Imbalance between tissue inhibitor of metalloproteinase 1 and matrix metalloproteinase 9 after

cardiopulmonary resuscitation^?

Jing-sha Li MD, Jing-quan Zhong MD, PhD?, Hong-zhen Liu MD, Qi-xian Zeng MD, Xiang-lin Meng MD, Dong-lin Liu MD, Guo-ying Su MD, Yun Zhang MD, PhD

Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Jinan 250012, China

Department of Cardiology, Qilu Hospital, Shandong University, Jinan 250012, China

Received 27 March 2011; revised 23 June 2011; accepted 17 July 2011

Abstract

Aims: This study aimed to determine whether (a) there was an imbalance between matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) after cardiopul- monary resuscitation (CPR) in a canine model of prolonged ventricular fibrillation (VF); (b) with the duration of VF, the degree of the imbalance would be greater; and (c) there was a relationship between the level of MMP-9 or TIMP-1 and the cardiac function.

Methods and Results: Ventricular fibrillation was electrically induced in 24 dogs. The animals were randomly divided into 3 groups (sham control, n = 8; 8-minute VF, n = 8; 12-minute VF, n = 8). Echocardiographic measurement and Hemodynamic variables were recorded before VF and after return of spontaneous circulation. Tissue inhibitor of metalloproteinase 1 (TIMP-1) and MMP-9 were analyzed by Western blot and immunohistochemistry. Compared with sham controls, dogs under VF and CPR showed significantly decreased level of TIMP-1 (P b .001), and with the duration of VF, the level of TIMP-1 declined (P b .01). The level of MMP-9 did not achieve statistical significance in the 3 groups (P N .05); however, they were higher in VF and longer duration VF groups. The ratios of TIMP-1/MMP- 9 were lower in VF groups (P b .05). There was a negative correlation between TIMP-1 and left atrium dimension and left ventricular diastolic dimensions (r = -0.83 and r = -0.96, respectively; P b .01) and a positive correlation between TIMP-1 and left ventricular ejection fraction (r = 0.85; P b .01).

Conclusions: There was an imbalance between TIMP-1 and MMP-9 after CPR. It may partly contribute to the postresuscitation cardiac dysfunction.

(C) 2012

Introduction

^? Conflict of interest: None declared.

* Corresponding author. Department of Cardiology, Qilu Hospital, Shandong University, Jinan, Shandong, 250012, China. Tel.: +86 531

82169429; fax: +1 86 531 86927544.

E-mail address: [email protected] (J. Zhong).

Ventricular fibrillation (VF) is one of the most common reasons for sudden cardiac death. The most effective therapies for VF are defibrillation and cardiopulmonary resuscitation (CPR). Although initial return of spontaneous circulation (ROSC) is achieved in about 58% of cases, only

0735-6757/$ - see front matter (C) 2012 doi:10.1016/j.ajem.2011.07.006

4.6% of the patients will be discharged with good outcome [1]. Cardiac dysfunction after CPR has been proven one of the most important reasons for the death in the early period of successful CPR [2]. Matrix metalloproteinases are a family of zinc-dependent endopeptidases, and they regulate the Extracellular matrix (ECM) turnover in a balance with tissue inhibitors of metalloproteinase (TIMPs) [3]. They are best known for their roles played in the chronic diseases such as ventricular remodeling of acute myocardial infarction, chronic heart failure, hypertension, occurrence or mainte- nance of atrial fibrillation, and dilated cardiomyopathy [4,5]. However, the imbalance between TIMPs and MMPs in acute Pathologic conditions could be also observed. Lalu et al [6] first proposed that there was a rapid (within 10 minutes) increase in MMP-2, MMP-9, and collagenase activities in the myocardium that was accompanied by a decrease in TIMP-1 and the enhanced MMP-2 and MMP-9 activities and that the loss of TIMP-1 correlated with the impaired left ventricular function during the process of ischemia-reperfusion. The process of CPR is equal to the pathologic process of ischemia-reperfusion [7]. The present study aimed to determine the changes in myocardial TIMP-1 and MMP-9 in the setting of different-duration VF episodes and after CPR and whether the imbalance between TIMP-1 and MMP- 9 correlated with the postresuscitation cardiac dysfunction.

Methods

Animal preparation

All animal care and experimental protocols complied with the approval of the Animal Care and Use Committee of Qilu Hospital, Shandong University, China. Twenty- four dogs (obtained from the Center for Experimental Animals of Qilu Hospital of Shandong University) of either sex weighing 11.5 to 17 kg were randomly divided to 3 groups: sham control (n = 8), 8-minute VF (n = 8), and 12-minute VF (n = 8). The animals were anesthetized with pentobarbital-Na (30 mg/kg intravenous, repetition when necessary). After anesthesia, dogs were placed in the supine position on the experimental table and restrained at the 4 extremities. A 5.0 cuffed tracheal tube was inserted into the trachea for intubation. The tube was attached to a ventilator (Newport E-100M; Newport Medical Instruments, Costa Mesa, CA). Venti- lation began at the tidal volume of 10 to 15 mL/kg, ventilator rate of 16 to 20 breaths per minute, and inspiration to expiration ratio of 1:1.5 to 2.0. Three surface electrodes were placed under 3 limbs separately to correspond to standard lead II electrocardiogram. The right femoral artery and bilateral femoral veins were cannulated. Six French catheters (Cordis Corp, Miami, FL) were positioned in the intrathoracic Ascending aorta and the right atrium under fluoroscopic guidance. The

remaining cannulated femoral vein was used for drug infusion. Heparin was administered at 100 U/kg for anticoagulation and added when necessary.

Creation of VF model

Ventricular fibrillation was induced by delivering a 5-s alternating current at 80 V across the thorax through 2 needles. Successful VF was defined as a decrease in aortic blood pressure below 25 mm Hg and the presence of VF waveform on the electrocardiogram. Dogs in the sham control group were only anesthetized and intubated trache- ally. Untreated VF lasted for 8 or 12 minutes in the other 2 groups. All animals were given 2 minutes of CPR (chest compression rate was 100 per minute; chest compressions- to-ventilations was 30:2) before every transthoracic counter- shock at 150 J. If VF persisted, additional epinephrine at doses of 0.02 mg/kg was given, CPR was continued, and shocks were repeated until the animals achieved ROSC or the whole process of rescue lasted for 30 minutes. Return of spontaneous circulation was defined as aortic systolic pressure (AOSP) 80 mm Hg or higher, lasting for at least 1 minute [8]. All measurements were performed by an investigator blinded to the experiment assignment. After finishing the experimental protocol, the survival animals were killed by infusing KCl.

Transthoracic echocardiographic study

All animals before VF and the animals attaining ROSC were examined by transthoracic echocardiographic (PHILIPS 7500 with a 2.5-3.5 MHz transducer, Horten, Norway) to measure the left atrium dimension (LAD), left ventricular diastolic dimensions (LVDds), and left ventricular ejection fraction (LVEF), respectively, in a parasternal long-axis and M-mode view.

Hemodynamic measurement

Catheter positions were confirmed by x-ray fluoroscopy. Both catheters in the intrathoracic ascending aorta and the right atrium were connected to the pressure transducers attached to a PRO EP recording system (PowerLab/16sp; AD Instruments, Castle Hill, Australia) by which AOSP, diastolic aortic pressure (AODP), and right atrial pressure (RAP) could be recorded during the whole study. coronary perfusion pressure was calculated as AODP minus RAP, and mean aortic pressure (MAP) was calculated as 1/3 AOSP plus 2/3 AODP.

Tissue collection

Tissue samples of the myocardium were collected within 15 seconds after the death and then immediately snap frozen in liquid nitrogen (stored at -80?C) to minimize time-

dependent effects of myocardial ischemia on the changes of TIMPs and MMPs.

Western blot analysis of TIMP-1 and MMP-9

Tissue samples (20 mg) were homogenized in 200 uL of ice-cold lysis buffer and 2-uL phenylmethylsulfonyl fluoride and centrifuged at 15000 rpm for 10 minutes at 4?C. Supernatants were collected. The levels of TIMP-1 and MMP-9 in the myocardium were detected by Western blot. Samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SD-PAGE) (10% polyacrylamide) and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). Tissue inhibitors of metalloproteinase I was identified by use of a polyclonal rabbit antidog TIMP-1 antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), and MMP-9 was identified by use of a polyclonal goat antidog MMP-9 antibody (1:500; Santa Cruz Biotechnology). monoclonal antibody against ?-actin (Santa Cruz Biotechnology) was used in every experiment for the internal control. Band densities were measured with Quantity One (Bio-Rad Laboratories, Inc, Shanghai, China).

Immunohistochemistry staining of TIMP-1 and MMP-9

Five-micrometer ventricular tissue sections were rehy- drated and blocked with 3% hydrogen peroxide followed by incubation in 20% (vol/vol) normal goat or rabbit serum (Santa Cruz Biotechnology). Tissue sections were incubated at 4?C overnight with an antibody specific for TIMP-1 or MMP-9 (Santa Cruz Biotechnology), followed by incubation with horseradish peroxidase-containing secondary antibody (Jinqiao, Zhongshan, China) for immunohistochemistry. Nuclei were counterstained with hematoxylin. To control for nonspecific signal, a duplicate sample was prepared as above but omitting primary antibody. Every digital image

Table 1 Baseline characteristics in the 3 groups

was analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, Inc, Beijing, China).

Statistical analysis

Data were presented as mean +- SD. A paired t test was used for within-group comparisons, an unpaired t test for comparing differences between 2 groups, and 1-way analysis of variance for differences between all 3 groups. Simple linear correlation analysis was used to evaluate the correlationship between the level of TIMP-1 and LAD, TIMP-1 and LVDd, and TIMP-1 and LVEF. Statistical significance was established at a 2-tailed P b .05. The statistical analysis was performed with SPSS 16.0 software (SPSS, Chicago, IL).

Results

Baseline characteristics

There were no statistically significant differences among the animals in body weight, heart rate, LAD, LVDd, LVEF, and hemodynamic variables at baseline (Table 1).

Effects of VF and CPR

There were 7 dogs attaining ROSC in the 8-minute VF group and 5 in the 12-minute VF group. Undoubtedly, significant differences in both hemodynamics and the parameters of left ventricular function, including LVEF, LAD, and LVDd, were detected before VF and after CPR in each VF group, that is, hemodynamic variables deteriorated, both LAD and LVDd enlarged, and LVEF decreased after ROSC (Table 2). Compared with the 8- minute VF group, the LAD was much larger (21.70 +- 1.31 mm vs 17.21 +- 1.35 mm; P = .001), and the LVEF

Contents	Sham group	8-minute VF group	12-minute VF group	P
Weight (kg)	13.54 +- 1.63	13.75 +- 1.74	12.71 +- 1.77	.456
Heart (beats per minute)	151 +- 7	152 +- 11	152 +- 14	.946
Echocardiography
LAD (mm)	16.65 +- 0.83	15.84 +- 1.47	17.33 +- 1.09	.057
LVDd (mm)	24.86 +- 1.90	25.40 +- 1.75	24.15 +- 1.43	.357
LVEF (%)	0.66 +- 0.05	0.69 +- 0.05	0.68 +- 0.08	.484
Hemodynamics
AOSP (mm Hg)	135.97 +- 15.14	131.48 +- 17.65	138.10 +- 20.32	.757
AODP (mm Hg)	118.91 +- 13.87	98.88 +- 32.43	119.10 +- 19.09	.174
MAP (mm Hg)	124.60 +- 13.66	109.75 +- 26.14	125.44 +- 19.40	.262
RAP (mm Hg)	8.41 +- 4.93	9.34 +- 3.26	6.28 +- 1.62	.218
CPP (mm Hg)	110.51 +- 12.62	89.54 +- 33.96	112.82 +- 19.95	.138
Values are expressed as mean +- SD.

Contents	8-minute VF				12-minute VF			P ?
	Baseline	ROSC ?	P		Baseline	ROSC ?	P

Echocardiography
LAD (mm)	15.84 +- 1.47	17.21	+- 1.35	.000	17.33 +- 1.09	21.70 +- 1.31	.028	.001
LVDd (mm)	25.40 +- 1.75	28.39	+- 2.13	.000	24.15 +- 1.43	31.23 +- 1.93	.032	.083
LVEF (%)	0.69 +- 0.05	0.55	+- 0.06	.000	0.68 +- 0.08	0.38 +- 0.07	.012	.004
Hemodynamics
AOSP (mm Hg)	131.48 +- 17.65	97.64	+- 14.27	.001	138.10 +- 20.32	86.86 +- 0.50	.033	.093
AODP (mm Hg)	98.88 +- 32.43	70.89	+- 24.27	.033	119.10 +- 19.09	64.42 +- 17.94	.066	.693
MAP (mm Hg)	109.75 +- 26.14	79.80	+- 18.25	.012	125.44 +- 19.40	71.91 +- 12.07	.049	.518
RAP (mm Hg)	9.34 +- 3.26	10.96	+- 4.52	.193	6.28 +- 1.62	7.78 +- 3.68	.342	.409
CPP (mm Hg)	89.54 +- 33.96	57.38	+- 24.98	.028	112.82 +- 19.95	47.75 +- 32.30	.052	.619
Values are expressed as mean +- SD. P represents the comparison between baseline and after ROSC in each VF group. * P represents the comparison between the 2 VF groups after ROSC.

was much lower (0.38 +- 0.07 vs 0.55 +- 0.06; P = .004) in the 12-minute VF group. Compared with sham controls, dogs under VF and CPR showed significantly decreased level of TIMP-1(Western blot, 0.86 +- 0.08 vs 0.65 +- 0.06 vs 0.48 +- 0.05; P = .00), and with the duration of VF, the level of TIMP-1 declined (Fig. 1A). The level of MMP-9 did not achieve statistical signifi- cance in the 3 groups (P = .57); however, they were higher in VF and longer duration VF groups (Western blot, 0.58 +- 0.03 vs 0.60 +- 0.08 vs 0.65 +- 0.16; Fig. 1B).

Table 2 Index of echocardiography and hemodynamic variables at baseline and after ROSC in the 2 VF groups

The ratios of TIMP-1/MMP-9 were lower in VF groups (1.48 +- 0.11 vs 1.11 +- 0.25 vs 0.79 +- 0.23; P = .001;

Fig. 1C). Fig. 2 showed the cellular localization of TIMP- 1 and MMP-9 within myocardium by immunohistochem- istry (40-fold amplified). They are mainly in the interstitium of the cardiomyocytes, and the expressions of TIMP-1 and MMP-9 in the 3 groups were accordant with the results of Western blot (TIMP-1, 0.082 +- 0.017 vs 0.025 +- 0.01 vs 0.0049 +- 0.001, P = .00; MMP-9,

0.039 +- 0.017 vs 0.047 +- 0.03 vs 0.05 +- 0.016, P = .74).

Correlation between TIMP-1 and cardiac function

The diameters of the left atrium and the left ventricular correlated negatively with the level of TIMP-1 (r = -0.83, r = -0.96, respectively, both P = .00; Fig. 3A and B), and there was a positive correlation between TIMP-1 and LVEF (r = 0.85; P = .00; Fig. 3C).

Discussion

There are many studies about the mechanisms of cardiac dysfunction after CPR, such as the release of oxygen radicals, cell adhesion molecules including selectins, immunoglobulin family, a variety of inflamma- tory cytokines such as interleukin 8, the involvement of

calcium overload, and so on [9,10]. However, as compared with the vast information on cardiac dysfunc- tion after CPR, there exists little information on whether the imbalance between TIMPs and MMPs plays a role in the cardiac dysfunction after CPR. Previous studies have shown that the roles played by TIMPs and MMPs are very extensive. Besides the participation in the synthesis and degradation of ECM in the chronic cardiovascular diseases [4,5], recently, several studies demonstrated that TIMPs and MMPs were also involved in acute pathologic or physiologic processes such as platelet aggregation, the regulation of vascular tone, inflamma- tion, and Ischemia-reperfusion injury [6,11-14]. For the process of CPR is equal to ischemia-reperfusion, the imbalance between TIMPs and MMPs after CPR and whether the imbalance contributed to the postresuscitation cardiac dysfunction were investigated. Tissue inhibitor of metalloproteinase 1 and MMP-9 were selected in the present study.

In this study, we found that the level of TIMP-1 decreased significantly in the VF groups as compared with the sham control group. The level of MMP-9 appeared higher in the VF groups compared with the sham control group, although the differences were not significant. The ratios of TIMP- 1/MMP-9 were lower in the VF groups. Meanwhile, our present findings also demonstrated that, with the duration of VF, the levels of TIMP-1 declined and MMP-9 increased. Echocardiographic study revealed that the LAD and LVDd were significantly larger and that the LVEF was lower after CPR in VF dogs, and with the duration of VF, the LAD became larger, and the LVEF was lower. The positive correlation between TIMP-1 and the left ventricular function suggested that the imbalance between TIMP-1 and MMP-9 probably contributed to the impairment of cardiac function after CPR.

The myocardial ECM is a dynamic and highly integrated structural supporting network that is essential for maintaining normal left ventricular geometry and pump

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Fig. 1 Western blot for TIMP-1 and MMP-9 obtained from the 3 groups. A, Western blot for TIMP-1: the level of TIMP-1 decreased after longer VF and CPR. Asterisk indicates P b .05 vs the sham control group; number sign indicates P b .05 vs the 8-minute VF group. B, Western blot for MMP-9: there was not a statistical significance in the 3 groups; P N .05. C, The ratios of TIMP-1/MMP-9 in the 3 groups: the ratios of TIMP-1/MMP-9 declined after CPR. Asterisk indicates P b .05 vs the sham control group.

function. In the ECM, the expression of MMP activities is specifically regulated by the endogenous inhibitors TIMPs (TIMP-1 to TIMP-4). Once the balance between them broke down, structural remodeling would occur, especially in the chronic diseases.

Tissue inhibitor of metalloproteinase 1 was found to be mostly colocalized with gelatinase activity including MMP-2 and MMP-9 [15]. It can form high-affinity noncovalent 1:1 enzyme-inhibitor complexes that prevent MMP-9 from degrading substrates [16]. A previous study

[6] has demonstrated that during reperfusion after warm blood cardioplegia, there was a Rapid increase in MMP-2 and MMP-9 and a decrease in TIMP-1 within 10 minutes, and the loss of TIMP-1 correlated with impairment of global left ventricular function. Thus, although CPR is an

acute pathologic process, a disruption in the balance between TIMP-1 and MMP-9, which was associated with the pathologic turnover of ECM components, may be one of the possible candidates responsible for the left heart dilatation in VF dogs after CPR because the process of VF-CPR can be understood as the process of ischemia- reperfusion. In the present study, we found a significant decrease in the level of myocardial TIMP-1 after CPR, and the level of TIMP-1 positively correlated with LVEF and inversely with the LAD and LVDd. These findings are supported by previous studies, which demonstrated de- creased myocardial TIMP-1 Messenger RNA expression after acute ischemia-reperfusion injury in isolated rabbit hearts [17] and decreased serum levels of TIMP-1 after acute myocardial infarction in humans [18]. Moreover,

Fig. 2 Cellular localization of TIMP-1 and MMP-9 within myocardium by immunohistochemistry (40-fold amplified). Yellow stainings indicate positive expressions of TIMP-1 and MMP-9 (arrow tracing). They are mainly in the interstitium of the cardiomyocytes. A, Expressions of TIMP-1 in the 3 groups (P b .05). B, Expressions of MMP-9 in the 3 groups (P N .05).

TIMP-1 is acutely regulated by several cytokines and growth factors [19]. Ventricular fibrillation could be understood as ischemia, and CPR is equal to the process of reperfusion. Reperfusion can produce a series of endotoxin and cytokine such as interleukins, tumor necrosis factor, and other inflammatory factors, which may result in the decreased TIMP-1. Previous studies demonstrated that local overexpression of TIMP-1 could prevent the expansion and rupture of aortic aneurysm in rats [20] or prevented cardiac injury and dysfunction during experimental viral myocarditis in mice [21]. All the results suggested that there may be an intrinsic protective role of TIMP-1 by inhibiting the activity of gelatinases including MMP-2 and MMP-9 in the normal heart.

Matrix metalloproteinase 9 is also a cytokine-inducible MMP. Previous studies have demonstrated that targeted deletion of MMP-9 could attenuate Left ventricular remodeling after myocardial infarction [22]. In addition, targeted deletion of MMP-9 could decrease the infarct size after ischemia-reperfusion injury [23]. In addition to destruction of the ECM, MMP-9 has been localized within the cardiomyocyte and may directly damage the contractile apparatus by cleavage of the myosin heavy chain [24], and MMP-9 may process other biologically active molecules, thereby activating detrimental signaling pathways that contribute to worsening left ventricular

function [25,26]. In the present study, although the difference in the protein level of MMP-9 did not achieve statistical significance in the sham control and VF groups, it was still higher in VF groups especially in the longer duration VF group. Meanwhile, the ratios of TIMP- 1/MMP-9 were significantly lower after CPR. In addition, the activity of MMP-9 would be enhanced because of the decreased level of TIMP-1. Thus, as mentioned above, the increased level and the enhanced activity of MMP-9 may play a critical role in the enlargement of the left side of the heart and the decreased LVEF by directly or indirectly destroying the myocardial cells and the structural supporting network of the ECM.

Several limitations should be considered in the interpre- tation of the present results. Because of the complexity of myocardial injury after CPR, it was difficult to conclude that the imbalance between TIMPs and MMPs must be involved in the postresuscitation cardiac dysfunction. Future studies will address this issue by either inhibiting MMPs or enhancing TIMP activity.

In conclusion, the loss of TIMP-1 and/or the increase in MMP-9 may contribute to the postresuscitation car- diac dysfunction. Zhang et al [27] proposed that Angiotensin II-converting enzyme inhibitors may have the potential to normalize the balance of MMP-9/TIMP-1 in atrial fibrillation model. Thus, for patients after CPR,

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Fig. 3 Correlation between TIMP-1 and cardiac function.

drugs such as angiotensin II-converting enzyme inhibi- tors may be used to ameliorate prognosis by normalizing the TIMP-1/MMP-9 balance, and a further study is needed to verify the question. In the present study, VF

was electrically induced in healthy animals; thus, the poten- tial confounders such as various chronic diseases, which may affect the expressions of TIMPs and MMPs, have been eliminated.

Acknowledgment

This study was supported by a grant from the National Basic Research Program of China (973 Program, 2007CB512001) and sponsored by the Natural Science Foundation of China (30871039).

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