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Bmx, a member of the Tec family of nonreceptor tyrosine kinases, is a novel participant in pharmacological cardioprotection. Am J Physiol Heart Circ Physiol 287: H2364-H2366, 2004. First published June 10, 2004; doi:10.1152/ajpheart.00416.2004.--Previous studies have indicated that PKC-[member of] is a central regulator of protective signal transduction in the heart. However, the signaling modules through which PKC-[member of] exerts its protective effects have only begun to be understood. We have identified a novel participant in the PKC-[member of] signaling system in cardioprotection, the nonreceptor tyrosine kinase Bmx. Functional proteomic analyses of PKC-[member of] signaling complexes identified Bmx as a member of these complexes. Subsequent studies in rabbits have indicated that Bmx is activated by nitric oxide (NO) in the heart, concomitant with the late phase of NO donor-induced protection, and provide the first analysis of Bmx expression/ distribution in the setting of cardioprotection. In addition, increased expression of Bmx induced by NO donors was blocked by the same mechanism that blocked cardioprotection: inhibition of PKC with chelerythrine. These findings indicate that a novel type of PKC-tyrosine kinase module (involving Bmx) is formed in the heart and may be involved in pharmacological cardioprotection by NO donors. myocardial ischemia; preconditioning; signaling complex; signaling module; nitric oxide

Recent studies have demonstrated that the adenosine [A.sub.1] receptor agonist 2-chloro-[N.sup.6]-cyclopentyladenosine (CCPA) and the adenosine [A.sub.3] receptor agonist [N.sup.6]-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) produce a delayed phase of protection against infarction similar to the late phase of ischemic preconditioning (PC). However, the mechanism for adenosine [A.sub.1] or [A.sub.3] receptor-induced late PC remains unknown. The goal of this study was to determine whether the delayed cardioprotective effects of adenosine [A.sub.1] or [A.sub.3] receptors are mediated by cyclooxygenase-2 (COX-2), which is an obligatory mediator of ischemic PC. We found that COX-2 protein expression (Western blotting) did not increase 24 h after the administration of either CCPA (100 [Mu]g/kg iv) or IB-MECA (300 [Mu]g/kg iv) compared with controls. To probe the role of constitutive COX-2 expression, conscious rabbits were subjected to 30-min coronary occlusion followed by 72-h reperfusion. Twenty-four hours before the occlusion, the rabbits were pretreated with CCPA (100 [Mu]g/kg iv) or IB-MECA (300 [Mu]g/kg iv). Both CCPA and IB-MECA resulted in a marked (~47%) reduction in infarct size vs. controls [36.2 [+ or -] 4.0% of the risk region (n = 9), 31.2 [+ or -] 4.7% (n = 9), and 59.5 [+ or -] 3.8% (n = 9), respectively; P [is less than] 0.05], similar to that induced by the late phase of ischemic PC [31.8 [+ or -] 3.2% (n = 9)]. The selective COX-2 inhibitor N-(2- [cyclohexyloxy]4-nitrophenyl)methanesulfonamide (NS-398, 5 mg/kg), which abolished the protective effect of ischemic late PC, failed to block the protection of either CCPA or IB-MECA, indicating that COX-2 does not mediate the delayed protection of either CCPA or IB-MECA [CCPA + NS-398, 29.1 [+ or -] 3.4% (n = 7); IB-MECA + NS-398, 34.9 [+ or -] 2.9% (n = 8)]. NS-398 in itself did not affect infarct size [54.9 [+ or -]_3.7% (n = 9)]. Taken together, these results demonstrate that, in contrast to ischemia-induced late PC, the mechanisms of adenosine [A.sub.1] or [A.sub.3] receptor-induced late PC is independent of COX-2. myocardial ischemia; myocardial reperfusion; 2-chloro-[N.sup.6]-cyclopentyl-adenosine- [N.sup.6]-(3-iodobenzyl)adenosin-5'-N-methyluronamide; NS-398

Takano, Hitoshi, Xian-Liang Tang, Eitaro Kodani, and Roberto Bolli. Late preconditioning enhances recovery of myocardial function after infarction in conscious rabbits. Am J Physiol Heart Circ Physiol 279: H2372-H2381, 2000.--It is unknown whether late preconditioning (PC) enhances the recovery of left ventricular (LV) function after a myocardial infarction. Thus 25 conscious rabbits were subjected to a 30-min coronary occlusion followed by 28 days of reperfusion after PC 24 h earlier with either ischemia or nitric oxide donor administration [S-nitroso-N-acetylpenicillamine (SNAP)]. The recovery of wall thickening (WTh) after reperfusion was significantly improved in the ischemic PC and SNAP PC groups compared with controls, both at rest and during dobutamine stress. Interestingly, neither ischemia- nor SNAP-induced late PC attenuated myocardial stunning from day 1 through day 14. Infarct size was smaller in the ischemic PC and SNAP PC groups compared with controls. In all groups, WTh at 28 days was positively and linearly related to the percentage of viable tissue in the region underlying the ultrasonic crystal (r = 0.90), indicating that the improvement in LV function after both ischemia-induced and NO donor-induced late PC can be fully explained by the reduction in infarct size; a separate effect of late PC on LV remodeling or LV contractility need not be invoked. In conclusion, in conscious rabbits late PC, induced either by ischemia or pharmacologically, not only limits infarct size but also enhances the recovery of LV function after myocardial infarction. This finding has important clinical implications and provides triphenyltetrazolium chloride-independent evidence that late PC limits myocellular death after sustained ischemia. ischemia-reperfusion injury; myocardial infarction, myocardial stunning, S-nitroso-N-acetylpenicillamine

Xuan, Yu-Ting, Xian-Liang Tang, Yumin Qiu, Supratim Banerjee, Hitoshi Takano, Hui Han, and Roberto Bolli. Biphasic response of cardiac NO synthase isoforms to ischemic preconditioning in conscious rabbits. Am J Physiol Heart Circ Physiol 279: H2360-H2371, 2000.--In conscious rabbits, a sequence of six 4-min coronary occlusion/4-min reperfusion cycles, which elicits late preconditioning (PC), caused rapid activation of calcium-dependent nitric oxide (NO) synthase (NOS) [cNOS; endothelial NOS (eNOS) and/or neuronal NOS (nNOS)], whereas calcium-independent NOS [inducible NOS (iNOS)] activity remained unchanged. The enhanced cNOS activity was associated with increased myocardial levels of [NO.sub.2] and/or [NO.sub.3] ([NO.sub.x]). Twenty-four hours after ischemic PC was induced, the opposite pattern was observed, i.e., there was a pronounced increase in cytosolic iNOS activity but no change in cNOS activity. The initial burst of ischemia-induced cNOS activity was not affected by pretreatment with the antioxidant N-2-mercaptopropionyl glycine (MPG), the protein kinase C (PKC) inhibitor chelerythrine, or the tyrosine kinase inhibitor lavendustin A, indicating that it is independent of the generation of oxidant species and the activation of PKC and tyrosine kinases. In contrast, the delayed upregulation of iNOS 24 h after PC was prevented by pretreatment with [N.sup.w]-nitro-L-arginine, MPG, or chelerythrine before the PC ischemia, indicating that it is triggered by a signaling mechanism that involves the generation of NO, the formation of oxidant species, and the activation of PKC. Taken together, these results demonstrate that, in conscious animals, ischemic PC elicits a biphasic response in cardiac NOS activity, i.e., an immediate activation of cNOS (most likely eNOS) followed 24 h later by a delayed upregulation of iNOS. To our knowledge, this is the first study to directly measure NOS activity after brief myocardial ischemia in vivo. In conjunction with previous functional studies, the data support a distinctive role of NOS isoforms in late PC, with eNOS serving as the trigger on clay 1 and iNOS as the mediator on day 2. nitric oxide synthase; chelerythrine; [N.sup.w]-nitro-L-arginine; reactive oxygen species; protein tyrosine kinases

Takano, Hitoshi, Xian-Liang Tang, and Roberto Bolli. Differential role of KATP channels in late preconditioning against myocardial stunning and infarction in rabbits. Am J Physiol Heart Circ Physiol 279: H2350-H2359, 2000.--The role of ATP-sensitive potassium ([K.sub.ATP]) channels in the late phase of ischemic preconditioning (PC) remains unclear. Furthermore, it is unknown whether [K.sub.ATP] channels serve as end effectors both for late PC against infarction and against stunning. Thus, in phase I of this study, conscious rabbits underwent a 30-min coronary occlusion (O) followed by 72 h of reperfusion (R) with or without ischemic PC (6 4-min O/4-min R cycles) 24 h earlier. Late PC reduced infarct size -46% versus controls. The [K.sub.ATP] channel blocker 5-hydroxydecanoic acid (5-HD), given 5 rain before the 30-rain O, abrogated the infarct-sparing effect of late PC but did not alter infarct size in non-PC rabbits. In phase II rabbits underwent six 4-rain O/4-min R cycles for 3 consecutive days (days 1, 2, and 3). In controls, the total deficit of systolic wall thickening (WTh) after the sixth reperfusion was reduced by 46% on day 2 and 54% on day 3 compared with day 1, indicating a late PC effect against myocardial stunning. Neither 5-HD nor glibenclamide, given on day 2, abrogated late PC. The [K.sub.ATP] channel opener diazoxide, given on day 1, attenuated stunning, and this effect was completely blocked by 5-HD. Thus the same dose of 5-HD that blocked the antistunning effect of diazoxide failed to block the antistunning effects of late PC. Furthermore, when diazoxide was administered in PC rabbits on day 2, myocardial stunning was further attenuated, indicating that diazoxide and late PC have additive anti-stunning effects. We conclude that [K.sub.ATP] channels play an essential role in late PC against infarction but not in late PC against stunning, revealing an important pathogenetic difference between these two forms of cardioprotection. myocardial ischemia; myocardial reperfusion; 5-hydroxyldecanoidic acid; glibenclamide; diazoxide

We examined the role of cyclooxygenase-2 (COX-2) in the late phase of ischemic preconditioning (PC). A total of 176 conscious rabbits were used. Ischemic PC (six cycles of 4-min coronary occlusions/4-min reperfusions) resulted in a rapid increase in myocardial COX-2 mRNA levels (+231 [+ or -] 64% at 1 h; RNase protection assay) followed 24 h later by an increase in COX-2 protein expression (+ 216 [+ or -] 79%; Western blotting) and in the myocardial content of prostaglandin (PG)[E.sub.2] and 6-keto-[PGF.sub.1[Alpha]] (+250 [+ or -] 85% and +259 [+ or -] 107%, respectively; enzyme immunoassay). Administration of two unrelated COX-2 selective inhibitors (NS-398 and celecoxib) 24 h after ischemic PC abolished the ischemic PC-induced increase in tissue levels of [PGE.sub.2] and 6-keto-[PGF.sub.1[Alpha]]. The same doses of NS-398 and celecoxib, given 24 h after ischemic PC, completely blocked the cardioprotective effects of late PC against both myocardial stunning and myocardial infarction, indicating that COX-2 activity is necessary for this phenomenon to occur. Neither NS-398 nor celecoxib lowered [PGE.sub.2] or 6-keto-[PGF.sub.1[Alpha]] levels in the nonischemic region of preconditioned rabbits, indicating that constitutive COX-1 activity was unaffected. Taken together, these results demonstrate that, in conscious rabbits, up-regulation of COX-2 plays an essential role in the cardioprotection afforded by the late phase of ischemic PC. Therefore, this study identifies COX-2 as a cardioprotective protein. The analysis of arachidonic acid metabolites strongly points to [PGE.sub.2] and/or [PGI.sub.2] as the likely effectors of COX-2-dependent protection. The recognition that COX-2 mediates the antistunning and antiinfarct effects of late PC impels a reassessment of current views regarding this enzyme, which is generally regarded as detrimental.

Shinmura, Ken, Xian-Liang Tang, Hitoshi Takano, Michael Hill, and Roberto Bolli. Nitric oxide donors attenuate myocardial stunning in conscious rabbits. Am. J. Physiol. 277 (Heart Circ. Physiol. 46): H2495-H2503, 1999.--Although previous studies suggested that the protection of late preconditioning (PC) against myocardial stunning is mediated by nitric oxide (NO), direct evidence that exogenous administration of NO attenuates myocardial stunning is lacking. Furthermore, although exogenous NO administration was shown to elicit a late PC phase, it is unknown whether NO donors also induce an early PC phase. Therefore, conscious rabbits underwent two experimental stages (3 days of six 4-min occlusion/4-min reperfusion cycles each) 2 wk apart. In study I, both stages were control stages (n = 7). In studies H and III, stage I was the control stage. On day 1 of stage H, seven rabbits received infusion of nitroglycerin (NTG; 2 [micro]g [multiplied by] kg-1 [multiplied by] [min.sup.-1] iv) during the ischemia-reperfusion sequence, starting 30 min before the 1st occlusion and ending 10 min after the 6th reperfusion (study II). Another seven rabbits received infusion of NTG (2 [micro]g [multiplied by] [kg.sup.-1] [multiplied by] [min.sup.-1] iv) for 1 h followed by a 30-min washout interval and then underwent six 4-min occlusion/4-min reperfusion cycles (study III). In the control stage of all three studies, recovery of wall thickening (WTh) after occlusion/reperfusion cycles was markedly enhanced on days 2 and 3 compared with day 1, indicating late PC. In study H, infusion of NTG during the occlusion/reperfusion cycles on day 1 resulted in significant and sustained enhancement in WTh recovery. A similar attenuation of stunning was observed in study IV in six rabbits given intravenous infusion of S-nitroso-N-acetylpenicillamine (SNAP) during occlusion/ reperfusion cycles. The magnitude of the protection afforded by NTG and SNAP was comparable to that afforded by the late ischemic PC phase. In contrast, in study III infusion of NTG before occlusion/reperfusion cycles did not enhance WTh recovery, indicating that NTG failed to induce an early PC effect against stunning. This study demonstrates that administration of hemodynamically inactive doses of two unrelated NO donors alleviates myocardial stunning in conscious rabbits, providing direct evidence for a protective action of NO in this setting. myocardial ischemia; reperfusion injury; left ventricular function; nitroglycerin

Banerjee, Supratim, Xian-Liang Tang, Yumin Qiu, Hitoshi Takano, Srinivas Manchikalapudi, Buddhadeb Dawn, Gregg Shirk, and Roberto Bolli. Nitroglycerin induces late preconditioning against myocardial stunning via a PKC-dependent pathway. Am. J. Physiol. 277 (Heart Circ. Physiol. 46): H2488-H2494, 1999.--Previous studies have shown that administration of nitric oxide (NO) donors induces a delayed cardioprotective effect indistinguishable from the late phase of ischemic preconditioning (PC). However, the ability of clinically relevant NO donors to elicit this phenomenon has not been evaluated. In this study we tested whether an NO-releasing agent that is nitroglycerin (NTG), which is widely used clinically, can mimic the late phase of ischemic PC. Four groups of conscious rabbits underwent six cycles of 4-min occlusion (O)/4-min reperfusion (R) for 3 consecutive days (days 1, 2, and 3). The severity of myocardial stunning was assessed as the total deficit of systolic wall thickening (WTh) after the last O/R cycle. In the control group (group I, n = 6), the total deficit of WTh was reduced by 50% and 51% on days 2 and 3 vs. day 1, respectively, indicating late PC against stunning. Pretreatment with NTG (2 [micro]g [multiplied by] [kg.sup.-1] [multiplied by] [min.sup.-1] iv over 1 h) on day 0 (group II, n = 6) was as effective as ischemic PC in mitigating myocardial stunning 24 h later (day 1); on days 2 and 3, no further reduction of stunning was seen. Coadministration of the PKC inhibitor chelerythrine (5 mg/kg) with NTG (group III, n = 6) completely abrogated the NTG-induced protection. Pretreatment with chelerythrine alone (group IV, n = 5) did not alter stunning. These results demonstrate that a relatively brief infusion of NTG induces a robust protective effect against stunning 24 h later via a protein kinase C (PKC)-dependent signaling mechanism. The magnitude of NTG-induced protection is equivalent to that observed during the late phase of ischemic PC. Late PC induced by brief treatment with NTG could be a useful therapeutic strategy for myocardial protection in patients with ischemic heart disease. nitric oxide; myocardial ischemia-reperfusion; nitrates; protein kinase C

The goal of this study was to interrogate the role of inducible NO synthase (iNOS) in the !ate 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 occlusion/4-min reperfusion exhibited a significant (P [is less than] 0.05) increase in myocardial iNOS protein content, iNOS activity (assessed as calcium-independent 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 occlusion/4-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 [is less than] 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 !ate 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.

Rizvi, Ali, Xian-Liang Tang, Yumin Qiu, Yu-Ting Xuan, Hitoshi Takano, Asad K. Jadoon, and Roberto Bolli. Increased protein synthesis is necessary for the development of late preconditioning against myocardial stunning. Am J. Physiol. 277 (Heart Circ. Physiol. 46): H874-H884, 1999.--In phase I of this study, the rate of protein synthesis was measured by the incorporation of [[sup.3]H]leucine into the protein pool in the heart of conscious rabbits. At 2 h after ischemic preconditioning (PC) with six 4-min occlusion/4-min reperfusion (O/R) cycles (group H), the [[sup.3]H]leucine content in the ischemic-reperfused region was increased by 82% compared with that in controls (group I), indicating increased protein synthesis. This increase was completely abrogated by pretreatment with cycloheximide (CH; group III). In phase H, rabbits underwent six O/R cycles for three consecutive days (clays 1-3). Controls (group IV) exhibited late PC against myocardial stunning on days 2 and 3. In group V, which received CH 30 min before the 1st O/R cycle on day 1 (same dose as group III), late PC against stunning on day 2 was completely abrogated. In group VI, pretreatment with CH 24 h before the 1st sequence of O/R cycles had no effect on myocardial stunning on day 1, indicating that the absence of late PC on day 2 in group V cannot be ascribed to delayed toxicity of CH. Taken together, these results demonstrate that, in the conscious rabbit, ischemic PC causes a rapid increase in myocardial protein synthesis and that this increased protein synthesis (or at least a fraction of it) is necessary for the development of the protection against myocardial stunning 24 h later. The late phase of ischemic PC is therefore dependent on the formation of new proteins in intact animals. cycloheximide; myocardial ischemia; myocardial reperfusion; [[sup.3]H]leucine; systolic wall thickening