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Generation of functional ion-channel tools by E3 targeting.
Here we describe a strategy for generating ion-channel inhibitors. It takes advantage of antibody specificity combined with a pattern recognition approach that targets the third extracellular region (E3) of a channel. To test the concept, we first focused on TRPC5, a member of the transient receptor potential (TRP) calcium channel family, the study of which has been hindered by poor pharmacological tools. Extracellular application of E3-targeted anti-TRPC5 antibody led to a specific TRPC5 inhibitor, enabling TRPC5 to be distinguished from its closest family members, and TRPC5 function to be explored in a relatively intractable physiological system. E3 targeting was further applied to voltage-gated sodium channels, leading to discovery of a subtype-specific inhibitor of Na(V)1.5. These examples illustrate the potential power of E3 targeting as a systematic method for producing gene-type specific ion-channel inhibitors for use in routine assays on cells or tissues from a range of species and having therapeutic potential.
A mathematical model of action potentials of mouse sinoatrial node cells with molecular bases.
Genetically modified mice are popular experimental models for studying the molecular bases and mechanisms of cardiac arrhythmia. A postgenome challenge is to classify the functional roles of genes in cardiac function. To unveil the functional role of various genetic isoforms of ion channels in generating cardiac pacemaking action potentials (APs), a mathematical model for spontaneous APs of mouse sinoatrial node (SAN) cells was developed. The model takes into account the biophysical properties of membrane ionic currents and intracellular mechanisms contributing to spontaneous mouse SAN APs. The model was validated by its ability to reproduce the physiological exceptionally short APs and high pacing rates of mouse SAN cells. The functional roles of individual membrane currents were evaluated by blocking their coding channels. The roles of intracellular Ca(2+)-handling mechanisms on cardiac pacemaking were also investigated in the model. The robustness of model pacemaking behavior was evaluated by means of one- and two-parameter analyses in wide parameter value ranges. This model provides a predictive tool for cellular level outcomes of electrophysiological experiments. It forms the basis for future model development and further studies into complex pacemaking mechanisms as more quantitative experimental data become available.
Arrhythmic substrate, slowed propagation and increased dispersion in conduction direction in the right ventricular outflow tract of murine Scn5a+/- hearts.
AIM: To test a hypothesis attributing arrhythmia in Brugada Syndrome to right ventricular (RV) outflow tract (RVOT) conduction abnormalities arising from Nav 1.5 insufficiency and fibrotic change. METHODS: Arrhythmic properties of Langendorff-perfused Scn5a+/- and wild-type mouse hearts were correlated with ventricular effective refractory periods (VERPs), multi-electrode array (MEA) measurements of action potential (AP) conduction velocities and dispersions in conduction direction (CD), Nav 1.5 expression levels, and fibrotic change, as measured at the RVOT and RV. Two-way anova was used to test for both independent and interacting effects of anatomical region and genotype on these parameters. RESULTS: Scn5a+/- hearts showed greater arrhythmic frequencies during programmed electrical stimulation at the RVOT but not the RV. The Scn5a+/- genotype caused an independent increase of VERP regardless of whether the recording site was the RVOT or RV. Effective AP conduction velocities (CV†s), derived from fitting regression planes to arrays of observed local activation times were reduced in Scn5a+/- hearts and at the RVOT independently. AP conduction velocity magnitudes derived by averaging MEA results from local vector analyses, CV*, were reduced by the Scn5a+/- genotype alone. In contrast, dispersions in conduction direction, were greater in the RVOT than the RV, when the atrioventricular node was used as the pacing site. The observed reductions in Nav 1.5 expression were attributable to Scn5a+/-, whereas increased levels of fibrosis were associated with the RVOT. CONCLUSIONS: The Scn5a+/- RVOT recapitulates clinical findings of increased arrhythmogenicity through reduced CV† reflecting reduced CV* attributable to reduced Nav 1.5 expression and increased CD attributable to fibrosis.
The effect of the sphingosine-1-phosphate analogue FTY720 on atrioventricular nodal tissue.
The sphingosine-1-phosphate (S1P) receptor modulator, fingolimod (FTY720), has been used for the treatment of patients with relapsing forms of multiple sclerosis, but atrioventricular (AV) conduction block have been reported in some patients after the first dose. The underlying mechanism of this AV node conduction blockade is still not well-understood. In this study, we hypothesize that expression of this particular arrhythmia might be related to a direct effect of FTY720 on AV node rather than a parasympathetic mimetic action. We, therefore, investigated the effect of FTY720 on AV nodal, using in vitro rat model preparation, under both basal as well as ischaemia/reperfusion conditions. We first look at the expression pattern of S1P receptors on the AV node using real-time PCR. Although all three S1P receptor isoforms were expressed in AVN tissues, S1P1 receptor isoform expression level was higher than S1P2 and S1P3. The effect of 25 nM FTY720 on cycle length (CL) was subsequently studied via extracellular potentials recordings. FTY720 caused a mild to moderate prolongation in CL by an average 9% in AVN (n = 10, P < 0.05) preparations. We also show that FTY720 attenuated both ischaemia and reperfusion induced AVN rhythmic disturbance. To our knowledge, these remarkable findings have not been previously reported in the literature, and stress the importance for extensive monitoring period in certain cases, especially in patients taking concurrently AV node blocker agents.
A novel immunomodulator, FTY-720 reverses existing cardiac hypertrophy and fibrosis from pressure overload by targeting NFAT (nuclear factor of activated T-cells) signaling and periostin.
BACKGROUND: Hypertension or aortic stenosis causes pressure overload, which evokes hypertrophic myocardial growth. Sustained cardiac hypertrophy eventually progresses to heart failure. Growing evidence indicates that restraining hypertrophy could be beneficial; here, we discovered that FTY-720, an immunomodulator for treating multiple sclerosis, can reverse existing cardiac hypertrophy/fibrosis. METHODS AND RESULTS: Male C57/Bl6 mice underwent transverse aortic constriction (TAC) for 1 week followed by FTY-720 treatment for 2 weeks under continuing TAC. Compared with vehicle-treated TAC hearts, FTY-720 significantly reduced ventricular mass, ameliorated fibrosis, and improved cardiac performance. Mechanistic studies led us to discover that FTY-720 appreciably inhibited nuclear factor of activated T-cells (NFAT) activity. Moreover, we found that in primary cardiomyocytes (rat and human) pertussis toxin (Gi-coupled receptor inhibitor) substantially blocked the antihypertrophic effect of FTY-720. This observation was confirmed in a mouse model of pressure overload. Interestingly, gene array analysis of TAC hearts revealed that FTY-720 profoundly decreased gene expression of a group of matricellular proteins, of which periostin was prominent. Analysis of periostin protein expression in TAC-myocardium, as well as in rat and human cardiac fibroblasts, confirmed the array data. Moreover, we found that FTY-720 treatment or knockdown of periostin protein was able to inhibit transforming growth factor-β responsiveness and decrease collagen expression. CONCLUSIONS: FTY-720 alleviates existing cardiac hypertrophy/fibrosis through mechanisms involving negative regulation of NFAT activity in cardiomyocytes and reduction of periostin expression allowing for a more homeostatic extracellular compartment milieu. Together, FTY-720 or its analogues could be a promising new approach for treating hypertrophic/fibrotic heart disease.
TGF-β1-mediated fibrosis and ion channel remodeling are key mechanisms in producing the sinus node dysfunction associated with SCN5A deficiency and aging.
BACKGROUND: Mutations in the cardiac Na(+) channel gene (SCN5A) can adversely affect electric function in the heart, but effects can be age dependent. We explored the interacting effects of Scn5a disruption and aging on the pathogenesis of sinus node dysfunction in a heterozygous Scn5a knockout (Scn5a(+/-)) mouse model. METHODS AND RESULTS: We compared functional, histological, and molecular features in young (3 to 4 month) and old (1 year) wild type and Scn5a(+/-) mice. Both Scn5a disruption and aging were associated with decreased heart rate variability, reduced sinoatrial node automaticity, and slowed sinoatrial conduction. They also led to increased collagen and fibroblast levels and upregulated transforming growth factor-β(1) (TGF-β(1)) and vimentin transcripts, providing measures of fibrosis and reduced Nav1.5 expression. All these effects were most noticeable in old Scn5a(+/-) mice. Na(+) channel inhibition by Nav1.5-E3 antibody directly increased TGF-β(1) production in both cultured human cardiac myocytes and fibroblasts. Finally, aging was associated with downregulation of a wide range of ion channel and related transcripts and, again, was greatest in old Scn5a(+/-) mice. The quantitative results from these studies permitted computer simulations that successfully replicated the observed sinoatrial node phenotypes shown by the different experimental groups. CONCLUSIONS: These results implicate a tissue degeneration triggered by Nav1.5 deficiency manifesting as a TGF-β(1)-mediated fibrosis accompanied by electric remodeling in the sinus node dysfunction associated with Scn5a disruption or aging. The latter effects interact to produce the most severe phenotype in old Scn5a(+/-) mice. In demonstrating this, our findings suggest a novel regulatory role for Nav1.5 in cellular biological processes in addition to its electrophysiologic function.
Mapping of reentrant spontaneous polymorphic ventricular tachycardia in a Scn5a+/- mouse model.
Two major mechanisms have been postulated for the arrhythmogenic tendency observed in Brugada Syndrome (BrS): delays in conduction or increased heterogeneities in repolarization. We use a contact mapping system to directly investigate the interacting roles of these two mechanisms in arrhythmogenesis using a genetic murine model for BrS for the first time. Electrograms were obtained from a multielectrode recording array placed against the left ventricle and right ventricle (RV) of spontaneously beating Langendorff-perfused wild type (WT) and Scn5a+/- mouse hearts. Scn5a+/- hearts showed activation waves arriving at the epicardial surface consistent with slowed conduction, which was exacerbated in the presence of flecainide. Lines of conduction block across the RV resulting from premature ventricular beats led to the formation of reentrant circuits and polymorphic ventricular tachycardia. WT hearts showed an inverse relationship between activation times and activation recovery intervals measured at the epicardial surface, which resulted in synchronicity of repolarization times. In contrast, Scn5a+/- hearts, despite having smaller mean activation recovery intervals, demonstrated a greater heterogeneity compared with WT. Isochronal maps showed that their normal activation recovery interval gradients at the epicardial surface were disrupted, leading to heterogeneity in repolarization times. We thus directly demonstrate the initiation of arrhythmia in the RV of Scn5a+/- hearts. This occurs as a result of the combination of repolarization heterogeneities leading to lines of conduction block and unidirectional conduction, with conduction slowing allowing the formation of reentrant circuits. The repolarization heterogeneities may also be responsible for the changing pattern of block, leading to the polymorphic character of the resulting ventricular tachycardia.
Mutation-specific effects of polymorphism H558R in SCN5A-related sick sinus syndrome.
INTRODUCTION: Mutations in SCN5A, the gene encoding alpha subunit of cardiac type sodium channel, Na(v)1.5, lead to familial sick sinus syndrome (SSS). Although several molecular mechanisms for this genetic condition have been explored, the underlying mechanisms for the variable genotype-phenotype relationships have not been well addressed. One of the important contributors to such relationships is the genetic background such as single-nucleotide polymorphisms. METHODS AND RESULTS: To clarify the effects of a common polymorphism in SCN5A gene, H558R, on SCN5A-related SSS phenotype, we investigated the electrophysiological properties of all of the 13 known SSS-related hNa(v)1.5 mutant channels on both H558 and R558 background. Electrophysiological properties of hNa(v)1.5 mutant channels were investigated by the whole-cell patch clamp technique in HEK293 cells. When peak currents were affected by the mutation, cell surface biotinylation was performed to quantify the fraction of correctly cell membrane-targeted mutant channels. Loss-of-function defect of D1275N in SCN5A was rescued by R558 through enhancing cell surface targeting and improving steady-state activation of the mutant channels. In contrast, the defects of mutants E161K, P1298L, and R1632H were aggravated in the R558 background, mainly due to the reduced steady-state availability of mutant channels. The electrophysiological properties of the remaining SSS-related hNa(v)1.5 mutants including the missense mutants (L212P, T220I, DelF1617, T187I, R878C, G1408R), and the truncated mutants (W1421X, K1578fs/52, R1623X) were not significantly affected by H558R. CONCLUSION: We conclude that polymorphism H558R has mutation-specific effects on SCN5A-related SSS. Our data highlight the importance of common genetic variants in modulating phenotypes of genetic diseases.
Novel bradykinin signaling in adult rat cardiac myocytes through activation of p21-activated kinase.
Although bradykinin (BK) is known to exert effects on the myocardium, its intracellular signaling pathways remain poorly understood. Experiments in other cell types indicated that p21-activated kinase-1 (Pak1), a Ser/Thr kinase downstream of small monomeric G proteins, is activated by BK. We previously reported that the expression of active Pak1 in adult cardiac myocytes induced activation of protein phosphatase 2A and dephosphorylation of myofilament proteins (Ke et al. Circ Res 94: 194-200, 2004). In experiments reported here, we tested the hypothesis that BK signals altered protein phosphorylation in adult rat cardiac myocytes through the activation and translocation of Pak1. Treatment of myocytes with BK resulted in the activation of Pak1 as demonstrated by increased autophosphorylation at Thr423 and a diminished striated localization, which is present in the basal state. BK induced dephosphorylation of both cardiac troponin I and phospholamban. Treatment of isolated myocytes with BK also blunted the effect of isoproterenol to enhance peak Ca(2+) and relaxation of Ca(2+) transients. Protein phosphatase 2A was demonstrated to associate with both Pak 1 and phospholamban. Our studies indicate a novel signaling mechanism for BK in adult rat cardiac myocytes and support our hypothesis that Pak 1 is a significant regulator of phosphatase activity in the heart.
Atrial arrhythmia, triggering events and conduction abnormalities in isolated murine RyR2-P2328S hearts.
AIM: RyR2 mutations are associated with catecholaminergic polymorphic tachycardia, a condition characterized by ventricular and atrial arrhythmias. The present experiments investigate the atrial electrophysiology of homozygotic murine RyR2-P2328S (RyR2(S/S)) hearts for ectopic triggering events and for conduction abnormalities that might provide a re-entrant substrate. METHODS: Electrocardiograph recordings were made from regularly stimulated RyR2(S/S) and wild type (WT) hearts, perfused using a novel modified Langendorff preparation. This permitted the simultaneous use of either floating intracellular microelectrodes to measure action potential (AP) parameters, or a multielectrode array to measure epicardial conduction velocity (CV). RESULTS: RyR2(S/S) showed frequent sustained tachyarrhythmias, delayed afterdepolarizations and ectopic APs, increased interatrial conduction delays, reduced epicardial CVs and reduced maximum rates of AP depolarization ((dV/dt)(max)), despite similar effective refractory periods, AP durations and AP amplitudes. Effective interatrial CVs and (dV/dt)(max) values of APs following ectopic (S2) stimulation were lower than those of APs following regular stimulation and decreased with shortening S1S2 intervals. However, although RyR2(S/S) atria showed arrhythmias over a wider range of S1S2 intervals, the interatrial CV and (dV/dt)(max) of S2 APs provoking such arrhythmias were similar in RyR2(S/S) and WT. CONCLUSIONS: These results suggest that abnormal intracellular Ca(2+) homoeostasis produces both arrhythmic triggers and a slow-conducting arrhythmic substrate in RyR2(S/S) atria. A similar mechanism might also contribute to arrhythmogenesis in other conditions, associated with diastolic Ca(2+) release, such as atrial fibrillation.
Conduction slowing contributes to spontaneous ventricular arrhythmias in intrinsically active murine RyR2-P2328S hearts.
INTRODUCTION: The familial condition catecholaminergic polymorphic ventricular tachycardia (CPVT) is characterized by episodic bidirectional ventricular tachycardia (BVT), polymorphic ventricular tachycardia (PVT), and ventricular fibrillation following adrenergic challenge. It is associated with mutations involving the cardiac ryanodine receptor (RyR2). METHODS AND RESULTS: We explored for a slowing of myocardial conduction that could potentially result in a substrate for the spontaneous arrhythmogenesis that was observed following introduction of isoproterenol and caffeine in intrinsically beating murine RyR2-P2328S hearts. Such pharmacological challenge increased the number of arrhythmic episodes in electrocardiographic recordings from intact anesthetized mice, with the greatest effects in the homozygote RyR2(S/S). Arrhythmias took the form of bigeminy, BVT, monomorphic ventricular tachycardia, and PVT, as found in human CPVT. Ventricular epicardial conduction velocities (CVs) measured using multielectrode array recordings and maximum action potential upstroke rates, (dV/dt)(max), measured using intracellular microelectrodes were indistinguishable in untreated wild-type (WT) and RyR2(S/S). Pharmacological challenge of RyR2(S/S), but not WT hearts, then reduced CV and (dV/dt)(max) and also revealed a strongly arrhythmic phenotype. There was no evidence of gross structural or fibrotic changes in either RyR2(+/S) or RyR2(S/S) hearts on light microscopy. CONCLUSIONS: We associate altered ventricular myocardial CV potentially resulting in arrhythmogenic substrate with arrhythmic properties associated with genetic RyR2 alterations for the first time.
FTY720 prevents ischemia/reperfusion injury-associated arrhythmias in an ex vivo rat heart model via activation of Pak1/Akt signaling.
Recent studies demonstrated a role of sphingosine-1-phosphate (S1P) in the protection against the stress of ischemia/reperfusion (I/R) injury. In experiments reported here, we have investigated the signaling through the S1P cascade by FTY720, a sphingolipid drug candidate displaying structural similarity to S1P, underlying the S1P cardioprotective effect. In ex vivo rat heart and isolated sinoatrial node models, FTY720 significantly prevented arrhythmic events associated with I/R injury including premature ventricular beats, VT, and sinus bradycardia as well as A-V conduction block. Real-time PCR and Western blot analysis demonstrated the expression of the S1P receptor transcript pools and corresponding proteins including S1P1, S1P2, and S1P3 in tissues dissected from sinoatrial node, atrium and ventricle. FTY720 (25 nM) significantly blunted the depression of the levels of phospho-Pak1 and phospho-Akt with ischemia and with reperfusion. There was a significant increase in phospho-Pak1 levels by 35%, 199%, and 205% after 5, 10, and 15 min of treatment with 25 nM FTY720 compared with control nontreated myocytes. However, there was no significant difference in the levels of total Pak1 expression between nontreated and FTY720 treated. Phospho-Akt levels were increased by 44%, 63%, and 61% after 5, 10, and 15 min of treatment with 25 nM FTY720, respectively. Our data provide the first evidence that FTY720 prevents I/R injury-associated arrhythmias and indicate its potential significance as an important and new agent protecting against I/R injury. Our data also indicate, for the first time, that the cardioprotective effect of FTY720 is likely to involve activation of signaling through the Pak1.
Delayed conduction and its implications in murine Scn5a(+/-) hearts: independent and interacting effects of genotype, age, and sex.
We explored for relationships between SCN5A haploinsufficiency, implicated in clinical arrhythmogenicity, and right ventricular (RV) conduction disorders in Langendorff-perfused, male and female, and young (3 months) and old (>12 month old) Scn5a ( +/-) and wild type (WT) hearts. The investigated conditions of genotype, age, and sex affected latencies but not repolarization time courses of RV monophasic action potentials. This prompted examination of the patterns of RV epicardial activation, its dispersion, and their interrelationships as possible arrhythmic mechanisms using a 64-channel, multi-electrode array. Mean ventricular activation times (T*(MEAN)), spatial dispersions (D* (S)) between recording channels/cardiac cycle, and maximum activation times (T* (MAX)) representing the slowest possible conduction in any given heart were all higher in old male Scn5a ( +/-) compared with young male and old female Scn5a ( +/-) and old male WT. Temporal dispersions (D*(T)) of recording channels were similarly higher in old male Scn5a (+/-) compared with old male WT. All groupings of D*(T), D*(S), and T*(MAX) nevertheless linearly correlated with T*(MEAN), with indistinguishable slopes. The variates explored thus influence D*(T), D*(S), and T*(MAX) through actions on T*(MEAN). These findings in turn correlated with increased levels of fibrosis in young male, young female, and old male Scn5a ( +/-) compared with the corresponding WTs. We thus demonstrate for the first time independent and interacting effects of genotype, age, and sex on epicardial conduction and its dispersions at least partially attributable to fibrotic change, resulting in the greatest effects in old male Scn5a ( +/-) in an absence of alterations in repolarization time courses. This directly implicates altered depolarization in the clinical arrhythmogenicity associated with Scn5a ( +/-).
Characterization of the effects of ryanodine, TTX, E-4031 and 4-AP on the sinoatrial and atrioventricular nodes.
AIMS: To characterize the effects of inhibition of Ryanodine receptor (RyR), TTX-sensitive neuronal Na+ current (iNa), "rapidly activating" delayed rectifier K+ current (iKr) and ultrarapid delayed rectifier potassium current (IKur) on the pacemaker activity of the sinoatrial node (SAN) and the atrioventricular node (AVN) in the mouse. METHODS: The structure of mouse AVN was studied by histology and immunolabelling of Cx43 and hyperpolarization-activated, cyclic nucleotide-binding channels (HCN). The effects of Ryanodine, TTX, E-4031 and 4-AP on pacemaker activities recorded from mouse intact SAN and AVN preparations have been investigated. RESULTS: Immuno-histological characterization delineated the structure of the AVN showing the similar molecular phenotype of the SAN. The effects of these inhibitors on the cycle length (CL) of the spontaneous pacemaker activity of the SAN and the AVN were characterized. Inhibition of RyR by 0.2 and 2 microM Ryanodine prolonged CL by 42+/-12.3% and 64+/-18.1% in SAN preparations by 163+/-72.3% and 241+/-91.2% in AVN preparations. Inhibition of TTX-sensitive iNa by 100 nM TTX prolonged CL by 22+/-6.0% in SAN preparations and 53+/-13.6% in the AVN preparations. Block of iKr by E-4031 prolonged CL by 68+/-12.5% in SAN preparations and 28+/-3.4% in AVN preparations. Inhibition of iKur by 50 microM 4-AP prolonged CL by 20+/-3.4% in SAN preparations and 18+/-3.0% in AVN preparations. CONCLUSION: Mouse SAN and AVN showed distinct different response to the inhibition of RyR, TTX-sensitive INa, IKr and iKur, which reflects the variation in contribution of these currents to the pacemaker function of the cardiac nodes in the mouse. Our data provide valuable information for developing virtual tissue models of mouse SAN and AVN.
Distribution and functional role of inositol 1,4,5-trisphosphate receptors in mouse sinoatrial node.
RATIONALE: Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) have been implicated in the generation of arrhythmias and cardiac muscle nuclear signaling. However, in the mammalian sinoatrial node (SAN), where the heart beat originates, the expression and functional activity of IP(3)Rs have not been investigated. OBJECTIVES: To determine whether SAN express IP(3)Rs and which isoforms are present. To examine the response of the SAN to IP(3)R agonists and antagonist, and the potential role played by IP(3)Rs in cardiac pacemaking. METHODS AND RESULTS: The expression and distribution of IP(3)Rs were studied by reverse-transcription polymerase chain reaction, Western blotting, and immunolabeling. Ca(2+) signaling and electric activity in intact mouse SAN were measured with Ca(2+)-sensitive fluorescent dyes. We found that although the entire SAN expressed three IP(3)R mRNA isoforms, the type II IP(3)R (IP(3)R2) was the predominant protein isoform detected by Western blot using protein extracts from the SAN, atrioventricular node, and atrial tissue. Immunohistochemistry studies also showed that IP(3)R2 was expressed in the central SAN region. Studies using isolated single pacemaker cells revealed that IP(3)R2 (but not IP(3)R1) was located with a similar distribution to the sarcoplasmic reticulum marker protein SERCA2a with some labeling adjacent to the surface membrane. The application of membrane-permeable IP(3) (IP(3)-butyryloxymethyl ester) increased Ca(2+) spark frequency and the pacemaker firing rate in single isolated pacemaker cells. In intact SAN preparations, IP(3)R agonists, endothelin-1 and IP(3)-butyryloxymethyl ester both increased intracellular Ca(2+) and the pacemaker firing rate, whereas the IP(3)R antagonist, 2-aminoethoxydiphenyl borate decreased Ca(2+) and the firing rate. Both of these effects were absent in the SAN from transgenic IP(3)R2 knockout mice. CONCLUSIONS: This study provides new evidence that functional IP(3)R2s are expressed in the mouse SAN and could serve as an additional Ca(2+)-dependent mechanism in modulating cardiac pacemaker activity as well as other Ca(2+)-dependent processes.