**3.1 Animal models related to the Notch signaling pathway**

The Notch signaling pathway is a highly conserved intercellular pathway involved with multisystem differentiation. Particularly in the cardiovascular system, Notch1 mediates ventricular morphogenesis, coronary vessel development, and communication between the endocardium and myocardium for cardiomyocyte proliferation and differentiation. Mutations in the Notch signaling pathway cause congenital heart disease [77]. Mammals have Notch 1–4, a group of transmembrane receptors with an extracellular domain and an intracellular domain. The Notch extracellular domain (NECD) interacts with ligands of the Delta and Jagged family, and these receptorligand interactions are modulated by manic fringe (Mfng) glycosyltransferase. Delta and Jagged ligands are ubiquitinated by Mib1, an E3 ubiquitin ligase, and trigger endocytosis of the ligand. Mib1 activity exposes the receptor to ADAM metalloproteases for Notch cleavage. In response, the Notch intracellular domain (NICD) translocates to the nucleus and acts as a transcription factor. The NICD is comprised of the RBPJ domain and ANK repeats [77].

The first murine model for LVNC was the Fkbp1a (or FKBP12)-deficient mouse. Deficiency in FKBP1a, a binding protein of the immunophilin family, cause**s** ventricular noncompaction, thin ventricular walls, hypertrabeculation, and ventricular septal defects [20, 78]. Fkbp1a is a negative modulator of activated Notch 1. In Fkbp1a-deficient mice, activated Notch1 is upregulated. In Fkb1a-deficient mice, Fkbp1a overexpression significantly reduced activated Notch1 [79]. Fkbp1a deficiency upregulates BMP10, a peptide growth factor in the TGF-β family involved in the cardiac compaction process. BMP10 upregulation in Fkbp1a-deficient mice indicates the importance of this gene in the trabeculation and compaction process.

In mouse embryogenesis, Bmp10 is transiently expressed in ventricular myocardium at E9.0–13.5 during myocardial maturation and in the atria E16.5–18.5 [74]. BMP10-deficient mice embryos appear unaffected at E8.5–9.0, arrest at E9.0–9.5, and die at E10.5. Immunostaining of mutant embryos exhibited thin ventricular walls and primitive trabecular ridges. The localization of BMP10 to the ventricular myocardium for the brief period at E9.0–13.5 suggests that BMP10 is crucial for continued myocardial development. Postnatal cardiac-specific BMP10 overexpression compromised cardiac growth, caused subaortic narrowing and concentric myocardial thickening [80]. Human atrial natriuretic factor (hANF) promoter can be used to overexpress BMP10 in mice. Overexpression of BMP10 demonstrated hypertrabeculation and severe heart failure [81]. Like in Fkbp1a-deficient mice and cardiac overexpression models, BMP10 is also upregulated in NUMB/NUMBLdeficient mice (myocardial double-knockout mice) [82]. NUMB and NUMBL proteins of the NUMB family are cell fate determinants for hemopoietic stem cells, muscle satellite cells, cancer stem cells, and hemangioblast progenitor cell types and maintain the fate of neural stem cells as well as regulate their differentiation [83]. Both NUMB and NUMBL inhibit Notch1 signaling and are crucial for trabeculation, cardiomyocyte proliferation and differentiation, and trabecular thickness [84]. On the other hand, inhibitory intracellular transducers such as Smad6 and Smad7 negatively regulate the BMP/ TGF-β signaling pathway [85]. Smad7 is expressed by endothelial cells in the major arteries in mice, and Smad7 deficiency causes increased Smad 1, 5, and 8 in the endocardial endothelium [81, 86]. Unsurprisingly, Smad7 mutant mice demonstrate ventricular noncompaction, thin ventricular walls, and ventricular septal defect. One of the key mediators of BMP10 signaling in ventricular myocardial development and maturation is TBX20, a member of the TBX1 subfamily of the T-box family transcription factors [87]. In murine embryos, Tbx20 can be detected in the cardiac precursor cells at E7·5 and the developing myocardium and endocardium at E8·0 [88]. Cardiac-specific overexpression of TBX20 results in severe DCM, ventricular hypertrabeculation, and abnormal muscular septum, consistent with the DCM type of LVNC [87].

Another Notch pathway element, Mib1, is associated with the LVNC phenotype of biventricular noncompaction with dilation and heart failure [20]. Genetic sequencing of 100 European patients revealed two autosomal dominant mutations—V943F and R530X. Injection of Mib1-mutated mRNA, corresponding to the V943F and R530X mutations, into zebrafish embryos disrupts Notch signaling [89]. Inactivation of Mib1 reduces Notch1 signaling and myocardial arrest. Mutant Mib1 mice produce an LVNC phenotype of immature trabeculae and noncompaction [89].

Vascular endothelial growth factor (VEGF) is produced by the myocardium and plays a role in endocardium-myocardium communication by binding to endocardial receptor Flk-1 [20]. Overexpression of VEGF-A in mice causes hypertrabeculation, abnormalities in cardiac morphology and coronary vessels, and embryonic lethality at E12.5–14.0 [90].

#### **3.2 Animal models related to the WNT signaling pathway**

The planar cell polarity (PCP) pathway is a β-catenin-independent Wnt pathway that was first studied in *Drosophila*. The PCP pathway plays a role in the epithelial orientation of hair and sensory bristles, apical-basolateral polarity, gastrulation, and neurulation [31, 91]. Disheveled-associated activator of morphogenesis I

*Left Ventricular Noncompaction Cardiomyopathy: From Clinical Features to Animal Modeling DOI: http://dx.doi.org/10.5772/intechopen.101085*

(Daam1) is a mediator of the PCP pathway and a formin protein found in the plasma membrane and cytoplasmic vesicles. Daam1 is involved with cardiac morphogenesis and is highly expressed in murine cardiac tissue. Daam1-deficient mice have been shown to cause an LVNC phenotype with ventricular noncompaction and the thin ventricular wall. It is likely that the abnormalities seen in Daam1-deficient mice are due to cytoskeletal dysfunction since Daam1 is involved with F-actin assembly and sarcomere organization. Interestingly, the absence of Daam1 does not alter BMP10 expression nor cardiomyocyte proliferation, which offers another pathogenic model of LVNC through disruption in myofibrillogenesis, cytoskeleton organization, and cardiomyocyte polarization [31, 92].

NUMB is also a component of the adherens junction by forming complexes with β-catenin to regulate cellular adhesion *via* Wnt signaling [82]. It also interacts with integrin-β subunits to regulate cell migration and promote their endocytosis for directional cell migration. Deletion of NUMB and NUMBL from mouse hearts results in LVNC with congenital heart disease with atrioventricular septal defects, truncus arteriosus, and double outlet right ventricle *in vivo* [82]. This model shows that NUMB family proteins regulate trabecular thickness by inhibiting Notch1 signaling and control cardiac morphogenesis in a Notch1-independent manner.

#### **3.3 Animal models related to other signaling pathways**

*NKX2–5*, a cardiac homeobox gene, is a transcription factor that regulates heart development, working along with MEF2, HAND1, and HAND2 transcription factors to direct heart looping during early heart development [93]. Genetic variants in *NKX2–5* are associated with progressive cardiomyopathy and conduction defects in humans [94, 95]. Ventricular-muscle-cell-restricted knockout of *NKX2–5* in mice leads to progressive atrioventricular block with conduction system cell dropout and fibrosis [96]. LVNC is a prominent feature in neonatal mice, with progressive biventricular dilation and heart failure developing early. It directly activates *MEF2* to control cardiomyocyte differentiation and operates in a positive feedback loop with GATA transcription factors to regulate cardiomyocyte formation. A high-level BMP10 expression in the adult ventricular myocardium has been also observed.

The expression of early response genes in lymphocytes is regulated by NFAT transcription factors [97]. *NFATC1* mutant mouse embryos have cardiac abnormalities including myocardial developmental abnormalities, narrowing or occlusion of the ventricular outflow tract, defective septum morphogenesis, and underdevelopment of valves [98, 99]. Ventricular hypertrophy and noncompaction with hypertrabeculation were seen in 40% of mutant mice, suggesting that NFAT signaling pathways are important for hypertrabeculation and noncompaction as well as the development of valves and the septum.

Barth syndrome is caused by mutations in the X-linked *TAZ* and is associated with LVNC and abnormal cardiolipin remodeling [12, 100]. Tafazzin catalyzes cardiolipin maturation reactions at the final stage of cardiolipin biosynthesis [101]. Inducible knockdown of *TAZ* (TAZKD) in murine models using short-hairpin RNA (shRNA) exhibited an adult-onset LVNC associated with abnormal cardiolipin profiles and mitochondrial structural abnormalities [102]. Knockout of *TAZ* at the embryonic stage leads to unique developmental cardiomyopathy characterized by ventricular myocardial hypertrabeculation and noncompaction and early lethality, suggesting that mitochondrial function is important for proper myocardial development [65].

Cytoskeletal and sarcomeric proteins encoding gene mutations have been shown to account for 20% or more of LVNC [7]. Truncating variants in *TTN* are welldocumented to cause skeletal myopathies and cardiomyopathies including LVNC

[48, 49, 103]. Homozygous mouse model carrying titin A178D mutation created by using CRISPR-Cas9 gene-editing displayed features of mild DCM, but not LVNC phenotype [104]. These mice showed complete loss of telethonin from the Z-disc and induction of a proteo-toxic response in the heart upon aging and adrenergic stress.
