From the Cincinnati Children's Hospital Medical
Center, The Children's Hospital Research Foundation, Division of
Molecular Cardiovascular Biology, MLC 7020, Cincinnati, Ohio
45229-3039, the ¶ Department of Molecular and Cellular
Physiology, University of Cincinnati, Cincinnati, Ohio 45267-0576, the
Department of Molecular Physiology & Biophysics, University of
Vermont, College of Medicine, Burlington, Vermont 05405, and the
** Department of Medicine, Division of Cardiology, University
of Colorado Health Science Center, Denver, Colorado 80262
Received for publication, October 22, 2002, and in revised form, March 4, 2003
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ABSTRACT |
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Comparison of mammalian cardiac Myosin, the molecular motor of the heart, generates force
and motion by coupling its ATPase activity to its cyclic interaction with actin. Myosin is a hexameric protein and is composed of two heavy
chains (MHC)1 and two
essential and two regulatory myosin light chains. Structurally, MHC is
composed of a number of discrete domains: a helical rod necessary for
thick filament formation, and a globular head that contains the
actin-binding site, catalytic, and motor domains (1).
In the mammalian heart, two functionally distinct MHC isoforms, termed
V1 and V3, are present. V1 is a
homodimer of two Although the proteins are functionally distinct, the primary amino acid
sequences of mouse In contrast to the abundance of detailed studies on in vitro
function of various MHC isoforms, our current knowledge of how differences found on the single molecule level are reflected in in vivo cardiac function is limited. Cardiac isoform shifts
can be achieved by endocrine intervention but hypothyroidism not only results in a nearly complete V1 The present study is the first to investigate directly the functional
significance of cardiac isoform diversity by using TG mice in which
ventricular V1 is largely replaced by V3. This
approach has the advantage of effecting isoform replacement in the
heart without the pleiotropic stimuli that are normally used to induce MHC isoform transitions, such as pressure-overload induced hypertrophy or changes in hormonal status (17, 18). A significant V1
Generation of TG Animals--
TG mice expressing full-length
mouse
Chimeric myosins in which either only the sequence of L2 or
of both Loop 1 and Loop 2 (L1+L2) of Immunohistochemistry--
Hearts were fixed in 4%
paraformaldehyde. 3-4-µm cryostat sections were probed with a
custom-made anti-Loop 2 ( ATPase Assays--
MHC was purified from individual mouse hearts
(21). F-actin was prepared from acetone powder of chicken pectoralis
muscle according to a protocol modified from Pardee and Spudich (22). Actin-activated ATPase activity was measured at actin concentrations ranging from 10 to 80 µM (21).
Ca2+-stimulated Mg2+-ATPase activity of
myofibrillar preparations from ventricular tissue (23) was determined
as previously described (24).
Miscellaneous Methods--
Transcript analysis was performed
with RNA blots with transcript-specific probes as described previously
(19). The two cardiac myosins were separated on denaturing gels in the
presence of glycerol, essentially as described by Reiser and co-workers
(25, 26). Fiber isolation and their analyses have been described in
detail (27). The in vitro motility assays (28) and
determination of cardiac hemodynamics in the isolated working heart
model and the closed chest intact mouse model were carried out as
described (29, 30).
Statistical Analysis--
All data are expressed as mean ± S.E. Comparisons between NTG and TG littermates were evaluated using
Student's t test, and a p value of <0.05 was
considered statistically significant.
Mediating an
In order to confirm that the transcript was the correct size, a
Northern blot was made using RNAs derived from each of the lines
ventricles, and hybridized against a myosin cDNA probe. All TG
lines showed only a single RNA species, which comigrated along with the
endogenous MHC RNA (Fig. 1B). Subsequently, the levels of
expression were quantitated using oligonucleotide probes hybridized to
RNA dot blots. Probes specific to either the Cardiac Myosin Heavy Chain Replacement--
TG mice were analyzed
for ventricular MHC protein content at 10-12 weeks. The
We reasoned that if a phenotype were likely to be present, it would be
most easily detected in line 102, and this line became the focus of our
analyses. Immunohistochemical staining using a V3-specific
antibody derived from the hypervariable Loop 2 region of Consequences of Isoform Replacement in Intact Fibers--
In light
of the unremarkable phenotype at the whole animal level, we wished to
confirm that changes in isoform content had affected the mechanical and
kinetic properties of the skinned myofibers. Ventricular papillary
muscles were isolated from line 102, PTU-treated and NTG mice. Line 102 heterozygotes have ~40%
We first wished to compare the effects of ~40% replacement
versus the fibers derived from PTU-treated animals, in which
~95% of the cardiac myosin consisted of
We confirmed both the trend and stability of these changes by
developing a cohort of heterozygotes and homozygotes over the course of
a year and subsequently carrying out fiber measurements comparing these
two populations to NTG fibers (Fig. 4).
Similar graded decreases in the unloaded shortening velocity (Fig.
4A), maximum shortening velocity (Fig. 4B), and
maximum power produced (Fig. 4C) were noted when the NTG,
heterozygotes, and homozygotes were compared. No changes in the
calcium-force relationship could be observed in any of the TG
fibers.2
Replacement of Cardiac Myosin with an In Vitro Motility Assays--
To determine the effects on motor
function, in vitro actin motility assays were performed
using MHC that had been isolated from heterozygous line 102 ATPase Assays--
The different myosins are characterized by
their unique enzymatic activities. We determined both the myofibrillar
Ca2+-stimulated Mg2+-ATPase and actin-activated
ATPase activities of myosins purified from NTG,
L1+L2-, L2-, and
To explore this phenomenon further, data were obtained for the more
physiologically relevant actin-activated Mg2+-ATPase
activity (Fig. 8). While MHC isolated
from line 102 homozygotes exhibited the expected decrease in enzymatic
activity (Fig. 8A) neither L1+L2 nor
L2 Cardiac Hemodynamics in Isolated Working Hearts and in
Vivo--
Although the V1
In vivo data from intact mice were also consistent with a
decrease in systolic and diastolic function in The aim of this study was to investigate the functional
consequences of myosin isoform diversity and how the different motor abilities influence cardiac contractile function. In mammalian adult
hearts, alterations in MHC isoform expression occur in response to
various pleiotropic stimuli such as hypertrophy, failure, hypo- or
hyperthyroidism. While these partial/complete isoform switches correlate with changes in cardiac performance, there are a myriad of
other structural and functional changes that accompany these processes
(17, 18), such that it is impossible to ascribe the changes in heart
function to modifications in MHC isoform content alone. To test the
hypothesis that changes in isoform content in the absence of such
global processes could alter heart function, we used transgenesis to
generate mice that overexpressed specifically in the heart the
Cardiac-specific transgenesis also was effective in replacing the
endogenous myosin with Changes in charge and length of Loop 2 in Dictyostelium
myosin can modulate MHC function (35). Neither mouse These seemingly contradictory data can be reconciled by hypothesizing
that the neighboring structures of the myosin backbone direct the
flexible loops into certain conformations and thereby promote or
ameliorate their influence on actin-myosin interactions or nucleotide
binding and release. Alignment of the amino acid sequences of the
backbones of Dictyostelium myosin II and mouse cardiac Functional differences between the cardiac MHC isoform were manifested
at the single motor, biochemical, and fiber levels, in a manner that
reflected the altered V1/V3 ratios. Thus, it is
clear that changes in myosin enzymatic activity are reflected by
concomitant changes in motor velocity, which, in turn, lead to changes
in fiber contractility. These changes are all consistent with the
correlations that have been previously noted (36-38). Comparing the
data with those studies, the unloaded shortening velocity decreased
30% in TG mice exhibiting 40% replacement with Our data also demonstrate how the in vitro mechanical and
kinetic differences of the MHC isoforms are reflected at the whole organ and intact animal levels. Although the unstressed animal is
overtly healthy, the 2-fold differences in actin filament sliding velocity and ATPase activity between Recently, contractile function of TG mouse hearts with low-level
expression of Myc-tagged rat The spontaneous heart rate of the isolated hearts, as well as heart
rate in propranolol-treated mice, was reduced in While it has been generally accepted that isoform shifts can result in
different functional endpoints, until very recently the potential of a
V1 - and
-myosin heavy chain isoforms reveals 93% identity. To date, genetic
methodologies have effected only minor switches in the mammalian
cardiac myosin isoforms. Using cardiac-specific transgenesis, we have
now obtained major myosin isoform shifts and/or replacements. Clusters
of non-identical amino acids are found in functionally important
regions, i.e. the surface loops 1 and 2, suggesting that
these structures may regulate isoform-specific characteristics. Loop 1 alters filament sliding velocity, whereas Loop 2 modulates
actin-activated ATPase rate in Dictyostelium myosin, but
this remains untested in mammalian cardiac myosins.
isoform
switches were engineered into mouse hearts via transgenesis. To assess
the structural basis of isoform diversity, chimeric myosins in which
the sequences of either Loop 1+Loop 2 or Loop 2 of
-myosin were
exchanged for those of
-myosin were expressed in vivo.
2-fold differences in filament sliding velocity and ATPase activity
were found between the two isoforms. Filament sliding velocity of the
Loop 1+Loop 2 chimera and the ATPase activities of both loop chimeras
were not significantly different compared with
-myosin. In
mouse cardiac isoforms, myosin functionality does not depend on Loop 1 or Loop 2 sequences and must lie partially in other non-homologous residues.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-MHC molecules, whereas V3 is a
-homodimer. Expression of V1 and V3 is
controlled both developmentally and hormonally. In the mouse,
-MHC
expression in the ventricles predominates prenatally. However, via thyroid hormone regulation,
-MHC expression is silenced at birth, and
-MHC is transcribed (2). The functional differences between V1 and V3 myosin in terms of shortening
velocity, force generation, and ATPase activity are profound. For
example, rabbit V1 myosin has a 2-3-fold faster actin
filament sliding velocity than V3, but generates only half
the average isometric force (3, 4). Likewise, both the
Ca2+-stimulated and actin-activated ATPase activities of
rabbit V1 myosin are ~2-3 times greater than for
V3 myosin (3, 5). Similar differences in actin velocity and
myofibrillar ATPase activity have been observed between mouse
V1 and V3 myosin, but there is no difference in
their average force generation (6).
- and
-MHC are 93% identical. Thus cardiac
isoform diversity must lie in the non-identical residues (127 of 1938 amino acids in mice). The differences in the enzyme kinetics and
mechanics of the myosin interactions that are observed between the two
cardiac isoforms are believed to reside in two, hypervariable
"loops," so called because their structures cannot be defined via
x-ray crystallography due to their relative disorder. Loop 1 (L1), which is located between residues 213 and 223, is at
the mouth of the nucleotide pocket while Loop 2 (L2), at
positions 624-646, cradles the long cleft running from the actin
binding site to the nucleotide binding pocket (1, 7). Comparison of MHC
sequences within the human sarcomeric MHC family shows that these
domains of sequence variability are conserved (8). Spudich and
co-workers (9-11) have proposed that Loop 1 modulates velocity through
ADP release, whereas Loop 2 helps regulate the actin-activated ATPase
rate. Data obtained from studies with chimeric Dictyostelium
and smooth muscle myosins corroborated the model. For example, chimeras
were constructed in which 9 amino acids in the L2 region of
Dictyostelium myosin II were substituted with the
corresponding residues from other myosins such as rabbit skeletal muscle myosin, chicken smooth muscle myosin, or rat cardiac myosin (11). The actin-activated ATPase activities of the chimeras correlated
well with the activities of the myosin from which the Loop 2 sequence
was derived. Thus, myosin's ATPase activity could be specifically
modulated depending on the sequence of Loop 2. However, a number of
studies indicate that the loops may not influence myosin kinetics and
mechanics as proposed and thus have varying roles depending on the
structure of the myosin backbone. Rat and pig
-MHC, which have
identical Loop 1 sequences apart from a single conservative
substitution, have 3-4-fold differences in ATPase activity and ADP
dissociation (12). Sweeney et al. (13) showed that the
properties of Loop 1 chimeras with a smooth muscle backbone are a
function of loop size/flexibility rather than related to the properties
of the myosin from which Loop 1 was derived. Furthermore, chimeric
myosins that consisted of a Dictyostelium MHC backbone with
carp loop sequences did not exhibit changes in sliding velocity if Loop
1 was substituted, although Loop 2 substitution did lead to the
expected modulation of actin-activated ATPase activity (14). Taken
together, these studies indicate that the role of the surface loops for
MHC functionality depends on the interplay of the surface loops with
other regions important for myosin mechanics and kinetics.
V3 shift in
rodent hearts, it also induces a number of structural changes including
mitochondrial swelling, as well as rupture and loss of continuity of
the myofilaments (15). Thus one cannot dissect MHC isoform shift
induced functional changes from contractile impairment due to
structural damage. Transgenesis avoids these issues. To date, only a
single transgenic study has dealt directly with cardiac myosin isoform
substitution, showing that contractile function of TG mouse hearts with
low-level expression of Myc-tagged rat
-MHC was reduced by 15%
(16). This disproportionate impairment of contractile function might be
due to the presence of a heterologous species (rat) cDNA being placed into the mouse context, resulting in a dominant negative effect
of the TG protein.
V3 shift resulted in the expected changes in the heart
at the single motor and biochemical levels as well as in fiber
mechanics and kinetics. However, in contrast to a
hypothyroidism-effected replacement, cellular structure appeared normal
and whole organ function was preserved with relatively minor effects on
systolic and diastolic hemodynamics in the intact animal. Furthermore, we generated transgenic (TG) mice in which we substituted the sequences
of Loop 1 and/or Loop 2 of mouse
-MHC with the respective sequences
of
-MHC and assessed the mechanical and enzymatic characteristics of
the chimeric MHCs. These experiments were designed to test whether a
sequence substitution in the Loop 1 and/or Loop 2 region is sufficient
to confer
-like activity to the
-MHC molecule. Complete
replacement of the endogenous
-MHC protein with the chimeric myosin
resulted in surprisingly minor differences in enzyme kinetics
indicating that, for these cardiac isoforms, other variable regions or
residues must play a predominant role in determining overall ATPase
activity and velocity of shortening.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-MHC were generated.
-MHC cDNA was produced using a
combination of a cDNA containing the 3' one-third of the RNA (19),
and RT-PCR with
-MHC-specific primers (GenBankTM
accession number AY056464). The product contained the entire
-MHC
cDNA and was linked to the mouse
-MHC promoter
(Fig. 1A). Multiple TG founders (FVB/N) were generated. Line
102 with the highest degree of MHC replacement (~40%) was bred to
homozygosity resulting in 73% replacement of
- with
-MHC (Fig.
1D). Neither the heterozygous or homozygous TG animals
showed an overt phenotype, and all animals had a normal life span. To
shift isoform expression in non-transgenic (NTG) hearts from
- to
-MHC, adult mice received an iodine-deficient diet containing 0.15%
propylthiouracil (PTU) for 8 weeks.
-MHC
was exchanged for the respective sequence of
-MHC were subsequently
constructed. A diagram of the TG constructs for the chimeric MHCs is
shown in Fig. 5A. Constructs for L2, and
L1+L2 chimeric MHC were made using standard PCR
methodology. Inner sets of oligonucleotides were designed so that the
overlap encoded the amino acids that form Loop 1 or Loop 2 of the
-isoform (Fig. 5B). Fragments generated by PCR
amplification were cloned back into full-length
-MHC and placed into
the mouse
-MHC promoter cassette (20). Finally, the DNA
was excised free of plasmid sequence and used to generate multiple TG
founders (FVB/N).
-L2) polyclonal antibody
(Genemed Synthesis Inc., San Francisco, CA) followed by incubation with
Alexa 488-conjugated secondary antibody and co-labeled with
phalloidin-Alexa 594 (Molecular Probes, Eugene, OR). Specimens were
examined using confocal microscopy.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
MHC Switch via Transgenesis--
To
understand the functional consequences of MHC isoform shifts in the
heart we used transgenesis to effect an
shift. A cDNA
encoding the mouse
-MHC cDNA was produced using a combination of
a cDNA that encoded the 3'-terminal one-third of the RNA (19), and
RT-PCR with
-MHC-specific primers. In our hands, for unknown reasons, the
-MHC cDNA full-length clone was difficult to
isolate, and, upon bi-directional sequencing, numerous errors were
apparent, particularly in the PCR-derived portion of the molecule even
though a "proof-reading" enzyme was used. Each error was repaired
using site-directed mutagenesis. Multiple clones of the final construct were sequenced in order to exclude any remaining PCR-induced errors and, in comparison to both the rat and human clones, a consensus sequence for the murine
-MHC was derived (GenBankTM
accession number AY056464).
-MHC is expressed in both the mouse
atria and ventricles in post-birth animals, and so the cDNA was
placed into the
-MHC promoter cassette, which contains
the full-length
-MHC promoter upstream of the single
cloning site, and a human growth hormone polyadenylation signal (hGH)
downstream (Fig. 1A).
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Fig. 1.
Transgenic expression of
-MHC in the heart. A, diagram of
the TG construct used. The 5.9-kb
-MHC cDNA was linked to the
mouse
-MHC promoter and the hGH polyadenylation signal.
The
-MHC cassette is flanked by the sequences comprising both the
5'- and 3'-UTR derived from
-MHC (exons, dark gray
boxes; introns, lines). B, Northern blot
showing that the TG mRNA is the same size as the endogenous message
(NTG). Line identities are shown above their respective
lanes. The probe corresponded to the mRNA 3'-UTR. C,
relative and absolute levels of overexpression at the transcript level
were quantitated from a series of dot blots. Probes included
oligonucleotides corresponding to either the hGH or 3'-UTR sequences.
The gray boxes denote the amount of TG MHC expression
relative to endogenous MHC mRNA levels. D, to induce a
V1
V3 shift in NTG hearts, mice were
treated with PTU. Ventricular protein was loaded onto a 5% glycerol
gel and electrophoresed to separate the
- and
-MHC-encoded isoforms. The PTU-treated hearts contained
>90% V3. Line 102 heterozygotes, which have 40%
replacement, were bred to homozygosity, and protein derived from those
hearts (lane 3,
-TG) contained 73-75%
V3.
-MHC 3'-UTR (which is
contained within the transgene, Fig. 1A) or an oligonucleotide specific for hGH were used. Both probes gave consistent results with respect to the lines' relative expression to one another,
while the
-MHC probe enabled us to determine the degree of
overexpression. For each of the 5 lines tested, TG transcript levels
were quite modest, ranging from 1.7 to 3.0-fold with respect to the
endogenous message (Fig. 1C). These levels were relatively low compared with those that are sometimes observed when other contractile proteins were overexpressed using transgenesis (27, 31,
32). However, we have noted in other related studies that high copy
numbers and high levels of MHC expression (>12-fold overexpression)
can be lethal, presumably because of the relatively insoluble nature of
the intact protein.
- and
-MHC proteins can be separated on 5% glycerol gels (Fig.
1D). Hypothyroidism resulting from PTU treatment resulted in
nearly complete replacement (90%) of
- with
-MHC. Our previous
contractile protein-based TG studies showed that the cardiomyocyte
rigidly controls the overall stoichiometry of the contractile protein
pool, such that TG overexpression at the mRNA level does not lead
to increases of overall protein content (30, 32, 33). That is, there is
no "overexpression." The steady state levels of endogenous protein
are down-regulated and replaced proportionally by the TG protein.
Therefore, we could achieve partial or even complete replacement of the
endogenous MHC with TG proteins. When the highest expressing line (Line
102) was bred to homozygosity, 73% of the total MHC was
-MHC (Fig. 1D).
-MHC (Fig.
5B) showed only traces of this isoform in NTG ventricles (Fig. 2A). Confocal analysis
confirmed that the PTU-treated animals showed significant accumulation
of
-MHC (Fig. 2B), but the characteristic striated
pattern was somewhat blunted, consistent with the major effects that
hypothyroidism has on cardiomyocyte morphology. In contrast with the
PTU-treated mice, striated morphology was well conserved in the
-MHC
TG cardiomyocytes, with the pattern of staining confirming the correct
incorporation of TG protein into the sarcomere (Fig. 2C).
Cardiac histology was examined using young adult animals (8-12 weeks)
(Fig. 2, D and E) and aged animals (1 and 2 years).2 No significant
differences in the gross morphology of either heterozygous or
homozygous TG hearts were observed, and no differences in heart rate or
chamber weight could be detected (Table
I). Quantitation of the molecular markers
of hypertrophy, which we have found to be a very sensitive marker of
any response at the cellular level, was carried out at the transcript
level as described previously (34), and no differences could be
detected.2 Similarly, we could not detect any obvious
differences in the cardiomyocytes from homozygous TG mouse hearts using
either light or electron microscopy (Fig. 2,
D-G). No early deaths or overt ill health was
noted in any of the TG animals during the first year and a half of life
as compared with the NTG experimental cohorts. We conclude that the
transition is benign in terms of the animals overall cardiac
morphology of the animals and that no early mortality or morbidity
presents under normal animal husbandry conditions.
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Fig. 2.
Incorporation of TG protein into the
sarcomere and cardiomyocyte structure. A-C,
representative images of longitudinal cryosections from 4-month-old
ventricles double-labeled with an antibody against mouse -MHC Loop 2 (green) and fluorescence-tagged phalloidin (red).
A, NTG; B, NTG treated with PTU; C,
line 102
-TG homozygotes. Note the lack of any
-MHC
(green) in the NTG cardiomyocyte and the relative lack of
striations in the PTU-treated animals when compared with either panels
A or C. D and E, NTG and
homozygous line 102 TG ventricles, respectively. Longitudinal sections
stained with hematoxylin-eosin display regularly aligned myofibrils
with distinct Z bands and M lines. The insets demonstrate
well-defined striations in both sets of cardiomyocytes.
F and G, thin sections from NTG and
homozygous line 102 TG ventricles, respectively, prepared for
transmission electron microscopy. No abnormalities could be detected by
observers who were blinded to sample identity.
Heart:body and chamber:body weights
-MHC while the homozygotes show ~73%
replacement. The skinned fiber is a complex system in which the
contractile machinery operates against the internal cytoskeletal
structures in both the cardiomyocytes and connective tissue. Therefore,
Vmax in a fiber is never truly unloaded, as is
assumed to be the case in the in vitro actin motility assay
(see below).
-MHC. Fibers were
isolated from 9-week-old animals in order to minimize the effects of
any secondary pathology that might develop later in life, and the unloaded shortening and maximum shortening velocities, as well as the
relative power that the fibers developed, were measured (Fig.
3). As expected on the basis of the
degree of
-MHC replaced by
-MHC, the values derived from line 102 heterozygotes were intermediate between the NTG (100%
-MHC) and PTU
(90%
-MHC) data. Significant, graded decreases in the unloaded
shortening velocity were noted (Fig. 3A) from NTG (3.80 ± 0.14 m. l./s, n = 7) to line 102 (2.72 ± 0.26 m. l./s, n = 4) to the PTU-derived fibers
(1.51 ± 0.24 m. l./s, n = 3). The same
gradual decreases were also observed in the force-velocity data used to
derive the maximum shortening velocities (Fig. 3B). The
power-force relationships, and maximum power produced followed the same
trend (Fig. 3C), and the data show that the shift in MHC
isoform content leads directly to changes in cross-bridge cycling
rates.
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Fig. 3.
Contractile properties of isolated
ventricular fibers I. A, slack test comparison between
TG (line 102 heterozygotes), NTG, and PTU-treated mice. The change in
length ( length) was plotted versus the time
lag between the onset of a release and the onset of tension recovery
(
time). Straight lines were then fitted by the
least-squared method. The maximum shortening velocities were also
determined using the slack test. Units are in muscle lengths per s
(m.l./s). B, force-velocity relationships and maximum
shortening velocities determined by isotonic quick releases under
constant load at pCa = 5. C, relative power
was extrapolated from the force-velocity relationships. Multiple fiber
preparations were derived from 3 to 7 animals per group. Power output
is defined as the relative power
(P/P0) multiplied by the velocity
(m.l./s). Values are expressed as means ± S.E. *,
p < 0.05; **, p < 0.01; ***,
p < 0.001 versus NTG; #, p < 0.05 versus
-TG heterozygous.
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Fig. 4.
Contractile properties of isolated
ventricular fibers II. A, slack test comparison between
NTG, TG line 102 heterozygotes, and TG line 102 homozygotes. The change
in length ( length) was plotted versus the time
lag between the onset of a release and the onset of tension recovery
(
time). Straight lines were then fitted by the
least-squared method. The maximum shortening velocities were also
determined using the slack test. Units are in muscle lengths per s
(m.l./s). B, force-velocity relationships and maximum
shortening velocities determined by isotonic quick releases under
constant load at pCa = 5. C, relative power
was extrapolated from the force-velocity relationships. Multiple fiber
preparations were derived from 3 to 4 animals per group. Power output
is defined as the relative power
(P/P0) multiplied by the velocity
(m.l./s). Values are expressed as means ± S.E. *,
p < 0.05; **, p < 0.01; ***,
p < 0.001 versus NTG. Absolute values
differ slightly from the data set in Fig. 3 because of minor
alterations in the fiber apparatus. However, relative differences are
conserved.
/
Chimera--
The above data clearly showed that the cardiomyocyte is
tolerant of significant MHC isoform shifts that are transgenically imposed. We next explored the structural basis of the different cardiac
myosins' unique functionalities by replacing the endogenous MHC with
/
chimeras, the working hypothesis being that the functional differences between the isoforms presumably are caused by the different
loop sequences. The structure of Loop 1 is thought to modulate the rate
of Mg2+-ATP binding and Mg2+-ADP release while
the structure of Loop 2 affects the rate of myosin attachment to actin
(9-11). Two constructs, in which the sequences of either Loop 1 and
Loop 2 (L1+L2) or only Loop 2 (L2) of mouse
-MHC were substituted by the corresponding
-MHC
sequences were made and used to generate TG mice (Fig.
5A). In order to detect the TG
protein, an antibody to the
-MHC Loop 2 sequence was generated (Fig.
5B). Quantification of protein replacement in hearts from
L1+L2- and L2-TG mice by Western
blotting (Fig. 5C) showed nearly complete replacement
(L1+L2, 100%; L2, 84%). This was
confirmed by mass spectroscopy, in which the tryptic peptide of the
endogenous protein (LMATLFSTYASADTGDSGK, mono-isotopic mass 1935.90)
was replaced by the respective fragment containing
-MHC-sequence (LLSNLFANYAGADAPADK, mono-isotopic mass 1850.93).
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Fig. 5.
Protein replacement in NTG and TG hearts
expressing chimeric MHCs. A, 5.9-kb -MHC cDNA
was modified to contain the
-MHC sequences of Loop 1 and Loop 2. B, variable loop sequences are shown and the peptide
fragment used to raise the Loop 2
-MHC-specific antibody is
indicated. C, Western blots of myofibrillar proteins derived
from NTG, PTU-treated, or TG mice carrying the chimeric myosins
(L1+L2 or L2) using the Loop 2
-MHC antibody. In the L1+L2 TG hearts,
replacement was 100%, in the L2 TG hearts, 84%.
-TG mice
(40% replacement), from high-replacement (100%)
L1+L2 TG hearts, and from NTG as well as from
PTU-treated hearts (Fig. 6).
V1
V3 replacement had a clear effect on
molecular motor velocity, with the isoform switches in the PTU and
-MHC TG preparations significantly decreasing the sliding velocity of myosin. For the 40% replacement of V1 with
V3 in line 102 heterozygotes, the observed values were
intermediate between the NTG and PTU-derived samples, as expected
considering the results of our previous studies in which we compared
filament sliding velocities of mouse V1/V3 mixtures in varying proportions, and observed a linear relationship between relative isoform content and filament sliding velocity (6). In
the present study, actin filament sliding velocity of the
L1+L2 chimeric MHC was not significantly
different from that of
-MHC, indicating that the loops did not
confer "
-like" activity on the molecule.
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Fig. 6.
In vitro motility assays. The
bars indicate the absolute velocities of actin filaments
(micrometers per second) translocated by the respective myosins. Values
are expressed as means ± S.E. *, p < 0.001 versus NTG.
-TG hearts. As
expected, a 2-fold difference in myofibrillar
Ca2+-stimulated Mg2+-ATPase activity was
observed between NTG and PTU hearts (Fig. 7A). Myosin isolated from the
-MHC-expressing TG line 102 homozygote hearts exhibited depressed
activities that were consistent with a 75% replacement of
V1 with V3 (Fig. 7B). The chimeric
myosins, while displaying somewhat diminished activities at the two
highest calcium concentrations tested, had myofibrillar ATPase values that were closer to V1 than V3 (Fig.
7C). For the L1+L2-TG hearts in
which a 100% replacement had been effected, enzymatic activity of this
myosin was more like V1 than V3, indicating
that the loops did not confer full "
-like" activity on the
molecule. There were no significant differences in the
pCa50 values between any of the groups. The slightly
different ATPase values between L1+L2 and
L2 myofibrils is most likely due to the higher degree of
protein replacement in the L1+L2-TG mouse
hearts.
View larger version (19K):
[in a new window]
Fig. 7.
In vitro Ca2+-stimulated
ATPase activities. A-C, Ca2+-stimulated
Mg2+-ATPase activities were determined from NTG and
PTU-treated (A), -TG (B), and
L1+L2 and L2-TG (C)
hearts. Panels B and C show the NTG and PTU data
as dotted lines to facilitate comparison. A 2-fold
difference in myofibrillar Ca2+-stimulated
Mg2+-ATPase activity was observed between the NTG and PTU
hearts. ATPase activity of L1+L2-TG myofibrils
was reduced, but not to the level of the PTU group. ATPase activity of
L2-TG myofibrils was also different from the PTU group, but
not from the NTG group. In contrast,
-TG myofibrils showed
significantly reduced ATPase activity close to that seen in the PTU
group. *, p < 0.05 versus NTG;
,
p < 0.05 versus PTU, n = 3-5, myofibrils from 3- to 6-month-old hearts.
/
chimeric MHCs showed any significant differences
from the V1 enzyme at any actin concentration (Fig. 8,
B and C).
View larger version (18K):
[in a new window]
Fig. 8.
In vitro actin-activated ATPase
activities. A-C, actin-activated ATPase activities of
myosin preparations from NTG and -TG (A),
L1+L2 (B), and L2
(C) myosin. For comparison, data from panel
A are shown again as dotted lines in
panels B and C.
-MHC, but not
L1+L2 and L2 chimeric myosin,
showed significantly reduced actin-activated ATPase activity. Myosin
was purified from 3- to 6-month-old hearts. *, p < 0.05 versus NTG.
V3 TG animals
appeared overtly healthy in the unstressed state and showed no signs of
morbidity or increased mortality, we reasoned that whole organ function
must be affected because of the slower motor velocity. To that end, we
determined cardiac hemodynamics for the line 102 homozygotes using the
isolated working heart preparation and in the intact animal (Table
II). The unpaced isolated working hearts,
with ~75% V3, displayed a significantly reduced heart
rate with concomitant reductions in both systolic and diastolic
parameters (Table II). We repeated the measurements under paced
conditions (397 bpm) so that heart rate would not directly affect
systolic or diastolic function and again noted significant decreases in
the values of both dP/dtmin and
dP/dtmax.
Hemodynamic measurements in the isolated working heart model and in
vivo
-TG). The unpaced
and paced models were carried out on slightly different apparati and
absolute values between these two groups cannot be directly compared;
however, NTG and TG cohorts within the unpaced or paced groups can be
compared directly. ND, not determined; MAP, mean arterial pressure;
LVPsys, left ventricular systolic peak pressure;
dP/dt, change in pressure in relation to time;
dP/dt40, dP/dt at
40 mm Hg developed pressure; Tau, time constant of relaxation.
-TG mice. Mean
arterial pressure, LV systolic pressure, and LV
dP/dtmax tended to be lower in TG
animals compared with wild type (Table II). Furthermore, dP/dt40 (dP/dt
at 40 mm Hg developed pressure) was also significantly lower in TG
mice, suggesting that the decreased rate of contraction could not be
accounted for by the observed differences in afterload and is more
likely due to actual differences in myocardial contractility. Likewise,
both dP/dtmin and the time constant
of relaxation (tau,
) also demonstrate significant impairment of
relaxation in the TG animals, consistent with data from the isolated
hearts. Interestingly, in contrast to the isolated unpaced working
heart, there was no difference in heart rate between the NTG and
-TG
mice, presumably because of compensation via neurohumoral mechanisms in
the intact animal. Supporting this hypothesis, we found that
administration of the
-adrenergic blocker propranolol revealed a
difference in heart rate between TG and wild type mice (434 ± 29 versus 474 ± 11). Finally it is important to note that
out of the 6 mice in the
-TG group, 2 were hemodynamically unstable
under anesthesia and died before completion of the protocol, further
underscoring their cardiovascular deficit.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-MHC-encoded isoform.
/
chimeric myosins, which could then be
used to investigate whether myosin functionality could be critically
altered by exchanging loop sequences between the cardiac MHC isoforms.
Based on chimeric studies in Dictyostelium myosin II,
Spudich et al. (9-11) postulated that the structures of the
myosin surface Loops 1 and 2 could serve as modulators of the enzymatic
and mechanical properties of the MHC. However, a number of other
investigators reported evidence that the structure of the myosin
backbone could profoundly influence surface loop function (12, 13). Rat
and pig
-MHC, which have identical Loop 1 sequences apart from a
single conservative substitution, have 3-4-fold differences in ATPase
activity and ADP dissociation (12). The present study addresses this
controversy in full-length mammalian cardiac MHC. Our data demonstrate
that the sequences of the surface loops of mouse cardiac MHC, in
isolation, are of minor importance in determining isoform-specific
characteristics. In light of the previous chimeric studies, the
sequences of the
- and
-loops in mouse cardiac MHC may be
dependent upon other variable amino acids for their ability to
influence both the kinetics and mechanics of cardiac muscle contraction.
- and
-Loop 1 nor mouse
- and
-Loop 2 differ in net charge (Fig.
5B), but charge distribution and spacing varies considerably
between the two isoforms. These differences in the three-dimensional
arrangement of charges may influence myosin-nucleotide and actin-myosin
interactions (8). This concept is supported by data demonstrating that
the structural differences between the rat
- and
-loops are
sufficient to alter Dictyostelium MHC function
(11). Loop 2 of Dictyostelium myosin II was
exchanged for Loop 2 of rat
- or
-MHC, resulting in
actin-activated ATPase activities of the two chimeras that closely
reflected ATPase activities of rat
- and
-MHC. Mouse and rat
-Loop 2 are identical, and mouse and rat
-Loop 2 differ only in
one amino acid (mouse A631V rat), a conservative substitution. Our data
indicate that Loop 2 exchanges between mouse
- and
-MHC do not
affect the physiologically relevant actin-activated
Mg2+-ATPase activity: neither the
L1+L2 nor L2 chimeras were
different from that of
-MHC. Similarly, ATPase activity of
myofibrils from L2-TG hearts was not altered. Only at
non-physiologically high calcium concentrations was the ATPase activity
of L1+L2 myofibrils reduced, indicating that
Loop 2 plays only a very minor role in regulating this aspect of
isoform functionality. Moreover, the present chimeric study also
demonstrates that switches in Loop 1 sequence do not alter ADP release
rate and thereby actin filament sliding velocity in mouse cardiac MHC,
in contrast to the previous Dictyostelium data (10). The
data, together with other chimeric-based studies indicate that the
structure of the MHC backbone influences whether or not loop exchanges
between isoforms can affect MHC function.
-MHC reveals only 33% identity (64% homology).
Consequently, it is not surprising that the Dictyostelium
myosin II backbone could provide different atomic interactions with a
chimeric loop than would the mouse cardiac MHC backbone. Furthermore,
it is well known that within the myosin head even widely separated
regions can critically influence one another, and this may underlie a partial explanation for the importance of the backbone sequence for
loop function. Therefore, alignment of more closely related MHC
isoforms in order to find regions that could influence isoform-specific characteristics appears to be the most feasible approach for
determining the critical amino acids that underlie the differing
functionalities of the unique isoforms. By comparing MHC isoform
sequences across various mammalian species in the context of all
available functional data, we identified only 8 non-conservative amino
acid substitutions in
-MHC (residues 2, 210, 442, 452, 801, 1092, 1637, and 1681) and only 4 residues in the
-MHC (residues 424, 573, 1201, and 1368) that may be responsible for species-specific
MHC-isoform functionality rather than surface Loops 1 and 2 (6).
Functional analysis of these residues may tell us which regions of the
backbone are of importance for MHC function and would provide the basis for further structure-function studies comparing
-MHC and
-MHC.
-MHC, by 56% in TG
mice with 73% replacement, and by 60% in the PTU-treated animals
(Figs. 3 and 4), a value in the same range as measured by Fitzsimons
et al. (39), who found an 80% decrease in rat single
cardiac myocytes that express essentially pure
-MHC. In our hands,
substituting the majority of
-MHC with
-MHC either via
transgenesis or by inducing hypothyroidism resulted in a similar impairment in fiber mechanics. This indicates that, in this setting, the MHC isoform switch has a predominant effect compared with other
pleiotropic effects of PTU.
- and
-MHC observed on the
molecular level resulted in altered systolic and diastolic function in
isolated hearts and in vivo. In unpaced isolated hearts, a
17% decrease in left ventricular pressure and a 31% decrease in
dP/dtmax together with a 45% increase in
dP/dtmin was seen, and when differences in heart
rate were removed by atrial pacing, these differences persisted. Left
ventricular pressure measurements from intact animals revealed similar
contractile deficits in
-TG mice.
-MHC was measured in Langendorff preparations (16). In those hearts,
dP/dtmax was reduced by 15% although
replacement of the endogenous MHC with the tagged
-MHC was only
12%. In light of our data, the relationship between contractility and
relative isoform content may be non-linear and supporting this
hypothesis, in hypothyroid hearts predominantly expressing
-MHC, a
small amount of
-MHC expression can significantly augment myocyte
power output (41).
-TG mice as
compared with NTG animals. A trend toward lower heart rates in
-TG
isolated hearts was also reported by Tardiff et al. (16).
These data support the concept that there might be an intrinsic
feedback mechanism adapting heart rate to the kinetic properties of the
MHC (42). In vivo, it is the autonomic nervous system that
regulates sinus node firing, even in anesthetized animals, and the
system is regulated through both the sympathetic and parasympathetic
neurons (43). Based on the normal histology, unremarkable sarcomeric
structure of the cardiomyocytes and the lack of hypertrophy in the
-TG mice, we conclude that as long as the heart is able to
adequately respond to autonomic regulation, and contractile function is
sufficiently compensated to maintain cardiac output, no overt or
subclinical phenotype will present.
V3 transition to affect human heart
disease remained problematic, as it was thought that the human
ventricle contained only the V3 isoform in either the
normal or diseased state. However, evidence now exists that an isoform
shift does occur in the failing human ventricle, with
-MHC mRNA accounting for as much as 34% of the total
MHC transcript in the normal heart (44). Down-regulation of
-MHC in the failing human ventricle at both the RNA and
protein levels occurs (45, 46). Our data show that a V1
V3 shift in mouse hearts reduces contractile function.
Taken together with the potential energy-conserving effect of a
V1
V3 shift and the fact that even small
amounts of V1 might impact favorably on cardiac function,
it is now critical to understand what role an isoform shift might play
in disease onset and progression. Crossing the V3 TG mice
into different mouse models of hypertrophy and failure should provide
insight into the potential role(s) the different cardiac MHCs may play in the pathogenesis of cardiac disease.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Mark W. Duncan, Ph.D, University of Colorado Health Sciences Center Biochemical Mass Spectrometry Facility, for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants HL69799, HL60546, HL52318, HL60546, HL56370 (to J. R.) and HL66157 (to D. W. and N. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a fellowship award from the American Heart Association.
To whom correspondence should be addressed: Division of
Molecular Cardiovascular Biology, MLC 7020, 3333 Burnet Ave.,
Cincinnati, OH 45229-3039. Tel.: 513-636-8098; Fax: 513-636-5958;
E-mail: jeff.robbins@cchmc.org.
Published, JBC Papers in Press, March 6, 2003, DOI 10.1074/jbc.M210804200
2 J. Robbins and M. Krenz, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MHC, myosin heavy chain; hGH, human growth hormone; L1, Loop 1; L2, Loop 2; NTG, non-transgenic; m.l./s, muscle lengths per second; TG, transgenic; PTU, propylthiouracil; UTR, untranslated region.
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