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In situ detection and measurement of intracellular reactive oxygen species in single isolated mature skeletal muscle fibers by real time fluorescence microscopy.

ABSTRACT: Reactive oxygen species (ROS) produced by skeletal muscle stimulate adaptive responses to activity and mediate some degenerative processes. ROS activity is usually studied by measuring indirect end-points of their reactions with various biomolecules. In order to develop a method to measure the intracellular ROS generation in real-time in mature skeletal muscle fibers, these were isolated from the flexor digitorum brevis (FDB) muscle of mice and cultured on collagen-coated plates. Fibers were loaded with 5- (and 6-) chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-DCFH DA) and measurements of 5- (and 6-) chloromethyl-2',7'-dichlorofluorescin (CM-DCF) fluorescence from individual fibers obtained by microscopy over 45 min. The sensitivity of this approach was demonstrated by addition of 1 microM H(2)O(2) to the extracellular medium. Contractions of isolated fibers induced by field electrical stimulation caused a significant increase in CM-DCF fluorescence that was abolished by pre-treatment of fibers with glutathione ethyl ester. Thus, CM-DCF fluorescence microscopy can detect physiologically relevant changes in intracellular ROS activity in single isolated mature skeletal muscle fibers in real-time, and contractions generated a net increase that was abolished when the intracellular glutathione content was enhanced. This technique has advantages over previous approaches because of the maturity of the fibers and the analysis of single cells, which prevent contributions from nonmuscle cells.

SUBMITTER: Palomero J

PROVIDER: S-EPMC2536563 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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In situ detection and measurement of intracellular reactive oxygen species in single isolated mature skeletal muscle fibers by real time fluorescence microscopy.

Palomero Jesus J Pye Deborah D Kabayo Tabitha T Spiller David G DG Jackson Malcolm J MJ

Antioxidants & redox signaling 20080801 8

Reactive oxygen species (ROS) produced by skeletal muscle stimulate adaptive responses to activity and mediate some degenerative processes. ROS activity is usually studied by measuring indirect end-points of their reactions with various biomolecules. In order to develop a method to measure the intracellular ROS generation in real-time in mature skeletal muscle fibers, these were isolated from the flexor digitorum brevis (FDB) muscle of mice and cultured on collagen-coated plates. Fibers were loa ...[more]

PMID: 18407749

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Project description:BackgroundFrustrated phagocytosis occurs when an asbestos fiber > 10 μm in length is engulfed imperfectly by a macrophage, and it is believed to be associated with chromosomal instability. Few studies have focused on dynamic cellular imaging to assess the toxicity of hazardous inorganic materials such as asbestos. One reason for this is the relative lack of fluorescent probes available to facilitate experimental visualization of inorganic materials. We recently developed asbestos-specific fluorescent probes based on asbestos-binding proteins, and achieved efficient fluorescent labeling of asbestos.ResultsLive-cell imaging with fluorescent asbestos probes was successfully utilized to dynamically analyze asbestos phagocytosis. The fluorescently labeled asbestos fibers were phagocytosed by RAW 264.7 macrophages. Internalized fibers of < 5 μm in length were visualized clearly via overlaid phase contrast and fluorescence microscopy images, but they were not clearly depicted using phase contrast images alone. Approximately 60% of the cells had phagocytosed asbestos fibers after 2 h, but over 96% of cells remained alive even 24 h after the addition of asbestos fibers. Immediate cell death was only observed when an asbestos fiber was physically pulled from a cell by an external force. Notably, at 24 h after the addition of asbestos fibers an approximately 4-fold increase in the number of binucleated cells was observed. Monitoring of individual cell divisions of cells that had phagocytosed asbestos suggested that binucleated cells were formed via the inhibition of cell separation, by asbestos fibers of > 10 μm in length that were localized in the proximity of the intercellular bridge.ConclusionsFluorescently labeled asbestos facilitated visualization of the dynamic biological processes that occur during and after the internalization of asbestos fibers, and indicated that (i) frustrated phagocytosis itself does not lead to immediate cell death unless the asbestos fiber is physically pulled from the cell by an external force, and (ii) macrophages that have phagocytosed asbestos can divide but sometimes the resulting daughter cells fuse, leading to the formation of a binucleated cell. This fusion only seemed to occur when a comparatively long asbestos fiber (> 10 μm) was shared by two daughter cells.

Project description:KRIT1 is a gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhage. Comprehensive analysis of the KRIT1 gene in CCM patients has suggested that KRIT1 functions need to be severely impaired for pathogenesis. However, the molecular and cellular functions of KRIT1 as well as CCM pathogenesis mechanisms are still research challenges. We found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS) homeostasis to prevent oxidative cellular damage. In particular, we demonstrate that KRIT1 loss/down-regulation is associated with a significant increase in intracellular ROS levels. Conversely, ROS levels in KRIT1(-/-) cells are significantly and dose-dependently reduced after restoration of KRIT1 expression. Moreover, we show that the modulation of intracellular ROS levels by KRIT1 loss/restoration is strictly correlated with the modulation of the expression of the antioxidant protein SOD2 as well as of the transcriptional factor FoxO1, a master regulator of cell responses to oxidative stress and a modulator of SOD2 levels. Furthermore, we show that the KRIT1-dependent maintenance of low ROS levels facilitates the downregulation of cyclin D1 expression required for cell transition from proliferative growth to quiescence. Finally, we demonstrate that the enhanced ROS levels in KRIT1(-/-) cells are associated with an increased cell susceptibility to oxidative DNA damage and a marked induction of the DNA damage sensor and repair gene Gadd45alpha, as well as with a decline of mitochondrial energy metabolism. Taken together, our results point to a new model where KRIT1 limits the accumulation of intracellular oxidants and prevents oxidative stress-mediated cellular dysfunction and DNA damage by enhancing the cell capacity to scavenge intracellular ROS through an antioxidant pathway involving FoxO1 and SOD2, thus providing novel and useful insights into the understanding of KRIT1 molecular and cellular functions.

Dataset Information

In situ detection and measurement of intracellular reactive oxygen species in single isolated mature skeletal muscle fibers by real time fluorescence microscopy.

Publications

In situ detection and measurement of intracellular reactive oxygen species in single isolated mature skeletal muscle fibers by real time fluorescence microscopy.

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