Intracellular Calcium Homeostasis Research Articles

Identification of a novel gene PpBCG1 that has a positive role in desiccation tolerance in the moss Physcomitrium patens.

Water-to-land transition is a hallmark of terrestrialization for land plants and requires molecular adaptation to resist water deficiency. Lineages- or species-specific genes are widespread across eukaryotes, and yet the majority of those are functionally unknown and not annotated. Recent studies have revealed that some of such genes could play a role in adapting to environmental stress responses. Here, we identified a novel gene PpBCG1 (Bryophyte Co-retained Gene 1) in the moss Physcomitrium patens that was responsive to dehydration and rehydration. Under de- and rehydration treatments, PpBCG1 was significantly co-expressed with the dehydrin-encoding gene PpDHNA. Microarray data revealed that PpBCG1 was highly expressed in tissues of spores, female organ archegonia, and mature sporophytes. In addition, the Ppbcg1 mutant showed reduced ability of dehydration tolerance, whose plants were accompanied by a relatively low level of chlorophyll content during recovery. Comprehensive transcriptomics uncovered a detailed set of regulatory processes that were affected by the PpBCG1 disruption. Moreover, experimental evidence showed that PpBCG1 might function in the antioxidant activity, abscisic acid (ABA) pathway, and intracellular calcium (Ca2+) homeostasis to resist desiccation. Together, our study provides insights into the roles of one bryophyte co-retained gene in the desiccation tolerance.

The effect of sodium-glucose cotransporter type 2 inhibitors on left ventricular diastolic function: current status and prospects

Sodium-glucose cotransporter-2 inhibitors (SGLT2 inhibitors) or gliflozins, are a new class of cardiovascular drugs with a proven clinical efficacy and a beneficial effect on prognosis in patients with heart failure with preserved ejection fraction (HFpEF). Impaired left ventricular (LV) diastolic function (DF) is an important element in the pathogenesis of HFpEF. Experimental studies have found intracellular mechanisms for the so-called diastolic effects in gliflozins. Studies using laboratory models of experimental HFpEF have demonstrated a positive effect of dapagliflozin and empagliflozin on the elastic properties of cardiomyocyte myofilaments, the dynamics of myocardial fibrosis, and intracellular sodium and calcium homeostasis. The significance of anti-inflammatory, antioxidant properties of gliflozins in improving the cardiomyocyte DF has been experimentally established. The effect of SGLT2 inhibitors on LV DF in patients at high risk for cardiovascular diseases and their complications, that has been demonstrated in relatively small clinical studies, is due to primary cardiac and secondary effects. Results of individual studies confirmed the protective (in relation to myocardial relaxation) properties of gliflozins in the conditions of a diastolic stress test. The regression of LV diastolic dysfunction associated with the SGLT2 inhibitor treatment found in small observational studies is important in the context of the significant beneficial effect of empagliflozin and dapagliflozin on the prognosis of cardiovascular diseases that has been demonstrated in large randomized clinical trials in patients with HFpEF.

Type I collagen-targeted liposome delivery of Serca2a modulates myocardium calcium homeostasis and reduces cardiac fibrosis induced by myocardial infarction

Fibrotic scarring and impaired myocardial calcium homeostasis serve as the two main factors in the pathology of heart failure following myocardial infarction (MI), leading to poor prognosis and death in patients. Serca2a is a target of interest in gene therapy for MI-induced heart failure via the regulation of intracellular calcium homeostasis and, subsequently, enhancing myocardial contractility. A recent study also reported that Serca2a ameliorates pulmonary fibrosis by blocking nuclear factor kB (NF-kB)/interleukin-6 (IL-6)-induced (SMAD)/TGF-β signaling activation, while the effect in MI-induced myocardial fibrosis remains to be addressed. Here, we loaded Serca2a plasmids into type 1 collagen-targeting nanoparticles to synthesize the GKWHCTTKFPHHYCLY-Serca2a-Liposome (GSL-NPs) for targeted treatment of myocardial infarction. We showed that GSL-NPs were effectively targeted in the scar area in MI-induced mice within tail-vein delivery for 48 h. Treatment with GSL-NPs improved cardiac functions and shrank fibrotic scars after MI in mice by up-regulating Serca2a. In cardiac fibroblasts, GSL-NPs alleviated hypoxia-induced fibrotic progression partly by inhibiting NF-kB activation. Furthermore, treatment with GSL-NPs protected cardiomyocyte calcium homeostasis and enhanced myocardial contractility during hypoxia. Together, we demonstrate that type I collagen-targeted liposome delivery of Serca2a may benefit patients with myocardial infarction by inhibiting fibrotic scarring as well as modulation of calcium homeostasis.

Therapeutic potential of transient receptor potential (TRP) channels in psychiatric disorders.

Psychiatric disorders such as Bipolar disorder, Anxiety, Major depressive disorder, Schizophrenia, Attention-deficit/hyperactivity disorder, as well as neurological disorders such as Migraine, are linked by the evidence of altered calcium homeostasis. The disturbance of intra-cellular calcium homeostasis disrupts the activity of numerous ion channels including transient receptor potential (TRP) channels. TRP channel families comprise non-selective calcium-permeable channels that have been implicated in variety of physiological processes in the brain, as well as in the pathogenesis of psychiatric disorders. Through a comprehensive review of current research and experimentation, this investigation elucidates the role of TRP channels in psychiatric disorders. Furthermore, this review discusses about the exploration of epigenetics and TRP channels in psychiatric disorders.

Intracellular cartilage oligomeric matrix protein augments breast cancer resistance to chemotherapy

Chemotherapy persists as the primary intervention for breast cancer, with chemoresistance posing the principal obstacle to successful treatment. Herein, we show that cartilage oligomeric matrix protein (COMP) expression leads to increased cancer cell survival and attenuated apoptosis under treatment with several chemotherapeutic drugs, anti-HER2 targeted treatment, and endocrine therapy in several breast cancer cell lines tested. The COMP-induced chemoresistance was independent of the breast cancer subtype. Extracellularly delivered recombinant COMP failed to rescue cells from apoptosis while endoplasmic reticulum (ER)-restricted COMP-KDEL conferred resistance to apoptosis, consistent with the localization of COMP in the ER, where it interacted with calpain. Calpain activation was reduced in COMP-expressing cells and maintained at a lower level of activation during treatment with epirubicin. Moreover, the downstream caspases of calpain, caspases -9, -7, and -3, exhibited significantly reduced activation in COMP-expressing cells under chemotherapy treatment. Chemotherapy, when combined with calpain activators, rendered the cells expressing COMP more chemosensitive. Also, the anti-apoptotic proteins phospho-Bcl2 and survivin were increased in COMP-expressing cells upon chemotherapy. Cells expressing a mutant COMP lacking thrombospondin repeats exhibited reduced chemoresistance compared to cells expressing full-length COMP. Evaluation of calcium levels in the ER, cytosol, and mitochondria revealed that COMP expression modulates intracellular calcium homeostasis. Furthermore, patients undergoing chemotherapy or endocrine therapy demonstrated significantly reduced overall survival time when tumors expressed high levels of COMP. This study identifies a novel role of COMP in chemoresistance and calpain inactivation in breast cancer, a discovery with potential implications for anti-cancer therapy.

STIM1 mediates methamphetamine-induced neuronal autophagy and apoptosis

Methamphetamine (METH) is a widely abused amphetamine-type psychoactive drug that causes serious health problems. Previous studies have demonstrated that METH can induce neuron autophagy and apoptosis in vivo and in vitro. However, the molecular mechanisms underlying METH-induced neuron autophagy and apoptosis remain poorly understood. Stromal interacting molecule 1 (STIM1) was hypothesized to be involved in METH-induced neuron autophagy and apoptosis. Therefore, the expression of STIM1 protein was measured and the effect of blocking STIM1 expression with siRNA was investigated in cultured neuronal cells, and the hippocampus and striatum of mice exposed to METH. Furthermore, intracellular calcium concentration and endoplasmic reticulum (ER) stress-related proteins were determined in vitro and in vivo in cells treated with METH. The results suggested that STIM1 mediates METH-induced neuron autophagy by activating the p-Akt/p-mTOR pathway. METH exposure also resulted in increased expression of Orai1, which was reversed after STIM1 silencing. Moreover, the disruption of intracellular calcium homeostasis induced ER stress and up-regulated the expression of pro-apoptotic protein CCAAT/enhancer-binding protein homologous protein (CHOP), resulting in classic mitochondria apoptosis. METH exposure can cause neuronal autophagy and apoptosis by increasing the expression of STIM1 protein; thus, STIM1 may be a potential gene target for therapeutics in METH-caused neurotoxicity.

A meta-analysis GWAS of taxane-induced peripheral neuropathy in patients with breast cancer.

12089 Background: Taxane-induced peripheral neuropathy (TIPN) is a common dose-limiting toxicity in the medical management of breast cancer patients. Despite the clinical efficacy of taxane treatment, there is significant heterogeneity in the development of neuropathy and its severity, and no clear genetic etiologies have yet been identified. We sought to discover genetic risk variants in two large electronic health record linked DNA biobanks. Methods: A meta-analysis genome wide association study (GWAS) was performed with participants in the All of Us Research Program and BioVU biobanks. All participants of the study population were of female sex at birth and had a breast cancer diagnosis. Cases were defined as taxane-exposed participants who received their first neuropathy diagnosis code within one year after first taxane exposure. Controls were defined as taxane-exposed participants without a neuropathy diagnosis code and without gabapentin initiation after taxane exposure. GWAS was performed with PLINK version 2.0 using age at taxane initiation, number of taxane cycles received, and the first 10 principal components as covariates. Expression quantitative trait loci (eQTL) analysis was performed with the GTEx 8.0 and EyeGEx databases. Results: The All of Us cohort had 196 cases and 254 controls. The BioVU cohort had 244 cases and 528 controls. In our meta-analysis GWAS, rs3065465 (chr11:69138693; closest gene by distance is LOC338694) was associated with TIPN (OR=2.14, P= 8.27E-09) and was similar across different genetic ancestries (Table). The eQTL analysis of rs3065465 shows significant association with mRNA expression of TPCN2(P= 6.44E-06), a cation channel involved in neuronal differentiation and intracellular calcium homeostasis. Conclusions: rs3065465 represents a potential novel genetic marker for TIPN risk in breast cancer patients.[Table: see text]

Inhibition of TRPA1-like alleviated unfolded protein response and apoptosis by regulating cytoplasmic Ca2+ in Yesso scallop Patinopecten yessoensis under high temperature stress

Transient receptor potential ankyrin subtype 1 (TRPA1) is a nonselective cation channel protein typically forms ion channels that regulate intracellular calcium homeostasis, and can be induced by temperature and various chemicals. In the present study, the involvement of PyTRPA1-like in regulating unfolded protein response (UPR) and apoptosis in Yesso Scallop Patinopecten yessoensis under high temperature stress was investigated. The mRNA transcripts of PyTRPA1-like were detected in haemocytes and all the examined tissues with the highest expression level in mantle. After TRPA1 activator (allyl-isothiocyanate, AITC) and high temperature (25°C) treatment, the expression level of PyTRPA1-like mRNA and the Ca2+ content in haemocytes increased significantly (p < 0.05) at 3 h, and then recovered to the normal level at 12 h, and the expression level of PyGRP78, PyIRE1, PyATF6β, PyPERK and PyCaspase-3 mRNA in haemocytes, and Caspase-3 activity and apoptosis rate were also significantly upregulated (p < 0.05). After TRPA1 antagonist (HC-030031) and high temperature (25°C) treatment, the intracellular Ca2+ content, the transcripts of PyGRP78, PyIRE1 and PyCaspase-3 in haemocytes, as well as the Caspase-3 activity and apoptosis rate decreased significantly compared to the control group (p < 0.05), while the Ca2+ distribution in haemocytes showed no difference with that in control group. These results collectively suggest that PyTRPA1-like plays important roles in regulating UPR and apoptosis by mediating calcium influx under high temperature stress in scallop P. yessoensis.

PFKFB3 controls acinar IP3R-mediated Ca2+ overload to regulate acute pancreatitis severity.

Acute pancreatitis (AP) is among the most common hospital gastrointestinal diagnoses; understanding the mechanisms underlying the severity of AP is critical for development of new treatment options for this disease. Here, we evaluate the biological function of phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) in AP pathogenesis in 2 independent genetically engineered mouse models of AP. PFKFB3 was elevated in AP and severe AP (SAP), and KO of Pfkfb3 abrogated the severity of alcoholic SAP (FAEE-SAP). Using a combination of genetic, pharmacological, and molecular studies, we defined the interaction of PFKFB3 with inositol 1,4,5-trisphosphate receptor (IP3R) as a key event mediating this phenomenon. Further analysis demonstrated that the interaction between PFKFB3 and IP3R promotes FAEE-SAP severity by altering intracellular calcium homeostasis in acinar cells. Together, our results support a PFKFB3-driven mechanism controlling AP pathobiology and define this enzyme as a therapeutic target to ameliorate the severity of this condition.

Endurance Exercise Prevented Diabetic Cardiomyopathy through the Inhibition of Fibrosis and Hypertrophy in Rats.

Exercise training could be essential in preventing pathological cardiac remodeling in diabetes. Therefore, the effects of moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT) singly or plus metformin on diabetes-induced cardiomyopathy were investigated in this study. Forty-nine Wistar rats (male) were recruited. Seven groups of animals were treated for six weeks as control, diabetes, MICT (15 m/min, 40 min/day), HIIT (20 m/min, 40 min/day), metformin (300 mg/kg), HIIT+metformin (Met-HIIT), and MICT+metformin (Met-MICT). The metformin was orally administered with an intragastrical needle, and the exercised rats were trained (5 days/week) with a motorized treadmill. Metabolic parameters, echocardiographic indices, histopathology evaluation, and assessment of gene expression connected with cardiac fibrosis, hypertrophy, mitochondrial performance, and intracellular calcium homeostasis were investigated. Our results demonstrated that all the interventions prevented weight loss and enhanced heart weight/body weight ratio and fasting plasma glucose in diabetic rats. Both types of exercise and their metformin combinations improved diabetic animals' echocardiography indices by enhancing heart rate, fractional shortening (FS), ejection fraction (EF) and reducing end-systolic and end-diastolic diameter of left ventricular (LVESD and LVEDD). Gene expression of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), transforming growth factor (TGF)- , and collagen increased in the diabetes group. In contrast, the gene expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1 ), AMP-activated protein kinase (AMPK), ryanodine receptors (RyR), and ATPase pump of the sarcoplasmic reticulum (SERCA) was reduced in diabetic animals. Exercise training alone or in combination with metformin reversed these changes. Moreover, diabetes-induced cardiac fibrosis was ameliorated in treated groups. All indicators of diabetic cardiomyopathy were improved more in the Met-HIIT group than in other groups. Exercise training, notably with metformin combination, alleviated diabetes-induced cardiac complications. The beneficial effects of exercise could be related to improving pathological cardiac remodeling and enhancing cardiac function.

Pharmacological SERCA activation limits diet-induced steatohepatitis and restores liver metabolic function in mice

Metabolic dysfunction-associated steatotic liver disease is the most common form of liver disease and poses significant health risks to patients who progress to metabolic dysfunction-associated steatohepatitis. Fatty acid overload alters endoplasmic reticulum (ER) calcium stores and induces mitochondrial oxidative stress in hepatocytes, leading to hepatocellular inflammation and apoptosis. Obese mice have impaired liver sarco/ER Ca2+-ATPase (SERCA) function, which normally maintains intracellular calcium homeostasis by transporting Ca2+ ions from the cytoplasm to the ER. We hypothesized that restoration of SERCA activity would improve diet-induced steatohepatitis in mice by limiting ER stress and mitochondrial dysfunction. WT and melanocortin-4 receptor KO (Mc4r−/−) mice were placed on either chow or Western diet (WD) for 8 weeks. Half of the WD-fed mice were administered CDN1163 to activate SERCA, which reduced liver fibrosis and inflammation. SERCA activation also restored glucose tolerance and insulin sensitivity, improved histological markers of metabolic dysfunction-associated steatohepatitis, increased expression of antioxidant enzymes, and decreased expression of oxidative stress and ER stress genes. CDN1163 decreased hepatic citric acid cycle flux and liver pyruvate cycling, enhanced expression of mitochondrial respiratory genes, and shifted hepatocellular [NADH]/[NAD+] and [NADPH]/[NADP+] ratios to a less oxidized state, which was associated with elevated PUFA content of liver lipids. In sum, the data demonstrate that pharmacological SERCA activation limits metabolic dysfunction-associated steatotic liver disease progression and prevents metabolic dysfunction induced by WD feeding in mice.

Buparvaquone Induces Ultrastructural and Physiological Alterations Leading to Mitochondrial Dysfunction and Caspase-Independent Apoptotic Cell Death in Leishmania donovani.

Leishmaniasis is a neglected tropical disease (endemic in 99 countries) caused by parasitic protozoa of the genus Leishmania. As treatment options are limited, there is an unmet need for new drugs. The hydroxynaphthoquinone class of compounds demonstrates broad-spectrum activity against protozoan parasites. Buparvaquone (BPQ), a member of this class, is the only drug licensed for the treatment of theileriosis. BPQ has shown promising antileishmanial activity but its mode of action is largely unknown. The aim of this study was to evaluate the ultrastructural and physiological effects of BPQ for elucidating the mechanisms underlying the in vitro antiproliferative activity in Leishmania donovani. Transmission and scanning electron microscopy analyses of BPQ-treated parasites revealed ultrastructural effects characteristic of apoptosis-like cell death, which include alterations in the nucleus, mitochondrion, kinetoplast, flagella, and the flagellar pocket. Using flow cytometry, laser scanning confocal microscopy, and fluorometry, we found that BPQ induced caspase-independent apoptosis-like cell death by losing plasma membrane phospholipid asymmetry and cell cycle arrest at sub-G0/G1 phase. Depolarization of the mitochondrial membrane leads to the generation of oxidative stress and impaired ATP synthesis followed by disruption of intracellular calcium homeostasis. Collectively, these findings provide valuable mechanistic insights and demonstrate BPQ's potential for development as an antileishmanial agent.

The VAMP-associated protein VAP27-1 plays a crucial role in plant resistance to ER stress by modulating ER–PM contact architecture in Arabidopsis

The endoplasmic reticulum (ER) and the plasma membrane (PM) form ER–PM contact sites (EPCSs) that allow the ER and PM to exchange materials and information. Stress-induced disruption of protein folding triggers ER stress, and the cell initiates the unfolded protein response (UPR) to resist the stress. However, whether EPCSs play a role in ER stress in plants remains unclear. VESICLE-ASSOCIATED MEMBRANE PROTEIN (VAMP)-ASSOCIATED PROTEIN 27-1 (VAP27-1) functions in EPCS tethering and is encoded by a family of 10 genes (VAP27-1–10) in Arabidopsis thaliana. Here, we used CRISPR-Cas9-mediated genome editing to obtain a homozygous vap27-1 vap27-3 vap27-4 (vap27-1/3/4) triple mutant lacking three of the key VAP27 family members in Arabidopsis. The vap27-1/3/4 mutant exhibits defects in ER–PM connectivity and EPCS architecture, as well as excessive UPR signaling. We further showed that relocation of VAP27-1 to the PM mediates specific VAP27-1-related EPCS remodeling and expansion under ER stress. Moreover, the spatiotemporal dynamics of VAP27-1 at the PM increase ER–PM connectivity and enhance Arabidopsis resistance to ER stress. In addition, we revealed an important role for intracellular calcium homeostasis in the regulation of UPR signaling. Taken together, these results broaden our understanding of the molecular and cellular mechanisms of ER stress and UPR signaling in plants, providing additional clues for improving plant broad-spectrum resistance to different stresses.

Vericiguat suppresses ventricular tachyarrhythmias inducibility in a rabbit myocardial infarction model.

The VICTORIA trial demonstrated a significant decrease in cardiovascular events through vericiguat therapy. This study aimed to assess the potential mechanisms responsible for the reduction of cardiovascular events with vericiguat therapy in a rabbit model of myocardial infarction (MI). A chronic MI rabbit model was created through coronary artery ligation. Following 4 weeks, the hearts were harvested and Langendorff perfused. Subsequently, electrophysiological examinations and dual voltage-calcium optical mapping studies were conducted at baseline and after administration of vericiguat at a dose of 5 μmol/L. Acute vericiguat therapy demonstrated a significant reduction in premature ventricular beat burden and effectively suppressed ventricular arrhythmic inducibility. The electrophysiological influences of vericiguat therapy included an increased ventricular effective refractory period, prolonged action potential duration, and accelerated intracellular calcium (Cai) homeostasis, leading to the suppression of action potential and Cai alternans. The pacing-induced ventricular arrhythmias exhibited a reentrant pattern, attributed to fixed or functional conduction block in the peri-infarct zone. Vericiguat therapy effectively mitigated the formation of cardiac alternans as well as the development of reentrant impulses, providing additional anti-arrhythmic benefits. In the MI rabbit model, vericiguat therapy demonstrates anti-ventricular arrhythmia effects. The vericiguat therapy reduces ventricular ectopic beats, inhibiting the initiation of ventricular arrhythmias. Furthermore, the therapy successfully suppresses cardiac alternans, preventing conduction block and, consequently, the formation of reentry circuits.

LIPUS regulates the progression of knee osteoarthritis in mice through primary cilia-mediated TRPV4 channels.

Osteoarthritis (OA) is a common disease in middle-aged and elderly people. An imbalance in calcium ion homeostasis will contribute to chondrocyte apoptosis and ultimately lead to the progression of OA. Transient receptor potential channel 4 (TRPV4) is involved in the regulation of intracellular calcium homeostasis. TRPV4 is expressed in primary cilia, which can sense mechanical stimuli from outside the cell, and its abnormal expression is closely related to the development of OA. Low-intensity pulsed ultrasound (LIPUS) can alleviate chondrocyte apoptosis while the exact mechanism is unclear. In this project, with the aim of revealing the mechanism of action of LIPUS, we proposed to use OA chondrocytesand animal models, LIPUS intervention, inhibition of primary cilia, use TRPV4 inhibitors or TRPV4agonist, and use Immunofluorescence (IF), Immunohistochemistry (IHC), Western Blot (WB), Quantitative Real-time PCR (QP) to detect the expression of cartilage synthetic matrix and endoplasmic reticulum stress markers.The results revealed that LIPUS altered primary cilia expression, promoted synthetic matrix metabolism in articular chondrocytes and was associated with primary cilia. In addition, LIPUS exerted a active effect on OA by activating TRPV4, inducing calcium inward flow, and facilitating the entry of NF-κB into the nucleus to regulate synthetic matrix gene transcription. Inhibition of TRPV4 altered primary cilia expression in response to LIPUS stimulation, and knockdown of primary cilia similarly inhibited TRPV4 function. These results suggest that LIPUS mediates TRPV4 channels through primary cilia to regulate the process of knee osteoarthritis in mice.

Mitochondria-Associated Membranes as Key Regulators in Cellular Homeostasis and the Potential Impact of Exercise on Insulin Resistance.

The communication between mitochondria and the endoplasmic reticulum (ER) is facilitated by a dynamic membrane structure formed by protein complexes known as mitochondria-associated membranes (MAMs). The structural and functional integrity of MAMs is crucial for insulin signal transduction, relying heavily on their regulation of intracellular calcium homeostasis, lipid homeostasis, mitochondrial quality control, and endoplasmic reticulum stress (ERS). This article reviews recent research findings, suggesting that exercise may promote the remodeling of MAMs structure and function by modulating the expression of molecules associated with their structure and function. This, in turn, restores cellular homeostasis and ultimately contributes to the amelioration of insulin resistance (IR). These insights provide additional possibilities for the study and treatment of insulin resistance-related metabolic disorders such as obesity, diabetes, fatty liver, and atherosclerosis.

Establishment and evaluation of targeted molecular screening model for the ryanodine receptor or sarco/endoplasmic reticulum calcium ATPase.

Endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) is crucial for maintaining intracellular calcium homeostasis due to the calcium-signaling-related proteins on its membrane. While ryanodine receptors (RyR) on insect ER/SR membranes are well-known as targets for diamide insecticides, little is known about other calcium channels. Given the resistance of diamide insecticides, the establishment of molecular screening models targeting RyR or sarco/endoplasmic reticulum calcium ATPase (SERCA) is conducive to the discovery of new insecticidal molecules. The morphological features of Mythimna separata SR have closed vesicles with integrity and high density. The 282 proteins in the SR component contained RyR and SERCA. A measurement model for the release and uptake of calcium was successfully established by detecting calcium ions outside the SR membrane using a fluorescence spectrophotometer. In vitro testing systems using SR vesicles found that diamide insecticides could activate dose-dependently RyR, with EC50 values of 0.14 μM (Chlorantraniliprole), 0.21 μM (Flubendiamide), and 0.57 μM (Cyantraniliprole), respectively. However, dantrolene inhibited RyR-mediated calcium release with an IC50 value of 353.9 μM, suggesting that dantrolene can weakly antagonize RyR. Moreover, cyclopiazonic acid significantly reduced the enzyme activity and calcium uptake capacity of SERCA. On the contrary, CDN1163 markedly activated the enzyme activity and improved the calcium transport capacity of SERCA. SR vesicles can be used to study the function of unknown proteins on the SR membranes, as well as for high-throughput screening of highly active compounds targeting RyR or SERCA. © 2024 Society of Chemical Industry.

HRAS-Mutant Cardiomyocyte Model of Multifocal Atrial Tachycardia.

Germline HRAS gain-of-function pathogenic variants cause Costello syndrome (CS). During early childhood, 50% of patients develop multifocal atrial tachycardia, a treatment-resistant tachyarrhythmia of unknown pathogenesis. This study investigated how overactive HRAS activity triggers arrhythmogenesis in atrial-like cardiomyocytes (ACMs) derived from human-induced pluripotent stem cells bearing CS-associated HRAS variants. HRAS Gly12 mutations were introduced into a human-induced pluripotent stem cells-ACM reporter line. Human-induced pluripotent stem cells were generated from patients with CS exhibiting tachyarrhythmia. Calcium transients and action potentials were assessed in induced pluripotent stem cell-derived ACMs. Automated patch clamping assessed funny currents. HCN inhibitors targeted pacemaker-like activity in mutant ACMs. Transcriptomic data were analyzed via differential gene expression and gene ontology. Immunoblotting evaluated protein expression associated with calcium handling and pacemaker-nodal expression. ACMs harboring HRAS variants displayed higher beating rates compared with healthy controls. The hyperpolarization activated cyclic nucleotide gated potassium channel inhibitor ivabradine and the Nav1.5 blocker flecainide significantly decreased beating rates in mutant ACMs, whereas voltage-gated calcium channel 1.2 blocker verapamil attenuated their irregularity. Electrophysiological assessment revealed an increased number of pacemaker-like cells with elevated funny current densities among mutant ACMs. Mutant ACMs demonstrated elevated gene expression (ie, ISL1, TBX3, TBX18) related to intracellular calcium homeostasis, heart rate, RAS signaling, and induction of pacemaker-nodal-like transcriptional programming. Immunoblotting confirmed increased protein levels for genes of interest and suppressed MAPK (mitogen-activated protein kinase) activity in mutant ACMs. CS-associated gain-of-function HRASG12 mutations in induced pluripotent stem cells-derived ACMs trigger transcriptional changes associated with enhanced automaticity and arrhythmic activity consistent with multifocal atrial tachycardia. This is the first human-induced pluripotent stem cell model establishing the mechanistic basis for multifocal atrial tachycardia in CS.

Sarcoma cell-specific radiation sensitization by titanate scrolled nanosheets: insights from physicochemical analysis and transcriptomic profiling

This study aimed to explore the potential of metal oxides such as Titanate Scrolled Nanosheets (TNs) in improving the radiosensitivity of sarcoma cell lines. Enhancing the response of cancer cells to radiation therapy is crucial, and one promising approach involves utilizing metal oxide nanoparticles. We focused on the impact of exposing two human sarcoma cell lines to both TNs and ionizing radiation (IR). Our research was prompted by previous in vitro toxicity assessments, revealing a correlation between TNs' toxicity and alterations in intracellular calcium homeostasis. A hydrothermal process using titanium dioxide powder in an alkaline solution produced the TNs. Our study quantified the intracellular content of TNs and analyzed their impact on radiation-induced responses. This assessment encompassed PIXE analysis, cell proliferation, and transcriptomic analysis. We observed that sarcoma cells internalized TNs, causing alterations in intracellular calcium homeostasis. We also found that irradiation influence intracellular calcium levels. Transcriptomic analysis revealed marked disparities in the gene expression patterns between the two sarcoma cell lines, suggesting a potential cell-line-dependent nano-sensitization to IR. These results significantly advance our comprehension of the interplay between TNs, IR, and cancer cells, promising potential enhancement of radiation therapy efficiency.

Puerarin Ameliorated PCOS through Preventing Mitochondrial Dysfunction Dependent on the Maintenance of Intracellular Calcium Homeostasis.

Polycystic ovarian syndrome (PCOS) is the major cause of infertility in reproductive women, but no universal drug is feasible. Although puerarin clinically treats cerebrovascular and cardiovascular diseases, its curative effect on PCOS remains elusive. The present study discovered that administration of puerarin restored estrous cycle of PCOS mice and diminished the number of cystic follicles with the concomitant recovery for circulating testosterone, LH and FSH levels, and LH/FSH ratio, indicating the therapeutic role of puerarin in PCOS. KEGG analysis of differential genes between PCOS and control revealed the enrichment in MAPK and calcium signaling pathway. Application of puerarin restricted the phosphorylation of ERK1/2 and JNK, whose activation neutralized the improvement of puerarin on the secretory function and apoptosis of ovarian granulosa cells (GCs). Meanwhile, puerarin alleviated the accumulation of cytosolic Ca2+ through restricting the opening of Ryr and Itpr channels, but this effectiveness was counteracted by the activatory ERK1/2 and JNK. Attenuation of cytosolic Ca2+ counteracted the antagonistic effects of ERK1/2 and JNK activation on puerarin's role in rescuing the calcineurin and Nfatc. Further analysis manifested that Mcu had been authenticated as a direct downstream target of Nfatc to mediate the amelioration of puerarin on mitochondrial Ca2+ uptake. Moreover, puerarin prevented the disorder of ATP content, mitochondrial membrane potential, and mitochondrial permeability transition pore opening through maintaining mitochondrial Ca2+ homeostasis. Collectively, puerarin might ameliorate the symptoms of PCOS mice through preventing mitochondrial dysfunction that is dependent on the maintenance of intracellular Ca2+ homeostasis after inactivation of ERK1/2 and JNK.