摘要:

Understanding the genetic control of cardiac development has been greatly assisted by the newly acquired ability to generate targeted mutations in the mouse. A number of mutations in genes that directly or indirectly affect cardiac development have now been reported, and the phenotypes of these mutations have suggested that cardiac development is under complex genetic control. Further analysis of the mouse model system should help elucidate the etiology of human congenital cardiovascular anomalies.(Circ Res. 1996;78:349-353.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Treatment of cultured neonatal cardiomyocytes with endothelin-1 and phorbol 12-myristate 13-acetate (PMA) results in cardiomyocyte hypertrophy. However, the signal transduction pathways involved in this process are poorly understood. Because increased ribosome biogenesis is a requisite for hypertrophy, we sought to (1) confirm the hypothesis that these two hypertrophic agents did indeed induce rRNA synthesis and (2) examine the mechanism through which this induction was accomplished. In this study, hypertrophy of contraction-arrested neonatal cardiomyocytes induced by treatment with either endothelin-1 or PMA was associated with increased rDNA transcription. Western blots demonstrated that the enhanced rates of rDNA transcription were not mediated by increased amounts of either RNA polymerase I or upstream binding factor (UBF), an rDNA transcription factor. However, immunoprecipitation of [sup 32 P]orthophosphate-labeled UBF from hypertrophying neonatal cardiomyocytes suggested that the increased rate of rDNA transcription may be due to the hyperphosphorylation of UBF, which would increase the activity of UBF. The increase in UBF phosphorylation occurred within 3 to 6 hours after exposure to either agent, was maximal at 12 hours, and was sustained for at least the first 24 hours of exposure. Phosphoamino acid analysis of UBF immunoprecipitated from control and treated cardiomyocytes demonstrated that UBF was phosphorylated exclusively on serine residues. Our previous studies have shown that the cellular UBF content increased in adrenergic- and contraction-induced models of cardiac hypertrophy. This study with endothelin-1 and PMA demonstrates that the modulation of UBF phosphorylation is an additional pathway by which ribosome biogenesis may be regulated in neonatal cardiomyocytes. These results support the hypothesis that UBF is an important regulatory factor during the initiation and maintenance of the accelerated rate of rDNA transcription observed during neonatal cardiomyocyte hypertrophy mediated by both phorbol esters and endothelin-1.(Circ Res. 1996;78:354-361.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Intercellular dehiscence is a common cardiocytic response to pathological conditions. However, little consideration has been given to the possibility of new intercellular junctions developing between cardiocytes within developed myocardium. To examine this possibility as it may relate to useful compensation for hemodynamic overloads, changes in cardiocytic connection were evaluated by scanning electron microscopy in hypertrophied myocardium of adult human hearts. Transmural myocardium of left ventricle was obtained at autopsy from five hearts with concentric hypertrophy, five hearts with eccentric hypertrophy, and five control hearts (noncardiac death). After formalin fixation, the number of cardiocytes connected to an individual cardiocyte was counted in tissues from the middle portion of the transmural samples by scanning electron microscopy. Cardiocytic diameter and connective tissue volume fraction were measured on the transmural sections by light microscopy. In concentrically hypertrophied hearts presenting both increased cardiocytic diameter and connective tissue volume fraction, the number of other cardiocytes connected to an individual cardiocyte (4.60 plus minus 0.10 [mean plus minus SE]) was significantly increased (P less than .05) compared with control hearts (4.19 plus minus 0.12) or eccentrically hypertrophied hearts (4.11 plus minus 0.10). The increase in junctions per cardiocyte in concentrically hypertrophied hearts suggests that new connections had been generated. More junctions developing during hypertrophy could add another structural advantage to those of cardiocytic hypertrophy and connective tissue proliferation as compensatory adjustments to hemodynamic overload in concentrically hypertrophied hearts.(Circ Res. 1996;78:362-370.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

In the adult mammalian myocardium, cellular Ca sup 2 plus entry is regulated by the sympathetic nervous system. L-type Ca2plus channel currents are markedly increased by beta-adrenergic (beta-A) agonists, which contribute to changes in pacing and contractile activity of the heart. In the developing mammalian heart, the regulation of Ca2plus entry by this enzyme cascade has not been clearly established, because changes in receptor density and coupling to downstream elements of the signaling cascade are known to occur during embryogenesis. In this study, we systematically investigated the regulation of L-type Ca2plus channel currents during development of the murine embryonic heart. We used conventional whole-cell and perforated-patch-clamp procedures to study modulation of L-type Ca2plus channel currents and to assay functional activity of distinct steps in the beta-A signaling cascade in murine embryonic myocytes at different stages of gestation. Our data indicate that L-type Ca2plus channels in early-stage (day-11 to minus 13) myocytes are unresponsive to either isoproterenol or cAMP. L-type Ca2plus channels in late-stage (day-17 to minus 19) murine myocytes, however, exhibit responses to isoproterenol and cAMP similar to responses in adult cells, providing evidence that the beta-A cascade becomes functionally active during this period of embryonic development. We found that L-type Ca2plus channel activity in early-stage cells is increased by cell dialysis with the catalytic subunit of cAMP-dependent protein kinase (cA-PK) and that dialysis of early-stage cells with the holoenzyme of cA-PK restores functional responses to forskolin and cAMP, but not to isoproterenol. Our results provide strong evidence that a key factor in the early-stage insensitivity of L-type Ca2plus channels to cAMP is the absence, or low expression level, of the holoenzyme of cA-PK but that in addition, another element in the signaling cascade upstream from adenylate cyclase is expressed at a nonfunctional level or is uncoupled from the cascade and thus contributes to L-type Ca2plus channel insensitivity to beta-A agonists in early stages of the developing murine heart.(Circ Res. 1996;78:371-378.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

To explore the compatibility of skeletal and cardiac programs of gene expression, transgenic mice that express a skeletal muscle myogenic regulator, bmyf5, in the heart were analyzed. These mice develop a severe cardiomyopathy and exhibit a significantly shorter life span than do their nontransgenic littermates. The transgene was expressed from day 7.5 post coitum forward, resulting in activation of skeletal muscle genes not normally seen in the myocardium. Cardiac pathology was not apparent at midgestation but was evident by day 2 of postnatal life, and by 42 days, hearts exhibited multifocal interstitial inflammation, fibrosis, cellular hypertrophy, and occasional myocyte degeneration. All four chambers of the heart were enlarged to varying degrees, with the atria demonstrating the most significant hypertrophy (more than 100% in 42-day-old mice). The transgene and several skeletal muscle-specific genes were expressed only in patchy areas of the heart in heterozygous mice. However, molecular markers of hypertrophy (such as alpha-skeletal actin and atrial myosin light chain-1) were expressed with a wider distribution, suggesting that their induction was secondary to the expression of the transgene. In older (28-week-old) mice, lung weights were also significantly increased, consistent with congestive heart failure. The life span of bmyf5 mice was significantly shortened, with an average life span of 109 days, compared with at least a twofold longer life expectancy for nontransgenic littermates. Expression of the transgene was associated with an increase in Ca2plus-stimulated myofibrillar ATPase in myofibrils obtained from the left ventricles of 42-day-old bmyf5 mice. Myocardial bmyf5 expression therefore induces a program of skeletal muscle gene expression that results in progressive cardiomyopathy that may be due to incompatibility of heart and skeletal muscle structural proteins.(Circ Res. 1996;78:379-387.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

After vascular injury, quiescent adult smooth muscle cells (SMCs) revert to a more immature synthetic-state phenotype concomitant with the onset of cell replication. The relationship between SMC proliferation and the reexpression of genes characteristic of immature SMCs (eg, tropoelastin [TE]), on an individual cell basis, has not been determined. Using a combined bromodeoxyuridine (BrdU) immunocyto-chemistry-TE in situ hybridization technique, we determined the relationship between DNA synthesis and TE gene expression in the rat vascular wall during development and after experimental injury. During the early development of the aortic media (embryonic days 13 to 18), low but detectable levels of TE expression occurred equally in both replicating and nonreplicating SMCs. TE message levels dramatically increased in the late fetal and early postnatal periods (fetal day 19 to 1 month postpartum), after a precipitous drop in SMC replication, and then decreased to undetectable levels by postpartum day 60. After balloon catheter injury in the adult, a developmental sequence of SMC replication followed by TE gene expression was reiterated in both the media and in the developing neointima. On an individual cell basis, adult SMCs replicating after injury expressed little or no TE message; detectable TE gene expression occurred only in nonreplicating SMCs. The most important implications of these data are that (1) adult SMCs replicating after injury appear to revert to a preelastogenic embryonic phenotype; (2) maximal TE expression occurs in SMCs only after the cessation of cell replication; and (3) in both the media and the neointima, adult SMCs responding to injury undergo temporally sequential changes in phenotype reflective of SMC development.(Circ Res. 1996;78:388-394.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Investigation of the molecular mechanisms that control smooth muscle cell (SMC) development and differentiation is a prerequisite in understanding the regulatory mechanisms of physiological and pathological SMC-associated vascular processes. The pluripotent murine embryonal carcinoma P19 cell, whose developmental potential resembles that of early embryonic cells, can develop into cell types derived from the neuroectoderm, mesoderm, and endoderm. In the present study, we have shown a unique strategy to enhance SMC differentiation in P19 cells. Under chemical induction of high concentrations of retinoic acid (1 mu mol/L), P19 cells showed optimum differentiation into SMCs. Because the P19 cells thus induced also showed differentiation into neuronal cells, a strategy to block neuronal lineage differentiation was developed using a stable transformant antisense RNA construct against Brn-2, a neuronal lineage-specific POU-domain transcription factor; thus, by specifically inhibiting neuronal differentiation, enhanced SMC differentiation by P19 cells was attained. SMC expression was confirmed by immunohistochemical staining, RNA analysis (RNase protection assay), and protein analysis (Western blot) using SMC-specific markers (eg, SM1 and calponin) and alpha-smooth muscle actin. Our results show that the pathway of SMC differentiation may provide an in vitro system useful in the investigation of SMC regulatory mechanisms (eg, transcriptional regulation) and in the further understanding of SMC development and differentiation.(Circ Res. 1996;78:395-404)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

The expression of plasminogen activators and inhibitors was examined in denuded arteries. Within 5 days, smooth muscle cells (SMCs) on the luminal surface expressed the mRNA for tissue-type plasminogen activator (TPA), urokinase-type plasminogen activator (UPA), the receptor for UPA (UPAR), and plasminogen activator inhibitor type-1 (PAI-1). Similar results were seen after 8 days. Six weeks later, only TPA mRNA was still expressed by SMCs on the luminal surface. En face casein zymograms revealed a net fibrinolytic activity in areas covered with luminal SMCs. Reverse zymography showed no antifibrinolytic activity in these zones. Quiescent endothelial cells did not express TPA, UPA, UPAR, or PAI-1 mRNA. Regenerating endothelium at the wound edge strongly expressed TPA, UPA, and UPAR, as well as PAI-1. UPA and UPAR expression was highly restricted to cells at the wound edge and was not present elsewhere. En face zymography showed no plasmin activity in endothelialized areas, and reverse zymography showed a net antifibrinolytic activity in endothelialized zones. These results suggest that plasminogen activator and inhibitor expression correlates with the migration of both SMCs and endothelial cells into an arterial wound.(Circ Res. 1996;78:405-414)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Endothelial cells release several compounds, including prostacyclin, NO, and endothelium-derived hyperpolarizing factor (EDHF), that mediate the vascular effects of vasoactive hormones. The identity of EDHF remains unknown. Since arachidonic acid causes endothelium-dependent relaxations of coronary arteries through its metabolism to epoxyeicosatrienoic acids (EETs) by cytochrome P450, we wondered if the EETs represent EDHFs. Precontracted bovine coronary arteries relaxed in an endothelium-dependent manner to methacholine. The cytochrome P450inhibitors, SKF 525A and miconazole, significantly attenuated these relaxations. They were also inhibited by tetraethylammonium (TEA), an inhibitor of Ca2plus-activated Kpluschannels, and by high [K sup plus]o(20 mmol/L). Methacholine also caused hyperpolarization of coronary smooth muscle (minus 27 plus minus 3.9 versus minus 40 plus minus 5.1 mV), which was completely blocked by SKF 525A and miconazole. In vessels prelabeled with [sup 3 H]arachidonic acid, methacholine stimulated the release of 6-ketoprostaglandin F1alpha, 12-HETE, and the EETs. Arachidonic acid relaxed precontracted coronary arteries, which were also blocked by TEA, charybdotoxin, another Ca2plus-activated Kpluschannel inhibitor, and high [Kplus]o. 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET relaxed precontracted coronary vessels (EC50, 1 times 10 sup minus 6 mol/L). The four regioisomers were equally active. TEA, charybdotoxin, and high [Kplus]oattenuated the EET relaxations. 11,12-EET hyperpolarized coronary smooth muscle cells from minus 37 plus minus 0.2 to minus 59 plus minus 0.3 mV. In the cell-attached mode of patch clamp, both 14,15-EET and 11,12-EET increased the open-state probability of a Ca2plus-activated Kpluschannel in coronary smooth muscle cells. This effect was blocked by TEA and charybdotoxin. These data support the hypothesis that the EETs are EDHFs.(Circ Res. 1996;78:415-423)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Involvement of a cGMP pathway in signal transduction stimulated by endothelins (ETs) and sarafotoxins (SRTXs) was examined in rat atrial slices. These peptides activated different receptor-binding sites (ET-1 and SRTX-b reacted with the picomolar binding sites of the ETAreceptor, and ET-3 and SRTX-c reacted with the nanomolar binding sites of the ETBreceptor) to produce cGMP. ET-1 and SRTX-b stimulated an increase in cGMP levels via a Ca2plus-dependent NO pathway involving a pertussis toxin-insensitive G protein, whereas ET-3 and SRTX-c elevated cGMP levels via a Ca sup 2 plus-independent CO pathway involving a pertussis toxin-sensitive G protein. These results can best be explained in terms of formation of different ligand-receptor-G-protein complexes. The ligands had no effect on ventricular slices, indicating that these signal transduction mechanisms are unique to the atria.(Circ Res. 1996;78:424-430.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID