CA2+ ACCUMULATION IN ISOLATED RAT HEART MITOCHONDRIA UNDER MAINTENANCE OF MITOCHONDRIAL POTENTIAL
A.Yu. Budko, N.A. Strutynska, I.Yu. Okhay, O.M. Semenykhina, V.F. Sagach
O.O. Bogomoletz Institute of Physiology, National Academy
of Sciences of Ukraine, Kyiv
It is known that mitochondria can accumulate calcium, which
regulates energy metabolism and cell death. About 90% of
energy of cardiomyocytes is synthesized in mitochondria.
Heart cells are also affected by the rapid changes in the Ca2+
concentration in the cytoplasm. Therefore, mitochondrial
Ca2+-accumulation ability is crucial. The aim of our work
was to study the accumulation of Ca2+ in isolated rat heart
mitochondria in the presence of mitochondrial potential and
different extramitochondrial Ca2+ concentrations. Isolated
organelles were loaded with fluorescent dye Fluo-4 AM (2,5
µmol/l) at a temperature of 26°C for 30 min. It has been
revealed that under these conditions high mitochondrial potential
was maintained sufficiently, which is necessary for the
functioning of the calcium transporting system in organelles.
We established that mitochondria have a limited ability to store
ionized calcium, as addition of Ca2+ ion in concentrations of
10, 20, 50 µmol/l ensures a certain level of accumulation in
organelles with further fluorescent signal growth cessation. Addition
of 100 µmol/l Ca2+ to isolated mitochondria resulted in
a significant increase in fluorescence intensity (46% in the fifth
minute, compared to the fluorescence when 20 µmol/l Ca2+ was
added) and likely to activation of cation release. It was shown
that ruthenium red (10-5 mol/l), an inhibitor of Ca2+ -uniporter,
prevented accumulation of calcium ions in organelles by 89%,
in the presence of 100 µmol/l Ca2+ . It was clearly seen that heart
mitochondria require Mg2+-ATP complex (3 mmol/l) to accumulate
Ca2 +, likely to maintain the inner membrane potential,
activity of Ca2+ uniporter and energetic processes in organelles.
Thus, the process of Ca2+ accumulation in rat heart mitochondria
requires the maintenance of mitochondrial potential, activity of
Ca2+-uniporter, depends on extramitochondrial Ca2 +concentration
and presence of Mg2+-ATP complex.
isolated mitochondria; heart; Ca2 +; flow cytometry; fluorescence probe Fluo-4 AM.
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