Activin A, a Novel Chemokine, Induces Mouse NK Cell Migration via AKT and Calcium Signaling.

Natural killer (NK) cells can migrate quickly to the tumor site to exert cytotoxic effects on tumors, and some chemokines, including CXCL8, CXCL10 or and CXCL12, can regulate the migration of NK cells. Activin A, a member of the transforming growth factor ß (TGF-ß) superfamily, is highly expressed in tumor tissues and involved in tumor development and immune cell activation. In this study, we focus on the effects of activin A on NK cell migration. In vitro, activin A induced NK cell migration and invasion, promoted cell polarization and inhibited cell adhesion. Moreover, activin A increased Ca2+, p-SMAD3 and p-AKT levels in NK cells. An AKT inhibitor and Ca2+ chelator partially blocked activin A-induced NK cell migration. In vivo, exogenous activin A increased tumor-infiltrating NK cells in NS-1 cell solid tumors and inhibited tumor growth, and blocking endogenous activin A with anti-activin A antibody reduced tumor-infiltrating NK cells in 4T-1 cell solid tumors. These results suggest that activin A induces NK cell migration through AKT signaling and calcium signaling and may enhance the antitumor effect of NK cells by increasing tumor-infiltrating NK cells.

Angiotensin II Directly Increases Endothelial Calcium and Nitric Oxide in Kidney and Brain Microvessels In Vivo With Reduced Efficacy in Hypertension.

BACKGROUND: The vasoconstrictor effects of angiotensin II via type 1 angiotensin II receptors in vascular smooth muscle cells are well established, but the direct effects of angiotensin II on vascular endothelial cells (VECs) in vivo and the mechanisms how VECs may mitigate angiotensin II-mediated vasoconstriction are not fully understood. The present study aimed to explore the molecular mechanisms and pathophysiological relevance of the direct actions of angiotensin II on VECs in kidney and brain microvessels in vivo. METHODS AND RESULTS: Changes in VEC intracellular calcium ([Ca2+]i) and nitric oxide (NO) production were visualized by intravital multiphoton microscopy of cadherin 5-Salsa6f mice or the endothelial uptake of NO-sensitive dye 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, respectively. Kidney fibrosis by unilateral ureteral obstruction and Ready-to-use adeno-associated virus expressing Mouse Renin 1 gene (Ren1-AAV) hypertension were used as disease models. Acute systemic angiotensin II injections triggered >4-fold increases in VEC [Ca2+]i in brain and kidney resistance arterioles and capillaries that were blocked by pretreatment with the type 1 angiotensin II receptor inhibitor losartan, but not by the type 2 angiotensin II receptor inhibitor PD123319. VEC responded to acute angiotensin II by increased NO production as indicated by >1.5-fold increase in 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate fluorescence intensity. In mice with kidney fibrosis or hypertension, the angiotensin II-induced VEC [Ca2+]i and NO responses were significantly reduced, which was associated with more robust vasoconstrictions, VEC shedding, and microthrombi formation. CONCLUSIONS: The present study directly visualized angiotensin II-induced increases in VEC [Ca2+]i and NO production that serve to counterbalance agonist-induced vasoconstriction and maintain residual organ blood flow. These direct and endothelium-specific angiotensin II effects were blunted in disease conditions and linked to endothelial dysfunction and the development of vascular pathologies.

Distinct neural activities of the cortical layer 2/3 across isoflurane anesthesia: A large-scale simultaneous observation of neurons.

Anesthesia inhibits neural activity in the brain, causing patients to lose consciousness and sensation during the surgery. Layers 2/3 of the cortex are important structures for the integration of information and consciousness, which are closely related to normal cognitive function. However, the dynamics of the large-scale population of neurons across multiple regions in layer 2/3 during anesthesia and recovery processes remains unclear. We conducted simultaneous observations and analysis of large-scale calcium signaling dynamics across multiple cortical regions within cortical layer 2/3 during isoflurane anesthesia and recovery in vivo by high-resolution wide-field microscopy. Under isoflurane-induced anesthesia, there is an overall decrease in neuronal activity across multiple regions in the cortical layer 2/3. Notably, some neurons display a paradoxical increase in activity during anesthesia. Additionally, the activity among multiple cortical regions under anesthesia was homogeneous. It is only during the recovery phase that variability emerges in the extent of increased neural activity across different cortical regions. Within the same duration of anesthesia, neural activity did not return to preanesthetic levels. To sum up, anesthesia as a dynamic alteration of brain functional networks, encompassing shifts in patterns of neural activity, homogeneousness among cortical neurons and regions, and changes in functional connectivity. Recovery from anesthesia does not entail a reversal of these effects within the same timeframe.

Pharmacological modulation of septins restores calcium homeostasis and is neuroprotective in models of Alzheimer's disease.

Abnormal calcium signaling is a central pathological component of Alzheimer's disease (AD). Here, we describe the identification of a class of compounds called ReS19-T, which are able to restore calcium homeostasis in cell-based models of tau pathology. Aberrant tau accumulation leads to uncontrolled activation of store-operated calcium channels (SOCCs) by remodeling septin filaments at the cell cortex. Binding of ReS19-T to septins restores filament assembly in the disease state and restrains calcium entry through SOCCs. In amyloid-ß and tau-driven mouse models of disease, ReS19-T agents restored synaptic plasticity, normalized brain network activity, and attenuated the development of both amyloid-ß and tau pathology. Our findings identify the septin cytoskeleton as a potential therapeutic target for the development of disease-modifying AD treatments.

Calcitonin gene­related peptide alleviates hyperoxia­induced human alveolar cell injury via the CGRPR/TRPV1/Ca2+ axis.

Although exogenous calcitonin gene­related peptide (CGRP) protects against hyperoxia­induced lung injury (HILI), the underlying mechanisms remain unclear. The present study attempted to elucidate the molecular mechanism by which CGRP protects against hyperoxia­induced alveolar cell injury. Human alveolar A549 cells were treated with 95% hyperoxia to establish a hyperoxic cell injury model. ELISA was performed to detect the CGRP secretion. Immunofluorescence, quantitative (q)PCR, and western blotting were used to detect the expression and localization of CGRP receptor (CGRPR) and transient receptor potential vanilloid 1 (TRPV1). Cell counting kit­8 and flow cytometry were used to examine the proliferation and apoptosis of treated cells. Digital calcium imaging and patch clamp were used to analyze the changes in intracellular Ca2+ signaling and membrane currents induced by CGRP in A549 cells. The mRNA and protein expression levels of Cyclin D1, proliferating cell nuclear antigen (PCNA), Bcl­2 and Bax were detected by qPCR and western blotting. The expression levels of CGRPR and TRPV1 in A549 cells were significantly downregulated by hyperoxic treatment, but there was no significant difference in CGRP release between cells cultured under normal air and hyperoxic conditions. CGRP promoted cell proliferation and inhibited apoptosis in hyperoxia, but selective inhibitors of CGRPR and TRPV1 channels could effectively attenuate these effects; TRPV1 knockdown also attenuated this effect. CGRP induced Ca2+ entry via the TRPV1 channels and enhanced the membrane non­selective currents through TRPV1 channels. The CGRP­induced increase in intracellular Ca2+ was reduced by inhibiting the phospholipase C (PLC)/protein kinase C (PKC) pathway. Moreover, PLC and PKC inhibitors attenuated the effects of CGRP in promoting cell proliferation and inhibiting apoptosis. In conclusion, exogenous CGRP acted by inversely regulating the function of TRPV1 channels in alveolar cells. Importantly, CGRP protected alveolar cells from hyperoxia­induced injury via the CGRPR/TRPV1/Ca2+ axis, which may be a potential target for the prevention and treatment of the HILI.

Regulation of T Lymphocyte Functions through Calcium Signaling Modulation by Nootkatone.

Recent advancements in understanding the intricate molecular mechanisms underlying immunological responses have underscored the critical involvement of ion channels in regulating calcium influx, particularly in inflammation. Nootkatone, a natural sesquiterpenoid found in Alpinia oxyphylla and various citrus species, has gained attention for its diverse pharmacological properties, including anti-inflammatory effects. This study aimed to elucidate the potential of nootkatone in modulating ion channels associated with calcium signaling, particularly CRAC, KV1.3, and KCa3.1 channels, which play pivotal roles in immune cell activation and proliferation. Using electrophysiological techniques, we demonstrated the inhibitory effects of nootkatone on CRAC, KV1.3, and KCa3.1 channels in HEK293T cells overexpressing respective channel proteins. Nootkatone exhibited dose-dependent inhibition of channel currents, with IC50 values determined for each channel. Nootkatone treatment did not significantly affect cell viability, indicating its potential safety for therapeutic applications. Furthermore, we observed that nootkatone treatment attenuated calcium influx through activated CRAC channels and showed anti-proliferative effects, suggesting its role in regulating inflammatory T cell activation. These findings highlight the potential of nootkatone as a natural compound for modulating calcium signaling pathways by targeting related key ion channels and it holds promise as a novel therapeutic agent for inflammatory disorders.

α-Hederin induces paraptosis by targeting GPCRs to activate Ca2+/MAPK signaling pathway in colorectal cancer.

BACKGROUND: Non-apoptotic cell death is presently emerging as a potential direction to overcome the apoptosis resistance of cancer cells. In the current study, a natural plant agent α-hederin (α-hed) induces caspase-independent paraptotic modes of cell death. PURPOSE: The present study is aimed to investigate the role of α-hed induces paraptosis and the associated mechanism of it. METHODS: The cell proliferation was detected by CCK-8. The cytoplasm organelles were observed under electron microscope. Calcium (Ca2+) level was detected by flow cytometry. Swiss Target Prediction tool analyzed the potential molecule targets of α-hed. Molecular docking methods were used to evaluate binding abilities of α-hed with targets. The expressions of genes and proteins were analyzed by RT-qPCR, western blotting, immunofluorescence, and immunohistochemistry. Xenograft models in nude mice were established to evaluate the anticancer effects in vivo. RESULTS: α-hed exerted significant cytotoxicity against a panel of CRC cell lines by inhibiting proliferation. Besides, it induced cytoplasmic vacuolation in all CRC cells. Electron microscopy images showed the aberrant dilation of endoplasmic reticulum and mitochondria. Both mRNA and protein expressions of Alg-2 interacting proteinX (Alix), the marker of paraptosis, were inhibited by α-hed. Besides, both Swiss prediction and molecular docking showed that the structure of α-hed could tightly target to GPCRs. GPCRs were reported to activate the phospholipase C (PLC)-ß3/ inositol 1,4,5-trisphosphate receptor (IP3R)/ Ca2+/ protein kinase C alpha (PKCα) pathway, and we then found all proteins and mRNA expressions of PLCß3, IP3R, and PKCα were increased by α-hed. After blocking the GPCR signaling, α-hed could not elevate Ca2+ level and showed less CRC cell cytotoxicity. MAPK cascade is the symbol of paraptosis, and we then demonstrated that α-hed activated MAPK cascade by elevating Ca2+ flux. Since non-apoptotic cell death is presently emerging as a potential direction to overcome chemo-drug resistance, we then found α-hed also induced paraptosis in 5-fluorouracil-resistant (5-FU-R) CRC cells, and it reduced the growth of 5-FU-R CRC xenografts. CONCLUSIONS: Collectively, our findings proved α-hed as a promising candidate for inducing non-apoptotic cell death, paraptosis. It may overcome the resistance of apoptotic-based chemo-resistance in CRC.

Control of astrocytic Ca2+ signaling by nitric oxide-dependent S-nitrosylation of Ca2+ homeostasis modulator 1 channels.

BACKGROUND: Astrocytes Ca2+ signaling play a central role in the modulation of neuronal function. Activation of metabotropic glutamate receptors (mGluR) by glutamate released during an increase in synaptic activity triggers coordinated Ca2+ signals in astrocytes. Importantly, astrocytes express the Ca2+-dependent nitric oxide (NO)-synthetizing enzymes eNOS and nNOS, which might contribute to the Ca2+ signals by triggering Ca2+ influx or ATP release through the activation of connexin 43 (Cx43) hemichannels, pannexin-1 (Panx-1) channels or Ca2+ homeostasis modulator 1 (CALHM1) channels. Hence, we aim to evaluate the participation of NO in the astrocytic Ca2+ signaling initiated by stimulation of mGluR in primary cultures of astrocytes from rat brain cortex. RESULTS: Astrocytes were stimulated with glutamate or t-ACPD and NO-dependent changes in [Ca2+]i and ATP release were evaluated. In addition, the activity of Cx43 hemichannels, Panx-1 channels and CALHM1 channels was also analyzed. The expression of Cx43, Panx-1 and CALHM1 in astrocytes was confirmed by immunofluorescence analysis and both glutamate and t-ACPD induced NO-mediated activation of CALHM1 channels via direct S-nitrosylation, which was further confirmed by assessing CALHM1-mediated current using the two-electrode voltage clamp technique in Xenopus oocytes. Pharmacological blockade or siRNA-mediated inhibition of CALHM1 expression revealed that the opening of these channels provides a pathway for ATP release and the subsequent purinergic receptor-dependent activation of Cx43 hemichannels and Panx-1 channels, which further contributes to the astrocytic Ca2+ signaling. CONCLUSIONS: Our findings demonstrate that activation of CALHM1 channels through NO-mediated S-nitrosylation in astrocytes in vitro is critical for the generation of glutamate-initiated astrocytic Ca2+ signaling.

Mature neurons from iPSCs unveil neurodegeneration-related pathways in mucopolysaccharidosis type II: GSK-3ß inhibition for therapeutic potential.

Mucopolysaccharidosis (MPS) type II is caused by a deficiency of iduronate-2-sulfatase and is characterized by the accumulation of glycosaminoglycans (GAGs). Without effective therapy, the severe form of MPS II causes progressive neurodegeneration and death. This study generated multiple clones of induced pluripotent stem cells (iPSCs) and their isogenic controls (ISO) from four patients with MPS II neurodegeneration. MPS II-iPSCs were successfully differentiated into cortical neurons with characteristic biochemical and cellular phenotypes, including axonal beadings positive for phosphorylated tau, and unique electrophysiological abnormalities, which were mostly rescued in ISO-iPSC-derived neurons. RNA sequencing analysis uncovered dysregulation in three major signaling pathways, including Wnt/ß-catenin, p38 MAP kinase, and calcium pathways, in mature MPS II neurons. Further mechanistic characterization indicated that the dysregulation in calcium signaling led to an elevated intracellular calcium level, which might be linked to compromised survival of neurons. Based on these dysregulated pathways, several related chemicals and drugs were tested using this mature MPS II neuron-based platform and a small-molecule glycogen synthase kinase-3ß inhibitor was found to significantly rescue neuronal survival, neurite morphology, and electrophysiological abnormalities in MPS II neurons. Our results underscore that the MPS II-iPSC-based platform significantly contributes to unraveling the mechanisms underlying the degeneration and death of MPS II neurons and assessing potential drug candidates. Furthermore, the study revealed that targeting the specific dysregulation of signaling pathways downstream of GAG accumulation in MPS II neurons with a well-characterized drug could potentially ameliorate neuronal degeneration.

Transient stimulation of TRPMLs enhance the functionality of hDPCs and facilitate hair growth in mice.

Autophagy is essential for eliminating aging and organelle damage that maintaining cellular homeostasis. However, the dysfunction of autophagy has been proven in hair loss such as AGA. Despite the crucial role of TRPML channels in regulating autophagy, their specific function in hair growth remains unclarified. To investigate the biological functions and associated molecular mechanisms of TRPMLs in hair growth, Animal experiments were conducted to confirm the function of TRLMLs activation in promoting hair growth. Subsequently, we analyzed molecular mechanisms in human dermal papilla cells (hDPCs) activated by TRPMLs through transcriptome sequencing analysis. MLSA1(a TRPML agonist) promoted hair regeneration and accelerated hair cycle transition in mice. The activation of TRPMLs upregulated calcium signaling inducing hDPCs to secrete hair growth promoting factors and decrease hair growth inhibiting factors. In addition, activation of TRPMLs triggered autophagy and reduced the generation of ROS, thereby delaying the senescence of hDPCs. All these findings suggested that TRPMLs activation could promote hair growth by regulating hDPCs secretion of hair growth-related factors. Moreover, it may play a prominent role in preventing hDPCs from ROS damage induced by H2O2 or DHT. Targeting TRPMLs may represent a promising therapeutic strategy for treating hair loss.

Amlodipine inhibits Synaptotagmin-4's oncogenic activity on gastric cancer proliferation by targeting calcium signaling.

BACKGROUND: Gastric cancer (GC) remains a leading cause of cancer mortality globally. Synaptotagmin-4 (SYT4), a calcium-sensing synaptic vesicle protein, has been implicated in the oncogenesis of diverse malignancies. PURPOSE: This study delineates the role of SYT4 in modulating clinical outcomes and biological behaviors in GC. METHODS: We evaluated SYT4 expression in GC specimens using bioinformatics analyses and immunohistochemistry. Functional assays included CCK8 proliferation tests, apoptosis assays via flow cytometry, confocal calcium imaging, and xenograft models. Western blotting elucidated MAPK pathway involvement. Additionally, we investigated the impact of the calcium channel blocker amlodipine on cellular dynamics and MAPK pathway activity. RESULTS: SYT4 was higher in GC tissues, and the elevated SYT4 was significantly correlated with adverse prognosis. Both univariate and multivariate analyses confirmed SYT4 as an independent prognostic indicator for GC. Functionally, SYT4 promoted tumorigenesis by fostering cellular proliferation, inhibiting apoptosis, and enhancing intracellular Ca2+ influx, predominantly via MAPK pathway activation. Amlodipine pre-treatment attenuated SYT4-driven cell growth and potentiated apoptosis, corroborated by in vivo xenograft assessments. These effects were attributed to MAPK pathway suppression by amlodipine. CONCLUSION: SYT4 emerges as a potential prognostic biomarker and a pro-oncogenic mediator in GC through a Ca2+-dependent MAPK mechanism. Amlodipine demonstrates significant antitumor effects against SYT4-driven GC, positing its therapeutic promise. This study underscores the imperative of targeting calcium signaling in GC treatment strategies.

Preventive effect of propolis on cognitive decline in Alzheimer's disease model mice.

Brazilian green propolis (propolis) is a chemically complex resinous substance that is a potentially viable therapeutic agent for Alzheimer's disease. Herein, propolis induced a transient increase in intracellular Ca2+ concentration ([Ca2+]i) in Neuro-2A cells; moreover, propolis-induced [Ca2+]i elevations were suppressed prior to 24-h pretreatment with amyloid-ß. To reveal the effect of [Ca2+]i elevation on impaired cognition, we performed memory-related behavioral tasks in APP-KI mice relative to WT mice at 4 and 12 months of age. Propolis, at 300-1000 mg/kg/d for 8 wk, significantly ameliorated cognitive deficits in APP-KI mice at 4 months, but not at 12 months of age. Consistent with behavioral observations, injured hippocampal long-term potentiation was markedly ameliorated in APP-KI mice at 4 months of age following repeated propolis administration. In addition, repeated administration of propolis significantly activated intracellular calcium signaling pathway in the CA1 region of APP-KI mice. These results suggest a preventive effect of propolis on cognitive decline through the activation of intracellular calcium signaling pathways in CA1 region of AD mice model.

Protective effect of ginsenoside Rb1 against aconitine cardiotoxicity studied by myocardial injury, action potential, and calcium signaling.

Aconitine is the main active component of Aconitum plants. Although aconitine has effects that include strengthening the heart, analgesia, anti-tumor, and immune-regulating effects, aconitine has both efficacy and toxicity, especially cardiotoxicity. Severe effects can include arrhythmia and cardiac arrest, which limits the clinical application of aconitine-containing traditional Chinese medicine. Ginsenoside Rb1(Rb1) is mainly found in plants, such as ginseng and Panax notoginseng, and has cardiovascular-protective and anti-arrhythmia effects. This study aimed to investigate the detoxifying effects of Rb1 on aconitine cardiotoxicity and the electrophysiological effect of Rb1 on aconitine-induced arrhythmia in rats. Pathological analysis, myocardial enzymatic indexes, and Western blotting were used to investigate the ameliorating effect of Rb1 on aconitine cardiotoxicity. Optical mapping was used to evaluate the effect of Rb1 on action potential and calcium signaling after aconitine-induced arrhythmia. Rb1 inhibited pathological damage caused by aconitine, decreased myocardial enzyme levels, and restored the balance of apoptotic protein expression by reducing the expression of Bax and cleaved caspase 3 and increasing the expression of Bcl-2, thereby reducing myocardial damage caused by aconitine. Rb1 also reduced the increase in heart rate caused by aconitine, accelerated action potential conduction and calcium signaling, and reduced the dispersion of action potential and calcium signal conduction. Rb1 reduced the cardiotoxicity of aconitine by attenuating aconitine-induced myocardial injury and inhibiting the aconitine-induced retardation of ventricular action potential and calcium signaling in rats.

Magnesium alleviates extracellular histone-induced apoptosis and defective bacterial phagocytosis in macrophages by regulating intracellular calcium signal.

Extracellular histones have been determined as important mediators of sepsis, which induce excessive inflammatory responses in macrophages and impair innate immunity. Magnesium (Mg2+), one of the essential nutrients of the human body, contributes to the proper regulation of immune function. However, no reports indicate whether extracellular histones affect survival and bacterial phagocytosis in macrophages and whether Mg2+ is protective against histone-induced macrophage damage. Our clinical data revealed a negative correlation between circulating histone and monocyte levels in septic patients, and in vitro experiments confirmed that histones induced mitochondria-associated apoptosis and defective bacterial phagocytosis in macrophages. Interestingly, our clinical data also indicated an association between lower serum Mg2+ levels and reduced monocyte levels in septic patients. Moreover, in vitro experiments demonstrated that Mg2+ attenuated histone-induced apoptosis and defective bacterial phagocytosis in macrophages through the PLC/IP3R/STIM-mediated calcium signaling pathway. Importantly, further animal experiments proved that Mg2+ significantly improved survival and attenuated histone-mediated lung injury and macrophage damage in histone-stimulated mice. Additionally, in a cecal ligation and puncture (CLP) + histone-induced injury mouse model, Mg2+ inhibited histone-mediated apoptosis and defective phagocytosis in macrophages and further reduced bacterial load. Overall, these results suggest that Mg2+ supplementation may be a promising treatment for extracellular histone-mediated macrophage damage in sepsis.

Compound SJ-12 attenuates streptozocin-induced diabetic cardiomyopathy by stabilizing SERCA2a.

Heart failure (HF) is one of the major causes of death among diabetic patients. Although studies have shown that curcumin analog C66 can remarkably relieve diabetes-associated cardiovascular and kidney complications, the role of SJ-12, SJ-12, a novel curcumin analog, in diabetic cardiomyopathy and its molecular targets are unknown. 7-week-old male C57BL/6 mice were intraperitoneally injected with single streptozotocin (STZ) (160 mg/kg) to develop diabetic cardiomyopathy (DCM). The diabetic mice were then treated with SJ-12 via gavage for two months. Body weight, fast blood glucose, cardiac utrasonography, myocardial injury markers, pathological morphology of the heart, hypertrophic and fibrotic markers were assessed. The potential target of SJ-12 was evaluated via RNA-sequencing analysis. The O-GlcNAcylation levels of SP1 were detected via immunoprecipitation. SJ-12 effectively suppressed myocardial hypertrophy and fibrosis, thereby preventing heart dysfunction in mice with STZ-induced heart failure. RNA-sequencing analysis revealed that SJ-12 exerted its therapeutic effects through the modulation of the calcium signaling pathway. Furthermore, SJ-12 reduced the O-GlcNAcylation levels of SP1 by inhibiting O-linked N-acetylglucosamine transferase (OGT). Also, SJ-12 stabilized Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase 2a (SERCA2a), a crucial regulator of calcium homeostasis, thus reducing hypertrophy and fibrosis in mouse hearts and cultured cardiomyocytes. However, the anti-fibrotic effects of SJ-12 were not detected in SERCA2a or OGT-silenced cardiomyocytes, indicating that SJ-12 can prevent DCM by targeting OGT-dependent O-GlcNAcylation of SP1.These findings indicate that SJ-12 can exert cardioprotective effects in STZ-induced mice by reducing the O-GlcNAcylation levels of SP1, thus stabilizing SERCA2a and reducing myocardial fibrosis and hypertrophy. Therefore, SJ-12 can be used for the treatment of diabetic cardiomyopathy.

Chronic nicotine exposure is associated with electrophysiological and sympathetic remodeling in the intact rabbit heart.

Nicotine is the primary addictive component of tobacco products. Through its actions on the heart and autonomic nervous system, nicotine exposure is associated with electrophysiological changes and increased arrhythmia susceptibility. To assess the underlying mechanisms, we treated rabbits with transdermal nicotine (NIC, 21 mg/day) or control (CT) patches for 28 days before performing dual optical mapping of transmembrane potential (RH237) and intracellular Ca2+ (Rhod-2 AM) in isolated hearts with intact sympathetic innervation. Sympathetic nerve stimulation (SNS) was performed at the first to third thoracic vertebrae, and ß-adrenergic responsiveness was additionally evaluated following norepinephrine (NE) perfusion. Baseline ex vivo heart rate (HR) and SNS stimulation threshold were higher in NIC versus CT (P = 0.004 and P = 0.003, respectively). Action potential duration alternans emerged at longer pacing cycle lengths (PCL) in NIC versus CT at baseline (P = 0.002) and during SNS (P = 0.0003), with similar results obtained for Ca2+ transient alternans. SNS shortened the PCL at which alternans emerged in CT but not in NIC hearts. NIC-exposed hearts tended to have slower and reduced HR responses to NE perfusion, but ventricular responses to NE were comparable between groups. Although fibrosis was unaltered, NIC hearts had lower sympathetic nerve density (P = 0.03) but no difference in NE content versus CT. These results suggest both sympathetic hypoinnervation of the myocardium and regional differences in ß-adrenergic responsiveness with NIC. This autonomic remodeling may contribute to the increased risk of arrhythmias associated with nicotine exposure, which may be further exacerbated with long-term use.NEW & NOTEWORTHY Here, we show that chronic nicotine exposure was associated with increased heart rate, increased susceptibility to alternans, and reduced sympathetic electrophysiological responses in the intact rabbit heart. We suggest that this was due to sympathetic hypoinnervation of the myocardium and diminished ß-adrenergic responsiveness of the sinoatrial node following nicotine treatment. Though these differences did not result in increased arrhythmia propensity in our study, we hypothesize that prolonged nicotine exposure may exacerbate this proarrhythmic remodeling.

The enhanced hepatotoxicity of isobavachalcone in depigmented zebrafish due to calcium signaling dysregulation and lipid metabolism disorder.

Isobavachalcone (IBC) is a flavonoid component derived from Psoraleae Fructus that can increase skin pigmentation and treat vitiligo. However, IBC has been reported to be hepatotoxic. Current studies on IBC hepatotoxicity are mostly on normal organisms but lack studies on hepatotoxicity in patients. This study established the depigmented zebrafish model by using phenylthiourea (PTU) and investigated the difference in hepatotoxicity between normal and depigmented zebrafish caused by IBC and the underlying mechanism. Morphological, histological, and ultrastructural examination and RT-qPCR verification were used to evaluate the effects of IBC on the livers of zebrafish larvae. IBC significantly decreased liver volume, altered lipid metabolism, and induced pathological and ultrastructural changes in the livers of zebrafish with depigmentation compared with normal zebrafish. The RNA-sequencing and RT-qPCR results showed that the difference in hepatotoxicity between normal and depigmented zebrafish caused by IBC was closely related to the calcium signaling pathway, lipid decomposition and metabolism, and oxidative stress. This work delved into the mechanism of the enhanced IBC-induced hepatotoxicity in depigmented zebrafish and provided a new insight into the hepatotoxicity of IBC.

Pathogenic soluble tau peptide disrupts endothelial calcium signaling and vasodilation in the brain microvasculature.

The accumulation of the microtubule-associated tau protein in and around blood vessels contributes to brain microvascular dysfunction through mechanisms that are incompletely understood. Delivery of nutrients to active neurons in the brain relies on capillary calcium (Ca2+) signals to direct blood flow. The initiation and amplification of endothelial cell Ca2+ signals require an intact microtubule cytoskeleton. Since tau accumulation in endothelial cells disrupts native microtubule stability, we reasoned that tau-induced microtubule destabilization would impair endothelial Ca2+ signaling. We tested the hypothesis that tau disrupts the regulation of local cerebral blood flow by reducing endothelial cell Ca2+ signals and endothelial-dependent vasodilation. We used a pathogenic soluble tau peptide (T-peptide) model of tau aggregation and mice with genetically encoded endothelial Ca2+ sensors to measure cerebrovascular endothelial responses to tau exposure. T-peptide significantly attenuated endothelial Ca2+ activity and cortical capillary blood flow in vivo. Further, T-peptide application constricted pressurized cerebral arteries and inhibited endothelium-dependent vasodilation. This study demonstrates that pathogenic tau alters cerebrovascular function through direct attenuation of endothelial Ca2+ signaling and endothelium-dependent vasodilation.

Nav1.7 as a chondrocyte regulator and therapeutic target for osteoarthritis.

Osteoarthritis (OA) is the most common joint disease. Currently there are no effective methods that simultaneously prevent joint degeneration and reduce pain1. Although limited evidence suggests the existence of voltage-gated sodium channels (VGSCs) in chondrocytes2, their expression and function in chondrocytes and in OA remain essentially unknown. Here we identify Nav1.7 as an OA-associated VGSC and demonstrate that human OA chondrocytes express functional Nav1.7 channels, with a density of 0.1 to 0.15 channels per µm2 and 350 to 525 channels per cell. Serial genetic ablation of Nav1.7 in multiple mouse models demonstrates that Nav1.7 expressed in dorsal root ganglia neurons is involved in pain, whereas Nav1.7 in chondrocytes regulates OA progression. Pharmacological blockade of Nav1.7 with selective or clinically used pan-Nav channel blockers significantly ameliorates the progression of structural joint damage, and reduces OA pain behaviour. Mechanistically, Nav1.7 blockers regulate intracellular Ca2+ signalling and the chondrocyte secretome, which in turn affects chondrocyte biology and OA progression. Identification of Nav1.7 as a novel chondrocyte-expressed, OA-associated channel uncovers a dual target for the development of disease-modifying and non-opioid pain relief treatment for OA.

The dietary sweetener sucralose is a negative modulator of T cell-mediated responses.

Artificial sweeteners are used as calorie-free sugar substitutes in many food products and their consumption has increased substantially over the past years1. Although generally regarded as safe, some concerns have been raised about the long-term safety of the consumption of certain sweeteners2-5. In this study, we show that the intake of high doses of sucralose in mice results in immunomodulatory effects by limiting T cell proliferation and T cell differentiation. Mechanistically, sucralose affects the membrane order of T cells, accompanied by a reduced efficiency of T cell receptor signalling and intracellular calcium mobilization. Mice given sucralose show decreased CD8+ T cell antigen-specific responses in subcutaneous cancer models and bacterial infection models, and reduced T cell function in models of T cell-mediated autoimmunity. Overall, these findings suggest that a high intake of sucralose can dampen T cell-mediated responses, an effect that could be used in therapy to mitigate T cell-dependent autoimmune disorders.