© 1999 American Heart Association, Inc.
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Mechanically Driven Contour-Length Adjustment in Rat Cardiac Titin's Unique N2B Sequence
Titin Is an Adjustable Spring
From the Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology (M.H., K.T., H.G.), Washington State University, Pullman, Wash; European Molecular Biology Laboratory (T.C., S.L.), Heidelberg, Germany; Department of Biophysics (M.K.), University Medical School of Pécs, Pécs, Hungary; Institut für Anästhesiologie und Operative Intensivmedizin (S.L.), Universitätsklinikum Mannheim, Mannheim, Germany; and Institute of Physiology II (W.A.L.), University of Heidelberg, Heidelberg, Germany.
Correspondence and reprint requests to Henk Granzier, Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, WA 99164-6520. E-mail: granzier{at}wsunix.wsu.edu
Abstract—The giant elastic
protein titin is largely responsible for passive forces in cardiac
myocytes. A number of different titin isoforms with distinctly
different structural elements within their central I-band region are
expressed in human myocardium. Their coexpression has so
far prevented an understanding of the respective contributions of the
isoforms to myocardial elasticity. Using isoform-specific antibodies,
we find in the present study that rat myocardium
expresses predominantly the small N2B titin isoform, which allows us to
characterize the elastic behavior of this isoform. The extensibility
and force response of N2B titin were studied by using immunoelectron
microscopy and by measuring the passive force–sarcomere length (SL)
relation of single rat cardiac myocytes under a variety of mechanical
conditions. Experimental results were compared with the predictions of
a mechanical model in which the elastic titin segment behaves as two
wormlike chains, the tandem immunoglobulin (Ig) segments and the PEVK
segment (rich in proline [P], glutamate [E], valine [V], and
lysine [K] residues), connected in series. The overall contour length
was predicted from the sequence of N2B cardiac titin. According to
mechanical measurements, above 
2.2 µm SL titin's elastic
segment extends beyond its predicted contour length. Immunoelectron
microscopy indicates that a prominent source of this contour-length
gain is the extension of the unique N2B sequence (located between
proximal tandem Ig segment and PEVK), and that Ig domain unfolding is
negligible. Thus, the elastic region of N2B cardiac titin consists of
three mechanically distinct extensible segments connected in series:
the tandem Ig segment, the PEVK segment, and the unique N2B sequence.
Rate-dependent and repetitive stretch-release experiments indicate that
both the contour-length gain and the recovery from it involve kinetic
processes, probably unfolding and refolding within the N2B segment. As
a result, the contour length of titin's extensible segment depends on
the rate and magnitude of the preceding mechanical perturbations. The
rate of recovery from the length gain is slow, ensuring that the
adjusted length is maintained through consecutive cardiac cycles and
that hysteresis is minimal. Thus, as a result of the extensible
properties of the unique N2B sequence, the I-band region of the N2B
cardiac titin isoform functions as a molecular spring that is
adjustable.
Key Words: elasticity • diastole • myocardial compliance • connectin • passive force
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