Cardiac troponin T N-domain variant destabilizes the actin interface resulting in disturbed myofilament function.

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca2+ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca2+ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament's relaxed state resulting in an increased number of crossbridges during Ca2+ activation. Additionally, in the low Ca2+-relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca2+, and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.

Short-term aerobic exercise prevents development of glucocorticoid myopathic features in aged skeletal muscle in a sex-dependent manner.

Older adults are vulnerable to glucocorticoid-induced muscle atrophy and weakness, with sex potentially influencing their susceptibility to those effects. Aerobic exercise can reduce glucocorticoid-induced muscle atrophy in young rodents. However, it is unknown whether aerobic exercise can prevent glucocorticoid myopathy in aged muscle. The objectives of this study were to define the extent to which sex influences the development of glucocorticoid myopathy in aged muscle, and to determine the extent to which aerobic exercise training protects against myopathy development. Twenty-four-month-old female (n = 30) and male (n = 33) mice were randomized to either sedentary or aerobic exercise groups. Within their respective groups, mice were randomized to either daily treatment with dexamethasone (DEX) or saline. Upon completing treatments, the contractile properties of the triceps surae complex were assessed in situ. DEX marginally lowered muscle mass and soluble protein content in both sexes, which was attenuated by aerobic exercise only in females. DEX increased sub-tetanic force and rate of force development only in females, which was not influenced by aerobic exercise. Muscle fatigue was higher in both sexes following DEX, but aerobic exercise prevented fatigue induction only in females. The sex-specific differences to muscle function in response to DEX treatment coincided with sex-specific changes to the content of proteins related to calcium handling, mitochondrial quality control, reactive oxygen species production, and glucocorticoid receptor in muscle. These findings define several important sexually dimorphic changes to aged skeletal muscle physiology in response to glucocorticoid treatment and define the capacity of short-term aerobic exercise to protect against those changes. KEY POINTS: There are sexually dimorphic effects of glucocorticoids on aged skeletal muscle physiology. Glucocorticoid-induced changes to aged muscle contractile properties coincide with sex-specific differences in the content of calcium handling proteins. Aerobic exercise prevents glucocorticoid-induced fatigue only in aged females and coincides with differences in the content of mitochondrial quality control proteins and glucocorticoid receptors.

The structure of the native cardiac thin filament at systolic Ca2+ levels.

Every heartbeat relies on cyclical interactions between myosin thick and actin thin filaments orchestrated by rising and falling Ca2+ levels. Thin filaments are comprised of two actin strands, each harboring equally separated troponin complexes, which bind Ca2+ to move tropomyosin cables away from the myosin binding sites and, thus, activate systolic contraction. Recently, structures of thin filaments obtained at low (pCa ∼9) or high (pCa ∼3) Ca2+ levels revealed the transition between the Ca2+-free and Ca2+-bound states. However, in working cardiac muscle, Ca2+ levels fluctuate at intermediate values between pCa ∼6 and pCa ∼7. The structure of the thin filament at physiological Ca2+ levels is unknown. We used cryoelectron microscopy and statistical analysis to reveal the structure of the cardiac thin filament at systolic pCa = 5.8. We show that the two strands of the thin filament consist of a mixture of regulatory units, which are composed of Ca2+-free, Ca2+-bound, or mixed (e.g., Ca2+ free on one side and Ca2+ bound on the other side) troponin complexes. We traced troponin complex conformations along and across individual thin filaments to directly determine the structural composition of the cardiac native thin filament at systolic Ca2+ levels. We demonstrate that the two thin filament strands are activated stochastically with short-range cooperativity evident only on one of the two strands. Our findings suggest a mechanism by which cardiac muscle is regulated by narrow range Ca2+ fluctuations.

Mechanical contribution to muscle thin filament activation.

Vertebrate striated muscle thin filaments are thought to be thermodynamically activated in response to an increase in Ca2+ concentration. We tested this hypothesis by measuring time intervals for gliding runs and pauses of individual skeletal muscle thin filaments in cycling myosin motility assays. A classic thermodynamic mechanism predicts that if chemical potential is constant, transitions between runs and pauses of gliding thin filaments will occur at constant rate as given by a Poisson distribution. In this scenario, rate is given by the odds of a pause, and hence, run times between pauses fit an exponential distribution that slopes negatively for all observable run times. However, we determined that relative density of observed run times fits an exponential only at low Ca2+ levels that activate filament gliding. Further titration with Ca2+, or adding excess regulatory proteins tropomyosin and troponin, shifted the relative density of short run times to fit the positive slope of a gamma distribution, which derives from waiting times between Poisson events. Events that arise during a run and prevent the chance of ending a run for a random interval of time account for the observed run time distributions, suggesting that the events originate with cycling myosin. We propose that regulatory proteins of the thin filament require the mechanical force of cycling myosin to achieve the transition state for activation. During activation, combinations of cycling myosin that contribute insufficient activation energy delay deactivation.

A comprehensive guide to genetic variants and post-translational modifications of cardiac troponin C.

Familial cardiomyopathy is an inherited disease that affects the structure and function of heart muscle and has an extreme range of phenotypes. Among the millions of affected individuals, patients with hypertrophic (HCM), dilated (DCM), or left ventricular non-compaction (LVNC) cardiomyopathy can experience morphologic changes of the heart which lead to sudden death in the most detrimental cases. TNNC1, the gene that codes for cardiac troponin C (cTnC), is a sarcomere gene associated with cardiomyopathies in which probands exhibit young age of presentation and high death, transplant or ventricular fibrillation events relative to TNNT2 and TNNI3 probands. Using GnomAD, ClinVar, UniProt and PhosphoSitePlus databases and published literature, an extensive list to date of identified genetic variants in TNNC1 and post-translational modifications (PTMs) in cTnC was compiled. Additionally, a recent cryo-EM structure of the cardiac thin filament regulatory unit was used to localize each functionally studied amino acid variant and each PTM (acetylation, glycation, s-nitrosylation, phosphorylation) in the structure of cTnC. TNNC1 has a large number of variants (> 100) relative to other genes of the same transcript size. Surprisingly, the mapped variant amino acids and PTMs are distributed throughout the cTnC structure. While many cardiomyopathy-associated variants are localized in α-helical regions of cTnC, this was not statistically significant χ2 (p = 0.72). Exploring the variants in TNNC1 and PTMs of cTnC in the contexts of cardiomyopathy association, physiological modulation and potential non-canonical roles provides insights into the normal function of cTnC along with the many facets of TNNC1 as a cardiomyopathic gene.

Mandibular muscle troponin of the Florida carpenter ant Camponotus floridanus: extending our insights into invertebrate Ca2+ regulation.

Ants use their mandibles for a variety of functions and behaviors. We investigated mandibular muscle structure and function from major workers of the Florida carpenter ant Camponotus floridanus: force-pCa relation and velocity of unloaded shortening of single, permeabilized fibres, primary sequences of troponin subunits (TnC, TnI and TnT) from a mandibular muscle cDNA library, and muscle fibre ultrastructure. From the mechanical measurements, we found Ca2+-sensitivity of isometric force was markedly shifted rightward compared with vertebrate striated muscle. From the troponin sequence results, we identified features that could explain the rightward shift of Ca2+-activation: the N-helix of TnC is effectively absent and three of the four EF-hands of TnC (sites I, II and III) do not adhere to canonical sequence rules for divalent cation binding; two alternatively spliced isoforms of TnI were identified with the alternatively spliced exon occurring in the region of the IT-arm α-helical coiled-coil, and the N-terminal extension of TnI may be involved in modulation of regulation, as in mammalian cardiac muscle; and TnT has a Glu-rich C-terminus. In addition, a structural homology model was built of C. floridanus troponin on the thin filament. From analysis of electron micrographs, we found thick filaments are almost as long as the 6.8 µm sarcomeres, have diameter of ~ 16 nm, and typical center-to-center spacing of ~ 46 nm. These results have implications for the mechanisms by which mandibular muscle fibres perform such a variety of functions, and how the structure of the troponin complex aids in these tasks.

Meta-analysis of cardiomyopathy-associated variants in troponin genes identifies loci and intragenic hot spots that are associated with worse clinical outcomes.

INTRODUCTION: Troponin (TNN)-encoded cardiac troponins (Tn) are critical for sensing calcium and triggering myofilament contraction. TNN variants are associated with development of cardiomyopathy; however, recent advances in genetic analysis have identified rare population variants. It is unclear how certain variants are associated with disease while others are tolerated. OBJECTIVE: To compare probands with TNNT2, TNNI3, and TNNC1 variants and utilize high-resolution variant comparison mapping of pathologic and rare population variants to identify loci associated with disease pathogenesis. METHODS: Cardiomyopathy-associated TNN variants were identified in the literature and topology mapping conducted. Clinical features were compiled and compared. Rare population variants were obtained from the gnomAD database. Signal-to-noise (S:N) normalized pathologic variant frequency against population variant frequency. Abstract review of clinical phenotypes was applied to "significant" hot spots. RESULTS: Probands were compiled (N = 70 studies, 224 probands) as were rare variants (N = 125,748 exomes; 15,708 genomes, MAF <0.001). TNNC1-positive probands demonstrated the youngest age of presentation (20.0 years; P = .016 vs TNNT2; P = .004 vs TNNI3) and the highest death, transplant, or ventricular fibrillation events (P = .093 vs TNNT2; P = .024 vs TNNI3; Kaplan Meir: P = .025). S:N analysis yielded hot spots of diagnostic significance within the tropomyosin-binding domains, α-helix 1, and the N-Terminus in TNNT2 with increased sudden cardiac death and ventricular fibrillation (P = .004). The inhibitory region and C-terminal region in TNNI3 exhibited increased restrictive cardiomyopathy (P =.008). HCM and RCM models tended to have increased calcium sensitivity and DCM decreased sensitivity (P < .001). DCM and HCM studies typically showed no differences in Hill coefficient which was decreased in RCM models (P < .001). CM models typically demonstrated no changes to Fmax (P = .239). CONCLUSION: TNNC1-positive probands had younger ages of diagnosis and poorer clinical outcomes. Mapping of TNN variants identified locations in TNNT2 and TNNI3 associated with heightened pathogenicity, RCM diagnosis, and increased risk of sudden death.

The intrinsically disordered C terminus of troponin T binds to troponin C to modulate myocardial force generation.

Aberrant regulation of myocardial force production represents an early biomechanical defect associated with sarcomeric cardiomyopathies, but the molecular mechanisms remain poorly defined. Here, we evaluated the pathogenicity of a previously unreported sarcomeric gene variant identified in a pediatric patient with sporadic dilated cardiomyopathy, and we determined a molecular mechanism. Trio whole-exome sequencing revealed a de novo missense variant in TNNC1 that encodes a p.I4M substitution in the N-terminal helix of cardiac troponin C (cTnC). Reconstitution of this human cTnC variant into permeabilized porcine cardiac muscle preparations significantly decreases the magnitude and rate of isometric force generation at physiological Ca2+-activation levels. Computational modeling suggests that this inhibitory effect can be explained by a decrease in the rates of cross-bridge attachment and detachment. For the first time, we show that cardiac troponin T (cTnT), in part through its intrinsically disordered C terminus, directly binds to WT cTnC, and we find that this cardiomyopathic variant displays tighter binding to cTnT. Steady-state fluorescence and NMR spectroscopy studies suggest that this variant propagates perturbations in cTnC structural dynamics to distal regions of the molecule. We propose that the intrinsically disordered C terminus of cTnT directly interacts with the regulatory N-domain of cTnC to allosterically modulate Ca2+ activation of force, perhaps by controlling the troponin I switching mechanism of striated muscle contraction. Alterations in cTnC-cTnT binding may compromise contractile performance and trigger pathological remodeling of the myocardium.

Cardiomyocyte nuclearity and ploidy: when is double trouble?

Considerable effort has gone into investigating mechanisms that underlie the developmental transition in which mammalian cardiomyocytes (CMs) switch from being able to proliferate during development, to essentially having lost that ability at maturity. This problem is interesting not only for scientific curiosity, but also for its clinical relevance because controlling the ability of mature CMs to replicate would provide a much-needed approach for restoring cardiac function in damaged hearts. In this review, we focus on the propensity of mature mammalian CMs to be multinucleated and polyploid, and the extent to which this may be necessary for normal physiology yet possibly disadvantageous in some circumstances. In this context, we explore whether the concept of the myonuclear domain (MND) in multinucleated skeletal muscle fibers might apply to cardiomyocytes, and whether cardio-MND size might be related to the transition of CMs to become multinuclear. Nuclei in CMs are almost certainly integrators of not only biochemical, but also-because of their central location within the myofibrils-mechanical information, and this multimodal, integrative function in adult CMs-involving molecules that have been extensively studied along with newly identified possibilities-could influence both gene expression as well as replication of the genome and the nuclei themselves.

Role of cardiac troponin I carboxy terminal mobile domain and linker sequence in regulating cardiac contraction.

Inhibition of striated muscle contraction at resting Ca(2+) depends on the C-terminal half of troponin I (TnI) in thin filaments. Much focus has been on a short inhibitory peptide (Ip) sequence within TnI, but structural studies and identification of disease-associated mutations broadened emphasis to include a larger mobile domain (Md) sequence at the C-terminus of TnI. For Md to function effectively in muscle relaxation, tight mechanical coupling to troponin's core-and thus tropomyosin-is presumably needed. We generated recombinant, human cardiac troponins containing one of two TnI constructs: either an 8-amino acid linker between Md and the rest of troponin (cTnILink8), or an Md deletion (cTnI1-163). Motility assays revealed that Ca(2+)-sensitivity of reconstituted thin filament sliding was markedly increased with cTnILink8 (∼0.9 pCa unit leftward shift of speed-pCa relation compared to WT), and increased further when Md was missing entirely (∼1.4 pCa unit shift). Cardiac Tn's ability to turn off filament sliding at diastolic Ca(2+) was mostly (61%), but not completely eliminated with cTnI1-163. TnI's Md is required for full inhibition of unloaded filament sliding, although other portions of troponin-presumably including Ip-are also necessary. We also confirm that TnI's Md is not responsible for superactivation of actomyosin cycling by troponin.

The functional significance of the last 5 residues of the C-terminus of cardiac troponin I.

The C-terminal region of cardiac troponin I (cTnI) is known to be important in cardiac function, as removal of the last 17 C-terminal residues of human cTnI has been associated with myocardial stunning. To investigate the C-terminal region of cTnI, three C-terminal deletion mutations in human cTnI were generated: Δ1 (deletion of residue 210), Δ3 (deletion of residues 208-210), and Δ5 (deletion of residues 206-210). Mammalian two-hybrid studies showed that the interactions between cTnI mutants and cardiac troponin C (cTnC) or cardiac troponin T (cTnT) were impaired in Δ3 and Δ5 mutants when compared to wild-type cTnI. Troponin complexes containing 2-[4'-(iodoacetamido) anilino] naphthalene-6-sulfonic acid (IAANS) labeled cTnC showed that the troponin complex containing cTnI Δ5 had a small increase in Ca(2+) affinity (P < 0.05); while the cTnI Δ1- and Δ3 troponin complexes showed no difference in Ca(2+) affinity when compared to wild-type troponin. In vitro motility assays showed that all truncation mutants had increased Ca(2+) dependent motility relative to wild-type cTnI. These results suggest that the last 5 C-terminal residues of cTnI influence the binding of cTnI with cTnC and cTnT and affect the Ca(2+) dependence of filament sliding, and demonstrate the importance of this region of cTnI.

Ca(2+)-regulatory function of the inhibitory peptide region of cardiac troponin I is aided by the C-terminus of cardiac troponin T: Effects of familial hypertrophic cardiomyopathy mutations cTnI R145G and cTnT R278C, alone and in combination, on filament sliding.

Investigations of cardiomyopathy mutations in Ca(2+) regulatory proteins troponin and tropomyosin provide crucial information about cardiac disease mechanisms, and also provide insights into functional domains in the affected polypeptides. Hypertrophic cardiomyopathy-associated mutations TnI R145G, located within the inhibitory peptide (Ip) of human cardiac troponin I (hcTnI), and TnT R278C, located immediately C-terminal to the IT arm in human cardiac troponin T (hcTnT), share some remarkable features: structurally, biochemically, and pathologically. Using bioinformatics, we find compelling evidence that TnI and TnT, and more specifically the affected regions of hcTnI and hcTnT, may be related not just structurally but also evolutionarily. To test for functional interactions of these mutations on Ca(2+)-regulation, we generated and characterized Tn complexes containing either mutation alone, or both mutations simultaneously. The most important results from in vitro motility assays (varying [Ca(2+)], temperature or HMM density) show that the TnT mutant "rescued" some deleterious effects of the TnI mutant at high Ca(2+), but exacerbated the loss of function, i.e., switching off the actomyosin interaction, at low Ca(2+). Taken together, our experimental results suggest that the C-terminus of cTnT aids Ca(2+)-regulatory function of cTnI Ip within the troponin complex.

Post-translational modifications of vertebrate striated muscle myosin heavy chains.

Post-translational modifications (PTMs) play a crucial role in regulating the function of many sarcomeric proteins, including myosin. Myosins comprise a family of motor proteins that play fundamental roles in cell motility in general and muscle contraction in particular. A myosin molecule consists of two myosin heavy chains (MyHCs) and two pairs of myosin light chains (MLCs); two MLCs are associated with the neck region of each MyHC's N-terminal head domain, while the two MyHC C-terminal tails form a coiled-coil that polymerizes with other MyHCs to form the thick filament backbone. Myosin undergoes extensive PTMs, and dysregulation of these PTMs may lead to abnormal muscle function and contribute to the development of myopathies and cardiovascular disorders. Recent studies have uncovered the significance of PTMs in regulating MyHC function and showed how these PTMs may provide additional modulation of contractile processes. Here, we discuss MyHC PTMs that have been biochemically and/or functionally studied in mammals' and rodents' striated muscle. We have identified hotspots or specific regions in three isoforms of myosin (MYH2, MYH6, and MYH7) where the prevalence of PTMs is more frequent and could potentially play a significant role in fine-tuning the activity of these proteins.

Nuclear tropomyosin and troponin in striated muscle: new roles in a new locale?

Tropomyosin and troponin have well known Ca(2+)-regulatory functions in the striated muscle sarcomere. In this review, we summarize experimental evidence that tropomyosin and troponin are localized, with as yet unidentified functional roles, in the striated muscle cell nucleus. We also apply bioinformatics approaches that predict localization of some tropomyosin and troponin to the nucleus, and that SUMOylation could be a covalent modification that modulates their nuclear localization and function. Further, we provide examples of cardiomyopathy mutations that alter the predicted likelihood of nuclear localization and SUMOylation of tropomyosin. These observations suggest novel mechanisms by which cardiomyopathy mutations in tropomyosin and troponin might alter not only cardiac contractility but also nuclear function.

Nuclear cardiac troponin and tropomyosin are expressed early in cardiac differentiation of rat mesenchymal stem cells.

Nuclear actin - which is immunologically distinct from cytoplasmic actin - has been documented in a number of differentiated cell types, and cardiac isoforms of troponin I (cTnI) and troponin T (cTnT) have been detected in association with nuclei of adult human cardiac myocytes. It is not known whether these and related proteins are present in undifferentiated stem cells, or when they appear in cardiomyogenic cells following differentiation. We first tested the hypothesis that nuclear actin and cardiac isoforms of troponin C (cTnC) and tropomyosin (cTm) are present along with cTnI and cTnT in nuclei of isolated, neonatal rat cardiomyocytes in culture. We also tested the hypothesis that of these five proteins, only actin is present in nuclei of multipotent, bone marrow-derived mesenchymal stem cells (BM-MSCs) from adult rats in culture, but that cTnC, cTnI, cTnT and cTm appear early and uniquely following cardiomyogenic differentiation. Here we show that nuclear actin is present within nuclei of both ventricular cardiomyocytes and undifferentiated, multipotent BM-MSCs. We furthermore show that cTnC, cTnI, cTnT and cTm are not only present in myofilaments of ventricular cardiomyocytes in culture but are also within their nuclei; significantly, these four proteins appear between days 3 and 5 in both myofilaments and nuclei of BM-MSCs treated to differentiate into cardiomyogenic cells. These observations indicate that cardiac troponin and tropomyosin could have important cellular function(s) beyond Ca(2+)-regulation of contraction. While the roles of nuclear-associated actin, troponin subunits and tropomyosin in cardiomyocytes are not known, we anticipate that the BM-MSC culture system described here will be useful for elucidating their function(s), which likely involve cardiac-specific, Ca(2+)-dependent signaling in the nucleus.

The duration of glucocorticoid treatment alters the anabolic response to high-force muscle contractions.

Glucocorticoids induce a myopathy that includes loss of muscle mass and strength. Resistance exercise may reverse the muscle loss because it induces an anabolic response characterized by increases in muscle protein synthesis and potentially suppressing protein breakdown. Whether resistance exercise induces an anabolic response in glucocorticoid myopathic muscle is unknown, which is a problem because long-term glucocorticoid exposure alters the expression of genes that may prevent an anabolic response by limiting activation of pathways such as the mechanistic target of rapamycin in complex 1 (mTORC1). The purpose of this study was to assess whether high-force contractions initiate an anabolic response in glucocorticoid myopathic muscle. The anabolic response was analyzed by treating female mice with dexamethasone (DEX) for 7 days or 15 days. After treatment, the left tibialis anterior muscle of all mice was contracted via electrical stimulation of the sciatic nerve. Muscles were harvested 4 h after contractions. Rates of muscle protein synthesis were estimated using the SUnSET method. After 7 days of treatment, high-force contractions increased protein synthesis and mTORC1 signaling in both groups. After 15 days of treatment, high-force contractions activated mTORC1 signaling equally in both groups, but protein synthesis was only increased in control mice. The failure to increase protein synthesis may be because baseline synthetic rates were elevated in DEX-treated mice. The LC3 II/I ratio marker of autophagy was decreased by contractions regardless of treatment duration. These data show duration of glucocorticoid treatment alters the anabolic response to high-force contractions.NEW & NOTEWORTHY Glucocorticoid myopathy is the most common, toxic, noninflammatory myopathy. Our work shows that high-force contractions increase protein synthesis in skeletal muscle following short-term glucocorticoid treatment. However, longer duration glucocorticoid treatment results in anabolic resistance to high-force contractions despite activation of the mechanistic target of rapamycin in complex 1 (mTORC1) signaling pathway. This work defines potential limits for high-force contractions to activate the processes that would restore lost muscle mass in glucocorticoid myopathic patients.

High-resolution cryo-EM structure of the junction region of the native cardiac thin filament in relaxed state.

Cardiac contraction depends on molecular interactions among sarcomeric proteins coordinated by the rising and falling intracellular Ca2+ levels. Cardiac thin filament (cTF) consists of two strands composed of actin, tropomyosin (Tm), and equally spaced troponin (Tn) complexes forming regulatory units. Tn binds Ca2+ to move Tm strand away from myosin-binding sites on actin to enable actomyosin cross-bridges required for force generation. The Tn complex has three subunits-Ca2+-binding TnC, inhibitory TnI, and Tm-binding TnT. Tm strand is comprised of adjacent Tm molecules that overlap "head-to-tail" along the actin filament. The N-terminus of TnT (e.g., TnT1) binds to the Tm overlap region to form the cTF junction region-the region that connects adjacent regulatory units and confers to cTF internal cooperativity. Numerous studies have predicted interactions among actin, Tm, and TnT1 within the junction region, although a direct structural description of the cTF junction region awaited completion. Here, we report a 3.8 Å resolution cryo-EM structure of the native cTF junction region at relaxing (pCa 8) Ca2+ conditions. We provide novel insights into the "head-to-tail" interactions between adjacent Tm molecules and interactions between the Tm junction with F-actin. We demonstrate how TnT1 stabilizes the Tm overlap region via its interactions with the Tm C- and N-termini and actin. Our data show that TnT1 works as a joint that anchors the Tm overlap region to actin, which stabilizes the relaxed state of the cTF. Our structure provides insight into the molecular basis of cardiac diseases caused by missense mutations in TnT1.

Micromechanical thermal assays of Ca2+-regulated thin-filament function and modulation by hypertrophic cardiomyopathy mutants of human cardiac troponin.

Microfabricated thermoelectric controllers can be employed to investigate mechanisms underlying myosin-driven sliding of Ca(2+)-regulated actin and disease-associated mutations in myofilament proteins. Specifically, we examined actin filament sliding-with or without human cardiac troponin (Tn) and α-tropomyosin (Tm)-propelled by rabbit skeletal heavy meromyosin, when temperature was varied continuously over a wide range (~20-63°C). At the upper end of this temperature range, reversible dysregulation of thin filaments occurred at pCa 9 and 5; actomyosin function was unaffected. Tn-Tm enhanced sliding speed at pCa 5 and increased a transition temperature (T(t)) between a high activation energy (E(a)) but low temperature regime and a low E(a) but high temperature regime. This was modulated by factors that alter cross-bridge number and kinetics. Three familial hypertrophic cardiomyopathy (FHC) mutations, cTnI R145G, cTnI K206Q, and cTnT R278C, cause dysregulation at temperatures ~5-8°C lower; the latter two increased speed at pCa 5 at all temperatures.

Detection of target ssDNA using a microfabricated Hall magnetometer with correlated optical readout.

Sensing biological agents at the genomic level, while enhancing the response time for biodetection over commonly used, optics-based techniques such as nucleic acid microarrays or enzyme-linked immunosorbent assays (ELISAs), is an important criterion for new biosensors. Here, we describe the successful detection of a 35-base, single-strand nucleic acid target by Hall-based magnetic transduction as a mimic for pathogenic DNA target detection. The detection platform has low background, large signal amplification following target binding and can discriminate a single, 350 nm superparamagnetic bead labeled with DNA. Detection of the target sequence was demonstrated at 364 pM (<2 target DNA strands per bead) target DNA in the presence of 36 µM nontarget (noncomplementary) DNA (<10 ppm target DNA) using optical microscopy detection on a GaAs Hall mimic. The use of Hall magnetometers as magnetic transduction biosensors holds promise for multiplexing applications that can greatly improve point-of-care (POC) diagnostics and subsequent medical care.