© 2000 American Heart Association, Inc.
Integrative Physiology |
Transgenic Modeling of a Cardiac Troponin I Mutation Linked to Familial Hypertrophic Cardiomyopathy
From the Department of Pediatrics, Division of Molecular Cardiovascular Biology, The Children’s Hospital Research Foundation, Cincinnati, Ohio.
Correspondence to J. Robbins, PhD, Division of Molecular Cardiovascular Biology, 3333 Burnet Ave, Cincinnati, OH 45229-3039. E-mail jeff.robbins{at}chmcc.org
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Abstract |
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Abstract—Multiple mutations in cardiac troponin I (cTnI) have been associated with familial hypertrophic cardiomyopathy. Two mutations are located in the cTnI inhibitory domain, a highly negatively charged region that alternately binds to either actin or troponin C, depending on the intracellular concentration of calcium. This region is critical to the inhibition of actin-myosin crossbridge formation when intracellular calcium is low. We modeled one of the inhibitory domain mutations, arginine145
glycine (TnI146Gly in the
mouse sequence), by cardiac-specific expression of the mutated protein
in transgenic mice. Multiple lines were generated with varying degrees
of expression to establish a dose relationship; the severity of
phenotype could be correlated directly with transgene expression
levels. Transgenic mice overexpressing wild-type cTnI were generated as
controls and analyzed in parallel with the
TnI146Gly animals. The control mice showed
no abnormalities, indicating that the phenotype of
TnI146Gly was not simply an artifact of
transgenesis. In contrast, TnI146Gly mice
showed cardiomyocyte disarray and interstitial fibrosis and suffered
premature death. The functional alterations that seem to be responsible
for the development of cardiac disease include increased skinned fiber
sensitivity to calcium and, at the whole organ level,
hypercontractility with diastolic dysfunction. Severely affected lines
develop a pathology similar to human familial hypertrophic
cardiomyopathy but within a dramatically shortened time frame. These
data establish the causality of this mutation for cardiac disease,
provide an animal model for understanding the resultant pathogenic
structure-function relationships, and highlight the differences in
phenotype severity of the troponin mutations between human and mouse
hearts.
Key Words: hypertrophic cardiomyopathy mouse cardiac troponin I sarcomere
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Introduction |
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Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease of the sarcomere with well over 100 associated mutations in 9 sarcomeric proteins, including cardiac troponin I (cTnI).1 2 cTnI is a small peptide (210 residues) that, together with troponin C and troponin T, forms the troponin complex. In striated muscle, this complex, which is associated with the thin filament, forms the major calcium sensor and, thus is responsible for controlling the thick-thin filament interactions that result in the contractile cycle. Troponin I is bound to cardiac troponin C (cTnC) in an antiparallel fashion such that its amino terminus associates with the carboxyl terminus of cTnC. cTnI differs from the slow skeletal isoform of TnI by a 32 amino acid extension at the cTnI amino terminus. Two serine residues that are substrates for protein kinase A (PKA) lie within this extension.3 4 cTnI is also sensitive to protein kinase C phosphorylation at serines 43 and 45 and threonine 143.5 The effect of cTnI phosphorylation is primarily a downward modulation of cardiac contractility, either by decreased ATPase activity, which is mediated by PKC, or by increased binding of cTnI to the thin filament, mediated by PKA. A truncation of cTnI (cTnI1–193) has been associated with myocardial stunning,6 7 and measuring the serum level of cTnI has become the standard of care in the diagnosis of myocardial injury.8 9 10 11 12
The so-called inhibitory region of cTnI is an 11 amino acid
motif that is evolutionarily conserved
(Figure 1
). This region alternatively binds to either the
thin filament or cTnC, depending on the intracellular concentration of
calcium
([Ca2+]i).
High [Ca2+]i values
promote Ca2+ binding to cTnC, facilitating a
shift of the inhibitory region of cTnI from the thin filament to cTnC.
This produces positional changes in the actin-tropomyosin conformation
and promotes the transition from weakly bound to strongly bound
actin-myosin crossbridges, leading to ATP
hydrolysis.13 During
diastole, when
[Ca2+]i is low, the
inhibitory region of cTnI remains bound to the thin filament,
suppressing the power stroke. Therefore, cTnI is a critical element,
transmitting the systolic and diastolic variations in
[Ca2+]i to the
sarcomere.
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Mutations that can cause FHC have been defined in 3 different domains of cTnI: 2 mutations in the inhibitory domain, 1 in the C-terminal domain, and 4 in the protein’s distal region. Patients with the inhibitory region mutations or one of the distal mutations (L206Q) develop the characteristic ventricular hypertrophy of FHC. In contrast, patients carrying the other cTnI mutations develop hypertrophy only at the cardiac apex.1 2 To begin exploring the functional significance of a single amino acid mutation in the inhibitory region of cTnI, we generated transgenic (TG) mice that express the R145G (R146G in the mouse sequence) mutation. This mutation replaces a positively charged arginine with an uncharged, polar glycine residue. The consequences of the altered structure-function relationships of the cardiac calcium sensor were subsequently determined at the molecular, cellular, and whole organ levels.
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Materials and Methods |
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Transgene Construction
All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee. Three TG constructs were used. The wild-type (wt) cDNA for mouse cTnI was obtained by reverse transcription–polymerase chain reaction using total RNA isolated from mouse cardiac ventricle as template. The 5' primer contained a portion of the cTnI 5' untranslated region (5'-GGGGAGGAGACAGCAGTATATTTAG-3'), whereas the 3' primer was made complementary to the 3' untranslated region (5'-GAGGTTTATTCTGCAGGGACGTCC-3'). The wt cDNA was subcloned into pBluescript and sequenced in full. The TnI146Gly mutation was created using standard polymerase chain reaction-based methods. A third cDNA was generated that included a FLAG tag14 15 inserted at the amino terminus just distal to the translational start site of the wt cDNA. The 3 cDNAs (TnI146Gly, wt, and wt-tagged) were separately subcloned into the mouse
-myosin
heavy chain (-MHC) promoter
(Figure 1
), and the purified DNA was used to generate
multiple lines of TG
mice.16
Phenotype Analyses
Except where noted, 3 to 6 animals of mixed gender
were used for our studies after preliminary experiments showed no
gender differences. For assessment of
cTnI146Gly and cTnI wt transcript size,
Northern blot analysis was performed using 7.5 µg of total RNA, as
described.17
Expression levels were determined by RNA dot blot analysis with a
32P-labeled oligonucleotide
(5'-GCGTGTGGCTCG-GTGGCATAGGCTCGGTAGTTGGCAGAGGAGCGGCGTCGGA
-CAGG-3'). Molecular markers of hypertrophy were examined by
RNA dot blot analysis using 32P-labeled
oligonucleotides, as
described.18 19 20
Two to three TG and non-TG (NTG) mice from each line were analyzed, and
statistically significant differences were determined by unpaired
Student’s t test, with significance defined as
P
0.05.
Western blot analysis of ventricular myofibrils was performed using a cTnI-specific affinity purified polyclonal antibody (Research Diagnostics, Inc) and a peroxidase-conjugated donkey antigoat IgG secondary antibody (Jackson ImmunoResearch Laboratories). Five micrograms of myofibrils was subjected to SDS-PAGE, transferred to polyvinylidene fluoride membrane (BioRad), and incubated with primary antibody (1:5000) and secondary antibody (1:10000). Immunofluorescence was analyzed with a STORM 820 imager (Molecular Dynamics). The percentage of TG replacement was calculated as FLAG-tagged cTnI/(FLAG-tagged cTnI+endogenous cTnI). The percentage of TG protein replacement was plotted as a function of transcript overexpression.
Structural analyses at the light and electron microscopy levels were performed as described.21 Using 2 to 4 mice of mixed gender, multiple sections were cut and >50 fields were observed. To assess the pattern of the FLAG-tagged cTnI incorporation into the sarcomere, isolated cardiomyocytes22 were examined by confocal microscopy with anti-FLAG M2 monoclonal antibody (Sigma), M.O.M. biotinylated antimouse IgG, and Texas Red Avidin D (Vector Laboratories).
Functional assays included skinned fibers analysis, described in detail elsewhere.23 Skinning was carried out essentially as described by Strang et al,24 who noted previously in these preparations that the myofibril proteins are not phosphorylated. All experiments were performed using a commercially available apparatus (Scientific Instruments). Strip tension (mN/mm2) was determined by dividing force by fiber cross-sectional area (calculated from widths measured at the major axis). Isolated working heart function was also determined, as previously described.25
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Results |
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Generation of Mice Carrying Troponin I Transgenes
Three constructs were used to generate multiple lines of TG mice (Figure 1
). To rule out any phenotype attributable to
overexpression of TnI, lines carrying the wt cDNA were made, and one
was selected for analysis (line 52) whose cTnI RNA level exceeded that
of the cTnI146Gly lines used. A total of 4
lines carrying the TnI146Gly transgene were
analyzed. Two of these, lines 135 and 141, could not be studied in
detail because of the paucity and early death of TG offspring. In fact,
the founders died prematurely and had grossly abnormal hearts (data not
shown). The remaining 2 lines (121 and 133) were selected for analysis.
RNA overexpression levels for lines 121 and 133 were 1.2-fold and
3.5-fold higher, respectively, compared with endogenous cTnI RNA. Line
52 expressed the wt transcript at 5-fold the endogenous level. In
previous experiments using the -MHC promoter to drive expression of
TG contractile transcripts, levels of transgene expression varied by 1-
to
15-fold16 18 23 24 25
and sometimes
more.21 The
relatively low and narrow range of TG expression in the
cTnI146Gly lines suggested that potential
founders with high TG expression may not have survived to birth.
Supporting this hypothesis, mice from the highest expressing line, 133
(with 3.5-fold greater expression relative to the endogenous
transcript), died between 13 and 17 days after birth and had markedly
abnormal hearts.
Despite a relatively narrow range of expression, differences
in morbidity and mortality presented. Line 121 had no overt phenotype
in early adulthood, although females stressed by pregnancy did develop
cardiac pathology (see below). However, life expectancy in this line
did not differ appreciably from normal animals
(Figure 2). Expression of wt cTnI at 5-fold the level of the
endogenous message (line 52) resulted in a cohort of mice with normal
survival
(Figure 2A). Breeding line 121 TG mice to one another doubled
transgene expression to 2.4-fold and resulted in death of the animals
carrying a double dose of the transgene at 18 to 26 days. Transgenic
mice from line 133, with 3.5-fold overexpression at the transcript
level, did not survive past 17 days. Although some aspects of a
hypertrophic response were observed at the molecular level, no
increases in the ventricle or body weights were detected in the adult
cTnI146Gly animals
(Figure 2B).
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RNA and Protein Expression in TG
Animals
We wished to determine whether animals displaying early
lethality showed any abnormalities in RNA expression in the heart,
reminiscent of the altered patterns that have been observed in other
models of cardiac
disease.20 At the
RNA level, line-133 mice at 10 days after birth (
3 to 7 days before
death) exhibited aspects of the expression pattern observed in adult
hearts undergoing a hypertrophic response at the molecular level.
Relative to NTG animals, atrial natriuretic factor and -skeletal
actin transcripts were elevated, whereas SERCA2 and
phospholamban transcripts were decreased
(Figure 3). Line 121 and the wt cTnI-expressing line 52 were
analyzed at 4 to 5 months of age. Line 121 only showed upregulation of
atrial natriuretic factor, consistent with the milder phenotype,
whereas the animals that expressed the wt TnI transgene did not differ
from NTG animals.
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Line 133 was analyzed at 10 days after birth because of early death
in this line, whereas lines 52 and 121 were analyzed at 3 to 4 months
after birth.
The changes observed in contractile protein isoform
expression, although striking, were not consistent with the resultant
mortality. We wished to see if any major changes in the contractile
apparatus had occurred as a result of
cTnI146Gly expression. Extensive experience
has shown that robust TG expression of a sarcomeric protein does not
affect myofibril
stoichiometry.16 18 21 23 24 25
This finding was confirmed in the TnI mice, with no alteration in the
absolute amounts of cTnI
(Figure 4A) or contractile protein stoichiometry in any of
the animals studied (data not shown). No changes in the MHC isoforms
were observed, consistent with the lack of a full hypertrophic response
(Figures 2 and 3). Attempts to separate
cTnI146Gly from the endogenous protein were
unsuccessful, prohibiting the direct determination of mutant cTnI
incorporation into the sarcomere. To correlate the amount of TG protein
substitution with the degree of overexpression at the transcript level,
an additional construct, wt cTnI with an epitope FLAG tag
(Figure 1), was used to generate TG mice. Previous
experiments in which sarcomeric proteins were expressed showed a linear
relationship between the level of TG RNA expression and the degree of
protein
replacement.18 Thus,
by comparing the level of TG overexpression determined by RNA dot blot
analysis to the percentage of replacement of endogenous cTnI by tagged
cTnI (determined by Western blotting), we were able to generate an
equation describing the relationship of RNA expression to TG protein
replacement
(Figures 4B and 4C). No unincorporated FLAG-tagged cTnI was
detected by confocal microscopy
(Figure 4D), indicating that Western blot analysis of
myofibrillar protein accumulation accurately represents the amount of
TG protein present in cardiomyocytes. Assuming that FLAG-tagged cTnI,
cTnI146Gly, and cTnI(wt) incorporate into
myofibrils with equal efficiency, we estimated that the cTnI complement
of line 133, with the highest level of
cTnI146Gly TG overexpression, consisted of
50% of mutant protein, whereas lines 121 and 52 had 40% and 60%
replacement, respectively, of endogenous cTnI with TG cTnI.
(Figure 4C).
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, FLAG-tagged cTnI replacement levels. 
,
interpolated cTnI146Gly replacement
levels.
At 18 days of gestation, the TG and NTG ventricles of the
highest expressor, line 133, were indistinguishable from one another, a
result consistent with the lack of TG expression in the ventricle at
this developmental stage
(Figure 5A). However, by 10 days after birth the ventricles
demonstrated cardiomyocyte disarray, interstitial and perivascular
fibrosis, and nuclear degeneration
(Figure 5B). This line could not be examined at later stages
because of death at 13 to 17 days. Male and nulliparous female TG mice
from the lower-expressing line 121, had no discernible pathology at any
age (data not shown). However, 13-month postpartum females showed
cardiomyocyte hypertrophy as well as patchy areas of interstitial and
perivascular fibrosis
(Figure 5C). Line-52 TG mice (wt overexpressors) were
indistinguishable from NTG littermates (data not shown). Thus, the
effects of cTnI146Gly on the myocardium seem
to be dose-dependent such that the highest expressing line showed
accelerated pathological changes and a decreased life expectancy,
whereas line 121 demonstrated abnormalities only after the
physiological stress of pregnancy.
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Ultrastructural changes in lines 121, 133, and 52 were
documented
(Figure 6). In comparison with age-matched NTG littermates
(Figure 6A), 9-day-old line-133 TG mice had abnormally short
sarcomeres (1 to 1.5 µm), with wide Z bands and no visible I bands
(Figure 6B). Sarcomeric organization was disturbed in many
areas and completely lacking at isolated foci. The sarcoplasmic
reticulum (SR) was swollen and disorganized, with disruption of the
normal spatial relationship between the sarcomeres and SR. The
mitochondrial ultrastructure and distribution was likewise abnormal. A
representative field from a line-121 parous female revealed features of
hypoxia in some cardiomyocytes, as evidenced by swollen mitochondria
and large gaps between the SR, mitochondria, and sarcomeres. The
interstitium contained small amounts of collagen fibers
(Figure 6C). Line 52 could not be distinguished from NTG
littermates (data not shown).
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We hypothesized that cTnI146Gly
expression would have direct effects on both fiber mechanics and
kinetics. The pCa-force relationship was determined using
dephosphorylated skinned papillary muscle strips from
cTnI146Gly line 133 at 10 days after birth.
A significant increase in Ca2+ sensitivity
compared with NTG littermate controls was observed, and maximum tension
(P0) was significantly depressed
(Figure 7A). There were no differences detected in unloaded
shortening velocity, maximum shortening velocity, or maximum relative
power (data not shown). We also studied the less affected animals, line
121, at 6 weeks of age and again found increased
Ca2+ sensitivity and significantly decreased
P0
(Figure 7B). As with line 133, line 121 did not have
alterations in unloaded shortening velocity, maximum shortening
velocity, or maximum relative power (data not shown). Line 52 was
indistinguishable from NTG animals
(Figure 7B).
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To examine the consequences of
cTnI146Gly on whole heart function, lines
121 and 52 were studied using the isolated working heart
preparation18
(Table).
Line 133 could not be analyzed because of early death. Line 52 did not
differ significantly from NTG littermate controls. However, the
striking differences in both +dP/dt and -dP/dt in line 121 was
surprising given the otherwise mild manifestations of cardiac pathology
at the molecular and microscopic levels. Contractility was
significantly enhanced, whereas relaxation was impaired. The time
constant of relaxation, 
, a load-independent measure of diastolic
function, was markedly prolonged in TG mice from line 121. Thus,
expression of cTnI146Gly in the mouse heart
results in enhanced systolic function but compromised diastolic
function.
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Discussion |
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There are few data concerning the relative morbidity and mortality of the cTnI mutations in humans. Three of the 7 mutations (R145G, R145Q, and L206Q) resulted in a typical hypertrophic cardiomyopathy, 1 (K183del) caused apical hypertrophy in one family member and typical hypertrophy in another, and 3 (R162W, S199N, and G203S) caused apical hypertrophy resulting in a spade-shaped ventricle.1 2 In 9 family members carrying S199N, 2 experienced sudden death in the sixth decade, 6 others had varying symptoms (arrhythmia or left ventricular dysfunction), and 1 was asymptomatic at 21 years of age.2
Our work describes the effects of the
cTnI146Gly mutation in mice. Consistent with
the presentation of many of the pathological features of typical FHC,
the mice exhibited myofibrillar disarray, and interstitial fibrosis;
some of the molecular markers of cardiac hypertrophy are activated
although no overt hypertrophy, as measured by increases in ventricle
mass/body mass were observed. At the whole organ level, contractile
function is enhanced but relaxation is compromised. Because cTnI binds
to both cTnC and actin, it seems likely that the primary etiology of
cardiac dysfunction in the cTnI146Gly mice
lies in altered cTnI interactions with cTnC, actin, or both. Certainly,
different cTnI isoforms can modulate the effective cross bridge
detachment rate.26
The binding of the inhibitory domain of cTnI to actin inhibits
actomyosin ATPase activity, depresses cross-bridge cycling, and
prevents contraction. The inhibition is released as cTnI binding shifts
from actin to cTnC, an action favored by an increase in
[Ca2+]i during
systole, when Ca2+ binds to cTnC. This
alters the tertiary structure of cTnC and exposes a hydrophobic pocket
in the amino terminus of protein. It is here that the inhibitory region
of cTnI binds, allowing movement of tropomyosin on actin, exposing the
myosin binding site, and promoting contraction. Augmented contractility
and depressed relaxation in the working heart are consistent with in
vitro data obtained using purified, recombinant human cTnI. The
decreases observed in maximal tension, although inherently
contradictory with the hypothesis that the switch is in the "on"
position, may well be accounted for by the myocyte dropout that we
observed in these animals
(Figure 5 and data not shown). Functional abnormalities,
which included a reduced ability to inhibit the
actin-tropomyosin–activated ATPase as well as an increase in
Ca2+
sensitivity,27
suggest that cTnI146Gly either does not
interact appropriately with actin or perhaps binds strongly to cTnC.
The net effect would be lack of ATPase inhibition, in essence leaving
the molecular switch in the "on" position. Decreased ATPase
inhibition attributable to the R145G mutation supports this
hypothesis.27 28
This model shows an accelerated disease course, with
very early death noted at 13 to 17 days after birth in mice having
50% TG cTnI replacement. The original report of the R145G mutation
provides scant clinical information regarding patient age at onset of
symptoms, severity of hypertrophy, or life expectancy but did not
emphasize a particularly malignant
course.1 This raises
the question of why mice expressing this mutation seem to be more
susceptible to cardiac dysfunction than humans. The answer may lie in
the intrinsic chronotropic demands placed on the mouse heart, which
must rapidly cycle through systole and diastole. The molecular switch
between contraction and relaxation depends on precise molecular
cooperation between thin and thick filament proteins, with the troponin
complex serving as a molecular clutch. Intuitively, it would seem
reasonable that as the heart cycles through systole and diastole at
increasingly rapid rates, the margin of error for compromised protein
interactions becomes ever smaller. The pathological outcome of an
abnormal molecular switch would thus be more pronounced as intrinsic
heart rate increases, with the most severe effects occurring at the
shortest cycle lengths.
Sensitivity of the mouse heart to abnormal troponin proteins
has been demonstrated in several experimental models. Targeted ablation
of cTnI was temporarily compensated by expression of the neonatal (slow
skeletal) isoform of
TnI.29 However, slow
skeletal TnI expression gradually ceased, and the mice died at 18 days
of age secondary to heart failure. Transgenic expression of a truncated
cTnI associated with human myocardial stunning resulted in diminished
contractility, decreased sensitivity to calcium, and ventricular
enlargement.30
Transgenic mice have also been used to model FHC-associated mutations
in cardiac troponin T (cTnT). Human patients develop little or no
ventricular hypertrophy yet have a high incidence of sudden death. When
a cTnT mutation was transgenically expressed in mice, animals with
<5% replacement of endogenous cTnT with mutated cTnT developed
diastolic dysfunction, decreased ventricular mass, and myocyte disarray
but had normal life spans. When these mice were bred to homozygosity,
yielding animals that had 10% replacement, they uniformly died
within 24 hours after birth, demonstrating that only small amounts of
truncated cTnT could be tolerated by the mouse
heart.31
The mouse seems to be preferentially sensitive to
several troponin mutations. Whether the
TnI146Gly mutation will be as virulent in a
larger animal remains to be determined. Differential pathologies
between mouse and human hypertrophic cardiomyopathy have been
documented in other sarcomeric gene mutations. Geisterfer-Lowrance et
al32 modeled the
human ß-MHCR403Q mutation in the mouse by
making the corresponding mutation in the mouse -MHC via gene
targeting. Although the model demonstrated cardiomyocyte hypertrophy,
interstitial fibrosis, and cardiomyocyte disarray, the
-MHC403/+ mice had depressed cardiac
output, a feature generally not seen in the human disease. Recently, a
transgenic rabbit expressing the
ß-MHCR403Q mutation was
created.33 The
rabbits accurately reproduced human FHC, demonstrating, in addition to
the typical histological features of the disease, increased ventricular
mass, preserved contractile function, and abnormal relaxation. The
differences in phenotype between the mouse and rabbit experiments are
not surprising given the distinctive biochemical properties of fast
-MHC and slow ß-MHC. Our ability to specifically express
transgenes in the rabbit heart has progressed
rapidly,34 and these
mutations can be generated in the larger animals whose contractile
apparatus more closely resembles that of the human. These experiments
are underway.
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Acknowledgments |
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This work was supported by National Institutes of Health Grants HL56370, HL41496, HL56620, HL52318, HL60546, and HL56620, National Institutes of Health Grant HL03769 (to J.J.), and the Marion Merrell-Dow foundation (to J.R.). We thank Lisa Martin for excellent technical assistance.
Received June 21, 2000; revision received August 31, 2000; accepted August 31, 2000.
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