E258K HCM-causing mutation in cardiac MyBP-C reduces contractile force and accelerates twitch kinetics by disrupting the cMyBP-C and myosin S2 interaction

Mutations in cardiac myosin binding protein C (cMyBP-C) are prevalent causes of hypertrophic cardiomyopathy (HCM). Although HCM-causing truncation mutations in cMyBP-C are well studied, the growing number of disease-related cMyBP-C missense mutations remain poorly understood. Our objective was to define the primary contractile effect and molecular disease mechanisms of the prevalent cMyBP-C E258K HCM-causing mutation in nonremodeled murine engineered cardiac tissue (mECT). Wild-type and human E258K cMyBP-C were expressed in mECT lacking endogenous mouse cMyBP-C through adenoviral-mediated gene transfer. Expression of E258K cMyBP-C did not affect cardiac cell survival and was appropriately incorporated into the cardiac sarcomere. Functionally, expression of E258K cMyBP-C caused accelerated contractile kinetics and severely compromised twitch force amplitude in mECT. Yeast two-hybrid analysis revealed that E258K cMyBP-C abolished interaction between the N terminal of cMyBP-C and myosin heavy chain sub-fragment 2 (S2). Furthermore, this mutation increased the affinity between the N terminal of cMyBP-C and actin. Assessment of phosphorylation of three serine residues in cMyBP-C showed that aberrant phosphorylation of cMyBP-C is unlikely to be responsible for altering these interactions. We show that the E258K mutation in cMyBP-C abolishes interaction between N-terminal cMyBP-C and myosin S2 by directly disrupting the cMyBP-C–S2 interface, independent of cMyBP-C phosphorylation. Similar to cMyBP-C ablation or phosphorylation, abolition of this inhibitory interaction accelerates contractile kinetics. Additionally, the E258K mutation impaired force production of mECT, which suggests that in addition to the loss of physiological function, this mutation disrupts contractility possibly by tethering the thick and thin filament or acting as an internal load.     

Pseudophosphorylation of cardiac myosin regulatory light chain: a promising new tool for treatment of cardiomyopathy

Many genetic mutations in sarcomeric proteins, including the cardiac myosin regulatory light chain (RLC) encoded by the MYL2 gene, have been implicated in familial cardiomyopathies. Yet, the molecular mechanisms by which these mutant proteins regulate cardiac muscle mechanics in health and disease remain poorly understood. Evidence has been accumulating that RLC phosphorylation has an influential role in striated muscle contraction and, in addition to the conventional modulation via Ca²⁺ binding to troponin C, it can regulate cardiac muscle function. In this review, we focus on RLC mutations that have been reported to cause cardiomyopathy phenotypes via compromised RLC phosphorylation and elaborate on pseudo-phosphorylation rescue mechanisms. This new methodology has been discussed as an emerging exploratory tool to understand the role of phosphorylation as well as a genetic modality to prevent/rescue cardiomyopathy phenotypes. Finally, we summarize structural effects post-phosphorylation, a phenomenon that leads to an ordered shift in the myosin S1 and RLC conformational equilibrium between two distinct states.     

Molecular Modeling of Disease Causing Mutations in Domain C1 of cMyBP-C

Cardiac myosin binding protein-C (cMyBP-C) is a multi-domain (C0–C10) protein that regulates heart muscle contraction through interaction with myosin, actin and other sarcomeric proteins. Several mutations of this protein cause familial hypertrophic cardiomyopathy (HCM). Domain C1 of cMyBP-C plays a central role in protein interactions with actin and myosin. Here, we studied structure-function relationship of three disease causing mutations, Arg177His, Ala216Thr and Glu258Lys of the domain C1 using computational biology techniques with its available X-ray crystal structure. The results suggest that each mutation could affect structural properties of the domain C1, and hence it’s structural integrity through modifying intra-molecular arrangements in a distinct mode. The mutations also change surface charge distributions, which could impact the binding of C1 with other sarcomeric proteins thereby affecting contractile function. These structural consequences of the C1 mutants could be valuable to understand the molecular mechanisms for the disease.     

Localization of the binding site of the C-terminal domain of cardiac myosin-binding protein-C on the myosin rod

cMyBP-C [cardiac (MyBP-C) myosin-binding protein-C)] is a sarcomeric protein involved both in thick filament structure and in the regulation of contractility. It is composed of eight IgI-like and three fibronectin-3-like domains (termed C0-C10). Mutations in the gene encoding cMyBP-C are a principal cause of HCM (hypertrophic cardiomyopathy). cMyBP-C binds to the LMM (light meromyosin) portion of the myosin rod via its C-terminal domain, C10. We investigated this interaction in detail to determine whether HCM mutations in beta myosin heavy chain located within the LMM portion alter the binding of cMyBP-C, and to define the precise region of LMM that binds C10 to aid in developing models of the arrangement of MyBP-C on the thick filament. In co-sedimentation experiments recombinant C10 bound full-length LMM with a K(d) of 3.52 microM and at a stoichiometry of 1.14 C10 per LMM. C10 was also shown to bind with similar affinity to LMM containing either the HCM mutations A1379T or S1776G, suggesting that these HCM mutations do not perturb C10 binding. Using a range of N-terminally truncated LMM fragments, the cMyBP-C-binding site on LMM was shown to lie between residues 1554 and 1581. Since it had been reported previously that acidic residues on myosin mediate the C10 interaction, three clusters of acidic amino acids (Glu1554/Glu1555, Glu1571/Glu1573 and Glu1578/Asp1580/Glu1581/Glu1582) were mutated in full-length LMM and the proteins tested for C10 binding. No effect of these mutations on C10 binding was however detected. We interpret our results with respect to the localization of the proposed trimeric collar on the thick filament.     

Mutations in beta-myosin S2 that cause familial hypertrophic cardiomyopathy (FHC) abolish the interaction with the regulatory domain of myosin-binding protein-C

The myosin filaments of striated muscle contain a family of enigmatic myosin-binding proteins (MyBP), MyBP-C and MyBP-H. These modular proteins of the intracellular immunoglobulin superfamily contain unique domains near their N termini. The N-terminal domain of cardiac MyBP-C, the MyBP-C motif, contains additional phosphorylation sites and may regulate contraction in a phosphorylation dependent way. In contrast to the C terminus, which binds to the light meromyosin portion of the myosin rod, the interactions of this domain are unknown. We demonstrate that fragments of MyBP-C containing the MyBP-C motif localise to the sarcomeric A-band in cardiomyocytes and isolated myofibrils, without affecting sarcomere structure. The binding site for the MyBP-C motif resides in the N-terminal 126 residues of the S2 segment of the myosin rod. In this region, several mutations in beta-myosin are associated with FHC; however, their molecular implications remained unclear. We show that two representative FHC mutations in beta-myosin S2, R870H and E924K, drastically reduce MyBP-C binding (Kd approximately 60 microM for R870H compared with a Kd of approximately 5 microM for the wild-type) down to undetectable levels (E924K). These mutations do not affect the coiled-coil structure of myosin. We suggest that the regulatory function of MyBP-C is mediated by the interaction with S2, and that mutations in beta-myosin S2 may act by altering the interactions with MyBP-C.     

Cardiac myosin-binding protein C interaction with actin is inhibited by compounds identified in a high-throughput fluorescence lifetime screen

Cardiac myosin-binding protein C (cMyBP-C) interacts with actin and myosin to modulate cardiac muscle contractility. These interactions are disfavored by cMyBP-C phosphorylation. Heart failure patients often display decreased cMyBP-C phosphorylation, and phosphorylation in model systems has been shown to be cardioprotective against heart failure. Therefore, cMyBP-C is a potential target for heart failure drugs that mimic phosphorylation or perturb its interactions with actin/myosin. Here we have used a novel fluorescence lifetime-based assay to identify small-molecule inhibitors of actin-cMyBP-C binding. Actin was labeled with a fluorescent dye (Alexa Fluor 568, AF568) near its cMyBP-C binding sites; when combined with the cMyBP-C N-terminal fragment, C0-C2, the fluorescence lifetime of AF568-actin decreases. Using this reduction in lifetime as a readout of actin binding, a high-throughput screen of a 1280-compound library identified three reproducible hit compounds (suramin, NF023, and aurintricarboxylic acid) that reduced C0-C2 binding to actin in the micromolar range. Binding of phosphorylated C0-C2 was also blocked by these compounds. That they specifically block binding was confirmed by an actin-C0-C2 time-resolved FRET (TR-FRET) binding assay. Isothermal titration calorimetry (ITC) and transient phosphorescence anisotropy (TPA) confirmed that these compounds bind to cMyBP-C, but not to actin. TPA results were also consistent with these compounds inhibiting C0-C2 binding to actin. We conclude that the actin-cMyBP-C fluorescence lifetime assay permits detection of pharmacologically active compounds that affect cMyBP-C-actin binding. We now have, for the first time, a validated high-throughput screen focused on cMyBP-C, a regulator of cardiac muscle contractility and known key factor in heart failure.     

The effect of the Asp175Asn and Glu180Gly TPM1 mutations on actin-myosin interaction during the ATPase cycle

Hypertrophic cardiomyopathy (HCM), characterized by cardiac hypertrophy and contractile dysfunction, is a major cause of heart failure. HCM can result from mutations in the gene encoding cardiac α-tropomyosin (TM). To understand how the HCM-causing Asp175Asn and Glu180Gly mutations in α-tropomyosin affect on actin-myosin interaction during the ATPase cycle, we labeled the SH1 helix of myosin subfragment-1 and the actin subdomain-1 with the fluorescent probe N-iodoacetyl-N′-(5-sulfo-1-naphtylo)ethylenediamine. These proteins were incorporated into ghost muscle fibers and their conformational states were monitored during the ATPase cycle by measuring polarized fluorescence. For the first time, the effect of these α-tropomyosins on the mobility and rotation of subdomain-1 of actin and the SH1 helix of myosin subfragment-1 during the ATP hydrolysis cycle have been demonstrated directly by polarized fluorimetry. Wild-type α-tropomyosin increases the amplitude of the SH1 helix and subdomain-1 movements during the ATPase cycle, indicating the enhancement of the efficiency of the work of cross-bridges. Both mutant TMs increase the proportion of the strong-binding sub-states, with the effect of the Glu180Gly mutation being greater than that of Asp175Asn. It is suggested that the alteration in the concerted conformational changes of actomyosin is likely to provide the structural basis for the altered cardiac muscle contraction.     

A high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein C to F-actin

Binding properties of actin-binding proteins are typically evaluated by cosedimentation assays. However, this method is time-consuming, involves multiple steps, and has a limited throughput. These shortcomings preclude its use in screening for drugs that modulate actin-binding proteins relevant to human disease. To develop a simple, quantitative, and scalable F-actin–binding assay, we attached fluorescent probes to actin''s Cys-374 and assessed changes in fluorescence lifetime upon binding to the N-terminal region (domains C0–C2) of human cardiac myosin-binding protein C (cMyBP-C). The lifetime of all five probes tested decreased upon incubation with cMyBP-C C0–C2, as measured by time-resolved fluorescence (TR-F), with IAEDANS being the most sensitive probe that yielded the smallest errors. The TR-F assay was compared with cosedimentation to evaluate in vitro changes in binding to actin and actin–tropomyosin arising from cMyBP-C mutations associated with hypertrophic cardiomyopathy (HCM) and tropomyosin binding. Lifetime changes of labeled actin with added C0–C2 were consistent with cosedimentation results. The HCM mutation L352P was confirmed to enhance actin binding, whereas PKA phosphorylation reduced binding. The HCM mutation R282W, predicted to disrupt a PKA recognition sequence, led to deficits in C0–C2 phosphorylation and altered binding. Lastly, C0–C2 binding was found to be enhanced by tropomyosin and binding capacity to be altered by mutations in a tropomyosin-binding region. These findings suggest that the TR-F assay is suitable for rapidly and accurately determining quantitative binding and for screening physiological conditions and compounds that affect cMyBP-C binding to F-actin for therapeutic discovery.     

Point Mutations in Human β Cardiac Myosin Heavy Chain Have Differential Effects on Sarcomeric Structure and Assembly: An ATP Binding Site Change Disrupts Both Thick and Thin Filaments, Whereas Hypertrophic Cardiomyopathy Mutations Display Normal Assembly

Hypertrophic cardiomyopathy is a human heart disease characterized by increased ventricular mass, focal areas of fibrosis, myocyte, and myofibrillar disorganization. This genetically dominant disease can be caused by mutations in any one of several contractile proteins, including β cardiac myosin heavy chain (βMHC). To determine whether point mutations in human βMHC have direct effects on interfering with filament assembly and sarcomeric structure, full-length wild-type and mutant human βMHC cDNAs were cloned and expressed in primary cultures of neonatal rat ventricular cardiomyocytes (NRC) under conditions that promote myofibrillogenesis. A lysine to arginine change at amino acid 184 in the consensus ATP binding sequence of human βMHC resulted in abnormal subcellular localization and disrupted both thick and thin filament structure in transfected NRC. Diffuse βMHC K184R protein appeared to colocalize with actin throughout the myocyte, suggesting a tight interaction of these two proteins. Human βMHC with S472V mutation assembled normally into thick filaments and did not affect sarcomeric structure. Two mutant myosins previously described as causing human hypertrophic cardiomyopathy, R249Q and R403Q, were competent to assemble into thick filaments producing myofibrils with well defined I bands, A bands, and H zones. Coexpression and detection of wild-type βMHC and either R249Q or R403Q proteins in the same myocyte showed these proteins are equally able to assemble into the sarcomere and provided no discernible differences in subcellular localization. Thus, human βMHC R249Q and R403Q mutant proteins were readily incorporated into NRC sarcomeres and did not disrupt myofilament formation. This study indicates that the phenotype of myofibrillar disarray seen in HCM patients which harbor either of these two mutations may not be directly due to the failure of the mutant myosin heavy chain protein to assemble and form normal sarcomeres, but may rather be a secondary effect possibly resulting from the chronic stress of decreased βMHC function.     

cAPK-phosphorylation controls the interaction of the regulatory domain of cardiac myosin binding protein C with myosin-S2 in an on-off fashion.

Myosin binding protein C is a protein of the myosin filaments of striated muscle which is expressed in isoforms specific for cardiac and skeletal muscle. The cardiac isoform is phosphorylated rapidly upon adrenergic stimulation of myocardium by cAMP-dependent protein kinase, and together with the phosphorylation of troponin-I and phospholamban contributes to the positive inotropy that results from adrenergic stimulation of the heart. Cardiac myosin binding protein C is phosphorylated by cAMP-dependent protein kinase on three sites in a myosin binding protein C specific N-terminal domain which binds to myosin-S2. This interaction with myosin close to the motor domain is likely to mediate the regulatory function of the protein. Cardiac myosin binding protein C is a common target gene of familial hypertrophic cardiomyopathy and most mutations encode N-terminal subfragments of myosin binding protein C. The understanding of the signalling interactions of the N-terminal region is therefore important for understanding the pathophysiology of myosin binding protein C associated cardiomyopathy. We demonstrate here by cosedimentation assays and isothermal titration calorimetry that the myosin-S2 binding properties of the myosin binding protein C motif are abolished by cAMP-dependent protein kinase-mediated tris-phosphorylation, decreasing the S2 affinity from a Kd of approximately 5 microM to undetectable levels. We show that the slow and fast skeletal muscle isoforms are no cAMP-dependent protein kinase substrates and that the S2 interaction of these myosin binding protein C isoforms is therefore constitutively on. The regulation of cardiac contractility by myosin binding protein C therefore appears to be a 'brake-off' mechanism that will free a specific subset of myosin heads from sterical constraints imposed by the binding to the myosin binding protein C motif.     

The effect of the Asp175Asn and Glu180Gly TPM1 mutations on actin-myosin interaction during the ATPase cycle

Hypertrophic cardiomyopathy (HCM), characterized by cardiac hypertrophy and contractile dysfunction, is a major cause of heart failure. HCM can result from mutations in the gene encoding cardiac α-tropomyosin (TM). To understand how the HCM-causing Asp175Asn and Glu180Gly mutations in α-tropomyosin affect on actin-myosin interaction during the ATPase cycle, we labeled the SH1 helix of myosin subfragment-1 and the actin subdomain-1 with the fluorescent probe N-iodoacetyl-N'-(5-sulfo-1-naphtylo)ethylenediamine. These proteins were incorporated into ghost muscle fibers and their conformational states were monitored during the ATPase cycle by measuring polarized fluorescence. For the first time, the effect of these α-tropomyosins on the mobility and rotation of subdomain-1 of actin and the SH1 helix of myosin subfragment-1 during the ATP hydrolysis cycle have been demonstrated directly by polarized fluorimetry. Wild-type α-tropomyosin increases the amplitude of the SH1 helix and subdomain-1 movements during the ATPase cycle, indicating the enhancement of the efficiency of the work of cross-bridges. Both mutant TMs increase the proportion of the strong-binding sub-states, with the effect of the Glu180Gly mutation being greater than that of Asp175Asn. It is suggested that the alteration in the concerted conformational changes of actomyosin is likely to provide the structural basis for the altered cardiac muscle contraction.     

Effects of Troponin T Cardiomyopathy Mutations on the Calcium Sensitivity of the Regulated Thin Filament and the Actomyosin Cross-Bridge Kinetics of Human β-Cardiac Myosin

Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) lead to significant cardiovascular morbidity and mortality worldwide. Mutations in the genes encoding the sarcomere, the force-generating unit in the cardiomyocyte, cause familial forms of both HCM and DCM. This study examines two HCM-causing (I79N, E163K) and two DCM-causing (R141W, R173W) mutations in the troponin T subunit of the troponin complex using human β-cardiac myosin. Unlike earlier reports using various myosin constructs, we found that none of these mutations affect the maximal sliding velocities or maximal Ca²⁺-activated ADP release rates involving the thin filament human β-cardiac myosin complex. Changes in Ca²⁺ sensitivity using the human myosin isoform do, however, mimic changes seen previously with non-human myosin isoforms. Transient kinetic measurements show that these mutations alter the kinetics of Ca²⁺ induced conformational changes in the regulatory thin filament proteins. These changes in calcium sensitivity are independent of active, cycling human β-cardiac myosin.     

Hypertrophic cardiomyopathy-related beta-myosin mutations cause highly variable calcium sensitivity with functional imbalances among individual muscle cells

Disease-causing mutations in cardiac myosin heavy chain (beta-MHC) are identified in about one-third of families with hypertrophic cardiomyopathy (HCM). The effect of myosin mutations on calcium sensitivity of the myofilaments, however, is largely unknown. Because normal and mutant cardiac MHC are also expressed in slow-twitch skeletal muscle, which is more easily accessible and less subject to the adaptive responses seen in myocardium, we compared the calcium sensitivity (pCa(50)) and the steepness of force-pCa relations (cooperativity) of single soleus muscle fibers from healthy individuals and from HCM patients of three families with selected myosin mutations. Fibers with the Arg723Gly and Arg719Trp mutations showed a decrease in mean pCa(50), whereas those with the Ile736Thr mutation showed slightly increased mean pCa(50) with higher active forces at low calcium concentrations and residual active force even under relaxing conditions. In addition, there was a marked variability in pCa(50) between individual fibers carrying the same mutation ranging from an almost normal response to highly significant differences that were not observed in controls. While changes in mean pCa(50) may suggest specific pharmacological treatment (e.g., calcium antagonists), the observed large functional variability among individual muscle cells might negate such selective treatment. More importantly, the variability in pCa(50) from fiber to fiber is likely to cause imbalances in force generation and be the primary cause for contractile dysfunction and development of disarray in the myocardium.     

The HCM-causing Y235S cMyBPC mutation accelerates contractile function by altering C1 domain structure

Mutations in cardiac myosin binding protein C (cMyBPC) are a major cause of hypertrophic cardiomyopathy (HCM). In particular, a single amino acid substitution of tyrosine to serine at residue 237 in humans (residue 235 in mice) has been linked to HCM with strong disease association. Although cMyBPC truncations, deletions and insertions, and frame shift mutations have been studied, relatively little is known about the functional consequences of missense mutations in cMyBPC. In this study, we characterized the functional and structural effects of the HCM-causing Y235S mutation by performing mechanical experiments and molecular dynamics simulations (MDS). cMyBPC null mouse myocardium was virally transfected with wild-type (WT) or Y235S cMyBPC (KO^Y235S). We found that Y235S cMyBPC was properly expressed and incorporated into the cardiac sarcomere, suggesting that the mechanism of disease of the Y235S mutation is not haploinsufficiency or poison peptides. Mechanical experiments in detergent-skinned myocardium isolated from KO^Y235S hearts revealed hypercontractile behavior compared to KO^WT hearts, evidenced by accelerated cross-bridge kinetics and increased Ca²⁺ sensitivity of force generation. In addition, MDS revealed that the Y235S mutation causes alterations in important intramolecular interactions, surface conformations, and electrostatic potential of the C1 domain of cMyBPC. Our combined in vitro and in silico data suggest that the Y235S mutation directly disrupts internal and surface properties of the C1 domain of cMyBPC, which potentially alters its ligand-binding interactions. These molecular changes may underlie the mechanism for hypercontractile cross-bridge behavior, which ultimately results in the development of cardiac hypertrophy and in vivo cardiac dysfunction.     

Regulatory light chain phosphorylation and N-terminal extension increase cross-bridge binding and power output in Drosophila at in vivo myofilament lattice spacing

The N-terminal extension and phosphorylation of the myosin regulatory light chain (RLC) independently improve Drosophila melanogaster flight performance. Here we examine the functional and structural role of the RLC in chemically skinned fibers at various thick and thin filament lattice spacings from four transgenic Drosophila lines: rescued null or control (Dmlc2⁺), truncated N-terminal extension (Dmlc2^Δ2-46), disrupted myosin light chain kinase phosphorylation sites (Dmlc2^S66A,S67A), and dual mutant (Dmlc2^{Δ2-46; S66A,S67A}). The N-terminal extension truncation and phosphorylation sites disruption mutations decreased oscillatory power output and the frequency of maximum power output in maximally Ca²⁺-activated fibers compressed to near in vivo inter-thick filament spacing, with the phosphorylation sites disruption mutation having a larger affect. The diminished power output parameters with the N-terminal extension truncation and phosphorylation sites disruption mutations were due to the reduction of the number of strongly-bound cross-bridges and rate of myosin force production, with the larger parameter reductions in the phosphorylation sites disruption mutation additionally related to reduced myosin attachment time. The phosphorylation and N-terminal extension-dependent boost in cross-bridge kinetics corroborates previous structural data, which indicate these RLC attributes play a complementary role in moving and orienting myosin heads toward actin target sites, thereby increasing fiber and whole fly power generation.     

Myocardial Infarction-induced N-terminal Fragment of Cardiac Myosin-binding Protein C (cMyBP-C) Impairs Myofilament Function in Human Myocardium

Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin-binding protein C, a cardiac-specific myofilament protein, is proteolyzed post-MI in humans, which results in an N-terminal fragment, C0-C1f. The presence of C0-C1f in cultured cardiomyocytes results in decreased Ca²⁺ transients and cell shortening, abnormalities sufficient for the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear. Here, we investigate the association between C0-C1f and altered contractility in human cardiac myofilaments in vitro. To accomplish this, we generated recombinant human C0-C1f (hC0C1f) and incorporated it into permeabilized human left ventricular myocardium. Mechanical properties were studied at short (2 μm) and long (2.3 μm) sarcomere length (SL). Our data demonstrate that the presence of hC0C1f in the sarcomere had the greatest effect at short, but not long, SL, decreasing maximal force and myofilament Ca²⁺ sensitivity. Moreover, hC0C1f led to increased cooperative activation, cross-bridge cycling kinetics, and tension cost, with greater effects at short SL. We further established that the effects of hC0C1f occur through direct interaction with actin and α-tropomyosin. Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed myofilament function and Ca²⁺ sensitivity in otherwise healthy human donor myocardium. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-tropomyosin, suggesting that cleaved C0-C1f could act as a poison polypeptide and disrupt the interaction of native cardiac myosin-binding protein C with the thin filament.     

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.     

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.     

Contributions of Ca2+-Independent Thin Filament Activation to Cardiac Muscle Function

Although Ca²⁺ is the principal regulator of contraction in striated muscle, in vitro evidence suggests that some actin-myosin interaction is still possible even in its absence. Whether this Ca²⁺-independent activation (CIA) occurs under physiological conditions remains unclear, as does its potential impact on the function of intact cardiac muscle. The purpose of this study was to investigate CIA using computational analysis. We added a structurally motivated representation of this phenomenon to an existing myofilament model, which allowed predictions of CIA-dependent muscle behavior. We found that a certain amount of CIA was essential for the model to reproduce reported effects of nonfunctional troponin C on myofilament force generation. Consequently, those data enabled estimation of ΔG_CIA, the energy barrier for activating a thin filament regulatory unit in the absence of Ca²⁺. Using this estimate of ΔG_CIA as a point of reference (∼7 kJ mol⁻¹), we examined its impact on various aspects of muscle function through additional simulations. CIA decreased the Hill coefficient of steady-state force while increasing myofilament Ca²⁺ sensitivity. At the same time, CIA had minimal effect on the rate of force redevelopment after slack/restretch. Simulations of twitch tension show that the presence of CIA increases peak tension while profoundly delaying relaxation. We tested the model’s ability to represent perturbations to the Ca²⁺ regulatory mechanism by analyzing twitch records measured in transgenic mice expressing a cardiac troponin I mutation (R145G). The effects of the mutation on twitch dynamics were fully reproduced by a single parameter change, namely lowering ΔG_CIA by 2.3 kJ mol⁻¹ relative to its wild-type value. Our analyses suggest that CIA is present in cardiac muscle under normal conditions and that its modulation by gene mutations or other factors can alter both systolic and diastolic function.     

The N-terminal domains of myosin binding protein C can bind polymorphically to F-actin

The regulation of vertebrate striated muscle contraction involves a number of different molecules, including the thin-filament accessory proteins tropomyosin and troponin that provide Ca²⁺-dependent regulation by controlling access to myosin binding sites on actin. Cardiac myosin binding protein C (cMyBP-C) appears to modulate this Ca²⁺-dependent regulation and has attracted increasing interest due to links with inherited cardiac diseases. A number of single amino acid mutations linked to clinical diseases occur in the N-terminal region of cMyBP-C, including domains C0 and C1, which previously have been shown to bind to F-actin. This N-terminal region also has been shown to both inhibit and activate actomyosin interactions in vitro. Using electron microscopy and three-dimensional reconstruction, we show that C0 and C1 can each bind to the same two distinctly different positions on F-actin. One position aligns well with the previously reported binding site that clashes with the binding of myosin to actin, but would force tropomyosin into an “on” position that exposes myosin binding sites along the filament. The second position identified here would not interfere with either myosin binding or tropomyosin positioning. It thus appears that the ability to bind to at least two distinctly different positions on F-actin, as observed for tropomyosin, may be more common than previously considered for other actin binding proteins. These observations help to explain many of the seemingly contradictory results obtained with cMyBP-C and show how cMyBP-C can provide an additional layer of regulation to actin-myosin interactions. They also suggest a redundancy of C0 and C1 that may explain the absence of C0 in skeletal muscle.