RESUMEN

OBJECTIVE: To explore the impact of CACNA1C rs58619945 genotype on the cortical thickness of attentional networks in patients with Bipolar 1 disorder type (BD-Ⅰ). METHODS: From August 2013 and August 2019, a total of 155 BD-Ⅰ patients were recruited from the outpatient and inpatient Departments of the Affiliated Brain Hospital of Guangzhou Medical University, along with 82 healthy controls (HC) from the community and university. Genotype for the CACNA1C rs58619945 locus was determined for all BD-I patients and HC subjects, followed by 3.0 T magnetic resonance imaging scans to measure the cortical thickness in the alert, orienting, and executive control subnetworks. General linear models (GLMs) were used to evaluate the impact of CACNA1C rs58619945 on the cortical thickness of attentional networks. Concurrently, attentional dimension functions were assessed using repeatable battery for the assessment of neuropsychological status (RBANS) and Cambridge neuropsychological test automated battery rapid visual information processing (CANTAB RVP) test. RESULTS: Compared with the HC group, the BD-I patients had shown reduced thickness in bilateral prefrontal cortex, bilateral posterior cingulate cortex, and bilateral superior temporal cortex. A significant interaction between the CACNA1C genotype and the cortical thickness of right prefrontal cortex, right posterior parietal cortex and right superior temporal cortex was noted. Partial correlation analysis has demonstrated a significant correlation between CANTAB RVP and RBANS attention indices and cortical thickness in the right prefrontal cortex, right posterior cingulate cortex, and right superior temporal cortex predominantly among carriers of the BD-I G allele. CONCLUSION: The G allele of CACNA1C rs58619945 is associated with cortical thickness of the right prefrontal cortex, right posterior cingulate cortex, and right superior temporal cortex in BD-Ⅰ, which are part of the alerting and orienting network.

Asunto(s)

Atención , Trastorno Bipolar , Canales de Calcio Tipo L , Genotipo , Humanos , Adulto , Trastorno Bipolar/genética , Trastorno Bipolar/fisiopatología , Trastorno Bipolar/diagnóstico por imagen , Masculino , Femenino , Canales de Calcio Tipo L/genética , Imagen por Resonancia Magnética , Corteza Cerebral/diagnóstico por imagen , Polimorfismo de Nucleótido Simple , Persona de Mediana Edad , Adulto Joven

RESUMEN

Variants in KCNMA1, encoding the voltage- and calcium-activated K+ (BK) channel, are associated with human neurological disease. The effects of gain-of-function (GOF) and loss-of-function (LOF) variants have been predominantly studied on BK channel currents evoked under steady-state voltage and Ca2+ conditions. However, in their physiological context, BK channels exist in partnership with voltage-gated Ca2+ channels and respond to dynamic changes in intracellular Ca2+ (Ca2+i). In this study, an L-type voltage-gated Ca2+ channel present in the brain, CaV1.2, was co-expressed with wild type and mutant BK channels containing GOF (D434G, N999S) and LOF (H444Q, D965V) patient-associated variants in HEK-293T cells. Whole-cell BK currents were recorded under CaV1.2 activation using buffering conditions that restrict Ca2+i to nano- or micro-domains. Both conditions permitted wild type BK current activation in response to CaV1.2 Ca2+ influx, but differences in behavior between wild type and mutant BK channels were reduced compared to prior studies in clamped Ca2+i. Only the N999S mutation produced an increase in BK current in both micro- and nano-domains using square voltage commands and was also detectable in BK current evoked by a neuronal action potential within a microdomain. These data corroborate the GOF effect of N999S on BK channel activity under dynamic voltage and Ca2+ stimuli, consistent with its pathogenicity in neurological disease. However, the patient-associated mutations D434G, H444Q, and D965V did not exhibit significant effects on BK current under CaV1.2-mediated Ca2+ influx, in contrast with prior steady-state protocols. These results demonstrate a differential potential for KCNMA1 variant pathogenicity compared under diverse voltage and Ca2+ conditions.

Asunto(s)

Canales de Calcio Tipo L , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Células HEK293 , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/genética , Canalopatías/genética , Canalopatías/metabolismo , Calcio/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Mutación

RESUMEN

Type 2 diabetes mellitus (T2DM) is a complex chronic disease characterized by decreased insulin secretion and the development of insulin resistance. Previous genome-wide association studies demonstrated that single-nucleotide polymorphisms (SNPs) present in genes coding for ion channels involved in insulin secretion increase the risk of developing this disease. We determined the association of 16 SNPs found in CACNA1D, KCNQ1, KCNJ11, and CACNA1E genes and the increased probability of developing T2DM. In this work, we performed a case-control study in 301 Mexican adults, including 201 cases with diabetes and 100 controls without diabetes. Our findings indicate a moderate association between T2DM and the C allele, and the C/C genotype of rs312480 within CACNA1D. The CAG haplotype surprisingly showed a protective effect, whereas the CAC and CGG haplotypes have a strong association with T2DM. The C allele and C/C genotype of rs5219 were significantly associated with diabetes. Also, an association was observed between diabetes and the A allele and the A/A genotype of rs3753737 and rs175338 in CACNA1E. The TGG and CGA haplotypes were also found to be significantly associated. The findings of this study indicate that the SNPs examined could serve as a potential diagnostic tool and contribute to the susceptibility of the Mexican population to this disease.

Asunto(s)

Canales de Calcio Tipo L , Diabetes Mellitus Tipo 2 , Predisposición Genética a la Enfermedad , Canal de Potasio KCNQ1 , Polimorfismo de Nucleótido Simple , Canales de Potasio de Rectificación Interna , Humanos , Diabetes Mellitus Tipo 2/genética , Canales de Calcio Tipo L/genética , Canal de Potasio KCNQ1/genética , Femenino , Masculino , Canales de Potasio de Rectificación Interna/genética , Persona de Mediana Edad , Estudios de Casos y Controles , Adulto , Haplotipos , Canales de Calcio Tipo R/genética , Alelos , México , Anciano , Estudios de Asociación Genética , Genotipo , Frecuencia de los Genes , Proteínas de Transporte de Catión

RESUMEN

Skeletal muscle contractions are initiated by action potentials, which are sensed by the voltage-gated calcium channel (CaV1.1) and are conformationally coupled to calcium release from intracellular stores. Notably, CaV1.1 contains four separate voltage-sensing domains (VSDs), which activate channel gating and excitation-contraction (EC-) coupling at different voltages and with distinct kinetics. Here we show that a single VSD of CaV1.1 controls skeletal muscle EC-coupling. Whereas mutations in VSDs I, II and IV affect the current properties but not EC-coupling, only mutations in VSD III alter the voltage-dependence of depolarization-induced calcium release. Molecular dynamics simulations reveal comprehensive, non-canonical state transitions of VSD III in response to membrane depolarization. Identifying the voltage sensor that activates EC-coupling and detecting its unique conformational changes opens the door to unraveling the downstream events linking VSD III motion to the opening of the calcium release channel, and thus resolving the signal transduction mechanism of skeletal muscle EC-coupling.

Asunto(s)

Canales de Calcio Tipo L , Calcio , Acoplamiento Excitación-Contracción , Simulación de Dinámica Molecular , Músculo Esquelético , Dominios Proteicos , Humanos , Potenciales de Acción/fisiología , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/química , Células HEK293 , Activación del Canal Iónico , Contracción Muscular/fisiología , Músculo Esquelético/metabolismo , Mutación

RESUMEN

Most neurons are not replaced after injury and thus possess robust intrinsic mechanisms for repair after damage. Axon injury triggers a calcium wave, and calcium and cAMP can augment axon regeneration. In comparison to axon regeneration, dendrite regeneration is poorly understood. To test whether calcium and cAMP might also be involved in dendrite injury signaling, we tracked the responses of Drosophila dendritic arborization neurons to laser severing of axons and dendrites. We found that calcium and subsequently cAMP accumulate in the cell body after both dendrite and axon injury. Two voltage-gated calcium channels (VGCCs), L-Type and T-Type, are required for the calcium influx in response to dendrite injury and play a role in rapid initiation of dendrite regeneration. The AC8 family adenylyl cyclase, Ac78C, is required for cAMP production after dendrite injury and timely initiation of regeneration. Injury-induced cAMP production is sensitive to VGCC reduction, placing calcium upstream of cAMP generation. We propose that two VGCCs initiate global calcium influx in response to dendrite injury followed by production of cAMP by Ac78C. This signaling pathway promotes timely initiation of dendrite regrowth several hours after dendrite damage.

Asunto(s)

Adenilil Ciclasas , Canales de Calcio Tipo L , Calcio , AMP Cíclico , Dendritas , Animales , Adenilil Ciclasas/metabolismo , Adenilil Ciclasas/genética , Axones/metabolismo , Axones/fisiología , Calcio/metabolismo , Canales de Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo T/metabolismo , Canales de Calcio Tipo T/genética , Señalización del Calcio/genética , AMP Cíclico/metabolismo , Dendritas/metabolismo , Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Regeneración Nerviosa/fisiología , Regeneración Nerviosa/genética , Neuronas/metabolismo , Regeneración/genética , Regeneración/fisiología , Transducción de Señal

RESUMEN

Vasodilation in response to low oxygen (O2) tension (hypoxic vasodilation) is an essential homeostatic response of systemic arteries that facilitates O2 supply to tissues according to demand. However, how blood vessels react to O2 deficiency is not well understood. A common belief is that arterial myocytes are O2-sensitive. Supporting this concept, it has been shown that the activity of myocyte L-type Ca2+channels, the main ion channels responsible for vascular contractility, is reversibly inhibited by hypoxia, although the underlying molecular mechanisms have remained elusive. Here, we show that genetic or pharmacological disruption of mitochondrial electron transport selectively abolishes O2 modulation of Ca2+ channels and hypoxic vasodilation. Mitochondria function as O2 sensors and effectors that signal myocyte Ca2+ channels due to constitutive Hif1α-mediated expression of specific electron transport subunit isoforms. These findings reveal the acute O2-sensing mechanisms of vascular cells and may guide new developments in vascular pharmacology.

Asunto(s)

Subunidad alfa del Factor 1 Inducible por Hipoxia , Mitocondrias , Oxígeno , Vasodilatación , Animales , Mitocondrias/metabolismo , Oxígeno/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Transducción de Señal , Masculino , Hipoxia/metabolismo , Ratones Endogámicos C57BL , Arterias/metabolismo , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/genética , Ratones Noqueados , Transporte de Electrón , Canales de Calcio/metabolismo , Canales de Calcio/genética

RESUMEN

Due to the widespread use of methamphetamine (METH) among reproductive-aged women, the effects of intrauterine exposure to METH need to be investigated, as previous studies on this topic have been limited. The goal of this study is to examine the influence of two regulatory genes (miRNA-151-3p and CACNA1C) on the intrauterine life of mice exposed to METH. Pregnant mice received doses of 2 and 5 mg/kg of METH and saline from day 10 of pregnancy until the end. Their offspring were then evaluated for miRNA-151-3p and CACNA1C gene expression levels using real-time PCR. The findings indicated that exposure to METH reduced the expression levels of both miRNA-151-3p and CACNA1C genes in offspring compared to the control group (p≤0.001). In conclusion, intrauterine exposure to METH leads to a decrease in expression levels of both miRNA-151-3p and CACNA1C genes, potentially disrupting regulatory pathways involving these genes and having an impact on male reproductive health.

Asunto(s)

Canales de Calcio Tipo L , Regulación hacia Abajo , Metanfetamina , MicroARNs , Efectos Tardíos de la Exposición Prenatal , Testículo , Animales , Metanfetamina/toxicidad , MicroARNs/genética , MicroARNs/metabolismo , Femenino , Masculino , Embarazo , Efectos Tardíos de la Exposición Prenatal/genética , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Testículo/efectos de los fármacos , Testículo/metabolismo , Ratas , Ratones

RESUMEN

Timothy syndrome, an extremely rare disease, is closely associated with a mutation in CACNA1C gene, which encodes the cardiac L-type voltage-gated calcium channel (Cav1.2). In this study, we generated a human induced pluripotent stem cell (iPSC) line from a Timothy syndrome infant carrying heterozygous CACNA1C mutation (transcript variant NM_000719.7c.1216G>A: p.G406R). The generated iPSC line showed typical stem cell morphology, positively expressed pluripotency and proliferation markers, normal karyotype, and trilineage differentiation potential. Therefore, this patient-specific iPSC can be of great significance in investigating the mechanisms underlying Timothy syndrome, and hence establishing effective intervention strategies.

Asunto(s)

Trastorno Autístico , Canales de Calcio Tipo L , Heterocigoto , Células Madre Pluripotentes Inducidas , Síndrome de QT Prolongado , Sindactilia , Humanos , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Sindactilia/genética , Sindactilia/patología , Síndrome de QT Prolongado/genética , Síndrome de QT Prolongado/patología , Síndrome de QT Prolongado/metabolismo , Trastorno Autístico/genética , Trastorno Autístico/patología , Mutación , Línea Celular , Diferenciación Celular , Lactante

RESUMEN

Long QT Syndrome type 8 (LQT8) is a cardiac arrhythmic disorder associated with Timothy Syndrome, stemming from mutations in the CACNA1C gene, particularly the G406R mutation. While prior studies hint at CACNA1C mutations' role in ventricular arrhythmia genesis, the mechanisms, especially in G406R presence, are not fully understood. This computational study explores how the G406R mutation, causing increased transmural dispersion of repolarization, induces and sustains reentrant ventricular arrhythmias. Using three-dimensional numerical simulations on an idealized left-ventricular model, integrating the Bidomain equations with the ten Tusscher-Panfilov ionic model, we observe that G406R mutation with 11% and 50% heterozygosis significantly increases transmural dispersion of repolarization. During S1-S4 stimulation protocols, these gradients facilitate conduction blocks, triggering reentrant ventricular tachycardia. Sustained reentry pathways occur only with G406R mutation at 50% heterozygosis, while neglecting transmural heterogeneities of action potential duration prevents stable reentry, regardless of G406R mutation presence.

Asunto(s)

Potenciales de Acción , Canales de Calcio Tipo L , Simulación por Computador , Síndrome de QT Prolongado , Sindactilia , Humanos , Síndrome de QT Prolongado/genética , Síndrome de QT Prolongado/fisiopatología , Canales de Calcio Tipo L/genética , Sindactilia/genética , Sindactilia/fisiopatología , Mutación , Trastorno Autístico/genética , Trastorno Autístico/fisiopatología , Ventrículos Cardíacos/fisiopatología , Modelos Cardiovasculares , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatología , Taquicardia Ventricular/genética , Taquicardia Ventricular/fisiopatología

RESUMEN

Dietary intake of omega-3 polyunsaturated fatty acids (eicosapentaenoic acid, EPA) exerts antiarrhythmic effects, although the mechanisms are poorly understood. Here, we investigated the possible beneficial actions of EPA on saturated fatty acid-induced changes in the L-type Ca2+ channel in cardiomyocytes. Cardiomyocytes were cultured with an oleic acid/palmitic acid mixture (OAPA) in the presence or absence of EPA. Beating rate reduction in cardiomyocytes caused by OAPA were reversed by EPA. EPA also retrieved a reduction in Cav1.2 L-type Ca2+ current, mRNA, and protein caused by OAPA. Immunocytochemical analysis revealed a distinct downregulation of the Cav1.2 channel caused by OAPA with a concomitant decrease in the phosphorylated component of a transcription factor adenosine-3',5'-cyclic monophosphate (cAMP) response element binding protein (CREB) in the nucleus, which were rescued by EPA. A free fatty acid receptor 4 (FFAR4) agonist TUG-891 reversed expression of Cav1.2 and CREB mRNA caused by OAPA, whereas an FFAR4 antagonist AH-7614 abolished the effects of EPA. Excessive reactive oxygen species (ROS) accumulation caused by OAPA decreased Cav1.2 and CREB mRNA expressions, which was reversed by an ROS scavenger. Our data suggest that EPA rescues cellular Cav1.2-Ca2+ channel decline caused by OAPA lipotoxicity and oxidative stresses via both free fatty acid receptor 4-dependent and -independent pathways.

Asunto(s)

Canales de Calcio Tipo L , Ácido Eicosapentaenoico , Miocitos Cardíacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Ácido Eicosapentaenoico/farmacología , Animales , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/genética , Ratas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Ácidos Grasos/metabolismo , Transducción de Señal/efectos de los fármacos , Células Cultivadas

RESUMEN

L-type CaV1.2 current (ICa,L) links electrical excitation to contraction in cardiac myocytes. ICa,L is tightly regulated to control cardiac output. Rad is a Ras-related, monomeric protein that binds to L-type calcium channel ß subunits (CaVß) to promote inhibition of ICa,L. In addition to CaVß interaction conferred by the Rad core motif, the highly conserved Rad C-terminus can direct membrane association in vitro and inhibition of ICa,L in immortalized cell lines. In this work, we test the hypothesis that in cardiomyocytes the polybasic C-terminus of Rad confers t-tubular localization, and that membrane targeting is required for Rad-dependent ICa,L regulation. We introduced a 3xFlag epitope to the N-terminus of the endogenous mouse Rrad gene to facilitate analysis of subcellular localization. Full-length 3xFlag-Rad (Flag-Rad) mice were compared with a second transgenic mouse model, in which the extended polybasic C-termini of 3xFlag-Rad was truncated at alanine 277 (Flag-RadΔCT). Ventricular cardiomyocytes were isolated for anti-Flag-Rad immunocytochemistry and ex vivo electrophysiology. Full-length Flag-Rad showed a repeating t-tubular pattern whereas Flag-RadΔCT failed to display membrane association. ICa,L in Flag-RadΔCT cardiomyocytes showed a hyperpolarized activation midpoint and an increase in maximal conductance. Additionally, current decay was faster in Flag-RadΔCT cells. Myocardial ICa,L in a Rad C-terminal deletion model phenocopies ICa,L modulated in response to ß-AR stimulation. Mechanistically, the polybasic Rad C-terminus confers CaV1.2 regulation via membrane association. Interfering with Rad membrane association constitutes a specific target for boosting heart function as a treatment for heart failure with reduced ejection fraction.

Asunto(s)

Canales de Calcio Tipo L , Miocitos Cardíacos , Animales , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/genética , Ratones , Miocitos Cardíacos/metabolismo , Membrana Celular/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Ratones Transgénicos , Proteínas ras

RESUMEN

The gene product of ocular albinism 1 (OA1)/G-protein-coupled receptor (GPR)143 is a receptor for L-3,4-dihydroxyphenylanine (l-DOPA), the most effective agent for Parkinson's disease. When overexpressed, human wild-type GPR143, but not its mutants, inhibits neurite outgrowth in PC12 cells. We investigated the downstream signaling pathway for GPR143-induced inhibition of neurite outgrowth. Nifedipine restored GPR143-induced neurite outgrowth inhibition to the level of control transfectant but did not affect outgrowth in GPR143-knockdown cells. Cilnidipine and flunarizine also suppressed the GPR143-induced inhibition, but their effects at higher concentrations still occurred even in GPR143-knockdown cells. These results suggest that GPR143 regulates neurite outgrowth via L-type calcium channel(s).

Asunto(s)

Canales de Calcio Tipo L , Proyección Neuronal , Nifedipino , Receptores Acoplados a Proteínas G , Células PC12 , Animales , Ratas , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/genética , Nifedipino/farmacología , Proyección Neuronal/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiología , Humanos , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Proteínas del Ojo/farmacología , Flunarizina/farmacología , Transducción de Señal/efectos de los fármacos , Levodopa/farmacología , Técnicas de Silenciamiento del Gen , Neuritas/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Glicoproteínas de Membrana

RESUMEN

To achieve the functional polarization that underlies brain computation, neurons sort protein material into distinct compartments. Ion channel composition, for example, differs between axons and dendrites, but the molecular determinants for their polarized trafficking remain obscure. Here, we identify mechanisms that target voltage-gated Ca2+ channels (CaVs) to distinct subcellular compartments. In hippocampal neurons, CaV2s trigger neurotransmitter release at the presynaptic active zone, and CaV1s localize somatodendritically. After knockout of all three CaV2s, expression of CaV2.1, but not CaV1.3, restores neurotransmitter release. We find that chimeric CaV1.3s with CaV2.1 intracellular C-termini localize to the active zone, mediate synaptic vesicle exocytosis, and render release sensitive to CaV1 blockers. This dominant targeting function of the CaV2.1 C-terminus requires the first EF hand in its proximal segment, and replacement of the CaV2.1 C-terminus with that of CaV1.3 abolishes CaV2.1 active zone localization and function. We conclude that CaV intracellular C-termini mediate compartment-specific targeting.

Asunto(s)

Hipocampo , Animales , Hipocampo/metabolismo , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/genética , Canales de Calcio/metabolismo , Canales de Calcio Tipo N/metabolismo , Canales de Calcio Tipo N/genética , Neuronas/metabolismo , Humanos , Ratones , Ratas , Vesículas Sinápticas/metabolismo , Exocitosis , Células HEK293

RESUMEN

Environmental endocrine disrupting chemical 4-tert-butylphenol (4-tBP), a widely-utilized surfactant in various industries, poses potential risks to aquatic organisms. Our previous sequencing results suggested that 4-tBP-induced common carp liver injury might be associated with Ca2+ signaling and autophagy. However, the intricate involvement of these pathways in 4-tBP-induced cytotoxic mechanisms remained unexplored. To bridge these knowledge gaps, this study focused on epithelioma papulosum cyprini (EPC) cells, a significant cell type in fish biology. Initial observations showed that 4-tBP induced a dose-dependent perturbation in Ca2+ levels. Further investigations, with siRNA and L-type Ca2+ channel agonist (BAYK8644), identified L-type calcium channel gene CACNA1D as a critical regulator of 4-tBP-induced Ca2+ overload. Predictive analysis using miRanda platform suggested a potential interaction between miR-363 and CACNA1D, which was subsequently verified through dual-luciferase reporter gene assays. We then established miR-363 mimic/inhibitor models, along with miR-363 and CACNA1D co-suppression models in EPC cells. Through TEM observation, immunofluorescence assay, Ca2+ staining, and qRT-PCR analysis, we evaluated the role of miR-363/CACNA1D axis in modulating the effects of 4-tBP on Ca2+ signaling and autophagy. Results showed that miR-363 inhibitor exacerbated 4-tBP-induced increase in CALM2, CAMKII, Calpain2, and p62 expression and also led to decrease in ATG5, ATG7, and LC3b expression. In contrast, miR-363 mimic notably alleviated these changes. Notably, siRNA CACNA1D effectively modulating miR-363 inhibitor's effect. Our study revealed that 4-tBP induced Ca2+ overload and subsequent autophagy impairment via miR-363/CACNA1D axis. These findings illuminated a profound understanding of molecular mechanisms underlying 4-tBP-induced cytotoxicity and spotlighted a potential therapeutic target.

Asunto(s)

Autofagia , Calcio , Disruptores Endocrinos , MicroARNs , Animales , Autofagia/efectos de los fármacos , MicroARNs/metabolismo , MicroARNs/genética , Calcio/metabolismo , Disruptores Endocrinos/toxicidad , Carpas/metabolismo , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/genética , Fenoles/toxicidad , Contaminantes Químicos del Agua/toxicidad , Proteínas de Peces/metabolismo , Proteínas de Peces/genética

RESUMEN

Cardiac channelopathies are a group of heritable disorders that affect the heart's electrical activity due to genetic variations present in genes coding for ion channels. With the advent of new sequencing technologies, molecular diagnosis of these disorders in patients has paved the way for early identification, therapeutic management and family screening. The objective of this retrospective study was to understand the efficacy of whole-genome sequencing in diagnosing patients with suspected cardiac channelopathies who were reported negative after whole exome sequencing and analysis. We employed a 3-tier analysis approach to identify nonsynonymous variations and loss-of-function variations missed by exome sequencing, and structural variations that are better resolved only by sequencing whole genomes. By performing whole genome sequencing and analyzing 25 exome-negative cardiac channelopathy patients, we identified 3 pathogenic variations. These include a heterozygous likely pathogenic nonsynonymous variation, CACNA1C:NM_000719:exon19:c.C2570G:p. P857R, which causes autosomal dominant long QT syndrome in the absence of Timothy syndrome, a heterozygous loss-of-function variation CASQ2:NM_001232.4:c.420+2T>C classified as pathogenic, and a 9.2 kb structural variation that spans exon 2 of the KCNQ1 gene, which is likely to cause Jervell-Lange-Nielssen syndrome. In addition, we also identified a loss-of-function variation and 16 structural variations of unknown significance (VUS). Further studies are required to elucidate the role of these identified VUS in gene regulation and decipher the underlying genetic and molecular mechanisms of these disorders. Our present study serves as a pilot for understanding the utility of WGS over clinical exomes in diagnosing cardiac channelopathy disorders.

Asunto(s)

Canalopatías , Secuenciación del Exoma , Linaje , Secuenciación Completa del Genoma , Humanos , Canalopatías/genética , Canalopatías/diagnóstico , Femenino , Masculino , Canal de Potasio KCNQ1/genética , Canales de Calcio Tipo L/genética , Adulto , Estudios Retrospectivos , Síndrome de QT Prolongado/genética , Síndrome de QT Prolongado/diagnóstico , Niño , Mutación , Exoma/genética , Predisposición Genética a la Enfermedad

RESUMEN

Resistance to inhibitors of cholinesterases (ric-8 proteins) are involved in modulating G-protein function, but little is known of their potential physiological importance in the heart. In the present study, we assessed the role of resistance to inhibitors of cholinesterase 8b (Ric-8b) in determining cardiac contractile function. We developed a murine model in which it was possible to conditionally delete ric-8b in cardiac tissue in the adult animal after the addition of tamoxifen. Deletion of ric-8b led to severely reduced contractility as measured using echocardiography days after administration of tamoxifen. Histological analysis of the ventricular tissue showed highly variable myocyte size, prominent fibrosis, and an increase in cellular apoptosis. RNA sequencing revealed transcriptional remodeling in response to cardiac ric-8b deletion involving the extracellular matrix and inflammation. Phosphoproteomic analysis revealed substantial downregulation of phosphopeptides related to myosin light chain 2. At the cellular level, the deletion of ric-8b led to loss of activation of the L-type calcium channel through the ß-adrenergic pathways. Using fluorescence resonance energy transfer-based assays, we showed ric-8b protein selectively interacts with the stimulatory G-protein, Gαs. We explored if deletion of Gnas (the gene encoding Gαs) in cardiac tissue using a similar approach in the mouse led to an equivalent phenotype. The conditional deletion of the Gαs gene in the ventricle led to comparable effects on contractile function and cardiac histology. We conclude that ric-8b is essential to preserve cardiac contractile function likely through an interaction with the stimulatory G-protein and downstream phosphorylation of myosin light chain 2.

Asunto(s)

Contracción Miocárdica , Animales , Ratones , Contracción Miocárdica/efectos de los fármacos , Cadenas Ligeras de Miosina/metabolismo , Cadenas Ligeras de Miosina/genética , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/genética , Miosinas Cardíacas/metabolismo , Miosinas Cardíacas/genética , Miocardio/metabolismo , Miocardio/patología , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Humanos , Inhibidores de la Colinesterasa/farmacología , Masculino , Apoptosis/efectos de los fármacos , Factores de Intercambio de Guanina Nucleótido

RESUMEN

Primary hypokalemic periodic paralysis (HypoPP) is a skeletal muscle channelopathy most commonly caused by pathogenic variants in the calcium channel gene, CACNA1S. HypoPP can present with attacks of paralysis and/or permanent muscle weakness. Previous studies have shown that patients with HypoPP can have impaired quality of life (QoL). In this cross-sectional study, we aimed to describe the QoL in patients with HypoPP caused by pathogenic variants in CACNA1S using The Individualized Neuromuscular Quality of Life (INQoL) questionnaire, a validated tool to measure the QoL of patients with neuromuscular diseases (higher score, worse QoL). We showed that muscle weakness and fatigue were the symptoms with the greatest impact on participants' lives and that "activities", in the life domain of the INQoL, was most affected by HypoPP. Furthermore, we showed that the total INQoL score increased with age. Low QoL was primarily driven by progressive permanent muscle weakness and not attacks of paralysis, although half of the participants reported that attacks of paralysis challenged their daily life. The results suggest that special attention should be given to muscle weakness and fatigue in patients with HypoPP.

Asunto(s)

Parálisis Periódica Hipopotasémica , Debilidad Muscular , Calidad de Vida , Humanos , Masculino , Parálisis Periódica Hipopotasémica/genética , Femenino , Adulto , Estudios Transversales , Persona de Mediana Edad , Encuestas y Cuestionarios , Adulto Joven , Fatiga , Canales de Calcio Tipo L/genética , Adolescente , Anciano

RESUMEN

The CACNA1C gene encodes the alpha-1c subunit of the Cav1.2 calcium channel, a regulator of neuronal calcium influx involved in neurotransmitter release and synaptic plasticity. Genetic data show a role for CACNA1C in depressive symptoms underlying different psychiatric diagnoses. However, the mechanisms involved still require further exploration. This study aimed to investigate sex and region-specific changes in the Cacna1c gene and behavioral outcomes in mice exposed to chronic stress. Moreover, we evaluated the Nuclear factor of activated T-cells 5 (Nfat5) and the Brain-derived neurotrophic factor (Bdnf) as potential upstream and downstream Cacna1c targets and their correlation in stressed mice and humans with depression. Male and female Swiss mice were exposed to chronic unpredictable stress (CUS) for 21 days. Animal-integrated emotionality was assessed using the sucrose splash test, the tail suspension, the open-field test, and the elevated-plus-maze. Gene expression analysis was performed in the amygdala, prefrontal cortex, and hippocampus. Human data for in silico analysis was obtained from the Gene Expression Omnibus. CUS-induced impairment in integrated emotional regulation was observed in males. Gene expression analysis showed decreased levels of Cacna1c and Nfat5 and increased levels of Bdnf transcripts in the amygdala of stressed male mice. In contrast, there were no major changes in behavioral responses or gene expression in female mice after stress. The expression of the three genes was significantly correlated in the amygdala of mice and humans. The strong and positive correlation between Canac1c and Nfat5 suggests a potential role for this transcription factor in Canac1c expression. These changes could impact amygdala reactivity and emotional responses, making them a potential target for psychiatric intervention.

Asunto(s)

Amígdala del Cerebelo , Factor Neurotrófico Derivado del Encéfalo , Canales de Calcio Tipo L , Estrés Psicológico , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/genética , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/genética , Estrés Psicológico/metabolismo , Masculino , Femenino , Ratones , Amígdala del Cerebelo/metabolismo , Humanos , Modelos Animales de Enfermedad , Conducta Animal/fisiología , Corteza Prefrontal/metabolismo , Hipocampo/metabolismo , Adulto , Expresión Génica , Depresión/metabolismo , Depresión/fisiopatología

RESUMEN

Importance: The risk of mental disorders is consistently associated with variants in CACNA1C (L-type calcium channel Cav1.2) but it is not known why these channels are critical to cognition, and whether they affect the layer III pyramidal cells in the dorsolateral prefrontal cortex that are especially vulnerable in cognitive disorders. Objective: To examine the molecular mechanisms expressed in layer III pyramidal cells in primate dorsolateral prefrontal cortices. Design, Setting, and Participants: The design included transcriptomic analyses from human and macaque dorsolateral prefrontal cortex, and connectivity, protein expression, physiology, and cognitive behavior in macaques. The research was performed in academic laboratories at Yale, Harvard, Princeton, and the University of Pittsburgh. As dorsolateral prefrontal cortex only exists in primates, the work evaluated humans and macaques. Main Outcomes and Measures: Outcome measures included transcriptomic signatures of human and macaque pyramidal cells, protein expression and interactions in layer III macaque pyramidal cells using light and electron microscopy, changes in neuronal firing during spatial working memory, and working memory performance following pharmacological treatments. Results: Layer III pyramidal cells in dorsolateral prefrontal cortex coexpress a constellation of calcium-related proteins, delineated by CALB1 (calbindin), and high levels of CACNA1C (Cav1.2), GRIN2B (NMDA receptor GluN2B), and KCNN3 (SK3 potassium channel), concentrated in dendritic spines near the calcium-storing smooth endoplasmic reticulum. L-type calcium channels influenced neuronal firing needed for working memory, where either blockade or increased drive by ß1-adrenoceptors, reduced neuronal firing by a mean (SD) 37.3% (5.5%) or 40% (6.3%), respectively, the latter via SK potassium channel opening. An L-type calcium channel blocker or ß1-adrenoceptor antagonist protected working memory from stress. Conclusions and Relevance: The layer III pyramidal cells in the dorsolateral prefrontal cortex especially vulnerable in cognitive disorders differentially express calbindin and a constellation of calcium-related proteins including L-type calcium channels Cav1.2 (CACNA1C), GluN2B-NMDA receptors (GRIN2B), and SK3 potassium channels (KCNN3), which influence memory-related neuronal firing. The finding that either inadequate or excessive L-type calcium channel activation reduced neuronal firing explains why either loss- or gain-of-function variants in CACNA1C were associated with increased risk of cognitive disorders. The selective expression of calbindin in these pyramidal cells highlights the importance of regulatory mechanisms in neurons with high calcium signaling, consistent with Alzheimer tau pathology emerging when calbindin is lost with age and/or inflammation.

Asunto(s)

Canales de Calcio Tipo L , Células Piramidales , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Humanos , Animales , Células Piramidales/fisiología , Células Piramidales/metabolismo , Masculino , Trastornos del Conocimiento/fisiopatología , Trastornos del Conocimiento/genética , Corteza Prefrontal/metabolismo , Corteza Prefrontal/fisiopatología , Corteza Prefontal Dorsolateral/fisiología , Macaca mulatta , Memoria a Corto Plazo/fisiología , Femenino , Transcriptoma

RESUMEN

Dominant mutations in CACNA1S gene mainly causes hypokalemic periodic paralysis (PP)(hypoPP). A 68-year-old male proband developed a progressive proximal weakness from the age of 35. Muscle biopsy showed atrophic fibers with vacuoles containing tubular aggregates. Exome sequencing revealed a heterozygous p.R528H (c.1583G>A) mutation in the CACNA1S gene. CACNA1S-related HypoPP evolving to persistent myopathy in late adulthood is a well-known clinical condition. However, isolated progressive myopathy (without PP) was only exceptionally reported and never with an early onset. Reporting a case of early onset CACNA1S-related myopathy in a patient with no HypoPP we intend to alert clinicians to consider it in the differential diagnosis of younger adult-onset myopathies especially when featuring vacuolar changes.

Asunto(s)

Canales de Calcio Tipo L , Mutación , Humanos , Masculino , Anciano , Canales de Calcio Tipo L/genética , Enfermedades Musculares/genética , Vacuolas/patología , Vacuolas/genética , Músculo Esquelético/patología , Edad de Inicio , Parálisis Periódica Hipopotasémica/genética