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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Circ Res. Author manuscript; available in PMC 2010 July 31.
Published in final edited form as:
PMCID: PMC2735228
NIHMSID: NIHMS139897

Why Don’t They Beat?

Cripto, Apelin/APJ and Myocardial Differentiation

The convoluted maze of transcription and signaling factors that underpin cardiomyocyte differentiation has taken two major paths of inquiry that inform and sometimes puzzle each other: embryonic in situ development of cardiomyocytes and differentiation of stem cells to cardiomyocytes. Cardiomyocyte differentiation in embryonic development has fascinated scientists for millennia. In the middle of the 20th century embryonic cardiomyocytes were tracked down in the embryo using extirpations and in vitro cultures of either cardiogenic or non-cardiogenic mesoderm. Cultures that beat were relegated to the cardiogenic region of the anterior lateral plate mesoderm1. Maps created in this way have been verified and refined in vivo. In the case of non-cardiogenic mesoderm explants, treatment with “cardiogenic” factors resulted in beating cultures2,3. These studies allowed us to begin to understand where the specified cardiogenic cells resided in the mesoderm, and later to determine what signaling factors promoted myocardial specification in non-cardiogenic mesoderm and subsequent differentiation. In an era when stem cell transplantation has become a reasonable goal of adult medical regenerative treatment, the ability to specify and differentiate large numbers of cardiomyocytes (or other pure populations of differentiated cells) from stem cells has become something of a holy grail.

In this regard, a novel signaling pathway promoting cardiomyocyte differentiation in embryonic stem (ES) cells is introduced in this issue of Circulation Research. D’Aniello et al, using cripto-null ES cells, which are unable to differentiate as cardiomyocytes, show that the apelin ligand and its receptor APJ (explained below) are able to partially rescue cardiomyocyte differentiation4. I say “partially” because the cripto-null, apelin/APJ-treated stem cells begin to express myocardial contractile proteins which are not expressed by untreated cripto-null stem cells5. Although the cells express contractile proteins, they do not beat even when left in culture for prolonged periods of time.

The apelin/APJ signaling pathway regulates adult blood pressure and positive inotropy in the heart69. The apelin ligand is translated as a 77 amino acid prepropeptide, expressed primarily in endothelial cells, that is cleaved to shorter activated C-terminal peptide fragments1012. Apelin peptides activate a 7-transmembrane G-protein-coupled receptor called APJ (angiotensin II receptor-like 1, Agtrl1, Xmsr in Xenopus)13. The APJ receptor is expressed in endothelial cells, vascular smooth muscle cells and cardiomyocytes14,15. Recent evidence suggests that the APJ receptor may function, in some contexts, independent of the apelin ligand16.

The role of apelin during embryonic development is not well studied and this new report showing that apelin/APJ signaling is involved in the cascade of signaling factors that promote myocardial differentiation makes it imperative to reevaluate the apelin/APJ studies that have been done in embryonic development. In zebrafish apelin expression is confined to unidentified cells in the embryonic midline while its receptor (agtrl1b) is expressed in lateral plate mesoderm17. Modulation of either the ligand or receptor (grinch mutant) causes a reduced number of cardiac progenitors to move inefficiently to the lateral plate mesoderm and subsequently to make a disorganized heart-like structure17. Interestingly, the apelin receptor in zebrafish does not colocalize with lateral plate mesoderm cells expressing cmlc217,18, suggesting that expression may be extinguished as the cells differentiate. Knockdown experiments in frog have shown that apelin is required for normal vascular and cardiac development19. In frog by the time the heart forms, expression of apelin is confined to the endothelium and endocardium13. In mouse embryos, APJ is expressed from E8 in lateral plate mesoderm20. Apelin-deficient mice are viable and fertile21, suggesting that other factors can rescue in vivo cardiac development in the absence of apelin.

In the case of the ES cell cultures reported by D’Aniello et al, apelin/APJ rescues expression of contractile proteins in cripto-null cells4. Cripto is a member of the EGF-CFC family of signaling factors named for Cripto, Frl1 (Xenopus) and Cryptic22. EGF-CFC genes encode extracellular proteins that share several domains including an N-terminal signal sequence, a variant EGF-like motif, a novel conserved cysteine-rich domain named the CFC (Cripto, FRL-1, and Cryptic) motif, and a C-terminal hydrophobic region22. Crypto is transcribed from a gene called teratocarcinoma-derived growth factor-123, which was identified and cloned from an embryonal carcinoma cell line. Cripto is an extracellular protein that is tethered to the surface of cells by a glycosyl-phosphatidylinositol (GPI) linkage to the cell membrane which appears to be important for its activity22.

In 8.5 dpc embryos cripto is expressed in the myocardium of the developing heart tubes and in the outflow tract of the heart at E9.5–10. The expression pattern suggests a role in cardiac morphogenesis and sure enough, cripto-null mice never show any signs of cardiomyocyte differentiation24.

The paper by D’Aniello et al in this issue4, builds on the fact that cripto-null cells express markers that indicate successful cardiomyocyte specification such as Nkx2.5, Mef2C, Gata4, ehand, dhand)4,5, but the cells fail to differentiate and so don’t express any of the cardiomyocyte contractile proteins and never beat even when cultured for an extended period of time. Wildtype clones give rise to a higher percentage of beating clones than the heterozygous cells suggesting some dose dependence5. Because other mesodermal cell types differentiate normally, this suggests a specific defect in cardiac differentiation5. Addition of crypto protein to stem cell cultures at 0–2 days restores the cardiomyocyte differentiation potential of crypto-null stem cells25. This is somewhat odd because it indicates that cripto signaling is needed very early perhaps even prior to specification of cardiomyocytes.

After establishing that apelin/APJ signaling is downstream of cripto, D’Aniello et al4 go on to show that apelin/APJ induces activation of ERKs and AKT leading to activation of p70S6 kinase. Blocking activation of MAPK prevents apelin rescue of cardiomyocyte differentiation.

So does any of this explain why they don’t beat? The simplest explanation is that cripto promotes cardiomyocyte differentiation via at least two different pathways: one through apelin/APJ to promote contractile protein expression and a second (or more) to promote expression of the membrane components needed for automaticity. Even though the paper shows that a few of the major contractile proteins are transcribed, it is unclear if they are all translated and if the whole battery needed for construction of normal sarcomeres is made. I think we can all agree that beating has been the traditional measure of cardiomyocyte differentiation and even though we are now able to identify molecular cardiomyocyte markers, beating must be included in considering differentiation of myocardial cells. That is why I have referred to the rescue reported by D’Aniello et al4 as “partial”. In any case, this paper brings us to some interesting new thoughts about cardiomyocyte differentiation. How many different signaling pathways converge to promote all the elements that lead to automaticity and beating including contractile protein expression, sarcomerogenesis, components that make an excitable membrane, excitation-contraction coupling? The fact that apelin/APJ represents one of these pathways is something of a surprise and there are likely more surprises to come.

Acknowledgments

Thanks to Thomas Quertermous and Mary Hutson for discussion and critical reading of the manuscript.

Sources of funding: Cincinnati Children’s Heart Foundation, NPRI at Duke University, NIH HL083240, HL084413

Footnotes

Disclosures: None

References

1. Rawles ME. The heart-forming areas of the early chick blastoderm. Physiol Zool. 1943;14:22–42.
2. Antin PB, Taylor RG, Yatskievych T. Precardiac mesoderm is specified during gastrulation in quail. Dev Dyn. 1994;200:144–154. [PubMed]
3. Barron M, Gao M, Lough J. Requirement for BMP and FGF signaling during cardiogenic induction in non-precardiac mesoderm is specific, transient, and cooperative. Dev Dyn. 2000;218(2):383–393. [PubMed]
4. D’Aniello C, Lonardo E, Iaconis S, Guardiola O, Liguoro AM, Liguori GL, Autiero M, Carmeliet P, Minchiotti G. G protein-coupled receptor APJ and its ligand apelin act downstream of cripto to specify embryonic stem cells toward the cardiac lineage through ERK/P70S6K signaling pathway. Cire Res. 2009 [PubMed]
5. Xu C, Liguori G, Adamson ED, Persico MG. Specific arrest of cardiogenesis in cultured embryonic stem cells lacking Cripto-1. Dev Biol. 1998;196(2):237–247. [PubMed]
6. Tatemoto K, Takayama K, Zou MX, Kumaki I, Zhang W, Kumano K, Fujimiya M. The novel peptide apelin lowers blood pressure via a nitric oxide-dependent mechanism. Regul Pept. 2001;99(2–3):87–92. [PubMed]
7. Ashley EA, Powers J, Chen M, Kundu R, Finsterbach T, Caffarelli A, Deng A, Eichhorn J, Mahajan R, Agrawal R, Greve J, Robbins R, Patterson AJ, Bernstein D, Quertermous T. The endogenous peptide apelin potently improves cardiac contractility and reduces cardiac loading in vivo. Cardiovasc Res. 2005;65(1):73–82. [PMC free article] [PubMed]
8. Berry MF, Pirolli TJ, Jayasankar V, Burdick J, Morine KJ, Gardner TJ, Woo YJ. Apelin has in vivo inotropic effects on normal and failing hearts. Circulation. 2004;110(11 Suppl 1):II187–193. [PubMed]
9. Szokodi I, Tavi P, Foldes G, Voutilainen-Myllyla S, Ilves M, Tokola H, Pikkarainen S, Piuhola J, Rysa J, Toth M, Ruskoaho H. Apelin, the novel endogenous ligand of the orphan receptor APJ, regulates cardiac contractility. Circ Res. 2002;91(5):434–440. [PubMed]
10. Habata Y, Fujii R, Hosoya M, Fukusumi S, Kawamata Y, Hinuma S, Kitada C, Nishizawa N, Murosaki S, Kurokawa T, Onda H, Tatemoto K, Fujino M. Apelin, the natural ligand of the orphan receptor APJ, is abundantly secreted in the colostrum. Biochim Biophys Acta. 1999;1452(1):25–35. [PubMed]
11. Kawamata Y, Habata Y, Fukusumi S, Hosoya M, Fujii R, Hinuma S, Nishizawa N, Kitada C, Onda H, Nishimura O, Fujino M. Molecular properties of apelin: tissue distribution and receptor binding. Biochim Biophys Acta. 2001;1538(2–3):162–171. [PubMed]
12. Lee DK, Cheng R, Nguyen T, Fan T, Kariyawasam AP, Liu Y, Osmond DH, George SR, O’Dowd BF. Characterization of apelin, the ligand for the APJ receptor. J Neurochem. 2000;74(1):34–41. [PubMed]
13. Devic E, Paquereau L, Vernier P, Knibiehler B, Audigier Y. Expression of a new G protein-coupled receptor X-msr is associated with an endothelial lineage in Xenopus laevis. Mech Dev. 1996;59(2):129–140. [PubMed]
14. Kleinz MJ, Davenport AP. Emerging roles of apelin in biology and medicine. Pharmacol Ther. 2005;107(2):198–211. [PubMed]
15. Kleinz MJ, Skepper JN, Davenport AP. Immunocytochemical localisation of the apelin receptor, APJ, to human cardiomyocytes, vascular smooth muscle and endothelial cells. Regul Pept. 2005;126(3):233–240. [PubMed]
16. Chun HJ, Ali ZA, Kojima Y, Kundu RK, Sheikh AY, Agrawal R, Zheng L, Leeper NJ, Pearl NE, Patterson AJ, Anderson JP, Tsao PS, Lenardo MJ, Ashley EA, Quertermous T. Apelin signaling antagonizes Ang II effects in mouse models of atherosclerosis. J Clin Invest. 2008;118(10):3343–3354. [PMC free article] [PubMed]
17. Zeng XX, Wilm TP, Sepich DS, Solnica-Krezel L. Apelin and its receptor control heart field formation during zebrafish gastrulation. Dev Cell. 2007;12(3):391–402. [PubMed]
18. Scott IC, Masri B, D’Amico LA, Jin SW, Jungblut B, Wehman AM, Baier H, Audigier Y, Stainier DY. The g protein-coupled receptor agtrl1b regulates early development of myocardial progenitors. Dev Cell. 2007;12(3):403–413. [PubMed]
19. Inui M, Fukui A, Ito Y, Asashima M. Xapelin and Xmsr are required for cardiovascular development in Xenopus laevis. Dev Biol. 2006;298(1):188–200. [PubMed]
20. Devic E, Rizzoti K, Bodin S, Knibiehler B, Audigier Y. Amino acid sequence and embryonic expression of msr/apj, the mouse homolog of Xenopus X-msr and human APJ. Mech Dev. 1999;84(1–2):199–203. [PubMed]
21. Kuba K, Zhang L, Imai Y, Arab S, Chen M, Maekawa Y, Leschnik M, Leibbrandt A, Markovic M, Schwaighofer J, Beetz N, Musialek R, Neely GG, Komnenovic V, Kolm U, Metzler B, Ricci R, Hara H, Meixner A, Nghiem M, Chen X, Dawood F, Wong KM, Sarao R, Cukerman E, Kimura A, Hein L, Thalhammer J, Liu PP, Penninger JM. Impaired heart contractility in Apelin gene-deficient mice associated with aging and pressure overload. Circ Res. 2007;101(4):e32–42. [PubMed]
22. Shen MM, Schier AF. The EGF-CFC gene family in vertebrate development. Trezds Genet. 2000;16:303–309. [PubMed]
23. Liguori G, Tucci M, Montuori N, Dono R, Lago CT, Pacifico F, Armenante F, Persico MG. Characterization of the mouse Tdgf1 gene and Tdgf pseudogenes. Mamm Genome. 1996;7(5):344–348. [PubMed]
24. Xu C, Liguori G, Persico MG, Adamson ED. Abrogation of the Cripto gene in mouse leads to failure of postgastrulation morphogenesis and lack of differentiation of cardiomyocytes. Development. 1999;126(3):483–494. [PubMed]
25. Parisi S, D’Andrea D, Lago CT, Adamson ED, Persico MG, Minchiotti G. Nodal-dependent Cripto signaling promotes cardiomyogenesis and redirects the neural fate of embryonic stem cells. J Cell Biol. 2003;163(2):303–314. [PMC free article] [PubMed]