**3. Left ventricular noncompaction (LVNC)**

Left ventricular noncompaction (LVNC), also knowns as left ventricular hyper trabeculation (LVHT) or spongy myocardium, is a rare congenital myocardial dysfunction resulting from the arrest of the compaction phase during embryogenesis. Gross histology of this heterogeneous disease condition is characterized by a spongy left ventricle (LV) myocardium, abnormal prominent trabeculations at the ventricular apex, and intra-trabeculae recesses adjacent to a well compacted LV myocardium during the final stages of cardiac development [27]. Within the ventricular cavity the double-layered ventricular myocardium consisting of epicardial and endocardial layers, there appears finger-like protrusions from the myocardium with intertrabecular recesses separating trabeculations. Pathologically these anatomical features of a noncompacted heart demonstrate a tremendously thickened endocardial layer with abnormal trabeculation and a compacted thin epicardial layer. In 1984, the first case of LVNC was described in a newborn that was also associated complications of atresia of semilunar valves and intact ventricular septum [28]. Since then, of the different phenotypes of LVNC (8 different subtypes identified to date) [29], isolated LVNCs (first reported in 1926 occurring as a result of trabeculae morphogenesis interference in the absence of any cardiac abnormalities) and congenital LVNCs are the most

commonly studied. With the latter type of LVNC being associated with systemic diseases including metabolic or mitochondrial diseases, cardiomyopathies such as hypertrophic, dilated, restrictive, and arrhythmogenic, congenital heart diseases, and complex syndromes affecting vast majority of the organs and tissue [30].

While rare forms of LVNC can occur from acquired mutations, commonly LVNCs are from genetic alterations transmitted through generations [31] with around 50% of the identified LVNC patients having either autosomal dominant, autosomal recessive, or X-linked genetic inheritance patterns with multiple genes modifications [32–34] (**Figure 3**). Predominate genetic variants associated with LV noncompaction include defects in mitochondrial, sarcomeric, and cardiac genes. In addition to mitochondrial DNA mutations [35], a wide variety of mitochondrial abnormalities significantly influence LVNC including but not limited to mitochondrial dysfunction [36], metabolic myopathy altered by mitochondrial energy production [37], dysmorphic mitochondria [31, 38], mitochondrial DNA transitions (a type of DNA mutation where a purine or pyrimidine nucleotide is replaced with its complementary purine or pyrimidines, respectively) [39, 40], deficiencies in mitochondrial respiratory chain function [41], mutations in overlapping regions [42], and mitochondrial genome mutations [43]. Further supporting mitochondrial involvement, mitochondrial numbers are enriched in the cardiac muscles of individuals with LVNC, and mitochondrial DNA (mtDNA) content and copy number are also largely altered in non-compacted hearts.

## **4. Diagnostics and therapeutic aspects of LVNC**

The occurrence of LVNC has increased in recent years with a prevalence of 0.01–0.3%, a mortality rate of 5–12%, and is predominately found in males [44]. While most LVNC patients are asymptomatic or noted to have mild symptoms of chest pain, later stages of LVNC can lead to sudden cardiac death due to compromised systolic function. Although the initial diagnosis can be at any stage in life, most individuals with LVNC go undiagnosed until the 5th decade of life. Symptoms are diverse in patients with LVNC and can include heart failure, cardiac arrhythmias, fatigue, excessive sweating, breathing difficulties, compromised growth, and abnormal weight gain, while others do not exhibit any symptoms [44–46]. Hence, early diagnosis is key for timely supportive care.

Common diagnostic tools available for LVNC include echocardiography (echo), cardiac magnetic resonance imaging (cardiac MRI or CMR), electrocardiogram (ECG), cardiac computed tomography (CT), coronary angiograms and myocardial biopsy based on the myocardial thickening [47]. In most cases the first-line diagnostic testing of any heart diseases is echo, due to its availability and being low-cost in nature. Echo criteria include (1) noncompacted/compacted ratio of the two-layered endocardium of >2, (2) left ventricular deep endomyocardial trabeculations, and (3) deep recesses filled with blood visualized on color Doppler [48, 49]. However, the detection of LVNC in its early stage of development are challenging, with only around 0.3% of the patients being referred for an echo [50, 51]. Additionally, echo diagnostics are sometimes considered to be a 'too sensitive of a method' as overdiagnosis frequently occurs, particularly in the black patient population. Therefore, serial echo diagnosis is recommended in LVNC [52].

Alternatively, CMR is a well-established, high resolution, noninvasive, albeit more expensive approach, to confirm LVNC after an initial diagnostic echo. CMR

#### **Figure 3.**

*Schematic overview of influencing factors in myocardial noncompaction: Differential contribution of mitochondria towards LVNC via mitochondrial dysfunction (mtDNA mutations, transitions, ETC defects, mt genome mutations) and involvement of genes associated with cardiomyopathies (sarcomeres, ion channels, cytoskeletal, chaperones, desmosomes).*

offers an enhanced sensitivity, as CMR can detect myocardial trabeculations that can be enhanced with steady-state free-precession (SSFP) cine imaging [53, 54]. In CMR, the myocardial trabeculations, apex region and end-diastole ratio can be well monitored over time. Although CMR is associated with many challenges including a wide diagnostic testing timeline, high costs, and availability, it still remains the best approach as a diagnostic tool for LVNC. Unlike echo and CMR, CT imaging is less commonly used due to the ionizing radiation and lower detection ability of myocardial tissues. Nevertheless, this tool is widely used in evaluating other disease processes such as coronary arteries. Apart from the above-mentioned diagnostic tools, advances using blood for whole exome sequencing (PGxome diagnostic exome test) and copy number variation detection can be performed to study single gene defect or defects in gene sets associated with LVNC [55].

In research settings and invasive cardiac catheterization techniques, a more invasive method of histologic examination of cardiac muscle tissue biopsies is assessed. Histological analysis can identify abnormalities in the mitochondrial appearance as ragged red fibers, indicative of mitochondrial dysfunction, are suggestive of mitochondrial anomalies (Gomori Trichrome stain, **Figure 4**). A typical drawback of Gomori Trichrome Staining is the absence of these ragged red fibers in children, and therefore, it fails to detect mitochondrial disorders in neonates at early stages [56, 57]. An alternative method of early detection is the examination of cardiac tissue under electron microscopy for swollen disorganized mitochondria containing irregular cristae [57].

Importantly, around 40% of patients identified with LVNC are determined to have a familial genetic defect that impacts only cardiac function. However, while many genetic syndromes, metabolic disorders, and mitochondrial related anomalies are also associated with LVNC, in many cases the cause of LVNC is still unknown. As there is no cure to date, routine cardiac screening is an important part of the treatment of familial LVNC, with supportive therapy for LVNC patients being dependent on individual symptoms. Supportive therapy targeted to improved quality of life and overcome cardiac dysfunction includes medications to treat heart failure such as angiotensin-converting enzyme inhibitor, beta blockers, blood thinners, diuretics, and digoxin. In severe cases of LVNC, pacemaker implantation, cardioverter defibrillator, or heart transplantation is recommended. A recent case study in 2018 by

#### **Figure 4.**

*Gomori Trichrome staining of cardiac tissue from an (a) hyper trabeculated heart and a (b) healthy heart: (a) arrows indicating dysmorphic mitochondria scattered as red-ragged fibers below the matrix of the membrane, (b) mitochondria appear organized and distributed across the cardiac tissue.*

Kimura et al. [58], showed successful treatment of a near-fatal ventricular arrhythmia in an infant with LVNC using cardiac resynchronization therapy (CRT). These studies determined that with the implantation of a cardioverter defibrillator (preventive measure for cardiac arrest), dual chamber pacing initiation and CRT, effectively treated the mechanical dyssynchrony of the heart, with subsequent improvement in cardiac function.

Currently, there are no mechanisms to prevent LVNC or to return gain of cardiac function. While genetic testing can be done to identify gene mutations (**Table 1**) and determine the risk of passing these mutations to offspring, there are not therapeutic options to actively treat these gene mutations [74]. Future therapy that promotes personalized gene therapy could be a useful tool in treating LVNC. However, this approach could be highly challenging due to the complex genetics of LVNC.

#### **5. Genes and related mutations associated with LVNC**

A variety of gene mutations studied concerning other cardiomyopathies such as genes coding for sarcomere proteins [75], ion channels, cytoskeletal genes [76], chaperone proteins [77], and cellular signaling pathways have a significant association with LVNC [11, 78]. Specific genetic alterations include genes encoding β-myosin heavy chain (MYH7) [34, 79], α-cardiac actin (ACTC1) [80], and cardiac troponin T (TNNT2) [81], lamin A/C (LMNA) [82], ZASP (LDB3) [83], and taffazin (TAZ) [83, 84], cardiac myosin-binding protein C (MYBPC3) [85], and cardiac troponin I (TNNI3) [86] are associated with LVNC (**Table 2**).

Sarcomere gene mutations can lead to noncompaction with reduced ventricular function and hyper trabeculation [110]. Alpha-cardiac actin gene (ACTC) codes for cardiac muscle-specific alpha-actin protein, present in cardiac sarcomeres and cytoskeletal proteins, and is responsible for heart muscle contraction and generation of force to support the contraction. Novel mutations in the ACTC gene are linked to LVNC cardiomyopathies. Although, the novel protein-level amino acid sequence (A21V) mutations of ACTC1 resulting in familial LVNC are rare, it causes diverse cardiac anomalies. This resulting missense mutation in the ACTC1 gene creates structural changes to sarcomeres and their anchorage. In addition to these changes, these variants also modify the highly conserved nature of actin-like domains of the protein resulting in its destabilization with pathogenic consequences [80]. The MYH7 gene encoding for myosin heavy chain beta (MyHC-β) isoform is another primary sarcomeric protein in the adult heart. The ATPase activity of MYH7 powers the myosin power stroke within the myosin heads to convert energy that propels shortening of the sarcomeres. A heterozygous missense mutation (I467T) in MYH7 found in inherited cardiomyopathies presenting with a high penetrance and sudden death, can result in LVNC and hypertrophic cardiomyopathy (HCM) [111]. As the mutation site in MyH7 is close to the hydrophobic ATP binding pocket of the motor domain, the amino acid replacement of hydrophobic isoleucine with a non-polar threonine alters the structure subsequently effecting the ATPase activity of the motor domain [79, 80]. Cardiac Myosin binding protein C (MYBPC3) is another LVNC linked sarcomere protein. Infants with pathogenic truncating mutations in *MYBPC3* die at birth from HCM and LVNC complications. These mutations are believed to cause alterations in the primary contractile function of the heart and septal defects. However, the absolute relationship of contractile dysfunction to sarcomere protein mutations in the progression and pathophysiology of LVNC remains poorly understood [112].



#### **Table 1.**

*Genetic details and characteristics identified in clinical studies.*

Notch signaling is a prime mediator of cardiac embryogenesis. Despite the anomalies in other signaling pathways, the dysfunction of the Notch pathway has a notable contribution to LVNC. Germline mutations in human myocardial MIB1 (mind bomb homolog 1) coding for E3 ubiquitin ligase that promotes the endocytosis of Delta and Jagged, (NOTCH ligands) are involved in LV noncompaction [113]. In these studies,



*\* https://omim.org/*

*ILVNC, isolated left ventricular noncompaction; NLVNC, non-isolated left ventricular noncompaction; LVHT, left ventricular hyper trabeculation; RVNC, right ventricular noncompaction; AVB, Atrio ventricular block; HF, heart failure; DCM, dilated cardiomyopathy; HCM, hypertrophic cardiomyopathy; VSD, ventricular septal defect; BTHS, Barth syndrome; DCMA, dilated cardiomyopathy with ataxia syndrome; HT, hyper trabeculation; MELAS, mitochondrial encephalomyopathy; MERRF, myoclonic epilepsy with ragged-red fibers disorder; ARVC, arrhythmogenic right ventricle cardiomyopathy; CHD, congenital heart diseases; LVD, left ventricular dilation; RCM, restrictive cardiomyopathy [33].*

**Table 2.**

*Genes and associated mutations in cardiomyopathies.*

MIB1 protein was noted to be vital in activating the NOTCH signaling pathway while cardiac deformity managed by two of the MIB1 autosomal dominant mutations (V943F and R530X) causes deregulation of Notch signaling resulting in LVNC. Furthermore, Luxan et al. [78] reported that myocardial inactivation of MIB1 results in the enlargement of the ventricular trabeculae and a thin compact myocardium.

Alterations in cytoskeletal genes have been linked with the LVNC development. The intermediate filament protein, lamin (lamin A/C) or LMNA, maintains the mechanical integrity of the nuclear envelope. Heterozygous LMNA gene mutations (R644C and R190W) are linked to familial and sporadic LVNC, although the mechanisms have not been elucidated to date. Furthermore, lamin A/C V445E mutations were found to be related to sudden death in LVNC patients [82]. Cardiac expressing alpha-dystrobrevin (DTNA) belongs to a dystrophin-related protein family that is significant in intracellular signal transduction. Genetic alterations in DTNA and dystrophin-associated glycoprotein complex (DAPC) located in sarcolemma provoke myocardial disorders targeting the systolic function of the heart [41]. Inactivation of DTNA leads to muscular dystrophy, skeletal myopathy, and cardiomyopathy characterized by deep trabeculations which are always observed in LVNC [76]. Congenital LVNC frequently accompanies neuromuscular disorders in patients with myotonic dystrophy type 1 (MD1), an autosomal dominant type of disorder resulting from a trinucleotide expansion (CTG nucleotide repetitions) in the dystrophia myotonica protein kinase (DMPK) gene [114].
