- Email: [email protected]
Rapamycin (Sirolimus): A two-edged sword?
Journal of Molecular and Cellular Cardiology 41 (2006) 226 – 227 www.elsevier.com/locate/yjmcc
Editorial
Rapamycin (Sirolimus): A two-edged sword? Rapamycin (Sirolimus) is an immunosuppressant macrolide antibiotic which has been approved for clinical use as a drug for the prevention of renal transplant rejection and heart transplant rejection and vasculopathy [1,2]. Rapamycin has also been included as a coating in stents used in coronary angioplasty to prevent restenosis and has been shown to be effective in preventing recurrent in-stent stenosis by intensive oral administration (OSIRIS Trial) [3]. In experimental studies, rapamycin has been shown to block the cell cycle progression at the G1/S step in human and rat smooth muscle cells [4]. Mechanistically, rapamycin is known to combine with the FKBP12 binding protein and subsequently inhibits the mTOR/p70S6K protein complex [5]. Activation of p70S6K results in the initiation of protein translational machinery in the nucleus which results in enhanced cell growth and cell size. If p70S6K stimulation is prolonged, cardiac or vascular smooth muscle hypertrophy is the end result [6]. Thus, inhibiting the mTOR/p70S6K complex will have beneficial effects to reduce restenosis and cardiac remodeling. In contrast, several papers in the literature have shown results which suggest that activating the mTOR/ p70S6K complex may mediate the beneficial effects of acute and delayed preconditioning to ischemia and several preconditioning mimetic drugs [7,8,9]. Thus, drugs which inhibit mTOR such as rapamycin may have either beneficial or detrimental effects depending on the experimental model and perhaps dose and timing of administration. In the present issue of the Journal of Molecular and Cellular Cardology, Khan and co-workers [10] have identified a new and exciting action of rapamycin in isolated mouse hearts and adult mouse myocytes. These investigators are the first to present novel data which suggest that rapamycin has a cardioprotective effect against ischemia/reperfusion injury in the isolated mouse heart and in cardiac myocytes subjected to hypoxia-reoxygenation injury. In the intact mouse studies, rapamycin or DMSO vehicle were administered ip to the mouse 30 min prior to the heart removal and isolation procedure. Once the hearts were mounted in the Langendorff apparatus, they were exposed to 20 min of global ischemia followed by 30 min of reperfusion. In one series of hearts, the mitochondrial KATP channel inhibitor 5-hydroxydecanoate (5-HD) was added to the perfusate 10 min prior to the ischemic period. Interestingly, the mice pretreated with rapamycin showed a marked reduction in myocardial infarct size from 0022-2828/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yjmcc.2006.04.013
28.2 ± 1.3% to 10.1 ± 2.8% and this protective effect was totally abrogated by pretreatment with 5-HD (32.2 ± 1.8%). 5-HD had no effect by itself. Surprisingly, the rapamycin-treated hearts did not demonstrate an enhanced recovery of function as assessed by the rate-force product in spite of the large reduction in infarct size. However, this lack of correlation between infarct size and recovery of function in isolated hearts is not without precedence and has been previously demonstrated by Jenkins et al. [11] in a similar model of global ischemia and reperfusion. The present investigators took these findings one step further and tested the effect of rapamycin to reduce necrosis and apoptosis in isolated mouse adult cardiac myocytes subjected to simulated ischemia and reoxygenation in which they assessed necrosis at 1 h after reoxygenation by trypan blue staining and assessed apoptosis by TUNEL 18 h after reoxygenation. In both instances, 3 concentrations of rapamycin (25, 50, and 100 nM) produced equivalent reductions in necrosis and apoptosis without any evidence of a concentration-dependent effect. These data would suggest that the 25 nM concentration is a maximal protective concentration and that rapamycin may still be effective at much lower concentrations. Unfortunately, in these studies, 5-HD was not included, so the role of the mitochondrial KATP channel in these 2 forms of myocardial injury remains unknown and requires further investigation. It also would have been interesting to determine if rapamycin administration was protective in an intact mouse model of infarction with 24–48 h of reperfusion to determine if this effect is permanent or only a delay in infarct development. The effect of rapamycin on apoptosis in vivo could also have been determined in the presence of this longer period of reperfusion. It is difficult to reconcile the present results where blocking mTOR with rapamycin is cardioprotective and those [7–9] which have shown that rapamycin blocks the protective effect of ischemic or pharmacological preconditioning. One possibility is that rapamycin may have opposing effects related to dose or concentration of drug used. In the present study by Khan et al. [10], these investigators used 0.25 mg/kg ip in the isolated heart studies and 25–100 nM in the myocyte studies. Similarly, Kis et al. [9] used 0.25 mg/kg, iv in their intact rabbit studies in which they showed that rapamycin blocked delayed ischemic preconditioning, thus, it is unlikely that the dose is responsible for the different results observed between the present study and that of Kis et al. [9] since 0.25 mg/kg was used in both studies
Editorial
although by different routes of administration. In contrast, 1 nM of rapamycin was used to block mTOR in the rat myocyte studies of Juhaszova et al. [12] and 1 μg/kg, iv was used in the intact rat studies by Gross et al. [13] to block mTOR. In both of these studies, blocking mTOR prevented the effect of preconditioning in myocytes and the cardioprotective effect of morphine in intact rats, respectively. Based on the dose and concentration used in these latter 2 studies, the results obtained suggest that a lower dose or concentration of rapamycin may have a different effect on cell survival than that of a larger dose or concentration similar to those used in the present study of Khan et al. [10]. Further dose–response studies are needed to confirm or deny this possibility. Another possibility concerns the timing of rapamycin administration in the various studies. In the majority of studies where rapamycin is shown to block cardioprotection, this compound was administered at or near the onset of reperfusion, whereas, in the present study of Khan et al. [10], rapamycin was administered as a pretreatment regimen. It may be quite probable that rapamycin is having a different effect during ischemia which may result in cardioprotection and another effect at reperfusion where it is blocking a survival kinase pathway. This possibility is easily testable and should be the focus of additional studies. A final possibility has been suggested by the present group who have explained the possible differences between the results of the present study and those of others by an effect of rapamycin to influence the cross-talk between the survival kinases in this complex pathway where inhibiting mTOR may result in the upregulation of other prosurvival kinases such as PI3K/Akt or the ERK 1/2 pathways. This is another interesting possibility that is a worthy area of future investigation. Of course, an obvious possibility is that the differences observed in the present study and those of previous studies may be related to a species difference since this is the first study to explore the effect of rapamycin in a mouse model of ischemia/reperfusion injury. The mechanism by which rapamycin results in the activation of the mito KATP channel is another question left unanswered by the present results. The authors of the present study suggest that nitric oxide (NO) may be involved but furnish no data to support an NO-induced opening of mitochondrial KATP channels as has been shown to occur in the presence of other drugs such as acetylcholine or bradykinin [14]. This is another fruitful area for future experiments. In conclusion, the present results which clearly show that rapamycin possesses novel cardioprotective effects in 2 mouse models of cardiac ischemia/reperfusion injury are of great clinical interest since this compound is already being used in the clinical arena in transplant medicine and as a coating for drugeluting stents. The finding that this antibiotic also possesses
227
cardioprotective properties suggests that this compound may have additional benefits besides its well-described antiproliferative effects in smooth muscle and heart. References [1] Kahan BD, For the rapamune US study group. Efficacy of sirolimus compared with azathioprine for reduction of acute renal allograft rejection: a randomized multicenter trial. Lancet 2000;356:194–202. [2] Keogh A, Richardson M, Ruygrok P, Spratt P, Galbraith A, Driscoll O, et al. Sirolimus in de novo heart transplant recipients reduces acute rejection and prevents coronary artery disease at 2 years: a randomized clinical trial. Circulation 2004;110:2694–700. [3] Hausleiter J, Kastrati A, Mehilli J, Vogeser M, Zohlnhofer D, Schuhlen H, et al. Randomized, double-blind placebo-controlled trial of oral sirolimus for restenosis prevention in patients with in-stent restenosis: the oral sirolimus to inhibit recurrent in-stent stenosis (OSIRIS) trial. Circulation 2004;110:790–5. [4] Marx SO, Jayaraman T, Go LO, Marks AR. Rapamycin-FKBP inhibits cell cycle regulators of proliferation in vascular smooth muscle cells. Circ Res 1995;76:412–7. [5] Asnaghi L, Bruno P, Priulla M, Nicolin A. mTOR: a protein kinase switching between life and death. Pharmacol Res 2004;50:545–9. [6] Kozma SC, Thomas G. Regulation of cell size in growth, development and human disease: P13K, PKB and S6K. BioEssays 2002;24:65–71. [7] Hausenloy DJ, Tsang A, Mocanu MM, Yellon DM. Ischemic preconditioning protects by activating prosurvival kinases at reperfusion. Am J Physiol 2005;288:H971–6. [8] Jonassen AK, Sack MN, Mjos OD, Yellon DM. Myocardial protection by insulin at reperfusion requires early administration and is mediated via Akt and p70s6 kinase cell-survival signaling. Circ Res 2001;89:1191–8. [9] Kis A, Yellon DM, Baxter GF. Second window of protection following myocardial preconditioning: an essential role for PI3K kinase and p70S6 kinase. J Mol Cell Cardiol 2003;35:1063–71. [10] Khan S, Salloum F, Das A, Xi L, Vetrovec GW, Kukreja RA. Rapamycin confers preconditioning-like protection against ischemia–reperfusion injury in isolated mouse heart and cardiomyocytes. J Mol Cell Cardiol. doi:10.1016/j.yjmcc.2006.04.014. [11] Jenkins DP, Pugsley WB, Yellon DM. Ischaemic preconditioning in a model of global ischaemia: infarct size limitation, but no reduction in stunning. J Mol Cell Cardiol 1995;27:1623–32. [12] Juhaszova M, Zorov DB, Kim SH, Pepe S, Fu Q, Fishbein KW, et al. Glycogen synthase kinase-3β mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore. J Clin Invest 2004;113:1535–49. [13] Gross ER, Hsu AK, Gross GJ. Opioid-induced cardioprotection occurs via glycogen synthase kinase β inhibition during reperfusion in intact rat hearts. Circ Res 2004;94:960–6. [14] Critz SD, Cohen MV, Downey JM. Mechanisms of acetylcholine and bradykinin-induced preconditioning. Vascul Pharmacol 2005;42:201–9.
Garrett J. Gross Medical College of Wisconsin, Milwaukee, WI 53226, USA E-mail address: [email protected]. 19 April 2006
Editorial
Rapamycin (Sirolimus): A two-edged sword? Rapamycin (Sirolimus) is an immunosuppressant macrolide antibiotic which has been approved for clinical use as a drug for the prevention of renal transplant rejection and heart transplant rejection and vasculopathy [1,2]. Rapamycin has also been included as a coating in stents used in coronary angioplasty to prevent restenosis and has been shown to be effective in preventing recurrent in-stent stenosis by intensive oral administration (OSIRIS Trial) [3]. In experimental studies, rapamycin has been shown to block the cell cycle progression at the G1/S step in human and rat smooth muscle cells [4]. Mechanistically, rapamycin is known to combine with the FKBP12 binding protein and subsequently inhibits the mTOR/p70S6K protein complex [5]. Activation of p70S6K results in the initiation of protein translational machinery in the nucleus which results in enhanced cell growth and cell size. If p70S6K stimulation is prolonged, cardiac or vascular smooth muscle hypertrophy is the end result [6]. Thus, inhibiting the mTOR/p70S6K complex will have beneficial effects to reduce restenosis and cardiac remodeling. In contrast, several papers in the literature have shown results which suggest that activating the mTOR/ p70S6K complex may mediate the beneficial effects of acute and delayed preconditioning to ischemia and several preconditioning mimetic drugs [7,8,9]. Thus, drugs which inhibit mTOR such as rapamycin may have either beneficial or detrimental effects depending on the experimental model and perhaps dose and timing of administration. In the present issue of the Journal of Molecular and Cellular Cardology, Khan and co-workers [10] have identified a new and exciting action of rapamycin in isolated mouse hearts and adult mouse myocytes. These investigators are the first to present novel data which suggest that rapamycin has a cardioprotective effect against ischemia/reperfusion injury in the isolated mouse heart and in cardiac myocytes subjected to hypoxia-reoxygenation injury. In the intact mouse studies, rapamycin or DMSO vehicle were administered ip to the mouse 30 min prior to the heart removal and isolation procedure. Once the hearts were mounted in the Langendorff apparatus, they were exposed to 20 min of global ischemia followed by 30 min of reperfusion. In one series of hearts, the mitochondrial KATP channel inhibitor 5-hydroxydecanoate (5-HD) was added to the perfusate 10 min prior to the ischemic period. Interestingly, the mice pretreated with rapamycin showed a marked reduction in myocardial infarct size from 0022-2828/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yjmcc.2006.04.013
28.2 ± 1.3% to 10.1 ± 2.8% and this protective effect was totally abrogated by pretreatment with 5-HD (32.2 ± 1.8%). 5-HD had no effect by itself. Surprisingly, the rapamycin-treated hearts did not demonstrate an enhanced recovery of function as assessed by the rate-force product in spite of the large reduction in infarct size. However, this lack of correlation between infarct size and recovery of function in isolated hearts is not without precedence and has been previously demonstrated by Jenkins et al. [11] in a similar model of global ischemia and reperfusion. The present investigators took these findings one step further and tested the effect of rapamycin to reduce necrosis and apoptosis in isolated mouse adult cardiac myocytes subjected to simulated ischemia and reoxygenation in which they assessed necrosis at 1 h after reoxygenation by trypan blue staining and assessed apoptosis by TUNEL 18 h after reoxygenation. In both instances, 3 concentrations of rapamycin (25, 50, and 100 nM) produced equivalent reductions in necrosis and apoptosis without any evidence of a concentration-dependent effect. These data would suggest that the 25 nM concentration is a maximal protective concentration and that rapamycin may still be effective at much lower concentrations. Unfortunately, in these studies, 5-HD was not included, so the role of the mitochondrial KATP channel in these 2 forms of myocardial injury remains unknown and requires further investigation. It also would have been interesting to determine if rapamycin administration was protective in an intact mouse model of infarction with 24–48 h of reperfusion to determine if this effect is permanent or only a delay in infarct development. The effect of rapamycin on apoptosis in vivo could also have been determined in the presence of this longer period of reperfusion. It is difficult to reconcile the present results where blocking mTOR with rapamycin is cardioprotective and those [7–9] which have shown that rapamycin blocks the protective effect of ischemic or pharmacological preconditioning. One possibility is that rapamycin may have opposing effects related to dose or concentration of drug used. In the present study by Khan et al. [10], these investigators used 0.25 mg/kg ip in the isolated heart studies and 25–100 nM in the myocyte studies. Similarly, Kis et al. [9] used 0.25 mg/kg, iv in their intact rabbit studies in which they showed that rapamycin blocked delayed ischemic preconditioning, thus, it is unlikely that the dose is responsible for the different results observed between the present study and that of Kis et al. [9] since 0.25 mg/kg was used in both studies
Editorial
although by different routes of administration. In contrast, 1 nM of rapamycin was used to block mTOR in the rat myocyte studies of Juhaszova et al. [12] and 1 μg/kg, iv was used in the intact rat studies by Gross et al. [13] to block mTOR. In both of these studies, blocking mTOR prevented the effect of preconditioning in myocytes and the cardioprotective effect of morphine in intact rats, respectively. Based on the dose and concentration used in these latter 2 studies, the results obtained suggest that a lower dose or concentration of rapamycin may have a different effect on cell survival than that of a larger dose or concentration similar to those used in the present study of Khan et al. [10]. Further dose–response studies are needed to confirm or deny this possibility. Another possibility concerns the timing of rapamycin administration in the various studies. In the majority of studies where rapamycin is shown to block cardioprotection, this compound was administered at or near the onset of reperfusion, whereas, in the present study of Khan et al. [10], rapamycin was administered as a pretreatment regimen. It may be quite probable that rapamycin is having a different effect during ischemia which may result in cardioprotection and another effect at reperfusion where it is blocking a survival kinase pathway. This possibility is easily testable and should be the focus of additional studies. A final possibility has been suggested by the present group who have explained the possible differences between the results of the present study and those of others by an effect of rapamycin to influence the cross-talk between the survival kinases in this complex pathway where inhibiting mTOR may result in the upregulation of other prosurvival kinases such as PI3K/Akt or the ERK 1/2 pathways. This is another interesting possibility that is a worthy area of future investigation. Of course, an obvious possibility is that the differences observed in the present study and those of previous studies may be related to a species difference since this is the first study to explore the effect of rapamycin in a mouse model of ischemia/reperfusion injury. The mechanism by which rapamycin results in the activation of the mito KATP channel is another question left unanswered by the present results. The authors of the present study suggest that nitric oxide (NO) may be involved but furnish no data to support an NO-induced opening of mitochondrial KATP channels as has been shown to occur in the presence of other drugs such as acetylcholine or bradykinin [14]. This is another fruitful area for future experiments. In conclusion, the present results which clearly show that rapamycin possesses novel cardioprotective effects in 2 mouse models of cardiac ischemia/reperfusion injury are of great clinical interest since this compound is already being used in the clinical arena in transplant medicine and as a coating for drugeluting stents. The finding that this antibiotic also possesses
227
cardioprotective properties suggests that this compound may have additional benefits besides its well-described antiproliferative effects in smooth muscle and heart. References [1] Kahan BD, For the rapamune US study group. Efficacy of sirolimus compared with azathioprine for reduction of acute renal allograft rejection: a randomized multicenter trial. Lancet 2000;356:194–202. [2] Keogh A, Richardson M, Ruygrok P, Spratt P, Galbraith A, Driscoll O, et al. Sirolimus in de novo heart transplant recipients reduces acute rejection and prevents coronary artery disease at 2 years: a randomized clinical trial. Circulation 2004;110:2694–700. [3] Hausleiter J, Kastrati A, Mehilli J, Vogeser M, Zohlnhofer D, Schuhlen H, et al. Randomized, double-blind placebo-controlled trial of oral sirolimus for restenosis prevention in patients with in-stent restenosis: the oral sirolimus to inhibit recurrent in-stent stenosis (OSIRIS) trial. Circulation 2004;110:790–5. [4] Marx SO, Jayaraman T, Go LO, Marks AR. Rapamycin-FKBP inhibits cell cycle regulators of proliferation in vascular smooth muscle cells. Circ Res 1995;76:412–7. [5] Asnaghi L, Bruno P, Priulla M, Nicolin A. mTOR: a protein kinase switching between life and death. Pharmacol Res 2004;50:545–9. [6] Kozma SC, Thomas G. Regulation of cell size in growth, development and human disease: P13K, PKB and S6K. BioEssays 2002;24:65–71. [7] Hausenloy DJ, Tsang A, Mocanu MM, Yellon DM. Ischemic preconditioning protects by activating prosurvival kinases at reperfusion. Am J Physiol 2005;288:H971–6. [8] Jonassen AK, Sack MN, Mjos OD, Yellon DM. Myocardial protection by insulin at reperfusion requires early administration and is mediated via Akt and p70s6 kinase cell-survival signaling. Circ Res 2001;89:1191–8. [9] Kis A, Yellon DM, Baxter GF. Second window of protection following myocardial preconditioning: an essential role for PI3K kinase and p70S6 kinase. J Mol Cell Cardiol 2003;35:1063–71. [10] Khan S, Salloum F, Das A, Xi L, Vetrovec GW, Kukreja RA. Rapamycin confers preconditioning-like protection against ischemia–reperfusion injury in isolated mouse heart and cardiomyocytes. J Mol Cell Cardiol. doi:10.1016/j.yjmcc.2006.04.014. [11] Jenkins DP, Pugsley WB, Yellon DM. Ischaemic preconditioning in a model of global ischaemia: infarct size limitation, but no reduction in stunning. J Mol Cell Cardiol 1995;27:1623–32. [12] Juhaszova M, Zorov DB, Kim SH, Pepe S, Fu Q, Fishbein KW, et al. Glycogen synthase kinase-3β mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore. J Clin Invest 2004;113:1535–49. [13] Gross ER, Hsu AK, Gross GJ. Opioid-induced cardioprotection occurs via glycogen synthase kinase β inhibition during reperfusion in intact rat hearts. Circ Res 2004;94:960–6. [14] Critz SD, Cohen MV, Downey JM. Mechanisms of acetylcholine and bradykinin-induced preconditioning. Vascul Pharmacol 2005;42:201–9.
Garrett J. Gross Medical College of Wisconsin, Milwaukee, WI 53226, USA E-mail address: [email protected]. 19 April 2006