Journal
JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY
Volume 46, Issue 6, Pages 891-901Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.yjmcc.2009.02.016
Keywords
Cardiomyocyte; Ca2+; Fluorescent Ca2+ indicator; Adenovirus Type 5; Mitochondria
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Funding
- Biotechnology and Biological Sciences Research Council (SK)
- BBSRC [BB/G020620/1, BB/D013852/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/G020620/1, BB/D013852/1, BB/C007697/1] Funding Source: researchfish
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In this study a Ca2+ sensitive protein was targeted to the mitochondria of adult rabbit ventricular cardiomyocytes using an adenovirus transfection technique. The probe (Mitycam) was a Ca2+-sensitive inverse pericam fused to subunit VIII of human cytochrome c oxidase. Mitycam expression pattern and Ca2+ sensitivity was characterized in HeLa cells and isolated adult rabbit cardiomyocytes. Cardiomyocytes expressing Mitycam were voltage-clamped and depolarized at regular intervals to elicit a Ca2+ transient. Cytoplasmic (Fura-2) and mitochondrial Ca2+ (Mitycam) fluorescence were measured simultaneously under a range of cellular Ca2+ loads. After 48 h post-adenoviral transfection, Mitycam expression showed a characteristic localization pattern in HeLa cells and cardiomyocytes. The Ca2+ sensitive component of Mitycam fluorescence was 12% of total fluorescence in HeLa cells with a K-d of similar to 220 nM. In cardiomyocytes, basal and beat-to-beat changes in Mitycam fluorescence were detected on initiation of a train of depolarizations. Time to peak of the mitochondrial Ca2+ transient was slower, but the rate of decay was faster than the cytoplasmic signal. During spontaneous Ca2+ release the relative amplitude and the time course of the mitochondrial and cytoplasmic signals were comparable. Inhibition of mitochondrial respiration decreased the mitochondrial transient amplitude by similar to 65% and increased the time to 50% decay, whilst cytosolic Ca2+ transients were unchanged. The mitochondrial Ca2+ uniporter (mCU) inhibitor Ru360 prevented both the basal and transient components of the rise in mitochondrial Ca2+. The mitochondrial-targeted Ca2+ probe indicates sustained and transient phases of mitochondrial Ca2+ signal, which are dependent on cytoplasmic Ca2+ levels and require a functional mCU. (C) 2009 Elsevier Inc. All rights reserved.
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