ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

To study the physiological effect of the overexpression of myocardial Gsalpha (protein levels increased by approximately threefold in transgenic mice), we examined the responsiveness to sympathomimetic amines by echocardiography (9 MHz) in five transgenic mice and five control mice (both 10.3 plus minus 0.2 months old). Myocardial contractility in transgenic mice, as assessed by left ventricular (LV) fractional shortening (LVFS) and LV ejection fraction (LVEF), was not different from that of control mice at baseline (LVFS, 40 plus minus 3% versus 36 plus minus 2%; LVEF, 78 plus minus 3% versus 74 plus minus 3%). LVFS and LVEF values in transgenic mice during isoproterenol (ISO, 0.02 mu g/kg per minute) infusion were higher than the values in control mice (LVFS, 68 plus minus 4% versus 48 plus minus 3%; LVEF, 96 plus minus 1% versus 86 plus minus 3%; P less than .05). Norepinephrine (NE, 0.2 mu g/kg per minute) infusion also increased LVFS and LVEF in transgenic mice more than in control mice (LVFS, 59 plus minus 4% versus 47 plus minus 3%; LVEF, 93 plus minus 2% versus 85 plus minus 3%; P less than .05). Heart rates of transgenic mice were higher than those of control mice during ISO and NE infusion. In three transgenic mice with heart rates held constant, LV dP/dt rose by 33 plus minus 2% with ISO (0.02 mu g/kg per minute) and by only 13 plus minus 2% in three wild-type control mice (P less than .01). NE (0.1 mu g/kg per minute) also induced a greater effect on LV dP/dt in the three transgenic mice with heart rates held constant compared with three wild-type control mice (65 plus minus 8% versus 28 plus minus 4%, P less than .05). Pathological and histological analyses of older transgenic mouse hearts (16.0 plus minus 0.8 months old) revealed hypertrophy, degeneration, atrophy of cells, and replacement fibrosis reflected by significant increases in collagen volume in the subendocardium (5.2 plus minus 1.4% versus 1.2 plus minus 0.3%, P less than .05) and in the cross-sectional area of myocytes (298 plus minus 29 versus 187 plus minus 12 micro meter2, P less than .05) compared with control mouse hearts. These results suggest that Gsalpha overexpression enhances the efficacy of the beta-adrenergic receptor-Gs-adenylylcyclase signaling pathway. This in turn leads to augmented inotropic and chronotropic responses to endogenous sympathetic stimulation. This action over the life of the animal results in myocardial damage characterized by cellular degeneration, necrosis, and replacement fibrosis, with the remaining cells undergoing compensatory hypertrophy. As a model, this transgenic mouse offers new insights into the mechanisms of cardiomyopathy and heart failure and provides a new tool for their study.(Circ Res. 1996;78:517-524.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

We have previously shown that extracellular ATP, like norepinephrine (NE) and many other hypertrophyinducing 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 [(14) C]phenylalanine (14) C-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 of14C-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 P2purinergic 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 Ca2plus-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.(Circ Res. 1996;78:525-535.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

To determine whether the attenuation in the growth capacity of myocytes in the overloaded aging heart is associated with an impairment in the activation of insulin-like growth factor-1 (IGF-1) and its receptor (IGF-1R) in the stressed cells, large myocardial infarcts were produced in Fischer 344 rats at 4 and 16 months of age, and the animals were killed 6 hours, 3 days, and 7 days later. After the documentation of cardiac failure, the unaffected myocytes were enzymatically dissociated, and the expression of IGF-1 and IGF-1R was measured at these three time points after surgery. The level of expression of IGF-1R mRNA increased at 3 days and remained elevated at 7 days in both age groups. In addition, an increase in IGF-1R protein in these cells was found, with no apparent difference with age. This phenomenon was coupled with an upregulation of IGF-1 mRNA of comparable magnitude in the younger and older animals. In contrast, the increases in the dimensional properties of myocytes were delayed and of smaller magnitude in the older infarcted rats. Moreover, the expression of atrial natriuretic factor, used as a molecular marker of myocyte cellular hypertrophy, was greater at 3 days in 4-month-old rats and at 7 days in 16-month-old rats. Thus, aging may affect the hypertrophic response of myocytes after infarction but has no impact on the ability of the cells to enhance the expression of IGF-1 and IGF-1R, which may sustain only in part the growth reserve mechanisms of the pathological heart.(Circ Res. 1996;78:536-546.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Mouse embryonic stem (ES) cells differentiate in vitro into a variety of cell types, including spontaneously contracting cardiac myocytes.The primary aim of this work was to use vital stain techniques for real-time detection of developing cardiac myocytes in ES cell differentiation cultures. The minus 440 to plus 6 human cardiac alpha-actin promoter was used to direct expression of the Escherichia coli reporter gene lacZ (pHCActlacZ) into ES cell-derived cardiac myocytes during cardiogenesis in vitro. Undifferentiated ES cells were electroporated with HCActlacZ together with a plasmid containing the neomycin gene under the direction of the phosphoglycerate kinase promoter, and stable transformants were selected in G418. Individual clones were screened for activation of lacZ gene expression in cardiac myocytes developing in vitro. Results showed that expression of the HCActlacZ reporter construct was activated very early during the ES cell differentiation program, at a time point before the appearance of spontaneous contractile activity. The earliest detection was at day 6 of differentiation, when approximate equal 25% of the differentiation cultures expressed the reporter construct, with expression increasing to approximate equal 70% at day 9 and continuing throughout the duration of spontaneous contractile activity exhibited by the ES cell-derived cardiac myocytes. Indirect immunofluorescence assays provide evidence that expression was restricted to the cardiac myocytes in culture. In the present study, we show vital staining of transgene expression in living cardiac myocytes using lipophilic fluorogenic beta-galactopyranoside substrates for real-time detection of the reporter gene during continuous contraction of the ES cell myocytes in vitro. The vital stain approach used in the present study will permit the identification of differentiating ES cells that are committed to the cardiac lineage for analysis of gene expression at early time points of ES cell cardiogenesis and, in addition, will aid in selecting genetically modified ES cell cardiac myocytes for use in functional studies.(Circ Res. 1996;78:547-552.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Previous studies established that thrombin stimulates phosphoinositide hydrolysis and modulates contractile function in neonatal rat ventricular myocytes.The present study further defines the signaling pathways activated by the thrombin receptor and their role in thrombin's actions in cardiac myocytes. The thrombin receptor-derived agonist peptide (TRAP, a portion of the tethered ligand created by thrombin's proteolytic activity) stimulates the rapid and transient accumulation of inositol bis- and tris-phosphates (IP2and IP3, respectively), which is followed by the more gradual and sustained accumulation of inositol monophosphate (IP1). TRAP elicits a larger and more sustained accumulation of IP1than does thrombin. Thrombin and TRAP also activate mitogen-activated protein kinase (MAPK) in cultured neonatal rat ventricular myocytes. Differences in the kinetics and magnitude of thrombin- and TRAP-dependent inositol phosphate (IP) accumulation are paralleled by differences in the kinetics and magnitude of thrombin- and TRAP-dependent activation of MAPK. Pretreatment with phorbol 12-myristate 13-acetate (PMA) to downregulate protein kinase C (PKC) attenuates thrombin- and TRAP-dependent activation of MAPK, although small and equivalent effects of thrombin and TRAP to stimulate MAPK persist in PMA-pretreated cells. These results support the notion that the thrombin receptor activates MAPK through PKC-dependent and PKC-independent pathways and that the incremental activation of MAPK by TRAP over that induced by thrombin is the consequence of enhanced activation through the PKC limb of the phosphoinositide lipid pathway. TRAP also increases the beating rate of spontaneously contracting ventricular myocytes and elevates cytosolic calcium in myocytes electrically driven at a constant basic cycle length. The effects of TRAP to modulate contractile function and elevate intracellular calcium are not inhibited by tricyclodecan-9-yl-xanthogenate (D609, to block TRAP-dependent IP accumulation) or pretreatment with PMA (to downregulate PKC). The TRAP-dependent rise in intracellular calcium also is not inhibited by verapamil or removal of extracellular calcium but is markedly attenuated by depletion of sarcoplasmic reticular calcium stores by caffeine. Patch-clamp experiments demonstrate that TRAP elevates intracellular calcium in cells held at a membrane potential of minus 70 mV. Taken together, these results support the conclusion that the thrombin receptor modulates contractile function by mobilizing intracellular calcium through an IP3-independentmechanism and that this response does not require activation of voltage-gated ion channels.(Circ Res. 1996;78:553-563.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

After a transient ischemic attack of the cardiac vascular system, reactive oxygen-derived free radicals, including the superoxide (O2minuscentered dot) and hydroxyl (centered) dot OH) radicals can be easily produced during reperfusion. These free radicals have been suggested to be responsible for reperfusion-induced cardiac stunning and reperfusion-induced arrhythmia. Hydrogen peroxide (H2O (2)) is often used as an experimental source of oxygen-derived free radicals. Using freshly dissociated single rat cardiac myocytes and the rat cardiac myoblast cell line, H9c2, we have shown, for the first time, that an intriguing pHiacidification (approximate equal 0.24 pH unit) is induced by the addition of 100 mu mol/L H2O2and that this dose is without effect on the intracellular free Ca2plus levels or viability of the cells. Using H9c2 as a model cardiac cell, we have shown that it is the intracellular production ofcentereddot OH, and not O2minuscentered dot or H2O2, that results in this acidification. We have excluded any involvement of (1) the three known cardiac pHiregulators (the Naplus-Hplusexchanger, the Clminus-HCO3exchanger, and the Naplus-HCO3cotransporter), (2) a rise in intracellular Ca2plus levels, and (3) inhibition of oxidative phosphorylation. However, we have found that H2O2-inducedacidosis is due to inhibition of the glycolytic pathway, with hydrolysis of intracellular ATP and the resultant intracellular acidification. In cardiac muscle and in skinned cardiac muscle fiber, it has been shown that a small intracellular acidification may severely inhibit contractility. Therefore, the sustained pHidecrease caused by hydroxyl radicals may contribute, in some part, to the well-documented impairment of cardiac mechanical function (ie, reperfusion cardiac stunning) seen during reperfusion ischemia.(Circ Res. 1996;78:564-572.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Endogenous catecholamine release may play a role in ischemic preconditioning either as a trigger or as a target within the process of myocardial preconditioning.Therefore, we investigated the effect of transient ischemia (TI) on norepinephrine release during sustained ischemia in isolated rat hearts. TI was induced by multiple cycles of global ischemia followed by reperfusion with a duration of 5 minutes each, comparable to ischemic preconditioning protocols. After TI, norepinephrine release was evoked by either sustained global ischemia, anoxia, cyanide intoxication, tyramine, or electrical stimulation. During TI, no washout of norepinephrine was observed, and tissue concentrations of norepinephrine were not changed. TI, however, reduced norepinephrine overflow after 20 minutes of sustained ischemia from 239 plus minus 26 pmol/g (control) to 79 plus minus 8 pmol/g (67% reduction, P less than .01). A similar reduction of ischemia-induced norepinephrine release from 192 plus minus 22 pmol/g (control) to 90 plus minus 15 pmol/g was observed when hearts underwent transient anoxia without glucose (P less than .05). When reperfusion between TI and sustained ischemia was prolonged from 5 to 90 minutes, the inhibitory effect of TI on norepinephrine release was gradually lost. Susceptibility to TI was a unique feature of norepinephrine release induced by sustained ischemia, since release of norepinephrine evoked by anoxia, cyanide intoxication, tyramine, or electrical stimulation remained unaffected by TI. We propose a protective effect of TI on neural tissue, which may reduce norepinephrine-induced damage during prolonged myocardial ischemia.(Circ Res. 1996;78:573-580.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

The thrombin receptor was the first cloned G protein-coupled receptor reported to be activated by proteolytic cleavage of its extracellular amino terminus. A second proteinase-activated receptor (PAR-2) was cloned recently and expressed in Xenopus laevis oocytes. PAR-2 was activated by trypsin and by a peptide (SLIGRL) derived from the new amino terminus. Since PAR-2 mRNA was detected in highly vascularized organs, we compared the physiological functions of the thrombin receptor and PAR-2 in vascular endothelium. Thrombin and trypsin both elicited endothelium-dependent relaxations in prostaglandin F2alpha (PGF2alpha)-contracted strips of porcine coronary artery. Whereas high doses of both thrombin or trypsin (10 U/mL) caused homologous desensitization, trypsin caused further relaxation of thrombin-desensitized tissues. Thrombin and PAR-2-derived peptides (SFLLRN and SLIGRL) both induced endothelium-dependent relaxations in PGF2alpha-contracted porcine coronary arteries. SFLLRN or SLIGRL (30 mu mol/L) also showed homologous desensitization but not cross desensitization. In the presence of the NO synthase inhibitor NG-monomethyl-L-arginine(1 mmol/L), both SFLLRN- and SLIGRL-induced relaxations were partially inhibited. SFLLRN elicited weak contraction in coronary arteries without endothelium, whereas SLIGRL had no effect. Intravenous injection of SFLLRN (1 mg/kg, bolus) into anesthetized rats elicited a transient depressor response followed by pronounced pressor response. In contrast, intravenous administration of SLIGRL (1 mg/kg, bolus) produced only a marked depressor response. Consistent with the in vivo data, SFLLRN contracted the endothelium-rubbed rat aortic rings and aggregated human platelets in vitro, whereas SLIGRL had no effect. The finding that both trypsin and SLIGRL induced endothelium-dependent relaxations indicates the presence of PAR-2 on endothelial cells. In addition, both trypsin and SLIGRL elicited relaxations in thrombin- or SFLLRN-desensitized tissue, suggesting that PAR-2 is distinct from thrombin receptor in vascular endothelium. The lack of PAR-2-mediated platelet aggregation or smooth muscle contraction suggested it might not share the pathogenic properties associated with the thrombin receptor in the vasculature.(Circ Res. 1996;78:581-588.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Ets-1 regulates the transcription of several genes encoding extracellular matrix proteins (ie, osteopontin and tenascin) as well as enzymes involved in degradation and remodeling of the extracellular matrix (ie, stromelysin and urokinase plasminogen activator). In the present study, we investigated the regulation of c-ets-1 in cultured rat vascular smooth muscle cells as well as in the arterial wall after balloon injury in vivo. Serum-starved smooth muscle cells exposed to serum for various time points express a major c-ets-1 mRNA transcript of 5.3 kb and minor bands of 4.0 and 2.5 kb with a peak at 2 hours after stimulation. These effects were concentration dependent. Western blotting revealed an increase in 55- and 40-kD immunoreactive ets-1 proteins in cells treated with serum for 2 hours, and binding to an oligonucleotide containing the ets-1 consensus cis-acting motif was demonstrated by electrophoretic mobility shift assay. Ets-1 mRNA abundance was induced with a peak at 2 hours after stimulation with platelet-derived growth factor-BB and with angiotensin II. There was a distinct increase of ets-1 immunoreactivity in the inner layer of the media 2 hours after balloon catheter injury of rat arteries, which declined after 6 hours and returned to the basal level 1 day after vessel wall damage. Arterial c-ets-1 mRNA content was induced with an identical time course. These findings suggest that c-ets-1 may be of importance in the mitogenic signaling pathway of smooth muscle cells grown in culture. In addition, ets-1 may play a role in the activation of smooth muscle cells in vivo after mechanical injury of the vessel wall. Because the ets-1 transcription factor activates the gene expression of a number of mRNA species involved in matrix deposition and degradation, these data are compatible with a role for ets-1 in vascular remodeling and/or cell migration.(Circ Res. 1996;78:589-595.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID