Heart and Circulatory Physiology

Targeted disruption of peroxiredoxin 6 gene renders the heart vulnerable to ischemia-reperfusion injury

Norbert Nagy, Gautam Malik, Aron B. Fisher, Dipak K. Das

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Peroxiredoxin 6 (Prdx6) is a novel peroxidase enzyme belonging to the Prdx family, which in mammals contains five more peroxiredoxins (Prdx1–Prdx5). Like glutathione peroxidase (GSHPx) and catalase, Prdx6 possesses H2O2-scavenging activities, and, like the former, it also removes hydroperoxides. Since significant amounts of catalase and GSHPx are present in the heart contributing toward the attenuation of H2O2 and hydroperoxides formed during ischemia-reperfusion injury and thereby providing cardioprotection, we asked whether Prdx6 also has any role in this process. In the present study we used Prdx6−/− mice to assess the role of Prdx6 in ischemic injury. Western blot analysis revealed the absence of any Prdx activity in the Prdx6−/− mouse heart, while the GSHPx-1 and catalase levels remained unchanged. Randomly selected hearts from Prdx6−/− mice and wild-type mice were subjected to 30 min of global ischemia followed by 120 min of reperfusion at normothermia. The hearts from the Prdx6−/− mice were more susceptible to ischemic reperfusion injury as evidenced by reduced recovery of left ventricular function, increased myocardial infarct size, and higher amount of apoptotic cardiomyocytes compared with wild-type mouse hearts. These Prdx6−/− hearts were also subjected to a higher amount of oxidative stress as evidenced by the presence of higher amount of malondialdehyde. The present study thus indicates a nonredundant role of Prdx6 in myocardial ischemic reperfusion injury as catalase, and GSHPx could not make up for the deficiency of Prdx6 activities.

  • redox signaling
  • reactive oxygen species
  • glutathione
  • catalase
  • glutathione peroxidase
  • peroxiredoxin gene knockout mice

recent studies have indicated a crucial role of glutathione peroxidase (GSHPx) in protection against myocardial ischemia-reperfusion injury (1, 15, 20). While transgenic mice overexpressing GSHPx were resistant to myocardial ischemia-reperfusion injury (23), mice devoid of GSHPx were susceptible to the same (22). Mammalian heart contains high amount of glutathione (GSH), which ensures the conversion of toxic lipid peroxides into nontoxic products utilizing the necessary reducing equivalents from the reduced GSH (18). GSH also detoxifies H2O2 and hydroperoxides that are produced in the ischemic myocardium. Oxidized glutathione is then reduced to GSH through GSHPx, thereby maintaining the supply of reduced glutathione.

Peroxiredoxin 6 (Prdx6) is a member of a relatively new family of antioxidant enzymes (other members are Prdx1 to Prdx5) that possesses peroxidase activity (19). Thus Prdx6 has the ability to reduce H2O2 and hydroperoxides into water and alcohol, respectively (13). Prdx6 is the only mammalian 1-Cys member of the peroxiredoxin superfamily that is expressed in all the vital organs, including the heart (8). Expression of Prdx6 mRNA (and protein) is reduced in mice that are susceptible to experimental atherosclerosis (24). A role of Prdx6 in neurodegenerative diseases has also been recognized (11). Mice with targeted mutation of Prdx6 are susceptible to oxidative stress (25). Transgenic mice overexpressing Prdx6 show increased resistance to lung injury in hyperoxia (26).

In view of the prevailing view that reperfusion of ischemic myocardium generates reactive oxygen species (ROS) (2, 4) and GSH is a potential target of ROS attack, we hypothesized that Prdx6 could play a role in ischemia-reperfusion injury. The results of our study indicated that the mouse hearts devoid of Prdx6 were indeed susceptible to ischemia-reperfusion injury despite the presence of significant amounts of catalase and GSHPx, thereby indicating a crucial nonredundant role of Prdx6 in myocardial ischemic-reperfusion injury.


Generation of Prdx6−/− mice.

The full-length BAC genomic clone (clone BACM-153C17 from a 129/SvJ mouse genomic library) containing functional 1-cysPrx gene (Genome Systems, St. Louis, MO) was used to construct the targeting vector as described elsewhere (12). The Prdx6−/− mice were bred at the University of Pennsylvania and housed at the University of Connecticut School of Medicine before use.

Expression analysis of the Prdx6 protein in the hearts of Prdx6−/− and wild-type mice.

Western blot analysis was performed to detect Prdx6 protein in the Prdx6−/− and wild-type mouse hearts.

Experimental protocol.

The study used isolated working Prdx6−/− and wild-type mouse hearts. All protocols were approved by the Institutional Animal Care Committee. All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 86–23, Revised 1996). The mice (25–34 g) were anesthetized with pentobarbital sodium (100 mg/kg body wt ip) (Abbott Laboratories, North Chicago, IL) and anticoagulated with heparin sodium (500 IU/kg body wt ip) (Elkin-Sinn, Cherry Hill, NJ) injection. The isolated hearts were randomly divided into two groups: Prdx6−/− or wild type. For baseline control, isolated hearts were perfused with Krebs-Henseleit bicarbonate (KHB) buffer for 2 h 45 min (results not shown). After sufficient depth of anesthesia was ensured, thoracotomy was performed, and aorta of heart was identified. After we excised the heart from the chest by the aorta, the lung and connective tissue were removed and the whole heart was transferred to ice-cold (4°C) modified KHB solution, which contained (in mM) 118 NaCl, 4.7 KCl, 1.7 CaCl2, 24 NaHCO3, 1.2 KH2PO4, 12 MgSO4, and 10 glucose, until contraction had ceased. Both the aorta and pulmonary vein were cannulated as quickly as possible and perfused in retrograde Langendorff mode at constant perfusion pressure of 50 cmH2O (5 kPa) for a standardization period (21). Immediate start of retrograde perfusion helped to wash blood and its component from the vascular system. Perfusate (KHB) temperature was maintained at 37°C and saturated with 95% O2-5% CO2 mixture during the entire experiment. The duration of the retrograde perfusion was 10 min; after this procedure, the heart was switched to an antegrade perfusion mode. In the antegrade perfusion mode, the buffer enters the cannulated left atrium at pressure equivalent to 10 cmH2O (1 kPa) and passes to the left ventricle, from which it is spontaneously ejected through the aortic cannula against a pressure equivalent to 100 cmH2O (10 kPa). After baseline measurements of heart rate, coronary flow, aortic flow, left ventricular developed pressure (LVDP), and its maximum first derivative, the antegrade perfusion line was closed, and the heart was subjected to 30 min ischemia. Before the initiation of 2-h reperfusion, the heart was perfused in retrograde mode to avoid the development of a high incidence of ventricular fibrillation. The measurements of the cardiac function were carried out at 15, 30, 60, and 120 min of the 2-h reperfusion. Any heart that showed any cardiac disturbance (ventricle arrhythmia and fibrillation) during the entire experiment was excluded from this study.

Measurement of the cardiac function.

The heart rate, LVDP (the difference between the maximum systolic and diastolic pressure), and the first derivative of LVDP were recorded by a Gould p23XL transducer (Gould Instrument System, Valley View, OH). The signal was amplified by using Gould 6600 series signal conditioner (Gould Instrument System) and monitored on Cordat II real-time acquisition system (Triton Technologies, San Diego, CA) (7). The aortic flow was measured by flowmeter. The coronary flow was measured by timed collection of the coronary effluent dripping from the heart.

Measurements of the infarct size.

At the end of each experiment, the heart was infused with 10% solution of triphenyltetrazolium (TTC) in phosphate buffer through the aortic cannula for 20 min (10). The left ventricle was removed and sliced into 1-mm thickness of cross-sectional pieces and weight. Each slice was scanned with computer-assisted scanner (ScanJet 5). The risk area of the whole myocardium was stained in red by TTC, while the infarct zone remained unstained by TTC. These were measured by using computerized software (Scion Image), and these areas were multiplied by the weight of the each section; these results were summed up to obtain the total of the risk zone and infarct zone. The infarct size was expressed as the ratio of the infarct zone to the risk zone.

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay for assessment of apoptotic cell death.

Immunohistochemical detection of apoptotic cells was carried out using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), in which residues of digoxigenin-labeled dUTP are catalytically incorporated into the DNA by terminal deoxynucleotidyl transferase II, an enzyme that catalyzes a template-independent addition of nucleotide triphosphate to the 3′-OH ends of double- or single-stranded DNA (14). The incorporated nucleotide was incubated with a sheep polyclonal anti-digoxigenin antibody followed by an FITC-conjugated rabbit anti-sheep IgG as a secondary antibody as described by the manufacturer (Apop Tag Plus, Oncor, Gaithersburg, MD). The sections were washed in PBS three times, blocked with normal rabbit serum, and incubated with mouse monoclonal antibody recognizing cardiac myosin heavy chain (Biogenesis, Poole, UK) followed by staining with TRIRC-conjugated rabbit anti-mouse IgG (200:1 dilution, Dako Japan, Tokyo). The fluorescence staining was viewed with a confocal laser microscope (Olympus, Tokyo). The number of apoptotic cells was counted and expressed as a percentage of total myocyte population.

Western blot analysis.

Left ventricles from the hearts were homogenized in a buffer containing 25 mM Tris·HCl, 25 mM NaCl, 1 mM orthovanadate, 10 mM NaF, 10 mM pyrophosphate, 10 mM okadaic acid, 0.5 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (15). Protein (100 μg) of each heart homogenate was incubated with 1 μg of antibody against Prdx6 (Abcam, Cambridge, MA), catalase (Santa Cruz, CA), or GSHPx-1 (Santa Cruz, CA) for 1 h at 4°C. The immune complexes were precipitated with protein A-Sepharose; immunoprecipitates were separated by SDS-PAGE and immobilized on polyvinylidene difluoride membrane. The membrane was then stripped and reblotted with specific antibodies against glucose-6-phosphate dehydrogenase, which served as loading control. The resulting blots were digitized, subjected to densitometric scanning using a standard NIH Image program, and normalized against loading control.

Measurement of malondialdehyde for assessment of oxidative stress.

Malondialdehyde (MDA) formation is considered a presumptive marker for oxidative stress. MDA was measured as MDA-2,4-dinitrophenylhydrazine (MDA-DNPH) derivative by HPLC (3). MDA was assayed as described previously to monitor the development of oxidative stress. In brief, coronary effluents were collected and derivatized with DNPH and extracted with pentane. Aliquots of 25 in acetonitrile were injected onto a Beckman Ultrasphere C18 (3 μm) column. The products were eluted isocratically with a mobile phase containing acetonitrile/water/acetic acid (40:60:0.1, vol/vol/vol) and measured at three different wavelengths (307, 325, and 356 nm) by using a Waters M-490 multichannel UV detector. The peak for MDA was identified by cochromatography with a DNPH derivative of the authentic standard, peak addition, UV pattern of absorption at the three wavelengths, and by gas chromatography-mass spectroscopy.

Statistical analysis.

The values for myocardial functional parameters, MDA, infarct sizes, and apoptotic cardiomyocytes are all expressed as means ± SE. The statistical analysis was performed by one-way ANOVA for any differences between the mean value of all groups. Differences between data were analyzed for significance by performing a Student's t-test. The results were considered significant if P < 0.05.


Characterization of Prdx6−/− mice.

As shown in Fig. 1, Prdx6−/− mouse hearts had no expression of Prdx6 while the wild-type mouse hearts possessed significant amount of Prdx6. Similar to GSHPx, Prdx6 scavenges both hydroperoxides and H2O2 (the latter can also be removed by catalase); it is thus presumed that Prdx6, GSHPx, and catalase together make an antioxidant module. We thus tested whether deletion of Prdx6 had any effects on catalase and GSHPx activities. Noticeably, in Prdx6−/− mouse heart GSHPx-1 and catalase levels remained unchanged vis-à-vis the wild-type hearts (Fig. 1).

Fig. 1.

Western blot analysis for the detection of glutathione peroxidase-1 (GSHPx-1), catalase, and peroxiredoxin 6 (Prdx6) proteins. Open bars, wild-type mice. Filled bars, Prdx6−/− mice. The Western blot was performed as described in materials and methods. The results of densitometric scanning are shown on the top of the protein blots. Results are expressed as means ± SE of 3 separate hearts per group. Prdx6 was not detected in the Prdx6−/− mouse hearts. *P < 0.05 vs. wild type.

Myocardial performance.

Throughout the study, the heart rates and coronary flows were not different between the two groups (Table 1), suggesting that Prdx6−/− had no effects on these parameters (the results for 15-min reperfusion are not shown). The aortic flow (AF) and LVDP, as well as the maximum first derivative of LVDP, were significantly lower in the Prdx6−/− mouse hearts vs. wild-type hearts during the reperfusion period, except for AF, which was significantly lower only at 60 and 120 min of reperfusion.

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Table 1.

Effects of ischemia-reperfusion on the left ventricular function of wild-type and Prdx6−/− mouse hearts

Myocardial infarction and cardiomyocyte apoptosis.

Myocardial infarct size expressed as infarct size/area of risk was significantly higher for the Prdx6−/− mouse hearts compared with that in wild-type controls (Fig. 2, left). However, both groups had a similar level of area of risk. Myocardial infarcted tissue was scattered throughout each ventricle in each heart from both groups. The white area that was not stained by TTC indicated irreversible ischemic injury. Mean value of infarct size in the Prdx6−/− group was 49.9 ± 1.7% vs. 36.5 ± 1.4% for the wild-type group. The apoptotic cardiomyocytes visualized by double-antibody staining (TUNEL in conjunction with a myosin heavy chain to detect myocytes) were present in significantly higher quantities (21.5 ± 0.9%) in the Prdx6−/− group compared with the wild-type (8.5 ± 0.8%) group (Fig. 2, right). Infarction was not developed or apoptosis was not detected in the hearts perfused for the same time period without subjecting them to ischemia-reperfusion (results not shown).

Fig. 2.

Myocardial infarct size (left) and cardiomyocyte apoptosis (right) are shown as means ± SE of 6 animals per group. Open bars, wild-type mice. Filled bars, Prdx6−/− mice. BL, baseline; I/R, ischemia-reperfusion. *P < 0.05 vs. wild-type.

ROS activity and oxidative stress.

MDA is the presumptive marker for lipid peroxidation and oxidative stress developed from ROS generated during the reperfusion of ischemic myocardium. MDA content of the heart determined at the end of each experiment showed significantly higher amount of MDA (39.6 ± 0.8 pg/g) compared with that in hearts from the wild-type controls (26.0 ± 0.9 pg/g), indicating development of higher amount of oxidative stress in the Prdx6−/− hearts (Fig. 3).

Fig. 3.

Malonaldehyde (MDA) formation in the hearts of wild-type (open bars) and Prdx6−/− mice (filled bars) at baseline and at the end of ischemia/reperfusion is shown as means ± SE of 6 animals per group. *P < 0.05 vs. wild-type.


The most noticeable and salient feature of the present study is that despite the presence of significant amount of catalase and GSHPx in the hearts of Prdx6−/− mice, these hearts were susceptible to ischemia-reperfusion injury, suggesting a nonredundant role of Prdx6 in cardioprotection. The hearts of Prdx6−/− mice had reduced postischemic ventricular recovery and increased myocardial infarct size and exhibited a greater number of apoptotic cardiomyocytes compared with those values in wild-type hearts. These hearts also had a significantly higher amount of MDA compared with that present in wild-type mouse hearts.

Peroxiredoxins, thioredoxin peroxidases, belong to a relatively new family of antioxidant enzymes. Six peroxiredoxins (Prdx1–Prdx6) have yet been identified, of which Prdx6 is found in the cytosolic fraction together with Prdx1, Prdx2, and Prdx4. Prdx4 is also found in mitochondria and peroxisome, while Prdx3 exists only in mitochondria (16). Prdx6 is abundantly present in most of the tissues, including the heart. The antioxidant activity of Prdx6 is attributed to its ability to reduce H2O2 and hydroperoxides. Unlike other members of the Prdx family, which have two catalytically active cysteines, Prdx6 contains only one NH2-terminal conserved cysteine (Cys47) and is, therefore, termed as 1-Cys Prdx (17). While oxidized Prdx1–Prdx5 are reduced through the electron transfer from thiol-containing donor thioredoxin, Prdx6 receives electron transfer from glutathione.

It has long been known that reperfusion of the ischemic heart produces ROS, thereby subjecting the hearts to an increased amount of oxidative stress. In normal hearts, because of the presence of an adequate amount of antioxidants, ROS are readily removed. In contrast, under pathophysiological conditions, O2· undergoes a sequence of reactions producing H2O2 and hydroxyl radicals (·OH). SOD scavenges O2· by catalyzing a dismutation reaction, where simultaneous oxidation (O2· to O2) and reduction (O2· to H2O2) reactions take place (5). The heme-containing enzyme catalase transforms H2O2 into H2O and molecular O2. The GSHPx reaction removes H2O2 at the expense of GSH. The maintenance of GSH levels thus appears to be crucial, and thiol (SH) groups are essential for the tissues to protect themselves against the ROS attack. Furthermore, the generated ·OH can attack the unsaturated lipids in the cell, causing lipid peroxidation and producing lipid hydroperoxides, which further exacerbates ischemia-reperfusion injury. GSHPx can also scavenge the hydroperoxides by converting them into hydroxy fatty acids. GSHPx reverses the thiol oxidation reaction, because GSHPx is a GSH-consuming enzyme. Similar to GSHPx, Prdx6 can also remove both H2O2 and hydroperoxides. However, Prdx6 can reduce phospholipid hydroperoxides while GSHPx (the type I or cytosolic enzyme) does not have that ability (9). We hypothesize that reduction of peroxidized membrane phospholipids by Prdx6 accounts for its unique antioxidant effect. In addition to GSH peroxidase activity, Prdx6 also possesses phospholipase A2 (PLA2) activity. A recent study showed a direct interaction between surfactant protein A and Prdx6, which provided a mechanism of regulation of the PLA2 activity of Prdx6 by surfactant protein A (28). The same authors demonstrated that Prdx6 null mice had reduced degradation of internalized dipalmitoylphosphatidylcholine (DPPC) in the lung epithelium and a decreased rate of DPPC synthesis by the remodeling pathway (8).

The results of our study showed the presence of significant amounts of catalase and GSHPx in the hearts of Prdx6−/− mice. It is interesting to note that despite the presence of catalase and GSHPx in these hearts, these hearts were subjected to an increased amount of oxidative stress and were vulnerable to cellular injury, suggesting a crucial role of Prdx6 in the ischemic reperfusion injury. Evidence is rapidly accumulating, suggesting a key role of Prdx6 in cellular injury. For example, transgenic mice overexpressing Prdx6 exhibited increased resistance to lung injury in hyperoxia (27). In this study, at 96 h of hyperoxia, transgenic mice had less epithelial cell necrosis, perivascular edema, and inflammatory cell recruitment, as well as lower thiobarbituric acid-reactive substances and protein carbonyls in lung homogenate, indicating increased cellular defense and providing evidence that Prdx6 functions as a lung antioxidant enzyme. In another study, the same authors showed an induction of Prdx6 in lung epithelial cells by oxidative stress (11). Increased lung expression of Prdx6 through adenoviral-mediated transfer of the Prdx6 gene protected against hyperoxic injury (27). Examination of the lungs indicated that Prdx6-overexpressing animals compared with wild type had less lipid peroxidation, less protein oxidation, less lung edema, and less lung inflammation when evaluated at 72 h of hyperoxia. Another recent study found that Prdx6−/− mice on a B6;129 background were significantly more susceptible to atherosclerosis compared with controls (24). However, Prdx6−/− mice on either 129 or B6 backgrounds were neither more susceptible nor more resistant to atherosclerosis than were their normal counterparts. In another related study, mice with targeted mutation of Prdx6 were found to develop normally but were susceptible to oxidative stress (25). This study showed that Prdx6−/− macrophages had higher H2O2 levels and lower survival rates, more severe tissue damage, and higher protein oxidation rates despite the fact that there were no differences in the mRNA expression levels of GSHPx and catalase. The results of the present study are therefore consistent with these reports that despite undiminished levels of catalase and GSHPx, Prdx6−/− hearts were more susceptible to ischemic injury. In summary, the results of the present study demonstrated a crucial role for Prdx6 in myocardial ischemia-reperfusion injury. Prdx6−/− mice devoid of Prdx6 exhibited reduced postischemic ventricular recovery and larger infarct size and a higher number of apoptotic cardiomyocytes compared with those in wild-type controls. It appears that these Prdx6−/− mouse hearts were exposed to a greater amount of oxidative stress as evidenced from the presence of higher amount of MDA in the hearts.


This study was supported in part from National Heart, Lung, and Blood Institute Grants HL-34360, HL-22559, and HL-33889 to D. K. Das and HL-79063 to A. B. Fisher.


  • The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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