A novel mutant cardiac troponin C disrupts molecular motions critical for calcium binding affinity and cardiomyocyte contractility

Troponin C (TnC) belongs to the superfamily of EF-hand (helix-loop-helix) Ca²⁺-binding proteins and is an essential component of the regulatory thin filament complex. In a patient diagnosed with idiopathic dilated cardiomyopathy, we identified two novel missense mutations localized in the regulatory Ca²⁺-binding Site II of TnC, TnC^(E59D,D75Y). Expression of recombinant TnC^(E59D,D75Y) in isolated rat cardiomyocytes induced a marked decrease in contractility despite normal intracellular calcium homeostasis in intact cardiomyocytes and resulted in impaired myofilament calcium responsiveness in Triton-permeabilized cardiomyocytes. Expression of the individual mutants in cardiomyocytes showed that TnC^D75Y was able to recapitulate the TnC^(E59D,D75Y) phenotype, whereas TnC^E59D was functionally benign. Force-pCa relationships in TnC^(E59D,D75Y) reconstituted rabbit psoas fibers and fluorescence spectroscopy of TnC^(E59D,D75Y) labeled with 2-[(4′-iodoacetamide)-aniline]naphthalene-6-sulfonic acid showed a decrease in myofilament Ca²⁺ sensitivity and Ca²⁺ binding affinity, respectively. Furthermore, computational analysis of TnC showed the Ca²⁺-binding pocket as an active region of concerted motions, which are decreased markedly by mutation D75Y. We conclude that D75Y interferes with proper concerted motions within the regulatory Ca²⁺-binding pocket of TnC that hinders the relay of the thin filament calcium signal, thereby providing a primary stimulus for impaired cardiomyocyte contractility. This in turn may trigger pathways leading to aberrant ventricular remodeling and ultimately a dilated cardiomyopathy phenotype.     

Cardiac Muscle Activation Blunted by a Mutation to the Regulatory Component,Troponin T

The striated muscle thin filament comprises actin, tropomyosin, and troponin. The Tn complex consists of three subunits, troponin C (TnC), troponin I (TnI), and troponin T (TnT). TnT may serve as a bridge between the Ca²⁺ sensor (TnC) and the actin filament. In the short helix preceding the IT-arm region, H1(T2), there are known dilated cardiomyopathy-linked mutations (among them R205L). Thus we hypothesized that there is an element in this short helix that plays an important role in regulating the muscle contraction, especially in Ca²⁺ activation. We mutated Arg-205 and several other amino acid residues within and near the H1(T2) helix. Utilizing an alanine replacement method to compare the effects of the mutations, the biochemical and mechanical impact on the actomyosin interaction was assessed by solution ATPase activity assay, an in vitro motility assay, and Ca²⁺ binding measurements. Ca²⁺ activation was markedly impaired by a point mutation of the highly conserved basic residue R205A, residing in the short helix H1(T2) of cTnT, whereas the mutations to nearby residues exhibited little effect on function. Interestingly, rigor activation was unchanged between the wild type and R205A TnT. In addition to the reduction in Ca²⁺ sensitivity observed in Ca²⁺ binding to the thin filament, myosin S1-ADP binding to the thin filament was significantly affected by the same mutation, which was also supported by a series of S1 concentration-dependent ATPase assays. These suggest that the R205A mutation alters function through reduction in the nature of cooperative binding of S1.     

Modulation of cardiac troponin C function by the cardiac-specific N-terminus of troponin I: influence of PKA phosphorylation and involvement in cardiomyopathies

The cardiac-specific N-terminus of cardiac troponin I (cTnI) is known to modulate the activity of troponin upon phosphorylation with protein kinase A (PKA) by decreasing its Ca²⁺ affinity and increasing the relaxation rate of the thin filament. The molecular details of this modulation have not been elaborated to date. We have established that the N-terminus and the switch region of cTnI bind to cNTnC [the N-domain of cardiac troponin C (cTnC)] simultaneously and that the PKA signal is transferred via the cTnI N-terminus modulating the cNTnC affinity toward cTnI_147-163 but not toward Ca²⁺. The K_d of cNTnC for cTnI_147-163 was found to be 600 μM in the presence of cTnI_1-29 and 370 μM in the presence of cTn1_1-29PP, which can explain the difference in muscle relaxation rates upon the phosphorylation with PKA in experiments with cardiac fibers. In the light of newly found mutations in cNTnC that are associated with cardiomyopathies, the important role played by the cTnI N-terminus in the development of heart disorders emerges. The mutants studied, L29Q (the N-domain of cTnC containing mutation L29Q) and E59D/D75Y (the N-domain of cTnC containing mutation E59D/D75Y), demonstrated unchanged Ca²⁺ affinity per se and in complex with the cTnI N-terminus (cTnI_1-29 and cTnI_1-29PP). The affinity of L29Q and E59D/D75Y toward cTnI_147-163 was significantly perturbed, both alone and in complex with cTnI_1-29 and cTnI_1-29PP, which is likely to be responsible for the development of malfunctions.     

Predicting Cardiomyopathic Phenotypes by Altering Ca2+ Affinity of Cardiac Troponin C

Cardiac diseases associated with mutations in troponin subunits include hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and restrictive cardiomyopathy (RCM). Altered calcium handling in these diseases is evidenced by changes in the Ca²⁺ sensitivity of contraction. Mutations in the Ca²⁺ sensor, troponin C (TnC), were generated to increase/decrease the Ca²⁺ sensitivity of cardiac skinned fibers to create the characteristic effects of DCM, HCM, and RCM. We also used a reconstituted assay to determine the mutation effects on ATPase activation and inhibition. One mutant (A23Q) was found with HCM-like properties (increased Ca²⁺ sensitivity of force and normal levels of ATPase inhibition). Three mutants (S37G, V44Q, and L48Q) were identified with RCM-like properties (a large increase in Ca²⁺ sensitivity, partial loss of ATPase inhibition, and increased basal force). Two mutations were identified (E40A and I61Q) with DCM properties (decreased Ca²⁺ sensitivity, maximal force recovery, and activation of the ATPase at high [Ca²⁺]). Steady-state fluorescence was utilized to assess Ca²⁺ affinity in isolated cardiac (c)TnCs containing F27W and did not necessarily mirror the fiber Ca²⁺ sensitivity. Circular dichroism of mutant cTnCs revealed a trend where increased α-helical content correlated with increased Ca²⁺ sensitivity in skinned fibers and vice versa. The main findings from this study were as follows: 1) cTnC mutants demonstrated distinct functional phenotypes reminiscent of bona fide HCM, RCM, and DCM mutations; 2) a region in cTnC associated with increased Ca²⁺ sensitivity in skinned fibers was identified; and 3) the F27W reporter mutation affected Ca²⁺ sensitivity, maximal force, and ATPase activation of some mutants.     

Engineered troponin C constructs correct disease-related cardiac myofilament calcium sensitivity

Aberrant myofilament Ca(2+) sensitivity is commonly observed with multiple cardiac diseases, especially familial cardiomyopathies. Although the etiology of the cardiomyopathies remains unclear, improving cardiac muscle Ca(2+) sensitivity through either pharmacological or genetic approaches shows promise of alleviating the disease-related symptoms. Due to its central role as the Ca(2+) sensor for cardiac muscle contraction, troponin C (TnC) stands out as an obvious and versatile target to reset disease-associated myofilament Ca(2+) sensitivity back to normal. To test the hypothesis that aberrant myofilament Ca(2+) sensitivity and its related function can be corrected through rationally engineered TnC constructs, three thin filament protein modifications representing different proteins (troponin I or troponin T), modifications (missense mutation, deletion, or truncation), and disease subtypes (familial or acquired) were studied. A fluorescent TnC was utilized to measure Ca(2+) binding to TnC in the physiologically relevant biochemical model system of reconstituted thin filaments. Consistent with the pathophysiology, the restrictive cardiomyopathy mutation, troponin I R192H, and ischemia-induced truncation of troponin I (residues 1-192) increased the Ca(2+) sensitivity of TnC on the thin filament, whereas the dilated cardiomyopathy mutation, troponin T ΔK210, decreased the Ca(2+) sensitivity of TnC on the thin filament. Rationally engineered TnC constructs corrected the abnormal Ca(2+) sensitivities of the thin filament, reconstituted actomyosin ATPase activity, and force generation in skinned trabeculae. Thus, the present study provides a novel and versatile therapeutic strategy to restore diseased cardiac muscle Ca(2+) sensitivity.     

Functional aberration of myofibrils by cardiomyopathy-causing mutations in the coiled-coil region of the troponin-core domain

Two cardiomyopathy-causing mutations, E244D and K247R, in human cardiac troponin T (TnT) are located in the coiled-coil region of the Tn-core domain. To elucidate effects of mutations in this region on the regulatory function of Tn, we measured Ca²⁺-dependent ATPase activity of myofibrils containing various mutants of TnT at these residues. The results confirmed that the mutant E244D increases the maximum ATPase activity without changing the Ca²⁺-sensitivity. The mutant K247R was shown for the first time to have the effect similar to the mutant E244D. Furthermore, various TnT mutants (E244D, E244M, E244A, E244K, K247R, K247E, and K247A) showed various effects on the maximum ATPase activity while the Ca²⁺-sensitivity was unchanged. Molecular dynamics simulations of the Tn-core containing these TnT mutants suggested that the hydrogen-bond network formed by the side chains of neighboring residues around residues 244 and 247 is important for Tn to function properly.     

Challenging Current Paradigms Related to Cardiomyopathies: ARE CHANGES IN THE Ca2+ SENSITIVITY OF MYOFILAMENTS CONTAINING CARDIAC TROPONIN C MUTATIONS (G159D AND L29Q) GOOD PREDICTORS OF THE PHENOTYPIC OUTCOMES?*

Two novel mutations (G159D and L29Q) in cardiac troponin C (CTnC) associate their phenotypic outcomes with dilated (DCM) and hypertrophic cardiomyopathy (HCM), respectively. Current paradigms propose that sarcomeric mutations associated with DCM decrease the myofilament Ca2+ sensitivity, whereas those associated with HCM increase it. Therefore, we incorporated the mutant CTnCs into skinned cardiac muscle in order to determine if their effects on the Ca2+ sensitivities of tension and ATPase activity coincide with the current paradigms and phenotypic outcomes. The G159D-CTnC decreases the Ca2+ sensitivity of tension and ATPase activation and reduces the maximal ATPase activity when incorporated into regulated actomyosin filaments. Under the same conditions, the L29Q-CTnC has no effect. Surprisingly, changes in the apparent G159D-CTnC Ca2+ affinity measured by tension in fibers do not occur in the isolated CTnC, and large changes measured in the isolated L29Q-CTnC do not manifest in the fiber. These counterintuitive findings are justified through a transition in Ca2+ affinity occurring at the level of cardiac troponin and higher, implying that the true effects of these mutations become apparent as the hierarchical level of the myofilament increases. Therefore, the contractile apparatus, representing a large cooperative machine, can provide the potential for a change (G159D) or no change (L29Q) in the Ca2+ regulation of contraction. In accordance with the clinical outcomes and current paradigms, the desensitization of myofilaments from G159D-CTnC is expected to weaken the contractile force of the myocardium, whereas the lack of myofilament changes from L29Q-CTnC may preserve diastolic and systolic function.     

The Ca/Mg sites of troponin C modulate crossbridge-mediated thin filament activation in cardiac myofibrils

The Ca²⁺/Mg²⁺ sites (III and IV) located in the C-terminal domain of cardiac troponin C (cTnC) have been generally considered to play a purely structural role in keeping the cTnC bound to the thin filament. However, several lines of evidence, including the discovery of cardiomyopathy-associated mutations in the C-domain, have raised the possibility that these sites may have a more complex role in contractile regulation. To explore this possibility, the ATPase activity of rat cardiac myofibrils was assayed under conditions in which no Ca²⁺ was bound to the N-terminal regulatory Ca²⁺-binding site (site II). Myosin-S1 was treated with N-ethylmaleimide to create strong-binding myosin heads (NEM-S1), which could activate the cardiac thin filament in the absence of Ca²⁺. NEM-S1 activation was assayed at pCa 8.0 to 6.5 and in the presence of either 1 mM or 30 μM free Mg²⁺. ATPase activity was maximal when sites III and IV were occupied by Mg²⁺ and it steadily declined as Ca²⁺ displaced Mg²⁺. The data suggest that in the absence of Ca²⁺ at site II strong-binding myosin crossbridges cause the opening of more active sites on the thin filament if the C-domain is occupied by Mg²⁺ rather than Ca²⁺. This finding could be relevant to the contraction–relaxation kinetics of cardiac muscle. As Ca²⁺ dissociates from site II of cTnC during the early relaxing phase of the cardiac cycle, residual Ca²⁺ bound at sites III and IV might facilitate the switching off of the thin filament and the detachment of crossbridges from actin.     

Ca2+-dependent Muscle Dysfunction Caused by Mutation of the Caenorhabditis elegans Troponin T-1 Gene

We have investigated the functions of troponin T (CeTnT-1) in Caenorhabditis elegans embryonic body wall muscle. TnT tethers troponin I (TnI) and troponin C (TnC) to the thin filament via tropomyosin (Tm), and TnT/Tm regulates the activation and inhibition of myosin-actin interaction in response to changes in intracellular [Ca²⁺]. Loss of CeTnT-1 function causes aberrant muscle trembling and tearing of muscle cells from their exoskeletal attachment sites (Myers, C.D., P.-Y. Goh, T. StC. Allen, E.A. Bucher, and T. Bogaert. 1996. J. Cell Biol. 132:1061–1077). We hypothesized that muscle tearing is a consequence of excessive force generation resulting from defective tethering of Tn complex proteins. Biochemical studies suggest that such defective tethering would result in either (a) Ca²⁺-independent activation, due to lack of Tn complex binding and consequent lack of inhibition, or (b) delayed reestablishment of TnI/TnC binding to the thin filament after Ca²⁺ activation and consequent abnormal duration of force. Analyses of animals doubly mutant for CeTnT-1 and for genes required for Ca²⁺ signaling support that CeTnT-1 phenotypes are dependent on Ca²⁺ signaling, thus supporting the second model and providing new in vivo evidence that full inhibition of thin filaments in low [Ca²⁺] does not require TnT.     

A troponin T mutation that causes infantile restrictive cardiomyopathy increases Ca2+ sensitivity of force development and impairs the inhibitory properties of troponin

Restrictive cardiomyopathy (RCM) is a rare disorder characterized by impaired ventricular filling with decreased diastolic volume. We are reporting the functional effects of the first cardiac troponin T (CTnT) mutation linked to infantile RCM resulting from a de novo deletion mutation of glutamic acid 96. The mutation was introduced into adult and fetal isoforms of human cardiac TnT (HCTnT3-DeltaE96 and HCTnT1-DeltaE106, respectively) and studied with either cardiac troponin I (CTnI) or slow skeletal troponin I (SSTnI). Skinned cardiac fiber measurements showed a large leftward shift in the Ca(2+) sensitivity of force development with no differences in the maximal force. HCTnT1-DeltaE106 showed a significant increase in the activation of actomyosin ATPase with either CTnI or SSTnI, whereas HCTnT3-DeltaE96 was only able to increase the ATPase activity with CTnI. Both mutants showed an impaired ability to inhibit the ATPase activity. The capacity of the CTnI.CTnC and SSTnI.CTnC complexes to fully relax the fibers after TnT displacement was also compromised. Experiments performed using fetal troponin isoforms showed a less severe impact compared with the adult isoforms, which is consistent with the cardioprotective role of SSTnI and the rapid onset of RCM after birth following the isoform switch. These data indicate that troponin mutations related to RCM may have specific functional phenotypes, including large leftward shifts in the Ca(2+) sensitivity and impaired abilities to inhibit ATPase and to relax skinned fibers. All of this would account for and contribute to the severe diastolic dysfunction seen in RCM.     

Fetal cardiac troponin isoforms rescue the increased Ca2+ sensitivity produced by a novel double deletion in cardiac troponin T linked to restrictive cardiomyopathy: a clinical, genetic, and functional approach

A novel double deletion in cardiac troponin T (cTnT) of two highly conserved amino acids (Asn-100 and Glu-101) was found in a restrictive cardiomyopathic (RCM) pediatric patient. Clinical evaluation revealed the presence of left atrial enlargement and marked left ventricle diastolic dysfunction. The explanted heart examined by electron microscopy revealed myofibrillar disarray and mild fibrosis. Pedigree analysis established that this mutation arose de novo. The patient tested negative for six other sarcomeric genes. The single and double recombinant cTnT mutants were generated, and their functional consequences were analyzed in porcine skinned cardiac muscle. In the adult Tn environment (cTnT3 + cardiac troponin I), the single cTnT3-ΔN100 and cTnT3-ΔE101 mutations had opposing effects on the Ca(2+) sensitivity of force development compared with WT, whereas the double deletion cTnT3-ΔN100/ΔE101 increased the Ca(2+) sensitivity + 0.19 pCa units. In addition, cTnT3-ΔN100/ΔE101 decreased the cooperativity of force development, suggesting alterations in intrafilament protein-protein interactions. In the fetal Tn environment, (cTnT1 + slow skeletal troponin I), the single (cTnT1-ΔN110) and double (cTnT1-ΔN110/ΔE111) deletions did not change the Ca(2+) sensitivity compared with control. To recreate the patient's heterozygous genotype, we performed a reconstituted ATPase activity assay. Thin filaments containing 50:50 cTnT3-ΔN100/ΔE101:cTnT3-WT also increased the myofilament Ca(2+) sensitivity compared with WT. Co-sedimentation of thin filament proteins indicated that no significant changes occurred in the binding of Tn containing the RCM cTnT mutation to actin-Tm. This report reveals the protective role of Tn fetal isoforms as they rescue the increased Ca(2+) sensitivity produced by a cTnT-RCM mutation and may account for the lack of lethality during gestation.     

A Functional and Structural Study of Troponin C Mutations Related to Hypertrophic Cardiomyopathy

Recently four new hypertrophic cardiomyopathy mutations in cardiac troponin C (cTnC) (A8V, C84Y, E134D, and D145E) were reported, and their effects on the Ca²⁺ sensitivity of force development were evaluated (Landstrom, A. P., Parvatiyar, M. S., Pinto, J. R., Marquardt, M. L., Bos, J. M., Tester, D. J., Ommen, S. R., Potter, J. D., and Ackerman, M. J. (2008) J. Mol. Cell. Cardiol. 45, 281–288). We performed actomyosin ATPase and spectroscopic solution studies to investigate the molecular properties of these mutations. Actomyosin ATPase activity was measured as a function of [Ca²⁺] utilizing reconstituted thin filaments (TFs) with 50% mutant and 50% wild type (WT) and 100% mutant cardiac troponin (cTn) complexes: A8V, C84Y, and D145E increased the Ca²⁺ sensitivity with only A8V demonstrating lowered Ca²⁺ sensitization at the 50% ratio when compared with 100%; E134D was the same as WT at both ratios. Of these four mutants, only D145E showed increased ATPase activation in the presence of Ca²⁺. None of the mutants affected ATPase inhibition or the binding of cTn to the TF measured by co-sedimentation. Only D145E increased the Ca²⁺ affinity of site II measured by 2-(4′-(2″-iodoacetamido)phenyl)aminonaphthalene-6-sulfonic acid fluorescence in isolated cTnC or the cTn complex. In the presence of the TF, only A8V was further sensitized to Ca²⁺. Circular dichroism measurements in different metal-bound states of the isolated cTnCs showed changes in the secondary structure of A8V, C84Y, and D145E, whereas E134D was the same as WT. PyMol modeling of each cTnC mutant within the cTn complex revealed potential for local changes in the tertiary structure of A8V, C84Y, and D145E. Our results indicate that 1) three of the hypertrophic cardiomyopathy cTnC mutants increased the Ca²⁺ sensitivity of the myofilament; 2) the effects of the mutations on the Ca²⁺ affinity of isolated cTnC, cTn, and TF are not sufficient to explain the large Ca²⁺ sensitivity changes seen in reconstituted and fiber assays; and 3) changes in the secondary structure of the cTnC mutants may contribute to modified protein-protein interactions along the sarcomere lattice disrupting the coupling between the cross-bridge and Ca²⁺ binding to cTnC.Hypertrophic cardiomyopathy (HCM)³ is typically inherited as an autosomal dominant disease that is caused by mutations in sarcomeric genes and is the most prevalent cause of sudden death in athletes and young people (1, 2). The clinical hallmark of HCM is an increased thickness of the left ventricular wall. Myocyte disarray, fibrosis, septal hypertrophy, and abnormal diastolic function can also be present in HCM patients (3). HCM mutations have been reported in 13 myofilament-related genes; however, the cardiac troponin C (cTnC) gene remained excluded from this list (4–7). The clinical and functional phenotypes may vary according to the gene and the location of the mutation (8). Recently our group has reported evidence that brings cTnC into focus as an HCM susceptibility gene (9). Interestingly the prevalence for cTnC HCM mutations was the same as other well characterized genes (i.e. actin and tropomyosin) (6). To date, prior to our recent report, only one mutation in cTnC (L29Q) had been linked to HCM (10). In vitro and in situ studies demonstrating changes in the functional parameters of cardiac muscle regulation suggest that this mutation is causative of the disease (11, 12).Analysis of a cohort of 1025 HCM patients from the Mayo Clinic revealed four new cTnC mutations (A8V, C84Y, E134D, and D145E) (9). The clinical records showed that the patients displayed left ventricle hypertrophy and significant left ventricular outflow obstruction managed by surgical myectomy. Symptoms such as dyspnea, syncope, and chest pain were also present. A8V, C84Y, and E134D patients did not present a familial history of HCM indicating that these were likely sporadic de novo mutations. The D145E mutation was observed in six family members suggesting genetic linkage. Functional analysis performed in skinned fibers showed increased Ca²⁺ sensitivity of force development (a characteristic of troponin (Tn) mutations related to HCM) for three of the four mutations. Additionally the A8V and D145E mutations that are located in different domains caused increases in maximal force in this system. These data strongly suggest that HCM mutations in distinct regions of cTnC can result in a similar functional phenotype (9).In cardiac muscle, the tropomyosin (Tm)·Tn complex, located in the thin filament, is responsible for muscle regulation (13, 14). Three Tn subunits are involved in this process: troponin T (TnT), which connects the Tn complex to the thin filament and is responsible for actomyosin ATPase activation in the presence of Ca²⁺ (8, 15); troponin I (TnI) is the subunit that binds to both TnT and TnC, inhibits muscle contraction, and is also implicated in HCM and restrictive cardiomyopathy (16); and TnC, a subunit that plays a crucial function in muscle regulation triggering contraction upon binding Ca²⁺ and is also considered an important intracellular Ca²⁺ buffer (17, 18). In the absence of Ca²⁺ binding to site II of cTnC, its N terminus is detached from the C terminus of cTnI, which under these conditions is bound to actin and inhibits muscle contraction. As Ca²⁺ binds to site II of cTnC, its N terminus binds to the C terminus of cTnI causing it to dissociate from actin. This is accompanied by the movement of cardiac Tm out of its inhibitory position on actin, thus relieving the inhibition of contraction (19–21). The dynamics of the interactions between Tn subunits and the thin filament that regulate contraction have been extensively studied (22–24).TnC consists of two globular regions that are connected by a long central helix (25). It is well known that cTnC has two EF-hands containing high affinity Ca²⁺ binding sites III and IV (∼10⁷ m⁻¹) in the C terminus and only one functional low affinity Ca²⁺ binding site II (∼10⁵ m⁻¹) in the N terminus (18). An additional feature of helix-loop-helix Ca²⁺-binding proteins is the presence of short segments of antiparallel β-sheets between the Ca²⁺ binding loops of each domain (25, 26). The C-terminal domain of cTnC can also bind Mg²⁺ competitively (∼10³ m⁻¹) and is termed the structural domain because it is essential to keep it bound to the thin filament. The N terminus is considered the regulatory domain because Ca²⁺ binding to site II initiates muscle contraction. When TnC is in the Tn complex, the Ca²⁺ binding affinity at all sites is increased by ∼10-fold (18, 27, 28). Several studies have shown that there is coupling between TnC and actomyosin ATPase. For example, bepridil and calmidazolium, two known Ca²⁺ sensitizers that bind to cTnC and enhance its Ca²⁺ binding affinity, also stimulate myofibrillar ATPase activity (29, 30). In addition, deletion of the N-helix of the TnC N-domain diminishes activation of regulated actomyosin ATPase activity (31, 32).The purpose of this study was to determine the functional effects of the four newly discovered HCM cTnC mutations not previously addressed and to investigate possible changes in their structure and Ca²⁺ binding properties. To answer these questions we performed reconstituted ATPase activity, co-sedimentation, and spectroscopy assays. In the presence of 100% HCM mutant or wild type (WT) cTnC, the ATPase activity rate measured by increasing the Ca²⁺ concentration in an actomyosin·Tm·Tn reconstituted complex showed increases in Ca²⁺ sensitivity similar to those obtained previously with cardiac skinned fibers (9). At a ratio of 50% mutant to 50% WT, only A8V had a diminished Ca²⁺ sensitivity. We also evaluated the ability of the Tn HCM mutants to activate and inhibit the ATPase activity in the presence and absence of Ca²⁺. Only cTnC-D145E showed higher levels of ATPase activation. Co-sedimentation did not show changes in the ability of the Tn complex containing the cTnC mutants to bind to actin·Tm. The Ca²⁺ binding properties of the regulatory site II of cTnC as estimated from fluorescence and measured at cTnC and cTn levels did not match the apparent affinity of this site in the fiber and reconstituted filaments. However, D145E showed increased Ca²⁺ affinity in the isolated and cTn states that was minimally affected in the presence of the thin filament (TF). In the presence of the TF, A8V was the only mutant that showed an increase in Ca²⁺ affinity that more closely approached the Ca²⁺ sensitivity measured in the fiber. However, the circular dichroism (CD) measurements suggest that significant structural changes exist in the secondary structure of the cTnC mutants A8V, C84Y, and D145E compared with wild type. All of these results considered together with the PyMol illustrations suggest that structural changes are present in at least three TnC HCM mutants that are likely to be crucial for protein-protein interactions but unable to affect the Ca²⁺ binding properties of TnC at the different levels of TF complexity. Here we show for the first time that the thick filament is probably essential to completely recreate the increased Ca²⁺ sensitivity produced by HCM TnCs and observed in ATPase and skinned fiber assays.     

In vivo effects of propyl gallate,a novel Ca sensitizer,in a mouse model of dilated cardiomyopathy caused by cardiac troponin T mutation

Aims

We have previously demonstrated that propyl gallate has a Ca^2 + sensitizing effect on the force generation in membrane-permeabilized (skinned) cardiac muscle fibers. However, in vivo beneficial effects of propyl gallate as a novel Ca^2 + sensitizer remain uncertain. In the present study, we aim to explore in vivo effects of propyl gallate.

Main methods

We compared effects of propyl gallate on ex vivo intact cardiac muscle fibers and in vivo hearts in healthy mice with those of pimobendan, a clinically used Ca^2 + sensitizer. The therapeutic effect of propyl gallate was investigated using a mouse model of dilated cardiomyopathy (DCM) with reduced myofilament Ca^2 + sensitivity due to a deletion mutation ΔK210 in cardiac troponin T.

Key findings

Propyl gallate, as well as pimobendan, showed a positive inotropic effect. Propyl gallate slightly increased the blood pressure without changing the heart rate in healthy mice, whereas pimobendan decreased the blood pressure probably through vasodilation via inhibition of phosphodiesterase and increased the heart rate. Propyl gallate prevented cardiac remodeling and systolic dysfunction and significantly improved the life-expectancy of knock-in mouse model of DCM with reduced myofilament Ca^2 + sensitivity due to a mutation in cardiac troponin T. On the other hand, gallate, a similarly strong antioxidant polyphenol lacking Ca^2 + sensitizing action, had no beneficial effects on the DCM mice.

Significance

These results suggest that propyl gallate might be useful for the treatment of inherited DCM caused by a reduction in the myofilament Ca^2 + sensitivity.     

Functional characterization of TNNC1 rare variants identified in dilated cardiomyopathy

TNNC1, which encodes cardiac troponin C (cTnC), remains elusive as a dilated cardiomyopathy (DCM) gene. Here, we report the clinical, genetic, and functional characterization of four TNNC1 rare variants (Y5H, M103I, D145E, and I148V), all previously reported by us in association with DCM (Hershberger, R. E., Norton, N., Morales, A., Li, D., Siegfried, J. D., and Gonzalez-Quintana, J. (2010) Circ. Cardiovasc. Genet. 3, 155-161); in the previous study, two variants (Y5H and D145E) were identified in subjects who also carried MYH7 and MYBPC3 rare variants, respectively. Functional studies using the recombinant human mutant cTnC proteins reconstituted into porcine papillary skinned fibers showed decreased Ca(2+) sensitivity of force development (Y5H and M103I). Furthermore, the cTnC mutants diminished (Y5H and I148V) or abolished (M103I) the effects of PKA phosphorylation on Ca(2+) sensitivity. Only M103I decreased the troponin activation properties of the actomyosin ATPase when Ca(2+) was present. CD spectroscopic studies of apo (absence of divalent cations)-, Mg(2+)-, and Ca(2+)/Mg(2+)-bound states indicated that all of the cTnC mutants (except I148V in the Ca(2+)/Mg(2+) condition) decreased the α-helical content. These results suggest that each mutation alters the function/ability of the myofilament to bind Ca(2+) as a result of modifications in cTnC structure. One variant (D145E) that was previously reported in association with hypertrophic cardiomyopathy and that produced results in vivo in this study consistent with prior hypertrophic cardiomyopathy functional studies was found associated with the MYBPC3 P910T rare variant, likely contributing to the observed DCM phenotype. We conclude that these rare variants alter the regulation of contraction in some way, and the combined clinical, molecular, genetic, and functional data reinforce the importance of TNNC1 rare variants in the pathogenesis of DCM.     

The control of myocardial contraction with skeletal fast muscle troponin C

The present study describes experiments on the myocardial trabeculae from the right ventricle of Syrian hamsters whose troponin C (TnC) moiety was exchanged with heterologous TnC from fast skeletal muscle of the rabbit. These experiments were designed to help define the role of the various classes of Ca2+-binding sites on TnC in setting the characteristic sensitivities for activations of cardiac and skeletal muscles. Thin trabeculae were skinned and about 75% of their troponin C extracted by chemical treatment. Tension development on activations by Ca2+ and Sr2+ was found to be nearly fully blocked in such TnC extracted preparations. Troponin C contents and the ability to develop tension on activations by Ca2+ and Sr2+ was permanently restored after incubation with 2-6 mg/ml purified TnC from either rabbit fast-twitch skeletal muscle (STnC) or the heart (CTnC, cardiac troponin C). The native (skinned) cardiac muscle is characteristically about 5 times more sensitive to activation by Sr2+ than fast muscle, but the STnC-loaded trabeculae gave response like fast muscle. Attempts were also made to exchange the TnC in psoas (fast-twitch muscle) fibers, but unlike cardiac muscle tension response of the maximally extracted psoas fibers could be restored only with homologous STnC. CTnC was effective in partially extracted fibers, even though the uptake of CTnC was complete in the maximally extracted fibers. The results in this study establish that troponin C subunit is the key in setting the characteristic sensitivity for tension control in the myocardium above that in the skeletal muscle. Since a major difference between skeletal and cardiac TnCs is that one of the trigger sites (site I, residues 28-40 from the N terminus) is modified in CTnC and has reduced affinity for Ca2+ binding, the possibility is raised that this site has a modulatory effect on activation in different tissues and limits the effectiveness of CTnC in skeletal fibers.     

The Functional Properties of Human Slow Skeletal Troponin T Isoforms in Cardiac Muscle Regulation

Human slow skeletal troponin T (HSSTnT) shares a high degree of homology with cardiac TnT (CTnT). Although the presence of HSSTnT has not been confirmed in the heart at the protein level, detectable levels of HSSTnT mRNA have been found. Whether HSSTnT isoforms are expressed transiently remains unknown. Because transient re-expression of HSSTnT may be a potential mechanism of regulating function, we explored the effect of HSSTnT on the regulation of cardiac muscle. At least three HSSTnT isoforms have been found to exist in slow skeletal muscle: HSSTnT1 (+exons 5 and 12), HSSTnT2 (+exon 5, −exon 12), and HSSTnT3 (−exons 5 and 12). Another isoform, HSSTnT hypothetical (Hyp) (−exon 5, +exon 12), has only been found at the mRNA level. Compared with HCTnT3 (adult isoform), Tn complexes containing HSSTnT1, -2, and -3 did not alter the actomyosin ATPase activation and inhibition in the presence and absence of Ca²⁺, respectively. HSSTnTHyp was not evaluated as it did not form a Tn complex under a variety of conditions. Porcine papillary skinned fibers displaced with HSSTnT1, -2, or -3 and reconstituted with human cardiac troponin I and troponin C (HCTnI·TnC) complex showed a decrease in the Ca²⁺ sensitivity of force development and an increase in maximal recovered force (HSSTnT1 and -3) compared with HCTnT3. In contrast, HSSTnTHyp showed an increase in the Ca²⁺ sensitivity of force development. This suggests that re- or overexpression of specific SSTnT isoforms might have therapeutic potential in the failing heart because they increase the maximal force of contraction. In addition, circular dichroism and proteolytic digestion experiments revealed structural differences between HSSTnT isoforms and HCTnT3 and that HSSTnT1 is more susceptible to calpain and trypsin proteolysis than the other HSSTnTs. Overall, HSSTnT isoforms despite being homologues of CTnT may display distinct functional properties in muscle regulation.     

Structural and functional properties of the non-muscle tropomyosins

Summary The non-muscle tropomyosins (TMs), isolated from such tissues as platelets, brain and thyroid, are structurally very similar to the muscle TMs, being composed of two highly -helical subunits wound around each other to form a rod-like molecule. The non-muscle TMs are shorter than the muscle TMs; sequence analysis demonstrates that each subunit of equine platelet TM consists of 247 amino acids, 37 fewer than for skeletal muscle TM. The major differences in sequence between platelet and skeletal muscle TM are found near the amino and carboxyl terminal ends of the proteins. Probably as the result of such alterations, the non-muscle TMs aggregate in a linear end-to-end manner much more weakly than do the muscle TMs. Since end-to-end interactions are responsible for the highly cooperative manner in which TM binds to actin, the non-muscle TMs have a lower affinity for actin filaments than do the muscle TMs. However, the attachment of other proteins to actin (e.g. the Tn-I subunit of skeletal muscle troponin or the S-1 subfragment of skeletal muscle myosin) can increase the affinity of actin filaments for non-muscle TM. The non-muscle TMs interact functionally with the Tn-I component of skeletal muscle troponin to inhibit the ATPase activity of muscle actomyosin and with whole troponin to regulate the muscle actomyosin ATPase in a Ca⁺⁺-dependent manner, even though one of the binding sites for troponin on skeletal TM is missing in non-muscle TM. A novel actomyosin regulatory system can be produced using Tn-I, calmodulin and non-muscle TM; in this case inhibition is released when the non-muscle TM detaches from the actin filament in the presence of Ca⁺⁺. Although it has not yet been demonstrated that the non-muscle TMs participate in a Ca⁺⁺-dependent contractile regulatory system in vivo it does appear that they are associated with actin filaments in vivo.     

A Mutation in TNNC1-encoded Cardiac Troponin C, TNNC1-A31S, Predisposes to Hypertrophic Cardiomyopathy and Ventricular Fibrillation

Defined as clinically unexplained hypertrophy of the left ventricle, hypertrophic cardiomyopathy (HCM) is traditionally understood as a disease of the cardiac sarcomere. Mutations in TNNC1-encoded cardiac troponin C (cTnC) are a relatively rare cause of HCM. Here, we report clinical and functional characterization of a novel TNNC1 mutation, A31S, identified in a pediatric HCM proband with multiple episodes of ventricular fibrillation and aborted sudden cardiac death. Diagnosed at age 5, the proband is family history-negative for HCM or sudden cardiac death, suggesting a de novo mutation. TnC-extracted cardiac skinned fibers were reconstituted with the cTnC-A31S mutant, which increased Ca(2+) sensitivity with no effect on the maximal contractile force generation. Reconstituted actomyosin ATPase assays with 50% cTnC-A31S:50% cTnC-WT demonstrated Ca(2+) sensitivity that was intermediate between 100% cTnC-A31S and 100% cTnC-WT, whereas the mutant increased the activation of the actomyosin ATPase without affecting the inhibitory qualities of the ATPase. The secondary structure of the cTnC mutant was evaluated by circular dichroism, which did not indicate global changes in structure. Fluorescence studies demonstrated increased Ca(2+) affinity in isolated cTnC, the troponin complex, thin filament, and to a lesser degree, thin filament with myosin subfragment 1. These results suggest that this mutation has a direct effect on the Ca(2+) sensitivity of the myofilament, which may alter Ca(2+) handling and contribute to the arrhythmogenesis observed in the proband. In summary, we report a novel mutation in the TNNC1 gene that is associated with HCM pathogenesis and may predispose to the pathogenesis of a fatal arrhythmogenic subtype of HCM.     

Troponin replacement in permeabilized cardiac muscle Reversible extraction of troponin I by incubation with vanadate

Calcium-dependent regulation of tension and ATPase activity in permeabilized porcine ventricular muscle was lost after incubation with 10 mM vanadate. After transfer from vanadate to a vanadate-free, low-Ca²⁺ solution (pCa> 8), the permeabilized muscle produced 84.8% ± 20.1% (± S.D., n=98) of the isometric force elicited by high Ca2²⁺ (pCa 4.5 prior to incubation with vanadate. Transfer back to a high Ca²⁺ solution elicited no additional force (83.2% ± 18.7% of control force). SDS-PAGE and immunoblot analysis of fibers and solutions demonstrated substantial extraction (>90%) of Troponin I (TnI). Calcium dependence was restored after incubation with solutions containing either whole cardiac troponin or a combination of TnI and troponin C subunits. This reversible extraction of troponin directly demonstrates the role of TnI in the regulation of striated muscle contractility and permits specific substitution of the native TnI with exogenously supplied protein.     

Preserved Close Linkage Between the Genes Encoding Troponin I and Troponin T, Reflecting an Evolution of Adapter Proteins Coupling the Ca2+ Signaling of Contractility

Ca²⁺-regulated motility is essential to numerous cellular functions, including muscle contraction. Systems with troponin C, myosin light chain, or calmodulin as the Ca²⁺ receptor have evolved in striated muscle and other types of cells to transduce the cytoplasm Ca²⁺ signals into allosteric conformational changes of contractile proteins. While these Ca²⁺ receptors are homologous proteins, their coupling to the responding elements is quite different in various cell types. The Ca²⁺ regulatory system in vertebrate striated muscle represents a highly specialized such signal transduction pathway consisting of the troponin complex and tropomyosin associated with the actin filament. To understand the molecular mechanism in the Ca²⁺ regulation of muscle contraction and cell motility, we have revealed a preserved ancestral close linkage between the genes encoding two of the troponin subunits, troponin I and troponin T, in the genome of mouse. The data suggest that the troponin I and troponin T genes may have originated from a single locus and evolved in parallel to encode a striated muscle-specific adapter to couple the Ca²⁺ receptor, troponin C, to the actin–myosin contractile machinery. This hypothesis views the three troponin subunits as two structure–function domains: the Ca²⁺ receptor and the signal transducing adapter. This model may help to further our understanding of the Ca²⁺ regulation of muscle contraction and the structure–function relationship of other potential adapter proteins which are converged to constitute the Ca²⁺ signal transduction pathways governing nonmuscle cell motility. Received: 15 April 1999 / Accepted: 15 July 1999