Figure 1. Isolation of neonatal pacemaker cells (PC) for assay for transposase-accessible chromatin (ATAC-seq).A, Isolation of neonatal PCs and right atrial cardiomyocytes (RACMs) from the sinoatrial node (SAN) using fluorescence-activated cell sorting (FACS) on samples derived from 3 biological replicates, where each biological replicate contained pooled SAN tissue from 5 neonatal mice. B, Ventral view of an Hcn4 (hyperpolarization-activated cyclic-nucleotide-gated ion channel 4)-green fluorescent protein (GFP)/Myh6-mCherry neonatal heart with SAN positive for both fluorescent markers (yellow). C, Enrichment of known PC genes (green bars) and depletion of known RACM genes (red bars) from bulk RNA sequencing on sorted PCs and RACMs (each biological replicate is shown [PC1, PC2, and PC3], data are displayed as log(2) fold change of SAN/RACM transcripts per million [TPM]). Blue bars show genes that are not differentially expressed. D and E, ATAC-seq signal for individual PC (n=4) and RACM (n=3) biological replicates at Hcn4 locus (D) and Nppa locus (E) Each biological replicate consisted of pooled SAN tissue from 1 to 2 litters of neonatal hearts. la indicates left atrium; lv, left ventricle; ra, right atrium; rv, right ventricle; and svc, superior vena cava.

Figure 2. Proximity of differential assay for transposase-accessible chromatin (ATAC-seq) peaks to differentially expressed genes and enrichment of cardiac TF (transcription factor) motifs and binding sites at ATAC-seq peaks.A, Clustered heatmap with Z scores for the top 500 differentially accessible (DA) ATAC-seq peaks across all 7 samples. B, Gene ontology terms for genes assigned to DA ATAC-seq peaks. C, Motif enrichment in DA ATAC-seq peaks. Motifs and false discovery rates (FDR) are for the underlined factors, encompassing several TF types, including LIM homeodomain proteins Isl1 (Islet-1), Lhx3 (LIM homeobox 3), and Lhx1 LIM homeobox 1); myocyte-specific enhancer factor 2 proteins; T-box (T-box transcription factors), Gata (GATA-binding proteins); and the TALE (3-amino acid-loop extension)-class proteins Meis1 (myeloid ecotropic viral insertion site 1), and Tgif2 and Tgif1 (TGFβ-induced factors 2 and 1). D, Overlap of published Mef2C (myocyte-specific enhancer factor 2C), Tbx5 (T-box transcription factor 5), Gata 4 (GATA-binding protein 4), Nkx2.5 (NK2 homeobox 5), and Shox2 (short stature homeobox 2) chromatin immunoprecipitation and sequencing peaks with DA ATAC-seq peaks. Abnl indicates abnormal; AP, action potential; Decr, decreased; RACM, right atrial cardiomyocytes; and SAN, sinoatrial node.

Figure 3. Selected pacemaker cell (PC)–enriched assay for transposase-accessible chromatin (ATAC-seq) peaks are sufficient to direct reporter activity to the cardiac venous inflow and sinoatrial node.A, ATAC-seq signal in PC (green track) and right atrial cardiomyocytes (RACM, red track) at the Rgs6 locus (top) and the Ptgfr locus (bottom) with differentially accessible (DA) ATAC-seq peaks highlighted in yellow. B, Three founders from transient transgenic enhancer-reporter mice generated with enSERT (enhancer inSERTion) and deposited in the VISTA Enhancer Browser (VISTA) at enhancer.lbl.gov for the DA ATAC-seq peak at Rgs6 locus (VISTA ID mm2011) and (C) Ptgfr locus (VISTA ID mm2010), harvested at embryonic day (E) 11.5 and stained with X-gal. Lower, Magnified view of the venous inflow (arrow) and right atrium (ra). D, DA ATAC-seq peak at the Isl1 locus is highlighted in yellow. E–J, Whole-mount images of ISE-LacZ embryos harvested and stained with salmon-gal at E8.5 (E) or bluo-gal at the developmental time points shown up to postnatal day 2 (P2; F–J). K, Double immunohistochemistry for Hcn4 (hyperpolarization-activated, cyclic nucleotide-gated ion channel 4) and β-gal (beta-galactosidase) in the sinoatrial node (SAN) of E14.5 ISE-LacZ embryo. L, Whole-mount image of an adult ISE-LacZ SAN stained with bluo-gal. a indicates atrium; la, left atrium; lv, left ventricle; oft, outflow tract; ra, right atrium; rv, right ventricle; sv, sinus venosus; and v, ventricle.

Figure 4. Sinoatrial node (SAN) enhancer activity co-localizes with Hcn4 expression and functional cardiac pacemaker cells.A and A’, Whole-mount fluorescence imaging of ISE-mCherry/Hcn4 (hyperpolarization-activated cyclic nucleotide-gated ion channel 4)–green fluorescent protein (GFP) embryos at embryonic day (E) 8.5 (arrows denote inflow tract), (B and B’) E10.5, and (C and C’) E14.5 imaged for mCherry alone (top row) and mCherry/GFP merge (bottom row). *mCherry+/GFP- SAN extension into the right atrium (ra). D, Postnatal ISE-mCherry activity at P2 and (E) in adult isolated pacemaker cells (PCs) with typical PC morphology. F, Spontaneous action potentials (APs) recorded from an adult mCherry+ isolated pacemaker cell. G, Firing rate, maximum diastolic potential, AP amplitude, early diastolic depolarization rate (EDDR), upstroke velocity (dV/dt max), and AP duration (APD) at 50% and 90% repolarization in 12 isolated adult mCherry+ pacemaker cells. Error bars indicate SD. H, Whole-cell voltage-clamp on an mCherry+ adult cell with funny current (I(f)) elicited by a series of hyperpolarizing voltage pulses from a holding potential of -40 mV to -130 mV in 10 mV increments. I, Mean I(f) current density at the indicated voltages in 11 mCherry+ PCs. Error bars indicate SEM. la indicates left atrium; and sv, sinus venosus.

Figure 5. Deletion of the Isl1 sinoatrial node (SAN) enhancer leads to reduced Isl1 expression in SAN and abnormal SAN development.A, Top, Deletion of a 2.7-Kb genomic segment containing the Isl1 locus sinoatrial node enhancer (ISE). Middle, Genotyping strategy to identify the mutant allele with an agarose gel (bottom) on an F4 intercross confirming germline transmission of the deletion allele. F denotes forward primer; and REV denotes reverse primer. B, Genotypes from 88 weaned pups of Isl1^ΔISE/+ intercrosses with the indicated frequencies. C, Immunohistochemistry of wild-type (WT; top) and Isl1^ΔISE/ΔISE (bottom) SAN at embryonic day (E) 14.5 stained for Hcn4 (hyperpolarization-activated cyclic nucleotide-gated ion channel 4; red), Isl1 (Islet-1; green), and DAPI (4',6-diamidino-2-phenylindole). Area of detail is shown in the right parts. Pacemaker cell nuclei in the WT are Isl1+ (turquoise, denoted by white arrows) whereas the Isl1^ΔISE/ΔISE has less Isl1 signal in pacemaker cell (PC) nuclei. D, Quantification of green fluorescent signal (Isl1) in 35 Hcn4+ pacemaker cell nuclei and 35 Hcn4- non-PC nuclei, normalized to tissue background green fluorescent signal, from 7 sections each from 2 different sets of WT and Isl1^ΔISE/ΔISE littermates (denoted replicate 1 and replicate 2). ****P<0.0001 and ***P<0.001. E, Junction of the superior vena cava (svc) and right atrium (ra) showing the location of the sinoatrial node (san) and the sectioning plane used to perform PC counts and cross-sectional area measurements. Z=0 denotes the center of the sinoatrial node head. F, Quantification of PC nuclei in 12-sections each at the indicated z-distance from the center of the SAN head. Counts were performed for 2 pairs of E14.5 littermates (denoted replicate 1 and replicate 2). G, Estimation of area of the SAN head for the sections analyzed in F. *P<0.05. NS indicates nonsignificant

Figure 6. Abnormal sinoatrial node (SAN) function in adult Isl1^ΔISE/ΔISE mice.A, Average hourly heart rate in adult Isl1^ΔISE/ΔISE (red, n=3 for female and n=6 for male) and wild-type (WT) littermate mice (black, n=6 for female and n=5 for male). Error bars denote SEM. The low activity period is highlighted in gray (B) Heart rates were averaged over 1-minute intervals for each mouse studied in (A; n=11 WT and 9 Isl1^ΔISE/ΔISE, analyzed separately by sex), and then binned into 10 bpm bins for generation of heart rate histograms for WT and Isl1^ΔISE/ΔISE females (top) and males (bottom) Vertical scale bar=25 counts. Comparison was made between WT and Isl1^ΔISE/ΔISE for total counts <400 bpm (male) or 500 bpm (female) for each mouse. *P<0.05 for Mann-Whitney test. C, A 2-second ECG tracing from a WT mouse (top) and with 2 examples of sinus arrhythmias observed in 2 different Isl1ΔISE/ΔISE mice: a sinus pause (middle tracing), and an episode of bradycardia (bottom tracing). D, Quantification of numbers of sinus arrhythmias recorded per hour over a 24-hour period in n=11 WT and n=9 Isl1^ΔISE/ΔISE mice. E, Quantification of total percentage of time spent with heart rate <250 bpm (bradycardia) for 11 WT and 9 mutant mice. Comparison of WT and Isl1^ΔISE/ΔISE distribution were made with a Mann-Whitney test with the P value shown.

Figure 7. Evolutionary conservation and upstream regulation of Isl1 sinoatrial node (SAN) enhancer function.A, View of Isl1 locus SAN enhancer (ISE) with alignment to human, mouse, and opossum genomes (above, areas in red indicate >70% conservation, scale from 50% to 100%) and previously published embryonic heart chromatin immunoprecipitation and sequencing (ChIP-seq) data for Gata4 (GATA-binding protein 4), Tbx5 (T-box transcription factor 5), and Tead1 (TEA domain transcription factor 1). B, Maximum intensity projections of confocal z-stacks of 48 hours postfertilization (hpf) Tg(myl7:mCherry-NTR) zebrafish hearts after injection of ISE:green fluorescent protein (GFP) reporter construct (top), or a 1.0-kb fragment (Fr1, lower). C, Enhancer-reporter analysis of founders from injections of ISE and a 1 kb fragment (Fr1 [fragment 1]) in 48 hpf zebrafish hearts with the indicated patterns of reporter activity. Numbers in parentheses indicate the embryos observed to have the indicated expression pattern/total embryos injected that survived to 48 hpf. a indicates atrium; ATAC-seq, assay for transposase-accessible chromatin; avc, av canal; F0, filial generation 0; PC, pacemaker cell; RACM, right atrial cardiomyocyte; san, sinoatrial node; sv, sinus venosus; and v, ventricle.

Figure 8. Association of pacemaker cell assay for transposase-accessible chromatin (ATAC-seq) peaks with resting heart rate associated single nucleotide polymorphisms.A, Manhattan plot showing association of 335 353 single nucleotide polymorphisms (SNPs) within or ±0.5 kb away from a region syntenic to any mouse ATAC-seq peak with human resting heart rate ascertained from the UK Biobank cohort. Blue line indicates significance threshold of Bonferroni adjusted P value of 0.01 (corresponding to uncorrected P value 1.2×10^-8; B) Manhattan plot similar to (A) including 239 SNPs within or ±0.5 kb away from the top 500 differentially accessible (DA) ATAC-seq peaks. Blue line indicates Bonferroni adjusted P value of 0.01 (corresponding to uncorrected P value 4.2×10^-5). C, Manhattan plot showing association of SNPs at the ISL1 locus with resting heart rate. The human genomic region syntenic to the mouse Isl1 sinoatrial node enhancer is highlighted. Chr indicates chromosome.