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Cardiovascular Research 2003 57(1):129-138; doi:10.1016/S0008-6363(02)00614-4
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Copyright © 2003, European Society of Cardiology

The functional role of the JAK–STAT pathway in post-infarction remodeling

Hala El-Adawia, Lili Denga,c, Anthony Tramontanob, Steven Smitha, Eduardo Mascarenoc, Kalyan Gangulya, Ricardo Castillob and Nabil El-Sherifa,b,*

aNew York Harbor VA Healthcare System, Brooklyn Campus, Brooklyn, NY 11209, USA
bDepartment of Medicine, State University of New York, Downstate Medical Center, Box 1199, 450 Clarkson Avenue, Brooklyn, NY 11203, USA
cDepartment of Anatomy and Cell Biology, State University of New York, Downstate Medical Center, Brooklyn, NY 11203, USA

* Corresponding author. Tel.: +1-718-270-4147; fax: +1-718-630-3740. nelsherif{at}aol.com

Received 18 April 2002; accepted 19 July 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Objectives: Recently, the Janus kinase/signal transducer and activator of transcription (JAK–STAT) signaling pathway was found to be prominently associated with activation of the autocrine loop of the heart tissue-localized renin angiotensin system (RAS). We investigated if the JAK–STAT pathway is activated in the post-myocardial infarction (MI) non-ischemic myocardium (NIM), destined to undergo remodeling and whether blockade of the pathway in vivo can modify early post-MI remodeling. Methods: We investigated the time course of tyrosine phosphorylation of JAK–STAT and gp130 proteins in the NIM of post-MI rat heart as well as the binding activity of STAT proteins to the St-domain of the angiotensinogen gene promoter. We further compared the effects of in vivo blockade of RAS by the AT1 receptor (AT1R) blocker losartan with the in vivo blockade of JAK–STAT pathway by the specific JAK2 blocker tyrphostin AG490 on certain aspects of early post-MI remodeling. Results: We showed that JAK2, STATs 1, 3, 5a and 6 and gp130 proteins are tyrosine phosphorylated as early as 5–30 min post-MI and that STATs 1, 3, and 5a remain activated up to 7 days post-MI. Gel mobility shift assay showed a strong binding activity of STAT proteins to the St-domain of angiotensinogen gene promoter in 1-day post-MI NIM. The binding was significantly reduced in rat hearts previously treated with losartan or tyrphostin AG490. Supershift experiments identified STATs 3 and 5a as specifically interacting with the St-domain. Both AT1R and JAK2 blockade resulted in significant amelioration of the increase of protein phosphatase 1 activity and decrease in basal level of p16-phospholamban that may underlie early diastolic dysfunction, as well as partial amelioration of early downregulation of Kv4.2 gene expression that may underlie increased arrhythmogenicity of 3-day post-MI heart. On the other hand, while blockade of AT1R significantly ameliorated apoptotic changes in 1-day post-MI border zone, blockade of JAK2 increased apoptosis. Conclusions: The study provides compelling evidence in favor of the linkage of the JAK–STAT pathway with the angiotensin II autocrine loop and uncovers a mechanism by which selective activation of a set of STAT proteins underlies mobilization of the gene activation program intrinsic to post-MI remodeling. It also suggests that drugs that inhibit JAK–STAT phosphorylation may provide a new approach to modify post-MI remodeling. This needs to be confirmed in long term in vivo studies in the post-MI heart.

KEYWORDS Apoptosis; Infarction; Remodeling; Renin angiotensin system; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
A plethora of experimental and clinical data show that the renin-angiotensin system (RAS) plays a major role in post-MI remodeling [1,2]. More recently, the Janus kinase/signal transducer and activator of transcription (JAK–STAT) signaling pathway was found to be prominently associated with activation of the autocrine loop of the heart tissue-localized RAS [3]. Angiotensin II (ANGII) uses a signal pathway in cardiac myocytes in which the promoter of the gene encoding its prohormone, angiotensinogen (ANG), serves as the target site for the STAT proteins. The JAK–STAT pathway is also a major signal transduction pathway of the cytokine superfamily through the gp130-dependent pathway [4]. Recently, the JAK–STAT pathway was shown to be activated in ischemic myocardium [5–7]. However, its role in the remodeling of the post-MI non-ischemic myocardium (NIM) remains largely unexplored. The present study will show that the JAK–STAT pathway is activated early in the post-MI NIM. Further, the functional role of the activated pathway was contrasted with that of RAS by investigating the effects of specific blockade of either pathways in vivo on selected functional aspects of early post-MI remodeling.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
2.1 Experimental model
The post-MI rat model: 2-month-old Sprague–Dawley rats weighing 200 to 250 g underwent left anterior descending artery (LAD) ligation or sham operation, as previously described [8]. The LAD was ligated ~2–3 mm from its origin, which resulted in a moderate size infarct (31–46% of left ventricle, LV). Animals were sacrificed at various time intervals after LAD ligation. Sham-operated animals underwent thoracotomy without LAD ligation and served as controls. For studies conducted up to 3 days post-MI the ischemic/infarcted zone and the non-ischemic zone were identified by means of injection of 1% Evans blue dye into the inferior vena cava before sacrifice. The viable area was stained blue, while the infarcted area remained pale. After 3 days post-MI, the infarct tissue was identified visually or by examination of Van-Giesen stained transverse LV sections. With the help of microdissection, tissue samples representing the NIM were obtained from the free LV wall excluding the ischemic/infarcted area plus the border zone. The border zone was defined as the 1-mm area next to the ischemic/infarcted area. Infarct size was determined planimetrically as the ratio of infarct tissue or scar to the length of the entire LV endocardial circumference as described previously [9]. It has been shown that infarct size significantly affects post-MI remodeling [9]. Data were collected only from rats with moderate (31–46%) or large (<46%) infarcts. The investigation conformed with the Guide for the Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 1996).

2.2 Drug protocol
One group of rats received the AT1R antagonist losartan. The drug was administered to rats 1 week prior to LAD ligation in a dose of 10 mg/kg/day [10]. The drug was added to the drinking water, with careful monitoring of water consumption and body weight to ensure proper drug dosage. In the second group of rats the JAK2 inhibitor tyrphostin AG490 was administered intraperitoneally in a total dose of 5 mg/kg/day, starting 2 h prior to LAD ligation [11].

2.3 Hemodynamic measurements
Hemodynamic measurements were obtained before the heart was removed from sham-operated rats (n=5), 3-day post-MI rats (n=5), and 3-day post-MI rats pretreated with losartan (n=5) or tyrphostin (n=5). A 2-F micromanometer-tipped catheter (model SPR-407 Miller Instrument, Houston, TX) was inserted through the right carotid artery into the aorta and subsequently advanced to the LV. LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), peak positive and peak negative first derivative of LV pressure (+dP/dt and –dP/dt) were calculated as previously reported by averaging 10 consecutive cycles [12].

2.4 Tyrosine phosphorylation studies
Monoclonal or polyclonal antibodies to JAK2, STAT proteins 1, 3, 5a and 6, gp130, and anti-phosphotyrosine were obtained from Upstate Biotechnology, Santa Cruz Biotechnology, and Sigma. Tissues were harvested from sham-operated LV, and from the post-MI non-ischemic LV at 5, 30, 120 min and 1, 3, and 7 days post-MI. To prepare cell extracts, cells were washed in PBS and then extracted in lysis buffer containing (in mmol/l) Tris–HCl (pH 7.4) 20, NaCl 100, EDTA 5, NaF 50, Na3P2O7 10, Na3VO4 1, phenylmethylsulfonyl fluoride 1 and 1.0% Triton X-100, 10% glycerol, 0.1% SDS, 1.0% deoxycholic acid, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. The lysates were centrifuged at 10 000xg for 15 min. Protein concentration was determined by the Bio-Rad protein assay. Cell lysates were incubated with 1 µg/ml of the respective antibodies overnight at 4 °C. Immunocomplexes were collected by incubating with 50 µl of protein A- or G-Sepharose for 2 h. Immunoprecipitates were washed four times with ice-cold lysis buffer. The pellets were re-suspended in 2x sample buffer containing 50 mmol/l Tris (pH 6.8), 2% SDS, 2% β-mercaptoethanol, 2% glycerol, and bromophenol blue. The samples were subjected to SDS–PAGE and were transferred to reinforced nitrocellulose membranes (Schleicher & Schuell). The membranes were blocked with 5% BSA in Tris-buffered saline–Tween solution (20 mmol/l Tris–HCl (pH 7.41), 150 mmol/l NaCl, and 0.05% Tween 20) for 2 h at room temperature. Blots were immunolabeled overnight at 4 °C with anti-phosphotyrosine antibody. Proteins were visualized by enhanced chemiluminescence (Amersham). The blots were stripped and reprobed with the same antibodies used for their immunoprecipitation, to ensure equal loading of the proteins.

2.5 St-domain binding studies
Tissues harvested from sham-operated LV and 1-day-old post-MI non-infarcted LV were dissociated into cells and the nuclei prepared by dounce homogenization in the appropriate lysis buffer. Nuclei were collected by centrifugation, treated with 0.5 M NaCl for 1 h at 4 °C, and re-centrifuged and aliquots of the nuclear lysates were frozen in liquid N2. The nuclear lysates were used in a gel mobility shift assay (GMSA) using as probe the St-domain of the ANG gene promoter. The St-domain is the promoter element that binds STAT proteins (the nucleotide sequence is: 5'-GGGTTCCTGGAAGGG-3') [3]. All radioactively labeled probes were incubated with nuclear lysate at 4 °C for 30 min. Protein–probe complexes were electrophoresed in 8% polyacrylamide gel and visualized by autoradiography. To identify which of the activated STAT proteins specifically bind to the St-domain of the ANG gene promoter supershift experiments were conducted as follows: prior to addition of St-domain probes, antibodies to the STAT proteins 1, 3, 5a and 6 were added to the nuclear lysates and incubated on ice for 4 h, then the St-domain probe was added and incubated as outlined above.

2.6 Protein phosphatase 1 activity
Assays were performed using the manufacturer's protocol (protein phosphatase assay system; Life Technologies, Rockville, MD, USA). Phosphatase activity was measured at 30 °C using P-labeled phosphorylase a as substrate in noninfarcted LV myocardium obtained from sham-operated animals, 3-day post-MI animals and 3-day post-MI animals pretreated with losartan or tyrphostin.

2.7 Phospholamban phosphorylation
To compare the level of phosphorylated phospholamban and total phospholamban in the same four groups of animals, the antibody p-16 that recognized phosphorylated Ser16 in phospholamban, which is known to be the main phosphorylation site for protein kinase A, was used. After a second antibody and luminescent detection, the membrane was stripped and relabeled with antibody A1, which recognized total phospholamban [12].

2.8 K+ channel gene expression
Details of the preparation of RNA from ventricular myocardium and RNase protection assay have been reported previously [13]. Noninfarcted LV myocardium from the LV free wall was dissected from sham-operated animals, 3-day post-MI animals and 3-day post-MI animals pretreated with losartan or tyrphostin. The tissues were rinsed in saline to remove excess blood, snap frozen in liquid nitrogen, and stored at –70 °C. Total RNA was extracted from the LV using the standard protocol of Chomczyniski and Sacchi of homogenization in acid guanidinium thiocyanate followed by phenol–chloroform extraction and ethanol precipitation [14]. The amount of RNA recovered in each sample was determined spectrophotometrically at a wavelength of 260 nm and the integrity of each sample confirmed by analysis on a denaturing agarose gel. Quantitative evaluation was performed using scanning densitometric analysis. For comparisons between groups, the arbitrary densitometric units were normalized to the value of the cyclophilin gene.

2.9 Measurement of caspase-3 activity
The activity of caspase-3 was determined in the 1-day post-MI border zone with the caspase-3 cellular activity assay kit (Biomol Research, PA, USA) according to the manufacturer's instructions. The activity was measured by means of detection of chromophore p-nitroanilide after cleavage from the labeled substrate Asp–Glu–Val–Asp (DEVD)–p-nitroanilide. Tissue samples (10 mg) were solubilized by using a Polytron homogenizer with a cell lysis buffer (50 mM HEPES, pH 7.4, 0.1% CHAPS, 1 mM DTT, 0.1 mM EDTA). Equal amounts of protein lysates were then reacted with 50 µM DEVD–p-nitroanilide for 1 h at 37 °C. The activity was determined in a microtiter plate-reader at 405 nm and the results were calibrated with known concentrations of p-nitroanilide.

2.10 Apoptosis assay
To visualize apoptotic nuclei in cardiac myocytes in the 1-day post-MI border zone, in situ labeling of fragmented DNA was performed using In Situ Death Detection Kit, Fluorescein (Roche Molecular Biochemicals). Sections for formalin fixed and paraffin-embedded rat left ventricular myocardium were placed on charged slides. Sections were dewaxed with microwave irrigation (370 W for 5 min in 200 ml of 0.1 M citrate buffer, pH 6). Slides were then cooled rapidly by immediately adding 80 ml of distilled water (20–25 °C). Slides were immersed for 30 min at room temperature in 0.1 M Tris–HCl, containing 3% BSA, pH 7.5 then rinsed twice with PBS at RT. Cell permeabilization was achieved by rinsing slides in 0.1% Triton X-100, 0.1% sodium citrate for 2 min on ice (4 °C) after which slides were then rinsed twice with PBS. TUNEL reaction mixture (100 µl) (terminal deoxynucleotidyl transferase from calf thymus mixed with nucleotide mixture in reaction buffer) was added to each slide after which a coverslip was applied. Slides were then incubated for 30 min at 37 °C in a humidified chamber, washed three times in PBS for 5 min, and evaluated under a fluorescence microscope. Normal myocardial tissue sections (Sham) were pretreated with Dnase I (IU/ML) dissolved in 50 mM Tris–HCl, pH 7.5, 1 mM MgCl2, 1 mg/ml BSA for 10 min at 25 °C to induce DNA strand breaks to serve as a positive control. Biotin-dUTP was omitted in negative controls. The sections were examined by light microscopy and an apoptotic index was calculated as the percentage of positive myocytes observed in 10-high power fields per section.

2.11 Measurement of apoptosis-related proteins
The expression of the apoptosis-related proteins Bax and Bcl-xL in the 1-day post-MI border zone was examined by Western blot analysis. Equal amounts of protein (30 µg) were separated by 20% SDS–PAGE gel and immunoblotted with anti-Bax and anti-Bcl-2 (Upstate Biotechnology).

2.12 Statistical analysis
Statistical analysis was performed by means of Student's t-test and one-way ANOVA followed by the Bonferroni procedure for multiple group comparisons. A value of P<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
Table 1 shows the effects of pretreatment with losartan or tyrphostin on hemodynamic parameters in 3-day post-MI rats. Compared to sham-operated animals, 3-day post-MI rats had significantly lower LVSP, higher LVEDP, and decreased +dP/dt and –dP/dt. Losartan-treated 3-day post-MI rats had a significantly lower LVEDP and higher –dP/dt compared to control post-MI rats. The +dP/dt was also higher than non-treated animals but the difference did not reach statistical significance. The tyrphostin-treated rats showed similar hemodynamic changes to the losartan-treated group. Thus both drugs seem to improve the early diastolic dysfunction in the 3-day post-MI rat.


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  		Table 1 Effects of pretreatment with losartan (L) or tyrphostin (T) on hemodynamic parameters in 3-day post myocardial infarction (MI) rat

 
Fig. 1A shows that phosphorylation of JAK2 in the NIM was detected as early as 30 min post-LAD ligation and peaked between 2 h and 1-day post-MI. Phosphorylation of STAT proteins 1, 3, 5a and 6 and gp130 protein followed closely the same time course of JAK2 phosphorylation. Fig. 1B–D shows that STATs 1, 3 and 5a phosphorylation remained significantly elevated up to 7 days post-MI. STAT 6 and gp130 phosphorylation returned to control levels in the 3-day post-MI samples (not shown).


Figure 1
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  		Fig. 1 (A–D) Time course of tyrosine phosphorylation of JAK–STATs and gp130 proteins in the remote nonischemic post-MI myocardium. (E) Tyrphostin (5 mg/kg/day) specifically inhibits JAK2 phosphorylation. Data were collected from five different experiments for each time frame. *P<0.05, **P<0.01.

 
The specificity of tyrphostin in a dose of 5 µmol/l/day as selective inhibitor of JAK2 phosphorylation was first shown in studies of acute lymphoblastic leukemia [11] and was later confirmed in rat cardiomyocyte culture [15]. We tested the specificity of tyrphostin in vivo on two other key kinases that are activated in the 1-day post-MI NIM and found that tyrphostin at 5 mg/kg/day has no effect on activated JNK and Akt but specifically inhibited JAK2 phosphorylation (Fig. 1E).

Fig. 2 shows the results of GMSA. Fig. 2A shows a strong St-domain/STAT binding activity in 1-day old post-MI NIM. Binding was significantly reduced in post-MI rat heart previously treated with either the AT1R blocker, losartan or the JAK2 blocker, tyrphostin. Specificity of binding by STAT proteins to the wild-type St-domain was demonstrated by competing out specific STAT–St-domain binding by co-incubating with a 100x excess of non-radioactive St-domain probe. A mutated form of the probe was also used as negative control to further ensure specificity of binding by STAT proteins. Fig. 2B illustrates a supershift experiment to identify the STAT proteins that specifically interact with the St-domain and shows that the STAT–DNA complexes were disrupted significantly by anti-STAT 3 and anti-STAT 5a.


Figure 2
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  		Fig. 2 Gel mobility shift assay in 1-day-old post-MI remote myocardium. The figure represents one of four different experiments each showing similar results. Los, losartan; Tyr, tyrphostin.

 
Fig. 3A shows that protein phosphatase 1 activity significantly increased in the NIM 3-day post-MI by 32% compared to sham. Three-day post-MI rats treated with losartan or tyrphostin showed significantly less increase of protein phosphatase 1 activity (15% and 12%, respectively, P<0.05). Fig. 3B compares the level of p16-phospholamban and total phospholamban in sham, 3-day post-MI rats and 3-day post-MI rats treated with losartan or tyrphostin. There was a 52% decrease in the basal level of p16-phospholamban in the NIM of 3-day post-MI rat. In 3-day post-MI rats treated with losartan or tyrphostin, the decrease in basal level of p16-phospholamban was significantly ameliorated (52% vs. 15% vs. 10% in sham, losartan group, and tyrphostin group, respectively, P<0.01).


Figure 3
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  		Fig. 3 (A) Protein phosphatase activity in preparations (membrane vesicles) from sham, 3-day post-MI, 3-day post-MI+losartan (MI+Los), and 3-day post-MI+tyrphostin (MI+Tyr). Data were collected from four different experiments for each group. (B) Representative Western plot analysis of p16-phospholamban (PLB) and total PLB protein in the same four groups. Data were collected from four different experiments for each group. *P<0.05, **P<0.01. See text for details.

 
Fig. 4 shows the Kv4.2 mRNA level in sham, 3-day post-MI rats, 3-day post-MI rats treated with losartan, and 3-day post-MI rats that received tyrphostin. The mRNA level of Kv4.2 was significantly decreased by 61% in the NIM of 3-day post-MI rats compared to sham (P<0.01). The decrease in Kv4.3 mRNA level was significantly ameliorated in rats treated with losartan (38%) or tyrphostin (39%), P<0.05.


Figure 4
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  		Fig. 4 Comparison of cardiac Kv4.2 gene expression by RNase protection assay in sham, 3-day post-MI, 3-day post-MI+tyrphostin (MI+Tyr), and 3-day post-MI+losartan (MI+Los) rat groups. The myocardial sample contained 10 µg total RNA and the negative sample contained 10 µg of yeast RNA. The bottom of the figure shows a bar graph of Kv4.2 mRNA level in the four groups (n=4 for each group); *P<0.05, **P<0.01. See text for details.

 
Fig. 5 shows caspase-3 activity in sham-operated LV, in the border zone of 1-day post-MI rat heart and 1-day post-MI rats treated with losartan or tyrphostin. The figure shows that caspase-3 activity in 1-day post-MI border zone increased 1.4 times compared to sham (P<0.05). In post-MI rats that received losartan the increase in caspase-3 activity was markedly reduced (1.1 times compared to sham). On the other hand, in rats treated with tyrphostin, caspase-3 activity was significantly higher (1.7 times compared to control post-MI rats, P<0.05). The specificity of the measurement was confirmed by the addition of a specific caspase-3 inhibitor DEVD-CHO (20 mm) to the sample reaction that resulted in suppression of caspase-3 activity to control level.


Figure 5
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  		Fig. 5 Caspase-3 activity in sham (S), sham+tyrphostin (S+T), and from the border zone of 1-day post-MI (MI), 1-day post-MI+tyrphostin (MI+T), and 1-day post-MI+losartan (MI+L) rat groups (n=4 for each group); *P<0.05. See text for details.

 
Fig. 6 shows TUNEL-positive nuclei from sham-operated LV, from the border zone of 1-day post-MI rats, and from rats treated with losartan or tyrphostin. There was a significant increase in TUNEL-positive cells in the border zone of 1-day post-MI rat (P<0.05). In losartan-treated rats the number of TUNEL-positive cells was markedly decreased and was not significantly different from sham. On the other hand, in rats treated with tyrphostin, there was significant increase of TUNEL-positive cells compared to control post-MI rats (P<0.05).


Figure 6
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  		Fig. 6 TUNEL-positive nuclei from sham, and from the border zone of 1-day post-MI (MI), 1-day post-MI+tyrphostin (MI+Tyr), and 1-day post-MI+losartan (MI+Los) rat groups (n=4 for each group). *P<0.05. See text for details.

 
To obtain some understanding of the molecular basis of tyrphostin- and losartan-related effects on apoptosis in the 1-day post-MI border zone we investigated the expression of apoptosis-related proteins in the border zone of sham-operated rats, 1-day post-MI rats, and 1-day post-MI rats treated with tyrphostin or losartan (Fig. 7). Both the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-xL increased in the border zone of control post-MI rats. Tyrphostin-treated rats showed an insignificant increase in Bax protein but a significant decrease in Bcl-xL protein compared to control post-MI rats resulting in an unfavorable Bax/Bcl-xL ratio. On the other hand, losartan-treated rats showed a significant decrease of Bax protein and a significant increase of Bcl-xL protein compared to control post-MI rats.


Figure 7
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  		Fig. 7 Western plot analysis of apotosis-related proteins, Bax and BcL-xL from sham and from the border zone of 1-day post-MI (MI), 1-day post-MI+tyrphostin (MI+Tyr), and 1-day post-MI+losartan (MI+Los) (n=4 for each group). *P<0.05, **P<0.01. See text for details.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
4.1 The JAK–STAT pathway and the heart
There has been increased interest in recent years in the role that the JAK–STAT pathway plays in cardiac pathology. A recent report showed that three of the four JAK proteins, JAK1, JAK2, and Tyk2 but not JAK3 and all seven STAT proteins, STAT 1, 2, 3, 4, 5A, 5B, and 6 are present in the mouse heart [6]. Pan et al. were the first to report that JAK1, JAK2, and Tyk2 were rapidly tyrosine-phosphorylated after the heart was exposed to pressure overload in vivo; subsequently, STAT 1, STAT 2 and STAT 3 were also tyrosine-phosphorylated [15]. These changes were partially reversed by AT1R blockade. Subsequently, Mascareno et al. reported that JAK2, STAT 3, and STAT 6 were selectively activated by ANGII treatment of cardiomyocytes in culture and that there was a significant increase in the St-domain binding activity of STAT proteins in the hypertrophied heart of the genetically hypertensive rat relative to that of age-matched normotensive rat [3].

Although recent reports have shown that the JAK–STAT pathway is induced in acute ‘ischemic’ myocardium, its role has been controversial. Negoro et al. have shown that activation of JAK–STAT pathway transduces the cytoprotective signal in rat acute MI [16]. This seems consistent with a recent report that demonstrated the beneficial role of the JAK–STAT pathway in the late phase of ischemic preconditioning in the mouse heart [6]. Ischemic preconditioning induced selective activation of JAK1, JAK2, STAT 1 and STAT 3 and ablation of this response impeded the up-regulation of iNOS and the concurrent acquisition of ischemic tolerance. On the other hand, Mascareno et al. have recently reported that JAK2, STATs 5a and 6 are activated in an ischemia–reperfusion rat model and that JAK2 inhibition by tyrphostin produced cytoprotective effects [7]. It thus appears that the role of the JAK–STAT pathway may differ according to the experimental model.

4.2 The JAK–STAT pathway is activated in the post-MI NIM
We have shown, for the first time, that JAK2–STATs 1, 3, 5a and 6 and gp130 proteins are tyrosine phosphorylated in the NIM early after regional myocardial ischemia. STAT 1, 3 and 5a remain activated at least up to 7 days post-MI and STATs 3 and 5a specifically bind to the St-domain of ANG gene promoter, thus resulting in continued activation and maintenance of the autocrine loop of ANGII. Mechanical stretch appears to be the immediate stimulus for activation of RAS and gp130-related pathways in the post-MI NIM. The loss of contractility of the ischemic zone within the first few minutes of ischemia results in abrupt increase in loading conditions in the non-ischemic zone, which becomes subjected to stretch [17]. This is somewhat similar to increased loading and stretch that the entire LV is subjected to following pressure or volume overload. Immunoreactive ANGII has been localized in secretory granules in ventricular myocytes, and it has been shown that stretching of cardiac myocytes in vitro causes release of ANGII acutely (10–30 min) and increases the expression of the ANGII gene in the long-term [18]. It was later reported that the JAK–STAT pathway is activated by mechanical stretch and that this activation was partially dependent on autocrine/paracrine-secreted ANGII but was mainly dependent on the interleukin-6 family of cytokines [19]. Similarly, phosphorylation of gp130 protein is rapidly detected at 2 min after stretching of cardiomyocytes in vitro [19]. Thus there seems to be a convergence of both the RAS pathway and the gp130-related pathway for early activation of the JAK/STAT system in the NIM.

4.3 Functional role of the JAK–STAT pathway on early post-MI remodeling of remote NIM
The present study has shown that activation of the JAK–STAT pathway in post-MI remote myocardium could be inhibited by specific blockade of the AT1R or of JAK2 phosphorylation. However, blockade of the two pathways is shown to have both similar and contrasting effects. In a previous study, we have shown that downregulation of key K+ channel genes and currents occurs as early as 3 days in the post-MI NIM and contributes to increased arrhythmogenicity in the early post-MI period [20]. Losartan and tyrphostin were equally effective in ameliorating the changes in K+ channel genes. Similarly, losartan and tyrphostin seem to be equally effective in counteracting the early decrease in basal level of p16-phospholamban and both drugs were shown to significantly ameliorate the early diastolic dysfunction in the 3-day post-MI rat. We have previously reported that in the 3-week post-MI heart, remodeled hypertrophy of NIM is at its maximum and the heart is usually in a compensated stage with no evidence of heart failure. However, significant diastolic dysfunction of the hypertrophied myocardium was present and was attributed, to a large extent, to decreased basal level of p16-PLB caused by increased protein phosphatase 1 activity [12]. The present study shows, for the first time, that the increase in protein phosphatase 1 activity and subsequent decreased level of p16-phospholamban, the main regulatory protein of SERCA2, occurs as early as 3 days post-MI in the remote NIM and seems to be dissociated from the hypertrophic response per se.

On the other hand, blockade of AT1R and JAK2 phosphorylation produced contrasting effects on apoptotic changes in the 1-day post-MI border zone. While there is no evidence that apoptosis in the border zone contributes significantly to post-MI remodeling, continued apoptosis in the remote myocardium with the resulting loss of cardiomyocytes may influence the remodeling process [21,22]. Little data are available on the complex interplay of hypertrophic, pro-apoptotic and anti-apoptotic signals on post-MI remodeling.

4.4 Study limitation
The present study does not address the specific molecular mechanisms and signaling pathways involved in the downregulation of K+ gene expression, the increase in protein phosphatase 1 activity, or apoptosis in the post-MI heart as it relates to either the ANGII–AT1R pathway or the JAK–STAT pathway. These areas of research should be of considerable interest.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
We have shown that the JAK–STAT pathway is activated in the post-MI remote NIM and that the activation could be blocked in vivo by a specific inhibitor of JAK2 phosphorylation. Blockade of the JAK–STAT pathway has beneficial functional consequences on early post-MI remodeling equivalent to that of the AT1R blockade. However, there are contrasting effects of blockade of the two pathways on at least one aspect of early post-MI remodeling, i.e. apoptotic changes in 1-day post-MI border zone. The similar/contrasting effects of blocking of either pathways on the various aspects of long term post-MI remodeling including hypertrophy, diastolic and systolic dysfunction, and survival remain unknown. The present findings indicate the need of in vivo studies of the long-term effects of blockade of the JAK–STAT pathway in the post-MI heart.

Time for primary review 26 days.


   Acknowledgements
 
Supported in part by VA MERIT and REAP grants to Nabil El-Sherif, MD.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 

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