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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
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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tumor necrosis factor; cell death; caspase; congestive heart failure; magnetic resonance imaging
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.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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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.
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RESULTS |
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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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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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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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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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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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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
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 (
70% of apoptotic cells), whereas the proportion of apoptotic cells that stained positively with -actinin was 10-fold less (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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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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(TNF). Circulation 97: 1375–1381, 1998.-induced expression of heat shock protein 72 in adult feline cardiac myocytes. Am J Physiol Heart Circ Physiol 270: H1231–H1239, 1996.This article has been cited by other articles:
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J.-F. Wang, A. Meissner, S. Malek, Y. Chen, Q. Ke, J. Zhang, V. Chu, T. G. Hampton, C. S. Crumpacker, W. H. Abelmann, et al. Propranolol ameliorates and epinephrine exacerbates progression of acute and chronic viral myocarditis Am J Physiol Heart Circ Physiol, October 1, 2005; 289(4): H1577 - H1583. [Abstract] [Full Text] [PDF]
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