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INTRODUCTION |
In microgravity, a significant stress on the cardiovascular system
is the redistribution of body fluid toward the head due to the lack of
hydrostatic pressure. Through neurohumoral regulations, this fluid
redistribution induces reductions of blood volume and central venous
pressure (1). Although decreased intrapleural pressure during space
flight may assist filling of the heart (2), prolonged exposure to
microgravity results in decreases in cardiac preload and function,
evident by echocardiography of astronauts showing decreases in left
ventricular end diastolic volume and ventricular stroke volume (3, 4).
Rats flown in space for 14 days showed decreased average
cross-sectional area of the myocytes in left ventricular muscle,
indicating myocardial atrophy (5). These observations suggest that
prolonged exposure to microgravity induces a decrease in cardiac
function. However, the regulation of cardiac muscle contractility in
microgravity is unclear. In addition to the health and safety of
astronauts during and after long space flight, a thorough understanding
of the adaptation of cardiac muscle in microgravity will also
contribute to the prevention and treatment of myocardial dysfunction in
chronic bedridden, paraplegic, and heart failure patients, since
similar changes are seen in their hearts (6-8).
The adaptation of myocardial contractility in microgravity may involve
structural and functional modifications of contractile proteins. The
contraction of cardiac muscle is based on actin-myosin interactions
regulated by intracellular Ca2+ via the thin filament-based
troponin-tropomyosin system (9). The regulation of thin filament
proteins may play a role in the functional adaptation of cardiac
muscle. The troponin complex contains three subunits: the
Ca2+-binding subunit troponin C
(TnC),1 the tropomyosin
(Tm)-binding subunit troponin T (TnT), and the inhibitory subunit
troponin I (TnI) (10, 11). During muscle contraction,
Ca2+-induced interactions between TnC and TnI, TnT, Tm, and
actin result in a series of allosteric conformational changes in the thin filament, translating the signal into the activation of actomyosin ATPase and development of force (10). A key step in this signaling mechanism is the release of inhibition of TnI on actin-myosin interaction (12).
Three homologous TnI genes (cardiac, fast skeletal muscle, and slow
skeletal muscle) have evolved in vertebrates to encode the muscle
type-specific TnI isoforms (13). Expression of TnI isoforms is
regulated during development. The embryonic heart expresses exclusively
slow skeletal muscle TnI. During perinatal heart development, the
expression level of slow TnI decreases, and expression of cardiac TnI
(cTnI) increases and becomes the only TnI isoform in the adult heart
(14-16). Primary structures of cardiac, slow, and fast skeletal muscle
TnI isoforms have been determined from cDNA and genomic cloning and
sequencing (15, 17-23). Post-translational regulation of TnI structure
and function has been found to involve both amino acid side chain
modification and cleavage of the primary structure. A proteolytic
truncation of 19 amino acids of the COOH terminus of cTnI has been
found during myocardial ischemia and reperfusion injury (24).
Expression of this cTnI fragment in transgenic mice produced myocardial
stunning (25). A significant difference between cTnI and skeletal
muscle TnIs is an NH2-terminal extension of 32 amino acids
in cTnI (12). The
-adrenergic signaling pathway controls
phosphorylation of two serine residues (Ser23 and
Ser24) in the NH2-terminal region of cTnI by
cAMP-dependent protein kinase (PKA) (26). This
phosphorylation of cTnI decreases myofilament Ca2+
sensitivity by reducing the Ca2+ binding affinity of TnC
(27). This mechanism plays an important role in the functional
adaptation of cardiac muscle to physiological or pathological stress
(28, 29).
The present study investigated the role of myofilament proteins in the
adaptation of myocardial contractility in a rat tail suspension model
of simulated microgravity (30). 4 weeks of tail suspension resulted in
decreases of cardiac muscle contractility without change in the
expression of contractile and regulatory protein isoforms. However, a
novel finding is an NH2-terminal truncated cTnI fragment
with increased amounts in the heart of tail suspension rats. This
proteolytic NH2-terminal modification of cTnI removes the
two serine residues that are PKA substrates. This post-translational
regulation in simulated microgravity suggests a role of the
NH2-terminal domain of cTnI in functional adaptations of
cardiac muscle.
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EXPERIMENTAL PROCEDURES |
Animal Model--
Male Harlan Sprague-Dawley rats weighing
180-210 g were randomly divided into control and tail suspension
groups. The rats were housed in a 22 ± 2 °C environment,
subjected to 12-h light/dark cycles, and fed water and Rat Chow
ad libitum. Tail suspension was carried out by a modified
Morey-Holton method (30) for 1, 2, 3, or 4 weeks. Care was taken to
protect the tail tissue, and the movement of the rats was not
restricted during the procedure.
Mechanical Recordings--
Rats were anesthetized with ether.
The heart was rapidly excised and rinsed in oxygenated Krebs-Henseleit
solution (120 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 20 mM NaHCO3,
1.2 mM NaH2PO4, and 10 mM glucose, pH 7.4). The left ventricular papillary muscle was removed and mounted in a continuous perfusion myograph chamber according to the method described previously (31). The muscle was
superfused with Krebs-Henseleit solution, and CaCl2 was
added to the solution at a series of steps to a final concentration of
6.5 mM. The solution was maintained at 30 °C and
oxygenated with 95% O2, 5% CO2. The
nontendinous end of the papillary muscle was held by a spring clip. The
tendinous end was tied to a stainless steel hook connected to an
isometric force transducer (TB-651; Kohden, Japan).
The muscles were electrically stimulated by square wave pulses (10-ms
duration) at 0.2 Hz. The length-tension relationship was recorded after
equilibration at a resting tension of 1 g for 60-90 min. The
length of the muscle was increased until a maximum developed force was
obtained (Lmax). The length-tension relationship was then measured by reducing the muscle length at 2% intervals from
the Lmax to 88% of Lmax
while recording the resting and developed forces. Each of the step
changes in length was after a reproducible sequence of 8-15
contractions to minimize effects of hysteresis.
At the end of each experiment, the Lmax was
measured again, and the muscle was then blotted dry and weighed. The
cross-sectional area of the muscle was calculated assuming the geometry
of a cylinder with a specific gravity of 1.0 (31). Tension was
normalized by cross-sectional area. The unloaded maximum velocity of
shortening (Vmax) was extrapolated from zero
loading condition according to the Maxwell model of muscle (32).
Skinned Cardiac Muscle Preparations--
Small cardiac muscle
bundles (~0.4 mm in diameter and 2.5-2.8 mm in length) were
dissected from the papillary muscle of left ventricle under a
dissection microscope. The bundles were mounted as above for the
measurement of isometric tension. After equilibration for 30 min, the
resting tension was adjusted to 100 mg. The bundles were then
chemically skinned with 1% Triton X-100 for 60 min in a relaxing
solution (pCa 9) containing 130 mM
potassium acetate, 1 mM MgCl2, 5 mM
EGTA, 5 mM Na2ATP, and 20 mM
imidazole-HCl, pH 7.0. A rigor solution (the relaxing solution without
ATP) was used to test whether the muscle was completely skinned. The
force-pCa relationship for the cardiac muscle was measured
by adding CaCl2 to the relaxing solution to achieve a
series of pCa (7.5, 7.0, 6.5, 6.3, 6.0, 5.5, 5.3 and 5.0).
All experiments were carried out at 20 °C. The free Ca2+
concentrations in the buffer were calculated using the apparent binding
constant of Ca2+ to EGTA under the experimental conditions
(33). The tension was normalized to the maximum developed tension at
pCa 5.0. The force-pCa relations were fitted to a
Hill equation, in which relative force = 100(Ca2+)n/(k + (Ca2+))n, where k and n are
constants (33).
ATPase Activity--
Rat cardiac myofibrils were isolated as
described (34). The protein concentration was determined by the
Bradford method (35). The Ca2+-activated ATPase activity of
the myofibrils was analyzed in a buffer containing 50 mM
KCl, 2 mM MgCl2, 1 mM EGTA, 2 mM NaN3, 1 mM CaCl2, 20 mM Tris-Cl, pH 7.0. The reaction was initiated by the
addition of 5 mM Na2ATP at 30 °C and was
terminated after 4 min by the addition of an equal volume of cold 10%
(w/v) trichloroacetic acid. After removing the precipitate, inorganic
phosphate (Pi) released was quantified as described
previously (36).
Western Blot Analysis of Myosin Heavy Chain, Tm, TnT, and TnI
Isoforms--
As described previously (37), total protein was
extracted from rat ventricular muscle by homogenization in
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer
containing 1% SDS. The myocardial protein extracts were resolved by
SDS-PAGE using Laemmli gels. 6% gel with an acrylamide/bisacrylamide
ratio of 180:1 was used for the analysis of myosin heavy chain (MHC);
14% gel with an acrylamide/bisacrylamide ratio of 180:1 was used for the examination of TnT and Tm; and 12% gel with an
acrylamide/bisacrylamide ratio of 29:1 was used for the examination of
TnI. The protein bands resolved by SDS-PAGE were electrically
transferred to nitrocellulose membrane (0.45-µm pore size) at 5 mA/cm2 for 25 min. The blotted nitrocellulose membrane was
blocked in Tris-buffered saline (TBS; 137 mM NaCl, 5 mM KCl, 25 mM Tris-HCl, pH 7.4) containing 1%
bovine serum albumin at room temperature for 1 h. The blocked
membrane was incubated with anti-MHC monoclonal antibodies (mAbs) FA1
and FA2 (38), an anti-cardiac TnT mAb CT3 (39), an anti-TnI mAb TnI-1
(40), a rabbit anti-TnI polyclonal antiserum (16), or an anti-Tm mAb
CH1 (a gift from Dr. Jim Lin, University of Iowa (41)) in TBS
containing 0.1% bovine serum albumin at 4 °C overnight. After
washes with TBS plus detergents (0.5% Triton X-100 and 0.05% SDS) and
TBS rinses, the membrane was incubated with alkaline
phosphatase-conjugated anti-mouse or anti-rabbit IgG second antibody
(from Sigma) in TBS containing 0.1% bovine serum albumin at room
temperature for 1.5 h. After washes as above,
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate
reaction was carried out as described previously (40) to reveal the
expression of MHC, TnT, TnI, and Tm isoforms in the cardiac muscle.
Immunoaffinity Chromatographic Isolation of cTnI--
Rat cTnI
was isolated by immunoaffinity chromatography using the TnI-1 mAb
against an epitope at the COOH terminus of TnI (40). The TnI-1 mAb
(IgG1) was purified from hybridoma ascites fluid using a Protein G
column (Amersham Pharmacia Biotech) and coupled to CNBr-activated
Sepharose 4B (Amersham Pharmacia Biotech) according to the
manufacturer's protocol. The immunoaffinity isolation of cTnI was then
carried out using a Sepharose 4B-TnI-1 mAb affinity column. Rat left
ventricular muscle was minced into 1-2-mm3 pieces and
extracted by 20 volumes (w/v) of Guba-Straub solution containing 300 mM KCl, 100 mM K2HPO4,
50 mM KH2PO4, 2.5 mM
MgCl2, 1 mM EGTA, and 0.1 mM
phenylmethylsulfonyl fluoride, pH 6.5, on ice for 15 min. After
centrifugation at 16,000 × g at 4 °C for 20 min,
the supernatant containing mainly myosin was removed. The pellet was
extracted in 20 volumes (w/v) of 1 M KCl, 10 mM Tris-HCl, pH 8.0, 0.1 mM phenylmethylsulfonyl fluoride by
stirring on ice for 30 min. After centrifugation as above, the extract was diluted 5-fold in TBS and loaded on the TnI-1 mAb affinity column
equilibrated in TBS. The column was washed with TBS, and the proteins
bound to the TnI-1 mAb affinity column were eluted with 50 mM glycine-HCl, pH 2.7. 0.5-ml fractions were collected into tubes containing 0.1 ml of neutralizing buffer containing 1 M Tris-HCl, 1.5 M NaCl, 1 mM EDTA,
pH 8.0. The fractions were analyzed by SDS-PAGE and Western blotting as
described above to identify the cTnI peak.
NH2-terminal Sequencing of cTnI Fragment--
The
cTnI fractions isolated by immunoaffinity columns were pooled and
dialyzed against 5 mM Tris-HCl, pH 7.5, and lyophilized. The concentrated protein was dissolved in SDS-PAGE sample buffer, resolved on 14% SDS-PAGE, and then electrophoretically transferred onto a polyvinylidene difluoride membrane as described above. The
membrane was stained by 0.5% Amido Black, and a stained protein band
representing the cTnI fragment was sliced out. NH2-terminal amino acid sequencing was carried out by Edman degradation using an
automated amino acid sequencer (performed at the Protein Sequencing Facility at the Medical University of South Carolina, Charleston, SC).
Densitometry--
Two-dimensional densitometric analysis of the
Western blots was performed to determine the relative amounts of the
Tm, TnT, TnI, and the TnI fragment in the cardiac muscle of tail
suspension and control rats. After the alkaline phosphatase substrate
color reaction, the nitrocellulose membranes were scanned on a SciScan 5000 densitometer (U.S. Biochemical Corp.) using the reflection mode.
The relative intensity of the protein bands recognized by the specific
antibodies was calculated from scans of multiple tail suspension and
control rat heart samples.
Data Analysis--
Values are presented as means ± S.E.
The statistical significance of differences between the mean values was
analyzed by Student's t test. The theoretical molecular
weight and isoelectric point of rat cTnI and fragments were calculated
from amino acid sequences using the DNAStar computer program.
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RESULTS |
Decreased Myocardial Function in the Tail Suspension Rats--
The
body weights of the tail-suspended rats were similar to age-matched
controls. The heart rate, mean arterial pressure, and maximal left
ventricular pressure of the 4-week tail suspension rats were not
significantly different from the controls. The heart weight/body weight
ratio and the size of papillary muscle of the tail suspension rats were
also not significantly different from those of the control rats (data
not shown).
Isometric force measurements on the papillary muscles of
tail-suspended and control rats showed no difference in resting tension (RT) (Fig. 1A). In
contrast, developed tension (DT) was decreased in the
cardiac muscle of the tail suspension rats (Fig. 1A,
p < 0.05-0.01). This decrease began at 2 weeks of
tail suspension (data not shown) and became significant at 4 weeks of
tail suspension. However, the length-tension relationship was preserved
in the cardiac muscle of tail suspension rats, similar to that seen in the failing cardiac muscle (42, 43). The calculated
Vmax of the papillary muscle was also decreased
after 4 weeks of tail suspension (p < 0.05) (Fig.
1B) together with a prolonged time to peak tension
development (data not shown).

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Fig. 1.
Tension-length relationship and velocity of
shortening in intact papillary muscle of control and tail suspension
rats. Lmax was determined as the muscle
length with a resting tension of 1 g/mm2.
n = 10 for each group. A, the results show
decreased developed tension (DT) for the 4-week tail
suspension rat (SUS) cardiac muscle, while resting tension
(RT) was not changed. The tension-length relationship was
preserved in the cardiac muscle of tail suspension rats. B,
a lowered Vmax was seen in the cardiac muscle of
tail suspension rats. Values are means ± S.E. *,
p < 0.05; **, p < 0.01 compared with
the control (CON).
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Despite the decreased force and velocity, the response curve and
EC50 of intact papillary muscle to the extracellular
Ca2+ concentration were not different from controls (data
not shown), indicating that the Ca2+ handling of
cardiomyocyte membranes was not significantly affected in the tail
suspension rats (44). The force-pCa curves of skinned cardiac muscle preparations showed no difference between the
pCa50 values obtained from the 4-week tail
suspension and control rats (Fig.
2A). The Hill coefficient
n obtained from the pCa50 values was
also not changed. However, consistent with the results from intact
cardiac muscle, the maximal isometric force
(Fmax) of the skinned cardiac muscle was
decreased in the tail suspension rats (p < 0.05) (Fig.
2B). Correspondingly, the Ca2+-activated ATPase
activity of the cardiac myofibrils of 4-week tail suspension rats was
significantly lower than that of the control rats (Fig. 2C,
p < 0.05).

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Fig. 2.
Ca2+ activation of skinned
cardiac muscle preparations. A, the
force-pCa curves show no difference in
pCa50 and Hill coefficient (n)
between the two groups. Fmax (B) and
Ca2+-activated MgATPase activity (C) of the left
ventricular myofibrils were decreased in the tail suspension rats
(SUS) as compared with the control (CON).
n = 6 rats for each group. Values are means ± S.E. *, p < 0.05.
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No Change in the Expression of Cardiac MHC, Tm, and TnT Isoforms in
the Heart of Tail Suspension Rats--
Expression of isoforms of the
contractile and regulatory proteins was examined to determine whether
any isoform switching could contribute to the decrease in the cardiac
muscle contractility in the tail suspended rats.
Using mAbs FA1 (specific to
-MHC) and FA2 (recognizing both
- and
-MHC) (38), the Western blots in Fig.
3 show no decrease in the normally
predominant
-MHC or detectable expression of
-MHC in the
ventricular muscle of 4-week tail suspension rats.

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Fig. 3.
Expression of cardiac myosin heavy chain in
the heart of control and tail suspension rats. SDS-PAGE
(top panel) and Western blots using FA1 (against
-MHC) and FA2 (against both - and -MHC) mAbs
(middle and lower panels) detected no
difference between the expression of cardiac MHC isoforms in the left
ventricle of 4-week tail suspension and control rats (two samples
each).
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The results in Fig. 6 show that the Western blots using the anti-Tm mAb
CH1 that recognizes both
- and
-Tm detected only the normally
occurring
-Tm in the heart of 3- and 4-week tail suspension rats
with no difference from the controls. The Western blots using the
anti-cardiac and slow skeletal muscle TnT mAb CT3 showed only adult
cardiac TnT, indicating no change in the expression of alternative RNA
splicing-generated cardiac TnT isoforms (45) in the heart of 3- and
4-week tail suspension rats. Western blots using the RATnT polyclonal
antibody against cardiac and skeletal muscle TnTs showed no expression
of fast or slow skeletal muscle TnT isoforms in the heart of tail
suspension rats. No proteolytic fragment of Tm and TnT was detected in
the tail suspension rat hearts by Western blot analysis.
An NH2-terminal Truncated cTnI Fragment Up-regulated in
the Heart of Tail Suspension Rats--
Western blots using mAb TnI-1
recognizing all three TnI isoforms (40) did not detect fast and slow
skeletal muscle TnI isoforms in the heart of tail suspension rats (Fig.
4). A novel finding was a ~22-kDa TnI
fragment with increased amounts in the heart of tail-suspended rats
(Fig. 4). In addition to the mAb TnI-1 from mouse ascites fluid,
supernatant from TnI-1 hybridoma cell culture recognizes this band on
Western blot, excluding nonspecific reaction from other immunoglobulins
in the mouse ascites fluid. This band is also recognized by a rabbit
polyclonal anti-TnI antibody, RATnI (16), confirming that it is indeed
a TnI fragment. The Western blots of hearts of tail-suspended and
control rats plus rat extensor digitorum longus (EDL; a fast skeletal
muscle), soleus (a slow skeletal muscle), and neonatal cardiac muscle
controls showed that the low Mr cTnI band
migrated faster than the slow skeletal muscle TnI but more slowly than
the fast skeletal muscle TnI (Fig. 4). Although slow skeletal muscle
TnI is expressed in embryonic and postnatal cardiac muscles (14-16),
the gel mobility indicates that this low Mr TnI
is not a reexpression of slow skeletal muscle TnI in the adult heart.
Although phosphorylation may also slightly alter the mobility of TnI in
SDS gel, we have confirmed by amino acid sequencing that this low
Mr TnI is a cTnI fragment. While a slight
increase was detected in the heart of 3-week tail suspension rats as
compared with controls, amounts of this cTnI fragment significantly
increased in the heart of 4-week tail suspension rats. The relative
levels of the low Mr cTnI band in hearts of 4-week tail suspension rats was quantified by two-dimensional densitometry of the TnI-1 mAb Western blots. The results demonstrate an
increase of the cTnI fragment from 13.1 to 16.3% of the total cTnI
(p < 0.05), reflecting an increase of its ratio to
intact cTnI from 1:6.61 to 1:5.15. Although the cTnI fragment increased 24.4%, this portion only represents 3.2% of the total cTnI.
Therefore, no significant change in the amounts of intact cTnI was
detected by densitometric analysis of the Western blots (Fig.
5).

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Fig. 4.
Expression of thin filament regulatory
proteins in the heart of tail suspension rats and a cTnI fragment.
Total protein extracts from the control and tail suspension (3 and 4 weeks) rat hearts and rat neonatal heart, adult soleus, and adult EDL
controls were resolved by SDS-PAGE and analyzed by Western blotting for
the expression of TnT, Tm, and TnI. While no difference in the
expression of TnT and Tm isoforms was found between the hearts of tail
suspension and control rats, a low Mr TnI band
was detected in the rat hearts with increased amounts in the 4-week
tail suspension rats. The gel mobility of the low
Mr cTnI differs from that of fast
(fsTnI) and slow (ssTnI) skeletal muscle TnIs
expressed in the control muscle samples. ssTnT and
fsTnT, slow and fast skeletal muscle TnT, respectively;
cTnTe and cTnTa, embryonic and adult cardiac TnT,
respectively.
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Fig. 5.
The relative amounts of Tm, cardiac TnT, and
cTnI in the heart of control and tail suspension rats.
A, densitometry of Western blots on total cardiac muscle
homogenate revealed significantly increased amounts of the cTnI
fragment in the heart of 4-week tail suspension (SUS) rats,
while no change in the levels of Tm, cardiac TnT (cTnT), and
intact cTnI was detected. The comparison was made using the blots of
control (CON) rat hearts as 100%. B, the ratios
between the cTnI fragment and intact cTnI in the heart of tail
suspension and control rats are shown. Values are means ± S.E.;
n = 6 for cTnT and cTnI; n = 5 for cTnI
fragment and Tm.
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Protein degradation during the post-mortem handling of the sample
could contribute to the different amounts of the cTnI fragment. To
exclude this possibility, Western blot analysis of mouse heart samples
taken at a series of post-mortem time points showed no difference in
the levels of the cTnI fragment during a period of up to 8-h storage at
room temperature (Fig. 6). We further showed that 1-h perfusion of rat heart in vitro as described
previously (46) did not change the amount of the cTnI fragment (Fig.
6). These results indicate that the cTnI fragment detected with an increased level in the heart of 4-week tail suspension rats is not a
product of nonspecific protein degradation. In contrast, it may reflect
a physiologically regulated proteolysis of cTnI in the cardiac muscle
and may play a role during the functional adaptation in simulated
microgravity.

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Fig. 6.
Unchanged levels of the cTnI fragment in
postmortem and in vitro perfused hearts.
8-week-old C57/BL6 mice were sacrificed by cervical dislocation, and
the bodies were placed at room temperature (22 °C) in a sealed
plastic bag to prevent dehydration. The hearts were removed at 0, 2, 4, and 8 h post mortem and immediately homogenized in SDS-PAGE sample
buffer. Demonstrated by the representative samples, SDS-PAGE and
Western blots using CT3 and TnI-1 mAbs showed no detectable degradation
of cardiac TnT or changes in the amounts of the cTnI fragment. There
was also no detectable change in the cTnI fragment in the rat heart
after 1-h in vitro perfusion.
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The isolation of the cTnI fragment by the anti-COOH terminus
TnI-1 mAb was as effective as that for the intact cTnI (Fig. 7). Since the TnI-1 mAb does not
recognize the cTnI-(1-192) fragment (40), this result indicates an
intact COOH terminus in the cTnI fragment. NH2-terminal
sequence analysis showed that there were three main species of cTnI
fragments generated by truncations at the COOH-end of amino acids
Asn26, Tyr27, or Tyr30 (Fig.
8). The main products were the deletions
of amino acids 1-26 and 1-27 (48 and 36%, respectively). These
truncation sites do not correspond to any exon boundaries of the cTnI
gene (Fig. 8). Therefore, the cTnI fragment is not generated by
alternative RNA splicing but, instead, by proteolytic cleavage. This
cleavage of cTnI polypeptide chain removes the exon 1 and 2-encoded
short segments at the very NH2 terminus and a large portion
of the exon 3-encoded cTnI-specific NH2-terminal extension.
The calculated Mr of the three
NH2-terminal truncated cTnI fragments (Table
I) is in agreement with the size range
shown in the SDS-PAGE (Figs. 4, 6, and 7) and further supports the
preservation of an intact COOH terminus.

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Fig. 7.
Immunoaffinity chromatographic isolation of
rat cTnI and cTnI fragment. A, thin filament proteins
extracted from rat ventricular muscle were fractionated on an anti-TnI
mAb affinity column. The proteins bound to the column were eluted with
low pH and analyzed by SDS-PAGE and Western blotting using anti-TnI
(TnI-1), TnT (CT3), and Tm (CH1) mAbs. The results show that both
intact cTnI and the cTnI fragment were recovered by the anti-TnI COOH
terminus mAb affinity column together with TnT, TnC, and trace amounts
of Tm and other myofibril proteins. B, the cTnI fractions
eluted from the affinity column were concentrated, resolved by
SDS-PAGE, and transferred to polyvinylidene difluoride membrane. Amido
Black staining revealed a good yield of the cTnI fragment, and the band
was sliced out for NH2-terminal amino acid
sequencing.
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Fig. 8.
NH2-terminal amino acid sequence
of the cTnI fragment. Structural maps of rat fast skeletal muscle
TnI, slow skeletal muscle TnI, and cTnI are aligned with the regions
for the binding of TnC, TnT, and actin as well as the inhibitory
peptide indicated. The segments encoded by different exons of the three
TnI genes are outlined by the boxes. The cTnI-specific exon
3 is shown by a filled box. The three
NH2-terminal sequences determined from the purified rat
cTnI fragment are shown and aligned with the sequence encoded by exons
1-3 of the rat cTnI gene (60). The arrowheads indicate the
three clustered cleavage sites. The two PKA substrate serine residues
(Ser23 and Ser24) are
highlighted.
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Table I
Physical properties of the NH2-terminal truncated rat cTnI
fragments
The sequence differences between the three NH2-terminal
truncated cTnIs and intact cTnI are shown in Fig. 8.
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Preserved Core Structure and Myofibril Incorporation of the
NH2-terminal Truncated cTnI--
The primary structural
alignment of cardiac, slow, and fast skeletal muscle TnIs (Fig. 8)
shows that the NH2-terminal truncated cTnI preserves the
regions homologous to the skeletal muscle TnIs containing all of the
identified binding sites for other thin filament proteins. It has been
shown that deletion of the NH2-terminal 32 amino acid did
not produce a significant change in the inhibitory function of cTnI
in vitro (47). Therefore, it may be assumed that the cTnI
fragment truncated by the NH2-terminal 26-30 amino acids would preserve its core function in the thin filament regulatory system. Consistent with this hypothesis, Western blot analysis of
extensively washed rat cardiac myofibrils showed that the incorporation of the NH2-terminal truncated cTnI fragment into the
myofilaments is comparable with that of intact cTnI, proportional to
their total levels detected in the cardiac muscle cells (Fig.
9A). The three truncated cTnIs
have very similar molecular weights and isoelectric points (Table I),
implying similarity in their three-dimensional structural and
functional features.

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Fig. 9.
Similar cTnI fragment in other vertebrate
hearts and incorporation into myofibrils. A, TnI-1 mAb
Western blots on protein extracts from intact cardiac muscle and
extensively washed myofibrils from rat heart showed that the
NH2-terminal truncated cTnI fragment was able to integrate
into the myofibril. B, total muscle homogenates of adult
heart of fish, turtles, mice, rats, rabbits, cats, dogs, and humans
were resolved by SDS-PAGE and examined by Western blotting using the
TnI-1 mAb against the COOH terminus of TnI. The results show low
Mr cTnI bands in all of the hearts
examined.
|
|
The Western blots in Figs. 6 and 8 demonstrate that normal rat and
mouse cardiac muscles contain significant amounts of the NH2-terminal truncated cTnI. Using the anti-TnI COOH
terminus mAb TnI-1, Western blot analysis of cardiac muscle samples
from a wide spectrum of vertebrate species revealed similar cTnI
fragments in all of the hearts examined (Fig. 9B). These
data suggest that the proteolytic NH2-terminal truncation
of cTnI occurs under physiological conditions, implying a
post-translational regulation of myocardial contraction. Together with
the removal of the NH2-terminal extension, an important
structural feature of the truncated cTnI is the loss of
Ser23 and Ser24 (Fig. 8), two PKA
phosphorylation sites in intact cTnI (26, 48).
 |
DISCUSSION |
The Tail Suspension Rat Model of Simulated Microgravity Produces
Changes in Myocardial Function--
The present study investigated the
adaptation of cardiac muscle in simulated microgravity using the tail
suspension rat model. The 30° head down tilt of the tail-suspended
rats produces a redistribution of body fluid toward the head. The
hemodynamic changes in the tail suspension rats are similar to those
found in astronauts during space flight (1). As a convenient model of
simulated weightlessness on the ground, tail-suspended rats have been
widely used to investigate the effects of microgravity environment on the cardiovascular system (1, 8, 49, 50). The modified tail suspension
method used in this study emphasized the protection of tail tissues to
reduce the nonspecific stress on the animal. In addition to permit a
long term tail suspension treatment, this precaution minimizes side
effects that may interfere with the physiological responses of the
cardiovascular system. The results demonstrate that this model of
simulated weightlessness produced detectable changes in the
contractility of cardiac muscle and in at least one of the cardiac
myofilament proteins, cTnI. Therefore, the tail suspension model
provides a valuable system to investigate the underlying mechanism for
physiological and pathological myocardial adaptation.
In our study, 1 week of tail suspension did not produce a significant
change in cardiac muscle contractile force (data not shown), similar to
the results from a previous study (8). In contrast, 4 weeks of tail
suspension resulted in a significant decrease in cardiac muscle
contractility. While the heart rate, mean arterial pressure, and
maximal left ventricular pressure were unchanged, the maximum developed
tension of the cardiac muscle was decreased in the tail suspension rats
(Fig. 1A). The Vmax was also decreased
(Fig. 1B), producing a prolonged time to peak tension
development. However, the length dependence of the developed tension of
cardiac muscle, which forms the basis for the Frank-Starling mechanism,
was preserved in the tail suspension rats (Fig. 1A). Therefore, the decreased myocardial contractility and volume preload may both contribute to the reduced cardiac function seen in long term
exposure to microgravity.
No Change in Heart-specific and Developmental Expression of Cardiac
Myofilament Protein Isoforms during the Adaptation to Simulated
Microgravity--
Although the Ca2+-activated ATPase
activity was reduced in the cardiac muscle of the tail suspension rats
(Fig. 2C), there was no change in the expression of MHC
isoforms (Fig. 3). Therefore, the decrease in contractility cannot be
attributed to changes in myosin isoenzymes in the heart. In addition,
no change in the expression of Tm, TnT, and TnI isoforms was found in
the heart of tail-suspended rats (Fig. 4). It has been shown by
numerous studies that these contractile and regulatory protein isoforms are sensitive markers for the change of muscle fiber types or developmental states (51). In contrast to the reexpression of fetal
cardiac genes in the unloaded adult ventricular muscle in heterotopic
transplanted heart (52), the lack of isoform switch of MHC, Tm, TnT,
and TnI in the hearts of tail suspension rats indicates no change in
cardiac muscle differentiation or developmental state during the
functional adaptation in simulated microgravity.
A Proteolytic Truncation of the cTnI-specific
NH2-terminal Segment Regulated during Functional Adaptation
of Cardiac Muscle--
A novel finding of the present study is the
NH2-terminal truncated cTnI fragment in both normal and
tail-suspended rat hearts (Figs. 4 and 6). Proteolytic modification of
cTnI has been shown with pathological effects on myocardial
contractility. For example, Ca2+ overload in cardiomyocytes
caused by ischemia-reperfusion may activate proteolytic cleavage of
cTnI at amino acid 192 to remove the COOH terminus (24). The
cTnI-(1-192) fragment reduces the maximal isometric tension of the
myocardium and causes a stunning phenotype in the hearts of transgenic
mice (25). The low molecular weight cTnI band identified in our study
has an intact COOH terminus as shown by its reactivity to the
anti-COOH terminus mAb TnI-1 (Fig. 4) that does not recognize
cTnI-(1-192) (40). Together with its presence in normal cardiac
muscle, the preserved core structure (Fig. 8) and apparently normal
integration into the myofilament (Fig. 9A) of this
NH2-terminal modified form of cTnI suggest a physiological
significance. This hypothesis is supported by the fact that a similar
cTnI fragment is also found in other vertebrate hearts (Fig.
9B). Therefore, the increased amounts of the
NH2-terminal truncated cTnI in the heart of tail-suspended rats may represent a functional adaptation of cardiac muscle in simulated microgravity and a proteolytic regulation of cardiac myofibrillar proteins. The regulatory mechanism of the
proteolytic NH2-terminal truncation of cTnI requires
further investigation. Calpain-catalyzed cTnI degradation that is
regulated by PKA and protein kinase C phosphorylation of cTnI (53) may
play an important role.
Structure-Function Significance of the
NH2-terminal Truncated cTnI--
The truncated cTnI has
lost almost all of the cTnI-specific NH2-terminal extension
(Fig. 8). Accordingly, the functional effects of the
NH2-terminal truncated cTnI may be 2-fold: to mimic the property of skeletal muscle TnI as well as to remove the PKA
phosphorylation-mediated regulation. Expression of slow skeletal muscle
TnI in the heart of transgenic mice resulted in an increased
sensitivity to Ca2+ activation, which was not reduced by in
the
-adrenergic stimulated PKA treatment (54). Such an effect may
increase the Ca2+ sensitivity of the cardiac muscle in the
tail-suspended rats. However, the effects of the
NH2-terminal truncated cTnI on cardiac muscle contraction
would not simply mimic that of slow skeletal muscle TnI, because over
50% sequence diversity is present between the homologous portion of
cTnI and slow TnI (40), which may also result in differences in
function. This is supported by the observation that a replacement of
the NH2-terminal half of the cTnI polypeptide chain with
the NH2-terminal domain of slow skeletal muscle TnI
resulted in contractility features differing from those of
either cTnI or slow TnI (55). Therefore, the NH2-terminal truncated cTnI may confer a unique functional change in the cardiac thin filament. Nevertheless, the NH2-terminal segment of
cTnI contains the two serine residues, Ser23 and
Ser24 (Fig. 8), which are substrates for PKA-catalyzed
phosphorylation (26, 27). Phosphorylation of Ser24 in
cTnI may occur constitutively, whereas phosphorylation of Ser23 produces a decrease in the sensitivity of the
myofilaments to Ca2+ via a modulation of
TnC-Ca2+ affinity (48). The presence of a dynamic pool of
the NH2-terminal truncated cTnI in the cardiac muscle may
provide a basal level of function that is not regulated by the PKA
signaling pathway. Therefore, the up-regulation of the
NH2-terminal truncated cTnI in the heart of tail suspension
rats may modulate cardiac muscle contractility by counteracting the
effect of PKA phosphorylation of Ser23 and
Ser24 on the sensitivity of thin filament to
Ca2+. While apparently complex neurohumoral regulation
during cardiac deconditioning reduces the contractility of the cardiac
muscle, this counteracting mechanism may have contributed to the
preserved Ca2+ sensitivity in the cardiac muscle of 4-week
tail suspension rats when the contractile force and velocity were
decreased (Figs. 1 and 2).
Role of Thin Filament Regulation in the Functional Adaptation of
Cardiac Muscle--
The up-regulation of the NH2-terminal
truncated cTnI in the heart of tail suspension rats indicates that the
function of thin filament may play a role in the adaptation of cardiac
muscle in microgravity. For example, the proteolytic modification of
cTnI may alter the responsiveness of the cardiac muscle to
-adrenergic regulation that plays an important role in myocardial
adaptations to physiological and pathological stresses (56-58). It is
worth noting that the arterial blood pressure and maximum left
ventricular pressure are not changed in the tail suspension rats, and
the afterload may stimulate a positive regulation at the myofilamental levels, while the systemic neurohumoral regulation in the
tail-suspended rat would reduce cardiac function in simulated
microgravity. The deletion of the NH2-terminal PKA
phosphorylation sites from cTnI may produce a
-adrenergic-independent domain of the cardiac muscle thin filament,
which may be important in preserving cardiac function during prolonged
exposure to microgravity. Therefore, the NH2-terminal truncation of cTnI represents a novel proteolytic regulation of cardiac
muscle contractility. This finding suggests that a selective inhibition
of the NH2-terminal phosphorylation of cTnI may be a
potential target for the prevention of cardiovascular dysfunction of
astronauts during and after long space flights. By increasing the
sensitivity of cardiac myofilament to Ca2+ activation under
elevated
-adrenergic stimulation, the proteolytic modification of
cTnI may also play a role in myocardial adaptation under other
physiological and pathological conditions, such as in chronic
bedridden, paraplegic, and heart failure patients (7, 28, 29, 59).