The Mechanism Of Cardiac Fatty Acid Binding Protein(FABP3) In The Regulation Of Calcium Channel RyR2 Activity

Objective:FABP3 inhibited calcium transients in mouse cardiomyocytes.Previously we have demonstrated that FABP3 lowers contractility of mouse ventricular myocytes by depleting SR Ca storage.However,the underlying mechanism is still not completely understood.Calcium removal rate is significantly slowed by FABP3,strongly indicating SERCA function is impaired by FABP3 perfusion.Additionally,we also showed that FABP3promotes binding between SERCA and its accessory protein PLB,a major mechanism causing reduced SERCA activity.Thus SERCA is very likely an intracellular target of FABP3.However,whether RyR2,another major intracellular calcium transporter,is affected by FABP3 is still under debate.Apparently FABP3 at max concentration lowers calcium spark frequency（insignificantly though）,a major indicator of RyR2 activity in vivo,it is accompanied with a depleted SR calcium content,which by itself may lead to reduces calcium spark frequency.This project is designed to uncover the underlying mechanism by which FABP3 depletes SR calcium content.Methods:1 Male C57BL/6 mice 12-14 weeks of age provided by Hebei Experimental Animal Center were used in this study;2 Western blot was used to detect the protein expression of Drd1 and Drd2in myocardial tissue;3 Calcium imaging was used to study the dynamic changes of calcium transients in mouse left ventricular cardiomyocytes;4 Observe the measurement of myocardial cellular calcium spark under FABP3 dosing condition;5 Immunofluorescence analysis of colocalization of FABP3 with Drd1and Drd2;6 Co-immunoprecipitation to study whether FABP3 binds to Drd1 and Drd2;7 Statistical analysis and related analysis:The data was represented by MEAN±SE.SPSS 13.0software was used for statistical analysis.The test was significant at P<0.05.Results:1.RyR2 is an intracellular target of FABP3 other than SERCA.1.1 In mouse ventricular myocytes,FABP3 inhibits SERCA activity in a dose dependent manner with an EC₅₀ of 0.435 nmol/L.1.2 FABP3 inhibition on calcium transient amplitude is also dose-dependent,with EC₅₀ being an order of magnitude lower.1.3 SR Ca content is depleted at max FABP3,but not a 0.005 nmol/L.1.4 calcium spark frequency is significantly increased at 0.005 nmol/L FABP3,with characteristics being significantly.1.5 furthermore,incidence of calcium waves is significantly increased as well.2 Dopamine receptor FABP3 interacts with Drd1 and Drd2.2.1 Expression of dopamine receptor in ventricular myocytes（Western blot and Immunostaining data）.2.2 Binding between FABP3 and Drd1,Drd2.2.3 Direct effect of dopamine on cardiomyocytes is negative instead of positive inotropic.Conclusions:1.Ry R2 is an intracellular target of FABP3 other than SERCA.2 Dopamine receptor FABP3 interacts with Drd1 and Drd2.