The earless monitor lizard, Lanthanotus borneensis, is a unique living fossil restricted to the island of Borneo and a possible key to understanding the evolution of the venom delivery system and secondary adaptation to water in lizards and snakes (Squamata). We sequenced and de novo assembled the genome of L. borneensis to a total size of 1.5 Gbp, 975 contigs with an N50 of 52 Mbp and an L50 of 9. The genome completeness is estimated to be 93% based on the Sauropsida OrthoDB core gene set. A genome-wide set of Lepidosauria orthologs was compiled to reconstruct and date their phylogeny, resulting in 966 protein-coding sequences amounting to a concatenated alignment of 356 kbp with 188 kbp parsimony-informative sites. Based on this phylogenomic analysis, one of the largest of its kind yet conducted for Squamata, we identified that a Toxicofera clade (comprising Serpentes, Anguimorpha, and Iguania) is supported by a plurality of gene trees, but critically, support for relationships within Toxicofera is almost equally distributed amongst the three possible topologies. Our tree-dating confirms a rapid divergence of all major squamate clades within the first 10% of squamate history, which may have contributed to rampant incomplete lineage sorting. While we did not identify positive selection on genes associated with venom components at the base of Toxicofera, our analyses found strong positive selection on the giant protein titin throughout the main clades of Toxicofera and especially in snakes. Genome-wide heterozygosity is low (HO = 0.0004), as is the effective population size towards the present. Future studies of the evolution of the venom delivery system in Toxicofera require a "true" species tree but also individual gene trees due to incomplete lineage sorting and the concomitant potential for hemiplasy. Titin-a key component of striated muscle elasticity-emerges as a target for future evolutionary studies in Toxicofera and especially in wide-gaped snakes ("Macrostomata"). The low observed genome-wide heterozygosity and the low but stable effective population size of L. borneensis during the large-scale habitat fluctuations on Sundaland in the Quaternary suggest an unexpected resilience to environmental perturbations but also a potentially lowered adaptive potential of this isolated lineage.

Missense variants in TTN pose a major challenge in genetic diagnostics due to their high frequency in the general population, the large size of the gene, and the complex multidomain architecture of the titin protein. While the contribution of truncating variants (TTNtv) to titinopathies is well established, the role of rare TTN missense variants remains poorly defined. Advances in computational prediction and functional testing offer new tools to assess their potential pathogenicity, which however are currently not fully utilized for clinical application. We analyzed an international cohort of unsolved myopathy cases selected based on the presence of a rare missense variant in trans with a TTNtv. Clinical data were collected from neuromuscular centers worldwide. In silico predictions were generated using AlphaMissense and complemented by minor allele frequency (MAF) and exon usage information. Additional inclusion criteria were based on a MAF < 0.010 and an AlphaMissense score ≥ 0.792 for the missense variants, in accordance with the latest ClinGen guidelines. Selected missense variants were characterized in vitro through protein expression and cell imaging assays to assess their effects on domain solubility and aggregation. Thirty patients with TTNtv/missense combinations were identified, presenting with heterogeneous myopathic phenotypes, ranging from congenital to adult onset. An in-depth analysis on AlphaMissense predictions highlighted those changes most frequently predicted as possibly pathogenic. Functional assays showed that three selected variants with changes to proline, located in β-sheets of Ig domains, led to impaired folding, cytoplasmic aggregation and co-localisation with proteostasis markers. In our cohort, all non-proline mutations occurred at buried sites, while some proline substitutions affected exposed residues. Notably, the variant p.(Gln7023Pro) was identified in 5 unrelated families sharing a conserved haplotype, indicating a common ancestor. This variant and the previously reported p.(Arg25480Pro) now meet ACMG criteria for classification as likely pathogenic. By integrating clinical, computational, and functional evidence, we propose a framework for interpreting TTN missense variants. Combining multiple lines of evidence is essential for variants' classification and interpretation, especially given TTN complexity. Advancing diagnostic accuracy will require tailored interpretation guidelines and a global effort in data sharing and functional validation.

Truncating variants in the TTN gene (TTNtv), encoding the giant sarcomeric protein titin, cause a range of human cardiac and skeletal muscle disorders of varying penetrance and severity. The effects of variant location on clinical manifestations are incompletely understood. We generated 6 zebrafish lines carrying truncating ttn.2 variants in the Z-disk, I-band, A-band, and M-band titin regions. Expression of titin transcripts and protein levels was evaluated using quantitative polymerase chain reaction and proteomics. Phenotype analysis was performed during embryonic development and in adult hearts. Homozygous embryos from all lines except the C-terminal line, e232, showed a significant reduction of Z-disk and I-band ttn.2 transcripts, but A-band and M-band transcript levels were reduced only in lines with truncations distal to the cronos promoter. These homozygous embryos uniformly died by 7 to 10 days postfertilization with marked impairment of cardiac morphology and function. Skeletal muscle motility and sarcomere organization were more disrupted in mutants with truncations distal to the cronos promoter compared with those proximal. In contrast, homozygous e232 embryos, which lacked only the titin kinase and M-band regions, had relatively preserved cardiac function with incorporation of truncated Ttn.2/Cronos protein and normal sarcomere assembly, but selective degradation of fast skeletal muscle sarcomeres. All heterozygous embryos were phenotypically indistinguishable from wild type. High-frequency echocardiography in adult heterozygous fish showed reduced ventricular contraction under resting conditions in A-band mutants. Heterozygous Z-disk and I-band mutants had no significant baseline impairment but were unable to augment ventricular contraction in response to acute adrenaline exposure, indicating a lack of cardiac reserve. Our data suggest that cardiac and skeletal muscle dysfunction associated with truncating ttn.2 variants is influenced by age, variant location, and the amount of functional titin protein. The distinctive phenotype associated with distal C-terminal truncations may reflect different requirements for C-terminal titin for maintenance of fast, slow, and cardiac muscle sarcomeres.

Since the 1950s, muscle contraction has been explained by the sliding filament and cross-bridge theories involving actin and myosin. However, these theories do not account for certain muscle properties, such as residual force enhancement (rFE). The sarcomeric protein titin has been proposed to contribute to active force and rFE, but its role remains unclear. A leading hypothesis suggests that titin binds to actin, thereby shortening its spring-like segment, with calcium regulating this interaction. We investigated the roles of calcium and cross-bridge formation in titin mechanics by measuring the length of titin's PEVK region in sarcomeres during (i) passive stretch, (ii) active stretch with cross-bridge inhibition (via 2,3-butanedione monoxime, BDM) and (iii) active isometric contraction. PEVK lengths were similar for passive and cross-bridge-inhibited conditions but were longer for active contractions. Our results suggest that cross-bridge engagement, not calcium alone, modulates titin extensibility and passive force under physiological conditions.

ObjectiveThe association, if any, between gene mutations, pathologic features of squamous cell carcinoma of the head and neck, and patient prognosis is unknown. This study investigates the association between common gene mutations in oral cavity squamous cell carcinoma, pathologic features of malignancy, and patient survival.Study DesignRetrospective database review.SettingUS hospitals.MethodsThe cBioPortal for Cancer Genomics database was queried for oral cavity squamous cell carcinoma patient data. Statistical analyses were conducted using IBM SPSS v29.ResultsOf the 423 patients included, the majority were male (66.6%), white (89.3%), and current/former smokers (74.1%). Tumor protein p53 (TP53), titin (TTN), and FAT atypical cadherin 1 (FAT1) mutations were present in 72.3%, 31.2%, and 22.0% of patients, respectively. Mutant TP53 was associated with positive extranodal extension compared to wild‐type (WT) TP53 (31.0% vs 18.8%) (P = .038) and perineural invasion (59.4% vs 44.3%, P = .021). High tumor mutational burden was present in 5.4% of cases. Gene mutations were not associated with differences in median overall survival or disease‐free survival. Multivariable analysis revealed an association between mutant TP53 and the presence of extranodal extension (odds ratio [OR] 2.61, 95% CI 1.05‐6.52, P = .039) and perineural invasion (OR 2.14, 95% CI 1.04‐4.42, P = .039).ConclusionMutant TP53 was associated with high‐risk pathologic features, including extranodal extension and perineural invasion, but not with inferior survival. A high tumor mutational burden (>10) is rare in oral cavity squamous cell carcinoma. Further research into the interplay between genetic mutations and patient outcomes is needed.

Pathogenic variants in the Titin gene (TTN) are implicated in a range of cardiac and musculoskeletal disorders. Among these, Titin truncating variants (TTNtv) represent a major genetic cause of dilated cardiomyopathy (DCM) and are frequently associated with various types of arrhythmias. We reviewed the medical records of a family presenting with frequent premature ventricular contractions (PVCs) as the primary clinical symptom. Clinical data included demographics, symptoms, 12-lead electrocardiograms (ECGs), transthoracic echocardiograms (TTEs), thyroid function tests, and 24-h Holter monitoring. For individuals exhibiting clinical phenotypes, cardiac magnetic resonance imaging (CMR) and late gadolinium enhancement (LGE) were also performed. Whole-exome sequencing was conducted for the proband and his daughter, and cascade screening of family members was performed via Sanger sequencing at an accredited genetic laboratory. We report a family with frequent PVCs. The proband, a 68-year-old male, was diagnosed with frequent PVCs through ECG and 24-h Holter monitoring. Following radiofrequency ablation, the frequency of PVCs was significantly reduced. However, he gradually developed a reduced left ventricular ejection fraction and ventricular dyskinesia, suggesting the progression toward cardiomyopathy. Two younger brothers and one daughter of the proband also exhibited frequent PVCs, with one brother showing evidence of cardiomyopathy. Genetic testing of all living relatives revealed that individuals with frequent PVCs shared a heterozygous frameshift mutation in TTN, resulting in a truncating variant in the titin protein. We hypothesize that frequent PVCs may represent a clinical phenotype of this TTN frameshift mutation and may be associated with titin-related cardiomyopathy.

The muscle protein titin spans half a sarcomere, from M‐line to Z‐disk, and is essential for both active and passive stretch. The N2A region of titin plays a critical role in various regulatory processes through its binding interactions. Located at the C‐terminus of the N2A region, adjacent to the PEVK region, are the I82 and I83 domains, which are key to binding calpain/p94. However, this interaction is absent in the mdm‐mouse model, which contains an 83‐amino acid deletion spanning the C‐terminus of the I83 domain and the N‐terminus of the PEVK region, leading to muscular dystrophy with myositis. This mdm‐deletion disrupts the structure of the I83 domain, preventing normal force enhancement in the presence of calcium and inhibiting eccentric contractions. Our lab has demonstrated that the I83 domain exhibits calcium sensitivity at concentrations similar to those found in active muscle. In this current study, we further demonstrate that the tandem I82‐I83 domains exhibit cooperative unfolding, as seen by a single unfolding event, and that calcium enhances the stability of the tandem I82‐I83 domains. The NMR structure of this construct exhibits a tighter interface between I82 and I83 than is observed in the crystal structure, suggesting that the two structures might represent the structure in the relaxed state versus the structure under force. The calcium response of these domains is hypothesized to affect the function of the N2A region during muscle activation.

This study investigates the influence of multilayer architecture on the mechanical, corrosion, and tribological properties of Ti/TiN coatings deposited on biomedical Ti-6Al-4V alloy. Nine multilayer configurations were prepared by DC/RF magnetron sputtering using metallic Ti and ceramic TiN targets, with a fixed TiN/Ti ratio of 3:1 and varying total numbers of layers (3, 5, and 7) and deposition times (30, 60, and 120 min). A strict application of the 10% indentation depth rule was implemented to eliminate substrate effects, which revealed significantly higher intrinsic hardness values (540–740 HV) and indentation moduli (124–143 GPa) compared to the substrate (353 HV; 114 GPa). In contrast, conventional higher-load testing underestimated coating performance due to substrate dominance. Among the investigated architectures, the Ti/TiN-7 configuration exhibited the best balance of properties, combining high hardness (~690 HV), modulus (~137 GPa), improved corrosion resistance (Ecorr up to −0.13 V, Icorr reduced by an order of magnitude), and stable abrasive wear behavior. These findings demonstrate that both bilayer number and deposition time critically determine the mechanical and functional response of Ti/TiN multilayers. The results provide practical guidelines for the reliable characterization and design of multilayer coatings for biomedical and aerospace applications.

Generation of two induced pluripotent stem cell lines from dilated cardiomyopathy patients harbouring TTN mutations

Elastic tethers connect telomeres of separating chromosomes in anaphase of animal cells. Immunofluorescence staining of titin in crane-fly spermatocytes, using 4 different antibodies, shows that the giant elastic protein titin seems to be a component of mitotic tethers: titin "strands" extend between separating chromosomes, connecting their telomeres, just as tethers do. Since titin is responsible for elastic forces in myofibrils, we suggest that titin is responsible for the backwards forces exerted on chromosome arms during anaphase.

Dephosphorylation-induced structural reorganization of skeletal muscle titin molecules.

Background: Tibial muscular dystrophy (TMD) is a type of muscle disease primarily affecting the lower legs. It typically begins in adulthood and is inherited through families in a dominant pattern, meaning that only one copy of the affected gene is required to pass the condition. It is associated with mutations in the TTN gene, located on chromosome 2q31, responsible for encoding the central sarcomeric protein titin. The TTN gene is a very large gene with 363 coding sections (exons). It provides instructions for making a huge protein called titin, which stretches across the sarcomere — the basic unit of muscle — from the Z disc to the M band. Changes (mutations) in the TTN gene can cause a variety of inherited conditions, including heart problems such as hypertrophic and dilated cardiomyopathy, as well as several muscle-related disorders. Methods: In this study, after collecting the patients' and parents' peripheral blood, DNA extraction was done using the salting-out method, and the sample was sequenced to check the exons. Results: Findings confirm the existence of the mutation NM_1333793 c.14952_14960 del (p.Leu4984_Gln4986del), which is heterozygous in the patient. Sequencing of the mother's genome also confirms this mutation. Conclusions: The results of this research show that due to the uncontrollable consequences of this mutation, the new whole exome sequencing (WES) method can be effective for screening before birth and marriage and reduce the psychological and treatment burden of such diseases in society.

Generation of iPSC lines (ICHi001-A, ICHi002-A, ICHi003-A, ICHi004-A) from four patients carrying Titin truncating variants associated with dilated cardiomyopathy.

The multi-domain muscle protein titin provides elasticity and mechanosensing functions to the sarcomere. Titin's PEVK domain is intrinsically disordered due to the presence of a large number of prolines and highly charged residues. Although PEVK does not have canonical actin-binding motifs, it has been shown to bind F-actin. Here, we explored whether the PEVK domain may also affect actin assembly. We cloned the middle, 733-residue-long segment (called PEVKII) of the full-length PEVK domain, expressed in E. coli and purified by using His- and Avi-tags engineered to the N- and C-termini, respectively. Actin assembly was monitored by the pyrene assay in the presence of varying PEVKII concentrations. The structural features of PEVKII-associated F-actin were studied with atomic force microscopy. The added PEVKII enhanced the initial and log-phase rates of actin assembly and the peak F-actin quantity in a concentration-dependent way. However, the critical concentration of actin polymerization was unaltered. Thus, PEVK accelerates actin polymerization by facilitating its nucleation. This effect was highlighted in the AFM images of F-actin-PEVKII adsorbed to the supported lipid bilayer. The sample was dominated by radially symmetric complexes of short actin filaments. PEVK's actin polymerization-modulating effect may, in principle, have a function in regulating sarcomeric actin length and turnover. Altogether, titin's PEVK domain is not only a non-canonical actin-binding protein that regulates sarcomeric shortening, but one that may modulate actin polymerization as well.

In contrast to invasive skeletal muscle biopsies and the associated complexity of tissue sampling techniques and potential detrimental side effects, the alternative application of liquid biopsy procedures has considerable advantages concerning minimal invasiveness, repeated sampling options, assay robustness and cost effectiveness. This article outlines the current status of serum biomarkers used for diagnosing and characterizing Duchenne muscular dystrophy (DMD), a primary muscle wasting disease of early childhood due to primary abnormalities in the extremely large DMD gene. Reviewed are important aspects of the discovery, characterization and diagnostic value of biofluid-based protein markers of dystrophinopathy. This includes an overview of traditional general skeletal muscle damage markers, such as creatine kinase, myoglobin and lactate dehydrogenase, which have been used for many decades in clinical applications to evaluate patients with muscular weakness. In addition, this article outlines the biochemical identification of novel biomarker candidates focusing on the usage of mass spectrometry-based proteomic surveys to establish comprehensive profiles of protein alterations in dystrophinopathy. Pathoproteomic serum markers of myonecrosis with great potential for improved patient screening, differential diagnosis, stage-specific prognosis and therapeutic monitoring include specific isoforms of muscle-derived cytosolic proteins, such as carbonic anhydrase isoform CA3 and fatty acid binding protein FABP3, as well as sarcomeric proteins, including specific isoforms of myosin light chain, myosin binding protein, troponin, and myomesin, in addition to peptide fragments derived from the giant protein titin. Biofluid-associated marker proteins of reactive myofibrosis include the extracellular matrix proteins fibronectin, osteopontin, collagen and matrix-metalloproteinases.

Pulmonary hypertension due to heart failure with preserved ejection fraction (PH-HFpEF) is a highly heterogeneous disease associated with right ventricular (RV) failure and adverse outcomes. Current diagnostic tools inadequately characterize pulmonary vascular disease and RV dysfunction, limiting treatment precision. We hypothesized that deep phenotyping-including invasive hemodynamics, advanced imaging, and myocardial transcriptomics-would identify high-risk PH-HFpEF phenotypes with distinct clinical, physiologic, and molecular characteristics. 42 PH-HFpEF participants (and 25 pre-capillary PH participants, as a comparison group) underwent clinical evaluation, echocardiography, cardiac MRI (cMRI), and invasive cardiopulmonary exercise testing (iCPET). K-means clustering considering clinical, iCPET, and cMRI data stratified participants into distinct clusters (phenogroups). A subset underwent further characterization by invasive pulmonary vascular mechanics (n=17 PH-HFpEF, n=5 pre-capillary PH) and 4D flow cMRI (n=10 PH-HFpEF, n=5 pre-capillary PH). Endomyocardial biopsies from 10 PH-HFpEF participants were analyzed using long-read RNA-sequencing for differential gene and transcript expression. Clustering revealed two PH-HFpEF phenogroups with significantly different outcomes at one-year follow-up (HR=11.96, CI: 2.66-53.86). High-risk participants had greater left ventricular mass, reduced RV ejection fraction, worse exercise-induced PH, impaired gas exchange (↓peak oxygen consumption, ↑slope of minute ventilation/carbon dioxide production), and lower myocardial strain. Pulmonary vascular mechanics showed higher proximal pulmonary artery stiffness (↑characteristic impedance), increased RV energy expenditure (↑compression waves), and abnormal distal vascular reflections (↓diastolic reflection index) in the high-risk group. 4D flow MRI revealed disturbed flow in pulmonary vasculature (both arteries and veins) in high-risk participants. Transcriptomic analysis implicated differences in post-transcriptional RNA processing and translational control, as well as mitochondrial function, in phenotypic divergence between these HFpEF subgroups. In particular, differential transcript usage for the TTN gene, encoding the giant sarcomeric protein titin, may be particularly relevant to elevated myocardial stiffness, which worsens vascular remodeling and precipitates RV dysfunction and failure. In PH-HFpEF, unsupervised clustering using deep physiologic and imaging data identified a high-risk group with impaired RV function and unique transcriptomic profiles. Our findings suggest that proximal and distal pulmonary vascular remodeling, as well as differences in titin isoform expression, may underlie RV failure in this phenogroup. This comprehensive approach provides a framework for mechanistically driven precision medicine strategies in PH-HFpEF. What is new?: 1.Unsupervised clustering integrating exercise hemodynamics, cardiac MRI, and clinical features revealed a distinct PH-HFpEF subgroup at significantly higher risk for adverse outcomes (HR = 11.96), independent of traditional IpcPH/CpcPH classification.Invasive wave mechanics (impedance and wave separation analyses) and non-invasive 4D flow cardiopulmonary MRI reveal unique patterns of proximal stiffness, RV energy inefficiency, and abnormal distal reflections in high-risk PH-HFpEF, providing insights into segmental vascular dysfunction.Long-read RNA sequencing of endomyocardial biopsies from PH-HFpEF patients revealed distinct transcriptomic signatures between high- and low-risk PH-HFpEF phenogroups.Long-read RNA-seq identified distinct transcript usage of gene TTN, encoding a giant sarcomeric protein (titin) that is a key determinant for myocardial stiffness, suggesting a potential underlying mechanism for RV dysfunction in PH-HFpEF.What are clinical implications?: 2.These findings offer a framework for more refined patient classification beyond traditional PVR-based methods, which may improve identification of high-risk individuals who require closer monitoring or targeted therapy.The integrative approach highlights titin isoform imbalance and vessel-specific stiffness as potential therapeutic targets, supporting the development of biology-informed, phenotype-specific interventions for PH-HFpEF with RV dysfunction.

Sarcomeres are the universal contractile units of muscles that enable animals to move. Insect muscles display a remarkable functional diversity: they operate at extremely different contraction frequencies (ranging from ~1 to 1000hertz) and amplitudes during flying, walking, and crawling. This is puzzling because sarcomeres are built from essentially the same actin-myosin components. Here, we address how functionally different sarcomeres are made. We show that the giant protein titin and the regulation of developmental contractility are key for the sarcomere specializations. I-band titin spans and determines the length of the sarcomeric I-band in a muscle type-specific manner. Unexpectedly, I-band titin also rules the length of the force-generating myosin filament using a feedback mechanism that is modulated by myosin contractility. We propose a model of how sarcomere specializations in insects are tuned, provide evidence for this model, and discuss its validity beyond insects.

Cardiac titin isoforms: Practice in interpreting results of electrophoretic analysis.

Marfan syndrome (MFS) is an autosomal dominant disease caused by mutations in the gene (FBN1) of fibrillin-1, a major determinant of the extracellular matrix (ECM). Functional impairment in the cardiac left ventricle (LV) of these patients is usually a consequence of aortic valve disease. However, LV passive stiffness may also be affected by chronic changes in mechanical load and ECM dysfunction. Passive stiffness is determined by the giant sarcomeric protein titin that has two main cardiac splice isoforms: the shorter and stiffer N2B and the longer and more compliant N2BA. Their ratio is thought to reflect myocardial response to pathologies. Whether this ratio and titin's sarcomeric layout is altered in MFS is currently unknown. Here, we studied LV samples from MFS patients carrying FBN1 mutation, collected during aortic root replacement surgery. We found that the N2BA:N2B titin ratio was elevated, indicating a shift toward the more compliant isoform. However, there were no alterations in the total titin content compared with healthy humans based on literature data. Additionally, while the gross sarcomeric structure was unaltered, the M-band was more extended in the MFS sarcomere. We propose that the elevated N2BA:N2B titin ratio reflects a general adaptation mechanism to the increased volume overload resulting from the valvular disease and the direct ECM disturbances so as to reduce myocardial passive stiffness and maintain diastolic function in MFS.

ABSTRACT Considering the great popularity of eccentric-based training, the purpose of this review is to first provide the scientific rationale for its use; second, summarize the eccentric-based training modalities that can be used; and finally, offer practical recommendations on how to implement eccentric-based modalities to enhance sports performance. The molecular and neural mechanisms underlying eccentric actions are partially distinct from those of concentric and isometric actions. During eccentric actions, theories suggest a strain-induced modulation of actin-myosin interactions at the cross-bridge level, activation of structural protein titin, and winding of titin on actin. Eccentric acute physiological responses differ from concentric exercise responses, including variations in neuromuscular, metabolic, hormonal, and anabolic signaling. Eccentric training elicits greater improvements in muscle strength, power, and stretch-shortening cycle function compared with concentric-only or traditional resistance training. Therefore, eccentric-based training can lead to unique neuromuscular (e.g., improved coordination of motor units) and morphological (e.g., increased muscle fascicle length and enhanced distal cross-sectional area) adaptations that could play a key role in sport performance. Practitioners may implement eccentric exercises with external loads, fully eccentric-based exercises (e.g., Nordic hamstring curl), accentuated eccentric loading, flywheel resistance exercise, and plyometrics to develop specific physical adaptations in line with their goals. Eccentric work (e.g., for hamstrings) can be obtained during other exercises such as downhill running tasks, decelerations, and sprinting activities. Practitioners need to be aware that no single “silver bullet” training modality exists; consequently, practitioners should use a combination of eccentric-based training approaches with their athletes to obtain the desired adaptations.