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Am J Physiol Heart Circ Physiol 287: H1303-H1311, 2004; doi:10.1152/ajpheart.00053.2004
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Cardiac myocyte apoptosis provokes adverse cardiac remodeling in transgenic mice with targeted TNF overexpression

David Engel,1 Ronald Peshock,2 Robert C. Armstong,3 Natarajan Sivasubramanian,1 and Douglas L. Mann1

1Winters Center for Heart Failure Research, Baylor College of Medicine, and Michael E. DeBakey Veterans Affairs Medical Center, Houston, 77030; 2Department of Radiology, University of Texas Southwestern Medical Center at Dallas, Texas 75235; and 3Idun Pharmaceuticals, San Diego, California 92121

Submitted 5 February 2004 ; accepted in final form 5 April 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although cardiac myocyte apoptosis has been detected in explanted hearts from patients with end-stage dilated and ischemic cardiomyopathy, the relative contribution of apoptotic cell death to left ventricular (LV) remodeling and cardiac decompensation is not known. To determine whether progressive cardiac myocyte apoptosis contributes to the transition from a hypertrophic to a dilated cardiac phenotype that is observed in transgenic myosin heavy chain secreted TNF (MHCsTNF) mice with cardiac restricted overexpression of tumor necrosis factor (TNF), we assessed cardiac myocyte apoptosis (using a DNA ligase technique) in MHCsTNF mice and littermate control mice in relation to serial changes in LV structure, which was assessed using MRI. The prevalence of cardiac myocyte apoptosis increased progressively from 4 to 12 wk as the hearts of the MHCsTNF mice underwent the transition from a concentric hypertrophic to a dilated cardiac phenotype. Treatment of the MHCsTNF mice with the broad-based caspase inhibitor N-[(1,3-dimethylindole-2-carbonyl)-valinyl]-3-amino4-oxo-5-fluoropentanoic acid significantly decreased cardiac myocyte apoptosis and significantly attenuated LV wall thinning and adverse cardiac remodeling. Additional studies suggested that the TNF-induced decrease in Bcl-2 expression and activation of the intrinsic mitochondrial death pathway were responsible for the cardiac myocyte apoptosis observed in the MHCsTNF mice. These studies show that progressive cardiac myocyte apoptosis is sufficient to contribute to adverse cardiac remodeling in the adult mammalian heart through progressive LV wall thinning.

tumor necrosis factor; cell death; caspase; congestive heart failure; magnetic resonance imaging

APOPTOSIS, OR PROGRAMMED CELL DEATH, is increasingly recognized as a potentially important pathophysiological process that occurs in a broad variety of disease states. In adult mammalian heart, cardiac myocyte apoptosis has been identified as a mechanism of cell death in acute myocardial infarction (17, 18, 23) and ischemia-reperfusion injury (12, 37) as well as in specific cardiac disorders such as arrhythmogenic right ventricular dysplasia (25). In addition, the presence of apoptotic cardiac myocytes has been demonstrated in the hearts of humans with end-stage dilated and ischemic cardiomyopathies (14, 28–30). Presently, however, it is not known whether continual loss of cardiac myocytes via apoptosis actually contributes to the development of the heart failure phenotype and/or progressive cardiac decompensation.

Recently we and others (3, 21, 31) have developed lines of mice with cardiac-restricted overexpression of TNF. These mice initially develop a concentric hypertrophic cardiac phenotype that transitions to a dilated cardiac phenotype and thus recapitulate the classic "transition to failure" that has been reported in many experimental models of cardiac decompensation. Although several of the potential downstream targets that are responsible for this transition have been identified in mice with targeted overexpression of TNF, the complete ensemble of basic mechanisms that underlie this transition has not been fully elucidated. Given that TNF can provoke cardiac myocyte apoptosis in certain settings (20), we sought to determine whether TNF-induced cardiac myocyte apoptosis was sufficient to contribute to adverse cardiac remodeling in a transgenic mouse line with cardiac-restricted overexpression of secreted TNF (MHCsTNF mice) as well as to delineate the possible mechanism(s) for cardiac myocyte apoptosis in this model.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All animals used in this study were treated humanely and in accordance with the guidelines set forth by the Baylor Animal Care and Use Committee.

Generation of MHCsTNF Mice

The generation and characterization of the line of transgenic mice with cardiac-restricted overexpression of secretable TNF that we refer to as MHCsTNF mice (hybrid C57/BL6 x ICR) has been described previously (31).

Cardiac Myocyte Apoptosis

DNA laddering and electron microscopy. To detect myocardial apoptosis, we examined myocardial DNA fragmentation in the hearts of the MHCsTNF and littermate control mice at 12 wk of age. Genomic DNA was isolated from hearts of the MHCsTNF and littermate control mice as previously described (26). To visualize the DNA fragments, we used a PCR-based technique that selectively amplifies DNA with double-stranded DNA breaks that are characteristic for apoptosis (ApoAlert LM-PCR Ladder Assay Kit; Clontech). After a period of overnight ligation with the supplied adaptors, 50 ng of ligated DNA was amplified with 25 cycles of PCR according to the manufacturer's protocol and was resolved on a 1.2% agarose-ethidium bromide gel. Transmission electron microscopy was performed on myocardial sections from 12-wk-old MHCsTNF and littermate control mice as previously described (32).

In situ DNA ligation. Hearts were excised from the MHCsTNF and littermate control mice at 4, 8, and 12 wk of age and were perfusion fixed in a retrograde fashion by cannulation of the aorta with 10% zinc-buffered formalin (Z-Fix; Anatech). Fixed hearts were embedded in paraffin, and myocardial sections of 6-µm thickness were fixed onto glass slides. Apoptotic cardiac myocytes were identified on paraffin-embedded myocardial sections using the in situ DNA ligation technique, which labels cell nuclei that contain double-stranded DNA breaks with single-base 3' overhangs that occur during apoptosis (6, 23). Apoptotic cell nuclei detected by the ligase assay were stained with fluorescein (excitation and transmission wavelengths, 495 and 525 nm, respectively). Sections were counterstained with the nucleic acid-binding dye 4',6'-diamidino-2-phenylindole hydrochloride (DAPI; Vector Laboratories; excitation and transmission wavelengths, 310 and 353 nm, respectively) to visualize the entire population of cell nuclei in the myocardial sections. To distinguish cardiac myocytes from nonmyocyte cell types within the myocardium, we labeled the myocardial sections with a primary anti-desmin antibody (1:20 dilution; Sigma) and subsequently with a rhodamine (excitation and transmission wavelengths, 550 and 570 nm, respectively)-conjugated secondary antibody (1:50 dilution; Sigma). This procedure was performed after the DNA labeling and thus enabled us to simultaneously label myocyte cell nuclei and cell cytoplasm. Only those nuclei that were labeled with the ligase technique and were identified as cardiac myocytes were included in our quantification of cardiac myocyte apoptosis. If two or more nuclei were labeled within the same myocyte, they were excluded from the analysis. Moreover, all ligase-positive interstitial cells or nonmyocytes were excluded from the formal analysis.

Quantification of cardiac myocyte apoptosis. We studied the prevalence of cardiac myocyte apoptosis in MHCsTNF and littermate control mice at ages 4, 8, and 12 wk. Myocardial sections were prepared using the in situ DNA ligation technique as described (6, 23) and were examined under fluorescence microscopy at x400 magnification using filters to permit the visualization of DAPI, fluorescein, and rhodamine. Images were digitally photographed (SPOT II camera system; Diagnostic Instruments) and analyzed with software that enabled us to enumerate cell nuclei (MetaView; Universal Imaging). In each microscopic field, the number of cardiac myocyte nuclei labeled by the in situ DNA ligation technique was divided by the total number of DAPI-stained nuclei. To quantify the prevalence of apoptosis, 10 predetermined radially arranged transmural left ventricular (LV) sections were chosen for evaluation in each animal. Each transmural slice through the LV wall was divided into thirds. Thus we examined the prevalence of apoptosis in the endocardium, the mid-wall, and the epicardium. Four LV cross-sections at the level of the papillary muscles were analyzed in this manner for each heart for a total of 120 microscopic fields per heart.

Cardiac Myocyte Necrosis

Insofar as TNF has been shown to provoke necrosis in certain cell types (34), we also examined the prevalence of myocyte necrosis in the MHCsTNF and littermate control mice.

For these studies, we injected 100 µg iv of an anti-cardiac myosin heavy-chain antibody (clone BGN/04/4481; Biogenesis) into the MHCsTNF and littermate control mice 24 h before death. The anti-myosin antibody binds to cardiac myosin and hence labels cardiac myocytes that have permeable cell membranes that are characteristic of necrotic cells (4). Mouse hearts were perfusion fixed and processed for paraffin sections as described (see In situ DNA ligation). To detect necrotic myocytes that had incorporated the anti-cardiac myosin antibody, we performed immunohistochemistry using an immunoperoxidase system (Vectastain ABC Elite; Vector Laboratories) as described previously (23).

Effects of Cardiac Myocyte Apoptosis on LV Remodeling

Magnetic resonance imaging. We used MRI to examine cardiac remodeling in the MHCsTNF and littermate control mice at 4 and 12 wk of age as described by Franco et al. (9, 10). Mice were anesthetized with a mixture of (in mg/ml) 1.4 acepromazine, 8.6 xylazine, and 42.8 ketamine before cardiac imaging was performed. The mice were killed immediately after MRI imaging, before they recovered from anesthesia, and heart and body weights were recorded. The hearts were then immediately perfusion fixed, and in situ DNA ligation assays were performed as described (see In situ DNA ligation).

Mechanisms for Cardiac Myocyte Apoptosis

Myocardial pro- and antiapoptotic gene expression. We examined the expression of a family of proapoptotic (Bak, Bax, and Bad) and antiapoptotic (Bcl-x and Bcl-2) genes in MHCsTNF and littermate control mouse hearts at ages 4, 8, and 12 wk. Ribonuclease protection assays were performed using a commercially available kit (RiboQuant; Pharmingen) as previously described (2). The ribosomal protein L32 and GAPDH were used as internal controls to ensure equal loading. The intensities of the bands corresponding to the pro- or antiapoptotic genes were quantified using a PhosphorImager system (Molecular Dynamics) and were normalized by corresponding bands for GAPDH. Final results were expressed as fold changes in proapoptotic gene expression in the MHCsTNF mice relative to littermate control mice at 4, 8, and 12 wk of age.

Bcl-2 protein expression. We performed two related experiments to analyze myocardial Bcl-2 protein levels in the MHCsTNF and littermate control mice. First, we examined total myocardial Bcl-2 protein levels using Western blot analysis in MHCsTNF and littermate control mice. Briefly, hearts were homogenized using a Dounce homogenizer in buffer (50 mM Tris·HCl, pH 7.4, 0.1% Triton X-100, 1% SDS, 250 mM NaCl, 15 mM MgCl2, 1 mM DTT, 2 mM EDTA, 2 mM EGTA, and 25 mM NaF) that contained 1 mM PMSF, 10 mg/ml leupeptin, and 10 mg/ml aprotinin. Equivalent amounts (50 µg) of myocardial protein lysates were then loaded onto 12% SDS-polyacrylamide gels, electrophoretically separated, and transferred to nitrocellulose membranes as previously described (19, 27). Western blot analysis was performed using a polyclonal anti-Bcl-2 antibody (1:1,000 dilution; Santa Cruz) as the primary antibody and a horseradish peroxidase-conjugated secondary antibody (1:1,000 dilution; Amersham), and the resulting protein levels were visualized by enhanced chemiluminescence (Amersham). The membranes were then washed and reprobed with an anti-actin polyclonal antibody (Santa Cruz) to ensure equal protein loading. Insofar as mitochondrial Bcl-2 is believed to be critical for stabilizing mitochondrial function and preventing cytochrome c release, we also examined Bcl-2 protein levels in mitochondrial extracts from hearts of the MHCsTNF and littermate control mice. Mitochondrial extracts were prepared according to the method of Yang et al. (36). To ensure equal protein loading, the membranes were washed and reprobed with an anti-cytochrome c oxidase subunit-IV antibody (clone 20E8-C12; Molecular Probes).

Cytochrome c release. We performed Western blot analysis for cytochrome c in myocardial mitochondrial and cytosolic extracts from MHCsTNF and littermate control hearts according to the method of Yang et al. (36) with the exception that cytosolic extracts were centrifuged at 30,000 g for 1 h. Equivalent amounts (20 µg) of mitochondrial and cytosolic myocardial protein lysates were then loaded onto 14% SDS-polyacrylamide gels, electrophoretically separated, and transferred to nitrocellulose membranes as previously described (19, 27). Western blot analysis was performed as described above using a polyclonal anti-cytochrome c antibody (1:1,000 dilution; Santa Cruz). The membranes were subsequently washed and reprobed for both GAPDH (clone 6C5; Advanced Immunochemical) and cytochrome c oxidase subunit IV (clone 20E8-C12; Molecular Probes) to ensure equal protein loading and relative purity of the mitochondrial and cytosolic extracts.

Caspase-3 activity. We performed fluorogenic assays for caspase-3 activity in the myocardium of the MHCsTNF and littermate control mice. Briefly, hearts were homogenized using a Dounce homogenizer in lysis buffer {10 mM HEPES, pH 7.4, 2 mM EDTA, 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, and 5 mM DTT} that contained 1 mM PMSF, 10 mg/ml pepstatin A, 10 mg/ml aprotinin, and 20 mg/ml leupeptin. These extracts were analyzed for caspase-3-like activity using a commercially available kit according to the manufacturer's suggestions (FluorAce Apopain Assay Kit; Bio-Rad) and a fluorogenic substrate that has a high specific activity for caspase-3 (acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethyl courmarin). To differentiate caspase-3-like activity from possible nonspecific protease activity, duplicate experiments were performed whereby each myocardial sample was pretreated with the specific caspase inhibitor Z-Asp-Glu-Val-Asp-fluoromethyl ketone (Z-DEVD-FMK). The values for Z-DEVD-FMK noninhibitable protease cleavage were subtracted from the values of the same samples obtained without pretreatment with Z-DEVD-FMK to obtain the specific capase-3-like activity for the MHCsTNF and littermate control mice.

Effects of Caspase Inhibition on LV Remodeling

MHCsTNF mice at 4 wk of age were anesthetized with subcutaneous injection of a mixture of xylazine (0.5 mg/ml) and ketamine (10 mg/ml) before receiving implantation of an osmotic minipump (Alzet 1002; Alza) in the peritoneal cavity. The osmotic minipumps were filled with either the broad-based caspase inhibitor N-[(1,3-dimethylindole-2-carbonyl)-valinyl]-3-amino-4-oxo-5-fluoropentanoic acid (IDN 1965, 30 mg/ml; Idun Pharmaceuticals) in 50% DMSO or with 50% DMSO alone (diluent), which has been shown to inhibit caspases-3, -6, and -8 at the doses employed in this study (16). The dose of IDN 1965 used in these experiments was determined by the manufacturer in previous studies of this compound in mice using the same Alzet minipump infusion system (16). MHCsTNF mice were treated with a continuous infusion of IDN 1965 or diluent for 14 days; after this they were reanesthetized, the original Alzet 1002 minipumps were surgically removed, and new Alzet minipumps were implanted peritoneally to extend treatment for an additional 14 days. Accordingly, mice received either diluent or IDN 1965 for a total of 28 days. The diluent- and IDN 1965-treated mice underwent cardiac MRI imaging on the second day after implantation of the minipumps and again at 8 wk of age (28 days of treatment). The mice were killed immediately after MRI imaging at 8 wk of age, and the hearts were perfusion fixed so in situ DNA ligation assays could be performed.

Statistical Analysis

Data are expressed as means ± SE. Two-way ANOVA was used to evaluate the mean differences in the age- and region-dependent prevalences of cardiac myocyte apoptosis in the MHCsTNF and littermate control mice as well as the differences in the heart weight-to-body weight ratios and the differences in cytochrome c release over time in the MHCsTNF and littermate control mice. One-way ANOVA was used to test for mean differences in the age-dependent prevalence of cardiac myocyte apoptosis, cytochrome c release, Bcl-2 family-member gene expression, and heart weight-to-body weight ratios in the MHCsTNF mice; post hoc ANOVA testing [Tukey's test (33)] was performed when appropriate. Student's t-tests were used to evaluate mean differences in the level of myocyte necrosis, Bcl-2 protein levels, caspase-3 activity, fold changes in end-diastolic volume (EDV), and the LV radius-to-wall thickness (r/h) ratios between the MHCsTNF and littermate control mice. Linear regression analysis was performed to relate the level of cardiac myocyte apoptosis with the parameters of LV remodeling obtained from the MRI analyses.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cardiac Myocyte Apoptosis

DNA laddering and electron microscopy. Figure 1A shows that DNA fragmentation was not detectable in the hearts of littermate control mice, whereas it was readily detectable in the hearts of MHCsTNF mice. The transmission electron micrograph depicted in Fig. 1B shows that cardinal features of apoptosis were present in cardiac myocytes, including the formation of apoptotic bodies, chromatin clumping, and margination along the nuclear envelope, and an intact sarcolemma.



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  		Fig. 1. Cardiac myocyte apoptosis in littermate control and transgenic mice with cardiac-restricted overexpression of secretable TNF (MHCsTNF mice). DNA laddering in littermate (LM) and MHCsTNF transgenic (TG) mice is shown (A). A transmission electron micrograph (B) shows an apoptotic body (arrow) in the nucleus of a cardiac myocyte from a MHCsTNF mouse heart. Representative myocardial sections (x200 magnification) show DNA ligase staining in littermate control (C) and MHCsTNF (D) mice. Apoptotic nuclei are shown by the bright green nuclear fluorescence (arrow). Staining with 4',6'-diamidino-2-phenylindole hydrochloride was used to identify the total number of cell nuclei in littermate control (E) and MHCsTNF (F) mice. An apoptotic nucleus was stained using the ligase technique (x600 magnification) in the absence (G) and presence (H) of anti-desmin immunostaining. Prevalence of cardiac myocyte apoptosis in littermate control and MHCsTNF mice at 4, 8, and 12 wk of age is shown (I); n = 6/group. Prevalence of cardiac myocyte apoptosis in the MHCsTNF mice within the endocardium, mid-wall, and epicardium at 4, 8, and 12 wk of age is indicated (J). The highest frequency of apoptosis in the MHCsTNF mice occurred in the endocardial region of the left ventricular (LV) wall; *P < 0.05.

 
In situ DNA ligation. Representative examples of the in situ DNA ligase staining in a MHCsTNF mouse and a littermate control mouse are shown in Fig. 1, C and D, respectively. Fluorescent green labeling of the nucleus indicates that cardiac myocyte apoptosis was detectable in the myocardial section from the MHCsTNF mouse but not the littermate control mouse. To confirm that the fluorescence-labeled nuclei were from cardiac myocytes, we performed anti-desmin immunostaining in myocardial sections that had also been stained with the in situ DNA ligase technique. Apoptotic nuclei were present in cardiac myocytes that were immunolabeled with the anti-desmin antibody (Fig. 1H). Results of the group data for MHCsTNF and wild-type mice are summarized in Fig. 1, I and J. The salient finding shown in Fig. 1I is that the prevalence of apoptosis was significantly (P < 0.001) greater in the MHCsTNF mice (n = 6 hearts/time) compared with littermate control mice (n = 6 hearts/time). Moreover, there were significant (P < 0.001) time-dependent changes in the prevalence of cardiac myocyte apoptosis in the MHCsTNF mice from 4 to 12 wk, whereas there was no change in the prevalence of apoptosis in cardiac myocytes from the littermate controls. Significant regional differences existed in the prevalence of apoptosis across the LV wall from 4 to 12 wk of age in the MHCsTNF mice (Fig. 1J). The prevalence of apoptosis was highest in the LV endocardium and lowest in the LV mid-wall. ANOVA indicated that there were significant time-dependent differences in the prevalence of apoptosis when the endocardium and epicardium were compared with the mid-wall (P < 0.001 for both); post hoc ANOVA (Tukey's test) indicated that the prevalence of apoptosis was significantly (P < 0.05) greater in the LV endocardium compared with the mid-wall and epicardium at 4, 8, and 12 wk of age.

Insofar as TNF has been shown to provoke necrosis in certain cell types (34), we also examined the prevalence of myocyte necrosis in the MHCsTNF and littermate control mice. The prevalence of necrosis was extremely low in the MHCsTNF and littermate control mice (0.09 ± 0.02 and 0.04 ± 0.02%, respectively; n = 2 hearts/group) at 12 wk and was not significantly different between the two groups (P = 0.33).

Effects of Cardiac Myocyte Apoptosis on LV Remodeling

Magnetic resonance imaging. To determine whether the progressive cardiac myocyte apoptosis in the MHCsTNF mice was associated with adverse cardiac remodeling, we examined LV structure using MRI. Figure 2A shows that at 4 wk of age, the MHCsTNF mice manifested a concentric hypertrophic phenotype compared with littermate controls. Analysis of group MRI data at 4 wk of age showed that LV wall thickness was significantly greater (P < 0.004) in the MHCsTNF mice (1.3 ± 0.04 mm; n = 6 hearts) than in littermate control mice (0.84 ± 0.04 mm; n = 5 hearts). However, the salient finding shown by Fig. 2A is that the MHCsTNF mice underwent a transition from a concentric hypertrophy phenotype to a dilated cardiac phenotype from 4 to 12 wk of age. As shown in Fig. 2B, the increase in LV EDV from 4 to 12 wk of age was approximately threefold greater (P = 0.008) in the MHCsTNF than the littermate control mice (97 ± 14 and 36 ± 12%, respectively). Importantly, the striking increase in LV dilation in the MHCsTNF mice was accompanied by a significant increase (P < 0.003) in LV wall thinning from 4 to 12 wk (1.3 ± 0.04 vs. 1.1 ± 0.02 mm, respectively). In contrast, the modest increase in LV volume in the littermate control mice was accompanied by a significant increase (P = 0.02) in LV wall thickness from 4 to 12 wk of age (0.84 ± 0.04 vs. 1.06 ± 0.02 mm, respectively). The increase in LV EDV and LV wall thinning resulted in adverse cardiac remodeling in the MHCsTNF mice as is shown by the significant increase (P < 0.001) in the r/h ratio (Fig. 2C) in the MHCsTNF mice (48 ± 7%). By way of comparison, the r/h ratio remained relatively unchanged (–9 ± 3%) in the littermate control mice from 4 to 12 wk of age, which is consistent with physiological hypertrophic growth. Linear regression analysis showed that there was a significant linear correlation between the prevalence of cardiac myocyte apoptosis and the fold change in the r/h ratio (y = 0.73x + 0.84; r = 0.77; P = 0.001), as well as the fold change in LV EDV (y = 28.9x + 28.5; r = 0.71; P = 0.001).



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  		Fig. 2. LV remodeling in littermate control and MHCsTNF mice. A: representative short-axis MRI images of the mice at 4 and 12 wk of age. B: group data for fold changes in LV end-diastolic volume (EDV) from 4 to 12 wk of age. *P = 0.008. C: group data for fold changes in the LV radius-to-LV wall (r/h) ratio at ages 4, 8, and 12 wk. *P < 0.001. D: heart weight-to-body weight ratios for mice at 4, 8, and 12 wk. *P < 0.01. E: body weights of mice at 4, 8, and 12 wk.

 
To complement the MRI data, we also performed standard morphometric analyses of the MHCsTNF and littermate control mice. Figure 2D shows two important findings with respect to the analysis of the heart weight-to-body weight ratios in these mice (n = 6 hearts/group). First, the heart weight-to-body weight ratio was significantly greater in the MHCsTNF than the littermate control mice (P < 0.01) at 4 wk of age, which is consistent with the initial cardiac hypertrophic phenotype observed in the MHCsTNF mice. Second, there was a significant decrease (P < 0.0001) in the overall heart weight-to-body weight ratio in MHCsTNF mice from 4 to 12 wk, whereas the heart weight-to-body weight ratio remained unchanged in littermate control mice during this time period. The observed decrease in the heart-weight-to-body weight ratio between MHCsTNF and littermate control mice was not secondary to differences in body weight between the groups of mice insofar as the body weights were not significantly (P > 0.05) different in MHCsTNF and littermate control mice from 4 to 12 wk of age (Fig. 2E).

Mechanisms for Cardiac Myocyte Apoptosis

Myocardial pro- and antiapoptotic gene expression. To explore the mechanism(s) for cardiac myocyte apoptosis in MHCsTNF mice, we examined changes in gene expression for the Bcl family of pro- and antiapoptotic genes. Representative ribonuclease protection assays for the Bcl family of pro- and antiapoptotic genes in both MHCsTNF and littermate control mice at ages 4 and 12 wk are depicted in Fig. 3A, and the corresponding group data (n = 6 hearts/group) are summarized in Fig. 3B. The salient finding shown by Fig. 3B is that the levels of Bcl-2 gene expression were significantly (P < 0.001) greater in MHCsTNF relative to littermate control mice at ages 4, 8, and 12 wk. In contrast, the relative levels of the other Bcl family members including Bak, Bax, Bad, and Bcl-x were not significantly different (P > 0.05 for each) in the MHCsTNF and littermate control mice at 4 to 12 wk of age. A second important aspect shown by Fig. 3B is that the relative Bcl-2 gene-expression level in the MHCsTNF mice decreased significantly (P < 0.05) from 4 to 12 wk. In contrast, this level did not change in the littermate control mice from 4 to 12 wk (data not shown).



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  		Fig. 3. Myocardial pro- and antiapoptotic gene expression in littermate control and MHCsTNF mice. A: representative ribonuclease protection panels for Bak, Bax, Bad, Bcl-x, and Bcl-2. B: group data for the mRNA levels for Bak, Bax, Bad, Bcl-x, and Bcl-2 at 4, 8, and 12 wk of age in MHCsTNF mice (expressed as fold changes) compared with wild-type mice at comparable time points. *P < 0.05.

 
Given that TNF has been shown to enhance Bcl-2 degradation through dephosphorylation and subsequent proteasomal degradation (7), we postulated that the increase in Bcl-2 gene expression in MHCsTNF mice may have been in response to increased Bcl-2 protein turnover. To explore this possibility, we measured Bcl-2 protein levels in the hearts of the MHCsTNF and littermate control mice at 12 wk of age. As shown by the Western blot in Fig. 4A, Bcl-2 protein levels were significantly lower (P = 0.02) in the hearts of MHCsTNF than littermate control mice at 12 wk of age. Interestingly, the polyclonal anti-Bcl-2 antibody we used detected 26- and 30-kDa immunoreactive bands in the whole cell myocardial extracts as was previously reported from extracts of rat and human myocardium (1, 5). Although Bcl-2 is classically described as a 26-kDa protein, it has been suggested that the 30-kDa form may represent a phosphorylated form of the protein (13) or possibly a separate Bcl-2 isoform (5). Figure 4C shows that a single immunoreactive 26-kDa band was detected in the mitochondrial extracts probed with the same polyclonal antibody used for the whole cell extracts. As shown by the group data in Fig. 4, B and D (n = 6/group), respectively, cytosolic and mitochondrial Bcl-2 levels were significantly lower in hearts of MHCsTNF than littermate control mice at 12 wk (P < 0.01 for both).



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  		Fig. 4. Myocardial Bcl-2 protein levels in littermate control and MHCsTNF mice at 12 wk of age. A: representative Western blots for Bcl-2 in whole heart myocardial extracts. B: group data for the ratio of Bcl-2 to actin in whole heart extracts. C: representative Western blot analysis for Bcl-2 in mitochondrial preparations isolated from whole heart extracts. D: group data for the ratio of Bcl-2 to cytochrome c oxidase in mitochondrial preparations isolated from whole heart extracts. *P < 0.05.

 
Cytochrome c release. The progressive loss of mitochondrial Bcl-2 protein suggested that the TNF-induced cardiac myocyte apoptosis was mediated by activation of the "intrinsic" or mitochondrial death pathway. Western blots for mitochondrial and cytosolic cytochrome c (Fig. 5B) show that there was an increase in release of cytochrome c from the mitochondria to the cytosol in the myocardial extracts from MHCsTNF compared with littermate control mice. Reprobing the membranes for both GAPDH and cytochrome c oxidase subunit IV confirmed that protein loading was equal and the mitochondrial and cytosolic extracts were relatively pure. As shown by the group data in Fig. 5B, there was a significant overall (P < 0.001) increase in the release of cytochrome c from the mitochondria to the cytosol in myocardium of MHCsTNF mice at 4 and 12 wk of age; post hoc ANOVA (Tukey's) testing indicated that the level of cytochrome c released from the mitochondria to the cytosol in MHCsTNF mice was significantly greater (P < 0.05) at 4 and 12 wk of age.



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  		Fig. 5. Cytochrome c release and caspase activation in littermate control and MHCsTNF mice. A: representative Western blots for cytochrome c in mitochondrial and cytosolic myocardial extracts obtained from 12-wk-old mice. B: group data for the cytosolic-to-mitochondrial cytochrome c ratio in mouse hearts at 4 and 12 wk of age. C: caspase-3 activity in myocardial extracts from mice at 12 wk of age. *P < 0.05.

 
Caspase-3-like activity. Because cytochrome c release is known to activate caspase-3, we next measured caspase-3 activity in the myocardial extracts from 12-wk-old wild-type and MHCsTNF mice (n = 6/group). The important finding shown by Fig. 5C is the significant increase (P = 0.007) in myocardial caspase-3-like activity in the hearts of TNF transgenic mice compared with littermate control animals.

Effects of Caspase Inhibition on LV Remodeling

Given that these studies suggested that cardiac myocyte apoptosis proceeds through a caspase-dependent pathway and that cardiac myocyte apoptosis is linearly related to cardiac remodeling, we next asked whether caspase inhibition is sufficient to attenuate cardiac myocyte apoptosis and/or LV remodeling in MHCsTNF mice from 4 to 8 wk of age. As shown in Fig. 6A, treatment with the broad-based caspase inhibitor IDN 1965 for 4 wk significantly attenuated cardiac myocyte apoptosis in MHCsTNF mice. Figure 6B shows that there was a small decrease in the fold change in LV EDV in IDN 1965-treated relative to diluent-treated MHCsTNF mice; however, these changes were not significant statistically (P = 0.14). In contrast, treatment with IDN 1965 resulted in a significant decrease in LV wall thinning in MHCsTNF mice when compared with diluent-treated MHCsTNF mice (1.18 ± 0.02 vs. 0.97 ± 0.08 mm, respectively). This decrease in LV wall thinning in the IDN 1965-treated MHCsTNF mice resulted in a significant attenuation in adverse cardiac remodeling as shown by the significant (P < 0.05) decrease in the r/h ratio in caspase-inhibitor-treated mice relative to diluent-treated mice.



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  		Fig. 6. Effects of caspase inhibition on cardiac myocyte apoptosis and LV remodeling. A: prevalence of apoptosis in MHCsTNF mice in the presence and absence of IDN 1965. B: fold change in LV EDV values in MHCsTNF mice in the presence and absence of IDN 1965. C: fold change in the r/h ratios for MHCsTNF mice in the presence and absence of IDN 1965. *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of this study, in which we used serial MRI to define the influence of cardiac myocyte apoptosis on structural heart remodeling, show that TNF-induced cardiac myocyte apoptosis contributes to adverse cardiac remodeling at least partly through progressive thinning of the LV wall. Several lines of evidence support this statement. First, the prevalence of cardiac myocyte apoptosis in MHCsTNF mice increased progressively from 4 to 12 wk of age (see Fig. 1I) as the hearts of MHCsTNF mice segued from a concentric hypertrophic phenotype to a dilated cardiac phenotype (see Fig. 2, A and C). Cell loss through cardiac myocyte necrosis is unlikely to have contributed to this process insofar as myocyte necrosis was barely detectable in the hearts of MHCsTNF mice and was not significantly different from littermate control mice. Additional evidence that cardiac myocyte apoptosis contributed to the LV structural remodeling in MHCsTNF mice is suggested by the finding that there was a relative loss of cardiac mass in these mice from 4 to 12 wk of age (see Fig. 2D). Second, there was a significant linear correlation between the prevalence of cardiac apoptosis and the increase in the r/h ratio of MHCsTNF mice. Third, when MHCsTNF mice were treated with the broad-based caspase inhibitor IDN 1965, we observed a significant decrease in cardiac myocyte apoptosis, significant attenuation of LV wall thinning, and complete abrogation of adverse remodeling (r/h ratio) in hearts of MHCsTNF mice (see Fig. 6). Importantly, caspase inhibition did not significantly attenuate the increase in LV EDV in the MHCsTNF mice, which we and others have shown to be secondary to increased activation of matrix metalloproteinases (MMPs; Refs. 8, 24).

A second interesting feature of this study was that TNF-induced activation of the intrinsic mitochondrial death pathway was responsible, at least in part, for the cardiac myocyte apoptosis observed in MHCsTNF mice. That is, we observed a loss of mitochondrial Bcl-2 protein levels (see Fig. 5, C and D), an increase in cytosolic release of cytochrome c from the mitochondria (see Fig. 5, A and B), and a concomitant increase in caspase-3-like activity (see Fig. 5C) in hearts of MHCsTNF relative to littermate control mice. Moreover, in parallel studies, we observed that crossing MHCsTNF mice with transgenic mice that overexpress Bcl-2 results in a decrease in cytochrome c release as well as a decrease in caspase-9 and -3 activation (S. B. Haudek and D. L. Mann, unpublished observations), which further supports a role for TNF-induced activation of the intrinsic mitochondrial death pathway in this model.

Cardiac Myocyte Apoptosis and Remodeling

Although cardiac myocyte apoptosis has been shown to occur in failing human hearts, the exact physiological significance and consequence(s) of this finding remain unknown for two reasons. First, at present there is tremendous uncertainty regarding the actual rate of cardiac myocyte apoptosis in failing human hearts (reviewed in Ref. 11). Indeed, our ability to precisely quantify the exact rate of apoptosis in human tissue is limited by the specificity of the existing methodologies for detecting apoptotic DNA strand breaks as well as uncertainty about the precise rate of removal of apoptotic myocytes in failing myocardium. Second, until recently the majority of studies of cardiac myocyte apoptosis were confined to ischemia-reperfusion and/or infarction models, which are known to lead to extensive cardiac remodeling through a variety of complex and interactive mechanisms including necrotic myocyte cell loss, extracellular matrix remodeling, neurohumoral activation, and loading-condition alterations. Two recent studies addressed this latter deficiency and show that activation of either the extrinsic pathway through targeted overexpression of caspase 8 (35) or activation of the intrinsic pathway through targeted overexpression of Nix/Bnip3L38 are both sufficient to engender progressive cardiac dilation and decompensation. Moreover, use of IDN 1965 was shown to prevent cardiac dilation, improve LV dysfunction, and abolish mortality in the peripartum cardiomyopathy of G{alpha}q transgenic mice. The results of the present study both confirm and expand upon these findings by demonstrating that targeted overexpression of a pathophysiologically relevant ligand results in progressive cardiac myocyte apoptosis, which in turn leads to adverse cardiac remodeling as a consequence of progressive LV wall thinning. Although the overall prevalence of cardiac myocyte apoptosis in the present study is consistent with a previous independent report in failing human hearts (28), the prevalence reported herein is higher than that reported in the majority of clinical studies (14, 29, 30). Nonetheless, it bears emphasis that the findings in the present study with respect to cardiac remodeling are entirely consistent with a recent experimental report in which the prevalence of cardiac myocyte apoptosis was similar to that observed in clinical studies (35). The results of the present study differ from a prior study in which cardiac-restricted overexpression of TNF was shown to lead to apoptosis primarily in infiltrating CD45+ cells ({approx}70% of apoptotic cells), whereas the proportion of apoptotic cells that stained positively with {alpha}-actinin was 10-fold less ({approx}7% of apoptotic cells; Ref. 22). Although the reasons for the discrepancy between the two studies is not known, they may relate to differences in genetic background (C57/BL6 x ICR vs. FVB21 mice), differences in the degree of TNF expression, differences in life expectancy [cumulative mortality at 6 mo of 35 (15) vs. 23% (21)], and/or the presence of myocarditis, which has been observed in the model studied by Kubota and colleagues (22) but not in the transgenic mouse model reported herein.

In conclusion, the results of this study contribute to the nascent body of literature that suggests that cardiac myocyte apoptosis contributes to adverse cardiac remodeling in adult mammalian hearts. From a pathophysiological standpoint, the increase in LV wall thinning that attends progressive cardiac myocyte apoptosis is expected to contribute to increased LV wall stress and, hence, sustained unfavorable loading conditions of the heart. On a more basic level, these studies may also provide insight into the basic mechanisms that govern LV remodeling. Whereas previous studies have suggested that LV wall thinning is engendered by transmural rearrangement of myocytes and/or myofibrillar bundles within the LV wall, the scientific evidence in favor of this point of view has largely been circumstantial. And indeed, the results of the present study suggest that this view may be only partially correct. That is, the observation that inhibition of myocyte apoptosis prevented LV wall thinning but did not significantly attenuate LV dilation in the MHCsTNF mice suggests that these two processes are not necessarily interdependent as was previously supposed. Moreover, when we examined the role of MMP inhibition in similar transgenic mouse models with cardiac-restricted overexpression of TNF, we observed that MMP inhibition abrogated LV dilation but had no effect on LV wall thickness (8). Taken together, these observations raise the interesting if not important question of understanding how LV wall thinning and dilation are interrelated during the initiation and/or reversal of adverse cardiac remodeling. Ongoing studies are being conducted to address this question.


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 ABSTRACT
 METHODS
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 DISCUSSION
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This research was supported by funds from the Department of Veterans Affairs and National Institutes of Health Grants P50 HL-O6H and RO1 HL-58081-01, RO1 HL-61543-01, and HL-42250-10/10.


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge the technical assistance of Dorellyn Lee Jackson and the secretarial support of Mary Helen Soliz. The authors also thank Dr. Francis Spinale for graciously providing the transmission electron micrographs of the apoptotic cardiac myocyte.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. L. Mann, Winters Center for Heart Failure Research, 6565 Fannin St., MS 524, Houston, TX 77030 (E-mail: dmann{at}bcm.tmc.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.


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