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Am J Physiol Heart Circ Physiol 287: H72-H80, 2004. First published March 4, 2004; doi:10.1152/ajpheart.00556.2003
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Alterations in apoptosis regulatory factors during hypertrophy and heart failure

Peter M. Kang, Patrick Yue, Zhilin Liu, Oleg Tarnavski, Natalya Bodyak, and Seigo Izumo

Cardiovascular Division, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachuesetts 02215

Submitted 12 June 2003 ; accepted in final form 25 February 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cardiac hypertrophy from pathological stimuli often proceeds to heart failure, whereas cardiac hypertrophy from physiological stimuli does not. In this study, physiological hypertrophy was created by a daily exercise regimen and pathological hypertrophy was created from a high-salt diet in Dahl salt-sensitive rats. The rats continued on a high-salt diet progressed to heart failure associated with an increased rate of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cardiomyocytes. We analyzed primary cultures of these hearts and found that only cardiomyocytes made hypertrophic by a pathological stimulus show increased sensitivity to apoptosis. Examination of the molecular changes associated with these distinct types of hypertrophy revealed changes in Bcl-2 family members and caspases favoring survival during physiological hypertrophy. However, in pathological hypertrophy, there were more diffuse proapoptotic changes, including changes in Fas, the Bcl-2 protein family, and caspases. Therefore, we speculate that this increased sensitivity to apoptotic stimulation along with proapoptotic changes in the apoptosis program may contribute to the development of heart failure seen in pathological cardiac hypertrophy.

pathological; physiological; caspases; Bcl-2

APOPTOSIS HAS BEEN ASSOCIATED with both human and animal models of heart failure and implicated as well in the pathogenesis of the transition from hypertrophy to heart failure (28). However, the association of cardiac hypertrophy to apoptosis is not clearly understood. Various hypertrophic signaling pathways have been associated with protecting the heart from apoptosis as well as predisposing it to apoptosis (2, 14, 17, 19, 26, 31, 33, 38). One potential explanation for the apparent incongruity of these results is the existence of both physiological and pathological forms of cardiac hypertrophy. Different types of cardiac hypertrophy, for example, may activate different molecular changes (20, 30). Physiological hypertrophy, such as exercise-induced cardiac hypertrophy, is a favorable adaptive change in the heart that accommodates it to increases in bodily demand and does not lead to heart failure. In contrast, pathological hypertrophy, such as pressure overload-induced hypertrophy, is a maladaptive response to pathological stimuli, and, if not ameliorated, usually leads to heart failure. However, the molecular changes that could explain why pathological hypertrophy is more apt to lead to the development of heart failure compared with physiological hypertrophy have not been elucidated.

On a cellular level, the induction of apoptosis may be mediated either by death receptors or by mitochondria. The death receptor-mediated pathway involves the binding of a death ligand, such as Fas ligand (FasL), to a membrane-bound death receptor, Fas, resulting in activation of caspase-8 (36). The mitochondrion-mediated pathway is initiated by the release of cytochrome c from mitochondria in response to apoptotic stimulation (34). The release of cytochrome c is tightly regulated by anti- and proapoptotic members of the Bcl-2 protein family (1). Once released, cytosolic cytochrome c binds to Apaf-1 and caspase-9 in the presence of dATP to form an active apoptosome complex (32, 48). Both pathways act by activating downstream effector caspases, such as caspase-3, which ultimately executes the biochemical and structural changes seen in apoptosis (32, 40).

In this study, physiological hypertrophy (via treadmill exercise) (25, 45) and pathological hypertrophy (via a high-salt diet) (22) were induced in Dahl salt-sensitive (DS) rats. Using these two distinct forms of cardiac hypertrophy, we sought to clarify the relationship between physiological and pathological hypertrophy in response to apoptotic stimulation and to investigate the molecular changes associated with these different modes of cardiac hypertrophy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Anti-caspase-9, anti-Bcl-xL, and anti-human X-chromosome-linked inhibitor of apoptosis protein (xIAP) antibodies and the rat APO-1 RNA protection assay kit were obtained from Pharmingen. Anti-caspase-3 antibodies (Oncogene) and anti-GAPDH antibodies (Research Diagnostic) were obtained from the specified sources.

Exercise- and high-salt diet-induced cardiac hypertrophy. All animal studies were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Revised 1996) and were approved by the Institutional Animal Care and Use Committee. DS rats (Harlan Sprague Dawley) were obtained at 5 wk of age. To generate exercise-induced cardiac hypertrophy, the rats were exercised daily beginning at 6 wk of age (EX group). The exercise protocol used was a modified version of one described previously (25, 45). Rats were exercised daily for 6 wk on a rodent treadmill (Exer-6M, Columbus Instruments). The exercise program consisted of 3 wk of progressively more strenuous exercise regimens, followed by a 3-wk maintenance period, during which the rats were exercised at 20 m/min at a 5° incline for 90 min/day. All rats completed the exercise protocol.

To generate pressure-overload cardiac hypertrophy, the rats were fed a 6% NaCl diet beginning at 6 wk of age (HS group) (22). The majority of the DS rats fed a high-salt diet developed clinical evidence of heart failure by the age of 19–21 wk. All rats died by age 21–23 wk if fed a high-salt diet. Control rats were sedentary and age-matched DS rats fed normal rat chow.

Adult rat cardiomyocyte culture and induction of apoptosis. Adult rat cardiomyocyte cultures were obtained from rat hearts according to a previously published protocol with modifications (27). The hearts were perfused for 45–60 min with 0.3% collagenase for adequate dissociation. The Percoll gradient separation step was omitted to minimize further stress on the dissociated cells after the longer enzymatic dissociation required in hypertrophic cells. The cells were used for experiments after 24 h of plating. A different heart was used for each experiment to minimize the variability between cultures. The induction of apoptosis by hypoxia/reoxygenation was done as described previously (27). Of note, there was a slight increase in the baseline apoptosis rate (7–10%) in these cultures, which was probably due to the omission of the Percoll gradient separation step. The induction of apoptosis was also achieved by exposing the cells to 0.1 mM hydrogen peroxide or 2 µM staurosporin for 24 h. Cell size was quantified using NIH Image software as described previously (39).

Quantitative analysis of cellular viability and apoptosis. The quantitative analysis of cell viability and apoptosis was done as described previously using propidium iodide and annexin V staining, respectively (27). DNA fragmentation assays were performed on equal numbers of cells from primary cardiac myocyte cultures using low-molecular-weight DNA extraction as published previously (27). Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis of frozen heart sections was done according to the published protocol (39).

Biochemical analysis and RNA protection assay. Protein extract was prepared from ventricle of the heart according to the published standard methods (27, 39). Total ventricular RNA was prepared via TRIzol reagent (Invitrogen). A RNA protection assay using APO-1 rat apoptosis kit was done according to the manufacturer's protocol (Pharmingen). APO-1 consisted of eight predetermined apoptosis genes (Fas, Bcl-xL, FasL, caspase-1, caspase-3, caspase-2, Bax, and Bcl-2) and two internal control DNA markers (L32 and GAPDH) (see Fig. 4A).



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  		Fig. 4. RNA protection assay of several apoptosis-related genes in control, hypertrophied, and failed hearts. A: representative RNA protection assay of hearts from various groups. Groups are unprotected probes (marker; lane 1), yeast tRNA (lane 2), control rat total RNA (lane 3), RNA from a control rat heart (lane 4), RNA from an exercised rat heart (lane 5), RNA from a HS hypertrophic rat heart (lane 6), RNA from a HS failed rat heart (lane 7). L32 and GAPDH were used as internal controls. The following are unprotected and protected nucleotide lengths corresponding to the specific genes, respectively: Fas (435 and 406 bp), Bcl-xL (393 and 363 bp), Fas ligand (FasL) (315 and 286 bp), caspase (Casp)-1 (282 and 253 bp), caspase-3 (255 and 226 bp), caspase-2 (231 and 202 bp), Bax (210 and 181 bp), Bcl-2 (189 and 160 bp), L32 (141 and 112 bp), and GAPDH (126 and 97 bp). B: quantitative analysis of various apoptosis-related genes from the RNA protection assay. Presented as the gene-to-L32 ratio. C1, caspase-1; C2, caspase-2; C3, caspase-3. n = 4. *P < 0.05.

 
Statistics. All data are expressed as means ± SE. Statistical analyses between two groups and among the groups were performed with an unpaired Student’s t-test and ANOVA with the Bonferroni method, respectively (44). P values of <0.05 were considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Physiological and pathological cardiac hypertrophy. Physiological cardiac hypertrophy was generated by a vigorous daily exercise regimen for 6 wk (EX group) and pathological cardiac hypertrophy was generated by feeding a 6% NaCl diet to DS rats starting at 6 wk of age (HS group) as described in MATERIALS AND METHODS. After several weeks on the high-salt diet, HS rats had significant hypertension as measured with a tail-cuff blood pressure monitor, and their hypertension gradually progressed and eventually reached over 200 mmHg after 6 wk on the diet (12 wk of age) (data not shown). Echocardiography of the EX and HS group rats showed significant increases in anterior and posterior wall thickness compared with the sedentary control rats fed normal rat chow (Table 1). These findings were consistent with significant exercise-induced cardiac hypertrophy with preserved cardiac function. Sections of EX and HS rat hearts also showed significant cardiac hypertrophy, concordant with the echocardiographic findings (Fig. 1A).


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  		Table 1. Echocardiographic findings of exercise and HS diet-induced hearts

 


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  		Fig. 1. Models of cardiac hypertrophy. A: hemotoxylin and eosin staining of hearts from control, exercised (EX), and high-salt diet-treated (HS) hypertrophic rats at 12 wk and failed rats at 20 wk. Significant hypertrophy is evident in EX and HS rat hearts. B: dissociated adult cardiomyocytes from control, EX, and HS rat hearts. *P < 0.05.

 
The body and tissue weights of EX and HS rats showed significant increases in heart weight without changes in other tissue weights (Table 2). To further demonstrate that cardiomyocytes were significantly hypertrophied in this model, we measured the surface area of dissociated cardiomyocytes. Cardiomyocytes from EX and HS rat hearts showed a significant increase in surface area compared with the control (Fig. 1B). Interestingly, the increase in cell surface area from the exercised rats was primarily due to an increase in the long axis without significant changes in the short-axis dimension. In comparison, the cardiomyocytes from HS rat hearts showed significant increases in both the long- and short-axis dimensions. Although an increase in cell surface area is not as good a measure of cellular hypertrophy as an increase in cell volume, we believe that in combination with echocardiogram and tissue weight, our models demonstrate significant cardiac hypertrophy via both physiological and pathological stimuli.


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  		Table 2. Tissue weights and TL in control sedentary and exercise rats

 
Hypertrophic cardiomyocytes from the HS group demonstrate increased sensitivity to various apoptotic stimulations. Next, we sought to determine the sensitivity of adult cardiomyocytes derived from these two models of hypertrophy to apoptotic stimulation. The apoptosis rate of hypertrophic cardiomyocytes from the EX group exposed to hydrogen peroxide, a known inducer of apoptosis (4), did not differ from the control (Fig. 2A, shaded bars). In fact, EX group cells were characterized by decreased apoptosis and increased viability compared with the control, although these effects did not reach statistical significance. In contrast, HS cardiomyocytes exposed to hydrogen peroxide showed a significant increase in apoptosis and a significant decrease in cellular viability compared with both the control and EX groups (Fig. 2A, solid bars). The HS group also demonstrated a greater increase in DNA laddering compared with control with or without hydrogen peroxide exposure (Fig. 2B). In fact, we found that the increased sensitivity to apoptosis in hypertrophic cardiac myocytes from the HS group was also evident with other types of apoptotic stimulation, such as staurosporin and hypoxia/reoxygenation (Fig. 2C). Of note, the present study does not show that exercise-induced hypertrophy is protective against apoptotic stimulation but rather just a trend toward decreased apoptosis and increased viability (Fig. 2A). Furthermore, to assess whether paracrine or neurohumoral factors are active during hypertrophy and heart failure, we checked to see whether nonmyocytes from various hypertrophic hearts are differentially sensitive to apoptotic stimulation. We found that primary nonmyocyte cultures from the control and HS group did not differ in their response to hydrogen peroxide exposure (percent apoptosis: control = 1.0 ± 0.3% vs. HS group = 1.4 ± 0.5%, P = not significant, n = 6).



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  		Fig. 2. Hypertrophic cardiomyocytes from the HS group, but not the EX group, demonstrate increased sensitivity to various apoptotic stimuli. A: apoptosis and cellular viability of control (Cont), EX, and HS rat cardiomyocytes after hydrogen peroxide exposure. NS, not significant. n = 6. *P < 0.05. B: DNA laddering assay induced by hydrogen peroxide in control (C) and HS cardiac myocyte cultures. When stimulated with hydrogen peroxide, there was a significant increase in DNA laddering in both control and HS, with a significantly greater increase seen in HS culture. MW, molecular weight marker. C: apoptosis and cellular viability of control and HS rat cardiac myocytes after various apoptotic stimulations. H/R, 6 h of hypoxia followed by 18 h of reoxygenation; ST, staurosporin. n = 6. *P < 0.05.

 
Pathological cardiac hypertrophy leads to heart failure that is associated with increased apoptosis in vivo. When the high-salt diet was continued after 12 wk, the rats developed either stroke or progressive heart failure between 14 and 18 wk or 19 and 21 wk, respectively. All the HS rats died by 21–23 wk (observed data), and only the rats that showed overt clinical evidence of heart failure were included in the study with an average age of 20 wk. Echocardiographic and heart section data from these rats demonstrated significant increases in wall thickness and left ventricular chamber diameter and a decrease in fractional shortening consistent with hypertensive dilated cardiomyopathy (Table 1). These rats had significant decreases in body weight due to cardiac cachexia and a nearly twofold increase in heart weight (Table 2). Furthermore, there were significant increases in the lung and liver weights consistent with left- and right-sided heart failure, respectively.

To test whether our model of pathological hypertrophy is associated with increased apoptosis in vivo, we checked for TUNEL staining in hypertrophied and failed rat heart sections (Fig. 3A). There was no significant increase in TUNEL-stained cardiomyocytes in either the EX group or in compensated hypertrophic hearts derived from the HS group (Fig. 3A). In contrast, there was a 15-fold increase in TUNEL-positive cardiomyocytes in failed hearts from the HS group (Fig. 3, A and B). Interestingly, there was increased nonmyocyte TUNEL staining in both hypertrophic and failed hearts, although the presence of TUNEL-positive nonmyoyctes was significantly greater in failed hearts (Fig. 3C).



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  		Fig. 3. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining of HS rats in the hypertrophic and failed states. A: representative TUNEL staining from frozen rat heart tissue sections of 12-wk control and HS-induced hypertrophic rats at 12 wk and HS-induced failed rats at 20 wk. Arrow indicates a TUNEL-positive cardiomyocyte. Green, TUNEL; red, propidium iodide; blue, troponin I. B and C: quantitative analysis of TUNEL-positive (pos) cardiac myocytes and nonmyocytes in control (C), hypertrophied (H), and failed (F) hearts.

 
These in vitro and in vivo findings suggest that pressure overload-induced pathological cardiac hypertrophy results in increased sensitivity to apoptotic stimulation, whereas exercise-induced physiological cardiac hypertrophy does not. Furthermore, because the HS group eventually progressed to heart failure with increased levels of apoptosis, we speculate that the increased sensitivity of this model to apoptosis might contribute to the development of heart failure in vivo.

Distinct activation of mitochondrion-dependent and -independent apoptotic pathways in hypertrophic cardiomyocytes. Given the differential sensitivity to apoptotic stimulation despite the similar degree of cardiac hypertrophy in physiological and pathological hypertrophy, we hypothesized that there may be molecular alterations that underlie these processes. Thus we examined the expression level of genes and proteins involved in apoptosis to define the molecular changes that are associated with these two models. First, we examined the mRNA expression level of various apoptosis-related genes using a RNA protection assay (Fig. 4A). Quantitative analysis of these genes relative to the internal control gene L32 showed that there were prosurvival changes in the EX group, such as an increased Bcl-xL-to-Bax ratio and a decrease in caspases. In contrast, the HS group was associated with proapoptotic gene changes in both the death receptor and mitochondrion-mediated apoptotic pathways (Fig. 4B). For example, there were increases in the death receptor Fas, proapoptotic Bax, and caspases. Conversely, the Bcl-xL-to-Bax ratio was decreased in the HS group. Continued pathological stimuli in this model resulting in heart failure caused secondary switching of the apoptotic gene program from a proapoptotic to an antiapoptotic state. This change in heart failure was generalized with an increase in Bcl-2 but decreases in Fas, caspase-2, and Bax, resulting in an antiapoptotic milieu.

Similar to mRNA expression changes, protein expression also revealed alternations in various apoptosis-related molecules (Figs. 5, A and B). The EX hypertrophy group was associated with antiapoptotic changes compared with the HS hypertrophy group, which was more apt to show proapoptotic molecular changes. Consistent with the mRNA levels, protein analysis of failed hearts showed a significant increase in Bcl-xL and a decrease in caspase-3 as well as a significant upregulation of xIAP (Figs. 5, A and B). Activated caspases were not detectable in either hypertrophic or failed heart samples. This is most likely due to the very low level of apoptosis, which was <1% even in failed heart in vivo.



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  		Fig. 5. Western blot analysis of several apoptosis-related genes in control, hypertrophied, and failed hearts. A: representative Western blot analysis in various heart tissues. E, exercised. GAPDH was used as an internal control. B: quantitative analysis of various apoptosis-related genes from Western blot analysis. Results are presented as the gene-to-GAPDH ratio. xIAP, human X-chromosome-linked inhibitor of apoptosis protein. n = 4. *P < 0.05. C: summary of RNA protection assays and Western blot analysis. Genes are separated into the Bcl-2 protein family, death receptor, and caspases. Gene/protein expressions are indicated compared with the control. ND, not done.

 
A summary of these findings for both the mRNA and protein analyses is shown in Fig. 5C. In general, the molecular changes in the EX group were antiapoptotic changes associated with alterations in Bcl-2 protein family and caspases but not the death receptor pathway. However, the changes in the HS group were predominantly proapoptotic changes associated with general alterations in apoptotic pathways. Interestingly, in failed hearts, there was a switch from a proapoptotic milieu to secondary antiapoptotic changes associated with alterations in the Bcl-2 family, caspases, and death receptor pathways.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we characterized two distinct models of cardiac hypertrophy. The first model develops hypertrophy secondary to moderate to high levels of exercise. The second model exhibits hypertrophy in the setting of hypertension caused by a high-salt diet. We believe that these two models represent examples of physiological and pathological cardiac hypertrophy, respectively. Furthermore, we found that despite developing similar degrees of cardiac hypertrophy in both models, there are significant differences in their sensitivity to apoptotic stimuli and the expression levels of various apoptotic modulators. Consistent with previous findings, we found that the HS model of hypertrophy ultimately leads to heart failure associated with an increased rate of apoptosis (21). We believe that these differences may explain why pathological hypertrophy tends to progress to heart failure, whereas physiological hypertrophy does not.

Currently, clear difference between physiological and pathological cardiac hypertrophy is still evolving. Physiological hypertrophy is usually associated with normal cardiac structure and relatively normal pattern of cardiac gene expression (15, 29). In comparison, most models of pathological hypertrophy are characterized by fibrosis, sarcomere disarrangement, and fetal gene activation (24, 41). A specific molecular pathway, such as phosphoinositide 3-kinase, has been shown to be involved in physiological hypertrophy (35). In contrast, several hypertrophic signaling molecules, such as G proteins, angiotensin II, protein kinase C, and MAPKs have been shown to be activated in pathological hypertrophy (6, 12, 13, 16). In thyroid hormone-induced physiological hypertrophy compared with pathological hypertrophy from pressure overload, opposite effects on several thyroid hormone-responsive genes, such as thyroid hormone receptors, {alpha}- and {beta}-myosin heavy chain, and sarco(endo)plasmic reticulum Ca2+-ATPase have been demonstrated (30). Ultimately, the critical functional difference between physiological and pathological hypertrophy is their propensity to progress to heart failure. Thus we believe that it is not hypertrophy per se that determines the progression to heart failure but rather the molecular mechanisms that are altered in a particular type of cardiac hypertrophy.

The effect of cardiac hypertrophy on apoptosis is controversial. Certain hypertrophic signaling factors such as cardiotrophin-1 and IGF-I seem to be protective against apoptosis (14, 19, 31, 33, 38). However, other hypertrophic signaling factors, such as heterotrimeric G proteins and angiotensin II (2, 17, 26), have been demonstrated to be predominately proapoptotic in nature. Further complicating these two contrasting views, some hypertrophic signaling molecules show both anti- and proapoptotic properties, such as JNK (3, 4, 42). We speculate that these contradictory findings may be due to the complicated nature of cardiac hypertrophic signaling and that the apoptotic response (or lack thereof) to a hypertrophic stimulus is most likely dependent on the cell type and the stimulus. For example, in DS rats on a high-salt diet, angiotensin II-induced expression of JNK and MAPK was increased in the hypertrophic stage but attenuated in the failed stage (18). There was no change in ERK expression, which had previously been shown to be associated with physiological hypertrophy in a transgenic model (7). Taken together, these data add to the notion that specific hypertrophic pathways are activated in physiological or pathological models of hypertrophy and may play a role in its propensity to undergo apoptosis. In this study, we report differential alterations in apoptotic molecules during cardiac hypertrophy induced by specific stimuli. These data provide in vitro evidence that molecular changes that occur in response to specific stimuli may be important in determining whether the hypertrophic cells are more sensitive or resistant to apoptosis. Further in vivo experiments, such as ischemia-reperfusion experiments in these hypertrophic hearts, may provide more direct evidence of increase apoptotic sensitivity during pathological hypertrophy.

The difference in propensity to undergo apoptosis in these models also suggests stimulus-dependent activation of specific apoptotic signaling. In the EX group, there was a significant downregulation of caspases, an increased Bcl-xL-to-Bax ratio, and no changes in Fas. Thus most of the changes were in molecules involved in the mitochondrion-mediated apoptosis pathway. In fact, in vivo and in vitro studies showed that the mitochondrial pathway appears predominant in settings where oxidative stress is the critical insult (e.g., ischemia-reperfusion-induced apoptosis) (27, 37, 43). In contrast, in the HS group, the changes were more diffuse, involving both mitochondrion- and death receptor-mediated pathways. There was a significant upregulation of caspases, a decreased Bcl-xL-to-Bax ratio, and an increase in Fas. Fas activation has been observed during pathological hypertrophic stimulations both in vitro and in vivo (5, 9). The death receptor pathway has been shown to be predominant in certain situations, most notably immune-mediated heart failure (23). It should be pointed out that the alterations in apoptotic factors in the experiments are not complete and direct causative association should not be made. We believe that a more targeted approach will be needed to confirm this finding.

In our model of heart failure, there was a reversal of early proapoptotic gene activation from compensated hypertrophy at 12 wk to the activation of a secondary survival gene program during decompensated heart failure at 20–21 wk. Because the analysis of apoptotic genes was done in parallel with the only difference being in the duration of the high-salt diet, the reversal of apoptotic modulators seen in heart failure rats, we believe, represents secondary change. Although other studies using aortic banding similarly observed proapoptotic changes in pathological hypertrophy, a secondary switch to antiapoptotic gene changes has not been observed (11). This may be explain by the fact that they are different models of heart failure and the aortic banding models do not reliably produce clinical heart failure compared with the DS model. Also, the secondary antiapoptotic changes that we see were at the very end stage of heart failure. Of note, in our experience, these cardiac myocytes from the failed hearts are more susceptible to further stress despite apparent activation of antiapoptotic molecules. This is based on the fact that isolating cardiac myocytes from the failed heart is extremely difficult even with extra careful digestion and handling. Thus we could speculate that the myocytes from decompensated hearts in this model may have had irreversible damage that prevented salvaging despite activation of the survival gene program. One should also interpret this finding with caution, because antiapoptotic molecules, such as Bcl-2 protein family members and IAP, could convert to proapoptotic molecules by enzymatic cleavage (8, 10). Also, the function of Bcl-2 family proteins could be modulated by phosphorylation, which was not addressed in this study (46, 47).

Our data suggest from both in vitro and in vivo models that pathological cardiac hypertrophy may progress to heart failure because the cardiomyocytes are more susceptible to apoptosis. However, we believe that more studies using other models of hypertrophy, as well as more complete analysis of molecular changes, will be needed to better address this hypothesis. Furthermore, although these findings provide possible strategies to modulate cardiac apoptosis, further investigations, especially of the transition from compensated hypertrophy to heart failure, are needed to better understand the complex and intricate balance that exists between hypertrophy, apoptosis, and heart failure.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by American Heart Association Grant 0030278N (to P. M. Kang) and National Institute on Aging Grant RO1 AG-61716 (to S. Izumo).


   ACKNOWLEDGMENTS
 
We thank Ellen Gower and Daniel Chen for editorial and technical assistance, respectively.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. M. Kang, Cardiovascular Div., Beth Israel Deaconess Medical Center, 330 Brookline Ave., SL-423C, Boston, MA 02215 (E-mail: pkang{at}bidmc.harvard.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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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