Myocyte cell death in the diseased heart.
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Apoptosis↗
Biomedical subjects
P Anversa
Publications and source records attributed to P Anversa.
Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.
Physical forces activate apoptosis and gene expression, but the mechanism is unknown. For this purpose, adult myocytes were stretched in an equibiaxial stretch apparatus and the magnitude of cell death was examined 4 and 24 h later. The possibility of stretch-mediated activation of p53 and p53-dependent genes was evaluated at 30 min, 2, 4, 8, and 24 h. Myocyte apoptosis increased by 4.4- and 7.6-fold at 4 and 24 h after stretch. p53 binding to the promoter of angiotensinogen, AT1 receptor, and Bax also increased. Expression of angiotensinogen, AT1 receptor, p53, and Bax increased and Bcl-2 decreased in stretched myocytes. The changes in AT1 receptor, p53, Bax, and Bcl-2 became more apparent with the duration of stretch. Angiotensin II concentration in the medium increased at 10 min, reaching maximal levels at 1 and 20 h. The AT1 blocker, losartan, abolished apoptosis in stretched myocytes. Myocyte volume was not influenced by stretch. In conclusion, stretch-mediated release of angiotensin II is coupled with apoptosis and the activation of p53 which may be responsible for the prolonged upregulation of the local renin-angiotensin system and the increased susceptibility of myocytes to undergo apoptosis.
Angiotensin II↗
Effects of constitutive overexpression of insulin-like growth factor-1 on the mechanical characteristics and molecular properties of ventricular myocytes.
Recently, insulin-like growth factor-1 (IGF-1) has been claimed to positively influence the cardiac performance of the decompensated heart. On this basis, the effects of constitutive overexpression of IGF-1 on the mechanical behavior of myocytes were examined in transgenic mice in which the cDNA for the human IGF-1B was placed under the control of a rat alpha-myosin heavy chain promoter. In mice heterozygous for the transgene and in nontransgenic littermates at 2.5 months of age, the alterations in Ca2+ sensitivity of tension development, unloaded shortening velocity, and sarcomere compliance were measured in skinned myocytes. The quantities and state of phosphorylation of myofilament proteins in these enzymatically dissociated ventricular myocytes were also examined. The overexpression of IGF-1 was characterized by a nearly 15% reduction in myofilament isometric tension at submaximum Ca2+ levels in the physiological range, whereas developed tension at maximum activation was unchanged. In contrast, unloaded velocity of shortening was increased 39% in myocytes from transgenic mice. Moreover, resting tension in these cells was reduced by 24% to 33%. Myocytes from nontransgenic mice pretreated with IGF-1 failed to reveal changes in myofilament Ca2+ sensitivity and unloaded velocity of shortening. The quantities of C protein, troponin I, and myosin light chain-2 were comparable in transgenic and nontransgenic mice, but their endogenous state of phosphorylation increased 117%, 100%, and 100%, respectively. Troponin T content was not altered, and myosin isozymes were essentially 100% V1 in both groups of mice. In conclusion, constitutive overexpression of IGF-1 may influence positively the performance of myocytes by enhancing shortening velocity and cellular compliance.
Actin Cytoskeleton↗
Pacing-induced heart failure in dogs enhances the expression of p53 and p53-dependent genes in ventricular myocytes.
BACKGROUND: Rapid ventricular pacing in dogs is characterized by a dilated myopathy in which myocyte cell death by apoptosis may play a significant role in the impairment of cardiac pump function. However, the molecular mechanisms implicated in the modulation of programmed cell death under this setting remain to be identified. Moreover, questions have been raised on the specificity and sensitivity of the histochemical detection of DNA strand breaks in nuclei by the terminal deoxynucleotidyl transferase (TdT) reaction. METHODS AND RESULTS: Changes in the expression of Bcl-2 and Bax and their transcriptional regulator, p53, were determined by Western blot analysis in myocytes isolated from dogs affected by pacing-induced heart failure. A mobility shift assay for p53 binding activity was also performed. In addition, apoptosis was measured by confocal microscopy, which allowed the simultaneous detection of chromatin alterations and DNA damage. p53 DNA binding activity to the bax promoter was increased in nuclear extracts from myocytes obtained from failing hearts, and this response was associated with enhanced expression of Bax protein, 52%, and attenuation of Bcl-2, -92%. Immunolabeling of p53 in myocyte nuclei, measured by confocal microscopy, was 100% higher in cells from paced hearts. The combination of the TdT assay and confocal microscopy demonstrated that 20 myocyte nuclei per 10(6) were undergoing apoptosis in control myocardium and 4000 per l0(6) after pacing. Moreover, DNA laddering was shown in myocytes by agarose gel electrophoresis of DNA fragments. CONCLUSIONS: The activation of p53 and p53-dependent genes may be critical in the modulation of myocyte apoptosis in pacing-induced heart failure.
Animals↗
Intracellular calcium, DNase activity and myocyte apoptosis in aging Fischer 344 rats.
Myocyte apoptosis increases with age in Fischer 344 rats, but the multiple molecular events implicated in this phenomenon remain to be identified. Several defects involving Ca2+ homeostasis, pH, and the expression of p53 and genes of the Bcl-2 protein family may contribute to the activation of myocyte death. Therefore, changes in intracellular pH, cytosolic Ca2+, DNase I and DNase II were measured in myocytes isolated by enzymatic digestion from rats of different ages. Moreover, the expression of p53, Bcl-2 and Bax in these cells was determined. Measurements of intracellular pH by BCECF fluorescence at 3, 12 and 24 months showed that this parameter did not change with age: 3 months, 7.20+/-0.05; 12 months, 7.21+/-0.07; 24 months, 7.18+/-0.09. In contrast, diastolic Ca2+ determined by the Fura 2-AM method increased progressively from 99.8+/-1.9 nm at 3 months to 136.3+/-9.6 nm at 24 months (P<0.001). Concurrently, DNase I activity evaluated by plasmid digestion assay in myocytes increased 3.2-fold from 3 to 24 months (P<0.02). Conversely, pH-dependent-DNase II remained essentially constant with age. Western blotting performed on ventricular myocytes did not detect significant changes in p53, Bax and Bcl-2 proteins with age. Similarly, immunocytochemically, the fraction of myocytes labeled by p53, Bax and Bcl-2 did not change from 3 to 24 months. In conclusion, myocyte aging is characterized by an increase in diastolic calcium which may activate DNase I triggering apoptosis, independently from the expression of p53, Bax and Bcl-2 in the cells.
Aging↗
Apoptosis and myocardial infarction.
Myocardial infarction was produced in rats and the contribution of apoptotic and necrotic myocyte cell death was measured quantitatively. Myocyte cell death by apoptosis involved 2.8 million cells at 2 hours after coronary artery occlusion and necrosis only 90,000 cells. Myocyte apoptosis continued to represent the major form of cell death, affecting 6.6 million cells at 4.5 hours, whereas myocyte necrosis peaked at 1 day, including 1.1 million cells. Apoptotic myocyte cell death was also present in the surviving portion of the wall adjacent to and remote from the infarcted myocardium where it peaked at 1-2 days. At this interval, 700/10(6) and 110/10(6) myocyte nuclei were undergoing apoptosis in the non-infarcted tissue bordering on and away from the ischemic area, respectively. Myocyte necrosis was absent in the viable myocardium after infarction. Since mechanical forces produced by pathologic loads may activate apoptosis, papillary muscles were exposed to high levels of resting tension in vitro and the magnitude of cell death in these samples was determined. Overstretching resulted in a 21-fold increase in apoptotic myocyte cell death which was coupled with the formation of reactive oxygen species, side-to-side slippage of myocytes, and depressed tension generation of the myocardium. In conclusion, apoptotic myocyte cell death plays a major role in ventricular remodeling after infarction, but whether physical forces, oxidant stress, architectural rearrangement of myocytes, and impaired force development of the myocardium in vivo are causaly related requires further investigation.
Animals↗
Myocyte apoptosis and heart failure.
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Apoptosis↗
Myocyte death and growth in the failing heart.
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Animals↗
Overexpression of insulin-like growth factor-1 in mice protects from myocyte death after infarction, attenuating ventricular dilation, wall stress, and cardiac hypertrophy.
To determine whether IGF-1 opposes the stimulation of myocyte death in the surviving myocardium after infarction, transgenic mice overexpressing human IGF-1B in myocytes (FVB.Igf+/-) and wild-type littermates at 1.5 and 2.5 mo of age were subjected to coronary ligation and killed 7 d later. Myocardial infarction involved an average 50% of the left ventricle, and produced cardiac failure. In the region proximate to infarction, myocyte apoptosis increased 4. 2-fold and 2.1-fold in nontransgenics at 1.5 and 2.5 mo, respectively. Corresponding increases in myocyte necrosis were 1. 8-fold and 1.6-fold. In contrast, apoptotic and necrotic myocyte death did not increase in FVB.Igf+/- mice at either age after infarction. In 2.5-mo-old infarcted nontransgenics, functional impairment was associated with a 29% decrease in wall thickness, 43% increase in chamber diameter, and a 131% expansion in chamber volume. Conversely, the changes in wall thickness, chamber diameter, and cavitary volume were 41, 58, and 48% smaller in infarcted FVB.Igf+/- than in nontransgenics. The differential response to infarction of FVB.Igf+/- mice resulted in an attenuated increase in diastolic wall stress, cardiac weight, and left and right ventricular weight-to-body wt ratios. In conclusion, constitutive overexpression of IGF-1 prevented activation of cell death in the viable myocardium after infarction, limiting ventricular dilation, myocardial loading, and cardiac hypertrophy.
Animals↗
Insulin-like growth factor-1 receptor and its ligand regulate the reentry of adult ventricular myocytes into the cell cycle.
To determine whether insulin-like growth factor-1 (IGF-1) stimulation in vitro of ventricular myocytes isolated from infarcted hearts is characterized by the reentry of cells into the cell cycle, the expression and kinase activity of cyclins E, A, and B and DNA synthesis were evaluated 5 days after coronary artery occlusion and 24 and 48 h following the addition of IGF-1. Myocytes surviving an acute myocardial infarction were employed because of their increase in surface insulin-like growth factor-1 receptors (IGF-1R). Western blot analysis documented that IGF-1 resulted in an upregulation of cyclins D1, E, A, and B in viable postinfarcted myocytes. Cyclin E- and A-associated histone H1 kinase activity and cyclin D1-associated retinoblastoma protein-associated kinase activity also increased, but cyclin B kinase activity was not enhanced by IGF-1. These changes in cyclins and kinase activities were characterized by a significant increase in the number of cells labeled by bromodeoxyuridine, from approximately 630/10(6) to nearly 9, 000/10(6) myocytes. This latter value was reduced by more than 50% by antisense oligodeoxynucleotide to IGF-1R mRNA. However, IGF-1 stimulation did not induce nuclear mitotic division and cytokinesis. In conclusion, the growth-promoting effect of IGF-1 on adult myocytes is regulated by the density of IGF-1R, which conditions the activation of the replicatory machinery of the cells. The failure of IGF-1 to enhance cyclin B kinase activity may be responsible for a block in the cell cycle and the inability of myocytes to progress through the M phase and subsequently divide.
Animals↗
p53 Induces myocyte apoptosis via the activation of the renin-angiotensin system.
The mechanism by which p53 activates apoptosis in various cell systems is unknown. In the absence of an external death stimulus, p53 and p53-dependent genes, bcl-2 and bax, cannot trigger apoptosis. However, p53 may enhance not only transcription of bax and repress bcl-2, but also may upregulate the local renin-angiotensin system, inducing the formation and secretion of angiotensin II from the cells. To test this hypothesis, adult rat ventricular myocytes were infected with AdCMV.p53, which resulted in downregulation of Bcl-2, upregulation of Bax, and death of 34% of the cells. Gel retardation assays demonstrated p53 binding in the promoters of angiotensinogen and angiotensin II AT1 receptor subtype. Angiotensinogen and AT1 mRNAs increased in AdCMV.p53 cells and this phenomenon was associated with a 14-fold increase in the secretion of angiotensin II. The AT1 receptor blocker losartan and angiotensin II antibody prevented p53-induced apoptosis. Thus, p53 enhances the myocyte renin-angiotensin-system and decreases the Bcl-2/Bax ratio in the cells, triggering apoptosis. The identification of this new pathway in p53-mediated apoptosis may be critical in the alterations of myocardial function in the pathologic heart.
Adenoviridae↗
Apoptosis in the failing human heart.
BACKGROUND: Loss of myocytes is an important mechanism in the development of cardiac failure of either ischemic or nonischemic origin. However, whether programmed cell death (apoptosis) is implicated in the terminal stages of heart failure is not known. We therefore studied the magnitude of myocyte apoptosis in patients with intractable congestive heart failure. METHODS: Myocardial samples were obtained from the hearts of 36 patients who underwent cardiac transplantation and from the hearts of 3 patients who died soon after myocardial infarction. Samples from 11 normal hearts were used as controls. Apoptosis was evaluated histochemically, biochemically, and by a combination of histochemical analysis and confocal microscopy. The expression of two proto-oncogenes that influence apoptosis, BCL2 and BAX, was also determined. RESULTS: Heart failure was characterized morphologically by a 232-fold increase in myocyte apoptosis and biochemically by DNA laddering (an indicator of apoptosis). The histochemical demonstration of DNA-strand breaks in myocyte nuclei was coupled with the documentation of chromatin condensation and fragmentation by confocal microscopy. All these findings reflect apoptosis of myocytes. The percentage of myocytes labeled with BCL2 (which protects cells against apoptosis) was 1.8 times as high in the hearts of patients with cardiac failure as in the normal hearts, whereas labeling with BAX (which promotes apoptosis) remained constant. The near doubling of the expression of BCL2 in the cardiac tissue of patients with heart failure was confirmed by Western blotting. CONCLUSIONS: Programmed death of myocytes occurs in the decompensated human heart in spite of the enhanced expression of BCL2; this phenomenon may contribute to the progression of cardiac dysfunction.
Apoptosis↗
Angiotensin II activates programmed myocyte cell death in vitro.
To determine whether angiotensin II (Ang II) can induce apoptosis of neonatal ventricular myocytes, these cells were exposed to 10(-9) M Ang II for 24 h in vitro and the effects of this intervention on programmed myocyte cell death were examined by the terminal deoxynucleotidyl transferase assay and DNA gel electrophoresis. Ang II resulted morphologically in a 2.5-fold increase in the percentage of myocytes with double strand cleavage of the DNA and biochemically in the formation of DNA fragments equal in size to mono- and oligonucleosomes. Moreover, Ang II stimulation was characterized by a 37% increase in resting level of intracellular calcium and the activation of calcium-dependent endogenous endonuclease. In contrast, pH-dependent endogenous endonuclease was not enhanced by the addition of Ang II. Ang II-induced DNA damage was inhibited by the AT1 receptor antagonist, losartan. Similarly, the calcium chelator, BAPTA-AM, prevented Ang II-mediated cell death. Conversely, the calcium ionophore, A23187, triggered programmed cell death. Finally, the selective AT2 receptor subtype blocker, PD123319, failed to reduce myocyte apoptosis. In conclusion, ligand binding of AT1 receptors may initiate programmed myocyte cell death via an elevation in cytosolic calcium and the stimulation of calcium-dependent endogenous endonuclease.
Angiotensin II↗
Angiotensin II induces apoptosis of adult ventricular myocytes in vitro.
To determine whether angiotensin II (Ang II) activates the suicide program of myocytes, primary cultures of adult rat ventricular myocytes were exposed to 10(-9) M of Ang II, for 24 h. Ang II resulted in a five-fold increase in programmed myocyte cell death (PMCD) documented by the terminal deoxynucleotidyl transferase assay and confirmed by DNA agarose gel electrophoresis. Ang II stimulation was associated with translocation of the epsilon and delta isoforms of protein kinase C (PKC) which was coupled with an increase in cytosolic Ca2+ in the cells. The PKC inhibitor chelerythrine abolished Ang II-mediated increases in cytosolic Ca2+ and PMCD. Similarly, pretreatment of cells with the intracellular Ca2+ chelator BAPTA/AM inhibited the formation of DNA strand breaks. Conversely, the Ca2+ ionophore A23187 markedly increased PMCD. Finally, the AT1 receptor antagonist, losartan, completely blocked Ang II-induced PMCD, whereas the AT2 receptor antagonist, PD123319, did not attenuate this phenomenon. In conclusion, ligand binding of AT1 receptors on myocytes triggers PMCD by a mechanism involving PKC-mediated increases in cytosolic calcium, which result in internucleosomal DNA fragmentation.
Alkaloids↗
Proliferating cell nuclear antigen (PCNA), DNA synthesis and mitosis in myocytes following cardiac transplantation in man.
Cardiac transplantation is characterized by rejection, myocyte loss, interstitial and replacement fibrosis, and loading abnormalities. These modifications contribute to enhance mural and muscle cell stress, activating reactive growth processes in myocytes and interstitial cells. However, it is unknown whether cell cycle related gene product, such as PCNA, and DNA synthesis are stimulated under these conditions. Therefore, 62 endomyocardial biopsies obtained from 17 patients who underwent cardiac transplantation were examined for the immunocytochemical detection of PCNA protein in myocyte and non-myocyte nuclei. In addition, tissue samples were labeled in vitro with bromodeoxyuridine (BrdU) to document ongoing DNA synthesis. The presence of mitotic images in myocytes and non myocytes were also examined. Biopsies were collected from 1-768 days after surgery. Histologic examination of tissue sections documented that PCNA labeling involved nearly 30% of myocyte nuclei in all patients. Similar percentages of PCNA labeling were detected in interstitial cells, lymphocytes and mononuclear infiltrates. DNA synthesis in myocytes and connective tissue cells was observed in nine and 14 subjects, respectively. BrdU positive lymphocytes and mononuclear infiltrates in 13 cases. Three mitotic figures in myocyte nuclei were identified. PCNA, BrdU labeling and mitosis were not detected in eight myocardial samples obtained from control hearts. These results suggest that the evolution of the transplanted heart involves the expression of a gene which is implicated in DNA replication. The presence of ongoing DNA synthesis and mitosis support the notion that proliferation of myocytes and non muscle cells may be a component of ventricular remodeling after cardiac transplantation.
Bromodeoxyuridine↗
Coronary artery stenosis in rats affects beta-adrenergic receptor signaling in myocytes.
OBJECTIVE: The purpose of this study was to determine whether the early chronic ischemic cardiomyopathy produced by non-occlusive coronary artery constriction was characterized by alterations in the regulation of beta-adrenoreceptor (beta-AR) signaling. METHODS: Coronary artery narrowing was surgically induced in rats and the animals sacrificed at 7 and 14 days. The changes in the biochemical properties of the multiple components of the beta-AR pathway were examined in enzymatically dissociated myocytes. RESULTS: Coronary stenosis, involving an average 55% reduction in luminal diameter, was associated with left ventricular failure and right ventricular dysfunction at both time intervals. A decrease in the quantity of beta-AR was detected at 7 days and preceded the loss of high-affinity binding sites. This regulatory modification was characterized by a reduction in beta 1 and beta 2 receptors and a shift in the isoproterenol dose response curve indicating a functional correlation between the decrease in beta-AR and attenuated inotropic support of the myocardium. The percentage of beta-AR binding agonist with high affinity decreased significantly at 14 days along with a further reduction in the density of beta 1 and beta 2 receptors. Reconstitution studies with cyc S49 lymphoma cells did not detect an impairment of Gs alpha functional activity, but the quantity of Gi alpha was increased at both intervals. Finally, activation of the catalytic unit of adenylyl cyclase by forskolin and GTP was not altered by coronary stenosis, however, basal cyclic AMP in myocytes was depressed at 14 days. CONCLUSIONS: Coronary stenosis induces distinct and progressive modifications in the beta-AR signaling cascade which may contribute to the impaired ventricular performance in this model of myocardial ischemia.
Adrenergic beta-Agonists↗
Myocyte cell death and ventricular remodeling.
The recognition that cell death in the myocardium is not only necrotic in nature but is also mediated by activation of the suicide program of myocytes has raised several questions concerning the magnitude of this phenomenon, and whether these two distinct forms of cell death are disease-dependent or coexist in the pathologic heart. Additionally, the times required for the completion of apoptotic and necrotic myocyte cell death are unknown, making the analysis of their respective rates in the myocardium impossible at present. The documentation that mechanical forces in vitro, mimicking diastolic Laplace overloading in vivo, can transmit a death signal to myocytes suggests that programmed cell death may be triggered in the stressed myocardium independently from the etiology of the overload. Because increasing pressure or volume loads, or both, in the failing heart induce myocyte hypertrophy and proliferation, a challenging question is whether the induction of genes regulating these cellular growth processes may activate programmed cell death as well. Finally, the identification of the mechanisms responsible for the translation of a diffuse environmental condition into a death signal in a limited number of cells scattered across the ventricular wall is a major challenge of future research.
Animals↗
Myocardial infarction alters myofilament calcium sensitivity and mechanical behavior of myocytes.
To determine whether myocardial infarction leads to alterations in myofilament isometric tension as a function of Ca2+ concentration, unloaded shortening velocity, and sarcomere compliance, these properties were examined in skinned myocytes 7 days after coronary artery occlusion. Changes in myofilament proteins were also evaluated Myocardial infarction was characterized by a 10-15% reduction in myofilament isometric tension at submaximum Ca2+ levels in the physiological range. However, developed tension at maximum activation was unaltered. Conversely, unloaded shortening velocity was decreased by 31% in the remaining viable cells, whereas resting tension was increased by 30-40%. The regulatory protein troponin I content was reduced, but phosphorylation of troponin I and troponin T was increased. Myosin isoenzymes and troponin T contents were not altered. In conclusion, molecular responses occurred acutely after myocardial infarction, and these adaptations may depress the mechanical behavior of the unaffected cells, contributing to acute impairment in global cardiac pump function beyond that resulting from myocyte loss.
Actin Cytoskeleton↗