ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Abstract—The spatial and dynamic properties of ventricular fibrillation (VF) may be random or related to cellular electrical properties of the normal heart. Local activation intervals (AIs) in VF may depend on the local refractory period (RP), and sustained VF may require a steep action potential (AP) restitution curve. In guinea pig hearts, AP durations (APDs) and RPs on the epicardium are shorter at the apex and progressively longer toward the base, producing gradients of RPs that may influence the spatial organization of VF. In the present study, the influence of APDs on VF dynamics is investigated in perfused guinea pig hearts stained with a voltage-sensitive dye by comparing APD gradients to the dynamics of VF elicited by burst pacing. In VF, AIs had no clear periodicity, but average AIs were shorter at the apex (57.5±8.1 ms) than the base (76.1±1.5 ms, n=6,P<0.05) and had gradients similar to APD gradients (correlation coefficient 0.71±0.04). Analysis of local velocity vectors showed no preferential directions, and fast Fourier transform (FFT) power spectra were broad (10 to 24 Hz) with multiple peaks (n=6). However, the selective inhibition of delayed K+rectifying currents,IKr(E4031; 0.5 &mgr;mol/L, n=3), shifted FFT spectra from complex to a lower dominant frequency (10 Hz) and altered repolarization but retained the correlation between mean AIs and RPs. Thus, VF dynamics are consistent with a multiple wave-make and wave-break mechanism, and the local RP influences VF dynamics by limiting the range of VF frequencies and AIs at each site. The full text of this article is available at http://www.circresaha.org.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Abstract—Proliferation and &agr;v&bgr;3integrin-dependent migration of vascular smooth muscle cells are suppressed on polymerized type I collagen. To identify genes specifically regulated in human smooth muscle cells by polymerized collagen, we used the suppressive subtraction hybridization technique. Compared with smooth muscle cells cultured on monomer collagen, polymerized collagen suppresses the following: (1) a number of other extracellular matrix proteins, including fibronectin, thrombospondin-1, tenascin-C, and cysteine-rich protein 61; (2) actin binding proteins including &agr;-actinin; (3) signaling molecules; (4) protein synthesis-associated proteins; and (5) genes with unknown functions. Some of the identified genes, including cysteine-rich protein 61, show unique kinetics of mRNA regulation by monomer or polymerized collagen distinct from growth factors, suggesting extracellular matrix-specific gene modulation. Moreover, in vivo balloon catheter-mediated injury to the rat carotid artery induces many of the genes that are suppressed by polymerized collagen. Protein levels of thrombospondin-1 and fibronectin are also suppressed by polymerized collagen. Thrombospondin-1-mediated smooth muscle cell migration on vitronectin is significantly inhibited after culture on polymerized collagen for 24 hours, which is associated with decreased &agr;-actinin accumulation at focal adhesions. Thus, polymerized type I collagen dynamically regulates gene expression, pericellular accumulation of extracellular matrix molecules, and the response to a given matrix molecule.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Abstract—Gene transfer with adenoviral vectors is an attractive approach for the treatment of atherosclerosis and restenosis. However, because expression of a therapeutic gene in nontarget tissues may have deleterious effects, artery-specific expression is desirable. Although expression vectors containing transcriptional regulatory elements of genes expressed solely in smooth muscle cells (SMCs) have proved efficient to restrict expression of the transgene, their use in the clinical setting can be limited by their reduced strength. In the present study, we show that low levels of transgene expression are obtained with the smooth muscle (SM)-specific SM22&agr; promoter compared with the viral cytomegalovirus (CMV) enhancer/promoter. We have generated chimeric transcriptional cassettes containing either a SM (SM-myosin heavy chain) or a skeletal muscle (creatine kinase) enhancer combined with the SM22&agr; promoter. With both constructs we observed significantly stronger expression that remains SM-specific. In vivo, reporter gene expression was restricted to arterial SMCs with no detectable signal at remote sites. Moreover, when interferon-&ggr; expression was driven by one of these two chimeras, SMC growth was inhibited as efficiently as with the CMV promoter. Finally, we demonstrate that neointima formation in the rat carotid balloon injury model was reduced to the same extent by adenoviral gene transfer of interferon-&ggr; driven either by the SM-myosin heavy chain enhancer/SM22&agr; promoter or the CMV promoter. These results indicate that such vectors can be useful for the treatment of hyperproliferative vascular disorders.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Abstract—Hypertrophied cardiac myocytes exhibit prolonged action potentials and decreased transient outward potassium current (Ito). Because Kv4.3 is a major contributor toIto, we studied regulation of its expression in neonatal rat cardiac myocytes in response to the known stimulators of cardiac myocyte hypertrophy, angiotensin II (Ang II) and phenylephrine (PE). RNase protection assays and immunoblots revealed that Ang II and PE each downregulate Kv4.3 mRNA and protein. However, although PE induces a faster and more extensive hypertrophic response than Ang II, the PE effect on Kv4.3 mRNA develops slowly and is sustained, whereas Ang II rapidly and transiently decreases Kv4.3 mRNA expression. Turnover measurements revealed that Kv4.3 mRNA is very stable, with a half-life >20 hours. This suggests that Ang II must destabilize the channel mRNA. In contrast, PE does not affect the rate of Kv4.3 mRNA degradation. To test for transcriptional regulation, the 5′ flanking region of the rat Kv4.3 gene was cloned, and Kv4.3 promoter-reporter constructs were expressed in cardiac myocytes. Whereas Ang II was found to have no effect on transcription, PE inhibits Kv4.3 promoter activity. Pharmacological experiments also indicate that PE and Ang II act independently to downregulate Kv4.3 gene expression. Thus, regulation of Kv4.3 gene expression is not a simple secondary response to hypertrophy. Rather, Ang II and PE use different mechanisms to decrease Kv4.3 channel expression in neonatal rat cardiac myocytes.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Expression of the voltage-gated K+channel Kv2.1, a possible molecular correlate for the cardiac delayed rectifier current (IK), has recently been shown to vary between individual ventricular myocytes. The functional consequences of this cell-to-cell heterogeneity in Kv2.1 expression are not known. Using multiplex single-cell reverse transcriptase–polymerase chain reaction (RT-PCR), we detected Kv2.1 mRNA in 47% of isolated midmyocardial myocytes from the rat left ventricular free wall that were positive for &agr;-myosin heavy chain mRNA (n= 74). Whole-cell patch-clamp recordings demonstrated marked differences in the magnitude ofIK(200 to 1450 pA atVPip=40 mV) between individual myocytes of the same origin. Furthermore, the tetraethylammonium (TEA)–sensitive outward current (ITEA), known to be partly encoded by Kv2.1 in mice, revealed a wide range of current magnitudes between single cells (150 to 1130 pA atVPip=40 mV). Combined patch-clamp recordings and multiplex single-cell RT-PCR analysis of the same myocytes, however, showed no differences inIKorITEAmagnitude or inactivation kinetics between myocytes expressing Kv2.1 mRNA and those that did not express Kv2.1 mRNA. In contrast, in all midmyocardial myocytes expressing the transient outward potassium current (Ito1), Kv4 mRNA, which has been shown to underlieIto1, was detected (n= 10). These results indicate thatIKheterogeneity among individual left ventricular myocytes cannot be explained by the distribution pattern of Kv2.1 mRNA. Other mechanisms besides Kv2.1 mRNA expression appear to determine magnitude and kinetics ofIKin rat ventricular myocytes.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

We tested the hypothesis that bacterial lipopolysaccharide (LPS) must be internalized to facilitate endotoxin-dependent signal activation in cardiac myocytes. Fluorescently labeled LPS was used to treat primary cardiomyocyte cultures, perfused heart preparations, and the RAW264.7 macrophage cell line. Using confocal microscopy and spectrofluorometry, we found that LPS was rapidly internalized in cardiomyocyte cultures and Langendorff-perfused hearts. Although LPS uptake was also observed in macrophages, only a fraction of these cells were found to internalize endotoxin to the extent seen in cardiomyocytes. Colocalization experiments with organelle or structure-specific fluorophores showed that LPS was concentrated in the Golgi apparatus, lysosomes, and sarcomeres. Similar intracellular localization was demonstrated in cardiomyocytes by transmission electron microscopy using gold-labeled LPS. The internalization of LPS was dependent on endosomal trafficking, because an inhibitor of microfilament reorganization prevented uptake in both cardiomyocytes and whole hearts. Inhibition of endocytosis specifically restricted early activation of extracellular signal-regulated kinase proteins and nuclear factor-&kgr;B as well as later tumor necrosis factor-&agr; production and inducible nitric oxide synthase expression. In conclusion, we have demonstrated that bacterial endotoxin is internalized and transported to specific intracellular sites in heart cells and that these events are obligatory for activation of LPS-dependent signal transduction.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID