Cardioprotective Effect Of Ischemic Preconditioning Research Articles

Nitric oxide is involved in the cardioprotection of neonatal rat hearts, but not in neonatal ischemic postconditioning.

The cardioprotective effect of ischemic preconditioning (IPC) and ischemic postconditioning (IPoC) in adult hearts is mediated by nitric oxide (NO). During the early developmental period, rat hearts exhibit higher resistance to ischemia-reperfusion (I/R) injury, contain higher levels of serum nitrates, and their resistance cannot be further increased by IPC or IPoC. NOS blocker (L-NAME) lowers their high resistance. Wistar rat hearts (postnatal Days 1 and 10) were perfused according to Langendorff and exposed to 40 min of global ischemia followed by reperfusion with or without IPoC. NO and reactive oxygen species donors (DEA-NONO, SIN-1) and L-NAME were administered. Tolerance to ischemia decreased between Days 1 and 10. DEA-NONO (low concentrations) significantly increased tolerance to I/R injury on both Days 1 and 10. SIN-1 increased tolerance to I/R injury on Day 10, but not on Day 1. L-NAME significantly reduced resistance to I/R injury on Day 1, but actually increased resistance to I/R injury on Day 10. Cardioprotection by IPoC on Day 10 was not affected by either NO donors or L-NAME. It can be concluded that resistance of the neonatal heart to I/R injury is NO dependent, but unlike in adult hearts, cardioprotective interventions, such as IPoC, are most likely NO independent.

Cardioprotective Effects of Sodium-Glucose Cotransporter Subtype Inhibition on Ischemic and Pharmacological Preconditioning.

Sodium-glucose cotransporter (SGLT) 2 inhibitors partially inhibit SGLT1 expression; however, whether a clinical dose of SGLT2 inhibitor abrogates ischemic preconditioning (IPC) is unknown, and the pharmacological cardioprotective effect under SGLT1 inhibition has not been examined. In this study, we investigated whether a clinical dose of tofogliflozin abrogates IPC and whether pharmacological preconditioning with olprinone has cardioprotective effects under SGLT1 inhibition. Male Wistar rats were divided into seven groups (seven rats per group) and subjected to the following treatments before inducing ischemia/reperfusion (I/R; 30 minutes of coronary artery occlusion followed by 120 minutes of reperfusion): saline infusion control treatment (Con); ischemic preconditioning(IPC); IPC after phlorizin infusion (IPC+Phl); IPC after low-dose tofogliflozin infusion (IPC+L-Tof); IPC after high-dose tofogliflozin infusion (IPC+H-Tof); olprinone infusion (Olp); and Olp infusion after phlorizin infusion (Olp+Phl). The infarct size was significantly decreased in the IPC group, but not in the IPC+Phl group. In contrast, the infarct size decreased in the IPC+L-Tof and IPC+H-Tof groups. Additionally, Olp reduced the infarct size, and the effect was preserved in Olp+Phl groups. Phosphorylated AMP-activated protein kinase (AMPK) expression was lower in the IPC+Phl group compared to that in the IPC group. The cardioprotective effect of IPC was attenuated by strong SGLT1 inhibition, but the effect was preserved under a clinical dose of highly selective SGLT2 inhibitor. Olprinone exerts a cardioprotective effect even under strong SGLT1 inhibition.

Ticagrelor enhances the cardioprotective effects of ischemic preconditioning in stable patients undergoing percutaneous coronary intervention: the TAPER-S randomized study.

Ticagrelor improves clinical outcomes in patients with acute coronary syndromes compared with clopidogrel. Ticagrelor also inhibits cell uptake of adenosine and has been associated with cardioprotective effects in animal models. We sought to investigate the potential cardioprotective effects of ticagrelor, as compared with clopidogrel, in stable patients undergoing percutaneous coronary intervention (PCI). This was a Prospective Randomized Open Blinded End-points (PROBE) trial enrolling stable patients with coronary artery disease (CAD) requiring fractional flow reserve-guided PCI of intermediate epicardial coronary lesions. ST-segment elevation at intracoronary electrocardiogram (IC-ECG) during a two-step sequential coronary balloon inflations in the reference vessel during PCI was used as an indirect marker of cardioprotection induced by ischemic preconditioning (IPC). The primary endpoint of the study was the comparison of the delta (Δ) (difference) ST-segment elevation measured by IC-ECG during two-step sequential coronary balloon inflations. Fifty-three patients were randomized to either clopidogrel or ticagrelor. The study was stopped earlier because the primary endpoint was met at a pre-specified interim analysis. ΔST-segment elevation was significantly higher in ticagrelor as compared to clopidogrel arms (P<0.0001). Ticagrelor was associated with lower angina score during coronary balloon inflations. There was no difference in coronary microvascular resistance between groups. Adenosine serum concentrations were increased in patients treated with ticagrelor as compared to those treated with clopidogrel. Ticagrelor enhances the cardioprotective effects of IPC compared with clopidogrel in stable patients with CAD undergoing PCI. Further studies are warranted to fully elucidate the mechanisms through which ticagrelor may exert cardioprotective effects in humans. http://www.clinicaltrials.gov. Unique Identifier: NCT02701140.

Caveolin-3 negatively regulates endocytic recycling of cardiac KATP channels.

Sarcolemmal ATP-sensitive potassium (KATP) channels play a vital role in cardioprotection. Cardiac KATP channels are enriched in caveolae and physically interact with the caveolae structural protein caveolin-3 (Cav3). Disrupting caveolae impairs the regulation of KATP channels through several signaling pathways. However, the direct functional effect of Cav3 on KATP channels is still poorly understood. Here, we used the cardiac KATP channel subtype, Kir6.2/SUR2A, and showed that Cav3 greatly reduced KATP channel surface density and current amplitude in a caveolae-independent manner. A screen of Cav3 functional domains revealed that a 25 amino acid region in the membrane attachment domain of Cav3 is the minimal effective segment (MAD1). The peptide corresponding to the MAD1 segment decreased KATP channel current in a concentration-dependent manner with an IC50 of ∼5 μM. The MAD1 segment prevented KATP channel recycling, thus decreasing KATP channel surface density and abolishing the cardioprotective effect of ischemic preconditioning. Our research identified the Cav3 MAD1 segment as a novel negative regulator of KATP channel recycling, providing pharmacological potential in the treatment of diseases with KATP channel trafficking defects.NEW & NOTEWORTHY Cardiac KATP channels physically interact with caveolin-3 in caveolae. In this study, we investigated the functional effect of caveolin-3 on KATP channel activity and identified a novel segment (MAD1) in the C-terminus domain of Caveolin-3 that negatively regulates KATP channel surface density and current amplitude by impairing KATP channel recycling. The peptide corresponding to the MAD1 segment abolished the cardioprotective effect of ischemic preconditioning.

Do reactive oxygen species damage or protect the heart in ischemia and reperfusion? Analysis on experimental and clinical data.

The role of reactive oxygen species (ROS) in ischemic and reperfusion (I/R) injury of the heart has been discussed for more than 40 years. It has been demonstrated that reperfusion triggers a multiple increase in free radical generation in the isolated heart. Antioxidants were found to have the ability to mitigate I/R injury of the heart. However, it is unclear whether their cardioprotective effect truly depends on the decrease of ROS levels in myocardial tissues. Since high doses and high concentrations of antioxidants were experimentally used, it is highly likely that the cardioprotective effect of antioxidants depends on their interaction not only with free radicals but also with other molecules. It has been demonstrated that the antioxidant N-2-mercaptopropionyl glycine or NDPH oxidase knockout abolished the cardioprotective effect of ischemic preconditioning. Consequently, there is evidence that ROS protect the heart against the I/R injury.

Preconditioning Using Naltrindole or its Analogues Exerts Robust Infarct‐Sparing Effects in Rat Myocardial Ischemia/Reperfusion

Ischemic heart disease remains the leading cause of death worldwide. Pharmacological agents that mimic the cardioprotective effects of ischemic preconditioning may have therapeutic potential as a secondary prevention strategy to resist infarction from subsequent cardiovascular events. Increased left ventricular end diastolic pressure (LVEDP) during ischemia, or ischemic peak pressure (IPP), is known to be correlated to infarct size. Recently our laboratory demonstrated that naltrindole (NTI), a selective delta opioid receptor antagonist, reduces IPP and infarct size when given prior to ischemia (preconditioning) in a Langendorff rat heart model. The purpose of this study was to examine the effects of NTI analogues naltriben (NTB, delta opioid receptor antagonist) and guanidinonaltrindole (GNTI, kappa opioid receptor antagonist) compared to NTI. Nor‐binaltrophine (BNI, kappa opioid receptor antagonist) and naloxone (NX, broad‐spectrum opioid receptor antagonist) were tested to evaluate cardioprotection by other opioid receptor antagonists.Isolated hearts from male Sprague‐Dawley rats (~300g) were subjected to 30‐min global ischemia (I)/45‐min reperfusion (R) with treatments infused for 5 min before I and during the first 5 min of R. LV cardiac function was measured using a pressure transducer. At the end of reperfusion, infarct size was assessed using 1% triphenyltetrazolium chloride staining and defined as infarcted tissue/total area at risk. Data were evaluated using ANOVA Student‐Neuman‐Keuls post‐hoc analysis.Control I/R hearts demonstrated an IPP of 39±3 mmHg compared to pre‐ischemic LVEDP of 9±1 mmHg at baseline (n=12, p&lt;0.01), resulting in substantial infarct at the end of 45 min R (32±4%). NTI (n=7) and NTB (n=6) elicited cardiodepressive effects during preconditioning by reducing the maximal rate in the rise of LV pressure (dP/dt max) to 1581±379 mmHg/s and 929±243 mmHg/s, respectively, compared to control (2471±72 mmHg/s, p&lt;0.01). IPP was reduced by NTI (18±3 mmHg/s) and NTB (15±3 mmHg/s) compared to all groups (p&lt;0.05). Post‐reperfused dP/dt max and infarct size was most improved following NTI (1830±90 mmHg/s, 7±2%) and NTB (1846±140 mmHg/s, 7±2%) pretreatment, compared to control (777±142 mmHg/s, 32±4%, p&lt;0.01). GNTI reduced infarct size (17 ± 4%, n=6, p&lt;0.05), but did not exert a negative inotropic effect. Cardiac function and infarct size did not improve with BNI (n=7) or NX (n=6) pretreatment.These results suggest that NTI and analogues, GNTI and NTB, are cardioprotective against myocardial I/R injury. The negative inotropic effects of NTI and NTB were associated with ~75% reduction in infarct size compared to control. GNTI decreased infarct size by ~50% and these results suggest that NTI, NTB, and GNTI exert tissue‐salvaging effects independent of delta or kappa opioid receptor antagonism. In future studies, we will examine different ischemic time points to administer NTI and its analogues to determine optimal cardioprotection and investigate downstream effects on calcium handling.

Role of ACE and ACE-2 in abrogated cardioprotective effect of ischemic preconditioning in ovariectomized rat heart

Ischemic heart disease is the leading cause of death in postmenopausal women. The activity of heart ACE increases whereas the activity of ACE-2 decreases after menopause. The present study was designed to investigate the role of ACE and ACE-2 in the abrogated cardioprotective effect of IPC in OVX rat heart. The heart was isolated from OVX rat and mounted on Langendorff’s apparatus for giving intermittent cycles of IPC. The infarct size was estimated using TTC stain, and coronary effluent was analyzed for LDH, CK-MB, and nitrite release. IPC induced cardioprotection was significantly attenuated in the ovariectomized rat heart as compared to the normal rat heart. However, this attenuated cardioprotection was significantly restored by perfusion of DIZE, an ACE-2 activator, and captopril, an ACE inhibitor, alone or in combination noted in terms of decrease in myocardial infarct size, the release of LDH and CK-MB, and also increase in the release of NO as compared to untreated OVX rat heart. Thus, it is suggested that DIZE and captopril, alone or in combination restore the attenuated cardioprotective effect of IPC in OVX rat heart which is due to an increase in ACE-2 activity and decrease in ACE activity after treatment.

Role of caveolin-eNOS platform and mitochondrial ATP-sensitive potassium channel in abrogated cardioprotective effect of ischemic preconditioning in postmenopausal women

Role of caveolin-eNOS platform and mitochondrial ATP-sensitive potassium channel in abrogated cardioprotective effect of ischemic preconditioning in postmenopausal women

Ischemic preconditioning protects the heart against ischemia-reperfusion injury in chronic kidney disease in both males and females

BackgroundUremic cardiomyopathy is a common cardiovascular complication of chronic kidney disease (CKD) characterized by left ventricular hypertrophy (LVH) and fibrosis enhancing the susceptibility of the heart to acute myocardial infarction. In the early stages of CKD, approximately 60% of patients are women. We aimed to investigate the influence of sex on the severity of uremic cardiomyopathy and the infarct size-limiting effect of ischemic preconditioning (IPRE) in experimental CKD.MethodsCKD was induced by 5/6 nephrectomy in 9-week-old male and female Wistar rats. Two months later, serum and urine laboratory parameters were measured to verify the development of CKD. Transthoracic echocardiography was performed to assess cardiac function and morphology. Cardiomyocyte hypertrophy and fibrosis were measured by histology. Left ventricular expression of A- and B-type natriuretic peptides (ANP and BNP) were measured by qRT-PCR and circulating BNP level was measured by ELISA. In a subgroup of animals, hearts were perfused according to Langendorff and were subjected to 35 min global ischemia and 120 min reperfusion with or without IPRE (3 × 5 min I/R cycles applied before index ischemia). Then infarct size or phosphorylated and total forms of proteins related to the cardioprotective RISK (AKT, ERK1,2) and SAFE (STAT3) pathways were measured by Western blot.ResultsThe severity of CKD was similar in males and females. However, CKD males developed more severe LVH compared to females as assessed by echocardiography. Histology revealed cardiac fibrosis only in males in CKD. LV ANP expression was significantly increased due to CKD in both sexes, however, LV BNP and circulating BNP levels failed to significantly increase in CKD. In both sexes, IPRE significantly decreased the infarct size in both the sham-operated and CKD groups. IPRE significantly increased the phospho-STAT3/STAT3 ratio in sham-operated but not in CKD animals in both sexes. There were no significant differences in phospho-AKT/AKT and phospho-ERK1,2/ERK1,2 ratios between the groups.ConclusionThe infarct size-limiting effect of IPRE was preserved in both sexes in CKD despite the more severe uremic cardiomyopathy in male CKD rats. Further research is needed to identify crucial molecular mechanisms in the cardioprotective effect of IPRE in CKD.

An inquest into regulatory mechanism of caveolin by ischemic preconditioning against orchidectomy-challenged rat heart.

Lower level of testosterone in men is related to major risks of cardiovascular diseases. This risk may increase due to the opening of mitochondrial permeability transition pore (mPTP). The mPTP is also regulated by ischemic preconditioning (IPC) and a membrane protein known as caveolin. The cardioprotective effect of IPC is the most effective methodologies used in testosterone deficiency. Daidzein (DDZ) a caveolin inhibitor shows cardioprotective action. The experiment has been designed to evaluate the pretreated DDZ effect in IPC-mediated cardioprotective action in orchidectomy (OCZ)-challenged rat heart. The experiment was designed on male Wistar rats with/without OCZ. The level of testosterone is decreased by OCZ which reduces general body growth. Isolated heart from normal and OCZ rat was tied up on Langendorff's perfused apparatus and followed by ischemic reperfusion (IR) and IPC cycle. To investigate the cardioprotective effect of DDZ in heart with testosterone deficiency, a total of nine groups, each consisting of six rats (n = 6) were as follows: Sham, IR, IPC, IPC + OCZ, IPC + DDZ, IPC + OCZ + DDZ, IPC + sodium nitrite, IPC + OCZ + sodium nitrite, IPC + OCZ + DDZ + sodium nitrite. Hemodynamic parameters, cellular injury (infarct size, LDH, CKMB and cardiac troponin-T), oxidative stress, mitochondrial function, integrity and immunoblot analysis were assessed for each group. The experimental data showed that DDZ potentiated IPC-mediated increase in the heart rate, left ventricular diastolic pressure, coronary flow; + dp/dtmax, and -dp/dtmax. The pretreated DDZ decreases the action of LDH and CKMB, myocyte size, cardiac collagen content, infarct size and ventricular fibrillation and attenuation in oxidative stress and mitochondrial dysfunction in OCZ-challenged rat heart in all sets of experiments. Sodium nitrite, a producer of nitric oxide (NO), enhanced potentiating effects of DDZ on IPC-mediated cardioprotection in OCZ-challenged rats. These observations show that the downregulation of caveolin through impaired opening of mPTP during reperfusion and caveolin might be a potential adjuvant to IPC against cardiac injury in OCZ-challenged rats.

An alternative viewpoint for the cardioprotective effects of ischemic preconditioning

An alternative viewpoint for the cardioprotective effects of ischemic preconditioning

Differences in intrinsic aerobic capacity alters sensitivity to ischemia-reperfusion injury but not cardioprotective capacity by ischemic preconditioning in rats.

IntroductionAerobic capacity is a strong predictor of cardiovascular mortality. Whether aerobic capacity influences myocardial ischemia and reperfusion (IR) injury is unknown.PurposeTo investigate the impact of intrinsic differences in aerobic capacity and the cardioprotective potential on IR injury.MethodsWe studied hearts from rats developed by selective breeding for high (HCR) or low (LCR) capacity for treadmill running. The rats were randomized to: (1) control, (2) local ischemic preconditioning (IPC) or (3) remote ischemic preconditioning (RIC) followed by 30 minutes of ischemia and 120 minutes of reperfusion in an isolated perfused heart model. The primary endpoint was infarct size. Secondary endpoints included uptake of labelled glucose, content of selected mitochondrial proteins in skeletal and cardiac muscle, and activation of AMP-activated kinase (AMPK).ResultsAt baseline, running distance was 203±7 m in LCR vs 1905±51 m in HCR rats (p<0.01). Infarct size was significantly lower in LCR than in HCR controls (49±5% vs 68±5%, p = 0.04). IPC reduced infarct size by 47% in LCR (p<0.01) and by 31% in HCR rats (p = 0.01). RIC did not modulate infarct size (LCR: 52±5, p>0.99; HCR: 69±6%, p>0.99, respectively). Phosphorylaion of AMPK did not differ between LCR and HCR controls. IPC did not modulate cardiac phosphorylation of AMPK. Glucose uptake during reperfusion was similar in LCR and HCR rats. IPC increased glucose uptake during reperfusion in LCR animals (p = 0.02). Mitochondrial protein content in skeletal muscle was lower in LCR than in HCR (0.77±0.10 arbitrary units (AU) vs 1.09±0.07 AU, p = 0.02), but not in cardiac muscle.ConclusionAerobic capacity is associated with altered myocardial sensitivity to IR injury, but the cardioprotective effect of IPC is not. Glucose uptake, AMPK activation immediately prior to ischemia and basal mitochondrial protein content in the heart seem to be of minor importance as underlying mechanisms for the cardioprotective effects.

A Possible Cardio-Protective Role of Early Remote Ischemia Preconditioning Against Ischemia Reperfusion Injury I’m Isolated Hearts of Adult Albino Rats: Possible Involvement of Hypoxia Inducible Factor

Abstract Background Inspite of the claimed cardio-protective effects of ischemic preconditioning (IPC), it is invasive and so using remote ischemic preconditioning (RIPC) may offer an alternative. Meanwhile, RIPC cardioprotective role is controversial, with an equivocal underlying mechanism. The hypoxia inducible factor 1 alpha (HIF-1-alpha) which is increased following ischemic insults is claimed as a humoral mediator for RIPC. Objective To investigate the effect of remote ischemic pre-conditioning on myocardial ischemia/reperfusion injury in rats, and to elucidate the possible role of hypoxia inducible factor in this protection. Materials and Methods The present study was performed on 28 adult female albino rats in the same estrus cycle evaluated by vaginal smear, and they were allocated into 3 groups: Group I: control rats subjected to ischemic/reperfusion injury (I/R) only, group II: early RIPC rats (RIPC 2 hours prior to I/R), group III: acriflavine-treated early RIPC rats. Acriflavine is a drug that binds directly to HIF-1 alpha and HIF-2 alpha subunits, thus inhibiting its dimerization and transcriptional activity, and it was injected IP 10 days prior to RIPC. On sacrifice day, ECG was recorded and isolated heart studies were performed. Later, cardiac chambers weight, serum HIF-1-alpha, myocardial perfusate lactate dehydrogenase, and cardiac oxidative markers: Malonaldehyde and glutathione perioxidase were measured. Results Compared to the control group, the early RIPC group showed significant increase in the heart rate (HR), QTc interval in the ECG recording, glutathione peroxidase and the HIF 1α levels together with reduction in the percent of decrease in PT and PT/LV, in the percent of prolongation in time to peak tension (TPT), perfusate lactate dehydrogenase and MDA levels, while no significant changes were recorded in the heart chronotropic activity, in the percent of half relaxation time (HRT) prolongation, or in the percent of decrease of MFR. Following acriflavine treatment, the effects of RIPC were abolished highlighting the role of HIF-1-alpha in mediating RIPC protective effects. Conclusion The non-invasive and non-pharmological remote ischemic preconditioning technique can ameliorate the cardiac ischemic reperfusion injury with an obvious role of HIF-1α in mediating these protective effects.

Effect of ischemic preconditioning and a Kv7 channel blocker on cardiac ischemia-reperfusion injury in rats

Recently, we found cardioprotective effects of ischemic preconditioning (IPC), and from a blocker of KCNQ voltage-gated K+ channels (KV7), XE991 (10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone), in isolated rat hearts. The purpose of the present study was to investigate the cardiovascular effects of IPC and XE991 and whether they are cardioprotective in intact rats. In conscious rats, we measured the effect of the KV7 channel blocker XE991 on heart rate and blood pressure by use of telemetry. In anesthetized rats, cardiac ischemia was induced by occluding the left coronary artery, and the animals received IPC (2 × 5 min of occlusion), XE991, or a combination. After a 2 h reperfusion period, the hearts were excised, and the area at risk and infarct size were determined. In both anesthetized and conscious rats, XE991 increased blood pressure, and the highest dose (7.5 mg/kg) of XE991 also increased heart rate, and 44% of conscious rats died. XE991 induced marked changes in the electrocardiogram (e.g., increased PR interval and prolonged QTC interval) without changing cardiac action potentials. The infarct size to area at risk ratio was reduced from 53 ± 2% (n = 8) in the vehicle compared to 36 ± 3% in the IPC group (P < 0.05, n = 9). XE991 (0.75 mg/kg) treatment alone or on top of IPC failed to reduce myocardial infarct size. Similar to the effect in isolated hearts, locally applied IPC was cardioprotective in intact animals exposed to ischemia-reperfusion. Systemic administration of XE991 failed to protect the heart against ischemia-reperfusion injury suggesting effects on the autonomic nervous system counteracting the cardioprotection in intact animals.

Role of fibrates in attenuated cardioprotective effect of ischemic preconditioning in hyperlipidemic rat heart

The present study has been designed to investigate the beneficial role of Fenofibrate in attenuated the cardioprotective effect of ischemic preconditioning (IPC) in hyperlipidemic (Hpl) rat heart. Experimental Hpl was produced by feeding high fat diet to rats for a period of 28 days. Isolated langendorff's perfused normal and Hpl rat hearts were subjected to global ischemia for 30 min followed by reperfusion for 120 min. The myocardial infarct size was assessed macroscopically using triphenyltetrazolium chloride staining. Coronary effluent was analyzed for lactate dehydrogenase (LDH) and creatine kinase-MB release to assess the extent of cardiac injury. Moreover, the oxidative stress in heart was assessed by measuring thiobarbituric acid reactive substance, superoxide anion generation and reduced form of glutathione. Moreover, I/R produced myocardial injury, which was assessed in terms of increase in myocardial infarct size, LDH and CK-MB release in coronary effluent and decrease in coronary flow rate. Four episodes of IPC (5 min each) afforded cardioprotection against I/R-induced myocardial injury in normal rat heart. On the other hand, IPC mediated myocardial protection against I/R-injury was abolished in Hpl rat heart. However, Treatment with Fenofibrate (100 mg/kg/day, i.p.) as agonists of PPAR-α have not affected the cardioprotective effect of IPC in normal rat heart, but its treatment markedly restored the cardioprotective potentials of IPC in Hpl rat heart. It is noted that the high degree of oxidative stress produced in Hpl rat heart during reperfusion and consequent down regulation of PPAR-α may be responsible to abolish the cardioprotectivepotentials of IPC.

Abstract 585: Role of Splice Variant Clic5B in Cardiac Mitochondrial Localization and Function

Blocking chloride channels (Cl - ) with indanyloxyacetic acid (IAA-94) abrogated the cardio-protective effects of ischemic preconditioning (IPC) and increased myocardial infarction suggesting the possible contribution of IAA-94 sensitive Cl - channels in IPC mediated cardio-protection. Unlike cation channels, insights into the role of Cl - channels in cardioprotection is limited due to lack of information on their molecular identity. Chloride intracellular channel (CLIC) proteins are one of the known targets of IAA-94. They exist in both soluble and integral membrane form. Amongst the six known mammalian paralogs, CLIC1, CLIC4 and CLIC5 are most abundant in heart. Previously, we showed differential localization of CLIC4 and CLIC5 to outer and inner mitochondrial membrane respectively where as CLIC1 is present in the endoplasmic reticulum. Both CLIC4 and CLIC5 are important for mitochondrial functional integrity as well as cardioprotection from ischemia-reperfusion injury. Interestingly, none of the CLICs possesses conventional mitochondrial targeting sequence, which intrigued us to understand the mechanism of their localization to mitochondria. In this study we demonstrate that the mitochondrial localization of CLIC5 is related with the presence of splice variant CLIC5B (~50 kDa) in heart but not in lung lysates. Two different bands of CLIC5 at ~30 kDa (CLIC5A) and ~50 kDa, were observed in the cardiac mitochondrial lysates. Lung lysates showed the presence of only CLIC5A. In addition, mass spectrometric analysis revealed a peptide coverage of ~30% corresponding to CLIC5B at 50 kDa in heart but not in lung lysates further confirming its presence in heart. Moreover CLIC5 localized to mitochondria in p3 neonatal cardiomyocytes isolated from mouse but not in mouse lung epithelial (11 ± 2.1 %) cells which lacked CLIC5B indicating that the splice variant CLIC5B has a role in IMM localization. Interestingly, cardiac mitochondria from clic5 -/- mice exhibited increased ROS (p&lt; 0.05, n=3) production whereas the clic5 -/- lung mitochondria did not show any change, suggesting the role of CLIC5B in regulating the mitochondrial function as well. Our study establishes splice variation as a mechanism for targeting ion channels to mitochondria.

Haemotherapy with Fibrinogen for Perioperative Bleeding Prevention-A View on Arterial Thrombogenesis and Myocardial Infarction in the Rat In Vivo.

Major blood loss during cardiac surgery is associated with increased morbidity and mortality. Clinical pilot studies indicated that preoperative fibrinogen supplementation reduces postoperative blood loss without increasing thrombotic complications. However, an increase in fibrinogen concentration might rather aggravate pre-existing thrombosis than increase the incidence of thrombotic events. Therefore, we investigated, in the present study, whether fibrinogen supplementation influences (1) arterial thrombus formation, (2) the extent of myocardial infarction and (3) the cardioprotective effect of ischaemic preconditioning. Arterial thrombogenesis of the femoral artery was induced by topic FeCl3 treatment in anaesthetised Wistar rats after pretreatment with 60 mg/kg (Fiblow), 120 mg/kg (Fibhigh) or vehicle (Con). Vessel blood flow was monitored, and time to vessel occlusion was analysed as a marker for arterial thrombogenesis. In addition, regional myocardial I/R injury was induced by temporary left coronary artery occlusion in rats pretreated with or without fibrinogen supplementation. In additional groups, ischaemic preconditioning (IPC) was induced by 3 cycles of 5 min of ischaemia/reperfusion. In all groups, myocardial infarct size was determined by triphenyltetrazoliumchlorid staining. Arterial thrombogenesis was not affected by fibrinogen pretreatment. No differences in time until vessel occlusion between Con, Fiblow and Fibhigh groups were observed. In addition, fibrinogen supplementation in low and high concentrations had no effect on infarct size after regional myocardial ischaemia and reperfusion (Fiblow: 66 ± 10%, Fibhigh: 62 ± 9%; each ns vs. Con). IPC reduced infarct size from 62 ± 14% to 34 ± 12% (p < 0.05 vs. Con). Furthermore, both fibrinogen concentrations did not affect cardioprotection by ischaemic preconditioning (Fiblow + IPC: 34 ± 11%, Fibhigh + IPC: 31 ± 13%; each ns vs. IPC). Haemotherapy with fibrinogen did not affect arterial thrombogenesis, myocardial infarction and the cardioprotective effect of ischaemic preconditioning.

Possible role of mitochondrial K-ATP channel and nitric oxide in protection of the neonatal rat heart

Cardioprotective effect of ischemic preconditioning (IPC) and ischemic postconditioning (IPoC) in adult hearts is mediated by mitochondrial-K-ATP channels and nitric oxide (NO). During early developmental period, rat hearts exhibit higher resistance to ischemia–reperfusion (I/R) injury and their resistance cannot be further increased by IPC or IPoC. Therefore, we have speculated, whether mechanisms responsible for high resistance of neonatal heart may be similar to those of IPC and IPoC. To test this hypothesis, rat hearts isolated on days 1, 4, 7, and 10 of postnatal life were perfused according to Langendorff. Developed force (DF) of contraction was measured. Hearts were exposed to 40 min of global ischemia followed by reperfusion up to the maximum recovery of DF. IPoC was induced by 5 cycles of 10-s ischemia. Mito-K-ATP blocker (5-HD) was administered 5 min before ischemia and during first 20 min of reperfusion. Another group of hearts was isolated for biochemical analysis of 3-nitrotyrosine, and serum samples were taken to measure nitrate levels. Tolerance to ischemia did not change from day 1 to day 4 but decreased on days 7 and 10. 5-HD had no effect either on neonatal resistance to I/R injury or on cardioprotective effect of IPoC on day 10. Significant difference was found in serum nitrate levels between days 1 and 10 but not in tissue 3-nitrotyrosine content. It can be concluded that while there appears to be significant difference of NO production, mito-K-ATP and ROS probably do not play role in the high neonatal resistance to I/R injury.

Influence of diabetes mellitus duration on the efficacy of ischemic preconditioning in a Zucker diabetic fatty rat model.

Augmented mortality and morbidity following an acute myocardial infarction in patients with diabetes mellitus Type 2 (T2DM) may be caused by increased sensitivity to ischemia reperfusion (IR) injury or altered activation of endogenous cardioprotective pathways modified by T2DM per se or ischemic preconditioning (IPC). We aimed to investigate, whether the duration of T2DM influences sensitivity against IR injury and the efficacy of IPC, and how myocardial glucose oxidation rate was involved. Male Zucker diabetic fatty rats (homozygote (fa/fa)) at ages 6-(prediabetic), 12- (onset diabetes) and 24-weeks of age (late diabetes) and their age-matched non-diabetic controls (heterozygote (fa/+) were subjected to IR injury in the Langendorff model and randomised to IPC stimulus or control. T2DM rats were endogenously protected at onset of diabetes, as infarct size was lower in 12-weeks T2DM animals than in 6- (35±2% vs 53±4%; P = 0.006) and 24-weeks animals (35±2% vs 72±4%; P<0.0001). IPC reduced infarct size in all groups irrespective of the presence of T2DM and its duration (32±3%; 20±2%; 36±4% respectively; (ANOVA P<0.0001). Compared to prediabetic rats, myocardial glucose oxidation rates were reduced during stabilisation and early reperfusion at onset of T2DM, but these animals retained the ability to increase oxidation rate in late reperfusion. Late diabetic rats had low glucose oxidation rates throughout stabilisation and reperfusion. Despite inherent differences in sensitivity to IR injury, the cardioprotective effect of IPC was preserved in our animal model of pre-, early and late stage T2DM and associated with adaptations to myocardial glucose oxidation capacity.

Angiotensin II-preconditioning is associated with increased PKCε/PKCδ ratio and prosurvival kinases in mitochondria.

Angiotensin II-preconditioning (APC) has been shown to reproduce the cardioprotective effects of ischaemic preconditioning (IPC), however, the molecular mechanisms mediating the effects of APC remain unknown. In this study, Langendorff-perfused rat hearts were subjected to IPC, APC or both (IPC/APC) followed by ischaemia-reperfusion (IR), to determine translocation of PKCε, PKCδ, Akt, Erk1/2, JNK, p38 MAPK and GSK-3β to mitochondria as an indicator of activation of the protein kinases. In agreement with previous observations, IPC, APC and IPC/APC increased the recovery of left ventricular developed pressure (LVDP), reduced infarct size (IS) and lactate dehydrogenase (LDH) release, compared to controls. These effects were associated with increased mitochondrial PKCε/PKCδ ratio, Akt, Erk1/2, JNK, and inhibition of permeability transition pore (mPTP) opening. Chelerythrine, a pan-PKC inhibitor, abolished the enhancements of PKCε but increased PKCδ expression, and inhibited Akt, Erk1/2, and JNK protein levels. The drug had no effect on the APC- and IPC/APC-induced cardioprotection as previously reported, but enhanced the post-ischaemic LVDP in controls. Losartan, an angiotensin II type 1 receptor (AT1-R) blocker, abolished the APC-stimulated increase of LVDP and reduced PKCε, Akt, Erk1/2, JNK, and p38. Both drugs reduced ischaemic contracture and LDH release, and abolished the inhibition of mPTP by the preconditioning. Chelerythrine also prevented the reduction of IS by APC and IPC/APC. These results suggest that the cardioprotection induced by APC and IPC/APC involves an AT1-R-dependent translocation of PKCε and survival kinases to the mitochondria leading to mPTP inhibition. In chelerythrine-treated hearts, however, alternate mechanisms appear to maintain cardiac function.