© 1999 American Heart Association, Inc.
Rapid Communication |
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
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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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Introduction |
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During diastole, while the ventricle fills, the myocardium is passively stretched. The rate of filling and the magnitude of the end-diastolic volume are significantly affected by the shape of the passive pressure–volume relation. Titin, a striated muscle–specific giant elastic protein, is largely responsible for the generation of the diastolic force in the cardiac myocyte.1 In addition to influencing ventricular filling, titin also helps maintaining the structural integrity of the contracting sarcomere (for reviews, see References 2 through 72 3 4 5 6 7 ).
Titin's force is derived from its extensible I-band region, which consists of two main segments: (1) a segment rich in proline (P), glutamate (E), valine (V), and lysine (K) residues (the so-called PEVK segment) and (2) the tandem immunoglobulin (Ig) segments (serially linked Ig-like domains) flanking the PEVK segment. Both segments are expressed in muscle type–specific length variants, with human cardiac muscle coexpressing the low molecular weight N2B isoform and larger isoforms containing the N2A element.8 The molecular mechanism of titin's elasticity has been investigated in dynamic light-scattering studies on titin in solution,9 mechanical studies on single titin molecules,10 11 12 and immunoelectron microscopic studies on skeletal muscle titin.13 14 15 A model has emerged in which the tandem Ig segments (containing folded Ig-like domains) and the PEVK segment (acting largely as an unfolded polypeptide) behave as serially linked wormlike chains (WLCs). A WLC is a deformable rod whose bending rigidity is expressed in terms of its persistence length (A), a distance within which thermally induced bending orientations are correlated.16 17 The more rigid the polymer, the longer its persistence length (low conformational entropy) and the smaller the force required for its extension (see Materials and Methods). Because the bending rigidity of the tandem Ig segments exceeds that of the PEVK,10 12 13 14 upon stretching the sarcomere, the tandem Ig extends first, followed by extension of the PEVK segment.13 14 15 18
The goal of the present study was to investigate (1) the titin isoform expressed in the rat cardiac myocyte, (2) the extensibility of rat cardiac titin's elastic segment, and (3) whether the recently established molecular properties of titin in vitro can explain the observed mechanical behavior of the passive myocyte. Rat myocardium was shown in the present study to express predominantly the N2B cardiac titin isoform, allowing us to characterize this isoform without confounding effects from the coexpression of other isoforms. The contour lengths of the tandem Ig and PEVK segments were estimated based on sequence data for N2B titin.8 These contour lengths were used in the simulation of the behavior of the elastic titin segment. Comparing the model predictions with the experimental observations in myocytes revealed that the elastic segment could be extended much further than the predicted contour length. Immunoelectron microscopy revealed that Ig domain unfolding is not a prominent process in contour-length gain. Instead, the contour-length gain is due mainly to extension of titin's unique N2B sequence, located between the proximal tandem Ig segment and the PEVK segment. Our work indicates that N2B cardiac titin contains three mechanically distinct molecular spring segments: (1) tandem Ig segments, (2) the PEVK segment, and (3) the unique N2B sequence.
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Preparation of Cardiac Myocytes
Care and treatment of animals in the present study were in accordance with protocol 2361 as approved by Washington State University. Single cardiac myocytes were isolated according to the method used by Granzier and Irving.1 Briefly, cells were isolated from rat (male Sprague-Dawley,
250 g; Simosen Labs,
Gilroy, Calif) heart by perfusing the coronary arteries with
oxygenated Krebs solution containing
collagenase. The atria were removed and discarded.
Ventricles were cut into small pieces, which were repeatedly drawn
through a plastic pipette tip to release isolated cells. The majority
of myocytes obtained this way had a normal, rod-like shape. The cells
were skinned by a 50-minute treatment with 1% Triton X-100 in relaxing
solution (in mmol/L: imidazole 40, EGTA 10, magnesium acetate 6.4,
sodium ATP 5.9, creatine phosphate 10, potassium propionate 80,
DTT 1.0 [pH 7.0 at 21°C]). The detergent was washed out by
extensive rinsing with relaxing solution. To prevent degradation,
solutions contained protease inhibitors (compare Reference
11 ). Preliminary experiments using intact and skinned
trabeculae indicate that the lattice expansion that occurs
upon skinning19 does not affect the passive
force–sarcomere length (SL) relation (I. Wu, H. Granzier,
unpublished observations, 1999), and lattice expansion was therefore
ignored in the present study.
Gel Electrophoresis and Western Blotting
Left ventricular myocardium of rat
and rabbit and skeletal muscle (human soleus) were frozen in liquid
nitrogen, pulverized to a fine powder, and then rapidly solubilized.
The samples were analyzed with SDS-PAGE (2% to 12%
acrylamide gradient gels) (for details, see Reference 11 ).
Western blotting with the anti-titin antibodies specific to the N2A and
N2B isoforms was performed as explained in our earlier
work.20 Both antibodies are affinity-purified polyclonal
antibodies. To raise the N2A antibody (X104–X105), the titin sequence
from the N2A splice pathway (base pairs 15607-15957 of human skeletal
cDNA entry [EMBL data library accession No. X90569]) was expressed in
Escherichia coli. For the N2B antibody (X150-X151),
the base pairs 11551 to 11928 of the human cardiac titin cDNA sequence
(EMBL data library accession No. X90568) were expressed. This sequence
locates within the nonrepetitive unique sequence of the N2B segment
(see Figure 1C
) that is found in only heart muscle. Protein
expression, purification, and antibody production were as
described earlier.13 15
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Mechanics
The instrumentation and the single-myocyte mechanical protocols
used have been described previously.1 Myocytes were added
to a temperature-controlled flow-through chamber (volume 300 µL)
mounted on the stage of a phase-contrast microscope. One end of a
single cell was glued to a motor.1 The free end was then
bent with a micromanipulator so that the myocyte axis aligned with the
microscope optical axis, and the myocyte cross-sectional area was
measured. Finally, the free end of the myocyte was glued to a force
transducer. Two types of force transducers were used. For experiments
with low stretch-release rates (< 1.6 lengths/sec), a model 406A force
transducer (Cambridge Technology) was used (resonance frequency 75
Hz). To accurately measure force in response to fast stretch-release
rates (>1.6 lengths/sec), a model 403A force transducer (Cambridge
Technology) was used (resonance frequency 400 Hz). To avoid the
disturbing effect of meniscus forces, the force transducer was
positioned entirely below the solution surface through the side of the
chamber. A gravity-based perfusion system was used. Experiments were
carried out at 20°C to 22°C.
Using potassium chloride and potassium iodide extraction,
we1 previously showed that the force–SL relation of
myocytes is largely titin-based with a small amount of force derived
from intermediate filaments (IFs). In the present work, extraction
experiments revealed that IF-based force at the longest SLs studied was
5% of the total force, and that the contribution was less at
shorter SLs. Considering the small force contribution of IFs, this
component was ignored in the present work.
SL Measurements
SL was measured by the method of Granzier and
Irving.1 Briefly, phase-contrast images of cells were
digitized, and density traces along the long axis of the cell were
measured. Density traces were restricted typically to a width of
4 µm and a length of 30 µm. The traces were
processed by discrete Fourier transformation, and the position of the
first-order peak in the power spectrum was used to calculate the SL.
Three different regions of the cell—encompassing 50% to 75% of the
cell width—were analyzed, and these results were averaged to
obtain a single representative value.
Immunolabeling
Both immunoelectron microscopy (IEM) and
immunofluorescence techniques were used. The
details of the IEM methods have been published
previously.21 22 Briefly, cells were glued in the
stretched state to the bottom of a minichamber that was used for
immunolabeling, fixing, and embedding of the cells. The time between
stretching and fixing of cells varied from 15 to 60 minutes. The
anti-titin antibodies T12, 9D10, Ti102, and N2B (X150-X151) were used.
(For the source of the T12, 9D10, and Ti102 antibodies, see Reference
2020 .) Their binding sites in the titin sequence are shown in Figure 1C
. Immunofluorescence was performed on
stretched single rat cardiac myofibrils according to Linke et
al.13
Actin Extraction Using Gelsolin
To obtain thin-filament free myocytes, actin was extracted by
using a gelsolin fragment. The extraction method is explained in
Trombitás and Granzier.23
Calculations
The elastic region of titin was modeled as two WLCs with
different elastic properties in series: the tandem Ig segment and the
PEVK segment. For a WLC, the external force (F) is related
to the chain's extension (z), as in References 16 and
1716 17 :
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Because the tandem Ig and PEVK segments are connected in series, they bear equal forces. Therefore, for a given F, the extension of each segment (zIg and zPEVK) can be calculated. We may then calculate, for that F, the total extension of titin's elastic segment (zIg+zPEVK). By adding the total length of nonextensible sarcomeric components (1800 nm; see References 20 and 2120 21 ), the SL (zIg+zPEVK+1800 nm) can be calculated.
The extension of a WLC approaches L as F-1/2 approaches zero.10 16 At high extensions, F-1/2 is a linear function of z and extrapolates to the length axis at the contour length. We used the feature of linearity to identify WLC behavior in our myocyte mechanical measurements and to estimate the contour length of titin's elastic segment in stretched myocytes. Increased contour length beyond the length estimated by the sequence of the elastic segment (see Results) was assumed to arise from conversion of folded domains into unfolded polypeptide. The effect of unfolding on the force versus SL relation was simulated by assuming that the persistence length of the unfolded polypeptide was 1.3 nm (based on our recent single-molecule mechanical experiments on N2B titin24 ) and adding the contour length of the unfolded segment to that of the PEVK segment.
Scaling Measured Myocyte Force to the Single Molecule
To scale the single myocyte measurements down to the single
titin molecule, the measured force per myocyte was divided by the
cross-sectional area of the cells. Considering that only part of the
cross-sectional area contains myofibrils, skinned cells were prepared
for electron microscopy, and cross sections were made and photographed.
Analysis of the cross sections indicated that 81±9% of the
cells (n=5) was taken up by myofibrils. This value was used to convert
passive stress per unit area of cell into stress per unit area of
myofibril. The number of thick filaments per
µm2 myofibril was taken as 540, and the number
of titin molecules per half-thick filament as six. For additional
details, see Granzier and Irving.1
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Composition of Titin's Elastic Region in Rat Myocardium
It has been shown in human myocardium that the N2B and N2A titin sequences located between the proximal tandem Ig and the PEVK segments are differentially expressed.8 Our SDS-PAGE analysis of rabbit myocardium is consistent with coexpression of two major titin isoforms because it revealed two T1 bands: a higher- and a lower-mobility band (Figure 1A
). In contrast, rat
myocardium contains a single T1 band, which comigrates with
the bottom T1 band of the rabbit (Figure 1A). To test for the
identity of the single T1 band in rat, Western blot experiments were
performed with the anti-titin antibodies N2B and N2A, raised against
unique sequences found in cardiac N2B and N2A isoforms, respectively
(Materials and Methods). The rat cardiac titin isoform, corresponding
to the lower T1 band of rabbit, was found to strongly react with the
N2B but not with the N2A antibody. Although these findings do not
exclude expression of a small amount of N2A titin that is below the
detection limit of our Western blot and SDS-PAGE methods, it is clear
that rat myocardium expresses predominantly the N2B titin
isoform.
The composition of the elastic region of rat cardiac titin can be
established from the known binding sites in the N2B titin sequence of
the T12 and Ti102 antibodies that demarcate the extensible region of
titin in rat cardiac myocytes.22 This indicates that the
elastic region of the N2B titin isoform contains 45
Ig/fibronectin-like domains, the 572-residue unique sequence in the N2B
element and a 163-residue PEVK segment (Figure 1C). A schematic
diagram showing our vision of how the elastic titin segment is
assembled from two WLCs (tandem Ig and PEVK segments) is shown in
Figure 1C (bottom).
Passive Properties of Cardiac Myocytes and Comparison With
Simulated WLC Curves
To determine the mechanical properties of titin's elastic segment
in single rat cardiac myocytes, relaxed cells were stretched at a
constant rate to a predetermined maximum length. Then the process was
reversed to obtain the release half-cycle. During this protocol, the SL
and the force (F) were measured. Between consecutive stretch-release
cycles, the myocyte was allowed to rest at the slack length (zero
force) for 15 minutes. Imposing the rest period ensured reproducibility
of the mechanical results. From the F and SL data, F versus SL plots
were constructed. From such F versus SL plots (Figure 2A), several initial observations can be
made. (1) In the early part of stretch, a nonlinear force response is
seen. (2) As the sarcomere is stretched to longer lengths (2.2
µm), the force response deviates from the initial nonlinearity and
increases almost linearly with increasing SL. (3) It was possible to
stretch the sarcomere to lengths greater than that allowed by the
contour length of titin's elastic segment (tandem Ig segments with
folded Ig domains plus unfolded PEVK segment, SL 2.35 µm).
(4) During release, the force response deviates from that seen during
stretch. In the initial part of release, force drops rapidly and
continues to decrease in a highly nonlinear fashion with decreasing
SL. (5) For a given SL, the stretch-force is always greater
than the release force. Thus, a force hysteresis is observed. (6)
Hysteresis is greater at longer maximum SLs. Hysteresis begins to
increase greatly at 2.1 µm and continues to increase
significantly with further increase in maximum SL (Figure 2A, inset, open symbols). Hysteresis is not affected by extracting thin
filaments using gelsolin (see Figure 2A, inset, closed
symbols).
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According to the analysis by Kellermayer et al,10
the single titin molecule may be modeled as a WLC. The WLC theory can
be more reliably and reproducibly applied to the release curves than to
the stretch curves. The release data obtained in the present study
on cardiac myocytes can indeed be well fit with WLC curves (Figure 3B and below). However, when the force
response of myocytes measured during stretch is scaled down to the
single titin molecule level, significant deviations from the prediction
based on the WLC model of titin's elastic segment can be seen, both at
SLs less than and greater than 2.2 µm. Below 2.2 µm
SL, the measured force exceeds the predicted one. In contrast, above
2.2 µm SL, the measured force is much less than predicted
(Figure 2B).
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The lower than predicted forces at SLs above 2.2 µm could conceivably be caused by an increase in titin's contour length due to structural transitions (eg, unfolding) within titin's elastic segment. The increased contour length may be reflected in characteristics of the release curve. Previous work on single titin molecules10 showed that at the beginning of release, a reversible WLC curve is seen with a contour length that reflects domain unfolding that took place during the preceding stretch. Refolding of these domains occurs only as the force is sufficiently lowered by shortening the molecule.10 The reversibility of the WLC curve at the beginning of the release indicates that the contour length stays constant, and its adjustment (increase/decrease) does not take place. Reversibility can be tested in partial release–restretch experiments. Accordingly, we tested whether contour-length adjustment is absent during the initial part of the release of cardiac myocytes, by partially releasing the stretched myocytes followed by restretching.
The force measured during restretching the myocyte after partial
release retraced the force curve obtained during release, ie,
hysteresis is absent (Figure 3A). The reversibility of the force
curve during this initial part of the release indicates that the
structural constraints that were broken during stretch and that gave
rise to contour-length adjustment did not reform during the partial
release. This observation supports previous suggestions that for
refolding to occur in the single titin molecule, the external force
must be decreased to low levels; before that, refolding is
precluded.10 Whether the extensible titin segment during
the initial part of the release behaves as a WLC was tested by plotting
F-1/2 versus SL for the release curve. The
F-1/2 versus SL curve obtained during release
reveals WLC behavior, because the experimental data linearly approached
the SL axis (Figure 3B). Linear extrapolation to the length axis
gives the SL at which the contour length of titin's elastic segment is
reached. Subtracting from the extrapolated SL the nonextensible
sarcomeric components (1800 nm) and dividing the result by two (to
account for the two half sarcomeres) will yield the contour length of
titin's elastic segment. The contour length thus obtained increased
with increasing maximum experimental SL (Figure 3C, left-hand
scale).
To estimate the effect of this contour-length gain on the simulated
F-SL curve, we assumed that the adjustment results from an increase in
the length of titin's unfolded region and determined the predicted
F–SL relation of the WLC model accordingly (for details, see Materials
and Methods). Results were compared with the average measured stretch
force scaled down to the single-molecule level. Findings indicate that
when contour-length gain is taken into account, predicted and measured
forces at SLs >2.2 µm agree more closely (Figure 2B, broken and thin solid lines, respectively).
In summary, our data suggest that the elastic region of titin behaves as a WLC with a contour length that increases with the amplitude of stretch. The length gain of the elastic segment may be explained by a progressive increase in the length of the unfolded segment. Increasing the length of the unfolded segment allows the sarcomeres to be stretched to long lengths while avoiding the extremely steep increase of force expected if contour-length adjustment were to be absent.
Time Course of Contour-Length Recovery
Absence of hysteresis in partial release–restretch experiments
(Figure 3A) suggests that contour-length recovery does not take
place at high force. To investigate the process of contour-length
recovery at low force, we determined the rate of hysteresis recovery
during successive stretch-release cycles. The myocyte was stretched
from its slack length to a predetermined amplitude (see inset of Figure 4A); the cell was then released to the
slack length in 2 ms, and, after a pause of preset duration, the
slow stretch/rapid release was repeated. Figure 4A shows an
example in which a cell was stretched to a SL of 2.4 µm, an
amplitude that is likely to result in contour-length adjustment, and in
which the pause durations were 0, 12, and 200 seconds. Recovery occurs
slowly and extends into the pause phase of the protocol. If recovery is
completed during the pause, then hysteresis is recovered, and the
successive stretch curve retraces the previous one. If, however,
recovery is incomplete, then hysteresis is not recovered completely,
and the second stretch curve lies below the first. The examples in
Figure 4A show that recovery is completed only after a
200-second pause. The time course of hysteresis recovery can be fitted
with a logarithmic function, an example of which is shown in Figure 4B.
Experiments were also conducted in which the amplitude of SL
stretch was varied. This indicated that the time for complete recovery
increases with increasing amplitude of stretch. For SL amplitudes below
2.0 µm, force recovery was completed within several seconds,
but for longer SLs, the time needed for 90% recovery ranged from 100
seconds at SL 2.15 µm to 400 seconds at SL 2.42 µm
(Figure 4C).
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The effect of slow hysteresis recovery on the F versus SL relation
during repeated stretch-release protocols was investigated by
subjecting the myocyte to stretch-release cycles with no pause between
the individual cycles. Repeated stretch-release cycles did not have a
significant effect on the F versus SL curve during release. However, a
dramatic effect of repeated stretch-release cycles was the collapse of
the F-SL curve during stretch onto the release curve, resulting in a
large reduction of force hysteresis (Figure 5A). The hysteresis recovered after a
20-minute rest period at the slack length (Figure 5A). Repeated
stretch-release cycles modestly reduced peak forces at SLs longer than
2.2 µm (Figure 5B) with a time course that was similar
to that of the hysteresis decrease (Figure 5C). Hysteresis
(determined as the area under the stretch curve minus the area under
the release curve) decreased significantly with the number of
stretch-release cycles, with 50% reduction in 5 cycles (Figure 5C).
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In summary, after a stretch release to a long SL, hysteresis recovery is a slow process, and, so is, by inference, contour-length recovery. Slow contour-length recovery minimizes hysteresis in repeated stretch-release cycles.
Effect of Stretch Rate on Contour-Length Gain
Single-molecule mechanical studies on titin revealed that
increasing the stretch rate reduces the contour-length length gain
through unfolding, as a result of the increase in the force at which
unfolding takes place with an increase in stretch rate.11
Conceivably, contour-length gain in the cardiac myocyte is similarly
reduced by increasing the stretch rate. If indeed the length gain by
the elastic segment is reduced under these conditions, a given SL can
be reached only at a greater fractional extension of the elastic
segment, hence at a greater force. The relationship between force and
stretch rate was studied by subjecting the myocyte to a series of
stretch-release cycles with increasing stretch rates. Figure 6A shows F versus SL stretch curves
obtained at different rates of stretch, spanning a 250-fold range (0.03
to 8 µm per sarcomere per second). The relationship between
force at the maximum experimental SL and the logarithm of stretch rate
is shown in Figure 6B. The force for a given SL increases with
stretch rate. The effect of stretch rate becomes more pronounced as the
SL exceeds 2.1 µm. For example, increasing stretch rate
250-fold at a SL of 2.45 µm increased the force by 50%
whereas doing so at a SL of 2.05 µm increased force by only
10%. Plotting the corresponding release curves of Figure 6A as
F-1/2 versus SL showed that at a SL of 2.45
µm, increasing the stretch rate 250-fold reduces the contour
length of the extensible titin segment by 30 nm. The effect of the
30-nm reduction in contour length was calculated, from the WLC
theory, to yield 45% increase in force—a value similar to the
measured value.
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In summary, the stretch force of the myocyte at a given SL increases with the stretch rate in a logarithmic manner. The increase in force with increase in stretch rate results from a reduction in contour-length gain of titin's elastic segment.
Sequence Within Titin Underlying Contour-Length Adjustment
To determine where along the elastic segment of titin
contour-length gain takes place, IEM was used. Using the antibodies T12
and Ti102 (see Figure 1C), it was shown previously that at SLs
<3.0 µm, rat cardiac titin's elastic segment is restricted to
the sequence demarcated by the T12 and Ti102 epitopes.20
Thus, contour-length gain does not result from recruiting titin from
the A-band or from near the Z-line to titin's extensible segment;
instead it results from within the segment demarcated by the T12 to
Ti102 epitopes. It is unlikely that the PEVK segment is a major source
of contour-length gain because studies with the anti-titin antibody
9D10, which labels the full length of this segment, indicate that the
PEVK does not extend much at SLs beyond 2.3
µm,25 whereas large contour-length gain takes place at
these SLs (Figure 3C). To explore the site of contour-length
adjustment, the T12, 9D10, and Ti102 antibodies were used to measure
the extension of (1) the proximal tandem Ig segment plus the unique N2B
element (T12 to edge of 9D10 labeled region) and (2) the distal tandem
Ig segment (edge of 9D10 labeled region to Ti102), both as a function
of SL. The predicted extension of the tandem Ig segments was also
calculated using the serially linked WLC model (Figure 1C; see
Materials and Methods).
The predicted extension of the proximal tandem Ig segment was close to
the measured T12-9D10 distance at SLs from 1.8 to 2.1 µm,
whereas at longer SLs, the measured extensions exceeded the predicted
extension (Figure 7B). For example, at a
SL of 2.9 µm, the measured length was 340±44 nm (n=13), or 265
nm longer than the predicted maximal length of the proximal tandem Ig
segment of 75 nm. The predicted extension of the distal tandem Ig
segment tracked reasonably well with the measured extensions at SLs
between 1.8 to 2.4 µm, although with further stretch, the
segment extended slightly beyond the predicted maximal contour length
of 140 nm (Figure 7C).
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The results of Figure 7 indicate that contour-length adjustment
takes place between T12 and 9D10 epitopes, a region that includes the
proximal tandem Ig segment and the N2B element (Figure 1C). To
determine which of these segments gives rise to contour-length
adjustment, experiments were performed with the N2B antibody
(X150-X151; see Materials and Methods), which was raised against the
unique N2B sequence located just outside the proximal tandem Ig segment
(Figure 8C, inset). Examples of IEM
images of rat cardiac myocytes labeled with the N2B antibody are shown
in Figure 8A. The antibody labels a well-defined epitope in the
I-band. The distance between this epitope and the T12 epitope was
measured in sarcomeres stretched to different lengths (Figure 8C, red symbols).
Initially, the segment tracked the predicted
length of the proximal tandem Ig segment (Figure 8C, curved
line); at SLs >2.1 µm, it exceeded the predicted length, and a
plateau was attained in sarcomeres longer than 2.3 µm (Figure 8C, Table). The plateau value
(85 nm) is slightly larger than the predicted contour length of the
proximal tandem Ig segment (75 nm).
Immunofluorescence on single myofibrils labeled
with N2B and 9D10 (Figure 8B and 8C) confirmed and extended the
IEM results.
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) as a
function of SL. Symbols with horizontal and vertical thin lines
indicate data obtained with immunofluorescence and
are the mean±SD of results in 100-nm-wide SL bins. Remaining symbols
indicate IEM data. Curved line is predicted extension of proximal
tandem Ig segment, and straight line is linear regression line of
T12-9D10 measurements (IEM). Closed circles are results from Gautel et
al
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The segment between the N2B epitope and the edge of the 9D10 labeled
region contains a large part of the unique N2B sequence as well as two
Ig domains, I18 and I19 (Figure 8C, inset). The length of this
segment can be obtained from the T12-9D10 length measurements (Figure 8C, open circles) minus the T12-N2B length measurements (Figure 8C, red symbols). The obtained values indicate that this segment
has a near zero length at SLs <2.2 µm, whereas at longer SLs,
it extends (Figure 8C, Table). It is unlikely that this
extension results from Ig domain unfolding (I18 and I19) because
superimposing results with an antibody against I18 (from Gautel et
al26 ) with 9D10 data indicates that the distance between
I18 and 9D10 does not increase with SL (Figure 8C, compare open
circles and closed black circles; see also legend to Figure 8).
Instead, our findings provide evidence that the unique N2B sequence is
extensible. Thus, a major source of contour-length gain in titin's
elastic segment is derived from the unique N2B sequence.
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In the beating heart, the myocytes, hence the sarcomeres within them, are subjected to repeated stretch-release cycles. During such a cycle, the passive force developed in the sarcomere changes: (1) during stretch (diastole), passive force increases because sarcomeric elastic elements are extended; (2) during release (systole), passive force decreases because the stretched elastic elements relax. The shape of the passive F–SL relation, which is determined by the properties of these elastic elements, is expected to influence ventricular filling during diastole and ventricular emptying during systole. It has previously been demonstrated that titin is the major sarcomeric elastic element that contributes to passive force of the cardiac sarcomere, with, as a minor source, the intermediate filaments.1 With novel insights about the mechanical features of the single, isolated titin molecule in hand,10 11 12 we explored the mechanical behavior of titin in situ, in the cardiac sarcomere, by subjecting the single isolated myocyte to a wide range of stretch-release protocols. We found that titin's elastic segment gains length during stretch, as a result of extension of the unique N2B sequence, and that both the rate and magnitude of the preceding mechanical perturbation significantly influence the shape of the passive F-SL relation.
Composition of Titin's Extensible Segment in Rat Cardiac
Myocytes
In human myocardium, the N2B and N2A titin sequences
located between the proximal tandem Ig and the PEVK segments are
differentially expressed.8 In contrast, our results
(Figure 1B) indicate that rat myocardium expresses
predominantly the N2B titin isoform. The composition and contour length
of the extensible region of rat cardiac titin can thus be established
from the known binding sites in the N2B titin sequence of the T12 and
Ti102 antibodies that demarcate the extensible region of titin in rat
cardiac myocytes.22 In N2B titin, this region contains
tandem Ig segments with a combined contour length of 215 nm
(assuming all Ig domains are folded) and a PEVK segment (assuming to be
completely unfolded) with a contour length of 60 nm (Figure 1B and 1C).
In addition, to tandem Ig and PEVK segments, the
extensible segment of N2B titin also contains a unique sequence of 572
residues located between the proximal tandem Ig segment and the PEVK
segment (see Figure 1B). Our findings indicate that this
sequence is extensible as well (Figure 8C). Thus, the extensible
segment of rat cardiac titin contains three types of subsegments: the
tandem Ig segments, the PEVK segment, and the unique N2B sequence.
Does Contour-Length Gain Result From Ig Domain Unfolding?
According to our mechanical measurements, above 2.2 µm
SL titin's elastic segment extends beyond the combined contour length
of tandem Ig and PEVK segments (Figure 3C). This contour-length
gain does not result from the unfolding of Ig domains in the distal
tandem Ig segment because this segment extends little at SLs
>2.2 µm (Figure 7C). This conclusion is
consistent with biophysical studies of genetically engineered
Ig domains from the distal tandem Ig segment27 28 that
found that these domains are very stable. Contour-length gain arises
instead from the region between the T12 and 9D10 epitopes (Figure 7B),
a region that includes Ig domains (proximal tandem Ig
segment) and the unique sequences of the N2B element. The proximal
tandem Ig segment is contained in the region demarcated by T12 and N2B
epitopes (Figure 1B). The distance between these epitopes
extends beyond the predicted contour length of the proximal tandem Ig
segment by 10 nm (Figure 8C), which may be explained by
unfolding of a just a single Ig domain. However, it is also possible
that the longer than expected T12-N2B distance results from the short
unique sequence contained within this region (Figure 8C, inset).
Future research with antibodies that more precisely demarcate the
proximal tandem Ig segment will be required to resolve this issue. It
is clear from the present work, however, that Ig domain unfolding
is, at best, of limited significance in explaining the large
contour-length adjustment that takes place in titin at SLs >2.2
µm. Although the titin elasticity model based on Ig domain unfolding,
proposed by Erickson,29 is attractive and has had great
impact, our studies of the largest titin isoform in skeletal muscle
(human soleus titin; Trombitás et al15 ) and the
smallest isoform in cardiac muscle (the present study) show that
large-scale Ig domain unfolding is unlikely under
physiological conditions.
The Unique N2B Sequence as Source of Contour-Length Gain
Results indicate that a prominent site of contour-length
adjustment in rat cardiac myocytes is the unique N2B sequence. The
unique N2B segment C-terminal of the N2B epitope contains between 310
and 436 residues (depending on where the N2B epitope is located within
the 126-residue-long unique N2B fragment that was used to raise the
antibody), and our results indicate that this segment has a short
end-to-end length at SLs <2.1 µm (Figure 8C). It is
likely therefore that in absence of external force, the unique sequence
has a compact structure. Our findings that this 310- to 436-residue
unique sequence extends to 70 nm at 2.4 µm SL in rat myocytes
(Figure 8C, Table) and the preliminary observations in
other species where extensions of 150 nm were attained (K.
Trombitás, S. Labeit, H. Granzier, unpublished data, 1999)
suggest that average residue spacings can be achieved that are only
compatible with a fully unfolded polypeptide. Thus, it is likely that
when the sarcomere is extended beyond a length of 2.2 µm, the
unique N2B sequence unfolds from a compact state to ultimately a
completely unfolded and extended polypeptide.
Unfolding-based extension of the unique N2B sequence is
consistent with the mechanical behavior of the myocyte. We
found that increasing the stretch rate increased the force at the
maximum experimental SL, a phenomenon that is most pronounced at long
SLs where extension of the unique N2B sequence takes place (Figure 6).
This stretch-rate dependence of force indicates that N2B
extension in the cardiac myocyte involves a rate-limited process.
Previous observations on single titin molecules have shown that the
unfolding within titin is a kinetic process that gives rise to a linear
relation between the unfolding force (the force at which domain
unfolding takes place in titin) and the logarithm of stretch
rate.11 Because we also find a linear relationship between
peak force and logarithm of stretch rate in isolated myocytes (Figure 6B),
it is likely that unfolding within the unique N2B sequence
is responsible for the length gain of titin's elastic segment.
Hysteresis
During stretch and release, the cardiac myocytes display force
hysteresis: for a given SL, the force measured during stretch is
greater than the force measured during release. The presence of
hysteresis in thin filament–extracted myocytes (Figure 2A, inset) suggests that titin–thin filament interaction does not greatly
contribute to force hysteresis; rather, the hysteresis arises from
within titin itself. Hysteresis begins to increase significantly as the
SL exceeds 2.1 µm and continues to increase with increasing
SL (Figure 2A). The onset of significant hysteresis is close to
the onset of extension of the unique N2B sequence, suggesting that
hysteresis depends on the kinetics of unfolding/refolding within the
unique N2B sequence.
The reproducibility of the stretch curve after a sufficient pause
indicates that unfolding is a completely reversible process in the
myocyte (Figures 2A and 4). However, refolding takes a
relatively long time to be completed. The time course of refolding is
reflected in the force recovery curves of Figure 4. The time
course can be fit with a logarithmic function, indicating that most of
the refolding is rapid but that complete refolding occurs more slowly
(Figure 4A and 4B). Because complete refolding is a slow
process, part of the N2B sequence is still in the unfolded state at the
completion of the stretch-release cycle. If a new stretch-release cycle
is initiated immediately, the N2B sequence will contain preunfolded
regions. Therefore, the force generated in the immediately following
stretch is less then in the previous one, bringing the stretch curve
closer to the release curve, and reducing hysteresis (Figure 5).
As a result of unfolding of the unique N2B sequence, sarcomeres can be
stretched to long lengths, whereas slow refolding during release gives
rise to strong hysteresis in the first stretch-release cycle, but it
minimizes hysteresis in subsequent cycles (Figure 5). If parts
of the extensible region of titin unfold and refold each time the
myocyte is stretched and released, an amount of energy equal to the
area within the hysteresis loop would be wasted as heat. Minimizing
passive force hysteresis in cells that are stretch-released
repetitively (Figure 5C), as is the case in the beating heart,
may be physiologically significant.
Cardiac Titin as an Adjustable Spring
It is well established that tandem Ig and PEVK segments extend
sequentially13 14 15 : extension is first dominated by
straightening of the tandem Ig segments and at longer SLs by that of
the PEVK polypeptide. Sequential extension can be simulated well by
modeling the PEVK and tandem Ig segments as serially linked WLCs with
different persistence lengths (bending rigidities).15
However, when slack cardiac myocytes are stretched, developed force is
initially slightly higher than predicted by the serially linked WLC
model (Figure 2B). We propose that this results from weak
noncovalent interactions between different regions of titin (see thin
black lines in Figure 9) that form when
titin is in a "contracted" state in slack sarcomeres. These
interactions are disrupted during stretch and reform only slowly during
release, giving rise to hysteresis at short SLs. Furthermore, when
cells are stretched to SLs >2.2 µm, contour-length gain
takes place within titin's extensible region, as a result of extension
of the unique N2B sequence (Figure 9 "adjustable spring
segment"), decreasing the fractional extension of tandem Ig and PEVK
segments and explaining why force at SLs longer than 2.2 µm is
much lower than predicted by the serially linked WLC model. Indeed,
when taking into account contour-length gain, the force–SL relation
predicted by the model more closely simulates measured forces (Figure 2B).
Thus, the unique N2B sequence provides cardiac N2B titin
with a third source of extensibility (in addition to straightening of
the tandem Ig segments with folded Ig domains and extension of the
largely unfolded PEVK segment) that allows sarcomeres to be stretched
to long lengths.
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Contour-length gain in the unique N2B sequence during stretch and slow recovery during release represent two levels of adjustment within titin that determine the shape of the F-SL curve, as well as the amount of hysteresis in repeated stretch-release cycles. Thus, N2B titin may be viewed as an adjustable spring. This property allows N2B titin to accommodate long SLs and to adjust titin's efficient working range with minimal energy loss due to hysteresis. Considering that human myocardium coexpresses length variants of titin,8 titin's adjustable spring property may also allow the efficient working range of isoforms to be adjusted to each other.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health National Heart, Lung, and Blood Institute (HL61497 and HL62881) to H.G., the Human Frontier Science Program (to S.L.), the Fortschungsfond fur Klinische Medizin Mannheim (to S.L.), the Deutsche Fortschungsgemeineschaft (La668/5-1 to S.L. and SFB 320 to S.L. and W.A.L.), and the Hungarian Science Foundation, OTKA F025353 (to M.K.). H.G. is an established investigator of the American Heart Association.
Received February 9, 1999; accepted April 19, 1999.
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