Activation of c-Jun N-terminal kinases and p38-mitogen-activated protein kinases in human heart failure secondary to ischaemic heart disease.

Three well-characterized mitogen-activated protein kinase (MAPK) subfamilies are expressed in rodent and rabbit hearts, and are activated by pathophysiological stimuli. We have determined and compared the expression and activation of these MAPKs in donor and failing human hearts. The amount and activation of MAPKs was assessed in samples from the left ventricles of 4 unused donor hearts and 12 explanted hearts from patients with heart failure secondary to ischaemic heart disease. Total MAPKs or dually phosphorylated (activated) MAPKs were detected by Western blotting and MAPK activities were measured by in gel kinase assays. As in rat heart, c-Jun N-terminal kinases (JNKs) were detected in human hearts as bands corresponding to 46 and 54 kDa; p38-MAPK(s) was detected as a band corresponding to approximately 40 kDa, and extracellularly regulated kinases, ERK1 and ERK2, were detected as 44- and 42-kDa bands respectively. The total amounts of 54 kDa JNK, p38-MAPK and ERK2 were similar in all samples, although 46-kDa JNK was reduced in the failing hearts. However, the mean activities of JNKs and p38-MAPK(s) were significantly higher in failing heart samples than in those from donor hearts (P<0.05). There was no significant difference in phosphorylated (activated) ERKs between the two groups. In conclusion, JNKs, p38-MAPK(s) and ERKs are expressed in the human heart and the activities of JNKs and p38-MAPK(s) were increased in heart failure secondary to ischaemic heart disease. These data indicate that JNKs and p38-MAPKs may be important in human cardiac pathology.     

Stage-specific differential activation of mitogen-activated protein kinases in hypertrophied and failing rat hearts

Mitogen-activated protein kinases (MAPKs) are involved in the early development of cardiac hypertrophy, but their roles in chronic left ventricular hypertrophy (LVH) are unclear. We studied the angiotensin (Ang) II-induced cardiac MAPK activation of the hypertensive Dahl salt-sensitive (DS) rats in the subacute developing LVH stage, the chronic compensated LVH stage, and the congestive heart failure (CHF) stage. In the isolated, coronary-perfused heart preparation, Ang II infusion (1x10(-6)mol/l) activated extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38-MAPK in the LV myocardium. No substantial differences were observed in the Ang II-induced ERK activation between the normotensive control DS rats and the hypertensive DS rats in either stage. In contrast, the Ang II-induced activation of JNK and p38-MAPK was augmented in the subacute LVH stage of the hypertensive DS rats, but then progressively attenuated in the chronic LVH and CHF stages. Chronic treatment with an angiotensin converting enzyme inhibitor, temocapril (20 mg/kg/day), ameliorated the responsiveness of the JNK/p38-MAPK activation, suggesting that the decreased JNK/p38-MAPK activation is a consequence of negative feedback regulation for the activated cardiac renin-angiotensin system in chronic LVH and CHF. Thus, the Ang II-induced activation of multiple cardiac MAPK pathways are differentially regulated, depending on the stages of chronic hypertrophic process. The JNK and p38-MAPK activation may be involved in the early development of adaptive LVH. However, the responsiveness of the cardiac JNK/p38-MAPK pathways progressively decreased in chronic LVH and CHF under the chronic activation of tissue renin-angiotensin system.     

Brief reoxygenation episodes during chronic hypoxia enhance posthypoxic recovery of LV function

Children with congenital cyanotic heart defects have worse outcomes after surgical repair of their heart defects compared with noncyanotic ones. Institution of extracorporeal circulation in these children exposes the cyanotic heart to reoxygenation injury. Mitogen-activated protein kinase (MAPK) signaling cascades are major regulators of cardiomyocyte function in acute hypoxia and reoxygenation. However, their roles in chronic hypoxia are incompletely understood. We determined myocardial activation of the three major MAPKs, c-Jun NH₂-terminal kinase (JNK), extracellular signal-regulated kinase-1/2 (ERK1/2), and p38-MAPK in adult rats exposed to hypoxia (FIO₂ = 0.10) for varying periods of time. Myocardial function was analyzed in isolated perfused hearts. Acute hypoxia stimulated JNK and p38-MAPK activation. Chronic hypoxia (2 weeks) was associated with increased p38-MAPK (but not JNK) activation, increased apoptosis, and impaired posthypoxic recovery of LV function. Brief normoxic episodes (1 h/day) during chronic hypoxia abolished p38-MAPK activation, stimulated MEK-ERK1/2 activation modestly, and restored posthypoxic LV function. In vivo p38-MAPK inhibition by SB203580 or SB202190 in chronically hypoxic rats restored posthypoxic LV function. These results indicate that sustained hypoxemia maintains p38-MAPK in a chronically activated state that predisposes to myocardial impairment upon reoxygenation. Brief normoxic episodes during chronic hypoxia prevent p38-MAPK activation and restore posthypoxic recovery of myocardial function. Returned for 1st revision: 25 November 2005 1st Revision received: 10 February 2006 Returned for 2nd revision: 28 November 2005 2nd Revision received: 3 March February 2006     

Activation of mitogen-activated protein kinases in the non-ischemic myocardium of an acute myocardial infarction in rats

As one of the signal transduction pathways related to myocardial remodeling, mitogen-activated protein kinases (MAPKs) possibly play an important role in ischemic heart disease, but it is still unknown whether myocardial MAPKs are activated in the non-ischemic region of an acute myocardial infarction (AMI). Therefore, the present study investigated the myocardial activity of extracellular signal-regulated kinases (ERKs), c-Jun NH2 terminal kinases (JNKs) and p38MAPK during the acute phase of an infarction of the rat heart, and measured the geometrical ventricular changes by echocardiography. All MAPKs were significantly activated in the ischemic myocardium (IM), non-ischemic septal wall (SW), and right ventricular wall (RV). Furthermore, the activation patterns of MAPKs differed in each region. The activation of p44ERK, JNKs and p38MAPK in the IM occurred rapidly after myocardial ischemia, followed by those in the SW and RV. The activator protein-1 DNA binding activities of the IM, SW and RV increased significantly at I day after coronary ligation. Echocardiography showed increased SW motion and RV dilatation. In conclusion, this is the first in vivo evidence that myocardial MAPKs are activated in the non-ischemic region of an AMI. Echocardiographic results suggest that acceleration of workload and/or stretch may partially induce the activation of MAPKs.     

Differential activation of mitogen-activated protein kinases in ischemic and nitroglycerin-induced preconditioning

Previous studies have shown that the cardioprotective effect of ischemic preconditioning (IPC) can be mimicked pharmacologically with clinically relevant agents, including nitric oxide (NO) donors. However, whether pharmacological preconditioning shares the same molecular mechanism with IPC is not fully elucidated. The present study aimed to determine the activation of mitogen-activated protein kinases (MAPKs) (ERK1/2, p38 MAPK and p46/p54 JNKs) during ischemia and at reperfusion in nitroglycerin-induced preconditioning as compared to IPC and to correlate this with the conferred cardioprotection in anesthetized rabbits. Sixty minutes of intravenous administration of nitroglycerin was capable of inducing both early and late phase preconditioning in anesthetized rabbits, as it was expressed by the reduction of infarct size. Despite the cardioprotective effect conferred by both ischemic and nitroglycerin-induced preconditioning, there was a differential phosphorylation of MAPKs between the studied groups. p38 MAPK was activated early in ischemia in both ischemic and the early nitroglycerin-induced preconditioning while JNKs were markedly increased only after IPC. Furthermore, in these groups, ERK1/2 were activated during reperfusion. A different profile was observed in the late preconditioning induced by nitroglycerin with increased p38 MAPK and ERK1/2 phosphorylation during late ischemia. No activation of JNKs was observed at any time point in this group. It seems that activation of individual MAPK subfamilies depends on the nature of preconditioning stimulus. Returned for 1st revision: 18 November 2005 1st revision received: 3 January 2006 Returned for 2nd revision: 19 January 2006 2nd revision received: 6 February 2006 Returned for 3rd revision: 22 February 2006 3rd revision received: 1 March 2006     

Protein kinase activation and myocardial ischemia/reperfusion injury

Myocardial ischemia and ischemia/reperfusion activate several protein kinase pathways. Protein kinase activation potentially regulates the onset of myocardial cell injury and the reduction of this injury by ischemic and pharmacologic preconditioning. The primary protein kinase pathways that are potentially activated by myocardial ischemia/reperfusion include: the MAP kinases, ERK 1/2, JNK 1/2, p38 MAPKalpha/beta; the cell survival kinase, Akt; and the sodium-hydrogen exchanger (NHE) kinase, p90RSK. The literature does not support a role for ischemia/reperfusion in the activation of the tyrosine kinases, Src and Lck, or the translocation and activation of PKC. This review will detail the role of these protein kinases in the onset of myocardial cell death by necrosis and apoptosis and the reduction of this injury by preconditioning.     

Activation of mitogen-activated protein kinases in in vivo ischemia/reperfused myocardium in rats.

In this study, we investigate the in vivo activation of mitogen-activated protein kinases (MAPK) as important signal transduction cascades observed after myocardial ischemia/reperfusion. Myocardial continuous ischemia and ischemia/reperfusion was produced in Wistar rats. The activities of MAPKs in the ischemic and ischemia/reperfused regions were measured using an in-gel kinase assay, an in vitro kinase assay and Western blot analysis. Activator protein-1 (AP-1) DNA binding activity was determined using an electrophoretic mobility shift assay. DNA fragmentation was detected as DNA ladders by agarose gel electrophoresis. The p46JNK and p55JNK activities of continuous ischemia were significantly increased at 30 min (5.9 and 4.2 fold, respectively P<0.05). Coronary reperfusion increased both p42ERK and p44ERK activities at 30 min (3.0 and 2.3 fold P<0.01), and both p46JNK and p55JNK activities at 30 min (1.4 and 1.7 fold P<0.05). The AP-1 DNA binding activities of continuous ischemia were significantly increased at 1, 3 and 7 days (28, 21 and 17 fold, respectively P<0.01). Coronary reperfusion markedly decreased AP-1 DNA binding activities at 1 (41%P<0.01) and 3 days (48%P<0.05). Myocardial DNA fragmentation was considerably more enhanced by reperfusion than continuous ischemia. In conclusion, our present work provides the first in vivo evidence that ERK and JNK are activated by reperfusion from the activities of continuous ischemia. These signal transduction mechanisms may be partially responsible for the myocardial injury.     

Cytoprotection by Jun kinase during nitric oxide-induced cardiac myocyte apoptosis

Nitric oxide (NO) induces apoptosis in cardiac myocytes through an oxidant-sensitive mechanism. However, additional factors appear to modulate the exact timing and rate of NO-dependent apoptosis. In this study, we investigated the role of mitogen-activated protein kinases (MAPKs) (extracellular signal-regulated kinase [ERK] 1/2, c-Jun N-terminal kinase [JNK] 1/2, and p38MAPK) in NO-mediated apoptotic signaling. The NO donor S:-nitrosoglutathione (GSNO) induced caspase-dependent apoptosis in neonatal rat cardiac myocytes, preceded by a rapid (<10-minute) and significant (approximately 50-fold) activation of JNK1/2. Activation of JNK was cGMP dependent and was inversely related to NO concentration; it was maximal at the lowest dose of GSNO (10 micromol/L) and negligible at 1 mmol/L. NO slightly increased ERK1/2 beginning at 2 hours but did not affect p38MAPK activity. Inhibitors of ERK and p38MAPK activation did not affect cell death rates. In contrast, expression of dominant-negative JNK1 or MKK4 mutants significantly increased NO-induced apoptosis at 5 hours (56.77% and 57.37%, respectively, versus control, 40.5%), whereas MEKK1, an upstream activator of JNK, sharply reduced apoptosis in a JNK-dependent manner. Adenovirus-mediated expression of dominant-negative JNK1 both eliminated the rapid activation of JNK by NO and accelerated NO-mediated apoptosis by approximately 2 hours. These data indicate that NO activates JNK as part of a cytoprotective response, concurrent with initiation of apoptotic signaling. Early, transient activation of JNK serves both to delay and to reduce the total extent of apoptosis in cardiac myocytes.     

Src family kinase and adenosine differentially regulate multiple MAP kinases in ischemic myocardium: modulation of MAP kinases activation by ischemic preconditioning.

Recent studies suggest that ischemia activates Src and members of the mitogen-activated protein (MAP) kinase superfamily and their downstream effectors, including big MAP kinase 1 (BMK1) and p90 ribosomal S6 kinase (p90RSK). It has also been reported that adenosine is released during ischemia and involved in triggering the protective mechanism of ischemic preconditioning. To assess the roles of Src and adenosine in ischemia-induced MAP kinases activation, we utilized the Src inhibitor PP2 (4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and the adenosine receptor antagonist 8-(p-sulfophenyl) theophylline (SPT) in perfused guinea pig hearts. PP2 (1 microm) inhibited ischemia-induced Src, BMK1 and JNK activation but not JAK2 and p38 activation. SPT inhibited ischemia-mediated p38 and JNK activation. These results demonstrate that Src family kinase and adenosine regulate MAP kinases by parallel pathways. Preconditioning significantly improved both recovery of developed pressure and dp/dt in isolated guinea pig hearts. Since the protective effect of preconditioning was blocked by PP2 (1 microm) and SPT (50 microm), we next investigated the regulation of Src, MAP kinases and p90RSK during preconditioning. The activity and time course of ERK1/2 was not changed, but p90RSK activation by reperfusion was completely inhibited by preconditioning. In contrast, the activation by ischemia of Src, BMK1, p38 and JNK was significantly faster in preconditioned hearts. Maximal BMK1 activation by ischemia was also significantly enhanced by preconditioning. These data suggest important roles for Src family kinases and adenosine in mediating preconditioning, and suggest specific roles for individual MAP kinases in preconditioning.     

Role of p38 and JNK in liver ischemia and reperfusion

Background/purpose

The signal transduction of mitogen-activated protein kinases (MAPKs) has appeared to be an important mediator of ischemic-related events. Because of this, we analyzed the participation of p38 and JNK in liver ischemia and reperfusion, as two individual members of the MAPK family of proteins.

Methods

All papers referred to in PubMed for the past 15 years were analyzed to determine how and when these MAPKs were considered to be an intricate part of the ischemic event. References were cross-studied to ascertain whether other papers could be found in the literature.

Results

The role of p38 and JNK in liver ischemia was confirmed in the literature. The activation of these mediators was associated with the induction of apoptosis and necrosis. Inhibitors of p38 and JNK reduced the liver ischemia and reperfusion damage, probably through the mechanisms mentioned before.

Conclusions

The development of effective inhibitors of p38 and JNK protein mediators is important for minimizing the harmful effects associated with liver ischemia and reperfusion.     

Src and multiple MAP kinase activation in cardiac hypertrophy and congestive heart failure under chronic pressure-overload: comparison with acute mechanical stretch

Activation of members of the mitogen-activated protein (MAP) kinase family and their downstream effectors has been proposed to play a key role in the pathogenesis of cell survival, ischaemic preconditioning, cardiac hypertrophy and heart failure. This study investigated the responses of Src kinase and multiple MAP kinases during the transition from compensated pressure-overload hypertrophy to decompensated congestive heart failure. Extracellular signal-regulated protein kinase (ERK) 1/2, p38, and Src were activated by chronic pressure-overload and their activity was sustained for 8 weeks after aortic banding. In contrast, while p90 ribosomal S6 kinase (90RSK) and big MAP kinase 1 (BMK1) were activated in compensated hypertrophy, their activities were significantly decreased in hearts with heart failure. No changes were found in C-Jun NH2 terminal kinase (JNK) activity after aortic banding. These data suggest that differential activation of MAP kinase family members may contribute to the transition from compensated to decompensated hypertrophy. We also examined acute effects of mechanical stretch on the activation of these kinases in normal and hypertrophied hearts. In the isolated coronary-perfused heart, a balloon in the left ventricle was inflated to achieve minimum end-diastolic pressure of 25 mmHg for 10-20 min. In normal guinea pig hearts, stretch activated ERK1/2, p90RSK, p38, Src, and BMK1 but not JNK. However in hypertrophied hearts, further activation of these kinases was not observed by acute mechanical stretch. Mechanical stretch-induced activation of ERK1/2 and p38 kinase in normal hearts was attenuated significantly by a protein kinase C inhibitor, chelerythrine. We demonstrate that ERK1/2, p90RSK, p38, Src, and BMK1 are activated by chronic pressure-overload and by acute mechanical stretch. These data suggest that Src, BMK1 and p90RSK play a role as novel signal transduction pathways leading to cardiac hypertrophy. In addition, the differential inhibition of p90RSK and BMK1 in hearts with congestive heart failure suggests the specific role of these two kinases to maintain cardiac function under chronic pressure-overload.     

Inhibition of extracellular signal-regulated kinase enhances Ischemia/Reoxygenation-induced apoptosis in cultured cardiac myocytes and exaggerates reperfusion injury in isolated perfused heart

Three major mammalian mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), p38, and c-Jun NH(2)-terminal protein kinase (JNK), have been identified in the cardiomyocyte, but their respective roles in the heart are not well understood. The present study explored their functions and cross talk in ischemia/reoxygenation (I/R)-induced cardiac apoptosis. Exposing rat neonatal cardiomyocytes to ischemia resulted in a rapid and transient activation of ERK, p38, and JNK. On reoxygenation, further activation of all 3 mitogen-activated protein kinases was noted; peak activities increased (fold) by 5.5, 5.2, and 6.2, respectively. Visual inspection of myocytes exposed to I/R identified 18.6% of the cells as showing morphological features of apoptosis, which was further confirmed by DNA ladder and terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL). Myocytes treated with PD98059, a MAPK/ERK kinase (MEK1/MEK2) inhibitor, displayed a suppression of I/R-induced ERK activation, whereas p38 and JNK activities were increased by 70.3% and 55.0%, respectively. In addition, the number of apoptotic cells was increased to 33.4%. With pretreatment of cells with SB242719, a selective p38 inhibitor, or SB203580, a p38 and JNK2 inhibitor, I/R+PD98059-induced apoptotic cells were reduced by 42.8% and 63.3%, respectively. Hearts isolated from rats treated with PD98059 and subjected to global ischemia (30 minutes)/reoxygenation (1 hour) showed a diminished functional recovery compared with the vehicle group. Coadministration of SB203580 attenuated the detrimental effects of PD98059 and significantly improved cardiac functional recovery. The data taken together suggest that ERK plays a protective role, whereas p38 and JNK mediate apoptosis in cardiomyocytes subjected to I/R, and the dynamic balance of their activities is critical in determining cardiomyocyte fate subsequent to reperfusional injury.     

Dissociation of stress-activated protein kinase (p38-MAPK and JNKs) phosphorylation from the protective effect of preconditioning in vivo

The aim of the present study was to examine and compare the role of the stress-activated protein kinases in ischemic and stretch-induced preconditioning. A model of anesthetized rabbits was used, and the preconditioning protocol included one or three cycles of short ischemia/reperfusion, or short mechanical stretch with acute pressure overload without or with the addition of the stretch blocker gadolinium. Infarct size was determined after 2h reperfusion and p38 MAPK and JNKs phosphorylation was determined after 20 min of prolonged ischemia. Preconditioning stimuli were equally effective in reducing the infarct size (14.2+/-3.4%, 12.9+/-3.0%, 15.9+/-3.3%, P<0.01 vs control). The addition of the stretch channel blocker gadolinium abrogated the effect of stretch preconditioning only, without any effect on ischemic preconditioning. Comparing p38-MAPK and p46/p54 JNKs phosphorylation in the ischemic and non-ischemic regions of the heart at the time of sustained ischemia, activation was observed in the ischemic or mechanically preconditioned groups compared with the control. The addition of gadolinium abolished this activation. The above results indicate that the phosphorylation of p38-MAPK and p46/p54 JNKs is increased in preconditioning but this effect can be dissociated from the protective effect of ischemic preconditioning. Activation of the stress-activated protein kinases may be related to the increased contracture, a characteristic of ischemic preconditioning.     

Activation of mitogen-activated protein kinases in different models of pancreatic acinar cell damage

Mitogen-activated protein kinase (MAPK) family members, namely MAPK, c-Jun NH2-terminal protein kinase (JNK), and p38MAPK, have been recently reported to have opposing effects on apoptosis. AIM: To determine the activity of MAPKs and the level of Bax, Bcl-2 and p53--proteins known to be involved in the regulation of apoptosis--in pancreatic acini subjected to stressful stimuli leading to cell death. METHODS AND RESULTS: Isolated pancreatic acini were irradiated for 30 min with ultraviolet B (UV-B) or stimulated with supraphysiological concentrations of cholecystokinin (CCK). As it was assessed by means of acridine orange/ethidium bromide staining, irradiation with UV-B induced predominantly apoptosis while necrosis predominated in CCK-stimulated acini. The activity of MAPK, JNK and p38MAPK was determined by means of Western-blotting, with the use of antibodies which recognize active, dually phosphorylated enzymes. Irradiation with UV-B induced a rapid, 3-fold increase in MAPK activity. It had a maximum at 30 min and then gradually declined to reach the normal level at 120 min. Concomitantly, early activation of p38-MAPK was found at 30 min. However, unlike MAPK, p38-MAPK activity was then gradually rising to reach a maximum (5-fold increase) at 180 min. UV-B-induced activation of both kinases was not affected by the pretreatment with antioxidant--N-acetylo-L-cysteine or protein kinase C inhibitor--GF-109203X. In UV-B-irradiated cells, we did not detect any significant JNK activation as well as any significant changes in Bax, Bcl-2 and p53 levels assessed by means of Western-blotting. CONCLUSION: It seems likely that a specific interaction between MAPK and p38MAPK signaling pathway may be involved in the determination of the cell death mechanism in pancreatic acini subjected to stressful stimuli.     

Activation of mitogen activated protein kinases in post-infarcted patients

Activation of mitogen-activated protein kinases (MAPKs) signaling cascade are important pathophysiologic regulators during the development of acute myocardial infarction (AMI). In present study, we designed to monitor the activity of these MAPKs in Iranian patients with AMI comparing with controls. The degree of activation (phosphorylation) of p38 kinase, p44/42 extracellular regulated kinase, and c-Jun N-terminal kinase (JNK1/2) and their corresponding activity levels were analyzed in 258 patients with AMI and 250 normal subjects. The expression of p38α mRNA was determined. These analysis were carried out immediately and 12 h after AMI. Activity of p38 and JNK1/2 MAPKs were significantly increased in patients with AMI than controls immediately after infarction. These activities were reduced during 12 h after AMI. However, there were no statistically differences in activation and activity of p44/42 in the patients and controls. The mRNA expression of p38α was increased in the patients comparing with controls. Results of this study indicate that these MAPKs signaling pathway might be activated by AMI which signal transduction involves kinase phosphorylation and play important roles in their activity. Elevated activity of p38 and JNK1/2 MAPKs suggests that they may potentially play significant roles in AMI.     

Angiotensin II-induced cardiac hypertrophy is associated with different mitogen-activated protein kinase activation in normotensive and hypertensive mice

OBJECTIVE: In addition to its haemodynamic effects, angiotensin II (AngII) is thought to contribute to the development of cardiac hypertrophy via its growth factor properties. The activation of mitogen-activated protein kinases (MAPK) is crucial for stimulating cardiac growth. Therefore, the present study aimed to determine whether the trophic effects of AngII and the AngII-induced haemodynamic load were associated with specific cardiac MAPK pathways during the development of hypertrophy. Methods The activation of the extracellular-signal-regulated kinase (ERK), the c-jun N-terminal kinase (JNK) and the p38 kinase was followed in the heart of normotensive and hypertensive transgenic mice with AngII-mediated cardiac hypertrophy. Secondly, we used physiological models of AngII-dependent and AngII-independent renovascular hypertension to study the activation of cardiac MAPK pathways during the development of hypertrophy. RESULTS: In normotensive transgenic animals with AngII-induced cardiac hypertrophy, p38 activation is associated with the development of hypertrophy while ERK and JNK are modestly stimulated. In hypertensive transgenic mice, further activation of ERK and JNK is observed. Moreover, in the AngII-independent model of renovascular hypertension and cardiac hypertrophy, p38 is not activated while ERK and JNK are strongly stimulated. In contrast, in the AngII-dependent model, all three kinases are stimulated. CONCLUSIONS: These data suggest that p38 activation is preferentially associated with the direct effects of AngII on cardiac cells, whereas stimulation of ERK and JNK occurs in association with AngII-induced mechanical stress.