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PubMed2001

Capillary endothelia and cardiomyocytes differ in vulnerability to ischemia/reperfusion during clinical heart transplantation.

OBJECTIVE: The development of accelerated graft arteriosclerosis is a major cause of late death after orthotopic heart transplantation. The influence and the extent of peritransplant injury, especially of cardiomyocyte or capillary endothelial cell edema is discussed. METHODS: A morphometric ultrastructural analysis of myocardial biopsies from 29 donor hearts (21 male, age 34+/-11 years) was performed. Right ventricular biopsies were obtained before cardioplegia (A), immediately following cardioplegia (B) (Custodiol, Dr. F. Köhler Chemie GmbH, Alsbach-Hähnlein, Germany), before implantation (C), after 30 (D) or 60 (E) min of reperfusion and 1 week after transplantation (F). Mean ischemic time was 185+/-68 min. Quantitative electron microscopy was carried out in five samples per heart and time point and in 30 test fields per sample by 'random systematic sampling' and 'point and intersection counting'. As parameters for cell edema the volume density of myofibrils in cardiomyocytes and the mean barrier thickness of capillary endothelia were analyzed. P-values of less than 0.05 were regarded as significant. Significant differences in contrast to the previous values are marked by *. RESULTS: The volume density of myofibrils (vol.%) was as follows: (B) 63.6+/-3.2, (C) 61.8+/-3.2, (D) 62.9+/-3.2, (E) 63.6+/-4.5. The mean barrier thickness (nm) was as follows: (A) 353+/-21, (B) 376+/-59, (C) 416+/-71*, (D) 473+/-45*; (E) 453+/-50*, (F) 379+/-39. CONCLUSIONS: Apart from a generally accepted edema of cardiomyocytes a relevant capillary endothelial cell edema develops during clinical heart transplantation. In contrast to cardiomyocytes the cell edema of endothelia shows a more pronounced and significant progression during cold ischemia and early reperfusion. After 60 min of reperfusion it is still significantly more pronounced than at the onset of ischemia. After 1 week there are no statistical differences compared to the initial values. Thus, an edema of capillary endothelia probably will trigger inhomogeneities in capillary perfusion. Peritransplant injury of endothelia may contribute to the later development of accelerated allograft arteriosclerosis.

Adult↗
PubMedDate not supplied

An ultrastructural study of cardiac myocytes in postmyocardial infarction ventricular aneurysm representative of chronic ischemic myocardium using semiquantitative and quantitative assessment.

To elucidate the characteristic myocytic changes in chronic ischemic myocardium, an electronmicroscopic (EM) study was carried out in surgically excised postmyocardial infarction left ventricular aneurysm (LVAN) (n = 15) using semiquantitative (in all 15 cases) and quantitative assessment methods (in 10 cases). The control group, representing normal ultrastructure, included left ventricular endomyocardial biopsy specimens of sick sinus syndrome (SSS) (n = 3), Wolff-Parkinson-White (WPW) Syndrome (n = 1), and intraoperative left ventricular endomyocardial biopsy in mitral stenosis (MS) (n = 3). Myocardial condition was assessed at the ultrastructural level according to the severity of morphologic changes, first semiquantitatively, thereafter with the use of the Image Processor-Analyzer LUZEX III for morphometric analysis. The most marked EM findings were mitochondrial regressive changes, glycogen accumulation, nuclear deformities, increased rough-surfaced endoplasmic reticulum at the perinuclear portion, lysis of myofibrils, and myofibrillar degeneration. The quantitative analysis revealed significant (p < 0.05) increase of glycogen deposition, only at the perinuclear portion in the LVAN group. The myofibril to mitochondria ratio at the intercalated disc (ID) portion of the cardiac myocytes significantly increased (p < 0.01) in the LVAN group as compared with the normal control group. Light microscopically evaluated quantitative analysis, using toluidine-blue stained semithin sections which underwent EM observation, showed that the fractional area of interstitial fibrous tissue was significantly increased in the LVAN group compared to the normal controls (p < 0.01). These results signify that in chronic ischemic myocardium, decreased consumption of glycogen in oxidative phosphorylation occurs in the surviving myocytes, and that hypertrophy of the myocytes appears. The results of this study may lead to the proper ultrastructural interpretation of biopsied human myocardium, regardless of etiology.

Adult↗
PubMed1999

Cryoenzymic studies on an organized system: myofibrillar ATPases and shortening.

We have exploited cryoenzymology, first, to probe the product release steps of myofibrillar ATPase under relaxing conditions and, second, to define the conditions for studying the contractile process in slow motion. Cryoenzymology implies perturbation by temperature and by the antifreeze added to allow for work at subzero temperatures. Here, we studied myofibrillar shortening and ATPases by the rapid quench flow method over a wide temperature range (-15 to 30 degrees C) in two antifreezes, 40% ethylene glycol and 20% methanol. The choice of solvent and temperature was dictated by the purpose of the experiment. Ethylene glycol (40%) is suitable for investigating the kinetics of the products release steps which is difficult in water. In this cryosolvent, the myofibrillar ATPase is not activated by Ca2+ nor is there shortening, except under special conditions, i.e., Ca2+ plus strong rigor bridges [Stehle, R., Lionne, C., Travers, F., and Barman, T. (1998) J. Muscl. Res. Cell Motil. 19, 381-392]. By the use of the glycol, we show that at low Ca2+ the kinetics of the ADP release are much faster with myofibrils than with S1. On the other hand, the kinetics of the Pi release were very similar for the two materials. Therefore, we suggest that, upon Ca2+ activation, only the Pi release kinetics are accelerated. In 20% methanol, in the presence of Ca2+, myofibrils shortened at temperatures above -2 degrees C but not below. At a given temperature above -2 degrees C, both the shortening and ATPase rates were reduced by the methanol. The temperature dependences of the myofibrillar ATPases (+/-Ca2+) converged with a decrease in temperature: at 20 degrees C, Ca2+ activated 30-fold, but at -15 degrees C, only about 5-fold. We suggest that studies in methanol may open the way for an investigation of muscle contraction in slow motion and, further, to obtain thermodynamic information on the internal forces involved in the shortening process.

Adenosine Diphosphate↗
PubMed2001

Identification of lipids as the main component of skeletal muscle Z-discs.

The existence of lipids in Z-discs of the vertebrate skeletal muscle was suggested by the results of microscopy using rhodamine 6G reagent, a fluorescent probe. After removal of lipids by treatment of myofibrils with 0.5% Trition X-100, intact configurations of Z-filaments composed of alpha-actinin were clearly visible under an electron microscope. These findings indicated that lipids were amorphous-matrix materials of the Z-disc. Lipids were proved to be the main component of Z-discs by their extraction from I-Z-I brushes with a mixture of methanol and chloroform and by analysing them. The total amount of lipids in Z-discs exceeded that of alpha-actinin and varied from 2.4 to 7.1 g per 100 g of myofibrillar proteins depending on the phenotype of skeletal muscle. The sum of the amounts of lipids and alpha-actinin was 4.5 g per 100 g of myofibrillar proteins in chicken pectoralis profundus muscle (fast type), while it was 10.1 g in chicken soleus muscle (slow type). Lipid classes were phospholipids, triacylglycerols, cholesterol and free fatty acids. Since the lipids extracted from I-Z-I brushes were hardly contaminated with those from the sarcoplasmic reticulum and mitochondria, their amounts and classes were considered to be characteristic of Z-discs. The lipids probably cement neighbouring Z-filaments electrostatically, reinforce the Z-disc structure, and play an important role in the force transmission of skeletal muscle myofibrils.

Actinin↗
PubMed2001

Regenerative and plastic insufficiency of cardiomyocytes during impairment of protein synthesis.

Regenerative and plastic insufficiency of cardiomyocytes comprises cell changes resulting from impaired DNA-dependent RNA synthesis and biosynthetic processes. It is accompanied by progressive involution of cytoplasmic structures and causes atrophy and partial elimination of cells without signs of coagulation and colliquative necroses. Segregation and fragmentation of nucleoli in cardiomyocyte nuclei, lysis of myofibrils, and focal degradation of the cytoplasm are ultrastructural signs of impaired protein synthesis. Intracellular cardiomyocyte regeneration is characterized by disorientation of newly formed myofibrils and their excessive elongation, which are related to DNA damages followed by changes in transcription and translation in cardiomyocytes. Calculation of the absolute number of cardiomyocytes is a new approach to evaluation of contractile myocardial insufficiency.

Animals↗
PubMed2002

Varieties of elastic protein in invertebrate muscles.

Elastic proteins in the muscles of a nematode (Caenorhabditis elegans), three insects (Drosophila melanogaster, Anopheles gambiae, Bombyx mori) and a crustacean (Procambus clarkii) were compared. The sequences of thick filament proteins, twitchin in the worm and projectin in the insects, have repeating modules with fibronectin-like (Fn) and immunoglobulin-like (Ig) domains conserved between species. Projectin has additional tandem Igs and an elastic PEVK domain near the N-terminus. All the species have a second elastic protein we have called SLS protein after the Drosophila gene, sallimus. SLS protein is in the I-band. The N-terminal region has the sequence of kettin which is a spliced product of the gene composed of Ig-linker modules binding to actin. Downstream of kettin, SLS protein has two PEVK domains, unique sequence, tandem Igs, and Fn domains at the end. PEVK domains have repeating sequences: some are long and highly conserved and would have varying elasticity appropriate to different muscles. Insect indirect flight muscle (IFM) has short I-bands and electron micrographs of Lethocerus IFM show fine filaments branching from the end of thick filaments to join thin filaments before they enter the Z-disc. Projectin and kettin are in this region and the contribution of these to the high passive stiffness of Drosophila IFM myofibrils was measured from the force response to length oscillations. Kettin is attached both to actin near the Z-disc and to the end of thick filaments, and extraction of actin or digestion of kettin leads to rapid decrease in stiffness; residual tension is attributable to projectin. The wormlike chain model for polymer elasticity fitted the force-extension curve of IFM myofibrils and the number of predicted Igs in the chain is consistent with the tandem Igs in Drosophila SLS protein. We conclude that passive tension is due to kettin and projectin, either separate or linked in series.

Animals↗
PubMed2002

Intracellular distribution of peroxynitrite during doxorubicin cardiomyopathy: evidence for selective impairment of myofibrillar creatine kinase.

Cardiac peroxynitrite and protein nitration are increased during doxorubicin cardiotoxicity, but the intracellular targets and functional consequences have not been defined. We investigated the intracellular distribution of protein nitration during doxorubicin cardiotoxicity in mice. Following in vivo cardiac function assessments by echocardiography, cardiac tissues were prepared for immunohistochemistry and electron microscopy 5 days after doxorubicin (20 mg kg(-1)) or vehicle control. Increased cardiac 3-nitrotyrosine was observed using light microscopy in doxorubicin treated animals. Immunogold electron microscopy (55,000x) revealed increased myofibrillar and mitochondrial 3-nitrotyrosine levels following doxorubicin, but cellular 3-nitrotyrosine density was 2 fold higher in myofibrils. We therefore investigated the actions of peroxynitrite on intact cardiac contractile apparatus. Skinned ventricular trabeculae were exposed to physiologically relevant peroxynitrite concentrations (50 or 300 nM) for 1 h, then Ca(2+) induced contractile responses were measured in the presence of ATP (4 mM) or phosphocreatine (12 mM) as high energy phosphate supplier. ATP maximal force generation was unaltered after 50 nM peroxynitrite, but phosphocreatine/ATP response was reduced (0.99+/-0.63 vs 1.59+/-0.11), suggesting selective inactivation of myofibrillar creatine kinase (MM-CK). Reduction of ATP maximal force was observed at 300 nM peroxynitrite and phosphocreatine/ATP response was further reduced (0.64+/-0.30). Western blotting showed concentration dependent nitration of MM-CK in treated trabeculae. Similarly, cardiac tissues from doxorubicin treated mice demonstrated increased nitration and inactivation of MM-CK compared to controls. These results demonstrate that peroxynitrite-related protein nitration are mechanistic events in doxorubicin cardiomyopathy and that the cardiac myofibril is an important oxidative target in this setting. Furthermore, MM-CK may be a uniquely vulnerable target to peroxynitrite in vivo.

Animals↗
PubMed1977

The proteins in the Z line of insect flight muscle.

Z discs were isolated from Lethocerus flight muscle by removing the contractile proteins from myofibrils with a solution of high ionic strength. The protein composition of the Z discs was analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the major proteins were alpha-actinin, actin and tropomyosin. Z lines were selectively removed from intact myofibrils by digestion with crude lipase and chymotrypsin, but not by purified lipase.

Animals↗
PubMed1980

New proline-rich proteins in isolated insect Z-discs.

Z-discs were isolated from Lethocerus (waterbug) flight muscle by removing the contractile proteins from myofibrils with a solution of high ionic strength. Sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis confirmed a previous report that major Z-disc proteins had subunit mol.wts of 200 000, 180 000, 105 000, 95 000, 42 000 and 35 000. A protein of subunit mol.wt 25 000 was found in once-washed Z-discs but was degraded or was removed by successive washes. In addition, a protein of high molecular weight (less than 300 000) was found in Z-discs. Proteins of subunit mol.wts. 42 000, 35 000 and 25 000 were individually sliced from SDS/polyacrylamide gels and eluted. Amino acid analysis showed that the 35 000-subunit-mol.wt. protein was not, as was previously suggested, tropomyosin, but was a distinct Z-disc protein rich in proline. Calculations based on the amino acid analysis showed that this protein contained substantial hydrophobic regions. Preliminary investigations into the isoelectric point and a method of isolation of the 35 000-subunit-mol.wt. Z-disc protein are described. This protein was found in slices cut from SDS/polyacrylamide-gel electrophoretograms of whole myofibrils. The protein of 42 000 subunit mol.wt. was shown by amino acid analysis to be actin and the 25 000-subunit-mol.wt. Z-disc protein was proline-rich.

Amino Acids↗
PubMed1981

Calmodulin-free skeletal-muscle troponin C prepared in the absence of urea.

A method is described for the rapid preparation of electrophoretically pure troponin C from rabbit skeletal-muscle myofibrils that avoids the use of urea. The three-step procedure includes extraction od the myofibrils with EDTA-containing buffers, one-step elution from DEAE-Sephadex and Sephadex G-100 chromatography in the presence of EDTA. The procedure gives yields comparable with those of currently used methods that involve dissociation of the troponin complex with urea. Except for the thiol-group reactivity, troponin C produced by our method is physicochemically and functionally indistinguishable from that obtained by the classical procedure. Purified troponin C always contains traces of calmodulin. However, this contamination can be decreased to less than 0.02% by means of a second Sephadex G-100 chromatography step in the presence of EDTA.

Animals↗
PubMed1996

Soft-X-ray damage to biological samples.

X-ray damage to biological samples was investigated in the wavelength region of 2.7-5 nm, which overlaps the so-called 'water-window', the wavelength range of 2.4-4.3 nm usually used in X-ray microscopy. Yeast cells and myofibrils were chosen as representatives of whole cell samples and motile protein systems, respectively. The samples were exposed to X-rays using an apparatus composed mainly of a laser-plasma X-ray source, a Wolter mirror condenser, and a sample cell. The yeast cells lost their dye exclusion ability when the X-ray flux was higher than 1 x 10(6) photons micron-2, while the myofibrils lost contractility when the X-ray flux was higher than 4 x 10(5) photons micron-2. These X-ray fluxes are lower than the flux required for the X-ray microscope observation of biological samples at a resolution higher than that of light microscopes.

Animals↗
PubMed1999

Red muscle development of gilthead sea bream Sparus aurata (L.): structural and ultrastructural morphometry.

The transverse red and white muscle area, the superficial red muscle fibres area and their percentages of mitochondria (%mit), myofibrils (%myof) and sarcoplasm (%sarc) were determined in the Mediterranean teleost gilthead sea bream, Sparus aurata (L.). Fish aged from hatching to 78 days were studied. The proportional growth of the red and white muscles was higher for the red muscle in the first half of the larval stage (1-35 days). Then the opposite relationship was observed. The hypertrophic growth of the superficial muscle fibres was continuous except in the first week after hatching. The percentage of mitochondria and percentage of myofibrils showed a significant change just after the half of the larval stage. Whereas the %mit of the superficial muscle fibres was higher than %myof from the first week after hatching to 35 days (average 66.64%), then the %mit decreased significantly and at 73-78 days both parameters were close to an average value of 50%. The meaning of these morphological changes is discussed in relation to the functional role of the red muscle of larvae and the onset of the gills respiration.

Aging↗
PubMed1999

Changes as the result of ageing in extraocular muscles: a post-mortem study.

BACKGROUND: Extraocular muscles differ from typical skeletal muscle in many respects such as smaller fibre size, high mitochondrial content, unusual contractile/innervation patterns and highly developed microvascular bed. Changes in typical skeletal muscle with ageing have been well documented yet the reports on ageing changes in extraocular muscles is limited. The aim of this study was to examine these changes. METHODS: The right inferior and medial rectus muscles were removed at post-mortem from patients with no history of prior ocular or neuromuscular disease. These included 25 patients aged between 23 and 88 years (14 male, 11 female). The median age of the patients was 69 years and 72% of patients were aged older than 66 years. The median post-mortem interval was 28 h, RESULTS: In young adults between 20 and 30 years of age, the muscle fibres were regular, of fairly uniform size with minimal endomysial connective tissue and no lipofuscin was detected in fibres. In adults between 40 and 50 years of age, a small amount of subsarcolemmal lipofuscin was detected. Between 50 and 60 years of age, a few fibres with reduced density of myofibrils were noted. All patients over 65 years had definite changes of ageing and those between 70 and 80 years of age showed similar features to those between 60 and 80 years of age. These changes included variation in fibre size, increased endomysial fibrous tissue and increased endomysial adipose tissue, and loss of myofibrils with some fibres devoid of fibrils and comprising 'bags' of mitochondria. There was increased lipofuscin in a number of fibres, increased 'degenerative' changes such as vacuolation of fibres, scattered 'eosinophilic intracytoplasmic bodies' and increased numbers of 'ringbinden'.

Adult↗
PubMed1986

Structural changes in skeletal muscle tissue with heavy-resistance exercise.

Muscle strength, muscle cross-sectional area, fiber size, fiber type distribution, capillarity, and mitochondrial volume were estimated before and after 6 weeks of heavy-resistance exercise. Isokinetic torque production of the knee extensor muscles increased by a total of 17.6%, mainly during the first half of the training period. The cross-sectional area of the vastus lateralis muscle increased by 8.4%, mainly during the second half of the training period. Morphometrically determined fiber size, fiber type distribution, and capillarity from biopsies of vastus lateralis did not change significantly with training. Likewise, the surface densities of inner and outer mitochondrial membranes as well as the volume density of myofibrils remained unchanged. In contrast, the volume density of mitochondria decreased by 9.6%. However, due to the increase in total muscle volume, the calculated absolute volume of mitochondria remained constant, whereas the absolute volume of myofibrils increased by 10%. It is concluded that strength training of short duration in previously untrained young male subjects does not change the ultrastructural composition of mitochondria and that the apparent dilution of mitochondria can quantitatively be accounted for by the increase in myofibrillar volume.

Adenosine Triphosphatases↗
PubMed2000

Desmin myopathy, a skeletal myopathy with cardiomyopathy caused by mutations in the desmin gene.

BACKGROUND: Myofibrillar myopathies, often referred to as desmin-related myopathies, are a heterogeneous group of inherited or sporadic distal-onset skeletal myopathies associated with cardiomyopathy. Among the myofibrillar proteins that characteristically accumulate within the muscle fibers of affected patients, the one found most consistently is desmin, a muscle-specific intermediate-filament protein responsible for the structural integrity of the myofibrils. Skeletal and cardiac myopathy develops in mice that lack desmin, suggesting that mutations in the desmin gene may be pathogenic. METHODS: We examined 22 patients from 8 families with dominantly inherited myofibrillar or desmin-related myopathy and 2 patients with sporadic disease and analyzed the desmin gene for mutations, using complementary DNA (cDNA) amplified from muscle-biopsy specimens and genomic DNA extracted from blood lymphocytes. Restriction-enzyme analysis was used to confirm the mutations. Expression vectors containing normal or mutant desmin cDNA were introduced into cultured cells to determine whether the mutant desmin formed intermediate filaments. RESULTS: Six missense mutations in the coding region of the desmin gene that cause the substitution of an amino acid were identified in 11 patients (10 members of 4 families and 1 patient with sporadic disease); a splicing defect that resulted in the deletion of exon 3 was identified in the other patient with sporadic disease. Mutations were clustered in the carboxy-terminal part of the rod domain, which is critical for filament assembly. In transfected cells, the mutant desmin was unable to form a filamentous network. Seven of the 12 patients with mutations in the desmin gene had cardiomyopathy. CONCLUSIONS: Mutations in the desmin gene affecting intermediate filaments cause a distinct myopathy that is often associated with cardiomyopathy and is termed "desmin myopathy." The mutant desmin interferes with the normal assembly of intermediate filaments, resulting in fragility of the myofibrils and severe dysfunction of skeletal and cardiac muscles.

Adult↗
PubMed2005

Paramyosin phosphorylation site disruption affects indirect flight muscle stiffness and power generation in Drosophila melanogaster.

The phosphoprotein paramyosin is a major structural component of invertebrate muscle thick filaments. To investigate the importance of paramyosin phosphorylation, we produced transgenic Drosophila melanogaster in which one, three, or four phosphorylatable serine residues in the N-terminal nonhelical domain were replaced by alanines. Depending on the residues mutated, transgenic lines were either unaffected or severely flight impaired. Flight-impaired strains had decreases in the most acidic paramyosin isoforms, with a corresponding increase in more basic isoforms. Surprisingly, ultrastructure of indirect flight muscle myofibrils was normal, indicating N-terminal phosphorylation is not important for myofibril assembly. However, mechanical studies of active indirect flight muscle fibers revealed that phosphorylation site mutations reduced elastic and viscous moduli by 21-59% and maximum power output by up to 42%. Significant reductions also occurred under relaxed and rigor conditions, indicating that the phosphorylation-dependent changes are independent of strong crossbridge attachment and likely arise from alterations in thick filament backbone properties. Further, normal crossbridge kinetics were observed, demonstrating that myosin motor function is unaffected in the mutants. We conclude that N-terminal phosphorylation of Drosophila paramyosin is essential for optimal force and oscillatory power transduction within the muscle fiber and is key to the high passive stiffness of asynchronous insect flight muscles. Phosphorylation may reinforce interactions between myosin rod domains, enhance thick filament connections to the central M-line of the sarcomere and/or stabilize thick filament interactions with proteins that contribute to fiber stiffness.

Analysis of Variance↗
PubMed2001

Synemin may function to directly link muscle cell intermediate filaments to both myofibrillar Z-lines and costameres.

Synemin is a large intermediate filament (IF) protein that has been identified in all types of muscle cells in association with desmin- and/or vimentin-containing IFs. Our previous studies (Bellin, R. M., Sernett, S. W., Becker, B., Ip, W., Huiatt, T. W., and Robson, R. M. (1999) J. Biol. Chem. 274, 29493-29499) demonstrated that synemin forms heteropolymeric IFs with major IF proteins and contains a binding site for the myofibrillar Z-line protein alpha-actinin. By utilizing blot overlay assays, we show herein that synemin also interacts with the costameric protein vinculin. Furthermore, extensive assays utilizing the Gal4 yeast two-hybrid system demonstrate interactions of synemin with desmin and vimentin and additionally define more precisely the protein subdomains involved in the synemin/alpha-actinin and synemin/vinculin interactions. The C-terminal approximately 300-amino acid region of synemin binds to the N-terminal head and central rod domains of alpha-actinin and the approximately 150-amino acid C-terminal tail of vinculin. Overall, these interactions indicate that synemin may anchor IFs to myofibrillar Z-lines via interactions with alpha-actinin and to costameres at the sarcolemma via interactions with vinculin and/or alpha-actinin. These linkages would enable the IFs to directly link all cellular myofibrils and to anchor the peripheral layer of myofibrils to the costameres.

Animals↗
PubMed2003

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.

Animals↗