Generation of dendritic Ca2+ oscillations as a consequence of altered ryanodine receptor function in AD neurons

Ryanodine receptor (RyR)-mediated Ca(2+) dysregulation is associated with Alzheimer's disease (AD) neuropathology. Using 2-photon Ca(2+) imaging and patch clamp recordings in brain slice preparations from young 3xTg-AD and NonTg control mice, we recently demonstrated that RyR-mediated Ca(2+) -induced Ca(2+) release (CICR) is substantially increased within dendrites from AD neurons, such that synaptic stimulation alone is sufficient to generate aberrant CICR. We also observed supra-additive Ca(2+) release upon coincident RyR activation with synaptic stimulation in 3xTg-AD mice. Here, we describe an additional observed phenomenon: generation of patterned Ca(2+) oscillations in the spines and dendrites from AD neurons upon coincident RyR and synaptic stimulation. As the temporal entrainment of Ca(2+) signals influences many downstream cellular and synaptic functions, these abnormal oscillatory patterns may be associated with the structural and functional breakdown of synapses in AD.     

Stabilizing ER Ca2+ Channel Function as an Early Preventative Strategy for Alzheimer’s Disease

Alzheimer’s disease (AD) is a devastating neurodegenerative condition with no known cure. While current therapies target late-stage amyloid formation and cholinergic tone, to date, these strategies have proven ineffective at preventing disease progression. The reasons for this may be varied, and could reflect late intervention, or, that earlier pathogenic mechanisms have been overlooked and permitted to accelerate the disease process. One such example would include synaptic pathology, the disease component strongly associated with cognitive impairment. Dysregulated Ca²⁺ homeostasis may be one of the critical factors driving synaptic dysfunction. One of the earliest pathophysiological indicators in mutant presenilin (PS) AD mice is increased intracellular Ca²⁺ signaling, predominantly through the ER-localized inositol triphosphate (IP₃) and ryanodine receptors (RyR). In particular, the RyR-mediated Ca²⁺ upregulation within synaptic compartments is associated with altered synaptic homeostasis and network depression at early (presymptomatic) AD stages. Here, we offer an alternative approach to AD therapeutics by stabilizing early pathogenic mechanisms associated with synaptic abnormalities. We targeted the RyR as a means to prevent disease progression, and sub-chronically treated AD mouse models (4-weeks) with a novel formulation of the RyR inhibitor, dantrolene. Using 2-photon Ca²⁺ imaging and patch clamp recordings, we demonstrate that dantrolene treatment fully normalizes ER Ca²⁺ signaling within somatic and dendritic compartments in early and later-stage AD mice in hippocampal slices. Additionally, the elevated RyR2 levels in AD mice are restored to control levels with dantrolene treatment, as are synaptic transmission and synaptic plasticity. Aβ deposition within the cortex and hippocampus is also reduced in dantrolene-treated AD mice. In this study, we highlight the pivotal role of Ca²⁺ aberrations in AD, and propose a novel strategy to preserve synaptic function, and thereby cognitive function, in early AD patients.     

Two ryanodine receptor isoforms in nonmammalian vertebrate skeletal muscle: possible roles in excitation-contraction coupling and other processes

The ryanodine receptor (RyR) is a Ca²⁺ release channel in the sarcoplasmic reticulum in vertebrate skeletal muscle and plays an important role in excitation–contraction (E–C) coupling. Whereas mammalian skeletal muscle predominantly expresses a single RyR isoform, RyR1, skeletal muscle of many nonmammalian vertebrates expresses equal amounts of two distinct isoforms, α-RyR and β-RyR, which are homologues of mammalian RyR1 and RyR3, respectively. In this review we describe our current understanding of the functions of these two RyR isoforms in nonmammalian vertebrate skeletal muscle. The Ca²⁺ release via the RyR channel can be gated by two distinct modes: depolarization-induced Ca²⁺ release (DICR) and Ca²⁺-induced Ca²⁺ release (CICR). In frog muscle, α-RyR acts as the DICR channel, whereas β-RyR as the CICR channel. However, several lines of evidence suggest that CICR by β-RyR may make only a minor contribution to Ca²⁺ release during E–C coupling. Comparison of frog and mammalian RyR isoforms highlights the marked differences in the patterns of Ca²⁺ release mediated by RyR1 and RyR3 homologues. Interestingly, common features in the Ca²⁺ release patterns are noticed between β-RyR and RyR1. We will discuss possible roles and significance of the two RyR isoforms in E–C coupling and other processes in nonmammalian vertebrate skeletal muscle.     

Superresolution Modeling of Calcium Release in the Heart

Stable calcium-induced calcium release (CICR) is critical for maintaining normal cellular contraction during cardiac excitation-contraction coupling. The fundamental element of CICR in the heart is the calcium (Ca²⁺) spark, which arises from a cluster of ryanodine receptors (RyR). Opening of these RyR clusters is triggered to produce a local, regenerative release of Ca²⁺ from the sarcoplasmic reticulum (SR). The Ca²⁺ leak out of the SR is an important process for cellular Ca²⁺ management, and it is critically influenced by spark fidelity, i.e., the probability that a spontaneous RyR opening triggers a Ca²⁺ spark. Here, we present a detailed, three-dimensional model of a cardiac Ca²⁺ release unit that incorporates diffusion, intracellular buffering systems, and stochastically gated ion channels. The model exhibits realistic Ca²⁺ sparks and robust Ca²⁺ spark termination across a wide range of geometries and conditions. Furthermore, the model captures the details of Ca²⁺ spark and nonspark-based SR Ca²⁺ leak, and it produces normal excitation-contraction coupling gain. We show that SR luminal Ca²⁺-dependent regulation of the RyR is not critical for spark termination, but it can explain the exponential rise in the SR Ca²⁺ leak-load relationship demonstrated in previous experimental work. Perturbations to subspace dimensions, which have been observed in experimental models of disease, strongly alter Ca²⁺ spark dynamics. In addition, we find that the structure of RyR clusters also influences Ca²⁺ release properties due to variations in inter-RyR coupling via local subspace Ca²⁺ concentration ([Ca²⁺]_ss). These results are illustrated for RyR clusters based on super-resolution stimulated emission depletion microscopy. Finally, we present a believed-novel approach by which the spark fidelity of a RyR cluster can be predicted from structural information of the cluster using the maximum eigenvalue of its adjacency matrix. These results provide critical insights into CICR dynamics in heart, under normal and pathological conditions.     

Homeostatic and stimulus-induced coupling of the L-type Ca2+ channel to the ryanodine receptor in the hippocampal neuron in slices

Activity-dependent increase in cytosolic calcium ([Ca²⁺]_i) is a prerequisite for many neuronal functions. We previously reported a strong direct depolarization, independent of glutamate receptors, effectively caused a release of Ca²⁺ from ryanodine-sensitive stores and induced the synthesis of endogenous cannabinoids (eCBs) and eCB-mediated responses. However, the cellular mechanism that initiated the depolarization-induced Ca²⁺-release is not completely understood. In the present study, we optically recorded [Ca²⁺]_i from CA1 pyramidal neurons in the hippocampal slice and directly monitored miniature Ca²⁺ activities and depolarization-induced Ca²⁺ signals in order to determine the source(s) and properties of [Ca²⁺]_i-dynamics that could lead to a release of Ca²⁺ from the ryanodine receptor. In the absence of depolarizing stimuli, spontaneously occurring miniature Ca²⁺ events were detected from a group of hippocampal neurons. This miniature Ca²⁺ event persisted in the nominal Ca²⁺-containing artificial cerebrospinal fluid (ACSF), and increased in frequency in response to the bath-application of caffeine and KCl. In contrast, nimodipine, the antagonist of the L-type Ca²⁺ channel (LTCC), a high concentration of ryanodine, the antagonist of the ryanodine receptor (RyR), and thapsigargin (TG) reduced the occurrence of the miniature Ca²⁺ events. When a brief puff-application of KCl was given locally to the soma of individual neurons in the presence of glutamate receptor antagonists, these neurons generated a transient increase in the [Ca²⁺]_i in the dendrosomal region. This [Ca²⁺]_i-transient was sensitive to nimodipine, TG, and ryanodine suggesting that the [Ca²⁺]_i-transient was caused primarily by the LTCC-mediated Ca²⁺-influx and a release of Ca²⁺ from RyR. We observed little contribution from N- or P/Q-type Ca²⁺ channels. The coupling between LTCC and RyR was direct and independent of synaptic activities. Immunohistochemical study revealed a cellular localization of LTCC and RyR in a juxtaposed configuration in the proximal dendrites and soma. We conclude in the hippocampal CA1 neuron that: (1) homeostatic fluctuation of the resting membrane potential may be sufficient to initiate functional coupling between LTCC and RyR; (2) the juxtaposed localization of LTCC and RyR has anatomical advantage of synchronizing a Ca²⁺-release from RyR upon the opening of LTCC; and (3) the synchronized Ca²⁺-release from RyR occurs immediately after the activation of LTCC and determines the peak amplitude of depolarization-induced global increase in dendrosomal [Ca²⁺]_i.     

A calcium-induced calcium release mechanism mediated by calsequestrin

Calcium (Ca²⁺)-induced Ca²⁺ release (CICR) is widely accepted as the principal mechanism linking electrical excitation and mechanical contraction in cardiac cells. The CICR mechanism has been understood mainly based on binding of cytosolic Ca²⁺ with ryanodine receptors (RyRs) and inducing Ca²⁺ release from the sarcoplasmic reticulum (SR). However, recent experiments suggest that SR lumenal Ca²⁺ may also participate in regulating RyR gating through calsequestrin (CSQ), the SR lumenal Ca²⁺ buffer. We investigate how SR Ca²⁺ release via RyR is regulated by Ca²⁺ and calsequestrin (CSQ). First, a mathematical model of RyR kinetics is derived based on experimental evidence. We assume that the RyR has three binding sites, two cytosolic sites for Ca²⁺ activation and inactivation, and one SR lumenal site for CSQ binding. The open probability (P_o) of the RyR is found by simulation under controlled cytosolic and SR lumenal Ca²⁺. Both peak and steady-state P_o effectively increase as SR lumenal Ca²⁺ increases. Second, we incorporate the RyR model into a CICR model that has both a diadic space and the junctional SR (jSR). At low jSR Ca²⁺ loads, CSQs are more likely to bind with the RyR and act to inhibit jSR Ca²⁺ release, while at high SR loads CSQs are more likely to detach from the RyR, thereby increasing jSR Ca²⁺ release. Furthermore, this CICR model produces a nonlinear relationship between fractional jSR Ca²⁺ release and jSR load. These findings agree with experimental observations in lipid bilayers and cardiac myocytes.     

Control of Sarcoplasmic Reticulum Ca2+ Release by Stochastic RyR Gating within a 3D Model of the Cardiac Dyad and Importance of Induction Decay for CICR Termination

The factors responsible for the regulation of regenerative calcium-induced calcium release (CICR) during Ca²⁺ spark evolution remain unclear. Cardiac ryanodine receptor (RyR) gating in rats and sheep was recorded at physiological Ca²⁺, Mg²⁺, and ATP levels and incorporated into a 3D model of the cardiac dyad, which reproduced the time course of Ca²⁺ sparks, Ca²⁺ blinks, and Ca²⁺ spark restitution. The termination of CICR by induction decay in the model principally arose from the steep Ca²⁺ dependence of RyR closed time, with the measured sarcoplasmic reticulum (SR) lumen Ca²⁺ dependence of RyR gating making almost no contribution. The start of CICR termination was strongly dependent on the extent of local depletion of junctional SR Ca²⁺, as well as the time course of local Ca²⁺ gradients within the junctional space. Reducing the dimensions of the dyad junction reduced Ca²⁺ spark amplitude by reducing the strength of regenerative feedback within CICR. A refractory period for Ca²⁺ spark initiation and subsequent Ca²⁺ spark amplitude restitution arose from 1), the extent to which the regenerative phase of CICR can be supported by the partially depleted junctional SR, and 2), the availability of releasable Ca²⁺ in the junctional SR. The physical organization of RyRs within the junctional space had minimal effects on Ca²⁺ spark amplitude when more than nine RyRs were present. Spark amplitude had a nonlinear dependence on RyR single-channel Ca²⁺ flux, and was approximately halved by reducing the flux from 0.6 to 0.2 pA. Although rat and sheep RyRs had quite different Ca²⁺ sensitivities, Ca²⁺ spark amplitude was hardly affected. This suggests that moderate changes in RyR gating by second-messenger systems will principally alter the spatiotemporal properties of SR release, with smaller effects on the amount released.     

Ca2+ Release through Ryanodine Receptors in the Sarcoplasmic Reticulum and Ca2+ Sequestration by the Mitochondria in Smooth Muscle Cells

The contribution of the Ca²⁺-induced Ca²⁺ release (CICR) mechanism in excitation-contraction (E-C) coupling and the tightness of the coupling between Ca²⁺ influx and Ca²⁺ release are still controversial in smooth muscle cells (SMC). In SMC isolated from the guinea-pig vas deferens or urinary bladder, a depolarizing stimulus initially induced spot-like increases in the intracellular Ca²⁺ concentration ([Ca²⁺] _i ), called “Ca²⁺ hot spots,” at several superficial areas in the cell. When a weak stimulus (a small or a short depolarizing step) was applied, only a few Ca²⁺ hot spots appeared transiently in the superficial area but did not spread into other regions, to trigger global [Ca²⁺] _i rise. Such depolarization-evoked local Ca2+ transients were distinctive from spontaneous Ca²⁺ sparks, since the former were susceptible to Ca²⁺ blockers, ryanodine, and inhibitors of the Ca²⁺ pump in the sarcoplasmic reticulum (SR), suggesting pivotal roles of Ca²⁺ influx through voltage-dependent Ca²⁺ channels (VDCC) and Ca²⁺ release from the SR through ryanodine receptors (RyR) for the activation of Ca²⁺ spots. Frequently discharging Ca²⁺ spark sites (FDS) under resting conditions were located exactly in the same areas as Ca²⁺ hot spots evoked by depolarization, indicating the existence of distinct local junction sites for tight coupling between VDCC in the plasmalemma and RyR in the SR. Co-localization of clusters of RyR and large-conductance Ca²⁺-activated K⁺ (BK) channels was also suggested. The fast and tight coupling for CICR in these junctional sites was triggered also by an action potential, whereas a slower spread of Ca²⁺ wave to the whole-cell areas suggests the loose coupling in propagating CICR to other cell areas. It can therefore be postulated that CICR may occur in two steps upon depolarization; the initial CICR in distinct junctional sites shows tight coupling between Ca²⁺ influx and release, and the following CICR may propagate slow Ca²⁺ waves to other areas. Ryanodine receptors form a multiprotein complex with molecules such as calsequestrin, junctin, triadin, junctophilins, and FK506-binding proteins, which directly or indirectly regulate the RyR activity and the tight coupling. Moreover, an evoked Ca²⁺ spot may enhance Ca²⁺ uptake by neighboring mitochondria and their ATP production to increase energy supply to the Ca²⁺ pump of the SR in the microdomain.     

CRISPR/Cas9 Gene editing of RyR2 in human stem cell-derived cardiomyocytes provides a novel approach in investigating dysfunctional Ca2+ signaling

Type-2 ryanodine receptors (RyR2s) play a pivotal role in cardiac excitation-contraction coupling by releasing Ca²⁺ from sarcoplasmic reticulum (SR) via a Ca²⁺ -induced Ca²⁺ release (CICR) mechanism. Two strategies have been used to study the structure-function characteristics of RyR2 and its disease associated mutations: (1) heterologous cell expression of the recombinant mutant RyR2s, and (2) knock-in mouse models harboring RyR2 point mutations. Here, we establish an alternative approach where Ca²⁺ signaling aberrancy caused by the RyR2 mutation is studied in human cardiomyocytes with robust CICR mechanism. Specifically, we introduce point mutations in wild-type RYR2 of human induced pluripotent stem cells (hiPSCs) by CRISPR/Cas9 gene editing, and then differentiate them into cardiomyocytes. To verify the reliability of this approach, we introduced the same disease-associated RyR2 mutation, F2483I, which was studied by us in hiPSC-derived cardiomyocytes (hiPSC-CMs) from a patient biopsy. The gene-edited F2483I hiPSC-CMs exhibited longer and wandering Ca²⁺ sparks, elevated diastolic Ca²⁺ leaks, and smaller SR Ca²⁺ stores, like those of patient-derived cells. Our CRISPR/Cas9 gene editing approach validated the feasibility of creating myocytes expressing the various RyR2 mutants, making comparative mechanistic analysis and pharmacotherapeutic approaches for RyR2 pathologies possible.     

Impaired TNF-α control of IP3R-mediated Ca2+ release in Alzheimer's disease mouse neurons

The misguided control of inflammatory signaling has been previously implicated in the pathogenesis of several neurological disorders, including Alzheimer's disease (AD). Induction of tumor necrosis factor-alpha (TNF-α), a central mediator of neuroinflammation, occurs commensurate with the onset of early disease in 3xTg-AD mice, which develop both amyloid plaque and neurofibrillary tangle pathologies in an age- and region-dependent pattern. Herein, we describe regulation inherent to 3xTg-AD neurons, which results in the loss of TNF-α mediated enhancement of inositol 1,4,5 trisphosphate (IP₃R)-mediated Ca²⁺ release. This modulation also leads to significant down-regulation of IP₃R signaling following protracted cytokine exposure. Through the experimental isolation of each AD-related transgene, it was determined that expression of the PS1^M146V transgene product is responsible for the loss of the TNF-α effect on IP₃R-mediated Ca²⁺ release. Furthermore, it was determined that the suppression of TNF-α receptor expression occurred in the presence of the presenilin transgene. Our findings attribute this familial AD mutation to suppressing a Ca²⁺-regulated signal cascade potentially intended to “inform” neurons of proximal neuroinflammatory events and trigger compensatory responses for protection of neural transmission.     

A Human Ventricular Myocyte Model with a Refined Representation of Excitation-Contraction Coupling

Cardiac Ca²⁺-induced Ca²⁺ release (CICR) occurs by a regenerative activation of ryanodine receptors (RyRs) within each Ca²⁺-releasing unit, triggered by the activation of L-type Ca²⁺ channels (LCCs). CICR is then terminated, most probably by depletion of Ca²⁺ in the junctional sarcoplasmic reticulum (SR). Hinch et al. previously developed a tightly coupled LCC-RyR mathematical model, known as the Hinch model, that enables simulations to deal with a variety of functional states of whole-cell populations of a Ca²⁺-releasing unit using a personal computer. In this study, we developed a membrane excitation-contraction model of the human ventricular myocyte, which we call the human ventricular cell (HuVEC) model. This model is a hybrid of the most recent HuVEC models and the Hinch model. We modified the Hinch model to reproduce the regenerative activation and termination of CICR. In particular, we removed the inactivated RyR state and separated the single step of RyR activation by LCCs into triggering and regenerative steps. More importantly, we included the experimental measurement of a transient rise in Ca²⁺ concentrations ([Ca²⁺], 10–15 μM) during CICR in the vicinity of Ca²⁺-releasing sites, and thereby calculated the effects of the local Ca²⁺ gradient on CICR as well as membrane excitation. This HuVEC model successfully reconstructed both membrane excitation and key properties of CICR. The time course of CICR evoked by an action potential was accounted for by autonomous changes in an instantaneous equilibrium open probability of couplons. This autonomous time course was driven by a core feedback loop including the pivotal local [Ca²⁺], influenced by a time-dependent decay in the SR Ca²⁺ content during CICR.     

Postulated role of interdomain interactions within the type 1 ryanodine receptor in the low gain of Ca2+-induced Ca2+ release activity of mammalian skeletal muscle sarcoplasmic reticulum

Ryanodine receptor (RyR) type 1 (RyR1) exhibits a markedly lower gain of Ca²⁺-induced Ca²⁺ release (CICR) activity than RyR type 3 (RyR3) in the sarcoplasmic reticulum (SR) of mammalian skeletal muscle (selective stabilization of the RyR1 channel), and this reduction in the gain is largely eliminated using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). We have investigated whether the hypothesized interdomain interactions within RyR1 are involved in the selective stabilization of the channel using [³H]ryanodine binding, single-channel recordings, and Ca²⁺ release from the SR vesicles. Like CHAPS, domain peptide 4 (DP4, a synthetic peptide corresponding to the Leu²⁴⁴²-Pro²⁴⁷⁷ region of RyR1), which seems to destabilize the interdomain interactions, markedly stimulated RyR1 but not RyR3. Their activating effects were saturable and nonadditive. Dantrolene, a potent inhibitor of RyR1 used to treat malignant hyperthermia, reversed the effects of DP4 or CHAPS in an identical manner. These findings indicate that RyR1 is activated by DP4 and CHAPS through a common mechanism that is probably mediated by the interdomain interactions. DP4 greatly increased [³H]ryanodine binding to RyR1 with only minor alterations in the sensitivity to endogenous CICR modulators (Ca²⁺, Mg²⁺, and adenine nucleotide). However, DP4 sensitized RyR1 four- to six-fold to caffeine in the caffeine-induced Ca²⁺ release. Thus the gain of CICR activity critically determines the magnitude and threshold of Ca²⁺ release by drugs such as caffeine. These findings suggest that the low CICR gain of RyR1 is important in normal Ca²⁺ handling in skeletal muscle and that perturbation of this state may result in muscle diseases such as malignant hyperthermia. malignant hyperthermia; 3-[(3-cholamidopropyl)dimethylammonio]propane sulfonic acid; domain peptide 4     

Up‐regulation of the type 3 ryanodine receptor is neuroprotective in the TgCRND8 mouse model of Alzheimer’s disease

The cellular pathology of Alzheimer’s disease is progressive and protracted leading eventually to considerable neuronal death. The underlying mechanisms of the pathology are complex but changes in the control of intracellular Ca²⁺ are believed to contribute to the demise of neurons. In this study, we investigated the functional consequences of an increase in the expression of the type 3 isoform of the ryanodine receptor (RyR3). We found that although cortical neurons from TgCRND8 mice secreted significantly more amyloid beta protein and showed significantly increased RyR3 expression, they were no more sensitive to cell stress than non‐transgenic neurons. Furthermore, despite increased intracellular Ca²⁺ release in response to ryanodine, we found that basal Ca²⁺, K⁺‐evoked Ca²⁺ responses, and capacitative Ca²⁺ entry were no different in TgCRND8 neurons compared with non‐transgenic neurons. Therefore, as RyR3 up‐regulation did not affect neuronal health or global Ca²⁺ homeostasis, we investigated the effect of reducing RyR3 expression using small interfering RNA. Surprisingly, a reduction of RyR3 expression in TgCRND8, but not in non‐transgenic, neurons increased neuronal death. These data reveal a new role for RyR3 and indicate a novel potential therapeutic target to delay or prevent the progression of Alzheimer’s disease.     

Mechanism of calsequestrin regulation of single cardiac ryanodine receptor in normal and pathological conditions

Release of Ca²⁺ from the sarcoplasmic reticulum (SR) drives contractile function of cardiac myocytes. Luminal Ca²⁺ regulation of SR Ca²⁺ release is fundamental not only in physiology but also in physiopathology because abnormal luminal Ca²⁺ regulation is known to lead to arrhythmias, catecholaminergic polymorphic ventricular tachycardia (CPVT), and/or sudden cardiac arrest, as inferred from animal model studies. Luminal Ca²⁺ regulates ryanodine receptor (RyR)2-mediated SR Ca²⁺ release through mechanisms localized inside the SR; one of these involves luminal Ca²⁺ interacting with calsequestrin (CASQ), triadin, and/or junctin to regulate RyR2 function.CASQ2-RyR2 regulation was examined at the single RyR2 channel level. Single RyR2s were incorporated into planar lipid bilayers by the fusion of native SR vesicles isolated from either wild-type (WT), CASQ2 knockout (KO), or R33Q-CASQ2 knock-in (KI) mice. KO and KI mice have CPVT-like phenotypes. We show that CASQ2(WT) action on RyR2 function (either activation or inhibition) was strongly influenced by the presence of cytosolic MgATP. Function of the reconstituted CASQ2(WT)–RyR2 complex was unaffected by changes in luminal free [Ca²⁺] (from 0.1 to 1 mM). The inhibition exerted by CASQ2(WT) association with the RyR2 determined a reduction in cytosolic Ca²⁺ activation sensitivity. RyR2s from KO mice were significantly more sensitive to cytosolic Ca²⁺ activation and had significantly longer mean open times than RyR2s from WT mice. Sensitivity of RyR2s from KI mice was in between that of RyR2 channels from KO and WT mice. Enhanced cytosolic RyR2 Ca²⁺ sensitivity and longer RyR2 open times likely explain the CPVT-like phenotype of both KO and KI mice.     

CaMKII‐dependent ryanodine receptor phosphorylation mediates sepsis‐induced cardiomyocyte apoptosis

Sepsis is associated with cardiac dysfunction, which is at least in part due to cardiomyocyte apoptosis. However, the underlying mechanisms are far from being understood. Using the colon ascendens stent peritonitis mouse model of sepsis (CASP), we examined the subcellular mechanisms that mediate sepsis‐induced apoptosis. Wild‐type (WT) CASP mice hearts showed an increase in apoptosis respect to WT‐Sham. CASP transgenic mice expressing a CaMKII inhibitory peptide (AC3‐I) were protected against sepsis‐induced apoptosis. Dantrolene, used to reduce ryanodine receptor (RyR) diastolic sarcoplasmic reticulum (SR) Ca²⁺ release, prevented apoptosis in WT‐CASP. To examine whether CaMKII‐dependent RyR2 phosphorylation mediates diastolic Ca²⁺ release and apoptosis in sepsis, we evaluated apoptosis in mutant mice hearts that have the CaMKII phosphorylation site of RyR2 (Serine 2814) mutated to Alanine (S2814A). S2814A CASP mice did not show increased apoptosis. Consistent with RyR2 phosphorylation‐dependent enhancement in diastolic SR Ca²⁺ release leading to mitochondrial Ca²⁺ overload, mitochondrial Ca²⁺ retention capacity was reduced in mitochondria isolated from WT‐CASP compared to Sham and this reduction was absent in mitochondria from CASP S2814A or dantrolene‐treated mice. We conclude that in sepsis, CaMKII‐dependent RyR2 phosphorylation results in diastolic Ca²⁺ release from SR which leads to mitochondrial Ca²⁺ overload and apoptosis.     

Loss of Ryanodine Receptor 2 impairs neuronal activity-dependent remodeling of dendritic spines and triggers compensatory neuronal hyperexcitability

Dendritic spines are postsynaptic domains that shape structural and functional properties of neurons. Upon neuronal activity, Ca²⁺ transients trigger signaling cascades that determine the plastic remodeling of dendritic spines, which modulate learning and memory. Here, we study in mice the role of the intracellular Ca²⁺ channel Ryanodine Receptor 2 (RyR2) in synaptic plasticity and memory formation. We demonstrate that loss of RyR2 in pyramidal neurons of the hippocampus impairs maintenance and activity-evoked structural plasticity of dendritic spines during memory acquisition. Furthermore, post-developmental deletion of RyR2 causes loss of excitatory synapses, dendritic sparsification, overcompensatory excitability, network hyperactivity and disruption of spatially tuned place cells. Altogether, our data underpin RyR2 as a link between spine remodeling, circuitry dysfunction and memory acquisition, which closely resemble pathological mechanisms observed in neurodegenerative disorders.Subject terms: Neuroscience, Neurological disorders     

Effects of ATP, Mg2+, and redox agents on the Ca2+ dependence of RyR channels from rat brain cortex

Despite their relevance for neuronal Ca²⁺-induced Ca²⁺ release (CICR), activation by Ca²⁺ of ryanodine receptor (RyR) channels of brain endoplasmic reticulum at the [ATP], [Mg²⁺], and redox conditions present in neurons has not been reported. Here, we studied the effects of varying cis-(cytoplasmic) free ATP concentration ([ATP]), [Mg²⁺], and RyR redox state on the Ca²⁺ dependence of endoplasmic reticulum RyR channels from rat brain cortex. At pCa 4.9 and 0.5 mM adenylylimidodiphosphate (AMP-PNP), increasing free [Mg²⁺] up to 1 mM inhibited vesicular [³H]ryanodine binding; incubation with thimerosal or dithiothreitol decreased or enhanced Mg²⁺ inhibition, respectively. Single RyR channels incorporated into lipid bilayers displayed three different Ca²⁺ dependencies, defined by low, moderate, or high maximal fractional open time (P_o), that depend on RyR redox state, as we have previously reported. In all cases, cis-ATP addition (3 mM) decreased threshold [Ca²⁺] for activation, increased maximal P_o, and shifted channel inhibition to higher [Ca²⁺]. Conversely, at pCa 4.5 and 3 mM ATP, increasing cis-[Mg²⁺] up to 1 mM inhibited low activity channels more than moderate activity channels but barely modified high activity channels. Addition of 0.5 mM free [ATP] plus 0.8 mM free [Mg²⁺] induced a right shift in Ca²⁺ dependence for all channels so that [Ca²⁺] <30 µM activated only high activity channels. These results strongly suggest that channel redox state determines RyR activation by Ca²⁺ at physiological [ATP] and [Mg²⁺]. If RyR behave similarly in living neurons, cellular redox state should affect RyR-mediated CICR. Ca²⁺-induced Ca²⁺ release; Ca²⁺ release channels; endoplasmic reticulum; thimerosal; 2,4-dithiothreitol; ryanodine receptor     

Profile for Amyloid-β and Tau Expression in Primary Cortical Cultures from 3xTg-AD Mice

Advances in transgenic technology as well as in the genetics of Alzheimer disease (AD) have allowed the establishment of animal models that reproduce amyloid-beta plaques and neurofibrillary tangles, the main pathological hallmarks of AD. Among these models, 3xTg-AD mice harboring PS1 _M146V, APP _Swe and tau _P301L human transgenes provided the model that most closely mimics human AD features. Although cortical cultures from 3xTg-AD mice have been shown to present disturbances in intracellular [Ca²⁺] homeostasis, the development of AD pathology in vitro has not been previously evaluated. In the current work, we determined the temporal profile for amyloid precursor protein, amyloid-β and tau expression in primary cortical cultures from 3xTg-AD mice. Immunocytochemistry and Western blot analysis showed an increased expression of these proteins as well as several phosphorylated tau isoforms with time in culture. Alterations in calcium homeostasis and cholinergic and glutamatergic responses were also observed early in vitro. Thus, 3x-TgAD cortical neurons in vitro provide an exceptional tool to investigate pharmacological approaches as well as the cellular basis for AD and related diseases.     

Developmental changes in the regulation of calcium-dependent neurite outgrowth

Intracellular calcium ions (Ca²⁺) have an essential role in the regulation of neurite outgrowth, but how outgrowth is controlled remains largely unknown. In this study, we examined how the mechanisms of neurite outgrowth change during development in chick and mouse dorsal root ganglion neurons. 2APB, a potent inhibitor of inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R), inhibited neurite outgrowth at early developmental stages, but not at later stages. In contrast, pharmacological inhibition with Ni²⁺, Cd²⁺, or dantrolene revealed that ryanodine receptor (RyR)-mediated Ca²⁺-induced Ca²⁺ release (CICR) was involved in neurite outgrowth at later stage, but not at early stages. The distribution of IP₃R and RyR in growth cones also changed during development. Furthermore, pharmacological inhibition of the Ca²⁺-calmodulin-dependent phosphatase calcineurin with FK506 reduced neurite outgrowth only at early stages. These data suggest that the calcium signaling that regulates neurite outgrowth may change during development from an IP₃R-mediated pathway to a RyR-mediated pathway.     

ARVC-related mutations in divergent region 3 alter functional properties of the cardiac ryanodine receptor

Two single-nucleotide polymorphisms in the type 2 ryanodine receptor (RyR2) leading to the nonsynonymous amino acid replacements G1885E and G1886S are associated with arrhythmogenic right ventricular cardiomyopathy in patients who are carrying both of the corresponding RyR2 alleles. The functional properties of HEK293 cell lines isogenically expressing RyR2 mutants associated with arrhythmogenic right ventricular cardiomyopathy, RyR2-G1885E, RyR2-G1886S, RyR2-G1886D (mimicking a constitutively phosphorylated Ser¹⁸⁸⁶), and the double mutant RyR2-G1885E/G1886S were investigated by analyzing the intracellular Ca²⁺ release activity resulting from store-overload-induced calcium release. The substitution of serine for Gly¹⁸⁸⁶ caused a significant increase in the cellular Ca²⁺ oscillation activity compared with RyR2 wild-type-expressing HEK293 cells. It was even more pronounced if glycine 1885 or 1886 was replaced by the acidic amino acids glutamate (G1885E) or aspartate (G1886D). Surprisingly, when both substitutions were introduced in the same RyR2 subunit (RyR2-G1885E/G1886S), the store-overload-induced calcium release activity was nearly completely abolished, although the Ca²⁺ loading of the intracellular stores was markedly enhanced, and the channel still displayed substantial Ca²⁺ release on stimulation by 5 mM caffeine. These results suggest that the adjacent glycines 1885 and 1886, located in the divergent region 3, are critical for the function and regulation of RyR2.