Absence of connexin43 and connexin45 does not disturb pre- and peri-implantation development.

Gap junctional intercellular communication is assumed to play an important role during pre- and peri-implantation development. In this study, we eliminated connexin43 (Cx43) and connexin45 (Cx45), major gap junctional proteins in the pre- and peri-implantation embryo. We generated Cx43 -/- Cx45 -/- embryos by Cx43 +/- Cx45 +/- intercrossing, because mice deficient in Cx43 (Cx43 -/-) exhibit perinatal lethality and those deficient in Cx45 (Cx45 -/-) exhibit early embryonic lethality. Wild-type, Cx43 -/-, Cx45 -/-, and Cx43 -/- Cx45 -/- blastocysts all showed similar outgrowths in in vitro culture. Moreover, Cx43 -/- Cx45 -/- embryos were obtained at the expected Mendelian ratio up to embryonic day 9.5, when the Cx45 -/- mutation proved lethal. The Cx43 -/- Cx45 -/- embryos seemed to have no additional developmental abnormalities in comparison with the single knockout strains. Thus, pre- and peri-implantation development does not require Cx43 and Cx45. Other gap junctional proteins are expressed around these stages and these may compensate for the lack of Cx43 and Cx45.

Characterization of NaV1.6-mediated Na+ currents in smooth muscle cells isolated from mouse vas deferens.

Patch-clamp experiments were performed to investigate the behavior of voltage-activated inward currents in vas deferens myocytes from Na(V)1.6-null mice (Na(V)1.6(-/-)) lacking the expression of the Na(+) channel gene, Scn8a, and their wild-type littermates (Na(V)1.6(+/+)). Immunohistochemistry confirmed expression of Na(V)1.6 in the muscle of Na(V)1.6(+/+), but not Na(V)1.6(-/-), vas deferens. PCR analysis revealed that the only beta(1)-subunit gene expressed in Na(V)1.6(+/+) vas deferens was Scn1b. In Na(V)1.6(+/+) myocytes, the threshold for membrane currents evoked by 20 msec voltage ramps (-100 mV to 60 mV) was -38.5 +/- 4.6 mV and this was shifted to a more positive potential (-31.2 +/- 4.9 mV) by tetrodotoxin (TTX). In Na(V)1.6(-/-) myocytes, the threshold was -30.4 +/- 3.4 mV and there was no TTX-sensitive current. The Na(+) current (I(Na)) in Na(V)1.6(+/+) myocytes had a bell-shaped current-voltage relationship that peaked at approximately -10 mV. Increasing the duration of the voltage ramps beyond 20 msec reduced the peak amplitude of I(Na). I(Na) displayed both fast (tau approximately 10 msec) and slow (tau approximately 1 sec) recovery from inactivation, the magnitude of the slow component increasing with the duration of the conditioning pulse (5-40 msec). During repetitive activation (5-40 msec pulses), I(Na) declined at stimulation frequencies > 0.5 Hz and at 10 Hz

Subcellular localization of Tektin2 in rat sperm flagellum.

Tektins are evolutionarily conserved filament-forming proteins localized in flagella and cilia, and have been reported to be involved in the stability and structural complexity of axonemal microtubules. Five mammalian Tektins (Tektin1-5) have been reported. Of these, Tektin2 (TEKT2) has been found to be required for normal flagellum structure and function. Tekt2-null sperm display flagellum bending and reduced motility, probably due to disruption of the dynein inner arm. However, the subcellular localization of TEKT2 in spermatozoa has not been clarified at the ultrastructural level. To elucidate the molecular localization of TEKT2 in flagella of rat spermatozoa, we performed confocal laser scanning microscopy, extraction of flagella followed by immunoblot analysis, and immunogold electron microscopy. Extraction of sperm flagella by SDS-EDTA resulted in complete extraction of axonemal tubulins, while TEKT2 was only partially released from flagella, suggesting that TEKT2 might be present in the peri-axonemal component, not directly associated with axonemal tubulins. Confocal laser scanning microscopy and pre-embedding immunoelectron microscopy revealed that TEKT2 is associated with the surface of outer dense fibers (ODFs). TEKT2 may function as an ODF-affiliated molecule required for flagellum stability and sperm motility.

[Gross anatomy dissection and the legal control].

In Japan, dissection of human body is generally prohibited by the Penal Code, i.e. the criminal law. However, the Postmortem Examination and Corpse Preservation Act allows for the dissection of the body in very limited situations, that include gross anatomy dissection and pathological and forensic autopsy in medical and dental schools. Growing numbers of co-medical schools have been founded more recently in Japan, and not a small number of co-medical schools try to adopt human body dissection in the course of anatomy education. The present short communication reminds us of the ways of thinking of the Postmortem Examination and Corpse Preservation Act and the Act on Body Donation for Medical and Dental Education in order that anatomy education in medical as well as co-medical schools takes place under the regulation by these two laws.

Targeted disruption of the cardiac troponin T gene causes sarcomere disassembly and defects in heartbeat within the early mouse embryo.

Cardiac troponin T (cTnT) is a component of the troponin (Tn) complex in cardiac myocytes, and plays a regulatory role in cardiac muscle contraction by anchoring two other Tn components, troponin I (TnI) and troponin C, to tropomyosin (Tm) on the thin filaments. In order to determine the in vivo function of cTnT, we created a null cTnT allele in the mouse TNNT2 locus. In cTnT-deficient (cTnT(-/-)) cardiac myocytes, the thick and thin filaments and alpha-actinin-positive Z-disk-like structures were not assembled into sarcomere, causing early embryonic lethality due to a lack of heartbeats. TnI was dissociated from Tm in the thin filaments without cTnT. In spite of loss of Tn on the thin filaments, the cTnT(-/-) cardiac myocytes showed regular Ca(2+)-transients. These findings indicate that cTnT plays a critical role in sarcomere assembly during myofibrillogenesis in the embryonic heart, and also indicate that the membrane excitation and intracellular Ca(2+) handling systems develop independently of the contractile system. In contrast, heterozygous cTnT(+/-) mice had a normal life span with no structural and functional abnormalities in their hearts, suggesting that haploinsufficiency could not be a potential cause of cardiomyopathies, known to be associated with a variety of mutations in the TNNT2 locus.

Freeze-fracture electron microscopic study of tight junction strands in HEK293 cells and MDCK II cells expressing claudin-1 mutants in the second extracellular loop.

Claudins constitute tight junction (TJ) strands. In order to examine the function of the second extracellular loop (ECL2), we constructed 1CLDeltaFY and 1CLDeltaPL in which highly conserved amino acids, FY or PL, in the ECL2 of mouse claudin-1 were deleted. They were then tagged with either EGFP at the NH(2)-terminus (EGFP1CLDeltaFY and EGFP1CLDeltaPL) or the myc-epitope at the COOH-terminus (1CLDeltaFYmyc and 1CLDeltaPLmyc). The expression of EGFP1CLDeltaFY and EGFP1CLDeltaPL in TJ-free HEK293 cells formed TJ strands resembling those formed by wild-type claudin-1. The expression of 1CLDeltaPLmyc in TJ-bearing MDCK II cells induced aberrant TJ strands in the lateral plasma membranes whose intramembranous particles were almost equally distributed in the P- and E-face. In contrast, 1CLDeltaFYmyc formed aggregates of short continuous strands which were frequently associated with vesicle-like structures. Coculture experiments with MDCK II cells showed that 1CLDeltaPLmyc was localized at heterotypic cell-cell junctions but 1CLDeltaFYmyc was not. These results suggest that changes in the TJ morphology due to the expression of either 1CLDeltaFYmyc or 1CLDeltaPLmyc may be caused by some factors specific to epithelial MDCK II cells including endogenous claudins.

Tektin5, a new Tektin family member, is a component of the middle piece of flagella in rat spermatozoa.

Tektins are composed of a family of filament-forming proteins localized in cilia and flagella. Four types of mammalian Tektins have been reported, and at least two types of Tektins, Tektin2 and Tektin4, have been verified to be present in sperm flagella. A new member of the TEKTIN gene family, which was designated as rat Tektin5, was obtained by PCR technique. Rat Tektin5 cDNA consists of 1,674 bp encoding a 62.8 kDa protein of 558 amino acids. Tektin5 protein contains a Tektin domain as well as a nonapeptide signature sequence that is a prominent feature of Tektin proteins. RT-PCR analysis indicated that Tektin5 was predominantly expressed in testis and that its expression was up-regulated during testis development. Immunoblot analyses revealed that Tektin5 is present in sperm flagella but not in heads and that it is completely released from rat spermatozoa by 6 M urea treatment, but not extracted by 1% Triton X-100 and 0.6 M potassium thiocyanate. Confocal laser scanning microscopy revealed that Tektin5 was located in the middle piece of flagella in rat spermatozoa with no immunolabeling in the heads and the principal piece. Immunogold electron microscopy adopting pre-embedding method discovered that Tektin5 is predominantly associated with the inner side of the mitochondrial sheath. Tektin5 might work as a middle piece component requisite for flagellar stability and sperm motility.

Progressive Atrial Conduction Defects Associated With Bone Malformation Caused by a Connexin-45 Mutation.

BACKGROUND: Inherited cardiac conduction disease is a rare bradyarrhythmia associated with mutations in various genes that affect action potential propagation. It is often characterized by isolated conduction disturbance of the His-Purkinje system, but it is rarely described as a syndromic form. OBJECTIVES: The authors sought to identify the genetic defect in families with a novel bradyarrhythmia syndrome associated with bone malformation. METHODS: The authors genetically screened 15 European cases with genotype-negative de novo atrioventricular (AV) block and their parents by trio whole-exome sequencing, plus 31 Japanese cases with genotype-negative familial AV block or sick sinus syndrome by targeted exon sequencing of 457 susceptibility genes. Functional consequences of the mutation were evaluated using an in vitro cell expression system and in vivo knockout mice. RESULTS: The authors identified a connexin-45 (Cx45) mutation (p.R75H) in 2 unrelated families (a de novo French case and a 3-generation Japanese family) who presented with progressive AV block, which resulted in atrial standstill without ventricular conduction abnormalities. Affected individuals shared a common extracardiac phenotype: a brachyfacial pattern, finger deformity, and dental dysplasia. Mutant Cx45 expressed in Neuro-2a cells showed normal hemichannel assembly and plaque formation. However, Lucifer yellow dye transfer and gap junction conductance between cell pairs were severely impaired, which suggested that mutant Cx45 impedes gap junction communication in a dominant-negative manner. Tamoxifen-induced, cardiac-specific Cx45 knockout mice showed sinus node dysfunction and atrial arrhythmia, recapitulating the intra-atrial disturbance. CONCLUSIONS: Altogether, the authors showed that Cx45 mutant p.R75H is responsible for a novel disease entity of progressive atrial conduction system defects associated with craniofacial and dentodigital malformation.

Mode and determination of the initial contraction stage in the mouse embryo heart.

The developing mammalian heart initiates spontaneous contractions shortly after the myocardium differentiates from the splanchnic mesoderm. The precise timing and mode of the onset of heartbeat, however, have not been statistically described in mice. We analyzed the patterns of contractive activity in video-recorded heart regions ranging from the pre-somite stage to day 10.5 (E10.5). The first sign was detected at the 3-somite stage (E8.25), when a few cardiac myocytes constituted small contracting groups on both sides of the heart tube. Fluctuations of the basal [Ca2+]i level were detected in dormant 3-somite-stage hearts, indicating the initiation of electrical activity before visible contractions. After weak and irregular contractions at the 3-somite stage, the contractions were almost coordinated as early as the 4-somite stage. Unidirectional blood flow through the atrioventricular canal was established around the 20-somite stage at E9.25, correlated with the development of the endocardial cushion. We propose that not only the endocardial cushion but also coordinated contractions are essential for unidirectional flow, because induced bradycardia due to short exposure at room temperature caused regurgitation at E10.5 when otherwise highly organized flow was observed. These findings complement and extend earlier observations on functional heart development, providing a reference for fundamental research on mammalian cardiogenesis.

Connexin45 contributes to global cardiovascular development by establishing myocardial impulse propagation.

Among gap junction-encoding genes, the loss of connexin (Cx) 45 most profoundly obstructs embryogenesis through an endocardial cushion defect and conduction block. However, the interdependence of these defects is not known, and the details of conduction block have not been elucidated. Here, we examined mouse embryos with a region-specific deletion of Cx45 in the myocardium (CA-Cre; Cx45(flox/flox)) or endothelium (Tie2-Cre; Cx45(flox/flox)). Although the deletion of Cx45 in the myocardium was heterogeneous, the CA-Cre; Cx45(flox/flox) embryos were lethal at the same stage as the constitutive Cx45-deficient (Cx45(-/-)) embryos. We determined the onset and patterns of their conduction block through point-tracking in video recordings of embryonic heart contractions. An incomplete conduction block at the atrioventricular canal appeared at embryonic day (E) 8.5 and was predominant around the lethal E9.5 stage in both the Cx45(-/-) and CA-Cre; Cx45(flox/flox) embryos. Although the Cx45(-/-) hearts showed a consistently severe reduction in atrioventricular conduction velocity, the CA-Cre; Cx45(flox/flox) hearts had delay times within the normal range and showed frequent retrograde conduction. As previously reported, the Cx45(-/-) endocardial cushion was consistently defective, and nuclear factor of activated T-cells cytoplasmic (NFATc)1 within the endocardium showed inactive cytoplasmic distribution. In CA-Cre; Cx45(flox/flox), however, the endocardial cushion was partially formed, with active NFATc1 within the endocardium. There was no developmental abnormality in the Tie2-Cre; Cx45(flox/flox) embryos. These results indicate that myocardial dysfunction is responsible for most of the reported defects in Cx45(-/-), which are alleviated by sporadic Cx45 expression in the CA-Cre; Cx45(flox/flox) myocardium.

Long-term inhibition of Rho-kinase suppresses neointimal formation after stent implantation in porcine coronary arteries: involvement of multiple mechanisms.

OBJECTIVE: We recently demonstrated that Rho-kinase, an effector of the small GTPase Rho, is substantially involved in the pathogenesis of arteriosclerosis. In this study, we examined whether Rho-kinase is also involved in in-stent restenosis and if so, what mechanism is involved. METHODS AND RESULTS: Pigs underwent stent implantation in the left coronary artery with or without administration of fasudil (30 mg/kg per day orally), a specific Rho-kinase inhibitor, starting 2 days before the procedure for a duration of 4 weeks. On day 28, reductions in coronary diameter and neointimal formation associated with macrophage accumulation, collagen deposition, and transforming growth factor (TGF)-beta1 expression were noted at the stent site, and all were significantly suppressed by fasudil. On day 7, fasudil significantly increased the frequency of TUNEL-positive apoptotic cells, while it tended to reduce that of bromodeoxyuridine-positive proliferating cells in the neointima. Western blot analysis on day 7 demonstrated that phosphorylations of the ezrin/radixin/moesin family (a marker of Rho-kinase activity in vivo) and protein expression of monocyte chemoattractant protein-1and bcl-2 were upregulated at the stent site and were significantly suppressed by fasudil. CONCLUSIONS: These results indicate that long-term inhibition of Rho-kinase suppresses in-stent neointimal formation by multiple mechanisms, including reduced vascular inflammation, enhanced apoptosis, and decreased collagen deposition.

Mice lacking connexin45 conditionally in cardiac myocytes display embryonic lethality similar to that of germline knockout mice without endocardial cushion defect.

The gap junction protein connexin45-deficient (Cx45-KO) mice die shortly after the hearts begin to beat. In addition to the heart defect, they also show defective vascular development which may be closely related with the cardiac phenotype. Therefore, we created mice whose floxed-Cx45 locus could be removed conditionally. We utilized cardiac alpha-actin-Cre transgenic mice to investigate the specific cardiac muscular function of Cx45 in vivo. The resultant conditional mutants were lethal, showing conduction block similar to that of the Cx45-KO mice. Unlike Cx45-KO, development of the endocardial cushion was not disrupted in the conditional mutants. X-gal staining was detected in the embryonic cardiac myocytes as a hallmark of Cre-loxP mediated floxed-Cx45 deletion. These results reconfirm the requirement of Cx45 for developing cardiac myocytes. These also indicate that establishing the first contractions is a crucial task for the early hearts.

Conduction abnormality in gap junction protein connexin45-deficient embryonic stem cell-derived cardiac myocytes.

In early-stage heart, the cardiac impulse does not propagate through the specialized conduction system but spreads from myocyte to myocyte. We hypothesized that the gap junction protein connexin45 (Cx45) regulates early-stage contractions, because it is the only gap junction protein described in early hearts. Cx45-deficient (Cx45(-/-)) mice die of heart failure, concomitantly displaying other complex defects in the cardiovascular system. In order to determine the specific cardiac muscular function of Cx45, we created Cx45(-/-) embryonic stem (ES) cells to be differentiated into cardiac muscle in vitro. Unlike the coordinated contractions of wild-type cells, differentiated Cx45(-/-) cardiac myocytes showed high and irregular pulsation rates. Alterations of the electrophysiological properties of the Cx45(-/-) cardiac myocytes were indicated both by extracellular recording on planar multielectrode array probes and by intracellular Ca(2+) recording of the fluorescent Ca(2+) indicator fura-2. The in vitro system minimizes an influence of hemodynamic factors that complicate the phenotypes of Cx45(-/-) mice. Our results indicate that Cx45 is an essential connexin for coordinated conduction through early cardiac myocytes. The Supplementary Material referred to in this article can be found at the Anatomical Record website (http://www.interscience.wiley.com/jpages/0003-276X/suppmat).

Molecular cloning of two guinea pig liver gap junction proteins, connexin32 and connexin26.

Intercellular coupling of hepatocytes through gap junctions facilitates exchange of small metabolites or ions, and contributes to maintenance of tissue homeostasis. As protein constituents of the liver gap junction channels, connexin32 (Cx32) and Cx26 have been identified. By use of rat cDNA probes, we cloned cDNAs for guinea pig homologs of Cx32 and Cx26, and compared their amino acid sequences with those of other species. The deduced primary structure of guinea pig Cx32 was 283 amino acids long and contained 98% identical amino acids to the rat and human Cx32. Only six amino acid exchanges were detected between the guinea pig and rat Cx32. On the contrary, the deduced amino acid sequence of guinea pig Cx26 (226 amino acids long) was 91 and 89% identical to the rat and human Cx26, respectively. Twenty-one amino acid exchanges were found between the guinea pig and rat, and the divergence was mostly located in cytoplasmic domains of Cx26. These results suggest that Cx26 shows structural diversity between species, while Cx32 is highly conserved.

Connexin mutant embryonic stem cells and human diseases.

Intercellular communication via gap junctions allows cells within multicellular organisms to share small molecules. The effect of such interactions has been elucidated using mouse gene knockout strategies. Although several mutations in human gap junction-encoding connexin (Cx) have been described, Cx mutants in mice do not always recapitulate the human disease. Among the 20 mouse Cxs, Cx26, Cx43, and Cx45 play roles in early cardiac or placental development, and disruption of the genes results in lethality that hampers further analyses. Embryonic stem cells (ESCs) that lack Cx43 or Cx45 have made analysis feasible in both in vitro differentiated cell cultures and in vivo chimeric tissues. The success of mouse ESCs studies is leading to the use of induced pluripotent stem cells to learn more about the pathogenesis of human Cx diseases. This review summarizes the current status of mouse Cx disruption models and ESC differentiation studies, and discusses their implication for understanding human Cx diseases.

The protoplasmic or exoplasmic face association of tight junction particles cannot predict paracellular permeability or heterotypic claudin compatibility.

Claudins constitute tight junction (TJ) strands and regulate paracellular permeability, which varies in the epithelial cells of various organs. Heterotypic claudin compatibility and/or the association of TJ particles to either the protoplasmic (P) or exoplasmic (E) face may be related to paracellular permeability. This study examined the relationship between the TJ morphology, heterotypic claudin compatibility and paracellular permeability using claudin-10b- or claudin-15-expressing HEK293 cells and MDCK I cells. Claudin-10b or -15 expressed in TJ-free HEK293 cells formed E-face- or P-face-associated TJ particles, respectively. The coculture of claudin-1-expressing HEK293 cells and either claudin-10b- or claudin-15-expressing HEK293 cells showed that claudin-10b and -15 were not compatible with claudin-1. The expression of claudin-10b or -15 in high-resistance MDCK I cells did not alter the expression of endogenous claudins except for claudin-3 and dramatically reduced transepithelial electrical resistance by increasing the permeability of Na(+) but it did not change that of Cl(-). The expression of claudin-10b or -15 in MDCK I cells either decreased or increased the flux of 4 kDa dextran, respectively. The coculture of MDCK I cells and either claudin-10b- or claudin-15-expressing MDCK I cells showed claudin-10b to be partly compatible, while claudin-15 was incompatible with the endogenous claudins in MDCK I cells. These results indicate that the TJ morphology cannot predict the properties of either paracellular permeability or heterotypic claudin compatibility.

Heterogeneous expression of endothelial connexin (Cx) 37, Cx40, and Cx43 in rat large veins.

Gap junctions are clusters of transmembrane protein channels for intercellular communication and are composed of connexin (Cx). The vascular endothelial cells express Cx37, Cx40, and Cx43. We herein examined the spatial distribution of the endothelial connexins Cx37, Cx40, and Cx43 in rat large veins including the cranial vena cava, thoracic section of the caudal vena cava, and abdominal section of the caudal vena cava. We also examined the mean size of the endothelial cells and quantified the protein expression levels of the endothelial connexins. We found that the large veins heterogeneously expressed Cx37, Cx40, and Cx43 as follows: Cx40 > Cx37 > > Cx43 in the cranial vena cava, Cx37 > Cx43 > > Cx40 in the thoracic section of the caudal vena cava, and Cx40 > Cx43 > > Cx37 in the abdominal section of the caudal vena cava. Double immunostaining of two of the endothelial connexins revealed that the gap-junction plaques were composed of various combinations of endothelial connexins. The mean size of the endothelial cells was large, moderate, or small in the cranial vena cava, the abdominal section of the caudal vena cava, or the thoracic section of the caudal vena cava, respectively. The heterogeneity of the endothelial cells of the rat large veins in terms of the connexin expression suggests that the endothelial cells are differently coupled in the large veins. The present data are useful for investigating, for example, disease-related alterations in expression of endothelial connexins in large veins.

Formation of aberrant TJ strands by overexpression of claudin-15 in MDCK II cells.

Claudins are one of the transmembrane proteins found at tight junctions (TJs); they constitute the backbone of TJ strands and comprise a multigene family. Claudins share a YV sequence at the COOH-termini, which is considered to be a ZO-binding motif. Overexpression of claudin-15 (15CL) or claudin-15 tagged with enhanced green fluorescent protein at the NH2-terminus (EGFP-15CL) induced aberrant strands in MDCK II cells, even though claudin-15 has the ZO-binding motif. Morphometric analysis by freeze-fracture electron microscopy revealed that the mean number of apical TJ strands increased by 47% in EGFP-1CL-expressing cells, 21% in EGFP-15CL-expressing cells, and 28% in 15CL-expressing cells. The number of free-ended apical strands increased remarkably in EGFP-15CL- and 15CL-expressing cells, but not in EGFP-1CL-expressing cells. When MDCK cells expressing EGFP-1CL, EGFP-15CL or 15CL were co-cultured with parent MDCK cells, which express claudin-1 but not claudin-15, EGFP-15CL and 15CL could not be concentrated at the apical junctional region between the heterotypic cells, though EGFP-1CL could. These results suggest that not only binding to ZO-1, but also head-to-head compatibility between claudin species, is involved in organizing claudin proteins at the apical junctional region.