EPAC1 and 2 inhibit K+ currents via PLC/PKC and NOS/PKG pathways in rat ventricular cardiomyocytes.

The Exchange Protein directly Activated by cAMP (EPAC) has been implicated in cardiac pro-arrhythmic signalling pathways including spontaneous diastolic Ca2+ leak from sarcoplasmic reticulum and increased Action Potential Duration (APD) in isolated ventricular cardiomyocytes. The AP lengthening following acute EPAC activation is mainly due to a decrease of repolarizing steady state K+ current (IKSS) but the mechanisms involved remain unknown. This study aimed to assess the role of EPAC1 and 2 in the decrease of IKSS and to investigate the underlying signalling pathways. AP and K+ currents were recorded with the whole cell configuration of the patch-clamp technique in freshly isolated rat ventricular myocytes. EPAC1 and 2 were pharmacologically activated with 8-CPTAM (10 µmol/L) and inhibited with R-Ce3F4 and ESI-05, respectively. Inhibition of EPAC1 and EPAC2 significantly decreased the effect of 8-CPTAM on APD and IKSS showing that both EPAC isoforms are involved in these effects. Unexpectedly, CaMKII inhibition by AIP or KN-93, and Ca2+ chelation by intracellular BAPTA, did not impact the response to 8-CPTAM. However, inhibition of PLC/PKC and NOS/PKG pathways partially prevents the 8-CPTAM-dependent decrease of IKSS. Finally, the cumulative inhibition of PKC and PKG blocked the 8-CPTAM effect, suggesting that these two actors work along parallel pathways to regulate IKSS upon EPAC activation. On the basis of such findings, we propose that EPAC1 and 2 are involved in APD lengthening by inhibiting a K+ current via both PLC/PKC and NOS/PKG pathways. This may have pathological implications since EPAC is upregulated in diseases such as cardiac hypertrophy.

Regional Differences in Ca2+ Signaling and Transverse-Tubules across Left Atrium from Adult Sheep.

Cardiac excitation-contraction coupling can be different between regions of the heart. Little is known at the atria level, specifically in different regions of the left atrium. This is important given the role of cardiac myocytes from the pulmonary vein sleeves, which are responsible for ectopic activity during atrial fibrillation. In this study, we present a new method to isolate atrial cardiac myocytes from four different regions of the left atrium of a large animal model, sheep, highly relevant to humans. Using collagenase/protease we obtained calcium-tolerant atrial cardiac myocytes from the epicardium, endocardium, free wall and pulmonary vein regions. Calcium transients were slower (time to peak and time to decay) in free wall and pulmonary vein myocytes compared to the epicardium and endocardium. This is associated with lower t-tubule density. Overall, these results suggest regional differences in calcium transient and t-tubule density across left atria, which may play a major role in the genesis of atrial fibrillation.

The calcium stored in the sarcoplasmic reticulum acts as a safety mechanism in rainbow trout heart.

Cardiomyocyte contraction depends on rapid changes in intracellular Ca(2+). In mammals, Ca(2+) influx as L-type Ca(2+) current (ICa) triggers the release of Ca(2+) from sarcoplasmic reticulum (SR) and Ca(2+)-induced Ca(2+) release (CICR) is critical for excitation-contraction coupling. In fish, the relative contribution of external and internal Ca(2+) is unclear. Here, we characterized the role of ICa to trigger SR Ca(2+) release in rainbow trout ventricular myocytes using ICa regulation by Ca(2+) as an index of CICR. ICa was recorded with a slow (EGTA) or fast (BAPTA) Ca(2+) chelator in control and isoproterenol conditions. In the absence of ß-adrenergic stimulation, the rate of ICa inactivation was not significantly different in EGTA and BAPTA (27.1 ± 1.8 vs. 30.3 ± 2.4 ms), whereas with isoproterenol (1 µM), inactivation was significantly faster with EGTA (11.6 ± 1.7 vs. 27.3 ± 1.6 ms). When barium was the charge carrier, inactivation was significantly slower in both conditions (61.9 ± 6.1 vs. 68.0 ± 8.7 ms, control, isoproterenol). Quantification revealed that without isoproterenol, only 39% of ICa inactivation was due to Ca(2+), while with isoproterenol, inactivation was Ca(2+)-dependent (∼65%) and highly reliant on SR Ca(2+) (∼46%). Thus, SR Ca(2+) is not released in basal conditions, and ICa is the main trigger of contraction, whereas during a stress response, SR Ca(2+) is an important source of cytosolic Ca(2+). This was not attributed to differences in SR Ca(2+) load because caffeine-induced transients were not different in both conditions. Therefore, Ca(2+) stored in SR of trout cardiomyocytes may act as a safety mechanism, allowing greater contraction when higher contractility is required, such as stress or exercise.

Left atrial cardiomyopathy: Pathophysiological insights, assessment methods and clinical implications.

Atrial cardiomyopathy is defined as any complex of structural, architectural, contractile or electrophysiological changes affecting atria, with the potential to produce clinically relevant manifestations. Most of our knowledge about the mechanistic aspects of atrial cardiomyopathy is derived from studies investigating animal models of atrial fibrillation and atrial tissue samples obtained from individuals who have a history of atrial fibrillation. Several noninvasive tools have been reported to characterize atrial cardiomyopathy in patients, which may be relevant for predicting the risk of incident atrial fibrillation and its related outcomes, such as stroke. Here, we provide an overview of the pathophysiological mechanisms involved in atrial cardiomyopathy, and discuss the complex interplay of these mechanisms, including aging, left atrial pressure overload, metabolic disorders and genetic factors. We discuss clinical tools currently available to characterize atrial cardiomyopathy, including electrocardiograms, cardiac imaging and serum biomarkers. Finally, we discuss the clinical impact of atrial cardiomyopathy, and its potential role for predicting atrial fibrillation, stroke, heart failure and dementia. Overall, this review aims to highlight the critical need for a clinically relevant definition of atrial cardiomyopathy to improve treatment strategies.

NGF increases Connexin-43 expression and function in pulmonary arterial smooth muscle cells to induce pulmonary artery hyperreactivity.

AIMS: Pulmonary hypertension (PH) is characterised by an increase in pulmonary arterial pressure, ultimately leading to right ventricular failure and death. We have previously shown that nerve growth factor (NGF) plays a critical role in PH. Our objectives here were to determine whether NGF controls Connexin-43 (Cx43) expression and function in the pulmonary arterial smooth muscle, and whether this mechanism contributes to NGF-induced pulmonary artery hyperreactivity. METHODS AND RESULTS: NGF activates its TrkA receptor to increase Cx43 expression, phosphorylation, and localization at the plasma membrane in human pulmonary arterial smooth muscle cells, thus leading to enhanced activity of Cx43-dependent GAP junctions as shown by Lucifer Yellow dye assay transfer and fluorescence recovery after photobleaching -FRAP- experiments. Using both in vitro pharmacological and in vivo SiRNA approaches, we demonstrate that NGF-dependent increase in Cx43 expression and activity in the rat pulmonary circulation causes pulmonary artery hyperreactivity. We also show that, in a rat model of PH induced by chronic hypoxia, in vivo blockade of NGF or of its TrkA receptor significantly reduces Cx43 increased pulmonary arterial expression induced by chronic hypoxia and displays preventive effects on pulmonary arterial pressure increase and right heart hypertrophy. CONCLUSIONS: Modulation of Cx43 by NGF in pulmonary arterial smooth muscle cells contributes to NGF-induced alterations of pulmonary artery reactivity. Since NGF and its TrkA receptor play a role in vivo in Cx43 increased expression in PH induced by chronic hypoxia, these NGF/Cx43-dependent mechanisms may therefore play a significant role in human PH pathophysiology.

Epac activator critically regulates action potential duration by decreasing potassium current in rat adult ventricle.

Sympathetic stimulation is an important modulator of cardiac function via the classic cAMP-dependent signaling pathway, PKA. Recently, this paradigm has been challenged by the discovery of a family of guanine nucleotide exchange proteins directly activated by cAMP (Epac), acting in parallel to the classic signaling pathway. In cardiac myocytes, Epac activation is known to modulate Ca(2+) cycling yet their actions on cardiac ionic currents remain poorly characterized. This study attempts to address this paucity of information using the patch clamp technique to record action potential (AP) and ionic currents on rat ventricular myocytes. Epac was selectively activated by 8-CPT-AM (acetoxymethyl ester form of 8-CPT). AP amplitude, maximum depolarization rate and resting membrane amplitude were unaltered by 8-CPT-AM, strongly suggesting that Na(+) current and inward rectifier K(+) current are not regulated by Epac. In contrast, AP duration was significantly increased by 8-CPT-AM (prolongation of duration at 50% and 90% of repolarization by 41±10% and 43±8% respectively, n=11). L-type Ca(2+) current density was unaltered by 8-CPT-AM (n=16) so this cannot explain the action potential lengthening. However, the steady state component of K(+) current was significantly inhibited by 8-CPT-AM (-38±6%, n=15), while the transient outward K(+) current was unaffected by 8-CPT-AM. These effects were PKA-independent since they were observed in the presence of PKA inhibitor KT5720. Isoprenaline (100nM) induced a significant prolongation of AP duration, even in the presence of KT5720. This study provides the first evidence that the cAMP-binding protein Epac critically modulates cardiac AP duration by decreasing steady state K(+) current. These observations may be relevant to diseases in which Epac is upregulated, like cardiac hypertrophy.

Critical Link between Calcium Regional Heterogeneity and Atrial Fibrillation Susceptibility in Sheep Left Atria.

BACKGROUND: Atrial fibrillation is the most sustained form of arrhythmia in the human population that leads to important electrophysiological and structural cardiac remodeling as it progresses into a chronic form. Calcium is an established key player of cellular electrophysiology in the heart, yet to date, there is no information that maps calcium signaling across the left atrium. OBJECTIVE: The aim of this study is to determine whether calcium signaling is homogenous throughout the different regions of the left atrium. This work tests the hypothesis that differences across the healthy left atrium contribute to a unique, region-dependent calcium cycling and participates in the pro-arrhythmic activity during atrial fibrillation. METHODS: An animal model relevant to human cardiac function (the sheep) was used to characterize both the electrical activity and the calcium signaling of three distinct left atrium regions (appendage, free wall and pulmonary veins) in control conditions and after acetylcholine perfusion (5 µM) to induce acute atrial fibrillation. High-resolution dual calcium-voltage optical mapping on the left atria of sheep was performed to explore the spatiotemporal dynamics of calcium signaling in relation to electrophysiological properties. RESULTS: Action potential duration (at 80% repolarization) was not significantly different in the three regions of interest for the three pacing sites. In contrast, the time to 50% calcium transient decay was significantly different depending on the region paced and recorded. Acetylcholine perfusion and burst pacing-induced atrial fibrillation when pulmonary veins and appendage regions were paced but not when the free wall region was. Dantrolene (a ryanodine receptor blocker) did not reduce atrial fibrillation susceptibility. CONCLUSION: These data provide the first evidence of heterogenous calcium signaling across the healthy left atrium. Such basal regional differences may be exacerbated during the progression of atrial fibrillation and thus play a crucial role in focal arrhythmia initiation without ryanodine receptor gating modification.

Inhibition of EPAC1 signaling pathway alters atrial electrophysiology and prevents atrial fibrillation.

Introduction: Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and is associated with increased mortality and morbidity. The Exchange Protein directly Activated by cAMP (EPAC), has been implicated in pro-arrhythmic signaling pathways in the atria, but the underlying mechanisms remain unknown. Methods: In this study, we investigated the involvement of EPAC1 and EPAC2 isoforms in the genesis of AF in wild type (WT) mice and knockout (KO) mice for EPAC1 or EPAC2. We also employed EPAC pharmacological modulators to selectively activate EPAC proteins (8-CPT-AM; 10 µM), or inhibit either EPAC1 (AM-001; 20 µM) or EPAC2 (ESI-05; 25 µM). Transesophageal stimulation was used to characterize the induction of AF in vivo in mice. Optical mapping experiments were performed on isolated mouse atria and cellular electrophysiology was examined by whole-cell patch-clamp technique. Results: In wild type mice, we found 8-CPT-AM slightly increased AF susceptibility and that this was blocked by the EPAC1 inhibitor AM-001 but not the EPAC2 inhibitor ESI-05. Consistent with this, in EPAC1 KO mice, occurrence of AF was observed in 3/12 (vs. 4/10 WT littermates) and 4/10 in EPAC2 KO (vs. 5/10 WT littermates). In wild type animals, optical mapping experiments revealed that 8-CPT-AM perfusion increased action potential duration even in the presence of AM-001 or ESI-05. Interestingly, 8-CPT-AM perfusion decreased conduction velocity, an effect blunted by AM-001 but not ESI-05. Patch-clamp experiments demonstrated action potential prolongation after 8-CPT-AM perfusion in both wild type and EPAC1 KO mice and this effect was partially prevented by AM-001 in WT. Conclusion: Together, these results indicate that EPAC1 and EPAC2 signaling pathways differentially alter atrial electrophysiology but only the EPAC1 isoform is involved in the genesis of AF. Selective blockade of EPAC1 with AM-001 prevents AF in mice.

Functional Epicardial Conduction Disturbances Due to a SCN5A Variant Associated With Brugada Syndrome.

BACKGROUND: Brugada syndrome is a significant cause of sudden cardiac death (SCD), but the underlying mechanisms remain hypothetical. OBJECTIVES: This study aimed to elucidate this knowledge gap through detailed ex vivo human heart studies. METHODS: A heart was obtained from a 15-year-old adolescent boy with normal electrocardiogram who experienced SCD. Postmortem genotyping was performed, and clinical examinations were done on first-degree relatives. The right ventricle was optically mapped, followed by high-field magnetic resonance imaging and histology. Connexin-43 and NaV1.5 were localized by immunofluorescence, and RNA and protein expression levels were studied. HEK-293 cell surface biotinylation assays were performed to examine NaV1.5 trafficking. RESULTS: A Brugada-related SCD diagnosis was established for the donor because of a SCN5A Brugada-related variant (p.D356N) inherited from his mother, together with a concomitant NKX2.5 variant of unknown significance. Optical mapping demonstrated a localized epicardial region of impaired conduction near the outflow tract, in the absence of repolarization alterations and microstructural defects, leading to conduction blocks and figure-of-8 patterns. NaV1.5 and connexin-43 localizations were normal in this region, consistent with the finding that the p.D356N variant does not affect the trafficking, nor the expression of NaV1.5. Trends of decreased NaV1.5, connexin-43, and desmoglein-2 protein levels were noted; however, the RT-qPCR results suggested that the NKX2-5 variant was unlikely to be involved. CONCLUSIONS: This study demonstrates for the first time that SCD associated with a Brugada-SCN5A variant can be caused by localized functionally, not structurally, impaired conduction.

Assessment of Cardiac Toxicity of Manganese Chloride for Cardiovascular Magnetic Resonance.

MRI is widely used in cardiology to characterize the structure and function of the heart. Currently, gadolinium-based contrast agents are widely used to improve sensitivity and specificity of diagnostic images. Recently, Manganese, a calcium analogue, has emerged as a complementary contrast agent with the potential to reveal remaining viable cells within altered tissue. Imaging applications may be limited by substantial toxicity of manganese. Indeed, cardiac safety of manganese is not yet comprehensively assessed. In this study we investigated the effect of MnCl2 (1-100 µM) on cardiac function. Hemodynamic function was determined ex vivo using an isolated working rat heart preparation. HL-1 cardiac myocytes were used to investigate cell viability (calcein AM) and calcium cycling (Cal-520 a.m.). Rat ventricular cardiomyocytes were dissociated by enzymatic digestion. Action potentials and calcium currents were recorded using the patch clamp technique. MRI experiments were performed at 1.5T on formalin-fixed rat hearts, previously perfused with MnCl2. MnCl2 perfusion from 1 up to 100 µM in isolated working hearts did not alter left ventricular hemodynamic parameters. Contractility and relaxation index were not altered up to 50 µM MnCl2. In HL-1 cardiac myocytes, incubation with increasing concentrations of MnCl2 did not impact cell viability. The amplitude of the calcium transients were significantly reduced at 50 and 100 µM MnCl2. In freshly isolated ventricular myocytes, action potential duration at 20, 50 and 90% of repolarization were not modified up to 10 µM of MnCl2. L-type calcium current amplitude was significantly decreased by 50 and 100 µM of MnCl2. MRI on heart perfused with 25 and 100 µM of MnCl2 showed a dose dependent decrease in the T1 relaxation time. In conclusion, our results show that low concentrations of MnCl2 (up to 25 µM) can be used as a contrast agent in MRI, without significant impact on cardiac hemodynamic or electrophysiology parameters.

TREK-1 in the heart: Potential physiological and pathophysiological roles.

The TREK-1 channel belongs to the TREK subfamily of two-pore domains channels that are activated by stretch and polyunsaturated fatty acids and inactivated by Protein Kinase A phosphorylation. The activation of this potassium channel must induce a hyperpolarization of the resting membrane potential and a shortening of the action potential duration in neurons and cardiac cells, two phenomena being beneficial for these tissues in pathological situations like ischemia-reperfusion. Surprisingly, the physiological role of TREK-1 in cardiac function has never been thoroughly investigated, very likely because of the lack of a specific inhibitor. However, possible roles have been unraveled in pathological situations such as atrial fibrillation worsened by heart failure, right ventricular outflow tract tachycardia or pulmonary arterial hypertension. The inhomogeneous distribution of TREK-1 channel within the heart reinforces the idea that this stretch-activated potassium channel might play a role in cardiac areas where the mechanical constraints are important and need a particular protection afforded by TREK-1. Consequently, the main purpose of this mini review is to discuss the possible role played by TREK -1 in physiological and pathophysiological conditions and its potential role in mechano-electrical feedback. Improved understanding of the role of TREK-1 in the heart may help the development of promising treatments for challenging cardiac diseases.

Phenanthrene impacts zebrafish cardiomyocyte excitability by inhibiting IKr and shortening action potential duration.

Air pollution is an environmental hazard that is associated with cardiovascular dysfunction. Phenanthrene is a three-ringed polyaromatic hydrocarbon that is a significant component of air pollution and crude oil and has been shown to cause cardiac dysfunction in marine fishes. We investigated the cardiotoxic effects of phenanthrene in zebrafish (Danio rerio), an animal model relevant to human cardiac electrophysiology, using whole-cell patch-clamp of ventricular cardiomyocytes. First, we show that phenanthrene significantly shortened action potential duration without altering resting membrane potential or upstroke velocity (dV/dt). L-type Ca2+ current was significantly decreased by phenanthrene, consistent with the decrease in action potential duration. Phenanthrene blocked the hERG orthologue (zfERG) native current, IKr, and accelerated IKr deactivation kinetics in a dose-dependent manner. Furthermore, we show that phenanthrene significantly inhibits the protective IKr current envelope, elicited by a paired ventricular AP-like command waveform protocol. Phenanthrene had no effect on other IK. These findings demonstrate that exposure to phenanthrene shortens action potential duration, which may reduce refractoriness and increase susceptibility to certain arrhythmia triggers, such as premature ventricular contractions. These data also reveal a previously unrecognized mechanism of polyaromatic hydrocarbon cardiotoxicity on zfERG by accelerating deactivation and decreasing IKr protective current.

Mechanisms underlying adaptation of action potential duration by pacing rate in rat myocytes.

Heart rate is an essential determinant of cardiac performance. In rat ventricular myocytes, a sudden increase in rate yields to a prolongation of the action potential duration (APD). The mechanism underlying this prolongation is controversial: it has been proposed that the longer APD is due to either: (1) a decrease in K+ currents only or (2) an increase in Ca2+ current only. The aim of this study was to quantitatively investigate the contribution of Ca2+ and K+ currents in the adaptation of APD to pacing rate. Simulation using a mathematical model of ventricular rat cardiac cell model [Pandit, S.V., Clark, R.B., Giles, W.R., Demir, S.S., 2001. A mathematical model of action potential heterogeneity in adult rat left ventricular myocytes. Biophys. J. 81, 3029-3051] predicted a role in the prolongation of APD for K+ currents only. In patch clamp experiments, increasing the pacing rate leads to a significant increase in APD in both control and detubulated myocytes, although it was more marked in control than detubulated myocytes. Supporting the model prediction, we observed that increasing stimulation frequency leads to a decrease in K+ currents in voltage clamped rat ventricular myocytes (square and action potential waveforms), and to a similar extent in both cell types. We have also observed that frequency-dependent facilitation of Ca2+ current occurred in control cells but not in detubulated cells (square and action potential waveforms). From these experiments, we calculated that the relative contribution of Ca2+ and K+ currents to the longer APD following an increase in pacing rate is approximately 65% and approximately 35%, respectively. Therefore, in contrast to the model prediction, Ca2+ current has a significant role in the adaptation of APD to pacing rate. Finally, we have introduced a simplistic modification to the Pandit's model to account for the frequency-dependent facilitation of Ca2+ current.

The role of mammalian cardiac t-tubules in excitation-contraction coupling: experimental and computational approaches.

The sarcolemmal membrane of mammalian cardiac ventricular myocytes is characterized by the presence of invaginations called transverse tubules (t-tubules). Transverse tubules occur at the Z-line as transverse elements with longitudinal extensions. While the existence of t-tubules has been known for some time, recent experimental studies have suggested that their structure and function are more complex than previously believed. There are, however, aspects of t-tubule function that are not currently amenable to experimental investigation, but can be investigated using computational and mathematical approaches. Such studies have helped elucidate further the possible role of t-tubules in cell function. This review summarizes recent experimental and complementary computational studies which highlight the important role of t-tubules in cardiac excitation-contraction coupling.

t-Tubules and sarcoplasmic reticulum function in cardiac ventricular myocytes.

Although the existence of t-tubules in mammalian cardiac ventricular myocytes has been recognized for a long time, it now appears that their structure and function are more complex than previously believed. Recent work has provided evidence that many of the key proteins underlying excitation-contraction coupling are located predominantly at the t-tubules. L-type Ca(2+) current (I(Ca)) flowing across the t-tubule membrane provides a rapidly inactivating Ca(2+) influx that triggers Ca(2+) release from the sarcoplasmic reticulum (SR), thereby allowing rapid and synchronous Ca(2+) release throughout the cell; I(Ca) at the t-tubules also appears to be more sensitive than that at the surface membrane to regulation by beta-adrenergic stimulation and intracellular Ca(2+). In contrast, although its density is lower, I(Ca) flowing across the surface membrane inactivates slowly, and thus may help load the SR with Ca(2+). There is also increasing evidence that many of the mechanisms that remove Ca(2+) from the cytoplasm are located predominantly at the t-tubules, which therefore play an important role in determining cellular, and hence SR, Ca(2+) content. Thus, the t-tubules appear to play a central role in the increase and subsequent decrease of Ca(2+) during the systolic Ca(2+) transient. Remodelling of the t-tubules has been reported in cardiac pathologies, and may play a role in the altered cellular, and hence cardiac, function observed in such conditions.

Transient receptor potential channels in cardiac health and disease.

Transient receptor potential (TRP) channels are nonselective cationic channels that are generally Ca2+ permeable and have a heterogeneous expression in the heart. In the myocardium, TRP channels participate in several physiological functions, such as modulation of action potential waveform, pacemaking, conduction, inotropy, lusitropy, Ca2+ and Mg2+ handling, store-operated Ca2+ entry, embryonic development, mitochondrial function and adaptive remodelling. Moreover, TRP channels are also involved in various pathological mechanisms, such as arrhythmias, ischaemia-reperfusion injuries, Ca2+-handling defects, fibrosis, maladaptive remodelling, inherited cardiopathies and cell death. In this Review, we present the current knowledge of the roles of TRP channels in different cardiac regions (sinus node, atria, ventricles and Purkinje fibres) and cells types (cardiomyocytes and fibroblasts) and discuss their contribution to pathophysiological mechanisms, which will help to identify the best candidates for new therapeutic targets among the cardiac TRP family.

Characterization of isolated ventricular myocytes from adult zebrafish (Danio rerio).

The zebrafish is widely used for human related disease studies. Surprisingly, there is no information about the electrical activity of single myocytes freshly isolated from adult zebrafish ventricle. In this study, we present an enzymatic method to isolate ventricular myocytes from zebrafish heart that yield a large number of calcium tolerant cells. Ventricular myocytes from zebrafish were imaged using light and confocal microscopy. Myocytes were mostly rod shaped and responded by vigorous contraction to field electrical stimulation. Whole cell configuration of the patch clamp technique was used to record electrophysiological characteristics of myocytes. Action potentials present a long duration and a plateau phase and action potential duration decreases when increasing stimulation frequency (as observed in larger mammals). Together these results indicate that zebrafish is a species ideally suited for investigation of ion channels related mutation screening of cardiac alteration important in human.

No apparent requirement for neuronal sodium channels in excitation-contraction coupling in rat ventricular myocytes.

The majority of Na channels in the heart are composed of the tetrodotoxin (TTX)-resistant (KD, 2 to 6 micromol/L) "cardiac" NaV1.5 isoform; however, TTX-sensitive (KD, 1 to 25 nmol/L) "neuronal" Na channel isoforms have recently been detected in several cardiac preparations. In the present study, we determined the functional subcellular localization of Na channel isoforms (according to their TTX sensitivity) in rat ventricular myocytes by recording INa in control and detubulated myocytes. We found that TTX-sensitive INa (KD, &8.8 nmol/L) makes up 14+/-3% of total INa in control and < or =4% in detubulated myocytes and calculated that &80% of TTX-sensitive INa is located in the t-tubules, where it generates &1/3 of t-tubular INa. In contrast, TTX-resistant INa is located predominantly (&78%) at the surface membrane. We also investigated the possible contribution of TTX-sensitive INa to excitation-contraction coupling, using 200 nmol/L TTX to selectively block TTX-sensitive INa. TTX decreased the rate of depolarization of the action potential by 10% but did not delay the rise of systolic Ca2+ in the center of the cell (transverse confocal line scan), suggesting that TTX-sensitive INa does not play a role in synchronizing Ca2+ release at the t-tubules; the amplitude of the Ca2+ transient and contraction were also unchanged by 200 nmol/L TTX. The quantity of charge entering via ICa elicited by control or TTX action potential waveforms was similar, suggesting that the trigger for Ca2+ release is not altered by blocking TTX-sensitive INa. We conclude that neuronal INa is concentrated at the t-tubules, but there is no evidence of a requirement for these channels in normal excitation-contraction coupling in ventricular myocytes.

Compartmentalized Structure of the Moderator Band Provides a Unique Substrate for Macroreentrant Ventricular Tachycardia.

Background Papillary muscles are an important source of ventricular tachycardia (VT). Yet little is known about the role of the right ventricular (RV) endocavity structure, the moderator band (MB). The aim of this study was to determine the characteristics of the MB that may predispose to arrhythmia substrates. Methods Ventricular wedge preparations with intact MBs were studied from humans (n=2) and sheep (n=15; 40-50 kg). RV endocardium was optically mapped, and electrical recordings were measured along the MB and septum. S1S2 pacing of the RV free wall, MB, or combined S1-RV S2-MB sites were assessed. Human (n=2) and sheep (n=4) MB tissue constituents were assessed histologically. Results The MB structure was remarkably organized as 2 excitable, yet uncoupled compartments of myocardium and Purkinje. In humans, action potential duration heterogeneity between MB and RV myocardium was found (324.6±12.0 versus 364.0±8.4 ms; P<0.0001). S1S2-MB pacing induced unidirectional propagation via MB myocardium, permitting sustained macroreentrant VT. In sheep, the incidence of VT for RV, MB, and S1-RV S2-MB pacing was 1.3%, 5.1%, and 10.3%. Severing the MB led to VT termination, confirming a primary arrhythmic role. Inducible preparations had shorter action potential duration in the MB than RV (259.3±45.2 versus 300.7±38.5 ms; P<0.05), whereas noninducible preparations showed no difference (312.0±30.3 versus 310.0±24.6 ms, respectively). Conclusions The MB presents anatomic and electrical compartmentalization between myocardium and Purkinje fibers, providing a substrate for macroreentry. The vulnerability to sustain VT via this mechanism is dependent on MB structure and action potential duration gradients between the RV free wall and MB.