Activation Of Ryanodine Receptors Research Articles

The diamide insecticide chlorantraniliprole increases the single-channel current activity of the mammalian skeletal muscle ryanodine receptor.

Very recently, the diamide insecticide chlorantraniliprole was shown to induce Ca2+-release from sarcoplasmic reticulum (SR) vesicles isolated from mammalian skeletal muscle through the activation of the SR Ca2+ channel ryanodine receptor. As this result raises severe concerns about the safety of this chemical, we aimed to learn more about its action. To this end, single-channel analysis was performed, which showed that chlorantraniliprole induced high-activity bursts of channel opening that accounts for the Ca2+-releasing action described before.

Saikosaponin d causes apoptotic death of cultured neocortical neurons by increasing membrane permeability and elevating intracellular Ca2+ concentration

Saikosaponins (SSs) are a class of naturally occurring oleanane-type triterpenoid saponins found in Radix bupleuri that has been widely used in traditional Chinese medicine. As the main active principals of Radix bupleuri, SSs have been shown to suppress mouse motor activity, impair learning and memory, and decrease hippocampal neurogenesis. In the present study, we investigated the effect of five SSs (SSa, SSb1, SSb2, SSc, and SSd) on neuronal viability and the underlying mechanisms in cultured murine neocortical neurons. We demonstrate that SSa, SSb1 and SSd produce concentration-dependent apoptotic neuronal death and induce robust increase in intracellular Ca2+ concentration ([Ca2+]i) at low micromolar concentrations with a rank order of SSd > SSa > SSb1, whereas SSb2 and SSc have no detectable effect on both neuronal survival and [Ca2+]i. Mechanistically, SSd-induced elevation in [Ca2+]i is the primary result of enhanced extracellular Ca2+ influx, which likely triggers Ca2+-induced Ca2+ release through ryanodine receptor activation, but not SERCA inhibition. SSd-induced Ca2+ entry occurs through a non-selective mechanism since blockers of major neuronal Ca2+ entry pathways, including L-type Ca2+ channel, NMDA receptor, AMPA receptor, Na+-Ca2+ exchanger, and TRPV1, all failed to attenuate the Ca2+ response to SSd. Further studies demonstrate that SSd increases calcein efflux and induces an inward current in neocortical neurons. Together, these data demonstrate that SSd elevates [Ca2+]i due to its ability to increase membrane permeability, likely by forming pores in the surface of membrane, which leads to massive Ca2+ influx and apoptotic neuronal death in neocortical neurons.

Intra-axonal Ca2+ mobilization contributes to triphenyltin-induced facilitation in glycinergic transmission of rat spinal neurons

Triphenyltin (TPT) is an organotin compound causing environmental hazard to many wild creatures. Our previous findings show that TPT increases of the frequency of spontaneous glycinergic inhibitory postsynaptic currents (sIPSCs) in rat spinal neurons without changing the amplitude and 1/e decay time. In our study, the effects of 2-aminoethoxydiphenyl borate (2-APB), dantrolene sodium, and thapsigargin on sIPSC frequency were examined to reveal the contribution of intra-axonal Ca2+ mobilization by adding TPT. 2-APB considerably attenuated the TPT-induced facilitation of sIPSC frequency while dantrolene almost completely masked the TPT effects, suggesting that the TPT-induced synaptic facilitation results from the activation of both IP3 and ryanodine receptors on endoplasmic reticulum (ER) membrane, though inositol triphosphate (IP3) receptor is less sensitive to TPT. Thapsigargin itself significantly increased the sIPSC frequency without affecting the current amplitude and decay time. Successive addition of TPT could not further increase the sIPSC frequency in the presence of thapsigargin, indicating that thapsigargin completely masked the facilitatory action of TPT. Results suggest that TPT activates the IP3 and ryanodine receptors while TPT inhibits the Ca2+-pump of ER membranes, resulting in the elevation of intra-axonal Ca2+ levels, leading to the increase of spontaneous glycine release from synaptic vesicles.

Lower sarcoplasmic reticulum Ca2+ threshold for triggering afterdepolarizations in diabetic rat hearts

Type 2 diabetes (T2D) increases arrhythmia risk through incompletely elucidated mechanisms. Ventricular arrhythmias could be initiated by delayed afterdepolarizations (DADs) resulting from elevated spontaneous sarcoplasmic reticulum (SR) Ca2+ release (SR Ca2+ leak). The purpose of this study was to test the role of DADs and SR Ca2+ leak in triggering arrhythmias in T2D hearts. We compared rats with late-onset T2D that display pancreatic and cardiac phenotypes similar to those in humans with T2D (HIP rats) and their nondiabetic littermates (wild type [WT]). HIP rats showed higher propensity for premature ventricular complexes and ventricular tachyarrhythmias, whereas HIP myocytes displayed more frequent DADs and had lower SR Ca2+ content than WT. However, the threshold SR Ca2+ at which depolarizing transient inward currents (Itis) are generated was also significantly decreased in HIP myocytes and was below the actual SR Ca2+ load, which explains the increased DAD incidence despite reduced Ca2+ in SR. In agreement with these findings, Ca2+ spark frequency was augmented in myocytes from HIP vs WT rats, which suggests activation of ryanodine receptors (RyRs) in HIP hearts. Indeed, RyR phosphorylation (by CaMKII and protein kinase A) and oxidation are enhanced in HIP hearts, whereas there is no RyR O-GlcNAcylation in either HIP or control hearts. CaMKII inhibition dissipated the difference in Ca2+ spark frequency between HIP and WT myocytes. The threshold SR Ca2+ for generating depolarizing Itis is lower in T2D because of RyR activation after hyperphosphorylation and oxidation, which favors the occurrence of DADs despite low SR Ca2+ loads.

Exploiting Peptidomimetics to Synthesize Compounds That Activate Ryanodine Receptor Calcium Release Channels.

Ryanodine receptor (RyR) Ca2+ -release channels are essential for contraction in skeletal and cardiac muscle and are prime targets for modification of contraction in disorders that affect either the skeletal or heart musculature. We designed and synthesized a number of compounds with structures based on a naturally occurring peptide (A peptides) that modifies the activity of RyRs. In total, 34 compounds belonging to eight different classes were prepared. The compounds were screened for their ability to enhance Ca2+ release from isolated cardiac sarcoplasmic reticulum (SR) vesicles, with 25 displaying enhanced Ca2+ release. Competition studies with the parent peptides indicated that the synthetic compounds act at a competing site. The activity of the most effective of the compounds, BIT 180, was further explored using Ca2+ release from skeletal SR vesicles and contraction in intact skeletal muscle fibers. The compounds did not alter tension in intact fibers, indicating that (as expected) they are not membrane permeable, but importantly, that they are not toxic to the intact cells. Proof in principal that the compounds would be effective in intact muscle fibers if rendered membrane permeable was obtained with a structurally related membrane-permeable scorpion toxin (imperatoxin A), which was found to enhance contraction.

Mitochondrial-Mediated Oxidative Ca2+/Calmodulin-Dependent Kinase II Activation Induces Early Afterdepolarizations in Guinea Pig Cardiomyocytes: An In Silico Study.

BackgroundOxidative stress–mediated Ca2+/calmodulin‐dependent protein kinase II (CaMKII) phosphorylation of cardiac ion channels has emerged as a critical contributor to arrhythmogenesis in cardiac pathology. However, the link between mitochondrial‐derived reactive oxygen species (mdROS) and increased CaMKII activity in the context of cardiac arrhythmias has not been fully elucidated and is difficult to establish experimentally.Methods and ResultsWe hypothesize that pathological mdROS can cause erratic action potentials through the oxidation‐dependent CaMKII activation pathway. We further propose that CaMKII‐dependent phosphorylation of sarcolemmal slow Na+ channels alone is sufficient to elicit early afterdepolarizations. To test the hypotheses, we expanded our well‐established guinea pig cardiomyocyte excitation‐contraction coupling, mitochondrial energetics, and ROS‐induced‐ROS‐release model by incorporating oxidative CaMKII activation and CaMKII‐dependent Na+ channel phosphorylation in silico. Simulations show that mdROS mediated‐CaMKII activation elicits early afterdepolarizations by augmenting the late Na+ currents, which can be suppressed by blocking L‐type Ca2+ channels or Na+/Ca2+ exchangers. Interestingly, we found that oxidative CaMKII activation–induced early afterdepolarizations are sustained even after mdROS has returned to its physiological levels. Moreover, mitochondrial‐targeting antioxidant treatment can suppress the early afterdepolarizations, but only if given in an appropriate time window. Incorporating concurrent mdROS‐induced ryanodine receptors activation further exacerbates the proarrhythmogenic effect of oxidative CaMKII activation.ConclusionsWe conclude that oxidative CaMKII activation–dependent Na channel phosphorylation is a critical pathway in mitochondria‐mediated cardiac arrhythmogenesis.

Ryanodine- and CaMKII-dependent release of endogenous CGRP induces an increase in acetylcholine quantal size in neuromuscular junctions of mice.

ObjectiveThe aim of this study was to identify the mechanism responsible for an increase in miniature endplate potentials (MEPPs) amplitude, induced by ryanodine as an agonist of ryanodine receptors in mouse motor nerve terminals.MethodsUsing intracellular microelectrode recordings of MEPPs and evoked endplate potentials (EPPs), the changes in spontaneous and evoked acetylcholine release in motor synapses of mouse diaphragm neuromuscular preparations were studied.ResultsRyanodine (0.1 μM) increased both the amplitudes of MEPPs and EPPs to a similar extent (up to 130% compared to control). The ryanodine effect was prevented by blockage of receptors of calcitonin gene‐related peptide (CGRP) by a truncated peptide CGRP 8‐37. Endogenous CGRP is stored in large dense‐core vesicles in motor nerve terminals and may be released as a co‐transmitter. The ryanodine‐induced increase in MEPPs amplitude may be fully prevented by inhibition of vesicular acetylcholine transporter by vesamicol or by blocking the activity of protein kinase A with H‐89, suggesting that endogenous CGRP is released in response to the activation of ryanodine receptors. Activation of CGRP receptors can, in turn, upregulate the loading of acetylcholine into synaptic vesicles, which will increase the quantal size. This new feature of endogenous CGRP activity looks similar to recently described action of exogenous CGRP in motor synapses of mice. The ryanodine effect was prevented by inhibitors of Ca/Calmodulin‐dependent kinase II (CaMKII) KN‐62 or KN‐93. Inhibition of CaMKII did not prevent the increase in MEPPs amplitude, which was caused by exogenous CGRP.ConclusionsWe propose that the activity of presynaptic CaMKII is necessary for the ryanodine‐stimulated release of endogenous CGRP from motor nerve terminals, but CaMKII does not participate in signaling downstream the activation of CGRP‐receptors followed by quantal size increase.

Anticancer Activity of Euplotin C, Isolated from the Marine Ciliate Euplotes crassus, Against Human Melanoma Cells.

Cutaneous melanoma is the most serious type of skin cancer, so new cytotoxic weapons against novel targets in melanoma are of great interest. Euplotin C (EC), a cytotoxic secondary metabolite of the marine ciliate Euplotes crassus, was evaluated in the present study on human cutaneous melanoma cells to explore its anti-melanoma activity and to gain more insight into its mechanism of action. EC exerted a marked cytotoxic effect against three different human melanoma cell lines (A375, 501Mel and MeWo) with a potency about 30-fold higher than that observed in non-cancer cells (HDFa cells). A pro-apoptotic activity and a decrease in melanoma cell migration by EC were also observed. At the molecular level, the inhibition of the Erk and Akt pathways, which control many aspects of melanoma aggressiveness, was shown. EC cytotoxicity was antagonized by dantrolene, a ryanodine receptor (RyR) antagonist, in a concentration-dependent manner. A role of RyR as a direct target of EC was also suggested by molecular modelling studies. In conclusion, our data provide the first evidence of the anti-melanoma activity of EC, suggesting it may be a promising new scaffold for the development of selective activators of RyR to be used for the treatment of melanoma and other cancer types.

Ryanodine receptor Ca2+ release channel post-translational modification: Central player in cardiac and skeletal muscle disease.

Calcium release from internal stores is a quintessential event in excitation-contraction coupling in cardiac and skeletal muscle. The ryanodine receptor Ca2+ release channel is embedded in the internal sarcoplasmic reticulum Ca2+ store, which releases Ca2+ into the cytoplasm, enabling contraction. Ryanodine receptors form the hub of a macromolecular complex extending from the extracellular space to the sarcoplasmic reticulum lumen. Ryanodine receptor activity is influenced by the integrated effects of associated co-proteins, ions, and post-translational phosphor and redox modifications. In healthy muscle, ryanodine receptors are phosphorylated and redox modified to basal levels, to support cellular function. A pathological increase in the degree of both post-translational modifications disturbs intracellular Ca2+ signalling, and is implicated in various cardiac and skeletal disorders. This review summarises our current understanding of the mechanisms linking ryanodine receptor post-translational modification to heart failure and skeletal myopathy and highlights the challenges and controversies within the field.

BH4 domain peptides derived from Bcl-2/Bcl-XL as novel tools against acute pancreatitis

Biliary acute pancreatitis (AP) is a serious condition, which currently has no specific treatment. Taurolithocholic acid 3-sulfate (TLC-S) is one of the most potent bile acids causing cytosolic Ca2+ overload in pancreatic acinar cells (PACs), which results in premature activation of digestive enzymes and necrosis, hallmarks of AP. The inositol 1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR) play major roles in intracellular Ca2+ signaling. Inhibition of these endoplasmic reticulum-located channels suppresses TLC-S-induced Ca2+ release and necrosis, decreasing the severity of AP. Anti-apoptotic B-cell lymphoma (Bcl)-2-family members, such as Bcl-2 and Bcl-XL, have emerged as important modulators of IP3Rs and RyRs. These proteins contain four Bcl-2 homology (BH) domains of which the N-terminal BH4 domain exerts critical roles in regulating intracellular Ca2+ release channels. The BH4 domain of Bcl-2, but not of Bcl-XL, binds to and inhibits IP3Rs, whereas both BH4 domains inhibit RyRs. Although clear cytoprotective effects have been reported for these BH4 domains, it remains unclear whether they are capable of inhibiting pathological Ca2+-overload, associated with AP. Here we demonstrate in PACs that the BH4 domains of Bcl-2 and Bcl-XL inhibit RyR activity in response to the physiological agonist cholecystokinin. In addition, these BH4 domains inhibit pathophysiological TLC-S-induced Ca2+ overload in PACs via RyR inhibition, which in turn protects these cells from TLC-S-induced necrosis. This study shows for the first time the therapeutic potential of BH4 domain function by inhibiting pathological RyR-mediated Ca2+ release and necrosis, events that trigger AP.

Omecamtiv Mecarbil: A Myosin Motor Activator Agent with Promising Clinical Performance and New in vitro Results.

Clinical treatment of heart failure is still suffering from limited efficacy and unfavorable side effects. The recently developed group of agents, the myosin motor activators, act directly on cardiac myosin resulting in an increased force generation and prolongation of contraction. The lead molecule, omecamtiv mecarbil is now in human 3 stage. In addition to the promising clinical data published so far, there are new in vitro results indicating that the effect of omecamtiv mecarbil on contractility is rate-dependent. Furthermore, omecamtiv mecarbil was shown to activate cardiac ryanodine receptors, an effect that may carry proarrhythmic risk. These new results, together with the controversial effects of the drug on cardiac oxygen consumption, are critically discussed in this review in light of the current literature on omecamtiv mecarbil. In therapeutically relevant concentrations the beneficial inotropic effect of the agent is not likely affected by these new results - in accordance with the good clinical data. At supratherapeutic concentrations, however, activation of cardiac ryanodine receptors may increase arrhythmia propensity, and the stronger effect on diastolic than systolic cell shortening, observed at higher pacing frequencies, may decrease or offset the inotropic effect of omecamtiv mecarbil. Further studies with definitely supratherapeutical concentrations of omecamtiv mecarbil should be designed to map the actual risk of these potentially harmful side-effects.

Intracellular Calcium Mobilization Is Required for Sonic Hedgehog Signaling

Graded Shh signaling across fields of precursor cells coordinates patterns of gene expression, differentiation, and morphogenetic behavior as precursors form complex structures, such as the nervous system, the limbs, and craniofacial skeleton. Here we discover that intracellular calcium mobilization, a process tightly controlled and readily modulated, regulates the level of Shh-dependent gene expression in responding cells and affects the development of all Shh-dependent cell types in the zebrafish embryo. Reduced expression or modified activity of ryanodine receptor (RyR) intracellular calcium release channels shifted the allocation of Shh-dependent cell fates in the somitic muscle and neural tube. Mosaic analysis revealed that RyR-mediated calcium mobilization is required specifically in Shh ligand-receiving cells. This work reveals that RyR channels participate in intercellular signal transduction events. As modulation of RyR activity modifies tissue patterning, we hypothesize that alterations in intracellular calcium mobilization contribute to both birth defects and evolutionary modifications of morphology.

Bifenthrin causes transcriptomic alterations in mTOR and ryanodine receptor-dependent signaling and delayed hyperactivity in developing zebrafish (Danio rerio)

Over the last few decades, the pyrethroid insecticide bifenthrin has been increasingly employed for pest control in urban and agricultural areas, putting humans and wildlife at increased risk of exposure. Exposures to nanomolar (nM) concentrations of bifenthrin have recently been reported to alter calcium oscillations in rodent neurons. Neuronal calcium oscillations are influenced by ryanodine receptor (RyR) activity, which modulates calcium-dependent signaling cascades, including the mechanistic target of rapamycin (mTOR) signaling pathway. RyR activity and mTOR signaling play critical roles in regulating neurodevelopmental processes. However, whether environmentally relevant levels of bifenthrin alter RyR or mTOR signaling pathways to influence neurodevelopment has not been addressed. Therefore, our main objectives in this study were to examine the transcriptomic responses of genes involved in RyR and mTOR signaling pathways in zebrafish (Danio rerio) exposed to low (ng/L) concentrations of bifenthrin, and to assess the potential functional consequences by measuring locomotor responses to external stimuli. Wildtype zebrafish were exposed for 1, 3 and 5 days to 1, 10 and 50 ng/L bifenthrin, followed by a 14 d recovery period. Bifenthrin elicited significant concentration-dependent transcriptional responses in the majority of genes examined in both signaling cascades, and at all time points examined during the acute exposure period (1, 3, and 5 days post fertilization; dpf), and at the post recovery assessment time point (19 dpf). Changes in locomotor behavior were not evident during the acute exposure period, but were observed at 19 dpf, with main effects (increased locomotor behavior) detected in fish exposed developmentally to bifenthrin at 1 or 10 ng/L, but not 50 ng/L. These findings illustrate significant influences of developmental exposures to low (ng/L) concentrations of bifenthrin on neurodevelopmental processes in zebrafish.

Catestatin: A Master Regulator of Cardiovascular Functions

Cardiovascular disease (CVD), the most common cause of death globally, accounts for ~30% of all deaths worldwide. Hypertension is a common contributor to morbidity and mortality from CVD. The plasma concentration of chromogranin A (CgA) is elevated in patients with CVD as well as patients with established human essential hypertension and heart failure (HF). In contrast, the plasma level of the CgA-derived peptide catestatin (CST) is diminished in human essential hypertension. Low conversion of CgA-to-CST has been associated with increased mortality in patients hospitalized with acute HF. Consistent with human findings, the lack of CST in CgA knockout (Chga-KO) mice eventuates in the development of hypertension and supplementation of CST to Chga-KO mice restores blood pressure, implicating CST as a key player in regulating hypertension. In the peripheral system, CST decreases blood pressure by stimulating histamine release, inhibiting catecholamine secretion, or causing vasodilation. Centrally, CST improves baroreflex sensitivity (BRS) and heart rate variability (HRV) by exciting GABAergic neurons in the caudal ventrolateral medulla (CVLM) and pyramidal neurons of the central amygdala; CST also decreases BRS by exciting glutamatergic rostral ventrolateral medulla (RVLM) neurons. In addition, CST provides cardioprotection by inhibiting inotropy and lusitropy; activating mitochondrial KATP channels, and stimulating reperfusion injury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) pathways and consequent inhibition of mitochondrial permeability transition pore (mPTP). CST modulates cardiomyocyte Ca2+ levels by direct inhibition of Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) activity and consequent reduction in phosphorylation of phospholamban and ryanodine receptor 2, thereby providing support for a direct functional role of CST in the failing myocardium. These multitude of effects establish CST as a master regulator of cardiovascular functions.

Cyclic Nucleotides Reduce Ryanodine Receptor Mediated Ca2+ Spark Activation Independent of Long Term Hypoxia in Ovine Fetal Pulmonary Arterial Myocytes

Ryanodine receptors (RyR) release Ca2+ from smooth muscle sarcoplasmic reticulum in rapid events known as Ca2+ sparks. These Ca2+ sparks activate voltage and Ca2+ gated potassium channels (BK channels) that cause cell repolarization. Ca2+ spark‐BK channel mediated cell repolarization dilates many types of vessels and is essential to pulmonary arterial (PA) vasodilation during the fetal transition to breathing air. Infants who do not successfully negotiate this transition are at risk of developing pulmonary hypertension, which has significant morbidity and mortality. Previous work shows that long term hypoxia (LTH) decreases Ca2+ spark events in fetal pulmonary arterial smooth muscle cells (PASMCs) even though the Ca2+ stores have functional RyRs. Additional studies illustrate that cyclic nucleotides can increase RyR activity, which led us to test the hypothesis that cyclic nucleotides could rescue RyR activity in myocytes of LTH fetuses. This was examined by treating PAs isolated from low (700m; LA) and high altitude (3801m; HA) near‐term fetal sheep with exogenous cyclic AMP (cAMP) and cyclic GMP (cGMP) or the methylxanthine IBMX, a non‐specific cAMP and cGMP phosphodiesterase inhibitor. RyR activity was measured by confocal imaging of Ca2+ sparks in fluo‐4 labeled PASMCs. The spatial and temporal aspects to the Ca2+ sparks were evaluated using SparkLab, a custom software package. Contrary to our hypothesis, the prevalence of Ca2+ sparks decreased in the presence of cAMP, cGMP, and IBMX for PASMC from both LA and HA fetal sheep. These data suggest that cyclic nucleotides reduce instead of increase vasodilatory capacity via RyR‐BK channel coupling mechanisms. Furthermore, increased cyclic nucleotides had no effect on the spatial‐temporal aspects to the Ca2+ sparks. Even though the signaling pathway remains unclear, the reduction of Ca2+ sparks by cyclic nucleotides is potentially a novel mechanism of RyR modulation in sheep fetal PASMC. This data suggests that increasing cyclic nucleotides may be contra‐indicated in newborn infants who are at risk of developing pulmonary hypertension.Support or Funding InformationThis work is supported by The National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development grant number HD083132, by the National Science Foundation under Grant No. MRI 0923559, and the Loma Linda University School of Medicine. SH was a Loma Linda University Summer Undergraduate Research Fellow.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

3,3′‐Dichlorobiphenyl (PCB 11) Promotes Dendritic Arborization in Primary Neurons via CREB‐Dependent Mechanisms

PCB 11 is a byproduct of contemporary paint pigment production that has been detected in environmental media around the world, including milk produced for human consumption. We previously reported that PCB 11 enhances dendritic arborization in primary rat neuronal cell cultures. Since increased dendritic arborization, or hyperconnectivity, is a shared characteristic of many neurodevelopmental disorders (NDDs), our findings suggest this chemical poses a risk to the developing brain. The goal of this study was to understand how PCB 11 promotes dendritic growth. We tested canonical mechanisms of PCB developmental neurotoxicity, including signaling mediated by the ryanodine receptor (RyR), aryl hydrocarbon receptor (AhR), thyroid hormone receptor (THR), and cAMP response element binding protein (CREB). [3H]Ryanodine binding assays were used to assess effects of PCB 11 on RyR activity; luciferase‐reporter cell lines, to assess effects on AhR or THR activation. To assess a functional role for these signaling molecules in PCB 11‐mediated dendritic growth, primary neuron‐glia co‐cultures dissociated from neocortices of neonatal Sprague Dawley rats were transfected at 6 days in vitro (DIV) with a MAP2B‐FusRed construct to label the dendritic arbors of individual neurons. At 7 DIV, cultures were pretreated with pharmacological antagonists and then co‐treated for 48 h with PCB 11 or vehicle (0.1% DMSO). Dendritic morphology was quantified in FusRed‐labeled neurons by quantifying the number of dendritic tips and primary dendrites. PCB 11 had no activity at RyR, AhR or THR, and pharmacological antagonism of these receptors had no significant effect on PCB 11‐induced dendritic growth. However, the dendritic promoting effect of PCB 11 was blocked by the selective inhibitor of CREB, 666‐15 (3‐(3‐Aminopropoxy)‐N‐[2‐[[3‐[[(4‐chloro‐2‐hydroxyphenyl)amino]carbonyl]‐2‐naphthalenyl]oxy]ethyl]‐2‐naphthalenecarboxamide), or by co‐transfection with a short hairpin RNA construct specific for CREB. These results indicate that PCB 11 promotes dendritic growth via interaction with CREB or CREB‐dependent mechanism(s), but not directly interfering with known molecular targets of the higher chlorinated PCBs. In light of emerging evidence implicating perturbations of CREB signaling in the etiology of many NDDs, these findings add to the weight of evidence suggesting that PCB 11 poses a threat to the developing human brain.Support or Funding InformationSupported by NIEHS (R01 ES014901 to PJL & INP; T32 ES007059 to SS; P01 ES013661 to HJL) & NICHD (F32 HD088016 to KPK and U54 HD079125).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acute Hypoxia Alters Ryanodine Receptor Activity in Pulmonary Arterial Myocytes of High Altitude Acclimatized Fetal and Adult Sheep

Intrauterine stress, such as long term high altitude hypoxia (LTH), can alter pulmonary function and reprogram fetal pulmonary vascular development, potentially causing respiratory distress and pulmonary hypertension after birth. Our recent data demonstrates that newborn LTH lambs exhibit exaggerated hypoxia‐induced pulmonary vasoconstriction (HPV). Previous evidence shows Ryanodine Receptor (RyR) activation is central to the HPV response. Furthermore, LTH disrupts RyR activated Ca2+ sparks in pulmonary arterial (PA) myocytes of sheep. Given the relationship between RyR and HPV responses, we tested the hypothesis that acute hypoxia (exposure to 4% O2) and LTH increase RyR mediated Ca2+ spark activity. This was tested by measuring the Ca2+ spark activity in PA myocytes of near‐term fetal lambs and adult sheep that resided at either low (335m, LA) or high (3801m, HA) altitude. Intracellular Ca2+ was recorded in myocytes of isolated PA tissues loaded with Fluo‐4 using line‐scan techniques on a confocal microscope. Line‐scan images were analyzed using custom software (SparkLab) to determine the number of cells displaying sparks, spark frequency, and spatial‐ temporal characteristics. Adult myocytes had greater prevalence and frequency of Ca2+ sparks relative to those of fetuses, confirming previous studies. Acute hypoxia significantly decreased spark prevalence and frequency in myocytes from both HA fetal and LA adult groups; however, acute hypoxia increased spark prevalence and frequency in HA adult sheep. Spark amplitude was decreased by acute hypoxia in the LA fetus but increased in the HA adult. Acute hypoxia also reduced spark duration in the fetus independent of altitude but increased the duration in HA adult. These results provide new insight into the influence of high altitude reprogramming of RyR function in PA of the fetus and adult and the roles their activity have during acute hypoxic exposures.Support or Funding InformationThis work is supported by The National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development grant number HD083132, by the National Science Foundation under Grant No. MRI 0923559, and the Loma Linda University School of Medicine. JT was a Loma Linda University Summer Undergraduate Research Fellow while TY was a Walter E Macpherson Society Medical Student Summer Research Fellow.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High-Fat-Diet-Induced Obesity Produces Spontaneous Ventricular Arrhythmias and Increases the Activity of Ryanodine Receptors in Mice.

Ventricular arrhythmias are a common cause of sudden cardiac death, and their occurrence is higher in obese subjects. Abnormal gating of ryanodine receptors (RyR2), the calcium release channels of the sarcoplasmic reticulum, can produce ventricular arrhythmias. Since obesity promotes oxidative stress and RyR2 are redox-sensitive channels, we investigated whether the RyR2 activity was altered in obese mice. Mice fed a high fat diet (HFD) became obese after eight weeks and exhibited a significant increase in the occurrence of ventricular arrhythmias. Single RyR2 channels isolated from the hearts of obese mice were more active in planar bilayers than those isolated from the hearts of the control mice. At the molecular level, RyR2 channels from HFD-fed mice had substantially fewer free thiol residues, suggesting that redox modifications were responsible for the higher activity. Apocynin, provided in the drinking water, completely prevented the appearance of ventricular arrhythmias in HFD-fed mice, and normalized the activity and content of the free thiol residues of the protein. HFD increased the expression of NOX4, an isoform of NADPH oxidase, in the heart. Our results suggest that HFD increases the activity of RyR2 channels via a redox-dependent mechanism, favoring the appearance of ventricular arrhythmias.

Pharmacological Targeting of Serca May Have Potential for Cellular Protection

We previously reported that CGP-37157 and K201, two benzothiazepines (BZT) with cardioprotective action, inhibit the Ca2+ ATPase of sarcoplasmic reticulum (SR) intracellular Ca2+ stores (SERCA). We tested if SERCA block could be part of the mechanism by which drugs protect cells from ischemic damage. We also screened structural characteristics in BZT that could affect their drug potency as SERCA inhibitors. SR microsomes isolated from rabbit skeletal muscle (SkM) and pig heart ventricle were utilized to measure modulation of SR Ca2+ loading and SERCA-mediated ATPase activity by drugs. Seven out of twenty cell-protective drugs tested (including pimozide, EGCG, KN-93 and carvedilol) inhibited, at least partially, SR Ca2+ loading and Ca2+-stimulated ATPase activity in SkM and heart SR microsomes. We also screened ten novel BZT derivatives and seven FDA-approved benzodiazepines (BZD; including bromazepam and clonazepam), which have close homology to CGP37157. Ca2+-dependent block of SERCA was found in four BZT's, which displayed higher (PH000995, PH000902) and similar potency (PH000902, PH006796) compared to CGP. BZD were all ineffective, which suggest that the sulfur atom in the BZT ring (substituted by nitrogen in BZD) is crucial for their SERCA blocking ability. All compounds above, were tested on ryanodine receptor (RyR) activity (planar bilayers, SR leak, [3H] ryanodine binding). None of these agents directly inhibited RyR function in heart and muscle. In contrast, some agents (BZT and BZD) had mild agonistic action on channel function. We think that SERCA block by these drugs, which persists at pH ∼6.5, may benefit ischemic cells by preventing SR Ca2+ overload, known to trigger, upon reperfusion, abnormal RyR-mediated Ca2+ leak associated with cell death and tissue injury. BZT have potential as templates for therapeutic targeting of SERCA (Supported by AHA and Eskridge Foundation).

The Role of Calmodulin Methionine Oxidation in Regulating Conformational Change

We have used molecular dynamics simulations to understand the role of calmodulin (CaM) methionine (Met) oxidation in disrupting calcium-induced conformational changes associated with CaM's regulation of its target proteins. Previous work suggests that C-terminal CaM methionines, particularly M109 and M124, are critical for functional interaction with the major calcium release channel in muscle, the ryanodine receptor (RyR). Methionine to glutamine (Gln) substitutions at M109 or M124 designed to mimic oxidation of Met to Met sulfoxide block CaM activation of RyR at low calcium levels and inhibition of RyR at high calcium levels (Journal of Biological Chemistry 278(18) 2003). Spectroscopic distance measurements across CaM's lobes suggest that these same Met to Gln substitutions altered the equilibrium between key CaM conformations (Biochemical and biophysical research communications 456(2) 2015). We carried out microsecond molecular dynamics simulations of the open and closed CaM structural states with Met to Gln substitutions at M109 or M124 to gain an atomic-level understanding of (1) the role of methionines in stabilizing CaM structure, and (2) how Met oxidation might influence conformational equilibria and regulatory interactions. We found that every CaM C-terminal Met participates in a Met-aromatic interaction with nearby Phe residues with interaction energies of ∼4-10 kcal/mol. M109 stably interacts with F92, but only in the open structure, and M124 stably interacts with F141, but only in the closed structure. Met to Gln substitution at M109 or M124 alters interaction energies with these nearby Phe and alters the stability of the open and closed structures. We expect that these results will be applicable to understanding the general mechanism through which Met oxidation orchestrates redox cell signaling.