Circulating tumor cell-derived exosome-transmitted long non-coding RNA TTN-AS1 can promote the proliferation and migration of cholangiocarcinoma cells.

BACKGROUND: Exosomes assume a pivotal role as essential mediators of intercellular communication within tumor microenvironments. Within this context, long noncoding RNAs (LncRNAs) have been observed to be preferentially sorted into exosomes, thus exerting regulatory control over the initiation and progression of cancer through diverse mechanisms. RESULTS: Exosomes were successfully isolated from cholangiocarcinoma (CCA) CTCs organoid and healthy human serum. Notably, the LncRNA titin-antisense RNA1 (TTN-AS1) exhibited a conspicuous up-regulation within CCA CTCs organoid derived exosomes. Furthermore, a significant elevation of TTN-AS1 expression was observed in tumor tissues, as well as in blood and serum exosomes from patients afflicted with CCA. Importantly, this hightened TTN-AS1 expression in serum exosomes of CCA patients manifested a strong correlation with both lymph node metastasis and TNM staging. Remarkably, both CCA CTCs organoid-derived exosomes and CCA cells-derived exosomes featuring pronounced TTN-AS1 expression demonstrated the capability to the proliferation and migratory potential of CCA cells. Validation of these outcomes was conducted in vivo experiments. CONCLUSIONS: In conclusion, our study elucidating that CCA CTCs-derived exosomes possess the capacity to bolster the metastasis tendencies of CCA cells by transporting TTN-AS1. These observations underscore the potential of TTN-AS1 within CTCs-derived exosomes to serve as a promising biomarker for the diagnosis and therapeutic management of CCA.

Skeletal Muscle Involvement in Patients With Truncations of Titin and Familial Dilated Cardiomyopathy.

BACKGROUND: Genetic variants in titin (TTN) are associated with dilated cardiomyopathy (DCM) and skeletal myopathy. However, the skeletal muscle phenotype in individuals carrying heterozygous truncating TTN variants (TTNtv), the leading cause of DCM, is understudied. OBJECTIVES: This study aimed to assess the skeletal muscle phenotype associated with TTNtv. METHODS: Participants with TTNtv were included in a cross-sectional study. Skeletal muscle fat fraction was evaluated by magnetic resonance imaging (compared with healthy controls and controls with non-TTNtv DCM). Muscle strength was evaluated by dynamometry and muscle biopsy specimens were analyzed. RESULTS: Twenty-five TTNtv participants (11 women, mean age 51 ± 15 years, left ventricular ejection fraction 45% ± 10%) were included (19 had DCM). Compared to healthy controls (n = 25), fat fraction was higher in calf (12.5% vs 9.9%, P = 0.013), thigh (12.2% vs 9.3%, P = 0.004), and paraspinal muscles (18.8% vs 13.9%, P = 0.008) of TTNtv participants. Linear mixed effects modelling found higher fat fractions in TTNtv participants compared to healthy controls (2.5%; 95% CI: 1.4-3.7; P < 0.001) and controls with non-TTNtv genetic DCM (n = 7) (1.5%; 95% CI: 0.2-2.8; P = 0.025). Muscle strength was within 1 SD of normal values. Biopsy specimens from 21 participants found myopathic features in 13 (62%), including central nuclei. Electron microscopy showed well-ordered Z-lines and T-tubuli but uneven and discontinuous M-lines and excessive glycogen depositions flanked by autophagosomes, lysosomes, and abnormal mitochondria with mitophagy. CONCLUSIONS: Mild skeletal muscle involvement was prevalent in patients with TTNtv. The phenotype was characterized by an increased muscle fat fraction and excessive accumulation of glycogen, possibly due to reduced autophagic flux. These findings indicate an impact of TTNtv beyond the heart.

Titin related myopathy with ophthalmoplegia. A novel phenotype.

Titin-related myopathy is an emerging genetic neuromuscular disorder with a wide spectrum of clinical phenotypes. To date, there have not been reports of patients with this disease that presented with extraocular muscle involvement. Here we discuss a 19-year-old male with congenital weakness, complete ophthalmoplegia, thoracolumbar scoliosis, and obstructive sleep apnea. Muscle magnetic resonance imaging revealed severe involvement of the gluteal and anterior compartment muscles, and clear adductor sparing, while muscle biopsy of the right vastus lateralis showed distinctive cap-like structures. Trio Whole Exome Sequencing (WES) showed compound heterozygous likely pathologic variants in the TTN gene. (c.82541_82544dup (p.Arg27515Serfs*2) in exon 327 (NM_001267550.2) and c.31846+1G>A (p.?) in exon 123 (NM_001267550.2). To our knowledge, this is the first report of a TTN-related disorder associated with ophthalmoplegia.

Titin as a potential novel therapeutic target in colorectal cancer.

Colorectal cancer (CRC) is identified as a primary cause of death around the world. The current chemotherapies are not cost-effective. Therefore, finding novel potential therapeutic target is urgent. Titin (TTN) is a muscle protein that is critical in hypertrophic cardiomyopathy. However, its role in CRC is not well understood. The study focused on exploring the possible role of TTN in CRC carcinogenesis. TTN mRNA and protein expression levels presented an obvious downregulation in CRC tissue samples, relative to normal control (p < 0.05). TTN expression significantly correlated with the clinical stage (normal vs. Stage 1, p < 0.05; normal vs. Stage 4, p < 0.05), node metastasis (normal vs. N1, p < 0.05; N1 vs. N2, p < 0.05), histological type (normal vs. adenocarcinoma, p < 0.05), race (Caucasian vs. Asian, p < 0.05; African-American vs. Asian, p < 0.05) and TP53 mutation (normal vs. TP53 mutation, p < 0.05), considering The Cancer Genome Atlas database. However, for patients who had higher TTN expression, the overall survival was remarkably shorter than patients who had low TTN expression. Furthermore, TTN was lowly expressed in four CRC cell lines. TTN overexpression facilitated CRC cells in terms of the proliferation, metastasis and invasion. Based on gene set enrichment analysis, the ERB pathway might be responsible for TTN-related CRC. Besides, TTN was involved in the response to azacitidine. Overall, TTN might serve as a potential novel therapeutic target for treating and overcoming chemotherapy resistance in CRC.

A novel mutation in the TTN gene resulted in left ventricular noncompaction: a case report and literature review.

BACKGROUND: Left ventricular noncompaction (LVNC) is a specific type of cardiomyopathy characterized by coarse trabeculae and interspersed trabecular crypts within the ventricles. Clinical presentation varies widely and may be nonsignificant or may present with progressive heart failure, malignant arrhythmias, and multiorgan embolism. The mode of inheritance is highly heterogeneous but is most commonly autosomal dominant. The TTN gene encodes titin, which is not only an elastic component of muscle contraction but also mediates multiple signalling pathways in striated muscle cells. In recent years, mutations in the TTN gene have been found to be associated with LVNC, but the exact pathogenesis is still not fully clarified. CASE PRESENTATION: In this article, we report a case of an adult LVNC patient with a TTN gene variant, c.87857G > A (p. Trp29286*), that has not been reported previously. This 43-year-old adult male was hospitalized repeatedly for heart failure. Echocardiography showed reduced myocardial contractility, dilated left ventricle with many prominent trabeculae, and a loose texture of the left ventricular layer of myocardium with crypt-like changes. During the out-of-hospital follow-up, the patient had no significant signs or symptoms of discomfort. CONCLUSION: This case report enriches the mutational spectrum of the TTN gene in LVNC and provides a basis for genetic counselling and treatment of this patient. Clinicians should improve their understanding of LVNC, focusing on exploring its pathogenesis and genetic characteristics to provide new directions for future diagnosis and treatment.

Social, Genetics and Histopathological Factors Related to Titin (TTN) Gene Mutation and Survival in Women with Ovarian Serous Cystadenocarcinoma: Bioinformatics Analysis.

Several factors may increase the risk of development of ovarian cancer. In this study, we investigated the relationship between social, genetic, and histopathologic factors in women with ovarian serous cystadenocarcinoma and titin (TTN) mutations, whether the TTN gene mutation may be a predictor, and its impact on mortality and survival in these patients. A total of 585 samples from patients with ovarian serous cystadenocarcinoma were collected from The Cancer Genome Atlas and PanCancer Atlas through the cBioPortal for analysis of social, genetic, and histopathological factors. Logistic regression was used to investigate whether TTN mutation could be a predictor, and the Kaplan-Meier method was applied to analyze survival time. TTN mutation frequency did not differ between age at diagnosis, tumor stage, and race, and was related to increased Buffa hypoxia score (p = 0.004), mutation count (p < 0.0001), Winter hypoxia Score (p = 0.030), nonsynonymous tumor mutation burden (TMB) (p < 0.0001), and reduced microsatellite instability sensor score (p = 0.010). The number of mutations (p < 0.0001) and winter hypoxia score (p = 0.008) were positively associated with TTN mutations, and nonsynonymous TMB (p < 0.0001) proved to be a predictor. Mutated TTN affects the score of genetic variables involved in cancer cell metabolism in ovarian cystadenocarcinoma.

Nanobodies combined with DNA-PAINT super-resolution reveal a staggered titin nanoarchitecture in flight muscles.

Sarcomeres are the force-producing units of all striated muscles. Their nanoarchitecture critically depends on the large titin protein, which in vertebrates spans from the sarcomeric Z-disc to the M-band and hence links actin and myosin filaments stably together. This ensures sarcomeric integrity and determines the length of vertebrate sarcomeres. However, the instructive role of titins for sarcomeric architecture outside of vertebrates is not as well understood. Here, we used a series of nanobodies, the Drosophila titin nanobody toolbox, recognising specific domains of the two Drosophila titin homologs Sallimus and Projectin to determine their precise location in intact flight muscles. By combining nanobodies with DNA-PAINT super-resolution microscopy, we found that, similar to vertebrate titin, Sallimus bridges across the flight muscle I-band, whereas Projectin is located at the beginning of the A-band. Interestingly, the ends of both proteins overlap at the I-band/A-band border, revealing a staggered organisation of the two Drosophila titin homologs. This architecture may help to stably anchor Sallimus at the myosin filament and hence ensure efficient force transduction during flight.

From ants to humans, the muscles that set an organism in motion are formed of bundles of fiber-like cells which can shorten and lengthen at will. At the microscopic level, changes in muscle cell lengths are underpinned by contractile filaments formed of multiple repeats of a basic unit, known as the sarcomere. Each unit is bookended by intricate 'Z-discs' and features an 'M-band' in its center. Three protein types give a sarcomere its ability to shorten and expand at will: two types of filaments (myosin and actin), which can slide on one another; and a spring-like molecule known as titin, which ensures that the unit does not fall apart by mechanically connecting myosin and actin. More specifically, actin filaments are anchored to the Z-discs and extend towards the M-band, while myosin filaments are centered around the M-band and extend towards the Z-discs. As myosin and actin slide alongside each other, the overlap between the two types of filaments increases or decreases and the whole unit changes its length. In vertebrates, one gigantic molecule of titin spans from the Z-disc to the M-band, linking together actin and myosin filaments and determining the length of the sarcomere. In insects and other invertebrates, however, this single molecule is replaced by two titin proteins known as Projectin and Sallimus. Understanding how these titins work together remains unclear and difficult to study. Traditional approaches are unable to precisely label titin in an environment teaming with other molecules, and they cannot offer the nanometer resolution required to dissect sarcomere organization. As a response, Schueder, Mangeol et al. combined super-resolution microscopy and a new toolbox of labelling molecules known as nanobodies to track the position of Sallimus and Projectin in the flight muscles of fruit flies. These experiments revealed that the two proteins are arranged in tandem along the length of the sarcomere, forming a structure that measures about 350 nm. Sallimus is anchored in the Z-disc and it runs alongside actin until it reaches the end of a myosin filament; there, it overlaps with Projectin for about 10 nm. Projectin then stretches for 250 nm along the length of the beginning myosin filament. These findings confirm the importance of titin in dictating the length of a sarcomere; they suggest that, in invertebrates, this role is split between two proteins, each possibly ruling over a section of the sarcomere. In addition, the work by Schueder, Mangeol et al. demonstrate the value of combining nanobodies and super-resolution microscopy to study complex structures in tissues.

TTN-AS1 delivered by gastric cancer cell-derived exosome induces gastric cancer progression through in vivo and in vitro studies.

Extracellular communication within the tumor microenvironment exerts critical functions in tumor progression. Moreover, exosomes are capable of packaging into long non-coding RNAs (lncRNAs) to regulate extracellular communication. We tried to discuss the role of exosomal lncRNA TTN-AS1 and its molecular mechanism on gastric cancer (GC) progression. Bioinformatics analysis depicted increased TTN-AS1 in GC which shared correlation with poor prognosis. Clinical tissue and cellular experiments also confirmed the elevation of TTN-AS1 in GC tissues and cells. GC cell (AGS)-derived Exo could be uptake by NCI-N87 cells to induce malignant features of GC cells. Functionally, TTN-AS1 could upregulate ZEB1 expression by binding to miR-499a-5p. In addition, in vitro experiments demonstrated that ZEB1 targeted and activated CDX2 transcription and promoted CDX2 expression; silencing CDX2 inhibited malignant phenotypes of AGS and NCI-N87 cells. Furthermore, Exo-TTN-AS1 promoted GC cell growth and migration by promoting CDX2 expression. Exosomal TTN-AS1 from GC cells could also promote metastasis of GC in vivo. In conclusion, our findings provided evidence describing that exosomes derived from GC cells transferred TTN-AS1 to GC cells, which aggravate GC through the miR-499a-5p/ZEB1/CDX2 axis. 1. Exo derived from GC cells promotes the growth and metastasis of GC cells by carrying TTN-AS1. 2. TTN-AS1 acts as a ceRNA to adsorb miR-499a-5p to regulate the expression of ZEB1. 3. ZEB1 targets and activates CDX2 transcription. 4. GC cell-derived Exo-TTN-AS1 enhances the growth and metastasis of GC cell xenografts in vivo.

Signaling network model of cardiomyocyte morphological changes in familial cardiomyopathy.

Familial cardiomyopathy is a precursor of heart failure and sudden cardiac death. Over the past several decades, researchers have discovered numerous gene mutations primarily in sarcomeric and cytoskeletal proteins causing two different disease phenotypes: hypertrophic (HCM) and dilated (DCM) cardiomyopathies. However, molecular mechanisms linking genotype to phenotype remain unclear. Here, we employ a systems approach by integrating experimental findings from preclinical studies (e.g., murine data) into a cohesive signaling network to scrutinize genotype to phenotype mechanisms. We developed an HCM/DCM signaling network model utilizing a logic-based differential equations approach and evaluated model performance in predicting experimental data from four contexts (HCM, DCM, pressure overload, and volume overload). The model has an overall prediction accuracy of 83.8%, with higher accuracy in the HCM context (90%) than DCM (75%). Global sensitivity analysis identifies key signaling reactions, with calcium-mediated myofilament force development and calcium-calmodulin kinase signaling ranking the highest. A structural revision analysis indicates potential missing interactions that primarily control calcium regulatory proteins, increasing model prediction accuracy. Combination pharmacotherapy analysis suggests that downregulation of signaling components such as calcium, titin and its associated proteins, growth factor receptors, ERK1/2, and PI3K-AKT could inhibit myocyte growth in HCM. In experiments with patient-specific iPSC-derived cardiomyocytes (MLP-W4R;MYH7-R723C iPSC-CMs), combined inhibition of ERK1/2 and PI3K-AKT rescued the HCM phenotype, as predicted by the model. In DCM, PI3K-AKT-NFAT downregulation combined with upregulation of Ras/ERK1/2 or titin or Gq protein could ameliorate cardiomyocyte morphology. The model results suggest that HCM mutations that increase active force through elevated calcium sensitivity could increase ERK activity and decrease eccentricity through parallel growth factors, Gq-mediated, and titin pathways. Moreover, the model simulated the influence of existing medications on cardiac growth in HCM and DCM contexts. This HCM/DCM signaling model demonstrates utility in investigating genotype to phenotype mechanisms in familial cardiomyopathy.

Kennedy's disease presented with mastication fatigue combined with positive titin antibody: a case report.

BACKGROUND: Spinal and bulbar muscular atrophy (SBMA) is an X-linked recessive hereditary neuromuscular disorder caused by the expanded trinucleotide repeat in the androgen receptors gene. The major clinical manifestations of SBMA consist of weakness in the bulbar and limb muscles, fasciculations, tremors, cramps, sensory impairment, and gynecomastia. However, atypical SBMA cases may lead to misdiagnosis. Muscular fatigue and decremental responses to repetitive nerve stimulation (RNS), despite being observed in some SBMA patients, are usually occurred in MG patients, and patient with the symptom of mastication fatigue was rarely reported. In addition, cardiological investigations have been performed in SBMA patients and several ECG alterations were identified. Here we report an SBMA patient presenting with a rare onset symptom of mastication fatigue, who has been detected with a positive titin antibody in the serum and showed a WPW pattern electrocardiogram. CASE PRESENTATION: The patient showed mildly progressive fatigue in the muscles of mastication over 3 years. Neurological examination showed facial muscle weakness and a wasting tongue with fasciculations, but the weakness, wasting, or fasciculations were not obvious in the limbs. 3-Hz RNS showed a decremental response in bilateral orbicularis oculi. The test of titin antibody was positive in the serum, and the electrocardiogram showed a WPW pattern ECG. Genetic analysis revealed an increased number (39 repeats) of tandem CAG repeats in the AR gene, which confirmed the diagnosis of SBMA. The fatigue symptom was significantly improved after oral pyridostigmine bromide treatment. CONCLUSION: This case calls for more attention to muscular fatigue as the onset symptoms of Kennedy's disease. ECG screening is of importance in SBMA patients and further studies are needed to investigate the titin antibody in SBMA patients as well as other neurogenic disorders.

Disclosing an In-Frame Deletion of the Titin Gene as the Possible Predisposing Factor of Anthracycline-Induced Cardiomyopathy: A Case Report.

Anthracycline-induced cardiomyopathy has been noted as a non-neglectable issue in the field of clinical oncology. Remarkable progress has been achieved in searching for inherited susceptible genetic deficits underlying anthracycline cardiotoxicity in the past several years. In this case report, we present the preliminary results of a genetic study in a young male patient who was treated with standard dose anthracycline-based chemotherapy for his acute myeloid leukemia and attacked by acute congestive heart failure after just two courses of therapy. After a survey of 76 target genes, an in-frame deletion of the titin gene was recognized as the most possible genetic defect responsible for his cardiomyopathy caused by anthracycline. This defect proved to pass down from the patient's mother and did not exist in seven unrelated chemotherapy-treated cancer patients without chemotherapy-induced cardiomyopathy and four other healthy volunteer DNA donors.

Generation of three TTN knock-out human induced pluripotent stem cell lines using CRISPR/Cas9 system.

TTN mutations are the common genetic cause for various types of cardiomyopathies (e.g., dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy) and skeletal myopathies. Here, we generated three TTN knock-out human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 system. These cell lines, which exhibit normal karyotype, typical morphology and pluripotency, could provide useful platform for investigating the role of TTN in associated disorders.

TTN mutations predict a poor prognosis in patients with thyroid cancer.

OBJECTIVE: We aimed to investigate the relationship between titin (TTN) gene mutations and thyroid cancer (THCA) and to explore the feasibility of the TTN gene as a potential prognostic indicator of THCA. METHODS: From TCGA-THCA cohort, we performed a series of analyses to evaluate the prognostic value and potential mechanism of TTN in THCA. These patients were divided into the mutant-type (MUT) group and the wild-type (WT) group. Differentially expressed genes (DEGs) in the two groups were screened using the 'DESeq2' R package. Functional enrichment analysis was performed, and the protein-protein interaction (PPI) network, transcription factor (TF)-target interaction networks, and competitive endogenous RNA (ceRNA) regulatory networks were established for the DEGs. The TIMER database was applied for immune cell infiltration. Survival analysis and Cox regression analysis were used to analyze the potential prognostic value of the TTN gene. RESULTS: Differential expression analysis showed that 409 genes were significantly up-regulated and 36 genes were down-regulated. Functional enrichment analysis revealed that TTN gene mutations played a potential role in the development of THCA. Analysis of the immune microenvironment indicated that TTN gene mutations were significantly associated with enrichment of M0 macrophages. Survival analysis showed that the MUT group predicted poorer prognosis than the WT group. Cox regression analysis demonstrated that TTN gene mutations were an independent risk factor for THCA. Nomograms also confirmed the prognostic values of the TTN gene in THCA. Conclusions In summary, our results demonstrated that TTN gene mutations predict poor prognosis in patients with THCA. This is the first study to research TTN gene mutations in THCA and to investigate their prognostic value in THCA.

SQLE facilitates the pancreatic cancer progression via the lncRNA-TTN-AS1/miR-133b/SQLE axis.

Studies have shown that SQLE is highly expressed in a variety of tumours and promotes tumour progression. However, the role of SQLE in pancreatic cancer (PC) has not been reported. Here, we aim to study the role and molecular mechanism of SQLE in PC. Immunohistochemistry and functional experiments showed that SQLE was highly expressed in PC tissues and promoted the proliferation and invasion of PC cells. Terbinafine, an inhibitor of SQLE, inhibited this effect. In order to further study the upstream mechanism that regulates SQLE, we used bioinformatics technology to lock miR-133b and lncRNA-TTN-AS. In situ hybridization was used to detect the expression of miR-133b and lncRNA-TTN-AS1 in PC tissues. The luciferase reporter gene experiment was used to confirm the binding of miR-133b and lncRNA-TTN-AS1. The results showed that miR-133b was down-regulated in PC tissues and negatively correlated with the expression of SQLE. LncRNA-TTN-AS1 was upregulated in pancreatic cancer tissues and positively correlated with the expression of SQLE. Luciferase gene reporter gene analysis confirmed lncRNA-TTN-AS1 directly binded to miR-133b. Therefore, we propose that targeting the lncRNA-TTN-AS1/miR-133b/SQLE axis is expected to provide new ideas for the clinical treatment of PC patients.

Novel compound heterozygous mutations in the TTN gene: elongation and truncation variants causing limb-girdle muscular dystrophy type 2J in a Han Chinese family.

INTRODUCTION: Limb-girdle muscular dystrophy (LGMD) is a group of clinically heterogeneous muscle disorders commonly manifesting proximal limb girdle muscle weakness. There have been more than 30 subtypes of LGMD associated with causative genes and limb-girdle muscular dystrophy type 2J (LGMD2J) is caused by mutations in the TTN gene. METHODS: We report a Han Chinese family with LGMD2J. The proband and his sister both presented with weakness in the proximal lower limbs bilaterally. Muscle biopsy and genetic analysis were performed. RESULTS: Muscle biopsy of the proband showed dystrophic changes accompanied by rimmed vacuoles. Whole-exome sequencing identified novel compound heterozygous mutations in the TTN gene, including elongation (c.107962_107963delAT, p.I35988Sfs*26) and truncation (c.99125_99128dupACAG, p.S33043Rfs*9) variants in the proband and his sister. Both two variants have never been reported. Notably, we are the first to identify an elongation mutation in the TTN gene, broadening the genetic mutation spectrum of LGMD2J. DISCUSSION: Several variants in the last exon of the TTN gene have been reported, one of which was associated with LGMD2J. Besides, LGMD2J should be distinguished from other myopathies caused by mutations in the TTN gene. The pathogenesis of and specific curative methods for LGMD2J remain to be further elucidated.

Identification of a Five-Gene Panel to Assess Prognosis for Gastric Cancer.

METHODS: Two datasets were used as training and validation cohorts to establish the predictive model. We used three types of screening criteria: background analysis, pathway analysis, and functional analysis provided by the cBioportal website. Fisher's exact test and multivariable logistic regression were performed to screen out related genes. Furthermore, we performed receiver operating characteristic (ROC) and Kaplan-Meier curve analyses to evaluate the correlation between the selected genes and overall survival. RESULT: We screened five genes (KNL1, NRXN1, C6, CCDC169-SOHLH2, and TTN) that were highly related to recurrence of GC. The area under the receiver operating characteristic (ROC) curve was 0.813, which was much higher than that of the baseline model (AUC = 0.699). This result suggested that the mutation of five selected genes had a significant effect on the prediction of recurrence compared with other factors (age, stages, history, etc.). Furthermore, the Kaplan-Meier estimator also revealed that the mutation of five genes positively correlated with patient survival. CONCLUSIONS: The patients who have mutations in these five genes may experience longer survival than those who do not have mutations. This five-gene panel will likely be a practical tool for prognostic evaluation and will provide another possible way for clinicians to determine therapy.

GSK-3ß Localizes to the Cardiac Z-Disc to Maintain Length Dependent Activation.

BACKGROUND: Altered kinase localization is gaining appreciation as a mechanism of cardiovascular disease. Previous work suggests GSK-3ß (glycogen synthase kinase 3ß) localizes to and regulates contractile function of the myofilament. We aimed to discover GSK-3ß's in vivo role in regulating myofilament function, the mechanisms involved, and the translational relevance. METHODS: Inducible cardiomyocyte-specific GSK-3ß knockout mice and left ventricular myocardium from nonfailing and failing human hearts were studied. RESULTS: Skinned cardiomyocytes from knockout mice failed to exhibit calcium sensitization with stretch indicating a loss of length-dependent activation (LDA), the mechanism underlying the Frank-Starling Law. Titin acts as a length sensor for LDA, and knockout mice had decreased titin stiffness compared with control mice, explaining the lack of LDA. Knockout mice exhibited no changes in titin isoforms, titin phosphorylation, or other thin filament phosphorylation sites known to affect passive tension or LDA. Mass spectrometry identified several z-disc proteins as myofilament phospho-substrates of GSK-3ß. Agreeing with the localization of its targets, GSK-3ß that is phosphorylated at Y216 binds to the z-disc. We showed pY216 was necessary and sufficient for z-disc binding using adenoviruses for wild-type, Y216F, and Y216E GSK-3ß in neonatal rat ventricular cardiomyocytes. One of GSK-3ß's z-disc targets, abLIM-1 (actin-binding LIM protein 1), binds to the z-disc domains of titin that are important for maintaining passive tension. Genetic knockdown of abLIM-1 via siRNA in human engineered heart tissues resulted in enhancement of LDA, indicating abLIM-1 may act as a negative regulator that is modulated by GSK-3ß. Last, GSK-3ß myofilament localization was reduced in left ventricular myocardium from failing human hearts, which correlated with depressed LDA. CONCLUSIONS: We identified a novel mechanism by which GSK-3ß localizes to the myofilament to modulate LDA. Importantly, z-disc GSK-3ß levels were reduced in patients with heart failure, indicating z-disc localized GSK-3ß is a possible therapeutic target to restore the Frank-Starling mechanism in patients with heart failure.

Comprehensive analysis of m6A regulator-based methylation modification patterns characterized by distinct immune profiles in colon adenocarcinomas.

Mounting evidences have indicated that RNA N6-methyladenosine (m6A) modification played important roles in tumor formation and growth. However, it is rarely reported that m6A modifications are involved in the immune regulation and tumor microenvironment (TME) formation. In this study, we aimed to investigate the correlation between m6A modifications and TME regulation of colon adenocarcinoma (COAD) by bioinformatic analysis. NMF algorithm was applied to carry out consensus molecular subtype analysis on 36 selected m6A regulators regarding methylation modification, to identify m6A modification patterns and characteristics of m6A related genes in colon adenocarcinoma (COAD). Further, the relative infiltration levels of different immune cell subsets were quantified by ssGSEA and CIBERSORT algorithms, and a m6Sig scoring scheme was constructed to predict the prognosis and evaluate the response to immunotherapy in the patients with COAD. Among 579 COAD samples, we identified three different m6A modification patterns which were related to different biological pathways and clinical outcomes. Then, a scoring scheme termed "m6Sig score" was developed based on m6A-related characteristic genes, and was utilized to score patients with COAD into groups. We found that COAD patients with lower m6Sig scores exhibited prolonged survival and potentiated immune infiltration, which were associated with higher tumor mutation load, lower PD-L1 expression, and higher mutation rates of SMG (such as TTN and KRAS). Moreover, analysis regarding evaluation of immune response revealed that the patients with lower m6Sig scores had higher Immunophenoscore. Collectively, our study provided in depth insight into the interactions between m6A modification and regulation of TME. In addition, the quantitative evaluation of m6A modification patterns in our results may have implications in further immunotherapy for individual COAD patients.

Titin-truncating mutations associated with dilated cardiomyopathy alter length-dependent activation and its modulation via phosphorylation.

AIMS: Dilated cardiomyopathy (DCM) is associated with mutations in many genes encoding sarcomere proteins. Truncating mutations in the titin gene TTN are the most frequent. Proteomic and functional characterizations are required to elucidate the origin of the disease and the pathogenic mechanisms of TTN-truncating variants. METHODS AND RESULTS: We isolated myofibrils from DCM hearts carrying truncating TTN mutations and measured the Ca2+ sensitivity of force and its length dependence. Simultaneous measurement of force and adenosine triphosphate (ATP) consumption in skinned cardiomyocytes was also performed. Phosphorylation levels of troponin I (TnI) and myosin binding protein-C (MyBP-C) were manipulated using protein kinase A and λ phosphatase. mRNA sequencing was employed to overview gene expression profiles. We found that Ca2+ sensitivity of myofibrils carrying TTN mutations was significantly higher than in myofibrils from donor hearts. The length dependence of the Ca2+ sensitivity was absent in DCM myofibrils with TTN-truncating variants. No significant difference was found in the expression level of TTN mRNA between the DCM and donor groups. TTN exon usage and splicing were also similar. However, we identified down-regulation of genes encoding Z-disk proteins, while the atrial-specific regulatory myosin light chain gene, MYL7, was up-regulated in DCM patients with TTN-truncating variants. CONCLUSION: Titin-truncating mutations lead to decreased length-dependent activation and increased elasticity of myofibrils. Phosphorylation levels of TnI and MyBP-C seen in the left ventricles are essential for the length-dependent changes in Ca2+ sensitivity in healthy donors, but they are reduced in DCM patients with TTN-truncating variants. A decrease in expression of Z-disk proteins may explain the observed decrease in myofibril passive stiffness and length-dependent activation.

Functional impact of titin (TTN) mutations in ocular surface squamous neoplasia.

Mutations in the titin (TTN) gene are among the most common genomic aberrations in ocular surface squamous neoplasia (OSSN), the most common cancer of the external eye. Further, TTN mutations are associated with resistance to standard therapy with topical interferon alpha-2b (IFN-α2b). However, it remains unclear how TTN mutations drive OSSN pathogenesis and treatment resistance. TTN encodes the largest protein in the human body and its best understood function is as a myofibril scaffold in striated muscle. However, recent evidence indicates that TTN has additional functions in non-muscle cells and in cancer. Here, we performed a disorder-based bioinformatics analysis which revealed that intrinsically disordered protein regions are abundant in TTN and provide mechanistic insights into its function as a nuclear protein in epithelial cells. Specific mutations found in OSSN are predicted to affect its intrinsically disordered protein regions (IDPRs), promoting chromosomal instability, oncogenesis, and altered response to IFN-α2b treatment.