Submitted to: Journal of Molecular Biology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 7/8/2010
Publication Date: 2/1/2011
Citation: Ceglia, L., Morais, M.D., Park, L., Morris, E., Harris, S.S., Bischoff-Ferrari, H., Fielding, R., Dawson-Hughes, B. 2011. Hip fracture risk in older US adults by treatment eligibility status based on new National Osteoporosis Foundation Guidance. Journal of Molecular Biology. 22(2):541-549. Interpretive Summary: Recent work indicates that vitamin D supplementation reduces the risk of falls and improves measures of muscle performance in older adults with low vitamin D status. Muscle biopsies in adults with profound vitamin D deficiency reveal wasting of selected Type 2 muscle fibers, which are the first fibers to be recruited when preventing a fall. While the actions of vitamin D on skeletal muscle are not well-understood, current research suggests that the effects of vitamin D may, at least in part, be mediated through the vitamin D receptor (VDR). It is important to identify the mechanisms by which vitamin D affects muscle in order to devise the most effective intervention strategy. In this research, we developed a multi-staining immunofluorescent method to detect vitamin D receptor expression and to define its location in specific muscle fibers. The measurements were made on serial sections cut from frozen samples obtained by needle biopsy of a leg muscle in frail older volunteers with low-normal vitamin D status. In this tissue we quantified intra-nuclear vitamin D receptor staining patterns and number of individual muscle fiber subtypes within a muscle section. This multi-staining immunofluorescent technique allows for measurement of many parameters in a single section. This method can thus be a useful approach to study potential relationships between muscle fiber subtypes and vitamin D receptor expression in humans with use of the small amounts of muscle tissue that can be obtained by needle biopsy.
Technical Abstract: Vitamin D receptors have been shown to be present in human skeletal muscle using different techniques. We developed a multi-staining immunofluorescent method to detect vitamin D receptor expression and colocalize it with myosin heavy chain isoform expression in skeletal muscle biopsies in older female subjects. Serial sections were cut from frozen samples obtained by needle biopsy of the Vastus Lateralis. Samples were probed with a primary vitamin D receptor monoclonal antibody and then re-probed with a type IIa myosin heavy chain isoformspecific antibody. Independent unfixed sections followed a similar protocol and were probed with type I and type IIx myosin heavy chain isoform-specific antibodies. Immunohistochemistry and fluorescent microscopy colocalized vitamin D receptor loci and myosin heavy chain isoforms in whole skeletal muscle sections. We quantified intranuclear vitamin D receptor staining patterns and number of individual muscle fiber subtypes within a muscle section. Immunohistochemical staining of the vitamin D receptor was confirmed by Western blot using the same monoclonal antibody. This multi-staining immunofluorescent technique allows for measurement of intranuclear vitamin D receptor expression in the context of the specific muscle fiber type profile in a single section. This method can thus be a useful approach to study potential relationships between muscle fiber subtypes and vitamin D receptor expression.