Cellular Calcium Homeostasis Research Articles

Mitochondrial Dynamics and Insulin Secretion.

Mitochondria are involved in the regulation of cellular energy metabolism, calcium homeostasis, and apoptosis. For mitochondrial quality control, dynamic processes, such as mitochondrial fission and fusion, are necessary to maintain shape and function. Disturbances of mitochondrial dynamics lead to dysfunctional mitochondria, which contribute to the development and progression of numerous diseases, including Type 2 Diabetes (T2D). Compelling evidence has been put forward that mitochondrial dynamics play a significant role in the metabolism-secretion coupling of pancreatic β cells. The disruption of mitochondrial dynamics is linked to defects in energy production and increased apoptosis, ultimately impairing insulin secretion and β cell death. This review provides an overview of molecular mechanisms controlling mitochondrial dynamics, their dysfunction in pancreatic β cells, and pharmaceutical agents targeting mitochondrial dynamic proteins, such as mitochondrial division inhibitor-1 (mdivi-1), dynasore, P110, and 15-oxospiramilactone (S3).

Decoding the biological toxicity of phenanthrene on intestinal cells of Eisenia fetida: Effects, toxicity pathways and corresponding mechanisms

Phenanthrene is frequently detected and exists extensively in the soil environment, and its residues inevitably impose a significant threat to soil organisms. Exposure to and toxicity of phenanthrene on earthworms has been extensively studied before, however, the possible mechanisms and related pathways associated with phenanthrene-triggered toxicity at the intestinal cell level remain unclear. Herein, primary intestinal cells isolated from Eisenia fetida (Annelida, Oligochaeta) intestine were used as targeted receptors to probe the molecular mechanisms involved in ROS-mediated damaging effects and the potential pathways of phenanthrene-induced toxicity at cellular and sub-cellular levels. Results indicated that phenanthrene exposure induced oxidative stress by activating intracellular ROS (elevated O2−, H2O2, and OH− content) bursts in E. fetida intestinal cells, causing various oxidative damage effects, including lipid peroxidation (increased MDA content), protein oxidation (enhanced PCO levels), and DNA damage (enhanced 8-OHdG levels). The enzymatic and non-enzymatic strategies in earthworm cells were activated to mitigate these detrimental effects by regulating ROS-mediated pathways involving defense regulation. Also, phenanthrene stress destroyed the cell membrane of E. fetida intestinal cells, resulting in cellular calcium homeostasis disruption and cellular energetic alteration, ultimately causing cytotoxicity and cell apoptosis/death. More importantly, the mitochondrial dysfunction in E. fetida cells was induced by phenanthrene-caused mitochondrial membrane depolarization, which in turn caused un-controlled ROS burst and induced apoptosis through mitochondria-mediated caspase-3 activation and ROS-mediated mitochondrial-dependent pathway. Furthermore, exposure to phenanthrene activated an abnormal mRNA expression profile associated with defense regulation (e.g., Hsp70, MT, CRT, SOD, CAT, and GST genes) in E. fetida intestinal cells, resulting in various cellular dysfunctions and pathological conditions, eventually, apoptotic cell death. Taken together, this study offers valuable insights for probing the toxic effects and underlying mechanisms posed by phenanthrene at the intestinal cell level, and is of great significance to estimate the detrimental side effects of phenanthrene on soil ecological health.

Prevention of noise-induced hearing loss by calpain inhibitor MDL-28170 is associated with upregulation of PI3K/Akt survival signaling pathway.

Noise-induced calcium overload in sensory hair cells has been well documented as an early step in the pathogenesis of noise-induced hearing loss (NIHL). Alterations in cellular calcium homeostasis mediate a series of cellular events, including activation of calcium-dependent protein kinases and phosphatases. Using cell-membrane- and blood-brain-barrier-permeable calpain-1 (μ-calpain) and calpain-2 (m-calpain) inhibitor MDL-28170, we tested the involvement of calpains, a family of calcium-dependent cysteine proteases, and the potential of MDL-28170 in preventing NIHL. CBA/J mice at the age of 12 weeks were exposed to broadband noise with a frequency spectrum from 2-20 kHz for 2 h at 101 dB sound pressure level to induce permanent hearing loss as measured by auditory brainstem response and distortion product otoacoustic emissions. Morphological damage was assessed by quantification of remaining sensory hair cells and inner hair cell synapses 2 weeks after the exposure. MDL-28170 treatment by intraperitoneal injection significantly attenuated noise-induced functional deficits and cochlear pathologies. MDL-28170 treatment also prevented noise-induced cleavage of alpha-fodrin, a substrate for calpain-1. Furthermore, MDL-28170 treatment prevented reduction of PI3K/Akt signaling after exposure to noise and upregulated p85α and p-Akt (S473) in outer hair cells. These results indicate that noise-induced calpain activation negatively regulates PI3K/Akt downstream signaling, and that prevention of NIHL by treatment with MDL-28170 is associated with upregulation of PI3K/Akt survival signaling pathways.

MAP Kinase FgHog1 and Importin β FgNmd5 Regulate Calcium Homeostasis in Fusarium graminearum.

Maintaining cellular calcium (Ca2+) homeostasis is essential for many aspects of cellular life. The high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway responsible for signal integration and transduction plays crucial roles in environmental adaptation, especially in the response to osmotic stress. Hog1 is activated by transient Ca2+ increase in yeast, but the functions of the HOG pathway in Ca2+ homeostasis are largely unknown. We found that the HOG pathway was involved in the regulation of Ca2+ homeostasis in Fusarium graminearum, a devastating fungal pathogen of cereal crops. The deletion mutants of HOG pathway displayed increased sensitivity to Ca2+ and FK506, and elevated intracellular Ca2+ content. Ca2+ treatment induced the phosphorylation of FgHog1, and the phosphorylated FgHog1 was transported into the nucleus by importin β FgNmd5. Moreover, the increased phosphorylation and nuclear accumulation of FgHog1 upon Ca2+ treatment is independent of the calcineurin pathway that is conserved and downstream of the Ca2+ signal. Taken together, this study reported the novel function of FgHog1 in the regulation of Ca2+ homeostasis in F. graminearum, which advance the understanding of the HOG pathway and the association between the HOG and calcineurin pathways in fungi.

Cardiac contractility modulation: an effective treatment strategy for heart failure beyond reduced left ventricular ejection fraction?

Heart failure (HF) with preserved ejection fraction (HFpEF) causes a progressive limitation of functional capacity, poor quality of life (QoL) and increased mortality, yet unlike HF with reduced ejection fraction (HFrEF) there are no effective device-based therapies. Both HFrEF and HFpEF are associated with dysregulations in myocardial cellular calcium homeostasis and modifications in calcium-handling proteins, leading to abnormal myocardial contractility and pathological remodelling. Cardiac contractility modulation (CCM) therapy, based on a pacemaker-like implanted device, applies extracellular electrical stimulation to myocytes during the absolute refractory period of the action potential, which leads to an increase in cytosolic peak calcium concentrations and thereby the force of isometric contraction promoting positive inotropism. Subgroup analysis of CCM trials in HFrEF has demonstrated particular benefits in patients with LVEF between 35% and 45%, suggesting its potential effectiveness also in patients with higher LVEF values. Available evidence on CCM in HFpEF is still preliminary, but improvements in terms of symptoms and QoL have been observed. Future large, dedicated, prospective studies are needed to evaluate the safety and efficacy of this therapy in patients with HFpEF.

Tumor Suppressor Candidate 2 (TUSC2): Discovery, Functions, and Cancer Therapy.

Tumor Suppressor Candidate 2 (TUSC2) was first discovered as a potential tumor suppressor gene residing in the frequently deleted 3p21.3 chromosomal region. Since its discovery, TUSC2 has been found to play vital roles in normal immune function, and TUSC2 loss is associated with the development of autoimmune diseases as well as impaired responses within the innate immune system. TUSC2 also plays a vital role in regulating normal cellular mitochondrial calcium movement and homeostasis. Moreover, TUSC2 serves as an important factor in premature aging. In addition to TUSC2's normal cellular functions, TUSC2 has been studied as a tumor suppressor gene that is frequently deleted or lost in a multitude of cancers, including glioma, sarcoma, and cancers of the lung, breast, ovaries, and thyroid. TUSC2 is frequently lost in cancer due to somatic deletion within the 3p21.3 region, transcriptional inactivation via TUSC2 promoter methylation, post-transcriptional regulation via microRNAs, and post-translational regulation via polyubiquitination and proteasomal degradation. Additionally, restoration of TUSC2 expression promotes tumor suppression, eventuating in decreased cell proliferation, stemness, and tumor growth, as well as increased apoptosis. Consequently, TUSC2 gene therapy has been tested in patients with non-small cell lung cancer. This review will focus on the current understanding of TUSC2 functions in both normal and cancerous tissues, mechanisms of TUSC2 loss, TUSC2 cancer therapeutics, open questions, and future directions.

Effect of Long Non-coding RNA and DNA Methylation on Gene Expression in Dental Fluorosis.

In the process of tooth development, the interaction between genetic information, epigenetic inheritance, and environment jointly affects the teeth formation. At present, the mechanism of dental fluorosis is rarely studied from transcriptomics, and there is no report on epigenetic perspective. In the study, SD rats were randomly divided into dental fluorosis group and control group fed with NaF (150mg/L) or distilled water for 8weeks. After 3.5days of birth, the RNAs or DNA of rat mandibular molars were detected by RNA-seq or MethylTarget, respectively. The results demonstrated that a total of 1723 differentially expressed genes (DEGs) and 2511 differential expression lncRNAs (DE-lncRNAs) were mainly involved in the ion channels, calcium ion transport, and immunomodulatory signaling pathways. ATP2C1 and Nr1d1, which were related to Ca2+ transport, cellular calcium homeostasis, endoplasmic reticulum stress and immunity, may be the key genes in the formation of dental fluorosis. Notably, we also found that the immune response plays an important role in the formation of dental fluorosis, and a large amount of DEGs was enriched in immune regulation and NF-κB signaling pathways. Furthermore, the methylation levels of 13 sites were increased in Ago4, Atf3, Atp2c1, Dusp1, Habp4, and Mycl, while methylation levels of 5 CpG sites decreased in Ago4, Atp2c1, Habp4, and Traf6, and conformably, the expression of these genes have been significantly changed. This study comprehensively analyzed the occurrence mechanism of dental fluorosis from transcriptomics and epigenetics, so as to provide theoretical reference for further research.

Restoring cellular calcium homeostasis to rescue ER stress by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester-loaded lipid-mPLGA hybrid-nanoparticles for acute kidney injury therapy

Early pathogenesis of ischemia-reperfusion (I/R)-induced acute kidney injury (AKI) is dominated by intracellular calcium overload, which induces oxidative stress, intracellular energy metabolism disorder, inflammatory activation, and a series of pathologic cascaded reactions that are closely intertwined with self-amplifying and interactive feedback loops, ultimately resulting in cell damage and kidney failure. Currently, most nanomedicines originate from the perspective of antioxidant stress, which can only quench existing reactive oxide species (ROS) but cannot prevent the continuous production of ROS, resulting in insufficient efficacy. As a safe and promising drug, BAPTA-AM is hydrolyzed into BAPTA by intracellular esterase upon entering cells, which can rapidly chelate with overloaded Ca2+, restoring intracellular calcium homeostasis, thus inhibiting ROS regeneration at the source. Here, we designed a KTP-targeting peptide-modified yolk-shell structure of liposome–poly(ethylene glycol)methyl ether-block-poly (l-lactide-co-glycolic) (mPLGA) hybrid nanoparticles (<100 nm), with the characteristics of high encapsulation rate, high colloid stability, facile modification, and prolonged blood circulation time. Once the BA/mPLGA@Lipo-KTP was targeted to the site of kidney injury, the cholesteryl hemisuccinate (CHEMS) in the phospholipid bilayer, as an acidic cholesterol ester, was protonated in the simulated inflammatory slightly acidic environment (pH 6.5), causing the liposomes to rupture and release the BA/mPLGA nanoparticles, which were then depolymerized by intracellular esterase. The BAPTA-AM was diffused and hydrolyzed to produce BAPTA, which can rapidly cut off the malignant loop of calcium overload/ROS generation at its source, blocking the endoplasmic reticulum (ER) apoptosis pathway (ATF4–CHOP–Bax/Bcl-2, Casp-12–Casp-3) and the inflammatory pathway (TNF-α–NF-κB–IL-6 axes), thus alleviating pathological changes in kidney tissue, thereby inhibiting the expression of renal tubular marker kidney injury molecule 1 (Kim-1) (reduced by 82.9%) and also exhibiting prominent anti-apoptotic capability (TUNEL-positive ratio decreased from 40.2% to 8.3%), significantly restoring renal function. Overall, this research holds huge potential in the treatment of I/R injury-related diseases.

Cryo-EM structures of human SPCA1a reveal the mechanism of Ca2+/Mn2+ transport into the Golgi apparatus.

Secretory pathway Ca2+/Mn2+ ATPase 1 (SPCA1) actively transports cytosolic Ca2+ and Mn2+ into the Golgi lumen, playing a crucial role in cellular calcium and manganese homeostasis. Detrimental mutations of the ATP2C1 gene encoding SPCA1 cause Hailey-Hailey disease. Here, using nanobody/megabody technologies, we determined cryo-electron microscopy structures of human SPCA1a in the ATP and Ca2+/Mn2+-bound (E1-ATP) state and the metal-free phosphorylated (E2P) state at 3.1- to 3.3-Å resolutions. The structures revealed that Ca2+ and Mn2+ share the same metal ion-binding pocket with similar but notably different coordination geometries in the transmembrane domain, corresponding to the second Ca2+-binding site in sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). In the E1-ATP to E2P transition, SPCA1a undergoes similar domain rearrangements to those of SERCA. Meanwhile, SPCA1a shows larger conformational and positional flexibility of the second and sixth transmembrane helices, possibly explaining its wider metal ion specificity. These structural findings illuminate the unique mechanisms of SPCA1a-mediated Ca2+/Mn2+ transport.

Mitochondrial microprotein PIGBOS1 is possibly associated with cellular calcium homeostasis and neuro-protection

Background: Calcium ion (Ca2+) plays a vital role in many physiological processes like memory formation, cell-cell communication,secretion, neurotransmission, cell survival, and death. The concerted activities of several transporters and ion channels maintain anoptimum concentration of Ca2+ inside the cell. Disruption of calcium homeostasis may cause abnormal protein aggregation, leading toseveral neurodegenerative diseases like Alzheimer’s disease and Parkinson’s disease (PD). Objective: Several pieces of evidence indicatethat altered calcium homeostasis, oxidative stress, and endoplasmic reticulum (ER) stress are intimately related to the development of PD.PIGBOS1, a recently discovered mitochondrial microprotein, has been shown to play an essential role in ER stress response through itsinteraction with an ER-resident chloride channel CLCC1. Materials and Methods: In the present study, we investigated the role of PIGBOS1in cellular calcium homeostasis by monitoring Ca2+ dynamics in the ER, cytoplasm, and mitochondria using genetically encoded Ca2+indicators. Results: In the resting state, PIGBOS1-overexpressed HEK-293T cells exhibited a higher level of basal Ca2+ in the ER. Further,cytosolic and mitochondrial Ca2+ rise following ionomycin/ histamine treatment was higher in PIGBOS1-overexpressed cells. Conclusion:Taken together, it implies that PIGBOS1 is an integral part of the cellular Ca2+ signaling network. We have discussed the possible role ofPIGBOS1 in neurodegenerative diseases like PD.

Non-interfacial self-assembly of synthetic protocells

BackgroundProtocell refers to the basic unit of life and synthetic molecular assembly with cell structure and function. The protocells have great applications in the field of biomedical technology. Simulating the morphology and function of cells is the key to the preparation of protocells. However, some organic solvents used in the preparation process of protocells would damage the function of the bioactive substance. Perfluorocarbon, which has no toxic effect on bioactive substances, is an ideal solvent for protocell preparation. However, perfluorocarbon cannot be emulsified with water because of its inertia.MethodsSpheroids can be formed in nature even without emulsification, since liquid can reshape the morphology of the solid phase through the scouring action, even if there is no stable interface between the two phases. Inspired by the formation of natural spheroids such as pebbles, we developed non-interfacial self-assembly (NISA) of microdroplets as a step toward synthetic protocells, in which the inert perfluorocarbon was utilized to reshape the hydrogel through the scouring action.ResultsThe synthetic protocells were successfully obtained by using NISA-based protocell techniques, with the morphology very similar to native cells. Then we simulated the cell transcription process in the synthetic protocell and used the protocell as an mRNA carrier to transfect 293T cells. The results showed that protocells delivered mRNAs, and successfully expressed proteins in 293T cells. Further, we used the NISA method to fabricate an artificial cell by extracting and reassembling the membrane, proteins, and genomes of ovarian cancer cells. The results showed that the recombination of tumor cells was successfully achieved with similar morphology as tumor cells. In addition, the synthetic protocell prepared by the NISA method was used to reverse cancer chemoresistance by restoring cellular calcium homeostasis, which verified the application value of the synthetic protocell as a drug carrier.ConclusionThis synthetic protocell fabricated by the NISA method simulates the occurrence and development process of primitive life, which has great potential application value in mRNA vaccine, cancer immunotherapy, and drug delivery.Graphical

Interaction of quercetin and its derivatives with Ca2+-ATPase from sarcoplasmic reticulum: Kinetic and molecular modeling studies.

Sarcoplasmic reticulum Ca2+-ATPases (SERCAs) regulate cellular calcium homeostasis and are targeted for age-related diseases. Among 14 SERCA mRNA splice variants, SERCA1a is specific to adult fast-twitch skeletal muscle. Quercetin derivatives (monochloropivaloylquercetin (CPQ), IC50 = 195.7 µM; 2-chloro-1,4-naphthoquinonequercetin (CHNQ), IC50 = 60.3 µM) were studied for their impact on SERCA1a using molecular modeling and enzyme kinetics. While there were some similarities in kinetic parameters and molecular modeling, the compounds exhibited diverse actions on SERCA1a. Quercetin reduced activity by 48% at 250 μM by binding to the cytosolic ATP-binding pocket with increased ATP affinity. CPQ bound near the Ca2+-binding site, possibly altering the transmembrane domain. CHNQ significantly reduced activity by 94% at 250 μM without binding to substrate sites. It was proposed that CHNQ induced global protein structure changes, inhibiting Ca2+-ATPase activity.

Metabolomic Analysis of Wooden Breast Myopathy Shows a Disturbed Lipid Metabolism.

Myopathies have risen strongly in recent years, likely linked to selection for appetite. For white striping (WS), causes have been identified; but for wooden breast (WB), the cause remains speculative. We used metabolomics to study the breast muscle of 51 birds that were scored for both at 35 days of age to better understand potential causes. A partial least square discriminant analysis revealed that WS and WB had distinct metabolic profiles, implying different etiologies. Arginine and proline metabolism were affected in both, although differently: WB increased arginine in breast muscle implying that the birds did not use this pathway to increase tissue blood flow. Antioxidant defenses were impeded as shown by low anserine and beta-alanine. In contrast, GSH and selenium concentrations were increased. Serine, linked to anti-inflammatory properties, was increased. Taurine, which can stabilize the cell's sarcolemma as well as modulate potassium channels and cellular calcium homeostasis, was also increased. Mineral data and depressed phosphatidylethanolamine, cAMP, and creatine-phosphate suggested compromised energy metabolism. WB also had drastically lower diet-derived lipids, suggesting compromised lipid digestion. In conclusion, WB may be caused by impaired lipid digestion triggered by a very high appetite: the ensuing deficiencies may well impair blood flow into muscle resulting in irreparable damage.

Disturbance of cellular calcium homeostasis plays a pivotal role in glyphosate-based herbicide-induced oxidative stress.

Glyphosate-based herbicides (GBHs) are the most worldwide used pesticides. The wide application of GBHs contaminates the soil and, consequently, water and food resources reaching human consumption. GBHs induce oxidative stress in non-target organisms, leading to a pro-inflammatory and pro-apoptotic cellular status, promoting tissue dysfunction and, thus, metabolic and neurobehavioral changes. This review presents evidence of oxidative damage induced by GBHs and the mechanism of cell damage and health consequences. To summarize, exposure to GBHs may induce disorders in calcium homeostasis related to the activation of ion channels. Also, alterations in pathways related to redox state regulation must have a primordial role in oxidative stress caused by GBHs.

Anti-malarial drugs: Mechanisms underlying their proarrhythmic effects.

Malaria remains the leading cause of parasitic death in the world. Artemisinin resistance is an emerging threat indicating an imminent need for novel combination therapy. Given the key role of mass drug administration, it is pivotal that the safety of anti-malarial drugs is investigated thoroughly prior to widespread use. Cardiotoxicity, most prominently arrhythmic risk, has been a concern for anti-malarial drugs. We clarify the likely underlying mechanisms by which anti-malarial drugs predispose to arrhythmias. These relate to disruption of (1) action potential upstroke due to effects on the sodium currents, (2) action potential repolarisation due to effects on the potassium currents, (3) cellular calcium homeostasis, (4) mitochondrial function and reactive oxygen species production and (5) cardiac fibrosis. Together, these alterations promote arrhythmic triggers and substrates. Understanding these mechanisms is essential to assess the safety of these drugs, stratify patients based on arrhythmic risk and guide future anti-malarial drug development.

The endoplasmic reticulum membrane protein Sec62 as potential therapeutic target in SEC62 overexpressing tumors.

The human SEC62 gene is located on chromosome 3q, was characterized as a tumor driver gene and is found to be overexpressed in an ever-growing number of tumors, particularly those with 3q26 amplification. Where analyzed, SEC62 overexpression was associated with poor prognosis. Sec62 protein is a membrane protein of the endoplasmic reticulum (ER) and has functions in endoplasmic reticulum protein import, endoplasmic reticulum-phagy and -in cooperation with the cytosolic protein calmodulin- the maintenance of cellular calcium homeostasis. Various human tumors show SEC62 overexpression in immunohistochemistry and corresponding cell lines confirm this phenomenon in western blots and immunofluorescence. Furthermore, these tumor cells are characterized by increased stress tolerance and migratory as well as invasive potential, three hallmarks of cancer cells. Strikingly, plasmid-driven overexpression of SEC62 in non-SEC62 overexpressing cells introduces the same three hallmarks of cancer into the transfected cells. Depletion of Sec62 from either type of SEC62 overexpressing tumor cells by treatment with SEC62-targeting siRNAs leads to reduced stress tolerance and reduced migratory as well as invasive potential. Where tested, treatment of SEC62 overexpressing tumor cells with the small molecule/calmodulin antagonist trifluoperazine (TFP) phenocopied the effect of SEC62-targeting siRNAs. Recently, first phase II clinical trials with the prodrug mipsagargin/G202, which targets cellular calcium homeostasis in prostate cells as well as neovascular tissue in various tumors were started. According to experiments with tumor cell lines, however, SEC62 overexpressing tumor cells may be less responsive or resistant against such treatment. Therefore, murine tumor models for tumor growth or metastasis were evaluated with respect to their responsiveness to treatment with a mipsagargin analog (thapsigargin), or trifluoperazine, which had previously been in clinical use for the treatment of schizophrenia, or with the combination of both drugs. So far, no additive effect of the two drugs was observed but trifluoperazine had an inhibitory effect on tumor growth and metastatic potential in the models. Here, we review the state of affairs.

Radiation-induced electrophysiological remodelling in neonatal rat ventricular cardiomyocytes

Abstract Background Ventricular tachycardia (VT) carries a relatively high risk of recurrence and significantly impacts patients' quality of life, despite state of the art treatment including antiarrhythmic drug therapy and catheter ablation. Cardiac stereotactic body radiotherapy (SBRT) has emerged as a promising non-invasive treatment option for refractory VT. A marked reduction in VT episodes is observed within days of treating high-risk patients with a single fraction SBRT. The cellular mechanisms underlying the clinically demonstrated antiarrhythmic effect are still poorly understood. Radiation-induced fibrosis is observed several months after irradiation, suggesting other mechanisms that explain the immediate benefit of radiotherapy on the VT substrate. Purpose We hypothesized that early antiarrhythmic effects of single-fraction radiation are produced by direct molecular and functional electrophysiological changes, independent of fibrosis. Our aim was to investigate the effects of 20 Gy ionizing photon radiation on the electrophysiology of ventricular neonatal rat cardiomyocytes at time periods from 24 hours to 96 hours after irradiation. Methods Ventricular neonatal rat cardiomyocytes were isolated, cultivated for 24 to 96 hours and then exposed to photon beam with a single dose of 20 Gy. Expression changes of ion channels, calcium handling proteins and structural proteins affecting cellular electrophysiology were assessed via RT-qPCR and Western blotting. Beat frequency was evaluated by video analysis and action potentials (APD) were recorded using the whole-cell voltage clamp technique in current-clamp mode. Intracellular calcium transients in Fura-2 AM loaded cardiomyocytes were recorded with an IonOptix recording system. Results Irradiation lead to significant changes in the mRNA and protein level of ion channels, with most distinct changes being observed 96h after radiation. Increased mRNA and protein expression of (Cav1.2) and of the potassium channels Kcnd3 (Kv4.3), Kcnh2 (Kv11.1), Kcnq1 (Kv7.1), Kcnk2 (K2P2.1) and Kcnj2 (Kir2.1) was observed. Additionally, Connexin 43 (Cx43) was upregulated. Irradiated cardiomyocytes showed a significant increase in beat rate over time. APD was significantly shortened, with a significantly increased upstroke velocity. Regarding calcium handling, significant upregulations of Ryr2 (RYR2) and Slc8a1 (NCX) transcript and protein level was detected. Additionally, calcium handling properties were altered. Conclusion Our results suggest that acute irradiation effects are associated with electrophysiological remodelling in cardiomyocytes. Changes of cellular ion channel expression and calcium homeostasis lead to normalization and shortening of heart-failure associated APD prolongation. These results reveal new insights into acute antiarrhythmic effects on cardiomyocytes after SBRT-therapy. Funding Acknowledgement Type of funding sources: None.

Lysosomal positioning diseases: beyond substrate storage.

Lysosomal storage diseases (LSDs) comprise a group of inherited monogenic disorders characterized by lysosomal dysfunctions due to undegraded substrate accumulation. They are caused by a deficiency in specific lysosomal hydrolases involved in cellular catabolism, or non-enzymatic proteins essential for normal lysosomal functions. In LSDs, the lack of degradation of the accumulated substrate and its lysosomal storage impairs lysosome functions resulting in the perturbation of cellular homeostasis and, in turn, the damage of multiple organ systems. A substantial number of studies on the pathogenesis of LSDs has highlighted how the accumulation of lysosomal substrates is only the first event of a cascade of processes including the accumulation of secondary metabolites and the impairment of cellular trafficking, cell signalling, autophagic flux, mitochondria functionality and calcium homeostasis, that significantly contribute to the onset and progression of these diseases. Emerging studies on lysosomal biology have described the fundamental roles of these organelles in a variety of physiological functions and pathological conditions beyond their canonical activity in cellular waste clearance. Here, we discuss recent advances in the knowledge of cellular and molecular mechanisms linking lysosomal positioning and trafficking to LSDs.

Androgen receptor signaling-mitochondrial DNA-oxidative phosphorylation: A critical triangle in early prostate cancer.

Mitochondria are more than just the cellular powerhouse. They also play key roles in vital functions such as apoptosis, metabolism regulation, and other intracellular interactions. The mitochondrial DNA (mtDNA) encodes for 12 subunits of the oxidative phosphorylation (OXPHOS) system. Depletion of mtDNA in androgen-dependent prostate cancer (PCa) cell lines renders them androgen-independent and more aggressive. Paradoxically, pharmaceutical inhibition of OXPHOS is lethal for subsets of PCa cells, whereas others become dependent on androgen receptor (AR) signaling for survival. Given that the AR-mitochondria interaction is critical for early PCa, it is crucial to understand the details of this interaction. Technical hurdles have made mitochondria traditionally difficult to study, with many techniques used for isolation masking the properties of given individual mitochondria. Although the isolation of mitochondria enables us to study OXPHOS, we miss the context in which mitochondria interact with the rest of the cell. Both AR signaling and mtDNA affect apoptosis, metabolism regulation, cellular calcium storage and homeostasis, intracellular calcium signaling, and redox homeostasis. In this review, we will attempt to understand how the crosstalk between AR-mtDNA-OXPHOS is responsible for "life or death" decisions inside the cells. Our aim is to point toward potential vulnerabilities that can lead to the discovery of novel therapeutic targets.

Mechanisms underlying the role of ankyrin-B in cardiac and neurological health and disease.

The ANK2 gene encodes for ankyrin-B (ANKB), one of 3 members of the ankyrin family of proteins, whose name is derived from the Greek word for anchor. ANKB was originally identified in the brain (B denotes “brain”) but has become most widely known for its role in cardiomyocytes as a scaffolding protein for ion channels and transporters, as well as an interacting protein for structural and signaling proteins. Certain loss-of-function ANK2 variants are associated with a primarily cardiac-presenting autosomal-dominant condition with incomplete penetrance and variable expressivity characterized by a predisposition to supraventricular and ventricular arrhythmias, arrhythmogenic cardiomyopathy, congenital and adult-onset structural heart disease, and sudden death. Another independent group of ANK2 variants are associated with increased risk for distinct neurological phenotypes, including epilepsy and autism spectrum disorders. The mechanisms underlying ANKB's roles in cells in health and disease are not fully understood; however, several clues from a range of molecular and cell biological studies have emerged. Notably, ANKB exhibits several isoforms that have different cell-type–, tissue–, and developmental stage– expression profiles. Given the conservation within ankyrins across evolution, model organism studies have enabled the discovery of several ankyrin roles that could shed important light on ANKB protein-protein interactions in heart and brain cells related to the regulation of cellular polarity, organization, calcium homeostasis, and glucose and fat metabolism. Along with this accumulation of evidence suggesting a diversity of important ANKB cellular functions, there is an on-going debate on the role of ANKB in disease. We currently have limited understanding of how these cellular functions link to disease risk. To this end, this review will examine evidence for the cellular roles of ANKB and the potential contribution of ANKB functional variants to disease risk and presentation. This contribution will highlight the impact of ANKB dysfunction on cardiac and neuronal cells and the significance of understanding the role of ANKB variants in disease.