Signal transducer and activator of transcription (STAT) 3 is involved in mediating a broad range of biological processes, including cell survival, proliferation, and immune response. Recent evidence has indicated that STAT3 in cardiomyocytes can be activated by ischemic-oxidative stress and exerts cardioprotection in the ischemic heart. There is no information, however, regarding the effect of endothelial cell-derived STAT3 on the myocardial response to ischemiareperfusion (I/R) injury. We hypothesized that the ablation of the STAT3 gene in endothelial cells would worsen postischemic myocardial function by affecting capillary network integrity, suppressing antiapoptotic signaling. Isolated hearts from wild-type and endothelial cell STAT3 knockout (STAT3KO) mice were subjected to 20 min of global ischemia followed by 60 min of reperfusion. Endothelial cell STAT3 deficiency decreased recovery of myocardial function in response to I/R, which was associated with higher levels of LDH release, decreased activation of myocardial STAT3, and elevated p38 MAPK activation in STAT3 endothelial knockout (KO) hearts. In addition, although no significant apoptosis was observed in wild-type and KO hearts, our results showed more expression of myocardial caspase-8 and more apoptosis in the myocardium around the capillary in STAT3KO mice subjected to I/R. Furthermore, endothelial cell STAT3 ablation resulted in increased myocardial expression of IL-6 and suppressor of cytokine signal 3. This study demonstrates that endothelial cell-derived STAT3 plays an important role in postischemic myocardial function.
- signal transducer and activator of transcription 3
- myocardial function
the signal transducer and activator of transcription (STAT) family genes are regulated by numerous of biological processes and have been implicated in a variety of cellular function (4, 5, 33, 38). There are seven known STATs (STAT 1, 2, 3, 4, 5a, 5b, and 6), of which the STAT3 pathway is most notably activated by the interleukin-6 (IL-6) family of cytokines via receptor glycoprotein 130 (6). The IL-6 cytokine family (IL-6, leukemia inhibitory factor, and cardiotrophin-1) binding results in receptor dimerization, thereby inducing tyrosine phosporylation and activation of STAT3 through Janus-activated kinases. Activated STAT3 then dimerizes and translocates to the nucleus to activate or repress downstream target gene expression.
The myocardium generates inflammatory mediators in response to ischemia-reperfusion (I/R) injury (18, 28, 35, 36, 41, 42). These inflammatory mediators contribute to myocardial functional depression and cardiomyocyte apoptosis. Since STAT3 signaling has been reported to play critical roles in the regulation of inflammatory response pathways (6, 7, 14), it is therefore assumed that the STAT3 pathway may be involved in mediating the myocardial response to I/R injury. Indeed, accumulated evidence has indicated that STAT3 can be activated by ischemic-oxidative stress and that this signaling pathway exerts cardioprotection in the ischemic heart (8, 21). Blockade of STAT3 pathway enhances myocardial injury after an infarction (22). Evidence from cardiomyocyte-restricted ablation of STAT3 mice further indicates that STAT3 protects the heart from ischemic injury through suppressing cardiomyocyte apoptosis, inducing local growth factor production (8).
Although recent evidence has indicated that STAT3 plays a cardioprotective role in the heart, no information exists regarding the effect of endothelial cell (EC)-derived STAT3 on the myocardial response to I/R injury. Indeed, there are numerous ECs present within the heart in addition to cardiomyocytes and fibroblasts, given that the heart has a large number of blood vessels. In addition, ECs may mediate the myocardial immune response through cell surface or local factor production during injury (26, 27). Furthermore, ECs have been reported to require STAT3 for protection against endotoxin-induced inflammation (12). A recent study has shown that STAT3 is involved in regulating many proinflammatory genes in the vascular endothelium (43). Therefore, it is necessary to elucidate whether ECs with STAT3 ablation affect myocardial dysfunction following I/R injury and, if so, by what mechanisms.
In this study, we hypothesized that ablation of the STAT3 gene in ECs might worsen postischemic myocardial function. The purposes of this study were to determine the effects of EC STAT3 on myocardial function and myocardial inflammatory pathways by using mice with a conditional deletion of STAT3.
MATERIALS AND METHODS
The STAT3-deficiency mouse line was of a C57B6 background and has been described previously (39). Four- to six-week-old wild-type (WT) and EC-restricted STAT3 knockout (STAT3KO) mice were maintained in a quiet quarantine room for 1 mo before the experiments. The animal protocol was reviewed and approved by the Indiana Animal Care and Use Committee of Indiana University. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996).
A total of 11 isolated mouse hearts (n = 6 for WT; n = 5 for STAT3KO) were subjected to the same I/R protocol: 15 min equilibration period, 20 min of global ischemia (37°C), and 60 min of reperfusion.
Isolated heart preparation (Langendorff) and measurement of cardiac function.
Experiments were performed with the use of a Langendorff apparatus as described previously for use in mouse heart. Briefly, mice were anesthetized (pentobarbital sodium, 60 mg/kg ip) and heparinized (500 units ip), and hearts were rapidly excised via median sternotomy and placed in 4°C Krebs-Henseleit solution. The aorta was cannulated, and the heart was perfused with oxygenated (95% O2-5% CO2t and −dP/dt
Lactate dehydrogenase assay.
Elevated lactate dehydrogenase (LDH) release indicates that cell membrane integrity has become damaged and is one of the most widely used indicators for cell viability and tissue injury. Coronary effluent was collected and stored in a −80°C freezer until enzymatic analysis for LDH activity with a commercially available kit (Cytotoxicity Detection Kit-LDH, Roche Diagnostics, Indianapolis, IN).
Western blot analysis.
Western blot analysis was performed to measure STAT3, p38 MAPKs, and caspase-8, -3, and Bcl-2 proteins. Heart tissue was homogenized in cold buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin, and 1 mM PMSF and centrifuged at 12,000 rpm for 10 min. The protein extracts (20 μg/lane) were subjected to electrophoresis on a 12% Tris·HCl gel from Bio-Rad and transferred to a nitrocellulose membrane, which was stained by Naphthol blue-black to confirm equal protein loading. The membranes were incubated in 5% dry milk for 1 h and then incubated with the following primary antibodies: STAT3, phosphor-STAT3 (Tyr705), p38 MAPK, phosphor-p38 MAPK antibody (Thr180/Tyr182) (Cell Signaling Technology, Beverly, MA), and caspase-8, -3, and Bcl-2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG secondary antibody and detection using supersignal West Pico stable peroxide solution (Pierce, Rockford, IL). Films were scanned using an Epson Perfection 3200 Scanner (Epson America, Long Beach, CA), and band density was analyzed using ImageJ software (NIH).
Perfused mouse hearts were rapidly frozen and embedded in optimum cutting temperature compound (Sakura Finetechnical). Twenty micrometer-thick sections were cut and stained with standard immunohistochemistry protocol (44, 45) for caspase-8, -3, Bcl-2, and suppressor of cytokine signal (SOCS)-3 antibodies (Santa Cruz Biotechnology). Peroxidase-conjugated secondary antibodies and peroxidase substrate kit (Vector, Burlingame, CA) were used for signal detection.
Apoptosis in the heart was assessed with a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Cell death detection ELISAPLUS; Roche Diagnostics) that detects mononucleosomes and oligonucleosomes. ELISA was performed according to the manufacture's instructions. Results are depicted as enrichment factor, the sample value (in milliunits). The tissue was also examined for apoptosis with a commercially available kit (DeadEnd Fluorimetric TUNEL System; Promega, Madison, WI). 4′,6-Diamidino-2-phenylindole stains the cell nuclei blue. Fluorescein-12-dUTP incorporation results in localized green fluorescence within the nucleus of apoptotic cells only.
Myocardial IL-6 in cardiac tissue were determined by ELISA using a commercially available ELISA set (R&D Systems, Minneapolis, MN). ELISA was performed according to the manufacturer's instructions. All samples and standards were measured in duplicate.
Total RNA was extracted from the left ventricle of each heart using RNA STAT-60 (TEL-TEST, Friendswood, TX). Total RNA (0.5 μg) was subjected to cDNA synthesis using cloned AMV first-strand cDNA synthesis kit (Invitrogen Life Technologies, Carlsbad, CA). cDNA from each sample was analyzed for 18S (assay ID No. Hs99999901_s1), TNF (assay ID No. Mm00443258_m1), and SOCS-3 (assay ID No. Mm00545913_s1) by using TaqMan gene expression assay (RT-PCR) (Applied Biosystems, Foster City, CA).
Presentation of data and statistical analysis.
All reported values are means ± SE. Data were compared using two-way ANOVA with post hoc Bonferroni test or Student's t-test. A two-tailed probability value of <0.05 was considered statistically significant.
Myocardial function on mice lacking STAT3 expression following I/R.
A mouse line, in which STAT3 was deleted from hematopoietic cells reported previously (39). To determine the effect of EC-derived STAT3 on myocardial function following I/R, the Langendorff model was performed on isolated hearts from this specific mouse line. I/R resulted in markedly impaired +dP/dt and −dP/dt in both WT and STAT3KO mouse hearts. However, recovery of +dP/dt and −dP/dt in the postischemic period was significantly lower in STAT3KO than in WT mice (Fig. 1). In addition, the heart of STAT3KO mice were developed normally, although their size was slightly reduced compared with the WT mice (WT, 17.3 ± 0.93 vs. STAT3KO, 15.7 ± 1.31 g) or heart weight (WT, 115 ± 8 vs. STAT3KO, 108 ± 4 mg) between WT and the conditional STAT3 deletion mice. This result may suggest that EC STAT3 ablation may not attribute to heart development.
Furthermore, elevated LDH release was observed in coronary effluent after 20 min of global ischemia. STAT3 deficiency resulted in more LDH leakage from injured myocardium compared with that in WT mice (Fig. 2), which is in line with impaired myocardial function in STAT3KO heart following I/R.
Activation of myocardial STAT3 and p38 MAPK following I/R.
Activation of myocardial p38 MAPK following I/R has been observed in animal and human studies (3, 15, 19), and inhibition of p38 MAPK activation results in improved myocardial function following I/R injury (11, 16, 17, 25). However, it is unknown whether EC-derived STAT3 deficiency affects myocardial p38 MAPK pathway following I/R. In this study, the myocardial phosphorylated/active STAT3 and p38 were assessed by Western blot analysis, as well as the nonphosphorylated/total STAT3 and p38 MAPK. Interestingly, we found that there was no significant difference in STAT3 activation in control hearts regardless of whether STAT3 was present in ECs. However, knockout (KO) of EC STAT3 decreased the I/R-induced phosphorylated/active form of STAT3 in mutant mouse heart compared with WT hearts (Fig. 3A). Additionally, higher levels of p38 MAPK activation were noticed in EC STAT3KO control hearts. I/R increased activation of myocardial p38 MAPK in WT (↑37.8%) mice but did so to a greater degree in EC STAT3KO hearts (Fig. 3B).
Effect of STAT3 on myocardial apoptotic signaling following I/R.
To elucidate the possible mechanisms that could be involved in I/R-induced myocardial dysfunction in STAT3KO hearts, we assessed the effect of STAT3 on myocardial apoptotic signaling. EC STAT3 deficiency increased basal levels of caspase-8 in control hearts. Additionally, I/R-induced caspase-8 expression was observed in both STAT3KO (↑91.1%) and WT (↑32.2%) hearts (Fig. 4A). However, there were higher levels of caspase-8 in KO hearts compared with WT hearts following I/R. Immunohistochemistry data showed an increase of caspase-8 expression in the myocardium around small vessels, as well as within the walls of those vessels (Fig. 4B). In addition, lower basal levels of Bcl-2 were observed in EC STAT3KO hearts. I/R increased Bcl-2 levels by 2.8 times in EC STAT3KO hearts, whereas there was a decrease in Bcl-2 expression (↓38%) in WT after I/R (Fig. 4C). Moreover, immunohistostaining indicated that I/R-induced Bcl-2 expression was present in the same tissue areas as caspase-8 expression in STAT3KO heart exposed to I/R (Fig. 4D). This phenomenon might be a feedback response. However, the detailed mechanisms regarding the relationship between EC STAT3 and Bcl-2 levels following I/R require further investigation.
Furthermore, cardiac myocyte apoptosis was observed around the capillary, which was consistent with increased caspase-8 expression in those areas (Fig. 5C). However, since the whole I/R procedure time (90 min) was too short to substantially initiate apoptosis in cardiac myocytes, no significant differences were noted in myocardial caspase-3 levels or overall myocardial apoptosis (Fig. 5, A and B).
Myocardial expression of IL-6, TNF, and SOCS-3 in mouse heart following I/R.
To determine whether EC STAT3 mediated myocardial inflammatory response following I/R, we assessed IL-6 protein levels, TNF gene expression, and SOCS-3 expression. STAT3 deficiency in ECs resulted in increased IL-6 production and TNF gene expression (Fig. 6, A and B), both of which may contribute to myocardial dysfunction. In addition, I/R-induced SOCS-3 expression was noted in WT animals but was noted more so in EC STAT3KO hearts. This result was in line with an observation of a twofold I/R-induced SOCS-3 gene expression in KO hearts compared with WT hearts (Fig. 6, C and D).
In the present study, we used EC-restricted STAT3KOs to analyze the effect of EC STAT3 on the myocardial response to I/R injury. Here we observed that 1) poorer postischemic myocardial functional recovery existed in mice with EC STAT3KO, which was associated with increased LDH release in KO heart following I/R; 2) ablation of STAT3 in ECs resulted in less activation of myocardial STAT3 and more activation of myocardial p38 MAPK response to I/R insult, which was consistent with a higher level of IL-6 in KO myocardium; and 3) I/R induced greater expression of caspase-8 in STAT3KO heart. Although there were no significant differences in caspase-3 or overall cardiomyocyte apoptosis, a higher level of apoptosis was observed in the myocardium around the capillary.
Previous studies have demonstrated that a systemic KO of STAT3 is embryonically lethal. This suggests that STAT3 plays an essential role in embryonic development (34). Given that STAT3 is involved in cell proliferation, differentiation, and cell survival, one might assume that the effect of EC STAT3 ablation on myocardial dysfunction might be a result of arrested heart development or vessel formation in KO mice. Although this specific STAT3KO strain results in reduced body and heart weight compared with WT control in this study, there are no significant differences between KOs and WTs with regard to these parameters. In addition, it has been demonstrated that embryonic cardiac tube formation exhibited no obvious differences between WT and STAT3 endothelium-conditioned KOs (12). Furthermore, since the systemic KO of STAT3 mice die before the formation of the embryonic vessel network (34), it can therefore be postulated that STAT3 may not be an essential angiogenic/developmental factor for vessel formation. In other words, EC STAT3 ablation does not affect normal heart development and vessel formation.
I/R results in cardiac stress, which is characterized by a reduction in oxygen supply, followed by the increase in reactive oxygen species (ROS), proinflammatory cytokine production, and myocardial dysfunction (37, 40). In the present study, we have demonstrated impaired myocardial contractile and compliance function following I/R. Interestingly, EC STAT3KO results in poorer postischemic functional recovery compared with WT, which is associated with less activation of myocardial STAT3 and higher level of p38 MAPK activation. Recent evidence has indicated that STAT3 plays a protective role in the myocardial response to various insults (7, 9). It has been reported that STAT3 can be activated by I/R injury not only in the ischemic infracted area but also in the healthy border area (21, 22). It is thought that activated STAT3 protects the myocardium from ischemic injury via upregulation of antiapoptotic signals, such as Bcl-2 (2, 30). In addition, STAT3 has been observed to provide cardioprotection through inhibiting antiangiogenic factors and promoting myocardial capillary formation (8). Furthermore, it has been shown that STAT3 exerts protective effects on hypoxic cardiomyocytes through the upregulation of manganese superoxide dismutase (21) or through the induction of metallothionein-1 and -2 (ROS scavengers) (23). Our data of decreased myocardial functional recovery with less myocardial STAT3 activation are in line with previous observations. In addition, increased myocardial p38 MAPK activation has been observed in animal and human studies after myocardial infarction (15, 29), and inhibition of p38 MAPK activation improves myocardial function following I/R injury (3, 11, 16, 31). Therefore, it is not surprising that more activation of myocardial p38 MAPK was observed in STAT3KO mice, which implicates that STAT3 in ECs exerts an indirect effect in regulation of p38 activation in cardiac myocytes.
Accumulated evidence has indicated that antiapoptotic signals are transduced via STAT3 in the myocardial response to multiple insults. Cardiomyocyte-restricted KO of STAT3 results in an increased apoptotic response to acute myocardial infarction (8). Inhibition of STAT3 activation increases caspase-3 activity and cardiac myocyte apoptosis in the heart subjected to I/R (22). Treatment with LPS enhances myocardial apoptosis in STAT3KO mice (10). STAT3-suppressed cardiomyocyte apoptosis appears to be mediated through 1) the upregulation of antiapoptotic protein Bcl-2 or Bcl-xL (2, 22, 30); 2) the induction of BCL2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3), a hypoxia-regulated inducer of cardiomyocyte death (8, 13); 3) the increase of heat shock proteins (1, 20, 32); 4) the production of angiogenic factors (24); and 5) the decrease of inflammatory cytokines (8, 10, 24). In the present study, our experimental period may be too brief (20 min of global ischemia followed by 60 min of reperfusion) to detect significant overall apoptosis in the heart. However, increased expression of caspase-8 was observed in STAT3KO mice compared with WT after I/R. In addition, our study has demonstrated that increased myocardial LDH release existed in STAT3KO mice in response to I/R. These results suggest that more damaged cells are present in the STAT3KO myocardium subjected to I/R, which likely correlates with increased myocardial dysfunction in KO mice. Furthermore, increased apoptosis was observed in the STAT3KO myocardium around the capillary compared with WT myocardium. This result is consistent with our data showing increased caspase-8 expression around small vessels, as well as in the walls of those vessels. This suggests that STAT3KO results in increased sensitivity of ECs to I/R injury, which may impair EC function and capillary integrity. In this regard, it can be postulated that impaired myocardial vessels/capillaries reduce perfusion supply, decrease tissue oxygenation, and result in decreased postischemic functional recovery in STAT3KO mice.
STAT3 has previously been reported to mediate proangiogenic paracrine circuits in the heart. Overexpression or activation of STAT3 in cardiomyocytes increases myocardial VEGF production (24). STAT3-dependent release of paracrine factors in cardiomyocytes has been reported to upregulate antiangiogenic factors (8). In addition, STAT3-controlled production of local factors from cardiomyocyte has been observed to directly mediate the phenotype of ECs, which is revealed by the observation that supernatant from cultured KO cardiomyocytes inhibits growth of ECs and increases apoptosis (8). Therefore, it is proposed, in turn, that STAT3-mediated paracrine factor production by ECs may affect cardiomyocyte function. Indeed, numerous ECs are present in the heart, and they can locally produce factors to protect the myocardium against pathophysiological stress (43). It has been shown that ECs mediate LPS-induced toxicity via the control of cytokine production, and this process is regulated by STAT3 (12). Although we do not show direct evidence of STAT3-mediated local factor production by ECs, we did observe observed higher levels of myocardial IL-6, as well as upregulated TNF and SOCS-3 expression in EC restricted STAT3 mouse hearts in response to I/R insult. All of those results suggest that EC STAT3 may mediate myocardial inflammation in response to I/R.
In summary, these results suggest that EC-derived STAT3 plays an important role in myocardial functional recovery following I/R injury by maintaining capillary integrity, protecting the myocardium from apoptosis, inhibiting protein kinases such as p38 MAPK, and improving myocardial function.
This work was supported in part by NIH Grants R01-GM-070628 (to D. R. Meldrum) and K99-HL-087607-01 (to M. Wang) and American Heart Association Postdoctoral Fellowship 0526008Z (to M. Wang) and grant-in-aid (to D. R. Meldrum). This investigation was conducted in a facility constructed with support from the Division of Research Resources Research Facilities Improvement Program Grant C06-RR-015481-01.
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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