Kiyomasa Nishii

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 Ca2+ sensitivity in force generation than those from wild-type mice. Peak amplitude of Ca2+ 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 Ca2+ transient was augmented to compensate for decreased myofilament Ca2+ 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 Ca2+ sensitivity could not be fully compensated by only increasing the intracellular Ca2+ transient. We found that a positive inotropic agent, pimobendan, which directly increases myofilament Ca2+ sensitivity, had profound effects of preventing cardiac enlargement, heart failure, and sudden death. These results verify the hypothesis that Ca2+ desensitization of cardiac myofilament is the absolute cause of the pathogenesis of dilated cardiomyopathy associated with this mutation and strongly suggest that Ca2+ sensitizers are beneficial for the treatment of dilated cardiomyopathy patients affected by sarcomeric regulatory protein mutations.

Diversity and molecular anatomy of gap junctions

In animal tissues, most cells are connected via intercellular cytoplasmic channels called gap junctions. Various electron microscopy techniques have made a crucial contribution to our understanding of the function and structure of gap junction channels. Tracer studies and freeze-fracture replica observations indicate that the connexon, the unit gap junction channel, is a pair of hemichannels apposed in the narrow intercellular gap between neighboring cell membranes. Recent advances in cellular biology have shown that connexon hemichannels are composed of hexamers of connexin proteins. Purification of the gap junction membrane and cDNA cloning analysis indicate the diversity of the connexin protein family, which contains more than 18 members, and their tissue- and cell type-specific distributions. Defects in some connexin genes may cause various hereditary diseases, such as X-linked Charcot-Marie-Tooth disease (Cx32), nonsyndromic autosomal deafness (Cx26), and cataract (Cx50). Analysis of gene knockout mice indicates that certain types of connexin play important roles in differentiation and development at crucial times in specific tissues and cell types.

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.

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 α-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.

Abnormalities of developmental cell death in Dad1‐deficient mice

Dad1, the defender against apoptotic cell death, comprises the oligosaccharyltransferase complex and is well conserved among eukaryotes. In hamster BHK21‐derived tsBN7 cells, loss of Dad1 causes apoptosis which cannot be prevented by Bcl‐2.

Regulation of the epithelial-mesenchymal transformation through gap junction channels in heart development.

Analyses of mice lacking the gap junction protein, connexin45 (Cx45), have provided new insights into the essential roles of gap junction channels in early embryogenesis. Of great surprise is the function of Cx45 in the endothelium, where it is essential for synchronized activation of the transcription factor Nfatc1. This laterally synchronized regulation model extends the generally accepted vertical model, in which interactions between the endocardium and the myocardium induce endocardial cushion formation through the epithelial-mesenchymal transformation.

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.

Identification of rab12 as a Secretory Granule Associated Small GTP-Binding Protein in Atrial Myocytes

A subfamily of small GTP-binding proteins, rab, has been shown to be involved in regulation of vesicular traffic in eukaryotic cells. The goal of this study was to identify the rab proteins associated with atrial secretory granules. A [32P]GTP-overlay assay showed the presence of multiple small GTP-binding proteins on the atrial granules. By biochemical analysis, we have demonstrated that one of the small GTP-binding proteins associated with the atrial granules is a rab12 protein (rab12p), one of the rab proteins that are most closely related to a Sec4 protein of yeast. Association of rab12p with the atrial granules was confirmed by immunogold electron microscopy. Immunoprecipitation followed by immunoblot analysis with anti-rab12 antibody showed that in addition to atria, rab12p was expressed in multiple other organs and cell lines. These results suggest that rab12p may function in vesicular traffic in multiple diverse types of cells.

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.

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.