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 ∼1.4 and ∼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. 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 ∼1.4 and ∼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. Titin forms a striated muscle-specific myofilament that develops passive force in response to sarcomere stretch (for a recent review with original citations, see Ref. 1.Granzier, H., and Labeit, S. (2002) @@email protected@J. Physiol. (Lond.)@@/email protected@, in pressGoogle Scholar). Titin's force is generated by serially linked and mechanically distinct spring elements (2.Freiburg A. Trombitas K. Hell W. Cazorla O. Fougerousse F. Centner T. Kolmerer B. Witt C. Beckmann J.S. Gregorio C.C. Granzier H. Labeit S. Circ. Res. 2000; 86: 1114-1121Crossref PubMed Scopus (287) Google Scholar). Tandem Ig segments (tandemly arranged Ig-like domains) and the PEVK domain (rich in proline, glutamate, valine, and lysine residues) are spring elements found in both cardiac and skeletal muscle titins (3.Linke W.A. Ivemeyer M. Olivieri N. Kolmerer B. Ruegg J.C. Labeit S. J. Mol. Biol. 1996; 261: 62-71Crossref PubMed Scopus (234) Google Scholar, 4.Trombitas K. Greaser M. Labeit S. Jin J.P. Kellermayer M. Helmes M. Granzier H. J. Cell Biol. 1998; 140: 853-859Crossref PubMed Scopus (200) Google Scholar) that vary in length in different isoforms of titin. For example, in human, the PEVK domain varies from 188 residues in the cardiac-specific N2B isoform to 2181 residues in skeletal soleus muscle (2.Freiburg A. Trombitas K. Hell W. Cazorla O. Fougerousse F. Centner T. Kolmerer B. Witt C. Beckmann J.S. Gregorio C.C. Granzier H. Labeit S. Circ. Res. 2000; 86: 1114-1121Crossref PubMed Scopus (287) Google Scholar). The cardiac-specific N2B unique sequence (N2B-Us) 1The abbreviations used are: N2B-UsN2B unique sequenceWLCworm-like chainAFMatomic force microscopy/microscopepNpiconewton(s)PPpolyproline forms a third spring element in cardiac titins and provides extensibility at the upper range of physiological sarcomere lengths in the heart (5.Helmes M. Trombitas K. Centner T. Kellermayer M. Labeit S. Linke W.A. Granzier H. Circ. Res. 1999; 84: 1339-1352Crossref PubMed Scopus (135) Google Scholar, 6.Trombitas K. Freiburg A. Centner T. Labeit S. Granzier H. Biophys. J. 1999; 77: 3189-3196Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 7.Linke W.A. Rudy D.E. Centner T. Gautel M. Witt C. Labeit S. Gregorio C.C. J. Cell Biol. 1999; 146: 631-644Crossref PubMed Scopus (198) Google Scholar). The three-spring system of cardiac titin results in a unique force-extension curve that underlies the majority of the physiological passive tensions of the myocardium, and the variable-length tandem Ig and PEVK elements allows passive tension to be adjusted so that it matches the mechanical demands placed on normal and diseased myocardium (8.Wu Y. Cazorla O. Labeit D. Labeit S. Granzier H. J. Mol. Cell. Cardiol. 2000; 32: 2151-2162Abstract Full Text PDF PubMed Scopus (169) Google Scholar, 9.Granzier H.L. Irving T.C. Biophys. J. 1995; 68: 1027-1044Abstract Full Text PDF PubMed Scopus (500) Google Scholar). N2B unique sequence worm-like chain atomic force microscopy/microscope piconewton(s) polyproline Immunoelectron microscopy has shown that in slack sarcomeres (no external force), the tandem Ig segments are in a “contracted” state. When the sarcomeres are stretched, the segments greatly extend (4.Trombitas K. Greaser M. Labeit S. Jin J.P. Kellermayer M. Helmes M. Granzier H. J. Cell Biol. 1998; 140: 853-859Crossref PubMed Scopus (200) Google Scholar) due to unbending of linkers between folded Ig domains (4.Trombitas K. Greaser M. Labeit S. Jin J.P. Kellermayer M. Helmes M. Granzier H. J. Cell Biol. 1998; 140: 853-859Crossref PubMed Scopus (200) Google Scholar) and possibly to domain A. M. Linke W.A. Biophys. J. Full Text Full Text PDF PubMed Scopus Google Scholar). The tandem Ig segments worm-like chain with a persistence length (measure of chain bending rigidity) that is nm on a recent single molecule J. J. Mol. Biol. PubMed Scopus Google tandem Ig segment extension in moderately stretched sarcomeres passive force is (4.Trombitas K. Greaser M. Labeit S. Jin J.P. Kellermayer M. Helmes M. Granzier H. J. Cell Biol. 1998; 140: 853-859Crossref PubMed Scopus (200) Google Scholar). The PEVK domain at forces (4.Trombitas K. Greaser M. Labeit S. Jin J.P. Kellermayer M. Helmes M. Granzier H. J. Cell Biol. 1998; 140: 853-859Crossref PubMed Scopus (200) Google and is to from of and polyproline that element K. K. PubMed Scopus Google Scholar). molecule Granzier H.L. C. PubMed Scopus Google Scholar) and (4.Trombitas K. Greaser M. Labeit S. Jin J.P. Kellermayer M. Helmes M. Granzier H. J. Cell Biol. 1998; 140: 853-859Crossref PubMed Scopus (200) Google Scholar) of skeletal muscle titin that the PEVK domain as a with a persistence length of recent single molecule on the cardiac PEVK domain persistence lengths that range from to nm H. M. S. A. PubMed Scopus Google Scholar). for cardiac-specific N2B-Us, microscopy has that sequence in the cardiac sarcomere (5.Helmes M. Trombitas K. Centner T. Kellermayer M. Labeit S. Linke W.A. Granzier H. Circ. Res. 1999; 84: 1339-1352Crossref PubMed Scopus (135) Google Scholar, 6.Trombitas K. Freiburg A. Centner T. Labeit S. Granzier H. Biophys. J. 1999; 77: 3189-3196Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 7.Linke W.A. Rudy D.E. Centner T. Gautel M. Witt C. Labeit S. Gregorio C.C. J. Cell Biol. 1999; 146: 631-644Crossref PubMed Scopus (198) Google the cardiac titin third spring The mechanical characteristics of spring we the extensibility of the cardiac titin PEVK element and N2B-Us atomic force for single the PEVK domain and N2B-Us displayed little force and force-extension curves followed behavior. length with the main at the length at ∼1.4 and ∼0.65 nm for the PEVK domain and N2B-Us, respectively. that these the persistence length of the single molecule to the extensible of the titin spring elements in the cardiac sarcomere as as the passive length of cardiac that the serially linked with mechanically distinct in closely the in of cardiac titin. titin were by derived from the cardiac titin N2B isoform were in and from the on with a from the cardiac N2B isoform to used the domain system of Centner T. F. M. M. Witt C.C. Labeit D. Gregorio C.C. Granzier H. Labeit S. Circ. Res. PubMed Scopus Google Scholar) to the of Labeit and Kolmerer S. Kolmerer B. 1995; PubMed Scopus Google Scholar) the PEVK domain with Ig domains and N2B-Us used the skeletal muscle-specific with residues soleus skeletal muscle titin, as the were and and at were stretched a force for The force on a of mechanical to of for for the PEVK domain, and for were to to for for for the PEVK domain, and for the N2B-Us were with were stretched in by the the and the with a curves were in stretch and release The results shown in were obtained at a of The of the obtained from bending as is obtained for by its bending and the the and is and the force at the used in curves were for to force length. The data obtained from the force response that to the the were for the obtained from the force response of the the length of the molecule calculated by the with bending as data were with the C. S. PubMed Scopus Google Scholar). The the molecule as a of persistence length (measure of bending rigidity) and length The length of the molecule is and the extension is The results in were obtained by a to release forces of force range to that used for PEVK and N2B-Us and it to the force-extension curves due to at force M. Gautel M. A. Biophys. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). that different force the of For we used of the in and The elastic region of cardiac titin in the sarcomere modeled as three serially linked with different and persistence the tandem Ig segments, the PEVK and The length of the tandem Ig segments calculated from of Ig domains Ig domains of the N2B element were by a domain of nm S. A. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) in a chain domains nm that that domains are For that see The persistence length of the tandem Ig segment as nm on the recent by and J. J. Mol. Biol. PubMed Scopus Google Scholar). The lengths of the PEVK domain and N2B-Us were obtained from of residues the in The of PEVK residues in titin N2B determined that the of the Ig domain is by the sequence and the of the by in 188 PEVK residues and a PEVK length of nm The of N2B-Us residues is in human, to a length. For of extensibility of cardiac titin (for we the N2B-Us and PEVK in in and used and nm as The persistence lengths of the PEVK domain and N2B-Us were at and the of tandem Ig segments, the PEVK domain, and N2B-Us, see Ref. S. Kolmerer B. 1995; PubMed Scopus Google and the tandem and N2B-Us segments are in for a the extension of segment and be for that force the extension of the titin elastic segment the length of Ref. K. Jin J.P. Granzier H. Circ. Res. 1995; 77: PubMed Scopus Google the sarcomere length be calculated and the predicted length The that the PEVK domain comprises it is that of the residues we calculated the persistence length of the PEVK segment N2B that the residues are for and Ref. K. K. PubMed Scopus Google Scholar) that of the 188 PEVK residues are of polyproline that is for the soleus PEVK domain, in which of the residues are predicted to be of the the of the residues in the titin used by K. K. PubMed Scopus Google The and for and the lengths of and to be that the persistence lengths of and are and values are with and nm and and that is between the H. M. S. A. PubMed Scopus Google The persistence length of the PEVK residues that a at nm and the length at nm calculated the force-extension curve of the region linked with the the The persistence length of the PEVK segment obtained by the curve with the (for see calculated the of of the on the force-extension curve of the PEVK we that these residues and persistence lengths of nm and respectively. of the to a we that the values were nm and respectively. calculated the and persistence lengths of the PEVK domain and used these values to the force of the PEVK element and The of extension and force in the sarcomere that Ig domain sarcomere is a we calculated the of domain by domain on the force-extension curve domain were on used Granzier H.L. C. PubMed Scopus Google Scholar, S. C. Granzier H.L. Biol. 2000; PubMed Scopus Google Scholar). For a force the of domains calculated to the is the in the of folded domains the and is the of folded The PubMed Scopus Google and The for and and the of the and were In we for to a of and nm on for H. M. Biol. 2000; PubMed Scopus Google Scholar). The values were obtained from and were calculated from at force titin, nm and M. Gautel M. A. Biophys. J. 1998; Full Text Full Text PDF PubMed Scopus Google nm and H. M. Biol. 2000; PubMed Scopus Google nm M. S. A. PubMed Scopus Google Scholar) and nm and C. M. S. Labeit, and H. for and nm and the domain on a of with a generated between and the of folded domains and the length of the folded segment by nm and the length of the segment by the of folded domains and the length of the folded segment by nm and the length of the segment by were a with a of the used force on cardiac see and a stretch determined by the force at and were and the of domains that The of as and were on a used determined passive length with a stretch of on cardiac cardiac titin (5.Helmes M. Trombitas K. Centner T. Kellermayer M. Labeit S. Linke W.A. Granzier H. Circ. Res. 1999; 84: 1339-1352Crossref PubMed Scopus (135) Google Scholar). passive tensions of of to force titin that of the (5.Helmes M. Trombitas K. Centner T. Kellermayer M. Labeit S. Linke W.A. Granzier H. Circ. Res. 1999; 84: 1339-1352Crossref PubMed Scopus (135) Google Scholar). the in the of the by a single calculated as Physiol. 1998; PubMed Scopus Google Scholar) of slack myocardium is see Ref. T.C. J. D. J. Physiol. 2000; PubMed Google and we that are titin O. Freiburg A. Helmes M. Centner T. M. Y. Trombitas K. Labeit S. Granzier H. Circ. Res. 2000; 86: PubMed Scopus Google Scholar, D. J. J. Mol. Biol. PubMed Scopus Google Scholar). are titin of and used to passive tensions (5.Helmes M. Trombitas K. Centner T. Kellermayer M. Labeit S. Linke W.A. Granzier H. Circ. Res. 1999; 84: 1339-1352Crossref PubMed Scopus (135) Google Scholar) to passive force single titin molecule the of tandem Ig segments, the PEVK domain, and N2B-Us, we used the results of earlier on cardiac K. Freiburg A. Centner T. Labeit S. Granzier H. Biophys. J. 1999; 77: 3189-3196Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, K. A. Centner T. Y. Labeit S. Granzier H. Biophys. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the of the tandem Ig segment determined with the which the K. Freiburg A. Centner T. Labeit S. Granzier H. Biophys. J. 1999; 77: 3189-3196Abstract Full Text Full Text PDF PubMed Scopus (81) Google we adjusted the that a domain nm to the length. The length data were in sarcomere length of and values were calculated used the and at at and of at C. PubMed Scopus Google Scholar). the mechanical of the cardiac PEVK domain and N2B-Us, we used the N2B-Us the PEVK domain with its Ig domains and curves were force for to the persistence lengths of single PEVK and N2B-Us we the of single protein that were so of the with the were we and from the and tandem Ig segments, force with a that is to be single and in M. H. Biophys. Mol. Biol. 2000; PubMed Scopus Google and the persistence length. of a curve of is shown in The stretch curve force that with force the of a Ig a stretch of the force with the of Ig domains H. J. S. A. 2000; PubMed Scopus Google Scholar). The curve in is from in which the force were on were of which is shown in determined the persistence length of Ig domains by the release data of The data were data stretch curves with force in and data stretch curves with force in found that the persistence length from data with force a at nm The obtained from curves with force to lengths and a at nm is that the curves are derived from in the force of the release curve and the persistence length the with earlier Granzier H.L. C. PubMed Scopus Google persistence length the single molecule persistence length to be from the in the at the length. we used to the PEVK domain and of curves obtained with the PEVK are shown in the PEVK domain and its Ig domains and we the stretch that the curves were in the force the of Ig domain The obtained force curves with those and the of the stretch curve with in force in these were possibly from between the molecule and the (for example, that the force were due to of PEVK domain of between the see length is that were to in the force range used in the force be the of the the PEVK domain release curves with the shown as in that the persistence lengths of the curves of curves with persistence lengths of nm and and ∼1.4 nm and The persistence length of results is shown in The with at and by that the persistence length at ∼1.4 nm the persistence length of the single molecule and that the at and nm the persistence lengths of and The in the persistence length determined in a recent by H. M. S. A. PubMed Scopus Google Scholar) the domain to as to the PEVK domain of the cardiac N2B isoform and serially linked three for persistence lengths the results of the are The length obtained from the is shown in the to the protein at a length with a length of nm PEVK see a of the values were nm from the in data at nm is with the length of the PEVK of force-extension curves obtained for N2B-Us are shown in for the PEVK the force-extension curve with the curve release In earlier we that N2B-Us be responsible for passive force in cardiac (5.Helmes M. Trombitas K. Centner T. Kellermayer M. Labeit S. Linke W.A. Granzier H. Circ. Res. 1999; 84: 1339-1352Crossref PubMed Scopus (135) Google and the as in of the a of to be for example, to the force and to and a of the of has to with The release curves of N2B-Us be with the on the release curves in Histograms of the derived persistence and lengths are shown in and with N2B-Us length of see the length has data at lengths The persistence length has a with at ∼0.65 and nm PEVK results that the are the persistence length of N2B-Us by that the persistence length at nm the persistence length of the single molecule and that the at nm that of the of the single molecule characteristics in the of the we modeled the extensible region of cardiac titin as three serially linked tandem Ig segments, the PEVK domain, and N2B-Us of Ig domains has to be the extensible region of titin, we the of domain as a function of force in characteristics at force and of the determined for of the Ig domains found in the extensible region of cardiac titin, we that domains characteristics those determined for titin, and (for see The results that in the of domain is domains curve curves a the curve curves it is that is domain and that domains in the titin extensible region a domains were to is the of of a single domain at the upper range of the physiological force is that of titin in the a single domain that that at physiological force domain is a and that it is to the extensible region of cardiac titin with tandem Ig segments folded calculated the extension of spring element that the titin extensible region as a function of sarcomere length and results with those experimentally determined in a for that in sarcomeres the tandem Ig segment extension predicted the N2B-Us extension is predicted in the physiological sarcomere length range and are used the serially linked of the cardiac titin extensible region to the force of a single titin molecule in the cardiac The results obtained were with the passive length of cardiac cardiac N2B to the single molecule for The results that the predicted passive forces are of those by the cardiac titin extensible region underlies the majority of the tension by the myocardium the of the heart (8.Wu Y. Cazorla O. Labeit D. Labeit S. Granzier H. J. Mol. Cell. Cardiol. 2000; 32: 2151-2162Abstract Full Text PDF PubMed Scopus (169) Google Scholar). In the myocardium, force by a of the between and O. Y. Irving T.C. Granzier H. Circ. Res. PubMed Scopus Google Scholar). that titin to protein and H., and Labeit, S. (2002) @@email protected@J. Physiol. (Lond.)@@/email protected@, in pressGoogle Scholar). range of it is to the of force by titin. we mechanical of the PEVK domain and N2B-Us, spring elements that the physiological levels of passive tension in moderately to highly stretched myocardium K. Freiburg A. Centner T. Labeit S. Granzier H. Biophys. J. 1999; 77: 3189-3196Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Titin a to M. Y. Trombitas K. M. Kellermayer Witt C. Labeit D. Labeit S. Greaser M. Granzier H. Biophys. J. Full Text Full Text PDF PubMed Scopus Google Scholar). in mechanical the data be derived from single and In earlier with skeletal muscle titin Granzier H.L. C. PubMed Scopus Google we that a of a persistence length be that is with the at the length the persistence length of the single in in which we and determined the persistence lengths of Ig domains from the release of the When results in a a obtained with at and The at the length derived from in which were that single were stretched are as a single molecule H. M. S. A. PubMed Scopus Google Scholar). The persistence length at the length from curves with that were derived from The that the length is to of the length that the is a length is of the and be due for example, between the that the that the the with the that the persistence length allows the single molecule to be In with a recent of a with three of the cardiac PEVK domain to in H. M. S. A. PubMed Scopus Google of the PEVK domain little and forces be with the the PEVK domain as spring with a The PEVK domain persistence length a with at and with derived from and respectively. The persistence length at lengths nm is to that by H. M. S. A. PubMed Scopus Google Scholar) the in the of data were found at lengths we found a at nm is that the protein that we used has a for the used by in the H. M. S. A. PubMed Scopus Google the to a single molecule its force of and those curves that were derived from from the were to the of the PEVK domain persistence length from that of H. M. S. A. PubMed Scopus Google that results of were derived from single that the persistence length of the single molecule vary greatly to is on the recent with a PEVK from skeletal muscle titin K. K. PubMed Scopus Google Scholar). Using and K. K. PubMed Scopus Google Scholar) that PEVK both and H. M. S. A. PubMed Scopus Google Scholar) that the polyproline be and that in the persistence length of the the persistence length range of the cardiac PEVK are to the persistence length a the the is in the a PEVK molecule with its in the a persistence length a molecule with for and Ref. K. K. PubMed Scopus Google Scholar) that of the 188 PEVK residues are of polyproline calculated the persistence length of the PEVK domain that the are (for see and obtained values of and respectively. The in values is the range of values in that of H. M. S. A. PubMed Scopus Google Scholar) The of the results different persistence lengths for and that nearly of PEVK residues are of is that a unique with a at nm F. M. PubMed Scopus Google Scholar) and that is in the of the PEVK is in see by K. K. PubMed Scopus Google the provides for as K. K. PubMed Scopus Google Scholar, M. Y. Trombitas K. M. Kellermayer Witt C. Labeit D. Labeit S. Greaser M. Granzier H. Biophys. J. Full Text Full Text PDF PubMed Scopus Google Scholar, K. J. Biol. Full Text Full Text PDF PubMed Scopus Google the persistence length that we for the PEVK domain we that persistence length is on the of and and the persistence length of the single molecule is ∼1.4 The cardiac-specific N2B-Us greatly the upper of the physiological sarcomere length range and the upper range of physiological forces K. Freiburg A. Centner T. Labeit S. Granzier H. Biophys. J. 1999; 77: 3189-3196Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). we characterized the mechanical of N2B-Us single molecule curves on force in were were in of the and be a of the the of force in of the curves be due to of we stretched a protein that has shown to force M. J. M. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). to those used for N2B-Us with force of that it is that force are from N2B-Us of are that levels of force The of force in the N2B-Us stretch curves and the that the were and followed N2B-Us a The of N2B-Us has experimentally that a residues of the in of the residues and of the residues be the that the majority of the residues be with earlier by and M. H. Biophys. Mol. Biol. 2000; PubMed Scopus Google Scholar) on the protein that the predicted of N2B-Us is to to force of that of its of that little and by of which displayed spring force M. H. Biophys. Mol. Biol. 2000; PubMed Scopus Google Scholar). be of J. M. D. M. M. J. Mol. Biol. PubMed Scopus Google Scholar) that to force at force (for example, to be to mechanical In the predicted for N2B-Us and is with the of force and the by The persistence length of N2B-Us is with at ∼0.65 and nm and be by a single molecule persistence length of nm and the of with a persistence length of The to be due for example, between of different that is to that the single molecule persistence length of N2B-Us is to that of Ig that of the PEVK domain values on force in the the persistence length of the PEVK domain be possibly due to the of the PEVK domain, the values of N2B-Us and Ig domains be of the it is that with that are for function M. PubMed Scopus Google Scholar, J. Mol. Biol. 1999; PubMed Scopus Google Scholar, K. PubMed Scopus Google Scholar, Biol. PubMed Scopus Google cardiac titin is a of a protein for which the is The extensible region of cardiac titin three mechanically distinct that are serially tandem Ig segments, N2B-Us, and the PEVK domain Using the characteristics of the PEVK domain and N2B-Us and those determined earlier for the tandem Ig segment with folded Ig domains we calculated the extension of the various spring elements and predicted the forces generated by titin, both as a function of sarcomere length. In the physiological sarcomere length range predicted values were of values and that the used in the closely those in the that of Ig domains is these at sarcomere lengths the tandem Ig segment extension that predicted and the physiological sarcomere length range, Ig is to that of domains the The of at physiological sarcomere lengths is by the calculated of domain in with force Ig domains in folded the tandem Ig segments a persistence length to extend force and and to the sarcomere length at which extension of the PEVK domain and N2B-Us to allows tandem Ig segments to function as a In that the PEVK domain and N2B-Us both as with different persistence length the single molecule persistence lengths to be and the obtained PEVK domain and N2B-Us a of the extensible region of cardiac titin be that closely the extension of titin in the sarcomere and the unique passive length of cardiac for