Xiulan Yang

Mechanism of Cardioprotection by Early Ischemic Preconditioning

A series of brief ischemia/reperfusion cycles (termed ischemic preconditioning, IPC) limits myocardial injury produced by a subsequent prolonged period of coronary artery occlusion and reperfusion. Over the last 2 decades our understanding of IPC’s mechanism has increased exponentially. Hearts exposed to IPC have a better metabolic and ionic status during prolonged ischemia compared to naïve hearts. However, this difference is not thought to be the main mechanism by which IPC protects against infarction. Signaling pathways that are activated by IPC distinguish IPC hearts from naïve hearts. During the trigger phase of IPC, adenosine, bradykinin and opioid receptors are occupied. Although these three receptors trigger signaling through divergent pathways, the signaling converges on protein kinase C. We have proposed that at the end of the index ischemia the activated PKC sensitizes the low-affinity A2b adenosine receptor (A2bAR) through phosphorylation of either the receptor or its coupling proteins so that A2bAR can be activated by endogenous adenosine released by the previously ischemic cardiomyocytes. The sensitized A2bAR would then be responsible for activation of the survival kinases including PI3 kinase, Akt and ERK which then act to inhibit lethal mitochondrial permeability transition pore formation which normally uncouples mitochondria and destroys many myocytes in the first minutes of reperfusion. Herein we review the evidence for the above mechanisms and their functional details.

Platelet P2Y12 Blockers Confer Direct Postconditioning-Like Protection in Reperfused Rabbit Hearts

Background: Blockade of platelet activation during primary percutaneous intervention for acute myocardial infarction is standard care to minimize stent thrombosis. To determine whether antiplatelet agents offer any direct cardioprotective effect, we tested whether they could modify infarction in a rabbit model of ischemia/reperfusion caused by reversible ligation of a coronary artery. Methods and Results: The P2Y12 (adenosine diphosphate) receptor blocker cangrelor administered shortly before reperfusion in rabbits undergoing 30-minute regional ischemia/3-hour reperfusion reduced infarction from 38% of ischemic zone in control hearts to only 19%. Protection was dose dependent and correlated with the degree of inhibition of platelet aggregation. Protection was comparable to that seen with ischemic postconditioning (IPOC). Cangrelor protection, but not its inhibition of platelet aggregation, was abolished by the same signaling inhibitors that block protection from IPOC suggesting protection resulted from protective signaling rather than anticoagulation. As with IPOC, protection was lost when cangrelor administration was delayed until 10 minutes after reperfusion and no added protection was seen when cangrelor and IPOC were combined. These findings suggest both IPOC and cangrelor may protect by the same mechanism. No protection was seen when cangrelor was used in crystalloid-perfused isolated hearts indicating some component in whole blood is required for protection. Clopidogrel had a very slow onset of action requiring 2 days of treatment before platelets were inhibited, and only then the hearts were protected. Signaling inhibitors given just prior to reperfusion blocked clopidogrel’s protection. Neither aspirin nor heparin was protective. Conclusions: Clopidogrel and cangrelor protected rabbit hearts against infarction. The mechanism appears to involve signal transduction during reperfusion rather than inhibition of intravascular coagulation. We hypothesize that both drugs protect by activating IPOC’s protective signaling to prevent reperfusion injury. If true, patients receiving P2Y12 inhibitors before percutaneous intervention may already be postconditioned thus explaining failure of recent clinical trials of postconditioning drugs.

A2B adenosine receptors inhibit superoxide production from mitochondrial complex I in rabbit cardiomyocytes via a mechanism sensitive to Pertussis toxin

BACKGROUND AND PURPOSE A2B adenosine receptors protect against ischaemia/reperfusion injury by activating survival kinases including extracellular signal‐regulated kinase (ERK) and phosphatidylinositol 3‐kinase (PI3K). However, the underlying mechanism(s) and signalling pathway(s) remain undefined.

A2b adenosine receptors can change their spots

Recently, a central role for the A2b adenosine receptor in a variety of cardiovascular functions including inflammation, erectile function, coronary artery dilation, asthma and cardioprotection has been demonstrated. Despite this evidence, the low‐affinity A2b adenosine receptor is still poorly understood. This receptor appears to be very promiscuous in its coupling. In most tissues, it couples to Gs much like its cousin, the A2a adenosine receptor, but in mast cells and now, most recently, in cardiac fibroblasts, the A2b receptor also couples to Gq. Because of its low affinity, this receptor was originally thought unlikely to play any important physiological role. But the sensitivity of A2b adenosine receptors can be greatly increased by interaction with protein kinase C (PKC) making this receptor, under various conditions, both an activator and a target of PKC. We have recently documented a third coupling involving Gi. This plasticity and versatility of A2b adenosine receptors position them as potential triggers of signalling in multiple signalling cascades in many physiological responses, making this a most interesting receptor indeed.

All Preconditioning-Related G Protein-Coupled Receptors Can Be Demonstrated in the Rabbit Cardiomyocyte:

G protein-coupled receptors for adenosine (A1, A3, A2A, and A2B), bradykinin (B1) and opioids (δ) are all involved in the mechanism of ischemic preconditioning. Although the heart is comprised of many tissue types, it has been assumed that preconditioning’s protective signaling occurs in the cardiomyocyte. We critically tested that hypothesis by testing for the presence of each of these receptors in isolated adult rabbit ventricular myocytes that had been transfected with cyclic nucleotide-gated (CNG) ion channels. Because subsarcolemmal cyclic adenosine monophosphate (cAMP) opens the CNG channels, we could monitor cAMP levels within a single cardiomyocyte by measuring channel current with a patch pipette. The presence of a receptor would be confirmed if we could alter cAMP in the cell with a selective agonist to the receptor being studied. Superfusion with the β-adrenergic Gs-coupled receptor agonist isoproterenol (50 nmol/L) transiently increased cAMP levels and, therefore, channel current. Pretreatment with selective agonists to A1 or A3 adenosine receptors (ARs) that are Gi-coupled markedly attenuated the response to isoproterenol, indicating inhibition of adenylyl cyclase by increased Gi activity. Agonists to bradykinin or δ-opioid receptors also attenuated isoproterenol’s response. A2AAR and A2BAR are Gs-coupled. The A2AAR–selective agonist CGS21680 increased current through CNG channels but only in the presence of phosphodiesterase (PDE) inhibitors, indicating low surface receptor activity and high intracellular PDE activity. As we previously reported, BAY 60-6583, an A2BAR-selective agonist which mimics preconditioning’s protection in rabbit heart, neither increased nor decreased membrane current in transfected cardiomyocytes, suggesting the absence or a markedly limited number of A2BAR in the sarcolemma. However, reverse transcription polymerase chain reaction (RT-PCR) of purified cardiomyocytes yielded an A2BAR band, implying that rabbit cardiomyocytes do indeed express A2BAR. These data reveal that all receptors reported to be involved in ischemic preconditioning do exist on or within the cardiomyocyte.

Augmented taurine release is not the mechanism of ischemic preconditioning’s cardioprotection

Summary.In ischemic preconditioning (IPC) a brief ischemic period protects the heart from a subsequent ischemic insult by an unknown mechanism. Osmotic swelling has been proposed to be a major cause of cell death when ischemic tissue is reperfused. The present study tests whether the preconditioned heart during reperfusion might release more taurine, an important osmolyte in the cardiac myocytes, to decrease cellular osmolarity, oppose swelling, and preserve viability. We collected the coronary effluent from isolated rabbit hearts for 10 min before and 10 min after preconditioning with 5 min of global ischemia. The heart then experienced 15 min of global ischemia and effluent was collected during reperfusion for 40 min. A control group was studied similarly but without the preconditioning ischemia. Fifteen min of ischemia was chosen to avoid any taurine release caused by ischemic cell death. Taurine was measured with HPLC. In the IPC group there was a postischemic release over baseline of 5.09 ± 1.51 μmol (approx 3.3% of the total taurine pool), whereas in the control group the release was not significantly different, 5.72 ± 1.67 μmol. The percent of the taurine pool lost from each heart during reperfusion was calculated based on an assumption of a total content of 20 μM taurine/gm wet weight. Since the amount of taurine released by the isolated rabbit heart following ischemia was not different in preconditioned and non-preconditioned hearts, we conclude that reduced swelling through taurine release is not the mechanism of the cardioprotective effects of IPC.