Research On The Roles Of HCN4in The Injury Of Rats’ Sinoatrial Node Induced By Microwave Exposure

Objective and Significance: With the advent of information era and the high speeddevelopment of microwave technology, people are accessible to the hazard of microwavepollution as well as the conveniences it brings about. Besides, microwave technology also saw itsincreasing development and application in military fields. It can not only severely damage theenemy’s electronic devices, but also do harm to the body. Conducting system of heart is one of thetargets sensitive to microwave radiation. However, as the essential component of heart conductingsystem, SAN’s pattern of damage from microwave radiation and their dose-effect relationshipremained unclear, and the damaging mechanism was uncovered. HCN4can regulate pacemakercells’ pacing current and maintains their physiologic functions. The abnormal expression of HCN4in the SAN tissues may be a molecular basis of a variety of SAN pathological changes, whileHCN4and its regulation may play an important role in rats’ SAN damage after microwaveradiation. It is followed that the study of the HCN4change, its regulation and significance in theSAN’s damage from microwave radiation will provide a new thought thread and target for thefurther study of molecular mechanism and protective measures of microwave radiation damage toheart. Meanwhile it can provide experimental evidence for the identification of sensitive diagnosisindex and the formulation of protective criteria.Materials and Methods:（1） Positioning of rats’ SANs and observation of their organizationalstructure: Twenty second-class adult Wistar rats were used to conduct the horizontal serial sectionsof the right atrium and the associated proximal segment of the superior vena cava. Serial sectionswere stained by HE and Masson staining and subsequently observed by light microscopy to locatethe position of SAN and to observe its organizational structure.（2） Observation of rats’ SAN’sultrastructure: Based on the SAN location and organizational characteristics,10second-class adultmale Wistar rats were taken to get the materials in two steps, the semi-thin sections werepre-stained by toluidine blue after the resin directed embedding and positioned through lightmicroscope. Following ultrathin sectioning, SAN ultrastructure was observed by the transmissionelectron microscopy.（3） Study of the effect of microwave radiation on rats’ SAN function andstructure:160Wistar rats were exposed to a pulse microwave field （0,5,10and50mW/cm²ï¼‰ for6minutes. Multi-channel polygraph was applied to detect the change of ECG in rats beforemicrowave exposure and immediately,7,14,28days and3,6,9months after microwave radiation.Light microscopy and electron microscopy were used to observe the organizational structure andultrastructure of rats’ SAN at1d,7d,14d,28d,3m,6m,9m,12m after microwave exposure.Masson staining, Sirius red staining and image analysis techniques were applied to observe thedynamic development of the content of collagen fiber in rats’ sinoatrial nodes.（4） Research on thechange of HCN4in rats’ SAN tissue and the regulation mechanism after microwave radiation: ISH, IHC and image analysis were used to detect the changes in the gene or protein expression ofHCN4, β1-AR, M2-AchR in rat’s SAN tissues after50mW/m2microwave radiation.（5） Researchon the damaging effect and mechanism of microwave radiation on primary culture SAN cells:SAN cells primarily cultured in vitro were taken to do the research. Inverted microscope, AFM,LSCM and IF were used to observe the effect of microwave radiation on cells’ morphosis,pulsation features, membrane’s structure, the content of Ca²⁺inside the cells and the expression ofHCN4.Results:（1） SAN’s position and histological feature of normal adult rat: Adult rats’ SANswere located in the junctional zone of superior vena cava and right atricle and on the inner-wall ofsuperior vena cava above the junctional zone. The adult rats’ SANs were shaped like the horseshoeor the letter ‘C’. The rats’ SANs, being loose in structure and rich in interstitial collagen fiber,mainly contain P cells, T cells and a small amount of atrial muscle cells.（2） Observation ofSAN’s ultrastructure of adult rats: The toluidine blue prestained semi-thin sections is a rapidand efficient method, which can effectively shorten the positioning time. The electron microscopyshowed that two types of cells were present in the rat SAN: P cells and T cells. With a small cellbody, P cell was rich in cytoplasm but poor in organelle. There were a small number of myofibrilswhich were messily distributed nearby the cell membrane and almost free of sarcomeres. T cellhad a bigger cell body and more organelles than P cell and the myofibrils. With visible sarcomeres,were often arranged along the cell longitudinal axis and close to the cell membrane.（3） Effect ofmicrowave radiation on SAN’s function. There were no significant change in the5mW/cm²group but appeared some damage to SAN’s functions in the10and50mW/cm²groups, whichwere mainly as follows: the heart rate tended to accelerate and then slow down; the amplitude of Pwave was decreased; the ECG showed sinus arrhythmia, sinoatrial node wandering heart rhythmand atrial arrhythmia, etc.（4） Structural changes in rats’ SAN tissues after microwaveradiation: There was no significant change in the group of5mW/cm². But in the10and50mW/cm²groups, changes could be observed like edema in P and T cells, wavy changes in T cells,increasing of acidophilia staining in the cytoplasm of P and T cells, and karyopyknosis andanachromasis from1to28days after microwave exposure. From3to6months, the damagestended to lessen and recover but there were still some cells in the edematous state, and from9to12months, parenchyma cells began to decrease and interstitial collagen fibers were increased, andthere appeared fatty infiltration. Changes above were remarkably observed in the group of50mW/cm².（5） SAN’s ultrastructural changes after exposure to microwave radiation. Therewas no significant change in the group of5mW/cm². In the period of1to28days, mitochondriaof T cells and P cells in the10and50mW/cm²groups were the earliest to be effected to appearswelling, crista breakage, and even cavitation; then came the focal dissolution and even breakageof myofibrils, and concentration of nuclear chromatin of P and T cells to the edge, followed by thewidening and edema of perivascular canal, and the reduction or disappearance of foveolas ofmembrane. In the period of6to12months, there came degeneration in parenchyma cells, increase of interstitial collagen fibrils and fatty infiltration.（6） Changes in HCN4, β1-AR, and M2-AchRin rats’ SAN tissues injured by50mW/cm²microwave radiation. The expression of HCN4mRNA began to be up-regulated at1to28days （P<0.05or P<0.01） and down-regulated at3months （P<0.05）.The expression of HCN4protein was increased at1to28days （P<0.05orP<0.01） and was decreased at3to6months （P<0.05）. The expression of β1-AR mRNA wasup-regulated at1d to3m （P<0.05or P<0.01） while the expression of β1-AR protein was increasedat1d to3m （P<0.05or P<0.01）. The expression of M2-AchR protein was increased after1d⁶m（P<0.05or P<0.01）.（7） Changes in cellular morphosis and pulsation feature of SAN cellscultured in vitro after microwave exposure. The normal cell of SAN was shaped like longspindle, sticking out pseudopodia or apophysis and connected with the cells around, and beatrhythmically in single with a high speed. But immediately after the exposure to10and50mW/cm²microwave, the cells of SAN began to beat significantly slower and irregular and tendedto become swollen and rounding, with pseudopodia or apophysis reducing. Besides, the contentsof Ca²⁺inside the cells of SAN rose immediately after50mW/cm²microwave exposure, andperforation could be observed in the surface of membrane.（8） Changes in the expression ofHCN4in the vitro cultured SAN cells after microwave radiation. The expression of HCN4protein was weakened significantly immediately after50mW/cm²microwave radiation （P＜0.01）and was strengthened significantly after12hours （P＜0.05）.Conclusion:（1） The special location of SANs in adult rats indicated that the proximal segmentof superior vena cava must be reserved when taking SANs from rats and the two-step methodbefore positioning was helpful for the making of electron microscope specimens of rats’ SANs.（2）Exposure to10and50mW/cm²microwave radiation can lead to dysfunction and damage toorganizational structure and ultrastructure of rats’ SANs. The above changes are positivelycorrelated to microwave radiation dose.（3） Exposure to10and50mW/cm²microwave can lead todamage to morphosis and reduction of pulsation ability of cultured SAN cells, which arepositively correlated to microwave radiation dose.（4） The abnormal expression of HCN4is likelyto be one of the important vulnerating molecules in the damaging process of microwave radiationto SANs.（5） In the tissue of SAN, the high expression of β1-AR is likely to aggratate SAN’sinjuries resulted from microwave radiation by stimulating the expression of HCN4while the highexpression of M2-AchR is likely to promote the recovery of SAN’s injuries from microwaveradiation by inhibiting the expression of HCN4.（6） The perforation of membrane, the overloadingof intracellular Ca²⁺, and the high expression of HCN4are important mechanisms leading to SANinjury resulted from microwave radiation.