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

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• 作者：Tatyana N. Andrienko ; Eckard Picht ; Donald M. Bers
• 关键词：Mitochondria ; Calcium transport ; Cardiac myocytes ; Sarcoplasmic reticulum
• 刊名：Journal of Molecular and Cellular Cardiology
• 出版年：2009
• 出版时间：June 2009
• 年：2009
• 卷：46
• 期：6
• 页码：1027-1036
• 全文大小：723 K

文摘

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.