Introduction: Adaptation to heat (acclimation [ACC]) and exercise training (EX) require global changes at all levels of body organization to enhance muscle performance. In this investigation, we combined these stressors and examined physiological and genomic aspects of adaptation in skeletal muscle (soleus).
Methods: Rats were divided into four groups: C (controls), ACC-acclimated to heat only at 34°C, EX-aerobic exercise on a treadmill at 24°C, and EXAC-acclimated to combined heat and aerobic training. The ACC period was 30 d. Isometric force generation was measured using isolated muscle preparations stimulated at 1-100 Hz, allowing assessment of muscle endurance. Global genomic responses of homeostatic genes were detected using a complementary DNA (cDNA) Atlas array (Rat 1.2; Clontech Laboratory, Palo Alto, CA).
Results: Significantly elevated force generation (P < 0.05) was only found in the EXAC group along with a marked decrease in relaxation velocity. Both heat-treated groups (ACC and EXAC) demonstrated less of a drop in power at stimulation frequencies above the highest force generation (P < 0.05). Gene reprogramming was noted in all treatment groups with stressor-specific dynamic profiles. Improved force generation in the EXAC soleus coincided with significant up-regulation in expression levels of genes encoding sarcoplasmic Ca2+-transporting proteins (SERCA2 and inositol triphospate receptor), glycolysis rate-limiting enzyme (phosphofructokinase), mitochondrial lipid metabolism (CPTII), and stress proteins with antiapoptotic or apoptotic activity.
Conclusions: Our data suggest that EXAC-specific gene up-regulation and cross talk between genes assigned to their gene ontology categories (transport, metabolism, and stress) differ in abundance and/or expression level (compared with other treatment groups) and contributed to the physiological advantage demonstrated by the EXAC soleus.
Laboratory of Environmental Physiology, Faculty of Dental Medicine, The Hebrew University, Jerusalem, ISRAEL
Address for correspondence: Michal Horowitz, Ph.D., or Einat Kodesh, Ph.D., Laboratory of Environmental Physiology, The Hebrew University, PO Box 12272, Jerusalem 91120, Israel; E-mail: email@example.com.
Submitted for publication July 2009.
Accepted for publication September 2009.
Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (http://www.acsm-msse.org).
Adaptation to stressors such as heat acclimation (ACC) or aerobic exercise training (EX) involves global changes at all levels of body organization to enhance effector performance (13). ACC improves heat tolerance and endurance during heat stress (20). Analogously, EX induces a battery of changes that boost muscle performance. Opposing features are required to adapt to each of these stressors. Whereas aerobic EX increases the basal metabolic rate to meet the needs for greater adenosine 5c-triphosphate production, it is markedly decreased in the ACC phenotype to counteract excessive heat production (20). In the heart, this dichotomy manifests in the remodeling of the myosin isoform profile, an important biochemical modality of muscle adaptation. In the ACC heart, the slow myosin V3 isoform with low adenosine triphosphatase (ATPase) activity predominates (17), whereas EX favors V1 fast myosin (27,38). Despite these differences, both ACC and EX lead to greater cardiac pressure generation (13), suggesting that different processes can induce similar outcomes. Furthermore, in the heart, combined heat ACC and EX (EXAC) greatly enhanced the capacity for force generation compared with ACC or training under normothermic conditions alone (20). The effect of the combined stress on skeletal muscle mechanics was not studied.
Given that exercise increases body temperature, the ambient temperature is a limiting factor for physical activity because both skin and muscles compete for adequate blood flow (4,12). Along this line, Thomas et al. (39) provided data that an increase in core temperature is a factor responsible for voluntary activation dysfunction. In addition, Fransesconi et al. (9) demonstrated that heat ACC under sedentary conditions limits exercise endurance in hyperthermic conditions. In contrast, EX in the heat improves performance endurance to adverse or extreme environmental heat, by and large via effecting thermoregulatory responses and body fluid balance (6,22). Using the double product as an index of cardiac performance in rats, Moran et al. (25) demonstrated that both heat ACC and EX improve cardiac index and, in turn, exercise endurance, with different underlying mechanisms. Interestingly, Cohen et al. (4) demonstrated that heat ACC improves cardiac contractility per se under both normothermic and elevated temperatures compared with nonacclimated hearts that are unable to restitute pressure at high ambient temperatures. Whether improved skeletal contractile response and muscular performance play an adaptive role in combined exercise and heat ACC is still under investigation.
Our knowledge of the cellular and molecular mechanisms underlying the physiological responses to EX and ACC is derived from animal experimentation. The accruing data are sporadic and confined to the special interests of particular research groups. Therefore, no comparisons between the adaptive responses of different contractile tissues of the same species (e.g., heart vs skeletal muscles) can be made. The effect of ACC on cellular and molecular contractile plasticity in mammals had been derived solely from studies on cardiac performance. These studies suggest improved force generation via remodeling of the EC-coupling pathway (23,24), with up-regulation of the expression of L-type Ca2+ channels, ryanodine receptors, and SERCA/PLB protein ratio, collectively increasing the calcium contractile signal (Ca2+ transient). Concomitantly, the responsiveness of the contractile elements to Ca2+ decreased (4). Our knowledge of the effect of ACC on skeletal muscle contractility is scarce, derived from studies on ectothermic animals (19) and birds (40) and confined to myosin isoform distribution. The cellular or molecular responses induced by training have only been examined under normothermic conditions. Similarly to ACC in the heart, exercise affects EC-coupling Ca2+ regulatory proteins and catecholamine secretion, increasing RyR1 via inhibition of FKBP12 (31). In contradistinction to ACC, EX enhances Ca2+ sensitivity (21).
Considering the different adaptive requirements of ACC and EX on muscular performance and the beneficial effects of combining these stressors (EXAC) on muscular performance, the underlying cellular and molecular mechanisms of contractility are intriguing. To this end, the effects of EXAC on cellular mechanisms have only been demonstrated in cardiac muscle. Given that transcript expression is one important level at which biological regulation occurs and affects the phenotype, the aim of the current investigation was twofold: (i) to characterize the skeletal muscle phenotype by measuring isometric force generation and muscular capacity to sustain performance during increased stimulation frequencies and (ii) to identify the phenotypic-specific global genomic responses in rats exposed to heat ACC, EX, and EXAC.
We showed that EXAC increases force generation in isolated skeletal muscle and that heat ACC increases contractile endurance after both sedentary and training conditions. Our data imply that the beneficial physiological response of heat ACC and EX is linked to the enhancement of cellular Ca2+ transport and metabolic and stress pathways.
MATERIALS AND METHODS
Animals and Experimental Procedures
Male rats (Rattus norvegicus, Sabra strain albino var), initially weighing 80-100 g, kept in a 12:12-h light-dark cycle, with food (Ambar lab chows) and water ad libitum, were used. Rats were divided into four experimental groups: controls (C)-maintained at 24°C with no further treatment, heat acclimated (ACC)-exposed to environmental heat at 34°C for 30 d, exercise groups (EX)-maintained at 24°C and trained on a treadmill using a progressively increasing exercise protocol for 30 d (Table 1), and heat acclimated, exercised trained (EXAC)-exposed to both environmental heat and aerobic EX as above. Body weight and colonic temperature (Tc) were measured weekly before and after an exercise session using a digital balance (Precisa; PAG, Zurich, Switzerland) and a YSI thermistor probes no. 402 (ITS-90; Eutech Instruments, Singapore) inserted 6 cm beyond the anal sphincter, respectively, as detailed by Horowitz et al. (15). All experimental procedures were conducted between 9:00 a.m. and 1:00 p.m. to avoid the effects of circadian rhythm. Experimental protocols were approved by the ethics committee for animal experimentation of The Hebrew University and adhere to the American College of Sports Medicine animal care standards.
Because of our interest in maximal force generation by a skeletal muscle and given our previous knowledge of the isovolumic force generation in the heart (23), we chose the analogous isometric force generation experimental model. Animals (7-11 per each treatment group) were anesthetized using ketamine and xylazine (8.5 mg·100 g−1 body weight ketamine in 0.5% xylazine, IP), left and right soleus muscles were isolated, and the tendons mounted using silk thread in a 20-mL stimulation chamber filled with continuously stirred Krebs-Henseleit (KHB) solution of the following composition (in mM): NaCl 118, NaHCO3 24, KCl 4.2, MgSO4 1.2, KH2PO4 1.2, CaCl2 2.4, and glucose 20. The solution was continuously bubbled with 95% O2 to 5% CO2, maintaining a pH of 7.4. All experiments were carried out at 28°C. The muscle was mounted between one fixed plastic hook and a second hook attached to a force transducer (MP100; Biopac Systems, Inc.). A Grass stimulation unit (GRASS S-48) was used to generate electrical pulses (duration = 5 ms, intensity = 20 W). The stimulation pulses were applied via two platinum plate electrodes placed on each side of the well and extending the whole length of the muscle. The resulting force was recorded using Biopac Systems Inc. MP100 software, and data were stored on a personal computer. After mounting, the muscle was stretched (1 g) and allowed to rest for at least 15 min in oxygenated KHB solution before the above mentioned measurements began. Each muscle was stimulated at frequencies of 1, 2, 4, 5, 8, 10, 20, 40, 50, 80, and 100 Hz, with a 2-min interval between each frequency, until peak force amplitude was achieved. The KHB solution was replaced four times during the measurements for each muscle. Measurements of the drop in isometric force generation during progressively increased stimulation frequency after the peak of tetanic contractility allowed assessment of the ability of the muscle to retain its mechanical performance. Velocities of contraction and relaxation were calculated for the 40-Hz frequency. At this frequency, the maximal muscle force generation was recorded using the Biopac software, and the velocities of contraction and relaxation were calculated as the positive and negative derivatives of the contractile force. The presented force generation was normalized by muscle mass.
Global genomic response.
Given the physiological phenotype of the EXAC soleus observed in our experiments, we attempted to find genes responding specifically to heat and exercise by analyzing cDNA arrays. Animals were anesthetized as above. The left and the right soleus muscles were removed, and the tissues were rapidly placed in liquid nitrogen (41). Frozen samples were stored at −80°C until analysis. For gene expression, we used a Clontech cDNA Atlas array containing 1187 stress genes representing a variety of homeostatic functional groups of the rat genome spotted on a nylon membrane (Rat 1.2, no. 634556; Clontech Laboratory, Palo Alto, CA). TRI Reagent (Molecular Research Center) was used to extract total RNA. For detection, we pooled the muscles of two animals. Three pools were prepared for each experimental group. The quantity and the quality of the RNA were estimated from its absorbance at 260 and 280 nm as well as by 1% agarose gel electrophoresis (14). The probes were labeled for 1 h by reverse transcription of total RNA (3 μg) at 42°C in a primer mix (Clontech) containing (32P) dATP (Amersham Biosciences, Buckinghamshire, UK). The reaction was terminated by adding 0.1 M of ethylenediaminetetraacetic acid and 1 mg·mL−1 of glycogen (Sigma). The unincorporated 32P-labeled nucleotides were removed using the Nucleo-Spin extraction columns (Clontech).
cDNA array hybridization.
The membranes were prehybridized for 1 h at 68°C in a hybridization solution (Clontech) containing 0.1 mg·mL−1 of sheared salmon testes DNA (Sigma) to block nonspecific binding. The synthesized radiolabeled cDNA probe (5-15 × 106 cpm) was applied to each membrane and hybridized overnight at 68°C. Cot-1 DNA (Clontech; 1 g·mL−1) was added to block nonspecific binding. Each membrane was used three times after stripping by boiling in 0.5% SDS, according to the manufacturer's instructions.
For analysis, the membranes were exposed for 4, 12, and 24 h to a phosphor screen and detected by using a Bio-imaging Analyzer BAS2000 (Fuji Photo Film). Atlas Image 2.01 software (Clontech) was used to record the pixel density of each spot and to perform background subtraction. The background-subtracted data were then further analyzed.
Calibration and normalization.
To avoid problems related to short- or long-term exposure to the phosphorimager (resulting in underestimation or saturation effects, respectively), we compared the intensities of phosphorimages recorded at different exposure times (between 3 and 48 h). To facilitate standardization of the different recordings of the same hybridization experiment, a linear regression was performed on the individual time points and averaged, with the recorded intensity at the middle time point. Finally, to use measurements from the different hybridizations, each expression value was rescaled using the average expression of all probes in that hybridization. The individual data sets attained were used to compare gene expression in the various experimental groups. The level of the expressed genes was presented versus the control (treat/C ratio). Control values were calculated using the geometric mean of a particular gene's rescaled expression values in all hybridizations in the control group. Relative expression of the visible genes was presented as treatment or control log2 ratio [relative expression of (1) or (−1) indicates a twofold change from average control]. Because previous studies (15,36) on heat ACC showed that a cutoff of 1.5-fold is reliable for the interpretation of acclimatory responses, we used that value (and beyond) significant.
Correlations and comparisons.
Before analysis, unexpressed genes (with an intensity of less than 1200 pixels) in any group were removed. Significant change was considered as +0.7 or −0.7 log2 ratio to control. Biological function analyses were only performed on genes with significantly altered expression (36). For an overall functional interpretation of the common responses, we sorted the genes into groups according to their gene ontology (GO) database categories for biological processes. The analysis was done using bioinformatics software such as the Intelligent System and Bioinformatics laboratories (http://vortex.cs.wayne.edu), the Database for Annotation Visualization and Integrated Discovery (http://david.abcc.ncifcrf.gov), and the Affymetrix (http://www.NetAffx.com). Pie charts were constructed to present the data. Using Spotfire DecisioSite 9.1.1 Data Analysis Package with the Functional Genomics Companion, we were able to detect the genes that were common to all treatments or responding specifically to a certain stressor. Uniqueness of gene activation in response to a specific treatment was defined as no change in expression [between −0.7 and 0.7 (log2)] in other groups. The data discussed in this publication have been deposited in the National Center for Biotechnology Information's Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE7802.
For statistical analysis of the physiological data, we used GraphPad Prism 4. Treatments were taken as the independent categorical variables, and the individual rats were considered a random sample from the population. To test the effects of treatment on the dependent feature, we used one-way ANOVA. For post hoc tests, we used Dunnett's test to compare each treatment to controls. Data are expressed as mean ± SE. Values of P < 0.05 were considered statistically significant.
For the gene expression experiments, we considered the pooled animal samples as random samples from the population. For genomic analysis, we used the hypergeometric distribution with the false discovery rate, the expected proportion of false rejections among all rejections, and the Fisher exact score correction. A P value of <0.05 was considered statistically significant.
Body and soleus weights and colonic temperatures, measured before and after exercise sessions and at the end of the ACC or training regimen, are presented in Table 2. All treated animals were smaller than controls (P < 0.01). Nevertheless, ACC rats increased muscle mass/body weight ratio significantly compared with C rats (P < 0.01). There was no significant change in basal Tc (colonic temperature) a mong the groups; however, during exercise, Tc elevation in the EXAC rats was 1°C less than that in the EX rats (P < 0.01).
Isometric Force Generation
The effect of ACC, EX, and EXAC on the isometric force generated by the soleus is presented in Figure 1. The amplitude of the force generated was significantly higher (by 44.8% vs control) in the group that underwent combined heat ACC and EX (EXAC) at stimulation frequencies of 10-100 HZ, peaking at 40 Hz. ACC muscles yielded significantly greater force than C muscles at a low stimulation frequency (10 HZ). In both heat-acclimated groups (ACC and EXAC), the drop in isometric force with increasing stimulation frequency beyond that of peak force generation was attenuated (by 10.9%, 3.2% in EXAC and ACC, respectively, P < 0.05) versus the normothermic groups (C and EX; Fig. 2). The velocities of contraction and relaxation, calculated using the positive and negative derivatives, are detailed in Table 3. EXAC rats demonstrated markedly slower relaxation compared with C muscles (P < 0.05).
Global Genomic Response
Out of 1185 genes spotted on the array, the EX group showed the highest and the EXAC group the lowest number (EX = 506, ACC = 302, EXAC = 282) of genes with altered expression. More genes were up-regulated after treatments than down-regulated. Two hundred and eighteen genes were significantly up- or down-regulated by at least 1.5-fold and were present in all the treatment groups. Previous studies using our models (14,36) showed that a 1.5-fold change in the genomic response has biological significance.
Analysis 1: Functional Analysis
Only genes with significant up- or down-regulation were analyzed. Given our interest in the adaptive features that maintain homeostasis and the contractile response, we focused on four gene ontology (GO) groups for biological processes, namely, metabolism, transport, stress, and signal transduction. Uniqueness of gene activation in response to a specific treatment was defined as no change in expression [between −0.7 and 0.7 (log2)] in other groups. This analysis provided evidence that EX alone induced changes primarily in genes associated with metabolic pathways, and ACC alone affected genes encoding proteins involved in transport processes. In the EXAC group, the stress-associated functional category had the greatest percentage of genes (15% vs 6% and 9% in EX and ACC, respectively). In both ACC and EXAC, the highest proportion was the transport category (37% and 23%, respectively, vs 15% in the EX group; Fig. 3, Table 4).
Analysis 2: Specific Pathways
To identify specific enriched pathways in response to each stressor, further analyses focusing on 1) EC-coupling process, 2) metabolism, and 3) cytoprotection were conducted. To pinpoint stress-specific genes, we also sorted genes that changed in response to one treatment only.
In the array, 61 genes were associated with EC coupling. These can be divided into two main categories: 1) "calcium regulation," including genes related to the excitation phase; and 2) "calcium signaling pathway," with genes involved in the contraction phase or upstream to proteins important to the coupling process (Supplemental digital content (SDC) Tables 1-3, http://links.lww.com/MSS/A13). Rats exposed to EX under normothermic conditions (EX) had the most genes associated with these two pathways, 51 compared with 22 in ACC and 26 in EXAC. The magnitude of changes in gene expression was similar in all groups (Fig. 4). Five genes from the calcium signaling pathway were common to all treatments: adenylyl cyclase type II, skeletal muscle sodium channel protein alpha subunit (SCN4A), MU-1, Janus tyrosine-protein kinase 2 (JAK2), and calcium- or calmodulin-dependent protein kinase type II beta subunit (CAM-kinase II beta; CAMK-II beta). In addition, the sodium or calcium exchanger NCX3, reacting similarly in all treatment groups, is associated with the calcium regulation pathway. The limited number of shared genes suggests that each of the stressors induces unique adaptations, for example, adenylate cyclase units (Nos. 3, 4, 6, and 8) only showed increased expression in the EX group. Notably, SERCA2 (SR Ca ATPase) and inositol triphospate receptor (ITPR) were only up-regulated in the EXAC group (the sole group with significantly greater force generation).
Genes involved with energy metabolism.
Fifty genes spotted on the array play a role in energy metabolism pathways. Carbohydrates and fats are the main substrates used for energy metabolism in muscle tissues. These substrates are used by glycolysis, beta oxidation, citric acid cycle, and electron transport chain. More genes on the array were associated with lipid metabolism. Given this precondition, our data imply that more genes associated with lipid metabolism changed than those involved in carbohydrate metabolism (for details, see SDC Table 2, http://links.lww.com/MSS/A13). The EXAC group had the least changed genes (EXAC = 7, EX = 17, and ACC = 11); however, the magnitude of up-regulation was the highest in this group (×4 vs control; Fig. 4). The genes are listed in SDC Table 2 (http://links.lww.com/MSS/A13), interestingly, their up-regulation in the EX and ACC groups (groups without improved force generation compared with controls) was also significant (×2-fold vs control).
Genes related to cytoprotection.
All treatments induced changes in stress-inducible genes. However, the balance between the various functional categories and the number of significantly reprogrammed genes varied among the groups. The least (14) and the greatest (26) number of altered genes were noted in the EXAC and ACC rats, respectively. No difference in the average expression levels of the altered genes was found (Fig. 4). We focused on genes associated with apoptosis, oxidation, DNA damage or repair, and heat shock response only because of their direct interaction with the integrative functions discussed in this study. Glutathione S-transferase (GST5-5) and plasma glutathione peroxidase (GSHPX-P; GPX3) were up-regulated in the heat-treated, ACC, and EXAC groups, whereas fas antigen, fasL receptor, apoptosis antigen 1, c-Jun N-terminal kinase (JNK) 1 and 2, major vault protein, and BCLX responded to exercise and were up-regulated in both EX and EXAC groups (SDC Table 3, http://links.lww.com/MSS/A13). Similarly, among members of the mitogen-activated protein kinase superfamily, important regulators of cytoprotection, the greatest number of genes was found in the exercising groups (SDC Table 3, http://links.lww.com/MSS/A13), including JNK1&2, important regulators of cell integrity upon stress (14) and AKT that, in its phosphorylated form, is a known activator of many adaptive signaling pathways 36. The absence of cytochrome p-450 units in the EXAC soleus only was notable.
After heat ACC and EX, improved exercise performance in hot environments has been mainly attributed to enhanced cardiovascular capacity, thermoregulatory responses, and fluid balance (22). Concomitantly, sporadic reports hinted that ACC and EX individually effect the plasticity of skeletal muscles. This investigation demonstrates for the first time that 1 month of combined heat ACC and EX (EXAC) enhances force generation per se in the soleus muscle. Improved isometric force generation and enhanced ability to retain the force at high stimulation frequencies, together with attenuated whole body heating during exercise, are important features of the EXAC phenotype. We suggest that significant elevations in the expression of genes linked with energy metabolism and EXAC-specific genes associated with Ca2+ regulation together with reprogramming of stress-specific inducible genes (e.g., with antioxidative, antiapoptotic activities) contributed to the consolidation of the EXAC phenotype. ACC and EX groups demonstrated altered gene expression as well. However, the reprogrammed gene expression profile in these groups did not enhance force generation.
Combined heat ACC and EX: force generation in an isometric system.
In the rat soleus, the highest force amplitude was recorded at 40 Hz. This stimulation frequency induced tetanic contraction. Our data provide evidence that the greater force generation in the EXAC group was not associated with significant muscle hypertrophy. It is therefore likely that the greater force generation was due to changes in the intrinsic properties of the muscle. This conclusion was confirmed by our finding that in the EXAC muscle, the velocity of relaxation (determined by the negative derivative df/dt) was only 46% of the value calculated for the control group. No significant changes in the negative derivatives were observed in the other experimental groups. These observations differ from studies demonstrating markedly shorter relaxation velocities in the rat soleus after endurance training versus the EX group in the current investigation (8,35) but match the negative lusitropic effect noted in heat-acclimated hearts (13). Differences in exercise protocols (2-3 months vs 1 month in the current study) partially explain the discrepancies.
Relaxation velocity depends on Ca2+ reuptake from the cytosol to the sarcoplasmic (SR) pools via phospholamban phosphorylation and SERCA (SR Ca2+ ATPase) activation. In the heart, attenuated SERCA activation contributes to a higher inotropic response and slower relaxation (2). Furthermore, in heat-acclimated hearts (24), because of sustained low plasma thyroxine levels, up-regulation of nonphosphorylated phospholamban, greater nonphosphorylated phospholamban/SERCA ratio, and calcium transients were associated with increased pressure generation and negative lusitropic effect (13). Slow myosin (V3) under these conditions decreases contractile velocity (24). Given that skeletal muscle is also targeted by thyroid hormones (32), the dichotomy between contraction and relaxation velocities in the EXAC soleus may imply that thyroxine has no effect, at least on myosin predominance. This agrees with our unpublished observation that combined heat ACC and swimming training does not effect the cardiac myosin isoform profile (Horowitz, M., unpublished) and with reports that EX overrides the effects of low thyroxine on myosin predominance (11). This is also congruent with Levy et al. (20), demonstrating greater pressure generation, without changes in contraction and relaxation velocities in EXAC hearts (20). Decreased relaxation velocity in EXAC skeletal muscle implies differences between the soleus and the heart. On the basis of our data in ACC and EXAC cardiomyocytes (4) and Kodesh et al. (unpublished) and this investigation, demonstrating negative lusitropic effect, we hypothesize that the decreased relaxation velocity of EXAC soleus is associated with the effects of heat ACC on Ca2+ turnover.
Together with greater force generation, the increase in Tc of the EXAC rats was less than that of the EX rats at the end of exercise bouts, suggesting improved contractile efficiency. Levy et al. (20) showed similar results after swimming training. Hyperthermia appears to be a key determinant of exercise performance in the heat. Thus, strategies that attenuate the rise in core temperature contribute to enhanced exercise performance. A whole animal exercise endurance test was beyond the scope of this investigation but has been demonstrated by Moran et al. (25,26).
In this investigation, we assessed endurance capacity by measuring the ability to retain power generation at stimulation frequencies beyond peak tetanic isometric force amplitude. We demonstrated that heat ACC improved endurance in both exercising and sedentary groups. The mechanism, however, remains unknown.
The global genomic approach helps to partially integrate the physiological phenotype and the underlying molecular mechanisms. Our results (i) highlight possible pathways leading to greater force generation in EXAC muscles and (ii) allow us to determine heat or training-specific-mediated genomic responses.
Calcium regulatory genes and force generation.
The principal physiological finding in this investigation is that exposure to combined heat ACC and EX (EXAC) enhances force generation of the soleus. Because of the positive correlation between enhanced EC-coupling pathway components and force generation, pathways linked with calcium regulation during EC coupling and calcium signaling were analyzed. Genes assigned to these pathways were up-regulated similarly in all groups. In addition to the shared genes, there was overexpression of two stress-specific genes in the EXAC group, namely (see SDC Table 1, http://links.lww.com/MSS/A13), SERCA2 (SR Ca2+ ATPase) and ITPR. SERCA and ITPR are involved in cellular Ca2+ regulation. Inositol triphosphate activates the ryanodine receptor to release calcium to the cytosol (7), whereas the SERCA pump is responsible for reuptake of calcium to the SR. Most of the up-regulated genes in this pathway were also detected in the EX group. Because no increase in force generation was measured in EX and ACC muscles, we suggest that enhanced expression of SERCA2 and ITPR in combination with the activated shared genes such as PKCδ, PKCζ, IP3k, c-AMP, and guanine nucleotide binding contributes to the EXAC force generation adaptive phenomenon (assigned to the ACC, EX, and EXAC shared genes; SDC Table 1, http://links.lww.com/MSS/A13).
Metabolic component and force generation.
Metabolic support is required to enhance force generation. In this study, the greatest expression of energy metabolism genes was detected in the EXAC group. The marked up-regulation of muscle 6-phosphofructokinase (PFKM), a rate-limiting enzyme in glycolysis and mitochondrial carnitine O-palmitoyltransferase (SDC Table 2, http://links.lww.com/MSS/A13) that mediates the transport of long chain fatty acids across the mitochondrial membrane for energy consumption in this group, is notable. This up-regulation may be responsible (at least in part) for the metabolic adaptation required for greater force generation in the EXAC group.
Stress and cytoprotection.
The stress-inducible genes we studied changed in a stress-specific manner. It is well accepted that stress-inducible genes are not only protective but serve as modulators of variety of integrative functions (16). We attempted to define cytoprotective genes linked with enhanced force generation seen in the EXAC rat soleus. An optimal cellular redox state exists for muscle force generation (29,30). Reactive oxygen species enhance force generation via their effect on L-type Ca2+ channels, ryanodine receptors, and SERCA (30). Animals frequently subjected to EX show less oxidative damage after exhaustive exercise than untrained ones. This is largely attributed to the up-regulation of endogenous antioxidant enzymes such as glutathione peroxidase and g-glutamylcysteine synthetase (10). In our experimental setup, parallel up-regulation of SERCA and antioxidant molecules and absence of cytochrome p-450 (28) in the EXAC soleus only are congruent with the enhanced force generation. Glutathione-related genes (glutathione peroxidase, which protects cells from oxidative damage, and glutathione S-transferase, defending the cell from toxicants via conjugation to glutathione 3) were up-regulated in both EXAC and ACC groups, suggesting specific heat effects.
The GO analysis also highlighted the up-regulation of cytoprotective genes such as heat shock proteins (HSP), various kinases, and genes involved in the apoptosis or antiapoptosis balance. HSP70 was detected in all experimental groups (ACC = 1.623, EX = 2.109, EXAC = 1.833 log2 vs control). HSP70 is associated with multimodal stresses and provides cytoprotection against metabolic and environmental stressors in variety of organs and tissues (e.g., see Arieli et al. (1) and Dillmann and Mestril (5)). Similar findings were previously shown in our studies on ACC hearts (see Horowitz and Robinson ; Assayag, unpublished, 2007) and the hypothalamus (36,37). This was reflected by less tissue damage after ischemic-reperfusion insult in ACC hearts and after hyperoxia in the brain (1). An important function of HSP70 is its involvement in inhibition and degradation of pro-apoptotic factors such as p53 and c-myc and up-regulation of antiapoptotic factors such as BCL-xl (34). In the current study, genes along the apoptotic FAS-mediated signaling pathway behaved similarly in the EX and EXAC groups. FAS-induced apoptosis can be effectively inhibited by BCL-xl (33). BCL-xl was up-regulated in the EX and EXAC groups; however, in the latter group, BAD and BAX, the pro-apoptotic members of the BCL family (up-regulated in EX rats) were absent. Together with our observations of enhanced salvage kinases activation (e.g., JNK, p38), our analysis indicates enhanced antiapoptotic activity and cytoprotection in the EXAC group. This analysis provides additional beneficial aspect to the EXAC muscles.
One possible limitation of the study was the use of a Clontech array, which differs somewhat from other platforms and the lack of representative genes for confirmatory qPCR. The same Clontech array was used by our group for hypothalamic gene profiling of short- and long-term heat-acclimated rats before and after hypohydration (36) and for the heart (unpublished). An additional Clontech array for stress gene profiling the heat-acclimated heart was also successfully used (15). qRT PCR and RT PCR confirmed the changes found in these arrays (15,36). Given the quality of the data from these published studies and the reproducibility of the results, we are confident in the data from the present study, although relatively few arrays were performed per treatment group (because of budget limitations). A match in HSP70 between the array and the qRT PCR in the current study (data not shown) was also obtained. In microarray experiments, samples are often pooled to reduce the effects of biological variations. We are aware of the fact that pooling the samples somewhat limits resolution among genes showing relatively small expression changes. However, the data will provide direction for future studies using qPCR of specific pathways or investigation of proteins of interest. In contrast to several studies, but in agreement with others, 1 month of exercise did not induce muscular hypotrophy, although swimming training for the same time resulted in cardiac hypertrophy (20). There is a divergence in reports about EX via treadmill on body and soleus muscle weight. For example, Jafari et al. (18) reported that body weight and soleus muscle weight are slightly greater in trained than that in untrained rats, but the difference is not significant. These data are in congruence with the present investigation. An additional potential difficulty is a direct extrapolation to humans. Differences in the consensus human ACC protocol and our rat ACC protocol limit comparisons. Nevertheless, because the features discussed here are conserved, we hypothesize that stress-specific gene activation [depending on ACC or training phase (short/long)] is maintained.
In sum, this investigation shows that animals exposed to combined heat ACC and EX have improved performance in the heat not only via enhanced cardiovascular or thermoregulatory integrative responses but also because of their capacity to augment and retain force generation at high stimulation frequencies. These phenotypic changes were studied in the soleus muscle. We determined possible molecular mechanisms for this response. We also detected stress-specific genes by comparing the genomic response in the various treatment groups. Given the large body of data demonstrating greater force generation in response to training and our evidence of reprogrammed gene expression in all groups, it is likely that a longer ACC period or a more intense exercise regimen is needed to enhance performance in the ACC and the EX rats.
This investigation was supported by the Israel Science Foundation founded by the Israel Academy of Sciences and Humanities grant no. 511/01-1.
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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ISOMETRIC CONTRACTION; FORCE GENERATION; AEROBIC TRAINING; GENE EXPRESSION; GENOMIC-RESPONSES SIGNALING; STRESS
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