ROS in diabetic atria regulate SK2 degradation by Atrogin-1 through the NF-κB signaling pathway.

One of the independent risk factors for atrial fibrillation is diabetes mellitus (DM); however, the underlying mechanisms causing atrial fibrillation in DM are unknown. The underlying mechanism of Atrogin-1-mediated SK2 degradation and associated signaling pathways are unclear. The aim of this study was to elucidate the relationship among reactive oxygen species (ROS), the NF-κB signaling pathway, and Atrogin-1 protein expression in the atrial myocardia of DM mice. We found that SK2 expression was downregulated comitant with increased ROS generation and enhanced NF-κB signaling activation in the atrial cardiomyocytes of DM mice. These observations were mimicked by exogenously applicating H2O2 and by high glucose culture conditions in HL-1 cells. Inhibition of ROS production by diphenyleneiodonium chloride or silencing of NF-κB by siRNA decreased the protein expression of NF-κB and Atrogin-1 and increased that of SK2 in HL-1 cells with high glucose culture. Moreover, chromatin immunoprecipitation assay demonstrated that NF-κB/p65 directly binds to the promoter of the FBXO32 gene (encoding Atrogin-1), regulating the FBXO32 transcription. Finally, we evaluated the therapeutic effects of curcumin, known as a NF-κB inhibitor, on Atrogin-1 and SK2 expression in DM mice and confirmed that oral administration of curcumin for 4 weeks significantly suppressed Atrogin-1 expression and protected SK2 expression against hyperglycemia. In summary, the results from this study indicated that the ROS/NF-κB signaling pathway participates in Atrogin-1-mediated SK2 regulation in the atria of streptozotocin-induced DM mice.

A single coiled-coil domain mutation in hIKCa channel subunits disrupts preferential formation of heteromeric hSK1:hIKCa channels.

The expression of IKCa (SK4) channel subunits overlaps with that of SK channel subunits, and it has been proposed that the two related subunits prefer to co-assemble to form heteromeric hSK1:hIKCa channels. This implicates hSK1:hIKCa heteromers in physiological roles that might have been attributed to activation of SK channels. We have used a mutation approach to confirm formation of heterometric hSK1:hIKCa channels. Introduction of residues within hSK1 that were predicted to impart sensitivity to the hIKCa current blocker TRAM-34 changed the pharmacology of functional heteromers. Heteromeric channels formed between wildtype hIKCa and mutant hSK1 subunits displayed a significantly higher sensitivity and maximum block to addition of TRAM-34 than heteromers formed between wildtype subunits. Heteromer formation was disrupted by a single point mutation within one COOH-terminal coiled-coil domain of the hIKCa channel subunit. This mutation only disrupted the formation of hSK1:hIKCa heteromeric channels, without affecting the formation of homomeric hIKCa channels. Finally, the Ca2+ gating sensitivity of heteromeric hSK1:hIKCa channels was found to be significantly lower than the Ca2+ gating sensitivity of homomeric hIKCa channels. These data confirmed the preferred formation of heteromeric channels that results from COOH-terminal interactions between subunits. The distinct sensitivity of the heteromer to activation by Ca2+ suggests that heteromeric channels fulfil a distinct function within those neurons that express both subunits.

Cross-species epigenetic regulation of nucleus accumbens KCNN3 transcripts by excessive ethanol drinking.

The underlying genetic and epigenetic mechanisms driving functional adaptations in neuronal excitability and excessive alcohol intake are poorly understood. Small-conductance Ca2+-activated K+ (KCa2 or SK) channels encoded by the KCNN family of genes have emerged from preclinical studies as a key contributor to alcohol-induced functional neuroadaptations in alcohol-drinking monkeys and alcohol-dependent mice. Here, this cross-species analysis focused on KCNN3 DNA methylation, gene expression, and single nucleotide polymorphisms, including alternative promoters in KCNN3, that could influence surface trafficking and function of KCa2 channels. Bisulfite sequencing analysis of the nucleus accumbens tissue from alcohol-drinking monkeys and alcohol-dependent mice revealed a differentially methylated region in exon 1A of KCNN3 that overlaps with a predicted promoter sequence. The hypermethylation of KCNN3 in the accumbens paralleled an increase in the expression of alternative transcripts that encode apamin-insensitive and dominant-negative KCa2 channel isoforms. A polymorphic repeat in macaque KCNN3 encoded by exon 1 did not correlate with alcohol drinking. At the protein level, KCa2.3 channel expression in the accumbens was significantly reduced in very heavy-drinking monkeys. Together, our cross-species findings on epigenetic dysregulation of KCNN3 represent a complex mechanism that utilizes alternative promoters to potentially impact the firing of accumbens neurons. Thus, these results provide support for hypermethylation of KCNN3 as a possible key molecular mechanism underlying harmful alcohol intake and alcohol use disorder.

A Novel KCNN2 Variant in a Family with Essential Tremor Plus: Clinical Characteristics and In Silico Analysis.

BACKGROUND: Essential tremor (ET) is one of the more common movement disorders. Current diagnosis is solely based on clinical findings. ET appears to be inherited in an autosomal dominant pattern. Several loci on specific chromosomes have been studied by linkage analysis, but the causes of essential tremor are still unknown in many patients. Genetic studies described the association of several genes with familial ET. However, they were found only in distinct families, suggesting that some can be private pathogenic variants. AIM OF THE STUDY: to characterize the phenotype of an Italian family with ET and identify the genetic variant associated. METHODS: Clinical and genetic examinations were performed. Genetic testing was done with whole-exome sequencing (WES) using the Illumina platform. Bidirectional capillary Sanger sequencing was used to investigate the presence of variant in all affected members of the family. In silico prediction of pathogenicity was used to study the effect of gene variants on protein structure. RESULTS: The proband was a 15-year-old boy. The patient was the first of two children of a non-consanguineous couple. Family history was remarkable for tremor in the mother line. His mother suffered from bilateral upper extremity kinetic tremors (since she was 20 years old), anxiety, and depression. Other relatives referred bilateral upper extremity tremors. In the index case, WES analysis performed supposing a dominant mode of inheritance, identified a novel heterozygous missense variant in potassium calcium-activated channel subfamily N member 2 (KCNN2) (NM_021614.3: c.1145G>A, p.Gly382Asp). In the pedigree investigation, all carriers of the gene variant had ET and showed variable expressivity, the elder symptomatic relative showing cognitive impairment and hallucinations in the last decade, in addition to tremor since a young age. The amino acid residue #382 is located in a transmembrane region and in silico analysis suggested a causative role for the variant. Modelling of the mutant protein structure showed that the variant causes a clash in the protein structure. Therefore, the variant could cause a conformational change that alters the ability of the protein in the modulation of ion channels Conclusions: The KCNN2 gene variant identified could be associated with ET. The variant could modify a voltage-independent potassium channel activated by intracellular calcium.

Small-conductance calcium-activated potassium channels in the heart: expression, regulation and pathological implications.

Ca2+-activated K+ channels are critical to cellular Ca2+ homeostasis and excitability; they couple intracellular Ca2+ and membrane voltage change. Of these, the small, 4-14 pS, conductance SK channels include three, KCNN1-3 encoded, SK1/KCa2.1, SK2/KCa2.2 and SK3/KCa2.3, channel subtypes with characteristic, EC50 ∼ 10 nM, 40 pM, 1 nM, apamin sensitivities. All SK channels, particularly SK2 channels, are expressed in atrial, ventricular and conducting system cardiomyocytes. Pharmacological and genetic modification results have suggested that SK channel block or knockout prolonged action potential durations (APDs) and effective refractory periods (ERPs) particularly in atrial, but also in ventricular, and sinoatrial, atrioventricular node and Purkinje myocytes, correspondingly affect arrhythmic tendency. Additionally, mitochondrial SK channels may decrease mitochondrial Ca2+ overload and reactive oxygen species generation. SK channels show low voltage but marked Ca2+ dependences (EC50 ∼ 300-500 nM) reflecting their α-subunit calmodulin (CaM) binding domains, through which they may be activated by voltage-gated or ryanodine-receptor Ca2+ channel activity. SK function also depends upon complex trafficking and expression processes and associations with other ion channels or subunits from different SK subtypes. Atrial and ventricular clinical arrhythmogenesis may follow both increased or decreased SK expression through decreased or increased APD correspondingly accelerating and stabilizing re-entrant rotors or increasing incidences of triggered activity. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

A sensory neuron-specific long non-coding RNA reduces neuropathic pain by rescuing KCNN1 expression.

Nerve injury to peripheral somatosensory system causes refractory neuropathic pain. Maladaptive changes of gene expression in primary sensory neurons are considered molecular basis of this disorder. Long non-coding RNAs (lncRNAs) are key regulators of gene transcription; however, their significance in neuropathic pain remains largely elusive.Here, we reported a novel lncRNA, named sensory neuron-specific lncRNA (SS-lncRNA), for its expression exclusively in dorsal root ganglion (DRG) and trigeminal ganglion. SS-lncRNA was predominantly expressed in small DRG neurons and significantly downregulated due to a reduction of early B cell transcription factor 1 in injured DRG after nerve injury. Rescuing this downregulation reversed a decrease of the calcium-activated potassium channel subfamily N member 1 (KCNN1) in injured DRG and alleviated nerve injury-induced nociceptive hypersensitivity. Conversely, DRG downregulation of SS-lncRNA reduced the expression of KCNN1, decreased total potassium currents and afterhyperpolarization currents and increased excitability in DRG neurons and produced neuropathic pain symptoms.Mechanistically, downregulated SS-lncRNA resulted in the reductions of its binding to Kcnn1 promoter and heterogeneous nuclear ribonucleoprotein M (hnRNPM), consequent recruitment of less hnRNPM to the Kcnn1 promoter and silence of Kcnn1 gene transcription in injured DRG.These findings indicate that SS-lncRNA may relieve neuropathic pain through hnRNPM-mediated KCNN1 rescue in injured DRG and offer a novel therapeutic strategy specific for this disorder.

Endothelial KCa3.1 and KCa2.3 Mediate S1P (Sphingosine-1-Phosphate)-Dependent Vasodilation and Blood Pressure Homeostasis.

BACKGROUND: S1P (sphingosine-1-phosphate) has been reported to possess vasodilatory properties, but the underlying pathways are largely unknown. METHODS: Isolated mouse mesenteric artery and endothelial cell models were used to determine S1P-induced vasodilation, intracellular calcium, membrane potentials, and calcium-activated potassium channels (KCa2.3 and KCa3.1 [endothelial small- and intermediate-conductance calcium-activated potassium channels]). Effect of deletion of endothelial S1PR1 (type 1 S1P receptor) on vasodilation and blood pressure was evaluated. RESULTS: Mesenteric arteries subjected to acute S1P stimulation displayed a dose-dependent vasodilation response, which was attenuated by blocking endothelial KCa2.3 or KCa3.1 channels. In cultured human umbilical vein endothelial cells, S1P stimulated immediate membrane potential hyperpolarization following activation of KCa2.3/KCa3.1 with elevated cytosolic Ca2+. Further, chronic S1P stimulation enhanced expression of KCa2.3 and KCa3.1 in human umbilical vein endothelial cells in dose- and time-dependent manners, which was abolished by disrupting either S1PR1-Ca2+ signaling or downstream Ca2+-activated calcineurin/NFAT (nuclear factor of activated T-cells) signaling. By combination of bioinformatics-based binding site prediction and chromatin immunoprecipitation assay, we revealed in human umbilical vein endothelial cells that chronic activation of S1P/S1PR1 promoted NFATc2 nuclear translocation and binding to promoter regions of KCa2.3 and KCa3.1 genes thus to upregulate transcription of these channels. Deletion of endothelial S1PR1 reduced expression of KCa2.3 and KCa3.1 in mesenteric arteries and exacerbated hypertension in mice with angiotensin II infusion. CONCLUSIONS: This study provides evidence for the mechanistic role of KCa2.3/KCa3.1-activated endothelium-dependent hyperpolarization in vasodilation and blood pressure homeostasis in response to S1P. This mechanistic demonstration would facilitate the development of new therapies for cardiovascular diseases associated with hypertension.

Loss-of-function KCa2.2 mutations abolish channel activity.

Small-conductance Ca2+-activated potassium channels subtype 2 (KCa2.2, also called SK2) are operated exclusively by a Ca2+-calmodulin gating mechanism. Heterozygous genetic mutations of KCa2.2 channels have been associated with autosomal dominant neurodevelopmental disorders including cerebellar ataxia and tremor in humans and rodents. Taking advantage of these pathogenic mutations, we performed structure-function studies of the rat KCa2.2 channel. No measurable current was detected from HEK293 cells heterologously expressing these pathogenic KCa2.2 mutants. When coexpressed with the KCa2.2_WT channel, mutations of the pore-lining amino acid residues (I360M, Y362C, G363S, and I389V) and two proline substitutions (L174P and L433P) dominant negatively suppressed and completely abolished the activity of the coexpressed KCa2.2_WT channel. Coexpression of the KCa2.2_I289N and the KCa2.2_WT channels reduced the apparent Ca2+ sensitivity compared with the KCa2.2_WT channel, which was rescued by a KCa2.2 positive modulator.

Channelopathy of small- and intermediate-conductance Ca2+-activated K+ channels.

Small- and intermediate-conductance Ca2+-activated K+ (KCa2.x/KCa3.1 also called SK/IK) channels are gated exclusively by intracellular Ca2+. The Ca2+ binding protein calmodulin confers sub-micromolar Ca2+ sensitivity to the channel-calmodulin complex. The calmodulin C-lobe is constitutively associated with the proximal C-terminus of the channel. Interactions between calmodulin N-lobe and the channel S4-S5 linker are Ca2+-dependent, which subsequently trigger conformational changes in the channel pore and open the gate. KCNN genes encode four subtypes, including KCNN1 for KCa2.1 (SK1), KCNN2 for KCa2.2 (SK2), KCNN3 for KCa2.3 (SK3), and KCNN4 for KCa3.1 (IK). The three KCa2.x channel subtypes are expressed in the central nervous system and the heart. The KCa3.1 subtype is expressed in the erythrocytes and the lymphocytes, among other peripheral tissues. The impact of dysfunctional KCa2.x/KCa3.1 channels on human health has not been well documented. Human loss-of-function KCa2.2 mutations have been linked with neurodevelopmental disorders. Human gain-of-function mutations that increase the apparent Ca2+ sensitivity of KCa2.3 and KCa3.1 channels have been associated with Zimmermann-Laband syndrome and hereditary xerocytosis, respectively. This review article discusses the physiological significance of KCa2.x/KCa3.1 channels, the pathophysiology of the diseases linked with KCa2.x/KCa3.1 mutations, the structure-function relationship of the mutant KCa2.x/KCa3.1 channels, and potential pharmacological therapeutics for the KCa2.x/KCa3.1 channelopathy.

Up-regulated small-conductance calcium-activated potassium currents contribute to atrial arrhythmogenesis in high-fat feeding mice.

AIMS: Metabolic syndrome (MetS) is associated with arrhythmias and cardiovascular mortality. Arrhythmogenesis in MetS results from atrial structural and electrical remodelling. The small-conductance Ca2+-activated K+ (SK) currents modulate atrial repolarization and may influence atrial arrhythmogenicity. This study investigated the regulation of SK current perturbed by a high-fat diet (HFD) to mimic MetS. METHODS AND RESULTS: Thirty mice were divided into two groups that were fed with normal chow (CTL) and HFD for 4 months. Electrocardiography and echocardiography were used to detect cardiac electrical and structure remodelling. Atrial action potential duration (APD) and calcium transient duration (CaTD) were measured by optical mapping of Langendorff-perfused mice hearts. Atrial fibrillation (AF) inducibility and duration were assessed by burst pacing. Whole-cell patch clamp was performed in primarily isolated atrial myocytes for SK current density. The SK current density is higher in atrial myocytes from HFD than in CTL mice (P ≤ 0.037). The RNA and protein expression of SK channels are increased in HFD mice (P ≤ 0.041 and P ≤ 0.011, respectively). Action potential duration is shortened in HFD compared with CTL (P ≤ 0.015). The shortening of the atrial APD in HFD is reversed by the application of 100 nM apamin (P ≤ 0.043). Compared with CTL, CaTD is greater in HFD atria (P ≤ 0.029). Calcium transient decay (Tau) is significantly higher in HFD than in CTL (P = 0.001). Both APD and CaTD alternans thresholds were higher in HFD (P ≤ 0.043), along with higher inducibility and longer duration of AF in HFD (P ≤ 0.023). CONCLUSION: Up-regulation of apamin-sensitive SK currents plays a partial role in the atrial arrhythmogenicity of HFD mice.

Calcium dependence of both lobes of calmodulin is involved in binding to a cytoplasmic domain of SK channels.

KCa2.1-3 Ca2+-activated K+-channels (SK) require calmodulin to gate in response to cellular Ca2+. A model for SK gating proposes that the N-terminal domain (N-lobe) of calmodulin is required for activation, but an immobile C-terminal domain (C-lobe) has constitutive, Ca2+-independent binding. Although structures support a domain-driven hypothesis of SK gate activation by calmodulin, only a partial understanding is possible without measuring both channel activity and protein binding. We measured SK2 (KCa2.2) activity using inside-out patch recordings. Currents from calmodulin-disrupted SK2 channels can be restored with exogenously applied calmodulin. We find that SK2 activity only approaches full activation with full-length calmodulin with both an N- and a C-lobe. We measured calmodulin binding to a C-terminal SK peptide (SKp) using both composition-gradient multi-angle light-scattering and tryptophan emission spectra. Isolated lobes bind to SKp with high affinity, but isolated lobes do not rescue SK2 activity. Consistent with earlier models, N-lobe binding to SKp is stronger in Ca2+, and C-lobe-binding affinity is strong independent of Ca2+. However, a native tryptophan in SKp is sensitive to Ca2+ binding to both the N- and C-lobes of calmodulin at Ca2+ concentrations that activate SK2, demonstrating that the C-lobe interaction with SKp changes with Ca2+. Our peptide-binding data and electrophysiology show that SK gating models need deeper scrutiny. We suggest that the Ca2+-dependent associations of both lobes of calmodulin to SKp are crucial events during gating. Additional investigations are necessary to complete a mechanistic gating model consistent with binding, physiology, and structure.

KCNN2 Mutation in Pediatric Tremor Myoclonus Dystonia Syndrome with Electrophysiological Evaluation.

BACKGROUND: Here we combine clinical, electrophysiological, and genetic findings to phenotype an unusual childhood movement disorder in a patient with a rare form of KCNN2 mutation. CASE REPORT: A 10-year-old male presented with a clinical syndrome of tremor and myoclonus. Electrophysiology demonstrated muscle activity indicative of myoclonus dystonia, an observation that was not appreciated clinically. Genetic testing revealed an abnormality in the KCNN 2 gene, not present in the parents, known to cause dystonia, as the etiology. DISCUSSION: The value of utilizing noninvasive, electrophysiological recording in pediatric movement disorders expands the precision of diagnosis, potentially informing treatment when correlated with clinical and genetic findings.

Histone deacetylase inhibitors (HDACi) increase expression of KCa2.3 (SK3) in primary microvascular endothelial cells.

The small conductance calcium-activated potassium channel (KCa2.3) has long been recognized for its role in mediating vasorelaxation through the endothelium-derived hyperpolarization (EDH) response. Histone deacetylases (HDACs) have been implicated as potential modulators of blood pressure and histone deacetylase inhibitors (HDACi) are being explored as therapeutics for hypertension. Herein, we show that HDACi increase KCa2.3 expression when heterologously expressed in HEK cells and endogenously expressed in primary cultures of human umbilical vein endothelial cells (HUVECs) and human intestinal microvascular endothelial cells (HIMECs). When primary endothelial cells were exposed to HDACi, KCa2.3 transcripts, subunits, and functional current are increased. Quantitative RT-PCR (qPCR) demonstrated increased KCa2.3 mRNA following HDACi, confirming transcriptional regulation of KCa2.3 by HDACs. By using pharmacological agents selective for different classes of HDACs, we discriminated between cytoplasmic and epigenetic modulation of KCa2.3. Biochemical analysis revealed an association between the cytoplasmic HDAC6 and KCa2.3 in immunoprecipitation studies. Specifically inhibiting HDAC6 increases expression of KCa2.3. In addition to increasing the expression of KCa2.3, we show that nonspecific inhibition of HDACs causes an increase in the expression of the molecular chaperone Hsp70 in endothelial cells. When Hsp70 is inhibited in the presence of HDACi, the magnitude of the increase in KCa2.3 expression is diminished. Finally, we show a slower rate of endocytosis of KCa2.3 as a result of exposure of primary endothelial cells to HDACi. These data provide the first demonstrated approach to increase KCa2.3 channel number in endothelial cells and may partially account for the mechanism by which HDACi induce vasorelaxation.

Channelopathy-causing mutations in the S45A/S45B and HA/HB helices of KCa2.3 and KCa3.1 channels alter their apparent Ca2+ sensitivity.

Small- and intermediate-conductance Ca2+-activated potassium (KCa2.x and KCa3.1, also called SK and IK) channels are activated exclusively by a Ca2+-calmodulin gating mechanism. Wild-type KCa2.3 channels have a Ca2+ EC50 value of ∼0.3 µM, while the apparent Ca2+ sensitivity of wild-type KCa3.1 channels is ∼0.27 µM. Heterozygous genetic mutations of KCa2.3 channels have been associated with Zimmermann-Laband syndrome and idiopathic noncirrhotic portal hypertension, while KCa3.1 channel mutations were reported in hereditary xerocytosis patients. KCa2.3_S436C and KCa2.3_V450L channels with mutations in the S45A/S45B helices exhibited hypersensitivity to Ca2+. The corresponding mutations in KCa3.1 channels also elevated the apparent Ca2+ sensitivity. KCa3.1_S314P, KCa3.1_A322V and KCa3.1_R352H channels with mutations in the HA/HB helices are hypersensitive to Ca2+, whereas KCa2.3 channels with the equivalent mutations are not. The different effects of the equivalent mutations in the HA/HB helices on the apparent Ca2+ sensitivity of KCa2.3 and KCa3.1 channels may imply distinct modulation of the two channel subtypes by the HA/HB helices. AP14145 reduced the apparent Ca2+ sensitivity of the hypersensitive mutant KCa2.3 channels, suggesting the potential therapeutic usefulness of negative gating modulators.

Antidepressant activity of pharmacological and genetic deactivation of the small-conductance calcium-activated potassium channel subtype-3.

RATIONALE: The voltage-insensitive, small-conductance calcium-activated potassium (SK) channel is a key regulator of neuronal depolarization and is implicated in the pathophysiology of depressive disorders. OBJECTIVE: We ascertained whether the SK channel is impaired in the chronic unpredictable stress (CUS) model and whether it can serve as a molecular target of antidepressant action. METHODS: We assessed the depressive-like behavioral phenotype of CUS-exposed rats and performed post-mortem SK channel binding and activity-dependent zif268 mRNA analyses on their brains. To begin an assessment of SK channel subtypes involved, we examined the effects of genetic and pharmacological inhibition of the SK3 channel using conditional knockout mice and selective SK3 channel negative allosteric modulators (NAMs). RESULTS: We found that [125I]apamin binding to SK channels is increased in the prefrontal cortex and decreased in the hippocampus, an effect that was associated with reciprocal levels of zif268 mRNA transcripts indicating abnormal regional cell activity in this model. We found that genetic and pharmacological manipulations significantly decreased immobility in the forced swim test without altering general locomotor activity, a hallmark of antidepressant-like activity. CONCLUSIONS: Taken together, these findings link depression-related neural and behavioral pathophysiology with abnormal SK channel functioning and suggest that this can be reversed by the selective inhibition of SK3 channels.

Zimmermann-Laband syndrome in monozygotic twins with a mild neurobehavioral phenotype lacking gingival overgrowth-A case report of a novel KCNN3 gene variant.

Zimmermann-Laband syndrome is a rare, heterogeneous disorder characterized by gingival hypertrophy or fibromatosis, aplastic/hypoplastic nails, hypoplasia of the distal phalanges, hypertrichosis, various degrees of intellectual disability, and distinctive facial features. Three genes are considered causative for ZLS: KCNH1, KCNN3, and ATP6V1B2. We report on a pair of female concordant monozygotic twins, both carrying a novel pathogenic variant in the KCNN3 gene, identified using exome sequencing. Only six ZLS patients with the KCNN3 pathogenic variant have been reported so far. The twins show facial dysmorphism, hypoplastic distal phalanges, aplasia or hypoplasia of nails, and hypertrichosis. During infancy, they showed mild developmental delays, mainly speech. They successfully completed secondary school education and are socio-economically independent. Gingival overgrowth is absent in both individuals. Our patients exhibited an unusually mild phenotype compared to published cases, which is an important diagnostic finding for proper genetic counseling for Zimmermann-Laband syndrome patients and their families.

Molecular and functional characterization of detrusor PDGFRα positive cells in spinal cord injury-induced detrusor overactivity.

Volume accommodation occurs via a novel mechanism involving interstitial cells in detrusor muscles. The interstitial cells in the bladder are PDGFRα+, and they restrain the excitability of smooth muscle at low levels and prevents the development of transient contractions (TCs). A common clinical manifestation of spinal cord injury (SCI)-induced bladder dysfunction is detrusor overactivity (DO). Although a myogenic origin of DO after SCI has been suggested, a mechanism for development of SCI-induced DO has not been determined. In this study we hypothesized that SCI-induced DO is related to loss of function in the regulatory mechanism provided by PDGFRα+ cells. Our results showed that transcriptional expression of Pdgfra and Kcnn3 was decreased after SCI. Proteins encoded by these genes also decreased after SCI, and a reduction in PDGFRα+ cell density was also documented. Loss of PDGFRα+ cells was due to apoptosis. TCs in ex vivo bladders during filling increased dramatically after SCI, and this was related to the loss of regulation provided by SK channels, as we observed decreased sensitivity to apamin. These findings show that damage to the mechanism restraining muscle contraction during bladder filling that is provided by PDGFRα+ cells is causative in the development of DO after SCI.

Circular RNA mmu_circ_0005019 inhibits fibrosis of cardiac fibroblasts and reverses electrical remodeling of cardiomyocytes.

BACKGROUND: Circular RNA (circRNA) have been reported to play important roles in cardiovascular diseases including myocardial infarction and heart failure. However, the role of circRNA in atrial fibrillation (AF) has rarely been investigated. We recently found a circRNA hsa_circ_0099734 was significantly differentially expressed in the AF patients atrial tissues compared to paired control. We aim to investigate the functional role and molecular mechanisms of mmu_circ_0005019 which is the homologous circRNA in mice of hsa_circ_0099734 in AF. METHODS: In order to investigate the effect of mmu_circ_0005019 on the proliferation, migration, differentiation into myofibroblasts and expression of collagen of cardiac fibroblasts, and the effect of mmu_circ_0005019 on the apoptosis and expression of Ito, INA and SK3 of cardiomyocytes, gain- and loss-of-function of cell models were established in mice cardiac fibroblasts and HL-1 atrial myocytes. Dual-luciferase reporter assays and RIP were performed to verify the binding effects between mmu_circ_0005019 and its target microRNA (miRNA). RESULTS: In cardiac fibroblasts, mmu_circ_0005019 showed inhibitory effects on cell proliferation and migration. In cardiomyocytes, overexpression of mmu_circ_0005019 promoted Kcnd1, Scn5a and Kcnn3 expression. Knockdown of mmu_circ_0005019 inhibited the expression of Kcnd1, Kcnd3, Scn5a and Kcnn3. Mechanistically, mmu_circ_0005019 exerted biological functions by acting as a miR-499-5p sponge to regulate the expression of its target gene Kcnn3. CONCLUSIONS: Our findings highlight mmu_circ_0005019 played a protective role in AF development and might serve as an attractive candidate target for AF treatment.

Targeting Kca3.1 Channels in Cancer.

The Kca3.1 channels, previously designated as IK1 or SK4 channels and encoded by the KCNN4 gene, are activated by a rise of the intracellular Ca2+ concentration. These K+ channels are widely expressed in many organs and involved in many pathologies. In particular, Kca3.1 channels have been studied intensively in the context of cancer. They are not only a marker and a valid prognostic tool for cancer patients, but have an important share in driving cancer progression. Their function is required for many characteristic features of the aggressive cancer cell behavior such as migration, invasion and metastasis as well as proliferation and therapy resistance. In the context of cancer, another property of Kca3.1 is now emerging. These channels can be a target for novel small molecule-based imaging probes, as it has been validated in case of fluorescently labeled senicapoc-derivatives. The aim of this review is (i) to give an overview on the role of Kca3.1 channels in cancer progression and in shaping the cancer microenvironment, (ii) discuss the potential of using Kca3.1 targeting drugs for cancer imaging, (iii) and highlight the possibility of combining molecular dynamics simulations to image inhibitor binding to Kca3.1 channels in order to provide a deeper understanding of Kca3.1 channel pharmacology. Alltogether, Kca3.1 is an attractive therapeutic target so that senicapoc, originally developed for the treatment of sickle cell anemia, should be repurposed for the treatment of cancer patients.

SK2 channel deletion reduces susceptibility to bupivacaine-induced cardiotoxicity in mouse.

Bupivacaine is frequently used for regional anesthesia and postoperative analgesia. However, an inadvertent intravenous injection can cause severe cardiotoxicity, manifesting as arrhythmia, hypotension, and even cardiac asystole. The mechanism of bupivacaine-mediated cardiotoxicity remains unclear. SK2 knockout mice (SK) and wild-type mice (WT) were divided into four groups, with 12 mice per group. We determined the difference in bupivacaine cardiotoxicity between SK2 knockout and WT mice by measuring the time to the first arrhythmia (Tarrhythmia) and the time to asystole (Tasystole). Secondary indicators of cardiotoxicity were the time from the beginning of bupivacaine infusion to 20% prolongation of the QT interval (TQT) and the time to 20% widening of the QRS complex (TQRS). Tarrhythmia and Tasystole were significantly longer in the SK-bupi group than in the WT-bupi group (both P < 0.05). TQT and TQRS were longer in the SK-bupi group than in the WT-bupi group (all P < 0.05). The time to 25%, 50%, and 75% reduction in HR in the SK-bupi group was significantly longer than in the WT-bupi group (all P < 0.05). Knocking out the SK2 channel can reduce bupivacaine-induced cardiotoxicity in the mouse.