Direct conversion of quiescent cardiomyocytes to pacemaker cells by expression of Tbx18

Abstract

The heartbeat originates within the sinoatrial node (SAN), a small structure containing <10,000 genuine pacemaker cells. If the SAN fails, the ∼5 billion working cardiomyocytes downstream of it become quiescent, leading to circulatory collapse in the absence of electronic pacemaker therapy. Here we demonstrate conversion of rodent cardiomyocytes to SAN cells in vitro and in vivo by expression of Tbx18, a gene critical for early SAN specification. Within days of in vivo Tbx18 transduction, 9.2% of transduced, ventricular cardiomyocytes develop spontaneous electrical firing physiologically indistinguishable from that of SAN cells, along with morphological and epigenetic features characteristic of SAN cells. In vivo, focal Tbx18 gene transfer in the guinea-pig ventricle yields ectopic pacemaker activity, correcting a bradycardic disease phenotype. Myocytes transduced in vivo acquire the cardinal tapering morphology and physiological automaticity of native SAN pacemaker cells. The creation of induced SAN pacemaker (iSAN) cells opens new prospects for bioengineered pacemakers.

Figure 1

Tbx18-transduced NRVMs become spontaneously beating cardiomyocytes. A. Tbx18-transduction significantly increases the number of spontaneously beating NRVM cultures compared to control and to several other transcription factors (Shox2, Tbx3, Tbx5 and Tbx20). Each n represents one well of a 24-well plate. More than five different cell batches (each batch comprising three litters of neonatal rat hearts) were examined. B. Representative action potential (AP) traces from GFP-(1B. left) and Tbx18-NRVMs (1B, right). Most GFP-NRVMs (7 of 9) were quiescent and fired an AP only after stimulation. In contrast, most Tbx18-NRVMs (7 of 9) exhibited spontaneous APs with prominent gradual phase-4 depolarization. C. Maximum diastolic potential, index of automaticity, and the total cell number of spontaneously-oscillating Ca²⁺ transients are summarized. Among the Tbx18-NRVMs, 73.8 ± 6.0% of the transduced cells beat spontaneously compared to 28.8 ± 6.1% in GFP-NRVMs (p <0.05). The beating rates of Tbx18-NRVMs were 95 ± 23 bpm compared to 46 ± 10 bpm of GFP-NRVMs (p <0.05). GFP- and Tbx18-groups are indicated by white and black bars, respectively, throughout the paper. D. Left: representative I_K1 raw traces (GFP, black trace; Tbx18, blue trace) elicited by a ramp protocol from −140 to −20 mV. The summarized I_K1 densities at −140 mV are shown in 1D right. E. HCN4 immunostaining image (HCN4-white, nuclei-blue) of GFP- or Tbx18-NRVMs are shown in left and middle, respectively. Scale bar: 200μm. Right panel: summary of the percent HCN4-positive cells per GFP- or Tbx18-transduced cells. F. Western blot indicates higher HCN4 protein level in Tbx18-NRVMs relative to control (left) comparable to the level HCN4 observed in adult rat SAN (right panel, n=3 samples). G. Tbx18-NRVMs exhibited robust I_f recorded in the presence of 2 mM Ba²⁺ to eliminate contaminating I_K1. H. Changes of relative mRNA levels of selected genes comparing Tbx18-NRVMs normalized to GFP-NRVMs (left) and SAN normalized to LV (right). SAN and Tbx18-NRVMs demonstrate similar pattern of normalized transcript levels (n=3 RT-PCR reactions, each reaction was performed with cells isolated from ≥4 wells from a 6-well plate).

Figure 2

Tbx18-transduced myocytes recapitulate major calcium clock characteristics of genuine SAN pacemakers. A. Representative confocal line-scan images of changes in [Ca²⁺]_i in Rhod2/AM loaded Tbx18-NRVMs 4 days post-transduction demonstrate LCRs preceding each whole cell Ca²⁺ transient. In contrast, only occasional Ca²⁺ sparks could be detected in GFP-NRVMs (B). C. The LCRs from Tbx18-NRVMs are longer and wider than spontaneous Ca²⁺ release events from GFP-NRVMs, measured as full duration at half-maximal width (FDHW) and full width at half-maximal duration (FWHD. Amplitudes of the Ca²⁺ signals (measured in arbitrary units of F/F_o, right panel) are similar between the two groups. D. LCR period is defined as the period between the start of a whole-cell Ca²⁺ transient to the beginning of the subsequent LCR. Cycle length is defined as the period between two consecutive whole cell Ca²⁺ transients. Tbx18-NRVMs exhibited spontaneous, rhythmic LCRs with an average period of 72±1% of that of the cycle length. E. Spatially averaged F/F₀ plots of changes in [Ca²⁺]_i demonstrate a 2.3-fold increase in 20 mM caffeine induced Ca²⁺ transients in Tbx18-NRVMs compared to control. F. Ryanodine (10 μM) suppressed the amplitudes of spontaneous Ca²⁺ transients by 47±6% in Tbx18-NRVMs and by 12±2% in paced GFP-NRVMs, suggesting larger subsarcolemmal Ca²⁺ stores in Tbx18-NRVMs. G. The ratio of phosphorylated phospholamban (p-PLB, Ser-16) to total PLB is significantly higher in Tbx18-NRVMs and in native SAN tissue compared to GFP-NRVMs and adult ventricular myocardium, respectively. H. No changes in the protein levels of SERCA2A, NCX1 and RyR were observed in the Tbx18-NRVMs compared to control. I. Tbx18-NRVMs showed 1.7-fold higher intracellular cAMP level compared to control. J. Higher intracellular cAMP concentration in Tbx18-NRVMs is complemented by cessation of spontaneous whole-cell Ca²⁺ transients upon PKA inhibition in Tbx18-NRVMs but not in GFP-NRVMs (15 μM PKI, p<0.05).

Figure 3

Morphological, epigenetic and functional features of Tbx18-transduced myocytes. Tbx18 leads to myofibrillar disorganization in NRVMs bearing resemblance to the unstructured myofibrillar network observed in the SAN in comparison to right atrium (RA). A. Neonatal rat SAN, demarcated by HCN4 expression (top middle), exhibits weaker and unstructured sarcomeric α-actinin (α-SA) expression (top panel). Bottom left: Zoomed-in image of the boxed area in top left. The pattern is faithfully recapitulated in Tbx18-NRVMs compared to GFP-NRVMs (bottom right and middle, respectively). Scale bar: 30μm. B. Comparison of cell area measurements (n=250 for each group) and cell membrane capacitance (n=16 for each group) indicate a 28% and 33% reduction (left and right, respectively) in Tbx18-NRVMs compared to control. C. Tri-methylation level on H3K27 indicates that Tbx18 increased inactivity of Cx43, Kir2.1, and α-SA promoters while relieving its repressive epigenetic pressure on HCN4 promoter normalized to control. Meanwhile, H3K4me3 levels indicate that ratio of active HCN4 promoter regions increased upon Tbx18 expression while the transcriptionally active promoter regions of Cx43, Kir2.1, and α-SA have decreased upon Tbx18 expression. (n=3 experiments. For each experiment, NRVMs from 6 wells of a 6-well plate were isolated for each group.) D. Focal expression of Tbx18 in the apex of guinea pig hearts in vivo created ectopic ventricular beats. Representative electrocardiographs (lead II) of GFP- or Tbx18-injected guinea pig (left and right, respectively, top panels). Ectopic ventricular pacemaker activity was observed in Tbx18-injected guinea pigs, but not in GFP-guinea pigs. Heart axis plots (bottom panels) suggest that the ectopic beats in Tbx18-guinea pig (right) originated near the site of gene injection (apex) and propagated towards the base. In contrast, the escape beats in control guinea pigs propagated toward the apex from the atrioventricular junction (left, n=5 for GFP-guinea pig and n=7 for Tbx18-guinea pig). E. The rate of ectopic ventricular beats in Tbx18-injected animals at day 3-5 after gene delivery is significantly higher than GFP-injected animals.

Figure 4

Tbx18 converts adult guinea pig ventricular myocytes into pacemaker cells in vivo. A. Left panel: representative images of freshly-isolated SAN myocytes, Tbx18-VMs (reported by GFP expression), and GFP-VMs. Right panel: immunostaining against α-SA reveals disorganized myofibrillar structure in Tbx18-VMs akin to what is observed in native SAN myocytes. Scale bar = 50 μm. B. Analyses of myocyte length-to-width ratio and whole-cell capacitance as a measure of cell shape and size from freshly-isolated, living myocytes. Tbx18-VMs are smaller in size and spindle-shaped compared to GFP-VMs (n=53 for Tbx18-VMs and 80 for GFP-VMs and non-transduced VMs, p<0.01), but similar to native SAN myocytes (n=24). C. Spontaneous action potentials recorded from freshly-isolated single Tbx18-VMs (n=5, middle panel) with perforated-patch current-clamp technique, showing robust and rhythmic APs with prominent diastolic depolarization recapitulating the electrophysiological hallmark of native SAN myocytes (left panel). The same recordings are expanded in the lower panel to show prominent diastolic depolarization in Tbx18-VMs and native SAN myocytes. Right panel: GFP-VMs displayed stable resting membrane potential and fired single action potential only upon electrical stimulation. D. Action potential parameters of Tbx18-VMs (n=5) were closer to native SAN myocytes (n=6) than to GFP-VMs (n=6). *p<0.05 vs. GFP-VMs.

Figure 5

De novo automaticity responds to autonomic regulation. A. A layout of 6-well multi-electrode array (MEA, left panel) and a representative image of NRVMs cultured as a monolayer on such a well. B. Average firing rates recorded from the MEAs demonstrate significantly faster baseline chronotropy in Tbx18-NRVMs compared to that in control. Firing rates of Tbx18-NRVMs increased significantly upon β-adrenergic stimulation by changing the basal media with one that containing1 μM isoproterenol (ISO). Subsequent cholinergic challenge with 1 μM acetylcholine (ACh) significantly slowed the firing rates of Tbx18-NRVMs. In contrast, the chronotropy of GFP-NRVMs responded little to the autonomic inputs. C. Representative raw traces from an electrode of a 6-well MEA plated with Tbx18-NRVMs. D. Immunostaining on Tbx18-NRVMs (GFP⁺ cells) demonstrates robust expression of β-adrenergic receptors and M₂ muscarinic receptors. E. Electrocardiographic recordings of an intact perfused heart injected with Tbx18 at the apex. Tbx18 was directly injected into the apex of a guinea pig heart in vivo. Seven days post-injection, the heart was harvest, perfused, and cryoablated at the AV junctional region. The polarity and morphology of the ectopic beats (left panel) is identical to those of electrode-paced beats at the site of transgene injection (right). F. In contrast, most control hearts (7/10) showed a narrow-QRS junctional escape rhythm (left panel), which were opposite in polarity and morphology to those of electrode-paced beats at the apex (right panel). G. Chronotropic response of Tbx18-injected hearts to autonomic inputs was assessed by changing the perfusate (normal Tyrode’s solution) to one that containing 1 μM isoproterenol for β-adrenergic stimulation followed by one that containing 1 μM acetylcholine for cholinergic suppression.

Figure 5

De novo automaticity responds to autonomic regulation. A. A layout of 6-well multi-electrode array (MEA, left panel) and a representative image of NRVMs cultured as a monolayer on such a well. B. Average firing rates recorded from the MEAs demonstrate significantly faster baseline chronotropy in Tbx18-NRVMs compared to that in control. Firing rates of Tbx18-NRVMs increased significantly upon β-adrenergic stimulation by changing the basal media with one that containing1 μM isoproterenol (ISO). Subsequent cholinergic challenge with 1 μM acetylcholine (ACh) significantly slowed the firing rates of Tbx18-NRVMs. In contrast, the chronotropy of GFP-NRVMs responded little to the autonomic inputs. C. Representative raw traces from an electrode of a 6-well MEA plated with Tbx18-NRVMs. D. Immunostaining on Tbx18-NRVMs (GFP⁺ cells) demonstrates robust expression of β-adrenergic receptors and M₂ muscarinic receptors. E. Electrocardiographic recordings of an intact perfused heart injected with Tbx18 at the apex. Tbx18 was directly injected into the apex of a guinea pig heart in vivo. Seven days post-injection, the heart was harvest, perfused, and cryoablated at the AV junctional region. The polarity and morphology of the ectopic beats (left panel) is identical to those of electrode-paced beats at the site of transgene injection (right). F. In contrast, most control hearts (7/10) showed a narrow-QRS junctional escape rhythm (left panel), which were opposite in polarity and morphology to those of electrode-paced beats at the apex (right panel). G. Chronotropic response of Tbx18-injected hearts to autonomic inputs was assessed by changing the perfusate (normal Tyrode’s solution) to one that containing 1 μM isoproterenol for β-adrenergic stimulation followed by one that containing 1 μM acetylcholine for cholinergic suppression.

Figure 6

Single-cell, quantitative RT-PCR of long-term Tbx18-VMs indicates persistent automaticity even after Tbx18 expression had waned. A. To validate the sensitivity of single-cell transcript detection by RT-qPCR, Tbx18-transduced ventricular myocytes were assayed three days after in vivo gene transfer. Tbx18 transcript levels could be reliably detected over a wide range from very low (2.6% of GAPDH, cell 1) to very high (168% of GAPDH, cell 8). B. Ventricular myocytes were freshly isolated from the guinea pigs 6-8 weeks after the initial in vivo gene transfer. RT-qPCR of spontaneously-beating cells demonstrate that the transcript levels of Tbx18 were either small (cells 1 and 2) or negligible (cells 3 and 4). A Tbx18-VM with a strong GFP signal (cell 5) exhibited larger relative amount of Tbx18. C. For negative control, ventricular myocytes expressing GFP alone were assayed for Tbx18.