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Pioglitazone ameliorates systolic and diastolic cardiac dysfunction in rat model of angiotensin II-induced hypertension
International Journal of Cardiology 167 (2013) 409–415
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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Pioglitazone ameliorates systolic and diastolic cardiac dysfunction in rat model of angiotensin II-induced hypertension Aiko Sakamoto, Makiko Hongo, Kyoko Furuta, Kan Saito, Ryozo Nagai, Nobukazu Ishizaka ⁎ Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, Japan
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Article history: Received 20 June 2011 Received in revised form 16 October 2011 Accepted 1 January 2012 Available online 23 January 2012 Keywords: Angiotensin II Lipid accumulation Peroxisome proliferator activated receptor Cardiac function
a b s t r a c t We previously showed that administration of angiotensin II to rats causes fibrosis and lipid accumulation in the heart. In the current study, we examined the effect of pioglitazone, an agonist of peroxisome proliferator activated receptor-γ, on angiotensin II-induced intracardiac lipid accumulation and cardiac dysfunction. Pioglitazone, given orally at a dose of 2.5 mg/kg/d, reduced cardiac triglyceride content and suppressed lipid deposition in the heart of angiotensin II-induced hypertensive rats without affecting angiotensin II-induced upregulation of lipogenic gene expression. Histological examination showed that pioglitazone reduced the area of cardiac fibrosis and iron deposition in the heart of angiotensin II-treated rats. Expression of an antioxidative molecule, heme oxygenase-1, was increased by angiotensin II infusion, and pioglitazone treatment preserved expression of HO-1. Angiotensin II increased the superoxide signals detected by dihydroethidium staining in myocardial cells with lipid deposition, and this increase was suppressed by pioglitazone. Cardiac function was analyzed in an ex vivo isolated cardiac perfusion system. It was found that pioglitazone improved both the systolic and diastolic cardiac performance, which was weakened by angiotensin II infusion, after transient ischemia and reperfusion. These findings collectively suggest that pioglitazone treatment ameliorated the histological and functional cardiac damage induced by angiotensin II infusion, the mechanism of which may be related to the antioxidative action of pioglitazone. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Several different mechanisms are considered to underlie the functional and histological cardiac damage caused by activation of the renin–angiotensin system (RAS) [1]; these include development of myocardial hypertrophy, interstitial fibrosis [2], enhancement of oxidative stress [3], and activation of inflammatory signaling pathways [4]. Excessive accumulation of triglycerides may occur in various non-adipose tissues, and can cause tissue damage, termed lipotoxicity [5,6]. Accumulation of lipids in the myocardium is known to occur in the conditions of pressure overload, metabolic syndrome [7], and diabetes, which may enhance cardiac damage [8]. The finding that inhibition of RAS reduced myocardial lipid deposition and improved cardiac function in a diabetic animal model [9] suggested that lipotoxicity may be another mechanism underlying the cardiac damage exerted by activated RAS. In a recent study, we demonstrated that administration of angiotensin II (Ang II) caused accumulation of lipids in the heart and kidney in rat model [10,11]. Ang II-induced cardiac and renal accumulation of lipids was, in part, a pressor-independent phenomenon, and was accompanied by the altered expression of lipid ⁎ Corresponding author at: Internal Medicine III, Division of Cardiology, Osaka Medical College, Faculty of Medicine, Takatsuki-shi Daigaku-machi 2-7, Osaka 569-8686, Japan. Tel.: + 81 72 683 1221; fax: + 81 72 684 6533. E-mail address: [email protected] (N. Ishizaka). 0167-5273/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2012.01.007
metabolism-related genes, such as sterol-regulatory element binding protein (SREBP)-1c, peroxisome proliferator activated receptor-γ (PPARγ), and fatty acid synthase (FAS). Several previous studies have shown that PPARγ agonists may act favorably in suppressing excess lipid accumulation [12] and preserving cardiac function in conditions that occur in response to certain noxious stimuli, such as myocardial ischemia [13,14] and abnormal glucose metabolism [15]. To this end, we here investigated whether administration of a PPARγ agonist, pioglitazone (Pio), would reduce the cardiac lipid content and suppress the cardiac injury induced by Ang II. Because increased superoxide production was demonstrated at the site of lipid deposition, we also examined whether the extent of superoxide production would be reduced by Pio in Ang II-treated animals.
2. Materials and methods 2.1. Animal models The experiments were performed in accordance with the Guidelines for Animal Experimentation approved by the Animal Center for Biomedical Research, Faculty of Medicine, University of Tokyo. Ang II-induced hypertension was induced in male Sprague–Dawley rats (250 to 300 g) by subcutaneous implantation of an osmotic minipump (Alza Pharmaceutical) as described previously [16]. Briefly, Val5-Ang II (Sigma Chemical) was infused at doses of 0.7 mg/kg/day via a subcutaneously implanted osmotic minipump that was continued for 7 days unless stated otherwise. In some
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A
B
Variables
Control
Pio
Ang II
Ang II + Pio
Weight Systolic BP (mm Hg) Total cholesterol (mg/dL) Triglycerides (mg/dL) Free fatty acids (μEq/L) Plasma fasting Glucose (mg/dL) Serum fasting insulin (mg/dL)
328 ± 3 112 ± 5 52 ± 2 24 ± 2 486 ± 27 146 ± 5 1.1 ± 0.3
326 ± 2 117 ± 2 48 ± 3 25 ± 2 487 ± 45 151 ± 8 1.7 ± 0.5
265 ± 4† 202 ± 7† 62 ± 4⁎ 32 ± 2⁎ 674 ± 30† 186 ± 8† 1.4 ± 0.2
255 ± 5† 194 ± 7† 54 ± 4 24 ± 2 688 ± 43† 159 ± 8 1.8 ± 0.5
Pio and Ang II indicate pioglitazone and angiotensin II, respectively. Number of samples was ≥ 7 in each group. ⁎ P b 0.05 versus untreated control. † P b 0.01 versus untreated control.
Tissue TG (µg/mg)
P<0.05
P<0.05
2 1 0
2.2. Measurement of lipid content in serum and the heart Serum levels of total cholesterol (TC) and triglycerides (TG), and nonesterified fatty acid (NEFA) were measured by enzymatic methods [10]. The amount of TG and TC in the heart tissue was measured in homogenated extracts by enzymatic colorimetric determination using the Triglyceride-E Test, Cholesterol-E Test, and Free cholesterol-E Test Wako, respectively (Wako Pure Chemicals). 2.3. Langendorff system and global ischemia and subsequent reperfusion Cardiac function of the excised heart was investigated in rats treated with Ang II for 14 days, because neither systolic nor diastolic cardiac function was found to be significantly reduced by 7 days of Ang II treatment (data not shown). The excised heart was put into ice-cold modified Krebs buffer and quickly perfused in the Langendorff apparatus (ADInstruments) at a constant perfusion pressure of 100 cm H2O at 37 °C with modified Krebs–Henseleit buffer solution (NaCl 118.5 mmol/L, KCl 4.8 mmol/L, KH2PO4 1.2 mmol/L, MgSO4 1.2 mmol/L, CaCl2 2.5 mmol/L, NaHCO3 25.0 mmol/L, and glucose 11 mmol/L, pH 7.4) that was continuously aerated with 95% O2 + 5% CO2. Systolic (+dP/dt) and diastolic (− dP/dt) left ventricular function was monitored and recorded continuously using a fluid-filled left ventricular balloon in line with a transducer (Powerlab, ADInstruments). The balloon volume was set to produce a left ventricular end diastolic pressure (LVEDP) of 5 mm Hg. Each heart was perfused for 15 min (control perfusion) and then subjected to 30 min of global ischemia by stopping the circulation, which was then followed by reperfusion for 60 min. 2.4. Histological analysis Oil red O staining was performed on sections of unfixed, freshly frozen heart samples (3 μm in thickness). The areas of lipid deposition were calculated by using the image analysis software, Photoshop (Adobe), and semiquantification of the lipid deposition was performed as described elsewhere [17]. Prussian blue staining was used to detect iron deposition within the cardiac tissue. The fibrosis area was calculated in the heart of rats given Ang II for 14 days. For quantification of the fibrous areas, heart sections subjected to Mallory-Azan staining were photographed and digitalized, and the number of pixels of blue-color was counted by using a photoimaging system. The
1
0
ratio of the area affected by fibrosis to the total cardiac area in each sample is expressed as the percentage of fibrosis [18]. Staining with the oxidative fluorescent dye dihydroethidium (DHE) was performed as described previously [10]. Images were obtained with a fluorescent microscope BX51 (Olympus, Tokyo, Japan), and the fluorescence intensity, which was determined from at least five fields for each section, is presented as the percentage of that of untreated controls.
2.5. Western blot analysis Western blot analysis was performed as described previously [19]. Antibodies against total and phosphorylated forms of AMP-activated protein kinase (Cell Signaling Technology, Danvers, MA), and total and phosphorylated forms of acetyl-CoA carboxylase (ACC) (Cell Signaling Technology) were used at a dilution of 1/1000. Polyclonal antibodies against rat ferritin (Panapharm, Kumamoto, Japan) and heme oxygenase1 (HO-1, StressGen, Victoria, BC, Canada), and monoclonal antibody against β-actin (Sigma) were used at dilutions of 1/1000, 1/2000 and 1/1000. The ECL Western blotting system (Amersham Life Sciences, Arlington Heights, IL) was used for detection. Bands were visualized by a lumino-analyzer (Fuji Photo Film, Tokyo, Japan). Band intensity was calculated and is expressed as a percentage of the control value.
2.6. Real time reverse transcription-polymerase chain reaction (RT-PCR) Expression of mRNA of lipid metabolism-related genes was analyzed by real time quantitative PCR performed by a LightCycler coupled with hybriprobe technology (Roche Diagnostics). Expression of each target gene was normalized to that of an endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The target genes encoded the following proteins: SREBP-1c, fatty acid synthase (FAS), carnitine palmitoyltransferase (CPT)-1 PPARγ, and Nox1. The forward and backward primers used have been described elsewhere [11].
2.7. Statistical analysis Data are expressed as the mean ± SEM. We used ANOVA followed by a multiple comparison test to compare raw data, before expressing the results as a percentage of the control value using the statistical analysis software SPSS Dr II. A value of p b 0.05 was considered to be statistically significant.
C
D
Oil red O-stained area (fold increase)
E B
P<0.05
2
Fig. 2. Tissue content of lipids. Content of triglycerides (TG) (A) and total cholesterol (TC) (B) in the heart. Data represent the mean ± SEM of the results from 5 to 7 rats in each group.
angiotensin II-infused rats, Pio (Takeda Pharmaceutical Co., Tokyo, Japan) was given orally at a dose of 2.5 mg/kg/day. These treatments were continued until sacrifice.
A
P<0.05
3
Tissue TC (µg/mg)
Table 1 Baseline conditions.
P<0.001
6
P<0.001 P<0.001
5 4 3 2 1 0
Fig. 1. Oil red O staining of the heart of rats given angiotensin II with or without pioglitazone administration. Shown are heart sections from a control rat (A), a rat given pioglitazone (Pio) (B), a rat given angiotensin II (Ang II) (C), and a rat given Ang II plus Pio (D). Original magnification, × 200. Scale bar indicates 100 μm. E. Semiquantification of the oil red O-stained area. Data represent the mean ± SEM of the results from 4 to 5 rats in each group.
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A B
C
150
P-AMPK (%)
AMPK (%)
B
411
100 50 0
*
150 100 50 0
E
D
* *
P-ACC (%)
ACC (%)
200 100 0
*
200 100 0
Fig. 3. Western blot analysis of AMP-activated protein kinase α (AMPK), acetyl-CoA carboxylase (ACC), the phosphorylated (activated) form of AMPKα (P-AMPK), and the phosphorylated (inactivated) form of ACC (P-ACC). A. Representative Western blots. B, C, D, E. Protein expression in treated rats relative to control rats. Data represent the mean ± SEM of the results from 4 to 5 rats in each group. Abbreviations are the same as in Fig. 2.
3. Results 3.1. Characteristics of experimental animals Ang II significantly elevated blood pressure (Table 1). On the other hand, Pio, administered either alone or concomitantly with Ang II, did not significantly alter the blood pressure level. Pio reduced serum TG and TC levels in Ang II-treated and untreated rats. On the other hand, Pio did not significantly reduce the levels of serum free fatty acids, which was increased by Ang II treatment. 3.2. Accumulation of lipids in the heart Oil red O staining of heart sections showed no apparent lipid deposition in the heart of untreated rats and rats treated with Pio alone (Fig. 1A, B). Accumulation of oil red O-stainable lipid was observed in the myocardium as well as in the arterial wall, of Ang
A
B
II-infused rats (Fig. 1C), and the extent of lipid deposition was substantially reduced in heart sections from rats treated with both Ang II and Pio (Fig. 1D, E). Tissue content of TG was found to be increased in the heart of Ang II-infused rats, and this increase was suppressed by Pio (Fig. 2). 3.3. Regulation of genes related to lipid metabolism The increase in mRNA expression of SREBP-1c and FAS in the heart of rats receiving Ang II infusion [11] was not inhibited by Pio. Pio suppressed the Ang II-induced upregulation of PPAR-γ, but not CPT-1. Ang II increased the amount of phospho-AMPKα, an active form of AMPKα, but not total AMPKα, and this increase in phospho-AMPKα was decreased by Pio treatment (Fig. 3). Similarly, Ang II increased the amount of the phosphorylated form of ACC, and this increase was suppressed by Pio, although Pio did not reduce the amount of total ACC protein in the heart of Ang II-infused rats.
C
G P=0.001 P<0.001 P<0.001
Control
D
Ang II
Pio
E
F
DHE signal intensity (% control)
300
200
100
0
Ang II+Pio
Ang II
Ang II
Fig. 4. Superoxide and lipid content in heart sections. Heart sections from a control rat (A), a rat treated with pioglitazone (Pio) (B), a rat treated with angiotensin (Ang) II (C, E, F) and a rat treated with both Ang II and Pio (D) are shown. A–D, F. Dihydroethidium (DHE) staining. E. Oil red O staining. E and F are serial sections. DHE signals were increased in the myocardium and vascular cells (arrowhead) of the heart from the Ang II-infused rat (C). Intense DHE staining can be observed in the area of the increased lipid deposits (E, F). Original magnification, × 200 (A–D), × 400 (E, F). Scale bars indicate 100 μm. G. Intensity of superoxide signals in treated rats relative to control rats. Data represent the mean ± SEM of the results from 4 to 5 rats in each group.
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(Fig. 6). On the other hand, Pio treatment in conjunction with Ang II infusion increased LVDP to a greater extent than in untreated control rats.
3.4. Superoxide production in the heart As compared to untreated controls, DHE staining-positive signals were increased by Ang II. In the heart of Ang II-infused rats, increased DHE signals were observed in the vessel wall and at the site of lipid deposition, as has been demonstrated previously [11]. Pioglitazone significantly reduced the superoxide signals. Ang II-induced upregulation of mRNA expression of Nox1, a component of NADPH oxidase, was found to be suppressed by Pio treatment (control 100 ± 10% [n = 7]; Ang II 473 ± 209% [n = 6], P b 0.05 versus control; Ang II ± Pio 39 ± 7% [n = 6], NS versus control, P b 0.05 versus Ang II).
3.7. Effect of Pio treatment on Ang II-induced cardiac fibrosis Ang II infusion significantly increased the area affected be fibrosis in both the right and the left ventricle (Fig. 7). Treatment of rats with Pio significantly suppressed the fibrotic effects of Ang II. 4. Discussion
3.5. Tissue iron deposition and expression of ferritin and HO-1 protein
Here we demonstrated that Pio reduced the increase in lipid content caused by Ang II infusion. In addition, it was found that Pio reduced the extent of cardiac fibrosis induced by Ang II and improved systolic and diastolic performance during the reperfusion period after the induction of 30-min global ischemia in the isolated perfused heart from Ang II-infused rats. As has been shown in a previous study [10], Ang II treatment upregulates the expression of lipogenic genes, SREBP-1c and FAS, in the heart. In the current study, however, Pio did not significantly alter the expression of lipogenic genes, although it reduced cardiac lipid content (Table 2). In addition, Pio also reduced the phosphorylated, thus activated, form of AMPK, and this might rather act to enhance intramyocardial lipid accumulation [21]. Therefore, it is possible that inhibition of lipid accumulation by Pio may be a direct consequence of its lipid lowering properties. This notion may be consistent with a recent report showing that PPARγ activation may not modulate the expression of genes involved in cardiac lipid metabolism [22]. Previous studies have shown that increased oxidative stress lowers cardiac contractility and/or promotes cardiac fibrosis. Enhanced production of reactive oxygen species (ROS) by the NADPH oxidase system is considered to play a role in the development of cardiomyocyte hypertrophy, interstitial fibrosis and the genesis of contractile dysfunction [23]. Overload of iron, which is associated
Ang II infusion caused iron deposition in the heart (Fig. 4), as has been described previously [20], and this deposition was suppressed by Pio. Western blot analysis showed that the upregulation of ferritin protein induced by Ang II was decreased by Pio. On the other hand, Pio did not significantly alter the upregulation of HO-1 expression that had been induced by Ang II. 3.6. Cardiac functions The mean left ventricular cardiac contractility (+ dP/dt) and rate of relaxation (− dP/dt) in rats treated with Ang II with or without Pio is shown in Fig. 5. Application of global ischemia for 30 min caused an immediate and marked decrease in both +dP/dt and −dP/dt. After reperfusion, +dP/dt was lower in the heart of Ang II-infused rats as compared with control rats; however, concomitant Pio treatment significantly improved the recovery in + dP/dt after reperfusion (Fig. 5A). Similarly, − dP/dt was found to be lower after reperfusion in the Ang II group, but Pio treatment recovered this parameter to the levels found in the control group (Fig. 5B). No significant difference was observed in left ventricle developed pressure (LVDP) between the untreated and Ang II-infused rat groups
A
E
B
C
D
F
G
P<0.01
P<0.05
150 100 50 0
P<0.01 P<0.05
P<0.05
Ferritin (%)
HO-1 (%)
200
200 100 0
Fig. 5. Iron deposition and expression of heme oxygenase-1 (HO-1) and ferritin protein. A–D. Prussian blue staining. Shown are heart sections from a control rat (A), a rat given pioglitazone (Pio) (B), a rat given angiotensin II (Ang II) (C), and a rat given Ang II plus Pio (D). Original magnification, × 200. Scale bar indicates 100 μm. E. Representative Western blots of HO-1 and ferritin. F, G. HO-1 and ferritin expression in treated rats relative to control rats. Data represent the mean ± SEM of the results from 4 to 6 rats in each group. *P b 0.05 versus control group.
A. Sakamoto et al. / International Journal of Cardiology 167 (2013) 409–415
A
global ischemia
4000
* * * * * * * * * * *
+dp/dt
3000
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*
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* * * * * * * * * *
2000
*
Control Ang II Ang II+Pio
1000 0 -10
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Time (min)
B
global ischemia
2500
-dp/dt
2000
*
† †
1500
* * * * * * * * * *
* * * * * * * * * *
*
1000
Control Ang II Ang II+Pio
500 0 -10
0 0
10
20
30
40
50
60
Time (min)
C LVDP (mmHg)
200
global ischemia
†
150
*
† † † † † † † †
100 Control Ang II Ang II+Pio
50 0 -10
0 0
10
20
30
40
50
60
Time (min) Fig. 6. Cardiac contractile and relaxation function and the left ventricle developed pressure (LVDP) of the isolated heart subjected to global ischemia and subsequent reperfusion. A, B. As compared with untreated control rats, both the maximum rate of rise (+dP/dt, A) and the maximum rate of drop (− dP/dt, B) in the developed pressure subjected to 30 min of global ischemia followed by reperfusion were reduced in the angiotensin II group (Ang II), and this reduction was recovered by pioglitazone treatment (Ang II + Pio). C. LVDP was not significantly different between the Angiotensin II group (Ang II, n = 9) and the untreated control group (n = 5). On the other hand, administration of pioglitazone to Ang II-infused rats (Ang II + Pio, n = 3) significantly increased LVDP. Data represent the mean ± SEM of the results from 5 to 9 rats in each group.*P b 0.01 and † P b 0.05 versus Ang II group.
with increased free radical production and elevated oxidative stress [24], worsens the cardiac fibrosis [20] and, conversely, chelation of iron suppresses the development of cardiac fibrosis and postischemic contractile dysfunction [25,26]. Furthermore, induction of antioxidative molecules, such as HO-1 [27] and superoxide dismutase [28], was shown to be effective in preventing myocardial injury following myocardial ischemia and conversely, myocardial ischemia/reperfusion injury in diabetic animals was exacerbated in the absence of HO-1 [29]. Several studies suggest that Pio may act to protect the heart by its antioxidative properties. Nakamura et al. showed that Pio downregulated the expression of NADPH components and reduced ROS production in the heart, which led to the protection of hypertensive cardiovascular damage [30]. In addition,
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Xu et al. showed that Pio decreased the expression of NADPH components in the rat heart and reduced age-related atrial fibrillation [31]. A recent study showed, albeit in the brain, that Pio acts protectively against iron-induced oxidative injury via its antiinflammatory action [32]. Several observations in the current study suggest that Pio reduced the extent of oxidative stress in the heart of Ang II-infused rats. Pio decreased both superoxide signals and iron deposition in the heart. In addition, Pio reduced the upregulation in Nox1 mRNA expression induced by Ang II. Of note, despite the presumed reduction in oxidative stress levels, Pio preserved the upregulation in HO-1 expression. Mersmann similarly observed that PPARγ agonist treatment reduced the infarct size of heart after ischemia/ reperfusion, although it preserved the upregulation of cardiac HO-1 expression induced by ischemia/reperfusion [33]. Induction of HO-1 expression observed by certain drugs, such as statins [34] and aspirin [35], is suggested to confer a cardioprotective effect. Therefore, the possibility exists that preservation, or upregulation, of HO-1 expression might represent Pio-induced protection from cardiac injury. This possibility might be further supported by the findings of Krönke et al. that PPARγ agonists transcriptionally upregulate HO-1 mRNA expression in vascular cells via a PPAR-responsive element [36]. Besides the possible antioxidative action of Pio, involvement of other cardioprotective mechanisms, such as PI3K/AKT activation [37] and mitochondrial KATP channel opening [38], in the Pio-induced suppression of Ang II-induced cardiac injury should be investigated in future studies. In the current study, Pio significantly improved systolic and diastolic cardiac function as well as developed pressure in the isolated heart from Ang II-infused rats. It has been demonstrated that by inducing the accumulation of salt and water, PPARγ agonists may worsen the extent of heart failure [39]. On the other hand, Reeba et al. reported that, in a mouse model of lipotoxic cardiomyopathy, rosiglitazone, which lowered both circulating and intra-cardiac triglyceride levels, decreased the expression of genes that reflect cardiac dysfunction [40]. In addition, Golfman et al. showed, in an animal model of insulin resistant diabetes, that PPARγ agonist treatment decreased myocardial lipid deposition and cardiac power in the isolated heart [41]. These observations suggest that the effects of a PPARγ agonist on cardiac function may differ depending on the circulating lipid levels as well as on whether cardiac function is tested in an in vivo or ex vivo system. A recent study also suggested that the extent of water retention may differ according to the PPARγ agonistic drugs that are used and their dosage [42]. In conclusion, Pio treatment reduced cardiac triglyceride content without affecting lipogenic gene expression, suppressed the development of cardiac fibrosis, and restored the systolic and diastolic dysfunction induced by Ang II infusion. These favorable actions of Pio were accompanied by a reduction of ROS and expression of an NADPH oxidase component, and by preservation of the antioxidative gene, HO-1. The causal or resultant relationship between the antioxidative and cardioprotective actions of Pio should be investigated in further studies.
Acknowledgments This work was supported by Grants in Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (grant 19590937) and Grant from the Takeda Science Foundation, the Sankyo Foundation of Life Science, Okinaka Memorial Institute for Medical Research, and Daiwa Securities Health Foundation. The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology.
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E
P<0.05
A
B
C
D
Fibrosis area (%)
20
P<0.05
P<0.05 P<0.05
15 10 5 0
Fig. 7. Effect of pioglitazone on angiotensin II-induced cardiac fibrosis. A–D. Mallory-Azan staining. Shown heart sections are from a control rat (A), a rat given pioglitazone (Pio) (B), a rat given angiotensin II (Ang II) (C), and a rat given Ang II plus Pio (D). Scale bar indicates 100 μm. E. Fibrotic area in the right (green bars) and left (orange bars) ventricles estimated by Mallory-Azan-staining. Data represent the mean ± SEM of the results from 4 to 6 rats in each group.
Table 2 Regulation of mRNA expression. Variables
Control
Pio
Ang II
Ang II + Pio
n SREBP-1 FAS CPT-1 PPAR-r
16 100 ± 4 100 ± 13 100 ± 11 100 ± 6
15 142 ± 30 98 ± 17 114 ± 14 83 ± 10
20 199 ± 13⁎ 184 ± 16⁎ 163 ± 37 181 ± 59⁎
16 252 ± 48† 230 ± 39⁎ 155 ± 26 108 ± 12
Pio and Ang II indicate pioglitazone and angiotensin II, respectively. ⁎ P b 0.05 versus untreated control. † P b 0.01 versus untreated control.
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[17] Ishizaka N, Matsuzaki G, Saito K, et al. Expression and localization of PDGF-B, PDGF-D, and PDGF receptor in the kidney of angiotensin II-infused rat. Lab Invest 2006;86:1285–92. [18] Ishizaka N, Aizawa T, Mori I, et al. Heme oxygenase-1 is upregulated in the rat heart in response to chronic administration of angiotensin II. Am J Physiol Heart Circ Physiol 2000;279:H672–8. [19] Aizawa T, Ishizaka N, Taguchi J, et al. Heme oxygenase-1 is upregulated in the kidney of angiotensin II-induced hypertensive rats : possible role in renoprotection. Hypertension 2000;35:800–6. [20] Ishizaka N, Saito K, Mitani H, et al. Iron overload augments angiotensin II-induced cardiac fibrosis and promotes neointima formation. Circulation 2002;106:1840–6. [21] Axelsen LN, Lademann JB, Petersen JS, et al. Cardiac and metabolic changes in long-term high fructose-fat fed rats with severe obesity and extensive intramyocardial lipid accumulation. Am J Physiol Regul Integr Comp Physiol 2010;298: R1560–70. [22] Gilde AJ, van der Lee KA, Willemsen PH, et al. Peroxisome proliferator-activated receptor (PPAR) alpha and PPARbeta/delta, but not PPARgamma, modulate the expression of genes involved in cardiac lipid metabolism. Circ Res 2003;92: 518–24. [23] Nabeebaccus A, Zhang M, Shah AM. NADPH oxidases and cardiac remodelling. Heart Fail Rev 2011;16:5–12. [24] Oudit GY, Trivieri MG, Khaper N, et al. Taurine supplementation reduces oxidative stress and improves cardiovascular function in an iron-overload murine model. Circulation 2004;109:1877–85. [25] van der Kraaij AM, van Eijk HG, Koster JF. Prevention of postischemic cardiac injury by the orally active iron chelator 1,2-dimethyl-3-hydroxy-4-pyridone (L1) and the antioxidant (+)-cyanidanol-3. Circulation 1989;80:158–64. [26] Pucheu S, Coudray C, Tresallet N, et al. Effect of iron overload in the isolated ischemic and reperfused rat heart. Cardiovasc Drugs Ther 1993;7:701–11. [27] L'Abbate A, Neglia D, Vecoli C, et al. Beneficial effect of heme oxygenase-1 expression on myocardial ischemia–reperfusion involves an increase in adiponectin in mildly diabetic rats. Am J Physiol Heart Circ Physiol 2007;293: H3532–41. [28] Hangaishi M, Nakajima H, Taguchi J, et al. Lecithinized Cu, Zn-superoxide dismutase limits the infarct size following ischemia–reperfusion injury in rat hearts in vivo. Biochem Biophys Res Commun 2001;285:1220–5. [29] Liu X, Wei J, Peng DH, et al. Absence of heme oxygenase-1 exacerbates myocardial ischemia/reperfusion injury in diabetic mice. Diabetes 2005;54:778–84. [30] Nakamura T, Yamamoto E, Kataoka K, et al. Beneficial effects of pioglitazone on hypertensive cardiovascular injury are enhanced by combination with candesartan. Hypertension 2008;51:296–301. [31] Xu D, Murakoshi N. IgarashiM, et al. PPAR-gamma activator pioglitazone prevents age-related atrial fibrillation susceptibility by improving antioxidant capacity and reducing apoptosis in a rat model. J Cardiovasc Electrophysiol 2012 Feb;23(2):209–17. [32] Yu HC, Feng SF, Chao PL, et al. Anti-inflammatory effects of pioglitazone on iron-induced oxidative injury in the nigrostriatal dopaminergic system. Neuropathol Appl Neurobiol 2010;36:612–22. [33] Mersmann J, Tran N, Zacharowski PA, et al. Rosiglitazone is cardioprotective in a murine model of myocardial I/R. Shock 2008;30:64–8. [34] Hsu M, Muchova L, Morioka I, et al. Tissue-specific effects of statins on the expression of heme oxygenase-1 in vivo. Biochem Biophys Res Commun 2006;343:738–44. [35] Grosser N, Abate A, Oberle S, et al. Heme oxygenase-1 induction may explain the antioxidant profile of aspirin. Biochem Biophys Res Commun 2003;308:956–60. [36] Kronke G, Kadl A, Ikonomu E, et al. Expression of heme oxygenase-1 in human vascular cells is regulated by peroxisome proliferator-activated receptors. Arterioscler Thromb Vasc Biol 2007;27:1276–82. [37] Wynne AM, Mocanu MM, Yellon DM. Pioglitazone mimics preconditioning in the isolated perfused rat heart: a role for the prosurvival kinases PI3K and P42/44MAPK. J Cardiovasc Pharmacol 2005;46:817–22.
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Contents lists available at SciVerse ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Pioglitazone ameliorates systolic and diastolic cardiac dysfunction in rat model of angiotensin II-induced hypertension Aiko Sakamoto, Makiko Hongo, Kyoko Furuta, Kan Saito, Ryozo Nagai, Nobukazu Ishizaka ⁎ Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, Japan
a r t i c l e
i n f o
Article history: Received 20 June 2011 Received in revised form 16 October 2011 Accepted 1 January 2012 Available online 23 January 2012 Keywords: Angiotensin II Lipid accumulation Peroxisome proliferator activated receptor Cardiac function
a b s t r a c t We previously showed that administration of angiotensin II to rats causes fibrosis and lipid accumulation in the heart. In the current study, we examined the effect of pioglitazone, an agonist of peroxisome proliferator activated receptor-γ, on angiotensin II-induced intracardiac lipid accumulation and cardiac dysfunction. Pioglitazone, given orally at a dose of 2.5 mg/kg/d, reduced cardiac triglyceride content and suppressed lipid deposition in the heart of angiotensin II-induced hypertensive rats without affecting angiotensin II-induced upregulation of lipogenic gene expression. Histological examination showed that pioglitazone reduced the area of cardiac fibrosis and iron deposition in the heart of angiotensin II-treated rats. Expression of an antioxidative molecule, heme oxygenase-1, was increased by angiotensin II infusion, and pioglitazone treatment preserved expression of HO-1. Angiotensin II increased the superoxide signals detected by dihydroethidium staining in myocardial cells with lipid deposition, and this increase was suppressed by pioglitazone. Cardiac function was analyzed in an ex vivo isolated cardiac perfusion system. It was found that pioglitazone improved both the systolic and diastolic cardiac performance, which was weakened by angiotensin II infusion, after transient ischemia and reperfusion. These findings collectively suggest that pioglitazone treatment ameliorated the histological and functional cardiac damage induced by angiotensin II infusion, the mechanism of which may be related to the antioxidative action of pioglitazone. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Several different mechanisms are considered to underlie the functional and histological cardiac damage caused by activation of the renin–angiotensin system (RAS) [1]; these include development of myocardial hypertrophy, interstitial fibrosis [2], enhancement of oxidative stress [3], and activation of inflammatory signaling pathways [4]. Excessive accumulation of triglycerides may occur in various non-adipose tissues, and can cause tissue damage, termed lipotoxicity [5,6]. Accumulation of lipids in the myocardium is known to occur in the conditions of pressure overload, metabolic syndrome [7], and diabetes, which may enhance cardiac damage [8]. The finding that inhibition of RAS reduced myocardial lipid deposition and improved cardiac function in a diabetic animal model [9] suggested that lipotoxicity may be another mechanism underlying the cardiac damage exerted by activated RAS. In a recent study, we demonstrated that administration of angiotensin II (Ang II) caused accumulation of lipids in the heart and kidney in rat model [10,11]. Ang II-induced cardiac and renal accumulation of lipids was, in part, a pressor-independent phenomenon, and was accompanied by the altered expression of lipid ⁎ Corresponding author at: Internal Medicine III, Division of Cardiology, Osaka Medical College, Faculty of Medicine, Takatsuki-shi Daigaku-machi 2-7, Osaka 569-8686, Japan. Tel.: + 81 72 683 1221; fax: + 81 72 684 6533. E-mail address: [email protected] (N. Ishizaka). 0167-5273/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2012.01.007
metabolism-related genes, such as sterol-regulatory element binding protein (SREBP)-1c, peroxisome proliferator activated receptor-γ (PPARγ), and fatty acid synthase (FAS). Several previous studies have shown that PPARγ agonists may act favorably in suppressing excess lipid accumulation [12] and preserving cardiac function in conditions that occur in response to certain noxious stimuli, such as myocardial ischemia [13,14] and abnormal glucose metabolism [15]. To this end, we here investigated whether administration of a PPARγ agonist, pioglitazone (Pio), would reduce the cardiac lipid content and suppress the cardiac injury induced by Ang II. Because increased superoxide production was demonstrated at the site of lipid deposition, we also examined whether the extent of superoxide production would be reduced by Pio in Ang II-treated animals.
2. Materials and methods 2.1. Animal models The experiments were performed in accordance with the Guidelines for Animal Experimentation approved by the Animal Center for Biomedical Research, Faculty of Medicine, University of Tokyo. Ang II-induced hypertension was induced in male Sprague–Dawley rats (250 to 300 g) by subcutaneous implantation of an osmotic minipump (Alza Pharmaceutical) as described previously [16]. Briefly, Val5-Ang II (Sigma Chemical) was infused at doses of 0.7 mg/kg/day via a subcutaneously implanted osmotic minipump that was continued for 7 days unless stated otherwise. In some
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Weight Systolic BP (mm Hg) Total cholesterol (mg/dL) Triglycerides (mg/dL) Free fatty acids (μEq/L) Plasma fasting Glucose (mg/dL) Serum fasting insulin (mg/dL)
328 ± 3 112 ± 5 52 ± 2 24 ± 2 486 ± 27 146 ± 5 1.1 ± 0.3
326 ± 2 117 ± 2 48 ± 3 25 ± 2 487 ± 45 151 ± 8 1.7 ± 0.5
265 ± 4† 202 ± 7† 62 ± 4⁎ 32 ± 2⁎ 674 ± 30† 186 ± 8† 1.4 ± 0.2
255 ± 5† 194 ± 7† 54 ± 4 24 ± 2 688 ± 43† 159 ± 8 1.8 ± 0.5
Pio and Ang II indicate pioglitazone and angiotensin II, respectively. Number of samples was ≥ 7 in each group. ⁎ P b 0.05 versus untreated control. † P b 0.01 versus untreated control.
Tissue TG (µg/mg)
P<0.05
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2.2. Measurement of lipid content in serum and the heart Serum levels of total cholesterol (TC) and triglycerides (TG), and nonesterified fatty acid (NEFA) were measured by enzymatic methods [10]. The amount of TG and TC in the heart tissue was measured in homogenated extracts by enzymatic colorimetric determination using the Triglyceride-E Test, Cholesterol-E Test, and Free cholesterol-E Test Wako, respectively (Wako Pure Chemicals). 2.3. Langendorff system and global ischemia and subsequent reperfusion Cardiac function of the excised heart was investigated in rats treated with Ang II for 14 days, because neither systolic nor diastolic cardiac function was found to be significantly reduced by 7 days of Ang II treatment (data not shown). The excised heart was put into ice-cold modified Krebs buffer and quickly perfused in the Langendorff apparatus (ADInstruments) at a constant perfusion pressure of 100 cm H2O at 37 °C with modified Krebs–Henseleit buffer solution (NaCl 118.5 mmol/L, KCl 4.8 mmol/L, KH2PO4 1.2 mmol/L, MgSO4 1.2 mmol/L, CaCl2 2.5 mmol/L, NaHCO3 25.0 mmol/L, and glucose 11 mmol/L, pH 7.4) that was continuously aerated with 95% O2 + 5% CO2. Systolic (+dP/dt) and diastolic (− dP/dt) left ventricular function was monitored and recorded continuously using a fluid-filled left ventricular balloon in line with a transducer (Powerlab, ADInstruments). The balloon volume was set to produce a left ventricular end diastolic pressure (LVEDP) of 5 mm Hg. Each heart was perfused for 15 min (control perfusion) and then subjected to 30 min of global ischemia by stopping the circulation, which was then followed by reperfusion for 60 min. 2.4. Histological analysis Oil red O staining was performed on sections of unfixed, freshly frozen heart samples (3 μm in thickness). The areas of lipid deposition were calculated by using the image analysis software, Photoshop (Adobe), and semiquantification of the lipid deposition was performed as described elsewhere [17]. Prussian blue staining was used to detect iron deposition within the cardiac tissue. The fibrosis area was calculated in the heart of rats given Ang II for 14 days. For quantification of the fibrous areas, heart sections subjected to Mallory-Azan staining were photographed and digitalized, and the number of pixels of blue-color was counted by using a photoimaging system. The
1
0
ratio of the area affected by fibrosis to the total cardiac area in each sample is expressed as the percentage of fibrosis [18]. Staining with the oxidative fluorescent dye dihydroethidium (DHE) was performed as described previously [10]. Images were obtained with a fluorescent microscope BX51 (Olympus, Tokyo, Japan), and the fluorescence intensity, which was determined from at least five fields for each section, is presented as the percentage of that of untreated controls.
2.5. Western blot analysis Western blot analysis was performed as described previously [19]. Antibodies against total and phosphorylated forms of AMP-activated protein kinase (Cell Signaling Technology, Danvers, MA), and total and phosphorylated forms of acetyl-CoA carboxylase (ACC) (Cell Signaling Technology) were used at a dilution of 1/1000. Polyclonal antibodies against rat ferritin (Panapharm, Kumamoto, Japan) and heme oxygenase1 (HO-1, StressGen, Victoria, BC, Canada), and monoclonal antibody against β-actin (Sigma) were used at dilutions of 1/1000, 1/2000 and 1/1000. The ECL Western blotting system (Amersham Life Sciences, Arlington Heights, IL) was used for detection. Bands were visualized by a lumino-analyzer (Fuji Photo Film, Tokyo, Japan). Band intensity was calculated and is expressed as a percentage of the control value.
2.6. Real time reverse transcription-polymerase chain reaction (RT-PCR) Expression of mRNA of lipid metabolism-related genes was analyzed by real time quantitative PCR performed by a LightCycler coupled with hybriprobe technology (Roche Diagnostics). Expression of each target gene was normalized to that of an endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The target genes encoded the following proteins: SREBP-1c, fatty acid synthase (FAS), carnitine palmitoyltransferase (CPT)-1 PPARγ, and Nox1. The forward and backward primers used have been described elsewhere [11].
2.7. Statistical analysis Data are expressed as the mean ± SEM. We used ANOVA followed by a multiple comparison test to compare raw data, before expressing the results as a percentage of the control value using the statistical analysis software SPSS Dr II. A value of p b 0.05 was considered to be statistically significant.
C
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Fig. 2. Tissue content of lipids. Content of triglycerides (TG) (A) and total cholesterol (TC) (B) in the heart. Data represent the mean ± SEM of the results from 5 to 7 rats in each group.
angiotensin II-infused rats, Pio (Takeda Pharmaceutical Co., Tokyo, Japan) was given orally at a dose of 2.5 mg/kg/day. These treatments were continued until sacrifice.
A
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Table 1 Baseline conditions.
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Fig. 1. Oil red O staining of the heart of rats given angiotensin II with or without pioglitazone administration. Shown are heart sections from a control rat (A), a rat given pioglitazone (Pio) (B), a rat given angiotensin II (Ang II) (C), and a rat given Ang II plus Pio (D). Original magnification, × 200. Scale bar indicates 100 μm. E. Semiquantification of the oil red O-stained area. Data represent the mean ± SEM of the results from 4 to 5 rats in each group.
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Fig. 3. Western blot analysis of AMP-activated protein kinase α (AMPK), acetyl-CoA carboxylase (ACC), the phosphorylated (activated) form of AMPKα (P-AMPK), and the phosphorylated (inactivated) form of ACC (P-ACC). A. Representative Western blots. B, C, D, E. Protein expression in treated rats relative to control rats. Data represent the mean ± SEM of the results from 4 to 5 rats in each group. Abbreviations are the same as in Fig. 2.
3. Results 3.1. Characteristics of experimental animals Ang II significantly elevated blood pressure (Table 1). On the other hand, Pio, administered either alone or concomitantly with Ang II, did not significantly alter the blood pressure level. Pio reduced serum TG and TC levels in Ang II-treated and untreated rats. On the other hand, Pio did not significantly reduce the levels of serum free fatty acids, which was increased by Ang II treatment. 3.2. Accumulation of lipids in the heart Oil red O staining of heart sections showed no apparent lipid deposition in the heart of untreated rats and rats treated with Pio alone (Fig. 1A, B). Accumulation of oil red O-stainable lipid was observed in the myocardium as well as in the arterial wall, of Ang
A
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II-infused rats (Fig. 1C), and the extent of lipid deposition was substantially reduced in heart sections from rats treated with both Ang II and Pio (Fig. 1D, E). Tissue content of TG was found to be increased in the heart of Ang II-infused rats, and this increase was suppressed by Pio (Fig. 2). 3.3. Regulation of genes related to lipid metabolism The increase in mRNA expression of SREBP-1c and FAS in the heart of rats receiving Ang II infusion [11] was not inhibited by Pio. Pio suppressed the Ang II-induced upregulation of PPAR-γ, but not CPT-1. Ang II increased the amount of phospho-AMPKα, an active form of AMPKα, but not total AMPKα, and this increase in phospho-AMPKα was decreased by Pio treatment (Fig. 3). Similarly, Ang II increased the amount of the phosphorylated form of ACC, and this increase was suppressed by Pio, although Pio did not reduce the amount of total ACC protein in the heart of Ang II-infused rats.
C
G P=0.001 P<0.001 P<0.001
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D
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Pio
E
F
DHE signal intensity (% control)
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Fig. 4. Superoxide and lipid content in heart sections. Heart sections from a control rat (A), a rat treated with pioglitazone (Pio) (B), a rat treated with angiotensin (Ang) II (C, E, F) and a rat treated with both Ang II and Pio (D) are shown. A–D, F. Dihydroethidium (DHE) staining. E. Oil red O staining. E and F are serial sections. DHE signals were increased in the myocardium and vascular cells (arrowhead) of the heart from the Ang II-infused rat (C). Intense DHE staining can be observed in the area of the increased lipid deposits (E, F). Original magnification, × 200 (A–D), × 400 (E, F). Scale bars indicate 100 μm. G. Intensity of superoxide signals in treated rats relative to control rats. Data represent the mean ± SEM of the results from 4 to 5 rats in each group.
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(Fig. 6). On the other hand, Pio treatment in conjunction with Ang II infusion increased LVDP to a greater extent than in untreated control rats.
3.4. Superoxide production in the heart As compared to untreated controls, DHE staining-positive signals were increased by Ang II. In the heart of Ang II-infused rats, increased DHE signals were observed in the vessel wall and at the site of lipid deposition, as has been demonstrated previously [11]. Pioglitazone significantly reduced the superoxide signals. Ang II-induced upregulation of mRNA expression of Nox1, a component of NADPH oxidase, was found to be suppressed by Pio treatment (control 100 ± 10% [n = 7]; Ang II 473 ± 209% [n = 6], P b 0.05 versus control; Ang II ± Pio 39 ± 7% [n = 6], NS versus control, P b 0.05 versus Ang II).
3.7. Effect of Pio treatment on Ang II-induced cardiac fibrosis Ang II infusion significantly increased the area affected be fibrosis in both the right and the left ventricle (Fig. 7). Treatment of rats with Pio significantly suppressed the fibrotic effects of Ang II. 4. Discussion
3.5. Tissue iron deposition and expression of ferritin and HO-1 protein
Here we demonstrated that Pio reduced the increase in lipid content caused by Ang II infusion. In addition, it was found that Pio reduced the extent of cardiac fibrosis induced by Ang II and improved systolic and diastolic performance during the reperfusion period after the induction of 30-min global ischemia in the isolated perfused heart from Ang II-infused rats. As has been shown in a previous study [10], Ang II treatment upregulates the expression of lipogenic genes, SREBP-1c and FAS, in the heart. In the current study, however, Pio did not significantly alter the expression of lipogenic genes, although it reduced cardiac lipid content (Table 2). In addition, Pio also reduced the phosphorylated, thus activated, form of AMPK, and this might rather act to enhance intramyocardial lipid accumulation [21]. Therefore, it is possible that inhibition of lipid accumulation by Pio may be a direct consequence of its lipid lowering properties. This notion may be consistent with a recent report showing that PPARγ activation may not modulate the expression of genes involved in cardiac lipid metabolism [22]. Previous studies have shown that increased oxidative stress lowers cardiac contractility and/or promotes cardiac fibrosis. Enhanced production of reactive oxygen species (ROS) by the NADPH oxidase system is considered to play a role in the development of cardiomyocyte hypertrophy, interstitial fibrosis and the genesis of contractile dysfunction [23]. Overload of iron, which is associated
Ang II infusion caused iron deposition in the heart (Fig. 4), as has been described previously [20], and this deposition was suppressed by Pio. Western blot analysis showed that the upregulation of ferritin protein induced by Ang II was decreased by Pio. On the other hand, Pio did not significantly alter the upregulation of HO-1 expression that had been induced by Ang II. 3.6. Cardiac functions The mean left ventricular cardiac contractility (+ dP/dt) and rate of relaxation (− dP/dt) in rats treated with Ang II with or without Pio is shown in Fig. 5. Application of global ischemia for 30 min caused an immediate and marked decrease in both +dP/dt and −dP/dt. After reperfusion, +dP/dt was lower in the heart of Ang II-infused rats as compared with control rats; however, concomitant Pio treatment significantly improved the recovery in + dP/dt after reperfusion (Fig. 5A). Similarly, − dP/dt was found to be lower after reperfusion in the Ang II group, but Pio treatment recovered this parameter to the levels found in the control group (Fig. 5B). No significant difference was observed in left ventricle developed pressure (LVDP) between the untreated and Ang II-infused rat groups
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E
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G
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150 100 50 0
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HO-1 (%)
200
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Fig. 5. Iron deposition and expression of heme oxygenase-1 (HO-1) and ferritin protein. A–D. Prussian blue staining. Shown are heart sections from a control rat (A), a rat given pioglitazone (Pio) (B), a rat given angiotensin II (Ang II) (C), and a rat given Ang II plus Pio (D). Original magnification, × 200. Scale bar indicates 100 μm. E. Representative Western blots of HO-1 and ferritin. F, G. HO-1 and ferritin expression in treated rats relative to control rats. Data represent the mean ± SEM of the results from 4 to 6 rats in each group. *P b 0.05 versus control group.
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Time (min) Fig. 6. Cardiac contractile and relaxation function and the left ventricle developed pressure (LVDP) of the isolated heart subjected to global ischemia and subsequent reperfusion. A, B. As compared with untreated control rats, both the maximum rate of rise (+dP/dt, A) and the maximum rate of drop (− dP/dt, B) in the developed pressure subjected to 30 min of global ischemia followed by reperfusion were reduced in the angiotensin II group (Ang II), and this reduction was recovered by pioglitazone treatment (Ang II + Pio). C. LVDP was not significantly different between the Angiotensin II group (Ang II, n = 9) and the untreated control group (n = 5). On the other hand, administration of pioglitazone to Ang II-infused rats (Ang II + Pio, n = 3) significantly increased LVDP. Data represent the mean ± SEM of the results from 5 to 9 rats in each group.*P b 0.01 and † P b 0.05 versus Ang II group.
with increased free radical production and elevated oxidative stress [24], worsens the cardiac fibrosis [20] and, conversely, chelation of iron suppresses the development of cardiac fibrosis and postischemic contractile dysfunction [25,26]. Furthermore, induction of antioxidative molecules, such as HO-1 [27] and superoxide dismutase [28], was shown to be effective in preventing myocardial injury following myocardial ischemia and conversely, myocardial ischemia/reperfusion injury in diabetic animals was exacerbated in the absence of HO-1 [29]. Several studies suggest that Pio may act to protect the heart by its antioxidative properties. Nakamura et al. showed that Pio downregulated the expression of NADPH components and reduced ROS production in the heart, which led to the protection of hypertensive cardiovascular damage [30]. In addition,
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Xu et al. showed that Pio decreased the expression of NADPH components in the rat heart and reduced age-related atrial fibrillation [31]. A recent study showed, albeit in the brain, that Pio acts protectively against iron-induced oxidative injury via its antiinflammatory action [32]. Several observations in the current study suggest that Pio reduced the extent of oxidative stress in the heart of Ang II-infused rats. Pio decreased both superoxide signals and iron deposition in the heart. In addition, Pio reduced the upregulation in Nox1 mRNA expression induced by Ang II. Of note, despite the presumed reduction in oxidative stress levels, Pio preserved the upregulation in HO-1 expression. Mersmann similarly observed that PPARγ agonist treatment reduced the infarct size of heart after ischemia/ reperfusion, although it preserved the upregulation of cardiac HO-1 expression induced by ischemia/reperfusion [33]. Induction of HO-1 expression observed by certain drugs, such as statins [34] and aspirin [35], is suggested to confer a cardioprotective effect. Therefore, the possibility exists that preservation, or upregulation, of HO-1 expression might represent Pio-induced protection from cardiac injury. This possibility might be further supported by the findings of Krönke et al. that PPARγ agonists transcriptionally upregulate HO-1 mRNA expression in vascular cells via a PPAR-responsive element [36]. Besides the possible antioxidative action of Pio, involvement of other cardioprotective mechanisms, such as PI3K/AKT activation [37] and mitochondrial KATP channel opening [38], in the Pio-induced suppression of Ang II-induced cardiac injury should be investigated in future studies. In the current study, Pio significantly improved systolic and diastolic cardiac function as well as developed pressure in the isolated heart from Ang II-infused rats. It has been demonstrated that by inducing the accumulation of salt and water, PPARγ agonists may worsen the extent of heart failure [39]. On the other hand, Reeba et al. reported that, in a mouse model of lipotoxic cardiomyopathy, rosiglitazone, which lowered both circulating and intra-cardiac triglyceride levels, decreased the expression of genes that reflect cardiac dysfunction [40]. In addition, Golfman et al. showed, in an animal model of insulin resistant diabetes, that PPARγ agonist treatment decreased myocardial lipid deposition and cardiac power in the isolated heart [41]. These observations suggest that the effects of a PPARγ agonist on cardiac function may differ depending on the circulating lipid levels as well as on whether cardiac function is tested in an in vivo or ex vivo system. A recent study also suggested that the extent of water retention may differ according to the PPARγ agonistic drugs that are used and their dosage [42]. In conclusion, Pio treatment reduced cardiac triglyceride content without affecting lipogenic gene expression, suppressed the development of cardiac fibrosis, and restored the systolic and diastolic dysfunction induced by Ang II infusion. These favorable actions of Pio were accompanied by a reduction of ROS and expression of an NADPH oxidase component, and by preservation of the antioxidative gene, HO-1. The causal or resultant relationship between the antioxidative and cardioprotective actions of Pio should be investigated in further studies.
Acknowledgments This work was supported by Grants in Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (grant 19590937) and Grant from the Takeda Science Foundation, the Sankyo Foundation of Life Science, Okinaka Memorial Institute for Medical Research, and Daiwa Securities Health Foundation. The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology.
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E
P<0.05
A
B
C
D
Fibrosis area (%)
20
P<0.05
P<0.05 P<0.05
15 10 5 0
Fig. 7. Effect of pioglitazone on angiotensin II-induced cardiac fibrosis. A–D. Mallory-Azan staining. Shown heart sections are from a control rat (A), a rat given pioglitazone (Pio) (B), a rat given angiotensin II (Ang II) (C), and a rat given Ang II plus Pio (D). Scale bar indicates 100 μm. E. Fibrotic area in the right (green bars) and left (orange bars) ventricles estimated by Mallory-Azan-staining. Data represent the mean ± SEM of the results from 4 to 6 rats in each group.
Table 2 Regulation of mRNA expression. Variables
Control
Pio
Ang II
Ang II + Pio
n SREBP-1 FAS CPT-1 PPAR-r
16 100 ± 4 100 ± 13 100 ± 11 100 ± 6
15 142 ± 30 98 ± 17 114 ± 14 83 ± 10
20 199 ± 13⁎ 184 ± 16⁎ 163 ± 37 181 ± 59⁎
16 252 ± 48† 230 ± 39⁎ 155 ± 26 108 ± 12
Pio and Ang II indicate pioglitazone and angiotensin II, respectively. ⁎ P b 0.05 versus untreated control. † P b 0.01 versus untreated control.
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