Cardiac effects of 5F-Cumyl-PEGACLONE.

Synthetic cannabinoids become increasingly popular as a supposedly safe and legal alternative to cannabis. In order to circumvent the German New Psychoactive Substances Law, producers of so-called herbal mixtures rapidly design new substances with structural alterations that are not covered by the law. Acting as full agonists not only at the cannabinoid receptors 1 and 2, synthetic cannabinoids might have not only desired mental but also serious physical adverse effects. However, knowledge of adverse effects of specific substances is sparse and incomplete. This also accounts for 5F-Cumyl-PEGACLONE, a synthetic cannabinoid, which has been detected regularly in Germany in recent years. By using an animal model, the isolated perfused Langendorff heart, the study at hand aimed on finding out more about possible cardiovascular adverse effects of 5F-Cumyl-PEGACLONE. Hearts of male Wistar rats, which were excised postmortem, were exposed to two different concentrations of 5F-Cumyl-PEGACLONE: 13 hearts were exposed to 50 ng/ml and 12 hearts were exposed to 100 ng/ml. Thirteen control hearts were merely exposed to an additional amount of buffer solution. Functional parameters heart rate, minimal and maximum left ventricular pressure and coronary flow were documented at pre-defined time points during and after the administration of 5F-Cumyl-PEGACLONE/additional buffer solution. Electrocardiograms (ECGs) were documented throughout the experiments and evaluated afterwards. Kruskal-Wallis analysis was performed for each functional parameter as well as for the duration of the QRS complexes and the duration of RR intervals as derived from the ECGs. Furthermore, a multivariate analysis, comprising all functional and ECG parameters, was performed. Kruskal-Wallis analysis revealed only single significant p-values for QRS duration and minimum left ventricular pressure that did not pass a Bonferroni test. The results of the multivariate approach were also comparably homogeneous, but still the model correctly recognized hearts exposed to 100 ng/ml of 5F-Cumyl-PEGACLONE more often than hearts exposed to the low concentration of 5F-Cumyl-PEGACLONE or additional buffer solution. Evaluation of the ECGs presented single cases of ST depression and QT prolongation. Though certainly not unambiguous, these findings support the assumption that 5F-Cumyl-PEGACLONE can cause severe, if not lethal, cardiac adverse effects like arrhythmias or myocardial infarctions especially if it is consumed in combination with other drugs like alcohol or if the consumer suffers from pre-existing heart diseases.

Pravastatin Improves Colonic and Hepatic Microcirculatory Oxygenation during Sepsis without Affecting Mitochondrial Function and ROS Production in Rats.

Microcirculatory and mitochondrial dysfunction are considered the main mechanisms of septic shock. Studies suggest that statins modulate inflammatory response, microcirculation, and mitochondrial function, possibly through their action on peroxisome proliferator-activated receptor alpha (PPAR-α). The aim of this study was to examine the effects of pravastatin on microcirculation and mitochondrial function in the liver and colon and the role of PPAR-α under septic conditions. This study was performed with the approval of the local animal care and use committee. Forty Wistar rats were randomly divided into 4 groups: sepsis (colon ascendens stent peritonitis, CASP) without treatment as control, sepsis + pravastatin, sepsis + PPAR-α-blocker GW6471, and sepsis + pravastatin + GW6471. Pravastatin (200 µg/kg s.c.) and GW6471 (1 mg/kg) were applied 18 h before CASP-operation. 24 h after initial surgery, a relaparotomy was performed, followed by a 90 min observation period for assessment of microcirculatory oxygenation (µHbO2) of the liver and colon. At the end of the experiments, animals were euthanized, and the colon and liver were harvested. Mitochondrial function was measured in tissue homogenates using oximetry. The ADP/O ratio and respiratory control index (RCI) for complexes I and II were calculated. Reactive oxygen species (ROS) production was assessed using the malondialdehyde (MDA)-Assay. Statistics: two-way analysis of variance (ANOVA) + Tukey's/Dunnett's post hoc test for microcirculatory data, Kruskal-Wallis test + Dunn's post hoc test for all other data. In control septic animals µHbO2 in liver and colon deteriorated over time (µHbO2: -9.8 ± 7.5%* and -7.6 ± 3.3%* vs. baseline, respectively), whereas after pravastatin and pravastatin + GW6471 treatment µHbO2 remained constant (liver: µHbO2 pravastatin: -4.21 ± 11.7%, pravastatin + GW6471: -0.08 ± 10.3%; colon: µHbO2 pravastatin: -0.13 ± 7.6%, pravastatin + GW6471: -3.00 ± 11.24%). In both organs, RCI and ADP/O were similar across all groups. The MDA concentration remained unchanged in all groups. Therefore, we conclude that under septic conditions pravastatin improves microcirculation in the colon and liver, and this seems independent of PPAR-α and without affecting mitochondrial function.

Gemfibrozil Improves Microcirculatory Oxygenation of Colon and Liver without Affecting Mitochondrial Function in a Model of Abdominal Sepsis in Rats.

Recent studies observed, despite an anti-hyperlipidaemic effect, a positive impact of fibrates on septic conditions. This study evaluates the effects of gemfibrozil on microcirculatory variables, mitochondrial function, and lipid peroxidation levels with regard to its potential role as an indicator for oxidative stress in the colon and liver under control and septic conditions and dependencies on PPARα-mediated mechanisms of action. With the approval of the local ethics committee, 120 Wistar rats were randomly divided into 12 groups. Sham and septic animals were treated with a vehicle, gemfibrozil (30 and 100 mg/kg BW), GW 6471 (1 mg/kg BW, PPARα inhibitor), or a combination of both drugs. Sepsis was induced via the colon ascendens stent peritonitis (CASP) model. Then, 24 h post sham or CASP surgery, a re-laparotomy was performed. Measures of vital parameters (heart rate (HR), mean arterial pressure (MAP), and microcirculation (µHbO2)) were recorded for 90 min. Mitochondrial respirometry and assessment of lipid peroxidation via a malondialdehyde (MDA) assay were performed on colon and liver tissues. In the untreated sham animals, microcirculation remained stable, while pre-treatment with gemfibrozil showed significant decreases in the microcirculatory oxygenation of the colon. In the CASP animals, µHbO2 levels in the colon and the liver were significantly decreased 90 min after laparotomy. Pre-treatment with gemfibrozil prevented the microcirculatory aberrations in both organs. Gemfibrozil did not affect mitochondrial function and lipid peroxidation levels in the sham or CASP animals. Gemfibrozil treatment influences microcirculation depending on the underlying condition. Gemfibrozil prevents sepsis-induced microcirculatory aberrances in the colon and liver PPARα-independently. In non-septic animals, gemfibrozil impairs the microcirculatory variables in the colon without affecting those in the liver.

Influence of Short and Long Hyperglycemia on Cardioprotection by Remote Ischemic Preconditioning-A Translational Approach.

The adverse impact of common diseases like diabetes mellitus and acute hyperglycemia on morbidity and mortality from myocardial infarction (MI) has been well documented over the past years of research. In the clinical setting, the relationship between blood glucose and mortality appears linear, with amplifying risk associated with increasing blood glucose levels. Further, this seems to be independent of a diagnosis of diabetes. In the experimental setting, various comorbidities seem to impact ischemic and pharmacological conditioning strategies, protecting the heart against ischemia and reperfusion injury. In this translational experimental approach from bedside to bench, we set out to determine whether acute and/or prolonged hyperglycemia have an influence on the protective effect of transferred human RIPC-plasma and, therefore, might obstruct translation into the clinical setting. Control and RIPC plasma of young healthy men were transferred to isolated hearts of young male Wistar rats in vitro. Plasma was administered before global ischemia under either short hyperglycemic (HGs Con, HGs RIPC) conditions, prolonged hyperglycemia (HGl Con, HGl RIPC), or under normoglycemia (Con, RIPC). Infarct sizes were determined by TTC staining. Control hearts showed an infarct size of 55 ± 7%. Preconditioning with transferred RIPC plasma under normoglycemia significantly reduced infarct size to 25 ± 4% (p < 0.05 vs. Con). Under acute hyperglycemia, control hearts showed an infarct size of 63 ± 5%. Applying RIPC plasma under short hyperglycemic conditions led to a significant infarct size reduction of 41 ± 4% (p < 0.05 vs. HGs Con). However, the cardioprotective effect of RIPC plasma under normoglycemia was significantly stronger compared with acute hyperglycemic conditions (RIPC vs. HGs RIPC; p < 0.05). Prolonged hyperglycemia (HGl RIPC) completely abolished the cardioprotective effect of RIPC plasma (infarct size 60 ± 7%; p < 0.05 vs. HGl Con; HGl Con 59 ± 5%).

A Toolbox to Investigate the Impact of Impaired Oxygen Delivery in Experimental Disease Models.

Impaired oxygen utilization is the underlying pathophysiological process in different shock states. Clinically most important are septic and hemorrhagic shock, which comprise more than 75% of all clinical cases of shock. Both forms lead to severe dysfunction of the microcirculation and the mitochondria that can cause or further aggravate tissue damage and inflammation. However, the detailed mechanisms of acute and long-term effects of impaired oxygen utilization are still elusive. Importantly, a defective oxygen exploitation can impact multiple organs simultaneously and organ damage can be aggravated due to intense organ cross-talk or the presence of a systemic inflammatory response. Complexity is further increased through a large heterogeneity in the human population, differences in genetics, age and gender, comorbidities or disease history. To gain a deeper understanding of the principles, mechanisms, interconnections and consequences of impaired oxygen delivery and utilization, interdisciplinary preclinical as well as clinical research is required. In this review, we provide a "tool-box" that covers widely used animal disease models for septic and hemorrhagic shock and methods to determine the structure and function of the microcirculation as well as mitochondrial function. Furthermore, we suggest magnetic resonance imaging as a multimodal imaging platform to noninvasively assess the consequences of impaired oxygen delivery on organ function, cell metabolism, alterations in tissue textures or inflammation. Combining structural and functional analyses of oxygen delivery and utilization in animal models with additional data obtained by multiparametric MRI-based techniques can help to unravel mechanisms underlying immediate effects as well as long-term consequences of impaired oxygen delivery on multiple organs and may narrow the gap between experimental preclinical research and the human patient.

Mediation of the Cardioprotective Effects of Mannitol Discovered, with Refutation of Common Protein Kinases.

The osmodiuretic agent Mannitol exerts cardioprotection against ischemia and reperfusion (I/R) injury when applied as a pre- and/or postconditioning stimulus. Previously, we demonstrated that these properties are mediated via the activation of mitochondrial ATP-sensitive potassium (mKATP) channels. However, considering Mannitol remains in the extracellular compartment, the question arises as to which receptor and intracellular signaling cascades are involved in myocardial protection by the osmodiuretic substance. Protein kinase B (Akt) and G (PKG), as part of the reperfusion injury salvage kinase (RISK) and/or endothelial nitric oxide (eNOS)/PKG pathway, are two well-investigated intracellular targets conferring myocardial protection upstream of mitochondrial potassium channels. Adenosine receptor subtypes have been shown to trigger different cardioprotective pathways, for example, the reperfusion injury. Further, Mannitol induces an increased activation of the adenosine 1 receptor (A1R) in renal cells conferring its nephroprotective properties. Therefore, we investigated whether (1) Akt and PKG are possible signaling targets involved in Mannitol-induced conditioning upstream of the mKATP channel and/or whether (2) cardioprotection by Mannitol is mediated via activation of the A1R. All experiments were performed on male Wistar rats in vitro employing the Langendorff isolated heart perfusion technique with infarct size determination as the primary endpoint. To unravel possible protein kinase activation, Mannitol was applied in combination with the Akt (MK2206) or PKG (KT5823) inhibitor. In further groups, an A1R blocker (DPCPX) was given with or without Mannitol. Preconditioning with Mannitol (Man) significantly reduced the infarct size compared to the control group. Co-administration of the A1R blocker DPXPC fully abolished myocardial protection of Mannitol. Interestingly and in contrast to the initial hypothesis, neither administration of the Akt nor the PKG blocker had any impact on the cardioprotective properties of Mannitol-induced preconditioning. These results are quite unexpected and show that the protein kinases Akt and PKG-as possible targets of known protective signaling cascades-are not involved in Mannitol-induced preconditioning. However, the cardioprotective effects of Mannitol are mediated via the A1R.

Influence of Hyperglycemia and Diabetes on Cardioprotection by Humoral Factors Released after Remote Ischemic Preconditioning (RIPC).

Remote ischemic preconditioning (RIPC) protects hearts from ischemia-reperfusion (I/R) injury in experimental studies; however, clinical RIPC trials were unsatisfactory. This discrepancy could be caused by a loss of cardioprotection due to comorbidities in patients, including diabetes mellitus (DM) and hyperglycemia (HG). RIPC is discussed to confer protective properties by release of different humoral factors activating cardioprotective signaling cascades. Therefore, we investigated whether DM type 1 and/or HG (1) inhibit the release of humoral factors after RIPC and/or (2) block the cardioprotective effect directly at the myocardium. Experiments were performed on male Wistar rats. Animals in part 1 of the study were either healthy normoglycemic (NG), type 1 diabetic (DM1), or hyperglycemic (HG). RIPC was implemented by four cycles of 5 min bilateral hind-limb ischemia/reperfusion. Control (Con) animals were not treated. Blood plasma taken in vivo was further investigated in isolated rat hearts in vitro. Plasma from diseased animals (DM1 or HG) was administered onto healthy (NG) hearts for 10 min before 33 min of global ischemia and 60 min of reperfusion. Part 2 of the study was performed vice versa-plasma taken in vivo, with or without RIPC, from healthy rats was transferred to DM1 and HG hearts in vitro. Infarct size was determined by TTC staining. Part 1: RIPC plasma from NG (NG Con: 49 ± 8% vs. NG RIPC 29 ± 6%; p < 0.05) and DM1 animals (DM1 Con: 47 ± 7% vs. DM1 RIPC: 38 ± 7%; p < 0.05) reduced infarct size. Interestingly, transfer of HG plasma showed comparable infarct sizes independent of prior treatment (HG Con: 34 ± 9% vs. HG RIPC 35 ± 9%; ns). Part 2: No infarct size reduction was detectable when transferring RIPC plasma from healthy rats to DM1 (DM1 Con: 54 ± 13% vs. DM1 RIPC 53 ± 10%; ns) or HG hearts (HG Con: 60 ± 16% vs. HG RIPC 53 ± 14%; ns). These results suggest that: (1) RIPC under NG and DM1 induces the release of humoral factors with cardioprotective impact, (2) HG plasma might own cardioprotective properties, and (3) RIPC does not confer cardioprotection in DM1 and HG myocardium.

Combination of Cyclosporine A and Levosimendan Induces Cardioprotection under Acute Hyperglycemia.

Prognosis of patients with myocardial infarction is detrimentally affected by comorbidities like diabetes mellitus. In the experimental setting, not only diabetes mellitus but also acute hyperglycemia is shown to hamper cardioprotective properties by multiple pharmacological agents. For Levosimendan-induced postconditioning, a strong infarct size reducing effect is demonstrated in healthy myocardium. However, acute hyperglycemia is suggested to block this protective effect. In the present study, we investigated whether (1) Levosimendan-induced postconditioning exerts a concentration-dependent effect under hyperglycemic conditions and (2) whether a combination with the mitochondrial permeability transition pore (mPTP) blocker cyclosporine A (CsA) restores the cardioprotective properties of Levosimendan under hyperglycemia. For this experimental investigation, hearts of male Wistar rats were randomized and mounted onto a Langendorff system, perfused with Krebs-Henseleit buffer with a constant pressure of 80 mmHg. All isolated hearts were subjected to 33 min of global ischemia and 60 min of reperfusion under hyperglycemic conditions. (1) Hearts were perfused with various concentrations of Levosimendan (Lev) (0.3-10 µM) for 10 min at the onset of reperfusion, in order to investigate a concentration-response relationship. In the second set of experiments (2), 0.3 µM Levosimendan was administered in combination with the mPTP blocker CsA, to elucidate the underlying mechanism of blocked cardioprotection under hyperglycemia. Infarct size was determined by tetrazolium chloride (TTC) staining. (1) Control (Con) hearts showed an infarct size of 52 ± 12%. None of the administered Levosimendan concentrations reduced the infarct size (Lev0.3: 49 ± 9%; Lev1: 57 ± 9%; Lev3: 47 ± 11%; Lev10: 50 ± 7%; all ns vs. Con). (2) Infarct size of Con and Lev0.3 hearts were 53 ± 4% and 56 ± 2%, respectively. CsA alone had no effect on infarct size (CsA: 50 ± 10%; ns vs. Con). The combination of Lev0.3 and CsA (Lev0.3 ± CsA) induced a significant infarct size reduction compared to Lev0.3 (Lev0.3+CsA: 35 ± 4%; p < 0.05 vs. Lev0.3). We demonstrated that (1) hyperglycemia blocks the infarct size reducing effects of Levosimendan-induced postconditioning and cannot be overcome by an increased concentration. (2) Furthermore, cardioprotection under hyperglycemia can be restored by combining Levosimendan and the mPTP blocker CsA.

Cardioprotective Properties of Mannitol-Involvement of Mitochondrial Potassium Channels.

Cardiac preconditioning (PC) and postconditioning (PoC) are powerful measures against the consequences of myocardial ischemia and reperfusion (I/R) injury. Mannitol-a hyperosmolar solution-is clinically used for treatment of intracranial and intraocular pressure or promotion of diuresis in renal failure. Next to these clinical indications, different organ-protective properties-e.g., perioperative neuroprotection-are described. However, whether Mannitol also confers cardioprotection via a pre- and/or postconditioning stimulus, possibly reducing consequences of I/R injury, remains to be seen. Therefore, in the present study we investigated whether (1) Mannitol-induced pre- and/or postconditioning induces myocardial infarct size reduction and (2) activation of mitochondrial ATP-sensitive potassium (mKATP) channels is involved in cardioprotection by Mannitol. Experiments were performed on isolated hearts of male Wistar rats via a pressure controlled Langendorff system, randomized into 7 groups. Each heart underwent 33 min of global ischemia and 60 min of reperfusion. Control hearts (Con) received Krebs-Henseleit buffer as vehicle only. Pre- and postconditioning was achieved by administration of 11 mmol/L Mannitol for 10 min before ischemia (Man-PC) or immediately at the onset of reperfusion (Man-PoC), respectively. In further groups, the mKATP channel blocker 5HD, was applied with and without Mannitol, to determine the potential underlying cardioprotective mechanisms. Primary endpoint was infarct size, determined by triphenyltetrazolium chloride staining. Mannitol significantly reduced infarct size both as a pre- (Man-PC) and postconditioning (Man-PoC) stimulus compared to control hearts (Man-PC: 31 ± 4%; Man-PoC: 35 ± 6%, each p < 0.05 vs. Con: 57 ± 9%). The mKATP channel inhibitor completely abrogated the cardioprotective effect of Mannitol-induced pre- (5HD-PC-Man-PC: 59 ± 8%, p < 0.05 vs. Man-PC) and postconditioning (5HD-PoC-Man-PoC: 59 ± 10% vs. p < 0.05 Man-PoC). Infarct size was not influenced by 5HD itself (5HD-PC: 60 ± 14%; 5HD-PoC: 54 ± 14%, each ns vs. Con). This study demonstrates that Mannitol (1) induces myocardial pre- and postconditioning and (2) confers cardioprotection via activation of mKATP channels.

Remote ischemic preconditioning does not induce activation of Akt and STAT5 in the rat heart.

Remote ischemic preconditioning (RIPC) is hypothesized to be a promising cardioprotective strategy to protect hearts against ischemia and reperfusion (I/R) injury; however, the current understanding of the underlying signal transduction pathways involved remains unclear. It has been previously demonstrated that protein kinase B/AKT, which is a crucial protein of the reperfusion injury salvage kinases pathway, and STAT5, which is a member of the survivor activating factor enhancement pathway, serve a pivotal role in cardioprotection. However, whether and at what time-points (TPs) RIPC leads to the activation of AKT and STAT5 in a rat model of RIPC and I/R injury remains to be determined. The present study hypothesized that RIPC may induce the phosphorylation of AKT and/or STAT5 immediately following RIPC and/or at a later TP with or without subsequent I/R. In the first set of experiments (part A), male Wistar rats were randomized into 2 groups (n=6 per group): The first group underwent RIPC via a hind limb tourniquet (4x5 min I/R episodes), while the second group received the respective sham treatment. In the second set of experiments (part B), the rats were randomized into 4 groups (n=6 per group) that either underwent RIPC or sham treatment prior to 35 min of ischemia by occlusion of the left anterior descending coronary artery followed by 120 min reperfusion or a respective sham treatment. At the end of the experiments, the heart tissue was isolated in order to analyze the phosphorylation levels of AKT and STAT5. The results revealed that RIPC did not induce the immediate or late phosphorylation of AKT or STAT5. In addition, following I/R, the activation of AKT and STAT5 was not modulated by RIPC. In conclusion, the findings of the present study suggested that RIPC-induced cardioprotection may not be mediated by the activation of AKT or STAT5 at the investigated TPs.

Dexmedetomidine Provides Cardioprotection During Early or Late Reperfusion Mediated by Different Mitochondrial K+-Channels.

BACKGROUND: Cardioprotective interventions-such as pharmacological postconditioning-are a promising strategy to reduce deleterious consequences of ischemia and reperfusion injury (I/RI) in the heart, especially as timing and onset of myocardial infarction are unpredictable. Pharmacological postconditioning by treatment with dexmedetomidine (Dex), an α2-adrenoreceptor agonist, during reperfusion protects hearts from I/RI, independently of time point and duration of application during the reperfusion phase. The mitochondrial ATP-sensitive K (mKATP) and mitochondrial large-conductance calcium-sensitive potassium channel (mBKCa) play a pivotal role in mediating this cardioprotective effect. Therefore, we investigated whether Dex-induced cardioprotection during early or late reperfusion is mediated variously by these mitochondrial K-channels. METHODS: Hearts of male Wistar rats were randomized into 8 groups and underwent a protocol of 15 minutes adaption, 33 minutes ischemia, and 60 minutes reperfusion in an in vitro Langendorff-system. A 10-minute treatment phase was started directly (first subgroup, early reperfusion) or 30 minutes (second subgroup, late reperfusion) after the onset of reperfusion. Control (Con) hearts received vehicle only. In the first subgroup, hearts were treated with 3 nM Dex, 100 µM mKATP-channel blocker 5-hydroxydecanoate (5HD) or 1 µM mBKCa-channel blocker Paxilline (Pax) alone or with respective combinations (5HD + Dex, Pax + Dex). Hearts of the second subgroup received Dex alone (Dex30') or in combination with the respective blockers (5HD + Dex30', Pax + Dex30'). Infarct size was determined with triphenyltetrazoliumchloride staining. Hemodynamic variables were recorded during the whole experiment. RESULTS: During early reperfusion (first subgroup), the infarct size reducing effect of Dex (Con: 57% ± 9%, Dex: 31% ± 7%; P< .0001 versus Con) was completely abolished by 5HD and Pax (52% ± 6%; Pax + Dex: 53% ± 4%; each P< .0001 versus Dex), while both blockers alone had no effect on infarct size (5HD: 54% ± 8%, Pax: 53% ± 11%). During late reperfusion (second subgroup) the protective effect of Dex (Dex30': 33% ± 10%, P< .0001 versus Con) was fully abrogated by Pax (Pax + Dex30': 58% ± 7%, P < .0001 versus Dex30'), whereas 5HD did not block cardioprotection (5HD + Dex30': 36% ± 7%). Between groups and within each group throughout reperfusion no significant differences in hemodynamic variables were detected. CONCLUSIONS: Cardioprotection by treatment with Dex during early reperfusion seems to be mediated by both mitochondrial K-channels, whereas during late reperfusion only mBKCa-channels are involved.

Effluent from ischemic preconditioned hearts confers cardioprotection independent of the number of preconditioning cycles.

Coronary effluent collected from ischemic preconditioning (IPC) treated hearts induces myocardial protection in non-ischemic-preconditioned hearts. So far, little is known about the number of IPC cycles required for the release of cardioprotective factors into the coronary effluent to successfully induce cardioprotection. This study investigated the cardioprotective potency of effluent obtained after various IPC cycles in the rat heart. Experiments were performed on isolated hearts of male Wistar rats, mounted onto a Langendorff system and perfused with Krebs-Henseleit buffer. In a first part, effluent was taken before (Con) and after each IPC cycle (Eff 1, Eff 2, Eff 3). IPC was induced by 3 cycles of 5 min of global myocardial ischemia followed by 5 minutes of reperfusion. In a second part, hearts of male Wistar rats were randomized to four groups (each group n = 4-5) and underwent 33 min of global ischemia followed by 60 min of reperfusion. The previously obtained coronary effluent was administered for 10 minutes before ischemia as a preconditioning stimulus. Infarct size was determined at the end of reperfusion by triphenyltetrazoliumchloride (TTC) staining. Infarct size with control effluent was 54±12%. Effluent obtained after IPC confers a strong infarct size reduction independent of the number of IPC cycles (Eff 1: 27±5%; Eff 2: 35±7%; Eff 3: 35±8%, each P<0.05 vs. Con). Effluent extracted after one cycle IPC is comparably protective as after two or three cycles IPC.

Perioperative Cardioprotection: General Mechanisms and Pharmacological Approaches.

Cardioprotection encompasses a variety of strategies protecting the heart against myocardial injury that occurs during and after inadequate blood supply to the heart during myocardial infarction. While restoring reperfusion is crucial for salvaging myocardium from further damage, paradoxically, it itself accounts for additional cell death-a phenomenon named ischemia/reperfusion injury. Therefore, therapeutic strategies are necessary to render the heart protected against myocardial infarction. Ischemic pre- and postconditioning, by short periods of sublethal cardiac ischemia and reperfusion, are still the strongest mechanisms to achieve cardioprotection. However, it is highly impractical and far too invasive for clinical use. Fortunately, it can be mimicked pharmacologically, for example, by volatile anesthetics, noble gases, opioids, propofol, dexmedetomidine, and phosphodiesterase inhibitors. These substances are all routinely used in the clinical setting and seem promising candidates for successful translation of cardioprotection from experimental protocols to clinical trials. This review presents the fundamental mechanisms of conditioning strategies and provides an overview of the most recent and relevant findings on different concepts achieving cardioprotection in the experimental setting, specifically emphasizing pharmacological approaches in the perioperative context.

Combination of the Phosphodiesterase Inhibitors Sildenafil and Milrinone Induces Cardioprotection With Various Conditioning Strategies.

Ischemic preconditioning and postconditioning are strong measures preserving the heart against ischemia-reperfusion injury in experimental setting but are too invasive and impractical for clinical routine. The cardioprotective effects of ischemic preconditioning and postconditioning can be imitated pharmacologically, for example, with the phosphodiesterase inhibitors sildenafil and milrinone. We hypothesize that sildenafil-induced preconditioning is concentration dependent and further that a combined treatment of "nonprotective" versus "protective" concentrations of sildenafil and milrinone leads to a significant infarct size reduction. Experiments were performed on isolated hearts of male Wistar rats, randomized into 12 groups, mounted onto a Langendorff system, and perfused with Krebs-Henseleit buffer. All hearts underwent 33 minutes ischemia and 60 minutes of reperfusion. For determination of a concentration-dependent effect of sildenafil, hearts were perfused with increasing concentrations of sildenafil (0.1-1 µM) over 10 minutes before ischemia. In a second series of experiments, hearts were treated with 0.3 µM sildenafil or 1 µM milrinone as the "protective" concentrations. A higher concentration of respective drugs did not further reduce infarct size. In addition, a combination of "protective" and "nonprotective" concentrations of sildenafil and milrinone was applied. Sildenafil and milrinone in lower concentrations led to significant infarct size reduction, whereas combining both substances in cardioprotective concentrations did not enhance this effect. Sildenafil in a concentration of 0.3 µM induces myocardial protection. Furthermore, treatment with sildenafil and milrinone in lower concentrations had an equally strong cardioprotective effect regarding infarct size reduction compared with the administration of "protective" concentrations.

Topical Melatonin Improves Gastric Microcirculatory Oxygenation During Hemorrhagic Shock in Dogs but Does Not Alter Barrier Integrity of Caco-2 Monolayers.

Systemic administration of melatonin exerts tissue protective effects in the context of hemorrhagic shock. Intravenous application of melatonin prior to hemorrhage improves gastric microcirculatory perfusion and maintains intestinal barrier function in dogs. The aim of the present study was to analyze the effects of a topical mucosal melatonin application on gastric microcirculation during hemorrhagic shock in vivo and on mucosal barrier function in vitro. In a randomized cross-over study, six anesthetized female foxhounds received 3.3 mg melatonin or the vehicle as a bolus to the gastric and oral mucosa during physiological and hemorrhagic (-20% blood volume) conditions. Microcirculation was analyzed with reflectance spectrometry and laser doppler flowmetry. Systemic hemodynamic variables were measured with transpulmonary thermodilution. For analysis of intestinal mucosal barrier function in vitro Caco-2 monolayers were used. The transepithelial electrical resistance (TEER) and the passage of Lucifer Yellow (LY) from the apical to the basolateral compartment of Transwell chambers were measured. Potential barrier protective effects of melatonin against oxidative stress were investigated in the presence of the oxidant H2O2. During physiologic conditions topical application of melatonin had no effect on gastric and oral microcirculation in vivo. During hemorrhagic shock, gastric microcirculatory oxygenation (µHbO2) was decreased from 81 ± 8% to 50 ± 15%. Topical treatment with melatonin led to a significant increase in µHbO2 to 60 ± 13%. Topical melatonin treatment had no effect on gastric microcirculatory perfusion, oral microcirculation or systemic hemodynamics. Incubation of H2O2 stressed Caco-2 monolayers with melatonin did neither influence transepithelial electrical resistance nor LY translocation. Topical treatment of the gastric mucosa with melatonin attenuates the shock induced decrease in microcirculatory oxygenation. As no effects on local microcirculatory and systemic perfusion were recorded, the improved µHbO2 is most likely caused by a modulation of local oxygen consumption. In vitro melatonin treatment did not improve intestinal barrier integrity in the context of oxidative stress. These results extend the current knowledge on melatonin's protective effects during hemorrhage in vivo. Topical application of melatonin exerts differential effects on local microcirculation compared to systemic pretreatment and might be suitable as an adjunct for resuscitation of hemorrhagic shock.

MiR-21-5p but not miR-1-3p expression is modulated by preconditioning in a rat model of myocardial infarction.

Isoflurane (Iso) preconditioning (PC) is known to be cardioprotective against ischemia/reperfusion (I/R) injury. It was previously shown that microRNA-21-5p (miR-21-5p) is regulated by Iso-PC. It is unclear, if expression of cardiac enriched miR-1-3p is also affected by Iso-PC, and associated with activation of HIF1α (hypoxia-inducible factor 1-alpha).  Male Wistar rats (n = 6-8) were randomly assigned to treatment with or without 1 MAC Iso for 30 min, followed by 25 min of regional myocardial ischemia, with 120 min reperfusion. At the end of reperfusion, myocardial expression of miR-1-3p, miR-21-5p and mRNAs of two HIF-1α-dependent genes, VEGF (vascular endothelial growth factor) and HO-1 (heme oxygenase-1), were determined by quantitative PCR. Protein expression of a miR-21 target gene, PDCD4 (programmed cell death protein 4), was assessed by western blot analysis. Infarct sizes were analyzed with triphenyltetrazoliumchloride staining. MiR-21-5p expression was increased by Iso, whereas expression of miR-1-3p was not altered. The expression of VEGF but not HO-1 was induced by Iso. Iso-PC reduced infarct sizes compared to untreated controls. No regulation of miRNA and mRNA expression was detected after I/R. PDCD4 protein expression was not affected after Iso exposure. Expression of miR-21-5p, in contrast to miR-1-3p, is altered during this early time point of Iso-PC. HIF1α signaling seems to be involved in miR-21-5p regulation.

Influence of Hyperglycemia on Dexmedetomidine-Induced Cardioprotection in the Isolated Perfused Rat Heart.

Pharmacological preconditioning (PC) and postconditioning (PoC), for example, by treatment with the α2-adrenoreceptor agonist Dexmedetomidine (Dex), protects hearts from ischemia-reperfusion (I/R) injury in experimental studies, however, translation into the clinical setting has been challenging. Acute hyperglycemia adversely affects the outcome of patients with myocardial infarction. Additionally, it also blocks cardioprotection by multiple pharmacological agents. Therefore, we investigated the possible influence of acute hyperglycemia on Dexmedetomidine-induced pre- and postconditioning. Experiments were performed on the hearts of male Wistar rats, which were randomized into 7 groups, placed in an isolated Langendorff system and perfused with Krebs-Henseleit buffer. All hearts underwent 33 min of global ischemia, followed by 60 min of reperfusion. Control (Con) hearts received Krebs-Henseleit buffer (Con KHB), glucose (Con HG) or mannitol (Con NG) as vehicle only. Hearts exposed to hyperglycemia (HG) received KHB, containing 11 mmol/L glucose (an elevated, but commonly used glucose concentration for Langendorff perfused hearts) resulting in a total concentration of 22 mmol/L glucose throughout the whole experiment. To ensure comparable osmolarity with HG conditions, normoglycemic (NG) hearts received mannitol in addition to KHB. Hearts were treated with 3 nM Dexmedetomidine (Dex) before (DexPC) or after ischemia (DexPoC), under hyperglycemic or normoglycemic conditions. Infarct size was determined by triphenyltetrazoliumchloride staining. Acute hyperglycemia had no impact on infarct size compared to the control group with KHB (Con HG: 56 ± 9% ns vs. Con KHB: 56 ± 7%). DexPC reduced infarct size despite elevated glucose levels (DexPC HG: 35 ± 3%, p < 0.05 vs. Con HG). However, treatment with Dex during reperfusion showed no infarct size reduction under hyperglycemic conditions (DexPoC HG: 57 ± 9%, ns vs. Con HG). In contrast, hearts treated with mannitol demonstrated a significant decrease in infarct size compared to the control group (Con NG: 37 ± 3%, p < 0.05 vs. Con KHB). The combination of Dex and mannitol presents exactly opposite results to hearts treated with hyperglycemia. While DexPC completely abrogates infarct reduction through mannitol treatment (DexPC NG: 55 ± 7%, p < 0.05 vs. Con NG), DexPoC had no impact on mannitol-induced infarct size reduction (DexPoC NG: 38 ± 4%, ns vs. Con NG). Acute hyperglycemia inhibits DexPoC, while it has no impact on DexPC. Treatment with mannitol induces cardioprotection. Application of Dex during reperfusion does not influence mannitol-induced infarct size reduction, however, administering Dex before ischemia interferes with mannitol-induced cardioprotection.

Activation of PKG and Akt Is Required for Cardioprotection by Ramelteon-Induced Preconditioning and Is Located Upstream of mKCa-Channels.

Ramelteon is a Melatonin 1 (MT1)-and Melatonin 2 (MT2)-receptor agonist conferring cardioprotection by pharmacologic preconditioning. While activation of mitochondrial calcium-sensitive potassium (mKCa)-channels is involved in this protective mechanism, the specific upstream signaling pathway of Ramelteon-induced cardioprotection is unknown. In the present study, we (1) investigated whether Ramelteon-induced cardioprotection involves activation of protein kinase G (PKG) and/or protein kinase B (Akt) and (2) determined the precise sequence of PKG and Akt in the signal transduction pathway of Ramelteon-induced preconditioning. Hearts of male Wistar rats were randomized and placed on a Langendorff system, perfused with Krebs-Henseleit buffer at a constant pressure of 80 mmHg. All hearts were subjected to 33 min of global ischemia and 60 min of reperfusion. Before ischemia, hearts were perfused with Ramelteon (Ram) with or without the PKG or Akt inhibitor KT5823 and MK2206, respectively (KT5823 + Ram, KT5823, MK2206 + Ram, MK2206). To determine the precise signaling sequence, subsequent experiments were conducted with the guanylate cyclase activator BAY60-2770 and the mKCa-channel activator NS1619. Infarct size was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Ramelteon-induced infarct size reduction was completely blocked by KT5823 (p = 0.0012) and MK2206 (p = 0.0005). MK2206 with Ramelteon combined with BAY60-2770 reduced infarct size significantly (p = 0.0014) indicating that PKG activation takes place after Akt. Ramelteon and KT5823 (p = 0.0063) or MK2206 (p = 0.006) respectively combined with NS1619 also significantly reduced infarct size, indicating that PKG and Akt are located upstream of mKCa-channels. This study shows for the first time that Ramelteon-induced preconditioning (1) involves activation of PKG and Akt; (2) PKG is located downstream of Akt and (3) both enzymes are located upstream of mKCa-channels in the signal transduction pathway.

The Melatonin Receptor Agonist Ramelteon Induces Cardioprotection that Requires MT2 Receptor Activation and Release of Reactive Oxygen Species.

PURPOSE: The melatonin receptor (MT) agonist ramelteon has a higher affinity to MT1 than for MT2 receptors and induces cardioprotection by involvement of mitochondrial potassium channels. Activation of mitochondrial potassium channels leads to release of free radicals. We investigated whether (1) ramelteon-induced cardioprotection is MT2 receptor specific and (2) if free radicals are involved in ramelteon-induced cardioprotection. METHODS: Hearts of male Wistar rats were randomized, placed on a Langendorff system, and perfused with Krebs-Henseleit buffer at a constant pressure of 80 mmHg. All hearts were subjected to 33 min of global ischemia and 60 min of reperfusion. Before ischemia hearts were perfused with ramelteon (Ram) with or without the MT2 receptor inhibitor 4-phenyl-2-propionamidotetralin (4P-PDOT+Ram, 4P-PDOT). In subsequent experiments, ramelteon was administered together with the radical oxygen species (ROS) scavenger N-2-mercaptopropionylglycine (MPG+Ram). To determine whether the blockade of ramelteon-induced cardioprotection can be restored, we combined ramelteon and MPG with mitochondrial permeability transition pore (mPTP) inhibitor cyclosporine A (CsA) at different time points. Infarct size was determined by triphenyltetrazolium chloride (TTC) staining. RESULTS: Ramelteon-induced infarct size reduction was completely blocked by 4P-PDOT and MPG. Ramelteon and MPG combined with CsA before ischemia were not cardioprotective but CsA at the onset of reperfusion could restore infarct size reduction. CONCLUSIONS: This study shows for the first time that despite the higher affinity to MT1 receptors, (1) ramelteon-induced cardioprotection involves MT2 receptors, (2) cardioprotection requires ROS release, and (3) inhibition of the mPTP can restore infarct size reduction.

Influence of Hyperglycemia During Different Phases of Ischemic Preconditioning on Cardioprotection-A Focus on Apoptosis and Aggregation of Granulocytes.

BACKGROUND: Ischemic preconditioning (IPC) protects the myocardium against ischemia/reperfusion injury. Evidence suggests that hyperglycemia inhibits IPC-induced cardioprotection. The effects of hyperglycemia initiated during different phases of IPC on myocardial injury were characterized with emphasis on apoptosis and aggregation of polymorphonuclear granulocytes (PMN). METHODS: Male Wistar rats were subjected to 35 min of myocardial ischemia and 2 h of reperfusion. Control animals were not further treated. IPC was induced by three cycles of 3 min ischemia and 5 min of reperfusion before major ischemia. Hyperglycemia (blood glucose more than 22.2 mmol/L) was induced by glucose administration with or without IPC during different phases (trigger- (before ischemia), mediator- (during ischemia), early reperfusion-phase). One additional group received an anti-PMN-antibody before ischemia. Infarct size was quantified by triphenyltetrazolium chloride staining. Cytochrome C release and B-cell lymphoma two (Bcl-2) expression were assessed by western blot analysis. Poly-ADP-Ribose staining and PMN accumulation were quantified with immunohistochemistry and histochemistry. RESULTS: IPC reduced infarct size compared with control. Hyperglycemia completely abolished IPC-induced cardioprotection independent of the time point of initiation. Hyperglycemia before and during major ischemia but without IPC also slightly reduced infarct size. IPC reduced the accumulation of PMNs. This effect was reversed by hyperglycemia during trigger- and mediator-phase but not by hyperglycemia during reperfusion. Hyperglycemia alone had no effect on PMN accumulation. In all treatment groups, signs of myocardial apoptosis were reduced compared with control. IPC alone, combined with hyperglycemia and anti-PMN treatment, reduced apoptosis by a Bcl-2-associated mechanism. Hyperglycemia alone reduced apoptosis by a Bcl-2-independent pathway. CONCLUSION: Hyperglycemia inhibits IPC-induced cardioprotection independent of its onset. Furthermore, hyperglycemia prevents apoptosis and IPC-induced reduction of PMN aggregation.