LAUSR

Transgenic (TG) mice overexpressing the human adenylate cyclase 8 (AC8) gene have markedly higher intrinsic (in the presence of dual autonomic blockade) heart rates than their wild type (WT) littermates. To understand the mechanisms underlying this, we performed RNA sequencing (RNASeq), comparing expression levels of important transcripts in the sinoatrial node (SAN) of AC8 TG mice with WT animals.RNA was extracted from the SAN of AC8 TG and WT mice (n=8 in each group), and after quality-control processed with SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian (Takara Bio USA, Inc.). 75 bp single end reads generated 30-40 million reads/library. Raw RNASeq reads were aligned and after quality trimming, mapped to the UCSC mm10 mouse reference genome and cDNA of human AC8, assembled using Tophat v2.0, resulting in sequence alignment data files for each sample. Cufflinks v.2.1.1 was used to calculate fragments per kilobase of transcript per million mapped reads (FPKM) for each sample. Differential gene expression analysis was performed with the Cufflinks v2.1.1, and the DESeq2 package of Bioconductor. We also performed Gene Set Enrichment Analysis (GSEA).Our results confirm that SAN from TG AC8 mice exhibit high levels of human AC8 transgene expression (∼330 FPKM), 2-fold higher than Ryr2 expression, and over 10-fold higher than HCN4 expression. Contrastingly, endogenous AC8 expression became reduced, accompanied by changes in expression of G-protein receptor coupled receptors and cAMP-PKA signaling molecules, including upregulation of phosphodiesterases (PDE types 4C, 4B and 3a), downregulation of Gng5, and downregulation of PKA regulatory subunit 2 (Prkag2). Contrastingly, Prkca was upregulated. GSEA revealed upregulation of Stim1 and Orai1, modulators of genes influencing Ca2+ signaling in SAN.These changes in the expression pattern of genes well known to modulate the coupled clock system of SAN cells is likely to substantially contribute to the witnessed tachycardia of AC8 TG mice.