Titin Isoform Research Articles

Revealing the global mechanism related to carnosine synthesis in the pectoralis major of slow-growing Korat chickens using a proteomic approach.

This study aimed to find global mechanisms related to carnosine synthesis in slow-growing Korat chickens (KRC) using a proteomic approach. M. pectoralis major samples were collected from 10-week-old female KRC including low-carnosine (LC, 2,756.6±82.88 μg/g; n = 5) and high-carnosine (HC, 4,212.5 ±82.88 μg/g; n = 5). We identified 152 common proteins, and 8 of these proteins showed differential expression between the LC and HC groups (p<0.05). Heat shock 70 kDa protein 8, Heat shock 70 kDa protein 2, protein disulfide isomerase family A, member 6, and endoplasmic reticulum resident protein 29 were significantly involved in protein processing in the endoplasmic reticulum pathway (false discovery rate<0.05), suggesting that the pathway is related to differential carnosine concentration in the M. pectoralis major of KRC. A high concentration of carnosine in the meat is mainly involved in low abundances of Titin isoform Ch12 and Connectin and high abundances of M-protein to maintain homeostasis during muscle contraction. These consequences improve meat characteristics, which were confirmed by the principal component analysis. Carnosine synthesis may occur when muscle cells need to recover homeostasis after being interfered with carnosine synthesis precursors, leading to improved muscle function. To the best of our knowledge, this is the first study to describe in detail the global molecular mechanisms in divergent carnosine contents in meat based on the proteomic approach.

Kbtbd13R408C-knockin mouse model reveals impaired relaxation kinetics as novel pathomechanism for NEM6 cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): VENI-Nederlandse organisatie voor Wetenschappelijk Onderzoek (NWO) We identified a novel cardiomyopathy gene: KBTBD13 encoding kelch repeat and BTB (POZ) domain containing 13. The Dutch founder variant KBTBD13R408C not only affects skeletal muscle function resulting in nemaline myopathy type 6 (NEM6), but also affects the heart. In the Dutch NEM6 cohort, various cardiac abnormalities were observed, including systolic dysfunction, diastolic dysfunction, atrial fibrillation, ventricular tachycardia and three patients died of sudden cardiac death. The majority of NEM6 patients harbors the Dutch founder variant, c.1222C&amp;gt;T, p.Arg408Cys (KBTBD13R408C). To provide insight in the mechanism underlying cardiac dysfunction in NEM6, here we assessed cardiac structure and function in Kbtbd13R408C-knockin mice, which closely recapitulate the well characterized skeletal muscle phenotype, i.e. impaired relaxation kinetics and mitochondrial dysfunction. Pressure-volume loop analyses showed that the end-diastolic pressure-volume relation was steeper in Kbtbd13R408C-knockin mice, indicating diastolic dysfunction. Histological evaluation of cardiac structure revealed no increase in fibrosis in Kbtbd13R408C-knockin mice. Also, no alterations in titin isoform composition or myosin heavy chain composition were observed that could account for the increased diastolic stiffness. Next, we studied the contraction and relaxation kinetics in wild type (WT) and Kbtbd13R408C-knockin mice at the intact single cardiomyocyte level by a high-throughput contractility set-up. Our data showed a prolonged relaxation time in Kbtbd13R408C-knockin mice compared to WT mice, indicating impaired relaxation kinetics. No differences in the magnitude of contraction (percentage of shortening) was found between cardiomyocytes of Kbtbd13R408C-knockin and WT mice. In parallel, calcium-handling was assessed by calcium indicator Fura-2AM. These studies reveal that calcium-release is not affected, but calcium-reuptake is slower in Kbtbd13R408C-knockin mice, which might contribute to the impaired relaxation kinetics. Current studies focus on how KBTBD13R408C affects calcium-reuptake kinetics and sarcomere kinetics in NEM6 cardiomyocytes. Hence, our studies provide the first insights in the pathomechanism underlying cardiac dysfunction in NEM6.

Relative expression of the N2B and N2BA isoforms of titin does not change markedly in heart failure with reduced ejection fraction in humans

Relative expression of the N2B and N2BA isoforms of titin does not change markedly in heart failure with reduced ejection fraction in humans

Human myocardium with ATTR amyloidosis exhibits increased fibrosis, amyloid localized microcalcifications, and downregulated Z-disc proteins with no change in titin isoform ratios

Human myocardium with ATTR amyloidosis exhibits increased fibrosis, amyloid localized microcalcifications, and downregulated Z-disc proteins with no change in titin isoform ratios

High-Intensity Exercise Promotes Deleterious Cardiovascular Remodeling in a High-Cardiovascular-Risk Model: A Role for Oxidative Stress.

Although the benefits of moderate exercise in patients at high cardiovascular risk are well established, the effects of strenuous exercise remain unknown. We aimed to study the impact of strenuous exercise in a very high cardiovascular risk model. Nephrectomized aged Zucker obese rats were trained at a moderate (MOD) or high (INT) intensity or were kept sedentary (SED) for 10 weeks. Subsequently, echocardiography and ex vivo vascular reactivity assays were performed, and blood, aortas, perivascular adipose tissue (PVAT), and left ventricles (LVs) were harvested. An improved risk profile consisting of decreased body weight and improved response to a glucose tolerance test was noted in the trained groups. Vascular reactivity experiments in the descending thoracic aorta demonstrated increased endothelial NO release in the MOD group but not in the INT group, compared with SED; the free radical scavenger TEMPOL improved endothelial function in INT rats to a similar level as MOD. An imbalance in the expression of oxidative stress-related genes toward a pro-oxidant environment was observed in the PVAT of INT rats. In the heart, INT training promoted eccentric hypertrophy and a mild reduction in ejection fraction. Obesity was associated with LV fibrosis and a transition toward β-myosin heavy chain and the N2Ba titin isoform. Exercise reverted the myosin imbalance, but only MOD reduced the predominance of the N2Ba titin isoform. In conclusion, moderate exercise yields the most intense cardiovascular benefits in a high-cardiovascular-risk animal model, while intense training partially reverts them.

Identification of differentially expressed genes and SNPs linked to harvest body weight of genetically improved rohu carp, Labeo rohita.

In most of the aquaculture selection programs, harvest body weight has been a preferred performance trait for improvement. Molecular interplay of genes linked to higher body weight is not elucidated in major carp species. The genetically improved rohu carp with 18% average genetic gain per generation with respect to harvest body weight is a promising candidate for studying genes' underlying performance traits. In the present study, muscle transcriptome sequencing of two groups of individuals, with significant difference in breeding value, belonging to the tenth generation of rohu carp was performed using the Illumina HiSeq 2000 platform. A total of 178 million paired-end raw reads were generated to give rise to 173 million reads after quality control and trimming. The genome-guided transcriptome assembly and differential gene expression produced 11,86,119 transcripts and 451 upregulated and 181 downregulated differentially expressed genes (DEGs) between high-breeding value and low-breeding value (HB & LB) groups, respectively. Similarly, 39,158 high-quality coding SNPs were identified with the Ts/Tv ratio of 1.23. Out of a total of 17 qPCR-validated transcripts, eight were associated with cellular growth and proliferation and harbored 13 SNPs. The gene expression pattern was observed to be positively correlated with RNA-seq data for genes such as myogenic factor 6, titin isoform X11, IGF-1 like, acetyl-CoA, and thyroid receptor hormone beta. A total of 26 miRNA target interactions were also identified to be associated with significant DETs (p-value < 0.05). Genes such as Myo6, IGF-1-like, and acetyl-CoA linked to higher harvest body weight may serve as candidate genes in marker-assisted breeding and SNP array construction for genome-wide association studies and genomic selection.

Comparative analysis of metabolic and structural changes in the skeletal muscles and myocardium of rats on the background of long-term administration of simvastatin

Objective: to conduct a comparative analysis of the dynamics of changes in the end products of glycolysis and the isoform composition of the giant protein - titin and nebulin in the skeletal and cardiac muscles of animals with HC during long-term administration of simvastatin.Materials and methods: the study was conducted on rats that were kept on a high-fat and high-carbohydrate diet for three months. After GC confirmation, the animals were divided into groups: group 1 received only the experimental diet; group 2 received simvastatin in the form of an aqueous suspension for two months at the rate of 0.012 g/kg of animal weight once a day. The control group of animals was kept on the general diet of the vivarium.Results: The formation of HCh was accompanied by the accumulation of pyruvate and lactic acid both in the myocardium and in muscle tissue. The revealed metabolic changes reflect structural disorders and indicate the formation of hypoxia. Against the background of the introduction of simvastatin, both in the myocardium and in the skeletal muscles, a decrease in the level of pyruvate and lactic acid was noted and these indicators approached the values of the control group. Such changes reflect a tendency to restore the integration of intracellular metabolic processes. However, in the study of titin and nebulin proteins against the background of the use of simvastatin, both in the myocardium and in skeletal muscles, structural changes were noted: accumulation of the proteolytic T-fragment, a decrease in the content of intact titin isoforms. Also, statin-induced structural abnormalities have been identified in earlier studies of rat muscle biopsies.Conclusion: The conducted comparative study clearly demonstrates that the myocardium, along with skeletal muscles, is the target organ of the toxic effect of statins. Based on this, the data obtained as a result of the study, it is important to take into account the pharmacotherapy with statins.

An isoform of the giant protein titin is a master regulator of human T lymphocyte trafficking

Response to multiple microenvironmental cues and resilience to mechanical stress are essential features of trafficking leukocytes. Here, we describe unexpected role of titin (TTN), the largest protein encoded by the human genome, in the regulation of mechanisms of lymphocyte trafficking. Human T and B lymphocytes express five TTN isoforms, exhibiting cell-specific expression, distinct localization to plasma membrane microdomains, and different distribution to cytosolic versus nuclear compartments. In T lymphocytes, the LTTN1 isoform governs the morphogenesis of plasma membrane microvilli independently of ERM protein phosphorylation status, thus allowing selectin-mediated capturing and rolling adhesions. Likewise, LTTN1 controls chemokine-triggered integrin activation. Accordingly, LTTN1 mediates rho and rap small GTPases activation, but not actin polymerization. In contrast, chemotaxis is facilitated by LTTN1 degradation. Finally, LTTN1 controls resilience to passive cell deformation and ensures T lymphocyte survival in the blood stream. LTTN1 is, thus, a critical and versatile housekeeping regulator of T lymphocyte trafficking.

Atrial cardiomyocytes from a canine model of atrial fibrillation demonstrate reduced force development and altered expression of contractile proteins.

Atrial fibrillation (AF) is the most common cardiac rhythm disturbance and associates with worse outcomes in cardiac contractile-related diseases including cardiomyopathy. As most studies surrounding AF focus on the electrophysiology of the disease, there is little known about cardiomyocyte contractile function in AF. We used atrial tissue from an established atrial tachypacing canine model of AF. Whole protein lysates were collected from the atria to conduct mass spectrometry analysis showing dysregulation of numerous molecular pathways, including contractility. Skinned atrial cardiomyocytes were isolated from the tissue for force calcium and passive tension experiments. Force calcium experiments showed a significant reduction in the maximum force of contraction of AF cardiomyocytes. To explain this, myosin heavy chain (MHC) isoform expression was evaluated because isoform switching is known to alter force of contraction. Decreased percent beta expression was found, explaining the reduction of force due to MHC alpha's capacity for reduced force of contraction. AF cardiomyocytes showed increased calcium sensitivity of force development, which may translate to prolonged contraction with slower relaxation in vivo. A significant reduction in S100A1 was seen in the AF samples via mass spectrometry, which is associated with increased calcium sensitivity. Prolonged contraction along with reduced force of each contraction could support the development of atrial clots. Atrial cardiomyocytes from the AF hearts demonstrated a significant reduction in the passive tension. Evaluation of titin isoform expression, which is known to alter passive tension, showed increased titin degradation, supporting the capacity for atrial dilation in vivo. Discerning the role of contractility in the pathology of atrial fibrillation could provide novel avenues of AF treatment, especially in instances where it is associated with contractile diseases.

Nonspecific Amyloid Aggregation of Chicken Smooth-Muscle Titin: In Vitro Investigations.

A giant multidomain protein of striated and smooth vertebrate muscles, titin, consists of tandems of immunoglobulin (Ig)- and fibronectin type III (FnIII)-like domains representing β-sandwiches, as well as of disordered segments. Chicken smooth muscles express several titin isoforms of ~500-1500 kDa. Using various structural-analysis methods, we investigated in vitro nonspecific amyloid aggregation of the high-molecular-weight isoform of chicken smooth-muscle titin (SMTHMW, ~1500 kDa). As confirmed by X-ray diffraction analysis, under near-physiological conditions, the protein formed amorphous amyloid aggregates with a quaternary cross-β structure within a relatively short time (~60 min). As shown by circular dichroism and Fourier-transform infrared spectroscopy, the quaternary cross-β structure-unlike other amyloidogenic proteins-formed without changes in the SMTHMW secondary structure. SMTHMW aggregates partially disaggregated upon increasing the ionic strength above the physiological level. Based on the data obtained, it is not the complete protein but its particular domains/segments that are likely involved in the formation of intermolecular interactions during SMTHMW amyloid aggregation. The discovered properties of titin position this protein as an object of interest for studying amyloid aggregation in vitro and expanding our views of the fundamentals of amyloidogenesis.

Rbm20ΔRRM Mice, Expressing a Titin Isoform with Lower Stiffness, Are Protected from Mechanical Ventilation-Induced Diaphragm Weakness.

Diaphragm weakness frequently develops in mechanically ventilated critically ill patients and is associated with increased morbidity, including ventilator weaning failure, mortality, and health care costs. The mechanisms underlying diaphragm weakness are incompletely understood but may include the elastic properties of titin, a giant protein whose layout in the muscle's sarcomeres makes it an ideal candidate to sense ventilation-induced diaphragm unloading, resulting in downstream signaling through titin-binding proteins. In the current study, we investigated whether modulating titin stiffness affects the development of diaphragm weakness during mechanical ventilation. To this end, we ventilated genetically engineered mice with reduced titin stiffness (Rbm20ΔRRM), and robust (TtnΔIAjxn) or severely (TtnΔ112-158) increased titin stiffness for 8 h, and assessed diaphragm contractility and protein expression of titin-binding proteins. Mechanical ventilation reduced the maximum active tension of the diaphragm in WT, TtnΔIAjxn and TtnΔ112-158 mice. However, in Rbm20ΔRRM mice maximum active tension was preserved after ventilation. Analyses of titin binding proteins suggest that muscle ankyrin repeat proteins (MARPs) 1 and 2 may play a role in the adaptation of the diaphragm to mechanical ventilation, and the preservation of diaphragm contractility in Rbm20ΔRRM mice. Thus, Rbm20ΔRRM mice, expressing titin isoforms with lower stiffness, are protected from mechanical ventilation-induced diaphragm weakness, suggesting that titin elasticity may modulate the diaphragm's response to unloading during mechanical ventilation.

Study of the expression of Cronos titin in TTN-truncation cardiomyopathy and heart development

Cronos is a recently discovered isoform of the giant protein titin driven by an alternative internal promoter. Cronos spans the half-A-band of a sarcomere and binds to myosin filaments, but its role in healthy or diseased hearts is largely unknown. Upregulation of Cronos has been suggested as a possible compensatory mechanism in titin-truncation cardiomyopathy, to help explain why truncations in titin's I-band region are less frequent and often cause a milder disease phenotype than truncations in the A-band region.

Abstract 13371: Beneficial Effects of Growth Hormone Releasing Hormone Agonist in a Murine Model of Cardiometabolic Heart Failure With Preserved Ejection Fraction

Introduction: Heart failure (HF) with preserved ejection fraction (HFpEF) represents a major unmet medical need due to its complex pathophysiology and lack of effective therapies. We previously showed that growth hormone-releasing hormone (GHRH)-agonists improve the phenotype of models of HF with reduced EF (HFrEF) and cardiorenal models of HFpEF. Hypothesis: We tested the hypothesis that the GHRH-agonist, MR-356 mitigates/reverses the cardio-metabolic HFpEF phenotype. Methods: C57BL6N mice received a high-fat diet (HFD) plus the nitric oxide synthase inhibitor (L-NAME) for 9 weeks. After 5 weeks of the HFD+L-NAME diet, animals were randomized to receive daily injections of MR-356 or placebo for 4-weeks. Control animals received no HFD+L-NAME or agonist treatment. Cardiac performance was assessed by echocardiography and hemodynamic measurements. Blood pressure, glucose tolerance, and exercise capacity, as well as cardiac hypertrophy, fibrosis, capillary density, and lung congestion were evaluated. Results: After 5 weeks of HFD+L-NAME diet, the placebo group showed impaired diastolic function (increased E/E’ratio, EDPVR, end-diastolic pressure), glucose intolerance and reduced global longitudinal strain and exercise capacity (p&lt;0.05). MR-356 treatment improved all of these parameters (p&lt;0.05). Cardiac hypertrophy, fibrosis, and capillary rarefaction in the placebo group (p&lt;0.05) were completely restored by MR-356 (p&lt;0.05). The increased expression of cardiac pro-BNP, and inducible nitric oxide synthase (iNOS) in the placebo group (p&lt;0.05) was brought to normal levels by MR-356 (p&lt;0.05). Hypophosphorylation of the titin isoforms in the placebo group (p&lt;0.05) was attenuated in the MR-356 group. Conclusions: Our data show the unique potential of MR-356 to treat several HFpEF-like features including diastolic dysfunction, cardiac hypertrophy, fibrosis, and exercise intolerance, suggesting that MR-356 reduces myocardial stress associated with metabolic inflammation in HFpEF. Thus, GHRH-agonists may be an effective therapeutic strategy for the treatment of the cardiometabolic HFpEF phenotype.

Recognizable Pattern of Arthrogryposis and Congenital Myopathy Caused by the Recurrent TTN Metatranscript-only c.39974-11T > G Splice Variant.

Arthrogryposis is characterized by the presence of multiple contractures at birth and can be caused by pathogenic variants in TTN (Titin). Exons and variants that are not expressed in one of the three major isoforms of titin are referred to as "metatranscript-only" and have been considered to be only expressed during fetal development. Recently, the metatranscript-only variant (c.39974-11T > G) in TTN with a second truncating TTN variant has been linked to arthrogryposis multiplex congenita and myopathy. Via exome sequencing we identified the TTN c.39974-11T > G splice variant in trans with one of three truncating variants (p.Arg8922*, p.Lys32998Asnfs*63, p.Tyr10345*) in five individuals from three families. Clinical presentation and muscle ultrasound as well as MRI images were analyzed. All five patients presented with generalized muscular hypotonia, reduced muscle bulk, and congenital contractures most prominently affecting the upper limbs and distal joints. Muscular hypotonia persisted and contractures improved over time. One individual, the recipient twin in the setting of twin-to-twin transfusion syndrome, died from severe cardiac hypertrophy 1 day after birth. Ultrasound and MRI imaging studies revealed a recognizable pattern of muscle involvement with striking fibrofatty involvement of the hamstrings and calves, and relative sparing of the femoral adductors and anterior segment of the thighs. The recurrent TTN c.39974-11T > G variant consistently causes congenital arthrogryposis and persisting myopathy providing evidence that the metatranscript-only 213 to 217 exons impact muscle elasticity during early development and beyond. There is a recognizable pattern of muscle involvement, which is distinct from other myopathies and provides valuable clues for diagnostic work-up.

The skeletal muscle circadian clock regulates titin splicing through RBM20.

Circadian rhythms are maintained by a cell-autonomous, transcriptional-translational feedback loop known as the molecular clock. While previous research suggests a role of the molecular clock in regulating skeletal muscle structure and function, no mechanisms have connected the molecular clock to sarcomere filaments. Utilizing inducible, skeletal muscle specific, Bmal1 knockout (iMSBmal1-/-) mice, we showed that knocking out skeletal muscle clock function alters titin isoform expression using RNAseq, liquid chromatography-mass spectrometry, and sodium dodecyl sulfate-vertical agarose gel electrophoresis. This alteration in titin's spring length resulted in sarcomere length heterogeneity. We demonstrate the direct link between altered titin splicing and sarcomere length in vitro using U7 snRNPs that truncate the region of titin altered in iMSBmal1-/- muscle. We identified a mechanism whereby the skeletal muscle clock regulates titin isoform expression through transcriptional regulation of Rbm20, a potent splicing regulator of titin. Lastly, we used an environmental model of circadian rhythm disruption and identified significant downregulation of Rbm20 expression. Our findings demonstrate the importance of the skeletal muscle circadian clock in maintaining titin isoform through regulation of RBM20 expression. Because circadian rhythm disruption is a feature of many chronic diseases, our results highlight a novel pathway that could be targeted to maintain skeletal muscle structure and function in a range of pathologies.

Contributions of Titin and Collagen to Passive Stress in Muscles from mdm Mice with a Small Deletion in Titin's Molecular Spring.

Muscular dystrophy with myositis (mdm) is a naturally occurring mutation in the mouse Ttn gene that results in higher passive stress in muscle fibers and intact muscles compared to wild-type (WT). The goal of this study was to test whether alternative splicing of titin exons occurs in mdm muscles, which contain a small deletion in the N2A-PEVK regions of titin, and to test whether splicing changes are associated with an increase in titin-based passive tension. Although higher levels of collagen have been reported previously in mdm muscles, here we demonstrate alternative splicing of titin in mdm skeletal muscle fibers. We identified Z-band, PEVK, and C-terminus Mex5 exons as splicing hotspots in mdm titin using RNA sequencing data and further reported upregulation in ECM-associated genes. We also treated skinned mdm soleus fiber bundles with trypsin, trypsin + KCl, and trypsin + KCL + KI to degrade titin. The results showed that passive stress dropped significantly more after trypsin treatment in mdm fibers (11 ± 1.6 mN/mm2) than in WT fibers (4.8 ± 1 mN/mm2; p = 0.0004). The finding that treatment with trypsin reduces titin-based passive tension more in mdm than in WT fibers supports the hypothesis that exon splicing leads to the expression of a stiffer and shorter titin isoform in mdm fibers. After titin extraction by trypsin + KCl + KI, mdm fibers (6.7 ± 1.27 mN/mm2) had significantly higher collagen-based passive stress remaining than WT fibers (2.6 ± 1.3 mN/mm2; p = 0.0014). We conclude that both titin and collagen contribute to higher passive tension of mdm muscles.

RBM20, a Therapeutic Target to Alleviate Myocardial Stiffness via Titin Isoforms Switching in HFpEF.

Increased myocardial stiffness is critically involved in heart diseases with impaired cardiac compliance, especially heart failure with preserved ejection fraction (HFpEF). Myocardial stiffness mainly derives from cardiomyocyte- and extracellular matrix (ECM)-derived passive stiffness. Titin, a major component of sarcomeres, participates in myocardial passive stiffness and stress-sensitive signaling. The ratio of two titin isoforms, N2BA to N2B, was validated to influence diastolic dysfunction via several pathways. RNA binding motif protein 20 (RBM20) is a well-studied splicing factor of titin, functional deficiency of RBM20 in mice profile improved cardiac compliance and function, which indicated that RBM20 functions as a potential therapeutic target for mitigating myocardial stiffness by modulating titin isoforms. This minor review summarized how RBM20 and other splicing factors modify the titin isoforms ratio, therefore providing a promising target for improving the myocardial compliance of HFpEF.

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

Altered kinase localization is gaining appreciation as a mechanism of cardiovascular disease. Previous work suggests GSK-3β (glycogen synthase kinase 3β) localizes to and regulates contractile function of the myofilament. We aimed to discover GSK-3β's in vivo role in regulating myofilament function, the mechanisms involved, and the translational relevance. Inducible cardiomyocyte-specific GSK-3β knockout mice and left ventricular myocardium from nonfailing and failing human hearts were studied. 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-3β. Agreeing with the localization of its targets, GSK-3β 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-3β in neonatal rat ventricular cardiomyocytes. One of GSK-3β'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-3β. Last, GSK-3β myofilament localization was reduced in left ventricular myocardium from failing human hearts, which correlated with depressed LDA. We identified a novel mechanism by which GSK-3β localizes to the myofilament to modulate LDA. Importantly, z-disc GSK-3β levels were reduced in patients with heart failure, indicating z-disc localized GSK-3β is a possible therapeutic target to restore the Frank-Starling mechanism in patients with heart failure.

Identification of the domains within the N2A region of titin that regulate binding to actin

Titin, the largest muscle protein, plays an important role in passive tension, sarcomeric integrity and cell signaling within the muscle. Recent work has also highlighted a role for titin in active muscle and the N2A region found in skeletal muscle titin and in some isoforms of cardiac titin has been linked to this function. The N2A region is a multi-domain region composed of four immunoglobulin domains (I80–I83) and a disordered region called the insertion sequence. Previously, our lab has shown that the N2A region binds F-actin in a calcium dependent manner, but it is not known which domains within this region are critical for this binding to occur. In this work, we have used co-sedimentation to demonstrate that only constructs containing the I80 domain are capable of binding F-actin. In addition, binding was only observed in constructs containing at least 3 immunoglobulin domains suggesting a length-dependence to binding. Finally, the calcium-dependence of N2A binding is lost when I83 is not present, consistent with the calcium stabilization that has been reported for this domain. Based on these results, we propose that I80 is critical for initiating binding to F-actin and that I83 is responsible for the calcium dependence.

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.