Titin Spring Research Articles

Titin takes centerstage among cytoskeletal contributions to myocardial passive stiffness.

Both diastolic filling and systolic pumping of the heart are dependent on the passive stiffness characteristics of various mechanical elements of myocardium. However, the specific contribution from each element, including the extracellular matrix, actin filaments, microtubules, desmin intermediate filaments, and sarcomeric titin springs, remains challenging to assess. Recently, a mouse model allowing for precise and acute cleavage of the titin springs was used to remove one mechanical element after the other from cardiac fibers and record the effect on passive stiffness. It became clear that the stiffness contribution from each element is context-dependent and varies depending on strain level and the force component considered (elastic or viscous); elements do not act in isolation but in a tensegral relationship. Titin is a substantial contributor under all conditions and dominates the elastic forces at both low and high strains. The contribution to viscous forces is more equally shared between microtubules, titin, and actin. However, the extracellular matrix substantially contributes to both force components at higher strain levels. Desmin filaments may bear low stiffness. These insights enhance our understanding of how different filament networks contribute to passive stiffness in the heart and offer new perspectives for targeting this stiffness in heart failure treatment.

Titin governs myocardial passive stiffness with major support from microtubules and actin and the extracellular matrix.

Myocardial passive stiffness is crucial for the heart's pump function and is determined by mechanical elements, including the extracellular matrix and cytoskeletal filaments; however, their individual contributions are controversially discussed and difficult to quantify. In this study, we targeted the cytoskeletal filaments in a mouse model, which enables the specific, acute and complete cleavage of the sarcomeric titin springs. We show in vitro that each cytoskeletal filament's stiffness contribution varies depending on whether the elastic or the viscous forces are considered and on strain level. Titin governs myocardial elastic forces, with the largest contribution provided at both low and high strain. Viscous force contributions are more uniformly distributed among the microtubules, titin and actin. The extracellular matrix contributes at high strain. The remaining forces after total target element disruption are likely derived from desmin filaments. Our findings answer longstanding questions about cardiac mechanical architecture and allow better targeting of passive myocardial stiffness in heart failure.

Decrypting cardiomyocyte passive stiffness through the specific manipulation of the microtubules, sarcolemma and titin springs in cardiac trabeculae

Decrypting cardiomyocyte passive stiffness through the specific manipulation of the microtubules, sarcolemma and titin springs in cardiac trabeculae

Mechanobiology of muscle and myofibril morphogenesis.

Muscles generate forces for animal locomotion. The contractile apparatus of muscles is the sarcomere, a highly regular array of large actin and myosin filaments linked by gigantic titin springs. During muscle development many sarcomeres assemble in series into long periodic myofibrils that mechanically connect the attached skeleton elements. Thus, ATP-driven myosin forces can power movement of the skeleton. Here we review muscle and myofibril morphogenesis, with a particular focus on their mechanobiology. We describe recent progress on the molecular structure of sarcomeres and their mechanical connections to the skeleton. We discuss current models predicting how tension coordinates the assembly of key sarcomeric components to periodic myofibrils that then further mature during development. This requires transcriptional feedback mechanisms that may help to coordinate myofibril assembly and maturation states with the transcriptional program. To fuel the varying energy demands of muscles we also discuss the close mechanical interactions of myofibrils with mitochondria and nuclei to optimally support powerful or enduring muscle fibers.

Unraveling the mysteries of the titin-N2A signalosome.

The sarcomeric titin springs and accessory proteins modulate muscle force and mechanical signaling at the N2A signalosome.

Regulation of titin-based cardiac stiffness by unfolded domain oxidation (UnDOx)

The relationship between oxidative stress and cardiac stiffness is thought to involve modifications to the giant muscle protein titin, which in turn can determine the progression of heart disease. In vitro studies have shown that S-glutathionylation and disulfide bonding of titin fragments could alter the elastic properties of titin; however, whether and where titin becomes oxidized in vivo is less certain. Here we demonstrate, using multiple models of oxidative stress in conjunction with mechanical loading, that immunoglobulin domains preferentially from the distal titin spring region become oxidized in vivo through the mechanism of unfolded domain oxidation (UnDOx). Via oxidation type-specific modification of titin, UnDOx modulates human cardiomyocyte passive force bidirectionally. UnDOx also enhances titin phosphorylation and, importantly, promotes nonconstitutive folding and aggregation of unfolded domains. We propose a mechanism whereby UnDOx enables the controlled homotypic interactions within the distal titin spring to stabilize this segment and regulate myocardial passive stiffness.

Deleting Titin's C-Terminal PEVK Exons Increases Passive Stiffness, Alters Splicing, and Induces Cross-Sectional and Longitudinal Hypertrophy in Skeletal Muscle.

The Proline, Glutamate, Valine and Lysine-rich (PEVK) region of titin constitutes an entropic spring that provides passive tension to striated muscle. To study the functional and structural repercussions of a small reduction in the size of the PEVK region, we investigated skeletal muscles of a mouse with the constitutively expressed C-terminal PEVK exons 219–225 deleted, the TtnΔ219–225 model (MGI: TtnTM 2.1Mgot). Based on this deletion, passive tension in skeletal muscle was predicted to be increased by ∼17% (sarcomere length 3.0 μm). In contrast, measured passive tension (sarcomere length 3.0 μm) in both soleus and EDL muscles was increased 53 ± 11% and 62 ± 4%, respectively. This unexpected increase was due to changes in titin, not to alterations in the extracellular matrix, and is likely caused by co-expression of two titin isoforms in TtnΔ219–225 muscles: a larger isoform that represents the TtnΔ219–225 N2A titin and a smaller isoform, referred to as N2A2. N2A2 represents a splicing adaption with reduced expression of spring element exons, as determined by titin exon microarray analysis. Maximal tetanic tension was increased in TtnΔ219–225 soleus muscle (WT 240 ± 9; TtnΔ219–225 276 ± 17 mN/mm2), but was reduced in EDL muscle (WT 315 ± 9; TtnΔ219–225 280 ± 14 mN/mm2). The changes in active tension coincided with a switch toward slow fiber types and, unexpectedly, faster kinetics of tension generation and relaxation. Functional overload (FO; ablation) and hindlimb suspension (HS; unloading) experiments were also conducted. TtnΔ219–225 mice showed increases in both longitudinal hypertrophy (increased number of sarcomeres in series) and cross-sectional hypertrophy (increased number of sarcomeres in parallel) in response to FO and attenuated cross-sectional atrophy in response to HS. In summary, slow- and fast-twitch muscles in a mouse model devoid of titin’s PEVK exons 219–225 have high passive tension, due in part to alterations elsewhere in splicing of titin’s spring region, increased kinetics of tension generation and relaxation, and altered trophic responses to both functional overload and unloading. This implicates titin’s C-terminal PEVK region in regulating passive and active muscle mechanics and muscle plasticity.

The Specific Cleavage of Titin Springs to Quantify the Contribution of Titin to Myocardial Passive Stiffness

The Specific Cleavage of Titin Springs to Quantify the Contribution of Titin to Myocardial Passive Stiffness

Posttranslational modifications of titin from cardiac muscle: how, where, and what for?

Titin is a giant elastic protein expressed in the contractile units of striated muscle cells, including the sarcomeres of cardiomyocytes. The last decade has seen enormous progress in our understanding of how titin molecular elasticity is modulated in a dynamic manner to help cardiac sarcomeres adjust to the varying hemodynamic demands on the heart. Crucial events mediating the rapid modulation of cardiac titin stiffness are post‐translational modifications (PTMs) of titin. In this review, we first recollect what is known from earlier and recent work on the molecular mechanisms of titin extensibility and force generation. The main goal then is to provide a comprehensive overview of current insight into the relationship between titin PTMs and cardiomyocyte stiffness, notably the effect of oxidation and phosphorylation of titin spring segments on titin stiffness. A synopsis is given of which type of oxidative titin modification can cause which effect on titin stiffness. A large part of the review then covers the mechanically relevant phosphorylation sites in titin, their location along the elastic segment, and the protein kinases and phosphatases known to target these sites. We also include a detailed coverage of the complex changes in phosphorylation at specific titin residues, which have been reported in both animal models of heart disease and in human heart failure, and their correlation with titin‐based stiffness alterations. Knowledge of the relationship between titin PTMs and titin elasticity can be exploited in the search for therapeutic approaches aimed at softening the pathologically stiffened myocardium in heart failure patients.

Specific Cleavage of the Titin Springs In Situ Uncovers the Role of Titin-Based Force in Sarcomere Structure and Muscle Contraction

The giant protein titin contributes to muscle force generation. However, titin's mechanical function is still incompletely understood, in part because no tool has been available to specifically cleave the titin springs in situ. In a new genetic mouse model, a tobacco etch virus (TEV) protease-recognition site and a HaloTag were cloned into elastic titin. The HaloTag-TEV cassette allowed for in-situ imaging of titin, specific proteolysis during myofiber mechanics and visualization of successful cleavage. Using permeabilized skeletal myofibers homozygous for HaloTag-TEV titin (knock-in; KI), we measured passive force over the sarcomere-length (SL) range 2.2-3.4 μm and Ca2+-triggered force (pCa 5.0) at 2.6 μm SL, in the absence or presence of TEV enzyme. Wildtype (WT) myofibres treated with TEV protease served as controls.Incubation of myofibers with TEV protease cleaved HaloTag-TEV titin specifically and completely in <20 min (room temperature), but had no effect on other proteins and on titin in WT myofibers. Titin cleavage affected myofiber ultrastructure (measured by electron microscopy) only little in the absence of a mechanical perturbation. However, mechanical stressing of TEV-treated KI fibers caused sarcomere destabilization. Both the Z-discs and the myosin A-bands became disordered. Passive tension dropped with titin-cleavage, on average by ∼70%. The remaining tension was attributed to other cytoskeletal structures/extracellular-matrix proteins. Active force at pCa 5.0 was reduced by titin-cleavage, on average by ∼50% vs. controls. We conclude that this model allows for the reliable quantitation of the contribution of titin-based force to passive and active muscle tension. We find that titin's spring force is not only necessary for most of the passive tension generated by skeletal muscles, but also for the regular structure of all sarcomere bands and for maintaining high Ca2+-dependent tension.

Titin as a force-generating muscle protein under regulatory control.

Titin has long been recognized as a mechanical protein in muscle cells that has a main function as a molecular spring in the contractile units, the sarcomeres. Recent work suggests that the titin spring contributes to muscle contraction in a more active manner than previously thought. In this review, we highlight this property, specifically the ability of the immunoglobulin-like (Ig) domains of titin to undergo unfolding-refolding transitions when isolated titin molecules or skeletal myofibrils are held at physiological force levels. Folding of titin Ig domains under force is a hitherto unappreciated, putative source of work production in muscle cells, which could work in synergy with the actomyosin system to maximize the energy delivered by a stretched, actively contracting muscle. This review also focuses on the mechanisms shown to modulate titin-based viscoelastic forces in skeletal muscle cells, including chaperone binding, titin oxidation, phosphorylation, Ca2+ binding, and interaction with actin filaments. Along the way, we discuss which of these modulatory mechanisms might contribute to the phenomenon of residual force enhancement relevant for eccentric muscle contractions. Finally, a brief perspective is added on the potential for the alterations in titin-based force to dynamically alter mechano-chemical signaling pathways in the muscle cell. We conclude that titin from skeletal muscle is a determinant of both passive and active tension and a bona fide mechanosensor, whose stiffness is tuned by various independent mechanisms.

Quantifying the Titin Contribution to Muscle Force Generation using a Novel Method to Specifically Cleave the Titin Springs in Situ

The giant protein titin contributes to muscle force generation. However, titin's mechanical role in muscle is still incompletely understood because of previous inability to specifically cleave the titin spring. To overcome this problem, we used a mouse model in which both a tobacco etch virus (TEV) protease-recognition site and a HaloTag were cloned into elastic I-band titin. The Halo-TEV cassette allowed for in-situ imaging of titin, specific proteolysis during myofiber mechanics, and visualization of successful cleavage on protein gels or tissue sections. Using permeabilized skeletal myofiber bundles dissected from mice homozygous for Halo-TEV-titin, we measured passive force over the sarcomere-length (SL) range 2.2-3.4 µm and maximum Ca2+-triggered force (pCa5) at 2.6 µm SL, in the absence or presence of TEV-enzyme. Wildtype myofibers treated with TEV-protease served as additional controls. We found that incubation of myofibers with TEV-protease at room temperature cleaved genetically modified titin specifically and completely in <1 hour, but had no effect on wildtype titin. Titin cleavage barely affected myofiber ultrastructure in the absence of a stretch or Ca2+-activation; however, mechanical stressing caused sarcomere destabilization and myosin-filament disarray. In both psoas and soleus myofiber bundles, passive forces dropped with titin cleavage by ∼50%, the remaining forces presumably being attributable to extracellular-matrix proteins. Subsequent treatment of the samples with high-salt buffer (0.6 M KCl/1.0 M KI) to dissolve the titin anchorage caused a further ∼25% drop in passive force, suggesting this frequently-used extraction method consistently overestimates the titin-contribution to passive tension. Active force development was reduced, on average by ∼50%, in titin-cleaved psoas or soleus fibers, compared to controls. Our approach thus allows for a direct and reliable quantitation of titin's contribution to passive and active forces in muscle.

Translocation of molecular chaperones to the titin springs is common in skeletal myopathy patients and affects sarcomere function

SummaryMyopathies encompass a wide variety of acquired and hereditary disorders. The pathomechanisms include structural and functional changes affecting, e.g., myofiber metabolism and contractile properties. In this study, we observed increased passive tension (PT) of skinned myofibers from patients with myofibrillar myopathy (MFM) caused by FLNC mutations (MFM-filaminopathy) and limb-girdle muscular dystrophy type-2A due to CAPN3 mutations (LGMD2A), compared to healthy control myofibers. Because the giant protein titin determines myofiber PT, we measured its molecular size and the titin-to-myosin ratio, but found no differences between myopathies and controls. All-titin phosphorylation and site-specific phosphorylation in the PEVK region were reduced in myopathy, which would be predicted to lower PT. Electron microscopy revealed extensive ultrastructural changes in myofibers of various hereditary myopathies and also suggested massive binding of proteins to the sarcomeric I-band region, presumably heat shock proteins (HSPs), which can translocate to elastic titin under stress conditions. Correlative immunofluorescence and immunoelectron microscopy showed that two small HSPs (HSP27 and αB-crystallin) and the ATP-dependent chaperone HSP90 translocated to the titin springs in myopathy. The small HSPs, but not HSP90, were upregulated in myopathic versus control muscles. The titin-binding pattern of chaperones was regularly observed in Duchenne muscular dystrophy (DMD), LGMD2A, MFM-filaminopathy, MFM-myotilinopathy, titinopathy, and inclusion body myopathy due to mutations in valosin-containing protein, but not in acquired sporadic inclusion body myositis. The three HSPs also associated with elastic titin in mouse models of DMD and MFM-filaminopathy. Mechanical measurements on skinned human myofibers incubated with exogenous small HSPs suggested that the elevated PT seen in myopathy is caused, in part, by chaperone-binding to the titin springs. Whereas this interaction may be protective in that it prevents sarcomeric protein aggregation, it also has detrimental effects on sarcomere function. Thus, we identified a novel pathological phenomenon common to many hereditary muscle disorders, which involves sarcomeric alterations.

Mechanically Unfolded Titin Immunoglobulin Domains Refold Faster and More Accurately in Presence of Chaperone Alpha-B-Crystallin

The spring region of the giant muscle protein titin contains many immunoglobulin-like (Ig) domains, which unfold and refold under low (physiological) stretch forces. Small heat shock proteins such as alphaB-crystallin (aBC) translocate under physiological or pathological stress to the titin springs. To better understand this protective function we studied the unfolding-refolding behavior of an 8-Ig-domain titin construct (I91)8 by single-molecule AFM force spectroscopy, in the absence/presence of recombinant aBC (pH7; pH6; or pH5) or ‘control’ protein of similar size. Titin Ig domains were unfolded at 175 pN constant force applied for a variable “denature” time (tD), then the force was set to zero for a variable “quench” time (tQ) to allow for domain refolding, and finally a “probe” pulse (175 pN; tP = 5 s) was applied to test how many domains had refolded. Interestingly, Ig domain unfolding kinetics were little affected by aBC. However, upon lowering pH from 7 to 6, the refolded fraction (number of refolded Ig domains during tQ indexed to number of unfolded Ig domains during tD) decreased slightly, indicating domain misfolding. At pH5, the refolded fraction dropped by half and ∼50% of titin Ig domains showed misfolding events, an effect independent of tD (variation, 2 - 40 s). Importantly, aBC (10 µM or 20 µM) normalized the refolded fraction to values observed at pH7, whereas control protein had no such protective effect. The refolded fraction depended strongly on tQ (variation, 0.5 - 10 s), under all experimental conditions. Ig domain refolding kinetics were greatly slowed by lowering pH from 7 to 5, as quantified on refolded fraction vs. tQ plots, on which means were fit by simple exponentials. Again, aBC normalized the refolding kinetics to those observed at pH7 in the absence of aBC, whereas control protein had no such effect. We conclude that aBC speeds up titin Ig domain refolding (novel foldase activity!) and protects from domain misfolding, especially under acidic stress, which is frequently encountered in muscle cells.

Aberrant developmental titin splicing and dysregulated sarcomere length in Thymosin β4 knockout mice

Sarcomere assembly is a highly orchestrated and dynamic process which adapts, during perinatal development, to accommodate growth of the heart. Sarcomeric components, including titin, undergo an isoform transition to adjust ventricular filling. Many sarcomeric genes have been implicated in congenital cardiomyopathies, such that understanding developmental sarcomere transitions will inform the aetiology and treatment. We sought to determine whether Thymosin β4 (Tβ4), a peptide that regulates the availability of actin monomers for polymerization in non-muscle cells, plays a role in sarcomere assembly during cardiac morphogenesis and influences adult cardiac function. In Tβ4 null mice, immunofluorescence-based sarcomere analyses revealed shortened thin filament, sarcomere and titin spring length in cardiomyocytes, associated with precocious up-regulation of the short titin isoforms during the postnatal splicing transition. By magnetic resonance imaging, this manifested as diminished stroke volume and limited contractile reserve in adult mice. Extrapolating to an in vitro cardiomyocyte model, the altered postnatal splicing was corrected with addition of synthetic Tβ4, whereby normal sarcomere length was restored. Our data suggest that Tβ4 is required for setting correct sarcomere length and for appropriate splicing of titin, not only in the heart but also in skeletal muscle. Distinguishing between thin filament extension and titin splicing as the primary defect is challenging, as these events are intimately linked. The regulation of titin splicing is a previously unrecognised role of Tβ4 and gives preliminary insight into a mechanism by which titin isoforms may be manipulated to correct cardiac dysfunction.

Alternative Splicing of Titin Restores Diastolic Function in an HFpEF-Like Genetic Murine Model (TtnΔIAjxn).

Patients with heart failure with preserved ejection fraction (HFpEF) experience elevated filling pressures and reduced ventricular compliance. The splicing factor RNA-binding motif 20 (RBM20) regulates the contour length of titin's spring region and thereby determines the passive stiffness of cardiomyocytes. Inhibition of RBM20 leads to super compliant titin isoforms (N2BAsc) that reduce passive stiffness. To determine the therapeutic potential of upregulating compliant titin isoforms in an HFpEF-like state in the mouse. Constitutive and inducible cardiomyocyte-specific RBM20-inhibited mice were produced on a Ttn(ΔIAjxn) background to assess the effect of upregulating compliant titin at the cellular and organ levels. Genetic deletion of the I-band-A-band junction (IAjxn) in titin increases strain on the spring region and causes a HFpEF-like syndrome in the mouse without pharmacological or surgical intervention. The increased strain represents a mechanical analog of deranged post-translational modification of titin that results in increased passive myocardial stiffness in patients with HFpEF. On inhibition of RBM20 in Ttn(ΔIAjxn) mice, compliant titin isoforms were expressed, diastolic function was normalized, exercise performance was improved, and pathological hypertrophy was attenuated. We report for the first time a benefit from upregulating compliant titin isoforms in a murine model with HFpEF-like symptoms. Constitutive and inducible RBM20 inhibition improves diastolic function resulting in greater tolerance to exercise. No effective therapies exists for treating this pervasive syndrome; therefore, our data on RBM20 inhibition are clinically significant.

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.

Titin stiffness modifies the force-generating region of muscle sarcomeres.

The contractile units of striated muscle, the sarcomeres, comprise the thick (myosin) and thin (actin) filaments mediating active contraction and the titin filaments determining “passive” elasticity. We hypothesized that titin may be more active in muscle contraction by directly modulating thick-filament properties. We used single-myofibril mechanical measurements and atomic force microscopy of individual sarcomeres to quantify the effects of sarcomere strain and titin spring length on both the inter-filament lattice spacing and the lateral stiffness of the actin-myosin overlap zone (A-band). We found that strain reduced the lattice spacing similarly in sarcomeres with stiff (rabbit psoas) or compliant titin (rabbit diaphragm), but increased A-band lateral stiffness much more in psoas than in diaphragm. The strain-induced alterations in A-band stiffness that occur independently of lattice spacing effects may be due to titin stiffness-sensing by A-band proteins. This mechanosensitivity could play a role in the physiologically important phenomenon of length-dependent activation of striated muscle.

I-Band Titin Interaction with Myosin in the Muscle Sarcomere during Eccentric Contraction: The Titin Entanglement Hypothesis

The mechanisms underlying muscle contraction have largely been defined within a two filament, actin and myosin based model. The discovery of a third filament decades later called titin, has yet to be adequately incorporated into contemporary thinking, despite mounting evidence for the significance of this molecular spring. Upon muscle stretch, this titin spring works to restore muscles to their resting length by developing a passive force. The magnitude of this passive force can be quite variable due to titin's dynamic nature, but one of the most significant changes to force development may arise from a reversible titin truncation through transient binding to neighboring actin filaments. When titin was fluorescently labelled within the muscle myofibril, segments of I-band titin were tracked during passive and active stretch in order to evaluate a titin-actin interaction. Interestingly, a titin-actin binding was not observed, but unexpectedly, a titin-myosin entanglement ensued whereby parts of I-band titin overlapped spatially with myosin filaments. When muscles were activated, the distal Ig and part of the PEVK region became firmly anchored to the myosin thick filament, while the proximal Ig and PEVK regions were forced to elongate dramatically to accommodate the muscle lengthening. This pattern remained until a threshold stress was achieved, after which point titin was rapidly freed from myosin, and the elongation pattern reversed. After this threshold, the pattern of active titin elongation resembled that of the passive titin elongation. This previously unimagined mechanism has the potential to explain some of the departures from theory that the two filament model cannot, and may have great implications for muscle contraction.

Direct Evidence for the Effect of Titin Stiffness on Thick-Filament Mechanical Properties in Stretched Muscle Sarcomeres

The titin springs of muscle sarcomeres determine tensile muscle stress. However, they run not strictly in parallel with the sarcomeric axis, being anchored to actin at Z-disks and myosin at A-bands. On tensile sarcomere strain, which stretches the titin springs, a force component arises both longitudinally and laterally. The latter could contribute to increased sarcomeric transversal stiffness, decreased lateral myofilament spacing, and length-dependent activation of stretched muscle. We aimed to quantify by AFM force-mapping the titin contribution to sarcomeric transversal stiffness in comparison to the contribution from electrostatic forces modulated by osmotic compression. Single myofibrils were isolated from skeletal muscles expressing stiff titin-isoform (rabbit psoas) or compliant titin-isoform (rabbit diaphragm). Myofibrils were placed in relaxing buffer under the MFP-3D-BIO-AFM (Asylum Research) and force curves (50x50) were performed over a region-of-interest encompassing a whole sarcomere. Measurements were done in the absence or presence (5%) of dextran, and in the non-stretched (‘slack’) or stretched state (140-150% of slack). Force mapping revealed distinct transversal stiffness patterns along the sarcomere, suggesting relatively higher Z-disk stiffness and somewhat lower A-band stiffness. A-band lateral stiffness did not differ between psoas and diaphragm sarcomeres at slack. With stretch, this stiffness was increased significantly greater in psoas than in diaphragm. Osmotic compression by 5% dextran at slack increased the A-band lateral stiffness to a degree matching (psoas) or exceeding (diaphragm) that seen after stretch in 0% dextran. The stretch-induced and compression-induced contributions to A-band lateral stiffness were additive. Thus, stiff titin contributes more to A-band stiffness than compliant titin, confirming a role for titin in lateral force generation during sarcomere stretching. The electrostatic and the titin-based lateral forces compressing the A-band lattice are of the same order of magnitude in stretched muscle.