Xilin Long

p53 and the hypoxia-induced apoptosis of cultured neonatal rat cardiac myocytes.

Myocyte cell loss is a prominent and important pathogenic feature of cardiac ischemia. We have used cultured neonatal rat cardiac myocytes exposed to prolonged hypoxia as an experimental system to identify critical factors involved in cardiomyocyte death. Exposure of myocytes to hypoxia for 48 h resulted in intranucleosomal cleavage of genomic DNA characteristic of apoptosis and was accompanied by increased p53 transactivating activity and protein accumulation. Expression of p21/WAF-1/CIP-1, a well-characterized target of p53 transactivation, also increased in response to hypoxia. Hypoxia did not cause DNA laddering or cell loss in cardiac fibroblasts. To determine whether the increase in p53 expression in myocytes was sufficient to induce apoptosis, normoxic cultures were infected with a replication-defective adenovirus expressing wild-type human p53 (AdCMV.p53). Infected cells expressed high intracellular levels of p53 protein and exhibited the morphological changes and genomic DNA fragmentation characteristic of apoptosis. In contrast, no genomic DNA fragmentation was observed in myocytes infected with the control virus lacking an insert (AdCMV.null) or in cardiac fibroblasts infected with AdCMV.p53. These results suggest that the intracellular signaling pathways activated by p53 might play a critical role in the regulation of hypoxia-induced apoptosis of cardiomyocytes.

Rapamycin Inhibits α1-Adrenergic Receptor–Stimulated Cardiac Myocyte Hypertrophy but Not Activation of Hypertrophy-Associated Genes Evidence for Involvement of p70 S6 Kinase

The 70-kD S6 kinase (p70S6K) has been implicated in the regulation of protein synthesis in many cell types and in the angiotensin II-stimulated hypertrophy of cardiac myocytes. Our purpose was to determine whether p70S6K plays a role in cardiomyocyte hypertrophy induced by the alpha 1-adrenergic receptor (alpha 1-AR) agonist phenylephrine (PE). PE stimulated the activity of p70S6K > 3-fold, and this increase was blocked by rapamycin, an immunosuppressant macrolide that selectively inhibits p70S6K. When administered for 3 days, PE stimulated a 30% increase in total protein content, a 2-fold increase in the incorporation of [14C]phenylalanine (14C-Phe) into protein, and a 50% increase in two-dimensional myocyte area. Rapamycin pretreatment (> or = 500 pg/mL) significantly inhibited each of these PE-stimulated changes. Two days of PE treatment resulted in a 1.6-fold increase in total RNA yield per dish, a 2-fold increase in incorporation of [14C]uridine into myocyte RNA, and increases in relative mRNA levels of the hypertrophy-associated atrial natriuretic factor (ANF, 2.1-fold) and skeletal alpha-actin (SK, 2.2-fold) genes. Although rapamycin abolished the PE-stimulated increases in total RNA and incorporation of [14C]uridine, it had no effect on the induction of the ANF and SK genes. LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3-K) activity, inhibited PE-stimulated increases in p70S6K activity and the incorporation of labeled precursors into myocyte protein and RNA. These results demonstrate that p70S6K is activated by the hypertrophic agent PE and that a PI3-K or PI3-K-like activity is required for p70S6K activation and myocyte hypertrophy. The data suggest that p70S6K activation may be required for PE-stimulated hypertrophy of cardiac myocytes. Our results demonstrate that intracellular signaling pathways responsible for transcriptional and translational responses diverge early after alpha 1-AR stimulation in cardiac myocytes.

Enhanced expression of p53 and apoptosis induced by blockade of the vacuolar proton ATPase in cardiomyocytes.

Activation of the vacuolar proton ATPase (VPATPase) has been implicated in the prevention of apoptosis in neutrophils and adult cardiac myocytes. To determine the role of the VPATPase in apoptosis of cardiac myocytes, we used a potent and specific inhibitor of the VPATPase, bafilomycin A1. Bafilomycin A1 alone caused increased DNA laddering of genomic DNA and increased nuclear staining for fragmented DNA in neonatal cardiomyocyte apoptosis in a dose- and time-dependent manner. Intracellular acidification in cardiac myocytes was also observed after 18 h of bafilomycin A1 treatment. Accordingly, bafilomycin A1-treated myocytes also showed increased accumulation of p53 protein and p53-dependent transactivation of gene expression, including a persistent upregulation of p21/wild-type p53 activated fragment 1/cyclin kinase inhibitor protein-1 mRNA. The bafilomycin A1-induced increase in p53 protein levels was accompanied by a marked increase in p53 mRNA accumulation. In contrast, cardiac fibroblasts treated with bafilomycin A1 showed no change in p53 protein expression or pHi and did not undergo apoptosis even after 24 h of treatment. Our data suggest that blockade of the VPATPase induces apoptotic cell death of cardiac myocytes and that this may occur through a p53-mediated apoptotic pathway.

Extracellular ATP Inhibits Adrenergic Agonist–Induced Hypertrophy of Neonatal Cardiac Myocytes

We have previously shown that extracellular ATP, like norepinephrine (NE) and many other hypertrophy-inducing agents, increases expression of the immediate-early genes c-fos and junB in cultured neonatal cardiac myocytes but that the intracellular signaling pathways activated by ATP and responsible for these changes differ from those stimulated by NE. Furthermore, whereas NE increases incorporation of [14C]phenylalanine (14C-Phe) and cell size in neonatal cardiomyocytes, ATP does not. Since ATP is coreleased with NE from sympathetic nerve endings in the heart, we investigated whether ATP could modulate cardiac hypertrophy induced by adrenergic agonists, such as NE. We report in the present study that extracellular ATP inhibited the increase in incorporation of 14C-Phe into cellular protein and the increase in cell size in neonatal rat cardiac myocytes that was induced by NE, phenylephrine (PE), basic fibroblast growth factor, or endothelin-1. This inhibition was dose dependent, occurred predominantly through P2 purinergic receptors, and was observed even when cells were treated with ATP for as little as 1 hour before the addition of the hypertrophy-inducing agent. ATP also selectively affected changes in gene expression associated with hypertrophy. It prevented PE-stimulated increases in atrial natriuretic factor and myosin light chain-2 mRNA levels, while appearing to augment basal and PE-stimulated skeletal alpha-actin mRNA levels. ATP alone increased sarcoplasmic reticulum Ca2+-ATPase mRNA levels but had no effect when added with PE. ATP did not significantly affect the level of the constitutively expressed mRNA for GAPDH. Neither the PE-stimulated increase in immediate-early gene expression nor the initial induction of mitogen-activated protein kinase activity by PE was inhibited by ATP. These results demonstrate that extracellular ATP can inhibit hypertrophic growth of neonatal cardiac myocytes and differentially alter the changes in gene expression that accompany hypertrophy.

Stimulation of P2Y receptors activates c-fos gene expression and inhibits DNA synthesis in cultured cardiac fibroblasts

OBJECTIVES The aims of this study were to determine (1) whether neonatal rat cardiac fibroblasts (CAFB) express P2Y receptors; (2) whether CAFB respond to extracellular ATP by inducing expression of c-fos mRNA; and (3) whether extracellular ATP modulates norepinephrine (NE)-stimulated cell growth in CAFB. METHODS Expression of P2Y1 and P2Y2 receptors and induction of c-fos were examined by Northern blot analysis. CAFB growth was assessed by measuring [3H]thymidine incorporation and DNA content. P2Y receptor pharmacology was studied using various ATP analogues. RESULTS Northern blot analysis of polyA enriched RNA confirmed that at least 2 subtypes of P2Y receptors (P2Y1 and P2Y2) are expressed in cultured CAFB. Extracellular ATP induced the expression of c-fos mRNA through a pathway that was sensitive to inhibitors of protein kinase C (PKC), but not to inhibitors of intracellular Ca2+ signaling. Extracellular ATP inhibited the NE-stimulated increases in DNA content and in [3H]thymidine incorporation into DNA. Whereas the potency order for stimulation of c-fos expression was ATP = UTP > ADP > adenosine, the potency order to inhibit the NE-induced increase of [3H]thymidine incorporation into DNA was ATP > ADP > UTP > adenosine. CONCLUSIONS These data demonstrate that CAFB express both P2Y1 and P2Y2 receptor mRNA and that CAFB respond to P2Y receptor stimulation by induction of c-fos and inhibition of DNA synthesis. These findings suggest that the effects of ATP on [3H]thymidine incorporation into DNA and on expression of c-fos mRNA are exerted via distinct P2Y receptor subtypes.

The Role of the Tumor Suppressor Gene p53 in Cardiomyocyte Apoptosis

The possibility that a significant fraction of cardiac myocyte loss in various disease states occurs through apoptosis has elicited considerable attention in recent years. Evidence from human studies as well as in vitro and animal models of disease has shown that cardiac myocyte apoptosis can be induced by a variety of stimuli and in a number of disease states, including hypoxia, ischemia–reperfusion, myocardial infarction, mechanical stretch, aortic constriction, and heart failure. Because adult cardiac myocytes are terminally differentiated cells, the effects of such loss can never be fully compensated. Interest in cardiomyocyte apoptosis has been fueled by the possibility that once the proximal and distal signals were defined that initiate this pathway of cell removal, it would be possible to develop stategies to selectively interfere with such signaling and prevent the loss of cardiac function. This article examines the evidence for possible proximal stimuli of apoptosis in the heart, including ligand-dependent activation of the membrane receptors for Fas ligand and tumor necrosis factor-α, and, in particular, activation of the tumor suppressor gene p53. It relates what is known about the mechanism by which these stimuli in other cells induce apoptosis and discusses possible strategies for inhibiting apoptosis in the heart.

Cardiac myocytes and fibroblasts exhibit differential sensitivity to apoptosis-inducing stimuli

Cell death in the cardiovascular system by apoptosis has received considerable attention in recent years. Cardiac and vascular cell death by apoptosis is not only a feature of embryologic and neonatal development, but can be elicited by a variety of diverse stimuli (Table I) and has been linked to virtually every major cardiovascular disease or disorder (Table II). In many of these disorders, particularly those leading to chronic heart failure, myocyte cell death/loss is usually accompanied by an increase in fibrous tissue content (1). Excessive fibrosis in the presence of myocyte loss has been advocated as a basis for impaired myocardial function in these disease states. The spontaneous hypertensive rat presents a clear example of the imbalance that exists as heart failure evolves (2). In this experimental model, increased cardiac myocyte apoptosis (Figure 1C) can be linked to the reduction in myocyte fractional mass (Figure 1A) as the hearts progress to failure. During this same time, there is a substantial increase in the fibrotic fractional area of the heart (Figure IB).

Expression Of aquaporin-1 (AQP-1) in rat heart

Abstract Background. AQP-1, a channel-forming integral membrane protein of 28 kDa prevalent in red blood cells and renal proximal tubules, was recently shown to be expressed in rat heart. Aims : Our purpose was to charaterise the experssion of the AQO-1 gene in rat heart with respect to cell type, developmental stage and pathophysiological condition. Methods: To determine in which heart cell type the water channel was expressed, we measured AQP-1 mRNA and protein levels and immunolocalised protein in freshly isolated myocytes from adult rats and cultured myocytes and fibroblasts from neonatal rats. Results: Northern blot analysis showed that AQP-1 mRNA is expressed in adult cardiac myocytes, neonatal myocytes, and neonatal cardiac fibroblasts of rats at levels nearly as high as those observed in rat kidney. Western blot analysis, however, detected AQP-1 protein only in purified adult and neonatal cardiac myocytes at levels markedly less than kidney. Immunohistochemistry and confocal imaging localised AQP-1 protein to the sarcolemmal membrane of myocytes. Since the expression of other membrane-spanning proteins is altered during hypertrophy, we determined the level of AQP-1 mRNA in hearts of aortic-constricted (AC) rats. The level of AQP-1 mRNA decreased progressively after AC, and was 42% less than the level in left ventricles (LVs) of sham-operated control rats after 3 days of AC. Conclusions: These data indicate that AQP-1 is expressed in cardiac myocytes of adult and neonatal rats, and its expression is modulated in the rat heart during pressure-overload hypertrophy.