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British Journal of Healthcare and Medical Research - Vol. 11, No. 6

Publication Date: December 25, 2024

DOI:10.14738/bjhmr.116.14733.

Maluze, U. O. (2024). Regulation of Muscle Contraction and How Mutations in the Muscle Proteins Cause Heart Disease. The

Laboratory Techniques Involves Molecular Biology, Protein Expression, Purification and Characterization Using Biochemical and

Biophyisical Techniques. British Journal of Healthcare and Medical Research, Vol - 11(6). 160-187.

Services for Science and Education – United Kingdom

Regulation of Muscle Contraction and How Mutations in the

Muscle Proteins Cause Heart Disease. The Laboratory Techniques

Involves Molecular Biology, Protein Expression, Purification and

Characterization Using Biochemical and Biophyisical Techniques

Ugochukwu Obinna Maluze

Department of Biomedical Engineering,

University of Bedfordshire, United Kingdom and

Family Health Hospital and Maternity, Lagos state, Nigeria

ABSTRACT

The expansion of the causative mutations to the rigid thin filament changed the

description of hypertrophic cardiomyopathy (HCM) from an illness of the cardiac

motor to a syndrome of the cardiac sarcomere and significantly extended the gasp

of the potential molecular pathogenic mechanism. An interesting hypotheses

concerning disease mechanism posted that the diverse medical prognoses in the

familial type of HCM may possibly be related to mutations in self-regulating protein

machinery of the sarcomere (Frank et al, 1968). The journal of the novel study in

1990 establishes the inherited association of the beta-myosin and tropomyosin

heavy chain genetic material to familial type of hypertrophic cardiomyopathy

(HCM). The current studies conducted by some researchers elaborated on the

various genetic alterations inside the genes encoding for the sarcomeric cardiac

proteins, alpha tropomyosin, troponin T, and myosin protein components. The

regularity of gene alteration in the alpha tropomyosin protein (TPM1) is lesser,

contributing to 5% of FHC. Currently, the D175N gene mutation has been recognized

in various unrelated populations, signifying that this spot could be an abnormal

gene “hot spot” for the disease. In this project research, a wild type of normal

protein and mutant genetic proteins (E180G and D175N) which are clinically

involved in familial hyper cardiomyopathy (FHCM) were produced. Having in mind

that the main effect of mutations E180G and D175N are mainly related to the

thermal stability of the protein; this research will also investigate the differences

between the thermal stability of wild type and mutated protein types using a Dye

base fluorescent method of analysis. Dye based fluorescent method was used to

monitor protein folding as a function of temperature for wild type tropomyosin and

for HCM mutant E180G and D175N proteins. The column chromatography method

of purification was used to purify the wild type and mutated proteins, and the

protein bands were separated using gel electrophoresis methods. A similar

assessment of folding stability and structural reports of several authors was in

consistency with this present report which suggested that such mutations might

alter protein folding. The results agree with previously published reports on the

impaired function of expressed E180G and D175N mutations suggesting that the

biochemical defects of the motor domain may affect myosin filament assembly in

the sarcomere. For future prospects, future biochemical analysis of several other

FHC mutations will be needed to establish a definite correlation between the

enzymatic impairment between different mutants and their clinical phenotype of

heart disease.

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161

Maluze, U. O. (2024). Regulation of Muscle Contraction and How Mutations in the Muscle Proteins Cause Heart Disease. The Laboratory Techniques

Involves Molecular Biology, Protein Expression, Purification and Characterization Using Biochemical and Biophyisical Techniques. British Journal of

Healthcare and Medical Research, Vol - 11(6). 160-187.

URL: http://dx.doi.org/10.14738/bjhmr.116.14733.

Keywords: hypertrophic cardiomyopathy, Tropomyosin, Troponin, Myosin, mutations,

autosomal-dorminant, inheritance, autosomal-recessive, Sarcomere, Arrhythmogenic,

Proteins, etc.

INRODUCTION

The expansion of the causative mutations to the rigid thin filament changed the description of

hypertrophic cardiomyopathy (HCM) from an illness of the cardiac motor to a syndrome of the

cardiac sarcomere and significantly extended the gasp of the potential molecular pathogenic

mechanism (Kimura et al, 1997; Olson et al, 2000). In the subsequent decade, causative

mutation was correlated to the genes programming the cardiac thin filament proteins (Kimura

et al, 1997, Olson et al, 2000; Landstrom et al, 2008). An interesting hypotheses concerning

disease mechanism posted that the diverse medical prognoses in the familial type of HCM may

possibly be related to mutations in self-regulating protein machinery of the sarcomere (Frank

et al, 1968).

In the 20 years, from the time when the landmark study of Geisterfer-Lowrance et al (Geisterfer

et al, 1990) that recognized the R403Q alteration in the genetic material programming the beta

cardiac myosin protein genes (MYH7) as contributing for hypertrophic cardiomyopathy (HCM),

100 of extra mutations in 10 diverse sarcomeric genetic material have been associated with the

illness (Xu et al, 2010; Konno et al, 2010), and bulk of these alteration in the genetic component

changes the beta myosin heavy chain, the power that co-ordinates cardiac muscle contraction,

and this serves as the principal element of sacomere thick filaments (Konno et al, 2010).

Interestingly, the journal of the novel study in 1990 establishes the inherited association of the

beta-myosin and tropomyosin heavy chain genetic material to familial type of hypertrophic

cardiomyopathy (HCM) (Teare, 1958). Belatedly in the 1950s, a mysterious illness drawed the

mind of cardiologists, surgeons, and pathologists (Brock, 1957). During the same year,

hypertrophic cardiomyopathy has involved deep attention with increasing understanding of its

prevalence, its function as the main source of unexpected cardiac death in juvenile individuals,

and its isolation as a mendelian autosomal dominant genes (Arndt et al, 2013). In 1958, it was

illustrated that alteration in the genetic component in the cardiac troponin and alpha

tropomyosin were the main basis of familiar ventricular cardiomyopathy, therefore switching

the disorder from a illness of the beta-myosin heavy chain to ailment of the cardiac sarcomere

(Teare, 1958). Hypertrophic cardiomyopathy has represented the model of monogenic cardiac

disorder, increasing the likelihood that its explanation would provide essential details into the

root of cardiac hypertrophy. Categorized as irregular septal hypertrophy (Teare, 1958),

efficient aortic stenosis (Brock, 1957; Marrow & Braunwald, 1959), hypertrophic obstreperous

cardiomyopathy (Goodwin et al, 1960), and idiopathic hypertrophic subaortic stenosis

(Braunwald & Ebert, 1962), early description described the remarkable medical trial associated

with the syndrome. Current biochemical research has revealed that HCM mutations in beta

MHC change the cycling speed of myosin heads (Lowey,2002) and, because patients are

heterozygous for these genetic alterations, their thick filaments will be poised of wild-type

myosin heads.

The current studies conducted by some researchers elaborated on the various genetic

alterations inside the genes encoding for the sarcomeric cardiac proteins, alpha tropomyosin,

troponin T, and myosin protein components. The regularity of gene alteration in the alpha

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British Journal of Healthcare and Medical Research (BJHMR) Vol 11, Issue 06, December-2024

Services for Science and Education – United Kingdom

tropomyosin protein (TPM1) is lesser, contributing to 5% of FHC. Different point mutations

resulting to alteration in gene sequence of the proteins have also beennoted: E62Q (Jongbloed

et al, 2003), A63V (Yamauchi et al, 1996; Nakajima-Taniguchi et al, 1995), K70T (Nakajima- Taniguchi et al, 1995), D175N (Thiefelder et al, 1994), E180G (Thiefelder et al), E180V (Regitz- Zagrosek et al, 2000) and L185R (Van Driest et al, 2002). Currently, the D175N gene mutation

has been recognised in various unrelated populations, signifying that this spot could be an

abnormal gene “hot spot” for the disease (Reed & Davies, 1994). ‘‘In vivo studies, using

transgenic mice as a model showed an impairment of cardiac function by altering the sensitivity

of myofilaments to calcium’’ (Evans et al, 2000). In vitro research conducted with recombinant

proteins expressing the gene mutations, demonstrated little changes on the whole stability of

the protein as detected by circular dichroism (Golitsina et al, 1997), and this showed abnormal

changes in the kinetics of contractile force production (Bing et al, 1997).

Various Forms of Mutation Involves in Cardiomyopathy

In the field of genetics, mutation is described as a permanent alteration of the genetic sequence.

Gene alterations in the muscle proteins can give rise to various types of abnormality in genetic

compositions and this can alter the genetic composition or stop the gene from performing

properly. At the molecular stage, different types of mutation have been noted as the aetiology

of cardiomyopathy. ‘‘Point mutations has been noted to cause hypertrophic cardiomyopathy,

and often caused by chemicals or malfunction of DNA replication and exchange of a single

nucleotide for another’’ (Freese & Emst, 1959). ‘‘These abnormal gene sequences are

categorised as transitions or transversions and the most common is the transitional state that

exchanges a purine for a purine (A ↔ G) or a pyrimidine for a pyrimidine, (C ↔ T)’’ (Freese &

Emst,1959).

Point mutations that take place in the protein coding region of DNA may be categorized into

three kinds of mutation (silent mutation that encode for the same amino acid, missense

mutation that programmed for a different amino acids and nonsense mutation which

programmed for a stop codon and therefore alter the genetic sequence of the protein, giving

rise to different types of cardiomyopathies (Boillee et al, 2006).

Splice site mutation or reading frame mutation is a type of gene mutation that occur due to

insertion or deletion of the genetic elements in the coding area of the genes, and this can change

the splicing of the mRA (splice site mutation, or lead to a change in the reading frame (frame

shift mutation); these two mutations can extensively alter the genetic component, alter the

protein structure and contributes to cardiomyopathy (Hogan & Michael, 2010).

Various Forms of Cardiomyopathy

Cardiomyopathies can be defined as a clinically diverse groups of heart muscle ailments, which

has a distinguishing feature of unusual myocardial structures. The present grouping of the

cardiomyopathies persisted to be based on phenotypic and clinical examinations of the affected

individuals. Familial hypertrophic cardiomyopathy (FHC) can be defined as an autosomal

dominant heart illness with prominent features of interventricular hypertrophy, mitral valve

disorders, monocyte hypertrophy, interstitial fibrosis and atrial fibrillation (Davies, 1984;

Maron et al., 1987; Olivotto et al, 2001). The illness is clinically inconsistent, and it starts as

benign condition and progresses to a severe devastating state which frequently give rise to

sudden deaths in young athletes (Maron et al., 1978 & 1986; Solomon et al., 1990; Dausse and

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163

Maluze, U. O. (2024). Regulation of Muscle Contraction and How Mutations in the Muscle Proteins Cause Heart Disease. The Laboratory Techniques

Involves Molecular Biology, Protein Expression, Purification and Characterization Using Biochemical and Biophyisical Techniques. British Journal of

Healthcare and Medical Research, Vol - 11(6). 160-187.

URL: http://dx.doi.org/10.14738/bjhmr.116.14733.

Schwartz, 1993; Watkins et al., 1995c). Every single mutation that causes FHC are structural

proteins of the sarcomere: b -myosin heavy chain (MyHC) (Geisterfer-Lowrance et al., 1990;

Watkins et al., 1993), a -tropomyosin (Watkins et al., 1995b), troponin T (Thierfelder et al.,

1994), myosin binding protein-C (Watkins et al., 1995a; Bonne et al., 1995), light chain 1 and

troponin I (Kimura et al., 1997).

Dilated Cardiomyopathy (DCM) is a heart disorders that take place as a result of gene alteration

on protein tropomyosin. Genetic abnormal changes have been connected with irregular onset

on DCM, and they have been recognized in families that frequently demonstrate an autosomal- dorminant inheritance prototype, with autosomal-recessive inheritance (Petretta et al, 2011).

A number of various genes have been implicated as a cause of DCM, and these genes codes for

various protein components of the ‘‘sarcomere, Z- disc, cytoskeleton, sarcolemma, and nucleus

‘’ (Fatkin et al, 1999, 2010). DCM has prominent features of enlarged chamber size and

abnormal systolic reduction of the two ventricles (Herman et al, 2012).

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is another type of cardiomyopathy

that is due to gene mutation on the protein tropomyosin, and relatives with ARVC usually

displayed autosomal-dominant inheritance patterns. Several researchers have identified about

nine abnormal genes as the genetic cause of ARVC; ‘‘five of these genes encode the desmosomal

proteins, plakophilin-2, plakoglobin, desmoplakin, desmocollin, and desmoglein-2’’

(Teekakirikul et al, 2013). About 50% of ARVC clinical manifestations have a desmosomal

genetic alteration, and 40% have a plakophilin-2-gene mutation (Van Tintelen et al, 2006). The

differential diagnosis of ARVC can be demanding, but some useful method of examination have

been devised which take into description of structural and functional disorders, tissue

characterizations, ECG disorders, arrhythmias, and genetic family history (McKenna et al,1994;

Marcus et al, 2010).

Restrictive Cardiomyopathy (RCM) is another type of cardiomyopathy linked with abnormal

ventricular diastolic features with a raised end-diastolic pressure that give rise to usual or

lowered ventricular magnitude (Cale-shu et al,2011). Genetic abnormal changes in seven

sarcomere protein genes encoding the cytoskeleton protein desmin have been implicated in

families diagnosed with RCM (Parvatiyar et al, 2010a; Sen-Chowdry et al, 2010; Caleshu et al,

2011). Generally, the diagnosis in RCM is poor, particularly in affected children, and heart

transplantation is frequently carried out (Sen-Chowdry et al, 2010).

Proteins Regulations in Muscle Contractions and Their Implications in

Cardiomyopathies

According to current research, there are different types of sarcomere proteins that are

implicated in cardiomyopathies. These includes alpha myosin heavy chain, titin, troponin C,

tropomyosin (Niimura et al,1997; Carniel et al, 2005; Satoh et al, 1999; Hoffmann et al, 2001);

Z-disc –associated proteins (actinin, ankyrin, myozenin 2) (56, 57,); muscle LIM proteins

(nexilin and telethonin) (Knoll et al, 2010; Wang et al, 2010; Bos et al, 2006; Hayashi et al, 2004)

and proteins implicated in other monocyte functions (phospholamban and viniculin)

(Landstrom et at, 2010; Vasile et al, 2006). These proteins are extremely essential in regulation

of muscle contractions, and mutations in these proteins can cause hypertrophic

cardiomyopathy (Osio et al, 2007).

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British Journal of Healthcare and Medical Research (BJHMR) Vol 11, Issue 06, December-2024

Services for Science and Education – United Kingdom

However, ‘Several genetic studies summarized the perception that abnormal gene changes on

the sarcomere proteins (alpha myosin, troponin C and tropomyosin) were the causes of familiar

hypertrophic cardiomyopathy’’ (Darsee et al, 1979). Schwartz and his co- researchers

recognized HCM locus on chromosome 11 where the thick filament myosin and tropomyosin

binding protein-C (MYBPC3) was mapped (Carrier et al, 1993) and abnormal gene identified

(Bonne et al, 1995; Watkins et al, 1995). The clinical examination of the myosin vital light chain

(MYL3) and regulatory chain (MYL2) genes by Epstein and colleaques (Poetter et al, 1996),

troponin I (TNN13) and tropomyosin by Sasazuki and colleaques (Kimura et al, 1997), and

cardiac actin (ACTC) by Olson and Fanana-pazir established a perfect role of gene mutation in

causing HCM.

Biochemistry of Tropomyosin, Its Characteristics and Definitive Roles in Muscle

Contractions

Tropomyosin (TMs) are classified as a family of extremely preserved proteins seen in most

eukaryotic cells that participate actively in muscle contractions (Fan anapazir, 1994; Satoh et

al, 1999; Lee-Miller, 1991). Tropomyosin and cardiac actin displayed a distinctive useful

element that plays a structural and active function in sarcomeric muscle protein. The genetic

constituent of TPM1 comprises of 14 exons and 4 isoforms (a and b -tropomyosins,

tropomyosin-4, and tropomyosin-30) (Schwartz et al, 1995; Lee-Miller, 1991). The striated

muscle isoform is made up of alpha helical protein, that produces a matching coiled-dimer

perverse in the area of the long axis of the actin filament. Each polypeptide chain contain about

284 amino acid residues (Lee-Miller, 1991), and each of the dimer is attached to seven actin

monomers and one troponin (Tn) complex (TnC, TnI and TnT) (Smillie, 1979) The

polymerization of head-to-tail pattern of the striated muscle cells with the troponin complex

control the calcium sensitivity of the actomyosin-ATPase complex (Smillie,1979).

The amino acid arrangement in Tm consists of seven-residue prototype (a to g) which are

recurring all over the whole sequence. Positions a and d, on the similar region of the segment,

are frequently taken by a polar amino acid which permit for hydrophobic exchanges among the

chains. Positions e and g are also taken by another exciting residue, and this add to the stability

and rigidity of the corresponding coiled-coil arrangement through their ionic exchanges with

residues at positions e and g of the former helical structures (Parry,1975). Positions b, c and f

are taken by glacial or ionic component which collaborate with other proteins (Smillie, 1971).

In Vitro (PCR) Site-directed Mutagenesis or Oligonucleotide-directed Mutagenesis

In molecular genetic medicine, invitro site-directed mutagenesis is a very useful method used

in studying protein structure-function interaction, gene composition, and also for vector

alteration (Kilbey, 1995). It is a genetic biology technique used to produce precise changes that

altered the DNA sequence (Kilbey, 1995; Shortle, 1981). Quite a lot of approaches to this

method have been documented, but these techniques usually involve single-stranded DNA

(ssDNA) serving as the template (Kunkel, 1985; Vandeyar, 1988; Sugimoto et al, 1989; Tayloy

et al, 1985) and they are laboured intensive or precisely complicated.The Stratagene’s Quik

Change site-directed mutagenesis technique is performed by means of pfuTurbo DNA

polymerase and a temperature cycler (Nelson, 1992). PfuTurbo DNA polymerase reproduce

with plasmid strands and uses a supercoiled double-stranded DNA (dsDNA) vector and two

artificial oligonucleotide primers displaying the desired mutation. The oligonucleotide primers,

each corresponding to reverse strands of the DNA gene, are extensive throughout the period of

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165

Maluze, U. O. (2024). Regulation of Muscle Contraction and How Mutations in the Muscle Proteins Cause Heart Disease. The Laboratory Techniques

Involves Molecular Biology, Protein Expression, Purification and Characterization Using Biochemical and Biophyisical Techniques. British Journal of

Healthcare and Medical Research, Vol - 11(6). 160-187.

URL: http://dx.doi.org/10.14738/bjhmr.116.14733.

temperature cycling by pfuTurbo DNA polymerase (Papworth et al, 1996). Inclusion of the

oligonucleotide primers produces a mutated genes comprising of staggered nicks (Papworth et

al, 1996). Subsequent to temperature cycling, the altered gene sequence is treated with Dpn 1.

The Dpn 1 endonuclease (target sequence: 5’-GmATC-3) which is very explicit for methylated

and hemimethylated DNA and is mainly utilized to process the parental DNA (Nelson &

McClelland, 1992). The genetic material produced from every E.coli bacteria is dam methylated

and consequently prone to Dpn 1 digestion. The nicked DNA gene containing the required

abnormal genetic sequence is then submerge into XL1-Blue supercompetent cells, which

contributes to a high-quality mutation efficiency (Nelson & McClelland, 1992).

Recombinant Protein Expression Using E.Coli

Increased level synthesis of recombinant proteins as a condition for immediate refinement has

grow to be a model technique. The laboratory synthesis of recombinant proteins involves

cloning of the suitable genetic material into an expression DNA vector under the control of an

inducible promoter (Marino 1989). But well-organized appearance of the recombinant gene is

based on a range of conditions such as most favourable displaying signals (both at the stage of

transcription and translation), accurate protein folding and cell expansion description (Marino,

1989). However, the use of bacterium E. coli has been the most popular means of producing

recombinant proteins for over two decades. The advantages of using E. coli are that it offers

short culturing time, easy genetic manipulation, low-cost media and it has unparalleled fast

growth kinetics (Sezonov et al, 2007). The factors influencing the expression level include

unique and subtle structural features of the gene sequence, the stability and efficiency of mRNA,

correct and efficient protein folding, codon usage, degradation of the recombinant protein by

ATP-dependent proteases and toxicity of the protein (Makrides,1996; Swartz, 2001).

In protein expression using E. coli, the regulated gene expression requires an inducible or

repressible system, and therefore, all expression systems are based on controllable promoters.

Four regulatable promoter systems are widely used, three are based on the repressors already

mentioned (LacI, TrpR and phage cI) and the fourth is based on a phage RNA polymerase (Rao

et al., 1994). The lac system consists of the promoter/operator region preceding the lac operon

and the LacI repressor encoded by the lacI gene. In the absence of an inducer, the Lac repressor

binds to its operator situated immediately downstream from the promoter as a homotetramer.

The wild-type lac promoter sequence contains one deviation in the -35 and two in the -10 box,

and the spacer region encompasses 18 nucleotides if compared to the consensus sequence. One

of the many promoter mutations isolated has been termed lacUV5. If its DNA sequence is

compared to that of the wild-type promoter, it becomes apparent that two nucleotides have

been exchanged. The promoter strength of lacUV5 has increased 2.5-fold, and mutations

increasing the promoter strength are called promoter-up mutations in general (de Boer et al.,

1983). In the case of the Plac, the PlacUV5 and the Ptac promoters, the repressor is inactivated

by addition of isopropyl- -D- thiogalactopyranoside (IPTG). This compound binds to the active

LacI repressor andcauses dissociation from its operator. IPTG has two advantages over lactose:

First, its uptake is not dependent on the Lac permease (it diffuses through the inner membrane)

and second, it cannot be cleaved by galactosidase preventing turn-off of transcription. The lacI

gene is either part of the expression plasmid or it is present within the chromosome. Since the

wild- type level of the LacI repressor is not sufficient to repress expression of the recombinant

gene in the absence of IPTG, two derivates have been isolated resulting in an increase in the