Deletion of a Genomic Segment Containing the Cardiac Troponin I Gene Knocks Down Expression of the Slow Troponin T Gene and Impairs Fatigue Tolerance of Diaphragm Muscle*


Deletion of a Genomic Segment Containing the Cardiac
Troponin I Gene Knocks Down Expression of the Slow
Troponin T Gene and Impairs Fatigue Tolerance of
Diaphragm Muscle*
Received for publication, May 14, 2009, and in revised form, September 2, 2009 Published, JBC Papers in Press, September 21, 2009, DOI 10.1074/jbc.M109.020826

Han-Zhong Feng, Bin Wei, and Jian-Ping Jin1

From the Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201

The loss of slow skeletal muscle troponin T (TnT) results in a
recessive nemaline myopathy in the Amish featured with lethal
respiratory failure. The genes encoding slow TnT and cardiac tro-
ponin I (TnI) are closely linked. Ex vivo promoter analysis sug-
gested that the 5�-enhancer region of the slow TnT gene overlaps
with the structure of the upstream cardiac TnI gene. Using trans-
genic expression of exogenous cardiac TnI to rescue the postnatal
lethality of a mouse line in which the entire cardiac TnI gene was
deleted, we investigated the effect of enhancer deletion on slow
TnTgeneexpressioninvivoandfunctionalconsequences.Thelev-
els of slow TnT mRNA and protein were significantly reduced in
the diaphragm muscle of adult double transgenic mice. The slow
TnT-deficient (ssTnT-KD) diaphragm muscle exhibited atrophy
and decreased ratios of slow versus fast isoforms of TnT, TnI, and
myosin. Consistent with the changes toward more fast myofila-
mentcontents,ssTnT-KDdiaphragmmusclerequiredstimulation
at higher frequency for optimal tetanic force production. The
ssTnT-KD diaphragm muscle also exhibited significantly reduced
fatigue tolerance, showing faster and more declines of force with
slower and less recovery from fatigue as compared with the wild
type controls. The natural switch to more slow fiber contents dur-
ing aging was partially blunted in the ssTnT-KD skeletal muscle.
The data demonstrated a critical role of slow TnT in diaphragm
function and in the pathogenesis and pathophysiology of Amish
nemaline myopathy.

Troponin T (TnT)2 is a subunit of the troponin complex in
striated muscles. Through interactions with troponin C, tropo-
nin I (TnI), and tropomyosin, TnT anchors the troponin com-
plex to actin thin filament and plays a central role in the Ca2�
regulation of muscle contraction (1). Three TnT genes are pres-
ent in the vertebrates: cardiac TnT, fast skeletal muscle TnT,
and slow skeletal muscle TnT (ssTnT). They encode three mus-
cle fiber type-specific TnT isoforms with differentiated expres-
sion and function (2).

A nonsense mutation at codon Glu180 in the ssTnT gene
(TNNT1) causes a postnatal lethal form of inherited nemaline
myopathy in the Amish (3). This mutation prematurely termi-
nates the ssTnT polypeptide, and the C-terminally truncated
ssTnT-(1–179) has a much decreased binding affinity for tro-
pomyosin and fails to incorporate into the myofilaments (4). As
a result, the truncated ssTnT protein was degraded and unde-
tectable in the muscle of Amish nemaline myopathy patients,
consistent with the recessive phenotype of the disease (5).
Accordingly, the pathogenesis and muscle pathophysiology of
Amish nemaline myopathy is due to the loss of slow TnT.
The genes encoding the muscle type-specific TnI and TnT

isoforms are linked in tandem in three pairs in the vertebrate
genome (2). The cardiac TnI (cTnI) (TNNI3) and ssTnT
genes are very closely linked. Shown in ex vivo promoter
analysis, the 5� upstream enhancer region of the ssTnT gene
overlaps with the structure of the cTnI gene (6). This struc-
tural integration of cTnI and ssTnT genes suggested that a
previously developed mouse line in which the entire Tnni3
gene was deleted through embryonic stem cell gene targeting
(7) would have lost a large portion of the 5�-enhancer region
of the Tnnt1 gene (Fig. 1).

Although the Tnni3-deleted mouse line is a potentially useful
model to study the phenotypes of ssTnT deficiency, a major
hurdle is that the Tnni3-deleted mice die 16–18 days after birth
from heart failure when the fetal expression of slow skeletal
muscle TnI in the heart ceases (7). This postnatal lethality pre-
cluded functional examination on adult skeletal muscle for the
phenotypes of ssTnT deficiency. However, adult skeletal mus-
cle is necessary for investigating the pathophysiology of Amish
nemaline myopathy because the fetal and neonatal expression
of cardiac TnT in skeletal muscles (8) may functionally com-
pensate for the loss of ssTnT in (5).
We previously developed two transgenic mouse lines

overexpressing an N-terminal truncated cTnI (cTnI-ND)
driven by an �-myosin heavy chain (MHC) promoter (9).
cTnI-ND was originally found as a product of restricted pro-
teolysis in cardiac adaptation to simulated microgravity,
which selectively removes the cTnI-specific N-terminal
extension and leaves the conserved structure intact (10).
cTnI-ND transgenic mice exhibit normal base-line life activ-
ity with an increased relaxation of the cardiac muscle (9).
Using this postnatally up-regulated (11) transgene allele
encoding a non-destructive exogenous TnI, we successfully

* This work was supported, in whole or in part, by National Institutes of Health
Grants AR-048816 and HL-078773 (to J.-P. J.).

1 To whom correspondence should be addressed: Dept. of Physiology, Wayne
State University School of Medicine, 540 E. Canfield, Detroit, MI 48201. Tel.:
313-577-1520; Fax: 313-577-5494; E-mail: jjin@med.wayne.edu.

2 The abbreviations used are: TnT, troponin T; TnI, troponin I; cTnI, cardiac TnI;
cTnI-KO, cTnI knockout; cTnI-ND, N-terminal truncated cTnI; mAb, mono-
clonal antibody; MHC, myosin heavy chain; PBS, phosphate-buffered
saline; ssTnT, slow skeletal muscle TnT; ssTnT-KD, ssTnT knockdown.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 46, pp. 31798 –31806, November 13, 2009
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

31798 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 46 • NOVEMBER 13, 2009
This is an Open Access article under the CC BY license.

http://creativecommons.org/licenses/by/4.0/


rescued the postnatal lethality of the Tnni3 gene-deleted
mice (12) to produce adult skeletal muscle for investigating
the functional effects of ssTnT deficiency.

Amish nemaline myopathy is a
rapidly progressive disorder, with
affected children dying primarily
from respiratory insufficiency due
to weakness of the respiratory mus-
cle, usually in the second year of life
(3). These clinical features suggest a
critical role of ssTnT in diaphragm
function. Therefore, we examined
in the present study the phenotypes
of diaphragm muscle of adult dou-
ble transgenic mice. The results
revealed significantly decreased
expression of ssTnT mRNA and
protein. The ssTnT deficiency pro-
duced atrophy and a change toward

more fast fibers in the diaphragm muscle. The slow TnT-defi-
cient diaphragm muscle had significantly decreased tolerance
to fatigue, demonstrating a critical role of ssTnT in respiratory
function.

MATERIALS AND METHODS

Tnni3-deleted/cTnI-ND Double Transgenic Mice—As de-
scribed recently (12), we crossed transgenic mouse line cTnI-
ND#7 (9) with heterozygotes of Tnni3-deleted mice (7), pro-
vided by Dr. Xupei Huang (Florida Atlantic University). The
genotyping of the double transgenic offspring was performed
on genome DNA isolated from tail biopsies using PCR, and the
compensation for the loss of endogenous cTnI by cTnI-ND in
cardiac muscle was confirmed by Western blotting as described
(7, 9) (Fig. 2).
The mice were maintained on a 12-h light/12-h dark cycle

(6:00 a.m./6:00 p.m.) and standard pellet diet. Except for aging
studies, mice at age 3–5 months of both sexes were used for
phenotype characterization. Immediately after euthanasia with
pentobarbital (intraperitoneally, 100 mg/kg), diaphragm mus-
cle strips were dissected from the 9 o’clock and 4 o’clock sectors
with the dorsal side as 12 and 1 o’clock positions (13) for use in
the experiments to avoid the difference between different por-
tions of the diaphragm. All animal procedures were approved
by the Institutional Animal Care and Use Committee.
Quantitative PCR—Total RNA was extracted from mouse

diaphragm muscle samples using the TRIzol reagent (Invitro-
gen). Integrity of the isolated RNA was verified using agarose
gel electrophoresis. One �g of each RNA was reverse tran-
scribed using an anchored oligo(dT) primer (TV20) at 42 °C for
2 h. Quantitative PCR was carried out with Power SYBR Green
PCR master mix (Applied Biosystems) to determine the level of
ssTnT cDNA relative to the level of glyceraldehyde-3-phos-
phate dehydrogenase, using an Applied Biosystems 7300 real
time PCR system (Applied Biosystems, Foster City, CA). The
PCR primers (Fig. 3A) were designed to target the common
sequence for all three alternatively spliced isoforms of ssTnT
mRNA (14) without cross-reaction with fast skeletal muscle
TnT cDNA (Fig. 3B). The quantitative PCR was carried out in a
20-�l volume with a 10-min preheating at 95 °C followed by 40
cycles of 15 s at 95 °C and 60 s at 60 °C. Melting curve analysis
was performed at the end to verify that there was no significant

FIGURE 1. Targeted deletion of cTnI gene in the mouse genome removes the 5� portion of the ssTnT gene
promoter-enhancer region. The cTnI gene and ssTnT gene are very closely linked in the mouse genome with
the 5� portion of the ssTnT gene promoter-enhancer region integrated in the structure of the cTnI gene (6). The
targeted deletion of the entire cTnI gene in a cTnI knock-out mouse line (7) had concurrently removed a
segment containing 5�-enhancer(s) of the ssTnT gene promoter, which would reduce transcriptional activity as
indicated by significantly decreased transcriptional activity of the �2.2 kb promoter construct versus the �5.4
kb control in previous ex vivo experiments (6).

FIGURE 2. Rescue of cTnI gene deletion mice by transgenic expression of
cTnI-ND. A, the two agarose gels demonstrated the PCR genotyping of dou-
ble transgenic mice using two pairs of specific primers recognizing wild type
and Tnni3 deletion alleles, respectively (7), to identify homozygote of cTnI
gene deletion (top) and the presence of �-MHC promoter-directed transgene
encoding cTnI-ND (bottom). B, the SDS-gel and Western blot using mAb TnI-1
showed that the cardiac muscle of adult double transgenic mice expressed
cTnI-ND in the absence of endogenous cTnI, demonstrating a successful res-
cue of the postnatal lethality of cTnI-KO mice. The mAb CT3 Western blot
showed normal expression of cardiac TnT (cTnT) in the double transgenic
mouse cardiac muscle.

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NOVEMBER 13, 2009 • VOLUME 284 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 31799



formation of primer dimmers under the PCR conditions. Dia-
phragm samples from three wild type and three double trans-
genic mice were examined, each with triplicate reactions. The
results were analyzed using the 2���Ct method as per the
Applied Biosystems user’s instructions.
SDS-PAGE and Western Blot—Endogenous cTnI and exoge-

nous cTnI-ND in the heart and TnT and TnI isoforms in the
diaphragm muscle were examined by SDS-PAGE and Western
blotting. Total protein was extracted from freshly isolated mus-
cle tissue by homogenization in SDS-PAGE sample buffer con-
taining 2% SDS to inactivate tissue proteases. The samples were
heated at 80 °C for 5 min, centrifuged at top speed in a micro-
centrifuge for 5 min to remove insoluble materials, and stored
at �80 °C until use.
The protein samples were resolved on 14% Laemmli gels with

an acrylamide/bisacrylamide ratio of 180:1. The resulting gels
were stained with Coomassie Blue R250 or electrically trans-
ferred to nitrocellulose membrane using a semidry transfer
apparatus (Bio-Rad). After blocking in Tris-buffered saline
containing 1% bovine serum albumin, the membranes were
probed using anti-TnI monoclonal antibody (mAb) TnI-1, anti-
slow and cardiac TnT mAb CT3 (14), or anti-fast TnT mAb
T12 (a gift from Prof. Jim Lin, University of Iowa) (15). The first
antibody reactions were in Tris-buffered saline containing 0.1%
bovine serum albumin at 4 °C overnight. The subsequent
washes, alkaline phosphatase-labeled anti-mouse IgG second
antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
incubation, and 5-bromo-4-chloro-3-indolylphosphate/nitro
blue tetrazolium substrate reaction were carried out as
described previously (16). The relative amounts of TnT and TnI
isoforms were quantified by two-dimensional densitometry of
the blots.
Myosin heavy chain isoforms were examined as described

previously (17). The muscle protein samples were resolved on
8% SDS-polyacrylamide gel containing 30% glycerol run in an
icebox overnight. The gels were stained with Coomassie Blue
R250, and the relative amounts of MHC isoforms were quanti-
fied by two-dimensional densitometry.
Hematoxylin-Eosin Staining of Paraffin Sections—After

euthanasia of the mice, diaphragm tissue was rapidly excised
and fixed in 3.7% formaldehyde in phosphate-buffered saline
(PBS). The diaphragm samples were embedded in paraffin with
precise orientation and thin cross sections were cut and pro-
cessed with hematoxylin-eosin staining at a service facility for
light microscopic examinations.
Immunohistochemistry—Mouse diaphragm muscle samples

were rapidly frozen in a small drop of O.C.T. compound in
isopentane at �159 °C and then embedded in O.C.T. for thin
frozen cross sectioning. The sections were fixed in 75% acetone,
25% ethanol for 5 min.
After blocking in PBS containing 0.05% Tween 20 (PBS-T)

and 1% bovine serum albumin for 30 min, the sections were
incubated with 1% H2O2 in PBS for 10 min to inactivate endog-
enous peroxidases. The sections were then washed with PBS-T
three times and incubated with anti-MHC-I mAb FA2 or SP2/0
myeloma culture supernatant at 4 °C overnight. After washes
with PBS-T to remove unbound mAb, the sections were incu-
bated with horseradish peroxidase-labeled anti-mouse second-

ary antibody at room temperature for 1 h, washed again, and
developed in 3,3�-diaminobenzidine-H2O2 substrate solution
in the dark for 30–60 s. The substrate reaction was stopped by
washes in 20 mM Tris-HCl, pH 7.6, for six changes.

After they were counterstained with hematoxylin for 5 min
and water washes, the sections were immersed in a drop of 50%
glycerol in PBS and mounted with Cytoseal. The slides were
examined under a Zeiss Observer microscope and photo-
graphed. FA2-positive (type 1) fibers were counted per unit of
cross-sectional area for comparisons.
Contractility Measurements—Mouse diaphragm muscle was

isolated immediately after euthanasia as above. Immersed in
Krebs solution (118 mM NaCl, 25 mM NaHCO3, 4.7 mM KCl, 1.2
mM KH2PO4, 2.25 mM MgSO4, 2.25 mM CaCl2, and 11 mM
D-glucose, pH 7.4) continuously bubbled with 95% O2 plus 5%
CO2 at room temperature, 2-mm-wide diaphragm muscle
strips were dissected with a piece of central tendon at one end
and a piece of the rib cage tissue at the other end. The rib cage
end of the muscle strip was tied to a triangle-shaped stainless
steel hook with a #5-0 suture. The muscle strip was then
mounted to the bottom pole of a vertical bath (Radnoti) con-
taining Krebs bubbled with 95% O2 plus 5% CO2 at 25 °C, and
the tendon end was tied to another stainless steel hook for con-
nection to a motorized force transducer (model 300B-LR,
Aurora Scientific, Inc.). Isometric twitch contractions were
stimulated by 20-V field electrical pulses of 0.2-ms duration
using an Aurora 701B stimulator. Force of the muscle strip was
measured, and length was controlled through the 300B-LR
transducer, and data were collected via a digital controller A/D
interface (model 604C, Aurora Scientific Inc.).
The mounted muscle strip was adjusted to an optimal length

for the production of maximal twitch force during 30 min of
equilibration. 500-ms tetanic contractions were then stimu-
lated every 1 min with 100-Hz 20-V field electrical pulses of
0.2-ms duration for 20 min. The force-frequency relationship
was determined from isometric tetanic contractions stimulated
with electrical pulses at various frequencies (60–240 Hz).
High Frequency and Intermittent Fatigue Protocols—The dia-

phragm muscle was examined for high frequency fatigability in
which tetanic contractions were stimulated with 20-V field
electrical pulses of 0.2-ms duration at the optimal frequency for
30 s, followed by stimulations with electrical pulses of 2.0-ms
duration for 5 s to bypass the membrane electrical fatigability
(18). The effect of bypassing membrane electrical fatigability
was verified by pulse stimulations of 2.0-ms duration for 30 s
followed by 3.0-ms pulses for 5 s. One minute after the high
frequency fatigue contractions, 500-ms tetanic contractions
were stimulated every 1 min with 20-V field electrical pulses of
0.2-ms duration at the optimal frequency for 20 min to monitor
the recovery.
Fatigability of the diaphragm muscle was also examined with

intermittent tetanic contractions in which 330 ms of tetanic
contraction was evoked every 2 s (a duty cycle of 16.5%), as
described previously (19, 20) with 20-V field electrical pulses of
0.2-ms duration at the optimal frequency for 7 min. One minute
after the intermittent fatigue contractions, 20 min of recovery
was monitored as above.

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31800 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 46 • NOVEMBER 13, 2009



All muscle strips used for the functional studies were recov-
ered at the end of the experiment and examined by SDS-PAGE
and Western blot to verify ssTnT and other myofilament pro-
tein contents.
Data Analysis—Two-dimensional densitometry was used to

quantify the SDS-gel and Western blots scanned at 600 dots/
inch using Image J software. Using Image J software, the average
thickness of the diaphragm was measured in 600-�m intervals
along the tissue sections, and the cross-sectional area of muscle
fibers was calculated. The data are shown as mean � S.E., and
statistical significance was determined using two-tailed
unpaired Student’s t test. Difference was considered significant
when p was �0.05.

RESULTS

Decreased ssTnT mRNA in Diaphragm Muscle of Adult Dou-
ble Transgenic Mice—Quantitative PCR with primers recogniz-
ing all three alternatively spliced isoforms of ssTnT but not fast
TnT mRNA showed significantly decreased expression of
ssTnT mRNA in diaphragm muscle of adult cTnI-KO/
cTnI-ND double transgenic mice as compared with that in wild
type mouse diaphragm muscles (Fig. 3). Therefore, the deletion
of the 5� portion of the ssTnT gene enhancer-promoter region
integrated in the cTnI gene resulted in a knockdown effect on
the transcription of the ssTnT gene in vivo.
Decreased ssTnT Protein in Diaphragm Muscle of Young

Adult Double Transgenic Mice—Western blots using mAb CT3
detected decreased ssTnT protein (down by �39%) in dia-
phragm muscle of cTnI-KO/cTnI-ND double transgenic mice
as compared with that in wild type control (Fig. 4A). Together
with the decreased ssTnT mRNA, the results demonstrated

that the double transgenic mice
provide a model of ssTnT deficiency
for investigating the function of
ssTnT in adult diaphragm muscle.
The double transgenic mice are
referred as ssTnT-KD in the follow-
ing phenotype characterizations.
Corresponding to the decreased

ssTnT, Western blots using mAb
T12 showed that fast TnT in the
ssTnT-KD diaphragm muscle was
increased by �15% as compared
with the level in wild type mouse
diaphragm muscle (Fig. 4A).
Decreases in the Slow Isoforms

of TnI and MHC in ssTnT-KD
Diaphragm—In 3–5-month-old
young adult ssTnT-KD mice,
Western blots with mAb TnI-1
recognizing all TnI isoforms
showed that slow TnI was signifi-
cantly decreased (down by �25%)
and fast TnI increased as com-
pared with the wild type controls
(Fig. 4A).
Glycerol SDS-PAGE showed

decreases of MHC-I in the adult
ssTnT-KD diaphragm muscle (�8.6% of total MHC versus
�13.8% in wild type control diaphragm) (Fig. 4B). Trends of
reduction of MHC-IIa and potentially compensatory increases
in MHC-IIx and MHC-IIb were also observed (Fig. 4B).
Decreased Number of Type 1 Slow Fibers and Atrophy in

ssTnT-KD Diaphragm Muscle—Type 1 slow fibers in mouse
diaphragm muscle were identified by staining frozen thin cross-
sections using anti-MHC-I mAb FA2. The results detected a
significantly decreased number of type 1 fibers in 3–5-month-
old ssTnT-KD mouse diaphragm muscle (�52% of the wild
type control normalized to unit area) (Fig. 5A). Correspond-
ingly, FA2-negative type 2 fibers were increased by �28% in the
ssTnT-KD diaphragm muscle as compared with that in wild
type control. These changes resulted in a significant decrease in
the ratio of slow/fast fiber in the young adult ssTnT-KD dia-
phragm muscle (�0.2) versus that in wild type control (�0.5)
(Fig. 5A).
Microscopic measurement of hematoxylin-eosin-stained

paraffin cross-sections showed that the young adult ssTnT-KD
mouse diaphragm was thinner than that of wild type control
(�0.33 versus �0.37 mm; Fig. 5B), indicating muscle atrophy.
Comparing the cross-sectional area of type 1 and type 2 fibers,
we further observed significant atrophy of type 1 fibers (�43%
of the wild type control; Fig. 5B). Interestingly, the cross-sec-
tional area of type 2 fibers was also decreased in ssTnT-KD
diaphragm muscle although less severely than that of the type 1
fibers (�88% of the wild type controls; Fig. 5B).
Higher Stimulating Frequency Required for the Production of

Maximum Tetanic Force in ssTnT-KD Diaphragm Muscle—We
studied the force-frequency relationships in tetanic contraction of
wildtypeandssTnT-KDmousediaphragmmusclestrips.Aright-

FIGURE 3. Decreased expression of ssTnT mRNA in diaphragm muscle of double transgenic mice. A, for
quantitative PCR, a pair of oligonucleotide primers were designed for a region identical in the three alterna-
tively spliced isoforms of ssTnT mRNA. B, the agarose gel verified the specificity of the primers in PCR on cloned
ssTnT and fast skeletal muscle TnT (fsTnT) cDNA templates. C, quantifications using real-time PCR detected a
significantly decreased level of ssTnT mRNA in the diaphragm muscle of cTnI-KO/cTnI-ND double transgenic
mice in comparison with the wild type control. Values are shown as mean � S.E., n � 3 mice in each group. ***,
p � 0.001 in two-tailed Student’s t test.

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NOVEMBER 13, 2009 • VOLUME 284 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 31801



ward shift of the force-frequency relationship curve was found in
young adult ssTnT-KD diaphragm muscle as compared with wild
type control (Fig. 6). The optimal frequencies at which the maxi-
mal tetanic force was generated in wild type and ssTnT-KD dia-
phragm muscles were around 120 and 140 Hz, respectively. This
shift indicates an increased dependence of the ssTnT-KD dia-

phragm muscle on stimulating frequency in tetanic force produc-
tion, consistent with a faster muscle phenotype.
Whereas most of the twitch contraction parameters did not

show significant difference between the ssTnT-KD and wild
type mouse diaphragm muscle strips, the shorter time of ten-
sion development (TPT and TP50) of the ssTnT-KD diaphragm
muscle was also consistent with a faster muscle phenotype
(Table 1). Although ssTnT-KD diaphragm muscle produced
lower tetanic force at the stimulating frequency optimal for wild
typediaphragmmuscle(Fig.6),tetaniccontractileforcegenerated
at its own optimal frequency of stimulation was not significantly
different from that of the wild type control (Table 1).
Unchanged Electrical Fatigability and Increased Myofila-

ment Fatigability of ssTnT-KD Diaphragm Muscle—Because
electrical pulses of short duration induce skeletal muscle con-
traction dependent on the propagation of action potential
along the plasma membrane, we used a high frequency fatigue
protocol to study the membrane electrical fatigability of the
mouse diaphragm muscle strips. The results in Fig. 7A showed
that wild type and ssTnT-KD young adult mouse diaphragm
muscles had no difference in membrane fatigability during a
30-s tetanic contraction stimulated with field electrical pulse of
0.2-ms duration at the optimal frequencies. Switching to elec-
trical pulse stimulations of 2-ms duration at the end of the pro-
tocol significantly increased the tetanic force production of
both wild type and ssTnT-KD groups to the same extent (Fig.
7A). The similar increases in force production upon bypassing
membrane fatigability using brief wider electrical pulse stimu-
lations (18) indicated similar extents of myofilament fatigability
in the wild type and ssTnT-KD mouse diaphragm muscle strips
when the contractile apparatus was protected by membrane
fatigability. Consistently, both wild type and ssTnT-KD dia-
phragm muscle strips completely recovered from the electrical
fatigue treatment (data not shown).
In a modified high frequency fatigue protocol using pulse

stimuli of 2.0-ms duration followed by a switch to 3.0-ms pulse
duration, we further investigated the fatigability of mouse dia-
phragm muscle bypassing the membrane electrical depend-
ence. The results in Fig. 7B revealed a significant difference
between ssTnT-KD diaphragm muscle strips and the wild type
controls. The faster and more extensive decreases in tetanic
force of ssTnT-KD diaphragm muscle indicated increased
myofilament fatigability in comparison with the wild type con-
trol. When the duration of electrical pulses was switched from
2.0 to 3.0 ms, there were small increases in tetanic force in both
ssTnT-KD and wild type groups, suggesting that the calcium
mobilization capacity (18) was not exhausted in the diaphragm
muscle fibers when stimulated by 20-V field pulses of 2.0-ms
duration.
Increased Fatigability and Decreased Recovery of ssTnT-KD

Diaphragm Muscle in Intermittent Tetanic Contractions—The
increased myofilament fatigability in ssTnT-KD diaphragm
muscle was further examined using a 7-min intermittent
fatigue protocol. The results in Fig. 8A showed that 210 cycles
of intermittent tetanic contraction at 16.5% duty cycle (330-ms
trains) stimulated with 0.2-ms electrical pulses at optimal fre-
quency exhibited force decreases to �40.5% of the prefatigue
level in ssTnT-KD diaphragm muscle as compared with

FIGURE 4. Decreased ssTnT and the slow isoforms of TnI and MHC in dia-
phragm muscle of young adult ssTnT-KD mice. A, SDS-gel and mAb CT3
Western blot densitometry detected significantly lower levels of ssTnT pro-
tein in diaphragm muscle of the ssTnT-KD mice as compared with the wild
type controls (down by 39.1 � 9.6% normalized to the level of actin in accom-
panying gels). Correspondingly, mAb T12 Western blot and densitometry
analysis detected higher levels of fast skeletal muscle TnT (fsTnT) in the
ssTnT-KD diaphragm muscle than the wild type controls (up by 14.99% nor-
malized to the level of actin in accompanying gels). Densitometry analysis of
mAb TnI-1 Western blot normalized to the amount of total TnI found
decreased the slow versus fast isoform ratio of TnI in the ssTnT-KD diaphragm
muscle as compared with the wild type controls. B, glycerol-SDS-gel and den-
sitometry analysis normalized to total MHC showed a decreased level of
MHC-I in ssTnT-KD diaphragm muscle (8.62% of total MHC versus 13.83% in
wild type control). *, p � 0.05 by two-tailed Student’s t test.

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31802 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 46 • NOVEMBER 13, 2009



�54.4% in wild type control muscle strips. The 20-min recovery
from fatigue treatment showed that tetanic force went back to
�98.5% of the prefatigue level in wild type diaphragm muscle
but only to �91.6% in the ssTnT-KD group (Fig. 8B). The data
indicated that the slow TnT deficiency resulted in significantly
increased myofilament fatigability in the young adult dia-
phragm muscle of ssTnT-KD mice.
Effects of ssTnT-KD on Contractility and Myofilament Pro-

tein Isoform Contents of Aging Diaphragm—We further exam-
ined diaphragm muscle from 13–20-month-old mice for con-

tractility and myofilament protein isoform expression to
investigate the effect of ssTnT-KD. The results in Fig. 9A
showed that aging wild type and ssTnT-KD diaphragm muscles
both switched toward slower fiber type, whereas the ssTnT-KD
group remained faster than the wild type control, consistent
with the previous observation that aging skeletal muscles
undergo a shift to more aerobic-oxidative metabolism in a
slower twitching fiber population (21, 22).
Slow TnT and slow TnI remained low in aging ssTnT-KD

diaphragm muscle (Fig. 9B). Consistent with the shift toward
slower muscle type, the slow/fast isoform ratio of TnI increased
in aging wild type diaphragm muscle but was insignificant in
ssTnT-KD diaphragm (Fig. 9B). The level of MHC-IIa
increased in aging diaphragm muscles of wild type mice with a
lesser degree in ssTnT-KD group (Fig. 9B). MHC-IIx decreased
during aging in both groups (Fig. 9B).

FIGURE 5. Atrophy and type 1 fiber reduction of ssTnT-KD diaphragm muscle. A, immunostaining of frozen sections using anti-MHC-I mAb FA2 showed
52.2 � 4.9% decrease in type 1 fibers and 28.0 � 5.2% increase of type 2 fibers in ssTnT-KD diaphragm muscle as compared with the wild type control.
Accordingly, the ratio of type 1/type 2 fibers was much lower in ssTnT-KD mouse diaphragm muscle (20.2 � 1.7%) than that in wild type control muscles (49.8 �
4.7%). B, hematoxylin-eosin-stained cross-sections showed that the thickness of adult ssTnT-KD mouse diaphragm was smaller (0.33 � 0.03 mm) than that of
wild type controls (0.37 � 0.03 mm), indicating muscle atrophy. Morphological analysis further found that the cross-sectional area (CSA) of type 1 fibers was
significantly decreased by 43.2 � 6.2% in ssTnT-KD diaphragm as compared with the wild type control. The cross-sectional area of type 2 fibers in ssTnT-KD
diaphragm was also decreased, although by a lesser extent (12.1 � 3.2%), as compared with the wild type control. *, p � 0.05 by two-tailed Student’s t test.

FIGURE 6. Shift of force-frequency relationship in ssTnT-KD diaphragm
muscle. Tetanic force production was examined with electrical pulse stimu-
lations of 0.2-ms duration at 60 –240 Hz in 20-Hz increments. The results
showed that ssTnT-KD diaphragm muscle had optimal frequency for maxi-
mum force at 140 Hz, increased from 120 Hz for the wild type mouse dia-
phragm muscle, consistent with increased fast fiber content. *, p � 0.05 by
two-tailed Student’s t test, n � 3 mice in each group.

TABLE 1
Parameters of twitch and tetanic contractions
Data are presented as mean � S.E. n � 10 mice for wild type and n � 11 mice for
ssTnT-KD groups. mN, millinewtons; dT/dt, develop tension; TP50, time to
reach 50% peak tension; TPT, time to reach peak tension; TR75, time to reach
75% relaxation.

Wild type ssTnT-KD

Developed twitch tension (mN/mm2) 46.05 � 3.23 40.23 � 4.84
�dT/dt (mN/s) 1683.6 � 20.6 1458.3 � 16.4
�dT/dt (mN/s) �851.8 � 82.0 �750.8 � 66.9
TP50 (ms) 10.73 � 0.19 10.08 � 0.23

a

TPT (ms) 28.91 � 0.79 25.50 � 0.68b
TR75 (ms) 32.25 � 2.84 30.75 � 2.06
Optimal tetanic tension (mN/mm2) 187.82 � 12.16 209.37 � 13.32

a p � 0.05 in two-tailed Student’s t test.
b p � 0.01 in two-tailed Student’s t test.

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In contrast to the significant decrease in MHC-IIb in aging
wild type mouse diaphragm muscle, the level of MHC-IIb
increased in ssTnT-KD mouse diaphragm during aging
(Fig. 9B). Although determination of its functional significance
requires follow up studies, this finding indicated that the
decrease in slow TnT induced adaptive gene regulations for the
expression of other myofilament proteins with differentiated
effects in young and aging skeletal muscles.

DISCUSSION

Troponin T abnormalities have been found in causing dis-
eases in both cardiac and skeletal muscles (23). We and others
have extensively studied the biochemical mechanisms for the
structure-function relationship of TnT isoforms (2, 24). It has
been shown that slow TnT has differentiated binding affinity
for TnI and tropomyosin and confers higher Ca2� sensitivity in
comparison with that of fast TnT (5, 13). The present study

demonstrated the importance of
slow TnT in the function of skeletal
muscle. Our findings provide some
new insights into the following
aspects.
The Structural Linkage of cTnI

and ssTnT Genes—Cardiac TnI and
ssTnT genes are very closely linked
in the vertebrate genome. We previ-
ously detected in ex vivo promoter
analysis that the kb �2.2 to �5.4
upstream region of the ssTnT gene,
which overlaps with the 3� region of
the cTnI gene, contained major
enhancer activity required for high
level transcription of the ssTnT
gene (6). The present study further
demonstrated that deletion of the
cTnI gene between the two BamHI
sites (Fig. 1A) (7), equivalent to the
�2.2 kb deletion of the upstream
region of ssTnT gene tested ex
vivo, had a destructive effect in
vivo on the promoter activity of
the ssTnT gene. Because a single
copy of a functional ssTnT gene
was sufficient in sustaining muscle
function in heterozygote careers
of Amish nemaline myopathy (5),
the decreased level of ssTnT in the
diaphragm muscle of ssTnT-KD
mice indicated a more than 50%
decrease of ssTnT gene promoter
activity due to deletion of the �2.2
kb upstream genomic enhancer
segment.
Although the ssTnT gene is iden-

tified to be the newest member
evolved among the three muscle
type TnT isoform genes (25), its
function is indispensable, and the

loss of ssTnT causes a lethal phenotype in Amish nemaline
myopathy (5). This functional importance might have been
responsible for the ssTnT gene remaining closely linked to
the upstream cTnI gene during evolution, because a destruc-
tion of either the 3�-coding exons of the cTnI gene or the
5�-enhancer region of the ssTnT gene would cause infantile
lethality (5, 7).
The Effects of ssTnT Deficiency on Myofilament Protein Iso-

forms and Atrophy of Diaphragm Muscle—The primary ssTnT
deficiency resulted a series of secondary changes in the dia-
phragm muscle of young adult ssTnT-KD mice. In addition to
an increased ratio of fast TnT, ssTnT-KD diaphragm muscle
showed decreased slow TnI and a trend of increase in fast TnI.
MHC-I was decreased together with trends of increases in
MHC-IIx and MHC-IIb. Therefore, the primary deficiency of
ssTnT resulted in reductions of the slow isoforms of both thin
and thick filament proteins. The decreased slow/fast isoform

FIGURE 7. High frequency fatigue analysis. A, a high frequency fatigue protocol was tested on diaphragm
muscle strips with field electrical stimuli of 0.2-ms duration for 30 s followed by 2.0-ms stimuli for 5 s. The results
showed no difference in electrical fatigability between the ssTnT-KD and wild type diaphragm muscles. Switch
of the electrical pulses from 0.2-ms duration to 2.0-ms duration generated similar partial recovery of the
contractile force in both groups, suggesting similar protection of the muscle by electrical fatigue. B, high
frequency fatigue analysis was modified to bypass the membrane dependence using electrical stimuli of
2.0-ms duration for 30 s followed by stimuli of 3.0-ms duration for 5 s. The results showed significantly higher
myofilament fatigability of ssTnT-KD diaphragm muscle as compared with the wild type control. *, p � 0.05 by
two-tailed Student’s t test, n � 6 mice in each group.

FIGURE 8. Intermittent fatigability analysis. A, an intermittent fatigue protocol was applied to examine wild
type and ssTnT-KD mouse diaphragm muscle strips. Tetanic contractions stimulated at optimum frequency
with 330-ms/2-s trains for 7 min demonstrated force declines to 54.4 � 2.6% in wild type and to 40.5 � 1.1% in
ssTnT-KD diaphragm muscle strips. B, during a 20-min recovery, the force of wild type diaphragm muscle
returned to 98.5 � 1.2% of the prefatigue level, whereas that of ssTnT-KD diaphragm muscle returned to 91.6 �
2.0% of the prefatigue level. *, p � 0.05 by two-tailed Student’s t test, n � 6 mice in each group.

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ratio of myofilament proteins formed a basis of the fast-ward
change of fiber types.
Because the diaphragm is a mixed fiber muscle (13), the

decreases in the slow isoform myofilament proteins could
reflect decreases in the number and/or size of slow fibers as well
as increases in the number and/or size of fast fibers as a com-
pensation for the decreases in ssTnT. Our results first showed
reductions of both number and size of type 1 slow muscle fibers,
which would directly contribute to the atrophy of ssTnT-KD
mouse diaphragm. Although the number of type 2 fibers
increased to compensate for the decreased number type 1
fibers, the cross-sectional area of type 2 fibers in ssTnT-KD
diaphragm was decreased (Fig. 5B). Therefore, the atrophy of
type 2 fibers that are the major fiber type in mouse diaphragm
muscle (Fig. 5A) further contributed to the atrophy of
ssTnT-KD diaphragm muscle. The mechanism for ssTnT defi-

ciency to cause atrophy of fast type 2
fibers in diaphragm muscle remains
to be investigated.
The effects of slow TnT defi-

ciency on diaphragm myofilament
protein contents exhibited aging
related changes. Although both wild
type and ssTnT-KD diaphragm
muscles turned toward a slower
fiber type in aging (Fig. 9), an inter-
esting adaptation to ssTnT-KD was
that MHC-IIb was significantly
decreased in wild type but increased
in ssTnT-KD diaphragm muscle.
This adaptive sustainment of MHC-
IIb that produces the fastest in vitro
motility among MHC isoforms (26)
mayhavecontributedtothelessslow-
wardshiftoftheagingssTnT-KDdia-
phragm muscle (Fig. 9A).
The finding that decreased

expression of slow TnT resulted in a
series of decreases in slow TnI and
MHC-I indicated a consequence of
fiber type switching. The finding
that a change in one thin filament
protein in skeletal muscle would
cause multiple secondary changes
in gene regulation suggests an
experimental model to investigate
the highly plastic nature of skeletal
muscle during physiological and
pathological adaptations.
Slow TnT Deficiency Reduces

Fatigue Tolerance of Diaphragm
Muscle—It has been established
that stimulating skeletal muscle
contraction with an electrical
pulse of short duration (0.1–0.2
ms) significantly depends on the
propagation of action potential
along the plasma membrane (18).

In contrast, electrical pulses with longer duration of 2.0 ms
can propagate directly to the transverse tubular membranes,
in which a majority of the L-type calcium channels are pres-
ent, to activate calcium mobilization mechanisms with less
dependence on plasma membrane action potential propaga-
tion, thus bypassing the fatigue mechanism of plasma mem-
brane inexcitability (27). Therefore, we applied a high fre-
quency fatigue stimulation protocol to dissect plasma
membrane electrical fatigability and myofibril fatigability.
The results clearly demonstrated that ssTnT deficiency
significantly increased myofilament fatigability with un-
changed plasma membrane excitability.
The increased myofilament fatigability of the ssTnT-KD

mouse diaphragm muscle was further demonstrated using an
intermittent tetanic fatigue protocol mimicking the respiratory
contractions. The results confirmed the physiological impor-

FIGURE 9. Effects of ssTnT-KD in aging diaphragm muscle. A (left), twitch and tetanic force measurements
found no significant difference between young and aging diaphragm muscles from wild type and ssTnT-KD
mice. Right, normalized twitch contraction curves revealed that wild type and ssTnT-KD mouse diaphragm
muscles both shifted toward slower fiber type in aging, whereas the ssTnT-KD group remained faster than the
wild type control. B (top), densitometry analysis of CT3, T12, and TnI-1 mAb Western blots showed that slow TnT
remained low in aging diaphragm muscle of ssTnT-KD mice along with the trend of higher fast TnT contents
and decreased slow versus fast isoform ratio of TnI as compared with the wild type controls. Bottom, glycerol-
SDS-gel and densitometry analysis showed that MHC-I went down in aging diaphragm muscle with much
more severity in the ssTnT-KD group than in the wild type control. MHC-IIa was increased during aging to a
lesser degree in ssTnT-KD than that in wild type diaphragms. There was a trend of decrease in MHC-IIx in both
wild type and ssTnT-KD diaphragm during aging. MHC-IIb was significantly decreased in aging wild type
mouse diaphragm muscle, whereas aging ssTnT-KD diaphragm had an increase in MHC-IIb. *, p � 0.05 aging
versus young groups of the same genotype; #, p � 0.05, ssTnT-KD versus wild type in the aging group, analyzed
by two-tailed Student’s t tests. mN, millinewtons.

Slow Troponin T and Diaphragm Fatigue

NOVEMBER 13, 2009 • VOLUME 284 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 31805



tance of ssTnT in diaphragm function. The fatigability of
ssTnT-KD diaphragm muscle shown by normalized force pro-
duction (Fig. 8) demonstrated that in addition to the overall
muscle atrophy, the qualitative changes of decreased number of
slow fibers and slow isoforms of myofilament proteins severely
impaired the fatigue tolerance of diaphragm muscle. In addi-
tion to explaining the terminal respiratory failure seen in virtu-
ally all Amish nemaline myopathy patients, the cTnI-KO/
cTnI-ND double transgenic mouse model provides a highly
valuable experimental system to study the pathogenesis, patho-
physiology, and treatment of this devastating and lethal genetic
disease.

Acknowledgments—We thank Hui Wang for PCR genotyping of trans-
genic mice, Dr. Xupei Huang (Florida Atlantic University) for provid-
ing the cTnI-KO mouse line, Dr. Jim Lin (University of Iowa) for pro-
viding the T12 mAb, and Dr. Jeffrey Robbins (University of
Cincinnati) for the mouse cardiac �-MHC gene promoter used in the
construction of cTnI-ND transgenic mouse lines.

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Slow Troponin T and Diaphragm Fatigue

31806 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 46 • NOVEMBER 13, 2009


	Deletion of a Genomic Segment Containing the Cardiac Troponin I Gene Knocks Down Expression of the Slow Troponin T Gene and Impairs Fatigue Tolerance of Diaphragm Muscle*
	MATERIALS AND METHODS
	RESULTS
	DISCUSSION
	REFERENCES