Molecular cloning, adenovirus construction and purification.

The human TBX18 gene with a C-terminal myc/FLAG tag was excised from pCMV6-Tbx18 (Origene, Rockville, MD) by digestion with FseI and SalI, then subcloned into a NotI- and XhoI-digested lentiviral expression vector with the desired reporter gene, pLVX-IRES-ZsGreen1 (Clontech, Mountain View, CA), to create pLV-Tbx18-IRES-ZsGreen (∼10.1 kb). We used ZsGreen1 as the reporter protein for Tbx18-transduced cardiomyocytes. Since ZsGreen1 has indistinguishable spectral characteristics as the commonly used GFP, we refer ZsGreen1 to GFP throughout this study. The recombinant target gene was then introduced to an adenovirus vector backbone by Gateway recombination cloning using Gateway-adapted vectors (Invitrogen, Carlsbad, CA). LR recombination reaction was performed between the entry clone and the destination vector, pAd/CMV/V5-DEST (∼36.7 kb), to generate the desired adenoviral expression construct, pAd-CMV-TBX18-IRES-GFP (∼39.8 kb). Positive constructs were verified to have the correct target gene by DNA sequencing (Laragen, Los Angeles, CA). The expression constructs were digested with PacI to expose inverted terminal repeats before transfecting into 293A cells to produce adenoviral stocks for use in subsequent expression of the transgene. Adenoviruses were plaque-purified, amplified, and affinity-column purified using Adenopure kit (Puresyn, Inc), and stored at −80 °C.

Electrophysiology.

Whole-cell electrophysiology recordings were performed as described below. Experiments were carried out using standard microelectrode whole-cell patch-clamp techniques with an Axopatch 200B amplifier (Axon instruments) with a sampling rate of 20 kHz and low-pass Bessel-filtered at 5 kHz. Experiments were performed at a room temperature for NRVMs or at 35 °C for freshly isolated adult guinea pigs cardiomyocytes. In experiments with NRVMs, cells were washed with a normal Tyrode's solution containing (mmol/L): NaCl 138, KCl 5, CaCl 2 2, glucose 10, MgCl 2 0.5, and HEPES 10; pH 7.4. The micropipette electrode solution was composed of (mmol/L): K-glutamate 130, KCl 9, NaCl 8, MgCl 2 0.5, HEPES 10, EGTA 2, and Mg-ATP 5; pH 7.2. Freshly isolated adult guinea pig cardiomyocytes (GFP-VMs, Tbx18-VMs, and SAN myocytes) were examined by nystatin-perforated,whole-cell patch-clamp, bathed in the Tyrode's with 1.8 mM CaCl 2 and 1.2 mM MgCl 2 . Pippete solution contained (mmol/L) K-glutamate 98, KCl 50, MgCl 2 1.0, HEPES 10, and 200 μg/ml nysattin, pH 7.2. Microelectrodes had tip resistances of 2 to 4 MΩ when filled with the internal recording solution. Voltage-clamp experiments were performed with an inter-episode interval of 2.5 s. Action potentials were either initiated by short depolarizing current pulses (2 to 3 ms, 500 to 800 pA) on GFP-NRVMs or recorded with I = 0 mode on Tbx18-NRVMs. I f currents were recorded by holding the resting membrane potential at −35 mV, then stepping to a test voltage to −140 mV for 2 s with 20 mV step in each sweep. Each test potential was followed by a step to −140 mV for 1 s to examine its activation kinetics, and then brought to the holding potential of –35 mV. Data were corrected for the estimated liquid junction potentials (−18 mV for NRVMs and −12.5 mV for guinea pig cardiomyocytes). A xenon arc lamp was used to view GFP fluorescence at 488/530 nm (excitation/emission).

Myocyte isolation and transduction.

NRVMs were isolated from 1- to 2-day-old neonatal rat pups and cultured as a monolayer as described previously7. Only the lower one third of the heart (from the apex to the midline) was excised in order to minimize contaminating atrioventricular nodal cells. A monolayer of NRVMs was transduced with either Ad-Tbx18-IRES-GFP or Ad-GFP one day after cell isolation, and cultured for 2-5 days. For all in vitro experiments, NRVMs were plated at a density of 210,000 cells per each cm2 of surface area. Unless indicated otherwise, the NRVMs were plated and transduced in 6-well plates. For intracellular Ca2+ recordings with Rhod2-AM, glass-bottom dishes of 35-mm diameter (MatTek Corp., Ashland, MA) were used. High-throughput measurements of spontaneous beating rates in Tbx18-transduced NRVMs were performed on 6-well multi-electrode arrays (Multi-Channel Systems, Reutlingen, Germany) with a surface area of about 0.9 cm2 per well. Adenoviral vector transduction was performed at multiplicity of infection (MOI) of 1 fluorescence forming unit (ffu) per cell. Transduction was performed in a routine NRVM culture media, based on M199 with the following components: 10 mM HEPES, 0.1 mM non-essential amino acids, 3.5 mg/mL glucose, 2 mM L-glutamine, 4 μg/ml vitamin B 12 , 100 U/ml penicillin and heat-inactivated FBS at 10% (first two days of culture) or 2% (after two days of culture) final concentration.

Rat SA node isolation.

SA nodal myocytes were isolated from adult Sprague-Dawley rats. Animals were anesthetized with isoflurane. Hearts were quickly removed, the atria separated from the ventricles, and the sinoatrial node region dissected in normal Tyrode's solution, which consisted of (in mM) 140 NaCl, 5.4 KCl, 1.2 KH 2 PO 4 , 5 HEPES, 5.55 glucose, 1 MgCl 2 , 1.8 CaCl 2 ; pH adjusted to 7.4 with NaOH. The rat sinoatrial node region was defined by the borders of the crista terminalis, the interatrial septum, and the superior and inferior vena cavae.

Intracellular calcium recordings and analyses.

For measurements of intracellular Ca2+ oscillations, 2 × 106 NRVMs were plated in 35-mm glass-bottom dishes (MatTek Cultureware) or 22 mm fibronectin-coated glass coverslips, transduced, and analyzed 4 days post-transduction. Cells were loaded with 2 μmol/L Rhod2-AM (Molecular Probes, Carlsbad, CA) for 18 min, washed once and subsequently placed in normal Tyrode's solution with 2 mmol/L Ca2+. Calcium transients were recorded at 37 °C from Ad-Tbx18-IRES-GFP and Ad-GFP transduced NRVMs. Whole cell Ca2+ transients for caffeine-induced Ca2+ measurements were obtained from 2-D confocal imaging with a spinning-disk confocal laser line-scan microscope (Perkin Elmer/Nikon). Offline analysis was performed using Ultraview (Perkin Elmer) and ImageJ. Calcium transients were analyzed by averaging the Rhod2 signal from the entire cell, and are presented as background-subtracted, normalized fluorescence (F/Fo, arbitrary units). Spontaneous and spatially restricted Ca2+ release events from GFP-NRVMs (Ca2+ sparks) and Tbx18-NRVMs (localized Ca2+ releases, LCRs) were analyzed in the following manner. With ImageJ, the background noise level was determined from each scanned images. Then, an LCR event (or a Ca2+ spark) was identified when the signal amplitude was greater than the mean value of [background noise + 3.8 × (s.d. of the background noise)] and when the signal duration was ≥ 25 ms as previously demonstrated55,56. For 2-D confocal Ca2+ imaging calcium transients were obtained by averaging the signal through the entire cell. Ryanodine and protein kinase A inhibitor were purchased from Tocris biosciences and caffeine was purchased from Sigma. Rhod-2/AM and was purchased from Invitrogen.

Immunostaining, immunoblotting, and morphometric analysis.

Frozen sections of neonatal rat SAN and NRVMs 4 days after adenoviral transduction were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X 100 and then incubated with the appropriate primary antibody: sarcomeric α-actinin (Sigma-Aldrich; A5044; 1:800), ANP (AbCam;ab-14348; 1:1,000), HCN4 (Abcam; ab85023; 1:500) and Alexa Fluor-conjugated secondary antibodies (Invitrogen). Western blots were performed using specific antibodies against to Cx43 (Sigma-Aldrich, C6219; 1:1,000), PLB (Alomone; A010-14: 1:5,000), p-PLB (Alomone; A010-12: 1:5,000) HCN4 (AbCam; ab85023; 1:500) Briefly, Ad-Tbx18, Ad-GFP transduced NRVMs, rat SAN and Left ventricle were homogenized in RIPA buffer containing a protease inhibitor cocktail (Sigma). Protein content was quantified by BCA assay and cell lysates (15 μg per lane) were run on a 12% SDS-PAGE gel and transferred onto a PVDF membrane. Then the transferred membrane was incubated with a primary antibody overnight at 4 °C, followed by 1-h incubation with a peroxidase-conjugated secondary antibody. Immunoreactivity was detected by chemi-luminescence (ECL Western Blotting Analysis System, Amersham). Equal protein loading of the gels was assessed by re-probing the membrane with monoclonal anti-GAPDH antibody (Abcam; ab9482; 1:10,000) or anti-β-actin (Sigma-Aldrich; A3848: 1:25,000). Morphometric assays were performed with ImageJ by measuring cell area and cell length (long-axis) of each cardiomyocytes. Then, an average cell width was calculated by dividing the cell area by longitudinal cell length. The ratio of the longitudinal cell length to the average cell width (length-to-width) was used to discern Tbx18-VMs with SAN-like morphology from Tbx18-VMs with unchanged morphology by setting an arbitrary threshold ratio of 12 above which no control VMs were found (Supplementary Fig. 4).

In vivo gene transfer.

Adenoviruses were injected into the left ventricular apex of guinea pigs. Adult female guinea pigs (weight, 250 to 300 g; Charles River) were anesthetized with 4% isoflurane, intubated, and placed on a ventilator with a vaporizer supplying 1.5% to 2% isoflurane. After lateral thoracotomy, a 30-gauge needle was inserted at the free wall apex of the left ventricle. 100 μl of adenovirus containing 1 × 109 fluorescence-forming units of Ad-Tbx18-IRES-GFP or Ad-GFP (control group) was injected into the left ventricle apex. We performed flow cytometry on live, freshly isolated adult guinea pig ventricular cardiomyocytes one week after direct intramyocardial injection of Tbx18-IRES-GFP adenovirus. The total number of Tbx18-transduced ventricular myocytes (gated for large cell size and GFP+) is estimated to be 23,000 ± 12,000 myocytes per heart (n = 3). Animals for the long-term studies were treated with cyclosporine A (10 mg/kg/24 h) by daily IP injection or with an implantable subcutaneous osmotic pump so as to lessen clearance of transduced cardiomyocytes by the animal's immune system.

In vivo and ex vivo electrocardiographic recordings.

Methacholine (0.1–0.5 mg per kg of body weight in saline, Sigma-Aldrich, St. Louis, MO) was delivered via the jugular vein in order to slow the animals' sinus rhythm before ECG recordings under general anesthesia (2% isoflurane, 98% O2). Lead I and Lead II ECGs were recorded analyzed at 2 kHz with a PowerLab Data Acquisition System and a LabChart software (ADInstruments Inc.). In all animals, methacholine was administered until complete heart block was achieved with accompanying reduction of the animals' sinus rhythm to <100 b.p.m. In most of the Tbx18-injected guinea pigs, ectopic ventricular rhythms were manifested well before the sinus rhythm reached 100 b.p.m. (Fig. 3d, right panel). In contrast, control animals exhibited no evidence of such ectopic ventricular beats even when the sinus rhythm was brought to <100 b.p.m. (Fig. 3d, left panel). Figure 3d highlights the lack of any ectopic ventricular rhythm in the control animals even at a very slow escape rate compared to Tbx18-injected animals.

For ex vivo, intact whole-heart ECG recordings, the heart was retrograde-perfused via aorta at 60 mm Hg with oxygenated Tyrode's solution at 36 °C. The perfused heart was placed in a sylgard-coated plate filled with warm Tyrode's solution. ECG leads were stationed at appropriate sites to record leads I and II (Supplementary Fig. 5). After a 20-min equilibration period, the region of atrioventricular node was ablated with a cryogun (Brymill Cryogenic Systems, Ellington, CT) filled with liquid N 2 . Electrode-pacing was performed at the site of transgene injection (anterior, left ventricular apex) at 200-ms intervals with a platinum electrode connected to an isolated pulse stimulator (Model 2100, A-M Systems, Carlsborg, WA).

RT-PCR for gene arrays.

Rat SAN, Left ventricle and Tbx18- and GFP-transduced NRVMs (4 days after transduction) were collected and mRNA was extracted (Qiagen mRNA Isolation Kit,). The mRNA samples were converted to first strand cDNA, using the RT2 First Strand Kit. (SA Biosciences). Then, the cDNA template was mixed with the RT2 qPCR Master Mix and the mixture was aliquoted into each well of the same plate containing pre-dispensed gene specific primer sets. PCR was performed on a 7900HT Fast Real-Time PCR System (Applied Biosystems/Life Technologies Corporation, Carlsbad, CA) and the relative expression of the genes was calculated.

cAMP assay.

The Cyclic AMP Elisa Assay Kit (catalog # STA-501; Cell Biolabs, INC) was used to determine cAMP levels in NRVMs transduced with Ad-Tbx18 or Ad-GFP. Briefly, 50 μl Tbx18- and GFP-NRVM cell lysates were added to the Goat Anti-Rabbit Antibody Coated Plate 96 well plate. 25 μL of diluted Peroxidase cAMP Tracer Conjugate was added to each tested well. Then, 50 μL of diluted Rabbit Anti-cAMP Polyclonal Antibody was added to each tested well and the plate was incubated at room temperature for 30 min with shaking. After washing 100 μL of Chemiluminescent Reagent was added to each well. After incubation at room temperature for 5 min on an orbital shaker, the plate was read for luminescence of each microwell on a plate luminometer. Measurement of light emission (RLU) allowed calculating the amount of cAMP in samples which were then normalized to β-actin for comparison of the samples.

Chromatin immunoprecipitation and qPCR.

NRVMs transduced with either Tbx18 or GFP were fixed two- to four-days after viral vector transduction with 10% formaldehyde for 8 min at room temperature. Cells were sheared using a sonicator with ten pulses of 20 s each, with a 30-s rest on ice between each pulse. Chromatin immunoprecipitation was performed using ChIP-IT Express Chromatin Immunoprecipitation kit (Active Motif, Carlsbad, CA) following the manufacturer's protocol. Primary antibodies for the H3K4me3 and H3K27me3 were purchased from Active Motif. Gene specific (Cx43, Kir2.1, α-SA, HCN4) primers, already validated for qPCR in rat, were purchased from SA Biosciences. For each gene, three sets of primers were employed corresponding to −2 kb (upstream), −1 kb, and +1 kb (downstream) of the transcription start site. The ΔCt values from the three tiles were averaged from each experiment for data analyses.

Single-cell quantitative real-time PCR.

Single myocytes were collected in PBS with a wide-opening patch pipette, placed on dry ice and then stored at −80 °C. Tbx18 mRNA levels in individual myocytes were examined by quantitative real-time PCR with an Ambion Single Cell-to-CT Kit (Life Technologies) according to manufacturer's instructions. Briefly, single cells were treated with cell lysis solution and DNase I for 5 min at room temperature. Reverse transcription was performed at 25 °C for 10 min, at 42 °C for 60 min and at 85 °C for 5 min after addition of SupersScript RT and VILO RT mix. Preamplification was performed with 14 cycles of 95 °C for 15 s and 60 °C for 4 min with addition of PreAmp mix and 0.2x TaqMan Gene Expression Assays, containing primers for human Tbx18 (assay ID:Hs01385457_m1), guinea pig GAPDH (assay ID:Cp03755742_g1), and guinea pig TnnT2 (assay ID:Cp04182357_g1).The custom primers were synthesized by Applied Biosystems (Carlsbad, CA). Preamplification products (1:20 dilution) were used for real-time PCR with TaqMan Gene Expression Assays using an Applied Biosystems 7900HT Fast Real-Time PCR System. Standard curves for each of the 3 primer sets were constructed with serial dilutions of input DNA templates (Supplementary Fig. 12) and validated comparable amplification efficiencies (curve slopes: −3.32 to −3.64). Relative mRNA levels of Tbx18, GAPDH, and TnnT2 were obtained by extrapolation of Ct values with the slopes of the standard curve for each primer sets. Tbx18 mRNA amount in each cell was then normalized to GAPDH or TnnT2 level.

Statistical analyses.

Data were analyzed for mean, s.d. and s.e.m. (SEM). The quantitative figures in this work represent the mean ± s.e.m. Data sets were statistically evaluated using an unpaired t test. Confidence level of P < 0.05 was considered significant unless indicated otherwise.

Determination of fold-change in gene expression level in Tbx18-NRVMs versus GFP-NRVMs, and SAN versus LV samples.

The original expression level (L) for each gene of interest is expressed as:

This is done because of the inverse relationship between the threshold cycle (Ct) and the gene expression level, and the assumed doubling of the amount of product with every cycle. To determine Tbx18-effected fold-change of each gene, we first normalized the expression level of Tbx18-NRVMs (or GFP-NRVMs) to a housekeeping gene (HKG):

Then Tbx18-NRVMs' gene expression level was normalized to that of GFP-NRVMs: