36 Chapter 2 size control and lethal cardiomegaly shortly after birth (von Gise et al., 2012). For example, mouse models harboring various embryonic deletions of the Hippo pathway members SAV1, MST1/2 and LATS2 display overgrown hearts and thickened ventricles owing to an excess of cardiomyocytes (Del Re, 2014). Downstream analysis in Hippo mutant hearts revealed that a lack of downregulation of canonical Wnt target genes and cell cycle regulators could explain the observed hyperplasia and cardiomegaly (Heallen et al., 2011). By contrast, upregulation of Hippo signaling leads to a downregulation of YAP and Wnt/β-catenin signaling to restrain heart size (Kim et al., 2017). Cardiac-specific deletion of YAP also impedes neonatal heart regeneration, resulting in a fibrotic response that generates scar tissue (Xin et al., 2013). Cytoplasmic YAP/TAZ can also increase cytosolic retention of β-catenin, and thereby negatively regulate the Wnt/β-catenin signaling pathway (Imajo et al., 2012). Chromatin immunoprecipitation sequencing (ChIP-seq) indicated that YAP/TAZ and β-catenin form a common regulatory complex at the SOX2 and SNAI2 gene loci (Heallen et al., 2011). Activating YAP also leads to upregulation of the IGF/Akt pathway, resulting in the inhibition of GSK-3β, stabilization of β-catenin and transcription of Wnt target genes to enhance cardiomyocyte proliferation (Xin et al., 2011) (Figure 1). During development, IGF signaling directs cardiomyocyte proliferation between E9.5 and E12.5, and IGF knockout mice exhibit decreased ventricular proliferation (Li et al., 2011). Interestingly, knockout of IGF is non-lethal and by E14.5 the ventricular wall size is comparable with that of wild-type mice (Li et al., 2011; Díaz Del Moral et al., 2021). The Wnt/β-catenin, IGF/Akt and Hippo/YAP pathways form a complex signaling network that regulates the balance between proliferation, differentiation and maturation of cardiomyocytes (Figure 1). It has generally been concluded that high Wnt, high IGF and low Hippo signaling results in proliferative growth of embryonic cardiomyocytes, whereas high Hippo signaling overrules Wnt and IGF to restrain cardiomyocyte proliferation and prevent cardiomegaly. Cardiomyocyte cell biology in vitro Developmental shortcuts for human pluripotent stem cell-derived cardiomyocyte differentiation Cardiomyocytes derived from pluripotent stem cells represent a promising source of cells for disease modeling, drug screens and/or cardiac regenerative medicine (Madonna et al., 2019). The untangling of cues that drive cardiac development has identified growth factors, transcriptional regulators and signaling cascades that can promote the differentiation of cardiac cells from human embryonic stem cells (hESCs). Initial protocols for embryonic stem cell differentiation required formation of embryoid bodies to generate various cell types (Rungarunlert et al., 2009), and downstream applications for cardiac cell biology were limited due to extremely low percentages of cardiomyocyte yield (Osafune et al., 2008). Previously, co-culture of hESCs with visceral endoderm-like cells induced cardiomyocyte differentiation, producing beating
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