Titin Research Articles

Modulating TTN gene expression in human iPSC-CMs to investigate mechanisms and therapies in TTN-associated dilated cardiomyopathy

Abstract Background Familial dilated cardiomyopathy (DCM) is a common and harmful condition, and no available therapies target the underlying genetic mechanisms. Truncating variants in TTN are the most commonly identified genetic cause of DCM. However, it remains unclear how TTN variants drive disease. Contemporary thinking identifies haploinsufficiency, dominant negative effects, or a combination of both as likely driving mechanisms. There is competing evidence as to whether I-band and A-band mutations differ in severity or in the relative roles of haploinsufficiency and dominant-negative effects. Clarifying disease mechanisms will help pave the way to targeted genetic therapies. Purpose To modulate TTN gene expression in human-induced stem cell-derived cardiomyocyte (hiPSC-CM) models of TTN-associated DCM carrying I-band or A-band variants to provide insight into the comparative roles of haploinsufficiency and dominant negative mechanisms in DCM, and to identify novel therapeutic strategies. Methods We used CRISPR-Cas9 gene-editing to generate hiPSC lines with known disease-causing heterozygous truncating mutations in the I-band or A-band of TTN. These were screened for off-target mutations and pluripotency. hiPSCs were differentiated into hiPSC-CMs for phenotyping. We assayed TTN mRNA and titin protein levels. We trialled approaches for modulating TTN expression including CRISPR activation. Results Two hiPSC lines with heterozygous mutations in the I-band and A-band of TTN were generated using CRISPR/Cas-9 gene-editing. These hiPSC lines were successfully differentiated into hiPSC-CMs. Both cell lines demonstrated unchanged TTN mRNA expression compared to wildtype controls, but had reduced expression of full-length titin protein and identifiable truncated protein products. TTN mRNA expression was subsequently increased in these models using modulators of gene expression. Conclusions hiPSC-CM models of DCM with truncating mutations in the I-band or A-band of TTN were generated and phenotyped. These models were then used to trial gene expression modulation methods. This provides a human cellular system that can be used to better understand disease mechanisms of TTN-associated DCM, with the ultimate aim of establishing gene therapies for TTN-associated DCM.

Influence of Nitride Coatings on Corrosion Resistance and the Biocompatibility of Titanium Alloy Products

The bioadhesion of bacteria to the surface of samples with Ti–TiN, Zr–ZrN, Zr–(Zr, Nb)N, and Zr–(Zr, Hf)N coatings was studied via incubation with gram-positive strains of Staphylococcus aureus. The samples were kept at 25 °C for 30 days in a 3% NaCl solution. The deposition of coatings slows, whereas oxidation processes intensify. The oxygen content on the TiN and (Zr, Nb)N coating surfaces was higher than that of the Ti sample without a coating. Samples with ZrN and, especially, (Zr, Hf)N coatings resist oxidation better. Regarding bioactivity toward S. aureus, the highest density of biological forms was observed on the surfaces of TiN and (Zr, Hf)N coatings. The lowest density was on the surfaces of uncoated, ZrN-coated, and (Zr, Nb)N-coated samples. On Ti–TiN, Zr–ZrN, and Zr–(Zr, Nb)N coatings, the formation of surface biostructures of a filamentary type was observed. In the uncoated sample, the biostructures have an island character, and in the sample with a Zr–(Zr, Hf)N coating, the formation of extensive areas of biostructures was observed. Between the biostructures and coating, a layer 5 to 15 nm thick was observed, presumably associated with bacterial adhesion. The presence of biostructures on the coating surface can activate or slow oxidation processes.

Structural features of skeletal muscle titin aggregates

Titin is a multidomain protein of striated and smooth muscles of vertebrates. The protein consists of repeating immunoglobulin-like (Ig) and fibronectin-like (FnIII) domains, which are β-sandwiches with a predominant β-structure, and also contains disordered regions. In this work, the methods of atomic force microscopy (AFM), X-ray diffraction and Fourier transform infrared spectroscopy were used to study the morphology and structure of aggregates of rabbit skeletal muscle titin obtained in two different solutions: 0.15 M glycine-KOH, pH 7.0 and 200 mM KCl, 10 mM imidazole, pH 7.0. According to AFM data, skeletal muscle titin formed amorphous aggregates of different morphology in the above two solutions. Amorphous aggregates of titin formed in a solution containing glycine consisted of much larger particles than aggregates of this protein formed in a solution containing KCl. The “KCl-aggregates” according to AFM data had the form of a “sponge”-like structure, while amorphous “glycine-aggregates” of titin formed “branching” structures. Spectrofluorometry revealed the ability of titin “glycine aggregates” to bind to the dye thioflavin T (TT), and X-ray diffraction revealed the presence of one of the elements of the amyloid cross β-structure, a reflection of ~4.6 Å, in these aggregates. These data indicate that the “glycine-aggregates” of titin are amyloid or amyloid-like. No similar structural features were found in titin “KCl-aggregates”; they also did not show the ability to bind to thioflavin T, indicating the non-amyloid nature of these titin aggregates. Fourier transform infrared spectroscopy revealed differences in the secondary structure of the two types of titin aggregates. The data obtained demonstrate the features of structural changes during the formation of intermolecular bonds between molecules of the giant titin protein during its aggregation. The data expand the understanding of the process of amyloid protein aggregation.

Exploring the etiology of dilated cardiomyopathy using Mendelian randomization.

Observational clinical studies suggest an association between dilated cardiomyopathy (DCM) and various factors including titin, cardiac troponin I (CTnI), desmocollin-2, the perinatal period, alcoholism, Behçet's disease, systemic lupus erythematosus, hyperthyroidism and thyrotoxicosis, hypothyroidism, carnitine metabolic disorder, and renal insufficiency. The causal nature of these associations remains uncertain. This study aims to explore these correlations using the Mendelian randomization (MR) approach. To investigate the etiology of DCM through Mendelian randomization analysis. Data mining was conducted in genome-wide association study databases, focusing on variant target proteins (titin, CTnI, desmocollin-2), the perinatal period, alcoholism, Behçet's disease, systemic lupus erythematosus, hyperthyroidism and thyrotoxicosis, hypothyroidism, carnitine metabolic disorder, and renal insufficiency, with DCM as the outcome. The analysis employed various regression models, namely, the inverse-variance weighted (IVW), MR-Egger, simple mode, weighted median, and weighted mode methods. The IVW results showed a correlation between titin protein and DCM, identifying titin as a protective factor [OR = 0.856, 95% CI (0.744-0.985), P = 0.030]. CTnI protein correlated with DCM, marking it as a risk factor [OR = 1.204, 95% CI (1.010-1.436), P = 0.040]. Desmocollin-2 also correlated with DCM and was recognized as a risk factor [OR = 1.309, 95% CI (1.085-1.579), P = 0.005]. However, no causal relationship was found between the perinatal period, alcoholism, Behçet's disease, systemic lupus erythematosus, hyperthyroidism and thyrotoxicosis, hypothyroidism, carnitine metabolic disorder, renal insufficiency, and DCM (P > 0.05). The MR-Egger intercept test indicated no pleiotropy (P > 0.05), affirming the effectiveness of Mendelian randomization in causal inference. Titin, CTnI, and desmocollin-2 proteins were identified as independent risk factors for DCM. Contrasting with previous observational studies, no causal relationship was observed between DCM and the perinatal period, alcoholism, Behçet's disease, systemic lupus erythematosus, hyperthyroidism and thyrotoxicosis, hypothyroidism, carnitine metabolic disorder, or renal insufficiency.

Fatiguing exercise reduces cellular passive Young's modulus in human vastus lateralis muscle.

Previous studies demonstrated that acute fatiguing exercise transiently reduces whole-muscle stiffness, which might contribute to increased risk of injury and impaired contractile performance. We sought to elucidate potential intracellular mechanisms underlying these reductions. To that end, the cellular passive Young's modulus was measured in muscle fibres from healthy, young males and females. Eight volunteers (four male and four female) completed unilateral, repeated maximal voluntary knee extensions until task failure, immediately followed by bilateral percutaneous needle muscle biopsy of the post-fatigued followed by the non-fatigued control vastus lateralis. Muscle samples were processed for mechanical assessment and separately for imaging and phosphoproteomics. Fibres were passively (pCa 8.0) stretched incrementally to 156% of initial sarcomere length to assess Young's modulus, calculated as the slope of the resulting stress-strain curve at short (sarcomere length=2.4-3.0µm) and long (sarcomere length=3.2-3.8µm) lengths. Titin phosphorylation was assessed by liquid chromatography followed by high-resolution mass spectrometry. The passive modulus was significantly reduced in post-fatigued versus control fibres from male, but not female, participants. Post-fatigued samples showed altered phosphorylation of five serine residues (four located within the elastic region of titin) but did not exhibit altered active tension or sarcomere ultrastructure. Collectively, these results suggest that acute fatigue is sufficient to alter phosphorylation of skeletal titin in multiple locations. We also found reductions in the passive modulus, consistent with prior reports in the literature investigating striated muscle stiffness. These results provide mechanistic insight contributing to the understanding of dynamic regulation of whole-muscle tissue mechanics in vivo. HIGHLIGHTS: What is the central question of this study? Previous studies have shown that skeletal muscle stiffness is reduced following a single bout of fatiguing exercise in whole muscle, but it is not known whether these changes manifest at the cellular level, and their potential mechanisms remain unexplored. What is the main finding and its importance? Fatiguing exercise reducescellular stiffnessin skeletal muscle from males but not females, suggesting thatfatigue alters tissue compliance in asex-dependent manner. The phosphorylation status of titin, a potential mediator of skeletal muscle cellular stiffness, is modified by fatiguing exercise. Previous studies have shown that passive skeletal muscle stiffness is reduced following a single bout of fatiguing exercise. Lower muscle passive stiffness following fatiguing exercise might increase risk for soft-tissue injury; however, the underlying mechanisms of this change are unclear. Our findings show that fatiguing exercise reduces the passive Young's modulus in skeletal muscle cells from males but not females, suggesting that intracellular proteins contribute to reduced muscle stiffness following repeated loading to task failure in a sex-dependent manner. The phosphorylation status of the intracellular protein titin is modified by fatiguing exercise in a way that might contribute to altered muscle stiffness after fatiguing exercise. These results provide important mechanistic insight that might help to explain why biological sex impacts the risk for soft-tissue injury with repeated or high-intensity mechanical loading in athletes and the risk of falls in older adults.

Abstract Tu097: Titin cleavage induces loss of myocardial tensional homeostasis driving a rapid TGF-beta-independent fibrotic response

Background: Cardiomyocytes (CMs) are subjected to different types of mechanical loads in the myocardium. Depending on its origin these loads can be classified as extracellular (extracellular matrix-ECM towards CMs), intracellular (exerted by the CM contraction machinery, i.e. the sarcomeres) or intercellular (exerted between CMs). In the myocardium, all these loads are actively maintained in tensional homeostasis (TH). In this context, the study of the sarcomere protein titin is essential, because its I-band region is considered as a hub for mechanotransduction, mostly of intracellular mechanical cues. Research Questions: It has been shown that mechanical disruption of the ECM affects the myocardial TH, leading to morphological changes inside CMs. However, potential alteration of TH resulting from defective mechanics of CM proteins remains unknown due to the lack of specific tools for in vivo studies. Aims: Here we explore the in vivo effects on myocardial TH of an abrupt tensional unloading of titin. Methods: We have experimentally challenged CMs to a tensional unloading by means of titin cleavage in vivo using TEVs-TTN mice. In this model, a cassette with a Tobacco Etch Virus protease (TEVp) recognition sequence has been included in the I-band of titin. Transducing TEVp with adeno-associated virus (AAV), we were able to cleave titin in vivo , ceasing only the mechanical properties of the protein. Samples were analyzed using an array of techniques including histology, immunofluorescence, and bulk and single nuclei RNA sequencing. Results: Our results show that a mosaic expression of TEVp, which does not affect cell viability, cleaving no more than 30% of total titin. However, this leads to a fast fibrotic response (less than six days), characterized by an increase in interstitial collagen and a response from cardiac fibroblasts population. Transcriptional and phospo-SMAD2/3 analysis results suggest that this ECM response occurs without noticeable activation of the TGFβ canonical pathway. Conclusion: Taken together, our results show experimentally for the first time that titin is not only a mechanotransducer of intracellular cues. In fact, mechanical abrogation of the I-band leads to a fast ECM response mediated by cardiac fibroblasts.

Imaging of Existing and Newly Translated Proteins Elucidates Mechanisms of Sarcomere Turnover.

How the sarcomeric complex is continuously turned over in long-living cardiomyocytes is unclear. According to the prevailing model of sarcomere maintenance, sarcomeres are maintained by cytoplasmic soluble protein pools with free recycling between pools and sarcomeres. We imaged and quantified the turnover of expressed and endogenous sarcomeric proteins, including the giant protein titin, in cardiomyocytes in culture and in vivo, at the single cell and at the single sarcomere level using pulse-chase labeling of Halo-tagged proteins with covalent ligands. We disprove the prevailing protein pool model and instead show an ordered mechanism in which only newly translated proteins enter the sarcomeric complex while older ones are removed and degraded. We also show that degradation is independent of protein age and that proteolytic extraction is a rate-limiting step in the turnover. We show that replacement of sarcomeric proteins occurs at a similar rate within cells and across the heart and is slower in adult cells. Our findings establish a unidirectional replacement model for cardiac sarcomeres subunit replacement and identify their turnover principles.

Titin: roles in cardiac function and diseases.

The giant protein titin is an essential component of muscle sarcomeres. A single titin molecule spans half a sarcomere and mediates diverse functions along its length by virtue of its unique domains. The A-band of titin functions as a molecular blueprint that defines the length of the thick filaments, the I-band constitutes a molecular spring that determines cell-based passive stiffness, and various domains, including the Z-disk, I-band, and M-line, serve as scaffolds for stretch-sensing signaling pathways that mediate mechanotransduction. This review aims to discuss recent insights into titin's functional roles and their relationship to cardiac function. The role of titin in heart diseases, such as dilated cardiomyopathy and heart failure with preserved ejection fraction, as well as its potential as a therapeutic target, is also discussed.

Exploring TTN variants as genetic insights into cardiomyopathy pathogenesis and potential emerging clues to molecular mechanisms in cardiomyopathies

The giant protein titin (TTN) is a sarcomeric protein that forms the myofibrillar backbone for the components of the contractile machinery which plays a crucial role in muscle disorders and cardiomyopathies. Diagnosing TTN pathogenic variants has important implications for patient management and genetic counseling. Genetic testing for TTN variants can help identify individuals at risk for developing cardiomyopathies, allowing for early intervention and personalized treatment strategies. Furthermore, identifying TTN variants can inform prognosis and guide therapeutic decisions. Deciphering the intricate genotype–phenotype correlations between TTN variants and their pathologic traits in cardiomyopathies is imperative for gene-based diagnosis, risk assessment, and personalized clinical management. With the increasing use of next-generation sequencing (NGS), a high number of variants in the TTN gene have been detected in patients with cardiomyopathies. However, not all TTN variants detected in cardiomyopathy cohorts can be assumed to be disease-causing. The interpretation of TTN variants remains challenging due to high background population variation. This narrative review aimed to comprehensively summarize current evidence on TTN variants identified in published cardiomyopathy studies and determine which specific variants are likely pathogenic contributors to cardiomyopathy development.

Digenic inheritance involving a muscle-specific protein kinase and the giant titin protein causes a skeletal muscle myopathy

In digenic inheritance, pathogenic variants in two genes must be inherited together to cause disease. Only very few examples of digenic inheritance have been described in the neuromuscular disease field. Here we show that predicted deleterious variants in SRPK3, encoding the X-linked serine/argenine protein kinase 3, lead to a progressive early onset skeletal muscle myopathy only when in combination with heterozygous variants in the TTN gene. The co-occurrence of predicted deleterious SRPK3/TTN variants was not seen among 76,702 healthy male individuals, and statistical modeling strongly supported digenic inheritance as the best-fitting model. Furthermore, double-mutant zebrafish (srpk3−/−; ttn.1+/−) replicated the myopathic phenotype and showed myofibrillar disorganization. Transcriptome data suggest that the interaction of srpk3 and ttn.1 in zebrafish occurs at a post-transcriptional level. We propose that digenic inheritance of deleterious changes impacting both the protein kinase SRPK3 and the giant muscle protein titin causes a skeletal myopathy and might serve as a model for other genetic diseases.

New mechanisms: From lactate to lactylation to rescue heart failure.

Lactylation of α-myosin heavy chain (α-MHC) has recently been reported to preserve sarcomeric structure and function and attenuate the development of heart failure. Specifically, lactylation enhanced the interaction of α-MHC with the sarcomeric protein Titin, thereby maintaining normal sarcomeric structure and myocardial contractile function. Furthermore, the administration of lactate or inhibition of lactate efflux potentially treats heart failure by restoring lactylation of α-MHC and the interaction of α-MHC with Titin. This finding highlights the significant role of α-MHC lactylation in myocardial diseases and presents a new therapeutic target for the treatment of heart failure.

The Structural Features of Skeletal Muscle Titin Aggregates

Titin is a multidomain protein of striated and smooth muscles of vertebrates. The protein consists of repeating immunoglobulin-like (Ig) and fibronectin-like (FnIII) domains, which are β-sandwiches with a predominant β-structure, and also contains disordered regions. In this work, the methods of atomic force microscopy (AFM), X-ray diffraction, and Fourier transform infrared spectroscopy were used to study the morphology and structure of aggregates of rabbit skeletal muscle titin obtained in two different solutions: 0.15 M glycine-KOH, pH 7.0 and 200 mM KCl, 10 mM imidazole, pH 7.0. According to AFM data, skeletal muscle titin formed amorphous aggregates of different morphologies in the above two solutions. Amorphous aggregates of titin formed in a solution containing glycine consisted of much larger particles than aggregates of this protein formed in a solution containing KCl. The "KCl-aggregates" according to AFM data had the form of a "sponge"-like structure, while amorphous "glycine-aggregates" of titin formed "branching" structures. Spectrofluorometry revealed the ability of "glycine-aggregates" of titin to bind to the dye thioflavin T (TT), and X-ray diffraction revealed the presence of one of the elements of the amyloid cross β-structure, a reflection of ~4.6 Å, in these aggregates. These data indicate that "glycine-aggregates" of titin are amyloid or amyloid-like. No similar structural features were found in "KCl-aggregates" of titin; they also did not show the ability to bind to thioflavin T, indicating the non-amyloid nature of these titin aggregates. Fourier transform infrared spectroscopy revealed differences in the secondary structure of the two types of titin aggregates. The data we obtained demonstrate the features of structural changes during the formation of intermolecular bonds between molecules of the giant titin protein during its aggregation. The data expand the understanding of the process of amyloid protein aggregation.

In-situ synthesis of spatial heterostructure Ti composites by laser powder bed fusion to overcome the strength and plasticity trade-off

Recent research has focused on laser in-situ additive manufacturing of metal matrix composites with spatially controllable microstructures (phases). This study, inspired by the process of inserting mesh fibers into reinforced concrete, synthesizes TiN in situ using laser powder bed fusion and N2 gas. The laser-melted track, embedded with TiN particles, formed a spatially heterostructured Ti composite (SHTC) with a three-dimensional, artificially controlled architecture in a pure Ti matrix. The influences of process parameters on the mechanical properties of the spatially heterostructured Ti composite and the microstructural evolution of TiN/Ti were investigated emphatically. The results showed that the growth direction of the microstructure was changed by laser powder bed fusion additive manufacturing with alternating N2–Ar gas under suitable N2 concentration and melting track spacing. Among all spatially heterostructured Ti composites, the TiN–Ti heterolayer net-like structure achieved a high ultimate tensile strength of ∼1.0 GPa and elongation of 27 %, demonstrating a superior strength-ductility combination than intrinsic pure Ti and uniform TiN composites, as well as traditional layered structure Ti-based composites. During the tensile test, the deformation behavior was monitored in situ using digital image correlation, and the fracture mechanism was investigated. Hetero-deformation induced strengthening and toughening potentially explains the mechanism behind the strength enhancement of spatially heterostructured Ti composites. Furthermore, this work may stimulate research and development in additive manufacturing of spatial heterostructures with configurable structures, targeting synergistic regulation of strength and ductility in the integration of structure-material-function.

Truncated titin protein in dilated cardiomyopathy incorporates into the sarcomere and transmits force.

Truncated titin protein in dilated cardiomyopathy incorporates into the sarcomere and transmits force.

Walking with giants: The challenges of variant impact assessment in the giant sarcomeric protein titin.

Titin, the so-called "third filament" of the sarcomere, represents a difficult challenge for the determination of damaging genetic variants. A single titin molecule extends across half the length of a sarcomere in striated muscle, fulfilling a variety of vital structural and signaling roles, and has been linked to an equally varied range of myopathies, resulting in a significant burden on individuals and healthcare systems alike. While the consequences of truncating variants of titin are well-documented, the ramifications of the missense variants prevalent in the general population are less so. We here present a compendium of titin missense variants-those that result in a single amino-acid substitution in coding regions-reported to be pathogenic and discuss these in light of the nature of titin and the variant position within the sarcomere and their domain, the structural, pathological, and biophysical characteristics that define them, and the methods used for characterization. Finally, we discuss the current knowledge and integration of the multiple fields that have contributed to our understanding of titin-related pathology and offer suggestions as to how these concurrent methodologies may aid the further development in our understanding of titin and hopefully extend to other, less well-studied giant proteins. This article is categorized under: Cardiovascular Diseases > Genetics/Genomics/Epigenetics Congenital Diseases > Genetics/Genomics/Epigenetics Congenital Diseases > Molecular and Cellular Physiology.

About the pathway of amyloid aggregation of titin

The process of amyloid aggregation is quite complex and poorly understood. In this work, having summarized previously obtained results on the aggregation of the multidomain smooth muscle protein titin, an attempt has been made to expand understanding of this process, and a new possible mechanism by which amyloid aggregation of titin may occur is delineated. Our main conclusion is that the ability of titin to form amorphous aggregates seems to be the only possible way of aggregation of this protein. Most likely, only separate parts of the molecules, but not the whole protein, are involved in the formation of the amyloid structure in amorphous aggregates of smooth muscle titin. This feature, given the large size of the protein molecule, distinguishes titin from other amyloid or amyloid-like proteins. The paper discusses the potential energy landscape underlying the formation of titin amyloid aggregates.

Titin domains with reduced core hydrophobicity cause dilated cardiomyopathy

The underlying genetic defect in most cases of dilated cardiomyopathy (DCM), a common inherited heart disease, remains unknown. Intriguingly, many patients carry single missense variants of uncertain pathogenicity targeting the giant protein titin, a fundamental sarcomere component. To explore the deleterious potential of these variants, we first solved the wild-type and mutant crystal structures of I21, the titin domain targeted by pathogenic variant p.C3575S. Although both structures are remarkably similar, the reduced hydrophobicity of deeply buried position 3575 strongly destabilizes the mutant domain, a scenario supported by molecular dynamics simulations and by biochemical assays that show no disulfide involving C3575. Prompted by these observations, we have found that thousands of similar hydrophobicity-reducing variants associate specifically with DCM. Hence, our results imply that titin domain destabilization causes DCM, a conceptual framework that not only informs pathogenicity assessment of gene variants but also points to therapeutic strategies counterbalancing protein destabilization.

Abstract 13979: Titin-Truncating Variants Predispose to Dilated Cardiomyopathy in Patients Genetically Similar to African Reference Populations

Introduction: Heterozygous mutations within the TTN gene can cause the translation of shortened forms of the titin protein known as titin-truncating variants (TTNtvs) leading to dilated cardiomyopathy (DCM) among certain individuals. We previously reported an odds ratio (OR) of 18.7 for DCM among individuals genetically similar to the European reference population (EUR) with high percentage spliced in (hiPSI) TTNtvs, however we were unable to show a similar relationship among individuals genetically similar to the African reference population (AFR). Methods: Penn Medicine Biobank volunteers with whole exome sequencing were screened for TTNtvs with minor allele frequency <0.001 and percent spliced in >0.9. Participants were also screened for a DCM diagnosis defined using International Classification of Diseases, ninth and tenth revision diagnoses codes. Left ventricular ejection fraction (LVEF) values were extracted from structured transthoracic echocardiography reports. Logistic and linear regression were employed to evaluate the association between hiPSI TTNtvs and DCM or LVEF adjusting for age, sex, and first five genetic principal components. Results: Of the 43,731 participants with sequencing data, 11,136 (27.1%) were genetically similar to the AFR reference population, 50.3% were male, and the median age was 57.8 years (IQR: 44.9-69.5 years). One percent (418 individuals) had a hiPSI TTNtv. The age- and sex-adjusted OR of having DCM was similar among AFR and EUR participants carrying hiPSI TTNtvs (AFR OR: 4.37, 95% CI 2.68 to 7.13, P < 0.001; EUR OR: 5.86, 95% CI 4.58 to 7.49, P < 0.001; meta OR: 5.48, 95% CI 4.31 to 6.97, P < 0.001). Among those individuals with echocardiography data available, hiPSI TTNtvs were associated with significantly decreased minimum LVEF independent of genetic reference population similarity (EUR β: -10.08, 95% CI -12.71 to -7.44, P < 0.001; AFR β: -12.83, 95% CI -18.39 to -7.27, P < 0.001; Meta β: -10.58, 95% CI -12.96 to -8.20, P < 0.001). Conclusions: Our analysis demonstrates a strong association between hiPSI TTNtv and both DCM and minimum LVEF in diverse populations suggesting that genetic testing and counseling recommendations should not differ between these populations.

The role of endosarcomeric cytoskeleton proteins in the mechanisms of left ventricular diastolic dysfunction: focus on titin

Recognizing the fact that isolated left ventricular (LV) diastolic dysfunction (DD) underlies approximately 50% of all heart failure cases requires a deep understanding of its principal mechanisms so that effective diagnostic and treatment strategies can be developed. Despite abundance of knowledge about the mechanisms underlying DD, many important questions regarding the pathophysiology of diastole remain unresolved. In particular, the role of endosarcomeric cytoskeleton pathology in the deterioration of the so-called active (relaxation of the LV myocardium and the atrioventricular pressure gradient at the beginning of diastole, closely related to it in a healthy heart) and passive (myocardial stiffness) characteristics of diastole needs to be clarified.The lecture briefly discusses the complex hierarchy of DD mechanisms (from the sarcomere to the whole heart) and covers the role of the giant protein titin in the latter, which is the main determinant of intracellular stiffness. Impairment of myocardial relaxation and deterioration of its wall compliance under a wide range of pathological conditions (pressure overload, ischemia, inflammation, cardiotoxic effects, oxidative stress, etc.) underlying DD can be explained by a shift in titin expression toward its more rigid N2B isoform, hypophosphorylation by protein kinases A and G or dephosphorylation by serine / threonine phosphatase 5 of its molecule in the extensible protein segment containing a unique N2B sequence, hyperphosphorylation of PEVK regions of titin by protein kinase C, as well as inhibition of the Ca2+-dependent titin – actin interaction.The results of deciphering these mechanisms can become a tool for developing new approaches to targeted therapy for diastolic heart failure that currently does not have effective treatment, on the one hand, and the key to understanding the therapeutic effects of drugs already used to treat chronic heart failure with preserved LV ejection fraction, on the other hand.

Proteostasis of heat shock protein 90 in skeletal muscles of the long-tailed ground squirrel during hibernation

We investigated changes in the content of heat shock protein 90 in m. soleus (comprised of mainly fibers expressing the MyHC slow isoform I) and m. gastrocnemius (composed of mainly fibers expressing the MyHC fast isoforms II) of the long-tailed ground squirrel Urocitellus undulatus in different periods of the annual cycle: summer activity (seasonal control), hypothermia/torpor, winter (interbout) activity. The content of the protein in both muscles was found not to change throughout the entire hibernation period despite the development of atrophic changes, more pronounced in fast m. gastrocnemius. The role of HSP90 in maintaining the stability of giant sarcomeric titin protein molecules is discussed with reference to animal's entry into and exit from hypothermia, when the activity of calpain proteases increases due to the increased content of Ca2+ in the cytosol of muscle cells; and with respect to the torpor, when the activity of calpains is, most likely, not inhibited completely. During the interbout activity with an observed increased titin turnover in squirrel's striated muscles, a constant content of HSP90 appears to be required for the correct folding of newly synthesized titin molecules and their integration into sarcomeres, as well as for the removal of misfolded titin molecules and other proteins. Thus, HSP90 proteostasis in skeletal muscles of the long-tailed ground squirrel can contribute to maintaining a steady-state level of titin and, possibly, other sarcomeric proteins during hibernation, which, in turn, will contribute to maintaining a highly ordered sarcomeric structure and the necessary level of muscle contractile activity in different phases of the torpor-arousal cycle.