Mechanics and structure of titin oligomers explored with atomic force microscopy

Titin is a giant polypeptide that spans half of the striated muscle sarcomere and generates passive force upon stretch. To explore the elastic response and structure of single molecules and oligomers of titin, we carried out molecular force spectroscopy and atomic force microscopy (AFM) on purified full-length skeletal-muscle titin. From the force data, apparent persistence lengths as long as approximately 1.5 nm were obtained for the single, unfolded titin molecule. Furthermore, data suggest that titin molecules may globally associate into oligomers which mechanically behave as independent wormlike chains (WLCs). Consistent with this, AFM of surface-adsorbed titin molecules revealed the presence of oligomers. Although oligomers may form globally via head-to-head association of titin, the constituent molecules otherwise appear independent from each other along their contour. Based on the global association but local independence of titin molecules, we discuss a mechanical model of the sarcomere in which titin molecules with different contour lengths, corresponding to different isoforms, are held in a lattice. The net force response of aligned titin molecules is determined by the persistence length of the tandemly arranged, different WLC components of the individual molecules, the ratio of their overall contour lengths, and by domain unfolding events. Biased domain unfolding in mechanically selected constituent molecules may serve as a compensatory mechanism for contour- and persistence-length differences. Variation in the ratio and contour length of the component chains may provide mechanisms for the fine-tuning of the sarcomeric passive force response.     

Are titin properties reflected in single myofibrils?

Titin is a structural protein in muscle that spans the half sarcomere from Z-band to M-line. Although there are selected studies on titin's mechanical properties from tests on isolated molecules or titin fragments, little is known about its behavior within the structural confines of a sarcomere. Here, we tested the hypothesis that titin properties might be reflected well in single myofibrils. Single myofibrils from rabbit psoas were prepared for measurement of passive stretch-shortening cycles at lengths where passive titin forces occur. Three repeat stretch-shortening cycles with magnitudes between 1.0 and 3.0μm/sarcomere were performed at a speed of 0.1μm/s·sarcomere and repeated after a ten minute rest at zero force. These tests were performed in a relaxation solution (passive) and an activation solution (active) where cross-bridge attachment was inhibited with 2,3 butanedionemonoxime. Myofibrils behaved viscoelastically producing an increased efficiency with repeat stretch-shortening cycles, but a decreased efficiency with increasing stretch magnitudes. Furthermore, we observed a first distinct inflection point in the force-elongation curve at an average sarcomere length of 3.5μm that was associated with an average force of 68±5nN/mm. This inflection point was thought to reflect the onset of Ig domain unfolding and was missing after a ten minute rest at zero force, suggesting a lack of spontaneous Ig domain refolding. These passive myofibrillar properties observed here are consistent with those observed in isolated titin molecules, suggesting that the mechanics of titin are well preserved in isolated myofibrils, and thus, can be studied readily in myofibrils, rather than in the extremely difficult and labile single titin preparations.     

Titin (Visco-) Elasticity in Skeletal Muscle Myofibrils

Titin is the third most abundant protein in sarcomeres and fulfills a number of mechanical and signaling functions. Specifically, titin is responsible for most of the passive forces in sarcomeres and the passive visco-elastic behaviour of myofibrils and muscles. It has been suggested, based on mechanical testing of isolated titin molecules, that titin is an essentially elastic spring if Ig domain un/refolding is prevented either by working at short titin lengths, prior to any unfolding of Ig domains, or at long sarcomere (and titin) lengths when Ig domain un/refolding is effectively prevented. However, these properties of titin, and by extension of muscles, have not been tested with titin in its natural structural environment within a sarcomere. The purpose of this study was to gain insight into the Ig domain un/refolding kinetics and test the idea that titin could behave essentially elastically at any sarcomere length by preventing Ig domain un/refolding during passive stretch-shortening cycles. Although not completely successful, we demonstrate here that titin’s visco-elastic properties appear to depend on the Ig domain un/refolding kinetics and that indeed, titin (and thus myofibrils) can become virtually elastic when Ig domain un/refolding is prevented.     

Titin Domains Progressively Unfolded by Force Are Homogenously Distributed along the Molecule

Titin is a giant filamentous protein of the muscle sarcomere in which stretch induces the unfolding of its globular domains. However, the mechanisms of how domains are progressively selected for unfolding and which domains eventually unfold have for long been elusive. Based on force-clamp optical tweezers experiments we report here that, in a paradoxical violation of mechanically driven activation kinetics, neither the global domain unfolding rate, nor the folded-state lifetime distributions of full-length titin are sensitive to force. This paradox is reconciled by a gradient of mechanical stability so that domains are gradually selected for unfolding as the magnitude of the force field increases. Atomic force microscopic screening of extended titin molecules revealed that the unfolded domains are distributed homogenously along the entire length of titin, and this homogeneity is maintained with increasing overstretch. Although the unfolding of domains with progressively increasing mechanical stability makes titin a variable viscosity damper, the spatially randomized variation of domain stability ensures that the induced structural changes are not localized but are distributed along the molecule''s length. Titin may thereby provide complex safety mechanims for protecting the sarcomere against structural disintegration under excessive mechanical conditions.     

Titin elasticity and mechanism of passive force development in rat cardiac myocytes probed by thin-filament extraction.

Titin (also known as connectin) is a giant filamentous protein whose elastic properties greatly contribute to the passive force in muscle. In the sarcomere, the elastic I-band segment of titin may interact with the thin filaments, possibly affecting the molecule's elastic behavior. Indeed, several studies have indicated that interactions between titin and actin occur in vitro and may occur in the sarcomere as well. To explore the properties of titin alone, one must first eliminate the modulating effect of the thin filaments by selectively removing them. In the present work, thin filaments were selectively removed from the cardiac myocyte by using a gelsolin fragment. Partial extraction left behind approximately 100-nm-long thin filaments protruding from the Z-line, whereas the rest of the I-band became devoid of thin filaments, exposing titin. By applying a much more extensive gelsolin treatment, we also removed the remaining short thin filaments near the Z-line. After extraction, the extensibility of titin was studied by using immunoelectron microscopy, and the passive force-sarcomere length relation was determined by using mechanical techniques. Titin's regional extensibility was not detectably affected by partial thin-filament extraction. Passive force, on the other hand, was reduced at sarcomere lengths longer than approximately 2.1 microm, with a 33 +/- 9% reduction at 2.6 microm. After a complete extraction, the slack sarcomere length was reduced to approximately 1.7 microm. The segment of titin near the Z-line, which is otherwise inextensible, collapsed toward the Z-line in sarcomeres shorter than approximately 2.0 microm, but it was extended in sarcomeres longer than approximately 2.3 microm. Passive force became elevated at sarcomere lengths between approximately 1.7 and approximately 2.1 microm, but was reduced at sarcomere lengths of >2.3 microm. These changes can be accounted for by modeling titin as two wormlike chains in series, one of which increases its contour length by recruitment of the titin segment near the Z-line into the elastic pool.     

Altered mechanical properties of titin immunoglobulin domain 27 in the presence of calcium

Titin (connectin) based passive force regulation has been an important physiological mechanism to adjust to varying muscle stretch conditions. Upon stretch, titin behaves as a spring capable of modulating its elastic response in accordance with changes in muscle biochemistry. One such mechanism has been the calcium-dependent stiffening of titin domains that renders the spring inherently more resistant to stretch. This transient titin-calcium interaction may serve a protective function in muscle, which could preclude costly unfolding of select domains when muscles elongate to great lengths. To test this idea, fluorescence spectroscopy was performed revealing a change in the microenvironment of the investigated immunoglobulin domain 27 (I27) of titin with calcium. Additionally, an atomic force microscope was used to evaluate the calcium-dependent regulation of passive force by stretching eight linked titin I27 domains until they unfolded. When stretching in the presence of calcium, the I27 homopolymer chain became stabilized, displaying three novel properties: (1) higher stretching forces were needed to unfold the domains, (2) the stiffness, measured as a persistence length (PL), increased and (3) the peak-to-peak distance between adjacent I27 domains increased. Furthermore, a peak order dependence became apparent for both force and PL, reflecting the importance of characterizing the dynamic unfolding history of a polymer with this approach. Together, this novel titin Ig-calcium interaction may serve to stabilize the I27 domain permitting titin to tune passive force within stretched muscle in a calcium-dependent manner.     

Hierarchical extensibility in the PEVK domain of skeletal-muscle titin

Titin is the main determinant of passive muscle force. Physiological extension of titin derives largely from its PEVK (Pro-Glu-Val-Lys) domain, which has a different length in different muscle types. Here we characterized the elasticity of the full-length, human soleus PEVK domain by mechanically manipulating its contiguous, recombinant subdomain segments: an N-terminal (PEVKI), a middle (PEVKII), and a C-terminal (PEVKIII) one third. Measurement of the apparent persistence lengths revealed a hierarchical arrangement according to local flexibility: the N-terminal PEVKI is the most rigid and the C-terminal PEVKIII is the most flexible segment within the domain. Immunoelectron microscopy supported the hierarchical extensibility within the PEVK domain. The effective persistence lengths decreased as a function of ionic strength, as predicted by the Odijk-Skolnick-Fixman model of polyelectrolyte chains. The ionic strength dependence of persistence length was similar in all segments, indicating that the residual differences in the elasticity of the segments derive from nonelectrostatic mechanisms.     

Stretching molecular springs: elasticity of titin filaments in vertebrate striated muscle

Titin, the giant protein of striated muscle, provides a continuous link between the Z-disk and the M-line of a sarcomere. The elastic I-band section of titin comprises two main structural elements, stretches of immunoglobulin-like domains and a unique sequence, the PEVK segment. Both elements contribute to the extensibility and passive force development of nonactivated muscle. Extensibility of the titin segments in skeletal muscle has been determined by immunofluorescence/immunoelectron microscopy of sarcomeres stained with sequence-assigned titin antibodies. The force developed upon stretch of titin has been measured on isolated molecules or recombinant titin fragments with the help of optical tweezers and the atomic force microscope. Force has also been measured in single isolated myofibrils. The force-extension relation of titin could be readily fitted with models of biopolymer elasticity. For physiologically relevant extensions, the elasticity of the titin segments was largely explainable by an entropic-spring mechanism. The modelling explains why during stretch of titin, the Ig-domain regions (with folded modules) extend before the PEVK domain. In cardiac muscle, I-band titin is expressed in different isoforms, termed N2-A and N2-B. The N2-A isoform resembles that of skeletal muscle, whereas N2-B titin is shorter and is distinguished by cardiac-specific Ig-motifs and nonmodular sequences within the central I-band section. Examination of N2-B titin extensibility revealed that this isoform extends by recruiting three distinct elastic elements: poly-Ig regions and the PEVK domain at lower stretch and, in addition, a unique 572-residue sequence insertion at higher physiological stretch. Extension of all three elements allows cardiac titin to stretch fully reversibly at physiological sarcomere lengths, without the need to unfold individual Ig domains. However, unfolding of a very small number of Ig domains remains a possibility.     

Molecular mechanics of cardiac titin's PEVK and N2B spring elements.

Titin is a giant elastic protein that is responsible for the majority of passive force generated by the myocardium. Titin's force is derived from its extensible I-band region, which, in the cardiac isoform, comprises three main extensible elements: tandem Ig segments, the PEVK domain, and the N2B unique sequence (N2B-Us). Using atomic force microscopy, we characterized the single molecule force-extension curves of the PEVK and N2B-Us spring elements, which together are responsible for physiological levels of passive force in moderately to highly stretched myocardium. Stretch-release force-extension curves of both the PEVK domain and N2B-Us displayed little hysteresis: the stretch and release data nearly overlapped. The force-extension curves closely followed worm-like chain behavior. Histograms of persistence length (measure of chain bending rigidity) indicated that the single molecule persistence lengths are approximately 1.4 and approximately 0.65 nm for the PEVK domain and N2B-Us, respectively. Using these mechanical characteristics and those determined earlier for the tandem Ig segment (assuming folded Ig domains), we modeled the cardiac titin extensible region in the sarcomere and calculated the extension of the various spring elements and the forces generated by titin, both as a function of sarcomere length. In the physiological sarcomere length range, predicted values and those obtained experimentally were indistinguishable.     

Unfolding of titin domains explains the viscoelastic behavior of skeletal myofibrils

The elastic section of the giant muscle protein titin contains many immunoglobulin-like domains, which have been shown by single-molecule mechanical studies to unfold and refold upon stretch-release. Here we asked whether the mechanical properties of Ig domains and/or other titin regions could be responsible for the viscoelasticity of nonactivated skeletal-muscle sarcomeres, particularly for stress relaxation and force hysteresis. We show that isolated psoas myofibrils respond to a stretch-hold protocol with a characteristic force decay that becomes more pronounced following stretch to above 2.6-microm sarcomere length. The force decay was readily reproducible by a Monte Carlo simulation taking into account both the kinetics of Ig-domain unfolding and the worm-like-chain model of entropic elasticity used to describe titin's elastic behavior. The modeling indicated that the force decay is explainable by the unfolding of only a very small number of Ig domains per titin molecule. The simulation also predicted that a unique sequence in titin, the PEVK domain, may undergo minor structural changes during sarcomere extension. Myofibrils subjected to 1-Hz cycles of stretch-release exhibited distinct hysteresis that persisted during repetitive measurements. Quick stretch-release protocols, in which variable pauses were introduced after the release, revealed a two-exponential time course of hysteresis recovery. The rate constants of recovery compared well with the refolding rates of Ig-like or fibronectin-like domains measured by single-protein mechanical analysis. These findings suggest that in the sarcomere, titin's Ig-domain regions may act as entropic springs capable of adjusting their contour length in response to a stretch.     

Individual Globular Domains and Domain Unfolding Visualized in Overstretched Titin Molecules with Atomic Force Microscopy

Titin is a giant elastomeric protein responsible for the generation of passive muscle force. Mechanical force unfolds titin’s globular domains, but the exact structure of the overstretched titin molecule is not known. Here we analyzed, by using high-resolution atomic force microscopy, the structure of titin molecules overstretched with receding meniscus. The axial contour of the molecules was interrupted by topographical gaps with a mean width of 27.7 nm that corresponds well to the length of an unfolded globular (immunoglobulin and fibronectin) domain. The wide gap-width distribution suggests, however, that additional mechanisms such as partial domain unfolding and the unfolding of neighboring domain multimers may also be present. In the folded regions we resolved globules with an average spacing of 5.9 nm, which is consistent with a titin chain composed globular domains with extended interdomain linker regions. Topographical analysis allowed us to allocate the most distal unfolded titin region to the kinase domain, suggesting that this domain systematically unfolds when the molecule is exposed to overstretching forces. The observations support the prediction that upon the action of stretching forces the N-terminal ß-sheet of the titin kinase unfolds, thus exposing the enzyme’s ATP-binding site and hence contributing to the molecule’s mechanosensory function.     

Positive feedback by a potassium-selective inward rectifier enhances tuning in vertebrate hair cells.

Titin (also known as connectin) is a muscle-specific giant protein found inside the sarcomere, spanning from the Z-line to the M-line. The I-band segment of titin is considered to function as a molecular spring that develops tension when sarcomeres are stretched (passive tension). Recent studies on skeletal muscle indicate that it is not the entire I-band segment of titin that behaves as a spring; some sections are inelastic and do not take part in the development of passive tension. To better understand the mechanism of passive tension development in the heart, where passive tension plays an essential role in the pumping function, we investigated titin's elastic segment in cardiac myocytes using structural and mechanical techniques. Single cardiac myocytes were stretched by various amounts and then immunolabeled and processed for electron microscopy in the stretched state. Monoclonal antibodies that recognize different titin epitopes were used, and the locations of the titin epitopes in the sarcomere were studied as a function of sarcomere length. We found that only a small region of the I-band segment of titin is elastic; its contour length is estimated at approximately 75 nm, which is only approximately 40% of the total I-band segment of titin. Passive tension measurements indicated that the fundamental determinant of how much passive tension the heart develops is the strain of titin's elastic segment. Furthermore, we found evidence that in sarcomeres that are slack (length, approximately 1.85 microns) the elastic titin segment is highly folded on top of itself. Based on the data, we propose a two-stage mechanism of passive tension development in the heart, in which, between sarcomere lengths of approximately 1.85 microns and approximately 2.0 microns, titin's elastic segment straightens and, at lengths longer than approximately 2.0 microns, the molecular domains that make up titin's elastic segment unravel. Sarcomere shortening to lengths below slack (approximately 1.85 microns) also results in straightening of the elastic titin segment, giving rise to a force that opposes shortening and that tends to bring sarcomeres back to their slack length.     

Towards a Molecular Understanding of the Elasticity of Titin

Vertebrate striated muscle behaves elastically when stretched and this property is thought to reside primarily within the giant filamentous protein, titin (connectin). The elastic portion of titin comprises two distinct structural motifs, immunoglobulin (Ig) domains and the PEVK titin, which is a novel motif family rich in proline, glutamate, valine and lysine residues. The respective contributions of the titin Ig and the PEVK sequences to the elastic properties of the molecule have been unknown so far. We have measured both the passive tension in single, isolated myofibrils from cardiac and skeletal muscle and the stretch-induced translational movement of I-band titin antibody epitopes following immunofluorescent labelling of sites adjacent to the PEVK and Ig domain regions. We found that with myofibril stretch, I-band titin does not extend homogeneously. The Ig domain region lengthened predominantly during small stretch, but such lengthening did not result in measurable passive tension and might be explained by straightening, rather than by unfolding, of the Ig repeats. At moderate to extreme stretch, the main extensible region was found to be the PEVK segment whose unravelling was correlated with a steady passive tension increase. In turn, PEVK domain transition from a linearly extended to a folded state appears to be principally responsible for the elasticity of muscle fibers. Thus, the length of the PEVK sequence may determine the tissue-specificity of muscle stiffness, whereas the expression of different Ig domain motif lengths may set the characteristic slack sarcomere length of a muscle type.     

Myomesin is a molecular spring with adaptable elasticity

The M-band is a transverse structure in the center of the sarcomere, which is thought to stabilize the thick filament lattice. It was shown recently that the constitutive vertebrate M-band component myomesin can form antiparallel dimers, which might cross-link the neighboring thick filaments. Myomesin consists mainly of immunoglobulin-like (Ig) and fibronectin type III (Fn) domains, while several muscle types express the EH-myomesin splice isoform, generated by the inclusion of the unique EH-segment of about 100 amino acid residues (aa) in the center of the molecule. Here we use atomic force microscopy (AFM), transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy for the biophysical characterization of myomesin. The AFM identifies the "mechanical fingerprints" of the modules constituting the myomesin molecule. Stretching of homomeric polyproteins, constructed of Ig and Fn domains of human myomesin, produces a typical saw-tooth pattern in the force-extension curve. The domains readily refold after relaxation. In contrast, stretching of a heterogeneous polyprotein, containing several repeats of the My6-EH fragment reveals a long initial plateau corresponding to the sum of EH-segment contour lengths, followed by several My6 unfolding peaks. According to this, the EH-segment is characterized as an entropic chain with a persistence length of about 0.3nm. In TEM pictures, the EH-domain appears as a gap in the molecule, indicating a random coil conformation similar to the PEVK region of titin. CD spectroscopy measurements support this result, demonstrating a mostly non-folded conformation for the EH-segment. We suggest that similarly to titin, myomesin is a molecular spring, whose elasticity is modulated by alternative splicing. The Ig and Fn domains might function as reversible "shock absorbers" by sequential unfolding in the case of extremely high or long sustained stretching forces. These complex visco-elastic properties of myomesin might be crucial for the stability of the sarcomere.     

Modeling AFM-induced PEVK extension and the reversible unfolding of Ig/FNIII domains in single and multiple titin molecules

Molecular elasticity is associated with a select number of polypeptides and proteins, such as titin, Lustrin A, silk fibroin, and spider silk dragline protein. In the case of titin, the globular (Ig) and non-globular (PEVK) regions act as extensible springs under stretch; however, their unfolding behavior and force extension characteristics are different. Using our time-dependent macroscopic method for simulating AFM-induced titin Ig domain unfolding and refolding, we simulate the extension and relaxation of hypothetical titin chains containing Ig domains and a PEVK region. Two different models are explored: 1) a series-linked WLC expression that treats the PEVK region as a distinct entropic spring, and 2) a summation of N single WLC expressions that simulates the extension and release of a discrete number of parallel titin chains containing constant or variable amounts of PEVK. In addition to these simulations, we also modeled the extension of a hypothetical PEVK domain using a linear Hooke's spring model to account for "enthalpic" contributions to PEVK elasticity. We find that the modified WLC simulations feature chain length compensation, Ig domain unfolding/refolding, and force-extension behavior that more closely approximate AFM, laser tweezer, and immunolocalization experimental data. In addition, our simulations reveal the following: 1) PEVK extension overlaps with the onset of Ig domain unfolding, and 2) variations in PEVK content within a titin chain ensemble lead to elastic diversity within that ensemble.     

Steered molecular dynamics studies of titin I1 domain unfolding

The cardiac muscle protein titin, responsible for developing passive elasticity and extensibility of muscle, possesses about 40 immunoglobulin-like (Ig) domains in its I-band region. Atomic force microscopy (AFM) and steered molecular dynamics (SMD) have been successfully combined to investigate the reversible unfolding of individual Ig domains. However, previous SMD studies of titin I-band modules have been restricted to I27, the only structurally known Ig domain from the distal region of the titin I-band. In this paper we report SMD simulations unfolding I1, the first structurally available Ig domain from the proximal region of the titin I-band. The simulations are carried out with a view toward upcoming atomic force microscopy experiments. Both constant velocity and constant force stretching have been employed to model mechanical unfolding of oxidized I1, which has a disulfide bond bridging beta-strands C and E, as well as reduced I1, in which the disulfide bridge is absent. The simulations reveal that I1 is protected against external stress mainly through six interstrand hydrogen bonds between its A and B beta-strands. The disulfide bond enhances the mechanical stability of oxidized I1 domains by restricting the rupture of backbone hydrogen bonds between the A'- and G-strands. The disulfide bond also limits the maximum extension of I1 to approximately 220 A. Comparison of the unfolding pathways of I1 and I27 are provided and implications to AFM experiments are discussed.     

Association of the chaperone alphaB-crystallin with titin in heart muscle

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

The mechanical stability of immunoglobulin and fibronectin III domains in the muscle protein titin measured by atomic force microscopy.

The domains of the giant muscle protein titin (connectin) provide interaction sites for other sarcomeric proteins and fulfill mechanical functions. In this paper we compare the unfolding forces of defined regions of different titin isoforms by single-molecule force spectroscopy. Constructs comprising six to eight immunoglobulin (Ig) domains located in the mechanically active I-band part of titin are compared to those containing fibronectin III (Fn3) and Ig domains from the A-band part. The high spatial resolution of the atomic force microscope allows us to detect differences in length as low as a few amino acids. Thus constructs of different lengths may be used as molecular rulers for structural comparisons with other modular proteins. The unfolding forces range between 150 and 300 pN and differ systematically between the constructs. Fn3 domains in titin exhibit 20% lower unfolding forces than Ig domains. Fn3 domains from tenascin, however, unfold at forces only half those of titin Fn3 domains. This indicates that the tightly folded titin domains are designed to maintain their structural integrity, even under the influence of stretching forces. Hence, at physiological forces, unfolding is unlikely unless the forces are applied for a long time (longer than minutes).     

Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments.

The passive tension-sarcomere length relation of rat cardiac muscle was investigated by studying passive (or not activated) single myocytes and trabeculae. The contribution of collagen, titin, microtubules, and intermediate filaments to tension and stiffness was investigated by measuring (1) the effects of KCl/KI extraction on both trabeculae and single myocytes, (2) the effect of trypsin digestion on single myocytes, and (3) the effect of colchicine on single myocytes. It was found that over the working range of sarcomeres in the heart (lengths approximately 1.9-2.2 microns), collagen and titin are the most important contributors to passive tension with titin dominating at the shorter end of the working range and collagen at longer lengths. Microtubules made a modest contribution to passive tension in some cells, but on average their contribution was not significant. Finally, intermediate filaments contributed about 10% to passive tension of trabeculae at sarcomere lengths from approximately 1.9 to 2.1 microns, and their contribution dropped to only a few percent at longer lengths. At physiological sarcomere lengths of the heart, cardiac titin developed much higher tensions (> 20-fold) than did skeletal muscle titin at comparable lengths. This might be related to the finding that cardiac titin has a molecular mass of 2.5 MDa, 0.3-0.5 MDa smaller than titin of mammalian skeletal muscle, which is predicted to result in a much shorter extensible titin segment in the I-band of cardiac muscle. Passive stress plotted versus the strain of the extensible titin segment showed that the stress-strain relationships are similar in cardiac and skeletal muscle. The difference in passive stress between cardiac and skeletal muscle at the sarcomere level predominantly resulted from much higher strains of the I-segment of cardiac titin at a given sarcomere length. By expressing a smaller titin isoform, without changing the properties of the molecule itself, cardiac muscle is able to develop significant levels of passive tension at physiological sarcomere lengths.     

The mechanical fingerprint of a parallel polyprotein dimer

We use the GCN4 oligomerization domain to engineer a covalently linked parallel polyprotein dimer based on the well-studied I27 domain of titin. We use single molecule atomic force microscopy techniques to stretch single polyprotein fibers and verify their mechanical properties. We find that the engineered polyprotein dimers extend in perfect register, doubling the unfolding force and halving the persistence length while keeping the contour length increase unchanged. These experiments directly confirm the mechanical scaling laws proposed for parallel bundles of modular proteins.