Cardiac origin of smooth muscle cells in the inflow tract

2.1. Generation of SLN-Cre mice

Exon 2 of the SLN locus including the 1st ATG was replaced with Cre cDNA. A correctly targeted R1 ES clone was screened by Southern blotting and genomic PCR. The recombination efficiency was 1/300 clones.

2.2. Preparation of cardiac mesenchymal feeder layer

Neonatal hearts were predigested with 0.5 mg/ml trypsin in HBSS at 4C overnight followed by strong digestion with collagenase at 37C for 1 hour (0.5 mg/ml in HBSS). Cardiac mesenchymal fibroblasts were separated from myocytes by differential plating for 1 hour twice. Fibroblasts from the first and the second differential plating were combined, grown until confluent and treated with 10μg/ml mitomycin C for 2 hours on the day before progenitors were seeded. The contamination of myocytes in the fibroblast fraction was less than 0.07% by cTnT staining.

2.3. Histology and immunostaining

Whole mount and section Xgal stainings were performed according to standard protocols. Double staining for Xgal and specific antibodies were performed as follows: 8μm frozen sections or cells were stained with Xgal followed by postfixation for 5min, 0.3% hydrogen peroxide treatment for 15min, blocking with 10% normal goat serum for 1 hour and antibody reaction in 3% normal goat serum at 4C overnight. Secondary antibody reaction was performed with Vectastain ABC kit (Vector lab) according to the manufacturer’s protocol. Section Xgal/Isl1 staining was performed as previously described [20]. The concentrations of the primary antibodies are; cTnT (1:200, Lab Vision Corp., Fremont, CA), smMHC (1:500, Biomedical Technologies Inc., Stoughton, MA), Isl1 (1:200, DSHB, Iowa City, IA), DsRed (1:500, Clontech, Mountain View, CA).

2.4. RT-PCR and qPCR

RNA was extracted with Trizol (Invitrogen, Carlsbad, CA) or Absolute nanoprep kit (Stratagene, Ceder Creek, TX) according to the manufacturer’s protocol, and cDNAs were synthesized with iScript kit (BioRad, Hercules, CA). Colony PCR was run for 35 cycles. Quantitative PCR was performed with the SYBR Green system and i-Cycler (BioRad, Hercules, CA).

2.5. Electron microscopic analysis

The SLN^cre/+; R26R hearts were dissected and fixed in 1%PFA and 2.5% glutaraldehyde in PBS for 3 hours, and stained for 4 hours in Bluo-gal staining solution; 1 mg/ml Bluo-gal (Sigma), 10mM ferro/ferri cyanide, 2mM MgCl2, 0.02% NP40 and 0.01% NaDOC. Stained tissues were post-fixed for 30 min in a mixture of 1% osmium tetroxide and 2% glutaraldehyde in 0.15 M cacodylate buffer (on ice), washed several times in PBS, and dehydrated in graded ethanol and acetone (all steps on ice). Preparations were left overnight in a 1:1 mixture of Epon and acetone and then for 5–10 h in unpolymerized Epon. They were transferred to molds, oriented and placed at 60°C for 24 h to permit polymerization of the Epon. Sections were mounted on net grids (Ted Pella) and treated with uranyl acetate and lead citrate.

3.1. SLN-Cre knockin strain is a sensitive and specific deleter line for the atrial lineage

To generate an atrial specific Cre line driven by an internal promoter, we introduced Cre recombinase by homologous recombination into exon 2 of the SLN locus (Fig. 1A, B, C). SLN^cre/+ heterozygotes displayed no morphological or fertility defects. While SLN mRNA is expressed at E10.0 (Fig. 2A), the βgal activity in SLN^cre/+; R26R embryos was first detected in the atria at around E10.5, when Isl1 is still positive in the atrial lineage (Fig. 2B). After E12.5, the atrial myocardium was broadly and strongly labeled by R26R and CAG-DsRed reporter lines [21,22] (Fig. 2C-H”, and Fig. S1A, B). Section Xgal staining of the neonatal hearts revealed that the vast majority of the atrial myocytes were labeled (Fig. 2E, F), whereas none of the endocardial or epicardial cells were stained (Fig. 2F, arrowheads; Supplemental Table 1). Interestingly, HCN4-positive SA nodal cells were also labeled by SLN-Cre (Fig. S2). These data suggest that the SLN-Cre line is a sensitive and specific deleter line for the atrial lineage. To our knowledge, this mouse is the first deleter line in which Cre recombinase is driven by an internal atrial specific promoter.

Xgal staining in the inflow region visualized the anatomical distribution of the myocardial sleeves of the venae cavae and pulmonary veins. The Xgal staining extended up to bifurcation of the internal jugular and subclavian veins in the cranial region, and down to the diaphragm in the thoracic cavity (Fig. 2I-K and S1C). The boundary of the right atrium and the venae cavae is demarcated by venous valves that also are derived from SLN-expressing cells (Fig. 3A, arrowhead). Whereas the muscular layer of the atrial chamber consists only of myocardial cells, the muscular wall of the venae cavae distal to the venous valves consist of two muscular layers – the outer myocardial layer derived from SLN-expressing cells and inner smooth muscle layer positive for smMHC, a definitive marker for vascular smooth muscle cells (Fig. 3A). The myocardial layer tapers off towards the periphery and generates myocardial sleeves in the great veins. Similar to the vena cavae, the proximal region of the pulmonary veins also showed a two layer-structure with outer myocardial sleeve and inner smooth muscle layer (Fig. S3).

3.2. SLN-positive cells give rise to smooth muscle cells in cardiac inflow tract

Further analysis of this two-layer structure revealed the close relationship between cardiac and smooth muscle lineages during cardiovascular development. Double staining of serial sections showed that about 5-10% of smMHC-positive cells in the inflow region are costained with Xgal (Fig. 3C, D, black arrows; Supplemental Table 1). To confirm this, the inflow region of the atrium from SLN^cre/+; R26R adult mice was enzymatically dissociated and cultured on a fibronectin-coated dish. Consistent with section stainings, a fraction of βgal-labeled cells were costained for smMHC (Fig. 3B). Furthermore, electron microscopic analysis of Bluo-gal-stained SLN^cre/+; R26R hearts revealed that the smooth muscle cells with non-striated myofilaments are labeled with Bluo-gal deposits on their membranes (Fig. 3E, E’, arrowheads). βgal-labeled smooth muscle cells were also found in myocardial sleeves in the pulmonary veins (Fig. S3). These data suggest the developmental contribution of SLN-positive cells to the smooth muscle cells in the cardiac inflow tract. It has been reported that SLN mRNA is expressed strongly in atria and esophageal muscle and least abundantly in skeletal muscle and bladder, but no expression has been detected in vascular smooth muscle [18]. Consistently, our SLN-cre lineage tracing experiments fail to detect any Xgal-positive smooth muscle cells in aortic or other major vasculature. Therefore, our findings raise the possibility that these two different cell types in the cardiac inflow region share a common cellular origin during cardiogenesis.

3.3. Isl1⁺/SLN⁺ cells represent a transient cell population in the venous pole

To examine the cellular origin of cardiac and smooth muscle cells in the inflow tract, we searched for Isl1-positive cells in the atrial lineage. As in situ hybridization and lineage tracing experiments indicated that SLN is expressed from E10.0 and on in the atria (Fig. 2A), and Isl1 expression continues until midgestation [15], we speculated that there is a spatial and temporal overlap of these two markers in the forming atria. Double staining for Xgal and Isl1 on SLN^cre/+ × R26R embryos revealed Isl1/SLN double positive cells (Fig. 4). At E10.5, immature cardiac progenitors with strong Isl1 expression were found in the splanchnic mesoderm (Fig. 4A, arrowheads). Cells in the primary atrial septum are also positive for Isl1 (Fig. 4A, white arrow). A subset of cardiac cells in the atrial chamber and sinus venosus have already started to express SLN, and some are positive for both Isl1 and SLN (Fig. 4A, black arrows). At E13.5, Isl1⁺ cells in the splanchnic mesoderm are still negative for SLN (Fig. 4B, black arrowheads), although most of the atrial myocytes were already positive for βgal activity. Isl1⁺/SLN⁺cells were still found in dorsal mesenchymal protrusion (Fig. 4B, black arrows) [15,23-28]. These cells gradually lose Isl1 expression as they migrate towards the cushion and progressively acquire SLN (Fig. 4B, black arrows). These data suggest that a subset of Isl1+ cells give rise to SLN⁺ atrial myocytes, and that Isl1⁺/SLN⁺ cells represent a transient cell population that are committed to the mature atrial myocyte fate.

3.4. Single Isl1+/SLN+ cells give rise to both cardiac and smooth muscle cells

To examine whether cardiac and smooth muscle lineages can originate from a single common Isl1⁺/SLN⁺ cell, we dissected forming atrial tissue from E9.5 SLN^cre/+; R26R embryos, dissociated them into single cells and cultured them at clonal density on cardiac mesenchymal feeder layers as previously described (Fig. 5A) [2,29,30]. βgal-labeled atrial cells grew to form colonies, and more than 90% of them were positive for Isl1 (Fig. 5B, D and Supplementary Table 2). Using a CAG-DsRed reporter line [22], the atrial progenitor colonies were visualized alive (Fig. 5C), picked up under the fluorescent microscope, and examined for their mRNA expression signature. After 3-4 days on feeders, the majority of the clones (early-stage colonies) were positive for early cardiac markers (Isl1, Nkx2.5, GATA4) and atrial markers (SLN, MLC2a) (Fig. 5D). However, the expression of ANF and smMHC were only occasionally found at this stage. Therefore, the feeder system is useful for propagating immature atrial cells in vitro.

After differentiation for 7~12 days in culture, cTnT-negative cells were found in the periphery of the βgal-labeled colonies (Fig. 5E, black arrow). These peripheral cells were co-stained with Xgal/smMHC (Fig. 5E, white arrows). RT-PCR revealed that no colony was positive for Isl1 at this stage (Fig. 5G). Whereas all the clones examined gave rise to cTnT-positive cardiomyocytes, 51.3% of them were capable of giving rise to smMHC-positive cells (Fig. 5G and Supplementary Table 2). Given that 90.3% of the colonies express both Isl1 and SLN, these data suggest that more than half of the Isl1⁺/SLN⁺ cells at E9.5 are capable of giving rise to both cardiac and smooth muscle cells. While more than a half of Isl1+/SLN+ cells are smooth muscle competent, not all of them give rise to smooth muscle cells. To examine the destination of Isl1+/SLN+ cells, DsRed-labeled cells were propagated for 7~12 days on feeders, and FACS-sorted onto glass slides by cytospin for smooth muscle staining. As shown in Fig. 5H, 3.1% of the progeny of atrial cells gave rise to smMHC-positive smooth muscle cells ex vivo consistent with in vivo lineage tracing data (Supplemental Table 1).

Interestingly, atrial colonies derived from later stage (E12.5 and 15.5) showed a decrease in smooth muscle differentiation capability (Supplementary Table 2), suggesting that smooth muscle competency is gradually restricted as the atrial myocytes mature.

Taken together, these data suggest that Isl1-positive cells in the atrial lineage contribute to cardiac as well as smooth muscle cells in the cardiac inflow tract during migration from splanchnic mesoderm.

In conclusion, we reported (1) a novel Cre mouse line which specifically and sensitively labels the atrial cardiac lineage, (2) the developmental contribution of the atrial cells into the smooth muscle cells in cardiac inflow tract, (3) the smooth muscle competency of immature atrial cells, and (4) the gradual loss of smooth muscle competency during cardiogenesis. Together, these findings provide an insight into the mechanism underlying the formation of the boundary between atrial myocardium and vascular smooth muscle layer. The myocardial-smooth muscle junction in the vena cava is often affected in some types of congenital heart disease. Moreover, the myocardial sleeve in pulmonary vein is highly arrhythmogenic in diseased and/or aged hearts. Elucidating how the plasticity of heart progenitors serves the cardiovascular morphogenesis will provide a better understanding of the pathogenesis of these diseases.

Diversification of atrial cell populations

The SHF has been characterized by several different techniques; dye injection, detection of specific markers and retrospective single cell fate tracing experiments. Different methods and different markers have determined slightly different populations of the cardiac lineages [8,10-12,31]. Atrial cells arise from the posterior part of Isl1-positive heart field [16,27,28]. The Isl1⁺/SLN⁺progenitors represent components of the posterior region of the secondary heart field [32], and retain proliferative activity [33] and bipotency until later stages of cardiogenesis, which apparently relates to their generation of the myocardial/smooth muscle sleeves that serve as the junctional boundary to connect the great vessels and the cardiac chambers into a functional syncytium. Interestingly, there are several diseases of the atrium and inflow tract, including a common form of congenital heart disease where the pulmonary venous inflow tract is ectopic or absent, and atrial fibrillation that relates to a reemergence of ectopic electrical activity in the pulmonary veins. The finding of bipotent Isl1+/SLN+ cells suggests the possibility that some of atrial diseases might arise from dys-regulation of the bipotency in atrial lineages.

The Isl1⁺/SLN⁺atrial progenitor population includes at least three populations; working atrial myocytes, Nkx2.5⁻/Tbx18⁺ cardiac progenitors in the sinus venosus and mediastinal myocardium in the dorsal mesocardium [16,23,24,33-35]. These three populations derive from Isl1⁺ posterior SHF progenitors and eventually acquire SLN expression, although our experiments cannot tell which population display smooth muscle competency. Discovery of earlier and later markers will help establish the lineage tree of the posterior SHF/venous pole population.

Lineage proximity of cardiac and smooth muscle cells

Smooth muscle cells appear to be recruited locally from a wide range of mesodermal tissues and differentiate in response to specific local signals. Our data suggest that during the migration from the posterior part of the SHF, Isl1+/SLN+ cells mainly contribute to mature atrial myocytes, but some cells escape from the cardiac lineage and give rise to the smooth muscle cells in the inflow tract (Fig. 6).

Given that SLN is relatively late marker of the atrial lineage, it is not unexpected that SLN+ cells contribute only to 5-10% of the inflow smooth muscle cells. It is rather significant in that atrial cells give rise to smooth muscle cells in vivo even after the expression of SLN gene, which is involved in atrial specific contractile function [17, 18].

It has been known that cardiac cells can transdifferentiate into smooth muscle cells even at the postnatal stage at low frequency upon certain culture conditions. However, little is known as to whether it is a dedifferentiation-redifferentiation process or a direct transition from differentiated cardiomyocyte to differentiated smooth muscle cell. Wu, et al. reported that Nkx2.5-positive cardiac progenitors downregulate Nkx2.5 before committing to smooth muscle lineage [3]. Since Nkx2.5 is also expressed in atrial lineage, it is likely that atrial cells shut of cardiac transcriptional program in order to give rise to smooth muscle cells. Although our study cannot give a clear answer to this question, it raises the possibility that the smooth muscle competency of the postnatal myocytes may be a trace of the developmental potential of the embryonic cardiac progenitors and myocytes.

A fundamental question relates to how the smooth muscle differentiation of cardiac progenitors serves to coordinate the morphogenesis of the atrial chamber and great vessels. An interesting speculation is that the smooth muscle competency of the atrial cells is required to anchor the great veins to the atrial chambers. Mal-connection of myocardium and vascular walls causes various types of congenital and adult heart diseases including anomalous pulmonary venous return and cor triatriatum [36]. In the aging and/or diseased hearts, the junction between pulmonary vein and left atria is highly arrhythmogenic and can be the foci of atrial fibrillation, one of the major cause of thromboembolic stroke [37-41]. Likewise, the heart-vessel junction at the arterial pole of the heart is often affected by various congenital malformations and acquired diseases including DeBakey Type I and II aortic dissection. It would be of great interest to analyze the pathogenesis of these congenital and acquired heart diseases from the viewpoint of progenitor cell plasticity.