AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 293: H246-H259, 2007. First published March 2, 2007; doi:10.1152/ajpheart.00734.2006
0363-6135/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/1/H246    most recent
00734.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Watson, P. A.
Right arrow Articles by Moore, R. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Watson, P. A.
Right arrow Articles by Moore, R. L.

Restoration of CREB function is linked to completion and stabilization of adaptive cardiac hypertrophy in response to exercise

Peter A. Watson,1,2 Jane E. B. Reusch,1,2 Sylvia A. McCune,3,4 Leslie A. Leinwand,3,4 Stephen W. Luckey,3 John P. Konhilas,3 David A. Brown,3 Adam J. Chicco,3 Genevieve C. Sparagna,3 Carlin S. Long,1 and Russell L. Moore3

1University of Colorado Health Sciences Center, and 2Denver Veterans Affairs Medical Center, Denver; and 3University of Colorado, and 4Myogen Inc., Boulder, Colorado

Submitted 10 July 2006 ; accepted in final form 27 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Potential regulation of two factors linked to physiological outcomes with left ventricular (LV) hypertrophy, resistance to apoptosis, and matching of metabolic capacity, by the transcription factor cyclic-nucleotide regulatory element binding protein (CREB), was examined in the two models of physiological LV hypertrophy: involuntary treadmill running of female Sprague-Dawley rats and voluntary exercise wheel running in female C57Bl/6 mice. Comparative studies were performed in the models of pathological LV hypertrophy and failure: the spontaneously hypertension heart failure (SHHF) rat and the hypertrophic cardiomyopathy (HCM) transgenic mouse, a model of familial idiopathic cardiomyopathy. Activating CREB serine-133 phosphorylation was decreased early in remodeling in response to both physiological (decreased 50–80%) and pathological (decreased 60–80%) hypertrophic stimuli. Restoration of LV CREB phosphorylation occurred concurrent with completion of physiological hypertrophy (94% of sedentary control), but remained decreased (by 90%) during pathological hypertrophy. In all models of hypertrophy, CREB phosphorylation/activation demonstrated strong positive correlations with 1) expression of the anti-apoptotic protein bcl-2 (a CREB-dependent gene) and subsequent reductions in the activation of caspase 9 and caspase 3; 2) expression of peroxisome proliferator-activated receptor-{gamma} coactivator-1 (PGC-1; a major regulator of mitochondrial content and respiratory capacity), and 3) LV mitochondrial respiratory rates and mitochondrial protein content. Exercise-induced increases in LV mitochondrial respiratory capacity were commensurate with increases observed in LV mass, as previously reported in the literature. Exercise training of SHHF rats and HCM mice in LV failure improved cardiac phenotype, increased CREB activation (31 and 118%, respectively), increased bcl-2 content, improved apoptotic status, and enhanced PGC-1 content and mitochondrial gene expression. Adenovirus-mediated expression of constitutively active CREB in neonatal rat cardiac recapitulated exercise-induced upregulation of PGC-1 content and mitochondrial oxidative gene expression. These data support a model wherein CREB contributes to physiological hypertrophy by enhancing expression of genes important for efficient oxidative capacity and resistance to apoptosis.

exercise; mitochondria; apoptosis; cyclic-nucleotide regulatory element binding protein

PHYSICAL ACTIVITY DECREASES cardiovascular and all-cause mortality (46) and is commonly used as a therapeutic modality for patients with cardiovascular disease, including cardiac failure. Left ventricular (LV) hypertrophy in response to physical exercise is limited in duration, sufficient to allow for the development and then stabilization of a hypertrophy that is commensurate with the exercise stimulus. Heart failure (and related failures), the leading cause of death and disability in the US (21), is characterized by unabated ventricular growth and is accompanied by ventricular dilation and failure. We are interested in the molecular mechanisms responsible for restricting cardiac hypertrophy to that required to match functional demand in response to exercise training.

Three critical, published observations indicate that cyclic-nucleotide regulatory element binding protein (CREB) function is important for the maintenance of normal physiological cardiac function. Transgenic ablation in the heart of the activity of the transcription factor CREB is sufficient to induce dilated cardiomyopathy (12). The absence of CREB function in this transgenic, dominant-negative CREB animal also prevents improvement of survival and cardiac pathology in response to exercise (53). Increased expression of the negative regulator of CREB transactivation, inducible cAMP early repressor, occurs during pathological hypertrophy induced by beta-adrenergic stimulation, angiotensin II, and hypertension, and results in diminished expression of bcl-2 and increased cardiac myocyte apoptosis in vivo (55). Overexpression of either inducible cAMP early repressor or dominant-negative CREB (Ser133 to Ala133 mutation) mimics the response to beta-adrenergic stimulation, inducing apoptosis in cultured cardiac myocytes (55). In addition, studies into the mechanisms underlying hypertrophy in response to ischemia/hypoxia indicate that activation of CREB occurs concurrent with reoxygenation and subsequent hypertrophy, and expression of dominant-negative CREB abrogates hypertrophy in response to hypoxia/reoxygenation (9).

Two mechanisms frequently evoked to account for the different outcomes occurring in response to exercise or pathological stimuli (such as hypertension) are 1) an increased myocardial cell apoptosis during pathological hypertrophy and failure and 2) a failure to increase metabolic energy production sufficiently to match increased energy demand during pathological hypertrophy. Mitochondrial dysfunction resulting from a number of causes in the heart has been shown to be seminal to the development of hypertrophic failure (for reviews, see Refs. 7, 15). CREB is a potent anti-apoptotic protein, primarily the result of its direct regulation of bcl-2 gene expression in the nuclear genome (37, 43, 44, 59), and as such it could impact this potential mechanism of hypertrophic failure. CREB has been shown in a number of tissues to regulate the expression of peroxisome proliferator-activated receptor-{gamma} coactivator-1 (PGC-1) (60). PGC-1 is crucial for regulation of contractile function and metabolic energy status in cardiac muscle (2, 30–32, 50), the expression of genes encoding both components of the oxidative machinery, and factors impacting transcription of these oxidative proteins in both the nuclear and mitochondrial genomes (13, 17, 30, 31).

Recently, studies in neurons indicate that CREB is present, functional, and regulated in the mitochondria of this cell type under proapoptotic conditions (6, 29, 48). This work demonstrates that CREB in the mitochondria is phosphorylated on serine-133, is capable of binding to sequences with homology to cyclic-nucleotide regulatory elements in the D-loop of the mitochondrial DNA, and is capable of regulating the expression of a number of genes encoding proteins in complex I of oxidative phosphorylation [NADH dehydrogenase (ND), complex I subunits 5 (ND5) and 6 (ND6)] (29, 48). Most convincingly, expression of dominant-negative CREB, targeted to be imported into mitochondria, results in a significant decrease in ND5 and ND6 mRNA (29). This strongly implies that CREB must be endogenously regulating the expression of these genes.

The studies described in this paper addressed the following hypothesis: restoration of CREB activation and function contributes to stabilizing stimulus-matched, physiological hypertrophy in response to exercise through mechanisms impacting both apoptosis and mitochondrial oxidative capacity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Rabbit polyclonal antisera for detection of CREB, phospho-Ser133 (pSer133) CREB, mouse-specific caspase 9, and BAX, as well as a mouse monoclonal antibody for pSer133 CREB, were obtained from Cell Signaling Technology (Beverly, MA). Additional antisera, obtained from other sources, included mouse monoclonal anti-bcl-2 (BD Transduction Laboratories), rabbit polyclonal anti-active caspase 3 (Biomol), beta-actin (mouse monoclonal; Sigma Chemical, St. Louis, MO), anti-PGC-1 antibody (rabbit polyclonal; Santa Cruz Biotech, Santa Cruz, CA), anti-cytochrome c antibody (mouse monoclonal; BD Pharmingen), and anti-pyruvate dehydrogenase E1{alpha} subunit (PDH-E1{alpha}) antibody (mouse monoclonal; MitoSciences, Eugene, OR). Antisera against the voltage-dependent anion channel (VDAC1, or Porin) (8, 38) and cytochrome oxidase IV (COX IV) (18, 25, 52) were obtained from Abcam (Cambridge, MA). These antisera have been used previously as markers of mitochondrial content as their contents parallel changes in mitochondrial density (8, 18, 25, 38, 52). The anti-nuclear respiratory factor-1 (Nrf-1) rabbit polyclonal antibody was the generous gift of Dr. Richard Scarpulla, Northwestern University. The rat-specific antibody against mitochondrial transcription factor A (TFAM) was the generous gift of Dr. Samson Jacob (The Ohio State University).

Animals. The use of animals, and all of the experimental interventions used in this study, received prior approval from the Institutional Animal Care and Use Committee at the University of Colorado at Boulder and were conducted under the guidelines accepted by the American Physiological Society.

Spontaneously hypertension heart failure rat samples. Frozen LV samples from true lean male spontaneously hypertension heart failure (SHHF) rat hearts were taken from young 2-mo (prehypertension), 7-mo (established hypertension), and 19 ± 2.5-mo SHHF rats in overt congestive heart failure. A second set of samples was from lean male SHHF with an accelerated heart failure, induced by an 8% sodium chloride diet given from 6 wk of age and euthanized at 6.5 mo of age (average heart weight, 2.34 ± 0.32 g), and was compared with animals of similar age with the established hypertension (above) as controls. The third set of samples was from a study of male SHHF rats that were treadmill trained at low intensity (14 m/min at a 10% grade) for 45-min sessions 3 days/wk for 6 mo, starting at 15.5 mo of age with sedentary age-matched counterparts. The results of this study showed that seven out of nine sedentary SHHF controls developed overt heart failure; heart failure was not observed in any of the run-trained SHHF rats (10).

Exercise training of rats. Female Sprague-Dawley rats were subjected to treadmill exercise training following acclimation, as previously described (5). Sedentary animals were placed on the nonmoving treadmill for the same amount of time each day for acclimation and training periods. Animals were euthanized 24 h after their last exercise bout by intraperitoneal injection of pentobarbital sodium, 35 mg/kg body wt. Hearts were removed, and LV were dissected, weighed, and either immediately frozen at the temperature of liquid nitrogen (Western blot analysis), or used immediately for preparation of mitochondria. Frozen samples were stored until preparation of analyses.

Hypertrophic cardiomyopathic transgenic mice. This mouse model of the most frequent form of familial cardiomyopathy in humans (16) has been shown previously to develop mitochondrial dysfunction concurrent with cardiac failure (35). Our laboratory has previously published work on the CREB phosphorylation and bcl-2 gene expression in hypertrophic cardiomyopathy (HCM) transgenic mice and nontransgenic control animals, both sedentary and voluntarily exercising on an exercise wheel (28). Tissue from the same samples used to obtain the CREB and bcl-2 data in our laboratory's previous paper (28) was reanalyzed by Western blot to assess expression of PGC-1, Nrf-1, and cytochrome c, as well as two markers of mitochondrial content, VDAC1 (8, 38) and COX IV (18, 25, 52).

Culture of neonatal rat cardiac myocytes and infection with CREB-recombinant adenoviruses. Cultured cardiac myocytes isolated from 1-day-old neonatal rats were obtained as described previously (22). Cultures were studied under serum-free MEM supplemented with transferrin, insulin, bovine serum albumen, bromodeoxyuridine, and PB12. Adenoviral constructs for DIEDML-CREB and beta-galactosidase were developed in our laboratory and have been described previously (26, 56, 57). Adenoviral infection was done at a multiplicity of infection of 5 to 10 on culture day 1 and incubated for 72 h. Protein extracts were prepared by scraping cells in Laemmli buffer, and protein concentration was determined and subsequent dilution into Laemmli sample buffer (LSB) for Western Blot analysis. RNA was isolated from cells in culture using the Qiagen RNAeasy Mini Kit (Qiagen, Valencia, CA), following the manufacturer's instructions. Total RNA was subjected to quantitative reverse transcription polymerase chain reaction to quantitative mRNA contents encoding for complex I ND subunits, as described below.

Preparation of LV tissue for Western blot analysis. Protein extracts from frozen heart tissue were prepared as described previously for aortic tissue (56). Protein in these extracts was analyzed by Bradford protein assay. Extracts were diluted with water and concentrated LSB to a final concentration of 1 µg/µl and 1x LSB. Samples were frozen and stored at –80°C until analysis. Sufficient protein was obtained from each of these preparations to perform all of the Western blot analyses reported on each experimental group in this paper.

Western blot analyses. Gel electrophoresis, transfer to polyvinylidene difluoride membranes, detection of specific proteins immobilized to membranes, and subsequent densitometric analysis were performed as previously described (56, 57). All samples had 40 µg of protein loaded per lane. Gel protein loading was assessed by Western analysis of samples for beta-actin content, which was not significantly different in any samples analyzed. Results are expressed as arbitrary densitometric units, with densitometric values for analysis of total protein content (e.g., PGC-1) corrected for beta-actin content before statistical analysis. All comparisons of phosphoprotein-to-protein ratios were not corrected for beta-actin, instead using only the ratio to represent the degree of phosphorylation of that protein on the specific amino acid.

Western blot membranes were sequentially blotted from proteins, which were separated significantly in molecular weight. Additionally, blots were stripped (45 min at 55°C in buffer containing 62.5 mM Tris·HCl, pH 6.7, 2% beta-mercaptoethanol, and 2% sodium dodecyl sulfate) and reprobed for other proteins. Sufficient protein was obtained in each LV extraction for multiple gels and Westerns, and all Western blot analyses reported within experimental groups were from the same samples. Samples for analyses of LV from HCM cardiomyopathic transgenic mice and their nontransgenic controls were the same as used for previously published results (28).

Coimmunoprecipitation experiments. Aliquots of extracts from hearts of sedentary rats, and rats run for 5 or 10 consecutive days that are undergoing hypertrophic remodeling, were subjected to immunoprecipitation with antibodies specific for either CREB (rabbit monoclonal, Cell Signaling, Beverly, MA) or PDH-E1{alpha} (MitoSciences, Eugene, OR). Immunoprecipitated proteins were run on 12% polyacrylamide-SDS gels and transferred to polyvinylidene difluoride membranes for subsequent Western blot analysis. Initial analysis was performed to see if the CREB antibody or the PDH-E1{alpha} antibody reacted with the other antibody's target, which they did not (data not shown). Immunoprecipitated CREB and PDH-E1{alpha} proteins were then probed to assess patterns of immunoreactivity to a pSer133 CREB (mouse monoclonal, Cell Signaling) to determine whether patterns of presumed CREB Ser133 phosphorylation were actually changes in PDH-E1{alpha} Ser300 phosphorylation (42). Briefly, 1.0 mg of LV tissue extracts were diluted to a concentration of 1 µg/µl in IP wash buffer 1 containing 1% Igepal (Sigma Chemical, St. Louis, MO) and 0.1% activated sodium orthovanadate in phosphate-buffered saline. Extracts were precleared with 18 µl of 50% slurry of protein A sepharose in IP buffer 1 for 30 min (all steps were performed at 4°C). Sepharose beads were pelleted by centrifugation for 1 min at 2,000 g. The extract was decanted and split into two 500-µl aliquots, and antisera for either CREB (2 µl of rabbit monoclonal, Cell Signaling) or PDH-E1{alpha} (4 µg of mouse monoclonal, MitoSciences, Eugene, OR) were added. Antibody/extract mixtures were incubated while rotating at 4°C overnight. One hundred microliters of a 50% slurry of protein A sepharose/IP buffer 1 were added to each tube and rotated at 4°C for 2 h. Sepharose beads were pelleted by centrifugation and washed four times each in IP buffer 1 and IP buffer 3 (10 mM Tris·HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 0.1 mM activated sodium orthovanadate). Proteins were eluted by addition of 60 µl LSB, 30 s of vortexing, boiling for 5 min, and centrifugation for 5 min at 2,000 g. Extracts were subjected to Western blot analysis, as described previously (see MATERIALS AND METHODS above).

Isolation of mitochondria from LV tissue. Subsarcolemmal and interfibrillar populations of mitochondria, which demonstrate different responses to aging (11) and ischemia (33), were isolated from fresh LV tissue removed from sedentary or exercised animals (24 h after their last exercise bout), used solely for this purpose, according to the method developed by Palmer et al. (40). A bicinchoninic acid protein assay (Pierce) is used to assess the concentration of mitochondrial protein in each sample.

Mitochondrial oxygen consumption. Determinations of mitochondrial respiration were performed using a Clark-type oxygen electrode and methods optimized for cardiac mitochondria by Weinberg et al. (58). Mitochondrial respiration measurements were initiated by the addition of either malate/pyruvate (to assess flux through complexes I, III, and IV) or succinate/rotenone (to assess flux through complexes II, III, and IV). Mitochondrial state 3 (ADP-dependent) respiration was obtained by adding ADP.

Activity of electron transport chain complex IV. Complex IV (cytochrome oxidase) was quantified by monitoring the KCN-sensitive oxidation of dithiothreitol-reduced cytochrome c (34). The rate of decrease in cytochrome c absorbance was monitored at 550 nm, and rates are expressed per minute.

Statistical analysis of densitometric data from Western blots. All data are presented as means ± SE. The differences between experimental groups were analyzed with a one-way ANOVA, which was followed by a Student's t-test with post hoc Bonferroni's correction to assess differences among mean values. The level of probability indicating significance between values was a priori set at P ≤ 0.05. Linear regression analysis was used to assess the relationship between LV mass and age in male SHHF rats progressing through genetic hypertension, subsequent hypertrophy, and failure (Fig. 1B).


Figure 1
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 1. Hypertrophy in response to exercise training is acute and limited, while hypertrophy in response to hypertension is continuous. A: female Sprague-Dawley (SD) rats were subjected to continuous, nonprogressive daily treadmill running, as described in MATERIALS AND METHODS. Sedentary and trained animals were euthanized at 0, 5, 10, and 120 days; n = 6 each group. #Statistically different than control (significance P < 0.05). B: male spontaneously hypertension heart failure (SHHF) rats undergoing genetic, age-dependent development of hypertension, hypertrophy, and failure. At times during the period before the development of hypertension (2 mo of age) and during the progression of hypertension (6–8 mo of age) and heart failure (14–21 mo), animals were euthanized. Left ventricular (LV) weight data are individual data points; n = 3 in each group.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
LV hypertrophy in response to exercise training is limited in duration, while hypertrophy in response to genetic hypertension is continuous. Female Sprague-Dawley rats were subjected to continuous, nonprogressive, daily treadmill running for 4 mo and demonstrated an accelerated, significant increase in LV weight following 10 days of exercise training (Fig. 1A). Progressive increases in LV weight were observed at 4 mo of training, but the increase in mass paralleled that seen with growth with the sedentary control animals (Fig. 1A).

Male SHHF rats undergoing genetic, age-dependent development of hypertension, hypertrophy, and failure (4, 10, 36) were assessed for increases in LV weight before the development of hypertension (2 mo of age), during the progression of hypertension (6–8 mo of age), and in overt heart failure (14–21 mo). LV weight demonstrated a continuous increase over the development of hypertrophy and failure from ages 2 mo to 21 mo of age (Fig. 1B) that exceeded the rate of LV growth seen in sedentary Sprague-Dawley control animals. The increase in LV weight observed in male SHHF rats over this time period was linear, with linear regression analysis indicating an r2 value of 0.874 (Fig. 1B).

CREB activation and function are significantly decreased during early remodeling in response to exercise training, but are restored concurrent with the cessation of hypertrophic growth. Female Sprague-Dawley rats demonstrated statistically significant (P < 0.05) LV hypertrophy after 10 days (LV mass; 612 ± 27 vs. 563 ± 46 mg in control animals) but not 5 days (522 ± 34 mg) of run training. CREB Ser133 phosphorylation, crucial for transcriptional activation, was diminished after 1, 2, and 5 days of exercise and remained diminished until completion of ventricular growth after 10 days of training (Fig. 2A, run 1–10 days). No further increase in heart weight was seen with additional training beyond 10 days (Fig. 1A). Immunoprecipitation/Western blot analyses indicate that our interpretation of the pSer133 CREB immunoblots is not confounded by PDH-E1{alpha} cross reactivity (42), as CREB (but not PDH-E1{alpha}) immunoprecipitated from LV of trained animals demonstrated changes in pSer133 CREB antibody reactivity (Fig. 1B).


Figure 2
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 2. Dynamic regulation of cyclic-nucleotide regulatory element binding protein (CREB) activation (Ser133 phosphorylation) and function in the heart during remodeling in response to exercise training in adult female rats. Female rats were subjected to a run training regimen, as described in MATERIALS AND METHODS. A: protein extracts were analyzed by Western blot analysis for phospho-Ser133 (pSer133) CREB (PCREB), CREB, and beta-actin as described in MATERIALS AND METHODS. Results in graph are from n = 6 in each group. B: duplicate aliquots of extracts from hearts of sedentary (Sed), 5-day (5d Ex), and 10-day trained (10d Ex) animals were subjected to immunoprecipitation (IP) with either a CREB mouse monoclonal or pyruvate dehydrogenase E1{alpha} subunit (PDH-E1{alpha}) mouse monoclonal antibody (MAb), and immunoprecipitated proteins were subjected to pSer133 CREB Western blot analysis. C: electrophoretic mobility shift/supershift analysis of nuclear proteins isolated from LV tissue of exercised female rats was performed as described in MATERIALS AND METHODS. The amount of CREB-DNA complex supershifted in response to the addition of a CREB-specific antibody parallels changes in protein-DNA complex content. pPDH-E1{alpha}, phospho-pyruvate dehydrogenase; Ab, antibody; polyAb, polyclonal antibody. #Statistically different than control (significance P ≤ 0.05).

 
The loss of CREB phosphorylation (activation) during exercise-induced remodeling and subsequent restoration at completion of hypertrophic growth is paralleled by the loss and subsequent restoration of binding of nuclear CREB to the cyclic-nucleotide regulatory elements in electrophoretic mobility shift assays and CREB antibody-induced supershift assays (Fig. 2C). The parallel losses of CREB activation (Ser133 phosphorylation) and DNA binding of CREB suggest that dynamic changes in CREB function occur during remodeling and subsequent compensation of exercise-induced, physiological LV hypertrophy.

We have recently published work on LV cardiac hypertrophy resulting from voluntary running of mice on exercise wheels (28). Animals develop significant LV physiological hypertrophy by day 21 of training. LV tissue of mice allowed to run voluntarily on an exercise wheel exhibited similar responses in CREB phosphorylation to those reported here in exercised rats, being reduced at 7 days by 50% (P ≤ 0.05), and returned to control levels by 21 days of exercise when statistically significant hypertrophy is achieved (28).

CREB activation is significantly diminished during both the early hypertrophic response and subsequent decompensated failure in response to hypertension. In male SHHF rats, a genetic model of hypertension, hypertrophy, and dilated cardiac failure (4, 10, 36), LV mass was significantly increased in male SHHF rats manifesting early genetic hypertension (6–8 mo of age; 1.45 ± 0.11 g) and subsequent dilated failure (16–22 mo of age; 2.64 ± 0.51 g) relative to control animals at 2 mo of age (0.67 ± 0.01 g). CREB Ser133 phosphorylation was decreased in the heart by 80% with the onset of hypertension (6–8 mo of age) and by 90% in animals developing cardiac hypertrophy and failure compared with hearts from 2-mo-old, pre-hypertensive animals (Fig. 3). Similar responses in CREB Ser133 phosphorylation were observed in SHHF male rats fed a high-salt diet starting at 2 mo of age to accelerate the onset of hypertension and failure at ages 4–6 mo (data not shown).


Figure 3
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 3. Dynamic regulation of CREB activation (Ser133 phosphorylation) during remodeling in the heart in response to hypertension in male SHHF rats. Western blot analysis of protein extracts were prepared from SHHF male rats with age and the development of genetic hypertension. Protein extracts were prepared from LV of SHHF rats before hypertension [young (Y); 2–3 mo of age], with genetic hypertension and hypertrophy [Hyperten (HT); 6–8 mo of age], and in cardiac failure [failure (F); 16–22 mo of age]. Western blot analysis of CREB activation was performed as described in MATERIALS AND METHODS. Graphed results are for n = 6 in all groups. #Statistically different than control (significance P < 0.05).

 
Preliminary studies in a nongenetic model of hypertension (thoracic aortic banding) have been performed in 4-mo-old female Sprague-Dawley rats. Animals were euthanized once short-axis two-dimensional and M-mode echocardiography (41, 49) indicated increased free wall thickening in animals with thoracic aortic banding relative to age-matched control animals (0.376 ± 0.008 vs. 0.306 ± 0.026 cm; P < 0.05), but before the development of LV dilation and failure. This increase in LV free wall thickness was accompanied by losses in CREB Ser133 phosphorylation (data not shown) similar to those seen early in LV hypertrophy with genetic hypertension in SHHF rats.

Pathological hypertrophy and failure are accompanied by decreased bcl-2 protein content and increases in markers of intrinsic death pathway apoptotic markers. Accelerated cardiac failure in the SHHF rat in response to high salt and aortic banding results in a decrease in the content of the anti-apoptotic protein bcl-2 (Fig. 4A), which occurs concurrently with the loss of CREB activity/Ser133 phosphorylation (Fig. 2). Loss of bcl-2, an anti-apoptotic component of the intrinsic mitochondrial death pathway, is accompanied by increases in caspase-9 cleavage products (Fig. 4B), the initial caspase in the intrinsic death pathway, and active cleavage products of caspase-3 (Fig. 4C). These changes in markers of cellular apoptosis are consistent with an early activation of myocyte apoptotic signaling, which may contribute to the progression to cardiac failure. Decreases in bcl-2 protein content and increases in active caspase-9 and active caspase-3 cleavage products are also seen in the nonaccelerated (normally progressing) SHHF rat heart failure model (data not shown).


Figure 4
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 4. Content of the CREB-dependent anti-apoptotic protein bcl-2 is decreased, and activation of caspase-9 and caspase-3 is enhanced concurrent with loss of Ser133 CREB in SHHF rat accelerated failure. Protein extracts prepared from female SHHF rats, some on a high-salt diet, were assessed by Western blot analysis for the contents of bcl-2 (A), caspase-9 active cleavage products (B), and caspase-3 active cleavage products (C), with materials and techniques described in MATERIALS AND METHODS. Results were similar to those observed in SHHF rat hearts undergoing genetic hypertension, hypertrophy, and failure (data not shown). Results in graph are from n = 6 in each group. #Statistically different than control (significance P ≤ 0.05).

 
Exercise-induced compensated hypertrophy is accompanied by increases in the anti-apoptotic protein bcl-2. Increases in the content of the bcl-2 in the heart accompany run training in rats (Fig. 5A). The protein content of BAX, the pro-apoptotic binding partner to bcl-2 (39), is decreased, resulting in an increase in the bcl-2-to-BAX ratio (Fig. 5A). A decrease occurs in the quantity of caspase-3 active products (Fig. 5B), reflective of decreased apoptosis in the heart with exercise training. Similarly, increases in the bcl-2-to-BAX ratio are observed in hearts of voluntarily run mice, with no change in the amount of caspase-3 active cleavage products (data not shown).


Figure 5
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 5. Bcl-2 protein content is increased, and activity of the mitochondrial death is decreased in response to run training in adult female rats. Protein extracts from hearts of female rats, trained on a treadmill, previously examined for CREB (Fig. 1B) were assess by Western blot analysis for bcl-2 protein content, BAX protein content, and the ratio of bcl-2 to BAX (A), as well as the generation of active caspase-3 cleavage products (B), using materials and techniques described in MATERIALS AND METHODS. Results in graph are from n = 6 in each group. #Statistically different than control (significance P ≤ 0.05). S, sedentary; 5d, 5-day trained; 10d, 10-day trained.

 
Exercise of old SHHF rats in cardiac failure, which improves survival, increases CREB activity/Ser133 phosphorylation, and bcl-2 protein content, and decreases caspase-3 activity. Mild exercise training of a group of old male SHHF rats in cardiac failure improved survival, with all animals in the training regimen surviving, but 21 out of 22 sedentary animals dying within the same time frame (10). Exercise did not reduce hypertension in SHHF rats (10), but did increase CREB Ser133 phosphorylation (Fig. 6A) and bcl-2 content (Fig. 6B) and diminished caspase-3 activity (Fig. 6C) relative to male SHHF rats of the same age in cardiac failure not subjected to exercise training.


Figure 6
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 6. Mild exercise training in female SHHF rats, manifesting hypertension and dilated failure, shown to improve survival, increases Ser133 CREB phosphorylation, increased bcl-2 content, and decreased caspase-3 activity. SHHF rats (22 mo old) with hypertension and dilated failure (16 mo of age) were either allowed to remain sedentary or were put on a program of mild exercise training, shown to drastically improve survival (10). Protein extracts from LV tissue were prepared and analyzed by Western blot for pSer133 CREB content (A), bcl-2 protein content (B), and caspase-3 active cleavage product content (C). Results in graph are from n = 6 in each group. #Statistically different than control (significance P ≤ 0.05).

 
Exercise training-induced restoration of CREB activation correlates strongly with increased expression of PGC-1, Nrf-1, TFAM, and accumulation of cytochrome c. The restoration of CREB activity (Ser133 phosphorylation) to levels seen in sedentary control rats observed in hypertrophied LV in female Sprague-Dawley rats after 10 days of exercise training (Figs. 1A and 7A) is accompanied by significant increases in the contents of PGC-1 and TFAM and restoration of Nrf-1 to contents seen in sedentary animals (Fig. 7, B, C, and E). These are proteins involved in the expression of genes encoding proteins either regulating or composing the oxidative phosphorylation machinery in the mitochondria (13, 14, 17, 47, 51). Additionally, expression of cytochrome c, known to be regulated by the coordinated action of Nrf-1 and CREB (20), also showed regulation parallel to changes observed in CREB Ser133 phosphorylation and Nrf-1 content (Fig. 7D).


Figure 7
View larger version (30K):
[in this window]
[in a new window]

  		Fig. 7. Exercise training in adult female rats induces changes in the contents in the LV of proteins encoded by nuclear genes involved in upregulation of mitochondrial oxidative capacity. Extracts prepared from female Sprague-Dawley rats undergoing treadmill run training (A; see Fig. 2) were subjected to Western blot analysis to assess changes in the content of phospho-CREB (A), peroxisome proliferator-activated receptor-{gamma} coactivator-1 (PGC-1; B), nuclear respiratory factor-1 (Nrf-1; C), cytochrome c (D), and mitochondrial transcription factor A (TFAM; E). Changes in the contents of these proteins parallel changes observed in CREB function (Fig. 2). Results in graph are from n = 6 in each group. #Statistically different than control (significance P ≤ 0.05).

 
Increased CREB activation, concurrent with exercise-induced rescue of pathological hypertrophy in SHHF rats and HCM mice, are paralleled by changes in PGC-1, Nrf-1, and cytochrome c content. Our laboratory has previously reported that exercise training results in improved cardiac function and increased CREB activation in the HCM cardiomyopathic transgenic mouse (28). Reanalysis of LV protein extracts from HCM transgenic mice in our laboratory's previously published study (28) indicates that training-induced increases in CREB activity are accompanied by increases in PGC-1, Nrf-1, and cytochrome c content (Fig. 8).


Figure 8
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 8. Voluntary exercise by hypertrophic cardiomyopathy (HCM) cardiomyopathic transgenic mice, shown to restore CREB activation in the LV of the heart, results in increased LV contents of PGC-1 (A and B) and Nrf-1 (A and C) to levels seen in nontransgenic (NTG) control animals. Decreased contents of PGC-1 and Nrf-1 are observed in HCM mice relative to NTG controls. Increases in these protein contents are observed in HCM animals undergoing exercise training. These increases parallel changes in CREB Ser133 phosphorylation reported previously (28). Results in graph are from n = 6 in each group. #Statistically different than control (significance P ≤ 0.05).

 
Similarly, mild exercise in male SHHF rats in cardiac failure improves survival and cardiac phenotype (10) and increases CREB Ser133 phosphorylation relative to SHHF rats of the same age in cardiac failure not subjected to exercise training (Figs. 2A and 9A). Concurrent with the restoration of CREB activation, we observe increases in the contents of PGC-1, Nrf-1, cytochrome c, and TFAM (Fig. 9, B, C, D, and E, respectively).


Figure 9
View larger version (29K):
[in this window]
[in a new window]

  		Fig. 9. Mild exercise training in SHHF rats, manifesting hypertension and dilated failure, shown to increase Ser133 CREB phosphorylation, results in increases in PGC-1, Nrf-1, and cytochrome c. Extracts were prepared from young female SHHF rats and older animals in cardiac failure (either sedentary or undergoing treadmill run training; see Fig. 5). Extracts were subjected to Western blot analysis to assess changes in the content of pSer133 CREB (A), PGC-1 (B), Nrf-1 (C), cytochrome c (D), and TFAM (E). Changes in the contents of these proteins parallel changes observed in CREB activation. Results in graph are from n = 6 in each group. #Statistically different than control (significance P ≤ 0.05). @Statistically different than Old SHHF, also in cardiac failure but not subjected to exercise training (significance P < 0.05).

 
We attempted to recapitulate the response to exercise on PGC-1, Nrf-1, cytochrome c, and TFAM protein contents by mimicking changes in CREB activity in cultures of neonatal rat cardiac myocytes (NRCM). NRCM, infected with recombinant, replication-deficient adenoviruses encoding either constitutively active CREB (DIEDML), demonstrate increases in content of PGC-1, Nrf-1, cytochrome c, and TFAM (Fig. 10). Alternatively, adenovirus-mediated expression of dominant-negative CREB resulted in decreases in Nrf-1 and cytochrome c protein contents (Fig. 10). These results imply, minimally, that Nrf-1 and cytochrome c protein contents are directly regulated by changes in CREB activity in cardiac myocytes.


Figure 10
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 10. Expression of constitutively active DIEDML-CREB in neonatal rat cardiac myocytes in culture results in upregulation of PGC-1, Nrf-1, cytochrome c, and TFAM protein contents. Cultures of neonatal rat cardiac myocytes were infected with recombinant, replication-deficient adenoviruses encoding either an active CREB mutant (DIEDML-CREB), a dominant-negative CREB mutant (ACREB), or a control virus expressing beta-galactosidase (beta-Gal; 1 x 109 multiplicity of infection). Cells were incubated for 36 h and subsequently processed to assess the contents of proteins. Protein extracts were prepared as described in MATERIALS AND METHODS. Proteins were analyzed by Western blot (A, representative blots; B, quantitation) as described in MATERIALS AND METHODS. Results in graph are from n = 6 in each group. #Statistically different than control (significance P < 0.05).

 
Exercise training results in increases in the content and activity or mitochondrial proteins and mitochondrial respiratory capacity in the LV of exercising female Sprague-Dawley rats that are commensurate with the increase in LV mass. Mitochondrial protein activity and mitochondrial function were assessed in mitochondria isolated from sedentary and 10 treadmill-trained female Sprague-Dawley rats. LV mitochondrial protein content was shown to be similar or increased per milligram of total LV protein in LV tissue from sedentary or 10-day exercise-trained animals. The contents of cytochrome c (Fig. 7D), COX IV (Table 1), and the VDAC1/Porin (Table 1) in LV tissue from 10-day trained animals were similar to or increased relative to contents observed in LV from sedentary controls. State 3 respiration in both interfibrillar and subsarcolemmal population of LV mitochondria of 10-day trained animals, assessed both for flux through complexes I, III, and IV (using malate/pyruvate as substrate) and through complexes II, III, and IV (using succinate as substrate and the complex I inhibitor Rotenone), was not statistically different than State 3 respiration assessed in LV mitochondria from sedentary control animals (Table 1). Additionally, cytochrome c oxidase/complex IV activity in mitochondria isolated from LV of sedentary and 10-day trained female Sprague-Dawley rats were not statistically different (Table 1). Mitochondrial oxygen consumption and cytochrome c oxidase activity per milligram of mitochondrial protein were not statistically different in mitochondria isolated from the LV of sedentary or 10-day exercise-trained animals, resulting from increases in mitochondrial protein content and activities that were commensurate with the observed increase in LV mass with exercise training.


View this table:
[in this window]
[in a new window]

  		Table 1. Exercise-induced changes in mitochondrial function and content in female Sprague-Dawley rats subjected to involuntary treadmill run training

 
Exercise training results in increases in the content and activity of mitochondrial proteins in the LV of animals manifesting cardiac failure. Mitochondrial protein content was examined in the female HCM cardiomyopathic transgenic mouse, shown in previous studies to manifest cardiac failure and mitochondrial dysfunction relative to its nontransgenic controls (35). When allowed to exercise voluntarily, these animals demonstrate improved cardiac phenotype, accompanied by increased CREB activation (28). Female HCM transgenic mice demonstrated a statistically significant (P < 0.05) reduction in mitochondrial protein content in LV tissue relative to nontransgenic control animals. In addition to the 55% decrease in LV content of cytochrome c observed in the LV of female HCM transgenic mice relative to nontransgenic controls (Fig. 8C), reductions in the LV content of VDAC1 (decreased 17%) and COX IV (decreased 32%) were also statistically significant (P < 0.05) relative to controls. Exercise training of HCM transgenic mice restored the content of cytochrome c (Fig. 8C), COX IV, and VDAC1 to levels not statistically different from those observed in LV tissue from nontransgenic controls.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Several significant conclusions derive from our data. First, dynamic regulation of CREB activation occurs during cardiac remodeling in response to exercise, with restoration of CREB function occurring concurrent with the development and then stabilization of a hypertrophy that is commensurate with the exercise stimulus. This is in contrast to models of hypertension-induced continuous cardiac hypertrophy and subsequent failure, in which CREB activation fails to be restored. Second, a strong correlation exists between exercise training-induced restoration of CREB activation in both healthy and cardiomyopathic animal models and increased expression of genes involved in mitochondrial oxidative capacity, resulting in restoration of mitochondrial respiratory capacity in the LV. Third, expression of the CREB-dependent protein bcl-2 is significantly increased in parallel with restoration of CREB activation and the development and then stabilization of a hypertrophy that is commensurate with the exercise stimulus. This is in contrast to suppressed CREB activation and bcl-2 expression during continuous hypertrophy, leading to failure in response to hypertension. Bcl-2 has been shown to exert a regulatory influence on two mechanisms that may contribute to remodeling of the myocardium during hypertrophic growth and mitochondria-mediated apoptosis (37).

As discussed in the Introduction, a role for CREB function in establishing and maintaining normal physiological cardiac function is well supported in the literature (9, 12, 53, 55). Our results demonstrate dynamic regulation of CREB function during hypertrophic remodeling in response to exercise training, with CREB function being restored to preexercise levels, concurrent with the completion of hypertrophic growth. CREB activation and function appear to be downregulated during early remodeling in response to both exercise and hypertension; CREB activation is restored only with compensation of exercise-induced hypertrophy. Our results also suggest that the transient loss of CREB function during hypertrophic remodeling may be permissive for cardiac remodeling. It is interesting to note that parallel studies in male Sprague-Dawley rats failed to elicit either a significant hypertrophy of the LV or any changes in CREB phosphorylation (data not shown), which may further support the need to modulate CREB activity to get hypertrophic remodeling in the LV. This gender difference in hypertrophy in response to exercise has also been reported in mice (27). We hypothesize that loss of CREB function may be permissive for hypertrophic growth, as well as by acting as a molecular "traffic cop," braking hypertrophic growth once the increase in ventricular mass restores mechanical homeostasis to the ventricle.

The physiological hypertrophy observed with nonprogressive exercise of female Sprague-Dawley rats was acute, being limited to the first 10 days of training (Fig. 1A), with subsequent increases in LV weight paralleling normal growth with aging observed in sedentary animals (Fig. 1A). In comparison, a continuous increase in LV weight was seen throughout the life of male SHHF rats developing hypertension, hypertrophy, and failure, which exceeded the rate of LV growth observed in healthy, control Sprague-Dawley rats (Fig. 1B). This accelerated increase in LV weight was fairly linear (Fig. 1B). Interestingly, the onset of failure is delayed in female SHHF rats compared with male SHHF rats, despite both sexes experiencing genetic hypertension (36).

Our observation of linear LV growth in male SHHF rats with time and disease onset is in contrast to the pattern of LV weight change in SHHF male rats reported by Reffelmann and Kloner (45), who report that LV weight is not significantly increased during the early phases of heart failure in male SHHF rats. LV mass was estimated by this group using transthoracic echocardiography, and this predicted LV mass was normalized to body weight (45). Even in this estimated, normalized data in the Reffelmann study (45), a trend toward increasing LV mass is observed later in the progression of heart failure. In the work reported in Fig. 1B and a previous study by one of the authors (4), where LV weight was assessed by direct weighing following dissection, a significant increase in LV mass was observed through the progression of heart failure.

Results from our experiments indicate that exercise in HCM transgenic cardiomyopathic mice increased CREB Ser133 phosphorylation and improved cardiac function (28). Exercise in male SHHF rats in cardiac failure, even of moderate intensity, also increased CREB Ser133 phosphorylation relative to SHHF rats of the same age in cardiac failure not subjected to exercise training (Fig. 6), which was accompanied by a significant improvement in cardiac myocyte length-to-width ratio, delayed the onset of decompensated heart failure, and increased survival compared with sedentary SHHF rats in heart failure (10). This effect of exercise on survival is likely to be true in female SHHF rats (S. A. McCune, unpublished observations). The idea that restoration of CREB function is necessary for the improvement of pathological hypertrophy with exercise training is supported by work in transgenic mice expressing dominant-negative (Ser133 to Ala133 mutation) CREB in a heart-specific manner. These animals normally develop dilated cardiomyopathy (12), and this is not improved in response to exercise training (53).

We speculate that increased LV CREB activity in HCM mice and SHHF rats in response to exercise may impact two frequently evoked mechanisms contributing to the conversion of hypertrophy to cardiac failure, cardiac cell apoptosis (1, 19, 23), and ineffective metabolic matching of energy production and energy demand (24). Regarding improved resistance to apoptosis, increased CREB function with exercise in these models of pathological hypertrophy (Ref. 28, Figs. 6 and 9A, respectively) is paralleled by increased expression of the CREB-dependent anti-apoptotic gene bcl-2 and decreased markers of apoptosis (Ref. 28, Fig. 6).

Our observations in rodent models of physiological and pathological hypertrophy demonstrate strong correlations between changes in CREB activation and expression of genes involved in mitochondrial respiration (Figs. 8 and 9). Genes in both the nuclear and mitochondrial genome seem to be expressed coordinately with changes in CREB activation in models of physiological hypertrophy, as well as in both genetic and nongenetic models of pathological, hypertensive LV hypertrophy. Direct evidence for CREB regulation of these genes in cardiac cells was obtained in cultured NRCM through infection of these cells with recombinant, replication-deficient adenoviruses expressing mutant CREB isoforms. These experiments provide direct evidence of CREB regulation in cardiac myocytes of the nuclear gene PGC-1 (Fig. 10), a crucial regulator of mitochondria content (30, 47) and function (2) in the heart, as well as Nrf-1, cytochrome c, and TFAM proteins (Fig. 10), nuclear genes that are responsive to changes in PGC-1 (30–32). These results may also indicate that CREB activity directly regulates both Nrf-1 and cytochrome c gene expression. Recent work in skeletal muscle cells supports a role for CREB function in regulation of PGC-1 expression and subsequent adaptive mitochondrial biogenesis (60). This work demonstrates CREB-dependent regulation of PGC-1 gene expression, mediated through transducer of regulated CREB-binding proteins, and a subsequent increase in cytochrome c accumulation (60) that bears marked similarity to the results from heart and cardiac myocytes presented here.

Preliminary studies in cultured cardiac myocytes indicate that, as observed in neuronal cells in culture (6, 29, 48), CREB activity may impact expression of complex I subunit genes in the mitochondrial genome. Manipulation of CREB activity using infection with recombinant adenoviruses expressing constitutively active DIEDML-CREB in cultured myocytes significantly increased expression of mRNA for complex I subunit genes ND4 and ND5 (data not shown). Preliminary examination of RNA from LV exercise-trained rats using quantitative RT-PCR also indicates that ND subunit mRNA contents parallel changes in CREB activity (data not shown).

The results presented in this paper lead us to propose the regulatory scheme presented in Fig. 11. Restoration of CREB function (Fig. 11A), in both nuclei and mitochondria, during the late stages of exercise-induced LV hypertrophy, is a critical factor in reestablishing physiologically balanced function to the myocardium. CREB function in both nuclei and mitochondria remains suppressed in response to pathological stimuli such as hypertension. Elevated CREB activity (Fig. 11B) during completion of physiological hypertrophy acts to increase metabolic capacity to match the increased energy demands of exercise by enhancing expression of genes required for increasing mitochondrial oxidative capacity. Elevated CREB activity also acts to increase bcl-2 gene expression and protein content, leading to increased resistance to apoptosis. The failure to upregulate CREB activity after the acute response to pathological stimuli results in failure to increase mitochondrial gene expression, leading to a metabolic mismatch where energy demand exceeds energy production (continuing pathological hypertrophy; Fig. 11). This metabolic mismatch leads to a greater risk of apoptotic death of cardiac myocytes, exacerbated by a decrease in CREB-dependent bcl-2 expression. We believed the combined effect of a negative energy balance and a reduced resistance to apoptosis increases the likelihood that the LV myocardium will transition from a compensatory hypertrophic response to cardiac failure. Indeed, studies in both animals (3) and humans (54) with cardiomyopathy, associated with decrements in mitochondrial oxidative potential and increased apoptosis, show improved cardiac function downstream of protection of mitochondrial respiratory function.


Figure 11
View larger version (46K):
[in this window]
[in a new window]

  		Fig. 11. Differences in CREB activation during physiological and pathological hypertrophy results in divergent responses in anti-apoptotic state and metabolic capacity. A; alterations in CREB phosphorylation, B; changes in gene expression.

 

   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Institutes of Health (NIH) Grants HL40306-15 (R. L. Moore), HL072790 (R. L. Moore), Veterans Affairs Merit and Research Enhancement Award Program Awards (J. E. B. Reusch, P. A. Watson), NIH RO1 DK06741 (J. E. B. Reusch), NIH DERC 57576, NIH PO1 56481, and an American Heart Association Regional Grant-in-Aid (J. E. B. Reusch).


   ACKNOWLEDGMENTS
 
We acknowledge the expert technical support of Joshua Lynch in the preparation of the rat models of exercise training, and Michelle Pedler for preparation of RNA and cDNA for quantitative RT-PCR.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Watson, Denver VA Medical Center, 111H, 1055 Clermont St., Denver CO 80220 (e-mail: pete.watson{at}uchsc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Anversa P, Kajstura J, Olivetti G. Myocyte death in heart failure. Curr Opin Cardiol 11: 245–251, 1996.[CrossRef][ISI][Medline]
  2. Arany Z, He H, Lin J, Hoyer K, Handschin C, Toka O, Ahmad F, Matsui T, Chin S, Wu PH, Rybkin II, Shelton JM, Manieri M, Cinti S, Schoen FJ, Bassel-Duby R, Rosenzweig A, Ingwall JS, Spiegelman BM. Transcriptional coactivator PGC-1 alpha control energy state and contractile function in cardiac muscle. Cell Metab 1: 259–271, 2005.[CrossRef][ISI][Medline]
  3. Ascensao A, Magalhaes J, Soares JMC, Ferreira R, Neuparth MJ, Marques F, Oliveira PJ, Duarte JA. Moderate endurance training prevents doxorubicin-induced in vivo mitochondriopathy and reduces the development of cardiac apoptosis. Am J Physiol Heart Circ Physiol 289: H722–H731, 2005.[Abstract/Free Full Text]
  4. Bergman MB, Kao RH, McCune SA, Holycross BJ. Myocardial tumor necrosis factor-{alpha} secretion in hypertensive and heart failure-prone rats. Am J Physiol Heart Circ Physiol 277: H543–H550, 1999.[Abstract/Free Full Text]
  5. Brown DA, Lynch JM, Armstrong CJ, Caruso NM, Ehlers LB, Johnson MS, Moore RL. Susceptibility of the heart to ischaemia-reperfusion injury and exercise-induced cardioprotection are sex-dependent in the rat. J Physiol 564: 619–630, 2005.[Abstract/Free Full Text]
  6. Cammarota M, Paratcha G, Bevilaqua LRM, Levi de Stein M, Lopez M, Pellegrino de Iraldi A, Izquierdo I, Medina JH. Cyclic AMP-response element binding protein in brain mitochondria. J Neurochem 72: 2272–2277, 1999.[CrossRef][ISI][Medline]
  7. Casademont J, Miro O. Electron transport chain defects in heart failure. Heart Fail Rev 7: 131–139, 2002.[CrossRef][Medline]
  8. De Oliveira F, Chauvin C, Ronot X, Mousseau M, Levere X, Fontaine E. Effects of permeability transition inhibition and decreases in cytochrome c content on doxorubicin toxicity in K562 cells. Oncogene 25: 2646–2655, 2006.[CrossRef][ISI][Medline]
  9. El Jamali A, Freund C, Rechner C, Scheidereit C, Dietz R, Bergmann MW. Reoxygenation after severe hypoxia induces cardiomyocyte hypertrophy in vitro: activation of CREB downstream of GSK3beta. FASEB J 18: 1096–1098, 2004.[Abstract/Free Full Text]
  10. Emter CA, McCune SA, Sparagna G, Radin MJ, Moore RL. Low-intensity exercise training delays onset of decompensated heart failure in spontaneously hypertensive heart failure rats. Am J Physiol Heart Circ Physiol 289: H2030–H2038, 2005.[Abstract/Free Full Text]
  11. Fannin SW, Lesnefsky EJ, Slabe TJ, Hassan MO, Hoppel CL. Aging selectively decreases oxidative capacity in rat heart interfibrillar mitochondria. Arch Biochem Biophys 372: 399–407, 1999.[CrossRef][ISI][Medline]
  12. Fentzke RC, Korcarz CE, Lang RM, Lin H, Leiden JM. Dilated cardiomyopathy in transgenic mice expressing a dominant-negative CREB transcription factor in the heart. J Clin Invest 101: 2415–2426, 1998.[ISI][Medline]
  13. Finck BN, Kelly DP. PGC-1 coactivators: inducible regulators of energy metabolism in health and disease. J Clin Invest 116: 615–622, 2006.[CrossRef][ISI][Medline]
  14. Finck BN, Lehman JJ, Barger PM, Kelly DP. Regulatory networks controlling mitochondrial energy production in development, hypertrophied, and diabetic heart. In: The Cardiovascular System, edited by Stillman B, Stewart DJ. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2002, p. 371–382.
  15. Fosslien E. Review: mitochondrial medicine-cardiomyopathy caused by defective oxidative phosphorylation. Ann Clin Lab Sci 33: 371–396, 2003.[Abstract/Free Full Text]
  16. Freeman K, Colon-Rivera C, Olsson MC, Moore RL, Weinberger HD, Grupp IK, Vikstrom KL, Iaccarino G, Koch WJ, Leinwand LA. Progression from hypertrophic to dilated cardiomyopathy in mice that express a mutant myosin transgene. Am J Physiol Heart Circ Physiol 280: H151–H159, 2001.[Abstract/Free Full Text]
  17. Garnier A, Fortin D, Delomenie C, Momken I, Veksler V, Venrua-Clapier R. Depressed mitochondrial transcription factors and oxidative capacity in rat failing cardiac and skeletal muscles. J Physiol 551: 491–501, 2003.[Abstract/Free Full Text]
  18. Hanson BJ, Carrozzo R, Piemonte F, Tessa A, Robinson BH, Capaldi RA. Cytochrome c oxidase-deficient patients have distinct subunit assembly profiles. J Biol Chem 276: 16296–16301, 2001.[Abstract/Free Full Text]
  19. Hayakawa MD, Chandra M, Miao W, Shirani J, Brown JH, Dorn GW II, Armstrong RC, Kitsis RN. Inhibition of cardiac myocyte apoptosis improves cardiac function and abolishes mortality in the peripartum cardiomyopathy of G{alpha}q transgenic mice. Circulation 108: 3036–3041, 2003.[Abstract/Free Full Text]
  20. Herzig RP, Scacco S, Scarpulla RC. Sequential serum-dependent activation of CREB and NRF-1 leads to enhanced mitochondrial respiration through the induction of cytochrome c. J Biol Chem 275: 13134–13141, 2000.[Abstract/Free Full Text]
  21. Hunt SA, Baker DW, Chin MH, Cinquegrani MP, Feldman AM, Francis GS, Ganiats TG, Goldstein S, Gregoratos G, Jessup MK, Noble RJ, Packer M, Silver MA, Stevenson LW. ACC/AHA guidelines for the evaluation and management of chronic heart failure in the adult: executive summary. Circulation 104: 2996–3007, 2001.[Free Full Text]
  22. Jeong MY, Kinugawa K, Vinson C, Long CS. AFos dissociates cardiac myocyte hypertrophy and expression of the pathological gene program. Circulation 111: 1645–1651, 2005.[Abstract/Free Full Text]
  23. Kang PM, Yue P, Lui Z, Tarnavski O, Bodyak N, Izumo S. Alterations in apoptosis regulatory factors during hypertrophy and heart failure. Am J Physiol Heart Circ Physiol 287: H72–H80, 2004.[Abstract/Free Full Text]
  24. Katz AM. Is the failing heart energy depleted? Cardiol Clin 16: 633–644, 1998.[CrossRef][Medline]
  25. Kim TH, Zhao Y, Ding WX, Shin JN, He X, Seo YM, Chen J, Rabinowich H, Amoscato AA, Yin XM. Bid-cardiolipin interaction at mitochondrial contact site contributes to mitochondrial cristae reorganization and cytochrome c release. Mol Biol Cell 15: 3061–3072, 2004.[Abstract/Free Full Text]
  26. Klemm DJ, Watson PA, Frid MG, Dempsey EC, Schaack J, Colton LA, Nesterova A, Stenmark KR, Reusch JE. cAMP response element-binding protein content is a molecular determinant of smooth muscle cell proliferation and migration. J Biol Chem 276: 46132–46141, 2001.[Abstract/Free Full Text]
  27. Konhilas JP, Maass AH, Luckey SW, Stauffer BL, Olson EN, Leinwand LA. Sex modifies exercise and cardiac adaptation in mice. Am J Physiol Heart Circ Physiol 287: H2768–H2776, 2004.[Abstract/Free Full Text]
  28. Konhilas JP, Watson PA, Maass A, Boucek DM, Horn T, Stauffer BL, Luckey SW, Rosenberg P, Leinwand LA. Exercise can prevent and reverse the severity of hypertrophic cardiomyopathy. Circ Res 98: 540–548, 2006.[Abstract/Free Full Text]
  29. Lee J, Kim CH, Simon DK, Aminova LR, Andreyev AY, Kushnareva YE, Murphy AN, Lonze BE, Kim KS, Ginty DD, Ferrante RJ, Ryu H, Ratan RR. Mitochondrial cyclic AMP response element-binding protein (CREB) mediates mitochondrial gene expression and neuronal survival. J Biol Chem 280: 40398–40401, 2005.[Abstract/Free Full Text]
  30. Lehman JJ, Barger PM, Kovacs A, Saffitz JE, Medeiros DM, Kelly DP. Peroxisome proliferators-activated receptor {gamma} coactivator-1 promotes cardiac mitochondrial biogenesis. J Clin Invest 106: 847–856, 2000.[ISI][Medline]
  31. Lehman JJ, Kelly DP. Transcriptional activation of energy metabolic switches in the developing and hypertrophied heart. Clin Exp Pharmacol Physiol 29: 339–345, 2002.[CrossRef][ISI][Medline]
  32. Lehman JJ, Kelly DP. Gene regulatory mechanisms governing energy metabolism during cardiac hypertrophic growth. Heart Fail Rev 7: 175–185, 2002.[CrossRef][Medline]
  33. Lesnefsky EJ, Slabe TJ, Stoll MS, Minkler PE, Hoppel CL. Myocardial ischemia selectively depletes cardiolipin in rabbit heart subsarcolemmal mitochondria. Am J Physiol Heart Circ Physiol 280: H2770–H2778, 2001.[Abstract/Free Full Text]
  34. Lesenfsky EJ, Tandler B, Ye J, Slabe TJ, Turkaly J, Hoppel CL. Myocardial ischemia decreases oxidative phosphorylation through cytochrome oxidase in subsarcolemmal mitochondria. Am J Physiol Heart Circ Physiol 273: H1544–H1554, 1997.[Abstract/Free Full Text]
  35. Lucas DT, Aryal P, Szweda LI, Kcoh WJ, Leinwand LA. Alterations in mitochondrial function in a mouse model of hypertrophic cardiomyopathy. Am J Physiol Heart Circ Physiol 284: H575–H583, 2003.[Abstract/Free Full Text]
  36. McCune SA, Park S, Radin MJ, Jurin RR. The SHHF/Mcc-facp rat model: a genetic model of congestive heart failure. In: Mechanisms of Heart Failure, edited by Singal PK, Dixon IMC, Beamish RE, Dhalla NS. Boston, MA: Kluwer Academic, 1995, chapt. 8, p. 91–106.
  37. Murphy E, Imahashi KI, Steenbergen C. Bcl-2 regulation of mitochondrial energetics. Trends Cardiovasc Med 15: 283–290, 2005.[CrossRef][ISI][Medline]
  38. Oliveira PJ, Wallace KB. Depletion of adenine nucleotide translocator protein in heart mitochondria from doxorubicin-treated rats; relevance for mitochondrial dysfunction. Toxicology 220: 160–168, 2006.[CrossRef][ISI][Medline]
  39. Otter I, Conus S, Ravn U, Rager M, Olivier R, Monney L, Fabbro D, Borner C. The binding properties and biological activities of Bcl-2 and Bax in cells exposed to apoptotic stimuli. J Biol Chem 273: 6110–6120, 1998.[Abstract/Free Full Text]
  40. Palmer JW, Tandler B, Hoppel CL. Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle. J Biol Chem 252: 8731–8739, 1977.[Abstract/Free Full Text]
  41. Pawlush DG, Moore RL, Musch TI, Davidson WR Jr. Echocardiographic evaluation of size, function, and mass of normal and hypertrophied rat ventricles. J Appl Physiol 74: 2598–2605, 1993.[Abstract/Free Full Text]
  42. Platenik J, Balcar V, Yoneda Y, Mioduszewska B, Buchal R, Hynek R, Kilianek L, Kuramoto N, Wilczynski G, Ogita K, Nakamura Y, Kaczmarek L. Apparent presence of Ser133-phosphorylated cyclic AMP response element binding protein (pCREB) in brain mitochondria is due to cross-reactivity of pCREB antibodies with pyruvate dehydrogenase. J Neurochem 95: 1446–1460, 2005.[CrossRef][ISI]