Postconditioning fails to improve no reflow or alter infarct size in an open-chest rabbit model of myocardial ischemia-reperfusion

Postconditioning (PoC) with brief intermittent ischemia after myocardial reperfusion has been shown to lessen some elements of postischemic injury including arrhythmias and, in some studies, the size of myocardial infarction. We hypothesized that PoC could improve reflow to the risk zone after reperfusion. Anesthetized, open-chest rabbits were subjected to 30 min of coronary artery occlusion followed by 3 h of reperfusion. In protocol 1, rabbits were randomly assigned to the control group (n = 10, no further intervention after reperfusion) or to the PoC group, which consisted of four cycles of 30-s reocclusions with 30 s of reperfusion in between starting at 30 s after the initial reperfusion (4 x 30/30, n = 10). In protocol 2, rabbits were assigned to the control group (n = 7) or the PoC group, which received PoC consisting of four cycles of 60-s intervals of ischemia and reperfusion starting at 30 s after the initial reperfusion (4 x 60/60, n = 7). No reflow was determined by injecting thioflavine S (a fluorescent marker of capillary perfusion), risk zone by blue dye, and infarct size by triphenyltetrazolium chloride. In protocol 1, there were no statistical differences in hemodynamics, ischemic risk zone, or infarct size (35 +/- 6% of the risk zone in the PoC group vs. 29 +/- 4% in the control group, P = 0.38) between the groups. Similarly, in protocol 2, PoC failed to reduce infarct size compared with the control group (45 +/- 4% of the risk zone in the PoC group vs. 42 +/- 6% in the control group, P = 0.75). There was a strong correlation in both protocols between the size of the necrotic zone and the portion of the necrotic zone that contained an area of no reflow. However, PoC did not affect this relationship. PoC did not reduce infarct size in this model, nor did it reduce the extent of the anatomic zone of no reflow, suggesting that this intervention may not impact postreperfusion microvascular damage due to ischemia.     

Nicorandil induces late preconditioning against myocardial infarction in conscious rabbits

Nicorandil has been shown to induce an infarct-limiting effect similar to that induced by the early phase of ischemic preconditioning (PC). The goals of this study were to determine whether nicorandil induces a delayed cardioprotection that is analogous to the late phase of ischemic PC and, if so, whether nicorandil-induced late PC is associated with upregulation of cardioprotective proteins. Chronically instrumented, conscious rabbits received vehicle (intravenous normal saline; control group, n = 10), nicorandil (100 microg/kg bolus + 30 microg x kg(-1) x min(-1) i.v. for 60 min; nicorandil group, n = 10), or ischemic PC (6 cycles of 4-min coronary occlusion/4-min reperfusion; PC group, n = 8). Twenty-four hours later, rabbits underwent a 30-min coronary occlusion, followed by 3 days of reperfusion. Myocardial infarct size was significantly reduced in rabbits pretreated with nicorandil (27.5 +/- 5.3% of the risk region) or with ischemia (30.3 +/- 4.2%) versus controls (59.1 +/- 4.7%, P < 0.05 vs. both). Furthermore, the expression of cyclooxygenase-2 (COX-2) and Bcl-2 was significantly elevated (+38% and +126%, respectively; P < 0.05) in myocardium of rabbits given nicorandil 24 h earlier versus controls. We conclude that nicorandil induces delayed cardioprotection against myocardial infarction similar to that afforded by the late phase of ischemic PC, possibly by upregulating COX-2 and Bcl-2.     

Cardioprotection by multiple preconditioning cycles does not require mitochondrial K(ATP) channels in pigs

To test whether cardioprotection induced by ischemic preconditioning depends on the opening of mitochondrial ATP-sensitive K(+) (K(ATP)) channels, the effect of channel blockade was studied in barbital-anesthetized open-chest pigs subjected to 30 min of complete occlusion of the left anterior descending coronary artery and 3 h of reflow. Preconditioning was elicited by two cycles of 5-min occlusion plus 10-min reperfusion before the 30-min occlusion period. 5-Hydroxydecanoate (5 mg/kg iv) was injected 15 min before preconditioning or pharmacological preconditioning induced by diazoxide (3.5 mg/kg, 1 ml/min iv). Infarct size (percentage of the area at risk) after 30 min of ischemia was 35.1 +/- 9.9% (n = 7). Preconditioning markedly limited myocardial infarct size (2.7 +/- 1.6%, n = 7), and 5-hydroxydecanoate did not abolish protection (2.4 +/- 0.9%, n = 8). Diazoxide infusion also significantly limited infarct size (14.6 +/- 7.4%, n = 7), and 5-hydroxydecanoate blocked this effect (30.8 +/- 8.0%, n = 7). Thus the opening of mitochondrial K(ATP) channels is cardioprotective in pigs, but these data do not support the hypothesis that opening of mitochondrial K(ATP) channels is required for the endogenous protection afforded by preconditioning.     

Prolonged transient acidosis during early reperfusion contributes to the cardioprotective effects of postconditioning

We have previously reported that the prolonged transient acidosis during early reperfusion mediates the cardioprotective effects in canine hearts. Recently, postconditioning has been shown to be one of the novel strategies to mediate cardioprotection. We tested the contribution of the prolonged transient acidosis to the cardioprotection of postconditioning. Open-chest anesthetized dogs subjected to 90-min occlusion of the left anterior descending coronary artery and 6-h reperfusion were divided into four groups: 1) control group; no intervention after reperfusion (n = 6); 2) postconditioning (Postcon) group; four cycles of 1-min reperfusion and 1-min reocclusion (n = 7); 3) Postcon + sodium bicarbonate (NaHCO(3)) group; four cycles of 1-min reperfusion and 1-min reocclusion with the administration of NaHCO(3) (n = 8); and 4) NaHCO(3) group; administration of NaHCO(3) without postconditioning (n = 6). Infarct size, the area at risk (AAR), collateral blood flow during ischemia, and pH in coronary venous blood were measured. The phosphorylation of Akt and extracellular signal-regulated kinase (ERK) in ischemic myocardium was assessed by Western blot analysis. Systemic hemodynamic parameters, AAR, and collateral blood flow were not different among the four groups. Postconditioning induced prolonged transient acidosis during the early reperfusion phase. Administration of NaHCO(3) completely abolished the infarct size-limiting effects of postconditioning. Furthermore, the phosphorylation of Akt and ERK in ischemic myocardium induced by postconditioning was also blunted by the cotreatment of NaHCO(3). In conclusion, postconditioning mediates its cardioprotective effects possibly via prolonged transient acidosis during the early reperfusion phase with the activation of Akt and ERK.     

Co-application of ischemic preconditioning and postconditioning provides additive neuroprotection against spinal cord ischemia in rabbits

Postconditioning can induce cardioprotection against ischemia. However, the data on postconditioning of the spinal cord is very limited. We investigated here whether co-application of ischemic preconditioning (IPC) and postconditioning can provide additive neuroprotection against prolonged spinal cord ischemia. Spinal cord ischemia was produced in rabbits by infrarenal aortic occlusion with a balloon catheter for 30 min. The four treatment groups were control (n=10): no intervention; IPC (n=10): a 5-minute aortic occlusion was performed 20 min before the prolonged ischemia; Postcon (n=10): postconditioning comprised of four cycles of 1-minute occlusion/1-minute reperfusion was applied one minute after the start of reperfusion. IPC+postcon (n=11): both IPC and postconditioning were applied. Functional evaluation with Tarlov score was performed during a 14-day observation period. Neurologic impairment was noticeably attenuated in the IPC+postcon group (compared with the control group, P<0.01, at day 1, day 2, day 7 and day 14, respectively), but not in either the IPC or Postcon group. Plasma malondialdehyde levels after reperfusion were significantly decreased to a similar extent in the IPC, Postcon and IPC+Postcon groups (compared with the control group (P<0.01). In the IPC+Postcon group, many more large motor neurons were preserved than in the control group (P<0.05) and white matter injury was also markedly attenuated as evidenced by reduction of the vacuolation area of the white matter (P<0.01) and decreased amyloid precursor protein immunoreactivity (P<0.01). From this, we conclude that the combination of IPC and postconditioning induces additive neuroprotective effects for spinal cord against ischemia and reperfusion injuries.     

Lovastatin interferes with the infarct size-limiting effect of ischemic preconditioning and postconditioning in rat hearts

Statins have been shown to be cardioprotective; however, their interaction with endogenous cardioprotection by ischemic preconditioning and postconditioning is not known. In the present study, we examined if acute and chronic administration of the 3-hydroxy-3-methylglutaryl CoA reductase inhibitor lovastatin affected the infarct size-limiting effect of ischemic preconditioning and postconditioning in rat hearts. Wistar rats were randomly assigned to the following three groups: 1) vehicle (1% methylcellulose per os for 12 days), 2) chronic lovastatin (15 mg.kg(-1).day(-1) per os for 12 days), and 3) acute lovastatin (1% methylcellulose per os for 12 days and 50 micromol/l lovastatin in the perfusate). Hearts isolated from the three groups were either subjected to a nonconditioning (aerobic perfusion followed by 30-min coronary occlusion and 120-min reperfusion, i.e., test ischemia-reperfusion), preconditioning (three intermittent periods of 5-min ischemia-reperfusion cycles before test ischemia-reperfusion), or postconditioning (six cycles of 10-s ischemia-reperfusion after test ischemia) perfusion protocol. Preconditioning and postconditioning significantly decreased infarct size in vehicle-treated hearts. However, preconditioning failed to decrease infarct size in acute lovastatin-treated hearts, but the effect of postconditioning remained unchanged. Chronic lovastatin treatment abolished postconditioning but not preconditioning; however, it decreased infarct size in the nonconditioned group. Myocardial levels of coenzyme Q9 were decreased in both acute and chronic lovastatin-treated rats. Western blot analysis revealed that both acute and chronic lovastatin treatment attenuated the phoshorylation of Akt; however, acute but not chronic lovastatin treatment increased the phosphorylation of p42 MAPK/ERK. We conclude that, although lovastatin may lead to cardioprotection, it interferes with the mechanisms of cardiac adaptation to ischemic stress.     

Increased mitochondrial calcium coexists with decreased reperfusion injury in postconditioned (but not preconditioned) hearts

Ca(2+) is the main trigger for mitochondrial permeability transition pore opening, which plays a key role in cardiomyocyte death after ischemia-reperfusion. We investigated whether a reduced accumulation of mitochondrial Ca(2+) might explain the attenuation of lethal reperfusion injury by postconditioning. Anesthetized New Zealand White rabbits underwent 30 min of ischemia, followed by either 240 (infarct size protocol) or 60 (mitochondria protocol) min of reperfusion. They received either no intervention (control), preconditioning by 5-min ischemia and 5-min reperfusion, postconditioning by four cycles of 1-min reperfusion and 1-min ischemia at the onset of reflow, or pharmacological inhibition of the transition pore opening by N-methyl-4-isoleucine-cyclosporin (NIM811; 5 mg/kg iv) given at reperfusion. Area at risk and infarct size were assessed by blue dye injection and triphenyltetrazolium chloride staining. Mitochondria were isolated from the risk region for measurement of 1) Ca(2+) retention capacity (CRC), and 2) mitochondrial content of total (atomic absorption spectrometry) and ionized (potentiometric technique) calcium concentration. CRC averaged 0.73 +/- 0.16 in control vs. 4.23 +/- 0.17 mug Ca(2+)/mg proteins in shams (P < 0.05). Postconditioning, preconditioning, or NIM811 significantly increased CRC (P < 0.05 vs. control). In the control group, total and free mitochondrial calcium significantly increased to 2.39 +/- 0.43 and 0.61 +/- 0.10, respectively, vs. 1.42 +/- 0.09 and 0.16 +/- 0.01 mug Ca(2+)/mg in sham (P < 0.05). Surprisingly, whereas total and ionized mitochondrial Ca(2+) decreased in preconditioning, it significantly increased in postconditioning and NIM811 groups. These data suggest that retention of calcium within mitochondria may explain the decreased reperfusion injury in postconditioned (but not preconditioned) hearts.     

Adenosine-enhanced ischemic preconditioning: adenosine receptor involvement during ischemia and reperfusion

Adenosine-enhanced ischemic preconditioning (APC) extends the cardioprotection of ischemic preconditioning (IPC) by both significantly decreasing myocardial infarct size and significantly enhancing postischemic functional recovery. In this study, the role of adenosine receptors during ischemia-reperfusion was determined. Rabbit hearts (n = 92) were used for Langendorff perfusion. Control hearts were perfused for 180 min, global ischemia hearts received 30-min ischemia and 120-min reperfusion, and IPC hearts received 5-min ischemia and 5-min reperfusion before ischemia. APC hearts received a bolus injection of adenosine coincident with IPC. Adenosine receptor (A(1), A(2), and A(3)) antagonists were used with APC before ischemia and/or during reperfusion. GR-69019X (A(1)/A(3)) and MRS-1191/MRS-1220 (A(3)) significantly increased infarct size in APC hearts when administered before ischemia and significantly decreased functional recovery when administered during both ischemia and reperfusion (P < 0.05 vs. APC). DPCPX (A(1)) administered either before ischemia and/or during reperfusion had no effect on APC cardioprotection. APC-enhanced infarct size reduction is modulated by adenosine receptors primarily during ischemia, whereas APC-enhanced postischemic functional recovery is modulated by adenosine receptors during both ischemia and reperfusion.     

Opening of mitochondrial K(ATP) channel occurs downstream of PKC-epsilon activation in the mechanism of preconditioning

We examined whether the mitochondrial ATP-sensitive K channel (K(ATP)) is an effector downstream of protein kinase C-epsilon (PKC-epsilon) in the mechanism of preconditioning (PC) in isolated rabbit hearts. PC with two cycles of 5-min ischemia/5-min reperfusion before 30-min global ischemia reduced infarction from 50.3 +/- 6.8% of the left ventricle to 20.3 +/- 3.7%. PC significantly increased PKC-epsilon protein in the particulate fraction from 51 +/- 4% of the total to 60 +/- 4%, whereas no translocation was observed for PKC-delta and PKC-alpha. In mitochondria separated from the other particulate fractions, PC increased the PKC-epsilon level by 50%. Infusion of 5-hydroxydecanoate (5-HD), a mitochondrial K(ATP) blocker, after PC abolished the cardioprotection of PC, whereas PKC-epsilon translocation by PC was not interfered with 5-HD. Diazoxide, a mitochondrial K(ATP) opener, infused 10 min before ischemia limited infarct size to 5.2 +/- 1.4%, but this agent neither translocated PKC-epsilon by itself nor accelerated PKC-epsilon translocation after ischemia. Together with the results of earlier studies showing mitochondrial K(ATP) opening by PKC, the present results suggest that mitochondrial K(ATP)-mediated cardioprotection occurs subsequent to PKC-epsilon activation by PC.     

Protective role of gap junctions in preconditioning against myocardial infarction

The aim of the present study was to examine the hypothesis that acceleration of gap junction (GJ) closure during ischemia contributes to anti-infarct tolerance afforded by preconditioning (PC). First, the effects of PC on GJ communication during ischemia were assessed. Isolated buffer-perfused rabbit hearts were subjected to 5-min global ischemia with or without PC with two cycles of 5-min ischemia/5-min reperfusion or a GJ blocker (2 mM heptanol), and then the tissue excised from the ischemic region was incubated in anoxic buffer containing lucifer yellow (LY; 2.5 mg/ml), a tracer of GJ permeability, for 20 min at 37 degrees C. PC and heptanol significantly reduced the area to which LY was transported in the ischemic myocardium by 39% and by 54%, respectively. In the second series of experiments, three GJ blockers (heptanol, 18beta-glycyrrhetinic acid, and 2,3-butanedione monoxime) infused after the onset of ischemia reduced infarct size after 30-min ischemia/2-h reperfusion to an extent equivalent to that in the case of PC. In the third series of experiments, Western blotting for connexin43 (Cx43) showed that PC shortened the time to the onset of ischemia-induced Cx43 dephosphorylation but reduced the extent of Cx43 dephosphorylation during a 30-min period of ischemia. Calphostin C, a protein kinase C (PKC) inhibitor, abolished preservation of phosphorylated Cx43 but not the early onset of Cx43 dephosphorylation after ischemia in the preconditioned myocardium. These results suggest that PC-induced reduction of GJ permeability during ischemia, presumably by PKC-mediated Cx43 phosphorylation, contributes to infarct size limitation.     

TNF-alpha antibodies are as effective as ischemic preconditioning in reducing infarct size in rabbits

Pretreatment with tumor necrosis factor-alpha (TNF-alpha) antibodies abolishes myocardial infarct size reduction by late ischemic preconditioning (IP). Whether or not TNF-alpha is also important for myocardial infarct size reduction by classic IP is unknown. Anesthetized rabbits were untreated (group 1, n = 7), classically preconditioned by 5 min left coronary artery occlusion/10 min reperfusion (group 2, n = 6), or pretreated with TNF-alpha antibodies without (group 3, n = 6) or with IP (group 4, n = 6) before undergoing 30 min of occlusion and 180 min of reperfusion. Infarct size in group 1 was 44 +/- 11 (means +/- SD)% of the area at risk. With a comparable area at risk, infarct size was reduced to 13 +/- 7%, 23 +/- 8%, and 19 +/- 12% (all P < 0.05) in groups 2, 3, and 4, respectively. The circulating TNF-alpha concentration was increased during ischemia in group 1 from 752 +/- 403 to 1,542 +/- 482 U/ml (P < 0.05) but remained unchanged in all other groups. Circulating TNF-alpha concentration during ischemia and infarct size correlated in all groups (r = 0.76). IP, TNF-alpha antibodies, and the combined approach reduced infarct size to a comparable extent. Therefore, the question of whether or not TNF-alpha is causally involved in the infarct size reduction by IP in rabbits could not be answered.     

Is microvascular protection by cariporide and ischemic preconditioning causally linked to myocardial salvage?

Two independent cardioprotective interventions, Na(+)/H(+) exchange inhibition and ischemic preconditioning (PC), were investigated with respect to differential effects on microvascular and myocardial salvage in anesthetized rabbits (30 min of ischemia, 180 min of reperfusion). Cariporide (Car, 300 microg/kg) administered before occlusion and PC reduced infarct size (IS) as measured by triphenyltetrazolium staining [control, 46.0 +/- 4.2% of risk area (RA); Car, 17.6 +/- 3.7% (P < 0.01); PC, 27.5 +/- 4.1% (P < 0.01)] and concomitantly decreased the area of anatomic no reflow (ANR) as measured by thioflavin S staining [control, 40.4 +/- 3.7%; Car, 19.0 +/- 2.9% (P < 0.01); PC, 26.9 +/- 3.4% (P < 0.05)]. Regional myocardial blood flow (RMBF, measured by radioactive microspheres) in the RA, which deteriorated between 30 and 180 min of reperfusion (control, from 79 +/- 6 to 26 +/- 2% of nonischemic flow), was shifted to higher values with both treatments [Car, from 110 +/- 12 to 49 +/- 7% (P < 0.05); PC, from 109 +/- 8 to 38 +/- 6% (P < 0.05)]. However, neither intervention uncoupled the close relationship between IS and ANR (r = 0.92-0.95) or RMBF. Car given at reperfusion did not alter IS, ANR, RMBF, or the close interrelationships. Because size and spatial distribution of no reflow and myocardial necrosis remained closely coupled with independent cardioprotective interventions, a potential causal connection between microvascular and myocardial salvage is discussed.     

Extent of damage in ischemic,nonreperfused, and reperfused myocardium of anesthetized rats

To investigate the localization of the earliest damage in ischemic and ischemic-reperfused myocardium, anesthetized rats were subjected to coronary occlusion for 15, 30, 45, or 90 min. One-half of the animals in each group had no reperfusion, whereas the other half was reperfused for 14 min. With the use of histological methods, preferentially in the periphery of the area at risk, localized zones were detected that lacked the hypoxia-specific increase in NADH fluorescence. The extent of these areas displaying injured tissue was found to be significantly smaller in the ischemic-nonreperfused hearts than in the ischemic-reperfused organs (15-min ischemia: 0.22 +/- 0.12% vs. 43.0 +/- 5.0%; 30-min ischemia: 5.7 +/- 2.7% vs. 64.6 +/- 2.9%; 45-min ischemia: 5.6 +/- 1.2% vs. 66.0 +/- 7.5%; 90-min ischemia: 39.3 +/- 5.5% vs. 86.7 +/- 1.8% of the area at risk). The results point to a localized initiation of the damage close to the surrounding oxygen-supplied tissue during ischemia and an expansion of this injury by intercellular actions into yet-intact areas upon reperfusion.     

Melatonin does not prevent the protection of ischemic preconditioning in vivo despite its antioxidant effect against oxidative stress

Free radicals are involved in the protective mechanism of preconditioning (PC), whereas antioxidant compounds abolish this benefit. Melatonin is a hormone with antioxidant properties. The aim of our study was to evaluate the effect of melatonin on infarct size in ischemic preconditioning in vivo. We randomly divided 33 male rabbits into four groups and subjected them to 30 min of myocardial ischemia and 3 h of reperfusion with the following prior interventions: (i) no intervention, (ii) iv melatonin at a total dose of 50 mg/kg, (iii) PC with two cycles of 5 min ischemia and 10 min reperfusion, and (iv) combined melatonin and PC. In a second series of experiments, another antioxidant agent N-acetylcysteine (NAC) was used in a control and in a PC group. Myocardial infarct size was determined and blood samples were drawn at different time points for the determination of lipid peroxidation products, total superoxide dismutase (SOD) activity, and (1)H-NMR spectra to evaluate the changes in the metabolic profile. Melatonin showed no effect on myocardial infarct size in the group of sustained ischemia (42.9 +/- 3.6% vs 47.4 +/- 4.9%) and it did not attenuate the reduction of myocardial infarct size in the PC group (13.6 +/- 2.4% vs 14.0 +/- 1.7%). A similar effect was found in NAC-treated groups (44.8 +/- 3.4% vs 14.3 +/- 1.3%). Lipid peroxidation product levels were significantly elevated in the control and PC groups, whereas melatonin decreased them in both groups. The SOD activity was enhanced in the PC group compared to controls; melatonin kept SOD activity unchanged during ischemia/reperfusion and enhanced its activity when it was combined with PC. Melatonin did not change the metabolic profile of the control and PC groups. Melatonin does not prevent the beneficial effect of ischemic PC on infarct size despite its antioxidant properties.     

Effect of 6-wk estrogen withdrawal or replacement on myocardial ischemic tolerance in rats

Menopausal status is a risk factor for coronary artery disease death, but the mechanism underlying this association is uncertain. To test whether estrogen ameliorates the effects of acute myocardial ischemia in ways likely to translate into a mortality difference, we compared the response to brief (6-min) and prolonged (45-min) coronary occlusion in vivo in five groups (each n = 16) of rats: ovariectomized females; ovariectomized females after 6 wk 17beta-estradiol replacement; male rats supplemented with estradiol for 6 wk; normal males; and normal females. Coronary occlusion produced a uniform ischemic risk area averaging 53 +/- 3% of left ventricular volume. After a brief occlusion, reperfusion ventricular tachycardia/fibrillation occurred with >85% frequency in all groups. During a prolonged occlusion, ischemic ventricular tachycardia occurred in 100% and sustained tachycardia requiring cardioversion in >75% of rats in all groups. Myocardial infarct size averaged 52 +/- 4% of the ischemic risk area and was similarly unaffected by gender or estrogen status. We conclude that neither short-term estrogen withdrawal, replacement, nor supplementation significantly affects the potentially lethal outcomes from acute coronary occlusion in this species.     

Preconditioning in the absence or presence of sustained ischemia modulates myocardial Cx43 protein levels and gap junction distribution

In the heart, brief repeated episodes of ischemia prior to a sustained occlusion (ischemic preconditioning; PC) significantly delay the onset of necrosis and arrhythmogenesis. Ischemia has been reported to influence gap junction organization and connexin43 (Cx43) content, but whether PC affects these structures is not known. We investigated the effect of PC (2 cycles of 5-min ischemia plus 10-min reperfusion) followed by prolonged reperfusion without concomitant regional coronary occlusion on the myocardial Cx43 content and its spatial distribution in rabbit hearts. We also compared the effect of sustained ischemia with or without PC on Cx43 spatial distribution. In experiments with PC only, there was an initial decrease in Cx43 levels within the ischemic zone followed by a progressive increase after 48 h reperfusion. End-to-end immunolabeling of Cx43 was augmented in the ischemic region between 24 and 48 h reperfusion; labeling was not uniquely confined to myocyte abutments, but was also dispersed along the sarcolemma. Cx43 immunolabelling was more intense and diffuse in hearts subjected to PC before sustained coronary occlusion (compared to non-PC). These data indicate that gap junctions are significantly altered during brief episodes of ischemia. Reorganization of the gap junction complex could contribute to PC-mediated reductions in cardiac arrhythmias.     

CRYAB and HSPB2 deficiency alters cardiac metabolism and paradoxically confers protection against myocardial ischemia in aging mice

The abundantly expressed small molecular weight proteins, CRYAB and HSPB2, have been implicated in cardioprotection ex vivo. However, the biological roles of CRYAB/HSPB2 coexpression for either ischemic preconditioning and/or protection in situ remain poorly defined. Wild-type (WT) and age-matched ( approximately 5-9 mo) CRYAB/HSPB2 double knockout (DKO) mice were subjected either to 30 min of coronary occlusion and 24 h of reperfusion in situ or preconditioned with a 4-min coronary occlusion/4-min reperfusion x 6, before similar ischemic challenge (ischemic preconditioning). Additionally, WT and DKO mice were subjected to 30 min of global ischemia in isolated hearts ex vivo. All experimental groups were assessed for area at risk and infarct size. Mitochondrial respiration was analyzed in isolated permeabilized cardiac skinned fibers. As a result, DKO mice modestly altered heat shock protein expression. Surprisingly, infarct size in situ was reduced by 35% in hearts of DKO compared with WT mice (38.8 +/- 17.9 vs. 59.8 +/- 10.6% area at risk, P < 0.05). In DKO mice, ischemic preconditioning was additive to its infarct-sparing phenotype. Similarly, infarct size after ischemia and reperfusion ex vivo was decreased and the production of superoxide and creatine kinase release was decreased in DKO compared with WT mice (P < 0.05). In permeabilized fibers, ADP-stimulated respiration rates were modestly reduced and calcium-dependent ATP synthesis was abrogated in DKO compared with WT mice. In conclusion, contrary to expectation, our findings demonstrate that CRYAB and HSPB2 deficiency induces profound adaptations that are related to 1) a reduction in calcium-dependent metabolism/respiration, including ATP production, and 2) decreased superoxide production during reperfusion. We discuss the implications of these disparate results in the context of phenotypic responses reported for CRYAB/HSPB2-deficient mice to different ischemic challenges.     

Early ischemic preconditioning against focal transient and permanent brain ischemia in rats: role of collateral circulation

We hypothesize that early ischemic preconditioning (IPC) can afford protection against focal brief and prolonged cerebral ischemia with subsequent reperfusion as well as permanent brain ischemia in rats by amelioration of regional cerebral blood flow. Adult male Wistar rats (n=97) were subjected to transient (30 and 60 minutes) and permanent middle cerebral artery (MCA) occlusion. IPC protocol consisted of two episodes of 5-min common carotid artery occlusion + 5-min reperfusion prior to test ischemia either followed by 48 hours of reperfusion or not. Triphenyltetrazolium chloride and Evans blue were used for delineation of infarct size and anatomical area at risk (comprises ischemic penumbra and ischemic core), respectively. Blood flow in the MCA vascular bed was measured with use of Doppler ultrasound. The IPC resulted in significant infarct size limitation in both transient and permanent MCA occlusion. Importantly, IPC caused significant reduction of area at risk after 30 min of focal ischemia as compared to controls [med(min-max) 11.4% (3.59-2 0.35%) vs. 2.47% (0.8-9.31%), p = 0.018] but it failed to influence area at risk after 5 min of ischemia [med(min-max) 7.61% (6.32-10.87%) vs. 8.2% (4.87-9.65%), p > 0.05]. No differences in blood flow were found between IPC and control groups using Doppler ultrasound. This is suggestive of the fact that IPC does not really influence blood flow in the large cerebral arteries such as MCA but it might have some effect on smaller arteries. It seems that, along with well established cytoprotective effects of IPC, IPC-mediated reduction of area at risk by means of improvement in local cerebral blood flow may contribute to infarct size limitation after focal transient and permanent brain ischemia in rats.     

VEGFR1 (Flt-1+/-) gene knockout leads to the disruption of VEGF-mediated signaling through the nitric oxide/heme oxygenase pathway in ischemic preconditioned myocardium

This report demonstrates that mice deficient in Flt-1 failed to establish ischemic preconditioning (PC)-mediated cardioprotection in isolated working buffer-perfused ischemic/reperfused (I/R) hearts compared to wild type (WT) subjected to the same PC protocol. WT and Flt-1+/- mice were divided into four groups: (1) WT I/R, (2) WT + PC, (3) Flt-1+/- I/R, and (4) Flt-1+/- + PC. Group 1 and 3 mice were subjected to 30 min of ischemia followed by 2 h of reperfusion and group 2 and 4 mice were subjected to four episodes of 4-min global ischemia followed by 6 min of reperfusion before ischemia/reperfusion. For both wild-type and Flt-1+/- mice, the postischemic functional recovery for the hearts was lower than the baseline, but the recovery for the knockout mice was less compared to the WT mice even in preconditioning. The myocardial infarction and apoptosis were higher in Flt-1+/- compared to wild-type I/R. Flt-1+/- KO mice demonstrated pronounced inhibition of the expression of iNOS, p-AKT & p-eNOS. Significant inhibition of STAT3 & CREB were also observed along with the inhibition of HO-1 mRNA. Results demonstrate that Flt-1+/- mouse hearts are more susceptible to ischemia/reperfusion injury and also document that preconditioning is not as effective as found in WT and therefore suggest the importance of VEGF/Flt-1 signaling in ischemic/reperfused myocardium.     

Ceramide in the antiapoptotic effect of ischemic preconditioning

Although the mechanism by which ischemic preconditioning (PC) inhibits myocardial apoptosis during ischemia-reperfusion is unclear, evidence indicates a role for the secondary messenger ceramide. We investigated in vivo whether PC may affect ceramide and sn-1,2-diacylglycerol (DAG) production, and attenuate apoptosis during ischemia. Rabbits underwent 30 min of ischemia, followed by 4 h of reperfusion. Before this, they received either no intervention (control group) or one episode of 5 min of ischemia, followed by 5 min of reperfusion (PC group), or an intravenous administration of the sphingomyelinase inhibitor D609. Myocardial content of ceramide and DAG was measured using the DAG kinase assay at different time points of the experiment. Apoptosis was detected and quantified by a sandwich enzyme immunoassay. Both AR and infarct size were measured using blue dye injection and triphenyltetrazolium chloride staining. Control hearts exhibited a peak of ceramide production at 5 min of the prolonged ischemia, with a mean value averaging 64 +/- 5 ng/mg tissue (P < 0.05 vs. 48 +/- 4 ng/mg at baseline). In contrast, ischemic PC and D609 prevented ceramide increase during the prolonged ischemia. Myocardial DAG content was increased only in PC hearts at 30 min of ischemia. Preconditioned and D609 groups developed less apoptosis, as well as a limited infarct size, compared with the control group. These results suggest that the antiapoptotic effect of PC may be due to a reduced ceramide production during sustained ischemia in the rabbit heart.