Inhibition of the activity of poly (ADP-ribose) polymerase reduces heart ischaemia/reperfusion injury via suppressing JNK-mediated AIF translocation

Poly (ADP-ribose) polymerase (PARP) has been proposed to play an important role in the pathogenesis of heart ischaemia/reperfusion (I/R) injury. However, the mechanisms of PARP-mediated heart I/R injury in vivo are still not thoroughly understood. Therefore, in this study, we investigate the effect of PARP inhibition on heart I/R injury and try to elucidate the underlying mechanisms. Studies were performed with I/R rats' hearts in vivo. Ischaemia followed by reperfusion caused a significant increase in Poly (ADP-ribose) (PAR), c-Jun NH2-terminal kinase (JNK) and apoptosis-inducing factor (AIF) activity. Administration of 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ), an inhibitor of PARP, decreased myocardial infarction size from 61.11±7.46%[0] to 38.83±5.67% (P<0.05) and cells apoptosis from 35±5.3% to 20±4.1% (P<0.05) and simultaneously improved the cardiac function. Western blot analysis showed that administration of DPQ reduced the activation of JNK and attenuated mitochondrial-nuclear translocation of AIF. Additionally, administration of SP600125, an inhibitor of JNK, attenuated mitochondrial-nuclear translocation of AIF. The results of the present study demonstrated that the inhibition of PARP was able to reduce heart I/R injury in vivo. Our results also suggested that JNK may be downstream of PARP activation and be required for PARP-mediated AIF translocation. Inhibition of the activity of PARP may reduce heart I/R injury via suppressing AIF translocation mediated by JNK.


Introduction
Poly (ADP-ribose) polymerase (PARP) is a chromatin-bound enzyme that plays a physiological role in the repair of strand breaks in DNA [1]. PARP locates in the nuclei of cells of various tissues, including the heart and skeletal muscle [2]. The breakage of DNA strands can activate PARP. The activation of PARP initiates an energy-consuming cycle by transferring ADP ribose units from NAD ϩ to nuclear proteins [3,4]. As a result, the damaged DNA was repaired. However, in many pathophysiological processes, such as inflammation, circulatory collapse and heart ischaemia/reperfusion (I/R) injury, massive amounts of reactive oxygen species, including superoxide anions [5], hydrogen peroxide and hydroxyl radicals [6], are generated, which cause the breakage of DNA strands and subsequently hyper-active of PARP. This process leads to the depletion of the intracellular nicotinamide adenine dinucleotide (NAD ϩ ) and ATP energetic pools which leading to cellular dysfunction and death.
Heart I/R may exacerbate myocardial injury by the release of massive of reactive oxygen and nitrogen species. These reactive oxygen and nitrogen species are able to lead to cell necrosis and apoptosis. PARP has been proposed to play a role in the pathogenesis of heart I/R injury [7,8]. Poly (ADP-ribose) (PAR) is the active morphous of PARP. Immunohistochemical detection of PAR formation demonstrated that PARP was activated in the reperfusion myocardium [9]. Myocardial reperfusion injuries in vitro or in vivo have been shown to ameliorate by pharmacological inhibitors of PARP [10,11], which indicated that the PARP pathway is involved in the pathogenesis of heart I/R injury.
Apoptosis-inducing factor (AIF) is a novel apoptotic factor that induces chromatin condensation and large-scale DNA fragmentation. AIF is a caspase-independent mitochondrial death factor [12]. AIF is strictly confined to mitochondria in normal cells. The most conspicuous feature of AIF in apoptosis is its mitochondrial-nuclear translocation, accompanied by nuclear condensation and large-scale DNA degradation [13]. In oxidantinduced cell, AIF transposes to the nucleus and induces cell death. Studies demonstrated that heart I/R in vivo were associated with the nuclear translocation of AIF [12]. Studies also demonstrated that PARP regulated the translocation of AIF, which contributed to myocardial injury and cardiac dysfunction in heart I/R [14,15].
Though many studies have reported that PARP hyper-activation prompted mitochondria dysfunction, which in turn released AIF and subsequently led to cell death [16], the mechanism by which PARP activation led to mitochondrial dysfunction and released of AIF was largely unknown [17,18]. Recently, Xu et al. [19] found that c-Jun NH2-terminal kinase (JNK) participated in PARP-mediated mitochondria dysfunction and subsequently cell death in mouse embryonic fibroblast cells. However, the role of JNK in PARP mediates mitochondria dysfunction and the releases of AIF in vivo are still unknown. Therefore, the aim of the present study is to investigate whether JNK participates in the PARP mediates release of AIF in I/R hearts in vivo.

Experimental groups
The following experimental groups were studied: (1)

Determination of myocardial infarct size
At the end of reperfusion, the coronary artery was re-occluded and the heart was perfused with 3 ml of Evans blue to delineate myocardial area at risk (AAR). Then the hearts were harvested and rinsed in normal saline. The atria, right ventricle and great vessels were removed. Then the tissue was semi-freezed for 30 min. in a Ϫ20ЊC freezer in order to be cut more easily. After that, the left ventricle was surgically isolated and cut into slices (1 mm thick). Nitro blue tetrazolium was obtained in powder form (Sigma, USA). The tetrazolium powder was diluted in a phosphate buffer. We used a two part buffer system consisting of NaH2PO4 (0.1 M) and Na2HPO4 (0.1 M). The ratio of the volume of NaH2PO4 and Na2HPO4 in the buffer was 77.4% and 22.6%. The pH of the buffer was 7.4. The slices were then incubated in the solution at a temperature of 37ЊC for 5 min. After evaluating the entire surface area, segments with blue staining were designated as viable, and those without staining were designated as non-viable (infarcted). Finally the different areas of the ventricle were weighed separately. Myocardial ischaemic area (AAR) was expressed as the percentage of the left ventricle. Infarct size was expressed as the percentage of the AAR [20].

Measurement of cardiac function
To monitor cardiovascular function during the I/R protocol, a fluid-filled cannula was introduced via the carotid artery into the left ventricle. LVDP, maximum rate of left ventricular pressure development (ϩdP/dt) and maximum rate of left ventricular pressure decline (-dP/dt) was measured using a pressure transducer interfaced with a computerized heart performance analysis system (Digi-Med, USA).

Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL)
To evaluate apoptotic activity, the TUNEL technique was used. Each section was de-paraffinized and re-hydrated with serial changes of xylene and ethanol. Proteinase K (20 mg/l) was applied to the section for 30 min. with the intention of producing optimal proteolysis. The endogenous peroxidase was inhibited with 3% hydrogen peroxide for 10 min. A commercial apoptosis detection kit (Roche, Germany) was used. The TdT reaction was carried out for 1 hr at 37ЊC, and then 3, 3Ј-diaminobenzidine (DAB) chromogen was applied. Haematoxylin-Eosin was used as a counter-stain. TUNELpositive cells were determined by randomly counting ten fields of the section and were expressed as a percentage of normal nuclei [21].

Preparation of the mitochondrial and nuclear fractions
Heart mitochondria and nuclear fractions were used to evaluate the activation of AIF by western blot analysis, respectively. They were prepared by differential centrifugation, which was described by Kim GT et al. [12]. Briefly, the ischaemic myocardium of killed rats were removed and cut into small pieces. The samples were suspended in six volumes of buffer (210 mM mannitol, 70 mM sucrose, 10 mM Tris-HCl, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 5 mM EDTA, 0.5 mM dithiothreitol, and 1 mM Na3VO4). After homogenized, the homogenates were centrifuged at 800 g at 4ЊC for 10 min. The pellets were nuclear fractions. The supernatant was centrifuged at 1,200 g at 4ЊC for 10 min. After decanting, it was recentrifuged at 15,000 g at 4ЊC for 20 min., the pellets were mitochondrial fractions.

Western blot
Myocardial PAR, JNK and AIF in the ischaemic myocardium after 180 min. reperfusion were determined by western blot analysis. Equal amounts of protein (50 g) were fractionated on 10% (for PAR) or 14% (for JNK and AIF) SDS-polyacrylamide gels in running buffer [25 mM Tris,192

Statistics
Values were expressed as means Ϯ SD. Statistical analysis of the results was carried out by one-way analysis of the variance followed by the Newman-Keuls test. PϽ0.05 was considered significant.

Result PARP, JNK and AIF were activated in the rat model of myocardial ischaemia and re-perfusion
Western blot analysis was performed to evaluate whether PARP, JNK and AIF can be activated in the myocardial I/R rat model. Six rats hearts were studied in this group. As shown in Fig. 1, PAR

Inhibition of PARP activity reduced myocardial infarct size
As shown in Fig. 2A and B, the AAR and infarct size of the heart were 52.95Ϯ1.28% and 61.11Ϯ7.46%, respectively in I/R group (n ϭ 6). These values showed no significant differences between I/R and I/Rϩ DMSO groups (n ϭ 6). Administration of DPQ or SP600125 caused significant reduction of AAR and infarct size. The AAR and infarct size of the heart was 31.12Ϯ1.58% and 38.83Ϯ5.67%, respectively in I/RϩDPQ group (n ϭ 7). In I/RϩSP600125 group (n ϭ 6), the AAR and infarct size of the heart was 32.61Ϯ 4.16% and 41.52Ϯ 4.20%, respectively. These data suggest that inhibition of PARP or JNK activity reduces myocardial infarct size in rat heart I/R injury.

Figures 2C-E contains the mean values of LVDP, ϩdP/dt and -dP/dt during preischaemia, ischaemia and re-perfusion in animals from all experimental groups. No group differences existed in any of these values before the introduction of ischaemia. However, during ischaemia and re-perfusion, the LVDP values of I/Rϩ
DMSO group (n ϭ 6) were significantly lower than those of the sham group (n ϭ 8) (PϽ0.05). The LV ϮdP/dt values of I/RϩDMSO group were also significantly lower than those of the sham group (PϽ0.05). Both LVDP and LV ϮdP/dt values of I/RϩDPQ group (n ϭ 7) were significantly higher than I/RϩDMSO group. These preliminary data indicate that inhibition of PARP activity may play an essential role in the protection of cardiac function in rat heart I/R injury.

Inhibition of PARP activity reduced the activation of JNK
Western blot analysis was performed to examine whether the activation of JNK in the rat model of myocardial I/R was related with PARP. As shown in Fig. 4A, phospho-JNK increased after reperfusion in non-inhibition of PARP rat. Ischaemia and reperfusion-induced activation of phospho-JNK was significantly attenuated in the inhibition of PARP. These data suggest that JNK may be downstream of PARP activation in rat heart I/R injury.

Inhibition of PARP or JNK activity reduces the AIF translocation
As shown in Fig. 4B, AIF increased after reperfusion in nuclear fractions. The activation of AIF was significantly attenuated by inhibition of PARP or JNK activity. On the contrary, AIF decreased in mitochondria fractions after reperfusion. Inhibition of PARP or JNK activity could increase the activation of AIF in mitochondria fractions. These data suggest that inhibition of PARP or JNK activity was able to reduce the mitochondrial-nuclear translocation of AIF. These preliminary data indicate AIF may be downstream of PARP and JNK activation. JNK may play an essential role in PARP-mediated AIF translocation.

DISCUSSION
In this experiment, we used DPQ to inhibit the activation of PARP in the rat model of myocardial ischaemia and reperfusion in vivo; as a result, the infarct size and the apoptosis of cells of hearts reduced, the cardiac function was protected. At the same time, the activation of JNK and the translocation of AIF from mitochondrial to nuclear were attenuated. We also found that inhibition of the activation of JNK was able to attenuate the translocation of AIF from mitochondrial to nuclear. The present study demonstrated that inhibition of PARP can reduce heart I/R injury in vivo. In this pathophysiological course, PARP may regulate the translocation of AIF by the activation of JNK.

Fig. 1 Western blot analysis of PAR (A), JNK (B) and AIF (C) in rats' hearts subjected to I/R. PAR and phospho-JNK increased
Coronary reperfusion after ischaemia results in a marked cellular injury and a large infarct rea, which ultimately leads to heart failure. Pathophysiological states, such as inflammation [22], circulatory shock [23] and I/R injury generate free radicals and oxidant species leads to DNA injury and the activation of PARP. In this experiment, western blot analysis showed that PARP increased during the reperfusion (up to 24 hrs) protocol. The results conform to those of other researches' [24]. Our experiment also showed that the peak activation of PARP was at 180 min. of re-perfusion. Several independent studies have confirmed that pharmacological inhibition of PARP [24][25][26] significantly reduced the infarct size and improved the cardiac function in I/R injury. In this experiment, we used DPQ to inhibit the activation of PARP. As a result, the infarct size of hearts reduced and the cardiac function was protected. DPQ is a very potent PARP inhibitor [27]. Xu et al. [19] in their experiment also used DPQ to inhibit PARP. The cardiac protection afforded by the PARP inhibitors was due to a selective inhibition of PARP. Though PARP was proposed to play a role in the pathogenesis of heart I/R injury, the precise mechanisms of PARP activation in heart I/R injury were not fully understood. Studies proved that mechanisms of PARP activation in heart I/R injury may involve several aspects. The fundamental pathologic effect of PARP activation is related to the myocardial energy metabolism [3,4]. In heart I/R injury the hyper-activation of PARP is able to disturb the energy generation process and the mitochondrial function. PARP can also regulate the expression of various proteins and cytokines such as ICAM-1 [28], AP-1 complex [29], NF-B [30], TNF-␣ and IL-10 [31]. PARP inhibition or PARP deficiency have been shown to suppress the cytokines expression in vitro or in vivo.
AIF is a 67 kD death promoting protein [32]. In healthy cells it is normally confined to mitochondria. Upon death stimuli, AIF transposes from the mitochondria to the nuclei. As a result, largescale DNA break, and cells die. However, van Empel et al. [33] reported that AIF exerts also cytoprotective effects on myocytes both in vitro and in vivo. They found that AIF has a role of antioxidant in stress-induced cell death and development of heart failure induced by pressure overload. But the precise mechanism for AIF protection in these cells was not known. van Empel presumed that AIF plays a critical role in mitochondrial respiration, by handling of reactive oxygen radicals that are normally released by the respiratory chain. In our study, we found that AIF had a proapoptotic role in rat heart I/R injury. Recent studies demonstrated that AIF transposed from the mitochondria to the nuclei required PARP activity [14,16]. These studies demonstrated that AIF was a downstream factor in PARP-mediated cell death. In this experiment, AIF increased after reperfusion in nuclear fractions. The activation of AIF was significantly attenuated by inhibition of PARP activity. These preliminary data indicate AIF may be downstream of PARP activation. However, how PARP regulates or induces AIF translocation or release from the mitochondria is not known. XU et al. [19] in their experiment found, based on genetic knockouts and pharmacological inhibition, that JNK, but not the other groups of mitogen activated protein kinase (MAPK), is required for PARP-induced mitochondrial dysfunction, AIF translocation and subsequent cell death in vitro. JNK has recently been demonstrated to be involved in cell apoptosis and necrosis [34]. JNK activation is also an important signaling step leading to cardiac myocytes apoptosis [35]. In vivo studies demonstrated that myocardial ischaemia followed by reperfusion in the heart were able to cause activation of JNK [36,37].
Our data of the present study also showed that JNK activated in I/R hearts and JNK may participate in the PARP-mediated release of AIF.
In this experiment, we found that the inhibition of PARP was able to reduce the heart infarct size and the apoptosis of cells in heart I/R injury in vivo. We further elucidated that the translocation of AIF from mitochondrial to nuclear attenuated after blocking PARP or JNK. Therefore, our results indicate that JNK may be downstream of PARP activation and JNK may be required for PARP mediates AIF translocation in a rat model of myocardial ischaemia and reperfusion in vivo. PARP/JNK/AIF pathway may be a new pathway of cardiac myocyte injury in vivo. This may provide a new management for patients suffering coronary heart diseases in the future.