Significant Difference In Ca2 Research Articles

Leaky RyR2 channels as pro-arrhythmic trigger in the PKP2-deficient atrial myocardium

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): American Heart Association National Institutes of Health Introduction Atrial arrhythmias, like atrial fibrillation, occur in 20–40% of patients with Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC). Atrial fibrillation in ARVC patients is often associated with an increased risk of sustained ventricular arrhythmias and inappropriate ICD shocks. However, the pathophysiology behind the development of atrial arrhythmias in ARVC is far from clear. A genome-wide association study, based on UK-biobank data, revealed a link between atrial fibrillation and variants in the desmosomal gene plakophilin-2 (PKP2). Previous studies show that PKP2 is necessary to maintain the transcription of genes that control intracellular calcium (Ca2+) cycling and that loss of PKP2 expression in adult mouse ventricular tissue leads to disruption of intracellular Ca2+ homeostasis. In this study, we test the hypothesis that loss of PKP2 expression leads to pro-arrhythmic changes in atrial myocytes. Methods Experiments were carried out using a cardiac-specific, tamoxifen (TAM)-activated PKP2 knockout murine model (PKP2-cKO). Cre-negative, flox-positive, TAM-injected littermates were used as controls. We used RNA sequencing, quantitative imaging modalities, as well as biochemical and electrophysiological methods to study the functional and structural properties of atrial PKP2cKO tissue. Studies were carried out 21 days post-tamoxifen injection, a time point at which an arrhythmogenic cardiomyopathy of right ventricular predominance is manifest. Results In contrast to the previously-reported observations in PKP2cKO ventricular myocytes, atrial PKP2cKO myocytes presented no significant differences in Ca2+ transient dynamics, SR load and action potential duration. However, PKP2cKO atrial myocytes showed an increased frequency and amplitude of Ca2+ sparks, in combination with increased diastolic Ca2+ levels as well as an increase in cellular ROS levels. RNAseq data showed an under-representation of mRNA for Integrinα7, a stabilizer of Ryr2 that reduces its phosphorylation. Separate studies showed that PKP2cKO atrial myocytes present impaired relaxation and enhanced sarcomere shortening, a finding consistent with a downregulation in the expression of Myosin Binding Protein C. Isoproterenol (ISO) exposure further increased the frequency of Ca2+ sparks and the levels of diastolic Ca2+, and these effects were reversed by acute (30 minute) in vivo treatment with Ryanodex (medical grade Dantrolene), a known RyR blocker. Conclusion Loss of PKP2 expression affects cellular Ca2+ handling and electrophysiology differently in the atrial versus ventricular myocardium. We speculate that an increased probability of opening of Ryr2 channels, caused by ROS-induced RyR2 oxidation and/or Integrinα7-induced RyR2 phosphorylation, serve as pro-arrhythmic trigger in the PKP2-deficient atrial myocardium, that these effects can be amplified by a catecholaminergic input and that RyR blockers can dampen these pro-arrhythmic effects.

Sex-dependent effect of aging on calcium signaling and expression of TRPM2 and CRAC channels in human neutrophils

The vulnerability of older adults to bacterial infections has been associated with age-related changes in neutrophils. We analyzed the consequences of aging on calcium (Ca2+) mobilization and TRPM2 and CRAC channels expression in human neutrophils. The percentages of granulocytes, mature neutrophils, and neutrophil precursors were equivalent between young and older adults. However, neutrophil chemotaxis towards IL-8, C5a, or fMLP was lower in older adults of both sexes. Interestingly, a stronger Ca2+ transient followed by an identical Ca2+ influx to IL-8 was observed in older adult females. In addition, the Ca2+ response to LPS was delayed and prolonged in neutrophils of older adult males. There was no significant difference in Ca2+ response to fMLP, C5a, or store-operated Ca2+ entry in the older adults. There were also no differences in the expression of CXCR2, CD88, FPLR1, and TLR4. Interestingly, TRPM2- and ORAI1-mRNA expression was lower in neutrophils of older adults, mainly in females. Both channels were detected intracellularly in the neutrophils. TRPM2 was in late endosomes in young adults and in lysosomes in older adult neutrophils. In summary, defective neutrophil chemotaxis in aging seemed not to stem from alterations in Ca2+ signals; nevertheless, the low TRPM2 and ORAI1 expression may affect other functions.

The allosteric gating mechanism of the MthK channel.

Allostery is a fundamental element during channel gating in response to an appropriate stimulus by which events occurring at one site are transmitted to distal sites to regulate activity. To address how binding of the first Ca2+ ion at one of the eight chemically identical subunits facilitates the other Ca2+-binding events in MthK, a Ca2+-gated K+ channel containing a conserved ligand-binding RCK domain, we analysed a large collection of MthK structures and performed the corresponding thermodynamic and electrophysiological measurements. These structural and functional studies led us to conclude that the conformations of the Ca2+-binding sites alternate between two quaternary states and exhibit significant differences in Ca2+ affinity. We further propose an allosteric model of the MthK-gating mechanism by which a cascade of structural events connect the initial Ca2+-binding to the final changes of the ring structure that open the ion-conduction pore. This mechanical model reveals the exquisite design that achieves the allosteric gating and could be of general relevance for the action of other ligand-gated ion channels containing the RCK domain.

Biofortification of Raphanus sativus through irrigation with Ca2+ solutions does not increase calcium content

Micronutrient deficiencies, which affect more than two billion people globally, occur when an individual does not receive adequate amounts of essential vitamins or minerals. Biofortification aims to increase the nutritional content of food crops, and it is an important tool in decreasing the effects of micronutrient deficiencies. Specifically, hypocalcemia, or calcium deficiency, causes a wide range of symptoms that affect many different body systems. In this study, we tested the hypothesis that increasing the amount of calcium available to the food crop during growth would increase the amount of calcium present in the food crop. A total of 180 radish (Raphanus sativus) plants were grown in a controlled environment and irrigated with water of varying Ca2+ concentrations. The height of each plant was measured every five days beginning at day 0 to track the growth. At maturity, a random sample of four plants from each group were tested for calcium and magnesium content using an atomic absorption spectrophotometer. The group that received the highest concentration of Ca2+ grew significantly smaller than most other groups. The Ca2+ contents of leaf samples had no significant difference in Ca2+ or Mg2+ content; however, the Ca2+ content of root samples showed a significant decrease from the control in both Ca2+ and Mg2+ content across several experimental groups. These results provide preliminary evidence that irrigation with Ca2+ solutions does not increase the calcium content of mature plants, but further testing is needed to confirm these results. If increased calcium in irrigation water significantly increases the calcium content of mature radish plants, this technique could be used to increase the calcium content of other crops in areas with high rates of hypocalcemia.

Identification of genes related to tipburn resistance in Chinese cabbage and preliminary exploration of its molecular mechanism

BackgroundTipburn, also known as leaf tip necrosis, is a severe issue in Chinese cabbage production. One known cause is that plants are unable to provide adequate Ca2+ to rapidly expanding leaves. Bacterial infection is also a contributing factor. Different cultivars have varying degrees of tolerance to tipburn. Two inbred lines of Chinese cabbage were employed as resources in this research.ResultsWe determined that the inbred line ‘J39290’ was the tipburn resistant material and the inbred line ‘J95822’ was the tipburn sensitive material based on the severity of tipburn, and the integrity of cell membrane structure. Ca2+ concentration measurements revealed no significant difference in Ca2+ concentration between the two materials inner leaves. Transcriptome sequencing technology was also used to find the differentially expressed genes (DEGs) of ‘J95822’ and ‘J39290’, and there was no significant difference in the previously reported Ca2+ uptake and transport related genes in the two materials. However, it is evident through DEG screening and classification that 23 genes are highly linked to plant-pathogen interactions, and they encode three different types of proteins: CaM/CML, Rboh, and CDPK. These 23 genes mainly function through Ca2+-CaM/CML-CDPK signal pathway based on KEGG pathway analysis, protein interaction prediction, and quantitative real-time PCR (qRT-PCR) of key genes.ConclusionsBy analyzing the Ca2+ concentration in the above two materials, the transcription of previously reported genes related to Ca2+ uptake and transport, the functional annotation and KEGG pathway of DEGs, it was found that Ca2+ deficiency was not the main cause of tipburn in ‘J95822’, but was probably caused by bacterial infection. This study lays a theoretical foundation for exploring the molecular mechanism of resistance to tipburn in Chinese cabbage, and has important guiding significance for genetics and breeding.

Differentiation between fresh and frozen-thawed scallop adductor muscle as raw materials for sashimi during cold storage.

The changes of sensory and biochemical characteristics on the fresh and frozen-thawed scallop adductor muscle during storage at 4°C were discussed in this study. The Quality Index Method (QIM) scheme for evaluating scallop adductor muscle as raw materials for sashimi was proposed for the first time. The results of sensory evaluation showed that frozen-thawed scallop adductor muscle within zero to one day of refrigeration can be happily accepted by consumers, indicating the superiority of freezing for long-distance transportation, although the triangle test confirmed that there are still sensorial differences between fresh and frozen-thawed scallop adductor muscle. The microscopic observation of myofibrils extracted from scallop adductor muscle suggested that the myofibrillar protein which constitutes myofibrils has suffered some extent change due to freezing and thawing, even though the head region of myosin remained stable judging by the fact that there was no significant difference in Ca2+ -ATPase activity (p > 0.05). The changes of adenosine triphosphate (ATP) and its related compounds, and pH value during storage can be regarded as indicators to differentiate fresh and frozen-thawed scallop adductor muscle. The changes of Mg2+ -ATPase activity indicated that the interaction between myosin and actin was weakened by the freezing and thawing process. Practical Application: The QIM scheme can be used to evaluate the scallop adductor muscle as raw materials for sashimi. The mechanism of quality changes in the frozen-thawed scallop adductor muscle was discussed in combination with the destruction of myofibrils, ATP degradation and the decrease of pH value. This study has positive significance for improving the quality of frozen-thawed scallop adductor muscle by combining the changes of sensory and biochemical characteristics.

RyR2 Gain-of-Function and Not So Sudden Cardiac Death

RyR2 Gain-of-Function and Not So Sudden Cardiac Death

Hypo-Osmotic Loading Induces Expression of IL-6 in Nucleus Pulposus Cells of the Intervertebral Disc Independent of TRPV4 and TRPM7.

Painful intervertebral disc (IVD) degeneration is an age-related process characterized by reduced tissue osmolarity, increased catabolism of the extracellular matrix, and elevated levels of pro-inflammatory molecules. With the aging population and constantly rising treatment costs, it is of utmost importance to identify potential therapeutic targets and new pharmacological treatment strategies for low back pain. Transient receptor potential (TRP) channels are a family of Ca2+ permeable cell membrane receptors, which can be activated by multitude of stimuli and have recently emerged as contributors to joint disease, but were not investigated closer in the IVD. Based on the gene array screening, TRPC1, TRPM7, and TRPV4 were overall the most highly expressed TRP channels in bovine IVD cells. We demonstrated that TRPV4 gene expression was down-regulated in hypo-osmotic condition, whereas its Ca2+ flux increased. No significant differences in Ca2+ flux and gene expression were observed for TRPM7 between hypo- and iso-osmotic groups. Upon hypo-osmotic stimulation, we overall identified via RNA sequencing over 3,000 up- or down-regulated targets, from which we selected aggrecan, ADAMTS9, and IL-6 and investigated whether their altered gene expression is mediated through either the TRPV4 or TRPM7 channel, using specific activators and inhibitors (GSK1016790A/GSK2193874 for TRPV4 and Naltriben/NS8593 for TRPM7). GSK1016790A induced the expression of IL-6 under iso-osmotic condition, alike to hypo-osmotic stimulation alone, indicating that this effect might be TRPV4-mediated. However, using the TRPV4 blocker GSK2193874 failed to prevent the increase of IL-6 under hypo-osmotic condition. A treatment with TRPM7-activator did not cause significant changes in the gene expression of tested targets. In conclusion, while TRPV4 and TRPM7 are likely involved in osmosensing in the IVD, neither of them mediates hypo-osmotically-induced gene expression changes of aggrecan, ADAMTS9, and IL-6.

Ca2+ flux through splice variants of the ATP-gated ionotropic receptor P2X7 is regulated by its cytoplasmic N terminus

Activation of ionotropic P2X receptors increases free intracellular Ca2+ ([Ca2+] i ) by initiating a transmembrane cation flux. We studied the "a" and "k" splice variants of the rat purinergic P2X7 receptor (rP2X7aR and rP2X7kR) to exhibit a significant difference in Ca2+ flux through this channel. This difference is surprising because the variants share absolute sequence identity in the area of the pore that defines ionic selectivity. Here, we used patch-clamp fluorometry and chimeric receptors to show that the fraction of the total current carried by Ca2+ is a function of the primary sequence of the cytoplasmic N terminus. Using scanning mutagenesis, we identified five sites within the N terminus that respond to mutagenesis with a decrease in fractional calcium current and an increase in permeability to the polyatomic cation, N-methyl-d-glucamine (NMDG+), relative to Na+ (PNMDG/PNa). We tested the hypothesis that these sites line the permeation pathway by measuring the ability of thiol-reactive MTSET+ to alter the current of cysteine-substituted variants, but we detected no effect. Finally, we studied the homologous sites of the rat P2X2 receptor (rP2X2R) and observed that substitutions at Glu17 significantly reduced the fractional calcium current. Taken together, our results suggest that a change in the structure of the N terminus alters the ability of an intra-pore Ca2+ selectivity filter to discriminate among permeating cations. These results are noteworthy for two reasons: they identify a previously unknown outcome of mutagenesis of the N-terminal domain, and they suggest caution when assigning structure to function for truncated P2X receptors that lack a part of the N terminus.

Cardiac-specific inducible overexpression of human plasma membrane Ca2+ ATPase 4b is cardioprotective and improves survival in mice following ischemic injury.

Background: Heart failure (HF) is associated with reduced expression of plasma membrane Ca2+-ATPase 4 (PMCA4). Cardiac-specific overexpression of human PMCA4b in mice inhibited nNOS activity and reduced cardiac hypertrophy by inhibiting calcineurin. Here we examine temporally regulated cardiac-specific overexpression of hPMCA4b in mouse models of myocardial ischemia reperfusion injury (IRI) ex vivo, and HF following experimental myocardial infarction (MI) in vivoMethods and results: Doxycycline-regulated cardiomyocyte-specific overexpression and activity of hPMCA4b produced adaptive changes in expression levels of Ca2+-regulatory genes, and induced hypertrophy without significant differences in Ca2+ transients or diastolic Ca2+ concentrations. Total cardiac NOS and nNOS-specific activities were reduced in mice with cardiac overexpression of hPMCA4b while nNOS, eNOS and iNOS protein levels did not differ. hMPCA4b-overexpressing mice also exhibited elevated systolic blood pressure vs. controls, with increased contractility and lusitropy in vivo In isolated hearts undergoing IRI, hPMCA4b overexpression was cardioprotective. NO donor-treated hearts overexpressing hPMCA4b showed reduced LVDP and larger infarct size versus vehicle-treated hearts undergoing IRI, demonstrating that the cardioprotective benefits of hPMCA4b-repressed nNOS are lost by restoring NO availability. Finally, both pre-existing and post-MI induction of hPMCA4b overexpression reduced infarct expansion and improved survival from HF.Conclusions: Cardiac PMCA4b regulates nNOS activity, cardiac mass and contractility, such that PMCA4b overexpression preserves cardiac function following IRI, heightens cardiac performance and limits infarct progression, cardiac hypertrophy and HF, even when induced late post-MI. These data identify PMCA4b as a novel therapeutic target for IRI and HF.

SNAP-25b-deficiency increases insulin secretion and changes spatiotemporal profile of Ca2+oscillations in \u03b2 cell networks

SNAP-25 is a protein of the core SNARE complex mediating stimulus-dependent release of insulin from pancreatic β cells. The protein exists as two alternatively spliced isoforms, SNAP-25a and SNAP-25b, differing in 9 out of 206 amino acids, yet their specific roles in pancreatic β cells remain unclear. We explored the effect of SNAP-25b-deficiency on glucose-stimulated insulin release in islets and found increased secretion both in vivo and in vitro. However, slow photo-release of caged Ca2+ in β cells within pancreatic slices showed no significant differences in Ca2+-sensitivity, amplitude or rate of exocytosis between SNAP-25b-deficient and wild-type littermates. Therefore, we next investigated if Ca2+ handling was affected in glucose-stimulated β cells using intracellular Ca2+-imaging and found premature activation and delayed termination of [Ca2+]i elevations. These findings were accompanied by less synchronized Ca2+-oscillations and hence more segregated functional β cell networks in SNAP-25b-deficient mice. Islet gross morphology and architecture were maintained in mutant mice, although sex specific compensatory changes were observed. Thus, our study proposes that SNAP-25b in pancreatic β cells, except for participating in the core SNARE complex, is necessary for accurate regulation of Ca2+-dynamics.

Neonatal neuronal apoptosis after betamethasone administration in pregnant Wistar rats

Objective: To analyze the apoptosis of cortical and hippocampal neurons in newborn following the intramuscular administration of betamethasone in pregnant Wistar rats.Methods: Betamethasone or placebo was administered to 10 pregnant rats. Subsequently, 98 newborns were analyzed in three different groups: therapeutic dose (TD, 20 mg/kg), triple therapeutic dose (3TD, 60 mg/kg), and nine times TD (9TD, 180 mg/kg). Forty-four newborns were injected with placebo (control subjects – CTR). Neuronal apoptosis was measured by immunofluorescence using the TUNEL assay. The one-way analysis of variance, Tukey–Kramer (parametric) test and Kruskal–Wallis (non-parametric) test were used for statistical analysis.Results: The CA1 area of the hippocampus of TD and 3TD groups showed significant differences from that of the CTR group (p < 0.001). Compared to the CTR group, there was increased neuronal apoptosis in the dentate gyrus (DG) of animals in TD and 3TD groups (p < 0.0001). There were no significant differences in CA2 and CA3 regions as well as in amygdala and cortex.Conclusion: Prenatal administration of betamethasone leads to significant changes in neuronal apoptosis in CA1 and DG regions.

L-type Ca2+ currents at CA1 synapses, but not CA3 or dentate granule neuron synapses, are increased in 3xTgAD mice in an age-dependent manner

Abnormal neuronal excitability and impaired synaptic plasticity might occur before the degeneration and death of neurons in Alzheimer's disease (AD). To elucidate potential biophysical alterations underlying aberrant neuronal network activity in AD, we performed whole-cell patch clamp analyses of L-type (nifedipine-sensitive) Ca2+ currents (L-VGCC), 4–aminopyridine-sensitive K+ currents, and AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid) and NMDA (N-methyl-D-aspartate) currents in CA1, CA3, and dentate granule neurons in hippocampal slices from young, middle-age, and old 3xTgAD mice and age-matched wild type mice. 3xTgAD mice develop progressive widespread accumulation of amyloid β-peptide, and selective hyperphosphorylated tau pathology in hippocampal CA1 neurons, which are associated with cognitive deficits, but independent of overt neuronal degeneration. An age-related elevation of L-type Ca2+ channel current density occurred in CA1 neurons in 3xTgAD mice, but not in wild type mice, with the magnitude being significantly greater in older 3xTgAD mice. The NMDA current was also significantly elevated in CA1 neurons of old 3xTgAD mice compared with in old wild type mice. There were no differences in the amplitude of K+ or AMPA currents in CA1 neurons of 3xTgAD mice compared with wild type mice at any age. There were no significant differences in Ca2+, K+, AMPA, or NMDA currents in CA3 and dentate neurons from 3xTgAD mice compared with wild type mice at any age. Our results reveal an age-related increase of L-VGCC density in CA1 neurons, but not in CA3 or dentate granule neurons, of 3xTgAD mice. These findings suggest a potential contribution of altered L-VGCC to the selective vulnerability of CA1 neurons to tau pathology in the 3xTgAD mice and to their degeneration in AD patients.

VSMC Ca2+ Signaling in Diabetes Associated Vascular Dysfunction

Ca2+ signaling plays a key role in regulating vascular smooth muscle cells (VSMC) contractility and as vascular tone. Hyperglycemia (HG)‐induced vascular dysfunction (VD) (i.e hypertension) is often manifested through alteration of VSMC function. Complex interactions affect vascular tone in diabetes, a fact that makes it difficult to dissect out the specific effect of HG on VSMC contractility. Here we use A7r5 rat aortic VSMC to test the effect of long term exposure to HG on Ca2+ signaling and VSMC contractility. We test the activity and expression levels of all the primary Ca2+ influx and release pathways in these cells, and show that culturing of A7r5 cells in 5mM (low glucose) or 25mM (high glucose, HG) for more than 4 weeks results in significant difference in Ca2+ signaling pathways. While several alterations were noted, one of the most dramatic was the inhibition of the endoplasmic reticulum Ca2+‐ATPase. This inhibition seems to be due to ROS production as evidenced by the reversal of the effect when the cells were cultured for 2 weeks in HG in the presence of the strong antioxidant N‐acetyl‐cysteine. The differential effect of glucose on Ca2+ signaling was also observed on the activity of the plasma membrane Ca2+‐ATPase, while no effect was found on the Na‐Ca2+ exchanger. In summary, this study offers a relevant model for the study of Ca2+ signaling in diabetes associated‐VD using physiological glucose concentration.

Effects of severe caloric restriction from birth on the hearts of adult rats

There has been increasing evidence suggesting that a severe caloric restriction (SCR) (above 40%) has beneficial effects on the hearts of rats. However, most of the reports have focused on the effects of SCR that started in adulthood. We investigated the consequences of SCR on the hearts of rats subjected to SCR since birth (CR50). From birth to the age of 3 months, CR50 rats were fed 50% of the food that the ad libitum group (AL) was fed. Thereafter, a maximal aerobic test was performed to indirectly evaluate global cardiovascular function. Indices of contractility (+dT/dt) and relaxation (-dT/dt) were analyzed in isolated heart preparation, and cardiomyocyte diameter, number, density, and myocardium collagen content were obtained through histologic analysis. Ventricular myocytes were isolated, using standard methods to evaluate phosphorylated AKT levels, and Ca(2+) handling was evaluated with a combination of Western blot analysis, intracellular Ca(2+) imaging, and confocal microscopy. CR50 rats exhibited increased aerobic performance and cardiac function, as shown by the increase in ±dT/dt. Despite the smaller cardiomyocyte diameter, CR50 rats had an increased heart-body weight ratio, increased cardiomyocyte density and number, and similar levels of myocardium collagen content, compared with AL rats. AKT was hyperphosphorylated in cardiomyocytes from CR50 rats, and there were no significant differences in Ca(2+) transient and SERCA2 levels in cardiomyocytes between CR50 and AL rats. Collectively, these observations reveal the beneficial effects of a 50% caloric restriction on the hearts of adult rats restricted since birth, which might involve cardiomyocyte AKT signaling.

Impaired Short-Term Plasticity in Mossy Fiber Synapses Caused by Mitochondrial Dysfunction of Dentate Granule Cells Is the Earliest Synaptic Deficit in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) in the early stages is characterized by memory impairment, which may be attributable to synaptic dysfunction. Oxidative stress, mitochondrial dysfunction, and Ca²⁺ dysregulation are key factors in the pathogenesis of AD, but the causal relationship between these factors and synaptic dysfunction is not clearly understood. We found that in the hippocampus of an AD mouse model (Tg2576), mitochondrial Ca²⁺ handling in dentate granule cells was impaired as early as the second postnatal month, and this Ca²⁺ dysregulation caused an impairment of post-tetanic potentiation in mossy fiber-CA3 synapses. The alteration of cellular Ca²⁺ clearance in Tg2576 mice is region-specific within hippocampus because in another region, CA1 pyramidal neuron, no significant difference in Ca²⁺ clearance was detected between wild-type and Tg2576 mice at this early stage. Impairment of mitochondrial Ca²⁺ uptake was associated with increased mitochondrial reactive oxygen species and depolarization of mitochondrial membrane potential. Mitochondrial dysfunctions in dentate granule cells and impairment of post-tetanic potentiation in mossy fiber-CA3 synapses were fully restored when brain slices obtained from Tg2576 were pretreated with antioxidant, suggesting that mitochondrial oxidative stress initiates other dysfunctions. Reversibility of early dysfunctions by antioxidants at the preclinical stage of AD highlights the importance of early diagnosis and antioxidant therapy to delay or prevent the disease processes.

Impaired Local Calcium Signaling in Primary Cultured Adult Rat Ventricular Myocytes

Although cultured adult cardiac myocytes have been adopted in studying protein functions in combination with cell-level genetic modifications, cellular alterations by culturing itself need to be clarified to understand real function of the protein genetically altered. We systematically compared contractile properties, calcium ion (Ca2+) signaling, transverse (t)-tubules, ryanodine receptor distributions between freshly isolated and two-days cultured adult rat ventricular myocytes. Density of t-tubules was remarkably decreased by culture. In cultured myocytes, cell shortenings were attenuated by ∼60% and relaxation was slowed. Consistently, magnitudes of action potential-induced Ca2+ transients were decreased to ∼50% and decays of the Ca2+ transients were retarded by culture. In cultured cells, density of L-type Ca2+ current was reduced to ∼40% and its inactivation was retarded. The latter is consistent with smaller Ca2+ transients in cultured group. However, sarcoplasmic reticulum Ca2+ contents were not different between two groups. To know the mechanism for smaller Ca2+ transient in cultured cells we examined Ca2+ sparks in these two groups of cells. The frequency of spontaneous Ca2+ sparks was significantly decreased by culturing. The amplitude, duration, and time-to-peak of individual Ca2+ sparks were not different between the two groups. Mean spark width was two-fold larger in cultured cells compared with freshly isolated cells. Quantitative analysis of immunofluorescence revealed shortening of longitudinal spacing between RyR2 clusters, and less dense and disorganized distributions of RyR2 clusters in cultured cells, which may be related to lower frequency of sparks in these cells. These results provide evidence on significant difference in Ca2+ sparks as well as excitation-contraction coupling in primary cultured adult ventricular myocytes. (This work was supported by National Research Foundation of Korea grants funded by the Ministry of Education, Science and Technolgy (2010-0000070).)

Endogenous SNAP-25 Regulates Native Voltage-gated Calcium Channels in Glutamatergic Neurons

In addition to its primary role as a fundamental component of the SNARE complex, SNAP-25 also modulates voltage-gated calcium channels (VGCCs) in various overexpression systems. Although these studies suggest a potential negative regulatory role of SNAP-25 on VGCC activity, the effects of endogenous SNAP-25 on native VGCC function in neurons are unclear. In the present study, we investigated the VGCC properties of cultured glutamatergic and GABAergic rat hippocampal neurons. Glutamatergic currents were dominated by P/Q-type channels, whereas GABAergic cells had a dominant L-type component. Also, glutamatergic VGCC current densities were significantly lower with enhanced inactivation rates and shifts in the voltage dependence of activation and inactivation curves compared with GABAergic cells. Silencing endogenous SNAP-25 in glutamatergic neurons did not alter P/Q-type channel expression or localization but led to increased VGCC current density without changes in the VGCC subtype proportions. Isolation of the P/Q-type component indicated that increased current in the absence of SNAP-25 was correlated with a large depolarizing shift in the voltage dependence of inactivation. Overexpressing SNAP-25 in GABAergic neurons reduced current density without affecting the VGCC subtype proportion. Accordingly, VGCC current densities in glutamatergic neurons from Snap-25(+/-) mice were significantly elevated compared with wild type glutamatergic neurons. Overall, this study demonstrates that endogenous SNAP-25 negatively regulates native VGCCs in glutamatergic neurons which could have important implications for neurological diseases associated with altered SNAP-25 expression.

Alterations in Intracellular Calcium Ion Concentrations in Cerebellar Granule Cells of the CACNA1A Mutant Mouse, Leaner, During Postnatal Development

Maintaining calcium ion (Ca²+) homeostasis is crucial for normal neuronal function. Altered Ca²+ homeostasis interferes with Ca²+ signaling processes and affects neuronal survival. In this study, we used homozygous leaner and tottering mutant mice, which carry autosomal recessive mutations in the gene coding for the α(1A) pore forming subunit of Ca(V)2.1 (P/Q-type) voltage-gated calcium channels (VGCC). Leaner mice show severe ataxia and epilepsy, while tottering mice are less severely affected. Leaner cerebellar granule cells (CGC) show extensive apoptotic cell death that peaks at postnatal (P) day 20 and continues into adulthood. Intracellular Ca²+ ([Ca²+](i)) concentrations in leaner and tottering mouse Purkinje cells have been described, but [Ca²+](i) concentrations have not been reported for granule cells, the largest neuronal population of the cerebellum. Using the ratiometric dye, Fura-2 AM, we investigated the role of Ca²+ homeostasis in CGC death during postnatal development by demonstrating basal [Ca²+](i), depolarization induced Ca²+ transients, and Ca²+ transients after completely blocking Ca(V)2.1 VGCC. From P20 onward, basal [Ca²+](i) levels in leaner CGC were significantly lower compared to age-matched wild-type CGC. We also compared basal [Ca²+](i) levels in leaner and wild-type CGC to basal [Ca²+](i) in tottering CGC. Potassium chloride induced depolarization revealed no significant difference in Ca²+ transients between leaner and wild-type CGC, indicating that even though leaner CGC have dysfunctional P/Q-type VGCC, Ca²+ transients after depolarization are the same. This suggests that other VGCC are compensating for the dysfunctional P/Q channels. This finding was further confirmed by completely blocking Ca(V)2.1 VGCC using ω-Agatoxin IV-A.

Endoplasmic reticulum Ca2+-homeostasis is altered in small and non-small cell lung cancer cell lines

BackgroundKnowledge of differences in the cellular physiology of malignant and non-malignant cells is a prerequisite for the development of cancer treatments that effectively kill cancer without damaging normal cells. Calcium is a ubiquitous signal molecule that is involved in the control of proliferation and apoptosis. We aimed to investigate if the endoplasmic reticulum (ER) Ca2+-homeostasis is different in lung cancer and normal human bronchial epithelial (NHBE) cells.MethodsThe intracellular Ca2+-signaling was investigated using fluorescence microscopy and the expression of Ca2+-regulating proteins was assessed using Western Blot analysis.ResultsIn a Small Cell Lung Cancer (H1339) and an Adeno Carcinoma Lung Cancer (HCC) cell line but not in a Squamous Cell Lung Cancer (EPLC) and a Large Cell Lung Cancer (LCLC) cell line the ER Ca2+-content was reduced compared to NHBE. The reduced Ca2+-content correlated with a reduced expression of SERCA 2 pumping calcium into the ER, an increased expression of IP3R releasing calcium from the ER, and a reduced expression of calreticulin buffering calcium within the ER. Lowering the ER Ca2+-content with CPA led to increased proliferation NHBE and lung cancer cells.ConclusionThe significant differences in Ca2+-homeostasis between lung cancer and NHBE cells could represent a new target for cancer treatments.