Figure 19. Schematic of Ras/Raf and Rho signaling. Growth, pro-death and pro-survival signals arc tranduced through these cascades. Transcription factors are activated and link extracellular stimuli to RNA synthesis in the nucleus. Stimulation of F.RK is pro-survival, JNK is pro-apoptotic and p3ii MAPK. can be either pro-survival or pro-apoptotic. See text for a more detailed explanation.
ing simulated ischemia decreased cell injury as measured by LDH release.144 Similarly, inhibition of p3S MAPK activation reduced myocardial injury in isolated rabbit and rat heart models of ischemia and reperfusion.'"15'146 Multiple cycle ischemic preconditioning attenuates ischemia and reperfusion induced p38 MAPK activation.147 148 However, transient activation of p38 MAPK during the brief episodes of ischemia and reperfusion seems to be an essential element of the preconditioning effect. In fact, treatment with SB203580 during preconditioning abrogated the protective effect.149 Lastly, an isoform specific function is identified for the p38 MAPK with the isoform p38a exhibiting a pro-apoptotic effect, while p38p displaying an anti-apoptotic effect. Transgenic mice expressing dominant-negative mutants of p38ct in the heart show reduced infarct size, TUNEL, and DNA laddering following ischemia and reperfusion.15".
Increased JNKs activation in response to ischemia and reperfusion is observed in an adult rabbit cardiomyocytes model of simulated ischemia and reoxygenadon. Attenuation of JNKs activity by transfection with a dominant negative mutant of the upstream activator of JNKs, JNK kinase-2 or JNK interacting protein-!, JIP-1, reduced cell death,151 Furthermore, ischemia and reperfusion induced JNKs activation was decreased in perfused hearts subjected to ischemic preconditioning or heat stress treatment.1"
2.3,4. The Ras/Rafsignaling
This signaling pathway is activated by tyrosine kinase receptors, Gi receptors, or non receptor stimuli. Figure 10. Ras is a small GTP binding protein. When GTP is bound to Ras, activation of the downstream kinases occurs. The cascade includes the MAP-KKK (MAP kinase kinase kinase) known as Raf-1, the MAP-KK, known as MEK and the MAPK, known as ERK. Activation of this pathway leads to the phosphorylation of target proteins in both cytosol and nucleus. Such molecules are the p90RSK ribosomal S6 kinase and the transcription factors ELK-1 and CREB. p90RSK is involved in NHE regulation and can also be activated by ERK independent pathways. ERK1/2 facilitates cell survival through several downstream mediators and end-effectors. ERK 1/2 forms a complex with PKCe in mitochondria, and increases the phosphorylation and inactivation of the pro-apoptotic Bcl-2 family member Bad. Similarly, inactivation of Bad occurs through the ERK/p90RSK ribosomal S6 kinase. ERK1/2 antagonizes the extrinsic apoptotic pathway through a mechanism involving inhibition of caspase-8 cleavage. Furthermore, ERK 1/2 activation in cardiac myocytes may provide protection through direct transcription linkage with GATA4.129 Ras/Raf signaling can interact with other signaling pathways (e.g JAK/STAT). Figure 19.
ERK is activated in response to ischemia and reperfusion and seems to be of cardioprotective nature. Inhibition of ERK activation by PD98059, an ERK inhibitor, increases apoptosis in neonatal cardiomyocytes exposed to simulated ischemia and re-oxygenation.153 Similarly, in an isolated perfused rat heart model of ischemia and reperfusion, postischemic functional recovery was reduced after treatment with PD98059.153 Furthermore, an interplay seems to exist between the ERK anti-apoptotic pathway and the p38 MAPK and JNKs pro-apoptotic pathways and cell fate is shown to depend on this balance. In fact, simultaneous inhibition of the p38 MAPK and JNKs pathways attenuated the increased ischemia and reoxygenation induced apoptosis that occurred by inhibition of the ERK pathway.153 Activation of ERK 1/2 occurs during preconditioning cycles and inhibition of its activity by PD98059, attenuated the preconditioning effect in an in vivo rat model.135 However, similar effect was not observed in the perfused rat heart model of ischemia and reperfusion.154
The PI3K signaling is mediated by receptors with intrinsic enzymatic activity (tyrosine kinase receptors) or non receptor stimuli. PI3K is activated by the receptor's association to Src-like tyrosine kinase or Ras kinase or to insulin receptor substrate-1 (IRS-1), an important docking site for several kinases and phosphatases. Figure 7. PI3K (a isoform) in turn activates PKD1/PKD2 and Akt while PI3K (y isoform) negatively regulates c-AMP and hence GPCRs related signaling. The physiological role of PI3K kinase is now revealed by studies on transgenic animals showing that overexpression of PI3Ka can result in hypertrophy without the activation of the fetal program, while overexpression of dnPBKa blocks hypertrophy without depressing contractility, reviewed by Abel.155 In contrast, deletion of PI3Ky increases contractility without an increase in cardiac size. PI3K activation leads to the activation of the protein kinase B, known as Akt, through the phosphorylation of PDK1/PDK2. Transgenic animals lacking Aktl isoform show a phenotype of generalized reduction in organ/body size, while deficiency of Akt2 results in type-2 diabetes. Overexpression of Akt induces hypertrophy and increases contractility both in vivo and in vitro.155 This signaling is considered to facilitate cell survival through several targets. Bad, Bax, GSK-3P, caspace-9, eNOS and KATp channels are downstream to Akt. p70S6K is a S6 kinase activated by Akt and is critical for the hypertrophic effect mediated by Akt. This kinase can also be activated by PKC
Transcription Faclors o
Figure 20, Schematic of the PI3K. signaling pathway, PJ3K mediates survival signaling Activation of this pathway results in inactivation of caspase 9, inactivation of the pro-apoptotic proteins Bad and Bnx, activation of eWOS, producing nitric oxide which may protect by inhibiting opening of the mitochondrial transition pore, and activation of p70S6K which inhibits Bad. A negative interaction between P13K. signaling and the pro-apoptotic INK and p3 8 MAPK pathways also exists. Growth factors and G-protein coupled ligands induce eardioproteclion by acting through the PI3K pathway.
in the absence of the PI3K activation.'^ An anti-apoptotic effect of P13K/Akt pathway may also be mediated through its modulatory interaction with the pro-apoptotic p38 MAPK and JNK pathways. This interplay seems to occur through the phosphorylation and inhibition of the apoptosis signal regulating kinase-1 (ASK1), the upstream activator of mitogen activated kinases.157 Figure 19, 20.
PI3K/Akt activation occurs during reperfusion and interventions that further enhance its activation limited ischemia and reperfusion injury, indicating a cardioprotective role of the PI3K/Akt pathway. Activation of the PI3K/Akt by insulin, insulin like growth factor—1, TGFp-l, cardiotrophin-1, urocortin, atorvastatm and bradykinin administration at reperfusion results in cardioprotection, reviewed by Hausenloy.!5S Activation of PI3K/Akt pathway is increased at the lime of reperfusion in the ischemic preconditioned isolated rat hearts subjected to sustained ischemia and reperfusion. Furthermore, PI3K/Akt acts in parallel with ERK1/2 cascade and seems that they are both required for the ischemic preconditioning effect. Interestingly, these pathways are shown to interact in such a way that inhibiting one kinase cascade upregulates the activity of the other pathway, reviewed by Hausenloy.158
2.3.6 The JAK/STATsignaling
JAK/STAT pathway is activated by the IL-6 cytokine family through the gpl30 receptor, G-protein coupled receptors and ischemia and reperfusion. Figure 8. JAK/STAT signaling has been recently linked to the cell response to ischemic stress. Simulated ischemia and reoxygenation in neonatal cardiomyocytes resulted in rapid induction and phosphorylation of STAT1 but not of the STAT3. Transfection of myocytes with STAT1 increased apoptosis following simulated ischemia, indicating a detrimental role of STAT1. On the contrary, transfection with STAT3 reduced ischemia induced apoptosis.159 JAK/STAT signaling is shown to mediate the late preconditioning cardioprotective effect in a mouse model of coronary occlusion and reperfusion. This effect was associated with transcriptional upregulation of the inducible nitric oxide synthase (iNOS) and the cyclooxygenase-2 (COX-2).160
Calcineurin (PP2B) is a calcium-calmodulin-activated, serine/threonine protein phosphatase that responds to sustained elevations of intracellular calcium. Calcineurin consists of a catalytic subunit (CnA), a calcium binding subunit (CnB) and calmodulin. Once activated, calcineurin directly dephosphorylates members of the NFAT transcription factor family in the cytoplasm promoting their nuclear translocation and the transcriptional induction of various genes. It is suggested that transient activation of calcineurin antagonizes myocyte apoptosis, whilst long standing activation induces cardiac hypertrophy and deleterious ventricular remodeling associated with heart failure, reviewed by Baines.129
A potential cardioprotective role for calcineurin is provided by studies in transgenic mice. Calcineurin A beta (-/-) gene targeted mice showed a greater loss of viable myocardium, enhanced DNA laddering and TUNEL and a greater loss of cardiac performance after ischemia and reperfusion compared with strain-matched wild-type control mice. This response was accompanied by reduced expression of the NFAT at baseline and after ischemia and reperfusion. Furthermore, expression of an activated NFAT mutant in cardiac myocytes resulted in protection while directed inhibition of NFAT augmented cell death.161
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