Myocardial Cell Death and Regeneration during Progression of Cardiac Hypertrophy to Heart Failure
2004; Elsevier BV; Volume: 279; Issue: 50 Linguagem: Inglês
10.1074/jbc.m402037200
ISSN1083-351X
AutoresSagartirtha Sarkar, Mamta Chawla‐Sarkar, David Young, Kazutoshi Nishiyama, Mary E. Rayborn, Joe G. Hollyfield, Subha Sen,
Tópico(s)Muscle Physiology and Disorders
ResumoCardiac hypertrophy and ensuing heart failure are among the most common causes of mortality worldwide, yet the triggering mechanisms for progression of hypertrophy to failure are not fully understood. Tissue homeostasis depends on proper relationships between cell proliferation, differentiation, and death and any imbalance between them results in compromised cardiac function. Recently, we developed a transgenic (Tg) mouse model that overexpress myotrophin (a 12-kDa protein that stimulates myocyte growth) in heart resulting in hypertrophy that progresses to heart failure. This provided us an appropriate model to study the disease process at any point from initiation of hypertrophy end-stage heart failure. We studied detailed apoptotic signaling and regenerative pathways and found that the Tg mouse heart undergoes myocyte loss and regeneration, but only at a late stage (during transition to heart failure). Several apoptotic genes were up-regulated in 9-month-old Tg hearts compared with age-matched wild type or 4-week-old Tg hearts. Cardiac cell death during heart failure involved activation of Fas, tumor necrosis factor-α, and caspases 9, 8, and 3 and poly(ADP-ribose) polymerase cleavage. Tg mice with hypertrophy associated with compromised functionshowedsignificantup-regulationofcyclins,cyclin-dependent kinases (Cdks), and cell regeneration markers in myocytes. Furthermore, in human failing and nonfailing hearts, similar observations were documented including induction of active caspase 3 and Ki-67 proteins in dilated cardiomyopathic myocytes. Taken together, our data suggest that the stress of extensive myocardial damage from longstanding hypertrophy may cause myocytes to reenter the cell cycle. We demonstrate, for the first time in an animal model, that cell death and regeneration occur simultaneously in myocytes during end-stage heart failure, a phenomenon not observed at the onset of the disease process. Cardiac hypertrophy and ensuing heart failure are among the most common causes of mortality worldwide, yet the triggering mechanisms for progression of hypertrophy to failure are not fully understood. Tissue homeostasis depends on proper relationships between cell proliferation, differentiation, and death and any imbalance between them results in compromised cardiac function. Recently, we developed a transgenic (Tg) mouse model that overexpress myotrophin (a 12-kDa protein that stimulates myocyte growth) in heart resulting in hypertrophy that progresses to heart failure. This provided us an appropriate model to study the disease process at any point from initiation of hypertrophy end-stage heart failure. We studied detailed apoptotic signaling and regenerative pathways and found that the Tg mouse heart undergoes myocyte loss and regeneration, but only at a late stage (during transition to heart failure). Several apoptotic genes were up-regulated in 9-month-old Tg hearts compared with age-matched wild type or 4-week-old Tg hearts. Cardiac cell death during heart failure involved activation of Fas, tumor necrosis factor-α, and caspases 9, 8, and 3 and poly(ADP-ribose) polymerase cleavage. Tg mice with hypertrophy associated with compromised functionshowedsignificantup-regulationofcyclins,cyclin-dependent kinases (Cdks), and cell regeneration markers in myocytes. Furthermore, in human failing and nonfailing hearts, similar observations were documented including induction of active caspase 3 and Ki-67 proteins in dilated cardiomyopathic myocytes. Taken together, our data suggest that the stress of extensive myocardial damage from longstanding hypertrophy may cause myocytes to reenter the cell cycle. We demonstrate, for the first time in an animal model, that cell death and regeneration occur simultaneously in myocytes during end-stage heart failure, a phenomenon not observed at the onset of the disease process. Withdrawal: Myocardial cell death and regeneration during progression of cardiac hypertrophy to heart failure.Journal of Biological ChemistryVol. 295Issue 45PreviewVOLUME 279 (2004) PAGES 52630–52642 Full-Text PDF Open AccessExpression of Concern: Myocardial cell death and regeneration during progression of cardiac hypertrophy to heart failure.Journal of Biological ChemistryVol. 295Issue 10PreviewVOLUME 279 (2004) PAGES 52630–52642 Full-Text PDF Open Access Cardiac hypertrophy and resulting heart failure are the most common cause of mortality in the world. The triggering mechanisms for progression of cardiac hypertrophy to heart failure are still not fully understood, but many observers have suggested that programmed cell death (PCD), 1The abbreviations used are: PCD, programmed cell death; Tg, transgenic; WT, wild-type; FADD, Fas-associated death domain; GAPDH, glyceraldehyde-3-phosphate dehydrogenase, PARP, poly(ADP-ribose) polymerase; pCNA, proliferation cell nuclear antigen; DCM, dilated cardiomyopathic; NF, nonfailing; TNF-α, tumor necrosis factor-α; RPA, RNase protection assays; DAPI, 4,6-diamidino-2-phenylindole; Cdk, cyclin-dependent kinases. that is, apoptosis, is a major contributor to heart failure. Although apoptosis in the myocardium is a complex process and difficult to recognize, there is evidence that potential mechanisms of induction of apoptosis at the cellular level may involve interplay between mechanical factors and elevated levels of neurohumoral factors. Volume overload and elevated end-diastolic left ventricular pressure may initiate the events of myocyte apoptosis (1Cheng W. Li B. Kajstura J. Li P. Wolin M.S. Sonnenblick E.H. Hintze T.H. Olivetti G. Anversa P. J. Clin. Investig. 1995; 96: 2247-2259Crossref PubMed Scopus (588) Google Scholar). Recently, myocyte apoptosis has been demonstrated after injury because of ischemia, reperfusion, myocardial infarction, ventricular pacing, cardiac aging, and coronary embolization (2Edwards D.R. Trends Pharmacol. Sci. 1994; 15: 239-244Abstract Full Text PDF PubMed Scopus (79) Google Scholar, 3Kajstura J. Cheng W. Sarangarajan R. Li P. Li B. Nitahara J.A. Chapnick S. Reiss K. Olivetti G. Anversa P. Am. J. Physiol. 1996; 271: H1215-H1228Crossref PubMed Google Scholar, 4Kajstura J. Cheng W. Reiss K. Clark W.A. Sonnenblick E.H. Krajewski S. Reed J.C. Olivetti G. Anversa P. Lab. Investig. 1996; 74: 86-107PubMed Google Scholar). Furthermore, Olivetti et al. (5Olivetti G. Abbi R. Quaini F. Kajstura J. Cheng W. Nitahara J.A. Quaini E. Di Loreto C. Beltrami C.A. Krajewski S. Reed J.C. Anversa P. N. Engl. J. Med. 1997; 336: 1131-1141Crossref PubMed Scopus (1486) Google Scholar) demonstrated that cell death accompanies congestive heart failure in humans. Tissue homeostasis depends on proper relationships between cell proliferation, differentiation, and death. The balance between proliferation and apoptosis must be maintained to sustain tissue homeostasis. As a cell progresses through the cell cycle, it must determine whether to complete cell division, arrest growth to repair cell damage, or undergo apoptosis if the damage is too severe to be repaired. Whether the heart can grow by multiplication of myocytes has been controversial. Recently, Beltrami et al. (6Beltrami A.P. Urbanek K. Kajstura J. Yan S.M. Finato N. Bussani R. Nadal-Ginard B. Silvestri F. Leri A. Beltrami C.A. Anversa P. N. Engl. J. Med. 2001; 344: 1750-1757Crossref PubMed Scopus (1300) Google Scholar) provided convincing proof of myocyte replication in the failing human heart and showed that this form of cell growth could compensate for exhaustion of myocyte hypertrophy. A myocyte mitotic index of 0.015% was measured in explanted hearts from patients in terminal stages of cardiac decompensation. One major limitation to examining molecular changes during the progression of hypertrophy to heart failure has been the availability of a suitable animal model. We have identified a factor, myotrophin, from spontaneously hypertensive rat hearts and cardiomyopathic human hearts, which stimulates myocyte growth (7Sen S. Kundu G. Mekhail N. Castel J. Misono K. Healy B. J. Biol. Chem. 1990; 265: 16635-16643Abstract Full Text PDF PubMed Google Scholar). Myotrophin is a novel gene, localized in human chromosome 7q33 (8Mitra S. Timor A. Gupta S. Wang Q. Sen S. Cytogenet. Cell Genet. 2001; 93: 151-152Crossref PubMed Google Scholar). Recently, we have developed a transgenic mouse model overexpressing myotrophin in the heart under the transcriptional regulation of the α-myosin heavy chain promoter. This model is associated with increased expression of proto-oncogenes, hypertrophy marker genes (β-myosin heavy chain and atrial natriuretic factor), and rapid organization of myofibrils (9Sarkar S. Leaman D.W. Gupta S. Sil P. Young D. Morerhead A. Mukherjee D. Ratliff N. Sun Y Rayborn M. Hollyfield J. Sen S. J. Biol. Chem. 2004; 279: 20422-20434Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). This transgenic mouse model showed hypertrophy as early as 4 weeks of age that progressively led to heart failure with severe compromised function (Fig. 1A). All the symptoms in this model mimic human heart failure. Using DNA microarray analysis, we compared a differential expression profile of several gene clusters in wild-type and transgenic animals during initiation (about 4 weeks of age) and transition of hypertrophy to heart failure (around 36 weeks of age). A cluster of apoptotic genes, as well as genes involved in cellular regeneration, was found to be significantly up-regulated in 36-week-old Tg mice heart samples but not those from 4-week-old mice (9Sarkar S. Leaman D.W. Gupta S. Sil P. Young D. Morerhead A. Mukherjee D. Ratliff N. Sun Y Rayborn M. Hollyfield J. Sen S. J. Biol. Chem. 2004; 279: 20422-20434Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Therefore, we chose to study the molecular changes for both cardiac cell death and regeneration during initiation of cardiac hypertrophy and during the transition from hypertrophy to heart failure, the later still being an open question. To establish the relevance of our findings in murine model, we also studied some key genes in these processes (active caspase 3 for cell death and Ki-67 for cell regeneration) in human dilated cardiomyopathic (DCM) and nonfailing (NF) hearts. Our data showed that both cell death and regeneration occur simultaneously during heart failure that is not evident during onset of this disease. Animals Used—All mice used in this study were obtained from Harlan Sprague-Dawley (Indianapolis, IN). This investigation conformed to the “Guide for the Care and Use of Laboratory Animals” (29United States National Institutes of Health Guide for the Care and Use of Laboratory Animals. National Institutes of Health, Bethesda, MD1996Google Scholar). For each experiment discussed in this article, at least five different animals, both wild-type (WT) and transgenic (Tg), from each age group (4 weeks and 9 months), were used. The Tg animals used represent all four founder lines that overexpress myotrophin protein in the heart. Our data represent both male and female Tg and WT mice. No difference was observed between males and females on the parameters studied. Human Samples—Human DCM and NF heart samples were obtained from the cardiac transplantation core at the Cleveland Clinic Foundation. NF human hearts were obtained from 5 organ donors not suitable for transplantation but with no history of cardiac diseases and were victims of either motor vehicle accidents or gunshot wounds. Failing hearts were obtained from 6 transplant patients diagnosed with DCM. All heart samples were transported to the laboratory in cold cardioplegia and were snap frozen instantly for future use. Protocols for tissue procurement were approved by the Cleveland Clinic Foundation Institutional Review Board. The clinical characteristics of these heart samples are tabulated in Table I.Table IClinical characteristics of the human heart samples used in this studyCodeDiagnosisAgeSexNonfailingA1MVAaMVA, motor vehicle accident52MaleA2MVA51FemaleA3MVA53FemaleA9MVA46FemaleA10MVA52MaleFailingA4DCM52MaleA5DCM57FemaleA7DCM58MaleA12DCM51FemaleA13DCM47Femalea MVA, motor vehicle accident Open table in a new tab TUNEL Assay—DNA fragmentation was detected in left ventricular sections of 9-month-old WT and Tg mice by TUNEL staining using the APO-BRDU™ kit (BD Pharmingen, San Diego, CA). Briefly, the sections were passed through graded alcohol and labeled with bromodeoxyribonucleotide triphosphate, washed twice with phosphate-buffered saline, and labeled with bromodeoxyribonucleotide triphosphate by the terminal deoxynucleotidyl transferase enzyme for 2 h at 37 °C. After labeling, sections were washed and stained with fluorescein isothiocyanate-conjugated anti-bromodeoxyuridine monoclonal antibody for 30 min in a low-light environment. RNase was added and samples were incubated for an additional 30 min at room temperature. Slides were rinsed 3–4 times with 1× phosphate-buffered saline before being mounted with Vectrashield (Vector Laboratories Inc., Burlingame, CA). The percentage of fluorescein isothiocyanate-positive cells was analyzed by fluorescence microscopy using an excitation wavelength in the range of 450–500 nm and detection in the range of 515–565 nm (green). Negative controls included sections incubated in the absence of substrate. RNase Protection Assay—Total RNA was isolated from WT and Tg mice hearts and human heart samples using TRIzol reagent (Invitrogen). RNase protection assays (RPAs) were done using the RiboQuant system with a multiprobe template set from BD Pharmingen. For mice, the mAPO-1, mAPO-2, mAPO-3, and mCYC-1 template sets were used for T7 polymerase directed synthesis of high specific activity [32P]UTP-labeled antisense RNA probes. The probe sets contained 13 probes including two housekeeping genes, GAPDH and L32. Probes (4 × 105 cpm) were hybridized with each RNA (10 μg) sample overnight at 56 °C. RNA samples were digested with RNase A and T1, purified, and resolved on 6% denaturing polyacrylamide gels. Internal housekeeping genes were analyzed to confirm equal RNA loading. For failing and nonfailing human heart samples (n = 5), multiprobe template sets hAPO-1c, hAPO-2c, hAPO-3, and hCYC-1 were used for RPA, following the manufacturer's protocol. Immunohistochemistry—Myocardial sections were stained with antibodies against Fas, Fas-associated death domain (FADD), the cleaved active form of caspase-8, -7, and -3, or the macrophage markers CD13 and CD14 (BD Pharmingen). The sections were counterstained with propidium iodide and analyzed by fluorescent microscopy (26Scarabelli T.M. Stephanou A. Pasini E. Comini L. Raddino R. Knight R.A. Latchman D.S. Circ. Res. 2002; 90: 745-748Crossref PubMed Scopus (149) Google Scholar). Active caspase-3 (BD Pharmingen) and Ki-67 (DAKO Corp., Carpinteria, CA) proteins were used for confocal microscopic analysis along with α-actinin antibody (Sigma) on myocardial sections with nuclear counterstaining agent (DAPI) by using an SP2 confocal laser scanning microscope (Leica, Heidelberg, Germany), equipped with 40, 60, and 100× infinity-adjusted oil immersion objectives and triple channel photodetectors. Both mice (WT and Tg) and human (NF and DCM) ventricular sections were used for confocal studies with active caspase-3 and Ki-67 (detected in green) and α-actinin (detected in red; n = 5). Each data set collected on the confocal microscope was processed with software included with the Leica Scan Wareoperating system, and used to construct an extended focus image, i.e. a computer-averaged assembly of some optical sections in the data set. Antibodies against cyclin B1 protein (Santa Cruz Biotechnologies) and phosphohistone H3 (Cell Signaling Technology, Beverly, MA) were used on 36-week-old Tg ventricular sections. Western Blotting—Heart samples were lysed in 1× lysis buffer (50 mm Tris-Cl, pH 8.0, 1% Triton X-100, 10% glycerol, 1 mm EDTA, 250 mm NaCl, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 10 μg/ml pepstatin) and aliquots containing 40 μg of protein were fractionated by 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were subsequently incubated with monoclonal antibody to Bax, Bcl2, Fas, tumor necrosis factor (TNF)-α RI and RII (Santa Cruz Biotechnology, Inc.), polyclonal antibody to active caspase-3 (BD Pharmingen), caspase-8 (Stressgen Biotechnologies Corp., Victoria, BC, Canada), Bcl-XL (Transduction Laboratories, San Diego, CA), or polyclonal antibody to cyclin A, B1, or B2 (Santa Cruz Biotechnologies) followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Pierce). Immunoreactive bands were visualized using enhanced chemiluminescence (PerkinElmer Life Sciences). Equal protein loading was confirmed by staining the gel with Coomassie Blue and probing with GAPDH antibody (Novus Biologicals Inc., Littleton, CO). Caspase Activity Assay—The activity of caspase-3, -8, and -9 was measured using a commercially available caspase assay kit (Clontech). Briefly, tissues were washed twice with cold phosphate-buffered saline and lysed on ice in 1× lysis buffer provided by the company. Tissue lysates were centrifuged at 10,000 × g for 10 min, and the total protein concentration was estimated using a protein assay reagent (Bio-Rad). The assay was performed in triplicate in 96-well plates. For each caspase-3 assay, 20 μg of protein extract, 200 μlof1× Hepes buffer, and 5 μl of Ac-DEVD-AFC (a fluorogenic substrate) were mixed and incubated at 37 °C for 1 h. As a control, cell lysates or substrate alone were incubated in parallel. Enzymatic hydrolysis of caspase-3 was measured by AMC liberation from Ac-DEVD-AFC at 380/460 nm using a spectrofluorometer. Relative fluorescence of substrate control was subtracted as background emission. Activity of caspase-8 and -9 was measured using Ac-IETD-AFC and Ac-LEHD-AMC substrates, as described for the caspase-3 assay. Cyclin-dependent Kinase Activity Assay—Six hundred micrograms of tissue lysate from Tg and WT hearts lysed in buffer (50 mm Hepes, pH 7.0, 150 mm sodium chloride, 10% glycerol, 0.1% Tween 20, protease inhibitor mixture (Calbiochem, San Diego, CA), 0.5 mm sodium pyrophosphate, 0.1 mm sodium orthovanadate, and 5 mm sodium fluoride) were immunoprecipitated with polyclonal antibody to Cdc2, Cdk2, or Cdk4 (Santa Cruz Biotechnologies) for 2 h at 4 °C. The protein A-Sepharose beads containing the immunocomplexes were incubated with 25 μl of kinase buffer, 2 μg of histone H1 (as substrate for Cdk2) or 0.5 μg of retinoblastoma pRb (as Cdk4 substrate, Santa Cruz Biotechnology Inc.) and 1 μl of 3000 Ci/mm [γ-32P]ATP (PerkinElmer Life Sciences) for 30 min at 30 °C. The samples were subjected to 10% SDS-PAGE, and the gel was exposed to autoradiographic film. To assess the background kinase activity, all the samples were immunoprecipitated with preimmune rabbit IgG and were run parallel on the gel. Background kinase activity was subtracted (27Reiss K. Cheng W. Giordano A. De Luca A. Li B. Kajstura J. Anversa P. Exp. Cell Res. 1996; 225: 44-54Crossref Scopus (47) Google Scholar). Isolation of Cardiac Myocytes from Hearts of WT and Tg Mice Overexpressing Myotrophin—As described previously (28Sil P. Kandaswamy V. Sen S. Circ. Res. 1998; 82: 1173-1188Crossref PubMed Scopus (45) Google Scholar), hearts were taken out from heparin-injected mice and cannulated via aorta (n = 6). Hearts were perfused with perfusion buffer (glucose, 1 g; NaHCO3, 0.58 g; and pyruvic acid, 0.27 g, pH 7.3) with 95% O2 and 5% CO2 on a Langendorff apparatus. After perfusing the heart for 10 min in EGTA-supplemented perfusion buffer, hearts were digested using collagenase (2 mg/ml) for 28 min, with gradual enhancement of CaCl2. After 28 min of digestion with collagenase, the heart was taken out and incubated in a diluted collagenase solution for 10 min in a shaking water bath at 37 °C. The ventricles were separated from the atria, triturated for 30 s, and subsequently filtered through cheesecloth. The filtrate was centrifuged at 400 rpm for 2 min, the supernatant was removed, and the pellet was resuspended in 4% bovine serum albumin solution and observed under a phase-contrast microscope. Preparations with 80–85% beating rod-shaped cells were used for experimental purposes. Isolation of Nuclear Protein from the Isolated Myocytes—Nuclear protein was prepared using NE-PER nuclear and cytoplasmic extraction reagents (Pierce), using the manufacturer's protocol. Both cytoplasmic and nuclear fractions were collected, the amount of protein was measured using standard techniques, and Western blots were performed as described earlier with primary antibodies. Twenty micrograms of nuclear protein was used to detect proteins using monoclonal antibodies to poly(ADP-ribose) polymerase (PARP) (Biomol, Plymouth Meeting, PA), pCNA (Santa Cruz Biotechnologies), and phosphohistone H3 (Cell Signaling Technology, Beverly, MA). Twenty micrograms of cytoplasmic proteins was used to detect c-kit and Sca-1 (R&D Systems, Minneapolis, MN) using the respective antibodies. These blots were probed with GAPDH antibody as a loading control. Statistical Analysis—Each experiment was repeated at least five times. Results were expressed as mean ± S.E. Data were analyzed by two-way analysis of variance, and differences between groups were determined by the least-square means test (SUPERNOVA). Significance was evaluated using the analysis of variance test. A value of p < 0.05 was considered significant. Cell death (apoptosis) was compared in heart tissue (n = 6) from 4-week-old and 9-month-old Tg mice with significant hypertrophy (heart weight:body weight ratio of 10.4 ± 0.4 compared with 4.7 ± 0.1 in WT) and age-matched WT mice by TUNEL staining. In 9-month-old WT hearts, only 5–8 nuclei per 105 cells were TUNEL positive (Fig. 1B). Apoptotic nuclei were absent in young (4-week-old) transgenic heart sections. This value was markedly increased in ventricular sections of failing hearts from 9-month-old Tg mice in which TUNEL-positive cells appeared to be distributed toward the distal end of the myocardium. The number of TUNEL-positive nuclei varied between 85 and 185 per 105 cells among different heart sections from 9-month-old Tg mice (n = 12). There were an average of 130 TUNEL-positive nuclei per 105 cells (Fig. 1B), resulting in an almost 12-fold increase in the number of apoptotic cells in failing hearts compared with nonfailing hearts of the same age group. RNA Profiling: by RNase Protection Assay—RPA studies were performed using RNA from 4-week-old and 9-month-old WT and Tg mouse hearts (n = 5) with mouse multiprobes mAPO1, mAPO2, and mAPO3 (BD Biosciences Pharmingen; Fig. 2). Several apoptosis-regulating genes were up-regulated in the 9-month-old Tg hearts compared with either the age-matched WT or 4-week-old Tg hearts. Transcript levels of genes involved in the death receptor pathway were analyzed using a mAPO1 probe set (caspase-8, Fas, FADD, Fas-associated phosphatase, Fas-associated factor, TNF-α-related apoptosis-inducing ligand, TNF-α Rp55, TNF-α receptor-1-associated death domain protein, RIP, L32, and glyceraldehyde-3-phosphate dehydrogenase). Four genes were up-regulated in 9-month-old Tg mice compared with the age-matched wild-type controls or 4-week-old Tg mice: Fas >5-fold, FADD >4-fold, TNF-related apoptosis-inducing ligand >2-fold, and TNF-α receptor (Rp55) >4-fold (Fig. 2A, p < 0.01). Changes in the expression of mitochondrial genes in the Bcl2 family (mAPO2) were compared in 4-week-old and 9-month-old Tg mice and WT controls. Bfl-1, Bcl-w, Bax, and Bcl-X transcripts were up-regulated significantly (>4-fold) in 9-month-old Tg mice compared with the WT or 4-week-old Tg mice (Fig. 2B, p < 0.01). Bcl2 transcripts were up-regulated by 2-fold in failing heart samples. No significant difference was observed in levels of Bak and Bad transcripts during initiation and transition phases of the disease. Significant up-regulation of the initiator and effector caspases, namely, caspase-3, -7, -8, and -12, were observed in failing hearts compared with normal or 4-week-old hearts during initiation of hypertrophy (Fig. 2C). No significant differences were observed in caspase-6, -2, and -1 transcripts between Tg and WT hearts. On the other hand, a >2-fold increase in caspase-X and -11 transcripts was observed in failing hearts only. L32 and GAPDH genes were used as internal loading controls (Fig. 2, A–C). Changes in Protein Expression: Immunoblot and Immunohistochemistry—(i) Immunohistological data showed increased expression of Fas and FADD in left ventricle sections of 9-month-old Tg heart compared with 4-week-old Tg mice or age-matched WT (n = 5; Fig. 3A). Immunoblot analyses detected a 2.5-fold increase in FAS and TNF-α expression and a 2-fold increase in TNF-α RI in 9-month-old Tg compared with WT mice (Fig. 3B). No change was observed during the initiation phase of hypertrophy in 4-week-old Tg mice, compared with age-matched WT for Fas, although a slight induction in TNF-α protein was observed in 4-week-old Tg. (ii) Bcl2 and Bax proteins were also up-regulated in failing Tg hearts compared with nonfailing hearts (Fig. 3, C and D). Immunohistological as well as immunoblot data showed a 2.5-fold increase in Bax protein in failing hearts, whereas Bcl2 was up-regulated by almost 5-fold in 9-month-old transgenic hearts (n = 5), compared with either age-matched WT or 4-week-old Tg mice. Induced expression of Bax protein was observed by Western blot analysis during the initiation phase of hypertrophy (4-week-old Tg compared with age-matched WT), although induced Bax protein was not observed in these hearts by immunohistological staining. (iii) Fig. 4A shows immunohistochemistry using antibodies against active fragments of caspase-3, -7, and -8 (n = 5). Data showed no difference in expression levels of the caspases between 4-week-old WT and Tg. However, a significant increase in expression of active caspases was observed in 9-month-old Tg mice hearts compared with either age-matched WT or 4-week-old Tg mice. Cleavage of caspase-3 and -8 was further confirmed by immunoblot analyses. Active fragments (p17 and p32) were detected in the failing hearts (9-month-old Tg) only but not in nonfailing hearts from WT or 4-week-old Tg mice (Fig. 4B). (iv) Induction of infiltrating macrophages, CD13 and CD14, was detected by immunohistological staining in failing heart sections only (9-month-old Tg; Fig. 4C, n = 5). Infiltration of macrophages was not detected in any of the nonfailing heart samples (9-month-old WT or 4-week-old Tg). High numbers of infiltrating macrophages in failing hearts may be involved in phagocytosis of dead cells in the tissue. Biochemical Analysis of Activity of Caspases in Cellular Extracts of Tg and WT Hearts—Because immunoblot analyses confirmed the presence of active caspases in the Tg hearts, caspase activity was measured in hearts from both Tg and WT mice using specific fluorogenic substrates (ApoAlert; Clontech, Palo Alto, CA) for caspase-3, -8, and -9. No difference was observed in caspase-3 activity levels between heart samples of 4-week-old WT and Tg mice. A slight but nonsignificant increase in caspase activity was also detected in 4-week-old Tg hearts compared with age-matched wild-type hearts, especially for caspase-8 and -9. Activity of the executor caspase, caspase-3 was increased 92.5% in hearts from 9-month-old Tg mice, whereas the activities of the initiator caspases, caspase-8 and -9, were increased by 59 and 79%, respectively, compared with age-matched WT samples or 4-week-old Tg mice hearts (n = 5; p < 0.01; Fig. 5). Despite significant cell death in Tg mice hearts, the heart weight:body weight ratio was >12, implicating either increased cell division (mitosis), cell enlargement, or both. To analyze how the transition from hypertrophy to heart failure in 9-month-old Tg hearts affects the expression of cell cycle regulatory genes, both RPA and immunoblot analyses were performed. RPA Analysis Using Cyclin Multiprobes—RPA studies using the mouse mCdk3b multiprobe of cell cycle regulatory genes showed significant up-regulation of different cyclin transcripts in the hearts of Tg mice. Maximum induction was observed for cyclin B1 and B2 transcripts (∼4-fold) in failing heart. The cyclin D family (D1, D2, and D3) was up-regulated by 2-fold, cyclin A2 by 3-fold, and cyclin C by 1.2-fold in hearts of 9-month-old Tg mice, compared with 9-month-old WT (n = 5; Fig. 6A). Some of the cyclin genes (cyclin C, D2, and D3) were induced in the 4-week-old Tg heart samples (although to a much lesser degree than 9-month-old Tg) when compared with age-matched WT samples. Immunoblot Analyses—Changes in the cell cycle regulator genes (cyclin A, B1, D1, and D3) between WT and Tg heart samples were further confirmed at the protein level. Western blot analysis showed maximum up-regulation of cyclin A as >4-fold, cyclin B1 as >3-fold, cyclin D1 as >5-fold, and cyclin D3 as ≥2-fold (n = 5; Fig. 6B) in 9-month-old Tg animals compared with age-matched WT or 4-week-old Tg hearts. Some induction (although nonsignficant) in the protein level of these cyclins was also observed in Tg mice at as early as 4 weeks, during initiation of hypertrophy, when compared with their age-matched WT. Changes in Cyclin-dependent Kinases—Cdks regulate the action of the cyclin genes. Cdk-1 (Cdc2), Cdk-2, and Cdk-4 bind to cyclin A, B, and D, respectively. Once it was determined that cyclin genes were up-regulated in hypertrophic heart, immunoblot analyses were done to analyze the protein expression of different Cdks. However, only Cdk-1 (not Cdk-2 and -4) showed a sig
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