Statins, the widely prescribed cholesterol-lowering drugs for the treatment of cardiovascular disease, cause adverse skeletal muscle side effects ranging from fatigue to fatal rhabdomyolysis. The purpose of this study was to determine the effects of simvastatin on mitochondrial respiration, oxidative stress, and cell death in differentiated primary human skeletal muscle cells (i.e. myotubes). Simvastatin induced a dose dependent decrease in viability of proliferating and differentiating primary human muscle precursor cells, and a similar dose-dependent effect was noted in differentiated myoblasts and myotubes. Additionally, there were decreases in myotube number and size following 48 h of simvastatin treatment (5 µM). In permeabilized myotubes, maximal ADP-stimulated oxygen consumption, supported by palmitoyl-carnitine + malate (PCM, complex I and II substrates) and glutamate + malate (GM, complex I substrates), was 32–37% lower (P<0.05) in simvastatin treated (5 µM) vs. control myotubes, providing evidence of impaired respiration at complex I. Mitochondrial superoxide and hydrogen peroxide generation were significantly greater in the simvastatin treated human skeletal myotube cultures compared to control. In addition, simvastatin markedly increased protein levels of Bax (pro-apoptotic, +53%) and Bcl-2 (anti-apoptotic, +100%, P<0.05), mitochondrial PTP opening (+44%, P<0.05), and TUNEL-positive nuclei in human skeletal myotubes, demonstrating up-regulation of mitochondrial-mediated myonuclear apoptotic mechanisms. These data demonstrate that simvastatin induces myotube atrophy and cell loss associated with impaired ADP-stimulated maximal mitochondrial respiratory capacity, mitochondrial oxidative stress, and apoptosis in primary human skeletal myotubes, suggesting mitochondrial dysfunction may underlie human statin-induced myopathy.

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The most common enzyme labels in enzyme-linked immunosorbent assays are alkaline phosphatase and horseradish peroxidase, which, however, have some limitations for use in electrochemical immunosensors. This Article reports that the small and thermostable DT-diaphorase (DT-D) and electrochemically inactive 4-nitroso-1-naphthol (4-NO-1-N) can be used as a bifunctional enzyme label and a rapidly reacting substrate, respectively, for electrochemical immunosensors. This enzyme-substrate combination allows high signal amplification via rapid enzymatic amplification and electrochemical redox cycling. DT-D can convert an electrochemically inactive nitroso or nitro compound into an electrochemically active amine compound, which can then be involved in electrochemical-chemical (EC) and electrochemical-enzymatic (EN) redox cycling. Six nitroso and nitro compounds are tested in terms of signal-to-background ratio. Among them, 4-NO-1-N exhibits the highest signal-to-background ratio. The electrochemical immunosensor using DT-D and 4-NO-1-N detects parathyroid hormone (PTH) in phosphate-buffered saline containing bovine serum albumin over a wide range of concentrations with a low detection limit of 2 pg/mL. When the PTH concentration in clinical serum samples is measured using the developed immunosensor, the calculated concentrations are in good agreement with the concentrations obtained using a commercial instrument. Thus, the use of DT-D as an enzyme label is highly promising for sensitive electrochemical detection and point-of-care testing.

T lymphocytes from patients with sarcoidosis respond weakly when stimulated with mitogen or antigen. However, the mechanisms responsible for this anergy are not fully understood. Here, we investigated the protein levels of nuclear transcription factor NF-B (p50, p65, and p105), IB␣ (inhibitor of NF-B), T-cell receptor (TCR) CD3-chain, tyrosine kinase p56 LCK , and nuclear factor of activated T cells c2 (NF-ATc2) in peripheral blood CD4 ؉ T cells from patients with sarcoidosis. Baseline expression of p65 in these lymphocytes was reduced in 50% of patients. The reduced levels of p65 in sarcoid CD4؉ T cells concurred with decreased levels of p50, p105, CD3, p56 LCK , IB␣, and NF-ATc2. Polyclonal stimulation of NF-B-deficient sarcoid T cells resulted in reduced expression of CD69 and CD154, decreased proliferation, and cytokine (i.e., interleukin 2 [IL-2] and gamma interferon [IFN-␥]) production. The clinical significance of these findings is suggested by the association between low p65 levels and the development of more severe and active sarcoidosis. Although correlative, our results support a model in which multiple intrinsic signaling defects contribute to peripheral T-cell anergy and the persistence of chronic inflammation in sarcoidosis.Sarcoidosis is a multisystem disease of unknown etiology characterized by noncaseating granuloma formation (15,32). It is associated with anergic responses to skin tests and depressed peripheral T-lymphocyte responses in vitro (16,34). Several studies have examined the mechanisms of peripheral anergy in sarcoidosis. Early reports concluded that the T-cell anergy in sarcoidosis patients was partly due to a decreased production of interleukin 1 (IL-1) by monocytes (28). It was also shown that monocytes contributed to the suppressed lymphocyte responses by releasing increased amounts of prostaglandins (24). More recently, it was demonstrated that expansion of regulatory T cells (Treg cells) and diminished dendritic cell function could be responsible for the peripheral T-cell anergy observed with sarcoidosis. The proposed mechanisms implicated in this suppression included inhibition of IL-2 production and T-cell proliferation by Treg cells and a decreased ability of myeloid dendritic cells to stimulate T lymphocytes (46, 50). Sarcoid patients, however, do not appear to develop significant clinical evidence of immunosuppression, as they are capable of mounting effective immune responses to bacterial, fungal, and viral infections (70). Compartmentalization of these effective responses to the affected organs (i.e., lungs) could also explain the peripheral anergy associated with this disease (30, 31). Although the T-cell anergy associated with sarcoidosis was recognized long ago, the underlying mechanism and implications of this phenomenon for the pathogenesis of sarcoidosis remain unclear.A key event in the induction of CD4 ϩ T-cell responses is the stimulation of the T-cell receptor (TCR)/CD3 complex on the membranes of T cells by major histocompatibility complex class II (MHC-II) mol...