Low-Dose Endotoxin Induces Late Preconditioning, Increases Peroxynitrite Formation, and Activates STAT3 in the Rat Heart.

Administration of low-dose endotoxin (lipopolysaccharide, LPS) 24 h before a lethal ischemia induces pharmacological late preconditioning. The exact mechanism of this phenomenon is not clear. Here we aimed to investigate whether low-dose LPS exerts late effects on peroxynitrite formation and activation of Akt, Erk, and STAT3 in the heart. Male Wistar rats were injected with LPS (S. typhimurium; 0.5 mg/kg i.p.) or saline. Twenty-four hours later, hearts were isolated, perfused for 10 min, and then used for biochemical analyses. LPS pretreatment enhanced cardiac formation of the peroxynitrite marker 3-nitrotyrosine. LPS pretreatment also increased cardiac levels of the peroxynitrite precursor nitric oxide (NO) and superoxide. The activities of Ca2+-independent NO synthase and xanthine oxidoreductase increased in LPS-pretreated hearts. LPS pretreatment resulted in significantly enhanced phosphorylation of STAT3 and non-significantly increased phosphorylation of Akt without affecting the activation of Erk. In separate experiments, isolated working hearts were subjected to 30 min global ischemia and 20 min reperfusion. LPS pretreatment significantly improved ischemia-reperfusion-induced deterioration of cardiac function. We conclude that LPS pretreatment enhances cardiac peroxynitrite formation and activates STAT3 24 h later, which may contribute to LPS-induced late preconditioning.

Novel, selective EPO receptor ligands lacking erythropoietic activity reduce infarct size in acute myocardial infarction in rats.

Erythropoietin (EPO) has been shown to protect the heart against acute myocardial infarction in pre-clinical studies, however, EPO failed to reduce infarct size in clinical trials and showed significant safety problems. Here, we investigated cardioprotective effects of two selective non-erythropoietic EPO receptor ligand dimeric peptides (AF41676 and AF43136) lacking erythropoietic activity, EPO, and the prolonged half-life EPO analogue, darbepoetin in acute myocardial infarction (AMI) in rats. In a pilot study, EPO at 100U/mL significantly decreased cell death compared to vehicle (33.8±2.3% vs. 40.3±1.5%, p<0.05) in rat neonatal cardiomyocytes subjected to simulated ischemia/reperfusion. In further studies (studies 1-4), in vivo AMI was induced by 30min coronary occlusion and 120min reperfusion in male Wistar rats. Test compounds and positive controls for model validation (B-type natriuretic peptide, BNP or cyclosporine A, CsA) were administered iv. before the onset of reperfusion. Infarct size (IS) was measured by standard TTC staining. In study 1, 5000U/kg EPO reduced infarct size significantly compared to vehicle (45.3±4.8% vs. 59.8±4.5%, p<0.05). In study 2, darbepoetin showed a U-shaped dose-response curve with maximal infarct size-reducing effect at 5µg/kg compared to the vehicle (44.4±5.7% vs. 65.9±2.7%, p<0.01). In study 3, AF41676 showed a U-shaped dose-response curve, where 3mg/kg was the most effective dose compared to the vehicle (24.1±3.9% vs. 44.3±2.5%, p<0.001). The positive control BNP significantly decreased infarct size in studies 1-3 by approximately 35%. In study 4, AF43136 at 10mg/kg decreased infarct size, similarly to the positive control CsA compared to the appropriate vehicle (39.4±5.9% vs. 58.1±5.4% and 45.9±2.4% vs. 63.8±4.1%, p<0.05, respectively). This is the first demonstration that selective, non-erythropoietic EPO receptor ligand dimeric peptides AF41676 and AF43136 administered before reperfusion are able to reduce infarct size in a rat model of AMI. Therefore, non-erythropoietic EPO receptor peptide ligands may be promising cardioprotective agents.

MicroRNAs associated with ischemia-reperfusion injury and cardioprotection by ischemic pre- and postconditioning: protectomiRs.

We aimed to characterize early changes in microRNA expression in acute cardioprotection by ischemic pre- and postconditioning in rat hearts. Hearts isolated from male Wistar rats were subjected to 1) time-matched nonischemic perfusion, 2) ischemia-reperfusion (30 min of coronary occlusion and 120 min of reperfusion), 3) preconditioning (3 × 5 min of coronary occlusion) followed by ischemia-reperfusion, or 4) ischemia-reperfusion with postconditioning (6 × 10 s of global ischemia-reperfusion at the onset of reperfusion). Infarct size was significantly reduced by both interventions. Of 350 different microRNAs assessed by microarray analysis, 147-160 microRNAs showed detectable expression levels. Compared with microRNA alterations induced by ischemia-reperfusion versus time-matched nonischemic controls, five microRNAs were significantly affected by both pre- and postconditioning (microRNA-125b*, microRNA-139-3p, microRNA-320, microRNA-532-3p, and microRNA-188), four microRNAs were significantly affected by preconditioning (microRNA-487b, microRNA-139-5p, microRNA-192, and microRNA-212), and nine microRNAs were significantly affected by postconditioning (microRNA-1, microRNA let-7i, microRNA let-7e, microRNA let-7b, microRNA-181a, microRNA-208, microRNA-328, microRNA-335, and microRNA-503). Expression of randomly selected microRNAs was validated by quantitative real-time PCR. By a systematic comparison of the direction of microRNA expression changes in all groups, we identified microRNAs, specific mimics, or antagomiRs that may have pre- and postconditioning-like cardioprotective effects (protectomiRs). Transfection of selected protectomiRs (mimics of microRNA-139-5p, microRNA-125b*, microRNA let-7b, and inhibitor of microRNA-487b) into cardiac myocytes subjected to simulated ischemia-reperfusion showed a significant cytoprotective effect. This is the first demonstration that the ischemia-reperfusion-induced microRNA expression profile is significantly influenced by both pre- and postconditioning, which shows the involvement of microRNAs in cardioprotective signaling. Moreover, by analysis of microRNA expression patterns in cardioprotection by pre- and postconditioning, specific protectomiRs can be revealed as potential therapeutic tools for the treatment of ischemia-reperfusion injury.

Moderate inhibition of myocardial matrix metalloproteinase-2 by ilomastat is cardioprotective.

Pharmacological inhibition of matrix metalloproteinase-2 (MMP-2) is a promising target for acute cardioprotection against ischemia/reperfusion injury. Therefore, here we investigated if the MMP inhibitor ilomastat administered either before ischemia or before reperfusion is able to reduce infarct size via inhibition of MMP-2, the most abundant MMP in the rat heart. Infarct-size limiting effect of ilomastat (0.3-6.0µmol/kg) was tested in an in vivo rat model of myocardial infarction induced by 30min coronary occlusion/120min reperfusion. Ilomastat at 0.75 and 1.5µmol/kg decreased infarct size significantly as compared to the vehicle-treated (dimethyl sulfoxide) group (from 66.1±4.6% to 45.3±7.0% and 46.7±5.5% of area at risk, p<0.0.5, respectively), when administered 5min before the onset of ischemia. Ilomastat at 6.0µmol/kg significantly reduced infarct size from its control value of 65.4±2.5% to 52.8±3.7% of area at risk (p<0.05), when administered 5min before the onset of reperfusion. Area at risk was not significantly affected by ilomastat treatments. To further assess the cytoprotective effect of ilomastat, primary cardiomyocytes isolated from neonatal rats were subjected to 240min simulated ischemia followed by 120min simulated reperfusion in the presence of ilomastat (5nM-5µM). Ilomastat at 500nM and 5µM significantly increased cell viability when compared to vehicle treated group. To assess the in situ MMP-2 inhibitory effect of ilomastat, in separate experiments in situ zymography was performed in cardiomyocytes. The cytoprotective concentration of ilomastat (500nM) showed a moderate (approximately 25%) inhibition of intracellular MMP-2 in ischemic/reperfused cardiomyocytes. In these cells, MMP-2 immunostaining showed a 90% colocalization with the in situ gelatinolytic activity. We conclude that the MMP inhibitor ilomastat reduces infarct size when administered either before the onset of ischemia or before the onset of reperfusion in vivo. Furthermore, this is the first demonstration that a moderate inhibition of intracellular MMP-2 is sufficient to confer cardiocytoprotection.

Metabolic syndrome influences cardiac gene expression pattern at the transcript level in male ZDF rats.

BACKGROUND: Metabolic syndrome (coexisting visceral obesity, dyslipidemia, hyperglycemia, and hypertension) is a prominent risk factor for cardiovascular morbidity and mortality, however, its effect on cardiac gene expression pattern is unclear. Therefore, we examined the possible alterations in cardiac gene expression pattern in male Zucker Diabetic Fatty (ZDF) rats, a model of metabolic syndrome. METHODS: Fasting blood glucose, serum insulin, cholesterol and triglyceride levels were measured at 6, 16, and 25 wk of age in male ZDF and lean control rats. Oral glucose tolerance test was performed at 16 and 25 wk of age. At week 25, total RNA was isolated from the myocardium and assayed by rat oligonucleotide microarray for 14921 genes. Expression of selected genes was confirmed by qRT-PCR. RESULTS: Fasting blood glucose, serum insulin, cholesterol and triglyceride levels were significantly increased, glucose tolerance and insulin sensitivity were impaired in ZDF rats compared to leans. In hearts of ZDF rats, 36 genes showed significant up-regulation and 49 genes showed down-regulation as compared to lean controls. Genes with significantly altered expression in the heart due to metabolic syndrome includes functional clusters of metabolism (e.g. 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2; argininosuccinate synthetase; 2-amino-3-ketobutyrate-coenzyme A ligase), structural proteins (e.g. myosin IXA; aggrecan1), signal transduction (e.g. activating transcription factor 3; phospholipase A2; insulin responsive sequence DNA binding protein-1) stress response (e.g. heat shock 70kD protein 1A; heat shock protein 60; glutathione S-transferase Yc2 subunit), ion channels and receptors (e.g. ATPase, (Na+)/K+ transporting, beta 4 polypeptide; ATPase, H+/K+ transporting, nongastric, alpha polypeptide). Moreover some other genes with no definite functional clusters were also changed such as e.g. S100 calcium binding protein A3; ubiquitin carboxy-terminal hydrolase L1; interleukin 18. Gene ontology analysis revealed several significantly enriched functional inter-relationships between genes influenced by metabolic syndrome. CONCLUSIONS: Metabolic syndrome significantly alters cardiac gene expression profile which may be involved in development of cardiac pathologies in the presence of metabolic syndrome.

Cardioprotection by farnesol: role of the mevalonate pathway.

PURPOSE: Farnesol is a key metabolite of the mevalonate pathway and known as an antioxidant. We examined whether farnesol treatment protects the ischemic heart. METHODS: Male Wistar rats were treated orally with 0.2, 1, 5, and 50 mg/kg/day farnesol/vehicle for 12 days, respectively. On day 13, the effect of farnesol treatment on cardiac ischemic tolerance and biochemical changes was tested. Therefore, hearts were isolated and subjected either to 30 min coronary occlusion followed by 120 min reperfusion to measure infarct size or to 10 min aerobic perfusion to measure cardiac mevalonate pathway end-products (protein prenylation, cholesterol, coenzyme Q9, coenzyme Q10, dolichol), and 3-nitrotyrosine (oxidative/nitrosative stress marker), respectively. The cytoprotective effect of farnesol was also tested in cardiomyocytes subjected to simulated ischemia/reperfusion. RESULTS: Farnesol pretreatment decreased infarct size in a U-shaped dose-response manner where 1 mg/kg/day dose reached a statistically significant reduction (22.3±3.9% vs. 40.9±6.1% of the area at risk, p<0.05). Farnesol showed a similar cytoprotection in cardiomyocytes. The cardioprotective dose of farnesol (1 mg/kg/day) significantly increased the marker of protein geranylgeranylation, but did not influence protein farnesylation, cardiac tissue cholesterol, coenzyme Q9, coenzyme Q10, and dolichol. While the cardioprotective dose of farnesol did not influence 3-nitrotyrosine, the highest dose of farnesol (50 mg/kg/day) tested did not show cardioprotection, however, it significantly decreased cardiac 3-nitrotyrosine. CONCLUSIONS: This is the first demonstration that oral farnesol treatment reduces infarct size. The cardioprotective effect of farnesol likely involves increased protein geranylgeranylation and seems to be independent of the antioxidant effect of farnesol.

Different administration schedules of darbepoetin alfa affect oxidized and reduced glutathione levels to a similar extent in 5/6 nephrectomized rats.

BACKGROUND: The development of erythropoiesis-stimulating agents (ESAs) with extended serum half-lives has allowed marked prolongation of the administration intervals. The level of oxidative stress is increased in chronic kidney disease, and is reportedly decreased after long-term ESA treatment. However, the effect of different dosing regimens of ESAs on oxidative stress has not been elucidated. METHODS: Five-sixths nephrectomized (NX) rats received either 0.4 µg/kg darbepoetin alfa (DA) weekly or 0.8 µg/kg DA fortnightly between weeks 4 and 10. NX animals receiving saline and a sham-operated (SHAM) group served as controls. The levels of oxidized and reduced glutathione (GSSG, GSH) were followed from blood samples drawn fortnightly. RESULTS: During the follow-up, the ratios GSSG/GSH showed similar trends in both DA groups, levels being significantly lower than those in the SHAM group at weeks 8 and 10. GSSG levels were lower than the baseline throughout the study in all groups except for NX controls. The GSH levels were increased in all three NX groups (weeks 6-10) compared with both the baseline and the SHAM group CONCLUSION: Our results suggest that the extent of oxidative stress is similar in response to different dosing regimens of DA in 5/6 NX rats when comparable hemoglobin levels are maintained. These findings remain to be confirmed in chronic kidney disease patients.

Preconditioning protects the heart in a prolonged uremic condition.

Metabolic diseases such as hyperlipidemia and diabetes attenuate the cardioprotective effect of ischemic preconditioning. In the present study, we examined whether another metabolic disease, prolonged uremia, affects ischemia/reperfusion injury and cardioprotection by ischemic preconditioning. Uremia was induced by partial nephrectomy in male Wistar rats. The development of uremia was verified 29 wk after surgery. Transthoracic echocardiography was performed to monitor cardiac function. At week 30, hearts of nephrectomized and sham-operated rats were isolated and subjected to a 30-min coronary occlusion followed by 120 min reperfusion with or without preceding preconditioning induced by three intermittent cycles of brief ischemia and reperfusion. In nephrectomized rats, plasma uric acid, carbamide, and creatinine as well as urine protein levels were increased as compared with sham-operated controls. Systolic anterior and septal wall thicknesses were increased in nephrectomized rats, suggesting the development of a minimal cardiac hypertrophy. Ejection fraction was decreased and isovolumic relaxation time was shortened in nephrectomized rats demonstrating a mild systolic and diastolic dysfunction. Infarct size was not affected significantly by nephrectomy itself. Ischemic preconditioning significantly decreased infarct size from 24.8 ± 5.2% to 6.6 ± 1.3% in the sham-operated group and also in the uremic group from 35.4 ± 9.5% to 11.9 ± 3.1% of the area at risk. Plasma ANG II and nitrotyrosine were significantly increased in the uremic rats. We conclude that although prolonged experimental uremia leads to severe metabolic changes and the development of a mild myocardial dysfunction, the cardioprotective effect of ischemic preconditioning is still preserved.

Cholesterol diet leads to attenuation of ischemic preconditioning-induced cardiac protection: the role of connexin 43.

Cardioprotection by ischemic preconditioning (IP) was abolished in connexin 43 (Cx43)-deficient mice due to loss of Cx43 located in mitochondria rather than at the sarcolemma. IP is lost in hyperlipidemic rat hearts as well. Since changes in mitochondrial Cx43 in hyperlipidemia have not yet been analyzed, we determined total and mitochondrial Cx43 levels in male Wistar rats fed a laboratory chow enriched with 2% cholesterol or normal chow for 12 wk. Hearts were isolated and perfused according to Langendorff. After a 10-min perfusion, myocardial tissue cholesterol, superoxide, and nitrotyrosine contents were measured and Cx43 content in whole heart homogenate and a mitochondrial fraction determined. In the cholesterol-fed group, tissue cholesterol and superoxide formation was increased (P < 0.05), while total Cx43 content remained unchanged. Mitochondrial total and dephosphorylated Cx43 content decreased. Hearts were subjected to an IP protocol (3 × 5 min ischemia-reperfusion) or time-matched aerobic perfusion followed by 30-min global ischemia and 5-min reperfusion. IP reduced infarct size in normal but not in cholesterol-fed rats. At 5-min reperfusion following 30-min global ischemia, the total and dephosphorylated mitochondrial Cx43 content was increased, which was abolished by IP in both normal and high-cholesterol diet. In conclusion, loss of cardioprotection by IP in hyperlipidemia is associated with a redistribution of both sarcolemmal and mitochondrial Cx43.

Role of iNOS and peroxynitrite-matrix metalloproteinase-2 signaling in myocardial late preconditioning in rats.

We have previously shown that the inhibition of myocardial nitric oxide (NO) and peroxynitrite-matrix metalloproteinase (MMP) signaling by early preconditioning (PC) is involved in its cardioprotective effect. Therefore, in the present study, we investigated the role of NO and peroxynitrite-MMP signaling in the development of late PC. PC was performed by five consecutive cycles of 4-min coronary occlusion and 4-min reperfusion in anesthetized rats in vivo. Twenty-four hours later, hearts were subjected to a 30-min coronary occlusion followed by 180-min reperfusion to measure infarct size. In separate experiments, heart tissue was sampled to measure biochemical parameters before and 3, 6, 12, or 24 h after the PC protocol, respectively. Late PC decreased infarct size, increased cardiac inducible NO synthase (iNOS) activity and gene expression, and decreased SOD activity at 24 h significantly compared with sham-operated controls. Late PC increased cardiac superoxide levels significantly at 24 h; however, it did not change cardiac NO levels. Cardiac peroxynitrite levels were significantly decreased. Downstream cellular targets of peroxynitrite, MMP-2 and MMP-9 activities were decreased in the late PC group at 24 h compared with the sham-operated group. To verify if PC-induced inhibition of MMPs had a causative role in the reduction of infarct size, in separate experiments, we measured infarct size after the pharmacological inhibition of MMPs by ilomastat and found a significant reduction of infarct size compared with the vehicle-treated group. In conclusion, this is the first demonstration that the inhibition of cardiac peroxynitrite-MMP signaling contributes to cardioprotection by late PC and that pharmacological inhibition of MMPs is able to reduce infarct size in vivo. Furthermore, increased expression of iNOS may play a role in the development of late PC; however, increased iNOS activity does not lead to increased NO production in late PC.

Expression of genes related to oxidative/nitrosative stress in mouse hearts: effect of preconditioning and cholesterol diet.

BACKGROUND: The aim of our study was to explore the effect of high-cholesterol diet and preconditioning on cardiac gene expression patterns in mouse hearts, focusing on genes involved in nitric oxide (NO) and free radical signaling and the mevalonate pathway. MATERIAL/METHODS: Mice were fed 2% high-cholesterol or normal diet for 8 weeks. Hearts isolated from both groups were subjected to either a preconditioning (PC) protocol (3 cycles of 5 min ischemia and 5 min aerobic perfusion) or a time-matched non-preconditioning protocol followed by 30 min global test ischemia and 2 hour reperfusion. RESULTS: PC altered gene expression only in the mice subjected to a normal diet, as shown in neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS) and the superoxide-producing enzymes xanthine oxidase (XO) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 1 and 4. The rate-limiting enzyme of the mevalonate pathway, 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase showed differential expression in the myocardium in response to I/R and PC in mice on normal diet but not in cholesterol-fed animals. CONCLUSIONS: We conclude that cholesterol-enriched diet leads to alterations in preconditioning-induced gene expression in the mouse heart, which might lead to marked changes of oxidative/nitrosative stress signaling and to the attenuation of the cardioprotective effect of preconditioning.

Gene and protein expression changes in response to normoxic perfusion in mouse hearts.

INTRODUCTION: Although crystalloid-perfused isolated heart models are widely used in cardiovascular research, there are several limitations of these techniques. Changes in cardiac gene expression pattern due to normoxic perfusion itself have not been studied, despite its potential importance to provide useful information on limitations of this model. Therefore, here we investigated the time-dependent effect of normoxic, normothermic perfusion on global gene expression at mRNA and protein levels. METHODS: Hearts from male CFLP mice were perfused according to the Langendorff technique. We assessed relative gene expression changes by DNA microarray analysis of 8000 genes after 0, 60 and 120 min perfusion. RESULTS: Twelve genes exhibited significant up-regulation and 27 showed repression in hearts perfused for 60 or 120 min as compared to 0 min controls. Expression changes of 17 selected genes were verified and an additional 19 genes were examined by real-time quantitative PCR. Genes with altered expression included those coding for Creatin kinase, Lactate dehydrogenase, Voltage-dependent anion channel 1, a Disintegrin and Metalloprotease domain 3, Integrin alpha 7, Long-chain acyl-CoA dehydrogenase, Casein kinase II, Ketohexokinase, Chloride ion current inducer protein, Matrix metalloproteinase 2 and 9, Superoxide dismutases and Nitric oxide synthases, etc. DISCUSSION: Our results show that normoxic crystalloid perfusion itself results in time-dependent changes in cardiac gene expression which should be considered when designing ex vivo perfusion protocols in the mouse heart to mimic cardiac pathologies as many of these genes have been suspected to influence several cardiovascular diseases.

Rapid ventricular pacing-induced postconditioning attenuates reperfusion injury: effects on peroxynitrite, RISK and SAFE pathways.

BACKGROUND AND PURPOSE: Rapid ventricular pacing (RVP) applied before an index ischaemia has anti-ischaemic effects. Here, we investigated whether RVP applied after index ischaemia attenuates reperfusion injury and whether peroxynitrite, reperfusion injury salvage kinase (RISK) and survival activating factor enhancement (SAFE) pathways as well as haem oxygenase 1 (HO1) are involved in the mechanism of RVP-induced postconditioning. EXPERIMENTAL APPROACH: Langendorff perfused rat hearts were subjected to 30 min regional ischaemia and 120 min reperfusion with or without ischaemic postconditioning (6 × 10/10 s reperfusion/ischaemia; IPost) or RVP (6 × 10/10 s non-pacing/rapid pacing at 600 bpm) applied at the onset of reperfusion. KEY RESULTS: Meta-analysis of our previous studies revealed an association between longer reperfusion-induced ventricular tachycardia/fibrillation with decreased infarct size. In the present experiments, we tested whether RVP is cardioprotective and found that both IPost and RVP significantly decreased infarct size; however, only RVP attenuated the incidence of reperfusion-induced ventricular tachycardia. Both postconditioning methods increased the formation of cardiac 3-nitrotyrosine and superoxide, and non-significantly enhanced Akt phosphorylation at the beginning of reperfusion without affecting ERK1/2 and STAT3, while IPost alone induced HO1. Application of brief ischaemia/reperfusion cycles or RVP without preceding index ischaemia also facilitated peroxynitrite formation; nevertheless, only brief RVP increased STAT3 phosphorylation. CONCLUSIONS AND IMPLICATIONS: Short periods of RVP at the onset of reperfusion are cardioprotective and increase peroxynitrite formation similarly to IPost and thus may serve as an alternative postconditioning method. However, downstream mechanisms of the protection elicited by IPost and RVP seem to be partially different. LINKED ARTICLES: This article is part of a themed section on Conditioning the Heart - Pathways to Translation. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-8.

The Absence of N-Acetyl-D-glucosamine Causes Attenuation of Virulence of Candida albicans upon Interaction with Vaginal Epithelial Cells In Vitro.

To better understand the molecular events underlying vulvovaginal candidiasis, we established an in vitro system. Immortalized vaginal epithelial cells were infected with live, yeast form C. albicans and C. albicans cultured in the same medium without vaginal epithelial cells were used as control. In both cases a yeast to hyphae transition was robustly induced. Whole transcriptome sequencing was used to identify specific gene expression changes in C. albicans. Numerous genes leading to a yeast to hyphae transition and hyphae specific genes were upregulated in the control hyphae and the hyphae in response to vaginal epithelial cells. Strikingly, the GlcNAc pathway was exclusively triggered by vaginal epithelial cells. Functional analysis in our in vitro system revealed that the GlcNAc biosynthesis is involved in the adherence to, and the ability to kill, vaginal epithelial cells in vitro, thus indicating the key role for this pathway in the virulence of C. albicans upon vulvovaginal candidiasis.

Measurement of myocardial infarct size in preclinical studies.

Ischemic heart disease is a major cause of morbidity and mortality worldwide. Myocardial ischemia followed by reperfusion results in tissue injury termed ischemia/reperfusion injury which is characterized by decreased myocardial contractile function, occurrence of arrhythmias, and development of tissue necrosis (infarction). These pathologies are all relevant as clinical consequences of myocardial ischemia/reperfusion injury and they are also important as experimental correlates and endpoints. The most critical determinant of acute and long-term mortality after myocardial infarction is the volume of the infarcted tissue. Therefore, development of cardioprotective therapies aims at reducing the size of the infarct developing due to myocardial ischemia/reperfusion injury. Different techniques are available to measure myocardial infarct size in humans and in experimental settings, however, accurate determination of the extent of infarction is necessary to evaluate interventions that may delay the onset of necrosis and/or limit the total extent of infarct size during ischemia/reperfusion. This paper highlights recent advances of the different techniques to measure infarct size.

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.