Protective effects of drag-reducing polymers on ischemic reperfusion injury of isolated rat heart

Abstract

Drag-reducing polymers (DRPs) are blood-soluble macromolecules that can increase blood flow and reduce vascular resistance. The purpose of the present study was to observe the effect of DRPs on ischemic reperfusion (I/R) injury of isolated rat hearts. Experiments were performed on isolated rat hearts subjected to 30 min of ischemia followed by 90 min of reperfusion in Langendorff preparations. Adult Wistar rats were divided into the following five groups: control group, I/R group, group III (I/R and 2×10⁻⁷ g/ml PEO reperfusion), group IV (I/R and 1×10⁻⁶ g/ml PEO reperfusion), and group V (I/R and 5×10⁻⁶ g/ml PEO reperfusion). Left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), maximum rate of ventricular pressure increase and decrease ( ± dp/dt_max), heart rate (HR) and coronary flow were measured. Lactate dehydrogenase (LDH) and creatine kinase (CK) activity and coronary flow, myocardial infarction size and cardiomyocytes apoptosis were also assayed. Our results showed that PEO decreased LVEDP and increased LVSP, ± dP/dt_max in group IV and group V compared with the I/R group (all P < 0.05). The coronary flow significantly increased and the activities of LDH and CK in the coronary flow significantly decreased in group IV and group V compared with those in the I/R group (all P < 0.05). Cell apoptosis and myocardial infarction size were reduced in group IV and group V compared with the I/R group (all P < 0.05). Collectively, these results suggested that DRPs had a protective effect on cardiac I/R injury of isolated rat hearts and it may offer a new potential approach for the treatment of acute ischemic heart diseases.

Keywords

Drag-reducing polymers ischemic reperfusion injury polyethylene oxide

1 Introduction

Drag-reducing polymers (DRPs) are long chain macromolecular polymers that reduce flow resistance without affecting the fluid viscosity in a pipe [29, 36]. It has been widely used in petroleum transportation, irrigation, navigation and other industrial pipelines [2 , 35]. In recent years, the potential medical application of DRPs had been explored in cardiovascular disease, atherosclerosis, shock and other fields [5 , 32]. Sakai et al. [30] demonstrated that DRP significantly improved the survival of rats with acute myocardial infarction. Pacella et al. [25] found that biocompatible DRP significantly improved myocardium perfusion and decreased microvascular resistance in dogs with flow-limiting coronary stenosis. Polyethylene oxide (PEO) is a type of blood soluble polymer which is consistent with the characteristics of DRPs. PEO is the most commonly researched DRP in medical science. We have recently demonstrated that PEO can significantly increase blood flow velocity and reduce flow resistance in arteries, small arteries and capillaries of rat limb skeletal muscle, and significantly increase the survival rate and improve the cardiac function in a rat model of myocardial infarction [3, 11].

Ischemic reperfusion injury often occurs after occluded coronary artery recanalization in acute myocardial infarction. Myocardial damage often induces arrhythmia, no reflow phenomenon, dysbolism and myocardial stunning in animal experiments and clinical observation [1 , 24]. Effective and rapid recovery of coronary artery blood supply is the most important method for the prevention of myocardial ischemic reperfusion injury. Blood flow in the microvascular is determined by vascular resistance and hemorheologic characteristics such as blood viscosity, erythrocyte aggregation and erythrocyte deformability [10]. Previous large sample clinical study has also found that plasma viscosity plays an important role in capillary perfusion [14]. DRPs have the unique characteristics of reducing vascular resistance and increasing capillary blood flow, and this is not simply via shear-induced vasodilation, but also due to tone-independent resistance lowering mechanisms [20 , 29]. Therefore, the present study aims to test the effects of DRPs in the prevention of myocardial ischemic reperfusion injury.

2 Methods

2.1 Animals

Fifty male Wistar rats (230–250 g) were obtained from the Experimental Animal Center of Yangzhou University (Yangzhou, China). All study procedures followed the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). The experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Yangzhou University.

2.2 Reagents

Polyethylene oxide with a molecular weight of 5000 kDa (Sigma-Aldrich, St Louis, MO, USA) was chosen to be the DRP in this study. PEO was dissolved in saline at a concentration of 1×10⁻³ g/ml and then dialyzed against saline for 24 h using a membrane with 50 kDa molecular weight (MW) cutoff (Spectrum Laboratories Inc.). After dialysis, the PEO solution was stored at 4°C.

2.3 Isolated rat heart perfusion

After anesthetization with intraperitoneal injection of pentobarbital sodium (45 mg/kg), rat hearts were removed, the left auricle was excised, and the hearts were placed on a Langendorff perfusion system (Powerlab, Australia). Then, the rat heart was perfused with Krebs-Henseleit (KH) solution through the aorta in a retrograde direction. Krebs-Henseleit (KH) solution consisted of: NaCl, 118.0 (mmol/L); KCl, 4.7 (mmol/L); KH₂PO₄, 0.93 (mmol/L); MgSO₄·7H₂O, 1.2 (mmol/L); CaCl₂, 1.5 (mmol/L); NaHCO₃, 25 (mmol/L); and C₆H₁₂O₆, 11.0 (mmol/L) at pH 7.4, 37°C and with a 100 cm H₂O constant pressure and pre-saturated mixed with 95% O₂ and 5% CO₂. A cardiac catheter with a balloon was inserted into the left ventricle from the atrioventricular valve. The balloon was filled and was linked to a pressure transducer connected to the physiological signal acquisition system (PowerLab, Australia) to monitor the cardiac function parameters. At the beginning of perfusion, the left ventricular end-diastolic pressure (LVEDP) maintained 0–10 mmHg (1 mmHg = 0.133 kPa). After pre-perfusion stabilization for 30 min, the LVEDP, left ventricular systolic pressure (LVSP), left ventricular developed pressure maximum increasing rate (+dP/dt_max), left ventricular developed pressure maximum decreasing rate (–dP/dt_max), and heart rate (HR) were recorded by the physiological signal acquisition system. The coronary flow was also collected and measured. The perfusion procedure is shown in Fig. 1.

2.4 Animal grouping

Fifty male Wistar rats were divided randomly into the following 5 groups. Group I (control group, n = 10): the isolated hearts were continuously perfused with K-H solution for 150 min during the whole experiment. Group II (I/R group, n = 10): after 30 min of stabilization, perfusion was stopped for 30 min (ischemia), and the isolated hearts were then reperfused for 90 min with KH solution. Group III (n = 10): the hearts were stabilized and perfused similarly to group II, but the reperfusion KH solution contained 2×10⁻⁷ g/ml of PEO. Group IV (n = 10): the hearts were stabilized and perfused similarly to group II, but the reperfusion KH solution contained 1×10⁻⁶ g/ml of PEO. Group V (n = 10): the hearts were stabilized and perfused similarly to group II, but the reperfusion KH solution contained 5×10⁻⁶ g/ml of PEO.

2.5 Measurement of CK and LDH activity

Samples of the coronary flow were collected and stored in liquid nitrogen before ischemia and at 30 min, 60 min and 90 min of reperfusion. The CK and LDH activity in the coronary flow was measured spectrophotometrically by commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.6 Determination of myocardium infarct size

After reperfusion, isolated hearts were frozen at −20°C for 2 h and then cut transversely into 5 slices. The slices were stained with 1% TTC in 0.1 mol/L phosphate buffer at 37°C for 10 min and then incubated in 10% formalin to identify the viable (red) and infarct (pale) tissue. The infarct size was determined by planimetry with Image J 1.37 software (NIH, Bethesda, MD) and is expressed as a percentage of the whole LV slices (all five slices were taken into account by summing the values).

2.7 Assay of myocardium apoptosis

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique was used to detect apoptotic cardiomyocytes after reperfusion. All of the procedures were performed according to the instructions of a TUNEL assay kit (Boster Bio-Engineering limited company, Wuhan, China). Normal nuclei were stained blue, while apoptotic nuclei were stained brownish yellow. TUNEL-positive myocytes were determined by randomly counting 500 cells in 10 fields (×100). The index of apoptosis was calculated as the number of apoptotic myocytes/total number of myocytes×100% .

2.8 Statistical analysis

All data were expressed as mean ± SD. Data were processed by SPSS13.0 (Chicago, IL, USA). Two-way ANOVA was used to analyze the changes in cardiac function data. All of the other data were analyzed by one-way ANOVA followed by post-hoc Tukey tests. A difference was accepted as significant at P < 0.05.

3 Results

3.1 Effect of DRP on cardiac function

3.1.1 Left ventricular systolic function

After a 30 min stabilization period, both LVSP and +dP/dt_max had no significant differences among all of the groups (all P > 0.05). Compared to the control group, LVSP and the +dP/dt_max in the I/R group were significantly decreased after 30 min, 60 min and 90 min of reperfusion (all P < 0.05). In Group III, both LVSP and +dP/dt_max showed no difference with the I/R group after 30 min, 60 min and 90 min of reperfusion. However, the LVSP in Group IV was higher than that in the I/R group after 30 min and 60 min of reperfusion (all P < 0.05). Also, the +dP/dt_max remained similar with the I/R after 30 min of reperfusion, but significantly increased after 60 min and 90 min of reperfusion compared with the I/R group (all P < 0.05). Consistently, both LVSP and +dP/dt_max in Group V were significantly higher than LVSP and +dP/dt_max in the I/R group after 30 min, 60 min and 90 min of reperfusion (all P < 0.05) (Fig. 2A and B).

3.1.2 Left ventricular diastolic function

LVEDP and –dp/dt_max are important parameters that represent the left ventricular diastolic function. After 30 min of stabilization, there were no significant differences among all of the groups (all P > 0.05). Compared to control group, LVEDP in the I/R group was increased significantly after 30 min, 60 min and 90 min of reperfusion (all P < 0.05), while –dP/dt_max in the I/R group was significantly decreased after 30 min, 60 min and 90 min of reperfusion (all P < 0.05). In group III, both the LVEDP and –dP/dt_max showed no difference with the I/R group after 30 min, 60 min and 90 min of reperfusion (all P > 0.05). In group IV, LVEDP showed no difference with the I/R group after 30 min of reperfusion, while LVEDP was lower than the I/R group after 60 min and 90 min of reperfusion (all P < 0.05). The –dP/dt_max was significantly increased after 30 min, 60 min and 90 min of reperfusion compared with the I/R group (all P < 0.05). In group V, the LVEDP significantly decreased, the –dP/dt_max increased after 30 min, 60 min and 90 min of reperfusion compared with the I/R group (all P < 0.05; Fig. 2C and D).

3.2 Effect of DRP on coronary flow

As shown in Fig. 3, the coronary flow in all of the groups showed a gradual reduction during the reperfusion period, and ischemic reperfusion injury made this progress more significant. However, the coronary flow in group IV and V was much higher than in the I/R group and group III throughoutthe reperfusion period.

3.3 Effect of DRP on heart rate

Compared to the control group, the HR after 30 min of ischemia in the other four groups was reduced during the reperfusion period. The HR was significantly increased in group IV and group V compared with the I/R group (all P < 0.05; Fig. 4).

3.4 Effect of DRP on CK and LDH in the coronary flow

The levels of LDH and CK in the coronary flow were similar prior to ischemia in all of the groups. Compared to the control group, the activity of LDH and CK in the coronary flow increased significantly in the I/R group (all P < 0.05). However, the activity of these enzymes was significantly decreased in group IV and group V compared with the I/R group (Tables 1 and 2).

3.5 Analysis of myocardial infarct size

After ischemia and reperfusion, the myocardial infarct size was significantly increased in the I/R group compared with the control group (P < 0.05). However, infarct size in group IV and group V decreased compared with the I/R group (all P < 0.05), while the infarct size in group III showed no difference relative to the infarct size in the I/R group (Fig. 5).

3.6 Analysis of myocardial apoptosis

The number of apoptotic cells in the I/R group was higher than in the control group (P < 0.05). There was no difference in the number of apoptotic cells between group III and the I/R group. There were less apoptotic cells in group IV and group V compared with the I/R group (all P < 0.05, Fig. 6).

4 Discussion

Myocardial ischemic reperfusion injury refers to a phenomenon of severe injury that occurs after timely restoration of coronary blood flow in acute myocardial infarction. The mechanism of myocardial ischemic reperfusion injury is very complex, and is associated with inflammatory response, vascular endothelial dysfunction, myocardium no reflow phenomenon, overload of intercellular calcium and oxygen free radicals [4 , 37]. Currently, many researchers focus their work on prevention and treatment of myocardial ischemic reperfusion injury [6, 38].

This study demonstrated that PEO can increase coronary flow, reduce the release of LDH and CK, suppress apoptosis, improve the cardiac functions and reduce myocardial infarct size in an isolated rat heart ischemic reperfusion model. These results suggest that DRPs may have potential application in reducing myocardial ischemic reperfusion injury in rats.

Our previous work had demonstrated that DRPs plays an important role in improving the LV function in a rat model of acute myocardial infarction, as characterized by a significant improvement in fractional shortening and ejection fraction [3]. In this study, we have observed that I/R injury immediately induced increases in LVEDP. We also showed that LVSP, +dP/dt_max and dP/dt_max decreased significantly following the ischemia and reperfusion process. LVSP and +dP/dt_max are important parameters of left ventricular systolic function, while LVEDP and –dp/dt_max represent left ventricular diastolic function. In agreement with previous in vivo studies, we have demonstrated that PEO decreased LVEDP and increased LVSP, +dP/dt_max, and −dP/dt_max in group IV and group V. PEO has a potential protective effect in left ventricular systolic and diastolic function.

DRPs potentiate the decrease in flow resistance and increase flow viscosity in a pipe. Previously, Pacella et al. [26] demonstrated that PEO reduced vascular resistance and increased capillary blood flow, not only due to some degree of shear-induced vasodilation, but also due to tone-independent resistance lowering mechanisms, which implied that PEO favorably altered the blood flow hydrodynamics. The Langendorff perfusion system can be considered a pipe system, such that coronary flow is the outflow of the pipe system. I/R injury significantly decreased coronary flow while PEO significantly increased coronary flow in group IV and group V. According to the formula that fluid resistance ∝ pressure/flow rate, as the perfusion pressure is consistent, the increase of coronary flow means that the fluid resistance of the Langendorff perfusion system had been decreased by PEO.

LDH and CK function as valuable markers of myocardial injury [12 , 21]. I/R injury significantly increased the activities of LDH and CK in coronary flow. In this study, the activities of LDH and CK in coronary flow significantly decreased in group IV and group V compared with the I/R group. Since the previous studies have shown that PEO can increase capillary blood flow velocity in the myocardium [25, 28], it is very likely that the cell injury can be protected by effective blood supply recovery.

Myocardial ischemic reperfusion injury leads to cell death, and both acute necrosis and apoptosis can be observed in acute myocardial infarction. TTC stain is a classic method for the detection of myocardial infarction size and the TUNEL assay can vividly show apoptotic cells. Pacella et al. tested DRP (PEO) in a dog model of coronary artery narrowing and found that DRP improved flow reserve during coronary stenosis by modulating capillary resistance [28]. Sakai et al. reported that DRPs prolonged survival time in rats following acute myocardial infarction and DRP-treated rats maintained higher mean arterial pressure and tissue perfusion [30]. In this study, PEO significantly decreased myocardial infarction size, which is consistent with previous in vivo studies [3]. More importantly, we are the first to demonstrate that PEO elicited a protective effect against cardiomyocytes apoptosis.

There are some limitations to this study. Firstly, isolated rat heart perfusion was used to create a model of ischemic reperfusion injury in our experiments. The parameters of cardiac function, hemodynamic and coronary flow and heart rate can avoid the effects of the nervous system and hormones in vivo. However, the mechanisms underlying the cardioprotective effects of DRPs need further investigations in vivo before it can be used in the development of clinical therapy. Secondly, resistance reducing phenomena is unique because it has a beneficial biological effect. While the flow resistance in the Langendorff perfusion system cannot be calculated accurately, we estimated the flow resistance according to the formula: fluid resistance ∝ pressure/flow rate. Since the driving pressure is consistent, the increase in the flow rate means a decrease of fluid resistance.

In summary, the results demonstrated that DRP had a protective effect on the heart in ischemic reperfusion injury, suggesting that DRP may offer a new potential approach for the treatment of acute ischemic heart diseases.

Footnotes

Acknowledgments

This work was partly supported by the National Natural Science Foundation of China (81200144, 81270197 and 81270198), and the Project of Prospering Health by Science and Education in Jiangsu Province (RC2011045) and Jiangsu “333” project funding (BRA2012095). The authors acknowledge Brian Davidson for his constructive suggestions on the manuscript. We also thank Lixin Chen and Xiaowei Xu for their technical support in the Langendorff perfusion experiments.

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