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

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.

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. 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. 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. 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. [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. 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.