•ChR2 optical stimulation induces a fine-tuned control of calcium activity in murine primary myotubes.•Calcium signature of primary myotubes, followed with Gcamp6, evolves with ...differentiation.•Mimicking myotube spontaneous calcium oscillations through ChR2 stimulation enhances their myogenic differentiation.
Calcium takes part in numerous cellular processes such as proliferation, migration, differentiation, or cell death and plays a particular role in myogenesis of skeletal muscle. Indeed, intracellular calcium signaling participates, in a non-negligeable manner, to the “on” signal of muscle differentiation from undifferentiated cells to differentiated myotubes. Therefore, this differentiation can be modulated by controlling calcium activity with electrical or optogenetic stimulation approaches. In this study, we used the optogenetic tool channelrhodopsin 2 (ChR2) to control calcium activity and to modulate skeletal muscle differentiation. Using primary cultures of mouse myotubes, we showed that ChR2 stimulation was well-adapted to control intracellular calcium activity at the single cell or whole culture scale. To modulate the calcium-dependent myotube differentiation, we used an optical stimulation protocol based on GCAMP6s-decoded spontaneous calcium activity patterns of differentiated myotubes. The optical training of myotubes increased the fusion index and their contractile ability. This study demonstrates that handling a mature calcium signature with such optogenetic tool improves the differentiation of primary murine myotubes.
The channelrhodopsin 2 (ChR2) constitutes a well-adapted tool to control calcium activity by activating excitation-contraction coupling of myotubes in differentiation. The application of an optical stimulation protocol developed on spontaneous calcium patterns of differentiated myotubes enhanced their differentiation state by increasing fusion processes and their contractile ability. Display omitted
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Cardiac Na(+) channels encoded by the SCN5A gene are essential for initiating heart beats and maintaining a regular heart rhythm. Mutations in these channels have recently been associated with atrial ...fibrillation, ventricular arrhythmias, conduction disorders, and dilated cardiomyopathy (DCM).We investigated a young male patient with a mixed phenotype composed of documented conduction disorder, atrial flutter, and ventricular tachycardia associated with DCM. Further family screening revealed DCM in the patient's mother and sister and in three of the mother's sisters. Because of the complex clinical phenotypes, we screened SCN5A and identified a novel mutation, R219H, which is located on a highly conserved region on the fourth helix of the voltage sensor domain of Na(v)1.5. Three family members with DCM carried the R219H mutation.The wild-type (WT) and mutant Na(+) channels were expressed in a heterologous expression system, and intracellular pH (pHi) was measured using a pH-sensitive electrode. The biophysical characterization of the mutant channel revealed an unexpected selective proton leak with no effect on its biophysical properties. The H(+) leak through the mutated Na(v)1.5 channel was not related to the Na(+) permeation pathway but occurred through an alternative pore, most probably a proton wire on the voltage sensor domain.We propose that acidification of cardiac myocytes and/or downstream events may cause the DCM phenotype and other electrical problems in affected family members. The identification of this clinically significant H(+) leak may lead to the development of more targeted treatments.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The intrinsic cardiac nervous system (ICNS) refers to clusters of neurons, located within the heart, that participate in the neuronal regulation of cardiac functions and that are involved in the ...initiation of cardiac arrhythmias. Therefore, deciphering its role in cardiac physiology and physiopathology is mandatory.
The aim of this study was to provide a phenotypic, electrophysiological, and pharmacological characterization of the mouse ICNS, which is still poorly characterized.
Global cardiac innervation and phenotypic diversity were investigated using immunohistochemistry on cleared murine hearts and on tissue sections. The patch clamp technique was used for the electrophysiological and pharmacological characterization of isolated mouse intracardiac neurons.
We have identified the expression of 7 distinct neuronal markers within the mouse ICNS, thus proving the neurochemical diversity of this network. Of note, it was the first time that the existence of neurons expressing the calcium-binding protein calbindin, neuropeptide Y, and cocaine and amphetamine regulated transcript peptide was described in the mouse. Electrophysiology studies also revealed the existence of 4 different neuronal populations on the basis of their electrical behavior. Finally, we showed that these neurons can be modulated by several neuromodulators.
This study showed that the mouse ICNS presents a molecular and functional complexity similar to other species and is therefore a suitable model to decipher the role of individual neuronal subtypes regarding the modulation of cardiac function and the initiation of cardiac arrhythmias.
Like many voltage-gated sodium channels, the cardiac isoform Nav1.5 is well known as a glycoprotein which necessarily undergoes N-glycosylation processing during its transit to the plasma membrane. ...In some cardiac disorders, especially the Brugada syndrome (BrS), mutations in Nav1.5 encoding gene lead to intracellular retention and consequently trafficking defect of these proteins. We used two BrS mutants as tools to clarify both Nav1.5 glycosylation states and associated secretory behaviors.
Patch-clamp recordings and surface biotinylation assays of HEK293T cells expressing wild-type (WT) and/or mutant Nav1.5 proteins were performed to assess the impact of mutant co-expression on the membrane activity and localization of WT channels. Enzymatic deglycosylation assays and brefeldin A (BFA) treatments were also employed to further characterize recombinant and native Nav1.5 maturation.
The present data demonstrate that Nav1.5 channels mainly exist as two differentially glycosylated forms. We reveal that dominant negative effects induced by BrS mutants upon WT channel current result from the abnormal surface expression of the fully-glycosylated forms exclusively. Furthermore, we show that core-glycosylated channels can be found at the surface membrane of BFA-treated or untreated cells, but obviously without generating any sodium current.
Our findings provide evidence that native and recombinant Nav1.5 subunits are expressed as two distinct matured forms. Fully-glycosylated state of Nav1.5 seems to determine its functionality whereas core-glycosylated forms might be transported to the plasma membrane through an unconventional Golgi-independent secretory route.
This work highlights that N-linked glycosylation processing would be critical for Nav1.5 membrane trafficking and function.
•Native and recombinant Nav1.5 channels exist as two distinct N-glycosylated forms.•Golgian maturation step is required for surface Nav1.5 full-functionality.•Defective trafficking Nav1.5 mutants alter mature wild-type channel distribution.•Immature channels could be transported through an unconventional secretory route.
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IJS, KILJ, NUK, OILJ, SAZU, SBCE, UL, UM, UPCLJ, UPUK
Key point
Fibroblasts play a major role in heart physiology. In pathological conditions, they can lead to cardiac fibrosis when they differentiate into myofibroblasts.
This differentiated status is ...associated with changes in expression profile leading to neo‐expression of proteins such as ionic channels.
The present study investigates electrophysiological changes associated with fibroblast differentiation focusing on voltage‐gated sodium channels in human atrial fibroblasts and myofibroblasts.
We show that human atrial fibroblast differentiation in myofibroblasts is associated with de novo expression of voltage gated sodium current. Multiple arguments support that this current is predominantly supported by the Nav1.5 α‐subunit which may generate a persistent sodium entry into myofibroblasts.
Our data revealed that Nav1.5 α‐subunit expression is not restricted to cardiac myocytes within the atrium. Since fibrosis is one of the fundamental mechanisms implicated in atrial fibrillation, it is of great interest to investigate how this channel could influence myofibroblasts function.
Fibroblasts play a major role in heart physiology. They are at the origin of the extracellular matrix renewal and production of various paracrine and autocrine factors. In pathological conditions, fibroblasts proliferate, migrate and differentiate into myofibroblasts leading to cardiac fibrosis. This differentiated status is associated with changes in expression profile leading to neo‐expression of proteins such as ionic channels. The present study investigates further electrophysiological changes associated with fibroblast differentiation focusing on the activity of voltage‐gated sodium channels in human atrial fibroblasts and myofibroblasts. Using the patch clamp technique we show that human atrial myofibroblasts display a fast inward voltage gated sodium current with a density of 13.28 ± 2.88 pA pF−1 whereas no current was detectable in non‐differentiated fibroblasts. Quantitative RT‐PCR reveals a large amount of transcripts encoding the Nav1.5 α‐subunit with a fourfold increased expression level in myofibroblasts when compared to fibroblasts. Accordingly, half of the current was blocked by 1 μm of tetrodotoxin and immunocytochemistry experiments reveal the presence of Nav1.5 proteins. Overall, this current exhibits similar biophysical characteristics to sodium currents found in cardiac myocytes except for the window current that is enlarged for potentials between −100 and −20 mV. Since fibrosis is one of the fundamental mechanisms implicated in atrial fibrillation, it is of great interest to investigate how this current could influence myofibroblast properties. Moreover, since several Nav1.5 mutations are related to cardiac pathologies, this study offers a new avenue on the fibroblasts involvement of these mutations.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Anomalies in constitutive calcium entry (CCE) have been commonly attributed to cell dysfunction in pathological conditions such as cancer. Calcium influxes of this type rely on channels, such as ...transient receptor potential (TRP) channels, to be constitutively opened and strongly depend on membrane potential and a calcium driving force. We developed an optogenetic approach based on the expression of the halorhodopsin chloride pump to study CCE in non-excitable cells. Using C2C12 cells, we found that halorhodopsin can be used to achieve a finely tuned control of membrane polarization. Escalating the membrane polarization by incremental changes in light led to a concomitant increase in CCE through transient receptor potential vanilloid 2 (TRPV2) channels. Moreover, light-induced calcium entry through TRPV2 channels promoted cell migration. Our study shows for the first time that by modulating CCE and related physiological responses, such as cell motility, halorhodopsin serves as a potentially powerful tool that could open new avenues for the study of CCE and associated cellular behaviors.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Cardiac arrhythmias, which occur in a wide variety of conditions where intracellular calcium is increased, have been attributed
to the activation of a transient inward current ( I ti ). I ti is the ...result of three different Ca i -sensitive currents: the Na + âCa 2+ exchange current, a Ca 2+ -activated chloride current and a Ca 2+ -activated non-selective cationic current. Using the cell-free configuration of the patch-clamp technique, we have characterized
the properties of a Ca 2+ -activated non-selective cation channel (NSC Ca ) in freshly dissociated human atrial cardiomyocytes. In excised inside-out patches, the channel presented a linear IâV relationship with a conductance of 19 ± 0.4 pS. It discriminated poorly among monovalent cations (Na + and K + ) and was slightly permeable to Ca 2+ ions. The channel's open probability was increased by depolarization and a rise in internal calcium, for which the K d for Ca 2+ i was 20.8 μ m . Channel activity was reduced in the presence of 0.5 m m ATP or 10 μ m glibenclamide on the cytoplasmic side to 22.1 ± 16.8 and 28.5 ± 8.6%, respectively, of control. It was also inhibited by
0.1 m m flufenamic acid. The channel shares several properties with TRPM4b and TRPM5, two members of the âTRP melastatinâ subfamily.
In conclusion, the NSC Ca channel is a serious candidate to support the delayed after-depolarizations observed in Ca 2+ overload and thus may be implicated in the genesis of arrhythmias.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Myotonic dystrophy type 1 (DM1), a muscular dystrophy due to CTG expansion in the DMPK gene, can cause cardiac conduction disorders and sudden death. These cardiac manifestations are similar to those ...observed in loss-of-function SCN5A mutations, which are also responsible for Brugada syndrome (BrS).
The purpose of this study was to investigate DM1 effects on clinical expression of a loss-of-function SCN5A mutation causing BrS.
We performed complete clinical evaluation, including ajmaline test, in 1 family combining DM1 and BrS. We screened the known BrS susceptibility genes. We characterized an SCN5A mutation using whole-cell patch-clamp experiments associated with cell surface biotinylation.
The proband, a 15-year-old female, was a survivor of out-of-hospital cardiac arrest with ventricular fibrillation. She combined a DMPK CTG expansion from the father's side and an SCN5A mutation (S910L) from the mother's side. S910L is a trafficking defective mutant inducing a dominant negative effect when transfected with wild-type Nav1.5. This loss-of-function SCN5A mutation caused a Brugada phenotype during the mother's ajmaline test. Surprisingly, in the father, a DM1 patient without SCN5A mutation, ajmaline also unmasked a Brugada phenotype. Furthermore, association of both genetic abnormalities in the proband exacerbated the response to ajmaline with a massive conduction defect.
Our study is the first to describe the deleterious effect of DM1 on clinical expression of a loss-of-function SCN5A mutation and to show a provoked BrS phenotype in a DM1 patient. The modification of the ECG pattern by ajmaline supports the hypothesis of a link between DM1 and Nav1.5 loss of -function.
Voltage-gated sodium channels play an essential biophysical role in many excitable cells such as neurons. They transmit electrical signals through action potential (AP) generation and propagation in ...the peripheral (PNS) and central nervous systems (CNS). Each sodium channel is formed by one alpha-subunit and one or more beta-subunits. There is growing evidence indicating that mutations, changes in expression, or inappropriate modulation of these channels can lead to electrical instability of the cell membrane and inappropriate spontaneous activity observed during pathological states. This review describes the biochemical, biophysical and pharmacological properties of neuronal voltage-gated sodium channels (VGSC) and their implication in several neurological disorders.
Hydrocotyle bonariensis is one of the medicinal plants used in traditional medicine for the management of hypertension in Africa, Asia, and Latin America. However, the real impact of the traditional ...use of this plant on arterial hypertension has not yet been the subject of conclusive scientific information in the literature. This study aimed essentially to evaluate the potential cardiomodulatory effect of the hydroethanolic extract of Hydrocotyle bonariensis. In other to do so, the hydroethanolic extract of H. bonariensis was studied in vivo on the Wistar rat ECG and then in vitro on the isolated perfused Wistar rat heart using the Langendorff system. The extract was also tested on isolated guinea pig atria kept alive in the organ-specific vessel under physiological conditions similar to those of a living organism. At the cellular level, the effects of the extract were evaluated on the human cardiac sodium current INav1.5 and on the human cardiac pacemaker current If. We noted that the extract caused a decrease in P wave and T wave amplitudes and heart rate and an increase in the duration of the RR interval on the in vivo rat ECG. On the isolated perfused Langendorff heart as well as on the isolated atria, a decrease in the RR interval and in the heart rate was noted. The extract had no effect on human cardiac sodium current, but it did reduce human cardiac pacemaker current. In conclusion, the present study demonstrated that Hydrocotyle bonariensis, a medicinal plant traditionally used to prevent and treat hypertension, has an overall cardiomoderating effect. This effect would contribute to the reduction of blood pressure.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK, VSZLJ
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