1-1. Single-nucleotide polymorphisms
Polymorphism represents natural sequence variants (alleles), which may occur in more than one form. These appear in at least 1% of a population and are considered biologically normal. Approximately 90% of DNA polymorphisms are single-nucleotide polymorphisms (SNPs) due to a single base exchange (Collins et al., 1998). A SNP represents an alternate nucleotide in a given and defined genetic location at a frequency exceeding 1% in a given population. This definition does not include other types of genetic variability like insertions and deletions, and variability in copy number of repeated sequences. They occur once in every 300 nucleotides on average, which means there are roughly 10 million SNPs in the human genome. Although the majority of DNA polymorphisms are probably functionally neutral, a proportion of them can exert allele-specific effects on the regulation of gene expression or function of the coded protein, which underlie individual differences in various biological traits and in susceptibility to disease (Brookes, 1999).
A SNP in which both alleles produce the same polypeptide sequence is called a
synonymous polymorphism (sometimes called a silent mutation); which produce a different polypeptide sequence is called a non-synonymous polymorphism (replacement
polymorphism). A non-synonymous polymorphism can differ from missense and nonsense, which the former results in a different amino acid and the latter results in a premature stop codon. Over half of all known disease mutations come from non-synonymous polymorphisms (Stenson, 2009). If gene expression is affected by a SNP this type is referred to as an eSNP (expression SNP) and may be upstream or downstream from the gene.
Analytical methods to discover novel SNPs and detect known SNPs include DNA sequencing; restriction fragment length polymorphism (RFLP); capillary electrophoresis;
mass spectrometry; single-strand conformation polymorphism (SSCP); electrochemical analysis; denaturating HPLC and gel electrophoresis; hybridization analysis. All of the references information of SNP in this study was obtain from National Center for Biotechnology Information (NCBI)-SNP database.
1-2. Congenital heart defects
1-2-1. Ventricular septal defects
Ventricular septal defects (VSDs) are the most common form of congenital heart disease.
The defect can be in any portion of the ventricular septum, and the physiologic consequences can range from trivial to severe. Approximately 20% of patients in congenital heart disease registries have VSD as a solitary lesion (Hoffman and Rudolph, 1965). Although historically the incidence of VSDs is cited as approximately 1.5 to 3.5 per 1,000 term infants and 4.5 to 7 per 1,000 premature infants (Moe and Guntheroth, 1987), recent echocardiographic studies demonstrated an incidence of VSD in newborns to be 5 to 50 per 1,000 (Tikanoja, 1995).
Rates of spontaneous closure for membranous and muscular VSDs in infant and children were 37% and 50%, respectively during a mean follow-up of 12 months (Moe and Guntheroth, 1987). The others have no such a fortunate course and the VSD persist. The development of echocardiography has provided insight into the mechanisms by which VSDs close
spontaneously in a gross point of view (Murphy et al., 1986).
Defects can generally be classified according to their location, either within the muscular septum (muscular defects) or at its margins. Ventricular septal defects at the margins of the muscular septum can be related to hinge-points of the leaflets of the atrioventricular valves (perimembranous), those of the arterial valves (juxta-arterial or subarterial), or both (Figure 1)
pouch or aneurysm derived from tissue from the septal leaflet of the tricuspid valve. Muscular defects appear to close by progressive growth of tissue from the right ventricular side of the circumference of the defect. Neither detail molecular insight of spontaneous VSDs closure has been studied, nor studies of association between extracellular matrix (ECM) and spontaneous VSDs closure have been clarified.
1-2-2. Atrial septal defects
Atrial septal defects (ASDs) represent 6 to 10% of all cardiac anomalies and are more frequent in females than males by about 2:1. ASDs occur in 1 child per 1,500 live births (Samanek, 1992). A prospective echocardiographic study suggested that as many as 24% of newborns have evidence of an opening (3 to 8 mm) in the atrial septum in the first week of life (Fukazawa et al., 1988). However, by a little more than 1 year of age, 92% of the patients were found to have spontaneous closure of the opening, and in most patients, there is
evidence of a valve-like opening of the atrial septum that is believed to contribute to closure.
Helgason and Jonsdottir (Helgason and Jonsdottir, 1999) reviewed the medical records of all patients in Iceland with a diagnosis of ASD born between 1984 and 1993. ASD was
confirmed by 2-D echocardiogram, and data only from patients with secundum ASDs were analyzed. A total of 84 children diagnosed at a mean age of 12 months were followed for 4 years. Spontaneous closure or decreased size was observed in 89% with a 4-mm ASD, 79%
with a 5- to 6-mm defect, and only 7% with a defect > 6 mm. Occasionally, spontaneous closure will occur as late as 16 years (Brassard et al., 1999). As the same condition mentioned in the paragraph of VSDs, the underlying mechanism of spontaneous closure of ASDs is till mysterious.
1-3. Matrix metalloproteinase
1-3-1. Structures and functions of MMPs
MMPs is a family of extracellular zinc-dependent neutral endopeptidases (Lombard et al., 2005), capable of degrading essentially all ECM components including fibrillar and
non-fibrillar collagens, fibronectin, laminin and basement membrane glycoproteins (Fedarko et al., 2004). MMPs not only play an important role in ECM remodeling in physiologic situations, such as embryonal development, tissue regeneration, and wound repair, also in pathological conditions including rheumatoid arthritis, osteoarthritis, atherosclerotic plaque rupture, tissue ulceration, and in cancer cell invasion and metastasis (Roeb and Matern, 2001;
Jones et al., 2003). The MMP family currently consists of 28 enzymes with somewhat different activities. The members are generally divided into six groups according to either structure or preferred substrates and referred to as interstitial collagenases (MMP-1, -8 and -13), stromelysins (MMP-3, -10, -11, and -12), matrilysins (MMP-7 and MMP-26),
gelatinases (MMP-2 and MMP-9), membrane-type MMPs (MMP-14, -15, -16, -17, -24, and -25) and others (Visse and Nagase, 2003; Bode and Maskos, 2003). Although MMPs are subclassified based on their ability to degrade 4 various proteins of the ECM, they also play other important roles such as the activation of cell surface receptors and chemokines
(Stefanidakis and Koivunen, 2006). In addition, MMP-2 has proteolytic activity to specific targets within the cell to cause acute, reversible contractile dysfunction in cardiac disease (Schulz, 2007).
The basic structures of MMPs can be approximately divided into three structurally well-preserved domain motifs, including a catalytic domain, an N-terminal domain and a C-terminal domain. Zinc-dependent catalytic domain of MMPs is similar with subtle
N-terminal domain (propeptide domain) contains a unique PRCG(V/N)PD sequence in which the cysteine residue interacts with the catalytic zinc atom in the active site, prohibiting
activity of the MMPs. Thus, the interaction has to be disrupted to “open” the cysteine switch in the process of MMPs activation (van Wart and Birkedal-Hansen, 1990), which is a critical step that leads to ECM breakdown (Carmeli et al., 2004). The C-terminal hemopexin domain of metalloproteinases has a four-bladed propeller structure and contributes to substrate specificity (Wallon and Overall, 1997). In membrane-type MMPs, the hemopexin domain contains a transmembrane domain for anchoring the protein in the membrane; besides, the hemopexin domain in MMP-2 also has a function in the activation of the enzyme (Morgunova et al., 1999; Overall et al., 1999). The regulation of MMPs occurs at many levels, including transcription (the major one), post-transcriptional modulation of mRNA stability, secretion, localization, zymogen (proenzyme) activation and inhibition of activity by natural inhibitors of MMPs, tissue inhibitor of metalloproteinases (TIMPs). In addition, there are more and more researchers turn their focus on emphasizing MMPs protein physical functions to the change of nucleotides and the SNPs of MMPs and TIMP correlated with different cardiac diseases and cancers (Table 1).
1-3-2. Gelatinase A (MMP-2, Type II collagenase)
In 1978, Sellers et al. were the first to separate a gelatinase activity from collagenase and stromelysin in culture medium from rabbit bone (Sellers et al., 1978). A similar enzyme, acting on basement membrane type IV collagen was reported by Liotta et al. the following year (Liotta et al., 1979). Gelatinase was purified from human skin, mouse tumor cells, rabbit bone, and human gingival. The completed sequence of the human enzyme except for the signal peptide was reported by Collier et al. (Collier et al., 2001). Gelatinase A had a triple
repeat of fibronectin type I domains inserted in the catalytic domain; these participate in binding to the gelatin substrates of the enzyme (Lee et al., 1997; Libson et al., 1995).
1-3-3. Gelatinase B (MMP-9, Type V collagenase)
Harrwas and Krane in 1972 detected a gelatinase activity in rheumatoid synovial fluid.
Sopata and Wize described a gelatinase from human polymorphonuclear leukocytes (Sopata and Wize, 1979). Rabbit macrophages produce a very similar enzyme which was able to digest type V collagen (Horwitz et al., 1977). The neutrophil collagenase and gelatinase were resolved in 1980 (Murphy et al., 1980). Purification was achieved in 1983 and sequencing of the cDNA in 1989. An interesting phenomenon, still not fully understood, is the binding of TIMP-1 to proMMP-9 to form a complex (Sakyo et al., 1983; Stetler-Stevenson et al., 1989).
Human neutrophil MMP-9 commonly occurs as a complex with lipocalin (Fernández et al., 2005). A series of papers concerned a 95 kDa protein in plasma that binds to gelatin
culminated in the identification of this protein as MMP-9 (Makowski and Ramsby, 1998).
1-4. The roles of MMPs in cardiac development
MMP-2 is the earliest MMP known to be expressed during heart development. The process of cardiac looping which converts the single, straight tubular heart into a S-shaped tube and re-positions the primitive heart chambers into their adult anatomical positions before cardiac septation is complete. During the process of making a single heart tube, MMP-2 is expressed in the endocardium, early differentiating cardiomyocytes, and dorsal mesocardium but is soon lost within the myocardium. Blocking MMP-2 activity prevents midline fusion of the primitive heart tubes leading to cardiac bifida (Cai et al., 2000; Linask et al., 2005).
Cell proliferation is more pronounced within the left splanchnic mesoderm and left dorsal mesocardium. Blocking MMP-2 activity not only disrupts this asymmetric pattern of proliferation, it also randomizes the direction of cardiac looping (Linask et al., 2005) and increases the incidence of dextrocardia (reversal of the normal anatomical position of the heart, i.e., right-sided heart). In other words, blocking MMP-2 activity prevents midline fusion of the primitive heart tubes leading to cardiac bifida (Linask et al., 2005). Therefore, MMP-2 mediated growth appears to be involved in the direction of cardiac looping.
Defects in cardiogenesis during the first three weeks of gestation are usually dangerous.
However, anomalous events occurring later in embryonic development often permit the embryo and fetus to make it to term. These anomalies most often manifest themselves as great vessel or cardiac septal defects in neonates. The septation of the atria and ventricles and
division of the cardiac outflow tract into the aorta and pulmonary artery requires the migration, proliferation, and differentiation of two distinct mesenchymal populations,
endocardial-derived cushion cells and invading neural crest (NC) cells (Creazzo et al.,1998).
MMPs have been implicated in regulating epithelial-to-mesenchymal transitions (EMTs) which responsible for forming both populations of cells. MMP-2 is expressed by endocardial cells prior to and during the EMT of the endocardium in both the atrioventricular and outflow tract regions of the developing heart (Cai et al., 2000, Alexander et al., 1997).
Hyaluronan is abundant in the ECM of the pre-mesenchymal heart (Markwald et al., 1979). Hyaluronan is an esssential mediator of cell migration and invasion for proper heart development (Camenisch et al., 2002). Hyaluronan relate to regulate the expression of
multiple MMPs in several cell types and directly induces EMT of the endocardium. Therefore, MMPs play pivotal roles for EMT of the endocardial and cardiac septation. Cells of septum primum adjacent ostium primum express MMP-2, TIMP-2, and TIMP-3 during the formation
and remodeling of the muscular septa. MMPs also involve in remodeling events which are responsible for transfiguring the primitive ventricular myocardial wall into a compact layer and inner trabecular layer. MMPs release bioactive VEGFs from the ECM (Belotti et al., 2003;
Sounni et al., 2004) and active VEGF-A increases the expression of several MMPs (Pufe et al., 2004; Wang and Keiser ,1998). Therefore, if MMP processing is required for erbB signaling in the developing heart, the specific temporal and tissue-specific expression of MMPs and TIMPs could dictate where and when particular growth factors modulate cardiac remodeling events.
In a nutshell, the heart undergoes remodeling events eliciting changes in MMP activity and ECM turnover as a part of the response to functionally compensate for the extra load.
Such responses may reflect MMP-driven remodeling events that occur during embryonic cardiac morphogenesis. All the evidences imply the MMPs play a vital role in cardiac developing.