**2.2.4 MicroRNA dysfunction**

130 Congenital Heart Disease – Selected Aspects

Haemodynamic forces have been demonstrated to play an important role in cardiac development. When these forces are impaired or when genes involved in growth and differentiation are not functioning correctly, malformations may arise. Shear stress is one of those haemodynamic forces, and the expression of many genes, including those of the endothelin pathway, changes in response to alterations in shear stress. For example, ligating the right lateral vitelline vein of chicken embryos results in cardiovascular malformations similar to those observed in knockout mice studies of components of the endothelin-1/endothelin-converting enzyme-1/ endothelin-A receptor pathway. In zebrafish, altering haemodynamics mechanically or genetically has profound consequences on heart morphology. In mice, a recent study pinpointed altered haemodynamics as a key intermediate between altered outflow tract morphogenesis and signaling events in branchial-arch artery remodeling. In human, complex congenital heart diseases with an outflow tract defect, such as tetralogy of Fallot, can be accompanied by 'accessory' congenital heart diseases, such as persistent right-sided aortic arch. Because the heart functions during its morphogenesis, haemodynamic forces might participate in cardiac

Fig. 3. Notch pathway

**2.2.3 Altered haemodynamics** 

morphogenesis [20, 71, 74] (Figure 4).

MicroRNAs are natural, single-stranded, non–protein-coding small RNA molecules (~22 nucleotides) that regulate gene expression by binding to target mRNAs and suppress its translation or initiate its degradation. Mature miRNAs are processed from -70 nucleotides long precursor miRNA (pre-miRNAs) that form hairpin secondary structures and that are often evolutionarily conserved. Pre-miRNAs are transcribed from miRNA genes. Although the specific biological roles of most miRNAs are still unknown, functional characterizations of a few of them suggest that these small RNA molecules are involved in many processes of animal development and physiology[75-77] .For example, miR-1 and miR-133 control cardiac and skeletal muscle development [78, 79]. Both genes are under the control of serum response factor, indicating that they are part of a developmental programme regulated by cardiac transcription factors. It has been shown that miR-1 targets the cardiac transcription factor HAND2. Deletion of *miR-1-2* results in heart defects that include VSDs; surviving mice have conduction system defects and increased cardiomyocyte proliferation. Dysregulation of miRNAs might result in congenital heart disease in human [80, 81](Figure 5).

Fig. 5. MicroRNA dysfunction results in CHD

#### **2.2.5 Epignetics**

Epigenetics refers to DNA and chromatin modifications that play a critical role in regulation of various genomic functions, and it was then redefined as the study of heritable traits that are not dependent on the primary sequence of DNA. Although the genotype of most cells of a given organism is the same (with the exception of gametes and the cells of the immune system), cellular phenotypes and functions differ radically, and this can be (at least to some extent) controlled by differential epigenetic regulation that is set up during cell differentiation and embryonic morphogenesis [82,83]. Once the cellular phenotype is established, genomes of somatic cells are 'locked' in tissue-specific patterns of gene expression, generation after generation. This heritability of epigenetic information

Molecular Mechanisms of Congenital Heart Disease 133

mutations revealed that some had aspects of cardiomyopathy, as predicted from the mouse data. Thus, embryonic patterning genes control structural components of the heart and can also have a separate role in heart function, for example by regulating *Serca2*.These genes can thus modulate important aspects of heart function that cause pathology in the postnatal heart when dysregulated. This concept has important implications for the clinical

The molecular mechanisms of congenital heart defects are so complex that we have to use diverse strategies to explore them. **Animal Models** Biomedical models have been defined as ''surrogates for a human being, or a human biologic system, that can be used to understand normal and abnormal function from gene to phenotype and to provide a basis for preventive or therapeutic intervention in human diseases'' . Because of the striking homology between mammalian genomes and the many similarities in anatomy, cell biology, and physiology, rat is an excellent animal model for studying of cardiac development and identifying novel genes that could contribute to human disease. The Human Genome Initiative is providing genetic information not only from humans, but also from animals traditionally used as models. In addition, related enabling technologies in transgenesis and animal cloning provide new approaches for designing and performing experiments to dissect complex biological systems. Because of these new technologies (e.g., transgenesis), scientists are no longer limited to the traditional methods of choosing naturally occurring models. Researchers can utilize genomic knowledge and available tools to create appropriate animal models. This approach is referred to as reverse genetics. In contrast to forward genetics in which the gene or genes responsible for a particular phenotype are identified by positional cloning (phenotype to genotype), the reverse genetics approach determines the function of a gene and predicts the phenotype of a cell, tissue, or organism (genotype to phenotype). **Genome-Wide Studies** Considerable progress has been made in understanding the pathophysiology of perioperative stress responses and their impact on the cardiovascular system; however, researchers are just beginning to unravel genetic and molecular determinants that predispose to increased risk for CHD. Recent improvements in genotyping technology and in our knowledge of human genetic variation have made it possible to carry out genome-wide genetic association studies to identify susceptibility genes for common disease. Multistage designs involving large numbers of coding sequence variants (300,000) and relatively large samples sizes (several hundred cases and control subjects) will be essential to reliably detect alleles with appreciable effect sizes (2-fold increase in relative risk). Direct sequencing of candidate genes in cases and control subjects provides an alternative approach that can reveal low-frequency alleles that influence disease susceptibility [88,89]. **Gene Expression (Microarrays)** Microarray analysis is a useful tool to obtain a gene expression profile of CHD. However, current estimates suggest that greater than 60% of human genes have more than one isoform. Alternatively, spliced isoforms from the same gene can produce proteins with different properties and distinct functions. The specific roles of gene expression and their splicing variants necessary for development need to be further delineated. **MicroRNA** Current research has revealed that the influence of RNA molecules on gene expression reaches beyond the realm of protein synthesis back into the nucleus, where it not only dictates the transcriptional activity of genes, but also shapes

management of adults with congenital heart disease [20,87].

**3. Strategies and future perspectives**

in somatic cells has been called an 'epigenetic inheritance system' [84]. Even after the epigenomic profiles are established, a substantial degree of epigenetic variation can be generated during the mitotic divisions of a cell in the absence of any specific environmental factors. Such variation is most likely to be the outcome of stochastic events in the somatic inheritance of epigenetic profiles. From the epigenetic point of view, phenotypic differences in monozygous twins could result, in part, from their epigenetic differences. It has recently become clear that epigenetic regulators play crucial roles in the global shaping and maintenance of developmental patterning. This involves dynamic tissue and cell type-specific changes during patterning, as well as the maintenance of the cellular memory that is required for developmental stability.BAF60C (also known as SMARCD3), a subunit of the Swi/Snf-like chromatin-remodelling complex BAF, physically links cardiac transcription factors to the BAF complex. Loss of BAF60C results in severe defects in cardiac morphogenesis and impaired activation of a subset of cardiac genes. The muscle-restricted histone methyltransferase SMYD1 (also known as BOP) is a crucial regulator of cardiac chamber growth and differentiation. Histone deacetylases have mostly been characterized as having an important role in heart hypertrophy and development [20] (Figure 6).

Fig. 6. Dysfunctions of epignetics leads to CHD

#### **2.2.6 Adult congenital heart diseases**

Individuals with congenital heart disease can suffer from secondary heart disease later in life, possibly as a result of corrective surgery during infancy. The sequelae are sometimes severe; for example, after closure of a septal defect, some patients can progress to heart failure. With improved surgical outcomes for those with congenital heart disease, the number of adults with such diseases now exceeds the number of children. The population of patients with adult congenital heart disease is approximately 800,000 in the U.S.A majority faces a lifetime of problems including arrhythmias, ventricular dysfunction, and one or more re-operations. Thus, it has become imperative to understand the postnatal consequences of congenital heart diseases [85]. Recent results suggest that these might be caused, at least in part, by the direct effects of mutations associated with congenital heart disease on postnatal heart morphology and function. For example, in a family with *GATA4*  mutations, apart from having heart structural defects, some individuals developed dilated cardiomyopathy later in life. Indeed, data from mouse models support a connection between *GATA4* mutations and adult cardiomyopathy [86]. Similarly, mutations in *TBX20* were identified in patients with cardiomyopathy as well as in those with structural congenital heart diseases. Mouse studies have also revealed roles for other congenital-heartdisease-associated genes in cardiac function. Studies of mice in which *Nkx2-5* had been deleted only in the ventricles suggest a role for this gene in the function of the postnatal conduction system and in myocardial structure, and examination of patients with *NKX2-5* 

in somatic cells has been called an 'epigenetic inheritance system' [84]. Even after the epigenomic profiles are established, a substantial degree of epigenetic variation can be generated during the mitotic divisions of a cell in the absence of any specific environmental factors. Such variation is most likely to be the outcome of stochastic events in the somatic inheritance of epigenetic profiles. From the epigenetic point of view, phenotypic differences in monozygous twins could result, in part, from their epigenetic differences. It has recently become clear that epigenetic regulators play crucial roles in the global shaping and maintenance of developmental patterning. This involves dynamic tissue and cell type-specific changes during patterning, as well as the maintenance of the cellular memory that is required for developmental stability.BAF60C (also known as SMARCD3), a subunit of the Swi/Snf-like chromatin-remodelling complex BAF, physically links cardiac transcription factors to the BAF complex. Loss of BAF60C results in severe defects in cardiac morphogenesis and impaired activation of a subset of cardiac genes. The muscle-restricted histone methyltransferase SMYD1 (also known as BOP) is a crucial regulator of cardiac chamber growth and differentiation. Histone deacetylases have mostly been characterized as having an important role in heart hypertrophy and

Individuals with congenital heart disease can suffer from secondary heart disease later in life, possibly as a result of corrective surgery during infancy. The sequelae are sometimes severe; for example, after closure of a septal defect, some patients can progress to heart failure. With improved surgical outcomes for those with congenital heart disease, the number of adults with such diseases now exceeds the number of children. The population of patients with adult congenital heart disease is approximately 800,000 in the U.S.A majority faces a lifetime of problems including arrhythmias, ventricular dysfunction, and one or more re-operations. Thus, it has become imperative to understand the postnatal consequences of congenital heart diseases [85]. Recent results suggest that these might be caused, at least in part, by the direct effects of mutations associated with congenital heart disease on postnatal heart morphology and function. For example, in a family with *GATA4*  mutations, apart from having heart structural defects, some individuals developed dilated cardiomyopathy later in life. Indeed, data from mouse models support a connection between *GATA4* mutations and adult cardiomyopathy [86]. Similarly, mutations in *TBX20* were identified in patients with cardiomyopathy as well as in those with structural congenital heart diseases. Mouse studies have also revealed roles for other congenital-heartdisease-associated genes in cardiac function. Studies of mice in which *Nkx2-5* had been deleted only in the ventricles suggest a role for this gene in the function of the postnatal conduction system and in myocardial structure, and examination of patients with *NKX2-5* 

development [20] (Figure 6).

Fig. 6. Dysfunctions of epignetics leads to CHD

**2.2.6 Adult congenital heart diseases** 

mutations revealed that some had aspects of cardiomyopathy, as predicted from the mouse data. Thus, embryonic patterning genes control structural components of the heart and can also have a separate role in heart function, for example by regulating *Serca2*.These genes can thus modulate important aspects of heart function that cause pathology in the postnatal heart when dysregulated. This concept has important implications for the clinical management of adults with congenital heart disease [20,87].
