder H, Varadharaj S, Velayutham M, Zweier J. Early ischemic preconditioning requires Akt- and PKA-mediated activation of eNOS via serine1176 phosphorylation. Cardiovasc Res 2013;97:33–43. Suzuki K, Murtuza B, Smolenski RT, Suzuki N, Yacoub MH. Development of an in vivo ischemia-reperfusion model in heterotopically transplanted rat hearts. Transplantation 2002;73:1398–402. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2ΔΔCt method. Methods 2001;25:402–8. Dignan RJ, Kadletz M, Dyke CM, Lutz HA, Yeh T Jr, Wechsler AS. Microvascular dysfunction after myocardial ischemnia. J Thorac Cardiovasc Surg 1995;109:892–8. Schramm R, Menger MD, Kirsch S, Langer F, Harder Y, Hamacher J, Schäfers HJ. The subepicardial microcirculation in heterotopically transplanted mouse hearts: an intravital multifluorescence microscopy study. J Thorac Cardiovasc Surg 2007;134:210–7. Eriksson EE. Intravital microscopy on atherosclerosis in Apolipoprotein E-deficient mice establishes microvessels as major entry pathways for leukocytes to advanced lesions. Circulation 2011;124:2129–38. Dutta P, Courties G, Wei Y, Leuschner F, Gorbatov R, Robbins CS, Iwamoto Y, Thompson B, Carlson AL, Heidt T, Majmudar MD, Lasitschka F, Etzrodt M, Waterman P, Waring MT, Chicoine AT, van der Laan AM, Niessen HW, Piek JJ, Rubin BB, Butany J, Stone JR, Katus HA, Murphy SA, Morrow DA, Sabatine MS, Vinegoni C, Moskowitz MA, Pittet MJ, Libby P, Lin CP, Swirski FK, Weissleder R, Nahrendorf M. Myocardial infarction accelerates atherosclerosis. Nature 2012;487:325–9. Fonouni H, Tahmasbi Rad M, Golriz M, Faridar A, Esmaeilzadeh M, Jarahian P, Hafezi M, Jafarieh S, Macher-Goeppinger S, Longerich T, Orakcioglu B, Sakowitz O, Schmidt J, Mehrabi A. Using microdialysis for early detection of vascular thrombosis after kidney transplantation in an experimental porcine model. Nephrol Dial Transplant 2012;27:541–7. Heinzel FR, Gres P, Boengler K, Duschin A, Konietzka I, Rassaf T, Snedovskaya J, Meyer S, Skyschally A, Kelm M, Heusch G, Schulz R. Inducible nitric oxide synthase expression and cardiomyocyte dysfunction during sustained moderate ischemia in pigs. Circ Res 2008;103:1120–7. Anttila V, Christou H, Hagino I, Iwata Y, Mettler BA, Fernandez-Gonzalez A, Zurakowski D, Jonas RA. Cerebral endothelial nitric oxide synthase expression is reduced after very-low-flow bypass. Ann Thorac Surg 2006; 81:2202–6. Martin C, Schulz R, Post H, Boengler K, Kelm M, Kleinbongard P, Gres P, Skyschally A, Konietzka I, Heusch G. Microdialysis-based analysis of interstitial NO in situ: NO synthase-independent NO formation during myocardial ischemia. Cardiovasc Res 2007;74:46–55. Hale SL, Dae MW, Kloner RA. Hypothermia during reperfusion limits ‘no-reflow’ injury in a rabbit model of acute myocardial infarction. Cardiovasc Res 2003;59:715.
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ORIGINAL ARTICLE
Confined ischemia may improve remote myocardial outcome after rat cardiac arrest
ZANXIN WANG1,2, HONGYAN LI1, VILMA VUOHELAINEN1, JYRKI TENHUNEN3, MARI HÄMÄLÄINEN4, TIMO RINNE5, EEVA MOILANEN4, TIMO PAAVONEN6, MATTI TARKKA1 & ARI MENNANDER1 1Heart
Center, Cardiac Research, Tampere University Hospital and Tampere University, Tampere, Finland, of Cardiovascular Surgery, Tianjin Medical University General Hospital, Tianjin, P. R. China, 3Department of Surgical Sciences/Anesthesiology and Intensive Care Medicine, University of Uppsala, Sweden and Critical Care Medicine Research Group in the Department of Intensive Care Medicine, Tampere University Hospital, Tampere, Finland, 4The Immunopharmacology Research Group, University of Tampere School of Medicine and Tampere University Hospital, Tampere, Finland, 5Department of Anesthesiology, Tampere University Hospital, Tampere, Finland, and 6Department of Pathology, Fimlab and Tampere University, Tampere, Finland 2Department
Abstract Background. Confined ongoing ischemia after ischemia-reperfusion injury (IRI) may alter myocardial recovery. We evaluated in a rat cardiac transplantation model whether distal persistent myocardial ischemia (dMI) and remote preconditioning (RPreC) have a remote myocardial impact after IRI. Material and methods. Syngeneic heterotopic cardiac transplantation was performed on 29 Fischer344 rats to induce IRI, including nine rats which underwent distal ligation of the left anterior coronary artery (LAD) to yield distal MI (IRI dMI). RPreC was applied by occluding the left renal artery 5 min prior to reperfusion in six rats with IRI (IRI RPreC) as well as in seven with distal MI (IRI dMI RPreC). Microdialysis, histology and qRT-PCR for inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were performed after graft harvesting. Results. In contrast to IRI dMI RPreC (39 7 μmol), glutamate decreased in IRI RPreC and IRI dMI as compared with IRI (26 3 and 31 8 vs 91 20, μmol respectively, p 0.007). The relative number of vacuolated intramyocardial artery nuclei decreased in IRI dMI as compared with IRI (0.02 0.01, range 0–12 vs. 0.42 0.31, range 0–3.25 PSU respectively, p 0.04). iNOS expression decreased in IRI RPreC as compared with IRI (p 0.04), and eNOS expression decreased in IRI dMI RPreC as compared with IRI dMI (p 0.006) along with increased glycerol release. Conclusions. dMI after IRI has a potentially beneficial myocardial impact after cardiac arrest, which is hampered by RPreC. Key Words: Cardiac arrest, ischemia-reperfusion, rat, remote preconditioning, distal myocardial infarction
Introduction Complete reperfusion is the gold standard of cardiac surgery after ischemia-reperfusion injury (IRI) [1], though it is often anticipated that after coronary artery bypass grafting a small ischemic myocardial area causes minimal, if any, remote myocardial changes. However, the effects of a persistent local cardiac ischemic area on remote myocardial microvasculature remain poorly understood after cardiac arrest causing IRI [2]. A distal myocardial ischemia
(dMI) may have an impact on the remote myocardium after IRI, even though the confined ischemic area nourished by the non-revascularized coronary artery is significantly small, or a relatively short duration of cardiac arrest necessary during surgery provides quick early functional recovery [1,3,4]. We have previously shown that myocardial infarct has a remote myocardial impact on increasing small intramyocardial artery edema [5,6]; we accomplished myocardial infarct by occluding the proximal part of
Correspondence: Ari A. Mennander, Cardiac Research, Heart Center, Tampere University Hospital, SDSKIR, Teiskontie 35, PL 2000 Tampere, Finland. Tel: 358 3 31164945. Fax: 358 3 31165756. E-mail: [email protected] (Received 15 February 2013 ; accepted 12 October 2013) ISSN 0036-5513 print/ISSN 1502-7686 online © 2014 Informa Healthcare DOI: 10.3109/00365513.2013.855944
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the left anterior descending coronary artery (LAD), in order to obtain a prominent myocardial ischemic area. In this study, we investigated whether dMI may alter the remote myocardium during IRI [4]. It has been shown earlier that abrupt reperfusion flow may cause ischemia reperfusion injury and remote preconditioning exerts a beneficial impact [7]. However, it is not clear whether a relatively short duration of remote preconditioning would afford sustainable cardioprotection after cold and warm ischemia. Using the rat cardiac transplantation model, we also evaluated whether remote preconditioning of the kidney (RPreC) may have a comparable effect, as well as whether or not RPreC, together with dMI, may have an additional remote myocardial impact during IRI. Ideally, decreased excessive energy consumption of the heart during cardiac surgery would enhance recovery after IRI. This principle is applied during heart surgery, while the heart is arrested and cardioprotection induced with cold fluid to decrease energy consumption. Nevertheless, an especially indispensable warm ischemic period during suturing phase of surgery increases the release of metabolic byproducts, such as increased lactate and glycerol after reperfusion of the heart. The metabolic changes have an effect on the development of edema and fluid retention [8], soon observed histologically, but later affecting functional recovery and, at least theoretically, enhancing the development of chronic myocardial arteriopathy and fibrosis [9]; the early histological changes caused by IRI are specifically found at the endothelial layer and inner intramyocardial artery wall structure, the primal targets after reperfusion [9,10]. To explore the remote myocardial outcome, we performed microdialysis to evaluate metabolic parameters associated with energy consumption, such as lactate and pyruvate after reperfusion. Together with energy consumption, functional recovery after IRI may also be predicted by evaluating the release of interstitial glutamate and glycerol that determine histopathological changes associated with edema [8]. We also investigated the expressions of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS), and studied intramyocardial artery histology. Nitric oxide synthases mirror the molecular cascade activation related to recovery from IRI [11], and these parameters were chosen to confirm the effect of IRI on the remote myocardium by seeking for delicate changes in gene expressions influencing the vascular endothelium. The aim of this study was to interfere with complete and abrupt reperfusion flow after 15 min of cold ischemia and 45 min of warm ischemia, both necessary for heart surgery. We hypothesized that dMI simulates RPreC, but dMI may offer a more sustainable increased remote myocardial protection after IRI as compared with RPreC. Reperfusion
injury may be decreased, while a small persistent and confined area of the heart is left without abrupt reperfusion flow. Methods Ethics The study was approved by the Finnish State Provincial Office. A total of 58 inbred Fischer 344 rats (F344/ NHsd, Harlan Laboratories, The Netherlands) weighing 200–270 g, served as donors (n 18) and recipients (n 29). The rats were kept in Tampere University vivarium and received humane care in compliance with the ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research and the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication No. 86-23, revised 1996). Surgical procedure Rats were anesthetized with sevoflurane (Baxter, USA) for inhalation, and a mixture of ketamine (Ketalar®; Orion Pharma Oy, Espoo, Finland; 7.5 mg/100g) and medetomidine (Dormitor®; Pfizer Oy Animal Health, Espoo, Finland; 0.05 mg/100 g) intraperitoneally. A modified heterotopic transplantation of the heart was performed as previously described [12]. Both donor and recipient rats breathed room air freely before and during surgery. The anesthetized donor rats were sacrificed after harvesting the heart graft. Likewise, the recipient rats were deeply anesthetized but breathed spontaneously during the whole experiment, and were sacrificed only after procurement of the heart. Briefly, before harvesting, cold infusion with cardioplegia fluid (4oC, Custodiol®; Bretschneider HTK solution for cardioplegia and miltiorgan protection, Germany) was infused into the donor aorta in order to arrest the heart and maximize myocardial protection. After harvesting, the cardiac graft was immersed into cold (4oC) temperature physiologic saline fluid with heparin (Heparin Leo®; Vianex S.A., Greece; 100 U). All 18 grafts underwent heterotopic cardiac transplantation intra-abdominally by joining the graft aorta to the aorta and the graft pulmonary artery to the inferior vena cava of the recipient, as previously described [5,7]. From the recipient aorta, the transplanted heart received oxygenated blood that was introduced into the coronary arteries of the graft. Via the coronary sinus, this blood circulated into the right atrium and eventually the right ventricle, from where deoxygenated blood recirculated to the recipient rat throughout the pulmonary artery. The nutritional flow of the myocardium consisted of oxygenated blood, and the transplanted heart was not ischemic
Confined ischemia and rat cardiac arrest after reperfusion upon transplantation. Since the aortic valve was competent, oxygenated blood was not allowed to fill the left ventricle, and therefore the transplanted heart simulated a non-working resting state of the left side of the graft. This heterotopic transplantation model allowed us to study ischemiareperfusion in-vivo without interferences of myocardial stress factors. Simulating the clinical setup during cardiac surgery, the heterotopic cardiac transplantation utilized cold 4°C cardioplegia to arrest the heart and cold 4°C liquid storage for cardiac protection before suturing during a warm 37°C ischemic period of 45 min. The model thus simulated the clinical concept of acute cardiac arrest resuscitated with initiation of cardiopulmonary bypass and left ventricle assist device [12]. After the procedure, carprofen (Rimadyl®; Pfizer Oy Animal Health, Helsinki, Finland) 0.1~0.15 ml was given subcutaneously for pain relief. Experimental groups The rats were randomized into four groups. Seven grafts underwent heart transplantation only to serve as controls with ischemia-reperfusion injury (IRI). In six transplanted cardiac grafts, the left renal artery of the recipient was occluded for 5 min immediately before reperfusion of the graft upon completion of surgery (IRI RPreC); the renal artery of the recipient was occluded under direct visualization immediately before onset of reperfusion of the heart graft. The occlusion of the left renal artery was kept for 5 min in accordance with the experimental protocol, and the occlusion was released thereafter again under direct visualization and ensured by checking the evident change of color of the left kidney from dark brown to light brown upon reperfusion of the kidney. In nine grafts, the LAD was ligated permanently but at its very distal part with a single 7-0 suture yielding a confined local and distal myocardial ischemia (IRI dMI); the ligation knot for LAD obstruction was placed immediately before the bifurcation of the 3rd diagonal artery branch of the LAD, after the onset of the 2nd diagonal branch before onset of reperfusion while the heart remained still. A confined blue distal ischemic area after reperfusion at the apex was ensured to confirm the desired anatomic area size of the completely ischemic myocardium. All hearts were used for surgery, and we approximated the confined ischemic area to 2 2 mm. In seven additional transplanted cardiac grafts together with the distal LAD occlusion, the left renal artery of the recipient was occluded 5 min immediately before reperfusion of the graft upon completion of surgery (IRI dMI RPreC). The baseline body weight was comparable among the rats (IRI: 339 66 g, IRI RPreC: 282 17 g, IRI dMI: 293 8 g, IRI dMI RPreC: 237 10 g, p 0.07). The overall cross-sectional length of the
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hearts was 0.77 0.1 cm. A detailed protocol of the experimental setting is provided on Figure 1. Microdialysis Glutamate, pyruvate, lactate and glycerol were investigated. Immediately after surgery, a single microdialysis probe (CMA 70; 20,000 Dalton cutoffs, 0.6 mm diameter; CMA Microdialysis AB) was implanted in the left ventricular wall of the graft tangentially to the LAD, as previously described [5]. Care was taken to place the microdialysis catheter away from the distal ischemic area, in the basal remote myocardial part of the heart. The microdialysis probe was connected to a CMA 107 microinfusion pump at a flow rate of 0.2 μl/min. Immediately after onset of reperfusion upon release of the flow of the abdominal vessels of the recipient, a waste microdialysis sample was discarded, as required for the technique utilizing microdialysis; this ensures that the microtrauma of the inserted microdialysis catheter interferes minimally with the collected parameters. After the 10-min tissue stabilization period, samples were collected without interruption for 10 min and for three cycles in 30 min. They were subsequently immediately stored at 20°C. Analysis was performed by a CMA 600 Microdialysis Analyzer (CMA Microdialysis AB).
Tissue samples After the procedure, the recipient rats were sacrificed after 40 min of reperfusion. The basal part of the cardiac graft was snap frozen in liquid nitrogen and stored at 70°C for further analysis. The middle half of the graft was embedded in paraffin and 5 μm sections were cut and stained with hematoxylin-eosin. Histology Evaluation of histology was performed blinded to the study protocol. The following variables were evaluated: presence of cardiomyocyte edema, hemorrhage and inflammation. These were graded according to an arbitrary scale from 0–2 and expressed as point score units (PSU): 0, no cardiomyocyte edema, hemorrhage or inflammation; 1, presence of occasional edematous cardiomyocytes, spots of hemorrhage or inflammatory cells; 2, groups of edematous cardiomyocytes, hemorrhage, inflammatory or proliferating cells. Vacuolated nuclei of the media layer of intramyocardial arteries reflected edema, and were counted in a representative cross-sectional intramyocardial artery chosen randomly from the left anterior ventricular wall. The inspected remote intramyocardial arteries were evenly distributed. One section and one artery from every individual hearts were included for an absolute measurement value.
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Figure 1. Schema of the experimental setting of the study. (A) IRI hearts undergoing transplantation with ischemia-reperfusion injury only. (B) IRI RPreC transplanted cardiac grafts with remote preconditioning. (C) IRI dMI transplanted grafts with distal LAD ligation. (D) IRI dMI RPreC transplanted cardiac grafts with distal LAD occlusion and remote preconditioning. The sequence of the experimental protocol was as follows: Infusion of cold (4oC) temperature cardioplegia (2 ml) into the aortic root of the surgically prepared donor rat heart (1) inducing immediate cardiac arrest (2) and onset of a 15 min cold ischemic time, during which the heart was surgically dissected and procured (3) and immersed into cold (4oC) temperature physiologic saline fluid (4), and the anesthetized recipient rat was surgically prepared to reveal the abdominal cavity with its aorta and vena cava (5). These vessels were clamped (6), and heterotopic heart transplantation was initiated (7), which defined the onset of a 45 min warm (37oC) ischemic time. Depending on the study group, the distal LAD was first ligated (IRI dMI and IRI dMI RPreC), after which the left kidney artery was clamped for 5 min (IRI RPreC and IRI dMI RPreC) before the application of the microdialysis catheter in all hearts and reperfusion. Warm ischemic time was terminated by releasing of the recipient abdominal aorta and vena cava clamps to initiate the onset of reperfusion. Immediately after the onset of reperfusion, a waste microdialysis sample was discarded during a 10-min stabilization period, as required for the technique utilizing microdialysis. The stabilization period ensures that the microtrauma of the inserted microdialysis catheter had a minimal effect to interfere with the collected parameters. Thereafter the three phases of the subsequent 10-min microdialysis samples were collected. Finally, after a 40-min reperfusion time, the heart was transected and the basal part was snap frozen in liquid nitrogen for RNA analysis, and the middle part of the heart was procured for histology.
Histology did not encompass the additional ischemic area in grafts with dMI, since distal occlusion of LAD was distal to the chosen cross-sectional area that represented remote myocardium. Round and smooth-edged blue nuclei of the media cells were defined as normal. The number of vacuolated nuclei was divided by the number of round smooth-edged media cell nuclei to obtain the relative number of vacuolated nuclei.
Quantitative RT-PCR analysis The frozen tissue of the base of the heart of six grafts randomly chosen from each group was homogenized and RNA extraction was carried out with GenElute™ Mammalian Total RNA Miniprep kit (Sigma– Aldrich, St. Louis, MO, USA) with proteinase K treatment. Total RNA was then reverse-transcribed to cDNA using TaqMan® Reverse Transcription reagents and random hexamers (Applied Biosystems,
Confined ischemia and rat cardiac arrest
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Figure 1. (Continued)
Foster City, CA, USA). The cDNA obtained from the RT reaction (amount corresponding to approximately 1 ng of total RNA) was subjected to quantitative PCR using QuantiTect® Primer Assays (Qiagen, Valencia, CA, USA) for eNOS, iNOS and GAPDH, Maxima® SYBR Green/ROX qPCR Master Mix (Thermo Scientific, Waltham, MA, USA) and ABI PRISM 7000 Sequence detection system (Applied Biosystems, Foster City, CA, USA). PCR reaction parameters for SYBR® Green detection were as follows: incubation at 50°C for 2 min, incubation at 95°C for 10 min, and thereafter 40 cycles of denaturation at 95°C for 15 sec and annealing and extension at 60°C for one minute. Each sample was determined in duplicate. Ct values were determined, and the relative quantification was calculated using the 2ΔΔCt method [13]. The values of six control samples were used as a calibrator, and the expression levels of eNOS and iNOS were normalized against GAPDH.
Statistical analysis Statistical analysis was performed using SPSS for Windows (version 19.0). The data is presented as mean standard error of the mean (SEM) in the text and the median is shown in figures. The data was analyzed by means of Kruskal-Wallis test and Mann-Whitney U test where appropriate for all groups and parameters. The p-values 0.05 were considered significant. For clarity, all statistically significant comparisons are shown on the figures.
Results Microdialysis (Figure 2) A total of 10 min was allowed for the heart to recover from the microtrauma associated with the insertion of the microdialysis catheter. At 10 min after this stabilization period, while the heart had
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Figure 2. Interstitial glutamate, pyruvate, lactate and glycerol in the IRI (), IRI RPreC (□), IRI dMI (䉬) and IRI dMI RPreC (䉫) groups, respectively. The median is shown with a horizontal line in each group. (A) Glutamate, (B) pyruvate, (C) lactate, and (D) glycerol.
been reperfused for 20 min, glutamate decreased in IRI RPreC and IRI dMI as compared with the IRI group (39 9 and 83 18 vs. 160 27, μmol, p 0.002 and p 0.01, respectively). The differences in glutamate prevailed throughout the study; at 20 min after stabilization, glutamate decreased in IRI RPreC and IRI dMI as compared with the IRI group (26 4 and 36 7 vs. 153 34, μmol, p 0.001 and p 0.0001, respectively). At 30 min after stabilization, glutamate continued to decrease in IRI RPreC and IRI dMI as compared with the IRI group (26 3 and 31 8 vs. 91 20, μmol, p 0.007 and p 0.0001, respectively). Instead, glutamate increased in IRI dMI RPreC as compared with the IRI dMI group at 20 min after stabilization (50 6 vs. 36 7, μmol, p 0.04). Concomitantly, at 20 min after stabilization, glycerol decreased in only IRI dMI as compared
with the IRI group (59 8 vs. 132 39, μmol, p 0.04). In contrast, glycerol increased in IRI dMI RPreC as compared with IRI dMI at every time point (at 10 min: 204 18 vs. 112 23, μmol, p 0.002; at 20 min: 159 26 vs. 59 8, μmol, p 0.001; at 30 min: 148 32 vs. 53 8, μmol, p 0.001). Statistically, the same pattern was observed with lactate, that decreased in only IRI dMI as compared with the IRI group at every time point after the stabilization period (at 10 min: 0.4 0.1 vs 1.7 0.5, mmol, p 0.002; at 20 min: 0.4 0.1 vs. 1.7 0.5, mmol, p 0.002; at 30 min: 0.3 0.1 vs. 1.0 0.3, mmol, p 0.005). In contrast, lactate increased in IRI dMI RPreC as compared with IRI dMI (at 10 min: 1.3 0.1 vs. 0.4 0.1, mmol, p 0.002; at 20 min: 1.3 0.1 vs. 0.4 0.1, mmol, p 0.001; at 30 min: 1.2 0.2 vs. 0.3 0.1, mmol, p 0.001).
Confined ischemia and rat cardiac arrest Similarily, pyruvate decreased in only IRI dMI as compared with the IRI group at every time point after stabilization period (at 10 min: 5 1 vs. 40 10, μmol, p 0.006; at 20 min: 5 0.1 vs. 34 12, μmol, p 0.005; at 30 min: 4 0.1 vs. 39 13, μmol, p 0.05); whereas pyruvate increased in IRI dMI RPreC as compared with IRI dMI (at 10 min: 25 7 vs. 5 1, μmol, p 0.005; at 20 min: 29 6 vs. 5 0.1, μmol, p 0.01; at 30 min: 23 6 vs. 4 0.1, μmol, p 0.005). Statistically, all other comparisons between groups remained non-significant in regard to microdialysis. Histology Cardiomyocyte edema was comparable among all grafts; 0.50 0.17 PSU in grafts with IRI, 0.67 0.21 PSU with IRI RPreC, 0.32 0.11 PSU with IRI dMI and 0.14 0.14 PSU with IRI dMI RPreC. Myocardial inflammation and hemorrhage were not present in the grafts (0 PSU). Instead, vacuolated non-clear nuclei of intra-myocardial artery cells of the left anterior ventricular wall were observed in grafts with IRI, whereas the majority of the nuclei of
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grafts with IRI RPreC, IRI dMI and IRI dMI RPreC remained round-shaped representing non-ischemic intramyocardial arteries (Figure 3). The relative number of vacuolated non-clear nuclei in intra-myocardial artery wall was comparable in grafts with IRI RPreC (0.01 0.01 PSU, range 0–0.03), IRI dMI (0.02 0.01 PSU, range 0–0.12) and IRI dMI RPreC (0.02 0.03 PSU, range 0–0.08). However, according to Mann-Whitney test, the relative number of vacuolated non-clear nuclei in intra-myocardial artery wall was higher in grafts with IRI (0.42 0.31 PSU, range 0–3.25) as compared with IRI dMI (0.02 0.01 PSU, range 0–0.12; p 0.04) (Figure 4). eNOS and iNOS expressions (Figure 5) After 40 min of reperfusion, eNOS expression in the grafts with IRI dMI RPreC decreased as compared to IRI dMI (0.63 0.2 vs. 1.71 0.1, fold change, respectively p 0.006); whereas grafts with IRI RPreC (0.46 0.1, fold change) remained comparable to the control IRI group. Concomitantly, iNOS expression was decreased in the grafts
Figure 3. Representative histology of the heart showing a left ventricular intramyocardial artery in cardiac grafts after 40 min of reperfusion in the IRI (A), IRI dMI (B) and IRI dMI RPreC groups (C). Hematoxylin-eosin staining 40. Arrows show vacuolated nuclei. No histological differences were observed in IRI RPreC as compared with IRI dMI.
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Figure 4. The relative number of the vacuolated nuclei versus normal clear nuclei in the intra-myocardial artery wall in the IRI (), IRI RPreC (□), IRI dMI (䉬) and IRI dMI RPreC (䉫) groups, respectively. The horizontal line indicates the median in each group. The value of 3.25 PSU in IRI is beyond the vertical axis and not shown.
with IRI RPreC (0.44 0, fold change, p 0.04) as compared to the control IRI group, and comparable in the grafts with IRI dMI and IRI dMI RPreC (1.79 0.7 vs. 1.06 0.2, fold change, respectively). Discussion We have demonstrated in this study that, during IRI, a confined distal ischemic area has an effect on remote intramyocardial arteries after cardiac arrest while reducing excessive myocardial metabolic activity. An additional remote ischemia established prior to IRI and IRI dMI decreased the cardiac iNOS and eNOS expressions respectively, but did not add to cardioprotection as compared with either IRI alone and IRI dMI. Instead, both IRI RPreC
and IRI dMI RPreC showed increased metabolic activity in terms of excessive release of pyruvate and lactate. Moreover, glycerol increased in the IRI dMI RPreC group, indicating poorer cardioprotection as compared with the IRI dMI grafts. The clinical setting of cardiac arrest implicates that even a distal and small ischemic area does not represent an inert tissue, but has an important role in controlling remote myocardium. It is anticipated that reperfusion ameliorates the fate of the whole myocardium after prolonged ischemia. However, microvascular endothelial dysfunction may contribute to the no-reflow phenomenon seen during IRI [14]. IRI may provoke myocardial capillary no-reflow and persistent tissue hypoxia [15]. Sensitive myocardial microvessels have an important impact on the progression of advance lesions observed after IRI [16,17]. The early intramyocardial injury associated with IRI is histologically observed in our study as an increased number of vacuolized nuclei. Nuclear vacuolization of the arterial wall is a primal indication of endothelial and smooth muscle cell injury after IRI [10]. The number of nuclear vacuolization may even predict the development of intramyocardial arteriopathy associated with arterial wall thickening and vacuoles of smooth muscle cells [9]. We and others have previously shown that a complete and proximal LAD obstruction leading to local myocardial infarction after IRI had an additional detrimental impact on increasing intramyocardial artery vacuolization [5,6,10]. In this study, we applied a distal LAD occlusion at the very apex of the graft that resulted in a small, approximately 2 2 mm confined ischemic area devoid of early reperfusion. Interestingly, even such a small local area without initial reperfusion injury induced a remote intramyocardial artery effect during IRI. It is tempting to speculate that an abrupt total IRI after ischemia causes remote intramyocardial changes that are
Figure 5. Endothelial nitric oxide synthase (eNOS, A) and inducible nitric oxide synthase (iNOS, B) mRNA expression of rat heart grafts with IRI (), IRI RPreC (□), IRI dMI (䉬) and IRI dMI RPreC (䉫) groups, respectively. Expression shown as fold change compared with the mean of IRI. Horizontal bars indicate the medians.
Confined ischemia and rat cardiac arrest susceptible to respond to early additional intervention regulating reperfusion flow. The outcome of the heart with dMI after cardiac arrest and IRI is not to be compared with the heart model devoid of IRI encompassing LAD obstruction of the thoracic heart without cardiac arrest; our experimental approach using the heterotopic transplantation model enables to simulate the clinical setting including the possibility to perform microdialysis of the heart. Microdialysis provided important information for early detection of myocardial metabolism that coincided with histological outcome during early IRI; in overall, increased levels of glutamate, glycerol, pyruvate and lactate were observed during IRI indicating increased metabolic activity of the heart after ischemia. As previously shown, decreased myocardial glutamate was associated with prevailing beneficial remote intramyocardial artery integrity [5]. Since glutamate is a crucial substrate in myocardial ischemia [8], decreased glutamate may reflect preservation of metabolism involved during cardioprotection in the IRI dMI group. In contrast, IRI dMI RPreC showed a tendency for increased metabolism, while increasing glutamate and therefore suggesting the relative loss of cardioprotection. However, though glutamate also decreased in IRI RPreC, other parameters of metabolic activity such as pyruvate, lactate and glycerol remained relatively identical with the IRI group alone. Similarily, in IRI dMI RPreC, pyruvate, lactate and glycerol were clearly increased, strongly confirming the detrimental metabolic milieu of these hearts as compared with the IRI dMI group. The opposite metabolic changes were consistently observed in IRI dMI as, not only glutamate but pyruvate, lactate and to a lesser extent glycerol were all decreased – reflecting cardioprotection and subsequent decreased nuclear vacuolization of remote intramyocardial arteries. We may therefore speculate that though both RPreC and dMI have metabolic effects after IRI, RPreC does not add to cardioprotection offered by dMI alone. It seems that the liberation of renal artery occlusion affords no additional myocardial protection after IRI [7,18]. Taken together, microdialysis confirmed that a confined myocardial ischemic area after IRI suffices to interfere with the remote myocardial metabolism. As an additional means to study remote myocardium, we evaluated eNOS and iNOS expression changes. Nitric oxide synthases mirror the cardioprotective state of the heart after IRI [11], and these parameters were chosen to confirm the effect of dMI and RPreC on the remote myocardium after the experiment setting. After 15 min of cold ischemia and 45 min of warm ischemia, the metabolic changes of the remote myocardium are demonstrated in hearts with IRI dMI RPreC as compared with IRI dMI (decrease of eNOS, Figure 5), as well as in hearts with IRI RPreC as compared with IRI alone (decrease of iNOS, Figure 5). On the other
35
hand – despite not being statistically significant – a slight increase of eNOS was observed in grafts with IRI dMI as compared with IRI alone. We therefore confirm that RPreC has an impact on the expression of nitric oxide synthases at the remote myocardium after IRI, but again RPreC did not add to the histological or metabolic protection after IRI dMI. Ongoing local ischemia induces myocardial iNOS expression after IRI [15]. In the brain, the association of glutamate metabolism and iNOS expression has been described [20]. In this study, there were no major differences in iNOS expression in hearts between the treatment groups, apart from the decrease observed in grafts with IRI RPrec. On the other hand, as also shown in the experimental brain study [20], decreased eNOS expression may predict worse outcome in grafts with IRI dMI RPreC. This is in line with the gradual increase of glycerol in these grafts, as discussed above. However, in a microdialysis-based analysis of interstitial NO, it was found that NOS-independent pathways are also involved in NO synthesis during myocardial ischemia [21]. We may cautiously state that the role of iNOS and eNOS remain speculative in terms of molecular mechanism, though NOS expression reflects early changes on remote intramyocardial arteries after IRI. Taken together, we were able to confirm that though RPreC has an impact after IRI, hearts with IRI dMI RPreC show no additional protection as compared with IRI dMI. Indeed, IRI dMI afforded decreased remote histological damage as evaluated by estimating vacuolization of intramyocardial arteries as well as metabolic parameters such as glutamate, pyruvate, lactate and glycerol after reperfusion. It is tempting to speculate that a confined local and persistent small ischemic area not only has a remote effect on the heart after IRI, but may even afford sustained early cardioprotection missing with RPreC alone due to the relatively short period of applying temporarily left renal artery occlusion. Interestingly, hemorrhage and myocardial edema did not show differences among the groups, and only the changes of intramyocardial arteries were distinctive histologically. This is in accord with previous studies, showing the impact of early ischemia on capillary and arteriole endothelium; the vascular endothelial layer being the primal target after IRI [9–11]. Limitations of the study are to be mentioned. Oxygen and saturation values were not included in the experimental protocol. In this acute setting, we did not measure left ventricle ejection fraction to describe cardiac function. IRI includes a dispersed range of histological findings necessitating careful statistical interpretation of the results. In this study, we wanted to simulate the clinical concept of IRI associated with cardiac arrest, and investigated the impact of a very distal local ischemia of the cardiac apex on the remote intramyocardial arteries during
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RPreC. Our results confirm the remote impact of confined ischemia after IRI on remote myocardium [22]; in a clinical setting with IRI, additional RPreC may not enhance the protection of remote myocardium changes caused by dMI. Regulating reperfusion flow during IRI may thus alter the remote myocardium outcome after cardiac arrest – a most important message for the clinician aiming to achieve complete reperfusion during coronary bypass grafting. The fundamental need in surgery with both cold and warm ischemic period may require the careful adjustment and timing of the reperfusion flow, and maneuvers relatively short in duration such as RPreC may not be sufficient to permanently abolish the remote myocardial effects of IRI. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This study was supported by research funding from The Competitive Research Funding of Pirkanmaa Hospital District, Finland, Tuberculosis Foundation, The Finnish Heart Association and The Finnish Cultural Foundation. Dr A. Mennander is the recipient of the Ingegeerd and Viking O. Bjork award for Scandinavian Cardiovascular Research. References [1] Mohammadi S, Kalavrouziotis D, Dagenais F, Voisine P, Charbonneau E. Completeness of revascularization and survival among octogenerians with triple-vessel disease. Ann Thorac Surg 2012;93:1432–8. [2] Bauer B, Simkhovich BZ, Kloner RA, Przyklenk K. Does preconditioning protect the coronary vasculature from subsequent ischemia/reperfusion injury? Circulation 1993; 88:659–72. [3] Harjai KJ, Boura J, Grines L, Goldstein J, Stone GW, Brodie B, Cox D, O’Neill WW, Grines C. Comparison of effectiveness of primary angioplasty for proximal versus distal right coronary artery culprit lesion during acute myocardial infarction. Am J Cardiol 2002;90:1193–7. [4] Przyklenk K, Bauer B, Ovize M, Kloner RA, Whittaker P. Regional ischemic ‘preconditioning’ protects remote virgin myocardium from subsequent sustained coronary occlusion. Circulation 1993;87:893–9. [5] Liu Z,Vuohelainen V, Tarkka M, Tenhunen J, Lappalainen RS, Narkilahti S, Paavonen T, Oksala N, Wu Z, Mennander A. Glutamate release predicts ongoing myocardial ischemia of rat hearts. Scand J Clin Lab Invest 2010;70:217–24. [6] Vuohelainen V, Raitoharju E, Levula M, Lehtimaki T, Pelto-Huikko M, Honkanen T, Huovila A, Paasonen T, Tarkka M, Mennander A. Myocardial infarction induces early increased remote ADAM8 expression of rat hearts after cadiac arrest. Sand J Clin Lab Invest 2011;71:553–62. [7] Bjornsson B, Windbladh A, Bojmar L, Trulsson LM, Olsson H, Sundquist T, Gulstrand P, Sandstrom P. Remote or conventional ischemic preconditioning – local liver metabolism in rats studied with microdialysis. JSR 2012;176:55–62. [8] Lofgren B, Povlsen JA, Rasmussen LE, Stottrup NB, Solskov L, Krarup P-M, Kristiansen SB, Botker HE,
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