Iron accumulation causes impaired myogenesis correlated with MAPK signaling pathway inhibition by oxidative stress

Skeletal muscle atrophy is caused by disruption in the homeostatic balance of muscle degeneration and regeneration under various pathophysiological conditions. We have previously reported that iron accumulation induces skeletal muscle atrophy via a ubiquitin ligase-dependent pathway. However, the potential effect of iron accumulation on muscle regeneration remains unclear. To examine the effect of iron accumulation on myogenesis, we used a mouse model with cardiotoxin (CTX)-induced muscle regeneration in vivo and C2C12 mice myoblast cells in vitro. In mice with iron overload, the skeletal muscles exhibited increased oxidative stress and decreased expression of satellite cell markers. Following CTX-induced muscle injury, these mice also displayed delayed muscle regeneration with a decrease in the size of regenerating myofibers, reduced expression of myoblast differentiation markers, and decreased phosphorylation of mitogen-activated protein kinase signaling pathways. In vitro, iron overload also suppressed the differentiation of C2C12 myoblast cells, but the suppression could be reversed by superoxide scavenging using tempol. Excess iron inhibits myogenesis via oxidative stress, leading to an imbalance in skeletal muscle homeostasis

Induction of Cytoplasmic Rods and Rings Structures by Inhibition of the CTP and GTP Synthetic Pathway in Mammalian Cells

Background: Cytoplasmic filamentous rods and rings (RR) structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined. Methodology/Principal Findings: Distinct cytoplasmic rods (,3–10 mm in length) and rings (,2–5 mm in diameter) in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1) and inosine monophosphate dehydrogenase 2 (IMPDH2) were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in.95 % of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (.95%); upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin

Henoch-Schönlein purpura (IgA vasculitis) developing after postoperative wound infection by methicillin-resistant Staphylococcus aureus

Henoch-Schönlein purpura (HSP) is an acute small-vessel leukocytoclastic vasculitis, affecting the skin, joints, gastrointestinal tract and kidneys. Its prognosis depends on the severity of nephritis. A wide variety of pathogens, drugs, and other environmental exposures have been associated with HSP. Although group A β-haemolytic streptococcus has been the most studied, the majority of cases showed no direct link to streptococcal infection. Here we report a case of methicillin-resistant Staphylococcus aureus (MRSA) infection-associated HSP. A 68-year-old woman underwent a coronary artery bypass surgery. After the surgery, a postoperative chest wound was infected by MRSA and sternal osteomyelitis developed. Palpable purpura then appeared on the extremities, followed by hematuria, proteinuria and increased serum creatine. Treatments with antibiotics and debridement of the infected wound and sequestrum resulted in rapid improvement of skin symptoms. Renal function partially recovered, however mild hematuria and proteinuria remained. Published work review and the present case suggest that Staphylococcal infection-associated HSP frequently involves kidney disease and its prognosis is likely to be poor compared to a common type of HSP. Further studies are needed to establish an appropriate treatment strategy for Staphylococcal infection-associated HSP

The distribution of cytoplasmic rods and rings was independent of the Golgi complex and centrosomes, and these structures were not enriched in tubulin or vimentin.

<p>(A) Merged image of HEp-2 co-stained with human anti-RR prototype serum 604/Alexa 488 goat anti-human Ig (green) and rabbit anti-giantin (Golgi marker)/Alexa 568 goat anti-rabbit Ig (red). Rods are often presented adjacent (short arrows) or perpendicular (long arrows) to the nucleus while rings (arrowheads) are found either 1 or 2 to a cell. M, mitotic cell. HEp-2 cells were also co-stained with serum 604/Alexa 488 goat anti-human Ig (green, B,E,H) and different cytoplasmic markers using mouse anti-tubulin (C), anti-vimentin (F), anti-pericentrin (I), followed by Alexa 568 goat anti-mouse Ig (red). Nuclei were counterstained with DAPI (blue). Bar, 10 µm.</p

The expression of rods versus rings was not correlated with the cell cycle.

<p>HEp-2 cells were co-stained with rabbit anti-CENP-F/Alexa 568 goat anti-rabbit IgG (A) and human anti-RR serum 604/Alexa 488 goat anti-human IgG (B). G1 cells had little or no CENP-F staining, whereas late S/G2 and mitotic (M) cells showed strong staining for CENP-F. Nuclei counterstained with DAPI (blue). Bar, 10 µm.</p

Inhibition of CTPS1-induced formation of RR in mouse primary cardiomyocytes, fibroblasts, and endothelial cells.

<p>Mouse primary cardiomyocytes prepared together with fibroblasts and endothelial cells, were treated with 2 mM DON and cultured for 24 h. Cells were co-stained with human anti-RR serum IT2006 (green) and mouse anti-actinin monoclonal antibody (red). Nuclei counterstained with DAPI (blue). Actinin-positive cardiomyocytes (A, B, red) as well as actinin-negative fibroblast or endothelial cells (A, C) all show distinct rods. The percentage of cells with RR displayed is shown in the lower right corner with the total number of cells counted indicated in parentheses (A, all cells; B, actinin-positive cardiomyocytes only; C; actinin-negative fibroblast and endothelial cells). Bar, 10 µm.</p

Concentration-dependent induction of RR in HEp-2 cells using different CTPS1 and IMPDH2 inhibitors for 24 h.

1<p>Ribavirin induced RR in nearly all cells regardless of the tested concentration (0.2 µM–2 mM). There is no statistical significance (ns) between concentrations after Bonferroni's adjustment except with untreated control (0 µM; p>0.005);</p>2<p>DON induced RR in most cells at concentrations higher than 0.5 mM (p<0.005, 0.5 mM or higher vs any lower concentration);</p>3<p>Acivicin started to induce RR at 15.6 µM and concentrations higher than 31.3 µM, nearly all cells had RR (p<0.005 vs lower concentration) and no difference between concentrations was observed;</p>4<p>ns between Ribavirin, DON, or Acivicin;</p>5<p>Ribavirin vs DON, p<0.0001, Ribavirin vs Acivicin, ns, DON vs Acivicin, p<0.0001;</p>6<p>Ribavirin vs DON, p<0.0001, Ribavirin vs Acivicin, p<0.0001, DON vs Acivicin, p = 0.024;</p>7<p>Ribavirin vs DON, p<0.0001, Ribavirin vs Acivicin, p<0.0001, DON vs Acivicin, ns.</p

Inhibition of CTPS1-induced formation of RR in several human cancer cell lines.

<p>Induction of RR observed in human cancer cell lines HeLa (A), CAL 27 (B), THP-1 (C), and HCT116 (D) when treated with 2 mM DON in culture for 24 h, fixed, and co-stained with human anti-RR serum It2006 (green) and rabbit anti-giantin (red). Untreated controls showed few or no RR. The percentage of cells with RR displayed for each condition is shown in the upper right corner with the total number of cells counted indicated in parentheses. Bar, 5 µm.</p