Caveolin and MAP kinase interaction in angiotensin II preconditioning of the myocardium.

Angiotensin II (Ang II) has been found to exert preconditioning (PC)-like effect in mammalian hearts. The present investigation reported for the first time a unique mitogen activated protein (MAP) kinase signalling in Ang II PC of the heart involving lipid rafts, which generated a survival signal by differentially associating MAP kinases with caveolin. A group of rat hearts was treated with Ang II in the absence or presence of NADPH oxidase inhibitor, apocynin or a cell permeable reactive oxygen species (ROS) scavenger, N-acetyl-cysteine (NAC). Ang II pre-treatment improved post-ischaemic ventricular recovery, myocardial infraction and decreased the number of cardiomyocyte apoptosis indicating PC effect of Ang II. Both apocynin and NAC abolished the PC ability of Ang II. In Ang II treated heart, there was a decreased association of p38MAPKbeta & extracellular-signal regulated kinase (ERK) 1/ 2 (anti-death signalling component) with caveolin while there was an increased association of p38MAPKalpha & Jun N-terminal kinase (JNK) (death signalling component) indicating reduced amount of death signal components and increased amount of anti-death signalling components being available to the Ang II treated heart to generate a survival signal, which was reversed with NAC or apocynin. The survival signal was also demonstrated by increased phosphorylation of serine/threonine-protein kinase B (AKT) and enhanced induction of expression of Bcl-2 during Ang II PC and its reversal with NAC & apocynin treated heart.

Introduction

A large number of studies have demonstrated the role of angiotensin II (Ang II) in cardiac preconditioning (PC) against ischaemia-reperfusion injury [1-5]. Generally, Ang II is a detrimental factor for the heart and its inhibition with angiotensin converting enzyme (ACE) inhibitor provides cardio protection [6]. There is no clear explanation for such paradoxical behaviors of Ang II. A number of different mechanisms of action have been put forward to explain the cardio protective ability of Ang II. Almost a decade ago, Ang II receptor stimulation was found to pre-condition rabbit myocardium [7]. Evidence is rapidly accumulating to support that Ang II can stimulate NADPH oxidase dependent superoxide generation by increasing activity of NADPH oxidase subunits (p22phox and gp91phos) [8]. A previous study showed the phosphorylaion of p47phox by Ang II with concomitant induction of reactive oxygen species (ROS) formation via NADPH oxidase [9].

The mitogen activated protein (MAP) kinases have been shown to play a crucial role in ischaemic PC [6, 10 and 11]. Several MAP kinases including ERK (1/2), JNK and p38MAPK are main targets of ROS signalling. Unlike ERK (1/2), which is activated by growth signal via Ras dependent signal trans-duction pathway, the activation of JNK and p38MAPK are potentiated by diverse stresses and proinflamatory cytokines [8]. To date, four members of the p38MAPK family have been identified: p38α, p38β, p38γ, p38δ[12]. Although these isoforms share functional similarities, difference exist in their upstream and downstream kinase specificity, sug-gesting that they may have non-overlapping functions [13]. In general, p38MAPKα is linked to death signal while p38MAPKβ linked with the survival signal [14]. Recent investigation from our laboratory showed that differential interaction and/or translocation of p38MAPKα/β with caveolin 1/3 play an important role in generation of survival signal during PC [15].

Since MAP kinases play a crucial role in ischaemic PC, we reasoned that Ang II might modulate MAP kinase signalling through its interaction and/or translocation with caveolin. Our results determined that Ang II indeed pre-conditioned the ischaemic heart by differentially regulating MAP kinase interaction with lipid raft thereby converting the death signal into a survival signal.

Materials and methods

Chemicals: Ang II and N-acetyl-cysteine (NAC) were obtained from Sigma (St Louis, MO, USA). Apocynin was obtained from Calbiochem, CA, USA. Antibodies against caveolin-1, caveolin-3, p38MAPKβ, eNOS and Bcl-2 were purchased from Santa Cruz Biotechnology, CA, Santa Cruz, USA. Antibodies against p38MAPKα, JNK, ERK, AKT, phospho-AKT were purchased from Cell Signalling technology (Danvers, MA, USA).

Experimental protocol

The study used isolated working rat hearts subjected to ischaemia/reperfusion protocol. Isolated rat hearts were randomly divided into seven groups: perfused with KHB buffer only for 15 min (group I); the hearts were perfused with Krebs-Henseleit biocarbonate buffer (KHB) buffer for 15 min in the presence of Ang II [100 nM] (group II); NAC [1 uM] (group III); Apocyanin [2mg/Kg, i.p. 4 hrs before experiment] (Group IV); Ang II + NAC (group V) and Ang II + apocynin (Group VI). All hearts were then subjected to 30 min global ischaemia followed by 2 hrs reperfusion with KHB buffer. Control hearts (group VII) after perfusion with KHB buffer for 15 min, were not exposed to ischaemia and reperfusion. They were only subjected to 2 hrs and 30 min continuous perfusion.

Isolated working rat heart preparation

Male Sprague Dawley rats of 250 gm body weight were used for this study. 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 Institute of Health (NIH Publication No. 85–23, revised 1996). The rats were anaesthetized with sodium pentobarbital (80 mg/kg b/w, ip.) (Abbott Laboratories, North Chicago, IL, USA) and anticoagulated with heparin sodium (500 IU/kg b.w, i.p) (Elkin-Sinn Inc., Cherry Hill, NJ, USA) injection.

After ensuring sufficient depth of anaesthesia, the hearts excised and then the isolated hearts were perfused in Langendorff mode (cannulation viaaorta) against at constant perfusion pressure of 100 cm of water (10 kPa) for stabilization period [16]. Any heart that showed any cardiac disturbance (ventricle arrhythmia, and fibrillation) during the entire experiment was excluded from this study. After 5 min perfusion in Langendroff mode the hearts were converted in working mode (i.e. left ventricular preparation) and perfused by working mode according to the protocol as described earlier. To ascertain the normal function of the heart, the heart rate, left ventricular developed pressure (the difference between the maximum systolic and diastolic pressure), left ventricular end-diastolic pressure and the first derivative of developed pressure were recorded by Gould p23XL transducer (Gould Instrument System Inc., Valley View, OH, USA). The signal was amplified by using Gould 6600 series signal conditioner (Gould Instrument System Inc.) and monitored on Cordat II real-time acquisition system (Triton technologies, San Diego, CA, USA) [17]. The aortic flow was measured by flow meter. The coronary flow was measured by time-collection of the coronary effluent dripping from the heart.

Measurements of the infarct size

After ischaemia/ reperfusion (I/R) procedure, the heart was infused with 10% solution of the Triphenyl tetrazolium (TTC) in phosphate buffer through the aortic cannula for 20 min [18]. The left ventricle was removed and sliced into 1-mm thickness of cross-sectional pieces and weighted. Each slice was scanned with computer-assisted scanner (Scanjet). The infarct zone remained unstained while the non-infract part of myocardium was stained red by TTC. These were measured by using of computerized software (Scion Image); areas were multiplied by the weight of each section, and summed up to obtain the total infarct zone. The infarct size was expressed as the ratio of the infarct zone to the non-infract zone.

TUNEL Assay for assessment of apoptotic cell death

Immunohistochemical detection of apoptotic cells was carried out by TUNEL assay using ApopTag in situ apoptosis detection kit (Intergen Company, Purchase, NY, USA) [19]. Tissue sections were prepared from left ventricular tissue of the heart. Cells were counted at 100X magnification and at least four fields per sample. The sections were incubated again with mouse monoclonal antibody recognizing cardiac myosin heavy chain to specifically recognize apoptotic cardiomyocytes. The fluorescence staining was viewed with a confocal laser microscope. The number of apoptotic cells was counted and expressed as a percent of total myocytes population.

Isolation of caveolin-rich membrane fractions

The hearts were homogenized in sodium carbonate buffer containing protease inhibitor cocktail, pH 11.0 using a Polytron homogenizer [three 10s bursts][Brinkman Instruments, Westbury, NY, USA]. The homogenate was sonicated [three 20s bursts], and adjusted to 45% sucrose by the addition of 2 ml of 90% sucrose prepared in MBS [25 mM Mes, pH 6.5, 0.15 M NaCl] and placed at the bottom of an ultracentrifuge tube as described previously [20]. A 35% discontinuous sucrose gradient was formed above [4 ml of 5% sucrose/4 ml of 35% sucrose–both in MBS containing 250 mM sodium carbonate] and centrifuged at 39,000 rpm for 16–20 hrs in an SW41 rotor [Beckman Instruments, Palo Alto, CA, USA]. From the top of each gradient, 1 ml gradient fractions were collected to yield a total of 12 fractions as described elsewhere. Caveolin migrates mainly in fractions 5 and 6 of these sucrose density gradients [20].

Immunoprecipitation with caveolin-1 and caveolin-3

Only caveolin rich fractions (fraction 5 & 6) were used for immunoprecipitation. Immunoprecipitation was performed with Protein-A sepharose CL-B4 (Pharmacia Biotech Inc.) using a polyclonal antibody against caveolin-1 or caveolin-3 [Santa Cruz Biotechnology] Incubation conditions were maintained as instructed by the supplier. Western blot analysis was then performed with antibodies against P38MAPKα P38MAPKβ, JNK, ERK, eNOS, AKT and Phospho-AKT and Bcl-2 according to established Method [21].

Statistical analysis

The values for number of apoptotic cardiomyocytes and infarct sizes as well as the functional parameters were all expressed as the Mean ± Standard Error of Mean (SEM) for at least six animals per group. The Western blot analyses were performed with at least three animals per group. The statistical analysis was performed by analysis of variance followed by Bonferroni's correction for any differences between the mean values of all groups. Differences between data were analysed for significance by performing a Student's t-test. The results were considered significant if P < 0.05.

Results

Effects of NAC and apocynin on Ang II pre-conditioning

As expected, pre-perfusion of the hearts with Ang II triggered the PC effect as evidenced by its ability to enhance post-ischaemic left ventricular function. As shown in Table I, Ang II group displayed better ventricular recovery as reperfusion progressed, and left ventricular developed pressure (LVDP), dp/dt, and aortic flow was significantly improved (P < 0.05) in the Ang II group compared to those of I/R group. This cardio protective ability of Ang II was abolished when Ang II treatment was done in presence of NAC or apocynin. There were no significant differences in coronary flow and heart rate among the groups at any time point. The groups of heart that received only NAC showed significant effects on the post-ischaemic ventricular recovery, but apocynin showed only better recovery (non-significant). However, both compounds abolished the PC effects of Ang II. The values of LVDP, dp/dt and aortic flow were significantly lower in the treated groups compared to Ang II group (Table 1).

1

Effect of NAC and Apocyanin on Ang II pre-conditioning. Isolated hearts were treated as mentioned in the methods section. The hearts were then subjected to 30 min ischaemia followed by 2 hrs reperfusion. Measurements were taken at the baseline and at the end of reperfusion. Results are expressed as mean ± SEM. *P < 0.05 versus I/R, †P < 0.05 versus Ang II. LVDP = Left Ventricular Developed Pressure, dp/dt = Max. First Derivative of Developed Pressure

	Control	I/R	Ang II	NAC	Apocynin	Ang II + NAC	Ang II + apocynin
Heart Rate (beats/min) % change from baseline	369 ± 17.3	414 ± 24.3	443 ± 12.5	421 ± 30.3	437 ± 26.8	422 ± 16.7	453 ± 19.2
12%↑		33%↑			20%↑				28%↑					30%↑						34%↑							32%↑
Aortic Flow (ml/min) % change from baseline	22.6 ± 4.1	4.2 ± 1.4	12.5 ± 2.7 *	7.9 ± 2.1 *	5.8 ± 0.5	4.7 ± 1.2†	3.7 ± 0.7†
30%↓		94%↓			83%↓				87%↓					90%↓						93%↓							94%↓
Coronary Flow (ml/min) % change from baseline	29.7 ± 1.9	21.5 ± 1.2	20.2 ± 1.7	22.4 ± 1.1	20.2 ± 0.9	21.9 ± 0.9	20.7 ± 1.4
12%↓		27%↓			25%↓				20%↓					25%↓						21%↓							21%↓
LVDP (mm Hg) % change from baseline	105 ± 5.8	42.5 ± 7.6	81 ± 3.4 *	70 ± 7.2 *	50.4 ± 2.7	69.8 ± 2.8†	58.6 ± 4.6†
16%↓		66%↓			36%↓				47%↓					50%↓						46%↓							56%↓
dp/dt (mm Hg/ Sec) % change from baseline	2772 ± 90	849 ± 70	1139 ± 93 *	988 ± 67	897 ± 78.4	908 ± 66.5†	885 ± 83†
40%↓		75%↓			60%↓				68%↓					69%↓						71%↓							69%↓

Effects of NAC and apocynin on infarct size lowering ability of Ang II

Figure 1A shows the effects of Ang II PC on the myocardial infarct size. The infarct size was about 37.6% for the I/R heart and Ang II reduced the infarct size by 31.2%. NAC and apocynin alone significantly reduced myocardial infarct size, but when used in conjunction with Ang II; they abolished infarct size lowering ability of Ang II.

1

Effect of Ang II, NAC & apocynin on the infract size of the heart and cardiomyocyte apoptosis. A:Infract size, B:car-diacmyocyte apoptosis.The results are mean ± SEM of six animal per group *P < 0.05 versus I/R, †P < 0.05 versus Ang II.

Effects of Ang II, NAC and apocynin on cardiomyocyte apoptosis

Ang II PC significantly lowered the number of apoptotic cardiomyocytes (Fig. 1B). The number of apoptotic cardiomyocytes was 24.7% in the I/R heart that had been subjected to 30 min ischaemia and 2 hr reper-fusion as compared to only 12.2% in the Ang II treated hearts. NAC and apocynin also significantly reduced cardiomyocyte apoptosis compared to control. When used in conjunction with Ang II, NAC and apocynin significantly increased the number of apoptotic cell compared to Ang II alone.

Effect of Ang II, NAC and apocyanin on differential interaction of caveolin & MAP kinase

Caveolin rich fractions (5 th and 6 th fraction of sucrose density gradient) of all seven groups of heart were immuno-precipitated with caveolin-1 and caveolin-3. The examination of the existence of p38MAPKα/β in the caveolin immunoprecipitated sample by Western blot revealed that high amount of p38MAPKα bind with caveolin-1 whereas interaction of caveolin-3 with p38MAPKα was very negligible. In contrast, high amount of p38MAPKβ bind with Caveolin-3 whereas its binding with caveolin-1 was very negligible. Earlier investigation of our laboratory showed the similar results [15]. JNK & ERK1/2 also bind with caveolin-1 whereas interaction with caveolin-3 is very negligible.

In I/R heart, there was increased association of p38MAPKβ & ERK1/2 (anti-death signalling component) with caveolin-3 and caveolin-1, respectively, while there was reduced association of p38MAPKα & JNK (death signalling component) with caveolin-1 indicating increased amount of death signalling component were available to I/R heart to generate a death signal. In contrast, in Ang II pre-conditioned heart, there was decreased association of anti-death signalling component (p38MAPKβ, ERK 1/2) with caveolin-3 and caveolin-1, respectively, while there was an increased association of death signalling components (p38MAPKα, JNK) with caveolin-1 indicating reduced amount of death signalling components and increased amount of anti-death signalling components were available to the Ang II treated heart to generate a survival signal. When Ang II pre–conditioned hearts were treated with NAC and apocynin, caveolin–MAPKs interaction showed a similar pattern like I/R heart making heart more abundance to death signalling component to induce death signal and suppress survival signal (Fig. 2).

2

Differential interaction of p38MAPK, JNK, ERK 1/2 and eNOS with caveolin-1 and caveolin-3 during ischaemia reperfusion, Ang II precondition in presence or absence of NAC or apocynin.The results are mean ± SEM of six animal per group *P < 0.05 versus I/R, †P < 0.05 versus Ang II.

In order to reconfirm our immunoprecipitation data, we tested all the 12 fraction (sucrose density gradient) of the Ang II pre-conditioned heart and found that p38MAPKβ and ERK was present in non-caveolin fraction and p38MAPKα and JNK was present in caveolin-rich zone. From these results it can be concluded that during Ang II PC, p38MAPKα and JNK (death signalling component) remain bound with caveolin and making them non-available to induce death signal. But at the same time p38MAPKβ and ERK (anti-death signalling component) remain in non-caveolin fraction and making the heart more abundance to them to induce survival signal (Fig. 3).

3

Distribution of p38MAPKα, p38MAPKβ, ERK and JNK in different fraction of the Ang II preconditioned heart.

Nitric oxide (NO) derived from vascular endothelium has many physiological effects related to the protection of the ischaemic-reperfused heart [22]. Similar to p38MAPKβ, interaction of eNOS with caveolin-3 was very high compared to its binding with caveolin-1. In Ang II treated heart, a negligible amount of eNOS bound with caveolin-3 (Fig. 2). Whereas more eNOS bound with caveolin-3 in I/R heart and NAC & apocynin treated heart, indicating more eNOS was available in Ang II pre-conditioned heart to induce cardio protection.

Effect of ANG II, NAC and apocynin on the expression of AKT, pAKT and Bcl-2

Bcl-2 and AKT are known to transmit survival signal. We did not find these proteins in the caveolin immunoprecipitated samples. The expression of these proteins was found in the cytosolic and crude fraction in of the heart. ANG II heart showed the upregulation of Bcl-2 compared to the control and I/R heart. NAC and apocynin abolished such increase in Bcl-2 protein (Fig. 4). Phospho-AKT was only present in the Ang II treated heart but not in the control, I/R, NAC and apocynin treated hearts.

4

Effect of Ang II, NAC & apocyanin on the induction of expression of Bcl-2, AKT and Phospho-AKT. The results are mean ± SEM of six animal per group *P < 0.05 versus I/R, †P < 0.05 versus Ang II.

Discussion

The results of the present study clearly demonstrated that Ang II could exert precondition-like cardioprotection confirming previous reports [1-6]. Ang II PC was associated with improved post-ischaemic ventricular recovery, reduced myocardial infarct size and decreased cardiomyocyte apoptosis [23]. Such PC effects of Ang II were almost completely abolished by ROS scavenger, NAC and NADPH oxidase inhibitor, apocynin as evidenced by depressed ventricular function and increased infarct size and cardiomyocyte apoptosis.

Although NAC and apocynin block the cardio protection offered by Ang II, cadioprotective abilities of NAC and apocynin were also demonstrated when they were treated alone (without Ang II). The dual nature of NAC and apocynin can be explained by the dual nature of ROS. ROS are central to cardiac ischaemic and reperfusion injury. They contribute to myocardial stunning, infarction and apoptosis, and possibly to the genesis of arrhythmias. Multiple laboratory studies and clinical trials have evaluated the use of scavengers of ROS to protect the heart from the effects of ischaemia and reperfusion. A possible role for oxygen radicals in PC and/or cardio protection was suggested by variety of laboratories. Administration of superoxide dismutase (SOD), an enzyme that removes superoxide anions, during the reperfusion period following the initial PC (short period of ischaemia), could prevent the phenomenon [24]. The authors hypothesized that myocardial reperfusion after the first, short ischaemic episode results in the generation of low amounts of oxygen free radicals, not sufficient to causes cell necrosis, but which could modify cellular activities and thus induce cardio protection. In another study, treatment with N-acetylcysteine negated the beneficial effect of PC on post-ischaemic recovery of contractile function [25]. Similarly, the beneficial effect of PC on reperfusion-induced arrhythmias was prevented by SOD administration during the PC [26]. Several mechanisms might explain the effects of oxygen radicals. Recently, it has become appreciated that exposure of cells to mild oxidative stress or low dose of oxygen radicals can reversibly modify several cellular activities, in the absence of cell damage, but secondary to changes in the activity of various enzymes and other cellular components [27, 28, 29]. Among others, low dose of ROS can modify some of the cellular activities that have been implicated in vivo as mediators of the signalling cascade of cardio protective phenomenon.

Earlier investigation from our laboratory showed that Ang II increased ROS activities, which were reduced by either NAC or apocynin. Most interestingly, Ang II induced the expression of two NADPH subunits, p22phox and gp91phox, which were completely inhibited with apocynin, and partially with NAC [8]. These results tend to suggest that Ang II PC be triggered by redox cycling of ROS, which are generated by both NADPH oxidase-dependent and independent pathway. [30, 23]. In another related study using renal homogenate, Ang II could stimulate NADPH oxidase dependent O2 generation [10]. NAD/NADPH oxidase is considered as the major source of ROS in VSMCs responsible for redox signalling [9]. ROS generated by Ang II could precondition the myocardium through the redoxregulated cell survival signalling pathways [11].

It is well established that ROS have either direct or indirect effect on MAPKs, under the family of serinethreonine kinase. MAPKs are considered as one of the major mediators of signal transduction in the stress activation pathway. Several recent studies have demonstrated that Ang II strongly phosphorylate ERK 1/2 [31-34]. In another study, Ang II induced robust phosphorylation of p38MAPK, ERK and JNK which was blocked by inhibition of NADPH oxidase, tyrosine kinase or ROS scavenger [35]. In another study using cardiac microvascular endothelial cells, Ang II activated ERK (1/2), but not p38MAPK in redox sensitive manner [36]. In smooth muscle cells, Ang II activated ERK 1/2 and p38 MAPK by a redox regulated mechanism [37]. The inhibition of JNK and p38MAPK activation was shown by using an antioxidant in the rat aortic smooth muscle cells, but interestingly the antioxidant did not change the activation of ERK (1/2) [38].

Lipid rafts are specialized membrane domain enriched with certain lipids, cholesterol and proteins. Caveolae, small plasma membrane invaginations that are coated with cholesterol binding protein, caveolin, are a subset of lipid raft. These lipid rafts serve as platforms for organizing and integrating signal transduction process. Recent investigation of our laboratory showed that p38MAPK translocation to caveolin during reperfusion and interacts differentially with caveolin-1 and caveolin-3. This differential interaction of p38MAPKα and p38MAPKβ with caveolin-1 and caveolin-3, respectively, function as a switch for conversion of I/R induced death signal to PC induced survival signal [13].

From the present results, it can be concluded that caveolin and MAPKs interaction play a very important role in generation of survival signal in Ang II preconditioned heart. In I/R heart anti-death signalling component (p38MAPKβ, ERK 1/2) bound with caveolin and binding of the death signalling component (p38MAPKα, JNK) with caveolin is less—exposing the heart to more abundance death signalling components thereby generating death signal. In contrast, in Ang II preconditioned heart binding of anti-death signalling component with caveolin is much less compared to the I/R heart and exposing the heart to more abundance anti-death signalling component, generating a survival signal. It appears that differential translocation and/or interaction of MAPKs with caveolin functions as a switch for the conversion of I/R induced death signal into Ang II PC induced survival signal.

NO derived from vascular endothelial has many physiological effects related to the protection of the ischaemic-reperfused heart [30]. Here, in the present investigation, only a negligible amount eNOS bound with caveolin-3 in Ang II pre-conditioned heart, whereas more eNOS bound with caveolin-3 in I/R heart indicating generation of survival signal by free eNOS in Ang II pre-conditioned heart. The survival signal was further confirmed by increased phosphorylation of AKT and enhanced induction of expression of Bcl-2 during Ang II pre-condition and its reversal with I/R, NAC & apocynin treated heart.

This result indicated that caveolin play a unique role in Ang II PC of the heart by interacting with different MAPKs and eNOS. In another ward, caveolin control the generation of survival signals in Ang II pre-conditioned heart by controlling the availability of survival signalling components to induce cardio protection.