Rapid Electrochemical Deprotection of the Iso­nicotinyl­oxycarbonyl Group from Carbonates and Thiocarbonates in a Microfluidic Reactor

Electro­reductive deprotection of the iso­nicotinyl­oxy­carbonyl (<i>i</i>Noc) group from hydroxy, thiol, and amino groups was carried out in an electro­chemical micro­reactor. The small distance of the platinum electrodes in the micro­reactor enables a rapid electro­chemical redox reaction without added electrolytes. As a result, the electro­chemical deprotection of <i>O</i>- and <i>S</i>-<i>i</i>Noc aromatic substrates was achieved in short reaction times (<2 min), while <i>N</i>-<i>i</i>Noc and non­aromatic substrates did not react under the same reaction conditions. This method enables a rapid and site-selective deprotection of <i>O</i>- or <i>S</i>-<i>i</i>Noc groups without removal of <i>N</i>-<i>i</i>Noc moieties

Cardiomyocyte undergo apoptosis only in later stages of IKK2-CA expression, but show early defects in proliferation.

<p>(A) TUNEL (terminal deoxynucleotidyltransferase-mediated UTP end labeling) assays were performed at E10.5, E12.5 and E14.5 for the indicated genotypes. TUNEL-positive cells show green fluorescence. An antibody against α-myosin heavy chain was used to specifically stain cardiomyocytes (red); nuclei were stained with DAPI (blue). RV, right ventricle; LV, left ventricle. Scale bar: 500 μm. The right panels show higher magnifications of the areas indicated in the middle panels. (B) Pregnant mice were injected with BrdU and their embryos (E10.5) were harvested 4 hours later. Cardiomyocytes were isolated and analyzed in FACS by staining with antibodies against the early murine cardiomyocyte marker Alcam (CD166)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141591#pone.0141591.ref013" target="_blank">13</a>] and against BrdU. IKK^MyHC cardiomyocytes (grey, filled) generally show a shift to the left in comparison to control cardiomyocytes (black line), indicating a lower BrdU staining and thus less proliferation.</p

Cardiomyocyte-specific IKK/NF-κB activation up-regulates cell-adhesion molecules and induces the recruitment of macrophages.

<p>(A) Immunofluorescence staining of heart cryosections from control and IKK^MyHC embryonic hearts shows infiltration with F4/80 positive macrophages. Macrophages were stained with an antibody against F4/80 (red) and cardiomyocytes were stained with an antibody against α-smooth muscle actin (αSMA, green). Scale bar: 100 μm. (B) Immunofluorescence staining of heart cryosections from control and IKK^MyHC embryonic hearts show Icam-1 expression in IKK^MyHC animals, but not in control animals at E11.5. Colocalization of Icam-1 (green) and the cardiomyocyte-specific cTNT (red) is visible as yellow staining. Scale bar: 100 μm.</p

Expression of constitutively active IKK2 in cardiomyocytes results in embryonic lethality.

<p>The table shows the absolute number and the percentage of animals born with the indicated genotypes for the transgenes MyHC.tTA and IKK2-CA (observed), and the corresponding expected number and percentage (expected). The observed number of animals positive for both MyHC-tTA and IKK2-CA (4) is much lower than expected (30.5).</p><p>Expression of constitutively active IKK2 in cardiomyocytes results in embryonic lethality.</p

Cardiomyocyte-specific expression of IKK2-CA is embryonically lethal.

<p>(A) Survival rate of IKK^MyHC embryos with expression of IKK2-CA transgene (i.e. in the absence of doxycycline). Pregnant mothers were killed at the indicated day of embryonic development, embryos were checked for viability by assessing the presence of a heart beat, and the genotype of the embryos was assessed both by PCR and by measuring luciferase activity. A total of 73 IKK^MyHC embryos were scored out of 288 embryos in total at the indicated days of embryonic development. Controls comprise both single transgenic and non-transgenic embryos. (B) Gross appearance of embryos at E10.5. Note the pericardial edema in IKK^MyHC embryos (arrow, lower right); the pericardium is filled with blood (lower right). A: atrium, V: ventricle, L: limb. Scale bar: 0.5 mm. (C) Immunofluorescence analysis of IKK2-CA transgene expression in control (upper left) and IKK^MyHC (upper right) embryos at E9.5. IKK2-CA expression (red) was exclusively detected in the heart of IKK^MyHC embryos. Nuclear staining with DAPI (blue). Scale bar 500 μm. Lower panel: Magnification of the ventricle of an IKK^MyHC embryo. Scale bar: 100 μm. A: atrium, V: ventricle, TA: truncus arteriosus. (D) Upper panel: Western blot analysis of heart extracts from E11.5 and E12.5 embryos. An antibody against human IKK (hIKK) was used to detect the IKK transgene, which is of human origin. IKK2-CA was exclusively detected in double transgenic embryos (IKK^MyHC), but not in control embryos (ctrl). Expression of Erk2 was used as a loading control. Lower panel: Western blot analysis of heart extracts of E14.5 animals with an antibody detecting both human (transgenic) and murine (endogenous) IKK. Erk2 was used as a loading control. (E) Immunofluorescence staining of E11.5 cryosections with antibodies against cardiac troponin T (cTNT, red), IKK and DAPI (blue), showing a colocalization of the cardiomyocyte-specific cTNT and the IKK transgene in IKK^MyHC embryos. Scale bar 200 μm. (F) Immunohistochemical analysis at E11.5 shows that RelA/p65 in IKK^MyHC cardiomyocytes, but not in control cardiomyocytes, is localized in the nucleus, suggesting NF-κB activation. Scale bar: 310 μm.</p

Cardiomyocyte-specific IKK/NF-κB activation induces myofiber degeneration and loss of typical cardiomyocyte proteins.

<p>(A) Control and IKK^MyHC sections were stained for sarcomeric α-actinin (green) and DAPI (blue). Scale bar: 100 μm. (B) Control and IKK^MyHC sections were stained for desmin (yellow) and DAPI (blue). Scale bar: 100 μm. (C) Electron microscopic analyses of control and IKK^MyHC heart sections. Cardiomyocytes of control animals show normally developed myofibers with typical sarcomeres (left). IKK^MyHC cardiomyocytes show general disorganization and a reduced width of myofibers (right). Scale bar: 1 μm.</p

Cardiomyocyte-specific expression of IKK2-CA in embryos.

<p>(A) In order to direct the expression of constitutively active IKK2 (IKK2-CA) to the heart, mice expressing the tetracycline-transactivator (tTA) under the α-myosin heavy chain promoter (α-MyHC) were crossed with mice bearing an IKK2-CA (IKK) and a luciferase allele under the control of a bidirectional tTA-responsive promoter (TRE, tetracycline response element). The transgene IKK2-CA is expressed only in the absence of doxycycline (-DOX), whereas its expression is blocked in the presence of doxycycline (+DOX). (B) Western blot of embryonic heart extracts at day E12.5. The transgene IKK2-CA is expressed exclusively in double transgenic (dtg) IKK^MyHC animals in the absence of doxycycline (-DOX). The antibody used also detects endogenous IKK, which migrates slightly below the transgene and is readily detectable in wild type animals (wt) as well as IKK^MyHC animals (dtg) treated with doxycycline. An antibody against ERK2 was used as a loading control.</p

Cytokine profile of IKK^MyHC hearts and IKK^MyHC cardiomyocytes.

<p>(A) Heat map of cytokines expressed in IKK^MyHC hearts at E12.5 (>2-fold regulation, p<0.05) as detected by microarray (N = 3 for wild type (wt), N = 4 for transgenics (IKK-MyHC). (B) Expression of the indicated cytokines as assessed by real-time quantitative PCR from IKK^MyHC (black bars) and control (grey bars) heart ventricles at E12.5. Shown are the means +SEM; N = 6, *P<0.05, **P<0.01, ***P<0.001 (t test), nd, not detected. (C) Expression of the indicated cytokines as assessed by real-time quantitative PCR from IKK^MyHC (black bars) and control (grey bars) cardiomyocytes isolated at E12.5. Shown are the means +SEM; N = 6, *P<0.05, **P<0.01, ***P<0.001 (t test), nd, not detected.</p

Cardiac-Specific Activation of IKK2 Leads to Defects in Heart Development and Embryonic Lethality

<div><p>The transcription factor NF-κB has been associated with a range of pathological conditions of the heart, mainly based on its function as a master regulator of inflammation and pro-survival factor. Here, we addressed the question what effects activation of NF-κB can have during murine heart development. We expressed a constitutively active (CA) mutant of IKK2, the kinase activating canonical NF-κB signaling, specifically in cardiomyocytes under the control of the α-myosin heavy chain promoter. Expression of IKK2-CA resulted in embryonic lethality around E13. Embryos showed defects in compact zone formation and the contractile apparatus, and overall were characterized by widespread inflammation with infiltration of myeloid cells. Gene expression analysis suggested an interferon type I signature, with increased expression of interferon regulatory factors. While apoptosis of cardiomyocytes was only increased at later stages, their proliferation was decreased early on, providing an explanation for the disturbed compact zone formation. Mechanistically, this could be explained by activation of the JAK/STAT axis and increased expression of the cell cycle inhibitor p21. A rescue experiment with an IκBα superrepressor demonstrated that the phenotype was dependent on NF-κB. We conclude that activation of NF-κB is detrimental during normal heart development due to excessive activation of pro-inflammatory pathways.</p></div

Dysregulation of regulators of proliferation and differentiation in embryonic IKK^MyHC hearts.

<p>(A) The expression of Stat1, phospho-Stat1, p21 and the transgenes IKK2 and luciferase was determined by Western blot, with Erk2 shown as loading control. The diagram on the right shows a quantification of the p21 immunoblot, normalized to the expression of ERK2. (B) Expression of the indicated cell cycle modulators was determined at the mRNA level using real-time quantitative PCR in purified cardiomyocytes from IKK^MyHC (black bars) and control (grey bars) embryos at E10.5. Shown are the means +SEM; N = 5, *P<0.05. (C) Upper panel: Staining of embryonic heart sections at E11.5 for Sca1 (green) and F4/80 (red). Lower panel: Staining of embryonic heart sections at E11.5 for the IKK2 transgene (green) and the progenitor marker Sca1 (red). DAPI was used to stain nuclei. Insets show a higher magnification of the indicated areas.</p