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. 2014 Sep 16;107(6):1289-301.
doi: 10.1016/j.bpj.2014.07.045.

Calcium movement in cardiac mitochondria

Liron Boyman  1 , Aristide C Chikando  1 , George S B Williams  2 , Ramzi J Khairallah  3 , Sarah Kettlewell  4 , Christopher W Ward  5 , Godfrey L Smith  4 , Joseph P Y Kao  1 , W Jonathan Lederer  6
Affiliations

Calcium movement in cardiac mitochondria

Liron Boyman et al. Biophys J. .

Abstract

Existing theory suggests that mitochondria act as significant, dynamic buffers of cytosolic calcium ([Ca(2+)]i) in heart. These buffers can remove up to one-third of the Ca(2+) that enters the cytosol during the [Ca(2+)]i transients that underlie contractions. However, few quantitative experiments have been presented to test this hypothesis. Here, we investigate the influence of Ca(2+) movement across the inner mitochondrial membrane during both subcellular and global cellular cytosolic Ca(2+) signals (i.e., Ca(2+) sparks and [Ca(2+)]i transients, respectively) in isolated rat cardiomyocytes. By rapidly turning off the mitochondria using depolarization of the inner mitochondrial membrane potential (ΔΨm), the role of the mitochondria in buffering cytosolic Ca(2+) signals was investigated. We show here that rapid loss of ΔΨm leads to no significant changes in cytosolic Ca(2+) signals. Second, we make direct measurements of mitochondrial [Ca(2+)] ([Ca(2+)]m) using a mitochondrially targeted Ca(2+) probe (MityCam) and these data suggest that [Ca(2+)]m is near the [Ca(2+)]i level (∼100 nM) under quiescent conditions. These two findings indicate that although the mitochondrial matrix is fully buffer-capable under quiescent conditions, it does not function as a significant dynamic buffer during physiological Ca(2+) signaling. Finally, quantitative analysis using a computational model of mitochondrial Ca(2+) cycling suggests that mitochondrial Ca(2+) uptake would need to be at least ∼100-fold greater than the current estimates of Ca(2+) influx for mitochondria to influence measurably cytosolic [Ca(2+)] signals under physiological conditions. Combined, these experiments and computational investigations show that mitochondrial Ca(2+) uptake does not significantly alter cytosolic Ca(2+) signals under normal conditions and indicates that mitochondria do not act as important dynamic buffers of [Ca(2+)]i under physiological conditions in heart.

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Figures

Figure 1
Figure 1
Turning off ΔΨm with light: effects on local cytosolic Ca2+ signals in cardiac ventricular myocytes. (A) A 9 × 3 μm ROI is identified in (i) by the box formed by a dashed white line. The polarized mitochondria throughout the rat ventricular myocyte are loaded with TMRM. (ii). Fluo-4 is imaged in the same cell to reveal the local and cell-wide [Ca2+]i signal. (iii) Images from panels (i) and (ii) are superimposed. (B) (i) The boxed region (dashed white line) indicates the depolarized (dark) mitochondria in the ROI. The ΔΨm, before (A) and after the laser illumination protocol (B). (i) TMRM signals, showing loss of ΔΨm only in the region that underwent sequential scans with the 543 nm laser (dashed lines) at 0.625 Hz until complete mitochondrial depolarization. (ii) Fluo-4 signals, showing the order and location of line-scan Ca2+ spark measurements. (iii) Overlay. (C) Surface plots of the average Ca2+ sparks measured as ΔF/F0 using Fluo-4 (n > 200 Ca2+ sparks). Ca2+ spark measurements are done before photon stress (Control), and resume 5 s postdepolarization in either the nondepolarized distal region (PDD Control) or within the depolarized ROI (Depolarized). Subsequent alternating measurements between PDD Control and Depolarized ROIs are every 15 s. (D) Ca2+ spark (i) profiles and (ii) properties affected by localized mitochondrial depolarization (n = 40 cells).
Figure 2
Figure 2
ROS production during TMRM laser illumination and the effect of ROS scavenging on Ca2+ sparks. (A) Confocal images of intact rat ventricular myocytes simultaneously loaded with TMRM and the ROS indicator DCF during laser illumination. Whole-cell images are acquired every 19 s, although the images of a region near the center of the cell (not shown, indicated by white box) are acquired faster (every ∼3 s). Top: Cell FTMRM showing spatially resolved ΔΨm. Bottom: DCF fluorescence at given time points. ROI-1 is the region scanned rapidly that is shown to depolarize. ROI-2 is a region distant from ROI-1 that does not depolarize. This is the region that is identified as the postdepolarization, distant (PDD control) region. (B) Time courses of changes in ΔΨm (red circles) and DCF fluorescence (green circles) outside (solid symbols) and within the more frequently illuminated region (open symbols), n = 9 cells. (C) Altered Ca2+ sparks properties in the presence of the ROS scavenger NAC (using the protocol described in Fig. 1). A p-value of <0.05 is indicated by either ∗ or # or &. The latter two symbols (# or &), are used to indicate statistical significance between ROI-1 and the Control region when the NAC concentration is 10 mM (#) or 50 mM (&). N = 40 cells for 0 mM NAC, 37 cells for 10 mM NAC, and 27 cells for 50 mM NAC. The timing of Ca2+ spark measurements within a cell are the same as in Fig. 1.
Figure 3
Figure 3
Ca2+ sparks in cardiac ventricular myocytes lacking polarized mitochondria. (A) Confocal images of intact rat ventricular myocyte simultaneously loaded with TMRM (left) and DCF (right). (B) Time course of whole-cell changes in FTMRM (red circles) and in the rates of ROS production measured as a baseline corrected DCF fluorescence slope (green circles). The pharmacological treatment to dissipate ΔΨm is carried out during the period highlighted in gray (n = 9 cells). (C) Top: Analysis of TMRM distribution before and after the pharmacological treatment. Values are normalized to FTMRM in the nucleus. Pixels with higher fluorescence intensity are indicative of TMRM accumulation in polarized mitochondria. Bottom: Simultaneously measured DCF distribution. (D) Ca2+ spark frequency before and after global ΔΨm dissipation with and without NAC (n = 11 cells). (E) Ca2+ spark amplitude before and after global ΔΨm dissipation (n = 11 cells).
Figure 4
Figure 4
[Ca2+]i transients in cardiac ventricular myocytes without polarized mitochondria. (A) Confocal line-scan images acquired from a voltage-clamped rat ventricular myocyte (whole-cell mode) simultaneously loaded with TMRM and 60 μM Fluo-4 K. [Ca2+]i transients were electrically elicited by two trains of voltage pulses applied at 0.5 Hz and separated by a rest period; with each pulse the membrane potential was stepped from a holding potential of −80 mV to 0 for 100 ms. The pharmacological treatment to dissipate ΔΨm is carried out during the rest period. (B) [Ca2+]i transients measured as ΔF/F0 before (black, n = 13 cells), after the pharmacological treatment (red, n = 7 cells), and at Train-2 in experiments done without depolarizing treatment (blue, n = 6 cells). (C) ΔΨm at the four time points (t1, t2, t3, and t4) indicated in Fig. 4A. Statistical comparisons indicate p-value <0.05 when compared to FTMRM at t1.
Figure 5
Figure 5
Spatially resolved effect of ΔΨm on [Ca2+]m. (A) Confocal images of a ventricular myocyte 48 h after infection with Ad-MityCam, shown are MityCam fluorescence (left), TMRM fluorescence (middle), and overlay (right). The cell region subjected to prolonged laser illumination is also marked (white box; inset) (B) The marked region in A was repeatedly imaged with the 488 nm and 543 nm lasers, sequentially (1.57 s/frame). MityCam (top) and TMRM (bottom) signals at four different time points. Time-dependent plots on the right are the fluorescent measurements from the regions indicated by dashed lines. (C) Spatially resolved signals of individual mitochondria are aligned with respect to the time of 50% decay in FTMRM, shown is the time course of normalized changes in FMityCam. Experiments were done either at [Ca2+]i = 100 nM (solid circles, n = 17 cells) or at [Ca2+]i = 0 nM (triangles, n = 12 cells, for more details on how cellular Ca2+ is depleted see Fig. S7). (D) [Ca2+]m changes in both conditions upon depolarization (10% increase in FMityCam is a significant [Ca2+]m decrease). For more details about the experimental and analytical procedures see Fig. S7.
Figure 6
Figure 6
Mitochondrial Ca2+ uptake and its influence on simulated Ca2+ sparks and [Ca2+]i transients. (A) Mitochondrial Ca2+ fluxes as a function of [Ca2+]i. Solid gray circles represent cardiac uptake rates reported in the literature (compiled in (6)). The red line represents theoretical formulations for mitochondrial Ca2+ fluxes for normal (1X) conditions (see (6)). For comparison, a 10X, 100X, and 1000X scaling of the mitochondrial Ca2+ fluxes are shown in blue, green, and magenta, respectively. Also shown are representative Ca2+ fluxes mediated by the sarcolemmal Na+/Ca2+ exchanger (NCX, dashed black line) and the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA, black line). (B and C) Simulations showing the influence of scaling mitochondrial Ca2+ fluxes on Fluo-4 fluorescence profiles (as ΔF/F0) during Ca2+ sparks (B) from a quiescent cell (similar to Fig. 1D) and during steady-state [Ca2+]i transients (C) from a paced cell (similar to Fig. 4B). See the Supporting Material for more details.
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