BackgroundDiaphragm 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.MethodsSkinned 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.ResultsPassive 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.DiscussionWe conclude that in the diaphragm of HF rats, passive elasticity is impaired, mainly resulting from titin loss.

When muscles are stretched, the giant protein titin develops passive force. Titin's force performs important functions that include maintaining the structural integrity of the sarcomere, and triggering signal transduction pathways. We propose that the mechanical properties of titin can be tuned according to the mechanical demands places on muscle, using mechanisms that include alternative splicing and posttranslational modifications.

We investigated the effect of protein kinase A (PKA) on passive force in skinned cardiac tissues that express different isoforms of titin, i.e., stiff (N2B) and more compliant (N2BA) titins, at different levels. We used rat ventricular (RV), bovine left ventricular (BLV), and bovine left atrial (BLA) muscles (passive force: RV > BLV > BLA, with the ratio of N2B to N2BA titin, approximately 90:10, approximately 40:60, and approximately 10:90%, respectively) and found that N2B and N2BA isoforms can both be phosphorylated by PKA. Under the relaxed condition, sarcomere length was increased and then held constant for 30 min and the peak passive force, stress-relaxation, and steady-state passive force were determined. Following PKA treatment, passive force was significantly decreased in all muscle types with the effect greatest in RV, lowest in BLA, and intermediate in BLV. Fitting the stress-relaxation data to the sum of three exponential decay functions revealed that PKA blunts the magnitude of stress-relaxation and accelerates its time constants. To investigate whether or not PKA-induced decreases in passive force result from possible alteration of titin-thin filament interaction (e.g., via troponin I phosphorylation), we conducted the same experiments using RV preparations that had been treated with gelsolin to extract thin filaments. PKA decreased passive force in gelsolin-treated RV preparations with a magnitude similar to that observed in control preparations. PKA was also found to decrease restoring force in skinned ventricular myocytes of the rat that had been shortened to below the slack length. Finally, we investigated the effect of the beta-adrenergic receptor agonist isoprenaline on diastolic force in intact rat ventricular trabeculae. We found that isoprenaline phosphorylated titin and that it reduced diastolic force to a degree similar to that found in skinned RV preparations. Taken together, these results suggest that during beta-adrenergic stimulation, PKA increases ventricular compliance in a titin isoform-dependent manner.

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.

A new dynamic model of left ventricular (LV) pressure-volume relationships in beating heart was developed by mathematically linking chamber pressure-volume dynamics with cardiac muscle force-length dynamics. The dynamic LV model accounted for >80% of the measured variation in pressure caused by small-amplitude volume perturbation in an otherwise isovolumically beating, isolated rat heart. The dynamic LV model produced good fits to pressure responses to volume perturbations, but there existed some systematic features in the residual errors of the fits. The issue was whether these residual errors would be damaging to an application where the dynamic LV model was used with LV pressure and volume measurements to estimate myocardial contractile parameters. Good agreement among myocardial parameters responsible for response magnitude was found between those derived by geometric transformations of parameters of the dynamic LV model estimated in beating heart and those found by direct measurement in constantly activated, isolated muscle fibers. Good agreement was also found among myocardial kinetic parameters estimated in each of the two preparations. Thus the small systematic residual errors from fitting the LV model to the dynamic pressure-volume measurements do not interfere with use of the dynamic LV model to estimate contractile parameters of myocardium. Dynamic contractile behavior of cardiac muscle can now be obtained from a beating heart by judicious application of the dynamic LV model to information-rich pressure and volume signals. This provides for the first time a bridge between the dynamics of cardiac muscle function and the dynamics of heart function and allows a beating heart to be used in studies where the relevance of myofilament contractile behavior to cardiovascular system function may be investigated.

Background— The role of the giant protein titin in patients with heart failure is not well established. We investigated titin expression in patients with end-stage heart failure resulting from nonischemic dilated cardiomyopathy, in particular as it relates to left ventricular (LV) myocardial stiffness and LV function. Methods and Results— SDS-agarose gels revealed small N2B (stiff) and large N2BA (compliant) cardiac titin isoforms with a mean N2BA:N2B expression ratio that was significantly ( P <0.003) increased in 20 heart failure patients versus 6 controls. However, total titin was unchanged. The coexpression ratio was highest in a subsample of patients with an impaired LV relaxation pattern (n=7), intermediate in those with pseudonormal filling (n=6), and lowest in the group with restrictive filling (n=7). Mechanical measurements on LV muscle strips dissected from these hearts (n=8) revealed that passive muscle stiffness was significantly reduced in patients with a high N2BA:N2B expression ratio. Clinical correlations support the relevance of these changes for LV function (assessed by invasive hemodynamics and Doppler echocardiography). A positive correlation between the N2BA:N2B titin isoform ratio and deceleration time of mitral E velocity, A wave transit time, and end diastolic volume/pressure ratio was found. These changes affect exercise tolerance, as indicated by the positive correlation between the N2BA:N2B isoform ratio and peak O 2 consumption (n=10). Upregulated N2BA expression was accompanied by increased expression levels of titin-binding proteins (cardiac ankyrin repeat protein, ankrd2, and diabetes ankyrin repeat protein) that bind to the N2A element of N2BA titin (studied in 13 patients). Conclusions— Total titin content was unchanged in end-stage failing hearts and the more compliant N2BA isoform comprised a greater percentage of titin in these hearts. Changes in titin isoform expression in heart failure patients with dilated cardiomyopathy significantly impact diastolic filling by lowering myocardial stiffness. Upregulation of titin-binding proteins indicates that the importance of altered titin expression might extend to cell signaling and regulation of gene expression.

We investigated the cellular and molecular mechanisms of systolic and diastolic dysfunction in a furazolidone (Fz)-induced model of dilated cardiomyopathy (DCM) in turkey poults. Serial echocardiograms disclosed marked systolic dysfunction in the Fz-treated poults, and ventricular weight and left ventricular (LV)/body weight ratio were significantly increased. Isolated heart experiments were performed to determine LV pressure-volume (P-V) relationships. In addition, LV sarcomere lengths (SLs) were measured after hearts had been fixed, and wall stress (sigma)-SL relationships were determined. When compared to control hearts, LV chamber volume in DCM hearts was approximately 3-fold increased, the active or developed LV P-V relationship was markedly depressed, the passive or diastolic P-V relationship was steeper, and SLs were significantly shorter. However, the developed sigma-SL relationships of DCM and control hearts were not different indicating that intrinsic myocardial capacity to generate active force is unaffected in this model of DCM. In contrast, passive sigma, and passive tension in trabecular muscle preparations increased much more steeply with SL in DCM than normal hearts. Trabecular muscle experiments disclosed that the increase in passive myocardial stiffness was primarily collagen based. Titin, the giant sarcomeric molecule, which is an important determinant of passive myocyte properties in normal myocardium, did not contribute significantly to increased passive myocardial stiffness in DCM. We conclude that increased collagen-based passive myocardial stiffness is the major cause of the steeper passive or diastolic P-V relationship in DCM. Further, altered passive myocardial properties and ventricular geometry in DCM play a critical role to reduce ventricular systolic function by limiting SL extension during diastole, thereby limiting the use of the myocardial length-tension relationship.

We studied the effects of Ca 2+ on titin (connectin)-based passive tension in skinned myocardium expressing either predominantly N2B titin (rat right ventricle, RRV) or predominantly N2BA titin (bovine left atrium, BLA). Actomyosin-based tension was abolished to undetectably low levels by selectively removing the thin filaments with a Ca 2+ -insensitive gelsolin fragment (FX-45). Myocardium was stretched in the presence and absence of Ca 2+ , and passive tension was measured. Ca 2+ significantly increased passive tension during and after stretch in the BLA. The increase was insensitive to the actomyosin inhibitor 2,3-butanedione 2-monoxime, supporting the conclusion that the effect is titin based. Passive tension did not respond to calcium in the RRV, indicating that passive tension developed by N2B titin is calcium insensitive. Western blot analysis and immunofluorescence studies indicated that N2BA titin expresses E-rich PEVK motifs, whereas they are absent from N2B titin, supporting earlier single molecule studies that reported that E-rich motifs are required for calcium sensitivity. We conclude that calcium affects passive myocardial tension in a titin isoform-dependent manner.

We have explored the role of the giant elastic protein titin in the Frank-Starling mechanism of the heart by measuring the sarcomere length (SL) dependence of activation in skinned cardiac muscles with different titin-based passive stiffness characteristics. We studied muscle from the bovine left ventricle (BLV), which expresses a high level of a stiff titin isoform, and muscle from the bovine left atrium (BLA), which expresses more compliant titin isoforms. Passive tension was also varied in each muscle type by manipulating the pre-history of stretch prior to activation. We found that the SL-dependent increases in Ca2+ sensitivity and maximal Ca2+-activated tension were markedly more pronounced when titin-based passive tension was high. Small-angle X-ray diffraction experiments revealed that the SL dependence of reduction of interfilament lattice spacing is greater in BLV than in BLA and that the lattice spacing is coupled with titin-based passive tension. These results support the notion that titin-based passive tension promotes actomyosin interaction by reducing the lattice spacing. This work indicates that titin may be a factor involved in the Frank-Starling mechanism of the heart by promoting actomyosin interaction in response to stretch.

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 ∼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.

HomeCirculationVol. 108, No. 4Variable Titin-Based Stiffness Adjustment in Heart Disease Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBVariable Titin-Based Stiffness Adjustment in Heart Disease Henk L. Granzier, Yiming Wu and Karoly Trombitas Christian Witt and Siegfried Labeit Stephen Bell and Martin LeWinter Henk L. GranzierHenk L. Granzier Washington State University, Pullman, Wash , Yiming WuYiming Wu Washington State University, Pullman, Wash and Karoly TrombitasKaroly Trombitas Washington State University, Pullman, Wash Christian WittChristian Witt Univeristatsklinikum Mannheim, Mannheim, Germany and Siegfried LabeitSiegfried Labeit Univeristatsklinikum Mannheim, Mannheim, Germany Stephen BellStephen Bell University of Vermont, Burlington, Vt and Martin LeWinterMartin LeWinter University of Vermont, Burlington, Vt Originally published29 Jul 2003https://doi.org/10.1161/01.CIR.0000081439.94575.E3Circulation. 2003;108:e23To the Editor:Several issues in a previous letter to the Editor1 concerning our work on titin in heart disease require clarification. We have studied titin isoform expression in the canine pacing model of heart failure2 and found that in controls, stiff (N2B) and compliant (N2BA) cardiac titin isoforms are coexpressed at ≈1:1 ratio, whereas after pacing, the stiff isoform is upregulated at the expense of the more compliant one.2 Using skinned muscle strips dissected from the midwall region of the left ventricle (LV) (note that we did not use myofibrils, as suggested by others1), we showed that titin-based passive stiffness is elevated in paced animals. The use of muscle strips provides more representative data (we studied ≈5 muscles per heart with each containing ≈105 myofibrils) than the use of single myofibrils. Considering the variation in isoform expression ratio (including variation within individual cells3), studying only a few myofibrils (3 to 5 per heart in the Neagoe et al4 study) has the potential for results that are not representative. Furthermore, gel electrophoresis can be performed with muscle strips but not single myofibrils, allowing the intactness of titin in muscle strip preparations (but not myofibrils) to be verified.1 Finally, muscle can be studied before and after abolishing titin-based passive stiffness (by extracting titin's anchors in the sarcomere), providing information about the contribution to passive stiffness of collagen as well as titin.2 Although each type of preparation (myofibril and muscle) has benefits and drawbacks, muscle is a suitable choice for studying the molecular basis of passive stiffness and its adjustments in disease.Upregulation of N2B titin in the pacing model is in contrast to the upregulation of N2BA titin reported by Neagoe et al4 to occur in human transplant hearts with coronary artery disease (CAD). Both canine and human models express similar levels of compliant N2BA and stiff N2B titins in control myocardium, and the titin-based stiffness will therefore be intermediate between that of sarcomeres that express solely N2B or solely N2BA titin. The equal amounts of N2BA and N2B titin and the resulting intermediate stiffness allow for considerable adjustment. Sarcomeres can greatly increase compliance by increasing the N2BA/N2B expression ratio (human transplant hearts with CAD4) or greatly increase stiffness by reducing this ratio (canine rapid pacing model2). Thus, processing of the titin pre-mRNA is subject to subtle regulatory mechanisms that control entry to either the N2B or N2BA splice pathways. We believe that the studies on canine2 and human4 models indicate that, depending on the disease state, treatment regimen, and (perhaps) species, a range of adjustments can occur, leading to either increased or decreased passive stiffness.1 Linke WA. Titin stiffness in heart disease. Circulation. 2003; 107: e73.LinkGoogle Scholar2 Wu Y, Bell SP, Trombitas K, et al. Changes in titin isoform expression in pacing-induced cardiac failure give rise to increased passive muscle stiffness. Circulation. 2002; 106: 1384–1389.LinkGoogle Scholar3 Trombitás K, Wu Y, Labeit D, et al. Co-expression of titin isoforms in the sarcomere of cardiac muscle: a mechanism to tune the diastolic properties of the heart. Am J Physiol Heart Circ Physiol. 2001; 281: 1793–1799.CrossrefMedlineGoogle Scholar4 Neagoe C, Kulke M, del Monte F, et al. Titin isoform switch in ischemic human heart disease. Circulation. 2002; 106: 1333–1341.LinkGoogle Scholar eLetters(0)eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate.Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.Sign In to Submit a Response to This Article Previous Back to top Next FiguresReferencesRelatedDetailsCited By Mukherjee R (2018) Frank(ly), Star(t)ling: A structural protein contributes to changes in left ventricular performance with cardiomyopathies?, The Journal of Thoracic and Cardiovascular Surgery, 10.1016/j.jtcvs.2017.12.063, 156:1, (215-216), Online publication date: 1-Jul-2018. Kontrogianni-Konstantopoulos A, Ackermann M, Bowman A, Yap S and Bloch R (2009) Muscle Giants: Molecular Scaffolds in Sarcomerogenesis, Physiological Reviews, 10.1152/physrev.00017.2009, 89:4, (1217-1267), Online publication date: 1-Oct-2009. July 29, 2003Vol 108, Issue 4 Advertisement Article InformationMetrics https://doi.org/10.1161/01.CIR.0000081439.94575.E3PMID: 12885736 Originally publishedJuly 29, 2003 PDF download Advertisement

A polymer strand can be influenced by an array of mechanical effects as it translates through a space with dimensions similar to its own. We summarize our experiments in which the mechanical behavior of single-molecules of the giant protein titin was characterized. Titin is a filamentous polypeptide embedded in the lattice space of the vertebrate muscle sarcomere. A single titin molecule stretches from the Z- to the M-line of the sarcomere, thereby spanning a distance of approximately I pin which is huge on the molecular scale. Physiologically, titin is important in generating passive muscle force and in maintaining the structural integrity of the sarcomere through its elastic properties. We analyzed the elastic properties of titin by stretching single molecules with laser tweezers. The force-response of the titin molecule revealed an entropic polymer whose behaviour can be well fitted with the wormlike chain model. Unfolding occurs in titin upon reaching high forces during stretch, and refolding takes place at low forces during the relaxation of the molecule. The force hysteresis implies folding non-equilibrium due to the slow unfolding and refolding rates relative to the stretch and release rates, respectively. Folding non-equilibrium as the source of force hysteresis can be demonstrated by the disappearance of hysteresis upon the addition of chemical denaturant. Repeated stretch and release cycles lead to the mechanical wear-out or fatigue of titin, which may have far-reaching implications for its physiological function.

Relaxed striated muscle cells exhibit mechanical fatigue when exposed to repeated stretch and release cycles. To understand the molecular basis of such mechanical fatigue, single molecules of the giant filamentous protein titin, which is the main determinant of sarcomeric elasticity, were repetitively stretched and released while their force response was characterized with optical tweezers. During repeated stretch-release cycles titin becomes mechanically worn out in a process we call molecular fatigue. The process is characterized by a progressive shift of the stretch-force curve toward increasing end-to-end lengths, indicating that repeated mechanical cycles increase titin's effective contour length. Molecular fatigue occurs only in a restricted force range (0-25 pN) during the initial part of the stretch half-cycle, whereas the rest of the force response is repeated from one mechanical cycle to the other. Protein-folding models fail to explain molecular fatigue on the basis of an incomplete refolding of titin's globular domains. Rather, the process apparently derives from the formation of labile nonspecific bonds cross-linking various sites along a pre-unfolded titin segment. Because titin's molecular fatigue occurs in a physiologically relevant force range, the process may play an important role in dynamically adjusting muscle's response to the recent history of mechanical perturbations.