Perturbation Of Ca2 Research Articles

Environmental endocrine disrupting chemical 4-tert-butylphenol induced calcium overload and subsequent autophagy impairment via miRNA-363/CACNA1D Axis in epithelioma papulosum cyprini cells

Environmental endocrine disrupting chemical 4-tert-butylphenol (4-tBP), a widely-utilized surfactant in various industries, poses potential risks to aquatic organisms. Our previous sequencing results suggested that 4-tBP-induced common carp liver injury might be associated with Ca2+ signaling and autophagy. However, the intricate involvement of these pathways in 4-tBP-induced cytotoxic mechanisms remained unexplored. To bridge these knowledge gaps, this study focused on epithelioma papulosum cyprini (EPC) cells, a significant cell type in fish biology. Initial observations showed that 4-tBP induced a dose-dependent perturbation in Ca2+ levels. Further investigations, with siRNA and L-type Ca2+ channel agonist (BAYK8644), identified L-type calcium channel gene CACNA1D as a critical regulator of 4-tBP-induced Ca2+ overload. Predictive analysis using miRanda platform suggested a potential interaction between miR-363 and CACNA1D, which was subsequently verified through dual-luciferase reporter gene assays. We then established miR-363 mimic/inhibitor models, along with miR-363 and CACNA1D co-suppression models in EPC cells. Through TEM observation, immunofluorescence assay, Ca2+ staining, and qRT-PCR analysis, we evaluated the role of miR-363/CACNA1D axis in modulating the effects of 4-tBP on Ca2+ signaling and autophagy. Results showed that miR-363 inhibitor exacerbated 4-tBP-induced increase in CALM2, CAMKII, Calpain2, and p62 expression and also led to decrease in ATG5, ATG7, and LC3b expression. In contrast, miR-363 mimic notably alleviated these changes. Notably, siRNA CACNA1D effectively modulating miR-363 inhibitor's effect. Our study revealed that 4-tBP induced Ca2+ overload and subsequent autophagy impairment via miR-363/CACNA1D axis. These findings illuminated a profound understanding of molecular mechanisms underlying 4-tBP-induced cytotoxicity and spotlighted a potential therapeutic target.

ER and SOCE Ca2+ signals are not required for directed cell migration in human iPSC-derived microglia

The central nervous system (CNS) is constantly surveilled by microglia, highly motile and dynamic cells deputed to act as the first line of immune defense in the brain and spinal cord. Alterations in the homeostasis of the CNS are detected by microglia that respond by extending their processes or – following major injuries – by migrating toward the affected area. Understanding the mechanisms controlling directed cell migration of microglia is crucial to dissect their responses to neuroinflammation and injury. We used a combination of pharmacological and genetic approaches to explore the involvement of calcium (Ca2+) signaling in the directed migration of human induced pluripotent stem cell (iPSC)-derived microglia challenged with a purinergic stimulus. This approach mimics cues originating from injury of the CNS. Unexpectedly, simultaneous imaging of microglia migration and intracellular Ca2+ changes revealed that this phenomenon does not require Ca2+ signals generated from the endoplasmic reticulum (ER) and store-operated Ca2+ entry (SOCE) pathways. Instead, we find evidence that human microglial chemotaxis to purinergic signals is mediated by cyclic AMP in a Ca2+-independent manner. These results challenge prevailing notions, with important implications in neurological conditions characterized by perturbation in Ca2+ homeostasis.

NOTCH1-mutated chronic lymphocytic leukemia displays high endoplasmic reticulum stress response with druggable potential.

Constitutive activation of NOTCH1-wild-type (NT1-WT) signaling is associated with poor outcomes in chronic lymphocytic leukemia (CLL), and NOTCH1 mutation (c.7541_7542delCT), which potentiates NOTCH1 signaling, worsens the prognosis. However, the specific mechanisms of NOTCH1 deregulation are still poorly understood. Accumulative evidence mentioned endoplasmic reticulum (ER) stress/unfolded protein response (UPR) as a key targetable pathway in CLL. In this study, we investigated the impact of NOTCH1 deregulation on CLL cell response to ER stress induction, with the aim of identifying new therapeutic opportunities for CLL. We performed a bioinformatics analysis of NOTCH1-mutated (NT1-M) and NT1-WT CLL to identify differentially expressed genes (DEGs) using the rank product test. Quantitative real-time polymerase chain reaction (qPCR), Western blotting, cytosolic Ca2+, and annexin V/propidium iodide (PI) assay were used to detect curcumin ER stress induction effects. A median-effect equation was used for drug combination tests. The experimental mouse model Eμ-TCL1 was used to evaluate the impact of ER stress exacerbation by curcumin treatment on the progression of leukemic cells and NOTCH1 signaling. Bioinformatics analysis revealed gene enrichment of the components of the ER stress/UPR pathway in NT1-M compared to those in NT1-WT CLL. Ectopic expression of NOTCH1 mutation upregulated the levels of ER stress response markers in the PGA1 CLL cell line. Primary NT1-M CLL was more sensitive to curcumin as documented by a significant perturbation in Ca2+ homeostasis and higher expression of ER stress/UPR markers compared to NT1-WT cells. It was also accompanied by a significantly higher apoptotic response mediated by C/EBP homologous protein (CHOP) expression, caspase 4 cleavage, and downregulation of NOTCH1 signaling in NT1-M CLL cells. Curcumin potentiated the apoptotic effect of venetoclax in NT1-M CLL cells. In Eμ-TCL1 leukemic mice, the administration of curcumin activated ER stress in splenic B cells ex vivo and significantly reduced the percentage of CD19+/CD5+ cells infiltrating the spleen, liver, and bone marrow (BM). These cellular effects were associated with reduced NOTCH1 activity in leukemic cells and resulted in prolonged survival of curcumin-treated mice. Overall, our results indicate that ER stress induction in NT1-M CLL might represent a new therapeutic opportunity for these high-risk CLL patients and improve the therapeutic effect of drugs currently used in CLL.

Secretagogin is a Ca2+-dependent stress-responsive chaperone that may also play a role in aggregation-based proteinopathies

Secretagogin (SCGN) is a three-domain hexa-EF-hand Ca2+-binding protein that plays a regulatory role in the release of several hormones. SCGN is expressed largely in pancreatic β-cells, certain parts of the brain, and also in neuroendocrine tissues. The expression of SCGN is altered in several diseases, such as diabetes, cancers, and neurodegenerative disorders; however, the precise associations that closely link SCGN expression to such pathophysiologies are not known. In this work, we report that SCGN is an early responder to cellular stress, and SCGN expression is temporally upregulated by oxidative stress and heat shock. We show the overexpression of SCGN efficiently prevents cells from heat shock and oxidative damage. We further demonstrate that in the presence of Ca2+, SCGN efficiently prevents the aggregation of a broad range of model proteins in vitro. Small-angle X-ray scattering (BioSAXS) studies further reveal that Ca2+ induces the conversion of a closed compact apo-SCGN conformation into an open extended holo-SCGN conformation via multistate intermediates, consistent with the augmentation of chaperone activity of SCGN. Furthermore, isothermal titration calorimetry establishes that Ca2+ enables SCGN to bind α-synuclein and insulin, two target proteins of SCGN. Altogether, our data not only demonstrate that SCGN is a Ca2+-dependent generic molecular chaperone involved in protein homeostasis with broad substrate specificity but also elucidate the origin of its altered expression in several cancers. We describe a plausible mechanism of how perturbations in Ca2+ homeostasis and/or deregulated SCGN expression would hasten the process of protein misfolding, which is a feature of many aggregation-based proteinopathies.

Mitochondria Fusion upon SERCA Inhibition Prevents Activation of the NLRP3 Inflammasome in Human Monocytes.

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) is a crucial component of the cellular machinery responsible for Ca2+ homeostasis. The selective inhibition of SERCA by thapsigargin (TG) leads to perturbations in Ca2+ signaling, which can trigger endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) pathway is activated in response to ER stress and induces an adaptive response to preserve cell survival or committee cells to programmed death, depending on stress duration and/or level. Early stages of ER stress stimulate mitochondrial metabolism to preserve survival but under chronic ER stress conditions, mitochondrial dysfunction is induced, which, in turn, can enhance inflammation through NLRP3 inflammasome activation. This study was aimed at investigating the role of SERCA inhibition on NLRP3 inflammasome activation in human monocytes, which was evaluated in primary monocytes isolated from healthy individuals and in the THP-1 human monocytic cell line. Findings obtained in both THP-1 and primary monocytes demonstrate that SERCA inhibition triggered by TG does not activate the NLRP3 inflammasome in these innate immune cells since IL-1β secretion was not affected. Results from THP-1 monocytes showing that SERCA inhibition increases mitochondrial Ca2+ content and fusion, in the absence of changes in ROS levels and membrane potential, support the view that human monocytes counteract ER stress that arises from inhibition of SERCA through modulation of mitochondrial morphology towards mitochondria fusion, thus preventing NLRP3 inflammasome activation. Overall, this work contributes to a better understanding of the molecular mechanisms that modulate the activity of the NLRP3 inflammasome leading to sterile inflammation, which are still poorly understood.

DMD – ANIMAL MODELS: EP.84 Cholesterol metabolism is a potential therapeutic target in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal muscle disease, caused by mutations in the DMD gene, encoding a protein that links the cytoskeleton and the extracellular matrix in skeletal and cardiac muscles. The primary consequences of disruption of the link due to lack of functional dystrophin involved sarcolemma destabilization, perturbation of Ca2+ homeostasis, activation of proteases, mitochondrial damage, leading to tissue degeneration. A recently emphasized secondary aspect of the dystrophic process is a progressive metabolic change of the dystrophic tissue.

Calcium dysregulation and compensation in cortical pyramidal neurons of the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) is a fatal, hereditary neurodegenerative disorder that predominantly affects striatal medium-sized spiny neurons and cortical pyramidal neurons (CPNs). It has been proposed that perturbations in Ca2+ homeostasis could play a role in CPN alterations. To test this hypothesis, we used the R6/2 mouse model of juvenile HD at different stages of disease progression; presymptomatic, early symptomatic, and late symptomatic. We combined whole-cell patch-clamp recordings of layer 2/3 CPNs with two-photon laser scanning microscopy to image somatic and dendritic Ca2+ transients associated with evoked action potentials (APs). We found that the amplitude of AP-induced Ca2+ transients recorded at the somata of CPNs was significantly reduced in presymptomatic and late symptomatic R6/2 mice compared with wild-type (WT) littermates. However, reduced amplitudes were compensated by increases in decay times, so that Ca2+ transient areas were similar between genotypes. AP-induced Ca2+ transients in CPN proximal dendrites were variable and differences did not reach statistical significance, except for reduced areas in the late symptomatic group. In late symptomatic mice, a specific store-operated Ca2+ channel antagonist, EVP4593, reduced somatic Ca2+ transient amplitude similarly in WT and R6/2 CPNs. In contrast, dantrolene, a ryanodine receptor (RyR) antagonist, and nifedipine, an L-type Ca2+ channel blocker, significantly reduced both somatic Ca2+ transient amplitude and area in R6/2 but not WT CPNs. These findings demonstrate that perturbations of Ca2+ homeostasis and compensation occur in CPNs before and after the onset of overt symptoms, and suggest RyRs and L-type Ca2+ channels as potential targets for therapeutic intervention.NEW & NOTEWORTHY We used two-photon microscopy to examine calcium influx induced by action potentials in cortical pyramidal neurons from a mouse model of Huntington's disease (HD), the R6/2. The amplitude of somatic calcium transients was reduced in R6/2 mice compared with controls. This reduction was compensated by increased decay times, which could lead to reduced calcium buffering capacity. L-type calcium channel and ryanodine receptor blockers reduced calcium transient area in HD neurons, suggesting new therapeutic avenues.

225-LB: Targeting ß-Cell Endoplasmic Reticulum (ER) TALK-1 Channels to Restore Ca2+ Homeostasis and Reduce ER Stress in Diabetes

Perturbations in Ca2+ homeostasis contribute to β-cell failure in diabetes, limiting insulin secretion and exacerbating hyperglycemia. TALK-1 is the most islet-restricted and most abundantly expressed K+ channel of the β-cell. ER TALK-1 channels provide K+ countercurrent for [Ca2+]ER release, thereby tuning [Ca2+]ER storage and release. During β-cell Ca2+ influx, TALK-1-enhanced [Ca2+]ER release leads to activation of the hyperpolarizing Ca2+ activated K+ channels, thus increasing the silent phase duration between Ca2+ oscillations. A gain-of-function mutation in TALK-1(L114P) causes MODY, by disrupting cytoplasmic and ER Ca2+ handling. However, the ability to probe ER TALK-1 channels as a therapeutic target to normalize β-cell Ca2+ handling and reduce ER stress in diabetes has not been determined. Here, using a high throughput fluorescence-based thallium (TI+) flux assay (z’ =0.64) in a TALK-1 overexpression cell line, we identified 80 TALK-1 selective inhibitors from a small molecule library of 50,000 compounds. 4 potent inhibitors that also inhibited the ER membrane TALK-1 channels were identified by monitoring cyclopiazonic acid-induced [Ca2+]ER release (e.g. K2P16-i7(IC50=5.3μM); 63.36% [Ca2+]ER recovery, p-value=0.02). We also tested these inhibitors for their efficacy in controlling mouse islet [Ca2+]ER storage as well as cytosolic Ca2+ oscillations. TALK-1 inhibitors significantly increased islet [Ca2+]ER storage and Ca2+ oscillation frequency (e.g.K2P16-i7; [Ca2+]ER AUC=60.9a.u., peaks/min=0.45 vs. DMSO; [Ca2+]ER AUC=49.7a.u., peaks/min= 0.17, p-value<0.05). Importantly, the inhibitors did not alter [Ca2+]ER and glucose-stimulated Ca2+ oscillation frequency in TALK-1 KO islets. In summary, we have identified the first ER TALK-1 selective inhibitors that can not only be utilized to assess the role of TALK-1 in human β-cell Ca2+ handling and GSIS, but also potentially reduce ER dysfunction during diabetes pathogenesis. Disclosure A. Y. Nakhe: None. P. Dadi: None. D. Westover: None. E. Days: None. J. Denton: None. D. Jacobson: None.

Cholesterol metabolism is a potential therapeutic target in Duchenne muscular dystrophy.

BackgroundDuchenne muscular dystrophy (DMD) is a lethal muscle disease detected in approximately 1:5000 male births. DMD is caused by mutations in the DMD gene, encoding a critical protein that links the cytoskeleton and the extracellular matrix in skeletal and cardiac muscles. The primary consequence of the disrupted link between the extracellular matrix and the myofibre actin cytoskeleton is thought to involve sarcolemma destabilization, perturbation of Ca2+ homeostasis, activation of proteases, mitochondrial damage, and tissue degeneration. A recently emphasized secondary aspect of the dystrophic process is a progressive metabolic change of the dystrophic tissue; however, the mechanism and nature of the metabolic dysregulation are yet poorly understood. In this study, we characterized a molecular mechanism of metabolic perturbation in DMD.MethodsWe sequenced plasma miRNA in a DMD cohort, comprising 54 DMD patients treated or not by glucocorticoid, compared with 27 healthy controls, in three groups of the ages of 4–8, 8–12, and 12–20 years. We developed an original approach for the biological interpretation of miRNA dysregulation and produced a novel hypothesis concerning metabolic perturbation in DMD. We used the mdx mouse model for DMD for the investigation of this hypothesis.ResultsWe identified 96 dysregulated miRNAs (adjusted P‐value <0.1), of which 74 were up‐regulated and 22 were down‐regulated in DMD. We confirmed the dysregulation in DMD of Dystro‐miRs, Cardio‐miRs, and a large number of the DLK1‐DIO3 miRNAs. We also identified numerous dysregulated miRNAs yet unreported in DMD. Bioinformatics analysis of both target and host genes for dysregulated miRNAs predicted that lipid metabolism might be a critical metabolic perturbation in DMD. Investigation of skeletal muscles of the mdx mouse uncovered dysregulation of transcription factors of cholesterol and fatty acid metabolism (SREBP‐1 and SREBP‐2), perturbation of the mevalonate pathway, and the accumulation of cholesterol in the dystrophic muscles. Elevated cholesterol level was also found in muscle biopsies of DMD patients. Treatment of mdx mice with Simvastatin, a cholesterol‐reducing agent, normalized these perturbations and partially restored the dystrophic parameters.ConclusionsThis investigation supports that cholesterol metabolism and the mevalonate pathway are potential therapeutic targets in DMD.

Perturbation of Ca2+ stores and store-operated Ca2+ influx by lidocaine in neuronal N2A and NG108-15 cells

In this report we examined the effects of lidocaine on Ca2+ homeostasis of neuronal cells using microfluorimetric measurement of cytosolic Ca2+ with fura 2 as probe. In mouse neuroblastoma N2A cells, 10 mM lidocaine caused Ca2+ release from the cyclopiazonic acid (CPA)-dischargeable pool and abolished ATP-triggered Ca2+ release. Lidocaine-triggered Ca2+ release was not affected by xestospongin C (XeC), an inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor. N2A cells did not have functional ryanodine receptors (RYR) (absence of caffeine response) and we used differentiated NG108-15 cells (presence of caffeine response) for further experiments. Caffeine-triggered Ca2+ release was unaffected by a brief lidocaine exposure, but was eliminated after a prolonged treatment of lidocaine, suggesting lidocaine abolished caffeine action possibly not by interfering caffeine binding but via Ca2+ store depletion. Lidocaine-elicited Ca2+ release was unaffected by XeC or a high concentration of ryanodine, suggesting Ca2+ release was not via IP3R or RYR. Lidocaine did not affect nigericin-dischargeable lysosomal Ca2+ stores. Lastly, we observed that lidocaine suppressed CPA-induced store-operated Ca2+ influx in both N2A cells and differentiated NG108-15 cells. Our results suggest two novel actions of lidocaine in neuronal cells, namely, depletion of Ca2+ store (via an IP3R- and RYR-independent manner) and suppression of store-operated Ca2+ influx.

Characteristics of the mitochondrial and cellular uptake of MPP+, as probed by the fluorescent mimic, 4'I-MPP.

The discovery that 1-methyl-4-phenylpyridinium (MPP+) selectively destroys dopaminergic neurons and causes Parkinson’s disease (PD) symptoms in mammals has strengthened the environmental hypothesis of PD. The current model for the dopaminergic toxicity of MPP+ is centered on its uptake into dopaminergic neurons, accumulation into the mitochondria, inhibition of the complex-I leading to ATP depletion, increased reactive oxygen species (ROS) production, and apoptotic cell death. However, some aspects of this mechanism and the details of the cellular and mitochondrial accumulation of MPP+ are still poorly understood. The aim of this study was to characterize a structural and functional MPP+ mimic which is suitable to study the cellular distribution and mitochondrial uptake of MPP+ in live cells and use it to identify the molecular details of these processes to advance the understanding of the mechanism of the selective dopaminergic toxicity of MPP+. Here we report the characterization of the fluorescent MPP+ derivative, 1-methyl-4-(4'-iodophenyl)pyridinium (4'I-MPP+), as a suitable candidate for this purpose. Using this novel probe, we show that cytosolic/mitochondrial Ca2+ play a critical role through the sodium-calcium exchanger (NCX) in the mitochondrial and cellular accumulation of MPP+ suggesting for the first time that MPP+ and related mitochondrial toxins may also exert their toxic effects through the perturbation of Ca2+ homeostasis in dopaminergic cells. We also found that the specific mitochondrial NCX (mNCX) inhibitors protect dopaminergic cells from the MPP+ and 4'I-MPP+ toxicity, most likely through the inhibition of the mitochondrial uptake, which could potentially be exploited for the development of pharmacological agents to protect the central nervous system (CNS) dopaminergic neurons from PD-causing environmental toxins.

Modulation of Ca2+ Influx at Hyperpolarized Membrane Potentials Alters Depolarization-Triggered Exocytosis in Bovine Chromaffin Cells

Perturbations in Ca2+ entry via neuronal transient receptor potential (TRP) and ORAI family proteins have been shown to alter synaptic signaling and architecture, and suggested to play a role in a spectrum of neuropathological conditions. Specific mechanisms by which of TRP/ORAI channels modulate synaptic transmission are not completely understood. Here we employed a combination of imaging and electrophysiological techniques to monitor changes in intracellular Ca2+ concentration ([Ca2+]i), membrane currents at hyperpolarized membrane potentials, voltage-gated Ca2+ currents, and changes in membrane capacitance to investigate effects of modulation of Ca2+ entry at hyperpolarized membrane potentials on exocytosis in bovine chromaffin cells. A small inwardly directed membrane current (I hold), whose amplitude was dependent on the concentration of extracellular Ca2+ and Na+, was present at resting membrane potential. Augmentation of I hold either by hyperpolarization (from −50 mV to −90 mV) or stimulation with bradykinin (1 μM), a catecholamine secretagogue, elevated [Ca2+]i at hyperpolarized potentials and facilitated exocytotic responses triggered by short depolarizations (<500 ms in duration). Exocytotic responses triggered by long depolarizations (>500 ms in duration) were smaller at −90 mV holding potential compared with those at −50 mV holding potential. Reversal of Na+/Ca2+ exchanger by removal of Na+ from bath solution did not cause depolarization-independent exocytosis at −50 mV holding potential. Membrane hyperpolarization from −50 mV to −90 mV in Na+ -free bath solution evoked depolarization-independent exocytosis with the maximal release rate of about 6 fF/s. Our findings provide first direct evidence that 1) Ca2+ influx via native Ca2+ channels operating at negative voltages is functionally coupled with Na+/Ca2+ exchanger and that 2) perturbation in Ca2+ influx an negative voltages with physiologically relevant stimuli significantly modulate exocytotic responses to depolarization-triggered Ca2+ influx in neuroendocrine cells.

Conditional Deletion of the L-Type Calcium Channel Cav1.2 in Oligodendrocyte Progenitor Cells Affects Postnatal Myelination in Mice.

Overall, it is clear that cells in the oligodendrocyte lineage exhibit remarkable plasticity with regard to the expression of Ca2+ channels and that perturbation of Ca2+ homeostasis likely plays an important role in the pathogenesis underlying demyelinating diseases. To determine whether voltage-gated Ca2+ entry is involved in oligodendrocyte maturation and myelination, we used a conditional knock-out mouse for voltage-operated Ca2+ channels in oligodendrocyte progenitor cells. Our results indicate that voltage-operated Ca2+ channels can modulate oligodendrocyte development in the postnatal brain and suggest that voltage-gated Ca2+ influx in oligodendroglial cells is critical for normal myelination. These findings could lead to novel approaches to intervene in neurodegenerative diseases in which myelin is lost or damaged.

Beta-Adrenoceptor Stimulation Reveals Ca2+ Waves and Sarcoplasmic Reticulum Ca2+ Depletion in Left Ventricular Cardiomyocytes from Post-Infarction Rats with and without Heart Failure.

Abnormal cellular Ca2+ handling contributes to both contractile dysfunction and arrhythmias in heart failure. Reduced Ca2+ transient amplitude due to decreased sarcoplasmic reticulum Ca2+ content is a common finding in heart failure models. However, heart failure models also show increased propensity for diastolic Ca2+ release events which occur when sarcoplasmic reticulum Ca2+ content exceeds a certain threshold level. Such Ca2+ release events can initiate arrhythmias. In this study we aimed to investigate if both of these aspects of altered Ca2+ homeostasis could be found in left ventricular cardiomyocytes from rats with different states of cardiac function six weeks after myocardial infarction when compared to sham-operated controls. Video edge-detection, whole-cell Ca2+ imaging and confocal line-scan imaging were used to investigate cardiomyocyte contractile properties, Ca2+ transients and Ca2+ waves. In baseline conditions, i.e. without beta-adrenoceptor stimulation, cardiomyocytes from rats with large myocardial infarction, but without heart failure, did not differ from sham-operated animals in any of these aspects of cellular function. However, when exposed to beta-adrenoceptor stimulation, cardiomyocytes from both non-failing and failing rat hearts showed decreased sarcoplasmic reticulum Ca2+ content, decreased Ca2+ transient amplitude, and increased frequency of Ca2+ waves. These results are in line with a decreased threshold for diastolic Ca2+ release established by other studies. In the present study, factors that might contribute to a lower threshold for diastolic Ca2+ release were increased THR286 phosphorylation of Ca2+/calmodulin-dependent protein kinase II and increased protein phosphatase 1 abundance. In conclusion, this study demonstrates both decreased sarcoplasmic reticulum Ca2+ content and increased propensity for diastolic Ca2+ release events in ventricular cardiomyocytes from rats with heart failure after myocardial infarction, and that these phenomena are also found in rats with large myocardial infarctions without heart failure development. Importantly, beta-adrenoceptor stimulation is necessary to reveal these perturbations in Ca2+ handling after a myocardial infarction.

Abstract 14156: Airway Gene Delivery of Serca2a Ameliorates Chronic Pulmonary Hypertension in a Large Animal Preclinical Model

Downregulation of sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) in the pulmonary vasculature leads to perturbations in Ca2+ homeostasis, endothelial dysfunction, and transition of pulmonary artery smooth muscle cells from a contractile to a proliferative phenotype. To assess the feasibility and benefits of sustained pulmonary vascular SERCA2a gene transfer, we created a model of chronic post-capillary PH in Yorkshire swine by partial pulmonary vein banding. Development of chronic PH was hemodynamically confirmed, and animals were randomized to intratracheal administration of adeno-associated virus type 1 (AAV1) carrying the human SERCA2a gene (n=10, AAV1.SERCA2a group) or Saline (n=10). Transduction efficacy after intratracheal delivery of AAV1 was confirmed by β-galactosidase detection in the distal pulmonary vasculature and significant overexpression of SERCA2a protein at the distal airway and small pulmonary arteries. Two months after gene delivery, disease progression was prevented in AAV1.SERCA2a treated animals as assessed by mean pulmonary pressure (29[5] vs 54[20] mmHg, p=0.027, Figure) and pulmonary vascular resistance index (3.2[2.2] vs 10.3[7.1] Wood units*m2, p=0.020) along with decreased small vessel remodeling in the AAV1.SERCA2a group (p&lt;0.05). RV ejection fraction (61[7] vs 50[7] % units, p=0.014) was preserved and improvement of RV end-diastolic pressure-volume relationship (0.12[0.08] vs 0.28[0.07] mmHg/ml, p=0.08) in AAV1.SERCA2a treated pigs versus Saline. In conclusion, airway-based delivery of AAV vectors to the pulmonary arteries is feasible, efficient, and safe in a clinically relevant chronic PH model. Selective vascular SERCA2a overexpression results in beneficial effects on pulmonary hemodynamics and RV performance, and may offer therapeutic benefit by modifying fundamental pathophysiology in pulmonary vascular diseases.

Ultrastructural and immunohistochemical localization of plasma membrane Ca2+-ATPase 4 in Ca2+-transporting epithelia

Plasma membrane Ca(2+)-ATPases (PMCAs) participate in epithelial Ca(2+) transport and intracellular Ca(2+) signaling. The Pmca4 isoform is enriched in distal nephron isolates and decreased in mice lacking the epithelial transient receptor potential vanilloid 5 Ca(2+) channel. We therefore hypothesized that Pmca4 plays a significant role in transcellular Ca(2+) flux and investigated the localization and regulation of Pmca4 in Ca(2+)-transporting epithelia. Using antibodies directed specifically against Pmca4, we found it expressed only in the smooth muscle layer of mouse and human intestines, whereas pan-specific Pmca antibodies detected Pmca1 in lateral membranes of enterocytes. In the kidney, Pmca4 showed broad localization to the distal nephron. In the mouse, expression was most abundant in segments coexpressing the epithelial ransient receptor potential vanilloid 5 Ca(2+) channel. Significant, albeit lower, expression was also evident in the region encompassing the cortical thick ascending limbs, macula densa, and early distal tubules as well as smooth muscle layers surrounding renal vessels. In the human kidney, a similar pattern of distribution was observed, with the highest PMCA4 expression in Na(+)-Cl(-) cotransporter-positive tubules. Electron microscopy demonstrated Pmca4 localization in distal nephron cells at both the basolateral membrane and intracellular perinuclear compartments but not submembranous vesicles, suggesting rapid trafficking to the plasma membrane is unlikely to occur in vivo. Pmca4 expression was not altered by perturbations in Ca(2+) balance, pointing to a housekeeping function of the pump in Ca(2+)-transporting epithelia. In conclusion, Pmca4 shows a divergent expression pattern in Ca(2+)-transporting epithelia, inferring diverse roles for this isoform not limited to transepithelial Ca(2+) transport.

Synergism of ochratoxin B and calcium-channel antagonist verapamil caused mitochondrial dysfunction

We examined the mechanism by which the ochratoxin B induced interaction with calcium-channel antagonist verapamil and mitochondrial dysfunction of the rat trachea in vitro experiment. The tracheas were cut into 2–3 mm wide rings and suspended in a tissue bath. Isometric tension was continuously measured with an isometric force transducer connected to a computer-based data acquisition system. Verapamil (1 × 10−6 M) produced a concentration-dependent contraction response in rat’s tracheal rings pre-contracted by acetylcholine. Incubation of rat’s tracheal rings with the ochratoxin B significantly potentiated the contraction responses of verapamil. Verapamil and OTB accelerate the overloading of Ca2+ in tracheal smooth muscle contributes the tissue toxicity as shown in electron microscopy and mitochondrial enzymes, through a mechanism that could involve perturbations of Ca2+ homeostasis. These results proved that ochratoxin B is a potential vasoconstrictor mycotoxin with the presence of calcium-channel antagonist. In conclusion, disturbance of Ca2+ homeostasis caused by OTA and plays a significant role in produces toxicity through mitochondrial enzyme inhibition.

Calcium signaling mediates the response to cadmium toxicity in Saccharomyces cerevisiae cells

The involvement of Ca2+ in the response to high Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Hg2+ was investigated in Saccharomyces cerevisiae. The yeast cells responded through a sharp increase in cytosolic Ca2+ when exposed to Cd2+, and to a lesser extent to Cu2+, but not to Mn2+, Co2+, Ni2+, Zn2+, or Hg2+. The response to high Cd2+ depended mainly on external Ca2+ (transported through the Cch1p/Mid1p channel) but also on vacuolar Ca2+ (released into the cytosol through the Yvc1p channel). The adaptation to high Cd2+ was influenced by perturbations in Ca2+ homeostasis. Thus, the tolerance to Cd2+ often correlated with sharp Cd2+-induced cytosolic Ca2+ pulses, while the Cd2+ sensitivity was accompanied by the incapacity to rapidly restore the low cytosolic Ca2+.

Physiological Function and Characterization of TRPCs in Neurons.

Ca2+ entry is essential for regulating vital physiological functions in all neuronal cells. Although neurons are engaged in multiple modes of Ca2+ entry that regulates variety of neuronal functions, we will only discuss a subset of specialized Ca2+-permeable non-selective Transient Receptor Potential Canonical (TRPC) channels and summarize their physiological and pathological role in these excitable cells. Depletion of endoplasmic reticulum (ER) Ca2+ stores, due to G-protein coupled receptor activation, has been shown to activate TRPC channels in both excitable and non-excitable cells. While all seven members of TRPC channels are predominately expressed in neuronal cells, the ion channel properties, mode of activation, and their physiological responses are quite distinct. Moreover, many of these TRPC channels have also been suggested to be associated with neuronal development, proliferation and differentiation. In addition, TRPCs also regulate neurosecretion, long-term potentiation and synaptic plasticity. Similarly, perturbations in Ca2+ entry via the TRPC channels have been also suggested in a spectrum of neuropathological conditions. Hence, understanding the precise involvement of TRPCs in neuronal function and in neurodegenerative conditions would presumably unveil avenues for plausible therapeutic interventions for these devastating neuronal diseases.

Exploring the Link between Ca2+ Signalling and ATP Dynamics in Cardiomyocyte Dysfunction

In heart failure the progressive deterioration of cardiac function is linked to Ca2+ handling abnormalities, altered metabolism and increased cardiomyocyte apoptosis. However the causal versus consequential nature of these linked phenomena remains to be determined. Here we aimed to investigate cellular ATP and Ca2+ environments and necrotic and apoptotic cell death in cellular models of Ca2+ dysfunction and metabolic perturbation. We constructed new ATP sensors via fusion of a red fluorescent protein (mCherry) with the pH-independent click-beetle luciferase in both mCherry-Luc and Luc-mCherry configurations. Chimeric proteins exhibited high specific activities of the luciferase partner in vitro (approximately 5 cps/amol protein) and we determined linear fluorescence-luminescence relationships (r2 > 0.88) following their expression in COS-7 cells. The dynamic range of the Luc-mCherry configuration was determined via exposure of COS-7 cells to manoeuvres that depleted ATP (e.g. 2-DOG, FCCP) and under conditions of elevated glucose availability (> 6g/L extracellular). In HL-1 cardiomyocyte monolayers, we monitored cellular ATP environments following pharmacologic manipulation of the amplitude and temporal patterning of Ca2+ signaling (e.g. using ouabain, staurosporine, BAPTA). Ca2+ and ATP environments were monitored through the transitions from spontaneously oscillatory syncytia to non-oscillatory phenotypes. Under these conditions, we measured cellular ATP effluxes and susceptibility to apoptosis using in situ DNA fragmentation and caspase activation assays. This approach reconciles specific perturbations in Ca2+ handling, the cellular ATP environment and phenotypic outcomes and builds a more complete picture of the events that contribute to the early stages of cellular dysfunction in chronic heart disease.