Mitochondrial free calcium regulation during sarcoplasmic reticulum calcium release in rat cardiac myocytes

Tatyana N. Andrienko, Eckard Picht, Donald M Bers

Research output: Contribution to journalArticle

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Abstract

Cardiac mitochondria can take up Ca2+, competing with Ca2+ transporters like the sarcoplasmic reticulum (SR) Ca2+-ATPase. Rapid mitochondrial [Ca2+] transients have been reported to be synchronized with normal cytosolic [Ca2+]i transients. However, most intra-mitochondrial free [Ca2+] ([Ca2+]mito) measurements have been uncalibrated, and potentially contaminated by non-mitochondrial signals. Here we measured calibrated [Ca2+]mito in single rat myocytes using the ratiometric Ca2+ indicator fura-2 AM and plasmalemmal permeabilization by saponin (to eliminate cytosolic fura-2). The steady-state [Ca2+]mito dependence on [Ca2+]i (with 5 mM EGTA) was sigmoid with [Ca2+]mito < [Ca2+]i for [Ca2+]i below 475 nM. With low [EGTA] (50 μM) and 150 nM [Ca2+]i (± 15 mM Na+) cyclical spontaneous SR Ca2+ release occurred (5-15/min). Changes in [Ca2+]mito during individual [Ca2+]i transients were small (∼ 2-10 nM/beat), but integrated gradually to steady-state. Inhibition SR Ca2+ handling by thapsigargin, 2 mM tetracaine or 10 mM caffeine all stopped the progressive rise in [Ca2+]mito and spontaneous Ca2+ transients (confirming that SR Ca2+ releases caused the [Ca2+]mito rise). Confocal imaging of local [Ca2+]mito (using rhod-2) showed that [Ca2+]mito rose rapidly with a delay after SR Ca2+ release (with amplitude up to 10 nM), but declined much more slowly than [Ca2+]i (time constant 2.8 ± 0.7 s vs. 0.19 ± 0.06 s). Total Ca2+ uptake for larger [Ca2+]mito transients was ∼ 0.5 μmol/L cytosol (assuming 100:1 mitochondrial Ca2+ buffering), consistent with prior indirect estimates from [Ca2+]i measurements, and corresponds to ∼ 1% of the SR Ca2+ uptake during a normal Ca2+ transient. Thus small phasic [Ca2+]mito transients and gradually integrating [Ca2+]mito signals occur during repeating [Ca2+]i transients.

Original languageEnglish (US)
Pages (from-to)1027-1036
Number of pages10
JournalJournal of Molecular and Cellular Cardiology
Volume46
Issue number6
DOIs
StatePublished - Jun 2009

Fingerprint

Sarcoplasmic Reticulum
Cardiac Myocytes
Calcium
Fura-2
Egtazic Acid
Tetracaine
Thapsigargin
Calcium-Transporting ATPases
Saponins
Sigmoid Colon
Caffeine
Cytosol
Muscle Cells
Mitochondria

Keywords

  • Calcium transport
  • Cardiac myocytes
  • Mitochondria
  • Sarcoplasmic reticulum

ASJC Scopus subject areas

  • Molecular Biology
  • Cardiology and Cardiovascular Medicine

Cite this

Mitochondrial free calcium regulation during sarcoplasmic reticulum calcium release in rat cardiac myocytes. / Andrienko, Tatyana N.; Picht, Eckard; Bers, Donald M.

In: Journal of Molecular and Cellular Cardiology, Vol. 46, No. 6, 06.2009, p. 1027-1036.

Research output: Contribution to journalArticle

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abstract = "Cardiac mitochondria can take up Ca2+, competing with Ca2+ transporters like the sarcoplasmic reticulum (SR) Ca2+-ATPase. Rapid mitochondrial [Ca2+] transients have been reported to be synchronized with normal cytosolic [Ca2+]i transients. However, most intra-mitochondrial free [Ca2+] ([Ca2+]mito) measurements have been uncalibrated, and potentially contaminated by non-mitochondrial signals. Here we measured calibrated [Ca2+]mito in single rat myocytes using the ratiometric Ca2+ indicator fura-2 AM and plasmalemmal permeabilization by saponin (to eliminate cytosolic fura-2). The steady-state [Ca2+]mito dependence on [Ca2+]i (with 5 mM EGTA) was sigmoid with [Ca2+]mito < [Ca2+]i for [Ca2+]i below 475 nM. With low [EGTA] (50 μM) and 150 nM [Ca2+]i (± 15 mM Na+) cyclical spontaneous SR Ca2+ release occurred (5-15/min). Changes in [Ca2+]mito during individual [Ca2+]i transients were small (∼ 2-10 nM/beat), but integrated gradually to steady-state. Inhibition SR Ca2+ handling by thapsigargin, 2 mM tetracaine or 10 mM caffeine all stopped the progressive rise in [Ca2+]mito and spontaneous Ca2+ transients (confirming that SR Ca2+ releases caused the [Ca2+]mito rise). Confocal imaging of local [Ca2+]mito (using rhod-2) showed that [Ca2+]mito rose rapidly with a delay after SR Ca2+ release (with amplitude up to 10 nM), but declined much more slowly than [Ca2+]i (time constant 2.8 ± 0.7 s vs. 0.19 ± 0.06 s). Total Ca2+ uptake for larger [Ca2+]mito transients was ∼ 0.5 μmol/L cytosol (assuming 100:1 mitochondrial Ca2+ buffering), consistent with prior indirect estimates from [Ca2+]i measurements, and corresponds to ∼ 1{\%} of the SR Ca2+ uptake during a normal Ca2+ transient. Thus small phasic [Ca2+]mito transients and gradually integrating [Ca2+]mito signals occur during repeating [Ca2+]i transients.",
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