Electrophysiological Characteristics Of Cardiac Sca-1 Positive Stem Cells In Mice

Background:Myocardial infarction is a type of cardiovascular disease caused by myocardial ischemia and necrosis due to coronary artery stenosis or occlusion.The current clinical treatment can only delay the occurrence of heart failure and reduce the clinical mortality rate by improving the symptoms of the acute phase,but it cannot fundamentally solve the problem of progressively reducing the number of myocardial cells after myocardial infarction.In recent years,stem cell treatments have brought new hope to solve this problem.Stem cells are the main seed cells in the body,which can proliferate and differentiate into different types of tissue cells.Due to this characteristic of stem cells,it is widely used in the treatment of myocardial infarction,which is mainly to increase the number of myocardial cells,improve the microenvironment for the treatment direction,and improve the heart failure after myocardial infarction.Therefore,it is considered to be one of the most promising methods for the treatment of myocardial infarction.At present,there are many types of stem cells used to treat myocardial infarction,including:embryonic stem cells（ESCs）,cardiac stem cells（CSCs）,and bone marrow mesenchymal stem cells（BMSCs）,adipose stem cells（Umbilical cord blood mesenchymal stem cells）,hematopoietic stem cells,and the like.A large number of studies have shown that stem cell transplantation can improve the heart structure and function of infarcted animals to varying degrees,but some studies have also demonstrated that stem cell transplantation may bring about the side effects of arrhythmia;analysis of its causes revealed that:（1）Stem cells used for transplantation Spontaneous electrical activity exists,and focal ectopic pacemaker sites can be formed,thus arousing the possibility of arrhythmia;（2）There is electrophysiological heterogeneity between the viable stem cells and the host myocardial cells,which is easy to form Reentry and trigger activities.Therefore,stem cell transplantation may lead to secondary arrhythmias in myocardial infarction animals;but different types of stem cell transplantation for the treatment of myocardial infarction,the side effects caused by arrhythmia may be different.The current study found that in many stem cell types for the treatment of myocardial infarction,cardiac stem cells are directed progenitor cell types,their differentiated cell types are limited to myocardial cells,fibroblasts and vascular endothelial cells.The cells have the same developmental conditions and microenvironment of survival,so their therapeutic effects on myocardial infarction are significantly better than other types of stem cells.So far,many subcategories of CSCs have been discovered,including major types of c-Kit,Sca-1,Isl1,and SP,among which Sca-1⁺stem cells occupy a comparative advantage in the heart.Therefore,whether this type of stem cell transplantation for the treatment of myocardial infarction will lead to the occurrence of arrhythmia,and what the possible mechanism of the occurrence of arrhythmia becomes our focus.Objective:By constructing the model of myocardial infarction and local injection of Lin^-Sca-1⁺CSCs in the border area of the myocardial infarction,it is observed whether the arrhythmia may occur after the transplantation of CSCs,and the similarities and differences of the ionic channel characteristics of the ventricular myocytes and the Lin^-Sca-1⁺CSCs,the main ion channel genotypes and the protein expression levels of the Lin^-Sca-1⁺CSCs are further compared.Objective to analyze the possible mechanism of arrhythmia induced by Lin^-Sca-1⁺CSCs and provide necessary theoretical basis for stem cell treatment for myocardial infarction.Methods:（1）Establish mouse acute myocardial infarction model（2）Isolation and purification of Lin^-Sca-1⁺CSCs（3）Direct myocardial injection of Lin^-Sca-1⁺CSCs for the treatment of myocardial infarctionLin^-Sca-1⁺CSCs were sorted by tissue block enzymatic method and immunomagnetic beads method;Lin^-Sca-1⁺heart stem cells were injected into the marginal area of myocardial infarction;electrocardiogram was collected by BL420biological function experiment system,and CSCs were analyzed for heart treatment.The occurrence of arrhythmias after infarction.（4）Acute isolation of high-quality mouse ventricular myocytesAfter retrograde intubation of the mouse aorta,the heart was enzymatically lysed on a langendorff perfusion device.C57 mouse ventricular myocytes were isolated and patch clamp experiments were performed after calcium reconstitution at room temperature.（5）Mouse ventricular myocyte membrane current recordsSealing by glass microelectrodes,membrane breakage and recording of resting potential（RP）,action potential（AP）,inward rectifier potassium current(I_k1),overdrive delayed outward rectification of mouse ventricular myocytes in Clamp 10.3 software Potassium current(I_kur),instantaneous outward potassium current（Ito）,sodium current(I_Na),L-type calcium current(I_Ca-L).（6）Ion channel current records of Lin^-Sca-1⁺CSCsThe Lin^-Sca-1⁺CSCs were subcultured to the third generation and their RP,AP,I_k1,I_kur,I_to,and I_Na were recorded using the patch clamp technique.（7）Transcriptome sequencing analysis of transcriptional levels of major ion channel genes in Lin^-Sca-1⁺CSCs.（8）Western blot analysis of the expression of different ion channel proteins in ventricular myocytes and Lin-Sca-1+CSCs.Results:（1）Four weeks after local injection of high-purity Lin-Sca-1+CSCs in the marginal region of the myocardial infarction,electrocardiographic monitoring showed that the incidence of arrhythmias was slightly higher in Lin-Sca-1+CSCs group than that in PBS group.（2）The ion channel current characteristics of mouse ventricular myocytes were recorded by patch clamp.The results showed that the resting potential（RP）of mouse cardiomyocytes was（-70.90±0.60 mV,n=8）;clamping voltage At 50 mV,the instantaneous outward potassium current（Ito）density was（IpA/pF=13.68±1.07,n=7）;the clamping current=0 mV at a sodium current（INa）density（IpA/pF=-11.12±0.66,n=5）;when the clamp voltage is-120 mV,the inward rectifier potassium current（Ik1）density is（IpA/pF=-27.41±1.39,n=8）;when the clamp voltage is 0 mV,the calcium current（ICa-L）density is（IpA/pF=-10.72±0.30,n=5）;Overvoltage-activated outward delayed rectifier potassium current（Ikur）density（IpA/pF=7.73±1.10,n=5）at clamping voltage=50 mV.（3）After studying the membrane current characteristics of mouse Lin-Sca-1+CSCs,the resting potential（RP）was（-21.03±1.96 mV,n=8）;when the clamping voltage was50mV,The outward potassium current（Ito）density was（Ip A/pF=2.83±0.57,n=8）;the clamped voltage=0 mV,the sodium current（INa）density was（Ip A/pF=-1.69±0.24,n=3）;When the clamp voltage is-120 mV,the density of the inward rectifier potassium current（Ik1）is（IpA/pF=-2.64±0.41,n=8）;when the clamp voltage is 50 mV,the overcurrent-activated outward delayed rectifier potassium current（Ikur）density is（IpA/pF=3.57±0.28,n=6）（4）Through transcriptome sequencing analysis it was found that:The main genes regulating the transcription of the above ion channels in Lin^-Sca-1⁺CSCs were as follows:I_Na ion channel gene SCN7A,I_to ion channel gene KCNA1,I_k1 ion channel gene KCNJ8,I_kur ion channel gene KCNA5.（5）Passed The expression of ion channel protein in C57 mouse ventricular myocytes and Lin^-Sca-1⁺CSCs was analyzed by Western blot.The results showed that:I_Na ion channel protein Scn7a,I_to ion channel protein Kv1.1,I_k1 ion channel protein Kir6.1.I_kur ion channel protein Kv1.5 is in both cardiomyocytes and stem cells In the expression,the I_Na ion channel protein Scn7a was more expressed in stem cells than that in ventricular myocytes,and there was no significant difference in the expression of other ion channel proteins between stem cells and ventricular myocytes.Conclusion:（1）Lin^-Sca-1⁺CSCs can induce low-risk arrhythmias to a certain extent in the treatment of myocardial infarction of mice.（2）There is a certain difference in ion channel current size,gene transcription level and ion channel protein expression level of Lin^-Sca-1⁺CSCs and ventricular myocyte membrane in mice.These differences may be the key factors to induce arrhythmia after stem cell transplantation.