Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, Nos. 12-13, pp. 2083-2106 The Author(s) 2024. This article is an open access publication.

2083

REVIEW

Troponins and Skeletal Muscle Pathologies

Agnessa P. Bogomolova

1,2,a

* and Ivan A. Katrukha

1,2,b

Faculty of Biology, Lomonosov Moscow State University, 119234 Moscow, Russia

Hytest Ltd., Turku, Finland

e-mail: bogomolova.agnessa@yandex.ru

e-mail: katrukhai@mail.ru

Received May 2, 2024

Revised November 19, 2024

Accepted December 1, 2024

Abstract— Skeletal muscles account for ~30-40% of the total weight of human body and are responsible for

its most important functions, including movement, respiration, thermogenesis, and glucose and protein me-

tabolism. Skeletal muscle damage negatively impacts the whole-body functioning, leading to deterioration of

the quality of life and, in severe cases, death. Therefore, timely diagnosis and therapy for skeletal muscle

dysfunction are important goals of modern medicine. In this review, we focused on the skeletal troponins

that are proteins in the thin filaments of muscle fibers. Skeletal troponins play a key role in regulation of

muscle contraction. Biochemical properties of these proteins and their use as biomarkers of skeletal muscle

damage are described in this review. One of the most convenient and sensitive methods of protein biomarker

measurement in biological liquids is immunochemical analysis; hence, we examined the factors that influence

immunochemical detection of skeletal troponins and should be taken into account when developing diagnostic

test systems. Also, we reviewed the available data on the skeletal troponin mutations that are considered

tobe associated with pathologies leading to the development of diseases and discussed utilization of troponins

as drug targets for treatment of the skeletal muscle disorders.

DOI: 10.1134/S0006297924120010

Keywords: skeletal muscle, troponin, ageing, myopathy, biomarker, monoclonal antibodies

Abbreviations: ALS, amyotrophic lateral sclerosis; cTnI, cardiac troponin I; cTnT, cardiac troponin T; fsTnC, fast skeletal

troponin C; fsTnI, fast skeletal troponinI; fsTnT, fast skeletal troponinT; ss/cTnC, slow skeletal/cardiac troponin C; ssTnI,

slow skeletal troponin I; ssTnT, slow skeletal troponin T.

* To whom correspondence should be addressed.

INTRODUCTION

In mammals, striated muscles are represented by

two tissue types: skeletal and cardiac. Skeletal mus-

cles accounts for ~30-40% of the total body weight and

perform important functions such as movement, res-

piration, and thermogenesis [1, 2]. They are involved

in the major share of glucose, lipid, and protein me-

tabolism. Thus, skeletal muscle damage or pathology

could have a significant impact on various aspects of

whole-body functioning, leading to impaired quality of

life and, in severe cases, disability or death.

Skeletal muscle dysfunction can be caused by

mechanical injuries, myopathies, and various diseases

that are accompanied by muscle atrophy and rhab-

domyolysis. The most common causes of mechanical

damage to skeletal muscles include vigorous physical

activity, surgery, trauma, and prolonged compression

syndrome  [3]. The term “myopathy” describes any

skeletal muscle disease of various etiologies. These pa-

thologies are frequently accompanied by abnormalities

of the skeletal muscle structure and development of

metabolic disorders in the tissue [4]. Myopathies are

classified as either congenital (inherited) or acquired

(secondary). Inherited myopathies include: muscular

dystrophies (associated with mutations in the genes

of certain cytoskeletal proteins of skeletal muscle, e.g.,

Duchenne muscular dystrophy or Becker muscular

dystrophy); congenital myopathies (e.g., nemaline my-

opathy, core myopathy); metabolic myopathies (associ-

ated with mutations in the genes of various enzymes

involved in carbohydrate and lipid metabolism);

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BIOCHEMISTRY (Moscow) Vol. 89 Nos. 12-13 2024

mitochondrial myopathies (associated with mutations

in mitochondrial oxidative phosphorylation proteins);

and channelopathies [5-8]. Muscular dystrophies are

considered as one of the most common and severe in-

herited myopathies, some of which, such as Duchenne

muscular dystrophy, are characterized by progressive

muscle weakness and atrophy, eventually leading

to death from cardiopulmonary arrest [9]. Acquired

myopathies include: inflammatory myopathies (most

commonly of autoimmune nature, such as derma-

tomyositis and polymyositis), myopathies associated

with various infections (caused by bacteria, fungi, or

viruses), toxic myopathies (e.g., caused by drug or al-

cohol poisoning or myotoxic drugs such as statins and

fibrates), myopathies associated with various diseases

(rheumatic diseases, malignant tumors, and endocrine

diseases), idiopathic myopathies, and endocrine myop-

athies [10-13].

Another serious pathology of skeletal muscle is

rhabdomyolysis. It is the process of skeletal muscle

breakdown accompanied by the release of intracel-

lular components into the bloodstream, which can

be life-threatening in severe cases. When more than

100  g of muscle tissue is simultaneously damaged,

concentration of myoglobin in the blood reaches a

critical value, and a significant amount of protein is

deposited in renal tubules. This process could provoke

acute renal failure, which is the main cause of fatal

outcomes in rhabdomyolysis [12, 14]. In children, the

major factors leading to rhabdomyolysis are viral my-

ositis, trauma, rheumatic diseases, high-intensity exer-

cise, and medications  [12]. In adults, rhabdomyolysis

most often occurs due to alcohol or drug abuse, med-

ications, trauma, malignant neuroleptic syndrome, or

immobility [5, 12].

Myopathies are accompanied by muscle atrophy,

a process of loss of muscle mass and strength when

catabolism of muscle proteins predominates over

anabolism. In addition to myopathies, other diseases

and pathologies such as chronic heart failure, chronic

obstructive pulmonary disease, cancer, chronic kidney

disease, Alzheimer’s disease, and infectious diseases

could also lead to the development of muscle atro-

phy [15, 16]. Muscle atrophy could also be caused by

immobility and muscle disuse due to paralysis or pro-

longed bed rest [3].

A loss of muscle mass also occurs during the

ageing process – sarcopenia [from Greek words sarx

(flesh) and penia (deficiency)]. This condition is man-

ifested by the decrease in the cross-sectional area and

number of muscle fibers, accumulation of adipose or

connective tissue in the skeletal muscle, and progres-

sive loss of the muscle mass and strength. All of this

entails poor physical performance and, if prolonged,

geriatric frailty [17]. Sarcopenia is characterized by

the increased risk of soreness, falls, limitations in daily

activities, poor prognosis after surgery and, ultimately,

high mortality [16]. Prevalence of sarcopenia reaches

a level up to 13% in the individuals aged 60-70 years

and up to 50% in the individuals aged over 80 [17].

Presence of diabetes increases the risk of sarcopenia

by 2-fold, and of geriatric frailty – by 1.5-4-fold [18].

Thus, importance of these problems grows with the

increase in life expectancy. To date, nutritional adjust-

ment and exercise have been used to stop progression

of sarcopenia, but also therapeutic strategies are being

developed to prevent muscle damage [16-18].

Skeletal muscle is the primary tissue responsible

for insulin-dependent glucose uptake; hence, loss of

muscle mass in sarcopenia could lead to the develop-

ment of insulin resistance, and ultimately type  2 dia-

betes. In addition, the skeletal muscle adiposis, which

accompanies sarcopenia, could also contribute to dys-

function [17, 19]. In sarcopenia and type  2 diabetes,

functional impairments could develop necessitating

diagnosis and timely treatment. So, monitoring the de-

crease in skeletal muscle mass is of high importance,

especially in the patients with diabetes.

Three types of skeletal muscle fibers have been

identified in humans: slow-twitch (type  1) and fast-

twitch (types 2A and 2X; other mammals also have

type  2B fast-twitch fibers) [20, 21]. The type  2A fast-

twitch muscle fibers are more resistant to fatigue than

the type  2X and contain more enzymes of oxidative

metabolism  [22]. Fiber composition of muscles de-

pends on the function they perform: muscles respon-

sible for maintaining body posture are mainly com-

posed of slow-twitch fibers, while muscles required

for movement are predominantly composed of fast-

twitch fibers [21].

Development of some pathologies could result in

the damage to muscle fibers of only certain type. For

example, predominantly fast-twitch muscle fibers are

affected, especially type 2X fibers, in the Duchenne

muscular dystrophy [23]. Muscle damage induced by ad-

ministration of statins primarily influences fast-twitch

fibers, while fibrates intake impacts slow-twitch fibers

[24,  25]. Intense eccentric muscle contractions also

primarily damage fast-twitch fibers  [26]. Many patho-

logical conditions entailing muscle atrophy are char-

acterized not only by deterioration of certain muscle

fiber types, but also by a fiber-shift from one type to

another. For instance, denervation or immobility of a

limb, spinal cord injury, or prolonged bed rest may

lead to slow-to-fast fiber type shift  [27]. The reverse

process, fast-to-slow fiber type shift, occurs as a result

of starvation, glucocorticoid administration, cachexia,

and sarcopenia [27]. In addition, fast-twitch and slow-

twitch muscle fibers have been shown to have differ-

ent abilities to regenerate after injury. Studies in rats

have shown that while muscles consisting mainly of

fast-twitch fibers (e.g., extensor digitorum) regenerate

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BIOCHEMISTRY (Moscow) Vol. 89 Nos. 12-13 2024

efficiently, muscles composed predominantly of slow-

twitch fibers (e.g., soleus) do not regenerate complete-

ly, developing fibrosis [28].

Various instrumental methods have been used

to diagnose skeletal muscle injuries. These include

magnetic resonance imaging, computed tomography,

and dual-energy X-ray absorptiometry [29]. These ap-

proaches allow non-invasive detection of changes in

the skeletal muscles (edema, replacement with adipose

and connective tissue, muscle atrophy), identification

of specific areas of damage, and even quantification

of changes in the structure of skeletal muscles [30-34].

However, disadvantage of using instrumental methods

is the need for expensive equipment and specialized

locations for manipulation. An alternative is determi-

nation of blood concentrations of biomarkers – most

commonly skeletal muscle proteins– that are released

into the bloodstream when muscle fibers are dam-

aged. Currently, the most widely used biomarkers of

skeletal muscle damage are creatine kinase, aspartate

aminotransferase, lactate dehydrogenase, and myo-

globin  [35]. However, it should be noted that all the

abovementioned proteins lack specificity: in addition

to skeletal muscle, they are expressed in other tissues,

that may reduce accuracy of the diagnosis. Therefore,

special studies have been conducted to seek and iden-

tify new and more specific biomarkers. These includ-

ed fatty acid binding protein  3, myosin light chain 3,

MM isoform of creatine kinase, and skeletal troponin I

(TnI) isoforms  [36]. Although all the above biomarkers

are comparable or superior in sensitivity to conven-

tional biomarkers, only the skeletal TnI isoforms are

specifically expressed in skeletal muscle [9, 36, 37].

TnI, together with troponin  C (TnC) and troponin  T

(TnT), constitutes the troponin complex that regulates

muscle contraction [38, 39]. TnT isoforms are also spe-

cific to skeletal muscle, but we were unable to find

information on their use as biomarkers of skeletal

muscle damage. The utilization of skeletal muscle TnT

as a biomarker requires further investigation.

For a long time, skeletal troponins have attracted

attention of the scientists, primarily due to the studies

reporting their role in regulation of muscle contrac-

tion. In this review, we describe role of these proteins

in the development of certain skeletal muscle diseas-

es and discuss applications of troponins for diagnosis

and therapy of skeletal muscle disorders.

BIOCHEMICAL PROPERTIES OF THE TROPONINS

Troponin I. In humans, TnI is represented by

three isoforms: cardiac TnI (cTnI) and two skeletal

isoforms, namely fast skeletal (fsTnI) and slow skele-

tal (ssTnI). fsTnI and ssTnI are expressed in fast-twitch

and slow-twitch muscle fibers, respectively (Table 1)

[23,  40]

. ssTnI is also expressed in cardiac muscle

during embryonic development and is replaced by

cTnI in the postnatal period [41]. cTnI is only ex-

pressed in cardiac muscle after birth [42, 43].

TnI inhibits interaction between myosin and actin

in the absence of Ca

. In the presence of Ca

, the in-

hibitory region of TnI dissociates from actin and this

facilitates actin-myosin interaction [44, 45]. The major

difference between the skeletal TnI isoforms and cTnI

is the presence of a unique 31 amino acid N-terminal

sequence (residues 2-32) in cTnI (Fig.  1). Phosphory-

lation of the Ser23 and Ser24 residues is considered

as a mechanism regulating muscle contraction [48].

For the skeletal TnI isoforms, the three-dimensional

(3D) structure has been resolved for fsTnI only. The

highest resolution 3D structure was obtained using

X-ray crystallography for the chicken fsTnI in complex

with TnC and for the portion of TnT in both Ca

-ac-

tivated state (3.00  Å resolution) and Ca

-free state

(7.00  Å resolution)  [46]. The 3D structure has been

resolved for the full-length fsTnI with the exception

of C-terminal part of the molecule. Several function-

al sites could be distinguished in the TnI structure:

the IT arm, inhibitory, switch, and C-terminal regions.

Similar sites for ssTnI have been denoted based on

their sequence similarity with fsTnI and cTnI [46,  47].

Numbering in all proteins of this review includes N-terminal Met.

Table 1. Some properties of human skeletal TnI isoforms

Isoforms

Fast skeletal TnI

(UniProt P48788)

Slow skeletal TnI

(UniProt P19237)

Gene TNNI2 TNNI1

Chromosomal

locus

11p15.5 1q32.1

Number

of exons

Number

of amino acid

residues

182 187

Molecular

mass (kDa)

21.3 21.7

Isoelectric

point

8.9 9.6

Tissue

specificity

fast-twitch

muscle fibers

slow-twitch

muscle fibers,

embryonic

cardiomyocytes

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Fig. 1. Protein sequence alignment of three TnI isoforms: fsTnI, ssTnI and cTnI. Amino acid sequences are derived from

the Uniprot database: fsTnI (TNNI2_HUMAN, P48788), ssTnI (TNNI1_HUMAN, P19237), cTnI (TNNI3_HUMAN, P19429). Clustal

Omega online software was used to make protein sequence alignments. Identical amino acid residues in the aligned se-

quences are colored in blue. Boxes show the borders of α-helices (numbering is for fsTnI sequence). Arrows indicate regions

and TnC- and TnT-binding sites, putative fsTnI and ssTnI phosphorylation (p) and fsTnI glutathionylation (glut) sites are

marked above the sequence [44-47].

The IT arm of TnI (~2-106 amino acid residue of

fsTnI; ~2-107 amino acid positions of ssTnI; number-

ing varies in different animal species) has a structur-

al function: it consists of the N-terminal region that

binds the C-terminal domain of TnC (~2-40 residues

of fsTnI; ~2-40 residues of ssTnI) and the TnT-bind-

ing site (~50-106 amino acid residues of fsTnI; ~50-107

residues of ssTnI). The IT-arm is formed by two op-

positely directed α-helices, H1 (~12-48 amino acids of

fsTnI; ~12-48 residues of ssTnI) and H2 (~58-103 res-

idues of fsTnI; ~59-104 residues of ssTnI), connected

by a linker (~49-57 residues of fsTnI; ~49-58 residues

of ssTnI). The inhibitory region (~107-115 residues of

fsTnI; ~108-116 residues of ssTnI) interacts with ac-

tin in the absence of Ca

ions, moving tropomyosin

molecule to the position where it blocks the inter-

action of myosin with actin thus preventing muscle

contraction. The switch region (~116-131 residues of

fsTnI; ~117-132 residues of ssTnI) includes the H3

α-helix (~118-127 residues of fsTnI; ~119-128 residues

of ssTnI), it binds to the N-terminal domain of TnC

when Ca

concentration increases, which leads to dis-

sociation of the inhibitory region of TnI from actin, dis-

placement of tropomyosin, and interaction of myosin

with actin.

The C-terminal portion of fsTnI could not be re-

solved by X-ray crystallography due to its mobility.

The nuclear magnetic resonance (NMR) data for the

chicken fast skeletal troponin complex shows that the

C-terminal region lacks secondary structure and is in

a disordered state, both in the absence and presence

of Ca

[49, 50]. According to the results of small-angle

neutron scattering of the chicken fast skeletal tropo-

nin complex, the C-terminal part of fsTnI is an elon-

gated structure at low Ca

concentrations that may

represent a supercoiled helix or β-layers, while in the

presence of Ca

the site adopts a compact structure

[51]. Other NMR data suggest that the C-terminal re-

gion of the chicken fsTnI has a secondary structure

consisting of an α-helix, two β-layers, and two more

α-helices [52-54]. The C-terminal region interacts with

actin in the absence of Ca

and thus participates in

inhibition of myosin binding to actin [52, 55, 56].

Troponin T. Like TnI, TnT is represented in the

human body by three isoforms: a cardiac (cTnT) and

two TnT skeletal isoforms, namely fast skeletal (fsTnT)

and slow skeletal (ssTnT) variants (Table 2). fsTnT and

ssTnT are only expressed in skeletal muscle, while cTnT

is expressed in both heart and skeletal muscle during

embryonic development and neonatal period [57, 58].

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Table 2. Some properties of human TnT isoforms

Isoforms

Fast skeletal TnT

(UniProt P45378)

Slow skeletal TnT

(UniProt P13805)

Gene TNNT3 TNNT1

Chromosomal

locus

11p15.5 19q13.42

Number

of exons

19 14

Number

of amino acid

residue

245-269 251-278

Molecular

mass (kDa)

29.1-31.8 30-33

Isoelectric

point

5.7-9 5.6-6.1

Tissue

specificity

skeletal muscles skeletal muscles

The function of TnT is to attach the troponin

complex to the actin and regulate the interaction of

the complex with thin filament proteins [59]. The TnT

molecule includes an N-terminal variable region and

conserved central and C-terminal regions (Fig. 2). The

N-terminal domain of TnT is variable not only in ami-

no acid composition but also in length, and diversi-

ty of the forms is the result of alternative splicing.

The conserved portion has been shown to contain

two α-helices, H1 (~162-188 residues of fsTnT) and

H2 (~196-240 residues of fsTnT). The H2 TnT α-helix

forms a supercoiled helix with H2 of TnI and interacts

with TnC via its C-terminus [46, 47, 60-62]. The con-

served portion also includes two tropomyosin bind-

ing sites, Tm1 and Tm2. While localization of Tm1

is determined with sufficient accuracy (~61-99 resi-

dues of fsTnT; ~65-103 residues of ssTnT), the data

on localization of the Tm2 are controversial. Jin and

Chong [60] presumed that Tm2 is located within the

~161-200 residues of fsTnT and ~165-204 residues of

ssTnT, whereas it was suggested in the other works

that Tm2 is formed by the last C-terminal residues of

TnT [63-66]. Studies describing structure of the cardiac

thin filament obtained with cryo-electron microscopy

(cryo-EM) and cryo-electron tomography (cryo-ET)

have suggested that cTnT is capable of binding two

Tm strands simultaneously. With the Tm2 site TnT in-

teracts with the same strand as TnI does, while with

the Tm1 region it binds to the neighboring thread of

tropomyosin [67, 68]. Investigation of the structure

of cardiac [67,  68] and skeletal muscle thin filaments

[69] by cryo-EM and cryo-ET also suggested that fsTnT

is likely to interact with the skeletal muscle protein

nebulin, which, like tropomyosin, binds to actin along

the entire thin filament. Although the exact structure

of skeletal TnT and nebulin has still not been resolved,

it is assumed that the R134-R179 portion of the mouse

fsTnT (R123-R169 in human fsTnT) has two nebulin

binding sites [69].

The TNNT3 gene encoding fsTnT consists of 19 ex-

ons. Eight exons among them can undergo alternative

splicing: namely exons 4, 5, 6, 7, 8, and the fetal exon

in the N-terminal portion of the molecule, as well as

exons 16 and 17 (or exons α and β) encoding a part

of the C-terminus [71]. The fetal exon, located between

the exons 8 and 9, is expressed in embryonic skeletal

muscle only [70,  72]. Expression of the exons 16 and

17 is mutually exclusive: exon 16 is expressed pre-

dominantly in adulthood, while exon 17 is expressed

primarily in embryonic and neonatal muscles [73,  74].

During embryonic development and in postnatal peri-

od, expression of the fsTnT splice variants is changing:

high molecular weight variants are replaced with low

molecular weight splice forms, low isoelectric point

(acidic) variants are replaced with high isoelectric

point (basic) splice forms (see below). The differences

in size and charge of these forms are due to alter-

native splicing of the N-terminal exons that encode

predominantly acidic residues [75].

In the adult rabbit muscle, expression of five

splice variants of fsTnT has been demonstrated by

Western blotting: dominant TnT1f (the longest splice

form, containing all exons except the fetal one), TnT2f,

TnT3f, and to a lesser extent TnT2fa and TnT4f [72,

76, 77]. It is important to note, however, that the dif-

ferences in the splice variant composition have been

observed in various muscle types. In the adult rat, six

splice variants of fsTnT have been identified by mass

spectrometry, and their composition also differed in

various muscle types  [78]. The same method detect-

ed six splice variants of fsTnT in the rhesus macaque

[79]. In the adult humans, only three fsTnT splice vari-

ants (fsTnT III, VI, and VII) have been identified by

the muscle top-down mass spectrometry to date, but

in that study, the authors analyzed only two types of

muscles – tibialis anterior and vastus lateralis – the

former of which contains more slow-twitch fibers, and

so some of the fsTnT splice variants may not have

been detected [80].

The TNNT1 gene encoding ssTnT consists of 14

exons. Unlike fsTnT, diversity of the splice forms

resulting from alternative splicing of ssTnT is low.

Presence of high molecular weight and low molecu-

lar weight splice variants of ssTnT has been shown by

Western blotting for mouse and sheep  [81]. Cloning of

the mouse cDNA demonstrated that the low molecular

weight ssTnT form appears due to alternative splic-

ing deletion of 11 residues of the N-terminal exon  5.

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Fig. 2. Protein sequence alignment of three TnT isoforms: fsTnT, ssTnT and cTnT. Amino acid sequences are derived from

the Uniprot database: fsTnT (TNNT3_HUMAN, P45378-3), ssTnT (TNNT1_HUMAN, P13805-1), cTnT (TNNT2_HUMAN, P45379-6).

The longest adult human splice variants were chosen; for fsTnT and cTnT, we selected those variants that do not contain

the fetal exon. Clustal Omega online software was used to make sequence alignments. The identically positioned amino acid

residues are marked with green. fsTnT and ssTnT exon structures are represented above. Boxes show borders of α-helices

(residues numeration is for fsTnT sequence), TnI and TnC binding site is marked above. Solid line oval indicates tropomy-

osin Tm1 binding site, dashed line ovals indicate putative tropomyosin Tm2 binding sites and potential nebulin binding

site. Possible fsTnT phosphorylation sites are marked above the sequence [46, 47, 60, 63-66, 69, 70].

Two high molecular weight ssTnT splice forms are

distinguished by a single residue deletion in the

exon  6 [81,  82]. Cloning of the human cDNA also re-

vealed three splice variants of ssTnT: a full-length

splice form, a splice form with a C-terminal deletion

of 16 residues derived from alternative splicing of the

exon 12, and a splice form with two deletions, the

aforementioned C-terminal deletion of the exon 12

and an N-terminal deletion of 11 residues (exon  5),

as in the mouse ssTnT [82-84]. At the protein level,

two splice variants of ssTnT (ssTnT II and III) with

deletions were identified in the human skeletal muscle

(vastus lateralis and tibialis anterior) by mass spec-

trometry [80].

Troponin  C. In contrast to TnI and TnT, TnC is

represented in humans by two isoforms: the slow

skeletal/cardiac isoform ss/cTnC and the fast skeletal

isoform fsTnC, which are expressed in the cardiac/

slow-twitch and fast-twitch muscle fibers, respective-

ly (Table 3) [85].

TnC provides the sensitivity of thin filament to

. It changes conformation of the troponin complex

after binding of Ca

, whose concentration increases

due to propagation of the nerve impulse along the

muscle fiber. TnC consists of a short N-terminal region

and four EF-hands (Ca

-binding domains) (Fig.  3).

These four EF-hands are joined in pairs to form N-ter-

minal and C-terminal domains connected by a linker.

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BIOCHEMISTRY (Moscow) Vol. 89 Nos. 12-13 2024

Table 3. Some properties of human TnC isoforms

Isoforms

Fast skeletal TnC

(UniProt P02585)

Slow skeletal/

cardiac TnC

(UniProt P63316)

Gene TNNC2 TNNC1

Chromosomal

locus

20q13.12 3p21.1

Number

of exons

Number

of amino acid

residue

160 161

Molecular

mass (kDa)

18.1 18.4

Isoelectric

point

4.1 4.0

Tissue

specificity

fast-twitch

muscle fibers

slow-twitch

muscle fibers,

cardiomyocytes

The N-terminal EF-hands (EF-hands I and II) have

low affinity, while the C-terminal hands (EF-hands

III and IV) have high affinity for Ca

. TheC-terminal

EF-hands are constantly saturated with Ca

or Mg

ions, whereas the N-terminal ones bind Ca

with

high selectivity only when its concentration increases

during propagation of action potential along the length

of the muscle fiber. In the fsTnC, all four EF-hands can

bind ions whereas in the ss/cTnC, the EF-hand  I has

lost this ability due to a single residue insertion and

substitution of two residues [47, 62, 89].

The 3D protein structure of fsTnC had been de-

ciphered by X-ray crystallography for rabbit fsTnC

[86], for chicken fsTnC [46] alone and as a part of

the troponin complex for four Ca

-binding and two

-binding states. Although the ss/cTnC isoform is

expressed in slow-twitch muscle fibers, most of the

studies on its 3D structure have been focused on its

place and role in the human cardiac troponin com-

plex [47].

The C-terminal globular domain of TnC is a part

of the IT arm that interacts with other troponins in

the supercoiled helix region (H2 TnI and H2 TnT)

and H1 helix of TnI. The N-terminal and C-terminal

Fig. 3. Protein sequence alignment of two TnC isoforms: fsTnC and ss/cTnC. Amino acid sequences are derived from the

Uniprot database: fsTnC (TNNC2_HUMAN, P02585), ss/cTnC (TNNC1_HUMAN, P63316). The identically positioned amino acid

residues are colored yellow. Amino acid residues that participate in binding of Ca

and Mg

ions are marked with bold.

Boxes show the borders of α-helices (N, A-H). Arrows indicate N-terminal and C-terminal domains and EF-hands. Grey

circles indicate the sites of Ca

and Mg

binding, solid line circles stand for a site that is specific to binding Ca

ions

in both TnC isoforms, dashed circle stands for the site of Ca

binding in fsTnC isoform (the first EF hand in ss/cTnC does

not bind ions) [46, 47, 62, 86-89].

BOGOMOLOVA, KATRUKHA2090

BIOCHEMISTRY (Moscow) Vol. 89 Nos. 12-13 2024

globular domains are connected by the central linker

[46, 47]. Although the TnC protein C-domain is always

occupied by ions, its N-domain can switch between

two states: closed conformation in the absence of Ca

or open conformation initiated by Ca

binding to its

EF-hand(s). Binding of Ca

ions induces formation of

a hydrophobic cleft that binds to the switch region of

TnI and moves the adjacent inhibitory region of TnI

away from actin [87, 88].

FUNCTIONAL DIFFERENCES

BETWEEN THE TROPONIN ISOFORMS

Different fibers of striated muscle – fast-twitch,

slow-twitch, and cardiac – vary in their response to

elevated Ca

concentrations. The slow-twitch fibers

have the highest Ca

sensitivity (threshold of Ca

concentration for activation is lower), while the fast-

twitch fibers have the lowest Ca

sensitivity [21, 90,

91]. The highest cooperativity of contraction (Hill co-

efficient) is observed in the fast-twitch fibers and the

lowest in the slow-twitch fibers. Evidence suggests

that these differences could be mediated by the tro-

ponin isoforms expressed in different fiber types. Re-

placement of ss/cTnC with fsTnC in the cardiac fibers

decreases Ca

sensitivity, while substitution of fsTnC

with ss/cTnC in the fast skeletal fibers increases it [90].

Replacement of the whole troponin complex in the

fast-twitch skeletal fibers with cardiac troponins re-

sults in the increased sensitivity to Ca

and decreased

cooperativity of contraction [92].

Decrease of intracellular pH (acidosis) leads to

contractile dysfunction in all types of striated mus-

cle and involves reduced Ca

sensitivity. In the heart

muscle, reduction in intracellular pH to 6.5 and below

is observed during prolonged ischemia and is caused

by accumulation of metabolic products, including lac-

tate [93, 94]. In the skeletal muscle, decrease of pH is

observed during exhaustive exercise, and although it

does affect skeletal muscle, the effect is not as signif-

icant as in the heart muscle. In the human skeletal

muscle, pH drops down to ~6.5 after high-intensity ex-

ercise, with typical average values of ~6.8-6.9 [95-97].

As with the response to Ca

, the extent of pH decrease

varies between the muscle fiber types, being most pro-

nounced in the cardiac muscle and least prominent in

the slow-twitch fibers [90, 98]. Experiments with iso-

lated muscle fibers have shown that when pH drops

from 7.0 to 6.5, the smallest decrease in Ca

sensi-

tivity is observed in the slow-twitch muscle fibers:

change in Ca

sensitivity is greater in the fast-twitch

and cardiac fibers, and it is approximately equal in

amplitude [90]. Moreover, further reduction in pH to

6.2 results in the strongest drop in Ca

sensitivity in

the cardiac fibers, but not in the skeletal muscle.

Sensitivity of the contractile apparatus to acidosis

is largely determined by the troponin complex, and

magnitude of this effect is determined, among other

things, by different troponin isoforms [99]. In par-

ticular, experiments performed with isolated mus-

cle fibers have shown that replacing of fsTnC with

ss/cTnC in the fast-twitch fibers leads to an increase

in pH sensitivity, and, conversely, replacing of ss/cTnC

with fsTnC in the slow-twitch fibers leads to a slight

decrease in pH sensitivity [100]. The latter effect is

even more pronounced when ss/cTnC is replaced with

fsTnC in the cardiac fibers  [99]. Thus, the ss/cTnC in-

creases pH-dependence and fsTnC decreasesit. Itwas

shown in another study that combinations of TnC

and TnI affect pH sensitivity  [90]. The authors isolat-

ed cardiac fibers and made substitutions of TnI and

TnC together and separately. In the fibers with cTnI

or fsTnI, ss/cTnC accounted for the increased pH sen-

sitivity compared to fsTnC. Moreover, replacement

of cTnI with ssTnI resulted in the reduced pH sensi-

tivity regardless of which TnC isoform was present.

Thus, the authors concluded that TnC is responsible

for pH sensitivity in the fast-twitch or cardiac fibers,

while TnI performs that function in the slow-twitch

fibers [90]. These findings are in good agreement with

the fact that the neonatal cardiomyocytes expressing

ssTnI are less sensitive to acidosis than the adult car-

diomyocytes expressing cTnI [101]. This effect has

been reproduced when cTnI was replaced with ssTnI

in the adult transgenic mice: sensitivity to acidosis

was reduced [102]. More recent studies have shown

that the effect could be associated with the residue

His131 (His131 in fsTnI, His132 in ssTnI) that is pres-

ent in ssTnI and fsTnI, but is replaced with Ala163

in cTnI. This residue is localized in the switch region

of TnI and participates in the interaction with TnC.

It has been shown that at lower pH, electrostatic in-

teraction between the His131 and Glu15 and Glu19

of TnC stabilizes conformation of the troponin com-

plex, leading to activation of the muscle contraction

[103-106].

Three isoforms of TnT differ in their isoelectric

points, with the adult fsTnT being the most basic iso-

form. Moreover, there is an acidic-to-basic fsTnT splice

variant shift that ensures expression of the basic splice

forms in the adult muscle. These differences are due

to the sequence variation in the TnT N-terminal re-

gion. There is a unique acidic fsTnT splice variant that

is synthesized in some of the adult chicken muscle

types  [107]. These muscles show lower sensitivity to

as well as higher tolerance to acidosis compared

to the other muscle types that express basic fsTnT

splice forms [108]. These findings are consistent with

the fact that expression of fsTnT in the transgenic

mouse hearts lowers tolerance of the cardiac muscle

to acidosis [109].

TROPONINS AND SKELETAL MUSCLE PATHOLOGIES 2091

BIOCHEMISTRY (Moscow) Vol. 89 Nos. 12-13 2024

SKELETAL TROPONINS AS BIOMARKERS

OF SKELETAL MUSCLE DAMAGE

One of the most sensitive and specific approaches

to detect and measure concentration of a biomarker is

the sandwich immunoassay [110]. In most cases, this

method is based on the use of two antibodies that rec-

ognize epitopes on the antigen surface, one of which

is an immobilized capture antibody and the other is

a detection antibody, conjugated with a label. To our

knowledge, there have been no registered immuno-

assays to detect skeletal TnI isoforms and all existing

studies have employed “research use only” assays.

Concentrations of the skeletal TnI isoforms in the

healthy individuals have been determined in a num-

ber of studies. The mean values of basal TnI concen-

trations have ranged between 1.74  ±  0.27  ng/mL and

2.5  ±  0.9  ng/mL when measured by the same method

in different studies [111-114]. In another study, con-

centrations of the skeletal TnI isoforms in the healthy

individuals were below the limit of detection (LOD)

(LOD  =  1.2  ng/mL)  [115]. In the studies where fsTnI

concentration was determined, its basal concentra-

tion was below the LOD of the detection method

(2.4  ng/mL) [9, 116]. Although further clarification is

needed, these data suggest that the median blood lev-

els of skeletal TnI isoforms in the healthy cohort are

below 1-2 ng/mL.

In cases of skeletal muscle damage, integrity of

the muscle fiber membrane is compromised, and in-

tracellular proteins are released into the bloodstream.

It has been shown that the circulating levels of skel-

etal TnI isoforms increase during skeletal muscle im-

pairment of various etiologies. In particular, the skele-

tal TnI isoform concentrations rise in the patients with

rhabdomyolysis [117, 118]; as well as after intensive

physical exercises: for example, after triathlon [113],

running [119], eccentric contractions [26, 120-122];

and in orthopedic and soft tissue injuries, including

those resulting from surgical operations [114, 119].

Elevated concentrations of the skeletal TnI isoforms

have also been observed in the patients with inflam-

matory myopathies such as polymyositis and dermato-

myositis [115, 119, 123]. Concentration of the skeletal

TnI isoforms correlates with the clinical picture in

muscular dystrophies (Duchenne, Becker, and limb-

girdle muscular dystrophy), and therefore it is pos-

sible to use this parameter to assess severity of the

disease and monitor response to the therapy [9].

One of the potential applications of skeletal TnI

isoforms as a biomarker is in determination of myo-

toxicity of drugs, where muscle fibers are destroyed

and troponin is released into the bloodstream [36,

37, 124, 125]. Feasibility of this application has been

demonstrated in rats as well as using 3D human mus-

cle tissue in  vitro [126, 127].

Presence of the TnI isoforms specific for different

types of skeletal fibers provides additional diagnostic

possibilities. As described in the previous section, a

number of skeletal muscle injuries/diseases could af-

fect a particular type of muscle fiber. For example,

eccentric muscle contractions damage predominant-

ly fast-twitch type fibers, and it is fsTnI rather than

ssTnI that is released into the bloodstream [26, 121,

122]. The increased level of fsTnI, but not the level of

ssTnI concentrations have also been shown for some

muscular dystrophies [128].

Maximum concentration of the skeletal TnI iso-

forms reached values of 500  ng/mL 6  h after complet-

ing a triathlon (4  km of swimming, 120  km bike-rid-

ing, 30  km running), with a median concentration of

62.2  ±  139  ng/mL [113]. Also, high skeletal TnI isoform

concentrations were reached 6 h after downhill run-

ning: the median value was 27.3  ng/mL (8.5-43  ng/mL

interquartile range), while lower concentrations were

found 6  h after level running: 6.6  ng/ml (3.7-9  ng/mL)

and after eccentric contractions of the quadriceps fem-

oris muscle: 6.8  ng/ml (3.1-14.9  ng/mL) [26]. Mean con-

centration levels of the skeletal TnI isoforms within

24  h of injury were: 15.3  ±  2.4  ng/mL after orthopedic

injury and 10.4  ±  1.8  ng/mL after soft tissue injury

[114]. Maximum concentration of the skeletal TnI iso-

forms in the patients with inflammatory myopathies

was 516  ng/mL and the median value was 8.6  ng/mL

(3.2-33.5  ng/mL interquartile range) [115].

We were not able to find any studies devoted to

the utilization of skeletal TnT isoforms as possible bio-

markers of muscle damage. Nevertheless, we assume

that, like cardiac troponinsI and T, which are markers

of cardiac muscle damage, the skeletal TnT isoforms,

along with the skeletal TnI isoforms, may also be a

potential marker for diagnosing skeletal muscle injury.

fsTnC has also been proposed as a biomarker

of skeletal muscle deterioration [129]. However, TnC

recognition would have less specificity than detection

of the skeletal TnI or TnT isoforms because fsTnC is

expressed in the fast-twitch muscle fibers only, while

c/ssTnC is expressed in the slow-twitch fibers and car-

diomyocytes.

FACTORS INFLUENCING IMMUNOCHEMICAL

DETECTION OF SKELETAL TROPONINS

Various factors could affect interaction of anti-

bodies with skeletal troponins. These include homol-

ogy with other troponin isoforms, presence of splice

variants, post-translational modifications, proteolysis,

autoantibodies binding, and shielding of epitopes by

proteins of the troponin complex. Such effects could

significantly distort the results of the measurements

and should be taken into account when studying

BOGOMOLOVA, KATRUKHA2092

BIOCHEMISTRY (Moscow) Vol. 89 Nos. 12-13 2024

the biochemical properties of the troponins and de-

veloping immunochemical systems.

Homology of isoforms. The degree of similar-

ity between the isoforms of human TnI and TnT is

~60% for the both TnI and TnT isoforms (when com-

paring the longest splice variants expressed in the

adults: P45378-3 for fsTnT, P13805-1 for ssTnT, and

P45379-6 for cTnT) (Figs. 1 and 2). Therefore, one of

the intricacies of immunochemical detection of skele-

tal troponins is cross-reactivity of antibodies with the

cardiac isoform of the protein or with the second skel-

etal isoform (in the case of differential detection of

slow- or fast-twitch skeletal troponin), and this could

reduce specificity of detection of the target biomarker.

High degree of troponin homology significantly reduc-

es the number of epitopes to which antibodies could

be generated for the specific detection of individual

protein isoforms. The most conserved sites for both

TnI and TnT are located in the C-terminal parts of

these proteins, while the most variable sites are in

their N-terminal portions.

Alternative splicing of TnT. As described above,

the skeletal TnT isoforms, especially fsTnT, are repre-

sented in the human muscle by several splice variants.

To develop immunodetection test systems that would

recognize all possible splice forms of fsTnT, antibod-

ies specific to the domains encoded by exons 1-3 and

9-15 should be utilized. For the fsTnT, antibodies spe-

cific to the N-terminal residues 11-38 as well as the

C-terminal region (residues 231-244 that may differ

between splice variants) should be avoided (residues

numbering is indicated for the sequence accession

number P45378-3). In the case of ssTnT, antibodies

recognizing residues 25-35 and 205-220 (numbering

is for the longest splice variant; sequence accession

number P13805-1), should be avoided because these

sequences may differ between the splice variants.

TnI and TnT phosphorylation. Phosphorylation

is a modification that changes charge of a protein and

could have significant effect on the antibody-antigen

interaction.

TnI phosphorylation. The rabbit fsTnI could be

phosphorylated by phosphorylase kinase, 3′,5′-cAMP-de-

pendent protein kinase, Ca

-phospholipid-dependent

protein kinase, and the human fsTnI by AMP-acti-

vated protein kinase in vitro [130-134]. The residues

that undergo phosphorylation have been identified as

Thr12 and Ser118 for the rabbit fsTnI and Ser118 for

the human protein [131, 132, 134]. Presence of the

Thr12 residue in the human fsTnI sequence as well

as in the rabbit fsTnI provides the possibility of its

phosphorylation in the human protein as well. It has

been shown that phosphorylation in  vitro is inhibited

by interaction with TnC, and this is consistent with

the data on the structure of troponin complex: both

of these residues are located in the sites of interac-

tion with TnC [46, 130]. Phosphorylation of ssTnI has

not been described in vitro, but there is one residue

homologous to the fsTnI phosphorylation site in the

ssTnI sequence, namely Ser118 (ssTnI) (Fig. 1).

The fsTnI isolated from the rabbit skeletal mus-

cle is present in the partially phosphorylated form,

and the degree of phosphorylation could depend on

the method of protein isolation [130, 131, 135]. The

fsTnI from the human muscle extract (vastus lateralis

and tibialis anterior) and rat muscle extract (7 muscle

types) analyzed by mass spectrometry were found to

be in the non-phosphorylated form, whereas the ssTnI

was present in both phosphorylated and non-phos-

phorylated forms [78, 80]. No phosphorylated forms of

the skeletal TnI isoforms were detected by mass spec-

trometry of the rhesus macaque muscle extract [79].

Based on the above data, it could be expected that

the proportion of phosphorylated forms of skeletal TnI

isoforms in blood would be negligible and, therefore,

would not affect their recognition by antibodies.

TnT phosphorylation. Using in vitro experimen-

tation, the fsTnT isolated from the rabbit skeletal

muscle has been shown to be a substrate for various

kinases including phosphorylase kinase, 3′,5′-cAMP-de-

pendent protein kinase, casein kinase2, and Ca

-phos-

pholipid-dependent protein kinase [59, 130, 132, 133,

136-140]. Three phosphorylation sites have been iden-

tified for the rabbit fsTnT: Ser2, Ser152, and Ser159

(numbers of residues are indicated for Uniprot acces-

sion number P45372-3). Ser2 is also represented in

the human fsTnT, while the Ser152 and Ser159 resi-

dues are in composition of the Ala-Leu-Ser-Ser-SerP-

Met-Gly-Ala-Asn-Tyr-Ser-SerP-Tyr sequence, which in

the rabbit fsTnT is completely consistent with the

human protein. This suggests the possibility of phos-

phorylation of these residues in the human fsTnT

as well [132].

Meanwhile, in a series of in  vivo studies, fsTnT

isolated from the rabbit skeletal muscle has been

shown to be present in a solely monophosphorylated

form [130, 132]. Moreover, phosphorylation occurred

at only one of the three residues identified in  vitro:

Ser2. These results are consistent with the mass spec-

trometry data for the proteins extracted from the

muscle tissue of various species. For human, rat, and

rhesus macaque fsTnT samples isolated from several

muscle types, unphosphorylated and monophosphor-

ylated forms (present in different splice variants) of

fsTnT were identified by mass spectrometry [78-80]

and Ser2 was the only site of phosphorylation deter-

mined in vivo.

There are no data on phosphorylation of the ssTnT

in  vitro. However, ssTnT contains Ser2 homologous to

the Ser2 in the fsTnT and cTnT, for which phosphor-

ylation has been shown both in  vitro and in  vivo [141,

142]. High probability of the ssTnT phosphorylation

TROPONINS AND SKELETAL MUSCLE PATHOLOGIES 2093

BIOCHEMISTRY (Moscow) Vol. 89 Nos. 12-13 2024

at Ser2 in the human muscle tissue was confirmed

by mass spectrometry [80]. Using this method, it was

found that in the human and rat skeletal muscle,

ssTnT was presented in a predominantly phosphory-

lated form, and in the rhesus macaque muscle in both

phosphorylated and non-phosphorylated forms [78-80].

It could be assumed that fsTnT and ssTnT are re-

leased into the bloodstream in a partially phosphory-

lated form, but phosphorylation site is located in the

terminal part of the protein and, therefore, could be

presumed not to affect immunochemical detection by

antibodies.

TnI glutathionylation. Lamb et al. showed in a

series of studies on isolated rat and human muscle

fibers that fsTnI, but not ssTnI, could undergo gluta-

thionylation [143-145]. This modification occurred at

one of the cysteine residues, Cys134. This is consistent

with the mass spectrometry data for the protein: two

proteoforms of fsTnI, with and without glutathione,

were detected in the skeletal muscles of rhesus ma-

caque and rat, although, abundance of the glutathio-

nylated form was quite low [78, 79].

TnI and TnT proteolysis. N-terminal and C-ter-

minal proteolysis has been demonstrated for cTnI:

various proteolyzed forms of cTnI were observed in

both necrotic cardiac tissue and in blood [146, 147].

cTnI was found to be a substrate of a number of pro-

teases: metalloproteinase-2, calpain 1 (µ-calpain) and

calpain  2 (m-calpain) [148-151]. Studies of the cTnI

proteolysis in blood of the patients with myocardial

infarction showed presence of at least 11 proteolyt-

ic fragments of different lengths [147, 152]. Due to

the sequence homology to cTnI, proteolysis of skeletal

TnI isoforms is also possible. The proteolyzed fsTnI

and ssTnI and their fragments have been identified

in the blood of patients, and amount of these frag-

ments in the blood varies depending on the type of

injury/disease [118]. Based on these results, the au-

thors suggested that proteolysis occurs directly in the

tissue and that such proteolyzed forms are released

into the bloodstream. Further studies on proteolysis of

the skeletal TnI isoforms and localization of the sites

of proteolysis are needed.

cTnT also undergoes proteolysis. In the apoptot-

ic cardiomyocytes, N-terminal cleavage of cTnT by

caspase-3 with formation of a 25-kDa fragment has

been demonstrated [153]. cTnT is also prone to limited

N-terminal proteolysis by calpain 1 (µ-calpain) during

myocardial ischemia-reperfusion [148, 154]. In the

blood of patients with myocardial infarction, at least

23 cTnT fragments of different lengths and carrying

several sites of proteolysis in the N-terminal, central,

and C-terminal parts of the molecule were found [155].

Similar studies have not yet been performed for the

skeletal TnT isoforms. For fsTnT, proteolysis has been

demonstrated only in the muscles after vigorous exer-

cise [156]. Further investigations are required to iden-

tify the forms of skeletal TnT isoforms present in the

bloodstream and to localize their sites of proteolysis.

Shielding of epitopes in the troponin complex.

cTnI and cTnT are present in the blood of patients

with myocardial infarction as part of a ternary tropo-

nin complex (TnI‒TnT‒TnC), a binary troponin com-

plex (TnI‒TnC), and as proteolytic fragments of TnT

[152, 157, 158]. Although there is no experimental data

describing the form in which skeletal troponins are

released into the blood, it can be assumed that they

would also be present in the samples as complexes.

For immunochemical detection, it is important to use

antibodies that recognize troponin epitopes that are

present in all forms that exist in blood. The skeletal

TnT isoforms could be covered and shielded from de-

tection by TnI and TnC at their binding sites, limited

to approximately 197-241 residues for fsTnT. In the

case of the skeletal TnI isoforms, a significant portion

of the molecules could be shielded from detection at

binding sites located at approximately 2-40, 50-106,

and 116-131 residues for fsTnI, and 2-40, 51-107, and

117-132 residues for ssTnI. Thus, small regions in the

central and C-terminal part of TnI molecule could

remain accessible to antibodies. In order to uncov-

er the epitopes shielded by other troponins in the

central part of TnI, an alternative method could be

to dissociate the troponin complex using EDTA [115,

116, 157].

Autoantibodies binding. One of the factors in-

fluencing the immunochemical detection of cardiac

troponins is the presence of autoantibodies that in-

teract with the analyzed protein in the blood. They

might interfere with the antibodies in composition of

the immunochemical test systems for detection of the

protein and lead to false negative results. It has been

demonstrated that approximately 10% of the healthy

individuals have autoantibodies to cTnI in their blood

[159]. These immunoglobulins have been shown to be

primarily specific for the conformational epitopes that

are formed when TnI and TnT combine into a com-

plex [160]. There are no studies on the presence of

autoantibodies interacting with the skeletal isoforms

of TnI; however, considering high degree of homolo-

gy, it can be assumed that they could also be present

in the blood of some patients.

Finally, we examined the sites that could be most

susceptible to various factors interfering with recogni-

tion of troponins by antibodies. The C-terminal regions

of TnI could be associated with the cross-reactivity

displayed by antibodies to other TnI isoforms, while

the central parts of the molecule could be shielded by

TnC or TnT. The effect of posttranslational modifica-

tions such as phosphorylation and glutathionylation is

considered to be negligible, while the role and extent

of proteolysis and influence of autoantibodies needs

BOGOMOLOVA, KATRUKHA2094

BIOCHEMISTRY (Moscow) Vol. 89 Nos. 12-13 2024

further investigation. Taking into account the above

factors, the most promising sites for the detection of

skeletal TnI isoforms are central parts of the mole-

cule, especially those that are not shielded by other

troponins.

In the case of TnT, the N-terminal (11-38 residues

for fsTnT, 25-35 residues for ssTnT) and C-terminal

(231-244 residues for fsTnT and 205-220 residues for

ssTnT) parts of the molecule are alternatively spliced,

while the C-terminal parts (~197-241 residues for

fsTnT) could be screened by TnI and TnC. The effect of

phosphorylation is considered to be negligible, while

the role and extent of N- and C-terminal proteolysis

needs further investigation. Therefore, central parts

of the skeletal TnT isoforms could be considered as

the most promising sites for their immunochemical

detection.

MUTATIONS IN TROPONINS AS CAUSES

OF SKELETAL MUSCLE PATHOLOGIES

The troponin complex is of high importance for

regulation of muscle contraction. Mutations could lead

to the changes in protein structure and functionality.

To date, a number of mutations have been identified

that are associated with the development of such skel-

etal muscle diseases as arthrogryposis and congenital

myopathies.

Mutations in the skeletal TnI genes. Mutations

in the TNNI2 gene (fsTnI) have been demonstrat-

ed to be one of the causes of distal arthrogryposis.

This is an autosomal dominant disease, in which

joints are affected and mobility of distal limbs is re-

duced. The TNNI2 mutations associated with distal

arthrogryposis are present in the following locations

within the C-terminal region of the molecule: R156X

[leading to expression of a truncated form of fsTnI

(1-156)], R162G, R162K, I165F, E167X, K168E, R174Q,

R174W, K175N, K175X, K176X, F178C, and F178L

[161-172].

For ssTnI (TNNI1), two pathogenic mutations have

been discovered to date [170,  173]. One is located in

the same region as mutations in the fsTnI and is as-

sociated with the development of proximal arthrogry-

posis, an autosomal dominant disease in which prox-

imal joints are affected. This mutation, K175X, results

in the expression of a truncated form of ssTnI [170].

Mutation in the N-terminal region of ssTnI, R37C, is

associated with expression of ssTnI in the heart for up

to two years after birth [173] that increases the risk

of sudden infant death.

Mutations in the skeletal TnT genes. Mutations

in the TNNT3 gene (fsTnT) lead to the development

of distal arthrogryposis. For TNNT3, several substi-

tutions affecting the same residue have been found:

R66C, R66H, and R66S [171, 174-177]

. Itis also worth

highlighting the TNNT3 (fsTnT) mutation, which caus-

es two diseases simultaneously: the aforementioned

distal arthrogryposis and the nemaline myopathy that

affects fast-twitch muscle fibers [178]. It is a muta-

tion in an intron that leads to the impaired splicing

and decreased expression of fsTnT, with compensa-

tory hypertrophy of slow-twitch muscle fibers being

observed.

Deletions in the TNNT1 gene (ssTnT) are associ-

ated with nemaline myopathy inherited in an autoso-

mal recessive pattern. This disease is manifested in

early childhood as a respiratory failure, slow-twitch

muscle fiber atrophy, and compensatory hypertrophy

of fast-twitch muscle fibers. Ultimately, nemaline my-

opathy leads to death in childhood from the respira-

tory failure. E180X (expression of the truncated form

of ssTnT 1-179), S108X (expression of the truncated

form of ssTnT 1-107), deletion of the exons 8 and 14,

and expression of the truncated form of ssTnT (1-203)

have been found [179-182]. These deletions affect tro-

pomyosin binding sites as well as TnI and TnC binding

sites.

Absence of the troponin and tropomyosin bind-

ing sites in ssTnT after E180X deletion impedes for-

mation of the troponin complex and its incorporation

into thin filaments [183]. While the ssTnT (1-179)

mRNA is still detectable in muscle fibers, the ssTnT

(1-179) protein molecule degrades rapidly and can no

longer be detected [184]. This fact explains recessive

nature of the mutation: one copy of the gene is suf-

ficient for expression of the full-length ssTnT and its

incorporation into the troponin complex, while ex-

pression of the mutant ssTnT (1-179) protein is not

cytotoxic, as the truncated ssTnT is immediately de-

graded [185].

Mutations in the TnC genes. Mutations in the

TNNC2 gene (fsTnC) are associated with congenital

myopathy inherited in an autosomal dominant pat-

tern. These mutations comprise the D34Y and M79Y

substitutions, which are located in the EF-hand  I and

near EF-hand  II, respectively. They affect Ca

bind-

ing and interaction with TnI switch region, so there is

reduced Ca

sensitivity in the presence of these mu-

tations [186, 187].

Since ss/cTnC is expressed in the slow-twitch skel-

etal muscle as well as in the cardiac muscle, mutations

in TNNC1 (ss/cTnS) are associated with cardiomy-

To specify this mutation, the authors of the studies used the fsTnT sequence corresponding to UniProt accession number

P45378-2, which is three residues shorter than the P45378-3 sequence used in this review. Therefore, this substitution

appears in the articles as R63.

TROPONINS AND SKELETAL MUSCLE PATHOLOGIES 2095

BIOCHEMISTRY (Moscow) Vol. 89 Nos. 12-13 2024

opathies and cardiac dysfunction. These genetic alter-

ations include missense mutations A8V, L29Q, A31S,

C84Y, E134D, and D145E, and frameshift mutations

Q122AfsX30 [188-191]. Along with cardiomyopathies,

some of the patients carrying these mutations also

exhibit skeletal muscle abnormalities, but more re-

search is needed to understand which mutations af-

fect skeletal muscle and exact mechanism involved in

the process [187, 192]. It was shown in the in vitro

experiments that all of the abovementioned mutations

except for C84Y had no effect on phenotype and func-

tion of the slow-twitch skeletal muscle fibers, while

the C84Y mutation increased Ca

sensitivity, as it does

in the cardiac muscle fibers. These differences in the

effects of the ss/cTnC mutations on slow-twitch and

cardiac muscle fibers could be due to the fact that in

the slow-twitch muscle fibers ss/cTnC forms complex

with the slow-twitch skeletal but not cardiac isoforms

of TnT and TnI [193].

TROPONINS AS DRUG TARGETS

In addition to being the source of pathologies,

skeletal troponins could also serve as drug targets

used in treatment of skeletal muscle diseases. These

include peripheral motor neuropathies– amyotrophic

lateral sclerosis (ALS), spinal muscle atrophy, Charcot–

Marie–Tooth disease – in which the motor neurons

are damaged, and nerve impulse transmission is

impaired, and also myasthenia gravis, in which the

neuromuscular junction is impaired. Disruption of

the skeletal muscle innervation ultimately leads to

muscle weakness, disability, and high mortality. One

of the therapeutic approaches for these pathologies is

to increase sensitivity of the muscle fibers to Ca

, so

that sufficient muscle activation is achieved even at

low Ca

concentrations. A molecule from the group

of first fast skeletal troponin activators, CK-2017357

(tirasemtiv), selectively interacts with the fast skeletal

troponin complex: binding occurs in the hydrophobic

pocket formed by the N-terminal domain of fsTnC

and the switch region of fsTnI [194]. Tirasemtiv bind-

ing has been shown to increase the degree of affin-

ity of low-affinity Ca

binding sites on fsTnC, there-

by increasing sensitivity of the fsTnC to Ca

[195].

Tirasemtiv has shown promising results in the patients

with ALS, patients with myasthenia gravis, and studies

even reached phase III clinical trials, but were halted

due to the discovery of side effects [196-202]. Today,

other drugs from the group of fast skeletal muscle

troponin activators, namely CK-2127107, CK-2066260,

and reldesemtiv are under development [203-206].

Reldesemtiv, a second-generation molecule that was

derived by optimization of tirasemtiv and has similar

mechanism of action, has been most successful [207,

208]. Reldesemtiv has reached phase II clinical trials

in spinal muscular atrophy and phaseIII in ALS, but,

unfortunately, trials on the patients with ALS were

stopped due to the lack of therapeutic success [206,

209, 210]. Although the ss/cTnC is expressed in both

slow-twitch skeletal and cardiac muscles, regulators of

the ss/cTnC Ca

sensitivity have been studied primar-

ily in the context of modulating heart function. These

drug molecules regulate Ca

sensitivity of muscle con-

traction by adjusting the Ca

-binding affinity of the

N-terminal domain of ss/cTnC. One of the most wide-

ly studied calcium sensitizers, levosimendan, binds

to the hydrophobic pocket formed by the N-terminal

domain of ss/cTnC and the switch region of cTnI– in

a manner similar to that of tirasemtiv [211]. Levosi-

mendan is approved for use in some countries, and

its analogs are intended to be used for treatment of

the heart diseases associated with depressed cardiac

function.

In contrast to the above drug molecules, the tro-

ponin inhibitor W7 acts by decreasing Ca

sensitivity,

and it could be used to treat contractile dysfunction

arising from the alkalosis-induced and inherited car-

diomyopathies [212, 213]. W7 also binds to the hy-

drophobic pocket between the N-terminal domain of

ss/cTnC and the cTnI switch region, but, unlike levosi-

mendan, it shifts the cTnI switch region from its bind-

ing site, which leads to the reduced Ca

sensitivity

[213, 214].

CONCLUSION

Skeletal muscle dysfunction has a negative impact

on the whole-body functioning and could be caused

by mechanical muscle injury, myopathies, and other

diseases accompanied by muscle atrophy, as well as by

rhabdomyolysis. Determination of the concentration

of protein biomarkers released into the bloodstream

when muscle fibers are damaged is a convenient

method of diagnosing and monitoring such pathol-

ogies. Skeletal troponins are potential candidates to

be used as biomarkers of skeletal muscle dysfunction

including various myopathies, traumas, and injuries

caused by vigorous physical exercise, as well as for

assessing severity of muscular dystrophies and deter-

mining myotoxicity of the drugs.

Structure and properties of troponins could af-

fect accuracy of their antibody-based detection in hu-

man blood. Main factors influencing immunochem-

ical detection of these proteins in clinical samples

are isoform homology, alternative splicing of TnT,

post-translational modifications (phosphorylation and

glutathionylation), proteolysis, shielding of TnI and

TnT epitopes within the complex, and autoantibodies

binding. Therefore, a careful selection of monoclonal

BOGOMOLOVA, KATRUKHA2096

BIOCHEMISTRY (Moscow) Vol. 89 Nos. 12-13 2024

antibodies is required for the development of immu-

noassays capable of reliable quantitative detection of

these proteins.

Certain mutations in skeletal troponins are associ-

ated with the development of arthrogryposis and con-

genital myopathies. Furthermore, skeletal troponins

could be used as drug targets for treatment of muscle

diseases such as peripheral motor neuropathies and

myasthenia gravis.

Open access. This article is licensed under a Cre-

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which permits use, sharing, adaptation, distribution,

and reproduction in any medium or format, as long

as you give appropriate credit to the original au-

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Commons license, and indicate if changes were made.

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are included in the article’s Creative Commons license,

unless indicated otherwise in a credit line to the mate-

rial. If material is not included in the article’s Creative

Commons license and your intended use is not permit-

ted by statutory regulation or exceeds the permitted

use, you will need to obtain permission directly from

the copyright holder. To view a copy of this license,

visit https://creativecommons.org/licenses/by/4.0/.

Contributions. A.P.B. and I.A.K. collected and an-

alyzed the data, prepared illustrations, wrote the man-

uscript, edited the manuscript; I.A.K. conceived and

supervised the study.

Funding. This work was supported by ongoing in-

stitutional funding. No additional grants to carry out

or direct this particular research were obtained.

Ethics declarations. This work does not con-

tain any studies involving human and animal sub-

jects. Theauthors of this work declare that they have

noconflicts of interest.

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