Sex Differences in Ion-Channel Promoter Expression of Healthy Human Heart Anna Gams1, Ndeye Rokhaya Faye1, Ruslan Deviatiiarov2, Aaron C. Koppel1, and Igor R. Efimov1* 1The George Washington University, Washington, DC 2Kazan Federal University, Kazan, Russian Federation Contact: [email protected]

Objective Results Not detectable Low Promoter Statistically Compare holistic ion-channel expression associated with sex differences using Cap Analysis of Name Type (16 ) expression identified significant Gene Expression (CAGE) analysis on left atrial (LA) and left ventricular (LV) human donor hearts. (13 genes) (17 genes) difference (4 genes) Introduction

Cardiovascular diseases remain the primary cause of death worldwide. Several epidemiologic and investigative studies have shown the evidence of sex hormones effect on cardiac electrophysiology through genomic regulation. However, very little is known about the molecular basis for gender- related discrepancies in cardiac electrophysiology. Due to physiologically distinct functions of atria (electrical impulse initiation) and ventricles (blood pumping) there is a difference in ion-channel expression within a heart that cause different disease susceptibilities between both sexes (Figure 1). Cap Analysis of Gene Expression (CAGE) is a method for promoter identification and transcription profiling. CAGE utilizes a “cap-trapping” technology based on the biotinylation of the 7-methylguanosine cap of Pol II transcripts, to pull down the 5’-complete cDNAs reversely transcribed from the captured transcripts. Through a massive parallel sequencing of the 5’ end of cDNA and analysis of the sequenced tags, transcription start sites Figure 1. Middle panel is a representation of typical atrial (TSS) and transcript counts are inferred on a genome-wide and ventricular action potentials of males and females scale. depicted in correlation with ECG wave. Methods

Conclusions In the past decade there has been a push towards sex-specific drug development since it has been established that medications affect males and females differently. 50 genes are known to code for ion-channel α- and β- subunits in human hearts. Interestingly, 14 genes were not detectable; 13 genes showed low expression which was insufficient for promoter identification with threshold of +/-500 bp regions (Figure 2). Male and female ion- Figure 2. Mind map of channel expression levels were significantly different from each other (p=0.0002). The highest upregulation in both promoter expression of cardiac sexes was detected in promoters of INa (SCN5A and SCN1B), Ito,f (KCNIP2), IK2P (KCNK1), and INCX (SLC8A1). ion-channels. Each box and Interestingly, SLC8A1, which is known to be cardioprotective from ischemic injury was significantly upregulated in whisker plot shows activity of a female atrium (p= 0.0408). Other promoter expression that was significantly different across sexes include: I single promoter of a specified to,f ion-channel. Normalization of (KCND3) (p=0.0186) and IKATP (KCNJ12) (p=0.049) in atrium and INa (SCN4B) (p=0.028) in ventricle. Our study raw CAGE tag count was reveals that there are sex-dependent gene expression differences in cardiac ion-channels and that CAGE performed as counts per approach allows high-throughput gene expression profiling which can be beneficial for gender-specific million. Statistical significance computational model development, drug testing, and personalized medicine overall. (p<0.05) was determined with Wilcoxon rank sum and Kruskal-Wallis tests. LA- left Acknowledgements References atrium, LV- left ventricle.

1. R. Tadros, A. T. Ton, C. Fiset, and S. Nattel, “Sex differences in cardiac This project was supported by NIH electrophysiology and clinical arrhythmias: Epidemiology, therapeutics, 5R01HL114395 and mechanisms,” Can. J. Cardiol., vol. 30, no. 7, pp. 783–792, 2014. 2. A. O. Grant, “Cardiac Ion Channels,” Circ. Arrhythmia Electrophysiol., vol. 2, no. 2, pp. 185–194, Apr. 2009.