Interaction Between Calcineurin And Autophagy In Hypertrophic Cardiomyocytes And The Intervention Mechanism Of Wenxin Granule

Myocardial hypertrophy(MH)is one of the main causes of malignant arrhythmia,and increases the incidence of sudden cardiac death(SCD).In recent years,many studies have shown that Calcineurin(CaN)signal pathway can inhibit the release of Ca2+,and keep the retention of Ca2+ in cardiomyocytes,causing the tissue remodeling of cardiomyocytes and electrophysiological changes.The increase of Ca2+ concentration will cause excessive autophagy in myocardial cells to damage the myocardium,resulting in the occurrence of MH,leading to arrhythmia,HF,and SCD.Wenxin granule(WXKL)is a new anti-arrhythmic Chinese medicine preparation.Many studies have shown that WXKL can not only inhibit myocardial hypertrophy,treat arrhythmia,but also play a role in the electrophysiological changes of hypertrophic cardiomyocytes.Therefore,this research was based on the pathological model of cardiac hypertrophy induced by overexpression of CaN signaling pathway protein.We observed the expression and fluorescence intensity of CaN protein,autophagy marker protein and autophagy-related protein,the changes of cytoskeleton,autophagy and L-type calcium current(ICa-L),by using laser confocal microscopy,electron microscopy and whole-cell patch clamp techniques for experiments to explore the relationship of CaN signal pathway and autophagy during the development of cardiac hypertrophy,and discussed the intervention of WXKL in its mechanism,in order to inhibit myocardial hypertrophy,treatment of arrhythmia,heart failure,reduce the incidence of SCD providing a new idea.Objectives:1.We observed that the effect of up-regulation/down-regulation of CaN channel protein on the autofluorescence marker protein LC3-II fluorescence intensity,the size of myocardial cytoskeleton,autophagy bodies,and myocardial ultrastructure and the effect of autophagy activator Rapamy cin(Rapa)and autophagy inhibitor 3-methyladenine(3-MA)on the fluorescence intensity of CaN channel proteinby,by using laser scanning confocal microscopy and scanning electron microscopy,to explore the autophagy and CaN signal pathway interactions during the development of MH,2.To explore the regulatory effect of WXKL on autophagy and CaN signaling pathway for the treatment on cardiac hypertrophy.Methods:1.Divide the cells into 8 groups:Control group,Rapa group,3-MA group,SiCaN group,SiCaN+Rapa group,OverexpressCaN group,OverexpressCaN+3-MA group and OverexpressCaN+WXKL group.After injection and dyeing,the fluorescence intensity and nuclear transfer rate of CaN protein,LC3 protein and NFATc4 protein were observed under confocal laser scanning microscope.2.The experiment was divided into 12 groups:Control group,Rapa group,3-MA groups,WXKL group,SiCaN group,SiCaN+Rapa group,SiCaN+3-MA group,SiCaN+WXKL group,OverexpressCaN group,OverexpressCaN+Rapa group,OverexpressCaN+3-MA group and OverexpressCaN+WXKL group.After drug treatment,cells were collected and stored in electron microscope stationary liquid.Then dehydration,embedding and ultrathin sections were performed.At last,ultrastructure of myocardial cells and autophagy bodies were observed by transmission electron microscope.3.Cell culture and group administration methods are the same as before.After treatment of the cells,the cells were stained with a phalloidin working solution and the changes in the cytoskeleton of the myocardium were observed under the laser confocal microscope.4.The experiment was divided into 4 groups.The methods of cultivation and dosing were the same as before:Control group,OverexpressCaN group,OverexpressCaN+3-MA group and OverexpressCaN+WXKL group.Whole-cell patch clamp technique was used to record ICa-L and current in voltage clamp mode.After the membrane capacitance was stable,the cell membrane capacitance and the potassium current was recorded.To eliminate errors caused by cell size,I values are expressed as current density(pA/pF).Results:1.The results of fluorescence intensity of laser confocal protein:compared with the Control group,the fluorescence intensity of LC3 protein,CaN protein and NFATc4 protein increased significantly in group OverexpressCaN,and the expression of CaN and NFATc4 in the nucleus and cytoplasm increased,which were consistent with the H9C2 cell effect after Rapa treatment.In the SiCaN group,compared with the Control group,the fluorescence intensity of LC3 protein decreased obviously and the fluorescence intensity and nuclear transfer of CaN and NFATc4 protein also decreased;after Rapa treatment,the fluorescence intensity and the nuclear transfer rate increased.The role of 5 g/L WXKL to OverexpressCaN myocardium was that it could obviously decrease the fluorescence intensity of LC3,CaN,NFATc4 protein and the nuclear transfer rate of CaN,NFATc4 protein,which is consistent with the effect in OverexpressCaN+3-MA group.2.The effect of up/down-regulation of CaN protein expression on autophagosomes and the intervention of WXKL:the mitochondrial bilayer membrane structure of control group was intact,with normal size and shape,and the internal iliac structure was intact;when Rapa was added,the mitochondrial bilayer membrane structure was destroyed,the internal hemorrhoidal structure was blurred,Intrinsic structure was ambiguous and disorderly.In SiCaN group,the shape of mitochondria was irregular and the structure of the double layer membrane was blurred;after adding Rapa,the mitochondria increased obviously,the internal structure of the vacuoles appeared in the interior,and the internal crista structure appeared incomplete.In overexpressCaN group,large vesicle-like structures surrounded mitochondria in mitochondria,and mitochondria were swollen,deformed,and ridges were broken or disappeared;after 3-MA treatment,the volume of mitochondria was significantly reduced,the structure of internal hemorrhoids was improved,and vacuoles disappeared.Mitochondrial structure improved after WXKL intervention,and lysosome color faded.3.Laser confocal measurement of cardiomyocyte protein skeleton results:The length and width of myocardial cells in Control group were 349.24±56.43 ?m and 125.59±18.74 ?m,respectively(n=10).Compared with Control,the length and width of myocytes induced by Rapa increased.The overexpressed CaN cell morphology significantly increased and the cell length and width respectively increased to 837.45±57.61 ?m and 268.83±64.16 ?m(n=10,P<0.01,vs.Control group,the difference was statistically significant);after stimulation with 3-MA and 5 g/L WXKL,the cell volume was significantly reduced.4.Compared with the Control group,the amplitude of ICa-L in OverexpressCaN group was significantly increased,and the current density was significantly increased in the range of-40 mV to+40 mV(n=3,P<0.01,vs.Control group,the difference was statistically significant);The steady-state activation curve and steady-state inactivation curve shifted to the left,The half-activated voltage(V1/2,act)and The half-inactivation voltage decreased.After the action of WXKL and autophagy inhibitor 3-MA,the current amplitude significantly reduced the current density in the range of-40 mV to+40 mV(n=3,P<0.01,vs.OverexpressCaN group,the difference was statistical significance),The SSA curve and SSI curve shifted to the right,V1/2,act and V1/2,inact were increased.Conclusions:1.In the process of hypertrophy of cardiac myocytes,CaN signaling pathway interacts with autophagy.Up-regulation of CaN expression activates abnormally active autophagy,and down-regulation inhibits autophagy.Activation of autophagy can increase the fluorescence intensity of CaN protein and NFATc4 protein,increase the rate of nuclear transfer,and the effects of inhibiting autophagy are opposite.WXKL can inhibit MH and treat arrhythmia by regulating CaN signaling pathway and autophagy of cardiac myocytes.2.The expression of CaN protein can activate autophagy in cardiac myocytes,causing ultrastructural damage and dysfunction,reducing the energy supply of the myocardium,increasing the oxygen consumption of the myocardium,destroying the structure and function of the myocardium,and causing MH.WXKL can inhibit the changes in the tissue structure of myocardial cells caused by the up-regulation of CaN protein,thus inhibit autophagy of cardiac myocytes to block the development of MH,which is a new way to protect the myocardium.3.Over-expression of CaN protein,the autophagy will start in cardiac myocyte,which causes the changes of myocardial cell structure,the enlargement of the skeleton,the increase of the stability of the cardiac myocytes,the decrease of the elasticity,the decrease of the contractile force and the decrease of the myocardial compliance.When the autophagy is suppressed,the CaN signal pathway can be counter regulated and the function of the myocardium can be protected.When WXKL acts on OverexpressCaN cells,the cytoskeleton changes significantly,indicating that WXKL can protect the myocardial function by regulating CaN signaling pathway.4.WXKL and autophagy regulate the CaN signaling pathway.At the same time,WXKL can also inhibit autophagy,protect mitochondria structure and function of cardiac myocytes,stabilize cell energy metabolism and calcium homeostasis,accelerate calcium circulation,stimulate calcium Exodus,and reduce intracellular Ca2+ concentration.This effect provides a new mechanism for WXKL treatment of MH,arrhythmia,and provides a new way of thinking for clinical practice.