- Research Article
- Open Access
ERK-MAPK Signaling Opposes Rho-Kinase to Reduce Cardiomyocyte Apoptosis in Heart Ischemic Preconditioning
- Hong-Jun Bian†1,
- Xiao-Xing Li1,
- Xiao-Bo Liu2,
- Jun-Ping Sun3,
- Yun-Zhang1 and
- Xiao-Ping Ji1Email author
© The Feinstein Institute for Medical Research 2010
- Received: 3 September 2009
- Accepted: 16 March 2010
- Published: 17 March 2010
We and others have reported that Rho-kinase plays an important role in the pathogenesis of heart ischemia/reperfusion (I/R) injury. Studies have also demonstrated that the activation of Rho-kinase is reversed in ischemic preconditioning (IPC). However, the mechanisms by which Rho-kinase is increased in I/R and reversed in IPC are not thoroughly understood. In female Wistar rats, we created I/R by ligating the left anterior-descending branch of the coronary artery (LAD) for 30 min and releasing the ligature for 180 min. IPC rats underwent IPC (two cycles of 5-min ligation of the LAD and 5-min reflow) before I/R. IPC caused a significant increase in extracellular signal-regulated kinase (ERK)1/2 activity and reduced Rho-kinase activity and cardiomyocyte apoptosis (P < 0.05 versus I/R). Administration of PD98059, an inhibitor of ERK-mitogen-activated protein kinase (MAPK), increased cardiomyocyte apoptosis, Caspase-3 activity and myocardial infarction size (P < 0.05 versus IPC). Western-blot analysis showed that administration of PD98059 increased Rho-kinase activity. Treatment with fasudil, an inhibitor of Rho-kinase, reversed cell apoptosis caused by treatment with PD98059 in IPC. In addition, ROCK1 (Rho-kinase 1) may be the major Rho-kinase isoform that is opposed by ERK-MAPK signaling in IPC. These results indicate that ERK-MAPK signaling is required in IPC to oppose Rho-kinase activity in cardiomyocyte apoptosis in vivo.
In the prototypical mechanism of RhoA GTPase signaling, various environmental cues, acting through G-protein-coupled receptors or receptor-dependent and -independent tyrosine kinases, activate guanine-nucleotide exchange factors, which induce exchange of guouosine diphosphate (GDP) for GTP binding and translocation of GTP-RhoA to the plasma membrane. Upon translocation to the plasma membrane, GTP-RhoA activates one or more of its effectors, including the ubiquitously expressed Rho-kinase. Rho-kinase plays crucial roles in various cellular functions, and mediates cellular events such as changes in cell morphology, cell motility, focal adhesions and cytokinesis (1,2). The myosin-phosphatase target subunit (MYPT-1) is the major effector of Rho-kinase. To date, two iso-forms of Rho-kinase have been cloned, Rho-kinase 1 (ROCK1) and ROCK2. Both isoforms are about 160 kD, with 92% homology at the kinase domain (3). Accumulating evidence demonstrates that Rho-kinase plays important roles in many major cardiovascular diseases such as hypertension, heart failure, myocardial infarction and atherosclerosis. Results of recent animal studies suggest that inhibition of Rho-kinase protects the heart against ischemia/reperfusion (I/R) injury (4). We demonstrated recently that the inhibition of Rho-kinase reduces cell apoptosis in I/R in vivo via suppression of the translocation of JNK (c-Jun NH2-terminal kinase)-mediated apoptosisinducing factor (5).
Ischemic preconditioning (IPC) has been exploited as a powerful endogenous form of cardioprotection. IPC was first discovered by Murry and associates (6), who demonstrated that a brief period of repetitive cardiac I/R exerts a protective effect against subsequent lethal periods of ischemia. IPC was found to similarly reduce cytosolic and mitochondrial Ca2+ overloading, to augment postischemic functional recovery and to decrease infarct size (7). In addition, IPC is known to decrease cardiomyocyte apoptosis during reperfusion. Previous studies have demonstrated that IPC causes a substantial decrease of Rho-kinase activation during sustained ischemia and reduces infarct size (8). In this study, we also observed that the activation of Rho-kinase induced by I/R was significantly attenuated by IPC. However, little is known about the mechanisms by which Rho-kinase activity is increased in I/R and decreased in IPC. Therefore, the aim of this study was to elucidate the mechanism of decreased Rho-kinase activity in IPC.
All procedures were performed in conformity with the Institutional Animal Care and Use Committee and National Institutes of Health guidelines.
Myocardial I/R and IPC
Female Wistar rats (body weight 250–300 g, from Shandong University, Shandong Province, China) were maintained under conditions of standard lighting (alternating 12-h light/dark cycles), temperature (22°C ± 0.5°C) and humidity (60% ± 10%) for at least 1 wk before the experiments. The rats were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally). The trachea was cannulated with a PE-90 catheter, and artificial respiration was provided by a respirator with an FiO2 (fraction of inspired oxygen) of 0.80, a frequency of 100 strokes/min and a tidal volume of 0.8 to 1.2 mL to maintain normal PO2 (partial pressure of oxygen), PCO2 (partial pressure of carbon dioxide) and pH. A left lateral thoracotomy was made in the fourth intercostal space; the skin, muscles and ribs were retracted; and the pericardial sac was removed. The left-anterior branch of the descending coronary artery (LAD) was occluded by ligation with a 4–0 silk suture. The LAD ligation was performed by using an easily opened knot set on a PE50 silicon tube lying over the LAD. After 30 min of ischemia, the ligation was loosened and reperfusion occurred. Rats were killed at 180 min of reperfusion. The sham control animals were subjected to the entire surgical procedure and the silk suture was passed beneath the coronary artery, but the LAD was not ligated.
IPC was introduced by two cycles of 5 min of ischemia followed by 5 min of reperfusion. The rats were then subjected to 30 min of LAD occlusion followed by 180 min of reperfusion similar to that performed in the I/R rats.
The experimental groups we studied (Figure 1) were as follows:
The IR group (control group; n = 12) underwent 30-min ischemia and 180-min reperfusion.
The IPC group (n = 12) underwent two cycles of 5-min ischemia followed by 5-min reperfusion before sustained ischemia.
The IPC + PD98059 group (n = 12) underwent two cycles of 5-min ischemia followed by 5-min reperfusion before sustained ischemia plus administration of PD98059, an inhibitor of extracellular signal-regulated kinase (ERK)1/2 (9). PD98059 was dissolved in 100 µL dimethylsulfoxide (DMSO), and 0.3 mg/kg was administered intravenously between the onset and two brief periods of ischemia.
The IPC + fasudil group (n = 12) underwent two cycles of 5-min ischemia followed by 5-min reperfusion before sustained ischemia plus administration of fasudil, an inhibitor of Rho-kinase, (10 mg/kg intravenously) (10).
The IPC + PD98059 + fasudil group (n = 12) underwent two cycles of 5-min ischemia followed by 5-min reperfusion before sustained ischemia plus administration of PD98059 and fasudil.
Determination of Myocardial Infarct Size
At the end of reperfusion, the LAD coronary artery was reoccluded and Evans blue dye solution (3 mL, 2% wt/vol) was injected into the left ventricle to enable us to identify ischemic (area at risk [AAR], unstained) and nonischemic myocardium (area not at risk, stained blue). Then the hearts were harvested and rinsed in normal saline. The atria, right ventricle and great vessels were removed. The tissues were semifreezed for 30 min in a −20°C freezer so they could be sliced more easily. After that, the left ventricle was surgically isolated and cut into slices (1-mm thick). The AAR was separated from the area not at risk and then incubated with nitro-blue tetrazolium (1% wt/vol, 15 min at 37°C), which enabled us to distinguish between ischemic and infarcted tissue. After we evaluated the entire surface area, we designated segments with blue staining as viable and those without staining as nonviable (infarcted). Finally, the AAR and infarct size were calculated after the respective tissue samples were weighed, and the infarct size was expressed as the percentage of the AAR (11).
Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling
To evaluate apoptotic activity, we used the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique. Each section was deparaffinized and rehydrated with serial changes of xylene and ethanol. Proteinase K (20 mg/L) was applied to the section for 30 min to produce optimal proteolysis. The endogenous peroxidase was inhibited with 3% hydrogen peroxide for 10 min. A commercial apoptosis detection kit (Roche, Mannheim, Germany) was used. The TdT reaction was carried out for 1 h at 37°C in a humidified chamber, and then 3,3′-diaminobenzidine tetrahydrochloride chromogen was applied. Hematoxylin was used as a counterstain. TUNEL-positive cells were determined by random counting of 10 fields of the section and were expressed as a percentage of normal nuclei (12).
Immunohistochemical Staining for Phospho-ERK1/2
The 5-µm-thick, formalin-fixed, paraffinembedded sections were cut, deparaffinized and then rehydrated with graded alcohol and xylene. Endogenous peroxidase was blocked by using 3% hydrogen peroxide for 10 min, followed by a brief wash in Tris buffer (pH 7.2). Sections were boiled in citrate buffer (pH 6.0) with microwaves at 92°C–98°C for 10 min to retrieve the antigen. After blocking of nonspecific binding with 5% bovine serum albumin, the slides were incubated with primary antibody overnight at 4°C (antimouse phospho-ERK1/2 1:100; Santa Cruz Biochemicals, Santa Cruz, CA, USA). Sections were thoroughly washed in phosphate-buffered saline (PBS) and incubated with a peroxidase-conjugated polymer that carried antibodies to mouse (1:200) immunoglobulin for 30 min. After being rinsed with PBS, the sections were exposed for 5–10 min to 3,3′-diaminobenzidine tetrahydrochloride. The slides were rinsed in water and counterstained with hematoxylin and eosin. We used sections that were incubated with PBS instead of the primary antiserum as the negative controls. The sections were examined by using light microscopy and analyzed with a computer-assisted color-image analysis system (Image-ProPlus 5.0, Media Cybernetics, Bethesda, MD, USA).
Nuclear and cytoplasmic fractions were extracted by using a kit from Bipec Biopharma (Cambridge, MA, USA) according to the manufacturer’s instructions. Membrane fractions were extracted by using the kit from Keygen Biotech (Nanjing, China) according to the manufacturer’s instructions. Equal amounts of protein (50 µg) were fractionated on 10% sodium dodecyl sulfate-polyacrylamide gels in running buffer (25 mmol/L Tris, 192 mmol/L glycine, 0.1% [wt/vol] sodium dodecyl sulfate, pH 8.3) at 90 V and then electroblotted to nitrocellulose membranes. Membranes were blocked at room temperature with 5% nonfat milk in Tris-buffered saline (25 mmol/L Tris, 137 mmol/L NaCl, and 2.7 mmol/L KCl) containing 0.05% Tween-20 and then incubated overnight at 4°C with the following primary antibodies: β-actin (Santa Cruz Biochemicals; dilutions, 1:1000; molecular weight of β-actin, 43 kD), rabbit monoclonal antirat cleaved Caspase-3 antibody (Cell Signaling, Danvers, MA, USA; dilutions, 1:1000; molecular weight 17 or 19 kD), rabbit polyclonal antirat phospho-MYPT-1 (Thr850) antibody (Millipore, Billerica, MA, USA; dilutions: 1:1500; molecular weight 80 kD), rabbit polyclonal MYPT-1 antibody (bioWORLD, Dublin, Ohio, USA; dilutions, 1:500; molecular weight 130 kD), rabbit polyclonal p-MYPT-1 antibody (bioWORLD; dilutions, 1:500; molecular weight 130 kD), rabbit polyclonal RhoA antibody (bioWORLD; dilutions, 1:500; molecular weight 23 kD), rabbit monoclonal antirat ROCK1 antibody (Abcam, Cambridge, UK; dilutions, 1:1000; molecular weight 158 kD), goat polyclonal ROCK2 antibody (Santa Cruz Biochemicals; dilutions, 1:1000; molecular weight 160 kD) mouse monoclonal antirat phospho-ERK1/2 antibody (Santa Cruz Biochemicals; dilutions, 1:1000; molecular weight 42 or 44 kD). Then the membranes were washed three times in Tween-20 and incubated with the corresponding secondary antibody (Santa Cruz Biochemicals; dilutions, 1:10000) conjugated to horseradish peroxidase at room temperature. Immunoreactive bands were visualized with the SuperSignal West Pico enhanced chemoluminescence kit (Piece, Rockford, IL, USA) according to the manufacturer’s instructions. Band intensities were quantified by using a densitometer analysis system (Quantity One, Bio-Rad, Hercules, CA, USA).
Real-time Quantitative Reverse-Transcription Polymerase Chain Reaction for ROCK1
Total RNA was isolated from frozen rat left-ventricular tissue with Trizol reagent (Invitrogen, Carlsbad, CA, USA) (13). The concentration of total RNA was quantified by spectrophotometry (Beckman, Fullerton, CA, USA) and reverse transcribed with the use of a Moloney murine leukemia virus reverse transcriptase system (Fermentas, Sankt Leon-Rot, Germany) and oligo(dT). The sequences of the real-time polymerase chain reaction (PCR) primers were designed by using Primer Premier 5 (Premier Biosoft, Palo Alto, CA, USA) and synthesized by BioAsia (Shanghai, China). Real-time PCR was performed with an ABI PRISM 7500 sequence-detection system (Applied Biosystems, Foster City, CA, USA) by using an SYBR reverse transcription-PCR (RT-PCR) kit (Toyobo, Osaka, Japan). Expression level of ROCK1 was determined by threshold cycle number (Ct) and, by use of the ΔΔCt method, was normalized against the internal standard gene β-actin and compared relative to the control.
The primer sequences used were: ROCK1 (78 bp), forward: 5′-TTT CGG ATT CAA CTA GTG TTG C-3′, reverse: 5′-TGC AGG CAG AAC CAA CTG-3′; β-actin (254 bp), forward: 5′-AGA CCT TCA ACA CCC CAG-3′, reverse: 5′-CAC GAT TTC CCT CTC AGC-3′.
Values were expressed as means ± SD. Statistical analysis of the results was carried out by one-way analysis of the variance followed by the Newman-Keuls test. Significance was defined at P < 0.05.
Effect of IPC on Cardiomyocyte Apoptosis, Caspase-3 Cleavage and Infarct Size
Effect of IPC on Activity of ERK1/2
Effect of IPC on Activity of RhoA, ROCK1, ROCK2 and Rho-Kinase
Inhibition of ERK1/2 Increased Cardiomyocyte Apoptosis, Caspase-3 Activity and Infarct Size during IPC
As shown in Figure 2, the TUNEL-positive cells were significantly increased with administration of PD98059 (29.83% ± 0.7%) in the IPC + PD98059 group (P < 0.05 versus IPC group). As shown in Figure 3, after administration of PD98059, cleaved Caspase-3 significantly increased. The increase in the amount of cleaved Caspase-3 was about 2.1-fold (P < 0.05 versus IPC group). As shown in Figure 4, administration of PD98059 caused a significant increase of myocardial infarct size. The infarct size of the heart was 44.88% ± 0.7% in the IPC + PD98059 group (P < 0.05 versus IPC group).
ERK1/2 Opposed Rho-Kinase during IPC
Inhibition of Rho-Kinase Rescued the Effect of Inhibition of ERK1/2 on Apoptosis
To test the hypothesis that ERK1/2 acts to inhibit Rho-kinase in IPC, we examined whether the effect of inhibition of ERK1/2 could be rescued by inhibition of Rho-kinase. As shown in Figure 2, fasudil treatment reversed cell apoptosis caused by treatment with PD98059 in IPC. The number of TUNEL-positive cells was 23.3% ± 0.67% in the IPC + PD98059 + fasudil group. The amount of cleaved Caspase-3 was also significantly reduced in the IPC + PD98059 + fasudil group (Figure 3). Fasudil resulted in a 22% reduction in cardiomyocyte apoptosis and a 30% reduction in cleaved Caspase-3 in the IPC + PD98059 + fasudil group (P < 0.05 versus the IPC + PD98059 group). In addition, as shown in Figure 4, the infarct size of the heart was 39.44% ± 0.92% in the IPC + PD98059 + fasudil group. The combination of PD98059 and fasudil also resulted in a 12% reduction in myocardial infarct size (P < 0.05 versus the IPC + PD98059 group).
The results of this study provided the experimental evidence that ERK-MAPK signaling opposed Rho-kinase to decrease cardiomyocyte apoptosis in IPC. We report for the first time that IPC induces the reduction of Rho-kinase activity via the activation of ERK-MAPK signaling in vivo.
Recent studies have shown that Rho-kinase is involved in the clearance of apoptotic cells through regulation of the actin cytoskeleton (14,15). Rho-kinase is recognized as a major regulator of the morphological events that occur during the execution phase of apoptosis, including cell contraction, dynamic membrane blebbing, nuclear disintegration and fragmentation of apoptotic cells into apoptotic bodies. Previous studies have demonstrated that I/R upregulates expression of RhoA in ischemic myocardium and subsequently increases Rho-kinase activity (4). The Rho-kinase inhibitor significantly inhibits the activation of Rho-kinase after I/R and reduces infarct size and the amount of cell apoptosis (5). In this study we found that both RhoA translocation and Rho-kinase activity increased in the myocardium following I/R, but only the activation of Rho-kinase was reversed in IPC. In the present study, we assessed the activity of Rho-kinase by examining phosphorylation of MYPT-1. IPC resulted in a 48.3% decrease in the amount of phospho-MYPT-1, indicating that the activation of Rho-kinase was reduced in the myocardium following IPC. We demonstrated that IPC produced an attenuation of myocardial infarction and cardiomyocyte apoptosis. In this study we also used the TUNEL assay to examine apoptotic myocardial cells. The percentage of TUNEL-positive cardiomyocytes after 30-min ischemia and 180-min reperfusion was 33.87% ± 1.57%; these data are similar to the data reported previously for rat hearts subjected to I/R (5,16). IPC resulted in a 49% reduction in apoptotic cardiomyocytes. In addition, Caspase-3 activation also attenuated in IPC group. These results suggest that Rho-kinase activity and cardiomyocyte apoptosis are reduced by IPC.
We attempted to elucidate the mechanism of the reduction of Rho-kinase activity in IPC. Rho-kinase activity can be regulated by several distinct mechanisms, such as the Rho/Rho-kinase pathway (17) and the Caspase-3/Rho-kinase pathway (18). Other negative regulators have been found bind to and inhibit Rho-kinase activity. Recent evidence indicates that there is selective cross-talk between Ras and Rho signaling pathways (19–21). Ras is a small GTP-binding protein that functions as a molecular switch and as a link between membrane tyrosine-kinase receptors and the downstream signal-transduction machinery. Ras-Raf-MEK-ERK1/2 is the prototypic MAP kinase cascade. Results of previous studies have shown that inhibition of ERK-MAPK signaling in cancer cells can lead to upregulation of Rho-kinase activity (22). ERK-MAPK signaling was found to oppose Rho-kinase to promote endothelial cell survival (23). We focused on investigating the role of ERK-MAPK in Rho-kinase activity in IPC.
The ERK-MAPK was first described in 1990 as a serine/threonine protein kinase that was tyrosine phosphorylated by various extracellular signals including insulin and nerve growth factor (24). ERK1/2, a prototypic member of the MAPK family, is the best candidate among the protein kinases that determine the specificity of cellular responses—including cell proliferation, cell differentiation and cell survival—and are activated by a large variety of extracellular agents (25). In cultured cardiomyocytes, peptide growth factors and other MAPK-inducing agents provide protective effects against serum starvation, hypoxia and reoxygenation-induced injuries. Inhibition of MEK or ERK activity with pharmacological reagents diminishes these protective effects (26,27). In vivo, MEK transgenic hearts are protected from I/R injury and cardiomyocyte apoptosis (28). In contrast, inhibition of Raf-1 by the expression of a dominant-negative mutant or cardiac-specific knockout promotes cardiomyocyte apoptosis and decompensated heart failure (29,30). Yue et al. have demonstrated that inhibition of ERK enhances ischemia/reoxygenation-induced apoptosis and exaggerates reperfusion injury (31). But the role of ERK1/2 as a potential mediator of protection in the setting of IPC has been controversial, with the majority of studies supporting its role in IPC (32), because several studies have failed to demonstrate a role for ERK1/2 in IPC (33). By use of immunohistochemical analysis and Western blot, we demonstrated that ERK-MAPK was strongly activated in IPC. Cardiomyocyte apoptosis was also significantly reduced in IPC. Inhibition of ERK-MAPK in IPC led to increased Rho-kinase activity and cardiomyocyte apoptosis. ERK1/2 activation has not been examined directly in this setting; its activation would be expected to reduce cell death through several different antiapoptotic mechanisms (34). We investigated the effect of ERK1/2 on cardiomyocyte apoptosis and myocardial infarct size following Rho-kinase activation. The effects of inhibition of ERK-MAPK could be rescued by inhibition of Rho-kinase in vivo. Inhibition of Rho-kinase reversed cardiomyocyte apoptosis and the size of infarcts caused by inhibition of ERK-MAPK in IPC. The combination of PD98059 and fasudil restored protection, as reflected by decreased cardiomyocyte apoptosis (22%) and infarct size (12%) (P < 0.05 versus the IPC + PD98059 group). The infarct size did not decrease as significantly as cardiomyocyte apoptosis. These data show that cardioprotection of Rho-kinase inhibition is mediated by antiapoptosis more than decreased infarct size after inhibition of ERK-MAPK. These results indicate that ERK-MAPK signaling is required in IPC to oppose the effect of Rho-kinase signaling on cell apoptosis.
Previous studies have demonstrated that sustained ERK-MAPK signaling might downregulate ROCK1 (19). In this study, we also examined ROCK1 and ROCK2 with Western blot analysis. We showed that ROCK1 and ROCK2 both increased in I/R, but only ROCK1 expression was downregulated in IPC. Inhibition of ERK-MAPK in IPC also leads to increased ROCK1 expression. Thus, ROCK1 may be the major Rho-kinase that is opposed by ERK-MAPK signaling in IPC.
In our study, inhibition of ERK1/2 and Rho-kinase partly reduced cell apoptosis, and ERK1/2 expression was not recovered. ERK-MAPK signaling leads to downregulation of Rho-kinase, but not RhoA, activity (19). I/R upregulated expression of RhoA in ischemic myocardium and subsequently increased Rho-kinase activity (4).
Overpression of RhoA in I/R might not be changed by preconditioning. Because RhoA is the main upstream activator of Rho-kinase, ERK-MAPK signaling partly decreased Rho-kinase activity. Thus cell apoptosis was reduced when fasudil was used in the IPC + PD98059 group. These data show that in IPC, Rho-kinase inhibition is mediated, at least in part, via ERK-MAPK signaling. Next we will study the cross-talk between ERK-MAPK signaling and thoroughly investigate Rho/Rho-kinase signaling in I/R and IPC.
In summary, we have demonstrated that Rho-kinase activity and cardiomyocyte apoptosis were reduced in IPC in vivo. Our results also indicated that ERK-MAPK activity increased in IPC, and inhibition of ERK-MAPK led to increased Rho-kinase activity and cardiomyocyte apoptosis. These results establish the relationship between ERK-MAPK and Rho-kinase, demonstrating that ERK-MAPK signaling is required in IPC to oppose the effect of Rho-kinase signaling on cell apoptosis in vivo. The elucidation of this process may lead to new treatment possibilities for patients suffering coronary heart diseases.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This work was supported by the National High-tech Research and Development Program of China Grant 2006AA02A406. We thank R. Wang, J-B Feng, C-X Liu, X-P Wang and H Jiang for their excellent technical help.
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