Using FGF13 Cardiac Conditional Knockout Mice To Study Effects And Mechanisms Of FGF13 In Cardiac Arrhythmia

Fibroblast growth factor homologous factors(FHFs), including FGF11-FGF14, are the novel modulators of Voltage-gated sodium channels(Nav channels), and can modulate cardiac excitability by regulating the expressing and function of Nav channels. Nav channel plays important role in excitability and conduction in excitable cells. In heart, the abnormalities of Nav channels are closely related with arrhythmias. Previous studies found FGF14 mutation and FGF12/FGF14 knockout mice show spinocerebellar ataxia phenotype. Our recent studies found FGF13 was abundantly expressed in murine heart, and regulated Nav channel function as well as conduction of excitability. However, so far there are absent of direct evidence about whether FHFs is related to cardiac arrhythmia, and which type of arrhythmia is related. To answer this scientific question, we detect the effects of knockout FGF13 on cardiac electrical activity by generating and using FGF13 cardiomyocyte conditional knockout mice. We also study the effects and related mechanism of FGF13 on cardiac action potential and sodium curents. The present study would provide important information to uncover the roles and mechanisms of FHFs on cardiac arrhythmias. Part 1 Generation of FGF13 cardiac conditional knockout miceObjective:(1)To generate FGF13 cardiac conditional knockout mice.(2)To evaluate the knockout efficiency of FGF13 cardiomyocyte conditional knockout mice.Methods:(1)Using electrotransformation, southern blot, micro- injection and implantation techniques to acquire the F1 generation heterozygous mice(FGF13-flox-neomycin).(2)In order to get ride of neomycin and FGF13-loxp mice, we use Flp-deleter mice crossing with FGF13-flox-neomycin mice.(3)Using myh6-Cre to cross with FGF13-flox mice and to generate FGF13-flox mice expressing Cre recombinase in cardiomyocytes.(4)Applying tamoxifen to generate temporally regulated FGF13 myocardial conditional knockout mice.(5)Using q-PCR, western-blot and immunostaining to evaluate knockout efficiency of FGF13 in FGF13 cardiac conditional knockout mice.Results:(1)Using Cre-Lox P gene targeting princilple, we generated the first generation FGF13-flox-neomycin heterozygous mice, then we use FGF13-flox-neomycin heterozygous mice crossed with Flp-deleter mice to get ride of the positive screening gene, neomycin. The FGF13-flox mice crossed with myh6-Cre, expressing Cre recombinase in myocardium, and generated FGF13-flox mice expressing cardiac-specific Cre recombinase, which can be used to generate the myocardial conditional knockout FGF13 mice after administration of tamoxifen.(2)Analyzed by q PCR technique, FGF13 m RNA level was significantly reduced in ventricular muscles in myh6-FGF13-/- mice, and was about 5% of wild type mice.(3)Comparing to control mice and analyzed by western blot, the protein level of FGF13 in ventricular muscle was significantly reduced in myh6-FGF13-/- mice.(4)Comparing to control mice, the expression of FGF13 protein was less in ventricular cardiomyocytesin myh6-FGF13-/- mice analyzed by immunofluorescence staining technique.Conclusion: We successfully generate FGF13 cardiac conditional knockout mice, and prove that m RNA and protein of FGF13 were successfully knocked out in the mice ventricular muscles, which suggests that FGF13 cardiac conditional knockout mice can be used in the following studies. Part 2 Study of roles and mechanism of FHFs in cardiac arrhythmiasby using FGF13 cardiac conditional knockout miceObjective:(1)To investigate whether FGF13 is related to some types of arrhythmia;(2)To investigate the mechanism accounting for FGF13 related in arrhythmia.Methods:(1)Using surface-ECG techniques to record mice ECG and detect whether and what type of arrhythmia occurs.(2)Using current- clamp technique to record action potential in ventricular myocytes and to observe the changes of action potential(AP) after FGF13 knockout.(3)Using voltage-clamp technique to detect the changes of sodium channel current density and gating properties.Results:(1)QRS interval was significantly prolonged in FGF13 cardiac conditional knockout mice. Flecainide can induce ECG phenotype of Brugada syndrome.(2)Amplitude of action potential(APA) was reduced, 0 phase of up-stroke was dramatically slowed and 50% repolarization was markedly prolonged in FGF13 cardiac conditional knockout mice.(3)In FGF13 cardiac conditional knockout mice, the sodium channel current density was significantly reduced, inactivation current shifts to depolarization direction, but there is no change in activation curves.Conclusion: FGF13 cardiac conditional knockout mice show Brugada syndrome phenotype, which is probably due to lose of FGF13 reducing sodium channel current density and changing action potential properties. All above suggest that FGF13 is a candidate gene for Brugada syndrome.