During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the homeodomain factor Nkx2-5. We now show that feedback repression of Bmp2/Smad1 signaling by Nkx2-5 critically regulates SHF proliferation and outflow tract (OFT) morphology. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification (including Bmp2) and maintenance of the progenitor state were upregulated, leading initially to progenitor overspecification, but subsequently to failed SHF proliferation and OFT truncation. In Smad1 mutants, SHF proliferation and deployment to the OFT were increased, while Smad1 deletion in Nkx2-5 mutants rescued SHF proliferation and OFT development. In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies were also rescued by Smad1 deletion. Our findings demonstrate that Nkx2-5 orchestrates the transition between periods of cardiac induction, progenitor proliferation, and OFT morphogenesis via a Smad1-dependent negative feedback loop, which may be a frequent molecular target in CHD.

Abstract-Migration and proliferation of arterial smooth muscle cells (SMCs) play a prominent role in the development of atherosclerotic plaques and restenosis lesions. Most of the growth-regulatory molecules potentially involved in these pathological conditions also demonstrate chemotactic properties. Extracellular purine and pyrimidine nucleotides have been shown to induce cell cycle progression and to elicit growth of cultured vascular SMCs. Moreover, the P2Y 2 ATP/UTP receptor was overexpressed in intimal thickening, suggesting a role of these nucleotides in vascular remodeling. Using the Transwell system migration assay, we demonstrate that extracellular ATP, UTP, and UDP exhibit a concentration-dependent chemotactic effect on cultured rat aortic SMCs. UTP, the most powerful nucleotide inducer of migration, elicited significant responses from 10 nmol/L. In parallel, UTP increased osteopontin expression dose-dependently. Key Words: extracellular nucleotides Ⅲ aortic smooth muscle cells Ⅲ migration Ⅲ osteopontin S everal studies suggest that migration and proliferation of arterial smooth muscle cells (SMCs) play a prominent role in the development of atherosclerotic plaques and restenosis lesions. 1,2 Although SMC proliferation is an important feature in experimental arterial injury models, however, only few proliferating SMCs have been detected in human primary or secondary atherosclerotic plaques, 3,4 thus underlining the prominent role of the migration process in these pathological conditions. SMC proliferation and migration are the result of multifactorial stimulation. Many growth-regulatory molecules and cytokines have been described in atherosclerotic plaques. 5 Extracellular nucleotides have also recently been shown to be involved in SMC growth. Extracellular purine and pyrimidine nucleotides induce cell cycle progression and elicit growth of cultured vascular SMCs. 6 -8 This mitogenic response involves the nucleotide binding to G protein-coupled P2Y receptor subtypes, 9 -11 including the P2Y 2 receptor, which is activated by UTP and ATP. 12 Furthermore, the P2Y 2 receptor is upregulated in cytokine-stimulated SMCs 13 and in rat aorta after balloon injury, 14 suggesting that extracellular ATP and UTP could play a critical role in intimal hyperplasia or vascular remodeling. Vascular P2Y receptors are activated in an autocrine or paracrine manner by nucleotides that are released in the vascular wall from perivascular nerves, activated platelets, and mechanically stretched cells. [15][16][17] Because many mitogenic compounds for SMCs also demonstrate a chemoattractant activity, 5 extracellular nucleotides could also exert a chemotactic effect on arterial SMCs. A potent role for extracellular nucleotides in SMC migration has also been suggested because it was previously shown that expression of the chemotactic protein osteopontin (OPN) is induced by ATP and UTP in cultured SMCs. 10,18 OPN is an RGD-containing extracellular matrix (ECM) protein involved in cell attachment and migration. Its activity necessi...

Abstract-Store-operated Ca 2ϩ entry was investigated in isolated mouse sinoatrial nodes (SAN) dissected from right atria and loaded with Ca 2ϩ indicators. Incubation of the SAN in Ca 2ϩ -free solution caused a substantial decrease in resting intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) and stopped pacemaker activity. Reintroduction of Ca 2ϩ in the presence of cyclopiazonic acid (CPA), a sarcoplasmic reticulum Ca 2ϩ pump inhibitor, led to sustained elevation of [Ca 2ϩ ] i , a characteristic of store-operated Ca 2ϩ channel (SOCC) activity. Two SOCC antagonists, Gd 3ϩ and SKF-96365, inhibited 72Ϯ8% and 65Ϯ8% of this Ca 2ϩ influx, respectively. SKF-96365 also reduced the spontaneous pacemaker rate to 27Ϯ4% of control in the presence of CPA. Because members of the transient receptor potential canonical (TRPC) gene family may encode SOCCs, we used RT-PCR to examine mRNA expression of the 7 known mammalian TRPC isoforms. Transcripts for TRPC1, 2, 3, 4, 6, and 7, but not TRPC5, were detected. Immunohistochemistry using anti-TRPC1, 3, 4, and 6 antibodies revealed positive labeling in the SAN region and single pacemaker cells. 4,5 In ventricular myocytes, PLC responses are generally modest with only small amounts of IP 3 being produced. 6 Thus release of Ca 2ϩ through IP 3 R is probably too small to modify excitation-contraction coupling. However, atria myocytes express functional IP 3 R at 6 to 10 times higher levels than those in ventricules, and Ca 2ϩ release from IP 3 R may be involved in the generation of arrhythmias. 7 Furthermore, recent studies have shown that activation of PLC in the heart leads to Ca 2ϩ release from perinuclear IP 3 R and thereby regulates nuclear-cytoplasmic cycling of transcription factors and alters gene expression. 8 A similar role may be played by IP 3 and the IP 3 R in skeletal muscle and SOCCs have been implicated in IGF-1 induced muscle hypertrophy. 5,9 The identity of the genes that encode SOCCs remains uncertain. Studies of the transient receptor potential (TRP) gene from Drosophila showed that it encodes a PLC-activated Ca 2ϩ permeable channel. 10 Subsequently, 7 TRP channel homologues in mammals, termed TRPC1-TRPC7, have been identified and there is considerable evidence to indicate that TRPC1 can encode a SOCC. 11 In addition, a recent study showed that overexpression of TRPC3 substantially enhanced SOCC activity in the heart. 5 The importance of Ca 2ϩ release from stores in cardiac pacemaking is now widely accepted. [12][13][14] In a previous study, we found that activation of the P2Y 1 purinergic receptor by ATP results in modulation of pacemaker firing attirbutable to receptor-coupled PLC activation and depletion of SR Ca 2ϩ stores. 15 Because activation of SOCCs also involves PLC, we speculated that SOCCs might be present in pacemaker tissue.In mammals, the sinoatrial node (SAN) is a heterogeneous tissue. The shape of the action potential and the rate of rise of the pacemaker potential change progressively from the periphery to the center, the latter being the leadin...