Kazuomi Nakamura

Cell cycle regulation in mouse heart during embryonic and postnatal stages

The regulation of cardiomyocyte proliferation is important for heart development and function. Proliferation levels of mouse cardiomyocytes are high during early embryogenesis and start to decrease at midgestation. Many cardiomyocytes undergo mitosis without cytokinesis, resulting in binucleated cardiomyocytes during early postnatal stages, following which the cell cycle arrests irreversibly. It remains unknown how the proliferation pattern is regulated, and how the irreversible cell cycle arrest occurs. To clarify the mechanisms, fundamental information about cell cycle regulators in cardiomyocytes and cell cycle patterns during embryonic and postnatal stages is necessary. Here, we show that the expression, complex formation, and activity of main cyclins and cyclin‐dependent kinases (CDKs) changed in a synchronous manner during embryonic and postnatal stages. These levels decreased from midgestation to birth, and then showed one wave in which the peak was around postnatal day 5. Detailed analysis of the complexes suggested that CDK activities were inhibited before the protein levels decreased. Analysis of DNA content distribution patterns in mono‐ and binucleated cardiomyocytes after birth revealed changes in cell cycle distribution patterns and the transition from mono‐ to binucleated cells. These analyses indicated that the wave of cell cycle regulator expression or activities during postnatal stages mainly produced binucleated cells from mononucleated cells. The data obtained should provide a basis for the analysis of cell cycle regulation in cardiomyocytes during embryonic and postnatal stages.

Histone H3 lysine 9 methyltransferases, G9a and GLP are essential for cardiac morphogenesis.

Lysine methylation of the histone tail is involved in a variety of biological events. G9a and GLP are known as major H3-K9 methyltransferases and contribute to transcriptional silencing. The functions of these genes in organogenesis remain largely unknown. Here, we analyzed the phenotypes of cardiomyocyte specific GLP knockout and G9a knockdown (GLP-KO/G9a-KD) mice. The H3-K9 di-methylation level decreased markedly in the nuclei of the cardiomyocytes of GLP-KO/G9a-KD mice, but not single G9a or GLP knockout mice. In addition, GLP-KO/G9a-KD mice showed neonatal lethality and severe cardiac defects (atrioventricular septal defects, AVSD). We also showed that hypoplasia in the atrioventricular cushion, which is a main part of the atrioventricular septum, caused AVSD. Expression analysis revealed downregulation of 2 AVSD related genes and upregulation of several non-cardiac specific genes in the hearts of GLP-KO/G9a-KD mice. These data indicate that G9a and GLP are required for sufficient H3-K9 di-methylation in cardiomyocytes and regulation of expression levels in multiple genes. Moreover, our findings show that G9a and GLP have an essential role in normal morphogenesis of the atrioventricular septum through regulation of the size of the atrioventricular cushion.

Repression of cyclin D1 expression is necessary for the maintenance of cell cycle exit in adult mammalian cardiomyocytes.

Background: How cell cycle exit is maintained in adult mammalian cardiomyocytes is largely unknown. Results: Cyclin D1 expression causes cell cycle reentry in >40% of adult mouse cardiomyocytes. Conclusion: Silencing the cyclin D1 expression is necessary for the maintenance of the cell cycle exit. Significance: One of the mechanisms regulating cell cycle exit in mammalian cardiomyocytes has been uncovered. The hearts of neonatal mice and adult zebrafish can regenerate after injury through proliferation of preexisting cardiomyocytes. However, adult mammals are not capable of cardiac regeneration because almost all cardiomyocytes exit their cell cycle. Exactly how the cell cycle exit is maintained and how many adult cardiomyocytes have the potential to reenter the cell cycle are unknown. The expression and activation levels of main cyclin-cyclin-dependent kinase (CDK) complexes are extremely low or undetectable at adult stages. The nuclear DNA content of almost all cardiomyocytes is 2C, indicating the cell cycle exit from G1-phase. Here, we induced expression of cyclin D1, which regulates the progression of G1-phase, only in differentiated cardiomyocytes of adult mice. In these cardiomyocytes, S-phase marker-positive cardiomyocytes and the expression of main cyclins and CDKs increased remarkably, although cyclin B1-CDK1 activation was inhibited in an ATM/ATR-independent manner. The phosphorylation pattern of CDK1 and expression pattern of Cdc25 subtypes suggested that a deficiency in the increase in Cdc25 (a and -b), which is required for M-phase entry, inhibited the cyclin B1-CDK1 activation. Finally, analysis of cell cycle distribution patterns showed that >40% of adult mouse cardiomyocytes reentered the cell cycle by the induction of cyclin D1. The cell cycle of these binucleated cardiomyocytes was arrested before M-phase, and many mononucleated cardiomyocytes entered endoreplication. These data indicate that silencing the cyclin D1 expression is necessary for the maintenance of the cell cycle exit and suggest a mechanism that involves inhibition of M-phase entry.

Lipopolysaccharide promotes the development of murine endometriosis-like lesions via the nuclear factor-kappa B pathway

Is lipopolysaccharide (LPS) involved in the development of endometriosis?

Exploring new gene integration sites for gene knock-in by gene-trapping strategy

The knock-in mouse is a powerful tool for biological research, but the stability of expression of an integrated gene strongly depends on where it is integrated in the mouse genome. At present, there are an insufficient number of loci suitable for gene knock-in, such as the Rosa26 locus. Therefore, in this study, we developed an efficient strategy for identifying genome loci suitable for gene knock-in and characterized the properties of such loci for gene integration. For efficient discovery and characterization, we constructed a new gene-trapping vector that enables monitoring of the expression of both trapped and integrated genes using fluorescence. We successfully obtained fluorescent–positive mouse embryonic stem cell (mESC) clones with the vector. Thorough analysis of the expression of fluorescent proteins in chimera embryos generated with the obtained mESC clones, some of the gene-trapped chimera embryos showed stable and ubiquitous expression of the integrated gene. Furthermore, adult mice derived from one of the gene-trapped mESC clones showed ubiquitous expression of the integrated gene in various tissues without any unusual phenotype. This indicated that the identified locus possesses high potential for foreign gene integration. Our strategy allows for efficient discovery and characterization of mouse genome loci for gene integration.

Inhibition of IAP (inhibitor of apoptosis) proteins represses inflammatory status via nuclear factor-kappa B pathway in murine endometriosis lesions

How is the role of inhibitor of apoptosis proteins (IAPs) in the development of murine endometriosis lesions?

Use of a Human Artificial Chromosome for Delivering Trophic Factors in a Rodent Model of Amyotrophic Lateral Sclerosis

A human artificial chromosome (HAC) is maintained as an episome within a cell and avoids random integration into the host genome. It can transfer multiple and/or large transgenes along with their regulatory elements thereby resembling native chromosomes. Using this HAC system, we established mesenchymal stem cells (MSCs) that simultaneously expressed hepatocyte growth factor, glial cell line-derived neurotrophic factor, and insulin-like growth factor 1, termed HAC-MSCs. This cell line provides an opportunity for stable transplantation and thorough analyses. We then introduced the cells for the treatment of a neurodegenerative disorder, amyotrophic lateral sclerosis. The HAC-MSCs were transplanted via the fourth cerebral ventricle (CV) or intravenous (i.v.) infusion at various ages of recipient mice. Littermate- and sex-matched mice underwent a sham procedure. Compared to the controls, there was an encouraging trend of increased life span via CV transplantation and delayed onset in i.v. infusion 60 days after transplantation. Further, we confirmed a statistically significant increase in life span via CV transplantation at 100 days. This effect was not seen in mice transplanted with MSCs lacking the HAC. We successfully enhanced the trophic potential of the MSCs using the HAC. This strategy could be a promising direction for the treatment of neurodegenerative disorders.

New insights into the efficacy of SR-16234, a selective estrogen receptor modulator, on the growth of murine endometriosis-like lesions

To evaluate the effects of SR‐16234 (SR), a selective estrogen receptor modulator (SERM), on murine endometriosis‐like lesions.