Abstract One of the main contributors to passive tension of the myocardium is titin. However, it is not exactly known what portions of this ≈1 μm-long molecule are anchored in the sarcomere (hence, are rendered inelastic) and what portions are elastic (hence, are mechanically active in developing passive tension). We assessed the length of the elastic domain of cardiac titin by ultrastructural and mechanical methods. Single cardiac myocytes were stretched by various amounts, and while in the stretched state, they were processed for immunoelectron microscopy. Several monoclonal anti-titin antibodies were used, and the locations of the titin epitopes in the sarcomere were studied as a function of sarcomere length. Only a small fraction (5% to 10%) of the ≈1000-nm-long molecule behaved elastically under physiological conditions. This mechanically active domain is located close to the A/I junction, and its contour length when unstretched is estimated at ≈50 to 100 nm. In sarcomeres that are slack (length ≈1.85 μm), the mechanically active domain is folded on top of itself, and the length of the domain reaches an elastic limit of ≈550 nm in sarcomeres that are ≈2.9 μm long.
When passive muscle is stretched, it develops tension (known as passive tension) that opposes the stretch and restores the original length of the muscle after release. The molecular basis of passive tension may lie in the organization and properties of the protein titin (also known as connectin) found in striated muscles (for reviews, see References 1 through 31 2 3 ). In skeletal muscle, a single titin molecule extends from the Z line to the M line of the sarcomere, a distance of ≈1 to 2 μm. While the segment of the titin molecule found in the I band behaves elastically when sarcomeres are stretched, the A-band segment is inelastic.4 5 6 The I-band domain of titin has been proposed to underlie passive tension of muscle by functioning as a molecular spring that develops a restoring force when stretched.1 Consistent with this proposal, passive tension is greatly reduced after titin has been destroyed.7 8 9 10
Most of our knowledge about titin and passive tension has been obtained from studies using skeletal muscle. However, titin and passive tension are likely to be more important in the myocardium. Passive tension is part of the heart’s wall tension that, during diastole, determines to what extent the heart will be filled. In support of this notion, the level of expression of titin in hearts of human patients with dilated cardiomyopathy has been reported to be reduced.11 Titin may thus play a critical role in the normal pumping function of the heart. In a recent study, we showed that over the working range of sarcomeres in the heart, titin is one of the main contributors to passive tension.7 Further, passive tension developed by titin increases much more steeply with sarcomere length in cardiac muscle than it does in skeletal muscle. It has been suggested that the steeper tension increase of cardiac muscle is a result of the smaller titin size variant expressed by cardiac muscle.7 12 The small size variant will result in a shorter I-band domain of titin, giving rise to a larger strain of this domain and thus a higher passive tension for a given degree of sarcomere stretch.7 A basic assumption in this proposal is that the full I-band domain of titin is elastic in both skeletal and cardiac muscle. However, recent studies on skeletal muscle indicate that only about half of the I-band domain is elastic in this muscle type.4 6 13 Thus, in order to compare the passive tension of cardiac and skeletal muscles and to understand better the passive tension of the heart in general, it is important to know the length of the titin domain that is elastic and mechanically active in developing passive tension.
We investigated the elastic behavior of different segments of the titin molecule by using immunoelectron microscopy on rat cardiac myocytes that had been stretched and labeled with anti-titin antibodies. Only a relatively small fraction (25% to 50%) of the unstrained I-band domain of titin turned out to be elastic and mechanically active in developing passive tension. We estimate that the unstretched mechanically active domain has a contour length of only 50 to 100 nm, or 5% to 10% of the total length of the titin molecule. In sarcomeres that are slack, we found evidence that the mechanically active domain is highly folded on top of itself.
Materials and Methods
Single myocytes were isolated from the ventricles of rat (male Sprague-Dawley, ≈250 g) hearts using the method of Granzier and Irving.7 To prevent titin degradation, cells were extensively washed before skinning, and all solutions contained protease inhibitors.7
The mechanical setup has been described in detail in Granzier and Irving.7 Briefly, cells were added to a temperature-controlled flow-through chamber (volume, ≈150 μL) that was attached to the microscope stage, and a single cell was glued at one end to the motor and at the other end to the force transducer. Sarcomere length was measured by using digital image analysis and Fourier transformation of videotaped cell images. For immunolabeling experiments, the cells were glued in the stretched state to the removable coverslip that functioned as the bottom of the chamber, by first applying two small droplets of glue to the coverslip, ≈100 μm apart, and then lowering the ends of the cell into this glue. The cells were surrounded by a shallow ring glued to the coverslip, which served as a minichamber (volume, ≈10 μL) for immunolabeling, fixing, and embedding of the cells.
Skeletal Muscle Fibers
The setup and the sarcomere length measuring technique have been described in detail by Granzier and Irving.7
Mechanical protocols were similar to those in Granzier and Irving.7 Because rat cardiac myocytes and rabbit semitendinosus muscle reach an elastic limit at a sarcomere length of ≈2.9 μm and ≈4.5 μm, respectively, which if exceeded may result in permanent mechanical damage,7 14 the stretch amplitude was kept below those limits. The obtained passive tension–sarcomere length curves were highly reproducible. All mechanical experiments were conducted at 20°C.
Immunolabeling and Electron Microscopy
Cardiac myocytes were fixed for 20 minutes in freshly prepared 3% paraformaldehyde in PBS containing (mmol/L) KCl 2.7, KH2PO4 1.5, NaCl 137, Na2HPO4 8.0, and EGTA 2, pH 7.2. After washing the cells three times with PBS for 30 minutes each, cells were blocked for 30 minutes in PBS/0.5% BSA. Cells were then incubated 24 to 48 hours with anti-titin monoclonal antibodies in PBS/BSA. The following antibodies were used: T12 (2 μg/mL), 9D10 (20 μg/mL), and Ti-102 (ascites 20× diluted). T12 is an antibody that was developed by Dr Weber (Max Planck Institute for Biophysical Chemistry, Goettingen, Germany) and is against skeletal muscle titin.4 T12 has been shown to be titin specific and to cross-react with cardiac titin.4 It is commercially available from Boehringer (No. 1248-634). The antibody 9D10 is against bovine cardiac titin and was developed by Dr Greaser (Muscle Biology Laboratory, University of Wisconsin). It has been shown to be titin specific.15 We obtained 9D10 from the Developmental Studies Hybridoma Bank maintained by the Department of Biological Sciences, University of Iowa, Iowa City. Finally, Ti-102 is a titin-specific monoclonal antibody against cloned rat cardiac titin class II motifs16 and was developed by Dr Jin (Department of Medical Biochemistry, University of Calgary). After washing cells three times with PBS/BSA, they were incubated for 24 to 48 hours in secondary antibody in PBS/BSA: for T12, rabbit anti-mouse IgG; for 9D10, goat anti-mouse IgG, IgA, and IgM; and for Ti-102, rabbit anti-mouse IgG whole molecule. In several experiments, the secondary antibody was nanogold Fab anti-mouse IgG (No. 2002, Nanoprobes Inc). Unbound antibody was removed by washing with PBS. Solutions contained 100 μg/mL leupeptin and were kept at 4°C.
Cells were fixed with glutaraldehyde/tannic acid, osmicated, and embedded in araldite (compare with Reference 66 ). The osmification step was omitted when nanogold Fab anti-mouse IgG was used. Instead, a silver-enhancement step was introduced (K. Trombitás, unpublished data, 1995) by using HQ silver (Nanoprobes Inc) as per the manufacturer’s protocol. The solutions for immunolabeling, fixing, and embedding were added while the cell remained glued to the glass coverslip inside a 10-μL chamber. Cells were embedded by inverting an araldite-filled beem capsule over the ring glued to the coverslip. After polymerization, the coverslip was removed by exposure to liquid nitrogen followed by an exposure to ≈60°C H2O. Ultrathin sections were cut with a Sorvall MT-2M ultramicrotome. They were stained with potassium permanganate and lead citrate and studied with a Hitachi H-600 electron microscope. Distances from the Z line to the epitope were obtained from electron micrographs. For spatial calibration, the A-band width (1.6 μm) was used as a standard.
Cells and skeletal muscle were solubilized and electrophoresed by using 2% to 12% acrylamide gradient gels that were stained with either Coomassie blue or ammoniacal silver stain.7
Results of the present study are given as the mean±SD, unless indicated otherwise. Significance in selected parameters was examined by Student’s t test (P<.05).
Titin in Cardiac Myocytes
To assess the integrity of titin in isolated cardiac myocytes, we measured the passive tension–sarcomere length relation of cardiac cells and compared it with results from skeletal muscle fibers. Because passive tension is derived not only from titin but also from intermediate filaments (IFs), we dissected the contribution of titin to passive tension using a KCl/KI treatment (compare with Reference 77 ), which abolishes titin-based tension while leaving that of IFs intact.7 12 17 18 We found that both IFs and titin developed much higher levels of tension in cardiac myocytes than in skeletal muscle fibers (Fig 1⇓). At a sarcomere length of 2.3 μm, tension developed by IFs and titin was 10 to 20 times higher in cardiac cells than in skeletal muscle fibers. Titin-based passive tension increased exponentially in both muscle types (Fig 1⇓). The increase was steepest in cardiac cells. The high levels of passive tension developed by cardiac titin confirm earlier findings7 and indicate that titin is not degraded by our cardiac myocyte isolation procedure.
We also electrophoresed solubilized cardiac myocyte proteins side by side with rabbit skeletal muscle. Cardiac titin consisted of a doublet: T1 and T2 (inset, Fig 1⇑). T1 of rat cardiac muscle migrated much further on the gels than did T1 of skeletal muscle, confirming the earlier finding that rat cardiac muscle expresses a low-molecular-mass size variant of titin.7 T2 is generally considered a product of degradation of the parent molecule, T1, a process occurring during sample preparation.1 The level of degradation in rat cardiac myocytes (inset, Fig 1⇑) is typical of the degradation seen in most skeletal muscles,12 indicating that collagenase digestion of the heart did not result in any additional titin degradation.
Immunolabeling of Myocytes
When cardiac cells were labeled with the anti-titin antibody T12, a single stripe appeared in the I band in close proximity to the Z line (Fig 2⇓). The distance between the center of the T12 epitope and the center of the Z line in slack sarcomeres was ≈100 nm. The same distance was maintained in sarcomeres that had shortened before immunolabeling to below the slack length, some of which were shorter than the A-band width (Fig 2C⇓). In sarcomeres that had been stretched, the T12 epitope was also found ≈100 nm from the Z line (Fig 2D⇓ and 2E⇓). For example, at a sarcomere length of 2.0 μm, the distance from the Z line to the epitope was 98.1±9.4 nm (n=9), and at 2.5 μm, the distance was 105±7 nm (n=13).
Under the experimental conditions used in this study, the antibody Ti-102 labeled a fine line at the edge of the A band (Fig 3⇓). The distance between the Ti-102 epitope and the middle of the A band was 841±40 nm (n=9) at a sarcomere length of 2.0 μm and 807±33 nm (n=13) at a length of 2.5 μm. The Ti-102 epitope is thus at the very edge of the A band, and its position with respect to the A band is independent of sarcomere length.
Double labeling with both Ti-102 and T12 antibodies revealed that the I-band segment of titin between the Ti-102 and T12 epitopes is elastic (Fig 3C⇑ and 3D⇑). The T12–to–Ti-102 segment is very short in sarcomeres near the slack length (bottom sarcomeres of Fig 3D⇑) and highly elongated when sarcomeres are stretched (top sarcomeres of Fig 3D⇑). At a length of ≈2.9 μm, both the T12 and Ti-102 epitopes lost their alignment in the sarcomere, and the epitope zones broadened and became fuzzy (Fig 4⇓). Similar phenomena have been reported for highly stretched skeletal muscle fibers6 19 and have been ascribed to detachment of the titin filament from the thick filament. Our results in Fig 4⇓ indicate that such detachment may also occur in cardiac muscle. Additionally, misalignment of thick filaments at high degrees of sarcomere stretch can also be expected to result in broadening of the epitope zones.
The anti-titin antibody 9D10 labeled a single epitope in the I band, approximately in the middle of the segment demarcated by T12 and Ti-102 (Fig 5⇓). The 9D10 epitope position was sensitive to sarcomere length and moved away from both the T12 and Ti-102 epitopes when sarcomeres were stretched (Fig 5⇓). This indicates that the titin segments between T12 and 9D10 (segment A) and the segment between 9D10 and Ti-102 (segment B) are both elastic. We measured the length of segment A and segment B at sarcomere lengths of 2.0 and 2.5 μm, respectively. Segment A increased in length from 37±11 (n=10) to 142±20 nm (n=11), whereas segment B increased from 64.3±21 (n=10) to 170±26 nm (n=11). In sarcomeres that were close to the slack length, 9D10, T12, and Ti-102 epitopes could not be distinguished separately, because the epitopes were merged together (Fig 5C⇓).
We studied the length of the domain of the titin molecule in cardiac muscle that is elastic and mechanically active in developing passive tension. Specific locations along the titin molecule were marked by monoclonal antibodies, and their positions were followed by immunoelectron microscopy after stretching the sarcomere to varying lengths. Only a relatively small fraction (25% to 50%) of the unstrained I-band domain of titin turned out to be elastic.
The T12 epitope is located ≈100 nm from the center of the Z line, and the Ti-102 epitope is located at the edge of the A band (Figs 2⇑ and 3⇑). It is likely that the titin molecule extends beyond these epitopes, because rat cardiac titin has been reported to cross-react with anti-titin antibodies that in skeletal muscle label either in the A band or in the region between the middle of the Z line and the T12 epitope (Reference 44 and K. Trombitás, unpublished data, 1995). Thus, it is likely that in cardiac muscle a single titin molecule spans from the Z line to the M line, as it does in skeletal muscle. The finding that both the distance from the Z line to T12 and the distance from Ti 102 to the M line are independent of sarcomere length (Figs 2⇑ and 3⇑) suggests that the ≈100-nm-long titin domain between the Z line and the T12 epitope and the ≈800-nm-long domain between the Ti-102 epitope and the M line are inelastic. Earlier, we estimated the contour length of unstretched rat cardiac titin to be ≈1000 nm,7 and subtracting the inelastic domains leaves ≈100 nm to span from the T12 to the Ti-102 epitopes.
In skeletal muscle, T12 labels titin ≈100 nm from the Z line, and the distance from the Z line to T12 is independent of sarcomere length,4 13 as we found for cardiac muscle. Further, those antibodies that label skeletal muscle either in the I band near the edge of the A band or in the A band maintain their positions relative to the M line over a wide range of sarcomere lengths.13 19 20 Therefore, it is likely that the ≈100-nm-long titin domain that starts at the Z line and the ≈800-nm-long titin domain in the A band are inelastic in all striated muscles.
It has been reported that the titin domain between the Z line and T12 epitope and the domain in the A band behave elastically in skeletal muscle stretched to extreme lengths. At sarcomere lengths of >≈4 μm, epitopes close to the edge of the A band move into the I band,6 12 whereas the T12 epitope moves away from the Z line.13 We found the same in cardiac myocytes stretched to lengths of >≈2.9 μm (Fig 4⇑). In those highly extended sarcomeres, the stress exerted on the T12 and Ti-102 epitopes by the elastic titin domain exceeds the force with which these epitopes are anchored, pulling them loose and allowing them to behave elastically. Thus, the inelastic titin domains are intrinsically elastic but are normally rendered inextensible by being anchored to other sarcomere structures. The A-band domain of titin may be anchored to the thick filament.1 2 3 Using cloned titin fragments, Jin16 recently reported binding between titin and F-actin, and this interaction may underlie the inelastic behavior of titin in the I band. The finding that only the first 100 nm of the I-band domain is inelastic suggests that the thin-filament region close to the Z line is unique. A similar conclusion was drawn in a study on tropomyosin localization in muscle.21
The ≈100-nm-long titin segment spanning from the T12 to the Ti-102 epitopes is elastic, as indicated by the translocation of the 9D10 epitope away from both the T12 and Ti-102 epitopes that occurs upon sarcomere stretch (Fig 5⇑). It cannot be excluded, however, that the T12–to–Ti-102 segment contains regions that are inelastic. It has been shown in skeletal muscle that the I-band domain of titin contains a 50-nm-long segment adjoining the A band that is inelastic.6 If such a region existed in cardiac muscle, then the remaining elastic domain would be only ≈50 nm long. Such domain would be able to elongate maximally about eightfold,22 allowing it to reach a length of ≈400 nm. However, we found that the T12 to Ti-102 segment can attain a length of ≈550 nm before the T12 and Ti-102 epitopes start to translocate (Fig 4⇑ and K. Trombitás, H. Granzier, unpublished data, 1995). If we assume that this 550-nm-long segment has been strained eightfold,22 then its unstrained length will be ≈70 nm. Although the exact length of the unstrained extensible titin segment remains to be established, it is likely to be somewhere between 50 and 100 nm, or 25% to 50% of the total I-band domain of titin. This is much shorter than the unstrained extensible I-band domain of titin in skeletal muscle. For example, in rabbit semitendinosus muscle, the total I-band domain when unstrained is ≈350 nm,12 of which ≈150 nm is inextensible4 6 13 and ≈200 nm is extensible. For a given degree of sarcomere stretch, the extensible I-band domain of titin will thus be strained to a much higher degree in cardiac than in skeletal muscle, and this is likely to underlie the much higher passive tensions developed by cardiac muscle (Fig 1⇑).
The T12, 9D10, and Ti-102 epitopes all merge in sarcomeres that are slack (Fig 5C⇑). Therefore, it is likely that in slack sarcomeres the elastic titin domain is folded on top of itself. This highly folded state is probably similar to that of the nodules that are often seen when purified native titin is viewed in the electron microscope23 24 and to that of the highly retracted titin seen in sarcomeres that have been “freeze-broken.”25 The entropy of such highly folded titin will be maximal, and force will have to be exerted to unfold it and lower its entropy.26 We propose that in cardiac muscle the titin-based passive tension at sarcomere lengths from ≈1.85 μm to ≈2.00 μm is developed by unfolding of the elastic titin domain. At a sarcomere length of ≈2.0 μm, the I-band width will be equal to the contour length of the total I-band domain of titin,7 and the elastic titin domain will be fully unfolded at this length. The domain will respond to further stretch by elongating. Elongation continues until a sarcomere length of ≈2.9 μm is reached (Fig 4⇑). At this length, the degree of elongation will be maximal (≈eightfold extension), and further sarcomere stretch results in recruitment of the inelastic A-band and Z-line domains to the elastic pool. We also propose that during shortening to below the slack length, titin develops a restoring force that brings sarcomeres back to the slack length. In sarcomeres below the slack length, the T12 epitope remains ≈100 nm away from the Z line (Fig 2C⇑). The ends of the thick filaments will thus move past the T12 epitope toward the Z line, extending the highly folded elastic titin domain in the process, just as occurs when the sarcomere length increases beyond slack. Unfolding of titin, both below and above the slack length, and titin elongation at lengths longer than ≈2.0 μm occur over the working range of sarcomeres in the heart (≈1.8 to 2.4 μm27 ) and are likely to play important roles in normal heart function.
This study was supported by a Grant-in-Aid from the American Heart Association, Washington State Affiliate, Inc, to Dr Granzier, by a Whitaker Foundation grant for biomedical research to Dr Granzier, by a grant-in-aid from the Heart and Stroke Foundation of Alberta to Dr Jin, by a development grant from the Medical Research Council of Canada to Dr Jin, and by a Hungarian OTKA T6280 grant to Dr Trombitás. We express our gratitude to Dr Miklós Kellermayer for critical reading of various drafts of the manuscript and to Bronislava Stockman for superb technical assistance.
Reprint requests to Dr Henk Granzier, Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, WA 99164-6520. E-mail email@example.com.
- Received May 19, 1995.
- Accepted August 1, 1995.
- © 1995 American Heart Association, Inc.
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