Autophagy Induced by Ischemic Preconditioning is Essential for Cardioprotection

Based on growing evidence linking autophagy to preconditioning, we tested the hypothesis that autophagy is necessary for cardioprotection conferred by ischemic preconditioning (IPC). We induced IPC with three cycles of 5 min regional ischemia alternating with 5 min reperfusion and assessed the induction of autophagy in mCherry-LC3 transgenic mice by imaging of fluorescent autophagosomes in cryosections. We found a rapid and significant increase in the number of autophagosomes in the risk zone of the preconditioned hearts. In Langendorff-perfused hearts subjected to an IPC protocol of 3 × 5 min ischemia, we also observed an increase in autophagy within 10 min, as assessed by Western blotting for p62 and cadaverine dye binding. To establish the role of autophagy in IPC cardioprotection, we inhibited autophagy with Tat-ATG5^K130R, a dominant negative mutation of the autophagy protein Atg5. Cardioprotection by IPC was reduced in rat hearts perfused with recombinant Tat-ATG5^K130R. To extend the potential significance of autophagy in cardioprotection, we also assessed three structurally unrelated cardioprotective agents—UTP, diazoxide, and ranolazine—for their ability to induce autophagy in HL-1 cells. We found that all three agents induced autophagy; inhibition of autophagy abolished their protective effect. Taken together, these findings establish autophagy as an end-effector in ischemic and pharmacologic preconditioning.     

Postkonditionierung: ein kurzer Überblick

Preconditioning is the most effective form of cardioprotection that can be induced via different interventions before a longer-lasting ischemia (= index ischemia). Preconditioning can be induced by short bouts of ischemia, several pharmaceuticals (e.g., adenosine), and volatile anesthetics. A brief ischemia of an organ other than the heart can likewise initiate protection of the heart, which has been called preconditioning at a distance or remote preconditioning. According to the more recent literature, short bouts of ischemia after an index ischemia can also initiate cardioprotection, e.g., improve postischemic dysfunction or reduce infarct size, which has been called postconditioning. Such a postconditioning can also be elicited at a distant organ, termed remote postconditioning. It is the aim of this short review to characterize preconditioning and in particular postconditioning, describe possible mechanisms, and call attention to the clinical relevance.     

HDL and its sphingosine-1-phosphate content in cardioprotection

Increasing evidence suggests that High-density lipoproteins (HDL) are a direct cardioprotective agent in the setting of acute myocardial ischemia/reperfusion injury, and that this cardioprotection occurs independently of their atheroprotective effect. Studies on the involved mechanisms have revealed that the biologically active HDL-compound sphingosine-1-phosphate (S1P) is responsible for the beneficial effect of HDL on the myocardium. There appears to be an intricate interplay between known preconditioning agents and components of the S1P synthesis machinery in the heart, which makes S1P signalling an attractive downstream convergence point of preconditioning and cardioprotection at the level of its G protein-coupled receptors. While local S1P production has been known to protect the heart against ischemia/reperfusion injury and to mediate preconditioning, systemic S1P supply via HDL adds a novel aspect to the regulation of cardioprotection. Thus the S1P-content of HDL may serve both as a potential cardiovascular risk marker and a novel therapeutic target. Strategies for short-term “acute” HDL elevation as well as S1P analogues may prove beneficial not only in the high-risk patient but also in any patient at risk of myocardial ischemia.     

Recycle or die: the role of autophagy in cardioprotection

Autophagy is a highly conserved cellular process responsible for the degradation of long-lived proteins and organelles. Autophagy occurs at low levels under normal conditions, but is upregulated in response to stress such as nutrient deprivation, hypoxia, mitochondrial dysfunction, and infection. Upregulation of autophagy may be beneficial to the cell by recycling of proteins to generate free amino acids and fatty acids needed to maintain energy production, by removing damaged organelles, and by preventing accumulation of protein aggregates. In contrast, there is evidence that enhanced autophagy can contribute to cell death, possibly through excessive self-digestion. In the heart, autophagy has an essential role for maintaining cellular homeostasis under normal conditions and increased autophagy can be seen in conditions of starvation, ischemia/reperfusion, and heart failure. However, the functional significance of autophagy in heart disease is unclear and controversial. Here, we review the literature and discuss the evidence that autophagy can have both beneficial and detrimental roles in the myocardium depending on the level of autophagy, and discuss potential mechanisms by which autophagy provides protection in cells.     

Innate immunity and cardiac preconditioning: a putative intrinsic cardioprotective program

Ischemic preconditioning is thought to evoke cell survival programs in the heart in large part via the activation of G(I)-protein coupled receptor signal transduction pathways. However, the identification and characterization of G(I)-protein coupled receptor independent pathways would enable researchers to pursue novel cellular events that could direct or promote preconditioning. In this regard recent work has begun to explore the role of the innate immune system in intrinsic cardioprotection against both viral myocarditis and ischemia. Interestingly, cytokines such as TNFalpha, IL-1beta and leukemia inhibitory factor, which are components of innate immunity, have been shown to mimic ischemic preconditioning. Thus as the innate immune system functions via a diverse array of G(I)-protein independent receptors, the study of this immunological system in the heart may provide new insight into mechanisms driving and promoting ischemic preconditioning. We propose that innate immunity is indeed an integral part of ischemic preconditioning. In this review, we provide an overview of the innate immune system, describe the studies whereby cytokines mimic ischemic preconditioning and finally postulate some mechanisms whereby innate immunity may promote cardioprotection as a component of preconditioning.     

Ischemic preconditioning: emerging evidence, controversy, and translational trials

Protection against ischemia by ischemic preconditioning (IP) is seen in many tissues and organs. However, the preconditioning ischemia must precede lethal ischemia for this effect to occur, and the creation of ischemia to treat heart disease does not seem to be a realistic strategy. Accordingly, the underlying mechanisms that confer cardioprotection should be identified. Early studies revealed that IP causes two windows of cardioprotection, and subsequent efforts to detect cardioprotective factors have identified various triggers, mediators, and potent effectors of IP, such as endogenous receptor agonists (adenosine, catecholamines, bradykinin, and opioids), intracellular messengers [protein kinase C (PKC), p38MAPK, PI-3K, and PKA], ion channels such as KATP channels, enzymes including heat shock proteins (HSPs), superoxide dismutase (SOD), and 5'-nucleotidase, and other factors [nitric oxide (NO), growth factors, free radicals, and products of the arachidonic acid cascade]. Some of these factors are involved in several different pathways and may have multiple roles in IP-induced cardioprotection. Recently, however, certain problems have arisen such as controversies related to increasing knowledge and the relative lack of clinical studies in contrast to the intensive performance of basic studies. To overcome these problems, the latest studies have followed three major trends: (1) investigation of mechanisms to explain the current controversies, (2) detection of other unknown potent mechanisms, and (3) promotion of clinical trials based on the evidence from experimental studies in larger animals. Here, we summarize recent investigations on IP, emphasizing on the controversial issues and emerging factors, and discuss current research on the prevention or treatment of ischemic heart disease including some relevant clinical studies.     

Adenosine and cardioprotection in the diseased heart.

Biological and mechanical stressors such as ischemia, hypoxia, cellular ATP depletion, Ca2+ overload, free radicals, pressure and volume overload, catecholamines, cytokines, and renin-angiotensin may independently cause reversible and/or irreversible cardiac dysfunction. As a defense against these forms of stress, several endogenous self-protective mechanisms are exerted to avoid cellular injury. Adenosine, a degradative substance of ATP, may act as an endogenous cardioprotective substance in pathophysiological conditions of the heart, such as myocardial ischemia and chronic heart failure. For example, when brief periods of myocardial ischemia precede sustained ischemia, infarct size is markedly limited, a phenomenon known as ischemic preconditioning. We found that ischemic preconditioning activates the enzyme responsible for adenosine release, ie, ecto-5'-nucleotidase. Furthermore, the inhibitor of ecto-5'-nucleotidase reduced the infarct size-limiting effect of ischemic preconditioning, which establishes the cause-effect relationship between activation of ecto-5'-nucleotidase and the infarct size-limiting effect. We also found that protein kinase C is responsible for the activation of ecto-5'-nucleotidase. Protein kinase C phosphorylated the serine and threonine residues of ecto-5'-nucleotidase. Therefore, we suggest that adenosine produced via ecto-5'-nucleotidase gives cardioprotection against ischemia and reperfusion injury. Also, we found that plasma adenosine levels are increased in patients with chronic heart failure. Ecto-5'-nucleotidase activity increased in the blood and the myocardium in patients with chronic heart failure, which may explain the increases in adenosine levels in the plasma and the myocardium. In addition, we found that further elevation of plasma adenosine levels due to either dipyridamole or dilazep reduces the severity of chronic heart failure. Thus, we suggest that endogenous adenosine is also beneficial in chronic heart failure. We propose potential mechanisms for cardioprotection attributable to adenosine in pathophysiological states in heart diseases. The establishment of adenosine therapy may be useful for the treatment of either ischemic heart diseases or chronic heart failure.     

远隔器官缺血预处理心脏保护作用的研究进展

心肌缺血预处理现象的发现,为心肌缺血再灌注损伤的预防开拓了一个新的研究领域。近年来,随着预处理研究的不断深入,其方法学也有一些新的进展。研究发现,心外组织如肾脏、小肠及骨骼肌短暂缺血不仅能减轻局部组织的再灌注损伤,对远隔的心脏也有保护作用,并将这种现象称为远隔预处理。现就远隔器官缺血预处理对心脏的保护作用及其机制作一简要综述。     

Protection in the aged heart: preventing the heart-break of old age?

The aged heart has a diminished functional and adaptive reserve capacity, an increased susceptibility to incur damage (e.g., as a result of ischemia), and a limited practical ability for repair/regeneration. Thus, there has been considerable interest to harness the heart's endogenous capacity to resist such damage, known as ischemic preconditioning (IPC), as well as other cardioprotective mechanisms. However, the translation of basic research findings into clinical practice has largely been inadequate because there have been few if any successful implementations in terms of viable therapies activating cardioprotection mechanisms to limit infarct size. Here, we provide an overview of the general mechanisms of cardioprotection, changes in the structure and function of the aged heart, and the current knowledge regarding cardioprotection in aged heart. The problems and opportunities for successful bench-to-bedside translation of cardioprotection in the elderly are discussed.     

Role of energy metabolism in the preconditioned heart--a possible contribution of mitochondria.

A brief period of ischemia and reperfusion has been shown to protect the myocardium against subsequent sustained ischemia and reperfusion injury, which is called "preconditioning". A great number of investigators have explored the mechanisms underlying this preconditioning-induced cardioprotection. This article dealt with possible mechanisms of energy metabolism and mitochondrial activity for preconditioning-induced cardioprotection. Particularly, the contribution of energy metabolites produced during a brief period of ischemia and reperfusion injury, as well as mitochondrial function that is modified by changes in mitochondrial ATPase activity, opening of mitochondrial ATP-dependent potassium channels and production of free radicals in mitochondria, to ischemic preconditioning is discussed.     

Exploring the Role of TRPV and CGRP in Adenosine Preconditioning and Remote Hind Limb Preconditioning-Induced Cardioprotection in Rats

The cardioprotective effects of remote hind limb preconditioning (RIPC) are well known, but mechanisms by which protection occurs still remain to be explored. Therefore, the present study was designed to investigate the role of TRPV and CGRP in adenosine and remote preconditioning-induced cardioprotection, using sumatriptan, a CGRP release inhibitor and ruthenium red, a TRPV inhibitor, in rats. For remote preconditioning, a pressure cuff was tied around the hind limb of the rat and was inflated with air up to 150 mmHg to produce ischemia in the hind limb and during reperfusion pressure was released. Four cycles of ischemia and reperfusion, each consisting of 5 min of inflation and 5 min of deflation of pressure cuff were used to produce remote limb preconditioning. An ex vivo Langendorff’s isolated rat heart model was used to induce ischemia reperfusion injury by 30 min of global ischemia followed by 120 min of reperfusion. RIPC demonstrated a significant decrease in ischemia reperfusion-induced significant myocardial injury in terms of increase in LDH, CK, infarct size and decrease in LVDP, +dp/dt_max and -dp/dt_min. Moreover, pharmacological preconditioning with adenosine produced cardioprotective effects in a similar manner to RIPC. Pretreatment with sumatriptan, a CGRP release blocker, abolished RIPC and adenosine preconditioning-induced cardioprotective effects. Administration of ruthenium red, a TRPV inhibitor, also abolished adenosine preconditioning-induced cardioprotection. It may be proposed that the cardioprotective effects of adenosine and remote preconditioning are possibly mediated through activation of a TRPV channels and consequent, release of CGRP.     

Metabolic plasticity and the promotion of cardiac protection in ischemia and ischemic preconditioning

The concept of metabolic protection of the ischemic myocardium is in constant evolution and has recently been supported by clinical studies. Historically, enhanced glucose metabolism and glycolysis were proposed as anti-ischemic cardioprotection. This hypothesis is supported by the sub-cellular linkage between key glycolytic enzymes and the activity of two survival-promoting membrane-bound pumps, namely the sodium-potassium ATPase, and the calcium uptake pump of the sarcoplasmic reticulum. Moreover, improved resistance against ischemia follows the administration of glucose-insulin-potassium in a variety of animal models and in patients following acute myocardial infarction. The metabolic plasticity paradigm has now been expanded to include (1) the benefit of improved coupling of glycolysis to glucose oxidation, which explains the action of anti-ischemic fatty acid inhibitors such as trimetazidine and ranolazine; (2) the role of malonyl CoA in the glucose-fatty acid interaction; and (3) the anti-apoptotic role of insulin. Furthermore, we argue for a protective role of increased glucose uptake in the preconditioning paradigm. Additionally, we postulate an adaptive role of mitochondrial respiration in the promotion of cardioprotection in the context of ischemic preconditioning. The mechanisms driving these mitochondrial perturbations are still unknown, but are hypothesized to involve an initial modest uncoupling of respiration from the production of mitochondrial ATP. These perturbations are in turn thought to prime the mitochondria to augment mitochondrial respiration during a subsequent ischemic insult to the heart. In this review we discuss studies that demonstrate how metabolic plasticity can promote cardioprotection against ischemia and reperfusion injury and highlight areas that require further characterization.     

It is time to ask what adenosine can do for cardioprotection

Prevention and attenuation of ischemia and reperfusion injury in patients with acute coronary syndrome are critically important for cardiologists. To save these patients from deleterious ischemic insults, there are three different strategies. The first strategy is to increase ischemic tolerance before the onset of myocardial ischemia; the second is to attenuate the ischemia and reperfusion injury when an irreversible process of myocardial cellular injury occurs; the third is to treat the ischemic chronic heart failure that is caused by acute myocardial infarction. Adenosine, which is known to be cardioprotective against ischemia and reperfusion injury, may merit being used for these three cardioprotection strategies. First of all, adenosine induces collateral circulation via induction of growth factors, and triggers ischemic preconditioning, both of which induce ischemic tolerance in advance. Secondly, endogenous adenosine may mediate the infarct size-limiting effect of ischemic preconditioning, and exogenous adenosine is known to attenuate ischemia and reperfusion injury. Thirdly, we also revealed that adenosine metabolism is changed in patients with chronic heart failure, and increases in adenosine levels may attenuate the severity of ischemic heart failure. Therefore, adenosine therapy may improve the pathophysiology of ischemic chronic heart failure. Taking these factors together, we hereby propose potential tools for cardioprotection attributable to adenosine in ischemic hearts, and we postulate the use of adenosine therapy before, during, and after the onset of acute myocardial infarction.     

Duration and reinstatement of myocardial protection against infarction by ischemic preconditioning in open chest dogs

These studies were undertaken to determine the duration of protection against myocardial infarction provided by ischemic preconditioning in the canine heart, and to learn if cardioprotection can be restored by another preconditioning stimulus when the initial effect is lost. Control and four preconditioning groups of anesthetized, open-chest dogs were compared. All underwent a test 60 min episode of ischemia, induced by occlusion of the anterior descending (LAD) artery, followed by 3 h of reperfusion. Preconditioning was induced by one 10 min LAD occlusion, followed by either 10 min, 2, 3, or 5 h of reperfusion. In order to test whether preconditioning could be reinstated, another group of dogs with preconditioning plus 3 h reperfusion underwent a second 10 min preconditioning stimulus with 10 min reperfusion before the 60 min test-occlusion. Infarct size (as percent of area-at-risk) was analyzed (using analysis of covariance) with respect to coronary collateral blood flow measured with radioactive microspheres. Infarct size was limited markedly by preconditioning (23+/-6 v 6+/-2%, P<0.05) but the protective effect was dissipated partially after 2 h reperfusion and was dissipated completely after 3 h reperfusion (20+/-4%, non-significant v Control and significant P<0.05 v preconditioning). Protection was restored in three of six dogs with preconditioning +5 h reperfusion, suggesting that the second window of protection appears early in some canine hearts. When preconditioning was repeated after 3 h reperfusion, cardioprotection was reinstated fully (7+/-2%, P<0.05 v Control and NS v preconditioning). The results show that maximal preconditioning cardioprotection is present in the dog heart after 10 min of reperfusion and is dissipated totally following 3 h of reperfusion. However, a second preconditioning stimulus of 10 min of ischemia followed by 10 min of reperfusion to the dissipated preconditioned heart reinstates full preconditioning. Thus, this model provides a system to test for theoretical causes of the preconditioned state. Final mediators should be present when preconditioning is present and absent when preconditioning is dissipated. It is noteworthy that a second window of protection appeared in 50% of dogs when the period of reperfusion was extended to 5 h.     

Cardioprotection from ischemia/reperfusion injury: basic and translational research

Because ischemic heart diseases (IHDs) are a major cause of mortality and heart failure, novel therapeutic approaches are expected to improve the clinical outcomes of patients with IHDs such as acute myocardial infarction and ischemic heart failure. Brief episodes of nonlethal ischemia and reperfusion before sustained ischemia or at the onset of reperfusion can reduce ischemia-reperfusion injury. These ischemic conditioning phenomena are termed "ischemic preconditioning" and "ischemic postconditioning", respectively. Furthermore, brief episodes of nonlethal ischemia and reperfusion applied to the organ or tissue distal to the heart reduce myocardial infarct size, known as "remote ischemic conditioning". The cardioprotection afforded by these ischemic conditionings can be used to treat patients with acute myocardial infarction or cardiac operations. Extensive research has determined that autacoids (eg, adenosine, bradykinin opioid) and cytokines, their respective receptors, kinase signaling pathways and mitochondrial modulation are involved in ischemic conditioning. Modification of these factors by pharmacological agents mimics the cardioprotection by ischemic conditioning and provides a novel therapeutic intervention for IHDs. Here, the potential mechanisms of ischemic conditioning and its "proof-of-concept" translational studies are reviewed. In the near future, large, multicenter, randomized, placebo-controlled, clinical trials will be required to determine whether pharmacological and ischemic conditioning can improve the clinical outcomes of patients with IHDs.     

Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy

Autophagy is an intracellular bulk degradation process for proteins and organelles. In the heart, autophagy is stimulated by myocardial ischemia. However, the causative role of autophagy in the survival of cardiac myocytes and the underlying signaling mechanisms are poorly understood. Glucose deprivation (GD), which mimics myocardial ischemia, induces autophagy in cultured cardiac myocytes. Survival of cardiac myocytes was decreased by 3-methyladenine, an inhibitor of autophagy, suggesting that autophagy is protective against GD in cardiac myocytes. GD-induced autophagy coincided with activation of AMP-activated protein kinase (AMPK) and inactivation of mTOR (mammalian target of rapamycin). Inhibition of AMPK by adenine 9-beta-d-arabinofuranoside or dominant negative AMPK significantly reduced GD-induced autophagy, whereas stimulation of autophagy by rapamycin failed to cause an additive effect on GD-induced autophagy, suggesting that activation of AMPK and inhibition of mTOR mediate GD-induced autophagy. Autophagy was also induced by ischemia and further enhanced by reperfusion in the mouse heart, in vivo. Autophagy resulting from ischemia was accompanied by activation of AMPK and was inhibited by dominant negative AMPK. In contrast, autophagy during reperfusion was accompanied by upregulation of Beclin 1 but not by activation of AMPK. Induction of autophagy and cardiac injury during the reperfusion phase was significantly attenuated in beclin 1(+/-) mice. These results suggest that, in the heart, ischemia stimulates autophagy through an AMPK-dependent mechanism, whereas ischemia/reperfusion stimulates autophagy through a Beclin 1-dependent but AMPK-independent mechanism. Furthermore, autophagy plays distinct roles during ischemia and reperfusion: autophagy may be protective during ischemia, whereas it may be detrimental during reperfusion.     

Autophagy: an affair of the heart

Whether an element of routine housekeeping or in the setting of imminent disaster, it is a good idea to get one’s affairs in order. Autophagy, the process of recycling organelles and protein aggregates, is a basal homeostatic process and an evolutionarily conserved response to starvation and other forms of metabolic stress. Our understanding of the role of autophagy in the heart is changing rapidly as new information becomes available. This review examines the role of autophagy in the heart in the setting of cardioprotection, hypertrophy, and heart failure. Contradictory findings are reconciled in light of recent developments. The preponderance of evidence favors a beneficial role for autophagy in the heart under most conditions.     

Delayed preconditioning of the human myocardium: signal transduction and clinical implications

OBJECTIVE: Ischemic preconditioning confers cardioprotection in early and delayed phases. We investigated the delayed window of pharmacological and ischemic preconditioning in human myocardium, and the involvement of mitoKATP, PKC and p38MAPK. METHODS: These studies were carried out using human right atrial tissue in a cell necrosis model. The tissue was obtained from patients undergoing coronary artery surgery. RESULTS: The second window triggered by ischemia, phenylephrine or adenosine resulted in similar cardioprotection between 24 and 72 h following the intervention. Atrial tissue taken from patients with a single episode of angina between 24 and 72 h prior to surgery were already protected and preconditioning with ischemia, phenylephrine or adenosine did not add to the protection. The trigger of preconditioning with mitoKATP channel opener diazoxide, PKC activator PMA and p38MAPK activator anisomycin produced similar delayed protection to that of ischemia or phenylephrine. Cardioprotection was lost when mitoKATP channels were blocked by 5HD, PKC by chelerythrine and p38MAPK by SB203580 24 h after the trigger of preconditioning. CONCLUSIONS: Ischemic and pharmacological preconditioning induce similar delayed cardioprotection of the human heart. This second window of protection that is seen between 24 and 72 h occurs in vitro and in vivo and requires opening of mitoKATP channels and activation of PKC and p38MAPK.     

Adenosine Transport Blockade Restores Attenuated Cardioprotective Effects of Adenosine Preconditioning in the Isolated Diabetic Rat Heart: Potential Crosstalk with Opioid Receptors

Considering the reduced ability of cardiac fibroblasts to release adenosine and increased ability of interstitial adenosine uptake during diabetes mellitus, the present study investigated the effect of adenosine preconditioning and the existence of cross-talk with opioid receptor activation in the diabetic rat heart subjected to ischemia–reperfusion (I/R). Langendorff-perfused normal and streptozotocin (65 mg/kg, i.p., once)-administered diabetic (after 8-weeks) rat hearts were subjected to 30-min global ischemia and 120-min reperfusion. Myocardial infarct size using triphenyltetrazolium chloride staining, markers of cardiac injury such as lactate dehydrogenase (LDH) and creatine kinase (CK-MB) release, coronary flow rate (CFR) and myocardial oxidative stress were assessed. The diabetic rat heart showed high degree of I/R injury with increased LDH and CK-MB release, high oxidative stress and reduced CFR as compared to the normal rat heart. The adenosine preconditioning (10 μM) afforded cardioprotection against I/R injury in the normal rat heart that was prevented by naloxone (100 μM) pre-treatment. Conversely, adenosine preconditioning-induced cardioprotection was abolished in the diabetic rat heart. However, co-administration of dipyridamole (100 μM), adenosine reuptake inhibitor, markedly restored the cardioprotective effect of adenosine preconditioning in the diabetic rat heart, and this effect was also abolished by naloxone pre-treatment. The reduced myocardial availability of extracellular adenosine might explain the inability of adenosine preconditioning to protect the diabetic myocardium. The pharmacological elevation of extracellular adenosine restores adenosine preconditioning-mediated cardioprotection in the diabetic myocardium by possibly involving opioid receptor activation.     

老年心肌缺血预适应延迟保护作用钝化机制的研究进展

缺血预适应是心肌抗缺血性损伤的最有效的内源性保护机制,但是老年人缺血预适应的延迟保护作用却明显削弱甚至消失,原因和产生心肌缺血预适应延迟保护作用环节的功能下降有关,其中线粒体功能的退化和障碍最为重要。