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. 2013 Feb 1;112(3):424-31.
doi: 10.1161/CIRCRESAHA.111.300501. Epub 2012 Dec 14.

Measuring local gradients of intramitochondrial [Ca(2+)] in cardiac myocytes during sarcoplasmic reticulum Ca(2+) release

Xiyuan Lu  1 , Kenneth S Ginsburg, Sarah Kettlewell, Julie Bossuyt, Godfrey L Smith, Donald M Bers
Affiliations

Measuring local gradients of intramitochondrial [Ca(2+)] in cardiac myocytes during sarcoplasmic reticulum Ca(2+) release

Xiyuan Lu et al. Circ Res. .

Abstract

Rationale: Mitochondrial [Ca(2+)] ([Ca(2+)](mito)) regulates mitochondrial energy production, provides transient Ca(2+) buffering under stress, and can be involved in cell death. Mitochondria are near the sarcoplasmic reticulum (SR) in cardiac myocytes, and evidence for crosstalk exists. However, quantitative measurements of [Ca(2+)](mito) are limited, and spatial [Ca(2+)](mito) gradients have not been directly measured.

Objective: To directly measure local [Ca(2+)](mito) during normal SR Ca release in intact myocytes, and evaluate potential subsarcomeric spatial [Ca(2+)](mito) gradients.

Methods and results: Using the mitochondrially targeted inverse pericam indicator Mitycam, calibrated in situ, we directly measured [Ca(2+)](mito) during SR Ca(2+) release in intact rabbit ventricular myocytes by confocal microscopy. During steady state pacing, Δ[Ca(2+)](mito) amplitude was 29±3 nmol/L, rising rapidly (similar to cytosolic free [Ca(2+)]) but declining much more slowly. Taking advantage of the structural periodicity of cardiac sarcomeres, we found that [Ca(2+)](mito) near SR Ca(2+) release sites (Z-line) versus mid-sarcomere (M-line) reached a high peak amplitude (37±4 versus 26±4 nmol/L, respectively P<0.05) which occurred earlier in time. This difference was attributed to ends of mitochondria being physically closer to SR Ca(2+) release sites, because the mitochondrial Ca(2+) uniporter was homogeneously distributed, and elevated [Ca(2+)] applied laterally did not produce longitudinal [Ca(2+)](mito) gradients.

Conclusions: We developed methods to measure spatiotemporal [Ca(2+)](mito) gradients quantitatively during excitation-contraction coupling. The amplitude and kinetics of [Ca(2+)](mito) transients differ significantly from those in the cytosol and are respectively higher and faster near the Z-line versus M-line. This approach will help clarify SR-mitochondrial Ca(2+) signaling.

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Figures

Figure 1
Figure 1. Mitycam in adult rabbit ventricular myocytes
(A) Mitycam, MitoTracker Red and merged signals exhibit a mitochondrial pattern (scale bar 8 µm), especially apparent in enlarged insets. (B) Lower magnification image of myocyte (scale bar, 100 µm). Scatter 2D plots of pixel intensities in red (MitoTracker) and green (Mityam) channels (right-bottom). Intensity thresholds were automatically determined by excluding dark pixels via algorithm in image analysis software, ZEN (Zeiss). Region 1 and 2 pixels represent signal in channel 1 or 2 only, respectively; region 3 represents colocalized pixels. (C) Mitycam signal is unaltered by saponin-permeabilization (typical images (left), average fluorescence before and after at (right); n=10; scale bar, 8 µm).
Figure 2
Figure 2. Kinetics of [Ca2+]mito in adult rabbit ventricular myocytes
(A) Kinetics of [Ca2+]cyto with and without 1 µM Ru360 during 0.2 Hz stimulation with 1.8 mM Ca (i) and mean Δ[Ca] and time to peak (ii). (B) [Ca2+]mito transient and mean Δ[Ca2+]mito with and without 1 µM Ru360. (C) Time to peak and decline tau of [Ca2+]mito and [Ca2+]i. (D) Influence of pacing frequency (as indicated above traces) on [Ca2+]mito signal with (right) and without 100 nM isoproterenol (left). (E) Amplitude and kinetics of [Ca2+]mito at different frequencies (±ISO). (n = 6, *P<0.05, ***P< 0.001).
Figure 3
Figure 3. Calibration and [Ca2+]mito transients in cardiomyocytes
(A) In situ Mitycam calibration in myocytes (n=8). (B) Average [Ca2+]mito transients, diastolic [Ca2+]mito and amplitude of transient during 0.2 Hz stimulation.
Figure 4
Figure 4. Spatial [Ca2+]mito signals at Z- and M-line regions
(A) Localization of T-tubule/Z-line (Di-Anepps, middle) and Mitycam. Merged image (with enlarged region) shows how Z- and M-line Mitycam signals are obtained by separating signals spatially. (B) Direct lateral application of 2 µM Ca2+ internal solution to permeabilized myocyte (pretreated with 5 µM thapsigargin and 40 µM cytochalasin D) and line scan image (right).
Figure 5
Figure 5. [Ca2+]mito transients at Z-line and M-line
Averaged [Ca2+]mito transients (A), diastolic and amplitude (B), time to peak (C) and decay time constant (τ; D). (E) Isochronal dependence of [Ca2+]mito on distance from Z-line at 50 ms before peak in the mean Z-line signal (horizontal line and large symbol indicate mean values for 0–0.5 and 0.5–1 µm as in A). (n = 6, *P < 0.05).
Figure 6
Figure 6. Sarcomeric expression of MCU
(A) MCU immunofluorescence, MitoTracker Red fluorescence, merged image and plot profiles (normalized to Z-line minimum). (B) Scheme of SR Ca2+ release and mitochondrial Ca2+ uptake.
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