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Summary: Neuronal hyperactivity is an early primary dysfunction in Alzheimer’s disease (AD) in humans and animal models, but effective neuronal hyperactivity-directed anti-AD therapeutic agents are lacking. Here we define a previously unknown mode of ryanodine receptor 2 (RyR2) control of neuronal hyperactivity and AD progression. We show that a single RyR2 point mutation, E4872Q, which reduces RyR2 open time, prevents hyperexcitability, hyperactivity, memory impairment, neuronal cell death, and dendritic spine loss in a severe early-onset AD mouse model (5xFAD). The RyR2-E4872Q mutation upregulates hippocampal CA1-pyramidal cell A-type K+ current, a well-known neuronal excitability control that is downregulated in AD. Pharmacologically limiting RyR2 open time with the R-carvedilol enantiomer (but not racemic carvedilol) prevents and rescues neuronal hyperactivity, memory impairment, and neuron loss even in late stages of AD. These AD-related deficits are prevented even with continued β-amyloid accumulation. Thus, limiting RyR2 open time may be a hyperactivity-directed, non-β-amyloid-targeted anti-AD strategy.

The endoplasmic reticulum (ER) plays a central role in cellular stress responses via mobilization of ER stress coping responses, such as the unfolded protein response (UPR). The inositol-requiring 1α (IRE1α) is an ER stress sensor and component of the UPR. Muscle cells also have a well-developed and highly subspecialized membrane network of smooth ER called the sarcoplasmic reticulum (SR) surrounding myofibrils and specialized for Ca(2+) storage, release, and uptake to control muscle excitation-contraction coupling. Here, we describe 2 distinct pools of IRE1α in cardiac and skeletal muscle cells, one localized at the perinuclear ER and the other at the junctional SR. We discovered that, at the junctional SR, calsequestrin binds to the ER luminal domain of IRE1α, inhibiting its dimerization. This novel interaction of IRE1α with calsequestrin, one of the highly abundant Ca(2+) handling proteins at the junctional SR, provides new insights into the regulation of stress coping responses in muscle cells.—Wang, Q., Groenendyk, J., Paskevicius, T., Qin, W., Kor, K. C., Liu, Y., Hiess, F., Knollmann, B. C., Chen, S. R. W., Tang, J., Chen, X.-Z., Agellon, L. B., Michalak, M. Two pools of IRE1α in cardiac and skeletal muscle cells.

The cardiac Ca(2+) release channel (ryanodine receptor, RyR2) plays an essential role in excitation-contraction coupling in cardiac muscle cells. Effective and stable excitation-contraction coupling critically depends not only on the expression of RyR2, but also on its distribution. Despite its importance, little is known about the distribution and organization of RyR2 in living cells. To study the distribution of RyR2 in living cardiomyocytes, we generated a knock-in mouse model expressing a GFP-tagged RyR2 (GFP-RyR2). Confocal imaging of live ventricular myocytes isolated from the GFP-RyR2 mouse heart revealed clusters of GFP-RyR2 organized in rows with a striated pattern. Similar organization of GFP-RyR2 clusters was observed in fixed ventricular myocytes. Immunofluorescence staining with the anti-α-actinin antibody (a z-line marker) showed that nearly all GFP-RyR2 clusters were localized in the z-line zone. There were small regions with dislocated GFP-RyR2 clusters. Interestingly, these same regions also displayed dislocated z-lines. Staining with di-8-ANEPPS revealed that nearly all GFP-RyR2 clusters were co-localized with transverse but not longitudinal tubules, whereas staining with MitoTracker Red showed that GFP-RyR2 clusters were not co-localized with mitochondria in live ventricular myocytes. We also found GFP-RyR2 clusters interspersed between z-lines only at the periphery of live ventricular myocytes. Simultaneous detection of GFP-RyR2 clusters and Ca(2+) sparks showed that Ca(2+) sparks originated exclusively from RyR2 clusters. Ca(2+) sparks from RyR2 clusters induced no detectable changes in mitochondrial Ca(2+) level. These results reveal, for the first time, the distribution of RyR2 clusters and its functional correlation in living ventricular myocytes.

The cardiac Ca2+ release channel [ryanodine receptor type 2 (RyR2)] is modulated by thiol reactive agents, but the molecular basis of RyR2 modulation by thiol reagents is poorly understood. Cys3635 in the skeletal muscle RyR1 is one of the most hyper-reactive thiols and is important for the redox and calmodulin (CaM) regulation of the RyR1 channel. However, little is known about the role of the corresponding cysteine residue in RyR2 (Cys3602) in the function and regulation of the RyR2 channel. In the present study, we assessed the impact of mutating Cys3602 (C3602A) on store overload-induced Ca2+ release (SOICR) and the regulation of RyR2 by thiol reagents and CaM. We found that the C3602A mutation suppressed SOICR by raising the activation threshold and delayed the termination of Ca2+ release by reducing the termination threshold. As a result, C3602A markedly increased the fractional Ca2+ release. Furthermore, the C3602A mutation diminished the inhibitory effect of N-ethylmaleimide on Ca2+ release, but it had no effect on the stimulatory action of 4,4′-dithiodipyridine (DTDP) on Ca2+ release. In addition, Cys3602 mutations (C3602A or C3602R) did not abolish the effect of CaM on Ca2+-release termination. Therefore, RyR2–Cys3602 is a major site mediating the action of thiol alkylating agent N-ethylmaleimide, but not the action of the oxidant DTDP. Our data also indicate that residue Cys3602 plays an important role in the activation and termination of Ca2+ release, but it is not essential for CaM regulation of RyR2.

Cardiac ryanodine receptors (RyR2s) are Ca2þ release channels clustering in the sarcoplasmic reticulum membrane. These clusters are believed to be the elementary units of Ca2þ release. The distribution of these Ca2þ release units plays a critical role in determining the spatio-temporal profile and stability of sarcoplasmic reticulum Ca2þ release. RyR2 clusters located in the interior of cardiomyocytes are arranged in highly ordered arrays. However, little is known about the distribution and function of RyR2 clusters in the periphery of cardiomyocytes. Here, we used a knock-in mouse model expressing a green fluorescence protein (GFP)-tagged RyR2 to localize RyR2 clusters in live ventricular myocytes by virtue of their GFP fluorescence. Confocal imaging and total internal reflection fluorescence microscopy was employed to determine and compare the distribution of GFP-RyR2 in the interior and periphery of isolated live ventricular myocytes and in intact hearts. We found tightly ordered arrays of GFP-RyR2 clusters in the interior, as previously described. In contrast, irregular distribution of GFP-RyR2 clusters was observed in the periphery. Time-lapse total internal reflection fluorescence imaging revealed dynamic movements of GFP-RyR2 clusters in the periphery, which were affected by external Ca2þ and RyR2 activator (caffeine) and inhibitor (tetracaine), but little detectable movement of GFP-RyR2 clusters in the interior. Furthermore, simultaneous Ca2þ- and GFP-imaging demonstrated that peripheral RyR2 clusters with an irregular distribution pattern are functional with a Ca2þ release profile similar to that in the interior. These results indicate that the distribution of RyR2 clusters in the periphery of live ventricular myocytes is irregular and dynamic, which is different from that of RyR2 clusters in the interior., This work was supported by research grants from the Canadian Institutes of Health Research, the Heart and Stroke Foundation of Canada, the Canada Foundation for Innovation, the Natural Sciences and Engineering Research Council of Canada, and the Heart and Stroke Foundation Chair in Cardiovascular Research (to S.R.W.C.). This study was also supported by the Spanish Ministry of Economy and Competitiveness SAF2014-58286-C2-1-R (to L.H.-M.) and DPI2013-44584-R (to R.B.).

Author(s): Kim, Do Young; Zhang, Fang-Xiong; Nakanishi, Stan T; Mettler, Timothy; Cho, Ik-Hyun; Ahn, Younghee; Hiess, Florian; Chen, Lina; Sullivan, Patrick G; Chen, SR Wayne; Zamponi, Gerald W; Rho, Jong M | Abstract: ObjectivesCarisbamate (CRS) is a novel monocarbamate compound that possesses antiseizure and neuroprotective properties. However, the mechanisms underlying these actions remain unclear. Here, we tested both direct and indirect effects of CRS on several cellular systems that regulate intracellular calcium concentration [Ca2+ ]i .MethodsWe used a combination of cellular electrophysiologic techniques, as well as cell viability, Store Overload-Induced Calcium Release (SOICR), and mitochondrial functional assays to determine whether CRS might affect [Ca2+ ]i levels through actions on the endoplasmic reticulum (ER), mitochondria, and/or T-type voltage-gated Ca2+ channels.ResultsIn CA3 pyramidal neurons, kainic acid induced significant elevations in [Ca2+ ]i and long-lasting neuronal hyperexcitability, both of which were reversed in a dose-dependent manner by CRS. Similarly, CRS suppressed spontaneous rhythmic epileptiform activity in hippocampal slices exposed to zero-Mg2+ or 4-aminopyridine. Treatment with CRS also protected murine hippocampal HT-22 cells against excitotoxic injury with glutamate, and this was accompanied by a reduction in [Ca2+ ]i . Neither kainic acid nor CRS alone altered the mitochondrial membrane potential (ΔΨ) in intact, acutely isolated mitochondria. In addition, CRS did not affect mitochondrial respiratory chain activity, Ca2+ -induced mitochondrial permeability transition, and Ca2+ release from the ER. However, CRS significantly decreased Ca2+ flux in human embryonic kidney tsA-201 cells transfected with Cav 3.1 (voltage-dependent T-type Ca2+ ) channels.SignificanceOur data indicate that the neuroprotective and antiseizure activity of CRS likely results in part from decreased [Ca2+ ]i accumulation through blockade of T-type Ca2+ channels.

CPVT (catecholaminergic polymorphic ventricular tachycardia) is an inherited life-threatening arrhythmogenic disorder. CPVT is caused by DADs (delayed after-depolarizations) that are induced by spontaneous Ca 2+ release during SR (sarcoplasmic reticulum) Ca 2+ overload, a process also known as SOICR (store-overload-induced Ca 2+ release). A number of mutations in the cardiac ryanodine receptor RyR2 are linked to CPVT. Many of these CPVT-associated RyR2 mutations enhance the propensity for SOICR and DADs by sensitizing RyR2 to luminal or luminal/cytosolic Ca 2+ activation. Recently, a novel CPVT RyR2 mutation, G230C, was found to increase the cytosolic, but not the luminal, Ca 2+ sensitivity of single RyR2 channels in lipid bilayers. This observation led to the suggestion of a SOICR-independent disease mechanism for the G230C mutation. However, the cellular impact of this mutation on SOICR is yet to be determined. To this end, we generated stable inducible HEK (human embryonic kidney)-293 cell lines expressing the RyR2 WT (wild-type) and the G230C mutant. Using single-cell Ca 2+ imaging, we found that the G230C mutation markedly enhanced the propensity for SOICR and reduced the SOICR threshold. Furthermore, the G230C mutation increased the sensitivity of single RyR2 channels to both luminal and cytosolic Ca 2+ activation and the Ca 2+ -dependent activation of [ 3 H]ryanodine binding. In addition, the G230C mutation decreased the thermal stability of the N-terminal region (amino acids 1–547) of RyR2. These data suggest that the G230C mutation enhances the propensity for SOICR by sensitizing the channel to luminal and cytosolic Ca 2+ activation, and that G230C has an intrinsic structural impact on the N-terminal domains of RyR2.

The cardiac ryanodine receptor (RyR2) is most abundantly expressed in the heart and brain. RyR2 is located in the sarco(endo)plasmic reticulum (ER/SR) membrane and functions as an intracellular calcium release channel, important for a number of fundamental processes, such as muscle contraction, secretion, neurotransmitter release, learning and memory. Proper function of RyR2 critically depends on its subcellular distribution. In ventricular myocyte of the heart, RyR2 is organized in highly-ordered arrays of clusters, which is thought to be important for synchronous, stable calcium release during excitation-contraction coupling. However, little is known about RyR2 distribution in other cardiac cells. In the brain, RyR2 is thought to play a central role in learning and memory, but the cellular/subcellular distribution of RyR2 in the brain remains largely undefined. Recently, we have generated a knock-in mouse model that expresses a green fluorescence protein (GFP)-tagged RyR2. This GFP-RyR2 mouse model allows us to directly and specifically monitor the cellular/subcellular distribution and expression of RyR2 in various cells and tissues. To improve the detection of GFP, we have also developed novel GFP-specific probes based on anti-GFP single domain antibodies (i.e. nanobodies). High-resolution confocal imaging of intact GFP-RyR2 heart sections and brain slices revealed highly ordered arrays of GFP-RyR2 in various regions of the heart, but disperse distribution of GFP-RyR2 in hippocampus and other regions of the brain. Using Alexa-Fluor 647 (AF647)-conjugated GFP-specific probes, we will perform super-resolution imaging to further define the subcellular distribution of GFP-RyR2 in the heart and brain (Supported by NSERC, CFI, CIHR, and LCIA).

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a life-threatening arrhythmia. Many congenital mutations in both the cardiac ryanodine receptor (RyR2) and calsequestrin (CASQ2) are known to be responsible for this disorder. It is now well established that CPVT is caused by delayed afterdepolarizations (DADs)-induced triggered activities, and that DADs are caused by spontaneous sarcoplasmic reticulum (SR) Ca release during Ca overload, a process also known as store overload induced Ca release (SOICR). A large body of evidence indicates that CPVT-causing RyR2 and CASQ2 mutations enhance the propensity for SOICR and DADs by increasing the response of RyR2 to SR luminal Ca. Recently, Marks and colleagues reported that a CPVT RyR2 mutation G230C increases the cytosolic Ca sensitivity (only after PKA phosphorylation) of single RyR2 channels in lipid bilayers, but has no effect on the luminal Ca sensitivity of the channel. These observations have led to the conclusion that SOICR is not involved in the disease mechanism of the RyR2-G230C mutation. However, the cellular impact of this mutation on SOICR has yet to be determined. To this end, we generated stable, inducible HEK293 cell lines expressing RyR2-WT and the RyR2-G230C mutant. We induced SOICR in these cells by elevating extracellular Ca, and found that the RyR2-G230C mutation markedly enhances the propensity for SOICR. Further, we employed single cell luminal Ca imaging to monitor the luminal Ca dynamics in RyR2-WT- and G230C-expressing cells during store Ca overload. We found that the G230C mutation significantly reduces the luminal Ca level at which spontaneous Ca release occurs (i.e. the SOICR threshold). Therefore, these results and those of previous studies demonstrate that reduced SOICR threshold is a common defect of CPVT-associated RyR2 mutations.

Mutations in ryanodine receptor 2 (RYR2) gene can cause catecholaminergic polymorphic ventricular tachycardia (CPVT). The novel RYR2-S4153R mutation has been implicated as a cause of CPVT and atrial fibrillation. The mutation has been functionally characterized via store-overload-induced Ca(2+) release (SOICR) and tritium-labelled ryanodine ([(3)H]ryanodine) binding assays. The S4153R mutation enhanced propensity for spontaneous Ca(2+) release and reduced SOICR threshold but did not alter Ca(2+) activation of [(3)H]ryanodine binding, a common feature of other CPVT gain-of-function RYR2 mutations. We conclude that the S4153R mutation is a gain-of-function RYR2 mutation associated with a clinical phenotype characterized by both CPVT and atrial fibrillation.

Background Nicotinic acid adenine dinucleotide phosphate (NAADP) has been identified as a calcium-mobilizing second messenger. NAADP is regularly enzymatically synthesized by ADP-ribosyl cyclases, in particular under acidic conditions. In nanomolar concentrations NAADP targets selectively the ryanodine receptor type 1 on the sarcoplasmic reticulum, and the two pore channels localized in dense-core secretory vesicles and lysosomes.

Highly ordered arrays of ryanodine receptor type 2 (RyR2) are believed to be critical for synchronous Ca release and effective, stable excitation-contraction coupling in adult cardiomyocytes. Altered RyR2 distribution and intracellular architectures have been implicated in the genesis of dyssynchronous Ca release often observed in disease hearts. To gain insights into the expression and distribution of RyR2 and their correlation with function in situ in adult cardiomyocytes, we generated a knock-in mouse model in which the green fluorescence protein (GFP) has been inserted into RyR2 after residue T1366. The GFP-tagged RyR2 mice show no gross structural and functional abnormalities. Confocal laser scanning microscopy of isolated cardiomyocytes from the GFP-tagged RyR2 mice revealed discrete clusters of GFP-RyR2 located along Z-lines. Confocal Ca imaging analysis of GFP-tagged RyR2 cardiomyocytes loaded with Rhod-2 AM showed that Ca sparks originate from GFP-RyR2 clusters and rarely occur in non-Z-line region. These observations suggest that the production of Ca sparks may require clustering of RyR2. To further define the distribution of GFP-RyR2 clusters, we employed a camelid single-domain antibody against GFP (GFP-nanobody) conjugated with the Alexa Fluor (AF) 647 fluorescent dye. The distribution of the GFP-nanobody staining in GFP-tagged RyR2 cardiomyocytes was found to be identical to that of GFP-RyR2 clusters. Further super-resolution imaging using the AF647-labelled GFP-nanobody should provide new and detailed insights into the nano-distribution of RyR2 in cardiomyocytes (Supported by CFI, CIHR, and LCIA).

Further understanding of calcium handling and excitation-contraction coupling in cardiac myocytes requires quantitative data analysis methods to characterize calcium release events in terms of structural properties of the cell. Such automatic methods provide a robust, consistent and reproducible characterization of physiological systems from observed experimental data. We present a multilevel analysis that simultaneously focuses on both the occurrence of spontaneous calcium sparks and the distribution of RyR clusters across a cardiac myocyte. The approach localizes the release events and determines the distance to the nearest ryanodine receptor cluster, therefore providing quantitative information on the spatio-temporal distribution of activation sites in the cell. The location of clusters takes into account motion artifacts produced by calcium waves or mini-waves. The method has been validated with line-scan confocal microscopy data from mouse ventricular myocytes and provides a full characterization of the spark morphology including its amplitude, baseline, decay time, upstroke time, Full-Width at Half-Maximum, Full-Duration at Half-Maximum and background noise.The processing is applied to linescan images of both RyR clusters and calcium fluorescence. It includes the following steps: i) Adaptive filtering of background fluorescence fluctuations using a robust estimation of the noise by means of the median absolute deviation of the basal fluorescence signal. ii) Individual sparks are detected by using a modified watershed segmentation method that includes stopping rules for both event size and shape. iii) Location of RyR clusters was implemented by thresholding a continuous wavelet transform of the cluster image (robust to motion and contraction). iV) Measurement of spark morphology properties, including the distance from each spark to the closest RyR cluster.