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Cardiac titin: Structure, functions and role in disease
Clinica Chimica Acta 375 (2007) 1 – 9 www.elsevier.com/locate/clinchim
Invited critical review
Cardiac titin: Structure, functions and role in disease☆ Martin M. LeWinter a,⁎, Yiming Wu b , Siegfried Labeit c , Henk Granzier b a
b
Department of Medicine and Cardiology Unit, University of Vermont, Burlington, VT, United States Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, WA, United States c Institut für Anästhesiologie und Operative Intensivmedizin, Universitätsklinikum Mannheim, Mannheim 68167, Germany Received 21 September 2005; received in revised form 21 June 2006; accepted 22 June 2006 Available online 7 July 2006
Abstract Titin is a giant sarcomeric protein found in both cardiac and skeletal muscle. In the heart, the structure, functions and role of titin in disease have begun to be elucidated over the last decade. Titin's N-terminus is anchored in the Z-disk while C-terminal domains are bound to the thick filament. The I-band segment is a complex molecular spring consisting of PEVK and tandem Ig segments as well as variable N2B and N2A elements. The latter determine titin's two isoforms. N2B alone is present in the smaller and stiffer N2B isoform and both N2A and N2B elements are present in the larger, more compliant N2BA isoform. Large mammals co-express both isoforms, while normal rodents have virtually exclusively N2B titin. With sarcomere stretch, titin's I-band segment elongates and develops passive tension. Titin is the predominant determinant of cardiomyocyte passive tension over the physiologic sarcomere length range. With contraction below slack length, the thick filament drags titin in the opposite direction such that extension of the spring results in generation of a restoring force resulting in elastic recoil. In addition to its mechanical properties, a role is emerging for titin as a major biomechanical sensing and signaling molecule. Moreover, recent studies indicate that titin undergoes dynamic isoform and possibly phosphorylation changes in disease. © 2006 Elsevier B.V. All rights reserved. Keywords: Titin; Passive myocardial stiffness; Restoring forces; Diastolic ventricular function
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . Structure of titin: relation to passive tension, restoring forces Modulation of titin-dependent passive tension . . . . . . . . 3.1. Isoform variation . . . . . . . . . . . . . . . . . . . 3.2. Phosphorylation . . . . . . . . . . . . . . . . . . . . 3.3. Ca2+ binding effects . . . . . . . . . . . . . . . . . . 4. Titin and active force development . . . . . . . . . . . . . . 5. Titin and biomechanical sensing . . . . . . . . . . . . . . . 6. Titin and heart disease . . . . . . . . . . . . . . . . . . . . 6.1. Adaptations and alterations during disease . . . . . . 6.2. Hereditary disease . . . . . . . . . . . . . . . . . . . 7. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Supported by NIH Grants HL61487 and HL62881. ⁎ Corresponding author. Cardiology Unit, Fletcher Allen Health Care, 111 Colchester Ave., Burlington, VT 05401, United States. Tel.: +1 802 847 2879; fax: +1 802 847 3637. E-mail address: [email protected] (M.M. LeWinter). 0009-8981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2006.06.035
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1. Introduction The giant sarcomeric protein titin found in both cardiac and skeletal muscle has been aptly termed the third filament of striated muscle. Although early electron microscopic studies during overstretch revealed the presence of additional filaments associated with the thick filaments [1–3], it was not until the 1980s that these were clearly identified as titin [4–8]. Contemporaneous with these observations, physiologists and cardiologists became increasingly interested in the determinants of the passive stiffness of the myocardium as it became evident that altered stiffness plays an important role in various forms of heart disease [9]. Before delineation of the functional role of titin, matrix collagen was thought to be the primary determinant of passive myocardial stiffness. Modern understanding of the structure and function of cardiac titin dates from the decade beginning in the late 1980s when a number of studies first elucidated its structure and detailed arrangement within the sarcomere as well as its elastic properties [10–19]. More recently, attention has focused on modulation of titin-based stiffness under physiologic and pathophysiologic conditions, the interaction of titin with shortening, and the potential role of titin in cell signaling. 2. Structure of titin: relation to passive tension, restoring forces and ventricular function Titin is the largest protein in mammals, ranging from 2970 to ∼ 3700 kDa depending on isoform composition. It is encoded by a single gene located on the long arm of chromosome 2, region 2q31 consisting of 363 exons [11,20–22]. As shown in Fig. 1, titin's N-terminus is anchored in the Z-disk and the protein extends to the M-line region of the sarcomere where it is bound to the myosin rod and also has attachments to or is associated with myosin binding protein C (not shown in figure). The near Zdisk segment in the I-band is bound to the thin filament. (As
discussed below, titin is also bound to multiple other proteins.) The behavior of titin as the sarcomere is passively stretched or shortens during contraction was delineated in large measure from immuno-electronmicroscopic experiments performed with antibodies to various epitopes [6,7,14,16–18]. In the absence of external forces, e.g. at slack length, the I-band region of titin is highly folded, with a near zero end-to-end length (Fig. 2, panel B). With stretch, much of the I-band segment gradually lengthens and functions as a spring, developing passive tension. In contrast, the A-band region as well as the near Z-disk I-band region is inextensible. The extensible I-band region is composed of multiple segments with differing spring properties, including tandemly arranged Ig segments and the PEVK segment, so named because it is rich in proline (P), glutamate (E), valine (V) and lysine (K). N2A (skeletal and cardiac muscle) and N2B (cardiac muscle only) segments function as additional spring elements [23–27]. The inextensible A-band segment of titin is composed of regular patterns of immunoglobulin (Ig)-like and fibronectin type 3 repeats. In some of the earliest studies delineating its physiologic role, degradation of skeletal muscle titin by ionizing radiation reduced the ability to generate passive tension [4]. Subsequent studies in which titin was degraded or dislodged from the thick filament by either gentle trypsin digestion or KCl treatment revealed that titin is the main determinant of cardiomyocyte passive tension over the physiologic sarcomere length (SL) range [13,14,16,24,25,28–30]). The behavior of the spring elements of titin during extension is complex, with Ig segments extending before PEVK and N2B/N2A segments (Fig. 2, panels C,D). This gives rise to distinct segments of the cardiomyocyte passive tension–SL relation, which is shallow near slack length but much steeper at longer SLs (Fig. 2) [16,25,28–30]. With contraction of the sarcomere below slack length the thick filament moves into the near Z-disk region of titin (Fig. 2) [31,32]. As a result, the extensible region is stretched in the opposite direction as that during passive elongation and a
Fig. 1. The positioning and structure of cardiac titin within the sarcomere (see text). Reprinted by permission of the American Heart Association (Granzier H, Labeit S, Circ Res 2004; 94: 284–95). See text for abbreviations.
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Fig. 2. Stretching of the sarcomere from slack length (panel B) results in sequential extension of tandem Ig (panel C) and then PEVK and N2B elements (panel C), each of which is responsible for a segment of the sarcomere length (SL)–passive force relation (right). Shortening below slack length (panel A) results in reverse extension of titin with development of a restoring force. Reprinted by permission of the American Heart Association (Granzier H, Labeit S, Circ Res 2004; 94: 284–95).
restoring force is established that pushes the Z-disks away from each other during relaxation. Thus, titin functions as a bidirectional spring and accounts for the bulk of the restoring force responsible for cardiomyocyte recoil after unloaded contraction. Myosin binding protein C may also play a role in the functional properties of titin in relation to passive stiffness. As indicated earlier, myosin binding protein C is closely associated with titin's myosin binding sites. In a transgenic mouse that fails to incorporate myosin binding protein C in the sarcomere because of a homozygous myosin binding protein C deletion mutation, we reported a dilated cardiomyopathy phenotype with a striking decrease in passive diastolic chamber stiffness [33]. These results suggest that the presence of myosin binding protein has an important influence on generation of passive tension by titin. Titin and collagen jointly determine passive tension during myocardial stretch and diastolic pressure during ventricular filling (Fig. 3) [34]. The relative contributions of the two have been delineated in detergent-treated (“skinned”) myocardial strips through the use of protocols similar to those employed in cardiomyocytes, where passive tension is measured before and after titin is chemically detached from the thick filament. At relatively short SLs in normal myocardium most passive tension is accounted for by titin. At longer SLs the influence of collagen progressively increases and is roughly equivalent with titin in determining passive tension. In view of its role in cardiac myocyte and myocardial passive tension and restoring forces, titin is certainly also a major
determinant of diastolic ventricular properties. In addition to determining passive chamber stiffness, a critical determinant of ventricular filling, the restoring forces generated by titin with contraction below slack length likely contribute toward the phenomenon of ventricular diastolic suction [27]. The latter results in part from reversal of complex three-dimensional deformation patterns (i.e., recoil) at the level of the ventricle and is an important mechanism of filling during early diastole, especially under conditions such as exercise where filling rate must be markedly increased. Finally, titin also plays a key role in determining the ventricular Frank–Starling relation, since it determines the amount of sarcomere stretch that occurs as the ventricle fills. 3. Modulation of titin-dependent passive tension 3.1. Isoform variation ∼90% of the titin gene exons are organized into modular repeats, including the Ig and PEVK segments [11,35,36] (Fig. 4, panel A). Exons 49–224 encode titin's central I-band region, with exon 49 accounting for the N2B spring element, 50–101 for tandemly repeated Ig segments, 102–108 for the N2A element and 109–224 for the PEVK region. This region undergoes complex differential splicing which provides for variable inclusion of the spring elements and corresponding variability of titin's spring properties. All titin isoforms contain the following elements [23,35,36]: 15 Ig domains near the Z-disk (the proximal tandem Ig segment), 22 Ig domains close to the A-band (the distal tandem Ig
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segment), and a ∼180 residue PEVK segment. Besides additional Ig and PEVK segments all skeletal muscle titin contains the N2A element. There are two classes of cardiac titin isoforms, the smaller 2970 kDa isoform known as N2B titin which contains the N2B element, and the larger N2BA isoform which contains both N2B and N2A elements as well as additional PEVK and a variable number of additional Ig domains (Fig. 4, panel B) [22–26,32]. N2BA isoforms range from ∼3200 to ∼3400 kDa. Large mammals co-express both isoform classes at the level of the halfsarcomere (Fig. 4, panel C) [23]. The atria normally express more N2BA than the ventricles and there are also normal transmural ventricular isoform gradients [25,27]. The large size of titin makes precise delineation of different isoforms with relatively small molecular weight differences very difficult, even with high resolution gel methods. Recently, the latter were combined with an exon microarray system that allows delineation of the simultaneous expression of all 363 exons simultaneously [37]. This approach provided more detailed insights into differential splicing of titin. A notable finding was that fetal myocardium contains a unique, large N2BA isoform that gradually disappears in post-natal life with a species-dependent time course (Fig. 5). The fetal isoform is associated with reduced cardiomyocyte passive tension. Because of their additional I-band spring elements N2BA titins are much less stiff than N2B titin [24,25,32]. Correspondingly, a given degree of sarcomere stretch results in less extension of
N2BA compared with N2B titin. As a result, cardiomyocytes expressing high proportions of the N2B isoform (e.g., from rodents, which have nearly exclusively N2B titin) have greater passive stiffness than those with appreciable N2BA titin (such as large mammals) [24]. Correspondingly, ventricular chamber stiffness is higher in rodents than in large mammals. High N2B titin expressing cardiomyocytes should also have greater restoring forces. It has been suggested that high proportions of N2B titin serve the function of allowing animals such as rodents who have very rapid heart rates to fill rapidly during diastole because of larger restoring forces [31]. The variable expression ratios of N2A and N2BA titin in large mammals provides for the possibility of gradations of passive stiffness (Fig. 6). It is also interesting to note that in different species collagen-dependent stiffness tends to parallel titin-dependent stiffness [34]. Thus, higher myocardial and ventricular stiffness in rodents is due to both higher titin- and higher collagen-dependent components of stiffness. 3.2. Phosphorylation The N2B sequence of cardiac titin functions as a substrate for protein kinase A (PKA) [38]. PKA-mediated phosphorylation of titin decreases passive tension (Fig. 6), as shown by Fukuda et al. [39] in intact and skinned right ventricular trabeculae treated with isoproterenol. It has been suggested that phosphorylation increases the functional length of the N2B element resulting in less fractional
Fig. 3. The contributions of titin and collagen to passive myocardial tension in mouse left ventricle (LV) (panel A) and dog LV (panel B). The fractional contributions of titin and collagen as a function of SL are shown in panel C for mouse (MLV) and dog (DLV). Increases in passive tension in conjunction with an increase in the ratio of N2B to N2BA titin in a model of tachycardia heart failure in the dog are shown in panel D. Reprinted by permission of the American Heart Association (Granzier H, Labeit S, Circ Res 2004; 94: 284–95).
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effect requires the presence of E-rich motifs containing ∼30– 50% of glutamic acid residues. Ca2+ binding experiments in skinned, E-rich mouse soleus muscle fibers following extraction of thin filaments reveal an augmented passive force response during stretch, suggesting a physiologically significant effect. In cardiac muscle, E-rich motifs are found in N2BA but not N2B titin isoforms. This is consistent with our finding that Ca2+ binding increases the passive stiffness of N2BA expressing myocardium, but has no effect on exclusively N2B myocardium (Fig. 6) [42]. Ca2+ also may have an indirect effect on passive force by modulating the interaction between titin and the thin filament. Thus, Yamasaki et al. [43] found that the PEVK domain in the extensible region of N2B titin binds to F-actin. This interaction may serve to retard thin filament sliding and contribute to passive cardiomyocyte passive stiffness (Fig. 6). While physiologic [Ca2+] does not influence the titin–actin interaction S100A1, a soluble Ca2+ binding protein found in high concentration in myocardium, inhibits the interaction in a Ca2+-dependent fashion. Physiologic significance of the titin–actin interaction is suggested by the finding that the stiffness of rat cardiac trabeculae decreases during diastole in parallel with the decline in [Ca2+] [44]. It is not known whether the PEVK segment of N2BA titin also interacts with actin, but experiments by Campbell et al. [45] suggest that the effect may be specific for N2B titin.
Fig. 4. The titin gene is outlined in panel A (see text). Panel B shows differences in the two major isoforms (see text). Panel C shows titin gels from bovine atrium and ventricle and rat left ventricle (LV) as well as immuno-electronmicroscopy demonstrating co-expression of both isoforms in bovine ventricle. Note that bovine atrium contains almost exclusively N2BA titin and rat LV almost exclusively N2B titin. T2 is a commonly observed faint band representing proteolysed titin. Reprinted by permission of the American Heart Association (Granzier H, Labeit S, Circ Res 2004; 94: 284–95).
extension at a given SL [38]. Reduction in passive tension by PKA phosphorylation is most pronounced in muscles with high expression levels of N2B titin (Fig. 6), which is consistent with the increased N2B length hypothesis [39]. It is uncertain how important PKA phosphorylation of titin is under physiologic conditions in the ventricle. We reported that intra-coronary dobutamine results in downward displacement of the fully-relaxed left ventricular pressure–length relation [40]. This result suggests that passive ventricular chamber stiffness can indeed be modulated by adrenergically-mediated phosphorylation of titin. 3.3. Ca2+ binding effects Recent experiments show that Ca2+ binding to titin directly lowers the bending rigidity of the PEVK segment [41]. This
Fig. 5. Microarray system used to delineate fetal N2BA titin isoform and associated fetal specific exons (panel A). High resolution gels were used to detect the large fetal isoform, which is associated with reduced passive tension of fetal cardiomyocytes (panel B). Reprinted by permission of the American Heart Association (Granzier H, Labeit S, Circ Res 2004; 94: 284–95).
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Fig. 6. Illustration of the difference in passive tension of cardiomyocytes containing varying proportions of N2B and N2BA titin, as well as other mechanisms whereby passive tension caused by titin can be modulated. Skeletal muscle passive tension is shown for comparison (see text). Reprinted by permission of the American Heart Association (Granzier H, Labeit S, Circ Res 2004; 94: 284–95).
4. Titin and active force development Recent studies suggest that titin modulates the SL-dependent change in Ca2+ sensitivity of active force that is an important component of the Frank–Starling relation, i.e., Ca2+ sensitivity increases in proportion to titin-dependent passive force [46,47]. Titin degradation with trypsin digestion reverses this effect [48]. Interestingly, the effect of titin on active force is most prominent in myocardium with high levels of titin-based passive force. It has been proposed that this effect is mediated by altering interfilament lattice spacing and/or effecting strains on the thick filament that influence cross-bridge motility [49]. Additional recent experiments suggest that titin's effects on the actomyosin interaction may be SL-dependent, with inhibitory effects at slack SLs or below and enhancement at SLs greater than slack length [48,50]. There are a number of unanswered questions about the interaction of titin with cross-bridge formation, suggesting that this should be a fruitful area of future investigation. 5. Titin and biomechanical sensing Recent evidence indicates that titin may function as both a key biomechanical sensor and an organizational element within the sarcomere. A priori, in view of its size and structural position within the sarcomere and its function in relation to passive, restoring and active forces titin is a logical candidate in this regard. A large number of structural and signaling proteins bind to titin (Fig. 1). Binding sites are found in and near the Z-disk, in the central I-band and the M-line regions. The Z-disk titincapping protein T-cap, or telethionin, binds to titin's N-terminus and links titin to signaling and structural molecules [51,52]. Tcap interacts with cytoplasmic domains of two membrane associated proteins, the K channel subunit minK/isk in the Ttubules [53] and small ankyrin-1 (sANK-1), a transmembrane sarcoplasmic reticulum (SR) protein [54]. The minK/isk interaction may function to modulate K channel function in response to stretch while the sANK-1 interaction may serve to fix the position of the SR around the Z-disk. An interaction between titin's near Z-disk domains and obscurin [55] suggests an additional role for titin in relation to organization of the SR
since obscurin in turn interacts with ankyrin isoform 1.5 [55,56]. The latter appears to link the SR to the sarcomere and regulate ryanodine receptor distribution in the SR [57]. These Z-disk region interactions suggest a general role for titin in positioning SR and T-tubule systems in close proximity to the I-band and perhaps ensuring that these systems move with the Z-disk, which may be important to their function and integrity. T-cap also interacts with muscle LIM protein (MLP) [58], a nuclear regulator of myogenic differentiation. Stretch-induced release of brain and atrial natriuretic factors by cardiomyocytes is absent in a MLP knockout mouse, suggesting a role for MLP in stretch sensing which could also be related to the dilated cardiomyopathy present in these animals [59]. The N2B and N2A elements in the central I-band region also appear to function in organization of the sarcomere and signaling. N2B interacts with the LIM-family protein DRAL/FHL-2, which binds the key metabolic enzymes creatine kinase, adenylate cyclase and phosphofructokinase [60]. Thus, titin may be involved in compartmentalization of these enzymes such that they are located in close proximity to sites where ATP utilization is high. The N2B element also interacts with the small heat shock protein αβcrystallin [61] which functions to maintain normal protein folding. Ig domains of the N2A element in the N2BA isoform as well as in skeletal muscle titin interact with calpain protease P94, and may modulate the function of P94 in protein degradation [62]. Additional Ig segments bind to three homologous ankyrin repeat proteins, cardiac ankyrin repeat protein (CARP), ankrd2 (or Arpp) and diabetes ankyrin repeat protein (DARP) [63]. One or more of these ankyrin repeat proteins are induced by cardiac injury, skeletal muscle denervation and stretch, and metabolic challenge [64–66]. Interestingly, levels of all three are increased in myocardium of patients with dilated cardiomyopathy [67]. Thus, these ankyrin repeat proteins may be a component of muscle stress responses in general. The I-band ligands of titin and their associated binding partners are also found in the nucleus, where they function in transcriptional and cell cycle regulation. It is intriguing to consider that this dual localization may link titin's cytoplasmic stretch-based sensing to gene expression via transcriptional regulation. Along these lines, a naturally occurring “muscular dystrophy with myositis” mouse has been found to contain an 83 amino acid deletion mutation in the N2A element along with marked changes in the expression of MLP, CARP and MURF-1 (a titin-ligand M-line protein) [68,69]. Thus, this deletion mutation appears to affect ligands in multiple domains. The M-line region of titin also contains a number of sites that may be involved in sensing and signaling. These include a serine/ threonine kinase domain [70], another DRAL/FHL-2 site [60], possible additional T-Cap and obscurin sites [55,71] and a binding site for the RING finger protein MURF-1 (muscle specific ring finger protein-1) [72]. MURF-1 in particular may be involved in multiple signaling pathways. MURF-1 is a sarcomere-associated skeletal and cardiac muscle protein that functions as an E3 ubiquitin ligase that conjugates ubiquitin moieties to proteins destined for proteolysis [73]. Thus, MURF-1 may play a regulatory role in degradation of muscle proteins. MURF-1 also binds to a number of other myofibrillar proteins including
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troponin I, which results in increased degradation and possibly modulation of contractility by MURF-1 [74]. Finally, it has been proposed that MURF-1 plays a role in hypertrophic signaling [75]. All of these relatively recent and as yet largely preliminary reports implicate diverse roles for titin in biomechanical sensing and signaling. This suggests that the protein has complex and important integrative functions beyond its purely mechanical properties and constitutes an exciting area for future research. 6. Titin and heart disease 6.1. Adaptations and alterations during disease Two early reports suggested a decrease in titin content in failing hearts [76,77]. In retrospect this may have resulted from unrecognized proteolysis after tissue removal, to which titin is very susceptible. However, because of its size titin is a good candidate not just for proteolysis but for other forms of damage in vivo, for example oxidation by free radicals, glycation and Xirradiation. Although there is little information available in this area, a recent report showed that titin is damaged early during the course of experimental exposure to the cancer chemotherapeutic drug doxorubicin in association with activation of calcium-dependent calpain proteolysis [78]. (Doxorubicin treatment is often associated with clinical cardiomyopathy.) Damage to titin in the course of various diseases is an area that also appears ripe for future investigations. Several investigators have determined isoform content in failing myocardium. We examined the pacing tachycardia model of dilated cardiomyopathy in dogs. After only two weeks of pacing, we detected exaggeration of the normal transmural N2B/N2BA isoform ratio gradient [27]. After four weeks of pacing, when decompensation is generally present, we reported an increase in the ratio of N2B/N2BA isoforms which, as would be predicted, was associated with increased titin-based myocardial stiffness as well as increased collagen-based stiffness [79]. In a recent paper [80], spontaneously hypertensive rats were reported to have reduced N2BA titin expression, which is consistent with increased passive stiffness of isolated cardiomyocytes from these animals. Titin isoform distribution has also been studied in explanted hearts from patients with end-stage, dilated cardiomyopathy. Here also changes in isoform distribution are reported but differ from those in animal models. Thus, in patients with ischemic cardiomyopathy the proportion of the more compliant N2BA isoform was found to be increased [81]. Because tissue studies in ischemic cardiomyopathy are potentially confounded by regional variations, it is of note that recent studies in hearts from patients with non-ischemic cardiomyopathy showed the same change in isoform ratio [67,82]. The isoform ratio was also correlated with echocardiographic-Doppler findings consistent with reduced myocardial stiffness [67]. In contrast to changes in several other cardiac proteins in heart failure, the increases in N2BA titin in end-stage, failing human hearts do not reflect reexpression of specifically fetal–neonatal isoforms. Although the observed changes in isoform ratios in both pacing tachycardia in dogs and human dilated cardiomyopathy were not very large, the
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difference in stiffness between N2B and N2BA isoforms is so marked that measurable changes in cardiomyocyte stiffness would be expected nonetheless. The reason(s) for the apparent difference in the nature of alterations in titin isoforms in experimental animals and humans is uncertain, but several possibilities may be suggested. Thus, it is possible that end-stage hearts are not representative of the spectrum of failing myocardium in humans and different results might be found in less severely diseased hearts. Alternatively, the pacing tachycardia model may be a specialized case that cannot be extrapolated to other forms of heart failure. Obviously, this could also represent a true biologic difference between species. Another interesting possibility in failing myocardium is that disease-associated changes in phosphorylation of titin may influence cardiomyocyte and myocardial stiffness, especially since end-stage, dilated heart failure is accompanied by marked depression of PKA activity. A fascinating recent report by Borbely et al. in patients with non-dilated, diastolic heart failure (i.e., heart failure with preserved contraction) demonstrated increased cardiomyocyte passive stiffness that was reversed by PKA treatment [83]. Increased ventricular chamber stiffness is the rule in these patients, who constitute somewhere between 40–50% of the heart failure population. These early reports in acquired forms of heart disease suggest the possibility of dynamic changes in the influence of titin on passive stiffness in disease as well as the possibility of a wide variety of other effects related to its role in mechanical sensing and signaling. 6.2. Hereditary disease Because of its size and diverse functions, titin would seem a prime candidate for disease causing mutations. Earlier linkage studies indeed identified titin as a candidate gene for certain inherited forms of dilated cardiomyopathy, but specific mutations were not detected on sequencing of a small number of exons [84]. The completion of titin's genomic structure has facilitated the search for mutations (Fig. 4). Thus, recent studies have identified two titin mutations that cause DCM (exons 18 and 326) [85]. Following sequencing of the entire gene, a mutation in exon 363 was reported in patients with a skeletal muscle dystrophy linked to titin (tibialis muscular dystrophy) [86]. Finally, a sequencing study recently identified two missense mutations in exons 2 and 14 in patients with hypertrophic cardiomyopathy [87]. It is intriguing that to date disease-causing titin mutations have been identified in regions present in both cardiac and skeletal muscle, yet phenotypic changes have been evident in only one muscle type. The ability to now sequence the entire gene should allow a full elucidation of the role of titin in genetically determined heart and skeletal muscle disease. 7. Summary The last decade has seen a rapid increase in our understanding of titin, a protein whose functions were unknown previously. Titin appears to have diverse and critical roles in the mechanical, organizational and signaling properties of cardiac and skeletal
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Invited critical review
Cardiac titin: Structure, functions and role in disease☆ Martin M. LeWinter a,⁎, Yiming Wu b , Siegfried Labeit c , Henk Granzier b a
b
Department of Medicine and Cardiology Unit, University of Vermont, Burlington, VT, United States Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, WA, United States c Institut für Anästhesiologie und Operative Intensivmedizin, Universitätsklinikum Mannheim, Mannheim 68167, Germany Received 21 September 2005; received in revised form 21 June 2006; accepted 22 June 2006 Available online 7 July 2006
Abstract Titin is a giant sarcomeric protein found in both cardiac and skeletal muscle. In the heart, the structure, functions and role of titin in disease have begun to be elucidated over the last decade. Titin's N-terminus is anchored in the Z-disk while C-terminal domains are bound to the thick filament. The I-band segment is a complex molecular spring consisting of PEVK and tandem Ig segments as well as variable N2B and N2A elements. The latter determine titin's two isoforms. N2B alone is present in the smaller and stiffer N2B isoform and both N2A and N2B elements are present in the larger, more compliant N2BA isoform. Large mammals co-express both isoforms, while normal rodents have virtually exclusively N2B titin. With sarcomere stretch, titin's I-band segment elongates and develops passive tension. Titin is the predominant determinant of cardiomyocyte passive tension over the physiologic sarcomere length range. With contraction below slack length, the thick filament drags titin in the opposite direction such that extension of the spring results in generation of a restoring force resulting in elastic recoil. In addition to its mechanical properties, a role is emerging for titin as a major biomechanical sensing and signaling molecule. Moreover, recent studies indicate that titin undergoes dynamic isoform and possibly phosphorylation changes in disease. © 2006 Elsevier B.V. All rights reserved. Keywords: Titin; Passive myocardial stiffness; Restoring forces; Diastolic ventricular function
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . Structure of titin: relation to passive tension, restoring forces Modulation of titin-dependent passive tension . . . . . . . . 3.1. Isoform variation . . . . . . . . . . . . . . . . . . . 3.2. Phosphorylation . . . . . . . . . . . . . . . . . . . . 3.3. Ca2+ binding effects . . . . . . . . . . . . . . . . . . 4. Titin and active force development . . . . . . . . . . . . . . 5. Titin and biomechanical sensing . . . . . . . . . . . . . . . 6. Titin and heart disease . . . . . . . . . . . . . . . . . . . . 6.1. Adaptations and alterations during disease . . . . . . 6.2. Hereditary disease . . . . . . . . . . . . . . . . . . . 7. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Supported by NIH Grants HL61487 and HL62881. ⁎ Corresponding author. Cardiology Unit, Fletcher Allen Health Care, 111 Colchester Ave., Burlington, VT 05401, United States. Tel.: +1 802 847 2879; fax: +1 802 847 3637. E-mail address: [email protected] (M.M. LeWinter). 0009-8981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2006.06.035
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1. Introduction The giant sarcomeric protein titin found in both cardiac and skeletal muscle has been aptly termed the third filament of striated muscle. Although early electron microscopic studies during overstretch revealed the presence of additional filaments associated with the thick filaments [1–3], it was not until the 1980s that these were clearly identified as titin [4–8]. Contemporaneous with these observations, physiologists and cardiologists became increasingly interested in the determinants of the passive stiffness of the myocardium as it became evident that altered stiffness plays an important role in various forms of heart disease [9]. Before delineation of the functional role of titin, matrix collagen was thought to be the primary determinant of passive myocardial stiffness. Modern understanding of the structure and function of cardiac titin dates from the decade beginning in the late 1980s when a number of studies first elucidated its structure and detailed arrangement within the sarcomere as well as its elastic properties [10–19]. More recently, attention has focused on modulation of titin-based stiffness under physiologic and pathophysiologic conditions, the interaction of titin with shortening, and the potential role of titin in cell signaling. 2. Structure of titin: relation to passive tension, restoring forces and ventricular function Titin is the largest protein in mammals, ranging from 2970 to ∼ 3700 kDa depending on isoform composition. It is encoded by a single gene located on the long arm of chromosome 2, region 2q31 consisting of 363 exons [11,20–22]. As shown in Fig. 1, titin's N-terminus is anchored in the Z-disk and the protein extends to the M-line region of the sarcomere where it is bound to the myosin rod and also has attachments to or is associated with myosin binding protein C (not shown in figure). The near Zdisk segment in the I-band is bound to the thin filament. (As
discussed below, titin is also bound to multiple other proteins.) The behavior of titin as the sarcomere is passively stretched or shortens during contraction was delineated in large measure from immuno-electronmicroscopic experiments performed with antibodies to various epitopes [6,7,14,16–18]. In the absence of external forces, e.g. at slack length, the I-band region of titin is highly folded, with a near zero end-to-end length (Fig. 2, panel B). With stretch, much of the I-band segment gradually lengthens and functions as a spring, developing passive tension. In contrast, the A-band region as well as the near Z-disk I-band region is inextensible. The extensible I-band region is composed of multiple segments with differing spring properties, including tandemly arranged Ig segments and the PEVK segment, so named because it is rich in proline (P), glutamate (E), valine (V) and lysine (K). N2A (skeletal and cardiac muscle) and N2B (cardiac muscle only) segments function as additional spring elements [23–27]. The inextensible A-band segment of titin is composed of regular patterns of immunoglobulin (Ig)-like and fibronectin type 3 repeats. In some of the earliest studies delineating its physiologic role, degradation of skeletal muscle titin by ionizing radiation reduced the ability to generate passive tension [4]. Subsequent studies in which titin was degraded or dislodged from the thick filament by either gentle trypsin digestion or KCl treatment revealed that titin is the main determinant of cardiomyocyte passive tension over the physiologic sarcomere length (SL) range [13,14,16,24,25,28–30]). The behavior of the spring elements of titin during extension is complex, with Ig segments extending before PEVK and N2B/N2A segments (Fig. 2, panels C,D). This gives rise to distinct segments of the cardiomyocyte passive tension–SL relation, which is shallow near slack length but much steeper at longer SLs (Fig. 2) [16,25,28–30]. With contraction of the sarcomere below slack length the thick filament moves into the near Z-disk region of titin (Fig. 2) [31,32]. As a result, the extensible region is stretched in the opposite direction as that during passive elongation and a
Fig. 1. The positioning and structure of cardiac titin within the sarcomere (see text). Reprinted by permission of the American Heart Association (Granzier H, Labeit S, Circ Res 2004; 94: 284–95). See text for abbreviations.
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Fig. 2. Stretching of the sarcomere from slack length (panel B) results in sequential extension of tandem Ig (panel C) and then PEVK and N2B elements (panel C), each of which is responsible for a segment of the sarcomere length (SL)–passive force relation (right). Shortening below slack length (panel A) results in reverse extension of titin with development of a restoring force. Reprinted by permission of the American Heart Association (Granzier H, Labeit S, Circ Res 2004; 94: 284–95).
restoring force is established that pushes the Z-disks away from each other during relaxation. Thus, titin functions as a bidirectional spring and accounts for the bulk of the restoring force responsible for cardiomyocyte recoil after unloaded contraction. Myosin binding protein C may also play a role in the functional properties of titin in relation to passive stiffness. As indicated earlier, myosin binding protein C is closely associated with titin's myosin binding sites. In a transgenic mouse that fails to incorporate myosin binding protein C in the sarcomere because of a homozygous myosin binding protein C deletion mutation, we reported a dilated cardiomyopathy phenotype with a striking decrease in passive diastolic chamber stiffness [33]. These results suggest that the presence of myosin binding protein has an important influence on generation of passive tension by titin. Titin and collagen jointly determine passive tension during myocardial stretch and diastolic pressure during ventricular filling (Fig. 3) [34]. The relative contributions of the two have been delineated in detergent-treated (“skinned”) myocardial strips through the use of protocols similar to those employed in cardiomyocytes, where passive tension is measured before and after titin is chemically detached from the thick filament. At relatively short SLs in normal myocardium most passive tension is accounted for by titin. At longer SLs the influence of collagen progressively increases and is roughly equivalent with titin in determining passive tension. In view of its role in cardiac myocyte and myocardial passive tension and restoring forces, titin is certainly also a major
determinant of diastolic ventricular properties. In addition to determining passive chamber stiffness, a critical determinant of ventricular filling, the restoring forces generated by titin with contraction below slack length likely contribute toward the phenomenon of ventricular diastolic suction [27]. The latter results in part from reversal of complex three-dimensional deformation patterns (i.e., recoil) at the level of the ventricle and is an important mechanism of filling during early diastole, especially under conditions such as exercise where filling rate must be markedly increased. Finally, titin also plays a key role in determining the ventricular Frank–Starling relation, since it determines the amount of sarcomere stretch that occurs as the ventricle fills. 3. Modulation of titin-dependent passive tension 3.1. Isoform variation ∼90% of the titin gene exons are organized into modular repeats, including the Ig and PEVK segments [11,35,36] (Fig. 4, panel A). Exons 49–224 encode titin's central I-band region, with exon 49 accounting for the N2B spring element, 50–101 for tandemly repeated Ig segments, 102–108 for the N2A element and 109–224 for the PEVK region. This region undergoes complex differential splicing which provides for variable inclusion of the spring elements and corresponding variability of titin's spring properties. All titin isoforms contain the following elements [23,35,36]: 15 Ig domains near the Z-disk (the proximal tandem Ig segment), 22 Ig domains close to the A-band (the distal tandem Ig
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segment), and a ∼180 residue PEVK segment. Besides additional Ig and PEVK segments all skeletal muscle titin contains the N2A element. There are two classes of cardiac titin isoforms, the smaller 2970 kDa isoform known as N2B titin which contains the N2B element, and the larger N2BA isoform which contains both N2B and N2A elements as well as additional PEVK and a variable number of additional Ig domains (Fig. 4, panel B) [22–26,32]. N2BA isoforms range from ∼3200 to ∼3400 kDa. Large mammals co-express both isoform classes at the level of the halfsarcomere (Fig. 4, panel C) [23]. The atria normally express more N2BA than the ventricles and there are also normal transmural ventricular isoform gradients [25,27]. The large size of titin makes precise delineation of different isoforms with relatively small molecular weight differences very difficult, even with high resolution gel methods. Recently, the latter were combined with an exon microarray system that allows delineation of the simultaneous expression of all 363 exons simultaneously [37]. This approach provided more detailed insights into differential splicing of titin. A notable finding was that fetal myocardium contains a unique, large N2BA isoform that gradually disappears in post-natal life with a species-dependent time course (Fig. 5). The fetal isoform is associated with reduced cardiomyocyte passive tension. Because of their additional I-band spring elements N2BA titins are much less stiff than N2B titin [24,25,32]. Correspondingly, a given degree of sarcomere stretch results in less extension of
N2BA compared with N2B titin. As a result, cardiomyocytes expressing high proportions of the N2B isoform (e.g., from rodents, which have nearly exclusively N2B titin) have greater passive stiffness than those with appreciable N2BA titin (such as large mammals) [24]. Correspondingly, ventricular chamber stiffness is higher in rodents than in large mammals. High N2B titin expressing cardiomyocytes should also have greater restoring forces. It has been suggested that high proportions of N2B titin serve the function of allowing animals such as rodents who have very rapid heart rates to fill rapidly during diastole because of larger restoring forces [31]. The variable expression ratios of N2A and N2BA titin in large mammals provides for the possibility of gradations of passive stiffness (Fig. 6). It is also interesting to note that in different species collagen-dependent stiffness tends to parallel titin-dependent stiffness [34]. Thus, higher myocardial and ventricular stiffness in rodents is due to both higher titin- and higher collagen-dependent components of stiffness. 3.2. Phosphorylation The N2B sequence of cardiac titin functions as a substrate for protein kinase A (PKA) [38]. PKA-mediated phosphorylation of titin decreases passive tension (Fig. 6), as shown by Fukuda et al. [39] in intact and skinned right ventricular trabeculae treated with isoproterenol. It has been suggested that phosphorylation increases the functional length of the N2B element resulting in less fractional
Fig. 3. The contributions of titin and collagen to passive myocardial tension in mouse left ventricle (LV) (panel A) and dog LV (panel B). The fractional contributions of titin and collagen as a function of SL are shown in panel C for mouse (MLV) and dog (DLV). Increases in passive tension in conjunction with an increase in the ratio of N2B to N2BA titin in a model of tachycardia heart failure in the dog are shown in panel D. Reprinted by permission of the American Heart Association (Granzier H, Labeit S, Circ Res 2004; 94: 284–95).
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effect requires the presence of E-rich motifs containing ∼30– 50% of glutamic acid residues. Ca2+ binding experiments in skinned, E-rich mouse soleus muscle fibers following extraction of thin filaments reveal an augmented passive force response during stretch, suggesting a physiologically significant effect. In cardiac muscle, E-rich motifs are found in N2BA but not N2B titin isoforms. This is consistent with our finding that Ca2+ binding increases the passive stiffness of N2BA expressing myocardium, but has no effect on exclusively N2B myocardium (Fig. 6) [42]. Ca2+ also may have an indirect effect on passive force by modulating the interaction between titin and the thin filament. Thus, Yamasaki et al. [43] found that the PEVK domain in the extensible region of N2B titin binds to F-actin. This interaction may serve to retard thin filament sliding and contribute to passive cardiomyocyte passive stiffness (Fig. 6). While physiologic [Ca2+] does not influence the titin–actin interaction S100A1, a soluble Ca2+ binding protein found in high concentration in myocardium, inhibits the interaction in a Ca2+-dependent fashion. Physiologic significance of the titin–actin interaction is suggested by the finding that the stiffness of rat cardiac trabeculae decreases during diastole in parallel with the decline in [Ca2+] [44]. It is not known whether the PEVK segment of N2BA titin also interacts with actin, but experiments by Campbell et al. [45] suggest that the effect may be specific for N2B titin.
Fig. 4. The titin gene is outlined in panel A (see text). Panel B shows differences in the two major isoforms (see text). Panel C shows titin gels from bovine atrium and ventricle and rat left ventricle (LV) as well as immuno-electronmicroscopy demonstrating co-expression of both isoforms in bovine ventricle. Note that bovine atrium contains almost exclusively N2BA titin and rat LV almost exclusively N2B titin. T2 is a commonly observed faint band representing proteolysed titin. Reprinted by permission of the American Heart Association (Granzier H, Labeit S, Circ Res 2004; 94: 284–95).
extension at a given SL [38]. Reduction in passive tension by PKA phosphorylation is most pronounced in muscles with high expression levels of N2B titin (Fig. 6), which is consistent with the increased N2B length hypothesis [39]. It is uncertain how important PKA phosphorylation of titin is under physiologic conditions in the ventricle. We reported that intra-coronary dobutamine results in downward displacement of the fully-relaxed left ventricular pressure–length relation [40]. This result suggests that passive ventricular chamber stiffness can indeed be modulated by adrenergically-mediated phosphorylation of titin. 3.3. Ca2+ binding effects Recent experiments show that Ca2+ binding to titin directly lowers the bending rigidity of the PEVK segment [41]. This
Fig. 5. Microarray system used to delineate fetal N2BA titin isoform and associated fetal specific exons (panel A). High resolution gels were used to detect the large fetal isoform, which is associated with reduced passive tension of fetal cardiomyocytes (panel B). Reprinted by permission of the American Heart Association (Granzier H, Labeit S, Circ Res 2004; 94: 284–95).
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Fig. 6. Illustration of the difference in passive tension of cardiomyocytes containing varying proportions of N2B and N2BA titin, as well as other mechanisms whereby passive tension caused by titin can be modulated. Skeletal muscle passive tension is shown for comparison (see text). Reprinted by permission of the American Heart Association (Granzier H, Labeit S, Circ Res 2004; 94: 284–95).
4. Titin and active force development Recent studies suggest that titin modulates the SL-dependent change in Ca2+ sensitivity of active force that is an important component of the Frank–Starling relation, i.e., Ca2+ sensitivity increases in proportion to titin-dependent passive force [46,47]. Titin degradation with trypsin digestion reverses this effect [48]. Interestingly, the effect of titin on active force is most prominent in myocardium with high levels of titin-based passive force. It has been proposed that this effect is mediated by altering interfilament lattice spacing and/or effecting strains on the thick filament that influence cross-bridge motility [49]. Additional recent experiments suggest that titin's effects on the actomyosin interaction may be SL-dependent, with inhibitory effects at slack SLs or below and enhancement at SLs greater than slack length [48,50]. There are a number of unanswered questions about the interaction of titin with cross-bridge formation, suggesting that this should be a fruitful area of future investigation. 5. Titin and biomechanical sensing Recent evidence indicates that titin may function as both a key biomechanical sensor and an organizational element within the sarcomere. A priori, in view of its size and structural position within the sarcomere and its function in relation to passive, restoring and active forces titin is a logical candidate in this regard. A large number of structural and signaling proteins bind to titin (Fig. 1). Binding sites are found in and near the Z-disk, in the central I-band and the M-line regions. The Z-disk titincapping protein T-cap, or telethionin, binds to titin's N-terminus and links titin to signaling and structural molecules [51,52]. Tcap interacts with cytoplasmic domains of two membrane associated proteins, the K channel subunit minK/isk in the Ttubules [53] and small ankyrin-1 (sANK-1), a transmembrane sarcoplasmic reticulum (SR) protein [54]. The minK/isk interaction may function to modulate K channel function in response to stretch while the sANK-1 interaction may serve to fix the position of the SR around the Z-disk. An interaction between titin's near Z-disk domains and obscurin [55] suggests an additional role for titin in relation to organization of the SR
since obscurin in turn interacts with ankyrin isoform 1.5 [55,56]. The latter appears to link the SR to the sarcomere and regulate ryanodine receptor distribution in the SR [57]. These Z-disk region interactions suggest a general role for titin in positioning SR and T-tubule systems in close proximity to the I-band and perhaps ensuring that these systems move with the Z-disk, which may be important to their function and integrity. T-cap also interacts with muscle LIM protein (MLP) [58], a nuclear regulator of myogenic differentiation. Stretch-induced release of brain and atrial natriuretic factors by cardiomyocytes is absent in a MLP knockout mouse, suggesting a role for MLP in stretch sensing which could also be related to the dilated cardiomyopathy present in these animals [59]. The N2B and N2A elements in the central I-band region also appear to function in organization of the sarcomere and signaling. N2B interacts with the LIM-family protein DRAL/FHL-2, which binds the key metabolic enzymes creatine kinase, adenylate cyclase and phosphofructokinase [60]. Thus, titin may be involved in compartmentalization of these enzymes such that they are located in close proximity to sites where ATP utilization is high. The N2B element also interacts with the small heat shock protein αβcrystallin [61] which functions to maintain normal protein folding. Ig domains of the N2A element in the N2BA isoform as well as in skeletal muscle titin interact with calpain protease P94, and may modulate the function of P94 in protein degradation [62]. Additional Ig segments bind to three homologous ankyrin repeat proteins, cardiac ankyrin repeat protein (CARP), ankrd2 (or Arpp) and diabetes ankyrin repeat protein (DARP) [63]. One or more of these ankyrin repeat proteins are induced by cardiac injury, skeletal muscle denervation and stretch, and metabolic challenge [64–66]. Interestingly, levels of all three are increased in myocardium of patients with dilated cardiomyopathy [67]. Thus, these ankyrin repeat proteins may be a component of muscle stress responses in general. The I-band ligands of titin and their associated binding partners are also found in the nucleus, where they function in transcriptional and cell cycle regulation. It is intriguing to consider that this dual localization may link titin's cytoplasmic stretch-based sensing to gene expression via transcriptional regulation. Along these lines, a naturally occurring “muscular dystrophy with myositis” mouse has been found to contain an 83 amino acid deletion mutation in the N2A element along with marked changes in the expression of MLP, CARP and MURF-1 (a titin-ligand M-line protein) [68,69]. Thus, this deletion mutation appears to affect ligands in multiple domains. The M-line region of titin also contains a number of sites that may be involved in sensing and signaling. These include a serine/ threonine kinase domain [70], another DRAL/FHL-2 site [60], possible additional T-Cap and obscurin sites [55,71] and a binding site for the RING finger protein MURF-1 (muscle specific ring finger protein-1) [72]. MURF-1 in particular may be involved in multiple signaling pathways. MURF-1 is a sarcomere-associated skeletal and cardiac muscle protein that functions as an E3 ubiquitin ligase that conjugates ubiquitin moieties to proteins destined for proteolysis [73]. Thus, MURF-1 may play a regulatory role in degradation of muscle proteins. MURF-1 also binds to a number of other myofibrillar proteins including
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troponin I, which results in increased degradation and possibly modulation of contractility by MURF-1 [74]. Finally, it has been proposed that MURF-1 plays a role in hypertrophic signaling [75]. All of these relatively recent and as yet largely preliminary reports implicate diverse roles for titin in biomechanical sensing and signaling. This suggests that the protein has complex and important integrative functions beyond its purely mechanical properties and constitutes an exciting area for future research. 6. Titin and heart disease 6.1. Adaptations and alterations during disease Two early reports suggested a decrease in titin content in failing hearts [76,77]. In retrospect this may have resulted from unrecognized proteolysis after tissue removal, to which titin is very susceptible. However, because of its size titin is a good candidate not just for proteolysis but for other forms of damage in vivo, for example oxidation by free radicals, glycation and Xirradiation. Although there is little information available in this area, a recent report showed that titin is damaged early during the course of experimental exposure to the cancer chemotherapeutic drug doxorubicin in association with activation of calcium-dependent calpain proteolysis [78]. (Doxorubicin treatment is often associated with clinical cardiomyopathy.) Damage to titin in the course of various diseases is an area that also appears ripe for future investigations. Several investigators have determined isoform content in failing myocardium. We examined the pacing tachycardia model of dilated cardiomyopathy in dogs. After only two weeks of pacing, we detected exaggeration of the normal transmural N2B/N2BA isoform ratio gradient [27]. After four weeks of pacing, when decompensation is generally present, we reported an increase in the ratio of N2B/N2BA isoforms which, as would be predicted, was associated with increased titin-based myocardial stiffness as well as increased collagen-based stiffness [79]. In a recent paper [80], spontaneously hypertensive rats were reported to have reduced N2BA titin expression, which is consistent with increased passive stiffness of isolated cardiomyocytes from these animals. Titin isoform distribution has also been studied in explanted hearts from patients with end-stage, dilated cardiomyopathy. Here also changes in isoform distribution are reported but differ from those in animal models. Thus, in patients with ischemic cardiomyopathy the proportion of the more compliant N2BA isoform was found to be increased [81]. Because tissue studies in ischemic cardiomyopathy are potentially confounded by regional variations, it is of note that recent studies in hearts from patients with non-ischemic cardiomyopathy showed the same change in isoform ratio [67,82]. The isoform ratio was also correlated with echocardiographic-Doppler findings consistent with reduced myocardial stiffness [67]. In contrast to changes in several other cardiac proteins in heart failure, the increases in N2BA titin in end-stage, failing human hearts do not reflect reexpression of specifically fetal–neonatal isoforms. Although the observed changes in isoform ratios in both pacing tachycardia in dogs and human dilated cardiomyopathy were not very large, the
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difference in stiffness between N2B and N2BA isoforms is so marked that measurable changes in cardiomyocyte stiffness would be expected nonetheless. The reason(s) for the apparent difference in the nature of alterations in titin isoforms in experimental animals and humans is uncertain, but several possibilities may be suggested. Thus, it is possible that end-stage hearts are not representative of the spectrum of failing myocardium in humans and different results might be found in less severely diseased hearts. Alternatively, the pacing tachycardia model may be a specialized case that cannot be extrapolated to other forms of heart failure. Obviously, this could also represent a true biologic difference between species. Another interesting possibility in failing myocardium is that disease-associated changes in phosphorylation of titin may influence cardiomyocyte and myocardial stiffness, especially since end-stage, dilated heart failure is accompanied by marked depression of PKA activity. A fascinating recent report by Borbely et al. in patients with non-dilated, diastolic heart failure (i.e., heart failure with preserved contraction) demonstrated increased cardiomyocyte passive stiffness that was reversed by PKA treatment [83]. Increased ventricular chamber stiffness is the rule in these patients, who constitute somewhere between 40–50% of the heart failure population. These early reports in acquired forms of heart disease suggest the possibility of dynamic changes in the influence of titin on passive stiffness in disease as well as the possibility of a wide variety of other effects related to its role in mechanical sensing and signaling. 6.2. Hereditary disease Because of its size and diverse functions, titin would seem a prime candidate for disease causing mutations. Earlier linkage studies indeed identified titin as a candidate gene for certain inherited forms of dilated cardiomyopathy, but specific mutations were not detected on sequencing of a small number of exons [84]. The completion of titin's genomic structure has facilitated the search for mutations (Fig. 4). Thus, recent studies have identified two titin mutations that cause DCM (exons 18 and 326) [85]. Following sequencing of the entire gene, a mutation in exon 363 was reported in patients with a skeletal muscle dystrophy linked to titin (tibialis muscular dystrophy) [86]. Finally, a sequencing study recently identified two missense mutations in exons 2 and 14 in patients with hypertrophic cardiomyopathy [87]. It is intriguing that to date disease-causing titin mutations have been identified in regions present in both cardiac and skeletal muscle, yet phenotypic changes have been evident in only one muscle type. The ability to now sequence the entire gene should allow a full elucidation of the role of titin in genetically determined heart and skeletal muscle disease. 7. Summary The last decade has seen a rapid increase in our understanding of titin, a protein whose functions were unknown previously. Titin appears to have diverse and critical roles in the mechanical, organizational and signaling properties of cardiac and skeletal
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