Dried blood spot eluates are suitable for testing of SARS-CoV-2 IgG antibodies targeting Spike protein 1 and Nucleocapsid protein.

Dried blood spots (DBS) provide easy handling and are thus a beneficial tool for data collection, e.g. for epidemiological studies. The suitability of DBS for the assessment of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was analyzed with regards to the use in future studies addressing seroprevalence in the population. 121 volunteers gave a venous blood sample and capillary blood samples on two DBS cards (PerkinElmer and Ahlstrom-Munksjö) via self-sampling under supervision. All samples were analyzed using the Anti-SARS-CoV-2 ELISA (IgG) and the Anti-SARS-CoV-2 NCP ELISA (IgG) from EUROIMMUN performed on the EUROIMMUN EUROLabWorkstation ELISA. Correlation coefficients between ELISA results based on the different sampling methods were calculated. Results of DBS analysis for SARS-CoV-2 IgG S1 and NCP highly correlated with the serum values (r = 0.96). In addition, the calculation of the phi coefficient showed no significant difference between the qualitative results of both sampling methods (rφ = 0.98-1.0). Further analysis of DBS eluates after prolonged storage of 6-8 h also showed a high correlation with serum results (r = 0.97 and r = 0.93, respectively). The study results indicate suitability of DBS for the analysis of antibodies against SARS-CoV-2 S1 and NCP. For DBS eluate, a stability of 6-8 h for measurement of SARS-CoV-2 antibodies can be assumed.

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.

Bilirubin-A Possible Prognostic Mortality Marker for Patients with ECLS.

Extracorporeal life support (ECLS) is a promising therapeutic option for patients with refractory cardiogenic shock. However, as the mortality rate still remains high, there is a need for early outcome parameters reflecting therapy success or futility. Therefore, we investigated whether liver enzyme levels could serve as prognostic mortality markers for patients with ECLS. The present study is a retrospective single-center cohort study. Adult patients >18 years of age who received ECLS therapy between 2011 and 2018 were included. Bilirubin, glutamic-oxaloacetic transaminase (GOT), and glutamic-pyruvic-transaminase (GPT) serum levels were analyzed at day 5 after the start of the ECLS therapy. The primary endpoint of this study was all-cause in-hospital mortality. A total of 438 patients received ECLS during the observation period. Based on the inclusion criteria, 298 patients were selected for the statistical analysis. The overall mortality rate was 42.6% (n = 127). The area under the curve (AUC) in the receiver operating characteristic curve (ROC) for bilirubin on day 5 was 0.72 (95% confidence interval (CI): 0.66-0.78). Cox regression with multivariable adjustment revealed a significant association between bilirubin on day 5 and mortality, with a hazard ratio (HR) of 2.24 (95% CI: 1.53-3.30). Based on the results of this study, an increase in serum bilirubin on day 5 of ECLS therapy correlates independently with mortality.

Intestinal Microbiota of Fattening Pigs Offered Non-Fermented and Fermented Liquid Feed with and without the Supplementation of Non-Fermented Coarse Cereals.

Introducing high numbers of lactic acid bacteria into the gastrointestinal tract of pigs via fermented liquid feed (FLF) could have an impact on intestinal bacterial ecosystems. Twenty piglets were allocated into four groups and fed a botanically identical liquid diet that was offered either non-fermented (twice), fully fermented or partially fermented but supplemented with 40% of non-fermented coarse cereals. Microbiota studies were performed on the small and large intestine digesta and faecal samples. A 16S rRNA gene amplification was performed within the hypervariable region V4 and sequenced with the Illumina MiSeq platform. R (version 3.5.2) was used for the statistical analyses. The digesta of the small intestines of pigs fed FLF were dominated by Lactobacillaceae (relative abundance up to 95%). In the colonic contents, the abundance of Lactobacillaceae was significantly higher only in the pigs fed the FLF supplemented with non-fermented coarse cereals. Additionally, the digesta of the small and large intestines as well as in the faeces of the pigs fed the FLF supplemented with non-fermented coarse cereals were significantly enriched for two operational taxonomic units (OTUs) belonging to the genus Lactobacillus and Bifidobacterium. The FLF supplemented with non-fermented coarse cereals had probiotic and prebiotic-like impacts on the intestinal and faecal bacterial composition of pigs.

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.

Characteristics of Dexmedetomidine Postconditioning in the Field of Myocardial Ischemia-Reperfusion Injury.

BACKGROUND: Timing and onset of myocardial ischemia are mostly unpredictable. Therefore, postconditioning could be an effective cardioprotective intervention. Because ischemic postconditioning is an invasive and not practicable treatment, pharmacological postconditioning would be a more suitable alternative cardioprotective measure. For the α2-adrenoreceptor agonist, dexmedetomidine postconditioning has been shown. However, data on a concentration-dependent effect of dexmedetomidine are lacking. Furthermore, it is unclear whether the time point and/or duration of dexmedetomidine administration in the reperfusion period is of relevance. We set out to determine whether infarct size reduction by dexmedetomidine is concentration dependent and whether time point and/or duration of dexmedetomidine application has an impact on the effect size of cardio protection. METHODS: Hearts of male Wistar rats were randomized and placed on a Langendorff system perfused with Krebs-Henseleit buffer at a constant pressure of 80 mm Hg. All hearts were subjected to 33 minutes of global ischemia and 60 minutes of reperfusion. In part I of the study, a concentration-response effect was determined by perfusing hearts with various concentrations of dexmedetomidine (0.3-100 nM) at the onset of reperfusion. Based on these results, part II of the study was conducted with 3 nM dexmedetomidine. Application of dexmedetomidine started directly at the onset of reperfusion (Dex60) and 15 minutes (Dex15), 30 minutes (Dex30), or 45 minutes (Dex45) after the start of reperfusion and lasted always until the end of the reperfusion period. Infarct size was determined by triphenyltetrazolium chloride staining. RESULTS: In part I, infarct size in control (Con) hearts was 62% ± 4%. Three-nanometer dexmedetomidine was the lowest most effective cardioprotective concentration and reduced infarct size to 24% ± 7% (P < .0001 versus Con). Higher concentrations did not confer stronger protection. Infarct size in control hearts from part II was 66% ± 6%. Different starting times and/or durations of application resulted in similar infarct size reduction (all P < .0001 versus Con). CONCLUSIONS: Postconditioning by dexmedetomidine is concentration dependent in ranges between 0.3 and 3 nM. Increased concentrations above 3 nM do not further enhance this cardioprotective effect. This cardioprotective effect is independent of time point and length of application in the reperfusion period.

Impact of Ca2+-Sensitive Potassium Channels in Levosimendan-Induced Postconditioning.

PURPOSE: Small and big conductance Ca2+-sensitive potassium (KCa) channels are involved in cardioprotective measures aiming at reducing myocardial reperfusion injury. For levosimendan, infarct size-reducing effects were shown. Whether activation of these channels is involved in levosimendan-induced postconditioning is unknown. We hypothesized that levosimendan exerts a concentration-dependent cardioprotective effect and that both types of Ca2+-sensitive potassium channels are involved. METHODS: In a prospective blinded experimental laboratory investigation, 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. At the onset of reperfusion, hearts were perfused with various concentrations of levosimendan (0.03-1 µM) in order to determine a concentration-response relationship. To elucidate the involvement of KCa-channels for the observed cardioprotection, in the second set of experiments, 0.3 µM levosimendan was administered in combination with the subtype-specific KCa-channel inhibitors paxilline (1 µM, big KCa-channel) and NS8593 (0.1 µM, small KCa-channel) respectively. Infarct size was determined by tetrazolium chloride (TTC) staining. RESULTS: Infarct size in controls was 60 ± 7% and 59 ± 6% respectively. Levosimendan at a concentration of 0.3 µM reduced infarct size to 30 ± 5% (P < 0.0001 vs. control). Higher concentrations of levosimendan did not induce a stronger effect. Paxilline but not NS8593 completely abolished levosimendan-induced cardioprotection while both substances alone had no effect on infarct size. CONCLUSIONS: Cardioprotection by levosimendan-induced postconditioning shows a binary phenomenon, either ineffective or with maximal effect. The cardioprotective effect requires activation of big but not small KCa channels.

Milrinone-Induced Pharmacological Preconditioning in Cardioprotection: Hints for a Role of Mitochondrial Mechanisms.

The activation of mitochondrial calcium-sensitive potassium (mBKCa) channels is crucially involved in cardioprotection induced by preconditioning. For milrinone (Mil)-induced preconditioning, the involvement of mBKCa-channels and further mitochondrial signaling is unknown. We hypothesize that (1) Mil-induced preconditioning is concentration-dependent and (2) that the activation of mBKCa-channels, release of reactive oxygen species (ROS), and the mitochondrial permeability transition pore (mPTP) could be involved. Isolated hearts of male Wistar rats were perfused with Krebs-Henseleit buffer and underwent 33 min of ischemia followed by 60 min of reperfusion. For determination of a concentration-dependent effect of Mil, hearts were perfused with different concentrations of Mil (0.3-10 µM) over 10 min before ischemia. In a second set of experiments, in addition to controls, hearts were pretreated with the lowest protective concentration of 1 µM Mil either alone or combined with the mBKCa-channel blocker paxilline (Pax + Mil), or paxilline alone (Pax). In additional groups, Mil was administered with and without the ROS scavenger N-2-mercaptopropionylglycine (MPG + Mil, MPG) or the mPTP inhibitor cyclosporine A (MPG + Mil + CsA, CsA + Mil), respectively. Infarct sizes were determined by triphenyltetrazolium chloride (TTC) staining. The lowest and most cardioprotective concentration was 1 µM Mil (Mil 1: 32 ± 6%; p < 0.05 vs. Con: 63 ± 8% and Mil 0.3: 49 ± 6%). Pax and MPG blocked the infarct size reduction of Mil (Pax + Mil: 53 ± 6%, MPG + Mil: 59 ± 7%; p < 0.05 vs. Mil: 34 ± 6%) without having an effect on infarct size when administered alone (Pax: 53 ± 7%, MPG: 58 ± 5%; ns vs. Con). The combined administration of CsA completely restored the MPG-inhibited cardioprotection of Mil (MPG + Mil + CsA: 35 ± 7%, p < 0.05 vs. MPG + Mil). Milrinone concentration-dependently induces preconditioning. Cardioprotection is mediated by the activation of mBKCa-channels, release of ROS and mPTP inhibition.

Impact of Anesthetics on Cardioprotection Induced by Pharmacological Preconditioning.

Anesthetics, especially propofol, are discussed to influence ischemic preconditioning. We investigated whether cardioprotection by milrinone or levosimendan is influenced by the clinically used anesthetics propofol, sevoflurane or dexmedetomidine. Hearts of male Wistar rats were randomised, placed on a Langendorff system and perfused with Krebs⁻Henseleit buffer (KHB) at a constant pressure of 80 mmHg. All hearts underwent 33 min of global ischemia and 60 min of reperfusion. Three different anesthetic regimens were conducted throughout the experiments: propofol (11 µM), sevoflurane (2.5 Vol%) and dexmedetomidine (1.5 nM). Under each anesthetic regimen, pharmacological preconditioning was induced by administration of milrinone (1 µM) or levosimendan (0.3 µM) 10 min before ischemia. Infarct size was determined by TTC staining. Infarct sizes in control groups were comparable (KHB-Con: 53 ± 9%, Prop-Con: 56 ± 9%, Sevo-Con: 56 ± 8%, Dex-Con: 53 ± 9%; ns). Propofol completely abolished preconditioning by milrinone and levosimendan (Prop-Mil: 52 ± 8%, Prop-Lev: 52 ± 8%; ns versus Prop-Con), while sevoflurane did not (Sevo-Mil: 31 ± 9%, Sevo-Lev: 33 ± 7%; p < 0.05 versus Sevo-Con). Under dexmedetomidine, results were inconsistent; levosimendan induced infarct size reduction (Dex-Lev: 36 ± 6%; p < 0.05 versus Dex-Con) but not milrinone (Dex-Mil: 51 ± 8%; ns versus Dex-Con). The choice of the anesthetic regimen has an impact on infarct size reduction by pharmacological preconditioning.

Cardioprotective Properties of Omecamtiv Mecarbil against Ischemia and Reperfusion Injury.

Omecamtiv mecarbil (OM) is a first-in-class myosin activator. It was developed as a new inotropic therapy option for heart failure and is currently the object of a phase 3 clinical trial program. OM activates ryanodine receptors, which were shown to be involved in cardioprotection induced by conditioning strategies. We hypothesize that OM exerts a concentration-dependent cardioprotective effect through pre- and postconditioning. Isolated male Wistar rat hearts underwent 33 min of global ischemia and 60 min of reperfusion. OM was administered in various concentrations (1, 3, 10, and 30 µM) over 10 min prior to ischemia. Based on these results, in subsequent experiments 3 and 10 µM OM were given over 10 min after ischemia. Infarct sizes were determined by TTC staining. In controls, the infarct size was 60% ± 10% and 59% ± 12%, respectively. Ten micromolar OM before ischemia reduced the infarct size to 33% ± 8%. The lower concentrations did not initiate cardioprotection, and the next highest concentration did not enhance the protective effect. Even if 10 µM OM was given in the early reperfusion phase, it significantly reduced the infarct size (31% ± 6%), whereas 3 µM OM did not trigger a protective effect (58% ± 15%). This study shows for the first time that OM induces cardioprotection by pre- and postconditioning with a binary phenomenon, which is either ineffective or has a maximal effect.

Cardioprotection by Humoral Factors Released After Remote Ischemic Preconditioning Depends on Anesthetic Regimen.

OBJECTIVES: Remote ischemic preconditioning (RIPC) is a practicable and noninvasive method to protect the heart against ischemia reperfusion injury. Unfortunately results from clinical studies are not convincing. Propofol is suggested to be an inhibiting factor of cardioprotection by RIPC, but the underlying mechanism is still unknown. We investigated whether after RIPC the release of humoral factors and/or the direct cardioprotective effect at the myocardium is inhibited by propofol. DESIGN: Randomized, prospective, blinded laboratory investigation. SETTING: Experimental laboratory. PATIENTS/SUBJECTS: Male Wistar rats. INTERVENTIONS: Repetitive hind limb ischemia in rats-blood plasma transfers to isolated rat heart. MEASUREMENTS AND MAIN RESULTS: In male Wistar rats (six groups, each n = 6/group), RIPC was induced by four cycles of 5 minutes bilateral hind limb ischemia alternately with 5 minutes of reperfusion. Blood samples were taken with (RIPC) and without RIPC (Con). Rats received continuous anesthesia with pentobarbital (Pento, 40 mg/kg body weight/hr) or propofol (Prop, 12 mg/kg body weight/hr), respectively. Cardioprotective properties of the blood plasma was investigated in the rat heart in vitro (six groups, each n = 6/group) perfused with Krebs-Henseleit buffer alone or with propofol (10 µM). Plasma was administered over 10 minutes before myocardial ischemia. All hearts underwent 33 minutes of global ischemia followed by 1 hour of reperfusion. At the end of the experiments, infarct size was determined by triphenyl-tetrazolium-chloride staining. RIPC plasma from pentobarbital anesthetized rats (Pento-RIPC) reduced infarct size from 64% (62-71%) (Pento-Con) to 34% (30-39%) (p < 0.0001). Infarct size with control plasma from propofol anesthetized rats was 59% (58-64%) (Prop-Con). RIPC plasma could not induce cardioprotection (Prop-RIPC: 63% [56-70%] ns vs Prop-Con). In contrast, RIPC plasma from pentobarbital anesthetized rats induced a significant infarct size reduction under propofol perfusion (Pento-RIPC: 34% [30-42%] vs Pento-Con: 54% [53-63%]; p < 0.0001). CONCLUSIONS: Loss of cardioprotection by RIPC during propofol anesthesia depends on inhibition of release of humoral factors.

Preconditioning by Levosimendan is Mediated by Activation of Mitochondrial Ca2+-Sensitive Potassium (mBKCa) Channels.

PURPOSE: Activation of mitochondrial large-conductance Ca2+-sensitive potassium (mBKCa)-channels is a crucial step for cardioprotection by preconditioning. Whether activation of these channels is involved in levosimendan-induced preconditioning is unknown. We investigated if cardioprotection by levosimendan requires activation of mBKCa-channels in the rat heart in vitro. METHODS: In a prospective blinded experimental laboratory investigation, 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 different concentrations of levosimendan (0.03-1 µM) for determination of a dose-effect curve. In a second set of experiments, 0.3 µM levosimendan was administered in combination with the mBKCa-channel inhibitor paxilline (1 µM). Infarct size was determined by TTC staining. RESULTS: In control, animal's infarct size was 58 ± 7%. Levosimendan at a concentration of 0.3 µM reduced infarct size to 30 ± 7% (P < 0.05 vs. control). Higher concentrations with 1 µM levosimendan did not confer stronger protection. Paxilline completely blocked levosimendan-induced cardioprotection while paxilline alone had no effect on infarct size. CONCLUSIONS: This study shows that activation of mBKCa-channels plays a pivotal role in levosimendan-induced preconditioning.

Melatonin Receptor Agonist Ramelteon Reduces Ischemia-Reperfusion Injury Through Activation of Mitochondrial Potassium Channels.

Activation of melatonin receptors induces cardioprotection. Mitochondrial potassium channels (mKCa and mKATP) are involved in the signaling cascade of preconditioning. The melatonin receptor agonist ramelteon is an approved oral medication for treatment of insomnia, but nothing is known about possible cardioprotective properties. We investigated whether (1) ramelteon induces cardioprotection mediated by the melatonin receptor; (2) this effect is concentration-dependent; and (3) mKCa and/or mKATP channels are critically involved in ramelteon-induced cardioprotection. Hearts of male Wistar rats were randomized and placed on a Langendorff system, perfused with Krebs-Henseleit buffer at a constant pressure of 80 mm Hg. All hearts were subjected to 33 minutes of global ischemia and 60 minutes of reperfusion. Before, ischemic hearts were perfused with different concentrations of ramelteon (0.01-5 µM) for determination of a concentration-effect curve. In subsequent experiments, the lowest protective concentration of ramelteon was administered together with paxilline (mKCa channel inhibitor) and 5-hydroxydecanoate (mKATP channel inhibitor). To determine whether the reduction of ischemia and reperfusion injury by ramelteon is mediated by melatonin receptor, we combined ramelteon with luzindole, a melatonin receptor antagonist. Infarct size was determined by triphenyltetrazolium chloride staining. In control animals, infarct size was 58% ± 6%. Ramelteon in a concentration of 0.03 µM reduced infarct size to 28% ± 4% (P < 0.0001 vs. Con). A lower concentration of ramelteon did not initiate cardioprotection, and higher concentrations did not further decrease infarct size. Paxilline, 5-hydroxydecanoate, and luzindole completely blocked the ramelteon-induced cardioprotection. This study shows for the first time that (1) ramelteon induces cardioprotection through melatonin receptor; (2) the effect is not concentration-dependent; and (3) activation of mKCa and mKATP channels is involved.

Impact of Anesthetic Regimen on Remote Ischemic Preconditioning in the Rat Heart In Vivo.

Remote ischemic preconditioning (RIPC) seems to be a promising cardioprotective strategy with contradictive clinical data suggesting the anesthetic regimen influencing the favorable impact of RIPC. This study aimed to investigate whether cardio protection by RIPC is abolished by anesthetic regimens. Male Wistar rats were randomized to 6 groups. Anesthesia was either maintained by pentobarbital (Pento) alone or a combination of sevoflurane (Sevo) and remifentanil or propofol (Prop) and remifentanil in combination with and without RIPC. RIPC reduced infarct size in Pento- and Sevo-anesthetized rats (Pento-RIPC: 30% ± 9% versus Pento-control [Con]: 65% ± 6%, P < .001; Sevo-RIPC: 31% ± 6% versus Sevo-Con: 61% ± 8%, P < .001), but RIPC did not initiate cardio protection in Prop-anesthetized animals (Prop-RIPC: 59% ± 6% versus Prop-Con: 59% ± 8%, P = 1.000). Cardio protection by RIPC is abolished by Prop.