Effects Of Aerobic Exercise On BK_Ca Channels In Arterial Myocytes From AKAP150 Gene Specific Knock-in Mouse

Objective:Large-conductance calcium-activated potassium channels play an important role in the maintenance of vascular myogenic tension.A-kinase anchoring protein 150(AKAP150)can coordinate spatiotemporally multiple signal transduction molecules,making signal transduction accurately,participating in the remodeling of ion channels in hypertension,and be involved in the regulation of arterial blood pressure and arterial tension.Our project previously established an AKAP150 gene smooth muscle specific knock-in model mouse(AKAP150 smKI)to explore the role of vascular smooth muscle AKAP150 in aerobic exercise improving vascular function in hypertension.Here,based on the latent bidirectional regulation of BKCa channels by vascular smooth muscle AKAP150,exploring the critical regulation of vascular function and blood pressure by vascular smooth muscle AKAP150,we further explain the mechanism of vascular smooth muscle AKAP150 in aerobic exercise-induced BKCa channel reverse remodeling of arterial smooth muscle in hypertension.Methods:(1)Using embryonic stem cell targeting technology,we established the mouse model of AKAP150 gene smooth muscle specific knock-in(AKAP 150 smKI).(2)The 3-month-old wild type(WT)mice and AKAP 150 smKI mice were randomly assigned to four groups:wild type quiet control group(WT-SED),wild type exercise group(WT-EX),AKAP150 gene smooth muscle specific knock-in quiet control group(AKAP 150 smKI-SED),and AKAP 150 gene smooth muscle specific knock-in exercise group(AKAP 150 smKI-EX).The exercise groups were performed with 12 weeks moderate intensity aerobic treadmill training.(3)After aerobic exercise,mesenteric arteries were harvested,and vascular smooth muscle cells were obtained by acute enzymatically isolation.The single-channel inside-out patch clamp recording technique was used to detect the biophysical characteristics of the BKCa channel of arterial smooth muscle.(4)The protein expressions of AKAP150,BKCa channel a subunit and β1 subunit in mesenteric artery were determined by Western blot analysis.Results:(1)The arterial blood pressure(systolic,diastolic,and mean arterial pressure)of 3-month-old AKAP150 smKI mice was higher than that of WT mice of the same age(P<0.01).After aerobic exercise,the blood pressure decreased in AKAP150 smKI-EX group(P<0.01),and the systolic blood pressure of WT-EX group decreased(P<0.01).(2)There was no difference in the single channel slope conductance of BKCa channel among four groups(P>0.05).(3)Compared with the WTSED group,the BKCa channel[(Normalized NPo)-V]relationship curve of the AKAP150smKI-SED group was right-shifted,with an enlarged V1/2 value(P<0.01);the curve in WT-EX group left-shifted,with a decreased V1/2 value(P<0.01).In contrast to AKAP150smKI-SED group,the curve in the AKAP150smKI-EX group left-shift with a decreased V1/2 value(P<0.01).(4)Compared with WT-SED group,the curve of the BKCa channel[(Normalized NPo)-Ca2+]relationship in AKAP150smKI-SED group was right-shifted with an increased Kd value(P<0.01);the curve of WT-EX group leftshifted,and the Kd value showed a downward trend(P>0.05).Relative to the AKAP150smKI-SED group,the curve in AKAP150smKI-EX group left-shifted,and the Kd value decreased(P<0.01).(5)Compared with WT-SED group,the open probability of BKCa channel in AKAP150 smKI-SED group decreased(P<0.01),with a shorter mean open time(P<0.01)and a longer mean closed time(P<0.01).The open probability of BKCa channel in WT-EX group was increased(P<0.01),the average open time was shortened(P>0.05)and the average closed time was prolonged(P<0.05);Meanwhile in AKAP150smKI-EX group,the open probability of BKCa channel was increased(P<0.01),average open time was prolonged(P<0.05)and average closed time was shortened(P<0.01).(6)Compared with WT-SED,the sensitivity of Tamoxifen in AKAP150smKI-SED group was attenuated(P<0.01).The sensitivity of Tamoxifen was greater in AKAP150smKI-EX group and WT-EX group after aerobic exercise(P<0.01).(7)There was no difference in the expression of a subunit among the four groups(P<0.01).Compared with WT-SED,the expression of AKAP150 was increased(P<0.01),β1 subunit expression was decreased(P<0.05),and β1/α ratio was downregulated in AKAP150 smKI-SED group.After aerobic exercise,the expression of AKAP150 was decreased(P<0.05),β1 subunit was increased(P<0.01)and β1/αratio was upregulated(P<0.05)in AKAP150smKI-EX group;in WT-EX group,AKAP150 protein expression was not significantly different(showing a decreasing trend),β1 subunit was increased(P<0.05),and the β1/α ratio was upregulated(P<0.05).Conclusion:AKAP150 gene smooth muscle specific knock-in can significantly increase the arterial blood pressure of mice,down regulate the expression of BKCa channel β1 subunit in arterial smooth muscle cell,and significantly decrease the function of BKCa channel.Aerobic exercise can effectively reverse the remodeling of BKCa channel by inhibiting the expression of AKAP150 in vascular smooth muscle cell,inducing blood pressure decreasing,suggesting that vascular smooth muscle AKAP150-BKCa does play a critical role in the improvement of vascular function by aerobic exercise in hypertension.