title: Diversity of mitochondrial Ca²⁺ signaling in rat neonatal cardiomyocytes: evidence from a genetically directed Ca²⁺ probe, mitycam-E31Q.
abstract: I(Ca)-gated Ca(2+) release (CICR) from the cardiac SR is the main mechanism mediating the rise of cytosolic Ca(2+), but the extent to which mitochondria contribute to the overall Ca(2+) signaling remains controversial. To examine the possible role of mitochondria in Ca(2+) signaling, we developed a low affinity mitochondrial Ca(2+) probe, mitycam-E31Q (300-500 MOI, 48-72h) and used it in conjunction with Fura-2AM to obtain simultaneous TIRF images of mitochondrial and cytosolic Ca(2+) in cultured neonatal rat cardiomyocytes. Mitycam-E31Q staining of adult feline cardiomyocytes showed the typical mitochondrial longitudinal fluorescent bandings similar to that of TMRE staining, while neonatal rat cardiomyocytes had a disorganized tubular or punctuate appearance. Caffeine puffs produced rapid increases in cytosolic Ca(2+) while simultaneously measured global mitycam-E31Q signals decreased more slowly (increased mitochondrial Ca(2+)) before decaying to baseline levels. Similar, but oscillating mitycam-E31Q signals were seen in spontaneously pacing cells. Withdrawal of Na(+) increased global cytosolic and mitochondrial Ca(2+) signals in one population of mitochondria, but unexpectedly decreased it (release of Ca(2+)) in another mitochondrial population. Such mitochondrial Ca(2+) release signals were seen not only during long lasting Na(+) withdrawal, but also when Ca(2+) loaded cells were exposed to caffeine-puffs, and during spontaneous rhythmic beating. Thus, mitochondrial Ca(2+) transients appear to activate with a delay following the cytosolic rise of Ca(2+) and show diversity in subpopulations of mitochondria that could contribute to the plasticity of mitochondrial Ca(2+) signaling.
authors.affiliation: Calcium Signaling Center of University of South Carolina, Medical University of South Carolina, and Clemson University, Charleston, SC 29425, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Haviland
authors.affiliation: Calcium Signaling Center of University of South Carolina, Medical University of South Carolina, and Clemson University, Charleston, SC 29425, USA.
authors.forename: Lars
authors.initials: L
authors.lastname: Cleemann
authors.affiliation: Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
authors.forename: Sarah
authors.initials: S
authors.lastname: Kettlewell
authors.affiliation: Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
authors.forename: Godfrey L
authors.initials: GL
authors.lastname: Smith
authors.affiliation: Calcium Signaling Center of University of South Carolina, Medical University of South Carolina, and Clemson University, Charleston, SC 29425, USA. Electronic address: moradm@musc.edu.
authors.forename: Martin
authors.initials: M
authors.lastname: Morad
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002147:Calmodulin / Q000235:genetics*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D005453:Fluorescence; D016257:Fura-2 / Q000031:analogs & derivatives*; D057809:HEK293 Cells; D006801:Humans; D008929:Mitochondria, Heart / Q000378:metabolism*; D015335:Molecular Probes / Q000235:genetics; D032383:Myocytes, Cardiac / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction
chemical_list: D002147:Calmodulin; D015335:Molecular Probes; C049925:fura-2-am; D002118:Calcium; D016257:Fura-2
keywords: Calcium (Ca(2+)); Calcium (Ca(2+)) imaging; Cardiomyocyte; Mitochondria