Optogenetic tissue-engineered cardiac pacemaker: demonstration of principle in an isolated rat heart

Percutaneous solution for a frequent complication after transcatheter aortic valve replacement: A case of atrioventricular leadless pacemaker implantation after transcatheter aortic valve replacement

Cardiolipin is a major mitochondrial membrane glycerophospholipid in the mammalian heart. In this study, the ability of the isolated intact rat heart to remodel cardiolipin and the mitochondrial enzyme activities that reacylate monolysocardiolipin to cardiolipin in vitro were characterized. Adult rat heart cardiolipin was found to contain primarily linoleic and oleic acids. Perfusion of the isolated intact rat heart in the Langendorff mode with various radioactive fatty acids, followed by analysis of radioactivity incorporated into cardiolipin and its immediate precursor phosphatidylglycerol, indicated that unsaturated fatty acids entered into cardiolipin mainly by deacylation followed by reacylation. The in vitro mitochondrial acylation of monolysocardiolipin to cardiolipin was coenzyme A-dependent with a pH optimum in the alkaline range. Significant activity was also present at physiological pH. With oleoyl-coenzyme A as substrate, the apparent K(m) for oleoyl-coenzyme A and monolysocardiolipin were 12.5 microm and 138.9 microm, respectively. With linoleoyl-coenzyme A as substrate, the apparent K(m) for linoleoyl-coenzyme A and monolysocardiolipin were 6.7 microm and 59.9 microm, respectively. Pre-incubation at 50 degrees C resulted in different profiles of enzyme inactivation for the two activities. Both activities were affected similarly by phospholipids, triacsin C, and various lipid binding proteins but were affected differently by various detergents and myristoyl-coenzyme A. [(3)H]cardiolipin was not formed from monolyso[(3)H]cardiolipin in the absence of acyl-coenzyme A. Monolysocardiolipin acyltransferase activities were observed in mitochondria prepared from various other rat tissues. In summary, the data suggest that the isolated intact rat heart has the ability to rapidly remodel cardiolipin and that rat heart mitochondria contain coenzyme A-dependent acyltransferase(s) for the acylation of monolysocardiolipin to cardiolipin. A simple and reproducible in vitro assay for the determination of acyl-coenzyme A- dependent monolysocardiolipin acyltransferase activity in mammalian tissues with exogenous monolysocardiolipin substrate is also presented.

See related article, pages 705–712 The past 20 years have seen tremendous progress in the medical treatment of cardiac diseases, a progress that actually translated into decreased cardiovascular morbidity and mortality in clinical practice.1 Yet evidence accumulates that interventions intended to slow down the progression of chronic heart diseases have approached their limit. This may be one of the reasons why the recent progress in stem cell biology has stirred so much attention. In addition, recent studies suggesting the existence of cell populations in the adult organism that, albeit at a low rate and insufficiently, endogenously replace cardiac myocytes and endothelial and smooth muscle cells have challenged the dogma of the heart as a nonregenerating organ.2–5 Such cell populations could be accessed in biopsies and principally serve as a source to replace cardiac myocytes and other cardiac cell types lost after myocardial infarction or other pathologies. Human embryonic stem cell lines, on the other hand, can nowadays be generated from human blastocysts (accessible from IVF leftovers6) at a high success rate and have been unambiguously shown to generate human cardiac myocytes.7–9 Thus, despite many open questions and some controversy in the field (reviews in references 10,11), cardiac regenerative therapy has become a realistic perspective. But what to do with stem cells or their derivatives, given that means will be provided to generate them at sufficient numbers? The most straightforward strategy is to infuse or inject these cells into the injured heart and to develop methods to increase the fraction of cells that home and survive in the heart and differentiate to the desired functional elements. This strategy has already been clinically evaluated, but gave mixed results,12–16 potentially also because the homing rate after infusion and the survival rate after intramyocardial injections are very low. …

We read the article by Keyser et al. with interest [1]. They investigated 718 patients (60.0 ± 14.2 years; male, n = 570) who were treated with a first implantable cardioverter defibrillator (ICD) at their institution analysed since 2005. Pacemaker and ICD implantation is often regarded as a simple and easy procedure, ideal for beginners in cardiac and general surgery. Pacemaker and ICD implantation has become a routine procedure in modern cardiology, and implantable cardioverter-defibrillators are implanted with increasing frequency. Although fatal complications are relatively rare, they may give rise to malpractice lawsuits against medical personnel [2] A frequently under-diagnosed complication of pacemaker and implantable cardioverter defibrillator lead implantation is the unintentional advancement of the leads into the systemic circulation. Despite standardized procedures and improvements in the technique of pacemaker implantation, life-threatening complications may still occur. Even in the hands of experienced physicians, pacemaker leads can be misplaced into the systemic circulation during implantation [3]. The most common complications of lead implantation through a coronary vein, however, are diaphragmatic stimulation, coronary sinus dissection, and lead dislodgement [4]. We think that detailed and careful consideration should be made during the process of installation of the device, not only to determine the appropriate defibrillation threshold, but also to determine the proper localization of ICD leads and mechanical complications. Accordingly, we agree with the authors that the testing of ICDs is a safe and efficient procedure. Conflict of interest: none declared

What is the central question of this study? The microtubule network is disrupted during myocardial ischaemia-reperfusion injury. It was suggested that prevention of microtubule disruption with paclitaxel might reduce cardiac infarct size; however, the effects on infarction have not been studied. What is the main finding and its importance? Paclitaxel caused a reduction in microtubule disruption and cardiomyocyte hypercontracture during ischaemia-reperfusion. However, it induced a greater increase in cytosolic calcium, which may explain the lack of effect against infarction that we have seen in isolated rat hearts. The large increase in perfusion pressure induced by paclitaxel in this model may have clinical implications, because detrimental effects of the drug were reported after its clinical application. Microtubules play a major role in the transmission of mechanical forces within the myocardium and in maintenance of organelle function. However, this intracellular network is disrupted during myocardial ischaemia-reperfusion. We assessed the effects of prevention of microtubule disruption with paclitaxel on ischaemia-reperfusion injury in isolated rat cardiomyocytes and hearts. Isolated rat cardiomyocytes were submitted to normoxia (1 h) or 45 min of simulated ischaemia (pH 6.4, 0% O2 , 37 °C) and reoxygenation, without or with treatment with the microtubule stabilizer, paclitaxel (10(-5) M), or the inhibitor of microtubule polymerization, colchicine (5 × 10(-6) M). Simulated ischaemia leads to microtubule disruption before the onset of ischaemic contracture. Paclitaxel attenuated both microtubule disruption and the incidence of hypercontracture, whereas treatment with colchicine mimicked the effects of simulated ischaemia and reoxygenation. In isolated normoxic rat hearts, treatment with paclitaxel induced concentration-dependent decreases in heart rate and left ventricular developed pressure and increases in perfusion pressure. Despite protection against hypercontracture, paclitaxel pretreatment did not modify infarct size (60.37 ± 2.27% in control hearts versus 58.75 ± 10.25, 55.44 ± 10.32 and 50.06 ± 10.14% after treatment with 10(-6) , 3 × 10(-6) and 10(-5) m of paclitaxel) after 60 min of global ischaemia and reperfusion in isolated rat hearts. Lack of protection was correlated with a higher increase in cytosolic calcium levels during simulated ischaemia in cardiomyocytes treated with paclitaxel (2.32 ± 0.15 versus 1.13 ± 0.16 a.u. in the presence or absence of 10(-6) m paclitaxel, respectively, P < 0.05), but not with changes in aortic reactivity. In conclusion, microtubule stabilization with paclitaxel reduces hypercontracture in isolated rat cardiomyocytes but does not protect against infarction in isolated rat hearts.

Background In the ICU the decision regarding site selection for central venous catheters (CVC) is largely influenced by the historic infection rates by site. It is thought that femoral placement has the highest incidence of line infections and therefore is often avoided. However, few studies have aimed to look at the overall risk to the patient regarding site placement taking not only risk of infection, but also risk of mechanical injury and complications into consideration. Methods We did a retrospective review of all patients who had a new central line (either triple lumen catheter or hemodialysis catheter) placed in the ICU for any indication between 1/31/2022 - 5/4/2023. Key information including demographics, Charlson Comorbidity index, type of catheter, site, date of insertion, and date of removal was obtained. Manual chart review was done for each patient in order to ascertain presence of CLABSI as well as mechanical complications. Results 472 lines were placed during the study period. The average age was 63.5 years. The average Charlson Comorbidity index was 5. There was no statistical significant difference in infection rates among femoral 5/196 (2.5%) and internal jugular (IJ) 9/247 (3.6%) CVCs, although IJ lines had a trend towards higher rates of mechanical complications 21/247 (8.5%) vs 11/195 (5.6%), respectively. Rate of infection was twice as high, 6/267 (2.3%) vs 9/205 (4.4%) in the BMI &amp;gt;30 group vs those with a BMI &amp;lt; 30. The 6-month all-cause mortality for patients requiring a central line in the ICU was &amp;gt;80%, regardless of the site of placement, presence of line infection, or mechanical complication. Conclusion Infection rates between IJ and femoral lines were comparable. Femoral lines had numerically less mechanical complications as compared to IJ lines. BMI &amp;gt; 30 had a trend towards an increased risk of CLABSI. Overall mortality was &amp;gt; 80% for patients who required a CVC for any reason in the ICU setting. Clinicians should take a balanced approach regarding infection and mechanical complication risk when placing a central line. Further research into determining infection rates for patients with higher BMI is needed. Intensivists should consider early palliative care consultation for patients requiring a central line. Disclosures All Authors: No reported disclosures

Transcatheter pacing systems (TPS) provide a novel, minimally invasive approach in which a miniaturized, leadless pacemaker (PM) is transfemorally implanted in the right ventricle. We evaluated the health-related quality of life (HRQoL) impact, patient satisfaction, and activity restrictions following TPS in a large prospective multicenter clinical trial. Patients who underwent a Micra TPS implantation between December 2013 and May 2015 were included. HRQoL impact was evaluated using the Short-Form-36 (SF-36) questionnaire at baseline, 3, and 12 months. Patient satisfaction was assessed using a three-item questionnaire determining recovery, activity level, and esthetic appearance at 3 months. Implanting physicians compared the patient activity restrictions for TPS to traditional PM therapy. A total of 720 patients were implanted with a TPS (76 ± 11 years; 59% male). Of these patients, 702 (98%), 681 (95%), and 635 (88%) completed the SF-36 at baseline, 3 and 12 months, respectively. Improvements were observed at 3 and 12 months in all SF-36 domains and all attained statistical significance. Of 693 patients who completed the patient satisfaction questionnaire, 96%, 91%, 74% were (very) satisfied with their esthetic appearance, recovery, and level of activity, respectively. TPS discharge instructions were rated less restrictive in 49%, equally restrictive in 47%, and more restrictive in 4% of cases compared with traditional PM systems. TPS resulted in postimplant HRQoL improvements at 3 and 12 months, and high levels of patient satisfaction at 3 months. Further, TPS was associated with less activity restrictions compared with traditional PM systems.

To investigate the myocardial electrophysiological effect and its underlying mechanisms of atorvastatin (Ator) on isolated rat hearts injured by ischemia/reperfusion (I/R). Isolated SD rat hearts were mounted on Langendorff system, and a local I/R was induced by ligation (30 min) and release (15 min) of the left anterior descending artery. During the reperfusion period, the effect of Ator on diastolic excitation threshold (DET), effective refractory period (ERP) and ventricular fibrillation threshold (VFT) on rat heart were measured. Compared with the control group, medium concentration of Ator prolonged the ERP in normal rat hearts; low, medium and high concentration of Ator significantly inhibited the decrease of DET, ERP and VFT induced by I/R. However, pretreatment with L-NAME cancelled these cardiac electrophysiological effects of Ator. Ator reduced electrophysiological alteration induced by I/R in isolated rat hearts, which may be mediated by activating nitric oxide pathway to enhance the myocardial electrophysiological stability.

ORAL PRESENTATION

AURICULAR SYSTOLE AND ITS RELATION TO VENTRICULAR OUTPUT

New FindingsWhat is the central question of this study?Rate–pressure product (RPP) is commonly used as an index of cardiac ‘effort’. In canine and human hearts (which have a positive force–frequency relationship), RPP is linearly correlated with oxygen consumption and has therefore been widely adopted as a species‐independent index of cardiac work. However, given that isolated rodent hearts demonstrate a negative force–frequency relationship, its use in this model requires validation.What is the main finding and its importance?Despite its widespread use, RPP is not correlated with oxygen consumption (or cardiac ‘effort’) in the Langendorff‐perfused isolated rat heart. This lack of correlation was also evident when perfusions included a range of metabolic substrates, insulin or β‐adrenoceptor stimulation.Langendorff perfusion of hearts isolated from rats and mice has been used extensively for physiological, pharmacological and biochemical studies. The ability to phenotype these hearts reliably is, therefore, essential. One of the commonly used indices of function is rate–pressure product (RPP); a rather ill‐defined index of ‘work’ or, more correctly, ‘effort’. Rate–pressure product, as originally described in dog or human hearts, was shown to be correlated with myocardial oxygen consumption (MV˙O2). Despite its widespread use, the application of this index to rat or mouse hearts (which, unlike the dog or human, have a negative force–frequency relationship) has not been characterized. The aim of this study was to examine the relationship between RPP and MV˙O2 in Langendorff‐perfused rat hearts. Paced hearts (300–750 beats min−1) were perfused either with Krebs–Henseleit (KH) buffer (11 mm glucose) or with buffer supplemented with metabolic substrates and insulin. The arteriovenous oxygen consumption (MV˙O2) was recorded. Metabolic status was assessed using 31P magnetic resonance spectroscopy and lactate efflux. Experiments were repeated in the presence of isoprenaline and in unpaced hearts where heart rate was increased by cumulative isoprenaline challenge. In KH buffer‐perfused hearts, MV˙O2 increased with increasing heart rate, but given that left ventricular developed pressure decreased with increases in rate, RPP was not correlated with MV˙O2, lactate production or phosphocreatine/ATP ratio. Although the provision of substrates or β‐adrenoceptor stimulation changed the shape of the RPP–MV˙O2 relationship, neither intervention resulted in a positive correlation between RPP and oxygen consumption. Rate–pressure product is therefore an unreliable index of oxygen consumption or ‘cardiac effort’ in the isolated rat heart.

Biochemical mechanisms in heart function

Preconditioning describes the phenomenon by which a traumatic or stressful stimulus confers protection against subsequent injury. Originally recognized in dog heart subjected to ischemic challenges, preconditioning has been demonstrated in multiple species, can be induced by various stimuli, and is applicable in different organ systems. Tremendous progress has been made elucidating the signal transduction cascade of preconditioning. Preconditioning represents a potent tissue-protective condition, and mechanistic understanding may allow safe clinical application. This review recalls the history of preconditioning and how it relates to the history of the investigation of endogenous adaptation; summarizes the current mechanistic understanding of acute preconditioning; outlines the signal transduction cascade leading to the development of delayed preconditioning; discusses preconditioning in noncardiac tissue; and explores the potential of using preconditioning clinically.

Preface R. Vetter, E.-G. Krause. A. Cardiac Development and Regulation. 1. Excitation-Contraction Coupling of the Developing Rat Heart M. Vornanen. 2. Developmental Changes of Calcium Transients and Contractility During the Cultivation of Rat Neonatal Cardiomyocytes B. Husse, M. Wussling. 3. Calcium Channels and Cation Transport ATPases in Cardiac Hypertrophy Induced by Aortic Constriction in Newborn Rats Lei Zheng, et al. 4. G Proteins, Adenylyl Cyclase and Related Phosphoproteins in the Developing Rat Heart S. Bartel, et al. 5. Localization of alpha 1,2,3-subunit isoforms of Na, K-ATPase in cultured neonatal and adult rat myocardium. The immunofluorescence and immunocytochemical study J. Slezak, et al. 6. Immediate Postnatal Rat Heart Development Modified by Abdominal Aortic Banding: Analysis of Gene Expression G.L. Engelmann, et al. 7. Early Postnatal Changes in Sarcoplasmic Reticulum Calcium Transport Function in Spontaneously Hypertensive Rats N. Freestone, et al. 8. Cardiac Phosphocreatine Deficiency Induced by GPA During Postnatal Development in Rat V. Pelouch, et al. 9. Role of Bradykinin in the Antihypertrophic effects of Enalapril in the Newborn Pig Heart C.J. Beinlich, et al. 10. Regulation of the Slow Ca+ Channels of Myocardial Cells N. Sperelakis, et al. 11. In Vivo Phosphorylation of the Cardiac L-type Calcium Channel Beta-Subunit in Response to Catecholamines H. Haase, et al. 12. Antibodies from T. cruzi Infected Mice Recognize the Second Extracellular Loop of the beta1-adrenergic and M2-muscarinic Receptors and Regulate CalciumChannels in Isolated Cardiomyocytes A. Mijares, et al. 13. b2-Adrenoceptor Activation by Zinterol Causes Protein Phosphorylation, Contractile Effects and Relaxant Effects Through a cAMP Pathway in Human Atrium A.J. Kaumann, et al. 14. Early After-Depolarisations Induced by Noradrenaline May be Initiated by Calcium Released from Sarcoplasmic Reticulum R. Janiak, B. Lewartowski. 15. Cardiac Pump Function of the Isolated Rat Heart at Two Modes of Energy Deprivation and Effect of Adrenergic Stimulation V.I. Kapelko, et al. 16. Regulation of b-adrenoceptor Properties and the Lipid Milieu in Heart Muscle Membranes During Stress S. Gudbjarnason, V. E. Benediktsdottir. 17. Heart Glycogen Content and Isoprenaline-Induced Myocardial Lesions M. Mraz, S. Hynie. 18. Role of Vascular Adrenergic Mechanisms in the Haemodynamic and PGI2 Stimulating Effects of Angiotensin in Diabetic Dogs M.Z. Koltai, et al. 19. Localization of a1-adrenoceptors in Rat and Human Hearts by Immunocytochemistry W. Schulze, M.L.X. Fu. 20. Inositol-1,4,5-trisphosphate Mass Content in Isolated Perfused Rat Heart During Alpha-1-Adrenoceptor Stimulation S. Hanem, et al. 21. Interstitial Noradrenaline Concentration of Rat Hearts as Influenced by Cellular Catecholamine Uptake Mechanisms O.O. Obst, et al. 22. Characterization of Catecholamine Uptake2 in Isolated Cardiac Myocytes O.O. Obst, et al. 23. Effects of Anti-Peptide Antibodies Against the Second Extracellular Loop of Human M2 Muscarinic Acetylcholine Receptors on Transmembrane Potentials and Currents in Guinea Pig Ventricular Myocy

Since the first description of bradykinin more than 50 years ago (Rocha e Silva et al., 1949), actions of the peptide in a variety of physiological and pathological responses have been extensively researched (reviewed in Bhoola et al., 1992; Wirth et al., 1997; Calixto et al., 2000). In the cardiovascular system, the classical action of bradykinin is vasodilatation, mediated in several vascular beds by the release of nitric oxide (NO) and prostacyclin (Hatta et al., 1997; Wirth et al., 1997). In the heart, exogenously-administered bradykinin is a potent coronary artery vasodilator substance, although the contribution of endogenous bradykinin to the regulation of coronary vascular tone is unclear. Several actions of bradykinin in the heart are of particular interest as they are independent of the vasodilator actions of the peptide. Such actions include the modulation of cell growth and division in the heart and the modulation of myocardial responses to ischaemia-reperfusion. The ability of bradykinin to act as an endogenous cytoprotective mediator in the ischaemic myocardium has received a great deal of attention in recent years. Much of this research on bradykinin has been fuelled by a growing appreciation of ischaemic preconditioning, an adaptive mechanism in which bradykinin plays an important role. This review focuses on the cytoprotective actions of bradykinin in the ischaemic and reperfused myocardium, discusses its role in the ischaemic preconditioning response, and examines the potential for manipulating endogenous bradykinin for therapeutic benefit in myocardial ischaemia. Acute thrombotic occlusion of a major coronary artery, leading to myocardial ischaemia, is a leading cause of death and morbidity in the industrialized and developing countries. Ischaemia rapidly produces profound metabolic, functional and morphological changes within myocardium, the severity of which are ultimately determined by the duration of impaired oxygenation and substrate delivery (Ganz & Braunwald, 1997). The principal metabolic changes centre around the failure of adequate adenosine triphosphate (ATP) generation by oxidative phosphorylation and the accumulation of byproducts of anaerobic glycolysis, particularly H+. The functional consequences of ATP depletion are rapidly manifested as a decrease in contractility and disturbances of a host of homoeostatic processes, including the activities of ion channels and exchangers, cell volume regulation and enzyme reactions. The electrical properties of ischaemic myocardium may be altered to the point where arrhythmogenic mechanisms can promote life theatening tachyarrhythmias. Ultrastructural changes may be detectable within several minutes of the onset of ischaemia. These alterations may be considered reversible if reperfusion of the tissue can be effected promptly. However, ischaemia lasting more than 20 – 30 min will result in irreversible cell injury (Schaper et al., 1992). Without reperfusion to salvage ischaemic myocardium, the most extreme manifestation of irreversible injury is tissue necrosis (myocardial infarction). Prompt reperfusion of the occluded vessel is required to save ischaemic myocardium from sustaining irreversible injury but, paradoxically, reperfusion may be associated with further cellular stress resulting in 'reperfusion injury'. The development of therapeutic strategies that can attenuate ischaemia-reperfusion injury has been a keen area of research for more than 30 years. Brief antecedent episodes of ischaemia enhance tissue tolerance to a subsequent longer episode of ischaemia. This phenomenon was formally described by Murry et al. (1986) who coined the term 'preconditioning with ischaemia'. They demonstrated in canine myocardium that four short non-injurious coronary artery occlusions, before a subsequent 40 min coronary occlusion, reduced the development of necrosis during the 40 min occlusion by almost 75% compared to the necrosis in non-preconditioned hearts. This powerful protective effect of antecedant ischaemia was not explained by changes in coronary collateral blood flow, suggesting a fundamental cellular alteration in the response to ischaemia. The protection conferred by preconditioning with ischaemia has been subsequently confirmed in many studies and has excited a huge effort to determine the underlying molecular mechanisms of protection (Cohen et al., 2000). Preconditioning protocols vary somewhat from one laboratory to another and many endpoints of ischaemic injury have been adopted to assess the extent of protection conferred by preconditioning, including development of necrosis, severity of arrhythmias, post-ischaemic recovery of contractile function and cardiac enzyme release. Striking features of preconditioning are the temporal aspects of the protection. The protection conferred by preconditioning is not absolute in as much as preconditioning in canine myocardium limits infarction produced by a 40 min coronary occlusion but does not protect against a 180 min occlusion (Murry et al., 1986). Protection is lost if the reperfusion period between the brief preconditioning ischaemia and the long ischaemic period is extended beyond 2 or 3 h (van winkle et al., 1991; Kuzuya et al., 1993). However, a further period of protection may be detected many hours later suggesting a biphasic pattern of protection. The early phase of protection, of rapid onset and short duration is the classic preconditioning effect, originally described by Murry et al. (1986), while the delayed phase occurring many hours later and lasting much longer, has been termed 'second window of protection', 'delayed preconditioning' or 'late preconditioning' (Bolli, 2000; Baxter & Ferdinandy, 2001). Extensive research has revealed that several endogenous mediators of myocyte, endothelial and neural origin, are generated during the brief preconditioning period, and these act as co-activators (or 'triggers') of a signal transduction cascade that rapidly results in the acquisition of tolerance to further ischaemia (see Figure 1). Detailed discussion of the involvement of multiple kinase families is beyond the scope of this review and has been comprehensively reviewed elsewhere (Cohen et al., 2000; Baines et al., 2001). The earliest mediator to be examined as an activator of preconditioning was adenosine. In several species, adenosine, released from myocytes as a consequence of ATP breakdown during preconditioning, acts on adenosine A1 receptors and possibly A3 receptors, initiating a signaling cascade. Early experimental evidence to support the involvement of adenosine came from Liu et al. (1991) who showed that adenosine receptor blockade with 8-p-(sulphophenyl)-theophylline (8-SPT) during preconditioning could abolish the infarct-limiting effect. Conversely, transient adenosine A1 receptor activation, but not A2 receptor activation, with selective agonists mimicked the preconditioning effect of brief coronary occlusion in the rabbit (Thornton et al., 1992; Tsuchida et al., 1993). A role for adenosine A3 receptor activation has been proposed by some workers (Armstrong & Ganote, 1994; Liu et al., 1994) but is not clearly resolved (Guo et al., 2001; Kilpatrick et al., 2001). It has since become clear that several other endogenously liberated autocrine/paracrine mediators contribute critically to initiating the process of cellular adaptation. All of these mediators are released or rapidly generated during relatively brief periods of myocardial ischaemia and they include catecholamines (Bankwala et al., 1994), opioid peptides (Schultz & Gross, 2001), reactive oxygen species (Baines et al., 1997) and bradykinin, which is the focus of this article. Schematic representation of the major identified pathways of early and delayed forms of preconditioning. Several autocrine/paracrine mediators released during the period of preconditioning ischaemia act on G-protein coupled receptors and are known to participate in the infarct-limiting effect of ischaemic preconditioning. These include adenosine released during ischaemia as a result of ATP breakdown, bradykinin released from vascular endothelium and mediators of neural origin (noradrenaline and opioid peptides). Reactive oxygen species, especially superoxide anion generated as a result of mitochondrial uncoupling, may also act as upstream mediators. A complex signal cascade is activated which involves activation of protein kinase C isoenzymes, tyrosine kinases and mitogen-activated protein kinases. The phosphorylation cascade is thought to result in activation of the ATP-sensitive potassium (KATP) channel on the mitochondrial inner membrane. At present it remains unknown how opening of this channel confers protection during ischaemia. The participation of other 'cytoprotective' proteins has been proposed, including proteins that suppress or modulate apoptosis and proteins associated with cytoskeletal integrity (αB-crystallin and 27 kDa heat shock protein). *The participation of endogenous NO (of endothelial or neural origin) in initiating the classical preconditioning mechanism may be model specific. Early protection against cell death and infarction is not NO-dependent whereas preconditioning against arrhythmias does involve NO generation. For delayed preconditioning, evidence for the involvement of NO (possibly as a signalling intermediate downstream of bradykinin) is more persuasive and consistent. The distinguishing feature of delayed preconditioning is the co-ordinated regulation of a gene transcription programme as a result of upstream kinase signalling. The delayed phase of protection is dependent on de novo synthesis of inducible proteins. Those thought to be particularly important in the acquisition of delayed tolerance to ischaemia include iNOS, cyclo-oxygenase-2 and intracellular antioxidant enzymes such as manganese – superoxide dismutase. For more detailed discussion see Baxter & Ferdinandy (2001). Bradykinin is one of several oligopeptides called kinins. The most important physiologically active kinins are kallidin (Lys-bradykinin), bradykinin, and des-Arg9-bradykinin. The interested reader is referred to a recent review by Blais et al. (2000) for a detailed account of kinin synthesis. Kallidin and bradykinin are synthesized by kallikreins acting on kininogen precursor molecules (summarized in Figure 2a). Precursors of kallikreins are found in plasma (pre-kallikreins) and in tissues (pro-kallikreins) and are activated by a variety of chemical and biological stimuli, including activated factor XII (Hageman factor). Circulating kininogens are synthesized primarily in liver and include a high molecular weight kininogen (88 – 115 kDa according to species) and a low molecular weight kininogen (50 – 68 kDa). (a) Synthesis of bradykinin. The activated form of Hageman factor promotes the conversion of prekallikrein to kallikrein. In rat, both plasma and tissue kallikrein catalyse the formation of bradykinin. In humans, however, plasma kallikrein generates bradykinin using high molecular weight kininogens and tissue kallikrein generates kallidin using low molecular weight kininogens. (b) Basic amino acid sequence of bradykinin and precursors. Arrows designate the peptide bonds cleaved by: 1 kininase-I; 2 angiotensin converting enzyme (kininase-II); 3 neutral endopeptidase; 4 aminopeptidase. Vascular endothelial cells are the primary source of bradykinin in the heart (Linz et al., 1996; Wirth et al., 1997). Enzymatic cleavage of pre-kallikrein generates kallikrein at the endothelial cell surface. Circulating kininogen is then cleaved by kallikrein to generate the kinin at the endothelial cell surface. The mechanisms by which pre-kallikrein is activated may be factor XII-dependent or -independent. In the absence of endothelial cell injury and hence contact binding of factor XII, the mechanism of kininogen attraction may involve a cell surface receptor complex. However, this mechanism is presently not well understood. It has been proposed that isolated cardiac myocytes can synthesize kinins (Matoba et al., 1999) but this possibility remains to be investigated more fully. A number of studies have provided evidence that even during brief preconditioning periods of ischaemia tissue and plasma bradykinin levels increase markedly (Linz et al., 1996; Schulz et al., 1998; Campbell, 2000; Pan et al., 2000). Bradykinin is generated in isolated tissues and endothelial cells in the absence of plasma (Baumgarten et al., 1993; Ahmad et al., 1996; Linz et al., 1996). Bradykinin released during ischaemia has been shown to primarily originate from endothelial cells (Linz et al., 1996; Wirth et al., 1997) but the precise molecular pathological mechanism leading to bradykinin generation during brief ischaemia is not understood. Once released, bradykinin is rapidly degraded into inactive metabolites (Bhoola et al., 1992) (Figure 2b). Enzymes that degrade bradykinin are generically referred to as 'kininases' or 'kinin peptidases'. The most important of these metalloproteases are angiotensin converting enzyme (ACE; syn. kininase II; EC 3.4.15.1), neutral endopeptidase (NEP; syn. NEP 24.11; enkephalinase; EC 3.4.24.11), kininase I (syn. carboxypeptidase N; EC 3.4.17.3), carboxypeptidase M (syn. membrane-bound kininase I), and aminopeptidase P (syn. prolyl-aminopeptidase; EC 3.4.11.9). Other enzymes include endopeptidase (EC 3.4.24.15), endothelin converting enzyme (ECE) and prolyl endopeptidase but their contribution is small (Brown & Vaughan, 1998; Erdos & Skidgel, 1997; Piedimonte et al., 1994). ACE is regarded as the most important kininase in most species (Ahmad et al., 1996; Dumoulin et al., 1998; Ersahin & Simmons, 1997; Hornig et al., 1997; Kuoppala et al., 2000). ACE has a higher affinity for bradykinin than for angiotensin I, resulting in more favourable kinetics for bradykinin than for angiotensin I degradation (Zisman, 1998). Hence ACE may be regarded as being primarily a kininase rather than an angiotensinase (Blais et al., 2000). The actions of kinins are mediated by two receptor subtypes, distinguishable on the basis of both pharmacological and molecular characterization (Hall, 1992; 1997). The bradykinin B2 receptor usually predominates, with the bradykinin B1 receptor only being expressed under pathological conditions (Bhoola et al., 1992). The B2 receptor belongs to the family of heptahelical G-protein coupled receptors, which initiate the generation of inositol triphosphate and diacylglycerol, with subsequent activation of PKC (Derian & Moskowitz, 1986; Minshall et al., 1995; Morgan-Boyd et al., 1987). Highly specific antagonists at the B2 receptor include the bradykinin-derivative icatibant (HOE140) and the non-peptide FR173657 (Aramori et al., 1997). Schoelkens et al. (1988) were the first to report the cardioprotective effects of exogenously administered bradykinin. In an isolated working rat heart preparation subjected to ischaemia followed by reperfusion, perfusion with bradykinin 10−10 mol l−1 resulted in better recovery of coronary flow and cardiac work during reperfusion, a reduction in the release of soluble markers of tissue injury, and improvement of metabolic efficiency. Following this report, intracoronary bradykinin administration, at a dose that did not induce coronary vasodilatation, was found to suppress both ischaemia- and reperfusion-induced arrhythmias in an anaesthetized canine model of epicardial coronary artery occlusion (Végh et al., 1991). In a porcine coronary occlusion model, infusion of bradykinin after the onset of coronary occlusion was found to attenuate plasma creatine kinase concentration. (Tio et al., 1991; Tobe et al., 1991). Subsequently, Végh et al. (1993) showed the anti-arrhythmic effect of bradykinin to be mediated by NO. These workers suggested that bradykinin might be a 'primary mediator' of ischaemic preconditioning. In 1994, two reports provided evidence for a primary role of endogenous bradykinin in mediating ischaemic preconditioning. Wall et al. (1994) reported that icatibant abolished the protective effects of preconditioning against infarction in an anaesthetized open-chest rabbit preparation with coronary artery occlusion. They also found that protection, comparable to that induced by preconditioning, could be produced by direct infusion of exogenous bradykinin (Wall et al., 1994). Almost simultaneously, Végh et al. (1994) documented the abrogation by icatibant of the anti-arrhythmic effects of preconditioning in the canine coronary occlusion model. Goto et al. (1995) subsequently confirmed the finding of Wall et al. (1994) that icatibant blocked the infarct-limiting effect of preconditioning in rabbit heart in vivo. However, they were unable to abolish the protective effect of preconditioning with icatibant in an isolated buffer-perfused rabbit heart preparation even when the preconditioning stimulus was increased from one to four 5 min cycles of ischaemia. This apparent non-participation of bradykinin in preconditioning of the buffer-perfused heart was attributed to the lack of blood-borne kininogens. Similarly, Bugge & Ytrehus (1996) found that icatibant did not block the protective effects of preconditioning in an isolated rat heart preparation. However, Bouchard et al. (1998) found that preconditioning of isolated rat heart attenuated post-ischaemic endothelial dysfunction. This effect was not abolished by icatibant but was reversed by Lys [Leu8] Des-Arg9-bradykinin, a bradykinin B1 receptor antagonist. Despite these inconsistencies in the literature examining the participation of endogenous bradykinin in isolated heart preparations, the ability of exogenously administered bradykinin to mimic ischaemic preconditioning has been confirmed by numerous investigators in a variety of models. These include the isolated rabbit heart with infarct size as the endpoint (Goto et al., 1995); the isolated rat heart with infarct size (Goto et al., 1995; Bugge & Ytrehus, 1996; Starkopf et al., 1997; Feng & Rosenkranz, 1999; Feng et al., 2000; Ebrahim et al., 2000) and ischaemia-reperfusion arrhythmias (Hassanabad et al., 1998) as endpoints; in pigs subjected to infarction (Schulz et al., 1998); in isolated human cardiac tissue subjected to hypoxia and reoxygenation (Brew et al., 1995) and in humans undergoing coronary angioplasty with ST segment shift as the endpoint (Leesar et al., 1999). Further evidence implying a central role for bradykinin in ischaemic preconditioning comes from mice with a targeted disruption of the bradykinin B2 receptor gene. Yang et al. (1997b), using B2 receptor knock-out mice, showed that ischaemic preconditioning did not confer protection against infarct size in these animals. These workers also demonstrated that rats deficient in high molecular weight kininogen did not display the preconditioning response (Yang et al., 1997b). A recent study provides compelling genetic evidence supporting a cytoprotective role of bradykinin in the ischaemic heart (Yoshida et al., 2000). The human tissue kallikrein gene was delivered into rats using adenoviral vector. One week following gene delivery, cardiac kinin levels were significantly increased. Hearts were subjected to coronary artery occlusion and reperfusion. It was observed that kallikrein gene delivery was associated with significant limitation of infarct size and attenuated severity of ventricular fibrillation. Finally, kallikrein gene delivery also attenuated apoptosis in the ischaemic zone, determined by terminal deoxynucleotidyl transferase-mediated nick end labelling. All the beneficial effects kallikrein overexpression were abolished by icatibant, implying a role for the bradykinin B2 receptor in the protection observed. Although the majority of work implicates involvement of bradykinin B2 receptor activation in mediating the cardioprotective actions of bradykinin during ischaemia-reperfusion, there is limited evidence implying a role for bradkinin B1 receptor activation. The bradykinin B1 receptor is not constitutively expressed in most tissues but is inducible under certain pathological conditions such as and (Bhoola et al., 1992). of this receptor has been proposed to the vascular protection by preconditioning. Bouchard et al. (1998) reported that the beneficial effects of ischaemic preconditioning on endothelial function was mediated by activation of the bradykinin B1 In to and found that bradykinin limited and reduced the of arrhythmias in the isolated rat heart model et al., 1993). This protective effect was not using but with a specific bradykinin B1 receptor Lys [Leu8] Des-Arg9-bradykinin, implying a role for the bradykinin B1 receptor as to the bradykinin B2 receptor et al., 1993). The mechanisms underlying the protective actions of bradykinin protection are not well understood. A number of have been proposed to participate in the protection including protein kinase C and tyrosine kinases et al., 1993; Goto et al., 1995; et al., 1995; Bugge & Ytrehus, 1996; Feng & Rosenkranz, 1999; Feng et al., 2000). is almost that B2 receptor activation is required for protection, since icatibant in most the protection by bradykinin. PKC activation is thought to be central in the preconditioning phenomenon and it has been proposed that it the phosphorylation of kinase and end proteins. et al. Bugge & Ytrehus (1996) and Goto et al. (1995) have evidence that exogenously administered bradykinin against ischaemia-reperfusion injury a mechanism in isolated rat and isolated rabbit The role of NO in mediating the cardioprotective properties of both endogenous and exogenously administered bradykinin has been the of some NO has been in some studies as a mediator of the early cardioprotective actions of bradykinin & 1992; Végh et al., 1993; et al., 1995; Feng et al., 2000). However, in to these Goto et al. (1995) found that infarct in rabbit heart was not by Bugge & Ytrehus (1996) found that in rat heart was not by a NO with a pharmacological to there may be important species and in the role of NO in mediating the cardioprotective actions of bradykinin, with some NO see although the infarct-limiting effect of bradykinin does not to be the delayed effect of bradykinin may be mediated by NO generation. of the mitochondrial channel has been proposed as mediator of preconditioning 2000) (see Figure 1). At present it is not known how opening of this channel might from ischaemia. pharmacological evidence the that bradykinin may protection a mechanism mitochondrial channel opening et al., 2000; et al., 2000). The mechanism by which this might is not Several have been proposed including the release of the generation of NO and the activation of a kinase downstream of PKC shown in Figure 1). bradykinin of Although have been shown to which have also been suggested to participate in classical preconditioning et al., 1994; et al., there is evidence at present to that mitochondrial channel Similarly, it is not clear to extent NO generated as a result of bradykinin B2 receptor activation on endothelium in or to mitochondrial channel activation. is some evidence that NO mitochondrial channel opening in cardiac myocytes et al., 2000) important by Goto et al. (1995) was the of involvement in preconditioning. were subjected to coronary artery occlusion with infarct size as the endpoint of protection. 5 min cycles of ischaemia were to the preconditioning response then icatibant did not block protection. However, if one 5 min of ischaemia was to the myocardium then protection was by These who provided evidence for the involvement of adenosine in ischaemic preconditioning, that a be in for the protective response of preconditioning to It was proposed that when only one brief of ischaemia is then bradykinin plays a primary role in protection such that B2 receptor blockade the effect. However, if cycles are other mediators are generated in such that the can be even in the of the B2 receptor (see Figure support for this important has been provided by several investigators in to the of ACE which is in more Schematic the multiple of classical preconditioning by Goto et al. The that the preconditioning response is only when a of intracellular kinase is All of the endogenous mediators known to act as of the preconditioning response can the intracellular signal cascade but they act in to the preconditioning a preconditioning is blockade of adenosine receptors with blockade of bradykinin B2 receptors with icatibant, blockade of opioid receptors with or of with will be to the of the intracellular signal the multiple preconditioning cycles are relatively more of the endogenous are these of may result in kinase activation to the which protection. The role of ACE as a kininase of primary is by studies in which ACE have been shown to and tissue bradykinin (Baumgarten et al., 1993; et al., 1994; Hornig & 1997). et al. (1993) showed that increased bradykinin from the isolated ischaemic rat This study showed that a kallikrein in the rat heart but that ACE are of bradykinin levels by its breakdown (see Figure this in several studies that ACE can a preconditioning stimulus by bradykinin levels et al., 1996; & 1997; Ebrahim et al., A preconditioning stimulus is regarded as a short ischaemic period which some or of the in classical preconditioning bradykinin) but their is to the protective response (see Figure et al. (1996) showed that with a preconditioning was to the preconditioning response in the open-chest rabbit coronary artery occlusion which was with icatibant, implying a role for the bradykinin B2 Similarly, & found that both and were to enhance preconditioning by bradykinin an effect also with Ebrahim et al. have shown that a preconditioning to induce limitation of infarct size in the isolated rat of the preconditioning response by angiotensin converting enzyme or neutral endopeptidase Bradykinin is and rapidly degraded by several enzymes especially ACE and of these enzymes the of bradykinin at B2 receptors on cardiac brief periods of ischaemia, bradykinin may be to initiate the preconditioning However, in the of an ACE or NEP of bradykinin is to initiate preconditioning. The ability of ACE to ischaemic has been demonstrated for both early and delayed forms of preconditioning (see The ability of ACE to confer protection in the absence of a preconditioning stimulus is more et al., 1997). In the studies of et al., 1996; & 1997; Ebrahim et al., or & 1997) to the ischaemic did not result in protection. Those are in to several other studies in which ACE was shown to protect against