title: Changes of intra-mitochondrial Ca2+ in adult ventricular cardiomyocytes examined using a novel fluorescent Ca2+ indicator targeted to mitochondria.
abstract: In this study a Ca(2+) sensitive protein was targeted to the mitochondria of adult rabbit ventricular cardiomyocytes using an adenovirus transfection technique. The probe (Mitycam) was a Ca(2+)-sensitive inverse pericam fused to subunit VIII of human cytochrome c oxidase. Mitycam expression pattern and Ca(2+) 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 Ca(2+) transient. Cytoplasmic (Fura-2) and mitochondrial Ca(2+) (Mitycam) fluorescence were measured simultaneously under a range of cellular Ca(2+) loads. After 48 h post-adenoviral transfection, Mitycam expression showed a characteristic localization pattern in HeLa cells and cardiomyocytes. The Ca(2+) sensitive component of Mitycam fluorescence was 12% of total fluorescence in HeLa cells with a K(d) of approximately 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 Ca(2+) transient was slower, but the rate of decay was faster than the cytoplasmic signal. During spontaneous Ca(2+) 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 approximately 65% and increased the time to 50% decay, whilst cytosolic Ca(2+) transients were unchanged. The mitochondrial Ca(2+) uniporter (mCU) inhibitor Ru360 prevented both the basal and transient components of the rise in mitochondrial Ca(2+). The mitochondrial-targeted Ca(2+) probe indicates sustained and transient phases of mitochondrial Ca(2+) signal, which are dependent on cytoplasmic Ca(2+) levels and require a functional mCU.
authors.affiliation: Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, UK.
authors.forename: S
authors.initials: S
authors.lastname: Kettlewell
authors.forename: P
authors.initials: P
authors.lastname: Cabrero
authors.forename: S A
authors.initials: SA
authors.lastname: Nicklin
authors.forename: J A T
authors.initials: JA
authors.lastname: Dow
authors.forename: S
authors.initials: S
authors.lastname: Davies
authors.forename: G L
authors.initials: GL
authors.lastname: Smith
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D004594:Electrophysiology; D006367:HeLa Cells; D006352:Heart Ventricles / Q000166:cytology*; D006801:Humans; D008929:Mitochondria, Heart / Q000378:metabolism*; D015335:Molecular Probes / Q000235:genetics / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D015335:Molecular Probes; D011993:Recombinant Fusion Proteins; D002118:Calcium