Direct Exposure to Ethanol Disrupts Junctional Cell-Cell Contact and Hippo-YAP Signaling in HL-1 Murine Atrial Cardiomyocytes.

Direct exposure of cardiomyocytes to ethanol causes cardiac damage such as cardiac arrythmias and apoptotic cell death. Cardiomyocytes are connected to each other through intercalated disks (ID), which are composed of a gap junction (GJ), adherens junction, and desmosome. Changes in the content as well as the subcellular localization of connexin43 (Cx43), the main component of the cardiac GJ, are reportedly involved in cardiac arrythmias and subsequent damage. Recently, the hippo-YAP signaling pathway, which links cellular physical status to cell proliferation, differentiation, and apoptosis, has been implicated in cardiac homeostasis under physiological as well as pathological conditions. This study was conducted to explore the possible involvement of junctional intercellular communication, mechanotransduction through cytoskeletal organization, and the hippo-YAP pathway in cardiac damage caused by direct exposure to ethanol. HL-1 murine atrial cardiac cells were used since these cells retain cardiac phenotypes through ID formation and subsequent synchronous contraction. Cells were exposed to 0.5-2% ethanol; significant apoptotic cell death was observed after exposure to 2% ethanol for 48 hours. A decrease in Cx43 levels was already observed after 3 hours exposure to 2% ethanol, suggesting a rapid degradation of this protein. Upon exposure to ethanol, Cx43 translocated into lysosomes. Cellular cytoskeletal organization was also dysregulated by ethanol, as demonstrated by the disruption of myofibrils and intermediate filaments. Coinciding with the loss of cell-cell adherence, decreased phosphorylation of YAP, a hippo pathway effector, was also observed in ethanol-treated cells. Taken together, the results provide evidence that cells exposed directly to ethanol show 1) impaired cell-cell adherence/communication, 2) decreased cellular mechanotransduction by the cytoskeleton, and 3) a suppressed hippo-YAP pathway. Suppression of hippo-YAP pathway signaling should be effective in maintaining cellular homeostasis in cardiomyocytes exposed to ethanol

Formation of high molecular weight p62 by CORM-3.

CORM-3 is a water-soluble carbon monoxide (CO)-releasing molecule developed for possible therapeutic use of CO. CORM-3 belongs to a group of metal carbonyl compounds that contain transition metals and carbonyls as the central scaffold and coordinated ligands, respectively. CORM-3 has been reported to be reactive with many proteins in eukaryotes including mammals. Among them, several extracellular proteins, such as lysozyme, as well as plasma albumin and fibronectin, have been shown to interact directly with CORM-3. p62 is an intracellular adaptor protein required for targeting ubiquitinated (Ub) proteins to lysosomal degradation through autophagy. p62 has been shown to undergo self-oligomerization via covalent crosslinking in response to treatment with verteporfin, a benzoporphyrin derivative used for photodynamic therapy. Here we show that CORM-3 also interacts directly with p62. When applied to mouse embryonic fibroblasts (MEFs) at a high concentration (1 mM), CORM-3 causes the formation of reduction- and detergent-resistant high molecular weight (HMW)-p62. HMW-p62 accumulates more in atg5-/- MEFs than in wild type (WT) MEFs, showing the elimination of HMW-p62 through autophagy. HMW-p62 is also generated in H9c2 rat cardiomyoblastoma as well as A549 human alveolar epithelial cells, suggesting that HMW-p62 formation is not specific to MEFs, but, rather, is a general event in mammalian cells. HMW-p62 formation by CORM-3 can be reproduced using purified p62 in vitro, demonstrating the direct interaction between CORM-3 and p62. These results show that p62 is a CORM-3-interactive intracellular protein

Factors Affecting the Career Continuation of Newly Graduated and Reinstated Dental Hygienists Who Participated in a Technical Training Program in Japan

This study aimed to identify (1) what newly graduated dental hygienists and reinstated dental hygienists consider important for preventing early turnover in their own professions and for encouraging them to continue in the profession more generally and (2) relevant factors among hygienists intending to continue working in the field. An anonymous, self-administered questionnaire survey was distributed to 215 Japanese dental hygienists who participated in the technical training programs (response rate: 72.6%). Of them, 143 participants were classified into two groups: newly graduated (NGDH, n = 32) and those reinstated to work (RDH, n = 111). Follow-up for lack of skills was most often selected as important for preventing early turnover among both groups and follow-up for lack of knowledge was significantly selected in the RDH group (p < 0.001). Regarding factors important for career continuation, NGDHs significantly selected gaining job satisfaction, whereas RDHs significantly selected working support. Relevant factors contributing to participants’ intentions to continue in the profession were job satisfaction (NGDH, OR = 8.37; RDH, OR = 8.83), career outlook (RDH, OR = 3.11), and job turnover experience because of marriage and parenting (RDH, OR = 2.70), thereby suggesting the importance of raising awareness regarding career progression and job-related rewards among dental hygienists, their educators, and the government through ongoing career education

Decreased activation of the hippo pathway and YAP phosphorylation by ethanol in HL-1 cells.

<p>(A) Phosphorylation of Mst1/2 and LATS1 and their suppression in HL-1 cells exposed to ethanol. Cells were treated with or without 2% ethanol for the indicated time periods. Levels of phosphorylated Mst1/2 (p-Mst1/2) and phosphorylated LATS1 (p-LATS1) were determined by western blot analysis. Hsc70 served as a loading control. (B) Phosphorylation of YAP and its suppression in HL-1 cells exposed to ethanol. Cells were treated with or without 2% ethanol for the indicated time periods. Levels of phosphorylated YAP (p-YAP) were determined by western blot analysis and normalized to Hsc70 levels. The graph shows mean±S.E. (n = 3). *, p<0.05 versus control. (C) Sub-cellular localization of YAP in cells treated with or without ethanol. Cells were treated with or without 2% ethanol for 24 hours, and the sub-cellular localization of YAP was examined by immunocytochemical analysis under fluorescence microscopy. The white arrow indicates a high-cell-density region where YAP is localized to the nucleus as well as cytoplasm. The yellow arrow indicates a low-cell-density region where YAP is localized to the nucleus.</p

Time-dependent and rapid decrease in Cx43 protein levels in ethanol-treated HL-1 cells.

<p>(A-D) Cells were incubated in medium containing 2% ethanol for the indicated time periods, and the protein levels of Cx43, Cx40 and Cx45 were determined by western blot analysis (A). Hsc70 served as a loading control. Quantitative analysis of Cx43 (B), Cx40 (C) and Cx45 (D) is shown. Each graph shows mean±S.E. (n = 4–6). **, p<0.01 versus 0 h. Relative levels to control (0 h) group were shown for Cx43 (B). (E) The levels of Cx43 mRNA were determined by qPCR. GAPDH served as an internal control. The graph shows mean±S.E. (n = 3). **, p<0.01, *, p<0.05 versus 0 h. (F) Ethanol disrupts cell-cell coupling. Cells were incubated in the medium containing 2% ethanol for 48 hours, and scrape-loading dye transfer assay was performed as described in the Materials and methods. Areas of dye spread (fluorescent areas) were quantified using ImageJ software. The graph shows mean±S.E. (n = 5). **, p<0.01 versus control.</p

Lysosomal localization of Cx43 in ethanol-treated HL-1 cells.

<p>(A and B) Change of Cx43 localization from the plasma membrane to lysosomes in HL-1 cells in response to ethanol exposure. Cells were transfected with combinations of vectors (Cx43-mApple and LAMP1-mGFP for (A) and Cx43-mApple and LMP2-GFP for (B)) and incubated in medium containing 2% ethanol for 24 hours. The cells were then observed under a fluorescence microscope. Arrows indicate gap-junctional cell-cell contact. (C) Proteasome inhibitors do not affect Cx43 protein levels. HL-1 cells were treated with 2% ethanol for 24 hours and Cx43 levels were determined. In some samples, MG132 (500 nM) or bortezomib (5 nM) was included in the medium during the last 6 hours of ethanol exposure. GAPDH served as a loading control. The graph shows mean±S.E. (n = 3). (D) A lysosome inhibitor affects Cx43 protein levels. HL-1 cells were treated with 2% ethanol with or without bafilomycinA1 (50 nM) for 48 hours, and Cx43 levels were determined. The graph shows mean±S.E. (n = 3).</p