Activation Of Voltage-gated Calcium Channels Research Articles

Holistic profiling of the venom from the Brazilian wandering spider Phoneutria nigriventer by combining high-throughput ion channel screens with venomics.

Introduction: Spider venoms are a unique source of bioactive peptides, many of which display remarkable biological stability and neuroactivity. Phoneutria nigriventer, often referred to as the Brazilian wandering spider, banana spider or "armed" spider, is endemic to South America and amongst the most dangerous venomous spiders in the world. There are 4,000 envenomation accidents with P. nigriventer each year in Brazil, which can lead to symptoms including priapism, hypertension, blurred vision, sweating, and vomiting. In addition to its clinical relevance, P. nigriventer venom contains peptides that provide therapeutic effects in a range of disease models. Methods: In this study, we explored the neuroactivity and molecular diversity of P. nigriventer venom using fractionation-guided high-throughput cellular assays coupled to proteomics and multi-pharmacology activity to broaden the knowledge about this venom and its therapeutic potential and provide a proof-of-concept for an investigative pipeline to study spider-venom derived neuroactive peptides. We coupled proteomics with ion channel assays using a neuroblastoma cell line to identify venom compounds that modulate the activity of voltage-gated sodium and calcium channels, as well as the nicotinic acetylcholine receptor. Results: Our data revealed that P. nigriventer venom is highly complex compared to other neurotoxin-rich venoms and contains potent modulators of voltage-gated ion channels which were classified into four families of neuroactive peptides based on their activity and structures. In addition to the reported P. nigriventer neuroactive peptides, we identified at least 27 novel cysteine-rich venom peptides for which their activity and molecular target remains to be determined. Discussion: Our findings provide a platform for studying the bioactivity of known and novel neuroactive components in the venom of P. nigriventer and other spiders and suggest that our discovery pipeline can be used to identify ion channel-targeting venom peptides with potential as pharmacological tools and to drug leads.

Investigation of PKA interactions with cardiac voltage gated calcium channel and Rad GTPase.

The β-adrenergic signaling pathway leads to PKA-mediated activation of L-type voltage-gated calcium channels (CaVs) in multiple tissues. Various sites have been proposed to be involved In CaV1.2. Notably, PKA-mediated phosphorylation of Ser1981 is reported in diabetes progression, synaptic plasticity, and the increase of cardiac calcium currents during the fight-or-flight response. However, the role of Ser1981 phosphorylation is heavily scrutinized, with alternative sites Ser1718 and Ser1535 proposed to be important during β-adrenergic activation of CaV1.2. Recently, a constitutive inhibitor of CaV1.2 called Rad was also identified as a target of PKA during cardiac excitation. However, it is unclear which of the four potential sites (Ser25, Ser38, Ser272, and Ser300) are preferentially phosphorylated by PKA. Using quantitative binding and kinetic experiments we measured significant binding and activity towards Ser1981 on CaV1.2 and Ser25 and Ser272 on Rad, with no measurable PKA binding or kinetic activity found for the other sites. We also captured multiple PKAc high resolution structures using X-ray crystallography. Our PKAc:AMP-PNP/Mg2+:CaV peptide ternary complex reveal a novel mode of PKAc substrate recognition, involving a phenylalanine at the P+1 position anchored into a hydrophobic pocket of the enzyme's active site. Our work thus provides valuable mechanistic insight into PKA substrate recognition and suggest the likely physiological target sites for PKA on CaV1.2 and Rad.

Disease-associated calmodulin variants impair regulation of neuronal voltage-gated calcium channels.

Calmodulin (CaM) is a regulatory protein that associates with numerous binding partners, and it participates in key mechanisms by which multiple types of excitable cells sense and respond to changing levels of calcium. In neurons and cardiomyocytes, calcium ions flux into the cytosol through voltage-gated calcium channels (VGCCs) and bind to CaM. These binding events trigger an array of important feedback processes, including calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF) of multiple VGCC subtypes. In this way, CaM modulates VGCC activity and participates in calcium signaling cascades. Calmodulinopathies are a group of related disorders that arise from pathogenic mutations in CaM. These mutations alter cardiac calcium dynamics in a way that leads to debilitating disorders, including long-QT syndrome (LQTS). LQTS in calmodulinopathy patients has been shown to arise from the mutant forms of CaM preventing the protein from mediating proper CDI of cardiac CaV1.2 L-type channels. However, CaM regulation of VGCCs is not exclusive to the heart, and disruption of these processes in neuronal VGCCs may contribute to the comorbid neurological symptoms frequently observed in calmodulinopathy patients, including autism spectrum disorder, developmental delay, and seizures. We investigated the impacts of calmodulinopathic mutations on VGCCs known to be important for neuronal function. We found that CaM mutants caused diminished CDI in CaV1.3 channels similar to the deficit of CDI seen in CaV1.2 channels, as well as disruption of CDF in CaV2.1 channels. Interestingly, the pattern of CaV2.1 CDF disruptions by certain mutations does not entirely match the pattern seen in CaV1.2 and CaV1.3, indicating a differential effect of select mutations in this neuronal channel. Overall, our results suggest that calmodulinopathic mutations may have consequences to calcium dynamics that extend into neural tissues, in addition to their well-described impacts on cardiac health.

The natural product argentatin C attenuates postoperative pain via inhibition of voltage-gated sodium and T-type voltage-gated calcium channels.

Postoperative pain occurs in as many as 70% of surgeries performed worldwide. Postoperative pain management still relies on opioids despite their negative consequences, resulting in a public health crisis. Therefore, it is important to develop alternative therapies to treat chronic pain. Natural products derived from medicinal plants are potential sources of novel biologically active compounds for development of safe analgesics. In this study, we screened a library of natural products to identify small molecules that target the activity of voltage-gated sodium and calcium channels that have important roles in nociceptive sensory processing. Fractions derived from the Native American medicinal plant, Parthenium incanum, were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion (DRG) neurons. Further separation of these fractions yielded a cycloartane-type triterpene identified as argentatin C, which was additionally evaluated using whole-cell voltage and current-clamp electrophysiology, and behavioural analysis in a mouse model of postsurgical pain. Argentatin C blocked the activity of both voltage-gated sodium and low-voltage-activated (LVA) calcium channels in calcium imaging assays. Docking analysis predicted that argentatin C may bind to NaV 1.7-1.9 and CaV 3.1-3.3 channels. Furthermore, argentatin C decreased Na+ and T-type Ca2+ currents as well as excitability in rat and macaque DRG neurons, and reversed mechanical allodynia in a mouse model of postsurgical pain. These results suggest that the dual effect of argentatin C on voltage-gated sodium and calcium channels supports its potential as a novel treatment for painful conditions.

S-13-5: UPDATES ON FAMILIAL HYPERALDSOTERONISM

The first reported (Sutherland et al. 1966) form of familial primary aldosteronism (PA) involved a father and son and was glucocorticoid-remediable (familial hyperaldosteronism type I, FH-I). In 1992, Lifton and colleagues elucidated its causation, a hybrid gene mutation composed of CYP11B1 (encoding 11beta-hydroxylase) regulatory sequences and CYP11B2 (aldosterone synthase) coding sequences causing ACTH-regulated aldosterone overproduction and hypertension, both readily controlled by low-dose glucocorticoid administration. Expression of aldosterone synthase activity within zona fasciculata (ZF) explained overproduction of hybrid steroids, 18-hydroxy- and 18-oxocortisol. Since 2011, PA-causing germline mutations in another four genes (three associated with FH) have been reported. While three encode cation channels (KCNJ5, CACNA1H and CACNA1D), one encodes an anion channel (CLCN2). FH-III is caused by inheritance of gain-of-function mutations in KCNJ5, encoding an inwardly rectifying potassium channel (GIRK-4). Depending on the exact mutation involved, phenotype varies from very severe (profound PA, hybrid steroid elevation and ZF hyperplasia and requiring bilateral adrenalectomy to control hypertension) to mild (absence of above with PA controllable with spironolactone). Most pedigrees demonstrate early-onset hypertension. Mutations reduce selectivity of GIRK-4 to potassium, permitting entry of sodium into the adrenal cortical cell, which results in cell depolarization and calcium influx, leading to increased expression of CYP11B2 and aldosterone synthesis. A gain-of-function mutation in CACNA1H, encoding a T-type voltage-gated calcium channel, was found in five of 40 subjects with early-onset PA and three relatives with early-onset hypertension (FH-IV). Adrenals appeared normal on imaging. The mutation shifts activation of zona glomerulosa (ZG) cells to a more hyperpolarized potential and slows inactivation, putatively resulting in increased calcium influx and aldosterone production. Germline mutations in CACNA1D, encoding an L-type voltage-gated calcium channel highly expressed in ZG, were identified in two of 100 individuals with early-onset PA. Both occurred de novo, and were associated with seizures and a variety of neurological abnormalities (PASNA). Both mutations cause channel activation at less depolarized membrane potentials, and one nearly abolished channel inactivation. In a large Australian family with FH-II, a mutation was found in CLCN2 encoding a chloride channel, ClC-2. CLCN2 mutations were found in another seven of 80 individuals with early-onset PA. A concurrent report found a CLCN2 mutation in one of 12 subjects with early-onset PA. PA due to CLCN2 mutations responds well to medical treatment. In the adrenal, ClC-2 localizes predominantly to ZG and mutations predispose cell depolarization, leading to calcium influx via activation of voltage-gated calcium channels and increased aldosterone production.

Regulation of Presynaptic Calcium Channels.

Voltage-gated calcium channels (VGCCs), especially Cav2.1 and Cav2.2, are the major mediators of Ca2+ influx at the presynaptic membrane in response to neuron excitation, thereby exerting a predominant control on synaptic transmission. To guarantee the timely and precise release of neurotransmitters at synapses, the activity of presynaptic VGCCs is tightly regulated by a variety of factors, including auxiliary subunits, membrane potential, G protein-coupled receptors (GPCRs), calmodulin (CaM), Ca2+-binding proteins (CaBP), protein kinases, various interacting proteins, alternative splicing events, and genetic variations.

Vascular Response of Tetrabromobisphenol a in Rat Aorta: Calcium Channels Inhibition and Potassium Channels Activation.

Tetrabromobisphenol A (TBBPA) is a flame retardant widely used to reduce flammability. It is an endocrine disruptor, and due to constant human exposure, some concerns have been raised regarding its impact on human health. Studies showed that TBBPA affects oxidative stress, cell proliferation and intracellular calcium levels. However, the vascular consequences of TBBPA exposure are still relatively unexplored. Hence, this work aimed to analyse TBBPA effects on rat aortic smooth muscle and its action mechanisms. Through an ex vivo approach, Wistar rat aortas were used in an organ bath to evaluate the vascular effect of TBBPA (0.01–100 μM). Additionally, TBBPA’s mode of action was studied through calcium and potassium channel inhibitors. Resorting to in vitro studies, A7r5 cells were used to analyse L-Type voltage-gated calcium channel (VGCC) activity through the whole-cell configuration of the patch clamp technique, and the mRNA expression of proteins and ion channels involved in vascular contractility. The results showed vasorelaxation of rat aorta induced by TBBPA exposure, involving the inactivation of L-Type VGCC and activation of potassium channels, and the modulation of mRNA expression of L-type calcium and large-conductance calcium 1.1 and the BKCa 1.1 α- and β1 -subunit channels, soluble guanylyl cyclase and protein Kinase G.

Genetic Alterations in Benign Adrenal Tumors

The genetic basis of most types of adrenal adenomas has been elucidated over the past decade, leading to the association of adrenal gland pathologies with specific molecular defects. Various genetic studies have established links between variants affecting the protein kinase A (PKA) signaling pathway and benign cortisol-producing adrenal lesions. Specifically, genetic alterations in GNAS, PRKAR1A, PRKACA, PRKACB, PDE11A, and PDE8B have been identified. The PKA signaling pathway was initially implicated in the pathogenesis of Cushing syndrome in studies aiming to understand the underlying genetic defects of the rare tumor predisposition syndromes, Carney complex, and McCune-Albright syndrome, both affected by the same pathway. In addition, germline variants in ARMC5 have been identified as a cause of primary bilateral macronodular adrenal hyperplasia. On the other hand, primary aldosteronism can be subclassified into aldosterone-producing adenomas and bilateral idiopathic hyperaldosteronism. Various genes have been reported as causative for benign aldosterone-producing adrenal lesions, including KCNJ5, CACNA1D, CACNA1H, CLCN2, ATP1A1, and ATP2B3. The majority of them encode ion channels or pumps, and genetic alterations lead to ion transport impairment and cell membrane depolarization which further increase aldosterone synthase transcription and aldosterone overproduction though activation of voltage-gated calcium channels and intracellular calcium signaling. In this work, we provide an overview of the genetic causes of benign adrenal tumors.

Inhibitory effects on L- and N-type calcium channels by a novel CaV\u03b21 variant identified in a patient with autism spectrum disorder

Voltage-gated calcium channel (VGCC) subunits have been genetically associated with autism spectrum disorders (ASD). The properties of the pore-forming VGCC subunit are modulated by auxiliary β-subunits, which exist in four isoforms (CaVβ1-4). Our previous findings suggested that activation of L-type VGCCs is a common feature of CaVβ2 subunit mutations found in ASD patients. In the current study, we functionally characterized a novel CaVβ1b variant (p.R296C) identified in an ASD patient. We used whole-cell and single-channel patch clamp to study the effect of CaVβ1b_R296C on the function of L- and N-type VGCCs. Furthermore, we used co-immunoprecipitation followed by Western blot to evaluate the interaction of the CaVβ1b-subunits with the RGK-protein Gem. Our data obtained at both, whole-cell and single-channel levels, show that compared to a wild-type CaVβ1b, the CaVβ1b_R296C variant inhibits L- and N-type VGCCs. Interaction with and modulation by the RGK-protein Gem seems to be intact. Our findings indicate functional effects of the CaVβ1b_R296C variant differing from that attributed to CaVβ2 variants found in ASD patients. Further studies have to detail the effects on different VGCC subtypes and on VGCC expression.

Disrupted regulation of voltage-gated calcium channels by pathogenic calmodulin variants

Calmodulin (CaM) is an essential sensor of intracellular calcium in multiple cell types, including cardiac myocytes and neurons. In these cells, calcium ions enter the cell through voltage-gated calcium channels (VGCCs) and bind to CaM in one of its four EF-hand domains. These binding events trigger an array of important downstream cellular processes, including calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF) of multiple VGCC subtypes. In this way, CaM tunes VGCC activity and facilitates calcium signaling cascades.

Magnetic Fields and Magnetically Stimulated Gold-Coated Superparamagnetic Iron Oxide Nanoparticles Differentially Modulate L-Type Voltage-Gated Calcium Channel Activity in Midbrain Neurons

Magnetic Fields and Magnetically Stimulated Gold-Coated Superparamagnetic Iron Oxide Nanoparticles Differentially Modulate L-Type Voltage-Gated Calcium Channel Activity in Midbrain Neurons

Neuroepithelial progenitors generate and propagate non-neuronal action potentials across the spinal cord

In the developing central nervous system, electrical signaling is thought to rely exclusively on differentiating neurons as they acquire the ability to generate and propagate action potentials. Accordingly, neuroepithelial progenitors (NEPs), which give rise to all neurons and glial cells during development, have been reported to remain electrically passive. Here, we investigated the physiological properties of NEPs at the onset of spontaneous neural activity (SNA) initiating motor behavior in mouse embryonic spinal cord. Using patch-clamp recordings, we discovered that spinal NEPs exhibit spontaneous membrane depolarizations during episodes of SNA. These rhythmic depolarizations exhibited a ventral-to-dorsal gradient with the highest amplitude located in the floor plate, the ventral-most part of the neuroepithelium. Paired recordings revealed that NEPs are coupled via gap junctions and form an electrical syncytium. Although other NEPs were electrically passive, we discovered that floor-plate NEPs generated large Na+/Ca2+ action potentials. Unlike in neurons, floor-plate action potentials relied primarily on the activation of voltage-gated T-type calcium channels (TTCCs). In situ hybridization showed that all 3 known subtypes of TTCCs are predominantly expressed in the floor plate. During SNA, we found that acetylcholine released by motoneurons rhythmically triggers floor-plate action potentials by acting through nicotinic acetylcholine receptors. Finally, by expressing the genetically encoded calcium indicator GCaMP6f in the floor plate, we demonstrated that neuroepithelial action potentials are associated with calcium waves and propagate along the entire length of the spinal cord. Our work reveals a novel physiological mechanism to generate and propagate electrical signals across a neural structure independently from neurons.

Urokinase plasminogen activator mediates changes in human astrocytes modeling fragile X syndrome.

The function of astrocytes intertwines with the extracellular matrix, whose neuron and glial cell-derived components shape neuronal plasticity. Astrocyte abnormalities have been reported in the brain of the mouse model for fragile X syndrome (FXS), the most common cause of inherited intellectual disability, and a monogenic cause of autism spectrum disorder. We compared human FXS and control astrocytes generated from human induced pluripotent stem cells and we found increased expression of urokinase plasminogen activator (uPA), which modulates degradation of extracellular matrix. Several pathways associated with uPA and its receptor function were activated in FXS astrocytes. Levels of uPA were also increased in conditioned medium collected from FXS hiPSC-derived astrocyte cultures and correlated inversely with intracellular Ca2+ responses to activation of L-type voltage-gated calcium channels in human astrocytes. Increased uPA augmented neuronal phosphorylation of TrkB within the docking site for the phospholipase-Cγ1 (PLCγ1), indicating effects of uPA on neuronal plasticity. Gene expression changes during neuronal differentiation preceding astrogenesis likely contributed to properties of astrocytes with FXS-specific alterations that showed specificity by not affecting differentiation of adenosine triphosphate (ATP)-responsive astrocyte population. To conclude, our studies identified uPA as an important regulator of astrocyte function and demonstrated that increased uPA in human FXS astrocytes modulated astrocytic responses and neuronal plasticity.

CACHD1-deficient mice exhibit hearing and balance deficits associated with a disruption of calcium homeostasis in the inner ear

CACHD1 recently was shown to be an α2δ-like subunit that can modulate the activity of some types of voltage-gated calcium channels, including the low-voltage activated, T-type CaV3 channels. CACHD1 is widely expressed in the central nervous system but its biological functions and relationship to disease states are unknown. Here, we report that mice with deleterious Cachd1 mutations are hearing impaired and have balance defects, demonstrating that CACHD1 is functionally important in the peripheral auditory and vestibular organs of the inner ear. The vestibular dysfunction of Cachd1 mutant mice, exhibited by leaning and head tilting behaviors, is related to a deficiency of calcium carbonate crystals (otoconia) in the saccule and utricle. The auditory dysfunction, shown by ABR threshold elevations and reduced DPOAEs, is associated with reduced endocochlear potentials and increased endolymph calcium concentrations. Paint-fills of mutant inner ears from prenatal and newborn mice revealed dilation of the membranous labyrinth caused by an enlarged volume of endolymph. These pathologies all can be related to a disturbance of calcium homeostasis in the endolymph of the inner ear, presumably caused by the loss of CACHD1 regulatory effects on voltage-gated calcium channel activity. Cachd1 expression in the cochlea appears stronger in late embryonic stages than in adults, suggesting an early role in establishing endolymph calcium concentrations. Our findings provide new insights into CACHD1 function and suggest the involvement of voltage-gated calcium channels in endolymph homeostasis, essential for normal auditory and vestibular function.

Regulation of GABAA Receptors Induced by the Activation of L-Type Voltage-Gated Calcium Channels.

GABAA receptors are pentameric ion channels that mediate most synaptic and tonic extrasynaptic inhibitory transmissions in the central nervous system. There are multiple GABAA receptor subtypes constructed from 19 different subunits in mammals that exhibit different regional and subcellular distributions and distinct pharmacological properties. Dysfunctional alterations of GABAA receptors are associated with various neuropsychiatric disorders. Short- and long-term plastic changes in GABAA receptors can be induced by the activation of different intracellular signaling pathways that are triggered, under physiological and pathological conditions, by calcium entering through voltage-gated calcium channels. This review discusses several mechanisms of regulation of GABAA receptor function that result from the activation of L-type voltage gated calcium channels. Calcium influx via these channels activates different signaling cascades that lead to changes in GABAA receptor transcription, phosphorylation, trafficking, and synaptic clustering, thus regulating the inhibitory synaptic strength. These plastic mechanisms regulate the interplay of synaptic excitation and inhibition that is crucial for the normal function of neuronal circuits.

Millimeter (MM) wave and microwave frequency radiation produce deeply penetrating effects: the biology and the physics.

Millimeter wave (MM-wave) electromagnetic fields (EMFs) are predicted to not produce penetrating effects in the body. The electric but not magnetic part of MM-EMFs are almost completely absorbed within the outer 1mm of the body. Rodents are reported to have penetrating MM-wave impacts on the brain, the myocardium, liver, kidney and bone marrow. MM-waves produce electromagnetic sensitivity-like changes in rodent, frog and skate tissues. In humans, MM-waves have penetrating effects including impacts on the brain, producing EEG changes and other neurological/neuropsychiatric changes, increases in apparent electromagnetic hypersensitivity and produce changes on ulcers and cardiac activity. This review focuses on several issues required to understand penetrating effects of MM-waves and microwaves: 1. Electronically generated EMFs are coherent, producing much higher electrical and magnetic forces then do natural incoherent EMFs. 2. The fixed relationship between electrical and magnetic fields found in EMFs in a vacuum or highly permeable medium such as air, predicted by Maxwell's equations, breaks down in other materials. Specifically, MM-wave electrical fields are almost completely absorbed in the outer 1mm of the body due to the high dielectric constant of biological aqueous phases. However, the magnetic fields are very highly penetrating. 3. Time-varying magnetic fields have central roles in producing highly penetrating effects. The primary mechanism of EMF action is voltage-gated calcium channel (VGCC) activation with the EMFs acting via their forces on the voltage sensor,rather than by depolarization of the plasma membrane. Two distinct mechanisms, an indirect and a direct mechanism, are consistent with and predicted by the physics, to explain penetrating MM-wave VGCC activation via the voltage sensor. Time-varying coherent magnetic fields, as predicted by the Maxwell-Faraday version of Faraday's law of induction, can put forces on ions dissolved in aqueous phases deep within the body, regenerating coherent electric fields which activate the VGCC voltage sensor. In addition, time-varying magnetic fields can directly put forces on the 20 charges in the VGCC voltage sensor. There are three very important findings here which are rarely recognized in the EMF scientific literature: coherence of electronically generated EMFs; the key role of time-varying magnetic fields in generating highly penetrating effects; the key role of both modulating and pure EMF pulses in greatly increasing very short term high level time-variation of magnetic and electric fields. It is probable that genuine safety guidelines must keep nanosecond timescale-variation of coherent electric and magnetic fields below some maximum level in order to produce genuine safety. These findings have important implications with regard to 5G radiation.

The Effect of Pulsed Electromagnetic Fields on Angiogenesis.

A pulsed electromagnetic field (PEMF) has been used to treat inflammation-based diseases such as osteoporosis, neurological injury, and osteoarthritis. Numerousanimal experiments and in vitro studies have shown that PEMF may affect angiogenesis. For ischemic diseases, in theory, blood flow may be richer by increasing the number of blood vessels which supply blood to ischemic tissue. PEMF plays a role in enhancing angiogenesis, and their clinical application may go far beyond the current scope. In this review, we analyzed and summarized the effects and possible mechanisms of PEMF on angiogenesis. Most studies have shown that PEMF with specific parameters can promote angiogenesis, which is manifested by an increased vascular growth rate and increased capillary density. The potential mechanisms consist of promoting vascular endothelial cell proliferation, migration, and tube formation, and increasing the expression level of vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), angiopoietin-2 (Ang-2), and other angiogenic growth factors. Additionally, PEMF has an impact on the activation of voltage-gated calcium channels (VGCC). Bioelectromagnetics. © 2021 Bioelectromagnetics Society.

Abstract 15494: Physiological Investigation of the C57bl6 Mouse Aorta Reveals a Critical Role for Voltage Gated Calcium Channel Activity in Spontaneous Arterial Ageing

Introduction: Arterial stiffness (AS) has gained much recognition as a hallmark and independent predictor of cardiovascular (CV) events. Although generally assumed to be an adaptive response to increased blood pressure (BP), AS precedes hypertension in at least two experimental mouse models, thus revealing an incomplete understanding of AS pathophysiology. Methods: The current study presents the longitudinal CV characterization of spontaneously ageing C57Bl6 mice (2, 4, 6, 9, 12 and 24-month old) (male, n>8). In vivo analysis of peripheral BP (Coda), aortic pulse wave velocity (aPWV, Vevo2100), and echocardiography (Vevo2100) was combined with ex vivo aortic studies of isometric reactivity (organ baths) and AS measurements in the Rodent Oscillatory Tension set-up for Arterial Compliance (ROTSAC). (Data are presented as mean ± SEM.) Results: In vivo and ex vivo characterisation confirms that aortic stiffness precedes peripheral BP alterations in spontaneously ageing C57Bl6 mice, with significantly and gradually increasing aPWV (Fig.A) and isobaric Peterson modulus (Ep) (Fig.B) from 6-month of age onward. Thereafter, cardiac hypertrophy was observed at 9-months of age (Fig.C), and peripheral BP measurement reveals elevated pulse pressure at 24-months (30% increase vs. all other ages) (Fig.D). Ex vivo investigation of the thoracic aorta shows increased contractions to phenylephrine (PE) in old (6-24 month) vs. young (2-4 month) mice (Fig.E,F) with an increased contribution of voltage-gated calcium channel (VGCC) (Fig.G,H) with age. Remarkably, no differences were observed on endothelial function, meaning that all changes occur on the level of the vascular smooth muscle cell (VSMC). Conclusions: Physiological ageing of VSMC results in high PE contractions, increased VGCC activity, and the development of significant arterial stiffening by 6-months of age. AS thereby precedes the development of peripheral BP alterations by 18 months.

Investigating the Transition of Pre-Symptomatic to Symptomatic Huntington's Disease Status Based on Omics Data.

Huntington’s disease is a rare neurodegenerative disease caused by a cytosine–adenine–guanine (CAG) trinucleotide expansion in the Huntingtin (HTT) gene. Although Huntington’s disease (HD) is well studied, the pathophysiological mechanisms, genes and metabolites involved in HD remain poorly understood. Systems bioinformatics can reveal synergistic relationships among different omics levels and enables the integration of biological data. It allows for the overall understanding of biological mechanisms, pathways, genes and metabolites involved in HD. The purpose of this study was to identify the differentially expressed genes (DEGs), pathways and metabolites as well as observe how these biological terms differ between the pre-symptomatic and symptomatic HD stages. A publicly available dataset from the Gene Expression Omnibus (GEO) was analyzed to obtain the DEGs for each HD stage, and gene co-expression networks were obtained for each HD stage. Network rewiring, highlights the nodes that change most their connectivity with their neighbors and infers their possible implication in the transition between different states. The CACNA1I gene was the mostly highly rewired node among pre-symptomatic and symptomatic HD network. Furthermore, we identified AF198444 to be common between the rewired genes and DEGs of symptomatic HD. CNTN6, DEK, LTN1, MST4, ZFYVE16, CEP135, DCAKD, MAP4K3, NUPL1 and RBM15 between the DEGs of pre-symptomatic and DEGs of symptomatic HD and CACNA1I, DNAJB14, EPS8L3, HSDL2, SNRPD3, SOX12, ACLY, ATF2, BAG5, ERBB4, FOCAD, GRAMD1C, LIN7C, MIR22, MTHFR, NABP1, NRG2, OTC, PRAMEF12, SLC30A10, STAG2 and Y16709 between the rewired genes and DEGs of pre-symptomatic HD. The proteins encoded by these genes are involved in various biological pathways such as phosphatidylinositol-4,5-bisphosphate 3-kinase activity, cAMP response element-binding protein binding, protein tyrosine kinase activity, voltage-gated calcium channel activity, ubiquitin protein ligase activity, adenosine triphosphate (ATP) binding, and protein serine/threonine kinase. Additionally, prominent molecular pathways for each HD stage were then obtained, and metabolites related to each pathway for both disease stages were identified. The transforming growth factor beta (TGF-β) signaling (pre-symptomatic and symptomatic stages of the disease), calcium (Ca2+) signaling (pre-symptomatic), dopaminergic synapse pathway (symptomatic HD patients) and Hippo signaling (pre-symptomatic) pathways were identified. The in silico metabolites we identified include Ca2+, inositol 1,4,5-trisphosphate, sphingosine 1-phosphate, dopamine, homovanillate and L-tyrosine. The genes, pathways and metabolites identified for each HD stage can provide a better understanding of the mechanisms that become altered in each disease stage. Our results can guide the development of therapies that may target the altered genes and metabolites of the perturbed pathways, leading to an improvement in clinical symptoms and hopefully a delay in the age of onset.

Impact of the DSP-H1684R Genetic Variant on Ion Channels Activity in iPSC-Derived Cardiomyocytes.

Mutations of desmosomal genes are known to cause arrhythmogenic cardiomyopathy characterized by arrhythmias and sudden cardiac death. Previously, we described a novel genetic variant H1684R in desmoplakin gene (DSP), associated with a progressive cardiac conduction disease (PCCD). In the present study, we aimed to investigate an effect of the DSP-H1684R genetic variant on the activity of ion channels. We used cardiomyocytes derived from induced pluripotent stem cells (iPSC cardiomyocytes) from a patient with DSP-H1684R genetic variant and from two healthy donors. Immunofluorescent staining and western blot analyses were used to characterize patient-specific cardiomyocytes. By the whole-cell voltage-clamp technique we estimated the activity of voltage-gated sodium, calcium, and potassium channels that are responsible for action potential generation and its shape. Action potentials' parameters were measured using whole-cell current-clamp technique. In patient-specific cardiomyocytes we observed both lower amplitudes of currents through sodium Nav1.5 channels and L-type calcium channels, but higher amplitude of current through transient-outward potassium channels in comparison to donor cardiomyocytes. Current-clamp measurements revealed shortening of action-potential in DSP-H1684R-carrying iPSC cardiomyocytes. Therefore, observed alterations in the channels activity might have a great impact on the properties of action potential and development of PCCD. Our results show that desmoplakin genetic variants, besides conduction slowing caused by structural heart remodeling, could affect multiple ion channel activity aggravating arrhythmia manifestation in PCCD.