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.

Evans Blue and Fluorescein Isothiocyanate-Dextran Double Labeling Reveals Precise Sequence of Vascular Leakage and Glial Responses After Exposure to Mild-Level Blast-Associated Shock Waves.

Abstract Blast-induced shock waves (BSWs) are responsible for several aspects of psychiatric disorders that are collectively termed mild traumatic brain injury (mTBI). The pathophysiology of mTBI includes vascular leakage resulting from blood-brain barrier (BBB) disruption. In this study, the precise sequence of BBB breakdown was examined using an Evans blue and fluorescein isothiocyanate (FITC)-dextran double labeling technique. Evans blue solution was injected into the tail vein of male C57BL6/J mice just before and 4 h, 1 day, 3 days, and 7 days after a single BSW exposure at as low as 25-kPa peak overpressure. In contrast, the FITC-dextran solution was transcardially injected just before perfusion fixation. Differences in the labeling time-point revealed that BBB breakdown was initiated after approximately 3 h, with significant remodeling by 1 day, and continued until 7 days after BSW exposure. BBB breakdown was upregulated in three distinct regions, namely the brain surface and subsurface areas facing the skull, regions closely associated with capillaries, and the circumventricular organ and choroid plexus. These regions showed distinct responses to BSW; moreover, clusters of reactive astrocytes were closely associated with the sites of BBB breakdown. In severe cases, these reactive astrocytes recruited activated microglia. Our findings provide important insights into the pathogenesis underlying mTBI and indicate that even mild BSW exposure affects the whole brain.

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.

The extracellular matrix controls gap junction protein expression and function in postnatal hippocampal neural progenitor cells.

BACKGROUND: Gap junction protein and extracellular matrix signalling systems act in concert to influence developmental specification of neural stem and progenitor cells. It is not known how these two signalling systems interact. Here, we examined the role of ECM components in regulating connexin expression and function in postnatal hippocampal progenitor cells. RESULTS: We found that Cx26, Cx29, Cx30, Cx37, Cx40, Cx43, Cx45, and Cx47 mRNA and protein but only Cx32 and Cx36 mRNA are detected in distinct neural progenitor cell populations cultured in the absence of exogenous ECM. Multipotential Type 1 cells express Cx26, Cx30, and Cx43 protein. Their Type 2a progeny but not Type 2b and 3 neuronally committed progenitor cells additionally express Cx37, Cx40, and Cx45. Cx29 and Cx47 protein is detected in early oligodendrocyte progenitors and mature oligodendrocytes respectively. Engagement with a laminin substrate markedly increases Cx26 protein expression, decreases Cx40, Cx43, Cx45, and Cx47 protein expression, and alters subcellular localization of Cx30. These changes are associated with decreased neurogenesis. Further, laminin elicits the appearance of Cx32 protein in early oligodendrocyte progenitors and Cx36 protein in immature neurons. These changes impact upon functional connexin-mediated hemichannel activity but not gap junctional intercellular communication. CONCLUSION: Together, these findings demonstrate a new role for extracellular matrix-cell interaction, specifically laminin, in the regulation of intrinsic connexin expression and function in postnatal neural progenitor cells.

Knock-in mouse model of dilated cardiomyopathy caused by troponin mutation.

We created knock-in mice in which a deletion of 3 base pairs coding for K210 in cardiac troponin (cTn)T found in familial dilated cardiomyopathy patients was introduced into endogenous genes. Membrane-permeabilized cardiac muscle fibers from mutant mice showed significantly lower Ca(2+) sensitivity in force generation than those from wild-type mice. Peak amplitude of Ca(2+) transient in cardiomyocytes was increased in mutant mice, and maximum isometric force produced by intact cardiac muscle fibers of mutant mice was not significantly different from that of wild-type mice, suggesting that Ca(2+) transient was augmented to compensate for decreased myofilament Ca(2+) sensitivity. Nevertheless, mutant mice developed marked cardiac enlargement, heart failure, and frequent sudden death recapitulating the phenotypes of dilated cardiomyopathy patients, indicating that global functional defect of the heart attributable to decreased myofilament Ca(2+) sensitivity could not be fully compensated by only increasing the intracellular Ca(2+) transient. We found that a positive inotropic agent, pimobendan, which directly increases myofilament Ca(2+) sensitivity, had profound effects of preventing cardiac enlargement, heart failure, and sudden death. These results verify the hypothesis that Ca(2+) desensitization of cardiac myofilament is the absolute cause of the pathogenesis of dilated cardiomyopathy associated with this mutation and strongly suggest that Ca(2+) sensitizers are beneficial for the treatment of dilated cardiomyopathy patients affected by sarcomeric regulatory protein mutations.

Molecular Hydrogen Prevents Social Deficits and Depression-Like Behaviors Induced by Low-Intensity Blast in Mice.

Detonation of explosive devices creates blast waves, which can injure brains even in the absence of external injuries. Among these, blast-induced mild traumatic brain injury (bmTBI) is increasing in military populations, such as in the wars in Afghanistan, Iraq, and Syria. Although the clinical presentation of bmTBI is not precisely defined, it is frequently associated with psycho-neurological deficits and usually manifests in the form of poly-trauma including psychiatric morbidity and cognitive disruption. Although the underlying mechanisms of bmTBI are largely unknown, some studies suggested that bmTBI is associated with blood-brain barrier disruption, oxidative stress, and edema in the brain. The present study investigated the effects of novel antioxidant, molecular hydrogen gas, on bmTBI using a laboratory-scale shock tube model in mice. Hydrogen gas has a strong prospect for clinical use due to easy preparation, low-cost, and no side effects. The administration of hydrogen gas significantly attenuated the behavioral deficits observed in our bmTBI model, suggesting that hydrogen application might be a strong therapeutic method for treatment of bmTBI.

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.

Gap junctional regulation of pressure, fluid force, and electrical fields in the epigenetics of cardiac morphogenesis and remodeling.

Epigenetic factors of pressure load, fluid force, and electrical fields that occur during cardiac contraction affect cardiac development, morphology, function, and pathogenesis. These factors are orchestrated by intercellular communication mediated by gap junctions, which synchronize action potentials and second messengers. Misregulation of the gap junction protein connexin (Cx) alters cardiogenesis, and can be a pathogenic factor causing cardiac conduction disturbance, fatal arrhythmia, and cardiac remodeling in disease states such as hypertension and ischemia. Changes in Cx expression can occur even when the DNA sequence of the Cx gene itself is unaltered. Posttranslational modifications might reduce arrhythmogenic substrates, improve cardiac function, and promote remodeling in a diseased heart. In this review, we discuss the epigenetic features of gap junctions that regulate cardiac morphology and remodeling. We further discuss potential clinical applications of current knowledge of the structure and function of gap junctions.

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).

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.

[Networks of pacemaker cells for gastrointestinal motility].

In the wall of the digestive tract, there are pacemaker and conduction systems which can be compared with those in the heart. The introduction of c-Kit as a specific marker of the cells, ICCs, have dramatically clarified morphological and functional understanding of the cells. Mutant animals that lack c-Kit lose or decrease intestinal motility. Four classes of ICCs have been identified and these are distributed along the digestive tract in an organ- and tissue-specific manner: 1) IC-MY locate along the myenteric plexus; 2) IC-DMP, along the deep muscular plexus of small intestine; 3) IC-SMP, along the interface between the submucosa and circular muscle layer of large intestine; and 4) IC-IM, within the muscular layer of the stomach and large intestine. Basically, IC-MY and IC-SMP have pacemaker functions, whereas IC-DMP and IC-IM link signals between the enteric nervous system and smooth muscle cells (SMC). All classes of the cells are connected by gap junctions. Immunocytochemical observations using specific antibodies against various gap junction proteins, connexins (Cx), revealed that Cx43 was localized in the gap junctions between SMC and ICCs, whereas Cx45 was specifically expressed in IC-DMP as it is in the cardiac conduction systems. Mutant animals that we produced enabled us to show cells expressing Cx45 mRNA by replacing the Cx45 locus with a LacZ reporter gene and revealed that most of SMC express Cx45, where so far gap junctions were not demonstrated by electron microscopy or immunocytochemistry, probably due to their small size.

Overexpression of Polycomb-group gene rae28 in cardiomyocytes does not complement abnormal cardiac morphogenesis in mice lacking rae28 but causes dilated cardiomyopathy.

The Polycomb-group genes (PcG) are widely conserved from Drosophila to mammals and are required for maintaining positional information during development. The rae28 gene (rae28) is a member of the mouse PcG. Mice deficient in rae28 (rae28(-/-)) demonstrated that rae28 has a role not only in anteroposterior patterning but also in cardiac morphogenesis. In this study we generated transgenic mice with ubiquitous or cardiomyocyte-specific exogenous rae28 expression. Genetic complementation experiments with these transgenic mice showed that ubiquitous expression of rae28 could reverse the cardiac anomalies in rae28(-/-), whereas cardiomyocyte-specific expression of rae28 could not, suggesting that rae28 is involved in cardiac morphogenesis through a noncardiomyocyte pathway. Interestingly, however, cardiomyocyte-specific overexpression of rae28 caused dilated cardiomyopathy, which was associated with cardiomyocyte apoptosis, abnormal myofibrils, and severe heart failure. Cardiac expression of rae28 was predominant in the early embryonic stage, whereas that of the other PcG members was relatively constitutive. Because rae28 forms multimeric complexes with other PcG proteins in the nucleus, it is presumed that constitutive cardiomyocyte-specific rae28 overexpression impaired authentic PcG functions in the heart. rae28-induced dilated cardiomyopathy may thus provide a clue for clarifying the direct role of PcG in the maintenance of cardiomyocytes.