Monitoring mitochondrial calcium and metabolism in the beating MCU-KO heart

Optical methods for measuring intracellular ions including Ca2+ revolutionized our understanding of signal transduction. However, these methods are not extensively applied to intact organs due to issues including inner filter effects, motion, and available probes. Mitochondrial Ca2+ is postulated to...

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Bibliographic Details
Published in:	Cell reports (Cambridge) 2021-10, Vol.37 (3), p.109846-109846, Article 109846
Main Authors:	Kosmach, Anna, Roman, Barbara, Sun, Junhui, Femnou, Armel, Zhang, Fan, Liu, Chengyu, Combs, Christian A., Balaban, Robert S., Murphy, Elizabeth
Format:	Article
Language:	eng
Subjects:	Adrenergic beta-Agonists - pharmacology Animals calcium Calcium - metabolism Calcium Channels - genetics Calcium Channels - metabolism Calcium Signaling - drug effects Energy Metabolism - drug effects Fluorescent Dyes heart HEK293 Cells Heterocyclic Compounds, 3-Ring Humans Isolated Heart Preparation isoproterenol Isoproterenol - pharmacology Mice Mice, Inbred C57BL Mice, Knockout mitochondria Mitochondria, Heart - drug effects Mitochondria, Heart - genetics Mitochondria, Heart - metabolism Mitochondrial Proteins - genetics Mitochondrial Proteins - metabolism Myocardial Contraction - drug effects Myocytes, Cardiac - drug effects Myocytes, Cardiac - metabolism Optical Imaging spectroscopy Time Factors
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Summary:	Optical methods for measuring intracellular ions including Ca2+ revolutionized our understanding of signal transduction. However, these methods are not extensively applied to intact organs due to issues including inner filter effects, motion, and available probes. Mitochondrial Ca2+ is postulated to regulate cell energetics and death pathways that are best studied in an intact organ. Here, we develop a method to optically measure mitochondrial Ca2+ and demonstrate its validity for mitochondrial Ca2+ and metabolism using hearts from wild-type mice and mice with germline knockout of the mitochondria calcium uniporter (MCU-KO). We previously reported that germline MCU-KO hearts do not show an impaired response to adrenergic stimulation. We find that these MCU-KO hearts do not take up Ca2+, consistent with no alternative Ca2+ uptake mechanisms in the absence of MCU. This approach can address the role of mitochondrial Ca2+ to the myriad of functions attributed to alterations in mitochondrial Ca2+. [Display omitted] •Rhod-2 loading in mouse hearts allows measurement of intraventricular mitochondrial Ca2+•Absorption spectra alternated with fluorescence are used to correct inner filter effects•Optical spectroscopy is used to monitor changes in mitochondrial Ca2+ and metabolism•Mitochondrial Ca2+ uptake is blocked in hearts with MCU ablation or inhibition Kosmach et al. develop an optical spectroscopy-based method using Ca2+ indicator dye Rhod-2-AM to monitor mitochondrial Ca2+ in the ex vivo perfused mouse heart. Using this method with hearts under beta-adrenergic stimulation, we find that there are no alternative Ca2+ uptake pathways in germline MCU-KO hearts.
ISSN:	2211-1247 2211-1247