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.     

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.     

Ischemic preconditioning exaggerates cardiac damage in PKC-delta null mice

Ischemic preconditioning confers cardiac protection during subsequent ischemia-reperfusion, in which protein kinase C (PKC) is believed to play an essential role, but controversial data exist concerning the PKC-delta isoform. In an accompanying study (26), we described metabolic changes in PKC-delta knockout mice. We now wanted to explore their effect on early preconditioning. Both PKC-delta(-/-) and PKC-delta(+/+) mice underwent three cycles of 5-min left descending artery occlusion/5-min reperfusion, followed by 30-min occlusion and 2-h reperfusion. Unexpectedly, preconditioning exaggerated ischemia-reperfusion injury in PKC-delta(-/-) mice. Whereas ischemic preconditioning increased superoxide anion production in PKC-delta(+/+) hearts, no increase in reactive oxygen species was observed in PKC-delta(-/-) hearts. Proteomic analysis of preconditioned PKC-delta(+/+) hearts revealed profound changes in enzymes related to energy metabolism, e.g., NADH dehydrogenase and ATP synthase, with partial fragmentation of these mitochondrial enzymes and of the E(2) component of the pyruvate dehydrogenase complex. Interestingly, fragmentation of mitochondrial enzymes was not observed in PKC-delta(-/-) hearts. High-resolution NMR analysis of cardiac metabolites demonstrated a similar rise of phosphocreatine in PKC-delta(+/+) and PKC-delta(-/-) hearts, but the preconditioning-induced increase in phosphocholine, alanine, carnitine, and glycine was restricted to PKC-delta(+/+) hearts, whereas lactate concentrations were higher in PKC-delta(-/-) hearts. Taken together, our results suggest that reactive oxygen species generated during ischemic preconditioning might alter mitochondrial metabolism by oxidizing key mitochondrial enzymes and that metabolic adaptation to preconditioning is impaired in PKC-delta(-/-) hearts.     

Ischemic postconditioning during reperfusion activates Akt and ERK without protecting against lethal myocardial ischemia-reperfusion injury in pigs

Transient episodes of ischemic preconditioning (PC) render myocardium protected against subsequent lethal injury after ischemia and reperfusion. Recent studies indicate that application of short, repetitive ischemia only during the onset of reperfusion after the lethal ischemic event may obtain equivalent protection. We assessed whether such ischemic postconditioning (Postcon) is cardioprotective in pigs by limiting lethal injury. Pentobarbital sodium-anesthetized, open-chest pigs underwent 30 min of complete occlusion of the left anterior descending coronary artery and 3-h reflow. PC was elicited by two cycles of 5-min occlusion plus 10-min reperfusion before the 30-min occlusion period. Postcon was elicited by three cycles of 30-s reperfusion, followed by 30-s reocclusion, after the 30-min occlusion period and before the 3-h reflow. Infarct size (%area-at-risk using triphenyltetrazolium chloride macrochemistry; means +/- SE) after 30 min of ischemia was 26.5 +/- 5.2% (n = 7 hearts/treatment group). PC markedly limited myocardial infarct size (2.8 +/- 1.2%, n = 7 hearts/treatment group, P < 0.05 vs. controls). However, Postcon had no effect on infarct size (37.8 +/- 5.1%, n = 7 hearts/treatment group). Within the subendocardium, Postcon increased phosphorylation of Akt (74 +/- 12%) and ERK1/2 (56 +/- 10%) compared with control hearts subjected only to 30-min occlusion and 15-min reperfusion (P < or = 0.05), and these changes were not different from the response triggered by PC (n = 5 hearts/treatment group). Phosphorylation of downstream p70S6K was also equivalent in PC and Postcon groups. These data do not support the hypothesis that application of 30-s cycles of repetitive ischemia during reperfusion exerts a protective effect on pig hearts subjected to lethal ischemia, but this is not due to a failure to phosphorylate ERK and Akt during early reperfusion.     

Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α(2)-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with dexmedetomidine before ischemia. The α(2)-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α(2)-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning (in vivo model) and peri-insult protection (ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α(2)-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α(2)-adrenergic receptor stimulation.     

Equal reduction in infarct size by ethylisopropyl-amiloride pretreatment and ischemic preconditioning in the in situ rabbit heart

Inhibition of Na⁺/H⁺ exchange with amiloride analogues has been shown to provide functional protection during ischemia and reperfusion and to reduce infarct size in isolated rat hearts. In rat hearts, treatment with ethylisopropyl-amiloride (EIPA, a selective Na⁺/H⁺ exchange inhibitor) was additive to the protection afforded by ischemic preconditioning. In addition, such compounds have been demonstrated to reduce infarct size in in situ rabbit hearts. The aim of the present study was to determine to what extent preischemic treatment with EIPA could reduce infarct size in an in situ rabbit model of regional ischemia and reperfusion. We also wished to determine if this effect was additive to the infarct reducing effect of ischemic preconditioning. Anaesthetized, open chest rabbits, were subjected to 45 min of regional ischemia and 150 min of reperfusion. The risk zone was determined by fluorescent particles and infarct size was determined by TTC staining. Four groups were investigated: control, ischemic preconditioned (IP) (5 min of ischemia followed by 10 min reperfusion), EIPA (0.65 mg/kg iv given preischemically) and EIPA + IP. The main results expressed as percent infarction of the risk zone ± S.E.M. for the different groups were: control 59.2 ± 3.3% (n = 6), IP 16.3 ± 2.1% (n = 6) (p < 0.001 vs. control), EIPA 16.9 ± 4.1% (n = 5) (p < 0.001 vs. control), EIPA + IP 22.5 ± 9.5% (n = 6) (p < 0.001 vs. control). In conclusion: EIPA, when administered prior to ischemia, caused a reduction in infarct size in the in situ rabbit heart which was similar to that seen with ischemic preconditioning, however, the effect was not additive to ischemic preconditioning.     

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.     

STAT subtype specificity and ischemic preconditioning in mice: is STAT-3 enough?

The role of other STAT subtypes in conferring ischemic tolerance is unclear. We hypothesized that in STAT-3 deletion alternative STAT subtypes would protect myocardial function against ischemia-reperfusion injury. Wild-type (WT) male C57BL/6 mice or mice with cardiomyocyte STAT-3 knockout (KO) underwent baseline echocardiography. Langendorff-perfused hearts underwent ischemic preconditioning (IPC) or no IPC before ischemia-reperfusion. Following ex vivo perfusion, hearts were analyzed for STAT-5 and -6 phosphorylation by Western blot analysis of nuclear fractions. Echocardiography and postequilibration cardiac performance revealed no differences in cardiac function between WT and KO hearts. Phosphorylated STAT-5 and -6 expression was similar in WT and KO hearts before perfusion. Contractile function in WT and KO hearts was significantly impaired following ischemia-reperfusion in the absence of IPC. In WT hearts, IPC significantly improved the recovery of the maximum first derivative of developed pressure (+dP/dtmax) compared with that in hearts without IPC. IPC more effectively improved end-reperfusion dP/dtmax in WT hearts compared with KO hearts. Preconditioned and nonpreconditioned KO hearts exhibited increased phosphorylated STAT-5 and -6 expression compared with WT hearts. The increased subtype activation did not improve the efficacy of IPC in KO hearts. In conclusion, baseline cardiac performance is preserved in hearts with cardiac-restricted STAT-3 deletion. STAT-3 deletion attenuates preconditioning and is not associated with a compensatory upregulation of STAT-5 and -6 subtypes. The activation of STAT-5 and -6 in KO hearts following ischemic challenge does not provide functional compensation for the loss of STAT-3. JAK-STAT signaling via STAT-3 is essential for effective IPC.     

TRPV1 gene knockout impairs preconditioning protection against myocardial injury in isolated perfused hearts in mice

Although the transient receptor potential vanilloid type 1 (TRPV1)-containing afferent nerve fibers are widely distributed in the heart, the relationship between TRPV1 function and cardiac ischemic preconditioning (PC) has not been well defined. Using TRPV1 knockout mice (TRPV1(-/-)), we studied the role of TRPV1 in PC-induced myocardial protection. Hearts of gene-targeted TRPV1-null mutant (TRPV1(-/-)) or wild-type (WT) mice were Langendorffly perfused in the presence or absence of CGRP(8-37), a selective calcitonin gene-related peptide (CGRP) receptor antagonist; or RP-67580, a selective neurokinin-1 receptor antagonist when hearts were subjected to three 5-min periods of ischemia PC followed by 30 min of global ischemia and 40 min of reperfusion (I/R). PC before I/R decreased left ventricular (LV) end-diastolic pressure and increased LV developed pressure, coronary flow (CF), peak-positive maximum rate of rise of LV pressure in WT mice (PC-WT) compared with PC-TRPV1(-/-), TRPV1(-/-), or WT hearts (P < 0.05), and PC also decreased LV end-diastolic pressure in PC-TRPV1(-/-) compared with TRPV1(-/-). CGRP(8-37) or RP-67580 abolished PC-induced protection in WT but not TRPV1(-/-) hearts (P < 0.05). Moreover, PC decreased lactate dehydrogenase release and infarct size in PC-WT compared with PC-TRPV1(-/-), TRPV1(-/-), or WT hearts, and it also lowered these parameters in PC-TRPV1(-/-) compared with TRPV1(-/-) hearts (P < 0.05). Radioimmunoassay showed that the release of substance P and CGRP after PC was higher in WT hearts than in TRPV1(-/-) hearts (P < 0.05), which was attenuated by capsazepine in WT but not TRPV1(-/-) hearts. Thus PC-induced protection of the heart was impaired in TRPV1(-/-) hearts, indicating that TRPV1 contributes to the beneficial effects of preconditioning against I/R injury through release substance P and CGRP.     

Role of Na+-Ca2+ exchanger in myocardial ischemia/reperfusion injury: evaluation using a heterozygous Na+-Ca2+ exchanger knockout mouse model

We used Na(+)-Ca(2+) exchanger (NCX) knockout mice to evaluate the effects of NCX in cardiac function and the infarct size after ischemia/reperfusion injury. The contractile function in NCX KO mice hearts was significantly better than that in wild type (WT) mice hearts after ischemia/reperfusion and the infarct size was significantly small in NCX KO mice hearts compared with that in WT mice hearts. NCX is critically involved in the development of ischemia/reperfusion-induced myocardial injury and therefore the inhibition of NCX function may contribute to cardioprotection against ischemia/reperfusion injury.     

Transgenic MMP-2 expression induces latent cardiac mitochondrial dysfunction

Matrix metalloproteinases (MMPs) are central to the development and progression of dysfunctional ventricular remodeling after tissue injury. We studied 6 month old heterozygous mice with cardiac-specific transgenic expression of active MMP-2 (MMP-2 Tg). MMP-2 Tg hearts showed no substantial gross alteration of cardiac phenotype compared to age-matched wild-type littermates. However, buffer perfused MMP-2 Tg hearts subjected to 30 min of global ischemia followed by 30 min of reperfusion had a larger infarct size and greater depression in contractile performance compared to wild-type hearts. Importantly, cardioprotection mediated by ischemic preconditioning (IPC) was completely abolished in MMP-2 Tg hearts, as shown by abnormalities in mitochondrial ultrastructure and impaired respiration, increased lipid peroxidation, cell necrosis and persistently reduced recovery of contractile performance during post-ischemic reperfusion. We conclude that MMP-2 functions not only as a proteolytic enzyme but also as a previously unrecognized active negative regulator of mitochondrial function during superimposed oxidative stress.     

Influence of ischemic preconditioning on intracellular sodium,pH, and cellular energy status in isolated perfused heart

The possible relationships between intracellular Na(+) (Na(i)(+)), bioenergetic status and intracellular pH (pH(i)) in the mechanism for ischemic preconditioning were studied using (23)Na and (31)P magnetic resonance spectroscopy in isolated Langendorff perfused rat heart. The ischemic preconditioning (three 5-min ischemic episodes followed by two 5-min and one 10-min period of reperfusion) prior to prolonged ischemia (20 min stop-flow) resulted in a decrease in ischemic acidosis and faster and complete recovery of cardiac function (ventricular developed pressure and heart rate) after 30 min of reperfusion. The response of Na(i) during ischemia in the preconditioned hearts was characterized by an increase in Na(i)(+) at the end of preconditioning and an accelerated decrease during the first few minutes of reperfusion. During post-ischemic reperfusion, bioenergetic parameters (PCr/P(i) and betaATP/P(i) ratios) were partly recovered without any significant difference between control and preconditioned hearts. The reduced acidosis during prolonged ischemia and the accelerated decrease in Na(i)(+) during reperfusion in the preconditioned hearts suggest activation of Na(+)/H(+) exchanger and other ion transport systems during preconditioning, which may protect the heart from intracellular acidosis during prolonged ischemia, and result in better recovery of mechanical function (LVDP and heart rate) during post-ischemic reperfusion.     

Extracellular Adenosine Formation by Ecto-5’-Nucleotidase (CD73) Is No Essential Trigger for Early Phase Ischemic Preconditioning

Background

Adenosine is a powerful trigger for ischemic preconditioning (IPC). Myocardial ischemia induces intracellular and extracellular ATP degradation to adenosine, which then activates adenosine receptors and elicits cardioprotection. Conventionally extracellular adenosine formation by ecto-5’-nucleotidase (CD73) during ischemia was thought to be negligible compared to the massive intracellular production, but controversial reports in the past demand further evaluation. In this study we evaluated the relevance of ecto-5’-nucleotidase (CD73) for infarct size reduction by ischemic preconditioning in in vitro and in vivo mouse models of myocardial infarction, comparing CD73^-/- and wild type (WT) mice.

Methods and Results

3x5 minutes of IPC induced equal cardioprotection in isolated saline perfused hearts of wild type (WT) and CD73^-/- mice, reducing control infarct sizes after 20 minutes of ischemia and 90 minutes of reperfusion from 46 ± 6.3% (WT) and 56.1 ± 7.6% (CD73^-/-) to 26.8 ± 4.7% (WT) and 25.6 ± 4.7% (CD73^-/-). Coronary venous adenosine levels measured after IPC stimuli by high-pressure liquid chromatography showed no differences between WT and CD73^-/- hearts. Pharmacological preconditioning of WT hearts with adenosine, given at the measured venous concentration, was evenly cardioprotective as conventional IPC. In vivo, 4x5 minutes of IPC reduced control infarct sizes of 45.3 ± 8.9% (WT) and 40.5 ± 8% (CD73^-/-) to 26.3 ± 8% (WT) and 22.6 ± 6.6% (CD73^-/-) respectively, eliciting again equal cardioprotection. The extent of IPC-induced cardioprotection in male and female mice was identical.

Conclusion

The infarct size limiting effects of IPC in the mouse heart in vitro and in vivo are not significantly affected by genetic inactivation of CD73. The ecto-5’-nucleotidase derived extracellular formation of adenosine does not contribute substantially to adenosine’s well known cardioprotective effect in early phase ischemic preconditioning.     

Ischemic and anesthetic preconditioning reduces cytosolic [Ca2+] and improves Ca(2+) responses in intact hearts

Ca(+) loading during reperfusion after myocardial ischemia is linked to reduced cardiac function. Like ischemic preconditioning (IPC), a volatile anesthetic given briefly before ischemia can reduce reperfusion injury. We determined whether IPC and sevoflurane preconditioning (SPC) before ischemia equivalently improve mechanical and metabolic function, reduce cytosolic Ca(2+) loading, and improve myocardial Ca(2+) responsiveness. Four groups of guinea pig isolated hearts were perfused: no ischemia, no treatment before 30-min global ischemia and 60-min reperfusion (control), IPC (two 2-min occlusions) before ischemia, and SPC (3.5 vol%, two 2-min exposures) before ischemia. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured at the left ventricular (LV) free wall with the fluorescent probe indo 1. Ca(2+) responsiveness was assessed by changing extracellular [Ca(2+)]. In control hearts, initial reperfusion increased diastolic [Ca(2+)] and diastolic LV pressure (LVP), and the maximal and minimal derivatives of LVP (dLVP/dt(max) and dLVP/dt(min), respectively), O(2) consumption, and cardiac efficiency (CE). Throughout reperfusion, IPC and SPC similarly reduced ischemic contracture, ventricular fibrillation, and enzyme release, attenuated rises in systolic and diastolic [Ca(2+)], improved contractile and relaxation indexes, O(2) consumption, and CE, and reduced infarct size. Diastolic [Ca(2+)] at 50% dLVP/dt(min) was right shifted by 32-53 +/- 8 nM after 30-min reperfusion for all groups. Phasic [Ca(2+)] at 50% dLVP/dt(max) was not altered in control but was left shifted by -235 +/- 40 nM [Ca(2+)] after IPC and by -135 +/- 20 nM [Ca(2+)] after SPC. Both SPC and IPC similarly reduce Ca(2+) loading, while augmenting contractile responsiveness to Ca(2+), improving postischemia cardiac function and attenuating permanent damage.     

Cardiac mTOR protects the heart against ischemia-reperfusion injury

Cardiac mammalian target of rapamycin (mTOR) is necessary and sufficient to prevent cardiac dysfunction in pathological hypertrophy. However, the role of cardiac mTOR in heart failure after ischemic injury remains undefined. To address this question, we used transgenic (Tg) mice with cardiac-specific overexpression of mTOR (mTOR-Tg mice) to study ischemia-reperfusion (I/R) injury in two animal models: 1) in vivo I/R injury with transient coronary artery ligation and 2) ex vivo I/R injury in Langendorff-perfused hearts with transient global ischemia. At 28 days after I/R, mortality was lower in mTOR-Tg mice than littermate control mice [wild-type (WT) mice]. Echocardiography and MRI demonstrated that global cardiac function in mTOR-Tg mice was preserved, whereas WT mice exhibited significant cardiac dysfunction. Masson's trichrome staining showed that 28 days after I/R, the area of interstitial fibrosis was smaller in mTOR-Tg mice compared with WT mice, suggesting that adverse left ventricular remodeling is inhibited in mTOR-Tg mice. In the ex vivo I/R model, mTOR-Tg hearts demonstrated improved functional recovery compared with WT hearts. Perfusion with Evans blue after ex vivo I/R yielded less staining in mTOR-Tg hearts than WT hearts, indicating that mTOR overexpression inhibited necrosis during I/R injury. Expression of proinflammatory cytokines, including IL-6 and TNF-α, in mTOR-Tg hearts was lower than in WT hearts. Consistent with this, IL-6 in the effluent post-I/R injury was lower in mTOR-Tg hearts than in WT hearts. These findings suggest that cardiac mTOR overexpression in the heart is sufficient to provide substantial cardioprotection against I/R injury and suppress the inflammatory response.     

Cardiac preconditioning with 4-h, 17 degrees C ischemia reduces [Ca(2+)](i) load and damage in part via K(ATP) channel opening

Brief ischemia before normothermic ischemia protects hearts against reperfusion injury (ischemic preconditioning, IPC), but it is unclear whether it protects against long-term moderate hypothermic ischemia. We explored in isolated guinea pig hearts 1) the influence of two 2-min periods of normothermic ischemia before 4 h, 17 degrees C hypothermic ischemia on cardiac cytosolic [Ca(2+)], mechanical and metabolic function, and infarct size, and 2) the potential role of K(ATP) channels in eliciting cardioprotection. We found that IPC before 4 h moderate hypothermia improved myocardial perfusion, contractility, and relaxation during normothermic reperfusion. Protection was associated with markedly reduced diastolic [Ca(2+)] loading throughout both hypothermic storage and reperfusion. Global infarct size was markedly reduced from 36 +/- 2 (SE)% to 15 +/- 1% with IPC. Bracketing ischemic pulses with 200 microM 5-hydroxydecanoic acid or 10 microM glibenclamide increased infarct size to 28 +/- 3% and 26 +/- 4%, respectively. These results suggest that brief ischemia before long-term hypothermic storage adds to the cardioprotective effects of hypothermia and that this is associated with decreased cytosolic [Ca(2+)] loading and enhanced ATP-sensitive K channel opening.     

Novel role of NADPH oxidase in ischemic myocardium: a study with Nox2 knockout mice

Several potential sources of reactive oxygen species (ROS) in cells exist. One source is NADPH oxidase, which is especially important for superoxide radical production. Nox2 is a primary regulatory subunit of NADPH oxidase. In the present study, we examined the role of ROS and NADPH oxidase in ischemic preconditioning (IP)-mediated cardioprotection by using Nox2(-/-) mice. Both wild-type (WT) and Nox2(-/-) mice were subjected to either 30?min of ischemia followed by 2?h of reperfusion (IR) or IP prior to 30?min ischemia and 2?h of reperfusion. Reduction in left ventricular developed pressure (60.1 versus 63?mmHg), dp/dt (max) (893 versus 1,027?mmHg/s), and aortic flow (0.9 versus 1.8?ml/min) was observed in Nox2(-/-)IPIR compared to WTIPIR along with increased infarct size (33% versus 22%) and apoptosis after 120?min of reperfusion. Differentially regulated genes were demonstrated by comparing gene expression in WTIPIR versus Nox2(-/-) IPIR hearts. Selected differentially regulated genes such as β-catenin, SRPK3, ERDR1, ACIN1, Syntaxin-8, and STC1 were validated by real-time PCR. Taken together, this is the first report identifying important, differentially expressed genes during ischemic preconditioning in Nox2(-/-) mice by using microarray analysis.     

Effect of age on alpha-calcitonin gene-related peptide-mediated delayed cardioprotection induced by intestinal preconditioning in rats

In the present study, we examined whether age-related reduction in cardioprotection of intestinal ischemic preconditioning is related to stimulation of the release and synthesis of calcitonin gene-related peptide (CGRP) in rats. Ischemia-reperfusion injury was induced by a 45-min coronary artery occlusion and 180-min reperfusion, and ischemic preconditioning was induced by six cycles of 4-min ischemia and 4-min reperfusion of the small intestine. The serum concentration of creatine kinase, infarct size, the expression of CGRP isoforms (alpha- and beta-CGRP) mRNA in lumbar dorsal root ganglia and CGRP concentration in plasma were measured. Pretreatment with intestinal ischemic preconditioning for 24 h significantly reduced infarct size and creatine kinase release concomitantly with a significant increase in the expression of alpha-CGRP mRNA, but not beta-CGRP mRNA, and plasma concentrations of CGRP at 6 months of age but not at 24 months of age. These results suggest that the delayed cardioprotective effect of intestinal ischemic preconditioning is decreased in senescent rats, and the age-related change is related to reduction of the synthesis and release of alpha-CGRP.