Ca Overload Research Articles

Perovskite-structured ultrasensitive CaMnO3 nanoenzyme enables highly efficient ultrasound-amplified and catalysis-involved synergistic tumor therapy

Catalytic nanoenzyme-mediated therapy with reactive oxygen species generation feature and tumor microenvironment regulation characteristic has demonstrated high therapeutic efficacy in oncological field, whereas its low enzyme-mimicking reaction efficacy typically results in dissatisfied therapeutic effect and outcome. Herein, an ultrasensitive CaMnO3 (CMO) nanoenzyme has been rationally designed and engineered for enzyodynamic-calcium overload synergistic tumor therapy. CMO nanoenzyme with oxidase (OXD)-, peroxidase (POD)- and glutathione peroxidase (GPx)-mimic activities is responsible for the generation of reactive oxygen species, the consumption of intra-tumor glutathione and mitochondrial damage. For the evaluation of OXD and POD-mimicking activities, CMO with ultra low concentration (1 μg ml−1) can rapidly catalyze the color reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) hydrochloride and reach the reaction termination in a very short time (1 s). Moreover, the release and overload of Ca2+ strengthen the tumor cell death. Exogenous ultrasound stimulation further amplifies the enzyodynamic-Ca2+ overload synergistic tumor-therapeutic efficacy. In vitro and in vivo results affirm the transcendent antineoplastic outcomes of CMO. Meanwhile, the existence of Mn2+ endows nanoenzyme with T1-weighted MR imaging capacity. This work provides new strategies for efficient enzyme-mimicking activity and tumor synergistic treatment based on new perspectives on the engineering of multifunctional therapeutic nanomedicine.

Chondroitin Sulfate-Modified Hydroxyapatite for Caspase-1 Activated Induced Pyroptosis through Ca Overload/ER Stress/STING/IRF3 Pathway in Colorectal Cancer.

Immune checkpoint inhibitors, are the fourth most common therapeutic tool after surgery, chemotherapy, and radiotherapy for colorectal cancer (CRC). However, only a small proportion (≈5%) of CRC patients, those with "hot" (immuno-activated) tumors, benefit from the therapy. Pyroptosis, an innovative form of programmed cell death, is a potentially effective means to mediate a "cold" to "hot" transformation of the tumor microenvironment (TME). Calcium-releasing hydroxyapatite (HAP) nanoparticles (NPs) trigger calcium overload and pyroptosis in tumor cells. However, current limitations of these nanomedicines, such as poor tumor-targeting capabilities and insufficient calcium (Ca) ion release, limit their application. In this study, chondroitin sulfate (CS) is used to target tumors via binding to CD44 receptors and kaempferol (KAE) is used as a Ca homeostasis disruptor to construct CS-HAP@KAE NPs that function as pyroptosis inducers in CRC cells. CS-HAP@KAE NPs bind to the tumor cell membrane, HAP released Ca in response to the acidic environment of the TME, and kaempferol (KAE) enhances the influx of extracellular Ca, resulting in intracellular Ca overload and pyroptosis. This is associated with excessive endoplasmic reticulum stress triggered activation of the stimulator of interferon genes/interferon regulatory factor 3 pathway, ultimately transforming the TME from "cold" to "hot".

Decreased eggshell strength caused by impairment of uterine calcium transport coincide with higher bone minerals and quality in aged laying hens

BackgroundDeteriorations in eggshell and bone quality are major challenges in aged laying hens. This study compared the differences of eggshell quality, bone parameters and their correlations as well as uterine physiological characteristics and the bone remodeling processes of hens laying eggs of different eggshell breaking strength to explore the mechanism of eggshell and bone quality reduction and their interaction. A total of 240 74-week-old Hy-line Brown laying hens were selected and allocated to a high (HBS, 44.83 ± 1.31 N) or low (LBS, 24.43 ± 0.57 N) eggshell breaking strength group.ResultsA decreased thickness, weight and weight ratio of eggshells were observed in the LBS, accompanied with ultrastructural deterioration and total Ca reduction. Bone quality was negatively correlated with eggshell quality, marked with enhanced structures and increased components in the LBS. In the LBS, the mammillary knobs and effective layer grew slowly. At the initiation stage of eggshell calcification, a total of 130 differentially expressed genes (DEGs, 122 upregulated and 8 downregulated) were identified in the uterus of hens in the LBS relative to those in the HBS. These DEGs were relevant to apoptosis due to the cellular Ca overload. Higher values of p62 protein level, caspase-8 activity, Bax protein expression and lower values of Bcl protein expression and Bcl/Bax ratio were seen in the LBS. TUNEL assay and hematoxylin-eosin staining showed a significant increase in TUNEL-positive cells and tissue damages in the uterus of the LBS. Although few DEGs were identified at the growth stage, similar uterine tissue damages were also observed in the LBS. The expressions of runt-related transcription factor 2 and osteocalcin were upregulated in humeri of the LBS. Enlarged diameter and more structural damages of endocortical bones and decreased ash were observed in femurs of the HBS.ConclusionThe lower eggshell breaking strength may be attributed to a declined Ca transport due to uterine tissue damages, which could affect eggshell calcification and lead to a weak ultrastructure. Impaired uterine Ca transport may result in reduced femoral bone resorption and increased humeral bone formation to maintain a higher mineral and bone quality in the LBS.Graphical

A ROS storm generating nanocomposite for enhanced chemodynamic therapy through H2O2 self-supply, GSH depletion and calcium overload.

Catalytic generation of toxic hydroxyl radicals (˙OH) from hydrogen peroxide (H2O2) is an effective strategy for tumor treatment in chemodynamic therapy (CDT). However, the intrinsic features of the microenvironment in solid tumors, characterized by limited H2O2 and overexpressed glutathione (GSH), severely impede the accumulation of intracellular ˙OH, posing significant challenges. To circumvent these critical issues, in this work, a CaO2-based multifunctional nanocomposite with a surface coating of Cu2+ and L-buthionine sulfoximine (BSO) (named CaO2@Cu-BSO) is designed for enhanced CDT. Taking advantage of the weakly acidic environment of the tumor, the nanocomposite gradually disintegrates, and the exposed CaO2 nanoparticles subsequently decompose to produce H2O2, alleviating the insufficient supply of endogenous H2O2 in the tumor microenvironment (TME). Furthermore, Cu2+ detached from the surface of CaO2 is reduced by H2O2 and GSH to Cu+ and ROS. Then, Cu+ catalyzes H2O2 to generate highly cytotoxic ˙OH and Cu2+, forming a cyclic catalysis effect for effective CDT. Meanwhile, GSH is depleted by Cu2+ ions to eliminate possible ˙OH scavenging. In addition, the decomposition of CaO2 by TME releases a large amount of free Ca2+, resulting in the accumulation and overload of Ca2+ and mitochondrial damage in tumor cells, further improving CDT efficacy and accelerating tumor apoptosis. Besides, BSO, a molecular inhibitor, decreases GSH production by blocking γ-glutamyl cysteine synthetase. Together, this strategy allows for enhanced CDT efficiency via a ROS storm generation strategy in tumor therapy. The experimental results confirm and demonstrate the satisfactory tumor inhibition effect both in vitro and in vivo.

Neurotoxicity study of lead-based perovskite nanoparticles

Lead-based halide perovskite nanoparticles (Pb-PNPs) are promising alternatives for the next-generation of optoelectronic materials, while their nonnegligible biological behavior after exposure are still an enigma. Here, we clarify the translocation, biotransformation, and biodistribution related neurotoxicity of representative CsPbBr3 PNPs in C57BL/6J mice after intranasal administration through a combination of the feat of advanced synchrotron radiation (SR) and traditional analytical techniques. SR-based microscopic X-ray fluorescence scanning, inductively coupled plasma mass spectrometry and behavioral data demonstrate that CsPbBr3 PNPs can be transported and accumulated in the hippocampus, easily triggering Ca overload, causing severe damage of hippocampus-dependent learning, memory, and cognition behavior. Meanwhile, SR–based X-ray absorption near-edge spectroscopy analysis also reveals that CsPbBr3 PNPs can transform into soluble and insoluble lead compounds in different physiological environments. The toxicity of CsPbBr3 PNPs is higher than that of soluble Pb(Ac)2 due to the sustained release of Pb ions. The CsPbBr3 PNPs cause nerve cell apoptosis through triggering intracellular Ca2+ overload, upregulating reactive oxygen species production, while disordering the mitochondrial membrane potential. Our work provides significant insights into the neurological effects and mechanism of Pb-PNPs.

Alzheimer's disease-related presenilin 1 M146L mutation disrupts SERCA2a regulation and increases propensity of Ca waves in ventricular cardiomyocytes.

Presenilin 1 (PS1) is a transmembrane protein expressed in the endoplasmic reticulum (ER) of different cell types. It has been shown that Ca mishandling due to PS1 mutations contributes to the Alzheimer's disease (AD)-related neurodegeneration. PS1 is also expressed in the heart. Several clinical studies revealed that PS1 mutations are associated with cardiomyopathies. We previously showed that PS1 interacts with the cardiac ER Ca ATPase (SERCA2a) and regulates its function. Here, we studied the role of AD-related PS1M146L mutation in regulation of intracellular Ca homeostasis. Fluorescent resonance energy transfer (FRET) experiments in HEK293 cells transfected with fluorescently labeled SERCA2a and PS1M146L revealed that the mutation does not disrupt the interaction between these two proteins. Measurements of SERCA2a-mediated Ca transport showed that at low ER Ca loads ([Ca]ER≤0.15 mM), both PS1WT and PS1M146L enhance ER Ca uptake by a similar level (∼40%) compared to control (no PS1 expression). At high ER Ca loads ([Ca]ER≥0.35 mM), however, PS1WT reduced ER Ca uptake and ER Ca load, whereas PS1M146L was less effective in regulating SERCA2a function. We used in vivo gene delivery in mouse cardiac wall to study the effect of PS1WT and PS1M146L overexpression on Ca regulation in ventricular myocytes. We found that both PS1WT and PS1M146L localized predominantly at the sarcoplasmic reticulum. Analysis of cytosolic Ca dynamics revealed that PS1WT overexpression in cardiomyocytes increases diastolic Ca and decreases Ca transient amplitudes. In contrast, PS1M146L expression increased propensity of spontaneous Ca waves. These results illustrate that the lack of SERCA2a regulation by PS1M146L at high ER Ca loads can lead to ER Ca overload and Ca waves. These defects in Ca regulation might contribute to the onset of cardiomyopathies in patients with PS1 mutations.

1295TiP Investigation of calcium electroporation (CaEP) therapy in malignant cutaneous and subcutaneous tumours: A non-randomized phase II clinical trial of a novel palliative therapy

Skin metastases can occur from any tumour entity and often cause distress. Surgery or radiation are options, limitations arise from size or location and prior treatments. Electroporation (EP) creates transient pores in cell membranes by short high-voltage pulses allowing diffusion of molecules into the cells. EP with systemic chemotherapy is a standard therapy for cutaneous tumours, causing cell death of tumour cells and skin tissue recovering. Calcium-ions (Ca) are part of various signalling pathways. Intracellular Ca overload induces celldeath through ATP depletion as demonstrated in vitro and vivo, with healthy cells recovering. Intratumoural Ca-injection before EP-treament of affected areas is a new, promising palliative treatment reducing use of chemotherapy and interactions anti-tumour-therapies. The purpose of this trial was to investigate CaEP as therapy for cutaneous and subcutaneous tumours. Approval of German and Danish Drug Oversight Agencies and local Ethics Committees was obtained prior to the start of the trial. It was supported from the European Fund for Regional Development as part of the “Changing Cancer Care”-project. This multi-centre non-randomized single-arm phase II trial investigates the use of CaEP in cutaneous and subcutaneous tumours of any cancer histology. All included patients receive CaEP. Primary endpoint is clinical response rate at two months. Response rate is defined as number of responding to treated lesions, evaluated by reduction in size, documented with clinical photography. Secondary endpoints include: treatment response at time points month 1, 3, 4, 6 and 12; histological tissue changes in biopsies one year after treatment; response on MRI on a subset of patients; quality-of-life assessments before treatment, after 2 and 12 months using the EORTC QLQ-C15-PAL questionnaire; examining systemic immune response through routine scans; response due to tumour histology; response rate of all tumours treated; response rate per patient; current of applied pulses and qualitative interviews of one subset of patients. The study is conducted at three cancer centres in Germany and Denmark. EudraCT 2019-004314-34; NCT04225767. The authors.

Abstract P1094: Sexual Dimorphism In Histone Deacetylases Activity Regulates Ventricular Calcium Handling Properties

Introduction: There is a sexual dimorphism in cardiac excitation contraction coupling (ECC) with female ventricular myocytes (F VM ) exhibiting an attenuated basal calcium concentration ([Ca] i ), Ca transient (CaT) amplitude, and a prolonged CaT decay constant (τ). Increased expression levels of the sodium-calcium exchanger (NCX) have been described in F VM however, the role NCX expression plays in sexual dimorphism of physiological and pathophysiological Ca handling properties, and how NCX expression is regulated in a sex specific manner remains to be determined. Here we tested the hypothesis, that a sexual dimorphism in HDAC activity regulates NCX expression and NCXs differential contribution to the CaT adds to the sexual dimorphism in ventricular ECC. Method: [Ca] i was quantified in field stimulated, freshly isolated ventricular myocytes (VMs) from male (M VM ) and female (F VM ) FVB/N mice loaded with the Ca sensitive fluorescent dye Fluo-4/AM. Changes in NCX expression were quantified by qPCR and western blotting. Results: Under basal conditions F VM exhibited an attenuated CaT amplitude (ΔF/F 0 ; M VM :1.8±0.08, F VM :1.4±0.06, p=0.038) and prolonged CaT decay constant (τ: M VM :0.37±0.01ms, F VM :0.46±0.02ms, p<0.01). The caffeine induced Ca transient (CaT caf ) amplitude was comparable in F VM and M VM , but the CaT caf decay constant (τ caf: M VM : 3.34±0.22s ; F VM :4.81±0.36s) was longer in F VM although increased NCX expression was determined on the protein and mRNA level. Consistent with increased NCX expression, F VM showed an attenuated increase in basal [Ca] i (F/F 0 ; M= 60%, F=44%, p=0.0033) and τ (M=0.37±0.01s, F=0.46±0.02s, p<0.0001) when SERCA activity was attenuated (CPA: 10μM). Also during conditions favoring NCX reverse mode ([Na] o =93, 46 mM) F VM exhibited a larger increase in CaT amplitude (ΔF/F 0 ; M=81%, F=133%, p<0.01) and an increased propensity for spontaneous Ca release events. Inhibition of HDAC class II (LMK: 5mg/kg/day) did not change Ca handling properties in F VM but slowed τ (M LMK : 0.44±0.03ms) and τ caf in M VM (M LMK =5.1±0.33s) making them comparable to F VM . Conclusion: The sexual dimorphism in ventricular ECC is mediated by attenuated HDAC activity in F VM s and facilitates NCX Ca overload.

Cardioprotection in ischemia-reperfusion injury achieved by a single amino acid substitution of NCX1 (H165A)

We used genetically modified mice to test the hypothesis that pH regulation of the cardiac sodium calcium-exchanger (NCX1) contributes to ischemia/reperfusion (I/R) injury. NCX1 is the dominant calcium (Ca) efflux mechanism of cardiomyocytes and is strongly regulated by pH. During ischemia, when pH is lowered by anaerobic glycolysis, inhibition of NCX1 by acidosis may potentiate the Na overload imposed by Na/K pump failure and Na-H exchange. Upon reperfusion and restoration of normal pH, this high intracellular Na concentration drives the now functional NCX1 into reverse mode resulting in Ca overload.

Abstract 11475: A Genetically Modified pH Resistant NCX1 Mouse Prevents Calcium Overload During Acidosis and is Cardioprotective

Acidosis inhibits the cardiac sodium(Na)/calcium(Ca) exchanger (NCX1), allegedly contributing to elevated cytoplasmic Na and Ca concentrations during ischemia. Upon reperfusion and restoration of normal pH, a reactivated NCX1 abruptly shifts into reverse (Ca influx) mode because of the abnormally high cellular Na concentration, thus producing massive Ca overload and cell damage. To test the dependence of ischemia/reperfusion (I/R) injury on NCX1’s pH sensitivity, we used CRISPR/Cas9 technology to create mice with a pH-resistant NCX1 by substituting Alanine for Histidine at position 165 (H165A). These mice live into adulthood with slightly reduced LV function (EF% H165A: 50 ± 5; Wildtype (WT): 67 ± 2; p<0.025, N=4-6). In enzymatically isolated adult ventricular myocytes, we used the patch clamp technique and voltage ramps from +80 to -120 mV to confirm that NCX1 current in H165A mice is resistant to pH reduction (from 7.4 to ~pH 6.4 by substituting 80 mM Na-Acetate for equimolar NaCl) when compared to WT animals (% reduction at +80mV: WT 77.7± 2.8, P<0.027 in 3 cells from 2 mice; H165A 0.21 ± 11.35, P=0.31 in 7 cells from 3 mice). We then performed I/R studies in explanted hearts perfused in Langendorff fashion with oxygenated Tyrode’s containing (in mM): 136 NaCl, 1.8 CaCl 2 , 5.4 KCl, 0.33 NaH 2 PO 4 , 1.0 MgCl 2 , 10 HEPES, and 10 glucose, pH 7.4, 37 o C. 20 minutes of stop-flow ischemia were followed by reperfusion for 120 minutes. Necrosis area (pixels 2 ) in WT was 26 ± 3 (N=11), compared to only 9 ± 1 (N=7) in H165A mice (P<0.0004). To compare effects of lower pH on isolated cardiomyocyte Ca, we used a photometric epifluorescence detection system to measure cytosolic Ca in enzymatically isolated ventricular myocytes loaded with the Ca indicator fura-2 AM (2 μM). When pH was lowered to ~6.4, Ca transients (ΔF340/F380) increased in WT from 0.6 to 0.9 (P < 0.0004, N=19 cells from 3 mice) but notably did not change in H165A (0.5 ± 0.03 @ pH 7.4, 0.5 ± 0.04 @ pH 6.4, P=0.307, N=21 cells from 3 mice). pH changes were verified in separate cells using the pH indicator SNARF-AM (10 μM). We conclude that eliminating pH sensitivity in NCX1 has major effects on intracellular Ca regulation, is cardioprotective, and a potential strategy for therapies in patients with prolonged ischemia prior to reperfusion.

Enteral Ca-Intake May Be Low and Affects Serum-PTH-Levels in Pre-school Children With Chronic Kidney Disease.

Treatment of chronic kidney disease (CKD) mineral bone disorder (MBD) is challenging in growing children due to the high amount of calcium needed for normal bone mineralization and the required dietary phosphate restriction, which often includes intake of calcium-rich products such as milk. Therefore, enteral calcium-intake (Ca-I) was calculated.Patients: We looked at pediatric CKD-Patients aged 0–6 years.Design: We used a retrospective analysis of Ca-I from dietary data collections. Ca-I below 60% or above 100% of the D-A-CH and the KDOQI reference values were considered as severe Ca deficiency or Ca overload, respectively.Results: We had 41 children, median age 1.1 (range 0-5.8) years, body weight 7.3 (2.4–19.9) kg, and length 68 (48-105) cm at the time of first dietary data collection. Renal function was classified as CKD stage III in 20, IV in 28, V in 44, and VD in 142 dietary data collections. At the first dietary data collection, 5 children were in the CKD stage III, 10 in IV, 9 in V, and 17 were on dialysis. Only one child progressed to a higher CKD stage. In total, 234 dietary data collections were analyzed, and 65 follow-up collections were available from 33 children after a time interval of 26 (1–372) days. The median caloric intake was 120 (47–217)% of D-A-CH RDI. In 149 (63.6%) of the dietary data collections, enteral Ca-I was below the target (<100% of the D-A-CH and KDOQI RDI). Severe Ca-deficiency was found in 11 (26%) and 4 (12%) of the children at the first and second dietary data collection, respectively. In total, 11 children were on Ca-containing phosphate binders. In dietary data collection 1 and 2, there were seven children. From these, 4/7 and 4/7 patients had an enteral total Ca-I above the 100% D-A-CH-limit or above the KDOQI limit, respectively. Absolute dietary Ca-I and Ca-I normalized to body weight correlated negatively with PTH (r = −0.196, p < 0.005 and r = −0.13, p < 0.05).Conclusion: Enteral Ca-I should repeatedly be monitored in CKD children because many may may otherwise be underexposed to enteral calcium and overexposed when calcium-containing phosphate binders are given. Our findings suggest a major impact of dietary calcium supply on bone health in pediatric CKD.

Increasing SERCA function promotes initiation of calcium sparks and breakup of calcium waves.

Increasing sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump activity enhances sarcoplasmic reticulum calcium (Ca) load, which increases both ryanodine receptor opening and driving force of Ca release flux. Both of these effects promote Ca spark formation and wave propagation. However, increasing SERCA activity also accelerates local cytosolic Ca decay as the wave front travels to the next cluster, which limits wave propagation. As a result, increasing SERCA pump activity has a biphasic effect on the propensity of arrhythmogenic Ca waves, but a monotonic effect to increase Ca spark frequency and amplitude. Waves of sarcoplasmic reticulum (SR) calcium (Ca) release can cause arrhythmogenic afterdepolarizations in cardiac myocytes. Ca waves propagate when Ca sparks at one Ca release unit (CRU) recruit new Ca sparks in neighbouring CRUs. Under normal conditions, Ca sparks are too small to recruit neighbouring Ca sparks where Ca sensitivity is also low. However, under pathological conditions such as a Ca overload or ryanodine receptor (RyR) sensitization, Ca sparks can be larger and propagate more readily as macro-sparks or full Ca waves. Increasing SERCA pump activity promotes SR Ca load, which promotes RyR opening and increases driving force of the Ca release flux from SR to cytosol, promoting Ca waves. However, high sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) activity can also decrease local cytosolic [Ca] as it approaches the next CRU, thereby reducing wave appearance and propagation. In this study, we use a physiologically detailed model of subcellular Ca cycling and experiments in phospholamban-knockout mice, to show how Ca waves are initiated and propagate and how different conditions contribute to the generation and propagation of Ca waves. We show that reducing diffusive coupling between Ca sparks by increasing SERCA activity prevents Ca waves by reducing [Ca] at the next CRU, as do Ca buffers, low intra-SR Ca diffusion and distance between CRUs. Increasing SR Ca uptake rate has a biphasic effect on Ca wave propagation; initially it enhances Ca spark probability and amplitude and CRU coupling, thereby promoting arrhythmogenic Ca wave propagation, but at higher levels SR Ca uptake can abort those arrhythmogenic Ca waves.

Effect of Oxygen Free Radicals on Cardiac Contractile Activity and Sarcolemmal Na(+)-Ca(2+) Exchange.

BACKGROUND: Although oxygen free radicals have been shown to induce myocardial cell damage and cardiac dysfunction, the exact mechanism by which these radicals affect the heart function is not clear. Since the occurrence of intracellular Ca(2+) overload is critical in the genesis of cellular damage and cardiac dysfunction, and since the sarcolemmal Na(+)-Ca(2+) exchange is intimately involved in Ca(2+) movements in myocardium, this study was undertaken to examine the effects of oxygen free radicals on the relationship between changes in cardiac contractile force development and sarcolemmal Na(+)-Ca(2+) exchange activity. METHODS AND RESULTS: Isolated rat hearts were perfused with a medium containing xanthine plus xanthine oxidase for different times, and changes in contractile force as well as sarcolemmal Na(+)-(2+) exchange activity were monitored. Perfusion of the heart with xanthine plus xanthine oxidase resulted in a transient increase followed by a marked decrease in contractile activity; the resting tension was markedly increased. The xanthine plus xanthine oxidase-induced depression in developed tension, rate of contraction, and rate of relaxation, except the transient increase in contractile activity, was prevented by the addition of catalase, but not by superoxide dismutase, in the perfusion medium. A time-dependent depression in sarcolemmal Na(+)-Ca(2+) was also evident upon perfusing the heart with xanthine plus xanthine oxidase. This depression in Na(+)-dependent Ca(2+) uptake was associated with a decrease in the maximal velocity of reaction without any changes in the affinity of Na(+)-Ca(2+) exchanger for Ca(2+). The presence of catalase, unlike superoxide dismutase, prevented the decrease in sarcolemmal Na(+)-Ca(2+) exchange activity in hearts perfused with xanthine plus xanthine oxidase. CONCLUSIONS: The results support the view that a depression in the sarcolemmal Na(+)-Ca(2+) exchange activity may contribute to the occurrence of intracellular Ca(2+) overload and subsequent decrease in contractile activity. Furthermore, these actions of xanthine plus xanthine oxidase in the whole heart appear to be a consequence of H(2)O(2) production rather than the generation of superoxide radicals.

MO055STABLE CALCIUM ISOTOPES: A NOVEL BIOMARKER OF BONE MINERAL CONTENT IN PATIENTS WITH CHRONIC KIDNEY DISEASE

Abstract Background and Aims In CKD dysregulated calcium (Ca) homeostasis is common and causally associated with reduced bone mineral density (BMD) and vascular calcification. Currently available radiological measures and biomarkers do not allow accurate evaluation of BMD. The aim of our study was to determine the sensitivity and specificity of stable Ca isotopes in determining bone mineral content. Method We measured stable Ca isotopes 44Ca and 42Ca by plasma-ionization mass-spectrometry in blood and urine. The relationship between bone Ca gain and loss is calculated using a compartment model based on Ca kinetics, and expressed as δ44/42Ca. Ca absorption from bones increases δ44/42CaBlood and δ44/42CaUrine, and resorption decreases these fractions. 104 children in CKD4-5 and on dialysis (CKD4-5D), 40 matched controls and 100 adults underwent Ca isotope measurement, bone biomarkers, dual energy x-ray absorptiometry (DXA) and tibial peripheral quantitative CT scan (pQCT), an accurate measure of cortical BMD. Results In healthy children the δ44/42CaBlood and δ44/42CaUrine were higher than in adults (p&amp;lt;0.0001), reflecting avid Ca uptake during bone formation. Since urinary Ca excretion is impaired in CKD, δ44/42CaBlood was higher and δ44/42CaUrine lower in children with CKD4-5D compared to controls (p&amp;lt;0.001 for both); Figure 1. In CKD2-5D δ44/42CaBlood positively correlated with cholecalciferol (p=0.01) and alfacalcidol (p=0.002) doses, implying increased bone Ca uptake when Ca bioavailability is increased. δ44/42CaBlood positively correlated with biomarkers of bone formation (alkaline phosphatase, p=0.05) and negatively with bone resorption markers (PTH, p=0.013; TRAP5b, p&amp;lt;0.001 and CTX, p=0.006). δ44/42CaBlood positively correlated with tibial cortical BMD-Z-score (p=0.006, R2=0.39), and DXA hip BMD-Z-score (p=0.02). On multivariable linear regression analysis significant and independent predictors of tibial cortical BMD-Z-score were δ44/42CaBlood (β=0.68, p=0.006) and PTH (β-0.39, p=0.04), together predicting 67% of the variability in BMD. Conclusion Ca isotope ratios provide a novel, non-invasive method of assessing bone mineralization. Further studies are in progress to define optimal levels of δ44/42CaBlood that can guide safe and effective treatment to prevent Ca deficiency or overload.

Dynamic Palmitoylation of the Sodium-Calcium Exchanger Modulates Its Structure, Affinity for Lipid-Ordered Domains, and Inhibition by XIP

SummaryThe transmembrane sodium-calcium (Na-Ca) exchanger 1 (NCX1) regulates cytoplasmic Ca levels by facilitating electrogenic exchange of Ca for Na. Palmitoylation, the only reversible post-translational modification known to modulate NCX1 activity, controls NCX1 inactivation. Here, we show that palmitoylation of NCX1 modifies the structural arrangement of the NCX1 dimer and controls its affinity for lipid-ordered membrane domains. NCX1 palmitoylation occurs dynamically at the cell surface under the control of the enzymes zDHHC5 and APT1. We identify the position of the endogenous exchange inhibitory peptide (XIP) binding site within the NCX1 regulatory intracellular loop and demonstrate that palmitoylation controls the ability of XIP to bind this site. We also show that changes in NCX1 palmitoylation change cytosolic Ca. Our results thus demonstrate the broad molecular consequences of NCX1 palmitoylation and highlight a means to manipulate the inactivation of this ubiquitous ion transporter that could ameliorate pathologies linked to Ca overload via NCX1.

Cardioprotection Conferred by a CRISPR/Cas9 Single Amino Acid Substitution of NCX1 (H165A): the PH Insensitive NCX Mouse

The cardiac sodium calcium-exchanger (NCX1), the dominant calcium efflux mechanism of cardiomyocytes, is strongly regulated by pH. This pH regulation is thought to contribute to ischemia/reperfusion injury by allowing Na ions to accumulate to very high levels during ischemia when NCX is disabled by low pH, resulting in massive Ca overload via reverse NCX when reperfusion suddenly restores normal pH and NCX function. We recently found in a heterologous system that substitution of a single Histidine for Alanine at position 165 (H165A) of NCX1 drastically decreases its pH sensitivity (John S et al., J Gen Physiol, 2018). We then used CRISPR/Cas9 technology (Ma X et al, Sci Reports, 2017) to produce the same single amino acid substitution in mice. We confirmed the mutation by sequencing and RT-qPCR. The mice live into adulthood and are fertile with reduced LV function. In enzymatically isolated adult ventricular myocytes, we used the patch clamp technique with voltage ramps to measure nickel sensitive NCX current. At normal pH, NCX current was the same in control and H165A mice. However, when pH was lowered to 6.4 using NH4Cl, NCX current in H165A mice was minimally inhibited, while NCX in control mice was reduced severely. In voltage mapping studies of control mouse hearts, acidosis to pH 6.4 resulted in the onset of abnormal cardiac rhythm, characterized by repetitive bursts of depolarization alternating with pauses. However, in H165A mice, rhythm was unaffected by lowering the pH. In preliminary whole heart ischemia/reperfusion studies using Langendorff perfusion, pH mice had a 58% reduction in the area of necrosis compared to WT. These results suggest that a pH insensitive NCX may be able to reduce ischemia/reperfusion injury and arrythmia.

Resveratrol Activates Neuronal Autophagy Through AMPK in the Ischemic Brain.

During cerebral ischemia, oxygen and glucose levels decrease, producing many consequences such as the generation of reactive oxygen species, tissue injury, and the general metabolism collapse. Resveratrol triggers signaling dependent on the protein kinase activated by adenosine monophosphate (AMPK), the sensor of cellular energy metabolism that regulates autophagy, eliminates damaged mitochondria, and increases energy sources. In the present study, we investigated the participation of AMPK activation in the protective effect of resveratrol on cerebral ischemia and excitotoxicity. We found that resveratrol increased the levels of phosphorylated AMPK in the cerebral cortex of rats subjected to middle cerebral artery occlusion (MCAO) and in primary cultured neurons exposed to glutamate-induced excitotoxicity. Resveratrol (1.8 mg/Kg; i. v.; administered at the beginning of reperfusion) decreased the infarct area and increased survival of rats subjected to MCAO. In neuronal cultures, resveratrol treatment (40 μM, after excitotoxicity) reduced the production of superoxide anion, prevented the overload of intracellular Ca+2 associated to mitochondrial failure, reduced the release of the lactate dehydrogenase enzyme, and reduced death. It also promoted mitophagy (increased Beclin 1 level, favored the recruitment of LC3-II, reduced LAMP1, and reduced mitochondrial matrix protein HSP60 levels). In both models, inhibition of AMPK activation with Compound C obstructed the effect of resveratrol, showing that its protective effect depends, partially, on the activation of the AMPK/autophagy pathway.

P1602Basic electrophysiological modifications induced by carvedilol in unstrectched and stretched ventricular myocardium

Abstract Background Acute regional ventricular stretch (ARVS) is a pathophysiologic event that may occur in certain situations, originating arrhythmogenic effects through the mechanoelectrical feedback. Mechanical effects of stretch originate calcium-related changes as sarcoplasmic recticulum Ca2+ overload that can trigger Ca2+ diastolic leaks (store-overload-induced Ca2+ release, SOICR), mediated by the cardiac ryanodine receptor (RyR2). SOICR seems to be implicated in the mechanisms underlying stretch-induced arrhythmias. Carvedilol can inhibit the overload of Ca2+ through blocking of beta-adrenergic receptors, and also suppress the release of Ca2+ induced by the SOICR. Purpose The aim of this investigation was to study the effects of carvedilol on the changes in ventricular refractoriness produced by AVRS, directly related with reentrant phenomenon and life-threatening arrhythmias. Methods Eleven adult male New Zealand White rabbits (3–3.5 kg) were heparinized (2500 IU) and euthanized by intravenous injection of sodium thiopental (100 mg/kg), according to European Ethic Guidelines. The hearts were excised, isolated and perfused in a Langendorff system. A pacing electrode and a recording multielectrode (121 electrodes) were placed on the left ventricle epicardium. The ARVS was produced by an “ad hoc” device introduced into the left ventricle. The ventricular effective and functional refractory periods (VERP, VFRP) were determined by the ventricular extrastimulus test with a basic cycle length of 250 ms, previously and at the third minute of ARVS, in control conditions and under Carvedilol (1 μM) infusion. The pacing threshold was determined for each situation and the stimulus amplitude was twice the diastolic threshold. A Student's t-test was used. Significance was reached when p&lt;0.05. Results Myocardial stretch reduced VERP and VFRP with respect to pre-stretch values under control conditions (VERP: 110±13 vs 99±16 ms, VFRP: 119±13 vs 113±13 ms, p&lt;0.05; n=10). No stretch-induced modifications of refractoriness were observed under carvedilol action (VERP: 139±18 vs 140±17 ms, VFRP: 161±29 vs 157±15 ms, ns; n=8). Before stretching, there were differences between control and carvedilol conditions (VERP: 110±13 vs 139±18 ms, VFRP: 119±13 vs 161±29 ms, p&lt;0.001), and during stretch VERP and VFRP were significantly higher under carvedilol perfusion than in control conditions (VERP: 99±16 vs 140±17 ms, VFRP: 113±13 vs 157±15 ms, p&lt;0.001). Conclusion The beta-adrenergic blocker and ryanodine receptor antagonist, carvedilol, attenuates the intrinsic electrophysiological modifications on refractoriness produced by myocardial acute local stretch.

Effects and mechanism of digoxin on atrium electrical remodeling and susceptibility of atrial fibrillation in aged rabbits

Objective: To investigate the effects and mechanism of digoxin on atrium electrical remodeling and susceptibility of atrial fibrillation (AF) in aged rabbits. Methods: Twenty aged male New Zealand rabbits were divided into aged group and aged plus digoxin group (n=10 each). Electrical parameters including heart rate (HR), RR and QT interval, ST segment and P wave dispersion from normal Ⅱ electrocardiogram, and the maximum upstroke velocity (Max(dv/dt)), plateau potential (plateau P), action potential duration of 10%, 20% and 90% (APD(10), APD(20), APD(90)) from recording of monophasic action potential (MAP), as well as atrial effective refractory period (AERP(200)) and dispersion (dERP(200)) with 200 ms of basic cycle length (BCL), and frequency self adaptation of AERP with 300 ms and 150 ms of BCLs (fERP) were recorded and compared between the 2 groups. BCLs and inducibility of AF post programmed electrical stimulation and Burst-pacing in left atrium tissue of rabbits in vivo were also analyzed. The L-type calcium current (I(Ca-L)) in 2 groups were recorded via whole-cell patch clamp technique, and the fluorescence intensity of intracellular free Ca(2+) was detected with Flup-3/AM loading by the laser scanning confocal microscope in enzymatically dissociated single rabbit atrial myocytes. Results: Compared with aged group, the heart rate was faster, RR and QT interval were obvious shorter, ST segment was raised and P wave dispersion was significantly increased in aged plus digoxin group (all P<0.05). Moreover, compared with aged group, the Max(dv/dt) and plateau P were obviously increased, APD(10) and APD(20) were significantly prolongated, and APD(9)0 was significantly shorter in aged plus digoxin group (all P<0.01). Otherwise, the fERP was markedly increased (0.81±0.15 vs. 0.67±0.05), and the induced rate of AF was obviously higher in aged plus digoxin group than in aged group (6/8 vs. 4/9) (all P<0.01). With voltage clamp model, digoxin significantly increased I(Ca-L) of atrial myocytes of aged rabbits, When command potential was 10 mV, the current densities of I(Ca-L) were significantly higher in digoxin group than that in aged group ((15.45±2.38) pA/pF vs. (7.03±1.69) pA/pF, P<0.01). Otherwise, the I-V curve of I(Ca-L) was downward shifted of all I-V curves in digoxin perfused aged atrial cells of rabbits. Moreover, the fluorescence intensities of intracellular free Ca(2+) was significantly higher in aged plus digoxin group than in aged group ((1 748±173) μmol/L vs. (478.13±87.63) μmol/L, P<0.01). Conclusion: Digoxin could aggravate the atrial electrical remodeling in atrium of aged rabbits, facilitate susceptibility of atrial fibrillation in aged rabbit, increased current density of I(Ca-L) and concentration of intracellular free Ca(2+), followed Ca(2+) overload and oscillations might be part of the underlying mechanisms.

Chronic ethanol exposure reduces the expression of NCX3 in the hippocampus of male C57BL/6 mice.

Chronic ethanol (EtOH) exposure can cause intracellular Ca overload by stimulating calcium channel receptors and trigger apoptosis of neurons. NCX3 may play a cytoprotective role in intracellular Ca excretion. In this study, the effect of EtOH on NCX3 was analyzed by observing NCX3 expression in the hippocampus of chronic EtOH-exposed male C57BL/6 mice. Mice were divided into a control group, a 10% EtOH group, and a 20% EtOH group for 30, 60, and 90 days. Behavioral changes were observed using the Morris water maze. The protein and mRNA expressions of NCX3 and their distribution in the hippocampus were observed by western blotting, quantitative PCR, and immunohistochemistry staining. The results showed that EtOH exposure exerted a significant adverse effect on the spatial memory capacity of mice. Increased expression of calpain-1 and cleaved caspase-3 proteins indicated increased apoptosis. The expression of NCX3 in the hippocampus was downregulated after exposure to EtOH (except 10% EtOH for 30 days) and this inhibition was time and dose dependent with EtOH exposure. The level of p-Akt, which is an upstream regulation factor of NCX3, showed a trend similar to that of NCX3 protein. Chronic EtOH exposure reduced the expression of NCX3 in the hippocampus of male C57BL/6 mice, increasing intracellular calcium and apoptosis, resulting in spatial memory impairment in mice.