Cardiac Troponin Complex: Cardiac Troponin C (TNNC1), Cardiac Troponin I (TNNI3), and Cardiac Troponin T (TNNT2)
The modern molecular understanding of muscle contraction took shape in the 1950s and 1960s. The sliding filament model was proposed in 1954, and it was known that muscle contraction was driven by myosin ATPase in the presence of actin. In the early 1960s, it was suspected, though not widely accepted, that muscle contraction is triggered by calcium. The idea was controversial, because while native preparations of actomyosin could be induced to relax by calcium-chelating agents, highly purified and reconstituted actomyosin did not display this behavior. In 1963, Ebashi demonstrated that the calcium dependence of skeletal muscle actomyosin activation was conferred by a third protein component, termed “native tropomyosin” or “tropomyosin-like protein” (Ebashi 1963). It resembled tropomyosin but was larger than the tropomyosin protein described by Bailey in 1946. In 1965, Ebashi and Kodama discovered that “native tropomyosin” was composed of tropomyosin and another component they named “troponin” (Ebashi and Kodama 1965). In 1971, Greaser and Gergely demonstrated that troponin is in fact made up of three separate subunits: calcium-binding troponin C (TnC), inhibitory troponin I (TnI), and tropomyosin-binding troponin T (TnT) (Greaser and Gergely 1971).
The troponin complex controls the calcium-dependent contraction and relaxation of striated muscle, which is either skeletal or cardiac (as opposed to smooth muscle). The characteristic striated appearance of these muscle types derives from the tandem arrangement of sarcomeres, the basic repeating units of contraction. Each sarcomere is bounded by two Z-discs, which anchor actin-containing thin filaments from neighboring sarcomeres. At the center of each sarcomere lies a hexagonally arranged lattice of bipolar myosin thick filaments. As put forth in the sliding filament model, activation of myosin ATPase powers muscle contraction by pulling the actin thin filaments towards the center of the sarcomere. This causes the thin filaments to slide against thick filaments, increasing their degree of overlap and reducing the length of the sarcomere.
Thin filaments are organized into repeating structural units, 38.5 nm long, each containing 14 actin monomers arranged as a twisted “double string of beads.” Tropomyosin forms a parallel coiled coil homodimer, with each homodimer lying across seven helically arranged actin monomers on each side of the thin filament. Thus, there are two diametrically opposed tropomyosin homodimers in a single 38.5 nm unit, each associated with a troponin complex. Tropomyosin further polymerizes in a head-to-tail manner to cover the entire length of the thin filament, except at the Z-disc.
Striated muscle contraction is regulated in a calcium-dependent manner through the action of tropomyosin and the troponin complex. At low Ca2+ concentration, troponin maintains tropomyosin in a position that blocks the interaction between myosin and actin, shutting off muscle contraction. The release of Ca2+ from the sarcoplasmic reticulum increases the cytoplasmic concentration of Ca2+, and Ca2+ binding to troponin results in a conformational change within the troponin complex that releases tropomyosin into a position that allows for actin-myosin interaction and muscle contraction.
The three isoforms of TnI are slow skeletal ssTnI (TNNI1), fast skeletal fsTnI (TNNI2), and cardiac cTnI (TNNI3) (Sheng and Jin 2016). During fetal development, the slow skeletal isoform is expressed in the heart, but it is completely replaced by cardiac isoform shortly after birth. TNNI3 is the sole isoform for the heart throughout adult life, and this does not change through pathologic states such as heart failure.
There are three muscle type-specific isoforms of TnT present in vertebrates: slow skeletal ssTnT (TNNT1), cardiac cTnT (TNNT2), and fast skeletal fsTnT (TNNT3) (Wei and Jin 2016). Their expression is fiber specific, with TNNT1 and TNNT3 expressed in slow and fast skeletal muscle, respectively. TNNT2 is expressed in cardiac muscle, but it is also expressed in embryonic skeletal muscle.
The presence of tissue-specific isoforms is clinically important. The measurement of elevated cTnI and cTnT levels in the serum is a very specific marker for myocardial damage. Detection of cTnI or cTnT has thus become the gold standard for diagnosing non-ST elevation myocardial infarction (NSTEMI). More recently, the detection of cTnI and cTnT has become so sensitive that low levels can be detected even in healthy individuals, and chronically elevated levels indicate increased cardiovascular risk (de Lemos et al. 2010).
Mutations in the cardiac isoforms of troponin give rise to heritable cardiomyopathies, all of which are associated with a risk of arrhythmias and progression to heart failure (Lu et al. 2013). Hypertrophic cardiomyopathy is the most common, with an estimated prevalence of 1:500. Its most prominent feature is abnormal hypertrophy of the ventricles, particularly in the ventricular septum. This process can lead to left ventricular outflow tract obstruction. There is some overlap between mutations causing hypertrophic cardiomyopathy and restrictive cardiomyopathy, with restrictive cardiomyopathy being a rarer and severe condition characterized by impaired relaxation and filling of the ventricles, leading to right-sided heart failure. Dilated cardiomyopathy is associated with enlarged thin-walled ventricles, generally leading to left-sided heart failure. A better understanding of the structure and function of the cardiac troponin complex is needed to explain the pathogenesis of the cardiomyopathies. One complicating factor is that the evidence linking some mutations to cardiomyopathies is poor, with some mutations only having been observed in a single individual. Without more data, it is difficult to determine whether some mutations are truly disease-causing alterations or just incidental benign polymorphisms.
Cardiac Troponin C (cTnC)
Structure and Function of cTnC
Cardiac troponin C is the calcium-binding subunit of the cardiac troponin complex, belonging to the EF hand superfamily of calcium-binding proteins. Each Ca2+-binding loop consists of 12 amino acid residues and is rich in aspartate and glutamate residues. Six residues 1(X), 3(Y), 5(Z), 7(-X), 9(-Y), and 12(-Z) in the loop provide oxygen ligands to coordinate the Ca2+ ion. X-ray and NMR studies reveal that cTnC adopts a dumbbell-shaped structure with two globular domains connected by a flexible linker (Sia et al. 1997; Takeda et al. 2003). NMR relaxation studies show that the two domains of cTnC tumble independently and are able to adopt various domain orientations in the absence of the other troponin subunits (Gaponenko et al. 1999).
Cardiomyopathy-Associated cTnC Mutations
Cardiac Troponin I (cTnI)
Structure and Function of cTnI
The cardiac-specific N-terminal extension (cTnI 1–31 ) is absent in fast and slow skeletal muscle TnI. The region is intrinsically disordered and invisible in the crystal structure of the cardiac troponin complex (Takeda et al. 2003). cTnI19–37 interacts electrostatically with cNTnC, which allows this region to remain disordered even within the troponin complex (Hwang et al. 2014). This interaction fixes the domain orientation of cNTnC relative to the rest of the troponin complex, positioning it optimally to bind the cTnI switch region. By promoting the interaction between cNTnC and the cTnI switch region, the Ca2+-bound open conformation of cNTnC is favored, and the apparent calcium affinity of cNTnC is enhanced.
The IT arm region (cTnI39–136) forms two α helices, cTnI39–79 and cTnI88–136. cTnI39–60 binds tightly to the hydrophobic cleft of cCTnC. cTnI88–136 forms a coiled coil with cTnT225–276, while cTnT225–276 binds to the other side of cCTnC (see Fig. 2). The IT arm constitutes the main structured core of the troponin complex. It is not known to bind directly to actin or tropomyosin.
The inhibitory region (cTnI135–147) interacts with actin to anchor the tropomyosin-troponin complex in a blocked state that shuts down muscle contraction.
The switch region (cTnI146–158) binds to cNTnC in a Ca2+-dependent manner, relieving the inhibition of adjacent sequences and facilitating the initiation of cardiac muscle contraction in systole.
- (e)The C-terminal actin-tropomyosin-binding site (cTnI159–209) enhances the inhibitory effect of the inhibitory region (cTnI135–147) (Tripet et al. 1997). The region is intrinsically disordered, although it may acquire some structure upon binding to actin-tropomyosin.
Posttranslational Modifications of cTnI
Posttranslational modifications of cTnI play a major role in regulating cardiac troponin activity. cTnI contains 3 tyrosine, 12 serine, and 8 threonine residues, many of which have been identified as phosphorylation sites, including Ser4, Ser5, Ser22, Ser23, Tyr25, Ser41, Ser43, Thr50, Ser76, Thr77, Thr142, Ser165, Thr180, and Ser198 (Zhang et al. 2012) (Fig. 5b). The most consistently identified phosphorylation sites in humans are Ser22/Ser23, which are phosphorylated by cyclic AMP-dependent protein kinase A (PKA) during β-adrenergic signaling (Solaro et al. 2013). However, these two serine residues can also be phosphorylated by several other protein kinases in vitro: protein kinase C (PKC), protein kinase D (PKD), and GMP-dependent protein kinase (PKG). Phosphorylation at these sites weakens the interaction between cTnI19–37 and cNTnC, promoting cardiac muscle relaxation by decreasing the calcium affinity of cNTnC (Zhang et al. 1995). Ser22 and Ser23 are highly phosphorylated in healthy individuals, whereas the levels of phosphorylation become reduced in heart failure (both heart failure with reduced ejection fraction and heart failure with preserved ejection fraction), as well as in hypertrophic and dilated cardiomyopathy (Wijnker et al. 2014).
Cardiomyopathy-Associated cTnI Mutations
Approximately 95% of the disease-causing mutations in cTnI are located in its C-terminal region, spanning residues 135 to 209 (Lu et al. 2013) (Fig. 5b). Almost all of these are associated with hypertrophic or restrictive cardiomyopathy (with the exception of N184K). This is consistent with the main physiologic role of this region, binding to actin-tropomyosin to maintain the thin filament in an inactive state. When this function is disrupted by mutation, the result is a hyperactivated thin filament with an associated increase in calcium sensitivity. Several cTnI mutations (L143Q, R144W, A170T, K177E, D189G, and R191H) in restrictive cardiomyopathy (RCM) patients showed greater Ca2+-sensitizing effects in cardiac muscle force generation compared to HCM mutations (Gomes et al. 2005b). At low Ca2+ concentration, these mutations also increased the force generation of cardiac muscle in the resting state. Interestingly, many of the mutations associated with RCM and HCM involve positively charged residues, underscoring the importance of electrostatic interactions, a recurrent theme for the intrinsically disordered segments of troponin.
Some mutations have also been reported in the N-terminal cardiac-specific extension of cTnI. One of these, R20C, has been linked to HCM (Gomes et al. 2005a). The mutation disrupts the phosphorylation consensus sequence, RRXS, of protein kinase A, abolishing the phosphorylation of Ser22 and Ser23. An additional four mutations (A1V, K35Q, N184K, and P15T) have been identified that have been associated with DCM in an autosomal dominant manner (Lu et al. 2013).
Cardiac Troponin (cTnT)
Variable Lengths of cTnT via Alternative Splicing
Structure and Function of cTnT
Classically, troponin T is divided into two functionally distinct fragments through partial proteolytic cleavage by chymotrypsin. The first fragment, T1, contains the N-terminal acidic hypervariable region followed by a tropomyosin-binding site (Fig. 6). There is a corresponding X-ray crystal structure of a short cTnT-derived peptide bound to the head-to-tail junction of tropomyosin (Murakami et al. 2008), but beyond this, precise structural details of the interaction between cTnT and tropomyosin are lacking. The second troponin T fragment, T2, contains a second tropomyosin-binding site, followed by a C-terminal region, which interacts with cTnC and cTnI to form the I-T arm of the troponin complex. It is known that the second tropomyosin-binding site of cTnT binds near Cys190 of tropomyosin (Ishii and Lehrer 1991), indicating that cTnT forms an elongated structure between its two tropomyosin-binding sites. cTnT224–274 forms a coiled coil with cTnI89–135, with a small stretch, cTnT259–270, also binding to the C-terminal domain of cTnC (Takeda et al. 2003) (Fig. 2). This arrangement places the C-terminal tail of cTnT, cTnT275–288, spatially adjacent to the key cTnI135–147 inhibitory region that is essential for binding to actin and shutting off muscle contraction in diastole. The exact role of cTnT275–288 is unknown, but it is also important in promoting relaxation (Franklin et al. 2012).
Phosphorylation Modifications of cTnT
Cardiomyopathy-Associated cTnT Mutations
Mutations in cTnT account for approximately 15% of familial HCM cases. Functional studies of HCM-associated mutations of cTnT (I79N, R92W, R92Q, R94L, A104V, R130C, ΔE160, and E244D, numbering from adult heart cTnT3) did not show significant perturbation in maximum force generation or maximum myosin ATPase activity, but showed increased Ca2+ sensitivity in skinned fibers (Lu et al. 2013). Most of the HCM-linked mutations of cTnT reside in the first tropomyosin-binding region and impair the inhibitory role of cTnI in the thin filament. Restrictive cardiomyopathy causing mutations (ΔE96 and E136K) showed significant and large increases in Ca2+ sensitivity in skinned fibers leading to severe diastolic dysfunction (Parvatiyar et al. 2010). A double deletion mutation (N100 and E101) found in a RCM pediatric patient also demonstrated a similar trend of increased Ca2+ sensitivity (Pinto et al. 2011) (Fig. 7). A deletion mutation ΔK210 was reported as a DCM-associated mutation in cTnT, which decreased the Ca2+ sensitivity in force development and also reduced the ATPase activity in cardiac myofibrils (Morimoto et al. 2002). To date, several DCM-linked mutations of cTnT have been reported (R131W, R141W, R205L, ΔK210, and D270N) (Lu et al. 2013). The R141W mutation of cTnT located in tropomyosin-binding site stabilizes the interaction of cTnT with tropomyosin, promoting cTnI-mediated inhibition of the thin filament and causing Ca2+ desensitization (Sommese et al. 2013).
Summary and Future Directions
The cardiac troponin complex plays a central role in turning cardiac contraction on and off with every heartbeat. While its central structural scaffold has been decisively determined by X-ray crystallography, the more disordered regions that interact with tropomyosin and actin need further characterization in order to fully understand the structure and function of the intact complex. Most of the splicing-induced and posttranslational modifications occur in these regions to modulate cardiac function. When these modifications occur in disease, it must be determined how they interact with cardiomyopathy-associated mutations and whether they are pathologic disease-promoting changes or useful physiologic adaptations. Small molecule pharmacotherapy can be used to modulate the functioning of the cardiac troponin complex, either directly through the globular regulatory cNTnC domain or indirectly through signaling pathways. Such treatments can be anticipated to be useful not only in the treatment of heritable cardiomyopathies, but also in a wide range of other cardiac diseases in which heart function is compromised.
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