Considerable controversy surrounds the role of protein kinase C (PKC) in ischemic preconditioning (PC). Previous studies have used pharmacological agents and/or measured total myocardial PKC activity; however, no information is available regarding the effects of PC on individual isoforms in vivo. We performed a comprehensive evaluation (using Western immunoblotting) of the expression and subcellular distribution of all 11 currently known PKC isoforms in the heart of conscious rabbits subjected to four different ischemic PC protocols known to induce early and/or late PC (one, three, or six cycles of 4-minute coronary occlusion [4'O]/4-minute reperfusion [4'R]; four cycles of 5-minute occlusion [5'O]/10-minute reperfusion [10'R]). Ten PKC isoforms (alpha, beta1/beta2, gamma, delta, epsilon, zeta, eta, iota, lambda, and mu) were found to be expressed in the rabbit heart. Quantitative immunoblotting demonstrated that as a subgroup, conventional PKCs (cPKCs) are more abundant than novel PKCs (nPKCs) (1445 versus 313 pg PKC/microg tissue protein, respectively) and that PKC alpha is the predominant isoform among the cPKCs (alpha, beta1, beta2, and gamma), representing 51% of this subgroup, and PKC epsilon is the most abundant among the nPKCs (delta, epsilon, zeta, and eta), accounting for 62% of this subgroup. None of the ischemic PC protocols examined caused appreciable changes in total PKC activity, in the subcellular distribution of total PKC activity, or in the subcellular distribution of PKC isoforms alpha, beta1/beta2, gamma, delta, zeta, iota, lambda, and mu. In contrast, all PC protocols caused significant translocation of PKC epsilon and PKC eta isoforms from the cytosolic to the particulate fraction. The particulate fraction of PKC epsilon increased in a dose-dependent fashion with the number of occlusion/reperfusion cycles performed, from 35+/-2% in the control group to 43+/-2% after one 4'O/5-minute reperfusion (5'R) cycle (P<.05), 52+/-2% after three cycles (P<.05 versus one cycle), and 66+/-3% after six cycles (P<.05 versus three cycles). The particulate fraction of PKC epsilon also increased, after four 5'O/10'R cycles, to 50+/-3% (P<.05 versus control). In contrast to PKC epsilon, the translocation of PKC eta was independent of the number of occlusion/reperfusion cycles performed. The particulate fraction of PKC eta increased from 67+/-3% in the control group to 84+/-2% after one 4'O/5'R cycle (P<.05), 84+/-2% after three 4'O/4'R cycles (P<.05), 86+/-3% after six 4'O/4'R cycles (P<.05), and 83+/-2% after four 5'O/10'R cycles (P<.05). When expressed as a percentage of control values, the increases in the particulate fraction of isoform epsilon were greater than those of isoform eta. The effects of 4'O without reperfusion were similar to those of one cycle of 4'O/5'R, indicating that 5'R did not attenuate isoform translocation. This is the first study to demonstrate PKC translocation after ischemic PC in vivo. The results indicate that in the conscious rabbit, ischemic PC causes selective translocation of ...

The goal of this study was to interrogate the role of inducible NO synthase (iNOS) in the late phase of ischemic preconditioning (PC) in vivo. A total of 321 mice were used. Wild-type mice preconditioned 24 h earlier with six cycles of 4-min coronary occlusion͞4-min reperfusion exhibited a significant (P < 0.05) increase in myocardial iNOS protein content, iNOS activity (assessed as calciumindependent L-citrulline formation), and nitrite ؉ nitrate tissue levels. In contrast, endothelial NOS protein content and calcium-dependent NOS activity remained unchanged. No immunoreactive neuronal NOS was detected. When wild-type mice were preconditioned 24 h earlier with six 4-min occlusion͞4-min reperfusion cycles, the size of the infarcts produced by a 30-min coronary occlusion followed by 24 h of reperfusion was reduced markedly (by 67%; P < 0.05) compared with sham-preconditioned controls, indicating a late PC effect. In contrast, when mice homozygous for a null iNOS allele were preconditioned 24 h earlier with the same protocol, infarct size was not reduced. Disruption of the iNOS gene had no effect on early PC or on infarct size in the absence of PC. These results demonstrate that (i) the late phase of ischemic PC is associated with selective up-regulation of iNOS, and (ii) targeted disruption of the iNOS gene completely abrogates the infarct-sparing effect of late PC (but not of early PC), providing unequivocal molecular genetic evidence for an obligatory role of iNOS in the cardioprotection afforded by the late phase of ischemic PC. Thus, this study identifies a specific protein that mediates late PC in vivo.

It is unknown whether ischemic preconditioning (PC; either early or late) occurs in the mouse. The goal of this study was to answer this question and to develop a reliable and physiologically relevant murine model of both early and late ischemic PC. A total of 201 mice were used. In nonpreconditioned open-chest animals subjected to 30 min of coronary occlusion followed by 24 h of reperfusion, infarct size (tetrazolium staining) averaged 52% of the region at risk. When the 30-min occlusion was performed 10 min after a PC protocol consisting of six cycles of 4-min occlusion and 4-min reperfusion, infarct size was reduced by 75%, indicating an early PC effect. When the 30-min occlusion was performed 24 h after the same PC protocol, infarct size was reduced by 48%, indicating a late PC effect. In mice in which the 30-min occlusion was followed by 4 h of reperfusion, infarct size was similar to that observed after 24 h of reperfusion, indicating that a 4-h reperfusion interval is sufficient to detect the final extent of cell death in this model. Fundamental physiological variables (body temperature, arterial oxygenation, acid-base balance, heart rate, and arterial pressure) were measured and found to be within normal limits. Taken together, these results demonstrate that, in the mouse, a robust infarct-sparing effect occurs during both the early and the late phases of ischemic PC, although the early phase is more powerful. This murine model is physiologically relevant, provides reliable measurements, and should be useful for elucidating the cellular mechanisms of ischemic PC in genetically engineered animals.

Taken together, these results indicate that in the conscious rabbit, the infarct-sparing effect of the late phase of ischemic PC is mediated by the activity of NOS and suggest that the specific isoform primarily responsible for this cardioprotective phenomenon is iNOS. Thus, NO appears to be a pivotal component of the pathophysiological cascade of late PC.

Abstract-Although it is recognized that late preconditioning (PC) results from upregulation of cardioprotective genes, the specific transcription factor(s) that govern this genetic adaptation remains unknown. The aim of this study was to test the hypothesis that the development of late PC is mediated by nuclear factor-B (NF-B) and to elucidate the mechanisms that control the activation of NF-B after an ischemic stimulus in vivo. A total of 152 chronically instrumented, conscious rabbits were used. A sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles, which elicits late PC, induced rapid activation of NF-B, as evidenced by a marked increase in p65 content (ϩ164%; Western immunoblotting) and NF-B DNA binding activity (ϩ306%; electrophoretic mobility shift assay) in nuclear extracts isolated 30 minutes after the last reperfusion. These changes were attenuated 2 hours after ischemic PC and resolved by 4 hours. Competition and supershift assays confirmed the specificity of the NF-B DNA complex signals.The mobility of the NF-B DNA complex was shifted by anti-p65 and anti-p50 antibodies but not by anti-c-Rel antibodies, indicating that the subunits of NF-B involved in gene activation after ischemic PC consist of p65-p50 heterodimers. Pretreatment with the NF-B inhibitor diethyldithiocarbamate (DDTC; 150 mg/kg IP 15 minutes before ischemic PC) completely blocked the nuclear translocation and increased DNA binding activity of NF-B. The same dose of DDTC completely blocked the cardioprotective effects of late PC against both myocardial stunning and myocardial infarction, indicating that NF-B activation is essential for the development of this phenomenon in vivo. The ischemic PC-induced activation of NF-B was also blocked by pretreatment with N -nitro-L-arginine (L-NA), a nitric oxide synthase (NOS) inhibitor, N-2-mercaptopropionyl glycine (MPG), a reactive oxygen species (ROS) scavenger, chelerythrine, a protein kinase C (PKC) inhibitor, and lavendustin A, a tyrosine kinase inhibitor (all given at doses previously shown to block late PC), indicating that ischemic PC activates NF-B via formation of NO and ROS and activation of PKC-and tyrosine kinase-dependent signaling pathways. A subcellular redistribution and increased DNA binding activity of NF-B quantitatively similar to those induced by ischemic PC could be reproduced pharmacologically by giving the NO donor diethylenetriamine/NO (DETA/NO) (at a dose previously shown to elicit late PC), demonstrating that NO in itself can activate NF-B in the heart. Taken together, these results provide direct evidence that activation of NF-B is a critical step in the signal transduction pathway that underlies the development of the late phase of ischemic PC in conscious rabbits. The finding that four different pharmacological manipulations (L-NA, MPG, chelerythrine, and lavendustin A) produced similar inhibition of NF-B suggests that this transcription factor is a common downstream pathway through which multiple signals elicited by ischemic stress (NO, ROS, PKC,...

The goal of this study was to test the hypothesis that the cardioprotective effects of the late phase of ischemic preconditioning (PC) can be mimicked by treatment with NO donors. In phase I (studies of myocardial stunning), conscious rabbits underwent a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles for 3 consecutive days (days 1, 2, and 3). In group I (controls, n=6), the total deficit of systolic wall thickening (WTh) after the sixth reperfusion was reduced by 54% on days 2 and 3 compared with day 1 (P<0.05), indicating a late PC effect against myocardial stunning. When rabbits were given the NO donors diethylenetriamine/NO (DETA/NO, 0.1 mg/kg IV, 4 times [group II, n=5]) or S-nitroso-N-acetylpenicillamine (SNAP, 2.5 µg • kg −1 • min −1 IV for 75 minutes [group III, n=5]) 24 hours before the first sequence of occlusion/ reperfusion cycles, the deficit of WTh on day 1 was 60% (group II) and 54% (group III) less than that observed in controls (P<0.05 for both). In both groups II and III, there was no further improvement in the deficit of WTh on days 2 and 3 compared with day 1. The protective effect of DETA/NO was completely abrogated when this agent was given in conjunction with the ONOO − and •OH scavenger mercaptopropionyl glycine (MPG) (group IV, n=5). In phase II (studies of myocardial infarction), conscious rabbits underwent a 30-minute coronary occlusion followed by 3 days of reperfusion. When rabbits were preconditioned 24 hours earlier with six 4-minute occlusion/4-minute reperfusion cycles, infarct size was reduced by 43% (33.2±2.7% versus 58.3 ±4.1% of the region at risk in controls, P<0.05), indicating a late PC effect against myocardial infarction. When rabbits were pretreated with DETA/NO (group VII, n=8) or SNAP (group IX, n=7) 24 hours before the 30-minute occlusion, infarct size was reduced by a similar degree (29.3 ±3.6% and 32.0±3.3% of the region at risk, respectively; P<0.05 versus controls). The degree of protection could not be increased by doubling the dose of DETA/NO (group VIII, n=5). Coadministration of MPG completely abrogated the infarct-sparing action of DETA/NO (group X, n=7). Taken together, these results demonstrate that in conscious rabbits the administration of 2 structurally unrelated NO donors induces protection 24 hours later against both reversible (stunning) and irreversible (infarction) ischemia/reperfusion injury and that the magnitude of this protection is indistinguishable from that observed during the late phase of ischemic PC. The fact that the late phase of ischemic PC can be mimicked by NO donors provides direct evidence that NO in itself is sufficient to elicit this cardioprotective mechanism. The fact that NO donor-induced late PC was abrogated by MPG indicates that the mechanism whereby NO induces this phenomenon involves the generation of oxidant species, possibly ONOO − and/or OH. Since a relatively brief treatment with hemodynamically inactive doses of NO donors can induce longlasting protective effects, these agents could be...

Recent studies in conscious pigs and rabbits have demonstrated that a series of brief coronary occlusions renders the heart relatively resistant to myocardial "stunning" 24 hours later (late preconditioning [PC] against stunning). The mechanism of this powerful cardioprotective response is unknown. The goal of the present study was to test the hypothesis that the development of late PC against stunning is triggered by increased generation of NO during the first ischemic challenge. Conscious rabbits underwent a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles for 3 consecutive days (days 1, 2, and 3). On day 1, rabbits received either an intravenous infusion of the NO synthase inhibitor NG-nitro-L-arginine (L-NA, 13 mg/kg before the first occlusion) (group II, n = 10) or vehicle (group I [control], n = 10). In the control group, on day 1 systolic wall thickening (WTh) in the ischemic/reperfused region remained significantly depressed for 4 hours after the sixth reperfusion, indicating myocardial stunning. On days 2 and 3, however, the recovery of WTh improved markedly, so that the total deficit of WTh decreased by 60% on day 2 and 55% on day 3 compared with day 1 (P < .01). In the L-NA-treated group, the total deficit of WTh on day 1 was similar to that observed in the control group. On day 2, however, the total deficit of WTh was not significantly different from that observed on day 1 and was 132% greater than that observed in control rabbits on day 2 (P < .01). On day 3, the total deficit of WTh was 66% less than that noted on day 2 (P < .01). Thus, in L-NA-treated rabbits the sequence of six coronary occlusions and reperfusions performed on day 1 failed to precondition against stunning on day 2, but the same sequence performed on day 2 did precondition against stunning on day 3. Another group of rabbits (group III, n = 6) received L-NA on day 1 in the absence of ischemia and was subjected to the occlusion/ reperfusion sequence on days 2 and 3. In these animals, the total deficit of WTh on day 2 did not differ from that observed in control rabbits on day 1, indicating that administration of L-NA did not exacerbate the severity of myocardial stunning 24 hours later; therefore, the absence of late PC against stunning on day 2 in group II cannot be ascribed to a delayed deleterious action of L-NA on WTh. In conclusion, these results demonstrate that the NO synthase inhibitor L-NA completely blocks the development of late PC against myocardial stunning in conscious rabbits, indicating that NO generated as a result of the PC ischemia triggers the development of the cardioprotective response observed 24 hours later. NO is known to exert numerous biological actions resulting in rapid but transient physiological responses. The present observations support a novel pathophysiological paradigm in which NO also plays a key role in the delayed myocardial adaptations to ischemic stress, acting as a signaling step in the transduction pathway that leads to increased resistance to subsequent ischemic injury.

Brief ischemic episodes confer marked protection against myocardial stunning 1-3 d later (late preconditioning [PC] against stunning). The mechanism of this powerful protective effect is poorly understood. Although protein kinase C (PKC) has been implicated in PC against infarction, it is unknown whether it triggers late PC against stunning. In addition, the entire PKC hypothesis of ischemic PC remains controversial, possibly because the effects of PKC inhibitors on PC protection have not been correlated with their effects on PKC activity and/or translocation in vivo. Thus, conscious rabbits underwent a sequence of six 4-min coronary occlusion (O)/4-min reperfusion (R) cycles for three consecutive days (days 1, 2, and 3). In the control group (group I, n ϭ 7), the recovery of systolic wall thickening after the six O/R cycles was markedly improved on days 2 and 3 compared with day 1, indicating the development of late PC against stunning. Administration of the PKC inhibitor chelerythrine at a dose of 5 mg/kg before the first O on day 1 (group II, n ϭ 10) abrogated the late PC effect against stunning, whereas a 10-fold lower dose (0.5 mg/kg; group III, n ϭ 7) did not. Administration of 5 mg/kg of chelerythrine 10 min after the sixth reperfusion on day 1 (group IV, n ϭ 6) failed to block late PC against stunning. When rabbits were given 5 mg/kg of chelerythrine in the absence of O/R (group V, n ϭ 5), the severity of myocardial stunning 24 h later was not modified. Pretreatment with phorbol 12-myristate 13-acetate (4 g/kg) on day 1 without ischemia (group VI, n ϭ 11) induced late PC against stunning on day 2 and the magnitude of this effect was equivalent to that observed after ischemic PC. In vehicle-treated rabbits (group VIII, n ϭ 5), the six O/R cycles caused translocation of PKC isoforms ⑀ and from the cytosolic to the particulate fraction without significant changes in total PKC activity, in the subcellular distribution of total PKC activity, or in the subcellular distribution of the ␣ , ␤ 1 , ␤ 2 , ␥ , ␦ , , , , and isoforms. The higher dose of chelerythrine (5 mg/kg; group X, n ϭ 5) prevented the translocation of both PKC ⑀ and induced by ischemic PC, whereas the lower dose (0.5 mg/kg; group XI, n ϭ 5) prevented the translocation of PKC but not that of ⑀ , indicating that the activation of ⑀ is necessary for late PC to occur whereas that of is not. To our knowledge, this is the first demonstration that a PKC inhibitor actually prevents the translocation of PKC induced by ischemic PC in vivo, and that this inhibition of PKC translocation results in loss of PC protection. Taken together, the results demonstrate that the mechanism of late PC against myocardial stunning in conscious rabbits involves a PKCmediated signaling pathway, and implicate ⑀ as the specific PKC isoform responsible for the development of this cardioprotective phenomenon.