摘要:

Abstract—Little is known regarding transcriptional regulatory mechanisms that control the sequential and coordinate expression of genes during smooth muscle cell (SMC) differentiation. To facilitate mechanistic studies of SMC differentiation, we established a novel P19-derived clonal cell line (designated A404) harboring a smooth muscle (SM) &agr;-actin promoter/intron-driven puromycin resistance gene. Retinoic acid plus puromycin treatment stimulated rapid differentiation of multipotential A404 cells into SMCs that expressed multiple SMC differentiation marker genes, including the definitive SM-lineage marker SM myosin heavy chain. Using this system, we demonstrated that various transcription factors were upregulated coincidentally with the expression of SMC differentiation marker genes. Of interest, the expression of serum response factor (SRF), whose function is critical for SMC-specific transcription, was high in undifferentiated A404 cells, and it did not increase over the course of differentiation. However, chromatin immunoprecipitation analyses showed that SRF did not bind the target sites of endogenous SMC marker genes in chromatin in undifferentiated cells, but it did in differentiated A404 cells, and it was associated with hyperacetylation of histones H3 and H4. The present studies define a novel cell system for studies of transcriptional regulation during the early stages of SMC differentiation, and using this system, we obtained evidence for the involvement of chromatin remodeling and selective recruitment of SRF to CArG elements in the induction of cell-selective marker genes during SMC differentiation.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Abstract—The roles of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF [FGF-2]) in early postnatal regulation of coronary angiogenesis were investigated by administering neutralizing antibodies to these growth factors between postnatal days 5 and 12. Immunohistochemistry and Western blotting both revealed decreases in VEGF protein in the hearts of rats treated with either antibody. In contrast, bFGF mRNA increased in both treated groups, whereas VEGF mRNA was unchanged. Using stereological assessment of perfusion-fixed hearts, we found that both anti-VEGF and anti-bFGF inhibited the rapid and marked capillary growth that occurs during this time period and that the effects of the two neutralizing antibodies are not additive. Arteriolar growth, as indicated by a lower length density, was inhibited by anti-bFGF, but not anti-VEGF. When both anti-VEGF and anti-bFGF were administered, arteriolar length density was not significantly lower, but the population of small arterioles (<15 &mgr;m) was markedly reduced, whereas the percentage of large arterioles (26 to 50 &mgr;m) more than doubled. Thus, inhibition of both growth factors negated or limited the formation of small arterioles and facilitated an expansion of the largest arterioles. These in vivo data are the first to document that during the early neonatal period, (1) both VEGF and bFGF modulate capillary growth, (2) bFGF facilitates arteriolar growth, and (3) the two growth factors interact to establish the normal hierarchy of the arteriolar tree.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Abstract—We investigated whether the heart, like other mammalian organs, possesses internal clocks, and, if so, whether pressure overload–induced hypertrophy alters the clock mechanism. Clock genes are intrinsically maintained, as shown by rhythmic changes even in single cells. Clocks are believed to confer a selective advantage by priming the cell for the expected environmental stimulus. In this way, clocks allow anticipation, thereby synchronizing responsiveness of the cell with the timing of the stimulus. We have found that in rat heart all mammalian homologues of knownDrosophilaclock genes (bmal1,clock,cry1,cry2,per1,per2,per3,dbp,hlf, andtef) show circadian patterns of expression and that the induction of clock output genes (the PAR [rich inproline andacidic amino acidresidues] transcription factorsdbp,hlf, andtef) is attenuated in the pressure-overloaded hypertrophied heart. The results expose a new dynamic regulatory system in the heart, which is partially lost with hypertrophy. Although the target genes of these PAR transcription factors are not known in the heart, the results provide evidence for a diminished ability of the hypertrophied heart to anticipate and subsequently adapt to physiological alterations during the day.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Abstract—Excitation-contraction coupling in heart muscle requires the activation of Ca2+-release channels/type 2 ryanodine receptors (RyR2s) by Ca2+influx. RyR2s are arranged on the sarcoplasmic reticular membrane in closely packed arrays such that their large cytoplasmic domains contact one another. We now show that multiple RyR2s can be isolated under conditions such that they remain physically coupled to one another. When these coupled channels are examined in planar lipid bilayers, multiple channels exhibit simultaneous gating, termed “coupled gating.” Removal of the regulatory subunit, the FK506 binding protein (FKBP12.6), functionally but not physically uncouples multiple RyR2 channels. Coupled gating between RyR2 channels may be an important regulatory mechanism in excitation-contraction coupling as well as in other signaling pathways involving intracellular Ca2+release.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Abstract—Ventricular arrhythmias and contractile dysfunction are the main causes of death in human heart failure (HF). In a rabbit HF model reproducing these same aspects of human HF, we demonstrate that a 2-fold functional upregulation of Na+-Ca2+exchange (NaCaX) unloads sarcoplasmic reticulum (SR) Ca2+stores, reducing Ca2+transients and contractile function. Whereas &bgr;-adrenergic receptors (&bgr;-ARs) are progressively downregulated in HF, residual &bgr;-AR responsiveness at this critical HF stage allows SR Ca2+load to increase, causing spontaneous SR Ca2+release and transient inward current carried by NaCaX. A given Ca2+release produces greater arrhythmogenic inward current in HF (as a result of NaCaX upregulation), and ≈50% less Ca2+release is required to trigger an action potential in HF. The inward rectifier potassium current (IK1) is reduced by 49% in HF, and this allows greater depolarization for a given NaCaX current. Partially blockingIK1in control cells with barium mimics the greater depolarization for a given current injection seen in HF. Thus, we present data to support a novel paradigm in which changes in NaCaX andIK1, and residual &bgr;-AR responsiveness, conspire to greatly increase the propensity for triggered arrhythmias in HF. In addition, NaCaX upregulation appears to be a critical link between contractile dysfunction and arrhythmogenesis.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Experimental and clinical evidence suggests a critical role for the left atrium (LA) in atrial fibrillation (AF). In animal models, repolarization is faster in the LA than in the right atrium (RA), leading to more stable reentry circuits with a shorter intrinsic period in the LA. The ionic mechanisms underlying LA-RA repolarization differences are unknown. Therefore, we evaluated ionic currents and action potentials (APs) with the whole-cell patch clamp in isolated canine atrial myocytes. The density of the rapid delayed rectifier current (IKr) was greater in the LA (eg, 1.83±0.10 pA/pF at +20 mV) than in the RA (1.15±0.07 pA/pF,P<0.01; n=16 cells per group). The slow and ultrarapid delayed rectifier, the inward rectifier, L-type Ca2+, and transient outward K+currents were all comparable in the LA and RA. There were no differences in kinetic or voltage-dependent properties of currents in LA versus RA. Western blots of ether-a-go-go–related gene (ERG) protein in three RA and corresponding LA regions showed significantly greater ERG expression in LA. AP duration (APD) was shorter in the LA versus RA in both isolated cells and multicellular preparations, and the effective refractory period (ERP) was shorter in the LA compared with the RA in vivo. Dofetilide had significantly larger APD- and ERP-increasing effects in the LA compared with RA, and LA-RA repolarization differences were eliminated by exposure to dofetilide. We conclude that LA myocytes have largerIKrthan do RA myocytes, contributing importantly to the shorter APD and ERP in LA. The larger LAIKrmay participate in the ability of the LA to act as a “driver region” for AF, with potentially important implications for understanding AF mechanisms and antiarrhythmic therapy.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Abstract—To elucidate the role of intracellular Na+kinetics in the mechanism for ischemic preconditioning (IPC), we measured intracellular Na+concentration ([Na+]i) using23Na–magnetic resonance spectroscopy in isolated rat hearts. IPC significantly delayed the initial [Na+]iincrease (d[Na+]i/dt) compared with non-IPC control, resulting in attenuation of Na+accumulation (&Dgr;[Na+]i) during 27 minutes of ischemia with better functional recovery. [Na+]iin IPC, but not in control, recovered to preischemic level during a 6-minute reperfusion. The Na+-H+exchange inhibitor further suppressed d[Na+]i/dt in both control and IPC hearts with concomitant improvement of functional recovery, suggesting little contribution to the mechanism of IPC. The mitochondrial ATP-sensitive K+(mito KATP) channel activator diazoxide (30 &mgr;mol/L) completely mimicked both [Na+]ikinetics and functional recovery in IPC without any additive effects to IPC. The mito KATPchannel blocker 5-hydroxydecanoic acid (100 &mgr;mol/L) lost protective effect as well as the attenuation of d[Na+]i/dt and [Na+]irecovery induced by diazoxide. However, 5-hydroxydecanoic acid also lost IPC-induced protection, but incompletely abolished the alteration of d[Na+]i/dt and the [Na+]irecovery. The Na+/K+-ATPase inhibitor ouabain (200 &mgr;mol/L) did not change d[Na+]i/dt in non-IPC hearts, but it abolished the IPC- or diazoxide-induced reduction of d[Na+]i/dt and the [Na+]irecovery, whereas IPC followed by ouabain treatment showed partial functional recovery with smaller &Dgr;[Na+]ithan other ouabain groups. In conclusion, alteration of Na+kinetics by preserving Na+efflux via Na+/K+-ATPase mediated by mito KATPchannel activation mainly contributes to functional protection in IPC hearts. The contribution of mito KATPchannel–independent pathway relating to Na+kinetics including reduced Na+influx is limited in functional protection of IPC.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Abstract—To clarify the function of the Na+-coupled glucose transporter in the regulation of cellular tone of cultured retinal pericytes, we investigated the effects of extracellular glucose concentration on cell size. The surface area and diameter of cultured bovine retinal pericytes under different glucose concentrations were measured by using a light microscope with a digital camera. We also examined the effects of extracellular Na+and Ca2+, inhibitors of the Na+-coupled glucose transporter and Na+-Ca2+exchanger, a Ca2+channel blocker, and nonmetabolizable sugars on cell size. The surface area and diameter of the cells changed according to extracellular glucose concentrations. &agr;-Methyl glucoside, which enters the cell through the Na+-coupled glucose transporter, induced cellular contraction. However, the cells did not contract in response to 2-deoxyglucose, which enters the cell through a facilitated glucose transporter. Glucose-induced cellular contraction was abolished in the absence of extracellular Na+and Ca2+. Moreover, phlorizin, an inhibitor of the Na+-coupled glucose transporter, and 2′,4′-dichlorobenzamil-HCl, an inhibitor of the Na+-Ca2+exchanger, also abolished glucose-induced cellular contraction, whereas nicardipine, a Ca2+channel blocker, did not. Our results indicate that high extracellular glucose concentrations induce contraction of bovine retinal pericytes via Na+entry through a Na+-coupled glucose transporter, suggesting that the Na+-coupled glucose transporter may act as a functional glucose sensor of retinal microvascular circulation.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

The reentrant pathways underlying different types of atrioventricular (AV) nodal reentrant tachycardia have not yet been elucidated. This study was performed to optically map Koch’s triangle and surrounding atrial tissue in an isolated canine AV nodal preparation. Multiple preferential AV nodal input pathways were observed in all preparations (n=22) with continuous (73%, n=16) and discontinuous (27%, n=6) AV nodal function curves (AVNFCs). AV nodal echo beats (EBs) were induced in 54% (12/22) of preparations. The reentrant circuit of the slow/fast EB (36%, n=8) started as a block in fast pathway (FP) and a delay in slow pathway (SP) conduction to the compact AV node, then exited from the AV node to the FP, and rapidly returned to the SP through the atrial tissue located at the base of Koch’s triangle. The reentrant circuit of the fast/slow EB (9%, n=2) was in an opposite direction. In the slow/slow EB (9%, n=2), anterograde conduction was over the intermediate pathway (IP) and retrograde conduction was over the SP. Unidirectional conduction block occurred at the junction between the AV node and its input pathways. Conduction over the IP smoothed the transition from the FP to the SP, resulting in a continuous AVNFC. A “jump” in AH interval resulted from shifting of anterograde conduction from the FP to the SP (n=4) or abrupt conduction delay within the AV node through the FP (n=2). These findings indicate that (1) multiple AV nodal anterograde pathways exist in all normal hearts; (2) atrial tissue is involved in reentrant circuits; (3) unidirectional block occurs at the interface between the AV node and its input pathways; and (4) the IP can mask the existence of FP and SP, producing continuous AVNFCs.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Connexin43 (Cx43) is the principal connexin isoform in the mouse ventricle, where it is thought to provide electrical coupling between cells. Knocking out this gene results in anatomic malformations that nevertheless allow for survival through early neonatal life. We examined electrical wave propagation in the left (LV) and right (RV) ventricles of isolated Cx43 null mutated (Cx43−/−), heterozygous (Cx43+/−), and wild-type (WT) embryos using high-resolution mapping of voltage-sensitive dye fluorescence. Consistent with the compensating presence of the other connexins, no reduction in propagation velocity was seen in Cx43−/−ventricles at postcoital day (dpc) 12.5 compared with WT or Cx43+/−ventricles. A gross reduction in conduction velocity was seen in the RV at 15.5 dpc (in cm/second, mean [1 SE confidence interval], WT 9.9 [8.7 to 11.2], Cx43+/−9.9 [9.0 to 10.9], and Cx43−/−2.2 [1.8 to 2.7;P<0.005]) and in both ventricles at 17.5 dpc (in RV, WT 8.4 [7.6 to 9.3], Cx43+/−8.7 [8.1 to 9.3], and Cx43−/−1.1 [0.1 to 1.3;P<0.005]; in LV, WT 10.1 [9.4 to 10.7], Cx43+/−8.3 [7.8 to 8.9], and Cx43−/−1.7 [1.3 to 2.1;P<0.005]) corresponding with the downregulation of Cx40. Cx40 and Cx45 mRNAs were detectable in ventricular homogenates even at 17.5 dpc, probably accounting for the residual conduction function. Neonatal knockout hearts were arrhythmic in vivo as well as ex vivo. This study demonstrates the contribution of Cx43 to the electrical function of the developing mouse heart and the essential role of this gene in maintaining heart rhythm in postnatal life.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID