Effects of thrombin and thrombin receptor activation on cardiac function after acute myocardial infarction

Abstract

Thrombin and thrombin receptor activation impact cardiomyocyte contraction and ventricular remodeling. However, there is some controversy regarding their effects in cardiac function, especially in cardiac dysfunction after acute myocardial infarction (AMI). A rat AMI model was created by left coronary artery ligation (LCA). Cardiac functional parameters, including the maximum left ventricular (LV) systolic pressure (LVSP_max), LV end-diastolic pressure (LVEDP), and the rise and fall rates in LV pressure (dp/dt _max and dp/dt _min, respectively), were measured. Hirudin decreased cardiac function within 120 minutes after AMI, whereas treatment with thrombin receptor-activating peptide (TRAP) reversed this hirudin-induced decrease in cardiac function. The mRNA and protein expression levels of inositol 1,4,5-trisphosphate receptor (IP₃R) subtypes in infarct area tissues were analyzed by reverse transcription-polymerase chain reaction and immunoreaction. Hirudin decreased the expression levels of IP₃R-1, -2, and -3 in the infarct area for up to 40 minutes after AMI, whereas TRAP treatment reversed these hirudin-induced effects. Treatment with the IP₃R antagonist 2-aminoethoxydiphenyl borate (2.5 mg/kg) eliminated the effect of TRAP on the hirudin-induced decrease in cardiac function after AMI. Finally, TRAP increased the maximum binding capacity of the three IP₃R subtypes, but only enhanced the affinity of IP₃R-2. Thrombin and thrombin receptor activation improved cardiac function after AMI by an IP₃R-mediated pathway, probably through the IP₃R-2 subtype.

Introduction

Cardiogenic shock occurs in ~7% of patients with acute myocardial infarction (AMI) [1,2] and is responsible for most cases of early mortality after myocardial infarction [3]. Cardiogenic shock complicating AMI remains an important clinical problem, despite advances in reperfusion therapy. Activation of the sympathetic nervous system and various neurohumoral regulatory mechanisms protect cardiac function during the acute stress period after AMI. However, other potential protection mechanisms after AMI have not been fully explored.

Thrombin formation plays an important role in the course of AMI. In addition to participating in platelet aggregation, thrombin activates cells through its receptor, impacting a wide range of physiological systems, such as the endothelial barrier, chemotaxis, inflammation, cell growth and division, cardiomyocyte contraction, ventricular remodeling, and so on [4,5]. Administration of thrombin receptor-activating peptide (TRAP) to mice in vivo caused rapid hypotension, followed by sustained moderated hypotension [6]. However, in thrombin receptor-deficient mice, the parameters of cardiac function and blood pressure (BP) were not different from levels in normal mice [7]. Therefore, there is some controversy regarding the role that thrombin plays in cardiac function.

The inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) is present in the sarcoplasmic reticulum (SR) of cardiomyocytes. When combined with IP₃, IP₃R can enhance cardiac contractile function through Ca²⁺ outflow from the SR [8]. Thrombin demonstrated a positive inotropic effect on rat myocytes in vitro, through a process involving increased cytosolic Ca²⁺ concentration in myocytes [9]. Thrombin receptor expression was increased after AMI within the ischemic myocardial tissue, but it is still unknown whether this expression will change the level of IP₃R and influence the cardiac function.

We previously demonstrated that thrombin and thrombin receptor activation can induce ventricular arrhythmia and ST-segment elevation after AMI [10,11]. The aim of the present study was to clarify whether thrombin receptor activation affects cardiac systolic function via the IP₃ pathway after AMI.

Materials and methods

Materials

Hirudin was purchased from Fudan University (Shanghai, China). Evans blue and TRAP were purchased from Sigma Chemical Co. (St. Louis, USA). TRAP was dissolved in 0.1% trifluoroacetic acid (TFA; Sigma Chemical Co.) as a stock solution, and diluted in 0.005% TFA to a 250-nM working solution. Hirudin was dissolved in a working solution of 0.01% mannitol (final concentration). The IP₃R antagonist, 2-aminoethoxydiphenyl borate (2-APB; Sigma Chemical Co.), was prepared for intravenous (i.v.) injection by reconstitution in dimethyl sulfoxide (DMSO; Sigma Chemical Co.) and diluted in normal saline to achieve a final concentration of 10% DMSO [12]. Animals received 2.5 mg/kg 2-APB injected directly into the iliac vein in a total of 0.5 ml of normal saline.

Animal model

All experimental protocols complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH) and the Animal Care and Use Committees of Sun Yat-sen University. Spague-Dawley rats (male, 200-250 g) were used for all experiments. The rat AMI model was created as described previously [10,11]. Briefly, each rat was anesthetized with an intraperitoneal injection of ketamine/xylazine (75/5 mg/kg), intubated, and ventilated on room air throughout the procedure. Under sterile conditions, a left thoracotomy was performed at the third intercostal space. The pericardium was opened, exposing the left atrial appendage and pulmonary cone in the heart. Gentle pressure was applied on the right side of the thorax to provide quick access to the heart. A suture was placed under the left coronary artery (LCA) between the pulmonary artery outflow tract and the left atrium. If the ST segment in lead I was elevated upon tightening of the suture but returned to normal when the suture was relaxed, then the suture was tightened and tied using a 6-0 sterile silk 5 minutes after i.v. administration. The animal model of AMI was considered successful if: 1) the color of the infarcted area changed from red to white, 2) the left atrium was enlarged just after the coronary artery was ligated, and 3) the ST segment was elevated by more than 0.2 mm after LCA ligation. Myocardial ischemia was confirmed by the presence of regional cyanosis and ST segment elevation on the ECG. It was further confirmed by Evans blue perfusion after every experiment.

Sham-operated rats served as controls. All rats were euthanized after the experiments by anesthetization with 100% O₂/5% isoflurane, followed by either decapitation or transcardial perfusion with 0.9% saline containing 4% formaldehyde, depending on the protocol.

Measurement of cardiac function

For left ventricular (LV) cannulation, all animals were adequately anesthetized, intubated in a supine position, and ventilated on room air with a small animal ventilator. To find the common carotid artery (CCA), the right CCA sheath was separated continuously, and the vagus nerve was freed. An attempt was made to insert the tube into the LV via the CCA. The LV pressure was monitored by the tube. If the LV diastolic BP rapidly fell to near zero, then the tube was considered to have entered the LV.

After the tube was fixed, it was connected to a tension transducer and the BL-420 biological function experimental system (Chengdu Taimeng Technology Co, Ltd. China). Hemodynamic parameters of the LV, including the LV systolic pressure (LVSP_max), LV end-diastolic pressure (LVEDP), and the rise and fall rates in the LV pressure (dp/dt _max and dp/dt _min, respectively), were obtained. Cardiac functional parameters were consecutively recorded within 2 hours, at 1 minute before and after intervention, and 1, 5, 10, 20, 40, 80, and 120 minutes after LCA ligation.

To prevent blood from clotting in the tube, before each data point was recorded, the tube was washed with 200 μl of low-concentration heparin solution (12,500 U heparin in 500 ml of saline) for 5 seconds. To determine the cardiac functional parameters, each segment constituted 20 waves, with a 10-second interval between segments. For each time point, the average of three segments was used to determine the average cardiac functional parameter.

qRT-PCR analysis of IP₃R in the local infarct tissue

Myocardial tissue (100 mg) was cleaned with sterilized double-distilled water containing 0.1% diethylpyrocarbonate (DEPC). The tissue was homogenized with 1 ml of Trizol reagent, followed by extraction with chloroform. RNA was precipitated with isopropanol, washed with 75% ethanol, mixed in 40 μl of DEPC-containing water, and stored at -80°C until use. RNA extracted from the myocardial tissue was subjected to agarose gel electrophoresis. A ThermoScientific NanoDrop 1000 spectrophotometer was used to quantify RNA and assess RNA purity.

Total RNA (1 μg) was reverse-transcribed into cDNA with a cDNA synthesis kit (Invitrogen). The cDNA levels of IP3R and β-actin (as a housekeeping gene) in the local infarct tissues were quantified by real-time PCR using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems) with the DyNAmoTM ColorFlash® SYBR Green qPCR kit (Finnzymes Oy, Espoo, Finland). The PCR protocol consisted of an initial step at 95°C for 5 minutes, followed by 95°C for 10 seconds, 55°C annealing for 20 seconds, and establishment of a melting curve from 65 to 9°C.

Expression levels were calculated via the comparative threshold cycle (CT) method and normalized to the expression of human β-actin (as a housekeeping gene). ΔΔCt was used to calculate the fold change in mRNA expression, as follows: ΔCt = Ct (target gene) - Ct (housekeeping gene), ΔΔCt = ΔCt (treatment) - ΔCt (control), where fold change = 2 - (ΔCt_sample - ΔCt_control). The primers used were as follows: for IP₃R-1, 5’-CGGAGTAGGA GATGTGCTCA G-3’ and 5’-CATCTCTGCC ACGTAGCTCT C-3’ (GenBank NM_001007235.1; 7967-7987 and 8304-8324); for IP3R-2, 5’-CTCTCTGGCC TCCAGATTCT T-3’ and 5’-GGTCCTAGTG TGTGCAGCAT T-3’ (GenBank NM_031046.3; 9559-9579 and 9800-9820); for IP₃R-3, 5’-AGCACTACAT TGTGGCTGTC C-3’ and 5’-AGAGAAAGTC CTGGGAGCAA G-3’ (GenBank NM_013138.1; 8463-8473 and 8637-8657); and for β-actin, 5’-CACGGCATTG TCACCAACTG -3’ and 5’-AGGGCAACAT AGCACAGCTT -3’ (GenBank NM_031144.2; 298-317 and 724-743).

Immunoblotting of IP₃R in the infarct area

At every time point in each group, the heart was cut, and the atrium and right ventricle were removed. The pale (infarcted) areas were frozen at -80°C. Tissues were grinded with liquid nitrogen while being washed with 1 × PBS. Radioimmunoprecipitation lysis buffer, consisting of 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS), was added to lyse the tissue. Aprotinin, pepstatin, leupeptin, and phenylmethylsulfonyl fluoride (PMSF) were added to inhibit protein decomposition. The tissue was homogenized completely and placed on ice for 30 minutes to complete the lysis process. The sample was centrifuged at 4°C and 15,000 × g for 20 minutes. The supernatant was mixed with 5 × loading buffer, consisting of 0.25 M Tris-HCl (pH 6.8), 15% SDS, 50% glycerol, 25% β-mercaptoethanol, and 0.01% bromophenol blue. It was heated for 5 minutes at 100°C and centrifuged at 4°C and 12000 × g for 5 minutes. Proteins (30 μg/lane) were separated by four 15% gradient SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes with a Mini Trans Blot Cell (Bio-Rad, Hercules, CA).

Membranes were incubated with polyclonal anti-IP₃R-1 (1:1,000 dilution), anti-IP₃R-2 (1:500 dilution), or monoclonal anti-IP₃R-3 (1:500 dilution; all Santa Cruz Biotechnology, TX) antibodies overnight at 4°C in Tris-buffered solution (TBS) with 0.1% Tween 20 (TBS-T) and 5% nonfat dry milk. After 3 washes with TBS-T, membranes were incubated for 1 hour with horseradish peroxidase (HP)-conjugated secondary antibody (1:10,000 dilution; Pierce Biotechnology) and washed with TBS-T. Membranes were developed using enhanced chemiluminescence (Amersham, Arlington Heights, IL). They were reprobed with monoclonal anti-actin antibody (1:10,000 dilution; Chemicon International), followed by HP-conjugated anti-mouse IgG. Band intensities were quantified by digital densitometry using Quantity One version 4.4.1 software. The IP₃R isoform band intensity was normalized to β-actin.

Preparation of cardiomyocytes

Ventricular cardiomyocytes were isolated from rats (200-250 g) using a modification of a previously described method [1,10]. Briefly, hearts were rapidly excised, cannulated, and subjected to retrograde perfusion on a Langendorff apparatus with Krebs-Henseleit (KH) buffer (in mmol/l: 10 HEPES, 118 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, 11 glucose, and 1 CaCl₂; pH 7.37) for 2 minutes. They were perfused with Ca²⁺-free KH buffer for 2 minutes, followed by Ca²⁺-free KH buffer containing 0.5 mg/ml collagenase type II and 1 mg/ml bovine serum albumin (BSA) for 25 minutes. The LV was removed, chopped into small pieces, and incubated in a 50-ml Falcon tube at 37°C for 3 minutes with shaking. Undigested tissue was allowed to settle for ~1 minute. The pellet containing undigested tissue was discarded, and the Ca²⁺ concentration in the supernatant was gradually increased to 1.2 mmol/l.

Isolated cardiomyocytes were pelleted by centrifugation at 50 × g for 2 minutes at room temperature and resuspended in a stabilizing buffer (in mmol/l: 20 HEPES, 137 NaCl, 4.9 KCl, 1.2 MgSO₄, 15 glucose, and 1.2 Ca²⁺; pH 7.4). The cell preparation was incubated in stabilizing buffer containing 1% BSA for ~15 minutes at 37°C. It was washed and resuspended in Medium 199 (Invitrogen, Guangzhou, China), supplemented with 100 IU/ml penicillin and 100 μg/ml streptomycin. This technique routinely yields 90% cardiomyocytes retaining a rod-shaped morphology [10,11]. Experiments were performed on the same day or 4 hours after isolation.

Purification of IP₃R subtypes 1, 2, and 3 from cardiomyocytes

Cardiomyocytes were incubated with TRAP (250 nM) or 0.1% TFA for 30 minutes. The supernatant was removed, and the cells were rinsed once with Hank’s Balanced Salt Solution (in mmol/l: 155 NaCl, 10 HEPES, and 1 EDTA; pH 7.4). The cells were placed on ice, and 3 to 6 ml of ice-cold lysis buffer (in mmol/l: 50 Tris-base, 150 NaCl, 1% Triton X-100, 1 EDTA, 0.2 PMSF, 1 dithiothreitol, 10 leupeptin, 10 pepstatin, and 0.2 soybean trypsin inhibitor; pH 8.0) were added to each dish. The cells were incubated for 30 minutes on ice and centrifuged at 10,000 × g for 20 minutes at 4°C. Supernatants in each dish were incubated at 4°C with polyclonal anti-IP₃R-1, -2, or -3 (1:40 dilution) and Agarose A/G beads (15 μl). They were resupinated overnight. Immune complexes were isolated by centrifugation at 10,000 × g for 2 minutes, and washed twice with lysis buffer. Finally, the washed beads were resuspended in 1.6 ml of 20 mM Tris-base and 1 mM EDTA at pH 8.0.

IP₃ binding to immunoprecipitated receptors

The Protein A bead/antibody/IP₃R complex was previously shown to remain intact during radioligand-binding studies [13,14]. Thus, washed beads (100 ml) were incubated at 4°C for 30 minutes with [³H]IP₃ (specific activity, 2-5 Ci/mmol Amersham Pharmacia Bio, UK) in 35 mmol/l Tris-base and 1.5 mmol/l EDTA, pH 8.0 (final volume, 200 ml). Bound ligand was isolated by vacuum filtration. Incubated mixtures were pipetted onto pre-moistured Whatman GF/B filters (Whatman Corp., UK) and washed twice with 4 ml of ice-cold 20 mmol/l Tris-base and 1 mmol/l EDTA, pH 8.0. Filters were added to vials with 0.5 ml of water and 5 ml of Ecoscint H. They were assessed for radioactivity after a 48-hour extraction.

Nonspecific binding was determined by including 10 μmol/l nonradioactive IP₃ in parallel incubations. Specific binding was analyzed with Prism (Packard Tri-Carb 2900TR, GMI, Inc. Meriden, CT, USA). Initially, the data were fitted to sigmoid curves of variable slope to determine the concentrations that gave half-maximal saturation. The maximum number of binding sites (B _max) obtained from these analyses was used to normalize data. Values of the equilibrium dissociation constant K_d for preparations of receptor types 1, 2, and 3 were determined by fitting the data to one-site saturation-binding curves.

Statistical analysis

Data are reported as means ± standard errors of the mean (SEMs). Differences between means at different time points before and after LCA ligation (a repeated measures analysis) were evaluated by using a linear mixed effects model with a random effect for each anima performed using SAS Statistical Software (version 9.2, SAS Institute Inc.). Differences between means, non repeated measures, were evaluated by analysis of variance (ANOVA) and Student’s t-test (paired data, unpaired data, and multiple data sets). Differences with P < 0.05 were considered statistically significant.

Results

Effect of thrombin on cardiac function after AMI

To determine the influence of thrombin receptor activation on cardiac function after AMI, the right iliac vein was injected with 200 U/kg hirudin (to eliminate thrombin) or vehicle (0.01% mannitol) 5 minutes before LCA ligation (Figure 1A). Compared to vehicle treatment, hirudin decreased LVSP_max, dp/dt _max, and dp/dt _min, but increased LVEDP, from 1 to 120 minutes after AMI (Figure 1B-E). When TRAP (270 mM, 100 μL) was injected into the iliac vein 1 minute before LCA ligation, the hirudin-induced effect of decreased cardiac function was reversed (Figure 2B-E). With each intervention, the peak effect on LVEDP and dp/dt _min occurred around 10 minutes after AMI. The levels gradually recovered, but remained different from those of the sham control groups.

Figure 1.

A: Experimental protocol. Sprague-Dawley rats were divided into three groups of five animals per group: sham group, hirudin group (200 U/kg), and vehicle group (0.01% mannitol). Hirudin or mannitol was injected into the right iliac vein 5 minutes before LCA ligation. The sham control involved an identical procedure, except that the LCA was not ligated. B and C: Percent changes in LVSP_max and dp/dt _max from basal values at different time points in each group. D and E: Percent changes in LVEDP and dp/dt _min from basal values at different time points in each group. Data are shown as the mean ± SEM. *P < 0.05 for vehicle vs. sham group. #P < 0.05 for hirudin vs. sham group. Mannitol is the vehicle of hirudin.

Figure 2.

A: Experimental protocol. Sprague-Dawley rats were divided into two groups of five animals per group: hirudin + TFA and hirudin + TRAP. Hirudin was administered intravenously via the left iliac vein 5 minutes before LCA ligation, followed by TFA/TRAP injection 1 minute before LCA ligation. B and C: Percent changes of LVSP_max and dp/dt _max from basal values at different time points in each group. D and E: Percent changes in LVEDP and dp/dt _min from basal values at different time points in each group. Data shown are the mean ± SEM. *P < 0.05 for hirudin + TFA vs. hirudin + TRAP group. TFA is the vehicle of TRAP.

Effect of hirudin on the expression of the IP₃R subtypes after AMI

Given the importance of IP₃ as a second messenger, we hypothesized that thrombin could exert its effects on cardiac function through IP₃R. Using RT-PCR and Western blot analyses, we measured the mRNA and protein expression levels, respectively, of IP₃R subtypes after neutralizing thrombin with hirudin in the infarct area. The results are shown in Figure 3 (data are reported as the fold change relative to the basal level).

Figure 3.

A: Experimental protocol. Sprague-Dawley rats were divided into two groups of five animals per group: vehicle and hirudin. Hirudin (200 U/kg) or 0.01% mannitol was injected into the right iliac vein before LCA ligation. B1, C1, and D1: Histograms showing the mRNA expression ratio of IP₃R-1, -2, or -3 relative to β-actin in the rat myocardium. Data (mean ± SEM) are expressed as a multiple of the basal level (fold), defined as the mRNA expression ratio at the LCA ligation time-point. *P < 0.05 for hirudin vs. vehicle group. B2, C2, and D2: Upper images show representative Western blot analyses for IP₃R-1, -2, and -3 and β-actin protein expressions in the rat AMI myocardium at different time points after AMI. The three bottom histograms show the protein expression ratios of rat myocardium IP₃R-1, -2, and -3 relative to β-actin. Data (mean ± SEM) are expressed as a multiple of the basal level (fold), defined as the protein expression ratio at the LCA ligation time point. *P < 0.05 for hirudin vs. vehicle group. Mannitol is the vehicle of hirudin.

The mRNA expression levels of IP₃R-1, -2, and -3 in the hirudin group were less than those in the vehicle group at 5 minutes (2.64 ± 0.06, 1.54 ± 0.03, 3.21 ± 0.02 vs. 4.36 ± 0.03, 5.45 ± 0.02, 2.78 ± 0.02 folds), 10 minutes (3.13 ± 0.02, 2.12 ± 0.02, 4.24 ± 0.04 vs. 5.12 ± 0.03, 3.84 ± 0.01, 6.32 ± 0.01 folds), 20 minutes (2.78 ± 0.05, 1.46 ± 0.03, 3.22 ± 0.03 vs. 3.97 ± 0.04, 2.62 ± 0.06, 5.17 ± 0.04 folds), and 40 minutes after AMI (1.04 ± 0.02, 1.13 ± 0.01, 1.65 ± 0.06 vs. 1.62 ± 0.04, 2.11 ± 0.06, 3.26 ± 0.02 folds). The mRNA expression level of IP₃R-2 in the hirudin group was less than that in the vehicle group at 80 minutes (0.78 ± 0.02 vs. 1.32 ± 0.04 folds) and 120 minutes (0.62 ± 0.03 vs. 1.21 ± 0.01 folds) after AMI (Figure 3B1-D1). The hirudin and vehicle groups displayed no significant difference in the mRNA expression level of IP₃R-1 or -3 at 80 minutes (0.91 ± 0.03, 0.82 ± 0.05 vs. 1.24 ± 0.04, 1.22 ± 0.01 folds) or 120 minutes after AMI (1.02 ± 0.05, 0.78 ± 0.04 vs. 1.03 ± 0.02, 0.82 ± 0.05 folds).

Similar changes to those observed for the mRNA expression were also observed for the protein expression levels of IP₃R-1, -2, and -3 between the two groups (Figure 3B2-D2). Immunohistochemistry for the protein expression levels of IP₃R-1, 2, and 3 in cardiomyocytes in the infarct areas showed similar differences between the two groups to those observed for the protein and mRNA expression levels (Supplementary Figure 1A-C).

Effect of TRAP on the expression of the IP₃R subtypes after AMI

To clarify the relationship between IP₃R and the thrombin receptor, hirudin was injected into the iliac vein 5 minutes before LCA ligation, followed by 100 μl of 270 mmol/L TRAP or 100 μl of 0.1% TFA (the vehicle of TRAP) 1 minute before LCA ligation. Using RT-PCR and Western blot analyses, we measured the mRNA and protein expression levels, respectively, of the IP₃R subtypes in the infarct area. The results are shown in Figure 4 (data are reported as the fold change relative to the basal level).

Figure 4.

Same experimental protocol as Figure 2A. A1, B1, and C1: Histograms showing mRNA expression ratios of rat myocardium IP₃R-1, -2, and -3 relative to β-actin. Data (mean ± SEM) are expressed as a multiple of the basal level (fold), defined as the mRNA expression ratio at the LCA ligation time point. *P < 0.05 for hirudin + TRAP vs. hirudin + TFA group. A2, B2, and C2: Upper images show representative Western blot analyses for IP₃R-1, -2, and -3 and β-actin protein expression in rat AMI myocardium at different time points after AMI. Three bottom histograms show the protein expression ratios of rat myocardium IP₃R-1, -2, and -3 relative to β-actin. Data (mean ± SEM) are expressed as a multiple of the basal level (fold), defined as the protein expression ratio at the LCA ligation time point. *P < 0.05 for hirudin + TRAP vs. hirudin + TFA group. TFA is the vehicle of TRAP.

The mRNA expression levels of IP₃R-1, -2, and -3 in the hirudin + TFA group were less than levels in the hirudin + TRAP group at 5 minutes (2.12 ± 0.04, 1.98 ± 0.04, 2.56 ± 0.03 vs. 3.52 ± 0.02, 3.14 ± 0.02, 4.02 ± 0.07 folds), 10 minutes (3.01 ± 0.02, 2.51 ± 0.01, 3.12 ± 0.02 vs. 4.76 ± 0.03, 4.01 ± 0.02, 5.64 ± 0.05 folds), 20 minutes (2.14 ± 0.06, 1.86 ± 0.07, 2.23 ± 0.06 vs. 3.42 ± 0.02, 3.17 ± 0.03, 4.42 ± 0.04 folds), and 40 minutes after AMI (0.98 ± 0.02, 0.98 ± 0.06, 1.02 ± 0.03 vs. 2.14 ± 0.03, 1.66 ± 0.04, 2.04 ± 0.01 folds). The IP₃R-2 mRNA expression in the hirudin + TFA group was less than that in the hirudin + TRAP group at 80 minutes (0.83 ± 0.04 vs. 1.41 ± 0.02 folds) and 120 minutes after AMI (0.68 ± 0.01 vs. 1.22 ± 0.03 folds; Figure 4A1-C1). The two groups showed no significant difference in the IP₃R-1 or -2 mRNA expression at 80 minutes (0.92 ± 0.02, 0.88 ± 0.04 vs. 0.89 ± 0.02, 0.81 ± 0.07 folds) or 120 minutes after AMI (0.88 ± 0.03, 0.82 ± 0.01 vs. 0.84 ± 0.04, 0.79 ± 0.03 folds).

Changes in the protein expression levels of IP₃R-1, -2, and -3 between the two groups were similar to those observed for mRNA expression (Figure 4A2-C2). Similar to the protein and mRNA expression differences, immunohistochemistry showed the same differences between the two groups in the protein expression levels of IP₃R-1, 2, and 3 in cardiomyocytes from the infarct areas (Supplementary Figure 2A-C).

Effect of 2-APB on cardiac function after AMI

To clarify whether the effect of thrombin on cardiac function is related to IP₃R, we used the IP₃R inhibitor, 2-APB. Treatment with 2-APB (2.5 mg/kg) in the hirudin + TRAP group eliminated the TRAP-induced improvement in the cardiac functional parameters (LVSP_max, dp/dt _max, LVEDP, and dp/dt _min) from 1 to 120 minutes after AMI compared to the hirudin + TFA group (P > 0.05, Figure 5).

Figure 5.

A: Experimental protocol. Sprague-Dawley rats were divided into two groups of five animals per group: hirudin + TFA, and hirudin +TRAP. 2-APB (2.5 mg/kg) or hirudin was administered intravenously via the iliac vein 10 or 5 minutes before LCA ligation, respectively, followed by TFA/TRAP 1 minute before LCA ligation. B and C: Percent changes in the LVSP_max and dp/dt _max from basal values at different time points in each group. D and E: Percent changes in the LVEDP and dp/dt _min from basal values at different time points in each group. Data are shown as the mean ± SEM. *P < 0.05 for hirudin +TFA vs. hirudin + TRAP group. TFA is the vehicle of TRAP.

Effect of TRAP on the affinity of IP₃ receptors in cardiomyocytes

As shown in Figure 6, the order of the basal affinity of the three subtypes of IP₃R in the TFA group was IP₃R-2 (1.8 ± 0.2 nM) > IP₃R-1 (3.9 ± 0.6 nM) > IP₃R-3 (14.1 ± 1.1 nM). The basal B _max values of IP₃R-1, -2, and -3 were 630 ± 115, 850 ± 150, and 210 ± 35 cpm, respectively. After incubation with TRAP for 20 minutes, the affinity of IP₃R-2 increased with time from 5 to 120 minutes compared to the basal TFA group (1.5 ± 0.3, 1.4 ± 0.1, 1.2 ± 0.3, 1.1 ± 0.2, 0.9 ± 0.1, and 0.7 ± 0.2 nM; P < 0.05), whereas the affinities of IP₃R-1 and -3 did not significantly change (IP₃R-1: 3.6 ± 0.8, 3.8 ± 0.5, 3.7 ± 0.9, 3.5 ± 0.4 , 3.6 ± 0.7, 3.7 ± 0.8 nM; IP₃R-3: 13.8 ± 0.9, 14 ± 1.2, 14.5 ± 0.7, 13.7 ± 1.2, 14.6 ± 0.8, 13.5 ± 1.3 nM). The B _max values of IP₃R-1, -2, and -3 increased with time from 5 to 40 minutes compared to the basal TFA group (1232 ± 210, 1940 ± 162, 2403 ± 185, 1800 ± 120 cpm; 1293 ± 216, 721 ± 300, 3123 ± 214, 2330 ± 320 cpm; and 386 ± 50, 586 ± 106, 700 ± 90, 560 ± 100 cpm, respectively; P < 0.05), but all of the values returned to their basal levels within 80 to 120 minutes after AMI (561 ± 135, 525 ± 150 cpm; 940 ± 104, 900 ± 98 cpm; and 192 ± 30, 184 ± 22 cpm, respectively; P > 0.05). These results also were illustrated by Scatchard analysis (Figure 7). TRAP had much stronger effects on the affinity of IP₃R-2 compared to IP₃R-1 and -3, and it had no effect on the B _max of IP₃R-1 or -3.

Figure 6.

Effect of TRAP on the maximum binding capacity (B _max) and affinity (K_d) of IP₃R-1 (A and B), IP₃R-2 (C and D), and IP₃R-3 (E and F). Data are shown as the mean ± SEM (n = 5). *P < 0.05 at different time points of TRAP incubation vs. basal time point. TFA is the vehicle of TRAP.

Figure 7.

Scatchard plots of the specific binding data (n = 5) after 20 (B) and 120 (C) minutes of TRAP incubation vs. data after 20 minutes of TFA incubation (A). TFA is the vehicle of TRAP.

Discussion

By eliminating thrombin, hirudin aggravated the cardiac dysfunction after AMI and decreased the expression of IP₃R subtypes, especially IP₃R-2. On the other hand, by activating the thrombin receptor, TRAP reversed the hirudin-induced cardiac dysfunction after AMI and increased the expression of all IP₃R subtypes, especially IP₃R-2. The ability of TRAP to improve cardiac function disappeared when rats were pretreated with 2-APB, an IP₃R antagonist. These findings reveal that the activated thrombin receptor elicited its effects on cardiac function after AMI via IP₃R.

For all interventions, the peak effects on LVEDP and dp/dt _min occurred around 10 minutes after AMI. The levels gradually recovered, although not to the levels of the sham control group. Thus, the peak effects on LVEDP and dp/dt _min after AMI probably did not come from the interventions themselves, but from the hemodynamic measurement methods. For example, when the cannulation tube was inserted into the LV via the common carotid artery went through the aorta and caused aorta valvular regurgitation, which may be one reason for our observation. The different interventions that we used in our experiments may have enhanced this effect.

The protease-activated receptor (PAR) superfamily of seven transmembrane G protein-coupled receptors includes PARs 1 to 4. The thrombin receptor is PAR-1 [15]. There is no direct evidence to prove that PAR-1 can affect cardiac function, but there is some indirect evidence. In the heart, PAR-1 is expressed by cardiomyocytes and cardiac fibroblasts [16,17]. PAR-1 expression was recently shown to be increased in the hearts of patients with ischemic and idiopathic dilated cardiomyopathy [18]. It was elevated in the LV in a mouse model of chronic heart failure [19]. In vitro studies using rat neonatal cardiomyocytes demonstrated that PAR-1 activation induced hypertrophy [17,20]. PAR-1-dependent changes included increases in intracellular Ca²⁺, protein content, cell size, and sarcomeric organization. Furthermore, activation of PAR-1 in cardiac fibroblasts induced cell proliferation [16]. Thrombin stimulates fibroblasts via activation of phospholipase C that hydrolyzes phosphatidylinositol 4,5-bisphosphate to IP₃ and diacylglycero [21], which act as intracellular second messengers. In cardiomyocytes, PAR-1 results in IP₃R-mediated Ca²⁺ outflow from the SR and increased cytosolic Ca²⁺, influencing cardiomyocyte contraction, electrophysiological changes, and the transcription of hypertrophy- and apoptosis-related nuclear factors [9]. IP₃ mobilizes Ca²⁺ from intracellular nonmitochondrial stores and elevates the intracellular Ca²⁺ concentration [22]. PAR-1 deficiency was shown to reduce LV dilation [23]. These results strongly suggest that PAR-1 may contribute to cardiac remodeling, which is the basis of heart functional changes, after injury [24]. However, PAR-1 has been reported to increase cardiac fibroblast proliferation and fibrotic activities [25]. Treatment with a PAR-1 inhibitor attenuated LV remodeling and infarct size in a rat myocardial ischemia-reperfusion model within 3 to 28 days after injury. The PAR-1 inhibitor did not cause any acute decrease in myocardial injury in the model [26,27]. Our results prove that thrombin and PAR-1 activation improved cardiac function within 120 minutes. The improvement likely derived from the increased cardiac systolic function by PAR-1 activation, a phenomenon that is not unique for the thrombin receptor, one of G protein-coupled receptors. Indeed, activation of the β-adrenergic receptor, another one of G protein-coupled receptors, in the heart also acutely increases cardiac systolic function, but chronically stimulates LV remodeling in chronic heart failure and AMI. The latter effects are the basis for the broad clinical application of β-adrenergic receptor blockers in chronical heart failure and AMI. One possibility is that PAR-1 temporarily compensates for the severely weakened neurohumoral regulation, which results from local blood and oxygen deficiencies in the infarcted area of the LV, for heart fuction after AMI.

Thrombin reportedly modulates phosphoinositide metabolism and cytosolic Ca²⁺ levels in the heart [28]. However, to the best of our knowledge, no study has reported that thrombin activation can induce improved cardiac function via IP₃R. PAR-1 activates signal transduction through a highly efficient process. Thrombin-activated myocardial cells generate IP₃ after 5 seconds, and the IP₃ level peaks within 1 minute [28]. Through its receptors, IP₃ elevates the intracellular Ca²⁺ concentration in the excitation-contraction coupling of cardiomyocytes, which is the basis of cardiac function. The increase in cytoplasmic free Ca²⁺, resulting from increased IP₃ and IP₃R, is an important mechanism for regulating cardiac contractile forces in response to hormones and pharmacological factors. A greater than 2-fold increase of IP₃R mRNA in the heart was observed during end-stage heart failure in humans [29].

IP₃R subtypes 1, 2, and 3 are expressed in the heart [29]. IP₃R-1 is mainly found in nonmyocytes of human atrial tissue and in rat Purkinje cells [30,31]. Most other species predominantly express IP₃R-2 in myocytes, with small amounts of IP₃R-1 and -3 [29,30]. At the organelle level, IP₃R is mainly found in the SR around the ryanodine receptor in myocytes. IP₃R has been reported to mediate release of Ca²⁺ from other intracellular organelles, including the nuclear envelope [8], Golgi, and secretory vesicles [34,35]. We found that all three IP₃R subtypes were expressed in the infarcted LV area, with IP₃R-2 being the most obvious . These findings are consistent with the previously reported literature.

Thrombin receptor activation induced IP₃R subtype changes within 40 minutes after AMI, although the effect of PAR-1 activation on cardiac function continued for more than 120 minutes. The effect of 2-APB on the TRAP-induced improvement in cardiac function also continued for more than 120 minutes. Most often, 2-APB is used as an experimental inhibitor of intracellular Ca²⁺ release through IP₃R [36,37]. However, several reports have demonstrated that 2-APB affects the cell Ca²⁺ homeostasis in a concentration-dependent manner by depressing IP₃R activity and store-operated channel-linked Ca²⁺ entry at low concentrations [38,39], while inducing Ca²⁺ leakage from isolated myocytes and nonexcitable cells at slightly higher concentrations [40]. Therefore, the effect of PAR-1 activation on cardiac function was not proven to occur through the IP₃R pathway. Since we can not find the same race of gene knockout animal (rat) and tried very hard with new techniques including miRNA and siRNA in cellular level, which did not give us convince data, so we have to conduct experiments to determine the effect of TRAP on the affinity of the IP₃R subtypes in cardiomyocytes because the IP₃R-mediated Ca²⁺ release from the endoplasmic reticulum is not only related to receptor expression and ligand number (B _max) but also to the IP₃R affinity. Thrombin receptor activation induced a change in the B _max values of all IP₃R subtypes for at least 80 minutes, but TRAP increased the affinity of the IP₃R-2 subtype for more than 120 minutes. As a result, the improvement in cardiac function by thrombin receptor activation might arise from the IP₃R-2 subtype.

In conclusion, thrombin and thrombin receptor activation improved cardiac function after AMI in rats. The improvement of cardiac function occurred through a pathway mediated by an IP₃R subtype (probably IP₃R-2).

Disclosure of conflict of interest

None to disclose.

Abbreviations

AMI

Acute myocardial infarction

TRAP

thrombin receptor-activating peptide

IP₃

inositol 1,4,5-trisphosphate

IP₃R

inositol 1,4,5-trisphosphate receptor

SR

sarcoplasmic reticulum

TFA

trifluoroacetic acid

2-APB

2-aminoethoxydiphenyl borate

DMSO

dimethyl sulfoxide

LCA

left coronary artery

CCA

common carotid artery

LV

left ventricular

LVSP_max

LV systolic pressure

LVEDP

LV end-diastolic pressure

dp/dt_max and dp/dt_min

and the rise and fall rates in the LV pressure

DEPC

diethylpyrocarbonate

SDS

sodium dodecyl sulfate