Heart Vessels (2005) 20:61–65 DOI 10.1007/s00380-004-0805-5
© Springer-Verlag 2005
ORIGINAL ARTICLE Peng Guo · Katsufumi Mizushige · Takahisa Noma Kazushi Murakami · Tsunetatsu Namba · Makoto Ishizawa Teppei Tsuji · Shoji Kimura · Masakazu Kohno
Association of uncoupling protein-2 expression with increased reactive oxygen species in residual myocardium of the enlarged left ventricle after myocardial infarction
Received: July 2, 2004 / Accepted: October 22, 2004
Abstract Left ventricular (LV) dilatation following myocardial infarction (MI) is a major determinant of the patient’s prognosis, and myocardial energy metabolism may play a key role in LV remodeling. We aimed to investigate the relative timing of LV dilatation to LV function, myocardial energy regulation by uncoupling protein (UCP)-2, and cellular damage in the noninfarct zone. Myocardial infarction was produced in Sprague-Dawley rats by ligation of the coronary artery. The LV end-diastolic dimension (mm) increased (8.9 ⫾ 0.3 vs 6.8 ⫾ 0.8 in sham-operated rats, P ⬍ 0.01) in association with elevation of the LV end-diastolic pressure (mmHg) (18 ⫾ 5 vs 6 ⫾ 2 in sham-operated rats) at 1 week following the ligation. At 4 weeks, the UCP-2 expression (180% of that in sham-operated rats) and LV enddiastolic dimension increased further (11.1 ⫾ 0.5, P ⬍ 0.01) but there was no change in the LV end-diastolic pressure. The mechanisms for LV dilatation were quite different between the early and late stages after MI. In the late stage, augmentation of UCP-2 expression in the noninfarct zone may be related to the LV dilatation. Further examinations regarding the possibility of the protective role of UCP-2 are needed. Key words Heart failure · Energy metabolism · Mitochondria · Left ventricular remodeling
P. Guo · T. Noma · T. Namba · M. Ishizawa · M. Kohno Second Department of Internal Medicine, School of Medicine, Kagawa University, Kagawa, Japan K. Mizushige (*) · K. Murakami · T. Tsuji Department of Cardiology, Takamatsuhigashi National Hospital 8 Otsu, Shinden-cho, Takamatsu, Kagawa 761-0193, Japan Tel. ⫹81-87-841-2146; Fax ⫹81-87-843-5545 e-mail: [email protected]
S. Kimura Department of Pharmacology, School of Medicine, Kagawa University, Kagawa, Japan
Introduction The morphology of the left ventricle following myocardial infarction is an important determinant of the patient’s prognosis. Preservation of cardiac function or left ventricular dilatation in the early or late stages after myocardial infarction is dependent on the size of the infarct zone or the contractile condition of the noninfarct myocytes.1,2 Expression of contractile proteins, and myocyte or mitochondrial membrane proteins was introduced as a molecular mechanism for the compensatory augmentation of regional cardiac function.3 Although impairment of the energy metabolism in the non-infarct myocardium was demonstrated, the molecular mechanisms of the energy dissipation in the noninfarct residual myocytes still remain unclear. Uncoupling protein (UCP)-2 is an isoform that is widely distributed throughout the body, with the most abundant expression in the heart.4–6 Using an experimental aortic regurgitation model, we have demonstrated that myocardial expression of UCP-2 is significantly increased in the enlarged left ventricle, which leads to a decrease in creatine phosphate.7,8 Mills et al. showed that an increase in UCP-2 expression leads to cell death in HeLa cells,9 and that the UCP-2 expression is upregulated and its uncoupling function is augmented by reactive oxygen species (ROS) in vitro.10 Therefore, it is hypothesized that UCP-2 expression is upregulated by the increase in oxidative stress in the noninfarct myocardium during the progressive left ventricular remodeling, which leads to left ventricular dysfunction. The purpose of this study was to examine the alterations in UCP-2 gene expression and ROS production in the noninfarct residual myocardium during the progressive deterioration of cardiac function after broad myocardial infarction in rats.
Materials and methods Animals Twenty-seven male Sprague-Dawley rats (6–8 weeks old, weight 220–250 g) were maintained at the Kagawa University animal experimental center. They were kept in a pathogen-free facility under controlled temperature (23° ⫾ 2°C) and humidity (55% ⫾ 5%) with a 12/12-h artificial light/dark cycle, and given free access to standard laboratory rat chow (MF, Oriental Yeast, Tokyo, Japan) and tap water. All procedures were in accordance with the institutional guidelines for animal research.
Hemodynamic studies One or 2 days after the echocardiographic recordings, the rats were anesthetized with sodium pentobarbital (50 mg/kg i.p.). A 1.4-F high-fidelity micromanometer (TCB-500, Millar, Houston, TX, USA) was inserted into the right carotid artery and then advanced into the left ventricle. Left ventricular pressure waveforms were directly input into an on-line computer and analyzed using calculating software (Acqknowledge) (Biopac, Santa Barbara, CA, USA). The left ventricular peak systolic and end-diastolic blood pressure, maximum dP/dt, and negative dP/dt were obtained. Following the left ventricular pressure measurement, arterial pressure was measured at the position of the ascending aorta.
Study protocol Thiobarbituric acid reactive substances assay Rats were anesthetized with ketamine (50 mg/kg intraperitoneal) and xylazine (10 mg/kg intraperitoneal). In 15 rats, myocardial infarction was produced by ligation of the left anterior descending coronary artery using the methods described by Litwin et al.11 Twelve sham-operated rats, in which the coronary artery was not ligated, were used as a control. Seven infarct and 6 sham-operated rats were killed at 1 week after the ligation (early stage after the infarction), and 8 infarct and 6 sham-operated rats were killed at 4 weeks after the ligation (late stage after the infarction). At the baseline (before the ligation), and 1 and 4 weeks after ligation, the body weight was measured, and echocardiography was performed for an assessment of the left ventricular geometry and function. At 1 and 4 weeks, hemodynamics, histopathology, thiobarbituric acid reactive substances, and UCP-2 expression were analyzed. Echocardiography Rats were anesthetized with ketamine (50 mg/kg intraperitoneal) and xylazine (10 mg/kg intraperitoneal), and transthoracic echocardiography was performed using an echocardiograph (Sonos 5500; Phillips Medical Systems, Andover, MA, USA) with a 12-MHz transducer. The left ventricular dimension was measured at end-diastole (LVDd) and end-systole (LVDs) on a left ventricular M-mode echocardiogram. Fractional shortening (FS) was calculated as (1 ⫺ LVDs/LVDd) ⫻ 100. The heart rate was determined from a simultaneously recorded electrocardiogram. After completion of the hemodynamic measurements, the rats were killed by deep anesthesia with sodium pentobarbital (50 mg/kg i.p.). The hearts were excised, and the atria were trimmed from the ventricles. The right ventricle and left ventricle including the septum were separated and weighed. The left ventricular wet weight-to-body weight ratio was calculated. The left and right ventricles were rapidly frozen in liquid nitrogen and stored at ⫺80°C until use for RNA extraction and thiobarbituric acid reactive substances (TBARS) assay.
The lipid peroxide content in the myocardium was determined using the TBARS method.12 Briefly, tissue was homogenized in (10% w/vl) 0.15 M KCl, 0.02 M Tris-HCl buffer (pH 7.4) and then 1 ml of homogenate was added to 2 ml TBA reagent. The tubes were then boiled for 15 min, cooled on ice to room temperature, and subsequently centrifuged at 3 500 rpm for 200 min. The absorbance of each supernatant was measured at 535 nm using a spectrophotometer. Commercially available malondialdehyde was used as the standard. RNA Extraction and Northern hybridization Total RNA was extracted from the noninfarct myocardium of the left ventricle using Isogen according to the manufacturer’s protocol (Nippon Gene, Toyama, Japan). Ten micrograms of total RNA was used for Northern hybridization with digoxigenin-dUTP-labeled cDNA probes synthesized by the polymerase chain reaction as described by the manufacturer (Roche Diagnostics, Basel, Switzerland). Statistics All values are shown as the mean ⫾ SEM. Values between the myocardial infarction rats at various stages and shamoperated rats were compared using one-way analysis of variance followed by Scheffe’s test. P ⬍ 0.05 was considered statistically significant.
Results Myocardial mass and hemodynamics The systolic blood pressure, mean blood pressure, and diastolic blood pressure of the myocardial infarction rats were lower than those of the sham-operated rats at 1 and 4 weeks (P ⬍ 0.01) (Table 1). The rats with myocardial infarction had significant systolic dysfunction, as evidenced by decreases in the left ventricular systolic pressure, mean blood
63 Table 1. Heart rate and blood pressure in the sham-operated and myocardial infarction model rats 1 week
Sham (n ⫽ 6) Heart rate (beats/min) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Mean blood pressure (mmHg) Left ventricular systolic pressure (mmHg) Left ventricular diastolic pressure (mmHg) ⫺dP/dt (mmHg/s) ⫹dP/dt (mmHg/s)
MI (n ⫽ 7)
302 ⫾ 5 109 ⫾ 8 98 ⫾ 7 92 ⫾ 3 125 ⫾ 8
298 ⫾ 4 100 ⫾ 3* 91 ⫾ 6 95 ⫾ 2 112 ⫾ 5†
18 ⫾ 5†
6 335 ⫾ 78 4 307 ⫾ 57
Sham (n ⫽ 6)
MI (n ⫽ 8)
⫾4 ⫾5 ⫾7 ⫾4 ⫾7
323 ⫾ 4 111 ⫾ 2# 95 ⫾ 1 87 ⫾ 5 131 ⫾ 4#
14 ⫾ 2#
319 123 102 92 164
4 573 ⫾ 48† 3 587 ⫾ 39†
6 613 ⫾ 76 5 113 ⫾ 49
4 777 ⫾ 19# 3 594 ⫾ 75#
MI, myocardial infarction * P ⬍ 0.01 vs sham-operated rats at 1 week; † P ⬍ 0.05 vs sham-operated rats at 1 week; # P ⬍ 0.01 vs sham-operated rats at 4 weeks
Table 2. Body weight and ventricular weights in the sham-operated and myocardial infarction model rats 1 week
Body weight (g) Left ventricular weight (mg) Right ventricular weight (mg) Left ventricular weight/ body weight (mg/g) Right ventricular weight/ body weight (mg/g)
Sham (n ⫽ 6)
MI (n ⫽ 7)
Sham (n ⫽ 6)
MI (n ⫽ 8)
351 ⫾ 11 624 ⫾ 8 159 ⫾ 5
340 ⫾ 9 612 ⫾ 7* 189 ⫾ 3*
414 ⫾ 9 878 ⫾ 6 251 ⫾ 4
441 ⫾ 6 963 ⫾ 10# 335 ⫾ 8#
1.78 ⫾ 0.03
1.80 ⫾ 0.07
2.12 ⫾ 0.01
2.27 ⫾ 0.02
0.45 ⫾ 0.02
0.56 ⫾ 0.01
0.61 ⫾ 0.06
0.75 ⫾ 0.04
* P ⬍ 0.01 vs sham-operated rats at 1 week; P ⬍ 0.01 vs sham-operated rats at 4 weeks #
Table 3. Echocardiographic measurements in the sham-operated and myocardial infarction model rats 1 week
Left ventricular end-diastolic dimension (mm) Left ventricular end-systolic dimension (mm) Fractional shortening (%) Ejection fraction (%)
Sham (n ⫽ 6)
MI (n ⫽ 7)
Sham (n ⫽ 6)
MI (n ⫽ 8)
6.8 ⫾ 0.8
8.9 ⫾ 0.3*
8.2 ⫾ 0.4
11.1 ⫾ 0.5#
4.6 ⫾ 0.5
7.1 ⫾ 0.4*
5.8 ⫾ 0.3
8.6 ⫾ 0.3#
40 ⫾ 3 78 ⫾ 5
21 ⫾ 5* 63 ⫾ 2*
34 ⫾ 5 69 ⫾ 7
17 ⫾ 8# 40 ⫾ 6#
* P ⬍ 0.01 vs sham-operated rats at 1 week; # P ⬍ 0.01 vs sham-operated rats at 4 weeks
pressure, and peak positive dP/dt. The rats with myocardial infarction also had severe diastolic dysfunction, as defined by the peak negative dP/dt and a marked elevation of the left ventricular end-diastolic pressure (Table 1). The left and right ventricular weights of the myocardial infarction rats were increased compared with those of the shamoperated rats at 4 weeks (P ⬍ 0.01) (Table 2).
those of the sham-operated rats (P ⬍ 0.01). Fractional shortening and the ejection fraction decreased remarkably in the myocardial infarction rats (P ⬍ 0.01). Further increases in the left ventricular dimensions and deteriorations of the left ventricular function were observed at 4 weeks (P ⬍ 0.05 vs the myocardial infarction rats at 1 week) (Table 3 and Fig. 1).
Assessment of oxidative stress
In the myocardial infarction rat, the anterior wall was akinetic and was progressively thinned, and the left ventricular cavity was progressively dilated. At 1 week, the left ventricular dimensions at end-diastole and end-systole were increased in the myocardial infarction rats compared with
At 1 week after the operation, the levels of TBARS were similar between the two groups. At 4 weeks, the myocardial TBARS level was significantly elevated (72 ⫾ 5 nmol/g) in the myocardial infarction rats compared with that in the sham-operated rats (38 ⫾ 3 nmol/g, P ⬍ 0.05) (Fig. 2).
Fig. 1. Echocardiograms after the operation in sham-operated and myocardial infarction rats. The left ventricular cavity is progressively dilated after myocardial infarction
Fig. 2. Lipid peroxidation as indicated by the thiobarbituric acid reactive substances (TBARS) level, in sham-operated and myocardial infarction (MI) rats. At 1 week after the operation, levels of TBARS are similar between the two groups. At 4 weeks, the myocardial TBARS level is significantly elevated in the myocardial infarction rats compared with that in sham-operated rats
Northern blotting analysis for UCP-2 mRNA Although the levels of UCP-2 mRNA expression were similar between the sham-operated and myocardial infarction rats at 1 week, a significant increase in UCP-2 mRNA expression was exhibited in the myocardial infarction rats at 4 weeks (1.8-fold the level in the sham-operated rats) (Fig. 3).
Discussion The Frank–Starling mechanism is well known, hemodynamically, as a compensatory mechanism for reduced left
Fig. 3. Uncoupling protein 2 (UCP-2) mRNA expression in the noninfarct zone of the left ventricular wall after myocardial infarction. A significant increase in UCP-2 mRNA expression is exhibited at 4 weeks
ventricular function. In this process, left ventricular dilatation occurs in association with an elevation of the left ventricular end-diastolic pressure, and this was observed at the early stage in our myocardial infarction model rats. Myocardial lipid oxidation as a marker of ROS and expression of UCP-2 were not observed during the early stage. The myocardial energetic condition or efficiency in this zone remained unaltered for at least 1 week after the myocardial infarction. Dilatation of the left ventricle and reduction of the left ventricular function were accelerated without an elevation of the left ventricular end-diastolic pressure in the late stage after myocardial infarction. A previous report demonstrated impairment of the energy metabolism in the intact
residual myocardium after myocardial infarction. This may contribute to the reduction in creatine kinase flux in association with re-establishment of the fetal isozyme profile and disturbance of mitochondria membrane proteins, including adenine nucleotide translocator-1 and the βsubunit of F1-ATPase. A relationship between the expression of cardiac UCP-2 or UCP-3 and cardiac dysfunction in rats with aortic regurgitation or diabetes mellitus has been reported.7,8,14 In these conditions, UCP-2 gene expression was usually up-regulated, which indicated a reduction in the mitochondrial membrane potential and energy dissipation. The reduction in the mitochondrial membrane potential may precede the morphological changes seen in apoptotic or oncotic cell death.15 An increase in ROS in the residual myocardium after myocardial infarction has been reported previously. Although the source of the ROS production in the residual myocardium is not clear, the level of UCP-2 expression regulates the ROS production generated by mitochondrial electron transport.10 In previous reports, depletion of UCP-2 resulted in an increase in the ROS production in immune cells.16 In contrast, ROS increased UCP-2 gene expression and activated the uncoupling function in several tissues.17,18 Subsequently, UCP-2 expression was increased by ROS, but UCP-2 reduced ROS production. This self-regulation linkage of ROS and UCP-2 may produce a balanced condition for myocardial energy metabolism, which shifts to reduced energy production in the residual myocytes. In the present study, left ventricular dilatation, impairment of left ventricular function, and UCP-2 expression were simultaneously observed at 4 weeks after the myocardial infarction. This temporal relationship probably indicates a possible decompensatory mechanism for left ventricular function after a large myocardial infarction. Reduction of the myocardial energy metabolism through UCP-2 gene expression in the noninfarct zone may play a key role in left ventricular dilatation in the late stage after myocardial infarction. In conclusion, we have demonstrated that upregulation of UCP-2 gene expression occurs in the residual myocardium associated with ROS production at the late stage after myocardial infarction. In these experiments using myocardial infarction model rats, we have not confirmed the direct effects of ROS production and UCP-2 gene expression on contractile dysfunction. Additionally, we measured UCP-2 mRNA levels, but did not measure its protein level. Recently, Hoerter et al.19 demonstrated that UCP-1 activity is induced during ischemic reperfusion and is protective against myocardial damage in an ischemic region. Further studies are required for a clear explanation regarding the contribution of UCP-2 to the pathophysiological changes after myocardial infarction, in-cluding the possibility of UCP-2 having a protective role. We believe that the upregulation of UCP-2 expression may reduce the energy efficiency in the residual myocardium, and may play an important role in left ventricular remodeling.
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