Ank-G Regulates The Nav1.5 Channel Of Hypoxic HL-1

Recently,some studies have found that myocardial cells are in a hypoxic environment during acute and strenuous exercise.At the same time,a series of changes in cardiomyocytes include changes in electrical activity,increased cardiac conduction and increased heart rate.These changes can easily lead to a series of heart diseases.Hypoxic environment plays a major regulatory role in the electrical activity of the myocardium and participates in the formation of tachyarrhythmia diseases.Ankyrin-G（ANK-G）is related to voltage-gated Na⁺channels（Nav1.5 channel）,Its specific role and mechanism have not yet been fully clarified.Objective:We used hypoxia to treat mouse atrial myocytes（HL-1）to simulate the environment of cardiomyocytes during cardiac hypoxia,explore the effect and mechanism of hypoxia on the electrophysiology of cardiomyocytes,and the intervention effect of Ankyrin-G.Methods:The mouse atrial muscle cell line HL-1 was used as the research object.Under the condition of hypoxia for 2 hours,the following groups were constructed using lentiviral vectors:normoxia group,hypoxia group,ANK-G NC group,ANK-G group,shANK-G NC group,shANK-G group,observe the relationship between Ankyrin-G and Nav1.5 channel and I_Na under hypoxic conditions.At the cellular level,through the whole-cell patch clamp technique,observe the characteristics of each group of I_Na,including the peak sodium current amplitude and density,IV curve,steady-state activation curve,steady-state inactivation curve,post-inactivation recovery curve,etc.Nav1.5The gating mechanism.At the molecular level,Western Blot（WB）was used to analyze the differences in the expression of Ankyrin-G and Nav1.5channel proteins in each group,and the distribution of Nav1.5 channel on HL-1 cells was observed by confocal microscope.Results:（1）The whole-cell patch clamp results showed that under hypoxic conditions,the amplitude of the I_Na peak current of HL-1 cell was significantly increased;hypoxic conditions made the I_Na peak current density significantly higher than that of the normoxia group.At-35 mV depolarization voltage,The peak current density increased from-162.3±12.4pA/pF to-272.5±9.86 pA/pF,（n=15,P<0.01）.The statistical Ⅰ-Ⅴ curve found that the current increase in the hypoxia group was more significant than that of the normoxia group.In the stimulation range of-50 mV to 0 mV,I_Na had a significant increase effect,especially near the peak value.（2）Draw the steady-state activation curve of I_Na current by stimulating HL-1 cells for a long time.It is found that compared with the normoxia group,the steady-state activation curve of the hypoxia group shifted slightly to the left,and hypoxia may accelerate the steady-state activation process slightly.,But there is no statistical difference.The dual stimulation mode was used to draw the steady-state inactivation curve of I_Na current.The results showed that compared with the normoxia group,the steady-state inactivation curve of the hypoxia group shifted significantly to the right.Using dual stimulation mode,drawing the recovery curve after I_Na current inactivation found that hypoxia treatment caused the recovery curve after inactivation to shift slightly to the left,but there was no statistical difference.（3）Compared with the normoxia group under hypoxia,the Ankyrin-G protein level increased significantly（n=3,P<0.01）,and the Nav1.5 protein level also increased significantly（n=3,P<0.01）;laser confocal results It also suggests that under hypoxic conditions,the expression of Ankyrin-G in the membrane and in the cell increases,and Nav1.5 channel has a significant increase and a tendency to redistribute to the membrane.（4）The WB results of each group showed that the increase of Ankyrin-G protein in the ANK-G group under hypoxia can significantly increase the level of Nav1.5 channel protein（n=3,P<0.01）,while the shANK-G group,Nav1.5 channel protein level was significantly reduced（n=3,P<0.05）.（5）The laser confocal results suggest that the increase of Ankyrin-G in the ANK-G group under hypoxia can promote the increase of the distribution of Nav1.5 channel toward the cell membrane,while the fluorescence of Nav1.5 channel in the shANK-G group is significantly reduced.（6）Under hypoxic conditions,overexpression of ANK-G increased the peak current amplitude and density of I_Na.At-35 mV depolarization voltage,the peak current density increased from-240.6±16.5 pA/pF to-332.1±16.3pA/pF（n=15,P<0.01）;Under the same conditions,silencing the ANK-G gene significantly reduced the peak current amplitude and density of I_Na.At-35 mV depolarization voltage,the peak current density was from-247.8±8.5pA/pF decreased to-178.3±10.4 pA/pF（n=15,P<0.01）.Conclusion:（1）Hypoxia conditions increase the I_Na amplitude and density of HL-1cell;（2）The gating mechanism of I_Na under hypoxic conditions is related to steady-state inactivation,but has nothing to do with steady-state activation and recovery after inactivation;（3）Hypoxia can promote the redistribution of Nav1.5 channel protein to the cell membrane,and under hypoxic conditions,increase the level of Ankyrin-G protein in HL-1 cells,which can promote the redistribution of Nav1.5 channel protein to the cell membrane,thereby increasing I_Na peak current amplitude and density.