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1 Department of Kinesiology, 2 School of Pharmacy, 3 Center for Neuroscience, 4 Molecular and Environmental Toxicology Center, and 5 Waisman Center, University of Wisconsin, Madison, Wisconsin 53706
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Previous studies have
shown that endurance exercise training increases myocardial
contractility. We have previously described training-induced
alterations in myocardial contractile function at the cellular level,
including an increase in the Ca2+ sensitivity of tension.
To determine the molecular mechanism(s) of these changes,
oligonucleotide microarrays were used to analyze the gene expression
profile in ventricles from endurance-trained rats. We used an 11-wk
treadmill training protocol that we have previously shown results in
increased contractility in cardiac myocytes. After the training, the
hearts were removed and RNA was isolated from the ventricles of nine
trained and nine control rats. With the use of an Affymetrix Rat Genome
U34A Array, we detected altered expression of 27 genes. Several genes
previously found to have increased expression in hypertrophied
myocardium, such as atrial natriuretic factor and skeletal 
-actin,
were decreased with training in this study. From the standpoint of
altered contractile performance, the most significant finding was an
increase in the expression of atrial myosin light chain 1 (aMLC-1) in
the trained ventricular tissue. We confirmed microarray results for
aMLC-1 using RT-PCR and also confirmed a training-induced increase in aMLC-1 protein using two-dimensional gel electrophoresis. aMLC-1 content has been previously shown to be increased in human cardiac hypertrophy and has been associated with increased Ca2+
sensitivity of tension and increased power output. These results suggest that increased expression of aMLC-1 in response to training may
be responsible, at least in part, for previously observed training-induced enhancement of contractile function.
atrial natriuretic factor; myosin regulatory light chain; calcium sensitivity
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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CHRONIC EXERCISE TRAINING is known to elicit a number of adaptations in the heart that result in improved cardiac performance. Increased maximal and submaximal stroke volume are thought to be the result, in part, of improvements in myocardial contractile performance (17). Altered force and shortening velocity properties have been demonstrated in myocardial muscle preparations (16, 36), and the contractile properties of single cardiac myocytes have been recently shown to be altered by training (5, 6, 18, 22, 39). We have recently shown that the Ca2+ sensitivity of tension was increased as a result of training in permeablized cardiac myocytes (6), a result also seen in intact myocytes (39). In addition, the length dependence of tension properties has been shown to be increased by exercise training in permeablized as well as intact myocytes (5, 22). These results suggest that training induces an adaptation within the myofibrillar contractile apparatus rather than, or in addition to, adaptations that have been hypothesized in other subcellular processes (for a review, see Ref. 17). However, there is no information available regarding training-induced changes within the contractile element that provides a mechanism for our results in single cardiac myocytes.
There are a number of molecular factors that are known to affect contractile properties in the myocardium and that might be altered in the course of an exercise training program. In addition to factors such as phosphorylation of regulatory proteins such as myosin regulatory light chain (RLC) (34) and troponin I (41), altered expression of contractile protein isoforms may provide a mechanism for training-induced enhancement of contractile performance. For example, the Ca2+ sensitivity of tension in cardiac muscle is known to be influenced by the presence of different isoforms of tropomyosin (40), troponin I and T (21, 31), and myosin light chains (MLCs) (19). There is currently no information regarding the effect of exercise training on the expression any of these contractile protein isoforms.
Analysis of gene expression using oligonucleotide microarrays allows for the simultaneous assessment of the expression levels for a large number of genes. Previous work has described the effects of exercise training on the expression of a small number of selected genes in the heart (1, 3, 12, 39), but the results of these studies have not clarified possible mechanisms for altered contractile function. In this study, we used microarray expression analysis to compare differences in expression levels for 8,800 genes in control rat ventricular myocardium compared with ventricular tissue from rats trained with an endurance training program. Results of the expression analysis revealed that training increases the expression of atrial MLC 1 (aMLC-1) in ventricular myocardium. This change in protein expression could account for a number of previously described training-induced changes in myocardial contractile function.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Exercise training protocol. Female Sprague-Dawley rats, 145-175 g initial body wt, were randomly divided into two groups (n = 9 animals/group): sedentary control and exercise trained. All animals were individually housed in a temperature- and light-controlled room (reversed 12:12-h light-dark cycle) with food and water ad libitum. Rats in the exercise-trained group underwent an 11-wk treadmill training protocol that has been previously shown to induce improvements in whole heart function (8), increases in papillary muscle isometric tension (36), and improvements in single myocyte contractile function (6). The rats were trained 5 days/wk with training initially for 15 min/day at 13 m/min and 10% grade. Duration and intensity increased weekly during weeks 1-5 until the animals were running for 1 h at 26 m/min and 20% grade. This workload level was maintained for the remainder of the 11-wk protocol. The University of Wisconsin-Madison Animal Use and Care Committee approved this protocol.
Oligonucleotide microarray experiments.
After the completion of the training program, rats were euthanized no
less than 72 h after the last exercise bout to avoid any acute
effects of exercise. Rats were anesthetized by inhalation of
methoxyflurane, and the hearts were quickly removed and placed in
ice-cold Ca2+-free relaxing solution. Atria were trimmed
away, and the ventricular tissue was blotted dry, weighed, and then
divided sagittally into three sections. The sections were quick frozen
in liquid nitrogen and stored at 
80°C for later RNA isolation. For
RNA isolation, a 30-mg piece was cut from each of the three sections
for each heart. Frozen sections were homogenized with a Tekmar
Tissumizer in TRIzol reagent (Invitrogen), and total RNA was
extracted. An RNEasy Mini Kit (Qiagen) was used to further purify total
RNA. RNA from three hearts from each group of animals was pooled to yield three control group RNA samples and three trained group samples.
Double-stranded cDNA was synthesized from the pooled total RNA using a
Superscript choice kit (Invitrogen) with a T7-(dT)24 primer
incorporating a T7 RNA polymerase promoter (Integrated DNA
Technologies). cRNA was prepared and biotin labeled by in vitro
transcription (Enzo Biochemical). Labeled RNA was fragmented by
incubation at 94°C for 35 min in the presence of 40 mM Tris-OAc (pH
8.1), 100 mM KOAc, and 30 mM MgOAc. Labeled, fragmented cRNA (15 µg)
was hybridized for 16 h at 45°C to a U34A rat genome array (Affymetrix). After hybridization, the gene chips were automatically washed and stained with streptavidin-phycoerythrin using a fluidics station. The probe arrays were scanned at 3-µm resolution using a
Genechip System confocal scanner made for Affymetrix by Aligent.
Microarray analysis.
Affymetrix Microarray Suite software (version 5.0) was used to scan and
analyze the relative abundance of each gene based on the intensity of
the signal from each probe set. Analysis parameters used by the
software were set to values corresponding to moderate stringency
(statistical difference threshold = 30, statistical ratio
threshold = 1.5). Output from the microarray analysis was merged with the Unigene or GenBank descriptor and saved as an Excel
data spreadsheet. We ran three arrays for each group (trained vs.
control). The RNA from three animals was pooled for each array, and the
comparisons were crossed such that each trained animal set was compared
with each control animal set for a total of nine comparisons (3 × 3 matrix). The comparison analysis compares each probe set on one array
to its counterpart on another array. For each comparison, the analysis
using the Affymetrix software generates a "difference call" of
either no change, marginal increase/decrease, or increase/decrease. We
then defined "increase, decrease, or no change" of expression for
individual genes based on ranking of these difference calls from the
3 × 3 matrix using a method previously described by Li and
Johnson (13). Briefly, we set no change = 0, marginal
increase = 1, increase = 2, marginal decrease = 1, and
decrease = 2. The final rank was determined as the sum of the
nine values corresponding to the difference calls, and the values
varied from 18 to 18. The cutoff value for increase or decrease was
set as ±n2 (n = 3) because of
the marginal calls. This means that a gene must be called a marginal
increase in all nine comparisons to rank 9 and be on the list.
Similarly, a gene called increased in five of nine comparisons (a
majority) would rank 10 and be on the list, whereas a gene called
increased in four of nine comparisons (a minority) would rank 8 and not
be on the list. Therefore, the cutoff in the 3 × 3 matrix would
be n2 = 9/9 (13, 14). The
reproducibility of paired comparisons was based on the coefficient of
variation (CV; SD/mean) for the fold change (FC) on the ranked genes. A
distribution curve of the CV was used to determine a CV cutoff value.
The cutoff value was CV < 0.7. Gene categorization was based on
the NetAffx database (http://www.NetAffx.com).
RT-PCR. Verification of changed expression was done by RT-PCR for two selected genes. Unique PCR primers specific for the genes of interest were designed using PRIMER3 software (available online at http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) and using sequence data from the National Center for Biotechnology Information database. Primers (Integrated DNA Technologies) were designed for aMLC-1 (5'-CCA AGC CTG AAG AGA TGA AT-3' and 5'-CCA GTA TGA GTC CAG TGC TC-3') and atrial natriuretic peptide (ANP; 5'-TTC AAG AAC CTG CTA GAC CA-3' and 5'-GCT CCA ATC CTG TCA ATC CT-3'). Single-stranded cDNA was created by RT (Promega Reverse Transcription System) from total RNA originally prepared for the microarrays. The reaction mix contained 1 µg total RNA, 0.5 µg oligo(dT)15 primer (provided), 1 mM each dNTP, and 15 units avian myeloblastosis virus reverse transcriptase. The RT proceeded for 1 h at 42°C. The PCR reaction mix contained 20% of the RT reaction, 50 pmol gene-specific primers, 200 µM dNTPs, 2 mM MgCl2, and 2.5 units Taq polymerase. DNA was amplified by an initial incubation at 94°C for 1.25 min followed by 21-27 cycles of 94°C for 0.5 min, 54°C for 0.5 min, 72°C for 0.5 min, and a final extension at 72°C for 6 min. The PCR products were separated by electrophoresis in a 1.2% agarose gel and visualized by ethidium bromide staining. Relative intensities were quantified using UN-SCAN-IT software (Silk Scientific).
Two-dimensional gel electrophoresis. To confirm the effect of training on aMLC-1 protein levels, we analyzed homogenates from control and trained heart samples using two-dimensional (2-D) gel electrophoresis. Pieces of frozen tissue from the same hearts from which RNA was isolated were homogenized at 100 mg/ml in sample buffer (8 M urea, 2 M thiourea, 75 mM DTT, and 10 mM Tris; pH 7.0) using a Fisher PowerGen 700 homogenizer with a 7-mm sawtooth generator at 20,000 rpm for 8-10 s. The protein concentration of this homogenate was determined using a Bio-Rad protein assay kit with BSA as the standard. Isoelectric focusing was performed using Bio-Rad Protean isoelectric focusing cell and 11-cm precast immobilized pH gradient (pI) gel strips (pH 3-10). Protein (300 µg) in sample buffer plus 1.85% CHAPS and 0.185% carrier ampholytes were loaded onto the strips via 1 h of passive rehydration and 12 h of active loading at 50 V and 20°C. The Bio-Rad IEF unit was programmed to rapidly ramp to 250 V for the first 15 min, rapidly ramp to 6,700-7,000 V for the next 2.5 h (limited to 50 mA/strip), and hold at peak voltage and 50 mA/strip for 35,000 V · h. Strips were held at 500 V at the conclusion of their run until removed from the power unit. Strips were incubated in equilibration buffer I [125 mM Tris · HCl (pH 6.8), 20% glycerol, 2% SDS, 6 M urea, and 2% DTT] and buffer II [125 mM Tris · HCl (pH 6.8), 20% glycerol, 2% SDS, 6 M urea, and 2.5% iodoacetamide] for 20 min each. After equilibration of the strips, second-dimension PAGE was performed using 12.5% Bio-Rad Criterion precast gels with IPG + one-well combs, run at 20 mA/gel for 45 min and 30 mA/gel for the duration of the run (2.5 h total). Gels were stained using a zinc stain (Pierce) and digitized using a Kodak Image Station 440CF. Sixteen exposures were summed to increase the signal-to-noise ratio. PDQuest software (version 6.0, Bio-Rad) was used for gel image analysis. The locations of the aMLC-1 and ventricular MLC-1 (vMLC-1) spots were determined based on the predicted isoelectric point and molecular mass of these proteins as well as by comparison to previously published 2-D gel analysis of these proteins (20, 25).
Citrate synthase assay.
Plantaris muscles were removed immediately after excision of the
heart. The muscles were trimmed of connective tissue, quick frozen in
liquid nitrogen, and stored at 80°C. The plantaris was thawed and
homogenized in a potassium phosphate buffer (pH 7.4) and assayed for
citrate synthase activity at 25°C as previously described
(32).
Solutions. The relaxing solution used during heart removal has been described previously (6) and contained 100 mM KCl, 1.75 mM EGTA, 10 mM imidazole, 4 mM ATP, and 5 mM MgCl2, adjusted to pH 7.0 with KOH.
Statistical analysis. Data for all experiments except microarray analysis are presented as means ± SD. Between-group comparisons (trained vs. control) were made using Student's t-test, with P < 0.05 considered to indicate a statistically significant difference.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The treadmill training program used in this study elicited typical
training effects in the rats, as shown in Table
1. There was no significant difference in
body weight between trained and control rats either before or after the
11-wk treadmill training program. However, training did elicit a 14%
increase in absolute heart mass and a 14.4% increase in the heart
weight-to-body weight ratio. In addition, the plantaris muscles taken
from the trained animals showed a 46% higher citrate synthase activity
compared with control plantaris muscle. The heart mass, heart
weight-to-body weight ratio, and plantaris muscle citrate synthase
activity were all significantly different than control and are
consistent with results of previous studies (5, 6, 18).
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The results of the microarray analysis are given in Table
2. The Affymetrix algorithm for detecting
differential expression considers several parameters described in the
Statistical Algorithms Reference Guide supplement to the Microarray
Suite version 5.0 User's Guide. With the use of the rank analysis
described above in METHODS, we originally identified 63 genes or expressed sequence tags whose expression was increased
or decreased in trained samples compared with control samples. We
eliminated genes with a mean FC of <1.5 as well as genes in which the
CV for the FC (SD/mean) was >0.7. These cutoff values provide a
conservative estimate of the numbers of genes whose expression level is
altered by exercise training. The genes that met the final exclusion
criteria are listed in Table 2.
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RT-PCR experiments.
To verify the results of the microarray analysis, we examined the
expression levels of two selected genes using RT-PCR. We chose the ANP
gene because previous studies had yielded conflicting results of the
effects of exercise training on the expression of this gene in
ventricular tissue (1-3, 12, 15). We chose the aMLC-1
gene because altered expression of this gene provides a potential
molecular mechanism for previously observed changes in cellular
contractile function. Figure 1 shows the
results of agarose gel electrophoresis of the products of the RT-PCR
using primers specific for ANP (A) and aMLC-1
(B). The results of the RT-PCR experiments confirmed the
microarray analysis results, that is, ANP gene expression is decreased
in trained compared with control hearts and aMLC-1 expression is
increased in trained compared with control ventricular tissue.
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2-D electrophoresis analysis.
To determine whether the increase in aMLC-1 expression demonstrated by
microarray analysis and RT-PCR actually resulted in increased aMLC-1
protein levels in the ventricles of trained animals, we performed 2-D
electrophoretic analysis. This method has been used previously to
separate the ventricular and atrial isoforms of MLC-1 in human
(19, 25) and porcine (20) myocardium. A
representative 2-D gel is shown in Fig.
2. The highlighted area was analyzed for
the presence of aMLC-1 and vMLC-1 based on the predicted pI and
molecular weight of these two isoforms. A magnified image of this
region of the gel is shown in Fig. 2B. We performed 2-D gel
analysis on atrial samples, a mixture of atrial and ventricular samples, and six trained and six control ventricular samples. Identification of spots corresponding to aMLC-1 and vMLC-1 was based on
pI and molecular weight information as well as previously published
determinations of aMLC-1 vs. vMLC-1 positions on 2-D gels (20,
25). The predicted pI and molecular mass values of rat aMLC-1
are 4.97 and 21,150.99 Da, respectively, whereas the pI and molecular
values of vMLC-1 are 5.03 and 22,025.01 Da (http://us.expasy.org). Densities of spots corresponding to
aMLC-1 and vMLC-1 were quantified using PDQuest software, and the
amount of each MLC-1 was expressed as a percentage of the total MLC-1 in that sample. Mean data for n = 6 animals from each
group showed that there was no detectable aMLC-1 protein in control
ventricles, whereas in trained animals aMLC-1 increased to 13.4 ± 2.1% of the total MLC-1. This difference is statistically significant (P < 0.05).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Previous studies indicate that endurance exercise training improves contractile function in the myocardium. One possible mechanism for this adaptation would be alterations in the expression of genes related to contractile function, but these possible molecular mechanism(s) have been difficult to elucidate. A number of recent studies have determined the effect of exercise training on expression of a small number of selected genes, but the results of these studies have not yielded a consistent view and have not provided a mechanism for the described changes in contractile function (6, 16, 22, 39). We undertook an oligonucleotide microarray analysis to determine the effects of exercise training on a large number of genes. With the use of our acceptance criteria, we found that 10 genes were increased and 17 genes were decreased in trained vs. control ventricular tissue. A number of the key changes are discussed below.
Genes related to cardiac hypertrophy.
One of the most common cardiac adaptations to an endurance exercise
training program is moderate cardiac hypertrophy. We found that
exercise training increased heart mass by 14% over control. Previous
reports have indicated that, under other hypertrophic stimuli such as
pressure overload, a so-called "fetal gene program" is induced
(24) that includes the downregulation of adult isoforms of
several cardiac proteins and the upregulation of genes such as
-myosin heavy chain (MHC), skeletal -actin, atrial natriuretic factor (ANF), the GLUT1 glucose transporter, and aMLC-1 (10, 11,
19, 20, 24, 26, 30). Despite clear evidence of hypertrophy in
response to the training program (Table 1), we found no evidence for
the induction of any of these "fetal genes" with the exception of
aMLC-1, which is discussed below. In fact, we found a decrease in the
expression of several of these genes previously related to hypertrophy
including ANF, skeletal -actin, and the GLUT1 glucose transporter.
Previous reports on the effect of training on the expression of ANF by
ventricular tissue are in conflict. ANF mRNA levels were found to be
increased by a rat swim training program (3), a dog
treadmill training program (15), and a mouse voluntary
wheel running program (1), but unchanged by a treadmill
training program in rats (2, 12). Because of this
uncertainty, we verified the results of our microarray analysis for ANF
using RT-PCR and confirmed that ANF expression was decreased in trained
compared with control tissue.
-MHC (9). We found an increase in IGF
binding protein 3 expression with training but a decrease in ANF
expression and no change in -MHC expression.
These results emphasize the complex relationship between cardiac
hypertrophy induced by a number of different stressors. Hypertrophic growth of the myocardium in response to stressors such as pressure overload is thought to initially be an adaptive response that temporarily augments or maintains cardiac output. However, this growth
eventually results in decreased cardiac function. Cardiac hypertrophy
as a result of exercise training is generally thought to improve
cardiac function, and this improved function is maintained. As
mentioned above, the changes in gene expression that we observed with
exercise training are substantially different, in some cases opposite,
than in hypertrophy associated with pressure overload or cardiac
myopathy. This would suggest that these different hypertrophic stimuli
operate by significantly different signaling pathways. However,
there are clearly some similarities in the hypertrophic response to
exercise training and pathological stimuli as evidenced by the
increases in aMLC-1 and IGF binding protein 3 expression.
Contractile protein genes. Previous studies (6, 16, 18, 39) of training-induced changes in myocardial contractile function suggest the possibility of alterations in contractile protein expression as a result of exercise training. From this standpoint, the most important result of our expression analysis was the increase in the expression of the atrial isoform of MLC-1 in ventricular tissue from trained hearts. To confirm the microarray results, we performed RT-PCR using a primer specific for aMLC-1 and also tested for the presence of aMLC-1 protein using 2-D gel electrophoresis. Both of these methods confirmed that aMLC-1 expression is increased in ventricular tissue at both the mRNA and protein levels as a result of the endurance exercise training program.
Myosin is a hexameric protein consisting of two heavy chains and two pairs of light chains. In the adult mammalian ventricle, two isoforms of MHC are expressed: and . The young adult rat ventricle
expresses predominantly the -MHC isoform. As mentioned above,
myocardial hypertrophy resulting from pressure overload results in an
increased expression of -MHC, but the effect of exercise training on
cardiac MHC expression is less clear. A number of previous studies have
suggested that exercise training induces an increase in -MHC isoform
expression (12, 23, 27). However, there are studies that
have found no evidence for a change in MHC expression in response to
exercise training (7, 36) as well as studies that have
found evidence for a training-induced increase in -MHC expression
(8). Our expression analysis indicated no change in MHC
isoform expression, but our results do show, for the first time, a
training-induced change in MLC isoform expression.
There are two subfamilies of light chains associated with myosin:
the essential light chains (ELC; also referred to as MLC-1 or MLC-3)
and the RLC (also referred to as MLC-2). There are multiple isoforms of both ELC and RLC expressed in rat striated muscle (for a
review, see Ref. 29). The expression of these MLC
isoforms changes throughout development. The fetal rat heart
expresses an embryonic form of the ELC that is identical to the aMLC-1
isoform. In the course of development, light chain expression changes
in ventricular tissue to the ventricular isoform (vMLC-1), which is
identical to the slow skeletal isoform of MLC-1. Thus, in the adult rat
heart, atrial tissue expresses exclusively aMLC-1, whereas ventricular
tissue expresses vMLC-1 (35).
This pattern of MLC-1 expression in ventricular tissue has been
shown to change under pathological conditions. In both human hypertrophic cardiomyopathy (19) and in a porcine model of
hypertension (20), aMLC-1 expression has been shown to be
increased in ventricular myocardium. In failing human hearts, the
amount of aMLC-1 ranged from 0% to 10.3% of the total MLC-1 pool
(i.e., aMLC-1 + vMLC-1), whereas no aMLC-1 was detected in control
human heart tissue (19). In porcine ventricular tissue,
the aMLC-1 content increased from undetectable in control tissue to
16.9% of the total MLC-1 content in hypertrophied ventricles
(20).
Expression of aMLC-1 in ventricular tissue has been shown to alter the
contractile properties of the myocardium. Studies on pathological
hypertrophy of human and porcine myocardium showed that the increase in
aMLC-1 was associated with increased Ca2+ sensitivity of
tension (19) and increased maximal shortening velocity
(20). The magnitude of these alterations in contractile properties were found to be significantly correlated with the magnitude
of the increase in aMLC-1 expression, suggesting this change in MLC-1
isoform expression represents an underlying molecular mechanism for the
changes in contractile function accompanying the pathological
condition. Sanbe et al. (28) used a transgenic mouse model
to overexpress ectopic MLC-1 isoforms and found that shortening
velocity and power output were increased when aMLC-1 was expressed in
ventricular tissue. The mechanism(s) for the effects of increased
aMLC-1 expression to alter myocardial contractility is not known. It
has been demonstrated that the NH2-terminal region of the
MLC-1 molecule interacts with the actin filament during cross-bridge
formation (37). Sequence differences between aMLC-1 and
vMLC-1 result in a difference in charge in this
NH2-terminal region that may affect the ability of the
light chain to bind to actin (19).
These effects on myocardial contractile properties of increased
aMLC-1 expression in ventricular myocardium are remarkably similar to
alterations in contractile properties associated with exercise
training. Previous studies have indicated that training increases the
Ca2+ sensitivity of tension in cardiac myocytes (6,
39) and increases the myocardial maximal shortening velocity and
power output (16). These results suggest that the
training-induced increase in aMLC-1 expression found in the present
study may be an underlying molecular mechanism for previously observed
contractile changes in response to endurance exercise training.
The results of our microarray analysis did not show a decrease in
vMLC-1 isoform expression to accompany the increase in aMLC-1 expression. This is consistent with the previous findings of Buttrick et al. (3), who found no effect of exercise training on
vMLC-1 expression. These data suggest that there may not be a shift in expression of MLC-1 isoforms, with aMLC-1 expression increased and
vMLC-1 expression decreased, but instead an overexpression of aMLC-1 in
ventricular tissue. The results of studies of hypertrophied myocardium
(19, 20) as well as transgenic mouse studies of overexpression of MLC-1 isoforms (28) suggest that
overexpression of aMLC-1 in ventricular myocardium results in
stoichiometric incorporation of the aMLC-1 isoform into the
myofilaments even in the absence of changes in expression of the vMLC-1 isoform.
Limitations to microarray analysis. Although oligonucleotide microarray analysis provides a powerful tool for determining changes in expression for a large number of genes, there are a number of important limitations to the interpretation of our results. First, the changes reported here represent only one time point in the process of myocardial adaptation to exercise, i.e., after 11 wk of progressive treadmill training. Other studies examining the effect of exercise training on cardiac gene expression have used training durations of 4 wk (1), 6 wk (3), 10 wk (2), 13 wk (12, 39), and 55 wk (15). It is likely that gene expression is altered along the entire time course of the training protocol, and the set of genes that we have identified represents only those genes with altered expression at this particular time point. Second, it is recognized that our ventricular samples are mixed tissue samples. It is likely that blood, neural tissue, and vascular tissue, as well as myocardial tissue, contributed to the total RNA pool on which expression analysis was done. Thus some of the gene expression changes that we have reported may not be adaptations of the myocardium per se. For example, the decrease in incoupling protein 2 expression that we report here and others have reported in the heart (4) may be due to training-induced changes in uncoupling protein 2 expression in coronary vascular tissue (38).
In conclusion, we have shown that endurance exercise training induces altered the expression of a number of genes in the heart including increased expression of the atrial isoform of MLC-1 in ventricular tissue. Previous reports indicate that increased aMLC-1 content has effects on the contractile properties of ventricular myocardial preparations similar to those seen with adaptation to exercise training. Thus we conclude that increased aMLC-1 expression in ventricular myocardium represents a possible molecular mechanism for the previously observed changes in myocardial contractile function associated with exercise training.| |
ACKNOWLEDGEMENTS |
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We thank Helga Ahrens and Dr. Santhanum Swaminathan in the Cell Markers Facility and Services Core of the University of Wisconsin-Madison Environmental Health Services Center for expert assistance with the 2-D electrophoresis experiments.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-61410 (to G. M. Diffee).
Address for reprint requests and other correspondence: G. M. Diffee, Biodynamics Laboratory, Univ. of Wisconsin, Dept. of Kinesiology, 2000 Observatory Drive, Madison, WI 53706 (E-mail: gmdiffee{at}facstaff.wisc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published November 7, 2002;10.1152/ajpheart.00761.2002
Received 30 August 2002; accepted in final form 28 October 2002.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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