Glucagon-like peptide-1 (GLP-1) mediates cardioprotection by remote ischaemic conditioning

This work was supported by the British Heart Foundation (Ref: RG/14/4/30736), Medical Research Council (MR/N02589X/1) and The Wellcome Trust (Ref: 200893/Z/16/Z). A.V.G. is a Wellcome Trust Senior Research Fellow. S.M. is a Marie Skłodowska-Curie Research Fellow (Ref: 654691)

Proteomic Analysis of Cardiac Adaptation to Exercise by High Resolution Mass Spectrometry

Regular exercise has many health benefits among which is a significant reduction of cardiovascular risk. Although many beneficial effects of exercise are well described, the exact mechanisms by which exercise confers cardiovascular benefits remain to be fully understood. In the current study, we have used high resolution mass spectrometry to determine the proteomic responses of the heart to exercise training in mice. The impact of exercise-induced oxidative stress on modifications of cardiomyocyte proteins with lipid peroxidation biomarker 4-hydroxynonenal (4-HNE) was examined as well. Fourteen male mice were randomized into the control (sedentary) group and exercise group subjected to a swim exercise training program for 5 days a week for 5 months. Proteins were isolated from the left ventricular tissue, fractionated and digested for shotgun proteomics. Peptides were separated by nanoliquid chromatography and analyzed on Orbitrap Fusion mass spectrometer using higher energy collision-induced dissociation and electron transfer dissociation fragmentation. We identified distinct ventricular protein signatures established in response to exercise training. Comparative proteomics identified 23 proteins upregulated and 37 proteins downregulated with exercise in addition to 65 proteins identified only in ventricular tissue samples of exercised mice. Most of the proteins specific for exercised mice are involved in respiratory electron transport, and/or implicated in glutathione conjugation. Additionally, 10 proteins were found to be modified with 4-HNE. This study provides new data on the effects of exercise on the cardiac proteome and contributes to our understanding of the molecular mechanisms underlying the beneficial effects of exercise on the heart

Distinct cardioprotective mechanisms of immediate, early and delayed ischaemic postconditioning

Cardioprotection against ischaemia/reperfusion injury in mice can be achieved by delayed ischaemic postconditioning (IPost) applied as late as 30 min after the onset of reperfusion. We determined the efficacy of delayed IPost in a rat model of myocardial infarction (MI) and investigated potential underlying mechanisms of this phenomenon. Rats were subjected to 20, 30 or 45 min of coronary artery occlusion followed by 120 min of reperfusion (I/R). Immediate and early IPost included six cycles of I/R (10/10 s) applied 10 s or 10 min after reperfusion onset. In the second series of experiments, the rats were subjected to 30 min of coronary occlusion followed by IPost applied 10 s, 10, 30, 45 or 60 min after the onset of reperfusion. Immediate and early IPost (applied 10 s or 10 min of reperfusion) established cardioprotection only when applied after a period of myocardial ischaemia lasting 30 min. Delayed IPost applied after 30 or 45 min of reperfusion reduced infarct sizes by 36 and 41 %, respectively (both P < 0.01). IPost applied 60 min after reperfusion onset was ineffective. Inhibition of RISK pathway (administration of ERK1/2 inhibitor PD-98059 or PI3K inhibitor LY-294002) abolished cardioprotection established by immediate IPost but had no effect on cardioprotection conferred by early IPost. Blockade of SAFE pathway using JAK/STAT inhibitor AG490 had no effect on the immediate or early IPost cardioprotection. Blockade of mitochondrial KATP (mitoKATP) channels (with 5-Hydroxydecanoate) abolished cardioprotection achieved by immediate and early IPost, but had no effect on cardioprotection when IPost was applied 30 or 45 min into the reperfusion period. Immediate IPost increased phosphorylation of PI3K-AKT and ERK1/2. Early or delayed IPost had no effect on phosphorylation of PI3K-AKT, ERK1/2 or STAT3. These data show that in the rat model, delayed IPost confers significant cardioprotection even if applied 45 min after onset of reperfusion. Cardioprotection induced by immediate and early postconditioning involves recruitment of RISK pathway and/or mitoKATP channels, while delayed postconditioning appears to rely on a different mechanism

A Critical Role for Purinergic Signalling in the Mechanisms Underlying Generation of BOLD fMRI Responses

The mechanisms of neurovascular coupling underlying generation of BOLD fMRI signals remain incompletely understood. It has been proposed that release of vasoactive substances by astrocytes couples neuronal activity to changes in cerebrovascular blood flow. However, the role of astrocytes in fMRI responses remains controversial. Astrocytes communicate via release of ATP, and here we tested the hypothesis that purinergic signaling plays a role in the mechanisms underlying fMRI. An established fMRI paradigm was used to trigger BOLD responses in the forepaw region of the somatosensory cortex (SSFP) of an anesthetized rat. Forepaw stimulation induced release of ATP in the SSFP region. To interfere with purinergic signaling by promoting rapid breakdown of the vesicular and/or released ATP, a lentiviral vector was used to express a potent ectonucleotidase, transmembrane prostatic acid phosphatase (TMPAP), in the SSFP region. TMPAP expression had no effect on resting cerebral blood flow, cerebrovascular reactivity, and neuronal responses to sensory stimulation. However, TMPAP catalytic activity markedly reduced the magnitude of BOLD fMRI responses triggered in the SSFP region by forepaw stimulation. Facilitated ATP breakdown could result in accumulation of adenosine. However, blockade of A(1) receptors had no effect on BOLD responses and did not reverse the effect of TMPAP. These results suggest that purinergic signaling plays a significant role in generation of BOLD fMRI signals. We hypothesize that astrocytes activated during periods of enhanced neuronal activity release ATP, which propagates astrocytic activation, stimulates release of vasoactive substances and dilation of cerebral vasculature

The importance of nitric oxide bioavailability and endothelial mechanisms for cardioprotection by pharmacological intervention during myocardial ischaemia and reperfusion

Myocardial ischaemia initiates complex severe cellular changes and, after a critical time period, myocardial cell death. Despite that restitution of coronary blood flow is an absolute pre-requisite for tissue survival, myocardial reperfusion per se will also increase irreversible damage in the jeopardised myocardium. Endothelial dysfunction is an early event during ischaemia and reperfusion, which is characterised by an impairment of endothelium-dependent relaxations, mainly due to reduced nitric oxide (NO) bioavailability and increased production of endothelin-1 (ET-1). Certain ET-I receptor antagonists and calcium channels blockers may in addition to their classical actions, affect NO bioavailability. The aim of the studies was to investigate the involvement and mechanisms of the endothelial factors NO and ET- I in the pathophysiology of myocardial ischaemia-reperfusion damage. Of particular interest was the importance of NO bioavailability for the cardioprotective effects of pharmacological intervention by an ETA receptor antagonist and a short-acting calcium antagonist. 1. Administration of the selective ETA receptor antagonist LU 135252 i.v. 10 min before ischaemia reduced infarct size (IS) and attenuated the increase in myocardial ET-like immunoreactivity in anaesthetised pigs subjected to 45 min of ischaemia and 4 h of reperfusion. These cardioprotective effects were abolished in the presence of NO synthase (NOS) inhibition by L-NNA and restored by addition of the NO precursor L-arginine. NOS inhibition did not affect IS per se. 2. Administration of ET- I i.v. during three 5 min infusion periods separated by 5 min before ischaemia reduced IS in anaesthetised rats subjected to 30 min of ischaemia followed by 2 h of reperfusion. This preconditioning (PC)-like cardioprotective effect was blocked either by the ETA receptor antagonist LU 135252, the nonselective ATP sensitive K+ channels (KATP) antagonist glibenclamide or the selective mithochondrial KATP channel antagonist 5-hydroxydecanoic acid. 3. Intracoronary administration of short-acting calcium antagonist clevidipine during the last 10 min of ischaemia and the first 5 min of reperfusion reduced IS and preserved endothelium-dependent vasodilation induced by substance P in anaesthetised pigs. The cardioprotective effect of clevidipine was abolished when it was administered together with the NOS inhibitor L-NMMA. Addition of L-arginine restored cardioprotection induced by clevidipine. The cardioprotective effect of clevidipine was also abolished, when it was given together with the bradykinin B2 receptor antagonist HOE 140. Addition of the NO donor S-nitroso-N-acetyl-D, L-penicillamine (SNAP) restored cardioprotection. 4. Intracoronary administration of the NO donor SNAP in a narrow concentration range for a brief period before ischaemia reduced IS, attenuated myeloperoxidase activity as index of neutrophil accumulation in the ischaemic/reperfused myocardium and improved endothelium-dependent coronary vasodilation induced by substance P in anaesthetised pigs. In conclusion, the cardioprotective effect of the ETA receptor antagonist during ischaemia and reperfusion is mediated via a mechanism related to no; under certain conditions exogenous ET-1 may also exert cardioprotection in vivo via the ETA receptor and activation of mitochondrial KATP channels; the cardioprotective effect of the short-acting calcium antagonist clevidipine during late ischaemia and early reperfusion is mediated via bradykinin- and NO-related mechanisms; NO exerts a classic PC-like cardioprotection against ischaemiareperfusion injury in vivo in a narrow concentration range

Identifying the Source of a Humoral Factor of Remote (Pre)Conditioning Cardioprotection.

Signalling pathways underlying the phenomenon of remote ischaemic preconditioning (RPc) cardioprotection are not completely understood. The existing evidence agrees that intact sensory innervation of the remote tissue/organ is required for the release into the systemic circulation of preconditioning factor(s) capable of protecting a transplanted or isolated heart. However, the source and molecular identities of these factors remain unknown. Since the efficacy of RPc cardioprotection is critically dependent upon vagal activity and muscarinic mechanisms, we hypothesized that the humoral RPc factor is produced by the internal organ(s), which receive rich parasympathetic innervation. In a rat model of myocardial ischaemia/reperfusion injury we determined the efficacy of limb RPc in establishing cardioprotection after denervation of various visceral organs by sectioning celiac, hepatic, anterior and posterior gastric branches of the vagus nerve. Electrical stimulation was applied to individually sectioned branches to determine whether enhanced vagal input to a particular target area is sufficient to establish cardioprotection. It was found that RPc cardioprotection is abolished in conditions of either total subdiaphragmatic vagotomy, gastric vagotomy or sectioning of the posterior gastric branch. The efficacy of RPc cardioprotection was preserved when hepatic, celiac or anterior gastric vagal branches were cut. In the absence of remote ischaemia/reperfusion, electrical stimulation of the posterior gastric branch reduced infarct size, mimicking the effect of RPc. These data suggest that the circulating factor (or factors) of RPc are produced and released into the systemic circulation by the visceral organ(s) innervated by the posterior gastric branch of the vagus nerve

Diagrammatic representation of the nervous control of hormone secretion by enteroendocrine cells of the gastrointestinal tract.

<p>Extrinsic vagal parasympathetic nerves either directly or via activation of the enteric neurones trigger release of hormones (hypothesised circulating cardioprotective factors) by releasing acetylcholine (among other transmitters). ACh, acetylcholine; AChR, acetylcholine receptor; BOM, bombesin; CCK, cholecystokinin; CGPR, calcitonin gene-related peptide; GliC, glicentin; GLP-1/2, glucagon-like peptide-1 and 2; OXM, oxyntomodulin; PYY, peptide YY; VIP, vasoactive intestinal peptide.</p

Cardioprotection established by remote ischaemic preconditioning (RPc) requires intact parasympathetic innervation of visceral organs.

<p><b>(a)</b> Illustration of the experimental protocols. RPc was induced by 15 min occlusion of both femoral arteries, followed by 10 min reperfusion. Sham-RPc procedure involved dissection of both femoral arteries without occlusion. Arrows indicate time of total subdiaphragmatic vagotomy, selective sectioning of individual visceral branches or sham surgery. <b>(b)</b> Total subdiaphragmatic vagotomy, bilateral gastric vagotomy and selective sectioning of the posterior gastric branch abolished the cardioprotective effect of RPc, whereas sectioning of the anterior gastric, celiac or hepatic branches had no effect on RPc cardioprotection. The infarct size is presented as the percentage of the area at risk. Individual data and means ± SEM are shown. P-values correspond to the Dunn’s post-hoc tests.</p