TBX18 overexpression enhances pacemaker function in a rat subsidiary atrial pacemaker model of sick sinus syndrome.

KEY POINTS: The sinoatrial node (SAN) is the primary pacemaker of the heart. SAN dysfunction, or 'sick sinus syndrome', can cause excessively slow heart rates and pauses, leading to exercise limitation and syncope, currently treated by implantation of an electronic pacemaker. 'Biopacemaking' utilises gene therapy to restore pacemaker activity by manipulating gene expression. Overexpressing the HCN pacemaker ion channel has been widely used with limited success. We utilised bradycardic rat subsidiary atrial pacemaker tissue to evaluate alternative gene targets: the Na+ /Ca2+ exchanger NCX1, and the transcription factors TBX3 and TBX18 known to be involved in SAN embryonic development. TBX18 overexpression restored normal SAN function, as assessed by increased rate, improved heart rate stability and restoration of isoprenaline response. TBX3 and NCX1 were not effective in accelerating the rate of subsidiary atrial pacemaker tissue. Gene therapy targeting TBX18 could therefore have the potential to restore pacemaker function in human sick sinus syndrome obviating electronic pacemakers. ABSTRACT: The sinoatrial node (SAN) is the primary pacemaker of the heart. Disease of the SAN, sick sinus syndrome, causes heart rate instability in the form of bradycardia and pauses, leading to exercise limitation and syncope. Biopacemaking aims to restore pacemaker activity by manipulating gene expression, and approaches utilising HCN channel overexpression have been widely used. We evaluated alternative gene targets for biopacemaking to restore normal SAN pacemaker physiology within bradycardic subsidiary atrial pacemaker (SAP) tissue, using the Na+ /Ca2+ exchanger NCX1, and the transcription factors TBX3 and TBX18. TBX18 expression in SAP tissue restored normal SAN function, as assessed by increased rate (SAN 267.5 ± 13.6 bpm, SAP 144.1 ± 8.6 bpm, SAP-TBX18 214.4 ± 14.4 bpm; P < 0.001), improved heart rate stability (standard deviation of RR intervals fell from 39.3 ± 7.2 ms to 6.9 ± 0.8 ms, P < 0.01; root mean square of successive differences of RR intervals fell from 41.7 ± 8.2 ms to 6.1 ± 1.2 ms, P < 0.01; standard deviation of points perpendicular to the line of identity of Poincaré plots (SD1) fell from 29.5 ± 5.8 ms to 7.9 ± 2.0 ms, P < 0.05) and restoration of isoprenaline response (increases in rates of SAN 65.5 ± 1.3%, SAP 28.4 ± 3.4% and SAP-TBX18 103.3 ± 10.2%; P < 0.001). These changes were driven by a TBX18-induced switch in the dominant HCN isoform in SAP tissue, with a significant upregulation of HCN2 (from 1.01 × 10-5  ± 2.2 × 10-6 to 2.8 × 10-5  ± 4.3 × 10-6 arbitrary units, P < 0.001). Biophysically detailed computer modelling incorporating isoform-specific HCN channel electrophysiology confirmed that the measured changes in HCN abundance could account for the observed changes in beating rates. TBX3 and NCX1 were not effective in accelerating the rate of SAP tissue.

Adenovirus-mediated gene transfer of fibromodulin inhibits neointimal hyperplasia in an organ culture model of human saphenous vein graft disease.

Poor long-term graft patency remains a major limitation of coronary artery bypass grafting using saphenous vein aortocoronary grafts. Neointimal hyperplasia (NIH) represents the principal mechanism of graft failure; a substantial body of evidence implicates transforming growth factor-beta1 (TGF-beta1) in the pathogenesis of NIH. The small leucine-rich proteoglycans decorin and fibromodulin possess TGF-beta-antagonist activity to differing extents and with differing avidities for the isoforms of TGF-beta. We compared their ability to inhibit NIH in an ex vivo model of human saphenous vein organ culture following adenovirus-mediated gene transfer. Surgically prepared human saphenous vein segments received adenovirus expressing fibromodulin (Ad5-Fmod), decorin (Ad5-Dcn), beta-galactosidase (Ad5-lacZ) or vehicle-only. Computerized morphometry 14 days after infection revealed significantly reduced neointimal area, neointimal thickness and intima/media ratio in Ad5-Fmod- and Ad5-Dcn-infected veins. Each parameter was significantly smaller in Ad5-Fmod- than in Ad5-Dcn-exposed segments. Fibrillar collagen content and levels of biologically active TGF-beta were lower in vessels receiving Ad5-Fmod or Ad5-Dcn than in those receiving Ad5-lacZ or vehicle-only. Fibromodulin is a more potent inhibitor of NIH in cultured human saphenous vein than decorin and offers potential therapeutic benefits in saphenous vein graft failure (and possibly in other forms of accelerated atherosclerosis) by reduction of associated neointima formation.

Upregulated TRPC1 channel in vascular injury in vivo and its role in human neointimal hyperplasia.

Occlusive vascular disease is a widespread abnormality leading to lethal or debilitating outcomes such as myocardial infarction and stroke. It is part of atherosclerosis and is evoked by clinical procedures including angioplasty and grafting of saphenous vein in bypass surgery. A causative factor is the switch in smooth muscle cells to an invasive and proliferative mode, leading to neointimal hyperplasia. Here we reveal the importance to this process of TRPC1, a homolog of Drosophila transient receptor potential. Using 2 different in vivo models of vascular injury in rodents we show hyperplasic smooth muscle cells have upregulated TRPC1 associated with enhanced calcium entry and cell cycle activity. Neointimal smooth muscle cells after balloon angioplasty of pig coronary artery also express TRPC1. Furthermore, human vein samples obtained during coronary artery bypass graft surgery commonly exhibit an intimal structure containing smooth muscle cells that expressed more TRPC1 than the medial layer cells. Veins were organ cultured to allow growth of neointimal smooth muscle cells over a 2-week period. To explore the functional relevance of TRPC1, we used a specific E3-targeted antibody to TRPC1 and chemical blocker 2-aminoethoxydiphenyl borate. Both agents significantly reduced neointimal growth in human vein, as well as calcium entry and proliferation of smooth muscle cells in culture. The data suggest upregulated TRPC1 is a general feature of smooth muscle cells in occlusive vascular disease and that TRPC1 inhibitors have potential as protective agents against human vascular failure.

Adenovirus-mediated gene transfer of a secreted transforming growth factor-beta type II receptor inhibits luminal loss and constrictive remodeling after coronary angioplasty and enhances adventitial collagen deposition.

BACKGROUND: Extracellular matrix (ECM) remodeling is central to the development of restenosis after coronary angioplasty (PTCA). As a regulator of ECM deposition by vascular cells, substantial evidence implicates transforming growth factor-beta1 (TGF-beta1) in the pathogenesis of restenosis. We investigated the effects of intracoronary expression of a transgenic antagonist of TGF-beta1 on luminal loss after PTCA. METHODS AND RESULTS: Porcine coronary arteries were randomized to receive a recombinant adenovirus expressing a secreted form of TGF-beta type II receptor (Ad5-RIIs), an adenovirus expressing beta-galactosidase (Ad5-lacZ), or vehicle only by intramural injection at the site of PTCA. Computerized morphometry 28 days after angioplasty revealed a greater minimum luminal area in Ad5-RIIs-injected arteries (1.71+/-0.12 mm(2)) than in the Ad5-lacZ (1.33+/-0.13 mm(2)) or vehicle-only (1.08+/-0.17 mm(2); P=0.010 by ANOVA) groups. This was accompanied by greater areas within the internal (P=0.013) and external (P=0.031) elastic laminae in Ad5-RIIs-treated vessels. Adventitial collagen content at the site of injury was increased in the Ad5-RIIs group, in contrast to decreases in the Ad5-lacZ and vehicle-only groups (P=0.004). CONCLUSIONS: Adenovirus-mediated antagonism of TGF-beta1 at the site of PTCA reduces luminal loss after PTCA by inhibiting constrictive remodeling. Antagonism of TGF-beta1 stimulates the formation of a dense collagenous adventitia, which prevents constrictive remodeling by acting as an external scaffold. These findings demonstrate the potential of gene therapy-mediated antagonism of TGF-beta1 as prophylactic therapy for restenosis.

Gene therapy for restenosis--what now, what next?

Late luminal loss after coronary angioplasty has resisted pharmacological and physical attempts at prevention for over twenty years. As a consequence of the resistance of restenosis to traditional therapeutic approaches it has become a popular target for DNA-based treatment modalities. In this review we consider what is currently known of the basic pathophysiology of restenosis and briefly outline the previous attempts to influence the long-term outcome after coronary intervention. We then discuss the animal models of vascular injury that have been used for studies of gene therapy and the vectors that have been applied to the setting of vascular gene transfer before considering the many studies in which the effects of specific gene transfer have been studied in the setting of vascular injury. These transgenes are considered in four broad groupings: those that act by the suppression of cellular proliferation in the vessel wall; those that inhibit cell migration; anti-thrombotic transgenes; and transgenes that have multiple effects within the vessel. We finally consider why, although more than eight years have passed since publication of the first studies of gene transfer to inhibit the vascular responses to endoluminal injury, little progress has been made in translating gene therapy for restenosis into the human setting. Principle reasons for the disappointingly slow clinical implementation of gene therapy for restenosis are an incomplete understanding of the vascular biology of restenosis, the difficulty of translating findings in animal models into the human setting and the technical difficulties incumbent upon localised gene delivery into coronary arteries.

Effects of hypothalamic lesions on courtship and copulatory behavior in sexual and unisexual whiptail lizards.

Lesions of the anterior hypothalamus-medial preoptic area (AH-MPOA) impair courtship and copulatory behavior in male Cnemidophorus inornatus, a sexually reproducing species of whiptail lizard, and male-like pseudosexual behaviors in C. uniparens, an all-female species. These results suggest that, since C. inornatus is a direct ancestor to C. uniparens, the neural structures responsible for mediating male-typical courtship and copulatory behavior in male whiptails are conserved in the evolution of the all-female parthenogen.

Coronary angiography and percutaneous coronary intervention in the porcine model: a practical guide to the procedure.

Assessment of safety and efficacy within the porcine coronary artery model remains a standard requirement for new therapies delivered to the coronary arteries before proceeding to clinical testing. Human coronary procedures carry a very low mortality rate; however, procedural mortality for porcine experiments is often high, despite these animals being young and free of atherosclerosis. Some of these deaths are due to poor technique, and therefore avoidable. However, despite the wide use of this model, a systematic description of the procedure has never been published. This article will detail how porcine angiography and stent implantation is performed in our institution and will discuss the relevant differences between humans and pigs with regard to anaesthesia, pharmacotherapy, vascular access, catheter selection and angiographic views. Important variations to the technique that have been reported are also covered.

Rat hippocampal neurons express genes for both rod retinal and olfactory cyclic nucleotide-gated channels: novel targets for cAMP/cGMP function.

Cyclic nucleotide-gated (CNG) channels are Ca(2+)-permeable, nonspecific cation channels that can be activated through direct interaction with cAMP and/or cGMP. Recent electrophysiological evidence for these channels in cultured hippocampal neurons prompted us to investigate the expression of CNG channel genes in hippocampus. PCR amplification detected the expression of transcripts for subunit 1 of both the rod photoreceptor (RCNGC1) and the olfactory receptor cell (OCNGC1) subtype of CNG channel in adult rat hippocampus. In situ hybridization detected expression of both channel subtypes in most principal neurons, including pyramidal cells of the CA1 through CA3 regions and granule cells of the dentate gyrus. From the hybridization patterns, we conclude that the two genes are colocalized in individual neurons. Comparison of the patterns of expression of type 1 cGMP-dependent protein kinase and the CNG channels suggests that hippocampal neurons can respond to changes in cGMP levels with both rapid changes in CNG channel activity and slower changes induced by phosphorylation. Future models of hippocampal function should include CNG channels and their effects on both electrical responses and intracellular Ca2+ levels.

Widespread expression of olfactory cyclic nucleotide-gated channel genes in rat brain: implications for neuronal signalling.

The cyclic nucleotides cAMP and cGMP are important intracellular messengers involved in a wide variety of signal transduction events in the nervous system. It has been proposed that cAMP/cGMP elicit some of their effects through direct gating of a novel class of Ca2+ -permeable ion channels that are termed cyclic nucleotide-gated (CNG) channels. Previous studies have identified the expression of a gene encoding one major CNG channel subtype, the olfactory receptor neuron alpha subunit, in the brain [El-Husseini et al. (1995) NeuroReport 6:1331-1335; Kingston et al. (1996a) Proc. Natl. Acad. Sci. U.S.A. 93:10440-10445; Bradley et al. (1997) J. Neurosci. 17:1993-2085]. We, therefore, proposed that the actions of cAMP/cGMP on neurons in the brain might occur through the activation of these CNG channels. To determine how widespread such a function might be, the regional and cellular distribution of the olfactory CNG channel alpha subunit has been examined in detail. Primers for multiple portions of the olfactory CNG channel were used in polymerase chain reaction (PCR) to amplify cDNA reverse-transcribed from several brain regions. The identities of PCR products were confirmed with Southern blots and by sequencing. In situ hybridization experiments demonstrated localization of CNG channel mRNA in discrete neuronal populations throughout the brain. In agreement with previous work, relatively strong hybridization signals are present in neuronal cell bodies of the cerebellum, olfactory bulb, cerebral cortex, and brainstem. Additionally, somewhat lesser signals are found in thalamus, hypothalamus, midbrain, and spinal cord while no hybridization signal was detectable in the caudate nucleus. This surprisingly wide distribution throughout the rat brain strengthens the hypothesis that CNG channels may influence numerous processes as downstream effectors of cyclic nucleotide cascades. Interestingly, the distribution of CNG channels is very similar to that of the nitric oxide/cGMP system, suggesting that one function of CNG channels in the brain could be to link diffusible messengers to elevated Ca2+ entry into neurons.

Gene transfer into neural cells in vitro using adenoviral vectors.

Adenoviruses (Ads) have become a very attractive and versatile vector system for delivering genes into brain cells in vitro and in vivo. One of the main attractions of Ads is that they can mediate gene transfer into post-mitotic cells, i.e. neurons. Ads are easy to grow and manipulate, stable, and their biology is very well understood. This unit is designed to help newcomers into the field, to design, prepare and grow replication-defective recombinant adenovirus vectors with the aim of transferring genes into neurons and glial cells in primary culture. It provides step-by-step methods describing the preparation of brain cell cultures, their infection using recombinant adenovirus vectors and also the assessment of transgene expression using a variety of techniques including fluorescence immunocytochemistry and fluorescence activated cell-sorting (FACS) analysis. The methods described will be useful to scientists wishing to enter the adenovirus field to construct adenovirus vectors to be used for gene transfer into neural cells.

A novel combination of promoter and enhancers increases transgene expression in vascular smooth muscle cells in vitro and coronary arteries in vivo after adenovirus-mediated gene transfer.

Recombinant adenoviruses are employed widely for vascular gene transfer. Vascular smooth muscle cells (SMCs) are a relatively poor target for transgene expression after adenovirus-mediated gene delivery, however, even when expression is regulated by powerful, constitutive viral promoters. The major immediate-early murine cytomegalovirus enhancer/promoter (MIEmCMV) elicits substantially greater transgene expression than the human cytomegalovirus promoter (MIEhCMV) in all cell types in which they have been compared. The Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) increases transgene expression in numerous cell lines, and fragments of the smooth muscle myosin heavy chain (SMMHC) promoter increase expression within SMC from heterologous promoters. We therefore, compared the expression of beta-galactosidase after adenovirus-mediated gene transfer of lacZ under the transcriptional regulation of a variety of combinations of the promoters and enhancers described, in vitro and in porcine coronary arteries. We demonstrate that inclusion of WPRE and a fragment of the rabbit SMMHC promoter along with MIEmCMV increases beta-galactosidase expression 90-fold in SMC in vitro and approximately 40-fold in coronary arteries, compared with vectors in which expression is regulated by MIEhCMV alone. Expression cassette modification represents a simple method of improving adenovirus-mediated vascular gene transfer efficiency and has important implications for the development of efficient cardiovascular gene therapy strategies.