Background:Full-length 16S rRNA gene sequencing using nanopore technology has become increasingly relevant for profiling complex microbial communities, including the human oral microbiome. Primer selection plays a critical role in amplification bias and taxonomic resolution, yet remains insufficiently investigated for oropharyngeal samples. Methods:We conducted a comparative analysis of two primer sets with differing degrees of degeneracy - Oxford Nanopores (ONT) standard 27F primer (27F-I) and a more degenerate variant (27F-II) - for full-length 16S rRNA gene sequencing of 80 human oropharyngeal swab samples using ONTs MinION Mk1C. Alpha diversity and taxonomic profiles were statistically compared between primer sets and benchmarked against a large-scale salivary microbiome dataset (n=1,989) from healthy individuals. Results:Primer choice significantly impacted microbial community composition and diversity. The more degenerate primer set 27F-II yielded significantly higher alpha diversity (Shannon index: 2.684 vs. 1.850; p < 0.001) and detected a broader range of taxa across all phyla. The taxonomic profiles generated with 27F-II strongly correlated with the reference dataset (Pearson's r = 0.86, p < 0.0001), whereas profiles generated with 27F-I showed weak correlation (r = 0.49, p = 0.06). 27F-I overrepresented Proteobacteria and underrepresented key genera such as Prevotella, Faecalibacterium, and Porphyromonas. Conclusion:Our findings demonstrate that primer degeneracy has a substantial effect on taxonomic resolution and biodiversity estimates in oropharyngeal 16S rRNA gene sequencing. The more degenerate 27F-II primer set seams to more faithfully captures the complexity of the human oropharyngeal microbiome and aligns more closely with population-level reference data. These results underscore the importance of careful primer selection and support the adoption of degenerate primers as a methodological standard in nanopore-based oral microbiome research.

Die Verkalkung des Mitralanulus ist die Folge einer atherosklerotischen Grunderkrankung und geht häufig mit einer erhöhten kardiovaskulären Morbidität und Mortalität einher. Ein chirurgischer Eingriff ist erforderlich, wenn Patienten aufgrund einer Herzklappenerkrankung Symptome entwickeln, deren Ursache in der Regel eine aufgetretene Mitralklappeninsuffizienz ist. In diesem Manuskript werden histopathologische Aspekte im Zusammenhang mit der Verkalkung des Mitralanulus erörtert und chirurgische Strategien für das Débridement und die Rekonstruktion des Anulus mit anschließender Reparatur oder Ersatz der Mitralklappe beschrieben. Neben den typischen Strategien werden auch seltene und eher experimentelle chirurgische Techniken diskutiert.

Mesenchymal stem cells (MSC) have been used to treat different clinical conditions although the mechanisms by which pathogenetic processes are affected are still poorly understood. We have previously analyzed the homing of bone marrow-derived MSC to diseased tissues characterized by a high degree of mononuclear cell infiltration and postulated that MSC might modulate inflammatory responses. Here, we demonstrate that MSC mitigate adverse tissue remodeling, improve organ function, and extend lifespan in a mouse model of inflammatory dilative cardiomyopathy (DCM). Furthermore, MSC attenuate Lipopolysaccharide-induced acute lung injury indicating a general role in the suppression of inflammatory processes. We found that MSC released sTNF-RI, which suppressed activation of the NFκBp65 pathway in cardiomyocytes during DCM in vivo. Substitution of MSC by recombinant soluble TNF-R partially recapitulated the beneficial effects of MSC while knockdown of TNF-R prevented MSC-mediated suppression of the NFκBp65 pathway and improvement of tissue pathology. We conclude that sTNF-RI is a major part of the paracrine machinery by which MSC effect local inflammatory reactions.

Full text Figures and data Side by side Abstract Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Migration of skeletal muscle precursor cells is a key step during limb muscle development and depends on the activity of PAX3 and MET. Here, we demonstrate that BRAF serves a crucial function in formation of limb skeletal muscles during mouse embryogenesis downstream of MET and acts as a potent inducer of myoblast cell migration. We found that a fraction of BRAF accumulates in the nucleus after activation and endosomal transport to a perinuclear position. Mass spectrometry based screening for potential interaction partners revealed that BRAF interacts and phosphorylates PAX3. Mutation of BRAF dependent phosphorylation sites in PAX3 impaired the ability of PAX3 to promote migration of C2C12 myoblasts indicating that BRAF directly activates PAX3. Since PAX3 stimulates transcription of the Met gene we propose that MET signaling via BRAF fuels a positive feedback loop, which maintains high levels of PAX3 and MET activity required for limb muscle precursor cell migration. https://doi.org/10.7554/eLife.18351.001 Introduction In vertebrates, skeletal muscles of the trunk and limbs originate from condensations of the paraxial mesoderm, the somites (Braun and Gautel, 2011; Buckingham and Relaix, 2007). Epaxial muscles are derived from the dorso-medial part of the somatic dermomyotome while the ventro-lateral part gives rise to hypaxial muscles (Ordahl and Le Douarin, 1992). Hypaxial muscles of the body wall and intercostal muscles are generated by elongation of the dermomyotomal epithelium. In contrast, muscles of the limbs, the diaphragm and the tongue are generated from a population of long-range migrating muscle precursor cells, which delaminate from the ventral dermomyotome at specific positions along the cranial-caudal axis after epithelial-mesenchymal transition, allowing them to form muscles far away from somites (Chevallier et al., 1977; Christ and Brand-Saberi, 2002; Christ et al., 1983). Several genes including Pax3 (Bober et al., 1994), Met (Bladt et al., 1995; Dietrich et al., 1999), Cxcr4 (Vasyutina et al., 2005), Gab1 (Sachs et al., 2000), Six1;Eya1 (Heanue et al., 1999) and Lbx1 (Brohmann et al., 2000; Gross et al., 2000; Schäfer and Braun, 1999) have been identified to control somite maturation and compartmentalization, delamination of muscle precursor cells from the dermomyotomal epithelium as well as muscle precursor cell migration, proliferation and differentiation. More specifically, Pax3 is required for correct formation of the ventro-lateral dermomyotome (Bober et al., 1994; Daston et al., 1996) as well as for survival (Relaix et al., 2005) and migration of limb muscle precursor cells (Daston et al., 1996). Met is necessary for de-epithelialization and migration of limb muscle precursor cells (Bladt et al., 1995) but also for myocyte fusion (Webster and Fan, 2013). It is also known that PAX3 controls expression of Met in the ventro-lateral dermomyotome (Relaix et al., 2005; Yang et al., 1996) by direct binding to the Met gene promoter (Epstein et al., 1996), thereby enabling delamination and migration of limb muscle precursor cells. However, the full complexity of the interactions within the genetic network orchestrating limb muscle precursor cell migration and the functional regulation of the activity of PAX3 and its multiple isoforms (Wang et al., 2006) has not been uncovered yet. MET signaling is highly complex and involves several scaffolding adaptors and surface signal modifiers, which allows MET to activate multiple different biochemical pathways including the MAPK (ERK, JNK and p38 MAPKs) pathway, the PI3K-AKT axis, the STAT pathway and the IkB-NFkB complex (reviewed in (Birchmeier et al., 2003; Trusolino et al., 2010)). Importantly, mutants of MET unable to bind the adaptor GRB2, which is considered to act as the primary mediator of RAS-RAF activation, does not affect migration of limb muscle precursor cells but inhibits proliferation of fetal myoblasts and formation of secondary myofibers (Maina et al., 1996). In contrast, inactivation of the adaptor Gab1 severely impairs migration of limb muscle precursor cells (Sachs et al., 2000). GAB1 acts as a docking platform for several molecules including PI3K, PLC, CRK, and SHP2 but also activates the RAS-RAF route after activation by the tyrosine phosphatase SHP2 (Birchmeier et al., 2003; Trusolino et al., 2010). This raises several questions: Does the RAS-RAF pathway contribute to migration of limb muscle precursor cells? If RAF is involved in regulation of limb muscle precursor cell migration, which of the three serine/threonine kinases (ARAF, BRAF, CRAF) does the job? Are potential effects of RAF transmitted via the canonical MEK-ERK pathway or by different means? To answer these questions we inactivated the Braf gene specifically in limb muscle precursor cells, since germ line inactivation of Braf results in embryonic lethality between E10.5 and E12.5 and causes multiple defects including growth retardation, vascular and neuronal defects (Wojnowski et al., 1997). We found that Braf is required for muscle precursor cell migration and skeletal muscle formation in the forelimbs. Protein-protein interaction studies revealed that BRAF phosphorylates and activates PAX3 after endosomal trafficking to a perinuclear position and translocation into the nucleus. Our results suggest a positive feedback loop, which drives skeletal muscle formation by maintaining high levels of PAX3 and MET activity in migrating limb muscle precursor cells. Results BRAF mediates growth factor induced muscle precursor cell migration in vitro The tyrosine kinase receptor MET is instrumental for delamination of limb muscle precursor cells from the dermomyotome and subsequent migration. To identify the branches of the MET signaling network driving migration of myogenic cells, we turned to the muscle cell line C2C12, since evaluation of signaling processes in migrating limb muscle precursor cells is difficult due to the small size of the cell population and its transient appearance. We found that HGF, the ligand of the MET receptor, robustly induced migration of C2C12 cells. Stimulation of migration was blocked by knockdown of Met demonstrating that C2C12 cells can be utilized to study the mechanisms of MET receptor signaling for migration of myogenic cells (Figure 1A). Systematic analysis of the role of potential downstream effectors of MET signaling by siRNA-mediated knockdown disclosed an important role of the serine/threonine-specific protein kinase BRAF, which essentially phenocopied the effects of Met receptor knockdown (Figure 1A). Interestingly, knockdown of Pax3 did also inhibit HGF-mediated stimulation of C2C12 cell migration suggesting a functional involvement of PAX3 in MET signaling (Figure 1A). To test whether expression of BRAF alone is sufficient to promote migration we transfected WT Braf and the constitutively active (CA) Braf (V600E) mutation into C2C12 cells. We observed a strong stimulation of migration by CA BRAF (V600E) and -to a lesser extent- WT BRAF while expression of CRAF had only minor effects on muscle cell migration (Figure 1A). CA BRAF (V600E) and WT BRAF also stimulated expression of Pax3 and Met in C2C12 cells adding further evidence to the putative role of BRAF in the regulatory loop controlling migration of myogenic cells (Figure 1A). Next, we investigated whether BRAF and PAX3 also promote migration of primary dermomyotomal cells (Mennerich et al., 1998). We found that RCAS virus-mediated expression of BRAF and PAX3 but not human alkaline phosphatase (AP) increased cellular migration out of somitic explants isolated from chicken embryos and cultivated in matrigel further corroborating the results obtained with C2C12 cells (Figure 1—figure supplement 1). Figure 1 with 1 supplement see all Download asset Open asset BRAF mediates muscle precursor cell migration independent of MEK/ERK signaling. (A) Immunofluorescence staining of migrating muscle cells (C2C12) after knock down of the hepatocyte growth factor (HGF) receptor (siMet), Braf (siBraf) and Pax3 (siPax3) or after transfection of cultures with Craf, Braf, a dominant negative form of Braf (DN Braf) and CA Braf (V600E) in the presence or absence of HGF. Con indicates control vector and siCon represents a scrambled siRNA control. Cultures were analyzed 4 hr after scratching excluding effects of cell proliferation. Cell numbers were determined by counting the number of DAPI-stained nuclei. A statistical assessment is shown in the upper part of the panel (n = 15; Mann-Whitney-U test, p*<0.05). siRNA knock-down efficiencies for Met (80%), Braf (70%) and Pax3 (85%) were determined by Western blot analysis. (B) Phosphorylation of ERK1/2 after transfection of C2C12 cells with control vector (Con), WT Braf and CA Braf (V600E) in the presence or absence of UO126 (5 µM) or cytochalasin D (5 µM; CytoD), noco (5 µM; nocodazole), Dynasore (80 µM; dyna), colchicine (0.01%; colch), methyl-β-cyclodextrin (3 mM; MβCD), and paclitaxel (5 µg/ml; paclit). Addition of DMSO served as an additional control. (n = 2). Cytochalasin D disrupts actin filaments. Nocodazole, colchicine, and paclitaxel interfere with microtubuli assembly or disassembly. U0126 inhibits the MEK/ERK pathway. Dynasore blocks dynamin-dependent endocytosis. Methyl-β-cyclodextrin removes cholesterol from cultured cells and disrupts lipid rafts. (C) Statistical assessment of the experiments shown in (D) (n = 15; Mann-Whitney-U test, p**<0.01). (D) Microscopic imaging of migrating C2C12 cells after transfection with a control vector (Con), WT Braf and CA Braf (V600E) in the presence or absence of DMSO, UO126 and Dynasore. Cultures were analyzed 4 hr after scratching excluding effects of cell proliferation. https://doi.org/10.7554/eLife.18351.002 Since BRAF activates the MEK-ERK signaling cascade in muscle cells (Figure 1B), we wanted to know whether inhibition of MEK1/2 blocks the effects of BRAF on migration. Surprisingly, addition of the MEK1/2 inhibitor UO126 had only minor effects on migration, although the increase in ERK1/2 phosphorylation after transfection of WT and CA BRAF (V600E) was efficiently prevented by the inhibitor (Figure 1C,D) indicating a MEK/ERK independent mechanism of BRAF signaling. Since MET undergoes rapid endocytosis in a process called ‘endosomal signaling’ and traffics through peripheral endosomes to a perinuclear localization (Barrow-McGee and Kermorgant, 2014), we investigated whether inhibition of endosomal trafficking abrogates the effects of BRAF on migration of C2C12 muscle cells. Interestingly, addition of Dynasore, a pharmacological inhibitor of endosomal trafficking, significantly prevented BRAF dependent stimulation of migration when the constitutively active form of BRAF was used in the experiments (Figure 1C,D). BRAF is required for migration of limb muscle precursor cells and formation of forelimb muscles Although C2C12 cells represent a useful model to study mechanistic aspects of muscle cell migration, the inhibition of HGF-stimulated migration after knockdown of Braf does not prove that Braf is also instrumental to regulate limb muscle formation in the embryo in vivo. We therefore took advantage of a mouse strain (Brafnfl) in which three loxP-sites had been inserted to flank exon three as well as a neomycin selection cassette (Figure 2A) (Pfeiffer et al., 2013). Breeding with MeuCre mice (Leneuve et al., 2003) yielded Braffl mice, in which the neomycin cassette was removed but exon three is flanked by loxP-sites, and Brafdel mice lacking exon 3, which encodes parts of the Ras-binding domain (Figure 2A). Deletion of exon three created a functional null allele of Braf leading to embryonic lethality of Brafdel/del mice consistent with previous reports (Pfeiffer et al., 2013; Wojnowski et al., 1997). Braffl mice were crossed to the Pax3-Cre strain allowing specific inactivation of Braf in the dermomyotome and limb muscle precursor cells (Figure 2B). Immunofluorescence staining using an anti-BRAF antibody confirmed the absence of BRAF protein in the dermomyotome of Pax3-Cre//Brafdel/del mutant embryos at E10.5 while the dermomyotome of WT control embryos was strongly positive for BRAF (Figure 3—figure supplement 1). Importantly, germ line inactivation (Brafdel/del) as well as deletion of Braf in Pax3-expressing limb muscle precursor cells (Pax3-Cre//Brafdel/del) resulted in arrest of cell migration as indicated by the absence of PAX3+-cells in the developing forelimb buds at E10.5 (Figure 3A,B), which was further validated by immunofluorescence analysis of PAX3+ cells (Figure 3—figure supplement 1) and RT-PCR analysis of Met and Pax3 expression (Figure 2C). Closer inspection of the PAX3 immunofluorescence staining revealed that only very few Braf-deficient PAX3+-cells delaminated from the dermomyotome and initiated migration suggesting a requirement of Braf for epithelia-mesenchymal transition of dermomyotomal cells and/or migration (Figure 3—figure supplement 1). Staining for Lbx1 expression, which is also expressed in limb muscle precursor cells and needed for targeted migration, confirmed this conclusion (Figure 3C). We also found a virtually complete absence of Myod and Myf5 expressing myogenic cells in limb buds of E10.5 Pax3-Cre//Brafdel/del embryos (Figure 3C), although we noted a reduced expression at later stages by Western blot analysis (Figure 2D) indicating that a small population of Braf-deficient limb muscle progenitor cells was able to reach its target. Accordingly, we observed a major reduction of forelimb muscles at later stages of development (E14.5), which was more pronounced in the distal compared to the proximal parts of the limb, where some residual muscle formation was seen (Figure 3D,E). In contrast, we did not observe a significant increase in the number of apoptotic cells in Braf mutant compared to WT control embryos at E10.5 suggesting that the loss of Braf in dermomyotomal cells does not lead to programmed cell death (Figure 3—figure supplement 1). Furthermore, we investigated the presence of endothelial precursor cells in Pax3-Cre//Brafdel/del mutant embryos at E10.5, since transplantation of Kdr-mutant mouse presomitic mesoderm into chicken embryos had revealed a crucial role of endothelial cells for muscle progenitor cell migration (Yvernogeau et al., 2012). KDR primarily signals via the PKC-MAPK and the PI3K pathway (Vieira et al., 2010), although some evidence exists that KDR can also activate MAPK via CRAF and MEK in some cells types (Takahashi et al., 1999), which raised the possibility that inactivation of Braf might indirectly disrupt muscle progenitor cell migration by preventing migration of endothelial precursor cells downstream of KDR. However, similar to the presence of normal numbers of endothelial progenitor cells in Pax3 mutant embryos (Yvernogeau et al., 2012), we did not observe a reduction of CD31-positive endothelial cells in Braf mutant limb buds (Figure 3—figure supplement 1) indicating that KDR does not critically rely on BRAF for promoting endothelial precursor cell migration. Figure 2 Download asset Open asset Inactivation of Braf in limb muscle precursor cells. (A) Strategy for generation of Braf floxed mice (Brafnfl). Breeding with MeuCre mice yielded Braffl mice, in which the neomycin cassette was removed but exon three is flanked by loxP-sites. (B) Braffl mice were bred with Pax3-Cre mice to generate animals lacking Braf in Pax3 expressing cells. Pax3-Cre//Brafdel/del embryos die around E15.5. Mutant embryos were analyzed between E10.5 and E14.5. (C) RT-PCR analysis of Met and Pax3 expression in limb buds of Pax3-Cre//Brafdel/del embryos at E10.5 n = 2. (D) Western blot analysis of expression of different markers in limb buds of Pax3-Cre//Brafdel/del embryos at E10.5 and E14.5 n = 2. (E) Expression of WT and CA Braf (VE600E) in C2C12 cells increases expression of Met and Pax3. n = 3. (F) BRAF enhances PAX3-dependent transcriptional responses. The pGl.3 Pax3BS luc reporter construct containing two PAX3 binding sites in front of a minimal promoter was co-transfected with different combinations of Pax3, Braf, and CA Braf (V600E) expression vectors into HEK293T cells. The activity of Firefly Luciferase was normalized by a co-transfected renilla luciferase in all experiments. Data represent the mean ± SEM and analyzed using ANOVA with a Tukey-Kramer post-hoc comparison test. ***p<0.001. https://doi.org/10.7554/eLife.18351.004 Figure 3 with 1 supplement see all Download asset Open asset BRAF is required for limb muscle precursor cell migration during mouse embryogenesis. (A) Pax3 whole mount in situ hybridization of WT, germ line Brafdel/del and Pax3-Cre//Brafdel/del mutant embryos at E10.5. (B) Transverse sections of WT and Pax3-Cre//Brafdel/del mutant embryos at E10.5 after Pax3 whole mount in situ hybridization. Inactivation of Braf results in loss of PAX3+ cells in forelimbs. NT: neural tube; dm: dermomyotome; fl: forelimb. (C) Whole mount in situ hybridization of E10.5 WT and Pax3-Cre//Brafdel/del mutant embryos using Lbx1, Myf5, Myod, and Myogenin probes. (D) Immunofluorescence staining of forelimbs from WT and Pax3-Cre//Brafdel/del mutants for myosin heavy chain (MHC) at E14.5. (E) Hematoxylin and eosin staining of forelimbs from WT and Pax3-Cre//Brafdel/del mutants at E14.5. https://doi.org/10.7554/eLife.18351.005 To further explore effects of BRAF on critical components of the regulatory network driving migration of myogenic cells we transfected WT Braf and CA Braf (V600E) into C2C12 myoblasts. RT-PCR analysis revealed a strong up-regulation of Met and Pax3 expression indicating that BRAF induced transcription of both genes, most likely by activation of key transcription factors (Figure 2E). In addition, we analyzed whether BRAF might directly increase the transcriptional activity of PAX3. We therefore constructed a luciferase reporter construct containing two PAX3 consensus binding sites in front of a minimal promoter. Co-transfection of the Pax3 reporter plasmid together with Pax3 and Braf or CA Braf (V600E) into HEK293T cells revealed a strong increase of transcriptional activity, resulting in an up to 15-fold change when CA Braf (V600E) was used (Figure 2F). Taken together our data indicate that BRAF is essential for limb muscle formation presumably by mediating MET signaling via PAX3 to enable migration of limb muscle precursor cells. BRAF physically interacts with PAX3 in muscle precursor cells in vivo and in vitro Since our data indicated that the effects of BRAF on migration of myogenic cells are not mediated via the canonical MEK-ERK signaling pathway, we decided to search for additional targets. Importantly, mass spectrometry analysis of immunoprecipitated samples from WT limb buds of E10.5 mouse embryos using an antibody against endogenous BRAF identified -among several other proteins- the nuclear transcription factors PAX3 and PAX7 as potential interaction partners of BRAF (Figure 4A, Figure 4—figure supplement 1). Similar observations were made when BRAF was precipitated from C2C12 cells transfected with the CA Braf (V600E). Reciprocal mass spectrometry experiments, in which we incubated protein extracts from WT limb buds of E10.5 mouse embryos with GST-PAX3 protein followed by immunoprecipitation with an anti-PAX3 antibody and immunoprecipitation of PAX3 from extracts of C2C12 cells transfected with HA-tagged Pax3 yielded identical results and also uncovered interactions of PAX3 and BRAF with components of the endocytosis pathway such as ARF5, six and DNM2 along with several other proteins (Figure 4A, Figure 4—figure supplement 1). Coupled immunoprecipitation-Western blot analysis of samples from E10.5 limb buds using either BRAF antibodies for IP and PAX3 antibodies for Western blot or vice versa further confirmed the physical interaction between BRAF and 53 kDa PAX3 (Figure 4B). Next, we wanted to know whether activation of BRAF leads to more efficient binding to PAX3. Comparative GST pull-down assays of extracts from C2C12 cells transfected either with WT Braf or CA Braf (V600E) revealed a significantly stronger interaction of the constitutively active compared to the WT form of BRAF with GST-PAX3 (Figure 4C). Identical results were obtained when endogenous PAX3 was immunoprecipitated from C2C12 cells after transfection with WT Braf or CA Braf (V600E) (Figure 4E). Finally, we performed cross-linking experiments with NHS-diazirine cross-linker (SD) in C2C12 cells transfected with CA Braf (V600E) followed by immunoprecipitation with BRAF or PAX3 antibodies. Western blot analysis using corresponding antibodies detected the 95 kDa BRAF and the 53 kDa PAX3 proteins as separate bands (Figure 4D). Moreover, either antibody detected an additional single band at ca. 150 kDa, which corresponds to the cross-linked BRAF-PAX3 heteromer. Cleavage of the spacer with DTT severed the cross-linked BRAF-PAX3 complex, further verifying identity of the band (Figure 4D). Figure 4 with 1 supplement see all Download asset Open asset BRAF directly interacts with PAX3 in migrating muscle precursor cells. (A) PAX3, GST-PAX3 and PAX3 were immunoprecipitated from protein extracts of E10.5 wild type embryos or from C2C12 muscle cells after transfection with HA-Pax3 or CA Braf (V600E). Immunoprecipitations were analyzed by mass spectrometry after SDS-PAGE and in gel digestions. A selected list of proteins identified by Mascot search analysis is presented. (B) Analysis of the interaction of PAX3 and BRAF in E10.5 and E12.5 WT embryos by coupled immunoprecipitation/Western blot analysis. n = 3. (C) Western blot analysis of GST and GST-PAX3 immunoprecipitations after transfection of C2C12 cells with empty vector, Braf and CA Braf (V600E). n = 3. (D) Western blot analysis of PAX3 or BRAF immunoprecipitations from C2C12 transfected with CA Braf (V600E) after chemical cross-linking (SD) and cleavage of the cross-linker with DTT (SD+DTT). None = no cross-linker added. n = 3. (E) Western blot analysis of PAX3 immunoprecipitations from C2C12 transfected with WT Braf, CA Braf (V600E) and vector control (vector). n = 3. https://doi.org/10.7554/eLife.18351.007 A fraction of activated BRAF accumulates in the nucleus after endosomal transport Although our co-immunoprecipiation experiments established a physical interaction between PAX3 and BRAF, the assumed location of BRAF in the cytoplasm seems to prevent a meaningful physiological association with PAX3, which is located in the nucleus. Hence, we analyzed specifically whether a fraction of BRAF is transported into the nucleus. Subcellular fractionation of C2C12 cells disclosed the presence of a minor amount of endogenous BRAF in the nucleus although the major share of BRAF was present in the cytoplasm (Figure 5A). In contrast, PAX3 was exclusively found in the nuclear fraction, even after expression of large amounts of HA-tagged Pax3. The concentration of BRAF in the nuclear fraction increased when CA Braf (V600E) was transfected into cells irrespective of the presence or absence of HGF. Interestingly, the nuclear level of BRAF decreased when endosomal trafficking was blocked by administration of Dynasore (Figure 5A). We also conducted additional co-immunoprecipitation experiments using only the nuclear fraction of transfected C2C12 cells, which corroborated the interaction of nuclear BRAF with PAX3 in the absence of cytosolic BRAF (Figure 5B). The co-immunoprecipitation experiments also revealed enhanced interaction of CA BRAF (V600E) with PAX3, which might be due to increased transport of CA BRAF (V600E) into the nucleus. Finally, we performed immunofluorescence staining for PAX3 and BRAF using mouse embryonic tissues or C2C12 cells transfected with Braf, CA Braf (V600E) and Pax3. We observed a striking co-localization of PAX3 and BRAF in nuclei of limb muscle precursor cells and strong signals for P-BRAF in nuclei of transfected cells although localization of P-BRAF in the cytoplasm dominated (Figure 5C,D, Figure 5—figure supplement 1). Co-staining for P-BRAF and the endosomal marker EEA1 revealed a close association of endosomal vesicle and P-BRAF in the cytoplasm but not in the nucleus (Figure 5—figure supplement 1). Figure 5 with 1 supplement see all Download asset Open asset A fraction of BRAF co-localizes with PAX3 in nuclei of muscle cells. (A) Western blot analysis of cytoplasmic (C) and nuclear fractions (N) of C2C12 cells transfected with CA Braf (V600E), WT Braf, HA-Pax3 or HA-Pax3-GFP. n = 3. Successful fractionation was monitored by cytoplasmic GAPDH and the nuclear protein LAMIN A/C. Some cultures were treated with Dynasore (Dyna) for 30 min before fractionation as indicated. (B) Western blot analysis of immunoprecipitations of nuclear fractions isolated from migrating C2C12 cells after transfection with WT Braf, CA Braf (V600E), or HA-Pax3. n = 3. (C) High resolution confocal images of C2C12 cells transfected with WT, CA Braf (V600E) and HA-Pax3-GFP. (D) High resolution confocal image of a PAX3 and BRAF positive forelimb muscle precursor cell at E10.5 (left panel). A lower magnification is shown in the right panel. https://doi.org/10.7554/eLife.18351.009 Intrigued by the fact that inhibition of endosomal trafficking by Dynasore abrogated the effects of BRAF on migration of myogenic cells and prevented accumulation of BRAF in the nucleus we targeted the early endosome antigen 1 (EEA1), an essential component of the endosomal pathway in C2C12 cells using siRNAs. Knockdown of Eea1 resulted in decreased levels of nuclear BRAF while siErk1/2 showed little effects (Figure 6A). Endosomal trafficking of MET has been described to be required for full activation of signals such as GAB1, ERK1/2, STAT3 and RAC1 (Barrow-McGee and Kermorgant, 2014). To explore whether disruption of endosomal trafficking does not only inhibit transport of BRAF into the nucleus but also activation of ERK1/2 we knocked down Eea1, dynamin-2 (Dnm2), clathrin heavy chain (Cltc), caveolin-1 (Cav1) and ADP ribosylation factor 6 (Arf6). In neither case we observed significant effects on the phosphorylation level of ERK1/2 while knock down of Braf or Erk1/2 resulted in a strong reduction of ERK1/2 phosphorylation (Figure 6B). Furthermore, knockdown of Eea1 but not of Erk1/2 significantly inhibited BRAF-mediated stimulation of C2C12 cell migration (Figure 6C) supporting our conclusion that signaling events downstream of MET necessary for limb muscle precursor migration rely on the translocation of BRAF into the nucleus and not on the activation of the ERK1/2 signaling cascade. Figure 6 Download asset Open asset Nuclear translocation of BRAF and migration of muscle cells depend on intact endosomal trafficking. (A) Western blot analysis of isolated subcellular fractions of C2C12 cells after transfection of CA Braf V600E and knockdown of Eea1 or Erk1/2. n = 3. Knockdown of Eea1 prevented accumulation of BRAF in the nucleus (N). C: cytoplasm. siRNA knock-down efficiencies for Eea1 (60%) and Erk (95%) were determined by Western blot analysis. (B) Knock down of Braf (siBRAF) but not of Eea1 (siEea1), Dnm-2 (siDnm2), Cltc (siCltc), Cav1 (siCav1) and Arf6 (siArf6) did prevent phosphorylation of ERK1/2. Western blot analyses of siRNA transfected C2C12 cells are shown. n = 3. Actin served as loading control. siRNA knock-down efficiencies for Pax3 (85%), Braf (70%), Arf6 (80%), Cav1 (75%), Cltc (70%), Dnm2 (75%) and Eea1 (60%) were determined by Western blot analysis. (C) Immunofluorescence staining of migrating C2C12 cells after knockdown of Eea1 or Erk-1/2. Cultures were transfected with control vector, Braf or CA Braf (V600E) as indicated. Con indicates control cultures and siCon means control siRNA. siRNA knock-down efficiencies for Eea1 (60%) and Erk (95%) were determined by Western blot analysis. Cell numbers were determined by counting the number of DAPI-stained nuclei. A statistical analysis of siEea1 versus siCon in CA Braf (VE600E) transfected cultures is shown. n = 12; Mann-Whitney-U test, (p*<0.05). https://doi.org/10.7554/eLife.18351.011 BRAF regulates muscle precursor cell migration through PAX3 phosphorylation The interaction of the BRAF kinase with PAX3 and the inhibition of HGF-mediated migration of myogenic cells by knockdown of Pax3 suggested that BRAF might exert its effects downstream of MET by phosphorylation and subsequent activation of PAX3. To characterize potential phosphorylation sites within the PAX3 protein, we isolated PAX3 by immunoprecipitation from C2C12 cells, HEK293T cells and from mouse embryonic limb buds at E10.5. Subsequent fractionation of FASP-digested PAX3 peptides by cation-exchange liquid chromatography followed by mass spectrometry analysis using a quadrupole-based Q Exactive instrument identified multiple new phosphorylation sites at serine and threonine residues as well as five phosphorylation sites (at Ser180, Ser187, Ser201, Ser205 and Ser209), which had already been reported before (Figure 7A). The majority of the phosphorylation events corresponded well to sites predicted by NetPhos 2.0 (www.cbs.dtu.dk/services/NetPhos), PHOSIDA (www.phosida.de) and PhosphoVariant (http://phosphovariant.ngri.go.kr). To understand whether enhanced BRAF act

fter myocardial infarction, dedifferentiated cardiomyocytes secrete the protein Reg3β, thereby recruiting macrophages required for neutrophil clearance and myocardial healing.

ObjectivesWe used immuhistochemistry and Western blot to study fibrillar and non-fibrillar collagens, collagen metabolism, matricellular proteins and regulatory factors of the ECM remodeling in left ventricular (LV) septum biopsies from 3 groups of patients with aortic valve stenosis (AS): (AS-1,n = 9): ejection fraction (EF) > 50%; AS-2,(n = 12): EF 30%–50%; AS-3,(n = 9): EF < 30%). Samples from 8 hearts with normal LV function served as controls.ResultsIn comparison with controls, fibrillar collagens I and III were progressively upregulated from compensated (AS-1) toward decompensated hypertrophy (AS-3). The collagenIII/collagen I ratio decreased 2-fold in the AS-2 and AS-3 groups as compared with AS-1 and controls. Non-fibrillar collagen IV was upregulated only in AS-3 patients, whereas collagen VI progressively increased from AS-1 to AS-3 group. Collagen synthesis in AS-3 was shifted to collagen I, while the maturation/degradation level was shifted to collagen III. RECK was downregulated only in AS-3 patients. Matricellular proteins tenascin and osteopontin were increased in all AS patients. However, thrombospondin 1, 4 and CTGF were increased only in AS-3. Only AS-3 patients were characterized by increased levels of TGFβ1 and downregulation of TGFβ3, TGFβ-activated kinase1 and Smad7. In contrast, Smad3 gradually increased from AS-1 toward AS-3. Similar trend of changes was observed for TNFα-R1 and TNFα-R2, whereas TNFα was diminished only in AS-2 and AS-3.ConclusionsDistinct changes in fibrillar collagen turnover, non-fibrillar collagens, matricellular proteins and the key regulatory profibrotic and anti-fibrotic factors of the myocardial ECM remodeling are involved in the transition from compensated to decompensated LV hypertrophy and HF in human patients with AS.

Heart failure (HF) is a common and potentially deadly condition, which frequently develops as a consequence of various diseases of the heart. The incidence of heart failure continuously increases in aging societies illustrating the need for new therapeutic approaches. We recently discovered that continuous activation of oncostatin M (OSM), a cytokine of the interleukin-6 family that induces dedifferentiation of cardiomyocytes, promotes progression of heart failure in dilative cardiomyopathy. To evaluate whether inhibition of OSM signaling represents a meaningful therapeutic approach to prevent heart failure we attenuated OSM-receptor (Oβ) signaling in a mouse model of inflammatory dilative cardiomyopathy. We found that administration of an antibody directed against the extracellular domain of Oβ or genetic inactivation of a single allele of the Oβ gene reduced cardiomyocyte remodeling and dedifferentiation resulting in improved cardiac performance and increased survival. We conclude that pharmacological attenuation of long-lasting Oβ signaling is a promising strategy to treat different types and stages of HF that go along with infiltration by OSM-releasing inflammatory cells.

Cardiomyocytes continuously generate the contractile force to circulate blood through the body. Imbalances in contractile performance or energy supply cause adaptive responses of the heart resulting in adverse rearrangement of regular structures, which in turn might lead to heart failure. At the cellular level, cardiomyocyte remodeling includes (1) restructuring of the contractile apparatus; (2) rearrangement of the cytoskeleton; and (3) changes in energy metabolism. Dedifferentiation represents a key feature of cardiomyocyte remodeling. It is characterized by reciprocal changes in the expression pattern of "mature" and "immature" cardiomyocyte-specific genes. Dedifferentiation may enable cardiomyocytes to cope with hypoxic stress by disassembly of the energy demanding contractile machinery and by reduction of the cellular energy demand. Dedifferentiation during myocardial repair might provide cardiomyocytes with additional plasticity, enabling survival under hypoxic conditions and increasing the propensity to enter the cell cycle. Although dedifferentiation of cardiomyocytes has been described during tissue regeneration in zebrafish and newts, little is known about corresponding mechanisms and regulatory circuits in mammals. The recent finding that the cytokine oncostatin M (OSM) is pivotal for cardiomyocyte dedifferentiation and exerts strong protective effects during myocardial infarction highlights the role of cytokines as potent stimulators of cardiac remodeling. Here, we summarize the current knowledge about transient dedifferentiation of cardiomyocytes in the context of myocardial remodeling, and propose a model for the role of OSM in this process.

Cardiomyocyte remodeling, which includes partial dedifferentiation of cardiomyocytes, is a process that occurs during both acute and chronic disease processes. Here, we demonstrate that oncostatin M (OSM) is a major mediator of cardiomyocyte dedifferentiation and remodeling during acute myocardial infarction (MI) and in chronic dilated cardiomyopathy (DCM). Patients suffering from DCM show a strong and lasting increase of OSM expression and signaling. OSM treatment induces dedifferentiation of cardiomyocytes and upregulation of stem cell markers and improves cardiac function after MI. Conversely, inhibition of OSM signaling suppresses cardiomyocyte remodeling after MI and in a mouse model of DCM, resulting in deterioration of heart function after MI but improvement of cardiac performance in DCM. We postulate that dedifferentiation of cardiomyocytes initially protects stressed hearts but fails to support cardiac structure and function upon continued activation. Manipulation of OSM signaling provides a means to control the differentiation state of cardiomyocytes and cellular plasticity.

OBJECTIVES:Microdialysis allows the in vivo biochemical analysis of interstitial fluids. Our aim was to reveal in vivo reliable data of the myocardium during open beating heart surgery.DESIGN:In ten patients undergoing routine beating coronary artery bypass grafting a microdialysis catheter was inserted into the left ventricle. Measurements were performed up to 45 min after anastomosis. Data were retrospectively compared with standard on-pump procedures.RESULTS:The myocardial lactate remained stable during anastomosis, followed by a significant decrease of lactate after revascularisation. Myocardial glucose levels showed a slight decrease, followed by a significant increase after revascularisation. Myocardial purines showed a slight increase during anastomosis, followed by a sharp decrease during reperfusion period.CONCLUSIONS:In contrast to on-pump procedures myocardial lactate and purines showed less increasing trend during the ischemic period, while myocardial glucose remained stable as a sign of preserved tissue blood flow. Myocardial microdialysis showed different values compared to the elective on-pump CABG and previous animal studies. This technique allows bedside monitoring of biochemical changes, suggesting its possible role as a clinical monitoring tool.

A commercially available catheter was used in a clinical trial of myocardial microdialysis. The myocardial lactate and pyruvate concentrations and the lactate-to-pyruvate ratio, as a sensitive marker of the myocardial redox status, showed profound changes in both off-pump and on-pump patients. These microdialysis data have to be interpreted in the context of a variety of clinical implications, and a great deal of effort needs to be invested to determine the precise role of microdialysis in CABG procedures and find whether it is possible to improve our clinical management of open heart surgery patients using this new technology.