Despite the central role of quantitative PCR (qPCR) in the quantification of mRNA transcripts, most analyses of qPCR data are still delegated to the software that comes with the qPCR apparatus. This is especially true for the handling of the fluorescence baseline. This article shows that baseline estimation errors are directly reflected in the observed PCR efficiency values and are thus propagated exponentially in the estimated starting concentrations as well as ‘fold-difference’ results. Because of the unknown origin and kinetics of the baseline fluorescence, the fluorescence values monitored in the initial cycles of the PCR reaction cannot be used to estimate a useful baseline value. An algorithm that estimates the baseline by reconstructing the log-linear phase downward from the early plateau phase of the PCR reaction was developed and shown to lead to very reproducible PCR efficiency values. PCR efficiency values were determined per sample by fitting a regression line to a subset of data points in the log-linear phase. The variability, as well as the bias, in qPCR results was significantly reduced when the mean of these PCR efficiencies per amplicon was used in the calculation of an estimate of the starting concentration per sample.

The sinoatrial node initiates the heartbeat and controls the rate and rhythm of contraction, thus serving as the pacemaker of the heart. Despite the crucial role of the sinoatrial node in heart function, the mechanisms that underlie its specification and formation are not known. Tbx3, a transcriptional repressor required for development of vertebrates, is expressed in the developing conduction system. Here we show that Tbx3 expression delineates the sinoatrial node region, which runs a gene expression program that is distinct from that of the bordering atrial cells. We found lineage segregation of Tbx3-negative atrial and Tbx3-positive sinoatrial node precursor cells as soon as cardiac cells turn on the atrial gene expression program. Tbx3 deficiency resulted in expansion of expression of the atrial gene program into the sinoatrial node domain, and partial loss of sinoatrial node-specific gene expression. Ectopic expression of Tbx3 in mice revealed that Tbx3 represses the atrial phenotype and imposes the pacemaker phenotype on the atria. The mice displayed arrhythmias and developed functional ectopic pacemakers. These data identify a Tbx3-dependent pathway for the specification and formation of the sinoatrial node, and show that Tbx3 regulates the pacemaker gene expression program and phenotype.[Keywords: Heart development; pacemaker; sinoatrial node; lineage; Tbx3; transgenic mice] Supplemental material is available at http://www.genesdev.org.

Abstract-The sinoatrial node, which resides at the junction of the right atrium and the superior caval vein, contains specialized myocardial cells that initiate the heart beat. Despite this fundamental role in heart function, the embryonic origin and mechanisms of localized formation of the sinoatrial node have not been defined. Here we show that subsequent to the formation of the Nkx2-5-positive heart tube, cells bordering the inflow tract of the heart tube give rise to the Nkx2-5-negative myocardial cells of the sinoatrial node and the sinus horns. Using genetic models, we show that as the myocardium of the heart tube matures, Nkx2-5 suppresses pacemaker channel gene Hcn4 and T-box transcription factor gene Tbx3, thereby enforcing a progressive confinement of their expression to the forming Nkx2-5-negative sinoatrial node and sinus horns. Thus, Nkx2-5 is essential for establishing a gene expression border between the atrium and sinoatrial node. Tbx3 was found to suppress chamber differentiation, providing an additional mechanism by which the Tbx3-positive sinoatrial node is shielded from differentiating into atrial myocardium. Pitx2c-deficient fetuses form sinoatrial nodes with indistinguishable molecular signatures at both the right and left sinuatrial junction, indicating that Pitx2c functions within the left/right pathway to suppress a default program for sinuatrial node formation on the left. Our molecular pathway provides a mechanism for how pacemaker activity becomes progressively relegated to the most recently added components of the venous pole of the heart and, ultimately, to the junction of the right atrium and superior caval vein. Key Words: heart development Ⅲ sinus node Ⅲ sinoatrial node Ⅲ Nkx2-5 Ⅲ Pitx2c Ⅲ Tbx3 Ⅲ Cx40 Ⅲ transgenic mice T he sinoatrial node (SAN) is the pacemaker of the heart that initiates the heartbeat and controls its rate of contraction. It is a specialized myocardial structure positioned at the junction of the right atrium and the superior caval vein. Disease, ageing, or gene defects may cause SAN dysfunction (sick sinus syndrome), a common cardiac disorder that currently requires the implantation of a pacemaker. SAN function depends on a complex tissue architecture and the expression and function of a set of ion-channel, sarcomeric and gap junction proteins expressed at levels distinct from the atrial working myocardium. 1,2 Hyperpolarization-activated pacemaker channel Hcn4 is expressed in the SAN and plays an important role in pacemaker activity in vivo. 2,3 Furthermore, the SAN expresses very low levels of connexin 40 (Cx40) and Cx43 gap-junctional subunits that are essential for the fast conduction of the electrical impulse in the atrial and ventricular working myocardium and ventricular conduction system. 4,5 The T-box transcription factor Tbx3, which, when mutated, causes ulnar-mammary syndrome of congenital malformation in human, 6 is expressed in the developing nodal tissues of the heart of mammals and chicken and is able to suppress promoter activity of atrial chamb...

Specific regions of the embryonic heart tube differentiate into atrial and ventricular chamber myocardium, whereas the inflow tract, atrioventricular canal, inner curvatures, and outflow tract do not. These regions express Tbx2, a transcriptional repressor. Here, we tested its role in chamber formation. The temporal and spatial pattern of Tbx2 mRNA and protein expression in mouse hearts was found to be complementary to that of chamber myocardiumspecific genes Nppa, Cx40, Cx43, and Chisel, and was conserved in human. In vitro, Tbx2 repressed the activity of regulatory fragments of Cx40, Cx43, and Nppa. Hearts of transgenic embryos that expressed Tbx2 in the prechamber myocardium completely failed to form chambers and to express the chamber myocardium-specific genes Nppa, Cx40, and Chisel, whereas other cardiac genes were normally expressed. These findings provide the first evidence that Tbx2 is a determinant in the local repression of chamber-specific gene expression and chamber differentiation.

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We identified the T-box transcription factor Tbx3 as a novel and accurate marker for the central conduction system. Our analysis implicates a role for Tbx3 in repressing a chamber-specific program of gene expression in regions from which the components of the central conduction system are subsequently formed.

Loss of muscle mass and function occurs in various diseases. Myostatin blocking can attenuate muscle loss, but downstream signaling is not well known. Therefore, to elucidate associated signaling pathways, we used the soluble activin receptor IIb (sActRIIB-Fc) to block myostatin and activins in mice. Within 2 wk, the treatment rapidly increased muscle size as expected but decreased capillary density per area. sActRIIB-Fc increased muscle protein synthesis 1–2 days after the treatment correlating with enhanced mTORC1 signaling (phosphorylated rpS6 and S6K1, r = 0.8). Concurrently, increased REDD1 and eIF2Bε protein contents and phosphorylation of 4E-BP1 and AMPK was observed. In contrast, proangiogenic MAPK signaling and VEGF-A protein decreased. Hippo signaling has been characterized recently as a regulator of organ size and an important regulator of myogenesis in vitro. The phosphorylation of YAP (Yes-associated protein), a readout of activated Hippo signaling, increased after short- and longer-term myostatin and activin blocking and in exercised muscle. Moreover, dystrophic mdx mice had elevated phosphorylated and especially total YAP protein content. These results show that the blocking of myostatin and activins induce rapid skeletal muscle growth. This is associated with increased protein synthesis and mTORC1 signaling but decreased capillary density and proangiogenic signaling. It is also shown for the first time that Hippo signaling is activated in skeletal muscle after myostatin blocking and exercise and also in dystrophic muscle. This suggests that Hippo signaling may have a role in skeletal muscle in various circumstances.

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