Original article
Rapamycin confers preconditioning-like protection against ischemia–reperfusion injury in isolated mouse heart and cardiomyocytes

https://doi.org/10.1016/j.yjmcc.2006.04.014Get rights and content

Abstract

Rapamycin (sirolimus) is an antibiotic that inhibits protein synthesis through mammalian target of rapamycin (mTOR) signaling and is used as an immunosuppressant in the treatment of organ rejection in transplant recipients. Recently, the antigrowth properties of rapamycin have been utilized for cardiovascular benefit as stents impregnated with rapamycin effectively reduce coronary restenosis. We report here a novel role of this drug in protection against ischemia/reperfusion (I/R) injury. Adult male ICR mice were treated with rapamycin (0.25 mg/kg, IP) or volume-matched DMSO (solvent for rapamycin). The hearts were subjected to 20 min of global ischemia and 30 min of reperfusion in Langendorff mode. The blocker of mitochondrial KATP channel, 5-hydroxydecanoate (5-HD, 100 μM) was given 10 min before ischemia. Infarct size in the DMSO treated group was 28.2  ±  1.3% and was reduced to 10.1  ±  2.8% in the rapamycin-treated mice (64% decrease, P  <  0.001). 5-HD blocked the protective effect (infarct area 32.2  ±  1.8%, P  <  0.001 vs. rapamycin). The infarct limiting effect of rapamycin was not associated with improved recovery of ventricular function. We further examined the effect of rapamycin in protection against necrosis and apoptosis in adult cardiomyocytes subjected to simulated ischemia and reoxygenation. Myocytes treated with rapamycin in doses from 25–100 nM demonstrated significantly lower trypan blue-positive necrotic cells and TUNEL-positive apoptotic nuclei, supporting the protective role of drug in the intact heart. These data suggest that rapamycin induces potent preconditioning-like effect against myocardial infarction through opening of mitochondrial KATP channels. We propose that rapamycin may be a novel therapeutic strategy to limit infarction, apoptosis, and remodeling following I/R injury in the heart.

Introduction

Since the initial findings by Murry et al. [1] that brief episodes of ischemia can paradoxically protect the heart from subsequent ischemic injury, many investigators have tried to discover a pharmacological solution to ischemic preconditioning (IPC). IPC works by activating endogenous protective mechanisms and is realized in two phases: the early phase, which lasts up to 2–3 h, and a late phase, which appears 12–24 h later with duration of up to 3–4 days. Considerable progress has been made toward identifying cellular triggers and signal transduction mechanisms involved in the process of preconditioning and many pharmacological agents have been shown to induce a preconditioning-like effect [2], [3]. However, despite significant advances in this field, no agent has yet gained widespread clinical use [4]. In the present study, we demonstrate a novel approach of preconditioning the heart by using rapamycin, an increasingly widespread staple of cardiac practice.

Rapamycin (sirolimus) is an antibiotic derived from Streptomyces hygroscopius, and for many years has been primarily used as an immunosuppressant in the treatment of organ rejection in transplant recipients [5]. Recently, rapamycin's antigrowth properties have been utilized for cardiovascular benefit as stents impregnated with rapamycin effectively reduce coronary restenosis [6]. The proposed mechanism for the antiproliferative effect of rapamycin is based on its ability to bind to its intracellular receptor, the FK506 binding protein (FKBP12) [7]. The rapamycin/FKBP12 complex is an inhibitor of the mammalian target of rapamycin (mTOR), a 290-kDa Ser/Thr kinase that controls mammalian protein translational processes that are central to cell growth [8]. Rapamycin prevents DNA and protein synthesis, in large part, by regulation of p70S6 kinase (p70S6K) phosphatase, leading to arrest of the cell cycle at the G1/S interface [9]. Additionally, rapamycin modulation of the mTOR kinase plays key roles in nutrient regulation [10], mitochondrial metabolism [11], [12], and growth-factor stimulated proliferation [9], [13].

In a short time, rapamycin-eluting stents have become a vital tool for native coronary artery revascularization and also demonstrate favorable outcomes for treatment of saphenous vein graft lesions [14] and in-stent restenosis [15]. Furthermore, rapamycin taken orally has also shown promise as a potential agent to inhibit restenosis after stenting of de novo coronary lesions [16], [17], [18] as well as slow down the progression of coronary artery disease in heart transplant recipients [19]. Although, the antigrowth effect of rapamycin on vascular tissue has been widely described [8], the full spectrum of action of rapamycin, particularly on cardiac tissue, is not known.

Chemical preconditioning with some immunosuppressants has been shown in the heart [20], brain [21], and liver [22]; however, the cardioprotective effect of rapamycin is unknown [23], [24], [25], [26]. In the present study, we show, for the first time, that rapamycin induces a preconditioning-like protective effect in the intact heart and adult cardiomyocyte subjected to ischemia/reperfusion. Furthermore, our results show that rapamycin-induced cardioprotection is mediated by opening of the mitochondrial ATP-sensitive potassium channel (mitoKATP channel).

Section snippets

Animals

Adult male outbred ICR mice were supplied by Harlan Inc. (Indianapolis, IN). Animal experimental protocols were approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University.

Drugs and chemicals

Rapamycin was purchased from Sigma-Aldrich (St. Loius, MO) and was dissolved in DMSO (Sigma-Aldrich) for intraperitoneal injection (final DMSO concentration  < 1%). Unless specified otherwise, all other chemicals including 5-hydroxydecanoate (5-HD), trypan blue dye, and triphenyltetrazolium

Baseline cardiovascular function

The adult male ICR mice used in the present study weighed an average of 34.7  ±  0.9 g (n  =  24). There was no significant difference in heart-weight/body-weight among the groups. Preischemic baseline values of the isolated perfused hearts are summarized in Table 1. Baseline function (developed force and rate–force product) was similar between the RAPA and DMSO pretreated groups. The DMSO pretreated group that received 5-HD infusion during stabilization (DMSO  +  5-HD) had an elevated developed

Discussion

The use of rapamycin has greatly increased during the past few years with the introduction of rapamycin-eluting coronary stents. The antihypertrophic effects of rapamycin have been well described [8]; however, other properties of this pharmacological agent are poorly understood. We report here our novel observation on the preconditioning-like effect of rapamycin in the mouse heart. More specifically, we have shown that intraperitoneal administration of rapamycin induces cardioprotection as

Conclusion

In summary, we have demonstrated, for the first time, that rapamycin induces preconditioning-like protective effects against myocardial infarction following ischemia–reperfusion injury through opening of mitoKATP channels. In addition, this drug reduced necrosis as well as apoptosis following simulated ischemia–reoxygenation injury in adult cardiomyocytes. We propose that the use of rapamycin may be a potentially novel therapeutic strategy to limit myocardial infarction and apoptosis following

Acknowledgments

The present study was supported in part by grants from National Institutes of Health (HL51045, HL59469, HL79424 to R.C.K) and American Heart Association, National Center (0530157N to L.X.).

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