Local calcium gradients during excitation-contraction coupling and alternans in atrial myocytes

Abstract

Subcellular Ca(2+) signalling during normal excitation-contraction (E-C) coupling and during Ca(2+) alternans was studied in atrial myocytes using fast confocal microscopy and measurement of Ca(2+) currents (I(Ca)). Ca(2+) alternans, a beat-to-beat alternation in the amplitude of the [Ca(2+)](i) transient, causes electromechanical alternans, which has been implicated in the generation of cardiac fibrillation and sudden cardiac death. Cat atrial myocytes lack transverse tubules and contain sarcoplasmic reticulum (SR) of the junctional (j-SR) and non-junctional (nj-SR) types, both of which have ryanodine-receptor calcium release channels. During E-C coupling, Ca(2+) entering through voltage-gated membrane Ca(2+) channels (I(Ca)) triggers Ca(2+) release at discrete peripheral j-SR release sites. The discrete Ca(2+) spark-like increases of [Ca(2+)](i) then fuse into a peripheral 'ring' of elevated [Ca(2+)](i), followed by propagation (via calcium-induced Ca(2+) release, CICR) to the cell centre, resulting in contraction. Interrupting I(Ca) instantaneously terminates j-SR Ca(2+) release, whereas nj-SR Ca(2+) release continues. Increasing the stimulation frequency or inhibition of glycolysis elicits Ca(2+) alternans. The spatiotemporal [Ca(2+)](i) pattern during alternans shows marked subcellular heterogeneities including longitudinal and transverse gradients of [Ca(2+)](i) and neighbouring subcellular regions alternating out of phase. Moreover, focal inhibition of glycolysis causes spatially restricted Ca(2+) alternans, further emphasising the local character of this phenomenon. When two adjacent regions within a myocyte alternate out of phase, delayed propagating Ca(2+) waves develop at their border. In conclusion, the results demonstrate that (1) during normal E-C coupling the atrial [Ca(2+)](i) transient is the result of the spatiotemporal summation of Ca(2+) release from individual release sites of the peripheral j-SR and the central nj-SR, activated in a centripetal fashion by CICR via I(Ca) and Ca(2+) release from j-SR, respectively, (2) Ca(2+) alternans is caused by subcellular alterations of SR Ca(2+) release mediated, at least in part, by local inhibition of energy metabolism, and (3) the generation of arrhythmogenic Ca(2+) waves resulting from heterogeneities in subcellular Ca(2+) alternans may constitute a novel mechanism for the development of cardiac dysrhythmias.

Figure 1. Ca²⁺ signalling during excitation-contraction (E-C) coupling in cat atrial myocytes

A, confocal images of a ventricular (left) and an atrial (right) myocyte from the same cat heart stained with the membrane-bound fluorescent dye Di-8-ANEPPS. The regular structures spaced in a sarcomeric pattern in the ventricular cell represent t-tubules. In contrast, the atrial myocyte is devoid of any t-tubular staining. B, [Ca²⁺]_i transient recorded in the confocal line-scan mode (scanning frequency was 250 Hz). The scanned line was positioned perpendicular to the longitudinal axis of the cell (c). Electrical stimulation of the cell during acquisition of the line-scan image triggered a ‘U’-shaped [Ca²⁺]_i transient (b), indicating that [Ca²⁺]_i increased first at the periphery of the cell (a) before propagating towards the centre of the myocyte. Panel d shows local [Ca²⁺]_i transients (top) measured in the subsarcolemmal space (ss) and the centre of the cell (ct) as well as averaged over the entire width of the cell (bottom). The arrow indicates the two components of the whole-cell [Ca²⁺]_i transient (see text). C, spatiotemporal pattern of an action potential (AP)-induced atrial [Ca²⁺]_i transient visualized by two-dimensional confocal microscopy using a Nipkow tandem-disk confocal unit in conjunction with a microchannel plate-intensified CCD camera. The images represent cellular fluo-4 fluorescence recorded at a temporal resolution of 60 Hz. [Ca²⁺]_i transients were elicited by extracellular electrical field stimulation. ‘0 ms’ refers to the image immediately preceding the first changes in fluo-4 fluorescence. [Ca²⁺]_i signals in B and C were recorded from different atrial myocytes. (A and B are modified from Hüser et al. 1996.)

Figure 2. Electromechanical and Ca²⁺ alternans in atrial myocytes

A, simultaneous recordings of APs (current-clamp method) and cell shortening (video edge detection) from a single cat atrial myocyte. Electrical stimulation at frequencies of 0.5-1.5 Hz evoked discordant electrical (top trace) and mechanical (bottom trace) alternans. To the right, two APs recorded during successive small- (open circle) and large-amplitude (filled circle) shortenings are superimposed to illustrate the differences in duration and kinetics. B, spatiotemporal characteristics of [Ca²⁺]_i transients during alternans in an atrial myocyte. Alternans (a) in atrial cells is characterized by marked variations in the spatial profile of the [Ca²⁺]_i transients (b). Local [Ca²⁺]_i profiles (panel c), recorded during large- and small-amplitude [Ca²⁺]_i transients, revealed [Ca²⁺]_i gradients directed from subsarcolemmal (ss) regions towards central regions (ct) of the cell. The small-amplitude [Ca²⁺]_i transients were spatially restricted to the subsarcolemmal regions. (Modified from Hüser et al. 2000.)

Figure 3. Ca²⁺ currents (I_Ca) and sarcoplasmic reticulum (SR) content during alternans

A, simultaneous measurements of cell shortening (Δl, top) and I_Ca (bottom) in an atrial myocyte. Holding potential = −40 mV, test potential = 0 mV. Despite large differences in cell shortening during alternans, peak I_Ca remained constant. B, SR Ca²⁺ content during alternans. SR Ca²⁺ content was evaluated with 10 mm caffeine pulses in atrial myocytes. Caffeine was applied twice to the same cell. The amplitude of the caffeine-induced [Ca²⁺]_i transient did not reveal any significant differences after small- (left panel) and large-amplitude (right panel) [Ca²⁺]_i transients, indicating that SR Ca²⁺ content remained constant and did not alternate. Whole-cell [Ca²⁺]_i transients were measured with the calcium-sensitive dye indo-1. Cellular indo-1 fluorescence was measured at 405 nm (F₄₀₅) and 485 nm (F₄₈₅). Changes in [Ca²⁺]_i are expressed as changes in the ratio F₄₀₅/F₄₈₅. (Modified from Hüser et al. 2000.)

Figure 4. [Ca²⁺]_i transient alternans induced by inhibition of glycolysis

Inhibition of glycolysis by exposure to 10 mm pyruvate (A) and 1 mm iodoacetic acid (B) caused [Ca²⁺]_i transient alternans in an atrial myocyte. Alternans were reversible upon washout of the inhibitors of glycolysis. [Ca²⁺]_i was measured with the calcium-sensitive dye indo-1. (Modified from Hüser et al. 2000.)

Figure 5. Subcellular [Ca²⁺]_i alternans

[Ca²⁺]_i signals recorded with the calcium-sensitive dye fluo-4 from a cat atrial myocyte with the aid of two-dimensional confocal microscopy. The images shown were recorded at intervals of 33 ms and represent surface plots of changes in [Ca²⁺]_i. The traces represent normalized subcellular [Ca²⁺]_i transients recorded from the regions of interest marked by the rectangles. The top and bottom region of the cell reveal Ca²⁺ alternans that is out of phase.