(Received for publication, February 1, 1996; and in revised form, March 26, 1996)
From the
Despite its potential as a key determinant of the functional
state of striated muscle, the impact of tropomyosin (Tm) isoform
switching on mammalian myofilament activation and regulation in the
intact lattice remains unclear. Using a transgenic approach to
specifically exchange -Tm for the native
-Tm in mouse hearts,
we have been able to uncover novel functions of Tm isoform switching in
the heart. The myofilaments containing
-Tm demonstrated an
increase in the activation of the thin filament by strongly bound
cross-bridges, an increase in Ca
sensitivity of
steady state force, and a decrease in the rightward shift of the
Ca
-force relation induced by cAMP-dependent
phosphorylation. Our results are the first to demonstrate the specific
effects of Tm isoform switching on mammalian thin filament activation
in the intact lattice and suggest an important role for Tm in
modulation of myofilament activity by phosphorylation of troponin.
The ability of myosin heads to react with actin in heart muscle
occurs with a transition of the thin filament from an ``off''
to an ``on'' state that depends on complex alterations
involving the tropomyosin (Tm) ()molecule (for reviews see (1) and (2) ). These alterations include possible
steric effects associated with changes in the position of Tm on the
thin filament, as well as allosteric and cooperative effects associated
with Tm-induced changes in actin structure and reactivity with
myosin(3, 4, 5) . The steric and
allosteric/cooperative alterations involving Tm are triggered by
Ca
binding to TnC, but also depend on myosin head
binding(6) . Ca
binding to TnC promotes
interactions with other Tn components, TnI(2) , an inhibitory
protein that binds to actin, as well as TnT, a TnI- and Tm-binding
protein. The steric model of activation (3) hypothesizes that
Ca
-TnC-induced movement of Tm or possibly TnI (2) reverses the off state by releasing actin sites for
reaction with myosin. The allosteric model(4, 5) proposes that Ca
-TnC itself cannot
activate the thin filament, but acts as a co-factor shifting the
equilibrium between off and on states of Tm such that strongly bound
cross-bridges more easily activate the thin filament. Although recent
considerations indicate that activation may involve both
processes(2) , the relative role of the steric and
allosteric/cooperative mechanisms in turning on the activity of
striated muscle remains unclear.
Our perception of the role of Tm in the regulation of striated muscle, as well as it's structure/function relations, has come from a variety of approaches. These include x-ray diffraction of muscle preparations (7) and crystals(8, 9) , reconstructions from electron micrographs(9, 10) , and reconstitution studies of soluble systems with Tm(11, 12, 13) , Tm peptides(14) , and mutants of Tm(15) . In some cases, inferences regarding structure/function relations have been made from comparisons of muscle fibers containing isoforms of Tm(16) . However, interpretation of these studies is difficult in that there are multiple changes in myofilament proteins that occur along with the natural variations in Tm. A clearer understanding of the structure/function relations of Tm has been hampered by an apparent lack of methods for reversibly extracting Tm from the myofilament lattice in a force-generating system, as has proved so successful in the case of Tn components such as TnC and TnI(17) . Thus, issues such as the role of Tm domains, covalent modifications, and the functional significance of isoform switching of Tm in the intact force-generating lattice of vertebrate-striated muscle have remained poorly understood. Delineating the functional differences between Tm isoforms has taken on new significance with the identification of missense mutations in the Tm gene causally linked to familial hypertrophic cardiomyopathy (18, 19, 20) .
In the present experiments, we used a transgenic approach to
overcome the difficulties of exchanging Tm isoforms in the intact
myofilament lattice. Transgenic mice, which overexpress -Tm in the
heart, were generated as described previously(21) . This has
permitted us to test explicitly the effects of alterations in Tm
isoforms on myofilament activation. Our results provide the first
unambiguous evidence that myofilament activation by Ca
and strong cross-bridges is affected by the population of Tm
isoforms present in the heart. Our results also indicate a role for Tm
in the modulation of myofilament activation by phosphorylation of Tn.
Some of our results have been published in abstract form(22) .
Figure 2:
The relation between pMgATP and
force of NTG and TG--Tm fiber preparations. Detergent-extracted
fiber preparations were sequentially exposed to solutions of decreasing
MgATP concentration at pCa 9.0. In both cases, the peak force
obtained at pMgATP 5.4 in the NTG fibers and pMgATP
5.0 in the TG-
-Tm fibers was only approximately half that obtained
under maximal Ca
-activated conditions at pCa
4.5 (data not shown). Values are expressed as the mean ± S.E. of n = 5 from 3 different hearts. *, p < 0.05
as determined by Student Newman Keul's post hoc t-test.
, NTG;
, TG-
-Tm.
Figure 3:
The effect of cAMP-dependent
phosphorylation on the pCa and force relation in NTG and
TG--Tm fiber preparations. A, pCa-force relation
of detergent-extracted NTG (
,
) and TG-
-Tm (
,
) fiber preparations under control conditions (A,
,
) and under phosphorylating conditions (B,
,
). Inset, protein phosphorylation profile of
detergent-extracted NTG and TG-
-Tm myofilament preparations
performed as described under ``Experimental Procedures.'' All
phosphorylation experiments represent at least 3 individual
determinations. Values are expressed as the mean ± S.E. In the
case of the NTG preparations, n = 3 from 3 different
hearts. In the case of the TG preparations, n = 5 from
3 different hearts. Each individual determination involved sequentially
immersing a fiber in two sets of solutions of varying pCa
values. The first set of Ca
solutions to which a
fiber was exposed (under control conditions) had no added cAMP. After
obtaining the initial measurements, the fiber bundle was then incubated
under phosphorylating conditions (see text and ``Experimental
Procedures'') and then reimmersed in solutions of varying pCa in the presence of 100 µM cAMP.
Fig. 1A illustrates the SDS-PAGE protein
profiles of detergent-treated myofilament preparations from NTG and
TG--Tm mouse hearts (lanes 1 and 2,
respectively). Tm was identified in the two preparations by Western
blot analysis as illustrated in Fig. 1B (NTG, lane
1, and TG-
-Tm, lane 2). This analysis demonstrated
that NTG hearts express only
-Tm in the myofilaments, whereas
-Tm is abundantly expressed in myofilaments from TG-
-Tm
hearts. We found no changes in the expression of other myofilament
proteins. The results illustrated in Fig. 1show that our
transgenic approach has made experiments explicitly testing the effects
of alterations in Tm isoforms on myofilament activation possible.
Figure 1:
SDS-PAGE and Western blot analysis of
NTG and TG--Tm cardiac myofibrillar preparations. A,
12.5% SDS-polyacrylamide gel onto which 50 µg of total cardiac
myofibrillar protein from NTG (lane 1) and TG-
-Tm (lane 2) samples was loaded. B, Western blot
performed with a monoclonal antibody which recognizes both Tm isoforms
and done specifically to identify the type(s) of Tm present in the
myofilaments (NTG, lane 1, and TG-
-Tm, lane 2).
Our results clearly demonstrate that NTG preparations contain only
-Tm whereas TG-
-Tm preparations contain an abundance of
-Tm.
As
a method for probing the effects of Tm isoform switching on the
cooperative activation of the thin filament, we controlled the
population of strongly bound cross-bridges by varying the MgATP
concentration at pCa 9.0 in detergent-treated fiber bundles (Fig. 2) from nontransgenic and transgenic preparations. At
relatively high concentrations of MgATP, force was low reflecting
relaxing conditions and a high population of cross-bridges with bound
nucleotide. As the MgATP concentration was lowered, force increased,
even at pCa 9.0, as strongly bound rigor cross-bridges
(nucleotide-free) activated the thin filament. Maximum force was a
function of the number of cycling and rigor cross-bridges as well as
the relative activation of the thin filament. As illustrated in Fig. 2, at pMgATP 5.0, myofilaments containing -Tm
developed significantly more force than myofilaments containing only
-Tm. Assuming that the number of rigor cross-bridges is the same
in both preparations, our results indicate that Tm isoform switching
increased the ability of strong cross-bridge binding to activate the
thin filament.
Myofilament activation via Ca binding to TnC was also measured in skinned fiber preparations.
Results presented in Fig. 3A indicate that the force
developed by TG-
-Tm myofilaments was significantly (p
0.05 two-way ANOVA) more sensitive to Ca
than NTG myofilaments. The pCa
was 5.72
± 0.01 for TG-
-Tm preparations and 5.57 ± 0.04 for
NTG preparations.
A unique property of cardiac TnI that may be
important in its reaction with Tm is that it is phosphorylated by
PKA(31) . In experiments reported in Fig. 3, we compared
the pCa-force relations for NTG and TG--Tm fiber bundles
before and after phosphorylation by cAMP/PKA as described under
``Experimental Procedures.'' Our results show that the
difference in Ca
sensitivity between NTG and
TG-
-Tm myofilaments was even more pronounced when the myofilaments
were phosphorylated by PKA (Fig. 3B). As expected from
previous experiments(32) , the NTG myofilaments responded to
cAMP-dependent phosphorylation of TnI by a rightward shift of the pCa-force relation. Yet, in TG-
-Tm myofilaments, there
was no significant rightward shift of the pCa-force relation
with cAMP-dependent phosphorylation. This resulted in a significant (p < 0.05, two-way ANOVA) magnification of the differences
in pCa
between NTG (5.46 ± 0.018) and
TG-
-Tm (5.69 ± 0.01) myofilaments. Levels of TnI
phosphorylation in NTG and TG-
-Tm preparations are illustrated by
the data shown in the inset of Fig. 3. The ratio of
autoradiogram band intensity to TnI staining was 3.56 for NTG
preparations and 3.78 for TG-
-Tm preparations. Based on these
results, we conclude that the stoichiometry of TnI phosphorylation was
the same in each case. These data are the first to demonstrate, in the
intact myofilament lattice, a role for Tm isoform switching in the
desensitization of myofilaments by phosphorylation of TnI.
Our data provide the first clear evidence that exchange of
-Tm for
-Tm alters thin filament activation by cross-bridge
binding. It is known that strong binding of cross-bridges is able to
``turn-on'' many actins through a cooperative process that
requires Tm(6) . Moreover, when overlap between adjacent Tm
molecules is removed, cooperative cross-bridge-actin binding is
significantly reduced(33) . In the mouse, isoform switching
from
- to
-Tm involves 39 amino acid substitutions. Twenty
five of these substitutions occur in the C-terminal half of the
molecule, (
)a region important for Tm head-to-tail
interactions. Previous studies (21) have demonstrated the
preferential formation of
-Tm heterodimers in these
transgenic cardiac myofilaments. This does not, however, preclude the
formation of
-Tm homdimers which were undoubtedly present due
to the abundance of
-Tm expressed. Importantly, Thomas and Smillie (13) have reported that
-Tm has a greater propensity
to form end-to-end interactions than either
- or
-Tm, which may significantly alter the cooperative potential
of the myofilaments containing
-Tm. Additionally, amino acid
substitutions Ser
Glu and His
Asn result in
-Tm having a(-2) charge change relative to
-Tm.
These changes could affect cooperative activation
of thin filaments: (i) by altering the interaction of Tm with the seven
actins under its control or (ii) by affecting end-to-end interactions
linking near neighbor functional units consisting of actin-Tm-Tn in a
7:1:1 ratio.
Based on previous studies, it is not surprising that
isoform switching from -Tm to
-Tm alters thin filament
activation by Ca
. Ca
binding
activates the thin filament by altering the interactions of TnC with
TnT (34) and TnI(35, 36) . These changes
induce changes in the binding of Tm to actin (10) and promote
cross-bridge binding to actin. Importantly, TnT has been shown to bind
more weakly to
-Tm than to
-Tm(37) . Thus, it would
be expected that activation may occur at a lower level of free
Ca
in myofilaments containing
-Tm.
Our
results also implicate a role for Tm isoform switching in the
modulation of thin filament activation by phosphorylation of TnI.
Although the inhibitory activity of TnI is potentiated by
Tm(6) , how phosphorylation of TnI might alter its interaction
with Tm has not been appreciated. In vitro studies of
Al-Hillawi et al.(38) demonstrated that cooperative
binding of TnI to actin-Tm is abolished when TnI is phosphorylated. Our
hypothesis is that isoform switching from -Tm to
-Tm itself
results in a weaker cooperative interaction between TnI, actin, and Tm.
Thus, the effect of TnI phosphorylation may be minimized or lost in
myofilaments containing
-Tm.
An increased Ca sensitivity and a reduced effect of TnI phosphorylation on
myofilaments containing
-Tm serves to explain results of studies
on isolated working hearts from NTG and TG-
-Tm mice. Muthuchamy et al.(21) showed that, compared to controls,
TG-
-Tm working heart preparations demonstrated an increase in the
time for half-maximal relaxation of ventricular pressure. This fits
with our findings of increased Ca
sensitivity in
myofilaments containing
-Tm. Moreover, the difference in
half-maximal relaxation time was increased with low level
-adrenergic stimulation. This is what we would expect given that
-adrenergic stimulation, which results in TnI phosphorylation, has
been shown to reduce myofilament Ca
sensitivity,
thereby enhancing relaxation(39) . Our results on skinned fiber
bundles indicate that this relaxant effect, associated with TnI
phosphorylation, would be much reduced or absent in the TG heart.
Our results also have important implications regarding the etiology
of FHC, which is genetically linked to sarcomeric mutations (for
review, see (40) ) Two of the three point mutations found in
the -Tm gene of patients with FHC involve charge changes in the
putative Ca
-dependent TnT binding
domain(18, 19, 20) . Our data show that
isoform switching involving charge changes near this domain enhance
myofilament Ca
sensitivity, an effect expected to
slow relaxation as demonstrated in working heart
preparations(21) . Interestingly, point mutations in myosin
heavy chain linked to FHC also slow contraction
dynamics(41, 42) . Thus, the mechanism for the slowing
need not involve myosin, but could occur through changes in myofilament
response to Ca
exacerbated during exercise. The
ability to test these ideas in transgenic animals brings us closer to
understanding the functional differences between Tm isoform populations
in physiological and pathological settings.