Shox2 regulates the pacemaker gene program in embryoid bodies.

The pacemaker tissues of the heart are a complex set of specialized cells that initiate the rhythmic heartbeat. The sinoatrial node (SAN) serves as the primary pacemaker, whereas the atrioventricular node can serve as a subsidiary pacemaker in cases of SAN failure or block. The elucidation of genetic networks regulating the development of these tissues is crucial for understanding the mechanisms underlying arrhythmias and for the design of targeted therapies. Here we report temporal and spatial self-organized formation of the pacemaker and contracting tissues in three-dimensional aggregate cultures of mouse embryonic stem cells termed embryoid bodies (EBs). Using genetic marker expression and electrophysiological analyses we demonstrate that in EBs the pacemaker potential originates from a localized population of cells and propagates into the adjacent contracting region forming a functional syncytium. When Shox2, a major determinant of the SAN genetic pathway, was ablated we observed substantial slowing of spontaneous contraction rates and an altered gene expression pattern including downregulation of HCN4, Cx45, Tbx2, Tbx3, and bone morphogenetic protein 4 (BMP4); and upregulation of Cx40, Cx43, Nkx2.5, and Tbx5. This phenotype could be rescued by adding BMP4 to Shox2 knockout EBs in culture from days 6 to 16 of differentiation. When wild-type EBs were treated with Noggin, a potent BMP4 inhibitor, we observed a phenotype consistent with the Shox2 knockout EB. Altogether, we have generated a reproducible in vitro model that will be an invaluable tool for studying the molecular pathways regulating the development of cardiac pacemaker tissues.

Embryonic stem cell-derived cardiomyocytes harbor a subpopulation of niche-forming Sca-1+ progenitor cells.

The adult mammalian heart is known to contain a population of cardiac progenitor cells. It has not been unambiguously determined, however, whether these cells form as part of the developmental program of the heart or migrate there by way of the circulatory system. This study was done in order to determine the origin of this population of cells. A population of cardiomyocytes was established from mouse embryonic stem (ES) cells using a genetic selection technique. In order to determine whether cardiac progenitor cells exist within this ES cell-derived cardiomyocyte population, the cells were analyzed by fluorescence activated cell sorting (FACS) using an antibody directed against stem cell antigen-1 (Sca-1). We observed that approximately 4% of the cardiomyocyte population was composed of Sca-1(+) cells. When the Sca-1(+) cells were isolated by magnetic cell sorting and differentiated as cellular aggregates, contractions were observed in 100% of the aggregates. Gene expression studies using quantitative RT-PCR showed that these cells expressed terminally differentiated cardiac-specific genes. When three-dimensional cellular aggregates were formed from ES cell-derived cardiomyocytes co-cultured with adult HL-1 cardiomyocytes, the Sca-1(+) cells were found to "sort out" and form niches within the cell aggregates. Our data demonstrate that cardiac progenitor cells in the adult heart originate as part of the developmental program of the heart and that Sca-1(+) progenitor cells can provide an important in vitro model system to study the formation of cellular niches in the heart.

Rab8 interacts with distinct motifs in alpha2B- and beta2-adrenergic receptors and differentially modulates their transport.

The molecular mechanism underlying the post-Golgi transport of G protein-coupled receptors (GPCRs) remains poorly understood. Here we determine the role of Rab8 GTPase, which modulates vesicular protein transport between the trans-Golgi network (TGN) and the plasma membrane, in the cell surface targeting of alpha(2B)- and beta(2)-adrenergic receptors (AR). Transient expression of GDP- and GTP-bound Rab8 mutants and short hairpin RNA-mediated knockdown of Rab8 more potently inhibited the cell surface expression of alpha(2B)-AR than beta(2)-AR. The GDP-bound Rab8(T22N) mutant attenuated ERK1/2 activation by alpha(2B)-AR, but not beta(2)-AR, and arrested alpha(2B)-AR in the TGN compartment. Co-immunoprecipitation revealed that both alpha(2B)-AR and beta(2)-AR physically interacted with Rab8 and glutathione S-transferase fusion protein pulldown assays demonstrated that Rab8 interacted with the C termini of both receptors. Interestingly, mutation of the highly conserved membrane-proximal C terminus dileucine motif selectively blocked beta(2)-AR interaction with Rab8, whereas mutation of residues Val(431)-Phe(432)-Asn(433)-Gln(434), Pro(447)-Trp(448), Gln(450)-Thr(451), and Trp(453) in the C terminus impaired alpha(2B)-AR interaction with Rab8. Furthermore, transport inhibition by Rab8(T22N) of a chimeric beta(2)-AR carrying the alpha(2B)-AR C terminus was similar to alpha(2B)-AR. These data provide strong evidence indicating that Rab8 GTPase interacts with distinct motifs in the C termini of alpha(2B)-AR and beta(2)-AR and differentially modulates their traffic from the TGN to the cell surface.

Enhancement of the recycling and activation of beta-adrenergic receptor by Rab4 GTPase in cardiac myocytes.

We investigate the role of Rab4, a Ras-like small GTPase coordinating protein transport from the endosome to the plasma membrane, on the recycling and activation of endogenous beta-adrenergic receptor (beta-AR) in HL-1 cardiac myocytes in vitro and transgenic mouse hearts in vivo. Beta1-AR, the predominant subtype of beta-AR in HL-1 cardiac myocytes, was internalized after stimulation with isoproterenol (ISO) and fully recycled at 4 h upon ISO removal. Transient expression of Rab4 markedly facilitated recycling of internalized beta-AR to the cell surface and enhanced beta-AR signaling as measured by ISO-stimulated cAMP production. Transgenic overexpression of Rab4 in the mouse myocardium significantly increased the number of beta-AR in the plasma membrane and augmented cAMP production at the basal level and in response to ISO stimulation. Rab4 overexpression induced concentric cardiac hypertrophy with a moderate increase in ventricle/body weight ratio and posterior wall thickness and a selective up-regulation of the beta-myosin heavy chain gene. These data provide the first evidence indicating that Rab4 is a rate-limiting factor for the recycling of endogenous beta-AR and augmentation of Rab4-mediated traffic enhances beta-AR function in cardiac myocytes.

The 21-day postnatal rat ventricular cardiac muscle cell in culture as an experimental model to study adult cardiomyocyte gene expression.

The purpose of this study was to develop and characterize a cardiomyocyte culture system for use as an experimental model to study the mechanism(s) by which cardiac muscle cells permanently exit the cell cycle during early neonatal life. Ventricular cardiomyocytes, isolated by retrograde perfusion of hearts from 21-day-old and adult rats, were compared through 10 days of culture. Expression patterns of genes encoding developmentally programmed proteins were determined to be similar between cardiomyocytes cultured from 21-day-old and adult rats, using the reverse transcription polymerase chain reaction. A lacZ-expressing reporter gene was used to test the efficiency of gene delivery in cultured cardiomyocytes. Transfections using cationic liposomes yielded 24+/-7, 25+/-7 and 10+/-1% cardiomyocytes positive for beta-galactosidase activity in cultured 1-day, 21-day and adult cardiomyocytes, respectively. Direct needle microinjection resulted in 48+/-7, 35+/-6 and 37+/-5% cardiomyocytes positive for enzymatic activity in 1-day, 21-day and adult cardiomyocytes, respectively. Cell cycle-specific cDNA arrays were used to analyze the expression pattern of cell cycle-related genes in 12-O-tetradecanoyl-phorbol-13-acetate (TPA)- and non-TPA-treated cultured 21-day cardiomyocytes. Based on the similarity of cultured 21-day to adult ventricular cardiomyocytes and their high transfection efficiencies, we propose the use of cultured cardiomyocytes from 21-day-old rat ventricles as an experimental model system for the study of adult cardiomyocyte gene expression and cell cycle machinery.