Changes of intra-mitochondrial Ca2+ in adult ventricular cardiomyocytes examined using a novel fluorescent Ca2+ indicator targeted to mitochondria

Kettlewell, S., Cabrero, P., Nicklin, S.A. , Dow, J.A.T., Davies, S.A. and Smith, G.L. (2009) Changes of intra-mitochondrial Ca2+ in adult ventricular cardiomyocytes examined using a novel fluorescent Ca2+ indicator targeted to mitochondria. Journal of Molecular and Cellular Cardiology, 46(6), pp. 891-901. (doi:10.1016/j.yjmcc.2009.02.016)

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Abstract

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. Methods: 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. Results: After 48hrs 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 Kd of ~220nM. In cardiomyocytes, basal and beat-to-beat changes in Mitycam fluorescence were detected on initiation of a train of depolarisations. 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 ~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+. Conclusion: The mitochondrial-targeted Ca2+probe indicates sustained and transient phases of mitochondrial Ca2+signal, which are dependent on cytoplasmic Ca2+levels and required a functional mCU

Item Type:Articles
Keywords:Cardiomyocyte, Ca2+, fluorescent Ca2+ indicator, adenovirus type 5, mitochondria
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Smith, Professor Godfrey and Nicklin, Professor Stuart and Dow, Professor Julian and Davies, Professor Shireen and Kettlewell, Dr Sarah
Authors: Kettlewell, S., Cabrero, P., Nicklin, S.A., Dow, J.A.T., Davies, S.A., and Smith, G.L.
Subjects:Q Science > QR Microbiology
College/School:College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Journal of Molecular and Cellular Cardiology
Publisher:Elsevier
ISSN:0022-2828
ISSN (Online):1095-8584
Published Online:26 February 2009

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
392411Investigation of calcium signalling in sarcoplasmic riticulum of cardiac myocytes using a novel targeted transporterShireen DaviesBiotechnology and Biological Sciences Research Council (BBSRC)BB/C007697/1Institute of Molecular Cell and Systems Biology
415661Spatiotemporal filtering in calcium control of mitochondrial functionJulian DowBiotechnology and Biological Sciences Research Council (BBSRC)BB/D013852/1Institute of Molecular Cell and Systems Biology