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Henk Granzier - One of the best experts on this subject based on the ideXlab platform.
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Titin‐based mechanosensing modulates muscle hypertrophy
Wiley, 2018Co-Authors: Robbert Van Derpijl, Siegfried Labeit, Henk Granzier, Joshua Strom, Stefan Conijn, Johan Lindqvist, Coen OttenheijmAbstract:Abstract Background Titin is an elastic sarcomeric filament that has been proposed to play a key role in mechanosensing and trophicity of muscle. However, evidence for this proposal is scarce due to the lack of appropriate experimental models to directly test the role of Titin in mechanosensing. Methods We used unilateral diaphragm denervation (UDD) in mice, an in vivo model in which the denervated hemidiaphragm is passively stretched by the contralateral, innervated hemidiaphragm and hypertrophy rapidly occurs. Results In wildtype mice, the denervated hemidiaphragm mass increased 48 ± 3% after 6 days of UDD, due to the addition of both sarcomeres in series and in parallel. To test whether Titin stiffness modulates the hypertrophy response, RBM20ΔRRM and TtnΔIAjxn mouse models were used, with decreased and increased Titin stiffness, respectively. RBM20ΔRRM mice (reduced stiffness) showed a 20 ± 6% attenuated hypertrophy response, whereas the TtnΔIAjxn mice (increased stiffness) showed an 18 ± 8% exaggerated response after UDD. Thus, muscle hypertrophy scales with Titin stiffness. Protein expression analysis revealed that Titin‐binding proteins implicated previously in muscle trophicity were induced during UDD, MARP1 & 2, FHL1, and MuRF1. Conclusions Titin functions as a mechanosensor that regulates muscle trophicity
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Novex-3, the tiny Titin of muscle
Biophysical Reviews, 2017Co-Authors: Dalma Kellermayer, John E. Smith, Henk GranzierAbstract:The giant multi-functional striated muscle protein Titin is the third most abundant muscle protein after myosin and actin. Titin plays a pivotal role in myocardial passive stiffness, structural integrity and stress-initiated signaling pathways. The complete sequence of the human Titin gene contains three isoform-specific mutually exclusive exons [termed novel exons (novex)] coding for the I-band sequence, named novex-1 (exon 45), novex-2 (exon 46) and novex-3 (exon 48). Transcripts containing either the novex-1 or novex-2 exons code for the novex-1 and novex-2 Titin isoforms. The novex-3 transcript contains a stop codon and polyA tail signal, resulting in an unusually small (∼700 kDa) isoform, referred to as novex-3 Titin. This ‘tiny Titin’ isoform extends from the Z-disc (N-terminus) to novex-3 (C-terminus) and is expressed in all striated muscles. Biochemical analysis of novex-3 Titin in cardiomyocytes shows that obscurin, a vertebrate muscle protein, binds to novex-3 Titin. The novex-3/obscurin complex localizes to the Z-disc region and may regulate calcium, and SH3- and GTPase-associated myofibrillar signaling pathways. Therefore, novex-3 Titin could be involved in stress-initiated sarcomeric restructuring.
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Cardiac Titin and heart disease.
Journal of cardiovascular pharmacology, 2014Co-Authors: Martin M. Lewinter, Henk GranzierAbstract:The giant sarcomeric protein Titin is a key determinant of myocardial passive stiffness and stress-sensitive signaling. Titin stiffness is modulated by isoform variation, phosphorylation by protein kinases, and, possibly, oxidative stress through disulfide bond formation. Titin has also emerged as an important human disease gene. Early studies in patients with dilated cardiomyopathy (DCM) revealed shifts toward more compliant isoforms, an adaptation that offsets increases in passive stiffness based on the extracellular matrix. Similar shifts are observed in heart failure with preserved ejection fraction. In contrast, hypophosphorylation of PKA/G sites contributes to a net increase in cardiomyocyte resting tension in heart failure with preserved ejection fraction. More recently, Titin mutations have been recognized as the most common etiology of inherited DCM. In addition, some DCM-causing mutations affect RBM20, a Titin splice factor. Titin mutations are a rare cause of hypertrophic cardiomyopathy and also underlie some cases of arrhythmogenic right ventricular dysplasia. Finally, mutations of genes encoding proteins that interact with and/or bind to Titin are responsible for both DCM and hypertrophic cardiomyopathy. Targeting Titin as a therapeutic strategy is in its infancy, but it could potentially involve manipulation of isoforms, posttranslational modifications, and upregulation of normal protein in patients with disease-causing mutations.
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shortening of the elastic tandem immunoglobulin segment of Titin leads to diastolic dysfunction
Circulation, 2013Co-Authors: Charles S. Chung, Siegfried Labeit, Xiuju Luo, John E. Smith, Kirk R Hutchinson, Mei Methawasin, Chandra Saripalli, Carlos Hidalgo, Caiying Guo, Henk GranzierAbstract:Background—Diastolic dysfunction is a poorly understood but clinically pervasive syndrome that is characterized by increased diastolic stiffness. Titin is the main determinant of cellular passive stiffness. However, the physiological role that the tandem immunoglobulin (Ig) segment of Titin plays in stiffness generation and whether shortening this segment is sufficient to cause diastolic dysfunction need to be established. Methods and Results—We generated a mouse model in which 9 Ig-like domains (Ig3–Ig11) were deleted from the proximal tandem Ig segment of the spring region of Titin (IG KO). Exon microarray analysis revealed no adaptations in Titin splicing, whereas novel phospho-specific antibodies did not detect changes in Titin phosphorylation. Passive myocyte stiffness was increased in the IG KO, and immunoelectron microscopy revealed increased extension of the remaining Titin spring segments as the sole likely underlying mechanism. Diastolic stiffness was increased at the tissue and organ levels, wi...
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Removal of Proximal IG Domains of Titin in Soleus Muscle Results in Differential Splicing of Titin MRNA
Biophysical Journal, 2013Co-Authors: Danielle Buck, John E. Smith, Charles S. Chung, Henk GranzierAbstract:Titin is the largest protein known that functions as a molecular spring in striated muscle. Its elastic properties are acquired through differential splicing of spring-like domains of Titin including the immunoglobuline-like (IG) and PEVK. Our lab has recently created a novel mouse model in which nine of the proximal IG domains (Titin exons 30-38, 90kDa reduction in protein size) have been deleted (IG KO). Surprisingly in the skeletal muscles from IG KO mice, Titin was found to undergo additional differential splicing to yield smaller Titin isoforms. These changes in splicing were found to be developmentally regulated and occur to different extents in various muscles. Most prominently, in the adult soleus muscle, two smaller Titin isoforms were present in the IG KO (3.42MDa ± 0.008 and 3.24MDa ± 0.027 respectively) as compared to one larger isoform found in wild-type mice (3.61MDa ± 0.057). Titin microarray analysis revealed IG KO mice had significant downregulation of exons in the PEVK region in addition to the lack of exons 30-38 as compared to wild-type. Consistent with a decrease in Titin size, IG KO soleus muscles produced an increased passive tension (19%, p=0.02) at 20% above slack length of the muscle as probed by intact mechanics. However, maximal active tension in the soleus and other muscles were not significantly different in the wildtype as compared to IG KO. Two Titin binding proteins were found to be differentially expressed by Affymetrix Array, CARP (7.23 fold increased in IG KO, p
Wolfgang A Linke - One of the best experts on this subject based on the ideXlab platform.
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Titin Gene and Protein Functions in Passive and Active Muscle.
Annual review of physiology, 2017Co-Authors: Wolfgang A LinkeAbstract:The thin and thick filaments of muscle sarcomeres are interconnected by the giant protein Titin, which is a scaffolding filament, signaling platform, and provider of passive tension and elasticity in myocytes. This review summarizes recent insight into the mechanisms behind how Titin gene mutations cause hereditary cardiomyopathy and how Titin protein is mechanically active in skeletal and cardiac myocytes. A main theme is the evolving role of Titin as a modulator of contraction. Topics include strain-sensing via Titin in the sarcomeric A-band as the basis for length-dependent activation, Titin elastic recoil and refolding of Titin domains as an energy source, and Ca2+-dependent stiffening of Titin stretched during eccentric muscle contractions. Findings suggest that Titin stiffness is a principal regulator of the contractile behavior of striated muscle. Physiological or pathological changes to Titin stiffness therefore affect contractility. Taken together, Titin emerges as a linker element between passiv...
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Tampering with springs: phosphorylation of Titin affecting the mechanical function of cardiomyocytes
Biophysical Reviews, 2017Co-Authors: Nazha Hamdani, Melissa Herwig, Wolfgang A LinkeAbstract:Reversible post-translational modifications of various cardiac proteins regulate the mechanical properties of the cardiomyocytes and thus modulate the contractile performance of the heart. The giant protein Titin forms a continuous filament network in the sarcomeres of striated muscle cells, where it determines passive tension development and modulates active contraction. These mechanical properties of Titin are altered through post-translational modifications, particularly phosphorylation. Titin contains hundreds of potential phosphorylation sites, the functional relevance of which is only beginning to emerge. Here, we provide a state-of-the-art summary of the phosphorylation sites in Titin, with a particular focus on the elastic Titin spring segment. We discuss how phosphorylation at specific amino acids can reduce or increase the stretch-induced spring force of Titin, depending on where the spring region is phosphorylated. We also review which protein kinases phosphorylate Titin and how this phosphorylation affects Titin-based passive tension in cardiomyocytes. A comprehensive overview is provided of studies that have measured altered Titin phosphorylation and Titin-based passive tension in myocardial samples from human heart failure patients and animal models of heart disease. As our understanding of the broader implications of phosphorylation in Titin progresses, this knowledge could be used to design targeted interventions aimed at reducing pathologically increased Titin stiffness in patients with stiff hearts.
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Impact of cGMP-PKG Pathway Modulation on Titin Phosphorylation and Titin-Based Myocardial Passive Stiffness
Biophysical Journal, 2016Co-Authors: Nazha Hamdani, Melissa Herwig, Soraya Hoelper, Marcus Krueger, Doris Koesling, Michaela Kuhn, Wolfgang A LinkeAbstract:RATIONALE: The crucial contribution of the giant myofilament protein Titin to diastolic stiffness and cardiomyocyte passive force(Fpassive) is dependent, in part, on Titin isoform composition and phosphorylation. Phosphorylation of Titin by cyclic guanosine monophosphate(cGMP)-dependent protein kinase G(PKG) lowers Titin-based stiffness, thus mediating a mechanical signaling process that is disturbed in heart failure. OBJECTIVE: To elucidate which elements of the nitric oxide (NO) cGMP-PKG signaling network are critical for Titin phosphorylation and stiffness in vivo. METHODS AND RESULTS: We employed genetic knockout(KO) mouse models deficient for cGMP-PKG pathway enzymes, including cardiomyocyte-specific deletion of the guanylyl cyclase(GC)-A receptor and cGMP-dependent-PKG(cGKI), and global deletion of soluble GC(sGC). We assessed Titin phosphorylation and Fpassive of single permeabilized cardiomyocytes recorded before/after PKG administration. In all three models, all-Titin phosphorylation was reduced compared to WT hearts. The important PKG-dependent phospho-S4080 site within Titin-N2-Bus was hypophosphorylated in all three KO-models. Unexpectedly, mass spectrometry analysis revealed that most class-1 Titin phospho-sites within the molecular spring segment, including the Ig-domain regions, were hyperphosphorylated. Only a few sites showed a phosphorylation deficit or remaining unchanged. Particularly in the cGKI model many class-1 phospho-sites were hyperphosphorylated compared to WT hearts, indicative of the presence of compensatory processes following loss of PKG; indeed, this was associated with upregulation of several kinases that phosphorylate Titin and a clear rise in Fpassive in KO vs. WT cardiomyocytes. While administration of PKG lowered Fpassive of WT and KO cardiomyocytes in all models, this effect was more pronounced in the cGKI KO. CONCLUSIONS: Multiple in vivo phosphorylated class I Titin phospho-sites were identified within the molecular spring segment, some of which depended on the cGMP-PKG pathway. While cGMP-activated PKG remains an important Titin-targeting kinase, many Titin phospho-sites may be regulated through a network of protein kinases/phosphatases.
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emerging importance of oxidative stress in regulating striated muscle elasticity
Journal of Muscle Research and Cell Motility, 2015Co-Authors: Lisa Beckendorf, Wolfgang A LinkeAbstract:The contractile function of striated muscle cells is altered by oxidative/nitrosative stress, which can be observed under physiological conditions but also in diseases like heart failure or muscular dystrophy. Oxidative stress causes oxidative modifications of myofilament proteins and can impair myocyte contractility. Recent evidence also suggests an important effect of oxidative stress on muscle elasticity and passive stiffness via modifications of the giant protein Titin. In this review we provide a short overview of known oxidative modifications in thin and thick filament proteins and then discuss in more detail those oxidative stress-related modifications altering Titin stiffness directly or indirectly. Direct modifications of Titin include reversible disulfide bonding within the cardiac-specific N2-Bus domain, which increases Titin stiffness, and reversible S-glutathionylation of cryptic cysteines in immunoglobulin-like domains, which only takes place after the domains have unfolded and which reduces Titin stiffness in cardiac and skeletal muscle. Indirect effects of oxidative stress on Titin can occur via reversible modifications of protein kinase signalling pathways (especially the NO-cGMP-PKG axis), which alter the phosphorylation level of certain disordered Titin domains and thereby modulate Titin stiffness. Oxidative stress also activates proteases such as matrix-metalloproteinase-2 and (indirectly via increasing the intracellular calcium level) calpain-1, both of which cleave Titin to irreversibly reduce Titin-based stiffness. Although some of these mechanisms require confirmation in the in vivo setting, there is evidence that oxidative stress-related modifications of Titin are relevant in the context of biomarker design and represent potential targets for therapeutic intervention in some forms of muscle and heart disease.
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Gigantic business: Titin properties and function through thick and thin.
Circulation research, 2014Co-Authors: Wolfgang A Linke, Nazha HamdaniAbstract:The giant protein Titin forms a unique filament network in cardiomyocytes, which engages in both mechanical and signaling functions of the heart. TTN, which encodes Titin, is also a major human disease gene. In this review, we cover the roles of cardiac Titin in normal and failing hearts, with a special emphasis on the contribution of Titin to diastolic stiffness. We provide an update on disease-associated Titin mutations in cardiac and skeletal muscles and summarize what is known about the impact of protein-protein interactions on Titin properties and functions. We discuss the importance of Titin-isoform shifts and Titin phosphorylation, as well as Titin modifications related to oxidative stress, in adjusting the diastolic stiffness of the healthy and the failing heart. Along the way we distinguish among Titin alterations in systolic and in diastolic heart failure and ponder the evidence for Titin stiffness as a potential target for pharmacological intervention in heart disease.
Siegfried Labeit - One of the best experts on this subject based on the ideXlab platform.
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Titin‐based mechanosensing modulates muscle hypertrophy
Wiley, 2018Co-Authors: Robbert Van Derpijl, Siegfried Labeit, Henk Granzier, Joshua Strom, Stefan Conijn, Johan Lindqvist, Coen OttenheijmAbstract:Abstract Background Titin is an elastic sarcomeric filament that has been proposed to play a key role in mechanosensing and trophicity of muscle. However, evidence for this proposal is scarce due to the lack of appropriate experimental models to directly test the role of Titin in mechanosensing. Methods We used unilateral diaphragm denervation (UDD) in mice, an in vivo model in which the denervated hemidiaphragm is passively stretched by the contralateral, innervated hemidiaphragm and hypertrophy rapidly occurs. Results In wildtype mice, the denervated hemidiaphragm mass increased 48 ± 3% after 6 days of UDD, due to the addition of both sarcomeres in series and in parallel. To test whether Titin stiffness modulates the hypertrophy response, RBM20ΔRRM and TtnΔIAjxn mouse models were used, with decreased and increased Titin stiffness, respectively. RBM20ΔRRM mice (reduced stiffness) showed a 20 ± 6% attenuated hypertrophy response, whereas the TtnΔIAjxn mice (increased stiffness) showed an 18 ± 8% exaggerated response after UDD. Thus, muscle hypertrophy scales with Titin stiffness. Protein expression analysis revealed that Titin‐binding proteins implicated previously in muscle trophicity were induced during UDD, MARP1 & 2, FHL1, and MuRF1. Conclusions Titin functions as a mechanosensor that regulates muscle trophicity
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shortening of the elastic tandem immunoglobulin segment of Titin leads to diastolic dysfunction
Circulation, 2013Co-Authors: Charles S. Chung, Siegfried Labeit, Xiuju Luo, John E. Smith, Kirk R Hutchinson, Mei Methawasin, Chandra Saripalli, Carlos Hidalgo, Caiying Guo, Henk GranzierAbstract:Background—Diastolic dysfunction is a poorly understood but clinically pervasive syndrome that is characterized by increased diastolic stiffness. Titin is the main determinant of cellular passive stiffness. However, the physiological role that the tandem immunoglobulin (Ig) segment of Titin plays in stiffness generation and whether shortening this segment is sufficient to cause diastolic dysfunction need to be established. Methods and Results—We generated a mouse model in which 9 Ig-like domains (Ig3–Ig11) were deleted from the proximal tandem Ig segment of the spring region of Titin (IG KO). Exon microarray analysis revealed no adaptations in Titin splicing, whereas novel phospho-specific antibodies did not detect changes in Titin phosphorylation. Passive myocyte stiffness was increased in the IG KO, and immunoelectron microscopy revealed increased extension of the remaining Titin spring segments as the sole likely underlying mechanism. Diastolic stiffness was increased at the tissue and organ levels, wi...
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eLS - Titin Gene (TTN)
eLS, 2011Co-Authors: Siegfried Labeit, Dietmar Labeit, Julius Bogomolovas, Henk GranzierAbstract:The Titin locus located on chromosome 2q24 in the human genome expresses about 100 kb full-length mRNAs, that are translated into giant up to 34.350-residue large polypeptides. Therefore, Titin is by far the largest known protein. The Titin protein is abundant in vertebrate muscles, where it spans half of the sarcomere. In situ, 1–2 μm long Titin polypeptides establish a sarcomeric filament system that is critical for myofibrillar integrity and elasticity. Biomechanically, Titin's intrinsic elasticity is fine-tuned in the different muscle tissues through alternative splicing, post-translational modifications and protein–protein interactions. Moreover, a plethora of molecular interactions with stress-regulated ligands positions Titin centrally in stretch-dependent signalling in muscle. Therefore, mutations in this filament system are important causes of hereditary cardiomyopathies and muscular dystrophies. Key Concepts: Sarcomeres consist of precisely assembled proteins that together form the basic functional units of striated muscle and give rise to efficient and finely tuned contraction. In muscle tissues, 1–2 μm single Titin polypeptide chains span half of the sarcomere. The intrasarcomeric filamentous Titin protein provides sarcomeres with intrinsic elasticity and couples stretch-dependent signalling together with muscle remodelling. Titin molecule is tailored to physiological requirements of different muscles through alternative splicing, post-translational modifications and protein–protein interactions. Mutations in the Titin gene are associated with different heart and skeletal muscle diseases. Keywords: muscle contraction; myofibrillar elasticity and signalling; sarcomere assembly
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Expression of distinct classes of Titin isoforms in striated and smooth muscles by alternative splicing, and their conserved interaction with filamins.
Journal of molecular biology, 2006Co-Authors: Siegfried Labeit, Sunshine Lahmers, Christoph Burkart, Chi Fong, Mark Mcnabb, Stephanie H. Witt, Christian C. Witt, Dietmar Labeit, Henk GranzierAbstract:While the role of Titin as a sarcomeric protein is well established, its potential functional role(s) in smooth muscles and non-muscle tissues are controversial. We used a Titin exon array to search for which part(s) of the human Titin transcriptional unit encompassing 363 exons is(are) expressed in non-striated muscle tissues. Expression profiling of adult smooth muscle tissues (aorta, bladder, carotid, stomach) identified alternatively spliced Titin isoforms, encompassing 80 to about 100 exons. These exons code for parts of the Titin Z-disk, I-band and A-band regions, allowing the truncated smooth muscle Titin isoform to link Z-disks/dense bodies together with thick filaments. Consistent with the array data, Western blot studies detected the expression of approximately 1 MDa smooth muscle Titin in adult smooth muscles, reacting with selected Z-disc, I-band, and A-band Titin antibodies. Immunofluorescence with these antibodies located smooth muscle Titin in the cytoplasm of cultured human aortic smooth muscle cells and in the tunica media of intact adult bovine aorta. Real time PCR studies suggested that smooth muscle Titins are expressed from a promoter located 35 kb or more upstream of the transcription initiation site used for striated muscle Titin, driving expression of a bi-cistronic mRNA, coding 5' for the anonymous gene FL39502, followed 3' by Titin, respectively. Our work showed that smooth muscle and striated muscle Titins share in their conserved amino-terminal regions binding sites for alpha-actinin and filamins: Yeast two-hybrid screens using Z2-Zis1 Titin baits identified prey clones coding for alpha-actinin-1 and filamin-A from smooth muscle, and alpha-actinin-2/3, filamin-C, and nebulin from skeletal muscle cDNA libraries, respectively. This suggests that the Titin Z2-Zis1 domain can link filamins and alpha-actinin together in the periphery of the Z-line/dense bodies in a fashion that is conserved in smooth and striated muscles.
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altered Titin expression myocardial stiffness and left ventricular function in patients with dilated cardiomyopathy
Circulation, 2004Co-Authors: Sherif F Nagueh, Siegfried Labeit, Sunshine Lahmers, Christian C. Witt, Gopi Shah, Guillermo Torreamione, Nicholas M P King, Katy BeckerAbstract:Background—The role of the giant protein Titin in patients with heart failure is not well established. We investigated Titin expression in patients with end-stage heart failure resulting from nonischemic dilated cardiomyopathy, in particular as it relates to left ventricular (LV) myocardial stiffness and LV function. Methods and Results—SDS-agarose gels revealed small N2B (stiff) and large N2BA (compliant) cardiac Titin isoforms with a mean N2BA:N2B expression ratio that was significantly (P0.003) increased in 20 heart failure patients versus 6 controls. However, total Titin was unchanged. The coexpression ratio was highest in a subsample of patients with an impaired LV relaxation pattern (n7), intermediate in those with pseudonormal filling (n6), and lowest in the group with restrictive filling (n7). Mechanical measurements on LV muscle strips dissected from these hearts (n8) revealed that passive muscle stiffness was significantly reduced in patients with a high N2BA:N2B expression ratio. Clinical correlations support the relevance of these changes for LV function (assessed by invasive hemodynamics and Doppler echocardiography). A positive correlation between the N2BA:N2B Titin isoform ratio and deceleration time of mitral E velocity, A wave transit time, and end diastolic volume/pressure ratio was found. These changes affect exercise tolerance, as indicated by the positive correlation between the N2BA:N2B isoform ratio and peak O2 consumption (n10). Upregulated N2BA expression was accompanied by increased expression levels of Titin-binding proteins (cardiac ankyrin repeat protein, ankrd2, and diabetes ankyrin repeat protein) that bind to the N2A element of N2BA Titin (studied in 13 patients). Conclusions—Total Titin content was unchanged in end-stage failing hearts and the more compliant N2BA isoform comprised a greater percentage of Titin in these hearts. Changes in Titin isoform expression in heart failure patients with dilated cardiomyopathy significantly impact diastolic filling by lowering myocardial stiffness. Upregulation of Titin-binding proteins indicates that the importance of altered Titin expression might extend to cell signaling and regulation of gene expression. (Circulation. 2004;110:155-162.)
Nazha Hamdani - One of the best experts on this subject based on the ideXlab platform.
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Tampering with springs: phosphorylation of Titin affecting the mechanical function of cardiomyocytes
Biophysical Reviews, 2017Co-Authors: Nazha Hamdani, Melissa Herwig, Wolfgang A LinkeAbstract:Reversible post-translational modifications of various cardiac proteins regulate the mechanical properties of the cardiomyocytes and thus modulate the contractile performance of the heart. The giant protein Titin forms a continuous filament network in the sarcomeres of striated muscle cells, where it determines passive tension development and modulates active contraction. These mechanical properties of Titin are altered through post-translational modifications, particularly phosphorylation. Titin contains hundreds of potential phosphorylation sites, the functional relevance of which is only beginning to emerge. Here, we provide a state-of-the-art summary of the phosphorylation sites in Titin, with a particular focus on the elastic Titin spring segment. We discuss how phosphorylation at specific amino acids can reduce or increase the stretch-induced spring force of Titin, depending on where the spring region is phosphorylated. We also review which protein kinases phosphorylate Titin and how this phosphorylation affects Titin-based passive tension in cardiomyocytes. A comprehensive overview is provided of studies that have measured altered Titin phosphorylation and Titin-based passive tension in myocardial samples from human heart failure patients and animal models of heart disease. As our understanding of the broader implications of phosphorylation in Titin progresses, this knowledge could be used to design targeted interventions aimed at reducing pathologically increased Titin stiffness in patients with stiff hearts.
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Impact of cGMP-PKG Pathway Modulation on Titin Phosphorylation and Titin-Based Myocardial Passive Stiffness
Biophysical Journal, 2016Co-Authors: Nazha Hamdani, Melissa Herwig, Soraya Hoelper, Marcus Krueger, Doris Koesling, Michaela Kuhn, Wolfgang A LinkeAbstract:RATIONALE: The crucial contribution of the giant myofilament protein Titin to diastolic stiffness and cardiomyocyte passive force(Fpassive) is dependent, in part, on Titin isoform composition and phosphorylation. Phosphorylation of Titin by cyclic guanosine monophosphate(cGMP)-dependent protein kinase G(PKG) lowers Titin-based stiffness, thus mediating a mechanical signaling process that is disturbed in heart failure. OBJECTIVE: To elucidate which elements of the nitric oxide (NO) cGMP-PKG signaling network are critical for Titin phosphorylation and stiffness in vivo. METHODS AND RESULTS: We employed genetic knockout(KO) mouse models deficient for cGMP-PKG pathway enzymes, including cardiomyocyte-specific deletion of the guanylyl cyclase(GC)-A receptor and cGMP-dependent-PKG(cGKI), and global deletion of soluble GC(sGC). We assessed Titin phosphorylation and Fpassive of single permeabilized cardiomyocytes recorded before/after PKG administration. In all three models, all-Titin phosphorylation was reduced compared to WT hearts. The important PKG-dependent phospho-S4080 site within Titin-N2-Bus was hypophosphorylated in all three KO-models. Unexpectedly, mass spectrometry analysis revealed that most class-1 Titin phospho-sites within the molecular spring segment, including the Ig-domain regions, were hyperphosphorylated. Only a few sites showed a phosphorylation deficit or remaining unchanged. Particularly in the cGKI model many class-1 phospho-sites were hyperphosphorylated compared to WT hearts, indicative of the presence of compensatory processes following loss of PKG; indeed, this was associated with upregulation of several kinases that phosphorylate Titin and a clear rise in Fpassive in KO vs. WT cardiomyocytes. While administration of PKG lowered Fpassive of WT and KO cardiomyocytes in all models, this effect was more pronounced in the cGKI KO. CONCLUSIONS: Multiple in vivo phosphorylated class I Titin phospho-sites were identified within the molecular spring segment, some of which depended on the cGMP-PKG pathway. While cGMP-activated PKG remains an important Titin-targeting kinase, many Titin phospho-sites may be regulated through a network of protein kinases/phosphatases.
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Gigantic business: Titin properties and function through thick and thin.
Circulation research, 2014Co-Authors: Wolfgang A Linke, Nazha HamdaniAbstract:The giant protein Titin forms a unique filament network in cardiomyocytes, which engages in both mechanical and signaling functions of the heart. TTN, which encodes Titin, is also a major human disease gene. In this review, we cover the roles of cardiac Titin in normal and failing hearts, with a special emphasis on the contribution of Titin to diastolic stiffness. We provide an update on disease-associated Titin mutations in cardiac and skeletal muscles and summarize what is known about the impact of protein-protein interactions on Titin properties and functions. We discuss the importance of Titin-isoform shifts and Titin phosphorylation, as well as Titin modifications related to oxidative stress, in adjusting the diastolic stiffness of the healthy and the failing heart. Along the way we distinguish among Titin alterations in systolic and in diastolic heart failure and ponder the evidence for Titin stiffness as a potential target for pharmacological intervention in heart disease.
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human myocytes are protected from Titin aggregation induced stiffening by small heat shock proteins
Journal of Cell Biology, 2014Co-Authors: Sebastian Kotter, Nazha Hamdani, Patrick Lang, Andreas Unger, M Vorgerd, Luitgard Nagelsteger, Wolfgang A LinkeAbstract:In myocytes, small heat shock proteins (sHSPs) are preferentially translocated under stress to the sarcomeres. The functional implications of this translocation are poorly understood. We show here that HSP27 and αB-crystallin associated with immunoglobulin-like (Ig) domain-containing regions, but not the disordered PEVK domain (Titin region rich in proline, glutamate, valine, and lysine), of the Titin springs. In sarcomeres, sHSP binding to Titin was actin filament independent and promoted by factors that increased Titin Ig unfolding, including sarcomere stretch and the expression of stiff Titin isoforms. Titin spring elements behaved predominantly as monomers in vitro. However, unfolded Ig segments aggregated, preferentially under acidic conditions, and αB-crystallin prevented this aggregation. Disordered regions did not aggregate. Promoting Titin Ig unfolding in cardiomyocytes caused elevated stiffness under acidic stress, but HSP27 or αB-crystallin suppressed this stiffening. In diseased human muscle and heart, both sHSPs associated with the Titin springs, in contrast to the cytosolic/Z-disk localization seen in healthy muscle/heart. We conclude that aggregation of unfolded Titin Ig domains stiffens myocytes and that sHSPs translocate to these domains to prevent this aggregation.
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Myocardial Titin: An Important Modifier of Cardiac Stiffness
Biophysical Journal, 2014Co-Authors: Nazha Hamdani, Wolfgang A LinkeAbstract:Background: A well-established function of Titin is the determination of passive tension (Fpassive) in myocardium. Modifications to the elastic Titin region have been suggested to contribute to left ventricular (LV) diastolic dysfunction in heart failure (HF). Titin-based stiffness can be modulated by isoform switch or phosphorylation.Results: We find that Titin-isoform switch accounts for a significant amount of myocardial stiffness modulation, giving rise to increased or reduced Fpassive in different types of heart failure. In addition, both acute and chronic modulations of cardiomyocyte Fpassive occur via altered Titin phosphorylation. Cyclic AMP-dependent protein kinase-A, cGMP-dependent protein kinase-G, and extracellular signal-regulated kinase-2 phosphorylate Titin at a cardiac-specific domain, the N2Bus; this phosphorylation results in a reduction in cardiomyocyte Fpassive in various species. PKCα phosphorylates the PEVK-domain of Titin, which increases Fpassive of normal mouse cardiomyocytes, but does not significantly alter Fpassive of cardiomyocytes obtained from a dog HF model. Calcium/calmodulin-dependent protein kinase-II (CaMKII) is the first kinase found to phosphorylate both the N2Bus and the PEVK-domain. This phosphorylation reduces cardiomyocyte Fpassive, as demonstrated in skinned mouse cardiomyocytes incubated with recombinant CaMKIIδ. Moreover, Fpassive is elevated in cardiomyocytes of CaMKIIγ/δ double knockout mice and reduced in those of CaMKIIδ-overexpressing transgenic mice. In both human and experimental HF, a global Titin phosphorylation deficit is observed, but site-specific Titin phosphorylation can be increased or decreased in HF, presumably depending on the activity and expression level of the relevant kinases.Conclusion: Titin phosphorylation may have beneficial effects in the heart via reducing myocardial diastolic stiffness and improving ventricular filling. Altered Titin phosphorylation in HF may severely affect Fpassive and compromise cardiac function. The degree, to which the different protein kinases contribute to alterations in diastolic passive stiffness, needs to be determined.
Zoltan Papp - One of the best experts on this subject based on the ideXlab platform.
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hypophosphorylation of the stiff n2b Titin isoform raises cardiomyocyte resting tension in failing human myocardium
Circulation Research, 2009Co-Authors: Attila Borbely, Nazha Hamdani, Ines Falcaopires, Loek Van Heerebeek, Istvan Edes, Cristina Gavina, Adelino F Leitemoreira, Jean G F Bronzwaer, Zoltan PappAbstract:High diastolic stiffness of failing myocardium results from interstitial fibrosis and elevated resting tension (F(passive)) of cardiomyocytes. A shift in Titin isoform expression from N2BA to N2B isoform, lower overall phosphorylation of Titin, and a shift in Titin phosphorylation from N2B to N2BA isoform can raise F(passive) of cardiomyocytes. In left ventricular biopsies of heart failure (HF) patients, aortic stenosis (AS) patients, and controls (CON), we therefore related F(passive) of isolated cardiomyocytes to expression of Titin isoforms and to phosphorylation of Titin and Titin isoforms. Biopsies were procured by transvascular technique (44 HF, 3 CON), perioperatively (25 AS, 4 CON), or from explanted hearts (4 HF, 8 CON). None had coronary artery disease. Isolated, permeabilized cardiomyocytes were stretched to 2.2-microm sarcomere length to measure F(passive). Expression and phosphorylation of Titin isoforms were analyzed using gel electrophoresis with ProQ Diamond and SYPRO Ruby stains and reported as ratio of Titin (N2BA/N2B) or of phosphorylated Titin (P-N2BA/P-N2B) isoforms. F(passive) was higher in HF (6.1+/-0.4 kN/m(2)) than in CON (2.3+/-0.3 kN/m(2); P<0.01) or in AS (2.2+/-0.2 kN/m(2); P<0.001). Titin isoform expression differed between HF (N2BA/N2B=0.73+/-0.06) and CON (N2BA/N2B=0.39+/-0.05; P<0.001) and was comparable in HF and AS (N2BA/N2B=0.59+/-0.06). Overall Titin phosphorylation was also comparable in HF and AS, but relative phosphorylation of the stiff N2B Titin isoform was significantly lower in HF (P-N2BA/P-N2B=0.77+/-0.05) than in AS (P-N2BA/P-N2B=0.54+/-0.05; P<0.01). Relative hypophosphorylation of the stiff N2B Titin isoform is a novel mechanism responsible for raised F(passive) of human HF cardiomyocytes.
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hypophosphorylation of the stiff n2b Titin isoform raises cardiomyocyte resting tension in failing human myocardium
Circulation Research, 2009Co-Authors: Attila Borbely, Nazha Hamdani, Ines Falcaopires, Istvan Edes, Cristina Gavina, Adelino F Leitemoreira, Jean G F Bronzwaer, Loek Van Heerebeek, Zoltan PappAbstract:High diastolic stiffness of failing myocardium results from interstitial fibrosis and elevated resting tension ( F passive) of cardiomyocytes. A shift in Titin isoform expression from N2BA to N2B isoform, lower overall phosphorylation of Titin, and a shift in Titin phosphorylation from N2B to N2BA isoform can raise F passive of cardiomyocytes. In left ventricular biopsies of heart failure (HF) patients, aortic stenosis (AS) patients, and controls (CON), we therefore related F passive of isolated cardiomyocytes to expression of Titin isoforms and to phosphorylation of Titin and Titin isoforms. Biopsies were procured by transvascular technique (44 HF, 3 CON), perioperatively (25 AS, 4 CON), or from explanted hearts (4 HF, 8 CON). None had coronary artery disease. Isolated, permeabilized cardiomyocytes were stretched to 2.2-μm sarcomere length to measure F passive. Expression and phosphorylation of Titin isoforms were analyzed using gel electrophoresis with ProQ Diamond and SYPRO Ruby stains and reported as ratio of Titin (N2BA/N2B) or of phosphorylated Titin (P-N2BA/P-N2B) isoforms. F passive was higher in HF (6.1±0.4 kN/m2) than in CON (2.3±0.3 kN/m2; P <0.01) or in AS (2.2±0.2 kN/m2; P <0.001). Titin isoform expression differed between HF (N2BA/N2B=0.73±0.06) and CON (N2BA/N2B=0.39±0.05; P <0.001) and was comparable in HF and AS (N2BA/N2B=0.59±0.06). Overall Titin phosphorylation was also comparable in HF and AS, but relative phosphorylation of the stiff N2B Titin isoform was significantly lower in HF (P-N2BA/P-N2B=0.77±0.05) than in AS (P-N2BA/P-N2B=0.54±0.05; P <0.01). Relative hypophosphorylation of the stiff N2B Titin isoform is a novel mechanism responsible for raised F passive of human HF cardiomyocytes.
