Ischemic preconditioning involves dual cardio-protective axes with p38MAPK as upstream target

The existing literature indicates a crucial role of p38 MAP (mitogen-activated protein) kinase (p38MAPK) and its downstream target MAPKAP kinase 2 (MK2) in ischemic preconditioning (IPC). Accordingly, deletion of MK2 gene should abolish the cardioprotective ability of IPC. Interestingly, we were able to partially precondition the hearts from MK2(-/-) knockout mice suggesting the existence of an as yet unknown alternative downstream target of p38MAPK. A recent study from our laboratory also determined a crucial role of CREB (cyclic AMP response element binding protein) in IPC. Since CREB is a downstream target of MSK-1 (mitogen- and stress-activated protein kinase-1) situated at the crossroad of ERK (extracellular receptor kinase) and p38MAPK signaling pathways, we reasoned that MSK-1 could be a downstream molecular target for p38MAPK and ERK signaling in the IPC hearts. To test this hypothesis, the rat hearts were subjected to IPC by four cyclic episodes of 5 min ischemia and 10 min reperfusion. As expected, IPC induced the activation of ERK1/2, p38MAPK, MK2 and HSP (heat shock protein) 27 as evidenced by their increased phosphorylation; and the inhibition of p38MAPK with SB203580 almost completely, and the inhibition of ERK1/2 with PD098059 partially, abolished cardioprotective effects of IPC. Inhibition of MSK-1 with short hairpin RNA (shRNA) also abolished the IPC-induced cardioprotection. SB203580 partially blocked the effects of MSK-1 suggesting that MSK-1 sits downstream of p38MAPK. shRNA-MSK-1 blocked the contribution of both p38MAPK and ERK1/2 as it is uniquely situated at the downstream crossroad of both of these MAP kinases. Although MSK-1 sits downstream of both ERK1/2 and p38MAPK, ERK1/2 activation appears to play less significant role compared to p38MAPK, since its inhibition blocked MSK activation only partially. Consistent with these results, shRNA-MSK-1 blocked the partial PC in MK2(-/-) hearts, and in combination with SB203580, completely abolished the PC effects in the wild-type hearts. The IPC-induced survival signaling was almost completely inhibited with SB203580, and only partially with PD 098059 as evidenced from the inhibition patterns of IPC induced activation of CREB, Akt and Bcl-2. Again SB203580 alone or in combination with shRNA-MSK-1 inhibited IPC induced survival signal comparatively, suggesting that MSK-1 exists downstream of p38MAPK. Taken together, these results indicate for the first time MSK-1 as an alternative (other than MK2) downstream target for p38MAPK, which also transmits survival signal through the activation of CREB.     

Remote vs. ischaemic preconditioning: the differential role of mitogen-activated protein kinase pathways

AIMS: Since mitogen-activated protein kinases (MAPKs) were found to be implicated in the signalling of ischaemic preconditioning (IPC), we tested the hypothesis of a contribution of these protein kinases to remote preconditioning (RPC). METHODS AND RESULTS: To determine the role of p38, ERK1/2, and JNK1/2 MAPKs in mediating cardiac protection, an in vivo model of myocardial infarction was applied in male Wistar rats. RPC or IPC was induced by occlusion of the superior mesenteric artery or the left coronary artery, respectively. Infarct size (IS) was determined based on 2,3,5-triphenyltetrazolium chloride staining. Phosphorylation of the various MAPKs was analysed by immunoblotting in samples of the small intestine and myocardium obtained after IPC or RPC procedures. The MAPK inhibitors SB203580 (p38), PD98059 (ERK1/2), and SP600125 (JNK1/2) were administered to assess the potential significance of MAPK signalling in RPC. Both preconditioning stimuli decreased myocardial IS significantly after a lethal period of ischaemia. Each of the applied MAPK inhibitors was capable of abrogating the RPC-induced cardioprotection. Western blot analysis of myocardial samples revealed an increase in phosphorylated amounts of ERK1/2 and JNK1 after IPC, whereas phosphorylation of p38 protein was decreased significantly. Likewise, RPC resulted in a considerable increase in phosphorylation of ERK1/2 and JNK1/2 proteins in the small intestine, whereas it did not alter the MAPK phosphorylation state in the myocardium. CONCLUSION: All investigated MAPK pathways appear to be involved in RPC-induced cardioprotection; however, they do not contribute to the alterations that define the preconditioned state of the myocardium prior to the infarction.     

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.     

SB 203580, an inhibitor of p38 MAPK, abolishes infarct-limiting effect of ischemic preconditioning in isolated rabbit hearts

There is debate concerning the involvement of p38 mitogen-activated protein kinase (MAPK) in ischemic preconditioning (PC). At the center of the controversy are data obtained after administration of SB 203580, a specific inhibitor of p38 MAPK. Whereas several studies have reported that SB 203580 abolishes the cardioprotective effect of PC, others claim that this compound is actually cardioprotective against ischemia. Many of these latter observations have been made in isolated myocardial cells. Accordingly the present study was designed to test the effect of SB 203580 in a model of preconditioning in intact rabbit hearts in which infarct size was the end-point. Isolated hearts experienced 30 min of regional ischemia followed by 120 min of reperfusion. Infarct size was measured with triphenyltetrazolium chloride. In control hearts infarction was 30.2 ± 3.3% of the risk zone. PC with 5 min of global ischemia and 10 min of reperfusion before the 30-min period of ischemia significantly reduced infarct size to 10.2 ± 2.4% (P < 0.05 vs. control). SB 203580 (2 μ M) added to the perfusate for 20 min starting 5 min before the index ischemia totally blocked the protection from PC (27.4 ± 3.3% infarction). SB 203580 alone had no effect on infarct size (28.6 ± 4.6% infarction). These results reveal that SB 203580 does not affect infarct size on its own, but selectively blocks preconditioning's anti-infarct effect in the intact rabbit heart. Received: 21 August 2000, Returned for revision: 30 August 2000, Revision received: 2000, Accepted: 6 September 2000     

Differential translocation or phosphorylation of alpha B crystallin cannot be detected in ischemically preconditioned rabbit cardiomyocytes

Alpha B Crystallin (alpha BC) is a putative effector protein of ischemic preconditioning (IPC), that is phosphorylated on Ser 45 by ERK1/2 and Ser 59 by the p38 MAPK substrate, MAPKAPK-2. Translocation and phosphorylation of alpha BC was determined in cytosolic and cytoskeletal fractions by 1D SDS-PAGE and IEF, or using Ser 45 and Ser 59 phospho-specific antibodies in: (1) control rabbit cardiomyocytes; (2) cells preconditioned by 10 min in vitro ischemia; or after pre-treatment with specific inhibitors of (3) Ser/Thr protein phosphatase 1/2A (calyculin A); (4) p38 MAPK (SB203580); or (5) ERK 1/2 (PD98059); all prior to 180 min ischemia. Ischemia induced a cytosolic to cytoskeletal translocation of alpha BC, which was similar in all the groups. Highly phosphorylated isoforms (D1/2) of alpha BC were present in cytosolic but not cytoskeletal fractions at 0 min ischemia. By 60-90 min ischemia, D1/2 isoforms had translocated to the cytoskeletal fraction. Calyculin A maintained D1/2 levels throughout prolonged ischemia. SB203580 decreased alpha BC phosphorylation. Neither PD98059 nor IPC altered alpha BC phosphorylation during prolonged ischemia. It is concluded that alpha BC phosphorylation during ischemia is regulated by p38 MAPK but not by ERK 1/2. The inability to detect a correlation between IPC protection and either alpha BC translocation or phosphorylation suggests that the proteins in the highly phosphorylated isoform bands of alpha BC quantitated in this study are not protective end effectors of classical IPC.     

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.     

The role of p38 mitogen-activated protein kinase in myocardial ischemia/reperfusion injury; relationship to ischemic preconditioning

Myocardial mitogen-activated protein kinases can be activated by ischemia and reperfusion, and they may play important roles in the evolution of ischemic injury. Considerable work has been performed to evaluate the role of different MAPK signaling pathways in ischemia/reperfusion injury. The focus of this review is the p38 MAPK pathway, specifically whether activation of the p38 MAPK signaling pathway is beneficial or detrimental. Different studies have come to conflicting conclusions. This review will examine the literature on the role of p38 MAPK in myocardial ischemia/reperfusion injury, highlight areas of controversy and areas of general agreement, examine possible downstream targets of p38 during acute ischemia, and attempt to draw some conclusions. Received: 25 February 2002/Returned for revision: 14 March 2002/Revision received: 4 April 2002/Accepted: 8 April 2002     

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     

5-HD abolishes ischemic preconditioning independently of monophasic action potential duration in the heart

Objective: Blocking of the K_ATP channel with either glibenclamide or 5-hydroxydecanoate (5-HD) has been shown to abolish the infarct reducing effect of ischemic preconditioning (IPC) in hearts from several species, but the results in rat and rabbit have been equivocal. In this study we investigated if 5-HD could abolish IPC in rat and rabbit and further if IPC or IPV + 5-HD were affecting action potential duration in the rabbit heart. Methods: The rat hearts were isolated and retrogradely perfused on a Langendorff perfusion apparatus with Krebs-Henseleit buffer. The rabbit experiments were performed in an in situ model. Rat and rabbit hearts were subjected to 30 min regional ischemia by ligating a coronary artery followed by 120 min (rat) or 150 min (rabbit) of reperfusion. The preconditioning protocol was one or three cycles of 5 min ischemia plus 5 min reperfusion in the rat and one cycle of 5 min ischemia plus 10 min reperfusion in the rabbit. In the rat 5-HD was added to the reservoir before ischemic preconditioning in different concentrations, and in the rabbit 5-HD was given as a bolus 5 mg/kg intraventricularly 2 min before the preconditioning ischemia. In the rabbit epicardial monophasic action potential duration at 50% repolarization (MAPD₅₀) was measured at 1, 2 and 5 min in each of the ischemic periods using a contact pressure electrode. Infarcts were measured with tetrazolium staining and risk zone volumes with fluorescent microspheres. Results: All data are presented as infarct size in % of risk zone volume (mean ± SEM). In the rat 200 μM of 5-HD abolished the protective effect of one cycle of IPC (28.6 ± 4.7 versus 8.4 ± 0.8) and 500M of 5-HD abolished three cycles of IPC (50.7 ± 7.8 versus 8.4 ± 2.0). Control was 40.9 ± 2.8. In the rabbit 5-HD abolished IPC (41.2 ± 7.2 versus 8.1 ± 3.2). Control was 53.5 ± 12.4. MAPD₅₀ were significantly more shortened compared to control at 1 and 2 min into the 30 min ischemia for the IPC and IPC+5-HD. Conclusions: We conclude that 5-HD abolishes ischemic preconditioning when given before the preconditioning ischemia in both rat and rabbit but does not abolish into ischemia induced shortening of the action potential duration in the rabbit; thus, a role for the mitochondrial K_ATP channel and not the sarcolemmal K_ATP channel in the protective mechanism behind IPC is probable. Received: 15 July 1999, Returned for 1. revision: 17 August 1999, 1. Revision received: 13 September 1999, Returned for 2. revision: 12 October 1999, 2. Revision received: 3 November 1999, Accepted: 17 November 1999     

Phosphorylation state of hsp27 and p38 MAPK during preconditioning and protein phosphatase inhibitor protection of rabbit cardiomyocytes

Small heat shock proteins (hsp) have been implicated in mediation of classic preconditioning in the rabbit, Hsp27 is a terminal substrate of the p38 MAPK cascade. One and 2D gel electrophoresis and immunoblotting of cell fractions was used to determine p38 MAPK and hsp27 phosphorylation levels, respectively, during in vitro ischemia in control, calyculin A (Cal A)-treated (protein phosphatase inhibitor), SB203580-treated (p38MAPK inhibitor) and preconditioned (IPC) isolated adult rabbit cardiomyocytes. The dual phosphorylation of p38 MAPK was increased by early ischemia (30-60 min), after which there was a loss of total cytosolic p38 MAPK. The ischemic increase of p38 MAPK dual phosphorylation was enhanced by IPC. Cal A strongly activated dual phosphorylation of p38 MAPK in oxygenated cells and this was maintained into early ischemia, SB203580 inhibited the dual phosphorylation of p38 MAPK and attenuated the loss of total cytosolic p38 MAPK. In each protocol, ischemia translocated hsp27 from the cytosolic fraction to the cytoskeletal fraction at similar rates and extents, Hsp27 phosphorylation was quantitated as the fraction of diphosphorylated hsp27, based on IEF mobility shifts of hsp27 phosphorylation isoforms. In oxygenated control cells, cytosolic and cytoskeletal hsp27 was highly phosphorylated. After 90 min ischemia, cytoskeletal hsp27 was markedly dephosphorylated. Cal A slightly increased control cytoskeletal hsp27 phosphorylation. During ischemic incubation, Cal A blocked ischemic dephosphorylation, SB203580 accelerated ischemic hsp27 dephosphorylation and injury, IPC insignificantly decreased the initial rate of ischemic dephosphorylation of hsp27, but not the extent of dephosphorylation in later ischemia. Phosphorylation is regulated by both kinase and phosphatase activities. IPC protection was not correlated with a significant increase in cytosolic or cytoskeletal hsp27 phosphorylation levels during prolonged (> 60-90 min) ischemia.     

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.     

Mitochondrial dysfunction as the cause of the failure to precondition the diabetic human myocardium

OBJECTIVES: We have shown previously that human diabetic myocardium cannot be preconditioned. Here, we have investigated the basis of this cardioprotective deficit. METHODS: Right atrial sections from four patient groups-non-diabetic, insulin-dependent diabetes mellitus (IDDM), non-insulin-dependent diabetes mellitus (NIDDM) receiving glibenclamide, and NIDDM receiving metformin-were subjected to one of the following protocols: aerobic control, simulated ischemia/reoxygenation, ischemic preconditioning before ischemia, and pharmacological preconditioning with alpha 1 agonist phenylephrine, adenosine, the mito-K(ATP) channel opener diazoxide, the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA), or the p38 mitogen-activated protein kinase (p38MAPK) activator anisomycin. Cellular damage was assessed using creatine kinase leakage and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction. In mitochondrial preparations from non-diabetic and diabetic myocardium, mitochondrial membrane potential (Psi(m)) was assessed using JC-1 dye, and production of reactive oxygen species was determined. RESULTS: Preconditioning with ischemia, phenylephrine, adenosine, or diazoxide failed to protect diabetic myocardium. However, activation of PKC or p38MAPK was still protective. In isolated non-diabetic mitochondria, diazoxide partially depolarized Psi(m), an effect not seen in diabetic mitochondria. Furthermore, diazoxide increased superoxide production in non-diabetic but not in diabetic mitochondria. CONCLUSIONS: Our results show that the cardioprotective deficit in diabetic myocardium arises upstream of PKC and p38MAPK. We suggest that mitochondrial dysfunction in diabetic myocardium, possibly dysfunctional mito-K(ATP) channels, leads to impaired depolarization and superoxide production, and that this causes the inability to respond to preconditioning.     

心肌缺血预适应延迟相的研究进展

心肌缺血预适应(ischemicpreconditioning,IPC)现象的发现,为心肌缺血再灌注损伤的预防开拓了一个新的研究领域。IPC分为快速相和延迟相,快速相IPC对心脏具有显著的保护作用,但这种保护作用在IPC后1~2h即消失。延迟相IPC在IPC后24h出现,心脏保护持续时间长,保护范围广。现对IPC延迟相的机制进展作一简要综述。     

Role of phasic dynamism of p38 mitogen-activated protein kinase activation in ischemic preconditioning of the canine heart

Although ischemic stress, including ischemic preconditioning (IP), activates p38 mitogen-activated protein kinase (MAPK), the relationship between p38 MAPK activation and the underlying cellular mechanisms of cardioprotection by IP is not verified in vivo. We examined the effects of the selective p38 MAPK inhibition on the cardioprotective effect of IP in the open-chest dogs. The coronary artery was occluded 4 times for 5 minutes, separated by 5 minutes of reperfusion (IP) followed by 90 minutes of occlusion and 6 hours of reperfusion. We infused SB203580 into the coronary artery during IP and 1 hour of reperfusion, during IP alone, and during sustained ischemia in the IP group. p38 MAPK activity markedly increased during IP but did not additionally increase at the onset of ischemia and was even attenuated at 15 minutes of sustained ischemia, and heat-shock protein (HSP) 27 was phosphorylated and translocated from cytosol to myofibril or nucleus without affecting total protein level at the onset of ischemia compared with the control group. SB203580 treatment (1 micromol/L) only during IP blunted the infarct size limitation by IP (37.3+/-6.3% versus 7.4+/-2.1% in the IP group, P:<0.01) and attenuated either phosphorylation or translocation of HSP27 during IP. Although the SB203580 treatment throughout the preischemic and postischemic periods had no significant effect on infarct size (33.3+/-9.4%) in this model, treatment with SB203580 only during ischemia partially mimicked the infarct size limitation by IP (26.8+/-3.5%). Thus, transient p38 MAPK activation during ischemic preconditioning mainly mediates the cardioprotection followed by HSP27 phosphorylation and translocation in vivo in the canine heart.     

Brief preconditioning ischemia alters diacylglycerol content and composition in rabbit heart

In order to give further insight into the potential role of PKC in beneficial effects of ischemic preconditioning, we have characterized the production of diacylglycerol, the endogenous activator of PKC, and its molecular species composition in ischemic control and preconditioned hearts. Preconditioning was induced by 1 cycle of 5 min of ischemia followed by 5 min of reperfusion. In control and preconditioned groups, hearts were harvested under deep anesthesia at baseline (preischemia) and at 2,5 and 10 min into the sustained coronary artery occlusion, i.e., preceding myocyte death. Diacylglycerol content and fatty acid composition were analysed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), respectively. Myocardial diacylglycerol content was increased at 2 min into the sustained ischemia in the control group (481 ± 34 vs 292 ± 64 ng.mg⁻¹ at baseline; p < 0.05), but was comparable to the baseline value at 5 and 10 min. In the preconditioned group, diacylglycerol production remained unchanged throughout the 10-min test ischemia (317 ± 17 at 2 min vs 312 ± 38 ng.mg⁻¹ at baseline; p = NS). A detailed analysis of the molecular species composition at the time of 2 min revealed a reduced contribution of phosphatidy-linositol to diacylglycerol production in preconditioned myocardium (global correlation coefficient 0.57 vs 0.66 in control myocardium) with a trend toward an enrichment of diacylglycerol composition with some species originating from phosphatidylcholine. Thus, our study revealed that brief preconditioning ischemia: (1) prevents the increase of diacylglycerol content in the early minutes of the sustained ischemia, and (2) emphasizes the contribution of phosphatidylcholine in diacylglycerol formation to the detriment of that of phosphatidylinositol. Received: 12 October 1999, Returned for 1.revision: 3 November 1999, 1.Revision received: 6 January 2000, Returned for 2.revision: 14 February 2000, 2.Revision received: 21 April 2000, Accepted: 9 May 2000     

Cardiac overexpression of monocyte chemoattractant protein-1 in transgenic mice mimics ischemic preconditioning through SAPK/JNK1/2 activation

OBJECTIVE AND METHODS: Although a beneficial association between innate immunity and ischemic preconditioning has recently been proposed, the mechanisms responsible for this link are poorly understood. To test the hypothesis that pro-inflammatory cytokines have a beneficial role in the activation of the cell survival pathway mediated by ischemic preconditioning, we have studied transgenic mice with cardiac myocyte specific overexpression of murine monocyte chemoattractant protein-1 (MCP-1). The resistance to ischemia was studied by performing 45-min (with or without injection of the SAPK/JNKs inhibitor D-JNKI1) and 3-day left coronary artery occlusions as well as 45-min left coronary artery occlusion followed by 3 days of reperfusion. In addition, quantitative Western blot analyses for TNF-alpha, and SAPK/JNK1/2, ERK1/2 and p38 activity were performed. RESULTS: Infarct size, expressed in percent of either the risk area or the left ventricle, was reduced in transgenic mice when compared with control after both, 45-min (14.7+/-2.6% vs. 52.0+/-2.4%; P<0.05) and 45-min occlusion followed by 3 days of reperfusion (23.2+/-1.8% vs. 30.0+/-1.8%; P<0.05) but it was not significantly different for 3-day occlusion. Western blot analyses showed significantly increased levels of TNF-alpha (1.8-fold) and phosphorylated-SAPK/JNK1/2 (1.5-fold) in transgenic hearts. Phosphorylated-ERK1/2, and phosphorylated-p38 levels were unchanged. Immunohistochemistry revealed that in transgenic mice monocytes/macrophages, lymphocytes, and fibroblasts are the source of TNF-alpha, whereas myocytes have increased phosphorylated-SAPK/JNK1/2 levels. In addition, injection of the SAPK/JNKs inhibitor D-JNKI1 partially abrogated the cardioprotective effect observed in untreated transgenic mice. CONCLUSION: Overexpression of MCP-1 by cardiomyocytes causes chronic infiltration and activation of leukocytes, resulting in elevated TNF-alpha secretion and SAPK/JNK1/2 activation. The activation of this pathway is in part responsible for the preconditioning effect of MCP-1 overexpression. These results show a possible beneficial link between innate immunity and ischemic preconditioning through MAP-kinase activation.     

Reciprocal modulation of mitogen-activated protein kinases and mitogen-activated protein kinase phosphatase 1 and 2 in failing human myocardium

BACKGROUND: Mitogen-activated protein kinases (MAPKs), consisting of the ERK1/2, JNKs, and p38-kinase families, play a key role in the regulation of myocyte growth and apoptosis in vitro. The activity of MAPKs is regulated by dual-specificity MAPK phosphatases (MKPs). Because myocardial failure is associated with myocyte hypertrophy and apoptosis, MAPKs may play a pathophysiologic role in human myocardial failure. METHODS AND RESULTS: We measured MAPKs activities and the protein levels of MAPKs and MKPs (MKP-1 and MKP-2) in the myocardium explanted at the time of transplantation from patients with end-stage failure caused by idiopathic dilated cardiomyopathy (n = 5-7). Nonfailing donor hearts (n = 5-7) were used for comparison. Although the protein levels for JNK1/2 and p38-kinase in failing hearts were not different from levels in nonfailing hearts, the activities of both were decreased (P <.05). Despite a >3-fold increase in the protein level for ERK1/2 in failing hearts, ERK1/2 activity was not increased. Expression of MKP-2 was significantly increased in failing hearts, while expression of MKP-1 was increased in 5 of 7 failing hearts as measured by Western analysis. CONCLUSIONS: JNK1/2 and p38 activities are decreased in failing human myocardium. Increased expression of MKPs may therefore contribute to decreased MAPKs activity in failing human myocardium.     

Autophagy Induced by Ischemic Preconditioning is Essential for Cardioprotection

Based on growing evidence linking autophagy to preconditioning, we tested the hypothesis that autophagy is necessary for cardioprotection conferred by ischemic preconditioning (IPC). We induced IPC with three cycles of 5 min regional ischemia alternating with 5 min reperfusion and assessed the induction of autophagy in mCherry-LC3 transgenic mice by imaging of fluorescent autophagosomes in cryosections. We found a rapid and significant increase in the number of autophagosomes in the risk zone of the preconditioned hearts. In Langendorff-perfused hearts subjected to an IPC protocol of 3 × 5 min ischemia, we also observed an increase in autophagy within 10 min, as assessed by Western blotting for p62 and cadaverine dye binding. To establish the role of autophagy in IPC cardioprotection, we inhibited autophagy with Tat-ATG5^K130R, a dominant negative mutation of the autophagy protein Atg5. Cardioprotection by IPC was reduced in rat hearts perfused with recombinant Tat-ATG5^K130R. To extend the potential significance of autophagy in cardioprotection, we also assessed three structurally unrelated cardioprotective agents—UTP, diazoxide, and ranolazine—for their ability to induce autophagy in HL-1 cells. We found that all three agents induced autophagy; inhibition of autophagy abolished their protective effect. Taken together, these findings establish autophagy as an end-effector in ischemic and pharmacologic preconditioning.     

Delayed attenuation of myocardial ischemia with repeated exercise in subjects with stable angina: a possible model for the second window of protection?

Aims: A delayed myocardial protection extends between 24 and 96 h after ischemic preconditioning in animals. To test this phenomenon in humans, subjects with stable angina were subjected to exercise test-induced myocardial ischemia and the effect of this “preconditioning” ischemic insult on the exercise-induced myocardial ischemia with the re-exercise after 24–96 hours was studied. Methods and results: Forty-eight males with a history of infarction and positive exercise test were recruited to the study. After baseline symptom-limited exercise test, the subjects were randomized to four experimental groups (n=12/group). The groups were allowed to recover for 24 h, 48 h, 72 h or 96 h before performing the second exercise test. Variables analyzed were heart rate-systolic blood pressure product at 1 mm ST segment depression, time to 1 mm ST segment depression, maximum ST segment depression, exercise duration, and the total ischemic time. There were no intergroup differences in baseline values for these variables. All variables were significantly improved at 24 h, the improvement peaked usually at 48 h (maximum increase in the variables by 31–46%), and the variables returned to baseline by 96 h after the first test. Conclusions: The exercise-induced ischemia caused transient attenuation of myocardial ischemia with re-exercise. Although the time-window and the time-course of this effect shows striking resemblance to those of the delayed preconditioning in animals, its mechanism remains speculative. The most probable mechanisms that may be involved include increased myocardial perfusion and/or some adaptive changes in the myocardium, the delayed preconditioning being one possibility. Received: 2 February 2000 Returned for 1. revision: 23 February 2000 1. Revision received: 3 April 2000 Returned for 2. revision: 3 May 2000 2. Revision received: 20 May 2000 Accepted: 26 May 2000