Microtubule polymerization state and clathrin-dependent internalization regulate dynamics of cardiac potassium channel: Microtubule and clathrin control of KV1.5 channel.

Ion channel trafficking powerfully influences cardiac electrical activity as it regulates the number of available channels at the plasma membrane. Studies have largely focused on identifying the molecular determinants of the trafficking of the atria-specific KV1.5 channel, the molecular basis of the ultra-rapid delayed rectifier current IKur. Besides, regulated KV1.5 channel recycling upon changes in homeostatic state and mechanical constraints in native cardiomyocytes has been well documented. Here, using cutting-edge imaging in live myocytes, we investigated the dynamics of this channel in the plasma membrane. We demonstrate that the clathrin pathway is a major regulator of the functional expression of KV1.5 channels in atrial myocytes, with the microtubule network as the prominent organizer of KV1.5 transport within the membrane. Both clathrin blockade and microtubule disruption result in channel clusterization with reduced membrane mobility and internalization, whereas disassembly of the actin cytoskeleton does not. Mobile KV1.5 channels are associated with the microtubule plus-end tracking protein EB1 whereas static KV1.5 clusters are associated with stable acetylated microtubules. In human biopsies from patients in atrial fibrillation associated with atrial remodeling, drastic modifications in the trafficking balance occurs together with alteration in microtubule polymerization state resulting in modest reduced endocytosis and increased recycling. Consequently, hallmark of atrial KV1.5 dynamics within the membrane is clathrin- and microtubule- dependent. During atrial remodeling, predominance of anterograde trafficking activity over retrograde trafficking could result in accumulation ok KV1.5 channels in the plasma membrane.

Dynamic of ion channel expression at the plasma membrane of cardiomyocytes.

Cardiac myocytes are characterized by distinct structural and functional entities involved in the generation and transmission of the action potential and the excitation-contraction coupling process. Key to their function is the specific organization of ion channels and transporters to and within distinct membrane domains, which supports the anisotropic propagation of the depolarization wave. This review addresses the current knowledge on the molecular actors regulating the distinct trafficking and targeting mechanisms of ion channels in the highly polarized cardiac myocyte. In addition to ubiquitous mechanisms shared by other excitable cells, cardiac myocytes show unique specialization, illustrated by the molecular organization of myocyte-myocyte contacts, e.g., the intercalated disc and the gap junction. Many factors contribute to the specialization of the cardiac sarcolemma and the functional expression of cardiac ion channels, including various anchoring proteins, motors, small GTPases, membrane lipids, and cholesterol. The discovery of genetic defects in some of these actors, leading to complex cardiac disorders, emphasizes the importance of trafficking and targeting of ion channels to cardiac function. A major challenge in the field is to understand how these and other actors work together in intact myocytes to fine-tune ion channel expression and control cardiac excitability.

Lateral Membrane-Specific MAGUK CASK Down-Regulates NaV1.5 Channel in Cardiac Myocytes.

RATIONALE: Mechanisms underlying membrane protein localization are crucial in the proper function of cardiac myocytes. The main cardiac sodium channel, NaV1.5, carries the sodium current (INa) that provides a rapid depolarizing current during the upstroke of the action potential. Although enriched in the intercalated disc, NaV1.5 is present in different membrane domains in myocytes and interacts with several partners. OBJECTIVE: To test the hypothesis that the MAGUK (membrane-associated guanylate kinase) protein CASK (calcium/calmodulin-dependent serine protein kinase) interacts with and regulates NaV1.5 in cardiac myocytes. METHODS AND RESULTS: Immunostaining experiments showed that CASK localizes at lateral membranes of cardiac myocytes, in association with dystrophin. Whole-cell patch clamp showed that CASK-silencing increases INa in vitro. In vivo CASK knockdown similarly increased INa recorded in freshly isolated myocytes. Pull-down experiments revealed that CASK directly interacts with the C-terminus of NaV1.5. CASK silencing reduces syntrophin expression without affecting NaV1.5 and dystrophin expression levels. Total Internal Reflection Fluorescence microscopy and biotinylation assays showed that CASK silencing increased the surface expression of NaV1.5 without changing mRNA levels. Quantification of NaV1.5 expression at the lateral membrane and intercalated disc revealed that the lateral membrane pool only was increased upon CASK silencing. The protein transport inhibitor brefeldin-A prevented INa increase in CASK-silenced myocytes. During atrial dilation/remodeling, CASK expression was reduced but its localization remained unchanged. CONCLUSION: This study constitutes the first description of an unconventional MAGUK protein, CASK, which directly interacts with NaV1.5 channel and controls its surface expression at the lateral membrane by regulating ion channel trafficking.

Shear stress triggers insertion of voltage-gated potassium channels from intracellular compartments in atrial myocytes.

Atrial myocytes are continuously exposed to mechanical forces including shear stress. However, in atrial myocytes, the effects of shear stress are poorly understood, particularly with respect to its effect on ion channel function. Here, we report that shear stress activated a large outward current from rat atrial myocytes, with a parallel decrease in action potential duration. The main ion channel underlying the increase in current was found to be Kv1.5, the recruitment of which could be directly observed by total internal reflection fluorescence microscopy, in response to shear stress. The effect was primarily attributable to recruitment of intracellular pools of Kv1.5 to the sarcolemma, as the response was prevented by the SNARE protein inhibitor N-ethylmaleimide and the calcium chelator BAPTA. The process required integrin signaling through focal adhesion kinase and relied on an intact microtubule system. Furthermore, in a rat model of chronic hemodynamic overload, myocytes showed an increase in basal current despite a decrease in Kv1.5 protein expression, with a reduced response to shear stress. Additionally, integrin beta1d expression and focal adhesion kinase activation were increased in this model. This data suggests that, under conditions of chronically increased mechanical stress, the integrin signaling pathway is overactivated, leading to increased functional Kv1.5 at the membrane and reducing the capacity of cells to further respond to mechanical challenge. Thus, pools of Kv1.5 may comprise an inducible reservoir that can facilitate the repolarization of the atrium under conditions of excessive mechanical stress.

SAP97 and dystrophin macromolecular complexes determine two pools of cardiac sodium channels Nav1.5 in cardiomyocytes.

RATIONALE: The cardiac sodium channel Na(v)1.5 plays a key role in excitability and conduction. The 3 last residues of Na(v)1.5 (Ser-Ile-Val) constitute a PDZ-domain binding motif that interacts with the syntrophin-dystrophin complex. As dystrophin is absent at the intercalated discs, Na(v)1.5 could potentially interact with other, yet unknown, proteins at this site. OBJECTIVE: The aim of this study was to determine whether Na(v)1.5 is part of distinct regulatory complexes at lateral membranes and intercalated discs. METHODS AND RESULTS: Immunostaining experiments demonstrated that Na(v)1.5 localizes at lateral membranes of cardiomyocytes with dystrophin and syntrophin. Optical measurements on isolated dystrophin-deficient mdx hearts revealed significantly reduced conduction velocity, accompanied by strong reduction of Na(v)1.5 at lateral membranes of mdx cardiomyocytes. Pull-down experiments revealed that the MAGUK protein SAP97 also interacts with the SIV motif of Na(v)1.5, an interaction specific for SAP97 as no pull-down could be detected with other cardiac MAGUK proteins (PSD95 or ZO-1). Furthermore, immunostainings showed that Na(v)1.5 and SAP97 are both localized at intercalated discs. Silencing of SAP97 expression in HEK293 and rat cardiomyocytes resulted in reduced sodium current (I(Na)) measured by patch-clamp. The I(Na) generated by Na(v)1.5 channels lacking the SIV motif was also reduced. Finally, surface expression of Na(v)1.5 was decreased in silenced cells, as well as in cells transfected with SIV-truncated channels. CONCLUSIONS: These data support a model with at least 2 coexisting pools of Na(v)1.5 channels in cardiomyocytes: one targeted at lateral membranes by the syntrophin-dystrophin complex, and one at intercalated discs by SAP97.

Cholesterol modulates the recruitment of Kv1.5 channels from Rab11-associated recycling endosome in native atrial myocytes.

Cholesterol is an important determinant of cardiac electrical properties. However, underlying mechanisms are still poorly understood. Here, we examine the hypothesis that cholesterol modulates the turnover of voltage-gated potassium channels based on previous observations showing that depletion of membrane cholesterol increases the atrial repolarizing current I(Kur). Whole-cell currents and single-channel activity were recorded in rat adult atrial myocytes (AAM) or after transduction with hKv1.5-EGFP. Channel mobility and expression were studied using fluorescence recovery after photobleaching (FRAP) and 3-dimensional microscopy. In both native and transduced-AAMs, the cholesterol-depleting agent MbetaCD induced a delayed ( approximately 7 min) increase in I(Kur); the cholesterol donor LDL had an opposite effect. Single-channel recordings revealed an increased number of active Kv1.5 channels upon MbetaCD application. Whole-cell recordings indicated that this increase was not dependent on new synthesis but on trafficking of existing pools of intracellular channels whose exocytosis could be blocked by both N-ethylmaleimide and nonhydrolyzable GTP analogues. Rab11 was found to coimmunoprecipitate with hKv1.5-EGFP channels and transfection with Rab11 dominant negative (DN) but not Rab4 DN prevented the MbetaCD-induced I(Kur) increase. Three-dimensional microscopy showed a decrease in colocalization of Kv1.5 and Rab11 in MbetaCD-treated AAM. These results suggest that cholesterol regulates Kv1.5 channel expression by modulating its trafficking through the Rab11-associated recycling endosome. Therefore, this compartment provides a submembrane pool of channels readily available for recruitment into the sarcolemma of myocytes. This process could be a major mechanism for the tuning of cardiac electrical properties and might contribute to the understanding of cardiac effects of lipid-lowering drugs.

Trafficking and Gating Cooperation Between Deficient Nav1.5-mutant Channels to Rescue INa.

BACKGROUND: Pathogenic variants in SCN5A, the gene encoding the cardiac Na+ channel α-subunit Nav1.5, result in life-threatening arrhythmias, e.g., Brugada syndrome, cardiac conduction defects and long QT syndrome. This variety of phenotypes is underlied by the fact that each Nav1.5 mutation has unique consequences on the channel trafficking and gating capabilities. Recently, we established that sodium channel α-subunits Nav1.5, Nav1.1 and Nav1.2 could dimerize, thus, explaining the potency of some Nav1.5 pathogenic variants to exert dominant-negative effect on WT channels, either by trafficking deficiency or coupled gating. OBJECTIVE: The present study sought to examine whether Nav1.5 channels can cooperate, or transcomplement each other, to rescue the Na+ current (INa). Such a mechanism could contribute to explain the genotype-phenotype discordance often observed in family members carrying Na+-channel pathogenic variants. METHODS: Patch-clamp and immunocytochemistry analysis were used to investigate biophysical properties and cellular localization in HEK293 cells and rat neonatal cardiomyocytes transfected respectively with WT and 3 mutant channels chosen for their particular trafficking and/or gating properties. RESULTS: As previously reported, the mutant channels G1743R and R878C expressed alone in HEK293 cells both abolished INa, G1743R through a trafficking deficiency and R878C through a gating deficiency. Here, we showed that coexpression of both G1743R and R878C nonfunctioning channels resulted in a partial rescue of INa, demonstrating a cooperative trafficking of Nav1.5 α-subunits. Surprisingly, we also showed a cooperation mechanism whereby the R878C gating-deficient channel was able to rescue the slowed inactivation kinetics of the C-terminal truncated R1860X (ΔCter) variant, suggesting coupled gating. CONCLUSIONS: Altogether, our results add to the evidence that Nav channels are able to interact and regulate each other's trafficking and gating, a feature that likely contributes to explain the genotype-phenotype discordance often observed between members of a kindred carrying a Na+-channel pathogenic variant.

Novel CALM3 Variant Causing Calmodulinopathy With Variable Expressivity in a 4-Generation Family.

BACKGROUND: CaM (calmodulin), encoded by 3 separate genes (CALM1, CALM2, and CALM3), is a multifunctional Ca2+-binding protein involved in many signal transduction events including ion channel regulation. CaM variants may present with early-onset long QT syndrome (LQTS), catecholaminergic polymorphic ventricular tachycardia, or sudden cardiac death. Most reported variants occurred de novo. We identified a novel CALM3 variant, p.Asn138Lys (N138K), in a 4-generation family segregating with LQTS. The aim of this study was to elucidate its pathogenicity and to compare it with that of p.D130G-CaM-a variant associated with a severe LQTS phenotype. METHODS: We performed whole exome sequencing for a large, 4-generation family affected by LQTS. To assess the effect of the detected CALM3 variant, the intrinsic Ca2+-binding affinity was measured by stoichiometric Ca2+ titrations and equilibrium titrations. L-type Ca2+ and slow delayed rectifier potassium currents (ICaL and IKs) were recorded by whole-cell patch-clamp. Cav1.2 and Kv7.1 membrane expression were determined by optical fluorescence assays. RESULTS: We identified 14 p.N138K-CaM carriers in a family where 2 sudden deaths occurred in children. Several members were only mildly affected compared with CaM-LQTS patients to date described in literature. The intrinsic Ca2+-binding affinity of the CaM C-terminal domain was 10-fold lower for p.N138K-CaM compared with wild-type-CaM. ICaL inactivation was slowed in cells expressing p.N138K-CaM but less than in p.D130G-CaM cells. Unexpectedly, a larger IKs current density was observed in cells expressing p.N138K-CaM, but not for p.D130G-CaM, compared with wild-type-CaM. CONCLUSIONS: The p.N138K CALM3 variant impairs Ca2+-binding affinity of CaM and ICaL inactivation but potentiates IKs. The variably expressed phenotype of this variant compared with previously published de novo LQTS-CaM variants is likely explained by a milder impairment of ICaL inactivation combined with IKs augmentation.

Kv4 potassium channels form a tripartite complex with the anchoring protein SAP97 and CaMKII in cardiac myocytes.

Membrane-associated guanylate kinase (MAGUK) proteins are major determinants of the organization of ion channels in the plasma membrane in various cell types. Here, we investigated the interaction between the MAGUK protein SAP97 and cardiac Kv4.2/3 channels, which account for a large part of the outward potassium current, I(to), in heart. We found that the Kv4.2 and Kv4.3 channels C termini interacted with SAP97 via a SAL amino acid sequence. SAP97 and Kv4.3 channels were colocalized in the sarcolemma of cardiomyocytes. In CHO cells, SAP97 clustered Kv4.3 channels in the plasma membrane and increased the current independently of the presence of KChIP and dipeptidyl peptidase-like protein-6. Suppression of SAP97 by using short hairpin RNA inhibited I(to) in cardiac myocytes, whereas its overexpression by using an adenovirus increased I(to). Kv4.3 channels without the SAL sequence were no longer regulated by Ca2+/calmodulin kinase (CaMK)II inhibitors. In cardiac myocytes, pull-down and coimmunoprecipitation assays showed that the Kv4 channel C terminus, SAP97, and CaMKII interact together, an interaction suppressed by SAP97 silencing and enhanced by SAP97 overexpression. In HEK293 cells, SAP97 silencing reproduced the effects of CaMKII inhibition on current kinetics and suppressed Kv4/CaMKII interactions. In conclusion, SAP97 is a major partner for surface expression and CaMKII-dependent regulation of cardiac Kv4 channels.

In vivo Dominant-Negative Effect of an SCN5A Brugada Syndrome Variant.

Loss-of-function mutations in the cardiac Na+ channel α-subunit Nav1.5, encoded by SCN5A, cause Brugada syndrome (BrS), a hereditary disease characterized by sudden cardiac death due to ventricular fibrillation. We previously evidenced in vitro the dominant-negative effect of the BrS Nav1.5-R104W variant, inducing retention of wild-type (WT) channels and leading to a drastic reduction of the resulting Na+ current (I Na ). To explore this dominant-negative effect in vivo, we created a murine model using adeno-associated viruses (AAVs). METHODS: Due to the large size of SCN5A, a dual AAV vector strategy was used combining viral DNA recombination and trans-splicing. Mice were injected with two AAV serotypes capsid 9: one packaging the cardiac specific troponin-T promoter, the 5' half of hSCN5A cDNA, a splicing donor site and a recombinogenic sequence; and another packaging the complementary recombinogenic sequence, a splicing acceptor site, the 3' half of hSCN5A cDNA fused to the gfp gene sequence, and the SV40 polyA signal. Eight weeks after AAV systemic injection in wild-type (WT) mice, echocardiography and ECG were recorded and mice were sacrificed. The full-length hSCN5A-gfp expression was assessed by western blot and immunohistochemistry in transduced heart tissues and the Na+ current was recorded by the patch-clamp technique in isolated adult GFP-expressing heart cells. RESULTS: Almost 75% of the cardiomyocytes were transduced in hearts of mice injected with hNav1.5 and ∼30% in hNav1.5-R104W overexpressing tissues. In ventricular mice cardiomyocytes expressing R104W mutant channels, the endogenous I Na was significantly decreased. Moreover, overexpression of R104W channels in normal hearts led to a decrease of total Nav1.5 expression. The R104W mutant also induced a slight dilatation of mice left ventricles and a prolongation of RR interval and P-wave duration in transduced mice. Altogether, our results demonstrated an in vivo dominant-negative effect of defective R104W channels on endogenous ones. CONCLUSION: Using a trans-splicing and viral DNA recombination strategy to overexpress the Na+ channel in mouse hearts allowed us to demonstrate in vivo the dominant-negative effect of a BrS variant identified in the N-terminus of Nav1.5.

Specificities of atrial electrophysiology: Clues to a better understanding of cardiac function and the mechanisms of arrhythmias.

The electrical properties of the atria and ventricles differ in several aspects reflecting the distinct role of the atria in cardiac physiology. The study of atrial electrophysiology had greatly contributed to the understanding of the mechanisms of atrial fibrillation (AF). Only the atrial L-type calcium current is regulated by serotonine or, under basal condition, by phosphodiesterases. These distinct regulations can contribute to I(Ca) down-regulation observed during AF, which is an important determinant of action potential refractory period shortening. The voltage-gated potassium current, I(Kur), has a prominent role in the repolarization of the atrial but not ventricular AP. In many species, this current is based on the functional expression of K(V)1.5 channels, which might represent a specific therapeutic target for AF. Mechanisms regulating the trafficking of K(V)1.5 channels to the plasma membrane are being actively investigated. The resting potential of atrial myocytes is maintained by various inward rectifier currents which differ with ventricle currents by a reduced density of I(K1), the presence of a constitutively active I(KACh) and distinct regulation of I(KATP). Stretch-sensitive or mechanosensitive ion channels are particularly active in atrial myocytes and are involved in the secretion of the natriuretic peptide. Integration of knowledge on electrical properties of atrial myocytes in comprehensive schemas is now necessary for a better understanding of the physiology of atria and the mechanisms of AF.

RNA interference targeting STIM1 suppresses vascular smooth muscle cell proliferation and neointima formation in the rat.

Our objective was to study the expression and function of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum protein recently identified as the calcium sensor that regulated Ca(2+)-released activated channels in T cells. STIM1 was found to be upregulated in serum-induced proliferating human coronary artery smooth muscle cells (hCASMCs) as well as in the neointima of injured rat carotid arteries. Growth factors-induced proliferation was significantly lower in hCASMC transfected with STIM1 siRNA than in those transfected with scrambled siRNA (increase relative to 0.1% S: 116 +/- 12% and 184 +/- 16%, respectively, P < 0.01). To assess the role of STIM1 in preventing vascular smooth muscle cells (VSMCs) proliferation in vivo, we infected balloon-injured rat carotid arteries with an adenoviral vector expressing a short hairpin (sh) RNA against rat STIM1 mRNA (Ad-shSTIM1). Intima/media ratios reflecting the degree of restenosis were significantly lower in Ad-shSTIM1- infected arteries than in Ad-shLuciferase-infected arteries (0.34 +/- 0.02 vs. 0.92 +/- 0.11, P < 0.006). Finally, we demonstrated that silencing STIM1 prevents activation of the transcription factor NFAT (nuclear factor of activated T cell). In conclusion, STIM1 appears as a major regulator of in vitro and in vivo VSMC proliferation, representing a novel and original pharmacological target for prominent vascular proliferative diseases.

Distinct calcium/calmodulin-dependent serine protein kinase domains control cardiac sodium channel membrane expression and focal adhesion anchoring.

BACKGROUND: Membrane-associated guanylate kinase proteins function as adaptor proteins to mediate the recruitment and scaffolding of ion channels in the plasma membrane in various cell types. In the heart, the protein calcium/calmodulin-dependent serine protein kinase (CASK) negatively regulates the main cardiac sodium channel NaV1.5, which carries the sodium current (INa) by preventing its anterograde trafficking. CASK is also a new member of the dystrophin-glycoprotein complex and, like syntrophin, binds to the C-terminal domain of the channel. OBJECTIVE: The purpose of this study was to unravel the mechanisms of CASK-mediated negative INa regulation and interaction with the dystrophin-glycoprotein complex in cardiac myocytes. METHODS: CASK adenoviral truncated constructs with sequential single functional domain deletions were designed for overexpression in cardiac myocytes: CASKΔCAMKII, CASKΔL27A, CASKΔL27B, CASKΔPDZ, CASKΔSH3, CASKΔHOOK, and CASKΔGUK. A combination of whole-cell patch-clamp recording, total internal reflection fluorescence microscopy, and biochemistry experiments was conducted in cardiac myocytes to study the functional consequences of domain deletions. RESULTS: We show that both L27B and GUK domains are required for the negative regulatory effect of CASK on INa and NaV1.5 surface expression and that the HOOK domain is essential for interaction with the cell adhesion dystrophin-glycoprotein complex. CONCLUSION: This study demonstrates that the multimodular structure of CASK confers an ability to simultaneously interact with several targets within cardiomyocytes. Through its L27B, GUK, and HOOK domains, CASK potentially provides the ability to control channel delivery at adhesion points in cardiomyocytes.

Protease-activated receptor-1 mediates thrombin-induced persistent sodium current in human cardiomyocytes.

After the thrombus formation in cardiac cavities or coronaries, the serine protease thrombin is produced and can therefore reach the myocardial tissue by the active process of extravasation and binds to the G protein-coupled protease-activated receptor-1 (PAR1) expressed in human myocardium. The role of PAR1 was investigated in the thrombin effect on sodium current (I(Na)). I(Na) was recorded in freshly isolated human atrial myocytes by the whole-cell patch-clamp method. Action potentials (AP) were recorded in guinea pig ventricular tissue by the conventional glass microelectrode technique. Thrombin-activated PAR1 induced a tetrodotoxin-blocked persistent sodium current, I(NaP), in a concentration-dependent manner with an apparent EC(50) of 28 U/ml. The PAR1 agonist peptide SFLLR-NH(2) (50 microM) was able to mimic PAR1-thrombin action, whereas PAR1 antagonists N(3)-cyclopropyl-7-((4-(1-methylethyl)-phenyl)methyl)-7H-pyrrolo(3,2-f)quinazoline-1,3-diamine (SCH 203099; 10 microM) and 1-(3,5-di-tert-butyl-4-hydroxy-phenyl)-2-[3-(3-ethyl-3-hydroxy-pentyl)-2-imino-2,3-dihydro-imidazol-1-yl]-ethanone (ER 112787) (1 microM), completely inhibited it. The activated PAR1 involves the calcium-independent phospholipase-A(2) signaling pathway because two inhibitors of this cascade, bromoenol lactone (50 microM) and haloenol lactone suicide substrate (50 microM), block PAR1-thrombin-induced I(NaP).Asa consequence of I(NaP) activation, in guinea pig right ventricle papillary muscle, action potential duration (APD) were significantly increased by 20% and 15% under the respective action of 32 U/ml thrombin and 50 microM SFLLR-NH(2), and these increases in APD were prevented by 1 microM tetrodotoxin or markedly reduced by application of 1 microM SCH 203099 or ER 112787. Thrombin, through PAR1 activation, increases persistent component of the Na(+) current resulting in an uncontrolled sodium influx into the cardiomyocyte, which can contribute to cellular injuries observed during cardiac ischemia.

Angiotensin II signaling pathways mediate expression of cardiac T-type calcium channels.

Recent studies indicate that cardiac T-type Ca2+ current (ICaT) reappears in hypertrophied ventricular cells. The aim of this study was to investigate the role of angiotensin II (Ang II), a major inducer of cardiac hypertrophy, in the reexpression of T-type channel in left ventricular hypertrophied myocytes. We induced cardiac hypertrophy in rats by abdominal aorta stenosis for 12 weeks and thereafter animals were treated for 2 weeks with losartan (12 mg/kg per day), an antagonist of type 1 Ang II receptors (AT1). In hypertrophied myocytes, we showed that the reexpressed ICaT is generated by the CaV3.1 and CaV3.2 subunits. After losartan treatment, ICaT density decreased from 0.40+/-0.05 pA/pF (n=26) to 0.20+/-0.03 pA/pF (n=27, P<0.01), affecting CaV3.1- and CaV3.2-related currents. The amount of CaV3.1 mRNA increased during hypertrophy and retrieved its nonhypertrophic level after losartan treatment, whereas the amount of CaV3.2 mRNA was unaffected by stenosis. In cultured newborn ventricular cells, chronic Ang II application (0.1 micromol/L) also increased ICaT density and CaV3.1 mRNA amount. UO126, a mitogen-activated protein kinase kinase-1/2 (MEK1/2) inhibitor, reduced Ang II-increased ICaT density and CaV3.1 mRNA amount. Bosentan, an endothelin (ET) receptor antagonist, reduced Ang II-increased ICaT density without affecting the amount of CaV3.1 mRNA. Finally, cotreatment with bosentan and UO126 abolished the Ang II-increased ICaT density. Our results show that AT1-activated MEK pathway and autocrine ET-activated independent MEK pathway upregulate T-type channel expression. Ang II-increased of ICaT density observed in hypertrophied myocytes may play a role in the pathogenesis of Ca2+ overload and arrhythmias seen in cardiac pathology.

Thrombin facilitation of voltage-gated sodium channel activation in human cardiomyocytes: implications for ischemic sodium loading.

BACKGROUND: Thrombin plays a role in mediating ischemic injury and cardiac arrhythmias, but the mechanisms involved are poorly understood. Because voltage-gated sodium channels (VGSCs) have not previously been considered, putative effects of thrombin on VGSC function were investigated in human isolated cardiomyocytes. METHODS AND RESULTS: Sodium current (I(Na)) was recorded by the whole-cell patch-clamp method. Thrombin increased peak I(Na) amplitude in an activity-dependent manner, from 1 to 100 U/mL, with an apparent EC50 of 91+/-16 U/mL. When tested at 32 U/mL, thrombin-increased I(Na) was abolished by tetrodotoxin (50 micromol/L). Thrombin effects on I(Na) were reversible and repeatable, and 100 U/mL doubled peak I(Na) amplitude. Thrombin (32 U/mL) shifted I(Na) activation to hyperpolarized potentials without affecting steady-state inactivation, producing unusually large increases in window current. Hirudin (320 U/mL) or haloenol lactone suicide substrate (10 micromol/L) failed to significantly affect these effects of thrombin. In current-clamped cardiomyocytes, thrombin (32 U/mL) depolarized resting membrane potential by 10 mV. CONCLUSIONS: Facilitation of VGSC activation causing large increases in window current is a major mechanism by which thrombin may promote ischemic sodium loading and injury.

The voltage-independent B-type Ca2+ channel modulates apoptosis of cardiac myocytes.

In cardiac myocytes, Ca2+ is the second messenger of various physiological processes, including the excitation-contraction coupling phenomenon. Here, we examined how Ca2+ regulates cardiac myocyte apoptosis. Using an adult rat ventricular myocyte culture model, we found that ceramide (Cer) induces an apoptotic process that is highly sensitive to calcium: it is inhibited by the Ca2+buffer BAPTA-AM and is potentiated by the Ca2+ ionophore ionomycin. Cer-treated myocytes maintained their Ca2+ homeostasis and normal activity of their voltage-dependent sarcolemmal Ca2+ entry pathways but showed enhanced activity of B-type Ca2+ channels that were stimulated by Cer in the inside-out patch clamp configuration. Pharmacological modulations of B-type Ca2+ channels modulated Cer-induced apoptosis. Mitochondria were involved in Cer-induced apoptosis, as indicated by the inhibitory effect of cyclosporine A and caspase-9 inhibitor. Both the loss of mitochondrial membrane potential and the caspase-9 activation in Cer-treated myocytes were attenuated by blocking B-type Ca2+ channels. The mitochondrial Ca2+ content was evaluated as changes in [Ca2+]i after a short application of a mitochondrial uncoupler, FCCP, was increased in Cer-treated cells. Together, these results show that Ca2+ modulates Cer-induced cardiac myocyte apoptosis through the coupling of B-type Ca2+ channels with mitochondria.

Abnormal sodium current properties contribute to cardiac electrical and contractile dysfunction in a mouse model of myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is the most common neuromuscular disorder and is associated with cardiac conduction defects. However, the mechanisms of cardiac arrhythmias in DM1 are unknown. We tested the hypothesis that abnormalities in the cardiac sodium current (INa) are involved, and used a transgenic mouse model reproducing the expression of triplet expansion observed in DM1 (DMSXL mouse). The injection of the class-I antiarrhythmic agent flecainide induced prominent conduction abnormalities and significantly lowered the radial tissular velocities and strain rate in DMSXL mice compared to WT. These abnormalities were more pronounced in 8-month-old mice than in 3-month-old mice. Ventricular action potentials recorded by standard glass microelectrode technique exhibited a lower maximum upstroke velocity [dV/dt](max) in DMSXL. This decreased [dV/dt](max) was associated with a 1.7 fold faster inactivation of INa in DMSXL myocytes measured by the whole-cell patch-clamp technique. Finally in the DMSXL mouse, no mutation in the Scn5a gene was detected and neither cardiac fibrosis nor abnormalities of expression of the sodium channel protein were observed. Therefore, alterations in the sodium current markedly contributed to electrical conduction block in DM1. This result should guide pharmaceutical and clinical research toward better therapy for the cardiac arrhythmias associated with DM1.