From the University of Miami School of Medicine,
Departments of Molecular and Cellular Pharmacology and
¶ Physiology and Biophysics, Miami, Florida 33136 and the
§ Department of Anesthesiology, Mayo Foundation,
Rochester, Minnesota 55905
Received for publication, July 27, 2000, and in revised form, September 28, 2000
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ABSTRACT |
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This study characterizes a transgenic animal
model for the troponin T (TnT) mutation (I79N) associated with familial
hypertrophic cardiomyopathy. To study the functional consequences of
this mutation, we examined a wild type and two I79N-transgenic mouse
lines of human cardiac TnT driven by a murine Contraction of vertebrate striated (skeletal and cardiac) muscle
is activated by the binding of Ca2+ to the
Ca2+-binding subunit
(TnC)1 of the troponin
complex, which together with TnI, TnT, tropomyosin, and actin
form the regulatory system of the contractile apparatus (1-4). The
exact function of TnT is still somewhat controversial, but it is
thought to stabilize the Tn complex and to affect the Ca2+
sensitivity of actomyosin ATPase activity, the level of ATPase activation, and/or force development (5-8). Recent studies have revealed that TnT is one of the sarcomeric proteins identified in
familial hypertrophic cardiomyopathy (FHC) (9, 10). FHC is an autosomal
dominant disease, characterized by left ventricular hypertrophy,
myofibril disarray, and sudden death. Numerous studies have shown that
FHC is caused by missense mutations in various genes that encode for
Until now, a transgenic model for the TnT-I79N has not been reported,
although other TnT transgenic mice have been described. A truncated
CTnT in transgenic mice studied by Tardiff et al. (31)
revealed sarcomeric disarray and significant diastolic dysfunction in
animals expressing protein at a very low level (<5%). Animals with
higher levels of transgene expression died within 24 h of birth.
Another TnT transgenic animal model reported by Marian's lab was
generated for the human cardiac TnT-R92Q mutation using a murine CTnT
promoter (32). The level of expression in transgenic lines (wild type
and R92Q) varied from 1 to 10% of the total CTnT pool. The authors
observed diastolic dysfunction and myocyte disarray in the mutant mice
as compared with wild type mice (32). The same TnT-R92Q mutant was
expressed in transgenic mice in Leinwand's laboratory where the level
of R92Q expression varied from 30 to 92% (33). A murine CTnT cDNA
and a rat To have an in vivo model of the TnT-I79N mutation and to
possibly clarify some of the conflicting in vitro results,
we have developed a transgenic model of this mutation. We examined a
wild type (Tg-WT) and two I79N-transgenic mouse lines (Tg-I79N) of HCTnT driven by a murine Clone Construction
The cDNA for wild type human cardiac troponin T (adult
isoform) was cloned by reverse transcription-PCR using primers based on
the published cDNA sequence (35) and standard methods (36): HCTnT,
5'-GACCATGGCTGACATAGAAGAGGT; HCTnT,
3'-GAGGATCCTATTTCCAGCGCCCGGTGACTT. The I79N mutant was made using
overlapping sequential PCR (36). Wild type and mutant cDNAs were
constructed to have an NcoI site at the amino-terminal ATG,
and a BamHI site following the stop codon to facilitate
cloning into pET-3d (Novagen), which was used for bacterial expression
of the proteins.
Transgene Construct
The wild type and mutant cDNAs were cloned into the unique
SalI site of the plasmid, Generation of Tg Mice
The transgene vector described above was purified on a cesium
chloride gradient and restricted with NotI to release a
7-kilobase fragment that was used for microinjection. This fragment was
purified by agarose gel electrophoresis, followed by electroelution
onto DEAE paper (37) and resuspended in 10 mM Tris-HCl, pH
7.4, 0.1 mM EDTA at a final concentration of 5 µg/ml.
Pronuclei were injected, and the surviving embryos were implanted using
standard methods (38). Founder mice were identified by preparing tail
clip DNA and analyzing its hybridization to a probe corresponding to
the human growth hormone 3'-untranslated region (a 630-base pair
HindIII/EcoRI fragment from the transgenic
construct). The PCR was also used to identify Tg mice. A forward/sense
primer (5'-TTCGACCTGCAGGAGAAGTT-3') was derived from HCTnT cDNA
sequence, and a reverse/antisense primer (5'-AGCAACTCAAATGTCCCACC-3')
was derived from human growth hormone sequence; these produced a
713-base pair fragment in mice harboring the transgene. Stable
transgenic lines were generated by breeding founder Tg mice to non-Tg
B6/SJL mice.
DNA and RNA Analysis of Tg Mice
Genomic Southern Blots
Large molecular weight genomic DNA was prepared from liver
according to Sambrook et al. (37), and the concentration was determined by measuring the fluorescence of the Hoechst 33258 (bisbenzimide) dye (Amersham Pharmacia Biotech). Copy number was estimated by quantitative densitometry (Molecular Dynamics
Densitometer) of Southern blots that had known amounts of the transgene
as standards.
Northern Blots
Northern blots were performed on RNA extracted from tissues from
non-Tg and Tg mice. All Tg mice hearts had an appropriately sized
transcript that hybridized to the human growth hormone 3'-untranslated probe, suggesting that the transgene was being transcribed correctly. This probe was not predicted to hybridize with RNA from nontransgenic hearts or transgenic liver.
Primer Extension
To quantify the relative levels of RNA from the transgene and
the endogenous mouse cardiac TnT gene, a primer extension experiment was designed (39). A radiolabeled oligonucleotide was used to prime
reverse transcriptase toward the 5' end of the RNA transcripts, resulting in radioactive products whose size reflects the length of the
primer from the 5' terminus of the RNA. The oligonucleotide primer
(5'-TCCTCCTCGTACTCYTCCACCACCT-3') was complementary to a conserved
region of both transcripts that extended from nucleotide +17 to +41
relative to the ATG (i.e. the region coding for amino acids
6-14 of both human and mouse CTnT). The template for the primer
extension experiment was total RNA isolated from 8-10-week-old Tg and
non-Tg animals.
Reverse Transcription-PCR
RNA was prepared from hearts of transgenic and nontransgenic
mice using the method of Chomczynski and Sacchi (40). Total cDNA
was synthesized using Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) according to the
manufacturer's recommendations.
Protein Analysis of Tg Mice (Western Blotting)
Mouse hearts and pieces of human hearts were homogenized in a
solution of 20% SDS and 10% Mice Exercise Protocol
A swimming protocol mainly described by Geisterfer-Lowrance
et al. (41) was utilized. Groups of four 2-month-old animals representing the different lines were exercised by swimming. Mice were
adapted to the swimming program by beginning with 10-min sessions two
times a day separated by 4 h. These were incremented by 10 min/day
until reaching 90 min/session. The program was completed in 4 weeks.
Animals were weighed weekly. During each session, they were monitored
for inability to sustain the exercise and/or sudden death. During
weekly intervals and at the conclusion of the program, mice were
sacrificed and heart to body weight ratio was determined.
Skinned Fibers Studies
Glycerinated Fibers
Steady State Force Development--
A bundle of 3-5 fibers
isolated from a batch of glycerinated fibers (stored for 1-2 weeks in
Ca2+ Dependence of Force Development--
After
measurements of the initial steady state force of the fibers, they were
relaxed in the pCa 8 buffer and then exposed to the solutions of
increasing Ca2+ concentrations (from pCa 8 to pCa 4). The
maximal force was measured in each "pCa" solution followed by the
short relaxation of the fibers in the pCa 8 solution. Data were
analyzed using the following equations: % Force Restored = 100 × (Force Restored Rates of Force Activation--
For the kinetic measurements the
bundle of 3-5 fibers was attached by tweezer clips to a force
transducer, placed in a 1-ml cuvette, and bathed in the pCa 8 solution.
The fibers were tested for steady state force development in the pCa 4 solution and relaxed in the pCa 8 solution. Then they were exposed to
2.5 mM DM-nitrophen, 1.002 mM
CaCl2, 100 mM, 1.2 mM
MgCl2, 1.4 mM ATP, 10 mM
glutathione, 29.4 mM
(1,6-hexamethylenediamine-N,N,N',N',-tetraacetic acid), and 20 mM creatine phosphate, pH 7.1. Subsequent to irridation by
a 1-ms UV light pulse from Xenon lamp (model
XFL-35S-30171), the Ca2+ chelator was cleaved,
releasing free Ca2+. Its high affinity for Ca2+
before photolysis, Kd decreased from 5.0 × 10 Rates of Force Relaxation--
The initial step of the fiber
preparation was the same as for the measurement of the activation
rates. To monitor the relaxation rates, a photolabile derivative of
O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid
tetrapotassium, Diazo-2, was used. Diazo-2 is able to rapidly chelate Ca2+ upon photolysis converting from a low affinity
(Kd = 2.2 µM) to high affinity
(Kd = 0.073 µM) for Ca2+.
After testing steady state force, the fibers were immersed in the
solution of 2 mM Diazo-2, 0.5 mM
CaCl2, 60 mM TES, pH 7.0, 5 mM
MgATP, 1 mM [Mg2+], and 10 mM
creatine phosphate along with 15 units/ml creatine phosphokinase (ionic
strength = 200 mM) adjusted with potassium propionate.
At the ratio of total added Ca2+ to Diazo-2 given above,
the resulting average initial force will be around 80% of the maximal
force measured in the pCa 4 solution. The ratio provides the greatest
extent of relaxation after photolysis of the Diazo-2. When force
reached equilibrium, the fibers were exposed to a UV flash from Xenon
lamp. The photolysis-induced relaxation was measured several times
during the fiber treatment. The rate constants of relaxation was
calculated according to the equation: y = Ae Simultaneous Force and ATPase Measurements in Fresh (Not
Glycerinated) Skinned Fibers
Small preparations (~1 mm long and 50-70 µm in diameter) of
mouse papillary muscle were dissected free in relaxing solution and
then treated with 1%Triton X-100 for 30 min. Subsequently they were
mounted in the Guth Muscle Research System, which allows for
simultaneous force and ATPase measurements (42, 43). The quartz cuvette
surrounding the preparation had a square cross-section of 1.0 mm2. The sarcomere length was set to 2.2 µm using a laser
diffraction pattern. The solution in the cuvette was changed every
20 s using a peristaltic pump triggered by a computer. The
hydrolysis of ATP was measured by the NADH fluorescence method, in
which ATP was regenerated from ADP and phospho(enol)pyruvate by the
enzyme pyruvate kinase (Reaction 1) (44). The reaction scheme is as follows.
Ca2+ Concentration Measurements--
The
Ca2+ concentration in the solution perfusing the skinned
preparation was varied by use of a gradient maker (Scientific
Instruments GmbH, Heidelberg, Germany) to mix two solutions of known
[Ca2+] and ionic composition together (42, 43). The
resulting [Ca2+] was calibrated using the fluorescent
Ca2+ indicator, Calcium Green-2 (Molecular Probes). The
Kd of Calcium Green-2 used to calculate pCa is 4.4 µM. The concentration of Calcium Green-2 in the gradient
solution was 1.0 µM. Calcium Green-2 changes its
fluorescence over the range of Ca2+ required for activation
of contraction and ATPase activity. The Calcium Green-2 fluorescence
was excited at 480 nm, and the fluorescence was measured with a cut-off
filter at 515 nm.
Solutions--
All fresh (not glycerinated) skinned fiber
solutions contained 85 mM K+ plus
Na+ added with Na2ATP, 2 mM
MgATP2 Computer Modeling of Experimental Data--
Computer simulations
were based on modified model of Robertson et al. (49) and a
two-state cross-bridge model (50) utilizing an exponential dependence
of the TnC off rate for Ca2+ (Ca2+-specific
site II) on force (51, 52). A detailed description of the model is
provided in the "Appendix."
Generation and Identification of HCTnT-WT and HCTnT-I79N Mutant Tg
Mice
A total of two wild type HCTnT (Tg-WT, lines 2 and 3) and six
HCTnT-I79N founder mice (Tg-I79N, lines 4-9) were identified by PCR
and Southern blot analysis (data not shown). Lines 3, 8, and 9 consistently produced expected Mendelian ratios of transgenic offspring
and were selected for further studies. The copy number of the
transgenes were 40 for line 3 (Tg-WT) and line 8 (Tg-I79N), and 48 for
line 9 (Tg-I79N) (data not shown). On Northern blot analysis, all Tg
mouse hearts had an appropriately sized transcript that hybridized to
the human growth hormone 3'-untranslated probe, suggesting that the
transgene was being transcribed correctly (data not shown). A primer
extension experiment was designed to quantify the relative levels of
RNA from the transgene and the endogenous mouse CTnT gene (Fig.
1). The predicted size of products from
the endogenous and transgenic transcripts was different, 113 nucleotides versus 144, respectively, mainly because the
transgene contained 5' exons coding for -myosin heavy chain
promoter. Extensive characterization of the transgenic I79N lines
compared with wild type and/or nontransgenic mice demonstrated: 1)
normal survival and no cardiac hypertrophy even with chronic exercise; 2) large increases in Ca2+ sensitivity of ATPase
activity and force in skinned fibers; 3) a substantial increase in the
rate of force activation and an increase in the rate of force
relaxation; 4) lower maximal force/cross-sectional area and ATPase
activity; 5) loss of sensitivity to pH-induced shifts in the
Ca2+ dependence of force; and 6) computer simulations that
reproduced experimental observations and suggested that the I79N
mutation decreases the apparent off rate of Ca2+ from
troponin C and increases cross-bridge detachment rate
g. Simulations for intact living fibers predict a higher
basal contractility, a faster rate of force development, slower
relaxation, and increased resting tension in transgenic I79N myocardium
compared with transgenic wild type. These mechanisms may contribute to
mortality in humans, especially in stimulated contractile states.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
-myosin heavy chain (11-14), ventricular myosin light chains 1 and
2 (15-17), myosin binding protein C (12), titin (18), actin (19),
-tropomyosin (9), troponin T (9, 10, 20), and troponin I (21, 22).
Whereas individuals with
-myosin heavy chain mutations, in general,
have a higher level of cardiac hypertrophy, those with TnT mutations
have less hypertrophy, but a higher incidence of sudden cardiac death
in young adults (10). To date, 15 human cardiac TnT mutations have been
associated with FHC: I79N, R92Q/W/L, R94L, A104V, F110I, R130C,
E160, E163K, E163K/R, E244D, R278C, and a mutation that arises from
abnormal splicing of Intron 16 (G1
A) (23). Among these
mutations, the I79N mutation is of special interest because it has been
found to cause the highest risk of sudden cardiac death in young adults
(10). At present, there is no clear understanding of why this TnT-I79N
mutation is associated with increased sudden cardiac death. Several
investigators have demonstrated an in vitro effect of the
TnT-I79N mutation on the contractile properties of cardiac and skeletal
muscle with conflicting results. Lin et al. (24), using rat
cardiac TnT containing a mutation in an equivalent position to the
TnT-I79N mutation in humans, showed that this mutant TnT had a normal
affinity for actin-tropomyosin and conferred normal Ca2+
sensitivity to acto-S1 ATPase activity. The regulated thin filaments, however, moved 50% faster over heavy meromyosin than control
filaments in an in vitro motility assay. Additional
measurements utilizing the same system carried out by Homsher et
al. (25) revealed that heavy meromyosin exerted reduced isometric
force on single thin filaments reconstituted with the TnT-I79N mutant.
Sweeney et al. (26) reported that TnT-I79N-transfected quail
skeletal muscle myotubes had decreased Ca2+ sensitivity of
force production, whereas the unloaded shortening velocity was
increased about 2-fold. An embryonic isoform of rat TnT-I79N expressed
in adult rat cardiac myocytes causes a decreased Ca2+
sensitivity of isometric force (27). Our results on
TnT-I79N-reconstituted porcine fibers (28) are in accord with those of
Morimoto et al. (29), who demonstrated that TnT-I79N
reconstituted skinned rabbit trabeculae increased the Ca2+
sensitivity of contraction. A very recent study of Yanaga et al. (30) confirmed increased Ca2+ sensitivity of
myofibrillar ATPase activity of TnT-I79N- reconstituted rabbit cardiac
myofibrils. Part of the disparity is likely due to the different
in vitro assays used by these investigators, which
illustrates the need to study the effect of the mutations in an
in vivo system.
-myosin heavy chain promoter were used in their study. The
R92Q hearts demonstrated significant induction of atrial natriuretic
factor and
-myosin heavy chain transcripts, interstitial fibrosis,
and mitochondrial pathology. Moreover, a basal sarcomeric activation and impaired relaxation were observed in the mutant mouse (33). In a
very recent paper of Lim et al. (34), a murine
-myosin heavy chain promoter was used to produce a transgenic mouse expressing human cardiac TnT-R92Q (34). The level of protein expression was
relatively low, and the mutant mice demonstrated myocyte disarray and
excess interstitial collagen. Interestingly, none of these transgenic
mice demonstrated significant cardiac hypertrophy.
-myosin heavy chain promoter. The levels of
expression of either Tg-WT or Tg-I79N, relative to mouse CTnT, were
71% (WT) or 35 and 52% in the two I79N lines. Extensive
characterization of the Tg-I79N lines compared with Tg-WT and/or non-Tg
mice demonstrated: 1) normal survival and no cardiac hypertrophy even
with chronic exercise in all groups; 2) large increases in the
Ca2+ sensitivity of the ATPase activity and force
development in skinned fibers; 3) a substantial increase in the rate of
force activation and an increase in the rate of force relaxation in
flash photolysis experiments; 4) significantly lower maximal
force/cross-sectional area and ATPase activity; 5) loss of sensitivity
to pH-induced shifts in the Ca2+ dependence of force
correlated with HCTnT-I79N expression levels; and 6) computer
simulations of force-pCa relations and of flash photolysis experiments
that reproduced the experimental observations and suggested that the
HCTnT-I79N mutation decreases the apparent off rate of Ca2+
from the Ca2+ specific site of TnC and increases the
cross-bridge apparent detachment rate g. Simulations for
intact living fibers predict a higher basal contractility, a faster
rate of force development, a slower isometric relaxation, and increased
resting tension in Tg-I79N myocardium compared with Tg-WT. A higher
basal contractility and residual resting tension limit the contractile
reserve and make the ventricle vulnerable to further diastolic
dysfunction. It is likely that these mechanisms contribute to the
mortality observed in patients with a TnT-I79N-induced FHC especially
in stimulated states of contractility such as seen during vigorous exercise or during inotropic therapy.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
-myosin heavy chain clone 26 (a
generous gift from Dr. Jeffrey Robbins), by filling in this
SalI site along with the NcoI and
BamHI sites of the cDNAs and ligating the blunt fragments. The resulting construct contains about 5.5 kilobases of the
mouse
-myosin heavy chain promoter, including the first 2 exons and
part of the third, followed by the HCTnT cDNA (876 base pairs), and
a 630-base pair 3'-untranslated region from the human growth hormone transcript.
-mercaptoethanol. Small amounts of
each homogenate were diluted, and their respective protein content was
determined by the Coomassie Plus Bio-Rad protein assay. Homogenates
were boiled in an equivalent volume of Laemmli loading buffer, mixed
together in defined ratios based on protein content, electrophoresed on
SDS-12.5% polyacrylamide (61:1 ratio) gels, and transferred to
nitrocellulose membranes (Idea Scientific, Minneapolis, MN). Human
cardiac TnT was detected using a human CTnT-specific monoclonal
antibody (clone 7G7, Research Diagnostics Inc., Flanders, NJ) at a
1:2000 dilution in BLOTTO (5% nonfat dry milk, 10 mM
Tris-HCl, pH 7.4, 140 mM NaCl). Total CTnT (i.e. mouse and human CTnT) was detected using a polyclonal CTnT antibody (at
a 1:3000 dilution in BLOTTO) produced in our lab. The relative reactivity of the polyclonal antibody to the human and mouse CTnTs were
the same based on Western blot analysis using the same protein amount
of human and mouse heart tissue. HCTnT in Tg-WT and two Tg-l79N lines
were determined by comparison of the immunoreactive products of the
electrophoresed samples with the standard curve. The standard curve was
generated from the signal intensity obtained from different ratios of
human and mouse tissues (by protein content) reacted with both the
monoclonal (clone 7G7) and polyclonal CTnT antibodies. Nearly identical
results were obtained for the relative levels of HCTnT protein in the
non-Tg and Tg mice from either a standard curve of the ratio of
polyclonal antibody band intensity to monoclonal band intensity
versus the percentage of HCTnT or the monoclonal band
intensity versus the percentage of HCTnT (see Fig. 2).
Immunoreactivity was detected using goat anti-mouse IgG labeled with
horseradish peroxidase or rabbit anti-goat IgG labeled with horseradish
peroxidase (both used at 1:3000 dilution; Sigma). Color was developed
using diaminobenzidine/H2O2 (Sigma).
Quantitative densitometry of Western blots were done using a Molecular
Dynamics Densitometer. Two hearts from each transgenic line were
independently analyzed on three different blots to assess the relative
levels of HCTnT protein in non-Tg, Tg-WT, and Tg-I79N mutant lines.
Human heart samples, obtained from a transplant patient at Jackson
Memorial Hospital adjacent to the University of Miami School of
Medicine, were rapidly frozen in liquid nitrogen and stored at
150 °C until use. These heart samples showed little or no sign of
degradation as based on Western blotting analysis.
20 °C) were attached by tweezer clips to a force transducer,
placed in a 1-ml cuvette and bathed in the pCa 8 solution
(10
8 M [Ca2+], 1 mM
[Mg2+], 7 mM EGTA, 5 mM
[MgATP2+], 20 mM imidazole, pH 7.0, 20 mM creatinine phosphate, and 15 units/ml of creatinine
phosphokinase; ionic strength = 150 mM), containing
1% Triton X-100. The length and diameter of each fiber were
immediately measured after mounting the fiber to the transducer. The
average length and diameter of the fibers selected for the experiment
were ~1.3-1.7 mm and 150-250 µm, respectively. The fibers were
tested for steady state force development in the pCa 4 solution
(composition is the same as pCa 8 buffer except the [Ca2+] was 10
4 M) and relaxed
in the pCa 8 solution. Steady state force developed by non-Tg, the wild
type Tg-WT and its mutant, Tg-I79N fibers were compared.
Residual Force)/Initial Force; % Change in Force = 100 × [Ca2+]n/([Ca2+]n + [Ca2+50] n) where
[Ca2+50] is the free Ca2+
concentration that produces 50% force and n is the Hill
coefficient (nH).
9 to 3.0 × 10
3 M,
following the UV flash. As a result of the rapid Ca2+
release, the fibers developed isometric tension, characterized by a two
exponential time course. The rate constants of activation, was
calculated according to the equation: y = A(1
e
k1t) + B(1
e
k2t) + C, where k1 and k2 are
the rate constants and A and B are the amplitudes
of the force transient. We believe that the major fast component is due
to the rapid activation of contraction and that the minor slow
component is due to a diffusion process related to re-equilibration of
the fiber with the bulk solution after the flash.
k1t + Be
k2t + C, where
k1 and k2 are the rate
constants and A and B are the amplitudes of the
force transient.
This reaction is coupled to the oxidation of NADH (fluorescent)
to NAD (nonfluorescent), and the reduction of pyruvate to lactate by
L-lactatic dehydrogenase (Reaction 2) (45, 46). In this
reaction 1 mol of phospho(enol)pyruvate and NADH are used to produce 1 mol of ATP and NAD. The solution surrounding the fiber in the quartz
cuvette was illuminated at 340 nm, and the decrease in NADH
concentration was detected by a decrease in the fluorescence signal at
wavelengths greater than 470 nm. The fluorescence change taking place
between each solution change was converted to rate of ATP hydrolysis by
comparison with NADH standards.
, 1 mM Mg2+, 7 mM EGTA, 10
9-10
3.4
M Ca2+, 5 mM phospho(enol)pyruvate,
l00 units/ml pyruvate kinase, and propionate as the major anion.
Solutions for ATPase measurements also contained 0.4 mM
NADH, 0.2 mM AP5A (to inhibit myokinase), and
140 units/ml L-lactatic dehydrogenase. Ionic strength was adjusted to 0.15 M, and the pH was maintained at 7.00 ± 0.02 with imidazole propionate. Relaxing solutions contained no
added Ca2+ (~10
9 M
Ca2+). The concentrations of the various ionic species were
determined by solving ionic equilibrium equations using published
binding constants (48).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
-myosin heavy chain
5'-untranslated RNA. This assay detected a consistent level of
endogenous CTnT transcript in Tg and non-Tg mice. Transgenic RNA
expression varied between lines, with considerably higher expression
levels in lines 3 (WT) and 8 (I79N) and lower levels in line 9 (I79N).
As expected, the level of transgenic RNA was very similar between
animals from the same transgenic line. The intense band of about 125 nucleotides in the Tg lines corresponds to a product that terminates
around the splice junction between exons 1 and 2 of the
-myosin
heavy chain gene and may reflect some unusual secondary structure. The total transgenic message was probably the sum of both the 125 nucleotide product and the full-length 144 nucleotide product, because
1) in this experiment, each RNA can only give rise to one cDNA
product and 2) reverse transcriptase has often been observed to pause
at secondary structures in RNA. To confirm that the transgene was
producing a correct transcript coding for HCTnT, RNA from Tg and non-Tg
mice was subjected to an reverse transcription-PCR experiment using
primers specific for either the endogenous mouse CTnT or the transgene.
Both primer pairs produced expected products (or lack of products) from
the Tg and non-Tg mice (data not shown).
View larger version (121K):
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Fig. 1.
Primer extension analysis of endogenous CTnT
and Tg-HCTnT transcripts. 20 µg of total cardiac RNA was used
for each analysis. Each lane represents primer extenstion products from
the indicated Tg lines (number of Tg line mouse number), non-Tg (NTG
mouse), or human cardiac RNA. The markers (69- and 99-mer) were used to
estimate the size indicated for the major primer extension products.
Note a consistent level of endogenous CTnT transcript in Tg and non-Tg
mice. Transgenic RNA expression varied between lines, with considerably
higher expression levels in lines 3 (WT) and 8 (I79N) and lower levels
in line 9 (I79N).
Protein Expression Levels
To determine the expression level of HCTnT in the non-Tg, Tg-WT,
and Tg-I79N lines, an HCTnT specific monoclonal antibody was utilized
as described under "Materials and Methods." In Fig. 2, the average data of two mice, each run
on three independent blots, are presented. The non-Tg and Tg samples
were compared with the standard curve (see "Materials and
Methods"), and the levels of HCTnT expression quantified: Tg-WT (line
3) contained 70.9% of the total TnT present in the mouse heart,
whereas Tg-I79N contained 52% (line 8) and 34.6% (line 9).
|
Heart Weight/Body Weight
Tg mice expressing HCTnT-I79N had normal survival and no cardiac
hypertrophy. A significantly decreased heart weight to body weight
ratio was observed for the TnT-I79N mice compared with Tg-WT (Fig.
3A). Chronic swimming exercise
(4 weeks), which has previously been used to induce cardiac hypertrophy
in another murine FHC model (41), did not affect survival in all groups of Tg lines. Surprisingly, heart to body weight ratio increased only in
the non-Tg and not in the Tg mice and remained significantly lower in
the Tg-I79N mice versus the Tg-WT mice (Fig.
3B).
|
Skinned Fibers Studies
Our previous results on TnT-I79N-reconstituted porcine fibers (28) demonstrated a significant increase in the Ca2+ sensitivity of force development but no change in the maximal force. The effect of the TnT-I79N mutation in vivo has been examined in two sets of skinned fiber experiments. The first set was performed on glycerinated bundles of mouse papillary muscles whose diameter was between 150 and 200 µm. The second set of experiments was performed on fresh (not glycerinated) thin papillary muscle, with a diameter of ~50-70 µm.
Glycerinated Fibers
Steady State Force Development--
After measurements of the
initial steady state force (pCa 4), non-Tg, Tg-WT, and Tg-I79N fibers
were exposed to solutions of increasing Ca2+ concentration,
and the force-pCa relationship for the different mice was established.
Fig. 4A shows a typical
force-pCa curves, and Fig. 4B summarizes the
pCa50 values for the sedentary and exercised mice. As can
be seen, the Tg-I79N mice had an increased Ca2+ sensitivity
of steady state force development in both the sedentary and exercised
groups compared with non-Tg or Tg-WT. The increase was
pCa50 0.2. No significant difference was observed
between the two mutant Tg-I79N lines (line 8 and line 9).
Interestingly, force per cross-sectional area was much lower for the
Tg-I79N fibers versus the Tg-WT in both sedentary and
exercised mice (Fig. 5).
|
|
Kinetics of Force Development--
To study the rates of force
activation and relaxation of the glycerinated fibers, we utilized flash
photolysis of either caged calcium (DM-nitrophen) or caged chelator
(Diazo-2), respectively. The Tg-I79N fibers demonstrated a
significantly increased rate of force activation and moderately
increased rate of relaxation compared with Tg-WT fibers. Fig.
6 illustrates representative experimental
traces and the double exponential fit curves of activation (panel
A) and relaxation (panel B) of force development for
Tg-WT and Tg-I79N fibers. The rates of activation and relaxation were acquired from different experiments utilizing several Tg lines and
averaged (only faster components with larger amplitudes). Activation of
the Tg-I79N fibers was 29.8 ± 1.4 s1
(n = 22), i.e. about 1.7-fold higher than
Tg-WT fibers, 18.0 ± 0.7 s
1 (n = 12). The rate of relaxation for Tg-I79N fibers was 33.5 ± 1.8 s
1 (n = 17) and 28.8 ± 2.3 s
1 (n = 9) for Tg-WT fibers. Data are the
averages of n experiments ± S.E.
|
Effect of pH on the Ca2+ Sensitivity of Force
Development--
Lowering pH in the physiological range (~ 7.0-6.5)
is known to shift the Ca2+ sensitivity of force development
from lower to higher calcium concentrations, especially in cardiac
muscle (53-55). The Tg-WT and non-Tg mice showed this clearly (Fig.
7). However, the Tg-I79N mice had a much
lower change in Ca2+ sensitivity to this change in pH.
Changing the pH from 7.0 to pH 6.5 decreased the Ca2+
sensitivity of force by pCa50
0.5 for the non-Tg
or Tg-WT fibers. Both lines (8 and 9) of Tg-I79N became less sensitive to acidic pH, and a much smaller decrease in Ca2+
sensitivity of force development was observed. For TnT-I79N line 9, the
change was
pCa50
0.39 and for line 8,
pCa50
0.2. Interestingly, line 8 had the highest
level of protein expression among the Tg-I79N lines (52%), and the
Ca2+ sensitivity change was only half of that observed for
line 9 (Fig. 7A) expressing
35% of HCTnT-I79N (Fig. 2).
The effect of chronic exercise on the Ca2+ sensitivity of
force in the two-pH conditions was examined for Tg-I79N line 9 compared
with Tg-WT (Fig. 7B). As shown, exercise did not affect the
level of change in Ca2+ sensitivity seen in the sedentary
mice.
|
Intact Fibers
The second set of experiments was performed on fresh (not
glycerinated) fibers whose diameter was between ~50 and 70 µm.
These freshly isolated fibers, skinned with Triton X-100, had a lower Ca2+ sensitivity of force development than the glycerinated
ones and had an even greater difference in Ca2+ sensitivity
(pCa50
0.44) when comparing the Tg-WT fibers with fibers from the Tg-I79N mice (Fig. 8).
Note that the force was also lower in the mutant mice compared with the
wild type (Fig. 8B). The same was true for the ATPase
measurement (Fig. 8A). The difference in Ca2+
sensitivity of the ATPase activity between Tg-WT and the Tg-I79N mice
was
pCa50
0.38. When the ATPase activity was
measured simultaneously with force, it was also shifted leftward.
Results of the ATPase and force measurements on intact fibers were very reproducible among the non-Tg (n = 16), Tg-WT
(n = 10), and Tg-I79N (n = 13) lines
with very little variation between individual measurements.
|
To assess the impact of changes incurred in force regulation in
cardiac skinned fibers by HCTnT-I79N, we used a quantitative computer
model that integrates Ca2+ binding to various buffers in
cardiac cells to predict the amplitude and time course of the
intracellular Ca2+ transient, of Ca2+ bound to
various Ca2+ buffers (TnC, calmodulin, sarcoplasmic
reticulum uptake), and of force in intact cardiac fibers (56, 57).
Table I illustrates the steps to derive
the values of koff(TnC·Ca), f, and g that would fit both the force-pCa relation and the
DM-nitrophen flash photolysis force transient in skinned cardiac
fibers. A decrease in koff(TnC·Ca) causes a
leftward shift in the force-pCa relationship with only a minute change
in peak force and of k, the rate constant of force
development after a pCa step from pCa 6.2 to 4.5. An increase in
g proved necessary to decrease peak force. The increase in
g also decreased the pCa50 left shift (from
0.611 to 0.471) and increased k to values seen
experimentally. Further fine tuning of
koff(TnC·Ca) gave values of k,
pCa50, and peak force that very closely fit our
experimental observations (see activation rates and Fig.
9). To determine whether this combination of values is unique or not, we attempted to produce the same results by
altering only f and g with no changes in
koff(TnC·Ca) (lower half of Table I). An
increase in f causes a leftward shift in pCa50,
an increase in k, but an increased peak force.
Increasing g to compensate for this balanced out the effects
of an increased f on
pCa50 and peak force. A
further increase in g caused a rightward shift of the
pCa50 and increased k way beyond values seen
experimentally. To cause a leftward shift in pCa50,
koff(TnC·Ca) was decreased (from 300 to 88 s
1), and this combination resulted in an excellent fit
for
pCa50 and peak force, yet k was almost
double the value seen experimentally. This is not surprising because in
skinned and intact fibers increases of f and/or g
increased k, the rate of force redevelopment after a quick
release-stretch cycle, ktr = f + g
(58). It therefore seems necessary to limit the combined values of
f and g not to exceed certain values to limit the
value of k. Furthermore, because f and
g effects on
pCa50 and peak force cancel each
other, it appeared unnecessary to change both f and
g. A change of g from 10 to 20 s
1
in addition to the aforementioned decrease in
koff(TnC·Ca) was sufficient to reproduce the
experimental results. In summary, a decrease in
koff(TnC·Ca) from 300 to 88 s
1
combined with an increase in g from 10 to 20 s
1 provided for an excellent fit to the experimentally
observed data and reproduced the DM-nitrophen flash photolysis force
transient and force-pCa relationship for both wild type and TnT-I79N.
Fig. 9A shows the results of the simulation of the
DM-nitrophen flash photolysis-induced force transient for both Tg-WT
and Tg-I79N. The rate constants of force development are very close to
those observed experimentally (WT 18.39 s
1 simulated
versus 18.0 s
1 observed; I79N 29.68 s
1 simulated versus 29.8 s
1
observed). Fig. 9B shows the simulation of a flash
photolysis experiment with Diazo-2. I79N myocardium relaxes faster than
WT with rate constants of 10.7 s
1 in the Tg-WT and 21.2 s
1 in the Tg-I79N myocardium. These values are of the
same order of magnitude as those seen experimentally (28.8 s
1 WT and 33.5 s
1 I79N). Although the
absolute values of the rate constant of relaxation of force were not
exactly reproduced, relaxation of I79N myocardium after a Diazo-2 flash
was faster both experimentally and with the current simulation
settings. Fig. 9C shows the force-pCa relation for both WT
and TnT-I79N mutant. By comparing these figures with the experimental
observations (Fig. 8B), it is obvious that the variables
used in the simulation reproduce the data of the skinned fibers quite
well. We also attempted to predict the behavior of intact ventricular
myocardium afflicted with the HCTnT-I79N mutation by using the same
variables of koff(TnC·Ca), f and
g and simulated the intracellular Ca2+ transient
and force in a continuous series of twitch contractions in steady state
conditions. Fig. 9D shows that the changes in koff(TnC·Ca) and g derived earlier
from skinned fibers data cause a smaller peak and slower decline of the
intracellular free Ca2+ transient (top panel),
an increased peak force, a delayed relaxation of force and an increased
rate of contraction (middle panel). To validly compare the
time course of isometric relaxation in WT and I79N, we normalized the
force traces to eliminate the well known effects of twitch amplitude on
relaxation time course. The bottom panel of Fig.
9D clearly demonstrates that isometric relaxation in I79N
myocardium is slower than in WT. If heart rate is not allowed to
change, the residual force at the end of one twitch is present at the
beginning of the next, and an increased end-diastolic force ensues.
|
|
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DISCUSSION |
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This study describes the first transgenic animal model for the
TnT-I79N mutation. We have examined three transgenic lines with high
levels of cardiac muscle specific expression of human cardiac TnT under
the control of the murine -myosin heavy chain promoter. There was no
obvious correlation between transgene copy number, mRNA expression
level, or endogenous TnT replacement with Tg-WT and both lines of
HCTnT-I79N. However, a correlation between the mRNA expression
level and endogenous TnT replacement was observed. The overall
stoichiometric ratio of all sarcomeric proteins in the total cardiac
extract was well preserved in all transgenic lines studied.
Interestingly, the animals tolerated exercise quite well, and there
were no deaths or any visible exercise-induced hypertrophy in the mice.
The heart weight to body weight ratio was slightly decreased in the
Tg-I79N animals compared with Tg-WT mice but not significantly
different from the non-Tg animals. These results are consistent with
the clinical data where no overall cardiac hypertrophy, increase in
maximal left ventricular wall thickness or collagen deposits were
associated with the TnT-I79N mutation in humans (10).
The skinned fiber experiments demonstrated that once mutant HCTnT-I79N was incorporated into the thin filaments of murine hearts, it caused several functional abnormalities compared with transgenic HCTnT-WT or nontransgenic muscle. The major finding of this report is that expression of mutant human TnT-I79N in mice increased Ca2+ sensitivity of the ATPase activity and force development in cardiac myofilaments. They were both shifted toward lower Ca2+ concentrations in the Tg-I79N mice. It should be mentioned that the pCa50 values of force development in the glycerinated thicker fibers were somewhat higher than those in the smaller diameter freshly skinned fibers. This could be due to various factors determining force-pCa dependence in these two different preparations, such as the fiber size and/or the glycerinating process. Moreover, the force-pCa dependence in the glycerinated fibers was determined in the pCa solutions whose free Ca2+ concentration was calculated using a computer program (59), whereas the simultaneous force and ATPase measurements in fresh (not glycerinated) skinned fibers utilized direct free Ca2+ determination with a fluorescent calcium indicator (42, 43). In any event, both methods demonstrated increased Ca2+ sensitivity in the Tg-I79N mice.
Interestingly, our previous experiments with HCTnT-I79N-reconstituted porcine muscle fibers demonstrated a similar increase in Ca2+ sensitivity of force (28). Yet, the effect seen in transgenic skinned HCTnT-I79N mouse fibers was larger than that observed in the reconstituted porcine filaments. Moreover, force/cross-sectional area was much lower for the Tg-I79N fibers versus the Tg-WT in both sedentary and exercised mice. Kinetics of force activation were also altered in the Tg-I79N mice. The rate of activation was about 1.7-fold higher for the Tg-I79N fibers compared with Tg-WT fibers, whereas the relaxation rates were only slightly different. The higher rates of activation in the Tg-I79N mice are in agreement with the results of Sweeney et al. (26), who showed that unloaded shortening velocity in TnT-I79N-transfected quail skeletal muscle myotubes was increased about 2-fold.
Transgenic studies are generally strengthened by inclusion of as many
independent lines as possible. Although only two Tg-I79N lines and one
Tg-WT line were extensively studied in this report, recent preliminary
data from our laboratory suggest the calcium effects are specific to
transgene expression, rather than insertional artifacts or epigenetic
effects. Specifically, administration of propyl-thiouracil, which
induces hypothyroidism and down-regulates the -myosin heavy chain
promoter (60, 61), causes the mutant phenotype to return to normal
(unpublished data). Furthermore, the
-myosin heavy chain promoter has proven to be very reliable in
driving cardiac-specific, developmentally regulated gene expression in
other transgenic systems (33, 34, 60).
These changes in the Ca2+ regulation of the ATPase and force development as well as changes in maximal force and rate of force activation could be critical in understanding abnormalities observed in humans that ultimately lead to catastrophic results and sudden death of individuals carrying the TnT-I79N mutation. One could speculate that this mutation in HCTnT leads to changes in the interactions between TnT and other troponin subunits, TnI and TnC, and/or to changes in their interactions with actin-tropomyosin. This could lead to changes in contractility and possibly affect the Ca2+ affinity of TnC. Because TnC is a major Ca2+ buffer within the muscle cell, changing its Ca2+ affinity would alter overall Ca2+ homeostasis as we observed in our computer simulation. This in turn might trigger numerous Ca2+-dependent cellular processes. Abnormalities seen in the level of force development for the Tg-I79N fibers suggest possible changes in inotropic responses in the working human heart. The increased rate of force activation in the Tg-I79N mice, with more force being produced, could indeed decrease the inotropic reserve, an effect that was observed in vivo in these transgenic HCTnT-I79N mice.2
Intracellular pH drops rapidly after the onset of ischemia in cardiac
muscle and may play some role in the rapid drop in force that ensues
(63-65). A decrease in pH results in the rightward shift of the
Ca2+ dependence of force development toward higher
Ca2+ concentrations. This effect is thought to be an
adaptive as well as protective mechanism of cardiac muscle to changes
in the acidic environment. Cardiac TnI, the inhibitory subunit of the
troponin complex, has been implicated as the Tn subunit responsible for the effect of pH on the Ca2+ sensitivity of contraction
(54, 66, 67). Our experiments suggest that TnT as well as TnI plays a
role in this process. Lowering pH in the physiological range
(~7.0-6.5) shifted the Ca2+ sensitivity of force
development from lower to higher calcium concentrations for non-Tg and
Tg-WT mice by pCa50
0.5. However, the Tg-I79N fibers
had a much smaller change in Ca2+ sensitivity over this
range of pH. Moreover, for the I79N line 8, which had the highest
protein expression among the Tg-I79N lines (52%), the Ca2+
sensitivity was essentially unaffected by this change in pH. Exercising
did not alter these properties. It has been postulated that the
rightward shift of the Ca2+ dependence of force development
toward higher Ca2+ concentrations at higher H+
concentration (lower pH) may result from a decrease in the affinity of
TnC for Ca2+ (55). This lack of Ca2+ response
in the Tg-I79N mice suggests that the interaction between TnT-I79N and TnC prevents TnC from lowering its affinity for
Ca2+ and therefore interferes with the adaptive and
protective mechanism of the muscle cell to function in the acidic
environment that ensues during myocardial ischemia.
To determine the possible impact of the HCTnT-I79N mutation on contraction and relaxation of intact ventricular myocardium, we used a simple mathematical model of intracellular Ca2+ buffering and of force generation based on Huxley's two-state cross-bridge model. The I79N mutation confers an increased Ca2+ sensitivity of force, a decreased peak force, and a faster development of force after a step in activation as achieved during a flash photolysis experiment with DM-nitrophen in skinned cardiac fibers. Relaxation of force during flash photolysis experiments with Diazo-2 was slightly faster in the Tg-I79N than in Tg-WT skinned fibers. Step by step simulation of these changes (Table I and Fig. 9) suggests that the dissociation of Ca2+ from TnC is slowed by the I79N mutation and that cross-bridge detachment is accelerated. These two opposing changes in a unique way modify peak force, pCa50, and the rate constant k of force development to exactly reproduce the experimental observations in skinned cardiac fibers. Therefore, in addition to increased Ca2+ affinity of TnC, it seems likely, based on theoretical considerations and deductive analysis of skinned fiber data, that cross-bridge kinetics are changed by HCTnT-I79N. The observed drop in force/cross-sectional area and the change in cross-bridge kinetics support this analysis. An increased rate of cross-bridge detachment was also inferred by others from their in vitro motility assays (24, 25) or TnT-I79N transfected myotubes (26), and a consistent picture regarding this mutation is beginning to emerge based on results from many different approaches. It also appears that the cross-bridge effects brought about by this mutation are distinct from the calcium effects, and it is possible that the former arise from an alteration in the interactions between TnT, tropomyosin, and F-actin, whereas the latter arise from altered TnT and TnC interactions.
Analysis of numerous simulations show that in intact cardiac fibers, a decrease in koff(TnC·Ca) is invariably accompanied by a change in the time course of the intracellular Ca2+ transient, whereas changes in the cross-bridge kinetics do not perceptibly change the Ca2+ transient. Relaxation of force is slowed the most by a decreased koff(TnC·Ca), whereas this effect is somewhat attenuated by an increase in cross-bridge detachment rate g. The twitch contraction operates from a pCa range of ~7.7 at rest to a peak systolic value of 5.7. Using these pCa values, simulated peak twitch force in steady state conditions in fibers containing Tg-I79N was higher than in fibers that contained Tg-WT (Fig. 9). The model further predicts an increased rate of force development, a slower isometric relaxation, and an increased residual force or resting tension at the onset of the next contraction. If twitch amplitudes in both Tg-WT and Tg-I79N were the same or are normalized, isometric relaxation of Tg-I79N myocardium is slower than in the WT as was observed in isovolumetrically contracting isolated heart.2 The predicted slower isometric relaxation in intact fibers may appear to contradict the relaxation flash photolysis results and simulations, which show a faster relaxation in Tg-I79N than in Tg-WT. However, intact fibers operate in the pCa 7.2-5.5 range, whereas skinned fiber results were obtained at lower pCa values, and intact fibers are driven by a time-varying Ca2+ transient, whereas in flash photolysis pCa changed in steps from one value to another fixed value. Because the pCa step in flash photolysis experiments is virtually instantaneous, the rate-limiting step for relaxation is the detachment of cross-bridges. Yet, in intact cardiac fibers, the slower decline of the intracellular Ca2+ transient (Fig. 9D, upper panel) may be the rate-limiting factor for relaxation and may account for the slower isometric relaxation in I79N myocardium compared with WT.
The higher basal contractile state, the increased rate of contraction, and slower relaxation2 in HCTnT-I79N myocardium carries with it several implications: 1) the contractile reserve would be diminished; 2) an increase in heart rate and/or of contractility, such as after isoproterenol administration, would jeopardize relaxation and lead to further diastolic dysfunction; 3) an increased contractility and heart rate would further increase diastolic [Ca2+]i and cause intracellular Ca2+ overload and dysrhythmias. These predictions seem to hold true for Tg-I79N mice challenged with isoproterenol,2 which demonstrate an impaired inotropic response, relaxation impairment, and fatal dysrhythmias. The simulations did not change the amount and time course of Ca2+ entry and release into the cytoplasm, i.e. myoplasmic Ca2+ delivery was held nearly constant in both WT and I79N myocardium. In intact ventricular myocardium, it is possible that myoplasmic Ca2+ is altered, and if for example transsarcolemmal Ca2+ entry and/or sarcoplasmic Ca2+ release were increased, this would only enhance the diastolic dysfunction in the intact heart.
In summary, this study demonstrates that transgenic expression of
mutant human troponin T (I79N) in mouse hearts significantly alters
contractile function and pH regulation at the myofilament level.
Computer simulation predicts for the Tg-I79N myocardium compared with
Tg-WT: 1) an increase in the apparent Ca2+ affinity of TnC
and an increase in the apparent cross-bridge detachment rate
g and 2) a higher basal contractility, impaired relaxation,
residual resting tension, and vulnerability to inotropic stimulation in
intact ventricular myocardium. It is likely that these mechanisms
contribute to the mortality observed in patients with a TnT-I79N-based
FHC.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants AR-45391 and HL-42325 (to J. D. P.), GM-36365 (to P. R. H.), and AR-40906 (to W. G. K.).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.
To whom correspondence should be addressed: Dept. of Molecular
and Cellular Pharmacology, University of Miami School of Medicine, 1600 N.W. 10th Ave., Miami, FL 33136. Tel.: 305-243-5874; Fax: 305-243-6233; E-mail: jdpotter@miami.edu.
Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M006746200
2 B. C. Knollmann, S. A. Blatt, K. Horton, F de Freitas, T. Miller, M. Bell, P. R. Housmans, N. J. Weissman, M. Morad, and J. D. Potter (2001) J. Biol. Chem., in press.
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ABBREVIATIONS |
---|
The abbreviations used are: TnC, troponin C; TnI, troponin I; TnT, troponin T; FHC, familial hypertrophic cardiomyopathy; CTnT, cardiac TnT; HCTnT, human cardiac TnT; PCR, polymerase chain reaction; Tg, transgenic; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.
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APPENDIX: Simulation of Flash Photolysis, Force-pCa Experiments, and Twitch Force in Intact Fibers |
---|
Mathematical Model of Intracellular Ca2+ Handling and Force Generation
The method consists of the following steps, each of which describe a time-varying function.
Step 1: Approximate a Free Intracellular Ca2+ Transient-- The free [Ca2+]i transient is assumed to have the following time course (49).
![]() |
(Eq. 1) |
Step 2: Calculate Ca2+ Bound to TnC (Ca·T) and to Calmodulin (Ca·C)-- From the law of mass action one obtains the following equations.
![]() |
(Eq. 2) |
![]() |
(Eq. 3) |
Step 3: Cross-bridge Model Values--
For the cross-bridge on and
off kinetics, we modified Huxley's 1957 model (68):
dn/dt = fx (1 n)
g × n, where
n = number of attached cross-rides.
![]() |
(Eq. 4) |
Step 4: Force Dependence of Affinity of TnC for
Ca2+--
The rate of release of Ca2+ from TnC
is slowed by the presence of cross-bridges.
koff(TnC·Ca) therefore becomes smaller as
cross-bridges form and force develops (47, 51, 69). The off rate of
Ca2+ from TnC was made to depend on force as follows.
![]() |
(Eq. 5) |
Step 5: Derivation of Myoplasmic Ca2+ Delivery--
To
simulate [Ca2+]i transients and force for a
variety of conditions in isolation or in combination, such as changes in the apparent affinity of TnC for Ca2+, of cross-bridge
rates f and g, and/or other variables, we need to
obtain the time course and amplitude of Ca2+ delivery into
the cytoplasm, mostly derived from release of Ca2+ from the
SR. This approach would be more valid than to assume a fixed pCa
transient, which in turn will be affected by changes in buffer
variables that one wishes to simulate. We used the deductive procedure
of Baylor et al. (62) to derive SR Ca2+ release
as follows: 1) The total amount of cytoplasmic (free and bound)
Ca2+ is given by the following equation.
![]() |
(Eq. 6) |
![]() |
(Eq. 7) |
![]() |
(Eq. 8) |
4) From Equation 7 it follows that cytoplasmic Ca2+
delivery equals
![]() |
(Eq. 9) |
Step 6: Simulation of [Ca2+]iTransient and Force-- At this stage, all components of the multi-compartment model are characterized including the time course and amplitude of myoplasmic Ca2+ delivery (mainly SR Ca2+ release). One can now change one or more components and assess the resultant changes in the intracellular Ca2+ transient and force generation. Intracellular calcium transients and force signals were simulated in control conditions and after changing one or more rate constants or variables by solving Equations 2-5 and 10 using Runge-Kutta fourth order numerical integration with a 50-µs step size.
![]() |
(Eq. 10) |
![]() |
Derivation of Initial Variables and of Changes Incurred by TnT-I79N Mutation
We used the results from skinned cardiac fiber studies, the flash
photolysis force transients (DM-nitrophen flash from an estimated pCa
6.2 to 4.5, and Diazo-2 flash for a pCa step from pCa 4.9 to 6.2) and
force-pCa relations to estimate the changes in the off rate of
Ca2+ from the Ca2+-specific site of TnC
(koff(TnC·Ca)) and the attachment and
detachment rate constants of actomyosin cross-bridges, f and g. To this effect, we simulated the force-pCa relationship
that would be obtained for several combinations of changes of
koff(TnC·Ca), f and g,
until a simulated force-pCa relationship was obtained that reproduced
the experimental results. Second, we also simulated the force transient
that would be observed during a step change in pCa from 6.2 to 4.5; at
pCa 6.2, already 15-20% of maximal force is developed in skinned
cardiac fibers. The mathematical approach to these two additional types
of simulation is identical to that outlined above for intact cardiac
fibers, with the following modifications: 1) For simulation of
force-pCa relations, all factors related to the sarcoplasmic reticulum
are removed. The intracellular Ca2+ transient becomes a
fixed value and only steady state conditions are taken into account.
The simulations are repeated for each pCa value from 8 to 4 in steps of
0.1 pCa unit, and steady state force was recorded and plotted as a
function of pCa. 2) For simulation of flash photolysis, all factors
related to the SR are removed. Force and Ca2+ buffers are
allowed to reach steady state at one pCa value (typically 6.2 in a
DM-nitrophen experiment), and pCa is suddenly changed to another pCa
(in this example 4.5). The transient change in force was recorded and
fitted to the equation F = Fo + a × (1 e
kt) by nonlinear
regression (Sigmaplot 5.03, SPSS Inc., Chicago, IL); k is
the rate constant of force (F) development,
Fo is force at the initial pCa, and a
is an amplitude factor. The transient change in force in the simulated
Diazo-2 experiment (pCa step from pCa 4.9 to 6.2) was fitted to the
equation F = Fo + a × e
bt, whereby a
is an amplitude factor and b is the rate constant of force decline.
Flash photolysis and force-pCa relations were simulated for a range of values for koff(TnC·Ca), f, and g to reproduce results found in experimental observations in skinned cardiac muscle. We found that a single unique set of values of koff(TnC·Ca), f, and g were able to reproduce the experimental results, and these values were then subsequently used in simulations to predict what the time course and amplitude of the twitch would be in intact cardiac muscle, both Tg-WT and the Tg-I79N mutation.
Prediction of Intracellular Ca2+ Transient and Force in Intact Cardiac Fibers
We simulated the intracellular Ca2+ transient and force during a twitch that would occur in wild type and with the HCTnT-I79N mutation by using the values of koff(TnC·Ca), f, and g found with the simulation of skinned fiber studies described above. This theoretical analysis predicts muscle contraction and relaxation as would be encountered in vivo and provides for a hypothesis that will be tested in the heart of animals transgenic for HCTnT-I79N.
All calculations were programmed in Microsoft Quickbasic 4.0 and
processed on a PC, and the results (saved as ASCII files) were
replotted with SigmaPlot 5.03 (SPSS, Inc., Chicago, IL). Further
development of this model and the analysis of
[Ca2+]i, Ca2+ buffers, and force
generation using this approach are the subject of a separate report.
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