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The thick filament is a key component of sarcomeres, the basic units of striated muscle 1 . Alterations in thick filament proteins are associated with familial hypertrophic cardiomyopathy and other heart and muscle diseases 2 . Despite the central importance of the thick filament, its molecular organization remains unclear. Here we present the molecular architecture of native cardiac sarcomeres in the relaxed state, determined by cryo-electron tomography. Our reconstruction of the thick filament reveals the three-dimensional organization of myosin, titin and myosin-binding protein C (MyBP-C). The arrangement of myosin molecules is dependent on their position along the filament, suggesting specialized capacities in terms of strain susceptibility and force generation. Three pairs of titin-α and titin-β chains run axially along the filament, intertwining with myosin tails and probably orchestrating the length-dependent activation of the sarcomere. Notably, whereas the three titin-α chains run along the entire length of the thick filament, titin-β chains do not. The structure also demonstrates that MyBP-C bridges thin and thick filaments, with its carboxy-terminal region binding to the myosin tails and directly stabilizing the OFF state of the myosin heads in an unforeseen manner. These results provide a foundation for future research investigating muscle disorders involving sarcomeric components. A cryo-electron tomography study reports the structure of thick myosin filaments of mouse cardiac muscle in the relaxed state in situ and the MyBP-C links that connect them with the surrounding thin actin filaments.

Pumping of the heart is powered by filaments of the motor protein myosin that pull on actin filaments to generate cardiac contraction. In addition to myosin, the filaments contain cardiac myosin-binding protein C (cMyBP-C), which modulates contractility in response to physiological stimuli, and titin, which functions as a scaffold for filament assembly 1 . Myosin, cMyBP-C and titin are all subject to mutation, which can lead to heart failure. Despite the central importance of cardiac myosin filaments to life, their molecular structure has remained a mystery for 60 years 2 . Here we solve the structure of the main (cMyBP-C-containing) region of the human cardiac filament using cryo-electron microscopy. The reconstruction reveals the architecture of titin and cMyBP-C and shows how myosin’s motor domains (heads) form three different types of motif (providing functional flexibility), which interact with each other and with titin and cMyBP-C to dictate filament architecture and function. The packing of myosin tails in the filament backbone is also resolved. The structure suggests how cMyBP-C helps to generate the cardiac super-relaxed state 3 ; how titin and cMyBP-C may contribute to length-dependent activation 4 ; and how mutations in myosin and cMyBP-C might disturb interactions, causing disease 5 , 6 . The reconstruction resolves past uncertainties and integrates previous data on cardiac muscle structure and function. It provides a new paradigm for interpreting structural, physiological and clinical observations, and for the design of potential therapeutic drugs. The intricate molecular architecture and interactions of the human cardiac myosin filament offer insights into cardiac physiology, disease and drug therapy.

Proteins that fold cotranslationally may do so in a restricted configurational space, due to the volume occupied by the ribosome. How does this environment, coupled with the close proximity of the ribosome, affect the folding pathway of a protein? Previous studies have shown that the cotranslational folding process for many proteins, including small, single domains, is directly affected by the ribosome. Here, we investigate the cotranslational folding of an all-β Ig domain, titin I27. Using an arrest peptide-based assay and structural studies by cryo-EM, we show that I27 folds in the mouth of the ribosome exit tunnel. Simulations that use a kinetic model for the force dependence of escape from arrest accurately predict the fraction of folded protein as a function of length. We used these simulations to probe the folding pathway on and off the ribosome. Our simulations—which also reproduce experiments on mutant forms of I27—show that I27 folds, while still sequestered in the mouth of the ribosome exit tunnel, by essentially the same pathway as free I27, with only subtle shifts of critical contacts from the C to the N terminus.

Peripartum cardiomyopathy shares clinical features with idiopathic dilated cardiomyopathy, a disorder associated with mutations in more than 40 genes. This study shows that mutations in some of these genes, notably TTN, also have a strong association with this condition. Peripartum cardiomyopathy is marked by the development of maternal systolic heart failure late in pregnancy or early in the postpartum period. 1 , 2 The incidence varies from 1 in 100 to 1 in 300 in geographic hot spots, including Nigeria and Haiti, to 1 in 1000 to 1 in 4000 in Europe and the United States. The strongest known risk factors are the presence of preeclampsia, twin gestation, and advanced maternal age. Among patients with peripartum cardiomyopathy, heart failure can resolve but often does not: rates of death of 5 to 10% are common, and 4% of cardiac transplantations in the . . .

Passive stiffness of the heart is determined largely by extracellular matrix and titin, which functions as a molecular spring within sarcomeres. Titin stiffening is associated with the development of diastolic dysfunction (DD), while augmented titin compliance appears to impair systolic performance in dilated cardiomyopathy. We found that myofibril stiffness was elevated in mice lacking histone deacetylase 6 (HDAC6). Cultured adult murine ventricular myocytes treated with a selective HDAC6 inhibitor also exhibited increased myofibril stiffness. Conversely, HDAC6 overexpression in cardiomyocytes led to decreased myofibril stiffness, as did ex vivo treatment of mouse, rat, and human myofibrils with recombinant HDAC6. Modulation of myofibril stiffness by HDAC6 was dependent on 282 amino acids encompassing a portion of the PEVK element of titin. HDAC6 colocalized with Z-disks, and proteomics analysis suggested that HDAC6 functions as a sarcomeric protein deacetylase. Finally, increased myofibril stiffness in HDAC6-deficient mice was associated with exacerbated DD in response to hypertension or aging. These findings define a role for a deacetylase in the control of myofibril function and myocardial passive stiffness, suggest that reversible acetylation alters titin compliance, and reveal the potential of targeting HDAC6 to manipulate the elastic properties of the heart to treat cardiac diseases.

Manmade high-performance polymers are typically non-biodegradable and derived from petroleum feedstock through energy intensive processes involving toxic solvents and byproducts. While engineered microbes have been used for renewable production of many small molecules, direct microbial synthesis of high-performance polymeric materials remains a major challenge. Here we engineer microbial production of megadalton muscle titin polymers yielding high-performance fibers that not only recapture highly desirable properties of natural titin (i.e., high damping capacity and mechanical recovery) but also exhibit high strength, toughness, and damping energy — outperforming many synthetic and natural polymers. Structural analyses and molecular modeling suggest these properties derive from unique inter-chain crystallization of folded immunoglobulin-like domains that resists inter-chain slippage while permitting intra-chain unfolding. These fibers have potential applications in areas from biomedicine to textiles, and the developed approach, coupled with the structure-function insights, promises to accelerate further innovation in microbial production of high-performance materials. Here, the authors engineer microbial production of muscle titin fibers with highly desirable mechanical properties and provide structural analyses that explain the molecular mechanisms underlying high performance of this polymer with potential uses in biomedicine and textile industries, among others.

Titin, essential for mechano-homeostasis in cardiac and skeletal sarcomere, contains numerous mechanosensitive immunoglobulin-like (Ig) domains in its I-band region. However, how proline isomerization and cysteine-mediated disulfide bond collectively regulate Ig domain dynamics within the physiological force range remains unclear. Here, we use single-molecule force spectroscopy to quantify the proximal Ig1 domain, revealing that proline isomerization leads to two native states–trans and cis states–with distinct mechanical and thermal stabilities. The trans-Ig1 unfolds at forces of  ~ 5 pN, which is over 50 pN lower than that of cis-Ig1, and unfolds 1000 times faster under physiological forces. Furthermore, such proline induced dual-state is likely shared feature across majority of I-band Ig domains. Additionally, reduced cis- and trans-Ig1 exhibit catch-slip bond unfolding, while oxidized forms display slip-catch-slip unfolding. This study offers insight into effective modulation of proline isomerization and disulfide bond in regulating mechanosensitive proteins within the physiological force range. Using single-molecule force spectroscopy, the authors demonstrate that proline isomerization and cysteine-mediated disulfide bonds lead to distinct mechanical states of the titin I-band immunoglobulin-like domains within the physiological force range.

Titin N2B unique sequence (N2B-us) is a 572 amino acid sequence that acts as an elastic spring to regulate muscle passive elasticity. It is thought to lack stable tertiary structures and is a force-bearing region that is regulated by mechanical stretching. In this study, the conformation of N2B-us and its interaction with four-and-a-half LIM domain protein 2 (FHL2) are investigated using AlphaFold2 predictions and single-molecule experimental validation. Surprisingly, a stable alpha/beta structural domain is predicted and confirmed in N2B-us that can be mechanically unfolded at forces of a few piconewtons. Additionally, more than twenty FHL2 LIM domain binding sites are predicted to spread throughout N2B-us. Single-molecule manipulation experiments reveals the force-dependent binding of FHL2 to the N2B-us structural domain. These findings provide insights into the mechano-sensing functions of N2B-us and its interactions with FHL2. Titin N2B unique sequence (N2B-us) is a 572 amino acid sequence that acts as an elastic spring to regulate muscle passive elasticity. Here the authors identify a mechanosensitive structural domain within the titin (N2B-us), and a force-dependent interaction between (N2B-us) and the protein FHL2.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations of the dystrophin gene, which spans 2.4 Mb on the X chromosome. Creatine kinase (CK) activity in blood and titin fragment levels in urine have been identified as biomarkers in DMD to monitor disease progression and evaluate therapeutic intervention. However, the difference in the sensitivity of these biomarkers in DMD remains unclear. Previously, we generated transchromosomic mice carrying the full-length human dystrophin gene on a human artificial chromosome (DYS-HAC1) vector. The human dystrophin derived from DYS-HAC1 improved pathological phenotypes observed in DMD-null mice, which lack the entire 2.4 Mb of the dystrophin gene. In this study, we compared the values of plasma CK activity and urine/plasma titin fragment levels in wild-type (WT), DYS-HAC1, DMD-null , and DYS-HAC1; DMD-null mice. Plasma CK activity and urine/plasma titin fragment levels in DMD-null mice were significantly higher than those in WT mice. Although plasma CK activity showed no significant difference between WT and DYS-HAC1; DMD-null mice, urine/plasma titin fragment levels in DYS-HAC1; DMD-null mice were higher than those in WT mice. Human dystrophin in DYS-HAC1; DMD-null mice drastically improved muscular dystrophy phenotypes seen in DMD-null mice; however, the proportion of myofibers with central nuclei in DYS-HAC1; DMD-null mice had a tendency to be slightly higher than that in WT mice. These results suggest that urine/plasma titin fragment levels could be a more sensitive biomarker than plasma CK activity.

Load-bearing tissues, such as muscle and cartilage, exhibit high elasticity, high toughness and fast recovery, but have different stiffness (with cartilage being significantly stiffer than muscle) 1 – 8 . Muscle achieves its toughness through finely controlled forced domain unfolding–refolding in the muscle protein titin, whereas articular cartilage achieves its high stiffness and toughness through an entangled network comprising collagen and proteoglycans. Advancements in protein mechanics and engineering have made it possible to engineer titin-mimetic elastomeric proteins and soft protein biomaterials thereof to mimic the passive elasticity of muscle 9 – 11 . However, it is more challenging to engineer highly stiff and tough protein biomaterials to mimic stiff tissues such as cartilage, or develop stiff synthetic matrices for cartilage stem and progenitor cell differentiation 12 . Here we report the use of chain entanglements to significantly stiffen protein-based hydrogels without compromising their toughness. By introducing chain entanglements 13 into the hydrogel network made of folded elastomeric proteins, we are able to engineer highly stiff and tough protein hydrogels, which seamlessly combine mutually incompatible mechanical properties, including high stiffness, high toughness, fast recovery and ultrahigh compressive strength, effectively converting soft protein biomaterials into stiff and tough materials exhibiting mechanical properties close to those of cartilage. Our study provides a general route towards engineering protein-based, stiff and tough biomaterials, which will find applications in biomedical engineering, such as osteochondral defect repair, and material sciences and engineering. The introduction of chain entanglements into protein-based hydrogels yields hydrogels with high stiffness, high toughness, fast recovery and ultrahigh compressive strength, with mechanical properties close to those of cartilage.