Original articleRapamycin protects against myocardial ischemia–reperfusion injury through JAK2–STAT3 signaling pathway
Highlights
► Rapamycin induced cardioprotection against ischemia–reperfusion injury. ► STAT3 is essential in rapamycin-induced cardioprotection. ► Rapamycin phosphorylates ERK, STAT3, eNOS and GSK3β.
Introduction
Rapamycin (Sirolimus®), an inhibitor of the mammalian target of rapamycin (mTOR), is a macrocyclic fermentation product isolated from Streptomyces hygroscopius, and has been widely used as an immunosuppressive agent for prophylaxis of allograft rejection [1]. Due to its antiproliferative property, rapamycin prevents intimal growth of graft coronary arteries and reduces the incidence of vasculopathy [2]. Rapamycin is currently used for coating drug-eluting stents to reduce restenosis after coronary angioplasty [3]. However, the therapeutic effects of rapamycin in patients with heart failure after ischemia injury remain unclear. Previous studies report that rapamycin can abolish the cardioprotective effect of ischemic or pharmacological preconditioning [4], [5], [6]. On the contrary, we first reported that rapamycin treatment reduced infarct size after ischemia-reperfusion (I/R) injury and also attenuated necrosis and apoptosis in cardiomyocytes following simulated ischemia/reoxygenation (SI/RO) [7]. We reported that attenuation of I/R injury with rapamycin was mediated through opening of ATP-sensitive K channels (mitoKATP channel). In addition, another mTOR inhibitor, everolimus, prevented left ventricular (LV) remodeling, limited infarct size, improved LV function and increased autophagy post myocardial infarction [8]. It appears that rapamycin concentrations and/or timing of its administration during I/R may contribute to such discrepant effects. Additional studies are needed to understand the differential effects of rapamycin treatment. Nevertheless, the signaling mechanisms in rapamycin-induced protection against I/R injury remain poorly understood.
Signal transducer and activator of transcription 3 (STAT3) is a central component of cardioprotection [9], [10]. The activation of JAK–STAT pathway by ischemic preconditioning up-regulates iNOS and thereby contributes to adaptation of the heart to ischemic stress [11], [12]. JAK–STAT pathway is composed of a family of receptor-associated cytosolic tyrosine kinases (JAKs) that phosphorylate a tyrosine residue in cognate of STATs [13]. Phosphorylation and activation of STAT in response to ischemic preconditioning confer cardioprotection via prosurvival signaling cascades or inhibition of proapoptotic factors [14]. The putative JAK2 inhibitor AG 490 abrogated ischemic preconditioning-induced acute cardioprotection after myocardial I/R [11]. Constitutive cardiomyocyte-restricted deletion of STAT3 has been shown to increase apoptosis [15], [16] and infarct size after I/R and cause loss of protection during ischemic postconditioning and pharmacological preconditioning [9], [17], [18]. Recent studies indicate that STAT3 is also present in the mitochondria, wherein it modulates mitochondrial respiration, regulates mitochondria-mediated apoptosis, and inhibits the opening of mitochondrial permeability transition pores (mPTP) [19], [20], [21]. Mitochondrial-targeted STAT3 overexpression in mice preserves complex 1 respiration during ischemia, reduces reactive oxygen species (ROS) production from complex I and blocks cytochrome c release into the cytosol [22]. However, it is unknown whether rapamycin induces acute cardioprotection through activation of JAK-STAT pathway.
Thus, considering the important role of JAK–STAT3 in preconditioning and cardioprotection, we undertook this investigation to determine the potential role of this signaling pathway in rapamycin-induced protection against I/R injury. The major aims of the present study were to 1) determine whether rapamycin would reduce infarct size and improve cardiac function following in situ I/R injury; 2) demonstrate whether rapamycin would affect cardioprotective signaling components, such as STAT3 and ERK1/2; and 3) determine the functional role of STAT3 in cardioprotection with rapamycin. Our results show that rapamycin induces ERK-dependent phosphorylation of STAT3, which is causatively involved in reducing I/R injury in the heart and cardiomyocytes.
Section snippets
Animals
Adult male outbred CD-1 mice (body weight ~ 30 g) were supplied by Charles River Laboratories. The animal care and experiments were approved by the Institutional Care and Use Committee of Virginia Commonwealth University.
Experimental groups
For global I/R protocol, we used six groups: the mice were injected (intraperitoneal, i.p.) with 1) DMSO (solvent for rapamycin, AG490 — JAK inhibitor and Stattic — STAT3 inhibitor), 2) rapamycin (0.25 mg/kg), 3) rapamycin + AG490 (40 mg/kg), 4) AG490 only, 5) rapamycin + stattic (20
Inhibition of JAK/STAT3 abolishes rapamycin-induced cardioprotection
Pretreatment with rapamycin reduced infarct size (% risk area) to 9.42 ± 1.22 compared with DMSO control at 33.52 ± 1.75 (n = 7, p < 0.001) following global I/R in Langendorff mode (Fig. 2A). This infarct-limiting effect was abolished by AG490 (37.58 ± 5.15%) and stattic (33.35 ± 2.16%), whereas treatment with AG490 (39.89 ± 2.43%) and stattic (26.17 ± 2.84%) alone had no effect on infarct size as compared to the control. The contractile function and post-ischemic coronary flow rate were not statistically
Discussion
In the present study, we investigated the signaling pathways by which rapamycin triggers preconditioning-like anti-infarct effect following I/R injury in mouse heart. Specifically, we focused on the potential role of JAK2–STAT3 pathway in rapamycin-induced protection. This is because inactivation of STAT3 or deletion of STAT3 appears to be a key event in the diminution of cardioprotection in response to various physiological stresses including I/R [9], [10], [27]. Moreover, it has been shown
Conflict of interest
None.
Acknowledgment
This study was supported by grants from the National Institutes of Health (HL51045, HL79424, and HL93685 to R.C.K.), the American Heart Association Mid-Atlantic Affiliate Beginning Grant-in-Aid (0765273U to A.D.), the National Scientist Development Grant (10SDG3770011 to F.N.S.) and the CTSA (UL1RR031990 from the National Center for Research Resources) and the AD Williams' Fund of the Virginia Commonwealth University (to A.D.). We thank Eric Mayton for technical assistance.
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