Transcriptome analysis of mouse and human sinoatrial node cells reveals a conserved genetic program.

The rate of contraction of the heart relies on proper development and function of the sinoatrial node, which consists of a small heterogeneous cell population, including Tbx3+ pacemaker cells. Here, we have isolated and characterized the Tbx3+ cells from Tbx3+/Venus knock-in mice. We studied electrophysiological parameters during development and found that Venus-labeled cells are genuine Tbx3+ pacemaker cells. We analyzed the transcriptomes of late fetal FACS-purified Tbx3+ sinoatrial nodal cells and Nppb-Katushka+ atrial and ventricular chamber cardiomyocytes, and identified a sinoatrial node-enriched gene program, including key nodal transcription factors, BMP signaling and Smoc2, the disruption of which in mice did not affect heart rhythm. We also obtained the transcriptomes of the sinoatrial node region, including pacemaker and other cell types, and right atrium of human fetuses, and found a gene program including TBX3, SHOX2, ISL1 and HOX family members, and BMP and NOTCH signaling components conserved between human and mouse. We conclude that a conserved gene program characterizes the sinoatrial node region and that the Tbx3+/Venus allele provides a reliable tool for visualizing the sinoatrial node, and studying its development and function.

Misexpression of Tbx18 in cardiac chambers of fetal mice interferes with chamber-specific developmental programs but does not induce a pacemaker-like gene signature.

Initiation of cardiac excitation depends on a specialized group of cardiomyocytes at the venous pole of the heart, the sinoatrial node (SAN). The T-box transcription factor gene Tbx18 is expressed in the SAN myocardium and is required for formation of a large portion of the pacemaker. Previous studies suggested that Tbx18 is also sufficient to reprogram ventricular cardiomyocytes into SAN cells in rat, guinea-pig and pig hearts. To evaluate the consequences of misexpression of Tbx18 for imposing a nodal phenotype onto chamber myocardial cells in fetal mice, we used two independent conditional approaches with chamber-specific cre driver lines and an Hprt(Tbx18) misexpression allele. Myh6-Cre/+;Hprt(Tbx18/y) mice developed dilated atria with thickened walls, reduced right ventricles and septal defects that resulted in reduced embryonic and post-natal survival. Tagln-Cre/+;Hprt(Tbx18/y) mice exhibited slightly smaller hearts with rounded trabeculae that supported normal embryonic survival. Molecular analyses showed that the SAN gap junction and ion channel profile was not ectopically induced in chamber myocardium but the working myocardial gene program was partially inhibited in atria and ventricles of both misexpression models. Left atrial expression of Pitx2 was strongly repressed in Myh6-Cre/+;Hprt(Tbx18/y) embryos. We conclude that exclusion of Tbx18 expression from the developing atria and (right) ventricle is important to achieve normal cardiac left-right patterning and myocardial differentiation, and that Tbx18 is not sufficient to induce full SAN differentiation of chamber cardiomyocytes in fetal mice.

Evidence for a Physiological Role of T-Type Ca Channels in Ventricular Cardiomyocytes of Adult Mice.

T-type Ca channels are strongly expressed and important in the developing heart. In the adult heart, these channels play a significant role in pacemaker tissues, but there is uncertainty about their presence and physiological relevance in the working myocardium. Here, we show that the T-type Ca channel isoforms Cav3.1 and Cav3.2 are expressed at a protein level in ventricular cardiomyocytes from healthy adult C57/BL6 mice. Myocytes isolated from adult wild-type and Cav3.2 KO mice showed considerable whole cell T-type Ca currents under beta-adrenergic stimulation with isoprenaline. We further show that the detectability of basal T-type Ca currents in murine wild-type cardiomyocytes depends on the applied experimental conditions. Together, these findings reveal the presence of functional T-type Ca channels in the membrane of ventricular myocytes. In addition, electrically evoked Ca release from the sarcoplasmic reticulum was significantly impaired in Cav3.2 KO compared to wild-type cardiomyocytes. Our work implies a physiological role of T-type Ca channels in the healthy adult murine ventricular working myocardium.

Gradual differentiation and confinement of the cardiac conduction system as indicated by marker gene expression.

The components of the cardiac conduction system, responsible for coordinated activation of the heart chambers, are well defined and their cells differ in gene expression profile and phenotype from those of the surrounding working myocardium. Yet, when and on what basis the myocardium of each of the conduction system components become distinguishable from other myocardium during heart development has not been well established. To identify and assess cell type-specific expression profiles and differentiation markers, we performed transcriptome analysis on fluorescence activated cell sorted purified conduction system (Venus+) and chamber myocardial cells (Katushka+) of Tbx3+/Venus;TgNppb(Katushka) double transgenic mouse fetuses. We found that transcripts associated with nervous system development and ion channel activity were enriched in Tbx3+ conduction system cells, whereas transcripts associated with mitochondrial function, muscle contraction and fatty acid metabolism were enriched in the Nppb+ working myocardium. We analyzed spatio-temporal expression patterns of several candidate markers (Cacna2d2, Cacna1g, Ephb3, Tnni1), reviewed those of established conduction system markers (Tbx3, Hcn4, Gja5, Cntn2), and placed the patterns in the context of conduction system development. The overview indicates that different properties of conduction system components develop gradually and at different developmental stages, and that chamber myocardium gradually differentiates and diverges from conduction system myocardium until after birth.