It has long been held that electrical excitation spreads from cell-to-cell in the heart via low resistance gap junctions (GJ). However, it has also been proposed that myocytes could interact by non-GJ-mediated “ephaptic” mechanisms, facilitating propagation of action potentials in tandem with direct GJ-mediated coupling. We sought evidence that such mechanisms contribute to cardiac conduction. Using super-resolution microscopy, we demonstrate that Nav1.5 is localized within 200 nm of the GJ plaque (a region termed the perinexus). Electron microscopy revealed close apposition of adjacent cell membranes within perinexi suggesting that perinexal sodium channels could function as an ephapse, enabling ephaptic cell-to-cell transfer of electrical excitation. Acute interstitial edema (AIE) increased intermembrane distance at the perinexus and was associated with preferential transverse conduction slowing and increased spontaneous arrhythmia incidence. Inhibiting sodium channels with 0.5 μM flecainide uniformly slowed conduction, but sodium channel inhibition during AIE slowed conduction anisotropically and increased arrhythmia incidence more than AIE alone. Sodium channel inhibition during GJ uncoupling with 25 μM carbenoxolone slowed conduction anisotropically and was also highly proarrhythmic. A computational model of discretized extracellular microdomains (including ephaptic coupling) revealed that conduction trends associated with altered perinexal width, sodium channel conductance, and GJ coupling can be predicted when sodium channel density in the intercalated disk is relatively high. We provide evidence that cardiac conduction depends on a mathematically predicted ephaptic mode of coupling as well as GJ coupling. These data suggest opportunities for novel anti-arrhythmic therapies targeting noncanonical conduction pathways in the heart.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-014-1675-z) contains supplementary material, which is available to authorized users.
Computational modeling indicates that cardiac conduction may involve ephaptic coupling – intercellular communication involving electrochemical signaling across narrow extracellular clefts between cardiomyocytes. We hypothesized that β1(SCN1B) –mediated adhesion scaffolds trans-activating NaV1.5 (SCN5A) channels within narrow (<30 nm) perinexal clefts adjacent to gap junctions (GJs), facilitating ephaptic coupling. Super-resolution imaging indicated preferential β1 localization at the perinexus, where it co-locates with NaV1.5. Smart patch clamp (SPC) indicated greater sodium current density (INa) at perinexi, relative to non-junctional sites. A novel, rationally designed peptide, βadp1, potently and selectively inhibited β1-mediated adhesion, in electric cell-substrate impedance sensing studies. βadp1 significantly widened perinexi in guinea pig ventricles, and selectively reduced perinexal INa, but not whole cell INa, in myocyte monolayers. In optical mapping studies, βadp1 precipitated arrhythmogenic conduction slowing. In summary, β1-mediated adhesion at the perinexus facilitates action potential propagation between cardiomyocytes, and may represent a novel target for anti-arrhythmic therapies.
Background-Although heart failure (HF) is closely associated with susceptibility to sudden cardiac death (SCD), the mechanisms linking contractile dysfunction to cardiac electrical instability are poorly understood. Cardiac alternans has also been closely associated with SCD, and has been linked to a mechanism for amplifying electrical heterogeneities in the heart. However, previous studies have focused on alternans in normal rather than failing myocardium.
We previously demonstrated that altering extracellular sodium (Nao) and calcium (Cao) can modulate a form of electrical communication between cardiomyocytes termed “ephaptic coupling” (EpC), especially during loss of gap junction coupling. We hypothesized that altering Nao and Cao modulates conduction velocity (CV) and arrhythmic burden during ischemia. Electrophysiology was quantified by optically mapping Langendorff-perfused guinea pig ventricles with modified Nao (147 or 155 mM) and Cao (1.25 or 2.0 mM) during 30 min of simulated metabolic ischemia (pH 6.5, anoxia, aglycemia). Gap junction-adjacent perinexal width ( WP), a candidate cardiac ephapse, and connexin (Cx)43 protein expression and Cx43 phosphorylation at S368 were quantified by transmission electron microscopy and Western immunoblot analysis, respectively. Metabolic ischemia slowed CV in hearts perfused with 147 mM Nao and 2.0 mM Cao; however, theoretically increasing EpC with 155 mM Nao was arrhythmogenic, and CV could not be measured. Reducing Cao to 1.25 mM expanded WP, as expected during ischemia, consistent with reduced EpC, but attenuated CV slowing while delaying arrhythmia onset. These results were further supported by osmotically reducing WP with albumin, which exacerbated CV slowing and increased early arrhythmias during ischemia, whereas mannitol expanded WP, permitted conduction, and delayed the onset of arrhythmias. Cx43 expression patterns during the various interventions insufficiently correlated with observed CV changes and arrhythmic burden. In conclusion, decreasing perfusate calcium during metabolic ischemia enhances perinexal expansion, attenuates conduction slowing, and delays arrhythmias. Thus, perinexal expansion may be cardioprotective during metabolic ischemia. NEW & NOTEWORTHY This study demonstrates, for the first time, that modulating perfusate ion composition can alter cardiac electrophysiology during simulated metabolic ischemia.
malities in calcium handling have been implicated as a significant source of electrical instability in heart failure (HF). While these abnormalities have been investigated extensively in isolated myocytes, how they manifest at the tissue level and trigger arrhythmias is not clear. We hypothesize that in HF, triggered activity (TA) is due to spontaneous calcium release from the sarcoplasmic reticulum that occurs in an aggregate of myocardial cells (an SRC) and that peak SCR amplitude is what determines whether TA will occur. Calcium and voltage optical mapping was performed in ventricular wedge preparations from canines with and without tachycardia-induced HF. In HF, steady-state calcium transients have reduced amplitude [135 vs. 170 ratiometric units (RU), P Ͻ 0.05] and increased duration (252 vs. 229 s, P Ͻ 0.05) compared with those of normal. Under control conditions and during -adrenergic stimulation, TA was more frequent in HF (53% and 93%, respectively) compared with normal (0% and 55%, respectively, P Ͻ 0.025). The mechanism of arrhythmias was SCRs, leading to delayed afterdepolarization-mediated triggered beats. Interestingly, the rate of SCR rise was greater for events that triggered a beat (0.41 RU/ms) compared with those that did not (0.18 RU/ms, P Ͻ 0.001). In contrast, there was no difference in SCR amplitude between the two groups. In conclusion, TA in HF tissue is associated with abnormal calcium regulation and mediated by the spontaneous release of calcium from the sarcoplasmic reticulum in aggregates of myocardial cells (i.e., an SCR), but importantly, it is the rate of SCR rise rather than amplitude that was associated with TA. heart failure; arrhythmia; delayed afterdepolarization; triggered activity HEART FAILURE (HF) is a serious public health problem that afflicts millions of people in the United States alone (32). HF is associated with impaired cardiac contractility and relaxation, as well as a high incidence of ventricular arrhythmias and sudden death. Abnormal calcium handling has been implicated as a source of both mechanical and electrical dysfunction observed in HF, making it a key target for investigation and clinical therapy.At the myocyte level, impaired ventricular contractility and relaxation in HF have been attributed to decreased calcium transient amplitude due to diastolic sarcoplasmic reticulum (SR) calcium leak (12,13,35) and reduced SR calcium uptake (3,8,22,24). Calcium dysregulation in human HF has been associated with a significant incidence of nonreentrant arrhythmias (28,30,33) that can occur as the result of early afterdepolarizations (EADs) or delayed afterdepolarizations (DADs). At the subcellular level, a DAD is caused by spontaneous calcium release from the SR that activates a transient inward current with a magnitude that depends on the amount of calcium flux (33). While studies in isolated myocytes have provided valuable insight into cellular pathophysiology, the translation of these results to arrhythmogenesis at the tissue level is not straightforward.The fact...
Katra RP, Oya T, Hoeker GS, Laurita KR. Ryanodine receptor dysfunction and triggered activity in the heart. Am J Physiol Heart Circ Physiol 292: H2144 -H2151, 2007. First published December 22, 2006; doi:10.1152/ajpheart.00924.2006.-Arrhythmogenesis has been increasingly linked to cardiac ryanodine receptor (RyR) dysfunction. However, the mechanistic relationship between abnormal RyR function and arrhythmogenesis in the heart is not clear. We hypothesize that, under abnormal RyR conditions, triggered activity will be caused by spontaneous calcium release (SCR) events that depend on transmural heterogeneities of calcium handling. We performed highresolution optical mapping of intracellular calcium and transmembrane potential in the canine left ventricular wedge preparation (n ϭ 28). Rapid pacing was used to initiate triggered activity under normal and abnormal RyR conditions induced by FKBP12.6 dissociation and -adrenergic stimulation (20 -150 M rapamycin, 0.2 M isoproterenol). Under abnormal RyR conditions, almost all preparations experienced SCRs and triggered activity, in contrast to control, rapamycin, or isoproterenol conditions alone. Furthermore, under abnormal RyR conditions, complex arrhythmias (monomorphic and polymorphic tachycardia) were commonly observed. After washout of rapamycin and isoproterenol, no triggered activity was observed. Surprisingly, triggered activity and SCRs occurred preferentially near the epicardium but not the endocardium (P Ͻ 0.01). Interestingly, the occurrence of triggered activity and SCR events could not be explained by cytoplasmic calcium levels, but rather by fast calcium reuptake kinetics. These data suggest that, under abnormal RyR conditions, triggered activity is caused by multiple SCR events that depend on the faster calcium reuptake kinetics near the epicardium. Furthermore, multiple regions of SCR may be a mechanism for multifocal arrhythmias associated with RyR dysfunction. spontaneous calcium release; sudden death; arrhythmia mechanisms ARRHYTHMIAS CAUSED BY ABNORMAL impulse formation (i.e., triggered activity) have been associated with (33, 34) and without (5, 9, 16, 27) structural heart disease. In addition, recent evidence suggests that such arrhythmias can be caused by spontaneous calcium release (SCR) from the sarcoplasmic reticulum (SR). SCR events may be a result of cardiac ryanodine receptor (RyR) dysfunction such as that associated with mutations in the human cardiac RyR channel (37), heart failure (25, 40), and/or alterations in the RyR (30) and accessory proteins (e.g., FKBP12.6) (45, 49); however, the underlying mechanisms are controversial. FKBP12.6 is an accessory protein to the tetrameric RyR channel with 4:1 stoichiometry that stabilizes the RyR in the closed/open state and prevents aberrant activation during diastole. Dissociation of the FKBP12.6 molecule is believed to cause RyR channels to gate independently and alter individual RyR channel gating kinetics, rendering the channel prone to SCR events (28). Alternatively, SCR associated with abn...
The relationship between cardiac conduction velocity (CV) and extracellular potassium (K+) is biphasic, with modest hyperkalemia increasing CV and severe hyperkalemia slowing CV. Recent studies from our group suggest that elevating extracellular sodium (Na+) and calcium (Ca2+) can enhance CV by an extracellular pathway parallel to gap junctional coupling (GJC) called ephaptic coupling that can occur in the gap junction adjacent perinexus. However, it remains unknown whether these same interventions modulate CV as a function of K+. We hypothesize that Na+, Ca2+, and GJC can attenuate conduction slowing consequent to severe hyperkalemia. Elevating Ca2+ from 1.25 to 2.00 mM significantly narrowed perinexal width measured by transmission electron microscopy. Optically mapped, Langendorff-perfused guinea pig hearts perfused with increasing K+ revealed the expected biphasic CV-K+ relationship during perfusion with different Na+ and Ca2+ concentrations. Neither elevating Na+ nor Ca2+ alone consistently modulated the positive slope of CV-K+ or conduction slowing at 10-mM K+; however, combined Na+ and Ca2+ elevation significantly mitigated conduction slowing at 10-mM K+. Pharmacologic GJC inhibition with 30-μM carbenoxolone slowed CV without changing the shape of CV-K+ curves. A computational model of CV predicted that elevating Na+ and narrowing clefts between myocytes, as occur with perinexal narrowing, reduces the positive and negative slopes of the CV-K+ relationship but do not support a primary role of GJC or sodium channel conductance. These data demonstrate that combinatorial effects of Na+ and Ca2+ differentially modulate conduction during hyperkalemia, and enhancing determinants of ephaptic coupling may attenuate conduction changes in a variety of physiologic conditions.
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