Cardiac Troponin C Research Articles

Essential Treatment of Covid19 Patients

I have recently seen the Italian study of cardiac troponinC in the blood of patients (1).

Cardiac troponins may be irreversibly modified by glycation: novel potential mechanisms of cardiac performance modulation

Dynamic movements of the cardiac troponin complex are an important component of the cardiac cycle. Whether cardiac troponins are subjected to irreversible advanced glycation end-product (AGE) modification is unknown. This study interrogated human and rat cardiac troponin-C, troponin-I and troponin-T to identify endogenous AGE modifications using mass spectrometry (LC-MS/MS). AGE modifications were detected on two amino acid residues of human troponin-C (Lys6, Lys39), thirteen troponin-I residues (Lys36, Lys50, Lys58, Arg79, Lys117, Lys120, Lys131, Arg148, Arg162, Lys164, Lys183, Lys193, Arg204), and three troponin-T residues (Lys107, Lys125, Lys227). AGE modifications of three corresponding troponin-I residues (Lys58, Lys120, Lys194) and two corresponding troponin-T residues (Lys107, Lys227) were confirmed in cardiac tissue extracts from an experimental rodent diabetic model. Additionally, novel human troponin-I phosphorylation sites were detected (Thr119, Thr123). Accelerated AGE modification of troponin-C was evident in vitro with hexose sugar exposure. This study provides the first demonstration of the occurrence of cardiac troponin complex AGE-modifications. These irreversible AGE modifications are situated in regions of the troponin complex known to be important in myofilament relaxation, and may be of particular pathological importance in the pro-glycation environment of diabetic cardiomyopathy.

Molecular Dynamics and Umbrella Sampling Simulations Elucidate Differences in Troponin C Isoform and Mutant Hydrophobic Patch Exposure.

Troponin C (TnC) facilitates muscle contraction through calcium-binding within its N-terminal region (NTnC). As previously observed using molecular dynamics (MD) simulations, this calcium-binding event leads to an increase in the dynamics of helices lining a hydrophobic patch on TnC. Simulation times of multiple microseconds were required to even see a partial opening of the hydrophobic patch, limiting the ability to thoroughly and quantitatively investigate these rare events. Here we describe the application of umbrella sampling to probe the TnC hydrophobic patch opening in a more targeted and quantitative fashion. Umbrella sampling was utilized to investigate the differences in the free energy of opening between cardiac (cTnC) and fast skeletal TnC (sTnC). We found that, in agreement with previous reports, holo (calcium-bound) sTnC had a lower free energy of opening compared with holo cTnC. Additionally, differences in the free energy of opening of hypertrophic (HCM) and dilated cardiomyopathy (DCM) cTnC systems were investigated. MD simulations and umbrella sampling revealed a lower free energy of opening for the HCM mutations A8V and A31S, as well as the calcium-sensitizing mutation L48Q. The DCM mutations, Y5H, Q50R, and E59D/D75Y, all exhibited a higher free energy of opening. An umbrella sampling simulation of cTnI-bound holo cTnC exhibited the lowest free energy in the open configuration, in agreement with experimental data. In conclusion, this study presents a novel and successful protocol for applying umbrella sampling simulations to quantitatively study the molecular basis of muscle contraction and proposes a mechanism by which HCM and DCM-associated mutations influence contraction.

Cardiomyopathy-Linked Mutation K15N in Tropomyosin Alters Calcium-Dependent Regulation of Reconstituted Cardiac Thin Filaments

In striated muscle, actin-based thin filaments and myosin-based thick filaments are organized into basic contractile units called sarcomeres. Tropomyosin (Tpm), a coiled-coil dimer, polymerizes head-to-tail along the thin filament, stabilizes the filament and, together with the troponin complex, regulates Ca2+-dependent actomyosin interactions which are molecular prerequisites for muscle contraction. Troponin, consisting of troponin I (TnI), troponin C (TnC), and troponin T (TnT), initiates muscle contraction through a series of structural changes within the complex, which is triggered by the binding of Ca2+ to TnC. The structural changes spread through TnT which in turn move Tpm's position on the thin filament, enabling actomyosin interactions. In this work, we studied two missense mutations in striated muscle tropomyosin (Tpm1.1), K15N and R21H, which are associated with dilated cardiomyopathy and hypertrophic cardiomyopathy, respectively. We investigated the mutations’ effects on Tpm1.1's affinity to F-actin and on the Ca2+-dependent regulation of reconstituted cardiac thin filaments using Fluorescence Resonance Energy Transfer (FRET). We labeled residues 151 and 167 of cardiac TnI with IAEDANS as FRET donors, and residue 89 of cardiac TnC with DDPM as FRET acceptor. We found that while both mutations reduced Tpm1.1's affinity to F-actin, the K15N mutation decreased Ca2+-sensitivity and Ca2+-dissociation-induced kinetics of reconstituted cardiac thin filaments. In the presence of the K15N mutant, the interprobe distances were greater than in the presence of wild type Tpm1.1, indicating that the mutation affected cation-induced conformational changes within troponin. Our data demonstrated that the K15N mutation in Tpm1.1 affected Ca2+-dependent thin filament regulation while the R21H mutation had almost no effect.

Key functional domains of human cardiac troponin-C are susceptible to advanced glycation end-product formation in vitro

Key functional domains of human cardiac troponin-C are susceptible to advanced glycation end-product formation in vitro

Molecular Dynamics-Based Predictions of the Structural and Functional Differences Between the Cardiac and Novel Slow-Skeletal Isoforms of Zebrafish Troponin C

Troponin C (TnC), the Ca2+-binding member of the Troponin complex, is responsible for initiating myocyte contraction. The sequence of TnC is highly conserved across vertebrates; however, TnC expressed in the hearts of many teleosts is the product of two distinct genes: cardiac TnC (cTnC) and the fish-specific slow skeletal TnC (ssTnC). In Zebrafish, ssTnC differs from cTnC by 18 residues, however changes can be measured the ΔG of simulated Ca2+ binding, and in the expression of each isoform in temperature acclimated Zebrafish.The cardiac and slow-skeletal isoforms of Zebrafish TnC were modeled using human cTnC as a template. The models were then equilibrated at either 18°C or 28°C, and the resultant structures were very similar. Individually, the N & C domains superimpose very well, although the relative positions of the domains are variable due to the flexibility of the D-E linker region of cTnC.The free energy of Ca2+ binding was calculated for each isoform at each temperature. The results indicate that there were small differences between the two isoforms at 18°C, the ΔG of binding for the slow-skeletal isoform is 11% lower than that of cardiac. At 28°C, the ΔG of binding for the slow-skeletal isoform was 43 % higher than that of the cardiac isoform.A series of qPCR experiments demonstrated that at 28°C, the ssTnC mRNA levels are twofold greater than cTnC mRNA levels, while at 18°C, cTnC expression is 1.5-fold higher over ssTnC in the Zebrafish atrium.Taken together, these results suggest that the increase in expression of the slow skeletal isoform at higher temperatures compensates for the decrease in the ability of the cardiac isoform to bind calcium.

Differences in Troponin C Dynamics Between Cardiac and Skeletal Muscle - A Molecular Dynamics Perspective

Troponin (Tn), part of the thin filament in cardiac as well as skeletal muscle, plays an important role in calcium signaling events in muscle contraction. It acts as a Ca2+-dependent switch, activating and deactivating the myofilament leading to contraction and relaxation of the muscle cell. An important mechanism in the regulation of contraction is the opening up of the Troponin C (TnC) hydrophobic patch to allow Troponin I (TnI) to bind. Two main structural differences are observed between cardiac TnC (cTnC) and skeletal TnC (sTnC): sTnC binds two calcium ions in its N-terminal regulatory domain, while cTnC only binds one calcium ion. The calcium binding site I in cTnC is inactive due to mutations. Additionally, the hydrophobic patch between helices A and B in the N-terminal regulatory domain is open in sTnC, while it is closed in cTnC. In previous work we performed microsecond molecular dynamics (MD) simulations of cTnC in different states of calcium binding to estimate the free energy difference for opening of the hydrophobic patch. Here we present long time-scale MD simulations of sTnC in its apo, one Ca2+-bound and two Ca2+-bound forms. We observe a 10,000-fold increased opening frequency in sTnC compared to cTnC. The dramatic difference is caused almost entirely by changes in the binding site I dynamics upon calcium binding. Investigations of sTnC mutations shown to prevent calcium binding to site I shed further light on the differences between the TnC isoforms. Additionally, Brownian dynamics simulations are used to investigate TnI association with TnC. Simulations of a full length troponin model elucidate the dynamical interplay between the TnC, TnI and TnT subunits.

Adult teleost heart expresses two distinct troponin C paralogs: cardiac TnC and a novel and teleost-specific ssTnC in a chamber- and temperature-dependent manner

The teleost-specific whole genome duplication created multiple copies of genes allowing for subfunctionalization of isoforms. In this study, we show that the teleost cardiac Ca2+-binding troponin C (TnC) is the product of two distinct genes: cardiac TnC (cTnC, TnnC1a) and a fish-specific slow skeletal TnC (ssTnC, TnnC1b). The ssTnC gene is novel to teleosts as mammals have a single gene commonly referred as cTnC but which is also expressed in slow skeletal muscle. In teleosts, the data strongly indicate that these are two TnC genes are different paralogs. Because we determined that ssTnC exists across many teleosts but not in basal ray-finned fish (e.g., bichir), we propose that these paralogs are the result of an ancestral tandem gene duplication persisting only in teleosts. Quantification of mRNA levels was used to demonstrate distinct expression localization patterns of the paralogs within the chambers of the heart. In the adult zebrafish acclimated at 28°C, ssTnC mRNA levels are twofold greater than cTnC mRNA levels in the atrium, whereas cTnC mRNA was almost exclusively expressed in the ventricle. Meanwhile, rainbow trout acclimated at 5°C showed cTnC mRNA levels in both chambers significantly greater than ssTnC. Distinct responses to temperature acclimation were also quantified in both adult zebrafish and rainbow trout, with mRNA in both chambers shifting to express higher levels of cTnC in 18°C acclimated zebrafish and 5°C acclimated trout. Possible subfunctionalization of TnC isoforms may provide insight into how teleosts achieve physiological versatility in chamber-specific contractile properties.

Temperature regulation of fish‐specific paralogs of cardiac TnC

Temperature‐dependent regulation of factors involved with cardiac contractility is crucial for ectothermic fish facing changes in temperature. The teleost‐specific whole genome duplication created multiple copies of genes allowing for sub‐functionalization of isoforms. The Ca2+‐binding troponin C (TnC), responsible for initiating myocyte contraction is highly conserved across vertebrates due to its crucial role in regulating contractility. TnC expressed in the hearts of many teleosts is the product of two distinct genes: cardiac TnC (cTnC) and the fish‐specific slow skeletal TnC (ssTnC). Tissue‐specific mRNA profiles in adult zebrafish revealed a distinct localization of gene expression, with ssTnC preferentially expressed in the atrium and cTnC is almost exclusively expressed in the ventricle. Chamber‐specific localization varies between teleost species, with trout preferentially expressing cTnC in both chambers of the heart. Isoform composition of the chambers is also modulated by changes in temperature. Possible sub‐functionalization of TnC isoforms may provide insight into how teleosts achieve physiological versatility in chamber‐specific contractile properties. Funded by NSERC.

Human Cardiac Troponin C undergoes Global Conformational Changes in Response to Divalent Cation Binding: Solution Studies of Fluorescent Protein Constructs by FRET and Analytical Ultracentrifugation

Troponin C (TnC), the Ca2+ binding subunit in striated muscle, is central to regulation of contraction. Crystal structures of skeletal TnC show a rigid, elongated central helix, whereas solution NMR shows it is flexible and unstructured in the absence of other troponin subunits. In Ca2+-saturated crystal structures of troponin complex core domain, sTnC's central helix is a well-defined, rigid α-helix, whereas the cardiac TnC isoform reveals a melted helix. This led to the hypothesis that the structural relationship between the N- and C-termini of cTnC depends more on interactions with other troponin subunits than divalent cation binding. We designed a series of novel FRET constructs using human cTnC, to examine the relative positions of the N- and C-termini upon divalent cation binding. Full length cTnC was flanked by FRET pairs of fluorescent proteins (mCerulean/mVenus, mTurquoise/mNeonGreen, mTurquoise/cpVenus). FRET was measured in the presence and absence of saturating Ca2+ and/or Mg2+. FRET increased substantially and reversibly upon Ca2+ binding to cTnC. Similar FRET changes were observed upon saturation of the C-terminus with Mg2+, suggesting that the structural changes detected are primarily attributable to occupancy of the C-terminal sites. Analytical ultracentrifugation (AUC) confirmed that constructs undergo global conformational changes to a more compact structure upon Mg2+ binding, with further compaction when Ca2+ occupies all 3 sites of cTnC. In summary: the ends of cTnC come closer upon binding divalent cations (FRET) yielding a more compact structure (AUC); the FRET signal is primarily attributed to the C-terminus binding of Ca2+ or Mg2+, whereas AUC studies suggest a more compact structure in the 3Ca2+ state compared to the 2Mg2+ state.

Significance of Troponin Dynamics for Ca2+-mediated Regulation of Contraction and Inherited Cardiomyopathy

Ca(2+) dissociation from troponin causes cessation of muscle contraction by incompletely understood structural mechanisms. To investigate this process, regulatory site Ca(2+) binding in the NH(2)-lobe of subunit troponin C (TnC) was abolished by mutagenesis, and effects on cardiac troponin dynamics were mapped by hydrogen-deuterium exchange (HDX)-MS. The findings demonstrate the interrelationships among troponin's detailed dynamics, troponin's regulatory actions, and the pathogenesis of cardiomyopathy linked to troponin mutations. Ca(2+) slowed HDX up to 2 orders of magnitude within the NH(2)-lobe and the NH(2)-lobe-associated TnI switch helix, implying that Ca(2+) greatly stabilizes this troponin regulatory region. HDX of the TnI COOH terminus indicated that its known role in regulation involves a partially folded rather than unfolded structure in the absence of Ca(2+) and actin. Ca(2+)-triggered stabilization extended beyond the known direct regulatory regions: to the start of the nearby TnI helix 1 and to the COOH terminus of the TnT-TnI coiled-coil. Ca(2+) destabilized rather than stabilized specific TnI segments within the coiled-coil and destabilized a region not previously implicated in Ca(2+)-mediated regulation: the coiled-coil's NH(2)-terminal base plus the preceding TnI loop with which the base interacts. Cardiomyopathy-linked mutations clustered almost entirely within influentially dynamic regions of troponin, and many sites were Ca(2+)-sensitive. Overall, the findings demonstrate highly selective effects of regulatory site Ca(2+), including opposite changes in protein dynamics at opposite ends of the troponin core domain. Ca(2+) release triggers an intramolecular switching mechanism that propagates extensively within the extended troponin structure, suggests specific movements of the TnI inhibitory regions, and prominently involves troponin's dynamic features.

Molecular basis of calcium-sensitizing and desensitizing mutations of the human cardiac troponin C regulatory domain: a multi-scale simulation study.

Troponin C (TnC) is implicated in the initiation of myocyte contraction via binding of cytosolic and subsequent recognition of the Troponin I switch peptide. Mutations of the cardiac TnC N-terminal regulatory domain have been shown to alter both calcium binding and myofilament force generation. We have performed molecular dynamics simulations of engineered TnC variants that increase or decrease sensitivity, in order to understand the structural basis of their impact on TnC function. We will use the distinction for mutants that are associated with increased affinity and for those mutants with reduced affinity. Our studies demonstrate that for GOF mutants V44Q and L48Q, the structure of the physiologically-active site II binding site in the -free (apo) state closely resembled the -bound (holo) state. In contrast, site II is very labile for LOF mutants E40A and V79Q in the apo form and bears little resemblance with the holo conformation. We hypothesize that these phenomena contribute to the increased association rate, , for the GOF mutants relative to LOF. Furthermore, we observe significant positive and negative positional correlations between helices in the GOF holo mutants that are not found in the LOF mutants. We anticipate these correlations may contribute either directly to affinity or indirectly through TnI association. Our observations based on the structure and dynamics of mutant TnC provide rationale for binding trends observed in GOF and LOF mutants and will guide the development of inotropic drugs that target TnC.

Long-Timescale Molecular Dynamics Simulations Elucidate the Dynamics and Kinetics of Exposure of the Hydrophobic Patch in Troponin C

Troponin (Tn) is an important regulatory protein in the thin-filament complex of cardiomyocytes. Calcium binding to the troponin C (TnC) subunit causes a change in its dynamics that leads to the transient opening of a hydrophobic patch on TnC’s surface, to which a helix of another subunit, troponin I (TnI), binds. This process initiates contraction, making it an important target for studies investigating the detailed molecular processes that underlie contraction. Here we use microsecond-timescale Anton molecular dynamics simulations to investigate the dynamics and kinetics of the opening transition of the TnC hydrophobic patch. Free-energy differences for opening are calculated for wild-type Ca2+-bound TnC (∼8 kcal/mol), V44Q Ca2+-bound TnC (3.2 kcal/mol), E40A Ca2+-bound TnC (∼12 kcal/mol), and wild-type apo TnC (∼20 kcal/mol). These results suggest that the mutations have a profound impact on the frequency with which the hydrophobic patch presents to TnI. In addition, these simulations corroborate that cardiac wild-type TnC does not open on timescales relevant to contraction without calcium being bound.

Cross-Talk between Neighboring Troponins in Ventricular Myofibrils

Troponin C (TnC) is responsible for Ca2+-dependent activation and inactivation of the thin filament during contraction and relaxation of the heart. To study the Ca2+ binding properties of TnC, we designed a human cardiac TnC (C35S T53C C84S) ± the L48Q mutation. L48Q TnC has an increased Ca2+ sensitivity and decreased rate of Ca2+ dissocation as previously shown in multiple biochemical systems. We exchanged IANBD labeled control or L48Q Tn into rabbit ventricular myofibrils and used a stopped-flow apparatus to measure the fluorescence change related to Ca2+ dissociation. Unlike the mono-phasic Ca2+ dissociation curve of control myofibrils (25.3/s ± 0.7), Ca2+ dissociation from L48Q TnC myofibrils was biphasic, with rates of 17.3 ± 0.5/s and 4.73 ± 0.07/s, at 15oC. The apparent Ca2+ affinities of the fast and slow phases for L48Q TnC corresponded to the steady-state Ca2+ binding affinities of control and L48Q myofibrils, respectively. We hypothesized that the biphasic rate of L48Q Tn exchanged myofibrils results from the influence of the un-exchanged endogenous Tn. Consistent with this hypothesis, reconstituted thin filaments containing 100% L48Q Tn had a mono-phasic Ca2+ dissociation curve. Whereas, Ca2+ dissociation from reconstituted thin filaments containing IANBD labeled L48Q Tn and unlabeled control Tn lead to a biphasic Ca2+ dissociation rate, confirming the crosstalk between Tn subunits. Interestingly, the biphasic characteristic of L48Q TnC was absent when incorporated into diseased human ventricular myofibrils, possibly due to an altered thin filament state. Fluorescently labeled L48Q TnC provides insight into the interactions occurring between neighboring Tn subunits. Thus, L48Q TnC may be a useful tool to probe myofilament protein interactions along the thin filament.

Mechanism of Regulation of Native Cardiac Muscle Thin Filaments by Rigor Cardiac Myosin-S1 and Calcium

We have studied the mechanism of activation of native cardiac thin filaments by calcium and rigor myosin. The acceleration of the rate of 2'-deoxy-3'-O-(N-methylanthraniloyl)ADP (mdADP) dissociation from cardiac myosin-S1-mdADP-P(i) and cardiac myosin-S1-mdADP by native cardiac muscle thin filaments was measured using double mixing stopped-flow fluorescence. Relative to inhibited thin filaments (no bound calcium or rigor S1), fully activated thin filaments (with both calcium and rigor-S1 bound) increase the rate of product dissociation from the physiologically important pre-power stroke myosin-mdADP-P(i) by a factor of ∼75. This can be compared with only an ∼6-fold increase in the rate of nucleotide diphosphate dissociation from nonphysiological myosin-mdADP by the fully activated thin filaments relative to the fully inhibited thin filaments. These results show that physiological levels of regulation are not only dependent on the state of the thin filament but also on the conformation of the myosin. Less than 2-fold regulation is due to a change in affinity of myosin-ADP-P(i) for thin filaments such as would be expected by a simple "steric blocking" of the myosin-binding site of the thin filament by tropomyosin. Although maximal activation requires both calcium and rigor myosin-S1 bound to the cardiac filament, association with a single ligand produces ∼70% maximal activation. This can be contrasted with skeletal thin filaments in which calcium alone only activated the rate of product dissociation ∼20% of maximum, and rigor myosin produces ∼30% maximal activation.

A Dilated Cardiomyopathy Troponin C Mutation Lowers Contractile Force by Reducing Strong Myosin-Actin Binding

In this study we explore the mechanisms by which a double mutation (E59D/D75Y) in cardiac troponin C (CTnC) associated with dilated cardiomyopathy reduces the Ca(2+)-activated maximal tension of cardiac muscle. Studying the single mutants (i.e. E59D or D75Y) indicates that D75Y, but not E59D, causes a reduction in the calcium affinity of CTnC in troponin complex, regulated thin filaments (RTF), and the Ca(2+) sensitivity of contraction and ATPase in cardiac muscle preparations. However, both D75Y and E59D are required to reduce the actomyosin ATPase activity and maximal force in muscle fibers, indicating that E59D enhances the effects of D75Y. Part of the reduction in force/ATPase may be due to a defect in the interactions between CTnC and cardiac troponin T, which are known to be necessary for ATPase activation. An additional mechanism for the reduction in force/ATPase comes from measurements of the binding stoichiometry of myosin subfragment-1 (S-1) to the RTF. Using wild type RTFs, 4.8 mol S-1 was bound per mol filament (seven actins), whereas with E59D/D75Y RTFs, the number of binding sites was reduced by approximately 23% to 3.7. Altogether, these results suggest that the reduction in force and ATPase activation is possibly due to a thin filament conformation that promotes fewer accessible S-1-binding sites. In the absence of any family segregation data, the functional results presented here support the concept that this is likely a dilated cardiomyopathy-causing mutation.

Magnesium Stabilizes the Closed Conformation of the C-Domain of Troponin C

Activation of the thin filament in striated muscles requires both the binding of Ca2+ to the N-domain of troponin C (TnC) and the binding of myosin crossbridges to actin, which has been shown to alter the C-domain conformation. Here we have evaluated the structural and functional consequences of divalent cation exchange in skeletal and cardiac TnC (sTnC and cTnC). We have used intrinsic tyrosine fluorescence, circular dichrosim (CD), and the fluorescent nonspecific hydrophobic probe bis-ANS to monitor changes in domain conformation in response to Ca2+ and Mg2+ binding in the sTnC, cTnC, and in a cTnC mutant in which the invariant Glu residue at the 12th position of the calcium binding loops III and IV were substituted with Asp (cTnCDD). Ca2+ binding causes an increase in Tyr fluorescence and α-helical content in sTnC and cTnC, but not in cTnCDD. Ca2+ induced C-domain opening characteristic of sTnC and cTnC was also greatly reduced in cTnCDD, as measured by bis-ANS fluorescence. Thus the Asp to Glu substitutions appear to prevent the C-domain from opening. Bis-ANS Ca2+ titrations also showed that high Ca2+ concentrations may be sufficient to open the N-domain of cTnC, which was reported to remain in the closed conformation in the Ca2+-bound state. Lastly, bis-ANS Mg2+ titrations indicate that Mg2+ does not cause domain opening in either cTnC or cTnCDD. The closed conformation of the Mg2+-bound C-domain of TnC implies a different mechanism of interaction with TnI than that in the presence of Ca2+ and suggests that the Mg2+- Ca2+exchange in TnC may contribute to the thin filament activation of muscle contraction. This conclusion is consistent with our observation that physiological concentrations of Mg2+ significantly lower the Ca2+-sensitivity of reconstituted cardiac thin filaments.

Comparison of the Binding of the Switch Regions of Cardiac Troponin-I and Skeletal Troponin-I to the Functional N-Domain of Human Cardiac Troponin-C

In the heart, there are two isoforms of troponin-I (TnI) that are developmentally regulated. The slow skeletal TnI (ssTnI) is the sole isoform expressed in the embryonic or neonatal heart, while cardiac TnI (cTnI) is expressed exclusively in the adult heart. One important distinction between ssTnI and cTnI relates to differences in myofilament Ca2+-sensitivity of force development under acidic pH conditions. Hearts expressing ssTnI show heightened Ca2+-sensitivity compared with hearts expressing cTnI under basal conditions. This isoform-specific difference is greatly accentuated during acidosis, which is often associated with ischemia of myocardial infarction. In vitro and in vivo functional studies have shown that replacement of cTnI with ssTnI results in a marked enhancement of myofilament Ca2+-sensitivity at acidic pH conditions. Recent reports have indicated that this effect can be ascribed to amino acid sequence differences in ssTnI and cTnI and in particular to a critical A162H substitution in the switch region. In this study, we have used NMR spectroscopy to examine the binding of the switch regions of ssTnI (sTnI115-131) and cTnI (cTnI147-163) to the N-domain of cardiac troponin-C (cNTnC) at physiological and acidic pH conditions. The results show that the affinity of sTnI115-131 for cNTnC•Ca2+ (KD ∼50uM) is ∼3-fold stronger than that of cTnI147-163 (KD ∼150uM), but neither are affected by a pH change from 7 to 6. The pKa of H130 in sTnI115-131 is 6.2 when free and 6.7 when bound to cNTnC•Ca2+. We have also used {1H, 15N}-HSQC NMR spectroscopy to monitor the pKa changes of cNTnC•Ca2+ from peptide free to peptide bound states. The implications of these results will be discussed in the context of structure and function of myofilament protein interactions.

Effect of Varying Mutant Troponin C Content on Contractile Properties of Striated Muscle

Genetic therapies show much potential as novel, long-term treatments for skeletal and cardiac muscle diseases. Troponin C (TnC), a subunit of troponin, is a particularly promising target for genetic therapies, as it is involved in thin filament activation, Ca2+ signaling and Ca2+ handling. Specifically, expression of engineered TnC mutants with altered Ca2+ binding properties has the potential to restore normal contractile function in diseased/damaged muscle with abnormal Ca2+ properties. We are, therefore, studying skeletal TnC (sTnC) and cardiac TnC (cTnC) mutants that we, and others, have demonstrated affect Ca2+ signaling: F27W, M80Q sTnC and I60Q sTnC, and cTnC L48Q and I61Q. Since various amounts of mutant proteins are incorporated into the myofilaments following viral vector administration of mutant TnC cDNA, the goal of this study was to determine the minimal amount of protein expression necessary to alter contractility in striated muscle. In preliminary studies, different ratios of F27W, M80Q to WT sTnC (100:1, N=4; 50:50, N=4; 25:75, N=4; 1:100, N=6; C0=1mg/mL for all TnCs) were reconstituted into demembranated skeletal muscle fibers. Following reconstitution, the maximal Ca2+ activated force (Fmax) was 90±0.02% (WT), 83±0.03% (100:1), 92±0.04% (50:50) and 90±0.01% (25:75) of pre-extracted Fmax (216±15 mN/mm2). Ca2+ sensitivity was significantly enhanced for all ratios (100:1, pCa50=6.17±0.02, p<0.001; 50:50, pCa50=5.90±0.03, p<0.05; 25:75, pCa50=6.01±0.01, p<0.01) as compared to WT (pCa50=5.76±0.04). These preliminary data indicate that <25% of the mutant protein is required to alter contractility without changing the maximal Ca2+ activated force. Continuing work will identify the minimal level of mutant TnC incorporation in striated muscle that significantly alters pCa50 and the rate force develops.

Calcium‐ and myosin‐dependent changes in troponin structure during activation of heart muscle

Each heartbeat is triggered by a pulse of intracellular calcium ions which bind to troponin on the actin-containing thin filaments of heart muscle cells, initiating a change in filament structure that allows myosin to bind and generate force. We investigated the molecular mechanism of calcium regulation in demembranated trabeculae from rat ventricle using polarized fluorescence from probes on troponin C (TnC). Native TnC was replaced by double-cysteine mutants of human cardiac TnC with bifunctional rhodamine attached along either the C helix, adjacent to the regulatory Ca(2+)-binding site, or the E helix in the IT arm of the troponin complex. Changes in the orientation of both troponin helices had the same steep Ca(2+) dependence as active force production, with a Hill coefficient (n(H)) close to 3, consistent with a single co-operative transition controlled by Ca(2+) binding. Complete inhibition of active force by 25 microM blebbistatin had very little effect on the Ca(2+)-dependent structural changes and in particular did not significantly reduce the value of n(H). Binding of rigor myosin heads to thin filaments following MgATP depletion in the absence of Ca(2+) also changed the orientation of the C and E helices, and addition of Ca(2+) in rigor produced further changes characterized by increased Ca(2+) affinity but with n(H) close to 1. These results show that, although myosin binding can switch on thin filaments in rigor conditions, it does not contribute significantly under physiological conditions. The physiological mechanism of co-operative Ca(2+) regulation of cardiac contractility must therefore be intrinsic to the thin filaments.