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

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 Tit 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. (C) 2017 Elsevier Inc. All rights reserve

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, 1 mu g/g bw) 10 min before ischemia, control animals (Con) received the vehicle. All animals underwent 45 min 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 +/- 12 mu l, NS: 45 +/- 8 mu l; TTEW4: Con: 82 +/- 31 mu l, NS: 44 +/- 8 mu l; 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 +/- 19 mu l, NS: 88 +/- 16 mu l; ESV: Con: 79 +/- 19 mu l, NS: 53 +/- 18 mu l; 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. (C) 2017 Elsevier B.V. All rights reserve

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

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. 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. 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. This study shows that in male rats protein kinase G-dependent opening of mBKCa channels plays a pivotal role in sildenafil-induced cardioprotectio

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 cardio-protection, 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 similar to 22%, whereas lower (0.1-1 nM) doses reduced infarct size to similar to 38%. Paxilline (1 mu M)and GAP27 (6 mu M) blocked the Dex-induced cardioprotection. NS1619 (10 mu M) 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 exposur

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 Pro

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% (P0.05 vs. Con). Also, atractyloside abolished infarct size reduction of morphine completely (65±9%; P0.05 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