Volume Prediction of Extended Latissimus Dorsi Musculocutaneous Flap for Breast Reconstruction Using a Computed Tomography Volume-Rendering Technique with an X-ray Contrast Thread Marking.

BACKGROUND: The extended latissimus dorsi (ELD) musculocutaneous flap is one of the surgical techniques used for breast reconstruction. Preoperative preparation to determine the exact amount of flap tissue to be harvested is important to achieve a good outcome with autologous tissue reconstruction. However, few reports exist on objective preoperative volume prediction of ELD flaps. The purpose of this study was to quantify the elevated ELD volume as a preoperative plan. METHODS: Patients who underwent immediate or delayed breast reconstruction with ELD flap after mastectomy between March 2015 and January 2022 are included. (1) The ELD flap was designed preoperatively, X-ray contrast thread was applied along the design, and CT imaging was performed in the same lateral supine position as the surgical position. 3D images were constructed, and the volume-rendering method was used to obtain the integrated volume. (2) Intraoperative ELD flap volume was calculated using the water displacement method. The correlation between (1) and (2) was examined. RESULTS: (1) The mean preoperative predicted value was 290.2 mL and (2) the mean intraoperative ELD flap volume was 298.3 mL. The correlation coefficient between the two volumes was 0.93, indicating that they were correlated. CONCLUSION: We could quantify the ELD flap volume using the volume-rendering method with X-ray contrast threads. This study could be a useful method for preoperative prediction planning of the ELD flap in breast reconstruction. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Expression analysis of Baf60c during heart regeneration in axolotls and neonatal mice.

Some organisms, such as zebrafish, urodele amphibians, and newborn mice, have a capacity for heart regeneration following injury. However, adult mammals fail to regenerate their hearts. To know why newborn mice can regenerate their hearts, we focused on epigenetic factors, which are involved in cell differentiation in many tissues. Baf60c (BRG1/BRM-associated factor 60c), a component of ATP-dependent chromatin-remodeling complexes, has an essential role for cardiomyocyte differentiation at the early heart development. To address the function of Baf60c in postnatal heart homeostasis and regeneration, we examined the detailed expression/localization patterns of Baf60c in both mice and axolotls. In the mouse heart development, Baf60c was highly expressed in the entire heart at the early stages, but gradually downregulated at the postnatal stages. During heart regeneration in neonatal mice and axolotls, Baf60c expression was strongly upregulated after resection. Interestingly, the timing of Baf60c upregulation after resection was consistent with the temporal dynamics of cardiomyocyte proliferation. Moreover, knockdown of Baf60c downregulated proliferation of neonatal mouse cardiomyocytes. These data suggested that Baf60c plays an important role in cardiomyocyte proliferation in heart development and regeneration. This is the first study indicating that Baf60c contributes to the heart regeneration in vertebrates.

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.

Coordinated regulation of differentiation and proliferation of embryonic cardiomyocytes by a jumonji (Jarid2)-cyclin D1 pathway.

In general, cell proliferation and differentiation show an inverse relationship, and are regulated in a coordinated manner during development. Embryonic cardiomyocytes must support embryonic life by functional differentiation such as beating, and proliferate actively to increase the size of the heart. Therefore, progression of both proliferation and differentiation is indispensable. It remains unknown whether proliferation and differentiation are related in these embryonic cardiomyocytes. We focused on abnormal phenotypes, such as hyperproliferation, inhibition of differentiation and enhanced expression of cyclin D1 in cardiomyocytes of mice with mutant jumonji (Jmj, Jarid2), which encodes the repressor of cyclin D1. Analysis of Jmj/cyclin D1 double mutant mice showed that Jmj was required for normal differentiation and normal expression of GATA4 protein through cyclin D1. Analysis of transgenic mice revealed that enhanced expression of cyclin D1 decreased GATA4 protein expression and inhibited the differentiation of cardiomyocytes in a CDK4/6-dependent manner, and that exogenous expression of GATA4 rescued the abnormal differentiation. Finally, CDK4 phosphorylated GATA4 directly, which promoted the degradation of GATA4 in cultured cells. These results suggest that CDK4 activated by cyclin D1 inhibits differentiation of cardiomyocytes by degradation of GATA4, and that initiation of Jmj expression unleashes the inhibition by repression of cyclin D1 expression and allows progression of differentiation, as well as repression of proliferation. Thus, a Jmj-cyclin D1 pathway coordinately regulates proliferation and differentiation of cardiomyocytes.

A jumonji (Jarid2) protein complex represses cyclin D1 expression by methylation of histone H3-K9.

Covalent modifications of histone tails have critical roles in regulating gene expression. Previously, we identified the jumonji (jmj, Jarid2) gene, the jmjC domain, and a Jmj family. Recently, many Jmj family proteins have been shown to be histone demethylases, and jmjC is the catalytic domain. However, Jmj does not have histone demethylase activity because the jmjC domain lacks conserved residues for binding to cofactors. Independently of these studies, we previously showed that Jmj binds to the cyclin D1 promoter and represses the transcription of cyclin D1. Here, we show the mechanisms by which Jmj represses the transcription of cyclin D1. We found that a protein complex of Jmj had histone methyltransferase activity toward histone H3 lysine 9 (H3-K9). We also found that Jmj bound to the H3-K9 methyltransferases G9a and GLP. Expression of Jmj recruited G9a and GLP to the cyclin D1 promoter and increased H3-K9 methylation. Inactivation of both G9a and GLP, but not of only G9a, inhibited the methylation of H3-K9 in the cyclin D1 promoter and repression of cyclin D1 expression by Jmj. These results suggest that Jmj methylates H3-K9 and represses cyclin D1 expression through G9a and GLP, and that Jmj family proteins can regulate gene expression by not only histone demethylation but also other histone modification.

Functions of a jumonji-cyclin D1 pathway in the coordination of cell cycle exit and migration during neurogenesis in the mouse hindbrain.

During development of the mouse central nervous system (CNS), most neural progenitor cells proliferate in the ventricular zone (VZ). In many regions of the CNS, neural progenitor cells give rise to postmitotic neurons that initiate neuronal differentiation and migrate out of the VZ to the mantle zone (MZ). Thereafter, they remain in a quiescent state. Here, we found many ectopic mitotic cells and cell clusters expressing neural progenitor or proneural marker genes in the MZ of the hindbrain of jumonji (jmj) mutant embryos. When we examined the expression of cyclin D1, which is repressed by jmj in the repression of cardiac myocyte proliferation, we found many ectopic clusters expressing both cyclin D1 and Musashi 1 in the MZ of mutant embryos. jmj is mainly expressed in the cyclin D1 negative region in the hindbrain, and cyclin D1 expression in the VZ was upregulated in jmj mutant mice. In jmj and cyclin D1 double mutant mice, the ectopic mitosis and formation of the abnormal clusters in the MZ were rescued. These results suggest that a jmj-cyclin D1 pathway is required for the precise coordination of cell cycle exit and migration during neurogenesis in the mouse hindbrain.

Cardiac abnormalities cause early lethality of jumonji mutant mice.

jumonji (jmj) mutant mice, obtained by a gene trap strategy, showed several morphological abnormalities including neural tube and cardiac defects, and died in utero around embryonic day 11.5 (E11.5). It is unknown what causes the embryonic lethality. Here, we demonstrate that exogenous expression of jmj gene in the heart of jmj mutant mice rescued the morphological phenotypes in the heart, and these embryos survived until E13.5. These results suggest that there are at least two lethal periods in jmj mutant mice, and that cardiac abnormalities may cause the earlier lethality. In addition, the rescue of the cardiac abnormalities by the jmj transgene provided solid evidence that the cardiac abnormalities resulted from mutation of the jmj gene.

Modifiers of the jumonji mutation downregulate cyclin D1 expression and cardiac cell proliferation.

Cell proliferation is an important factor in various developmental processes in tissue morphogenesis, and is strictly regulated spatiotemporally. jumonji (jmj) deficient mice with a C3H/He background show hyperproliferation of cardiac myocytes and die probably of the phenotype around embryonic day 11.5. Analyses of the abnormalities revealed that repression of cyclin D1 expression by jmj is necessary for downregulation of cardiac myocyte proliferation. On the other hand, jmj mutant mice with a BALB/c background die around E14.5, suggesting that genetic background modifies hyperproliferation in the heart and timing of lethality. Here, we demonstrated that the hyperproliferation was not observed, and that cell proliferation and expression of cyclin D1 were downregulated properly in the cardiac ventricles of jmj mutant mice with a BALB/c background. These results suggest the modifier(s) of the jmj mutation can downregulate cardiac cell proliferation by repressing cyclin D1 expression in the same way as jmj.

jumonji downregulates cardiac cell proliferation by repressing cyclin D1 expression.

Spatiotemporal regulation of cell proliferation is necessary for normal tissue development. The molecular mechanisms, especially the signaling pathways controlling the cell cycle machinery, remain largely unknown. Here, we demonstrate a negative relationship between the spatiotemporal patterns of jumonji (jmj) expression and cardiac myocyte proliferation. cyclin D1 expression and cell proliferation are enhanced in the cardiac myocytes of jmj-deficient mutant embryos. In contrast, jmj overexpression represses cyclin D1 expression in cardiac cells, and Jmj protein binds to cyclin D1 promoter in vivo and represses its transcriptional activity. cyclin D1 overexpression causes hyperproliferation in the cardiac myocytes, but the absence of cyclin D1 in jmj mutant embryos rescues the hyperproliferation. Therefore, Jmj might control cardiac myocyte proliferation and consequently cardiac morphogenesis by repressing cyclin D1 expression.