Hai-Long Fan*,Yu-Lan Ren,Li-Zhen Yang,Fei Wu

1Hohhot Hospital of Mongolian Medicine and Traditional Chinese Medicine, Hohhot, China.2School of Acupuncture Moxibustion and Tuina,Chengdu University of Traditional Chinese Medicine,Chengdu,China.

#Hai-Long Fan and Yu-Lan Ren are the co-first authors of this paper.

Abstract

Keywords: MiR-214/NCX1, Acupuncture preconditioning, Coronary heart disease, Myocardial ischemia,Reperfusion injury

Background

Myocardial ischemia, the main mechanism of angina pectoris, usually causes the symptoms of severe chest pain and discomfort in the left anterior chest or adjacent areas among coronary heart disease (CHD)patients [1].It is believed that the most effective treatment to improve ischemic cardiac tisse after acute myocardial infarction is early reperfusion.However,in the process of myocaridial reperfusion, the number of intracellular Ca2+may be probably increased abnormally, which may cause the death of cardiomyocytes and consequent loss of cardiac function, or myocardial ischemia/reperfusion (I/R)injury.Myocardial I/R injury, caused by myocardial ischemia, cardiac surgery or circulatory arrest, may finally rise up to adverse cardiovascular outcomes [2].MicroRNA-214 (miR-214) was recorded to be able to protect cardiomyocytes from I/R injury by the inhibition of the mRNA that encodes Na+/Ca2+exchanger (NCX) 1 and consequently controls Ca2+overload-mediated cell death[3].These findings reveal a significant role for the miR-214/NCX1 mRNA co-expression network model as a regulator of cardiomyocyte Ca2+balance and survival during myocardial I/R injury.

Among several strategies that are being studied to modulate miR-214, acupuncture preconditioning could represent a new non-pharmacological approach of early intervention and prevention of CHD for modulating gene expression of the anti-apoptosis signaling [4-10].The concept of acupuncture preconditioning is described in the ancient bookFan Wang Fang(Eastern Jin Dynasty of China).It refers to the application of acupuncture in advance to stimulate the Qi (Qi is believed to be a vital force forming part of any living entity) of meridians and collaterals(Meridians and collaterals system is a concept in traditional Chinese medicine about a path through which the life-energy known as Qi flows) before the occurrence of disease or when the disease is relatively mild, so as to enhance the body's disease resistance,prevent the occurrence of disease, and alleviate the damage degree of subsequent disease.Acupuncture preconditioning follows the principles of disease prevention in the ancient bookHuang Di Nei Jing(Qin Han Period of China,475 B.C.-221 B.C.).

However, there are no available data regarding the relationship between acupuncture preconditioning and modulation of the miR-214/NCX1mRNA co-expression in myocardial I/R injury.This paper will try to present evidence for the miR-214/NCX1mRNA co-expression after acupuncture preconditioning and argue how this interaction may represent an crucial link in the puzzle of myocardial I/R injury.

Hypothesis

We hypothesize that by evoking miRNA-214 in the heart, the cardioprotective roles of acupuncture preconditioning at Neiguan (PC6) point (an acupoint located on the anterior forearm, two cun above to the wrist crease, between the tendons of palmaris longus and flexor carpi radialis muscles, along the pericardium meridian) are attributed to the repression of NCX1 mRNA expression,to the inhibition of NCX1 and some Ca2+downstream effector proteins [B-cell leukemia/lymphoma 2 (BCL2) and calmodulindependent protein kinase (CaMK) IIδ] that regulate cell death pathways, and to a cardioprotective role for I/R injury; acupuncture preconditioning at Neiguan(PC6) point may be a non-pharmacological option of early intervention and prevention of CHD for activating the miRNA-214/NCX1 mRNA co-expression network model and blocking myocardial apoptosis for myocardial I/R injury.

Hypothesis evaluation

MiRNA-214/NCX1 mRNA co-expression network and myocardial I/R injury

The role of Ca2+, a core of cardiac contraction and signaling networks, in the regulation of pathological cardiac growth and remodeling is crucial, yet the overload of intracellular Ca2+often occurs when cardiomyocyte suffer from ischemic injury and other stresses [11, 12], and is accompanied by increasing expression of both proapoptotic BCL2 family members[11] and CaMK [13]; these changes lead to cardiac contractile dysfunction and ultimately cell death thanks to the signals transducted through the effector proteins in the Ca2+downstream singnaling pathway [11-14].A series of proteins (eg.the LTCC-SERCA2 pump and NCX1)modulate Ca2+handling[3].NCX, particularly,is considered to be the main route for Ca2+influx during I/R injury [14, 15].Blocking NCX-mediated Ca2+influx can offer good myocardial protection [16,17].

Furthermore, accumulating evidence suggests that many microRNAs (miRNAs) are associated with I/R injury [18-23].It’s reported that miRNAs may plays key roles in the regulation of cellular processes of cardiac remodeling and disease, including myocyte hypertrophy [24-28] as well as apoptosis [18-20, 29].MiRNAs are a class of endogenous and non-coding short-length RNAs (17-25 nt).And they may mediate target mRNAs to reduce or inhibit protein translation[30].It has been studied that it is in this way that miRNAs act an essential part in the several cardiac functions [31].MiR-214 can protect cardiac myocytes[3, 4], the expression of which was dramatically depressed in myocardial I/R injury [5].As outlined above, miR-214 knocked-out mice suffered depletion of cardiac contractility, excessive fibrosis, and increased apoptosis in answer to I/R injury [3].MiR-214 was found contributing to the repression of mRNA that encodes NCX1, which regulating of Ca2+influx, as well as the repression of Ca2+downstream signaling effectors (BCL2, CaMKIIδ)that mediate cell death [3].These findings show the importantance of the miR-214/ NCX1 mRNA co-expression network model to regulate cardiomyocyte Ca2+balance and survival during myocardial I/R injury(Figure 1).

Figure 1 A miRNA-214/NCX1 mRNA co-expression network model for blocking myocardial apoptosis

Acupuncture preconditioning and myocardial I/R injury

In 1986, Murry [32] identified myocardial ischemic preconditioning (IPC) for the first time; the term refers to cardiac muscle with an enhanced tolerance for subsequent lethal I/R injury after repeated brief I/R injuries.For early intervention and prevention of CHD,myocardial IPC provides a compelling new strategy:an activated,endogenous protective mechanism for the heart to improve damage resistance of myocardial ischemia and anoxia,with the least damage to ischemic tissue cells.The theory behind myocardial IPC is similar to that of prevention in traditional Chinese medicine (TCM).A detailed interpretation of acupuncture preconditioning was described in the ancient bookZhen Jiu Ju Yingwritten by Gao Wu in the Ming Dynasty of China, which referred the acupuncture maniupulation applied to patients with sub-health or sickness might stimulate Qi, enhance disease resistance and alleviate stress.According to the record of antient bookHuang Di Nei Jing(Qin Han Period of China, 475 B.C.-221 B.C.), the clinical prevention and control of angina pectoris in CHD by acupuncture at Neiguan(PC6)point can be traced back to 3,000 years ago.Additional detailed applications of a cardioprotective effect of Neiguan (PC6) point were described in other TCM classics, such as theZhen Jiu Zi Sheng Jingin 1220 A.D., theZhen Jiu Ju Yingin 1537 A.D.and theZhen Jiu Da Chengin 1601 A.D.Besides, the World Health Organization reported:“Acupuncture: Review and Analysis of Reports on Controlled Clinical Trials in 2002,” recommended acupuncture as one of the alternative and complementary therapies for early intervention,prevention and treatment of angina pectoris in CHD.Recent animal studies indicate that acupuncture preconditioning at Neiguan (PC6) point has a cardioprotective effect on ischemia-reperfusion in the myocardium [33].Methods of preparing acupuncture preconditioning for myocardial I/R injury rats model are ligating anterior descending branch of the left coronary artery for 40 min and re-perfusing the artery for 60 min.The myocardial I/R injury rats model in an acupuncture preconditioning group were pretreated with acupuncture applied at bilateral Neiguan (PC6)point for 20 min once a day for 5 consecutive days before ischemia.The Neiguan (PC6) point of rats is located on the point in the midline and 3mm above the tranverse crease of forelimbs.

Acupuncture preconditioning and miRNA-214/NCX1 mRNA co-expression network

Studies in laboratory animals have shown [7-8, 10,33-35] that myocardial protective effects of acupuncture preconditioning at Neiguan (PC6) point for myocardial I/R injury [8-10] are analogous to IPC,but the mechanism behind the effects remains unclear.Several possibilities exist, including the repression of oxygen-free radicals, release of endogenous active substances (e.g.adenosine), up-regulation of Ca2+-ATPase mRNA and Na+-K+-ATPase mRNA,decrease of Ca2+content in the myocardial cell, or stimulation of the BCL2 protein that represses cell death [36].Acupuncture preconditioning at Neiguan(PC6) point promotes the expression of miR-214 in myocardial I/R injury [4, 6].MiR-214 repression directly targets on NCX1 [3].Tests showed that the relevant protein levels of NCX1 and CaMK IIδ were also decreased in cells of myocardial I/R injury treated with acupuncture preconditioning at Neiguan (PC6)point[6].The miR-214 binding sites are in the 3?-UTR of NCX1 mRNA.The present findings indicate that acupuncture preconditioning at Neiguan (PC6) point mediates many target mRNAs related to Ca2+handling[36].Here, we focus on the relationship between acupuncture preconditioning at Neiguan (PC6) point and modulation of the miR-214/NCX1 mRNA co-expression in myocardial I/R injury.These findings reveal a pivotal role for the miR-214/NCX1 mRNA co-expression network model as a regulator of cardiomyocyte Ca2+homeostasis and survival during myocardial I/R injury.We show that the essential mechanism is that by evoking miRNA-214 in the heart,acupuncture preconditioning at Neiguan (PC6) point regulates NCX1 mRNA expression, and inhibits the expression of NCX1 and several Ca2+downstream signaling effectors (BCL2, CaMKIIδ)that mediate cell death pathways to play a cardioprotective role for myocardial I/R injury.The mechanism of acupuncture preconditioning that influences the miRNA-214/NCX1 mRNA co-expression network model may prevent and treat myocardial I/R injury(Figure 2).

Figue 2 A miRNA-214/NCX1 mRNA co-expression network model for blocking myocardial apoptosis:a novel hypothesis for acupuncture’s protective mechanism against myocardial I/R injury

Consequences of the hypothesis and discussion

Our hypothesis suggests that acupuncture preconditioning at Neiguan (PC6) point may be a non-pharmacological option of early intervention and prevention of CHD for activating the miRNA-214/NCX1 mRNA co-expression network model and blocking myocardial apoptosis for myocardial I/R injury.Acupuncture preconditioning at Neiguan (PC6)point appears to have myocardial protective effects.If our hypothesis is confirmed by further studies, the scientific evidence will support acupuncture preconditioning for myocardial I/R injury by acupoint selection along the meridian.We hope that our paper stimulates research on this topic, which may have important clinical and public health implications.