N2B Region Research Articles

Deletion of the titin N2B region accelerates myofibrillar force development but does not alter relaxation kinetics

Cardiac titin is the main determinant of sarcomere stiffness during diastolic relaxation. To explore whether titin stiffness affects the kinetics of cardiac myofibrillar contraction and relaxation, we used subcellular myofibrils from the left ventricles of homozygous and heterozygous N2B-knockout mice which express truncated cardiac titins lacking the unique elastic N2B region. Compared with myofibrils from wild-type mice, myofibrils from knockout and heterozygous mice exhibit increased passive myofibrillar stiffness. To determine the kinetics of Ca(2+)-induced force development (rate constant kACT), myofibrils from knockout, heterozygous and wild-type mice were stretched to the same sarcomere length (2.3 µm) and rapidly activated with Ca(2+). Additionally, mechanically induced force-redevelopment kinetics (rate constant kTR) were determined by slackening and re-stretching myofibrils during Ca(2+)-mediated activation. Myofibrils from knockout mice exhibited significantly higher kACT, kTR and maximum Ca(2+)-activated tension than myofibrils from wild-type mice. By contrast, the kinetic parameters of biphasic force relaxation induced by rapidly reducing [Ca(2+)] were not significantly different among the three genotypes. These results indicate that increased titin stiffness promotes myocardial contraction by accelerating the formation of force-generating cross-bridges without decelerating relaxation.

A Novel Mechanism Involving Four-and-a-half LIM Domain Protein-1 and Extracellular Signal-regulated Kinase-2 Regulates Titin Phosphorylation and Mechanics

Understanding mechanisms underlying titin regulation in cardiac muscle function is of critical importance given recent compelling evidence that highlight titin mutations as major determinants of human cardiomyopathy. We previously identified a cardiac biomechanical stress-regulated complex at the cardiac-specific N2B region of titin that includes four-and-a-half LIM domain protein-1 (Fhl1) and components of the mitogen-activated protein signaling cascade, which impacted muscle compliance in Fhl1 knock-out cardiac muscle. However, direct regulation of these molecular components in mediating titin N2B function remained unresolved. Here we identify Fhl1 as a novel negative regulator of titin N2B levels and phosphorylation-mediated mechanics. We specifically identify titin N2B as a novel substrate of extracellular signal regulated-kinase-2 (Erk2) and demonstrate that Fhl1 directly interferes with Erk2-mediated titin-N2B phosphorylation. We highlight the critical region in titin-N2B that interacts with Fhl1 and residues that are dependent on Erk2-mediated phosphorylation in situ. We also propose a potential mechanism for a known titin-N2B cardiomyopathy-causing mutation that involves this regulatory complex. These studies shed light on a novel mechanism regulating titin-N2B mechano-signaling as well as suggest that dysfunction of these pathways could be important in cardiac disease states affecting muscle compliance.

Titin Phosphorylation

See related articles, pages 631–638 Diastolic performance is regulated by net myocardial stiffness, which is determined by the mechanosensitive protein network comprised of extracellular proteins such as collagen, intracellular sarcomeric proteins, and cell surface integrins. Mechanical force and its regulation are sensed and propagated by each of these components in a concerted fashion.1 During diastole, titin filaments serve as tensiometers and passive force generators. This is accomplished by modulation via directional signaling of multiple linkages between different regions within the titin molecule and the cellular contractile apparatus. An intricate network of signaling molecules coordinates the extracellular and intracellular components in the contraction of a sarcomere. In this issue of Circulation Research , Hidalgo et al present an elegant set of experiments that reveal a novel mechanism whereby altering the phosphorylation state of titin modulates myocardial passive stiffness.2 Specifically, they demonstrated that titin, in addition to being a protein kinase (PK)A and PKG substrate, is also a PKCα substrate. They further identified the PEVK region of titin as the prominent site of PKCα phosphorylation, and showed that phosphorylation at this site increased passive tension. Titin is an approximately 3000-kDa molecular-mass sarcomeric protein that spans the Z disk to the M line of the sarcomere.3 Originally thought to only provide structural scaffolding to link the many regulatory, contractile, and structural proteins within the sarcomere, titin is now recognized to be a major regulator of intracellular myocyte stiffness. Titin determines the passive tension of the intracellular component of cardiomyocytes, which, together with collagen, determines the total myocardial passive stiffness. Although the immunoglobulin-like domain and fibronectin type III repeats make up 90% of the titin molecule, titin also includes a unique I-band region that is flexible and specifically serves as a molecular spring to determine passive stiffness. …

Truncation of Titin’s Elastic PEVK Region Leads to Cardiomyopathy With Diastolic Dysfunction

The giant protein titin plays key roles in myofilament assembly and determines the passive mechanical properties of the sarcomere. The cardiac titin molecule has 2 mayor elastic elements, the N2B and the PEVK region. Both have been suggested to determine the elastic properties of the heart with loss of function data only available for the N2B region. The purpose of this study was to investigate the contribution of titin's proline-glutamate-valine-lysine (PEVK) region to biomechanics and growth of the heart. We removed a portion of the PEVK segment (exons 219 to 225; 282 aa) that corresponds to the PEVK element of N2B titin, the main cardiac titin isoform. Adult homozygous PEVK knockout (KO) mice developed diastolic dysfunction, as determined by pressure-volume loops, echocardiography, isolated heart experiments, and muscle mechanics. Immunoelectron microscopy revealed increased strain of the N2B element, a spring region retained in the PEVK-KO. Interestingly, the PEVK-KO mice had hypertrophied hearts with an induction of the hypertrophy and fetal gene response that includes upregulation of FHL proteins. This contrasts the cardiac atrophy phenotype with decreased FHL2 levels that result from the deletion of the N2B element. Titin's PEVK region contributes to the elastic properties of the cardiac ventricle. Our findings are consistent with a model in which strain of the N2B spring element and expression of FHL proteins trigger cardiac hypertrophy. These novel findings provide a molecular basis for the future differential therapy of isolated diastolic dysfunction versus more complex cardiomyopathies.

Targeted deletion of titin N2B region leads to diastolic dysfunction and cardiac atrophy

Titin is a giant protein that is in charge of the assembly and passive mechanical properties of the sarcomere. Cardiac titin contains a unique N2B region, which has been proposed to modulate elasticity of the titin filament and to be important for hypertrophy signaling and the ischemic stress response through its binding proteins FHL2 and alphaB-crystallin, respectively. To study the role of the titin N2B region in systole and diastole of the heart, we generated a knockout (KO) mouse deleting only the N2B exon 49 and leaving the remainder of the titin gene intact. The resulting mice survived to adulthood and were fertile. Although KO hearts were small, they produced normal ejection volumes because of an increased ejection fraction. FHL2 protein levels were significantly reduced in the KO mice, a finding consistent with the reduced size of KO hearts. Ultrastructural analysis revealed an increased extension of the remaining spring elements of titin (tandem Ig segments and the PEVK region), which, together with the reduced sarcomere length and increased passive tension derived from skinned cardiomyocyte experiments, translates to diastolic dysfunction as documented by echocardiography. We conclude from our work that the titin N2B region is dispensable for cardiac development and systolic properties but is important to integrate trophic and elastic functions of the heart. The N2B-KO mouse is the first titin-based model of diastolic dysfunction and, considering the high prevalence of diastolic heart failure, it could provide future mechanistic insights into the disease process.

Functional analysis of titin/connectin N2-B mutations found in cardiomyopathy

Hypertrophic cardiomyopathy and dilated cardiomyopathy are two major clinical phenotypes of "idiopathic" cardiomyopathy. Recent molecular genetic analyses have now revealed that "idiopathic" cardiomyopathy is caused by mutations in genes for sarcomere components. We have recently reported several mutations in titin/connectin gene found in patients with hypertrophic cardiomyopathy or dilated cardiomyopathy. A hypertrophic cardiomyopathy-associated titin/connectin mutation (Arg740Leu) was found to increase the binding to actinin, while other dilated cardiomyopathy-associated titin/connectin mutations (Ala743Val and Val54Met) decreased the binding to actinin and Tcap/telethonin, respectively. We also reported several other mutations in the N2-B region of titin/connectin found in hypertrophic cardiomyopathy and dilated cardiomyopathy. Since the N2-B region expresses only in the heart, it was speculated that functional alterations due to the mutations cause cardiomyopathies. In this study, we investigated the functional changes caused by the N2-B region mutations by using yeast-two-hybrid assays. It was revealed that a hypertrophic cardiomyopathy-associated mutation (Ser3799Tyr) increased the binding to FHL2 protein, whereas a dilated cardiomyopathy-associated mutation (Gln4053ter) decreased the binding. In addition, another TTN mutation (Arg25618Gln) at the is2 region was found in familial DCM. Because FHL2 protein is known to tether metabolic enzymes to N2-B and is2 regions of titin/connectin, these observations suggest that altered recruitment of metabolic enzymes to the sarcomere may play a role in the pathogenesis of cardiomyopathies.

Association of the Chaperone αB-crystallin with Titin in Heart Muscle

alphaB-crystallin, a major component of the vertebrate lens, is a chaperone belonging to the family of small heat shock proteins. These proteins form oligomers that bind to partially unfolded substrates and prevent denaturation. alphaB-crystallin in cardiac muscle binds to myofibrils under conditions of ischemia, and previous work has shown that the protein binds to titin in the I-band of cardiac fibers (Golenhofen, N., Arbeiter, A., Koob, R., and Drenckhahn, D. (2002) J. Mol. Cell. Cardiol. 34, 309-319). This part of titin extends as muscles are stretched and is made up of immunoglobulin-like modules and two extensible regions (N2B and PEVK) that have no well defined secondary structure. We have followed the position of alphaB-crystallin in stretched cardiac fibers relative to a known part of the titin sequence. alphaB-crystallin bound to a discrete region of the I-band that moved away from the Z-disc as sarcomeres were extended. In the physiological range of sarcomere lengths, alphaB-crystallin bound in the position of the N2B region of titin, but not to PEVK. In overstretched myofibrils, it was also in the Ig region between N2B and the Z-disc. Binding between alphaB-crystallin and N2B was confirmed using recombinant titin fragments. The Ig domains in an eight-domain fragment were stabilized by alphaB-crystallin; atomic force microscopy showed that higher stretching forces were needed to unfold the domains in the presence of the chaperone. Reversible association with alphaB-crystallin would protect I-band titin from stress liable to cause domain unfolding until conditions are favorable for refolding to the native state.

Subcellular targeting of metabolic enzymes to titin in heart muscle may be mediated by DRAL/FHL-2.

During sarcomere contraction skeletal and cardiac muscle cells consume large amounts of energy. To satisfy this demand, metabolic enzymes are associated with distinct regions of the sarcomeres in the I-band and in the M-band, where they help to maintain high local concentrations of ATP. To date, the mechanism by which metabolic enzymes are coupled to the sarcomere has not been elucidated. Here, we show that the four and a half LIM-only protein DRAL/FHL-2 mediates targeting of the metabolic enzymes creatine kinase, adenylate kinase and phosphofructokinase by interaction with the elastic filament protein titin in cardiomyocytes. Using yeast two-hybrid assays, colocalisation experiments, co-immunoprecipitation and protein pull-down assays, we show that DRAL/FHL-2 is bound to two distinct sites on titin. One binding site is situated in the N2B region, a cardiac-specific insertion in the I-band part of titin, and the other is located in the is2 region of M-band titin. We also show that DRAL/FHL-2 binds to the metabolic enzymes creatine kinase, adenylate kinase and phosphofructokinase and might target these enzymes to the N2B and is2 regions in titin. We propose that DRAL/FHL-2 acts as a specific adaptor protein to couple metabolic enzymes to sites of high energy consumption in the cardiac sarcomere.

Weakness of a giant: mutations of the sarcomeric protein titin

In 2002, three reports described for the first time mutations in the sarcomeric protein titin associated with dilated cardiomyopathy in humans. Despite different locations (Z-line region, Z-I transitional zone, N2B region, half A band region) all mutations resulted in heart failure. In addition, an N2B mutation was found in zebrafish embryos with ventricular dilatation and cardiac insufficiency. It is concluded that titin mutations have significant functional consequences and need to be studied intensively in the future.

Titin Mutations as the Molecular Basis for Dilated Cardiomyopathy

Dilated cardiomyopathy (DCM) is a heterogeneous cardiac disease characterized by ventricular dilatation and systolic dysfunction. Recent genetic studies have revealed that mutations in genes for cardiac sarcomere components lead to DCM. The cardiac sarcomere consists of thick and thin filaments and a giant protein, titin. Because one of the loci of familial DCM was mapped to the region of the titin gene, we searched for titin mutations in the patients and identified four possible disease-associated mutations. Two mutations, Val54Met and Ala743Val, were found in the Z-line region of titin and decreased binding affinities of titin to Z-line proteins T-cap/telethonin and α–actinin, respectively, in yeast two-hybrid assays. The other two mutations were found in the cardiac-specific N2-B region of titin and one of them was a nonsense mutation, Glu4053ter, presumably encoding for a truncated nonfunctional molecule. These observations suggest that titin mutations may cause DCM in a subset of the patients.

I-band titin in cardiac muscle is a three-element molecular spring and is critical for maintaining thin filament structure.

In cardiac muscle, the giant protein titin exists in different length isoforms expressed in the molecule's I-band region. Both isoforms, termed N2-A and N2-B, comprise stretches of Ig-like modules separated by the PEVK domain. Central I-band titin also contains isoform-specific Ig-motifs and nonmodular sequences, notably a longer insertion in N2-B. We investigated the elastic behavior of the I-band isoforms by using single-myofibril mechanics, immunofluorescence microscopy, and immunoelectron microscopy of rabbit cardiac sarcomeres stained with sequence-assigned antibodies. Moreover, we overexpressed constructs from the N2-B region in chick cardiac cells to search for possible structural properties of this cardiac-specific segment. We found that cardiac titin contains three distinct elastic elements: poly-Ig regions, the PEVK domain, and the N2-B sequence insertion, which extends approximately 60 nm at high physiological stretch. Recruitment of all three elements allows cardiac titin to extend fully reversibly at physiological sarcomere lengths, without the need to unfold Ig domains. Overexpressing the entire N2-B region or its NH(2) terminus in cardiac myocytes greatly disrupted thin filament, but not thick filament structure. Our results strongly suggest that the NH(2)-terminal N2-B domains are necessary to stabilize thin filament integrity. N2-B-titin emerges as a unique region critical for both reversible extensibility and structural maintenance of cardiac myofibrils.

Assembly of the cardiac I-band region of titin/connectin: expression of the cardiac-specific regions and their structural relation to the elastic segments.

The giant molecule titin (also called connectin) provides an elastic connection in the I-band between the Z-disk and A-band of striated muscle. This region is assembled in a tissue-specific way by extensive differential splicing events. We have raised monoclonal antibodies against the two N2-line isoforms of titin and demonstrate that both forms of cardiac I-band titin are constitutively co-expressed in atrial and ventricular muscle. In developing mouse embryos, the expression of the cardiac N2-B isoform remains strictly cardiac-specific and is linked to the expression of the ubiquitous N2-A isoform. The mechanical function of the cardiac N2-line region was investigated ultrastructurally. Immunoelectron microscopy reveals that the N2-B region separates two mechanically distinct sections of titin with a hyperextensible segment spanning the distance to the Z-disk. The formation of a plateau in the extension of cardiac titin rules out that Ig-domains can be unfolded as a mechanism of elasticity.