Expression Of DPP6 In The Hearts Of Different Animal Species

The transient outward potassium current(I_to)is the main component of the repolarization phase 1 of cardiomyocyte action potential,which can affect the action potential morphology and action potential duration.The transient outward potassium channel is composed of homotetramer formed by Kv4?Kv4.3 or Kv4.2?in the pore region subunit and auxiliary subunit.The recognized auxiliary subunits currently are Potassium channel interacting protein 2?KChIP2?and Dipeptidyl peptidase-like protein subtype 6?DPP6?.KChIP2 has deeper understanding of cardiac expression distribution and function in the I_to channel.DPP6 is a single transmembrane protein and discovered in the central nervous system originally,which can accelerate the attenuation of Kv4 current and the reactivation of channel.In recent years,it has been reported that DPP6 gene mutation can induce family idiopathic ventricular fibrillation.Our previous experiment found that DPP6intermediate channel regulator NS5806 that had different pharmacological response to I_too of different species,suggesting that DPP6 auxiliary subunit played an important role in the I_to channel of hearts.However,little is known about the distribution of DPP6 in animal hearts.DPP6 is known to have multiple splice isoforms,the most common of which are DPP6-L?long?and DPP6-S?short?.This study examined the expression of DPP6-L and DPP6-S in hearts of different animal species and different regions of the hearts,which is expected to provide an experimental basis for revealing the physiological function of DPP6.Methods:1.By qRT-PCR method firstly,detect the mRNA expression of DPP6-L and DPP6-S in the left ventricle of mouse.Because the mRNA of DPP6 was found to be significantly expressed in brain tissues,so we used the brain tissues as a positive control.PCR primers are designed based on different sequence of the N-terminal intracellular domain.2.Western blot was used to observe the protein expression of DPP6-L and DPP6-S in mice,rats,rabbits,canine hearts?different parts?and human induced pluripotent stem cell differentiated cardiomyocytes?hiPSC-CMs?.The antibody used in this experiment was a polyclonal antibody of 316³66amino acid residues of rabbit anti-human DPP6,which could detect DPP6-L and DPP6-S simultaneously.3.To observe the effect of I_to regulator NS5806 on the QTc?QT after heart rate correction?interval of isolated hearts,and further verify the regulation of DPP6 on I_to channel.Adult SD rats were anesthetized with intraperitoneal injection of pentobarbital sodium.The hearts were quickly opened and removed,then the Langendorff perfusion device was used to perfuse the electrocardiogram?corresponding to the II guide of the surface electrocardiogram?.After stabilization,we observed the influence of 10?M NS5806 on the electrocardiogram,measured the QT and RR intervals,calculated the average of 10 consecutive QRS complexes,and obtaind the change of QTc?QT after heart rate correction?.Results:1.mRNA expression of DPP6 in the left ventricle of miceThe mRNA expression of DPP6-S in mice brain tissues were higher,and DPP6-L had certain mRNA expression,which was consistent with previous literature report.However,the mRNA expression level of DPP6-L and DPP6-S in the left ventricle of mouse was low and almost undetectable.2.Protein expression of DPP6 in the hearts of different species of animalsDue to the lack of specific antibody of DPP6-L and DPP6-S,we have known that the brain tissues have DPP6-L and DPP6-S expression,mainly DPP6-S.Therefore,brain tissues were used as a positive control.In the brain tissues,the band with low expression and slightly larger molecular weight is DPP6-L,the molecular weight is 115 KDa approximately;the band with high expression and slightly smaller molecular weight is DPP6-S,the molecular weight is 105 KDa approximately.The results showed that DPP6-S was predominantly expressed in mice brain tissues,and DPP6-L expression was less,which was consistent with previous literature report.DPP6-L was predominantly expressed in the left ventricle of mice,DPP6-S was hardly expressed;DPP6-L was predominantly expressed in hiPSC-CMs,and DPP6-S was less expressed;The expression of DPP6-S is predominant in the left ventricle of canines,with little expression of DPP6-L.It is known that there is a significant regional difference in the I_to current density of the hearts,and we further examined the expression of DPP6 in different parts of the hearts.The results showed that the rats hearts?left subventricular,middle and subepithelial tissue,right ventricle,ventricular septum,atrium?,mice hearts?left subventricular and subepithelial tissue,right ventricle,ventricular septum,atrium?,rabbits hearts?left ventricle,right ventricle,left atrium,right atrium?were mainly DPP6-L,and the protein expression was not significantly different in different parts of the hearts.3.Effect of NS5806 on cardiac electrocardiogram of Langendorff isolated rats perfusion 10?M NS5806 significantly prolonged the QTc interval of isolated ECG in rats,from 139 ms before dosing to 265 ms after dosing.Paired T-test was used before and after administration,calculated mean and standard deviation,P<0.01,the difference was considered statistically significant.Conclusion:DPP6 had significant protein expression in animal hearts,but there was species difference.DPP6-L was expressed in rats,mice and rabbits hearts.DPP6-L and DPP6-S were simultaneously expressed in human cardiomyocytes,but the former was mainly;the ventricle of canines mainly expressed DPP6-S oppositly.There was no significant difference in the expression of DPP6-L in different parts of rats,mice and rabbits hearts.