Part two. BLOOD OR YOUNG PLASMA AND REGENERATION OF THE CARDIAC MUSCLE (Factors Promoting Cardiogenesis in Vertebrates)

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TAK1 and the SMAD pathways, activated by BMPs, act in parallel to promote Cardiogenesis by activating several transcription factors like ATF2 (Activating Transcription Factor-2), NKX2.5 and GATA4. ATF2 is critical for Cardiomyocyte differentiation. ATF2 stimulates the Beta-MHC (Beta-Myosin Heavy Chain) promoter activity synergistically with SMAD1, SMAD4 and TAK1 and promotes terminal Cardiomyocyte differentiation. ATF2-induced transactivation of Beta-MHC gene depends on both the SMAD and the TAK1 pathways. Single amino acid changes in Beta-MHC result in abnormal actomyosin interactions, confirming the primary role of missense mutations in Beta-MHC gene in the etiology of hypertrophic Cardiomyopathy. Apart from stimulating Beta-MHC, ATF2 also plays a pivotal role in transactivation of some Cardiac-specific genes like NPPA (Natriuretic Peptide Precursor-A) and NPPB (Natriuretic Peptide Precursor-B). The hetero-oligomer of SMAD3 and SMAD4 bind directly to ATF2 through the MH1 (MAD Homology-1) region of SMAD3 and SMAD4 and the bZIP region of ATF2 and enhance the transactivating capacity of ATF2. NPPB is the major heart secretory product that is also accepted as clinical markers of the diseased Heart and it is also controlled by GATA factors. Other cardiac transcription factors like NKX2.5 (NK2 Transcription Factor Related Locus-5) and GATA4 (GATA Binding Protein-4) also plays pivotal role in the Cardiogenic BMP signaling pathway. Transactivation of NKX2.5 and GATA4 is mediated by TAK1. Subsequently, NKX2.5 and GATA4 induce differentiation into Cardiomyocytes cooperatively with unknown factor(s) induced by DMSO (Dimethyl sulfoxide). Although some cardiac-specific genes, such as MEF2C (MADS Box Transcription Enhancer Factor-2 Polypeptide-C) and MLC2v (Ventricular Myosin Light Chain Type-2), are upregulated by NKX2.5 and GATA4 alone, differentiation into beating cardiomyocytes requires the cooperative effects of both NKX2.5 and GATA4. MLC2v and MEF2C are positively regulated by NKX2.5. MEF2C binds to the AT-rich element in regulatory regions of numerous muscle-specific genes. GATA4 binds to the WGATAR motif in promoter regions of Cardiac- or Gut-specific genes. Both MEF2C and GATA4 are expressed simultaneously in the precardiac mesoderm along with NKX2.5 and plays an important role in cardiac differentiation. Other members of the TGF-Beta (Transforming Growth Factor-Beta) Superfamily have also been involved in induction of the mesoderm in Xenopus, Zebrafish, Chicken and Mouse. TGF-Beta is expressed early in the Cardiac region of the mesoderm (Ref.6 & 7). 

Besides BMPs, another protein involved in the induction of Cardiogenesis includes Cripto. Cripto is the original member of a family of vertebrate signaling molecules, the EGF-CFC family which includes: Human, Mouse and Chick Cripto; Human and mouse Cryptic, Xenopus FRL1 and Zebrafish one-eyed pinhead. All EGF-CFC proteins contain a signal sequence for extracellular secretion, a characteristic EGF-like domain, a second cysteine-rich region called the CFC domain, and a hydrophobic C-terminus. Nodal signaling is required to support Cripto-regulated Cardiac induction and differentiation in ESCs (Embryonic Stem Cells). Cripto interacts with ALK4 (Activin Receptor-Like Kinase-4) to permit Nodal binding to the ALK4/ Act-RII (Activin type II serine/threonine kinase Receptor) complex, leading to SMAD phosphorylation. In addition, Cripto is also implicated in Nodal signaling via the orphan receptor ALK7 (Activin Receptor-Like Kinase-7), since its expression enhances the ability of ALK7 and ActRIIB to respond to Nodal ligands. Membrane-bound Cripto recruit Nodal to an Activin type I receptor (ALK4 or ALK7) and to ActRII receptors; upon receptor activation, intracellular effectors SMAD2 and/or SMAD3 are phosphorylated and accumulate together with SMAD4 in the nucleus. The intracellular mediators, in combination with co-factors then switch on transcription of Cardiac specific genes. Addition of Nodal-antagonist Cer1 (Cerberus Short), which specifically blocks Nodal by direct binding to the ligand, results in a strong inhibition of Cripto activity in promoting Cardiogenesis and provides direct evidence for a functional role of ALK4/Nodal pathway in Cripto-mediated specification of the Cardiac lineage (Ref.8, 9 & 10).