Titin Research Articles

Genetically based atrial fibrillation: Current considerations for diagnosis and management.

Atrial fibrillation (AF) is the most common atrial arrhythmia and is subcategorized into numerous clinical phenotypes. Given its heterogeneity, investigations into the genetic mechanisms underlying AF have been pursued in recent decades, with predominant analyses focusing on early onset or lone AF. Linkage analyses, genome-wide association studies (GWAS), and single gene analyses have led to the identification of rare and common genetic variants associated with AF risk. Significant overlap with genetic variants implicated in dilated cardiomyopathy syndromes, including truncating variants of the sarcomere protein titin, have been identified through these analyses, in addition to other genes associated with cardiac structure and function. Despite this, widespread utilization of genetic testing in AF remains hindered by the unclear impact of genetic risk identification on clinical outcomes and the high prevalence of variants of unknown significance (VUS). However, genetic testing is a reasonable option for patients with early onset AF and in those with significant family history of arrhythmia. While many knowledge gaps remain, emerging data support genotyping to inform selection of AF therapeutics. In this review, we highlight the current understanding of the complex genetic basis of AF and explore the overlap of AF with inherited cardiomyopathy syndromes. We propose a set of criteria for clinical genetic testing in AF patients and outline future steps for the integration of genetics into AF care.

Heterozygous missense variant in the\xa0TTN\xa0gene causing Tibial muscular dystrophy

BackgroundTibial muscular dystrophy (TMD), tardive, is a dominantly inherited mild degenerative disorder of anterior tibial muscles. Mutations of Titin (TTN) have been reported in patients with different phenotypes such as skeletal muscular abnormalities or complex overlapping disorders of muscles. Titin (TTN) is a large 363 exon gene that encodes an abundant protein (the longest polypeptide known in nature) expressed in the heart and skeletal muscles.MethodsDNA from peripheral blood sample was extracted, whole exome sequencing (WES) was performed, and a neuromuscular disorders related gene-filtering strategy was used to analyse the disease-causing mutations. Further, sanger sequencing was applied to confirm the variant.ResultsA novel missense variant (c.41529G > C;p.Arg13843Ser) of TTN gene was identified in a patient with lower limb weakness, occasional tongue fasciculation and mild scoliosis. This variant leads to a substitution of arginine with serine, causing structural changes in titin protein that is responsible for the TMD disease.ConclusionThe novel variant detected has widened the genetic spectrum of TTN-associated diseases, further functional studies will aid in establishing the clinical diagnosis.

Clinical and molecular genetic analysis of early-onset myopathy with fatal cardiomyopathy: Novel biallelic M-line TTN mutation and review of the literature

Early-onset myopathy with fatal cardiomyopathy (EOMFC), also known as Salih myopathy (SALMY) is a rare, heterogeneous, and severe form of titinopathies with autosomal recessive inherited neuromuscular disorders that affects both skeletal and cardiac muscles. It was previously identified only in the Arab population with unknown incidence. Titin (TTN) mutations that have been reported in congenital myopathies are associated with a variety of phenotypic spectrum of titinopathies, which are scattered along with the 364 exons of the gene. In this study, we report a consanguineous Moroccan female child aged 29 months who presented with congenital myopathy associated to dilated cardiomyopathy. Clinical Exome Sequencing identified a novel homozygous truncating mutation c.106541delA p.(Asp35514Valfs*32) in exon 361 of the TTN gene. Sanger sequencing confirmed the mutation in a homozygous state in the proband and in a heterozygous state in both of her parents. Clinical and molecular data of the proband were correlated with 15 patients reported in the literature for congenital myopathy associated to a heart defect or development of dilated cardiomyopathy with at least one mutant allele in the M-band titin protein as inclusion criteria. In conclusion, the application of NGS in rare genetic heterogeneous forms as EOMFC provides more evidently an increasing proportion of congenital myopathies than currently recognized and expands the mutation spectrum of the TTN gene for better guiding the genetic diagnosis with adequate genetic counseling to the Moroccan families.

From the Literature

From the Literature

Passive and active mechanical properties of titin studied in intact frog muscle fibers upon inhibition of myosin motors by para-nitro-blebbistatin

In the striated muscle thousands of serially linked half-sarcomeres (the force generating units) make macroscopic the shortening but also imply instability, with lengthening of the weaker half-sarcomeres caused by shortening of the stronger ones. The giant protein titin spans the whole half-sarcomere, through the I-band, connecting the Z-line at the end of the sarcomere to the tip of the thick filament and through the A-band, associated to thick filament, up to M-line at the centre of the sarcomere.

Truncated titin is incorporated into the sarcomere in human cardiac samples with dilated cardiomyopathy

The most common genetic cause of dilated cardiomyopathy (DCM) is mutations in titin gene, TTN, with truncating variants (TTNtv). TTN encodes the giant protein titin, a large (3-4 MDa) and abundant protein that forms the third myofilament type of striated muscle. Titin spans half the sarcomere, from the Z-disk to the M-line. The underlying mechanisms by which titin mutations induce DCM are poorly understood.

Calmodulin interactions within the N2A region of titin

One of the most interesting elastic proteins is the muscle protein titin, which exists in the sarcomere of the muscle and plays a critical role in maintaining myofibril integrity and in passive force. Within titin, there are several types of structural elements that work synergistically to produce the various functions of the protein. Our lab has recently been focused on the N2A region, which is located in the center of the I-band region of titin. Structurally, the N2A region is comprised of four immunoglobulin domains and a unique region that consists of 117 amino acids in both ordered and disordered regions.

RBM20 ASOs improve ventricular filling in a mouse model with increased titin-based stiffness

The giant protein titin provides the elastic backbone of the myofilament and is extensively spliced to adapt the mechanical properties of the ventricle. The titin splice factor RBM20 determines not only the elastic but also the contractile properties of the cardiac ventricle. Animal models that replicate phenotypic features of heart failure with preserved ejection fraction (HFpEF), such as increased stiffness of the cardiac ventricle, respond to a 50% reduction in RBM20 levels with improved diastolic filling and improved cardiac function.

Truncated titin protein in dilated cardiomyopathy

Truncating variations in the gene coding for titin (TTNtv) have long been known to cause dilated cardiomyopathy (DCM), but efforts to detect direct evidence of either haploinsufficiency or dominant negative mechanisms have left the mechanism open to controversy, with the default assumption being that TTNtv proteins are absent in the myocardium. By analyzing a collection of 184 post-transplant human hearts, 22 of which bear TTNtv’s, we show evidence supporting both dominant-negative gain of toxic function due to truncated titin protein associated with the sarcomeric fraction of the myocardium, as well as haploinsufficiency due to lack of sufficient full length titin.

Single Nucleus RNA-sequencing Reveals Altered Intercellular Communication and Dendritic Cell Activation in Nonobstructive Hypertrophic Cardiomyopathy.

End stage, nonobstructive hypertrophic cardiomyopathy (HCM) is an intractable condition with no disease-specific therapies. To gain insights into the pathogenesis of nonobstructive HCM, we performed single nucleus RNA-sequencing (snRNA-seq) on human HCM hearts explanted at the time of cardiac transplantation and organ donor hearts serving as controls. Differential gene expression analysis revealed 64 differentially expressed genes linked to specific cell types and molecular functions. Analysis of ligand-receptor pair gene expression to delineate potential intercellular communication revealed significant reductions in expressed ligand-receptor pairs likely affecting the extracellular matrix, growth factor binding, peptidase regulator activity, platelet-derived growth factor binding and protease binding in the HCM tissue. Changes in Integrin-β1 receptor expression were responsible for many observed changes related to extracellular matrix interactions, by increasing in dendritic, smooth muscle and pericyte cells while decreasing in endothelial and fibroblast cells, suggesting potential mechanisms for fibrosis and microvascular disease in HCM and a potential role for dendritic cells. In contrast, there was an increase in ligand-receptor pair expression associated with adenylate cyclase binding, calcium channel molecular functions, channel inhibitor activity, ion channel inhibitor activity, phosphatase activator activity, protein kinase activator activity and titin binding, suggesting important shifts in various signaling cascades in nonobstructive, end stage HCM.

Mechanochemical Evolution of Disulfide Bonds in Proteins.

Disulfide bonds play a pivotal role in the mechanical stability of proteins. Numerous proteins that are known to be exposed to mechanical forces in vivo contain disulfide bonds. The presence of cryptic disulfide bonds in a protein structure may be related to its resistance to an applied mechanical force. Disulfide bonds in proteins tend to be highly conserved but their evolution might be directly related to the evolution of the protein mechanical stability. Hence, tracking the evolution of disulfide bonds in a protein can help to derive crucial stability/function correlations in proteins that are exposed to mechanical forces. Phylogenic analysis and ancestral sequence reconstruction (ASR) allow tracking the evolution of proteins from the past ancestors to our modern days and also establish correlations between proteins from different species. In addition, ASR can be combined with single-molecule force spectroscopy (smFS) to investigate the mechanical properties of proteins including the occurrence and function of disulfide bonds. Here we present a detailed protocol to study the mechanochemical evolution of proteins using a fragment of the giant muscle protein titin as example. The protocol can be easily adapted to AFS studies of any resurrected mechanical force bearing protein of interest.

Truncated titin proteins in the pathophysiology of DCM.

Truncated titin proteins in the pathophysiology of DCM.

Abstract 10240: Key Pathomechanisms of Cardiomyopathy Due to TTN Truncation

Introduction: Heterozygous truncating variants in TTN (TTNtv) coding for titin cause dilated cardiomyopathy (DCM) but the underlying pathomechanisms are unclear and disease management remains uncertain. Here, we aim to elucidate the key pathomechanisms of TTNtv-DCM. Hypothesis: Stable expression of truncated titin proteins and titin haploinsufficiency characterize TTNtv-DCM and represent potential targets for therapy. Methods and Results: We studied left ventricular tissues from 14 nonfailing donor hearts and 113 endstage failing DCM patients and identified a TTNtv in 22 DCM patients (19.5%) by next generation sequencing. Using titin protein gel electrophoresis and western blot we demonstrate, for the first time, titin haploinsufficiency in TTNtv-DCM hearts. Strikingly, all 21 adult TTNtv-DCM hearts of our cohort showed stable expression of truncated titin proteins. Expression was variable, up to one-half of the total titin protein pool, and negatively correlated to patient age at heart transplantation. Truncated titin proteins were not detected in sarcomeres but were present in intracellular aggregates. Deregulated ubiquitin-dependent protein quality control was apparent. Next, we produced human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), comparing wildtype controls to cells with a patient-derived, prototypical A-band TTNtv or a CRISPR/Cas9-edited M-band TTNtv. TTNtv hiPSC-CMs showed reduced wildtype titin expression and contained truncated titin proteins, whose proportion increased upon inhibition of proteasomal activity. In engineered heart muscle generated from hiPSC-CMs, depressed contractility caused by TTNtv could be reversed by correction of the mutation using CRISPR/Cas9, thereby eliminating truncated titin proteins and raising wildtype titin content. Functional improvement also occurred when titin protein content was increased by proteasome inhibition. Conclusions: Our study reveals the major pathomechanisms of TTNtv-DCM, which include titin haploinsufficiency as a life-long condition and truncated titin protein enrichment with aggregate formation, along with aberrant protein quality control. Results can be exploited for new therapies of TTNtv cardiomyopathies.

Abstract 12802: Intron-Mediated Enhancement of Titin ( TTN ) Regulates Sarcomere Formation and Function

Background: Heterozygous truncating variants in the sarcomere protein titin (TTN) are the most common genetic cause of heart failure, a major cause of morbidity and mortality. This causality indicates that even two-fold changes in the amount of TTN can profoundly disturb cardiac physiology. Although a critical role of TTN in sarcomere formation and cardiomyocyte contractility is well established, the mechanisms regulating transcription of the TTN gene remain poorly understood. Methods: We performed bioinformatics analysis to identify a putative transcriptional enhancer of TTN . Next, we created biallelic deletion of the enhancer in human induced pluripotent stem cell derived cardiomyocytes and performed enhancer reporter assays both in vitro and in vivo to demonstrate necessity and sufficiency of the enhancer in TTN gene expression, respectively. Furthermore, we performed massive parallel reporter assay to define critical transcriptional factors of the TTN enhancer activity and analyzed whole genome sequencing (WGS) data of human patients with unexplained dilated cardiomyopathy (DCM). Results: We identified an intron mediated enhancer that promotes cardiac-specific TTN expression. Global deletion of this element downregulated TTN expression in cardiomyocytes and impaired sarcomere development, while transgenic expression promoted cardiac expression in mice. Using mutational scanning we defined key transcription factor binding sites, including NKX2-5 and MEF2 motifs that promote TTN expression in cardiomyocytes. Consistent with these functions, analyses of WGS data in 69 patients with unexplained DCM revealed one rare variant that disrupted the conserved MEF2 transcriptional factor binding motif. Conclusions: Discovery of a TTN enhancer advances our understanding of cardiomyocyte development, provides an opportunity to modulate TTN transcriptional activity, and ultimately develop therapeutic strategies to treat dilated cardiomyopathy caused by TTN haploinsufficiency.

Truncated titin proteins in dilated cardiomyopathy.

Truncating variants in TTN (TTNtvs) are the most common known cause of nonischemic dilated cardiomyopathy (DCM), but how TTNtvs cause disease has remained controversial. Efforts to detect truncated titin proteins in affected human DCM hearts have failed, suggesting that disease is caused by haploinsufficiency, but reduced amounts of titin protein have not yet been demonstrated. Here, we leveraged a collection of 184 explanted posttransplant DCM hearts to show, using specialized electrophoretic gels, Western blotting, allelic phasing, and unbiased proteomics, that truncated titin proteins can quantitatively be detected in human DCM hearts. The sizes of truncated proteins corresponded to that predicted by their respective TTNtvs; the truncated proteins were encoded by the TTNtv-bearing allele; and no degradation fragments from protein encoded by either allele were detectable. In parallel, full-length titin was less abundant in TTNtv+ than in TTNtv− DCM hearts. Disease severity or need for transplantation did not correlate with TTNtv location. Transcriptomic profiling revealed few differences in splicing or allelic imbalance of the TTN transcript between TTNtv+ and TTNtv− DCM hearts. Studies with isolated human adult cardiomyocytes revealed no defects in contractility in cells from TTNtv+ compared to TTNtv− DCM hearts. Together, these data demonstrate the presence of truncated titin protein in human TTNtv+ DCM, show reduced amounts of full-length titin protein in TTNtv+ DCM hearts, and support combined dominant-negative and haploinsufficiency contributions to disease.

Truncated titin proteins and titin haploinsufficiency are targets for functional recovery in human cardiomyopathy due to TTN mutations.

Heterozygous truncating variants in TTN (TTNtv), the gene coding for titin, cause dilated cardiomyopathy (DCM), but the underlying pathomechanisms are unclear and disease management remains uncertain. Truncated titin proteins have not yet been considered as a contributor to disease development. Here, we studied myocardial tissues from nonfailing donor hearts and 113 patients with end-stage DCM for titin expression and identified a TTNtv in 22 patients with DCM (19.5%). We directly demonstrate titin haploinsufficiency in TTNtv-DCM hearts and the absence of compensatory changes in the alternative titin isoform Cronos. Twenty-one TTNtv-DCM hearts in our cohort showed stable expression of truncated titin proteins. Expression was variable, up to half of the total titin protein pool, and negatively correlated with patient age at heart transplantation. Truncated titin proteins were not detected in sarcomeres but were present in intracellular aggregates, with deregulated ubiquitin-dependent protein quality control. We produced human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs), comparing wild-type controls to cells with a patient-derived, prototypical A-band-TTNtv or a CRISPR-Cas9–generated M-band-TTNtv. TTNtv-hiPSC-CMs showed reduced wild-type titin expression and contained truncated titin proteins whose proportion increased upon inhibition of proteasomal activity. In engineered heart muscle generated from hiPSC-CMs, depressed contractility caused by TTNtv could be reversed by correction of the mutation using CRISPR-Cas9, eliminating truncated titin proteins and raising wild-type titin content. Functional improvement also occurred when wild-type titin protein content was increased by proteasome inhibition. Our findings reveal the major pathomechanisms of TTNtv-DCM and can be exploited for new therapies to treat TTNtv-related cardiomyopathies.

Investigation of phase formations and mechanical properties in the binary systems of Ti–TiB<sub>2</sub>, Ti–TiN and ternary system of Ti–TiB<sub>2</sub>–TiN

Investigation of phase formations and mechanical properties in the binary systems of Ti–TiB<sub>2</sub>, Ti–TiN and ternary system of Ti–TiB<sub>2</sub>–TiN

E3-ligase knock down revealed differential titin degradation by autophagy and the ubiquitin proteasome system

The sarcomere protein titin is a major determinant of cardiomyocyte stiffness and ventricular distensibility. The constant mechanical stress on titin requires well-controlled protein quality control, the exact mechanisms of which have not yet been fully elucidated. Here, we analyzed E3-ligases potentially responsible for cardiac titin ubiquitination and specifically studied the involvement of the autophagosomal system in titin degradation. Pharmacological inhibition of autophagy and the proteasome in cultured primary rat cardiomyocytes significantly elevated titin ubiquitination and increased titin degradation. Using in-vitro pull down assays we identified binding of E3-ligases MuRF1-3, CHIP and Fbx32 to several titin domains. Immunofluorescence analysis showed sarcomeric localization of the E3-ligases. siRNA-mediated knock-down of the E3-ligases MuRF-1, -3 and a combination of CHIP/Fbx32 significantly reduced autophagy-related titin ubiquitination, whereas knock-down of MuRF-2 and -3 reduced proteasome-related titin ubiquitination. We demonstrated that the proteasomal and the autophagosomal-lysosomal system participate in degradation of the titin filament. We found that ubiquitination and degradation of titin are partially regulated by E3-ligases of the MuRF family. We further identified CHIP and Fbx32 as E3-ligases involved in titin ubiquitination.

Efficiency of Application of (Mo, Al)N-Based Coatings with Inclusion of Ti, Zr or Cr during the Turning of Steel of Nickel-Based Alloy

The article compares the properties of multilayer composite wear-resistant coatings of Zr–ZrN–(Zr, Mo, Al)N, Ti–TiN–(Ti, Mo, Al)N, and Cr–CrN–(Cr, Mo, Al)N. The investigation was focused on hardness, resistance to fracture during scratch tests, elemental composition, and structure of the coatings. Experiments were carried out to study the wear resistance of coated carbide tools during the turning of 1045 steel and of NiCr20TiAl heat-resistant nickel alloy. With the elemental compositions identical in the content of molybdenum (Mo) and aluminium (Al), identical thicknesses and nanolayer periods of λ, the coatings being studied demonstrated a noticeable difference in wear resistance. Both during the turning of steel and nickel-based alloy, the highest wear resistance was detected for tools with the Zr–ZrN–(Zr, Mo, Al)N coating (the tool life was 3–5 times higher than for uncoated tools). The good wear resistance of the Zr–ZrN–(Zr, Mo, Al)N coating may be related to the optimal combination of hardness and plasticity and the active formation of molybdenum oxide (MoO3) on the coating surface during the cutting, with good tribological and protective properties.

Increased Expression of N2BA Titin Corresponds to More Compliant Myofibrils in Athlete's Heart.

Long-term exercise induces physiological cardiac adaptation, a condition referred to as athlete’s heart. Exercise tolerance is known to be associated with decreased cardiac passive stiffness. Passive stiffness of the heart muscle is determined by the giant elastic protein titin. The adult cardiac muscle contains two titin isoforms: the more compliant N2BA and the stiffer N2B. Titin-based passive stiffness may be controlled by altering the expression of the different isoforms or via post-translational modifications such as phosphorylation. Currently, there is very limited knowledge about titin’s role in cardiac adaptation during long-term exercise. Our aim was to determine the N2BA/N2B ratio and post-translational phosphorylation of titin in the left ventricle and to correlate the changes with the structure and transverse stiffness of cardiac sarcomeres in a rat model of an athlete’s heart. The athlete’s heart was induced by a 12-week-long swim-based training. In the exercised myocardium the N2BA/N2B ratio was significantly increased, Ser11878 of the PEVK domain was hypophosphorlyated, and the sarcomeric transverse elastic modulus was reduced. Thus, the reduced passive stiffness in the athlete’s heart is likely caused by a shift towards the expression of the longer cardiac titin isoform and a phosphorylation-induced softening of the PEVK domain which is manifested in a mechanical rearrangement locally, within the cardiac sarcomere.