A Mouse Model Of Light-Controlled Cardiac Pacing Based On Optogenetics

Objective:Electrical stimulation is a standard technique for artificially controlling cardiac beating frequency.However,in experimental studies,this method has great limitations,such as local electrolysis effect and limited stimulation frequency and duration.Moreover,electrical pacing stimulates all cells in the target range,and can not selectively act on myocardial cells.Experimental research,on the other hand,need to get the myocardial cells.Traditional technology of cardiomyocyte isolation requires special device to perform retrograde perfusion in the main blood vessels of the heart.Experimenters need professional training to proficiently use the equipment,and have a lot of technical problems to overcome during the isolation process,which seriously hindered the progress of experiment.In this study,we experimentally demonstrated the optogenetics-based light-controlled cardiac pacing technology and explored a more convenient method to isolate cardiomyocytes.This is very important in the study of cardiac electrophysiology,especially arrhythmia.Method:1.Crossbreeding of transgenic mice:(1)the?MHC-Cre transgenic mice used in this study express Cre recombinase driven by the?MHC promoter specifically in cardiac muscle.ChR2(H134R)-tdTomato transgenic mice contain two lox P sites and a STOP sequence inserted between the universal CAG promoter and the ChR2(H134R)-tdTomato fusion gene.In the absence of Cre recombinase,there is no expression of ChR2(H134R)-tdTomato.After crossing the two transgenic mice,the Cre recombinase expressed in cardiomyocytes cleaved and recombined the two lox P sites,and the STOP sequence in the middle was removed,resulting in the expression of ChR2(H134R)-tdTomato fusion protein.The mice were divided into two experimental groups:?MHC-Cre~+/ChR2-tdTomato~+group(8-12 weeks)and control group?MHC-Cre~-/ChR2-tdTomato~+(8-12 weeks).(2)Genotype identification of transgenic mice.(3)Fluorescence phenotype identification of mouse hearts.2.Transgenic mice with cardiomyocytes specifically expressing the photoactivated cationic channel channelrhodopsin-2(ChR2)were stimulated by blue light at a specific frequency to depolarize the membrane potential of cardiomyocytes,thereby initiating action potential and controlling cardiac beating.3.A simple method without Langendorff perfusion was used to isolate adult mouse cardiomyocytes.Results:1.By crossing?MHC-Cre mice and ChR2-tdTomato mice,?MHC-Cre~+/ChR2-tdTomato~+(myocardium specifically expressing Cre and ChR2-tdTomato),?MHC-Cre~+/ChR2-tdTomato~-(myocardium specifically expressing Cre but not carrying ChR2-tdTomato gene)and?MHC-Cre~-/ChR2-tdTomato~+(no Cre,with ChR2-tdTomato gene but unable to be expressed due to the presence of upstream termination sequence)were obtained in this study.2.(1)Fluorescence phenotype of transgenic mouse hearts:under the excitation of561 nm LED light source,the hearts of?MHC-Cre~+/ChR2-tdTomato~+mice showed bright red fluorescence in the hearts and no fluorescence in other parts,indicating that the fusion protein ChR2-tdTomato had a high level of tissue specific expression in the hearts of mice with this genotype.Under the same condition,the?MHC-Cre~-/ChR2-tdTomato~+mouse did not have obvious fluorescence in the heart and other parts,indicating that there was no expression of ChR2-tdTomato.(2)Light-controlled cardiac pacing in mice with autonomic heart beating:The spontaneous heart rate of the?MHC-Cre~+/ChR2-tdTomato~+mice that express ChR2-tdTomato in myocardium was about 4 Hz in the absence of blue light stimulation after being connected to the ventilator and thoracostomy.When the mice were stimulated by blue light at 2 Hz,the original autonomic rhythm was disturbed and obvious arrhythmias appeared.When the blue light stimulation frequency was the same as the autonomic pulse frequency,the heart rate was maintained at 4 Hz.When the blue light stimulation frequency of 6 Hz was used,the heart beat was completely dominated by the light and showed sinus rhythm.When the blue light stimulus frequency was increased to 8 Hz,the heart rate was also changed to 8 Hz,but there was a brief arrhythmia.When stimulated with blue light at 10 Hz,the myocardium of the mice could not withstand such rapid depolarization.Although the heart beat was still relatively regular,the electrocardiogram waveform was seriously disturbed.When the blue light stimulus was removed,the hearts of the mice quickly returned to the autonomic pulse rate of 4 Hz,regardless of the stimulation frequency previously used.(3)Light-controlled cardiac pacing in mice with cardiac arrest:in order to examine the response to blue light stimulus in the hearts that stop beating due to ischemic damage,?MHC-Cre~+/ChR2-tdTomato~+mice were not connected to the ventilator after anesthesia,and subjected to direct thoracic opening.Blue light stimulation was applied after the heart stopped beating.It was found that when the stimulus frequency is 1-5 Hz,blue light stimulation can effectively trigger heart beating in mice,exactly at the same frequency as that of the stimulus.When the blue light stimulated at a higher frequency(7.5-10 Hz),the heart rate of the mice was only maintained in the 4-5 Hz range and could not be further increased due to possible myocardial damage.These results indicate that optogenetic technique has the ability of pacing arrested heart,and its pacing effect may be closely related to the degree of myocardial injury.(4)Control mice have no response to blue light stimulation:In order to confirm that the light-controlled beating of?MHC-Cre~+/ChR2-tdTomato~+mouse heart is caused by ChR2 expression in myocardium,this study also conducted the same experiment on?MHC-Cre~+/ChR2-tdTomato~-and?MHC-Cre~-/ChR2-tdTomato~+control mice that do not express ChR2-tdTomato,and found that both mice did not respond to blue light stimulation at 5 Hz and 10 Hz,and their heart rates were maintained at 6Hz.(5)Light-controlled contraction and ChR2 currents of isolated cardiomyocytes:both blue light and electric stimulation could induce the contraction of?MHC-Cre~+/ChR2-tdTomato~+mouse cardiomyocytes,while the?MHC-Cre~-/ChR2-tdTomato~+mouse cardiomyocytes did not respond to the light stimulation.The photostimulated currents were exclusively recorded in the?MHC-Cre~+/ChR2-tdTomato~+cardiomyocytes by patch clamp technique,suggesting that the expression of ChR2 is the determinant of light-controlled cardiac pacing.Conclusion:1.Transgenic mice that specifically express the light-sensitive channel ChR2 in the myocardium can use blue light irradiation for cardiac pacing.When the blue light stimulation frequency is lower than the autonomic heart beating frequency of the mice,blue light stimulation will interfere with sinus rhythm,resulting in temporary arrhythmia.2.When the blue light stimulation frequency was equal to or higher than the spontaneous heartbeat frequency of ChR2 expressing mice,the heart rate of the mice was completely dominated by the blue light stimulation and returned to the spontaneous heartbeat state after the blue light irradiation was removed.3.The hearts of mice expressing ChR2 can also be stimulated by blue light to initiate pacing after they lose their ability to beat autonomically due to ischemic injury.4.The hearts of control mice without ChR2 expression did not respond to blue light stimulation under any circumstances.5.Blue light stimulation can activate the ChR2 channel expressed on mouse cardiomyocytes,produce photocurrent and induce cell contraction.6.The simple method of isolating adult mouse cardiomyocytes without Langendorff perfusion is easier to operate than the traditional Langendorff perfusion method and the cardiomyocytes are in good condition.