Acute Adenoviral Infection Elicits an Arrhythmogenic Substrate Prior to Myocarditis.

BACKGROUND: Viral cardiac infection represents a significant clinical challenge encompassing several etiological agents, disease stages, complex presentation, and a resulting lack of mechanistic understanding. Myocarditis is a major cause of sudden cardiac death in young adults, where current knowledge in the field is dominated by later disease phases and pathological immune responses. However, little is known regarding how infection can acutely induce an arrhythmogenic substrate before significant immune responses. Adenovirus is a leading cause of myocarditis, but due to species specificity, models of infection are lacking, and it is not understood how adenoviral infection may underlie sudden cardiac arrest. Mouse adenovirus type-3 was previously reported as cardiotropic, yet it has not been utilized to understand the mechanisms of cardiac infection and pathology. METHODS: We have developed mouse adenovirus type-3 infection as a model to investigate acute cardiac infection and molecular alterations to the infected heart before an appreciable immune response or gross cardiomyopathy. RESULTS: Optical mapping of infected hearts exposes decreases in conduction velocity concomitant with increased Cx43Ser368 phosphorylation, a residue known to regulate gap junction function. Hearts from animals harboring a phospho-null mutation at Cx43Ser368 are protected against mouse adenovirus type-3-induced conduction velocity slowing. Additional to gap junction alterations, patch clamping of mouse adenovirus type-3-infected adult mouse ventricular cardiomyocytes reveals prolonged action potential duration as a result of decreased IK1 and IKs current density. Turning to human systems, we find human adenovirus type-5 increases phosphorylation of Cx43Ser368 and disrupts synchrony in human induced pluripotent stem cell-derived cardiomyocytes, indicating common mechanisms with our mouse whole heart and adult cardiomyocyte data. CONCLUSIONS: Together, these findings demonstrate that adenoviral infection creates an arrhythmogenic substrate through direct targeting of gap junction and ion channel function in the heart. Such alterations are known to precipitate arrhythmias and likely contribute to sudden cardiac death in acutely infected patients.

Mannitol and hyponatremia regulate cardiac ventricular conduction in the context of sodium channel loss of function.

Scn5a heterozygous null (Scn5a+/-) mice have historically been used to investigate arrhythmogenic mechanisms of diseases such as Brugada syndrome (BrS) and Lev's disease. Previously, we demonstrated that reducing ephaptic coupling (EpC) in ex vivo hearts exacerbates pharmacological voltage-gated sodium channel (Nav)1.5 loss of function (LOF). Whether this effect is consistent in a genetic Nav1.5 LOF model is yet to be determined. We hypothesized that loss of EpC would result in greater reduction in conduction velocity (CV) for the Scn5a+/- mouse relative to wild type (WT). In vivo ECGs and ex vivo optical maps were recorded from Langendorff-perfused Scn5a+/- and WT mouse hearts. EpC was reduced with perfusion of a hyponatremic solution, the clinically relevant osmotic agent mannitol, or a combination of the two. Neither in vivo QRS duration nor ex vivo CV during normonatremia was significantly different between the two genotypes. In agreement with our hypothesis, we found that hyponatremia severely slowed CV and disrupted conduction for 4/5 Scn5a+/- mice, but 0/6 WT mice. In addition, treatment with mannitol slowed CV to a greater extent in Scn5a+/- relative to WT hearts. Unexpectedly, treatment with mannitol during hyponatremia did not further slow CV in either genotype, but resolved the disrupted conduction observed in Scn5a+/- hearts. Similar results in guinea pig hearts suggest the effects of mannitol and hyponatremia are not species specific. In conclusion, loss of EpC through either hyponatremia or mannitol alone results in slowed or disrupted conduction in a genetic model of Nav1.5 LOF. However, the combination of these interventions attenuates conduction slowing.NEW & NOTEWORTHY Cardiac sodium channel loss of function (LOF) diseases such as Brugada syndrome (BrS) are often concealed. We optically mapped mouse hearts with reduced sodium channel expression (Scn5a+/-) to evaluate whether reduced ephaptic coupling (EpC) can unmask conduction deficits. Data suggest that conduction deficits in the Scn5a+/- mouse may be unmasked by treatment with hyponatremia and perinexal widening via mannitol. These data support further investigation of hyponatremia and mannitol as novel diagnostics for sodium channel loss of function diseases.

Extracellular Perinexal Separation Is a Principal Determinant of Cardiac Conduction.

BACKGROUND: Cardiac conduction is understood to occur through gap junctions. Recent evidence supports ephaptic coupling as another mechanism of electrical communication in the heart. Conduction via gap junctions predicts a direct relationship between conduction velocity (CV) and bulk extracellular resistance. By contrast, ephaptic theory is premised on the existence of a biphasic relationship between CV and the volume of specialized extracellular clefts within intercalated discs such as the perinexus. Our objective was to determine the relationship between ventricular CV and structural changes to micro- and nanoscale extracellular spaces. METHODS: Conduction and Cx43 (connexin43) protein expression were quantified from optically mapped guinea pig whole-heart preparations perfused with the osmotic agents albumin, mannitol, dextran 70 kDa, or dextran 2 MDa. Peak sodium current was quantified in isolated guinea pig ventricular myocytes. Extracellular resistance was quantified by impedance spectroscopy. Intercellular communication was assessed in a heterologous expression system with fluorescence recovery after photobleaching. Perinexal width was quantified from transmission electron micrographs. RESULTS: CV primarily in the transverse direction of propagation was significantly reduced by mannitol and increased by albumin and both dextrans. The combination of albumin and dextran 70 kDa decreased CV relative to albumin alone. Extracellular resistance was reduced by mannitol, unchanged by albumin, and increased by both dextrans. Cx43 expression and conductance and peak sodium currents were not significantly altered by the osmotic agents. In response to osmotic agents, perinexal width, in order of narrowest to widest, was albumin with dextran 70 kDa; albumin or dextran 2 MDa; dextran 70 kDa or no osmotic agent, and mannitol. When compared in the same order, CV was biphasically related to perinexal width. CONCLUSIONS: Cardiac conduction does not correlate with extracellular resistance but is biphasically related to perinexal separation, providing evidence that the relationship between CV and extracellular volume is determined by ephaptic mechanisms under conditions of normal gap junctional coupling.

Reevaluating methods reporting practices to improve reproducibility: an analysis of methodological rigor for the Langendorff whole heart technique.

In recent decades, the scientific community has seen an increased interest in rigor and reproducibility. In 2017, concerns about methodological thoroughness and reporting practices were implicated as significant barriers to reproducibility within the preclinical cardiovascular literature, particularly in studies using animal research. The Langendorff, whole heart technique has proven to be an invaluable research tool, being modified in a myriad of ways to probe questions across the spectrum of physiological and pathophysiological functions of the heart. As a result, significant variability in the application of the Langendorff technique exists. This literature review quantifies the different methods employed in the implementation of the Langendorff technique and provides brief examples of how individual parametric differences can impact the outcomes and interpretation of studies. From 2017 to 2020, significant variability of animal models, anesthesia, cannulation time, perfusate composition, pH, and temperature demonstrate that the technique has diversified to meet new challenges and answer different scientific questions. The review also reveals which individual methods are most frequently reported, even if there is no explicit agreement upon which parameters should be reported. The analysis of methods related to the Langendorff technique suggests a framework for considering methodological approach when interpreting seemingly contradictory results, rather than concluding that results are irreproducible.

Hypernatremia and intercalated disc edema synergistically exacerbate long-QT syndrome type 3 phenotype.

Cardiac voltage-gated sodium channel gain-of-function prolongs repolarization in the long-QT syndrome type 3 (LQT3). Previous studies suggest that narrowing the perinexus within the intercalated disc, leading to rapid sodium depletion, attenuates LQT3-associated action potential duration (APD) prolongation. However, it remains unknown whether extracellular sodium concentration modulates APD prolongation during sodium channel gain-of-function. We hypothesized that elevated extracellular sodium concentration and widened perinexus synergistically prolong APD in LQT3. LQT3 was induced with sea anemone toxin (ATXII) in Langendorff-perfused guinea pig hearts (n = 34). Sodium concentration was increased from 145 to 160 mM. Perinexal expansion was induced with mannitol or the sodium channel ß1-subunit adhesion domain antagonist (ßadp1). Epicardial ventricular action potentials were optically mapped. Individual and combined effects of varying clefts and sodium concentrations were simulated in a computational model. With ATXII, both mannitol and ßadp1 significantly widened the perinexus and prolonged APD, respectively. The elevated sodium concentration alone significantly prolonged APD as well. Importantly, the combination of elevated sodium concentration and perinexal widening synergistically prolonged APD. Computational modeling results were consistent with animal experiments. Concurrently elevating extracellular sodium and increasing intercalated disc edema prolongs repolarization more than the individual interventions alone in LQT3. This synergistic effect suggests an important clinical implication that hypernatremia in the presence of cardiac edema can markedly increase LQT3-associated APD prolongation. Therefore, to our knowledge, this is the first study to provide evidence of a tractable and effective strategy to mitigate LQT3 phenotype by means of managing sodium levels and preventing cardiac edema in patients.NEW & NOTEWORTHY This is the first study to demonstrate that the long-QT syndrome type 3 (LQT3) phenotype can be exacerbated or concealed by regulating extracellular sodium concentrations and/or the intercalated disc separation. The animal experiments and computational modeling in the current study reveal a critically important clinical implication: sodium dysregulation in the presence of edema within the intercalated disc can markedly increase the risk of arrhythmia in LQT3. These findings strongly suggest that maintaining extracellular sodium within normal physiological limits may be an effective and inexpensive therapeutic option for patients with congenital or acquired sodium channel gain-of-function diseases.

The conduction velocity-potassium relationship in the heart is modulated by sodium and calcium.

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.

Attenuating loss of cardiac conduction during no-flow ischemia through changes in perfusate sodium and calcium.

Myocardial ischemia leads to conduction slowing, cell-to-cell uncoupling, and arrhythmias. We previously demonstrated that varying perfusate sodium (Na+) and calcium (Ca2+) attenuates conduction slowing and arrhythmias during simulated ischemia with continuous perfusion. Cardioprotection was selectively associated with widening of the perinexus, a gap junction adjacent nanodomain important to ephaptic coupling. It is unknown whether perfusate composition affects the perinexus or ischemic conduction during nonsimulated ischemia, when coronary flow is reduced or halted. We hypothesized that altering preischemic perfusate composition could facilitate perinexal expansion and attenuate conduction slowing during global ischemia. To test this hypothesis, ex vivo guinea pig hearts (n = 49) were Langendorff perfused with 145 or 153 mM Na+ and 1.25 or 2.0 mM Ca2+ and optically mapped during 30 min of no-flow ischemia. Altering Na+ and Ca2+ did not substantially affect baseline conduction. Increasing Na+ and decreasing Ca2+ both lowered pacing thresholds, whereas increasing Ca2+ narrowed perinexal width (Wp). A least squares mean estimate revealed that reduced perfusate Na+ and Ca2+ resulted in the most severe conduction slowing during ischemia. Increasing Na+ alone modestly attenuated conduction slowing, yet significantly delayed the median time to conduction block (10 to 16 min). Increasing both Na+ and Ca2+ selectively widened Wp during ischemia (22.7 vs. 15.7 nm) and attenuated conduction slowing to the greatest extent. Neither repolarization nor levels of total or phosphorylated connexin43 correlated with conduction slowing or block. Thus, perfusate-dependent widening of the perinexus preserved ischemic conduction and may be an adaptive response to ischemic stress.NEW & NOTEWORTHY Conduction slowing during acute ischemia creates an arrhythmogenic substrate. We have shown that extracellular ionic concentrations can alter conduction by modulating ephaptic coupling. Here, we demonstrate increased extracellular sodium and calcium significantly attenuate conduction slowing during no-flow ischemia. This effect was associated with selective widening of the perinexus, an intercalated disc nanodomain and putative cardiac ephapse. These findings suggest that acute changes in ephaptic coupling may serve as an adaptive response to ischemic stress.

The adhesion function of the sodium channel beta subunit (ß1) contributes to cardiac action potential propagation.

Computational modeling indicates that cardiac conduction may involve ephaptic coupling - intercellular communication involving electrochemical signaling across narrow extracellular clefts between cardiomyocytes. We hypothesized that ß1(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 ß1 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, ßadp1, potently and selectively inhibited ß1-mediated adhesion, in electric cell-substrate impedance sensing studies. ßadp1 significantly widened perinexi in guinea pig ventricles, and selectively reduced perinexal INa, but not whole cell INa, in myocyte monolayers. In optical mapping studies, ßadp1 precipitated arrhythmogenic conduction slowing. In summary, ß1-mediated adhesion at the perinexus facilitates action potential propagation between cardiomyocytes, and may represent a novel target for anti-arrhythmic therapies.

Electrophysiologic effects of the IK1 inhibitor PA-6 are modulated by extracellular potassium in isolated guinea pig hearts.

The pentamidine analog PA-6 was developed as a specific inward rectifier potassium current (IK1) antagonist, because established inhibitors either lack specificity or have side effects that prohibit their use in vivo. We previously demonstrated that BaCl2, an established IK1 inhibitor, could prolong action potential duration (APD) and increase cardiac conduction velocity (CV). However, few studies have addressed whether targeted IK1 inhibition similarly affects ventricular electrophysiology. The aim of this study was to determine the effects of PA-6 on cardiac repolarization and conduction in Langendorff-perfused guinea pig hearts. PA-6 (200 nm) or vehicle was perfused into ex-vivo guinea pig hearts for 60 min. Hearts were optically mapped with di-4-ANEPPS to quantify CV and APD at 90% repolarization (APD90). Ventricular APD90 was significantly prolonged in hearts treated with PA-6 (115 ± 2% of baseline; P < 0.05), but not vehicle (105 ± 2% of baseline). PA-6 slightly, but significantly, increased transverse CV by 7%. PA-6 significantly prolonged APD90 during hypokalemia (2 mmol/L [K+]o), although to a lesser degree than observed at 4.56 mmol/L [K+]o In contrast, the effect of PA-6 on CV was more pronounced during hypokalemia, where transverse CV with PA-6 (24 ± 2 cm/sec) was significantly faster than with vehicle (13 ± 3 cm/sec, P < 0.05). These results show that under normokalemic conditions, PA-6 significantly prolonged APD90, whereas its effect on CV was modest. During hypokalemia, PA-6 prolonged APD90 to a lesser degree, but profoundly increased CV Thus, in intact guinea pig hearts, the electrophysiologic effects of the IK1 inhibitor, PA-6, are [K+]o-dependent.

Reduced Arrhythmia Inducibility With Calcium/Calmodulin-dependent Protein Kinase II Inhibition in Heart Failure Rabbits.

RATIONALE: Calcium/calmodulin-dependent protein kinase II (CaMKII) is activated in heart failure (HF) and can contribute to arrhythmias induced by ß-adrenergic receptor-mediated sarcoplasmic reticulum calcium leak. OBJECTIVE: To evaluate the effect of CaMKII inhibition on ventricular tachycardia (VT) induction in conscious HF and naive rabbits. METHODS AND RESULTS: Nonischemic HF was induced by aortic insufficiency and constriction. Electrocardiograms were recorded in rabbits pretreated with vehicle (saline) or the CaMKII inhibitor KN-93 (300 µg/kg); VT was induced by infusion of increasing doses of norepinephrine (1.56-25 µg·kg⁻¹·min⁻¹) in naive (n = 8) and HF (n = 7) rabbits. With saline, median VT dose threshold in HF was 6.25 versus 12.5 µg·kg⁻¹·min⁻¹ norepinephrine in naive rabbits (P = 0.06). Pretreatment with KN-93 significantly increased VT threshold in HF and naive rabbits (median = 25 µg·kg⁻¹·min⁻¹, P < 0.05 vs. saline for both groups). Mean cycle length of VT initiation was shorter in HF (221 ± 20 milliseconds) than naive (296 ± 23 milliseconds, P < 0.05) rabbits with saline; this difference was not significant after treatment with KN-93. CONCLUSIONS: KN-93 significantly reduced arrhythmia inducibility and slowed initiation of VT, suggesting that CaMKII inhibition may have antiarrhythmic effects in the failing human heart.

Sodium channels in the Cx43 gap junction perinexus may constitute a cardiac ephapse: an experimental and modeling study.

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.

Sex differences in ß-adrenergic responsiveness of action potentials and intracellular calcium handling in isolated rabbit hearts.

Cardioprotection in females, as observed in the setting of heart failure, has been attributed to sex differences in intracellular calcium handling and its modulation by ß-adrenergic signaling. However, further studies examining sex differences in ß-adrenergic responsiveness have yielded inconsistent results and have mostly been limited to studies of contractility, ion channel function, or calcium handling alone. Given the close interaction of the action potential (AP) and intracellular calcium transient (CaT) through the process of excitation-contraction coupling, the need for studies exploring the relationship between agonist-induced AP and calcium handling changes in female and male hearts is evident. Thus, the aim of this study was to use optical mapping to examine sex differences in ventricular APs and CaTs measured simultaneously from Langendorff-perfused hearts isolated from naïve adult rabbits during ß-adrenergic stimulation. The non-selective ß-agonist isoproterenol (Iso) decreased AP duration (APD90), CaT duration (CaD80), and the decay constant of the CaT (τ) in a dose-dependent manner (1-316.2 nM), with a plateau at doses ≥31.6 nM. The Iso-induced changes in APD90 and τ (but not CaD80) were significantly smaller in female than male hearts. These sex differences were more significant at faster (5.5 Hz) than resting rates (3 Hz). Treatment with Iso led to the development of spontaneous calcium release (SCR) with a dose threshold of 31.6 nM. While SCR occurrence was similar in female (49%) and male (53%) hearts, the associated ectopic beats had a lower frequency of occurrence (16% versus 40%) and higher threshold (100 nM versus 31.6 nM) in female than male hearts (p<0.05). In conclusion, female hearts had a decreased capacity to respond to ß-adrenergic stimulation, particularly under conditions of increased demand (i.e. faster pacing rates and "maximal" levels of Iso effects), however this reduced ß-adrenergic responsiveness of female hearts was associated with reduced arrhythmic activity.

Spontaneous calcium release in tissue from the failing canine heart.

Abnormalities 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 < 0.05] and increased duration (252 vs. 229 s, P < 0.05) compared with those of normal. Under control conditions and during beta-adrenergic stimulation, TA was more frequent in HF (53% and 93%, respectively) compared with normal (0% and 55%, respectively, P < 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 < 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 enhances susceptibility to arrhythmogenic cardiac alternans.

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. OBJECTIVE: This study sought to investigate the hypothesis that HF enhances susceptibility to arrhythmogenic cardiac alternans. METHODS: High-resolution transmural optical mapping was performed in canine wedge preparations from normal (n = 8) and HF (n = 8) hearts produced by rapid ventricular pacing. RESULTS: HF significantly (P < .004) lowered the heart rate (HR) threshold for action potential duration alternans (APD-ALT) from 236 +/- 25 beats/min to 185 +/- 25 beats/min. In dual optical mapping of action potentials and intracellular Ca experiments (n = 16), HF lowered the HR threshold for Ca-ALT (beat-to-beat alternations of cellular Ca cycling) from 238 +/- 35 to 177 +/- 26 beats/min (P < .005). Importantly: (1) Ca-ALT always either developed at slower HR or simultaneously with APD-ALT in the same cells, and (2) the magnitude of Ca-ALT and APD-ALT were closely correlated (P < .05). HF similarly lowered the HR threshold for Ca-ALT in isolated myocytes under nonalternating action potential clamp, indicating that HF enhances susceptibility to cellular alternans independent of HF-associated changes in repolarization. Importantly, HF significantly (P < .02) lowered the HR threshold for spatially discordant arrhythmogenic alternans (different regions of cells alternating in opposite phase, DIS-ALT). Ventricular fibrillation (VF) was induced in 88% of HF preparations, but only 12% of normal preparations (P < .003) and was uniformly preceded by development of DIS-ALT. CONCLUSION: Heart failure increases the susceptibility to arrhythmogenic cardiac alternans, which arises from HF-induced impairment in calcium cycling.

Ryanodine receptor dysfunction and triggered activity in the heart.

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 high-resolution optical mapping of intracellular calcium and transmembrane potential in the canine left ventricular wedge preparation (n = 28). Rapid pacing was used to initiate triggered activity under normal and abnormal RyR conditions induced by FKBP12.6 dissociation and beta-adrenergic stimulation (20-150 microM rapamycin, 0.2 microM 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 < 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.