P652The cardioprotective effect of exogenous sphingosine-1-phosphate requires the activation of endogenous sphingosine-1-phosphate via the sphingosine kinase 1

Purpose: Exogenous administration of sphingosine-1-phosphate (S1P) alone, or as part of high density lipoprotein, protects against myocardial infarction. S1P-induced cardioprotection targets the inhibition of the mitochondrial permeability transition pore via mechanisms that remain unclear. In the cell, the endogenous production of S1P from sphingosine is dependent on the activation of sphingosine kinases (SphK) 1 and 2. These two kinases play a role in cardioprotection against ischemia-reperfusion (IR) injury. Therefore, we hypothesised that the cardioprotective effect of exogenous S1P requires the activation of endogenous S1P via SphK. Methods: Isolated cardiomyocytes from adult wildtype mice were exposed to 2 hours of simulated ischemia (SI) in the presence of S1P (10nM) with/without N,N-dimethylsphingosine (DMS, a SphK1 and 2 inhibitor, 10μM) or SKI (a specific SphK1 inhibitor, 15μM). Cell viability was assessed using trypan blue staining and normalised to the normoxic control. Isolated perfused hearts from adult wildtype mice were exposed to 35 minutes of global ischemia followed by 45 minutes of reperfusion (IR) in the presence of S1P (10nM) with/without SKI (10μM). Infarct size (IS) was assessed using tripheyltetrazolium chloride staining and SphK1 activity using a specific biochemical fluorescence based assay kit. Both parameters were normalised to the IR control. Results: In isolated cardiomyocytes, viability under normoxic conditions was 76±1%. SI reduced viability to 52±1% (p< 0.001 vs. normoxia). Pre-treatment with S1P restored the viability to 75±1% (p<0.001 vs. SI). The beneficial effect of S1P was partially inhibited in the presence of DMS (67±4%) (ns vs. S1P) and totally abrogated with SKI pre-treatment (54±2%). Similarly, pre-treatment with S1P in isolated hearts reduced IS following IR from 50±1% (IR control) to 31±2% (S1P) (p<0.001 vs. control). Pre-treatment with SKI abrogated the cardioprotective effect of S1P (56±8%) (p<0.05 vs. S1P) as well as the S1P-induced increase in SphK1 activity (from S1P: 196±79 arbitrary units (AU) to SKI+S1P: 53±27 AU, p<0.05 vs. S1P). Conclusions: Our data, performed in both isolated cardiomyocytes and isolated hearts subjected to an ischemia/reperfusion insult, strongly suggest that exogenous sphingosine-1-phosphate-induced cardioprotection is dependent on the activation of endogenous sphingosine-1-phosphate via sphingosine kinase

Platelet inhibitors influence cardioprotection: importance in preclinical study design: reply

We would like to thank Professors Cohen and Downey for highlighting platelet inhibitors, in particular platelet P2Y12 receptor antagonists, as an important confounder to take into consideration in pre-clinical studies designed to study novel cardioprotective strategies against ischaemia/reperfusion injury

IMproving Preclinical Assessment of Cardioprotective Therapies (IMPACT) criteria : guidelines of the EU-CARDIOPROTECTION COST Action

Full list of the EU-CARDIOPROTECTION COST Action CA16225 Working group members is provided at the end of the article in Acknowledgements section. Funding Information: This article is based on the work from COST Action EU-CARDIOPROTECTION CA16225 supported by COST (European Cooperation in Science and Technology). DJH is supported by the Duke-National University Singapore Medical School, Singapore Ministry of Health’s National Medical Research Council under its Clinician Scientist-Senior Investigator scheme (NMRC/CSA-SI/0011/2017) and Collaborative Centre Grant scheme (NMRC/CGAug16C006). SL is supported by grants from the South African Department of Science and Technology and the South African National Research Foundation. SMD is supported by grants from the British Heart Foundation (PG/19/51/34493 and PG/16/85/32471). GH is supported by the German Research Foundation (SFB 1116 B8). MRM is supported by the Spanish Institute of Health Carlos III (FIS PI19/01196 and CIBER-CV). RS is supported by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) [Project number 268555672—SFB 1213, Project B05]. PF is supported by the National Research, Development and Innovation Office of Hungary (Research Excellence Program—TKP, National Heart Program NVKP 16-1-2016-0017) and by the Higher Education Institutional Excellence Program of the Ministry of Human Capacities in Hungary, within the framework of the Therapeutic Development thematic program of the Semmelweis University. Funding Information: The IMPACT criteria were presented for approval to the Management Committee of the EU-CARDIOPROTECTION COST Action CA16225: Pavle Adamovski, Ioanna Andreadou, Saime Batirel, Monika Bartekov?, Luc Bertrand, Christophe Beauloye, David Biedermann, Vilmante Borutaite, Hans Erik Botker, Stefan Chlopicki, Maija Dambrova, Sean Davidson, Yvan Devaux, Fabio Di Lisa, Dragan Djuric, David Erlinge, Ines Falcao-Pires, P?ter Ferdinandy, Eleftheria Galatou, Alfonso Garcia-Sosa, Henrique Girao, Zoltan Giricz, Mariann Gyongyosi, Derek J Hausenloy, Donagh Healy, Gerd Heusch, Vladimir Jakovljevic, Jelena Jovanic, George Kararigas, Risto Kerkal, Frantisek Kolar, Brenda Kwak, Przemys?aw Leszek, Edgars Liepinsh , Jacob Lonborg, Sarah Longnus, Jasna Marinovic, Danina Mirela Muntean, Lana Nezic, Michel Ovize, Pasquale Pagliaro, Clarissa Pedrosa Da Costa Gomes, John Pernow, Andreas Persidis, S?ren Erik Pischke, Bruno Podesser, Ines Poto?njak, Fabrice Prunier, Tanya Ravingerova, Marisol Ruiz-Meana, Alina Serban, Katrine Slagsvold, Rainer Schulz, Niels van Royen, Belma Turan, Marko Vendelin, Stewart Walsh, Nace Zidar, Coert Zuurbier, Derek Yellon. Publisher Copyright: © 2021, The Author(s).Acute myocardial infarction (AMI) and the heart failure (HF) which may follow are among the leading causes of death and disability worldwide. As such, new therapeutic interventions are still needed to protect the heart against acute ischemia/reperfusion injury to reduce myocardial infarct size and prevent the onset of HF in patients presenting with AMI. However, the clinical translation of cardioprotective interventions that have proven to be beneficial in preclinical animal studies, has been challenging. One likely major reason for this failure to translate cardioprotection into patient benefit is the lack of rigorous and systematic in vivo preclinical assessment of the efficacy of promising cardioprotective interventions prior to their clinical evaluation. To address this, we propose an in vivo set of step-by-step criteria for IMproving Preclinical Assessment of Cardioprotective Therapies (‘IMPACT’), for investigators to consider adopting before embarking on clinical studies, the aim of which is to improve the likelihood of translating novel cardioprotective interventions into the clinical setting for patient benefit.publishersversionPeer reviewe

Improving Reconstituted HDL Composition for Efficient Post-Ischemic Reduction of Ischemia Reperfusion Injury.

BACKGROUND: New evidence shows that high density lipoproteins (HDL) have protective effects beyond their role in reverse cholesterol transport. Reconstituted HDL (rHDL) offer an attractive means of clinically exploiting these novel effects including cardioprotection against ischemia reperfusion injury (IRI). However, basic rHDL composition is limited to apolipoprotein AI (apoAI) and phospholipids; addition of bioactive compound may enhance its beneficial effects. OBJECTIVE: The aim of this study was to investigate the role of rHDL in post-ischemic model, and to analyze the potential impact of sphingosine-1-phosphate (S1P) in rHDL formulations. METHODS AND RESULTS: The impact of HDL on IRI was investigated using complementary in vivo, ex vivo and in vitro IRI models. Acute post-ischemic treatment with native HDL significantly reduced infarct size and cell death in the ex vivo, isolated heart (Langendorff) model and the in vivo model (-48%, p&lt;0.01). Treatment with rHDL of basic formulation (apoAI + phospholipids) had a non-significant impact on cell death in vitro and on the infarct size ex vivo and in vivo. In contrast, rHDL containing S1P had a highly significant, protective influence ex vivo, and in vivo (-50%, p&lt;0.01). This impact was comparable with the effects observed with native HDL. Pro-survival signaling proteins, Akt, STAT3 and ERK1/2 were similarly activated by HDL and rHDL containing S1P both in vitro (isolated cardiomyocytes) and in vivo. CONCLUSION: HDL afford protection against IRI in a clinically relevant model (post-ischemia). rHDL is significantly protective if supplemented with S1P. The protective impact of HDL appears to target directly the cardiomyocyte

Correction to: RIC in COVID-19—a Clinical Trial to Investigate Whether Remote Ischemic Conditioning (RIC) Can Prevent Deterioration to Critical Care in Patients with COVID-19

Purpose: Coronavirus disease 19 (COVID-19) has, to date, been diagnosed in over 130 million persons worldwide and is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Several variants of concern have emerged including those in the United Kingdom, South Africa, and Brazil. SARS-CoV-2 can cause a dysregulated inflammatory response known as a cytokine storm, which can progress rapidly to acute respiratory distress syndrome (ARDS), multi-organ failure, and death. Suppressing these cytokine elevations may be key to improving outcomes. Remote ischemic conditioning (RIC) is a simple, non-invasive procedure whereby a blood pressure cuff is inflated and deflated on the upper arm for several cycles. “RIC in COVID-19” is a pilot, multi-center, randomized clinical trial, designed to ascertain whether RIC suppresses inflammatory cytokine production. Methods: A minimum of 55 adult patients with diagnosed COVID-19, but not of critical status, will be enrolled from centers in the United Kingdom, Brazil, and South Africa. RIC will be administered daily for up to 15 days. The primary outcome is the level of inflammatory cytokines that are involved in the cytokine storm that can occur following SARS-CoV-2 infection. The secondary endpoint is the time between admission and until intensive care admission or death. The in vitro cytotoxicity of patient blood will also be assessed using primary human cardiac endothelial cells. Conclusions: The results of this pilot study will provide initial evidence on the ability of RIC to suppress the production of inflammatory cytokines in the setting of COVID-19. Trial Registration: NCT04699227, registered January 7th, 2021

Effect of Remote Ischaemic Conditioning on the Inflammatory Cytokine Cascade of COVID-19 (RIC in COVID-19): a Randomized Controlled Trial

Purpose: Patients hospitalized with COVID-19 may develop a hyperinflammatory, dysregulated cytokine “storm” that rapidly progresses to acute respiratory distress syndrome, multiple organ dysfunction, and even death. Remote ischaemic conditioning (RIC) has elicited anti-inflammatory and cytoprotective benefits by reducing cytokines following sepsis in animal studies. Therefore, we investigated whether RIC would mitigate the inflammatory cytokine cascade induced by COVID-19. Methods: We conducted a prospective, multicentre, randomized, sham-controlled, single-blind trial in Brazil and South Africa. Non-critically ill adult patients with COVID-19 pneumonia were randomly allocated (1:1) to receive either RIC (intermittent ischaemia/reperfusion applied through four 5-min cycles of inflation (20 mmHg above systolic blood pressure) and deflation of an automated blood-pressure cuff) or sham for approximately 15 days. Serum was collected following RIC/sham administration and analyzed for inflammatory cytokines using flow cytometry. The endpoint was the change in serum cytokine concentrations. Participants were followed for 30 days. Results: Eighty randomized participants (40 RIC and 40 sham) completed the trial. Baseline characteristics according to trial intervention were overall balanced. Despite downward trajectories of all cytokines across hospitalization, we observed no substantial changes in cytokine concentrations after successive days of RIC. Time to clinical improvement was similar in both groups (HR 1.66; 95% CI, 0.938–2.948, p 0.08). Overall RIC did not demonstrate a significant impact on the composite outcome of all-cause death or clinical deterioration (HR 1.19; 95% CI, 0.616–2.295, p = 0.61). Conclusion: RIC did not reduce the hypercytokinaemia induced by COVID-19 or prevent clinical deterioration to critical care. Trial Registration: ClinicalTrials.gov Identifier: NCT04699227

Analysis of a Local HIV-1 Epidemic in Portugal Highlights Established Transmission of non-B and -G Subtypes

The existing data supports Portugal as the Western Europe country with highest HIV-1 subtype diversity. However, detailed phylogenetic studies of Portuguese HIV-1 epidemics are still scarce. Thus, our main goal was to analyze the phylodynamics of a local HIV-1 infection in the Portuguese region of Minho. Molecular epidemiological analysis was applied to data from 289 HIV-1 infected individuals followed in the reference Hospital of the province of Minho, Portugal, in which isolated viruses had been sequenced between 2000 and 2012. Viruses of the G (29.1%) and B (27.0%) subtypes were the most frequent, followed by recombinant forms (17.6%), C (14.5%), F1 (7.3%) and A1 (4.2%) subtypes. Multinomial logistic regression revealed that the odds of being infected with A1 and F1 subtype increased over the years when compared with B, G, C or recombinant viruses. As expected, polyphyletic patterns suggesting multiple and old introductions of subtypes B and G were found. However, transmission clusters of non-B and -G viruses among native individuals were also found with the dates of the most recent common ancestor estimated to the early 2000s. Our study supports that the HIV-1 subtype diversity in the Portuguese region of Minho is high and has been increasing in a manner that is apparently driven by factors other than immigration and international travel. Infections with A1 and F1 viruses in the region of Minho are becoming established and were mainly found in sexually transmitted clusters, reinforcing the need for more efficacious control measures targeting this infection route

Extracellular vesicles in diagnostics and therapy of the ischaemic heart: Position Paper from the Working Group on Cellular Biology of the Heart of the European Society of Cardiology

Extracellular vesicles (EVs)-particularly exosomes and microvesicles (MVs)-are attracting considerable interest in the cardiovascular field as the wide range of their functions is recognized. These capabilities include transporting regulatory molecules including different RNA species, lipids, and proteins through the extracellular space including blood and delivering these cargos to recipient cells to modify cellular activity. EVs powerfully stimulate angiogenesis, and can protect the heart against myocardial infarction. They also appear to mediate some of the paracrine effects of cells, and have therefore been proposed as a potential alternative to cell-based regenerative therapies. Moreover, EVs of different sources may be useful biomarkers of cardiovascular disease identities. However, the methods used for the detection and isolation of EVs have several limitations and vary widely between studies, leading to uncertainties regarding the exact population of EVs studied and how to interpret the data. The number of publications in the exosome and MV field has been increasing exponentially in recent years and, therefore, in this ESC Working Group Position Paper, the overall objective is to provide a set of recommendations for the analysis and translational application of EVs focussing on the diagnosis and therapy of the ischaemic heart. This should help to ensure that the data from emerging studies are robust and repeatable, and optimize the pathway towards the diagnostic and therapeutic use of EVs in clinical studies for patient benefit

The 10th Biennial Hatter Cardiovascular Institute workshop: cellular protection—evaluating new directions in the setting of myocardial infarction, ischaemic stroke, and cardio-oncology

Due to its poor capacity for regeneration, the heart is particularly sensitive to the loss of contractile cardiomyocytes. The onslaught of damage caused by ischaemia and reperfusion, occurring during an acute myocardial infarction and the subsequent reperfusion therapy, can wipe out upwards of a billion cardiomyocytes. A similar program of cell death can cause the irreversible loss of neurons in ischaemic stroke. Similar pathways of lethal cell injury can contribute to other pathologies such as left ventricular dysfunction and heart failure caused by cancer therapy. Consequently, strategies designed to protect the heart from lethal cell injury have the potential to be applicable across all three pathologies. The investigators meeting at the 10th Hatter Cardiovascular Institute workshop examined the parallels between ST-segment elevation myocardial infarction (STEMI), ischaemic stroke, and other pathologies that cause the loss of cardiomyocytes including cancer therapeutic cardiotoxicity. They examined the prospects for protection by remote ischaemic conditioning (RIC) in each scenario, and evaluated impasses and novel opportunities for cellular protection, with the future landscape for RIC in the clinical setting to be determined by the outcome of the large ERIC-PPCI/CONDI2 study. It was agreed that the way forward must include measures to improve experimental methodologies, such that they better reflect the clinical scenario and to judiciously select combinations of therapies targeting specific pathways of cellular death and injury

Inhibition of permeability transition pore opening by mitochondrial STAT3 and its role in myocardial ischemia/reperfusion

The signal transducer and activator of transcription 3 (STAT3) contributes to cardioprotection by ischemic pre- and postconditioning. Mitochondria are central elements of cardioprotective signaling, most likely by delaying mitochondrial permeability transition pore (MPTP) opening, and STAT3 has recently been identified in mitochondria. We now characterized the mitochondrial localization of STAT3 and its impact on respiration and MPTP opening. STAT3 was mainly present in the matrix of subsarcolemmal and interfibrillar cardiomyocyte mitochondria. STAT1, but not STAT5 was also detected in mitochondria under physiological conditions. ADP-stimulated respiration was reduced in mitochondria from mice with a cardiomyocyte-specific deletion of STAT3 (STAT3-KO) versus wildtypes and in rat mitochondria treated with the STAT3 inhibitor Stattic (STAT3 inhibitory compound, 6-Nitrobenzo[b]thiophene 1,1-dioxide). Mitochondria from STAT3-KO mice and Stattic-treated rat mitochondria tolerated less calcium until MPTP opening occurred. STAT3 co-immunoprecipitated with cyclophilin D, the target of the cardioprotective agent and MPTP inhibitor cyclosporine A (CsA). However, CsA reduced infarct size to a similar extent in wildtype and STAT3-KO mice in vivo. Thus, STAT3 possibly contributes to cardioprotection by stimulation of respiration and inhibition of MPTP opening