Structural Effects of Disease-Related Mutations in Actin-Binding Period 3 of Tropomyosin

Tropomyosin (Tpm) is an actin-binding coiled-coil protein. In muscle, it regulates contractions in a troponin/Ca2+-dependent manner and controls the thin filament lengths at the pointed end. Due to its size and periodic structure, it is difficult to observe small local structural changes in the coiled coil caused by disease-related mutations. In this study, we designed 97-residue peptides, Tpm1.164–154 and Tpm3.1265–155, focusing on the actin-binding period 3 of two muscle isoforms. Using these peptides, we evaluated the effects of cardiomyopathy mutations: I92T and V95A in Tpm1.1, and congenital myopathy mutations R91P and R91C in Tpm3.12. We introduced a cysteine at the N-terminus of each fragment to promote the formation of the coiled-coil structure by disulfide bonds. Dimerization of the designed peptides was confirmed by gel electrophoresis in the presence and absence of dithiothreitol. Using circular dichroism, we showed that all mutations decreased coiled coil stability, with Tpm3.1265–155R91P and Tpm1.164–154I92T having the most drastic effects. Our experiments also indicated that adding the N-terminal cysteine increased coiled coil stability demonstrating that our design can serve as an effective tool in studying the coiled-coil fragments of various proteins.

Structure of the thin filament in native skeletal muscles reveals its interaction with nebulin and two distinct conformations of myosin

Nebulin is a major structural protein of skeletal sarcomeres and is essential for proper assembly and contraction of skeletal muscle1. It stabilises and regulates the length of thin filaments,2 but the structural mechanism remains nebulous. Using electron cryotomography and sub-tomogram averaging, we present the first structure of native nebulin bound to thin filaments within the A-band and I-band of intact sarcomeres. This in-situ reconstruction reveals unprecedented detail of interaction at pseudo-atomic resolution between nebulin and actin, providing the basis for understanding the structural and regulatory roles of nebulin. The position of nebulin on the thin filament indicates that there is no contact to tropomyosin or myosin, but an unexpected interaction with a troponin-T linker, possibly through two binding motifs on nebulin. In addition, our structure of myosin bound to the thin filaments reveals different conformations of the neck domain, both within the same sarcomere and when compared to purified structures, highlighting an inherent structural variability in muscle. We provide a complete description of cross-bridge formation on fully native, nebulin-containing thin filaments at near-atomic scale. Our structures establish the molecular basis for the role of nebulin as a thin filament “molecular ruler” and the impact of nemaline myopathies mutations that will aid future development of therapies.

Single-molecule imaging reveals the concerted release of myosin from regulated thin filaments

Regulated thin filaments (RTFs) tightly control striated muscle contraction through calcium binding to troponin, which enables tropomyosin to expose myosin-binding sites on actin. Myosin binding holds tropomyosin in an open position, exposing more myosin-binding sites on actin, leading to cooperative activation. At lower calcium levels, troponin and tropomyosin turn off the thin filament; however, this is antagonised by the high local concentration of myosin, questioning how the thin filament relaxes. To provide molecular details of deactivation, we used single-molecule imaging of green fluorescent protein (GFP)-tagged myosin-S1 (S1-GFP) to follow the activation of RTF tightropes. In sub-maximal activation conditions, RTFs are not fully active, enabling direct observation of deactivation in real time. We observed that myosin binding occurs in a stochastic step-wise fashion; however, an unexpectedly large probability of multiple contemporaneous detachments is observed. This suggests that deactivation of the thin filament is a coordinated active process.

The titin N2B and N2A regions: biomechanical and metabolic signaling hubs in cross-striated muscles

Muscle specific signaling has been shown to originate from myofilaments and their associated cellular structures, including the sarcomeres, costameres or the cardiac intercalated disc. Two signaling hubs that play important biomechanical roles for cardiac and/or skeletal muscle physiology are the N2B and N2A regions in the giant protein titin. Prominent proteins associated with these regions in titin are chaperones Hsp90 and αB-crystallin, members of the four-and-a-half LIM (FHL) and muscle ankyrin repeat protein (Ankrd) families, as well as thin filament-associated proteins, such as myopalladin. This review highlights biological roles and properties of the titin N2B and N2A regions in health and disease. Special emphasis is placed on functions of Ankrd and FHL proteins as mechanosensors that modulate muscle-specific signaling and muscle growth. This region of the sarcomere also emerged as a hotspot for the modulation of passive muscle mechanics through altered titin phosphorylation and splicing, as well as tethering mechanisms that link titin to the thin filament system.

Abstract 112: The 3CL Protease Of SARS-CoV2 Induces Sarcomeric Degradation Through Cleavage Of The Giant Protein Obscurin

We hypothesized that the 3CL protease (3CLPro) is responsible for the sarcomere degradation observed in cardiomyocytes infected by SARS-COV2. Overexpression of 3CLPro, but not a catalytically inactive mutant, resulted in breakdown of sarcomeres characterized by intact Z-disk/thin filament subunits that has been recently reported (Perez-Bermejo, et al, 2021) in SARS-COV2 infection. To identify potential host protein targets of 3CLPro in an unbiased fashion we screened the human proteome using a cut-site scoring algorithm that we developed. Scoring, ie likelihood of 3CL protease cleavage, was based off experimental data (Chuck et al, 2010) previously published on the highly homologous (96%) 3CL protease from SARS-COV. This scoring was followed by refinement by secondary structure prediction to identify cut-sites that lie in unstructured regions that are thus thus more likely to be accessible to the protease. Using this method, we identified >1000 potential high-likelihood cut sites across the proteome. Further filtering by proteins with cardiomyocyte expression showed 5 high-likelihood sites within the giant sarcomeric protein, Obscurin (OBSCN), as well as many other structural and signaling proteins which we experimentally validated. Expression of 3CLPro in IPSC cardiomyocytes resulted in significantly reduced OBSCN staining without alterations in Z-disk, thin filament, or thick filament proteins by both western blot and immunocytochemistry. In addition, imaging showed loss of OBSCN at sites with intact Z-disk/thin filament subunits. Thus we propose that activity of 3CLPro is a significant contributor to sarcomere breakdown in SARS-COV2 infection via degradation of Obscurin.

Abstract 402: Defining Diverse Disease Pathomechanisms Across Thick And Thin Filament Hypertrophic Cardiomyopathy Variants.

Hypertrophic cardiomyopathy (HCM) affects as many as ~1 in 500 individuals, and is often typified by hyperdynamic contraction and poor cellular relaxation. HCM can be caused by mutations in a variety of key contractile proteins of the sarcomere. A large proportion of these variants are found in MYBPC3, MYH7, TNNT2, and TNNI3. These genes encode proteins that control cardiac muscle contraction at the thick (MYBPC3 and MYH7) and thin filaments (TNNT2 and TNNI3) of the sarcomere. In this study we use human induced pluripotent stem cell derived cardiomyocytes to model HCM across all of these genes. We do this to define key mechanistic differences between thick and thin filament HCM. We define sarcomeric contractility (SarcTrack) calcium transients (CalTrack) and myosin states using the mant-ATP assay. We use the parametric data from these experimental studies in iPSC-CMs to model possible disease mechanisms in silico. Our experimental analysis highlights that both thick and thin filament HCM variants cause cellular hypercontractility, with slowed cellular relaxation. We find that thick filament HCM variants drive cellular HCM phenotypes by destabilising the myosin interacting heads motif (IHM), showing a marked reduction in the super relaxed state of myosin. Counterintuitively thin filament based HCM variants show a reduction in DRX myosin. When applying Mavacamten the allosteric myosin ATPase inhibitor to our thin and thick filament HCM variant iPSC-CMs we find a dichotomy of cellular responses. The thick filament variants studied all show a clear resolution of cellular HCM. However, not all cellular phenotypes of thin filament HCM are corrected by Mavacamten treatment, although there is benefit. We conclude that causal mechanisms of thick filament HCM are well corrected at the molecular and cellular level by Mavacamten, but these causal mechanisms in thin filament based HCM are not suitably corrected. We highlight key mechanistic pharmacological targets for thin filament variants that could add cellular benefit to HCM phenotype resolution.

Abstract P439: Harnessing Multiscale Models Of A Dilated Cardiomyopathy Mutation For Precision Medicine

Familial dilated cardiomyopathy (DCM) is a leading cause of both adult and pediatric heart failure. Currently, there is no cure for DCM, and the 5-year transplant free survival rate is <50%. There is therefore an outstanding need to develop new therapeutics. Prior studies have established a strong genetic basis for DCM and identified causative genetic mutations. These observations provide unique opportunities to apply precision medicine approaches that target and circumvent the effects of deleterious mutations. Here, we used a multiscale approach to study the consequences of a human mutation in troponin T that causes DCM, ΔK210. We found that at the molecular scale ΔK210 changes the positioning of tropomyosin along the thin filament, leading to molecular hypocontractility. Using genome edited human stem cell derived cardiomyocytes heterozygous for the mutation, we show reduced cellular contractility at the single cell and tissue levels. Importantly, we demonstrate that mutant tissues show a reduced Frank-Starling response, increased stiffness, and misaligned myocytes. Based on our molecular mechanism, we hypothesized that treatment of ΔK210 with Omecamtiv Mecarbil (OM), a thin filament activator in clinical trials for heart failure, would improve the function of mutant tissues. We found that treatment of ΔK210 molecular complexes and tissues with OM causes a dose-dependent increase in cardiac function, reversing the mutation-induced contractile defect. Taken together, our study demonstrates how mechanistic molecular studies can be harnessed to identify precision medicine therapeutics.

Titin switches from an extensible spring to a mechanical rectifier upon muscle activation

In contracting striated muscle titin acts as a spring in parallel with the array of myosin motors in each half-sarcomere and could prevent the intrinsic instability of thousands of serially linked half-sarcomeres, if its stiffness, at physiological sarcomere lengths (SL), were ten times larger than reported. Here we define titin mechanical properties during tetanic stimulation of single fibres of frog muscle by suppressing myosin motor responses with Para-Nitro-Blebbistatin, which is able to freeze thick filament in the resting state. We discover that thin filament activation switches I-band titin spring from the large SL-dependent extensibility of the OFF-state to an ON-state in which titin acts as a SL-independent mechanical rectifier, allowing free shortening while opposing stretch with an effective stiffness 4 pN nm-1 per half-thick filament. In this way during contraction titin limits weak half-sarcomere elongation to a few % and, also, provides an efficient link for mechanosensing-based thick filament activation.