Heart Vessels DOI 10.1007/s00380-014-0589-1
ORIGINAL ARTICLE
Effects of intracoronary melatonin on ischemia–reperfusion injury in ST-elevation myocardial infarction Sarah V. Ekeløf · Natalie L. Halladin · Svend E. Jensen · Tomas Zaremba · Jens Aarøe · Benedict Kjærgaard · Carsten W. Simonsen · Jacob Rosenberg · Ismail Gögenur
Received: 2 June 2014 / Accepted: 3 October 2014 © Springer Japan 2014
Abstract Acute coronary occlusion is effectively treated by primary percutaneous coronary intervention. However, myocardial ischemia–reperfusion injury is at the moment an unavoidable consequence of the procedure. Oxidative stress is central in the development of ischemia–reperfusion injury. Melatonin, an endogenous hormone, acts through antioxidant mechanisms and could potentially minimize the myocardial injury. The aim of the experimental study was to examine the cardioprotective effects of melatonin in a porcine closed-chest reperfused infarction model. A total of 20 landrace pigs were randomized to a dosage of 200 mg (0.4 mg/mL) melatonin or placebo (saline). The intervention was administered intracoronary and intravenous. Infarct size, area at risk and microvascular obstruction were determined ex vivo by cardiovascular magnetic resonance imaging. Myocardial salvage index was calculated. The plasma levels of high-sensitive troponin T were assessed repeatedly. The experimenters were blinded with regard to treatment regimen. Melatonin did not significantly
increase myocardial salvage index compared with placebo [melatonin 21.8 % (16.1; 24.8) vs. placebo 20.2 % (16.9; 27.0), p = 1.00]. The extent of microvascular obstruction was similar between the groups [melatonin 3.8 % (2.7; 7.1) vs. placebo 3.7 % (1.3; 7.7), p = 0.96]. The area under the curve for high-sensitive troponin T release was insignificantly reduced by 32 % in the melatonin group [AUC melatonin 12,343.9 (6,889.2; 20,147.4) ng h/L vs. AUC placebo 18,285.3 (5,180.4; 23,716.8) ng h/L, p = 0.82]. Combined intracoronary and intravenous treatment with melatonin did not reduce myocardial reperfusion injury. The lack of a positive effect could be due to an ineffective dose of melatonin, a type II error or the timing of administration. Keywords Acute myocardial infarction · Reperfusion injury · Animal experimentation · Oxidative stress · Antioxidant
Introduction S. V. Ekeløf (*) · N. L. Halladin · J. Rosenberg · I. Gögenur Department of Surgery, Herlev Hospital, Centre for Perioperative Optimization, University of Copenhagen, Herlev Ringvej 75, 2730 Herlev, Denmark e-mail: [email protected] S. E. Jensen · T. Zaremba · J. Aarøe Department of Cardiology, Aalborg University Hospital, Hobrovej 18, 9000 Aalborg, Denmark B. Kjærgaard Department of Cardiothoracic Surgery, Aalborg University Hospital, Hobrovej 18, 9000 Aalborg, Denmark C. W. Simonsen Department of Diagnostic Imaging, Aalborg University Hospital, Hobrovej 18, 9000 Aalborg, Denmark
Acute ST-elevation myocardial infarction (STEMI) is effectively treated by early reperfusion of the ischemic myocardium by primary percutaneous coronary intervention. This quick revascularization reduces the myocardial infarct and improves the clinical outcomes following a STEMI [1]. However, reperfusion itself may induce reversible and irreversible damage to the myocardium, a phenomenon referred to as myocardial ischemia–reperfusion injury, IRI [2, 3]. The pathogenesis of IRI is multifactorial but evidence has proven that oxidative stress including the production of reactive oxygen and nitrogen species is of crucial importance [4–10]. Reactive oxygen and nitrogen species are involved in several detrimental processes during reperfusion involving DNA damage, peroxidation of lipid
13
membranes, macromolecule oxidation, alteration of the intracellular calcium homeostasis and opening of the mitochondrial permeability transition pores leading to apoptosis [4, 11, 12]. Therefore, a major concern during myocardial reperfusion is to ameliorate the damage of the reactive oxygen and nitrogen species. Melatonin, an endogenously produced circadian hormone, is a potent direct and indirect antioxidant in physiological as well as pharmacological concentrations [13–16]. Melatonin is a potent scavenger of a wide variety of reactive oxygen and nitrogen species including the hydroxyl radical, peroxyl radical, hydrogen peroxide and nitric oxide [13, 15, 17]. Melatonin’s second- and third-generation metabolites formed in the reaction with free radicals also have the scavenging ability [18–20]. Moreover, melatonin stimulates the intracellular antioxidant enzymes including superoxide dismutase and glutathione peroxidase and acts synergistically with other endogenous antioxidants [21, 22]. At the mitochondrial level, melatonin reduces electron leakage during reperfusion leading to a reduction in generation of free radicals [23] and acts antiapoptotic by inhibiting the opening of mitochondrial permeability transition pores [24, 25]. The cardioprotective effect of melatonin has been documented in several experimental studies of myocardial ischemia and reperfusion [15, 26–29]. However, evidence of a cardioprotective effect of melatonin in large animal models is sparse. The aim of the present study was to examine the cardioprotective effect of intracoronary (ic) and intravenous (iv) melatonin in a clinically relevant in vivo porcine model of myocardial ischemia and reperfusion.
Methods Ethical standards The present experimental study was performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 1996) and approved by the Danish Animal Experiments Inspectorate, license no. 2012-15-2934-00583. Experimental procedure A total of 20 normally fed, female, Danish Landrace pigs were used in the closed-chest porcine model of myocardial ischemia and reperfusion. The pigs were premedicated with 4 mL im Zoletil (ketamin 6.25 mg/mL, tiletamin 6.25 mg/ mL, zolazepam 6.25 mg/mL, butorphanol 1.25 mg/mL and xylazin 6.5 mg/mL), intubated with cuffed endotracheal tubes and mechanically ventilated with FiO2 0.5. The ventilation was adjusted to obtain normocapnia (pCO2 4–6 kPa).
13
Heart Vessels
Anesthesia was maintained by inhalation of 1 % isoflurane and analgesia was assured by continuous infusion of fentanyl 5 μL/mL and midazolam 5 μL/mL at a rate of 8 mL/ min and adjusted if needed. The pigs were continuously monitored by electrocardiography (ECG), pulse oximetry and central body temperature. A central venous access was established with a 7Fr introducer sheath in the femoral vein. A single bolus of unfractionated heparin (5000 Units) was administered iv prior to the arterial catheterization. Baseline values were recorded during the pre-ischemic period. A 6Fr percutaneous coronary intervention (PCI)-guiding catheter was inserted through a 9Fr introducer sheath in the femoral artery and under X-ray guidance placed in the left coronary ostium. A baseline coronary angiogram was performed with Iomeron 300 mg/mL. Under X-ray guidance an over-the-wire 6Fr PCI catheter (Apex OTW balloon catheter, Boston Scientific) was placed in the left anterior descending artery (LAD) or the circumflex coronary artery (CX). Under angiographic control, ischemia was induced by inflation of the angioplasty balloon. The occlusion was verified by ECG changes. The vessel was kept occluded for 45 min. Ischemia was followed by 4 h of reperfusion. At the end of the experimental protocol the hearts were explanted and analyzed ex vivo by cardiovascular magnetic resonance (CMR) imaging for left ventricle mass, infarct size (IS), area at risk (AAR) and microvascular obstruction (MVO). An overview of the experimental protocol is shown in Fig. 1. In case of ventricular fibrillation a biphasic defibrillator (360 J) was used for cardioversion. Plasma levels of highsensitive troponin T (hs-TnT) were analyzed at baseline, 30 min after reperfusion and at 1, 2, 3 and 4 h after reperfusion. The plasma level of hs-TnT was analyzed using the Cobas e601 (Roche Diagnostic). An oxidative marker, plasma malondialdehyde and inflammatory markers were assessed repeatedly. Data are reported elsewhere [30]. Intervention procedure The animals were randomized to receive iv and ic melatonin (0.4 mg/mL) or iv and ic placebo (isotonic saline, 0.9 mg/mL). Five minutes prior to reperfusion an iv infusion of 198 mg melatonin diluted in isotonic saline (0.4 mg/ mL) or placebo, 495 mL isotonic saline, was started. The infusion lasted 30 min. One minute prior to reperfusion a bolus of 2 mg melatonin diluted in isotonic saline (0.4 mg/ mL) or placebo, 5 mL isotonic saline, was slowly injected through the central lumen of the over-the-wire catheter directly into the coronary artery. The duration of the ic infusion was 2 min and the infusion lasted during the first minute of reperfusion, see Fig. 1. The experimenters were blinded with regard to treatment regimen.
Heart Vessels
Myocardial edema was visualized by T2-weighted fast spin-echo sequence. Post gadolinium contrast T2-weighted assessment of myocardial edema has been previously validated by Ubachs et al. [34]. In the present study, the following parameters were applied: repetition time (TR) 2000 ms, echo time (TE) 105 ms, slice thickness 4 mm, slice gap 0.4 mm, field of view 24 × 19 cm, image matrix 384 × 256, number of excitations (NEX) 4. Afterwards, the infarct size and area of no-reflow (MVO) were imaged using T1-weighted IR-prepped Fast Gradient sequence—standard GE Late Gadolinium Enhancement sequence with slice thickness 4 mm, slice gap 0.4 mm, field of view 24 × 19 cm, image matrix 224 × 192, NEX 2. Inversion time was adjusted visually to null normal myocardium and ranged 180–220 ms. Fig. 1 Study protocol. a Experimental procedure. Ischemia was induced by inflation of an angioplasty balloon located in the coronary artery. After 45 min, the balloon was deflated and the ischemic myocardium was reperfused for 4 h before the heart was explanted and scanned by ex vivo cardiovascular magnetic resonance imaging. b Intervention procedure. The pigs (n = 20) were randomized to a total dose of 200 mg melatonin or placebo (saline). The intravenous infusion was started 5 min prior to reperfusion and the intracoronary bolus was administered 1 min prior to reperfusion. CMR cardiovascular magnetic resonance imaging, iv intravenous, ic intracoronary
Dose of melatonin The dose of melatonin was based on an in vivo experimental study on myocardial ischemia and reperfusion [26] and on a clinical randomized study on lower body ischemia and reperfusion [31]. The dose of melatonin was estimated to be 200 mg (5 mg/kg, expected weight of 40 kg/pig) diluted in 500 mL of isotonic saline (0.4 mg/mL). Cardiovascular magnetic resonance imaging protocol A gadolinium-based contrast agent (0.4 mmol/kg) was administered iv 30 min prior to explantation of the hearts. After explantation, the hearts were rinsed in cold saline and the atriums were removed. To shape the hearts anatomically correct the ventricles were filled with thin rubber balloons containing a solution of sodium chloride, magnesium and manganese. The ex vivo CMR scan was undertaken according to previously described experimental protocols [32, 33]. The imaging was performed using a 1.5-tesla GE Discovery 450 MRI scanner (General Electric Company, Milwaukee, WI, USA) with a standard 8-channel phased-array head coil. After acquiring localizer images, the hearts were scanned in the short-axis direction to cover the entire left ventricle.
Image analysis CMR images were analyzed using dedicated software ReportCARD 4.0 (GE Healthcare). The CMR analysis was performed by a blinded observer. Myocardial mass of the left ventricle was defined by manually tracing the epicardial and endocardial borders. The papillary muscles were included in the left ventricle mass. Area at risk was defined as an area of hyper intense signal relative to normal myocardium on the T2-weighted images and was traced manually. The infarct size was quantified on inversion recovery images. An automatic algorithm of the analysis software was applied, using a threshold of 50 % between normal dark and enhanced bright pixels. If needed, the area of delayed gadolinium enhancement (LGE) was corrected manually by the observer. MVO was defined as a hypointense core within the area of LGE. It was traced manually and was included in the infarct size calculation. Myocardial salvage (MS) was defined as the difference between AAR and IS. To adjust for any differences in AAR, MS was indexed to AAR and was reported as myocardial salvage index, MSI = (AAR − IS)/AAR × 100 [35]. Statistics The sample size was calculated based on a risk of a type I error at 5 %, type II error at 10 % and a standard deviation on IS assessed by ex vivo CMR imaging at 8.2 [33]. The power calculation revealed that seven pigs were needed in each group. We included 10 pigs in each treatment group, since this was the first study to investigate the effect of melatonin in the closed-chest porcine model and due to an expected waste of 1–2 pigs in each arm. Calculations and statistics were performed with the SPSS/PC+ package 20.0 (SPSS, Chicago, IL, USA). Nonparametric tests were used after testing for normality with
13
Heart Vessels
39.9 (39.2; 40.9) 39.5 (39.0; 40.8) 39 (38.6; 40.1)
40.1 (39.5; 41.7)
127.0 (111.3; 183.8) 121.0 (94.0; 143.5) 111.0 (85.5; 136.0) 117.0 (78.0; 138.5)
Parameter
Melatonin (n = 8)
Placebo (n = 9)
p value
ISa AARa MSIb
26.5 (24.0; 31.4) 32.1 (29.5; 41.0) 21.8 (16.1; 24.8)
24.1 (20.0; 32.9) 34.5 (26.5; 41.8) 20.2 (16.9; 27.0)
0.54 0.74 1.00
MVOa
3.8 (2.7; 7.1)
3.7 (1.3; 7.7)
0.96
The values are expressed as median (quartiles). Groups were compared by Mann–Whitney U test IS infarct size, AAR area at risk, MSI myocardial salvage index, MVO microvascular obstruction a
Percent of left ventricle
b
MSI = (AAR − IS)/AAR × 100
the Kolmogorov–Smirnov test. The Mann–Whitney U test was used to test for statistical significance for two independent samples. Values were presented as median (quartiles). Statistical significance was accepted where p
ORIGINAL ARTICLE
Effects of intracoronary melatonin on ischemia–reperfusion injury in ST-elevation myocardial infarction Sarah V. Ekeløf · Natalie L. Halladin · Svend E. Jensen · Tomas Zaremba · Jens Aarøe · Benedict Kjærgaard · Carsten W. Simonsen · Jacob Rosenberg · Ismail Gögenur
Received: 2 June 2014 / Accepted: 3 October 2014 © Springer Japan 2014
Abstract Acute coronary occlusion is effectively treated by primary percutaneous coronary intervention. However, myocardial ischemia–reperfusion injury is at the moment an unavoidable consequence of the procedure. Oxidative stress is central in the development of ischemia–reperfusion injury. Melatonin, an endogenous hormone, acts through antioxidant mechanisms and could potentially minimize the myocardial injury. The aim of the experimental study was to examine the cardioprotective effects of melatonin in a porcine closed-chest reperfused infarction model. A total of 20 landrace pigs were randomized to a dosage of 200 mg (0.4 mg/mL) melatonin or placebo (saline). The intervention was administered intracoronary and intravenous. Infarct size, area at risk and microvascular obstruction were determined ex vivo by cardiovascular magnetic resonance imaging. Myocardial salvage index was calculated. The plasma levels of high-sensitive troponin T were assessed repeatedly. The experimenters were blinded with regard to treatment regimen. Melatonin did not significantly
increase myocardial salvage index compared with placebo [melatonin 21.8 % (16.1; 24.8) vs. placebo 20.2 % (16.9; 27.0), p = 1.00]. The extent of microvascular obstruction was similar between the groups [melatonin 3.8 % (2.7; 7.1) vs. placebo 3.7 % (1.3; 7.7), p = 0.96]. The area under the curve for high-sensitive troponin T release was insignificantly reduced by 32 % in the melatonin group [AUC melatonin 12,343.9 (6,889.2; 20,147.4) ng h/L vs. AUC placebo 18,285.3 (5,180.4; 23,716.8) ng h/L, p = 0.82]. Combined intracoronary and intravenous treatment with melatonin did not reduce myocardial reperfusion injury. The lack of a positive effect could be due to an ineffective dose of melatonin, a type II error or the timing of administration. Keywords Acute myocardial infarction · Reperfusion injury · Animal experimentation · Oxidative stress · Antioxidant
Introduction S. V. Ekeløf (*) · N. L. Halladin · J. Rosenberg · I. Gögenur Department of Surgery, Herlev Hospital, Centre for Perioperative Optimization, University of Copenhagen, Herlev Ringvej 75, 2730 Herlev, Denmark e-mail: [email protected] S. E. Jensen · T. Zaremba · J. Aarøe Department of Cardiology, Aalborg University Hospital, Hobrovej 18, 9000 Aalborg, Denmark B. Kjærgaard Department of Cardiothoracic Surgery, Aalborg University Hospital, Hobrovej 18, 9000 Aalborg, Denmark C. W. Simonsen Department of Diagnostic Imaging, Aalborg University Hospital, Hobrovej 18, 9000 Aalborg, Denmark
Acute ST-elevation myocardial infarction (STEMI) is effectively treated by early reperfusion of the ischemic myocardium by primary percutaneous coronary intervention. This quick revascularization reduces the myocardial infarct and improves the clinical outcomes following a STEMI [1]. However, reperfusion itself may induce reversible and irreversible damage to the myocardium, a phenomenon referred to as myocardial ischemia–reperfusion injury, IRI [2, 3]. The pathogenesis of IRI is multifactorial but evidence has proven that oxidative stress including the production of reactive oxygen and nitrogen species is of crucial importance [4–10]. Reactive oxygen and nitrogen species are involved in several detrimental processes during reperfusion involving DNA damage, peroxidation of lipid
13
membranes, macromolecule oxidation, alteration of the intracellular calcium homeostasis and opening of the mitochondrial permeability transition pores leading to apoptosis [4, 11, 12]. Therefore, a major concern during myocardial reperfusion is to ameliorate the damage of the reactive oxygen and nitrogen species. Melatonin, an endogenously produced circadian hormone, is a potent direct and indirect antioxidant in physiological as well as pharmacological concentrations [13–16]. Melatonin is a potent scavenger of a wide variety of reactive oxygen and nitrogen species including the hydroxyl radical, peroxyl radical, hydrogen peroxide and nitric oxide [13, 15, 17]. Melatonin’s second- and third-generation metabolites formed in the reaction with free radicals also have the scavenging ability [18–20]. Moreover, melatonin stimulates the intracellular antioxidant enzymes including superoxide dismutase and glutathione peroxidase and acts synergistically with other endogenous antioxidants [21, 22]. At the mitochondrial level, melatonin reduces electron leakage during reperfusion leading to a reduction in generation of free radicals [23] and acts antiapoptotic by inhibiting the opening of mitochondrial permeability transition pores [24, 25]. The cardioprotective effect of melatonin has been documented in several experimental studies of myocardial ischemia and reperfusion [15, 26–29]. However, evidence of a cardioprotective effect of melatonin in large animal models is sparse. The aim of the present study was to examine the cardioprotective effect of intracoronary (ic) and intravenous (iv) melatonin in a clinically relevant in vivo porcine model of myocardial ischemia and reperfusion.
Methods Ethical standards The present experimental study was performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 1996) and approved by the Danish Animal Experiments Inspectorate, license no. 2012-15-2934-00583. Experimental procedure A total of 20 normally fed, female, Danish Landrace pigs were used in the closed-chest porcine model of myocardial ischemia and reperfusion. The pigs were premedicated with 4 mL im Zoletil (ketamin 6.25 mg/mL, tiletamin 6.25 mg/ mL, zolazepam 6.25 mg/mL, butorphanol 1.25 mg/mL and xylazin 6.5 mg/mL), intubated with cuffed endotracheal tubes and mechanically ventilated with FiO2 0.5. The ventilation was adjusted to obtain normocapnia (pCO2 4–6 kPa).
13
Heart Vessels
Anesthesia was maintained by inhalation of 1 % isoflurane and analgesia was assured by continuous infusion of fentanyl 5 μL/mL and midazolam 5 μL/mL at a rate of 8 mL/ min and adjusted if needed. The pigs were continuously monitored by electrocardiography (ECG), pulse oximetry and central body temperature. A central venous access was established with a 7Fr introducer sheath in the femoral vein. A single bolus of unfractionated heparin (5000 Units) was administered iv prior to the arterial catheterization. Baseline values were recorded during the pre-ischemic period. A 6Fr percutaneous coronary intervention (PCI)-guiding catheter was inserted through a 9Fr introducer sheath in the femoral artery and under X-ray guidance placed in the left coronary ostium. A baseline coronary angiogram was performed with Iomeron 300 mg/mL. Under X-ray guidance an over-the-wire 6Fr PCI catheter (Apex OTW balloon catheter, Boston Scientific) was placed in the left anterior descending artery (LAD) or the circumflex coronary artery (CX). Under angiographic control, ischemia was induced by inflation of the angioplasty balloon. The occlusion was verified by ECG changes. The vessel was kept occluded for 45 min. Ischemia was followed by 4 h of reperfusion. At the end of the experimental protocol the hearts were explanted and analyzed ex vivo by cardiovascular magnetic resonance (CMR) imaging for left ventricle mass, infarct size (IS), area at risk (AAR) and microvascular obstruction (MVO). An overview of the experimental protocol is shown in Fig. 1. In case of ventricular fibrillation a biphasic defibrillator (360 J) was used for cardioversion. Plasma levels of highsensitive troponin T (hs-TnT) were analyzed at baseline, 30 min after reperfusion and at 1, 2, 3 and 4 h after reperfusion. The plasma level of hs-TnT was analyzed using the Cobas e601 (Roche Diagnostic). An oxidative marker, plasma malondialdehyde and inflammatory markers were assessed repeatedly. Data are reported elsewhere [30]. Intervention procedure The animals were randomized to receive iv and ic melatonin (0.4 mg/mL) or iv and ic placebo (isotonic saline, 0.9 mg/mL). Five minutes prior to reperfusion an iv infusion of 198 mg melatonin diluted in isotonic saline (0.4 mg/ mL) or placebo, 495 mL isotonic saline, was started. The infusion lasted 30 min. One minute prior to reperfusion a bolus of 2 mg melatonin diluted in isotonic saline (0.4 mg/ mL) or placebo, 5 mL isotonic saline, was slowly injected through the central lumen of the over-the-wire catheter directly into the coronary artery. The duration of the ic infusion was 2 min and the infusion lasted during the first minute of reperfusion, see Fig. 1. The experimenters were blinded with regard to treatment regimen.
Heart Vessels
Myocardial edema was visualized by T2-weighted fast spin-echo sequence. Post gadolinium contrast T2-weighted assessment of myocardial edema has been previously validated by Ubachs et al. [34]. In the present study, the following parameters were applied: repetition time (TR) 2000 ms, echo time (TE) 105 ms, slice thickness 4 mm, slice gap 0.4 mm, field of view 24 × 19 cm, image matrix 384 × 256, number of excitations (NEX) 4. Afterwards, the infarct size and area of no-reflow (MVO) were imaged using T1-weighted IR-prepped Fast Gradient sequence—standard GE Late Gadolinium Enhancement sequence with slice thickness 4 mm, slice gap 0.4 mm, field of view 24 × 19 cm, image matrix 224 × 192, NEX 2. Inversion time was adjusted visually to null normal myocardium and ranged 180–220 ms. Fig. 1 Study protocol. a Experimental procedure. Ischemia was induced by inflation of an angioplasty balloon located in the coronary artery. After 45 min, the balloon was deflated and the ischemic myocardium was reperfused for 4 h before the heart was explanted and scanned by ex vivo cardiovascular magnetic resonance imaging. b Intervention procedure. The pigs (n = 20) were randomized to a total dose of 200 mg melatonin or placebo (saline). The intravenous infusion was started 5 min prior to reperfusion and the intracoronary bolus was administered 1 min prior to reperfusion. CMR cardiovascular magnetic resonance imaging, iv intravenous, ic intracoronary
Dose of melatonin The dose of melatonin was based on an in vivo experimental study on myocardial ischemia and reperfusion [26] and on a clinical randomized study on lower body ischemia and reperfusion [31]. The dose of melatonin was estimated to be 200 mg (5 mg/kg, expected weight of 40 kg/pig) diluted in 500 mL of isotonic saline (0.4 mg/mL). Cardiovascular magnetic resonance imaging protocol A gadolinium-based contrast agent (0.4 mmol/kg) was administered iv 30 min prior to explantation of the hearts. After explantation, the hearts were rinsed in cold saline and the atriums were removed. To shape the hearts anatomically correct the ventricles were filled with thin rubber balloons containing a solution of sodium chloride, magnesium and manganese. The ex vivo CMR scan was undertaken according to previously described experimental protocols [32, 33]. The imaging was performed using a 1.5-tesla GE Discovery 450 MRI scanner (General Electric Company, Milwaukee, WI, USA) with a standard 8-channel phased-array head coil. After acquiring localizer images, the hearts were scanned in the short-axis direction to cover the entire left ventricle.
Image analysis CMR images were analyzed using dedicated software ReportCARD 4.0 (GE Healthcare). The CMR analysis was performed by a blinded observer. Myocardial mass of the left ventricle was defined by manually tracing the epicardial and endocardial borders. The papillary muscles were included in the left ventricle mass. Area at risk was defined as an area of hyper intense signal relative to normal myocardium on the T2-weighted images and was traced manually. The infarct size was quantified on inversion recovery images. An automatic algorithm of the analysis software was applied, using a threshold of 50 % between normal dark and enhanced bright pixels. If needed, the area of delayed gadolinium enhancement (LGE) was corrected manually by the observer. MVO was defined as a hypointense core within the area of LGE. It was traced manually and was included in the infarct size calculation. Myocardial salvage (MS) was defined as the difference between AAR and IS. To adjust for any differences in AAR, MS was indexed to AAR and was reported as myocardial salvage index, MSI = (AAR − IS)/AAR × 100 [35]. Statistics The sample size was calculated based on a risk of a type I error at 5 %, type II error at 10 % and a standard deviation on IS assessed by ex vivo CMR imaging at 8.2 [33]. The power calculation revealed that seven pigs were needed in each group. We included 10 pigs in each treatment group, since this was the first study to investigate the effect of melatonin in the closed-chest porcine model and due to an expected waste of 1–2 pigs in each arm. Calculations and statistics were performed with the SPSS/PC+ package 20.0 (SPSS, Chicago, IL, USA). Nonparametric tests were used after testing for normality with
13
Heart Vessels
39.9 (39.2; 40.9) 39.5 (39.0; 40.8) 39 (38.6; 40.1)
40.1 (39.5; 41.7)
127.0 (111.3; 183.8) 121.0 (94.0; 143.5) 111.0 (85.5; 136.0) 117.0 (78.0; 138.5)
Parameter
Melatonin (n = 8)
Placebo (n = 9)
p value
ISa AARa MSIb
26.5 (24.0; 31.4) 32.1 (29.5; 41.0) 21.8 (16.1; 24.8)
24.1 (20.0; 32.9) 34.5 (26.5; 41.8) 20.2 (16.9; 27.0)
0.54 0.74 1.00
MVOa
3.8 (2.7; 7.1)
3.7 (1.3; 7.7)
0.96
The values are expressed as median (quartiles). Groups were compared by Mann–Whitney U test IS infarct size, AAR area at risk, MSI myocardial salvage index, MVO microvascular obstruction a
Percent of left ventricle
b
MSI = (AAR − IS)/AAR × 100
the Kolmogorov–Smirnov test. The Mann–Whitney U test was used to test for statistical significance for two independent samples. Values were presented as median (quartiles). Statistical significance was accepted where p
