Advances in molecular mechanism of myocardial ischemia/reperfusion injury

WEI Cheng-lu, FENG Qing-min, CHEN Song-cheng, ZHUO Huai-mi, LI Ji-ke

Hainan Medical University, Haikou 571199, China

Keywords:Ischemia/reperfusion injury Myocardial Molecular mechanism Signal transduction pathway

ABSTRACT Myocardial ischemia/reperfusion (I/R) injury is a pathological change that occurs during the restoration of blood supply to ischemic or occluded coronary arteries after coronary heart disease, stroke, cardiac arrest and resuscitation, organ transplantation, shock and other events.Myocardial I/R injury is often accompanied by cardiovascular adverse events, which seriously affect the prognosis of myocardial ischemia. The potential mechanism of myocardial I/R injury is complex, involving many pathological processes, such as oxygen free radical injury,calcium overload, inflammation, apoptosis, activation, immune imbalance, endoplasmic reticulum stress, autophagy, myocardial energy metabolism disorder, myocardial microvascular endothelial cell injury and so on. Ischemic preconditioning is an effective preventive and therapeutic measure to reduce ischemia-reperfusion (I/R) injury. At present, there are many experimental studies on the pathogenesis and prevention and treatment measures, but the clinical application is still limited, so the prevention and treatment of I/R injury is still a major challenge. The pathogenesis of myocardial I/R injury is not completely clear, and the treatment methods and drugs are limited, so this paper summarizes the molecular mechanism and related signal pathways involved in its injury, as well as the emerging targeted therapy, to provide strategy and theoretical basis for clinical prevention and treatment of myocardial I/R injury.

1. Introduction

Myocardial ischemia-reperfusion injury (MIRI) refers to the phenomenon that the cardiac function is not improved but aggravated immediately after the ischemic myocardium resumes blood perfusion[1]. Myocardial ischemia caused by coronary artery occlusion is characterized by persistent and severe retrosternal pain, resulting in myocardial infarction, shock, arrhythmia or heart failure. Early restoration of blood flow in ischemic area is the most commonly used treatment strategy, such as coronary angioplasty,percutaneous coronary intervention and coronary artery bypass grafting, which can restore myocardial oxygen and nutrition supply,save ischemic myocardium and save patients' lives. Jones et al.[2]found that MIRI is not only a key factor leading to cardiomyocyte apoptosis, but also an important factor affecting the prognosis of patients. Some patients inevitably suffer from reperfusion injury,which is characterized by persistent cardiomyocyte death, further deterioration of cardiac function, and low long-term survival rate,which seriously limits the clinical efficacy and prognosis. Reducing the injury of cardiomyocytes caused by ischemia-reperfusion (I/R) is an important measure to improve the therapeutic effect and prognosis of patients with myocardial ischemia. Since Jennings first reported MIRI, MIRI has been the focus of cardiovascular disease research[3]. Despite the achievements of new treatments(thrombolysis, percutaneous coronary revascularization,percutaneous coronary intervention, bypass, etc.), there is still no way to completely prevent the additional damage caused by reperfusion itself. At present, the drug treatment measures for clinical reperfusion injury have not been effective. Therefore, it is of great significance to understand the potential molecular mechanism of MIRI and develop new therapeutic strategies for the prevention and treatment of cardiac I/R injury.

2. Injury mechanism and target blocker

2.1 Excessive inflammatory response

Effective inflammation is necessary for the host to resist injury and tissue repair. However, excessive or chronic myocardial inflammation is reported to cause severe damage to the myocardium and is associated with many heart diseases, such as myocarditis,myocardial infarction, I/R injury, heart failure, aortic valve disease,atherosclerosis and hypertension. Reperfusion is inevitably accompanied by a special aseptic inflammation, which has been widely studied as the main cause of further myocardial injury and dysfunction[4]. The early stage of I/R is mainly acute inflammatory reaction. More and more evidence shows that I/R injury produces a large number of inflammatory mediators and chemokines, which activate leukocytes, platelets and vascular endothelial cells to express a large number of adhesion molecules, such as selectin and integrin, promote leukocyte adhesion to vascular endothelial cells and leukocyte aggregation in blood vessels. At the same time,activated neutrophils can secrete cytokines,such as tumor necrosis factor-α, interleukin-1 β and interleukin-6[5]. These cytokines play an important role in cell injury, such as inducing apoptosis, and then the inflammatory response continues to expand, causing further damage to cardiomyocytes. Inflammatory reaction exposes vascular endothelial cell adhesion molecules, increases the infiltration of inflammatory cells to vascular endothelial cells, further aggregates neutrophils and platelets and adheres to the surface of vascular endothelium, aggravates vascular endothelial injury, and results in no reflow after reperfusion,lead to tissue edema, vascular edema,promote microcirculation disturbance, and further aggravate myocardial injury[6]. This may lead to metabolic dysfunction of cardiomyocytes, degeneration and necrosis of cardiomyocytes and oxidative stress response.

2.1.1 p38 MAPK signal pathway

Mitogen-activated protein kinases (MAPK) are composed of extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-jun N-terminal kinases (JNK) and p38[7]. Studies have shown that reactive oxygen species produced by I/R injury activate MAPK, to cause cardiomyocyte apoptosis and necrosis, activate neutrophils,increase the expression of cytokines and adhesion molecules, and phosphorylate cytoplasmic proteins and reverse transcription factors,thus aggravating MIRI. Activation of ERK1/ERK2 and inhibition of p38/JNK protect myocardium from myocardial injury by reducing oxidative stress and inflammation and maintaining cytoskeletal structure[8].

Isoflurane can effectively restore the cardiac function of MIRI rats, improve the pathological changes of cardiomyocytes, reduce the inflammatory reaction, and reduce the degree of myocardial infarction and ischemia by inhibiting p38MAPK signal pathway[9].Prazosin can down-regulate the activities and expression levels of NF-AT, AP-1 and NF-κB in cardiomyocytes by stimulating the expression and activity of ERK, and reduce the inflammation and oxidative stress of cardiomyocytes injured by I/R[10].

2.1.2 PI3K-Akt-mTOR signal pathway

Phosphatidylinositol 3-kinase (PI3K) / protein kinase B (Akt) /rapamycin target protein (MTOR) signal pathway is an important membrane receptor pathway. MTOR is located in the downstream of PI3K/AKT pathway and is positively regulated by PI3K/Akt pathway to alleviate MIRI. During myocardial ischemia-reperfusion, the activation of PI3K/Akt pathway can phosphorylate mTOR,mTOR as a key regulator of autophagy. Phosphorylated mTOR has been reported to protect I/R injury by reducing autophagy and promoting cardiac recovery[11].

Hesperidin attenuates myocardial I/R injury by activating PI3K/Akt/mTOR pathway to inhibit autophagy, inhibit inflammation and oxidative stress[12], and recombinant human brain natriuretic peptide(rhBNP) attenuates myocardial I/R injury by promoting PI3K/AKT/mTOR phosphorylation, inhibiting JurkatT cell proliferation,inhibiting the expression of pro-inflammatory related genes, and alleviating I/R injury[13].

2.1.3 TLR4/NF- κ B/NLRP3 signal pathway

Toll-like receptor (TLRs) is the receptor of exogenous pathogens,which initiates inflammation through immune cells. Activated B nuclear factor kappa light chain enhancer (NF- κB) is a nuclear transcription factor, which normally binds to the inhibitory protein(IkB) and is sensitive to redox response. It is the common hub of most regulatory pathways, such as p38, trak, IKK, Pi, and so on.The activation of TLR4 promotes the increase of NF-kB, and NFkB regulates the expression of proinflammatory cytokines. NLRP3 inflammatory body is a member of the (NLR) family of Nodlike receptors, which consists of a nuclear side-bound oligomeric domain-like receptor (NLRP3), a caspase recruitment domain (ASC)and caspase-1. NLRP3 protein is polymerized and bound to the ASC junction to induce caspase-1 translocation and activation. In addition,the activated caspase-1 is responsible for triggering the secretion of mature forms of proinflammatory cytokines. TLR4/NF-kB signaling pathway mediates inflammatory response and the pathogenesis of MIRI by regulating pro-inflammatory cytokines[14].

Biochanin A inhibits inflammation by negatively regulating TLR4/NF- κB/NLRP3 signal pathway, thus alleviating MIRI [6].

2.1.4 AMPK/JNK/NF- κ B signal pathway

Adenylate-activated protein kinase (AMPK) is activated by upstream kinases such as liver kinase B1 (LKB1) through phosphorylated Tr172 residues. AMPK responds to an increase in AMP/ATP ratio by shutting down energy consumption pathways and stimulating ATP production pathways, such as fatty acid β oxidation and glycolysis[15]. P38MAPK, ERK and JNK, in MAPK are considered to be the upstream factors of NF-κB. Under hypoxia and reoxygenation stress, the activation of AMPK significantly weakens the JNK-NF- κB signal transduction pathway, suppresses the gene and protein levels of proinflammatory cytokines[16], and protects cardiomyocytes from injury during ischemia and reperfusion.

Metformin, an AMPK agonist, decreased JNK activation and downstream NF- κB activation and inflammation by up-regulating TNF α and IL-6 mRNA[16].

2.1.5 TLR4/TRIF signal pathway

The signal transduced by TLR4, which contains leucine-rich repeat(LRR) domain and Toll/IL-1 receptor (TIR) domain, plays a key role in inducing inflammation during MIRI. TLR4 binds to its chaperone molecule, coreceptor and junction protein and is transported to the cis-Golgi matrix in the vesicles coated by the coat protein complex II. TLR4 is then output to the plasma membrane, where it reacts to its ligands and triggers a series of inflammatory cascades[17].Activation of TLR4 by lipopolysaccharide (LPS) induces two signaling pathways: myeloid differentiation factor 88 (MyD88)-dependent pathway and MyD88-independent pathway. TLR4/MYD88 and TLR4/TRIF activate the transcriptional activity of NF- kappa B and interferon regulatory factor 3 (IRF3) respectively,induce a series of inflammatory factors and aggravate MIRI[18].

The synergistic protective effect of remote ischemic preconditioning and sevoflurane preconditioning on rat MIRI is achieved by inhibiting the TLR4/MyD88/NF- κB signal pathway[19].

2.1.6 Toll-like receptor 9 signal pathway

Mitochondrial DNA released from necrotic cardiomyocytes can activate TLR9, mitochondrial DNA and H/R synergistically induce NF- kappa B activity through TLR9-dependent mechanism. Ablation of myocardial TLR9 signaling pathway can reduce inflammatory response and myocardial I/R injury, but it has also been reported that TLR9 stimulation reduces energy substrate, increases AMP/ATP ratio, and then activates AMP-dependent kinase, thus improving cardiomyocyte tolerance to hypoxia. Without causing a typical inflammatory response[20].

The combination of DNaseI and mitochondrial targeted endonuclease III can maintain the integrity of mitochondria in ischemic cardiomyocytes and reduce the activation of TLR9, which has an additional protective effect on myocardial I/R injury [21].

2.1.7 A20/NF-κ B signal pathway

Zinc finger protein A20, also known as tumor necrosis factor alpha inducible protein 3 (TNFAIP3), is an anti-inflammatory, NF- κB inhibitory and anti-apoptotic molecule[22]. A20 is considered to be the key link of inflammation throughout the whole pathological process of myocardial ischemia / reperfusion injury[23]. A20 is the center of NF- kappa B, which can induce negative regulatory factors and regulate a variety of inflammatory signal transduction pathways.Silencing A20 can significantly activate IKK-β, promote NF- κ Bp65 translocation, IκB-α phosphorylation,Pmns infiltration,and overexpression of intercellular adhesion molecule-1 (ICAM-1),vascular cell adhesion molecule-1 (VCAM-1) and inducible nitric oxide synthase (iNOS) in ventricular myocytes, resulting in proinflammatory state[24].

Ginkgolide B(GB) can increase the expression of A20, improve the ultrastructural characteristics of the heart of MIRI rats, reduce the content of serum inflammatory cytokines and alleviate the inflammatory response induced by MIRI through A20 murine NFκB signal pathway[24].

2.1.8 PI3K/Akt/HO-1 signal pathway

Heme oxygenase (HO-1) is an important antioxidant stress and tissue protective enzyme. When cardiomyocytes are in a state of oxidative stress, nuclear factor E2-related factor 2 (Nrf2) is phosphorylated, dissociated, activated and transferred to the nucleus by Akt, where it binds to the antioxidant stress response element(ARE) and promotes the expression of antioxidant proteins such as HO-1 and SOD to combat oxidative stress induced by ischemia and hypoxia [25]. The gene regulation of HO-1 is also negatively regulated by some Nrf2 suppressors, such as Bach1, and the deletion of Bach1 gene will lead to the increase of HO-1 expression. PI3K is an intracellular phosphatidylinositol kinase and the second messenger located on the plasma membrane. Sun et al pointed out that inhibition of PI3K/Akt pathway can significantly reduce the expression of HO-1 protein, thus weakening the protective effect of HO-1 on cardiomyocytes[26], that is, PI3K/Akt up-regulates Nrf2-ARE pathway and mediates the expression of HO-1.

Ginkgo biloba extract-761 (EGb761) can induce Akt phosphorylation to a certain extent, activate Akt, to promote the transfer of Nrf2 into the nucleus, up-regulate the expression of HO-1,reduce oxidative stress and inflammation, inhibit cardiomyocyte apoptosis, and protect myocardium[27].

2.1.9 NLRP3 inflammatory body signaling pathway

The formation of reactive oxygen species and oxidative stress have been proved to be important promoters of inflammatory body activation[28].NOX4, the source of cellular superoxide anions, has been shown to mediate the activation of NLRP3 inflammatory bodies by regulating carnitine palmitoyltransferase 1A (CPT1A),a key enzyme involved in fatty acid oxidation[29].The activation of caspase-1, by NLRP3 leads to the processing and secretion of proinflammatory cytokines IL-1β and IL-18, and aggravates MIRI.Ethyl pyruvate (EP) could inhibit the activation of NLRP3 inflammatory bodies and significantly reduce myocardial I/R injury[30].

2.1.10 Notch1/PTEN/AKT signal pathway

MicroRNA-21 (miR-21) is a highly specific miRNA in the heart.MIRI significantly decreased the expression of miR-21, and the overexpression of miR-21 effectively inhibited cardiomyocyte apoptosis and the release of inflammatory factors[31]. Notch pathway controls the expression of miR-21, and myocardial hypoxiareperfusion reduces the expression of Notch1 protein[32],thus reducing the expression of miR-21. PTEN on chromosome 10 with homologous deletion of phosphatase and tensin is a tumor suppressor gene. PTEN can be negatively regulated by Notch1 signal, which is the upstream and negative regulator of AKT pathway[13]. PTEN/Akt signaling pathway plays an important role in myocardial remodeling,myocardial hypertrophy, myocardial fibrosis and MIRI[33].

Kaempferol pretreatment can significantly increase the expression of miR-21 in the H/R process of H9c2 cells, and promote the Notch1/PTEN/Akt signaling pathway in a miR-21-dependent manner, that is, promote the expression of Notch1 and inhibit the expression of PTEN. Enhance the phosphorylation of Akt, thereby reducing the H/R damage of H9c2 cells [32].

2.2 Excessive free radical damage

In many diseases characterized by ischemia, hypoxia-induced oxidative stress leads to irreversible damage. Oxidative stress can cause cell membrane rupture, swelling or death through cell homeostasis, mitosis, cell differentiation and intracellular signal transduction. Serum malondialdehyde (MDA), superoxide dismutase(SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) levels can reflect the state of myocardial oxidative stress, I/R The content of serum MDA increased significantly, and the content of SOD, CAT and GSH-Px decreased significantly.

In cardiomyocytes, reactive oxygen species (ROS) are mainly produced by two ways: the first way is to consume most of the oxygen through the mitochondrial respiratory chain of cardiomyocytes, produce mitochondrial ROS, and release a large amount of ATP for cell life activities; The two ways are to generate ROS through enzymatic reactions in cells[34]. ROS includes superoxide anion (O2·-), hydroxyl radical (·OH), peroxynitrite(ONOODNA) and hydrogen peroxide (H2O2). Under normal circumstances, cells use transient increases in intracellular ROS levels as a mechanism to activate growth and proliferation. The increase of free radical production and the decrease of antioxidant enzyme activity are closely related to myocardial I/R damage. After myocardial ischemia, a small amount of oxygen free radicals can be observed in myocardial tissue, and the rapid increase in the number of oxygen free radicals occurs a few seconds to 1 minute after reperfusion. Activated neutrophils, cardiomyocytes and vascular endothelial cells can all produce and release oxygen free radicals.The lack of ATP secondary to hypoxia-ischemia and the subsequent increase in anaerobic metabolism lead to a decrease in cell pH and intracellular calcium overload. At the beginning of reperfusion, the rapid restoration of intracellular pH and oxygen leads to increased mitochondrial ROS production, which leads to the opening of mitochondrial permeability transition pores through oxidation, DNA damage and lipid peroxidation, which lead to gene mutation and cell death[35]. Oxidative stress caused by excessive oxygen free radicals is a key factor of reperfusion injury.

Allopurinol, edaravone, VitE, SOD, amifostine, metformin,carvedilol, etc. can reduce free radicals and resist lipid peroxidation.

2.3 Calcium overload

In the MIRI process, in the absence of oxygen, cell metabolism is converted to anaerobic glycolysis, leading to the accumulation of lactic acid and cytoplasmic acidification. Low pH and abnormal ATPdependent pump/exchange ion activity lead to the net accumulation of calcium ions in cardiomyocytes and the production of reactive oxygen species. Oxygen free radicals cause increased permeability of the cardiomyocyte membrane and excessive extracellular calcium influx, excessive cells Internal calcium will enter the mitochondria, causing mitochondrial calcium overload, which will inhibit the production of ATP, hinder the signal transmission in the cell, aggravate the energy metabolism disorder, and aggravate the myocardial reperfusion[36]. Oxygen free radicals can also damage the sarcoplasmic reticulum, and ultimately increase intracellular calcium levels, further aggravating calcium overload. Intracellular calcium can also activate some phospholipases, mainly protein kinase C and phospholipase A, destroy the cell membrane skeleton,and at the same time promote excessive contraction of myocardial fibers, forming a transient inward current through the exchange of Na+/Ca2+, and forming a delay after the myocardial action potential After depolarization, it causes arrhythmia. In addition, this reaction will also produce some toxic substances, such as free fatty acids,leukotrienes, prostaglandins and oxygen free radicals[37]. Myocardial calcium overload can also cause changes in the structure and function of coronary blood vessels and microvascular endothelial cells. It can cause the adhesion, aggregation and infiltration of neutrophils, release a series of inflammatory factors, and further damage the cardiovascular tissues. Disorders of energy metabolism caused by calcium overload can also cause myocardial spasm and cause pathological changes. Blocking the increase in [Ca2+]i can reduce or delay irreversible myocardial damage[38].

Astragalus polysaccharides, adenosine and calcium channel blocker (CCBs) can reduce the level of intracellular calcium in cardiomyocytes, which leads to a series of physiological effects against a variety of diseases. In clinical studies, the administration of calcium antagonists during myocardial reperfusion did not achieve beneficial effects[39].

2.4 Autophagy

Autophagy begins with the formation of autophagosomes.Autophagosomes are a double-layered intracellular structure of reticular origin, which engulf cytoplasmic contents and finally fuse with lysosomes to degrade substances. The substances degraded in these newly formed autolysosomes are recruited into anabolic reactions to maintain energy levels and provide macromolecules for the synthesis of nucleic acids, proteins or organelles, thereby maintaining cell metabolism, homeostasis and survival[40].The specific type of mitochondrial autophagy, that is, mitotic phagocytosis, removes damaged mitochondria that are toxic to cells and cause an immune response. In I/R injury, the activation of autophagy helps maintain energy balance by promoting the production of ATP during ischemia, and then switches to clear the damaged organelles and proteins during the reperfusion phase, and the amount of autophagy during maintenance reperfusion can be reduced. The infarct size protects the heart from I/R damage[41].However, the excessive activation of autophagy may also mediate cell death through the process of autophagy cell death.

2.5 Apoptosis

Cell shrinkage, increased cytoplasmic density, chromatin condensation, nuclear DNA degradation, and the formation of apoptotic bodies are the characteristics of apoptotic cells[42]. It is known that apoptosis is triggered shortly after myocardial infarction and is significantly increased during reperfusion[43]. Bcl-2 family proteins play an important role in the regulation of cell apoptosis,including anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax.The ratio of Bcl-2/Bax has become a marker reflecting the degree of apoptosis. Caspase-3 is a pro-apoptotic protein, which is believed to play an important role in the cascade of apoptosis. When activated in the early stages of apoptosis, it becomes lysed caspase-3. In recent years, people have recognized three major apoptosis signaling pathways, including mitochondrial signaling pathway, death receptor signaling pathway, and endoplasmic reticulum signaling pathway.In acute myocardial I/R injury, apoptosis is stimulated by the mechanical targets of PI3K/AKT/MTOR signaling pathway, thereby reducing the survival rate of cardiomyocytes. JAK/STAT pathway is involved in regulating cell growth, proliferation, differentiation and apoptosis. The activation of JAK-STAT signaling pathway can alleviate MIRI.

Remifentanil inhibits the Fas/FasL signal transduction pathway,reduces cardiomyocyte apoptosis, and reduces oxidative stress and inflammation caused by I/R[44].

3. Treatment progress

At present, the intervention methods for the molecular mechanism of MIRI include: ischemic preconditioning, drug-induced preconditioning, ischemic post-conditioning, drug post-treatment,remote ischemic treatment, drugs to reduce inflammation, antioxygen free radicals, and reduce calcium overload. Intervention,the above intervention methods have not entered the clinical stage or the clinical application effect is not good. Hypothermic targeted body temperature management is the only clinical treatment that can reduce reperfusion injury by upregulating the survival pathway after cardiac arrest[45]. However, its efficacy is often limited by the following factors: the target body temperature cannot be reached within the treatment window. Side effects and delayed neural prediction[46].

4. Summary and Outlook

The mechanism of MIRI is complex and involves the participation of multiple molecules and multiple pathways. The specific mechanism still needs further study. Our previous studies have confirmed that hypoxia induces oxidative stress in cardiomyocytes,induces autophagy and apoptosis, and affects cell survival and growth[47]; Reoxygenation can further aggravate the oxidative stress damage of H9c2 cardiomyocytes caused by hypoxia.Activate the ROS/MAPKs pathway[48]; Pigment epithelium-derived factor (PEDF) increases cardiomyocyte apoptosis during hypoxia through Fas, and PEDF receptors are expressed on the myocardial cell membrane[49]. By studying the mechanism of MIRI, a better understanding of potential signal transduction pathways will help in-depth exploration of relevant therapeutic targets. However,most of the current studies are limited to the experimental stage of animals in vivo and in vitro, and its clinical effects need to be further verified. However, with the mutual application of molecular biology, biomarker technology,nanotechnology,proteomics,organic chemistry and other multidisciplinary developments, the research on the molecular mechanism and pathway of MIRI gradually becomes clear, and the development of pathway target blockers is about to open up new possibilities. field. Strengthen multidisciplinary and related research cooperation. We look forward to the development of new therapies to reduce MIRI and include them in clinical trials as soon as possible to improve the prognosis of patients with acute myocardial infarction and extracardiac surgery.

Author's contribution: Cheng-lu Wei conceives and designs the article, writes and revises the thesis. Qing-min Feng, Huai-mi Zhuo, and Song-cheng Chen are members of this research group who participated in the compilation of the article, and Ji-ke Li is responsible for the overall management of the article.

This article has no conflict of interest.