摘要:

&NA;Cardiac myocytes are electrically coupled by gap junctions, clusters of low‐resistance intercellular channels composed of connexins. Variations in the quantities and spatial distribution of different connexin types have been implicated in regional differentiation of electrophysiological properties in the heart. Although independent studies have demonstrated that connexin43 is abundant in working ventricular myocardium and that connexin40 is preferentially expressed in the atrioventricular conduction system of a number of species, information on the spatial distribution of connexin45 in the heart is limited to data obtained using an antibody raised to a single peptide sequence. In the present study, we report on the production and characterization of a new anti‐connexin45 antibody and its application to the investigation of connexin45 expression in mouse and rat myocardium. The affinity‐purified antiserum, raised in guinea pig to residues 354 to 367 of human connexin45, recognized a single 45‐kD band on Western blots of HeLa cells transfected to express connexin45 and gave punctate immunolabeling at the cell borders, demonstrated by freeze‐fracture cytochemistry to represent gap junctions. Only low levels of connexin45 mRNA were detected on Northern blots of mouse and rat cardiac tissues, and connexin45 protein levels were below the limit of detection on Western blots. Confocal microscopy of immunolabeled ventricular tissue revealed that the major part of the working myocardium was immunonegative for connexin45. A clearly defined zone containing connexin45‐expressing cells was, however, localized to the endocardial surface, overlapping with connexin40‐expressing myocytes of the conduction system. As these results contrast with the prevailing view that connexin45 is widely distributed in working ventricular myocytes, we compared the immunolabeling pattern obtained with a commercially supplied anti‐connexin45 antiserum raised against the same peptide that was used in previous studies. The commercial connexin45 antiserum gave widespread labeling throughout the ventricular myocardium, but this labeling was inhibited by a six‐amino acid peptide matching part of the connexin43 sequence, indicating cross‐reaction of the commercial connexin45 antiserum with connexin43 in the tissue. Further evidence for such cross‐reactivity came from observations on connexin43‐transfected cells, which gave positive immunolabeling with the commercial anti‐connexin45 antiserum. Our demonstration of a specific association of connexin45 with connexin40‐expressing myocytes in rat and mouse ventricle raises the possibility that connexin45 contributes to the modulation of electrophysiological properties in the ventricular conduction system and highlights the need for reappraisal of the distribution and role of connexin45 in other species. (Circ Res. 1998;82:232‐243.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;This study was designed to demonstrate the characteristic pattern of angiotensin II‐induced JAK/STAT (indicating just another kinase/signal transducer and activator of transcription) activation in cultured rat cardiomyocytes by comparing it with leukemia inhibitory factor (LIF)‐induced activation. Angiotensin II (10‐7mol/L) induced rapid phosphorylation of JAK2 and Tyk2, but not JAK1, and phosphorylated STAT1 and STAT2, but not STAT3, in the early stage up to 30 minutes. The time course of JAK/STAT activation by angiotensin II was apparently slower than that by LIF. Interestingly, angiotensin II phosphorylated STAT3 and rephosphorylated STAT1 in the late stage at 120 minutes. We also found that angiotensin II induced the formation of interferon‐stimulating gene factor (ISGF) complexes biphasically, in the early stage at 15 to 30 minutes and in the late stage at 120 minutes, and that angiotensin II induced delayed activation of the sis‐inducing factor (SIF) complex at 120 minutes. Formation of ISGF and SIF complexes in response to angiotensin II paralleled the phosphorylation pattern of STAT1 and STAT3 and was quite different from those obtained in response to LIF. The phosphorylation of STAT1 was suppressed by pretreatment with the angiotensin II type‐1 (AT1) receptor antagonist CV11974, but the delayed addition of CV11974 failed to suppress phosphorylation of STAT3 at 120 minutes. In conclusion, angiotensin II‐induced JAK/STAT activation in rat cardiomyocytes is biphasic and entirely different from LIF‐induced activation. (Circ Res. 1998;82:244‐250.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;To determine how myocardial terminal differentiation is regulated by cell cycle control genes, we studied cdc2 expression in rat cardiac muscle and found that cdc2 mRNA and protein levels were reduced in neonatal compared with fetal ventricles and became undetectable in juvenile and adult ventricles. To further determine whether cdc2 downregulation is attributed to a decrease in transcription, transient expression assay was performed using the progressively truncated 6.2‐, 1.8‐, 1.1‐, 0.7‐, and 0.1‐kb human cdc2 5 prime flanking regions. All five fragments activated reporter expression in fetal myocytes and were significantly less active in neonatal myocytes. The 0.1‐kb fragment showed 65% of the activity of the 6.2‐kb fragment. A protein binding site that contains an inverted CCAAT box was identified within the 0.1‐kb fragment by DNase I footprint assay and named the cdc2 promoter binding factor (CPBF) site. Point mutations within the CPBF site that abolish CPBF binding significantly decreased both 0.1‐ and 6.2‐kb promoter activities. Competition and antibody supershift assays suggested that CPBF was identical or related to the transcription factor, nuclear factor Y (NF‐Y). The 0.1‐kb promoter activity was suppressed by a dominant‐negative NF‐Y mutant in fetal myocytes. Taken together, our results demonstrate that cardiac cdc2 expression is downregulated after birth and turned off when the juvenile stage is attained. A 0.1‐kb promoter fragment of cdc2 contains major information for both cdc2 transcriptional activation and suppression in fetal and neonatal myocytes, respectively. NF‐Y or its related factor plays a critical role in activating the 0.1‐kb cdc2 promoter. (Circ Res. 1998;82:251‐260.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;Our objective in experiments reported here was to identify myofilament proteins of rat hearts either lost or degraded by cardiac ischemia (15‐ or 60‐minute duration) with and without 45 minutes of reperfusion. We correlated these changes with alterations in myofilament sensitivity to Ca2+and maximum force generation. Protein degradation and loss were assessed by high‐performance liquid chromatography, SDS‐PAGE, Western blotting analysis, and amino acid sequencing. Compared with nonischemic control hearts, bundles of skinned fibers from hearts subjected to ischemia alone demonstrated a decrease in maximum force generation and an increase in sensitivity to Ca2+. These changes in function were increased with the duration of the ischemia and with reperfusion. With increasing duration of ischemia, there was an increased loss and degradation of myofibrillar alpha‐actinin and troponin I (TnI) at its C‐terminus. alpha‐Actinin and TnI were most susceptible to ischemia, but with 60 minutes of ischemia/reperfusion, there was also degradation of myosin light chain‐1 (MLC1) involving a clip of residues 1 to 19. The MLC1 degradation product was detected in the reperfusion effluent (along with troponin T, tropomyosin, and alpha‐actinin) but not in the tissue with 60 minutes of ischemia with no reperfusion. Moreover, with ischemia the following proteins became associated with the myofibrils: GAPDH and proteins of the mitochondrial ATP synthase complex. Our results provide new evidence regarding the mechanism by which ischemia/reperfusion causes myocardial injury and support the hypothesis that an important element in the injury is altered activity and structure of the myofilaments. (Circ Res. 1998;82:261‐271.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;A defining property of ATP‐sensitive K+(KATP) channels is inhibition by sulfonylurea drugs, yet the response of cardiac KATPchannels toward sulfonylureas during myocardial ischemia is not consistent. Altered channel sensitivity toward sulfonylureas has, in part, been ascribed to antagonism by cytosolic nucleotide diphosphates, although the mechanism of interaction remains unclear. Herein, in inside‐out patches excised from cardiomyocytes, we observed a dual response of KATPchannels toward the sulfonylurea drug, glyburide, in the presence of cytosolic UDP. Specifically, glyburide failed to inhibit spontaneous KATPchannel activity in the presence of UDP but inhibited UDP‐induced channel activity after rundown of spontaneous channel openings. Such behavior of KATPchannels cannot be explained by differences in the level of channel activity or by UDP‐induced displacement of glyburide. Rather, the dual response toward the sulfonylurea could be attributed to a property of KATPchannels to switch between operative conditions (spontaneous versus UDP‐induced) each associated with a distinct responsiveness toward ligands. Conversion of post‐rundown KATPchannels to the spontaneously operative channel condition, by Mg‐ATP, restored the ability of UDP to antagonize the inhibitory action of glyburide lost after rundown, suggesting that the response of the channel to glyburide is phosphorylation dependent. The existence of distinct operative conditions of cardiac KATPchannels could be the basis for the inconsistent response of the channel toward sulfonylurea drugs and should be considered when sulfonylureas are used to implicate the opening of KATPchannels in the myocardium. (Circ Res. 1998;82:272‐278.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;The excitable gap of a reentrant circuit has both temporal (time during the cycle length that the circuit is excitable) and spatial (length of the circuit that is excitable at a given time) properties. We determined the temporal and spatial properties of the excitable gap in reentrant circuits caused by nonuniform anisotropy. Myocardial infarction was produced in canine hearts by ligation of the left anterior descending coronary artery. Four days later, reentrant circuits were mapped in the epicardial border zone of the infarcts with a multielectrode array during sustained ventricular tachycardia induced by programmed stimulation. During tachycardia, premature impulses were initiated by stimulation at sites around and in the reentrant circuits, and their conduction characteristics in the circuit were mapped. All circuits had a temporal excitable gap in at least part of the circuit, which allowed premature impulses to enter the circuit. Completely and partially excitable segments of the temporal gap were identified by measuring conduction velocity of the premature impulses; conduction was equal to the native reentrant wave front in completely excitable regions and slower than the reentrant wave front in partially excitable regions. In some circuits, a temporal gap existed throughout the circuit, permitting the entire circuit to be reset over a range of premature coupling intervals, although the size of the gap varied at different sites. In other circuits, the gap became so small at local sites that even though premature impulses could enter the circuit, the circuit could not be reset. Premature impulses could terminate reentry in circuits that could be reset or not. We also found a significant spatial gap, which was identified by determining the distance between the head of the circulating wave front, which could be located on the activation map, and its tail, which was the site most distal from the head as located by the site of entry of the premature wave front into the circuit. The spatial gap could also vary in different parts of the circuit. Therefore, nonuniform anisotropic reentrant circuits have both a temporal and spatial excitable gap with fully and partially excitable components that change in different parts of the circuit. (Circ Res. 1998;82:279‐293.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID