Animal models of LVNC.
Cardiomyopathy or disease of the heart muscle involves abnormal enlargement and a thickened, stiff, or spongy-like appearance of the myocardium. As a result, the function of the myocardium is weakened and does not sufficiently pump blood throughout the body nor maintain a normal pumping rhythm, leading to heart failure. The main types of cardiomyopathies include dilated hypertrophic, restrictive, arrhythmogenic, and noncompaction cardiomyopathy. Abnormal trabeculations of the myocardium in the left ventricle are classified as left ventricular noncompaction cardiomyopathy (LVNC). Myocardial noncompaction most frequently is observed at the apex of the left ventricle and can be associated with chamber dilation or muscle hypertrophy, systolic or diastolic dysfunction, or both, or various forms of congenital heart disease. Animal models are incredibly important for uncovering the etiology and pathogenesis involved in this disease. This chapter will describe the clinical and pathological features of LVNC in humans and present the animal models that have been used for the study of the genetic basis and pathogenesis of this disease.
- animal models
- cardiac development
Left ventricular noncompaction (LVNC) is a unique form of cardiomyopathy that is distinguished by a distinctive (“spongy”) morphological appearance of the left ventricle (LV) myocardium . This “spongy” appearance encompasses hypertrabeculation, deep intertrabecular recesses or sinusoids, and a bilayered ventricular myocardium with a noncompacted endocardium and compacted epicardium . Although LVNC is rare, the prevalence of LVNC is reported to be <0.3% in adults and < 0.0001% in children. It is the third most common form of inherited cardiomyopathies and accounts for 9% of all pediatric cardiomyopathy cases . Prevalence was reported as 0.014–1.3% in adult patients who underwent echocardiography. In heart failure patients, the prevalence of LVNC is estimated at 3–4% . LVNC is characterized as genetic, primary cardiomyopathy by the 2006 American Heart Association classification model, whereas the European Society of Cardiology has not classified LVNC as distinct cardiomyopathy due to its phenotypic heterogeneity [5, 6].
Clinical presentation of LVNC is variable, and Towbin et al. have described nine distinct subtypes: 1) benign, 2) arrhythmogenic, 3) dilated, 4) hypertrophic, 5) mixed hypertrophic and dilated, 6) restrictive, 7) right ventricular with the normal left ventricle, 8) biventricular, and 9) associated with congenital heart disease . Some of the LVNC phenotypes are shown in Figure 1. Complications of LVNC include chronic heart failure, arrhythmias, cardioembolism, chest pain, dyspnea, syncope, myocardial infarction, and sudden cardiac death (SCD) . Patients with LVNC associated with neuromuscular disease may present with exercise intolerance, fatigue, muscle pain, muscle stiffness, and muscle weakness. Heart failure associated with LVNC is often due to either systolic or diastolic ventricular dysfunction. Electrocardiogram (ECG) abnormalities are very common (88–94% and 88%, respectively) in both adult and pediatric cases. Common arrhythmias are atrial fibrillation, atrial flutter, paroxysmal supraventricular tachycardia, and atrioventricular block. Heart failure and arrhythmias are the greatest cause of concern for mortality in LVNC patients .
The etiology, pathogenesis, and natural history of LVNC are not clearly understood. The genetic causes of LVNC are heterogeneous [10, 11], involving final common pathways initiated by primary (the sarcomere) and developmental (NOTCH pathway) genetic abnormalities, often
2. Embryonic development of compacted myocardium
The underlying mechanisms for LVNC remain largely unknown, but many studies associate it with the failure of compaction of trabecular myocardium during embryogenesis . The development of the functionally competent, compacted, and multilayered myocardial wall is a two-part process consisting of trabeculation followed by a compaction process set at the midgestational period of cardiogenesis . When the myocardial spiral system enfolds, myocyte recruitment and proliferation lead to myocardial maturation with the development of protrusions into the lumen. Endocardial cells invaginate, and cardiomyocytes in specific regions along the inner wall of the heart form sheet-like protrusions into the lumen to give rise to the trabecular myocardium . The intertrabecular recesses communicate with the blood-filled cavity of the heart tube to increase the surface area for gas exchange and blood. This mechanism favors the concomitant increase in myocardial mass despite the absence of a distinct epicardial coronary circulation .
The trabecular myocardium starts undergoing compaction between weeks 5 and 8 of human embryonic development and coincides with the invasion of the developing coronary vasculature from the epicardium. Compaction is gradual, from the epicardial to the endocardial surface and from the basal segments of the ventricle moving toward the apex . Vascular endothelial growth factor (VEGF) and angiopoietin-1 may be involved with triggering compaction . As a result of the compaction process, the intertrabecular recesses disappear almost entirely leaving a smooth endocardial ventricular surface. Compaction is more pronounced in the LV than in the right ventricle (RV), therefore the RV endomyocardial surface is more heavily trabeculated (Figure 2) . On the other hand, noncompaction indicates failure of compact myocardium formation, leaving spongy myocardium and deep intertrabecular recesses .
2.1 Etiopathophysiology of LVNC and genotype: phenotype correlation
While the etiology of LVNC is not clearly understood, it is largely considered that hypertrabeculation or noncompaction in LVNC has a genetic origin with typically autosomal dominant inheritance if the implicated genes encode components of the sarcomere, Z-disc, or cytoskeleton . Autosomal recessive, X-linked, and mitochondrial inheritance patterns have also been found [3, 22]. One large retrospective multicenter study showed that nearly one-third of the LVNC patients had genetic variants in at least one cardiomyopathy-causative gene . LVNC has also been reported in many complex syndromes [23, 24] and neuromuscular disorders [25, 26, 27]. LVNC can also be considered congenital or acquired, and several hypotheses have been proposed for the development of LVNC . The primary hypothesis for congenital LVNC is the embryonic hypothesis, which attributes the hypertrabeculation of LVNC to the arrest in normal ventricular compaction during myocardial embryogenesis . The etiology of LVNC can be described as having two components, congenital and modifier factors.
Genetically, LVNC is heterogeneous and has been associated with chromosomal defects and genetic mutations in myosin heavy chain 7 (
The causal nature of genetic defects is further complicated by the overlap of genetic mutations in distinct cardiomyopathies. LVNC can be categorized based on its association with dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), and other forms of heart muscle disease. Using next-generation sequencing, several groups revealed a wide range of pathogenic variants in LVNC patients and an association between pathogenic variants and poor prognoses, especially in those patients harboring multiple pathogenic variants [10, 11, 37, 38]. Variants in
The embryonic hypothesis does not explain acquired LVNC that presents after birth, some forms of which are potentially reversible. Acquired LVNC, has been identified in athletes, pregnant women, and patients with sickle cell anemia, myopathies, and chronic renal failure . The etiology of acquired LVNC is merely speculative. One such hypothesis argues that mild LVNC can remain undetected until transient LV dilation allows LVNC to become visible under precise and accurate imaging . It is also speculated that acquired LVNC may be due to cardiac remodeling from increased preload and altered hemodynamics . Ventricular trabeculation in athletes, particularly in the LV apex, allows for increased compliance which reduces wall stress and strain . Given the high risk of possible cardiac embolic events from thrombus formation in the intertrabecular recesses, clinical trials for thromboembolic events in isolated LVNC have been suggested [53, 54, 55].
2.2 Diagnosis and therapeutic strategy
The clinical manifestations of LVNC vary widely, including no symptoms, thromboembolic events (ventricular or systemic arterial), LV dilation, impaired contractility with heart failure leading to pulmonary edema, arrhythmia including ventricular tachycardia and atrial fibrillation, and sudden cardiac death. Patients with neuromuscular disorder and LVNC may present with elevation in muscle form of creatine kinase, CK-MM (creatine kinase, muscle isoform) consistent with skeletal myopathy .
Echocardiography is the first-line diagnostic routine and an accessible technique to detect abnormal trabeculations or a “spongy” appearance of the myocardium. Several diagnostic criteria have been developed to define LVNC through echocardiographic analysis. A ratio of >2:1 in thickness of noncompacted to compacted layers during diastole is deemed diagnostic for LVNC [56, 57]. Compared with echocardiography, cardiac magnetic resonance (CMR) imaging offers more in-depth anatomic and functional features of the noncompacted myocardium. Late gadolinium enhancement specifically provides detection of cardiac fibrosis. CMR criteria developed by Petersen et al. to accurately diagnose pathologic noncompaction is based on a noncompaction to compaction ratio at end-diastole of >2.3 . Quantitative CMR criteria by Jacquier
Genetic testing in patients with LVNC and family members has been important in identifying genetic causes of cardiac dysfunction. Testing can be done on genes known to be associated with LVNC and other forms of cardiomyopathy as well as genes involved in syndromic diseases such as metabolic abnormalities, mitochondrial dysfunction, Barth syndrome, and storage diseases. Identification of pathogenic variants in probands and family members can be followed by segregation studies in the family .
Treatment strategy in LVNC depends on clinical presentations and complications, and clinical needs are managed according to corresponding guidelines . The key targets of clinical management are the treatment of heart failure (including beta-blockers, angiotensin-converting enzyme inhibitors, angiotensin-II receptor blockers, aldosterone antagonists, diuretics, and heart transplantation), arrhythmias (including ablation and implantation of an implantable cardioverter-defibrillator in patients with life-threatening events), and oral anticoagulation. In patients with congenital heart disease and LVNC, surgery for the congenital abnormalities takes precedence when feasible. In many cases, palliative surgery ultimately fails because of the myocardial abnormality, and cardiac transplantation is required .
3. Animal models of LVNC
LVNC is an overlap disorder and it appears that any of these “final common pathways” can be involved depending on the specific form of LVNC . Combining information about disease-causing genes with murine models is crucial in identifying pathways involved in ventricular noncompaction. For instance, Barth syndrome is caused by tafazzin mutations, and tafazzin knockdown mice were engineered using a short-hairpin RNA-inducible transgenic approach. These mice demonstrated hypertrabeculation and noncompaction, and the knockdown mice died prenatally at E12.5–14.5 . New powerful cutting-edge gene-editing technologies using transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) have been used for modeling LVNC [66, 67, 68, 69]. Patient-specific induced pluripotent stem cells (iPSCs) derived cardiomyocytes have emerged as a useful tool for investigating pathological mechanisms of many cardiovascular diseases including LVNC [70, 71]. Several signaling pathways such as the Dll4-NOTCH , MIB1 , BMP , and TGF-β  have been demonstrated to regulate myocardial trabecular compaction as well as to be involved in the development of LVNC. While zebrafish and
|Gene||Animal model||Signaling||Ref #|
|FKBP12||KO mouse||Decrease in Notch1 activity, increase in BMP10|||
|BMP10||KO E9.0–13.5 mouse||TGF-b|||
|BMP10||Overexpression adult mouse||TGF-b|||
|NUMB/NUMBL||Double KO mouse||Inhibition of Notch1, Smad6 and Smad7, WNT|||
|SMAD7||Mutant mouse||BMP10, TGF-b|||
|TBX20||Overexpression mouse||T-box family, TBX1|||
|MIB1||Mutant zebrafish||Reduces Notch1|||
|MIB1||Decifient zebrafish||Reduces Notch1|||
|VEGF||Overexpression mouse||Notch1, Flk-1|||
|Daam1||Decifient mouse||PCP and WNT|||
|NKX2–5||KO mouse||MEF2, HAND1, HAND2, GATA, BMP10|||
|TAZ||Inducible knockdown||Cardiolipin remodeling|||
|TAZ||KO mouse||Cardiolipin remodeling|||
|TTN||Mutant mouse||Telethonin loss|||
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 . 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 receptor-ligand 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 .
The first murine model for LVNC was the Fkbp1a (or FKBP12)-deficient mouse. Deficiency in FKBP1a, a binding protein of the immunophilin family, cause
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 . 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 . Human atrial natriuretic factor (hANF) promoter can be used to overexpress BMP10 in mice. Overexpression of BMP10 demonstrated hypertrabeculation and severe heart failure . Like in Fkbp1a-deficient mice and cardiac overexpression models, BMP10 is also upregulated in NUMB/NUMBL-deficient mice (myocardial double-knockout mice) . 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 . Both NUMB and NUMBL inhibit Notch1 signaling and are crucial for trabeculation, cardiomyocyte proliferation and differentiation, and trabecular thickness . On the other hand, inhibitory intracellular transducers such as Smad6 and Smad7 negatively regulate the BMP/ TGF-β signaling pathway . 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 . In murine embryos, Tbx20 can be detected in the cardiac precursor cells at E7·5 and the developing myocardium and endocardium at E8·0 . Cardiac-specific overexpression of TBX20 results in severe DCM, ventricular hypertrabeculation, and abnormal muscular septum, consistent with the DCM type of LVNC .
Another Notch pathway element, Mib1, is associated with the LVNC phenotype of biventricular noncompaction with dilation and heart failure . 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 . Inactivation of Mib1 reduces Notch1 signaling and myocardial arrest. Mutant Mib1 mice produce an LVNC phenotype of immature trabeculae and noncompaction .
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 . Overexpression of VEGF-A in mice causes hypertrabeculation, abnormalities in cardiac morphology and coronary vessels, and embryonic lethality at E12.5–14.0 .
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
NUMB is also a component of the adherens junction by forming complexes with β-catenin to regulate cellular adhesion
3.3 Animal models related to other signaling pathways
The expression of early response genes in lymphocytes is regulated by NFAT transcription factors .
Barth syndrome is caused by mutations in the X-linked
Cytoskeletal and sarcomeric proteins encoding gene mutations have been shown to account for 20% or more of LVNC . Truncating variants in
The pathogenesis of LVNC, a recently classified cardiomyopathy, remains largely unclear. With over 40 genes linked to LVNC, both genotype variation and phenotype variation are vast. Genetic testing in families and individuals with LVNC shas proved useful in identifying disease-causing variants. While the Notch signaling pathway is implicated in the pathogenesis of LVNC, there may be other pathways leading to congenital and acquired forms of LVNC. Identifying specific pathway elements is crucial for diagnosis and treatment. Modeling LVNC variants can help us to determine the genetic basis and pathogenesis of the disease.
Conflict of interest
The authors have no conflicts of interest.