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Abstract
Objective— Granulocyte colony stimulating factor (G-CSF) has been reported recently to prevent cardiac remodeling and dysfunction after acute myocardial infarction through signal transducer and activator of transcription 3 (STAT3). In this study, we examined acute effects of G-CSF on the heart against ischemia-reperfusion injury.
Methods and Results— Rat hearts were subjected to global 35-minute ischemia and 120-minute reperfusion in Langendorff system with or without G-CSF (300 ng/mL). G-CSF administration was started at the onset of reperfusion. Triphenyltetrazolium chloride staining revealed that G-CSF markedly reduced the infarct size. G-CSF strongly activated Janus kinase 2 (Jak2), STAT3, extracellular signal-regulated kinase (ERK), Akt, and endothelial NO synthase (NOS) in the hearts subjected to ischemia followed by 15-minute reperfusion. The G-CSF–induced reduction in infarct size was abolished by inhibitors of phosphatidylinositol 3-kinase, Jak2, and NOS but not of mitogen-activated protein kinase kinase (MEK).
Conclusions— These results suggest that G-CSF acts directly on the myocardium during ischemia-reperfusion injury and has acute nongenomic cardioprotective effects through the Akt–endothelial NOS pathway.
Myocardial infarction (MI) is the most common cause of cardiac morbidity and mortality in many countries. Reperfusion therapy is beneficial to prevent cardiomyocyte death and contractile dysfunction after MI. However, numerous studies have shown that reperfusion itself may enhance the injury, resulting in extension of infarct size after ischemia (ie, ischemia-reperfusion [IR] injury). Although ischemic preconditioning and pharmacological preconditioning mimetics strongly protect the heart against IR injury, the requirement for pretreatment has greatly limited their clinical relevance. Meanwhile, it has been reported recently that brief intermittent ischemia applied after the onset of reperfusion, termed “postconditioning,” reduces myocardial injury to an extent comparable to preconditioning.1 Various cardioprotective mechanisms have been reported including activation of reperfusion injury salvage kinases pathway consisting of phosphatidylinositol 3-kinase (PI3K)–Akt and extracellular signal-regulated kinase (ERK).2 Therefore, pharmacological postconditioning by administration of agents that activate the reperfusion injury salvage kinase pathway seems to be beneficial to IR injury.
Granulocyte colony stimulating factor (G-CSF) has been reported recently to prevent left ventricular (LV) remodeling and dysfunction after acute MI.3–6 G-CSF is a hematopoietic cytokine that promotes proliferation and differentiation of neutrophil progenitors. Because G-CSF has been used widely to induce mobilization of hematopoietic stem cells for transplantation, and the safety has been established, G-CSF could be used to treat MI if the efficacy has been established and the mechanisms of its beneficial effects have been elucidated. We demonstrated that G-CSF phosphorylates and activates various signaling pathways such as Akt, ERK, and Janus kinase 2 (Jak2)–signal transducer and activator of transcription 3 (STAT3) through G-CSF receptors on cardiac myocytes and protects cardiomyocytes from death at least in part through STAT3-induced upregulation of antiapoptotic proteins.7 Furthermore, pretreatment with G-CSF reduced myocardial IR injury using Langendorff perfusion model.7 In that study, G-CSF was administered from 20 minutes before ischemia to 120 minutes after reperfusion to show the preconditioning effects of G-CSF on myocardium. Although the transcriptional regulation plays a critical role in preventing LV remodeling, G-CSF has acute, probably nongenomic effects on the heart.7 In the present study, we therefore examined whether G-CSF has the postconditioning-like effects on myocardial IR injury using isolated perfused hearts and clarified the molecular mechanism.
Materials and Methods
Animals
Male Wistar rats (300±50 g) were used for these studies. All animals were obtained from Takasugi Experimental Animals Supply Co Ltd, Japan. All experimental protocols were approved by the institutional animal care and use committee of Chiba University.
Isolated Perfused Rat Heart
Hearts were excised rapidly and mounted on a Langendorff perfusion system. All isolated hearts were stabilized for 30 minutes by perfusion of Krebs-Henseleit (KH) buffer followed by 35 minutes of global normothermic ischemia and reperfusion (IR) for either 120 minutes to measure infarct size or 15 minutes to measure kinase activities as described previously.2
Treatment Protocols
The experimental protocols for these studies are presented in Figure 1A. All the kinase inhibitors were dissolved in dimethyl sulfoxide (DMSO) and added to KH buffer so that the final DMSO concentration was <0.005%. The hearts were randomly assigned to one of the following treatment groups: (1) administration of 0.005% DMSO vehicle (control group; n=5); (2) administration of G-CSF (300 ng/mL) was started at the onset of reperfusion and continued throughout reperfusion (G-CSF group; n=5); (3) G-CSF+Jak2 inhibitor AG490 (5 μmol/L; G+AG group; n=5); (4) G-CSF+PI3K inhibitor LY294002 (5 μmol/L; G+LY group; n=5); (5) G-CSF+mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 (10 μmol/L; G+PD group; n=5); (6) G-CSF+NO synthase (NOS) inhibitor Nω-nitro-l-arginine methyl ester (l-NAME; 30 μmol/L; G+l-NAME group; n=5). The administration of each inhibitor was started 15 minutes before ischemia and continued throughout reperfusion. Additionally, hearts of 4 to 5 rats in each group were used to perform Western blot analysis, and hearts of 6 rats in each group were used to analyze the dose dependence of G-CSF–induced cardioprotection.
Figure 1. Protocol, LV pressure, and infarct size. A, Experimental protocol. B, %LVDP. Data shown indicate the mean percentage of LVDP recovery±SEM (each group n=6). G10, G-CSF 10 ng/mL; G50, G-CSF 50 ng/mL; G300, G-CSF 300 ng/mL. *P<0.05 vs control group. C, Infarct size. Results are given as mean±SEM (each group n=6). G10, G-CSF 10 ng/mL; G50, G-CSF 50 ng/mL; G300, G-CSF 300 ng/mL. *P<0.05 compared with control group. #P<0.01 compared with control group.
LV Pressure
To evaluate the contractile function, a polyethylene film balloon was inserted into the cavity of the left ventricle through the left atrium. The balloon was filled with saline to adjust the baseline end-diastolic pressure to 5 to 10 mm Hg. LV pressure was measured continuously. LV developed pressure (LVDP) was designated as difference between systolic and diastolic pressures of LV. %LVDP represents percentage of LVDP recovery.
Infarct Size
Hearts were sliced into 4 transverse sections and each was weighed, incubated for 3 minutes at 37°C in 1% triphenyltetrazolium chloride (TTC), and photographed, and the area of infarcted (unstained) and viable (stained) tissue was measured by computed planimetry. The mass-weighted average of ratio of infarct area to total cross-sectional area of LV from each slice was determined (% infarct size).
Western Blot Analysis
At 15 minutes after reperfusion, the LV was excised and freeze-clamped in liquid nitrogen before being stored at −80°C. We probed the membranes with antibodies to phospho-Jak2 (Tyrosine 1007/1008), phospho-STAT3 (Tyrosine 705), phospho-ERK (Threonine 202/Tyrosine 204), phospho-Akt (Serine 473), phospho-endothelial NOS (eNOS; phospho-eNOS; Serine 1177; Cell Signaling), Jak2, STAT3, Akt (Santa Cruz Biotechnology), ERK (Zymed Laboratories Inc), or eNOS (BD Biosciences). We used the enhanced chemiluminescence system (Amersham Biosciences Corp) for detection.
Measurement of NO Release
A sealed reaction chamber was placed around the hearts to collect the effluent from perfused hearts by KH solution, and the chamber was filled with N2 gas and maintained at 37°C. In the bottom of chamber, the collecting space was placed and filled with the effluent, which was replaced continuously. NO release was measured using an NO-selective sensor (amiNO-700; Innovative Instruments Inc) as described previously.8 The sensor was placed in the effluent to measure directly and quick NO concentration released from the hearts. The level of NO concentration (nmol/L) in the effluent was measured from preischemia throughout reperfusion. The sensor was calibrated by producing standard concentrations of NO based on dilutions of NO-equilibrated solutions (Innovative Instruments Inc). Data were digitally recorded and analyzed with APOLLO 4000 free radical analyzer (World Precision Instruments). NO concentration (% of preischemia)=NO concentration at each time after reperfusion (nmol/L)/NO concentration at preischemia (nmol/L)×100 (%).
Statistical Analysis
All data are presented as means±SEM. All data were analyzed by 1-way ANOVA followed by the Fisher procedure for comparison of means. A P value <0.05 was considered statistically significant.
Results
G-CSF Protects Reperfused Myocardium
Isolated hearts were subjected to global ischemia for 35 minutes and then exposed to G-CSF during reperfusion. There was no difference in heart rate between control and G-CSF–treated groups. After reperfusion, LVDP was significantly higher in G-CSF–treated hearts than control hearts, and there was a dose dependency of G-CSF (Figure 1B). TTC staining revealed that infarct size was significantly reduced by G-CSF in a dose-dependent manner (control group 55.8±3.2% versus G-CSF 10 ng/mL; 45.2±3.1%, G-CSF 50 ng/mL; 39.3±2.1%, G-CSF 300 ng/mL, 35.2±3.7%; Figure 1C).
G-CSF Activates Various Signaling Pathways in Reperfused Myocardium
In the previous study, we demonstrated that cardiomyocytes have G-CSF receptors and that G-CSF activates Jak2, Akt, and ERK in cultured cardiomyocytes.7 In this study, we examined whether the same signals were phosphorylated and activated by G-CSF in whole hearts during reperfusion. Western blot analysis using phosphoprotein-specific antibodies demonstrated that G-CSF (300 ng/mL) phosphorylated Jak2, STAT3, ERK, Akt, and eNOS as early as 7 minutes (data not shown), and the phosphorylation level reached a peak at 15 minutes (Figure 2A through 2E). Phosphorylation of the amino acid of each protein has been reported to associate with activation of each protein.
Figure 2. G-CSF–induced signaling pathway and involvement of G-CSF in NO production. A through E. Phosphorylation of various signal pathways in reperfused myocardium. Zero on the x axis indicates the onset of reperfusion. Results are given as means±SEM (each group n=4 to 5). G+PD, G-CSF+PD98059; G+AG, G-CSF+AG490; G+LY, G-CSF+LY294002; G+l-NAME, G-CSF+l-NAME. *P<0.05 compared with control group. Representative results of 5 independent experiments were shown. F, NO concentration (% of preischemia) at 30 minutes after reperfusion. Results are given as means±SEM (each group n=5). *P<0.05 compared with control group.
Akt/eNOS Pathway Mediates G-CSF–Induced Cardioprotective Effects
To assess the signaling pathways involved in G-CSF–induced cardioprotection, hearts were treated with G-CSF in the presence of various inhibitors such as a MEK inhibitor PD98059, a Jak2 inhibitor AG490, a PI3K inhibitor LY294002, or a NOS inhibitor l-NAME. PD98059, AG490, and LY294002 completely inhibited G-CSF–induced phosphorylation of ERK, Jak2, and Akt, respectively (Figure 2A through 2E). Phosphorylation of eNOS by G-CSF was inhibited in the presence of AG490 or LY294002 but not PD98059 (Figure 2C). Furthermore, AG490 inhibited G-CSF–induced phosphorylation of Akt (Figure 2B).
To evaluate whether G-CSF increases NO production through eNOS phosphorylation, we measured NO concentration in the effluent by using an NO-selective sensor and its analyzer. NO concentration of the hearts was measured at the time of preischemia and 30 minutes after reperfusion. G-CSF (300 ng/mL) significantly increased NO production in the reperfused hearts (control group 99.7±1.7% versus G-CSF group 109.1±2.0%; P<0.05; Figure 2F). The increase in NO production induced by G-CSF was suppressed completely by l-NAME (G+l-NAME group; 96.6±1.0%; Figure 2F).
G-CSF–induced reduction in infarct size (control group 55.8±3.2%; G-CSF group 35.2±3.7%) was blocked by AG490 and LY294002 (G+AG group 63.0±3.6%; G+LY group 56.8±8.2%), whereas the pretreatment with PD98059 had no effect on G-CSF–induced reduction in infarct size (37.9±1.5%; Figure 3A and 3B). These results suggest that G-CSF protects the heart from IR injury by phosphorylating the Jak2–PI3K/Akt pathway but not the ERK pathway. To elucidate the downstream pathway of PI3K/Akt, we examined the role of eNOS using l-NAME. l-NAME treatment inhibited G-CSF–induced reduction in infarct size (G+l-NAME group 52.4±6.9%; Figure 3A and 3B). Each inhibitor alone did not influence the infarct size (data not shown).
Figure 3. Signals in G-CSF–induced cardioprotection. A, TTC staining of hearts in each group (each group n=5). B, Infarct size. Results are given as means±SEM (each group n=5). G+PD, G-CSF+PD98059; G+AG, G-CSF+AG490; G+LY, G-CSF+LY294002; G+l-NAME, G-CSF+l-NAME. *P<0.05 compared with control group. C, Hypothetical scheme postulating the possible signaling pathways induced by G-CSF.
Discussion
In the present study, we demonstrated that G-CSF has direct and acute protective effects on myocardium against IR injury by activating Akt–eNOS pathway in the isolated perfused rat hearts. Recently, G-CSF has been reported to prevent LV remodeling and dysfunction after acute MI in animal models.4–7 Orlic et al reported that G-CSF induces myocardial regeneration by promoting mobilization of bone marrow stem cells into the injured region after MI.3 However, recent studies demonstrated that bone marrow hematopoietic stem cells cannot transdifferentiate into cardiomyocytes in MI hearts.9,10 We demonstrated recently that G-CSF acts directly on cardiomyocytes and induces the survival signals in post-MI hearts.7 G-CSF–induced phosphorylation of Jak2–STAT3 pathway plays a critical role in upregulating the expression of antiapoptotic proteins and angiogenetic factors on post-MI hearts. Although transcriptional regulation by STAT3 is important for preventing LV remodeling at chronic stage, Langendorff experiments revealed that other mechanisms may also be involved in the acute stage. G-CSF actually activated various signaling pathways such as Akt, ERK, and Jak2–STAT3 in cultured cardiomyocytes.7
In the present study, we examined whether G-CSF administered at the onset of reperfusion has acute postconditioning-like effects on myocardial IR injury. G-CSF phosphorylated and activated ERK, Jak2, STAT3, Akt, and eNOS and significantly reduced the infarct size. Because Jak2 inhibitor AG490 inhibited G-CSF–induced phosphorylation of Jak2, STAT3, Akt, and eNOS but not ERK, and PI3K inhibitor LY294002 suppressed G-CSF–induced phosphorylation of Akt and eNOS but not Jak2, STAT3, and ERK, the signals are activated by the order of Jak2>PI3K>Akt>eNOS, and the signaling pathways of ERK may be different. G-CSF increased NO production in reperfused hearts, and its effect was inhibited by l-NAME. Furthermore, the reduction of infarct size afforded by G-CSF administration was completely abolished in the presence of AG490, LY294002, and l-NAME but not PD98059. These results suggest that G-CSF acts directly on the myocardium during IR injury and has cardioprotective effects even if G-CSF administration is started after reperfusion, and that G-CSF–induced activation of Akt-eNOS and production of NO are important for its acute cardioprotective effects (Figure 3C).
Transcriptional regulation by the phosphorylation of Jak2–STAT3 pathway is one of the key mechanisms in G-CSF–mediated cardioprotection against MI heart in the chronic stage, whereas the Akt-eNOS pathway may be very important in the acute stage. eNOS has been reported to be phosphorylated by Akt, and activated eNOS-producing NO has been reported to play a pivotal role in the cardioprotection of preconditioning by preserving ischemic blood flow and attenuating platelet aggregation and neutrophil–endothelium interaction after IR.11 NO is known to be the trigger for ischemic preconditioning, especially in the late phase of preconditioning, and to activate downstream pathways including protein kinase G, mitochondrial ATP-sensitive K+ channels, free radicals, and protein kinase C. However, it remains unknown whether NO acts as the trigger for postconditioning as well as preconditioning. It has been reported recently that postconditioning inhibits the opening of mitochondrial permeability transition pore (mPTP), which is involved in IR injury,12 and that NO inhibits mPTP opening.11 Because glycogen synthase kinase-3β, another downstream molecule of Akt, has been reported to induce opening mPTP,13 glycogen synthase kinase-3β may also be involved in G-CSF–induced inhibition of IR injury. Further studies are needed to clarify the downstream of NO in the postconditioning-like effects of G-CSF against IR injury. The present study suggests that G-CSF can be used as a novel and valuable postconditioning agent.
Acknowledgments
This work was supported by a grant-in-aid for scientific research, developmental scientific research, and scientific research on priority areas from the Ministry of Education, Science, Sports, and Culture and by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Drug ADR Relief, R&D Promotion and Product Review of Japan. The authors thank E. Fujita, R. Kobayashi, M. Ikeda, Y. Ohtsuki, A. Furuyama, and M. Tamagawa for excellent technical assistance.
- Received October 12, 2005.
- Accepted March 9, 2006.
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- Granulocyte Colony Stimulating Factor Directly Inhibits Myocardial Ischemia-Reperfusion Injury Through Akt–Endothelial NO Synthase Pathway
