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

Abstract

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

Figure 1. Mitochondrial localization of fura-2 in permeabilized myocytes

Confocal images of intact rat ventricular cardiomyocyte simultaneously loaded with fura-2 and Mitotracker Orange before (A) and after sarcolemmal permeabilization by saponin (B). Top panels (a) are fura-2 signals, middle panels (b) are Mitotracker Orange, showing typical mitochondrial pattern and lower panels (c) show merged images. Fura-2 only colocalizes with mitochondria after permeabilization. Scale bars are equal 10 μm.

FIGURE 2. Mitochondrial free [Ca²⁺] measurements with fura-2

A, Top traces show background-corrected fura-2 fluorescence at 340 and 380 nm excitation after permeabilization. The inset shows plot of F₃₄₀ vs. F₃₈₀ as [Ca²⁺]_mito varies to determine αand allows measurement of F_c (Ca²⁺-independent fura-2 fluorescence) in bottom normalized trace showing loss of 70% of fura-2 upon permeabilization. B, Rise in [Ca²⁺]_mito in the presence of 800 nM Ca²⁺ and in the absence of Na⁺ (±2.5 μM FCCP added 2 min prior to increasing [Ca²⁺]_i). C, In situ calibration of background subtracted fura-2 fluorescence in permeabilized cells in the presence of 3 μM ionomycin (Iono), 10 μM FCCP and 20 μg/mL oligomycin. D, Fura-2 ratio dependence on [Ca²⁺]_i for data in C fit to [Ca²⁺] = K_dβ(R-R_min)/(R_max-R), where EC₅₀ = K_dβ and β = F_380-max/F_380-min.

FIGURE 3. Steady-state dependence of [Ca²⁺]_mito on [Ca²⁺]_i

A, example of background-corrected raw F₃₄₀ and F₃₈₀ 380 nm signals, where [Ca²⁺]_i was raised from 0 to 429 nM, with R_min and R_max measured at the end using ionophores (Iono). B, calibrated [Ca²⁺]_mito calculated from A. C, Steady-state dependence of [Ca²⁺]_mito on [Ca²⁺]_i in the presence of 10 mM Na⁺ (Mean ±SD, n=3–7 myocytes).

FIGURE 4. [Ca²⁺]_mito during cyclical SR Ca²⁺ release

A, Simultaneous recording of [Ca²⁺]_mito (grey) and contractions (black) in the presence of 150 nM Ca²⁺ and 15 mM Na⁺. 40 μM cytochalasin D was included to limit contraction. Inset shows expanded superimposed traces for the 7^th contraction (traces smoothed by fast Fourier transform). B, [Ca²⁺]_mito under control conditions and after pretreated with 1 μM thapsigargin for 30 min. Mitochondrial Ca²⁺ uptake was halted by acute application of 10 mM caffeine (C), 2 mM tetracaine (D), or 1 μM Ru360 (E). Each experiment ended with Ca²⁺-free solution with ionophores (Iono) to start calibration.

FIGURE 5. Calibrated rise in [Ca²⁺]_mito per beat and [Na⁺]_i-dependence

A, 10 mM caffeine stops [Ca²⁺]_mito, but with 15 mM Na⁺ [Ca²⁺]_mito declines (filled symbols); mitochondrial Ca²⁺ uptake in presence of 5 mM EGTA (empty symbols). B, examples of [Ca²⁺]_mito increase per contraction (±15 mM Na⁺). C, Average increase of [Ca²⁺]_mito per contraction (±15 mM Na⁺). D, Blockade of NCX_m (5 μM CGP-37157) increases rate of [Ca²⁺]_mito rise in presence of 15 mM Na⁺. Each experiment ended with Ca²⁺-free solution with ionophores (Iono) to start calibration.

FIGURE 6. Spatially resolved [Ca²⁺]_mito during SR Ca²⁺ release

A, confocal image of permeabilized rat ventricular myocyte loaded with 10 μM rhod-2 AM, after 5 min of spontaneous Ca²⁺ transients in 15 mM Na⁺ (with 80 μM cytochalasin D) with 4 regions of interest (ROI) indicated. Scale bar is equal 10 μm. B-C, Time courses of [Ca²⁺]_mito in 4 ROIs, either as fluorescence (F in arbitrary units, B) or calibrated Δ[Ca²⁺]_mit (C) using averaged Δ[Ca²⁺]_mito/beat from Fig. 5C (see B and text). C, ROI 3 and 4 contain 3 mitochondria and inset shows the indicated [Ca²⁺]_mito transients expanded.

FIGURE 7. Mitochondrial [Ca²⁺] balance during SR Ca²⁺ release

Aa, [Ca²⁺]_mito measured in line-scan images in permeabilized myocyte loaded with 5 μM rhod-2 AM in 15 mM Na⁺ (with 80 μM cytochalasin D; subjected to 5 point smoothing). Ab, Amplitudes of rise (filled symbols) and decay phase (empty symbols). Ac, Net gain in [Ca²⁺]_mito per wave. Ad, Integral of change in [Ca²⁺]_mito. B, Expanded and curve fitting for the transients indicated in A.

FIGURE 8. Kinetics of [Ca²⁺]_mito vs. [Ca²⁺]_i signals

Aa, [Ca²⁺]_mito and [Ca²⁺]_i measured in line-scan images in permeabilized myocyte with mitochondrial rhod-2 and 5 μM cytosolic fura-2 salt (15 mM Na⁺, 80 μM cytochalasin D, 5 point smoothing). Ab, Time-to-Peak (rise time for either [Ca²⁺]_mito or [Ca²⁺]_i). Ac, Delay of [Ca²⁺]_mito after [Ca²⁺]_i as start-to-start and peak-to-peak, Ad, time constant τof decline. B, Curve fitting and expanded mitochondrial (black) and cytosolic (grey) transients indicated in Aa. C, Difference in mitochondrial and cytosolic kinetics (n=3 cells, 25 transients).