摘要:

Abstract—The humanether-a-gogo–related gene (hERG) encodes the &agr; subunit of the cardiac potassium currentIKr. Several mutations in hERG produce trafficking-deficient channels that may cause hereditary long-QT syndrome and sudden cardiac death. Although hERG currents have been studied extensively, little is known about the proteins involved in maturation and trafficking of hERG. Using immunoprecipitations, we show that the cytosolic chaperones heat shock protein (Hsp) 70 and Hsp90, but not Grp94, interact with hERG wild type (WT) during maturation. The specific Hsp90 inhibitor geldanamycin prevents maturation and increases proteasomal degradation of hERG WT, while reducing hERG currents in heterologous expression systems. In ventricular myocytes, inhibition of Hsp90 also decreasesIKr, whereas geldanamycin had no effect onIKsor heterologously expressed Kv2.1 and Kv1.5 currents. Both Hsp90 and Hsp70 interact directly with the core-glycosylated form of hERG WT present in the endoplasmic reticulum but not the fully glycosylated, cell-surface form. For the trafficking-deficient LQT2 mutants, hERG R752W and hERG G601S, interactions with Hsp90 and Hsp70 are increased as both mutants remained tightly associated with Hsp90 and Hsp70 in the endoplasmic reticulum. Incubation at lower temperature for R752W or with the hERG blocker astemizole for G601S dissociates channel-chaperone complexes and restores trafficking. In contrast, nonfunctional but trafficking-competent hERG G628S is released from chaperone complexes during maturation comparable to WT. We conclude that Hsp90 and Hsp70 are crucial for the maturation of hERG WT as well as the retention of trafficking-deficient LQT2 mutants. The full text of this article is available online at http://www.circresaha.org.

ISSN:0009-7330    

出版商:OVID    

年代:2003

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2003

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2003

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2003

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2003

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

Endothelial cells (ECs) undergo a finite number of cell divisions before growth arrest or replicative senescence, modulated in part by the proinflammatory cytokine, interleukin-1 (IL-1). IL-1 and its family members are expressed in human atherosclerotic vessels, mainly in the endothelium. EC replicative senescence and IL-1 have been associated with atherosclerosis. Genetic variants at the IL-1 locus have been associated with a variety of coronary phenotypes. In this study, we examined the relationship between the interleukin-1 receptor antagonist variable number tandem repeat allele 2 (IL-1RN*2*2) and EC replicative capacity. A significant decrease in EC cumulative population doublings (CPDs) was associated with the rare allele (IL-1RN*2*2) at IL-1RN, 8.56±0.97 (n=7) versus 13.14±1.00 (IL-1RN*1*1, n=20),P=0.0118. Proliferation of IL-1RN*2*2 ECs detected by Ki67 expression was also significantly reduced particularly at later passage, passage 6: 21.76±0.93% (n=6) versus 48.10±8.81% (IL-1RN*1*1, n=7) (P=0.0323) and passage 8: 22.48±3.08% (n=6) versus 42.29±3.06% (IL-1RN*1*1, n=7) (P=0.0028). IL-1RN*2 carriage was associated with increased numbers of senescent ECs. Basal apoptosis, telomerase activity, and telomere length were not different with respect to IL-1RN genotype. Addition of exogenous IL-1ra (1 ng/mL) increased CPDs in a number of human umbilical vein endothelial cell cultures and increased proliferating cells from 12.11±1.21% to 27.82±2.82% (P=0.0216, IL-1RN*2*2, passage 8, n=2). These data suggest genetic control of EC proliferation and life span by the IL-1 locus and imply that IL-1ra may have a function connected with EC growth.

ISSN:0009-7330    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

Abstract—Endothelin-1 (ET-1) is a 21–amino-acid potent vasoconstrictor peptide that is mainly produced by vascular endothelial cells. Expression of the ET-1 gene is subject to complex regulation by numerous factors, among which transforming growth factor-&bgr; (TGF-&bgr;) is one of the most important. It has been widely documented that TGF-&bgr; increases ET-1 mRNA and peptide levels. We have explored the mechanism by which TGF-&bgr; upregulates ET-1 expression in endothelial cells. Transcriptional activation of the ET-1 promoter accounted for the TGF-&bgr;–induced increase in ET-1 mRNA levels. We have identified within the ET-1 promoter two DNA elements indispensable for TGF-&bgr;–mediated induction of ET-1: an activator protein-1 (AP-1) site at −108/−102, known to be important for constitutive and induced expression, and a novel regulatory sequence located at −193/−171, which constitutes a specific binding site for Smad transcription factors. Mutation of both elements abolished TGF-&bgr; responsiveness. Binding of Smad3/Smad4 and c-Jun to their corresponding DNA elements was evidenced by electrophoretic mobility shift assays. Furthermore, the coactivator CREB-binding protein (CBP)/p300 was found to play an essential role in the induction of the gene. The simultaneous requirement for two distinct and independent DNA elements suggests that Smads and activator protein-1 functionally cooperate through CBP/p300 to mediate TGF-&bgr;–induced transcriptional activation of the ET-1 gene.

ISSN:0009-7330    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

Abstract—Previous studies have shown that integrin activation and fluid shear stress can modulate the activity of sterol regulatory element binding proteins (SREBPs) in vascular endothelial cells. We investigated the role of small GTPase Rho-mediated signal transduction pathway in this mode of SREBP activation. Fluid shear stress activates the Rho downstream effectors ROCK, LIM kinase (LIMK), and cofilin. The various negative mutants of RhoA, ROCK, LIMK, and cofilin can block the shear stress activation of SREBPs. The shear stress–activated SREBP depends on S2P proteases but not caspase-3. Mechanistically, the endoplasmic reticulum-to-Golgi transport of SREBP cleavage–activating protein requires the actin-based cytoskeleton and is enhanced by the Rho-ROCK-LIMK-cofilin pathway. By enhancing the SREBP-mediated cholesterol metabolism, this unique mechanism may contribute to endothelial cell functions under flow.

ISSN:0009-7330    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

Abstract—Activation of the nuclear factor of activated T-cell (NFAT) family of transcription factors is associated with changes in gene expression and myocyte function in adult cardiac and skeletal muscle. However, the role of NFATs in normal embryonic heart development is not well characterized. In this report, the function of NFATc3 and NFATc4 in embryonic heart development was examined in mice with targeted disruption of bothnfatc3andnfatc4genes. Thenfatc3−/−nfatc4−/−mice demonstrate embryonic lethality after embryonic day 10.5 and have thin ventricles, pericardial effusion, and a reduction in ventricular myocyte proliferation. Cardiac mitochondria are swollen with abnormal cristae, indicative of metabolic failure, but hallmarks of apoptosis are not evident. Furthermore, enzymatic activity of complex II and IV of the respiratory chain and mitochondrial oxidative activity are reduced innfatc3−/−nfatc4−/−cardiomyocytes. Cardiac-specific expression of constitutively active NFATc4 innfatc3−/−nfatc4−/−embryos prolongs embryonic viability to embryonic day 12 and preserves ventricular myocyte proliferation, compact zone density, and trabecular formation. The rescued embryos also maintain cardiac mitochondrial ultrastructure and complex II enzyme activity. Together, these data support the hypothesis that loss of NFAT activity in the heart results in a deficiency in mitochondrial energy metabolism required for cardiac morphogenesis and function.

ISSN:0009-7330    

出版商:OVID    

年代:2003

数据来源: OVID