Research progress of TRP channel's protective effect on myocardial ischemia-reperfusion injury

XIE Feng, DUAN Guang-jing, QU Xin-liang, ZHAO Bo, JIANG Ya-ni, YAN Ruo-nan,ZHANG Jia-hao, OU Li, GAO Feng, LI Min

1.College of Pharmacy, Shaanxi University of Traditional Chinese Medicine, Xianyang 712046, China

Keywords:Transient receptor channel Myocardial ischemia reperfusion injury Protective effects

ABSTRACT Transient receptor potential (TRP) channels are a type of cation channel located on the cell membrane. TRP channels are divided into 7 subfamilies (TRPC, TRPA, TRPM, TRPV, TRPN,TRPP and TRPML) and widely expressed in myocardial tissue. In recent years, with the application of gene knockout and transgenic model animals, it has been found that members of the TRP channel subfamilies TRPM, TRPC and TRPV are closely related to myocardial ischemia-reperfusion injury. The activation or inhibition of TRP channels participates in the regulation of myocardial ischemia-reperfusion injury, reduces the infarct area of the myocardium, and exerts a protective effect. Therefore, this paper first summarizes the structural characteristics of TRPM, TRPC, and TRPV and their distribution in the cardiovascular system,and then summarizes the mechanisms of TRPM, TRPC, and TRPV that regulate myocardial ischemia and reperfusion, which will provide a certain theoretical basis for treatment of myocardial ischemia-reperfusion injury.?Corresponding author: LI Min, Professor, Postgraduate Tutor, Ph.D.,E-mail: 413159921@qq.com.

1. Introduction

Ischemic heart disease (IHD) is a common and serious disease in the cardiovascular system. In recent years, the incidence rate has increased year by year, which seriously threatens the safety of human life[1] .With the development of society and the changes in people’s lifestyles, ischemic heart disease has gradually become younger and its growth trend is obvious. The number of deaths related to cardiovascular diseases in China accounts for about 45% of the total number of deaths of residents, far exceeding other diseases such as malignant tumors and ranking first[2] .Myocardial ischemia is one of the main causes of death of human diseases today. Timely reperfusion can reduce the damage caused by myocardial ischemia, but it will also bring serious reperfusion injury while restoring blood flow, which may lead to illness worsen[3].This tissue damage caused by the restoration of blood flow due to myocardial ischemia is called myocardial ischemia reperfusion injury (MI/RI), which can irreversibly damage the structure and function of the myocardium.[4]Among members of the transient receptor potential (TRP) channel channel superfamily, TRPM,TRPC and TRPV are closely related to myocardial ischemia and reperfusion[5-7].During MI/RI, the TRPM subfamily is involved in cell proliferation and apoptosis; TRPC channels are involved in mediating Ca2+influx in the cytoplasm; TRPV channels can be activated by physical, mechanical, and chemical stimulation[8-9] and participate in the myocardium.A large number of studies in recent years have also confirmed that TRP channels play an important role in the regulation of MI/RI,This article summarizes the structural characteristics of TRPM (TRPM2, TRPM4, TRPM7),TRPC (TRPC3, TRPC6) and TRPV (TRPV1, TRPV2, TRPV4) in the TRP family and their respective protective mechanisms for the myocardium.

2. TRPM, TRPC, TRPV structural characteristics and their distribution in the cardiovascular system

TRPM, TRPC, TRPV channels have six transmembrane domains(TM), with a channel hole between TM5 and TM6[10].In the TRP subfamily, the similarity of the primary amino acid sequence is mainly restricted by the transmembrane fragments, and different structural domains are formed by the difference in the length of the -NH2 and -COOH ends in the cell[11].Each TRP channel has a different amount of ankyrin at the end of -NH2: for example, there are 3-4 TRPCs, 6 TRPVs, 14-15 TRPAs, and 29 TRPNs[12].The-NH2 end of TRPMs consists of four segments of residues, which are designated as TRPM homology domains (MHD).In TRPCs and TRPMs, there is a small area extending from the -COOH end to TM6, the so-called TRP domain, Participate in the activation and desensitization of PIP2 regulation channels[13-14].It is precisely because of the diversity of its domains that the TRP family can intervene in myocardial ischemia-reperfusion injury through multiple channels. Some scholars have used TR-PCR, immunohistochemistry and other methods to find that TRP channels are expressed in different types of cardiomyocytes and fibroblasts[15-16].

3. TRPMs, TRPCs, TRPVs and myocardial ischemia reperfusion injury

3.1 TRPMs' regulation of MI/RI

TRPM channels are a subgroup of the TRP channel superfamily and play an important role in cardiomyocyte proliferation.TRPM2 is the second subfamily member to be cloned and is expressed in tissues such as the heart, vascular system and hematopoietic cells[17].Literature studies have found that TRPM2 activation protects or damages myocardial ischemia-reperfusion injury, and there is still some controversy.Miller BA[18] et al. ligated the left anterior descending coronary artery in TRPM2 knockout group(TRPM2-/-) mice and normal group (WT) mice. After 30 minutes,they found that the infarct area was similar,however, the +dp/dt of the TRPM2-/- group was lower than that of the normal group(WT), compared with the WT group TRPM2-/- group, the calcium influx in the myocardial cells of mice was reduced.Increased tissue reactive oxygen species (ROS) ROS levels, hypoxia-inducible factor 1α (HIF-1α) HIF-1α, Forkhead box O (FoxOs) transcription factors, and superoxide dismutase (SOD) levels reduce,Prompt that TRPM2 channel activation has a protective effect on MI/RI.Recent studies have found that the protective mechanism of TRPM2 against myocardial ischemia-reperfusion injury may be through maintaining mitochondrial function and reducing the production of mitochondrial ROS, promoting calmodulin-RACK1 related physiological signals,which are all related to Ca2+influx[19].On the contrary, Hiroi[20] et al. found that compared with WT mice, TRPM2-/- knockout mice had reduced myocardial infarction area and improved myocardial contractility after MI/RI.Neutrophils (PMNs) were obtained from WT mice and TRPM2-/-, respectively, and infected with WT and TRPM2-/- mice. It was found that only PMNs from WT mice caused the infarct area of WT and TRPM2-/-Increased, suggesting that the cause of MI/RI may be the accumulation of neutrophils in the reperfusion zone mediated by TRPM2 activation.The above results suggest that TRPM2 channel is involved in the regulation of MI/RI,but whether TRPM2 activation has a protective effect on myocardial ischemia remains to be further studied.

Activation of TRPM4 channel participates in the formation of myocardial ischemia-reperfusion injury, leading to myocardial infarction.TRPM4 inhibitor (9-Phe) pretreatment can alleviate myocardial damage after ischemia-reperfusion in rats, and significantly improve the contractile function of damaged ventricles.Further research found that MI/RI rats treated with 9-Phe showed a significant reduction in the area of myocardial infarction.The above results suggest that the regulation of TRPM4 plays an important role in reversing myocardial damage caused by MI/RI, and its mechanism may be related to improving the contractile function of the damaged left ventricle and resisting arrhythmia[21].The expression levels of TRPM1, TRPM2, TRPM3, TRPM4, TRPM5,TRPM6, TRPM7, TRPM8 genes in myocardial tissue after MI/RI were detected by reverse transcription PCR (reverse transcription PCR) RT-PCR,Set up a control group, an ischemia group, and an ischemia-reperfusion group. Compared with the control group, it was found that the expression levels of TRPM2, TRPM4, and TRPM6 in the ischemia group and the ischemia-reperfusion group did not change significantly,the expression level of TRPM7 was significantly increased, and the expression of TRPM7 was positively correlated with the degree of myocardial MI/RI; TRPM1, TRPM3, TRPM5 and TRPM8 were not expressed in myocardial tissue[22].In summary,the activation of TRPM channels may not have a protective effect on myocardial ischemia-reperfusion, and reducing the expression level of TRPM7 channel protein through the application of its antagonists may reduce the damage caused by myocardial reperfusion.

3.2 Regulation of MI/RI by TRPCs

A large number of studies have clearly confirmed that the realtime fine regulation of cellular calcium ions (Ca2+) participates in or dominates various physiological activities of the myocardium.If the regulation occurs, a series of pathologies such as myocardial injury or death, cell electrophysiological disorders, etc. may occur Physiological changes[23].When myocardial ischemia occurs,calcium ions in mitochondria increase, and myocardial reperfusion is aggravated,A large amount of calcium ions are deposited in mitochondria, affecting oxidative phosphorylation, resulting in abnormal energy conversion and cell death.Experimental studies have found that cell membrane calcium channel antagonists used in myocardial ischemia and reperfusion can reduce the area of myocardial infarction by about 50%[24-25].These conclusions indicate that calcium overload is an important cause of tissue damage after ischemia, and TRPC3 and TRPC6 are the main factors that mediate Ca2+influx, leading to ischemia/reperfusion injury (MI/RI).Studies have found that blocking TRPC activity or TRPC gene knockout has a significant protective effect on myocardial tissue or cells after ischemia-reperfusion[26].Down-regulation of TRPC5 can reduce the damage caused by myocardial ischemia and reperfusion and reduce the area of myocardial infarction[27].Studies have verified the effect of TRPC3 inhibitor Pyr3 on myocardial I/R injury in mice.By temporarily blocking the left anterior descending artery (LAD)coronary artery, a mouse I (30 minutes)/R (24 hours) injury model was established.Pyr3 was administered via the right jugular vein at a concentration of 0, 2.5, 5, or 10 mg/kg 5 minutes before reperfusion.We have observed that the selective TRPC3 inhibitor 10 mg/kg Pyr3 can significantly reduce the infarct size of the left ventricle in mice,and reduce the apoptotic rate and inflammatory response of mouse cardiomyocytes.In short, TRPC3 can be used as a candidate target to prevent I/R injury. Pyr3 can directly bind to TRPC3 channel protein,inhibit TRPC3 channel activity, and improve myocardial I/R injury related to TRPC3[28].So what is the role of TRPC in myocardial ischemia?Some scholars are exploring whether the protective mechanism of neurotrophic factor (BDNF) on myocardial infarction rats is related to TRPC. They used left coronary ligation to establish a rat MI/RI model and divided it into a normal group and a TRPC3/6 siRNAs group,compared with the normal group, the cell survival rate of TRPC3/TRPC6 siRNA group was significantly reduced, and the number of cardiomyocyte apoptosis increased.It suggests that the protective effect of BDNF on cardiomyocyte apoptosis may be related to the TRPC3/6 channel[29].

3.3 TRPVs' regulation of MI/RI

After TRPV1 channel is activated, it stimulates the release of calcitonin gene-related peptide (CGRP) and substance P (SP), and plays a myocardial protective effect during ischemia-reperfusion injury.After capsaicin (CPZ) interferes with hypoxia/reoxygenation(H/R) H9C2 cells, the intracellular calcium ion content increases,the number of apoptosis increases, the mitochondrial superoxide content decreases, the mitochondrial membrane potential decreases and the inhibition of mitochondrial biological activity(Take the expression of ATP synthase β as a reference)[30]Conversely, using capsaicin (CAP) to interfere with H/R H9C2 cardiomyocytes can attenuate H/R-induced apoptosis.CGRP837 (a CGRP receptor antagonist) and RP67580 (an SP receptor antagonist) were used to eliminate the confounding effects of neuropeptides, and TRPV1 was activated after using CGRP837 and RP67580, and it was found that there was no significant difference with the TRPV1-/- group., The results suggest that TRPV1 may participate in the protection of H/R through CGRP and substance P.Whether this kind of regulation also exists in animals remains to be further explored, and its regulation mechanism and pathway are still unclear.The above conclusions provide a new understanding of the role of TRPV1 channel in ischemia/reperfusion injury.TRPV2 can play an important role in calcium homeostasis[31]. Some scholars found through gene chip analysis that compared with the sham operation control group, the expression of TRPV2 mRNA in the left ventricle (LV) after acute myocardial infarction (MI) in rats was significantly and specifically up-regulated.Acute myocardial infarction was caused by ligation of the left anterior descending branch of the rat heart. LV sections were made with anti-TRPV2 antibody and anti-mononuclear/macrophage antibody, immunohistochemistry (IHC) and immunofluorescence(IFC) staining, and the source of LV. The cells were analyzed by flow cytometry, TRPV2siRNA was transiently transfected into rat alveolar macrophages NR8383, and the transpore migration method was used to make them migrate to the hypoxia-conditioned medium of the rat cardiomyocyte cell line H9C2.It was found that TRPV2 expression rats have macrophage infiltration around the infarct area.After inhibiting the TRPV2 channel, the number of macrophages migrating to the hypoxic cardiomyocyte conditioned medium is significantly reduced, and in the early stage of myocardial infarction,there are some around the infarct area.Massive inflammatory cell infiltration[32].In some macrophages around the infarcted area,the cation channel TRPV2 was significantly overexpressed.This increase is unique to TRPV2 and is not observed in other members of the TRP gene superfamily.It is suggested that the overexpression of TRPV2 gene may damage the myocardium by enhancing the phagocytic activity of macrophages around the infarct area.

TRPV4 is highly expressed in the heart and blood vessels and can be activated during MI/RI.TRPV4 antagonist (HC-067047G) interferes with myocardial MI/RI in mice, which can significantly reduce the infarct size, reduce the level of troponin T, and improve heart function[33-34].Some researchers used HC-067047 and TRPV4-/- for research, and the results showed that it can significantly reduce the infarct size, reduce the expression of troponin T and improve heart function[35].In addition, TRPV4 mediated excessive Ca2+ influx can trigger the changes in the levels of apoptotic factors caspase-3 and Bax/Bcl-2, leading to cell apoptosis[36-40].The protective effect of HC-067047 on cardiomyocytes in MI/RI was blocked by specific drug blockers of the reperfusion injury salvage kinase (RISK) pathway, indicating that the cardioprotective effect of TRPV4 blockers may be related to the RISK pathway regulation is related [41].But there is no further research to confirm.

4. Summary and outlook

There are many members of TRP channel family, among which TRPC (TRPC3、TRPC4、TRP5、TRPC6、TRPC7),TRPV(TRPV1、TRPV2、TRPV4),TRPM(TRPM2、TRPM2、TRPM7),and TRPP2 are expressed in cardiomyocytes. The main ones that are closely related to MIRI are TRPM (TRPM2, TRPM4,TRPM7), TRPC (TRPC3, TRPC6) and TRPV (TRPV1, TRPV2,TRPV4). The role of TRP channels in MI/RI is not all protective.For example, the activation or overexpression of TRPV4, TRPM4,TRPM2, and TRPM7 can lead to different degrees of damage to the myocardium, while the activation of TRPV2 and TRPV1 can reduce ischemia and recurrence protects against myocardial damage after perfusion. This discovery may provide a new entry point for studying the protective mechanism of myocardial ischemia in perfusion injury,and the screening of TRP channels can be used as a new type of drug target for the treatment of ischemic heart disease. As TRP channel is a new field and direction for conquering ischemic heart disease at this stage, the following scientific issues still need to be further explored:(1) The research on myocardial ischemia mostly stays at the shallow level of correlation, and the regulation mechanism and pathway of TRP channel on MI/RI are not clear. (2) Existing studies are still in the study of a single TRP subfamily and MI/RI,but different subfamilies have shown the same pharmacological effects in the research. For example, antagonizing TRPV4, TRPM4,TRPM2, and TRPM7 respectively can reduce the damage to the myocardium during perfusion and play a protective effect. Therefore,the combined application of multiple TRP channels in the treatment of MI/RI is also very necessary, and further exploration is needed.In addition, TRPA1 is also expressed in cardiomyocytes, and through the use of its blocker ASP7663, it was found that compared with the model group, the myocardial infarction area of the blocker group was significantly reduced and there were significant differences[42].TRPV1 is also involved in the production of related pain induced by temperature [43], chemical [44] and mechanical stimulation [45], which is related to the pain caused by myocardial infarction.Therefore,further exploration of the role and mechanism of TRPA1 and the rational application of antagonists have far-reaching significance for myocardial ischemia-reperfusion injury.

Author contribution:Document collection and collation: Xie Feng, Duan Guangjing, Qu Xinliang, Zhao Bo, Jiang Yani, Yan Ruonan, Zhang Jiahao; Writing:Gao Feng, Xie Feng.Modification: Oli, Li Min