© 1999 American Heart Association, Inc.
Original Contribution |
Cardiac Troponin I Gene Knockout
A Mouse Model of Myocardial Troponin I Deficiency
From the Department of Physiology, University of Wisconsin, Madison, Wis. The current affiliation for Dr Gregg is the Departments of Biochemistry and Ophthalmology, University of Louisville, Louisville, Ky.
Correspondence to Dr Jeffery W. Walker, Department of Physiology, 1300 University Ave, Madison, WI 53706. E-mail jwalker{at}physiology.wisc.edu
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Abstract |
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Abstract—Troponin I is a subunit of the thin filament–associated troponin-tropomyosin complex involved in calcium regulation of skeletal and cardiac muscle contraction. We deleted the cardiac isoform of troponin I by using gene targeting in murine embryonic stem cells to determine the developmental and physiological effects of the absence of this regulatory protein. Mice lacking cardiac troponin I were born healthy, with normal heart and body weight, because a fetal troponin I isoform (identical to slow skeletal troponin I) compensated for the absence of cardiac troponin I. Compensation was only temporary, however, as 15 days after birth slow skeletal troponin I expression began a steady decline, giving rise to a troponin I deficiency. Mice died of acute heart failure on day 18, demonstrating that some form of troponin I is required for normal cardiac function and survival. Ventricular myocytes isolated from these troponin I–depleted hearts displayed shortened sarcomeres and elevated resting tension measured under relaxing conditions and had a reduced myofilament Ca sensitivity under activating conditions. The results show that (1) developmental downregulation of slow skeletal troponin I occurs even in the absence of cardiac troponin I and (2) the resultant troponin I depletion alters specific mechanical properties of myocardium and can lead to a lethal form of acute heart failure.
Key Words: ischemia • heart failure • cardiac development
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Contraction of heart muscle is triggered by calcium binding to the troponin-tropomyosin regulatory complex on cardiac muscle filaments. One protein subunit of this regulatory complex is troponin I (TnI), a 24-kDa phosphoprotein thought to play a central role in communicating calcium binding to activation of the actin filament.1 2 3 Understanding TnI function in the heart is important, because TnI may be modified in certain cardiac-diseased states. Selective proteolysis of TnI has been reported to underlie the pathology of stunned myocardium.4 Moreover, TnI may become depleted in ischemic,5 infarcted,6 7 8 and possibly failing myocardium.9 A cardiac-specific TnI isoform (cTnI) is released from damaged myocardial tissue, providing a clinically useful serum marker for diagnosis of some acute coronary (or ischemic) syndromes.10 Importantly, this TnI loss may not be restricted to necrotic myocardial tissue but may also occur in tissue that survives and attempts to maintain organ function.7 8 9 Interpretation of the effects of TnI loss in animal models of cardiac disease is often complicated by other cellular changes that occur in parallel.5 6 7 8 On the other hand, selective removal of TnI has been reported,11 12 but this approach limits investigation to permeabilized fiber bundles. A more complete understanding of the physiological consequences of myocardial TnI depletion in vivo is needed.
To better understand and control TnI expression in the heart, we knocked out the gene for cTnI in mice. Two main TnI genes are expressed in the mammalian heart under the control of a developmentally regulated program.13 Fetal TnI, which is identical to slow skeletal TnI (ssTnI), is expressed first and predominates throughout embryonic and fetal development. Around embryonic day 10, cTnI begins to be expressed. Soon after birth, cTnI accounts for roughly half of the total thin-filament TnI content, and then cTnI predominates throughout adulthood in most mammals, including humans.13 14 15 Since the mechanism of the fetal to adult TnI isoform switch is not well understood, we considered 2 possible outcomes of deleting the cTnI gene. The fetal ssTnI isoform could compensate, resulting in effective replacement of cTnI with ssTnI in the adult mouse heart. Alternatively, ssTnI could be downregulated as usual during the TnI isoform switch, creating a mammalian model with a myocardial TnI deficiency.
Homologous recombination in embryonic stem (ES) cells was used to delete the entire cTnI gene from the mouse genome. ssTnI was found to compensate for the absence of cTnI, but only temporarily, as ssTnI was eventually downregulated by an as-yet-unidentified mechanism. This provided an opportunity to assess the effects on cardiac function of TnI loss in a system that should be relatively free of other confounding cellular changes known to occur in cardiac-diseased states, such as altered TnI phosphorylation8 16 and defective excitation-contraction coupling.17 The results clearly show that TnI depletion alters both resting and active mechanical properties of ventricular myocytes and causes a lethal phenotype when TnI content drops below a critical threshold. The results also show for the first time that expression of the adult cTnI isoform is not an essential component of the switch that terminates expression of the fetal ssTnI isoform in the developing heart. This mouse strain with its predictable time-dependent loss of TnI should be useful in future investigations to establish the effects of TnI depletion on cardiac function in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gene Targeting
A P1 clone containing the mouse cardiac TnI gene was obtained from a mouse 129 P1 genomic library (Genome Systems, St. Louis, MO). An 11-kb SalI fragment from the P1 clone was isolated, subcloned, and found to contain the entire cardiac TnI gene (Figure 1A
). A 2.5-kb
SalI/BamHI fragment located upstream of exon 1
and a 2.2-kb BamHI fragment located downstream of exon 8
were used as the 5' and 3' homology units, respectively. These
fragments were cloned into a pNT3 vector containing a neomycin
(neo) resistance gene linked to a thymidine kinase promoter
flanked by multiple cloning sites. The vector also contained a herpes
simplex thymidine kinase cassette for negative selection of
nonrecombinant ES cell clones. The resulting targeting vector (Figure 1A) contained a unique SalI restriction site for
linearization of the plasmid before electroporation into ES cells.
Homologous recombination between the targeting vector and the cognate
cTnI locus deleted exons 1 to 8, or the entire cTnI allele (Figure 1A). Colony selection and target clone identification were as
described.18 Targeting vector (25 µg) was
introduced into 5x106 ES cells. Of 256 G418 and
1-(2'-deoxy-2'-fluoro-ß-D-arabinofuranosyl)-5-iodouracil–resistant
clones, 96 were analyzed and 12 were found to be correctly
targeted by Southern blotting with probe 1 and subsequently with probe
2. Three correctly targeted clones were microinjected into C57B1/6J
blastocysts, and blastocysts were implanted in pseudopregnant 129 mice.
Appropriate breeding was used to produce mice either heterozygous or
homozygous for the targeted cTnI allele. Southern blot
analysis was initially used for genotyping (Figure 1B).
Genomic DNA was isolated from tail biopsies using Purgene DNA isolation
(Gentra Systems). Five micrograms of genomic DNA was digested and then
size fractionated on agarose gels, transferred to nylon membranes, and
probed with DNA labeled with 32P by a random
oligonucleotide protocol. Probe 1 detected a 6.5-kb
EcoRI fragment from the wild-type cTnI allele and a
4.8-kb EcoRI fragment from the targeted allele (Figure 1B). A polymerase chain reaction (PCR)–based assay was also
developed for rapid screening. Primers were designed to produce a
630-bp and a 390-bp fragment for the wild-type and targeted
alleles, respectively.
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Immunoblotting
TnI protein levels were determined by Western blotting of
ventricular tissue homogenates after
electrophoresis in 12% SDS-PAGE gels and transfer onto nitrocellulose
paper. An anti-TnI monoclonal antibody (clone 6F9, Advanced
ImmunoChemical Inc) that recognized both mouse cTnI and ssTnI was used
at a dilution of 1:1000. The identities of positive bands were
confirmed by isoform-specific TnI monoclonal antibodies from the same
commercial source (clone 7F4 for cTnI and clone A9 for ssTnI). ssTnI
was further confirmed by comigration with an antibody-positive band
(clones A9 and 6F9) in mouse soleus muscle. Antibodies on
immunoblots were visualized by enhanced chemiluminescence
(ECL). For quantification of TnI content, ECL bands were scanned by
densitometry and compared with a range of pure cTnI on the same blot to
ensure linearity. Protein loads were standardized by a bicinchoninic
acid protein assay before electrophoresis and by quantitative
densitometry of Coomassie blue–stained gels as
described.19
Histology
Care and handling of mice were carried out according to
institutional guidelines approved by the Association for Assessment and
Accreditation of Laboratory Care (AAALAC) International. Mice were
killed by cervical dislocation. For light microscopy, hearts were
quickly excised and immersed in 10% formaldehyde solution at room
temperature. Fixed hearts were sectioned into 50-µm-thick slices,
stained with hematoxylin and eosin, and then viewed under a Nikon
Diaphot inverted microscope equipped with a 5x objective and National
Institutes of Health Image software. Lung tissue was rapidly frozen in
liquid nitrogen after lungs were inflated with air to 
50 mm
hydrostatic pressure. Frozen tissue was sectioned into 5- to
10-µm-thick slices and viewed under the light microscope. For
electron microscopy (EM) analysis, excised hearts were rapidly
immersed in PBS containing 2% (vol/vol)
paraformaldehyde and 2% (vol/vol)
glutaraldehyde. Blocks of tissue 1
mm2 were dissected, embedded in resin, sectioned,
and viewed on a Philips CM120 transmission electron
microscope.
Force Measurements
Excised hearts were depleted of blood by massage in Ringer's
solution, and then ventricular tissue was diced and
homogenized for 3 to 5 s in 5 mL of relaxing solution
using a Polytron homogenizer. Cells were collected by
differential centrifugation on a tabletop
centrifuge and then incubated for 6 minutes at room temperature
in relaxing solution containing 0.3% Triton X-100 and 0.5 mg/mL BSA.
Skinned single myocytes (or small bundles of up to 4 cells) were
attached to a Cambridge model 403 force transducer, and force output
was monitored as described.20 A passive length-tension
relationship was determined in relaxing solution containing no added
calcium (nominally pCa 9, where
pCa-log[Ca2+]). Sarcomere length (SL)
was monitored by video microscopy and varied from 1.7 to 2.7 µm
in 0.2 to 0.4–µm increments. Active tension was recorded at
2.2 µm by transferring the attached myocyte into activating
solution at pCa 4.5 (32 µmol/L free Ca2+)
as described.20 All tension measurements were carried out
at 20°C to 22°C and normalized to myocyte cross-sectional area.
Solutions
The composition of Ringer's solution was as follows (in
mmol/L): NaCl 118, KCl 4.8, HEPES 25 (adjusted to pH 7.4 with NaOH),
KH2PO4 2,
MgCl2 1.2, pyruvate 5, and glucose 11. The
composition of relaxing solution was (in mmol/L): KCl 80, MgATP 4,
MgCl2 7.4 (1 free Mg2+),
EGTA 7, creatine phosphate 11, and imidazole 25 (adjusted to pH 7.0
with KOH). Measurements of maximum active tension were performed with
an activating solution of essentially the same composition as relaxing
solution but containing CaCl2 (added before pH
adjustment) to achieve 32 µmol/L free Ca (pCa 4.5), and KCl was
adjusted to maintain a constant ionic strength of 0.18 mol/L. For
intermediate Ca levels between pCa 4.5 and 9, a mixing table was used
to determine the amount of relaxing and activating solution to achieve
pCa values of 4.8, 5.0, 5.2, 5.5, 5.8, 6.0, and
6.5.20
Statistics
Data are expressed as mean±SEM and statistically
analyzed by both paired and unpaired Student's t
tests. P<0.05 was considered to be a significant
difference. Data fitting was carried out by use of Marquardt's
nonlinear regression method. The quality of fits was taken to be
acceptable when the SEs in the estimated pCa50
and nH values were 
12% and
P<0.005.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cTnI gene was deleted by using homologous recombination in murine ES cells. A vector containing a neomycin resistance cassette was used for positive selection of targeted ES cells, and a thymidine kinase gene was used for negative selection against random insertion (Figure 1
). Mice heterozygous for targeted cTnI (cTnI+/–) were
indistinguishable from wild-type mice in body size, general appearance,
and life span (Table 1). Heterozygote
crosses showed normal fertility (60/64 pairs, 94%) with low neonatal
mortality (8/448, 1.8%) and produced normal litter sizes (5 to 14
pups/litter, mean=7.4±2.6). The genotype distribution of live
neonates was 27:48:25 (wild type:cTnI+/–:cTnI–/–) as determined by
Southern blots (Figure 2, top), a ratio
consistent with mendelian segregation. The absence of the cTnI
protein in cTnI–/– animals was confirmed by immunoblots
with anti-TnI antibodies (Figure 2, bottom). Remarkably,
cTnI–/– animals were also indistinguishable from wild-type animals on
the basis of body weight and heart/body weight ratio measured up to 18
days postnatally (Table 1).
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A time course of protein expression levels showed the expected fetal to
adult TnI isoform switch in wild-type mouse hearts, but the switch was
delayed by 3 days in heterozygotes (cTnI+/–) (Figure 2). In
cTnI–/– mice, ssTnI remained elevated for at least 10 days beyond the
normal time taken for the isoform switch (Figure 2), apparently
compensating for the absence of adult cTnI and accounting for the
normal appearance of cTnI–/– animals. At day 15 after birth, ssTnI
levels began an abrupt decline that continued over the next 3 days,
reaching 36±9% of its original value (Figure 2). During this
decline, there was no evidence of proteolytic fragments of TnI with the
antibodies used. On day 18, dyspnea (difficulty breathing) and lethargy
developed, and cTnI–/– animals died within hours of the onset of
these overt signs. Both the extreme nature and the
consistency of the phenotype were striking, as
>90% of cTnI–/– animals died on day 18 after birth, with the other
10% having died on day 17 (Table 1).
Initiation of a hypertrophic growth program and other forms of remodeling including infiltration and fibrosis are common responses to cardiac insufficiency.21 22 Heart tissue sections prepared from cTnI–/– animals and stained with hematoxylin and eosin revealed neither hypertrophy nor ventricular dilation even in 18-day-old animals with advanced symptoms (not shown). Gomori's trichrome staining for fibrosis also showed no differences between wild-type and cTnI-null hearts (not shown). Examination of ventricular tissue from 17- to 18-day-old cTnI–/– mice by EM showed few changes in muscle ultrastructure. Myofibrillar disarray, fibrosis, and macrophage infiltration were minimal. The lack of obvious indications of remodeling in cTnI–/– mice may be a characteristic of this type of myofilament defect, or more likely it is a reflection of how suddenly TnI loss occurs. Regardless of the correct explanation, these observations suggest that TnI depletion is the principal defect in cTnI–/– hearts and that secondary effects, because of tissue remodeling, contribute little to the pathology. Comparison of wild-type and null cardiac myofibrils by SDS-PAGE with silver staining revealed no obvious differences in protein profiles (other than TnI), but given uncertainties in this analysis, we cannot rule out the possibility that other myofilament proteins were altered as a consequence of TnI depletion.
Analysis of myofibrillar SL in EM sections did reveal 1
significant change in cTnI–/– animals. SL measured from Z-line to
Z-line was greatly reduced in cTnI–/– mice compared with wild-type
littermates (cTnI–/– SL=1.2±0.2 µm, n=10; wild-type
SL=2.1±0.1 µm, n=10) (Figure 3).
Shortening of sarcomeres was not observed in cTnI–/– mice up to day
14 after birth, but developed later, in parallel with the loss of
ssTnI. This shortening of sarcomeres under relaxed conditions indicates
the presence of Ca-independent forces in the direction of normal active
muscle contraction. The nature of this force is currently unclear, but
EM images showed changes in mitochondria including a 47±9%
(P<0.01) increase in numbers and a trend toward larger
individual mitochondria. These observations are consistent with
an increase in O2/ATP consumption, suggesting
that ATP-dependent active forces are responsible for shortened
sarcomeres in Figure 3.
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Direct functional measurements in isolated hearts have thus far been
precluded by the small size of 17- to 18-day-old mouse hearts.
Histological analysis of lung tissue revealed
grossly enlarged and congested pulmonary capillaries (not
shown), consistent with left ventricular failure.
To determine the functional defects at the cellular level, cardiac
myocytes were isolated and attached to a force transducer after the
surface membrane was removed by detergent skinning.20 In
myocytes from 17-day-old cTnI–/– mice, isometric tension measured
under relaxing conditions was significantly elevated compared with
wild-type myocytes at all SLs examined (Figure 4A). Resting tension was not elevated in
14-day-old cTnI–/– myocytes (Figure 4A), so the increased
resting tension at 17 days cannot be attributed to the presence of
ssTnI rather than cTnI, but it is clearly the result of TnI depletion.
These steady-state force measurements directly demonstrate the presence
of a force responsible for the shortened sarcomeres observed in
ventricular tissue sections.
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) and day 17 (
) after birth. 
, Data from
17-day-old wild-type myocytes. Data are mean±SEM.
#P<0.02; *P<0.005. Solid lines are fits
to
f(x)=A*exp[B*(x–xo)/xo)]
(Reference 34), with A=0.79, B=2.92, and
xo=1.52 µm (
Finally, the effects of TnI depletion on Ca-activated tension
were examined. To assess the effects of TnI loss, tension-pCa curves
were compared in myocytes from 14-day-old and 17-day-old cTnI–/– mice
(Figure 4B). During this 3-day period, TnI content was reduced
from its maximum level to <40% of maximum. This loss of TnI did not
greatly affect maximum tension (Po) because, although
Po increased somewhat, a similar increase
was observed in wild-type myocytes over this same time period (Table 2). The largest effect of TnI loss was on
the pCa50 value, which decreased from pCa 5.70 to
5.36 (Figure 4B). It is not likely that developmental changes in
other myofilament proteins contributed to this time-dependent change in
pCa50, because shifts in the tension-pCa
relationship were considerably less in wild-type myocytes (Table 2). Thus, TnI depletion is associated with a decrease in the Ca
sensitivity of tension development in this system.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We deleted the cardiac-specific TnI gene, cTnI, with the goal of manipulating the content or the species of TnI expressed in the mouse heart. Because there are 2 main TnI genes expressed in the mammalian heart, ssTnI and cTnI (located on different chromosomes), it was unclear how ssTnI expression would change in response to deletion of cTnI. In wild-type ventricular tissue, ssTnI and cTnI are reciprocally expressed, giving rise to a time-dependent isoform switch, as follows. ssTnI is expressed first during development and then is gradually replaced by cTnI, beginning at about embryonic day 10 and finishing around postnatal day 10.13 14 15 At one extreme, the ssTnI expression pattern could have been completely unaffected by the absence of cTnI and the heart would be effectively depleted of ssTnI by postnatal day 10. At the other extreme, ssTnI could have remained at a high level of expression throughout development and growth, thereby fully compensating for the absence of cTnI. What we observed was somewhere between these extremes. ssTnI initially compensated for the absence of cTnI, but beginning around day 15 after birth a steady loss of ssTnI occurred, giving rise to a condition of TnI deficiency.
It is clear from the present results that TnI depletion has deleterious effects on the mechanical properties of cardiac muscle, including changes in both resting and active tension. One observed defect was elevation of resting tension, but the nature of this force in the direction of sarcomere shortening is presently unclear. TnI extraction experiments in isolated ventricular trabeculae have shown that active cross-bridges are recruited (even in the absence of Ca) as TnI is removed.11 In the present study, EM sections of cTnI-null ventricular tissue revealed proliferation and enlargement of mitochondria, suggesting increased ATP consumption, consistent with the possibility that the force is due to recruitment of unregulated active cross-bridges as TnI is lost. In this respect the effect of TnI depletion appears to be similar to cTnI extraction in skinned cardiac muscle.11
Another defect in TnI depleted cTnI–/– myocytes was a reduced responsiveness of the regulatory system to Ca during the development of active force. This observation is different from what has been reported in skeletal muscle following extraction of TnI,23 in which an increase in Ca sensitivity was observed. This may be an indication that cardiac and skeletal muscle are fundamentally different in this respect. TnI extraction experiments have been performed in cardiac muscle, but the effects of TnI removal per se on Ca sensitivity of tension have not been presented in detail.11 12 We consider it unlikely that developmental changes in other myofilament proteins occurring between days 14 and 17 are responsible for the observed rightward shift in the tension-pCa relationship, because there was little change in the tension-pCa curve in wild-type myocytes measured in this time window. Moreover, a candidate myofilament protein has not been identified in the literature that both undergoes a significant switch during this brief period and would be able to account for such a large desensitization of the myofilaments to Ca.
The observed changes in myocyte contractility with TnI
loss are important in view of the possibility that TnI is altered in
certain cardiac-diseased states.4 5 6 7 8 In dog
myocardium subjected to coronary occlusion, all 3
troponin subunits were degraded with TnI showing the most marked
change.5 Myofilament Ca sensitivity assessed by
superprecipitation of actomyosin was depressed by this
treatment.5 In rat myocardium subjected to 60
minutes of complete global ischemia, both cTnI and TnT were
reduced by 40% to 50%, whereas other myofilament proteins were
unaffected.6 The myofilament Ca sensitivity of actomyosin
ATPase activity was enhanced in this experimental system,6
but this may be due to binding of cytosolic proteins to the
myofilaments.24 In pig hearts subjected to acute
myocardial infarction, cTnI and TnT levels were reduced by 40% to 80%
in myocardial tissue remote from the infarction zone after 2 months of
remodeling.7 In rat experimental myocardial infarction,
cTnI content was reduced by 53% in myocytes remaining 7 days after
surgery.8 In the latter case, assessment of the mechanical
properties of ventricular myocytes revealed an increase in
resting tension and a decrease in Ca sensitivity of active
tension.8 However, substantial increases in
phosphorylation of cTnI and TnT were also
observed,8 which could contribute to the observed decrease
in myofilament Ca responsiveness.3 16 In the present
study, any influence of TnI phosphorylation by protein
kinase A can be ruled out, because the TnI isoform present in these
hearts is lacking protein kinase A sites. Also, in the present
study there was no evidence of new proteins bound to the myofilaments
or of secondary changes due to fibrosis or tissue remodeling, which can
confound interpretation of mechanical measurements in diseased tissue.
These considerations strengthen our conclusion that TnI depletion by
50% to 60% is responsible for the mechanical defects observed in
cTnI–/– myocytes including as much as a 2-fold increase in resting
tension and a decrease in Ca sensitivity of tension by more than 0.3
pCa units.
Myocardial stunning is another disease state in which TnI may be selectively modified. Evidence has been presented in a rat model of stunned myocardium that Ca-dependent proteolysis of cTnI, but not other myofilament proteins, underlies the pathology.4 Physiological measurements in intact ventricular muscle demonstrated a decreased myofilament Ca responsiveness and altered diastolic tone after stunning.25 A reduction in myofilament Ca sensitivity has also been observed in stunned porcine ventricular myocytes.26 27 It should be recognized, however, that changes in TnI in stunned myocardium may not be analogous to those in the cTnI knockout mice. Proteolysis in stunned rat myocytes produced a polypeptide fragment of TnI4 that presumably remained associated with the myofilaments; therefore, this condition cannot be considered equivalent to TnI depletion. No proteolytic fragments of TnI were detected in the present study or in other studies reporting reduced TnI levels,7 8 but such fragments could have gone undetected by the antibodies used. A different proteolytic fragment of TnI was reported in the rat complete global ischemia model,6 but evidence has been presented that this polypeptide is unrelated to TnI.23 Establishing the mechanism of ssTnI loss in the knockout mice and the relationship of this process to cTnI proteolysis in stunned myocardium and to cTnI loss during ischemic cardiac disease must await further investigation.
At this point, the cTnI knockout mouse should not be considered a model of any specific human cardiac disease. It should nevertheless contribute to our understanding of the consequences of myocardial TnI deficiency and thereby facilitate understanding of certain diseased states. Ischemic heart disease can be associated with diastolic dysfunction and depressed contractile function,7 8 21 22 28 and the results with TnI knockout myocytes are consistent with the notion that TnI modification contributes to the pathology of postischemic heart disease. Specifically, we propose, on the basis of the increase in resting tension observed here, that TnI depletion could contribute to impaired relaxation, increased myocardial stiffness, and altered ventricular filling during diastole. Moreover, the observed reduction in Ca sensitivity of active tension would be expected to depress contractility in surviving regions of the beating heart. Indeed, it seems quite possible that degradation and/or depletion of TnI occurs not only in necrotic myocardial tissue, but also in myocytes that survive and contribute to dynamic contractile function.4 7 8
Current evidence argues against the possibility that TnI depletion is a common defect in heart failure,14 15 16 29 although serum TnI levels can be elevated in severe forms.9 It remains a formal possibility that TnI insufficiency either due to mutation or to altered gene expression could be a factor in some forms of idiopathic cardiomyopathy in humans. The frequency of "severe" TnI mutations in the human population remains to be established, but several point mutations in TnI have recently been shown to be associated with some forms of human hypertrophic cardiomyopathy.30 The line of mice described here may complement transgenic approaches for creating mice with interesting TnI mutations (eg, by eliminating complications arising from endogenous cTnI expression). Moreover, the striking phenotype of the cTnI-null mice, including the sudden onset and highly predictable time of death, can be exploited as a bioassay for optimizing delivery of TnI genes, TnI peptides, or peptidomimetics into myocardium with potentially broad implications for treating cardiac dysfunction.
Targeted ablation of the cTnI gene has shed light on the mechanism of the TnI isoform switch by showing that the appearance of cTnI in the developmental program influences ssTnI expression but is not required for the disappearance of ssTnI from the heart. These observations suggest that cardiac expression of the ssTnI gene is "set" in the developmental program to switch off, and it does so even in the absence of the adult cTnI isoform. The time course of the TnI isoform switch was delayed in cTnI+/– heterozygotes and even further delayed in homozygous cTnI–/– mice. These observations can be rationalized by a model in which a competition exists between TnI isoforms for a fixed number of binding sites on the thin filament strand (X.P.H. and J.W.W., unpublished observations, 1998). TnI molecules that do not find a site are presumably rapidly degraded by proteolysis. This simple model provides a straightforward explanation for the apparent gene dosage effect on the time course of the TnI isoform switch in heterozygous cTnI+/– mice; a reduced amount of cTnI protein would be less effective competing with ssTnI, resulting in a delayed transition point during the isoform switch. It also offers an explanation for the apparent compensation by ssTnI. In this case, the absence of cTnI prevents it from displacing ssTnI from filament sites. Eventually, however, ssTnI expression ceases, and when normal turnover reduces the filament TnI content, a condition of TnI depletion prevails.
It is remarkable in the case of these cTnI mutant mice that apparent ssTnI gene inactivation occurs regardless of the consequences to the animals in terms of survival. This lack of plasticity in fetal TnI expression in the juvenile mouse heart is reminiscent of the observation in failing hearts that the fetal TnI gene, unlike many other contractile proteins,31 32 fails to become reactivated during the end stages of heart failure.14 15 16 29 Taken together, these observations indicate that ssTnI expression in the mammalian myocardium is developmentally programmed to switch off and remain off. Mechanisms responsible for this inactivation of ssTnI expression are currently unclear.
It is worth noting that differences in the behavior of the
thin-filament regulatory system due to the presence of ssTnI versus
cTnI are apparent in the data. At 14 days after birth, when wild-type
myocytes have nearly a full complement of cTnI and null myocytes have
nearly a full complement of ssTnI, a difference of 0.5 pCa units was
observed in pCa50 values (Table 2). Thus,
myocytes expressing primarily ssTnI displayed an enhanced Ca
sensitivity compared with myocytes expressing primarily cTnI,
consistent with previous reports.1 33 This
supports the suggestion that TnI isoforms are responsible for a large
part of the difference in the Ca responsiveness of neonatal versus
adult cardiac muscle.1 33
In summary, the consequences of targeted ablation of the cTnI gene include partial compensation by altered expression of ssTnI, followed by a state of TnI depletion when ssTnI expression declines. Changes in physiological properties that develop in parallel with TnI depletion include elevated Ca-independent force, suggesting impaired cardiomyocyte relaxation, a substantial reduction in the Ca sensitivity of force development, and ultimately a lethal phenotype due to acute heart failure. Because loss of ssTnI is rapid and there is little evidence of fibrosis, infiltration, or hypertrophy, we conclude that these changes in cardiac function are the result of TnI depletion. The cTnI knockout mice represent an important model system for further investigation into the mechanisms and consequences of TnI depletion and for controlling the molecular species of TnI to elucidate its role in cardiac function and in the etiology of cardiac disease.
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Acknowledgments |
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This work was supported by National Institutes of Health grant P01 HL-47053 (to R.G.G., P.A.P., and J.W.W.). Y.Q.P. was supported by a postdoctoral fellowship from the American Heart Association, Wisconsin Affiliate, Inc. K.J.L. was supported by a Scientist Development Grant from the American Heart Association, Inc. J.W.W. was supported by a National Institutes of Health Research Career Development Award (K04 HL-03119). The authors wish to thank Drs Gary Lyons, Matthew Wolff, Timothy Kamp, Marion Greaser, and Richard Moss for helpful discussions and comments on an earlier version of this manuscript. The technical assistance of Kara Kemnitz (PCR), Inge Sigglekow (heart tissue sections), and Dr Robert Conhaim (lung tissue sections and analysis) is gratefully acknowledged.
Received July 14, 1998; accepted October 22, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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