Alexander M. Simon

Diverse functions of vertebrate gap junctions

Gap junctions are clusters of intercellular channels between adjacent cells. The channels are formed by the direct apposition of oligomeric transmembrane proteins, permitting the direct exchange of ions and small molecules (< 1 kDa) between cells without involvement of the extracellular space. Vertebrate gap junction channels are composed of oligomers of connexins, an enlarging family of proteins consisting of perhaps > 20 members. This article reviews recent advances in understanding the structure of intercellular channels and describes the diverse functions attributable to gap junctions as a result of insights gained from targeted gene disruptions in mice and genetic disease in humans.

Mice lacking connexin40 have cardiac conduction abnormalities characteristic of atrioventricular block and bundle branch block

Activation of cardiac muscle is mediated by the His-Purkinje system, a discrete pathway containing fast-conducting cells (Purkinje fibers) which coordinate the spread of excitation from the atrioventricular node (AV node) to ventricular myocardium [1]. Although pathologies of this specialized conduction system are common in humans, especially among the elderly [2], their molecular bases have not been defined. Gap junctions are present at appositions between Purkinje fibers and could provide a mechanism for propagating impulses between these cells [3]. Studies of the expression of connexins - the family of proteins from which gap junctions are formed - reveal that connexin40 (Cx40) is prominent in the conduction system [4]. In order to study the role of gap junction communication in cardiac conduction, we generated mice that lack Cx40. Using electrocardiographic analysis, we show that Cx40 null mice have cardiac conduction abnormalities characteristic of first-degree atrioventricular block with associated bundle branch block. Thus, gap junctions are essential for the rapid conduction of impulses in the His-Purkinje system.

Luteinizing hormone causes MAP kinase-dependent phosphorylation and closure of connexin 43 gap junctions in mouse ovarian follicles: one of two paths to meiotic resumption

Luteinizing hormone (LH) acts on ovarian follicles to reinitiate meiosis in prophase-arrested mammalian oocytes, and this has been proposed to occur by interruption of a meioisis-inhibitory signal that is transmitted through gap junctions into the oocyte from the somatic cells that surround it. To investigate this idea, we microinjected fluorescent tracers into live antral follicle-enclosed mouse oocytes, and we demonstrate for the first time that LH causes a decrease in the gap junction permeability between the somatic cells, prior to nuclear envelope breakdown (NEBD). The decreased permeability results from the MAP kinase-dependent phosphorylation of connexin 43 on serines 255, 262 and 279/282. We then tested whether the inhibition of gap junction communication was sufficient and necessary for the reinitiation of meiosis. Inhibitors that reduced gap junction permeability caused NEBD, but an inhibitor of MAP kinase activation that blocked gap junction closure in response to LH did not prevent NEBD. Thus, both MAP kinase-dependent gap junction closure and another redundant pathway function in parallel to ensure that meiosis resumes in response to LH.

High-Resolution Optical Mapping of the Right Bundle Branch in Connexin40 Knockout Mice Reveals Slow Conduction in the Specialized Conduction System

Connexin40 (Cx40) is a major gap junction protein that is expressed in the His-Purkinje system and thought to be a critical determinant of cell-to-cell communication and conduction of electrical impulses. Video maps of the ventricular epicardium and the proximal segment of the right bundle branch (RBB) were obtained using a high-speed CCD camera while simultaneously recording volume-conducted ECGs. In Cx40–/– mice, the PR interval was prolonged (47.4±1.4 in wild-type [WT] [n=6] and 57.5±2.8 in Cx40–/– [n=6];P <0.01). WT ventricular epicardial activation was characterized by focused breakthroughs that originated first on the right ventricle (RV) and then the left ventricle (LV). In Cx40–/– hearts, the RV breakthrough occurred after the LV breakthrough. Additionally, Cx40–/– mice showed RV breakthrough times that were significantly delayed with respect to QRS complex onset (3.7±0.7 ms in WT [n=6] and 6.5±0.7 ms in Cx40–/– [n=6];P <0.01), whereas LV breakthrough times did not change. Conduction velocity measurements from optical mapping of the RBB revealed slow conduction in Cx40–/– mice (74.5±3 cm/s in WT [n=7] and 43.7±6 cm/s in Cx40–/– [n=7];P <0.01). In addition, simultaneous ECG records demonstrated significant delays in Cx40–/– RBB activation time with respect to P time (P-RBB time; 41.6±1.9 ms in WT [n=7] and 55.1±1.3 ms in [n=7];P <0.01). These data represent the first direct demonstration of conduction defects in the specialized conduction system of Cx40–/– mice and provide new insight into the role of gap junctions in cardiac impulse propagation.

Central Role of Connexin40 in the Propagation of Electrically Activated Vasodilation in Mouse Cremasteric Arterioles In Vivo

&NA; When a short segment of arteriole is stimulated, vasomotor responses spread bidirectionally along the vessel axis purportedly via gap junctions. We used connexin40 knockout (Cx40‐/‐) mice to study vasomotor responses induced by 10‐second trains of electrical stimulation (30 Hz, 1 ms, 30 to 50 V) in 2nd or 3rd order arterioles of the cremaster muscle. Measurements were made at the stimulation site (local) and at conducted sites (500, 1000, and 2000 &mgr;m upstream). In wild‐type (Cx40+/+) animals, electrical stimulation evoked a local vasoconstriction and a conducted vasodilation that spread very rapidly along the vessel length without detectable decay. In Cx40‐/‐ mice, the conducted dilation was converted into either vasoconstriction or a slowly developing vasodilation that decayed along the vessel length. Tetrodotoxin (TTX, 1 &mgr;mol/L) had no effect on the local vasoconstriction in either Cx40+/+ or Cx40‐/‐ mice, but enhanced the conducted vasodilation in Cx40+/+ animals. In Cx40‐/‐ mice, TTX abolished the conducted vasoconstriction when present and revealed a small vasodilation that decayed with distance. In the group of Cx40‐/‐ mice in which electrical stimulation elicited a conducted vasodilation, TTX had no effect. Immunocytochemistry revealed Cx40 only in the endothelial layer of arterioles from Cx40+/+ mice and complete elimination of this connexin in the Cx40‐/‐ animals. These results indicate that focal current stimulation causes vasoconstriction by a combination of perivascular nerve stimulation and smooth muscle activation. Moreover, electrical stimulation activates a nonneuronal, Cx40‐dependent vasodilator response that spreads along the vessel length without decay. (Circ Res. 2003;92:793–800.)

Decreased intercellular dye-transfer and downregulation of non-ablated connexins in aortic endothelium deficient in connexin37 or connexin40

Vascular endothelial cells are coupled by gap junctions that permit cell-to-cell transfer of small molecules, including signals that may be important for vasomotor responses. Connexin37 (Cx37) and connexin40 (Cx40) are the predominant gap-junction proteins present in mouse endothelium. We examined the effect of eliminating Cx37, Cx40, or both, on interendothelial communication in mouse aorta. Intercellular transfer of biocytin and [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium (NBD-TMA) was used to assess gap-junction-mediated coupling. Ablation of Cx40 generally had a greater effect on dye-transfer than ablation of Cx37. The effect of Cx40 ablation on dye-transfer was age dependent. There was a 27-fold reduction in biocytin transfer in embryonic Cx40–/– aortic endothelium, a much larger change than in aortas of 6-7-week-old Cx40–/– animals, which showed a 3.5-fold reduction. By contrast, there was no reduction in biocytin transfer in embryonic Cx37–/– endothelium. Embryonic aortas lacking both Cx37 and Cx40 showed a complete loss of endothelial dye-transfer. Surprisingly, elimination of Cx40 resulted in up to a 17-fold drop in endothelial Cx37 on western blots, whereas deletion of Cx37 reduced endothelial Cx40 up to 4.2-fold. By contrast, in the medial layer, both Cx37 and Cx43 increased∼ fourfold in Cx40–/– aortas. Declines in non-ablated endothelial connexins were not mediated by changes in connexin mRNA levels, suggesting a post-transcriptional effect. Our results indicate that Cx37 and Cx40 are the only functional connexins expressed in mouse aortic endothelium and are collectively crucial for endothelial communication. Furthermore, Cx37 and Cx40 are codependent on each other for optimal expression in vascular endothelium.

Connexin37 and Connexin43 deficiencies in mice disrupt lymphatic valve development and result in lymphatic disorders including lymphedema and chylothorax.

Intraluminal valves are required for the proper function of lymphatic collecting vessels and large lymphatic trunks like the thoracic duct. Despite recent progress in the study of lymphvasculogenesis and lymphangiogenesis, the molecular mechanisms controlling the morphogenesis of lymphatic valves remain poorly understood. Here, we report that gap junction proteins, or connexins (Cxs), are required for lymphatic valvulogenesis. Cx37 and Cx43 are expressed early in mouse lymphatic development in the jugular lymph sacs, and later in development these Cxs become enriched and differentially expressed by lymphatic endothelial cells on the upstream and downstream sides of the valves. Specific deficiencies of Cx37 and Cx43 alone or in combination result in defective valve formation in lymphatic collecting vessels, lymphedema, and chylothorax. We also show that Cx37 regulates jugular lymph sac size and that both Cx37 and Cx43 are required for normal thoracic duct development, including valve formation. Another Cx family member, Cx47, whose human analog is mutated in some families with lymphedema, is also highly enriched in a subset of endothelial cells in lymphatic valves. Mechanistically, we present data from Foxc2-/- embryos suggesting that Cx37 may be a target of regulation by Foxc2, a transcription factor that is mutated in human lymphedema-distichiasis syndrome. These results show that at least three Cxs are expressed in the developing lymphatic vasculature and, when defective, are associated with clinically manifest lymphatic disorders in mice and man.

Connexin channels provide a target to manipulate brain endothelial calcium dynamics and blood-brain barrier permeability.

The cytoplasmic Ca2+ concentration ([Ca2+]i) is an important factor determining the functional state of blood-brain barrier (BBB) endothelial cells but little is known on the effect of dynamic [Ca2+]i changes on BBB function. We applied different agonists that trigger [Ca2+]i oscillations and determined the involvement of connexin channels and subsequent effects on endothelial permeability in immortalized and primary brain endothelial cells. The inflammatory peptide bradykinin (BK) triggered [Ca2+]i oscillations and increased endothelial permeability. The latter was prevented by buffering [Ca2+]i with BAPTA, indicating that [Ca2+]i oscillations are crucial in the permeability changes. Bradykinin-triggered [Ca2+]i oscillations were inhibited by interfering with connexin channels, making use of carbenoxolone, Gap27, a peptide blocker of connexin channels, and Cx37/43 knockdown. Gap27 inhibition of the oscillations was rapid (within minutes) and work with connexin hemichannel-permeable dyes indicated hemichannel opening and purinergic signaling in response to stimulation with BK. Moreover, Gap27 inhibited the BK-triggered endothelial permeability increase in in vitro and in vivo experiments. By contrast, [Ca2+]i oscillations provoked by exposure to adenosine 5’ triphosphate (ATP) were not affected by carbenoxolone or Gap27 and ATP did not disturb endothelial permeability. We conclude that interfering with endothelial connexin hemichannels is a novel approach to limiting BBB-permeability alterations.

A targeted disruption in connexin40 leads to distinct atrioventricular conduction defects.

AbstractIntroduction: Gap junctions consist of connexin (Cx) proteins that enable electrical coupling of adjacent cells and propagation of action potentials. Cx40 is solely expressed in the atrium and His-Purkinje system. The purpose of this study was to evaluate atrioventricular (AV) conduction in mice with a homozygous deletion of Connexin40 (Cx40−/−). Methods: Surface ECGs, intracardiac electrophysi-ology (EP) studies, and ambulatory telemetry were performed in Cx40−/− mutant mice and wild-type (WT) controls. Atrioventricular (AV) conduction parameters and arrhythmia inducibility were evaluated using programmed stimulation. Analysis of heart rate variability was based on results of ambulatory monitoring. Results: Significant findings included prolonged measures of AV refractoriness and conduction in connexin40-deficient mice, including longer PR, AH, and HV intervals, increased AV refractory periods, and increased AV Wenckebach and 2:1 block cycle lengths. Connexin40-deficient mice also had an increased incidence of inducible ventricular tachycardia, decreased basal heart rates, and increased heart rate variability. Conclusion: A homozygous disruption of Cx40 results in prolonged AV conduction parameters due to abnormal electrical coupling in the specialized conduction system, which may also predispose to arrhythmia vulnerability.

Reduced Connexin 43 Expression and Its Effect on the Development of Vascular Lesions in Retinas of Diabetic Mice

PURPOSE. To examine whether diabetes-induced connexin 43 downregulation promotes retinal vascular lesions characteristic of diabetic retinopathy (DR). METHODS. Two animal models, streptozotocin-induced diabetic mice and Cx43 heterozygous knockout (Cx43(+/-)) mice, were studied to directly assess whether diabetes reduces the expression of retinal Cx43, which, in turn, contributes to retinal vascular cell loss by apoptosis. Retinal Cx43 protein levels were assessed in nondiabetic control mice, diabetic mice, and Cx43(+/-) mice by Western blot analysis, and Cx43 localization and distribution in the retinal vascular cells were studied by immunostaining of retinal trypsin digests (RTDs). In parallel, RTDs were stained with hematoxylin and periodic acid Schiff to determine pericyte loss (PL) and acellular capillaries (AC), and TUNEL assays were performed to determine retinal vascular cell apoptosis. RESULTS. Western blot analysis indicated significant reductions in retinal Cx43 protein levels in diabetic mice and Cx43(+/-) mice compared with those of nondiabetic mice. Similarly, a significant reduction in Cx43 immunostaining was observed in the retinal capillaries of diabetic mice and Cx43(+/-) mice compared with those of control mice. Both diabetic and age-matched Cx43(+/-) mice exhibited increased amount of PL, AC, and TUNEL-positive cells compared with control mice. CONCLUSIONS. Diabetes-induced inhibition of Cx43 expression contributes to vascular cell apoptosis in retinas of diabetic mice. This suggests that reduced Cx43 expression plays a critical role in the development of AC and PL associated with DR.