Focus on cardiac troponin complex: From gene expression to cardiomyopathy

The cardiac troponin complex (cTn) is a regulatory component of sarcomere. cTn consists of three subunits: cardiac troponin C (cTnC), which confers Ca2+ sensitivity to muscle; cTnI, which inhibits the interaction of cross-bridge of myosin with thin filament during diastole; and cTnT, which has multiple roles in sarcomere, such as promoting the link between the cTnI-cTnC complex and tropomyosin within the thin filament and influencing Ca2+ sensitivity of cTn and force development during contraction. Conditions that interfere with interactions within cTn and/or other thin filament proteins can be key factors in the regulation of cardiac contraction. These conditions include alterations in myofilament Ca2+ sensitivity, direct changes in cTn function, and triggering downstream events that lead to adverse cardiac remodeling and impairment of heart function. This review describes gene expression and post-translational modifications of cTn as well as the conditions that can adversely affect the delicate balance among the components of cTn, thereby promoting contractile dysfunction.


Introduction
Cardiac muscle is composed of a contractile apparatus arranged in a pattern that is similar but not identical to the arrangement observed in skeletal muscle.Thin filaments of actin, together with structural and regulatory proteins, interdigitate with thick filaments composed of myosin to form the sarcomere, the functional unit of the contractile apparatus.The "sliding filament theory" proposes that the hydrolysis of adenosine triphosphate (ATP) by the myosin head powers the strong attachment between myosin and actin followed by sliding of the thin filament to generate contraction. 1 In addition to ATP, which provides the energy necessary for muscle contraction, Ca 2þ is the essential element for sarcomere function.The isometric tension of cardiac muscle fibers is low in the absence of Ca 2þ but achieves 50% of the maximum tension in the presence of w2 Â 10 6 M À1 Ca 2þ . 2,3At the beginning of 1900, it was observed in a frog animal model, that the contraction of the isolated heart was blocked in the absence of Ca 2þ .4e6 Around 1930, it was suggested that, in addition to the extracellular space, the Ca 2þ is intracellularly stored in the "cortex of the muscle fiber", and it is quickly released after stimulation, thus regulating muscle contraction. 7Later, in 1961, the structure of the sarcoplasmic reticulum was evidenced and the classical "two-state" theory of the contraction and relaxation of the striated muscle was proposed. 8Generally, in the classical two-state model of contraction, the increase of cytoplasmic Ca 2þ triggered by the activation of the action potential induces the release of further Ca 2þ from the sarcoplasmic reticulum (Ca 2þ -induced Ca 2þ release).Increased Ca 2þ levels promote the binding of Ca 2þ to the "regulatory domain" of the sarcomere, the cardiac troponin complex (cTn), resulting in a cascade of structural changes within the thin filaments, 9 including the shift of tropomyosin across the surface of the actin filament and the opening of the binding sites for myosin heads.14e18 In the nineties, the two-step activation/relaxation model was extended to the "three-state model". 19In this model, the states are in dynamic equilibrium and are regulated by the cTn binding to actin, the Ca 2þ binding to cTn, the myosin binding to actin, the cTn-tropomyosin-actin interaction, and the ATP hydrolysis by myosin (Fig. 1).20e22 Recently, the three-state model was confirmed by cryogenic-electron microscopy (cryo-EM).23e26 In striated skeletal and cardiac muscles, the thin filament is composed of a repeating pattern of actin, Tm, and cTn in 7:1:1 stoichiometry.Comparison between Tn subunits of cardiac muscles with those of striated skeletal muscles is reported in Table S1e4 of Supplementary Material.The cTn has three subunits: the cardiac troponin C (cTnC), the subunit that confers Ca 2þ sensitivity to muscle; the cTnI, which inhibits the actomyosin cross-bridge formation during diastole; and the cTnT, the tropomyosin (Tm) binding subunit. 27 list of genes encoding cTn subunits is reported in Table 1.The X-ray crystallography and the cryo-EM of native or reconstituted thin filaments obtained by cardiac myocytes were essential to establish the structure of the single domains of each cardiac troponin and the orientation of cTn on Tm and actin filament.To date, the cTn crystal structure consists of total TnC and fragments of cTnI and cTnT, covering just 65% of the total cTn lengths (Fig. 2). 26,28The 3D map of the thin filament suggested that cTn could be highly elongated over the seven actin subunits.In this case, the upper four actin subunits are bound by the C-terminal domain of cTnI followed by the cTn "core domain" (cTnC, Nterminal domain of cTnI, and C-terminal domain of cTnT), whilst the bottom three actin subunits of the opposite strand are bound by N-terminal domain of cTnT. 26,29mong the cTn subunits, cTnI and cTnT are well-known for their pivotal role as biomarkers of myocardial injury.High circulating levels of cTnI and cTnT are commonly associated with diseased states such as acute myocardial Figure 1 Structure of human cardiac thin filament in the calcium free state (A) and in the calcium bound state (B).The monomers of actin are in red and the tropomyosins are in grey; the blue structure represents cTnC, the green element is cTnI, and the yellow protein is cTnT.Figure modified from www.ncbi.nlm.nih.gov/Structure/pdb/6KN8. infarction, coronary microembolization, 30,31 aortic stenosis, 32 heart failure (HF), myopericarditis, 33 and atrial fibrillation, or in healthy subjects, in the condition of prolonged exercise.Moreover, a reduction in cTnI and cTnT circulating levels can reflect the efficacies of cardioprotective and prognostic procedures as remote ischaemic preconditioning. 34However, the relationship between cTn and myocardial disease extends beyond the cell damage and release of proteins in the blood.Mutations of cTn genes, 35,36 the re-expression of fetal cTn variants after birth, 37 or chronic post-translational modification (PTM) of cTn proteins, 27 can modify the Ca 2þ sensitivity of cTn, affecting the duration of systole/diastole phase and contributing to heart disease, including cardiomyopathies and HF. 38In this review, the gene expression and the PTMs of cTn were described together with those conditions able to adversely affect the fine balance among the elements of complex finally promoting heart dysfunction.

Cardiac troponin C Gene structure and expression
The gene encoding for cTnC is located on chromosome 3p21.1 in humans, on chromosome 14, in mice, and on chromosome 16p16 in rats.The TNNC1 gene is organized in six exons and five introns which encode for cTnC, 18,20 a protein of 161 amino acids (aa) (18 kDa) (Fig. 3A). 39,40In contrast to cTnI and cTnT, cTnC is the only isoform present in the heart during both fetal and adult life. 39,40otein structure and functional domains X-ray crystallography studies showed that the 161 aa of cTnC are arranged in 9 a-helices Helix-N (3e9 aa) and Nlobe helices AeD (aa 14e87) plus C-lobe helices EeH (aa 92e161) (Fig. 4A).The helices A to H create four EF-hands consisting of 30e40-aa motifs from two a-helices (E-helix and F-helix) connected by a loop that forms a unique structure able to bind to divalent cations (as the C-lobe sites are occupied by Mg 2þ rather than Ca 2þ ). 25,39In addition, EF-hand I (16e51 aa) does not bind to divalent cations in cTnC, whilst EF-hand II (52e87 aa) is the regulatory region of cTnC that is highly selective for Ca 2þ and has a low Ca 2þ affinity. 41In physiological conditions, EF-hand I never binds to Ca 2þ , whilst EF-hand II is linked with Ca 2þ during systole, thus the EF-hand II is involved in contraction.The EF-hand III (92e127 aa) and IV (128e161 aa) show a high affinity for bivalent ion Ca 2þ /Mg 2þ and are always occupied by ions.The N-terminal domain (1e87 aa) is the regulatory element of cTnC and, in contrast, the C-terminal domain is considered the cTnC-structural region (92e161 aa) that keeps cTnC anchored to cTn.When Ca 2þ concentration (cells range: 10 À7 to 10 À5 M) increases in the cytoplasm and binds to the EF-hand II, the cTnC changes conformation, leading to the opening of a hydrophobic patch able to bind to the switch region on cTnI and to induce cTn changes, with consequent sarcomere contraction. 41

Cardiac troponin I Gene structure and expression
The gene encoding for cTnI is located on chromosome 19q13.42 in humans, on chromosome 7 in mice, and on chromosome 1q12 in rats.The TnI is expressed in the human heart in two age-dependent-isoforms that change during development.Slow skeletal troponin I (ssTnI) is the expression product of the TNNI1 gene (chromosome 1q32.1) and is predominantly synthesized in the slow skeletal muscle but also the heart during embryonic life.The TNNI1 gene, characterized by 9 exons and 8 introns, encodes a protein of 187 aa.ssTnI is completely replaced by cTnI in 9e24 months after birth in the human heart, 42e44 while in an animal model, it was found that TNNI1 gene was expressed up to 15 days after birth. 45In humans, the cTnI gene (TNNI3) includes 8 exons and 7 introns and encodes for a flexible protein of 210 aa (24 kDa).It was suggested that the replacement of ssTnI with cTnI during newborn life could be associated with increased responsiveness to the badrenergic system. 44The Ser 23/24 at the N-terminal domain of cTnI is regulated by protein kinase A (PKA) which is, in turn, activated by the b-adrenergic system, 46 but the N-terminal domain is missing in ssTnI.

Epigenetic regulation
In recent years, epigenetic regulation of TnI expression has been studied.Epigenetics mechanisms may fine-tune the gene expression by chromatin remodeling (acetylation/ deacetylation and methylation/demethylation of histone) or by silencing the mRNA (long non-coding RNA and microRNA/ miRNA).Hence, the role of miRNA-449 in regulating cTnI expression was demonstrated. 47Low levels of acetylation of cTnI promoter are associated with a low cTnI expression in elderly mice. 48In in vivo and in vitro studies, high levels of miR-449 could indirectly increase the cTnI levels through the binding and degradation of histone deacetylase 1 mRNA. 26In this way, acetylation of the GATA element and recruitment of transcription factor to the TNNI3 promoter region was favored. 49On the other hand, non-coding RNA, such as miRNA-208b and miRNA-499, can regulate the expression of "fast skeletal troponins" TNNT3 and TNNI2, troponins of striate skeletal muscle, also in the mouse heart. 50,51otein structure and functional domains cTnI is organized into five domains with different functions (Fig. 3B).The N-terminal domain (2e32 aa) is a finely regulated region characterized by two adjacent serine residues (Ser 23-Ser 24), that can be phosphorylated by PKA or other kinases, under stress conditions and/or exercise, thus affecting the time of Ca 2þ release from cTnC and, consequently, the sarcomere contraction. 10,13he second domain of cTnI, the IT arm (42e136 aa), is composed of two a-helices, Helix H1 (43e79 aa) and Helix H2 (90e135 aa), linked by a flexible-U-turn sequence. 41The IT arm is the structural domain of cTnI able to bind to cTnT by a rigid coil-to-coil interaction 52 and is a valid anchoring site for the C-terminal domain of cTnC. 10,13,53The classical model describes that the C-and N-terminal parts of the IT arm are near and parallel to the actin-tropomyosin filament. 54The cryo-EM map resolution showed that only the C-terminal part of the IT arm is close to the actin-tropomyosin, whereas the N-terminal end is perpendicular to the long axis of thin filament together with the Helix D of cTnC. 9,54,55eyond the IT arm, the C-terminal domain of cTnI is composed of the inhibitory-peptide region (137e148 aa) and the switch peptide (149e164 aa) (Fig. 4B).53e58 Biochemical studies demonstrated that, in the condition of low Ca 2þ concentration, the EF-hand II of cTnC is closed and the switch peptide is far from cTnC and tightly bound to actin-Tm. 26In this state, the inhibitory peptide stabilizes the Tm on the actin filament in a "blocked position", preventing the entrance of the myosin head into the thin filament. 26,59,60In addition to cTnIeTm interaction, the molecular model of human cardiac thin filament studied by cryo-EM suggested an intimate electrostatic interaction between arginine residues 145, 146, and 148 of TnI inhibitory peptide and Asp 24 and 25 of actin filament. 21,2661e64 Thus, the C-terminal domain of cTnI can reduce the regulatory activity of cTn on actin-Tm, facilitating the shift of Tm to a "closed" position and, consequently, the access of myosin heads to actin. 21,26The Tm transition in the "three-state" model of contraction and the exact binding sites of Tm and cTn on actin are still partially unclear. 21

Post-translational modifications
PTMs could change the properties of cTn by proteolytic cleavage or by the addition of biochemical groups (phosphorylation or glycosylation mechanisms) to one or more amino acids.Under physiological conditions, PTMs play a key role in numerous biological processes.The main PTMs observed for cTnI is the phosphorylation of functionally relevant amino acid residues, scattered throughout the protein, such as Ser 23/24, tyrosine 26, Ser 42/44, threonine (Thr) 5, Ser 77, and Thr 78/143.Among them, the Ser 23/24 phosphorylation could be considered as a mechanism useful to accelerate the diastolic relaxation period, thus improving cardiac function. 65,66Conversely, other modifications of cTnI, such as Ser 150 phosphorylation, could increase myofilament Ca 2þ sensitivity and blunt the Ca 2þ desensitization induced by Ser 23/24 phosphorylation. 67he effect of cTnI phosphorylation on cardiac contraction has emerged from biophysics studies in isolated + MODEL myofibrils. 68The phosphorylation of Ser 23/24 is a physiological event activated by the increase of catecholamine circulating levels.Specifically, catecholamines induce the cTnI Ser 23/24 phosphorylation by PKA activation, 69,70 thus improving heart performance by the reduction of the heart contraction-relaxation period. 71The classical theory of Ser 23/24 phosphorylation suggests that Ser 24 is modified before Ser 23 and that only the bi-phosphorylation reduces the switch peptide's ability to bind to the hydrophobic patch of cTnC, thus inducing the release of Ca 2þ from EFhand II in an indirect way. 72,73Even if the bi-phosphorylation of Ser 23/24 mediated by PKA is considered the essential condition required for Ca 2þ sensitivity reduction of the thin filament, PKD-mediated phosphorylation of only Ser 24 can largely reproduce the PKA effects. 19esides PKA and PKD, Ser 23/24 can be phosphorylated by other kinases, such as PKG and PKC.However, whilst PKA, PKD, and PKG can phosphorylate only Ser 23/24 on cTnI, PKC phosphorylates also other sites, such as Ser 42/44 or Thr 143. 74In mice, improvement in cardiac contraction dependent on Ser 23/24 phosphorylation and PTMs of Ser 43/45 or Thr 144 (equivalent to Ser 42/44 and Thr 143 in human) could reduce myofilament Ca 2þ sensitivity and develop a reduction of pump function. 75,76Molecular mechanisms and conformational changes activated by the phosphorylation of these sites have not yet been adequately studied and there is still a lot to understand about their potential effect in heart disease.
In addition to phosphorylation, cTnI could be subject to other PTMs.Elevation of glucose modifies various proteins by O-linked b-N-acetylglucosamine glycosylation of serine or threonine residue.Specific O-linked b-N-acetylglucosamine glycosylation at Ser 150 of cTnI was found in cardiac trabeculae isolated from mice that could modify the myofilament Ca 2þ sensitivity. 7

Cardiac troponin T Gene structure and expression
The gene encoding for cTnT is located on chromosome 1q32.1 in humans, on chromosome 1 in mice, and on chromosome 13q13 in rats.The cTnT is recognized as the structural component of cTn that firmly fixes the complex to the thin filament of the sarcomere, via tropomyosin.Moreover, cTnT has also an active role in the "three-state model" of muscle contraction, interacting with TnC, TnI, and tropomyosin, and, in particular, regulating the strong cross-bridge attachment to actin to generate force. 25,54,76he cTnT is encoded by the TNNT2 gene and is composed of 17 exons and 16 introns.During the development of the human heart, alternative splicing of exons 4 and 5 generates up to four isoforms: cTnT1 (with exons 4 and 5), cTnT2 (with only exon 5), cTnT3 (with only exon 4), and cTnT4 (without both exons). 37The fetal heart predominantly expresses cTnT1 (298 aa) and, to a lesser extent, cTnT2 (293 aa) and cTnT4 (283 aa).After birth, a regulated switch occurs from the embryonic isoforms to the cTnT3 (288 aa), the only expressed isoform in the adult heart. 37Besides the canonical isoforms, cTnT1, cTnT2, cTnT3, and cTnT4, generated by the variants 5, 8, 2e7, and 4, respectively, further non-canonical cTnT isoforms, cTnT10, cTnT11, and cTnT12, are produced by other variants (Table 2).However, the role of non-canonical isoforms of cTnT within sarcomere is still unknown.

Protein structure and functional domains
Isoforms of cTnT are organized in three domains (Fig. 3C): the N-terminal region, the flexible linker, and the C-terminal region.Within the N-terminal region, the T1 domain (1e168 aa for cTnT3) is a highly flexible domain that binds cTnT to Tm. 37,77 Cryo-EM studies showed that a small part of the N-terminal region of cTnT interacts with the N-terminal end of Tm located at the same contractile subunit (7 actins, 1 cTn, and 1 Tm). 26,29The rest of the T1 domain crosses the tropomyosin N-/C-terminal overlapping domain and then extends over the C-terminal part of the adjacent Tm molecule. 26,29Whitin the cTn core, 77 the T2 domain (201e288 aa for cTnT3) is characterized by two a-helices (Helix H1 and Helix H2) and by the C terminal peptide (278e288 aa) (Fig. 4C). 77Whilst the Helix H2 forms an antiparallel coiled-coil with the Helix H2 to cTnI (IT arm), interacting also with the C-terminal domain of cTnC, 13,41 the C-terminal peptide of cTnT (16 aa, commonly unstructured) can bind to the inhibitory-peptide of cTnI, the N-terminal peptides of cTnT in the hinge region, and, less frequently, the TnT Helix 2 and the C-terminal domain of TnC. 13 cTnT can modulate Ca 2þ sensitivity.The expression of exon 5 makes the N-terminal region of cTnT1 and cTnT2 highly acidic and negatively charged at physiological pH.Moreover, compared with human cTnT4 with an isoelectric point Z 5.2, the human cTnT1 has an isoelectric point Z 4.94, showing a higher Ca 2þ sensitivity. 78Embryonic cardiac TnT with more negative charges in the N-terminal region and a lower isoelectric point can improve the Ca 2þ sensitivity of cTn in an environment with low Ca 2þ concentration and increase the force of muscle contraction, compared with the adult isoform of cTnT.77e79 Thus, in the context of development or cardiac disease, cardiac troponin undergoes functional adaptations by isoform switches to respond to an altered cardiac demand.Indeed, fetal isoforms of cTnT are re-expressed in the heart of HF patients, in particular the cTnT4 isoform. 37,80Specifically, the TnT N-terminal charge could alter the affinity to cTnI and Tm and the more acid TnT (isoelectric point Z 6.33)

Post-translational modifications
At the beginning of the 1980s, different research groups found an enzyme indicated as troponin T kinase able to phosphorylate bovine or dog cTnT in a single site (Ser 2) located at the N-terminal domain. 52,82,83Subsequently, other phosphorylation sites modified by PKC activity have been found in purified cTnT, such as Ser 208, Thr 213, and Ser 285. 59Moreover, the number of phosphorylated sites on cTnT increased after prolonged incubation of cTnT with PKC. 74n addition to PKC, other kinases such as apoptosis signal-regulating kinase 1, calmodulin-dependent protein kinase II, Rho-A-dependent protein kinase II, and Raf-1 proto-oncogene serine/threonine-protein kinase can phosphorylate "purified" cTnT in Thr 204, Thr 206, Ser 208, Thr 213, Ser 285, and Thr 294, suggesting that PTM of cTnT could be regulated from different pathways. 84,85In in vitro studies, purified or recombinant cTnT is exposed to kinases for phosphorylation, and to cTnI, cTnC, and Tm for assembling cTn, and to hook the complex to thin filament. 86hese phosphorylations avoid the binding of cTnT to Tm but not to cTn, thus reducing the ability of sarcomere contraction.In particular, the phosphorylation of Thr 213 is associated with a reduction of Ca 2þ sensitivity and maximum tension development by myofilament.Unlike, in vitro studies using top-down protein mass spectrometry applied to rat and human heart samples showed that cTnT was predominantly monophosphorylated (Ser 2) in physiological conditions.42e87

Cardiac troponin in cardiovascular disease
Considering the key role of cTn in the regulation of cardiac contraction, conditions able to modify cTn gene expression, such as mutations in the cTn genes and re-expression of fetal cTn isoforms, could affect the sarcomere function.These molecular changes might affect cardiomyocytes leading to progressive dysfunction.How specific molecular changes could lead to different disease phenotypes, including dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), restrictive cardiomyopathy (RCM), and left ventricular non-compaction cardiomyopathy, is still under investigation.Moreover, besides regulation of gene expression, disruption or chronic activation of PTMs could contribute to the starting and progression of pathological states with long-term consequences that lead to cardiac dysfunction and HF (Fig. 5).

Gene mutations
Mutations in TNNC1, TNNI3, and TNNT2 have been identified as relatively uncommon causes of cardiomyopathy, such as DCM, HCM, and RCM, when compared with the frequency of mutations in other sarcomere genes, 36,37,88,89 probably because most of them are not compatible with life.Known mutations into the cTnC-cTnI-cTnT genes were listed in Tables 3e5, respectively, together with the molecular effect and the clinical condition related to each mutation (as indicated by the American College of Medical Genetics and Genomics classification).The molecular effect was not reported in the tables for those variants identified only by genetic testing and then recorded in the ClinVar database without any pathophysiological Cardiac troponins in physiological and pathophysiological condition 7  Cardiac troponins in physiological and pathophysiological condition 9  At least 115 variants of TNNC1 due to point mutations that vary from wildtype protein (161 aa) by a single amino acid are reported.Approximately, 26% of these variants can compromise the Ca 2þ sensitivity of cTnC or the ability of troponin to interact properly with the other cTn components.However, the possible adverse effects of all variants are unknown. 40The identified cTnC variants, the associated cardiomyopathies, and the relative molecular effects are reported in Table 3.Among them, the cTnC-A8V variant was associated with HCM and RCM. 40,90In in vitro studies, a delay of mechanical relaxation and intracellular Ca 2þ decay was observed in cardiomyocytes with cTnC-A8V substitution. 90This is probably due to the replacement of alanine with valine in position 8 which may increase the Ca 2þbinding affinity of cTnC, thus inducing a delay in the Ca 2þ dissociation rate from the thin filament. 40,90Similarly, in an animal model of HCM containing the human cTnC-A8V substitution, an increase in Ca 2þ sensitivity of cTnC was sufficient to induce cardiac remodeling and myofilament dysfunction. 91cTnC-A8V mice showed a thicker left ventricular wall and a lower diastolic diameter and volume compared with the control. 92Moreover, the in vivo study suggested that these adverse morphological changes affected more females than males. 92Alternatively, a reduction of Ca 2þ sensitivity could be the primary consequence of those cTnC variants that are associated with DCM, such as D75Y, E59D, and E32K. 93,94In particular, the D75Y substitution, but not E59D, causes a reduction of the Ca 2þ affinity.However, both D75Y and E59D are required to reduce the actomyosin ATPase activity and maximal force development in muscle fibers, indicating that E59D substitution can be considered as a booster for D75Y effects. 94NNI3 mutations account for about 5% of genotyped families with HCM.94e98 A list of TNNI3 mutations together with the associated molecular and pathophysiological effects (when known) is reported in Table 4.The main mutations of the cTnI gene are located in the inhibitory/switch peptide and the C-terminal domain, thus interfering with the regulatory property of cTnI.It has been observed that the mutations R145G, R145Q, R162W, R21C, K206Q, and DK178 have the ability to interfere with Ca 2þ sensitivity and force development by sarcomere fibers. 53The cTnI-R145G variant, located within the inhibitory peptide of cTnI, is one of the most studied TNNI3 mutations.The cTnI-R145G variant is historically associated with HCM, however, its effect on the cTn function is not fully clear. 36Enhanced Ca 2þ sensitivity and actomyosin ATPase activities were observed in thin filaments reconstituted with human skeletal muscle, actin, tropomyosin, and recombinant human cTn, including cTnI with mutation R145G when compared with filaments with cTnI wild-type. 95However, when human recombinant cTnI-R145G was incorporated into murine or guinea-pig myofibrils, Ca 2þ sensitivity did not change, 95e98 whilst if mouse recombinant cTnI-R145G was merged in the murine sarcomere, a decrease in Ca 2þ sensitivity could be observed. 95The cTnI-R21C substitution, also extensively studied, 99 is located at the cTnI N-terminal region, near Ser 23 and Ser 24. 46The contractile fibers containing the TnI-R21C substitution are characterized by an increase in Ca 2þ sensitivity and force development, and by a reduction in phosphorylation of Ser 23/24 by PKA when compared with wild-type fibers. 100,101Accordingly, in cTnI-R21C transgenic mice, a reduction in Ser 23/24 phosphorylation, an alteration in the b-adrenergic pathway, and an activation of hypertrophic molecular mechanisms were observed compared with control mice. 100,101Moreover, abnormalities in calcium regulation were observed using CalTrack, a MatLab-based algorithm able to monitor fluorescent calcium changes in living cardiomyocytes.In particular the cTnI-R21C þ/À variant caused a significant acceleration of time to reach calcium peak and a longer duration of time for Ca 2þ transient decay and Ca 2þ transient duration.Such abnormalities were only partially corrected by dedicated allosteric modulators of thick filament drugs, such as mavacamten. 102utations of cTns affecting the contact site can disrupt the normal troponin complex interaction, leading to a decreased affinity of binding between cTnI and cTnT. 103,104TnI-C11R is a dominant negative mutation responsible for a decreased binding affinity of cTnI for TnT, thus reducing the regulatory function of the entire complex on the sarcomere.104 The known pathogenic/likely pathogenic mutations of TNNT2, including deletions (protein change in DE163, DE173, DK210, DK177, etc.) and single nucleotide substitutions (protein change in R134G, R134S, R92L, R92W, R94C, K103Q, K88Q, I79N, I89N, etc.), are reported in Table 5.Most of the cTnT mutations induced changes either in the flexibility of cTnT or in the Ca 2þ affinity for the cTnC calcium-binding site, thus contributing to HCM, RCM, DCM, or left ventricular non-compaction cardiomyopathy phenotypes.105e110 The location of the cTnT mutation contributes to the type of alterations in protein properties.108,109 Mutations in the T2 domain, such as the cTnT-DK210 mutation, historically associated with dilated ventricular chamber and reduced systolic function, can directly contribute to impairment in Ca 2þ affinity of cTnC.98 Ca 2þ sensitivity of the recombinant thin filaments containing an equimolar mix of cTnT-DK210 and cTnT-wildtype decreased compared with control filaments.71 Moreover, the thin filament sliding speed was reduced in recombinant thin filaments containing cTnT-DK210 compared with wild-type cTnT.108 Considering that most of the TNNT2 mutations are heterozygous, these data could suggest that cTnT-DK210 mutation contributes in a dose-dependent fashion to DCM development.108 In a recent study using human embryonic stem cell-derived cardiomyocytes and mouse heart tissues, it was observed that cTnT-DK210 mutation was associated with a reduced expression of Xin actin-binding repeat-containing protein genes.111 The XIN proteins are localized predominantly at the intercalated discs and are responsible for signal transduction involved in cardiac remodeling. 111 reduction of XIN proteins is associated with impaired contractility and attenuated muscle repair.111 In vivo and in vitro studies showed that the overexpression of XIN proteins improved sarcomere organization and contraction force of TNNT2-DK210, and significantly improved the DCM phenotypes, suggesting a possible therapeutic target for subjects carrying the cTnT-DK210 mutation.111 Cardiac troponins in physiological and pathophysiological condition 13 Mutations in the T1 domain could be associated with an aberrant interaction with tropomyosin, as observed for cTnT-R94C, leading to a reduced binding affinity of cTnT for thin filaments. 112The mutation cTnT-R134G, located in the T1 domain of cTnT, is typically associated with DCM.In an in vitro study, cardiomyocytes with cTnT-R134G substitution showed a reduction in Ca 2þ sensitivity and contractile function together with sarcomere disorganization when compared with the control. 112However, the regular thin filament with cTnT-R134G substitution showed an increased cTnC Ca 2þ affinity when compared with the wild-type. 112iscrepancies in these results could depend on the ability of cTnT-R134G to promote shifting of the Tm in a permanent locking state independently from Ca 2þ concentration, thus allowing a reduction of the contractile function of the sarcomere. 112Again, cTnT-R173W substitution is typically associated with DCM.This mutation modifies the interaction between the N-terminal domain of cTnT and Tm, thus reducing the troponin anchoring on sarcomere filaments and destabilizing the sarcomere protein alignment.Moreover, TnT-R173W substitution decreases PKA-mediated TnI phosphorylation at Ser 23/24 by limiting the binding of PKA to sarcomere.PKA-mediated phosphorylation is a critical regulatory step for the modulation of cardiac contraction. 108

Regulation of gene expression
During HF, fetal isoforms of cTnT or TnI are expressed together with the adult isoforms.The co-presence of different cTnT and TnI isoforms is associated with a reduction of cardiac performance, a decrease in stroke volume, and slower velocities of contraction and relaxation, thus inducing a progression of HF. 37,79,80 A recent study reported the re-expression of fetal isoforms of cTnT and TnI in cardiac biopsies collected from adult and children patients with HF before ventricular assist device implant. 80However, fetal isoforms were not modified by ventricular assist device implant, while an increased expression of adult isoforms, such as cTnT3, cTnT4, and cTnI, was observed in pediatric patients. 80he effects of different isoforms of cTnT (1/3/4) in combination with ssTnI or cTnI on Ca 2þ sensitivity and force of contraction of sarcomere were studied. 79In the presence of ssTnI, the absence of exon 5 in cTnT (cTnT3 or cTnT4) decreased the Ca 2þ sensitivity compared with cTn with cTnT1.Interestingly, fibers with cTnT4-ssTnI and cTnT4-cTnI developed similar maximal force.In contrast, the maximal force of contraction was higher in cTnT3-ssTnI fiber than in cTnT3-cTnI recombinant skinned fiber. 63It is unlikely that the increase of maximal force was due to ssTnI alone, but rather it was dependent on some complex interplay between the cTnT isoforms, TnI isoforms, and the rest of the thin filament proteins. 79he ability of cTnI to regulate the actomyosin ATPase activity was similar in the presence either of cTnT3 or of cTnT4, while ssTnI linked with cTnT3 showed an increased ATPase activity when compared with the complex ssTnI-cTnT4. 79TnC plays a critical role in regulating muscle contraction by its ability to bind Ca 2þ to the EF-hand II regulatory site. 113The effects of cTnT or TnI isoforms on the rate of Ca 2þ removal from the high-or low-affinity sites on cTnC were investigated.cTnT1 had a greater effect on the kinetics of the Ca 2þ dissociation rate from site II of cTnC in the cTnI-cTnC complex than in the ssTnI-cTnC complex.In particular, the cTn containing ssTnI had a 2-to-3-fold slower rate of Ca 2þ dissociation from cTnC (site II) than the complex containing cTnI. 79These results suggest that in the normal fetal heart, a contemporary presence of cTnT1 isoform, which showed a high Ca 2þ sensitivity, and ssTnI, which had a slower rate of Ca 2þ dissociation, increased the duration of the systolic phase.These characteristics of fetal cTn (cTnT1-ssTnI-cTnC) could be associated with the lower Ca 2þ levels in the fetal heart.Thus, the re-expression of the fetal gene of troponin in HF patients and their substitution to functional cTn complex could be associated with the disarray of sarcomere and reduction of left ventricular ejection fraction typically observed in these patients. 37,80,99The causal relationship between the reexpression of fetal cTn genes and sarcomere disarray is one of the possibilities to be explored to better understand the molecular mechanisms underlying HF.
Moreover, TNNT2 is expressed in cardiomyocytes during heart and skeletal muscle development and it can be reexpressed in the muscle during neuromuscular diseases. 114,115In these patients, in the absence of myocardial injury, a persistent blood elevation of cTnT without cTnI was observed.Re-expressed cTnT in diseased skeletal muscle is considered the source of the elevated cTnT detected in the circulation of these patients. 114ery recently, an epigenetic regulation of cTn genes in pediatric patients with HF supported by a ventricular assist device was reported.Specifically, gene expression of troponins, found to be differentially expressed in cardiac biopsies at post-ventricular assist device when compared with pre-ventricular assist device, were identified as putative targets of miR-199b-5p, miR-19a-3p, and miR-1246. 80oreover, regulation of troponin expression during translation has been reported.A very recent study in RPL3L-deficient mice suggested that loss of RPL3L ribosomes determined a reduction of cardiac contractility as a consequence of loss of the overall transcriptome.In particular, transcripts from genes related to sarcomere including cTnT-mRNA were affected by RPL3L mutation, 3,116 thus facilitating the heart remodeling and dysfunction.During HF an increase in 2-oxoglutarate-and iron-dependent oxygenase domain-containing protein 1 (OGFOD1), a member of the 2-oxoglutarate-dependent dioxygenase family, that regulates several aspects of gene expression including translation, was found to be up-regulated.In vitro and in vivo studies reported that loss of OGFOD1 (in iPCS-CMs OGFOD1-KO or OGFOD1-KO mice) resulted in a significantly increased level of sarcomere proteins, such as titin, cTnC, cTnI, and cTnT, compared with control. 117,118Moreover, OGFOD1-KO mice were protected from hypertrophy under chronic stress conditions. 117

Post-translational modifications
As previously described, the phosphorylation of Ser 23/24 can be considered among the main PTM associated with cTnI, possibly leading to functional alteration.During HF, the pathway catecholamine/b-adrenergic receptor/PKA decreased as a consequence of b-adrenergic receptor down-regulation.In particular, a reduction of both badrenergic receptors I and II was observed in cardiac biopsies obtained from end-stage DCM patients compared with control.Thus, in DCM patients, the turn-off of the badrenergic receptors was directly related to the reduction of PKA activity and Ser 23/24 phosphorylation. 119Consequently, this reduction induced a slow sarcomere relaxation and an impaired heart function. 111Conversely, the expression of the N-terminal-deleted cTnI (cTnI-ND) decreased the development of the cardiomyopathy-like phenotype in a b-adrenergic-deficient transgenic aged mouse model. 120,121Thus, the removal of cTnI N-terminal extension induces structural and functional modifications in cTnI molecules such as those typically observed in case of low Ser 23/24 phosphorylation.These data suggested that the removal of the N-terminal region may maintain cardiac function in aged mice during stress. 120,121esides reduction in PKA activity, the hyperactivation of Ca 2þ /calmodulin-dependent protein kinase II during longlasting hyperglycemia causes phosphorylation of cTnI and decreases myofilament Ca 2þ sensitivity. 59o evaluate the involvement of cTnT phosphorylation in pathological conditions, such as HF, the PTMs of cTnT and other sarcomere proteins were compared between endstage HF patients and non-failing donor hearts. 84,19cTnI and myosin light chain 2 phosphorylation decreased in failing human myocardium, while cTnT phosphorylation was unaltered. 122The Ca 2þ sensitivity and the myofilament isometric force increased in HF patients compared with the control. 123,124n rats with congestive HF, phosphorylation of cTnT and cTnI was greater in the failing heart compared with the control and higher levels of phosphorylated cTnT were present in the failing left ventricle compared with the right ventricle.In addition, the phosphorylation of cTnT is associated with a reduction of Ca 2þ sensitivity and, consequently, with dysfunction of myofilament.Overall, these studies showed a discrepancy between human and animal models, emphasizing the need for other specific studies dedicated to humans.

Small molecules: A new era of cardiomyopathy therapy
The development of drugs targeting the contractile apparatus of cardiac muscle is considered the new frontier for DCM, HCM, and, consequently, HF treatment.Five classes of "small molecules" have been identified and classified based on their effects.Myosin inhibitors, Ca 2þ -desensitizers, and recoupler were designed to treat hypercontractility, 125e127 and myosin activators and Ca 2þsensitizers were proposed as positive inotrope agents. 128,129mong the agents for treating hypercontractility, mavacamten is the first and only reversible cardiac myosin inhibitor approved by the FDA. 130This small molecule can influence cardiac muscle contractility, reducing dynamic left ventricular outflow tract obstruction and improving cardiac filling pressures in patients with HCM.cTn is suggested as a target of Ca 2þ -desensitizer small molecules in hypercontractility conditions.Among them, in vitro studies suggested a cardioprotective role for epigallocatechin-3-gallate and epicatechin gallate 131e133 which were able to improve the diastolic function in isolated working mouse hearts, binding cTnC and reducing Ca 2þ sensitivity of troponin. 132However, these molecules showed also pleiotropic effects that make them inadequate for in vivo use.Another class of small molecules able to interfere with cTn function are recouplers.These new small molecules demonstrated the ability to restore the phosphorylation-dependent Ca 2þ -sensitivity in HCM and DCM conditions dependent on cTnI mutations. 17he hypo-contractility of the heart is a complex condition associated with HF.Considering the complex nature of hypo-contractility appears evident, several elements of the contractile apparatus should be at the same time targeted by new treatments to have an efficacy clinical response.Currently, a fair number of myosin activators and Ca 2þsensitizers were proposed as positive inotrope agents. 128,129mecamtiv mecarbil is a first class of myosin activators, a small molecule designed to increase the number of active actin-myosin cross bridges during the cardiac cycle.Even if an improvement in cardiac contraction was observed in preclinical studies, to date no clinical trials have obtained FDA approval. 112,134Among Ca 2þ -sensitizers, bepridil and EMD57033 in a different way, were able to improve the force contractility of sarcomere modulating the interaction between the N-terminal region of cTnI and C-terminal domain of cTnC, independently from Ser 23/24 PTMs on cTnI. 135The AGM 594 novel small-molecule that acts as a troponin activator can increase contractility ex vivo and in vivo in anesthetized normal rats without negative effects on myocardial energetics. 129Among them, only AMG 594 demonstrated more positive effects than adverse effects in preclinical trials suggesting that more investigation is needed before replacing the current inotrope therapy with these troponin activators.

Conclusion
Because cardiac troponins are regulatory elements of sarcomere function, any conditions that may affect their synergic interaction could interfere with a proper myofilament function, resulting in heart dysfunction, cardiomyopathy, or death.Further investigations are needed to complete the knowledge of the regulatory complex of sarcomere.It is still questioned how a modification within the thin filament could modulate the contractile function and whether a specific change in cTn is directly responsible or is indirectly an effector of additional downstream events for adverse remodeling and dysfunction.
To date, several mutations of TNNC1, TNNI3, and TNNT2 genes have been identified, but the physio-pathological effect of each mutation has not yet been clarified.Even if sarcomere variants generally have high penetrance, they produce a variable expression of clinical manifestations. 71utations in the cTnI gene have been identified in families with HCM, RCM, and recessively inherited idiopathic dilated cardiomyopathy 136e139 reflecting the important and diverse functional role of the cTnI protein in normal cardiac Cardiac troponins in physiological and pathophysiological condition biology.The diversity of the phenotypic expression of cTns mutations is not explained by the current knowledge of their molecular and functional impact and suggests that additional environmental, genetic, and epigenetic factors may interact and affect clinical disease expression leading to phenotypic diversity of cardiomyopathies.A better knowledge of molecular mechanisms affected by these mutations has important implications for understanding disease pathogenesis and could be useful for the development of targeted therapeutics for those patient subpopulations with myofilament defects.
Accordingly, the switch of cTnI and cTnT from fetal to adult isoforms during physiological development or the reexpression of fetal troponin isoforms during heart diseases, such as HF, has been documented and a possible regulatory role for epigenetic mechanisms (i.e., miRNA) has been proposed.However, only a few studies investigated the link between epigenetic mechanisms and cardiac troponin gene expression.A complete understanding of how and which miRNAs are involved in cardiac troponin expression during physiological and pathological conditions is crucial for hypothesizing the use of miRNA therapy in HF patients.
Moreover, X-ray crystallography and CryoEM technology have improved the knowledge of the structure and spatial orientation of every single domain of cTn, but gaps in understanding the cTn switching mechanism and movement across the actin during cardiac muscle activation are still present.
In addition to the well-known role of cTns as regulatory components of the sarcomere, recent studies have demonstrated the presence of cTnI and cTnT in the nucleus of cardiomyocytes.Actually, the mechanisms that regulate the transport of cTnI and cTnT from the cytoplasm into the nucleus are not known, while an interaction of cTnI with a histone deacetylase 140 and of cTnT with histone de-methylases was observed in the nucleus of cardiomyocytes. 141hus, these results indicate a novel role of cTns and might provide new aspects for investigations in heart development and cardiovascular diseases.
Furthermore, many additional questions remain unsolved: how many PTMs ascertained in vitro could be detected in vivo?What are their functions?What is the role of non-canonical cTnT?Answers to these questions could help to identify new drugs targeting the pathways involved in post-translational cTn modifications, opening a new era for cardiomyopathy therapy, with the final aim of improving heart function.

Figure 2
Figure 2 Crystal structure of human cardiac troponin in the calcium saturated.(A) Cardiac troponin complex in calcium bound state.(B) The cTnC (blue) and its Ca 2þ -binding sites (highlighted in yellow).(C) The cTnI (green) and its phosphorylated aa (red).(D) The cTnT (yellow) and its phosphorylated aa (red).Figure modified from www.ncbi.nlm.nih.gov/Structure/pdb/1J1D.

Figure 3
Figure 3 Primary structure of cardiac troponins.(A) Cardiac troponin C (cTnC).Schematic representation of cTnC, the sub-unity of cardiac troponin complex (cTn) responsible for binding to Ca 2þ .The rectangular in green represents the N-terminal and the Cterminal domain, whilst the red circle shows the flexible region.The aa involved in the alpha-helix structure are highlighted in yellow (Helix AeH) and dark green (Helix N). (B) Cardiac troponin I (cTnI).Schematic representation of cTnI, the inhibitory subunity of cardiac troponin complex (cTn).The rectangular in green represents the N-terminal, the IT arm, and the C-terminal domain, whilst the blue and the orange rectangular show the inhibitory peptide region and the switch peptide region respectively.The aa involved in the alpha-helix structure is highlighted in yellow (Helix H1eH4).The post-translational modifications (phosphorylation) are indicated with a "P" within a circle in correspondence with phosphorylation sites.(C) Cardiac troponin T (cTnT).Schematic representation of adult cTnT isoform.The rectangular in green represents the T1 and T2 domains, and the blue rectangular shows the linker peptide.The aa involved in the alpha-helix structure is highlighted in yellow (Helix H1 and H2).The posttranslational modification (phosphorylation) is indicated with a "P" within a circle in correspondence with the phosphorylation site.

Figure 4
Figure 4 The detailed 3D reconstruction of each component of cardiac troponin complex (cTn).(A) The cTnI molecule (from Thr 31 to Ser 166; green) with its resolved structural regions: IT-arm, inhibitory peptide region (yellow rhombus), and switch peptide.The phosphorylated amino acids (post-translational modifications) are reported in circle blue.(B) The cTnT (from Leu 99 to Gln 272, in yellow) with its resolved domains (T1 and T2) highlighted.The phosphorylated amino acids (post-translational modifications) are reported in circle blue.(C) The cTnC molecule (from Asp 2 to Glu 161, blu 2) with its nine helices and relative EF-hand highlighted.

Figure 5
Figure 5 Molecular conditions that can influence the cardiac troponin complex expression and function.(A) The action of epigenetic regulation and post-translational modifications in physiological conditions.(B) Effects of gene mutation, epigenetic deregulation, and post-translational modifications in pathophysiological conditions.

Table 2
Canonical and non-canonical isoforms of human cardiac troponin T (cTnT).

Table 5
(continued )in experimental models.Conversely, mutations whose molecular effects have been studied at the molecular/pathophysiological level by dedicated studies are described in column 2 of each table.