Challenges in the Separation of Purified Cardiac Mitochondrial Membranes

Abstract

BackgroundMitochondria are the bioenergetic engines of the cell, thus maintaining mitochondrial health is essential for meeting energy demands. Mitochondrial function is achieved upon the balance of structural processes that involve mitochondrial biogenesis, fusion and fission. Structural and functional integrity of the mitochondrial membranes, the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM) is important for the maintenance of the mitochondrial quality control and mitochondrial metabolism. Therefore, elucidation of the individual contribution of each membrane to mitochondrial metabolism and function, as well as mitochondria‐mediated cell survival pathways and cell death is important. In this study, we attempted to set up the methods for isolation of mitoplasts (mitochondria without OMM) and the OMM from the rat heart.MethodsCardiac mitochondria were isolated from Sprague Dawley rats (250–300g) by differential centrifugation and purified via Percoll gradient (continuous layers of 19%, 30% and 60% concentration). Isolation of mitoplasts was performed by incubation in different hypotonic and hypertonic buffers, sonication (20% output/power), and separation of membranes by ultracentrifugation using sucrose continuous gradients (20–80%). Mitochondrial swelling assay was made to confirm the swelling/shrinking method used. Mitoplast formation was validated by monoamine oxidase (MAO) activity, oxygen consumption in the presence and absence of cytochrome‐c and Western Blotting against specific inner and outer mitochondrial membrane proteins.ResultsWestern Blot analysis showed pure isolated mitochondria, validated by the absence of GAPDH (cytosolic marker) upon Percoll gradient. In addition, more than 30% decrease in TOMM20 (OMM specific protein) expression in isolated mitoplasts in comparison to mitochondria. Mitochondrial respiratory rates and MAO activity suggests changes in mitochondrial membrane distribution. Mitochondrial swelling assay showed that cardiac mitochondria were resistant to swelling in hypotonic solution, therefore other methods involving the addition of Ca+2 or EGTA were used to induced separation of mitochondrial membranes.ConclusionDespite application of different preparative approaches, we were not able to isolate purified OMM, and only one method was proved to make a slight reduction of OMM contamination in mitoplasts.Support or Funding InformationSupported by the NIGMS NIH grant SC1GM128210This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.