Intra-coronary administration of tacrolimus markedly attenuates infarct size and preserves heart function in porcine myocardial infarction

All animal experimental procedures were approved by the Institute of Animal Care and Use Committee at our institute and performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85–23, National Academy Press, Washington, DC, USA, revised 1996).

Each male mini-pig (Taitung Animal Propagation Station, Livestock Research Institute, Taiwan), weighing 16–18 kg, was anesthetized by intramuscular injection of ketamine (15 mg/kg) and maintained in anesthetized condition using an inhalation of 1.5% isoflurane for the whole procedure. After being shaved on the chest, the mini-pig was placed in supine position on a warming pad at 37 °C and then received endotracheal intubation with positive-pressure ventilation (180 mL/min) with room air using a ventilator support (Sn: Q422ZO, SIMS PneuPAC, Ltd.) during the procedure. Electrocardiogram (ECG) monitor and defibrillator were connected to the chest wall of each mini-pig. One ample of amiodarone (150 mg) was intravenously given to each animal before the AMI-induction procedure to prevent the occurrence of malignant arrhythmia.

Under sterile conditions, the heart was exposed through mid-thoracotomy. The pericardium was gently removed and the mid-portion of LAD was ligated with 5–0 prolene suture just distal to the first diagonal branch. Regional myocardial ischemia is confirmed by typical changes in waveform on ECG monitor and the observation of rapid discoloration of myocardium from pink to gray over the anterior surface of left ventricle, together with the rapid development of akinesia and dilatation of at-risk area. AMI was confirmed by complete ECG following the procedure.

Mid-LAD ligations were performed in 16 mini-pigs. Two mini-pigs in each group succumbed to either ventricular tachycardia or ventricular fibrillation even after defibrillation. The remaining 12 mini-pigs recruited for this study were then categorized into AMI plus normal saline treatment (MI-only) group (n = 6) and AMI plus tacrolimus therapy (MI-Tac) group (n = 6). For the purpose of comparison at molecular-cellular levels, normal cardiac tissues were obtained from a group of six mini-pigs without receiving any treatment that served as the normal controls (N_C).

To determine the optimal drug dosage for maximal efficacy and acceptable safety, three different dosages of tacrolimus were adopted for pilot study, including: 1) 1.0 mg; 2) 0.5 mg; and 3) 0.25 mg. Each dosage was tried on two mini-pigs. The highest dosage induced fatal malignant ventricular tachyarrhythmia, whereas the second and third dosages were found to be safe. Since the infarct size was remarkably smaller in animals receiving the second dosage compared with the third one, the second dosage (i.e. 0.5 mg/kg) was utilized in the current study. By 30 minutes after LAD ligation, intra-coronary injections of physiological saline (3.0 mL) in MI-only group and tacrolimus (0.5 mg in 2.5 mL physiological saline) in MI-Tac group were given through the LAD beyond the point of ligation. The muscle and skin of the chest wall were then closed in layers. The animals were allowed to recover on the warming pad under close observation.

The echocardiographic study was performed using iE33 (Philips Medical System, Bothell, WA) with S5 transducers. The end-systolic dimension (EDS) and end-diastolic dimension (EDD) were measured at mitral valve and papillary levels of left ventricle. Recordings were stored for off-line two-dimensional image analysis using a computer software (Q-lab v6; Philips Medical System). For each mini-pig, three consecutive beats were measured and averaged for each variable. With the animals in a supine position, left ventricular internal dimensions [i.e. end-systolic diameter (ESD) and end-diastolic diameter (EDD)] were measured according to the American Society of Echocardiography leading-edge method using at least three consecutives cardiac cycles. The LV ejection fraction (LVEF) was calculated as: LVEF (%) = [(LVEDd³-LVEDs³)/LVEDd³] x 100. All measurements were performed by an animal cardiologist blind to treatment and non-treatment groups.

Intra-observer variability was evaluated in a series of six mini-pigs by one observer who examined the recordings from each animal in a consecutive way. Intra-observer variability for LVEF was 2.5 ± 2.1%. All measurements were performed by a cardiologist blinded to the treatment and non-treatment groups.

The heart was removed from each mini-pig following intravenous injection of an overdose of potassium chloride. Repeated flushing of the coronary artery with normal saline for washing out the red blood cells was performed immediately following heart removal. Three cross-sections (1 cm in thickness) from the basal, middle, and apical level, respectively, were used for infarct area (IA) analysis in IM-only and MI-Tac animals after being stained with 2% triphenyltetrazolium chloride (TTC). Briefly, all heart sections were placed on a tray with a scaled vertical bar to which a digital camera was attached. The sections were photographed from directly above at a fixed height. The images obtained were then analyzed using Image Tool 3 (IT3) image analysis software (University of Texas, Health Science Center, San Antonio, UTHSCSA; Image Tool for Windows, Version 3.0, USA). The calculated IA was expressed as arbitrary unit for comparison. The rest of the cardiac tissue was then cut into pieces for specific studies.

To determine the impact of tacrolimus therapy on thickness of the infarcted LV wall, three cross-sections of left ventricle at papillary muscle level were made for each animal and three measurements were recorded on the thickest regions for each section. The mean thickness was obtained for each animal. All measurements were performed by a technician blinded to the treatment and non-treatment groups.

Reverse transcription polymerase chain reaction (RT-PCR) was conducted using LighCycler TaqMan Master (Roche, Germany) in a single capillary tube according to the manufacturer’s guidelines for individual component concentrations. Forward and reverse primers were each designed based on individual exons of the target gene sequence to avoid amplifying genomic DNA.

The myocardium from IA was excised and washed with buffer A (100 mM Tris–HCl, 70 mM sucrose, 10 mM EDTA and 210 mM mannitol, pH 7.4). Samples were minced finely in cold buffer A and then incubated for 10 minutes. All samples were homogenized in an additional 3 mL of buffer A using a motor-driven grinder. The homogenate was centrifuged twice at 700 g for 10 minutes at 4 °C. The supernatant was again centrifuged at 8,500 g for 15 minutes, and the pellets were then washed with buffer B (10 mM Tris–HCl, 70 mM sucrose, 1 mM EDTA, and 230 mM mannitol, pH 7.4). The mitochondria-rich pellets were then collected and stored at −70 °C.

Equal amounts (50μg) of protein extracts were loaded and separated by SDS-PAGE using 12% acrylamide gradients. After electrophoresis, the separated proteins were transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences). Nonspecific sites were blocked by incubation of the membrane in blocking buffer [5% nonfat dry milk in T-TBS (TBS containing 0.05% Tween 20)] for overnight. The membranes were incubated with the indicated primary antibodies [Bcl-2 (1: 200, Abcam), Caspase 3 (1: 4000, Abcam), α-smooth muscle actin (SMA) (1: 1000, Sigma), Bax (1: 1000, Abcam), connexin (Cx)43 (1: 2000, Chemicon), matrix metalloproteinase (MMP)-9 (1: 5000, Abcam), tumor necrotic factor (TNF)-α (1: 1000, Cell Signaling), nuclear factor (NF)-κB (1: 600, Abcam), endothelial nitric oxide synthase (eNOS) (1: 1000, Abcam), NAD(P)H:quinone oxidoreductase (NQO1) (1: 1000, Abcam), heme oxygenase 1 (HO-1) (1: 1000, Abcam), Actin (1: 10000, Chemicon )] for 1 hr at room temperature. Horseradish peroxidase -conjugated anti-rabbit immunoglobulin IgG (1: 2000, Cell Signaling) was used as a second antibody for 1 hr at room temperature. The washing procedure was repeated eight times within 1 hr, and immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences) and exposure to Biomax L film (Kodak). For purposes of quantitation, ECL signals were digitized using Labwork software (UVP).

For each mini-pig, six sections (three longitudinal and three transverse sections of LV myocardium) were analyzed by an in situ Cell Death Detection Kit [TdT-FragEL^TM DNA Fragmentation (Calbiochem)] according to the manufacture's guidelines. The TUNEL-positive cells was examined in three randomly chosen high-power field (400x) and normalized to the total number of cells divided by 18.

Paraffin sections (3 μm thick) were obtained from LV myocardium of each animal. For identification of CD40+ cells, the sections were initially incubated in 3% hydrogen peroxide for blocking the action of endogenous peroxidase, and then further processed using BEAT Blocker Kit (Zymed Company, #50-300) with immersion in solutions A and B for 30 minutes and 10 minutes, respectively, at room temperature. Polyclonal rabbit antibodies against CD40 (dilution 1/100; Spring Bioscience) were then used, followed by application of SuperPicTure^TM Polymer Detection Kit (Zymed) for 10 minutes at room temperature. Finally, the sections were counterstained with hematoxylin. IHF staining was performed using primary antibodies to recognize CD68+ cells (dilution 1/100; Abcam) or troponin I (dilution 1/100; Abcam), followed by the application of FITC-conjugated secondary antibodies (dilution 1/200; molecular probe). For negative control experiments, the primary antibodies were omitted.

The Oxyblot Oxidized Protein Detection Kit was purchased from Chemicon (S7150). The oxyblot procedure was performed as previously described [19]. The DNPH derivatization was carried out on 6 μg of protein for 15 minutes according to the manufacturer’s instructions. One-dimensional electrophoresis was carried out on 12% SDS/polyacrylamide gel after DNPH derivatization. Proteins were transferred to nitrocellulose membranes which were then incubated in the primary antibody solution (anti-DNP 1: 150) for 2 h, followed by incubation in second antibody solution (1:300) for 1 h at room temperature. The washing procedure was repeated eight times within 40 minutes. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences) which was then exposed to Biomax L film (Kodak). For quantification, ECL signals were digitized using Labwork software (UVP). For oxyblot protein analysis, a standard control was loaded on each gel.

Masson's trichrome staining was used for studying fibrosis of LV myocardium. Three serial sections of LV myocardium were prepared at 4 μm thickness by Cryostat (Leica CM3050S). The integrated area (μm²) of fibrosis in the slides was calculated using Image Tool 3 (IT3) image analysis software (University of Texas, Health Science Center, San Antonio, UTHSCSA; Image Tool for Windows, Version 3.0, USA). Three selected sections were quantified for each animal. Three randomly selected HPFs (400x) were analyzed in each section. After determining the number of pixels in each fibrotic area per HPF, the numbers of pixels obtained from the three HPFs were summed. The procedure was repeated in two other slides for each animal. The mean pixel number per HPF for each animal was then determined by summating all pixel numbers and dividing by 9. The mean the integrated area (μm²) of fibrosis in LV myocardium per HPF was obtained using a conversion factor of 19.24 (1 μm² represented 19.24 pixels).

Data are expressed as mean values ± SD or percentage (%). The significance of group differences was evaluated with t-test. Continuous variables among 3 groups were compared using one-way ANOVA followed by Tukey’s multiple comparison procedure. Statistical analysis was performed using SAS statistical software for Windows version 8.2 (SAS institute, Cary, NC). A probability value < 0.05 was considered statistically significant.

Table 1 shows the echocardiographic findings prior to the procedure and at day 14 after AMI induction. By day 0 prior to AMI induction, at both mitral valve and papillary levels, the left ventricular end-diastolic dimension (LVEDd), left ventricular end-systolic dimension (LVEDs), left ventricular end-diastolic volume (LVEVd), left ventricular end-systolic volume (LVEVs), left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) did not differ between animals without treatment (MI-Only) and those with tacrolimus treatment (MI-Tac). Additionally, these parameters at mitral valve level were also similar between these two groups of animals at day 14 after AMI induction. Furthermore, no difference was noted in LVEDs and LVEVs at papillary level between these two groups of animals at day 14 after AMI induction. However, at papillary level, LVEDd and LVEVd were significantly higher, whereas LVFS and LVEF were significantly lower in MI-only group than in MI-Tac group at day 14 after AMI induction. These findings suggest that tacrolimus treatment attenuated LV remodeling and preserved LV function following AMI.

Upper Panel of Figure 1 shows the results of TTC staining by day 14 following AMI. As shown on the left panel (Figure 1A), IA was notably larger in MI-only than in MI-Tac group at basal, middle, and apical levels. Accordingly, quantification of the IA (Figure 1B) demonstrated significantly larger infarct at all three levels in the former. Additionally, the wall thickness at papillary level was significantly reduced in MI-only than in MI-Tac (Figure 1C).

The lower panel of Figure 1 shows the results of H. & E. stain of the IA at middle level of left ventricle by day 14 after AMI induction. As demonstrated on Figure 1D, 1E and 1F, the IA was remarkably larger in MI-only than in MI-Tac group. Accordingly, quantification of the IA (Figure 1G) revealed significantly larger infarct at middle level of left ventricle in the former.

These findings imply that, as compared with AMI without treatment, therapy using tacrolimus markedly limited infarct size and preserved infarcted wall thickness of the animals on day 14 after AMI.

Figure 2A, 2B and 2C indicates troponin-I staining of normal and IA for detecting viable myocardium, respectively. The immunofluorescent imaging study demonstrated that the distribution of troponin-I positive myocardium in IA was markedly decreased in MI-only group compared with that in the MI-Tac group on day 14 following AMI (Figure 2D).

To determine the impact of tacrolimus therapy on fibrosis in IA, Masson Trichrome staining was performed (Figure 2E-G). As expected, the mean fibrotic area in IA was remarkably higher in MI-only than in MI-Tac animals on day 14 after AMI induction (Figure 2H).

The mRNA expressions of plasminogen activator inhibitor (PAI)-1 (Figure 3A) and matrix metalloproteinase (MMP)-9 (Figure 3B), two indexes of inflammation, were remarkably higher in MI-only compared to normal control animals (N_C) on day 14 after AMI. However, these two markers were significantly lower in the MI-Tac group than in MI-only animals. These findings suggest that tacrolimus plays an essential role in suppressing the inflammatory reaction after AMI.

Interestingly, the mRNA expression of interleukin (IL)-10 (Figure 3C), an anti-inflammatory marker, was notably higher in MI-only than in N_C animals at day 14 after AMI induction. This biomarker was further increased in MI-Tac group compared with that in the MI-only group. Possible explanation may be a triggering of inherent self-protection in response to myocardial injury that was further enhanced by the suppression of immune/inflammatory reactions through tacrolimus therapy.

The gene and protein expressions of endothelial nitric oxide synthase (eNOS) (Figure 3D and E), an indicator of the integrity of endothelial function/vasodilatation as well as an anti-inflammatory contributor, were remarkably reduced in MI-only animals than in N_C, but they were substantially increased in MI-Tac animals compared to their MI-only counterparts. Besides, the protein expressions of NF-κB (Figure 3F) and TNF-α (Figure 3G), two indicators of inflammation, were significantly increased in MI-only group than in N_C, but they were markedly reduced in MI-Tac group as compared with MI-only animals. Furthermore, oxidative stress (Figure 4A and B) was significantly increased in MI-only group than in N_C, but it was markedly reduced in MI-Tac than in MI-only animals by day 14 after AMI. Moreover, HO-1 (Figure 4C) and NQO1 (Figure 4D), two anti-oxidative biomarkers, were remarkably higher in MI-Tac animals than in MI-only and N_C animals, and significantly higher in MI-only group than in N_C animals on day 14 after AMI induction. These findings further support that AMI-elicited vigorous inflammatory reaction and oxidative stress, which cause further myocardial damage and deterioration of heart function, were effectively suppressed by tacrolimus.

Both mRNA and protein expressions of Bax (Figure 5A and D) and caspase 3 (Figure 5B and E), two indicators of apoptosis, were notably higher in MI-only group than in N_C, but were much lower in MI-Tac than in MI-only animals at day 14 after AMI induction. Conversely, both mRNA and protein expressions of Bcl-2 (Figure 5C and F), an index of anti-apoptosis, were remarkably lower in MI-only group than in N_C, but were significantly higher in MI-Tac as compared with MI-only animals. Besides, TUNEL assay (Figure 6) showed substantially higher number of apoptotic nuclei in peri-IA in MI-only group than in N_C, but notably lower number in MI-Tac compared with MI-only animals. These findings imply that tacrolimus suppressed myocardial damage through inhibiting cellular apoptosis after AMI.

IHC staining demonstrated that the protein expression of Cx43 (Figure 7A), a component of gap junction for providing pathways with minimal resistance for inter-cellular electrical coupling, was markedly reduced in MI-only group than in N_C, but was notably up-regulated in MI-Tac compared with MI-only animals by day 14 after AMI. In contrast to Cx43 expression, the protein expression of α-SMA (Figure 7B), indicator of cardiac fibroblast proliferation in response to AMI, was substantially increased in MI-only animals than in N_C, but it was significantly attenuated in MI-Tac than in MI-only group.

To evaluate whether CD40+ cells (Figure 8A-D) and CD68+ cells (Figure 8E-H) (macrophage surface marker), two surface markers of inflammatory cells, were up-regulated in the IA, IHC and IHF staining were performed, respectively. As anticipated, the numbers of CD40-positive and CD68-positive cells were substantially higher in MI-only group than in N_C. However, they were found to be notably suppressed in MI-Tac animals than in those with MI only.

This study did not performed cyclosporine-treated AMI group. Thus, we utilized the results from our recent report [18] to compare the impact of cyclosporine and tacrolimus therapy on ameliorating the inflammatory, apoptotic and oxidative-stress responses after AMI. The results in Table 2 exhibited the superior effects of tacrolimus than that of cyclosporine on suppressing majority of these biomarkers.

This study has limitations. First, the therapeutic benefits of cyclosporine, a calcineurin inhibitor similar to tacrolimus, in limiting myocardial infarction, preserving LV function, and improving clinical outcome have been previously reported [17, 18], however, since we did not include the positive control group of cyclosporine in the present study, whether the mechanisms of tacrolimus treatment involved in the protection of myocardium from ischemic injury are similar to those of cyclosporine therapy remains unclear. Further experimental investigation comparing the effects of these immunosuppressants on the progression of myocardial infarction, therefore, would strengthen the significance and interest of the present study. Second, the mechanisms of tacrolimus therapy on reducing the inflammation and cellular apoptosis were not explored in the current study. However, previous study has shown that tacrolimus therapy suppressed T cell activation through the inhibition of NF-AT activation which, in turn, attenuated inflammatory reaction [14, 15].