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Synergistic Roles of Neuregulin-1 and Insulin-like Growth Factor-I in Activation of the Phosphatidylinositol 3-Kinase Pathway and Cardiac Chamber Morphogenesis*

Open AccessPublished:December 24, 1999DOI:https://doi.org/10.1074/jbc.274.52.37362
      Cardiac chamber morphogenesis requires the coordinated growth of both cardiac muscle and endocardial cell lineages. Paracrine growth factors may modulate the coordinated cellular specification and differentiation during cardiac chamber morphogenesis, as suggested by the essential role of endothelial-derived growth factors, neuregulin-1, and insulin-like growth factor-I. Using the whole mouse embryo culture system for delivery of diffusible factors into the cardiac chamber, neuregulin-1 was shown to promote trabeculation of the ventricular wall. Another factor, insulin-like growth factor-I, had no apparent effect by itself. Combined treatment with neuregulin-1 and insulin-like growth factor-I strongly induced DNA synthesis of cardiomyocytes and expansion of both the ventricular compact zone and the atrioventricular cushions leading to chamber growth and maturation. In cultured cardiomyocytes, combined neuregulin-1 and insulin-like growth factor-I also had a synergistic effect to promote DNA synthesis and cellular growth, which were prevented by wortmannin, an inhibitor of phosphatidylinositol 3-kinase. Adenoviral delivery of dominant negative Rac1, which acts downstream of phosphatidylinositol 3-kinase, blocked the effect of combined neuregulin-1/insulin-like growth factor-I treatment. These studies support the concept that the interaction of neuregulin-1 and insulin-like growth factor-I pathways plays an important role in coordinating cardiac chamber morphogenesis and may occur through convergent activation of phosphatidylinositol 3-kinase.
      NRG-1
      neuregulin-1
      IGF-1
      insulin-like growth factor-1
      En
      embryonic dayn post-coitum
      MAP
      mitogen-activated protein
      PI
      phosphatidylinositol
      PBS
      phosphate-buffered saline
      AchE
      acetylcholinesterase
      BrdUrd
      bromodeoxyuridine
      GFP
      green fluorescent protein
      Av
      atrioventricular
      DiI
      chloromethylbenzamido octadecyl indocarbocyanine
      The formation, maturation, and septation of distinct cardiac chambers during heart development may require the interplay of signals between myocardial and endocardial cell lineages (
      • Fishman M.C.
      • Chien K.R.
      ,
      • Olson E.N.
      • Srivastava D.
      ). These distinct cell types present in the cardiogenic mesoderm will give rise to the double-layered heart tube, with myocardial cells forming the outer layer and endocardial cells forming the inner layer (
      • Cohen G.L.
      • Mikawa T.
      ,
      • Sugi Y.
      • Markwald R.R.
      ). Within the looped cardiac tube, positional signals in the ventricular segment induce specific maturational steps in ventricular muscle cells, including trabeculation, expansion of the ventricular compact zone and atrioventricular cushions, and development of the interventricular septum. Another set of cues leads to the generation of cushion mesenchyme derived from endocardial cells, which contributes to septation of the conotruncus and outflow tract and remodeling of the atrioventricular canal, which ultimately separates the atrial and ventricular chambers (
      • Fishman M.C.
      • Chien K.R.
      ,
      • Olson E.N.
      • Srivastava D.
      ,
      • Eisenberg L.M.
      • Markwald R.R.
      ). It has been previously shown byin vitro assay systems that cushion formation depends on extracellular matrix, and signals emanating from the adjacent cardiomyocytes (
      • Eisenberg L.M.
      • Markwald R.R.
      ,
      • Ramsdell A.F.
      • Markwald R.R.
      ). However, the potential effect of endocardial-derived signals in cushion morphogenesis has not yet been explored. The maturational steps in ventricular muscle and endocardial cells might be mutually coordinated. However, the interplaying signals involved at these different steps of the developing embryonic heart are still unknown.
      Recent studies from targeted gene inactivation in the mouse indicate that two likely candidates for exerting paracrine effects on ventricular chamber and cushion development are neuregulin-1 (NRG-1)1 and insulin-like growth factor-I (IGF-I) (
      • Meyer D.
      • Birchmeier C.
      ,
      • Powell B.L.
      • Hollingshead P.
      • Giltinan D.
      • Pitts M.S.
      • Stewart T.
      ). Both NRG-1 and IGF-I are endothelial-derived signals expressed early in embryonic development that bind and activate receptors expressed throughout the cardiac chamber (
      • Meyer D.
      • Birchmeier C.
      ,
      • Corfas G.
      • Rosen K.M.
      • Aratake H.
      • Krauss R.
      • Fischbach G.D.
      ,
      • Bondy C.A.
      • Werner H.
      • Roberts C.J.
      • LeRoith D.
      ). Targeted ablation of these growth factors or their cognate receptors severely affects murine cardiac morphogenesis. Homozygous null embryos for NRG-1 or the cognate receptors erbB2 or erbB4 display a lack of trabeculation of the ventricular wall, whereas individual knockouts for IGF-I or IGF-I receptor die perinatally with deficient myofibrillogenesis of ventricular myocytes (
      • Meyer D.
      • Birchmeier C.
      ,
      • Powell B.L.
      • Hollingshead P.
      • Giltinan D.
      • Pitts M.S.
      • Stewart T.
      ,
      • Gassmann M.
      • Casagranda F.
      • Orioli D.
      • Simon H.
      • Lai C.
      • Klein R.
      • Lemke G.
      ,
      • Lee K.F.
      • Simon H.
      • Chen H.
      • Bates B.
      • Hung M.C.
      • Hauser C.
      ,
      • Erickson S.
      • Shea K.
      • Ghaboosi N.
      • Loverro L.
      • Frantz G.
      • Bauer M.
      • Lu L.
      • Moore M.
      ,
      • Liu J.P.
      • Baker J.
      • Perkins A.S.
      • Robertson E.J.
      • Efstratiadis A.
      ,
      • Lemke G.
      ,
      • D'Ercole A.J.
      • Ye P.
      • Gutierrez O.G.
      ).
      To assess the role of NRG-1 and IGF-I in cardiac chamber maturation and morphogenesis in intact embryos, we have utilized the whole mouse embryo culture system for injection of these peptide growth factors through the ventricular wall of mouse embryos. The distinct advantage of the ex vivo mouse embryo culture system is that it allows functional studies of exogenous growth factors during cardiac morphogenesis in the context of the intact embryo. The present study documents that NRG-1 induces trabeculation of the ventricular wall without a significant increase in the proliferation of cardiomyocytes, in agreement with an essential role of NRG-1 to initiate trabeculae (
      • Meyer D.
      • Birchmeier C.
      ). Another secreted growth factor, IGF-I, which is essential for cardiac growth in neonatal heart, had no apparent effect on development. However, combined injection of NRG-1 and IGF-I induced DNA synthesis in ventricular myocytes and significant growth of the ventricular compact zone. The potentiation of NRG-1 and IGF-I on cardiomyocyte proliferation was corroborated in cultured cardiac muscle cells. To dissect downstream signals activated by NRG-1 and IGF-I, we used specific inhibitors or dominant negative mutants to block either the MAP kinase or phosphatidylinositol (PI) 3-kinase signaling pathways in cultured cardiomyocytes. Our results indicate that the synergistic effect was mediated by convergent activation of PI 3-kinase rather than the MAP kinase pathway, which was activated by each growth factor alone. Taken together, these results support a synergistic role for NRG-1 and IGF-I in coordinating ventricular chamber growth and maturation, which may occur through convergent signaling pathways at the level of PI 3-kinase activation.

      ACKNOWLEDGEMENTS

      We thank Dr. Cary Lai for fruitful discussions and cDNA probes, Dr. Mark Sliwkowski for recombinant heregulin β, Dr. Kuo-Fen Lee for recombinant neuregulin-1 α, Dr. David Luo for acetylcholinesterase cDNA probe, Dr. Robert Price for sharing the scanning electron microscope, and Dr. Tristan Bahnson for use of the Pipette puller. We are indebted to Mahmoud Itani and Janelle Stricker for skillful technical assistance and Julie Anderson for maintaining the mouse colony and specially grateful to Dr. Pilar Ruiz-Lozano for practical advice and Dr. Sylvia Evans for insightful discussions and critical review of the manuscript.

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