Published online before print May 10, 2001, doi: 10.1161/hh1001.090876
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2001 American Heart Association, Inc.
Cellular Biology |
Titin-Based Modulation of Calcium Sensitivity of Active Tension in Mouse Skinned Cardiac Myocytes
From the Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology (O.C., Y.W., H.G.), Washington State University, Pullman, Wash, and the Center for Synchrotron Radiation Research and Instrumentation and Department of Biological, Chemical, and Physical Sciences (T.C.I.), Illinois Institute of Technology, Chicago, Ill. Present address of O.C. is INSERM U390/IFR3, Physiopathologie Cardiovasculaire, Montpellier, France.
Correspondence to Dr Henk Granzier, Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Wegner Hall, 205, Pullman, WA 88164. E-mail granzier{at}wsunix.wsu.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract—We studied the effect of titin-based passive force on the length dependence of activation of cardiac myocytes to explore whether titin may play a role in the generation of systolic force. Force-pCa relations were measured at sarcomere lengths (SLs) of 2.0 and 2.3 µm. Passive tension at 2.3 µm SL was varied from
1 to 10
mN/mm2 by adjusting the characteristics of
the stretch imposed on the passive cell before activation. Relative to
2.0 µm SL, the force-pCa curve at 2.3 µm SL and low passive tension
showed a leftward shift (
pCa50 [change in
pCa at half-maximal activation]) of 0.09±0.02 pCa units while
at 2.3 µm SL and high passive tension the shift was increased to
0.25±0.03 pCa units. Passive tension also increased
pCa50 at reduced interfilament lattice
spacing achieved with dextran. We tested whether titin-based passive
tension influences the interfilament lattice spacing by measuring the
width of the myocyte and by using small-angle x-ray diffraction of
mouse left ventricular wall muscle. Cell width and
interfilament lattice spacing varied inversely with passive tension, in
the presence and absence of dextran. The passive tension effect on
length-dependent activation may therefore result from a radial
titin-based force that modulates the interfilament lattice
spacing.
Key Words: x-ray diffraction • myofilament lattice • collagen • Frank-Starling
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The precise mechanisms by which the heart is able to enhance its contractile performance in response to an increase in volume (Frank-Starling mechanism) remain to be resolved. The cellular basis of the Frank-Starling mechanism involves the sarcomere length (SL) dependence of the Ca2+ sensitivity of tension.1 Length-dependent Ca2+ sensitivity is revealed by the leftward shift of the force-pCa (-log[Ca2+]) relation as SL is increased. The mechanisms that underlie this shift may involve a length-dependent increase in affinity of the regulatory site of troponin C for Ca2+, as well as an increase in the number of strongly binding crossbridges.2 3 The enhanced active force response when muscle is stretched may be explained by the myofilaments moving closer together,4 thereby increasing the probability of crossbridge binding to actin.5 6 Experiments in which the Ca2+ sensitivity was increased by osmotically compressing the filament spacing5 6 support the idea that changes in myofilament spacing contribute to length-dependent activation.
Recent studies identified titin (connectin) as a possible factor involved in length-dependent Ca2+ sensitivity.7 8 The I-band segment of titin functions as a molecular spring that underlies the passive force of cardiac myocytes.9 10 This force is the main contributor to overall passive force of cardiac muscle, except toward the upper limit of the physiological SL range where collagen dominates.9 11 A passive force–based increase in the number of crossbridges bound to actin has been suggested by earlier work on insect flight muscle12 and rabbit psoas muscle.13 Here we investigated the involvement of titin-based passive tension in the SL dependence of Ca2+ activation of skinned cardiac myocytes and in modulating the interfilament lattice spacing. Force-pCa curves were measured at SL 2.0 and 2.3 µm, and the pCa50 (pCa at half-maximal activation, an index of Ca2+ sensitivity) was determined at various levels of passive tension achieved by varying the stretch characteristics imposed on the cell before activation. We found that passive tension significantly influences both the length dependence of activation and the myofilament lattice spacing in normal and compressed muscle. Thus, titin is not just a passive spring that is independent of active force development, which is the conventional view, but titin also influences actomyosin interaction, possibly via modulating interfilament lattice spacing.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Preparations and Solutions
Myocytes and muscles were isolated from mice and skinned as previously described.11 14 Solution composition was as described,8 as were all contained protease inhibitors.9 For osmotic compression, dextran (T500) was used. For additional details see, online expanded Materials and Methods section available at http://www.circresaha.org.
Experimental Setup
The setup was as
described.11 SL was measured
online at 15 Hz (Ionoptix Corp). The system uses a
pseudo–2-dimensional FFT analysis of the digitized striation
images of the attached
cell.15 SL could be
controlled during activation via adjusting the motor input voltage so
as to keep SL at a constant value. Force was normalized by the
cross-sectional area of the
cell.9 The force-pCa relation
of freshly isolated cells was measured at 2.0 µm and then at 2.3 µm
SL (15°C). Passive force was varied as explained below. Active forces
at submaximal activations were normalized to that produced at pCa 4.5
at the same SL. (Force at pCa 4.5 slightly diminished with the number
of imposed contractions; the mean reduction during the measurement of 2
force-pCa curves was 23±2% [n=41].) The relation between relative
force and pCa was fitted to the following equation: force =
[Ca2+]nH/(K+[Ca2+]nH),
where nH
is the Hill coefficient and pCa50 is -(log
K)/nH.
Values in the
Table
are the average values from all cells studied under a certain
condition. All shown curves were fitted to the average force produced
at each pCa.
|
X-Ray Diffraction
The BioCAT undulator-based beamline at the Argonne
National Laboratory (Argonne, IL) was used. Muscles were mounted to a
force transducer and a motor in a small trough with windows for
collection of x-ray patterns and viewing of striations (SL was
determined as described above). X-ray patterns were collected and
spacings of the 1,0 and 1,1 equatorial reflections were measured and
converted to
d1,0
values.4
Titin Degradation
X-ray experiments were performed on skinned muscle
preparations that had been trypsinized (0.25 µg/mL; 25 minutes at
25°C) to degrade
titin.10 11 To
determine the amount of intact titin, preparations were solubilized
after completion of the x-ray experiment and analyzed with
SDS-PAGE. For details, see References 9 and 169 16 .
Statistics
Results are mean±SE (unless indicated otherwise).
Significant differences were assigned using the paired or unpaired
Student t test (as appropriate)
or 1-way ANOVA and Tukey multiple comparison with
P<0.05.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The Ca2+ sensitivity of active force was studied at various levels of passive force. To determine active force, we subtracted the passive force before activation from total force during activation. However, this is only valid if during activation sarcomeres are isometric, because SL shortening reduces passive force and stretching has the opposite effect. We measured SL online and observed (as have others6 8 15 ) that although cells were kept isometric during contraction, sarcomeres typically changed length, especially when activation was maximal. The SL change varied somewhat from cell to cell, and for each cell we first performed a test contraction at pCa 4.5 and only continued with those cells that were well attached with minimal (<0.1 µm) SL changes. When required, cell length was varied during contraction so as to keep SL constant (see Figure 1
). The differences in SL between the start and the
peak of contraction of all cells used in this study were small
(0.03±0.02 µm). Thus, changes in passive force during contraction
are negligible, and active force equals the activation-induced force
increase.
|
Passive force was adjusted by varying the SL history before
activation. High levels of passive force were obtained by rapidly
stretching the cell to 2.3 µm with activation following immediately
(Figure 1
). Low to intermediate levels of passive force were
obtained by (1) stretching sarcomeres to a length that exceeded 2.3
µm (typically to 2.5 µm), (2) holding cell length constant, (3)
releasing to 2.3 µm SL, and (4) holding the cell at 2.3 µm SL and
then activating. The central panel of
Figure 2A shows an example of this protocol. During the hold
phase at long SL,2 passive
force rapidly decayed, most likely because of contour length gain of
subdomains within the extensible region of
titin.17 Recovery takes
place if the cell is completely released to the slack length and held
there for several minutes. A partial release is insufficient for full
refolding, and passive force will therefore be
lower.17 These protocols
allowed us to vary passive tension at SL 2.3 µm between 1 and
10 mN/mm2.
|
) and at 2.3 µm SL with high (8.7±0.3 mN/mm2; •) and low (1.7±0.3 mN/mm2; 
) passive tension. Note that curves at 2.3 µm SL are shifted leftward and that this shift is largest at high passive tension. C, Effect of passive tension on
When cells were submaximally activated at 2.3 µm
SL, active force was significantly lower if passive force was low
(compare
Figure 2A, middle and right). This reduction in active force
did not result from protocol-induced damage to either titin or the
contractile apparatus, as both passive and active forces
recovered after a 10-minute rest period at the slack length (compare
Figure 2A, left and right). To ensure that all contractions
were induced at the same passive force level, between contractions
cells were always released to their slack length followed by a
10-minute rest, and then the exact same stretch-activation protocol
was imposed.
Force-pCa relations were determined at 2.0 and 2.3 µm SL
at high (8.7±0.3 mN/mm2) and low (1.7±0.3
mN/mm2) passive tension. At both passive
tension levels, the force-pCa curves at 2.3 µm were shifted leftward
relative to the curve at 2.0 µm SL. The shift was significantly
larger at high passive tension
(Figure 2B and
Table).
The pCa for half-maximal activation (pCa50)
increased by 0.25±0.02 pCa units and 0.09±0.01 pCa units for high and
low passive tensions, respectively. Results were independent of the
order in which the experiments were performed (high versus low passive
tension), indicating that no permanent damage was done by these
protocols. We measured the maximal active tension (pCa 4.5) in
experiments in which 3 contractions were induced (in random order), as
follows: (1) 2.0 µm SL, (2) 2.3 µm SL (low passive tension), and
(3) 2.3 µm SL (high passive tension). Maximal active tensions (in
mN/mm2) were 30.7±2.3 (n=18), 31.6±1.7
(n=13), and 34.3±1.8 (n=18), respectively. Only results at 2.3 µm SL
(high passive tension) were significantly higher than at SL 2.0 µm
(ANOVA, P<0.05).
Varying the amplitude and the duration of prestretch
resulted in intermediate passive tension levels at SL 2.3 µm. Results
from 29 cells were pooled in passive tension bins of 2.0
mN/mm2, and their corresponding
pCa50 (pCa50 at SL
2.3-pCa50 at 2.0 µm) values were averaged.
Figure 2C shows that at passive tensions between 0 and 2
mN/mm2, pCa50 is
0.09 pCa units and that at higher tensions
pCa50 increases until it reaches 0.25 pCa
units at a passive tension of 10 mN/mm2.
Linear regression (broken line in
Figure 2C) shows that the length dependence of activation
(pCa50) has a passive tension–independent
component (0.08 pCa units in size) and a component that varies with
passive tension.
Considering that the large prestretch/partial release
protocol requires stretch to nonphysiological SLs,
we also measured the force-pCa relation of cells in which passive
tension was reduced with trypsin, as an independent method. Because of
the high trypsin sensitivity of the PEVK domain of titin, a mild
trypsin treatment can be used to specifically degrade the I-band region
of titin and to thereby lower passive tension at a given
SL10 11 (see also
below and online Materials and Methods available at
http://www.circresaha.org). Trypsin-treated cells were stretched to SL
2.3 µm and then immediately activated. Because of degradation
of titin, passive tension was now low (1.7±0.6
mN/mm2; n=6). The force-pCa relation so
obtained was shifted to the right compared with the curve measured at
high passive tension
(Figure 2D). The pCa50 (5.79±0.04)
and maximal active tension (33.1±2.8
mN/mm2) are indistinguishable from that at
low passive tension obtained after the large prestretch/partial release
protocol (5.78±0.03, and 31.6±1.7 mN/mm2,
respectively). Thus, reduction of passive tension via either trypsin
treatment or a large stretch/partial release leads to the same
force-pCa relation.
We studied whether the passive tension–induced shift of the
force-pCa relation involves a passive tension effect on the myofilament
lattice spacing as indicated by the cell width at 2.3 µm SL. Cell
width was significantly smaller at high passive tension than at low
passive tension
(Figure 3C [a]). Furthermore, during stress recovery
(Figure 3C [b]), cells shrank significantly.
|
It is well known that the myofilament lattice spacing of
striated muscles expands during skinning as a result of the loss of the
osmotic constraint to swelling imposed by the
sarcolemma.4 18 19
It has been reported that the myofilament lattice on skinning of
cardiac muscle expands19 and
that this may be countered with 2.5% (wt/vol) dextran T-500. To
test whether passive tension affects the length dependence of
activation in conditions in which skinning-induced swelling was
compensated, we measured the force-pCa relations at high and low
passive tension in the presence of 2.5% dextran.
In the presence of dextran, the cell width at SL 1.9 µm
was reduced by 8%
(Figure 4A), consistent with the 7% to 8%
reduction reported by
others.6 20 The
force-pCa relations at 2.0 µm SL in the presence of dextran were
shifted to the left (relative to no dextran) to a position similar to
the one obtained without dextran at 2.3 µm SL
(Figure 4B and
Table).
This is consistent with the results of others obtained on
cardiac myocytes6 that showed
that dextran sensitizes the cells at short SL. To our knowledge, the
effect of dextran on the force-pCa relation of cardiac myocytes at 2.3
µm SL has not been investigated before. Relative to the curve at 2.0
µm SL (with dextran), our experiments revealed a
pCa50 of 0.08 pCa units at low passive
tension and 0.15 pCa units at high passive tension
(Figures 4B and 4C,
Table).
|
We measured the maximal active tension in experiments in which 3 maximal active contractions were induced, at SL 2.0, SL 2.3 (low passive tension), and SL 2.3 (high passive tension) µm, in a randomized order. Maximal active tensions (in mN/mm2) were 32.4±2.5 (n=10), 36.6±2.3 (n=10), and 34.9±2.0 (n=10), respectively. Results comparing high versus low passive tension at 2.3 µm were not significantly different. The maximum tensions at 2.3 µm SL (low passive tension) and those at 2.3 µm SL (high passive tension) were both significantly higher than at 2.0 µm.
To further probe the mechanism underlying these findings, we
studied the effect of passive tension on myofilament lattice spacing by
using low-angle x-ray diffraction. Because x-ray diffraction on cells
is currently not feasible, because of their low x-ray scattering mass,
for these studies we used mouse skinned myocardium
dissected from the left ventricular free wall.
Figure 5A, panel 1, shows a typical x-ray pattern from a
skinned preparation in relaxing solution with clearly resolved
equatorial reflections. (SDS-PAGE showed that titin was unaffected by
the x-ray exposure.) The separation of these reflections allowed us to
measure lattice spacing with 0.1-nm resolution. Myofilament lattice
spacing decreased significantly as SL was increased
(Figure 6A), which was consistent with recent
measurements on rat cardiac
trabeculae.4
|
|
To vary passive tension, we were unable to use the "large
prestretch protocol," as collagen in mouse myocardium
limits the maximal SL with reversible mechanical characteristics to
2.3 µm.11 Again, we
varied passive tension by degrading titin with trypsin (0.25 µg
trypsin/mL, 25 minutes at 25°C). Treating mouse skinned
myocardium with trypsin greatly degraded titin
(T1) without significantly affecting
other proteins
(Figure 5B). To test whether trypsin affects collagen-based
force, we treated collagen strips as well as myocardium
with trypsin that had been extracted with high salt to abolish titin as
a source of passive force.11
The force of collagen was unaffected by trypsin
(Figures 5C and 5E). We also tested whether trypsin affects
intermediate filament–based force and found that this force also is
not affected by trypsin
(Figures 5D and 5E). Finally, we measured the force-pCa
relation at a SL of 1.9 µm and found that the relation was unaffected
by trypsin
(Figure 5F). Neither pCa50 (control,
5.71±0.03 [n=12]; trypsin treated, 5.73±0.04 [n=12]) nor maximal
active tension (control, 25.7±2.0 mN/mm2
[n=12]; trypsin treated, 23.3±1.7 mN/mm2
[n=12]) were significantly affected by trypsin. Thus, under
our experimental conditions trypsin specifically abolishes titin-based
passive force.
An example of an x-ray diffraction pattern after trypsin
treatment is shown in
Figure 5A, panel 2, and the SL dependence of the
d1,0
spacing before and after trypsin treatment in
Figure 6A. Degradation of titin significantly increased the
lattice spacing with an average increase of 3 nm. The large increase
in d1,0
at SL 1.9 µm and our finding that trypsin treatment does not affect
calcium sensitivity at SL 1.9 µm
(Figure 5F) suggests that calcium sensitivity is independent
of d1,0
spacing in the range of 44 to 47 nm. Although support for the idea that
changes in myofilament lattice spacing contribute to length-dependent
activation5 6 is
compelling, this finding may be viewed as a cautionary
note.
We also studied whether degrading titin had an effect on the
osmotically compressed lattice achieved by adding dextran. Definitive
studies of the amount of dextran required to restore the in vivo
lattice spacing in skinned cardiac muscle have not been reported;
earlier studies used a range from 2% to 4%
dextran,4 19 20
and we chose 4% for these experiments. An example of an x-ray pattern
is shown in
Figure 5A, panel 3, and the SL dependence of
d1,0 in
Figure 6B. Results indicate that at a given SL, dextran
greatly reduces the lattice spacing. For example, at SL 2.1 µm
dextran reduced
d1,0
from 42.4 to 35.4 nm. (Note that this spacing in dextran is close to
that of intact cardiac muscle at SL 2.1 µm; for rat, reported values
are 34.84 and
35.619 nm). The effect of
degrading titin with trypsin in the presence of dextran was then
studied. An example of an x-ray pattern is shown in
Figure 5A, panel 4, and measurements are shown in
Figure 6B. Degrading titin significantly increased the
lattice spacing, this increase being largest at 1.9 µm SL with a
gradual decrease with SL. These studies support the notion that titin
modulates the interfilament lattice spacing in both the presence and
the absence of dextran.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Numerous studies have shown that an important component of the Frank-Starling mechanism is the length dependence of the calcium sensitivity of force. A now widely held explanation for length-dependent activation is that the change in interfilament spacing that accompanies SL change modulates the probability of actomyosin interaction at the same calcium concentration. The mechanism by which interfilament spacing affects actomyosin interaction may involve an interfilament-spacing effect on weakly bound crossbridges by affecting their number21 or the rate of transition from the weak to strong binding states.22 Here we report that titin modulates interfilament spacing and that titin-based passive tension influences length-dependent activation.
Effect of Titin-Based Passive Tension on
Calcium Sensitivity
Because of stress relaxation, titin-based passive
tension at a given SL is not constant but decreases with time. We took
advantage of the high degree of passive stress relaxation at long SL
and its slow recovery on partially releasing the cell, to vary passive
tension at a SL of 2.3 µm and study its effect of the force-pCa
relation. Results indicate that the length dependence of activation
(pCa50) has a passive tension–independent
component of 0.08 pCa units (intercept of line in
Figure 2C) that is likely to involve thin- and
thick-filament–based processes independent of titin. The passive
tension–independent component is somewhat less than the 0.12
pCa50 values reported by other laboratories
studying the
mouse.23 24 25
Although passive tension was not reported in these previous studies,
they used protocols generally consisting of a slow stretch followed by
a long wait period before activation, and passive tensions are
therefore likely to have been relatively low (our
Figure 2C indicates that a pCa50
of 0.12 is accompanied by 2.5 mN/mm2
passive tension). Thus, the pCa50 values at
low passive tension found here are in general agreement with those of
others. High passive tensions were achieved by rapidly stretching cells
to SL 2.3 µm and then immediately activating them
(Figure 1). We found that passive tension significantly
enhances the length dependence of activation with a
pCa50 of 0.25 pCa units at the highest
passive tensions used. Thus, for maximal calcium sensitivity of skinned
cardiac myocytes, a high level of titin-based passive tension is
required.
Effect of Titin on Interfilament
Spacing
For cardiac myocytes, we used the cell width as an
indicator of interfilament spacing. Although cell width is an imperfect
indicator, it nevertheless provides qualitative insights into
myofilament lattice
behavior.20 We found that
passive tension correlates negatively with cell width
(Figure 3), suggesting that titin modulates myofilament
lattice spacing. In agreement with this are the low-angle x-ray
diffraction studies on cardiac muscle that showed that degradation of
titin significantly increases
d1,0.
These findings are consistent with results on mechanically
skinned skeletal muscle fibers in which a close correlation is found
between titin-based passive tension and
d1,026
and in which, after degradation of titin,
d1,0 is
found to be independent of
SL.27 Thus, titin is a
modulator of interfilament lattice spacing in skeletal and cardiac
muscle.
After titin degradation, the myofilament lattice was still
responsive to SL
(Figure 6). This suggests that in addition to titin, other
modulators of myofilament lattice spacing exist. A possible candidate
is collagen. By comparing
d1,0 of
chemically and mechanically skinned skeletal muscle fibers, it has been
shown that at long SL collagen compresses the myofilament lattice in
skeletal muscle.26 In
myocardium, collagen is the main source of passive tension
at long SL, whereas titin is the main source at short
SL.9 11 If
collagen and titin both affect lattice spacing, eliminating the force
of titin will have the largest effect on spacing at short SL and the
smallest at long SL, giving rise to steeper
d1,0–
SL relations after elimination of titin. This expectation is in
agreement with our findings
(Figure 6) and supports the idea that the myofilament lattice
spacing is under the influence of both collagen and titin.
The segment of titin near the Z line binds strongly to the
thin filament,28 and the
A-band segment of titin attaches to the thick
filament.29 Thus, the
elastic region of titin runs obliquely to the thin and thick filaments,
with an angle that depends on thin- and thick-filament spacing and on
the end-to-end length of the extensible region of titin (see
Figure 7A). As a result, titin is expected to develop
a longitudinal force
(FL) and
radial
(Fr)
force, the latter of which compresses the lattice
(Figure 7A, inset).
|
The major repulsive force that separates myofilaments in
skinned muscle (under relaxing conditions) is the electrostatic force
(FES)
that arises from the net negative charge carried by the thin and thick
filaments.18
FES
values at the measured lattice spacings of
Figure 6A (control curve) were calculated as described in
the expanded online Materials and Methods available at
http://www.circresaha.org. Results shown in
Figure 7B reveal that the repulsive force,
FES, and
the compressive component of titin-based passive force,
Fr, are
of similar magnitude. Thus, titin develops a radial force that is
sufficiently large for it to play a role in counteracting the repulsive
FES,
which supports the concept that titin can modulate the myofilament
lattice spacing of passive muscle.
The proposal that titin gives rise to a radial force is
consistent with the reduced effect of titin on myofilament
lattice spacing in the presence of dextran
(Figure 6). Compressing the myofilament lattice increases the
repulsive force between
filaments,30 whereas the
radial force will be reduced. The latter results from the more shallow
angle adopted by the titin filament (
in
Figure 7A) when thin and thick filaments move closer
together. Calculations show that the 6-nm
d1,0
reduction in dextran (control curves of
Figure 6) reduces
Fr by
15%. Thus, in the presence of dextran, the effect of titin on
myofilament lattice spacing is expected to be reduced,
consistent with
d1,0
measurements
(Figure 6B) and the reduced effect of passive tension on
pCa50(Figure 4).
Our work suggests that titin-based passive force modulates the myofilament lattice spacing, and it seems reasonable to propose that this underlies at least part of the effect of the passive force of titin on the length dependence of calcium sensitivity. It is also possible that a role is played by a mechanism proposed earlier,12 13 in which crossbridge disorder is enhanced by passive force–induced thick filament strain, which leads to an increased likelihood of actomyosin interaction and an increase in calcium sensitivity. The notion that the crossbridge order can vary in passive muscle is supported by studies that investigated thick filament structure in response to changes in passive stretch,31 temperature,32 phosphorylation of myosin light chains,33 and C protein.34 Thus, thick filament strain as well as interfilament spacing may be involved in linking titin to calcium sensitivity, and their relative importance and interrelationship remains to be established.
In conclusion, titin influences the length dependence of calcium sensitivity of active force in cardiac myocytes, and the underlying mechanism may involve an effect of titin on myofilament lattice spacing. These findings challenge the conventional notion that titin is independent of actomyosin interaction, and they suggest that titin has the potential to enhance systolic performance as the ventricular volume is increased.
|
Acknowledgments |
|---|
This work was supported by National Institutes of Health (NIH) Grants HL67274 and HL62881 (to H.G.), American Heart Association (AHA) Grant 9950459N (to T.C.I.), and an AHA fellowship (to O.C.). Use of Advanced Photon Source was supported by the Department of Energy (Grant W-31-109-ENG-38). BioCAT is an NIH-supported research center (Grant RR08630). We acknowledge Dr P.P. de Tombe and laboratory members for assistance and use of their mechanics and setup for x-ray work and Drs S. Labeit, K. Campbell, B. Slinker, and P.P. de Tombe for discussions. We acknowledge Dr K. Bischoff for assistance with data analysis and Jennifer Eldridge for gel electrophoresis.
|
Footnotes |
|---|
Original received December 18, 2000; revision received April 2, 2001; accepted April 2, 2001.
1 Both authors contributed equally to this study. 
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Kentish JC, TerKeurs H, Ricciardi L, Bucx JJJ, Noble MIM. Comparison between the sarcomere length-force relations of intact and skinned trabeculae from rat right ventricle: influence of calcium concentrations. Circ Res. 1986;58:755–768.
2. Metzger J. Myosin binding-induced cooperative activation of the thin filament in cardiac myocytes and skeletal muscle fibers. Biophys J. 1995;68:1430–1442.
3. Wang YP, Fuchs F. Length, force, and Ca2+-troponin C affinity in cardiac and slow skeletal muscle. Am J Physiol. 1994;266:C1077–C1082.
4. Irving T, Konhilas J, Perry D, Fischetti R, de Tombe P. Myofilament lattice spacing as a function of sarcomere length in isolated rat myocardium. Am J Physiol. 2000;279:H2568–H2573.
5. Fuchs F, Wang YP. Sarcomere length versus interfilament spacing as determinants of cardiac myofilament Ca2+ sensitivity and Ca2+ binding. J Mol Cell Cardiol. 1996;28:1–9.
6. McDonald K, Moss R. Osmotic compression of single cardiac myocytes eliminates the reduction in calcium sensitivity of tension at short sarcomere length. Circ Res. 1995;77:199–205.
7. Cazorla O, Pascarel C, Garnier D, Le Guennec JY. Resting tension participates in the modulation of active tension in isolated guinea pig ventricular myocytes. J Mol Cell Cardiol. 1997;29:1629–1637.
8. Cazorla O, Vassort G, Garnier D, Le Guennec JY. Length modulation of active force in rat cardiac myocytes: is titin the sensor? J Mol Cell Cardiol. 1999;31:1215–1227.
9. Granzier H, Irving T. Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments. Biophys J. 1995;68:1027–1044.
10. Helmes M, Trombitas K, Granzier H. Titin develops restoring force in rat cardiac myocytes. Circ Res. 1996;79:619–626.
11. Wu Y, Cazorla O, Labeit D, Labeit S, Granzier H. Changes in titin and collagen underlie diastolic stiffness diversity of cardiac muscle. J Mol Cell Cardiol. 2000;32:2151–2161.
12. Granzier H, Wang K. Interplay between passive tension and strong and weak binding cross-bridges in insect indirect flight muscle. J Gen Physiol. 1993;101:235–270.
13. Granzier H, Wang K. Passive tension and stiffness of vertebrate skeletal and insect flight muscles: the contribution of weak cross-bridges and elastic filaments. Biophys J. 1993;65:2141–2159.
14. Cazorla O, Freiburg A, Helmes M, Centner T, McNabb M, Wu Y, Trombitás K, Labeit S, Granzier H. Differential expression of cardiac titin isoforms and modulation of cellular stiffness. Circ Res. 2000;86:59–67.
15. Fan D, Wannenburg T, de Tombe PP. Decreased myocyte tension development and calcium responsiveness in rat right ventricular pressure overload. Circulation. 1997;95:2312–2317.
16. Granzier H, Wang K. Gel electrophoresis of giant proteins: solubilization and silver-staining of titin and nebulin from single muscle fiber segments. Electrophoresis. 1993;14:56–64.
17. Helmes M, Trombitas K, Centner T, Kellermayer M, Labeit S, Linke A, Granzier H. Mechanically driven contour-length adjustment in rat cardiac titin’s unique N2B sequence: titin is an adjustable spring. Circ Res. 1999;84:1139–1352.
18. Millman BM. The filament lattice of striated muscle. Physiol Rev. 1998;78:359–391.
19. Matsubara I, Maughan D, Saeki Y, Yagi N. Cross-bridge movement in rat cardiac muscle as a function of calcium concentration. J Physiol. 1989;417:555–565.
20. Roos KP, Brady A. Osmotic compression and stiffness changes in relaxed skinned cardiac myocytes in PVP-4 and dextran T-500. Biophys J. 1990;58:1273–1283.
21. Smith S, Fuchs F. Length-dependence of cross-bridge mediated activation of the cardiac thin filament. J Mol Cell Cardiol. 2000;32:831–838.
22. McDonald K, Wolff M, Moss R. Sarcomere length dependence of the rate of tension redevelopment and submaximal tension in rat and rabbit skinned skeletal muscle fibers. J Physiol. 1997;501:607–621.
23. Wolska B, Keller R, Evans C, Palmiter K, Phillips R, Muthuchamy M, Oehlenschlager J, Wieczorek D, de Tombe P, Solaro J. Correlation between myofilament response to calcium and altered dynamics of contraction and relaxation in transgenic cells that express ß-tropomyosin. Circ Res. 1999;84:745–751.
24. Arteaga G, Palmiter K, Leiden J, Solaro J. Attenuation of length dependence of calcium activation in myofilaments of transgenic mouse hearts expressing slow skeletal troponin I. J Physiol. 2000;526:541–549.
25. McDonald K, Field LJ, Parmacek MS, Soonpaa MS, Leiden JM, Moss RL. Length dependence of Ca2+ sensitivity of tension in mouse cardiac myocytes expressing skeletal troponin C. J Physiol. 1995;483:131–139.
26. Higuchi H, Umazuma Y. Lattice shrinkage with increasing resting tension in stretched, single skinned fibers of frog muscle. Biophys J. 1986;50:385–389.
27. Higuchi H. Lattice swelling with the selective digestion of elastic components in single-skinned fibers from frog muscle. Biophys J. 1987;52:29–32.
28. Trombitas K, Granzier H. Actin removal from cardiac myocytes shows that near Z line titin attaches to actin while under tension. Am J Physiol. 1997;273:C662–C670.
29. Trombitas K, Jin JP, Granzier H. The mechanically active domain of titin in cardiac muscle. Circ Res. 1995;77:856–861.
30. Millman B, Irving T. Filament lattice of frog striated muscle: radial forces, lattice stability, and filament compression in the A-band of relaxed and rigor muscle. Biophys J. 1988;54:437–447.
31. Wakabayashi K, Sugimoto Y, Tanaka H, Ueno Y, Takezawa Y, Amemiya Y. X-ray diffraction evidence for the extensibility of actin and myosin filaments during muscle contraction. Biophys J. 1994;67:2422–2435.
32. Malinchik S, Xu S, Yu LC. Temperature-induced structural changes in the myosin thick filament of skinned rabbit psoas muscle. Biophys J. 1997;73:2304–2312.
33. Levine RJ, Yang Z, Epstein ND, Fananapazir L, Stull JT, Sweeney HL. Structural and functional responses of mammalian thick filaments to alterations in myosin regulatory light chains. J Struct Biol. 1998;122:149–161.
34. Weisberg A, Weingrad S. Alteration of myosin cross bridges by phosphorylation of myosin-binding protein C in cardiac muscle. Proc Natl Acad Sci U S A. 1996;93:8999–9003.
This article has been cited by other articles:
 |
|
 |
|
  D. Matsuba, T. Terui, J. O-Uchi, H. Tanaka, T. Ojima, I. Ohtsuki, S. Ishiwata, S. Kurihara, and N. Fukuda Protein kinase A-dependent modulation of Ca2+ sensitivity in cardiac and fast skeletal muscles after reconstitution with cardiac troponin J. Gen. Physiol., June 1, 2009; 133(6): 571 - 581. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. A. Shiels and E. White The Frank-Starling mechanism in vertebrate cardiac myocytes J. Exp. Biol., July 1, 2008; 211(13): 2005 - 2013. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. M. Hanft, F. S. Korte, and K. S. McDonald Cardiac function and modulation of sarcomeric function by length Cardiovasc Res, March 1, 2008; 77(4): 627 - 636. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Terui, M. Sodnomtseren, D. Matsuba, J. Udaka, S. Ishiwata, I. Ohtsuki, S. Kurihara, and N. Fukuda Troponin and Titin Coordinately Regulate Length-dependent Activation in Skinned Porcine Ventricular Muscle J. Gen. Physiol., February 25, 2008; 131(3): 275 - 283. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Udaka, S. Ohmori, T. Terui, I. Ohtsuki, S. Ishiwata, S. Kurihara, and N. Fukuda Disuse-induced Preferential Loss of the Giant Protein Titin Depresses Muscle Performance via Abnormal Sarcomeric Organization J. Gen. Physiol., December 31, 2007; 131(1): 33 - 41. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Granzier, M. Radke, J. Royal, Y. Wu, T. C. Irving, M. Gotthardt, and S. Labeit Functional genomics of chicken, mouse, and human titin supports splice diversity as an important mechanism for regulating biomechanics of striated muscle Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2007; 293(2): R557 - R567. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. E. Petre, M. P. Quaile, E. I. Rossman, S. J. Ratcliffe, B. A. Bailey, S. R. Houser, and K. B. Margulies Sex-based differences in myocardial contractile reserve Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2007; 292(2): R810 - R818. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. K. Frazier, K. S. Stone, D. Moser, R. Schlanger, C. Carle, L. Pender, J. Widener, and H. Brom Hemodynamic Changes During Discontinuation of Mechanical Ventilation in Medical Intensive Care Unit Patients Am. J. Crit. Care., November 1, 2006; 15(6): 580 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. S. Chung, A. Strunc, R. Oliver, and S. J. Kovacs Diastolic ventricular-vascular stiffness and relaxation relation: elucidation of coupling via pressure phase plane-derived indexes Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2415 - H2423. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. P. Farman, J. S. Walker, P. P. de Tombe, and T. C. Irving Impact of osmotic compression on sarcomere structure and myofilament calcium sensitivity of isolated rat myocardium Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1847 - H1855. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Cazorla, Y. Ait Mou, L. Goret, G. Vassort, M. Dauzat, A. Lacampagne, S. Tanguy, and P. Obert Effects of high-altitude exercise training on contractile function of rat skinned cardiomyocyte Cardiovasc Res, September 1, 2006; 71(4): 652 - 660. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. A. MacGowan, J. A. Kirk, C. Evans, and S. G. Shroff Pressure-calcium relationships in perfused mouse hearts Am J Physiol Heart Circ Physiol, June 1, 2006; 290(6): H2614 - H2624. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. C. Ottenheijm, L. M. A. Heunks, T. Hafmans, P. F. M. van der Ven, C. Benoist, H. Zhou, S. Labeit, H. L. Granzier, and P. N. R. Dekhuijzen Titin and Diaphragm Dysfunction in Chronic Obstructive Pulmonary Disease Am. J. Respir. Crit. Care Med., March 1, 2006; 173(5): 527 - 534. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Cazorla, S. Szilagyi, N. Vignier, G. Salazar, E. Kramer, G. Vassort, L. Carrier, and A. Lacampagne Length and protein kinase A modulations of myocytes in cardiac myosin binding protein C-deficient mice Cardiovasc Res, February 1, 2006; 69(2): 370 - 380. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Hayashi, T. Arimura, M. Itoh-Satoh, K. Ueda, S. Hohda, N. Inagaki, M. Takahashi, H. Hori, M. Yasunami, H. Nishi, et al. Tcap gene mutations in hypertrophic cardiomyopathy and dilated cardiomyopathy J. Am. Coll. Cardiol., December 7, 2004; 44(11): 2192 - 2201. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Fujita, D. Labeit, B. Gerull, S. Labeit, and H. L. Granzier Titin isoform-dependent effect of calcium on passive myocardial tension Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2528 - H2534. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. C. Lim, C. Zuppinger, X. Guo, G. M. Kuster, M. Helmes, H. M. Eppenberger, T. M. Suter, R. Liao, and D. B. Sawyer Anthracyclines Induce Calpain-dependent Titin Proteolysis and Necrosis in Cardiomyocytes J. Biol. Chem., February 27, 2004; 279(9): 8290 - 8299. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. L. Granzier and S. Labeit The Giant Protein Titin: A Major Player in Myocardial Mechanics, Signaling, and Disease Circ. Res., February 20, 2004; 94(3): 284 - 295. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Fukuda, Y. Wu, G. Farman, T. C Irving, and H. Granzier Titin isoform variance and length dependence of activation in skinned bovine cardiac muscle J. Physiol., November 15, 2003; 553(1): 147 - 154. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P Konhilas, T. C Irving, B. M Wolska, E. E Jweied, A. F Martin, R John Solaro, and P. P de Tombe Troponin I in the murine myocardium: influence on length-dependent activation and interfilament spacing J. Physiol., March 15, 2003; 547(3): 951 - 961. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Helmes, C. C. Lim, R. Liao, A. Bharti, L. Cui, and D. B. Sawyer Titin Determines the Frank-Starling Relation in Early Diastole J. Gen. Physiol., February 3, 2003; 121(2): 97 - 110. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. D Stuyvers, A. D McCulloch, J. Guo, H. J Duff, and H. E D J ter Keurs Effect of stimulation rate, sarcomere length and Ca2+ on force generation by mouse cardiac muscle J. Physiol., November 1, 2002; 544(3): 817 - 830. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P Konhilas, T. C Irving, and P. P de Tombe Length-dependent activation in three striated muscle types of the rat J. Physiol., October 1, 2002; 544(1): 225 - 236. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Wu, S. P. Bell, K. Trombitas, C. C. Witt, S. Labeit, M. M. LeWinter, and H. Granzier Changes in Titin Isoform Expression in Pacing-Induced Cardiac Failure Give Rise to Increased Passive Muscle Stiffness Circulation, September 10, 2002; 106(11): 1384 - 1389. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Lindstedt, T. E. Reich, P. Keim, and P. C. LaStayo Do muscles function as adaptable locomotor springs? J. Exp. Biol., August 1, 2002; 205(15): 2211 - 2216. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Yamasaki, Y. Wu, M. McNabb, M. Greaser, S. Labeit, and H. Granzier Protein Kinase A Phosphorylates Titin's Cardiac-Specific N2B Domain and Reduces Passive Tension in Rat Cardiac Myocytes Circ. Res., June 14, 2002; 90(11): 1181 - 1188. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Granzier and S. Labeit Cardiac titin: an adjustable multi-functional spring J. Physiol., June 1, 2002; 541(2): 335 - 342. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Watanabe, P. Nair, D. Labeit, M. S. Z. Kellermayer, M. Greaser, S. Labeit, and H. Granzier Molecular Mechanics of Cardiac Titin's PEVK and N2B Spring Elements J. Biol. Chem., March 22, 2002; 277(13): 11549 - 11558. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Moss and D. P. Fitzsimons Frank-Starling Relationship: Long on Importance, Short on Mechanism Circ. Res., January 11, 2002; 90(1): 11 - 13. [Full Text] [PDF]
|
|
|
|
|
|
J. L. Sutko, N. G. Publicover, and R. L. Moss Titin: An Elastic Link Between Length and Active Force Production in Myocardium Circulation, October 2, 2001; 104(14): 1585 - 1587. [Full Text] [PDF]
|
|
|
|
|
|
N. Fukuda, D. Sasaki, S.'i. Ishiwata, and S. Kurihara Length Dependence of Tension Generation in Rat Skinned Cardiac Muscle: Role of Titin in the Frank-Starling Mechanism of the Heart Circulation, October 2, 2001; 104(14): 1639 - 1645. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Trombitas, Y. Wu, D. Labeit, S. Labeit, and H. Granzier Cardiac titin isoforms are coexpressed in the half-sarcomere and extend independently Am J Physiol Heart Circ Physiol, October 1, 2001; 281(4): H1793 - H1799. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P. Konhilas, T. C. Irving, and P. P. de Tombe Myofilament Calcium Sensitivity in Skinned Rat Cardiac Trabeculae: Role of Interfilament Spacing Circ. Res., January 11, 2002; 90(1): 59 - 65. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||