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Physiol. Genomics 29: 246-252, 2007. First published January 23, 2007; doi:10.1152/physiolgenomics.00188.2006
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Received 25 August 2006; accepted in final form 16 January 2007.
Physiological Genomics 29:246-252 (2007)
1094-8341/07 $8.00 © 2007 American Physiological Society

Exercise training improves the net balance of cardiac Ca2+ handling protein expression in heart failure

Natale P. L. Rolim1, Alessandra Medeiros1, Kaleizu T. Rosa2, Katt C. Mattos1, Maria C. Irigoyen2, Eduardo M. Krieger2, Jose E. Krieger2, Carlos E. Negrão1,2 and Patricia C. Brum1

1 School of Physical Education and Sport
2 Heart Institute (InCor), Medical School, University of São Paulo, São Paulo, Brazil


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The molecular basis of the beneficial effects associated with exercise training (ET) on overall ventricular function (VF) in heart failure (HF) remains unclear. We investigated potential Ca2+ handling abnormalities and whether ET would improve VF of mice lacking {alpha}2A- and {alpha}2C-adrenoceptors ({alpha}2A/{alpha}2CARKO) that have sympathetic hyperactivity-induced HF. A cohort of male wild-type (WT) and congenic {alpha}2A/{alpha}2CARKO mice in a C57BL/J genetic background (5–7 mo of age) was randomly assigned into untrained and trained groups. VF was assessed by two-dimensional guided M-mode echocardiography. Cardiac myocyte width and ventricular fibrosis were evaluated with a computer-assisted morphometric system. Sarcoplasmic reticulum Ca2+ ATPase (SERCA2), phospholamban (PLN), phospho-Ser16-PLN, phospho-Thr17-PLN, phosphatase 1 (PP1), and Na+-Ca2+ exchanger (NCX) were analyzed by Western blotting. ET consisted of 8-wk running sessions of 60 min, 5 days/wk. {alpha}2A/{alpha}2CARKO mice displayed exercise intolerance, systolic dysfunction, increased cardiac myocyte width, and ventricular fibrosis paralleled by decreased SERCA2 and increased NCX expression levels. ET in {alpha}2A/{alpha}2CARKO mice improved exercise tolerance and systolic function. ET slightly reduced cardiac myocyte width, but unchanged ventricular fibrosis in {alpha}2A/{alpha}2CARKO mice. ET significantly increased the expression of SERCA2 (20%) and phospho-Ser16-PLN (63%), phospho-Thr17-PLN (211%) in {alpha}2A/{alpha}2CARKO mice. Furthermore, ET restored NCX and PP1 expression in {alpha}2A/{alpha}2CARKO to untrained WT mice levels. Thus, we provide evidence that Ca2+ handling is impaired in this HF model and that overall VF improved upon ET, which was associated to changes in the net balance of cardiac Ca2+ handling proteins.

calcium; hemodynamics


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
THERE IS CONVINCING EVIDENCE for the benefits of regular exercise training in heart failure (HF) patients (33). The improvement in peak oxygen uptake, quality of life, and functional class after exercise training in HF patients strongly suggests that this nonpharmacological strategy plays an important role for the treatment of HF (16, 33). However, the underlying mechanism by which exercise training improves clinical outcome in HF patients is still under investigation. In a previous study we demonstrated a significant reduction in muscle sympathetic nerve activity after exercise training in chronic HF patients (33). Other studies show that exercise training caused a significant increase in endothelial function (40). These neural and vascular improvements result in less vasoconstriction and increased peripheral oxygen supply (38), possibly leading to a decreased intracellular TNF-{alpha} and interleukin-1 expression (11). The consequence of this peripheral adaptation is the increase in muscle oxidative capacity and exercise tolerance in HF patients.

The cardiac effects of exercise training in HF are less understood. Some investigators have reported that exercise training has no effect in cardiac output (39, 26). In contrast, recent studies have shown that exercise training increases stroke volume and, hence, cardiac output in patients with HF (7, 9).

Ventricular function is highly coupled with Ca2+ transients in the heart, and myocardial dysfunction observed in severe HF is caused mainly by alterations in phosphorylation status of sarcomeric proteins (19) and a diminished sarcoplasmic reticulum Ca2+ load that arises from enhanced activity and expression of Na+-Ca2+ exchanger (41), reduced sarcoplasmic reticulum Ca2+ ATPase (SERCA2) function (32), and increased diastolic Ca2+ leak via ryanodine receptors (1, 22). In addition, alterations of SERCA2 activity have been attributed to a phosphorylatable protein, phospholamban (PLN) (8, 13), which in its dephosphorylated form decreases the apparent Ca2+ affinity of SERCA2. PLN protein levels are unchanged in HF (25) patients. However, PLN phosphorylation at Ser16 by protein kinase A seems to be decreased, while Thr17 phosphorylation by Ca2+-calmodulin-dependent protein kinase II is increased in aortic stenosis-induced HF (1, 24). Thus, Ca2+ handling protein abnormalities often accompany the development of HF and may be considered markers or potential new therapeutic targets.

We have previously reported that mice lacking both {alpha}2A/{alpha}2C-adrenoceptors ({alpha}2A/{alpha}2CARKO) develop sympathetic hyperactivity-induced HF (4). Therefore, these mice provide a model system for better understanding the mechanisms underlying the cardiac deleterious effect of sympathetic hyperactivity, as well as, to test different therapeutic strategies for HF.

In the present study, we tested three hypotheses: 1) that ventricular dysfunction of {alpha}2A/{alpha}2CARKO is associated with abnormalities in the cardiac expression of Ca2+ handling proteins, 2) that exercise training can increase overall cardiac function in a genetic model of sympathetic hyperactivity-induced HF, and 3) that exercise training can improve the net balance of cardiac Ca2+ handling proteins involved in transsarcolemmal flux and sarcoplasmic reticulum reuptake of Ca2+ in this genetic model of HF.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sampling
Animals' care.
A cohort of male wild-type (WT) and congenic {alpha}2A/{alpha}2CARKO mice in a C57Bl6/J genetic background was studied from 5 to 7 mo of age. At this age, {alpha}2A/{alpha}2CARKO mice display advanced stage of cardiomyopathy as previously described (4). Genotypes were determined by polymerase chain reaction on genomic DNA obtained from tail biopsies using primers to detect the intact and disrupted genes.

Mice were maintained in a light (12-h light cycle)- and temperature (22°C)-controlled environment and were fed a pellet rodent diet (Nuvital Nutrientes, Curitiba, PR Brazil) ad libitum and had free access to water. WT and {alpha}2A/{alpha}2CARKO mice were randomly assigned into untrained and exercise-trained groups. This study was conducted in accordance with the ethical principles in animal research adopted by the Brazilian College of Animal Experimentation (www.cobea.org.br). The animal care and protocols in this study were reviewed and approved by the Ethical Committee of the University of São Paulo (CEP04).

Measurements and Procedures
Graded treadmill exercise test.
Exercise capacity, estimated by total distance run, was evaluated with a graded treadmill exercise protocol for mice. After being adapted to treadmill exercises over 1 wk (10 min of exercise session), mice were placed in the exercise streak and allowed to acclimatize for at least 30 min. Exercise began at 6 m/min with no grade and increased by 3 m/min every 3 min thereafter until exhaustion. WT and {alpha}2A/{alpha}2CARKO mice performed the graded treadmill exercise test before and after the experimental period.

Exercise training protocol.
Low intensity exercise training was performed on a motor treadmill over 8 wk, 5 days/wk (mo 5–7). The running speed and duration of exercise were progressively increased to elicit 60% of maximal speed, achieved during a graded treadmill exercise protocol, for 60 min at the fourth week. This intensity was maintained during the rest of the 8-wk training period. All untrained mice were exposed to treadmill exercise (5 min) three times a week to become accustomed to exercise protocol and handling.

Cardiovascular measurements.
Heart rate (HR) was determined noninvasively with a computerized tail-cuff system (BP 2000 Visitech Systems) described elsewhere (15). Mice were acclimatized to the apparatus during daily sessions over 6 days, 1 wk before the experimental period started. HR measurements were obtained serially in WT and {alpha}2A/{alpha}2CARKO mice once a week throughout the 8 wk of experiment.

Noninvasive cardiac function was assessed by two-dimensional guided M-mode echocardiography, in halothane-anesthetized WT and {alpha}2A/{alpha}2CARKO mice, before and after the experimental period. Briefly, mice were positioned in the supine position with front paws wide open, and an ultrasound transmission gel was applied to the precordium. Transthoracic echocardiography was performed using an Acuson Sequoia model 512 echocardiographer equipped with a 14-MHz linear transducer. Left ventricle systolic function was estimated by fractional shortening as follows: Fractional Shortening (%) = [(LVEDD – LVESD)/LVEDD] x 100, where, LVEDD means left ventricular end-diastolic dimension, and LVESD means left ventricular end-systolic dimension.

Estimation of cardiac sympathetic tone.
To confirm that {alpha}2A/{alpha}2CARKO mice have increased cardiac sympathetic tone, we measured the HR (electrocadiogram) after pharmacological blockade of muscarinic receptors with methylatropine (1 mg/kg, Sigma Chemical) and ß-adrenergic receptors with propranolol (3 mg/kg, Sigma Chemical) at the end of the experimental protocol. The sympathetic tonus was analyzed as the difference between the maximum HR after methylatropine injection and the intrinsic HR (HR after muscarinic and ß-adrenergic receptor blockade) (10, 23, 28).

Structural analysis.
Twenty-four hours after the last exercise training session, untrained and exercise-trained {alpha}2A/{alpha}2CARKO and WT control mice were killed and their tissues harvested. Cardiac chambers were then fixed by immersion in 4% buffered formalin and embedded in paraffin for routine histological processing. Sections (4 µm) were stained with hematoxylin and eosin for examination by light microscopy. Only nucleated cardiac myocytes from areas of transversely cut muscle fibers were included in the analysis. Quantification of left ventricular fibrosis was achieved by Sirius red staining. Cardiac myocyte width and ventricular fibrosis were measured in the LV free wall with a computer-assisted morphometric system (Leica Quantimet 500, Cambridge, UK).

Antibodies.
Mouse monoclonal antibodies to SERCA2 (1:2,500), PLN (1:500), and Na+-Ca2+ exchanger (1:2,000) were obtained from Affinity BioReagents (Golden, CO); rabbit polyclonal antibody to protein phosphatase type 1 (PP1, 1:1,000) was obtained from Upstate Biotechnology (Lake Placid, NY); phospho-Ser16-PLN (1:5,000) and phospho-Thr17-PLN (1:5,000) were obtained from Badrilla (Leeds, UK). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:2,000) was obtained from Advanced Immunochemical (Long Beach, CA). Targeted bands were normalized to cardiac GAPDH.

Western blot analysis.
Left ventricular homogenates were analyzed by Western blotting to compare SERCA2, PLN, phospho-Ser16-PLN, phospho-Thr17-PLN, PP1, and Na+-Ca2+ exchanger. Briefly, liquid nitrogen frozen ventricles isolated from WT and {alpha}2A/{alpha}2CARKO mice were homogenized in a buffer containing 50 mM potassium phosphate buffer (pH 7.0), 0.3 M sucrose, 0.5 mM DTT, 1 mM EDTA (pH 8.0), 0.3 mM PMSF, 10 mM NaF, and phosphatase inhibitor cocktail (1:100; Sigma-Aldrich, St. Louis, MO). Samples were subjected to SDS-PAGE in polyacrylamide gels (6 or 10% depending on protein molecular weight). After electrophoresis, proteins were electro-transferred to nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ). Equal loading of samples (50 µg) and even transfer efficiency were monitored with the use of 0.5% Ponceau S staining of the blot membrane. The blotted membrane was then blocked (5% nonfat dry milk, 10 mM Tris·HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20) for 2 h at room temperature and incubated with specific antibodies overnight at 4°C. Binding of the primary antibody was detected with the use of peroxidase-conjugated secondary antibodies (rabbit or mouse depending on the protein, 1:10,000, for 1:30 h at room temperature) and developed by enhanced chemiluminescence (Amersham Biosciences) detected by autoradiography. Quantification analysis of blots was performed with the use of Scion Image software (Scion based on NIH Image).

Statistical Analysis
Data are presented as means ± SE. For distance run, fractional shortening, and HR measurements, comparisons of untrained and exercise-trained {alpha}2A/{alpha}2CARKO and WT control mice were performed by two-way ANOVA for repeated measurements with post hoc testing by Duncan (Statistica software; StatSoft, Tulsa, OK). For cardiac structural analysis, cardiac sympathetic tonus, and protein expression levels, comparison among all groups were performed by two-way ANOVA with post hoc testing by Duncan (Statistica Software, StatSoft). Probability values <0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of Exercise Training on Exercise Tolerance and HR
{alpha}2A/{alpha}2CARKO mice displayed exercise intolerance compared with WT control mice (288 ± 14 vs. 359 ± 9 m, P = 0.05). Exercise-trained {alpha}2A/{alpha}2CARKO mice increased exercise capacity toward untrained WT mice levels (365 ± 8 vs. 359 ± 9 m). As expected, exercise training increased exercise tolerance in trained WT mice. Baseline HR was significantly higher in {alpha}2A/{alpha}2CARKO mice than age-matched WT control mice (Fig. 1A). Exercise training significantly decreased HR in WT and {alpha}2A/{alpha}2CARKO mice. The reduction of HR in {alpha}2A/{alpha}2CARKO mice was dramatic, reaching untrained WT control mice levels at the seventh week of training (Fig. 1A). Cardiac sympathetic tone was significantly increased in untrained {alpha}2A/{alpha}2CARKO mice compared with age-matched untrained WT control mice (Fig. 1B). In contrast, cardiac sympathetic tone in exercise-trained {alpha}2A/{alpha}2CARKO mice was comparable to WT control mice and significantly lower than in untrained {alpha}2A/{alpha}2CARKO mice (Fig. 1B). Exercise-trained WT mice displayed cardiac sympathetic tone similar to untrained WT mice.


Figure 1
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  		Fig. 1. Heart rate (HR, A) and cardiac sympathetic tone (ST, B) in untrained and exercise-trained wild-type (WTUN and WTT, respectively) and {alpha}2A/{alpha}2C-adrenoceptor knockout (ARKO; DKOUN and DKOT, respectively) mice. Note that exercise training decreased HR and ST in {alpha}2A/{alpha}2CARKO mice to untrained WT control mice levels. As expected, WTT displayed resting bradycardia. Data are presented as means ± SE. *P < 0.05 vs. basal levels, {dagger}P < 0.05 vs. untrained group.

 
Cardiac Contractility and Myocyte Structure
Baseline fractional shortening was significantly lower in untrained {alpha}2A/{alpha}2CARKO mice compared with age-matched untrained WT control mice (Fig. 2). While exercise training did not change fractional shortening in WT mice, it significantly increased fractional shortening toward normal levels in {alpha}2A/{alpha}2CARKO mice (Fig. 2). The quantitative morphometrical analysis showed that cardiac myocyte cross-sectional diameter was significantly greater in untrained {alpha}2A/{alpha}2CARKO mice compared with untrained WT control mice (Fig. 3A). Cardiac myocyte cross-sectional diameter was significantly lower in exercise-trained {alpha}2A/{alpha}2CARKO mice than in untrained {alpha}2A/{alpha}2CARKO mice, but significantly greater than in untrained WT control mice (Fig. 3A). Increased cardiac myocyte cross-sectional diameter in untrained {alpha}2A/{alpha}2CARKO mice was paralleled by an increased ventricular fibrosis, represented by a threefold increase in cardiac collagen volume fraction of {alpha}2A/{alpha}2CARKO mice when compared with untrained WT control mice (Fig. 3B). Exercise training tended to reduce left ventricular fibrosis in {alpha}2A/{alpha}2CARKO mice, but this result did not reach significantly values (Fig. 3B). Exercise-trained WT mice did not change either myocyte cross-sectional diameter or cardiac collagen volume fraction when compared with untrained WT mice.


Figure 2
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  		Fig. 2. Fractional shortening (FS) used as an index of systolic function evaluated at 5 ({square}) and 7 ({blacksquare}) mo of age in WTUN and WTT, respectively, and {alpha}2A/{alpha}2CARKO. Note that exercise training significantly improved FS in {alpha}2A/{alpha}2CARKO mice. Data are presented as means ± SE. *P < 0.05 vs. WT groups, {dagger}P < 0.05 vs. DKOUN group.

 

Figure 3
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  		Fig. 3. Cardiac myocyte cross-sectional diameter (A) and cardiac collagen volume (B) in untrained ({square}) and exercise-trained ({blacksquare}) wild-type (WT) and {alpha}2A/{alpha}2CARKO (DKO) mice. Exercise training partially reduced cardiac myocyte cross-sectional diameter in {alpha}2A/{alpha}2CARKO and had no impact on ventricular fibrosis as measured by collagen volume fraction. Data are presented as means ± SE. *P < 0.05 vs. WT groups, {dagger}P < 0.05 vs. untrained DKO group.

 
Expression of Proteins Involved in Intracellular Ca2+ Regulation
Since it has been demonstrated that the expression of calcium-handling proteins is altered in HF (17, 37), we investigated whether the expression of these proteins is altered in our {alpha}2A/{alpha}2CARKO mice and whether exercise training would change their expression profile.

GAPDH protein levels remained unchanged among the four groups studied and were used to normalize the Ca2+ handling protein levels. SERCA2 expression levels were significantly reduced in untrained {alpha}2A/{alpha}2CARKO mice compared with untrained WT control mice (Fig. 4, A and B). In contrast, no significant differences were found in SERCA2 expression between exercise-trained {alpha}2A/{alpha}2CARKO mice and WT control mice. Na+-Ca2+ exchanger expression levels were significantly increased in untrained {alpha}2A/{alpha}2CARKO mice (Fig. 4, A and C). Exercise training significantly reduced Na+-Ca2+ exchanger expression in {alpha}2A/{alpha}2CARKO mice to same level observed in untrained WT control mice (Fig. 4, A and C). In contrast, exercise training in WT mice had no impact on either SERCA2 or Na+-Ca2+ exchanger expression levels. As the sarcoplasmic Ca2+ content depends on Ca2+ reuptake by SERCA2 relative to transsarcolemmal Ca2+ elimination by Na+-Ca2+ exchanger, we calculated the SERCA2/Na+-Ca2+ exchanger ratio for all mice studied (Fig. 4D). SERCA2/Na+-Ca2+ exchanger ratio tended to be reduced in untrained {alpha}2A/{alpha}2CARKO mice (P = 0.08). Exercise training increased SERCA2/Na+-Ca2+ exchanger ratio in {alpha}2A/{alpha}2CARKO mice to the level observed in WT control mice (Fig. 4D).


Figure 4
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  		Fig. 4. Scarcoplasmic reticulum Ca2+ ATPase (SERCA2) and Na+-Ca2+ exchanger (NCX) expression and SERCA2/NCX ratio in untrained ({square}) and exercise-trained ({blacksquare}) WT and DKO mice. Data are presented as means ± SE. A: representative bands of SERCA2, NCX, and GAPDH (selected from the same gel) from untrained and exercise-trained WT and DKO mice. B, C, and D: quantitative average results from all individual samples of SERCA2, NCX expression, and SERCA2/NCX ratio, respectively. Targeted bands were normalized to cardiac GAPDH. *P < 0.05 vs. WT groups, {dagger}P < 0.05 vs. untrained DKO group.

 
Despite the fact that SERCA2 activity is under PLN control, PLN expression levels were similar among the three groups studied (data not shown), while the expression of phosphorylated PLN was changed by both genotype and exercise training (Fig. 5). In untrained {alpha}2A/{alpha}2CARKO mice, the expression of phospho-Ser16-PLN normalized to total PLN was not significantly different from untrained WT control mice (Fig. 5, A and B). However, exercise training significantly increased phospho-Ser16-PLN in {alpha}2A/{alpha}2CARKO, while it did not change it in WT mice. The expression of phospho-Thr17-PLN normalized to total PLN was higher in untrained {alpha}2A/{alpha}2CARKO mice than in untrained WT control mice. Exercise training further increased phospho-Thr17-PLN expression levels in {alpha}2A/{alpha}2CARKO (Fig. 5, A and C). This response was also observed in exercise-trained WT mice, which displayed increased phospho-Thr17-PLN expression levels compared with untrained WT mice.


Figure 5
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  		Fig. 5. Phospho-Ser16-phospholamban (PLN) and Phospho-Thr17-PLN expression normalized to total PLN and protein phosphatase type 1 (PP1) expression in untrained ({square}) and exercise-trained ({blacksquare}) WT and DKO mice. Data are presented as means ± SE. A: representative bands of Phospho-Ser16-PLN, Phospho-Thr17-PLN, PLN, PP1, and GAPDH (selected from the same gel) from untrained and exercise-trained WT and DKO mice. B,C and D: quantitative average results from all individual samples of Phospho-Ser16-PLN, Phospho-Thr17-PLN, and PP1, respectively. Targeted bands were normalized to cardiac GAPDH. *P < 0.05 vs. untrained WT group, {dagger}P < 0.05 vs. untrained DKO group, and {ddagger}P < 0.05 vs. exercise-training WT mice.

 
PLN phosphorylation is mainly regulated by PP1, and the expression and activity of PP1 seem to be increased in end-stage HF (29, 42). The expression of PP1 was increased in untrained {alpha}2A/{alpha}2CARKO mice compared with untrained WT control mice (Fig. 5, A and D). Exercise training significantly reduced PP1 expression to levels similar to WT control mice (Fig. 5, A and D), while no changes in PP1 expression levels were observed in exercise-trained WT mice. (The online version of this article contains supplemental material.)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
A great body of evidence indicates that exercise training has a beneficial effect on the treatment of HF patients including an improvement in stroke volume and cardiac output (7, 9). However, the exact mechanism by which exercise training improves cardiac function remains elusive. In the present study we demonstrate first that this genetic model of HF is also accompanied by Ca2+ handling abnormalities, second, that exercise training improves ventricular function in these mice, and finally we provided direct evidence for the involvement of Ca2+ handling proteins in this process.

Sympathetic exacerbation is the hallmark of HF and has been associated with high morbidity and mortality (34). Usually this is a secondary phenomenon, but under the conditions of the present study sympathetic augmentation was directly achieved by the use of genetically engineered mice. Interestingly, even though the experimental conditions suppressed the effects of negative feedback signals, we still observed some degree of reduction in cardiac sympathetic tone upon exercise training.

The novel finding of the present study is that exercise training increases the cardiac expression of SERCA2 and phosphorylation of PLN at Ser16 and Thr17 in {alpha}2A/{alpha}2CARKO mice. In addition, exercise training restored Na+-Ca2+ exchanger and PP1 expression levels in these mice. Presumably, both changes tend to improve Ca2+ handling under this condition. Interestingly, the effect of exercise training on SERCA2, Na+-Ca2+ exchanger, and PP1 was only observed in {alpha}2A/{alpha}2CARKO mice; no changes were observed in WT mice. These responses suggest that training effects are restricted to proteins with altered baseline expression levels.

Previous studies have shown that exercise training improves sarcoplasmic reticulum Ca2+ uptake paralleled by a normalized Na+-Ca2+ exchanger current and increased expression of SERCA2 in ischemia-induced HF rats (20, 43). Our findings extend these observations to show that exercise training also is associated to an increased phosphorylation of PLN at Ser16 and Thr17 and decreased PP1 expression levels in {alpha}2A/{alpha}2CARKO mice with sympathetic hyperactivity-induced HF. Under this scenario one may consider the possibility that phosphorylation of PLN at either Ser16 or Thr17 removes the inhibitory effect of PLN on SERCA2. Furthermore, PLN acts as an integrator of ß-adrenergic and Ca2+-dependent signaling pathways to promote increased myocardium contractility. Thus, it is reasonable to suggest that exercise training-induced augmentation of phosphorylated PLN cardiac levels improves Ca2+ reuptake and fractional shortening in {alpha}2A/{alpha}2CARKO mice. It is important to note that the phosphorylation of PLN at Ser16 or Thr17 is differentially affected by training and genotype. While the effects of exercise training on phosphorylation of PLN at Ser16 are specific to {alpha}2A/{alpha}2CARKO mice, its effect at Thr17 occurred in both WT and {alpha}2A/{alpha}2CARKO mice, even with the latter presenting higher phospho-Thr17-PLN baseline expression levels than WT controls. These responses suggest that the increased expression of phosphorylated PLN at Ser16 might play a role improving ventricular function of exercise trained {alpha}2A/{alpha}2CARKO mice, since increased phospho-Thr17-PLN expression levels were not related to an improved fractional shortening in exercise-trained WT mice.

We also observed a reduction in PP1 expression in exercise-trained {alpha}2A/{alpha}2CARKO mice. PP1 regulates the phosphorylation status of PLN by dephosphorylating PLN at both Ser16 and Thr17 residues. In HF, the overall activity and expression of PP1 are exaggerated (1, 12). The consequence of this abnormal increased expression is a reduction in Ca2+ reuptake by SERCA2 followed by a decrease in sarcoplasmic reticulum Ca2+ load, which deteriorates ventricular function (36). Interestingly, we provide evidence that exercise training reverses the increased PP1 expression levels in {alpha}2A/{alpha}2CARKO to WT control mice levels. Moreover, it may contribute, at least in part, to the improvement in fractional shortening after exercise training.

Kubo et al. (18) demonstrated that treatment with ß-blocker normalizes the abundance of cardiac myocyte Ca2+ regulatory proteins and Ca2+ handling. It is possible that our strategy based on exercise training has similar effects. In this case, exercise training would restore the depressed ß-adrenergic receptor signaling observed in persistent sympathetic activation, which, in turn, improves the balance of adrenergic-mediated regulation of kinases and phosphatases that control intracellular Ca2+ homeostasis.

HF is characterized by progressive myocardial remodeling associated with cardiac myocyte loss and ventricular fibrosis (2). In fact, we observed cardiac myocyte hypertrophy associated with increased collagen volume fraction in {alpha}2A/{alpha}2CARKO mice. The mechanisms underlying the maladaptative cardiac remodeling in HF are not completely understood, but alterations in proteins that regulate Ca2+ homeostasis have been reported (14, 31). In addition, other subcellular abnormalities such as mitochondrial remodeling, apoptosis, changes in myosin isozyme composition, and alterations in troponin phosphorylation and Ca2+-binding affinity have also been found (5, 6, 27, 35) in HF. Exercise training partially decreased cardiac myocyte cross-sectional diameter but had little impact on ventricular fibrosis. The mechanisms by which exercise training reverses cardiac ultrastructural abnormalities in {alpha}2A/{alpha}2CARKO mice is beyond the scope of the present study, but it is undoubtedly an interesting topic for future investigations.

Study Limitations
Our study shows that exercise training causes both an increase in fractional shortening and alterations in the expression of Ca2+ handling proteins. However, it does not provide direct evidence to support the cause-effect relationship between Ca2+ handling proteins expression and cardiac function. Although Ca2+ transients tend to parallel changes in the expression of cardiac Ca2+ handling proteins and cardiac function (1, 3, 21, 30), we have not directly assessed Ca2+ transients. The data are consistent with the idea that the changes observed by exercise training in the expression of Ca2+ handling proteins and ventricular function can be attributed to an improvement of cardiac Ca2+ transient. Furthermore, it is important to emphasize that the exercise training protocol was sufficient to improve exercise tolerance and elicit resting bradycardia, which paralleled the improvement in ventricular function in this genetic model of sympathetic hyperactivity-induced HF.

Even though the present model is characterized by a sympathetic hyperactivity as seen in human HF, one may argue that in this case the hyperactivity is not secondary, but primary, and the levels reached may be too high. However, HR that is highly influenced by sympathetic tone responded favorably to 8 wk exercise training, indicating that, although high, the sympathetic tone can still be modulated by a physiological intervention.

In conclusion, we provided evidence that Ca2+ handling is impaired in this HF model. Furthermore, the benefits of exercise training in this genetic model of sympathetic hyperactivity-induced HF include improvement in the net balance of myocardial Ca2+ handling proteins. These findings provide additional insight into the improvement in cardiac function associated with exercise training in HF.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
N. P. L. Rolim holds a doctoral scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. A. Medeiros holds a doctoral scholarship from FAPESP (04/00745-7). P. C. Brum holds a scholarship from Conselho Nacional de Pesquisa e Desenvolvimento - Brasil (CNPq, BPQ).


   ACKNOWLEDGMENTS
 
The authors thank the Fundação de Amparo à Pesquisa do Estado de São Paulo, São Paulo - SP (FAPESP N.° 2002/04588-8) for funding this present investigation. We also express our gratitude to Fundação Zerbini, São Paulo - SP, for the support of this study.


   FOOTNOTES
 
Address for reprint requests and other correspondence: P. C. Brum, Escola de Educação Física e Esporte da Universidade de São Paulo, Departamento de Biodinâmica do Movimento do Corpo Humano, Av. Professor Mello Moraes, 65 - Butantã - São Paulo - SP, 05508-900 Brazil (e-mail: pcbrum{at}usp.br).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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