Spatial Nonuniformity of ExcitationeCContraction Coupling Causes Arrhythmogenic Ca2+ Waves in Rat Cardiac Muscle
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循环研究杂志 2005年第6期
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
Biophysics (B.D.S., H.E.D.J.t.K.), Health Sciences Centre, University of Calgary, Canada
the Department of Pharmacology (P.A.B.), Columbia University, New York.
Abstract
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 excitationeCcontraction 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 e) 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
Ischemic and failing hearts are both prone to ventricular arrhythmias and commonly show regional differences in contractile strength caused by heterogeneous impairment of excitationeCcontraction 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 shortening5eC7 associated with a [Ca2+]i transient that propagates in a wave-like manner along the muscle.8eC10 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
Measurements of Force, SL, and [Ca2+]i in Rat Trabeculae
Trabeculae (n=15; length: 2.30±0.09 mm, width: 262±25 e, thickness: 103±4 e in slack conditions) were dissected from the right ventricle of Lewis Brown Norway rats5eC14 (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).
Measurement of [Ca2+]i has been described previously.8eC11 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 F360//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 e; Figure 1) using a syringe pump connected to a glass pipette (100 e 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
Nonuniformity and Sarcomere Mechanics
The jet reached one short muscle segment (300 e) (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/Fmax) decreased (eC11±3% with low [Ca2+]jet (n=5), eC28±5% with caffeine (n=6), and eC36±7% with BDM (n=5) (Figure 2C; Table). Low [Ca2+]jet solution did not affect resting SL (SLo), whereas caffeine and BDM slightly decreased (eC2.2±0.7%) and increased (+3.1±0.6%) SLo, 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 e 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 e) beyond the jet-exposed region (Figure 2B). The diffraction pattern of sarcomeres in BZ illuminated by a 150 e 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).
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 (CT) and increased diastolic [Ca2+]i ([Ca2+]diast or CD) 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 eC36±6, eC17±3, and eC37±7%, and [Ca2+]diast changed by +82±28, +48±13, eC44±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+]ieC 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+]jeteC, 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.
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 e 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).
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 (CW/CT; r=0.61, P<0.001; see supplemental Figure IIs) in all jet exposures.
Propagation of Ca2+ Waves
Propagation velocity of the Ca2+ waves (Vprop) (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).8eC10 Vprop correlated with the [Ca2+]i increase seen in the BZ during the initial Ca2+ surge (CW) (r=0.66, P<0.0001; n=40; Figure 5C). Furthermore, Vprop 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).
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 Vprop and with decrement (14/27 waves Vprop=0.81±0.13 mm/s) but completely through the region of observation (450 e). The slowest waves stopped after 200 to 300 e (7/21 waves; Vprop=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 e 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.
Discussion
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 ECC19,20 because of a reduced SR-Ca2+ content21,22 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 e 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 channels10eC14 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 myofilaments11,28 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-releaseeCinduced 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-releaseeCinduced Ca2+ dissociation from TnC caused by the decline of force in the shortening BZ sarcomeres led to the local Ca2+ surge.11,28eC32 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 TnC33,34 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 e (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 e 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 (eCdF/dtmax 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 (CW) and latency of the Ca2+ transient (t[CTeCCW]) (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.4eC6,8,13,34,36eC38 A lower cellular Ca2+ load would explain why Ca2+ waves were both smaller and Vprop lower and propagated with a gradual decline of their amplitude and disappeared after a few hundred e. 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 Vprop.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+eCCa2+ exchange and Ca2+-sensitive inward currents7,10,39eC41 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.42eC44 In addition, increased open probability of the SR-Ca2+ release channels45 owing to channel gene mutation40,42,46 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 motion2,46 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).
References
Antzelevitch C. Electrical heterogeneity, cardiac arrhythmias, and the sodium channel. Circ Res. 2000; 87: 106eC117.
Young AA, Dokos S, Powell KA, Sturm B, McCulloch AD, Starling RC, McCarthy PM, White RD. Regional heterogeneity of function in nonischemic dilated cardiomyopathy. Cardiovasc Res. 2001; 49: 308eC318.
Babuty D, Lab MJ. Mechanoelectric contributions to sudden cardiac death. Cardiovasc Res. 2001; 50: 270eC279.
ter Keurs HEDJ, Zhang YM, Miura M. Damage induced arrhythmias: reversal of excitation-contraction coupling. Cardiovasc Res. 1998; 40: 444eC455.
Daniels MCG, ter Keurs HEDJ. Spontaneous contractions in rat cardiac trabeculae; trigger mechanism and propagation velocity. J Gen Physiol. 1990; 95: 1123eC1137.
Mulder BJM, de Tombe PP, ter Keurs HEDJ. Spontaneous and propagated contractions in rat cardiac trabeculae. J Gen Physiol. 1989; 93: 943eC961.
Daniels MCG, Fedida D, Lamont C, ter Keurs HEDJ. Role of the sarcolemma in triggered propagated contractions in rat cardiac trabeculae. Circ Res. 1991; 68: 1408eC1421.
Miura M, Boyden, PA, ter Keurs HEDJ. Ca2+ waves during triggered propagated contractions in intact trabeculae; determinants of the velocity of propagation. Circ Res. 1999; 84: 1459eC1468.
Miura M, Boyden, PA, ter Keurs, HEDJ. Ca2+ waves during triggered propagated contractions in intact trabeculae. Am J Physiol. 1998; 274: H266eCH276
Wakayama Y, Miura M, Sugai Y, Kagaya Y, Watanabe J, ter Keurs HEDJ. Stretch and quick release of cardiac trabeculae accelerates Ca2+ waves and triggered propagated contractions. Am J Physiol Circ Physio. 2001; 281: H2133eCH2142.
Backx PH, ter Keurs HEDJ. Fluorescent properties of rat cardiac trabeculae microinjected with fura-2 salt. Am J Physiol. 1993; 264: H1098eCH1110.
ter Keurs HEDJ, Rijnsburger WH, van Heuningen R, Nagelsmit MJ. Tension development and sarcomere length in rat cardiac trabeculae. Evidence of length-dependent activation. Circ Res. 1980; 46: 703eC714.
Zhang Y, Miura M, ter Keurs HEDJ. Triggered propagated contractions in rat cardiac trabeculae; inhibition by octanol and heptanol. Circ Res. 1996; 79: 1077eC1085.
Zhang Y, ter Keurs HEDJ. Effects of gadolinium on twitch force and triggered propagated contractions in rat cardiac trabeculae. Cardiovasc Res. 1996; 32: 180eC188.
Bers DM. Excitation-contraction coupling and cardiac contractile force, 2nd edition. 2001. Kluwer Academic Publishers, Dordrecht, The Netherlands.
Trafford AW, Diaz ME, Eisner DA. Coordinated control of cell Ca(2+) loading and triggered release from the sarcoplasmic reticulum underlies the rapid inotropic response to increased L-type Ca(2+) current. Circ Res. 2001; 88: 195eC201.
Konishi M, Kurihara S, Sakai T. The effects of caffeine on tension development and intracellular calcium transients in rat ventricular muscle. J Physiol. 1984; 355: 605eC618.
Sitsapesan R, Williams AJ. Mechanisms of caffeine activation of single calcium-release channels of sheep cardiac sarcoplasmic reticulum. J Physiol. 1990; 423: 425eC439.
Sellin LC, McArdle JJ. Multiple effects of 2,3-butanedione monoxime. Pharmacol and Toxicol. 1994; 74: 305eC313.
Backx PH, Gao WD, Azan-Backx MD, Marban E. Mechanism of force inhibition by 2,3-butanedione monoxime in rat cardiac muscle: roles of [Ca2+]i and cross-bridge kinetics. J Physiol. 1994; 476: 487eC500.
Adams W, Trafford AW, Eisner DA. 2,3-Butanedione monoxime (BDM) decreases sarcoplasmic reticulum Ca content by stimulating Ca release in isolated rat ventricular myocytes. Pflugers Arch. 1998; 436: 776eC781.
Phillips RM, Altschuld RA. 2,3-Butanedione 2-monoxime (BDM) induces calcium release from canine cardiac sarcoplasmic reticulum. Biochem Biophys Res Commun. 1996; 229: 154eC157.
Tripathy A, Xu L, Pasek DA, Meissner G. Effects of 2,3-butanedione 2-monoxime on Ca2+ release channels (ryanodine receptors) of cardiac and skeletal muscle. J Membr Biol. 1999; 169: 189eC198.
Banijamali H, Gao WD, MacIntosh B, ter Keurs HEDJ. Force-interval relations of twitches and cold contractures in rat cardiac trabeculae; effect of ryanodine. Circ Res. 1998; 69: 937eC948.
Trafford AW, Sibbring GC, Diaz ME, Eisner DA. The effects of low concentrations of caffeine on spontaneous Ca release in isolated rat ventricular myocytes. Cell Calcium. 2000; 28: 269eC276.
Davidoff AW, Boyden PA, Schwartz K, Michel JB, Zhang YM, Obayashi M, Crabbe D, ter Keurs HE. Congestive heart failure after myocardial infarction in the rat: cardiac force and spontaneous sarcomere activity. Ann N Y Acad Sci. 2004; 1015: 84eC95.
Jiang Y, Patterson MF, Morgan DL, Julian FJ. Basis for late rise in fura 2 R signal reporting [Ca2+]i during relaxation in intact rat ventricular trabeculae. the Am Physiological Society. 2004; 43: C1273eCC1282.
Housmans PR, Lee NKM, Blinks JR. Active shortening retards the decline of the intracellular calcium transient in mammalian heart muscle. Science. 1983; 221: 159eC161.
Allen DG, Kentish JC. Calcium concentration in the myoplasm of skinned ferret ventricular muscle following changes in muscle length. J Physiol. 1988; 407: 489eC503.
Kurihara S, Komukai K. Tension-dependent changes of the intracellular Ca2+ transients in ferret ventricular muscles. J Physiol. 1995; 489: 617eC625.
Allen DG, Kurihara S. The effects of muscle length on intracellular calcium transients in mammalian cardiac muscle. J Physiol. 1982; 327: 79eC94.
Lab MJ, Allen DG, Orchard CH. The effects of shortening on myoplasmic calcium concentration and on the action potential in mammalian ventricular muscle. Circ Res. 1984; 55: 825eC829.
Fabiato A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol. 1983; 245: C1eCC14.
Backx PHM, de Tombe PP, van Deen JHK, Mulder BJM, ter Keurs HEDJ. A model of propagating calcium-induced calcium release mediated by calcium diffusion. J Gen Physiol. 1989; 93: 963eC977.
Raisaro A, Villa A, Recusani F, Rossi A, Bargiggia G, Tronconi L, Campani R, Montemartini C. [The remodelling of the heart in heart failure: from thoracic radiography to magnetic resonance]. G Ital Cardiol. 1993; 23: 167eC175.
Lamont C, Luther PW, Balke CW, Wier GW. Intercellular Ca2+ waves in rat heart muscle. J Physiol. 1998; 512: 669eC676.
Wier WG, ter Keurs HEDJ, Marban E, Gao WD, Balke W. Ca2+‘sparks’ and waves in intact ventricular muscle resolved by confocal imaging. Circ Res. 1997; 81: 462eC469.
Kaneko T, Tanaka H, Oyamada M, Kawata S, Takamatsu T. Three distinct types of Ca2+ waves in Langendorff-perfused rat heart revealed by real-time confocal microscopy. Circ Res. 2000; 86: 1093eC1099.
Miura M, Ishide N, Oda H, Sakurai M, Shinozaki T, Takishima T. Spatial features of calcium transients during early and delayed afterdepolarizations. Am J Physiol. 1993; 265: H439eCH444.
Schlotthauer K, Bers DM. Sarcoplasmic reticulum Ca(2+) release causes myocyte depolarization. Underlying mechanism and threshold for triggered action potentials. Circ Res. 2000; 87: 774eC780.
Lakatta EG. Functional implications of spontaneous sarcoplasmic reticulum Ca2+ release in the heart. Cardiovasc Res. 1992; 26: 193eC214.
Laitinen PJ, Brown KM, Piippo K, Swan H, Devaney JM, Brahmbhatt B, Donarum EA, Marino M, Tiso N, Viitasalo M, Toivonen L, Stephan DA, Kontula K. Mutations of the cardiac ryanodine receptor (RyR2) gene in familial polymorphic ventricular tachycardia. Circulation. 2001; 103: 485eC490.
Marks AR. Ryanodine receptors/calcium release channels in heart failure and sudden cardiac death. J Mol Cell Cardiol. 2001; 33: 615eC624.
Postma AV, Denjoy I, Joorntje TM, Lupoglazoff J-M, DaCosta A, Sebillon P, Mannens M, Wilde AAM, Guicheney P. Absence of calsequestrin 2 causes severe forms of catecholaminergic polymorphic ventricular tachycardia. Circ Res. 2002; 91: e21eCe26.
Boyden PA, ter Keurs HEDJ. Reverse excitation contraction coupling: Ca2+ ions as initiators of arrhythmias. J Cardiovasc Electrophysiol. 2001; 12: 382eC385.
Siogas K, Pappas S, Graekas G, Goudevenos J, Liapi G, Sideris DA. Segmental wall motion abnormalities alter vulnerability to ventricular ectopic beats associated with acute increases in aortic pressure in patients with underlying coronary artery disease. Heart. 1998; 79: 268eC273.(Yuji Wakayama, Masahito M)
the Departments of Medicine, Physiology
Biophysics (B.D.S., H.E.D.J.t.K.), Health Sciences Centre, University of Calgary, Canada
the Department of Pharmacology (P.A.B.), Columbia University, New York.
Abstract
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 excitationeCcontraction 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 e) 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
Ischemic and failing hearts are both prone to ventricular arrhythmias and commonly show regional differences in contractile strength caused by heterogeneous impairment of excitationeCcontraction 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 shortening5eC7 associated with a [Ca2+]i transient that propagates in a wave-like manner along the muscle.8eC10 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
Measurements of Force, SL, and [Ca2+]i in Rat Trabeculae
Trabeculae (n=15; length: 2.30±0.09 mm, width: 262±25 e, thickness: 103±4 e in slack conditions) were dissected from the right ventricle of Lewis Brown Norway rats5eC14 (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).
Measurement of [Ca2+]i has been described previously.8eC11 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 F360//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 e; Figure 1) using a syringe pump connected to a glass pipette (100 e 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
Nonuniformity and Sarcomere Mechanics
The jet reached one short muscle segment (300 e) (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/Fmax) decreased (eC11±3% with low [Ca2+]jet (n=5), eC28±5% with caffeine (n=6), and eC36±7% with BDM (n=5) (Figure 2C; Table). Low [Ca2+]jet solution did not affect resting SL (SLo), whereas caffeine and BDM slightly decreased (eC2.2±0.7%) and increased (+3.1±0.6%) SLo, 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 e 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 e) beyond the jet-exposed region (Figure 2B). The diffraction pattern of sarcomeres in BZ illuminated by a 150 e 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).
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 (CT) and increased diastolic [Ca2+]i ([Ca2+]diast or CD) 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 eC36±6, eC17±3, and eC37±7%, and [Ca2+]diast changed by +82±28, +48±13, eC44±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+]ieC 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+]jeteC, 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.
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 e 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).
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 (CW/CT; r=0.61, P<0.001; see supplemental Figure IIs) in all jet exposures.
Propagation of Ca2+ Waves
Propagation velocity of the Ca2+ waves (Vprop) (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).8eC10 Vprop correlated with the [Ca2+]i increase seen in the BZ during the initial Ca2+ surge (CW) (r=0.66, P<0.0001; n=40; Figure 5C). Furthermore, Vprop 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).
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 Vprop and with decrement (14/27 waves Vprop=0.81±0.13 mm/s) but completely through the region of observation (450 e). The slowest waves stopped after 200 to 300 e (7/21 waves; Vprop=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 e 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.
Discussion
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 ECC19,20 because of a reduced SR-Ca2+ content21,22 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 e 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 channels10eC14 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 myofilaments11,28 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-releaseeCinduced 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-releaseeCinduced Ca2+ dissociation from TnC caused by the decline of force in the shortening BZ sarcomeres led to the local Ca2+ surge.11,28eC32 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 TnC33,34 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 e (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 e 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 (eCdF/dtmax 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 (CW) and latency of the Ca2+ transient (t[CTeCCW]) (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.4eC6,8,13,34,36eC38 A lower cellular Ca2+ load would explain why Ca2+ waves were both smaller and Vprop lower and propagated with a gradual decline of their amplitude and disappeared after a few hundred e. 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 Vprop.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+eCCa2+ exchange and Ca2+-sensitive inward currents7,10,39eC41 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.42eC44 In addition, increased open probability of the SR-Ca2+ release channels45 owing to channel gene mutation40,42,46 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 motion2,46 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).
References
Antzelevitch C. Electrical heterogeneity, cardiac arrhythmias, and the sodium channel. Circ Res. 2000; 87: 106eC117.
Young AA, Dokos S, Powell KA, Sturm B, McCulloch AD, Starling RC, McCarthy PM, White RD. Regional heterogeneity of function in nonischemic dilated cardiomyopathy. Cardiovasc Res. 2001; 49: 308eC318.
Babuty D, Lab MJ. Mechanoelectric contributions to sudden cardiac death. Cardiovasc Res. 2001; 50: 270eC279.
ter Keurs HEDJ, Zhang YM, Miura M. Damage induced arrhythmias: reversal of excitation-contraction coupling. Cardiovasc Res. 1998; 40: 444eC455.
Daniels MCG, ter Keurs HEDJ. Spontaneous contractions in rat cardiac trabeculae; trigger mechanism and propagation velocity. J Gen Physiol. 1990; 95: 1123eC1137.
Mulder BJM, de Tombe PP, ter Keurs HEDJ. Spontaneous and propagated contractions in rat cardiac trabeculae. J Gen Physiol. 1989; 93: 943eC961.
Daniels MCG, Fedida D, Lamont C, ter Keurs HEDJ. Role of the sarcolemma in triggered propagated contractions in rat cardiac trabeculae. Circ Res. 1991; 68: 1408eC1421.
Miura M, Boyden, PA, ter Keurs HEDJ. Ca2+ waves during triggered propagated contractions in intact trabeculae; determinants of the velocity of propagation. Circ Res. 1999; 84: 1459eC1468.
Miura M, Boyden, PA, ter Keurs, HEDJ. Ca2+ waves during triggered propagated contractions in intact trabeculae. Am J Physiol. 1998; 274: H266eCH276
Wakayama Y, Miura M, Sugai Y, Kagaya Y, Watanabe J, ter Keurs HEDJ. Stretch and quick release of cardiac trabeculae accelerates Ca2+ waves and triggered propagated contractions. Am J Physiol Circ Physio. 2001; 281: H2133eCH2142.
Backx PH, ter Keurs HEDJ. Fluorescent properties of rat cardiac trabeculae microinjected with fura-2 salt. Am J Physiol. 1993; 264: H1098eCH1110.
ter Keurs HEDJ, Rijnsburger WH, van Heuningen R, Nagelsmit MJ. Tension development and sarcomere length in rat cardiac trabeculae. Evidence of length-dependent activation. Circ Res. 1980; 46: 703eC714.
Zhang Y, Miura M, ter Keurs HEDJ. Triggered propagated contractions in rat cardiac trabeculae; inhibition by octanol and heptanol. Circ Res. 1996; 79: 1077eC1085.
Zhang Y, ter Keurs HEDJ. Effects of gadolinium on twitch force and triggered propagated contractions in rat cardiac trabeculae. Cardiovasc Res. 1996; 32: 180eC188.
Bers DM. Excitation-contraction coupling and cardiac contractile force, 2nd edition. 2001. Kluwer Academic Publishers, Dordrecht, The Netherlands.
Trafford AW, Diaz ME, Eisner DA. Coordinated control of cell Ca(2+) loading and triggered release from the sarcoplasmic reticulum underlies the rapid inotropic response to increased L-type Ca(2+) current. Circ Res. 2001; 88: 195eC201.
Konishi M, Kurihara S, Sakai T. The effects of caffeine on tension development and intracellular calcium transients in rat ventricular muscle. J Physiol. 1984; 355: 605eC618.
Sitsapesan R, Williams AJ. Mechanisms of caffeine activation of single calcium-release channels of sheep cardiac sarcoplasmic reticulum. J Physiol. 1990; 423: 425eC439.
Sellin LC, McArdle JJ. Multiple effects of 2,3-butanedione monoxime. Pharmacol and Toxicol. 1994; 74: 305eC313.
Backx PH, Gao WD, Azan-Backx MD, Marban E. Mechanism of force inhibition by 2,3-butanedione monoxime in rat cardiac muscle: roles of [Ca2+]i and cross-bridge kinetics. J Physiol. 1994; 476: 487eC500.
Adams W, Trafford AW, Eisner DA. 2,3-Butanedione monoxime (BDM) decreases sarcoplasmic reticulum Ca content by stimulating Ca release in isolated rat ventricular myocytes. Pflugers Arch. 1998; 436: 776eC781.
Phillips RM, Altschuld RA. 2,3-Butanedione 2-monoxime (BDM) induces calcium release from canine cardiac sarcoplasmic reticulum. Biochem Biophys Res Commun. 1996; 229: 154eC157.
Tripathy A, Xu L, Pasek DA, Meissner G. Effects of 2,3-butanedione 2-monoxime on Ca2+ release channels (ryanodine receptors) of cardiac and skeletal muscle. J Membr Biol. 1999; 169: 189eC198.
Banijamali H, Gao WD, MacIntosh B, ter Keurs HEDJ. Force-interval relations of twitches and cold contractures in rat cardiac trabeculae; effect of ryanodine. Circ Res. 1998; 69: 937eC948.
Trafford AW, Sibbring GC, Diaz ME, Eisner DA. The effects of low concentrations of caffeine on spontaneous Ca release in isolated rat ventricular myocytes. Cell Calcium. 2000; 28: 269eC276.
Davidoff AW, Boyden PA, Schwartz K, Michel JB, Zhang YM, Obayashi M, Crabbe D, ter Keurs HE. Congestive heart failure after myocardial infarction in the rat: cardiac force and spontaneous sarcomere activity. Ann N Y Acad Sci. 2004; 1015: 84eC95.
Jiang Y, Patterson MF, Morgan DL, Julian FJ. Basis for late rise in fura 2 R signal reporting [Ca2+]i during relaxation in intact rat ventricular trabeculae. the Am Physiological Society. 2004; 43: C1273eCC1282.
Housmans PR, Lee NKM, Blinks JR. Active shortening retards the decline of the intracellular calcium transient in mammalian heart muscle. Science. 1983; 221: 159eC161.
Allen DG, Kentish JC. Calcium concentration in the myoplasm of skinned ferret ventricular muscle following changes in muscle length. J Physiol. 1988; 407: 489eC503.
Kurihara S, Komukai K. Tension-dependent changes of the intracellular Ca2+ transients in ferret ventricular muscles. J Physiol. 1995; 489: 617eC625.
Allen DG, Kurihara S. The effects of muscle length on intracellular calcium transients in mammalian cardiac muscle. J Physiol. 1982; 327: 79eC94.
Lab MJ, Allen DG, Orchard CH. The effects of shortening on myoplasmic calcium concentration and on the action potential in mammalian ventricular muscle. Circ Res. 1984; 55: 825eC829.
Fabiato A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol. 1983; 245: C1eCC14.
Backx PHM, de Tombe PP, van Deen JHK, Mulder BJM, ter Keurs HEDJ. A model of propagating calcium-induced calcium release mediated by calcium diffusion. J Gen Physiol. 1989; 93: 963eC977.
Raisaro A, Villa A, Recusani F, Rossi A, Bargiggia G, Tronconi L, Campani R, Montemartini C. [The remodelling of the heart in heart failure: from thoracic radiography to magnetic resonance]. G Ital Cardiol. 1993; 23: 167eC175.
Lamont C, Luther PW, Balke CW, Wier GW. Intercellular Ca2+ waves in rat heart muscle. J Physiol. 1998; 512: 669eC676.
Wier WG, ter Keurs HEDJ, Marban E, Gao WD, Balke W. Ca2+‘sparks’ and waves in intact ventricular muscle resolved by confocal imaging. Circ Res. 1997; 81: 462eC469.
Kaneko T, Tanaka H, Oyamada M, Kawata S, Takamatsu T. Three distinct types of Ca2+ waves in Langendorff-perfused rat heart revealed by real-time confocal microscopy. Circ Res. 2000; 86: 1093eC1099.
Miura M, Ishide N, Oda H, Sakurai M, Shinozaki T, Takishima T. Spatial features of calcium transients during early and delayed afterdepolarizations. Am J Physiol. 1993; 265: H439eCH444.
Schlotthauer K, Bers DM. Sarcoplasmic reticulum Ca(2+) release causes myocyte depolarization. Underlying mechanism and threshold for triggered action potentials. Circ Res. 2000; 87: 774eC780.
Lakatta EG. Functional implications of spontaneous sarcoplasmic reticulum Ca2+ release in the heart. Cardiovasc Res. 1992; 26: 193eC214.
Laitinen PJ, Brown KM, Piippo K, Swan H, Devaney JM, Brahmbhatt B, Donarum EA, Marino M, Tiso N, Viitasalo M, Toivonen L, Stephan DA, Kontula K. Mutations of the cardiac ryanodine receptor (RyR2) gene in familial polymorphic ventricular tachycardia. Circulation. 2001; 103: 485eC490.
Marks AR. Ryanodine receptors/calcium release channels in heart failure and sudden cardiac death. J Mol Cell Cardiol. 2001; 33: 615eC624.
Postma AV, Denjoy I, Joorntje TM, Lupoglazoff J-M, DaCosta A, Sebillon P, Mannens M, Wilde AAM, Guicheney P. Absence of calsequestrin 2 causes severe forms of catecholaminergic polymorphic ventricular tachycardia. Circ Res. 2002; 91: e21eCe26.
Boyden PA, ter Keurs HEDJ. Reverse excitation contraction coupling: Ca2+ ions as initiators of arrhythmias. J Cardiovasc Electrophysiol. 2001; 12: 382eC385.
Siogas K, Pappas S, Graekas G, Goudevenos J, Liapi G, Sideris DA. Segmental wall motion abnormalities alter vulnerability to ventricular ectopic beats associated with acute increases in aortic pressure in patients with underlying coronary artery disease. Heart. 1998; 79: 268eC273.(Yuji Wakayama, Masahito M)