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Aims The occurrence of connexin40 (Cx40) minor polymorphism (−44 G → A) was increased in patients with idiopathic atrial fibrillation (AF), although its effect on atrial Cx40 protein expression is unknown. We aimed to evaluate whether alterations in Cx40 are directly linked to the development of AF, we studied the effect of this polymorphism on Cx40 expression and distribution in patients without any history of AF and in patients who developed post-operative AF. Methods and Results Hundred and eight patients (mean age 67 ± 9 years), without a history of AF or conditions that predispose to AF, were included. During heart surgery, 10 cc blood was collected for DNA genotyping and the right atrial appendage was partly excised. Ten patients (9%) were homozygous for the minor allele (AA, Group 1), 30 (28%) were heterozygous (AG, Group 2), and 68 (63%) were non-carriers (GG, Group 3). Ten age- and sex-matched tissue samples per group were analysed for Cx40 expression by: (i) real-time quantitative polymerase chain reaction (Q-PCR), (ii) western blotting, and (iii) immunohistochemistry on cryosections. Real-time quantitative polymerase chain reaction showed no significant differences of Cx40 mRNA among the groups. Western blot analysis, however, revealed a reduction in Cx40 protein in Groups 1 (−36.4%) and 2 (−39.5%) as compared with Group 3. Immunohistochemistry confirmed this reduction but indicated an unaltered subcellular distribution of the remaining Cx40. Incidence of post-operative AF (28%) was age-dependent but unrelated to the presence of the polymorphism or fibrosis. Conclusion Presence of the Cx40 minor allele (−44 G → A) results in a uniform down-regulation of right atrial appendage Cx40 protein which was not significantly related to development of post-operative AF.

In the chronic complete atrioventricular (AV) block dog (CAVB) model, both bradycardia and altered ventricular activation due to the uncontrolled idioventricular rhythm contribute to ventricular remodeling and the enhanced susceptibility to Torsade de Pointes (TdP) arrhythmias. We investigated the effect of permanent bradycardic right ventricular apex (RVA) pacing on mechanical and electrical remodeling and TdP. In 23 anesthetized dogs, serial experiments were performed at sinus rhythm (SR), acutely after AV block (AAVB) and 3 weeks of remodeling CAVB at a fixed pacing rate of 60/min. ECG, and left (LV) and right ventricular (RV) monophasic action potentials durations (MAPD) were recorded; activation time (AT) and activation recovery interval (ARI) were determined from ten distinct LV electrograms; interventricular mechanical delay (IVMD) and time-to-peak strain (TTP) of the LV septal and lateral wall (ΔTTP: lateral wall minus septal wall) were obtained echocardiographically. Dofetilide (25 μg/kg/5 min) was infused to study TdP inducibility. In baseline AAVB, in comparison to SR, RVA bradypacing acutely increased QT interval, LV, and RVMAPD. Echocardiographic IVMD and ΔTTP were initially increased, which was partially corrected after 3 weeks of RVA pacing (IVMD: 22 ± 13 vs. 42 ± 11 vs. 31 ± 6 ms; ΔTTP: -2 ± 47 vs. -114 ± 38 vs. -36 ± 22 ms). QT interval (362 ± 23 vs. 373 ± 29 ms), LVMAPD (245 ± 18 vs. 253 ± 22 ms), RVMAPD (226 ± 26 vs. 238 ± 31 ms), and mean LV-ARI (268 ± 5 vs. 267 ± 6 ms) were not significantly changed after 3 weeks of RVA pacing. During AAVB, dofetilide increased mean LV-ARI (381 ± 11 ms) with largest increases in the later activated basal areas (slope AT-ARI: +0.96). In contrast with acute RVA pacing, 3 week pacing increased TdP inducibility (0/13 vs. 11/21) and mean LV-ARI (484 ± 18 ms), while the slope of AT-ARI responded differently on dofetilide (-2.37), with larger APD increases in the early region. The latter was supported at the molecular level: reduced RNA expressions of three repolarization-related ion channel genes in early (KCNQ1, KCNH2, and KCNJ2) versus two in late regions (KNCQ1 and KCNJ2). In conclusion, bradycardic RVA pacing acutely induced LV intra- and interventricular mechanical dyssynchrony, which was partially reversed after 3 weeks of pacing (remodeling). The latter occurred without apparent baseline electrical effects. However, dofetilide clearly unmasked (region-specific) arrhythmic consequences of remodeling.

Na(v)1.5, the pore forming α-subunit of the voltage-dependent cardiac Na(+) channel, is an integral membrane protein involved in the initiation and conduction of action potentials. Mutations in the gene-encoding Na(v)1.5, SCN5A, have been associated with a variety of arrhythmic disorders, including long QT, Brugada, and sick sinus syndromes as well as progressive cardiac conduction defect and atrial standstill. Moreover, alterations in the Na(v)1.5 expression level and/or sodium current density have been frequently noticed in acquired cardiac disorders, such as heart failure. The molecular mechanisms underlying these alterations are poorly understood, but are considered essential for conception of arrhythmogenesis and the development of therapeutic strategies for prevention or treatment of arrhythmias. The unravelling of such mechanisms requires critical molecular insight into the biology of Na(v)1.5 expression and function. Therefore, the aim of this review is to provide an up-to-date account of molecular determinants of normal Na(v)1.5 expression and function. The parts of the Na(v)1.5 life cycle that are discussed include (i) regulatory aspects of the SCN5A gene and transcript structure, (ii) the nature, molecular determinants, and functional consequences of Na(v)1.5 post-translational modifications, and (iii) the role of Na(v)1.5 interacting proteins in cellular trafficking. The reviewed studies have provided valuable information on how the Na(v)1.5 expression level, localization, and biophysical properties are regulated, but also revealed that our understanding of the underlying mechanisms is still limited.

The sodium channel isoform Na(v)1.5 mediates sodium current, excitability, and electrical conduction in the human heart. Recent studies have indicated alternative splicing within the protein-coding portion of its gene, SCN5A, as a mechanism to generate diversity in Na(v)1.5 protein structure and function. In the present study we identified several novel SCN5A transcripts in human heart, displaying distinct 5′-untranslated regions but identical protein-coding sequences. These transcripts originated from the splicing of alternative exons 1 (designated 1A, 1B, 1C, and 1D) to the translational start codon-containing exon 2, and were preferentially expressed in the heart as compared to other tissues. Comparison of their expression level between adult and fetal heart demonstrated that exon 1C- and 1D-derived sequences were more prominent in adult than in fetal heart. Two new promoters (designated P2 and P3) for the SCN5A gene were identified and functionally characterized in myocardial- and nonmyocardial-derived cell lines. Translation of the transcript containing exon 1D-derived sequences proved to be significantly impaired in these cell lines, which could be restored by mutation of an upstream translational start codon. These results implicate the usage of alternative promoters and 5′-untranslated regions as new mechanisms in the regulation of human Na(v)1.5 expression.

Aims Sudden arrhythmogenic cardiac death is a major cause of mortality in patients with congestive heart failure (CHF). To investigate determinants of the increased arrhythmogenic susceptibility, we studied cardiac remodelling and arrhythmogenicity in CHF patients and in a mouse model of chronic pressure overload. Methods and results Clinical and (immuno)histological data of myocardial biopsies from CHF patients with (VT+) and without (VT−) documented ventricular arrhythmia were compared with controls. In CHF patients, ejection fraction was decreased and QRS duration was increased. Cell size and interstitial fibrosis were increased, but Connexin43 (Cx43) levels, the most abundant gap junction in ventricular myocardium, were unchanged. No differences were found between VT+ and VT− patients, except for the distribution pattern of Cx43, which was significantly more heterogeneous in VT+. Mice were subjected to transverse aortic constriction (TAC) or sham operated. At 16 weeks, cardiac function was determined by echocardiography and epicardial ventricular activation mapping was performed. Transverse aortic constriction mice had decreased fractional shortening and prolonged QRS duration. Right ventricular conduction velocity was reduced, and polymorphic VTs were induced in 44% TAC and 0% sham mice. Interstitial fibrosis was increased and Cx43 quantity was unchanged in TAC mice with and without arrhythmias. Similar to CHF patients, heterogeneous Cx43 distribution was significantly associated with arrhythmias in TAC mice and with spatial heterogeneity of impulse conduction. Conclusion Heterogeneous Cx43 expression during CHF is associated with dispersed impulse conduction and may underlie enhanced susceptibility to ventricular tachyarrhythmias.

In the mammalian heart, the gap junction protein connexin45 (Cx45) has a characteristic spatiotemporal expression pattern and is involved in mediating the rapid spreading of the electrical impulse that precedes coordinated contraction. The aim of this study was to isolate and characterize the rat Cx45 gene and to investigate its expression pattern in various tissues and cell lines. The gene consists of four exons (termed E1a, E1b, E2, and E3), of which the complete protein-coding sequence as well as a small part of the 5' -untranslated region (5'-UTR) reside on E3. 5' -Rapid amplification of cDNA ends (5' -RACE) analysis demonstrated the existence of four transcripts, which all contained the same coding region (derived from E3) but differed in the composition of their 5'-UTR. Analysis of Cx45 RNA expression in various rat tissues and cultured cell lines revealed that the transcripts composed of either E1a, E2, and E3 (i.e., E1a/2/3) or of E1b, E2, and E3 (E1b/2/3) sequences are both ubiquitously expressed. Comparison of the rat Cx45 gene structure with its murine ortholog indicated both similarities and species-specific differences in Cx45 gene organization. These findings will allow for the mapping and characterization of the rat Cx45 gene regulatory regions.

Embryonic stem (ES) cells and embryonal carcinoma (EC) cells express high amounts of functional connexin43 (Cx43). During mesoderm formation and subsequent cardiac differentiation, Cx43 is initially down-regulated but is up-regulated again as the emerging cardiomyocytes mature. In this study, we investigated the regulation of Cx43 expression during early phases of differentiation in F9 and P19 EC cells. We found a striking inverse correlation between the expression of Cx43 and that of the transcriptional repressor Snail1. No clear relationship was found with Smad-interacting-protein1 (SIP1), another transcription factor inducing epithelium-to-mesenchyme transition (EMT). Promoter-reporter assays indicated Cx43 repression at the promoter level by ectopically expressed Snail1. To establish whether the Cx43 down-regulation depends on endogenous Snail1, MES-1 cells, differentiated derivatives of P19 EC, were stably transfected by an siRNA construct silencing Snail1 expression. This resulted in a mesenchyme-to-epithelium transition, which was accompanied by increased levels of Cx43 mRNA and protein and enhanced metabolic and electrical coupling. We conclude that Snail1-mediated EMT results in a Cx43 repression.

Background: Renal failure is associated with adverse cardiac remodeling and sudden cardiac death. The mechanism leading to enhanced arrhythmogenicity in the cardiorenal syndrome is unclear. The aim of this study was to characterize electrophysiological and tissue alterations correlated with enhanced arrhythmogenicity in two distinct mouse models of renal failure. Methods: Thirty-week-old 129Sv mice received a high-salt diet and deoxycorticosterone acetate (DOCA) for 8 weeks, followed by an additional period of high-salt diet for 27 weeks (DOCA-salt aged model). Adult CD-1 mice were submitted to 5/6-subtotal nephrectomy (SNx) and treated for 11 weeks with a high-salt diet (SNx-salt adult model). Vulnerability to arrhythmia as well as conduction velocities (CVs) of the hearts were determined ex vivo with epicardial mapping. Subsequently, the hearts were characterized for connexin 43 (Cx43) and fibrosis. Results: DOCA-salt and SNx-salt mice developed renal dysfunction characterized by albuminuria. Heart, lung and kidney weights were increased in DOCA-salt mice. Both DOCA-salt and SNx-salt mice were highly susceptible to ventricular arrhythmias. DOCA-salt mice had a significant decrease in both longitudinal and transversal CV in the left ventricle. Histological analysis revealed a significant reduction in Cx43 expression as well as an increase in interstitial fibrosis in both DOCA-salt and SNx-salt mice. Conclusion: DOCA-salt and SNx-salt treatment induced renal dysfunction, which resulted in structural and electrical cardiac remodeling and enhanced arrhythmogenicity. The reduced Cx43 expression and increased fibrosis levels in these hearts are likely candidates for the formation of the arrhythmogenic substrate.

Cardiac fibrosis is a major hallmark of cardiac diseases. For evaluation of cardiac fibrosis, the development of highly specific and preferably noninvasive methods is desired. Our aim was to evaluate CNA35, a protein known to specifically bind to collagen, as a specific marker of cardiac fibrosis. Fluorescently labeled CNA35 was applied ex vivo on tissue sections of fibrotic rat, mouse, and canine myocardium. After quantification of CNA35, sections were examined with picrosirius red (PSR) and compared to CNA35. Furthermore, fluorescently labeled CNA35 was administered in vivo in mice. Hearts were isolated, and CNA35 labeling was examined in tissue sections. Serial sections were histologically examined with PSR. Ex vivo application of CNA35 showed specific binding to collagen and a high correlation with PSR (Pearson r = .86 for mice/rats and r = .98 for canine; both p < .001). After in vivo administration, CNA35 labeling was observed around individual cardiomyocytes, indicating its ability to penetrate cardiac endothelium. High correlation was observed between CNA35 and PSR ( r = .91, p < .001). CNA35 specifically binds to cardiac collagen and can cross the endothelial barrier. Therefore, labeled CNA35 is useful to specifically detect collagen both ex vivo and in vivo and potentially can be converted to a noninvasive method to detect cardiac fibrosis.

Cardiac hypertrophic remodelling, initiated by signalling cascades in response to increased workload, injury or intrinsic disease, is initially adaptive. However, prolonged hypertrophy as a consequence of pathological stress leads to maladaptive changes that increase the risk for fatal ventricular arrhythmias. One of these changes is the remodelling of myocardial gap junctions, which provide for electrical coupling of adjacent cardiomyocytes. Myocardial gap junctions are composed of three connexin isotypes, connexin40 (Cx40), -43 (Cx43), and -45 (Cx45) and each display a characteristic developmental and regional expression pattern. Alterations in the distribution and expression of Cx43, the predominant isoform in the adult ventricles, has been the main focus of examination in humans, experimental animal models and cultured cardiomyocytes in response to hypertrophy. The molecular mechanisms and signalling pathways underlying these changes have been studied less thoroughly. In this review we summarize what is known about the remodelling of myocardial gap junctions during hypertrophy, the putative underlying mechanisms and functional consequences thereof.

Rapid spreading of the electrical impulse throughout the heart is essential for coordinated contraction and is mediated by electrical coupling of cardiomyocytes through gap junction channels composed of connexin40 (Cx40), connexin43 (Cx43) or connexin45 (Cx45). Each of these connexin proteins has a characteristic developmental and regional expression pattern in the heart. Alterations in this pattern may result in abnormal cellular coupling and consequently contribute to irregularities in cardiac rhythm. Indeed, alterations in cardiac connexin expression have been correlated with cardiovascular disease for which the molecular mechanisms, however, are largely unknown. Transcription factors and their target elements in the genome regulate the expression of genes during development and in response to extracellular signals in a cell type-specific and quantitative manner. Altered transcriptional regulation of gene expression is a characteristic feature in the development of cardiac disease which may influence the connexin expression pattern as well. In this review, we will summarize what is known on transcriptional regulation of the Cx40, Cx43 and Cx45 genes in general, with an emphasis on the heart.

Altered transcriptional control is likely to contribute to the down-regulation of connexin 43 (Cx43) expression observed in many forms of heart disease. However, little is known about the factors regulating Cx43 transcription in the heart under (patho)physiological conditions. Therefore, a systematic study of rat Cx43 (rCx43) proximal promoter regulation in rat primary neonatal ventricular cardiomyocytes (NCM) and, for comparison, different cell types was initiated. Luciferase assays revealed that, in NCM, the proximal promoter is preserved in a conserved region extending from 148 nucleotides upstream towards 281 nucleotides downstream relative to the transcription initiation site (TIS). Further deletional analysis suggested the involvement of four putative Sp- and two AP1-binding sites. The binding of both Sp1 and Sp3 to the Sp-binding elements and AP1 to the AP1-binding elements was demonstrated by electrophoretic mobility shift assays (EMSA). Promoter-luciferase assays using the natural rCx43 proximal promoter and mutated derivatives in NCM, HL-1 and A7r5 cells revealed that all sites contribute to basal promoter activity. Trans-activation of the Cx43 proximal promoter with Sp1 and Sp3 in Drosophila Schneider line 2 (SL2) cells demonstrated that Sp1 and, to a lesser extent, Sp3 determine rCx43 promoter activation. Thus Sp1, Sp3 and AP1 determine basal Cx43 expression. In addition, we studied the effect of the cardiac transcription factor Nkx2.5 on Cx43 regulation. NCM were infected with adenovirus encoding either beta-galactosidase (control) or Nkx2.5. Cx43 protein and mRNA were significantly decreased after Nkx2.5 infection as shown by Western and Northern blot analyses. Promoter-reporter assays demonstrated that the rCx43 promoter was down-regulated approximately twofold upon Nkx2.5 overexpression. Therefore, in NCM, Nkx2.5 appears to play a role in the regulation of Cx43 expression.

Objectives: The gap junction protein connexin40 (Cx40) is differentially expressed during embryonic development and in adult tissues, for which the molecular basis is unknown. In order to elucidate the molecular mechanisms controlling Cx40 expression, we set out to map and characterize its promoter. Methods: The transcriptional activity of individual rat Cx40 (rCx40)-derived promoter fragments fused to the luciferase reporter gene was determined by transfection/reporter assays in Cx40-expressing (A7r5, rat smooth muscle embryonic thoracic aorta cells, and BWEM, v- myc transformed rat fetal cardiomyocytes) and Cx40-nonexpressing cells (N2A, mouse neuroblastoma cells). The nature of DNA–protein interactions was investigated by a combination of standard electrophoretic-mobility-shift assays (EMSA) and EMSA/antibody supershift assays. Results: Quantification of luciferase activity in cell lysates revealed that a 235-base-pair fragment, in between map positions −150 and +85 relative to the transcription initiation site, is able to provide for a significant level of transcription in both Cx40-expressing (A7r5, BWEM) and -nonexpressing (N2A) cells. These results indicate that this region contains the basal promoter but is not sufficient to completely determine the endogenous Cx40-expression pattern within these cell types. In search for the responsible transcriptional regulatory element(s), additional segments of the (−150, +85) region were deleted and the remaining fragments were tested for transcriptional activity. These studies established that the regions in between map positions (−96, −71) and (+58, +85) contribute to promoter activity. EMSA with these regions revealed that predominantly two DNA–protein complexes are formed upon incubation with either A7r5, BWEM or N2A nuclear extracts, which could be both inhibited by including excess oligonucleotide containing the Sp1 consensus binding site in the binding reaction. Purified recombinant human Sp1 provided also for a shift in the EMSA using these promoter regions as target fragments. When the DNA–protein complexes formed with nuclear extract were subsequently incubated with either an anti-Sp1 or an anti-Sp3 antibody clear supershifts in the EMSA were obtained, indicating Sp1 and Sp3 binding to both the (−98, −64) and (+53, +87) regions. The introduction of mutations within the core sequence of the putative Sp1/Sp3 binding sites present in these regulatory elements reduced the level of transcriptional activity and abrogated Sp1/Sp3 binding to these sites. Conclusion: The results indicate that at least two Sp1/Sp3 binding sites in the rCx40 promoter contribute to the transcriptional activation of its gene in cultured cells.

The effects of cytokine stimulation during retroviral transduction on in vivo reconstitution of mouse hematopoietic stem cells was tested in a murine competitive repopulation assay with alpha-thalassemia as a marker to distinguish donor and recipient red blood cells (RBCs) and the enhanced green fluorescent protein (EGFP) as a marker for gene transfer. After transplantation, EGFP was detected in up to 90% of circulating RBCs, platelets, and leukocytes, and in primitive progenitors in bone marrow (BM), spleen, and thymus of individual transplanted mice for observation periods of more than 6 months. Large quantitative differences in reconstitution were observed after transplantation with graded numbers (1000-30, 000) of EGFP(+) cells preconditioned with various combinations of Kit ligand (KL), FLT-3 ligand (FL), thrombopoietin (TPO), interleukin 3 (IL-3), and IL-11. Relative to nonmanipulated BM cells, repopulation of EGFP(+) cells was maintained by KL/FL/TPO stimulation, but approximately 30-fold reduced after KL/FL/TPO/IL-3, or KL/FL/IL-3/IL-11. These differences were not caused by changes in the ability of immature hematopoietic cells to home to the BM, which was only moderately reduced. In conclusion, these quantitative transplantation studies of mice demonstrate the importance of optimal ex vivo cytokine stimulation for gene transfer to stem cells with retention of their in vivo hematopoietic potential, and also emphasize that overall in vitro transduction frequency does not predict gene transfer to repopulating stem cells.

The feasibility of using the enhanced green fluorescent protein (EGFP) as a selectable reporter molecule of retroviral-mediated gene transfer in immature rhesus monkey and human CD34+ hematopoietic cells was examined. Retroviral transduction with the MFG-EGFP retroviral vector resulted in readily detectable EGFP expression in 27% of human and 11-35% of rhesus monkey bone marrow cells, and in 17-38% of rhesus monkey peripheral blood cells mobilized with FLT3 ligand (FL) and granulocyte colony-stimulating factor (G-CSF). In addition, we used the human CD34+ KG1A cell line as a model to study viability and growth of successfully transduced cells. Cultures of mock- and EGFP-transduced KG1A cells generated equal viable cell numbers for at least 1 month, indicating the absence of a cytotoxic effect of EGFP expression in these cells. FACS selection on the basis of EGFP and CD34 expression resulted in enriched subsets (> or = 87%) of CD34+ EGFP-negative and CD34+ EGFP-positive KG1A, rhesus monkey and human bone marrow cells, demonstrating the potential of obtaining almost pure populations of transduced immature hematopoietic cells. EGFP expression was also readily demonstrated in erythroid and granulocyte/macrophage colonies derived from the CD34+ EGFP-positive rhesus monkey and human bone marrow cells by either inverted fluorescence microscopy or flow cytometry. Using four-color flow cytometry, EGFP expression could also be demonstrated in viable and phenotypically defined immature subpopulations of the CD34+ cells, ie those expressing little or no HLA-DR (rhesus monkey) or CD38 (human) antigens at the cell surface. These results demonstrate that EGFP is a very useful marker to monitor gene transfer efficiency in phenotypically defined immature rhesus monkey and human hematopoietic cell types and to select for these cells by multicolor flow cytometry prior to transplantation.

Purified CD34+ and CD34+CD38− human umbilical cord blood (UCB) cells were transduced with the recombinant variant of Moloney murine leukemia virus (MoMLV) MFG-EGFP or with SF-EGFP, in which EGFP expression is driven by a hybrid promoter of the spleen focus-forming virus (SFFV) and the murine embryonic stem cell virus (MESV). Infectious MFG-EGFP virus was produced by an amphotropic virus producer cell line (GP+envAm12). SF-EGFP was produced in the PG13 cell line pseudotyped for the gibbon ape leukemia virus (GaLV) envelope proteins. Using a 2-day growth factor prestimulation, followed by a 2-day, fibronectin fragment CH-296–supported transduction, CD34+ and CD34+CD38− UCB subsets were efficiently transduced using either vector. The use of the SF-EGFP/PG13 retroviral packaging cell combination consistently resulted in twofold higher levels of EGFP-expressing cells than the MFG-EGFP/Am12 combination. Transplantation of 105 input equivalent transduced CD34+ or 5 × 103input equivalent CD34+CD38− UCB cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in median engraftment percentages of 8% and 5%, respectively, which showed that the in vivo repopulating ability of the cells had been retained. In addition, mice engrafted after transplantation of transduced CD34+ cells using the MFG-EGFP/Am12 or the SF-EGFP/PG13 combination expressed EGFP with median values of 2% and 23% of human CD45+ cells, respectively, which showed that the NOD/SCID repopulating cells were successfully transduced. EGFP+ cells were found in all human hematopoietic lineages produced in NOD/SCID mice including human progenitors with in vitro clonogenic ability. EGFP-expressing cells were also detected in the human cobblestone area–forming cell (CAFC) assay at 2 to 6 weeks of culture on the murine stromal cell line FBMD-1. During the transduction procedure the absolute numbers of CAFC week 6 increased 5- to 10-fold. The transduction efficiency of this progenitor cell subset was similar to the fraction of EGFP+ human cells in the bone marrow of the NOD/SCID mice transplanted with MFG-EGFP/Am12 or SF-EGFP/PG13 transduced CD34+ cells, ie, 6% and 27%, respectively. The study thus shows that purified CD34+ and highly purified CD34+CD38− UCB cells can be transduced efficiently with preservation of repopulating ability. The SF-EGFP/PG13 vector/packaging cell combination was much more effective in transducing repopulating cells than the MFG-EGFP/Am12 combination.

The further improvement of gene transfer into hematopoietic stem cells and their direct progeny will be greatly facilitated by markers that allow rapid detection and efficient selection of successfully transduced cells. For this purpose, a retroviral vector was designed and tested encoding a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP). Murine cell lines (NIH 3T3, Rat2) and bone marrow cells transduced with this retroviral vector demonstrated a stable green fluorescence signal readily detectable by flow cytometry. Functional analysis of the retrovirally transduced bone marrow cells showed EGFP expression in in vitro clonogenic progenitors (GM-CFU), day 13 colony-forming unit-spleen (CFU-S), and in peripheral blood cells and marrow repopulating cells of transplanted mice. In conjunction with fluorescence-activated cell sorting (FACS) techniques EGFP expression could be used as a marker to select for greater than 95% pure populations of transduced cells and to phenotypically define the transduced cells using antibodies directed against specific cell-surface antigens. Detrimental effects of EGFP expression were not observed: fluorescence intensity appeared to be stable and hematopoietic cell growth was not impaired. The data show the feasibility of using EGFP as a convenient and rapid reporter to monitor retroviral-mediated gene transfer and expression in hematopoietic cells, to select for the genetically modified cells, and to track these cells and their progeny both in vitro and in vivo.

In mice, the calcium-dependent phosphatase calcineurin A (CnA) induces a transcriptional pathway leading to pathological cardiac hypertrophy. Interestingly, induction of CnA has been frequently noticed in human hypertrophic and failing hearts. Independently, the arrhythmia vulnerability of such hearts has been regularly associated with remodeling of parameters determining electrical conduction (expression level of connexin43 (Cx43) and NaV1.5, connective tissue architecture), for which the precise molecular basis and sequence of events is still unknown. Recently, we observed reduced Cx43 and NaV1.5 expression in 4-week old mouse hearts, overexpressing a constitutively active form of CnA (MHC-CnA model), but the order of events is still unknown. Therefore, three key parameters of conduction (Cx43, NaV1.5 and connective tissue expression) were characterized in MHC-CnA ventricles versus wild-type (WT) during postnatal development on a weekly basis. At postnatal week 1, CnA overexpression induced cardiac hypertrophy in MHC-CnA. Moreover, protein and RNA levels of both Cx43 and NaV1.5 were reduced by at least 50% as compared to WT. Cx43 immunoreactive signal was reduced at week 2 in MHC-CnA. At postnatal week 3, Cx43 was less phosphorylated and RNA level of Cx43 normalized to WT values, although the protein level was still reduced. Additionally, MHC-CnA hearts displayed substantial fibrosis relative to WT, which was accompanied by increased RNA levels for genes previously associated with fibrosis such as Col1a1, Col1a2, Col3a1, Tgfb1, Ctgf, Timp1 and microRNA miR-21. In MHC-CnA, reduction in Cx43 and NaV1.5 expression thus coincided with overexpression of CnA and hypertrophy development and preceded significant presence of fibrosis. At postnatal week 4 the alterations in conductional parameters observed in the MHC-CnA model lead to abnormal conduction and arrhythmias, similar to those observed in cardiac remodeling in heart failure patients. The MHC-CnA model, therefore, provides for a unique model to resolve the molecular origin of conductional remodeling in detail.

Connexin 43 (Cx43) is the predominant isoform of gap junction proteins in the working myocardium. In the heart, mitogen-activated protein (MAP) kinases are implicated in regulating Cx43 remodeling; however, their precise roles remain obscure. Mitogen-activated protein kinase kinase 4 (MKK4) is a critical component of the stress-activated MAP kinase signaling pathway. We have previously demonstrated that MKK4 antagonizes cardiomyocyte hypertrophy. Herein, we investigate the role of MKK4 in regulating Cx43 expression in cardiomyocytes. We found that knockdown of MKK4 expression or inhibition of its kinase activity in neonatal rat cardiomyocytes (NRCMs) significantly reduced phenylephrine-induced Cx43 expression. Furthermore, two activator protein-1 (AP-1) binding elements in the Cx43 promoter region were identified as being responsible for the MKK4-regulated Cx43 expression. Consistently, we also detected heterogeneously reduced Cx43 expression and attenuated zonula occludens-1 (ZO-1) content in the hearts of MKK4 cardiomyocyte-specific knockout mice (MKK4cko) following pressure overload. To test whether heterogeneously reduced Cx43 expression contributes to ventricular arrhythmic vulnerability, MKK4cko and control mice were subjected to pressure overload followed by programmed electrical stimulation (PES). 6 of 13 MKK4cko mice, but none of the controls, developed ventricular tachycardia. Epicardial activation mapping recorded from the MKK4cko hypertrophied heart showed ventricular activation delay. Mathematical models have simulated that the spatially heterogeneous decrease in Cx43 causes slowed ventricular conduction and fragmented wave propagations leading to re-entrant excitations. Collectively, these data reveal a novel role for MKK4 in regulating Cx43 expression and preventing hypertrophy-associated arrhythmogenesis.

Alterations in expression levels of Na(v)1.5, Cx43 and Cx40 have been frequently reported in cardiac disease and are associated with the development of arrhythmias, but little is known about the underlying molecular mechanisms. In this study we investigated electrical conduction and expression of Na(v)1.5, Cx43 and Cx40 in hearts of transgenic mice overexpressing a constitutively active form of calcineurin (MHC-CnA). ECG recordings showed that atrial, atrioventricular and ventricular activation were significantly prolonged in MHC-CnA hearts as compared to wildtype (WT) littermates. Epicardial activation and arrhythmia susceptibility analysis revealed increased ventricular activation thresholds and arrhythmia vulnerability. Moreover, epicardial ventricular activation patterns in MHC-CnA mice were highly discontinuous with multiple areas of block. These impaired conduction properties were associated with severe reductions in Na(v)1.5, Cx43 and Cx40 protein expression in MHC-CnA hearts as visualized by immunohistochemistry and immunoblotting. Real-time RT-PCR demonstrated that the decreased protein levels for Na(v)1.5 and Cx40, but not for Cx43, were accompanied by corresponding reductions at the RNA level. Cx43 RNA isoform analysis indicated that the reduction in Cx43 protein expression is caused by a post-transcriptional mechanism rather than by RNA isoform switching. In contrast, RNA isoform analysis for Cx40 and Na(v)1.5 provided additional evidence that in calcineurin-induced hypertrophy the downregulation of these proteins originates at the transcriptional level. These results provide the molecular rationale for Na(v)1.5, Cx43 and Cx40 downregulation in this model of hypertrophy and failure and the development of the pro-arrhythmic substrate.

Expression of the tissue-specific gap junction protein connexin(Cx)40 is regulated by the interaction of ubiquitous and tissue-specific factors such as Sp1 and GATA4. Cardiac Cx40 expression is altered under pathological conditions such as atrial fibrillation. A human promoter polymorphism, a G → A change at position − 44 that has been associated with atrial-specific arrhythmias, is located between the TBE-NKE-Sp and GATA consensus transcription factor binding sites important for the regulation of the mouse Cx40 gene. The presence of the A-allele at position − 44 in promoter–reporter constructs significantly reduces promoter activity. Using electrophoretic mobility shift assays and luciferase reporter assays in various cell types, we show that Sp1 and GATA4 are important regulators of human Cx40 gene transcription and that the − 44 G → A polymorphism negatively affects the promoter regulation by the transcription factors Sp1 and GATA4.

The rat gap junction protein connexin40 (rCx40) has a characteristic developmental and regional expression pattern, for which the exact regulatory mechanisms are not known. To identify the molecular factors controlling Cx40 expression, its proximal promoter was characterized. The proximal rCx40 promoter is the most conserved noncoding region within the Cx40-gene known thus far and contains five potential binding sites for Sp-family transcription factors. The binding of both Sp1 and Sp3 to each of these DNA elements was demonstrated by EMSA. Luciferase assays of the natural rCx40 proximal promoter or mutated derivatives in Cx40-expressing (NCM, primary rat neonatal cardiomyocytes and A7r5, rat smooth muscle embryonic thoracic aorta cells) and -nonexpressing cells (N2A, mouse neuroblastoma cells) revealed that all sites are contributing to basal promoter activity. Trans-activation assays in Drosophila Schneider line 2 cells demonstrated that Sp1 and Sp3 activate the rCx40 proximal promoter in a dose-dependent and additive manner.

Background Cardiac fibrosis has severe adverse effects on systolic and diastolic function and increases the risk of arrhythmias. To detect cardiac fibrosis, the development of highly specific and preferably noninvasive methods is desired. In this study, the potential of labeled CNA35, a protein that specifically binds to vascular collagen, is investigated as a specific marker of cardiac fibrosis. Picrosirius red (PSR) staining served as reference for cardiac fibrosis detection. Methods Fluorescently labeled CNA35 (2.5 µM) was applied to frozen tissue sections of dog myocardium (n = 36). After CNA35 quantification, sections were histologically examined with PSR and compared to CNA35. Furthermore, fluorescently labeled CNA35 (2 × 38 µM) was administered intravenously in mice (n = 8). Hearts were isolated, and CNA35 labeling was examined in frozen tissue sections (n = 34). Serial sections were examined with PSR. Results CNA35 application on frozen canine myocardium shows specific CNA35 binding to collagen. CNA35 labeling highly correlates with PSR (r = 0.98, P in vivo administration in mice, myocardial CNA35 labeling is clearly observed, suggesting that labeled CNA35 is able to penetrate the cardiac endothelium. Strong correlation is observed between CNA35 and PSR (r = 0.91, P Conclusions CNA35 specifically binds to cardiac collagen when applied to frozen sections and after in vivo application of CNA35. Strong correlation has been observed between CNA35 and PSR. These data indicate that CNA35 is useful for quantification and localization of myocardial fibrosis and warrants further research into its use for monitoring cardiac fibrosis in vivo .

In many mammalian cells, efficient introduction of genetic material can be accomplished with recombinant retroviral vectors. Retrovirus vectors have also been used as vehicles for gene therapy studies, since many therapeutic applications require integration of genes into cellular DNA (15, 35). Important potential targets for gene modification are hematopoietic stem cells that have the ability to establish long-lived and multilineage reconstitution of hematopoiesis in mammals (23, 40). Despite advantages of both retroviral vectors and hematopoietic stem cells as tools for genetic therapy, retroviral gene transfer into primitive stem cells of large animals via retrovirus vectors has been problematic, and the potential therapeutic use of gene transfer technology in human diseases remains largely unfulfilled (30). Retrovirus-mediated gene transfer into human hematopoietic stem cells is influenced by multiple factors, including low viral titer (12, 25, 44) and the cycle status of the target stem cells (36). Recently, increasing attention has been focused on the level of expression of viral receptors in these primitive cells. Two different retroviral receptors have been extensively used for targeting cells of the human hematopoietic system: the amphotropic murine leukemia virus (MLV) receptor and the gibbon ape leukemia virus (GALV) receptor (31). Both receptors have been characterized as sodium-dependent phosphate symporters (5, 45). Significant sequence similarity exists between the amphotropic receptor and the GALV receptor, although these receptors bind virion particles without cross-interference (37). The level of expression of the amphotropic receptor, Pit-2, has been measured by mRNA levels and appears to be low in human and primate CD34+ hematopoietic cells (21, 43, 51) and in murine hematopoietic stem cells (42). This has led to speculation that a low level of expression of the receptor protein is responsible, at least in part, for low-level transduction with amphotropic-packaged vectors (21, 42, 49). In contrast, the mRNA level of the GALV receptor, Pit-1, appears to be higher than that of the amphotropic receptor in CD34+ cells isolated from nonhuman primates (21). Thus the levels of mRNA of these two receptors appear to correlate with the respective levels of infection in different cells by recombinant retroviral vectors (21, 49). Kiem et al. (22) have exploited this difference to improve transduction of primate hematopoietic stem cells with GALV-pseudotyped vectors. Previous studies have demonstrated that the capacity of retroviruses to bind their cognate receptor is sensitive to a variety of manipulations. During infection with replication-competent retrovirus, receptor occupancy by envelope protein interferes with subsequent reinfection of cells, a process termed superinfection interference (48). Transduction with replication-defective viruses, which lack envelope protein coding sequences, has rarely been reported to be associated with receptor interference (28). However, receptor interference may be particularly important with respect to the functional consequences of low receptor density in hematopoietic cells, a major target population for gene therapy applications. Since viral occupancy of the cognate receptor is a critical component of efficient infection, our laboratory has exploited colocalization of viral particles and target cells via interaction with fragments of the extracellular matrix protein fibronectin (FN) to increase transduction of a variety of mammalian cells, including hematopoietic cells (17, 39, 46). In a recent study, we noted that continuous exposure of human hematopoietic cells to virus in the presence of the peptide fragment FN CH-296 prior to induction of cell cycling led to a transduction efficiency significantly inferior to that when the cells were stimulated with cytokines prior to virus exposure (16). Since current clinical protocols frequently utilize multiple infections targeting the same receptor, the possibility that receptor expression or receptor interference may play a role in inefficient transduction of hematopoietic cells, particularly in the presence of fibronectin, could be critical to gene therapy protocols. In the study reported here, hematopoietic cell lines and primary human CD34+ hematopoietic cells were infected on FN CH-296 simultaneously with distinguishable retroviral vectors pseudotyped with either the same or different envelope proteins. Simultaneous infection with two viruses pseudotyped with the same envelope protein was associated with inhibition of transduction both in human erythroleukemia (HEL) cells and in human CD34+ primary hematopoietic cells. This inhibition was attributable to receptor interference, since no inhibition could be demonstrated during simultaneous infection by two vectors utilizing different receptors for cell entry. Receptor interference continued for up to 24 h after the initial exposure to virus. In contrast, simultaneous infection with MLV amphotropic and GALV-pseudotyped vectors yielded transduction efficiencies consistently higher than in infection with either vector alone in both target cells. These studies demonstrate that, in the presence of FN CH-296, functional receptor density limits transduction of hematopoietic cells and sequential infection of these cells within a short period of time provides little additional quantitative transduction benefit. Finally, simultaneous infection with vectors targeting different receptors may prove to be an additional approach for further improving transduction of hematopoietic targets for gene therapy protocols.

Retroviral vectors are widely used for the introduction of exogenous genetic material into hematopoietic cells. Here we report the generation of retroviral vectors containing the Aequorea victoria green fluorescent protein (GFP) gene and improved versions thereof. Murine fibroblasts transduced with the mutant GFP genes demonstrated a distinct green fluorescent signal in fluorescence-activated cell sorter (FACS) analysis. The relative intensities of peak green fluorescence observed with different GFP mutants were in the order EGFP>hGFP(S65T)> GFP-PTS1 or RSGFP>wildtype GFP (wtGFP). Furthermore, GFP-PTS1 expression was observed in murine (3T3, Rat2, and freshly-cultured bone marrow) and human (K562) cells transduced with the corresponding retroviral vector. The GFP-PTS1 positive phenotype could be selected for by FACS and appeared to be stable for at least 1 month in murine fibroblasts and human K562 cells. Therefore, these GFP variants are convenient selectable markers to monitor retroviral-mediated gene transfer and expression in mammalian hematopoietic cells.

The largest genome segment, RNA 1, of alfalfa mosaic virus forms complexes with viral coat protein. These complexes were subjected to digestion with ribonucleases T1 or A and filtered onto Millipore filters. Specific fragments were collected from the filters by phenol extraction. After electrophoretic separation in denaturing polyacrylamide gels, these fragments were sequenced. Besides extracistronic fragments originating from the 3'-terminal region of RNA 1, fragments were found originating from an intracistronic region of the RNA. A striking phenomenon is that the intracistronic fragments were not found when ribonuclease A was used to degrade RNA/protein complexes. The findings are in agreement with the postulation of Houwing and Jaspars (1978), that a conformational change at the 3' ends of the genome RNAs induced by the coat protein is a prerequisite to start an infection cycle.

Trypsinized coat protein of alfalfa mosaic virus lacking 25 amino acids at its N terminus still has the capability to form complexes with RNA which are detectable by sedimentation in sucrose gradients. However, it does not protect specific sites on the RNA against degradation by ribonuclease, as the native coat protein does (D. Zuidema, M. F. A. Bierhuizen, B. J. C. Cornelissen, J. F. Bol, and E. M. J. Jaspars (1983) Virology 125, 361-369.). The trypsinized coat protein has lost the capacity of the native coat protein to make the genome RNAs of alfalfa mosaic virus infectious or to interfere with the infectivity brought about by the native coat protein. These findings suggest that genome activation occurs via binding of the N-terminal part of the coat protein to specific sites on the RNAs.