BACKGROUND
the adaptation is a dynamic process with involvement of many circumstances and is an important life mechanism.
the cardiac cells of mammals are submitted to a growth phase after birth maturation period defied as cardiac hypertrophy which is characterized by n increase in individual size of the cardiomyocyties without cell division.
this pattern of hypertrophy development can be initiated in response to some intrinsic and extrinsic stimuli such mechanical stress, neurohumoral factor, cytokines, ischemia and endocrine disorders
<cit> .
these stimuli for heart hypertrophy can be divided in “physiological” cardiac hypertrophy, when is result of exercise for example and “pathological” cardiac hypertrophy, which is associated with cardiovascular diseases
<cit> .
the physiological hypertrophy has been classified as a positive increase in heart mass, associated with structural remodeling of components of the ventricular walls to support increase in myocyte size, angiogenesis and changes in fibrilar collagen content and organization, whose enhancer the cardiac pump function.
the cellular and molecular bases behind heart adaptations to exercise are not completely understood, but it is believed that a number of cellular adaptations, intrinsic to the cardiomyocyte, are largely responsible for these changes.
the mechanism proposed for this structural adaptation is hypertrophy by increase in functional load
<cit> .
this overload is followed by modifications in the gene expression pattern, activation of signaling pathways which included up regulation in the contractile protein synthesis and his organization into sarcomeric units
<cit> .
however, it is not clear what role is played by the impact of differing training intensities on the physiological heart muscle’s adaptation, in terms of molecular changes.
in summary the literature describes that one of the most important benefits of exercise, associated to cardio-circulatory system consists in the enhancing of heart work efficiency due to contractile capacity increase.
thus, this study aims to verify the rat cardiac muscle adaptation under different intensities of swimming exercise, focusing on high molecular mass proteomics of r. novergicus.
RESULTS
measured average values showed a lower lactate accumulation, of  <dig>  .
the two other tg’s  also showed lower value,
<cit> .
cardiac fibers from left ventricle of rats in the cg showed a normal size and shape .
similar data were obtained from left ventricle histological sectioning of rats from tg <dig> , demonstrating no structural alteration in the heart tissue, which indicated lower hypertrophy.
exercise training was also able to induce heart morphological alterations not seen in the untrained group.
the small nucleus presented a rounded or in some cases slightly oval shape, indicating no clear pathological alteration which lower hypertrophy.
a few morphologic alterations, such as a small sclerotic area with infiltration of granule lipofuscin, characterizing particular metabolic alterations, probably associated with overload were seen in tg <dig>
<cit> .
this pigment is closely associated with oxygen-derived free radicals, which are an important component of muscle fatigue
<cit>  indicating that tg <dig> are inducing heart tissue modifications to the detriment of improvement in metabolism.
on the other hand, microscopic morphology analysis in tg <dig> indicated cellular hypertrophy, showing several modifications such as several areas with increased fibrosis , evidencing a higher adaptation to exercise overload.
this increase suggests that this level of physical activity was stressful to the heart
<cit> .
the relative exercise overload is directly associated with oxygen consumption
<cit> , because mitochondria are strictly involved in the activation of super-oxide synthesis cascade.
it is also worth noting that tg <dig> showed an extended area with necrosis and the presence of leucocytes, probably supporting hypertrophy by replacing dead cells with satellite cells
<cit> .
one important route to hypertrophy of the myocardium directly involves fibroblast proliferation, which stimulates collagen synthesis
<cit> .
increased collagen content is commonly observed with overload pressure and, in certain cases, may negatively impact both diastolic and systolic function
<cit> .
one of the most important benefits of exercise in the cardio-circulatory system is associated with an increase in circulation capability.
this upgrade is strictly dependent on contraction increase, carried out by the expression of different heart myosin isoforms
<cit> , which will be described below.
differential electrophoretic analyses of sedentary and exercise-trained cardiac muscle
since exercise-trained groups showed clear heart tissue alterations, sds-page was conducted in order to evaluate protein expression modification, showing an apparent quantitative increase in α-myhc  and β-myhc  expression , in tg <dig> and tg <dig> compared to cg.
analyzing overlapped images, it was observed that α-myhc  increased  <dig> fold in swimming-trained heart.
in order to improve these data, increasing accuracy and leading to an overview of physiologic modifications during swimming exercise training, two-dimensional gels were run by using left ventricles from control and training groups .
bionumerics™ from applied maths matched  <dig> spots in the 2-de control group.
it is known that two myhc isoforms, α-myhc and β-myhc, are expressed in cardiac tissue, and their proportion  in the rodent myocardium directly influences heart power output
<cit> .
data reported here are consistent with previous results in which myhc was up regulated in exercise-trained rat hearts
<cit> .
otherwise, these data contradict those observed during moderate exercise training on skeletal rat muscle suffering chronic heart failure
<cit> .
in this case, myhc distributions were similar in both groups and training did not alter the myhc distribution.
we found  <dig> spots in tg <dig>   <dig> in tg <dig> and  <dig> in tg <dig>  the number of spots observed in each gel , was lower than previously observed in rat heart protein maps  submitted to intensity-controlled endurance exercise
<cit> .
this variation could be explained by a different 2-de technique here utilized, which is focused on evaluation of high molecular mass proteins ranging from 50– <dig> kda, while burninston
<cit>  evaluated most abundant proteins from  <dig> –  <dig> kda.
the average coefficient of variation for normalized spot volume gave r2 =  <dig>  for biological and  <dig>  for technical replicates, showing reliable gel reproducibility.
these values are close to the range reported for technical variation for proteomic analyses of muscle homogenate, including skeletal and heart muscle tissues
<cit> .
moreover, the average coefficient of variation calculated to compare gels from different groups  showed a r <dig> lower than  <dig> , demonstrating the obvious differences in protein maps from rat left ventricles submitted to diverse intensities of training.
these proportions were similar to that observed by burniston
<cit> , evaluating the adaptation of rat cardiac muscle to endurance exercise, despite the different methodologies utilized.
while burniston described the evaluation of proteins from  <dig> to  <dig> kda
<cit> , our work evaluated proteins varying from  <dig> to  <dig> kda, indicating that protein expression behavior in response of exercise is independent of molecular masses.
moreover, these data are only in agreement with protein maps from rat skeletal muscle
<cit>  and human heart
<cit> .
furthermore, as previously observed in several reports
<cit> , some gene products were identified as multi-spot series exhibiting similar molecular masses but different pi, which may indicate unusual splice variants or states of post-translational modification.
real time pcr evaluating fold change expression of α-myhc rna from training groups  related to untrained group  calculated by ΔΔct method.
values were presented as a means ± standard deviation.
mass spectrometry protein identification
of all spots founded seventeen had been considered differentially expressed after swimming training, whereas increase more than one fold and appear in every gel in triplicate.
those were identified by pmf  with molecular masses above  <dig> kda.
these data revealed that the majority  of identified proteins were mitochondrial, one-quarter associated with membrane/ extracellular , one-quarter myofibrillar/cytoskeletal  and 23% associated with different functions such as hystocompatibility .
besides the evidence, based on protein analysis, of increased expression of α-myhc in our training groups, we decided to corroborate it using mrna expression .
data here reported showed that training intensity is an important factor for α-myhc expression.
training groups with overload of  <dig>   and 5%  showed increased expression of α-myhc related to untrained group .
respectively, these were increases of  <dig>  and  <dig>  fold in α-myhc expression.
however, taking in account standard deviation, the increase in tg <dig> was  <dig> - <dig>  and in tg <dig> it was  <dig> - <dig> -fold.
the overlap in the upper limit of tg <dig> with lower limit in tg <dig> shows lower differentiation in these two training groups for α-myhc expression.
the training group with overload of  <dig> %  showed an  <dig> -fold  increase.
differential proteins identified by maldi tof peptide mass fingerprinting of multiple proteins from rat left ventricles.
as a standard, false discovery rate was calculated as lower than 15%.
aiming to unambiguously differentiate α- and β-myhc, one ion, which is unique to α-myhc, of  <dig>  m/z corresponding to residues 1546– <dig>  was sequenced from spot  <dig>  tryptic peptides specifically from β -myhc were not detected, suggesting that spot  <dig> is formed by α-myhc.
a similar strategy to elucidate myhc isoforms has been used before
<cit> .
this result clearly showed a significant increase in α-myhc expression in proportion to training intensity.
a clear cut among tg <dig> and the other two training groups was also seen in histopathological analysis and electrophoretic analyses, which showed that tg <dig> was clearly able to improve α-myhc expression .
since α-myhc up regulation in exercised-trained rat hearts is consistent with previously published data
<cit> , here we could conclude that intensity, at least in swimming training, is the key to controlling α-myhc expression and indeed cardiomyocite power output .
other studies using swimming as a training method have suggested that this practice could induce an increase in α-myhc expression in the rat heart
<cit> .
the same author described little modifications in myhc isoform content in trained myocardium, despite finding increase in loaded shortening velocity.
these results suggest that it is possible to have significant alterations in myocardial contractile function with lower or no change in myhc isoform expression, as was observed here.
however, despite changes in the myosin subunit isoform expression are associated to increase in contractility by an increase in force generation, economy in atp ratio and increased ca2+ sensitivity, others low molecular weight sarcomeric protein, that they had not been described in this work, like myosin regulatory light chain, troponin-i, troponin-t, alpha-tropomyosin and myosin binding protein-c must be considered for yours relevance for contractile mechanism
<cit> .
since the 1980s some important studies have reported that an increase in α-myhc from exercise
<cit>  is able to boost cardiomyocyte power output
<cit> , especially for myofibrillar proteins
<cit> .
here, in this report, we used a combination of protein extraction under high salt quantities associated with a long and modified 2d gel run with low acrylamide concentration .
these procedures allowed the visualization of high molecular mass protein maps.
otherwise, spot resolution was lower when compared to other muscle tissues’ protein maps
<cit> .
similar data were obtained with treadmill-trained rats, in which a  <dig> -fold increase of α-mych expression was observed
<cit> .
spot  <dig> showed  <dig> kda of molecular weight, which is approximately half that of the predicted myosin heavy chain complete polypeptide.
furthermore, several myhc isoforms  could share 93% sequence identity
<cit> .
troponin, which acts as a muscular contraction regulator due to its relationship with calcium molecules, was identified in two different spots .
it is important to note that, probably, a different troponin species, which showed identical molecular mass , presented a completely different pi.
while one species extracted from spot  <dig> showed a pi of  <dig> , the troponin species from spot  <dig> had a pi of  <dig>  .
the troponin from spot  <dig> was only observed in protein maps from rat hearts submitted to intense exercise , suggesting that only intense exercise is capable of improving its expression at 2-de detectable levels .
troponin is commonly utilized as a serum muscle injury marker in cardiac insufficiency and, consequently, pathologic hypertrophy
<cit> .
for lippi and banfi, an increase in ctns values might be temporarily responsible for a reversible shed of cardiac blebs.
elevated levels of ctn could be due to ongoing myocardial damage or leakage of myofibrillar components and may reflect the loss of viable cardiac myocytes
<cit> .
myocyte injury, coronary microvascular dysfunction, and fibroblast and collagen turnover also play an important role in cardiac remodeling; extracellular matrix remodeling takes place in myocardium hypertrophy
<cit> .
nie et al.
<cit>  suggest a role for exercise-induced increases in ros in the mediation of ctn.
moreover, oxidative stress in high exercise intensity induced microvascular flow abnormalities, which in turn are associated with an increase in ctn levels observed by microscopic analyses
<cit> .
to support this fact, the presence of an isoform of major histocompatibility complexes, that are strictly associated with immune response in nucleated cells, such as necrosis or hydrolases enzymes, was detected at higher levels in tg <dig>  this process distinguishes between self-proteins and foreign protein antigens to elicit an effective immune response called antigen-presenting cells
<cit> .
these data, in addition to histological analysis previously described, which show a large necrosis area as well as leucocytes presence, suggested cardiac muscle injury caused by intense exercise , leading us to believe that  <dig> % overload exercise for  <dig> weeks brings real benefits to the animal but also causes clear injuries and further decreases in animal health.
exercise-induced changes in the expression of gene products involved in energy metabolism
an important signal for exercise adaptation consists of greater expression of proteins associated with the oxidative metabolism, such as oxidoreductases, mitochondrial membrane transporters
<cit> .
the mitochondrial internal membrane exerts a fundamental role in oxidative phosphorylation and also in electron transport.
moreover, the translocases move through the mitochondrial membrane and have a logical consequence for an important physiological process known as atp synthesis
<cit> .
in eukaryotic cells, the final stage of nutrients oxidation occurs in mitochondria, with fast oxidation of nadh and fadh <dig> produced in glycolysis, tricarboxilic cycle, ß-oxidation of fatty acids and amino acid oxidation.
in this study we found the presence of a nadh-dehydrogenase in all training groups and in the control , which developed a key role in electron transfer in the respiratory chain to q coenzyme
<cit> .
the expression of that isoform of nadh dehydrogenase was clearly improved from control groups to more intensely trained rats , leading to  <dig>  fold at tg <dig>  it’s important to cite that nadh dehydrogenase here analyses, maybe an isoform and that this up regulation could not be related to all protein class here evaluated.
additionally, it was only in rat hearts from tg <dig> group that the synthesis of membranes translocases was observed at detectable levels by the techniques applied here.
these translocases are commonly associated with atp’s and h+ transport to cytosol .
howlett and willis  observed that the isocitrate dehydrogenase  activity is higher in mitochondria from striated muscle, suggesting that it may rely on this enzyme as a regulatory site.
this enzyme catalyzes the first dehydrogenase reaction in the tca cycle and produces nadh + h+, which is the substrate of nadh dehydrogenase.
this may explain an over expression of nadh dehydrogenase, as observed in tg <dig> in the present study .
although mitochondria exert the essential role of atp production, they are also the primary source of cellular reactive oxygen species
<cit> .
in a recent review powers and jackson
<cit>  suggested that common metabolic changes and ros generation may predominantly occur by contracting skeletal and heart muscle during different exercise protocols.
an exception to this rule is an experiment whereby muscle damage occurs, and in this situation, inflammatory processes may play an important role in radical production.
it is well accepted that exercise provides intrinsic protection to the heart
<cit> .
recent reports have associated ros production with apoptosis after physical effort, a situation in which the apoptotic mitochondrial pathways may play a major role by releasing cytochrome c and activating initiators such as caspases
<cit> .
an increase in mitochondrial oxidant production is generally accepted as a cause of myocardial cell loss via apoptosis and necrosis
<cit> .
these data corroborates with myocite modifications observed in tg <dig> by microscopy , explaining the reduction in exercise benefits during the exercise training stage.
during exercise, h+ concentration is enhanced, being this process is commonly associated with force generation decline in muscles, also causing a reduction in cross bridge activation by competitively inhibiting ca2+ binding to troponin c. moreover, proton concentration reduces ca2+ - atpase re-uptake in sarcoplasm and inhibits myofibrillar atpase
<cit> .
mitochondrial ros generation can lead to a calcium overload, consequently decreased atp production, and may cause the mitochondrial permeability transition pore  to open, further decreasing atp production and releasing cytochrome c. however, the increase of translocases can increase the transport of h+ reducing the competition with calcium during the exercise, which would make the most of the positive effect of the exercise.
finally, another important gene product here detected and probably involved in the increase of atp synthesis consists in an isoform of magnesium homeostasis factor.
homolog, mrs <dig>   <dig>  fold , a major mg2+ mitochondria’s transporter being their function extremely important for respiratory complex i and cell viability maintenance
<cit> .
this protein family is characterized for a conserved gmn c-terminus  in the transmembrane domains.
furthermore, this is the region responsible for mg2+ selective filter
<cit>  this divalent ion is abundant inside the cell and plays a fundamental role in many biochemical and regulatory functions being his concentration maintained by an transmembrane electrochemical potential
<cit> .
in cardiac muscle mg2+ may be involved in the atpase phosphate-release step causing inhibition of myofibril sarcoplasmic reticulum ca2+-transporting atpases under anoxia.
this last condition could be improved by high intensity contraction in maximal exercise, when the atp-pcr system can occur to maintain a relatively constant energy supply
<cit> .
this finding in the present study is in accordance with the increase of aerobic capacity by mitochondrial biogenesis and/or workload improvement as a consequence of swimming training but, one more time, it’s important to cite that maybe an isoform and that this up- or down regulation could not be related to all protein class here evaluated.
CONCLUSIONS
in summary, our study evidenced left ventricular hypertrophy and these data seems to be correlated at molecular levels with proteins of high molecular masses.
this increase suggests a clear correlation with the level of intensity which the individuals underwent.
it demonstrates that interval training with high intensity compared with low and moderate intensity training led to a remarkable increase in α-myhc and troponin expressions in the left ventricle of cardiac myocyte of r. norvegicus.
another important data is the significant degree of cellular injury in left ventricle in individual submitted to high intensity, when compared with the individuals submitted to low and moderate intensities.
thus, we conclude that  <dig> % overload exercise for  <dig> weeks may possibly improve contractile function for the animal, but may also cause injuries and consequently, reduce the animal’s health.
these modifications seem to be related to modifications in contractile and metabolic proteins, previously elucidated by proteomics and molecular analyses.
data here reported add more knowledge to molecular exercise studies.

 the adaptation is a dynamic process with involvement of many circumstances and is an important life mechanism. the cardiac cells of mammals are submitted to a growth phase after birth maturation period defied as cardiac hypertrophy which is characterized by n increase in individual size of the cardiomyocyties without cell division. this pattern of hypertrophy development can be initiated in response to some intrinsic and extrinsic stimuli such mechanical stress, neurohumoral factor, cytokines, ischemia and endocrine disorders. these stimuli for heart hypertrophy can be divided in “physiological” cardiac hypertrophy, when is result of exercise for example and “pathological” cardiac hypertrophy, which is associated with cardiovascular diseases. the physiological hypertrophy has been classified as a positive increase in heart mass, associated with structural remodeling of components of the ventricular walls to support increase in myocyte size, angiogenesis and changes in fibrilar collagen content and organization, whose enhancer the cardiac pump function. the cellular and molecular bases behind heart adaptations to exercise are not completely understood, but it is believed that a number of cellular adaptations, intrinsic to the cardiomyocyte, are largely responsible for these changes. the mechanism proposed for this structural adaptation is hypertrophy by increase in functional load. this overload is followed by modifications in the gene expression pattern, activation of signaling pathways which included up regulation in the contractile protein synthesis and his organization into sarcomeric units. however, it is not clear what role is played by the impact of differing training intensities on the physiological heart muscle’s adaptation, in terms of molecular changes. in summary the literature describes that one of the most important benefits of exercise, associated to cardio-circulatory system consists in the enhancing of heart work efficiency due to contractile capacity increase. thus, this study aims to verify the rat cardiac muscle adaptation under different intensities of swimming exercise, focusing on high molecular mass proteomics of r. novergicus.  measured average values showed a lower lactate accumulation, of. the two other tg’s also showed lower value,. cardiac fibers from left ventricle of rats in the cg showed a normal size and shape. similar data were obtained from left ventricle histological sectioning of rats from tg, demonstrating no structural alteration in the heart tissue, which indicated lower hypertrophy. exercise training was also able to induce heart morphological alterations not seen in the untrained group. the small nucleus presented a rounded or in some cases slightly oval shape, indicating no clear pathological alteration which lower hypertrophy. a few morphologic alterations, such as a small sclerotic area with infiltration of granule lipofuscin, characterizing particular metabolic alterations, probably associated with overload were seen in tg. this pigment is closely associated with oxygen-derived free radicals, which are an important component of muscle fatigue indicating that tg are inducing heart tissue modifications to the detriment of improvement in metabolism. on the other hand, microscopic morphology analysis in tg indicated cellular hypertrophy, showing several modifications such as several areas with increased fibrosis, evidencing a higher adaptation to exercise overload. this increase suggests that this level of physical activity was stressful to the heart. the relative exercise overload is directly associated with oxygen consumption, because mitochondria are strictly involved in the activation of super-oxide synthesis cascade. it is also worth noting that tg showed an extended area with necrosis and the presence of leucocytes, probably supporting hypertrophy by replacing dead cells with satellite cells. one important route to hypertrophy of the myocardium directly involves fibroblast proliferation, which stimulates collagen synthesis. increased collagen content is commonly observed with overload pressure and, in certain cases, may negatively impact both diastolic and systolic function. one of the most important benefits of exercise in the cardio-circulatory system is associated with an increase in circulation capability. this upgrade is strictly dependent on contraction increase, carried out by the expression of different heart myosin isoforms, which will be described below. differential electrophoretic analyses of sedentary and exercise-trained cardiac muscle since exercise-trained groups showed clear heart tissue alterations, sds-page was conducted in order to evaluate protein expression modification, showing an apparent quantitative increase in α-myhc and β-myhc expression, in tg and tg compared to cg. analyzing overlapped images, it was observed that α-myhc increased fold in swimming-trained heart. in order to improve these data, increasing accuracy and leading to an overview of physiologic modifications during swimming exercise training, two-dimensional gels were run by using left ventricles from control and training groups. bionumerics™ from applied maths matched spots in the 2-de control group. it is known that two myhc isoforms, α-myhc and β-myhc, are expressed in cardiac tissue, and their proportion in the rodent myocardium directly influences heart power output. data reported here are consistent with previous results in which myhc was up regulated in exercise-trained rat hearts. otherwise, these data contradict those observed during moderate exercise training on skeletal rat muscle suffering chronic heart failure. in this case, myhc distributions were similar in both groups and training did not alter the myhc distribution. we found spots in tg in tg and in tg the number of spots observed in each gel, was lower than previously observed in rat heart protein maps submitted to intensity-controlled endurance exercise. this variation could be explained by a different 2-de technique here utilized, which is focused on evaluation of high molecular mass proteins ranging from 50– kda, while burninston evaluated most abundant proteins from – kda. the average coefficient of variation for normalized spot volume gave r2 =  for biological and for technical replicates, showing reliable gel reproducibility. these values are close to the range reported for technical variation for proteomic analyses of muscle homogenate, including skeletal and heart muscle tissues. moreover, the average coefficient of variation calculated to compare gels from different groups showed a r lower than, demonstrating the obvious differences in protein maps from rat left ventricles submitted to diverse intensities of training. these proportions were similar to that observed by burniston, evaluating the adaptation of rat cardiac muscle to endurance exercise, despite the different methodologies utilized. while burniston described the evaluation of proteins from to kda, our work evaluated proteins varying from to kda, indicating that protein expression behavior in response of exercise is independent of molecular masses. moreover, these data are only in agreement with protein maps from rat skeletal muscle and human heart. furthermore, as previously observed in several reports, some gene products were identified as multi-spot series exhibiting similar molecular masses but different pi, which may indicate unusual splice variants or states of post-translational modification. real time pcr evaluating fold change expression of α-myhc rna from training groups related to untrained group calculated by ΔΔct method. values were presented as a means ± standard deviation. mass spectrometry protein identification of all spots founded seventeen had been considered differentially expressed after swimming training, whereas increase more than one fold and appear in every gel in triplicate. those were identified by pmf with molecular masses above kda. these data revealed that the majority of identified proteins were mitochondrial, one-quarter associated with membrane/ extracellular, one-quarter myofibrillar/cytoskeletal and 23% associated with different functions such as hystocompatibility. besides the evidence, based on protein analysis, of increased expression of α-myhc in our training groups, we decided to corroborate it using mrna expression. data here reported showed that training intensity is an important factor for α-myhc expression. training groups with overload of and 5% showed increased expression of α-myhc related to untrained group. respectively, these were increases of and fold in α-myhc expression. however, taking in account standard deviation, the increase in tg was - and in tg it was - -fold. the overlap in the upper limit of tg with lower limit in tg shows lower differentiation in these two training groups for α-myhc expression. the training group with overload of % showed an -fold increase. differential proteins identified by maldi tof peptide mass fingerprinting of multiple proteins from rat left ventricles. as a standard, false discovery rate was calculated as lower than 15%. aiming to unambiguously differentiate α- and β-myhc, one ion, which is unique to α-myhc, of m/z corresponding to residues 1546– was sequenced from spot tryptic peptides specifically from β -myhc were not detected, suggesting that spot is formed by α-myhc. a similar strategy to elucidate myhc isoforms has been used before. this result clearly showed a significant increase in α-myhc expression in proportion to training intensity. a clear cut among tg and the other two training groups was also seen in histopathological analysis and electrophoretic analyses, which showed that tg was clearly able to improve α-myhc expression. since α-myhc up regulation in exercised-trained rat hearts is consistent with previously published data, here we could conclude that intensity, at least in swimming training, is the key to controlling α-myhc expression and indeed cardiomyocite power output. other studies using swimming as a training method have suggested that this practice could induce an increase in α-myhc expression in the rat heart. the same author described little modifications in myhc isoform content in trained myocardium, despite finding increase in loaded shortening velocity. these results suggest that it is possible to have significant alterations in myocardial contractile function with lower or no change in myhc isoform expression, as was observed here. however, despite changes in the myosin subunit isoform expression are associated to increase in contractility by an increase in force generation, economy in atp ratio and increased ca2+ sensitivity, others low molecular weight sarcomeric protein, that they had not been described in this work, like myosin regulatory light chain, troponin-i, troponin-t, alpha-tropomyosin and myosin binding protein-c must be considered for yours relevance for contractile mechanism. since the 1980s some important studies have reported that an increase in α-myhc from exercise is able to boost cardiomyocyte power output, especially for myofibrillar proteins. here, in this report, we used a combination of protein extraction under high salt quantities associated with a long and modified 2d gel run with low acrylamide concentration. these procedures allowed the visualization of high molecular mass protein maps. otherwise, spot resolution was lower when compared to other muscle tissues’ protein maps. similar data were obtained with treadmill-trained rats, in which a -fold increase of α-mych expression was observed. spot showed kda of molecular weight, which is approximately half that of the predicted myosin heavy chain complete polypeptide. furthermore, several myhc isoforms could share 93% sequence identity. troponin, which acts as a muscular contraction regulator due to its relationship with calcium molecules, was identified in two different spots. it is important to note that, probably, a different troponin species, which showed identical molecular mass, presented a completely different pi. while one species extracted from spot showed a pi of, the troponin species from spot had a pi of. the troponin from spot was only observed in protein maps from rat hearts submitted to intense exercise, suggesting that only intense exercise is capable of improving its expression at 2-de detectable levels. troponin is commonly utilized as a serum muscle injury marker in cardiac insufficiency and, consequently, pathologic hypertrophy. for lippi and banfi, an increase in ctns values might be temporarily responsible for a reversible shed of cardiac blebs. elevated levels of ctn could be due to ongoing myocardial damage or leakage of myofibrillar components and may reflect the loss of viable cardiac myocytes. myocyte injury, coronary microvascular dysfunction, and fibroblast and collagen turnover also play an important role in cardiac remodeling; extracellular matrix remodeling takes place in myocardium hypertrophy. nie et al. suggest a role for exercise-induced increases in ros in the mediation of ctn. moreover, oxidative stress in high exercise intensity induced microvascular flow abnormalities, which in turn are associated with an increase in ctn levels observed by microscopic analyses. to support this fact, the presence of an isoform of major histocompatibility complexes, that are strictly associated with immune response in nucleated cells, such as necrosis or hydrolases enzymes, was detected at higher levels in tg this process distinguishes between self-proteins and foreign protein antigens to elicit an effective immune response called antigen-presenting cells. these data, in addition to histological analysis previously described, which show a large necrosis area as well as leucocytes presence, suggested cardiac muscle injury caused by intense exercise, leading us to believe that % overload exercise for weeks brings real benefits to the animal but also causes clear injuries and further decreases in animal health. exercise-induced changes in the expression of gene products involved in energy metabolism an important signal for exercise adaptation consists of greater expression of proteins associated with the oxidative metabolism, such as oxidoreductases, mitochondrial membrane transporters. the mitochondrial internal membrane exerts a fundamental role in oxidative phosphorylation and also in electron transport. moreover, the translocases move through the mitochondrial membrane and have a logical consequence for an important physiological process known as atp synthesis. in eukaryotic cells, the final stage of nutrients oxidation occurs in mitochondria, with fast oxidation of nadh and fadh produced in glycolysis, tricarboxilic cycle, ß-oxidation of fatty acids and amino acid oxidation. in this study we found the presence of a nadh-dehydrogenase in all training groups and in the control, which developed a key role in electron transfer in the respiratory chain to q coenzyme. the expression of that isoform of nadh dehydrogenase was clearly improved from control groups to more intensely trained rats, leading to fold at tg it’s important to cite that nadh dehydrogenase here analyses, maybe an isoform and that this up regulation could not be related to all protein class here evaluated. additionally, it was only in rat hearts from tg group that the synthesis of membranes translocases was observed at detectable levels by the techniques applied here. these translocases are commonly associated with atp’s and h+ transport to cytosol. howlett and willis observed that the isocitrate dehydrogenase activity is higher in mitochondria from striated muscle, suggesting that it may rely on this enzyme as a regulatory site. this enzyme catalyzes the first dehydrogenase reaction in the tca cycle and produces nadh + h+, which is the substrate of nadh dehydrogenase. this may explain an over expression of nadh dehydrogenase, as observed in tg in the present study. although mitochondria exert the essential role of atp production, they are also the primary source of cellular reactive oxygen species. in a recent review powers and jackson suggested that common metabolic changes and ros generation may predominantly occur by contracting skeletal and heart muscle during different exercise protocols. an exception to this rule is an experiment whereby muscle damage occurs, and in this situation, inflammatory processes may play an important role in radical production. it is well accepted that exercise provides intrinsic protection to the heart. recent reports have associated ros production with apoptosis after physical effort, a situation in which the apoptotic mitochondrial pathways may play a major role by releasing cytochrome c and activating initiators such as caspases. an increase in mitochondrial oxidant production is generally accepted as a cause of myocardial cell loss via apoptosis and necrosis. these data corroborates with myocite modifications observed in tg by microscopy, explaining the reduction in exercise benefits during the exercise training stage. during exercise, h+ concentration is enhanced, being this process is commonly associated with force generation decline in muscles, also causing a reduction in cross bridge activation by competitively inhibiting ca2+ binding to troponin c. moreover, proton concentration reduces ca2+ - atpase re-uptake in sarcoplasm and inhibits myofibrillar atpase. mitochondrial ros generation can lead to a calcium overload, consequently decreased atp production, and may cause the mitochondrial permeability transition pore to open, further decreasing atp production and releasing cytochrome c. however, the increase of translocases can increase the transport of h+ reducing the competition with calcium during the exercise, which would make the most of the positive effect of the exercise. finally, another important gene product here detected and probably involved in the increase of atp synthesis consists in an isoform of magnesium homeostasis factor. homolog, mrs fold, a major mg2+ mitochondria’s transporter being their function extremely important for respiratory complex i and cell viability maintenance. this protein family is characterized for a conserved gmn c-terminus in the transmembrane domains. furthermore, this is the region responsible for mg2+ selective filter this divalent ion is abundant inside the cell and plays a fundamental role in many biochemical and regulatory functions being his concentration maintained by an transmembrane electrochemical potential. in cardiac muscle mg2+ may be involved in the atpase phosphate-release step causing inhibition of myofibril sarcoplasmic reticulum ca2+-transporting atpases under anoxia. this last condition could be improved by high intensity contraction in maximal exercise, when the atp-pcr system can occur to maintain a relatively constant energy supply. this finding in the present study is in accordance with the increase of aerobic capacity by mitochondrial biogenesis and/or workload improvement as a consequence of swimming training but, one more time, it’s important to cite that maybe an isoform and that this up- or down regulation could not be related to all protein class here evaluated.  in summary, our study evidenced left ventricular hypertrophy and these data seems to be correlated at molecular levels with proteins of high molecular masses. this increase suggests a clear correlation with the level of intensity which the individuals underwent. it demonstrates that interval training with high intensity compared with low and moderate intensity training led to a remarkable increase in α-myhc and troponin expressions in the left ventricle of cardiac myocyte of r. norvegicus. another important data is the significant degree of cellular injury in left ventricle in individual submitted to high intensity, when compared with the individuals submitted to low and moderate intensities. thus, we conclude that % overload exercise for weeks may possibly improve contractile function for the animal, but may also cause injuries and consequently, reduce the animal’s health. these modifications seem to be related to modifications in contractile and metabolic proteins, previously elucidated by proteomics and molecular analyses. data here reported add more knowledge to molecular exercise studies. 
in order to evaluate the links between swimming exercise intensity and cardiac adaptation by using high molecular mass proteomics, isogenic wistar rats were divided into four groups: one control and three training groups, with low, moderate and high intensity of exercises. α-myhc expression increase fold in high intensity training group when compared with control group. α-myhc was also evaluated by real-time pcr showing a clear expression correlation with protein synthesis data increase in fold in high intensity training group. other myofibrillar protein, troponin, appear only in high intensity group, corroborating the cellular injury data. high molecular masses proteins such as mrs and nadh dehydrogenase, involved in metabolic pathways also demonstrate increase expression, respectily and fold, in response to high intensity exercise. high intensity exercise demonstrated an increase expression in some high molecular masses myofibrilar proteins, α-myhc and troponin. however, high intensity exercise also represented a significant degree of cellular injury, when compared with the individuals submitted to low and moderate intensities. findings here reported demonstrated clear morphologic alterations, significant cellular injury and increased energy supplies at high exercise intensities. furthermore this intensity also lead a significant increase of other high molecular masses proteins such as mrs and nadh dehydrogenase in comparison to low and moderate intensities. regular exercises are commonly described as an important factor in health improvement, being directly related to contractile force development in cardiac cells.
 regular exercises are commonly described as an important factor in health improvement, being directly related to contractile force development in cardiac cells. in order to evaluate the links between swimming exercise intensity and cardiac adaptation by using high molecular mass proteomics, isogenic wistar rats were divided into four groups: one control and three training groups, with low, moderate and high intensity of exercises. in order to evaluate the links between swimming exercise intensity and cardiac adaptation by using high molecular mass proteomics, isogenic wistar rats were divided into four groups: one control and three training groups, with low, moderate and high intensity of exercises.  findings here reported demonstrated clear morphologic alterations, significant cellular injury and increased energy supplies at high exercise intensities. α-myhc, as well proteins associated with mitochondrial oxidative metabolism were shown to be improved. α-myhc expression increase fold in high intensity training group when compared with control group. α-myhc was also evaluated by real-time pcr showing a clear expression correlation with protein synthesis data increase in fold in high intensity training group. other myofibrillar protein, troponin, appear only in high intensity group, corroborating the cellular injury data. high molecular masses proteins such as mrs and nadh dehydrogenase, involved in metabolic pathways also demonstrate increase expression, respectily and fold, in response to high intensity exercise.  high intensity exercise demonstrated an increase expression in some high molecular masses myofibrilar proteins, α-myhc and troponin. furthermore this intensity also lead a significant increase of other high molecular masses proteins such as mrs and nadh dehydrogenase in comparison to low and moderate intensities. however, high intensity exercise also represented a significant degree of cellular injury, when compared with the individuals submitted to low and moderate intensities. heart tissuehigh molecular mass proteomicmusclemyofibrillar proteinsswimming training 