Articles in PresS. J Appl Physiol (January 15, 2009). doi:10.1152/japplphysiol.90660.2008
Effects
of
fatiguing
jumping
exercise
on
mRNA
expression of titin-complex proteins and calpains
Maarit Lehti1, Riikka Kivelä1,2, Paavo Komi2, Jyrki Komulainen1,2,
Heikki Kainulainen1,2, Heikki Kyröläinen2
LIKES research Center for Sport and Heath Sciences, Rautpohjankatu 8, FIN-40700
Jyväskylä, Finland
2
Neuromuscular Research Center, Department of Biology of Physical Activity, P.O: Box
35, FIN-40014 University of Jyväskylä, Finland
Running head:
Exercise, titin-complex proteins and calpains
Correspondence:
Maarit Lehti
LIKES Research Center
Rautpohjankatu 8
FIN-40700 Jyväskylä
Finland
e-mail: [email protected]
phone: +358 14 260 2056
fax: +358 14 260 1571
Copyright © 2009 by the American Physiological Society.
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1
Abstract
Eccentric exercise induced by electrostimulation increases mRNA expression of titincomplex proteins in rodent skeletal muscle. In this study, mRNA expression of titin,
muscle LIM protein (MLP), cardiac ankyrin repeat protein (CARP), ankyrin repeat
domain protein 2 (Ankrd2), diabetes-related ankyrin repeat protein (DARP) and calciumactivated proteinases, calpains, were investigated in human skeletal muscle after fatiguing
jumping exercise. Fatiguing jumping exercise did not change mRNA expression of titin,
2 days post exercise. CARP mRNA level was already elevated 30 min and remained
elevated 2 days post exercise. Increased mRNA expression of MLP, CARP, and Ankrd2
– observed for the first time in human skeletal muscle - may be part of the signaling
activated by physical exercise. The rapid increase in the level of CARP mRNA level
nominates CARP as one of the first genes to respond to exercise. The increase in the
mRNA level of calpain 2 suggests its involvement in myofiber remodeling after
strenuous jumping exercise.
Key words: titin, muscle LIM protein, muscle ankyrin repeat protein, calpain, stretchshortening cycle, mRNA expression
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DARP, calpain 1 or calpain 3. MLP, Ankrd2 and calpain 2 mRNA levels were increased
1
Introduction
Physical exercise induces remodeling process in skeletal muscle tissue. One of the
triggers of adaptation is mechanical loading. Recent studies have shown titin and titininteracting proteins to participate in the sensing of mechanical stress and strain during
muscle action (16, 30, 40). Titin is the only molecule that extends over half a sarcomere
and during muscle action it maintains temporal and spatial assembly of the contractile
filaments (48). At Z-disc, titin interacts with titin cap (T-cap), which is a linking protein
as CSRP3) and myostatin (29). Interaction of MLP with myogenic regulatory factors
(MRFs) in the nucleus and its effect on expression of brain natriuretic peptide (BNP) and
atrial natriuretic factor (ANF) makes MLP a possible stretch regulatory protein (16). The
central I-band of titin interacts with calpain 3 and with three muscle ankyrin repeat
proteins (MARPs): cardiac ankyrin repeat protein (CARP; also known as Ankrd1),
ankyrin repeat domain protein 2 (Ankrd2), and diabetes-related ankyrin repeat protein
(DARP; also known as Ankrd23). MARPs are induced under different stress conditions
(16). Titin-interacting calpains (3 and 1) as well as calpain 2 are calcium-activated
proteinases. The ubiquitous calpains (1 and 2) are thought to be responsible for the
release of myofibrillar proteins (e.g. actin and myosin) from sarcomeres, which is
followed by ubiquitination and degradation by proteasome (6). In contrast to ubiquitous
calpains, calpain 3 activity correlates negatively with muscle degradation (13). Calpain 3
is thought to have a role in sarcomere maintenance in mature muscle cells (13).
In the context of exercise, titin and titin-complex proteins have been little studied. The
response of these proteins to the exercise is interesting already because of their location
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for several signaling and structural proteins, e.g. muscle LIM protein (MLP; also known
2
in the center of muscle action and their regulative possibilities related to that. It has been
shown that in animal muscles mRNA levels of MLP, CARP, Ankrd2, and DARP are
already elevated a few hours after exercise (19, 31), and knock-out studies of these
proteins suggest their involvement in structural and regulatory roles in skeletal muscle
(4). However, little is known about the effect of physical exercise on these proteins in
human muscle. Titin staining has been found to disappear from some myofibers within 3
days after eccentrically-biased exercise (54) and protein content to be reduced by 30 % in
human skeletal muscle 24 h post exercise (47). Calpains are known to respond
exercise on MARPs or MLP has not been studied in human skeletal muscle.
The purpose of the present study was to examine the effects of high mechanical loading,
which was induced, in particular in the thigh muscles, by jumping exercise, on the
expression of titin, calpains and four other titin-complex proteins, MLP, CARP, Ankrd2,
and DARP, in human skeletal muscle. The stretch-shortening cycle type of exercise was
selected, because it well represents natural movement. We hypothesized that mechanical
stress would induce a myofiber remodeling process, which would include increased MLP
and MARPs expression, as found after pure eccentric muscle actions in animal muscles,
and changes in the expression of calpains as the proteolytic modelers of myofibers.
Materials and methods
Subjects
Eight male subjects (mean ± SD age 26 ± 4 years) volunteered for the study. Their body
mass and height were 78 ± 9 kg and 1.79 ± 0.07 m, respectively. None of the subjects
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differentially to eccentric muscle action at the mRNA level (14). The effect of physical
3
were involved in systematic training during the study or the year immediately before it.
All subjects were informed of the purpose, nature, and potential risks of the study prior to
giving their written consent. The study was performed according to the Declaration of
Helsinki and was approved by the Ethical Committee of the University of Jyväskylä.
Exercise protocol
After a 5-min warm-up period on a bicycle ergometer maximum voluntary contraction of
both legs was measured unilaterally. Thereafter, optimal dropping height on a special
subjects performed 100 maximal unilateral drop jumps at a frequency of one every 5 s.
Maximal jumps were immediately followed by continuous jumping to a submaximal
height (50 % of individual maximum) until exhaustion. Exercise was performed by the
right leg, while the left leg served as a control. Immediately and 48 h post exercise the
isometric maximal force of both legs was measured with a motorized isokinetic
dynamometer. Serum creatine kinase (CK) activity was measured immediately and 48 h
post exercise with a commercial test kit (Biochemical Boehringer, Mannheim, Germany)
from venous blood. Lactate concentration was analyzed (LactatePro, Minami-Ku, Kyoto,
Japan) from fingertip blood samples before and immediately after exercise.
Muscle biopsies
Muscle needle biopsies were obtained 30 min and 2 d post exercise from the middle
region of the vastus lateralis muscle of the exercised right leg under local anesthesia
(Lidocain-epinephrine, 1%). Time points were chosen to study immediate response to
exercise and response during recovery phase after possible micro-injury. The control
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sledge ergometer (28) (inclined at 20.3˚ from the horizontal) was determined and the
4
biopsy was taken from the non-exercised left vastus lateralis muscle 2 d post exercise.
Muscle samples were immediately frozen in liquid nitrogen and stored at –80 °C for later
analysis.
Total RNA extraction and quantitative PCR measurements
Total RNA was extracted from the biopsy sample with Trizol Reagent (Invitrogen,
Carlsbad, CA, USA) according to the manufacture’s instructions. Total RNA
concentration was measured photometrically at 260 nm and the purity of RNA assessed
transcribed using a High-Capacity cDNA Archive kit (Applied Biosystems, Foster City,
CA, USA) according to the manufacturer’s instructions. TaqMan Universal PCR Master
Mix (Applied Biosystems) and TaqMan Gene Expression assays (Applied Biosystems)
were used to analyze mRNA levels of titin (Hs00399225_m1), MLP (Hs00185787_m1),
CARP (Hs00173317_m1), Ankrd2 (Hs00220469_m1), DARP (Hs00329135_m1),
calpain
1
(Hs00559804_m1),
calpain
2
(Hs00156251_m1),
and
calpain
3
(Hs00544982_m1). The ABI Prism 7300 Sequence Detection System (Applied
Biosystems) was used to perform real-time PCR reactions.
Expression data were
normalized to the mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH;
Hs99999905_m1) and 18S rRNA (Hs99999901_s1). These two normalizations gave
nearly identical results. Previously GAPDH was found to be the most stable of the
housekeeping genes normally used in exercise studies (10, 20). The data normalized to
the mRNA level of GAPDH is presented in results. All samples were analyzed in
triplicate.
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on the basis of a 260 nm 280 nm-1 absorption ratio of ~2,0. Total RNA was reverse-
5
Titin SDS-PAGE
The distribution of titin isoforms was analyzed as previously described (27). In brief, a
sample of thin sections from the biopsies were treated in 60˚C lysing buffer for 10 min
and loaded on a SDS-PAGE gel (3.3% to 12.0% linear gradient, Fairbanks buffer). Gels
were run at 70 V for 24 h at RT and silver stained (Silver Stain Plus, Bio-Rad
Laboratories, Hercules, CA). Titin bands were identified with titin antibody (Sigma) and
an avidin-biotin peroxidase kit (Vector Laboratories, USA ) from the PVDF membrane
Statistical methods
Statistical comparisons between the control and exercise samples were performed with
the Friedman and Wilcoxon signed ranks tests. The level of statistical significance chosen
for the analyses was 0.05.
Results
The subjects performed an average of 876 ± 609 jumps (maximal + submaximal) during
the exercise protocol which lasted on average 19.3 ± 13.2 min. The isometric maximal
force of the exercised leg showed a decrease (P < 0.05) at 30 min post exercise (847 ± 92
vs. 584 ± 86 N), returning close to the preexercise levels at 48 h after the exercise (727 ±
125 N; P = 0.05). There were no changes in isometric maximal force in the control leg.
Blood lactate concentration increased from 1.4 ± 0.2 mM pre exercise to 9.9 ± 3.5 mM
post exercise (P < 0.05). Increased mean serum CK activity was observed immediately
(350 ± 244 pre and 443 ± 306 IU/l, P < 0.05) and 48 h (824 ± 843 IU/l; P = 0.069) post
the exercise.
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after overnight blotting (25 V).
6
The titin SDS-PAGE results revealed that one titin band was observed in seven subjects,
whereas two titin bands were observed only in one subject. Fatiguing jumping exercise
did not induce statistically significant change in titin mRNA expression 30 min or 2 d
post exercise (figure 1A). The mRNA expression of MLP varied between subjects at the
30 min time point and was significantly increased 2 d post exercise (P < 0.05; figure 1B).
The mRNA expression of CARP was already increased 30 min post exercise and
remained elevated 2 d after the exercise (figure 1C). The CARP mRNA level was higher
CARP mRNA was at least 4.6-fold higher in the experimental compared to control
subjects, and in one subject the peak level was over 30-fold greater than the control level
at the 2 d. Ankrd2 transcription was activated 2 d post exercise (figure 1D). The mRNA
level of Ankrd2 increased by at least 2-fold from 30 min to 2 d post exercise in all
subjects rising to a mean of 4-fold over the control level 2 d after the exercise. In DARP
mRNA expression no statistically significant change was observed at any time point,
although large variation in the mRNA levels at the 30 min and 2 d time points were found
(figure 1E).
Statistically significant changes were not observed in the mRNA expression of calpain-1
or calpain-3 at the studied time points (figure 2A, 2C). However, individual changes of
over 4-fold were observed in the mRNA level of calpain-1. The mRNA expression of
calpain 2 showed a significant increase 2 d post exercise (figure 2B).
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at the 30 min than 2 d time point in five of the eight subjects. The measured peak level of
7
Discussion
The present exercise model was chosen to exert high mechanical loading on muscle
fibers. In jumping exercise, both eccentric and concentric muscle work is present as the
fibers undergo repeated stretch-shortening cycles. Similar muscle work also occurs in
natural human motion, e.g. walking. In the present exercise the loading was, however,
high and exercise can be considered strenuous, since immediately after the exercise blood
lactate concentration was elevated and isometric maximal force was markedly decreased.
(21). Slight swelling of the myofibers observed 2 d post exercise may be a sign of the
ongoing remodeling of muscle fibers (54) in exercised muscle. The present results
showed for the first time that high mechanical loading induced mRNA expression of
MLP, CARP, Ankrd2, in addition to, calpain 2 in the human vastus lateralis muscle. The
response of CARP was extremely rapid since the increased expression was observed as
early as 30 min post exercise.
Protein adaptation was studied at the transcriptional level assuming that changes in
transcriptional program reflect well adaptation to altered environment, and, as shown in a
previous animal study (5), mRNA changes were expected after mechanical loading.
Expression and activity of these proteins may be regulated also at the translational level
both by phosphorylation/dephosphorylation and by localization in the cell. Therefore,
present results open the discussion of the presumable importance of MLP, CARP and
Ankrd2 in human skeletal muscle after the physical exercise.
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In the histological examination of the muscle biopsies, no damaged fibers were noted
8
No changes observed in titin mRNA expression
Titin is located in between the contractile filaments in the muscle sarcomere (48). During
muscle contractions it maintains the order of thin and thick filaments and is subjected to
high stretching forces during eccentric muscle action. Thus it is not surprising that after
eccentric muscle action remodeling of titin structures is observed in animal and human
skeletal muscle (15, 32, 54). After lengthening contractions abnormal staining of titin was
observed consistently with unorganized contractile filaments in animal studies (32, 45).
after eccentrically-biased exercise (54) and protein content falls by 30 % 24 h post
exercise (47). In this study, we did not see any changes in the mRNA expression of titin.
In a recent study, however, decrease in titin mRNA expression was observed 1 h after
mild eccentric exercise (22). The present results are well in line with our previous
observation from an animal study where titin mRNA expression did not change between
3 and 96 h after a single downhill running session but was increased 3 h after repeated
exercise (31). The results together suggest that titin mRNA level is first decreased shortly
after the eccentric exercise and possibly increased much later. Reappearance of titin can
be clearly observed by microscopy after 7-8 days post exercise (54).
Titin isoform composition is interesting since it determines the passive mechanical
properties of the sarcomere (48). In the present as in our previous study (27), we
observed that one of the 8 subjects had a different titin isoform composition (represented
by two bands on SDS-PAGE). The subject with a different titin isoform composition
showed the largest change in titin mRNA expression (1.4 times the control value) as well
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In human skeletal muscle, titin staining disappears from some myofibers within 3 days
9
as CARP mRNA level at 30 min and Ankrd2 mRNA level at 2 d. This result is interesting
in the light of a recent suggestion that MARPs may alter titin isoform expression (4).
CARP response to exercise is extremely rapid
As suggested in recent reviews (16, 23, 35), titin is the probable backbone of striated
muscle stress and stretch-sensing structures. One member of stretch-sensing structures is
CARP which interacts with titin at the I-band. This is the first study to show increased
CARP mRNA expression after physical exercise in human skeletal muscle. In rodents,
actions (5) and 3 hours after prolonged concentric contractions (34). We observed
increased CARP mRNA expression as early as 30 min post exercise, which can be
regarded as an extremely rapid response. Increase in CARP mRNA expression is as fast
as that of the immediate early genes c-Jun and c-Fos (38) or that of some ubiquitin ligases
(33). This suggests a role for CARP early in adaptation after physical exercise.
The up-regulation of CARP is known to activate angiogenesis (43) and muscle
hypertrophy is known to be accompanied by capillary growth (17). Our previous
observation of increased angiogenic and ECM remodeling proteins Cyr 61 and CTGF in
the same muscle samples from the same experiment (21) supports the idea that the
angiogenesis is activated.
Increase in MLP mRNA expression may be a part of myogenic
response
MLP interacts with titin through T-cap In rodents, increased mRNA expression of MLP
has been observed 6 hours after lengthening muscle actions (5) and 3 hours after
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increased mRNA expression CARP has been observed 6 hours after lengthening muscle
10
prolonged concentric contractions (34). In the present study, the increase in the mRNA
levels of MLP was observed 2 d post exercise. This is the first study to show increased
MLP mRNA expression after physical exercise in human skeletal muscle. A recent study
additionally suggests that MLP promotes myosin heavy chain expression and especially
slow myosin heavy chain is up-regulated if MLP is co-expressed with calsineurin (12).
Since MLP is also located in the nucleus (1, 2) it may regulate the transcriptional
processes involved in regeneration and/or adaptation to increased physical loading. In the
nucleus, MLP enhances the binding of MRFs to DNA (25). Interestingly, a few hours
Myf-5 and MyoD are early markers of myogenesis (42). Thus, the increase in MLP
mRNA levels may be a step towards myogenesis after physical exercise.
Ankrd2 mRNA expression is activated after exercise in human
skeletal muscle
Ankrd2, which is another member of the MARP protein family in addition to CARP, is
recently shown to translocate from I-band to the nucleus in response to muscle damage
(49). In the nucleus, Ankrd2 can interact with nuclear proteins including p53 and YB-1
(24). Furthermore, a recent cell culture study suggests that Ankrd2 may coordinate
myocyte differentiation and apoptosis during myoblast proliferation and also stabilize
forming myotubes (7). Previously, mRNA expression of Ankrd2 was shown to increase
after lengthening contractions in mouse skeletal muscle (5). In this study, the increase
was observed for the first time in human muscle after exercise. The increase in Ankrd2
mRNA level may be a part of myogenic response after physical exercise.
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after exercise, the transcription of MRFs is activated (3, 9, 39, 46, 53). MRFs such as
11
No changes in DARP mRNA expression
DARP is the third member of the MARP protein family known to be induced in different
stress conditions (16). Expression of DARP is activated in insulin-resistant skeletal
muscle (18). The authors suggest that DARP expression is activated in insulin-resistant
animals to compensate for fat accumulation in muscle. In this study, there was no change
in DARP mRNA expression. In a previous study, DARP mRNA level was observed to
increase after lengthening contractions followed by a decrease 2 days later in mouse
lateral leg, which may explain the difference between the present and their results.
Increase in calpain 2 mRNA expression may be related to exercise
induced muscle remodeling
Nonlysosomal Ca2+ -activated cystein proteases, calpains, are known to degrade
cytoskeletal and myofibrillar proteins (8). It has been proposed that calpain-induced
myofibril cleavage is a preliminary step in muscle remodeling conditions (like atrophy or
hypertrophy), allowing either further degradation of myofibrillar proteins or the
qualitative remodeling of the muscle (6). Of three calpains, expressed in skeletal muscle,
calpains 1 and 2 (also called µ- and m-calpain) are ubiquitous calpains and calpain 3 (also
called p94) is an inducible member of this protein family. In the previous studies, calpain
3 mRNA expression has been shown to decrease after eccentric muscle action (14) but
increase 24 h after a bout of eccentric exercise (36). In this study no changes were
observed in the mRNA levels of calpain 1 and calpain 3, although a decreasing trend in
calpain 3 mRNA expression was seen. Calpain 2 mRNA expression increased 2 d after
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muscle (5). These results also showed changes in DARP mRNA expression in the contra
12
the exercise. Previously, increased calpain 2 mRNA and protein expression has been
detected after muscle unloading followed by reloading and also 1 day after eccentric
muscle action (14, 44). In exercising muscle, ubiquitous calpains may be activated by
phosphorylation even though the Ca2+ environment may not reach the level required to
activate them (51, 52). Interestingly, these phosphorylating kinases are known to be
activated in stretch and physical training in skeletal muscle (26, 37). In addition to
degrading cytoskeletal proteins (6), calpain 2 is involved in myoblast fusion as a part of
satellite cell activation (8). Increased ubiquitous calpain activity is redistributed to the
proliferating satellite cells (6). We suggest that the observed increase in calpain-2 mRNA
may be a part of the muscle remodeling process (e.g. myoblast fusion) activated by the
jumping exercise.
Summary
The main novel finding of this study is the increased mRNA expression of MLP, CARP
and Ankrd2 in human skeletal muscle induced by physical exercise. This observation
concur with previous studies of eccentric muscle action in rodents. Increased mRNA
expression of these genes and calpain-2 suggests that they may constitute a part of the
signaling cascades activated by physical exercise. This idea is supported by the
localization of all these proteins in the nucleus in addition to cytosol (1, 2, 24, 41). The
notably rapid increase in CARP mRNA level nominates CARP as one of the first genes
to respond to acute exercise. In a recent study, myogenesis and angiogenesis were shown
to co-exist and were suggested to be co-regulated (11). Interestingly, increased MLP and
CARP expression is reported in angiogenic as well as myogenic conditions (43, 50).
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cell membrane instead of cytosol after running exercise and in the mucleus of the
13
These observations together with ours nominate MLP and CARP as possible coregulators of angiogenesis and myogenesis after strenuous physical loading.
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14
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Figure legends
Figure 1. Quantitative PCR measurements of titin (Ttn, A), muscle LIM protein (MLP,
B), cardiac ankyrin repeat protein (CARP, C), ankyrin repeat domain 2 (Ankrd2, D), and
diabetes-related ankyrin repeat protein (DARP, E) mRNAs presented as fold change from
control values. Values are mean ± SD. * (P<0.05) compared to control; # (P<0.05)
compared to mRNA expression at 30 min.
Figure 2. Quantitative PCR measurements of calpains 1 (CAPN1, A), 2 (CAPN2, B), and
SD. * (P<0.05) compared to control; # (P<0.05) compared to mRNA expression at 30
min.
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3 (CAPN3, C) mRNAs presented as fold change from control values. Values are mean ±
A
B
#
C
D
#
E
A
B
#
C