β3-adrenergic receptor polymorphism-dependent regulation of L-type calcium current and force of contraction in human atrium

Abstract

Introduction. β3-adrenergic receptor (β3-AR) agonists stimulate L-type Ca²+ current (I Ca,L) and force of contraction in human atrium. contraction force of the heart muscle depends on Ca²+ ion current through L-type calcium channels in plasma membrane of single myocytes, which is predominantly regulated by β-adrenergic receptors and cAMP-dependent phosphorylation. One nonsynonymous single nucleotide polymorphism Trp64Arg of β3-AR has been reported, which causes weaker cAMP accumulation comparing to Trp64Trp one. Therefore, we examined a role of β3-AR polymorphism in β3-AR agonist CGP12177 and BRL37344 induced stimulation of ICa,L and contraction force in isolated human atrial myocytes and atrial trabeculae, respectively. Methods and Materials. Genotyping of β3-AR Trp64Arg polymorphism was performed by the restriction fragment lenght polymorphism method. The PCR reaction was set up as follows (50μl): 20 ng of genomic DNA extracted from the whole blood ("Nucleospin Blood L", Macherey-Nagel GmbH&Co), 1XTaq buffer (100mM Tris-HCl (ph 8.8), 500 mM KCl, 0.8% (v/v) Nonidet P40), 1.5 mM MgCl2, 5% DMSO, 0.2 mM each of dNTP (Fermentas), 0.4 μM each of the primer (5'-CGCCCAATACCAACAC-3'; 5'-CCACCAGGAGATCACC-3, "Biomers"), 1 u of recombinant Taq DNA polymerase (Fermentas). The PCR reaction began with initial denaturation at 940C for 3 minutes, followed by 30 cycles of denaturation at 950C for 45 seconds, annealing at 600C for 45 seconds, extension at 720C for 30 seconds, with a final extension at 720C for 10 minutes. The amplified PCR products were digested with MvaI restriction endonuclease and analyzed by 10% non-denaturing polyacrylamide gel electrophoresis. Specimens of right atrial trabeculae were obtained from patients undergoing heart surgery or transplantation in Kaunas University Hospital (KUH), and all protocols for obtaining human cardiac tissues were approved by the ethics committee of KUH. The whole-cell configuration of the patch-clamp technique was used to record ICa,L in enzymatically isolated human atrial myocytes. Isometric contraction of atrial trabeculae was recorded using a mechanoelectrical force transducer [1]. Results We examined genotype of 65 control DNA and 64 DNA samples obtained from KUH patients. The rate of Trp64Trp and Trp64Arg genotype was 10% and 8%, respectively. In case of Trp64Trp, CGP12177 and BRL37344 stimulated ICa,L by 64±19% (n=10) and 122±27% (n=22) over control, respectively, and force of contraction by 13±3.9% (n=9) and 26±2.7% (n=4) over control, respectively. In case of Trp64Arg, CGP12177 and BRL37344 stimulated ICa,L by 33±4.9% (n=7) and 94±11% (n=3) over control, respectively, and force of contraction by 4.2±2.2% (n=2) and 5.3±1.8% (n=2) over control, respectively. Figure1 demonstrates typical experiments when ICa,L and force of contraction were stimulated by 1 mM and 10 mM of BRL37344, respectively. The weaker stimulation of ICa,L by β3-AR agonists in case of Trp64Arg genotype correlates with results of other authors [3] demonstrating the reduced ability of b3-AR Trp64Arg to generate cAMP accumulation. Conclusions Our preliminary results indicate that the efficacy of β3-AR-dependent stimulation of ICa,L and contraction force may depend on Trp64Arg polymorphism, however to make a final conclusion a number of Trp64Arg cases must be significantly increased.