Repolarization alternans is a harbinger of sudden cardiac death, particularly when it becomes spatially discordant. Alternans, a beat-to-beat alternation in the action potential duration (APD) and intracellular Ca (Cai), can arise from either tissue heterogeneities or dynamic factors. Distinguishing between these mechanisms in normal cardiac tissue is difficult because of inherent complex three-dimensional tissue heterogeneities. To evaluate repolarization alternans in a simpler two-dimensional cardiac substrate, we optically recorded voltage and/or Cai in monolayers of cultured neonatal rat ventricular myocytes during rapid pacing, before and after exposure to BAY K 8644 to enhance dynamic factors promoting alternans. Under control conditions ( n = 37), rapid pacing caused detectable APD alternans in 81% of monolayers, and Cai transient alternans in all monolayers, becoming spatially discordant in 62%. After BAY K 8644 ( n = 28), conduction velocity restitution became more prominent, and APD and Cai alternans developed and became spatially discordant in all monolayers, with an increased number of nodal lines separating out-of-phase alternating regions. Nodal lines moved closer to the pacing site with faster pacing rates and changed orientation when the pacing site was moved, as predicted for the dynamically generated, but not heterogeneity-based, alternans. Spatial APD gradients during spatially discordant alternans were sufficiently steep to induce conduction block and reentry. These findings indicate that spatially discordant alternans severe enough to initiate reentry can be readily induced by pacing in two-dimensional cardiac tissue and behaves according to predictions for a predominantly dynamically generated mechanism.
Background-Electrophysiological changes promoting arrhythmias during acute regional ischemia/reperfusion are challenging to study in intact cardiac tissue because of complex 3-dimensional myocardial and vascular geometry. We characterized electrophysiological alterations and arrhythmias during regional ischemia/reperfusion in a simpler 2-dimensional geometry of cultured neonatal rat ventricular myocyte monolayers. Methods and Results-Optical mapping of intracellular Ca (Ca i ) and voltage was performed with the use of Rhod 2-AM and Rh-237, respectively. Regional ischemia was mimicked by covering the central portion of monolayer with a glass coverslip, and reperfusion was mimicked by removing the coverslip. Monolayers were stained with fluorescent antibodies to detect total and dephosphorylated connexin-43 at various time points. During coverslip ischemia, action potential duration shortened, Ca i transient duration was prolonged, and local conduction velocity (CV) slowed progressively, with loss of excitability after 10.6Ϯ3.6 minutes. CV slowing was accompanied by connexin-43 dephosphorylation. During ischemia, spontaneous reentry occurred in 5 of 11 monolayers, initiated by extrasystoles arising from the border zone or unidirectional conduction block of paced beats. On reperfusion, excitability recovered within 1.0Ϯ0.8 minutes, but CV remained depressed for 9.0Ϯ3.0 minutes, promoting reentry in the reperfused zone. As connexin-43 phosphorylation recovered in the reperfused zone, CV normalized, and arrhythmias resolved. Conclusions-Acute regional ischemia/reperfusion in neonatal rat ventricular myocyte monolayers recapitulates electrophysiological alterations and arrhythmias similar to those observed during acute coronary occlusion/reperfusion in intact hearts. During early reperfusion, slow recovery from connexin-43 dephosphorylation leads to persistent CV slowing, creating a highly arrhythmogenic substrate.
conduction block and reentry in neonatal rat ventricular myocyte monolayers. Am J Physiol Heart Circ Physiol 300: H271-H278, 2011. First published October 29, 2010 doi:10.1152/ajpheart.00758.2009.-Anisotropy can lead to unidirectional conduction block that initiates reentry. We analyzed the mechanisms in patterned anisotropic neonatal rat ventricular myocyte monolayers. Voltage and intracellular Ca (Ca i) were optically mapped under the following conditions: extrastimulus (S1S2) testing and/or tetrodotoxin (TTX) to suppress Na current availability; heptanol to reduce gap junction conductance; and incremental rapid pacing. In anisotropic monolayers paced at 2 Hz, conduction velocity (CV) was faster longitudinally than transversely, with an anisotropy ratio [AR ϭ CV L/CVT, where CVL and CVT are CV in the longitudinal and transverse directions, respectively], averaging 2.1 Ϯ 0.8. Interventions decreasing Na current availability, such as S1S2 pacing and TTX, slowed CV L and CVT proportionately, without changing the AR. Conduction block preferentially occurred longitudinal to fiber direction, commonly initiating reentry. Interventions that decreased gap junction conductance, such as heptanol, decreased CV T more than CVL, increasing the AR and causing preferential transverse conduction block and reentry. Rapid pacing resembled the latter, increasing the AR and promoting transverse conduction block and reentry, which was prevented by the Ca i chelator 1,2-bis oaminophenoxy ethane-N,N,N=,N=-tetraacetic acid (BAPTA). In contrast to isotropic and uniformly anisotropic monolayers, in which reentrant rotors drifted and self-terminated, bidirectional anisotropy (i.e., an abrupt change in fiber direction exceeding 45°) caused reentry to anchor near the zone of fiber direction change in 77% of monolayers. In anisotropic monolayers, unidirectional conduction block initiating reentry can occur longitudinal or transverse to fiber direction, depending on whether the experimental intervention reduces Na current availability or decreases gap junction conductance, agreeing with theoretical predictions.anisotropy; reentry; myocyte monolayers; optical mapping CARDIAC TISSUE IS INHERENTLY anisotropic, with faster conduction velocity (CV) along fiber direction than across it (24, 29, 31), a property implicated in arrhythmogenesis. Spach et al. (31) showed that anisotropy can lead to preferential longitudinal conduction block initiating reentry, due to a lower safety factor for longitudinal than transverse conduction. Other investigators, however, found preferential transverse conduction block in anisotropic tissue (7,8,22,24,26), particularly when gap junction conductance was pharmacologically decreased by heptanol (8). Although theoretical mechanisms for both preferential longitudinal block (due to reduced safety factor) (27, 31) and preferential transverse block [due to reduced gap junction conductance (13) and certain low excitability conditions (8)] have been proposed, the experimental evidence for these mechanisms in intact thre...
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