Necrostatin: A Potentially Novel Cardioprotective Agent?

Background Necrostatin-1 (Nec-1), a small tryptophan-based molecule, was recently reported to protect the cerebral cortex against ischemia-reperfusion (I/R) injury. We investigated the actions of Nec-1 and its so-called inactive analog, Nec-1i, in the setting of myocardial I/R injury. Materials and methods The actions of Nec-1 and Nec-1i were examined in cultured C2C12 and H9c2 myocytes, cardiomyocytes isolated from male Sprague–Dawley rats, Langendorff isolated perfused C57Bl/6J mouse hearts and an in vivo open-chest C57Bl/6J mouse heart model. Results Nec-1 at 30 μM and 100 μM (but not 100 μM Nec-1i) reduced peroxide-induced cell death in C2C12 cells from 51.2 ± 1.1% (control) to 26.3 ± 2.9% (p < 0.01 vs control) and 17.8 ± 0.9% (p < 0.001), respectively. With H9c2 cells cell death was also reduced from 73.0 ± 0.4% (control) to 56.7 ± 0% (30 μM Nec-1, p < 0.05) and 45.4 ± 3.3% (100 μM Nec-1, p < 0.01). In the isolated perfused heart Nec-1 (30 μM) reduced infarct size (calculated as a percentage of the risk area) from 48.0 ± 2.0% (control) to 32.1 ± 5.4% (p < 0.05). Nec-1i (30 μM) also reduced infarct size (32.9 ± 5.1%, p < 0.05). In anesthetized C57Bl/6J mice Nec-1 (1.65 mg/kg), given intraperitoneally to coincide with reperfusion following left anterior descending artery ligation (30 min), also reduced infarct size from 45.3 ± 5.1% (control) to 26.6 ± 4.0% (p < 0.05), whilst Nec-1i (1.74 mg/kg) was ineffective (37.8 ± 6.0%). Stimulus-induced opening of the mitochondrial permeability transition pore (MPTP) in rat cardiomyocytes, as reflected by the time until mitochondrial depolarisation, was unaffected by Nec-1 or Nec-1i at 30 μM but increased at 100 μM i.e. 91% (p < 0.05 vs control) and 152% (p < 0.001) for Nec-1 and Nec-1i, respectively. Conclusion This is the first study to demonstrate that necrostatins inhibit myocardial cell death and reduce infarct size, possibly via a mechanism independent of the MPTP.     

Inhibition of RIP1-dependent necrosis prevents adverse cardiac remodeling after myocardial ischemia–reperfusion in vivo

Accumulating evidence indicatesthat programmed necrosis plays a critical role in cell death during ischemia–reperfusion. Necrostatin-1 (Nec-1), a small molecule capable of inhibiting a key regulator of programmed necrosis (RIP1), was shown to prevent necrotic cell death in experimental models including cardiac ischemia. However, no functional follow-up was performed and the action of Nec-1 remains unclear. Here, we studied whether Nec-1 inhibits RIP1-dependent necrosis and leads to long-term improvements after ischemia–reperfusion in vivo. Mice underwent 30 min of ischemia and received, 5 min before reperfusion, 3.3 mg/kg Nec-1 or vehicle treatment, followed by reperfusion. Nec-1 administration reduced infarct size to 26.3 ± 1.3 % (P = 0.001) compared to 38.6 ± 1.7 % in vehicle-treated animals. Furthermore, Nec-1 inhibited RIP1/RIP3 phosphorylation in vivo and significantly reduced necrotic cell death, while apoptotic cell death remained constant. By using MRI, cardiac dimensions and function were assessed before and 28 days after surgery. Nec-1-treated mice displayed less adverse remodeling (end-diastolic volume 63.5 ± 2.8 vs. 74.9 ± 2.8 μl, P = 0.031) and preserved cardiac performance (ejection fraction 45.81 ± 2.05 vs. 36.03 ± 2.37 %, P = 0.016). Nec-1 treatment significantly reduced inflammatory influx, tumor necrosis factor-α mRNA levels and oxidative stress levels. Interestingly, this was accompanied by significant changes in the expression signature of oxidative stress genes. Administration of Nec-1 at the onset of reperfusion inhibits RIP1-dependent necrosis in vivo, leading to infarct size reduction and preservation of cardiac function. The cardioprotective effect of Nec-1 highlights the importance of necrotic cell death in the ischemic heart, thereby opening a new direction for therapy in patients with myocardial infarction.     

A novel role for mitochondrial sphingosine-1-phosphate produced by sphingosine kinase-2 in PTP-mediated cell survival during cardioprotection

Although mitochondria are key determinants of myocardial injury during ischemia–reperfusion (I/R), their interaction with critical cytoprotective signaling systems is not fully understood. Sphingosine-1-phosphate (S1P) produced by sphingosine kinase-1 protects the heart from I/R damage. Recently a new role for mitochondrial S1P produced by a second isoform of sphingosine kinase, SphK2, was described to regulate complex IV assembly and respiration via interaction with mitochondrial prohibitin-2. Here we investigated the role of SphK2 in cardioprotection by preconditioning. Littermate (WT) and sphk2 ^−/− mice underwent 45 min of in vivo ischemia and 24 h reperfusion. Mice received no intervention (I/R) or preconditioning (PC) via 5 min I/R before the index ischemia. Despite the activation of PC-cytoprotective signaling pathways in both groups, infarct size in sphk2 ^−/− mice was not reduced by PC (42 ± 3% PC vs. 43 ± 4% I/R, p = ns) versus WT (24 ± 3% PC vs. 43 ± 3% I/R, p < 0.05). sphk2 ^−/− mitochondria exhibited decreased oxidative phosphorylation and increased susceptibility to permeability transition (PTP). Unlike WT, PC did not prevent ischemic damage to electron transport or the increased susceptibility to PTP. To evaluate the direct contribution to the resistance of mitochondria to cytoprotection, SphK2, PHB2 or cytochrome oxidase subunit IV was depleted in cardiomyoblasts. PC protection was abolished by each knockdown concomitant with decreased PTP resistance. These results point to a new action of S1P in cardioprotection and suggest that the mitochondrial S1P produced by SphK2 is required for the downstream protective modulation of PTP as an effector of preconditioning protection.     

Long-term Follow-up of Patients Undergoing Postconditioning During ST-Elevation Myocardial Infarction

Reperfusion injury may offset the optimal salvage of myocardium achieved during primary coronary angioplasty. Thus, coronary reperfusion must be combined with cardioprotective adjunctive therapies in order to optimize myocardial salvage and minimize infarct size. Forty-three patients with their first ST-elevation myocardial infarction were randomized to myocardial postconditioning or standard of care at the time of primary coronary angioplasty. Postconditioning was performed immediately upon crossing the lesion with the guide wire and consisted of four cycles of 30 s occlusion followed by 30 s of reperfusion. End-points included infarct size, myocardial perfusion grade (MPG), left-ventricular ejection fraction (LVEF), and long-term clinical events (death and heart failure). Despite similar ischemic times (≅4.5 h) (p = 0.9) a reduction in infarct size was observed among patients treated with the postconditioning protocol. Peak creatine phosphokinase (CPK), as well as its myocardial band (MB) fraction, was significantly lower in the postconditioning group when compared with the control group (CPK—control, 2,444 ± 1,928 IU/L vs. PC, 2,182 ± 1,717 IU/L; CPK-MB—control, 242 ± 40 IU/L vs. PC, 195 ± 33 IU/L; p = 0.64 and p < 0.01, respectively). EF in the postconditioning group was improved when compared with the control group (control, 43% ± 15 vs. PC, 52% ± 9; p = 0.05). After a mean follow-up of 3.4 years, a 6-point absolute difference in LVEF was still evident in the postconditioning group (p = 0.18). MPG was better among patients treated with the postconditioning protocol compared with control (2.5 ± 0.5 vs. 2.1 ± 0.6; p = 0.02). Due to the small sample size no significant differences in clinical events were detected (p value for death = 0.9; p value for heart failure = 0.2). A simple postconditioning protocol applied at the onset of mechanical reperfusion, resulted in reduction of infarct size, better epicardial and myocardial flow, and improvement in left ventricular function. The beneficial effects of postconditioning on cardiac function persist beyond 3 years.     

Nitric oxide/cGMP signalling mediates the cardioprotective action of adrenomedullin in reperfused myocardium

We demonstrated previously that adrenomedullin (AM), when given during early reperfusion, limited infarct size in rat heart. The present study was undertaken to provide direct evidence of the NO-dependency of AM’s cardioprotective action by assessing NO biosynthesis and involvement of the soluble guanylyl cyclase (sGC) pathway. Perfused hearts from male CD-1 mice were subjected to 30-min left coronary occlusion and 60-min reperfusion. Infarct size was determined by tetrazolium staining. AM 10 nM was administered from 20 min after coronary occlusion until 10 min after reperfusion. Coronary effluent was analysed for NO₂ ⁻ and NO₃ ⁻, and myocardial samples were analysed for NO₂ ⁻, NO₃ ⁻, nitroso-adducts and cGMP concentration. To examine the role of NO/sGC signalling in the infarct-limiting action of AM, further hearts received the sGC inhibitor ODQ 2 μM. AM treatment stimulated NO synthesis, indicated by increased NO₂ ⁻ efflux in coronary effluent throughout reperfusion (summarised as area under curve, AM 29.2 ± 3.9 vs. control 14.4 ± 2.8 μmol min² mL⁻¹, P < 0.05). AM limited infarct size (35.4 ± 2.7 vs. 12.2 ± 2.3%, P < 0.01), associated with a 2.45-fold increase (P < 0.05) in myocardial cGMP concentration at 10 min after reperfusion. ODQ abolished the infarct size-limiting effect of AM (28.9 ± 4.3%). These data provide the first evidence that AM increases NO bioavailability in intact murine myocardium and confirm that the NO/sGC/cGMP pathway is central to the cytoprotective action of AM against ischaemia–reperfusion injury.     

Prostaglandin D<Subscript>2</Subscript> DP1 receptor is beneficial in ischemic stroke and in acute exicitotoxicity in young and old mice

The cardiovascular complications reported to be associated with cyclooxygenase inhibitor use have shifted our focus toward prostaglandins and their respective receptors. Prostaglandin D₂ and its DP1 receptor have been implicated in various normal and pathologic conditions, but their role in stroke is still poorly defined. Here, we tested whether DP1 deletion aggravates N-methyl-d-aspartic acid (NMDA)-induced acute toxicity and whether DP1 pharmacologic activation protects mice from acute excitotoxicity and transient cerebral ischemia. Moreover, since the elderly are more vulnerable to stroke-related damage than are younger patients, we tested the susceptibility of aged DP1 knockout (DP1^−/−) mice to brain damage. We found that intrastriatal injection of 15 nmol NMDA caused significantly larger lesion volumes (27.2 ± 6.4%) in young adult DP1^−/− mice than in their wild-type counterparts. Additionally, intracerebroventricular pretreatment of wild-type mice with 10, 25, and 50 nmol of the DP1-selective agonist BW245C significantly attenuated the NMDA-induced lesion size by 19.5 ± 5.0%, 39.6 ± 7.7%, and 28.9 ± 7.0%, respectively. The lowest tested dose of BW245C also was able to reduce middle cerebral artery occlusion-induced brain infarction size significantly (21.0 ± 5.7%). Interestingly, the aggravated NMDA-induced brain damage was persistent in older DP1^−/− mice as well. We conclude that the DP1 receptor plays an important role in attenuating brain damage and that selective targeting of this receptor could be considered as an adjunct therapeutic tool to minimize stroke damage.     

Myocardial Ischaemia-reperfusion Injury is Attenuated by Intact Glucagon Like Peptide-1 (GLP-1) in the In Vitro Rat Heart and may Involve the p70s6K Pathway

Background and methods Glucagon Like Peptide-1 (GLP-1), one of the most potent incretin hormones, has potential beneficial actions on the ischaemic and failing heart. This study sought to further identify the mechanisms of action of GLP-1 on the ischaemic heart using an in vitro isolated perfused rat heart model of ischaemic-reperfusion injury (measuring infarct size to area of risk (%)) subjected to 35 min regional ischaemia and 2 h reperfusion. To examine the effect of intact GLP-1 we used an inhibitor of GLP-1 breakdown, Valine pyrrolidide (VP). The downstream target of phosphatidylinositol 3-kinase includes the mTOR/p70s6 kinase pathway which was pharmacologically inhibited by rapamycin. Results and conclusion GLP-1 alone did not decrease myocardial infarction (54.4 ± 3.1%). VP alone did not decrease myocardial infarction (52.5 ± 4%). GLP-1 in the presence of VP produced significant reduction in myocardial infarction compared to control hearts (28.4 ± 2.7% vs. 56.4 ± 3.9% vs. P < 0.05). Inhibiting p70s6 Kinase with rapamycin completely abolished GLP-1 induced protection (57.1 ± 4.9% vs. 28.4 ± 2.7% P < 0.05). There was no detectable increase in the phosphorylated p70s6k after either 5 or 10 min of treatment with GLP-1/VP or with VP alone in comparison to control blots. In conclusion we show for the first time that the protective effects of GLP-1 are mediated by intact GLP-1 and can be inhibited by blocking the p70s6 kinase.     

Influence of the angiotensin II AT1 receptor antagonist irbesartan on ischemia/reperfusion injury in the dog heart

The aim of the present study was to investigate whether the non-peptide angiotensin II type 1 (AT₁) receptor antagonist irbesartan (SR 47436, BMS 186295, 2-n-butyl-3[2‘-(1H-tetrazol-5-yl)-biphenyl-4-yl)methyl]-1,3-diaza-spiro[4,4]non-1-en-4-one) has myocardial protective effects during regional myocardial ischemia/reperfusion in vivo. Eighteen anesthetized open-chest dogs were instrumented for measurement of left ventricular and aortic pressure (tip manometer and pressure transducer, respectively), and coronary flow (ultrasonic flowprobes). Regional myocardial function was assessed by Doppler displacement transducers as systolic wall thickening (sWT) in the antero-apical and the postero-basal wall. The animals underwent 1 h of left anterior descending coronary artery (LAD) occlusion and subsequent reperfusion for 3 hours. Irbesartan (10 mg kg⁻¹, n=9) or the vehicle (KOH, control, n=9) was injected intravenously 30 min before LAD occlusion. Regional myocardial blood flow (RMBF) was measured after irbesartan injection and at 30 min LAD occlusion using colored microspheres. Infarct size was determined by triphenyltetrazolium chloride staining after 3 h of reperfusion. There was no recovery of sWT in the LAD perfused area in both groups at the end of the experiments (systolic bulging, −15.1±6.1% of baseline (irbesartan) vs. −12.3±3.0% (control), mean±SEM). Irbesartan led to an increase in RMBF in normal myocardium (2.47±0.40 vs. 1.35±0.28 ml min⁻¹ g⁻¹, P<0.05), and also to an increase in collateral blood flow to the ischemic area (0.27±0.04 vs. 0.17±0.02 ml min⁻¹ g⁻¹, P=<0.05). Infarct size (percent of area at risk) was 24.8±3.2% in the treatment group compared with 26.9±4.8% in the control group (P=0.72). These results indicate that a blockade of angiotensin II AT₁ receptors with irbesartan before coronary artery occlusion led to an increase in RMBF, but did not result in a significant reduction of myocardial infarct size. Received: 6 October 1999 Returned for revision: 16 November 1999 Revision received: 7 February 2000 Accepted: 29 March 2000     

Ischemia-reperfusion injury and cardioprotection: investigating PTEN,the phosphatase that negatively regulates PI3K,using a congenital model of PTEN haploinsufficiency

Activation of the PI3K/Akt pathway protects the heart from ischemia-reperfusion injury (IRI). The phosphatase PTEN is the main negative regulator of this pathway. We hypothesized that reduced PTEN levels could protect against IRI. Isolated perfused mouse hearts from PTEN^+/− and their littermates PTEN^+/+ (WT), were subjected to 35 min global ischemia and 30 min reperfusion, with and without 2, 4 or 6 cycles ischemic preconditioning (IPC). The end point was infarct size, expressed as a percentage of the myocardium at risk (I/R%). PTEN and Akt levels were determined using Western blot analysis. Unexpectedly, there were no significant differences in infarction between PTEN^+/− and WT (42.1 ± 5.0% Vs. 45.6 ± 3.3%). However, the preconditioning threshold was significantly reduced in the PTEN^+/− Vs. WT, with 4 cycles of IPC being sufficient to reduce I/R%, compared to 6 cycles in the WT (4 cycles IPC: 29.8. ± 3.69% in PTEN^+/− Vs. 45.5. ± 5.08% in WT, P < 0.01). In addition, the ratio between the phospho/total Akt (Ser473 and Thr308) was slightly but significantly increased in the PTEN^+/− indicating an upregulation of PI3K/Akt pathway. Interestingly, the levels of the other phosphatases that may negatively regulate the PI3K/Akt pathway (PP2A, SHIP2 and PHLPP) were not significantly different between littermates and PTEN^+/−. In conclusion, PTEN haploinsufficiency alone does not induce cardioprotection in this model; however, it reduces the threshold of protection induced by IPC. Returned for 1. Revision: 29 November 2007 1. Revision received: 5 May 2008 Returned for 2. Revision: 2 June 2008 2. Revision received: 5 June 2008     

Milrinone Administered Before Ischemia or Just After Reperfusion, Attenuates Myocardial Stunning in Anesthetized Swine

Purpose We assessed the dose or timing effect of milrinone administered against myocardial stunning in 37 anesthetized open-chest swine. Methods All swine were subjected to 12-min ischemia followed by reperfusion to produce myocardial stunning. Group A (n = 12) received saline in place of milrinone both before and after ischemia. Group B (n = 9) and C (n = 9) received intravenous milrinone at a rate of 5 μg/kg/min for 10 min followed by 0.5 μg/kg/min for 10 min and 10 μg/kg/min for 10 min followed by 1 μg/kg/min for 10 min, respectively, until 30 min before coronary occlusion. Group D (n = 7) received the same dose of milrinone as group B starting 1 min after reperfusion. Myocardial contractility was assessed by percentage segment shortening (%SS). Results Five swine in group A and two swine in groups B and C each had ventricular fibrillation or tachycardia after reperfusion, and were thus excluded from further analysis. The percentage changes of %SS from the baseline 90 min after reperfusion in groups B, C, and D were 78 ± 9%, 82 ± 13%, and 79 ± 7%, respectively, which were significantly higher than those in group A (43 ± 13%). Conclusion We conclude that milrinone administered before ischemia or just after reperfusion attenuates myocardial stunning.     

The Effect of the Phytoestrogen Genistein on Myocardial Protection, Preconditioning and Oxidative Stress

Introduction We have previously shown that estrogen administered in ovariectomized female rabbits significantly reduce myocardial infarct size. We now investigated whether the phytoestrogen genistein similarly protects ischemic myocardium and whether this is associated with its antioxidant properties. In addition, we examined whether genistein abolishes preconditioning, since at high doses, it inhibits tyrosine kinase. Materials and methods We studied five groups of New Zealand white female rabbits. Group A (n = 12) were normal controls, group B (n = 14) were ovariectomized 4 weeks prior to the experiment, group C (n = 10) were ovariectomized and treated with genistein (0.2 mg kg⁻¹ day⁻¹ subcutaneously) for 4 weeks before the experiment, group D (n = 12) had intact gonads and were treated with genistein (0.2 mg kg⁻¹ day⁻¹ subcutaneously) for 4 weeks before the experiment and group E (n = 8) were ovariectomized 4 weeks prior to the experiment and treated with a single dose of genistein (0.2 mg kg⁻¹ day⁻¹ subcutaneously) just prior to the experiment. All animals underwent 30 min of heart ischemia and 120 min of reperfusion, with (subgroup I) or without (subgroup II) preconditioning. Malondialdehyde (MDA) concentration just before the experiment was determined. Results We found significant differences between the groups—p < 0.0001 in factorial ANOVA. The groups with preconditioning had significant smaller infarcts compared to those without—AI vs AII (10.66 ± 1.42% vs 43.22 ± 2.67%), BI vs BII (18.53 ± 2.36% vs 43.05 ± 8.37%), CI vs CII (10.17 ± 2.07% vs 44.5 ± 5.47%), DI vs DII (14.98 ± 2.36% vs 37.79 ± 3.92%) and EI vs EII (17.11 ± 3.24% vs 42.08 ± 3.42%), p < 0.0005. Ovariectomy was not associated with larger myocardial infarctions—AII vs BII, p = NS. Genistein, for 4 weeks, did not protect ischemic myocardium in either ovariectomized or non-ovariectomized animals—BII vs CII and AII vs DII, p = NS. There was no significant difference between the preconditioned animals, with intact gonads or ovariectomized (AI vs BI, p = NS), ovariectomized with or without genistein (BI vs CI, p = NS) and non-ovariectomized whether treated with genistein or not (AI vs DI, p = NS). A single dose of genistein did not offer any protection (EII vs BII, p = NS), nor did it modify the preconditioning effect (EI vs BI, p = NS). We found no significant difference in MDA plasma levels between the groups. Conclusion Genistein, at this dose, does not reduce infarct size per se nor abolishes the protection induced by preconditioning, in both ovariectomized and non-ovariectomized animals. Preconditioning offers myocardial protection in animals with intact gonads as well as estrogen deprived; bilateral ovariectomy, at least during short-term, is not associated with larger myocardial infarcts compared to control animals. In addition estrogen deprivation, during short term, as well as genistein do not modify oxidative stress.     

Chronic Erythropoietin Treatment Limits Infarct-size in the Myocardium in Vitro

Summary It has been well established that erythropoietin (EPO) can limit myocardial ischemia/reperfusion injury in a variety of acute settings. However, despite EPO being used chronically to treat anemia the infarct limiting effects of long term treatment (chronic) have never been fully investigated. In this study we examined the effects of a 3 week treatment of EPO (5,000 IU/Kg) in male Sprague Dawley rats in limiting myocardial infarction after 35 min ischemia and 2 h reperfusion in an in vitro isolated heart perfusion model. Treating the animals ‘once a week’ failed to limit infarct size significantly compared to a saline control (54.1% ± 3.5 v 52.3% ± 4.4), whereas a ‘3 times a week’ regime succeeded in significantly reducing infarct size (36.2% ± 3.2 v 52.3% ± 4.4, p < 0.05). To demonstrate that the effect was not due to improved oxygen supply caused by a raised hematocrit level, we also administered EPO 24 h prior to ischemia/reperfusion. This treatment again reduced infarct size compared to a saline control (39.9% ± 4.4 v 58.4% ± 5.0, p < 0.05). To examine the mechanism of protection we used the PI3K inhibitor wortmannin and the nitric oxide synthase inhibitor L-NAME to try to abrogate EPO mediated protection. Where wortmannin failed to block the effects of EPO (31.7% ± 6.0 v 36.2% ± 3.2), L-NAME did abrogate protection (51.6% ± 5.6 v 36.2% ± 3.2, p < 0.05). We demonstrate that chronic EPO treatment limits infarct size and that it does so in a nitric oxide dependent manner.     

Antiapoptosis and Mitochondrial Effect of Pioglitazone Preconditioning in the Ischemic/reperfused Heart of Rat

Purpose  Pioglitazone, used clinically in the treatment of type 2 diabetes mellitus, has been implicated as a regulator of cellular inflammatory and ischemic responses. The present study examined whether pioglitazone could inhibit cadiocyte apoptosis and reduce mitochondrial ultrastructure injury and membrane potential loss in the ischemic/reperfused heart of the rat. Furthermore, we investigated whether the protective effect of pioglitazone was related to opening of the mitochondrial_ATP-sensitive potassium channels. Methods  Adult male Sprague–Dawley rats were subjected to 30 min of ischemia followed by 4 h of reperfusion. At 24 h before ischemia, rats were randomized to receive 0.9% saline, 5-hydroxydecanoate (5-HD, 10 mg kg⁻¹, i.v.) plus pioglitazone (3 mg kg⁻¹, i.v.) or pioglitazone (3 mg kg⁻¹, i.v.). One group served as sham control. We investigated mitochondrial structure, apoptosis rate and Bcl-2, Bax and Caspase-3 proteins by immunohistochemistry staining. RT-PCR was used to determine the expression of P38MAPKmRNA and JNKmRNA. Western blotting was used to measure the expression of P38MAPK, JNK and NFκB P65. A second group of rats were randomly divided into sham-operated, ischemia/reperfusion (I/R), pioglitazone treatment, 5-HD + pioglitazone and 5-HD groups and the size of myocardial infarction was determined. Primary cultured cardiomyocytes of neonatal Sprague–Dawley rats were divided into control, hypoxia reoxygenation, different concentrations of pioglitazone and 5-HD + pioglitazone groups. JC-1 staining flowcytometry was used to examine mitochondrial membrane potential (ΔΨm). Results  Pioglitazone decreased mitochondrial ultrastructural damage compared to I/R, and reduced infarct size from 34.93 ± 5.55% (I/R) to 20.24 ± 3.93% (P < 0.05). Compared with the I/R group, the apoptosis rate and positive cell index (PCI) of Bax and Caspase-3 proteins in the pioglitazone group were significantly decreased (P < 0.05), while the PCI of Bcl-2 protein was increased (P < 0.05). There was no significant difference between the I/R and 5-HD + pioglitazone groups. Compared with the sham-operated group, the expression of P38MAPK mRNA, JNK mRNA and protein of P38MAPK, JNK and NFκB P65 in I/R was increased (P < 0.05). Pioglitazone did inhibit the increase in expressions vs I/R (P < 0.05). The rate of loss ΔΨm cells in the pioglitazone group was significantly lower than in the hypoxia reoxygenation group, while the addition of 5-HD inhibited the effect of pioglitazone. Conclusion  Pioglitazone inhibited cadiocyte apoptosis and reduced mitochondrial ultrastructure injury and membrane potential loss in the ischemic/reperfused heart of rat. These protective effects of pioglitazone may be related to opening mitochondrial_ATP-sensitive potassium channels.     

The acute cardioprotective effect of glucocorticoid in myocardial ischemia–reperfusion injury occurring during cardiopulmonary bypass

The purpose of this study was to evaluate the acute cardioprotective effect of high-dose methylprednisolone (25 mg/kg) in the controlled in vivo model of myocardial ischemia–reperfusion injury occurring during cardiopulmonary bypass. Forty nondiabetic male patients with three-vessel disease undergoing first-time bypass surgery were enrolled for this double-blind prospective study. Patients were randomized to be given 25 mg/kg methylprednisolone (Group I) and saline (Group II) 1 h before cardiopulmonary bypass. The levels of cardiac troponin-I (cTnI) were used as a marker of myocardial tissue damage in myocardial ischemia–reperfusion injury. The cTnI levels were measured before surgery, at the second hour after cardiopulmonary bypass, at the 6th and 24th hours, and 5th day postoperatively. There was no significant difference between the two groups in respect to the duration of ischemia and reperfusion. The preoperative cTnI levels were 0.22 ± 0.29 ng/ml in Group I and 0.23 ± 0.28 ng/ml in Group II. cTnI levels increased to 2.40 ± 1.0 ng/ml in Group I and 3.19 ± 0.88 ng/ml in Group II at the 2nd hour after cardiopulmonary bypass. When the differences between T1 and T0 level that showed the amount of troponin release occurring due to ischemia–repefusion injury was calculated and then compared, there was a significant difference between Groups I and II (P = 0.024). The cTnI levels measured at 6 h after CPB were 1.98 ± 0.63 ng/ml in Group I and 2.75 ± 1.15 ng/ml in Group II (P = 0.049). cTnI levels decreased to 0.22 ± 0.10 ng/ml in Group I and 0.49 ± 0.25 ng/ml in Group II on the postoperative day 5 (P = 0.0001). Univalent regression analysis showed that preoperative high-dose corticosteroid usage decreased the troponin release in about 12% and this effect was statistically significant (R² = 0.12, P < 0.05). A single dose of intravenous methylpredisolone (25 mg/kg) given 1 h before ischemia reduced myocardial ischemia–reperfusion injury. These results demonstrated that the acute cardioprotective effect of corticosteroids has much potential in the future for reducing ischemia–reperfusion injury occurring during cardiopulmonary bypass when it is inevitable.     

Ischemic preconditioning targets the reperfusion phase

Emerging studies suggest that signaling during the myocardial reperfusion phase contributes to ischemic preconditioning (IPC). Whether the activation of PKC, the opening of the mK_ATP channel, redox signaling and transient acidosis specifically at the time of myocardial reperfusion are required to mediate IPC-induced protection is not known. Langendorff-perfused rat hearts were subjected to 35 min ischemia followed by 120 min reperfusion at the end of which infarct size was determined by tetrazolium staining. Control and IPC-treated hearts were randomized to receive for the first 15 min of reperfusion: (1) DMSO (0.02%) vehicle control; (2) chelerythrine (10 μmol/l), a PKC antagonist; (3) 5 hydroxydecanoate (5- HD,100 μmol/l), a mK_ATP channel blocker; (4) N-mercaptopropionylglycine (MPG,1 mmol/l), a reactive oxygen species scavenger; (5) NaHCO₃ (pH 7.6), to counteract any acidosis. Interestingly, all four agents given at the time of myocardial reperfusion abolished the infarct reduction elicited by IPC (N > 6/group): (1) DMSO at reperfusion: 49.3 ± 3.6% in control versus 21.0 ± 3.6% with IPC:P < 0.05; (2) chelerythrine at reperfusion: 57.1 ± 2.5% in control versus 60.1 ± 3.3% with IPC:P = NS; (3) 5-HD at reperfusion: 53.4 ± 6.5 % in control versus 42.6 ± 4.4% with IPC:P = NS; (4) MPG at reperfusion: 55.3 ± 4.6% in control versus 43.9 ± 5.2% with IPC:P = NS; (5) NaHCO₃ at reperfusion 53.4 ± 2.5% in control versus 59.0 ± 3.3% with IPC:P = NS. In conclusion, we report for the first time that PKC activation, mK_ATP channel opening, redox signaling and a low pH at the time of myocardial reperfusion are required to mediate the cardioprotection elicited by ischemic preconditioning.     

Critical role of inducible nitric oxide synthase in degeneration of retinal capillaries in mice with streptozotocin-induced diabetes

Aims/hypothesis Diabetes results in the upregulation of the production of several components of the inflammatory response in the retina, including inducible nitric oxide synthase (iNOS). The aim of this study was to investigate the role of iNOS in the pathogenesis of the early stages of diabetic retinopathy using iNOS-deficient mice (iNos ^−/−). Materials and methods iNos ^−/− mice and wild-type (WT; C57BL/6J) mice were made diabetic with streptozotocin or kept as non-diabetic controls. Mice were killed at different time points after the induction of diabetes for assessment of vascular histopathology, cell loss in the ganglion cell layer (GCL), retinal thickness, and biochemical and physiological abnormalities. Results The concentrations of nitric oxide, nitration of proteins, poly(ADP-ribose) (PAR)-modified proteins, endothelial nitric oxide synthase, prostaglandin E₂, superoxide and leucostasis were significantly (p < 0.05) increased in retinas of WT mice diabetic for 2 months compared with non-diabetic WT mice. All of these abnormalities except PAR-modified proteins in retinas were inhibited (p < 0.05) in diabetic iNos ^−/− mice. The number of acellular capillaries and pericyte ghosts was significantly increased in retinas from WT mice diabetic for 9 months compared with non-diabetic WT controls, these increases being significantly inhibited in diabetic iNos ^−/− mice (p < 0.05 for all). Retinas from WT diabetic mice were significantly thinner than those from their non-diabetic controls, whereas diabetic iNos ^−/− mice were protected from this abnormality. We found no evidence of cell loss in the GCL of diabetic WT or iNos ^−/− mice. Deletion of iNos had no beneficial effect on diabetes-induced abnormalities on the electroretinogram. Conclusions/interpretation We demonstrate that the inflammatory enzyme iNOS plays an important role in the pathogenesis of vascular lesions characteristic of the early stages of diabetic retinopathy in mice. An erratum to this article can be found at     

Deficiency in TIMP-3 increases cardiac rupture and mortality post-myocardial infarction via EGFR signaling: beneficial effects of cetuximab

Cardiac rupture is a fatal complication of myocardial infarction (MI); however, its underlying molecular mechanisms are not fully understood. This study investigated the role of tissue inhibitor of metalloproteinase-3 (TIMP-3)/matrix metalloproteinase (MMP)/epidermal growth factor (EGF)/transforming growth factor (TGF)-β1 pathway in infarct healing and effects of cetuximab on cardiac rupture after MI. Induction of MI was achieved by left coronary artery ligation in wild-type (WT) and TIMP-3^−/− mice. TIMP-3 deficiency resulted in a fourfold increase in cardiac rupture and 50% decrease in survival after MI. Hydroxyproline content, collagen synthesis and myofibroblast cell number in the infarct region, and the force required to induce rupture of the infarct scar were significantly decreased, while MMP activity was increased in TIMP-3^−/− mice. EGF proteins were increased by threefold in TIMP-3^−/− mice following MI, while TGF-β1 mRNA levels were decreased by 68%. Cell proliferation of cultured adult cardiac myofibroblasts was significantly decreased in TIMP-3^−/− compared to WT myofibroblasts. EGF treatment significantly decreased collagen synthesis and TGF-β1 expression. Conversely, TGF-β1 treatment increased collagen synthesis in cardiac myofibroblasts. Treatment with cetuximab significantly decreased the incidence of cardiac rupture and improved survival post-MI in TIMP-3^−/− mice. We conclude that deficiency in TIMP-3 increases cardiac rupture post-MI via EGF/epidermal growth factor receptor (EGFR) signaling which downregulates TGF-β1 expression and collagen synthesis. Inhibition of EGFR by cetuximab protects against cardiac rupture and improves survival post-MI.     

Effects of Levosimendan on Myocardial Infarct Size and Hemodynamics in a Closed-Chest Porcine Ischemia–Reperfusion Model

Introduction Levosimendan is a positive inotropic drug with vasodilator action and proposed myocardioprotective properties. In a canine model, levosimendan increased coronary collateral flow and reduced myocardial infarct size (IS). We investigated the effect of levosimendan on IS and hemodynamics in the closed-chest porcine ischemia–reperfusion model, which is devoid of coronary collaterals. Methods Infusion with levosimendan (0.2 μg/kg/min following a bolus of 24 μg/kg) or saline was initiated 30 min prior to ischemia in anaesthetized pigs (n = 10 in both groups). Balloon occlusion of the left anterior descending coronary artery for 45 min was followed by 2 1/2 h of reperfusion. Hemodynamics were monitored with a Swan-Ganz catheter and a left ventricular pressure micromanometer. Left ventricular systolic and diastolic function was estimated by dP/dt_max and τ, respectively. Myocardial area at risk (AAR) and IS were assessed in vivo by myocardial perfusion imaging (MPI) and ex vivo by histopathology (fluorescein staining for AAR, tetrazolium staining for IS). Results Prior to ischemia, levosimendan improved left ventricular systolic and diastolic function with coincident preload and afterload reduction. Cardiac output increased by 10 ± 4% (p = 0.04), dP/dt_max by 15 ± 5% (p = 0.01). Pulmonary capillary wedge pressure decreased by 18 ± 3% (p = 0.04), τ by 11 ± 2% (p = 0.001), and mean arterial pressure by 11 ± 2% (p < 0.001). A similar trend was observed during ischemia–reperfusion. The ratio of IS/AAR was not reduced by levosimendan compared to saline as evaluated by histopathology (76 ± 4% vs. 64 ± 7%, p = 0.12) and by MPI (94 ± 2% vs. 87 ± 5%, p = 0.14). Conclusion Levosimendan improves hemodynamics but does not reduce IS in an ischemia–reperfusion model without coronary collaterals.