Igor Khaliulin

Inhibition of Mitochondrial Permeability Transition Pore Opening by Ischemic Preconditioning Is Probably Mediated by Reduction of Oxidative Stress Rather Than Mitochondrial Protein Phosphorylation

Inhibition of mitochondrial permeability transition pore (MPTP) opening at reperfusion is critical for cardioprotection by ischemic preconditioning (IP). Some studies have implicated mitochondrial protein phosphorylation in this effect. Here we confirm that mitochondria rapidly isolated from preischemic control and IP hearts show no significant difference in calcium-mediated MPTP opening, whereas IP inhibits MPTP opening in mitochondria isolated from IP hearts following 30 minutes of global normothermic ischemia or 3 minutes of reperfusion. Analysis of protein phosphorylation in density-gradient purified mitochondria was performed using both 2D and 1D electrophoresis, with detection of phosphoproteins using Pro-Q Diamond or phospho-amino-specific antibodies. Several phosphoproteins were detected, including voltage-dependent anion channels isoforms 1 and 2, but none showed significant IP-mediated changes either before ischemia or during ischemia and reperfusion, and neither Western blotting nor 2D fluorescence difference gel electrophoresis detected translocation of protein kinase C (&agr;, ϵ, or &dgr; isoforms), glycogen synthase kinase 3&bgr;, or Akt to the mitochondria following IP. In freeze-clamped hearts, changes in phosphorylation of GSK3&bgr;, Akt, and AMP-activated protein kinase were detected following ischemia and reperfusion but no IP-mediated changes correlated with MPTP inhibition or cardioprotection. However, measurement of mitochondrial protein carbonylation, a surrogate marker for oxidative stress, suggested that a reduction in mitochondrial oxidative stress at the end of ischemia and during reperfusion may account for IP-mediated inhibition of MPTP. The signaling pathways mediating this effect and maintaining it during reperfusion are discussed.

Temperature preconditioning of isolated rat hearts – a potent cardioprotective mechanism involving a reduction in oxidative stress and inhibition of the mitochondrial permeability transition pore

We investigate whether temperature preconditioning (TP), induced by short‐term hypothermic perfusion and rewarming, may protect hearts against ischaemic/reperfusion injury like ischaemic preconditioning (IP). Isolated rat hearts were perfused for 40 min, followed by 25 min global ischaemia and 60 min reperfusion (37°C). During pre‐ischaemia, IP hearts underwent three cycles of 2 min global ischaemia and 3 min reperfusion at 37°C, whereas TP hearts received three cycles of 2 min hypothermic perfusion (26°C) interspersed by 3 min normothermic perfusion. Other hearts received a single 6 min hypothermic perfusion (SHP) before ischaemia. Both IP and TP protocols increased levels of high energy phosphates in the pre‐ischaemic heart. During reperfusion, TP improved haemodynamic recovery, decreased arrhythmias and reduced necrotic damage (lactate dehydrogenase release) more than IP or SHP. Measurements of tissue NAD+ levels and calcium‐induced swelling of mitochondria isolated at 3 min reperfusion were consistent with greater inhibition of the mitochondrial permeability transition at reperfusion by TP than IP; this correlated with decreased protein carbonylation, a surrogate marker for oxidative stress. TP increased protein kinase Cɛ (PKCɛ) translocation to the particulate fraction and pretreatment with chelerythrine (PKC inhibitor) blocked the protective effect of TP. TP also increased phosphorylation of AMP‐activated protein kinase (AMPK) after 5 min index ischaemia, but not before ischaemia. Compound C (AMPK inhibitor) partially blocked cardioprotection by TP, suggesting that both PKC and AMPK may mediate the effects of TP. The presence of N‐(2‐mercaptopropionyl) glycine during TP also abolished cardioprotection, indicating an involvement of free radicals in the signalling mechanism.

Protection of myocardium by cyclosporin A and insulin: In vitro simulated ischemia study in human myocardium

BACKGROUND The efficacy of myocardial protection by cyclosporin A (CSA) and insulin was tested in human right atrial myocardial slices subjected to simulated ischemia and reoxygenation. METHODS Slices of right atrial trabeculae were obtained from patients undergoing elective cardiac surgery. Trabeculae were incubated with oxygenated glucose containing phosphate buffered saline (O(2), G-PBS). After 30 minutes of stabilization the sections were exposed to 90 minutes of simulated ischemia (N(2), PBS without glucose) followed by 90 minutes reoxygenation (O(2), G-PBS). Cyclosporin A (0.2 micromol/L) or insulin (5 mU/mL) was added during the stabilization period prior the ischemia. Cell viability was measured by using 3-[4.5 dimethylthiazol 2-yl]-2,5-diphenyltetrazolium bromide (MTT), which is cleaved by active mitochondrial dehydrogenases of living cells. RESULTS The viability of untreated slices (control) was 30.45% +/- 2.5% versus 52.65% +/- 4.4% in the CSA treated slices, p less than 0.001. The extent of protection by CSA was affected by oral antiglycemic drugs (glibenclamide). The effect obtained by CSA was inhibited by 5-hydroxydecanoate (5HD), a specific blocker of mitochondrial K(ATP) channels. Protection of the myocardial slices with insulin appears to be superior and not affected by the medication before surgery. This protection was maximal when insulin was present during both preischemic equilibration and reoxygenation periods (68.9% +/- 9.3% viability with insulin versus 33.2% +/- 6.9% in the control, p < 0.001). CONCLUSIONS Protection of right atrial trabeculae slices with insulin is superior to that obtained with CSA and is independent of preoperative medication.

Consecutive pharmacological activation of PKA and PKC mimics the potent cardioprotection of temperature preconditioning

Aims Temperature preconditioning (TP) provides very powerful protection against ischaemia/reperfusion. Understanding the signalling pathways involved may enable the development of effective pharmacological cardioprotection. We investigated the interrelationship between activation of protein kinase A (PKA) and protein kinase C (PKC) in the signalling mechanisms of TP and developed a potent pharmacological intervention based on this mechanism. Methods and results Isolated rat hearts were subjected to TP, 30 min global ischaemia, and 60 min reperfusion. Other control and TP hearts were perfused with either sotalol (β-adrenergic blocker) or H-89 (PKA inhibitor). Some hearts were pre-treated with either isoproterenol (β-adrenergic agonist) or adenosine (PKC activator) that were given alone, simultaneously, or sequentially. Pre-treatment with isoproterenol, adenosine, and the consecutive isoproterenol/adenosine treatment was also combined with the PKC inhibitor chelerythrine. Cardioprotection was evaluated by haemodynamic function recovery, lactate dehydrogenase release, measurement of mitochondrial permeability transition pore opening, and protein carbonylation during reperfusion. Cyclic AMP and PKA activity were increased in TP hearts. H-89 and sotalol blocked the cardioprotective effect of TP and TP-induced PKC activation. Isoproterenol, adenosine, and the consecutive treatment increased PKC activity during pre-ischaemia. Isoproterenol significantly reduced myocardial glycogen content. Isoproterenol and adenosine, alone or simultaneously, protected hearts but the consecutive treatment gave the highest protection. Cardioprotective effects of adenosine were completely blocked by chelerythrine but those of the consecutive treatment only attenuated. Conclusion The signal transduction pathway of TP involves PKA activation that precedes PKC activation. Pharmacologically induced consecutive PKA/PKC activation mimics TP and induces extremely potent cardioprotection.

Temperature preconditioning is optimal at 26°C and confers additional protection to hypothermic cardioplegic ischemic arrest

We have recently shown that brief episodes of hypothermic perfusion interspersed with periods of normothermic perfusion, referred to as temperature preconditioning (TP), are cardioprotective and can be mimicked by consecutive isoproterenol/adenosine treatment. Here we investigate the optimal temperature for TP and whether TP further enhances protection provided by hypothermic ischemia with or without polarized cardioplegic arrest. Three experimental groups of Langendorff-perfused rat hearts were used. In the first group, hearts were subjected to three episodes of hypothermic perfusion at 7, 17, 26 and 32°C during the TP protocol, followed by 30 min normothermic index ischemia and 60 min reperfusion (37°C). Protein kinase A (PKA) activity and cyclic AMP (cAMP) concentrations were measured prior to index ischemia. In the second group, TP (26°C) hearts were subjected to two hours hypothermic index ischemia at 26°C and two hours normothermic reperfusion. In the third group, TP (26°C) hearts or hearts treated with isoproterenol/adenosine (pharmacological simulation of TP) were subjected to four hours hypothermic index ischemia with procaine-induced polarized cardioplegia at 26°C followed by two hours normothermic reperfusion. Hemodynamic function recovery, lactate dehydrogenase release and infarct size were used to assess cardioprotection. TP at 26°C resulted in highest cardioprotection, increased cAMP concentration and PKA activity, while TP at 7°C exacerbated ischemia/reperfusion damage, and had no effect on cAMP concentration or PKA activity. TP at 26°C also protected hearts during hypothermic ischemia with or without polarized cardioplegia. Isoproterenol/adenosine treatment conferred additional protection similar to TP. In conclusion, the study shows that TP-induced cardioprotection is temperature dependent and is optimal at 26°C; TP confers additional protection to hypothermia and polarized cardioplegia; and that the pharmacological treatment based on the mechanism of TP (consecutive isoproterenol/adenosine treatment) is a potential cardioprotective strategy that can be used during heart surgery and transplantation.

Activation of Glibenclamide-Sensitive ATP-Sensitive K + Channels During β-Adrenergically Induced Metabolic Stress Produces a Substrate for Atrial Tachyarrhythmia

Background— Cardiac ATP-sensitive K+ channels have been suggested to contribute to the adaptive physiological response to metabolic challenge after &bgr;-adrenoceptor stimulation. However, an increased atrial K+-conductance might be expected to be proarrhythmic. We investigated the effect of ATP-sensitive K+ channel blockade on the electrophysiological responses to &bgr;-adrenoceptor-induced metabolic challenge in intact atria. Methods and Results— Atrial electrograms were recorded from the left atrial epicardial surface of Langendorff-perfused rat hearts using a 5×5 electrode array. Atrial effective refractory period and conduction velocity were measured using an S1–S2 protocol. The proportion of hearts in which atrial tachyarrhythmia was produced by burst-pacing was used as an index of atrial tachyarrhythmia-inducibility. Atrial nucleotide concentrations were measured by high performance liquid chromatography. Perfusion with ≥10–9 mol/L of the &bgr;-adrenoceptor agonist, isoproterenol (ISO), resulted in a concentration-dependent reduction of atrial effective refractory period and conduction velocity. The ISO-induced changes produced a proarrhythmic substrate such that atrial tachyarrhythmia could be induced by burst-pacing. Atrial [ATP] was significantly reduced by ISO (10–6 mol/L). Perfusion with either of the ATP-sensitive K+ channel blockers, glibenclamide (10–5 mol/L) or tolbutamide (10–3 mol/L), in the absence of ISO had no effect on basal atrial electrophysiology. On the other hand, the proarrhythmic substrate induced by 10–6 mol/L ISO was abolished by either of the sulfonylureas, which prevented induction of atrial tachyarrhythmia. Conclusions— Atrial ATP-sensitive K+ channels activate in response to &bgr;-adrenergic metabolic stress in Langendorff-perfused rat hearts, resulting in a proarrhythmic substrate.

Prospects for Creation of Cardioprotective Drugs Based on Cannabinoid Receptor Agonists

Cannabinoids can mimic the infarct-reducing effect of early ischemic preconditioning, delayed ischemic preconditioning, and ischemic postconditioning against myocardial ischemia/reperfusion. They do this primarily through both CB1 and CB2 receptors. Cannabinoids are also involved in remote preconditioning of the heart. The cannabinoid receptor ligands also exhibit an antiapoptotic effect during ischemia/reperfusion of the heart. The acute cardioprotective effect of cannabinoids is mediated by activation of protein kinase C, extracellular signal-regulated kinase, and p38 kinase. The delayed cardioprotective effect of cannabinoid anandamide is mediated via stimulation of phosphatidylinositol-3-kinase-Akt signaling pathway and enhancement of heat shock protein 72 expression. The delayed cardioprotective effect of another cannabinoid, Δ9-tetrahydrocannabinol, is associated with augmentation of nitric oxide (NO) synthase expression, but data on the involvement of NO synthase in the acute cardioprotective effect of cannabinoids are contradictory. The adenosine triphosphate-sensitive K+ channel is involved in the synthetic cannabinoid HU-210-induced cardiac resistance to ischemia/reperfusion injury. Cannabinoids inhibit Na+/Ca2+ exchange via peripheral cannabinoid receptor (CB2) activation that may also be related to the antiapoptotic and cardioprotective effects of cannabinoids. The cannabinoid receptor agonists should be considered as prospective group of compounds for creation of drugs that are able to protect the heart against ischemia–reperfusion injury in the clinical setting.

Functional and cardioprotective effects of simultaneous and individual activation of protein kinase A and Epac

Myocardial cAMP elevation confers cardioprotection against ischaemia/reperfusion (I/R) injury. cAMP activates two independent signalling pathways, PKA and Epac. This study investigated the cardiac effects of activating PKA and/or Epac and their involvement in cardioprotection against I/R.

Activation of Glibenclamide-Sensitive KATP Channels during β-Adrenergically-Induced Metabolic Stress Produces a Substrate for Atrial Tachyarrhythmia

Background— Cardiac ATP-sensitive K+ channels have been suggested to contribute to the adaptive physiological response to metabolic challenge after &bgr;-adrenoceptor stimulation. However, an increased atrial K+-conductance might be expected to be proarrhythmic. We investigated the effect of ATP-sensitive K+ channel blockade on the electrophysiological responses to &bgr;-adrenoceptor-induced metabolic challenge in intact atria. Methods and Results— Atrial electrograms were recorded from the left atrial epicardial surface of Langendorff-perfused rat hearts using a 5×5 electrode array. Atrial effective refractory period and conduction velocity were measured using an S1–S2 protocol. The proportion of hearts in which atrial tachyarrhythmia was produced by burst-pacing was used as an index of atrial tachyarrhythmia-inducibility. Atrial nucleotide concentrations were measured by high performance liquid chromatography. Perfusion with ≥10–9 mol/L of the &bgr;-adrenoceptor agonist, isoproterenol (ISO), resulted in a concentration-dependent reduction of atrial effective refractory period and conduction velocity. The ISO-induced changes produced a proarrhythmic substrate such that atrial tachyarrhythmia could be induced by burst-pacing. Atrial [ATP] was significantly reduced by ISO (10–6 mol/L). Perfusion with either of the ATP-sensitive K+ channel blockers, glibenclamide (10–5 mol/L) or tolbutamide (10–3 mol/L), in the absence of ISO had no effect on basal atrial electrophysiology. On the other hand, the proarrhythmic substrate induced by 10–6 mol/L ISO was abolished by either of the sulfonylureas, which prevented induction of atrial tachyarrhythmia. Conclusions— Atrial ATP-sensitive K+ channels activate in response to &bgr;-adrenergic metabolic stress in Langendorff-perfused rat hearts, resulting in a proarrhythmic substrate.

Cardioprotective and Antioxidant Effects of Apomorphine

Apomorphine is a potent antioxidant that infiltrates through biological membranes. We studied the effect of apomorphine (2 μM) on myocardial ischemic-reperfusion injury in the isolated rat heart. Since iron and copper ions (mediators in formation of oxygen-derived free radicals) are released during myocardial reperfusion, apomorphine interaction with iron and copper and its ability to prevent copper-induced ascorbate oxidation were studied. Apomorphine perfused before ischemia or at the commencement of reperfusion demonstrated enhanced restoration of hemodynamic function (i.e. recovery of the work index (LVDP × HR) was 69.2±4.0% with apomorphine pre-ischemic regimen vs. 43.4±9.01% in control hearts, p<0.01, and 76.3±8.0% with apomorphine reperfusion regimen vs. 30.4±11.1% in controls, p<0.001). This was accompanied by decreased release of proteins in the effluent and improved coronary flow recovery in hearts treated with apomorphine after the ischemia. Apomorphine forms stable complexes with copper and with iron, and inhibits the copper-induced ascorbate oxidation. It is suggested that these iron and copper chelating properties and the redox-inactive chelates formed by transition metals and apomorphine play an essential role in post-ischemic cardioprotection.