1Faculty of Life Sciences, University of Manchester, 2School of Biological Sciences, Queen's University Belfast
Gostimskaya, I., Galkin, A. Preparation of Highly Coupled Rat Heart Mitochondria. J. Vis. Exp. (43), e2202, doi:10.3791/2202 (2010).
The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.
Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism. They can be obtained quickly and reliably in great quantity from wild type or knock-out animals. The general procedure consists of tissue digestion by trypsin, fractionation and differential centrifugation.
There are several conditions that have to be kept during Isolation of mitochondria from various tissues, including the heart (Fig. 1).
Materials and instruments
Solutions (calculated per 2 rats):
The general scheme of the procedure is shown on Fig. 1. Hearts should be cooled and washed in Washing buffer, ventricular tissue excised, minced and homogenised during photolytic treatment with trypsin. The suspension is centrifuged at low speed and the obtained supernatant is centrifuged again at higher speed to collect mitochondria. Depending on the planned experiments the resuspending buffer can be replaced by any other isotonic solution.
Animals were terminated in accordance with the U.K. "Animals (Scientific Procedures) Act." by cervical dislocation followed by decapitation. It is necessary to extract the heart immediately after decapitation of the animal, so that there is no delay between animal termination and heart extraction. If parallel isolation of liver mitochondria is expected the rats should be fasted overnight prior to the experiment.
Figure 1. Scheme demonstrating the step by step procedure of isolation of rat heart mitochondria. Top part, Stages 1-9: tissue disruption, trypsin treatment and homogenisation; bottom part, stages 10-15: differential centrifugation.
Detailed rapid protocol for isolation of intact mitochondria with a high degree of coupling is described. Obtained preparation can be used for functional or structural research on cellular bioenergetics, proteomic studies or analysis of mitochondrial DNA and lipid content. Biophysical studies of active transport of ions and metabolic intermediates can also be performed. It is possible to further process the mitochondrial preparation in order to isolate submitochondrial particles, outer membrane or purify separate components of oxidative phosphorylation. The described method is a modification of the standard protocol of isolation by Jacobus and Saks1. The expected yield of the prepared mitochondria is approximately 10-15 mg of mitochondrial protein per heart and the respiratory control ratio on malate/glutamate of such preparation varies between 8 to 12 with State IV respiration of around 0.12 μmol O2xmin-1xmg protein-1 at 23°C in the medium comprising 0.25 M sucrose, 10 mM KCl, 0.2 mM EDTA, 5 mM potassium phosphate (pH 8.0) with 150 μM ADP for initiation State III respiration (for the exact experimental conditions and additions, see ref. 2).
Special attention should be given to several technical details before the starting of the isolation procedure. It is crucial to use clean polycarbonate or polypropylene centrifuge tubes, therefore tubes should be thoroughly washed with a clean brush and hot water, however the detergent treatment should be kept at the minimum. Also, it is better to collect all necessary glassware and instruments in the cold room a day before the isolation.
During the isolation when transferring buffers from flasks to beakers, а special attention should be given to prevent drops of ice water into the solutions. When handling tissues it should be stressed that heart extraction should be carried out as soon as possible since even short-time delay between decapitation of the animal and cooling the tissue would result in partially uncoupled mitochondria. Rapid cooling and thorough washing of removed hearts is essential to obtain standard preparation, free from haemoglobin and erythrocyte NADase.
Trypsin is used for connective proteins degradation to increase susceptibility of the tissue to shearing force during homogenisation. Prolonged exposure of the tissue to protease should be avoided since it results in mitochondria of inferior quality.
After high speed centrifugations the walls of the centrifuge tubes should be wiped with tissue paper to remove any deposits of fat material accumulated on the walls.
For the final resuspension it is better to use a low volume of the final buffer in order to minimise loss of mitochondrial functions during storage (150 to 200 μl of the buffer per heart). For studies of the transport of divalent cations no chelating agents must be present in the final preparation thus mitochondria should be washed for several times with EGTA-free medium and used within first hours after isolation.
No conflicts of interest declared.
We thank Prof. A. Vinogradov and Dr Vera Grivennikova for introduction of the authors to the field of mitochondrial biology and many useful insights and advice in this procedure. The authors are grateful to Mrs Amanda McKintock from the Queens University media office for the help with the video preparation.