Kirsti Ytrehus

Exogenous hydrogen sulfide (H2S) protects against regional myocardial ischemia–reperfusion injury

Abstract Hydrogen sulfide (H2S) is a gaseous mediator, produced by the metabolic pathways that regulate tissue concentrations of sulfur–containing amino acids. Recent studies indicate that endogenous or exogenous H2S exerts physiological effects in the cardiovascular system of vertebrates, possibly through modulation of KATP channel opening. The present study was undertaken to examine the hypothesis that H2S is cytoprotective against myocardial ischemia–reperfusion injury and that this protective action is mediated by KATP opening. Rat isolated hearts were Langendorff–perfused and underwent 30 min left main coronary artery occlusion and 120 min reperfusion. The resulting injury was assessed as infarct size, determined by tetrazolium staining. Treatment of hearts with the H2S–donor, NaHS, commencing 10 min prior to the onset of coronary occlusion and maintained until 10 min reperfusion, resulted in a concentration–dependent limitation of infarct size (control, 41.0 ± 2.6% of risk zone; NaHS 0.1 μM, 33.9 ± 2.1%, [0.05 > P < 0.1]; NaHS 1 μM, 20.2 ± 2.1% [P < 0.01]). Pretreatment with the KATP channel blockers glibenclamide 10 μM or sodium 5–hydroxydecanoate (5HD) 100 μM led to abrogation of the infarct–limiting effect of NaHS 1 μM (glibenclamide + NaHS 42.5 ± 3.6%; 5HD + NaHS 44.7 ± 2.2%). No statistically significant effects of NaHS treatment on coronary flow, heart rate or left ventricular developed pressure were observed in this experimental preparation. These data provide the first evidence that exogenous H2S protects against irreversible ischemia–reperfusion injury in myocardium and support the involvement of KATP opening in the mechanism of action. Further work is required to elucidate the potential role of endogenous H2S as a cytoprotective mediator against myocardial ischemia–reperfusion injury, the mechanisms regulating its generation, and the nature of its interaction with protein targets such as the KATP channel.

Inhibition of sodium-hydrogen exchange reduces infarct size in the isolated rat heart — a protective additive to ischaemic preconditioning

OBJECTIVES Inhibition of Na+/H+ exchange with amiloride analogues has been shown to protect the ischaemic and reperfused heart. The aim of this study was to examine if preischaemic or postischaemic treatment with the selective Na+/H+ exchange inhibitor ethyl-isopropyl amiloride (EIPA, 1 microM) influenced infarct size in an isolated rat heart model of regional ischaemia and reperfusion, and if any such protection was additive to the protection afforded by ischaemic preconditioning. METHODS Langendorff perfused rat hearts were subjected to 30 or 45 min of regional ischaemia and 120 min of reperfusion. The risk zone was determined by fluorescent particles and infarct size was determined by staining with triphenyltetrazolium chloride. RESULTS Treatment with EIPA for 20 min before 30 min regional ischaemia significantly reduced infarct size (in % of the risk zone) compared to untreated controls [3.1 (SEM 1.0)% v 38.1(5.8)%, P < 0.001], a protection similar to that afforded by ischaemic preconditioning [6.1(2.5)%]. Combination of preischaemic EIPA treatment and ischaemic preconditioning also reduced infarct size [5.2(2.0)%, P < 0.01 v control group]. When EIPA was added to the buffer only during the first 30 min of reperfusion, no protection was observed [infarct size = 37.8(5.8)%, NS v control group]. In order to clarify if the protection observed with EIPA treatment was additive to protection by ischaemic preconditioning, another set of experiments was performed. In these experiments regional ischaemia was extended to 45 min. Preischaemic EIPA treatment reduced infarct size also in this model compared to controls [15.3(2.9)% v 64.3(2.9)%, P < 0.001], as did ischaemic preconditioning [23.5(4.2)%, P < 0.001 v controls, NS v EIPA treated hearts]. Combination of ischaemic preconditioning and preischaemic EIPA treatment further reduced infarct size significantly [3.9(0.6)%, P < 0.05 v all other groups with 45 min regional ischaemia]. CONCLUSIONS Inhibition of Na+/H+ exchange reduces infarct size in the isolated rat heart infarct model if the exchanger is inhibited during the ischaemic period, and this protection is additive to the protection afforded by ischaemic preconditioning.

Novel targets and future strategies for acute cardioprotection: Position Paper of the European Society of Cardiology Working Group on Cellular Biology of the Heart

Ischaemic heart disease and the heart failure that often results, remain the leading causes of death and disability in Europe and worldwide. As such, in order to prevent heart failure and improve clinical outcomes in patients presenting with an acute ST-segment elevation myocardial infarction and patients undergoing coronary artery bypass graft surgery, novel therapies are required to protect the heart against the detrimental effects of acute ischaemia/reperfusion injury (IRI). During the last three decades, a wide variety of ischaemic conditioning strategies and pharmacological treatments have been tested in the clinic-however, their translation from experimental to clinical studies for improving patient outcomes has been both challenging and disappointing. Therefore, in this Position Paper of the European Society of Cardiology Working Group on Cellular Biology of the Heart, we critically analyse the current state of ischaemic conditioning in both the experimental and clinical settings, provide recommendations for improving its translation into the clinical setting, and highlight novel therapeutic targets and new treatment strategies for reducing acute myocardial IRI.Ischaemic heart disease and the heart failure that often results, remain the leading causes of death and disability in Europe and worldwide. As such, in order to prevent heart failure and improve clinical outcomes in patients presenting with an acute ST-segment elevation myocardial infarction and patients undergoing coronary artery bypass graft surgery, novel therapies are required to protect the heart against the detrimental effects of acute ischaemia/reperfusion injury. During the last three decades, a wide variety of ischaemic conditioning strategies and pharmacological treatments have been tested in the clinic - however, their translation from experimental to clinical studies for improving patient outcomes has been both challenging and disappointing. Therefore, in this Position Paper of the European Society of Cardiology Working Group on Cellular Biology of the Heart, we critically analyse the current state of ischaemic conditioning in both the experimental and clinical settings, provide recommendations for improving its translation into the clinical setting, and highlight novel therapeutic targets and new treatment strategies for reducing acute myocardial ischaemia/reperfusion injury.

Position Paper of the European Society of Cardiology Working Group Cellular Biology of the Heart: Cell-based therapies for myocardial repair and regeneration in ischemic heart disease and heart failure

Despite improvements in modern cardiovascular therapy, the morbidity and mortality of ischaemic heart disease (IHD) and heart failure (HF) remain significant in Europe and worldwide. Patients with IHD may benefit from therapies that would accelerate natural processes of postnatal collateral vessel formation and/or muscle regeneration. Here, we discuss the use of cells in the context of heart repair, and the most relevant results and current limitations from clinical trials using cell-based therapies to treat IHD and HF. We identify and discuss promising potential new therapeutic strategies that include ex vivo cell-mediated gene therapy, the use of biomaterials and cell-free therapies aimed at increasing the success rates of therapy for IHD and HF. The overall aim of this Position Paper of the ESC Working Group Cellular Biology of the Heart is to provide recommendations on how to improve the therapeutic application of cell-based therapies for cardiac regeneration and repair.

Ischaemic preconditioning is protein kinase C dependent but not through stimulation of α adrenergic or adenosine receptors in the isolated rat heart

OBJECTIVE The aim was (1) to clarify whether alpha adrenoceptor and adenosine receptor stimulation is involved in the anti-infarct effect of ischaemic preconditioning in the rat heart, and (2) to test the hypothesis that signal transduction through membrane bound protein kinase C is essential for the protection. METHODS Isolated, buffer perfused rat hearts were subjected to 30 min of regional ischaemia and 120 min of reperfusion. The risk zone was determined by fluorescent particles, and infarct size was determined by staining with triphenyltetrazolium chloride. RESULTS Ischaemic preconditioning with three cycles of 5 min ischaemia plus 5 min of reperfusion significantly reduced infarct size as compared to non-preconditioned group [4.5(SEM 0.6)% of the risk zone v 45.5(5.7)%, P < 0.001]. Blockade of alpha adrenoceptors alone and simultaneous blockade of alpha adrenoceptors with phenoxybenzamine (10 microM) and adenosine receptors with sulphophenyltheophylline (100 microM) did not prevent the protective effect of ischaemic preconditioning [infarct size = 2.4(0.4) and 5.6(1.9)% respectively, NS v the non-treated preconditioned group]. Blocking either the membrane binding of protein kinase C with polymyxin B (1 microM) or direct inhibition of protein kinase C activity with chelerythrine (2 microM) completely abolished the infarct size reducing effect of ischaemic preconditioning [32.4(3.3)% and 48.2(4.0)% respectively, P < 0.005 v non-treated preconditioned group: NS v the non-preconditioned group]. CONCLUSIONS In the rat heart infarct model the protective effect of ischaemic preconditioning is not mediated through stimulation of alpha adrenoceptors alone or the combined stimulation of alpha adrenergic and adenosine receptors, and it is dependent on activation of membrane bound protein kinase C.

Short- and long-term effects of melatonin on myocardial post-ischemic recovery

Abstract:  Melatonin, the chief secretory product of the pineal gland, has been shown to protect the heart against ischemia–reperfusion injury. This was attributed to its free radical scavenging and broad‐spectrum antioxidant properties. The possibility that melatonin may act via its receptor and intracellular signaling, has not yet been addressed in this regard. In all previous studies, only the acute effects of melatonin on the heart, were evaluated. The aims of the present study were to: (i) compare the acute and long‐term effects of melatonin on infarct size and functional recovery of the ischemic heart, and (ii) evaluate the role of the melatonin receptor in cardioprotection. For evaluation of the short‐term effects of melatonin on contractile recovery and infarct size, the isolated perfused working rat heart was subjected to 20 min global ischemia or 35 min regional ischemia respectively, and melatonin (25–50 μm) administered either before and during reperfusion, or before ischemia or during reperfusion after ischemia. The melatonin receptor was manipulated using luzindole and N‐acetyltryptamine. The long‐term effects of melatonin were evaluated 24 hr after melatonin administration (2.5 or 5.0 mg/kg, i.p.) or after oral administration for 7 days (20 or 40 μg/mL). Infarct size and mechanical recovery during reperfusion of the working heart were used as endpoints. Melatonin (50 μm), when administered either before and during reperfusion after ischemia or during reperfusion only, significantly improved cardiac output and work performance and reduced infarct size compared with untreated controls. Luzindole (5 μm), a melatonin receptor antagonist, abolished these cardioprotective effects. Long‐term administration of melatonin (i.p. or orally for 7 days) caused a significant reduction in infarct size of hearts subjected to 35 min regional ischemia. The cardioprotection persisted for 2–4 days after discontinuation of treatment. In summary, the results obtained suggest that melatonin induces short‐ as well as long‐term protection and that the melatonin receptor is also involved in its cardioprotective actions.

Ultrastructural changes induced in the isolated rat heart by enzymatically generated oxygen radicals

This study describes the effect of oxygen radicals on the ultrastructure of the isolated Langendorff-perfused rat heart. Oxygen radicals were enzymatically generated by xanthine oxidase (0.025 U/ml) and hypoxanthine (0.96 mM). Hearts were perfusion-fixed for electron microscopy and stereological technique was performed to obtain estimates of volume fractions (Vv) of different tissue components. Perfusion with oxygen radicals resulted in areas with severely damaged myocardial cells. These changes included swelling and cristolysis of mitochondria, disruption of filaments, development of intracellular edema and focal disruption of the sarcolemma. Stereological examination revealed few alterations after 5 min perfusion with oxygen radicals. After 10 min perfusion with oxygen radicals, however, the Vv (myocyte/myocardium) increased from 0.542 +/- 0.042 (mean +/- S.D.) to 0.663 +/- 0.144, and this paralleled the development of Vv (cellular edema/myocyte) being 0.047 +/- 0.028. Vv (capillary wall/capillary) increased from 0.215 +/- 0.046 to 0.411 +/- 0.123 indicating endothelial swelling. Although the mitochondria appeared swollen, Vv (mitochondria/myocyte) remained constant. The effect of a 35 min recovery period on the ultrastructure was minor. The application of SOD and catalase together with xanthine oxidase and hypoxanthine reduced the observed changes significantly, thus proving the participation of oxygen radicals. This study confirms that oxygen radicals can induce major alterations in myocardial ultrastructure.

Melatonin receptor‐mediated protection against myocardial ischaemia/reperfusion injury: role of its anti‐adrenergic actions

Abstract:  Melatonin has potent cardioprotective properties. These actions have been attributed to its free radical scavenging and anti‐oxidant actions, but may also be receptor mediated. Melatonin also exerts powerful anti‐adrenergic actions based on its effects on contractility of isolated papillary muscles. The aims of this study were to determine whether melatonin also has anti‐adrenergic effects on the isolated perfused rat heart, to determine the mechanism thereof and to establish whether these actions contribute to protection of the heart during ischaemia/reperfusion. The results showed that melatonin (50 μm) caused a significant reduction in both isoproterenol (10−7 m) and forskolin (10−6 m) induced cAMP production and that both these responses were melatonin receptor dependent, since the blocker, luzindole (5 × 10−6 m) abolished this effect. Nitric oxide (NO), as well as guanylyl cyclase are involved, as l‐NAME (50 μm), an NO synthase inhibitor and ODQ (20 μm), a guanylyl cyclase inhibitor, significantly counteracted the effects of melatonin. Protein kinase C (PKC), as indicated by the use of the inhibitor bisindolylmaleimide (50 μm), also play a role in melatonin’s anti‐adrenergic actions. These actions of melatonin are involved in its cardioprotection: simultaneous administration of l‐NAME or ODQ with melatonin, before and after 35 min regional ischaemia, completely abolished its cardioprotection. PKC, on the other hand, had no effect on the melatonin‐induced reduction in infarct size. Cardioprotection by melatonin was associated with a significant activation of PKB/Akt and attenuated activation of the pro‐apoptotic kinase, p38MAPK during early reperfusion. In summary, the results show that melatonin‐induced cardioprotection may be receptor dependent, and that its anti‐adrenergic actions, mediated by NOS and guanylyl cyclase activation, are important contributors.

Endothelin-1 can reduce infarct size through protein kinase C and KATP channels in the isolated rat heart

OBJECTIVE Protection from ischaemic preconditioning (IP) is dependent on activation of protein kinase C (PKC), and preconditionings protection can be mimicked by stimulation of various membrane receptors which are known to activate PKC. It is well known that KATP channel activation is cardioprotective. We tested the hypothesis that preischaemic treatment with endothelin-1 (ET-1) can protect against infarction by a PKC-dependent mechanism and by activating KATP channels. METHODS Buffer-perfused isolated rat hearts were subjected to 30 min regional ischaemia and 120 min reperfusion. Risk zone was determined by fluorescent particles, and infarct size by TTC staining. RESULTS Treatment with ET-1 in a dose of 1 nM prior to ischaemia significantly reduced infarct size in % of the risk zone compared to the control group (infarct size: 14.1 +/- 2.6 vs. 41.9 +/- 3.4%), while ET-1 0.1 nM did not protect (infarct size: 40.9 +/- 3%). AS the protective dose of ET-1 resulted in a significant reduction of coronary flow, a control group with a similar preischaemic flow-reduction was included (infarct size: 48.1 +/- 4.2%). Both the nonselective ETA/ETB receptor antagonist bosentan (1 microM) and the ET(A)-receptor-selective antagonist BQ 123 (2 microM) abolished protection from ET-1 (infarct size: 43.3 +/- 3.5 and 41.3 +/- 3.3%, respectively), as did the PKC inhibitor chelerythrine (2 microM) (infarct size: 41.1 +/- 5.2%) and the KATP blocker 5-hydroxydecanoate (infarct size: 41.7 +/- 2.9%). None of the ET receptor antagonists bosentan and BQ-123 influenced infarct size alone (infarct size: 42.7 +/- 2.5 and 41.3 +/- 3.3%, respectively). IP, similarly to ET-1, reduced infarct size (infarct size: 6.1 +/- 1.4%), but the nonselective ET receptor antagonist bosentan did not interfere with preconditionings protection (infarct size: 13.2 +/- 4.3%). CONCLUSIONS ET-1 treatment prior to ischaemia can protect against infarction via ETA receptors by a PKC-dependent mechanism and by activating KATP channels, but ET does not mediate IP in the isolated rat heart.

Lipid peroxidation, arachidonic acid and products of the lipoxygenase pathway in ischaemic preconditioning of rat heart

OBJECTIVE Preconditioning with brief intermittent periods of ischaemia is known to provide protection against ischaemic injury. It has been suggested that myocardial ischaemia also activates phospholipase A2, which releases arachidonic acid from phospholipids. In the present study the possible role of phospholipid peroxidation, arachidonic acid and products of the lipoxygenase pathway in cellular mechanisms of ischaemic preconditioning was examined. METHODS Isolated, buffer-perfused rat hearts were freeze-clamped at the end of preconditioning (a cycle of 5 min global ischaemia +5 min reperfusion) and at the end of 30 min global ischaemia and analysed for non-esterified fatty acids and fatty acids in the 2-position of phospholipid. In a separate set of experiments, hearts pretreated with a lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), were subjected to 30 min regional ischaemia and 120 min reperfusion. Infarct size was determined by tetrazolium staining and the ischaemic risk zone with fluorescent particles. RESULTS Myocardial levels of arachidonic as well as of linoleic and docosahexaenoic acid were significantly elevated by preconditioning. Also, the level of peroxidized polyunsaturated fatty acids (measured as hydroxy conjugated dienes) in myocardial phospholipid was significantly increased: 101.4 +/- 16.8 nmol/g versus 51.2 +/- 7.3 nmol/g tissue dw in the control group, p < 0.05. Pre-treatment of hearts with 5 microM NDGA blocked the infarct limiting effects of preconditioning: infarct size was 37.4 +/- 6.4% of risk zone in control, 9.0 +/- 0.9% in the preconditioning group and 27.7 +/- 3.8% in the preconditioning + NDGA group (p < 0.05 vs. i.p., n.s. vs. control). CONCLUSION Our findings provide evidence for the involvement of phospholipase A2 and lipoxygenase derived lipid second messengers in ischaemic preconditioning of the isolated rat heart.