Recombinant adenoviruses were used to overexpress green fluorescent protein (GFP)‐fused auxiliary Ca2+ channel β subunits (β1‐β4) in cultured adult rat heart cells, to explore new dimensions of β subunit functions in vivo. Distinct β‐GFP subunits distributed differentially between the surface sarcolemma, transverse elements, and nucleus in single heart cells. All β‐GFP subunits increased the native cardiac whole‐cell L‐type Ca2+ channel current density, but produced distinctive effects on channel inactivation kinetics. The degree of enhancement of whole‐cell current density was non‐uniform between β subunits, with a rank order of potency β2aαβ4 > β1b > β3. For each β subunit, the increase in L‐type current density was accompanied by a correlative increase in the maximal gating charge (Qmax) moved with depolarization. However, β subunits produced characteristic effects on single L‐type channel gating, resulting in divergent effects on channel open probability (Po). Quantitative analysis and modelling of single‐channel data provided a kinetic signature for each channel type. Spurred on by ambiguities regarding the molecular identity of the actual endogenous cardiac L‐type channel β subunit, we cloned a new rat β2 splice variant, β2b, from heart using 5′ rapid amplification of cDNA ends (RACE) PCR. By contrast with β2a, expression of β2b in heart cells yielded channels with a microscopic gating signature virtually identical to that of native unmodified channels. Our results provide novel insights into β subunit functions that are unattainable in traditional heterologous expression studies, and also provide new perspectives on the molecular identity of the β subunit component of cardiac L‐type Ca2+ channels. Overall, the work establishes a powerful experimental paradigm to explore novel functions of ion channel subunits in their native environments.

Krü ppel-like factor 4 (KLF4) is an epithelial cell-enriched, zinc finger-containing transcription factor, the expression of which is associated with growth arrest. Previous studies show that constitutive expression of KLF4 inhibits DNA synthesis but the manner by which KLF4 exerts this effect is unclear. In the present study, we developed a system in which expression of KLF4 is controlled by a promoter that is induced upon treatment of cells containing the receptors for the insect hormone, ecdysone, with ponasterone A, an ecdysone analogue. The rate of proliferation of a stably transfected colon cancer cell line, RKO, was significantly decreased following addition of ponasterone A when compared with untreated cells. Flow cytometric analyses indicated that the inducible expression of KLF4 caused a block in the G 1 /S phase of the cell cycle. A similar block was observed when ecdysone receptor-containing RKO cells were infected with a replication-defective recombinant adenovirus containing an inducible KLF4 and treated with ponasterone A. Results of these studies provide evidence that the inhibitory effect of KLF4 on cell proliferation is mainly exerted at the G 1 /S boundary of the cell cycle.

The cyclin D1 gene is overexpressed in human breast cancers and is required for oncogene-induced tumorigenesis. Peroxisome proliferator-activated receptor ␥ (PPAR␥) is a nuclear receptor selectively activated by ligands of the thiazolidinedione class. PPAR␥ induces hepatic steatosis, and liganded PPAR␥ promotes adipocyte differentiation. Herein, cyclin D1 inhibited ligand-induced PPAR␥ function, transactivation, expression, and promoter activity. PPAR␥ transactivation induced by the ligand BRL49653 was inhibited by cyclin D1 through a pRB-and cdk-independent mechanism, requiring a region predicted to form an helix-loop-helix ( The cyclin-dependent kinase holoenzymes are a family of serine/threonine kinases that play a pivotal role in controlling progression through the cell cycle (38,47). Dysregulation of the cell cycle control apparatus is an almost uniform aberration in tumorigenesis (48). The cyclins encode regulatory subunits of the kinases which phosphorylate specific proteins, including the retinoblastoma (pRB) protein, to promote transition through specific cell cycle checkpoints (47, 57). Cyclin D1 plays a pivotal role in G 1 /S phase cell cycle progression in fibroblasts and is rate limiting in growth factor-or estrogen-induced mammary epithelial cell proliferation (29, 67). Cyclin D1 overexpression is found in Ͼ30% of human breast cancers, correlating with poor prognosis (23). Several different oncogenic signals induce cyclin D1 expression, including mutations of the Ras and Wnt/APC/␤-catenin pathway (2, 49). Mammary-targeted expression of cyclin D1 is sufficient for the induction of mammary adenocarcinoma, and cyclin D1 Ϫ/Ϫ mice are resistant to ErbB2-induced tumorigenesis (53,64).In addition to binding cyclin-dependent kinases 4 and 6 (cdk4 and cdk6) and pRB, cyclin D1 forms physical associations with P/CAF (p300/CBP-associated factor), Myb, MyoD, and the cyclin D1 myb-like binding protein (DMP1) (16,20,31,39). Binding of cyclin D1 to the estrogen receptor alpha (ER␣) enhances ligand-independent reporter gene activity, and liganded androgen receptor reporter gene activity is inhibited by cyclin D1 (33, 39, 68). The in vivo or genetic evidence indicating a requirement for cyclin D1 in nuclear receptor function remained to be determined. The peroxisome proliferator-activator receptors, including PPAR␣, PPAR␥, and PPAR␦, are ligand-activated nuclear receptors (42). Their modular structure resembles those of other nuclear hormone receptors with N-terminal AF-1, a DNA binding domain, and a carboxyl-terminal ligand-binding domain (LBD). PPAR␥ was cloned as a transcription factor involved in fat cell differentiation and is required for the induction of adipocyte differentiation (41, 51). Adenoviral delivery of PPAR␥ to the livers of mice induces hepatic steatosis, consistent with an important role for PPAR␥ in hepatocellular lipid biosynthesis (65). The PPAR␥ ligands include eicosanoids, such as 15-deoxy-⌬12,14-prostaglandin J2 (15d-PGJ 2 ), and synthetic ligands of the thiazolidinedione (TZD) class. PPAR␥ ...

Graded, reversible suppression of neuronal excitability represents a logical goal of therapy for epilepsy and intractable pain. To achieve such suppression, we have developed the means to transfer "electrical silencing" genes into neurons with sensitive control of transgene expression. An ecdysone-inducible promoter drives the expression of inwardly rectifying potassium channels in polycistronic adenoviral vectors. Infection of superior cervical ganglion neurons did not affect normal electrical activity but suppressed excitability after the induction of gene expression. These experiments demonstrate the feasibility of controlled ion channel expression after somatic gene transfer into neurons and serve as the prototype for a novel generalizable approach to modulate excitability.

Uncoupling protein 2 (UCP-2) mRNA expression has been shown to be altered by metabolic conditions such as obesity in humans, but its functional significance is unknown. The expression of UCP-2 mRNA and protein in normal rat islets was established by reverse transcriptase-polymerase chain reaction and immunocytochemistry in pancreatic islets and tissue, respectively. Intense immunostaining of UCP-2 correlated with insulin-positive ,-cells. Overexpression of UCP-2 in normal rat islets was accomplished by infection with an adenovirus (AdEGI-UCP-2) containing the full-length human UCP-2 coding sequence. Induction of the AdEGI-UCP-2 gene resulted in severe blunting of glucose-stimulated insulin secretion (GSIS) without affecting islet insulin content or the ability of the calcium ionophore A23187 to increase insulin secretion from AdEGI-UCP-2-expressing islets. Therefore, UCP-2 overexpression affects signal transduction proximal to Ca2+-mediated steps, including exocytosis. Insulin secretion from single beta-cells to 16.5 mmol/l glucose examined by reverse hemolytic plaque assay was nearly ablated if UCP-2 was overexpressed. Thus, a direct, causal relationship between overexpression of UCP-2 and inhibition of GSIS in normal islets has been established. These data suggest that increased expression of UCP-2 has the potential to cause the lack of a glucose effect on insulin secretion in type 2 diabetes.

Abstract-Previous studies have demonstrated a role for Kv4 ␣ subunits in the generation of the fast transient outward K ϩ current, I to,f , in the mammalian myocardium. The experiments here were undertaken to explore the role of homomeric/heteromeric assembly of Kv4.2 and Kv4.3 and of the Kv channel accessory subunit, KChIP2, in the generation of mouse ventricular I to,f . Western blots reveal that the expression of Kv4.2 parallels the regional heterogeneity in I to,f density, whereas Kv4.3 and KChIP2 are uniformly expressed in adult mouse ventricles. Antisense oligodeoxynucleotides (AsODNs)

To probe the molecular identity of transient outward (A-type) potassium currents, we expressed a truncated version of Kv4.2 in heart cells and neurons. The rat Kv4.2-coding sequence was truncated at a position just past the first transmembrane segment and subcloned into an adenoviral shuttle vector downstream of a cytomegalovirus promoter (pE1Kv4.2ST). We hypothesized that this construct would act as a dominant-negative suppressor of currents encoded by the Kv4 family by analogy to Kv1 channels. Cotransfection of wild-type Kv4.2 with a ␤-galactosidase expression vector in Chinese hamster ovary (CHO)-K1 cells produced robust transient outward currents (I to ) after two days (14.0 pA/pF at 50 mV, n ‫؍‬ 5). Cotransfection with pE1Kv4.2ST markedly suppressed the Kv4.2 currents (0.8 pA/pF, n ‫؍‬ 6, p < 0.02; cDNA ratio of 2:1 Kv4.2ST:wild type), but in parallel experiments, it did not alter the current density of coexpressed Kv1.4 or Kv1.5 channels. Kv4.2ST also effectively suppressed rat Kv4.3 current when coexpressed in CHO-K1 cells. We then engineered a recombinant adenovirus (AdKv4.2ST) designed to overexpress Kv4.2ST in infected cells. A-type currents in rat cerebellar granule cells were decreased two days after AdKv4.2ST infection as compared with those infected by a ␤-galactosidase reporter virus (116.0 pA/pF versus 281.4 pA/pF in Ad ␤-galactosidase cells, n ‫؍‬ 8 each group, p < 0.001). Likewise, I to in adult rat ventricular myocytes was suppressed by AdKv4.2ST but not by Ad␤-galactosidase (8.8 pA/pF versus 21.4 pA/pF in ␤-galactosidase cells, n ‫؍‬ 6 each group, p < 0.05). Expression of a GFP-Kv4.2ST fusion construct enabled imaging of subcellular protein localization by confocal microscopy. The protein was distributed throughout the surface membrane and intracellular membrane systems. We conclude that genes from the Kv4 family are the predominant contributors to the A-type currents in cerebellar granule cells and I to in rat ventricle. Overexpression of dominant-negative constructs may be of general utility in dissecting the contributions of various ion channel genes to excitability.

Gene therapy for common myocardial diseases will require effective and homogeneous gene delivery throughout the intact heart. We created two experimental models to identify and optimize parameters important for adenovirus-mediated cardiac gene transfer. In cultured rabbit ventricular myocytes, the percentage of infected cells increased with higher absolute numbers of virus particles, longer durations of virus exposure, physiological temperatures, and specific culture media compositions. Simulating the in vitro conditions, we delivered adenovirus to intact rabbit hearts by intracoronary perfusion. The percentage of infected cells increased with higher coronary f low rates, longer virus exposure times, and higher virus concentrations. Under optimal conditions, nearly 100% of myocytes expressed the reporter gene ␤-galactosidase after ex vivo infection. This novel delivery method, the first to demonstrate virtually complete transduction of any intact organ, could be adapted to achieve widespread gene transfer in vivo.Diseases such as congestive heart failure, familial hypertrophic cardiomyopathy, and the long QT syndrome result from alterations of myocardial function on a cellular or subcellular level, and effecting a cure will necessitate modification of a majority of the diseased cells. Adenovirus-mediated gene transfer has been used to introduce recombinant genes to cardiac myocytes, offering the potential to treat both rare and common cardiac disorders. Previous attempts utilized intramyocardial injection of viral vectors and achieved locally intense gene delivery, limited to an area within 1-2 mm of the needle track (1, 2). More diffuse but less effective gene transfer was demonstrated with coronary arterial delivery, either by percutaneous in vivo delivery or in explanted hearts prior to cardiac transplantation (3)(4)(5). Despite all of these attempts, no strategy yet devised has achieved high levels of homogeneous gene expression throughout the intact heart. These observations motivated us to evaluate infection conditions in a more controlled environment to identify parameters that would increase infection efficiency.Our work identifies several key variables that influence recombinant adenoviral gene transfer in cultured cardiac myocytes and intact hearts. We first evaluated infection conditions in primary cultures of adult rabbit ventricular myocytes using recombinant adenoviruses encoding the reporter gene for either ␤-galactosidase (␤-gal) (Ad␤gal) or luciferase (AdLuc). Infection of cultured cells is most effective at 37ЊC in crystalloid solutions with elevated virus concentrations and virus-to-cell ratios. Using such conditions in intact hearts, we achieve reporter gene expression in 96% of cardiac myocytes infected by coronary perfusion. If appropriately modified for in vivo applications, this delivery strategy would be a viable method for gene therapy in the heart and other solid organs. MATERIALS AND METHODSAdenovirus Vectors. Ad␤gal contained the Escherichia coli lac Z gene driven by the huma...