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(Circulation Research. 2005;96:1266.)
© 2005 American Heart Association, Inc.

Cellular Biology

Spatial Nonuniformity of Excitation–Contraction Coupling Causes Arrhythmogenic Ca²⁺ Waves in Rat Cardiac Muscle

Yuji Wakayama, Masahito Miura, Bruno D. Stuyvers, Penelope A. Boyden, Henk E.D.J. ter Keurs

From the First Department of Internal Medicine (Y.W., M.M.), Tohoku University School of Medicine, Cardiovascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan; the Departments of Medicine, Physiology, and Biophysics (B.D.S., H.E.D.J.t.K.), Health Sciences Centre, University of Calgary, Canada; and the Department of Pharmacology (P.A.B.), Columbia University, New York.

Correspondence to Henk E.D.J. ter Keurs, Department of Medicine, University of Calgary, 3330 Hospital Dr NW, Calgary, Alberta T2N4N1 Canada. E-mail terkeurs{at}ucalgary.ca

	Abstract

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Ca²⁺ waves underlying triggered propagated contractions (TPCs) are initiated in damaged regions in cardiac muscle and cause arrhythmias. We studied Ca²⁺ waves underlying TPCs in rat cardiac trabeculae under experimental conditions that simulate the functional nonuniformity caused by local mechanical or ischemic local damage of myocardium. A mechanical discontinuity along the trabeculae was created by exposing the preparation to a small jet of solution with a composition that reduces excitation–contraction coupling (ECC) in myocytes within that segment. The jet solution contained either caffeine (5 mmol/L), 2,3-butanedione monoxime (BDM; 20 mmol/L), or low Ca²⁺ concentration ([Ca²⁺]; 0.2 mmol/L). Force was measured with a silicon strain gauge and sarcomere length with laser diffraction techniques in 15 trabeculae. Simultaneously, [Ca²⁺]_i was measured locally using epifluorescence of Fura-2. The jet of solution was applied perpendicularly to a small muscle region (200 to 300 µm) at constant flow. When the jet contained caffeine, BDM, or low [Ca²⁺], during the stimulated twitch, muscle-twitch force decreased and the sarcomeres in the exposed segment were stretched by shortening normal regions outside the jet. Typical protocols for TPC induction (7.5 s-2.5 Hz stimulus trains at 23°C; [Ca²⁺]_o=2.0 mmol/L) reproducibly generated Ca²⁺ waves that arose from the border between shortening and stretched regions. Such Ca²⁺ waves started during force-relaxation of the last stimulated twitch of the train and propagated (0.2 to 2.8 mm/sec) into segments both inside and outside of the jet. Arrhythmias, in the form of nondriven rhythmic activity, were induced when the amplitude of the Ca²⁺-wave was increased by raising [Ca²⁺]_o. Arrhythmias disappeared rapidly when uniformity of ECC throughout the muscle was restored by turning the jet off. These results show, for the first time, that nonuniform ECC can cause Ca²⁺ waves underlying TPCs and suggest that Ca²⁺ dissociated from myofilaments plays an important role in the initiation of Ca²⁺ waves.

Key Words: rat trabeculae • nonuniformity • troponin C • Ca²⁺ waves • arrhythmias

	Introduction

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Ischemic and failing hearts are both prone to ventricular arrhythmias and commonly show regional differences in contractile strength caused by heterogeneous impairment of excitation–contraction coupling (ECC). It is generally accepted that lethal arrhythmias are frequently associated with alterations of the excitation step of ECC.¹ It is less well known what role nonuniform ECC² plays in initiating arrhythmias.^3,4

We have previously investigated the triggered propagated contractions (TPCs) phenomenon in rat cardiac trabeculae. TPCs probably result from local damage and the ensuing nonuniform ECC.⁴ TPCs consist of local sarcomere shortening^5–7 associated with a [Ca²⁺]_i transient that propagates in a wave-like manner along the muscle.^8–10 Ca²⁺ waves underlying TPCs cause delayed after-depolarizations (DADs) and triggered arrhythmias.^6,7,10 In the model of damaged muscle, TPCs and underlying Ca²⁺ waves started invariably in regions located near the dissected end of the muscle or near cut branches.^4,5 The regions bordering the damaged areas exhibit elevated cytosolic and sarcoplasmic reticulum (SR)-Ca²⁺ and constitute a source of nonuniformity in ECC.⁶ However, a detailed study of the role of these regions in the initiation of arrhythmogenic Ca²⁺ waves is hampered by the difficulty in controlling the extent and severity of damage, and as such neither sarcomere length (SL) nor [Ca²⁺]_i can be measured reliably.

Here, we developed a novel model of controlled nonuniformity in rat trabeculae. Using this model, we show that controlled initiation of Ca²⁺ waves underlying TPCs can trigger nondriven regular spontaneous contractions in cardiac muscle. The initiation of arrhythmogenic Ca²⁺ waves can be explained by nonuniform ECC and Ca²⁺-dissociation from the contractile filaments occurring during relaxation of nonuniform cardiac muscle.

	Materials and Methods

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Measurements of Force, SL, and [Ca²⁺]_i in Rat Trabeculae
Trabeculae (n=15; length: 2.30±0.09 mm, width: 262±25 µm, thickness: 103±4 µm in slack conditions) were dissected from the right ventricle of Lewis Brown Norway rats^5–14 (Charles River Canada, Saint Constant, QC, Canada) and mounted between a motor arm and force (F) transducer in a bath perfused by HEPES solution on an inverted microscope. SL was measured by laser diffraction techniques¹² (Figure 1A).

View larger version (58K): [in this window] [in a new window]   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.

Measurement of [Ca²⁺]_i has been described previously.^8–11 Briefly, Fura-2 salt was microinjected iontophoretically into the trabecula.¹¹ Excitation light of 340, 360, or 380 nm was used and fluorescence was collected using an image intensified CCD camera (IIC) at 30 frames/s to assess local [Ca²⁺]_i (Figure 1A). ¹¹ We calculated [Ca²⁺]_i in a region of interest along the trabeculae from the calibrated ratio of F_360//F₃₈₀ (see Miura et al for details⁹ and Figure Is, available online at http://circres.ahajournals.org).

Reduction of Local Contraction
To produce nonuniform ECC, a restricted region was exposed to a small jet of solution (0.06 mL/min) that had been directed perpendicularly to a small muscle segment (300 µm; Figure 1) using a syringe pump connected to a glass pipette (100 µm diameter) (Figure 1A and 1B; see also supplemental Movie 1 in the online data supplement). The jet was positioned with respect to the muscle using a neutral colorant (Figure 1B) or fluorescein (<0.01 mg/mL). To reduce contraction in the exposed region by modified ECC, the jet solution was composed of standard HEPES solution containing either: (1) Caffeine (CF; 5 mmol/L); (2) 2,3-butanedione monoxime (BDM; 20 mmol/L); or (3) low [Ca²⁺] (low [Ca²⁺]_jet. The Ca²⁺ concentration in the jet ([Ca²⁺]_jet) was usually identical to the bath solution ([Ca²⁺]_o), except for low [Ca²⁺]_jet solution or unless mentioned otherwise.

Induction of Ca²⁺ Waves
During exposure to the jet, Ca²⁺ waves underlying TPCs were induced by stimulation of the muscle at 2.5 Hz for 7.5 s every 15 s at [Ca²⁺]_o of 2 mmol/L (caffeine, BDM) or 2.7±0.2 mmol/L (low [Ca²⁺]_jet) at 23.7±0.2°C.^4,7,10,13,14 Measurement of [Ca²⁺]_i commenced when the amplitude of stimulated twitches, TPCs, and underlying Ca²⁺ waves were constant (within 10 minutes).

Data Analysis
Data were expressed as mean±SEM. Statistical analysis was performed using ANOVA followed by a Post-hoc test. Differences were considered significant when P<0.05 (see online data supplement).

	Results

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Nonuniformity and Sarcomere Mechanics
The jet reached one short muscle segment (300 µm) (Figure 1A and 1B). Figure 1C shows that the fluid flow from the pipette using a solution with composition similar to the bath solution (HEPES) had no effect on F or SL by itself.

When a jet containing either BDM (Figures 1C and 2A), caffeine (Figure 2A), or low [Ca²⁺] (Figure 2A) was applied to the stimulated trabeculae, sarcomere stretch rapidly replaced the normal active shortening in the exposed segment (Figure 2B), whereas peak force (F/F_max) decreased (–11±3% with low [Ca²⁺]_jet (n=5), –28±5% with caffeine (n=6), and –36±7% with BDM (n=5) (Figure 2C; Table). Low [Ca²⁺]_jet solution did not affect resting SL (SL_o), whereas caffeine and BDM slightly decreased (–2.2±0.7%) and increased (+3.1±0.6%) SL_o, respectively. All effects were rapidly reversible (Figure 1C). Sarcomere dynamics along muscles exposed to a jet revealed 3 distinct regions during the twitch (Figure 2B): (1) a region located >200 µm from the jet where sarcomeres exhibited typical shortening (see [1] in Figure 2B); (2) the segment exposed to the jet where sarcomeres were stretched (see [2] in Figure 2B); (3) in a region between [1] and [2] sarcomeres shortened early during the twitch and then were stretched although less than in segment [2]; we denoted this region [3] the Border Zone (BZ). The BZ extended 1 to 2 cell lengths (100 to 200 µm) beyond the jet-exposed region (Figure 2B). The diffraction pattern of sarcomeres in BZ illuminated by a 150 µm diameter laser beam showed a clear single peak during both shortening and lengthening; similar changes in regional sarcomere dynamics were observed in caffeine and low [Ca²⁺]_jet experiments (data not shown).

View larger version (30K): [in this window] [in a new window]   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.

View this table: [in this window] [in a new window]   Table 1. Characteristics of Initiation and Propagation of Ca2+ Waves

Nonuniformity and [Ca²⁺]_i Transients
In contrast to the similarity of the effects of the various jet solutions on sarcomere dynamics, jets of caffeine, BDM, or low [Ca²⁺]_o solution had distinct effects on [Ca²⁺]_i (Figure 3A, 3B, and 3C respectively). Robust electrically driven [Ca²⁺]_i-transients occurred in regions ([1]) outside the jet independent of the composition of the jet. The caffeine-jet decreased the peak of the stimulated [Ca²⁺]_i-transient (C_T) and increased diastolic [Ca²⁺]_i ([Ca²⁺]_diast or C_D) in the jet-region (Figure 3A), whereas BDM decreased the [Ca²⁺]_i-transient only slightly (Figure 3B). Low [Ca²⁺]_jet decreased both [Ca²⁺]_i-transient and [Ca²⁺]_diast (Figure 3C). Average [Ca²⁺]_i-transients decreased by –36±6, –17±3, and –37±7%, and [Ca²⁺]_diast changed by +82±28, +48±13, –44±14% respectively in segments exposed to caffeine (n=9), BDM (n=9), and low [Ca²⁺]_jet (n=9), compared with [Ca²⁺]_i-transients and [Ca²⁺]_diast outside the jet (see Table and online data supplement). The [Ca²⁺]_i– changes were smaller in BZ, consistent with a gradient between regions caused by diffusion of the contents of the jet. Ca²⁺ waves (caffeine: n=9; BDM: n=9; and low [Ca²⁺]_jet: n=9 [15 muscles]) started systematically in the BZ after the decline of the last stimulated Ca²⁺ transient. These waves propagated into the regions outside and, in the cases of BDM and low [Ca²⁺]_jet–, inside the jet exposed region (Figure 3; supplemental Movie 2 in the online data supplement). Figure 3B (BDM jet) clearly shows 2 initiation sites of Ca²⁺ waves in the BZ and symmetric propagation into regions outside and inside the jet.

View larger version (65K): [in this window] [in a new window]   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, 000703BDM2(1)1, C: 010516T2LC1.

Lowering [Ca²⁺] to 0.2 mmol/L in the jet also triggered Ca²⁺ waves if [Ca²⁺]_o in the main solution was slightly increased (from 2 to 2.5 mmol/L). These waves started in the BZ and propagated inside and outside the jet exposed region at different velocities, with waves in the jet region being the slowest (Figure 3C).

Initiation of Ca²⁺ Waves
Figure 4 shows initiation of Ca²⁺ waves in the BZ of a BDM exposed trabecula. All muscles responded reproducibly to increasing [Ca²⁺]_o; at [Ca²⁺]_o=1 mmol/L (Figure 4A), only a localized transient in [Ca²⁺]_i (300 nmol/L), denoted as initial Ca²⁺ surge (see arrow), occurred along 100 to 150 µm of the BZs without apparent propagation of Ca²⁺ waves. The initiating Ca²⁺ surge took place 325 ms after stimulation; ie, late during twitch relaxation, when F had declined by 70% to 80% (Figure 4B). Increasing [Ca²⁺]_o (Figure 4B and 4C) accelerated, increased the Ca²⁺ surge (Figure 4A), and induced bidirectional propagating Ca²⁺ waves. The initial Ca²⁺ surge always occurred in the BZs late during relaxation of the last stimulated Ca²⁺ transient (see arrows). Increasing [Ca²⁺]_o also further accelerated propagation of the Ca²⁺ waves (Figure 4).

View larger version (67K): [in this window] [in a new window]   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, 000703BDM2(1)1, 0703BDM4-1, and 010309T2bm5-1a.

Similar observations were made in low [Ca²⁺]_jet-exposed muscles. In caffeine-exposed muscles, Ca²⁺ waves did not propagate into the jet region (Figure 3A). This precluded determination of the site of origin of Ca²⁺ waves, but the earliest Ca²⁺ surge was again observed in the BZ. These observations suggest strongly that the initial surge in [Ca²⁺]_i in the BZ initiated Ca²⁺ waves.

Ca²⁺ waves induced by exposure to either BDM, caffeine, or low [Ca²⁺]_jet started late during relaxation, ie, 40±5 ms after the maximal rate of sarcomere shortening in the stretched segment and 35±15 ms after twitch force had decline below 30% of peak force (F₃₀ in Figure 4C; Table). The delay between peak of the last stimulated Ca²⁺ transient and the start of the propagating Ca²⁺ transient in BZ decreased inversely with the amplitude of the initial Ca²⁺-surge (C_W/C_T; r=0.61, P<0.001; see supplemental Figure IIs) in all jet exposures.

Propagation of Ca²⁺ Waves
Propagation velocity of the Ca²⁺ waves (V_prop) (Figure 5), outside (n=27) and inside (n=13) the jet region, ranged from 0.2 to 2.8 mm/s, ie, comparable to Ca²⁺ waves observed in our damaged muscle studies (0.34 to 5.47 mm/s).^8–10 V_prop correlated with the [Ca²⁺]_i increase seen in the BZ during the initial Ca²⁺ surge (C_W) (r=0.66, P<0.0001; n=40; Figure 5C). Furthermore, V_prop correlated with the amplitude of the waves both inside and outside the jet⁸ (data not shown). These correlations were strongest in BDM experiments (r=0.86, P<0.0001; n=16; Figure 5C).

View larger version (44K): [in this window] [in a new window]   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.

Propagation into the normal region occurred often with a gradual decline in amplitude. Only fast waves propagated (6/27 waves; 1.56±0.27 mm/s) into the normal region outside the jet with a negligible decrease in amplitude. Small waves propagated at lower V_prop and with decrement (14/27 waves V_prop=0.81±0.13 mm/s) but completely through the region of observation (450 µm). The slowest waves stopped after 200 to 300 µm (7/21 waves; V_prop=0.47±0.11 mm/s).

Frequently, waves propagating inside the jet region collided with the wave arriving from the opposite BZ and then terminated (5/7 waves; see white arrow in Figure 4B). All waves moving inside low [Ca²⁺]_jet propagated slowly over 100 to 200 µm with a steep decline in amplitude (see b and c, in Figure 5A and 5B), sometimes gradually slowing down before terminating. This contrasted with waves observed in the region outside the low [Ca²⁺]_jet; such waves propagated rapidly with little decline in amplitude (see a in Figure 5A and 5B).

Nonuniformity and Arrhythmias
Nonuniformity of ECC created by the jet induced nondriven rhythmic activity. The arrhythmia consisted of spontaneous twitches at regular intervals starting after an after-contraction that followed the last stimulated contraction. The arrhythmia continued until the next stimulus train (7.5 s; Figure 6 [muscle exposed to BDM]). The intervals between nondriven contractions were usually slightly longer than those of the preceding stimulus train. As shown in Figure 6, these arrhythmias terminated abruptly when the jet was turned off and the uniformity of ECC restored.

View larger version (33K): [in this window] [in a new window]   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.

	Discussion

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The Model of Nonuniform ECC
We have used in this study a novel model of nonuniform ECC in cardiac muscle to study arrhythmogenic Ca²⁺ waves underlying TPCs in cardiac muscle. We created nonuniformity in ECC by exposing a small segment of the muscle to caffeine, BDM, or low [Ca²⁺]_o. We expected that (1) low [Ca²⁺]_jet would reduce Ca²⁺ current and the Ca²⁺ transient attributable to ECC¹⁵ despite increased SR-Ca²⁺ content,¹⁶ (2) caffeine would open SR-Ca²⁺ release channels and thereby deplete the SR,^15,17,18 and (3) BDM would modestly affect Ca²⁺ transients attributable to ECC^19,20 because of a reduced SR-Ca²⁺ content^21,22 and potentiation of RyR²³ and inhibited cross-bridge cycling.²⁰ Consistent with these expectations, the amplitude of stimulated Ca²⁺ transients decreased dramatically in regions exposed to caffeine and low [Ca²⁺]_jet but only slightly with BDM (Figure 3).

Each of these perturbations reduced muscle force because of creation of a muscle segment which developed less twitch force than the normal cells remote from the jet, as is witnessed by stretch of the weakened sarcomeres in the jet by the fully activated sarcomeres outside the exposed region (Figure 2C). These regions were connected mechanically by a border zone of 1 to 2 cells, where the sarcomeres first contracted and, then, were stretched (Figure 2B, region [3]. The diffraction pattern of sarcomeres in the BZ illuminated by an 150 µm diameter laser beam (ie, 1.5 cell lengths) showed a clear single peak during both shortening and lengthening, strongly suggesting that sarcomere contraction in BZ was also partially suppressed, probably owing to diffusion of the contents of each jet solution.

These observations confirm that this method causes nonuniform ECC along the muscle and affects specifically a selected region of the trabeculae, which results in regional decrease of contractile force of the sarcomeres.

Nonuniform ECC and Initiation of Ca²⁺ Waves
Ca²⁺ waves and TPCs have been closely related to Ca²⁺ overload in damaged regions and the resultant nonuniformity of muscle contraction.⁴ However, in that model it is difficult to investigate underlying mechanisms because damage is difficult to control. This study shows clearly that Ca²⁺ waves are reversibly initiated in regions without damage and, more specifically, from the BZ, in which contraction is partially suppressed. The common effect of the 3 protocols was to suppress contraction and reduce sarcomere force (Figure 2); the latter occurred with (caffeine and low [Ca²⁺]_jet) or without change of the Ca²⁺ transient (BDM), and with (caffeine) or without change of diastolic [Ca²⁺]_i (BDM).

The observation that the effect of force was common to all 3 interventions whereas the effect on [Ca²⁺]_i and on the Ca²⁺ transient was dramatically different between the interventions makes it reasonable to assume that Ca²⁺ waves are initiated as a result of nonuniformity of sarcomere force generation and the resultant sequence of stretch and release of sarcomeres in the BZ by contraction of normal cells in regions remote from the jet.

By varying [Ca²⁺]_o, we detected a small localized [Ca²⁺]_i surge in the BZ, which developed into a propagating wave when [Ca²⁺]_o was increased (Figure 4A). Once initiated, these Ca²⁺ waves traveled from the region with the localized [Ca²⁺]_i rise proving that this region constitutes the initiation site for Ca²⁺ waves.

The initiating Ca²⁺-surge took place late during twitch relaxation when both F and free Ca²⁺ in the cytosol had decayed by 70% to 80% (Figure 4B). By this time the SR-Ca²⁺ channels have partially recovered²⁴ and are able to support Ca²⁺-induced Ca²⁺ release (CICR) and Ca²⁺ wave generation.¹⁵ However, the delay between the stimulus-moment and Ca²⁺-surge makes it highly unlikely that Ca²⁺ entry via L-type Ca²⁺-channels causes CICR from the SR and the initial Ca²⁺ surge. Furthermore, Ca²⁺ waves never started in jet-exposed regions where sarcomeres were maximally stretched even if the amplitude of the stimulated Ca²⁺ transients witnessed a robust SR-Ca²⁺ content.¹⁵ Ca²⁺ waves never started simultaneously with the peak of stretch (Figure 4C) making it unlikely that a stretch-related mechanism such as activation of Gd³⁺-sensitive stretch-activated channels^10–14 is involved in the initial Ca²⁺-surge.

It has been shown that caffeine²⁵ and BDM increase the open probability of SR-Ca²⁺ release channels (RyR),^21,23 whereas low [Ca²⁺]_jet could theoretically do so by increasing the SR-Ca²⁺ load.¹⁶ However, several observations make it unlikely that potentiation of RyR caused spontaneous Ca²⁺ release in the BZ: (1) the same interventions cause no spontaneous Ca²⁺ release in uniform muscle²⁶ or myocytes²¹; (2) the effect of these interventions must have been maximal in the jet exposed region, whereas Ca²⁺ waves never started in this region.

A Novel Mechanism Underlying Arrhythmias
We suggest that Ca²⁺ that is bound to Troponin C (TnC) during this phase of twitch underlies the Ca²⁺-surge that initiates Ca²⁺ waves.²⁷ It is well known that a quick release of Ca²⁺-activated cardiac muscle induces a surge of Ca²⁺ ions dissociating from myofilaments^11,28 because of rapid reduction of the TnC affinity for Ca²⁺ owing to a reduction in the number of Ca²⁺-activated cross-bridges.²⁹ The concept of quick-release–induced Ca²⁺ dissociation from TnC, demonstrated in uniform cardiac muscle, is applicable to the chain of cells in the nonuniform muscle exposed to the jet. Rapid sarcomere shortening during the force decline occurred both in the jet region and in the BZ, but led only to a Ca²⁺ surge and Ca²⁺ wave initiation in the BZ (Figure 4B), making it probable that quick-release–induced Ca²⁺ dissociation from TnC caused by the decline of force in the shortening BZ sarcomeres led to the local Ca²⁺ surge.^11,28–32 The region inside the jet, where ECC was all but abolished, probably contained either little TnC-Ca²⁺ (caffeine or low [Ca²⁺]_jet) or only few Ca²⁺activated force generating cross-bridges (BDM), which would render a quick release of this region unable to generate a Ca²⁺ surge and Ca²⁺ wave. The BZ, on the other hand, could generate a Ca²⁺ surge that is large enough to induce local CICR and thus a Ca²⁺ wave even if only a fraction of TnC^33,34 were occupied with Ca²⁺.⁴

A quantitative analysis of the relation between quick release dynamics and onset of the Ca²⁺ surge or waves may shed further light on this mechanism of initiation of Ca²⁺ waves. Our present study allows for an estimate of the latency (40 ms) between rapid sarcomere shortening and the initial rise of [Ca²⁺]_i during the Ca²⁺ surge in a small BZ region 100 µm (Figure 4A), although the precision of this estimate is limited by the temporal resolution of the IIC camera (33 ms/frame). Furthermore, focusing the laser beam to 100 to 200 µm for SL measurements reduced signal to noise ratio thereby precluding more accurate measurement of the time of quick release of BZ sarcomeres. Force derived parameters of twitch relaxation (–dF/dt_max and F₃₀; see Figure 4B and 4C) also preceded the onset of Ca²⁺ waves in the BZ by minimally 35 ms (Table). This measure probably still overestimated the true latency because the onset of the Ca²⁺ surge was measured from the nadir between the last stimulated Ca²⁺ transient and the initial [Ca²⁺]_i rise in the BZ (Figure 4 and 5). Nevertheless, both estimates allow a minimum delay of 30 to 40 ms between Ca²⁺ dissociation from TnC and SR-Ca²⁺ release. Despite methodological limitations, the inverse relationship between amplitude of the initiating Ca²⁺ transient (C_W) and latency of the Ca²⁺ transient (t[C_T–C_W]) (see Table and the online data supplement) is consistent with the hypothesis that magnitude and rate of SR-Ca²⁺ release depends on the amount of triggering Ca²⁺ released from TnC and responsiveness of the SR-Ca²⁺ release channels.⁸

Propagation of Ca²⁺ Waves
Ca²⁺ waves propagated in this model at slightly lower velocity (0.2 to 2.8 mm/s) than those of previous studies of regionally damaged muscles.^9,10,35 In this study the amplification of the Ca²⁺ signal by SR-Ca²⁺ release required for propagation may have been lower in the absence of Ca²⁺ loading of the muscle by damaged areas.^{4–6,8,13,34,36–38} A lower cellular Ca²⁺ load would explain why Ca²⁺ waves were both smaller and V_prop lower and propagated with a gradual decline of their amplitude and disappeared after a few hundred µm. Differences of propagation pattern of Ca²⁺ waves in caffeine, BDM, or low [Ca²⁺]_jet (Figure 3) are consistent with the assumption that regional SR function and SR Ca²⁺ content determine V_prop.⁸ Ca²⁺ waves did propagate into regions with normal SR-Ca²⁺ release such as inside the BDM jet (Figure 3B) and propagated slowly inside the low [Ca²⁺] jet where [Ca²⁺]_i is reduced (Figure 3C).⁸ Ca²⁺ waves did not propagate through regions exposed to caffeine (Figure 3A), which is consistent with the effect of caffeine to deplete SR-Ca²⁺ required for wave propagation.⁸

Implication: Nonuniform ECC, Ca²⁺ Waves, and Arrhythmias
One striking finding of this study is the arrhythmogenic nature of mechanically nonuniform myocardium independent of any damage, which is clearly caused by the induction of Ca²⁺ waves (Figure 6). Diastolic [Ca²⁺]_i transients are known to cause transient depolarizations caused by electrogenic Na⁺–Ca²⁺ exchange and Ca²⁺-sensitive inward currents^7,10,39–41 and alter action potential configuration.³² Several compartments which normally only release Ca²⁺ during the cardiac cycle in response to the action potential can release Ca²⁺ spontaneously during diastole. Such compartments include the SR in which abnormal Ca²⁺ storage may be arrhythmogenic.^42–44 In addition, increased open probability of the SR-Ca²⁺ release channels⁴⁵ owing to channel gene mutation^40,42,46 or to posttranslational channel changes in heart failure may cause arrhythmias.⁴³

In this study of controlled nonuniformity of muscle contraction, we identify nonuniform ECC in cardiac muscle for the first time as a possible arrhythmogenic mechanism. Whether this mechanism plays a role in the wall of the ventricles remains to be proven, although the arrangement of the cardiac wall in muscle fascicles, which transmit force longitudinally and therefore are subject to comparable constraints as trabeculae in this study, makes this possibility highly likely. This mechanism may contribute to arrhythmogenesis in diseased heart where nonuniform segmental wall motion^2,46 may result from ischemia, nonuniform electrical activation, or nonuniform adrenergic activation.⁴⁶

	Acknowledgments

 
This work was supported by grants from Alberta Heart and Stroke Foundation and National Institutes of Health (HL 58860). H.E.D.J. ter Keurs is a Medical Scientist of the Alberta Heritage Foundation for Medical Research (AHFMR).

	Footnotes

 
Original received December 27, 2004; revision received May 9, 2005; accepted May 24, 2005.

	References

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