Abstract
Mitochondria play a critical role in the cardiomyocyte physiology by generating majority of the ATP required for the contraction/relaxation through oxidative phosphorylation (OXPHOS). Aging is a major risk factor for cardiovascular diseases (CVD) and mitochondrial dysfunction has been proposed as potential cause of aging. Recent technological innovations in Seahorse XFe24 Analyzer enhanced the detection sensitivity of oxygen consumption rate and proton flux to advance our ability study mitochondrial function. Studies of the respiratory function tests in the isolated mitochondria have the advantages to detect specific defects in the mitochondrial protein function and evaluate the direct mitochondrial effects of therapeutic/pharmacological agents. Here, we provide the protocols for studying the respiratory function of isolated murine cardiac mitochondria by measuring oxygen consumption rate using Seahorse XFe24 Analyzer. In addition, we provide details about experimental design, measurement of various respiratory parameters along with interpretation and analysis of data.
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Funding
This work was supported by the National Institutes of Health grants NS094834 (P.V. Katakam: funded by National Institute of General Medical Sciences, NIGMS, and National Institute of Neurological Disorders and Stroke, NINDS) and DK107694 (R. Satou: National Institute of Diabetes and Digestive and Kidney Diseases, NIDDK), American Heart Association National Center Scientist Development grant (14SDG20490359 to P.V. Katakam), American Heart Association Greater Southeast Affiliate Predoctoral Fellowship grant (16PRE27790122 to V.N. Sure), American Heart Association Greater Southeast Affiliate Scientist Development Grant (17SDG33410366 to Ibolya Rutkai), and LACaTS (supported in part by U54 GM104940 from the National Institute of General Medical Sciences of the National Institutes of Health, which funds the Louisiana Clinical and Translational Science Center to Ibolya Rutkai).
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Animal procedures and protocols were performed in accordance with institutional and federal guidelines. All procedures from this study have been approved by the Institution of Animal Care and Use Committee (IACUC) at Tulane University School of Medicine.
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Sakamuri, S.S.V.P., Sperling, J.A., Sure, V.N. et al. Measurement of respiratory function in isolated cardiac mitochondria using Seahorse XFe24 Analyzer: applications for aging research. GeroScience 40, 347–356 (2018). https://doi.org/10.1007/s11357-018-0021-3
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DOI: https://doi.org/10.1007/s11357-018-0021-3