A theoretical study of miRNA155-DNA methylation mediated mitokATP regulating mitochondrial autophagy in the improvement of myocardial ischemia-reperfusion injury by Qishen Yiqi Dropping Pills

Gui-Xin He, Yu-Fei Feng, Wei-Bin Qin, Lin Lin, Meng-Xian Hu, Guo-Kun Zheng, Li-Yan Yu, Zi-Yong Jia, Juan Wei, Qi Wen, Zhen-Le Huang, Shi-Hai Liu

1. The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530022, China

2. Guangxi University of Chinese Medicine, Nanning 530299, China

Keywords:Qishen Yiqi Dropping Pills Myocardial ischemia MiRNA155-DNA methylation Mitochondrial autophagy MitoKATP

ABSTRACT In the state of acute myocardial ischemia, miRNA expression can regulate related genes and proteins, reduce myocardial cell damage, and thus play a protective role in the myocardium.However, the specific mechanism still needs to be further explored. Recent studies have found that the opening of the mitoKATP channel can regulate mitochondrial autophagy, and the initiation of miRNA-DNA methylation plays a regulatory role in inducing cell autophagy. The applicant research team previously found that Qishen Yiqi Dropping Pills could significantly improve myocardial ischemia by mediating MitokATP channels to regulate mitochondrial autophagy., and animal experiments have confirmed that miR-155 plays a significant role in the aspect of autophagy regulates, inflammatory reaction and Vascular smooth muscle cell migration. Therefore, the applicant innovatively proposed that Qishen Yiqi Dropping Pills can regulate miRNA155-DNA methylation to mediate the opening of mitoKATP, thereby regulating mitochondrial autophagy and improving myocardial ischemia. In this paper, the association between mitochondrial autophagy and oxidative stress injury after myocardial ischemia was described, and the possible mechanism of Qisen Yiqi dropping pills regulating mitochondrial autophagy by regulating miRNA155-DNA methylation to mediate MitokATP to improve myocardial ischemia reperfusion injury was discussed, so as to provide theoretical ideas for related research.✉Corresponding author: HE Gui-xin, M.D., Chief Physician, Professor, Postgraduate Supervisor.

According to China Cardiovascular Disease Report [1], the mortality rate of myocardial infarction has been increasing rapidly in recent years, which is one of the major diseases threatening people's lives and has become a major public health problem. The reperfusion strategy can reopen the occluded vessel and restore the oxygen supply to the ischemic myocardium. Recovery process can cause a variety of pathological processes, such as oxidative stress, cardiomyocyte apoptosis, autophagy and the release of inflammatory cytokines, which further aggravate myocardial damage, that is, myocardial ischemia-reperfusion injury (MIRI).Therefore, the active and effective inhibition of reperfusion injury after blood flow recovery in patients with myocardial infarction can increase the resistance of myocardial cells to MIRI injury, and further rescue the damaged myocardium. It is significant to improve the prognosis of patients[2].

Studies have shown that the impairment of mitochondrial function during myocardial ischemia is an important factor in aggravating cell necrosis. Myocardial ischemia can reduce the activity of various complexes in subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM), damage the electron transport chain (ETC),and affect the function of electron transport. Ischemic damage to ETC increases the net production of reactive oxygen species(ROS) by mitochondria,Excessive ROS triggers further myocardial injury to form a vicious circle[3-4]. After ischemia-reperfusion,not only ETC, but also many metabolic pathways are damaged by ETC. These damaged mitochondria are potent stimulators of the transition to cardiomyopathy during sustained injury and prolonged reperfusion. Therefore, timely removal of damaged mitochondria can maintain the homeostasis of myocardial tissue.It is of great significance for the normal function of myocardial cells. Autophagy has been shown to be a major regulator of cardiac homeostasis and function. Autophagy, which preserves cardiac structure and function under baseline conditions, is activated upon stress, limiting damage under most conditions to preserve cardiac function. By limit that accumulation of misfolded proteins, mitochondrial dysfunction and oxidative stress,Reduce chronic ischemic remodeling and regulate cardiac adaptation to pressure overload[5]. Therefore, the cardioprotective strategy targeting mitophagy is a new direction to explore the treatment of cardiovascular diseases.

MiRNAs are differentially expressed in diseased tissues and can be released into the circulation to affect the course of disease.Therefore, it has become an important research field to explore the diagnostic and therapeutic effects of miRNAs, and to find new regulatory mechanisms and biomarkers of disease. It has been reported that miRNAs are the core regulators of gene expression,and their abnormal regulation can affect the pathogenesis of coronary heart disease[6].Although many miRNAs have been proved to play an important role in cardiovascular diseases, there are many kinds of miRNAs with a wide range of roles, and the current understanding and mastery of miRNA regulatory genes and their mechanisms involved in diseases are only the tip of the iceberg of the huge miRNA family, and more regulatory mechanisms still need to be further explored.Previous miRNA-related studies have found that the change of DNA methylation level of miRNA promoter can directly lead to the change of its expression level, participate in its post-transcriptional regulation, and play an important role in the pathogenesis, diagnosis and development of diseases[7-9]. In recent years, it has been found that miRNA-DNA methylation plays an important role in the development of myocardial ischemia[10-11].As prove by animal experiments,Some traditional Chinese medicine compounds can significantly reduce myocardial necrosis area after myocardial ischemia-reperfusion injury, regulate autophagy and inhibit the formation of atherosclerotic plaques by regulating miRNA or DNA methylation, but the specific mechanism is not clear[12]. Previous studies have found that Qishen Yiqi Dropping Pills can significantly improve MIRI and regulate autophagy.And cell experiment prove that that Qishen Yiqi dripping pill can activate a mitoKATP signal pathway so as to improve myocardial ischemia.In addition, previous studies have confirmed that exosomal miR-155 plays an important role in the regulation of endothelial cell autophagy, inflammatory response, oxidative stress injury, and vascular smooth muscle cell migration. However, whether miR-155 promoter DNA methylation is involved in MIRI has not been reported.Therefore, the author proposed the hypothesis that Qishen Yiqi Dropping Pill regulates mitochondrial autophagy by regulating miRNA155-DNA methylation, thereby alleviating MIRI. The related theories are discussed as follows:

1. Oxidative stress injury after myocardial ischemia

When myocardial ischemia occurs, mitochondrial reactivation and oxygen regeneration can lead to oxidative stress, which mediates myocardial injury by stimulating a variety of destructive factors.Reactive oxygen species (ROS) as a key factor in the induction of oxidative stress, when they are produced too much and exceed the ability of the body to scavenge peroxides, the balance between the oxidative system and the antioxidant system will be disrupted.Resulting in oxidative damage to the tissue. MIRI activates neutrophils and mitochondrial respiratory chain dysfunction through the xanthine oxidase system, producing an excess of anaerobic free radicals. Oxidative stress induced by a large amount of ROS is an important initiator of myocardial cell death and decline in systolic and diastolic function[13]. In fact, after myocardial ischemia, only a small amount of oxygen free radicals can be observed.The explosive growth of oxygen free radicals occurred several seconds to one minute after reperfusion. ROS can directly attack the unsaturated fatty acids on the biomembrane to cause lipid peroxidation and damage the structural integrity of myocardial cells and endothelial cell membranes. In addition, ROS can trigger cytosolic Ca2 +overload, resulting in an excessive increase in mitochondrial Ca2 +, which in turn induces mPTP to open and depolarize the mitochondrial membrane potential.Opening of mPTP results in loss of ATP, mitochondrial swelling, and release of cytochrome C,leading to apoptosis[14]. In addition, oxidative stress can induce DNA damage in cardiomyocytes. Luo Jiaming[15]et al. Found that hypoxia/reoxygenation H9C2 cells with antioxidants significantly reduced DNA damage, and inhibited myocardial injury and apoptosis to improve MIRI.DNA damage mediated by oxidative stress may be closely involved in the pathogenesis of MIRI. In addition,ROS can also promote inflammatory signal transduction, regulate cardiomyocyte apoptosis and participate in myocardial remodeling after myocardial infarction[16]. Therefore, effective scavenging of excess ROS may provide a theoretical basis and therapeutic targets for alleviating MIRI.

2. Mitochondrial Autophagy and Oxidative Stress Injury after Ischemia

Myocardium is a highly oxidized tissue, and mitochondria play a central role in maintaining the optimal performance of the heart.Mitochondria is a two-membrane organelle, the inner membrane is an ion permeable membrane and the outer membrane is a factor permeable membrane. This complex membrane structure produces adenosine triphosphate (ATP) through mitochondrial oxidative phosphorylation (OXPHOS). In that heart of an adult mammal,OXPHOS is responsible for 95% of ATP production. The creatine phosphate shuttle system transports high-energy phosphate groups from sites of mitochondrial production to myofibrils to regenerate ATP consumed during contraction. Therefore, the normal survival of cells depends on the integrity of mitochondrial function and structure.

Autophagy is a lysosome-dependent degradation process.Mitochondrial autophagy is a typical selective autophagy, which eliminates damaged or dysfunctional mitochondria to maintain mitochondrial homeostasis and cellular homeostasis. "Mitochondrial autophagy" was first proposed by Lemasters[17]in 2005, emphasizing the non-stochastic nature of the process after recognition of the Uth1 gene in yeast.Mitochondrial autophagy was proved to be selective.With the deepening of research, the understanding of the process and molecular mechanism of mitophagy has been gradually strengthened.Mitochondrial autophagy pathways that are currently broadly accepted include the ubiquitin-type, receptor-type, and atypical mitochondrial autophagy pathways[18].Mitochondrial autophagy is divided into four stages: autophagy induction, autophagosome formation, autophagosome formation and degradation. Damaged mitochondria can cause changes in their permeability and activation of related proteins. Proteins on the mitochondrial membrane are linked to autophagosomes in the cytoplasm, gradually wrapping the whole mitochondria, and then transported to lysosomes.Encapsulated mitochondria fuse with lysosomes, which are then degraded and used by the cell to synthesize new organelles and proteins. In cardiac tissue, autophagy decreases with age, and misfolded proteins and dysfunctional mitochondria accumulate in the aging heart.Therefore, mitochondrial autophagy is an important mediator in the control of mitochondrial quality in cardiomyocytes. Examination of Mito-Keima mice indicates that the basal mitochondrial autophagy activity in the heart is greater than in other organs, such as the thymus[19], possibly reflecting high levels of oxidative phosphorylation, ROS, and myocardial cell mitochondrial damage. Mitochondrial autophagy is activated during stress, allowing the removal of damaged mitochondria.At the same time, autophagy is tightly coupled with mitochondrial biogenesis,so the removed mitochondria are constantly replaced by new ones.Mitochondrial autophagy disorders have been found to result in persistent loss of calcium homeostasis, excessive production of ROS,impaired production of cellular energy and ATP, and ultimately cell death[20]. In heart failure caused by pressure overload,Downregulation of mitophagy mediates mitochondrial dysfunction, which is an important cause of disease, and effective increase of mitophagy can improve cardiac dysfunction[21]. In the process of myocardial ischemia and hypoxia, the damage of mitochondria in myocardial cells produces a large number of apoptosis-promoting factors such as ROS.Mitochondrial autophagy activation exerts quality control by degrading malfunctioning mitochondria as well as reducing ROS levels, while promoting ATP production to provide energy to maintain cellular homeostasis[22]. In addition, mitophagy can reduce the inflammatory response of cardiomyocytes and inhibit the process of myocardial injury[23].Therefore, effective mitophagy plays a positive role in myocardial protection after myocardial ischemia.

3. ATP-sensitive potassium channels and myocardial ischemia-reperfusion injury

ATP-sensitive potassium channels are inwardly rectifying potassium channels that are regulated by the concentration of intracellular ATP.KATP channel is a heterogeneous membrane protein complex[25]composed of four internally rectifying potassium channel 6 (Kir6,Kir6.1, or Kir6.2) subunits and four ABCC (ATP binding cassette,subfamily C) family members sulfonylurea receptors (SUR,SUR1, SUR2A, or SUR2B). KATP channels can be divided into sarcolemmal (sarcKATP) and mitochondrial (mitoKATP) types.KATP channels are abundant in cardiac tissue and play a crucial role in the signal transduction pathways of cardiac myocytes[26]. Studies have shown that opening KATP channels can protect myocardium by reducing cardiomyocyte injury and apoptosis[27]. The activity of KATP channels is closely related to cellular metabolism and is regulated by intracellular ATP concentration.ATP has a high concentration in the cell, at which time the KATP channel is closed;When myocardial cells are subjected to pathological stimuli such as ischemia, reperfusion injury, cell stress and apoptosis, intracellular ATP will be consumed and its concentration will decrease, thus inducing the opening of KATP channels, the outflow of potassium ions, stabilizing the resting potential of cell membrane, shortening the action potential time and reducing the inflow of calcium ions.Reduce the calcium overload of myocardial cells, reduce the energy consumption of myocardial cells, increase stress tolerance, and improve myocardial injury[28]. KATP channels have also been shown to have antiarrhythmic effects during MIRI[29]. Previous studies have shown that mitoKATP may be able to prevent the long-term opening of MPTP, thereby maintaining the integrity of mitochondria, ensuring the energy status of cells and promoting survival.Meanwhile, mitoKATP channels play an important role in cardioprotection involving autophagy-regulated processes[30-31].Previous studies have found that mitoKATP signaling pathway can promote the improvement of myocardial ischemia, and Qishen Yiqi Dropping Pill can effectively activate mitoKATP channel.

4. MiRNA-DNA methylation is involved in myocardial ischemia-reperfusion injury

Mirna is a kind of endogenous non-coding small ribonucleic acid,consisting of 18-24 nucleotides. MiRNAs can induce and regulate physiological processes or signaling pathways by binding to the 3 ′ -untranslated region (3 ′ -UTR) of target mRNAs after transcription, resulting in the degradation or silencing of target mRNAs. It is estimated that miRNAs control the activity of 30 to 50% of protein-coding genes,Unlike transcriptional regulators that have an on and off function in controlling gene expression,miRNAs can fine-tune protein expression levels in response to changes in environmental conditions. As micromanagers, miRNAs are involved in cell survival, stress response, proliferation, and apoptosis[32]. For example, miR-145 directly targets angiopoietin-2,1,Inhibition of Angpt2 expression by increasing miR-145 levels protects cardiomyocytes from I/R injury[33]. MiR-17-3p promotes cardiomyocyte hypertrophy, proliferation, and survival.Negative exercise-induced heart growth, including cardiomyocyte hypertrophy and the expression of cardiomyocyte proliferation markers [34], confirms the direct and indirect regulation of miRNA on cardiomyocytes.

Previous studies have found that miRNA, due to its high degree of conservation, acts as an epigenetic regulator in the body and is also regulated by other epigenetic regulators. In the mechanism of epigenetic regulation, miRNA binds to DNMT1, 3A, 3B through miRNA targets.TET2 or Drosha promotes DNA methylation modification to indirectly cause changes in methylation levels of target gene promoters and gene expression levels[35-36].Hypermethylation on miRNA promoters may indicate reduced miRNA function and biogenesis. In hcy-induced atherosclerosis,the expression of miR-124 in foam cells was decreased, while the methylation level of miR-124 promoter DNA was increased in a dose-dependent manner.Therefore, it is speculated that promoter DNA methylation may be an important mechanism of atherosclerosis[37]. It has been shown that miRNA biogenesis depends on DNA methylation. When regions on both sides of the miRNA coding sequence are highly methylated, miRNAs are expressed at higher levels with greater sequence conservation and are more likely to drive a cancer-associated phenotypethan miRNAs encoded by unmethylated sites[38].

MiR-155 is a multifunctional microRNA that was originally thought to have immunomodulatory functions because of its critical role in innate and adaptive immune responses[39]. With the deepening of research, the function of miR-155 has been gradually revealed. Exosomes enriched for mir-155 inhibit fibroblast proliferation and promote fibroblast inflammation[40]. Deletion of mir-155 significantly reduces the incidence of cardiac rupture after acute myocardial infarction (AMI) and improves cardiac function.Mir-155 also occurs in macrophage-derived exosomes, through which it can be transferred into cardiac fibroblasts. Mimetics of mir-155 or exosomes containing mir-155 inhibit cardiac fibroblast proliferation by down-regulating Son of Sevenless 1 expression,And promotes inflammatory responses by reducing the expression of suppressor of cytokine signaling 1[41]. Many studies have found that miR-155 also plays an important role in the regulation of endothelial cell autophagy, inflammatory response, vascular smooth muscle cell migration, and then affects the pathogenesis of ischemic cardiomyopathy and atherosclerosis[42].Previous studies by the team have shown that miR-155 has a cardioprotective effect, but the effect of DNA methylation of miR155 on myocardial ischemia has not been reported. Therefore, the author boldly suspected that MiRNA155-DNA methylation played an important role in the regulation of myocardial ischemia-reperfusion injury.

5. Effect of Qishen Yiqi Dripping Pill on Myocardial Ischemia Reperfusion Injury

There is no description of "myocardial ischemia" in traditional Chinese medicine theory, but combined with the clinical manifestations of patients, myocardial ischemia should belong to the category of "chest pain", "true pain", "headache", "vertigo" and"stroke". Qishen Yiqi Dropping Pill is a compound preparation of traditional Chinese medicine composed of Astragalus membranaceus, Salvia miltiorrhiza, Panax notoginseng and Lignum Dalbergiae Odoriferae, which has the effects of invigorating qi,promoting blood circulation and relieving pain.At present, Qishen Yiqi Dropping Pills are widely used in the adjuvant treatment of myocardial infarction and heart failure. Animal experiments show that Qishen Yiqi Dropping Pill has myocardial protective effect,which can regulate 7nAChR anti-inflammatory pathway and SIRT3/β-catenin/PPARγ signaling to reduce the inflammatory response,apoptosis and ventricular remodeling caused by AMI injury, thereby improving cardiac function[43-44].From the perspective of evidencebased medicine, Dai Qianqian[45]et al confirmed that Qishen Yiqi Dropping Pill can effectively improve adverse cardiovascular events after PCI, improve clinical efficacy, improve quality of life and prognosis, and is safe and reliable. Previous studies by the author's team also confirmed that Qishen Yiqi Dropping Pills also improved myocardial microcirculation disorders after myocardial infarction.Has obvious effect on resisting myocardial ischemia-reperfusion injury[46-48].

The network pharmacology research shows that the Qishen Yiqi Dropping Pill has the action characteristics of multiple paths,multiple targets and multiple ways, and the main active components of the Qishen Yiqi Dropping Pill comprise astragaloside IV,calycosin, formononetin, ginsenoside Rg1, tanshinone IIA and the like, and involve multiple paths such as a calcium signal path, a cAMP signal path, CGMP-PKG and the like,It plays a role in energy metabolism, oxidative stress, inflammation and apoptosis, fibrosis,mitochondrial function regulation, and protects cardiomyocytes and improves cardiac remodeling[49]. Astragaloside IV has a cardioprotective effect on acute myocardial infarction, which promotes angiogenesis by regulating the PTEN/PI3K/Akt signaling pathway [50]. In addition, astragaloside IV can reduce oxidative stress.Alleviate myocardial damage[51]. Tanshinone Ⅱa can improve the symptoms of reperfusion injury in rats with coronary heart disease and blood stasis syndrome by regulating AngⅡ- (AT-1R)-Cx43 signaling pathway[52]. Sodium tanshinone IIA sulfonate attenuates I/R-induced myocardial injury, improves cardiac function by reducing inflammation and apoptosis, and enhances autophagy[53]. The ginsenoside 1 can improve that myocardial infarction area aft MIRI,The decrease of myocardial blood flow(MBF) and cardiac function, as well as the change of myocardial structure, play an inhibitory role in myocardial injury. At the same time, ginsenoside 1 inhibits myocardial apoptosis and regulates energy metabolism by binding to RhoA[54]. The author found in the previous experimental study that Qishen Yiqi Dropping Pill could reduce the infarction area of myocardial ischemia-reperfusion injury, alleviate myocardial injury and improve cardiac function.MiR-155 plays an important role in the regulation of endothelial cell autophagy, inflammatory response and vascular smooth muscle cell migration. Therefore, the authors speculate that Yiqi Dropping Pills may affect mitochondrial autophagy through miRNA155-DNA methylation regulatory network, and ultimately achieve the purpose of improving myocardial ischemia-reperfusion injury.

6. Summary and significance

Myocardial ischemia-reperfusion injury after emergency revascularization of myocardial infarction is an important factor affecting the short-term and long-term prognosis of myocardial infarction, and it is also an important direction for further study. On the basis of previous studies, we put forward the hypothesis that Qishen Yiqi Dropping Pill regulates miR155-DNA methylation mediated miKATP channel.It can inhibit myocardial ischemiareperfusion injury and improve cardiac function after AMI by regulating mitochondrial autophagy. This theory will lay a solid theoretical foundation for effectively inhibiting myocardial ischemiareperfusion injury and improving the effect of traditional Chinese medicine in the treatment of myocardial infarction.