Mitochondrial Calcium Retention Capacity

CRC assays were performed on freshly isolated mitochondria from adult non-tg mouse brain and heart (n = 6) after GNX-4728 was administered intravenously by tail vein injection (15 mg/kg in 20% DMSO and 40% PEG400) followed by a survival of 5 min. Control mice (n = 6) were injected with vehicle. Brain and heart mitochondria were isolated using a similar procedure as described (Wong et al., 2013 (link)). Mitochondrial CRC was assessed fluorimetrically in the presence of the fluorescent Ca²⁺ indicator Calcium Green 5N (Invitrogen Molecular Probes) using a temperature controlled Perkin-Elmer LS 55 spectrofluorimeter as described (Fancelli et al., 2014 (link)). Briefly, purified organ mitochondria were pulse-loaded with 10 mM calcium and then challenged with increasing concentrations of calcium until mitochondrial permeability transition was triggered as evidenced by complete release of mitochondrially-stored calcium due to mPTP opening.

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Martin L.J., Fancelli D., Wong M., Niedzwiecki M., Ballarini M., Plyte S, & Chang Q. (2014). GNX-4728, a novel small molecule drug inhibitor of mitochondrial permeability transition, is therapeutic in a mouse model of amyotrophic lateral sclerosis. Frontiers in Cellular Neuroscience, 8, 433.

Publication 2014

Calcium Green 5N LS 55 spectrofluorimeter

Adult Assays Brain Calcium Calcium green Dmso Heart Heart mitochondria Mice Mitochondria Mitochondrial Mitochondrial permeability transition Molecular probes Mptp Peg400 Pulse Tail Vein

Corresponding Organization : Johns Hopkins University

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Variable analysis

independent variables

Administration of GNX-4728 (15 mg/kg in 20% DMSO and 40% PEG400) by intravenous tail vein injection

dependent variables

Mitochondrial calcium retention capacity (CRC) assessed fluorimetrically in the presence of Calcium Green 5N

control variables

Freshly isolated mitochondria from adult non-tg mouse brain and heart
Isolation procedure for brain and heart mitochondria
Temperature-controlled spectrofluorimeter
Pulse-loading of mitochondria with 10 mM calcium

controls

Positive control: Mitochondria from GNX-4728-treated mice (n = 6)
Negative control: Mitochondria from vehicle-injected control mice (n = 6)

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