Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Mitochondria are subcellular organelles that establish cytoplasmic energetic conditions optimized for specific cell functions. While cellular, tissue, and organism-level studies can provide insights into mitochondrial function, isolating the organelles offers a level of experimental control not possible otherwise. Mitochondrial isolations have been performed since the 1940s, allowing mechanistic studies of metabolism and respiration across a variety of cells and tissues^1,2. The historical relevance of mitochondria is also well-documented³. As the main producers of ATP, mitochondria play many key roles that are vital for optimal cellular and organ function⁴. Within the mitochondrial matrix, substrates are oxidized by the TCA cycle, producing reducing equivalents and mobile electron carriers such as NADH and UQH₂^5,6. Cytochrome C is the third main mobile electron carrier in the mitochondrial biochemical reaction network⁷. These molecules are then oxidized by the transmembrane complexes of the electron transport system (ETS) embedded in the inner mitochondrial membrane⁸. Redox reactions of the ETS are coupled with proton translocation from the matrix to the intermembrane space. These processes establish an electrochemical proton gradient that is used to phosphorylate ADP with Pi by F₁F₀ ATP synthase to produce ATP^9,10. The individual processes that occur at each complex can be explored with high-resolution respirometry using Clark-type electrodes or microplate oxygen consumption assays^11,12. Additionally, disease models and treatments using isolated mitochondria can determine the impact or importance of mitochondrial function in the progression of certain pathologies. This has proven fruitful in the field of cardiology, where alterations in fuel and substrate delivery have been used to elucidate how mitochondrial dysfunction influences heart failure^13,14,15,16. Mitochondria are also known to impact the development of other disease states such as diabetes, cancer, obesity, neurological disorders, and myopathies^17,18. Therefore, the use of isolated or purified mitochondria enables mechanistic investigations of oxidative metabolism and ATP production in the source tissue.

There is no shortage of mitochondrial isolation protocols due to their importance in bioenergetic research. Additionally, highly specific methods tailored to subpopulations of mitochondria within tissues and cells can be found^19,20. The basic procedural steps are similar between isolation methods, but variations can be made to buffer composition, homogenization steps, and centrifugation spins to improve the amount and quality of mitochondria. Changes to these aspects are based on the metabolic demand of the tissue, overall organ function, mitochondrial density, and other factors. In tissues such as the liver and skeletal muscle, handheld homogenizers are used to preserve mitochondrial integrity and limit damage to the mitochondrial membranes²¹. However, when isolating from kidneys, some protocols suggest using manually driven homogenization or commercial kits to yield better results²². Although both methods yield functional mitochondria, the quality of the organelles can become compromised by the additional time it takes to complete isolations using these protocols. Centrifugation is also vital to the extraction of mitochondrial protein, as it separates cellular components such as nuclei and other organelles from mitochondria²³. During the isolation process, it is debated whether differential or density-based centrifugation should be implemented to obtain purer isolates²⁴. While density centrifugation can separate mitochondria from organelles of similar specific gravity, such as peroxisomes, it is not well-established if mitochondria from these methods better represent in situ organelle function compared to mitochondria isolated using differential centrifugation. In the field of mitochondrial physiology, density-based centrifugation is preferred and can be easily altered to increase isolate purity. Whether changes to g-forces, centrifugation duration, and the number of centrifugation spins are incorporated should be considered before experimentation due to their influence on mitochondrial purification. Furthermore, mitochondrial resuspension, arguably the most crucial step during isolation, differs greatly between studies, with the use of scraping, vortex-based mixing, or homogenization^23,25,26. Mechanical resuspension of these types can be too abrasive and impair the membrane integrity of mitochondria. For this reason, gentle washing should be performed to correct this. Despite the plethora of modulations and suggested methodologies, there are fewer comprehensive protocols with high reproducibility and adaptability for rodent models.

The methods described herein outline a detailed, robust, and highly reproducible protocol that yields purified, well-coupled mitochondria from small animal cardiac tissue. As demonstrated, this method can be easily adapted to accommodate the specific needs of each experiment and/or laboratory environment for isolating mitochondria from kidneys, liver, and cultured cells. Further modifications can be made to isolate mitochondria from tissues and other animals not listed here. Buffer recipes used for all isolations are provided and can be modified if needed. Similar to other published protocols, motorized homogenization, and differential centrifugation are implemented; however, adjustments are made to both the shearing time and the force at which the samples are centrifuged to consistently deliver high-quality mitochondrial isolates, depending on the isolation source. Notably, this protocol differs from others by using gentle washing via pipetting to resuspend pelleted mitochondria, which helps preserve mitochondrial membrane integrity and maintain the overall functionality of the organelles. Mitochondrial protein is quantified either by total protein determination or by measuring citrate synthase activity. The utility and broad applicability of this isolation method are further supported by the values of respiratory control ratios (RCR) achieved across various organisms and tissue sources.