Cardiovascular Research - current issue

Aims and Scope

2009-06-25

Contents Page

2009-06-25

Editorial Board

2009-06-25

SPOTLIGHT ISSUE ON Cardiac Protection

2009-06-25

Announcement Spotlight Issue on Mechanisms of Vascular Inflammation

2009-06-25

Cardiac protection takes off

Piper, H. M., Garcia-Dorado, D. — 2009-06-25

Lethal reperfusion injury in acute myocardial infarction: facts and unresolved issues

Garcia-Dorado, D., Ruiz-Meana, M., Piper, H. M. — 2009-06-25

Orphan targets for reperfusion injury

Inserte, J., Barrabes, J. A., Hernando, V., Garcia-Dorado, D. — 2009-06-25

Cardiomyocyte death secondary to transient ischaemia occurs mainly during the first minutes of reperfusion in the form of contraction band necrosis. Research on the mechanisms leading to sarcolemmal rupture and necrosis during initial reperfusion identified several promising pharmacological targets directed either to correct the alterations in Ca²⁺ handling occurring during this period (Na⁺/H⁺-exchanger, reverse mode of Na⁺/Ca²⁺-exchanger, sarcoplasmic reticulum) or to interfere with its consequences [hypercontracture, calpain activation, and mitochondrial permeability transition pore (mPTP) opening]. However, despite the fact that pharmacological tools against some of these targets have consistently demonstrated that it is possible to reduce infarct size in experimental studies by interventions applied at the time of reperfusion, the translation of these approaches to clinical practice has failed due in part to the lack of drugs able to be tested in humans. Recently, the benefits of both post-conditioning and inhibition of mPTP have been supported by proof-of-concept trials demonstrating the clinical applicability of strategies aimed at preventing lethal reperfusion injury. These promising results should stimulate efforts to develop drugs testable in humans against known, unexploited targets involved in reperfusion injury and to identify and validate additional ones.

Cardioprotective growth factors

Hausenloy, D. J., Yellon, D. M. — 2009-06-25

Many of the originally identified cardiovascular ‘growth factors’ have been demonstrated to exert a diverse variety of actions within the cardiovascular system, the majority of which are unrelated to their initially proposed mechanism of action. Interestingly, several of these growth factors have been demonstrated to protect the cardiomyocyte from the detrimental effects of acute ischaemia–reperfusion injury, through the activation of a variety of cell-surface receptors and the subsequent recruitment of a number of intracellular signal transduction pathways, which include components of the reperfusion injury salvage kinase pathway. This article will review several of these cardioprotective growth factors with respect to their ability to confer direct myocardial protection, focusing on the underlying signalling pathways involved and their potential for clinical application.

Myocardial protection by nitrite

Calvert, J. W., Lefer, D. J. — 2009-06-25

Nitrite has long been considered to be an inert oxidative metabolite of nitric oxide (NO). Recent work, however, has demonstrated that nitrite represents an important tissue storage form of NO that can be reduced to NO during ischaemic or hypoxic events. This exciting series of discoveries has created an entirely new field of research that involves the investigation of the molecular, biochemical, and physiological activities of nitrite under a variety of physiological and pathophysiological states. This has also led to a re-evaluation of the role that nitrite plays in health and disease. As a result there has been an interest in the use of nitrite as a therapeutic strategy for the treatment of acute myocardial infarction. Nitrite therapy has now been studied in several animal models and has proven to be an effective means to reduce myocardial ischaemia–reperfusion injury. This review article will provide a brief summary of the key findings that have led to the re-evaluation of nitrite and highlight the evidence supporting the cardioprotective actions of nitrite and also highlight the potential clinical application of nitrite therapy to cardiovascular diseases.

Phosphodiesterase 5 inhibitors: are they cardioprotective?

Reffelmann, T., Kloner, R. A. — 2009-06-25

A growing body of animal studies provides evidence for potential cardioprotective effects of inhibitors of the enzyme phosphodiesterase isoform 5. Infarct size reduction by administration of phosphodiesterase 5 inhibitors was described in various experimental models of ischaemia and reperfusion. Furthermore, potential beneficial effects were demonstrated in experimental models of congestive heart failure and left ventricular hypertrophy. Some of the observed effects resemble the basic mechanisms of ischaemic pre-conditioning, mimicking both acute and delayed effects. Other effects may be due to action on systemic and cardiac haemodynamics. Mechanisms and signalling pathways, characterized in some of the experimental models, appear to be complex: for instance, the rate of cyclic guanosine monophosphate (cGMP) synthesis and the functional compartmentalization of intracellular cGMP metabolism as well as interaction with ß-adrenergic and nitric oxide signalling may influence effects in different experimental settings. In this review, we discuss mechanisms, signalling pathways, and experimental limitations and touch on considerations for translation into potentially useful applications in the clinical arena.

Regulation and pharmacology of the mitochondrial permeability transition pore

Zorov, D. B., Juhaszova, M., Yaniv, Y., Nuss, H. B., Wang, S., Sollott, S. J. — 2009-06-25

The ‘mitochondrial permeability transition', characterized by a sudden induced change of the inner mitochondrial membrane permeability for water as well as for small substances (≤1.5 kDa), has been known for three decades. Research interest in the entity responsible for this phenomenon, the ‘mitochondrial permeability transition pore’ (mPTP), has dramatically increased after demonstration that it plays a key role in the life and death decision in cells. Therefore, a better understanding of this phenomenon and its regulation by environmental stresses, kinase signalling, and pharmacological intervention is vital. The characterization of the molecular identity of the mPTP will allow identification of possible pharmacological targets and assist in drug design for its precise regulation. However, despite extensive research efforts, at this point the pore-forming core component(s) of the mPTP remain unidentified. Pivotal new genetic evidence has shown that components once believed to be core elements of the mPTP (namely mitochondrial adenine nucleotide translocator and cyclophilin D) are instead only mPTP regulators (or in the case of voltage-dependent anion channels, probably entirely dispensable). This review provides an update on the current state of knowledge regarding the regulation of the mPTP.

Inhibition of mitochondrial permeability transition pore opening: translation to patients

Gomez, L., Li, B., Mewton, N., Sanchez, I., Piot, C., Elbaz, M., Ovize, M. — 2009-06-25

A large body of experimental evidence indicates that during an acute myocardial infarction (AMI), tissue injury occurring after reperfusion represents a significant amount of the whole, irreversible damage. It is now recognized that mitochondrial permeability transition pore opening plays a crucial role in this specific component of myocardial infarction. Ischaemic postconditioning and cyclosporine A (CsA) have been shown to dramatically reduce infarct size in many animal species. Recent proof-of-concept clinical trials support the idea that lethal myocardial reperfusion injury is also of significant importance in patients with ongoing AMI, and that targeting mitochondrial permeability transition by either percutaneous coronary intervention postconditioning or CsA can reduce infarct size and improve the recovery of contractile function after reperfusion. Large-scale trials are ongoing to address whether these new treatments may improve clinical outcome in reperfused AMI patients.

Protective ischaemia in patients: preconditioning and postconditioning

Granfeldt, A., Lefer, D. J., Vinten-Johansen, J. — 2009-06-25

Infarct size can be limited by reducing the determinants of infarct size or increasing collateral blood flow by treatment initiated before the ischaemic event. Reperfusion is the definitive treatment for permanently reducing infarct size and restoring some degree of contractile function to the affected myocardium. Innate survival mechanisms in the heart can be stimulated by short, non-lethal periods of ischaemia and reperfusion, applied either before or after the ischaemic event. Preconditioning, a series of transient intervals of ischaemia and reperfusion applied before the lethal ‘index’ ischaemic event, sets in motion molecular and cellular mechanisms that increase cardiomyocyte survival to a degree that had not hitherto been seen before. The cardioprotective ischaemic-reperfusion protocol applied at onset of reperfusion, termed ‘postconditioning’ (Postcon), is also associated with significant cardioprotection that can be applied at the point of reperfusion treatment in the catheterization laboratory or operating room. Both preconditioning and Postcon have been successfully applied to the clinical setting and have been found to reduce infarct size and other attributes of post-ischaemic injury. This review will summarize the physiological preclinical data on preconditioning and Postcon that are relevant to their translation to clinical therapeutics and treatment.

Loss of cardioprotection with ageing

Boengler, K., Schulz, R., Heusch, G. — 2009-06-25

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.

The use of controlled reperfusion strategies in cardiac surgery to minimize ischaemia/reperfusion damage

Beyersdorf, F. — 2009-06-25

Ischaemia and reperfusion occur during almost every cardiac operation, and one of the key elements to achieve a successful operation is to counteract the detrimental effects of induced ischaemia and reperfusion during the operation. The cardiac surgeon is in a unique position to protect the heart before ischaemia is induced and to avoid further damage during the reperfusion period. The surgeon can alter the composition of the reperfusate and the conditions of reperfusion so that the ischaemia/reperfusion injury is minimal, even after very complex procedures that require long aortic cross-clamp periods. This in turn allows him to perform a near-perfect surgical repair of the underlying disease without the pressure of time. The vast knowledge gained in this field over the years has led to application in other organs, such as the limbs (acute limb ischaemia), lungs (lung transplantation), kidney and liver (kidney and liver transplantation), and more recently even for the brain [acute cerebral artery occlusion (stroke)] and the whole body (cardiopulmonary resuscitation). Further improvements in reperfusion strategies will allow salvage of tissue and even whole body after ischaemic periods thought previously to be irreversibly damaged.

Benefits of reperfusion beyond infarct size limitation

Takemura, G., Nakagawa, M., Kanamori, H., Minatoguchi, S., Fujiwara, H. — 2009-06-25

The most critical determinant of prognosis in patients with acute myocardial infarction (MI) is infarct magnitude, which can be established within several hours of an attack. The importance of the subsequent healing process is not negligible, however. In fact, much experimental and clinical evidence suggests that late reperfusion of the infarct-related coronary artery—i.e. at times too late to salvage the myocardium within the area at risk—is beneficial for reducing left ventricular remodelling and decreasing mortality (‘open artery hypothesis’). For instance, one recent study highlighted the beneficial effects of late reperfusion therapy on the infarct tissue cell dynamics following acute MI. Nonetheless, several recent large, randomized clinical trials have failed to provide evidence of such benefits, refuting the clinical efficacy of late reperfusion. In addition, they also underscore the need for revised clinical studies in which there is less heterogeneity in the timing of reperfusion and in the initial infarct size, as well as the need for sustained patency of the recanalized artery. This review focuses on the effects of late reperfusion on the pathophysiology of MI in the context of the infarct tissue dynamics and clinical outcomes. We also discuss the issues that need to be resolved to improve clinical application.

Pharmacological postconditioning effect of muramyl dipeptide is mediated through RIP2 and TAK1

Sicard, P., Jacquet, S., Kobayashi, K. S., Flavell, R. A., Marber, M. S. — 2009-06-25

Aims

Despite their ability to cause septic shock and myocardial dysfunction, components of Gram-negative bacterial cell walls, like lipopolysaccharide, have been shown in numerous studies to induce myocardial protection during ischaemia–reperfusion injury. Muramyl dipeptide (MDP) is another such component recognized by an intracellular receptor, nucleotide-binding oligomerization domain 2. Receptor activation leads to intracellular signals through receptor interacting protein-2 (RIP2) and tumour growth factor-β-activated kinase-1 (TAK1). However, little is known about the RIP2/TAK1 pathway in the heart. The aim of this study was to determine whether the RIP2/TAK1 pathway has a cardioprotective role in a mouse model of myocardial infarction.

Methods and results

We isolated and subjected wild-type (WT) and RIP2^–/– mouse hearts to 30 min of global ischaemia and 120 min of reperfusion with or without perfusion of MDP (10 µg/mL) before or after the ischaemic period and determined the infarct size. We examined activation of the TAK1/nuclear factor B (NFB) signalling pathway. The effect of TAK1 inhibition on MDP-induced cardioprotection was also evaluated. Exposure to MDP during reperfusion significantly reduced infarct size in WT hearts (from 51.7 ± 5.6% in control to 38.1 ± 6.7%, P < 0.05), but not in RIP2^–/– hearts or in WT hearts with coincident pharmacological inhibition of TAK1. MDP treatment significantly increased the levels of p-TAK1 and p-JNK (Jun N-terminal kinase) and led to NFB activation via phosphorylation and degradation of IkappaB in the WT, but not in the RIP2^–/–, myocardium.

Conclusion

These results indicate that MDP at reperfusion induced cardioprotection through an RIP2/TAK1-dependent mechanism.

Protective effects of sphingosine-1-phosphate receptor agonist treatment after myocardial ischaemia-reperfusion

2009-06-25

Aims

Several experimental studies have demonstrated protection against cardiac ischaemia–reperfusion injury achieved by pre-treatment with exogenous sphingosine-1-phosphate (S1P). We tested the hypothesis that pharmacological S1P receptor agonists improve recovery of function when applied with reperfusion.

Methods and results

Isolated rat cardiomyocytes were stimulated with exogenous S1P, the selective S1P1 receptor agonist SEW2871, or the S1P1/3 receptor agonist FTY720. Western blot analysis was performed to analyse downstream signalling pathways. Ischaemia–reperfusion studies were conducted in rat cardiomyocytes, isolated Langendorff-perfused rat hearts, and in human myocardial muscle strip preparations to evaluate the effect of S1P receptor agonists on cell death and recovery of mechanical function. All S1P receptor agonists were able to activate Akt. This was associated with transactivation of the epidermal growth factor receptor. In isolated cardiomyocytes, selective stimulation of the S1P1 receptor by SEW2871 induced protection against cell death when administered either before or after ischaemia–reperfusion. In isolated rat hearts, treatment with FTY720 during reperfusion attenuated the rise in left ventricular end-diastolic pressure (LVEDP) and improved the recovery of left ventricular developed pressure without limiting infarct size. However, selective S1P1 receptor stimulation did not improve functional recovery but rather increased LVEDP. Additional experiments employing a human myocardial ischaemia–reperfusion model also demonstrated improved functional recovery induced by FTY720 treatment during reperfusion.

Conclusion

Pharmacological S1P receptor agonists have distinct effects on ischaemia–reperfusion injury. Their efficacy when applied during reperfusion makes them potential candidates for pharmaceutical postconditioning therapy after cardiac ischaemia.

High glucose sensitizes adult cardiomyocytes to ischaemia/reperfusion injury through nitrative thioredoxin inactivation

Luan, R., Liu, S., Yin, T., Lau, W. B., Wang, Q., Guo, W., Wang, H., Tao, L. — 2009-06-25

Aims

Ischaemic cardiac injury is significantly increased in diabetic patients, but its underlying mechanisms remain incompletely understood. The current study attempted to identify new molecular mechanisms potentially contributive to hyperglycaemic-exaggeration of myocardial ischaemic injury.

Methods and results

Adult mouse cardiomyocytes were cultured in normal-glucose (NG, 5.5 mM) or high-glucose (HG, 25 mM) medium. Twelve hours after NG or HG pre-culture, cardiomyocytes were subjected to 3 h of simulated ischaemia (SI), followed by 3 h of reperfusion (R) in NG medium. Prior to and after SI/R, the following were determined: cardiomyocyte death and apoptosis, sustained oxidative/nitrative stress and thioredoxin (Trx) activity, expression, and nitration. Compared with NG-cultured cardiomyocytes, 12 h HG culture significantly increased superoxide and peroxynitrite production, increased Trx-1 nitration, and reduced Trx activity (P < 0.01). Despite being subject to identical SI/R procedures and conditions, cells pre-cultured in HG sustained greater injury, evidenced by elevated lactate dehydrogenase release and caspase-3 activation (P < 0.01). Moreover, SI/R induced greater superoxide/peroxynitrite overproduction and greater Trx-1 nitration and inactivation in HG pre-cultured cardiomyocytes than in NG pre-cultured cardiomyocytes. Finally, the supplementation of human Trx-1, superoxide scavenger, or peroxynitrite decomposition catalyst in HG pre-cultured cells reduced Trx-1 nitration, preserved Trx-1 activity, and normalized SI/R injury to levels observed in NG pre-cultured cardiomyocytes.

Conclusion

High glucose sensitized cardiomyocytes to ischaemia/reperfusion injury through nitrative Trx-1 inactivation. Interventions restoring Trx-1 activity in the diabetic heart may represent novel therapies attenuating cardiac injury in diabetic patients.

Growth hormone-releasing hormone promotes survival of cardiac myocytes in vitro and protects against ischaemia-reperfusion injury in rat heart

2009-06-25

Aims

The hypothalamic neuropeptide growth hormone-releasing hormone (GHRH) stimulates GH synthesis and release in the pituitary. GHRH also exerts proliferative effects in extrapituitary cells, whereas GHRH antagonists have been shown to suppress cancer cell proliferation. We investigated GHRH effects on cardiac myocyte cell survival and the underlying signalling mechanisms.

Methods and results

Reverse transcriptase–polymerase chain reaction analysis showed GHRH receptor (GHRH-R) mRNA in adult rat ventricular myocytes (ARVMs) and in rat heart H9c2 cells. In ARVMs, GHRH prevented cell death and caspase-3 activation induced by serum starvation and by the β-adrenergic receptor agonist isoproterenol. The GHRH-R antagonist JV-1-36 abolished GHRH survival action under both experimental conditions. GHRH-induced cardiac cell protection required extracellular signal-regulated kinase (ERK)1/2 and phosphoinositide-3 kinase (PI3K)/Akt activation and adenylyl cyclase/cAMP/protein kinase A signalling. Isoproterenol strongly upregulated the mRNA and protein of the pro-apoptotic inducible cAMP early repressor, whereas GHRH completely blocked this effect. Similar to ARVMs, in H9c2 cardiac cells, GHRH inhibited serum starvation- and isoproterenol-induced cell death and apoptosis through the same signalling pathways. Finally, GHRH improved left ventricular recovery during reperfusion and reduced infarct size in Langendorff-perfused rat hearts, subjected to ischaemia–reperfusion (I/R) injury. These effects involved PI3K/Akt signalling and were inhibited by JV-1-36.

Conclusion

Our findings suggest that GHRH promotes cardiac myocyte survival through multiple signalling mechanisms and protects against I/R injury in isolated rat heart, indicating a novel cardioprotective role of this hormone.

Leptin attenuates cardiac apoptosis after chronic ischaemic injury

McGaffin, K. R., Zou, B., McTiernan, C. F., O'Donnell, C. P. — 2009-06-25

Aims

We have previously shown that activation of leptin signalling in the heart reduces cardiac morbidity and mortality after myocardial infarction (MI). In the present study, we tested the hypothesis that leptin signalling limits cardiac apoptosis after MI through activation of signal transducer and activator of transcription (STAT)-3 responsive anti-apoptotic genes, including B-cell lymphoma (bcl)-2 and survivin, that serve to downregulate the activity of caspase-3.

Methods and results

Hearts from C57BL/6J and three groups of leptin-deficient Ob/Ob mice (food-restricted, ad libitum, and leptin-repleted) were examined 4 weeks after permanent left coronary artery ligation or sham operation. Inflammatory and apoptotic cell number was determined in cardiac sections by immunostaining. Expression of cardiac bcl-2, survivin, and pro and active caspase-3 was determined and correlated with in vitro caspase-3 activity. In the absence of MI, both lean and obese leptin-deficient mice exhibited increased cardiac apoptosis compared with wild-type mice. After MI, the highest rates of apoptosis were seen in the infarcted tissue of lean and obese Ob/Ob mice. Further, leptin-deficient hearts, as well as hearts from wild-type mice treated with the STAT-3 inhibitor WP1066, exhibited blunted anti-apoptotic bcl-2 and survivin gene expression, and increased caspase-3 protein expression and activity. The increased caspase-3 activity and apoptosis in hearts of leptin-deficient mice after MI was significantly attenuated in Ob/Ob mice replete with leptin, reducing apoptosis to levels comparable to that observed in wild-type mice after MI.

Conclusion

These results demonstrate that intact leptin signalling post-MI acts through STAT-3 to increase anti-apoptotic bcl-2 and survivin gene expression and reduces caspase-3 activity, consistent with a cardioprotective role of leptin in the setting of chronic ischaemic injury.

Parstatin: a cryptic peptide involved in cardioprotection after ischaemia and reperfusion injury

2009-06-25

Aims

Thrombin activates protease-activated receptor 1 by proteolytic cleavage of the N-terminus. Although much research has focused on the activated receptor, little is known about the 41-amino acid N-terminal peptide (parstatin). We hypothesized that parstatin would protect the heart against ischaemia–reperfusion injury.

Methods and results

We assessed the protective role of parstatin in an in vivo and in vitro rat model of myocardial ischaemia–reperfusion injury. Parstatin treatment before, during, and after ischaemia decreased infarct size by 26%, 23%, and 18%, respectively, in an in vivo model of ischaemia–reperfusion injury. Parstatin treatment immediately before ischaemia decreased infarct size by 65% and increased recovery in ventricular function by 23% in an in vitro model. We then assessed whether parstatin induced cardioprotection by activation of a Gi-protein-dependent pathway. Gi-protein inactivation by pertussis toxin completely abolished the cardioprotective effects. The cardioprotective effects were also abolished by inhibition of nitric oxide synthase (NOS), extracellular signal-regulated kinases 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK), and K_ATP channels in vitro. Furthermore, parstatin increased coronary flow and decreased perfusion pressure in the isolated heart. The vasodilatory properties of parstatin were confirmed in rat coronary arterioles.

Conclusion

A single treatment of parstatin administered prior to ischaemia confers immediate cardioprotection by recruiting the Gi-protein activation pathway including p38 MAPK, ERK1/2, NOS, and K_ATP channels. Parstatin exerts effects on both the cardiomyocytes and the coronary circulation to induce cardioprotection. This suggests a potential therapeutic role of parstatin in the treatment of cardiac injury resulting from ischaemia and reperfusion.

The targeting of cyclophilin D by RNAi as a novel cardioprotective therapy: evidence from two-photon imaging

2009-06-25

Aims

An opening of the mitochondrial permeability transition pore (MPTP), which leads to the loss of mitochondrial membrane potential (_m), is the earliest event that commits cells to death, and this process is potentially a prime target for therapeutic intervention against myocardial ischaemia/reperfusion. We aimed to investigate the protective effects of RNA interference (RNAi)-mediated gene silencing of cyclophilin D (CypD), one of the putative components of the MPTP, against myocardial ischaemia/reperfusion using two-photon laser scanning microscopy.

Methods and results

We created an adenovirus carrying short-interfering RNA (siRNA) that inactivates CypD. Transduction of CypD-siRNA in rat cardiomyocytes achieved a 61% reduction in CypD mRNA and a 63% reduction in protein levels as well as protection against oxidant-induced _m loss and cytotoxicity. To further investigate the effects in vivo, we monitored the spatio-temporal changes of _m in perfused rat hearts subjected to ischaemia/reperfusion using two-photon imaging. Adult rats received direct intramyocardial injections of the adenovirus. Two to three days after injection, rat hearts were perfused by the Langendorff method and _m levels of individual cells were monitored. The progressive loss of _m during ischaemia/reperfusion was significantly suppressed in CypD-siRNA-transduced cells compared with non-transduced cells. Furthermore, the protective effect of CypD-siRNA was dose-dependent.

Conclusion

Therapeutic interventions designed to inactivate CypD may be a promising strategy for reducing cardiac injury against myocardial ischaemia/reperfusion. The two-photon imaging technique provides deeper insight into cardioprotective therapy that targets mitochondria.

Rapid cooling preserves the ischaemic myocardium against mitochondrial damage and left ventricular dysfunction

2009-06-25

Aims

We investigated whether rapid cooling instituted by total liquid ventilation (TLV) improves cardiac and mitochondrial function in rabbits submitted to ischaemia-reperfusion.

Methods and results

Rabbits were chronically instrumented with a coronary artery occluder and myocardial ultrasonic crystals for assessment of segment length-shortening. Two weeks later they were re-anaesthetized and underwent either a normothermic 30-min coronary artery occlusion (CAO) (Control group, n = 7) or a comparable CAO with cooling initiated by a 10-min hypothermic TLV and maintained by a cold blanket placed on the skin. Cooling was initiated after 5 or 15 min of CAO (Hypo-TLV and Hypo-TLV_15' groups, n = 6 and 5, respectively). A last group underwent normothermic TLV during CAO (Normo-TLV group, n = 6). Wall motion was measured in the conscious state over three days of reperfusion before infarct size evaluation and histology. Additional experiments were done for myocardial sampling in anaesthetized rabbits for mitochondrial studies. The Hypo-TLV procedure induced a rapid decrease in myocardial temperature to 32–34°C. Throughout reperfusion, segment length-shortening was significantly increased in Hypo-TLV and Hypo-TLV_15' vs. Control and Normo-TLV (15.1 ± 3.3%, 16.4 ± 2.3%, 1.8 ± 0.6%, and 1.1 ± 0.8% at 72 h, respectively). Infarct sizes were also considerably attenuated in Hypo-TLV and Hypo-TLV_15' vs. Control and Normo-TLV (4 ± 1%, 11 ± 5%, 39 ± 2%, and 42 ± 5% infarction of risk zones, respectively). Mitochondrial function in myocardial samples obtained at the end of ischaemia or after 10 min of reperfusion was improved by Hypo-TLV with respect to ADP-stimulated respiration and calcium-induced opening of mitochondrial permeability transition pores (mPTP). Calcium concentration opening mPTP was, e.g., increased at the end of ischaemia in the risk zone in Hypo-TLV vs. Control (157 ± 12 vs. 86 ± 12 µM). Histology and electron microscopy also revealed better preservation of lungs and of cardiomyocyte ultrastructure in Hypo-TLV when compared with Control.

Conclusion

Institution of rapid cooling by TLV during ischaemia reduces infarct size as well as other sequelae of ischaemia, such as post-ischaemic contractile and mitochondrial dysfunction.

Nrf2-dependent upregulation of antioxidative enzymes: a novel pathway for proteasome inhibitor-mediated cardioprotection

2009-06-25

Aims

We have shown previously that non-toxic inhibition of the ubiquitin–proteasome system upregulates antioxidative defence mechanisms and protects endothelial cells from oxidative stress. Here, we have addressed the question whether the induction of antioxidative enzymes contributes to cardioprotection by non-toxic proteasome inhibition.

Methods and results

Treatment with 0.5 µmol/L MG132 for 48 h proved to be non-toxic and protected neonatal rat cardiac myocytes against H₂O₂-mediated oxidative stress in lactate dehydrogenase assays. This correlated with reduced levels of intracellular reactive oxygen species as determined by loading myocytes with dichlorofluorescein. Immunoblots showed significant upregulation of superoxide dismutase 1 (SOD1), haem oxygenase 1, and catalase upon proteasome inhibition. Luciferase assays using a reporter driven by the SOD1 promoter revealed proteasome inhibitor-mediated induction of luciferase activity. Deletion and mutation analyses identified an antioxidant response element (ARE) in the SOD1 promoter to be not only essential but also sufficient for transcriptional upregulation by proteasome inhibition. An essential role for the antioxidative transcription factor NF-E2-related factor 2 (Nrf2)—which was stabilized by proteasome inhibition—in ARE-mediated transcriptional activation was revealed in cardiac myocytes from Nrf2 wild-type and knockout mice: proteasome inhibition upregulated antioxidative enzymes and conferred protection against H₂O₂-mediated oxidative stress in Nrf2 wild-type cells. In contrast, the induction of antioxidative enzymes and cytoprotection were completely abolished in cardiac myocytes from Nrf2 knockout mice.

Conclusion

Non-toxic proteasome inhibition upregulates antioxidative enzymes via an Nrf2-dependent transcriptional activation of AREs and confers cardioprotection.

Role of B-type natriuretic peptide in epoxyeicosatrienoic acid-mediated improved post-ischaemic recovery of heart contractile function

2009-06-25

Aims

This study examined the functional role of B-type natriuretic peptide (BNP) in epoxyeicosatrienoic acid (EET)-mediated cardioprotection in mice with targeted disruption of the sEH or Ephx2 gene (sEH null).

Methods and results

Isolated mouse hearts were perfused in the Langendorff mode and subjected to global no-flow ischaemia followed by reperfusion. Hearts were analysed for recovery of left ventricular developed pressure (LVDP), mRNA levels, and protein expression. Naïve hearts from sEH null mice had similar expression of preproBNP (Nppb) mRNA compared with wild-type (WT) hearts. However, significant increases in Nppb mRNA and BNP protein expression occurred during post-ischaemic reperfusion and correlated with improved post-ischaemic recovery of LVDP. Perfusion with the putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid prior to ischaemia reduced the preproBNP mRNA in sEH null hearts. Inhibitor studies demonstrated that perfusion with the natriuretic peptide receptor type-A (NPR-A) antagonist, A71915, limited the improved recovery in recombinant full-length mouse BNP (rBNP)- and 11,12-EET-perfused hearts as well as in sEH null mice. Increased expression of phosphorylated protein kinase C and Akt were found in WT hearts perfused with either 11,12-EET or rBNP, while mitochondrial glycogen synthase kinase-3β was significantly lower in the same samples. Furthermore, treatment with the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin abolished improved LVDP recovery in 11,12-EET-treated hearts but not did significantly inhibit recovery of rBNP-treated hearts.

Conclusion

Taken together, these data indicate that EET-mediated cardioprotection involves BNP and PI3K signalling events.

Myocardial microvascular function during acute coronary artery stenosis: effect of hypertension and hypercholesterolaemia

2009-06-25

Aims

Coronary collateral arteries (CCA) reduce cardiovascular events. We tested the hypothesis that new microvessels that proliferate in early atherosclerosis may be associated with myocardial protection during acute subtotal coronary artery obstruction (CAO).

Methods and results

Acute left anterior descending CAO was induced by a balloon catheter in pigs after 12 weeks of high-cholesterol (HC) diet, renovascular hypertension (HTN), or normal control. Cardiac structure, myocardial perfusion, and functional response to iv adenosine and CAO were studied in vivo using electron beam computed tomography (CT). The intra-myocardial microvessels were subsequently evaluated ex vivo using micro-CT, and myocardial expression of growth factors using immunoblotting. Basal myocardial perfusion and microvascular permeability were similar among the groups, whereas their responses to adenosine were attenuated in HC and HTN. A significant decline in myocardial perfusion in normal pigs during acute CAO was attenuated in HC and abolished in HTN. CAO also elicited an increase in normal anterior wall microvascular permeability (+202 ± 59%, P < 0.05), which was attenuated in HC and HTN (+55 ± 9 and +31 ± 8%, respectively, P < 0.05 vs. normal). Microvascular (<200 µm) spatial density was significantly elevated in HC and HTN, accompanied by increased myocardial growth factor expression.

Conclusion

This study demonstrates that early exposure to the cardiovascular risk factors HC and HTN protects the heart from decreases in myocardial perfusion during acute subtotal CAO. This protective effect is associated with and potentially mediated by pre-emptive development of intra-myocardial microvessels that might serve as recruitable CCA.

Stimulation of cGMP signalling protects coronary endothelium against reperfusion-induced intercellular gap formation

2009-06-25

Aims

Ischaemia–reperfusion provokes barrier failure of the coronary microvasculature, impeding functional recovery of the heart during reperfusion. The aim of the present study was to investigate whether the stimulation of cGMP signalling by activation of soluble guanylyl cyclase (sGC) can reduce reperfusion-induced endothelial intercellular gap formation and to determine whether this is due to an influence on endothelial cytosolic Ca²⁺ homeostasis during reperfusion.

Methods and results

Experiments were performed with cultured coronary endothelial monolayers and isolated saline-perfused rat hearts. HMR1766 (1 µmol/L) or DEAnonoate (0.5 µmol/L) were used to activate sGC. After exposure to simulated ischaemic conditions, reperfusion of endothelial cells led to a pronounced increase in cytosolic calcium levels and intercellular gaps. Stimulation of cGMP signalling during reperfusion increased Ca²⁺ sequestration in the endoplasmic reticulum (ER) and attenuated the reperfusion-induced increase in cytosolic [Ca²⁺]. Phosphorylation of phospholamban was also increased, indicating a de-inhibition of the ER Ca²⁺ pump (SERCA). Reperfusion-induced intercellular gap formation was reduced. Reduction of myosin light chain phosphorylation indicated inactivation of the endothelial contractile machinery. Effects on cytsolic Ca²⁺ and gaps were abrogated by inhibition of cGMP-dependent protein kinase (PKG) with KT5823. In reperfused hearts, stimulation of cGMP signalling led to decreased oedema development.

Conclusion

sGC/PKG activation during reperfusion reduces reperfusion-induced endothelial intercellular gap formation by attenuation of cytosolic calcium overload and reduction of contractile activation in endothelial cells. This mechanism protects the heart against reperfusion-induced oedema.

Antithrombin reduces shedding of the endothelial glycocalyx following ischaemia/reperfusion

2009-06-25

Aims

Antithrombin is an important inhibitor of the coagulation system, additionally exerting specific anti-inflammatory effects on endothelial cells. Healthy vascular endothelium is coated by the endothelial glycocalyx, diminution of which increases capillary permeability, e.g. after ischaemia. Antithrombin is known to infiltrate the glycocalyx, binding to glycosaminoglycans, and to preserve the glycocalyx after application tumour necrosis factor-. We investigated the influence of antithrombin on glycocalyx subjected to ischaemia/reperfusion.

Methods and results

Isolated guinea pig hearts were perfused with Krebs–Henseleit buffer (KHB). Antithrombin was applied to achieve physiological levels (1 U/mL) before inducing 20 min of ischaemia (37°C). Hearts were reperfused for 20 min at constant flow (baseline perfusion pressure 70 cmH₂O) with KHB or KHB plus 2 g% hydroxyethyl starch (130 kDa). Coronary net fluid filtration was assessed directly by measuring transudate formation on the epicardial surface. Post-ischaemic coronary release of syndecan-1 and heparan sulfate was quantified by ELISA. Hearts were perfusion-fixed to visualize the glycocalyx by electron microscopy. Ischaemia/reperfusion caused degradation of the glycocalyx, enhanced coronary perfusion pressure, and increased vascular permeability. Antithrombin significantly reduced post-ischaemic glycocalyx shedding, coronary perfusion pressure, coronary leak, and tissue oedema formation compared to untreated hearts. Additional application of colloid augmented these actions of antithrombin. Electron microscopy revealed a mostly intact glycocalyx after antithrombin treatment.

Conclusion

Antithrombin preserves the endothelial glycocalyx, sustaining the vascular barrier function and reducing interstitial oedema. The potentiated effect of colloid in these hearts suggests that the prevention of shedding should be of functional benefit also in vivo.

Development and characterization of a mouse in vitro model of ischaemia-induced ventricular fibrillation

Stables, C. L., Curtis, M. J. — 2009-06-25

Aims

We sought to generate a mouse Langendorff model of ischaemia-induced ventricular fibrillation (VF) that does not depend on triggers such as programmed electrical stimulation.

Methods and results

Hearts from male Tuck Ordinary mice were perfused with Krebs solution (modified to contain low-normal K⁺, 3 mmol/L, and high Ca²⁺, 2.4 mmol/L) containing different combinations of catecholamines (epinephrine 313 nmol/L plus norepinephrine 75 nmol/L) and/or angiotensin II (100 pmol/L) designed to mimic the in vivo milieu. VF was absent during 30 min regional ischaemia (and during 10 min reperfusion) in Krebs-perfused hearts. Catecholamines unmasked ischaemia-induced VF (50%; P < 0.05) and reperfusion-induced VF (50%; P < 0.05). Co-perfusion with angiotensin II did not facilitate VF. Supraventricular pacing (600 b.p.m.) stabilized pre-ischaemic sinus rhythm and partially mimicked the VF-unmasking effect of catecholamines. Arrhythmia susceptibility was greatest with supraventricular pacing plus catecholamines (57% VF during ischaemia and 71% during reperfusion).

Conclusion

For the first time, regional ischaemia-induced VF was consistently evoked in a mouse Langendorff preparation, unmasked by simple periphysiological manipulation of the perfusion conditions. The model is suitable for functional genomic studies.

Human cardiac gap-junction coupling: effects of antiarrhythmic peptide AAP10

Hagen, A., Dietze, A., Dhein, S. — 2009-06-25

Aims

Ventricular arrhythmia is one of the most important causes of death in industrialized countries and often accompanies myocardial infarction and heart failure. In recent years modification of gap-junctional coupling has been proposed as a new antiarrhythmic principle. We wanted to examine whether the gap junction modulator (antiarrhythmic peptide) AAP10 exerts effects on human cardiac gap junctions, whether the effect might be enhanced in uncoupled cells, whether it affects electrical and metabolic coupling, and which of the cardiac connexin isoforms (Cx40, Cx43, Cx45) may be affected.

Methods and results

We determined the influence of 50 nM AAP10 (H₂N-Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH₂) on macroscopic gap junction conductance by dual whole-cell voltage clamping in human and rat cardiomyocytes. Cells were partially uncoupled by CO₂-mediated acidosis (pH 6.3) or kept at ‘normal’ conditions (pH 7.4, T 36°C). Furthermore, we investigated effects of AAP10 in HeLa cells stably transfected with connexin 40, 43, or 45 and on metabolic coupling determined by dye transfer (Lucifer yellow). AAP10 (50 nM)-enhanced gap-junctional intercellular coupling in human and rat cardiomyocytes, completely prevented CO₂-acidosis-induced uncoupling and improved metabolic coupling. The coupling effect of AAP10 was significantly enhanced in previously uncoupled cells. Regarding the connexin isoforms, AAP10-enhanced electrical and metabolic coupling in HeLa cells expressing Cx43 or Cx45, but not in HeLa cells expressing Cx40.

Conclusion

We conclude that the antiarrhythmic peptide AAP10, which improves gap-junctional intercellular coupling and prevents uncoupling by acidification in human cardiomyocytes, might be useful for antiarrhythmic strategies regarding arrhythmias caused by uncoupling of Cx43 and Cx45, but not Cx40.