Nidhi Kapoor

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

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.

Transcriptional Suppression of Connexin43 by TBX18 Undermines Cell-Cell Electrical Coupling in Postnatal Cardiomyocytes

T-box transcription factors figure prominently in embryonic cardiac cell lineage specifications. Mesenchymal precursor cells expressing Tbx18 give rise to the hearts pacemaker, the sinoatrial node (SAN). We sought to identify targets of TBX18 transcriptional regulation in the heart by forced adenoviral overexpression in postnatal cardiomyocytes. Neonatal rat cardiomyocytes (NRCMs) transduced with GFP showed sarcolemmal, punctate Cx43 expression. In contrast, TBX18-transduced NRCMs exhibited sparse Cx43 expression. Both the transcript and protein levels of Cx43 were greatly down-regulated within 2 days of TBX18 transduction. Direct injection of TBX18 in the guinea pig heart in vivo inhibited Cx43 expression. The repressor activity of TBX18 on Cx43 was highly specific; protein levels of Cx45 and Cx40, which comprise the main gap junctions in the SAN and conduction system, were unchanged by TBX18. A reporter-based promoter assay demonstrated that TBX18 directly represses the Cx43 promoter. Phenotypically, TBX18-NRCMs exhibited slowed intercellular calcein dye transfer kinetics (421 ± 54 versus control 127 ± 43 ms). Intracellular Ca2+ oscillations in control NRCM monolayers were highly synchronized. In contrast, TBX18 overexpression led to asynchronous Ca2+ oscillations, demonstrating reduced cell-cell coupling. Decreased coupling led to slow electrical propagation; conduction velocity in TBX18 NRCMs slowed by more than 50% relative to control (2.9 ± 0.5 versus 14.3 ± 0.9 cm/s). Taken together, TBX18 specifically and directly represses Cx43 transcript and protein levels. Cx43 suppression leads to significant electrical uncoupling, but the preservation of other gap junction proteins supports slow action potential propagation, recapitulating a key phenotypic hallmark of the SAN.

Spatially Defined InsP3-Mediated Signaling in Embryonic Stem Cell-Derived Cardiomyocytes

The functional role of inositol 1,4,5-trisphosphate (InsP3) signaling in cardiomyocytes is not entirely understood but it was linked to an increased propensity for triggered activity. The aim of this study was to determine how InsP3 receptors can translate Ca2+ release into a depolarization of the plasma membrane and consequently arrhythmic activity. We used embryonic stem cell-derived cardiomyocytes (ESdCs) as a model system since their spontaneous electrical activity depends on InsP3-mediated Ca2+ release. [InsP3]i was monitored with the FRET-based InsP3-biosensor FIRE-1 (Fluorescent InsP3 Responsive Element) and heterogeneity in sub-cellular [InsP3]i was achieved by targeted expression of FIRE-1 in the nucleus (FIRE-1nuc) or expression of InsP3 5-phosphatase (m43) localized to the plasma membrane. Spontaneous activity of ESdCs was monitored simultaneously as cytosolic Ca2+ transients (Fluo-4/AM) and action potentials (current clamp). During diastole, the diastolic depolarization was paralleled by an increase of [Ca2+]i and spontaneous activity was modulated by [InsP3]i. A 3.7% and 1.7% increase of FIRE-1 FRET ratio and 3.0 and 1.5 fold increase in beating frequency was recorded upon stimulation with endothelin-1 (ET-1, 100 nmol/L) or phenylephrine (PE, 10 µmol/L), respectively. Buffering of InsP3 by FIRE-1nuc had no effect on the basal frequency while attenuation of InsP3 signaling throughout the cell (FIRE-1), or at the plasma membrane (m43) resulted in a 53.7% and 54.0% decrease in beating frequency. In m43 expressing cells the response to ET-1 was completely suppressed. Ca2+ released from InsP3Rs is more effective than Ca2+ released from RyRs to enhance INCX. The results support the hypothesis that in ESdCs InsP3Rs form a functional signaling domain with NCX that translates Ca2+ release efficiently into a depolarization of the membrane potential.

Regulation of calcium clock‐mediated pacemaking by inositol‐1,4,5‐trisphosphate receptors in mouse sinoatrial nodal cells

Inositol‐1,4,5‐trisphosphate receptors (IP3Rs) modulate pacemaking in embryonic heart, but their role in adult sinoatrial node (SAN) pacemaking is uncertain. We found that stimulation of IP3Rs accelerates spontaneous pacing rate in isolated mouse SAN cells, whereas inhibition of IP3Rs slows pacing. In atrial‐specific sodium‐calcium exchanger (NCX) knockout (KO) SAN cells, where the Ca2+ clock is uncoupled from the membrane clock, IP3R agonists and antagonists modulate the rate of spontaneous Ca2+ waves, suggesting that IP3R‐mediated Ca2+ release modulates the Ca2+ clock. IP3R modulation also regulates Ca2+ spark parameters, a reflection of ryanodine receptor open probability, consistent with the effect of IP3 signalling on Ca2+ clock frequency. Modulation of Ca2+ clock frequency by IP3 signalling in NCX KO SAN cells demonstrates that the effect is independent of NCX. These findings support development of IP3 signalling modulators for regulation of heart rate, particularly in heart failure where IP3Rs are upregulated.

Regulation of Calcium Clock-Mediated Pacemaking by Inositol-1,4,5-Trisphosphate Receptors in Mouse Sinoatrial Nodal Cells

Sinoatrial node (SAN) automaticity is attributable to the interplay of several membrane currents such as funny current (If) and the Na-Ca exchanger (NCX) current activated in response to ryanodine receptor (RyR) mediated Ca release from the sarcoplasmic reticulum (SR). Whether another SR Ca release channel, the inositol-1,4,5-triphosphate receptor (IP3R), can influence automaticity in SAN is controversial, in part due to the confounding influence of periodic Ca flux through the sarcolemma accompanying each beat. We used atrial-specific NCX knockout (KO) SAN cells to study IP3 signaling in a system where periodic [Ca]i cycling persists despite the absence of depolarization or Ca flux across the sarcolemma. We recorded confocal line scans of spontaneous Ca release in WT and NCX KO SAN cells, in the presence or absence of an IP3R blocker (2-APB) or during inhibition of phospholipase C by U73122. We found that superfusion with 2APB (2 µM) decreased the frequency of Ca transients in WT by 82.7% (n=9, p 0.05) and Ca wave frequency in KO cells (n=9, p <0.05) that was reversed by 2-APB. To determine if IP3Rs exert their modulatory effect on pacemaking via RyRs, we recorded Ca transients during application of PE in the continued presence of ryanodine at a blocking concentration (100 µm) that does not deplete SR stores. Under these conditions PE was unable to restore Ca transients. Thus, Ca release from IP3Rs can modulate the “Ca clock” processes that regulate pacemaker frequency in the murine SAN via Ca-induced Ca release through the RyRs.