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b-Blocker Improves Cardiac Function by Reducing Oxidative Stress and Metalloproteinase Activity After Myocardial Infarction*

b-Blocker Improves Cardiac Function by Reducing Oxidative Stress and Metalloproteinase Activity After Myocardial Infarction*

 

Mauricio Bernstein, MD

Suresh C. Tyagi, PhD

 

Department of Physiology and Biophysics,

School of Medicine

The University of Mississippi Medical Center,

Jackson, MS 39216

 

 

*This work was supported in part by NIH grant GM-48595, HL-51971, and a grant-in-aid from American Heart Association-Mississippi Affiliate. A part of this study was presented at the 53rd Annual Fall Conference and Scientific Sessions of the American Heart Association Council of High Blood Pressure Research, September 13-16, 1999, Orlando, FL.

 

KEY WORDS: extracellular matrix, MMP, TIMP-4, collagen, remodeling, muscarinic receptor, catecholamine, norepinephrine, diastolic dysfunction, heart failure

 

 

ABSTRACT

b-blockers have been shown to reduce oxidative stress and improve cardiovascular function. It is unclear, however, whether b-blockers improve cardiac remodeling by inhibiting metalloproteinase and oxidative stress. The aims of this study were to determine the effects of a b-adrenergic blocker (metoprolol [M]) on matrix metalloproteinase (MMP) expression and activity, tissue inhibitor of metalloproteinase-4 (TIMP-4) expression, collagen degradation, and diastolic function in a rat model of myocardial infarction (MI). Sprague Dawley rats were divided into four groups: (1) sham, (2) sham+M, (3) MI, and (4) MI+M (n=6). Two days prior and continuously for 2 weeks after ligating the left anterior descending artery, rats were given 10 mg/day metoprolol by gavage. Hemodynamic parameters, serum, and cardiac specific creatine phosphokinases were measured. Left ventricle (LV) systolic and diastolic function were measured in anaesthetized rats. LV MMP-2 was measured by Northern-blot and zymography. The levels of TIMP-4 were measured by Northern-blot and Western-blot analyses. The scanned intensity data of MMP-2 and TIMP-4 mRNA and protein were normalized with 18S RNA and b-actin, respectively. In sham, sham+M, MI, and MI+M groups, respectively, the following data were obtained: The mean arterial pressures (MAP) were 111+3, 109+8, 99+16, and 88+21 mm Hg (P > .05). The end-diastolic pressures were 4.15+1.4, 3.85+0.95, 12.9+2.0, and 9.9+1.0 mm Hg (P < .05). Heart rates were 339+13, 331+9, 310+43, and 256+29 beats/min. The (-dP/dt)/MAP were 45+9, 46+6, 30+3, and 38+4 sec-1. The 66 kDa MMP-2 activity was 0.04+0.02, 0.03+0.01, 0.57+0.02, and 0.25+0.03 (scan unit). The 72 kDa gelatinase was 0.28+0.08, 0.24+0.03, 0.68+0.07, and 0.38+0.08 (scan unit). There was no significant change in the protein levels of MMP-9 and TIMP-4 activity in either group. Total reduction-oxidation (redox) disulfides, a marker of oxidative stress, were 0.066+0.05, 0.06+0.02, 0.095+0.02, and 0.055+0.01 mg/mg of tissue. A negative correlation between total MMP-2/TIMP-4 activity and (-dP/dt)/MAP was obtained. The results suggested that treatment with metoprolol improves cardiac function by inhibiting metalloproteinase and decreasing oxidative stress after MI.

 

INTRODUCTION

During protracted cycles of ischemia/reperfusion,1 the oxygen produces oxyradical: 2O2 + 2H2O = 2H2O2 + O2- (toxic oxygen), dependent or independent of NADH/NAD oxidase,2 and masks the activity of superoxide dismutase and catalase.3-5 Therefore, during myocardial infarction (MI) oxygen tension and oxidative stress are robustly increased.6 Oxidative stress increases the levels of cytokines, growth factors, and neurohormones.7,8 This starts a vicious cycle of oxidative stress in which neurohormone such as angiotensin II10 increases further oxidative stress by decreasing the levels of bradykinin and prostaglandins. Otherwise, these molecules mediate antioxidation by increasing nitric oxide production.11 In parallel, angiotensin II also induces NADH/NAD oxidase.12 Therefore, increased oxidative stress may be the initial cause of alteration in remodeling, structure, and function.

A critical balance between matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) is necessary for normal tissue constitution.13 In the normal heart most of the MMPs are latent, and the MMP:TIMP ratio is 0.15 to 0.6,14 but in end-stage heart failure this ratio increases to approximately 6 to 715. In vitro treatment of normal cardiac extract with oxidants and inflammatory cell proteinase increases the ratio to approximately 5 to 6.14 In ischemic heart disease, inflammatory cytokines, neurohumoral factors and oxidative stress are elevated.16,17 Oxidative stress and norepinephrine produce collagenolysis and cardiac dilatation.18,19 The sympathometric blockade is associated with decreased oxidative stress and improved cardiac function after MI.20-23 It is unclear, however, whether beta-sympathometric blockade improves cardiac function, in part, by inhibiting oxidative stress and matrix remodeling. The hypothesis is that b-adrenergic blockade improves cardiac function, in part, by inhibiting MMP after MI.

 

MATERIALS AND METHODS

Experimental model of myocardial infarction: Harlan Sprague-Dawley rats, weighing 300 to 375 g, were intubated, and respiration was maintained during thoracotomy by intermittent positive pressure. Ventilation was delivered by a small animal ventilator. Rats were anesthetized with pentobarbital (50 mg/kg intraperitoneal). The left side thorax was opened through 3 to 4 ribs, the heart was exteriorized, and the left main coronary artery was ligated 2 mm from its origin29. The chest was closed, intrathoracic negative pressure was reestablished, and the animals were allowed to recover. Rats were divided into four groups: (1) rats were subjected to sham-operation, and surgery was performed but the ligature was not tied around the coronary artery (Sham); (2) sham received daily gavage with metoprolol (Sham+M); (3) surgery was performed, and the ligature was tied around the coronary artery (MI); and (4) rats were given metoprolol (10 mg/day) gavage 2 days prior to creation of MI and continued for 2 weeks (MI+M). A minimum of 6 rats (n = 6) was used in each group. At the end of 2 weeks treatment the serum creatine phosphokinase (CK) and degree of MI were measured. All studies conformed to the principles of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the protocols were approved by our Institution's Animal Care and Use Committee.

Molecular assessment of heart failure: To determine plasma CK MB (myocardial) activity, blood (0.25 mL was collected from the tail vein by producing a small excision in the tail of each animal on days 1 and 12 following coronary ligation. Because specific

CK MB isoenzyme is released in plasma following myocardial injury in patients with MI,24,25 we measured CK MB activity by agarose substrate gel electrophoresis.26 The bands due to CK MB activity were scanned and the intensity (I) was used to estimate the amount of CK MB. The amount of CK MB in the serum of MI rats (sample) was determined as follows: Amountsample=AmountST/IST x Isample; where ST is standard isoenzyme MB (Sigma). A criteria of serum CK activity >10 mg/mL was applied to determine the severity of MI.

Measurements of plasma redox-thiols: Total plasma redox-thiols were measured by titrating the -SH group with dithio-bis-nitrobenzoate (DTNB) in the presence of a minimal reducing agent. After incubation at 37oC for 3.5 hours, all thiol and disulfides were exchanged to DTNB and produced color at 412 nm. The concentration of thiols was calculated using an extinction coefficient of 13,600 M-1cm-1 at 412 nm.27

In vivo assessment of hemodynamic parameters and left ventricular function: Mean arterial pressure (MAP), heart rate (HR), systolic and diastolic blood pressure (SBP and DBP), left ventricular pressure (LVP) and maximum fall in LVP, -dP/dt, were measured in anesthetized rats with Inactin (Sigma Chemical Co, St. Louis, MO) (100 mg/kg intraparitoneal)26. This anesthesia had minimal effect on cardiovascular function.28 For MAP, a fluid-filled catheter (PE-50 tubing) was inserted into the right femoral artery. The arterial catheter was connected to a pressure transducer (Micro-Med, Corp) positioned at the level of the heart. Pulsatile arterial pressure was recorded by a personal computer using customized software. For LVP, the catheter was advanced to LV via right carotid artery, and the LVP was recorded. The pressure transducers were calibrated and electronically interfaced to a personal computer for analog-to-digital conversion, storage, and analysis of data. Ten minutes after insertion of the arterial and ventricular catheters, respectively, resting measurements of arterial BP, heart rate, and LVP were taken. The maximum derivative of fall in LVP (-dP/dt) and rise (+dP/dt) were estimated. The -dP/dt is afterload-dependent; to correct for the afterload, -dP/dt was divided by MAP.26 After functional measurements, anesthetized animals were prepared for excision of the heart.

Identification of infarction: The heart was removed under sterile conditions and weighed. The LV and RV were separated. The ratio LV weight (g)/body weight (g) was employed to evaluate the increase in mass of the ventricle. The MI was identified by observing discoloration and Evan's blue staining of the ischemic muscle. The MI tissue was dissected and weighed. The percentage of MI was calculated as follows: MI tissue weight/LV weight x 100. The rest of the tissue was divided into three subgroups: (1) fixed in 10% buffered formalin for histologic analysis, (2) tissue cooled in isopentane and stored at -80oC for immuno- and in situ labeling, and (3) directly frozen tissue for mRNA analyses.

Zymography: The MMP activity was measured by zymography as described.14 In brief, LV extract were prepared. The extracts (25 mg of protein in each lane) were loaded onto nonreducing (1%) gelatin-SDS-PAGE. Gels were incubated overnight and stained with Coomassie blue. To ensure that no significant gel-to-gel variation occurred, standard rat heart MMP-2 (purified in our laboratory) was used in each gel. The lytic bands were scanned and normalized with actin. Actin was detected by coomassie blue at 42 kDa.

Western blots: The levels of TIMP-4 were specifically measured by Western-blot analysis using anti-TIMP-4 antibody (Chemicon Corp). Intrinsic activation of MMP produces soluble collagen fragments. Collagen-degradation was measured by immuno-blot analysis using anti-collagen antibody (Sigma Chemical Co). Cardiac extracts were prepared. The LV extracts were loaded onto reducing SDS-PAGE. The gels were transferred onto nitrocellulose membrane. The membranes were blotted with anti-TIMP-4 antibody (1:200) or anti-collagen antibody (1:150). To detect the labeling, secondary antibody conjugated with alkaline phosphatase was used. The bands below 100 kDa for alpha chain of collagen were identified as collagen breakdown fragments.15 The scanned value of 3/4 fragment was used as measure of collagen degradation.

Expression of MMP-2 and TIMP-4: The mRNA analysis of cardiac MMP-2 and TIMP-4 was performed at days 1, 4, and 12 following coronary ligation and sham operation at day 12 by Northern-blots as described.29 The plasmid-containing collagenase IV cDNA probe was obtained from ATCC. The cDNA probe for MMP-2 was 2.119 kb and was isolated by EcoRI enzyme digestion. The mRNA levels for TIMP-4 were analyzed by Northern-blot using cDNA probe isolated from RT-PCR. Primers for TIMP-4 were: sense 5'-GTGACGAGAAGGAGGTGGATTCC and anti-sense 5'-CTTGATGCAGGCAAAGAACTTGGC (GenBank No. U76456). Isolated cDNA was radio-labeled by random primer labeling using [32P]ATP and used as a probe for mRNA labeling. A 4.5 kb EcoRI fragment of 18SR gene (a gift from Dr. R. Guntaka) was used as an internal control. Same blots were stripped and reprobed for different mRNA analysis.

Statistical Analysis: Because the data in each experimental group was collected from different animals (ie, sham; sham+M; MI; and MI+M), the significance of data between the groups was determined using a 2-way ANOVA analysis of variance. In each group, a minimum of six animal data was used to determine the significance. The SEM and mean are reported.

 

RESULTS

LVH, oxidative stress, and cardiac injury after MI: The LV and RV weights/body weight ratios were increased in MI. Metoprolol had no significant effect on LV and RV weights/body weight (Table 1). There was robust increase in the oxidative thiols in the MI group. The treatment with metoprolol decreases the levels of oxidative stress (Table 1). To determine the degree of cardiac muscle injury, plasma levels of CK MB activity after MI were measured. Day 1 after MI, CK MB levels were minimal. However, day 12 CK MB as well as MM and BB were increased. This suggested severe tissue injury after MI (Figure 1). The treatment with metoprolol decreases cardiac injury by reducing CK MB activity (Table 1).

MMP-2 and TIMP-4 after MI: The levels of TIMP-4 were elevated at day 1 after MI (Figure 2). TIMP-4 was induced early in response to ischemic insult. The results may suggest compensatory response of TIMP-4 after MI. However, at days 4 through 12 MMP-2/TIMP-4 ratio increased significantly (P < .01) in the MI group compared with the sham control (Figure 2). The treatment with metoprolol decreases MMP-2/TIMP-4 ratio after MI (Table 2). The MMP-2 expression was decreased in MI rats treated with metoprolol for 12 days (data not shown). The metoprolol had no significant effect on sham animals (Table 2).

Collagen degradation: To determine whether interstitial fibrillar collagen is degraded after MI, collagenolytic activity was determined by measuring 3/4 and 1/4 collagen fragments in cardiac tissue homogenates at 12 days after MI. Results suggested collagen degradation after MI. The generation of these fragments was inhibited in rats treated with metoprolol after MI (Figure 3). MMP-2 and TIMP-4 activity was measured by zymography and Western blot analysis, respectively (Figure 4). The levels of TIMP-4 protein did not change significantly in either groups compared with sham controls. MMP-2 activity at 66 and 72, and MMP-9 at 92 kDa was increased significantly after MI (P < .05). The activity at 66 and 72 kDa MMP was significantly decreased in rats treated with metoprolol (P < .05). However, there was no significant effect of metoprolol on MMP activity at 92 kDa (P > .05; Figure 5).

Hemodynamic and cardiac function: MI rats with >10 mg/mL of plasma CK MB activity demonstrated significant alterations in hemodynamic and LV function (Tables 1 and 2). Arterial pressure was reduced in rats with MI. Metoprolol has no significant effect on MAP but decreases heart rate in MI rats compared with controls (Table 1). LV function, -dP/dt, and end-diastolic pressure were impaired after MI and improved following metoprolol treatment (Table 2). Based on MMP activity and LV function, a relationship between (-dP/dt)/MAP versus levels of MMP-2/TIMP-4 ratio was delineated (Figure 6). The cardiac MMP-2/TIMP-4 ratio was reduced, from 1.10 + 0.17 in the MI group to 0.61 + 0.08 in the MI+M group. This decrease was associated with improved LV function by metoprolol.

 

DISCUSSION

Our results suggest that the b-blocker prevents oxidative damage to the myocardium and alters ventricular loading conditions so that the stimulus for remodeling was attenuated. There is an association between b-blocker, oxidative stress, and MMP-2 activity. Metoprolol decreases MMP-2 in virtue of an antioxidant effect, and the decrease of oxidative stress and MMP-2 were parallel.

All three creatine phosphokinase isoforms (BB, MB, MM) were induced after MI (Figure 1). These enzymes are also induced in hypertrophic heart disease.26,30 In humans, the functional exercise capacity of survivors of MI patients was inversely related to serum creatine phosphokinase.31,32 Our results suggested that treatment with metoprolol decreases the levels of CK isoenzymes and reduces the cardiac injury after MI.

Rhode and colleagues33 treated MI mice with either a placebo or nonspecific MMP inhibitor. The results were mixed in that both groups had mice that experienced no change (30% of placebo versus 18% receiving drug), an increase (50% placebo versus 30% drug), or a decrease (20% placebo and 52% drug) in the chamber area. Unfortunately, infarct size was not measured in that study and one could never be certain that the differences between the two groups were not the result of variations in infarct size. Peterson and coworkers34 have demonstrated that some MMPs were significantly elevated during the first week after MI and another was not until 16 weeks after MI. Previously in the human heart15 a dissociation was demonstrated between LV MMP/TIMP mRNA and protein levels, suggesting posttranslational processing after MI in which MMP is activated. Cleutjens and colleagues,35 using a rat model of MI, demonstrated that TIMP-1 mRNA was increased 6 hours after coronary ligation and reached a maximum level on day 2, remaining high by day 7. They also showed that mRNA for MMP-1 was transiently induced only at day 7 after MI. Our data suggest that MMP-2 induced between days 4 and 7 after MI and cardiospecific TIMP-436 induced day 1 up to day 12 (Figure 2). MMP-2 activity increased and TIMP-4 protein levels remained constant up to 2 weeks after MI (Figure 4), suggesting an increased MMP-2/TIMP-4 ratio after MI.

Despite significant fibrosis in both ventricles after MI, collagen content is reduced and collagen is continuously being degraded in 3- to 8-month-old infarcted rat heart.37 Since rat heart does not contain MMP-138 and MMP-2 degrades interstitial collagen,39 we analyzed degradation of cardiac collagen into 3/4 and 1/4 fragments. Results suggested collagen degradation (Figure 3) and activation of MMP-2 in MI heart (Figure 4). A 48-hours tachypacing model in dogs showed that increased LV chamber stiffness and increased gelatinase activity can be inhibited by b-blockade.40 However, this study did not elucidate the interplay of oxidative stress, MMP-2 and -9 activation, levels of TIMP-4, and collagenolysis in regard to cardiac function. To determine whether MMP activity and collagen degradation in MI heart can be inhibited by metoprolol, we analyzed cardiac extracts from MI rats treated with metoprolol. Results suggested that metoprolol was able to inhibit collagenolytic activity after MI (Figure 3 and 4). Rats on metoprolol showed reduced MMP-2 activity (Figures 4 and 5). The levels of TIMP-4 did not change significantly by metoprolol treatment (Table 2). Cardiac MMP-2 at 66 and 72 kDa (ie, gelatinase a) were inhibited in metoprolol treated rats, and there was no effect on gelatinase b (92 kDa; Figure 5, Table 2), suggesting that metoprolol treatment decreases MMP-2/TIMP-4 ratio after MI.

In humans, serum levels of MMP-1 and TIMP-1 were negatively correlated to left ventricle end-systolic volume index.41 A negative relationship between cardiac MMP-2/TIMP-4 ratio and cardiac index, (-dP/dt)/MAP, in a rat model of MI is shown in Figure 6, suggesting activation of MMP may be one of the causes of cardiac dysfunction after MI, and metoprolol improves cardiac index in part by inhibiting MMP.

Perspective: A study of the treatment of animal with catecholamine demonstrated collagenolysis and LV dilatation.18 Our results suggest that MMP-2/TIMP-4 ratio is reduced in the metoprolol group compared with MI and may in part be responsible for the improvement of cardiac function. However, it does not differentiate whether MMP inhibition by metoprolol is, in part, due to its effect on the reduction of blood pressure or decreased levels of norepinephrine. It would be of great interest to determine whether other b-blockers, independent of lowering blood pressure, and antioxidants improve cardiac function by inhibiting extracellular matrix (ECM) remodeling. These studies are in progress.

 

Figure 1. Plasma creatinine phosphokinase isoform in sham control and day 1 and 12 following ligation: Lanes 1 and 2 are plasma from rats on day 12 following ligation. Lane 3 is standard CK isoforms. Lanes 4 through 7 are day 1 after ligation. Lanes 8 through 11, plasma from sham controls. CK MM (skeletal), MB (cardiac) and BB (nerve and kidney) activity was analyzed on agarose gel electrophoresis using kinase substrate as described in Materials and Methods.

 

Figure 2. Northern-blot analysis of temporal changes in MMP-2 and TIMP-4 mRNA following MI: Lane 1, sham surgery; lane 2, 1 day after creation of MI; lane 3, 4 days after MI; and lane 4, 12 days after MI. The 18S RNA was used as internal control. The size of transcript was 3.5 kb for MMP-2 and 1.5 kb for TIMP-4.

 

Figure 3. Cardiac collagen degradation in tissue extract prepared from MI, metoprolol treated, and sham rats: After SDS-PAGE immuno-blot was performed using anti-collagen I antibody. Collagen 3/4 and 1/4 fragments are representative of collagen degradation. Lanes 1 through 4, extracts from MI; lanes 5 through 6, metoprolol treated-MI; lane 7, sham. Relative amounts of collagen-degraded fragments are reported on top of the blot.

 

Figure 4. Zymographic and Western-blot analysis of MMP-2 and TIMP-4, respectively, in cardiac tissue extracts from lane 1, sham; lanes 2 through 5, MI; and lanes 6 and 7, MI rats treated with metoprolol for 2 weeks. Actin was used as internal control. Molecular weight marker for MMP are shown on the right.

 

Figure 5. Histographic presentation of MMP 66 kDa, 72 kDa, and 92 kDa in the sham, MI, and MI+metoprolol groups: The bands from zymographic gels were scanned. To minimize gel-to-gel variation, a standard gelatinase was loaded onto each gel. The intensity (arbitrary unit) was compared with standard gelatinase and was normalized for actin. The mean + SEM is reported.

 

Figure 6. A correlation between MMP-2/TIMP-4 ratio versus active cardiac diastolic function, (-dD/dt)/MAP (r = 0.90; P = .01): Cardiac function in sham, sham+M, MI, and MI plus metoprolol, was recorded. The MMP-2 and TIMP-4 activity (intensity) was measured in cardiac tissue homogenates prepared from respective animals. Data from all groups were combined. The metoprolol group is on the left. The mean + SEM is reported.

 


 

Table 1. Body, LV and RV weights, cardiac tissue reduction-oxidation (redox)-thiols, hemodynamic parameters, and activity of plasma creatine phosphokinase cardiac isoform (CK MB) in sham, sham + M, MI, and MI + M groups of rats at day 12: Each rat was anesthetized by Inactin (100 mg/kg intraparitoneal). Heart rate (HR), mean arterial pressure (MAP), systolic blood pressure (SBP), and diastolic blood pressure (DBP) were measured. After hemodynamic and cardiac function measurements, tissue was isolated and MI was identified. The mean + SEM are reported.

__________________________________________________________________________________

Sham Sham+M MI MI+M

n 6 6 6 6

Body Weight (BW), g 392+9 385+12 335+34 313+47

Left Ventricle Weight (LVW), g 0.86+0.04 0.85+0.02 0.85+0.05 0.84+0.07

Right Ventricle Weight (RVW), g 0.19+0.03 0.18+0.04 0.18+0.03 0.16+0.06

LVW/BW, x 103 2.19+0.04 2.20+0.06 2.54+0.05 2.68+0.07

RVW/BW, x 103 0.48+0.03 0.47+0.02 0.54+0.03 0.51+0.06

% MI 0-5% 0-5% 27.6+9.3 18.4+6.0*

Redox-thiols, mg/mg of tissue 0.066+0.050 0.06+0.02 0.095+0.02 0.055+0.01*

CKMB, mg/mL of plasma 0.010+0.008 0.011+0.006 12.5+2.7 4.8+1.2*

 

Hemodynamic Parameters

 

MAP, mm Hg 111+3 109+8 99+16 89+21

HR, beats/min 339+13 331+9 310+43 256+28*

SBP, mm Hg 135+8 132+6 126+16 108+27

DBP, mm Hg 93+3 91+7 83+12 72+17

__________________________________________________________________________________

*P value was < .05 when MI+M group was compared with MI group.

 

 

 


Table 2. Cardiac MMP activity at 66, 72, and 92 kDa, TIMP-4 (arbitrary unit), end-diastolic pressure (EDP) and cardiac output, (-dP/dt)/MAP of sham, sham+M, MI and MI+M groups of rats: Each rat was anesthetized with Inactin (100 mg/kg, intraparitoneal). Left ventricle-EDP and -dP/dt were measured. The mean + SEM are reported.

__________________________________________________________________________________

Sham Sham+M MI MI+M

n 6 6 6 6

MMP-2; 66 kDa 0.04 + 0.02 0.03 + 0.01 0.57 + 0.02 0.25 + 0.03*

MMP-2; 72 kDa 0.28 + 0.08 0.24 + 0.03 0.67 + 0.07 0.38 + 0.08*

MMP-9; 92 kDa 0.01 + 0.01 0.01 + 0.01 0.06 + 0.07 0.09 + 0.11

TIMP-4 0.53 + 0.04 0.55 + 0.06 0.62 + 0.05 0.62 + 0.07

 

Active Cardiac Diastolic Function

 

(-dP/dt)/MAP, sec-1 45+9 46+6 30+3 39+4*

EDP, mm Hg 4+1 3.85+0.95 13+2 9.9+1.0*

 

__________________________________________________________________________________

*P value was < .05 when the MI+M group was compared with MI group.

 


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