1. Spatial Nonuniformity of Excitation–Contraction Coupling Causes Arrhythmogenic Ca2+ Waves in Rat Cardiac Muscle
by Yuji Wakayama, Masahito Miura, Bruno D. Stuyvers, Penelope A. Boyden, and Henk E.D.J. ter Keurs
Circulation Research
Volume 96(12):
June 24, 2005
Copyright © American Heart Association, Inc. All rights reserved.

2. Figure 1. A, Set-up for measurements of Force, SL, and [Ca2+]i (Cai).
Figure 1. A, Set-up for measurements of Force, SL, and [Ca2+]i (Cai). Force is measured with a silicon strain gauge and SL by diffraction of a Helium-Neon (He-Ne) laser beam. A glass pipette (P) is placed perpendicularly to the trabecula (T) and a jet solution is applied to a short segment of the muscle; the solution is discarded together with the main solution. Xe indicates xenon arc lamp; IIC, image intensified charge-coupled device (CCD) camera. B, Photomicrograph showing the jet of solution projected from the pipette (P) (tip diameter=60 μm) to the trabecula (T); the solution was identical to the main solution and was made visible with a neutral colorant; the jet is dispersed slightly by the muscle (200 to 300 μm) and afterward does not come in contact with the muscle anymore; arrows indicate direction of jet (0.06 mL/min) and main stream solution (4 mL/min), respectively. C, Chart recordings of Force (F) and SL in the region affected by a jet containing a control solution (HEPES; [Ca2+]o=0.7 mmol/L, 0.5Hz stimulus; upper) or BDM (lower), whereas the jet is turned OFF, ON, and OFF.
Yuji Wakayama et al. Circ Res. 2005;96:
Copyright © American Heart Association, Inc. All rights reserved.

3. Figure 2. Effect of local jet exposure on F and SL
Figure 2. Effect of local jet exposure on F and SL. A, Typical F and SL tracings in the jet-exposed segment before (gray) and during (black) exposure to a jet of HEPES (control), low [Ca2+]jet (LC), caffeine (CF), and BDM solution.
Figure 2. Effect of local jet exposure on F and SL. A, Typical F and SL tracings in the jet-exposed segment before (gray) and during (black) exposure to a jet of HEPES (control), low [Ca2+]jet (LC), caffeine (CF), and BDM solution. F is normalized to the maximal force (Fmax). B, Spatial effects of local exposure to BDM on SL patterns; SL tracings recorded from 3 different segments along the muscle: [1] outside the jet, [2] inside the jet, [3] in a border zone (BZ) between [1] and [2] (BZ). The traces compare effects of BDM exposure on resting SL (▪) and SL during peak twitch (•) in [1], [2], and [3]. C, Summary of effects of HEPES (n=7), caffeine (CF; n=6), BDM (n=5), and low [Ca2+]jet (LC; n=5) on Force (F/Fmax), resting SL (SL0), and SL at peak-twitch (DSLpeak) in the absence and presence of the jet-flow. F/Fmax and SL in the segment that had been exposed to the jet for 5 minutes (ON) are compared with F and SL before exposure to the jet-flow (OFFpre; †P<0.05) and compared with F and SL both (*P<0.01) before and 5 minutes after cessation of the jet (OFFpost). [Ca2+]o=0.7 mmol/L, stimulation rate=0.5 Hz; 25°C.
Yuji Wakayama et al. Circ Res. 2005;96:
Copyright © American Heart Association, Inc. All rights reserved.

4. Figure 3. Three-Dimensional (left) and corresponding 2-Dimensional (right) spatio-temporal representations of [Ca2+]i during induction of TPCs whereas a small segment of the trabecula is exposed to caffeine (CF; A), BDM (B), or low [Ca2+]jet (LC; C) solutions.
Figure 3. Three-Dimensional (left) and corresponding 2-Dimensional (right) spatio-temporal representations of [Ca2+]i during induction of TPCs whereas a small segment of the trabecula is exposed to caffeine (CF; A), BDM (B), or low [Ca2+]jet (LC; C) solutions. Images show the 2 last stimulated Ca2+ transients (see arrowheads for moments of electrical stimulation) and Ca2+ events occurring subsequently inside (indicated by the dashed lines) and outside the jet-exposed segment. X-axis: time; Y-axis: position along the long axis of the trabecula (Figure Is in online supplement); Z-axis or color bar: [Ca2+]i. [Ca2+]jet and [Ca2+]o were 2.0 mmol/L except when low [Ca2+]jet ([Ca2+]jet=0.2 mmol/L) was used, [Ca2+]o was 2.5 mmol/L; bath temperature: 23.8 (CF), 23.1 (BDM), and 23.3°C (LC). A, Exp000809cf5-2; B, BDM2(1)1, C: T2LC1.
Yuji Wakayama et al. Circ Res. 2005;96:
Copyright © American Heart Association, Inc. All rights reserved.

5. Figure 4. A, Initiating events of Ca2+ waves induced by local BDM exposure at [Ca2+]o=1, 2, and 4 mmol/L.
Figure 4. A, Initiating events of Ca2+ waves induced by local BDM exposure at [Ca2+]o=1, 2, and 4 mmol/L. At low [Ca2+] in the bath (1 mmol/L, top) only a local Ca2+-surge (starting 360 ms) after stimulation is observed. Increasing [Ca2+]o (2 and 4 mmol/L; middle and bottom) led to the initiation of bi-directional Ca2+ waves, which propagate into the segment inside the jet and into the normal muscle. Both amplitude of the initial and propagating transient as well as propagation velocity increased with increase of [Ca2+]o, whereas the latency of onset of the Ca2+ transient decreased (300 ms). Arrows indicate initiation sites of propagating waves. B, Ca2+ waves in a region exposed to BDM with [Ca2+]o=2 mmol/L; white arrow in the upper figure indicates termination of 2 opposite waves after they collide. C, Comparison between [Ca2+]i, F and SL in the BZ detected in the BDM experiment of panel B; [Ca2+]i traces results from the average of profiles indicated by the square bracket along the top. The onset (see arrow) of initial [Ca2+]i rise (defined as the moment of the nadir between the last stimulated Ca2+ transient and [Ca2+]i rise) corresponded with the time at which the twitch had relaxed to 10% (Fonset). The times of peak force (Fpeak), −dF/dtmax, and F30 at which the sarcomere shortening rate is maximal are indicated. [Ca2+]jet=2.0 mmol/L; temperature was 23.1 (A), 23.0°C (B). Exp0703BDM1-1, BDM2(1)1, 0703BDM4-1, and T2bm5-1a.
Yuji Wakayama et al. Circ Res. 2005;96:
Copyright © American Heart Association, Inc. All rights reserved.

6. Figure 5. A, Low[Ca2+]jet-induced Ca2+ waves and corresponding traces of F and of regional [Ca2+]i (averaged from regions indicated by the brackets) inside (green) and outside (blue) the low [Ca2+] jet and in the BZ (pink).
Figure 5. A, Low[Ca2+]jet-induced Ca2+ waves and corresponding traces of F and of regional [Ca2+]i (averaged from regions indicated by the brackets) inside (green) and outside (blue) the low [Ca2+] jet and in the BZ (pink). Propagation velocities (Vprop) were calculated using a linear regression through peak values (yellow circle) of the Ca2+ wave (a: 2.84, b: 0.22, and c: 0.16 mm/s). The outward wave started (see arrow) at the moment of 21% twitch force. CT indicates peak of the last Ca2+-transient; CW, peak of the initial [Ca2+]i rise in the BZ; CD reflects diastolic [Ca2+]i and corresponds to the minimum [Ca2+]i between CT and CW; t(CT−CW) is the latency between CT and CW. B, Peak [Ca2+]i of the low [Ca2+]jet-induced Ca2+ waves (a, b, and c) were plotted as a function of position along the trabecula. [Ca2+]o=2.5 mmol/L; temperature, 23.3°C. Exp010516T2LC1.
Yuji Wakayama et al. Circ Res. 2005;96:
Copyright © American Heart Association, Inc. All rights reserved.

7. Figure 6. Nonuniform ECC causes arrhythmias.
Figure 6. Nonuniform ECC causes arrhythmias. A continuous chart recording of force showing that stimulus trains during local exposure to BDM (gray bars above the tracings) repeatedly induced arrhythmias. An expanded force tracing showing that spontaneous contractions were both preceded and followed by after-contractions induced by the stimulus train. OFF (arrow) indicates when the jet was turned off; S, stimulus trains (2.5Hz-7.5s) repeated every 15 s. [Ca2+]o=3.5 mmol/L; temperature 25.8°C. Exp000519ArBDM.
Yuji Wakayama et al. Circ Res. 2005;96:
Copyright © American Heart Association, Inc. All rights reserved.