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Real-time analysis of the induction of apoptosis in duchene muscular dystrophy


Duchenne muscular dystrophy is characterized by the absence of dystrophin resulting in an altered membrane permeability of muscle fibers. This entails an impaired homeostasis of the intracellular calcium level. In mayo tubes of mdx mice, a model system for DMD, this impairment is amplified in mitochondria. Such an elevation of free calcium in mitochondria is known to serve as a trigger for the onset of apoptosis, which is characterized by the release of a number of factors like cytochrome c and several procaspases from the mitochondria into the cytoplasm. Amongst these is caspase 3 which is then activated and acts as a key player in several apoptotic processes. To directly address whether the rise in mitochondrial calcium leads to the induction of apoptosis, I here propose a set of biochemical and morphological research. First, I want to establish a protocol for the preparation of mature mdx myotubes in which apoptosis is reproducibly inducible. Apoptosis I want to show by TUNEL-staining and screening for DNA clustering. Second, by the co-expression of fusion proteins based on the green fluorescent protein and fluorescence resonance energy transfer technology, I want to simultaneously observe the mitochondrial calcium level and localization of caspase 3 in mdx myotubes in viva after induction of apoptosis This should reveal that caspase 3 exits mitochondria upon rise in mitochondrial calcium level. Third, I want to monitor online the kinetics and localization of activation and activity of caspase 3 in these myotubes. This study would show for the first time a direct connection between calcium rise and apoptosis induction in rndx myotubes and would allow to measure the kinetics and sub cellular localization of these processes.


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