Remote ischaemic conditioning: defining critical criteria for success-report from the 11th Hatter Cardiovascular Workshop.

The Hatter Cardiovascular Institute biennial workshop, originally scheduled for April 2020 but postponed for 2 years due to the Covid pandemic, was organised to debate and discuss the future of Remote Ischaemic Conditioning (RIC). This evolved from the large multicentre CONDI-2-ERIC-PPCI outcome study which demonstrated no additional benefit when using RIC in the setting of ST-elevation myocardial infarction (STEMI). The workshop discussed how conditioning has led to a significant and fundamental understanding of the mechanisms preventing cell death following ischaemia and reperfusion, and the key target cyto-protective pathways recruited by protective interventions, such as RIC. However, the obvious need to translate this protection to the clinical setting has not materialised largely due to the disconnect between preclinical and clinical studies. Discussion points included how to adapt preclinical animal studies to mirror the patient presenting with an acute myocardial infarction, as well as how to refine patient selection in clinical studies to account for co-morbidities and ongoing therapy. These latter scenarios can modify cytoprotective signalling and need to be taken into account to allow for a more robust outcome when powered appropriately. The workshop also discussed the potential for RIC in other disease settings including ischaemic stroke, cardio-oncology and COVID-19. The workshop, therefore, put forward specific classifications which could help identify so-called responders vs. non-responders in both the preclinical and clinical settings.

Ectopic expression of S28A-mutated Histone H3 modulates longevity, stress resistance and cardiac function in Drosophila.

Histone H3 serine 28 (H3S28) phosphorylation and de-repression of polycomb repressive complex (PRC)-mediated gene regulation is linked to stress conditions in mitotic and post-mitotic cells. To better understand the role of H3S28 phosphorylation in vivo, we studied a Drosophila strain with ectopic expression of constitutively-activated H3S28A, which prevents PRC2 binding at H3S28, thus mimicking H3S28 phosphorylation. H3S28A mutants showed prolonged life span and improved resistance against starvation and paraquat-induced oxidative stress. Morphological and functional analysis of heart tubes revealed smaller luminal areas and thicker walls accompanied by moderately improved cardiac function after acute stress induction. Whole-exome deep gene-sequencing from isolated heart tubes revealed phenotype-corresponding changes in longevity-promoting and myotropic genes. We also found changes in genes controlling mitochondrial biogenesis and respiration. Analysis of mitochondrial respiration from whole flies revealed improved efficacy of ATP production with reduced electron transport-chain activity. Finally, we analyzed posttranslational modification of H3S28 in an experimental heart failure model and observed increased H3S28 phosphorylation levels in HF hearts. Our data establish a critical role of H3S28 phosphorylation in vivo for life span, stress resistance, cardiac and mitochondrial function in Drosophila. These findings may pave the way for H3S28 phosphorylation as a putative target to treat stress-related disorders such as heart failure.

Pleiotropic, heart rate-independent cardioprotection by ivabradine.

BACKGROUND AND PURPOSE: In pigs, ivabradine reduces infarct size even when given only at reperfusion and in the absence of heart rate reduction. The mechanism of this non-heart rate-related cardioprotection is unknown. Hence, in the present study we assessed the pleiotropic action of ivabradine in more detail. EXPERIMENTAL APPROACH: Anaesthetized mice were pretreated with ivabradine (1.7 mg · kg(-1) i.v.) or placebo (control) before a cycle of coronary occlusion/reperfusion (30/120 min ± left atrial pacing). Infarct size was determined. Isolated ventricular cardiomyocytes were exposed to simulated ischaemia/reperfusion (60/5 min) in the absence and presence of ivabradine, viability was then quantified and intra- and extracellular reactive oxygen species (ROS) formation was detected. Mitochondria were isolated from mouse hearts and exposed to simulated ischaemia/reperfusion (6/3 min) in glutamate/malate- and ADP-containing buffer in the absence and presence of ivabradine respectively. Mitochondrial respiration, extramitochondrial ROS, mitochondrial ATP production and calcium retention capacity (CRC) were assessed. KEY RESULTS: Ivabradine decreased infarct size even with atrial pacing. Cardiomyocyte viability after simulated ischaemia/reperfusion was better preserved with ivabradine, the accumulation of intra- and extracellular ROS decreased in parallel. Mitochondrial complex I respiration was not different without/with ivabradine, but ivabradine significantly inhibited the accumulation of extramitochondrial ROS, increased mitochondrial ATP production and increased CRC. CONCLUSION AND IMPLICATIONS: Ivabradine reduces infarct size independently of a reduction in heart rate and improves ventricular cardiomyocyte viability, possibly by reducing mitochondrial ROS formation, increasing ATP production and CRC.

Extracellular signalling molecules in the ischaemic/reperfused heart - druggable and translatable for cardioprotection?

In patients with acute myocardial infarction, timely reperfusion is essential to limit infarct size. However, reperfusion also adds to myocardial injury. Brief episodes of ischaemia/reperfusion in the myocardium or on organ remote from the heart, before or shortly after sustained myocardial ischaemia effectively reduce infarct size, provided there is eventual reperfusion. Such conditioning phenomena have been established in many experimental studies and also translated to humans. The underlying signal transduction, that is the molecular identity of triggers, mediators and effectors, is not clear yet in detail, but several extracellular signalling molecules, such as adenosine, bradykinin and opioids, have been identified to contribute to cardioprotection by conditioning manoeuvres. Several trials have attempted the translation of cardioprotection by such autacoids into a clinical scenario of myocardial ischaemia and reperfusion. Adenosine and its selective agonists reduced infarct size in a few studies, but this benefit was not translated into improved clinical outcome. All studies with bradykinin or drugs which increase bradykinin's bioavailability reported reduced infarct size and some of them also improved clinical outcome. Synthetic opioid agonists did not result in a robust infarct size reduction, but this failure of translation may relate to the cardioprotective properties of the underlying anaesthesia per se or of the comparator drugs. The translation of findings in healthy, young animals with acute coronary occlusion/reperfusion to patients of older age, with a variety of co-morbidities and co-medications, suffering from different scenarios of myocardial ischaemia/reperfusion remains a challenge.

Myocardial protection by remote ischaemic pre-conditioning is abolished in sulphonylurea-treated diabetics undergoing coronary revascularisation.

BACKGROUND: Remote ischaemic pre-conditioning attenuates myocardial injury. Because sulphonylurea drugs interfere with ischaemic and anaesthetic pre-conditioning, we assessed whether remote ischaemic pre-conditioning effects are altered in sulphonylurea-treated diabetics. METHODS: Using the database of our ongoing randomised, placebo-controlled study (ClinicalTrials.gov NCT01406678), we assessed the troponin I concentration area under curve (measurements: baseline, 1, 6, 12, 24, 48, and 72 h post-operatively) in sulphonylurea-treated diabetics (n = 27) and non-diabetics (n = 230) without and with remote ischaemic pre-conditioning (three 5-min periods of left upper arm ischaemia with 5-min reperfusion each) during isoflurane anaesthesia before two- to three-vessel coronary artery surgery. RESULTS: Remote ischaemic pre-conditioning in non-diabetic patients evoked a 41% decrease in the troponin I concentration area under curve (514 ng/ml × 72 h ± 600 vs. 302 ± 190, P = 0.001) but no change (404 ng/ml × 72 h ± 224 vs. 471 ± 383, P = 0.62) in sulphonylurea-treated diabetics. There was no significant correlation between the troponin I concentration area under curve and arterial glucose concentrations, and the latter was not an independent confounder. CONCLUSION: Cardioprotection by remote ischaemic pre-conditioning during isoflurane anaesthesia is abolished in sulphonylurea-treated diabetics.

Impaired red blood cell deformability in patients with coronary artery disease and diabetes mellitus.

Patients with diabetes mellitus (DM) have an increased cardiovascular morbidity and mortality. There is increasing evidence that diabetes mellitus is associated with pathological hemorheological alterations, which might contribute to impaired coronary blood flow in coronary artery disease (CAD). We hypothesize that red blood cell (RBC) deformability is impaired in diabetic patients with CAD in comparison to nondiabetic patients with CAD. RBC deformability was measured in 21 patients with CAD and type 2 diabetes mellitus (CAD + DM) and 24 patients with CAD (CAD - DM). RBC deformability was measured by the Laser-assisted optical rotational cell analyzer by determining the elongation index (EI). RBC deformability was reduced in patients with CAD + DM in comparison to patients with CAD - DM (EI @ 1.12 Pa 0.236 ± 0.008 vs. 0.260 ± 0.005, p=0.007). Inverse univariate correlations were found between the EI @ 1.12 Pa and plasma glucose concentration (r= - 0.57; p<0.001) and HbA1c (r= - 0.45; p=0.002). Multivariate linear regression analysis identified plasma glucose concentration as the independent predictor of RBC deformability (ß= - 0.58; p=0.007) thereby indicating that increased glucose concentrations determine RBC deformability in diabetic patients with CAD. In patients with CAD, diabetes mellitus leads to an impairment of RBC deformability which might contribute to increased morbidity of diabetic patients with CAD.

L-arginine preferentially dilates stenotic segments of coronary arteries thereby increasing coronary flow.

BACKGROUND AND OBJECTIVES: Oxidized LDL cholesterol and cytokines increase arginase and decrease nitric oxide (NO) synthase expression in human endothelial cells, leading to a decrease in NO production. In arteriosclerotic plaques, characterized by increased oxidized LDL and cytokine levels, a sustained local NO reduction might enhance sensitivity of the downstream guanylyl cyclase system towards an acute NO increase. We tested whether application of the NO synthase substrate l-arginine (l-arg, 150 micromol min(-1)) or the NO donor isosorbide dinitrate (ISDN; 0.3 mg) preferentially dilates stenotic coronary artery segments (CS) subsequently increasing poststenotic coronary blood flow (CBF) in patients with coronary artery disease (CAD). DESIGN, SETTING AND SUBJECTS: Changes in coronary diameter and circumferential surface area were assessed by quantitative coronary angiography (QCA) in a nonstenotic upstream segment, the CS, downstream the CS and in a reference vessel (n = 24). CBF was estimated in a subset of 13 patients by QCA and intracoronary Doppler. RESULTS: CS ranged from 62% to 89% (77 +/- 5%). l-arg increased minimal luminal diameter of the stenotic segment from 0.98 +/- 0.06 to 1.14 +/- 0.07 mm (P < 0.05) without affecting other coronary segments. Poststenotic CBF increased by 24 +/- 3%. ISDN dilated all segments again with a predominance of CS (25 +/- 4%) and increased poststenotic CBF by 38 +/- 9%. In a multifactorial anova, a medication with an angiotensin-converting enzyme inhibitor (decreasing inflammation and radical formation) and a ratio of LDL/HDL <3.5 were predictive for an l-arg-induced dilation. CONCLUSION: The increase in poststenotic CBF without affecting nondiseased arteries highlights the therapeutic potential of l-arg in patients with CAD.