Responses of muscle mass, strength and gene transcripts to long-term heat stress in healthy human subjects
Katsumasa Goto . Hideshi Oda . Hidehiko Kondo . Michihito Igaki . Atsushi Suzuki .
Shuichi Tsuchiya . Takatoshi Murase . Tadashi Hase . Hiroto Fujiya . Ichiro Matsumoto .
Hisashi Naito . Takao Sugiura . Yoshinobu Ohira . Toshitada Yoshioka
Abstract The present study was performed to investigate
the effects of long-term heat stress on mass, strength and
gene expression profile of human skeletal muscles without
exercise training. Eight healthy men were subjected to
10-week application of heat stress, which was performed
for the quadriceps muscles for 8 h/day and 4 days/week by
using a heat- and steam-generating sheet. Maximum
isometric force during knee extension of the heated leg
significantly increased after heat stress (*5.8%, P\0.05).
Mean cross-sectional areas (CSAs) of vastus lateralis (VL,
*2.7%) and rectus femoris (*6.1%) muscles, as well as
fiber CSA (8.3%) in VL, in the heated leg were also
significantly increased (P\0.05). Statistical analysis of
microarrays (SAM) revealed that 10 weeks of heat stress
increased the transcript level of 925 genes and decreased
that of 1,300 genes, and gene function clustering analysis
(Database for Annotation, Visualization and Integrated
Discovery: DAVID) showed that these regulated transcripts
stemmed from diverse functional categories. Transcript
level of ubiquinol-cytochrome c reductase binding
protein (UQCRB) was significantly increased by 10 weeks
of heat stress (*3.0 folds). UQCRB is classified as one of
the oxidative phosphorylation-associated genes, suggesting
that heat stress can stimulate ATP synthesis. These results
suggested that long-term application of heat stress could be
effective in increasing the muscle strength associated with
hypertrophy without exercise training.
Introduction
Skeletal muscles exhibit a remarkable plasticity to adapt to
muscular exercise training resulting in hypertrophy (Flu¡§ck
and Hoppeler 2003; Schiaffino et al. 2007). Molecular
mechanisms responsible for skeletal muscle hypertrophy in
response to various mechanical stimuli (stresses), such as
physical exercise, strength training and/or stretch, have not
been fully elucidated; nevertheless, several hypotheses
have been proposed. It has been proposed that mechanical
loading, such as stretch, is one of the primary factors
regulating the synthesis of proteins in skeletal muscles
(Goldspink et al. 1983; Ohira and Edgerton 1997;
Vandenburg 1987). Mechanical overloading and/or some
other unknown exercise stimuli would stimulate protein
biosynthesis (Adams and Haddad 1996), resulting in
muscular hypertrophy. Although several molecules involving
stress-induced muscle hypertrophy have been proposed,
the role of elevated muscle temperature during muscular
training in muscular hypertrophy is not still clear.
Muscular exercise causes an increase in core body and
muscle temperature (Harris and Starnes 2001). Elevation of
muscle temperature during muscle contraction is induced by
free energy derived from the hydrolysis of adenosine triphosphate,
independent of exercise stimuli and mechanical
loading. Expression of heat shock proteins (HSPs) in skeletal
muscles is also up-regulated by muscular exercise (Febbraio
and Koukoulas 2000; Naito et al. 2001; Puntschart et al.
1996). However, the precise mechanism responsible for the
exercise-induced HSP expression has not been elucidated yet.
HSPs, such as HSP72, the inducible form of HSP70, and
HSP90, are also induced by heat stress (Craig et al. 1993;
Morimoto 1993; Skidmore et al. 1995). HSP72 and HSP90
function as important molecular chaperones (Becker and
Craig 1994; Burel et al. 1992; Kilgore et al. 1998; Sass et al.
1996; Wegele et al. 2004; Welch 1991; Welch 1993).
Therefore, cellular proteins can be stabilized following the
up-regulation of HSP72 and HSP90, and protein synthesis
may be facilitated. Over-expression of HSPs is thought to be
beneficial for the protection against occlusion.reperfusioninduced
cardiac injuries (Jayakumar et al. 2001; Latchman
2001; Okubo et al. 2001; Senf et al. 2008). It is also reported
that heat stress could partially prevent muscular atrophy
induced by unloading (Naito et al. 2000).
Recently, we have confirmed that light-intensity exercise
combined with heat stress increased the volume and
strength of human biceps muscles (Goto et al. 2007). It was
also reported that the application of heat stress-induced
hypertrophy in cultured muscle cells (Goto et al. 2003), as
well as the rat skeletal muscles in vivo (Kobayashi et al.
2005; Uehara et al. 2004), facilitated the recovery of muscle
atrophy following unloading (Goto et al. 2004). Evidences
obtained in our previous study suggested that the application
of heat stress may stimulate the protein synthesis via
insulin-related signaling pathway (Uehara et al. 2004). It
has been reported that gene reprogramming of stressorspecific
dynamic profiles, without improved force generation,
in soleus muscle was induced by heat stress at 34C for
30 days (Kodesh and Horowitz 2010). However, the
mechanism responsible for the heat stress-associated muscle
hypertrophy in humans is not clearly known.
It was hypothesized that muscle hypertrophy, which may
improve strength development, might be induced, if heat
stress could activate a biological pathway, such as insulinrelated
signaling pathway. Therefore, the present study was
performed to test our hypothesis that application of heat
stress could induce hypertrophy and increase the strength
development in human skeletal muscles, even without
exercise training. Changes of the cross-sectional area (CSA)
in quadriceps muscles and isometric torque with or without
10-week application of heat stress were investigated.
Differential expression of genes in muscle tissue in vivo
was also investigated to study the mechanisms responsible
for the muscular adaptation to heat stress.
Materials and methods
Subjects
Eight healthy men participated in the study
Discussion
This study showed that long-term heat stress induced an
increase of the maximum isometric torque of knee
extension, associated with increased muscle and muscle
fiber CSA and changes in gene expression pattern in
quadriceps muscle of human male subjects. During heat
application, the temperature of vastus lateralis muscle
was increased up to *38C using a heat- and steamgenerating
sheet.
Heat stress-associated muscle hypertrophy
It has been generally believed that exercise training with
intensity less than 65% of 1 repetition maximum (1 RM) is
not useful for the improvement of muscular size and
strength (McDonagh and Davies 1984). However, increase
of force development and muscular hypertrophy were
induced by application of heat stress, even without exercise
training, in the present study. This result is consistent with
our previous studies that showed the heat stress-associated
muscle hypertrophy in cultured cells and muscle of rats
without other stimulations (Kobayashi et al. 2005; Uehara
et al. 2004). Heat stress-associated muscle hypertrophy is
caused by increased CSA of single muscle fibers (Uehara
et al. 2004). These results suggest that heat stress might
stimulate the intracellular signaling(s) contributing to
protein synthesis. On the other hand, heat stress at 42C
inhibits muscle hypertrophy induced by functional overloading
(Frier and Locke 2007). This discrepancy might be
derived from the level of temperature. It has been also
reported that muscle hypertrophy was induced in response
to heating at over *38C for more than 45 min in rats
(Goto et al. 2005). Such phenomena were related to the
activation of satellite cells (Uehara et al. 2004; Kojima
et al. 2007; Piestun et al. 2009).
Muscular strength and size were increased 5.8% and
2.7.6.1% significantly following 10 weeks of heat stress,
respectively. It has been also reported that muscle hypertrophy
was induced in response to heating at over*38C for
more than 45 min in rats (Goto et al. 2005). It was also
reported that the size and strength of quadriceps muscle
increased by*3 and*19% following 12 weeks of strength
training at 15.5% of 1RM in healthy young men (*25 years
old) (Holm et al. 2008). These values are comparable. But,
the strength training at 70% of 1 RMincreased the muscular
size and strength by *8 and *36% (Holm et al. 2008).
Following long-term (10-week) heat stress, hypertrophy
of muscle and muscle fibers was observed. However, it was
suggested that such hypertrophy may be closely related to
the increase of myonuclear number (Fig. 2a). Further, the
functional capacity (maximum isometric torque) was also
increased. Although, the water content in muscle was not
measured because of the small sample size (*10 mg), it
was suggested that the heat stress-related retention of water
might be minor (Goto et al. 2005; Kobayashi et al. 2005;
Uehara et al. 2004), and the fibers are not swollen abnormally,
either (Fig. 2). Therefore, the heat stress-related
improvement of morphological and functional properties of
muscle is physiologically significant.
In the present study, the insignificant increases of the
mean maximum isometric torque during knee extension
(*4%) in the non-heated leg were observed. It has been
also reported that unilateral knee extension resistance
training (8 weeks, 6.12 RM) induced an increase of
muscle strength not only in the trained, but also in the
contralateral leg (Holm et al. 2008). Therefore, the crosstransfer
of heat stress effect might be induced in the present
study also, though it might be minor.
Genes in response to long-term heat stress
Long-term heat stress induced a significant increase in the
relative transcriptional level of 925 genes and a significant
decrease in that level of 1,300 genes in heated muscles,
compared with pre-heating. However, the up-regulations of
the genes related to both myofibrillar and heat shock proteins
were not observed after 10 weeks of heat stress in the
present study.
Myofibril-associated genes
It has been reported that resistance training, as well as
endurance training, could up-regulate gene transcription of
myosin heavy chain (Balagopal et al. 2001; Friedmann
et al. 2004; Willoughby and Nelson 2002). On the contrary,
the other report described that myosin heavy chain IIx
(MYH1) gene was down-regulated following a long-term
resistance training (Balagopal et al. 2001). Although there
was no report indicating the long-term heat stress-associated
up-regulation of myofibril-related genes in skeletal
muscles, heat stress (at 47C for 30 min) on C2C12 mouse
myotubes induced acute and transient up-regulation in
transcriptional level of the myofibril-related genes within
24 h (Szustakowski et al. 2007). These observations suggested
that the up-regulation of the myofibril-related genes
might be induced in an early phase (*several days or
weeks) of heating. However, these phenomena were not
evaluated in the present study, because gene expression
analyses were performed in muscle tissues sampled 1 day
after the last bout of heating. It was thus speculated that the
major cause of these adaptations might be due to chronic
effects of heating, because hypertrophy and increased
strength of muscles were clearly noted.
Transcriptional up-regulation of the genes involved in
both protein synthesis and the proteolysis was observed in
the phase of muscle damage during reloading of atrophied
rat soleus muscle (Flu¡§ck et al. 2005). Nevertheless, during
the regrowth phase in response to reloading, the genes
involved in oxidative metabolism, such as fatty acid
transporter, mitochondrial respiratory chain constitutions,
beta-oxidation and voltage-gated cation channels, were
up-regulated and correlated with muscle mass during the
regrowth from disuse-related muscle atrophy (Flu¡§ck et al.
2005). The present study was performed to investigate the
differential gene expression in response to the long-term
heat stress at a steady state, but not acute phase. Therefore,
it was speculated that any up-regulated changes in
transcriptional level of protein synthesis genes, e.g.,
ribosomal factors, which would induce an increase in
muscle mass, might occur in the early phase during the
10-week heating.
Heat shock proteins
None of the HSP genes was significantly up-regulated by
long-term heat stress. The HSPs function as molecular
24 Eur J Appl Physiol (2011) 111:17.27
123
chaperons and repair the proteins having mis-folding errors
(Becker and Craig 1994; Burel et al. 1992; Wegele et al.
2004; Welch 1991; Welch 1993). It has been reported that
there was no up-regulation of HSP72 protein in skeletal
muscles of rats, if the colonic temperature was less than
38C during 1-h heating (Goto et al. 2005). The results
seemed to be consistent with the ones from the current
study, since it has been generally considered and reported
that the up-regulation of HSP72 protein in cells would be
induced when the body temperature was elevated by 3.5C
(Kilgore et al. 1998), and since the temperature of the
vastus lateralis muscle was increased up to*38C and was
maintained at least for 5 h in our study.
Genes that were affected significantly
Transcription level of ubiquinol-cytochrome c reductase
binding protein (UQCRB) gene was significantly increased
by 10 weeks of heat stress (*3.0-fold). UQCRB is classified
as one of the oxidative phosphorylation-associated
genes, suggesting that the long-term heat stress could have
stimulated ATP synthesis. It was unclear why the oxidative
phosphorylation-related gene was up-regulated, but one
possibility might be that this kind of gene response triggered
the increased potential of ATP supply for prolonged
muscle activity (Table 4).
Interestingly, titin (TTN) and insulin-like growth factor
2 receptor (IGF2R) gene expressions were significantly
decreased by the long-term heating (*0.5-fold). Titin,
so-called connectin, is sarcomeric structural filaments of
muscle cells. Influences due to hypertrophic stimuli on
the transcription on TTN gene are still unknown. There
were several reports explaining that exercise training
depressed transcription of TTN gene in mammalian
skeletal muscles (Lehti et al. 2007; Velders et al. 2008),
and these results might be consistent with our current
study. On the other hand, the IGF2R-dependent signaling
pathway was suspected to be involved in the induction of
cell apoptosis in the myocardium (Chu et al. 2009).
Overall, the physiological significance(s) of the decrease
in the transcription level of TTN and IGF2R genes is still
unclear.
The genes involved in transcriptional regulation, such as
PHF20 (plant homeodomain finger protein 2) and TFDP2
(transcription factor Dp-2/E2F dimerization partner 2),
were also affected by long-term heat stress and their gene
transcription levels were increased. TFDP2 is known to
associate with E2F and is involved in DNA synthesis
(Hitchens and Robbins 2003).
One of the cytoskeleton regulating genes, Traf2- and
Nck-interacting kinase (TNIK) was also affected and its
transcription was increased by *2 folds. TNIK interacts
with both TNF (tumor necrosis factor) receptor-associated
factor 2 (TRAF2) and the adapter protein Nck (Fu et al.
1999). TNIK was shown to phosphorylate gelsolin, the
principal intracellular and extracellular actin-severing
protein, in vitro. An optimistic speculation could be made
that in such a way long-term heat stress might enhance the
synthesis of cytoskeleton-related proteins.
Conclusion
Long-term heat stress without exercise training induced
increases of the maximum isometric torque in knee
extension and CSA of quadriceps muscle and muscle fibers
of human male subjects. Heat stress also increased the
transcription of 925 genes and decreased transcription of
1,300 genes, among them were found some obviously
useful genes. Future studies may help to further understand
mechanisms of mass increase and strength development of
muscles. Additionally, the results and findings obtained
from this study suggested that long-term heat stress might
be a useful tool for prevention of muscle atrophy caused by
inactivity and/or sarcopenia.
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