Florence van Tienen

In 25% of patients with mitochondrial myopathies, pathogenic mitochondrial DNA (mtDNA) mutation are the cause. For heteroplasmic mtDNA mutations, symptoms manifest when the mutation load exceeds a tissue-specific threshold. Therefore, lowering the mutation load is expected to ameliorate disease manifestations. This can be achieved by fusing wild-type mesoangioblasts with mtDNA mutant myotubes. We have tested this in vitro for female carriers of the m.3271T>C or m.3291T>C mutation (mutation load >90%) using wild-type male mesoangioblasts. Individual fused myotubes were collected by a newly-developed laser capture microdissection (LCM) protocol, visualized by immunostaining using an anti-myosin antibody. Fusion rates were determined based on male-female nuclei ratios by fluorescently labelling the Y-chromosome. Using combined ‘wet’ and ‘air dried’ LCM imaging improved fluorescence imaging quality and cell yield. Wild-type mesoangioblasts fused in different ratios with myotubes containing either the m.3271T>C or the m.3291T>C mutation. This resulted in the reduction of the mtDNA mutation load proportional to the number of fused wild-type mesoangioblasts for both mtDNA mutations. The proportional reduction in mtDNA mutation load in vitro after fusion is promising in the context of muscle stem cell therapy for mtDNA mutation carriers in vivo, in which we propose the same strategy using autologous wild-type mesoangioblasts.