Dronedarone does not affect infarct volume as assessed by magnetic resonance imaging in a porcine model of myocardial infarction

  • Authors:
    • Josefine Linke
    • Kirsten Utpatel
    • Carmen Wolke
    • Matthias Evert
    • Jens‑Peter Kühn
    • Alicja Bukowska
    • Andreas Goette
    • Uwe Lendeckel
    • Barbara Peters
  • View Affiliations

  • Published online on: July 13, 2015     https://doi.org/10.3892/mmr.2015.4077
  • Pages: 5169-5178
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Abstract

Dronedarone has been demonstrated to be harmful in patients with recent decompensated heart failure. Furthermore, a PALLAS study reported that dronedarone therapy increases mortality rates in patients with permanent atrial fibrillation. Although a pathophysiological explanation for these finding remains to be elucidated, the long term effects of dronedarone on myocardial structure and stability have been suggested. The aim of the present study was to determine whether dronedarone therapy affects left ventricular (LV) function in a chronic model of myocardial infarction (MI). An anterior MI was induced in 16 pigs. Of these animals, eight pigs were then treated with dronedarone for 1 week prior to, and 4 weeks following MI, the remaining pigs served as controls. LV angiography was performed 4 weeks after MI to determine the LV ejection fraction (LVEF). A post‑mortem magnetic resonance imaging scan of the LV was then performed on the two groups (n=6) to determine the volume and size of the induced MI. Dronedarone therapy did not affect systemic and intracardiac hemodynamic parameters or LVEF during the follow‑up assessment. Of note, dronedarone had no negative effect on the total infarct volume and size and did not induce lethal proarrhythmic effects following the induced anterior MI. Therefore, the results suggested that dronedarone did not increase the volume or size of induced anterior MI and did not affect LV performance. Thus, dronedarone therapy was observed to be safe in a porcine model of anterior MI.

Introduction

Atrial fibrillation (AF) is associated with a high risk for subsequent ischemic events (1). Data from the previous ATHENA study demonstrated that dronedarone therapy reduces cardiovascular mortality rates, hospitalization and the incidence of myocardial infarction (MI) in patients with AF (2). Separate subgroup analyses revealed that the incidence of acute coronary syndromes is also reduced in patients with coronary artery disease at baseline (3). The pathological mechanisms underlying these observations remain to be elucidated.

In patients with AF, the coronary flow reserve (CFR) is reduced (4,5). A compromised CFR has also been described in an acute model of rapid atrial pacing (RAP) in vivo, in which the fractional flow reserve appears to be unaffected (6,7). Calcium overload and oxidative stress are likely to contribute to these effects (6,7), as the two are associated with increased inflammation, fibrosis, adipocyte infiltration and myocardial hypertrophy and, thus, promote structural remodeling of the myocardium (810). In line with previous data demonstrating the potential of benzofuranes, including dronedarone (or amiodarone) to increase coronary flow (11,12), our previous study demonstrated that dronedarone largely prevents the AF-dependent decline of the CRF during acute RAP in vivo (7). The capability of dronedarone to inhibit α-adrenergic vasoconstriction (13,14) may well contribute to the improved flow. In addition, as dronedarone reduces heart rate, an increase in diastolic duration and a reduction in oxygen demand may also contribute to its pleiotropic effects (14,15). Of note, dronedarone has demonstrated cardioprotective activity in a porcine model of myocardial infarction/reperfusion, which is independent of the remaining subendocardial blood flow during ischemia (16). Although dronedarone has been observed to attenuate redox-activated and ischemia-associated gene expression (7), the mechanisms underlying its potential cardioprotective action remain to be fully elucidated. To confirm the hypothesis that the administration of dronedarone prevents functional changes in the coronary arteries and damage to the adjacent myocardial tissue, in vivo experiments are required, which allow for a pharmacological intervention. Consequently, the present study analyzed the possible effects of dronedarone on infarct size in a porcine model of acute MI with a subsequent 4-week recovery period. This aimed to detect late structural/contractile alterations, which may result from the possible interference of dronedarone with structural remodeling and repair processes in the myocardium and infarct scar. These are known to be slow processes due to the long half-life time of the components involved, including matricellular proteins (17).

In this context, magnetic resonance imaging (MRI) may be a suitable approach to evaluate infarct size, as histological quantification is time consuming and its accuracy depends on the slice thickness. The 7 Tesla MRI method enables the potential to acquire three dimensional (3D) data with a thin slice thickness (100 µm) and to quantify the 3D infarct structures at an almost microscopic level (18). Usually, contrast agents are used to identify necrotic tissue (19). However, these contrast agents may interfere with further biochemical or molecular biological analysis. To evaluate the capability of 7 Tesla MRI to differentiate between infarcted and non-infarcted myocardium at microscopic levels without the use of contrast agents, the present study performed 3D-MRI analysis and compared the results with microscopic findings of the corresponding regions.

Materials and methods

Anterior MI model

All animal care and experimental procedures were approved by the Landesverwaltungsamt Sachsen-Anhalt (Az42502-2-1117-IMTR). A total of 16 male castrated pigs (German Landrace/German Pietrain, mean weight 30 kg, mean age 3 months; Schweinezucht Gladau GmbH, Kleindemsin, Germany) were used in the current study. Animal health status was monitored daily prior to study and twice/day during the study. Animals were maintained between 15 and 20°C with a 12 h light/dark cycle, were fed with conventional piglet raising food (Deuka Deutsche Kraftfutterwerke GmbH & Co., Düsseldorf, Germany) and had ad libitum access to water. The pigs were assigned into two study groups, both received the surgery-associated standard medication, described below, and one received dronedarone (Sanofi-Aventis, Paris, France). The dronedarone treatment (200 mg) was administered orally twice a day, to attempt to match the quantity of 400 mg used in the ATHENA trial (2), commenced 7 days prior to the experimental MI and was continued for 4 weeks until explantation of the heart. The experiments were performed in three temporally separated rounds, each containing six or four pigs each.

The premedication provided to the animals consisted of an oral administration of 100 mg acetylsalicylic acid (Ratiopharm, Ulm, Germany) and 75 mg clopidogrel (Plavix®, Sanofi Pharma, Berlin, Germany) 2 days prior to surgery, as well as a single dose of 25 mg atenolol (Ratiopharm) 1 day prior to surgery. Atenolol is known to prevent mortality caused by severe arrhythmia during the induction phase of acute MI. For sedation, intramuscular injections of 0.2 ml kg−1 ketamine (10% ketamine; Ursotamin®; Serumwerk Bernburg AG, Bernburg, Germany) and 0.1 ml kg−1 xylazine (2% xylazine hydrochloride; Xylazin®; Riemser Arzneimittel GmbH, Greifswald, Germany) were administered. Anesthesia was induced using 6 ml propofol intravenously (1% Recofol, Curamed Pharma GmbH, Baden-Wurttemberg, Germany) and maintained with 1–2 vol% isoflurane (Isofluran Curamed, Curamed Pharma GmbH) in a mixture of O2:N2O (30:70 vol%) via an endotracheal tube. Oxygen saturation and electrocardiogram (ECG) were monitored continuously throughout the procedure. At the beginning of surgery, the animals received 0.04 ml kg−1 meloxicam subcutaneously (20 mg/ml−1 Metacam®, Boehringer-Ingelheim GmBH, Ingelheim, Germany), 0.2 ml/kg−1 oxytetracycline subcutaneously (10% Ursocyclin®, Serumwerk Bernburg AG), 250 mg acetylsalicylic acid and 5,000 IU heparin intra-arterially (B. Braun Melsungen AG, Melsungen, Germany). Anticoagulatory prevention was prolonged with intra-arterial injections of 2,500 IU heparin during the procedure.

The MI was induced by temporary occlusion of the left anterior descending (LAD) artery using a balloon catheter (SeQuent®II; 3.0×15 mm; B. Braun Melsungen AG). Prior to occlusion, ventricular angiography and coronary angiography of the left main coronary artery were performed to examine the positions of its branches. To attain comparable infarct sizes, the occlusion was performed distal to the first septal branch. The correct position of the balloon catheter was confirmed using angiography (Artis Zee Biplane System; Siemens AG Healthcare Sector, Erlangen, Germany) (Fig. 1A). Prior to inflation of the balloon, 100 mg lidocaine (Bela-pharm Arzneimittelfabrik, Vechta, Germany) and 0.5 mg buprenorphine (Temgesic®, Essex Pharma GmBH, Munich, Germany) were administered intravenously. The blood flow of the LAD artery was interrupted for a total of 90 min. Subsequently, the balloon catheter was deflated and the reperfusion phase was monitored for the first 60 min.

After 4 weeks, the hearts were explanted for determination of the infarct size using MRI imaging (7.1 Tesla Animal MR Scanner; Bruker; Ettlingen, Germany). For surgery, the pigs were sedated, anesthetized and subjected to analgesia as described above. In addition, 1 mg pancuronium (Ratiopharm) was administered intravenously for muscle relaxation. Following ventricular angiography, the pigs were sacrificed with 10 ml KCl intravenous administration. Following explantation, the hearts were fixed in 4.5% formalin for MRI imaging.

ECG monitoring and analysis

Throughout the entire infarction and explantation surgical procedures, the cardiac function of the pigs was monitored using a 6-lead-ECG (Axiom Sensis XP Monitoring System; Siemens AG Healthcare Sector) and by measurement of the intraaortic bloodpressure. In the resting ECG analysis, heart rate, PQ-duration (beginning of the P-wave to the beginning of the QRS-complex), QRS-duration and the QT-interval (beginning of the QRS-complex to the end of the T-wave) were investigated using an ECG lead II. The QT-duration was adjusted to the heart rate using Fridericia's formula (QTc = QT/3√RR), as QTc, which appears to provide more accurate results, particularly at higher heart rates (20).

Blood and tissue collection

Blood samples were collected from the pigs prior to occlusion of the LAD, 60 min following reperfusion, and 28 days following infarction, as shown in Fig. 1B. Blood was collected into a BD Vacutainer® EDTA (6 ml; violet) or SST (5 ml; gold), respectively (BD Diagnostics, Heidelberg, Germany). The serum and plasma samples were prepared according to standard surgical procedures and were aliquoted, shock-frozen in liquid nitrogen and stored at −80°C until further use. The plasma levels of troponin I (TnI), creatinine kinase (CK)-MB, alanine transaminase, aspartate aminotransferase (ASAT) and C-reactive protein were analyzed at the Institute of Clinical Chemistry and Laboratory Diagnostics of the University Medicine Greifswald (Greifswald, Germany) using an established platform and diagnostic kits, which are regularly applied in clinical routine analyses. Laboratory measurements were performed on a Siemens Vista 1500 analyzer (Siemens Healthcare Diagnostics, Eschborn, Germany). Aspartate (Siemens AST) and alanine aminotransferase (Siemens ALTI) activities were measured by adapted enzymatic methods of International Federation of Clinical Chemistry and Laboratory Medicine (Milan, Italy) recommended assays (21). The creatinine kinase isoenzyme MB activity was determined by an enzymatic method (Siemens CKMB) using an immunological inhibition of creatinine kinase M subunit activity (22). The cardiac troponin I concentration was measured by a homogenic, high-sensitivity sandwich chemiluminescence immunoassay based on the LOCI™ technology (Luminescent oxygen channeling immunoassay) (23).

Serum concentrations of dronedarone and its metabolite, SR35021, were measured following methanol/acetonitrile extraction by liquid chromatography-tandem mass spectrometry (LC-MS/MS), using a Shimadzu UFLC-XR (Shimadzu Corporation, Kyoto, Japan) coupled with an API4000 AB Sciex mass spectrometer).

For further biochemical and expression analyses, tissue samples of ~0.5 cm3 were cut from the healthy left ventricular (LV) area and the right ventricular area, the border zone and the infarct area (MI). The precise localization of the samples to the indicated areas was confirmed using MRI and histological analyses. The tissue samples were then frozen in liquid nitrogen and stored at −80°C for further use.

Determination of infarct size: Workflow, tissue processing and histopathological analysis

The general workflow of the investigation performed is outlined in Fig. 1C. Initially, the diagnostic criteria for MI required definition in the MRI images. In order to perform this verification, spatial correspondence between the MR images and the tissue-derived histology images is required. This is in general challenging due to the deformations involved during resection of the tissue and histological processing, and the difficulty of accurately registering the histological slices to the MR images (24). Therefore, following acquisition of the MRI 3D dataset, the hearts were cut into 5–7 slices, according to the areas of MI identified in the MRI short axis views. The slice thickness was 1.5 cm. Biopsy sites were used to provide landmarks between MRI and pathology. Images were captured of each slice (Olympus E-410; Olympus, Tokyo, Japan) and the whole macroscopic infarct area, containing a rim of vital myocardium, was embedded in 2–6 paraffin blocks. Sections of 2 µm were then cut vertically and mounted on glass slides (SuperFrost®, R. Langenbrinck, Emmendingen, Germany). The sections were subsequently deparaffinized with xylene and ethanol and stained with hematoxylin and eosin (HE) and acid fuchsin orange G (AFOG), according to standard protocols. The slices were examined using a Zeiss microscope (Axioplan2, Zeiss, Jena, Germany). To outline the infarcted myocardium, AFOG staining was selected, as it not only distinguishes between normal myocardial cells and areas of fibrosis, but also detects non-vital myocardial cells. In AFOG staining, fibrosis of the infarcted area stains blue. Residual red-stained non-vital cardiomyocytes were occasionally found within the fibrotic areas, but not in the surrounding tissue. The border between the infarcted and vital tissues were sharp and readily identified in each sample. The AFOG-stained specimens were then scanned (Leica SCN 400 slide scanner; Leica Microsystems GmbH, Wetzlar, Germany) and the images pasted into the images of the respective slice using Adobe Photoshop CS6 (Adobe Systems Europe, Ltd., Maidenhead, UK). A significant difficulty encountered was that of tissue deformation caused during histological processing. Therefore, the pasted histology slices were aligned to fit to the macroscopic image. In the resultant image, as well as in the corresponding MRI section, the infarct area was measured using ImageJ software (National Institutes of Health Bethesda, MA, USA). A good correlation between the relative area of the infarct (% of LV area) in the MRI images and the corresponding histological images was observed (R=0.98; n=40).

Hemodynanic data

The LV end-diastolic volume, stroke volume and ejection fraction were determined using LV angiography. The ventricular angiography was performed at a 30° right anterior oblique projection using a 6F pigtail catheter (B. Braun Melsungen AG), which was moved into the left ventricle through an arterial 7F port (B. Braun Melsungen AG). The injection of the contrast medium, iopromid (Ultravist®-370; Bayer Healthcare, Berlin, Germany) was performed with an angiomat (Angiomat 3000; Liebel-Flarsheim Company LLC, Cincinnati, OH, USA), using a volume of 20 ml contrast medium and a flow rate of 12 ml/sec.

Quantitative analysis of the LV volumes was performed using the image data processing software of the Artis Zee (Siemens Healthcare, Berlin, Germany) X-ray system for interventional radiology. For this purpose, all images were automatically calibrated with an isocenter calibration. Subsequently, the end-diastolic and end-systolic volume were determined by an automatic contour detection and subsequent manual correction, based on images captured of the left ventricle in end-diastole end-systole. Using these data, the Artis Zee software (Siemens Healthcare) calculated the LV end-diastolic and end-systolic volumes using the biplane area length method, the stroke volume as the difference of the end-diastolic volume, and the end-systolic volume and ejection fraction as a quotient of the stroke volume and the end-diastolic volume. All data were collected in an analysis report, which was stored digitally together with the angiography images.

Calculation of infarct volume and size using 3D MRI

Imaging of the formalin fixed hearts was performed on a 7.1 Tesla Animal MR Scanner. A three-dimensional gradient echo sequence was used; repitition time/echo time, 30/5 ms; interpolated matrix, 1,024×1,024 mm; field of view, 110 mm; 144 slices, slice thickness, 0.7-mm.

From the 3D MRI dataset, a stack of 1 mm thick short axis slices, rectangular to the ventricular septum and covering the whole ventricle, were generated. A total of 70–80 slices were evaluated, depending on the size of the heart, using Osirix software (Pixameo, Bernex, Switzerland) by two observers in a blinded-manner. All measurements were performed manually. LV volume and mass, determined as mass (g) = volume (ml) × myocardial density (1.05 g/ml), was calculated from the difference between the LV pericardial volume and endocardial volume. The infarct size was expressed as the absolute volume and as the LV infarct index, denoting the infarct mass as a percentage of the LV mass. Biopsy had been performed on all hearts in the infarct and peri-infarct regions. The infarct region was estimated in these areas using different image planes and alike planes, tangential to the myocardial wall.

Thinning of the infarcted ventricle wall is not always accompanied by complete scarring of the region (25). For this reason, the area of LV infarct as a percentage of the LV area was measured in addition to the infarct volume associated with the LV volume.

Statistical analysis

The values in Table I are presented as the median with 1st and 3rd quartiles, normalized to the median of the experimental round. The Mann-Whitney-U test for unpaired group comparison was performed to determine any differences between the control and dronedarone at the baseline, during the procedure (5, 30, 60 and 90 min of infarction; 5, 30 and 60 min of reperfusion) and after 4 weeks (explantation). To determine time-dependent changes within one experimental groups, the Wilcoxon-test for paired group comparisons was performed. A Bland-Altman plot was used for analyzing the associations between infarct size determination by MRI and pathohistology. All statistical analyses was two-tailed. P<0.05 was considered to indicate a statistically significant difference, without α-adjustment. All data are expressed as the median with box (1st and 3rd quartile) and whisker (range) plots. Statistical analyses were performed using IBM® SPSS® 20 (IBM SPSS, Somers, NY, USA).

Table I

Hemodynamic parameters during infarction and reperfusion.

Table I

Hemodynamic parameters during infarction and reperfusion.

ParameterTimeControl median (1st; 3rd quartile)Dronedarone median (1st; 3rd quartile)P-value (Mann-Whitney-U)
Body weight (kg)Baseline31.78 (30.31; 33.18)30.65 (29.54; 31.82)0.050
Infarction38.73 (37.38; 39.25)39.24 (37.62; 40.14)0.505
Heart rate (beats/min)Baseline98.50 (95.99; 104.09)97.50 (91.76; 99.89)0.645
5 min Inf91.67 (83.92; 110.64)93.00 (88.62; 99.73)0.574
30 min Inf98.11 (90.86; 115.68)97.00 (91.66; 102.34)1.000
60 min Inf93.48 (83.98; 105.35)94.52 (91.42; 102.78)0.779
90 min Inf89.93 (82.18; 100.53)93.03 (88.90; 95.10)0.328
5 min Reperf123.83 (115.36; 134.27)120.52 (106.82; 126.77)0.536
30 min Reperf138.13 (123.09; 143.59)143.68 (131.41; 156.44)0.414
60 min Reperf119.13 (108.30; 123.93)132.11 (107.89; 154.13)0.336
Explantation87.50 (76.25; 109.25)95.72 (89.05; 123.81)0.132
Mean arterial blood pressure (mmHg)Baseline101.07 (95.96; 114.66)102.42 (94.82; 108.13)0.959
5 min Inf60.39 (56.70; 83.33)65.61 (56.67; 72.10)0.798
30 min Inf70.66 (63.66; 75.73)74.16 (71.84; 76.74)0.336
60 min Inf70.50 (62.32; 74.21)66.70 (61.65; 70.70)0.345
90 min Inf64.50 (57.50; 66.27)66.05 (63.95; 71.82)0.397
5 min Reperf62.00 (56.50; 78.34)59.00 (48.27; 72.00)0.536
30 min Reperf65.00 (59.50; 74.50)66.00 (52.70; 71.44)0.573
60 min Reperf65.97 (62.56; 82.60)63.06 (60.15; 76.17)0.336
Explantation98.50 (58.00; 118.25)97.38 (78.23; 122.44)0.818
PQ-duration (ms)Baseline100 (100; 113)100 (96; 104)0.442
Explantation100 (100; 109)100 (100; 110)0.878
QRS-duration (ms)Baseline55.0 (54.1; 57.9)57.0 (54.1; 57.9)0.798
Explantation60.0 (57.0; 63.0)60.0 (60.0; 64.0)0.574
QTc-duration (ms)Baseline362 (348; 381)376 (353; 396)0.382
Explantation399 (391; 405)410 (398; 430)0.161
LV end-diastolic volume (ml)Baseline80.64 (77.13; 91.57)75.58 (70.47; 79.57)0.105
Explantation101.40 (91.36; 109.94)108.88 (96.86; 123.38)0.328
LV stroke volume (ml)Baseline36.26 (32.09; 39.54)35.92 (32.24; 38.42)0.878
Explantation25.11 (24.25; 30.59)32.51 (29.30; 38.53)0.050
LV ejection fraction (%)Baseline42.26 (41.28; 49.29)46.89 (44.55; 48.10)0.065
Explantation29.78 (25.00; 31.81)30.22 (29.47; 35.35)0.574
Infarct volume (cm3)9.28 (5.45; 10.45)8.79 (8.19; 11.02)1.000
LV volume (cm3)78.24 (75.86; 87.21)90.42 (84.69; 98.04)0.065
LV mass (g)81.36 (78.89; 90.70)94.03 (88.08; 101.96)0.065
LV mass/body weight (g/kg)2.16 (2.04; 2.37)2.39 (2.29; 2.47)0.093
Infarct volume, vs. LV volume (%)10.10 (6.53; 10.91)9.20 (7.71; 10.97)0.937

[i] Inf, infarction; reperf, reperfusion; LV, left ventricular.

Results

Dronedarone serum levels

To prevent any failure of treatment due to too low blood levels of dronedarone, a high dosage (200 mg) was administered. Blood levels of dronedarone or its metabolite, SR35021, were 58.9±6.9 or 8.1±0,73 ng/ml, respectively, in the dronedarone-treated group and, thus confirmed to be in the therapeutic range (Cmax=111 ng/ml). As expected, in the serum of the control animals, the dronedarone and SR35021 concentrations were below the limit of detection.

Hemodynamic parameters

No statistically significant differences were identified in the heart rate or mean arterial blood pressure between the animals treated with or without dronedarone. In the two groups, the heart rate increased by ~40% during reperfusion of the infarcted area, compared with baseline. The mean arterial blood pressure decreased in the two groups by ~30% during MI and reperfusion, compared with baseline. Dronedarone treatment did not impair the stability of the mean arterial blood pressure or increase the risk of ventricular arrhythmias and tachycardia during MI and reperfusion (Table I).

Compared with baseline, the dronedarone-treated animals and the control animals exhibited a significant prolongation of the QRS-duration on the resting ECG following 4 weeks of infarct healing (control, 55 vs. 60 ms; dronedarone, 57 vs. 60 ms, respectively; P=0.012) and of the QTc-duration (adjusted to the heart rate; control, 362 vs. 399 ms; dronedarone, 376 vs. 410 ms, respectively; P=0.025). Dronedarone treatment had no significant effect on the PQ-, QRS- or QT-durations on the resting ECGs of the pigs.

Plasma markers of MI

As expected, the plasma levels of TnI, CK-MB, and ASAT were significantly increased in response to MI (blood sample #A2; Fig. 1). All parameters decreased to baseline 28 days after infarction. No differences were identified in plasma levels between the dronedarone-treated group and the control group (Fig. 2A).

The data indicated that the severity of infarction/tissue damage induced by 90 min LAD occlusion did not differ between the groups.

LV function

LV end-diastolic volume, stroke volume and ejection fraction were determined as parameters for LV function using LV angiography.

All animals in the control group exhibited a statistically significant decrease in LV stroke volume by an average of 31% following 4 weeks of infarct healing, as a result of impaired myocardial contraction in the area of the infarct (P=0.021). However, the stroke volume of the dronedarone-treated pigs did not decrease significantly on the day of the explantation, compared with the baseline. Therefore, LV stroke volume tended to increase by 7.4 ml in the dronedarone-treated group, compared with the stroke volume of the control group, following 4 weeks of infarct healing (P=0.05).

Prior to experimental MI, the dronedarone-treated animals were observed to have a reduced end-diastolic volume, compared with the control animals, although this was not significant (P=0.105). As a result of LV dilatation, the end-diastolic volume of the dronedarone-treated animals was significantly increased by 44% following 4 weeks of infarct healing (P=0.012). The end-diastolic volume of the control animals was also significantly increased, compared with baseline, although only by 26% (P=0.017). Following 4 weeks of infarct healing, the end-diastolic volumes of the control animals and the dronedarone-treated animals were similar in size at 101.4 and 108.88 ml (P=0.328; Fig. 2B).

Compared with baseline measurements, the LVEF was significantly reduced by 30% in the control group (P=0.012) and 35% in the dronedarone-treated group (P=0.012) following 4 weeks of infarct healing (control, 42.26 vs. 29.78%; dronedarone, 46.89 vs. 30.22%). However, the dronedarone-treated animals tended to have a higher ejection fraction prior to experimental MI (P=0.065), although this was not detected following 4 weeks of infarct healing, However, dronedarone treatment did not impair LV function 4 weeks after experimental MI (Fig. 2B).

Histopathology

Evidence of an MI was present in all the hearts. The infarcted myocardium was replaced by granulation tissue and young fibrosis, corresponding to the alterations, which are expected 4 weeks after ischemia, termed 'myocardial infarct in organization' (17). The adjacent myocardium was vital and several cardiomyocytes were hypertrophic. Post-infarct edema was observed in all control and dronedarone-treated porcine hearts.

Evaluating myocardial infarct size using 3D MRI

Infarcted regions were identified, even without the use of contrast agent, on MRI (Fig. 3A). In line with the corresponding histological specimens, it was demonstrated that MRI was able to detect viable myocardium in the infarct region (Fig. 3A). Vital regions inside the infarct zone were mostly found in the first layers of the subendocardial myocardium, but also in other areas of the infarcted area. The right ventricular myocardium in the region of the sulcus interventricularis was preserved. This was clearly delineated in the MRI and in the AFOG-stained histological sections. Thus, this region was used as a reference for vital myocardium inside the infarct zone. Measurement of the area of infarction in the pathological specimens and the corresponding MRI sections revealed a high level of correlation between the two methods (R2=0.98). In addition, no proportional bias was noted between the percentage of LV infarct areas, calculated from the segmented images obtained by MRI and histology, as demonstrated by the corresponding Bland-Altman analysis (Fig. 3B). The average inter-individual variation in the measured infarct size was 5.91%.

The shape of the left ventricle on the MRI compared with the angiography demonstrated that the configuration of the hearts in vitro was similar to the shape of end-systolic LV.

The determination of the LV infarct size revealed no differences between the dronedarone group and the control group. Neither the absolute infarct volume nor the infarct size normalized to the LV volume were significantly altered by dronedarone treatment. Dronedarone did not enlarge the area affected by MI (Fig. 3C).

Discussion

Coronary heart disease (CHD) is common among patients with AF (26) and contributes to an increased risk for cardiovascular events. In addition, CHD promotes ventricular arrhythmia, limiting the use of antiarrhythmic drugs in a particular fraction of AF patients with concomitant CHD. Dronedarone has been demonstrated to largely prevent the development of microcirculatory abnormalities in the ventricles, which is observed following a few hours of rapid atrial pacing (7). This is suggestive of dronedarone being particularly effective in the early stages of AF. Dronedarone reduces the recurrence of AF in patients with paroxysmal and persistent AF (27). The ATHENA trial revealed that dronedarone reduced cardiovascular hospitalization and mortality rates in patients with paroxysmal and persistent AF with ≥1 risk factor for vascular events (2). Post-hoc analysis of a 2.5 year follow-up of the ATHENA trial analyzed the safety and cardiovascular outcome of dronedarone in >1,400 patients with persistent or paroxysmal AF and concomitant CHD. As observed previously in the ATHENA trial, dronedarone reduced cardiovascular hospitalization and mortality rates, and reduced first acute coronary syndrome (3). Of note, no increase of proarrhythmias in the dronedarone group was observed. In contrast to these positive effects of dronedarone, the PALLAS study had to be terminated early due to the risk of stroke, MI, systemic embolism or fatalities in patients with permanent AF, who were treated with dronedarone. Therefore, dronedarone is not recommended for patients with permanent AF at high risk for cardiovascular events or in patients with severe heart failure (New York Heart Association class ≥III) (28). The reasons for the discrepancy remain to be fully elucidated, however, it is reasonable to suggest that particularly advanced structural alterations of the myocardium in permanent AF and severe heart failure contribute to the lack of beneficial effects of dronedarone. A previous study reporting data from 4,856 Swedish patients with symptomatic, non-permanent AF treated with dronedarone revealed that the mortality rates in the patients treated with dronedarone were lower than in the AF patients who did not receive dronedarone. This effect remained following adjustment for a number of baseline variables (29). Hohnloser et al (30) also noted that the prescription of dronedarone to patients with AF does not expose patients to an increased risk of mortality or liver disease.

Experimental data has suggested that dronedarone may prevent microcirculatory flow abnormalities in the ventricles during AF (7). These flow abnormalities cause angina pectoris in patients following the initiation of an AF episode. Furthermore, microcirculatory alterations may lead to low grade ischemia, causing the release of troponin in the systemic circulation. The protective effects of dronedarone in the case of ischemia are not limited to AF-dependent changes of the microcirculatory system, rather they appear to reflect a more general, pleiotropic action of dronedarone (14,15,24). Accordingly, dronedarone has been reported to exhibit protective activity in an acute MI model (24), in which dronedarone reduces the size of the infarcted tissue in an acute setting. It remained to be elucidated, however, whether the potential protective effects of dronedarone in reducing infarct size and preservng LV performance, are maintained long-term following an induced MI. The existence of longer-lasting effects is suggested by evidence that, in a rat model of cerebral infarction, a reduction in infarct size in the dronedarone-treated groups is observed 7 days post-infarction (31). This effect, however, was obtained with 3–7.5-fold higher dosages of dronedarone than those used in the present study. Notably, these beneficial effects of dronedarone on infarct size remained, although to a lesser extent, when dronedarone was administered 2 h following infarction. This indicates that dronedarone affects different phases and mechanisms during infarction in repair. Previous studies have indicated that the protective mechanisms may include the improvement/preservation of microvascular flow (7), reduction of heart rate (32) and direct cardioprotection, which may result from the induction of a redox-associated gene expression panel (7) or the protection from calcium-overload (16,19). It has been suggested that the first effect may be partly due to the attenuation of AF-dependent α-adrenergic vasoconstriction (16,33). In an acute infarction model, the reduction in infarct size by dronedarone 2 h following reperfusion was found to be independent of the remaining blood flow (16).

In an experimental rat model, dronedarone has been revealed to reduce the incidence of ischemia- and reperfusion-induced arrhythmia and mortality rates (34). This may be due to a reduction of extrasystoles, as was observed in a rabbit right ventricle 'border zone' model (35). A meta-analysis of the effects of dronedarone on the occurrence of stroke or transient ischemic attack indicated a reduced risk in patients with paroxysmal and persistent AF treated with dronedarone (36). Of note, a previous study also demonstrated the potential of a combined administration of low dose dronedarone and ranolazine together reducing ischemia-induced AF vulnerability and AF duration, as well as ventricular tachyarrhythmias (37).

In conclusion, data from experimental and clinical studies indicate the existence of acute beneficial effects of dronedarone in the setting of ischemia/reperfusion, although the underlying mechanisms remain to be fully elucidated. The results of the present study do not support the hypothesis that dronedarone exerts beneficial long-term effects (28 days post infarct) on infarct size following acute MI. In the present study, the area of the MI was not associated with the area at risk, as it was intended to preserve the tissue for further gene expression investigations, which may have contributed to the discrepancy between the observations. LAD occlusion, however, was performed by highly experienced personnel following a standardized protocol to minimize experimental variation. The LAD was occluded directly following the first diagonal branch. More proximal occlusion of the LAD directly following the left main coronary artery is known to produce refractory ventricular fibrillation in pigs, and, therefore, the aforementioned technique and location was selected. Of note, other investigations using the same experimental protocol of LAD occlusion did not detect a significant variation in the size of the area at risk (16). Another limitation of the present study is the relatively small number of animals included, which is the reason the data required careful interpretation. However, the data suggested, that the use of dronedarone is safe for patients with AF who suffer an acute anterior MI.

How dronedarone affects myocardial gene expression in healthy, infarcted or border zone myocardium and how it may improve myocardial function and stability in the long-term, remain to be elucidated in future investigations.

In the present study, in vitro MRI was used for the determination of infarct size, in addition to in vivo angiography. In vitro MRI for the quantification of pathological changes is an expanding field, as high-resolution 3D data sets may be acquired and analyzed at an almost microscopic level. Usually, contrast media are applied to delineate the pathological tissue, however, as they are taken up by the cells, changes in gene expression may occur, including monocytes (38), and distort results of interest.

In the present study, it was demonstrated that, even without the use of contrast agents, in vitro MRI is a suitable approach to delineate infarcted regions from vital myocardium, as judged in comparison to the pathohistology. In several sections, the first layers of the subendocardial myocardium were found to be vital. This was clearly delineated by the MRI and in the AFOG-stained histological sections. Not only this subendocardial vital zone, but also intermingled areas of viable cells surrounded by fibrosis were visualized in the MRI. Thus, compared with the pathohistological investigations, the complete pig heart can be readily analyzed in sections as small as 1 mm, or even less, producing accurate data on infarct size. In the present study, no reduced scar formation was detected in the dronedarone-treated animals, which is in accordance with the angiographic finding that EF was reduced to almost the same degree in the control and dronedarone group.

In summary, the results of the current study demonstrate the suitability of MRI for infarct size determination without the need for contrast agents. Although we a reduction of infarct size by dronedarone was not observed, the data suggest that dronedarone is not harmful in patients who suffer from acute anterior MI. Future studies are required to assess the consequences of dronedarone-dependent alterations in myocardial gene expression on cardiac structure and function in the long-term.

Acknowledgments

The authors would like to thank Mrs. Manja Möller and Mrs. Ines Schultz (Institute of Medical Biochemistry and Molecular Biology, University Medicine Greifswald, Ernst-Moritz-Arndt-University, Greifswald, Germany), Mrs. Cathleen Brandenburg and Mrs. Christine Karrer-Hoffmann (Institute of Pathology, University Medicine Greifswald, Ernst-Moritz-Arndt-University, Greifswald, Germany) and Mr. Stefan Hadlich (Department of Diagnostic Radiology and Neuroradiology, University Medicine Greifswald, Ernst-Moritz-Arndt-University, Greifswald, Germany) for their technical assistance. The authors would also like to thank Sanofi-Aventis for financial support of this investigator-sponsored trial.

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October-2015
Volume 12 Issue 4

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Spandidos Publications style
Linke J, Utpatel K, Wolke C, Evert M, Kühn JP, Bukowska A, Goette A, Lendeckel U and Peters B: Dronedarone does not affect infarct volume as assessed by magnetic resonance imaging in a porcine model of myocardial infarction. Mol Med Rep 12: 5169-5178, 2015
APA
Linke, J., Utpatel, K., Wolke, C., Evert, M., Kühn, J., Bukowska, A. ... Peters, B. (2015). Dronedarone does not affect infarct volume as assessed by magnetic resonance imaging in a porcine model of myocardial infarction. Molecular Medicine Reports, 12, 5169-5178. https://doi.org/10.3892/mmr.2015.4077
MLA
Linke, J., Utpatel, K., Wolke, C., Evert, M., Kühn, J., Bukowska, A., Goette, A., Lendeckel, U., Peters, B."Dronedarone does not affect infarct volume as assessed by magnetic resonance imaging in a porcine model of myocardial infarction". Molecular Medicine Reports 12.4 (2015): 5169-5178.
Chicago
Linke, J., Utpatel, K., Wolke, C., Evert, M., Kühn, J., Bukowska, A., Goette, A., Lendeckel, U., Peters, B."Dronedarone does not affect infarct volume as assessed by magnetic resonance imaging in a porcine model of myocardial infarction". Molecular Medicine Reports 12, no. 4 (2015): 5169-5178. https://doi.org/10.3892/mmr.2015.4077