Kabaeva, Zhyldyz: Genetic analysis in hypertrophic cardiomyopathy: missense mutations in the ventricular myosin regulatory light chain gene


Chapter 1. Introduction

Hypertrophic Cardiomyopathy (HCM) is a heart disorder characterized by unexplained ventricular myocardial hypertrophy and a high risk of sudden cardiac death.1 Myocardial hypertrophy is predominantly confined to the left ventricle (LV) and generally easily detectable by conventional echocardiography. The main diagnostic criterion for HCM is an increased LV wall thickness (normal le12mm) in the absence of other possible causes of myocardial hypertrophy as arterial hypertension, valvular disease, and others. HCM is also diagnosed pathologically by the presence of myocyte disarray and interstitial fibrosis along with myocyte hypertrophy.2 The disease is caused by mutations in genes encoding for sarcomeric proteins. It can either be transmitted as an autosomal-dominant trait from an ill parent to a child or develop due to a de novo mutation.3

HCM is a relatively common genetically transmitted cardiovascular disease with a prevalence in the general population of about 0.2% (or 1 in 500).3,4 The annual mortality rate for all HCM related deaths (sudden cardiac death, heart failure, and stroke) has been estimated as 1.4%, where the rate for sudden death is as high as 0.7%.5 Young HCM patients are more prone to sudden cardiac death, however, elder patients are also in substantial risk of dying unexpectedly.5


1.1 Molecular genetics and pathogenesis of HCM

Since the first detailed description of HCM in 1958 by Teare,6 research has been mainly directed on elucidating causes and pathogenesis of this disorder that could provide clues in earlier diagnosis, treatment, and prevention of the disease. Major advances have been made in understanding the etiologic factors of this disease. To date, mutations in nine genes all encoding for the cardiac sarcomeric proteins have been shown to cause HCM, however, mechanisms by which they lead to the disease are still not completely understood.

The first described gene was the one encoding for cardiac beta-myosin heavy chain, the major contractile protein of the cardiac sarcomere.7 Identification of mutations in two more sarcomeric components, alpha-tropomyosin and cardiac troponin T,8 in HCM patients led to the postulation that HCM results from defects in the sarcomeric proteins. HCM was subsequently referred to as a “disease of the sarcomere“.8 Later, this postulation was supported by identification of mutations in the next six genes also encoding for the proteins of the cardiac sarcomere, namely, cardiac myosin binding protein-C,9 ventricular myosin essential and regulatory light chains,10 cardiac troponin I,11 cardiac alpha-actin,12 and titin.13

The genes encoding for the sarcomeric proteins involved in HCM are located on different chromosomes and listed in table 1.1. As shown, the contribution of single gene mutations to HCM varies from less than 5% to 30%.3,14 The most common causes are mutations in the beta-myosin heavy chain, myosin binding protein-C, and cardiac troponin T genes accounting for approximately 70% of all HCM cases (table 1.1). Mutations in other genes are much less common. In total, more than 130 causal mutations have been identified, most of them in the beta-myosin heavy chain gene. So far, only few mutations have been found in the titin, cardiac alpha-actin, and ventricular myosin essential light chain genes.


Table 1.1. Sarcomeric proteins and genes responsible for HCM

Sarcomeric protein




Number of mutations

beta -Myosin heavy chain



~ 30%


Myosin binding protein-C



~ 20%


Cardiac troponin T



~ 20%





~ 5%


Cardiac troponin I



~ 5%


Cardiac alpha-actin



< 5%





< 5%


Myosin light chain, regulatory (RLC)



1 - 7%*


Myosin light chain, essential (ELC)



~ 1%*


Note: adapted from ref. 3. *From ref. 10, 15, and 16.

Genetically engineered animal models have been used efficiently to confirm the causality of sarcomeric protein mutations in HCM. Phenotypes similar to those found in human HCM were induced in transgenic mice expressing a sarcomeric protein carrying a certain human mutation, and in "knockout" mice, in which a particular sarcomeric protein gene was ablated by gene targeting.17 The cardiac expression of the common beta-myosin heavy chain mutation (Arg403Gln) in transgenic rabbits also induced hypertrophy, myocyte and myofibrillar disarray, interstitial fibrosis, and premature death, phenotypes observed in HCM patients carrying this mutation.18 Development of animal models, in which disease progression can be studied closely over the lifespan of an animal, has also shed significant light into the pathogenesis of HCM.

The sarcomere is the contractile unit of striated muscle. As shown in figure 1.1, cardiac myocytes contain numerous myofibrils. Each myofibril is in turn composed of repeating sarcomere units separated by Z discs. Each sarcomere is a highly ordered complex array of numerous proteins, the precise organisation and alignment of which are essential for proper muscle function.19 The overall organisation of the sarcomere is similar in all striated muscles, although the proteins constituting it have a number of isoforms, which are differentially expressed depending on the muscle type.


Figure 1.1. Microscopic structure of heart muscle. A) Myocardium as seen under the light microscope. Myocytes contain a centrally located nucleus and are connected across intercalated disks. B) Myocardial cell reconstituted from electron micrographs. Each myocyte is composed of multiple parallel fibrils. Each fibril is composed of serially connected sarcomeres (N, nucleus). C) Sarcomere from a myofibril, with diagrammatic representation of myofilaments. Thick filaments (1.5 µm long, composed of myosin) from the A band, and thin filaments (1µm long, composed primarily of actin) extend from the Z line through the I band into the A band. The overlapping of thick and thin filaments is seen only in the A band. D) Cross sections of the sarcomere indicate the specific lattice arrangements of the myofilaments. In the center of the sarcomere only the thick, or myosin, filaments arranged in a hexagonal array are seen. In the distal portions of the A band, both thick and thin, or actin, filaments are found, with each thick filament surrounded by six thin filaments. In the I band only thin filaments are present. From ref. 20.


Figure 1.2. Schematic diagram of sarcomere organisation and contraction process. The thin filament is made up of actin, the troponin complex (T,C and I) and alpha-tropomyosin. The thick filament is composed of myosin heavy and light chains. The sarcomere produces muscle contraction by sliding of myofilaments: the myosin heads interact with actin and pull it towards the center of the sarcomere resulting in shortening of the sarcomere. From ref. 2.

The sarcomere consists of overlapping arrays of thick and thin filaments, which shorten the length of the sarcomere during contraction by sliding past each other (figure 1.2). The thin filaments are attached to the Z discs. The thick filaments extend from the centre of the sarcomere in either direction towards the Z lines and are supported by binding to the protein-C and titin molecules. The major components of the thin filaments are cardiac alpha-actin, alpha-tropomyosin, and the troponin complex consisting of three subunits: troponin C, troponin I and troponin T. The thick filaments are composed of several hundreds of myosin molecules assembled together.

Myosin is called "molecular motor" of the sarcomere due to its ability to hydrolyse adenosine triphosphate (ATP) and thereby to transfer chemical energy into contraction force and motion.21 Each myosin molecule is made up of two myosin heavy chains and two pairs of light chains (figure 1.3 A).


Figure 1.3. A) Schematic representation of a myosin molecule constituting the thick filaments of the sarcomere. The myosin molecule is a hexamer consisting of two heavy chains (orange), two essential light chains (blue) and two regulatory light chains (yellow). The myosin heavy chains are dimerized through their coiled-coil tails. Adapted from ref. 22.
B) Three-dimensional (crystal) structure of a chicken skeletal myosin head. The catalytic and light-chain-binding domains are indicated. The heavy chain is shown in red, green and blue. The essential (yellow) and regulatory (purple) light chains wrap around the heavy chain alpha-helix (blue). Adapted from ref. 10 and 30.

The myosin heavy chain is a highly asymmetric molecule with a predominantly globular head and a rod like tail. The latter is formed by a coiled-coil structure of two alpha-helices and accounts for the formation of the thick filament backbone. The globular head contains a light-chain-binding domain and a catalytic domain with actin- and ATP-binding sites as shown in figure 1.3 B depicting the three-dimensional structure of a chicken skeletal myosin head.21


The regulatory and essential light-chain-binding domain of myosin is also referred to as the neck region, because it connects the head with the myosin tail. As shown in figure 1.3 B, the essential light chain wraps around the amino terminal half of the myosin neck, whereas the regulatory light chain lies closer to the head-rod junction. Myosin light chains have several isoforms with some of them encoded by different genes. The genes encoding for the ventricular myocardium isoforms of myosin light chains were analysed for HCM causal mutations in the present study and, therefore, will be considered in detail later in this chapter.

The actin- and ATP-binding sites are crucial for the myosin function. During contraction, the myosin heads attach to actin, forming so called "cross-bridges" between the thick and thin filaments. Subsequently, an ATP molecule binds up to a myosin head. Following ATP hydrolysis along with the release of products of this hydrolysis causes conformational changes in the myosin heads, which result in the displacement of the thin filament along the thick filament causing contraction.

The force generating myosin-actin interaction is regulated by tropomyosin, the troponin complex, and calcium ions.23 In a relaxed muscle, tropomyosin, troponin T and troponin I inhibit the attachment of the myosin heads to actin. With the beginning of a contraction event, myoplasmic Ca2+ concentration increases from 10-7 to about 10-5 M. Troponin C subsequently binds up to four calcium ions and relieves the inhibition of the actin-myosin interaction produced by tropomyosin, troponin T and troponin I. This enables the myosin heads to form cross-bridges and to draw the actin filament towards the centre of the sarcomere. Cycling formation of cross-bridges occurs until myoplasmic concentration of Ca2+ decreases, and troponin C relieves the Ca2+ molecules bound to it.

The mechanisms by which sarcomeric protein mutations lead to HCM are still unclear. However, the evidences accumulated from diverse functional studies, including animal modelling, have led to a hypothesis, which considers myocyte disarray, hypertrophy and interstitial fibrosis as a compensatory response to the alteration of the sarcomere contractile function by mutated proteins.3,24 In the case of missense mutations, it is assumed that mutated proteins are incorporated into the myofibrils and act as "poison peptides" affecting the function of the normal proteins (dominant-negative effect). Truncation mutations are assumed to result in an insufficient amount of functional proteins by either complete inactivation of a mutated allele or production of truncated proteins unable to incorporate into the myofibrils ("haploinsufficiency" or "null


allele" effect). Both cases lead to the impairment of force generation by the contractile units. The contractile deficit further provides the primary stimulus for increased expression of trophic and mitotic factors in the heart (such as insulin-like growth factor 1, transformic growth factor, and endotelin 1), which leads to hypertrophy, disarray and interstitial fibrosis characteristic of HCM. However, despite already available supporting evidences such as the observation of impaired mechanical performance of cardiac myocytes expressing mutated sarcomeric proteins, decreased LV end-systolic stress-volume ratio, and upregulation of the above stated trophic factors in patients with HCM, more studies are needed to prove the accuracy of this hypothesis.

1.2 The ventricular myosin regulatory and essential light chains

The human MYL2 gene encoding for the ventricular myosin regulatory light chain (RLC or also called MLC-2s/v) is located on chromosome 12q23-q24.3.25 Seven coding exons of this gene encode for a polypeptide of 166 amino acids. Apart from ventricular myocardium, the RLC is also expressed in slow skeletal muscle fibers.

The ventricular myosin essential light chain (ELC or MLC-1s/v) is encoded in humans by the MYL3 gene. It is located on chromosome 3p21.3-p21.2 and is also composed of seven exons, of which the last one is noncoding.26 MYL3 encodes for a polypeptide of 195 amino acids, which is, similar to the RLC, expressed in ventricular myocardium and slow skeletal muscle.

The RLC and ELC belong to a family of calcium-binding proteins like calmodulin and troponin C. The common feature of these proteins is the presence of structural motifs made up of a bivalent-cation-binding loop flanked by alpha-helices. These motifs are also called EF-hand domains. Calmodulin and troponin C have four functionally active EF-hand domains, which are essential for striated and smooth muscle contraction.23,27 It has been shown that deletions and non-conserved amino acid substitutions inactivate all EF-hand domains of the ELC and three of the RLC.28 Only one N terminal EF-hand domain of the RLC retains the ability to bind a bivalent cation. 28

Besides the EF-hand domain, the RLC possesses a putative phosphorylation site on a single serine residue at the amino termini (Ser15),29 while the ELC has an actin-binding site also at its amino terminal half.30


The function of myosin light chains in striated muscle is only partially understood. Based on the three-dimensional structure, it has been initially suggested that a major function of the striated muscle myosin light chains is to stabilize and elongate the 8.5 nm alpha-helical neck region of myosin.21 It is thought that a swinging motion of this neck relative to the catalytic domain is essential in amplifying generated power stroke (the lever arm model).21,31 Further functional studies, however, have suggested that the striated muscle myosin light chains also regulate and modulate the myosin-actin interaction.30,32-34

The ELC is thought to modulate the force production by binding with its N-terminus to the C-terminus domain of actin and thereby acting as a tether between the thin and thick filaments.30

The RLC might influence the myosin-actin interaction through phosphorylation and/or calcium binding. It was shown that RLC phosphorylation increases the rate of cross-bridges and, hence, increases the force production in cardiac and skeletal muscles at low levels of calcium.35,36 The mechanism of such effects of RLC phosphorylation might involve the conformational change of the entire myosin head due to a change in the charge of the N-terminal region of the RLC that occurs upon phosphorylation.37,38

It was also shown that a definite link exists between RLC phosphorylation and calcium binding.29,39 Szczesna et al.39 demonstrated that inactivation of the RLC calcium-binding site causes removal of all effects of phosphorylation. Furthermore, both phosphorylation and calcium binding properties as well as their relationship have been shown to be altered due to HCM-causing RLC mutations suggesting that alteration of exactly these properties could contribute to the pathogenesis of this disorder.29

1.3 Clinical features and diagnosis of HCM

HCM is a clinically heterogeneous and unpredictable disease. Its clinical manifestations vary from a benign course to that of severe heart failure and peripheral embolisation.5 The importance of recognising this disorder in patients as early as possible is highlighted by a high rate of sudden cardiac death in young people.


Premature death can occur even in completely asymptomatic patients as the first manifestation of the disease.

Myocardial hypertrophy generally develops during adolescence, however, in severe cases it can occur in an infant or even during foetal life. Simultaneously with hypertrophy, some HCM patients become symptomatic, while others exhibit no symptoms over long periods. HCM patients generally present with dyspnea, angina pectoris, palpitations, fatigue, presyncope, and syncope. Although these symptoms are common in all HCM patients, their onset and severity show great variability.

In approximately 25% of cases, myocardial hypertrophy leads to dynamic LV outflow or midventricular obstruction and, consequently, to the development of a pressure gradient.40 In case of LV outflow tract obstruction, apart from hypertrophy, systolic anterior motion of the mitral valve and mitral valve-septal contact contribute to the development of the pressure gradient. The value of the pressure gradient varies among the patients. If pressure gradients of >30 mm Hg (at rest) are present, the potential for further hypertrophy and deterioration is very likely.40 In such patients, operative reduction of the pressure gradient by means of septal myectomy (Morrow procedure) or nonsurgical septal reduction has been shown to be effective.40 Patients with the obstructive form of HCM usually exhibit a number of clinical signs, which are not seen in the non-obstructive form of the disease. Among them are systolic ejection murmur, bifid arterial pulse, double systolic impulse, and paradoxically split second heat sound.41

The minimal investigations needed for the diagnosis of HCM include ECG and transthoracic echo Doppler examination. Electrocardiogram is generally abnormal in HCM, although entirely normal electrocardiograms are seen in about 15% of patients and usually are found in the presence of only localized LV hypertrophy.42 The most common abnormalities are evidence of LV hypertrophy, negative T-waves, ST abnormalities, and pathological Q-waves. All these abnormalities can be absent in children and become evident over time with development of LV hypertrophy. However in some cases, especially in the young, ECG may be abnormal, even when echocardiography reveals no LV hypertrophy.43

Transthoracic echo Doppler examination is the most important diagnostic test in HCM. These combined techniques allow the assessment of extent and distribution of hypertrophy, systolic and diastolic function, the presence of systolic anterior motion of mitral valve, and the severity of the pressure gradient. The magnitude of LV wall


thickness can be very variable (13 - 30 mm or more).44 The location of hypertrophy is also diverse, although four frequent patterns of LV hypertrophy distribution have been reported.45 Type I is confined to the anterior portion of the interventricular septum (IVS), whereas type II involves the entire IVS. Type III, the most common, is characterized by hypertrophy of substantial portions of both interventricular septum and LV anterolateral free wall. Hypertrophy identified in regions of the LV other than basal IVS belongs to type IV.

Among other investigations, Holter ECG monitoring is a valuable tool in assessing the type and severity of cardiac arrhythmias. Chest X-ray, heart catheterisation, and magnetic resonance imaging can be helpful in the differential diagnosis of HCM, revealing the particular hypertrophy pattern and the stage of congestive heart failure.

Clinical heterogeneity of HCM makes it difficult to predict the outcome of the disease and to diagnose subjects who are in a high risk of premature death. According to clinical studies, a family history of premature sudden cardiac deaths, magnitude of hypertrophy more than 30 mm, an abnormal blood pressure response to exercise testing, and nonsustained/sustained ventricular tachycardia could be used as markers for sudden cardiac death in HCM and justify prophylactic therapy with amiodaron or implantation of cardioverter defibrillator.44,46,47 However, the accuracy of these risk factors is still subject of discussion. For instance, it has been argued that such risk factor as magnitude of hypertrophy is not accurate, since sudden cardiac death also occurs in the presence of little hypertrophy as in HCM caused by mutations in cardiac troponin T.48,49 A recent study showed that combination of the several risk factors increases the likelihood of sudden death.46 In addition to the investigation of the clinical risk factors, attempts have also been directed towards establishing genetic markers for assessing the severity of HCM phenotypes.


1.4 Genotype-phenotype correlation studies

Genotype-phenotype correlation studies have revealed that HCM phenotype is substantially influenced by the nature of the causative genetic defect. The causal gene as well as the type and localization of a mutation play the primary role. Thus, mutations in the beta-myosin heavy chain gene are generally associated with more significant hypertrophy and severe disease course than those in the other genes.50,51 Myosin binding protein-C gene mutations are mostly characterized by late clinical manifestation and a relatively benign disease course.52 High incidences of sudden cardiac death but little LV hypertrophy are features of cardiac troponin T mutations.53 Mutations in the cardiac troponin I gene have been shown to cause LV apical hypertrophy,11 whereas those in the ventricular myosin light chain genes have been initially associated with left midventricular hypertrophy.10 Concerning the causal mutations, protein truncation mutations or those located in highly important protein domains are generally associated with a severe course of HCM.54,55

The diversity in disease appearance in individuals bearing exactly the same mutation suggested that phenotypic expression of HCM is also influenced by factors other than the basic genetic defect, such as modifier genes or environmental influences.56 Amongst the known potential modifier genes are those encoding for functional variants of angiotensin-1 converting enzyme, angiotensinogen, endotelin-1, and several trophic factors.3,57

Correlation studies have also revealed that causal mutations carry prognostic significance.58 Some of them were associated with poor prognosis and a high incidence

of sudden cardiac death and could be therefore used as genetic markers for sudden death in HCM. Table 1.2 lists some mutations associated with a high, intermediary and low risk of sudden cardiac death in HCM.


Table 1.2. Mutations and prognosis in HCM





Sarcomeric protein
























Cardiac troponin T



















Ser179Phe (homozygous)


All unless listed







Myosin light chains


Insufficient data


Note: beta-MHC, beta-myosin heavy chain; MyBP-C, myosin binding protein-C. *Splice acceptor site mutation in intron 20. From ref. 58.

One should also keep in mind that the number of families identified with each specific mutation is relatively small, and the described phenotypes may be unique to the particular family and not generally applicable. More studies are needed to draw strong and accurate conclusions regarding the prognostic significance of a given genetic defect. However, identification of a malignant mutation along with the clinical risk factors can be useful in revealing patients with an adverse disease phenotype and the need for preventive measures.


1.5 Aims of the present study

In comparison with other disease genes, only few studies concerning the MYL2 and MYL3 genes have been performed so far. As mentioned above, mutations in these genes have been initially associated with a particular phenotype with massive hypertrophy of papillary muscles and adjacent LV tissue causing midventricular obstruction.10 However, further investigations have shown that typical septal hypertrophy can be also caused by ELC and RLC mutations.15,16,59 In contrast to other genes, phenotypic characterisation of HCM caused by defects in MYL2 and MYL3 has mainly dealt with the pattern of hypertrophy, and very little data are available regarding the disease course and prognosis.

Considering the limited information on HCM related to the ELC/RLC, this study was aimed to detect disease-causing mutations in the MYL2 and MYL3 genes in a group of clinically well-characterized HCM patients. Further purpose was to assess whether the detected mutations are associated with malignant or benign phenotype in the respective families.

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