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.

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.

The release of cardioprotective humoral factors after remote ischemic preconditioning in humans is age- and sex-dependent.

BACKGROUND: Preclinical and proof-of-concept studies suggest a cardioprotective effect of remote ischemic preconditioning (RIPC). However, two major clinical trials (ERICCA and RIPHeart) failed to show cardioprotection by RIPC. Aging and gender might be confounding factors of RIPC affecting the inter-organ signalling. Theoretically, confounding factors might prevent the protective potency of RIPC by interfering with cardiac signalling pathways, i.e. at the heart, and/or by affecting the release of humoral factor(s) from the remote organ, e.g. from the upper limb. This study investigated the effect of age and sex on the release of cardioprotective humoral factor(s) after RIPC in humans. METHODS: Blood samples were taken from young and aged, male and female volunteers before (control) and after RIPC (RIPC). To investigate the protective potency of the different plasma groups obtained from the human volunteers, isolated perfused hearts of young rats were used as bioassay. For this, hearts were perfused with the volunteer plasma (0.5% of coronary flow) before hearts underwent global ischemia and reperfusion. In addition, to characterize the protective potency of humoral factor(s) after RIPC to initiate protection not only in young but also aged hearts, plasma from young male volunteers were transferred to isolated hearts of aged rats. At the end of the experimental protocol, infarct sizes were determined by TTC-staining (expressed as % of left ventricle). RESULTS: RIPC plasma of young male volunteers reduced infarct size in young rat hearts from 47 ± 5 to 31 ± 10% (p = 0.02). In contrast, RIPC plasma of aged male volunteers had no protective effect. Infarct size after application of control plasma of young female volunteers was 33 ± 10%, and female RIPC plasma did not lead to an infarct size reduction. RIPC plasma of old female initiated no cardioprotection. RIPC plasma of young male volunteers reduced infarct size in isolated hearts from aged rats (41 ± 5% vs. 51 ± 5%; p < 0.001). CONCLUSIONS: The release of humoral factor(s) into the blood after RIPC in humans is affected by both age and sex. In addition, these blood borne factor(s) are capable to initiate cardioprotection within the aged heart.

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.

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.

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.

Activation of Melatonin Receptors by Ramelteon Induces Cardioprotection by Postconditioning in the Rat Heart.

Activation of melatonin receptors protects the heart against ischemia-reperfusion injury. Ramelteon, a clinically used drug for insomnia, acts via activation of melatonin receptors. We investigated whether ramelteon induces acute infarct size reduction by postconditioning. Male Wistar rats were randomized to 6 groups. Hearts were treated with melatonin and ramelteon at the beginning of reperfusion. The melatonin receptor inhibitor luzindole was administered with and without melatonin and ramelteon, respectively. Ramelteon reduced infarct size to the same extent as melatonin. Both effects were completely abolished by luzindole. The results show for the first time that ramelteon induces cardioprotection by postconditioning.

Tranexamic Acid Does Not Influence Cardioprotection by Ischemic Preconditioning and Remote Ischemic Preconditioning.

Prior studies have suggested that the antifibrinolytic drug aprotinin increases the infarct size after ischemia and reperfusion (I/R) and attenuates the effect of ischemic preconditioning (IPC). Aprotinin was replaced by tranexamic acid (TXA) in clinical practice. Here, we investigated whether TXA influences I/R injury and/or cardioprotection initiated by IPC and/or remote ischemic preconditioning (RIPC). Anesthetized male Wistar rats were randomized to 6 groups. Control animals were not further treated. Administration of TXA was combined with and without IPC and RIPC. Estimated treatment effect was 20%. Compared to control group (56% ± 11%), IPC reduced infarct size by 46% (30% ± 6%; mean difference, 26%; 95% confidence interval, 19-33; P < .0001), and RIPC reduced infarct size by 29% (40% ± 8%; mean difference, 16%; 95% confidence interval, 9-24; P < .011). Additional application of TXA had no effect on I/R injury and cardioprotection by IPC or RIPC. TXA does not abolish infarct size reduction by IPC or RIPC.

Milrinone-Induced Postconditioning Requires Activation of Mitochondrial Ca2+-sensitive Potassium (mBKCa) Channels.

OBJECTIVES: Cardioprotection by postconditioning requires activation of mitochondrial large-conductance Ca2+-sensitive potassium (mBKCa) channels. The involvement of these channels in milrinone-induced postconditioning is unknown. The authors determined whether cardioprotection by milrinone-induced postconditioning involves activation of mBKCa channels in the rat heart in vitro. DESIGN: Randomized, prospective, blinded laboratory investigation. SETTING: Experimental laboratory. PARTICIPANTS: Male Wistar rats. INTERVENTIONS: 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 minutes of global ischemia and 60 minutes of reperfusion. At the onset of reperfusion, hearts were perfused with different concentrations of milrinone (0.3-100 µM) for determination of a dose-effect curve. In a second set of experiments, 3 µM milrinone was administered in combination with the mBKCa channel inhibitor paxilline (1 µM). Infarct size was determined by triphenyltetrazoliumchloride staining. MEASUREMENTS AND MAIN RESULTS: In control animals, infarct size was 37 ± 7%. Milrinone at a concentration of 3 µM reduced infarct size to 22 ± 7% (p < 0.05 v control). Higher milrinone concentrations did not confer stronger protection. Paxilline completely blocked milrinone-induced cardioprotection whereas paxilline alone had no effect on infarct size. CONCLUSIONS: This study shows that activation of mBKCa channels plays a pivotal role in milrinone-induced postconditioning.

The Cardioprotective Effect of Dexmedetomidine in Rats Is Dose-Dependent and Mediated by BKCa Channels.

The alpha-2 receptor agonist Dexmedetomidine (Dex) protects the heart against ischemia-reperfusion injury. We investigated the signaling cascade underlying Dex-induced acute cardioprotection, with special emphasis on large-conductance Ca2+-sensitive potassium (BKCa) channels. Rats were anesthetized with pentobarbital. Hearts were isolated, mounted on a Langendorff system and perfused with Krebs-Henseleit buffer. Hearts underwent 33 minutes of ischemia followed by 60 minutes of reperfusion. Before the beginning of ischemia, Dex was administered at different doses (0.1-30 nM) for characterization of a dose-effect relationship. In another set of experiments, Dex (3 nM) was administered together with the BKCa channel inhibitor paxilline and the connexin-43 inhibitor peptide Gap27. Also, the BKCa channel opener NS1619 was administered. In control animals, infarct size was 49% ± 5%. Dex at 3-30 nM reduced infarct size to ∼22%, whereas lower (0.1-1 nM) doses reduced infarct size to ∼38%. Paxilline (1 µM) and GAP27 (6 µM) blocked the Dex-induced cardioprotection. NS1619 (10 µM) reduced infarct size to about the same magnitude as did the higher doses of Dex. Functional heart parameters and coronary flow were not different between the study groups. In male rats, the Dex-induced protection against ischemia-reperfusion injury involves connexin-43 and activation of BKCa channels.

Extended Second Window of Protection of Sevoflurane-induced Preconditioning.

Late preconditioning (LPC) can be induced by volatile anesthetics and initiates cardioprotection against ischemia/reperfusion injury for 3-4 days. We investigated the possibility to extend the time window of sevoflurane-induced LPC by repeated sevoflurane administration. An in vivo rat model of regional myocardial ischemia/reperfusion injury was used. Myocardial infarct size was determined by triphenyltetrazolium chloride staining at the end of the experiment. In the first series of experiments, male Wistar rats were randomized to 5 groups (each n = 8). Control animals were not treated further. Animals in the preconditioning groups inhaled sevoflurane for 60 minutes (1 MAC) 24, 48, 72, and 96 hours, respectively, before myocardial ischemia. Based on the findings of the first experimental series, another 6 groups of animals were investigated. Again, control animals were left untreated; all other animals received a second sevoflurane stimulus 72 hours after the first sevoflurane treatment, and myocardial ischemia was induced 24, 48, 72, and 96 hours, respectively, after the second sevoflurane treatment to investigate, whether the cardioprotective effect could be extended. Sevoflurane reduced infarct size after 24, 48, and 72 hours (each P < 0.05 vs. control) but not after 96 hours. The repeated administration of sevoflurane 72 hours after the first stimulus extended the time window of protection for additional 72 hours (each P < 0.05 vs. control). There was no myocardial protection 4 days after the second preconditioning stimulus. The time window of sevoflurane-induced LPC can be extended by an additional sevoflurane stimulus up to 72 hours after the initial sevoflurane exposure.

Cardioprotection by Remote Ischemic Preconditioning is Blocked in the Aged Rat Heart in Vivo.

OBJECTIVES: In animal studies, remote ischemic preconditioning (RIPC) is a powerful tool to protect the heart from ischemia and reperfusion injury. Unfortunately, this effect was not seen consistently in recent large clinical trials. Aging was shown to be a confounding factor for the effect of direct preconditioning in experimental studies, but whether aging also can influence the effect of RIPC and thus be responsible for the contradictory clinical effect is unknown. The aim of this study was to investigate whether the cardioprotective effect of RIPC was abolished by aging. DESIGN: Randomized, prospective, blinded laboratory investigation. SETTING: Experimental laboratory. PARTICIPANTS: Male Wistar rats. INTERVENTIONS: Anesthetized young (Y, 2-3 months) and aged (A, 22-24 months) male Wistar rats were randomized to 4 groups (n = 6 per group). Control animals (Y-Con and A-Con) were not treated further; RIPC groups (Y-RIPC and A-RIPC) received 4 cycles of 5 minutes of bilateral hind limb ischemia interspersed with 5 minutes reperfusion before myocardial ischemia and reperfusion. All animals underwent 25 minutes of regional myocardial ischemia and 120 minutes of reperfusion. At the end of reperfusion, infarct size was determined by TTC staining. MEASUREMENTS AND MAIN RESULTS: In the control group of young rats, infarct size was 56±9% of the area at risk. RIPC reduced infarct size to 31±9% (p<0.05 v Y-Con). Cardioprotection by RIPC was abolished completely in the aged rat heart (A-RIPC: 62±8%, A-Con: 63±4%; ns). CONCLUSIONS: The results of the authors' study showed that cardioprotection induced by remote ischemic preconditioning was blocked in the aged rat heart.

Estimated Long-Term Benefits of Finerenone in Heart Failure: A Prespecified Secondary Analysis of the FINEARTS-HF Randomized Clinical Trial.

Importance: People living with heart failure (HF) with mildly reduced or preserved ejection fraction have substantially curtailed life expectancy free from clinical events compared with their peers of comparable age. The nonsteroidal mineralocorticoid receptor antagonist, finerenone, was recently shown to reduce risks of cardiovascular events in this population over a median follow-up of 2.6 years; as patients with HF typically continue treatment beyond this time frame, estimating the potential long-term benefits of finerenone could inform shared clinical decision-making. Objective: To estimate the projected long-term treatment effects of finerenone in patients with HF with mildly reduced or preserved ejection fraction if treated over a patient's lifetime. Design, Setting, and Participants: Prespecified analyses were conducted of the FINEARTS-HF trial, a phase 3 randomized clinical trial conducted across 653 sites in 37 countries. Adults 40 years and older with symptomatic HF and left ventricular ejection fraction of 40% or greater were randomized from September 2020 to January 2023. Median (IQR) follow-up was 2.6 (1.9-3.0) years. Interventions: Finerenone (titrated to either 20 mg or 40 mg) or placebo. Main Outcomes and Measures: The primary composite outcome was time to cardiovascular death or worsening HF event. The long-term gains in survival free from a primary end point with finerenone were iteratively estimated with age-based Kaplan-Meier curves using age at randomization rather than time from randomization. Differences in areas under the survival curves between the finerenone and placebo arms represented event-free survival gains. Results: Among 6001 participants (median [IQR] age, 73 [66-79] years; 3269 male [54.5%]), mean survival free from the primary end point for a 55-year-old participant was 13.6 years (95% CI, 11.9-15.2 years) with finerenone and 10.5 years (95% CI, 6.8-11.3 years) with placebo, representing a gain in event-free survival of 3.1 years (95% CI, 0.8-5.4 years; P = .007). Mean event-free survival for a 65-year-old participant was 11.0 years (95% CI, 10.1-11.9 years) with finerenone and 8.9 years (95% CI, 8.1-9.8 years) with placebo, representing a gain of 2.0 years (95% CI, 0.8-3.3 years; P = .001). Projected mean event-free survival was numerically greater with finerenone than with placebo for every starting age between 50 to 80 years. Lifetime gains in event-free survival were observed even among individuals already treated with a sodium-glucose cotransporter 2 inhibitor (65-year-old participant: 3.1 years; 95% CI, 0.1-6.0 years; P = .04). Conclusions and Relevance: In this prespecified secondary analysis of the FINEARTS-HF randomized clinical trial, long-term treatment with finerenone was estimated to extend event-free survival by up to 3 years among people with HF with mildly reduced or preserved ejection fraction. Trial Registration: ClinicalTrials.gov Identifier: NCT04435626.

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.

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.

Matrixmetalloproteinases and tissue inhibitors of metalloproteinases: Immunhistochemical markers in the diagnosis of lethal myocardial infarctions?

Matrixmetalloproteinases (MMP) 2 and 9 as well as tissue inhibitor of metalloproteinases (TIMP) 1 were tested as markers of myocardial early ischemia/infarctions. Experiments with an animal model, the isolated Langendorff heart, and analysis of human tissue samples drawn during autopsies were performed. Results of the experiments with the Langendorff model implied that the detectable amount of the markers might increase early after the onset of ischemia, in less than one hour, under ideal conditions. The results of the examined human cases showed that MMP-2 is constantly detectable in human myocardial tissue with an increased amount in case of an infarction with longer survival times. MMP-9 and TIMP-1 were negative in control cases, distinct positive staining results were obtained mainly in cases of infarctions with longer survival times and only rarely in those with a short survival time. According to these results MMPs and TIMPs do not qualify as first choice markers of myocardial infarctions. As an interesting side finding in the Langendorff experiments, positive staining results for all three markers were seen in myocardial areas that were mechanically traumatized by ECG-electrodes or ligation of blood vessels. These findings make the markers interesting for forensic wound age estimation.

Echocardiographic Analysis of Cardiac Function after Infarction in Mice: Validation of Single-Plane Long-Axis View Measurements and the Bi-Plane Simpson Method.

Although echocardiography is commonly used to analyze cardiac function in small animal models of cardiac remodeling after myocardial infarction, the different echocardiographic methods are validated poorly. End-diastolic volume, end-systolic volume and ejection fraction were analyzed using either standard single-plane analysis from parasternal long-axis B-mode views (PSLAX) or the bi-plane Simpson method (using PSLAX and three short-axis views) and validated using magnetic resonance imaging as standard. Ejection fraction measured by PSLAX was moderately correlated with a coefficient of R2 = 0.49. The standard deviation of residuals was 9.91. Simpson analysis revealed an improved correlation coefficient of R2 = 0.77 and a reduction in standard deviation of residuals by 45% (5.45 vs. 9.92, p = 0.014). Subgroup analysis revealed that the high variation in PSLAX is due to changes in ventricular geometry after myocardial infarction. Our results indicate that the bi-plane Simpson method is advantageous for the assessment of cardiac function after myocardial infarction.

Opening of calcium-activated potassium channels improves long-term left-ventricular function after coronary artery occlusion in mice.

BACKGROUND: Opening of mitochondrial calcium-activated potassium channels (BKCa) reduces infarct size after myocardial ischemia/reperfusion injury (I/R). It is unknown if targeting BKCa-channels improves cardiac performance in the long-term after I/R. METHODS: Experiments were conducted in compliance with institutional and national guidelines in C57BL/6 mice (n=7-8/group). Animals were randomized into two groups. Preconditioning was induced by intraperitoneal application of NS1619 (NS, 1µg/g bw) 10min before ischemia, control animals (Con) received the vehicle. All animals underwent 45min of myocardial ischemia and four weeks of reperfusion. Transthoracal Echocardiography (TTE) was conducted one and four weeks after ischemia (TTEW1/TTEW4) and additionally a cardiac MRI was done in week four. At the end of experiments the infarction scar was determined by AZAN staining. RESULTS: TTE revealed that NS1619 improved ejection fraction one week (Con: 36±4%, NS: 45±4%; P<0.05) and four weeks after I/R (Con: 33±11%, NS: 46±8%; P<0.05). Preconditioning with NS1619 reduced end-diastolic volume at both time points (TTEW1: Con: 60±12µl, NS: 45±8µl; TTEW4: Con: 82±31µl, NS: 44±8µl; each P<0.05) and increased fractional shortening after four weeks (TTEW4: Con: 12±6%, NS: 24±8%; P<0.05). MRI-analysis after four weeks confirmed the echocardiographic results. NS1619 increased ejection fraction by 45% (MRI: Con: 29±6%, NS: 42±9%; P<0.05 vs. Con) and reduced end-diastolic and -systolic volume (EDV, ESV) compared to control (MRI: EDV: Con: 110±19µl, NS: 88±16µl; ESV: Con: 79±19µl, NS: 53±18µl; each P<0.05). Preconditioning reduced infarction scar after four weeks by 25% (Con: 12±3%, NS: 9±2%; P<0.05). CONCLUSIONS: Preconditioning by opening of BKCa-channels with NS1619 improves cardiac performance after four weeks of reperfusion and reduces myocardial infarction scar.

Morphine-Induced Preconditioning: Involvement of Protein Kinase A and Mitochondrial Permeability Transition Pore.

BACKGROUND: Morphine induces myocardial preconditioning (M-PC) via activation of mitochondrial large conductance Ca2+-sensitive potassium (mKCa) channels. An upstream regulator of mKCa channels is protein kinase A (PKA). Furthermore, mKCa channel activation regulates mitochondrial bioenergetics and thereby prevents opening of the mitochondrial permeability transition pore (mPTP). Here, we investigated in the rat heart in vivo whether 1) M-PC is mediated by activation of PKA, and 2) pharmacological opening of the mPTP abolishes the cardioprotective effect of M-PC and 3) M-PC is critically dependent on STAT3 activation, which is located upstream of mPTP within the signalling pathway. METHODS: Male Wistar rats were randomised to six groups (each n = 6). All animals underwent 25 minutes of regional myocardial ischemia and 120 minutes of reperfusion. Control animals (Con) were not further treated. Morphine preconditioning was initiated by intravenous administration of 0.3 mg/kg morphine (M-PC). The PKA blocker H-89 (10 µg/kg) was investigated with and without morphine (H-89+M-PC, H-89). We determined the effect of mPTP opening with atractyloside (5 mg/kg) with and without morphine (Atr+M-PC, Atr). Furthermore, the effect of morphine on PKA activity was tested in isolated adult rat cardiomyocytes. In further experiments in isolated hearts we tested the protective properties of morphine in the presence of STAT3 inhibition, and whether pharmacological prevention of the mPTP-opening by cyclosporine A (CsA) is cardioprotective in the presence of STAT3 inhibition. RESULTS: Morphine reduced infarct size from 64±5% to 39±9% (P<0.05 vs. Con). H-89 completely blocked preconditioning by morphine (64±9%; P<0.05 vs. M-PC), but H-89 itself had not effect on infarct size (61±10%; P>0.05 vs. Con). Also, atractyloside abolished infarct size reduction of morphine completely (65±9%; P<0.05 vs. M-PC) but had no influence on infarct size itself (64±5%; P>0.05 vs. Con). In isolated hearts STAT3 inhibitor Stattic completely abolished morphine-induced preconditioning. Administration of Stattic and mPTP inhibitor cyclosporine A reduced infarct size to 31±6% (Stat+CsA, P<0.05 vs. Con). Cyclosporine A alone reduced infarct size to 26±7% (CsA P<0.05 vs. Con). In cardiomyocytes, PKA activity was increased by morphine. CONCLUSION: Our data suggest that morphine-induced cardioprotection is mediated by STAT3-activation and inhibition of mPTP, with STA3 located upstream of mPTP. There is some evidence that protein kinase A is involved within the signalling pathway.

Impact of Mitochondrial Ca2+-Sensitive Potassium (mBKCa) Channels in Sildenafil-Induced Cardioprotection in Rats.

BACKGROUND: Mitochondrial large-conductance Ca2+-sensitive potassium (mBKCa) channels are involved in myocardial ischemic preconditioning. Their role in sildenafil-induced cardioprotection is unknown. We investigated whether sildenafil-induced acute cardioprotection is mediated by activation of mBKCa channels in the rat heart in vitro. METHODS: Male Wistar rats (n = 8 per group) were randomized and anesthetized with pentobarbital (90 mg/kg). Hearts were isolated, mounted on a Langendorff system and perfused with Krebs-Henseleit buffer at a constant pressure of 80 mmHg. Hearts underwent 30 min of global ischemia followed by 60 min of reperfusion. At the end of the experiments infarct size was determined by TTC staining. In the control group rats were not further treated. Sildenafil (3 µM) was administered over 10 min before the beginning of ischemia. The mBKCa channel inhibitor paxilline (1 µM) was administered with and without sildenafil before the onset of ischemia. The pathway underlying sildenafil-induced cardioprotection was further investigated with the protein kinase G blocker KT5823 (1 µM). Myocardial cGMP concentration was measured by ELISA. Data (mean±SD) were analysed with a one and two-way analysis of variance as appropriate. RESULTS: In control animals infarct size was 52±8%. Sildenafil increased cGMP concentration and reduced infarct size to 35±6% (P<0.05 vs. control). Paxilline and KT5823 completely blocked sildenafil-induced cardioprotection (paxilline+sildenafil: 50±8%, KT5823+sildenafil: 45±8%; both P<0.05 vs. sildenafil). Functional heart parameters and coronary flow were not different between the study groups. CONCLUSION: This study shows that in male rats protein kinase G-dependent opening of mBKCa channels plays a pivotal role in sildenafil-induced cardioprotection.