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) (PMID:19249308)
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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 > School of Cardiovascular & Metabolic Health College of Medical Veterinary and Life Sciences > School of Molecular Biosciences |
| 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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