P161 – 2452: HIF1 alpha abnormal distribution in muscle fibers in the cases of nemaline myopathy

Abstract

Objective The comparative study of immunohistochemical distribution of hypoxia marker HIF1 alpha and mitochondrial markers in striated muscle fibers of patients suffering from nemaline myopathy (NM) was performed for evaluation of tissue hypoxia's role in the pathogenesis of NM, characterized by rod-shaped structures, which originated from the Z-disks of the sarcomeres. Methods Fluorescence immunohistochemistry was performed. Primary mouse mitochondria monoclonal antibody, biotin conjugate (clone MTCO2) and Alexa Fluor 488 goat anti-mouse IgG as secondary antibody were used for mitochondria visualization. Visual image of HIF1 alpha was achieved by using primary rabbit polyclonal antibodies to HIF1 alpha and Alexa Fluor 555 goat anti-rabbit IgG as secondary antibody. Paraffin embedded muscle tissue section slides obtained from 3 NM patients were stained. Evaluation of the products is carried out by The EVOS® FL Imaging System. Results Mitochondria and abnormal mitochondrial accumulations were visualized in subsarcolemmal zones of some muscle fibers. In the same muscle fibers we observed increased expression of HIF1 alpha; its localization matched with location of detected mitochondrial clusters, resulted in fluorescent lights overlapping and color change of subsarcolemmal zones luminescence. Furthermore, the most intensive fluorescence and clearly delineated HIF1 alpha clusters were observed in the areas of rods accumulation, pointing pronounced tissue hypoxia in these regions. Conclusion While the diffuse subsarcolemmal zones of tissue hypoxia and increased number of mitochondrial clusters are often found in other myopathies, location of HIF1 alpha clusters in the rods areas is very unusual. Due to their brightness, HIF1 alpha clusters can be used in the quantification of muscular disorders severity at low magnification of fluorescence microscopy. No evidence whether these changes are secondary to rods accumulation or vice versa was found in the literature. In this way immunomorphological characterisation of energy muscle tissue metabolism of patients suffering from NM is of the current interest.