Synchronization of Intracellular Ca2+ Release in Multicellular Cardiac Preparations

Figures

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  FIGURE 1. Di-4-ANEPPS loaded trabeculae show the myocyte borders in a rat trabecula in field-scan mode on a confocal microscope. The red line is drawn to illustrate where a typical line-scan would be conducted in experiments. Given the spatial and temporal resolution used, the line-scan typically covered five to seven adjacent myocytes.

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  FIGURE 2. Representative force tracings of a trabecula paced at 1 Hz, with the stimulation indicated by the arrows, applicable to all panels. (A) The control trabecula shows no ESCs following relaxation. (B) ESCs are observed with the addition of 0.5 mM caffeine and 100 nM isoproterenol. (C) Same trabecula after 5 min: the incidence and amplitude of ESCs increase, reaching a maximum of three contractions per cycle.

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  FIGURE 3. Distribution of the ESCs amplitude as a percentage of the amplitude of the developed force in stimulated contraction. The ESCs were divided into three separate groups according to their temporal order. SC, stimulated contraction.

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  FIGURE 4. The ESCs are not randomly spread throughout diastole, as they displayed a high probability of occurrence around three specific time points. (A–C) Show the temporal distribution of the first, second, and third ESC, occurring at 310 ± 4 ms, 578 ± 10 ms, and 702 ± 10 ms, respectively. The plots are overlaid in D. SC, stimulated contraction.

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  FIGURE 5. (Left) Confocal line-scan images of a cardiac trabecula loaded with the Ca2+ dye Rhod-2 and paced at 1 Hz (stimulation indicated by the arrows). The black dotted lines represent myocyte borders, and they were manually drawn based on the deflection of the Ca2+ waves. The electrical stimulation triggers SR Ca2+ release simultaneously across each of the myocytes in the field of view (control). Following the addition of both 0.5 mM caffeine and 100 nM isoproterenol, the trabecula shows Ca2+ waves that occur between stimulated Ca2+ release. The Ca2+ waves occur only in four out of six cells. However, after 5 min, the Ca2+ waves synchronize as the Ca2+ release occurs simultaneously across all six myocytes. (Right) Confocal line-scan images with the corresponding Ca2+ fluorescence plot profiles for each of the six myocytes in the field of view. Top: the Ca2+ release is not simultaneous across the myocytes, as the fluorescence peaks occur at different time points and not in all cells. Lower: the fluorescence peaks at the same time in each one of the six cells in the field of view, showing synchronization.

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  FIGURE 6. (A) Typical example of time of onset of largest ESC at 1 and 5 min after addition of isoproterenol and caffeine. Time of onset is highly variable in six adjacent myocytes after 1 min (spread of onset 100’s of ms), but becomes more synchronized at 5 min (spread of onset of ∼30 ms). (B) A non-externally stimulated action potential causes the spread of onset to virtually disappear, i.e., the spread of onset is 0–1 ms between adjacent cells (four in this example) in a triggered event.

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  FIGURE 7. Latency distribution of Ca2+ waves. (A,C) Latency distribution of the first Ca2+ wave at 0.2 and 0.5 mM caffeine, respectively. (B,D) Distribution of all waves at 0.2 and 0.5 mM caffeine, respectively.

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  FIGURE 8. Role of systolic Ca2+ release in the synchronization of Ca2+ waves in the multicellular trabeculae (0.5 mM caffeine): effects of several pulses on the timing of the Ca2+ waves. (A) Last three stimulated Ca2+ transients before the stimulation stopped. A synchronized Ca2+ release event occurred immediately following the last stimulated Ca2+ release; however, over time the amplitude and temporal homogeneity of Ca2+ waves decreased. (B) A synchronized SR Ca2+ release occurred long after the last stimulated Ca2+ release in an area of the muscle that was previously completely quiescent, strongly suggesting of triggered activity elsewhere on the preparation that propagated via AP to this quiescent area. SC, stimulated contraction.