Nilanjana Maulik

Ischemic preconditioning triggers the activation of MAP kinases and MAPKAP kinase 2 in rat hearts

While much is known about the beneficial effects of myocardial stress adaptation, relatively less information is available about the adaptive mechanisms. To explore the signaling pathways of stress adaptation, isolated working rat hearts were divided into three groups. Group I was adapted to stress by conventional technique of repeated ischemia and reperfusion consisting of 5 min of ischemia followed by 10 min of reperfusion, repeated four times. Group II was treated with 100 μM of genistein, a tyrosine kinase inhibitor, followed by preconditioning as described for group I. The third group, perfused with buffer only for 60 min, served as control. All hearts were subjected to 30 min of ischemia followed by 30 min of reperfusion. The results of our study demonstrated better postischemic myocardial functions in the preconditioned hearts as evidenced by increased aortic flow, coronary flow, developed pressure and lesser amount of tissue injury as evidenced by the decreased creatine kinase release. The preconditioning effects were associated with enhancement of phospholipase D activity in the heart. The preconditioning effect was almost abolished by the genistein treatment which also prevented the enhancement of phospholipase D activities. Additionally, preconditioning of the rat hearts stimulated protein kinase C, MAP kinase, and MAPKAP kinase 2 activities which were inhibited by genistein. The results identifies for the first time tyrosine kinase‐phospholipase D as potential signaling pathway for ischemic preconditioning, and implicates the involvement of multiple protein kinases in myocardial adaptation to ischemia.

An essential role of NFκB in tyrosine kinase signaling of p38 MAP kinase regulation of myocardial adaptation to ischemia

We have recently demonstrated that myocardial adaptation to ischemia triggers a tyrosine kinase regulated signaling pathway leading to the translocation and activation of p38 MAP kinase and MAPKAP kinase 2. Since oxidative stress is developed during ischemic adaptation and since free radicals have recently been shown to function as an intracellular signaling agent leading to the activation of nuclear transcription factor, NFκB, we examined whether NFκB was involved in the ischemic adaptation process. Isolated perfused rat hearts were adapted to ischemic stress by repeated ischemia and reperfusion. Hearts were pretreated with genistein to block tyrosine kinase while SB 203580 was used to inhibit p38 MAP kinases. Ischemic adaptation was associated with the nuclear translocation and activation of NFκB which was significantly blocked by both genistein and SB 203580. The ischemically adapted hearts were more resistant to ischemic reperfusion injury as evidenced by better function recovery and less tissue injury during post‐ischemic reperfusion. Ischemic adaptation developed oxidative stress which was reflected by increased malonaldehyde formation. A synthetic peptide containing a cell membrane‐permeable motif and nuclear sequence, SN 50, which blocked nuclear translocation of NFκB during ischemic adaptation, significantly inhibited the beneficial effects of adaptation on functional recovery and tissue injury. In concert, SN 50 reduced the oxidative stress developed in the adapted myocardium. These results demonstrate that p38 MAP kinase might be upstream of NFκB which plays a role in ischemic preconditioning of heart.

Redox signaling in vascular angiogenesis

Angiogenesis is thought to be regulated by several growth factors (EGF, TGF-alpha, beta-FGF, VEGF). Induction of these angiogenic factors is triggered by various stresses. For instance, tissue hypoxia exerts its pro-angiogenic action through various angiogenic factors, the most notable being vascular endothelial growth factor, which has been mainly associated with initiating the process of angiogenesis through the recruitment and proliferation of endothelial cells. Recently, reactive oxygen species (ROS) have been found to stimulate angiogenic response in the ischemic reperfused hearts. Short exposure to hypoxia/reoxygenation, either directly or indirectly, produces ROS that induce oxidative stress which is associated with angiogenesis or neovascularization. ROS can cause tissue injury in one hand and promote tissue repair in another hand by promoting angiogenesis. It thus appears that after causing injury to the cells, ROS promptly initiate the tissue repair process by triggering angiogenic response.

Oxidative stress developed during the reperfusion of ischemic myocardium induces apoptosis.

Apoptosis or programmed cell death is a genetically controlled response for cells to commit suicide and, is associated with DNA fragmentation or laddering. The common inducers of apoptosis include oxygen free radicals/oxidative stress and Ca2+ which are also implicated in the pathogenesis of myocardial ischemic reperfusion injury. To examine whether ischemic reperfusion injury is mediated by apoptotic cell death, isolated perfused rat hearts were subjected to 15, 30 or 60 min of ischemia as well as 15 min of ischemia followed by 30, 60 or 120 min of reperfusion. At the end of each experiment, hearts were processed for the evaluation of apoptosis, DNA laddering. Apoptosis was studied by visualizing the apoptotic cardiomyocytes by direct fluorescence detection of digoxigenin-labeled genomic DNA using APOPTAG in situ apoptosis detection kit. DNA laddering was evaluated by subjecting the DNA obtained from the hearts to 1.8% agarose gel electrophoresis and photographed under UV illumination. The results of our study revealed apoptotic cells only in the 60 and 120 min reperfused hearts as demonstrated by the intense fluorescence of the immunostained digoxigenin-labeled genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results corroborated with the findings of DNA fragmentation which showed increased ladders of DNA bands in the same reperfused hearts representing integer multiples of the intenucleosomal DNA length (about 180 bp). The presence of apoptotic cells and DNA fragmentation in the myocardium were abolished by preperfusing the hearts in the presence of ebselen, which also removed the oxidative stress developed in the heart. Taken together, these results clearly demonstrate that oxidative stress developed in the ischemic reperfused myocardium induces apoptosis.

Gene expression on acute myocardial stress. Induction by hypoxia, ischemia, reperfusion, hyperthermia and oxidative stress

It is apparent from the above discussion that acute stress, such as ischemia and reperfusion, hypoxia and reoxygenation, hyperthermia and oxidative stress, can rapidly potentiate the induction of genes for certain members of the HSP families and for antioxidants/antioxidant enzymes. Whether the stress response and induction of these genes have a direct role in myocardial protection is not known, but the induction of the expression of these genes are mostly associated with the preservation of myocardial cells from subsequent injury resulting from ischemia, hypoxia and reperfusion. The ubiquitous presence of some of these stress genes, such as for HSP 70 and catalase, in normal unstressed myocardium further suggests a role of these genes in many basic and essential biochemical and metabolic pathways. It is reasonable to speculate that the cells respond to the stress as a consequence of perturbations of one or more of the metabolic pathways by stimulating the induction of the stress genes of that particular pathway in which they participate. Thus, these genes are likely to be involved both in the protection and recovery/repair mechanisms. The precise mechanism by which myocardial cell recognizes and responds to a particular stress agent such as ischemia, hypoxia, hyperthermia or oxidative stress is not clear. While it is tempting to speculate that a generalized mechanism exists, applying to all different modes of stress response and gene induction, whether these agents induce the response via independent pathways or converge within a single point is entirely unclear. However, from the striking resemblance between the pattern of gene expression, especially with regard to HSP and antioxidant genes, it is reasonable to hypothesize the existence of a common and essential pathway of molecular signaling that leads to the expression of these stress genes (Fig. 2). The identification and characterization of the transcription factors that regulate the expression of the genes induced by these forms of stress should greatly facilitate our future understanding of the mechanism of stress response.

Reactive oxygen species function as second messenger during ischemic preconditioning of heart.

Ischemic preconditionining has been shown to trigger a signaling pathway by potentiating tyrosine kinase phosphorylation leading to the activation of p38 MAP kinase and MAPKAP kinase 2. Recently, the nuclear transcription factor, NFκB, was found to play a role in the signaling process. Since NFκB is a target of oxygen free radicals, we hypothesized that reactive oxygen species might play a role in the signaling process. To test this hypothesis, isolated rat hearts were perfused in the absence or presence of either dimethyl thiourea (DMTU), a OH· radical scavenger, or SN 50 peptide, a NFκB blocker. Hearts were then subjected to ischemic preconditioning by four repeated episodes of 5 min ischemia each followed by 10 min reperfusion. All hearts were then made globally ischemic for 30 min followed by 2 h of reperfusion. The results of our study demonstrated enhanced tyrosine kinase phosphorylation during ischemic preconditioning which was blocked by DMTU. DMTU also inhibited preconditioning mediated increased phosphorylation of p38 MAP kinase and MAPKAP kinase 2 activity. However, DMTU had no effect on the translocation and activation of protein kinase C (PKC) resulting from preconditioning. Preconditioning reduced myocardial infarct size as expected. This cardioprotective effect of preconditioning was abolished by both DMTU and SN 50. Preconditioning resulted in the nuclear translocation and activation of NFκB. Increased NFκB binding was blocked by both DMTU and SN 50. The results of this study demonstrate that reactive oxygen species play a crucial role in signal transduction mediated by preconditioning. This signaling process appears to be potentiated by tyrosine kinase phosphorylation resulting in the activation of p38 MAP kinase and MAPKAP kinase 2 leading to the activation of NFκB suggesting a role of oxygen free radicals as second messenger. Free radical signaling seems to be independent of PKC although PKC is activated during preconditioning process suggesting the role of two separate signaling pathways in ischemic preconditioning.

Ischemic preconditioning triggers tyrosine kinase signaling: a potential role for MAPKAP kinase 2

Myocardial adaptation to ischemia has been shown to activate protein tyrosine kinase, potentiating activation of phospholipase D, which leads to the stimulation of mitogen-activated protein (MAP) kinases and MAP kinase-activated protein (MAPKAP) kinase 2. The present study sought to further examine the signal transduction pathway for the MAPKAP kinase 2 activation during ischemic adaptation. Isolated perfused rat hearts were adapted to ischemic stress by repeated ischemia and reperfusion. Hearts were pretreated with genistein to block tyrosine kinase, whereas SB-203580 was used to inhibit p38 MAP kinases. Western blot analysis demonstrated that p38 MAP kinase is phosphorylated during ischemic stress adaptation. Phosphorylation of p38 MAP kinase was blocked by genistein, suggesting that activation of p38 MAP kinase during ischemic adaptation is mediated by a tyrosine kinase signaling pathway. MAPKAP kinase 2 was estimated by following in vitro phosphorylation with recombinant human heat shock protein 27 as specific substrate for MAPKAP kinase 2. Again, both genistein and SB-203580 blocked the activation of MAPKAP kinase 2 during myocardial adaptation to ischemia. Immunofluorescence microscopy with anti-p38-antibody revealed that p38 MAP kinase is primarily localized in perinuclear regions. p38 MAP kinase moves to the nucleus after ischemic stress adaptation. After ischemia and reperfusion, cytoplasmic striations in the myocytes become obvious, indicating translocation of p38 MAP kinase from nucleus to cytoplasm. Corroborating these results, myocardial adaptation to ischemia improved the left ventricular functions and reduced myocardial infarction that were reversed by blocking either tyrosine kinase or p38 MAP kinase. These results demonstrate that myocardial adaptation to ischemia triggers a tyrosine kinase-regulated signaling pathway, leading to the translocation and activation of p38 MAP kinase and implicating a role for MAPKAP kinase 2.

JAK/STAT Signaling Is Associated With Cardiac Dysfunction During Ischemia and Reperfusion

Background—Activation of the heart renin-angiotensin system (RAS) under pathophysiological conditions has been correlated with the development of ischemic injury. The binding of angiotensin II to its receptors triggers induction of several, perhaps multifunctional, intracellular signaling pathways, notable among them the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. In this study, we investigated whether the JAK/STAT signaling is involved in the ischemia/reperfusion injury in adult rat myocardium. Methods and Results—We report here that 2 components of the JAK/STAT signaling pathway, namely STAT 5A and STAT 6, are selectively activated in the rat heart subjected to ischemia/reperfusion. The activated STATs bind to a conserved nucleotide sequence (St domain) in the promoter of the angiotensinogen (ANG) gene and consequently upregulate the level of ANG mRNA. Treatment of the hearts with losartan (4.5 &mgr;mol/L), an AT1 blocker, or with tyrphostin AG490 (5 &mgr;mol/L), an inhibitor of JAK 2 phosphorylation, results in loss of the STAT/ANG promoter binding activity and an upregulated level of ANG mRNA. Hearts treated with the JAK 2 inhibitor tyrphostin AG490 showed a reduction in myocardial infarct size and in number of cardiomyocytes undergoing apoptosis. The treated hearts also showed a recovery in functional hemodynamics of the myocardium. Conclusions—These findings suggest that activation of the JAK/STAT signaling pathway is a significant contributing factor to the pathogenesis of myocardial ischemia and that interference in activation of the pathway potentiates recovery in cardiac function.

Antioxidant effectiveness in ischemia-reperfusion tissue injury.

In summary, much evidence supports the formation of toxic oxygen metabolites in ischemic reperfused tissue. Tissues are equipped with both an intracellular and extracellular antioxidant defense system. The defense system can also be divided into enzymatic and nonenzymatic defenses. Important components of a nonenzymatic antioxidant include alpha-tocopherol, ascorbic acid, and beta-carotene as well as other compounds that can react with radicals to form less reactive products such as sulfur-containing amino acids. Extracellular fluid comprises a second line of defense against oxidant injury. These extracellular antioxidants include ceruloplasmin, albumin, transferrin, haptoglobin, and uric acid. The oxidant injury can potentially occur during ischemia and reperfusion due to (1) an excess production of oxygen free radicals, (2) a decrease in antioxidant defenses, or (3) both. Because antioxidants function by removing the toxic oxygen metabolites, they are generally highly effective in reducing ischemia-reperfusion injury.

Resveratrol enhances GLUT-4 translocation to the caveolar lipid raft fractions through AMPK/Akt/eNOS signalling pathway in diabetic myocardium

Homeostasis of blood glucose by insulin involves stimulation of glucose uptake by translocation of glucose transporter Glut‐4 from intracellular pool to the caveolar membrane system. In this study we examined resveratrol (RSV)‐mediated Glut‐4 translocation in the streptozotocin (STZ)‐induced diabetic myocardium. The rats were randomized into three groups: Control (Con), Diabetes Mellitus (DM) (STZ 65 mg/kg b.w., i.p.) & DM + RSV (2.5 mg/kg b.wt. for 2 weeks orally) (RSV). Isolated rat hearts were used as per the experimental model. RSV induced glucose uptake was observed in vitro with H9c2 cardiac myoblast cells. Decreased blood glucose level was observed after 30 days (375 mg/dl) in RSV‐treated rats when compared to DM (587 mg/dl). Treatment with RSV demonstrated increased Adenosine Mono Phosphate Kinase (AMPK) phosphorylation compared to DM. Lipid raft fractions demonstrated decreased expression of Glut‐4, Cav‐3 (0.4, 0.6‐fold) in DM which was increased to 0.75‐and 1.1‐fold on RSV treatment as compared to control. Increased Cav‐1 expression (1.4‐fold) in DM was reduced to 0.7‐fold on RSV treatment. Increased phosphorylation of endothelial Nitric Oxide Synthase (eNOS) & Akt was also observed in RSV compared to DM (P< 0.05). Confocal microscopy and co‐immunoprecipitation studies demonstrated decreased association of Glut‐4/Cav‐3 and increased association of Cav‐1/eNOS in DM as compared to control and converse results were obtained on RSV treatment. Our results suggests that the effect of RSV is non‐insulin dependent and triggers some of the similar intracellular insulin signalling components in myocardium such as eNOS, Akt through AMPK pathway and also by regulating the caveolin‐1 and caveolin‐3 status that might play an essential role in Glut‐4 translocation and glucose uptake in STZ‐ induced type‐1 diabetic myocardium.