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. 2015;9(3):129-38.
doi: 10.1080/19336950.2015.1040966.

Cytosolic and nuclear calcium signaling in atrial myocytes: IP3-mediated calcium release and the role of mitochondria

Felix Hohendanner  1 , Joshua T Maxwell, Lothar A Blatter
Affiliations

Cytosolic and nuclear calcium signaling in atrial myocytes: IP3-mediated calcium release and the role of mitochondria

Felix Hohendanner et al. Channels (Austin). 2015.

Abstract

In rabbit atrial myocytes Ca signaling has unique features due to the lack of transverse (t) tubules, the spatial arrangement of mitochondria and the contribution of inositol-1,4,5-trisphosphate (IP3) receptor-induced Ca release (IICR). During excitation-contraction coupling action potential-induced elevation of cytosolic [Ca] originates in the cell periphery from Ca released from the junctional sarcoplasmic reticulum (j-SR) and then propagates by Ca-induced Ca release from non-junctional (nj-) SR toward the cell center. The subsarcolemmal region between j-SR and the first array of nj-SR Ca release sites is devoid of mitochondria which results in a rapid propagation of activation through this domain, whereas the subsequent propagation through the nj-SR network occurs at a velocity typical for a propagating Ca wave. Inhibition of mitochondrial Ca uptake with the Ca uniporter blocker Ru360 accelerates propagation and increases the amplitude of Ca transients (CaTs) originating from nj-SR. Elevation of cytosolic IP3 levels by rapid photolysis of caged IP3 has profound effects on the magnitude of subcellular CaTs with increased Ca release from nj-SR and enhanced CaTs in the nuclear compartment. IP3 uncaging restricted to the nucleus elicites 'mini'-Ca waves that remain confined to this compartment. Elementary IICR events (Ca puffs) preferentially originate in the nucleus in close physical association with membrane structures of the nuclear envelope and the nucleoplasmic reticulum. The data suggest that in atrial myocytes the nucleus is an autonomous Ca signaling domain where Ca dynamics are primarily governed by IICR.

Keywords: 2-APB, 2-aminoethoxydiphenyl borate; AP, action potential; CICR, Ca-induced Ca release; CRU, Ca release units; CT, central; CaT, Ca transient; ECC, excitation-contraction coupling; IICR; IICR, IP3R-induced Ca release; IP3; IP3R, Inositol-1,4,5-trisphosphate receptor; LCC, L-type Ca channels; MCU, mitochondrial Ca uniporter; NE, nuclear envelope; NFAT, nuclear factor of activated T cells; NPR, nucleoplasmic reticulum; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; SS, subsarcolemmal; TF50, time to half-maximal amplitude; TZ, transition zone.; [Ca]i, cytosolic Ca concentration; [Ca]mito, mitochondrial Ca concentration; atria; excitation-contraction coupling; j-SR, junctional SR; mitochondria; nj-SR, non-junctional SR; nuclear calcium; t-tubule, transverse tubule.

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Figures

Figure 1.
Figure 1.
AP-induced CaTs and cell shortening in intact ventricular, atrial and detubulated ventricular cells. (A, a) membrane and t-tubule staining with WGA-Alexa 594 in a rabbit ventricular myocyte. (b) line scan image of an AP-induced CaT. The confocal line scan image was recorded along the line shown in (a). (c) subcellular CaTs recorded from 1 µm wide susbsarcolemmal (gray; SS in panel b) and central (black; CT in panel b) regions. (d) cell shortening. Arrow heads indicate time of electrical field stimulation. (B, C) same type of data recorded in an atrial (B) and detubulated ventricular (C) myocyte. Detubulation occurred by exposure to 1.5 M formamide. (D, E) average twitch-induced cell shortening (D) and change in sarcomere length (E) in intact ventricular, atrial and detubulated ventricular myocytes. (F) analysis of subcellular synchrony of AP-induced Ca release. (a, b and c) graph of time elapsed between onset of electrical stimulation and the time the local CaT has reached half-maximal amplitude (TF50) along the transverse cell axis in an intact ventricular (a), atrial (b) and detubulated ventricular (c) myocyte. (d) average standard deviation of all local TF50 values (SDTF50) at single pixel resolution across the transverse cell axis for intact ventricular, atrial and detubulated ventricular myocytes. Square brackets indicate significant differences at P < 0.05, one-way ANOVA. Numbers in columns indicate number of cells.
Figure 2.
Figure 2.
Mitochondria and Ca propagation. (A) mitochondrial distribution in atrial (a) and ventricular (b) myocytes visualized with the mitochondria targeted fluorescent probe Mitycam. TZ, transition zone. (B) TF50 profile across an atrial myocyte. The dashed lines represent the slope of the TF50 curve in the j-SR/TZ (2—2) and the nj-SR (1—1) space. The slopes represent 1/velocity of propagation of Ca release from the cell periphery to the cell center, thus a shallow slope indicates fast propagation and vice versa. (C) average propagation velocities of AP-induced Ca release in atrial (TZ vs nj-SR), detubulated ventricular myocytes and spontaneous Ca waves in ventricular myocytes. Square brackets indicate significant differences at P < 0.05, one-way ANOVA.
Figure 3.
Figure 3.
Mitochondrial effects on CaTs. (A, a) effect of rapidly increasing [Ca]i from 0 to 5 μM on mitochondrial Ca uptake in permeabilized atrial cells under control conditions and in the presence of the MCU inhibitor Ru360 (10 µM). [Ca]mito was measured with the mitochondria-targeted fluorescent probe Mitycam. (b) Average time-to-peak of the [Ca]mito signal after elevation of extramitochondrial [Ca] to 5 µM in control and in the presence of Ru360. (B, a) TF50 profile along the transverse cell axis in control and in the presence of Ru360. (b) Average Ca propagation velocities in the TZ and central cell regions occupied by nj-SR in control, and in the presence of Ru360 and FCCP (5 µM), respectively. (C) line scan images (top) and subcellular (SS, CT) CaTs (bottom) induced by electrical field stimulation in control and in the presence of Ru360. Arrow heads indicate electrical stimulation. (D) average normalized SS and CT CaT amplitudes in the presence of Ru360. Dashed line (100%) indicates normalized SS and CT CaT amplitudes under control conditions. Numbers in columns indicate number of cells. Square brackets indicate significant differences at P < 0.05, one-way ANOVA. *P < 0.05, student´s t-test.
Figure 4.
Figure 4.
IP3-dependent effects on subcellular CaTs. (A) AP-induced subcellular CaTs under control conditions (top) and after photolysis of caged IP3 (bottom) recorded from 1 µm wide regions of interest. The traces represent subsarcolemmal (SS) cytosolic, central (CT) cytosolic and nuclear changes of [Ca]i. IP3 was uncaged by illumination of the entire cell with 405 nm laser light (2 ms). Arrow heads indicate electrical stimulation. (B) Summary data of averaged subcellular CaT amplitudes under control conditions and after IP3 photolysis (n = 4).
Figure 5.
Figure 5.
Differential effects of IP3 on cytosolic and nuclear [Ca]. From top to bottom: cytosolic CaT recorded from a cytosolic (C) region immediately adjacent to the nucleus (N), longitudinal line scan image with C and N regions of interest marked and time, duration and location where IP3 uncaging occurred (flash symbol), image of a ‘mini’-Ca wave that was restricted to the nuclear compartment at higher magnification and intensity setting, and nuclear CaT. Arrow heads indicate electrical stimulation. Nuclear ‘mini’-Ca waves are marked by stars.
Figure 6.
Figure 6.
Ca puff activity. (A) protocol to isolate IICR-mediated Ca puffs in atrial myocytes. Longitudinal line scan images from a permeabilized atrial myocyte under control (CTRL) conditions (a), after application of 4 mM tetracaine (b), tetracaine + IP3 (5 μM) (c), and tetracaine +IP3 + 2-APB (10 μM) (d). (B) Ca puff frequency in the presence of teracaine + IP3 in the cytosol vs. nuclear compartment. (C) average frequencies of nuclear elementary Ca release events under control conditions, and in the presence of tetracaine, tetracaine + IP3, and tetracaine + IP3 + 2-APB. (n = 15 cells). (D) 2-D (x-y, left) and line scan (x-t, right) confocal image of the nuclear and perinuclear regions of an atrial myocytes loaded with X-rhod-1/AM. The line in the x-y image marks the position of the scan line. The horizontal black lines in the x-t image indicate the nuclear envelope (NE) and membrane structures of the nucleoplasmic reticulum (NPR). (E) simultaneously recorded line scan images of X-rhod-1 (left) and fluo-4 (middle) fluorescence signals from a permeabilized atrial myocyte in the presence of tetracaine + IP3. Right: F/F0 image of a Ca puff originating from the nuclear envelope. F0 represents resting cellular Fluo-4 fluorescence measured at the beginning of the recording. (F) Average frequencies of nuclear Ca puffs measured close to (<1 µm) and distant (>1 µm) from nuclear membrane structures (n = 15 cells).
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