Channels In Cardiac Myocytes Research Articles

3,5-Dimethoxyphenol identification from urine by GC-MS after a lethal intoxication with Taxus baccata: A case report

Taxus baccata, also known as English or European yew, is an evergreen conifer shrub or tree that is traditionally used in ornamental landscaping, woodwork and pharmaceutical purposes. The toxic nature of the yew plant has been known for more than 2000 years. All parts of the plant, except for the red pulp of its berries, contain taxine alkaloids. Minimum lethal doses are reported in the range of 0.6-1.3 g of leaf/kg body weight, which means that for a 70-kg adult this vary between 42 and 91 g of yew leaf. Intoxication is known to be resistant to standard treatments with no effective antidote. The aim of this study was the confirmation of Taxus poisoning by 3,5-dimethoxyphenol determination. A case of an intentional fatal poisoning of a 39-year-old man is presented, hypothesized by the forensic autopsy and confirmed by application of gas chromatography-mass spectrometry. Postmortem urine sample was analysed, after liquid-liquid extraction with dichloromethane from acidic and basic environment. The autopsy has not revealed any significant traumatic injuries or specific pathological lesions. Suggestive content was found in the oral cavity, esophagus and stomach, containing partially digested food mixed with fragmented needle-shaped, green leaves. During toxicological analysis, 3,5-dimethoxyphenol (3,5-DMP), the aglycone of taxicatine metabolite, considered as a marker of Taxus poisoning, was identified in the urine using the NIST spectra library database. Taxine alkaloids are the active constituents of Taxus sp. that cause cardiotoxicity. This occurs via the dose-dependent blockade of sodium and calcium channels in cardiac myocytes, leading to atrioventricular block, evidenced by prolonged PR and QRS intervals on ECG. The clinical severity is dependent on quantity, type an route of exposure, clinical signs usually occur within 3-4 h after ingestion. Yew tree poisoning has a high mortality rate, but remains a rare occurrence, with only a few case reports in literature. Analysis of taxine alkaloids can be used to confirm the diagnosis.

Propranolol modulation of tetrodotoxin-resistant Na+ channels in dural afferent neurons

Propranolol, a representative adrenergic β-receptor antagonist, is widely used to prevent migraine attacks. Although propranolol is well known to inhibit tetrodotoxin-resistant (TTX-R) Na+ channels in cardiac myocytes, it is unclear whether the drug modulates these channels expressed in dural afferent neurons. In this study, we examined the effects of propranolol on TTX-R Na+ channels in medium-sized dural afferent neurons identified by the fluorescent dye DiI. The TTX-R Na+ currents (INa) were recorded from acutely isolated DiI-positive neurons using a whole-cell patch clamp technique under voltage-clamp conditions. Propranolol inhibited the noninactivating steady-state component more potently than the peak component of transient TTX-R INa. Propranolol also potently inhibited the slow voltage ramp-induced TTX-R INa in a concentration-dependent manner, suggesting that it preferentially inhibited the noninactivating or persistent INa in DiI-positive neurons. Propranolol had little effect on voltage dependence, but it increased the extent of the use-dependent inhibition of TTX-R Na+ channels. Propranolol also accelerated the onset of inactivation and retarded recovery from inactivation in these channels. Under current-clamp conditions, propranolol decreased the number of action potentials elicited by depolarizing current stimuli. In conclusion, the propranolol-mediated preferential inhibition of persistent INa and modulation of the inactivation kinetics of TTX-R Na+ channels might represent additional mechanisms for migraine prophylaxis.

Super-Resolution Imaging Using a Novel High-Fidelity Antibody Reveals Close Association of the Neuronal Sodium Channel NaV1.6 with Ryanodine Receptors in Cardiac Muscle.

The voltage-gated sodium channel [pore-forming subunit of the neuronal voltage-gated sodium channel (NaV1.6)] has recently been found in cardiac myocytes. Emerging studies indicate a role for NaV1.6 in ionic homeostasis as well as arrhythmogenesis. Little is known about the spatial organization of these channels in cardiac muscle, mainly due to the lack of high-fidelity antibodies. Therefore, we developed and rigorously validated a novel rabbit polyclonal NaV1.6 antibody and undertook super-resolution microscopy studies of NaV1.6 localization in cardiac muscle. We developed and validated a novel rabbit polyclonal antibody against a C-terminal epitope on the neuronal sodium channel 1.6 (NaV1.6). Raw sera showed high affinity in immuno-fluorescence studies, which was improved with affinity purification. The antibody was rigorously validated for specificity via multiple approaches. Lastly, we used this antibody in proximity ligation assay (PLA) and super-resolution STochastic Optical Reconstruction Microscopy (STORM) studies, which revealed enrichment of NaV1.6 in close proximity to ryanodine receptor (RyR2), a key calcium (Ca2+) cycling protein, in cardiac myocytes. In summary, our novel NaV1.6 antibody demonstrates high degrees of specificity and fidelity in multiple preparations. It enabled multimodal microscopic studies and revealed that over half of the NaV1.6 channels in cardiac myocytes are located within 100 nm of ryanodine receptor Ca2+ release channels.

Expression and Roles of N-type Ca<sup>2+</sup> Channel in Cardiac Myocytes

Expression and Roles of N-type Ca<sup>2+</sup> Channel in Cardiac Myocytes

Nucleoporin 107 facilitates the nuclear export of Scn5a mRNA to regulate cardiac bioelectricity.

Nucleoporins (Nups) are known to be functional in nucleo‐cytoplasmic transport, but the roles of nucleoporins in nonproliferating cells, such as cardiac myocytes, are still poorly understood. In this study, we report that Nup107 regulates cardiac bioelectricity by controlling the nucleo‐cytoplasmic trafficking of Scn5a mRNA. Overexpression of Nup107 induced the protein expression of Scn5a rather than that of other ion channels, with no effects of their mRNA levels. The analysis for the protein production demonstrated Nup107‐facilitated transport of Scn5a mRNA. Using RIP‐PCR and luciferase assay, we found that the 5′‐UTR of Scn5a mRNA was not involved in the interaction, whereas the spatial interaction between Nup107 protein and Scn5a mRNA was formed when Scn5a mRNA passing through the nuclear pore. Functionally, Nup107 overexpression in neonatal rat ventricle myocytes significantly increased the currents of Scn5a‐encoded INa channel. Moreover, the close correlation between Nup107 and Nav1.5 protein expression was observed in cardiomycytes and heart tissues subjected to hypoxia and ischaemic insults, suggesting a fast regulation of Nup107 on Nav1.5 channel in cardiac myocytes in a posttranscriptional manner. These findings may provide insights into the emergent control of cardiac electrophysiology through Nup‐mediated modulation of ion channels.

The AKAP Cypher/Zasp contributes to β-adrenergic/PKA stimulation of cardiac CaV1.2 calcium channels.

Stimulation of the L-type Ca2+ current conducted by CaV1.2 channels in cardiac myocytes by the β-adrenergic/protein kinase A (PKA) signaling pathway requires anchoring of PKA to the CaV1.2 channel by an A-kinase anchoring protein (AKAP). However, the AKAP(s) responsible for regulation in vivo remain unknown. Here, we test the role of the AKAP Cypher/Zasp in β-adrenergic regulation of CaV1.2 channels using physiological studies of cardiac ventricular myocytes from young-adult mice lacking the long form of Cypher/Zasp (LCyphKO mice). These myocytes have increased protein levels of CaV1.2, PKA, and calcineurin. In contrast, the cell surface density of CaV1.2 channels and the basal Ca2+ current conducted by CaV1.2 channels are significantly reduced without substantial changes to kinetics or voltage dependence. β-adrenergic regulation of these L-type Ca2+ currents is also significantly reduced in myocytes from LCyphKO mice, whether calculated as a stimulation ratio or as net-stimulated Ca2+ current. At 100 nM isoproterenol, the net β-adrenergic-Ca2+ current conducted by CaV1.2 channels was reduced to 39 ± 12% of wild type. However, concentration-response curves for β-adrenergic stimulation of myocytes from LCyphKO mice have concentrations that give a half-maximal response similar to those for wild-type mice. These results identify Cypher/Zasp as an important AKAP for β-adrenergic regulation of cardiac CaV1.2 channels. Other AKAPs may work cooperatively with Cypher/Zasp to give the full magnitude of β-adrenergic regulation of CaV1.2 channels observed in vivo.

Trypanosoma cruzi Infection Induces Pannexin-1 Channel Opening in Cardiac Myocytes.

Trypanosoma cruzi, the etiological agent of Chagas diseases, invades the cardiac tissue causing acute myocarditis and heart electrical disturbances. In T. cruzi invasion, the parasite induces [Ca2+]i transients in the host cells, an essential phenomenon for invasion. To date, knowledge on the mechanism that elicits transients of [Ca2+]i during the infection of cardiac myocytes has not been fully characterized. Pannexin1 (Panx1) channel are poorly selective channels found in all vertebrates that serve as a pathway for ATP release. In this article, we demonstrate that T. cruzi infection results in the opening of Panx1 channels in cardiac myocytes. We show that pharmacological blockade of Panx1 channels inhibits T. cruzi-induced [Ca2+]i transients and invasion in cardiac myocytes. Our results indicate that opening of Panx1 channels are required for T. cruzi invasion in cardiac myocytes, and we propose that targeting Panx1 channel could provide new potential therapeutic approaches to treat Chagas disease.

Dexamethasone-induced upregulation of CaV3.2 T-type Ca2+ channels in rat cardiac myocytes

Glucocorticoids are widely used to treat acute and chronic diseases. Unfortunately, their therapeutic use is associated with severe side effects. Glucocorticoids are known to regulate several ion channels in cardiac myocytes, including voltage-dependent Ca2+ channels. Low-voltage-activated T-type Ca2+ channels are expressed in ventricular myocytes during the fetal and perinatal period, but are practically absent in the adult. However, these channels can be re-expressed in adult cardiomyocytes under some pathological conditions. We have investigated the glucocorticoid regulation of T-type Ca2+ channels in rat cardiomyocytes. Molecular studies revealed that dexamethasone induces the upregulation of CaV3.2 mRNA in neonatal rat ventricular myocytes, whereas CaV3.1 mRNA is only slightly affected. Patch-clamp recordings confirmed that T-type Ca2+ channel currents were upregulated in dexamethasone treated cardiomyocytes, and the addition of 50 μmol/L NiCl2 demonstrated that the CaV3.2 channel is responsible for this upregulation. The effect of dexamethasone on CaV3.2 is mediated by the activation and translocation to the cell nucleus of the glucocorticoid receptor (GR). We have isolated the upstream promoter of the Cacna1h gene and tested its activity in transfected ventricular myocytes. The initial in silico analysis of Cacna1h promoter revealed putative glucocorticoid response elements (GREs). Transcriptional activity assays combined with deletion analyses and chromatin immunoprecipitation assays demonstrated that GR binds to a region a GRE located in -1006/-985 bp of Cacna1h promoter. Importantly, upregulation of the CaV3.2 channel is also observed in vitro in adult rat ventricular myocytes, and in vivo in a rat model of excess of glucocorticoids.

The S1 helix critically regulates the finely tuned gating of Kv11.1 channels

Congenital mutations in the cardiac Kv11.1 channel can cause long QT syndrome type 2 (LQTS2), a heart rhythm disorder associated with sudden cardiac death. Mutations act either by reducing protein expression at the membrane and/or by perturbing the intricate gating properties of Kv11.1 channels. A number of clinical LQTS2-associated mutations have been reported in the first transmembrane segment (S1) of Kv11.1 channels, but the role of this region of the channel is largely unexplored. In part, this is due to problems defining the extent of the S1 helix, as a consequence of its low sequence homology with other Kv family members. Here, we used NMR spectroscopy and electrophysiological characterization to show that the S1 of Kv11.1 channels extends seven helical turns, from Pro-405 to Phe-431, and is flanked by unstructured loops. Functional analysis suggests that pre-S1 loop residues His-402 and Tyr-403 play an important role in regulating the kinetics and voltage dependence of channel activation and deactivation. Multiple residues within the S1 helix also play an important role in fine-tuning the voltage dependence of activation, regulating slow deactivation, and modulating C-type inactivation of Kv11.1 channels. Analyses of LQTS2-associated mutations in the pre-S1 loop or S1 helix of Kv11.1 channels demonstrate perturbations to both protein expression and most gating transitions. Thus, S1 region mutations would reduce both the action potential repolarizing current passed by Kv11.1 channels in cardiac myocytes, as well as the current passed in response to premature depolarizations that normally helps protect against the formation of ectopic beats.

The Cardiac Sodium Channel and Its Protein Partners.

Activation of the electrical signal and its transmission as a depolarizing wave in the whole heart requires highly organized myocyte architecture and cell-cell contacts. In addition, complex trafficking and anchoring intracellular machineries regulate the proper surface expression of channels and their targeting to distinct membrane domains. An increasing list of proteins, lipids, and second messengers can contribute to the normal targeting of ion channels in cardiac myocytes. However, their precise roles in the electrophysiology of the heart are far from been extensively understood. Nowadays, much effort in the field focuses on understanding the mechanisms that regulate ion channel targeting to sarcolemma microdomains and their organization into macromolecular complexes. The purpose of the present section is to provide an overview of the characterized partners of the main cardiac sodium channel, NaV1.5, involved in regulating the functional expression of this channel both in terms of trafficking and targeting into microdomains.

Regulation of human cardiac potassium channels by full-length KCNE3 and KCNE4

Voltage-gated potassium (Kv) channels comprise pore-forming α subunits and a multiplicity of regulatory proteins, including the cardiac-expressed and cardiac arrhythmia-linked transmembrane KCNE subunits. After recently uncovering novel, N-terminally extended (L) KCNE3 and KCNE4 isoforms and detecting their transcripts in human atrium, reported here are their functional effects on human cardiac Kv channel α subunits expressed in Xenopus laevis oocytes. As previously reported for short isoforms KCNE3S and KCNE4S, KCNE3L inhibited hERG; KCNE4L inhibited Kv1.1; neither form regulated the HCN1 pacemaker channel. Unlike KCNE4S, KCNE4L was a potent inhibitor of Kv4.2 and Kv4.3; co-expression of cytosolic β subunit KChIP2, which regulates Kv4 channels in cardiac myocytes, partially relieved Kv4.3 but not Kv4.2 inhibition. Inhibition of Kv4.2 and Kv4.3 by KCNE3L was weaker, and its inhibition of Kv4.2 abolished by KChIP2. KCNE3L and KCNE4L also exhibited subunit-specific effects on Kv4 channel complex inactivation kinetics, voltage dependence and recovery. Further supporting the potential physiological significance of the robust functional effects of KCNE4L on Kv4 channels, KCNE4L protein was detected in human atrium, where it co-localized with Kv4.3. The findings establish functional effects of novel human cardiac-expressed KCNE isoforms and further contribute to our understanding of the potential mechanisms influencing cardiomyocyte repolarization.

Impact Of Hypoxia In The Expression And Regulation Of The TASK‐1 Potassium Channel In Cardiac Myocytes

TASK‐1 is a member of the two‐pore domain potassium channel family and strongly expressed in the heart. However, little is known about the roles and regulation of TASK‐1 channels in cardiac hypoxia. The present study was designed to investigate whether hypoxia modulated the expression and function of TASK‐1 and whether protein kinase C (PKC) was critical in hypoxia‐mediated processes in adult rat cardiac myocytes. The effects of acute hypoxia on the TASK‐1 channel function were chemically induced by sodium cyanide (NaCN). Activity of TASK‐1 in cardiomyocytes was investigated by the whole‐cell patch‐clamp technique. The current flowing through TASK‐1 channels in cardiac myocytes was quantified by analyzing the effects of extracellular pH or the selective TASK‐1 inhibitor methanandamide on the steady‐state current‐voltage relation. The voltage protocol, which included voltage ramps from +40 to −100 mV preceded by a test pulse from 0 to +40 mV, was designed to inactivate voltage‐dependent ion channels. In addition, a blocker cocktail, that had no effect on TASK‐1 channels expressed in HEK293T cells, was used to eliminate a number of ion channels in cardiac myocytes. Our data show that exposure of cardiac myocytes to NaCN for 5 minutes reduced the steady‐state outward current at +40 mV, an effect that was abolished by pretreatment with methanandamide. Either methanandamide or extracellular acidification (from pH 7.4 to 6.4) decreased the outward current, identifying TASK‐1 as the channel mediating this effect. Interestingly, the NaCN‐induced reduction in the outward current was largely prevented by pretreatment with the PKC selective inhibitor bisindolylmaleimide. To assess the expression and protective role of TASK‐1 in hypoxia, the prolonged hypoxia (4 hours) was employed by incubating cells in an airtight chamber in which O2 was replaced by N2 with glucose‐free Tyrode's solution. While 4 hours of hypoxia caused a significant reduction in the viability of cardiac myocytes, the inhibition of cardiac TASK‐1 channels by methanandamide further increased hypoxia‐induced cell death. Western blot analysis showed that 4 hours of hypoxia led to a decrease in the protein expression of TASK‐1 in cardiac myocytes. In summary, we provided the evidence that TASK‐1 channels in cardiomyocytes is regulated by hypoxia‐induced PKC signaling in cardiomyocytes. Our data are also supportive for a possible role of TASK‐1 channels in the cardioprotection against hypoxia.

Implications of Kir/Nav1.5 Reciprocity for Propagating Action Potentials in Cardiac Cells

The main ion channels involved in the propagation of action potentials in cardiac cells are the voltage-gated sodium channels, the inward rectifier potassium channels, and the inter-cellular gap junction channels. Sodium channels have been shown to be localized to the intercalated disks along with Cx43 channels. Recent evidence of reciprocity in the co-localized expression of Kir2.1 and Nav1.5 channels in cardiac myocytes suggest that ionic currents due to these two channels are in some way calibrated to each other. In isolated cells, the fast sodium current is much larger than IK1 resulting in a large depolarization reserve. Using simulations of chains of cardiac cells, we show that the depolarization reserve for conducting action potentials is significantly smaller than in isolated cells. We also studied the changes in the depolarization reserve with variations in gap junction channel density and explored the conditions under which propagation slowed or failed. These insights will allow a better understanding of the effects of Na channel blockers as well as regional differences in action potential conduction in the heart.

Characterization of Disopyramide derivative ADD424042 as a non-cardiotoxic neuronal sodium channel blocker with broad-spectrum anticonvulsant activity in rodent seizure models

It was reported that antiarrhythmic drugs (AADs) can be useful in controlling refractory seizures in humans or in enhancing the action of antiepileptic drugs (AEDs) in animal models.Disopyramide phosphate (DISO) is an AAD that blocks sodium channels in cardiac myocytes. We evaluated a DISO derivative, 2-(2-chlorophenyl)-2-(pyridin-2-yl)acetamide (ADD424042) for its anticonvulsant activity in a battery of rodent models of epileptic seizures. The compound displayed a broad spectrum of activity in the ‘classical’ models as well as in the models of pharmacoresistant seizures. Furthermore, ADD424042 showed good therapeutic indices between the anticonvulsant activity and the motor impairment. On the contrary, no anticonvulsant effects but severe lethality were observed in the primary anticonvulsant testing of the parent DISO. By performing the whole-cell voltage-clamp experiments in dispersed cortical neurons we demonstrated that ADD424042 decreased the maximal amplitude of voltage-gated sodium channels with an IC50 value in nM range. Moreover, the compound enhanced use-dependent block and decreased excitability in pyramidal neurons in the current-clamp experiments in cortical slices. Importantly, we found that ADD424042 possessed either no, or very small cardiotoxic effect. In contrast to DISO, ADD424042 did not produce any apparent changes in electrocardiogram (ECG) and arterial blood pressure recordings. ADD424042 had no effect on QT and corrected QT intervals, at a dose which was 15 times higher than ED50 for the anticonvulsant effect in the MES model. Taken together, these data suggest that ADD424042 has the potential to become a lead structure for novel broadly acting AEDs with wide margin of cardiac safety.

Proarrhythmic effect of sustained EPAC activation on TRPC3/4 in rat ventricular cardiomyocytes

The Exchange Protein directly Activated by cAMP (EPAC) participates to the pathological signaling of cardiac hypertrophy and heart failure, in which the role of Ca2+ entry through the Transient Receptor Potential Canonical (TRPC) channels begin to be appreciated. Here we studied whether EPAC activation could influence the activity and/or expression of TRPC channels in cardiac myocytes. In adult rat ventricular myocytes treated for 4 to 6h with the selective EPAC activator, 8-pCPT (10μM), we observed by Fluo-3 confocal fluorescence a Store-Operated Ca2+ Entry (SOCE) like-activity, which was blunted by co-incubation with EPAC inhibitors (ESI-05 and CE3F4 at 10μM). This SOCE-like activity, which was very small in control incubated cells, was sensitive to 30-μM SKF-96365. Molecular screening showed a specific upregulation of TRPC3 and C4 protein isoforms after 8-pCPT treatment. Moreover, sustained EPAC activation favored proarrhythmic Ca2+ waves, which were reduced either by co-incubation with EPAC inhibitors or bath perfusion with TRPC inhibitors.Our study provides the first evidence that sustained selective EPAC activation leads to an increase in TRPC3 and C4 protein expression and induces a proarrhythmic SOCE-like activity in adult rat ventricular cardiomyocytes, which might be of importance during the development of cardiac diseases.

Statistical Metamodeling and Sequential Design of Computer Experiments to Model Glyco-Altered Gating of Sodium Channels in Cardiac Myocytes.

Glycan structures account for up to 35% of the mass of cardiac sodium ( Nav ) channels. To question whether and how reduced sialylation affects Nav activity and cardiac electrical signaling, we conducted a series of in vitro experiments on ventricular apex myocytes under two different glycosylation conditions, reduced protein sialylation (ST3Gal4(-/-)) and full glycosylation (control). Although aberrant electrical signaling is observed in reduced sialylation, realizing a better understanding of mechanistic details of pathological variations in INa and AP is difficult without performing in silico studies. However, computer model of Nav channels and cardiac myocytes involves greater levels of complexity, e.g., high-dimensional parameter space, nonlinear and nonconvex equations. Traditional linear and nonlinear optimization methods have encountered many difficulties for model calibration. This paper presents a new statistical metamodeling approach for efficient computer experiments and optimization of Nav models. First, we utilize a fractional factorial design to identify control variables from the large set of model parameters, thereby reducing the dimensionality of parametric space. Further, we develop the Gaussian process model as a surrogate of expensive and time-consuming computer models and then identify the next best design point that yields the maximal probability of improvement. This process iterates until convergence, and the performance is evaluated and validated with real-world experimental data. Experimental results show the proposed algorithm achieves superior performance in modeling the kinetics of Nav channels under a variety of glycosylation conditions. As a result, in silico models provide a better understanding of glyco-altered mechanistic details in state transitions and distributions of Nav channels. Notably, ST3Gal4(-/-) myocytes are shown to have higher probabilities accumulated in intermediate inactivation during the repolarization and yield a shorter refractory period than WTs. The proposed statistical design of computer experiments is generally extensible to many other disciplines that involve large-scale and computationally expensive models.

Expression of TRP Ion Channels in Cardiac Myocytes

The overall goal of this investigation was to determine the extent to which transient receptor potential (TRP) ion channels of the A1 and V1 subtypes are expressed in the murine heart and whether their expression patterns are altered in the setting of diabetes. TRP ion channels are well‐established ion channel receptors found throughout the body and have significant roles in regulation of the cardiovascular system. Surprisingly little is known about coexpression patterns of TRPA1 and TRPV1 in diabetic and healthy cardiac myocytes or the role they play in mediating intracellular events in the heart. Preliminary data from our lab shows for the first time that TRPA1 is expressed at the protein level in isolated mouse cardiac myocytes. Furthermore, we have found that TRPA1 and TRPV1 in cardiac myocytes show significant (P<0.05) dose‐dependent calcium responses when exposed to allyl isothiocyanate (AITC) and capsaicin, respectively. Moreover, the expression levels of these channels in diabetic cardiac myocytes are significantly lower (P<0.05) than those in healthy cardiac myocytes. In conclusion, TRPA1 and TRPV1 ion channels in cardiac myocytes respond to AITC and capsaicin, respectively, and are down‐regulated in diabetic cardiac myocytes.

Endostatin inhibits T-type Ca2+ channel current in guinea pig ventricular myocyte.

Endostatin, a fragment of collagen XVIII, is known as an endogenous angiogenesis inhibitor, and its serum concentration increases in various cardiovascular diseases. T-type Ca2+ channel, low voltage-activated Ca2+ channel, is not expressed in adult ventricular myocytes. Re-expression of T-type Ca2+ channels in cardiac myocytes is thought to be involved in the development of cardiac hypertrophy. We examined the effects of endostatin on T-type Ca2+ channel current by whole-cell patch clamp technique in freshly isolated adult guinea pig ventricular myocytes, which exceptionally express T-type Ca2+ channels. Although endostatin 300 ng/ml had no effect on L-type Ca2+ current, it significantly inhibited T-type Ca2+ current. These data indicate that endostatin can be an endogenous inhibitor of T-type Ca2+ channels in the cardiac myocytes.

Wnt Signaling Selectively Inhibits Sodium Channels in Cardiac Myocytes

Background: Wnt signaling plays crucial roles in heart development but is normally suppressed postnatally. In arrhythmogenic conditions, such as cardiac hypertrophy and heart failure, Wnt signaling is reactivated. We explored the potential role of Wnt signaling in arrhythmogenic electrical remodeling by examining ion channel changes in postnatal cardiomyocytes with activated Wnt signaling.Methods: Neonatal rat ventricular myocytes (NRVMs) were treated with either recombinant Wnt3a protein (0.1-1.0 μg/ml) or CHIR-99021 (CH, 3 μM, a glycogen synthase kinase-3β inhibitor) to activate canonical Wnt signaling. Gene expression was assayed by real-time RT-PCR. Western blot and immunocytostaining were used to investigate protein changes. Ionic currents were recorded using whole-cell patch-clamp.Results: Treatment of NRVMs with either Wnt3a or CH caused a dose-dependent increase in Wnt target gene expression (Axin2 and Lef1), indicating activation of the Wnt/β-catenin pathway. Cardiac Na+ current (INa) density was reduced (p 10) by Wnt3a (−20±4 vs. control −59±7 pA/pF, at −30 mV) or CH (−22±5 pA/pF), without changes in steady-state inactivation or repriming kinetics. Wnt3a and CH also produced dose-dependent reductions in the mRNA level of Scn5a (the cardiac Na+ channel α subunit gene), as well as a 60% reduction in Nav1.5 protein level. Consistent with INa reduction, action potentials in Wnt3a-treated NRVMs had lower (p<0.01, n=5) upstroke amplitude (91±3 vs. control 137±2 mV) and decreased maximum upstroke velocity (70±10 vs. control 163±15 V/s). In contrast, inward rectifier K+ current and L-type Ca2+ current were not affected by Wnt3a treatment (n=8).Conclusions: The Wnt/β-catenin pathway selectively suppresses INa expression in postnatal cardiomyocytes and may contribute to arrhythmogenic remodeling in heart failure. Thus, the Wnt/β-catenin pathway may be a novel therapeutic target for heart failure and cardiac arrhythmias.

Functional interaction with filamin A and intracellular Ca2+ enhance the surface membrane expression of a small-conductance Ca2+-activated K+ (SK2) channel.

For an excitable cell to function properly, a precise number of ion channel proteins need to be trafficked to distinct locations on the cell surface membrane, through a network and anchoring activity of cytoskeletal proteins. Not surprisingly, mutations in anchoring proteins have profound effects on membrane excitability. Ca(2+)-activated K(+) channels (KCa2 or SK) have been shown to play critical roles in shaping the cardiac atrial action potential profile. Here, we demonstrate that filamin A, a cytoskeletal protein, augments the trafficking of SK2 channels in cardiac myocytes. The trafficking of SK2 channel is Ca(2+)-dependent. Further, the Ca(2+) dependence relies on another channel-interacting protein, α-actinin2, revealing a tight, yet intriguing, assembly of cytoskeletal proteins that orchestrate membrane expression of SK2 channels in cardiac myocytes. We assert that changes in SK channel trafficking would significantly alter atrial action potential and consequently atrial excitability. Identification of therapeutic targets to manipulate the subcellular localization of SK channels is likely to be clinically efficacious. The findings here may transcend the area of SK2 channel studies and may have implications not only in cardiac myocytes but in other types of excitable cells.