Rationale Treatment of sinus node disease with regenerative or cell-based therapies will require a detailed understanding of gene regulatory networks in cardiac pacemaker cells (PCs). Objective To characterize the transcriptome of PCs using RNA sequencing, and to identify transcriptional networks responsible for PC gene expression. Methods and Results We used laser capture micro-dissection (LCM) on a sinus node reporter mouse line to isolate RNA from PCs for RNA sequencing (RNA-Seq). Differential expression and network analysis identified novel SAN-enriched genes, and predicted that the transcription factor Islet-1 (Isl1) is active in developing pacemaker cells. RNA-Seq on SAN tissue lacking Isl1 established that Isl1 is an important transcriptional regulator within the developing SAN. Conclusions (1) The PC transcriptome diverges sharply from other cardiomyocytes; (2) Isl1 is a positive transcriptional regulator of the PC gene expression program.

Regional differences in cardiomyocyte automaticity permit the sinoatrial node (SAN) to function as the leading cardiac pacemaker and the atrioventricular (AV) junction as a subsidiary pacemaker. The regulatory mechanisms controlling the distribution of automaticity within the heart are not understood. To understand regional variation in cardiac automaticity, we carried out an in vivo analysis of cis-regulatory elements that control expression of the hyperpolarization-activated cyclic-nucleotide gated ion channel 4 (Hcn4). Using transgenic mice, we found that spatial and temporal patterning of Hcn4 expression in the AV conduction system required cis-regulatory elements with multiple conserved fragments. One highly conserved region, which contained a myocyte enhancer factor 2C (Mef2C) binding site previously described in vitro, induced reporter expression specifically in the embryonic non-chamber myocardium and the postnatal AV bundle in a Mef2c-dependent manner in vivo. Inhibition of histone deacetylase (HDAC) activity in cultured transgenic embryos showed expansion of reporter activity to working myocardium. In adult animals, hypertrophy induced by transverse aortic constriction, which causes translocation of HDACs out of the nucleus, resulted in ectopic activation of the Hcn4 enhancer in working myocardium, recapitulating pathological electrical remodeling. These findings reveal mechanisms that control the distribution of automaticity among cardiomyocytes during development and in response to stress.

Mammalian cardiac Purkinje fibers (PFs) are specified from ventricular trabecular myocardium during mid-gestation and undergo limited proliferation before assuming their final form. MicroRNA-1 (miR-1), a negative regulator of proliferation, is normally expressed in the heart at low levels during the period of PF specification and outgrowth, but expression rises steeply after birth, when myocardial proliferation slows and postnatal cardiac maturation and growth commence. Here, we test whether premature up-regulation and overexpression of miR-1 during the period of PF morphogenesis influences PF development and function. Using a mouse model in which miR-1 is expressed under the control of the Myh6 promoter, we demonstrate that premature miR-1 expression leads to PF hypoplasia that persists into adulthood, and miR-1 TG mice exhibit delayed conduction through the ventricular myocardium beginning at neonatal stages. In addition, miR-1 transgenic embryos showed reduced proliferation within the trabecular myocardium and embryonic ventricular conduction system (VCS), a source of progenitor cells for the PF. This repression of proliferation may be mediated by direct translational inhibition by miR-1 of the cyclin dependent kinase Cdk6, a key regulator of embryonic myocardial proliferation. Our results suggest that altering the timing of miR-1 expression can regulate PF development, findings which have implications for our understanding of conduction system development and disease in humans.

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