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Stromal interaction molecule 1 (STIM1)-mediated store-operated Ca2+ entry (SOCE) is now recognized as the main mechanism of the majority of nonexcitable cell calcium influx. Calcium overload is a primary mechanism of endothelial cell injury during systemic inflammatory response and sepsis. Whether STIM1-mediated SOCE plays a role in calcium overload in vascular endothelial cell injury remains unclear. To explore the role of STIM1-gated SOCE in vascular endothelial cell calcium overload and inflammation, we established a human septic serum or lipopolysaccharide (LPS)-induced human umbilical vein endothelial cell (HUVEC) experimental system and derived ribonucleic acid interference (RNAi)-mediated STIM1, ORAI1 (orai gene [HGNC: 25896 Entrez Gene: 84876] coding protein, ORAI Calcium Release-Activated Calcium Modulator 1), and transient receptor potential channel 1 (TRPC1) (core components of store-operated Ca2+[SOC]) downregulated HUVECs, as well as STIM1 overinduced HUVECs. Our results show that sepsis serum or LPS stimulation increased STIM1 in HUVECs and increased all cytokines except for VEGF and the inflammatory mediators tumor necrosis factor, intercellular cell adhesion molecule-1, and endothelin-1 in a time-dependent manner. RNAi-mediated knockdown of STIM1 significantly inhibited serum or LPS-induced inflammatory cytokine expression, and STIM1 overexpression in HUVECs promoted LPS-mediated induction of these cytokines. Meanwhile, similar to the blocking effect of the specific SOC inhibitors Gd3+ and La3+ on LPS-induced calcium influx, RNAi-mediated depletion of STIM1 or the SOC proteins TRPC1 and ORAI1 could significantly inhibit serum or LPS-induced extracellular calcium influx, as well as the expression of the inflammatory cytokines tumor necrosis factor, intercellular cell adhesion molecule-1, and endothelin-1. Simultaneous downregulation of the SOCE core units TRPC1 and ORAI1 inhibited LPS-induced calcium influx and cytokine expression, which could not be restored by inducing STIM1. Forced expression of nuclear factor-κB (NF-κB) in HUVECs significantly induced STIM1 expression, whereas RNAi-mediated depletion of NF-κB significantly inhibited STIM1 mRNA levels and significantly reduced the thapsigargin-mediated SOCE calcium influx, which was similar to results with the NF-κB inhibitor wogonin. Septic serum stimulates the expression of STIM1, cytokines, and inflammatory mediators in HUVECs. STIM1-mediated SOCE is required for Ca2+ influx induced by LPS or septic serum and contributes cytokines and inflammatory mediators in septic serum–stimulated HUVECs. In addition, STIM1-mediated SOCE on Ca2+ influx by septic serum or LPS involves NF-κB signaling. [ABSTRACT FROM AUTHOR]

Background: Sepsis is a systemic inflammatory response to a local severe infection that may lead to multiple organ failure and death. Previous studies have shown that 40–50% of patients with sepsis have diverse myocardial injuries and 70 to 90% mortality rates compared to 20% mortality in patients with sepsis without myocardial injury. Therefore, uncovering the mechanism of sepsis-induced myocardial injury and finding a target-based treatment are immensely important. Objective: The present study elucidated the mechanism of sepsis-induced myocardial injury and examined the value of human umbilical cord mesenchymal stem cells (huMSCs) for protecting cardiac function in sepsis. Methods: We used cecal ligation and puncture (CLP) to induce sepsis in mice and detect myocardial injury and cardiac function using serological markers and echocardiography. Cardiomyocyte apoptosis and heart tissue ultrastructure were detected using TdT-mediated dUTP Nick-End Labeling (TUNEL) and transmission electron microscopy (TEM), respectively. Fura-2 AM was used to monitor Ca2+ uptake and efflux in mitochondria. FQ-PCR and Western blotting detected expression of mitochondrial Ca2+ distribution regulators and PTEN-induced putative kinase 1 (PINK1). JC-1 was used to detect the mitochondrial membrane potential (Δψm) of cardiomyocytes. Results: We found that expression of PINK1 decreased in mouse hearts during sepsis, which caused cardiomyocyte mitochondrial Ca2+ efflux disorder, mitochondrial calcium overload, and cardiomyocyte injury. In contrast, we found that exosomes isolated from huMSCs (huMSC-exo) carried Pink1 mRNA, which could be transferred to recipient cardiomyocytes to increase PINK1 expression. The reduction in cardiomyocyte mitochondrial calcium efflux was reversed, and cardiomyocytes recovered from injury. We confirmed the effect of the PINK1-PKA-NCLX axis on mitochondrial calcium homeostasis in cardiomyocytes during sepsis. Conclusion: The PINK1-PKA-NCLX axis plays an important role in mitochondrial calcium efflux in cardiomyocytes. Therefore, PINK1 may be a therapeutic target to protect cardiomyocyte mitochondria, and the application of huMSC-exo is a promising strategy against sepsis-induced heart dysfunction. [ABSTRACT FROM AUTHOR]

Chemotherapy remains one of the most widely used cancer treatment modalities in clinical practice. However, the characteristic microenvironment of solid tumors severely limits the anticancer efficacy of chemotherapy. In addition, a single treatment modality or one death pathway reduces the antitumor outcome. Herein, tumor-targeting O 2 self-supplied nanomodules (CuS@DOX/CaO 2 -HA) are proposed that not only alleviate tumor microenvironmental hypoxia to promote the accumulation of chemotherapeutic drugs in tumors but also exert photothermal effects to boost drug release, penetration and combination therapy. CuS@DOX/CaO 2 -HA consists of copper sulfide (CuS)-loaded calcium peroxide (CaO 2) and doxorubicin (DOX), and its surface is further modified with HA. CuS@DOX/CaO 2 -HA underwent photothermal treatment to release DOX and CaO 2. Hyperthermia accelerates drug penetration to enhance chemotherapeutic efficacy. The exposed CaO 2 reacts with water to produce Ca2+, H 2 O 2 and O 2 , which sensitizes cells to chemotherapy through mitochondrial damage caused by calcium overload and a reduction in drug efflux via the alleviation of hypoxia. Moreover, under near infrared (NIR) irradiation, CuS@DOX/CaO 2 -HA initiates a pyroptosis-like cell death process in addition to apoptosis. In vivo, CuS@DOX/CaO 2 -HA demonstrated high-performance antitumor effects. This study provides a new strategy for synergistic enhancement of chemotherapy in hypoxic tumor therapy via combination therapy and multiple death pathways. [ABSTRACT FROM AUTHOR]

[Display omitted] • DOX&ICG@H-ZIF-8@CaCO 3 -PEG NPs very high drug loading due to hollow structure. • π-π stacking effect of ICG and DOX increases drug stability. • The system has shown synergistic effects of pH sensitivity, photothermal, and calcium overload synergistic effects in vitro. In this study, we synthesized DOX-loaded H-ZIF-8@CaCO 3 -PEG nanocarrier, which combined with calcium overload and photothermal therapy (PTT) for synergistic therapy. The nanocarrier comprised a PEG modified calcium carbonate (CaCO 3) shell and a hollow imidazolate zeolite skeleton (H-ZIF-8) core, in which the chemotherapy drug doxorubicin hydrochloride (DOX) and photosensitizer indocyanine green (ICG) were simultaneously co-loaded. In vitro cellular experiments revealed that the lethality of DOX&ICG@H-ZIF-8@CaCO 3 -PEG against tumor cells was significantly higher than that of the single chemotherapy owing to the combination with chemotherapy and photothermal therapy as well as calcium overloading. This drug-loaded nanocarrier exhibits notable characteristics such as exceptional stability, elevated drug loading capacity and efficient release of the loaded drug, which provides a promising perspective for efficient synergistic therapy. [ABSTRACT FROM AUTHOR]

Mitochondrial calcium (Ca2+) uptake augments metabolic processes and buffers cytosolic Ca2+ levels; however, excessive mitochondrial Ca2+ can cause cell death. Disrupted mitochondrial function and Ca2+ homeostasis are linked to numerous neurodegenerative diseases (NDs), but the impact of mitochondrial Ca2+ disruption is not well understood. Here, we show that Drosophila models of multiple NDs (Parkinson's, Huntington's, Alzheimer's, and frontotemporal dementia) reveal a consistent increase in neuronal mitochondrial Ca2+ levels, as well as reduced mitochondrial Ca2+ buffering capacity, associated with increased mitochondria-endoplasmic reticulum contact sites (MERCs). Importantly, loss of the mitochondrial Ca2+ uptake channel MCU or overexpression of the efflux channel NCLX robustly suppresses key pathological phenotypes across these ND models. Thus, mitochondrial Ca2+ imbalance is a common feature of diverse NDs in vivo and is an important contributor to the disease pathogenesis. The broad beneficial effects from partial loss of MCU across these models presents a common, druggable target for therapeutic intervention. [Display omitted] • Drosophila models of multiple neurodegenerative diseases have increased neuronal MERCs • Disease model mitochondria have elevated basal Ca2+ and reduced Ca2+ buffering capacity • Reducing mCa2+ uptake via MCU loss rescues phenotypes across a broad range of disease models • Increasing mCa2+ efflux via NCLX overexpression is also beneficial across disease models Twyning et al. show using a single in vivo platform that reducing mitochondrial calcium uptake via MCU or increasing efflux via NCLX is beneficial across a variety of different disease models. These findings underscore the pathogenic role of mitochondrial calcium in neurodegeneration and highlight its potential as a therapeutic target. [ABSTRACT FROM AUTHOR]

Abstract Background Chondrocyte apoptosis is a central feature in the progression of osteoarthritis (OA), and would be triggered by sustained elevation of intracellular calcium ion (Ca2+), also known as a cellular second messenger. Transient receptor potential ankyrin 1 (TRPA1) is a membrane-associated cation channel, and the activation of which causes an influx of cation ions, in particularly Ca2+, into the activated cells. Therefore, we investigate the potential role of TRPA1 in mediating Ca2+ influx to promote chondrocyte apoptosis in OA. Methods The expression of TRPA1 in interleukin (IL)-1β-treated rat chondrocytes was assessed by Polymerase chain reaction (PCR) and Western blot (WB), and the functionality of TRPA1 channel by Ca2+ influx measurements. Meanwhile, the chondrocyte apoptosis in IL-1β-treated cells was measured by TUNEL assay and flow cytometry. The measurement of mitochondrial membrane potential and apoptosis-associated proteins after inhibition of TRPA1 were also performed in IL-1β-treated rat chondrocytes. Results After being induced by IL-1β, the gene and protein expression of TRPA1 was increased in the dose-dependent manner. Meanwhile, Ca2+ influx mediated by TRPA1 in rat chondrocytes was also enhanced. Pharmacological inhibition of TRPA1 downregulated the apoptotic rate in IL-1β-treated rat chondrocytes. In addition, the membrane potential depolarization was improved and significantly increased expression of apoptosis-associated proteins also reduced by the TRPA1 antagonist. Conclusions We found the IL-1β caused the increased functional expression of TRPA1, the activation of which involved IL-1β-induced apoptosis in rat chondrocytes. The potential mechanism may be linked to the intracellular calcium overload mediated by TRPA1 and attendant mitochondrial dysfunction.

Background/Aims: Myocardial ischemia/reperfusion (I/R) or hypoxia/reoxygenation (H/R) injury is always characterized by Ca2+ overload, energy metabolism disorder and necrocytosis of cardiomyocytes. We showed previously that chronic intermittent hypobaric hypoxia (CIHH) improves cardiac function during I/R through improving cardiac glucose metabolism. However, the underlying cellular and molecular mechanisms of CIHH treatment improving energy metabolism in cardiomyocytes are still unclear. In this study, we determined whether and how CIHH protects cardiomyocytes from Ca2+ overload and necrocytosis through energy regulating pathway. Methods: Adult male Sprague-Dawley rats were randomly divided into two groups: control (CON) and CIHH group. CIHH rats received a hypobaric hypoxia simulating 5,000-m altitude for 28 days, 6 hours each day, in hypobaric chamber. Rat ventricular myocytes were obtained by enzymatic dissociation. The intracellular calcium concentration ([Ca2+]i) and cTnI protein expression were used to evaluate the degree of cardiomyocytes injury during and after H/R. The mRNA and protein expressions involved in cardiac energy metabolism were determined using quantitative PCR and Western blot techniques. PGC-1α siRNA adenovirus transfection was used to knock down PGC-1α gene expression of cardiomyocytes to determine the effect of PGC-1α in the energy regulating pathway. Results: H/R increased [Ca2+]i and cTnI protein expression in cardiomyocytes. CIHH treatment decreased [Ca2+]i (p< 0.01) and cTnI protein expression (p< 0.01) in cardiomyocytes after H/R. Both mRNA and protein expression of PGC-1α increased after CIHH treatment, which was reversed by PGC-1α siRNA adenovirus transfection. Furthermore, CIHH treatment increased the expression of HIF-1α, AMPK and p-AMPK in cardiomyocytes, and pretreatment with AMPK inhibitor dorsomorphin abolished the enhancement of PGC-1α protein expression in cardiomyocytes by CIHH (p< 0.01). In addition, PGC-1α knock down also abolished the increased protein level of GLUT4 (p< 0.01) and decreased the protein level of CPT-1b (p< 0.05) in cardiomyocytes by CIHH treatment. Conclusion: CIHH treatment could reduce the calcium overload and H/R injury in cardiomyocytes by up-regulating the expression of PGC-1α and regulating the energy metabolism of glucose and lipid. The HIF-1α-AMPK signaling pathway might be involved in the process.

BACKGROUND: The previous research of our group has shown that DAIa2GIP, a new analogue of glucose dependent insulin stimulating polypeptide, has a protective effect on chondrocytes, but its mechanism is not clear. OBJECTIVE: To observe the effect of DAIa2GIP on interleukin-1β (IL-1β) induced apoptosis of chondrocytes, and to explore its molecular biological mechanism. METHODS: The costal chondrocytes of Sprague-Dawley rats were extracted and identified by SABC immunohistochemistry. The third generation cells were divided into six groups: (1) normal control group; (2) IL-1β induction group; (3) DAIa2GIP+IL-1β induction group; (4) DAIa2GIP+IL-1β+pro3GIP (GIPR antagonists) induction group; (5) DAIa2GIP induction group; (6) pro3GIP induction group. After 48 hours of drug treatment, mitochondrial membrane potential was tested, cell apoptosis was detected using Annexin-V FITC Kit (phosphatidylserine eversion analysis), calcium overload of cells was determined under laser confocal microscope, and cytochrome C content were measured by ELISA. The study protocol was approved by the Animal Ethics Committee of Shanxi Medical University. RESULTS AND CONCLUSION: SABC immunohistochemical staining showed that collagen II could be developed as chondrocytes; the maintenance degree of mitochondrial membrane potential was significantly higher in the IL-1β+DAIa2GIP incubation group than the IL-1β induction group, but the potential in both groups was lower than that in the normal control group. The apoptotic rate of chondrocytes in the IL-1β+DAIa2GIP incubation group was significantly lower than that in the IL-1β induction group, but the apoptotic rate in both groups was higher than that in the normal control group. The degree of calcium overload was compared with fluorescence intensity, and the result showed that the IL-1β+DAIa2GIP incubation group had a significantly higher intensity than the IL-1β induction group, and the fluorescence intensity in both groups was higher than that in the normal control group. The release of chondrocyte mitochondrial cytochrome C in the IL-1β+DAIa2GIP incubation group was significantly lower than that in the IL-1β induction group (P < 0.01), and the release amount in both groups was significantly higher than that in the normal control group (P < 0.01). The above four indicators showed no significant difference between the IL-1β induction group and the DAIa2GIP+IL-1β+pro3GIP group, but the values in both groups were lower than those in the control group. There was no significant difference among the normal control group, DAIa2GIP induction group, and pro3GIP induction group. To conclude, DALa2GIP can effectively antagonize IL-1β induced mitochondrial dysfunction of rat chondrocytes, thus antagonizing chondrocyte apoptosis. In this process, DALa2GIP can effectively reduce the degree of calcium overload and the release of cytochrome C. [ABSTRACT FROM AUTHOR]

Ions are essential to body, but sometimes can evolve into weapons to attack and destroy cells without systematic toxicity and drug resistance. Inspired by nitric oxygen as neurotransmitter in mediating Ca2+ release, NO nanodonors with high photoreactivity and stability are constructed with upconversion nanoparticles (UCNPs) coated by zeolitic nitro‐/nitrile‐imidazole framework‐82 (ZIF‐82), capable of near‐infrared light (NIR) triggered NO generation and berbamine (BER) release, to achieve cancer therapy with the stored Ca2+ in cells. The spatial confinement effect of 2‐nitroimidazole in ZIF‐82 enables NO‐releasing with tunable release kinetics. NO turns on the ryanodine receptors overexpressed in cancer cells for abrupt Ca2+ elevation; meanwhile, berbamine (BER) turns Ca2+‐excretion pumps off to inhibit calcium efflux, resulting in intracellular Ca2+ overload induced apoptosis. This work provides the first example of regulating endogenous ions for cell killing, which holds promise as an effective cancer therapeutics that is complementary to traditional chemotherapeutics. [ABSTRACT FROM AUTHOR]

Mitochondrial cristae are dynamic invaginations of the inner membrane and play a key role in its metabolic capacity to produce ATP. Structural alterations caused by either genetic abnormalities or detrimental environmental factors impede mitochondrial metabolic fluxes and lead to a decrease in their ability to meet metabolic energy requirements. While some of the key proteins associated with mitochondrial cristae are known, very little is known about how the inner membrane dynamics are involved in energy metabolism. In this study, we present a computational strategy to understand how cristae are formed using a phase-based separation approach of both the inner membrane space and matrix space, which are explicitly modeled using the Cahn–Hilliard equation. We show that cristae are formed as a consequence of minimizing an energy function associated with phase interactions which are subject to geometric boundary constraints. We then extended the model to explore how the presence of calcium phosphate granules, entities that form in calcium overload conditions, exert a devastating inner membrane remodeling response that reduces the capacity for mitochondria to produce ATP. This modeling approach can be extended to include arbitrary geometrical constraints, the spatial heterogeneity of enzymes, and electrostatic effects to mechanize the impact of ultrastructural changes on energy metabolism. [ABSTRACT FROM AUTHOR]

Cells undergoing hypoxia experience intense cytoplasmic calcium (Ca2+) overload. High concentrations of intracellular calcium ([Ca2+]i) can trigger cell death in the neural tissue, a hallmark of stroke. Neural Ca2+ homeostasis involves regulation by the Na+/Ca2+ exchanger (NCX). Previous data published by our group showed that a product of the enzymatic depolymerization of heparin by heparinase, the unsaturated trisulfated disaccharide (TD; ΔU, 2S-GlcNS, 6S), can accelerate Na+/Ca2+ exchange via NCX, in hepatocytes and aorta vascular smooth muscle cells. Thus, the objective of this work was to verify whether TD could act as a neuroprotective agent able to prevent neuronal cell death by reducing [Ca2+]i. Pretreatment of N2a cells with TD reduced [Ca2+]i rise induced by thapsigargin and increased cell viability under [Ca2+]I overload conditions and in hypoxia. Using a murine model of stroke, we observed that pretreatment with TD decreased cerebral infarct volume and cell death. However, when mice received KB-R7943, an NCX blocker, the neuroprotective effect of TD was abolished, strongly suggesting that this neuroprotection requires a functional NCX to happen. Thus, we propose TD-NCX as a new therapeutic axis for the prevention of neuronal death induced by [Ca2+]i overload. [ABSTRACT FROM AUTHOR]

Hexavalent chromium [Cr(VI)] is a pervasive environmental pollutant that can enter the body through a variety of routes and cause organ toxicity, genetic damage, and cancer. Cr(VI)-induced apoptosis is a toxicant mechanism of Cr(VI). Studies have shown that Cr(VI) can induce p53-independent apoptosis, but the mechanisms are not fully understood. The intracellular calcium concentration affects cellular life. Apoptosis-inducing factor (AIF), a caspase-independent apoptotic effector, can induce DNA degradation. Using p53-null Hep3B cells, we investigated the effects of cytoplasmic calcium homeostasis and AIF on Cr(VI)-induced apoptosis. We found that 20 µM of Cr(VI) induced DNA damage and mitochondrial permeability transition pore (MPTP) openings, causing calcium overload that was accompanied by decreased Ca2+-Mg2+-ATPase and Na+-K+-ATP activities, downregulation of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CAMKII) mRNA, and increased expression of p-CaMKII/CaMKII protein. After treatment with calcium chelating agent BAPTA-AM, Cr(VI)-induced DNA damage, calcium overload, and apoptosis were reduced. AIF was released from the mitochondria and translocated into the nuclei. As the Cr(VI) treatment time progressed, the mRNA and protein expression of B cell lymphoma 2 (Bcl-2) and heat-shock protein 70 (HSP70) decreased, whereas the mRNA and protein expression of Bcl-2-associated X (Bax), cyclophilin A (CypA), and endonuclease G (EndoG) were upregulated. These results indicated that Cr(VI)-induced apoptosis of Hep3B cells (p53-null) was closely associated with calcium overload, and was accompanied by the activation of Ca2+/CaM/CaMKII signaling pathway. Besides, Cr(VI) triggered AIF nuclear translocation in Hep3B cells, accompanied by the changes in the levels of apoptosis-associated factors. These results provide additional experimental evidence of the molecular mechanisms involved in Cr(VI)-induced p53-independent apoptosis.

Overweight or obese women seeking pregnancy is becoming increasingly common. Human maternal obesity gives rise to detrimental effects during reproduction. Emerging evidence has shown that these abnormities are likely attributed to oocyte quality. Oxidative stress induces poor oocyte conditions, but whether mitochondrial calcium homeostasis plays a key role in oocyte status remains unresolved. Here, we established a mitochondrial Ca2+ overload model in mouse oocytes. Knockdown gatekeepers of the mitochondrial Ca2+ uniporters Micu1 and Micu2 as well as the mitochondrial sodium calcium exchanger NCLX in oocytes both increased oocytes mitochondrial Ca2+ concentration. The overload of mitochondria Ca2+ in oocytes impaired mitochondrial function, leaded to oxidative stress, and changed protein kinase A (PKA) signaling associated gene expression as well as delayed meiotic resumption. Using this model, we aimed to determine the mechanism of delayed meiosis caused by mitochondrial Ca2+ overload, and whether oocyte-specific inhibition of mitochondrial Ca2+ influx could improve the reproductive abnormalities seen within obesity. Germinal vesicle breakdown stage (GVBD) and extrusion of first polar body (PB1) are two indicators of meiosis maturation. As expected, the percentage of oocytes that successfully progress to the germinal vesicle breakdown stage and extrude the first polar body during in vitro culture was increased significantly, and the expression of PKA signaling genes and mitochondrial function recovered after appropriate mitochondrial Ca2+ regulation. Additionally, some indicators of mitochondrial performance—such as adenosine triphosphate (ATP) and reactive oxygen species (ROS) levels and mitochondrial membrane potential—recovered to normal. These results suggest that the regulation of mitochondrial Ca2+ uptake in mouse oocytes has a significant role during oocyte maturation as well as PKA signaling and that proper mitochondrial Ca2+ reductions in obese oocytes can recover mitochondrial performance and improve obesity-associated oocyte quality.

Calcium overload, a notable instigator of acute pancreatitis (AP), induces oxidative stress and an inflammatory cascade, subsequently activating both endogenous and exogenous apoptotic pathways. However, there is currently lack of available pharmaceutical interventions to alleviate AP by addressing calcium overload. In the present study, the potential clinical application of liposome nanoparticles (LNs) loaded with 1,2‑bis(2‑aminophenoxy)ethane‑N,N,N',N'‑tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA‑AM), a cell‑permeant calcium chelator, was investigated as a therapeutic approach for the management of AP. To establish the experimental models in vitro , AR42J cells were exposed to high glucose/sodium oleate (HGO) to induce necrosis, and in vivo , intra‑ductal taurocholate (TC) infusion was used to induce AP. The findings of the present study indicated that the use of BAPTA‑AM‑loaded LN (BLN) effectively and rapidly eliminated excessive Ca 2+ and reactive oxygen species, suppressed mononuclear macrophage activation and the release of inflammatory cytokines, and mitigated pancreatic acinar cell apoptosis and necrosis induced by HGO. Furthermore, the systemic administration of BLN demonstrated promising therapeutic potential in the rat model of AP. Notably, BLN significantly enhanced the survival rates of rats subjected to the TC challenge, increasing from 37.5 to 75%. This improvement was attributed to the restoration of pancreatic function, as indicated by improved blood biochemistry indices and alleviation of pancreatic lesions. The potential therapeutic efficacy of BLN in rescuing patients with AP is likely attributed to its capacity to inhibit oxidative stress, prevent premature activation of zymogens and downregulate the expression of TNF‑α, IL‑6 and cathepsin B. Thus, BLN demonstrated promising value as a novel therapeutic approach for promptly alleviating the burden of intracellular Ca 2+ overload in patients with AP.

Ischemic stroke is currently the most common type of stroke, and the key pathological link is cerebral ischemia-reperfusion injury (CIRI), while the key factor leading to apoptosis and necrosis of ischemic nerve cells is calcium overload. Current studies have confirmed that acupuncture therapy has a good modulating effect on calcium homeostasis and can reduce cerebral ischemia-reperfusion induced damage of neuronal cells by inhibiting calcium overload. After reviewing the relevant literature published in the past 15 years, we find that acupuncture plays a role in regulating the pathological mechanism of calcium overload after CIRI by inhibiting the opening of connexin 43 hemichannels, regulating the intracellular free calcium ion concentration, suppressing the expression of calmodulin, and blocking the function of L-type voltage-gated calcium channels, thereby inhibiting calcium overload, regulating calcium homeostasis and antagonizing neuronal damage resulted from cerebral ischemia-reperfusion, which may provide ideas for future research.

Regarded as one of the hallmarks of tumorigenesis and tumor progression, the evasion of apoptotic cell death would also account for treatment resistance or failure during cancer therapy. In this study, a Ca 2+ /Cu 2+ dual-ion "nano trap" to effectively avoid cell apoptosis evasion by synchronously inducing paraptosis together with apoptosis was successfully designed and fabricated for breast cancer treatment. In brief, disulfiram (DSF)-loaded amorphous calcium carbonate nanoparticles (NPs) were fabricated via a gas diffusion method. Further on, the Cu 2+ -tannic acid metal phenolic network was embedded onto the NPs surface by self-assembling, followed by mDSPE-PEG/lipid capping to form the DSF-loaded Ca 2+ /Cu 2+ dual-ions "nano trap". The as-prepared nanotrap would undergo acid-triggered biodegradation upon being endocytosed by tumor cells within the lysosome for Ca 2+ , Cu 2+ , and DSF releasing simultaneously. The released Ca 2+ could cause mitochondrial calcium overload and lead to hydrogen peroxide (H 2 O 2 ) overexpression. Meanwhile, Ca 2+ /reactive oxygen species-associated mitochondrial dysfunction would lead to paraptosis cell death. Most importantly, cell paraptosis could be further induced and strengthened by the toxic dithiocarbamate (DTC)-copper complexes formed by the Cu 2+ combined with the DTC, the metabolic products of DSF. Additionally, the released Cu 2+ will be reduced by intracellular glutathione to generate Cu + , which can catalyze the H 2 O 2 to produce a toxic hydroxyl radical by a Cu + -mediated Fenton-like reaction for inducing cell apoptosis. Both in vitro cellular assays and in vivo antitumor evaluations confirmed the cancer therapeutic efficiency by the dual ion nano trap. This study can broaden the cognition scope of dual-ion-mediated paraptosis together with apoptosis via a multifunctional nanoplatform.

Sonodynamic therapy (SDT) is an emerging cancer treatment method in recent years. However, the ultrasound signal utilized for SDT is usually located at a low-frequency spectrum (<2 MHz), and in the field of SDT research, few studies have focused on the exploration and development of ultrasound frequency. Studies have shown that the GHz-level ultrasound can increase cell membrane permeability and have a negligible effect on cell vitality. Herein, we reported the study of a GHz thin film bulk acoustic resonator as an ultrasound source for synergistic treatment with nanoscale calcium peroxide (CaO 2 ). It was discovered that this ultrasound source ultimately achieved an efficient therapeutic outcome on mouse breast cancer cell line 4T1. Such GHz-level ultrasound application in SDT is of high significance to broaden the cognition and application scope of SDT., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Tumor therapy presents significant challenges, and conventional treatments exhibit limited therapeutic effectiveness. Imbalance of calcium homeostasis as a key cause of tumor cell death has been extensively studied in tumor therapy. Calcium overload therapy has garnered significant interest as a new cancer treatment strategy. This study involves the synthesis of a transformable nanosonosensitizer with a shell of a calcium ion nanomodulator. The nanosystem is designed to induce mitochondrial dysfunction by combining the calcium ion nanomodulator, nanosonosensitizer, and chemotherapeutic drug. Under ultrasound-activated conditions, CaCO 3 dissolves in the tumor microenvironment, causing the nanosonosensitizer to switch from the "off" to the "on" state of ROS generation, exacerbating mitochondrial calcium overload. A two-dimensional Ti 3 C 2 /TiO 2 heterostructure generates reactive oxygen species (ROS) under ultrasound and exhibits an efficient sonodynamic effect, enhancing calcium overload. Under ultrasound irradiation, Ti 3 C 2 /TiO 2 @CaCO 3 /KAE causes multilevel damage to mitochondria by combining the effects of rapid Ca 2+ release, inhibiting Ca 2+ efflux, enhancing tumor inhibition, and converting a "cold" tumor into a "hot" tumor. Therefore, this study proposes a method to effectively combine mitochondrial Ca 2+ homeostasis and sonodynamic therapy (SDT) by the preparing pH-sensitive, double-activated, and multifunctional Ti 3 C 2 /TiO 2 -based nanosystems for cancer therapy.

Low selectivity and tumor drug resistance are the main hinderances to conventional radiotherapy and chemotherapy against tumor. Ion interference therapy is an innovative anti-tumor strategy that has been recently reported to induce metabolic disorders and inhibit proliferation of tumor cells by reordering bioactive ions within the tumor cells. Calcium cation (Ca 2+ ) are indispensable for all physiological activities of cells. In particular, calcium overload, characterized by the abnormal intracellular Ca 2+ accumulation, causes irreversible cell death. Consequently, calcium overload-based ion interference therapy has the potential to overcome resistance to traditional tumor treatment strategies and holds promise for clinical application. In this review, we 1) Summed up the current strategies employed in this therapy; 2) Described the outcome of tumor cell death resulting from this therapy; 3) Discussed its potential application in synergistic therapy with immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Fan, Wang, He, Xu and Li.)

Hyperphosphatemia is the primary risk factor for vascular calcification, which is closely associated with cardiovascular morbidity and mortality. Recent evidence showed that oxidative stress by high inorganic phosphate (Pi) mediates calcific changes in vascular smooth muscle cells (VSMCs). However, intracellular signaling responsible for Pi-induced oxidative stress remains unclear. Here, we investigated molecular mechanisms of Pi-induced oxidative stress related with intracellular Ca2+ ([Ca2+]i) disturbance, which is critical for calcification of VSMCs. VSMCs isolated from rat thoracic aorta or A7r5 cells were incubated with high Pi-containing medium. Extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin were activated by high Pi that was required for vascular calcification. High Pi upregulated expressions of type III sodium-phosphate cotransporters PiT-1 and -2 and stimulated their trafficking to the plasma membrane. Interestingly, high Pi increased [Ca2+]i exclusively dependent on extracellular Na+ and Ca2+ as well as PiT-1/2 abundance. Furthermore, high-Pi induced plasma membrane depolarization mediated by PiT-1/2. Pretreatment with verapamil, as a voltage-gated Ca2+ channel (VGCC) blocker, inhibited Pi-induced [Ca2+]i elevation, oxidative stress, ERK activation, and osteogenic differentiation. These protective effects were reiterated by extracellular Ca2+-free condition, intracellular Ca2+ chelation, or suppression of oxidative stress. Mitochondrial superoxide scavenger also effectively abrogated ERK activation and osteogenic differentiation of VSMCs by high Pi. Taking all these together, we suggest that high Pi activates depolarization-triggered Ca2+ influx via VGCC, and subsequent [Ca2+]i increase elicits oxidative stress and osteogenic differentiation. PiT-1/2 mediates Pi-induced [Ca2+]i overload and oxidative stress but in turn, PiT-1/2 is upregulated by consequences of these alterations. [ABSTRACT FROM AUTHOR]

Quercetin has attracted more attention in recent years due to its protective role against ischemia/reperfusion injury. Quercetin can alleviate oxidative stress injury through the inhibition of NADPH oxidase and xanthine oxidase, blockage of the Fenton reaction, and scavenging of reactive oxygen species. Quercetin can also exert anti-inflammatory and anti-apoptotic effects by reducing the response to inflammatory factors and inhibiting cell apoptosis. Moreover, it can induce vasodilation effects through the inhibition of endothelin-1 receptors, the enhancement of NO stimulation and the activation of the large-conductance calcium-activated potassium channels. Finally, Quercetin can also antagonize the calcium overload. These multifaceted activities of Quercetin make it a potential therapeutic alternative for the treatment of ischemia/reperfusion injury.

Exertional heat stroke (EHS) is a life-threatening disease characterized by high mortality and incidence of rhabdomyolysis (RM). It would therefore be valuable to establish a stable EHS-induced RM model that accurately reflects the clinical characteristics of EHS patients and provides an objective animal model for further study of the pathogenesis of RM. In the current study, 8∼9-week-old, male, wild-type C57BL/6J mice, at the stage of sexual maturity, were randomly divided into four groups: the EHS group, the classical heat stroke (CHS) group, the sham heat exercise group, and sham heat rest group. The survival rate of mice was determined under relatively high levels of temperature and humidity (37.5°C, 65% relative humidity (RH); 37.5°C, 70% RH; 39.5°C, 65% RH; and 39.5°C, 70% RH) as well as a high core temperature (Tc; 42, 42.5, and 43°C). Results showed that the environmental condition of 39.5°C and 65% RH was most suitable for EHS modeling. The end point of EHS evaluation was exhaustion or an individual’s core temperature reaching 43°C. The survival rate of mice in the EHS group within 24 h under these conditions was 37.34%, which is consistent with the high mortality characteristics noted in EHS patients. Severe RM was observed in the EHS group by H&E staining and transmission electron microscopy. Creatine kinase levels in the EHS group mostly exceeded 10,000 U/L, which was approximately 10 times higher than that in the sham heat rest group. Renal tubules of the EHS group exhibited severe necrosis, and calcium overload in the skeletal muscles of this group was also observed using intravital 2-photon microscopy. In conclusion, we made improvements to a stable EHS-induced RM animal model to truly reflect the clinical characteristics of EHS patients. This new model should be helpful in the further study of RM pathogenesis.

Ca2+, a ubiquitous but nuanced modulator of cellular physiology, is meticulously controlled intracellularly. However, intracellular Ca2+ regulation, such as mitochondrial Ca2+ buffering capacity, can be disrupted by 1O2. Thus, the intracellular Ca2+ overload, which is recognized as one of the important cell pro‐death factors, can be logically achieved by the synergism of 1O2 with exogenous Ca2+ delivery. Reported herein is a nanoscale covalent organic framework (NCOF)‐based nanoagent, namely CaCO3@COF‐BODIPY‐2I@GAG (4), which is embedded with CaCO3 nanoparticle (NP) and surface‐decorated with BODIPY‐2I as photosensitizer (PS) and glycosaminoglycan (GAG) targeting agent for CD44 receptors on digestive tract tumor cells. Under illumination, the light‐triggered 1O2 not only kills the tumor cells directly, but also leads to their mitochondrial dysfunction and Ca2+ overload. An enhanced antitumor efficiency is achieved via photodynamic therapy (PDT) and Ca2+ overload synergistic therapy. [ABSTRACT FROM AUTHOR]

Introduction: Hypocalcemia has been widely recognized in sepsis patients. However, the cause of hypocalcemia in sepsis is still not clear, and little is known about the subcellular distribution of Ca2+ in tissues during sepsis. Methodology: We measured the dynamic change in Ca2+ levels in body fluid and subcellular compartments, including the cytosol, endoplasmic reticulum and mitochondria, in major organs of cecal ligation and puncture (CLP)-operated rats, as well as the subcellular Ca2+ flux in HUVECs which treated by endotoxin and cytokines. Results: In the model of CLP-induced sepsis, the blood and urinary Ca2+ concentrations decreased rapidly, while the Ca2+ concentration in ascites fluid increased. The Ca2+ concentrations in the cytosol, ER, and mitochondria were elevated nearly synchronously in major organs in our sepsis model. Moreover, the calcium overload in CLP-operated rats treated with calcium supplementation was more severe than that in the non-calcium-supplemented rats but was alleviated by treatment with the calcium channel blocker verapamil. Similar subcellular Ca2+ flux was found in vitro in HUVECs and was triggered by lipopolysaccharide (LPS)/TNF-a. Conclusions: Ca2+ influx from the blood into the intercellular space and Ca2+ release into ascites fluid may cause hypocalcemia in sepsis and that this process may be due to the synergistic effect of endotoxin and cytokines. [ABSTRACT FROM AUTHOR]

Quercetin has attracted more attention in recent years due to its protective role against ischemia/reperfusion injury. Quercetin can alleviate oxidative stress injury through the inhibition of NADPH oxidase and xanthine oxidase, blockage of the Fenton reaction, and scavenging of reactive oxygen species. Quercetin can also exert anti-inflammatory and anti-apoptotic effects by reducing the response to inflammatory factors and inhibiting cell apoptosis. Moreover, it can induce vasodilation effects through the inhibition of endothelin-1 receptors, the enhancement of NO stimulation and the activation of the large-conductance calcium-activated potassium channels. Finally, Quercetin can also antagonize the calcium overload. These multifaceted activities of Quercetin make it a potential therapeutic alternative for the treatment of ischemia/reperfusion injury. [ABSTRACT FROM AUTHOR]

Exertional heat stroke (EHS) is a life-threatening disease characterized by high mortality and incidence of rhabdomyolysis (RM). It would therefore be valuable to establish a stable EHS-induced RM model that accurately reflects the clinical characteristics of EHS patients and provides an objective animal model for further study of the pathogenesis of RM. In the current study, 8∼9-week-old, male, wild-type C57BL/6J mice, at the stage of sexual maturity, were randomly divided into four groups: the EHS group, the classical heat stroke (CHS) group, the sham heat exercise group, and sham heat rest group. The survival rate of mice was determined under relatively high levels of temperature and humidity (37.5°C, 65% relative humidity (RH); 37.5°C, 70% RH; 39.5°C, 65% RH; and 39.5°C, 70% RH) as well as a high core temperature (Tc; 42, 42.5, and 43°C). Results showed that the environmental condition of 39.5°C and 65% RH was most suitable for EHS modeling. The end point of EHS evaluation was exhaustion or an individual's core temperature reaching 43°C. The survival rate of mice in the EHS group within 24 h under these conditions was 37.34%, which is consistent with the high mortality characteristics noted in EHS patients. Severe RM was observed in the EHS group by H&E staining and transmission electron microscopy. Creatine kinase levels in the EHS group mostly exceeded 10,000 U/L, which was approximately 10 times higher than that in the sham heat rest group. Renal tubules of the EHS group exhibited severe necrosis, and calcium overload in the skeletal muscles of this group was also observed using intravital 2-photon microscopy. In conclusion, we made improvements to a stable EHS-induced RM animal model to truly reflect the clinical characteristics of EHS patients. This new model should be helpful in the further study of RM pathogenesis. [ABSTRACT FROM AUTHOR]

Background: Silica nanoparticles (nanoSiO2) are promising systems that can deliver biologically active compounds to tissues such as the heart in a controllable manner. However, cardiac toxicity induced by nanoSiO2 has been recently related to abnormal calcium handling and energetic failure in cardiomyocytes. Moreover, the precise mechanisms underlying this energetic debacle remain unclear. In order to elucidate these mechanisms, this article explores the ex vivo heart function and mitochondria after exposure to nanoSiO2. Results: The cumulative administration of nanoSiO2 reduced the mechanical performance index of the rat heart with a half-maximal inhibitory concentration (IC50) of 93 μg/mL, affecting the relaxation rate. In isolated mitochondria nanoSiO2 was found to be internalized, inhibiting oxidative phosphorylation and significantly reducing the mitochondrial membrane potential (ΔΨm). The mitochondrial permeability transition pore (mPTP) was also induced with an increasing dose of nanoSiO2 and partially recovered with, a potent blocker of the mPTP, Cyclosporine A (CsA). The activity of aconitase and thiol oxidation, in the adenine nucleotide translocase, were found to be reduced due to nanoSiO2 exposure, suggesting that nanoSiO2 induces the mPTP via thiol modification and ROS generation. In cardiac cells exposed to nanoSiO2, enhanced viability and reduction of H2O2 were observed after application of a specific mitochondrial antioxidant, MitoTEMPO. Concomitantly, CsA treatment in adult rat cardiac cells reduced the nanoSiO2-triggered cell death and recovered ATP production (from 32.4 to 65.4%). Additionally, we performed evaluation of the mitochondrial effect of nanoSiO2 in human cardiomyocytes. We observed a 40% inhibition of maximal oxygen consumption rate in mitochondria at 500 μg/mL. Under this condition we identified a remarkable diminution in the spare respiratory capacity. This data indicates that a reduction in the amount of extra ATP that can be produced by mitochondria during a sudden increase in energy demand. In human cardiomyocytes, increased LDH release and necrosis were found at increased doses of nanoSiO2, reaching 85 and 48%, respectively. Such deleterious effects were partially prevented by the application of CsA. Therefore, exposure to nanoSiO2 affects cardiac function via mitochondrial dysfunction through the opening of the mPTP. Conclusion: The aforementioned effects can be partially avoided reducing ROS or retarding the opening of the mPTP. These novel strategies which resulted in cardioprotection could be considered as potential therapies to decrease the side effects of nanoSiO2 exposure. [ABSTRACT FROM AUTHOR]

Purpose: Vepoloxamer (VEPO), a rheologic agent, repairs damaged cell membranes, thus inhibiting unregulated Ca2+ entry into cardiomyocytes. This study examined the effects of i.v. infusion of VEPO on LV function in dogs with coronary microembolization–induced heart failure (HF) (LV ejection fraction, EF ~ 30%). Methods: Thirty-five HF dogs were studied. Study 1: 21 of 35 dogs were randomized to 2-h infusion of VEPO at dose of 450 mg/kg (n = 7) or VEPO at 225 mg/kg (n = 7) or normal saline (control, n = 7). Hemodynamics were measured at 2 h, 24 h, 1 week, and 2 weeks after infusion. Study 2: 14 HF dogs were randomized to 2-h infusions of VEPO (450 mg/kg, n = 7) or normal saline (control, n = 7). Each dog received 2 infusions of VEPO or saline (pulsed therapy) 3 weeks apart and hemodynamics measured at 24 h, and 1, 2, and 3 weeks after each infusion. In both studies, the change between pre-infusion measures and measures at other time points (treatment effect, Δ) was calculated. Results: Study 1: compared to pre-infusion, high dose VEPO increased LVEF by 11 ± 2% at 2 h, 8 ± 2% at 24 h (p < 0.05), 8 ± 2% at 1 week (p < 0.05), and 4 ± 2% at 2 weeks. LV EF also increased with low-dose VEPO but not with saline. Study 2: VEPO but not saline significantly increased LVEF by 6.0 ± 0.7% at 2 h (p < 0.05); 7.0 ± 0.7%% at 1 week (p < 0.05); 1.0 ± 0.6% at 3 weeks; 6.0 ± 1.3% at 4 weeks (p < 0.05); and 5.9 ± 1.3% at 6 weeks (p < 0.05). Conclusions: Intravenous VEPO improves LV function for at least 1 week after infusion. The benefits can be extended with pulsed VEPO therapy. The results support development of VEPO for treating patients with acute on chronic HF. [ABSTRACT FROM AUTHOR]

• In the absence of disease, calcium overload decreases net ROS emission. • Calcium overload inhibits NADH-dependent pathways. • ROS emission is primarily controlled by membrane potential. • The relationship between net ROS emission and oxygen concentration is hyperbolic. Elevated calcium and reactive oxygen species (ROS) are responsible for the bulk of cell death occurring in a variety of clinical settings that include acute coronary events, cerebrovascular accidents, and acute kidney injury. It is commonly believed that calcium and ROS participate in a viscous cycle during these events. However, the precise feedback mechanisms are unknown. We quantitatively demonstrate in this study that, on the contrary, calcium does not stimulate free radical production but suppresses it. Isolated mitochondria from guinea pig hearts were energized with a variety of substrates and exposed to calcium concentrations designed to induce moderate calcium overload conditions associated with ischemia/reperfusion injury but do not elicit the well-known mitochondrial permeability transition phenomenon. Metabolic function and free radical emission were simultaneously quantified using high-resolution respirometry and fluorimetry. Membrane potential, high amplitude swelling, and calcium dynamics were also quantified in parallel. Our results reveal that calcium overload does not lead to excessive ROS emission but does decrease ADP stimulated respiration rates for NADH-dependent pathways. Moreover, we developed an empirical model of mitochondrial free radical homeostasis to identify the processes that are different for each substrate and calcium condition. In summary, we show that in healthy guinea pig mitochondria, calcium uptake and free radical generation do not contribute to a viscous cycle and that the relationship between net free radical production and oxygen concentration is hyperbolic. Altogether, these results lay out an important foundation necessary to quantitatively determine the role of calcium in IR injury and ROS production. [ABSTRACT FROM AUTHOR]

Background and Purpose: Intracellular calcium concentration ([Ca2+]i) overload occurs in myocardial ischemia and -reperfusion. The augmentation of the late sodium current (INaL) causes intracellular Na+ accumulation and subsequent [Ca2+]i overload via the reverse mode of the Na+/Ca2+ exchange current (reverse-INCX), which can lead to arrhythmia and cardiac dysfunction. Thus, inhibition of INaL is a potential therapeutic approach for ischemic heart disease. The aim of this study was to investigate the effects of thyroid hormone on augmented INaL, reverse-INCX, altered action potential duration (APD), and [Ca2+]i concentration in hypoxia/reoxygenation (H/R)-induced ventricular myocytes in vitro. Methods: The transient Na+ current (INaT), INaL, reverse-INCX, and APs were recorded using a whole-cell patch-clamp technique in neonatal mouse ventricular myocytes. [Ca2+]i concentration alteration were, respectively, observed by confocal microscopy and flow cytometry. Results: Triiodothyronine (T3) pretreatment decreased the INaL in a concentration-dependent manner. H/R injury aggravated the INaL, INaT, and reverse-INCX in cardiomyocytes and increased the continuous accumulation of [Ca2+]i (p < 0.05). The application of T3 prior to H/R injury significantly decreased the increased INaL, INaT, and reverse-INCX and blunted the [Ca2+]i increase. Furthermore, T3 pretreatment shortened the APD induced by H/R injury. Conclusion: T3 inhibited H/R-increased INaL and reverse INCX augmentation, shortened the APD, and diminished [Ca2+]i overload, indicating a potential therapeutic use of T3 as an INaL inhibitor to maintain Ca2+ homeostasis and protect cardiomyocytes against H/R injury. [ABSTRACT FROM AUTHOR]

The etiology of Alzheimer's disease (AD) has been intensively studied. However, little is known about the molecular alterations in early-stage and late-stage AD. Hence, we performed RNA sequencing and assessed differentially expressed genes (DEGs) in the hippocampus of 18-month and 7-month-old APP/PS1 mice. Moreover, the DEGs induced by treatment with nicotine, the nicotinic acetylcholine receptor agonist that is known to improve cognition in AD, were also analyzed in old and young APP/PS1 mice. When comparing old APP/PS1 mice with their younger littermates, we found an upregulation in genes associated with calcium overload, immune response, cancer, and synaptic function; the transcripts of 14 calcium ion channel subtypes were significantly increased in aged mice. In contrast, the downregulated genes in aged mice were associated with ribosomal components, mitochondrial respiratory chain complex, and metabolism. Through comparison with DEGs in normal aging from previous reports, we found that changes in calcium channel genes remained one of the prominent features in aged APP/PS1 mice. Nicotine treatment also induced changes in gene expression. Indeed, nicotine augmented glycerolipid metabolism, but inhibited PI3K and MAPK signaling in young mice. In contrast, nicotine affected genes associated with cell senescence and death in old mice. Our study suggests a potential network connection between calcium overload and cellular signaling, in which additional nicotinic activation might not be beneficial in late-stage AD. [ABSTRACT FROM AUTHOR]

Exaggerated Ca2+ signaling might be one of primary causes of neural dysfunction in Alzheimer's disease (AD). And the intracellular Ca2+ overload has been closely associated with amyloid-β (Aβ)-induced endoplasmic reticulum (ER) stress and memory impairments in AD. Here we showed for the first time the neuroprotective effects of Xestospongin C (XeC), a reversible IP3 receptor antagonist, on the cognitive behaviors and pathology of APP/PS1 AD mice. Male APP/PS1-AD mice (n = 20) were injected intracerebroventricularly with XeC (3μmol) via Alzet osmotic pumps for four weeks, followed by cognition tests, Aβ plaque examination, and ER stress-related protein measurement. The results showed that XeC pretreatment significantly improved the cognitive behavior of APP/PS1-AD mice, raising the spontaneous alteration accuracy in Y maze, decreasing the escape latency and increasing the target quadrant swimming time in Morris water maze; XeC pretreatment also reduced the number of Aβ plaques and the overexpression of ER stress proteins 78 kDa glucose-regulated protein (GRP-78), caspase-12, and CAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) in the hippocampus of APP/PS1 mice. In addition, in vitro experiments showed that XeC effectively ameliorated Aβ1 - 42-induced early neuronal apoptosis and intracellular Ca2+ overload in the primary hippocampal neurons. Taken together, IP3R-mediated Ca2+ disorder plays a key role in the cognitive deficits and pathological damages in AD mice. By targeting the IP3 R, XeC might be considered as a novel therapeutic strategy in AD. [ABSTRACT FROM AUTHOR]

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[Display omitted] • Hydrophobic silibinin was linked to the N-acetylgalactosamine-modified hydrophilic dextran backbone via a phenylboronic acid linkage to produce an amphiphilic polymer. • ROS-responsive liver-targeting NPs for efficient encapsulation of hydrophobic cell-permeable Ca2+ chelator BAPTA-AM agents via hydrophobic/hydrophilic interactions. • The released BAPTA-AM blocked the sterile inflammatory reaction and apoptosis signaling in the upstream link of acute liver injury. • The silibinin exerted synergistic hepatoprotective effects after the ROS-responsive cleavage of phenylboronic acid. Calcium overload in hepatocytes mediates the crosstalk and amplification loop with oxidative stress and severe inflammation that initiate pro-apoptotic and necrotic signaling pathways in acute liver injury (ALI). In this study, an N-acetylgalactosamine-modified amphiphilic dextran-silibinin conjugate (GalNAc-Dex-Sil) with ROS-sensitive phenylboronic acid (PBA) linkage was synthesized. The as-prepared amphiphilic polymer was self-assembled into GalNAc-Dex-Sil nanoparticles, which efficiently encapsulated the membrane-permeable calcium chelator BAPTA-AM (BA), achieving GalNAc-mediated hepatocyte-specific targeting and ROS-responsive release in injured hepatocytes through PBA cleavage. Accordingly, a single low-dose injection of formulation (BA: 150 μg·kg−1) significantly alleviated hepatocyte oxidative stress and inflammation, rescued dying hepatocytes, prevented further hepatocyte damage, and ultimately restored liver function in the ALI mouse model by 1) rapidly and effectively eliminating the overloaded calcium and scavenging excessive ROS via released BA to block the feed-forward vicious cycles of the cell damage, 2) inhibiting the inflammatory cascade by decreasing the expression of the key factor of the NF-κB/NLPR3 axis, 3) inhibiting the key branch of the mitochondrial apoptotic pathway (Cyt C/Bcl-2 family/caspase-3 axis) and death receptor pathway (TNFα/Caspase-8 axis), and 4) stabilizing hepatocyte membranes through the synergistic hepatoprotective effect of silibinin. Taken together, this formulation exhibited great potential as a potent and effective ALI treatment strategy. [ABSTRACT FROM AUTHOR]

The treatment efficacies of conventional medications against colorectal cancer (CRC) are restricted by a low penetrative, hypoxic, and immunosuppressive tumor microenvironment. To address these restrictions, we developed an innovative antitumor platform that employs calcium overload-phototherapy using mitochondrial N770-conjugated mesoporous silica nanoparticles loaded with CaO 2 (CaO 2 –N770@MSNs). A loading level of 14.0 wt% for CaO 2 –N770@MSNs was measured, constituting an adequate therapeutic dosage. With the combination of oxygen generated from CaO 2 and hyperthermia under near-infrared irradiation, CaO 2 –N770@MSNs penetrated through the dense mucus, accumulated in the colorectal tumor tissues, and inhibited tumor cell growth through endoplasmic reticulum stress and mitochondrial damage. The combination of calcium overload and phototherapy revealed high therapeutic efficacy against orthotopic colorectal tumors, alleviated the immunosuppressive microenvironment, elevated the abundance of beneficial microorganisms (e.g. , Lactobacillaceae and Lachnospiraceae), and decreased harmful microorganisms (e.g. , Bacteroidaceae and Muribaculaceae). Moreover, together with immune checkpoint blocker (α PD-L1), these nanoparticles showed an ability to eradicate both orthotopic and distant tumors, while potentiating systemic antitumor immunity. This treatment platform (CaO 2 –N770@MSNs plus α PD-L1) open a new horizon of synergistic treatment against hypoxic CRC with high killing power and safety. [Display omitted] [ABSTRACT FROM AUTHOR]

This review summarizes the applications of metal-ion-delivered nanomaterials in cancer therapy and diagnostic imaging. The metal-ion-induced therapies (e.g., chemodynamic therapy, immunotherapy, etc.) and cell death pathways (e.g., ferroptosis, cuproptosis, etc.) are detailed. Mono-/Multi-model tumor imaging technologies depending on metal ions are also the emphasis. The future perspectives of metal-ions-based theranostic nanoplatforms in preclinic or clinics are recommended. [Display omitted] • Discussing biological effects of metal ions in tumor cells and tumor treatment strategies based on responsive nanomaterials. • Introducing the advanced cell death strategies caused by regulating intracellular metal-ion balance through nanomaterials. • Summarizing application and importance of metal-ion-based nanomaterials in tumor guided therapy and diagnostic imaging. • Presenting the challenges and emerging strategies for metal-ion-delivered nanomaterials. Nanomaterials have been extensively used for cancer therapy and diagnosis due to their designability, multifunctionality, and unique in vivo distribution. Metal ions are increasingly seen in cancer therapy and diagnostics as more and more biological effects of metal ions are revealed. Metal-ion-based antitumor strategies, as well as metal-ion-based designed nanomaterials, are continuously being developed with exciting and emerging impacts. Meanwhile, metal-ion-based nanomaterials are forming drug-free theranostic nanoplatforms, where metal ions, as part of building nanomaterials, replace conventional drugs. In this review, we discuss recent advances in multi-functional designed nanomaterials with the ability to deliver metal ions and focus on the role of metal ions in cancer therapy, including chemotherapy, chemodynamic therapy, immunotherapy, etc. The metal-ion-induced cell death, such as apoptosis, ferroptosis, and cuproptosis, is detailed in this review. Mono-/Multi-model imaging depending on metal ions for tumor diagnosis and guided tumor treatment is also the emphasis. By discussing the applications, challenges, and opportunities, we expect the metal-ions-based theranostic nanoplatforms have a far-reaching development in preclinic or clinics. [ABSTRACT FROM AUTHOR]

Cytotoxic effects of cannabidiol (CBD) and tamoxifen (TAM) have been observed in several cancer types. We have recently shown that CBD primarily targets mitochondria, inducing a stable mitochondrial permeability transition pore (mPTP) and, consequently, the death of acute lymphoblastic leukemia (T-ALL) cells. Mitochondria have also been documented among cellular targets for the TAM action. In the present study we have demonstrated a synergistic cytotoxic effect of TAM and CBD against T-ALL cells. By measuring the mitochondrial membrane potential (ΔΨm), mitochondrial calcium ([Ca2+]m) and protein-ligand docking analysis we determined that TAM targets cyclophilin D (CypD) to inhibit mPTP formation. This results in a sustained [Ca2+]m overload upon the consequent CBD administration. Thus, TAM acting on CypD sensitizes T-ALL to mitocans such as CBD by altering the mitochondrial Ca2+ homeostasis.

Ca2+ channel blockers have been shown to protect neurons from ischemia, and aerobic exercise has significant protective effects on a variety of chronic diseases. The present study injected huwentoxin-I (HWTX-I), a spider peptide toxin that blocks Ca2+ channels, into the caudal vein of a chronic cerebral ischemia mouse model, once every 2 days, for a total of 15 injections. During this time, a subgroup of mice was subjected to treadmill exercise for 5 weeks. Results showed amelioration of cortical injury and improved neurological function in mice with chronic cerebral ischemia in the HWTX-I + aerobic exercise group. The combined effects of HWTX-I and exercise were superior to HWTX-I or aerobic exercise alone. HWTX-I effectively activated the Notch signal transduction pathway in brain tissue. Aerobic exercise up-regulated synaptophysin mRNA expression. These results demonstrated that aerobic exercise, in combination with HWTX-I, effectively relieved neuronal injury induced by chronic cerebral ischemia via the Notch signaling pathway and promoting synaptic regeneration.

Intracellular Ca2+ overload, prolongation of the action potential duration (APD), and downregulation of inward rectifier potassium (IK1) channel are hallmarks of electrical remodeling in cardiac hypertrophy and heart failure (HF). We hypothesized that enhancement of IK1 currents is a compensation for IK1 deficit and a novel modulation for cardiac Ca2+ homeostasis and pathological remodeling. In adult Sprague-Dawley (SD) rats in vivo, cardiac hypertrophy was induced by isoproterenol (Iso) injection (i.p., 3 mg/kg/d) for 3, 10, and 30 days. Neonatal rat ventricular myocytes (NRVMs) were isolated from 1 to 3 days SD rat pups and treated with 1 μmol/L Iso for 24 h in vitro. The effects of zacopride, a selective IK1/Kir2.1 channel agonist, on cardiac remodeling/hypertrophy were observed in the settings of 15 μg/kg in vivo and 1 μmol/L in vitro. After exposing to Iso for 3 days and 10 days, rat hearts showed distinct concentric hypertrophy and fibrosis and enhanced pumping function (P < 0.01 or P < 0.05), then progressed to dilatation and dysfunction post 30 days. Compared with the age-matched control, cardiomyocytes exhibited higher cytosolic Ca2+ (P < 0.01 or P < 0.05) and lower SR Ca2+ content (P < 0.01 or P < 0.05) all through 3, 10, and 30 days of Iso infusion. The expressions of Kir2.1 and SERCA2 were downregulated, while p-CaMKII, p-RyR2, and cleaved caspase-3 were upregulated. Iso-induced electrophysiological abnormalities were also manifested with resting potential (RP) depolarization (P < 0.01), APD prolongation (P < 0.01) in adult cardiomyocytes, and calcium overload in cultured NRVMs (P < 0.01). Zacopride treatment effectively retarded myocardial hypertrophy and fibrosis, preserved the expression of Kir2.1 and some key players in Ca2+ homeostasis, normalized the RP (P < 0.05), and abbreviated APD (P < 0.01), thus lowered cytosolic [Ca2 +]i (P < 0.01 or P < 0.05). IK1channel blocker BaCl2 or chloroquine largely reversed the cardioprotection of zacopride. We conclude that cardiac electrical remodeling is concurrent with structural remodeling. By enhancing cardiac IK1, zacopride prevents Iso-induced electrical remodeling around intracellular Ca2+ overload, thereby attenuates cardiac structural disorder and dysfunction. Early electrical interventions may provide protection on cardiac remodeling.

BackgroundThe dual Na+ and cardiac Ca2+-release channel inhibitor, Flecainide (FLEC) is effective in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by mutations in cardiac Ca2+-release channels (RyR2), calsequestrin (Casq2), or calmodulin. FLEC suppresses spontaneous Ca2+ waves in Casq2-knockout (Casq2−/−) cardiomyocytes, a CPVT model. However, a report failed to find FLEC efficacy against Ca2+ waves in another CPVT model, RyR2-R4496C heterozygous mice (RyR2R4496C+/−), raising the possibility that FLEC efficacy may be mutation dependent.ObjectiveTo address this controversy, we compared FLEC in Casq2−/− and RyR2R4496C+/− cardiomyocytes and mice under identical conditions.MethodsAfter 30 min exposure to FLEC (6 μM) or vehicle (VEH), spontaneous Ca2+ waves were quantified during a 40 s pause after 1 Hz pacing train in the presence of isoproterenol (ISO, 1 μM). FLEC efficacy was also tested in vivo using a low dose (LOW: 3 mg/kg ISO + 60 mg/kg caffeine) or a high dose catecholamine challenge (HIGH: 3 mg/kg ISO + 120 mg/kg caffeine).ResultsIn cardiomyocytes, FLEC efficacy was dependent on extracellular [Ca2+]. At 2 mM [Ca2+], only Casq2−/− myocytes exhibited Ca2+ waves, which were strongly suppressed by FLEC. At 3 mM [Ca2+] both groups exhibited Ca2+ waves that were suppressed by FLEC. At 4 mM [Ca2+], FLEC no longer suppressed Ca2+ waves in both groups. Analogous to the results in myocytes, RyR2R4496C+/− mice (n = 12) had significantly lower arrhythmia scores than Casq2−/− mice (n = 9), but the pattern of FLEC efficacy was similar in both groups (i.e., reduced FLEC efficacy after HIGH dose catecholamine challenge).ConclusionFLEC inhibits Ca2+ waves in RyR2R4496C+/− cardiomyocytes, indicating that RyR2 channel block by FLEC is not mutation-specific. However, FLEC efficacy is reduced by Ca2+ overload in vitro or by high dose catecholamine challenge in vivo, which could explain conflicting literature reports.

Circadian mechanisms have been associated with the pathogenesis of a variety of cardiovascular diseases, including myocardial ischemia-reperfusion injury (I-R). Myocardial ischemia resulting from impaired oxygen delivery to cardiac muscle sets into motion a cascade of cellular events that paradoxically triggers greater cardiac dysfunction upon reinstitution of coronary blood supply, a phenomenon known as I-R. I-R injury has been attributed to a number of cellular defects including increased reactive oxygen species (ROS), increased intracellular calcium and impaired mitochondrial bioenergetics that ultimately lead to cardiac cell death, ventricular remodeling and heart failure. Emerging evidence has identified a strong correlation between cellular defects that underlie I-R and the disrupted circadian. In fact, recent studies have shown that circadian dysfunction exacerbates cardiac injury following MI from impaired cellular quality control mechanisms such as autophagy, which are vital in the clearance of damaged cellular proteins and organelles such as mitochondria from the cell. The accumulation of cellular debris is posited as the central underlying cause of excessive cardiac cell death and ventricular dysfunction following MI. The complexities that govern the interplay between circadian biology and I-R injury following MI is at its infancy and understanding how circadian misalignment, such as in shift workers impacts I-R injury is of great scientific and clinical importance toward development of new therapeutic strategies using chronotherapy and circadian regulation to mitigate cardiac injury and improve cardiac outcomes after injury. In this review, we highlight recent advances in circadian biology and adaptive cellular quality control mechanisms that influence cardiac injury in response to MI injury with a specific focus on how circadian biology can be utilized to further cardiovascular medicine and patient care., (Copyright © 2022. Published by Elsevier Inc.)

Increasing evidence points to the critical role of calcium overload triggered by mitochondrial dysfunction in the development of alcoholic liver disease (ALD). As an important organelle for aerobic respiration with a double-layered membrane, mitochondria are pivotal targets of alcohol metabolism-mediated lipid peroxidation, wherein mitochondria-specific phospholipid cardiolipin oxidation to 4-hydroxynonenal (4-HNE) ultimately leads to mitochondrial integrity and function impairment. Therefore, it is absolutely essential to identify effective nutritional intervention targeting mitochondrial redox function for an alternative therapy of ALD, in order to compensate for the difficulty in achieving alcohol withdrawal due to addiction. In this study, we confirmed the significant advantages of astaxanthin (AX) against alcohol toxicity among various carotenoids via cell experiments and identified the potential in mitochondrion morphogenesis and calcium signaling pathway by bioinformatics analysis. The ALD model of Sprague-Dawley (SD) rats was also generated to investigate the effectiveness of AX on alcohol-induced liver injury, and the underlying mechanisms were further explored. AX intervention attenuated alcohol-induced oxidative stress and lipid peroxidation as well as mitochondrial dysfunction characterized by degenerative morphology changes and collapsed membrane potential. Also, AX reduced the production of 4-HNE by activating the Nrf2-ARE signaling pathway, which is closely associated with the redox balance of mitochondria. In addition, relieved mitochondrial Ca 2+ accumulation caused by AX was observed both in vivo and in vitro. Furthermore, we revealed the structure-activity relationship of AX and mitochondrial membrane channel proteins MCU and VDAC1, implying potential acting targets. Altogether, our data indicated a new mechanism of AX intervention which protects against alcohol-induced liver injury through restoring redox balance and Ca 2+ homeostasis in mitochondria, as well as provided novel insights into the development of AX as a therapeutic option for the management of ALD.

Background: Caveolin-1, the scaffolding protein of cholesterol-rich invaginations, plays an important role in store-operated Ca 2+ influx and its phosphorylation at Tyr14 (p-caveolin-1) is vital to mobilize protection against myocardial ischemia (MI) injury. SOCE, comprising STIM1, ORAI1 and TRPC1, contributes to intracellular Ca 2+ ([Ca 2+ ] i ) accumulation in cardiomyocytes. The purified extract of steamed Panax ginseng (EPG) attenuated [Ca 2+ ] i overload against MI injury. Thus, the aim of this study was to investigate the possibility of EPG affecting p-caveolin-1 to further mediate SOCE/[Ca 2+ ] i against MI injury in neonatal rat cardiomyocytes and a rat model., Methods: PP2, an inhibitor of p-caveolin-1, was used. Cell viability, [Ca 2+ ] i concentration were analyzed in cardiomyocytes. In rats, myocardial infarct size, pathological damages, apoptosis and cardiac fibrosis were evaluated, p-caveolin-1 and STIM1 were detected by immunofluorescence, and the levels of caveolin-1, STIM1, ORAI1 and TRPC1 were determined by RT-PCR and Western blot. And, release of LDH, cTnI and BNP was measured., Results: EPG, ginsenosides accounting for 57.96%, suppressed release of LDH, cTnI and BNP, and protected cardiomyocytes by inhibiting Ca 2+ influx. And, EPG significantly relieved myocardial infarct size, cardiac apoptosis, fibrosis, and ultrastructure abnormality. Moreover, EPG negatively regulated SOCE via increasing p-caveolin-1 protein, decreasing ORAI1 mRNA and protein levels of ORAI1, TRPC1 and STIM1. More importantly, inhibition of the p-caveolin-1 significantly suppressed all of the above cardioprotection of EPG., Conclusions: Caveolin-1 phosphorylation is involved in the protective effects of EPG against MI injury via increasing p-caveolin-1 to negatively regulate SOCE/[Ca 2+ ] i ., Competing Interests: The authors declare that there are no conflicts of interest.A conflicting interest exists when professional judgement concerning a primary interest (such as patient's welfare or the validity of research) may be influenced by a secondary interest (such as financial gain or personal rivalry). It may arise for the authors when they have financial interest that may influence their interpretation of their results or those of others. Examples of potential conflicts of interest include employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding., (© 2023 The Korean Society of Ginseng. Publishing services by Elsevier B.V.)

Mitochondrial cristae are dynamic invaginations of the inner membrane and play a key role in its metabolic capacity to produce ATP. Structural alterations caused by either genetic abnormalities or detrimental environmental factors impede mitochondrial metabolic fluxes and lead to a decrease in their ability to meet metabolic energy requirements. While some of the key proteins associated with mitochondrial cristae are known, very little is known about how the inner membrane dynamics are involved in energy metabolism. In this study, we present a computational strategy to understand how cristae are formed using a phase-based separation approach of both the inner membrane space and matrix space, which are explicitly modeled using the Cahn–Hilliard equation. We show that cristae are formed as a consequence of minimizing an energy function associated with phase interactions which are subject to geometric boundary constraints. We then extended the model to explore how the presence of calcium phosphate granules, entities that form in calcium overload conditions, exert a devastating inner membrane remodeling response that reduces the capacity for mitochondria to produce ATP. This modeling approach can be extended to include arbitrary geometrical constraints, the spatial heterogeneity of enzymes, and electrostatic effects to mechanize the impact of ultrastructural changes on energy metabolism.

Synovial sarcoma is a rare but aggressive soft-tissue sarcoma associated with translocation t(X;18). Metastasis occurs in approximately 50% of all patients, and curative outcomes are difficult to achieve in this group. Since the efficacies of current therapeutic approaches for metastatic synovial sarcoma remain limited, new therapeutic agents are urgently needed. Tilapia piscidin 4 (TP4), a marine antimicrobial peptide, is known to exhibit multiple biological functions, including anti-bacterial, wound-healing, immunomodulatory, and anticancer activities. In the present study, we assessed the anticancer activity of TP4 in human synovial sarcoma cells and determined the underlying mechanisms. We first demonstrated that TP4 can induce necrotic cell death in human synovial sarcoma AsKa-SS and SW982 cells lines. In addition, we saw that TP4 initiates reactive oxygen species (ROS) production and downregulates antioxidant proteins, such as uncoupling protein-2, superoxide dismutase (SOD)-1, and SOD-2. Moreover, TP4-induced mitochondrial hyperpolarization is followed by elevation of mitochondrial ROS. Calcium overload is also triggered by TP4, and cell death can be attenuated by a necrosis inhibitor, ROS scavenger or calcium chelator. In our experiments, TP4 displayed strong anticancer activity in human synovial sarcoma cells by disrupting oxidative status, promoting mitochondrial hyperpolarization and causing calcium overload.

Intracellular Ca2+ overload, prolongation of the action potential duration (APD), and downregulation of inward rectifier potassium (IK1) channel are hallmarks of electrical remodeling in cardiac hypertrophy and heart failure (HF). We hypothesized that enhancement of IK1 currents is a compensation for IK1 deficit and a novel modulation for cardiac Ca2+ homeostasis and pathological remodeling. In adult Sprague-Dawley (SD) rats in vivo , cardiac hypertrophy was induced by isoproterenol (Iso) injection (i.p., 3 mg/kg/d) for 3, 10, and 30 days. Neonatal rat ventricular myocytes (NRVMs) were isolated from 1 to 3 days SD rat pups and treated with 1 μmol/L Iso for 24 h in vitro. The effects of zacopride, a selective IK1/Kir2.1 channel agonist, on cardiac remodeling/hypertrophy were observed in the settings of 15 μg/kg in vivo and 1 μmol/L in vitro. After exposing to Iso for 3 days and 10 days, rat hearts showed distinct concentric hypertrophy and fibrosis and enhanced pumping function (P < 0.01 or P < 0.05), then progressed to dilatation and dysfunction post 30 days. Compared with the age-matched control, cardiomyocytes exhibited higher cytosolic Ca2+ (P < 0.01 or P < 0.05) and lower SR Ca2+ content (P < 0.01 or P < 0.05) all through 3, 10, and 30 days of Iso infusion. The expressions of Kir2.1 and SERCA2 were downregulated, while p -CaMKII, p -RyR2, and cleaved caspase-3 were upregulated. Iso-induced electrophysiological abnormalities were also manifested with resting potential (RP) depolarization (P < 0.01), APD prolongation (P < 0.01) in adult cardiomyocytes, and calcium overload in cultured NRVMs (P < 0.01). Zacopride treatment effectively retarded myocardial hypertrophy and fibrosis, preserved the expression of Kir2.1 and some key players in Ca2+ homeostasis, normalized the RP (P < 0.05), and abbreviated APD (P < 0.01), thus lowered cytosolic [Ca2 +]i (P < 0.01 or P < 0.05). IK1channel blocker BaCl2 or chloroquine largely reversed the cardioprotection of zacopride. We conclude that cardiac electrical remodeling is concurrent with structural remodeling. By enhancing cardiac IK1, zacopride prevents Iso-induced electrical remodeling around intracellular Ca2+ overload, thereby attenuates cardiac structural disorder and dysfunction. Early electrical interventions may provide protection on cardiac remodeling. [ABSTRACT FROM AUTHOR]

Background: The dual Na+ and cardiac Ca2+-release channel inhibitor, Flecainide (FLEC) is effective in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by mutations in cardiac Ca2+-release channels (RyR2), calsequestrin (Casq2), or calmodulin. FLEC suppresses spontaneous Ca2+ waves in Casq2-knockout (Casq2−/−) cardiomyocytes, a CPVT model. However, a report failed to find FLEC efficacy against Ca2+ waves in another CPVT model, RyR2-R4496C heterozygous mice (RyR2R4496C+/−), raising the possibility that FLEC efficacy may be mutation dependent. Objective: To address this controversy, we compared FLEC in Casq2−/− and RyR2R4496C+/− cardiomyocytes and mice under identical conditions. Methods: After 30 min exposure to FLEC (6 μM) or vehicle (VEH), spontaneous Ca2+ waves were quantified during a 40 s pause after 1 Hz pacing train in the presence of isoproterenol (ISO, 1 μM). FLEC efficacy was also tested in vivo using a low dose (LOW: 3 mg/kg ISO + 60 mg/kg caffeine) or a high dose catecholamine challenge (HIGH: 3 mg/kg ISO + 120 mg/kg caffeine). Results: In cardiomyocytes, FLEC efficacy was dependent on extracellular [Ca2+]. At 2 mM [Ca2+], only Casq2−/− myocytes exhibited Ca2+ waves, which were strongly suppressed by FLEC. At 3 mM [Ca2+] both groups exhibited Ca2+ waves that were suppressed by FLEC. At 4 mM [Ca2+], FLEC no longer suppressed Ca2+ waves in both groups. Analogous to the results in myocytes, RyR2R4496C+/− mice (n = 12) had significantly lower arrhythmia scores than Casq2−/− mice (n = 9), but the pattern of FLEC efficacy was similar in both groups (i.e., reduced FLEC efficacy after HIGH dose catecholamine challenge). Conclusion: FLEC inhibits Ca2+ waves in RyR2R4496C+/− cardiomyocytes, indicating that RyR2 channel block by FLEC is not mutation-specific. However, FLEC efficacy is reduced by Ca2+ overload in vitro or by high dose catecholamine challenge in vivo , which could explain conflicting literature reports. [ABSTRACT FROM AUTHOR]