Titin Size Research Articles

Protein Quality Control at the Sarcomere: Titin Protection and Turnover and Implications for Disease Development.

Sarcomeres are mainly composed of filament and signaling proteins and are the smallest molecular units of muscle contraction and relaxation. The sarcomere protein titin serves as a molecular spring whose stiffness mediates myofilament extensibility in skeletal and cardiac muscle. Due to the enormous size of titin and its tight integration into the sarcomere, the incorporation and degradation of the titin filament is a highly complex task. The details of the molecular processes involved in titin turnover are not fully understood, but the involvement of different intracellular degradation mechanisms has recently been described. This review summarizes the current state of research with particular emphasis on the relationship between titin and protein quality control. We highlight the involvement of the proteasome, autophagy, heat shock proteins, and proteases in the protection and degradation of titin in heart and skeletal muscle. Because the fine-tuned balance of degradation and protein expression can be disrupted under pathological conditions, the review also provides an overview of previously known perturbations in protein quality control and discusses how these affect sarcomeric proteins, and titin in particular, in various disease states.

Abstract 440: The Cardiomyocyte Diastolic Stiffness and Contractility are Inversely Related to the Size of Titin: A Cellular Work Loop Study of Single Intact Cardiomyocytes

Studying cardiac titin under physiological conditions can be at the level of the left ventricular (LV) chamber and the multicellular muscle level. However, those preparations are influenced by neurohormonal reflex, preload-afterload status and extracellular matrix. In this study, cellular work loop force measurement of the single intact cardiomyocyte was conducted under physiological conditions. Intact cell experiments were performed at 37°C under field-stimulation at 2 Hz frequency. The cell was attached to the force transducer and the piezo length controller which modulates the cell length by the feedback control system based on developed force. The force length relation (cellular work loop) of the single cell was generated. The end diastolic force length relation (EDFLR), the end systolic force length relation (ESFLR) and the preload recruitable stroke work (PRSW) were obtained. The Ttn ΔIAjxn -/- ( IA -/- ) mice, in which the I-band/A-band region of titin’s spring has been deleted were studied in comparison to α-MHC-Cre ; cRbm20 ΔRRM +/- ( cRbm20 +/- ) mice which are deficient in titin splicing and express large titin isoforms in the heart. The cellular diastolic stiffness (slope of EDFLR) was increased in IA -/- and was reduced in cRbm20 +/- (151.3, 104.0 and 91.5 μN/mm 2 in IA -/- , WT and cRbm20 +/- respectively). The contractility (slopes of ESFLR and PRSW) were 233.5 and 1215.6 uN/mm 2 in WT, and were increased in IA -/- (371.2 and 2076 μN/mm 2 ) but attenuated in cRbm20 +/- (202.9 and 907.7 μN/mm 2 ). Cellular work loop studies were also conducted in intact cells isolated from the mice that underwent transverse aortic constriction (TAC) surgery with deoxycorticosterone acetate (DOCA) implantation. TAC DOCA mice developed diastolic dysfunction at the LV chamber level. The result showed that the cells from TAC DOCA hearts had increased slopes of EDFLR, ESFLR and PRSW ( 157.9, 378.9 and 1699.0 μN/mm 2 ) compared to the cells from sham mice (98.6, 230.3, and 1203.9 μN/mm 2 ). Conclusions: Cellular work loops recapitulated the cardiac physiology at the LV chamber level. The result showed that the cellular diastolic stiffness and contractility were inversely related to the size of titin and that titin stiffness plays an important roles in both diastolic and systolic function.

Abstract 393: Cronos Titin in Human Heart Development

Titin is a giant myofibrillar protein that provides passive tension and facilitates sarcomere formation in striated muscle, where it spans from the Z-disk to the M-line. Recently, an alternative start site has been identified in the titin gene that is predicted to allow for expression of a novel isoform, Cronos, that is ~2/3 the size of full-length titin and lacks the Z-disk and most of the I-band domains. Because the start site for this isoform is spliced out of full-length titin transcripts, we are able to determine the expression levels of Cronos compared to full-length titin. Reverse-transcription followed by quantitative PCR using primers specific to this region indicate Cronos transcript levels are higher in human fetal ventricle and atrial tissue (days 90-105) than in adult samples, while full-length titin transcripts are higher in adult than fetal cardiac tissue. Additionally, human ventricular tissue stained with a custom antibody raised to recognize this specific region of Cronos titin indicated high levels of protein expression and integration into myofibrils in early fetal samples (days 54 and 81), while this novel isoform was nearly undetectable in later fetal (day 130) and adult tissue. The Cronos titin transcript and protein were also detected in high quantities in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), which are believed to have a fetal level of maturity. Based on these data, we conclude that Cronos is predominantly a fetal isoform of titin. Future experiments will investigate the effects of a Cronos titin knockdown in hiPSC-CMs during and after differentiation to establish its functional role in human heart development.

Thin filament length in the cardiac sarcomere varies with sarcomere length but is independent of titin and nebulin

Thin filament length (TFL) is an important determinant of the force-sarcomere length (SL) relation of cardiac muscle. However, the various mechanisms that control TFL are not well understood. Here we tested the previously proposed hypothesis that the actin-binding protein nebulin contributes to TFL regulation in the heart by using a cardiac-specific nebulin cKO mouse model (αMHC Cre Neb cKO). Atrial myocytes were studied because nebulin expression has been reported to be most prominent in this cell type. TFL was measured in right and left atrial myocytes using deconvolution optical microscopy and staining for filamentous actin with phalloidin and for the thin filament pointed-end with an antibody to the capping protein Tropomodulin-1 (Tmod1). Results showed that TFLs in Neb cKO and littermate control mice were not different. Thus, deletion of nebulin in the heart does not alter TFL. However, TFL was found to be ~0.05μm longer in the right than in the left atrium and Tmod1 expression was increased in the right atrium. We also tested the hypothesis that the length of titin's spring region is a factor controlling TFL by studying the Rbm20ΔRRM mouse which expresses titins that are ~500kDa (heterozygous mice) and ~1000kDa (homozygous mice) longer than in control mice. Results revealed that TFL was not different in Rbm20ΔRRM mice. An unexpected finding in all genotypes studied was that TFL increased as sarcomeres were stretched (~0.1μm per 0.35μm of SL increase). This apparent increase in TFL reached a maximum at a SL of ~3.0μm where TFL was ~1.05μm. The SL dependence of TFL was independent of chemical fixation or the presence of cardiac myosin-binding protein C (cMyBP-C). In summary, we found that in cardiac myocytes TFL varies with SL in a manner that is independent of the size of titin or the presence of nebulin.

Abstract 215: The Role of Titin in Cardiac Function: Studies in Two Genetically Engineered Mouse Models With Disparate Titin’s Size

Titin, a myofilament that acts as a molecular spring in the sarcomere, is considered the main contributor to passive stiffness of cardiomyocytes and is responsible for cardiac diastolic function. Increased titin stiffness is related to diastolic dysfunction and HFpEF (Heart Failure with preserved Ejection Fraction). Alteration in size of titin’s spring region leads to changes in cardiomyocyte and left ventricular (LV) chamber stiffness. We tested the effect of alteration in titin’s size in two genetically engineered mouse models. We investigated the effect of shortening titin’s spring region in a mouse model in which I-band/A-band region of titin’s spring has been deleted (TtnΔIAjxn ), in comparison to the effect of lengthening titin’s spring region in a mouse model deficient in titin splicing factor (Rbm20ΔRRM). Integrative approaches were used from single cardiomyocyte mechanics to pressure-volume analysis and exercise study. Study of skinned LV cardiomyocytes revealed that cellular passive stiffness was inversely related to the size of titin. Cellular passive stiffness was increased in TtnΔIAjxn homozygous (-/-) (~ 110 % higher than wildtype (WT)) and was reduced in a graded manner in Rbm20ΔRRM heterozygous (+/-) and -/- cardiomyocytes (~61% and ~87% less than WT). This effect was carried through at the LV chamber level which could be demonstrated in pressure volume (PV) analysis as an increased end-diastolic pressure-volume relationship (EDPVR) in TtnΔIAjxn -/- (~110% higher than WT’s hearts) and reduced EDPVR in Rbm20ΔRRM +/- and -/- (~57% and ~48% less than WT’s hearts). Free-wheel running studies revealed a running deficiency in TtnΔIAjxn -/- mice but an increase in exercise capacity in Rbm20ΔRRM +/– mice. Conclusions: Functional studies from the cellular to in-vivo LV chamber levels showed that mice with shortening of titin’s spring region had increased LV stiffness, diastolic dysfunction and reduced exercise capacity, while mice with lengthening titin’s spring region had compliant LV and increased exercise capacity. Thus, our work supports titin’s important roles in LV diastolic function and suggests that modification of the size of titin’s spring region could be a potential therapeutic strategy for HFpEF.

Of cilia, titin, and neurosteroids

Of cilia, titin, and neurosteroids

Titin isoform size is not correlated with thin filament length in rat skeletal muscle

The mechanisms controlling thin filament length (TFL) in muscle remain controversial. It was recently reported that TFL was related to titin size, and that the latter might be involved in TFL determination. Titin plays several crucial roles in the sarcomere, but its function as it pertains to the thin filament has not been explored. We tested this relationship using several muscles from wild type rats and from a mutant rat model (Greaser et al., 2008) which results in increased titin size. Myofibrils were isolated from skeletal muscles [extensor digitorum longus (EDL), external oblique (EO), gastrocnemius (GAS), longissimus dorsi (LD), psoas major (PM), and tibialis anterior(TA)] using both adult wild type (WT) and homozygous mutant (HM) rats (n = 6 each). Phalloidin and antibodies against tropomodulin-4 (Tmod-4) and nebulin's N-terminus were used to determine TFL. The WT rats studied express skeletal muscle titin sizes ranging from 3.2 to 3.7 MDa, while the HM rats express a giant titin isoform sized at 3.8 MDa. No differences in phalloidin based TFL, nebulin distance, or Tmod distance were observed across genotypes. However, the HM rats demonstrated a significantly increased (p < 0.01) rest sarcomere length relative to the WT phenotype. It appears that the increased titin size, predominantly observed in HM rats' middle Ig domain, allows for increased extensibility. The data indicates that, although titin performs many sarcomeric functions, its correlation with TFL and structure could not be demonstrated in the rat.

Removal of Proximal IG Domains of Titin in Soleus Muscle Results in Differential Splicing of Titin MRNA

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<0.001) and myomesin (2.13 fold decreased in IG KO, p<0.001). The reason for the differential expression of titin binding proteins and the muscle specific changes in alternative splicing need to be further studied.

Human skeletal muscle biochemical diversity

The molecular components largely responsible for muscle attributes such as passive tension development (titin and collagen), active tension development (myosin heavy chain, MHC) and mechanosensitive signaling (titin) have been well studied in animals but less is known about their roles in humans. The purpose of this study was to perform a comprehensive analysis of titin, collagen and MHC isoform distributions in a large number of human muscles, to search for common themes and trends in the muscular organization of the human body. In this study, 599 biopsies were obtained from six human cadaveric donors (mean age 83 years). Three assays were performed on each biopsy - titin molecular mass determination, hydroxyproline content (a surrogate for collagen content) and MHC isoform distribution. Titin molecular mass was increased in more distal muscles of the upper and lower limbs. This trend was also observed for collagen. Percentage MHC-1 data followed a pattern similar to collagen in muscles of the upper extremity but this trend was reversed in the lower extremity. Titin molecular mass was the best predictor of anatomical region and muscle functional group. On average, human muscles had more slow myosin than other mammals. Also, larger titins were generally associated with faster muscles. These trends suggest that distal muscles should have higher passive tension than proximal ones, and that titin size variability may potentially act to 'tune' the protein's mechanotransduction capability.

Comprehensive analysis of titin protein isoform and alternative splicing in normal and mutant rats

Titin is a giant protein with multiple functions in cardiac and skeletal muscles. Rat cardiac titin undergoes developmental isoform transition from the neonatal 3.7 MDa N2BA isoform to primarily the adult 2.97 MDa N2B isoform. An autosomal dominant mutation dramatically altered this transformation. Titins from eight skeletal muscles: Tibialis Anterior (TA), Longissimus Dorsi (LD) and Gastrocnemius (GA), Extensor Digitorum Longus (ED), Soleus (SO), Psoas (PS), Extensor Oblique (EO), and Diaphram (DI) were characterized in wild type and in homozygous mutant (Hm) rats with a titin splicing defect. Results showed that the developmental reduction in titin size is eliminated in the mutant rat so that the titins in all investigated skeletal muscles remain large in the adult. The alternative splicing of titin mRNA was found repressed by this mutation, a result consistent with the large titin isoform in the mutant. The developmental pattern of titin mRNA alternative splicing differs between heart and skeletal muscles. The retention of intron 49 reveals a possible mechanism for the absence of the N2B unique region in the expressed titin protein of skeletal muscle.

Removal of Ig Domains of Titin Alters Contractility in Mouse Soleus Muscle

The giant protein titin provides elasticity to the skeletal muscle sarcomere by acting as a molecular spring. We created a mouse model in which 9 immunoglobulin (Ig) domains (Ig3-11) have been deleted from titin's spring region, providing a novel model to test the effect of a shorter spring on the physiology of skeletal muscle. The soleus muscle was studied by using gel electrophoresis and functional measurements on contractility made via an intact muscle mechanics system on six month old male mice. An additional set of mice were exposed to exercise training on a treadmill to determine exercise capacity and response to exercise. Soleus mass was lower in the IGKO (8.3±0.2mg) vs WT (10.2±1.2mg). IGKO mice showed a significant decrease in titin size on 1% agarose gels greater than the 88KDa loss predicted by the deletion if Ig3-11. Furthermore, IGKO mice have a reduced myosin II/ myosin I ratio (2.1 vs. 8.0, p=0.002). Intact muscle mechanics measurements probed both passive and active tension. IG KO muscles produced an increased passive tension (19% p=0.02) at 20% above slack length of the muscle. Active force measurements probed at the optimal twitch length (Lo) revealed a significantly decreased twitch/tetanus ratio in IGKO's (12.4% vs 8.1%, p<0.001) and decreased active force at all frequencies examined (1-150Hz). IGKO also experienced less fatigue (28% vs 67% decrease, p=0.01). Exercise testing in mice showed a significantly reduced maximum running speed between IGKO and WT (18.8 vs 22.0 m/min, p=0.005), which persisted after two weeks of training. The IgKO mouse provides a unique model for elucidating the effect of titin's spring elements on contractility in skeletal muscle.

Postmortem titin proteolysis is influenced by sarcomere length in bovine muscle1

The calpain protease system, in particular, μ-calpain is involved in the disassembly of specific myofibrillar proteins, resulting in tenderization of meat postmortem. Given the size, complexity, and integral nature of titin to the structure of the sarcomere, it is plausible that the length of a sarcomere may alter the susceptibility of various domains of titin to cleavage by the calpains. Therefore, we hypothesized titin degradation differs in a sarcomere-length-dependent manner in beef. After slaughter, beef carcasses were split and sides were either suspended by the Achilles tendon (normal suspension, NS) or by the aitchbone (hip suspension, HS). Immediately after suspension, samples were dissected from the LM, psoas major (PM), and semitendinosus (STN) muscles to serve as 0-d controls. After 24 h, 4 steaks were removed from each muscle and randomly assigned to 1-, 4-, 7-, or 10-d aging treatments. After the assigned aging period, myofibrils were purified for determination of sarcomere length. Warner-Bratzler shear force analysis was also performed to evaluate differences in tenderness. Muscle proteins were solubilized and subjected to SDS-VAGE (vertical agarose gel electrophoresis) to evaluate titin degradation. Sarcomere lengths differed (P < 0.0001) between contralateral muscles of NS and HS carcasses. Quantification of SDS-VAGE gels revealed less (P < 0.05) intact titin in the PM muscle of NS carcasses at each aging period compared with the PM of HS carcasses. No significant differences (P > 0.05) were detected in the disappearance of intact titin among suspension methods in the LM or STN. These data demonstrate that suspension method alters proteolysis of titin and suggest an increase in sarcomere length may contribute to the susceptibility of titin to postmortem proteolysis in beef.

Cardiac Titin

Titin constitutes the third myofilament of cardiac muscle, with a single giant polypeptide spanning from Z-disk to the M-band region of the sarcomere1 (Fig. 1). The ∼1.0 MDa region in the I-band is extensible and consists of tandemly arranged immunoglobulin (Ig)-like domains that make up proximal (near Z-disk) and distal (near A-I junction) segments, interspersed by the PEVK sequence (rich in proline, glutamate, valine, and lysine residues) and the N2B element2. Each functions as a distinct spring element3. The C-terminal ∼2 MDa of titin is located in the A-band and is inextensible. It is composed of regular arrays of Ig and fibronectin type 3 (Fn3) modules forming so-called super-repeats2. A-band titin may function as a molecular ruler, regulating assembly of the thick filament2,4,5. Titin's ∼250-kDa COOH-terminal region is an integral part of the M-band and contains a kinase domain6,7. As in the Z-disk, where titin filaments from opposite sarcomeres overlap, titin filaments from opposite half-sarcomeres overlap within the M-band, where they are interconnected by M-band proteins8. Thus, titin filaments with opposite polarity overlap in both Z-disk and M-band, forming a contiguous filament along the myofibril. In this review, we discuss titin's functions in the heart, with an emphasis on its role in diastolic function and the various mechanisms whereby passive stiffness can be tuned. Due to space constraints, it has not been possible to provide inclusive references to all original articles in the field. Figure 1 Schematic of titin in sarcomere. Differential splicing Titin is encoded by a single gene containing 368 exons. Multiple splice pathways in the I-band encoding region (∼230 exons) give rise to isoforms with different spring composition9. The three cardiac isoform classes are shown in Fig. 1. The relatively small ∼3.0 MDa isoform is known as N2B titin (it contains the N2B element)9. A second class also contains the N2A element, and is termed N2BA titin. N2BA titins have a longer PEVK segment and a variable number of additional Ig domains resulting in a ∼3.3-3.5 MDa size9. The third class includes isoforms that predominate in fetal-neonatal life which contain additional spring elements in both tandem Ig and PEVK regions, resulting in a ∼3.6-3.8 MDa size protein10-12. These isoforms gradually disappear during postnatal development. Regulation of the spring composition of titin in fetal-neonatal myocardium allows adjustment of diastolic filling properties during development.

Titin Isoform Size is not Correlated with Thin Filament Length in Rat Skeletal Muscle

The striated muscle sarcomere is dependent upon the precise interactions of a variety of myofibrillar proteins for its proper formation. As the largest and third most abundant protein in this milieu, titin plays a number of functions in the sarcomere, including assembly of the thick filaments and preventing overstretch. The titin gene is expressed as multiple splice variants in skeletal muscle, generating a continuum of titin protein sizes. Recently it was reported that thin filament length was related to titin size, and that the latter might be involved in determining thin filament length. We tested this hypothesis using several muscles from wild type rats and from a mutant rat model (Greaser et al J Mol Cellul Cardiol 44:983, 2008) which results in increased titin size. Myofibrils were isolated from skeletal muscles (diaphragm, extensor digitorum longus, gastrocnemius, longissimus dorsi, psoas major, rectus abdominis, and tibialis anterior) using both adult wild type (WT) and homozygous mutant (HM) rats (n=6 each). Thin filament length was estimated using fluorescent dye labeled phalloidin and relaxed sarcomere length was determined by phase contrast microscopy after adding ATP and BDM. No differences in thin filament lengths were found between WT muscles with titin sizes ranging from 3.2 to 3.7 MDa. Similarly the thin filament lengths in the mutant rats did not differ from the paired WT muscles in spite of large differences in titin size with several muscles. However, the relaxed sarcomere length was correlated to titin size in muscles from WT rats, and it was significantly increased relative to WT for within muscle comparisons. The data indicates that, although titin performs many functions, its relationship to thin filament length could not be demonstrated in the rat. Supported by HL77196.

Heart failure decreases passive tension generation of rat diaphragm fibers

Background Diaphragm dysfunction is well-known to limit quality of life and prognosis of patients with heart failure (HF), but its underlying mechanisms are not well understood. In an animal model for HF we recently showed that impaired diaphragm contractility arises at the single fiber level and is associated with sarcomeric injuries. For optimal muscle function and sarcomeric stability passive elastic structures, like titin, are indispensable. The current study aimed to investigate if impaired passive elasticity contributes to diaphragm dysfunction in rats with heart failure. Methods Skinned muscle fibers were isolated from the diaphragm and soleus of rats with chronic HF, induced by left coronary artery ligation and of sham-operated rats. Passive tension–length relationships were determined by applying segmental extension tests. Immunofluorescence was performed on muscle cryosections using antibodies (T12) against a titin epitope near the Z-line. Titin content was determined by SDS-agarose-gel electrophoresis. Titin's mobility on gel was studied to detect changes in titin size. Results Passive tension generation upon stretch was significantly reduced (> 35%) in HF diaphragm fibers compared to sham. Immunostaining intensities against titin were reduced in diaphragm cryosections of HF rats compared to sham. Soleus fibers from HF and sham rats did not display differences, neither in passive tension nor in immunostaining. No differences in titin's size were detected in HF and sham diaphragm. Titin content, however, was significantly reduced (∼ 25%) in HF diaphragm. Discussion We conclude that in the diaphragm of HF rats, passive elasticity is impaired, mainly resulting from titin loss.

Isoform Diversity of Giant Proteins in Relation to Passive and Active Contractile Properties of Rabbit Skeletal Muscles

The active and passive contractile performance of skeletal muscle fibers largely depends on the myosin heavy chain (MHC) isoform and the stiffness of the titin spring, respectively. Open questions concern the relationship between titin-based stiffness and active contractile parameters, and titin's importance for total passive muscle stiffness. Here, a large set of adult rabbit muscles (n = 37) was studied for titin size diversity, passive mechanical properties, and possible correlations with the fiber/MHC composition. Titin isoform analyses showed sizes between ∼3300 and 3700 kD; 31 muscles contained a single isoform, six muscles coexpressed two isoforms, including the psoas, where individual fibers expressed similar isoform ratios of 30:70 (3.4:3.3 MD). Gel electrophoresis and Western blotting of two other giant muscle proteins, nebulin and obscurin, demonstrated muscle type–dependent size differences of ≤70 kD. Single fiber and single myofibril mechanics performed on a subset of muscles showed inverse relationships between titin size and titin-borne tension. Force measurements on muscle strips suggested that titin-based stiffness is not correlated with total passive stiffness, which is largely determined also by extramyofibrillar structures, particularly collagen. Some muscles have low titin-based stiffness but high total passive stiffness, whereas the opposite is true for other muscles. Plots of titin size versus percentage of fiber type or MHC isoform (I-IIB-IIA-IID) determined by myofibrillar ATPase staining and gel electrophoresis revealed modest correlations with the type I fiber and MHC-I proportions. No relationships were found with the proportions of the different type II fiber/MHC-II subtypes. Titin-based stiffness decreased with the slow fiber/MHC percentage, whereas neither extramyofibrillar nor total passive stiffness depended on the fiber/MHC composition. In conclusion, a low correlation exists between the active and passive mechanical properties of skeletal muscle fibers. Slow muscles usually express long titin(s), predominantly fast muscles can express either short or long titin(s), giving rise to low titin-based stiffness in slow muscles and highly variable stiffness in fast muscles. Titin contributes substantially to total passive stiffness, but this contribution varies greatly among muscles.

Developmentally regulated switching of titin size alters myofibrillar stiffness in the perinatal heart.

Before birth, the compliance of the heart is limited predominantly by extracardiac constraint. Reduction of this constraint at birth requires that myocardial compliance be determined mainly by the heart's own constituents. Because titin is a principal contributor to ventricular passive tension (PT), we studied the expression and mechanics of cardiac-titin isoforms during perinatal rat heart development. Gel electrophoresis and immunoblotting revealed a single, 3.7-MDa, N2BA isoform present 6 days before birth and an additional, also previously unknown, N2BA isoform of 3.5 to 3.6 MDa expressed in the near-term fetus. These large isoforms rapidly disappear after birth and are replaced by a small N2B isoform (3.0 MDa) predominating in 1-week-old and adult rats. In addition, neonatal pig hearts showed large N2BA-titin isoforms distinct from those present in the adult porcine myocardium. By quantitative reverse transcriptase-polymerase chain reaction, developmentally expressed titin-mRNA species were detected in rat heart. Titin-based PT was much lower (approximately 15 times) in fetal than adult rat cardiomyocytes, and measured PT levels were readily predictable with a model of worm-like chain titin elasticity. Immunofluorescence microscopy showed the extensibility of the differentially spliced molecular spring regions of fetal/neonatal titin isoforms in isolated rat cardiomyofibrils. Whereas the titin-isoform shift by 700 kDa ensures high passive stiffness of the postnatal cardiac myofibrils, the expression of specific fetal/neonatal cardiac-titin isoforms may also have important functions for contractile properties, myofibril assembly or turnover, and myocardial signaling during perinatal heart development.

Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins.

The electrophoretic separation of high-molecular-weight proteins (> 500 kDa) using polyacrylamide is difficult because gels with a large enough pore size for adequate protein mobility are mechanically unstable. A 1% vertical sodium dodecyl sulfate (SDS)-agarose gel electrophoresis (VAGE) system has been developed that allows titin (a protein with the largest known SDS subunit size of 3000-4000 kDa) to migrate over 10 cm in a approximately 13 cm resolving gel. Such migration gives clear and reproducible separation of titin isoforms. Proteins ranging in size from myosin heavy chain ( approximately 220 kDa) up to titin can be resolved on this gel system. Electroblotting of these very large proteins was nearly 100% efficient. This VAGE system has revealed two titin size variants in rabbit psoas muscle, two N2BA bands in rabbit cardiac muscle, and species differences between titins from rat and rabbit muscle. Agarose electrophoresis should be the method of choice for separation and blotting of proteins with very large subunit sizes.

Titin Isoforms and Kinematics of Fast Swimming Carp Larvae (Cyprinus Carpio L.)

Titin, a striated-muscle specific protein spanning the distance between Z- and M-lines of sarcomeres, is held responsible for developing passive tension and for maintaining the central position of thick filaments in contracting sarcomeres. Different muscles express titin isoforms of different molecular mass. To improve the insight in the relation between titin isoforms and kinematics of fast swimming at different ages the presence of carp larval muscle titin (Cyprinus carpio L.) was investigated and compared with data of adult carp. Gel-electrophoresis revealed that titin isoforms were larger in adult than in larval muscle. Apparently the molecular structure of titin changed during ontogeny. A previous study showed that the size of titin is correlated with the functioning of different muscles during swimming. Fish larvae (6.5-8 mm total length), subjected to low Reynolds-number regimes during swimming (Re < 500), require special features to overcome frictional effects. Fibres with smaller titin isoforms require more passive tension when being stretched. During fast swimming of larvae, passively stretched fibres at the convex side of the body axis absorb energy, generated by activity of fibres at the concave side, that is released in the successive opposite bending.

Dissecting titin into its structural motifs: identification of an alpha-helix motif near the titin N-terminus.

Titin, also known as connectin, is a giant modular protein specifically found in vertebrate striated muscle. Since the huge size of titin does not allow a direct structure determination, we have started a long-term project to characterize the protein by cutting it into smaller domains or structural units. The major part of the titin sequence is assembled by modules approximately 100 amino acids long that belong to two major protein superfamilies. Most of these modules are linked together by stretches of variable length with unique sequence. No direct structural characterization has been achieved so far for any of these linkers. We present here a study of a stretch located in the titin N-terminus and part of a linker between two modules. Our attention was drawn toward this region because it shows 100% probability to form a coiled coil when analyzed by a prediction program. A synthetic 38 amino acid peptide spanning such a sequence was studied in aqueous solution by circular dichroism, nuclear magnetic resonance, and analytical ultracentrifugation at various pH, salt, and peptide concentrations. Under all conditions, it shows a strong tendency to form alpha-helical structures. In the presence of salt, this conformation is associated with the formation of helical bundles below pH 5. Above pH 5, any aggregate breaks, and the titin peptide is a monomeric helix in equilibrium with its random coil conformation. We discuss the factors which stabilize the helical conformation and the possible role of this stretch in vivo.