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In the last 20 years there has been a revolution in our understanding of how blood flow is regulated in many tissues. Whereas it used to be thought that essentially all blood flow control occurred at the arteriole level, it is now recognised that control of capillary blood flow by contractile pericytes plays a key role both in regulating blood flow physiologically and in reducing it in clinically-relevant pathological conditions. In this article we compare and contrast how brain and cardiac pericytes regulate cerebral and coronary blood flow, focusing mainly on the pathological events of cerebral and cardiac ischemia. The cerebral and coronary capillary beds differ dramatically in morphology, yet in both cases pericyte-mediated capillary constriction plays a key role in restricting blood flow after ischemia and possibly in other pathological conditions. We conclude with suggestions for therapeutic approaches to relaxing pericytes, which may prove useful in the long term for reducing pericyte-induced ischemia., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Competing Interests: Conflict of interest: None declared.

Aims: Female sex has been proposed to be cardioprotective per se. Studies with myocardial ischaemia/reperfusion and infarct size as endpoint have demonstrated cardioprotection in female, castrated male, and male pigs. These studies are difficult to compare, given the different pig strains, models, durations of ischaemia, and methods of infarct size quantification. The few studies using both female and male pigs reported no differences in infarct size and cardioprotection. We, therefore, prospectively compared infarct size in Göttingen minipigs undergoing ischaemia/reperfusion (I/R) without and with ischaemic pre-conditioning (IPC) between female, castrated male, and male pigs., Methods and Results: In a prospective, randomized approach, 28 Göttingen open-chest, anaesthetized minipigs underwent 60 min ischaemia by distal left anterior descending artery (LAD) occlusion and 180 min reperfusion without and with IPC by three cycles of 5 min LAD occlusion/10 min reperfusion. Infarct size with I/R was not different between female, castrated male, and male pigs (45 ± 8 vs. 45 ± 13 vs. 41 ± 9% area at risk), as was the reduction in infarct size with IPC (25 ± 11 vs. 30 ± 8 vs. 19 ± 10% area at risk). In addition, the area of no-reflow was not different between female, castrated male, and male pigs with I/R (57 ± 13 vs. 35 ± 7 vs. 47 ± 26% infarct size) or IPC (4 ± 10 vs.12 ± 20 vs. 0 ± 0% infarct size). Phosphorylation of signal transducer and activator of transcription 3 was increased at 10 min reperfusion by IPC but not by I/R to the same extent in female, castrated male, and male pigs (198 ± 30 vs. 230 ± 165 vs. 179 ± 107% of baseline)., Conclusion: Our data do not support the notion of sex- or castration-related differences in infarct size, coronary microvascular injury, and cardioprotection by IPC., Translational Perspective: The translation of successful preclinical studies on cardioprotection to the benefit of patients with reperfused myocardial infarction has been difficult. The difficulties have been attributed to confounders such as co-morbidities and co-medications which patients typically have but animals don´t, but also to age and sex. Notably, female sex has been considered as protective per se. We have now, using our established and clinically relevant pig model of reperfused acute myocardial infarction and ischaemic preconditioning as the most robust cardioprotective intervention looked for sex-related differences of infarct size, no-reflow and cardioprotection by ischaemic preconditioning in a prospectively powered approach but found none such difference., Competing Interests: Conflict of interest: none declared, (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Obstructive disease of the epicardial coronary arteries is the main cause of angina. However, a number of patients with anginal symptoms have normal coronaries or non-obstructive coronary artery disease (CAD) despite electrocardiographic evidence of ischaemia during stress testing. In addition to limited microvascular vasodilator capacity, the coronary microcirculation of these patients is particularly sensitive to vasoconstrictor stimuli, in a condition known as microvascular angina. This review briefly summarizes the determinants and control of coronary blood flow (CBF) and myocardial perfusion. It subsequently analyses the mechanisms responsible for transient myocardial ischaemia: obstructive CAD, coronary spasm and coronary microvascular dysfunction in the absence of epicardial coronary lesions, and variable combinations of structural anomalies, impaired endothelium-dependent and/or -independent vasodilation, and enhanced perception of pain. Lastly, we exemplify mechanism of angina during tachycardia. Distal to a coronary stenosis, coronary dilator reserve is already recruited and can be nearly exhausted at rest distal to a severe stenosis. Increased heart rate reduces the duration of diastole and thus CBF when metabolic vasodilation is no longer able to increase CBF. The increase in myocardial oxygen consumption and resulting metabolic vasodilation in adjacent myocardium without stenotic coronary arteries further acts to divert blood flow away from the post-stenotic coronary vascular bed through collaterals., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

The coronary circulation is both culprit and victim of acute myocardial infarction. The rupture of an epicardial atherosclerotic plaque with superimposed thrombosis causes coronary occlusion, and this occlusion must be removed to induce reperfusion. However, ischaemia and reperfusion cause damage not only in cardiomyocytes but also in the coronary circulation, including microembolization of debris and release of soluble factors from the culprit lesion, impairment of endothelial integrity with subsequently increased permeability and oedema formation, platelet activation and leucocyte adherence, erythrocyte stasis, a shift from vasodilation to vasoconstriction, and ultimately structural damage to the capillaries with eventual no-reflow, microvascular obstruction (MVO), and intramyocardial haemorrhage (IMH). Therefore, the coronary circulation is a valid target for cardioprotection, beyond protection of the cardiomyocyte. Virtually all of the above deleterious endpoints have been demonstrated to be favourably influenced by one or the other mechanical or pharmacological cardioprotective intervention. However, no-reflow is still a serious complication of reperfused myocardial infarction and carries, independently from infarct size, an unfavourable prognosis. MVO and IMH can be diagnosed by modern imaging technologies, but still await an effective therapy. The current review provides an overview of strategies to protect the coronary circulation from acute myocardial ischaemia/reperfusion injury. This article is part of a Cardiovascular Research Spotlight Issue entitled 'Cardioprotection Beyond the Cardiomyocyte', and emerged as part of the discussions of the European Union (EU)-CARDIOPROTECTION Cooperation in Science and Technology (COST) Action, CA16225., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.)

Acute myocardial infarction (AMI) and the heart failure (HF) that often complicates this condition, are among the leading causes of death and disability worldwide. To reduce myocardial infarct (MI) size and prevent heart failure, novel therapies are required to protect the heart against the detrimental effects of acute ischaemia/reperfusion injury (IRI). In this regard, targeting cardiac innervation may provide a novel therapeutic strategy for cardioprotection. A number of cardiac neural pathways mediate the beneficial effects of cardioprotective strategies such as ischaemic preconditioning and remote ischaemic conditioning, and nerve stimulation may therefore provide a novel therapeutic strategy for cardioprotection. In this article, we provide an overview of cardiac innervation and its impact on acute myocardial IRI, the role of extrinsic and intrinsic cardiac neural pathways in cardioprotection, and highlight peripheral and central nerve stimulation as a cardioprotective strategy with therapeutic potential for reducing MI size and preventing HF following AMI. This article is part of a Cardiovascular Research Spotlight Issue entitled 'Cardioprotection Beyond the Cardiomyocyte', and emerged as part of the discussions of the European Union (EU)-CARDIOPROTECTION Cooperation in Science and Technology (COST) Action, CA16225., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

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., (The last two authors contributed equally to the paper as joint senior authors. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.)

Ischaemic heart disease (IHD) remains the leading cause of death and disability worldwide. As a result, novel therapies are still needed to protect the heart from the detrimental effects of acute ischaemia-reperfusion injury, in order to improve clinical outcomes in IHD patients. In this regard, although a large number of novel cardioprotective therapies discovered in the research laboratory have been investigated in the clinical setting, only a few of these have been demonstrated to improve clinical outcomes. One potential reason for this lack of success may have been the failure to thoroughly assess the cardioprotective efficacy of these novel therapies in suitably designed preclinical experimental animal models. Therefore, the aim of this Position Paper by the European Society of Cardiology Working Group Cellular Biology of the Heart is to provide recommendations for improving the preclinical assessment of novel cardioprotective therapies discovered in the research laboratory, with the aim of increasing the likelihood of success in translating these new treatments into improved clinical outcomes., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Coronary heart disease (CHD) is the leading cause of death and disability worldwide. Despite current therapy, the morbidity and mortality for patients with CHD remains significant. The most important manifestations of CHD arise from acute myocardial ischaemia-reperfusion injury (IRI) in terms of cardiomyocyte death and its long-term consequences. As such, new therapeutic interventions are required to protect the heart against the detrimental effects of acute IRI and improve clinical outcomes. Although a large number of cardioprotective therapies discovered in pre-clinical studies have been investigated in CHD patients, few have been translated into the clinical setting, and a significant number of these have failed to show any benefit in terms of reduced myocardial infarction and improved clinical outcomes. Because of this, there is currently no effective therapy for protecting the heart against the detrimental effects of acute IRI in patients with CHD. One major factor for this lack of success in translating cardioprotective therapies into the clinical setting can be attributed to problems with the clinical study design. Many of these clinical studies have not taken into consideration the important data provided from previously published pre-clinical and clinical studies. The overall aim of this ESC Working Group Cellular Biology of the Heart Position Paper is to provide recommendations for optimizing the design of clinical cardioprotection studies, which should hopefully result in new and effective therapeutic interventions for the future benefit of CHD patients.

Cardioprotection, i.e. the reduction in infarct size by pre-, post-, or remote conditioning, has originally been characterized in young and healthy experimental animals. Over the last two decades many signalling steps of cardioprotection have been identified, again in young and healthy animals. Although proof-of-concept studies unequivocally demonstrated the recruitment of all forms of cardioprotection in humans, the translation of cardioprotection to clinical routine has been poor. The apparent lack of translation has been attributed to poor design of clinical trials, but also to the neglect of confounders, such as age, sex, comorbidities, and comedications, in experimental studies. The present opinionated review focuses on the coronary circulation as a major determinant of cardioprotection. Coronary occlusion and the restoration of coronary blood flow are the causes of myocardial ischaemia and reperfusion injury from which protection is sought. On the other hand, brief cycles of coronary occlusion and reperfusion are also the stimulus for protection from myocardial ischaemia/reperfusion injury. The recruitment of collateral blood flow also contributes to protection from infarction. Finally, the coronary microcirculation is also a target of both injury by myocardial ischaemia/reperfusion and protection from it. Different manifestations of coronary artery disease, such as coronary stenosis or coronary microembolization, impact on both injury and protection.

Ischaemic postconditioning (brief periods of ischaemia alternating with brief periods of reflow applied at the onset of reperfusion following sustained ischaemia) effectively reduces myocardial infarct size in all species tested so far, including humans. Ischaemic postconditioning is a simple and safe manoeuvre, but because reperfusion injury is initiated within minutes of reflow, postconditioning must be applied at the onset of reperfusion. The mechanisms of protection by postconditioning include: formation and release of several autacoids and cytokines; maintained acidosis during early reperfusion; activation of protein kinases; preservation of mitochondrial function, most strikingly the attenuation of opening of the mitochondrial permeability transition pore (MPTP). Exogenous recruitment of some of the identified signalling steps can induce cardioprotection when applied at the time of reperfusion in animal experiments, but more recently cardioprotection was also observed in a proof-of-concept clinical trial. Indeed, studies in patients with an acute myocardial infarction showed a reduction of infarct size and improved left ventricular function when they underwent ischaemic postconditioning or pharmacological inhibition of MPTP opening during interventional reperfusion. Further animal studies and large-scale human studies are needed to determine whether patients with different co-morbidities and co-medications respond equally to protection by postconditioning. Also, our understanding of the underlying mechanisms must be improved to develop new therapeutic strategies to be applied at reperfusion with the ultimate aim of limiting the burden of ischaemic heart disease and potentially providing protection for other organs at risk of reperfusion injury, such as brain and kidney.

Aims: Reperfusion is mandatory to salvage ischaemic myocardium from infarction, but also induces additional reperfusion injury and contributes to infarct size (IS). Gentle reperfusion (GR) has been proposed to attenuate reperfusion injury, but this remains contentious. We now investigated whether (i) GR reduces IS and (ii) GR is associated with the activation of reperfusion injury salvage kinases (RISK)., Methods and Results: Anaesthetized pigs were subjected to 90 min left anterior descending coronary artery hypoperfusion and 120 min reperfusion. GR was induced by slowly increasing coronary inflow back to baseline over 30 min, using an exponential algorithm [F(t) = F(i)+e(-(0.1)(t)((min)-3)).(F(b)-F(i)); F(b), coronary inflow at baseline; F(i), coronary inflow during ischaemia; n = 12]. Pigs subjected to immediate full reperfusion (IFR; n = 13) served as controls. IS was determined by triphenyl tetrazolium chloride staining. The expression level of phosphorylated RISK proteins was determined by western blot analysis in myocardial biopsies taken at baseline, after 80-85 min ischaemia and at 10, 30, and 120 min reperfusion. In additional experiments with IFR (n = 3) and GR (n = 3), the PI3-AKT and MEK1/2-ERK1/2 pathways were pharmacologically blocked (BL). IS was 37 +/- 2% (mean +/- SEM) of the area at risk with IFR and 29 +/- 1% (P < 0.05) with GR. RISK phosphorylation was similar between GR and IFR at baseline and 85 min ischaemia. At 10 min reperfusion, RISK phosphorylation was increased with IFR, but not with GR. At 30 and 120 min reperfusion, RISK phosphorylation was still greater with IFR than GR. RISK blockade did not abolish the IS reduction by GR (BL-IFR: 27 +/- 4% of the area at risk; BL-GR: 42 +/- 5%; P < 0.05)., Conclusion: Gentle reperfusion reduces infarct size in pigs, but RISK activation is not causally involved in this infarct size reduction.

Aims: Connexin43 is present at the inner membrane of cardiomyocyte mitochondria (mCx43), but its function remains unknown., Methods and Results: In this study we verified the presence of mCx43 by a mass spectrometry-based proteomic approach in purified mitochondrial preparations from mouse myocardium and determined by western blot analysis that the C-terminus of mCx43 is oriented towards the intermembrane space. Cross-linking studies with dimethylsuberimidate indicated the presence of Cx43 hexamers in mitochondrial membranes. The contribution of Cx43 to both mitochondrial dye uptake and K(+) flux was assessed in wild-type mice using hemichannel blockers and Cx43KI32 mice in which Cx43 had been replaced by Cx32. Uptake of the Cx43 hemichannel-permeant dye Lucifer Yellow was reduced in mitochondria from wild-type mice by two hemichannel blockers (carbenoxolone and heptanol) and in Cx43KI32 compared with wild-type mice. Mitochondrial K(+) influx (PBFI fluorescence) was decreased in digitonin-permeabilized cardiomyocytes from Cx32 mutants compared with wild-type mice, and addition of the Cx43 hemichannel blocker 18alpha-glycyrrhetinic acid had an inhibitory effect on mitochondrial K(+) influx in wild-type cardiomyocytes, but not in cardiomyocytes from Cx32 mutants., Conclusion: These results indicate that mCx43 contributes to mitochondrial K(+) flux in cardiomyocytes, potentially by forming hemichannel-like structures.

Not only the prevalence, but also the mortality due to ischaemic cardiovascular disease is higher in older than in young humans, and the demographic shift towards an ageing population will further increase the prevalence of age-related cardiovascular disease. In order to develop strategies aimed to limit reversible and irreversible myocardial damage in older patients, there is a need to better understand age-induced alterations in protein expression and cell signalling. Cardioprotective phenomena such as ischaemic and pharmacological pre and postconditioning attenuate ischaemia/reperfusion injury in young hearts. Whether or not pre and postconditioning are still effective in aged organs, animals, or patients, i.e. under conditions where such cardioprotection is most relevant, is still a matter of debate; most studies suggest a loss of protection in aged hearts. The present review discusses changes in protein expression and cell signalling important to ischaemia/reperfusion injury with myocardial ageing. The efficacy of cardioprotective manoeuvres, e.g. ischaemic pre and postconditioning in aged organs and animals will be discussed, and the development of strategies aimed to antagonize the age-induced loss of protection will be addressed.

Connexins are transmembrane proteins whose best known function is to form gap junction channels. Ventricular cardiomyocytes express the connexin isoform Cx43 and are rich in gap junctions essential for the normal propagation of the action potential. In addition to this physiological role, cardiomyocyte gap junctions contribute to the pathophysiology of ischaemia-reperfusion injury, mainly by synchronizing the onset of rigour contracture during ischaemia and cell-to-cell propagation of hypercontracture during reperfusion. More recently, it has been recognized that Cx43 plays a role in protection during ischaemic and pharmacological preconditioning and that this role is independent from gap junction-mediated communication. It was demonstrated that Cx43 is localized in cardiomyocyte mitochondria, at least in part in the inner mitochondrial membrane, where it is imported by the general mitochondrial membrane translocase system. Interfering with this import system or genetic ablation of Cx43 abolishes diazoxide-induced protection, indicating that mitochondrial Cx43 participates in the preconditioning cascade. The role of mitochondrial Cx43 in preconditioning appears to be related to reactive oxygen species signalling, but its molecular mechanisms have not been elucidated in detail. The present article reviews available evidence on the localization of Cx43 in cardiomyocyte mitochondria, its role in protection by preconditioning, and the potential molecular mechanisms involved. These data may help to understand the pathophysiology of myocardial ischaemia-reperfusion and ischaemic preconditioning and to identify new strategies for cardioprotection, and they may be particularly relevant to situations such as aging in which total and mitochondrial Cx43 contents have been shown to be reduced.

Objectives: Nitric oxide (NO) synthesis by NO synthases (NOS) requires oxygen. However, although counterintuitive, NO synthesis is increased in ischemic myocardium. Accordingly, mechanisms independent of the NOS pathway have been suggested to contribute to NO synthesis during ischemia. NO initiates detrimental as well as protective mechanisms in a concentration-dependent manner, thus aggravating or improving the outcome of ischemia. The aim of this study was to measure in situ interstitial NO concentrations in parallel to infarct size in anaesthetized pigs subjected to myocardial ischemia/reperfusion. The contribution of NOS-independent pathways to NO synthesis was studied using NOS blockade., Methods: Interstitial NO measurements, based on microdialysis combined with the oxyhemoglobin method, were made during 90 min of moderate or severe ischemia and subsequent reperfusion. To examine the effect of NOS inhibition, an initial 30-min ischemic period was followed 60 min later by a second 30-min ischemic period with intracoronary infusion of S-ethyl-isothiourea., Results: During ischemia, the interstitial NO concentration increased for about 30 min and then remained constant at this elevated level. The increase in NO concentration by 253+/-82 nmol/L during moderate and 565+/-169 nmol/L during severe ischemia correlated inversely with subendocardial blood flow (r=-0.76). NOS inhibition increased coronary arterial pressure and decreased the interstitial basal NO concentration and tissue nitrite content. However, it did not diminish the increase in interstitial NO concentration during ischemia., Conclusion: NOS-independent pathways are significantly involved in NO synthesis during myocardial ischemia.

Objective: Reactive oxygen species (ROS) play an ambivalent role in cardiomyocytes: low concentrations are involved in cellular signaling, while higher concentrations contribute to cellular injury. We studied ROS formation during increases in contraction frequency in isolated cardiomyocytes., Methods: Rat ventricular cardiomyocytes were loaded with dichlorodihydrofluorescein and electrically stimulated (37 degrees C). ROS formation was assessed by the rate of oxidation-dependent fluorescence increase (OxR). Oxygen consumption (VO(2)) and NAD(P)H autofluorescence were measured in parallel experiments., Results: Increases in contraction frequency were accompanied by an increase in VO(2) and a decrease in NAD(P)H fluorescence. OxR increased to 124+/-4%, 146+/-8%, 204+/-25% and 256+/-29% of OxR at baseline during 1, 2, 3 and 4 Hz stimulation, and subsequently returned to baseline values with 0.2 Hz. The OxR increase was dose-dependently inhibited by the antioxidant NAC (10 and 100 mM), but unaffected by the NO synthase inhibitor l-NAME (200 microM and 10 mM). The OxR increase was attenuated when myosin ATPase activity was inhibited by butanedione monoxime (BDM; 5 mM)., Conclusion: Increased contraction frequency induces ROS formation in rat cardiomyocytes.

Objective: The extracellular matrix (ECM) of the atherosclerotic lesion is a crucial determinant of its stability, while its degradation by matrix metalloproteinases (MMPs) has been implied in plaque rupture. As accumulation of both MMP-derived collagen fragments and apoptotic smooth muscle cells (SMC) is observed at sites of plaque rupture, we tested the effect of polymerized and degraded type I collagen on the susceptibility of SMC to apoptosis., Methods: Human SMC were cultured on monomeric or polymerized collagen, and collagen gels were degraded by collagenase. Apoptosis was evaluated using antibodies to active caspases and their substrates. Calpain and caspase activity were measured using fluorogenic substrates., Results: Culture of SMC on polymerized collagen led to increased apoptosis compared to culture on monomeric collagen. In addition, we observed a distinct proteolytic degradation of the endogenous caspase inhibitor X-chromosome-linked inhibitor of apoptosis (xIAP). As MMP-1 was strongly activated in SMC on polymerized collagen, we examined the effect of degraded collagen fragments on xIAP cleavage and apoptosis. Degraded collagen induced rapid proteolytic processing of xIAP identical to that on polymerized collagen. We identified calpains as the proteolytic enzymes responsible for xIAP processing as: i) they were rapidly activated by degraded collagen; ii) recombinant calpain II processed xIAP in an identical manner, and iii) inhibition of calpains by BAPTA or calpeptin abrogated xIAP degradation in intact cells. The functional consequence of xIAP processing by calpains was a loss of its caspase-inhibitory potential. Calpain activation distinctly preceded caspase activation, and inhibition of calpains suppressed apoptosis., Conclusions: Collagen fragments proteolytically released from the ECM by MMPs may propagate apoptosis of SMC by calpain-mediated inactivation of anti-apoptotic proteins such as xIAP. This may be a novel mechanism of SMC apoptosis in biological settings of enhanced collagen degradation such as vascular remodeling, neointima formation, and atherosclerotic plaque rupture.

Objective: Connexin 43 (Cx43) is involved in infarct size reduction by ischemic preconditioning (IP); the underlying mechanism of protection, however, is unknown. Since mitochondria have been proposed to be involved in IP's protection, the present study analyzed whether Cx43 is localized at mitochondria of cardiomyocytes and whether such localization is affected by IP., Methods and Results: Western blot analysis on mitochondrial preparations isolated from rat, mouse, pig, and human hearts showed the presence of Cx43. The preparations were not contaminated with markers for other cell compartments. The localization of Cx43 to mitochondria was also confirmed by FACS sorting (double staining with MitoTracker Red and Cx43) and immuno-electron and confocal microscopy. To study the role of Cx43 in IP, mitochondria were isolated from the ischemic anterior wall (AW) and the control posterior wall (PW) of pig myocardium at the end of 90 min low-flow ischemia without (n=13) or with (n=13) a preceding preconditioning cycle of 10 min ischemia and 15 min reperfusion. With IP, the mitochondrial Cx43/adenine nucleotide transporter ratio was 3.4+/-0.7 fold greater in AW than in PW, whereas the ratio remained unchanged in non-preconditioned myocardium (1.1+/-0.2, p<0.05). The enhancement of the mitochondrial Cx43 protein level occurred rapidly, since an increase of mitochondrial Cx43 was already detected with two cycles of 5 min ischemia/reperfusion in isolated rat hearts to 262+/-63% of baseline., Conclusion: These data demonstrate that Cx43 is localized at cardiomyocyte mitochondria and that IP enhances such mitochondrial localization.

Objectives: In human end-stage heart failure as well as in experimental animal models of heart failure, G-protein-coupled receptor kinase activity (GRK) is increased while beta-adrenoceptor responsiveness is diminished. In animal studies, beta-adrenoceptor blockers reverse the GRK-mediated desensitization and down-regulation of myocardial beta-adrenoceptors. The aim of this study was to investigate whether alterations in GRK activity are an early or late accompaniment of human heart failure and whether also in humans beta-adrenoceptor blocker treatment is able to influence myocardial GRK activity., Methods: We assessed in right atria, obtained from patients at different stages of heart failure, treated with or not treated with beta-adrenoceptor blockers, and in the four chambers of explanted hearts, obtained from patients with end-stage heart failure, beta-adrenoceptor density (by (-)-[(125)I]-iodocyanopindolol binding) and GRK activity (by an in vitro rhodopsin phosphorylation assay)., Results: With increasing severity of heart failure, plasma noradrenaline levels increased while myocardial beta-adrenoceptor density decreased with a maximum in GRK activity in end-stage heart failure. However, in relation to the progression of heart failure, we found that GRK activity transiently increased at an early stage of heart failure (NYHA I and II) but decreased back to control values in patients at NYHA III and IV. beta-Adrenoceptor blockers were able to reduce the early increase in GRK activity at NYHA I and II to control levels, whereas in those patients who did not have increased GRK activity (NYHA III and IV), they had only a marginal effect., Conclusion: According to our results, an increase in GRK activity is an early and transient event in the course of heart failure that can be prevented by beta-adrenoceptor blocker treatment.

Objective: After coronary microembolization (ME) adenosine is released from ischemic areas of the microembolized myocardium. This adenosine dilates vessels in adjacent nonembolized myocardium and increases coronary blood flow. For ischemic preconditioning (IP) to protect the myocardium against infarction, an increase in the interstitial adenosine concentration (iADO) prior to the subsequent ischemia/reperfusion is necessary. We hypothesized that the adenosine release after ME is sufficient to increase iADO and protect the myocardium against infarction from subsequent ischemia/reperfusion. We have therefore compared myocardial protection by either coronary microembolization or ischemic preconditioning prior to ischemia/reperfusion., Methods: In anesthetized pigs, the left anterior descending (LAD) was cannulated and perfused from an extracorporeal circuit. In 11 pigs, sustained ischemia was induced by 85% inflow reduction for 90 min (controls). Two other groups of pigs were subjected either to IP (n = 8; 10-min ischemia/15-min reperfusion) or coronary ME (n = 9; i.c. microspheres; 42 microm Ø; 3000 x ml(-1) x min inflow) prior to sustained ischemia. Coronary venous adenosine concentration (vADO) and iADO (microdialysis) were measured. Infarct size was determined after 2-h reperfusion by triphenyl tetrazolium chloride staining., Results: In pigs subjected to IP, infarct size was reduced to 2.6 +/- 1.1% (mean +/- S.E.M.) vs. 17.0 +/- 3.2% in controls. iADO was increased from 2.4 +/- 1.3 to 13.1 +/- 5.8 micromol x l(-1) during the reperfusion following IP. In pigs subjected to ME, at 10 min after ME, coronary blood flow (38.6 +/- 3.6 to 53.6 +/- 4.3 ml x min(-1)) and vADO (0.25 +/- 0.04 to 0.48 +/- 0.07 micromol x l(-1)) were increased. However, iADO (2.0 +/- 0.5 at baseline vs. 2.3 +/- 0.6 micromol x l(-1) at 10 min after ME) did not increase. Infarct size induced by sustained ischemia following ME (22.5 +/- 5.2%) was above that of controls for any given subendocardial blood flow., Conclusion: ME released adenosine into the vasculature and increased coronary blood flow. The failure of iADO to increase with ME possibly explains the lack of protection against infarction after ME., (Copyright 2004 European Society of Cardiology)

Aims: Inhibition of the Na+/H+-exchanger (NHE) preserves myocardial morphology and function in rat and mouse models of hypertrophy and failure. The mechanism(s) involved in such cardioprotective effects remain(s) unclear, but might involve blockade of increased protein kinase activity as observed in untreated hearts., Methods and Results: We investigated the functional, morphological and biochemical consequences of NHE-inhibition with BIIB722 in rabbits with pacing-induced heart failure (HF). In sham rabbits treated with placebo (n = 9) or BIIB722 (30 mg/kg/day po, n = 9), LV end-diastolic diameter (LVEDD) and systolic fractional shortening (FS, %) remained unchanged. In HF rabbits (n = 9), LVEDD increased and FS decreased from 31.5 +/- 1.4 to 8.1 +/- 0.9 (p < 0.05) at 3 weeks of LV pacing (400 bpm). Apoptosis, fibrosis and myocyte cross-sectional area as well as p38MAPK phosphorylation and iNOS protein expression were significantly increased in HF compared to sham rabbits. The activity of the 90 kDa NHE-kinase was greater in HF than in sham rabbits. In HF rabbits receiving BIIB722 prior to (18.1 +/- 2.2, n = 9) or following 1 week (15.5 +/- 1.6, n = 7) of pacing, FS at 3 weeks was better preserved than in untreated HF rabbits (p < 0.05). Apoptosis, fibrosis, myocyte cross-sectional area, p38MAPK phosphorylation and iNOS protein expression were significantly reduced in HF rabbits receiving BIIB722., Conclusion: NHE-inhibition attenuates the functional, morphological and biochemical derangements of pacing-induced HF in rabbits., (Copyright 2004 European Society of Cardiology)

Connexin 43 (Cx43) is the essential protein to form hemichannels and gap junctions in the myocardium. The phosphorylation status of Cx43 which is regulated by a variety of protein kinases and phosphatases determines hemichannel and/or gap junction conductance and permeability. Gap junctions are involved in cell-cell coupling while hemichannels contribute to cardiomyocyte volume regulation. Cx43-formed channels are involved in ischemia/reperfusion injury, since blockade of a large portion of Cx43-formed channels attenuates ischemic hypercontracture, infarct development and post myocardial infarction remodeling. Ischemic preconditioning's protection also depends on functional Cx43-formed channels, since uncoupling of channels or genetic Cx43 deficiency abolishes infarct size reduction by ischemic preconditioning. The exact underlying mechanism(s) how Cx43 mediates protection remain to be established.

Administration of nitric oxide (NO), NO donors or drugs that enhance NO release (statins, calcium antagonists, ACE-inhibitors, dexamethasone) prior to ischemia protects the myocardium against ischemia/reperfusion injury. While this exogenous administration of NO prior to ischemia can initiate a preconditioning-like phenomenon, endogenous NO-synthase (NOS)-derived NO is not involved in triggering or mediating the early phase of ischemic preconditioning's protection, but does play a pivotal role for initiating and mediating the delayed phase of ischemic preconditioning's protection. The present review now summarizes the importance of endogenous and exogenous NO when given at the time of reperfusion for vascular and myocardial function and morphological outcome following ischemia/reperfusion. Given the inconsistency of the published data, potential confounding factors that might affect experimental results on the role of NO in myocardial ischemia/reperfusion were identified, such as (1) the lack of characterization of the involved NOS isoforms in myocardial ischemia/reperfusion injury in different animal species, (2) the lack of direct measurements of myocardial NO concentration and/or NOS activity to assure sufficient NOS inhibition, (3) the lack of consideration of nonenzymatic NO production as a potential source of NO, and (4) the absence of plasma or blood components in in vitro studies influencing NO delivery and metabolism. Future research on the importance of NO in ischemia/reperfusion injury will have to focus more precisely on the identification and standardization of potential confounding experimental factors that influence synthesis, transport, and interaction of NO with various targets in blood and tissue.

Objective: In pigs, the infarct size (IS) reduction achieved by a strong preconditioning stimulus (IPs) of 10 min ischemia and 15 min reperfusion is greater than that by a weaker preconditioning stimulus (IPw) of 3 min ischemia and 15 min reperfusion. The cardioprotection achieved by IPw is completely abolished by blockade of bradykinin-B(2)-receptors. Since activation of bradykinin-B(2)-receptors subsequently activates cyclooxygenase, we now tested whether or not inhibition of cyclooxygenase with indomethacin interferes with IS reduction by IP., Methods and Results: In 42 enflurane-anesthetized pigs, the LAD coronary artery was cannulated, and subendocardial blood flow (ENDO, microspheres, ml/min/g) and IS (%, TTC-staining) were determined. Following 90 min ischemia and 120 min reperfusion, IS averaged 25.5+/-3.8 (S.E.M.) (ENDO: 0.05+/-0.01). IS was reduced by IPw to 6.3+/-2.1 (ENDO: 0.07+/-0.01) and further reduced by IPs to 2.4+/-1.0 (ENDO: 0.06+/-0.01). Indomethacin (10 mg/kg i.v.) did not alter IS per se (20.9+/-5.4, ENDO: 0.06+/-0.02), but completely abolished the IS reduction by IPw (23.2+/-5.9, ENDO: 0.06+/-0.01). In contrast, indomethacin abolished the IS reduction by IPs in only two of seven pigs (16.1+/-7.4, ENDO: 0.05+/-0.01)., Conclusion: Prostaglandins are involved in IP. However, with stronger IP stimuli other triggers/mediators can compensate for the lack of prostaglandins.

Objective: The role of p38MAPK in ischemic preconditioning (IP) is still equivocal, insofar as the p38MAPK-inhibitor SB203580 abolished IP in rats, rabbits and dogs, but not in pigs. Blockade of p38MAPK prior to the sustained ischemia also generated contradictory findings, insofar as p38MAPK acted as trigger in dogs but as mediator in rats. We have now tested whether the two structurally unrelated p38MAPK-inhibitors, BIX-645 and SB203580, abolished infarct size (IS) reduction by IP in pigs and whether their effects depended on the time of administration., Methods: Sixty-five enflurane-anesthetized pigs underwent 90 min low-flow ischemia and 120 min reperfusion without or with one preceding cycle of 10 min preconditioning ischemia and 15 min reperfusion. Pigs received BIX-645 (1 mg/kg, i.v.) or SB203580 (10 microM, i.c.) prior to either IP or the sustained ischemia., Results: IS (% TTC-staining) was reduced by IP [4.8+/-3.1(S.E.M.), P<0.05] compared to placebo (25.8+/-5.5). BIX-645 or SB203580 per se had no effect on IS (23.5+/-5.2 and 21.8+/-4.4, respectively). IS reduction by IP was abolished by BIX-645 (26.2+/-6.4 or 25.5+/-4.7) and SB203580 (19.9+/-4.3 or 16.7+/-4.7), given either prior to IP or the sustained ischemia, respectively. The supernatant of homogenized myocardial biopsies taken during the sustained ischemia from preconditioned pigs receiving either BIX-645 or SB203580 inhibited the anisomycin-stimulated ATF-2 phosphorylation in cultured Rat1 fibroblasts. This in vitro inhibition of ATF-2 phosphorylation correlated to the actual IS., Conclusion: The attenuation of the IS-reducing effect of IP depends on the effectiveness of blockade of p38MAPK activity. p38MAPK is a mediator of IP in pigs.

Objective: Short-term myocardial hibernation is characterized by an adaptation of contractile function to the reduced blood flow, the recovery of creatine phosphate content and lactate balance back towards normal, whereas ATP content remains reduced at a constant level. We examined the hypothesis that, despite the absence of ATP recovery, the short-term hibernating myocardium regains an energetic balance., Methods: An enzymatic method was modified for the measurement of inorganic phosphate (Pi) in transmural myocardial drill biopsies (about 5 mg). In 12 anaesthetized swine, moderate ischemia was induced by reduction of coronary inflow into the cannulated left anterior descending coronary artery to decrease regional myocardial function (sonomicrometry) by 50%., Results: The development of short-term hibernation was verified by the recovery of creatine phosphate content, the persistence of inotropic reserve in response to dobutamine and the absence of necrosis (triphenyl tetrazolium chloride). At 5-min ischemia, Pi was increased from 3.6 +/- 0.3 (SD) to 8.1 +/- 1.1 mumol/gwet wt (p < 0.05). The free energy of ATP hydrolysis (delta GATP) was decreased from -57.8 +/- 0.8 to -52.2 +/- 1.4 kJ/mol (p < 0.05). The relationships between function and Pi (r = -0.81) and delta GATP (r = -0.83), respectively, during control and at 5-min ischemia became invalid at 90-min ischemia, as myocardial blood flow and function remained reduced at a constant level, but Pi decreased back to 4.9 +/- 0.9 mumol/g (p < 0.05 vs. control and 5-min ischemia), and delta GATP fully recovered back to -57.2 +/- 1.3 kJ/mol (p < 0.05 vs. 5-min ischemia)., Conclusions: In short-term hibernating myocardium, myocardial inorganic phosphate content recovers partially and the free energy change of ATP hydrolysis returns to control values. Contractile function remains reduced by mechanisms other than an energetic deficit.

Background: Myocardial hibernation and stunning are characterised by a reversible contractile dysfunction during and after ischaemia, respectively. Calcium homeostasis might be disturbed in hibernation and stunning due to altered expression of cardiac proteins involved in calcium handling., Methods: In enflurane-anaesthetised swine the coronary blood flow through the left anterior descending coronary artery was decreased to reduce regional contractile function (microsonometry) by approximately 50%. In transmural biopsies obtained during ischaemia and reperfusion creatine phosphate as well as the expression of sarcoplasmic reticulum calcium ATPase (SERCA), phospholamban (PLB), calsequestrin (CSQ), and troponin inhibitor (TnI) were determined., Results: During ischaemia creatine phosphate, after an initial reduction, recovered back to control values, and necrosis was absent (hibernation). After 90 min of ischaemia the myocardium was reperfused for 120 min but regional contractile function continued to be depressed (stunning). PLB, SERCA, CSQ, and TnI proteins were unchanged during ischaemia as well as reperfusion. Likewise, levels of PLB and SERCA mRNAs were unchanged., Conclusion: It is concluded that other mechanisms than altered expression of these regulating proteins underlie the contractile dysfunction observed during acute ischaemia, short-term hibernation and stunning.

Objective: In isolated cardiac preparations of non-failing hearts from different species, including man, there is a positive force-frequency relation which is reversed into a negative relation in preparation from failing hearts. Whether or not such relations between ventricular function and heart rate hold true in the in situ heart is not clear at present. Mechanical restitution and postextrasystolic potentiation might serve as alternative measures of excitation-contraction coupling., Methods: Eleven dogs were instrumented with a left ventricular micromanometer, ultrasonic crystals for the measurement of regional wall thickness, two hydraulic occluders around the descending aorta and the inferior caval vein, and left atrial and ventricular pacing leads with a subcutaneous pacemaker. Left ventricular dP/dtmax, as an isovolumic phase index, and systolic wall thickening, as an ejection phase index, were plotted versus heart rate, and heart rate was increased by left atrial pacing from rest to 200 min-1 in increments of 25 min-1. In a subset of dogs, left ventricular filling was controlled and the frequency range expanded by the bradycardic agent UL-FS 49. Measurements were performed in the presence and absence of autonomic blockade (hexamethonium, atropine). Mechanical restitution and postextrasystolic potentiation were determined as normalized dP/dtmax and systolic wall thickening, respectively, of the extra- and postextrasystolic beat versus defined variations of the extrasystolic time interval (250-550 ms). Following control studies, heart failure was induced by rapid left ventricular pacing at 250 min-1 for 20 days +/- 6 (SD) and measurements repeated. Isolated left ventricular trabeculae from non-failing and failing hearts were studied during stimulation at 0.2-4 Hz., Results: Only with filling control and in the absence of autonomic blockade, was there a slightly positive relation between dP/dtmax and heart rate in the control state. Otherwise, the relation of dP/dtmax to heart rate was flat both in the control state and in heart failure. The relation between systolic wall thickening and heart rate in the control state was negative, unless filling was controlled, and it was flat in heart failure. In contrast, the time constants of mechanical restitution and postextrasystolic potentiation were increased significantly with heart failure from 91 +/- 25 (SD) to 164 +/- 13 ms and from 107 +/- 18 to 156 +/- 4 ms, respectively, for dP/dtmax and from 76 +/- 22 to 162 +/- 10 ms and from 101 +/- 17 to 160 +/- 17 ms, respectively, for systolic wall thickening. These time constants were, however, insensitive to UL-FS 49 and autonomic blockade. There was a negative force-frequency relation in left ventricular trabeculae from non-failing hearts at higher calcium concentrations, where it was flat in trabeculae from failing hearts., Conclusion: Time constants of mechanical restitution and postextrasystolic potentiation are more sensitive than the steady state relation of ventricular function and heart rate to characterize the impairment of excitation-contraction coupling in heart failure.

Preconditioning the myocardium using short episodes of sublethal ischaemia will delay the onset of necrosis during a subsequent lethal ischaemic insult. This powerful protective adaptation of the myocyte has also been observed in other cell types. The potential for clinical application to benefit patients with a variety of pathological conditions has led to an expansion in our knowledge concerning the pathophysiology of ischemia-reperfusion injury and the regulatory mechanisms underlying cellular metabolism. We feel it is timely to assess the current position in this field and provide a critical appraisal to facilitate future research.

Objective: The calcium antagonist nisoldipine has recently been reported to induce rather than to attenuate ischaemia in some patients with stable angina. The aim of the study was to investigate the mechanisms underlying this proischaemic effect., Methods: In 20 anaesthetised dogs systemic haemodynamic variables, regional myocardial blood flow (coloured microspheres), and systolic wall thickening (sonomicrometry) were measured during control conditions and following severe stenosis on the left circumflex coronary artery, before and after intravenous administration of equihypotensive doses of either nisoldipine (group I, n = 10) or dipyridamole (group II, n = 10). Finally, measurements were performed while the drug induced decreases in mean aortic pressure--18 (SD 6) mmHg in group I and 14(6) mm Hg in group II--were reversed by inflation of an intra-aortic balloon., Results: The stenosis decreased posterior wall thickening to 50% of control, and posterior subendocardial blood flow from 1.48(0.27) to 0.61(0.19) ml.min-1.g-1 in group I and from 1.49(0.23) to 0.62(0.18) ml.min-1.g-1 in group II. Subendocardial blood flow was further decreased after administration of either nisoldipine [0.37(0.20) ml.min-1.g-1, p < 0.05 v stenosis] or dipyridamole [0.22(0.11) ml.min-1.g-1, p < 0.05 v stenosis]. Regional myocardial blood flow in the anterior region was increased. The drug induced reduction of subendocardial blood flow decreased posterior wall thickening further from 9.3(2.1) to 6.2(3.9)% (p < 0.05 v stenosis, group I) and from 9.1(1.7) to 4.3(2.4)% (p < 0.05 v stenosis, group II). When the drug induced decrease in aortic pressure was reversed, subendocardial blood flow again increased in group I [0.63(0.19) ml.min-1.g-1, p < 0.05 v stenosis and nisoldipine] whereas in group II it remained decreased [0.40(0.29) ml.min-1.g-1, NS v stenosis and dipyridamole]. There was restoration of posterior wall thickening in group I [10.4(3.8)%, p < 0.05 v stenosis and nisoldipine], but not in group II [5.2(3.5)%, NS v stenosis and dipyridamole]., Conclusions: Nisoldipine and dipyridamole decrease subendocardial blood flow and contractile function distal to a severe stenosis when aortic pressure is decreased. No aggravation of ischaemia by nisoldipine is seen when hypotension is prevented. In contrast, dipyridamole in the absence of hypotension still induces a redistribution of flow at the expense of the ischaemic region.

Objective: Short term hibernating myocardium is characterised by a decrease in contractile function in proportion to the reduced blood flow, the recovery of creatine phosphate despite ongoing ischaemia, a recruitable inotropic reserve, and the absence of necrosis. During acute myocardial ischaemia systolic wall thickening decreases and post-ejection wall thickening develops. The extent of post-ejection thickening during severe ischaemia correlates with the recovery of contractile function during reperfusion. Whether the extent of post-ejection wall thickening can also distinguish short term hibernating myocardium from more severely ischaemic, infarcting myocardium and thus predict the amount of viable tissue was tested in 13 anaesthetised pigs., Methods: The left anterior descending coronary artery (LAD) was cannulated and perfused at constant flow. After control measurements of regional myocardial blood flow (with radiolabelled microspheres) and wall thickening (sonomicrometry), coronary inflow was reduced to produce a reduction in regional contractile function by 60-100%. After 85 minutes of ischaemia, dobutamine was infused into the LAD for five minutes to determine the extent of inotropic reserve. Transmural biopsies were taken to measure regional myocardial creatine phosphate content and infarct size was determined after two hours of reperfusion by staining with triphenyl tetrazolium chloride., Results: The extent of post-ejection wall thickening after 85-90 minutes of ischaemia correlated with the myocardial creatine phosphate content (r = 0.812, n = 11, p < 0.01) and the extent of the dobutamine recruitable inotropic reserve (r = 0.783, n = 7, p < 0.05). A negative correlation existed between the extent of post-ejection wall thickening and % infarct size (r = -0.699, n = 10, p < 0.05 for the transmural piece of tissue containing the ultrasonic crystals; r = -0.743, n = 10, p < 0.05 for the area of the left ventricle at risk). Finally, post-ejection wall thickening after 85-90 minutes of ischaemia correlated with the recovery of contractile function at 30 minutes reperfusion (r = 0.657, n = 10, p < 0.05)., Conclusion: The extent of post-ejection wall thickening may indicate the amount of viable tissue after 85-90 minutes of low flow ischaemia. The greater the post-ejection wall thickening, the more myocardium is successfully hibernating.