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Although the initiation, development, and maintenance of atrial fibrillation (AF) have been linked to alterations in myocyte redox state, the field lacks a complete understanding of the impact these changes may have on cellular signalling, atrial electrophysiology, and disease progression. Recent studies demonstrate spatiotemporal changes in reactive oxygen species production shortly after the induction of AF in animal models with an uncoupling of nitric oxide synthase activity ensuing in the presence of long-standing persistent AF, ultimately leading to a major shift in nitroso-redox balance. However, it remains unclear which radical or non-radical species are primarily involved in the underlying mechanisms of AF or which proteins are targeted for redox modification. In most instances, only free radical oxygen species have been assessed; yet evidence from the redox signalling field suggests that non-radical species are more likely to regulate cellular processes. A wider appreciation for the distinction of these species and how both species may be involved in the development and maintenance of AF could impact treatment strategies. In this review, we summarize how redox second-messenger systems are regulated and discuss the recent evidence for alterations in redox regulation in the atrial myocardium in the presence of AF, while identifying some critical missing links. We also examine studies looking at antioxidants for the prevention and treatment of AF and propose alternative redox targets that may serve as superior therapeutic options for the treatment of AF. [ABSTRACT FROM AUTHOR]

Aim TRPC3 is a non-selective cation channel, which forms a Ca2+ entry pathway involved in cardiac remodelling. Our aim was to analyse acute electrophysiological and contractile consequences of TRPC3 activation in the heart. Methods and results We used a murine model of cardiac TRPC3 overexpression and a novel TRPC3 agonist, GSK1702934A, to uncover (patho)physiological functions of TRPC3. GSK1702934A induced a transient, non-selective conductance and prolonged action potentials in TRPC3-overexpressing myocytes but lacked significant electrophysiological effects in wild-type myocytes. GSK1702934A transiently enhanced contractility and evoked arrhythmias in isolated Langendorff hearts from TRPC3-overexpressing but not wild-type mice. Interestingly, pro-arrhythmic effects outlasted TRPC3 current activation, were prevented by enhanced intracellular Ca2+ buffering, and suppressed by the NCX inhibitor 3',4'-dichlorobenzamil hydrochloride. GSK1702934A substantially promoted NCX currents in TRPC3-overexpressing myocytes. The TRPC3-dependent electrophysiologic, pro-arrhythmic, and inotropic actions of GSK1702934A were mimicked by angiotensin II (AngII). Immunocytochemistry demonstrated colocalization of TRPC3 with NCX1 and disruption of local interaction upon channel activation by either GSK1702934A or AngII. Conclusion Cardiac TRPC3 mediates Ca2+ and Na+ entry in proximity of NCX1, thereby elevating cellular Ca2+ levels and contractility. Excessive activation of TRPC3 is associated with transient cellular Ca2+overload, spatial uncoupling between TRPC3 and NCX1, and arrhythmogenesis. We propose TRPC3-NCX micro/nanodomain communication as determinant of cardiac contractility and susceptibility to arrhythmogenic stimuli. [ABSTRACT FROM AUTHOR]

Aims Cardiac function depends on the highly regulated and co-ordinate activity of a large ensemble of potassium channels that control myocyte repolarization. While voltage-gated K+ channels have been well characterized in the heart, much less is known about regulation and/or targeting of two-pore K+ channel (K2P) family members, despite their potential importance in modulation of heart function. Methods and results Here, we report a novel molecular pathway for membrane targeting of TREK-1, a mechano-sensitive K2P channel regulated by environmental and physical factors including membrane stretch, pH, and polyunsaturated fatty acids (e.g. arachidonic acid). We demonstrate that βIV-spectrin, an actin-associated protein, is co-localized with TREK-1 at the myocyte intercalated disc, associates with TREK-1 in the heart, and is required for TREK-1 membrane targeting. Mice expressing βIV-spectrin lacking TREK-1 binding (qv4J) display aberrant TREK-1 membrane localization, decreased TREK-1 activity, delayed action potential repolarization, and arrhythmia without apparent defects in localization/function of other cardiac potassium channel subunits. Finally, we report abnormal βIV-spectrin levels in human heart failure. Conclusions These data provide new insight into membrane targeting of TREK-1 in the heart and establish a broader role for βIV-spectrin in organizing functional membrane domains critical for normal heart function. [ABSTRACT FROM PUBLISHER]

Aims Alterations in cardiac acid–base balance can produce profound impact on excitation–contraction coupling and precipitate cardiac dysfunction and arrhythmias. A member of the solute carrier (SLC) family, Slc26a6, has been shown to be a chloride-hydroxyl exchanger and the predominant chloride-bicarbonate exchanger in the mouse heart. However, the exact isoforms and functional characteristics of cardiac Slc26a6 remain unknown. The objective of the present study is to determine the molecular identity of cardiac Slc26a6 isoforms, to examine their cellular expressions in the heart, and to test the function of Slc26a6 in cardiomyocytes. Methods and results We examined the expression and function of slc26a6 in mouse cardiomyocytes using RT–PCR, immunofluorescence confocal microscopy, and patch-clamp technique coupled with the fast solution exchange system. We identified four cardiac Slc26a6 isoforms, denoted C-a, C-b, C-c, and C-d, and detected significant expression of Slc26a6 in the plasma membrane of both atrial and ventricular myocytes. Isoforms C-a and C-b share the same sequence with the previously reported murine Slc26a6a and Slc26a6b, respectively. Isoform C-c lacks an alternate in-frame exon 12, whereas C-d is a C-terminal truncated form resulting from 102 bp exon insertion between exons 15 and 16 compared with C-b. Patch-clamp recordings demonstrated electrogenic Cl−/oxalate and electrogenic Cl−/HCO3− exchange activities in cardiomyocytes. Conclusion We demonstrate that cardiac myocytes express different isoforms of Slc26a6, which encode electrogenic Cl−/HCO3− and Cl−/oxalate exchangers. The electrogenic nature of the Cl−/HCO3− exchange of cardiac Slc26a6 suggests important roles in regulating acid–base balance in the heart. [ABSTRACT FROM PUBLISHER]

Early afterdepolarizations (EADs) are secondary voltage depolarizations during the repolarizing phase of the action potential, which can cause lethal cardiac arrhythmias. The occurrence of EADs requires a reduction in outward current and/or an increase in inward current, a condition called reduced repolarization reserve. However, this generalized condition is not sufficient for EAD genesis and does not explain the voltage oscillations manifesting as EADs. Here, we summarize recent progress that uses dynamical theory to build on and advance our understanding of EADs beyond the concept of repolarization reserve, towards the goal of developing a holistic and integrative view of EADs and their role in arrhythmogenesis. We first introduce concepts from nonlinear dynamics that are relevant to EADs, namely, Hopf bifurcation leading to oscillations and basin of attraction of an equilibrium or oscillatory state. We then present a theory of phase-2 EADs in nonlinear dynamics, which includes the formation of quasi-equilibrium states at the plateau voltage, their stabilities, and the bifurcations leading to and terminating the oscillations. This theory shows that the L-type calcium channel plays a unique role in causing the nonlinear dynamical behaviours necessary for EADs. We also summarize different mechanisms of phase-3 EADs. Based on the dynamical theory, we discuss the roles of each of the major ionic currents in the genesis of EADs, and potential therapeutic targets. [ABSTRACT FROM PUBLISHER]

Aims Cardiac hypertrophy is accompanied by reprogramming of gene expression, where the altered expression of ion channels decreases electrical stability and increases the risk of life-threatening arrhythmias. However, the underlying mechanisms are not fully understood. Here, we analysed the role of the depolarizing current If which has been hypothesized to contribute to arrhythmogenesis in the hypertrophied ventricle. Methods and results We used transverse aortic constriction in mice to induce ventricular hypertrophy. This resulted in an increased number of If positive ventricular myocytes as well as a strongly enhanced and accelerated If when compared with controls. Of the four HCN (hyperpolarization-activated cyclic nucleotide-gated channels) isoforms mediating If, HCN2 and HCN4 were the predominantly expressed subunits in healthy as well as hypertrophied hearts. Unexpectedly, only the HCN1 transcript was significantly upregulated in response to hypertrophy. However, the combined deletion of HCN2 and HCN4 disrupted ventricular If completely. The lack of If in hypertrophic double-knockouts resulted in a strong attenuation of pro-arrhythmogenic parameters characteristically observed in hypertrophic hearts. In particular, prolongation of the action potential was significantly decreased and lengthening of the QT interval was reduced. Conclusions We suggest that the strongly increased HCN channel activity in hypertrophied myocytes prolongs the repolarization of the ventricular action potential and thereby may increase the arrhythmogenic potential. Our results provide for the first time a direct link between an upregulation of ventricular If and a diminished repolarization reserve in cardiac hypertrophy. [ABSTRACT FROM AUTHOR]

Aims The transient outward potassium current (Ito) plays important roles in action potential (AP) morphology and dynamics; however, its role in the genesis of early afterdepolarizations (EADs) is not well understood. We aimed to study the effects and mechanisms of Ito on EAD genesis in cardiac cells using combined experimental and computational approaches. Methods and results We first carried out patch-clamp experiments in isolated rabbit ventricular myocytes exposed to H2O2 (0.2 or 1 mM), in which EADs were induced at a slow pacing rate. EADs were eliminated by either increasing the pacing rate or blocking Ito with 2 mM 4-aminopyridine. In addition to enhancing the L-type calcium current (ICa,L) and the late sodium current, H2O2 also increased the conductance, slowed inactivation, and accelerated recovery from the inactivation of Ito. Computer simulations showed that Ito promoted EADs under the condition of reduced repolarization reserve, consistent with the experimental observations. However, EADs were only promoted in the intermediate ranges of the Ito conductance and the inactivation time constant. The underlying mechanism is that Ito lowers the AP plateau voltage into the range at which the time-dependent potassium current (namely IKs) activation is further slowed and ICa,L is available for reactivation, leading to voltage oscillations to manifest EADs. Further experimental studies in cardiac cells of other species validated the theoretical predictions. Conclusion In cardiac cells, Ito, with a proper conductance and inactivation speed, potentiates EADs by setting the AP plateau into the voltage range where ICa,L reactivation is facilitated and IKs activation is slowed. [ABSTRACT FROM AUTHOR]

Aims Female gender is a risk factor for long QT-related arrhythmias, but the underlying mechanisms remain uncertain. Here, we tested the hypothesis that gender-dependent function of the post-depolarization ‘late’ sodium current (INa-L) contributes. Methods and results Studies were conducted in mice in which the canonical cardiac sodium channel Scn5a locus was disrupted, and expression of human wild-type SCN5A cDNA substituted. Baseline QT intervals were similar in male and female mice, but exposure to the sodium channel opener anemone toxin ATX-II elicited polymorphic ventricular tachycardia in 0/9 males vs. 6/9 females. Ventricular INa-L and action potential durations were increased in myocytes isolated from female mice compared with those from males before and especially after treatment with ATX-II. Further, ATX-II elicited potentially arrhythmogenic early afterdepolarizations in myocytes from 0/5 male mice and 3/5 female mice. Conclusion These data identify variable late INa as a modulator of gender-dependent arrhythmia susceptibility. [ABSTRACT FROM AUTHOR]

Aims Fibrosis is known to promote cardiac arrhythmias by disrupting myocardial structure. Given recent evidence that myofibroblasts form gap junctions with myocytes at least in co-cultures, we investigated whether myofibroblast–myocyte coupling can promote arrhythmia triggers, such as early afterdepolarizations (EADs), by directly influencing myocyte electrophysiology. Methods and results Using the dynamic voltage clamp technique, patch-clamped adult rabbit ventricular myocytes were electrotonically coupled to one or multiple virtual fibroblasts or myofibroblasts programmed with eight combinations of capacitance, membrane resistance, resting membrane potential, and gap junction coupling resistance, spanning physiologically realistic ranges. Myocytes were exposed to oxidative (1 mmol/L H2O2) or ionic (2.7 mmol/L hypokalaemia) stress to induce bradycardia-dependent EADs. In the absence of myofibroblast–myocyte coupling, EADs developed during slow pacing (6 s), but were completely suppressed by faster pacing (1 s). However, in the presence of myofibroblast–myocyte coupling, EADs could no longer be suppressed by rapid pacing, especially when myofibroblast resting membrane potential was depolarized (−25 mV). Analysis of the myofibroblast–myocyte virtual gap junction currents revealed two components: an early transient-outward Ito-like current and a late sustained current. Selective elimination of the Ito-like component prevented EADs, whereas selective elimination of the late component did not. Conclusion Coupling of myocytes to myofibroblasts promotes EAD formation as a result of a mismatch in early vs. late repolarization reserve caused by the Ito-like component of the gap junction current. These cellular and ionic mechanisms may contribute to the pro-arrhythmic risk in fibrotic hearts. [ABSTRACT FROM PUBLISHER]

Aims: We have shown that failing human and rabbit left ventricle (LV) exhibits downregulation and dephosphorylation of connexin43 (Cx43) and that Cx43 dephosphorylation in heart failure (HF) contributes to reduced cell coupling. However, the role of Cx43 downregulation per se in impaired coupling in HF is unclear. [ABSTRACT FROM PUBLISHER]

Gap junctions form the cell-to-cell pathways for propagation of the precisely orchestrated patterns of current flow that govern the regular rhythm of the healthy heart. As in most tissues and organs, multiple connexin types are expressed in the heart: connexin43 (Cx43), Cx40 and Cx45 are found in distinctive combinations and relative quantities in different, functionally-specialized subsets of cardiac myocyte. Mutations in genes that encode connexins have only rarely been identified as being a cause of human cardiac disease, but remodelling of connexin expression and gap junction organization are well documented in acquired adult heart disease, notably ischaemic heart disease and heart failure. Remodelling may take the form of alterations in (i) the distribution of gap junctions and (ii) the amount and type of connexins expressed. Heterogeneous reduction in Cx43 expression and disordering in gap junction distribution feature in human ventricular disease and correlate with electrophysiologically identified arrhythmic changes and contractile dysfunction in animal models. Disease-related alterations in Cx45 and Cx40 expression have also been reported, and some of the functional implications of these are beginning to emerge. Apart from ventricular disease, various features of gap junction organization and connexin expression have been implicated in the initiation and persistence of the most common form of atrial arrhythmia, atrial fibrillation, though the disparate findings in this area remain to be clarified. Other major tasks ahead focus on the Purkinje/working ventricular myocyte interface and its role in normal and abnormal impulse propagation, connexin-interacting proteins and their regulatory functions, and on defining the precise functional properties conferred by the distinctive connexin co-expression patterns of different myocyte types in health and disease. [ABSTRACT FROM PUBLISHER]

Aims: KCNQ1 (alias KvLQT1 or Kv7.1) and KCNE1 (alias IsK or minK) co-assemble to form the voltage-activated K+ channel responsible for IKs—a major repolarizing current in the human heart—and their dysfunction promotes cardiac arrhythmias. The channel is a component of larger macromolecular complexes containing known and undefined regulatory proteins. Thus, identification of proteins that modulate its biosynthesis, localization, activity, and/or degradation is of great interest from both a physiological and pathological point of view. [ABSTRACT FROM PUBLISHER]

Aims: Alteration of connexin43 (Cx43)-mediated intercellular communication is known to promote susceptibility to ventricular tachyarrhythmias. However, the precise mechanism of the altered Cx43 responsible for arrhythmogenesis remains unclear. We sought to understand changes in impulse propagation of ventricular myocytes under dominant-negative (DN) inhibition of Cx43 in the development of arrhythmias. [ABSTRACT FROM PUBLISHER]

There is much evidence showing that some lethal ventricular arrhythmias arise from waves of Ca2+ release from the sarcoplasmic reticulum (SR) that propagate along cardiac cells. The purpose of this review is to discuss the mechanism of production of these waves and how they depend on the properties of the SR Ca2+ release channel or ryanodine receptor (RyR). The best-known method of producing Ca2+ waves is by increasing the Ca2+ content of the cell by either increasing Ca2+ influx or decreasing efflux. Once SR Ca2+ content reaches a threshold level a Ca2+ wave is produced. Altering the properties of the RyR affects the threshold level of Ca2+ required to produce a wave. Patients with a mutation in the RyR suffer from catecholaminergic polymorphic ventricular tachycardia, and this may be due to a decrease in the SR Ca2+ threshold for wave production. Heart failure has also been suggested to result in Ca2+ waves due to a leak of Ca2+ through the RyR. We review the finding that these changes in RyR function will only result in Ca2+ waves in the steady state if some other mechanism maintains the SR Ca2+ content. The review concludes with a description of potential mechanisms for treating arrhythmias produced by Ca2+ waves. [ABSTRACT FROM PUBLISHER]

Abstract: Objective: Cellular Ca2+ waves are understood as reaction–diffusion systems sustained by Ca2+-induced Ca2+ release (CICR) from Ca2+ stores. Given the recently discovered sensitization of Ca2+ release channels (ryanodine receptors; RyRs) of the sarcoplasmic reticulum (SR) by luminal SR Ca2+, waves could also be driven by RyR sensitization, mediated by SR overloading via Ca2+ pump (SERCA), acting in tandem with CICR. Methods: Confocal imaging of the Ca2+ indicator fluo-3 was combined with UV-flash photolysis of caged compounds and the whole-cell configuration of the patch clamp technique to carry out these experiments in isolated guinea pig ventricular cardiomyocytes. Results: Upon sudden slowing of the SERCA in cardiomyocytes with a photoreleased inhibitor, waves indeed decelerated immediately. No secondary changes of Ca2+ signaling or SR Ca2+ content due to SERCA inhibition were observed in the short time-frame of these experiments. Conclusions: Our findings are consistent with Ca2+ loading resulting in a zone of RyR ‘sensitization’ traveling within the SR, but inconsistent with CICR as the predominant mechanism driving the Ca2+ waves. This alternative mode of RyR activation is essential to fully conceptualize cardiac arrhythmias triggered by spontaneous Ca2+ release. [Copyright &y& Elsevier]

Abstract: Background: AVE0118 (2′-{[2-(4-Methoxy-phenyl)-acetylamino]-methyl}-biphenyl-2-carboxylic acid (2-pyridin-3-yl-ethyl)-amide) blocks atrial ultrarapid delayed rectifier currents (I Kur) and prolongs the atrial action potential (AP) plateau without affecting ventricular repolarisation. In patients with atrial contractile dysfunction due to atrial tachyarrhythmias, this response might increase atrial contractility without risk of ventricular proarrhythmia. This study was designed to evaluate the inotropic mechanisms of AVE0118. Methods and results: In isometrically contracting atrial trabeculae, AVE0118 increased contractile force by 55.4% in sinus rhythm patients (n =9) and by 107.4% in patients with atrial fibrillation (n =8). In freshly isolated canine atrial myocytes studied under perforated patch current clamp (37 °C), AVE0118 increased myocyte fractional shortening from 3.8±0.6 to 9.6±0.8% and prolonged action potential duration at 30% repolarisation from 9±2 to 102±11 ms. Clamping cells to an AP waveform recorded during exposure to AVE0118 produced the same inotropic response as the drug itself. In action potential clamp, peak Ca2+ inward current (I CaL) current declined from 5.5±1.3 pA/pF during control to 4.1±0.7 pA/pF when an AP recorded in the presence of AVE0118 was used as command waveform. However, I CaL was more sustained with AVE0118 and the time integral did not change (135±37 vs. 173±30 pA/pF⁎ms, p =ns). Importantly, blockade of reverse mode Na+/Ca2+-exchanger activity with 5 μM KBR7943 or using a Na+-free pipette solution abolished the positive inotropic effect of the AP recorded in the presence of AVE0118. In ventricular myocytes AVE0118 did not elicit a positive inotropic response. Conclusions: Block of I Kur by AVE0118 enhances atrial contractility both in patients with sinus rhythm and atrial fibrillation. The positive inotropic effect is atrial-specific and due to the changes of the action potential configuration which enhances Ca2+ entry via reverse mode Na+/Ca2+ exchange. [Copyright &y& Elsevier]

Abstract: Background: The epicardial border zone (EBZ) of surviving myocytes in the healing, 4- to 5-day-old canine infarct is an arrhythmogenic substrate characterized by both structural and functional remodeling of Cx43. Unknown is whether the remodeling of gap junction conductance is heterogeneous in the EBZ like that of sarcolemmal ion channel remodeling and how remodeling of the gap junction influences conduction and anisotropy. Methods and results: Ventricular tachycardia was initiated by programmed stimulation in healing canine infarcted hearts. Reentrant circuits were mapped and the central common pathway (CCP) and outer pathway (OP) regions localized. Epimyocardium removed from the CCP was disaggregated to generate myocyte pairs for conductance measurements. Cx43 distribution was determined by immunofluorescent confocal microscopy. While transverse coupling (gap junction conductance) was markedly decreased in OP cells, CCP cells with lateralized Cx43 gap junctions showed normal conductance. Longitudinal coupling in both OP and CCP was no different than normal. Consistent with conductance measurements, the anisotropic ratio in the CCP was similar to that of normal tissue. In the OP it was increased. Despite normal longitudinal and transverse conductance and anisotropic ratio, longitudinal and transverse conduction velocities were decreased in the CCP with respect to normal epicardium, possibly as a result of the remodeling of sarcolemmal ion channels in this region. Conclusions: Gap junction conductance and distribution is heterogeneous in different regions of reentrant circuits. Lateralization of Cx43 gap junctions in CCP of reentrant circuits is associated with normal transverse conductance between cell pairs. In contrast, absence of lateralization in OP is associated with reduced transverse conductance. Despite normal anisotropic ratio, conduction velocity in CCP region remains slower than normal. This suggests that the effects of Cx43 remodeling in the infarcted heart should be interpreted in conjunction with other types of remodeling occurring in the EBZ (i.e. sarcolemmal ion channels). [Copyright &y& Elsevier]

Abstract: Objective: Sphingosine-1-phosphate (S-1-P), a potent lysophospholipid mediator which is released from platelets during clotting, activates a G-protein-gated inwardly rectifying K+ current (GIRK) in atrial and sino-atrial node myocytes. We denote this current I K(S-1-P). A similar GIRK, which is activated by acetylcholine (ACh) and denoted I K(ACh), is expressed in atrium. It shortens the action potential duration (APD) and reduces the effective refractory period (ERP). We have examined the effect of S-1-P on APD in guinea pig atrial myocytes by characterizing the rectification properties of I K(S-1-P) and evaluating whether I K(S-1-P) and I K(ACh) exhibit convergence/occlusion. Methods: Membrane potential and K+ currents were recorded from guinea pig atrial myocytes. Inwardly rectifying K+ currents were recorded using a ramp voltage clamp waveform between +30 to −130mV from a holding potential of −7mV. Agonist-induced current changes were obtained by subtracting the control current. Results: S-1-P (1 and 10nM) altered both passive and active properties of atrial myocytes. S-1-P increased the threshold current for excitation and decreased the time constant of the subthreshold electrotonic potentials. In addition, both APD50 and APD90 were decreased substantially. Voltage clamp analysis showed that the outward conductance of I K(IR) (G K(IR)out) was 134.8±11.3pS pF−1 (n =19) in S-1-P (100nM), and 207.0±19.6pS pF−1 (n =18) in ACh (10μM). The ratio of G K(IR)out : G K(IR)in was about 0.7 for both S-1-P and ACh. The EC50 values for the activation of G K(IR)out and G K(IR)in by S-1-P were 1.6 and 1.3nM, respectively. Addition of S-1-P (100nM) after the effect of ACh (10μM) had developed fully caused very little additional change. Conclusion: I K(S-1-P) is carried by weakly inwardly-rectifying K+ channels that are the same as those activated by ACh. This K+ current can markedly shorten APD in guinea pig atrial myocytes. This effect would be expected to increase the incidence of atrial rhythm disturbances. [Copyright &y& Elsevier]

Abstract: Background: Brugada syndrome (BrS) is an inherited electrical disorder associated with a high incidence of sudden death. In a minority of patients, it has been linked to mutations in SCN5A, the gene encoding the pore-forming α-subunit of the cardiac Na+ channel. Other causally related genes still await identification. We evaluated the role of HERG (KCNH2), which encodes the α-subunit of the rapid delayed rectifier K+ channel (I Kr), in BrS. Methods and results: In two unrelated SCN5A mutation-negative patients, different amino acid changes in the C-terminal domain of the HERG channel (G873S and N985S) were identified. Voltage-clamp experiments on transfected HEK-293 cells show that these changes increase I Kr density and cause a negative shift of voltage-dependent inactivation, resulting in increased rectification. Action potential (AP) clamp experiments reveal increased transient HERG peak currents (I peak) during phase-0 and phase-1 of the ventricular AP, particularly at short cycle length. Computer simulations demonstrate that the increased I peak enhances the susceptibility to loss of the AP-dome typically in right ventricular subepicardial myocytes, thereby contributing to the BrS phenotype. Conclusion: Our study reveals a modulatory role of I Kr in BrS. These findings may provide better understanding of BrS and have implications for diagnosis and therapy. [Copyright &y& Elsevier]

Abstract: Objective: Autologous skeletal muscle cell (SM) transplantation into the in vivo heart sometimes induces serious arrhythmias. The purpose of this study was to investigate the electrophysiology of cardiomyocyte (CM) and SM in direct contact and to study the mechanism underlying the cause of arrhythmia using the recently developed cell sheet engineering technique. Methods: Primary cultured rat neonatal SM and CM were prepared, and cell sheets were fabricated using temperature-responsive culture dishes. The action potential was recorded by a conventional microelectrode. Intracellular calcium concentration and optical mapping image of the action potential were recorded using Fluo-3 and di-4-ANEPPS, respectively. A video motion-detecting system was used for the detection of arrhythmias. Results: SM myotubes occasionally displayed automaticity. SM sheets did not display synchronized contraction, but instead groups of myotubes contracted independently. The action potential of SM, induced by artificial pacing, did not expand to the entire sheet but was limited within a restricted, small area around the electrode, and it was unfeasible to generate an electrical connection or propagate an action potential between CM and SM sheets. SM sheets, in which some of the myotubes displayed automaticity, caused fibrillation-like contraction in the co-cultured CM sheets, and this arrhythmia was specifically blocked by the stretch-activated channel blocker GsMTx-4. Conclusions: These findings show that SM sheets do not contract synchronously or generate functional syncytia with the surrounding CM sheets and that stretch-induced arrhythmias due to spontaneous contraction of SM may occur in the CM sheet. [Copyright &y& Elsevier]

Abstract: Background: Cardiac dyssynchrony due to intraventricular conduction delay produces heterogeneous regional wall stress and worsens arrhythmia susceptibility in failing hearts. We examined whether chronic dyssynchrony per se induces regionally heterogeneous electrophysiological remodeling. Methods and results: Adult dogs (n =9) underwent left bundle branch radiofrequency ablation (QRS duration increased from 50±7 to 104±7 ms); 6 untreated dogs served as controls. A subset of ablated (n =3) and control (n =4) dogs underwent tagged MR imaging to confirm ablation-induced left ventricular (LV) dyssynchrony. Four weeks later, hearts were excised and early (anterior)- and late (lateral)-activated myocardial segments were isolated. Conduction velocity (CV), action potential duration (APD), and refractory period (RP) of paced, arterially perfused myocardial wedges were studied by extracellular and optical mapping, and arrhythmia susceptibility was assessed by programmed stimulation. Regional stress-response kinase, calcium cycling, and gap junction protein expression were assayed by Western blotting, and the subcellular distribution of connexin43 was analyzed by immunofluorescence microscopy. CV, APD, and RP were significantly reduced in the late-activated, lateral wall of dyssynchronous hearts compared to the anterior wall. Normal differences in CV (endocardial>epicardial) were reversed in the dyssynchronous lateral LV. While the total expression of connexin43 was unaltered in dyssynchronous models, its subcellular location was redistributed in late-activated myocardium from intercalated discs to lateral myocyte membranes. Arrhythmias were rare in tissue from normal and dyssynchronous models. Total expression of calcium-cycling proteins (sarcoplasmic reticulum Ca2 +-ATPase and phospholamban) and the stress-response kinase phospho-ERK did not vary regionally in either model. Conclusions: Dyssynchrony even in the absence of LV dysfunction induces regionally specific changes in conduction and repolarization. These changes support a novel mechanism linking mechanical dyssynchrony to persistent electrophysiological remodeling and heterogeneity. [Copyright &y& Elsevier]

Abstract: Objectives: Altered gap junctional coupling of ventricular myocytes plays an important role in arrhythmogenesis in ischemic heart disease. Since hypoxia is a major component of ischemia, we tested the hypothesis that hypoxia causes gap junctional remodeling accompanied by conduction disturbances. Methods: Cultured neonatal rat ventricular myocytes were exposed to hypoxia (1% O2) for 15 min to 5 h, connexin43 (Cx43) expression was analyzed, and conduction velocity was measured using the Micro-Electrode Array data acquisition system. Results: After 15 min of hypoxia, conduction velocity was unaffected, while total Cx43, including the phosphorylated and nonphosphorylated isoforms, was increased. After 5 h of hypoxia, total Cx43 protein was decreased by 50%, while the nonphosphorylated Cx43 isoform was unchanged. Confocal analyses yielded a 55% decrease in the gap junctional Cx43 fluorescence signal, a 55% decrease in gap junction number, and a 26% decrease in size. The changes in Cx43 were not accompanied by changes in mRNA levels. The reduction in Cx43 protein levels was associated with a ∼20% decrease in conduction velocity compared to normoxic cultures. Conclusions: Short-term hypoxia (5 h) decreases Cx43 protein and conduction velocity, thereby contributing to the generation of an arrhythmogenic substrate. [Copyright &y& Elsevier]

Abstract: Objective: Overexpression of calcineurin causes cardiac hypertrophy and arrhythmic deaths. During disease development, sinus bradycardia followed by high degree atrioventricular (AV) block finally culminating in ventricular asystole has been observed over time in calcineurin hearts. AV block is associated with the development of pleomorphic ventricular tachycardia in mice and downregulation of potassium currents in ventricular myocytes. We tested the hypothesis that the abnormalities of AV block and propensity to ventricular tachycardia relate to overexpression of calcineurin independent of the development of hypertrophy. Methods: Cardiac electrophysiologic properties were compared in isolated perfused hearts with ventricular hypertrophy due to overexpression of calcineurin or NF-AT3 and in their corresponding wild types at 15 or 30 days of age. Results: Compared to wild-type hearts, significant prolongation of sinus node recovery times was noted in both NF-AT3 and calcineurin hearts. Compared to wild-type hearts, Wenckebach cycle length (WCL) and the left ventricular effective refractory period (LVERP) were significantly prolonged in the calcineurin hearts (p<0.05) but not NF-AT3 hearts. In calcineurin mice, left ventricular effective refractory period impinged on Wenckebach cycle length resulting in a significant correlation between left ventricular effective refractory period and Wenckebach cycle length (r2=0.56). No such correlation was observed for wild type or NF-AT3 hearts. At 30 days of development, ventricular tachycardia developed in 70% of calcineurin hearts compared to 0% wild-type hearts (p=0.003), whereas ventricular tachycardia was observed in 33% of NF-AT3 hearts and 10% of corresponding wild-type hearts (p=NS). Conclusions: The prolonged ventricular refractoriness, seen only in calcineurin hearts, impinges on Wenckebach cycle length resulting in heart block and is associated with propensity to ventricular tachycardia. [Copyright &y& Elsevier]

Objective: To determine the characteristics of a TTX-sensitive Ca2+ current that occurred only following remodelling after myocardial infarction in Wistar rat. Methods: Using the whole-cell patch-clamp technique, we studied ionic inward current in myocytes isolated from four different ventricular regions of control Wistar rat hearts, or from hearts 4 to 6 months after ligation of the left coronary artery. Inward current characteristics were also analysed in Xenopus laevis oocytes that heterologously expressed the human sodium channel α-subunit Nav1.5. The effects of oxidative stress by hydrogen peroxide or tert-butyl-hydroxyperoxide as well as those of PKA-dependent phosphorylation, which partly mimic the pathological conditions, were investigated on control cardiomyocytes and Nav1.5-expressing oocytes. Results: In Na-free solution, a low-threshold, tetrodotoxin-sensitive inward current was found in 20 out of 78 cells isolated from 16 post-myocardial infarcted (PMI) cardiomyocytes but not in cardiomyocytes from young and sham rat hearts. This current exhibited kinetics and pharmacological properties similar to the ICa(TTX) current previously reported. ICa(TTX)-like current was critically dependent on extracellular Na+ and was reduced by micromolar Na+ concentrations. Neither in normal rat cardiomyocytes nor in Nav1.5-expressing oocytes could a ICa(TTX)-like current be elicited in Na+-free extracellular solution, even after oxidative stress or PKA-dependent phosphorylation. Conclusions: Our data suggest that ICa(TTX)-like current in PMI myocytes does not arise from classical Na+ channels modified by oxidative stress or PKA phosphorylation and most probably represents a different Na+ channel type re-expressed in some cells after remodelling. [Copyright &y& Elsevier]

Objective: To compare gap junction expression and intercellular coupling in wildtype neonatal cardiac myocytes to those from mice lacking the most abundant cardiac gap junction protein (connexin43, Cx43). Methods: Northern and Western blots compared connexin mRNA and protein levels, immunocytochemistry evaluated connexin distribution in neonatal Cx43 null(−/−), heterozygous(+/−) and wildtype(+/+) mouse hearts. Ca2+ imaging, dye coupling and electrophysiological methods evaluated intercellular communication. Results: Similar levels of Cx40 and Cx45 were detected in all genotypes, although in adult cardiac tissue from wildtype mice, Cx43 expression was higher than in heterozygotes. After culturing dissociated cells for 3–4 days, cardiocytes beat spontaneously; in Cx43(+/+) and (+/−) cultures, the beating was generally quite synchronous. In Cx43(−/−) mice, interbeat intervals were on average twice as long and more variable than in Cx43(+/+) or Cx43(+/−) cultures. Junctional conductance was lower by about 60% in Cx43(−/−) as compared to Cx43(+/−) and (+/+) littermates; Lucifer Yellow dye coupling was virtually absent in Cx43(−/−) cardiomyocytes but was comparably strong in wildtype and heterozygous siblings. Macroscopic junctional conductance measurements on Cx43(−/−) cardiocytes showed slightly stronger voltage sensitivity in these cells than in Cx43(+/+) cardiocytes. Unitary junctional conductance measurements revealed distinct populations of channels contributing to macroscopic conductance for Cx43(+/+) and Cx43(−/−) genotypes. Conclusions: In Cx43-deficient cardiac myocytes, the expression of other connexins only partially compensates for the functional loss, with dye coupling and spontaneous beating being strongly impaired. [Copyright &y& Elsevier]

Objective: After-depolarization associated arrhythmias are frequently observed in heart failure and associated with spontaneous calcium release from sarcoplasmic reticulum (SR), calcium after-transients. We hypothesize that disturbed SR calcium handling underlies calcium after-transients in heart failure (HF). Methods: We measured the stimulation rate dependence (0.2–3 Hz) of diastolic calcium, calcium transient amplitude and SR calcium content in left ventricular myocytes isolated from hearts of rabbits with pressure and volume overload-induced HF and age-matched control animals. Cytosolic calcium was measured with indo-1. In some experiments, delayed after-depolarizations (DADs) were monitored with the voltage sensitive dye di-4-Annepps. SR calcium content was estimated from the response to rapid cooling (RC). After-transients were elicited in the presence of norepinephrine (100 nmol/l) after cessation of burst pacing. Results: With increasing stimulation rate (0.2–3.0 Hz): (1) steady state diastolic [Ca]i increased from 102 to 174 nmol/l in HF and from 44 to 103 nmol/l in control, (2) calcium transient amplitudes decreased from 310 to 254 nmol/l in HF and increased from 186 to 429 nmol/l in control, (3) SR calcium content decreased from 1.25 to 1.09 mmol/l in HF and increased from 1.51 to 2.48 mmol/l in control, (4) in HF and control, the end diastolic SR membrane calcium gradient decreased by about 30%; at any stimulation rate, the magnitude of gradient in HF was one-third of control, (5) systolic depletion of SR was 85% in HF and 60% in control. In HF, noradrenaline (100 nmol/l) increased SR calcium content and SR membrane gradient by 40% versus about 7% in control. Calcium after-transients were observed in 14 out of 18 HF rabbits, and none in eight control animals and were associated with DADs. Calcium after-transients were associated with a 35% decrease in SR calcium content. The frequency of occurrence of calcium after-transients was related to diastolic calcium. Conclusions: in HF, diastolic calcium is increased and both SR calcium content and SR membrane calcium gradient are decreased in a stimulation rate-dependent manner. In HF, β-adrenergic stimulation can partly restore the SR calcium content and SR membrane gradient at higher stimulation rates in a meta-stable condition; upon transition to low stimulation rates, the SR membrane can no longer maintain this high unbalanced SR calcium load at increased diastolic calcium, the magnitude of which is causally related to the occurrence of calcium after-transients. [Copyright &y& Elsevier]

Objective: The aim of this study was to characterize the spatio-temporal dynamics of slow Ca2+ waves (SCW's) with cellular resolution in the arterially-perfused rat heart. Methods: Wister rat hearts were Langendorff-perfused with Tyrode solution containing bovine-albumine and Dextran. The heart was loaded with the Ca2+ sensitive dye Fluo-3 AM. Intracellular fluorescence changes reflecting changes in [Ca2+]i were recorded from subepicardial tissue layers using a slit hole confocal microscope with an image intensified video camera system at image rates of up to 50/s. Results: SCW's appeared spontaneously during cardiac rest or after trains of electrical stimuli. They were initiated preferentially in the center third of the cell and propagated to the cell borders, suggesting a relation between the cell nucleus and wave initiation. They were suppressed by Ca2+ transients and their probability of occurrence increased with the Ca2+ resting level. Propagation velocity within myocytes (40 to 180 μm/s) decreased with the resting Ca2+ level. Intercellular propagation was mostly confined to two or three cells and occurred bi-directionally. Intercellular unidirectional conduction block and facilitation of SCW's was occasionally observed. On average 10 to 20% of cells showed non-synchronized simultaneous SCW's within a given area in the myocardium. Conclusions: SCW's occurring at increased levels of [Ca2+]i in normoxic or ischemic conditions are mostly confined to two or three cells in the ventricular myocardium. Spatio-temporal summation of changes in membrane potential caused by individual SCW's may underlie the generation of triggered electrical ectopic impulses. [ABSTRACT FROM PUBLISHER]

The authors discuss the link between sarcoplasmic reticulum (SR) and mitochondria in cardiac pathophysiology. They state that alterations in the function of the SR are very significant for the development of hypertrophy and heart failure as well as in the genesis of arrhythmias. They mention that the interaction between SR and mitochondria does not only determine cardiomyocyte function under physiological conditions, but also in the development of cardiomyocyte dysfunction or death under pathophysiological conditions.

Aims Reentrant arrhythmias often develop in the setting of myocardial infarction and ensuing slow propagation. Increased Na+ channel expression could prevent or disrupt reentrant circuits by speeding conduction if channel availability is not limited by membrane depolarization within the diseased myocardium. We therefore asked if, in the setting of membrane depolarization, action potential (AP) upstroke and normal conduction can be better preserved by the expression of a Na+ channel isoform with altered biophysical properties compared to the native cardiac Na+ channel isoform, namely having a positively shifted, voltage-dependent inactivation. Methods and results The skeletal Na+ channel isoform (SkM1) and the cardiac Na+ channel isoform (Nav1.5) were expressed in newborn rat ventricular myocyte cultures with a point mutation introduced in Nav1.5 to increase tetrodotoxin (TTX) sensitivity so native and expressed currents could be distinguished. External K+ was increased from 5.4 to 10 mmol/L to induce membrane depolarization. APs, Na+ currents, and conduction velocity (CV) were measured. In control cultures, elevated K+ significantly reduced AP upstroke (∼75%) and CV (∼25%). Expression of Nav1.5 did not protect AP upstroke from K+ depolarization. In contrast, in SkM1 expressing cultures, high K+ reduced AP upstroke Conclusion Expression of SkM1 but not Nav1.5 preserves AP upstroke and CV in a K+-depolarized syncytium. The higher AV and shorter excitable gap observed during reentry excitation around a void in SkM1 cultures would be expected to facilitate reentry self-termination. SkM1 Na+ channel expression represents a novel gene therapy for the treatment of reentrant arrhythmias. [ABSTRACT FROM AUTHOR]