Methods to identify modulators of RyR calcium channels
10281476 ยท 2019-05-07
Assignee
Inventors
- Razvan L. Cornea (Minneapolis, MN, US)
- Donald M. Bers (Davis, CA, US)
- David D. Thomas (Minneapolis, MN, US)
- Gregory D. Gillispie (Minneapolis, MN, US)
Cpc classification
International classification
G01N31/00
PHYSICS
G01N33/53
PHYSICS
Abstract
Provided herein are methods for identifying a compound that modulates a Ryanodine receptor (Ryr). Fluorescence resonance energy transfer between an FKBP bound to an RyR and fluorescent derivatives of RyR binding partners (e.g., calmodulin) or domain-peptide biosensors is used to provide a readout dependent on the RyR functional state. The methods permit measurement of RyR present in a permeabilized cell or in a purified membrane.
Claims
1. A method for identifying a compound that modulates a ryanodine receptor (RyR) homotetrameric calcium channel comprising: providing a complex comprising an RyR homotetrameric calcium channel (RyR molecule), an FK-binding protein (FKBP), and a modulatory protein, wherein the FKBP comprises a covalently attached donor probe, and wherein the modulatory protein comprises a covalently attached acceptor probe, and wherein the donor probe and the acceptor probe can be used for energy transfer; contacting the complex with a test compound to form a mixture; measuring the fluorescence lifetime or intensity of the donor probe, wherein measuring fluorescence lifetime comprises direct waveform recording detection.
2. The method of claim 1 wherein the molar concentration of the FKBP is at, or within one order of magnitude, of the dissociation constant (K.sub.d) of the FKBP for the RyR molecule, and the molar concentration of the modulatory protein is at, or within one order of magnitude, of the K.sub.d of the modulatory protein for the RyR molecule.
3. The method of claim 1 wherein the modulatory protein is calmodulin, S100A1, or sorcin.
4. The method of claim 1 wherein the fluorescence lifetime or intensity of the donor probe is changed in the presence of the test compound.
5. The method of claim 4 wherein the change between the fluorescence lifetime in the presence of the test compound and the fluorescence lifetime in the absence of the test compound is a E of greater than 3 times the E standard deviation of a control.
6. The method of claim 1 wherein the measuring comprises high throughput screening.
7. The method of claim 1 wherein the RyR molecule is an RyR1molecule or an RyR2 molecule.
8. The method of claim 1 wherein the complex is present in a permeabilized cell.
9. The method of claim 1 wherein the complex is present in a purified membrane.
10. The method of claim 1 wherein the mixture comprises a compound selected from H.sub.2O.sub.2 and oxidized glutathione.
11. The method of claim 1 wherein the complex further comprises a domain peptide.
12. The method of claim 11 wherein the domain peptide is DPc-10.
13. The method of claim 11 wherein the concentration of the domain peptide is at, or within one order of magnitude, of the K.sub.d of the domain peptide for the RyR molecule.
14. The method of claim 11 wherein the concentration of the FKBP is a saturating concentration, the concentration of the domain peptide is a saturating concentration, or the combination thereof.
15. The method of claim 1 wherein the FKBP is FKBP12 or FKBP12.6.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
(23)
(24)
(25)
(26)
(27)
(28)
(29)
(30)
(31)
(32)
(33)
(34)
(35)
(36)
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
(37) Provided herein are methods for identifying compounds that modulate a ryanodine receptor (RyR) intracellular Ca.sup.2+ release channel. Calcium plays a central role in muscle contraction. In muscle contraction, Ca.sup.2+ is released through the RyR from the sarcoplasmic reticulum (SR) intracellular reservoir, then pumped back into the SR by sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. Therefore, RyR gating is critical to normal muscle function. In a resting cell (e.g., myocyte in diastole, relaxed muscle) elevated Ca.sup.2+ in the cytoplasm due to leak through the RyR (and also insufficient Ca.sup.2+ transport by SERCA pumps) is a hallmark dysfunction directly underlying severe diseases of the striated muscle, including skeletal (e.g., Duchenne muscular dystrophy (MD), limb girdle MD, geriatric muscle dysfunctions) and cardiac (e.g., ischemic heart failure (HF), catecholaminergic ventricular tachycardia (CPVT)). Moreover, evidence increasingly points to RyR dysregulation as a therapeutic target for widely spread non-muscle diseases e.g., type 2 diabetes and Alzheimer's disease (AD). Elevated intracellular Ca.sup.2+ is part of a vicious cycle generating these severe and widespread pathologies in muscle, as well as other types of tissue. This vicious cycle may be interrupted by actions on the main Ca.sup.2+ cycling players, the RyR channel and the SERCA pumps, to restore normal intracellular calcium concentration.
(38) RyR Protein
(39) The RyR protein is a homotetrameric complex (565 kDa/protomer) with most of its mass in a cytosolic headpiece that serves as scaffold for a multitude of smaller soluble partners that regulate the RyR Ca.sup.2+ channel function (Bers D M, J Mol Cell Cardiol. 2004; 37:417-429). Three RyR isoforms have been identified in mammals. RyR1 is expressed in skeletal muscle, which provides an abundant source for in vitro studies. RyR2 is expressed mainly in the heart, while RyR3 is more ubiquitous. The three dimensional structures of the three RyR isoforms are highly similar, and structural studies of RyR1 and RyR2 show highly similar accessory protein topologies within RyR complexes (Cornea et al., Proc Natl Acad Sci USA. 2009; 106:6128-6133; Cornea et al., J Biol Chem. 2010; 285:19219-19226; and Guo et al., Biophys J. 2011; 101:2170-2177).
(40) The smaller soluble partners that regulate the RyR Ca.sup.2+ channel function include, but are not limited to, FK506 binding protein (FKBP12 and 12.6, 12 kDa), which promotes the closed RyR state, and calmodulin (CaM, 16 kDa), which integrates intracellular [Ca.sup.2+] into target-specific functional signals.
(41) There is general agreement that sarcoplasmic reticulum calcium leak is increased in numerous pathological conditions, and several mechanisms are under consideration. Relevant for the methods described herein, for pathologically active (leaky) RyR channels (1) calmodulin binding is decreased (Ai et al., Circ Res. 2005; 97:1314-1322; Guo et al., Circ Res. 2006; 99:398-406; and Aracena et al., Antioxid Redox Signal. 2005; 7:870-881) and (2) structural interactions between N-terminal and central RyR domains are disrupted (a phenomenon often referred to as unzipping) (Kobayashi et al., J Biol Chem. 2005; 280:6580-6587; Kimura et al., Cell Calcium. 2009; 45:264-274; Circ J. 2010; 74:2579-2584; Oda et al., Circulation. 2005; 111:3400-3410; and Uchinoumi et al., Circ Res. 2010; 106:1413-1424). As described in Example 1, using FRET the inventors have found that access of a fluorescently-labeled unzipping domain peptide (F-DPc10, where F refers to the fluorescent label) to its RyR2 binding site is sterically hindered in the resting normal RyR2 (zipped state, (
(42) Without being bound by theory, in one embodiment, modulation of RyR by a test compound may have an effect on the conformation of an RyR molecule. The altered conformation may be a change in the secondary structure of the RyR, the tertiary structure of the RyR, or a combination thereof. In one embodiment, modulation of RyR by a test compound may alter the movement of calcium through the RyR channel. In one embodiment, modulation of RyR by a test compound may alter the binding of a domain peptide, a modulatory protein, a ryanodine variant, or a combination thereof, to an RyR molecule.
(43) In one embodiment, the RyR protein used in the methods described herein is wild-type, and in some embodiments the RyR molecule includes one or more mutations that are present in RyR molecules associated with RyR dysfunction. In certain embodiments, an RyR protein is RyR1, RyR2, RyR3, or a combination thereof. The amino acid sequences of examples of each of these isoforms in various animals (including human, porcine, rat, and mouse), and the amino acid mutations associated with RyR dysfunction, are known to the person skilled in the art and are readily available.
(44) In one embodiment, the RyR molecule used in the methods is a fusion protein, where the fusion is with a chromophore, such as a fluorescent protein. The fusion can be in any location within the amino acid sequence of an RyR monomer, provided it does not abolish the activity of an RyR channel, and provided the location of the fusion permits FRET between the chromophore and a second chromophore. Most known RyR constructs with fluorescent protein tags have altered function in some way. RyR fusion proteins useful herein may have altered function, but preserve essential channel characteristics, such as maintaining the RyR response to caffeine. The first chromophore may be a donor or an acceptor, and in one embodiment is a donor. In one embodiment, the second chromophore is also present in an RyR monomer as a fusion.
(45) In one embodiment, an RyR protein includes a chromophore, and the chromophore (donor, acceptor, or both) is inserted into the RyR amino acid sequence to result in a fusion protein. Various RyR2 constructs with green fluorescent protein (GFP)-like inserts have been described (e.g., Liu et al., J Cell Sci. 2010; 123:1775-1784; Wang et al., J Biol Chem. 2011; 286:12202-12212; Huang et al., J Cell Sci. 2013; 126:4527-4535). Similar constructs that are near the 3 dimensional position of the FKBP binding site, or CaM binding site, or DPc10 binding site may be used herein. The optical properties of the GFP-like inserts can be optimized for brightness and separation from cell autofluorescence. In one embodiment, a GFP-like tag at position 1 (as described for RyR1 by Raina et al., PLoS One. 2012; 7:e38594), is used as FRET donor for acceptor-labeled fluorescent ryanodine variants. In one embodiment, a non-protein chromophore can be attached to the RyR.
(46) An RyR protein used in the methods described herein can be produced using recombinant techniques, chemical or enzymatic synthesis, or be naturally occurring. An RyR protein may be isolated, or may be present in a cell.
(47) FKBP
(48) In one embodiment, the methods include the use of FKBP. In some embodiments, FKBP is FKBP12 and/or FKBP12.6. The FKBP12 and FKBP12.6 proteins are normally expressed in cardiac myocytes and can form tight complexes with RyR at a stoichiometry of 4 FKBPs per tetrameric RyR channel (Bers, J Mol Cell Cardiol. 2004; 37:417-429.). These FKBP isoforms are considered important RyR2 subunits and have been reported to promote the closed channel state. In some embodiments, the use of an FKBP variant with lower RyR-binding affinity is desirable. Examples of such FKBP variant proteins are known and include, but are not limited to, D41C (Cornea et al., J Biol Chem. 2010; 285:19219-19226), and the isoform FKBP12, which binds to RyR2 with 100-fold lower affinity than FKBP12.6 (and only 4-fold lower binding affinity for RyR1). In other cases, single point mutations, within regions of FKBPs that interface with RyRs, can significantly reduce FKBP-RyR binding affinity. In one embodiment, an FKBP variant has a 5 to 100-fold decrease in affinity, which would provide appropriate sensitivity. The amino acid sequences of examples of each of these isoforms, and the amino acid sequences of FKBP variants with decreased RyR-affinity, are known to the person skilled in the art and are readily available.
(49) In one embodiment, an FKBP or a variant thereof includes a chromophore. The chromophore may be a donor or an acceptor. In one embodiment, the chromophore is a donor. When an FKBP includes a chromophore, it provides an RyR-specific fluorescent signal that maintains association with the RyR over the time-course of a typical FRET experiment. A molecule that includes a chromophore is referred to herein as being labeled with that chromophore. The chromophore and an FKBP protein or variant thereof may be attached by, for instance, an ionic bond, a hydrogen bond, a Van der Waals force, a hydrophobic interaction, a covalent bond, or a combination thereof. In one embodiment, the attachment includes a covalent bond. The chromophore may be attached to any location of an FKBP or variant thereof, provided the attached chromophore does not prevent the FKBP protein or variant thereof from binding to an RyR. In one embodiment an F-FKBP variant having unaltered RyR binding affinity and having reduced potency to inhibit RyR, namely ALEXA FLUOR488-labeled N32C/C22A/C76I-hFKBP12.6, (Cornea et al., J Biol Chem. 2010;285:19219-19226) may be used. Suitable locations for attachment of a chromophore to an FKBP protein or variant thereof are known.
(50) An FKBP protein used in the methods described herein can be produced using recombinant techniques, chemical or enzymatic synthesis, or be naturally occurring. An FKBP protein may be isolated, or may be present in a cell.
(51) Modulatory Proteins
(52) In one embodiment, the methods include the use of a protein that modulates the activity of an RyR protein, but is not an FKBP. In one embodiment, the modulatory protein is a calmodulin protein. Calmodulin (CaM) is an RyR modulator which integrates intracellular calcium concentration into target-specific functional signals. Other examples of modulatory proteins include, but are not limited to, soluble globular proteins like S100A1 and sorcin, membrane proteins like triadin and junctin, or venom peptides like imperatoxins A. The amino acid sequences of examples of calmodulin, S100A1, and sorcin are known to the person skilled in the art and are readily available. Mutants of these proteins that induce the pathological leaky state of RyR might be particularly useful, for example, Ca-insensitive CaMs (Cornea et al., Proc Natl Acad Sci USA. 2009; 106:6128-6133), or a CaM carrying the N54I or N98S mutations (Hwang et al., Circ Res. 2014; 114:1114-1124) linked to cathecolaminergic polymorphic ventricular tachycardia.
(53) In one embodiment, a modulatory protein includes a chromophore. The chromophore may be a donor or an acceptor. In one embodiment, the chromophore is an acceptor. The interaction between a modulatory protein and a chromophore may be, for instance, an ionic bond, a hydrogen bond, a Van der Waals force, a hydrophobic interaction, a covalent bond, or a combination thereof. In one embodiment, the interaction includes a covalent bond. The chromophore may be attached to any location of a modulatory protein, provided the attached chromophore does not prevent the modulatory protein from binding to an RyR protein.
(54) A modulatory protein used in the methods described herein can be produced using recombinant techniques, chemical or enzymatic synthesis, or be naturally occurring. A modulatory protein may be isolated, or may be present in a cell.
(55) Domain Peptides
(56) In one embodiment, the methods include the use of a domain peptide. An RyR molecule includes an N-terminal region and a central region that interact to stabilize and close the channel and prevent the movement of calcium through the channel. A domain peptide, also referred to as DP, specifically associates with the N-terminal region or the central region of an RyR molecule to destabilize the interaction between the two regions (a process referred to as unzipping) and increase movement of calcium through the channel.
(57) In one embodiment, a domain peptide is a protein that has a sequence identical to a series of 8 to 50 consecutive amino acids in an RyR polypeptide, such as residues 2460-2495 of RyR2 (Tateishi et al., Cardiovasc Res. 2009; 81:536-545) and the equivalent region of RyR1 (e.g., residues 2442-2477, Yamamoto & Ikemoto, Biochemistry 2002; 41:1492-1501). In one embodiment, a domain peptide has a sequence that is identical to a wild-type RyR. The amino acid sequences of examples of domain peptides are known to the person skilled in the art and are readily available. The skilled person can easily design other domain peptides that are a subset of known domain peptides and determine if they specifically associate with the N-terminal region or the central region of an RyR molecule to destabilize the interaction between the two regions. An example of a domain peptide is DcP10, which corresponds to RyR2 residues 2460-2495 (Yamamoto and Ikemoto, Biochem Biophys Res Commun. 2002; 291:1102-1108). Another example of a domain peptide is DP163-195, which corresponds to RyR2 residues 163-195 (Tateishi et al., Cardiovasc Res. 2009; 81:536-545). In one embodiment, a domain peptide has a sequence that includes a mutation that is present in RyR molecules associated with RyR dysfunction, such as heart failure and lethal arrhythmias (e.g., catecholaminergic polymorphic ventricular tachycardia [CPVT]) (Tateishi et al., Cardiovasc Res. 2009; 81:536-545).
(58) In one embodiment, a domain peptide includes a chromophore. The chromophore may be a donor or an acceptor. In one embodiment, the chromophore is an acceptor. The interaction between a domain peptide and a chromophore may be an ionic bond, a hydrogen bond, a Van der Waals force, a covalent bond, or a combination thereof. In one embodiment, the interaction includes a covalent bond. In one embodiment, the chromophore is present at the N-terminus. The chromophore may be attached to any location of a domain peptide, provided the attached chromophore does not prevent the domain peptide from binding to an RyR protein and inhibiting zipping.
(59) A domain peptide used in the methods described herein can be produced using recombinant techniques, chemical or enzymatic synthesis, or be naturally occurring. A domain peptide may be isolated, or may be present in a cell.
(60) Ryanodine
(61) In one embodiment, the methods include the use of a ryanodine molecule. In one embodiment, ryanodine is labeled with a chromophore. The chromophore may be a donor or an acceptor. In one embodiment, the chromophore is an acceptor. Methods for attaching a chromophore to a ryanodine molecule are known to the skilled person and are routine.
(62) Chromophores
(63) Any appropriately selected two chromophores can be used as a donor-acceptor pairs in the methods described herein, provided that the energy emitted by a donor (the emission spectrum) overlaps with the energy absorbed by an acceptor (the excitation spectrum), e.g., an energy transfer process (FRET) occurs between two chromophores. A donor and an acceptor that meet this overlap are referred to as a donor-acceptor pair. In one embodiment, donor-acceptor pairs are chosen such that interference from cell autofluorescence or test-compound fluorescence is minimized. Accordingly, in some embodiments donors that can be excited at longer wavelengths are superior to those excitable at shorter wavelengths (e.g., ALEXA FLUOR 488 is superior to IAEDANS). Also, in some embodiments probes with longer fluorescence lifetime (FLT) (more than 3 nanoseconds (ns)) will be superior to probes with shorter FLT.
(64) Chromophores suitable for the methods described herein are known to the skilled person and are routinely used. Methods for attaching a chromophore to a selected protein are known and routine. In one embodiment, examples include those suitable for analysis by conventional flow cytometry. Donor-acceptor pairs which can be used for detection by most conventional flow cytometers are discussed in, for example, Szollosi et al., Communications in Clinical Cytometry, 1998; 34:159-179). Chromophores as used herein may be fluorescent or non-fluorescent (e.g., luminescent components, or 4-((4-(dimethylamino)phenyl)azo)benzoic acid (DABCYL)). In certain embodiments, combinations of chromophores as used herein include those used in the classical tandem conjugates (see van Dongen et al., U.S. Pat. No. 7,413,862).
(65) Examples of fluorescent components that are suitable for FRET assays disclosed herein include, but are not limited to, fluorescein, rhodamine, Green Fluorescent Protein (and variants thereof), 4-nitrobenzo-2-oxa-1 ,3-diazole (NBD); cascade blue, 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3- propionic acid; 4,4-difluoro-5,p-methoxyphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-propionic acid; 6-carboxy-Xrhodamine, N,N,N,N-tetramcthyl-6-carboxyrhodamine; iodoacetyl-directed probes such as 5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid (1AEDANS, used interchangeably with AEDANS); 5-carboxyfluorescein; 6-carboxyfluorescein; 6-(fluorescein-5-carboxamide)hexanoic acid; fluorescein isothiocyanate (FITC); tetramethylrhodamine isothiocyanate (TRJTC); TEXAS RED (TR); eosin; a phycobiliprotein; cyanine dye; coumarin; R-phycoerythrin; allophycoerythrin (APC); a R-phycoerythrin (R-PE) conjugate; an ALEXA FLUOR dye; a HILYTE FLUOR dye; a quantum dot dye; maleimide-directed probes such as 4-dimethylaminoazobenzne-4-maleimide (DABmal) and fluorescein-5-maleimide (Fmal); or a combination thereof (e.g., tandem conjugates) (see Thomas et al., WO 2010/085514).
(66) In one embodiment, a chromophore used herein is a nucleotide analog such as an ATP-, ADP- or AMP-analog (see, e.g. Bagshaw, J of Cell Science 2001; 114:459-460). In certain embodiments, a nucleotide analog is fluorescent. Examples of fluorescent nucleotide analogs include, but are not limited to, 2-(or -3)-0-(trinitrophenyl)adenosine 5-triphosphate (TNP-A TP), 2-(or -3)-0-(trinitrophenyl)adenosine 5-diphosphate (TNP-ADP), e-A TP, e-aza-A TP, FTP, 2AP-TP, ant-ATP. Mant-ATP, DEDAA TP, FEDA-ATP, RED A-A TP and Cys3-EDA-ATP.
(67) In one embodiment, examples of luminescent chromophores that are suitable for the methods disclosed herein include, but are not limited to, the luminescent ions of europium and terbium introduced as lanthanide chelates (Thomas et al.,Proc Nat/ Acad Sci USA, 1978;75:5746-5750). In one embodiment, examples of donor-acceptor pairs include fluorescein and rhodamine, NBD and rhodamine, NBD and eosin, NBD and erythrosine, fluorescein and eosin, fluorescein and erythrosin, dansyl and rhodamine, acridine orange and rhodamine, lanthanide ion and fluorescein, IAEDANS and TNP-ADP, IAEDANS and FITC, IAEDANS and DABmal, IAEDANS and Fmal, IAEDANS and DABCYL, ALEXA FLUOR 488 and either ALEXA FLUOR 568 or a HILYTE FLUOR 647. In one embodiment, the donor and acceptor pair as used herein is ALEXA FLUOR 488 and either ALEXA FLUOR 568 or a HILYTE FLUOR 647.
(68) Methods
(69) In the methods described herein, the experimental observations indicate that FRET detection of RyR binding of modulatory proteins like calmodulin and domain peptides like DPc10 provide measurable indications of the functional state of an RyR molecule. In some embodiments, the methods described herein will detect the effects of compounds on binding of FKBP to RyR, binding of calmodulin to RyR, access of domain peptides to their RyR binding sites, and/or binding of ryanodine-like molecules to RyR.
(70) In preferred embodiments, the methods use the lifetime of a chromophore instead of its intensity. Methods for measuring fluorescence lifetime (FLT) are known to the skilled person and are routine. In one embodiment, FLT is measured using a plate reader (FLT-PR). FLT-PRs useful in the methods described herein are readily available (e.g., the FLT-PR available at the Biophysical Spectroscopy Center, University of Minnesota, Minneapolis, Minn.). The measurement of FLT by using direct wave recording detection technology in a plate reader provides the precision to resolve small changes in FRET, and can scan the plate rapidly (around 2 minutes/384-well plate). Advantages of the FLT readout include its insensitivity to variability in the donor concentration and to sample inhomogeneity (e.g., whether cells or membranes settle at the bottom of the well or are uniformly dispersed throughout the volume of the sample). Further, in some embodiments noise is decreased 30-fold, which greatly decreases both false positives and negatives. The conventional wisdom has been that FLT detection at high precision was just too slow for high throughput screening. Typical lifetime plate-readers require greater than an hour to scan a 384-well plate at a signal/noise (S/N) of 100, while a lifetime plate-reader such as those described herein can scan a 384-well plate in approximately 2 minutes at S/N of 100. The result is a surprising and unexpected improvement of a factor of 100,000. In contrast, steady-state fluorescence readers measure intensity and provide much less information and less precision (
(71) A measuring instrument useful in the methods disclosed herein is a spectrometer that is compatible with FRET assays and can perform direct waveform recording to detect the entire time course of a time-resolved fluorescence decay with high quality (signal/noise>100) within 1 ms or less, in a microplate format that allows for the analysis of several hundred samples per minute. An example of such an instrument is described by Cornea et al.(J Biomol Screen. 2013;18:97-107). In certain embodiments, the FRET assays disclosed herein are measured at a single emission wavelength. In certain embodiments, the entire emission spectrum of the FRET is measured. For example, when ALEXA FLUOR 488 is the fluorescent donor, the emission wavelength is scanned from 500 nm to 650 nm.
(72) RyR molecules are normally present in the membrane of the sarcoplasmic reticulum. RyR molecules useful in the methods described herein may be present in membranes purified from cells, or may be present in permeabilized cells. The cells that are used as a source of purified membranes and the cells that are permeabilized may be cells that normally have a large amount of sarcoplasmic reticulum. Such cells include myocytes, for example cardiomyocytes, such as ventricular myocytes. In some embodiments, myocytes from muscle that is dysfunctional, e.g., failing heart, pathologically stressed, or dystrophic, may be used as a source of RyR molecules. Cells may be vertebrate cells, such as human, murine (including mouse and rat), canine, or porcine, or from invertebrates, such as Army worm (e.g., Sf21 cells). Other cells include those engineered to express RyR molecules. The RyR used may be the isoform RyR1, RyR2, or RyR3
(73) In some embodiments, the conditions used to assay RyR functional states may be modified to mimic the environment present in dystrophic or heart failure myocytes. For instance, the conditions may include supplementation with H.sub.2O.sub.2 or oxidized glutathione, an FKBP mutant with attenuated potency to inhibit RyR, a CaM mutant that activates RyR2 in nanomolar calcium or with attenuated potency to inhibit RyR2 in nanomolar calcium, e.g., the CaM with N54I or N98S mutations (Hwang et al., Circ Res. 2014; 114:1114-1124).
(74) In one embodiment, the methods use permeabilized myocytes, such as ventricular myocytes. The myocytes may be adult myocytes. Prior to running the experiment, it was uncertain and unexpected that changes in FRET could be detected in cells when the subject protein was RyR, as it may be present in certain cells at higher levels, but it was unclear whether such levels were high enough to permit detection of FRET and FRET changes.
(75) To achieve 3-10 nM [D-FKBP] bound, it was estimated that 3,000-30,000 cells/well were needed. To test feasibility, permeabilized adult rat myocytes were used, treated with D-FKBP12.6 (unbound removed), with subsaturating and saturating acceptor-labeled CaM (
(76) In
(77) The sources for compounds that may modulate an RyR molecule include, but are not limited to, for instance, chemical compound libraries, fermentation media of Streptomycetes, other bacteria and fungi, and cell extracts of plants and other vegetations. Small molecule libraries are available, and include AMRI library, AnalytiCon, BioFocus DPI Library, Chem-X-Infinity, ChemBridge Library, ChemDiv Library, Enamine Library, The Greenpharma Natural Compound Library, Life Chemicals Library, LOPAC1280, MicroSource Spectrum Collection, Pharmakon, The Prestwick Chemical Library, SPECS, NIH Clinical Collection, Chiral Centers Diversity Library. In some embodiments, the number of compounds evaluated in an assay includes between 1 and 200,000 compounds, between 1 and 100,000 compounds, between 1 and 1,000 compounds, or between 1 and 100 test compounds. In one embodiment, during testing a compound may be present a micromolar concentrations, such as 10 micromolar.
(78) Provided herein are methods for identifying a compound that modulates RyR. In one embodiment, the method includes providing a complex that includes an RyR molecule, an FKBP, and a domain peptide. The FKBP includes an attached chromophore that is a donor or an acceptor. The domain peptide also includes an attached chromophore that can be used with the chromophore attached to the FKBP for energy transfer. In one embodiment, the FKBP chromophore is a donor and the domain peptide chromophore is an acceptor. The method includes contacting the complex with a test compound to form a mixture, and measuring the fluorescence lifetime or intensity of the donor chromophore and/or the acceptor chromophore. A difference between the fluorescence lifetime or intensity in the presence of the test compound and the fluorescence lifetime or intensity in the absence of the test compound indicates that the test compound modulates RyR.
(79) In one embodiment, the method includes providing a complex that includes an RyR molecule, an FKBP, and a modulator. The FKBP includes an attached chromophore that is a donor or an acceptor. The modulator also includes an attached chromophore that can be used with the chromophore attached to the FKBP for energy transfer. In one embodiment, the FKBP chromophore is a donor and the modulator chromophore is an acceptor. The method includes contacting the complex with a test compound to form a mixture, and measuring the fluorescence lifetime or intensity of the donor chromophore and/or the acceptor chromophore. A difference between the fluorescence lifetime or intensity in the presence of the test compound and the fluorescence lifetime or intensity in the absence of the test compound indicates that the test compound modulates RyR.
(80) In one embodiment, the method includes providing a complex that includes an RyR molecule, an FKBP, a domain peptide, and a modulator. The domain peptide includes an attached chromophore that is a donor or an acceptor. The modulator also includes an attached chromophore that can be used with the chromophore attached to the domain peptide for energy transfer. In one embodiment, the domain chromophore is an acceptor and the modulator chromophore is a donor. The method includes contacting the complex with a test compound to form a mixture, and measuring the fluorescence lifetime or intensity of the donor chromophore and/or the acceptor chromophore. A difference between the fluorescence lifetime or intensity in the presence of the test compound and the fluorescence lifetime or intensity in the absence of the test compound indicates that the test compound modulates RyR.
(81) In the methods described herein, the fluorescence lifetime or intensity of a chromophore may increase or decrease. In general, a test compound that decreases FRET between an FKBP and a domain peptide, such as DPc10, is considered an inhibitor of RyR1, RyR2, and RyR3. A test compound that increases FRET between an FKBP and a modulator such as calmodulin is considered an RyR2 inhibitor. For RyR1, the correlation between increased FRET from FKBP to calmodulin and RyR1 channel function may be different, because calmodulin has an activating effect on RyR1 at nanomolar Ca.sup.2+, and an inhibitory effect at micromolar Ca.sup.2+. Nevertheless, it is expected that a predictable correlation exists between test compound effects on FRET and RyR1 function. Such test compounds may be useful as therapeutic agents and/or as lead compounds in the development of therapeutic agents.
(82) In some embodiments, the order of addition of the components may vary. For instance, in one embodiment, the complex includes an RyR molecule and the FKBP, and the test compound is added to the mixture before the domain peptide is added. In another embodiment, the complex includes an RyR molecule and the FKBP, and the test compound is added to the mixture before the modulator is added.
(83) In certain embodiments, methods provided herein use FRET based methods to specifically detect RyR binding of modulatory proteins (e.g., CaM) and domain peptides which act as biosensors of channel unzipping (e.g., DPc10). FRET donor-probes are targeted to the cytoplasmic domain of RyR2 by using a FKBP or a variant thereof that is labeled with an appropriate chromophore, such as ALEXA FLUOR 488 or 568. As an RyR protein is typically the most prevalent FKBP target in the sarcoplasmic reticulum membranes and cells used in the methods, labeled FKBP provides an RyR specific fluorescent signal that is associated with its target over the time-course of a typical FRET experiment (
(84) In certain embodiments, the experimental design includes assays for RyR-specific FRET from D-FKBP (where D refers to donor probe) to both A-Donor Peptide (A-DP) and/or A-Calmodulin (A-CaM) (where A refers to acceptor probe) at subsaturating concentrations of the acceptor. In this embodiment, a hit-compound is a potential RyR inhibitor, and will both reduce DP binding and enhance CaM binding. Assays may use, for instance, isolated sarcoplasmic reticulum or permeabilized cells expressing RyR, such as the isoform RyR1, RyR2, or RyR3. In one embodiment, the amount of protein per well, the concentration of A-CaM, and the concentration of A-DP is used to give 50% of maximal FRET. Precision (CV) may be assessed in full plates, with DMSO (the typical solvent in chemical libraries), with a test library and appropriate positive controls. In some embodiments, controls compared to conditions that mimic the environment in dystrophic or heart failure myocytes. Dystrophic or heart failure myocytes can be produced in a laboratory setting by exposure of cardiac myocytes to low concentrations of H.sub.2O.sub.2 or oxidized glutathione (GSSG)). As shown in Example 2, oxidative modification by H.sub.2O.sub.2 promotes RyR leakiness, reduces the affinity of calmodulin for RyR, has no effect on the affinity of FKBP for RyR, and causes domain unzipping. These lead to untimely and potentially arrhythmogenic RyR channel opening, however, domain peptides and modulators such as calmodulin are useful biosensors of this pathophysiological state of RyR. Further, H.sub.2O.sub.2 treatment to decrease CaM affinity and increase DP access can be used to widen the dynamic range (hence sensitivity) of the assays described herein.
(85) In one embodiment, the method includes providing a complex that includes an RyR molecule, an FKBP protein, and a domain peptide. In one embodiment, the FKBP protein includes a donor probe, and the domain peptide includes an acceptor probe. The donor probe and the acceptor probe are matched in that they can be used for energy transfer from the donor to the acceptor that can be detected as a decrease in fluorescence lifetime or intensity. The complex is contacted with a test compound to form a mixture, and the fluorescence lifetime or intensity of the donor probe is measured. A difference between the fluorescence lifetime or intensity in the presence of the test compound and the fluorescence lifetime or intensity in the absence of the test compound indicates that the test compound modulates RyR. In one embodiment, the fluorescence lifetime or intensity of the donor probe is increased in the presence of the test compound, indicating that the test compound inhibits binding of the domain peptide to RyR.
(86) Changes in FRET between D-FKBP and A-DPc10 were detectable after 2 hours incubation with 10-20 M A-DPc10. In preliminary studies, the inventors have found that DPc10 binding to RyR2 is decreased by CaM (via allosteric interaction). Indeed, CaM inhibits A-DPc10 binding in the presence of nanoMolar (nM) Ca.sup.2+ (relaxation), and in microMolar (M) Ca.sup.2+ (contraction) (
(87) In one embodiment, the method includes providing a complex that includes an RyR molecule and an FKBP protein, where the FKBP protein includes a donor probe. The complex is contacted with a test compound to form a mixture. A domain peptide is added to the mixture, where the domain peptide includes an acceptor probe. The donor probe and the acceptor probe are matched in that they can be used for energy transfer from the donor to the acceptor that can be detected as a decrease in fluorescence lifetime or intensity. The fluorescence lifetime or intensity of the donor probe is measured. A difference between the fluorescence lifetime or intensity in the presence of the test compound and the fluorescence lifetime or intensity in the absence of the test compound indicates that the test compound modulates RyR. In one embodiment, the modulation is alteration of access of the domain peptide for the RyR molecule. In one embodiment, the fluorescence lifetime or intensity of the donor probe is increased in the presence of the test compound, indicating that the test compound decreases access of the domain peptide for the RyR molecule.
(88) Instead of equilibrium D-FKBP/A-DPc10 FRET (as described herein) the kinetics of A-DPc10 binding to RyR1 can be assessed. For instance, a an assay can be read at various times after A-DPc10 addition, such as 0 and 30 min after A-DPc10 addition). Data in myocytes shows slow F-DPc10 wash-in (80 min) in controls, and 50% faster wash-in after H.sub.2O.sub.2 treatment, and this is restored by the positive-control compound (RyR inhibitor) dantrolene (
(89) In one embodiment, the method includes providing a complex that includes an RyR molecule, an FKBP protein, and a modulatory protein. The FKBP protein includes a donor probe and the modulatory protein includes an acceptor probe. The donor probe and the acceptor probe are matched in that they can be used for energy transfer from the donor to the acceptor that can be detected as a decrease in fluorescence lifetime or intensity. The complex is contacted with a test compound to form a mixture, and the fluorescence lifetime or intensity of the donor probe is measured. A difference between the fluorescence lifetime or intensity in the presence of the test compound and the fluorescence lifetime or intensity in the absence of the test compound indicates that the test compound modulates RyR.
(90) Preliminary data show a 50% reduction of RyR2 A-CaM binding (at subsaturating concentrations of A-CaM) in myocyte from failing hearts (and similarly by 50 M H.sub.2O.sub.2). However, CaM binding is unaltered at saturating A-CaM concentrations (
(91) In some embodiments, a FKBP variant is used to detect changes in FKBP binding by using FRET between the FKBP variant and CaM. Wild-type FKBP12.6 binds too tightly, associates too fast, dissociates too slow to make it a practical probe in measuring changes in FKBP binding in some embodiments; however, FKBP variants having lowered RyR-binding affinity can be used. Such FKBP variants, e.g., D41C (Cornea et al., J Biol Chem. 2010; 285:19219-19226), have been reported. Screens in the presence of subsaturating D-FKBP concentration (i.e., near the K.sub.d) and saturating A-CaM concentration can be conducted. In one embodiment, an FKBP has a K.sub.d of 5 to 1000 times higher than wild type FKBP. In one embodiment, a FKBP is at or within 1, 2, or 3 orders of magnitude of the K.sub.d of the FKBP for an RyR molecule. Compounds that significantly change FRET at equilibrium (i.e., alter the D-FKBP affinity) or in kinetic mode (i.e., alter association or dissociation constants) are expected to affect RyR channel function. Compounds that enhance D-FKBP/RyR affinity or association rate, or slow the dissociation rate do so presumably by promoting the closed RyR.
(92) In one embodiment, the RyR molecule used in the methods is a fusion protein, where the fusion is with a fluorescent protein. Instead of the donor assays or the acceptor assays described above, an RyR-fluorescent fusion protein of optical properties suitable for a FRET partner is inserted at appropriate discrete locations within the RyR structure. The acceptor probe is attached to a modulatory protein or a domain peptide. Preliminary data using a Yellow Fluorescent Protein-RyR1 construct indicate that this is a perfectly feasible approach, as enabled by the FLT readout.
(93) In yet another embodiment, the donor is an RyR-fluorescent fusion protein, where the fusion protein is inserted at the N-terminus of RyR. The acceptor is attached to a ryanodine molecule. Alterations in FRET in the presence and absence of a test compound indicate that the test compound modulates RyR. For example, a fusion with Yellow Fluorescent Protein at position 1 of an RyR would function as donor for an ALEXA FLUOR 568-ryanodine acceptor.
(94) Kits
(95) Also provided herein are kits for identifying a compound that modulates RyR. A kit may include, in any combination, a FKBP protein (optionally labeled with a donor or an acceptor probe), a modulatory protein such as calmodulin (optionally labeled with an acceptor probe), and/or a domain peptide (optionally labeled with an acceptor probe). A kit may optionally include a RyR molecule fusion protein, where the fusion is with a fluorescent protein. A kit may optionally include a ryanodine molecule that is attached to an acceptor probe.
(96) In certain embodiments, a kit may further include buffers and reagents useful for the procedure, and instructions for carrying out the assay. In certain embodiments, a kit may further include other useful agents, such as positive and negative control reagents, and the like.
(97) Methods and kits disclosed herein may be carried out in numerous formats known in the art. In certain embodiments, the methods provided herein are carried out using solid-phase assay formats. In certain embodiments, the methods provided herein are carried out in a well of a plate with a plurality of wells, such as a multi-well plate or a multi-domain multi-well plate. The use of multi-well assay plates allows for the parallel processing and analysis of multiple samples distributed in multiple wells of a plate. Multi-well assay plates (also known as microplates or microtiter plates) can take a variety of forms, sizes and shapes (for instance, round- or flat-bottom multi-well plates). Examples of multi-well plate formats that can be used in the methods provided herein include those found on 96-well plates (128 array of wells), 384-well plates (2416 array of wells), 1536-well plate (483 2 array of well), 3456-well plates and 9600-well plates. Other formats that may be used in the methods provided herein include, but are not limited to, single or multi-well plates comprising a plurality of domains. In certain embodiments, the plates are opaque-wall, opaque-bottom plates. In certain embodiments, the plates are black-wall, black-bottom plates. In certain embodiments, the plates have black walls and clear bottoms in order to allow bottom excitation and reading of the fluorescence signals. In certain embodiments, the plates are chosen with minimal and uniform intrinsic fluorescence intensity within the range used in the method to avoid interference with the FRET signals.
(98) The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
EXAMPLE 1
In Cardiomyocytes, Binding of Unzipping Peptide Activates Ryanodine Receptor 2 and Reciprocally Inhibits Calmodulin Binding
(99) DPc10 and related RyR2 peptides therefore may, serve as useful molecular probes to study the channel's structure-function relationship. However, the details of DPc10 binding to RyR2, including affinity and kinetics, are still unknown. In the present study, our goal was to characterize the binding of DPc10 to the RyR2 in the relatively intact environment of saponin-permeabilized rat ventricular myocytes. We used fluorescent DPc10 to measure the affinity and kinetics of DPc10 binding to RyR2 and its influence on CaM and FKBP12.6 binding and function. Furthermore, we used fluorescence resonance energy transfer (FRET) among fluorescent FKBP12.6, DPc10 and CaM to determine how DPc10 alters CaM and FKBP12.6 binding and to assess where the DPc10-binding site on RyR2 is in relation to CaM-binding and FKBP12.6-binding sites.
(100) Materials and Methods
(101) Rat ventricular myocytes were isolated and permeabilized as previously described (Li et al., Circ Res, 2002; 90:309-316). All procedures were performed according to the Guiding Principles in the Care and Use of the Animals and were approved by the Council of American Physiological Society. DPc10, FKBP12.6, and CaM were labeled at specific sites with small fluorescence probes, similar to our previous studies (Guo et al., Circ Res, 2010; 106:1743-1752; Cornea et al., PNAS USA, 2009; 106:6128-6133; Guo et al., Biophys J, 2011; 101:2170-2177). Competitive inhibition of fluorescent DPc10 (F-DPc10) binding to RyR2 by nonfluorescent DPc10 (nonfluorescent [NF]-DPc10) showed that both bind to RyR2 at the same site and same affinity (
(102) Rat Cardiac Myocyte Isolation
(103) Single ventricular myocytes were isolated from rat hearts as described previously (Li et al., Circ Res. 2002; 90:309-316). Briefly, after anesthesia (isoflurane, 5%), hearts were excised and perfused (5 min, 37 C.) with the minimal essential medium (MEM, GIBCO Life Technologies) gassed with 95% O2/5% CO2 before inclusion of collagenase B (0.5 mg/ml, Boehringer Mannheim) and protease (0.02 mg/ml, Sigma). Triturates were incubated (10 min, 37 C.) in the same enzyme solution, washed and kept in 100 M Ca2+MEM solution.
(104) Domain Peptide
(105) DPc10 peptides unlabeled and labeled with 5-carboxyfluorescein or HiLyte Fluor647 were synthesized at AnaSpec (Fremont, Calif.). The DPc10 sequence is: 2460-GFCPDHKAAMVLFLDRVYGIEVQDFLLHLLEVGFLP-2495 (SEQ ID NO:1).
(106) Fluorescent Labeling of Single-Cysteine Mutants of FKBP12.6 and CaM
(107) A single-cysteine variant of the human FKBP12.6-isoform (T14C-C22A-C76I-FKBP12.6) was labeled using the thiol-specific maleimide derivatives of ALEXA FLUOR 488 and ALEXA FLUOR 568 (Invitrogen), as described previously (Cornea et al., PNAS USA, 2009; 106:6128-6133; Cornea et al., JBio Chem, 2010; 285:19219-19226). A single-cysteine CaM (T34CCaM) was labeled with ALEXA FLUOR 568 maleimide as described previously (Cornea et al., PNAS USA, 2009; 106:6128-6133; Cornea et al., JBio Chem, 2010; 285:19219-19226).
(108) Laser scanning confocal microscopy
(109) Confocal images was measured using a Biorad RADIANCE2100 laser scanning confocal microscope equipped with an Argon ion laser, Green HeNe laser and with a Nikon Fluo x40 oil lens. FRET (fluorescence resonance energy transfer) experiments between CaM and DPc10, and FKBP12.6 and DPc10 were performed using an Olympus FV1000 confocal microscope. All experiments were done at room temperature.
(110) Ca2+Sparks in Permeabilized Cells Using Confocal Microscopy
(111) Myocytes were permeabilized with saponin (50g/mL) for 60 seconds and placed in internal solution composed of EGTA 0.5 mmol/L; HEPES 10 mmol/L; Kaspartate 120 mmol/L; ATP 5 mmol/L; free MgCl2 1 mmol/L, reduced glutathione 10 mmol/L; and free [Ca2+] 50nmol/L (calculated using MaxChelator), creatine phosphokinase 5 U/mL, phosphocreatine 10 mmol/L, dextran 4% Fluo-4 0.025 mmol/L, pH 7.2. Ca2+sparks were recorded by a laser scanning confocal microscope (RADIANCE2000 MP, Bio-Rad, UK) as previously described (Li et al., Circ Res, 2002; 90:309-316). Fluo-4 was excited at 488 nm and emission was recorded using 500/530 nm bandpass filter. To assess SR Ca2+content, caffeine (15 mmol/L) was rapidly perfused. Ca2+spark were analyzed as using SparkMaster (Picht et al., Am J Physiol Cell Physiol, 2007; 293:C1073-C1081).
(112) FRET Measurements
(113) For the FRET between CaM and DPc10, and FKBP12.6 and DPc10, we used Alexa Fluor 568 attached at the C-lobe of CaM (AF568-110-CaM) (Yano et al., Circulation, 2000; 102:2131-2136), ALEXA FLUOR 488 attached at the N-lobe of CaM (AF488-34-CaM), ALEXA FLUOR 568-, or ALEXA FLUOR 488-FKBP12.6 (Ai et al., Circ Res, 2005; 97:1314-1322; Wehrens et al., PNAS USA, 2006; 103:511-518) (AF488-FKBP12.6, AF568-FKBP12.6) and HiLyte Fluor647-DPc10 (HF647-DPc10) as a donor-acceptor pair. AF488-, AF568- and HF647- were excited with separate laser channels of 488 nm, 543 nm and 635 nm, respectively. Emission fluorescence intensity data were obtained at 505-605 nm for AF488-FKBP12.6/AF488-34-CaM, 560-620 nm for AF568-FKBP12.6/AF568-110-CaM and 655-755 nm for HF647-DPc10. We used two experimental approaches, (1) comparing the donor fluorescence intensities before and after equilibration with acceptor (donor quenching) and (2) monitoring the increase in donor fluorescence after acceptor photobleaching (acceptor photobleaching), to detect and measure FRET signals in the permeabilized cardiomyocytes.
(114) For the donor quenching method, FRET is indicated by a decrease in the donor fluorescence at wavelengths 505-605 nm (AF488-FKBP12.6) or 560-620 nm (AF568-FKBP12.6). The FRET efficiency (E) was calculated according to:
E=1F.sub.DA/F.sub.D,
Where F.sub.D and F.sub.DA are the fluorescence intensities of the donor-only and donor-acceptor samples, respectively.
(115) Complete acceptor (HF647) photobleaching was achieved by repeated scans of a defined area of the myocyte with the 635 nm laser at maximum power, for 60 sec. E was calculated according to:
E=[(I.sub.donor-postI.sub.donor-pre)/I.sub.donor-post]100%,
where I.sub.donor-post and I.sub.donor-pre are donor fluorescence intensities before and after acceptor photobleach. Donor-acceptor distances, R, were calculated from the equation:
R=R.sub.0(E.sup.11).sup.1/6,
where the Frster distance, R.sub.0 is defined as the distance at which E=0.5. R.sub.0 is calculated from
R.sub.0=9780(J.sup.2n.sup.4.sub.D).sup.1/6,
where n is the refractive index of protein in aqueous solution (1.4), is the orientation factor (set to , corresponding to random orientation), .sub.D is the fluorescence quantum yield of the donor (0.92 for AF488, and 0.69 for AF568.sup.5). J is the normalized spectral-overlap integral of donor emission F.sub.D() and acceptor absorbance () and is calculated from
J=(F.sub.D()().sup.4d)/F.sub.D()d,
by numerical integration using a Microcal Origin template. For the AF488-HF647 and AF568-AF647 donor-acceptor pairs we used .sub.H647 (652 nm)=250,000 (mol.sup.1 cm.sup.1) to calculate R.sub.0 values of 54 A and 75 A, respectively.
Statistics
(116) Data were expressed as meanSEM, and significance was evaluated using student's t test or one-way ANOVA. A P-value below 0.05 was considered statistically significant.
(117) Results
(118) Localization and Binding Isotherms of F-DPc10 in Permeabilized Myocytes
(119)
(120) Binding Kinetics of F-DPc10 in Permeabilized Cardiac Myocytes
(121) To characterize DPc10-binding kinetics at Z-lines, we performed F-DPc10 wash-in (500 nmol/L) and washout experiments in permeabilized myocytes (
k.sub.wash-in=[F-DPc10]k.sub.on+k.sub.off
where k.sub.washoutk.sub.off and k=1/ (s.sup.1). Accordingly, k.sub.on=20220 (L mol.sup.1 s.sup.1) and k.sub.off=0.110.01 (10.sup.3 s.sup.1). Based on these values and K.sub.d=k.sub.off/k.sub.on, F-DPc10 binds at the Z-line with K.sub.d=58069 nmol/L, consistent with the steady-state K.sub.d measurements (
(122) We were intrigued by the slow k.sub.wash-in and conducted measurements to further understand the basis of this slow association. We tested the hypothesis that at resting [Ca.sup.2+].sub.i, DPc10 access to its RyR2 binding site is sterically hindered. If the N-terminal and central domains are tightly zipped to each other, then this interaction may occlude the DPc10-binding site on the RyR2, thus limiting the k.sub.on for DPc10. Alternatively, a limiting factor may be the rate at which DPc10 adopts a conformation that can bind to RyR2. To discern between these mechanisms, we determined the effect of [F-DPc10] on .sub.wash-in. If the small fraction of DPc10 in the right conformation limits binding rate, then .sub.wash-in should be faster at higher [F-DPc10], according to Equation.
(123) In our working model, under resting conditions, the RyR2 closed state may be stabilized by the interaction between the N-terminal and central domain in the zipped state. We hypothesized that conditions that promote RyR2 opening might enhance the rate of unzipping and accelerate .sub.wash-in for F-DPc10. To test this, we first monitored F-DPc10 wash-in at elevated Ca.sup.2+ (500 nmol/L). However, the 13% faster mean .sub.wash-in was not significant (
(124) Both FKBP12.6 and CaM bind to the RyR2 and can reduce channel opening, which might alter DPc10 binding.
(125) Next, we asked whether RyR2 is activated by DPc10 and whether FKBP12.6 or CaM can prevent this. We assessed Ca.sup.2+ sparks in permeabilized myocytes perfused with internal solution containing 50 nmol/L free Ca.sup.2+ plus 1 mol/L autocamtide-2-related inhibitory peptide (to inhibit CaMKII activity). Line-scan images were recorded after 3-hour incubations under control conditions and in the presence of 5 mol/L DPc10, with or without 1 mol/L CaM or 100 nmol/L FKBP12.6 (
(126) TABLE-US-00001 TABLE I Ca2.sup.+ spark characteristics in permeabilized cardiomyocytes. No. of Peak FWHM FDHM Sparks (F/F.sub.0) (m) (ms) Control 1761 0.499 0.004 1.28 0.01 55.7 0.67 +DPc10 1467 0.475 0.004* 1.34 0.01* 58.5 1.00* +FKBP12.6/ 1921 0.439 0.002* 1.21 0.01* 60.5 0.73* DPc10 +CaM/DPc10 2195 0.467 0.003* 1.23 0.01* 56.9 0.74
(127) Because CaSpF strongly depends on the SR Ca.sup.2+ content, we also measured SR Ca.sup.2+ content as the amplitude of caffeine-induced Ca.sup.2+ release (
(128) Effect of DPc10 on FKBP12.6 and CaM Binding in Permeabilized Myocytes
(129) To examine the converse influence that DPc10 may have on FKBP12.6 and CaM binding to RyR2 in situ, we used fluorescent FKBP12.6 and CaM variants labeled with ALEXA FLUOR 488 or 568 (AF488 and AF568, respectively). These fluorescent proteins were added to saponin-permeabilized myocytes with or without pre-equilibration with saturating DPc10 concentration. First, we found that AF488-FKBP12.6 at 1 nmol/L (near its K.sub.d) (Guo et al., Circ Res, 2010; 106:1743-1752) forms a striated pattern that is not affected by preincubation with 5 mon DPc10 (
(130) FRET Between CaM and DPc10
(131) We used a fluorescence donor probe (AF568) at the C-lobe of CaM (Cornea et al., PNAS USA, 2009; 106:6128-6133) (AF568-110-CaM) and HiLyte Fluor 647 (HF647) as the acceptor probe on the N terminus of DPc10 (HF647-DPc10). We used the acceptor photobleach approach with measurement of the resultant increase in donor (AF568-110-CaM) fluorescence in saponin-permeabilized myocytes (
(132) Our results show that it is impractical to saturate RyR2 with both CaM and DPc10 (
(133) To rule out the possibility that there is energy transfer between a donor and multiple acceptors, we measured the relationship between donor fluorescence enhancement and acceptor photobleach and found a linear relationship (
(134) FRET efficiency between AF568-110-CaM and HF647-DPc10 on 98.2%0.2% acceptor photobleach was 0.890.01 (n=8). This corresponds to a distance of 531 (
(135) FRET Between FKBP12.6 and DPc10
(136) To gain further information about the topology of the DPc10-binding site on RyR2, we used the location of FKBP12.6 as a reference point (Guo et al., Biophys J, 2011; 101:2170-2177; Sams et al., J Mol Biol, 2006; 356:917-927; Cornea et al., J Biol Chem, 2010; 285:19219-19226). FKBP12.6 was labeled at position 14 (Cornea et al., PNAS USA, 2009; 106:6128-6133) with the fluorescent donor AF488 (AF488-FKBP12.6) or AF568 (AF568-FKBP12.6), whereas DPc10 was labeled with the acceptor HF647. We used the same 2 methods to measure FRET in permeabilized myocytes.
(137) To ensure that FRET between FKBP12.6 and DPc10 accurately reflects interprobe distance, we performed several controls. As shown in
(138) The average FRET efficiency between AF568/488-FKBP12.6 and HF647-DPc10 was used to estimate the distance between FKBP12.6 and DPc10. The FRET efficiency between AF568-FKBP12.6 and HF647-DPc10 measured by the donor quench method was E=0.920.01 (n=31), whereas that measured by acceptor photobleach method was E=0.910.01 (n=19;
(139) Discussion
(140) We used fluorescent DPc10, FKBP12.6, CaM, and confocal microscopy of permeabilized cardiomyocytes and found the following: (1) DPc10 access to its binding site is sterically hindered in resting (zipped) RyR2; (2) F-DPc10 wash-in kinetics provides a sensitive measure of the RyR2 unzipped state in permeabilized myocytes; (3) DPc10 and CaM binding to RyR2 are mutually inhibitory (via allosteric rather than competitive interaction); and (4) DPc10, CaM, and FKBP12.6 are physically 50 to 60 from each other as vertices of an approximately equilateral triangle on RyR2.
(141) RyR2 is the Main Target of DPc10 Binding at Z-Lines
(142) To assess DPc10 binding affinity and concentration at Z-lines, we used equilibrium and kinetic binding methods. Both methods (
(143) Access of DPc10 to its RyR2-Binding Site is Restricted
(144) We found that both the wash-in and washout kinetics of F-DPc10 binding (k.sub.wash-in and k.sub.washout) are extremely slow (
(145) We also found that enhancing RyR2 open state (by caffeine plus ryanodine) hastened the F-DPc10 association (
(146) Relationship Between FKBP12.6 and DPc10 Binding to RyR2
(147) FKBP12.6 has been found to quiet RyR2 channel opening (Prestle et al., Circ Res, 2001; 88:188-194), but this is an intensely controversial issue (Bers, Circ Res, 2012; 110:796-799; Xiao et al., J Biol Chem, 2007; 282:34828-34838), and FKBP12.6 may only inhibit pathologically leaky RyRs (Oda et al., Circulation, 2005; 111:3400-3410). Because >80% of the RyRs in the cardiomyocytes have no natively bound FKBP12.6 (Guo et al., Circ Res, 2010; 106:1743-1752), adding saturating concentrations of exogenous FKBP12.6 should decrease Ca.sup.2+ leak caused by DPc10-induced unzipping. Here, we found that FKBP12.6 has no effect on either DPc10 binding (B.sub.max or .sub.wash-in) or vice versa (
(148) Relationship Between CaM and DPc10 Binding to RyR2
(149) In myocytes containing a CPVT-linked RyR2 mutation, -adrenergic stimulation decreases CaM binding at the Z-lines, and this effect is mimicked in healthy myocytes by treatment with DPc10 (Xu et al., Biochem Biophys Res Commun, 2010; 394:660-666). Here, we used methods designed to monitor CaM and DPc10 binding specifically at the RyR2 in myocytes, aiming to understand the structural basis of the inhibition of CaM-RyR2 binding by DPc10. One important finding in the present study is that saturating CaM binding at the RyR2 dramatically reduced F-DPc10 binding and Ca.sup.2+ spark activation (
(150) Our novel FRET-based method allows direct assessment of CaM-RyR2 binding in the native cardiac myocyte environment (using FKBP12.6-CaM FRET) (Guo et al., Biophys J, 2011; 101:2170-2177). Using this method, we found that unzipping the RyR2 by treatment with saturating [DPc10] reciprocally inhibits CaM binding to RyR2 (
(151) To discern between these possibilities, we assessed whether CaM and DPc10 can coexist on RyR2. In
(152) Our working hypothesis (
(153) Topology of the DPc10-Binding Site on RyR2
(154) Although our aim here was not a detailed mapping of the DPc10-binding site within the cryo-EM 3-dimensional structure of RyR2, our FRET measurements help narrow the range of possible locations. The location of FKBP12.6 and CaM on the RyR2 structure is known from cryo-EM structural analysis, and their relative positions agree with our previous FKBP-CaM FRET studies. (Cornea et al., PNAS USA, 2009; 106:6128-6133; Guo et al., Biophys J, 2011; 101:2170-2177; Sams et al., J Mol Biol, 2006; 356:917-927; Cornea et al., J Biol Chem, 2010; 285:19219-19226). These are represented by the centers of the blue and red spheres in
(155) We represent the FKBP12.6, CaM, and DPc10 sites all on the same face of the RyR2 tetramer. We previously showed that this is true for the FKBP-CaM FRET pair, (Cornea et al., PNAS USA, 2009; 106:6128-6133; Guo et al., Biophys J, 2011; 101:2170-2177) but we also tested whether the potential DPc10 site could be between CaM and FKBP sites on adjacent RyR2 faces.
(156) Relevance to Heart Failure
(157) Until now, methods to monitor local conformational changes occurring in the interacting regulatory domains of RyR have relied on a large fluorescence quencher (used in isolated SR vesicles) (Oda et al., Circulation, 2005; 111:3400-3410; Tateishi et al., Cardiovasc Res. 2009; 81:536-545) or on FRET between a yellow fluorescent protein inserted into the N-terminal region and a cyan fluorescent protein inserted into the central region of RyR2 (in HEK293 cells) (Liu et al., J Cell Sci, 2010; 123:1775-1784). In this study, we show how the F-DPc10 wash-in kinetics can be used in the more native environment of permeabilized cardiomyocytes to evaluate domain interaction between the N-terminal and central domains of RyR2. This could serve as a powerful and versatile investigative tool in preclinical and clinical studies with respect to the domain unzipping hypothesis. For example, the time course of F-DPc10 wash-in can be monitored in myocytes from failing hearts in which unzipping has already occurred (Oda et al., Circulation, 2005; 111:3400-3410; Ono et al., Cardiovasc Res, 2010; 87:609-617) or can be monitored to gauge RyR function under pathological conditions (eg, oxidative stress, phosphorylation state) and in the evaluation (or validation) of drug candidates that act to stabilize the RyR2 zipped state (Oda et al., Circulation, 2005; 111:3400-3410; Kobayashi et al., J Am Coll Cardiol, 2009; 53:1993-2005).
EXAMPLE 2
(158) This example investigates directly in cardiac myocytes how oxidation alters RyR2 conformation, as detected by measuring the fluorescently labeled DPc10 (F-DPc10) binding kinetics, and the binding affinity of fluorescent CaM and FKBP12.6. Also assessed is how dantrolene affects RyR2 conformation change and the binding affinity of CaM-RyR2, and FKBP12.6-RyR2, and DPc-10.
(159) Materials and Methods
(160) Cardiac Myocytes Isolation
(161) Rat ventricular myocytes were isolated and permeabilized as previously described (Li et al., Circ Res. 2002; 90:309-316). Briefly, after euthanasia, the heart was quickly removed and perfused with 95% O.sub.2/5% CO.sub.2 gassed with 0.8 mg/ml collagenase B (Boehringer Mannheim) containing minimal essential medium (MEM, GIBCO Life Technologies). Left ventricle was minced with scissors, filtered, suspended, rinsed to prepare the rod-shaped cardiomyocytes. Isolated cardiomyocytes were placed in a laminin-coated glass culture dishes with normal Tyrode's (NT) solution. Experiments were done at room temperature.
(162) Domain Peptide
(163) DPc10 peptide was labeled with HiLyte Fluor647 at AnaSpec (Fremont, Calif.). The DPc10 sequence is: 2460-GFCPDHKAAMVLFLDRVYGIEVQDFLLHLLEVGFLP-2495 (SEQ ID NO:1).
(164) Fluorescent FKBP12.6 and CaM Probes
(165) Single-cysteine variants of FKBP12.6 and CaM were labeled with ALEXA FLUOR 488 (Invitrogen), as described previously (Cornea et al., Proc Natl Acad Sci USA. 2009;106:6128-6133; Cornea et al., J Bio Chem. 2010;285:19219-19226).
(166) Cell Permeabilization
(167) Isolated myocytes were superfused with a relaxing solution containing (mM): EGTA 0.1, HEPES 10, K-aspartate 120, free MgCl.sub.2 1, ATP 5, phosphocreatine di-Tris 5 at pH 7.4. After myocytes were permeabilized by saponin (50 g/ml), myocytes were placed in an internal solution containing (mM): EGTA 0.5, HEPES 10, K-aspartate 120, free MgCl.sub.2 1, ATP 5, phosphocreatine di-Tris 5, creatine phosphokinase 5 U/ml, phosphocreatine 10, dextran (MW: 40,000) 4% at pH 7.2, and CaCl.sub.2, added to obtain 50 nM free [Ca.sup.2+] (calculated by MaxChelator).
(168) Monitoring of Ca.sup.2+ Sparks, SR Ca.sup.2+ Content and Spontaneous Ca.sup.2+ Waves
(169) Intact cardiomyocytes were loaded with Fluo-4 AM (5 mol/L, Molecular Probes) for 15 min in NT solution (in mM): NaCl 140, KCl 4, MgCl.sub.2 1, CaCl.sub.2 2, glucose 10, HEPES 5, pH 7.4. Fluo-4 AM was excited at 488 nm and emission was recorded using a 500-530 nm bandpass filter. Ca.sup.2+sparks were recorded after field stimulation at 1 Hz, and caffeine (10mmol/L) was rapidly perfused for assessing the SR Ca.sup.2+content. Both Ca.sup.2+sparks and SR Ca.sup.2+content were recorded by using a laser scanning confocal microscope (RADIANCE2000 MP, Bio-Rad, UK). Data were analyzed using SparkMaster, an automated analysis program that allows rapid and reliable spark analysis (Picht et al., J. Am J Physiol Cell Physiol. 2007;293:C1073-C1081). Spontaneous Ca.sup.2+waves were recorded using a BZ9000 fluorescence digital microscope (Keyence, Japan), after 10 sec from field electric stimulation at 1,2,3,4 and 5 Hz, as described previously (Fukuda et al., Biochem Biophys Res Commun. 2014;448:1-7).
(170) Intracellular ROS and Oxidation of RyR2
(171) Intracellular reactive oxygen species (ROS) formation was measured in rat cardiac myocytes using a fluorescent probe, 5-(and -6)-chloromethyl-2,7-dichlorodihydrofluoroscein diacetate (H.sub.2DCFDA, 10 mol/L) (Terentyev et al., Circ Res. 2008; 103:1466-1472). This assay is widely used as a reliable method for the measurement of intracellular ROS such as hydrogen peroxide, hydroxyl radicals, and hydroperoxide. The H.sub.2DCFDA dye was excited with the 488 nm and emission was collected at 500-530 nm. The content of free thiols in RyR2 was determined based on the monobromobimane (mBB, Calbiochem) fluorescence intensity, as previously described (Terentyev et al., Circ Res. 2008; 103:1466-1472; Ho et al., J Physiol. 2011; 19:4697-4708). In brief, myocytes were incubated with mBB for 1 hr. Myocytes were then lysed in lysis buffer and loaded on 6% SDS-PAGE gels. The fluorescence emission intensity of the RyR2-bound mBB was measured at 482 nm by exciting at 382 nm. mBB fluorescence was normalized to the RyR2 amount, which was determined using quantitative densitometry of Coomassie Blue-stained gels.
(172) FRET Measurements Between FKBP12.6 and DPc10
(173) The FRET pair we used in this consisted of ALEXA FLUOR 488 (donor) covalently attached to FKBP12.6 (F-FKBP) and HiLyte Fluor647 (acceptor) covalently attached to DPc10 (F-DPc10)..sup.8 To detect F-DPc10 specifically bound to RyR2, we measured donor fluorescence intensities through a 500-530 nm bandpass filter before and after equilibration with acceptor (donor quenching method). The FRET efficiency (E) was calculated according to:
E=1-F.sub.DA/F.sub.D,
Where F.sub.D and F.sub.DA are the fluorescence intensities of the donor-only and donor-plus-acceptor samples, respectively (Oda et al., Circ Res. 2013;112:487-497).
Statistics
(174) Data are expressed as meanSEM, with significance assessed using student's t-test or one-way ANOVA. A p-value <0.05 was considered statistically significant.
(175) Results
(176) Activated RyR2 Function by Oxidation in Myocytes
(177) Oxidation induced by hydrogen peroxide (H.sub.2O.sub.2) is expected to activate SR Ca.sup.2+ leak through RyR2 at diastolic [Ca].sub.i (Wagner et al., Circ Res. 2011; 108:555-65; Yan et al., Cardiovasc Res. 2008; 77:432-41). We used line-scan images to measure the effects of 50 M/L H.sub.2O.sub.2 on Ca.sup.2+ spark frequency (CaSpF) and SR Ca.sup.2+ content (evaluated via rapid caffeine application) in intact cardiomyocytes after 1 Hz electric field stimulation (
(178) TABLE-US-00002 TABLE 2 Ca.sup.2+ transient characteristics in rat intact cardiomyocytes No. of [Ca].sub.i Tau [Ca].sub.i cells twitch (F/F.sub.0) decline (s) Control 16 2.38 0.13 0.32 0.01 +DAN 17 2.35 0.16 0.31 0.01 +H.sub.2O.sub.2 10 2.56 0.19 0.32 0.01 +H.sub.2O.sub.2 + DAN 7 2.32 0.23 0.32 0.03
(179) TABLE-US-00003 TABLE 3 Ca.sup.2+ spark characteristics in rat intact cardiomyocytes No. of Amplitude FWHM FDHM sparks (F/F.sub.0) (m) (ms) Control 108 0.53 0.01 1.86 0.08 .sup.55.56 2.56.sup.# +DAN 125 0.55 0.01 1.93 0.09 61.98 3.35 +H.sub.2O.sub.2 200 0.57 0.01 2.04 0.07 67.05 3.25* +H.sub.2O.sub.2 + DAN 53 0.53 0.02 1.87 0.11 54.56 3.12 FWHM: full width at half maximum, FDHM: full duration at half maximum. *p < 0.05 vs. Control, .sup.#P < 0.05 vs H.sub.2O.sub.2
(180) We also tested whether H.sub.2O.sub.2 enhanced the propensity for arrhythmogenic DADs, as measured by spontaneous Ca.sup.2+ waves (SCW) in intact cardiac myocytes.
(181) Dantrolene Quiets Oxidation-Induced RyR2 Activation
(182) Dantrolene can prevent abnormal Ca.sup.2+ leak in CPVT KI and HF models (Uchinoumi et al., Circ Res. 2010; 106:1413-1424; Kobayashi et al., J Am Coll Cardiol. 2009; 53:1993-2005; Kobayashi et al., Circ J. 2010; 74:2579-84 Maxwell et al., Am J Physiol Heart Circ Physiol. 2012; 302:H953-63). To test whether dantrolene could prevent H.sub.2O.sub.2-induced increase in CaSpF, dantrolene was added to myocytes before H.sub.2O.sub.2 treatment. Dantrolene pretreatment had no effect on control myocytes, but suppressed the H.sub.2O.sub.2-induced increase in frequency of Ca.sup.2+ sparks and SCW (
(183) H.sub.2O.sub.2-Induced Intracellular and RyR2 Redox Modification
(184) To monitor intracellular ROS level, we used H.sub.2DCFDA (10 mol/L).
(185) Effect of H.sub.2O.sub.2 on FKBP12.6 Binding to Z-Line in Permeabilized Myocytes
(186) We assessed the molecular mechanism by which RyR2 activities were increased by H.sub.2O.sub.2-induced oxidative stress. FKBP12.6 can bind to and stabilize RyR2 channel gating, but details are controversial (Bers et al., Circ Res. 2012; 110:796-799). We measured the effect of H.sub.2O.sub.2 on FKBP12.6-RyR2 binding affinity in saponin-permeabilized myocytes using F-FKBP (Yang et al., Circ Res. 2014; 114:295-306; Cornea et al., Proc Natl Acad Sci USA. 2009; 106:6128-6133; Guo et al., Circ Res. 2010; 106:1743-1752; Guo et al., Biophys J. 2011; 101:2170-2177; Cornea et al., J Biol Chem. 2010; 285:19219-19226).
(187) Effect of H.sub.2O.sub.2 on CaM Binding to Z-Line in Permeabilized Myocytes
(188) CaM also binds to RyR2 and reduces RyR2 open probability, and works as a regulatory protein for RyR2 channel gating (Yamaguchi et al., J Biol Chem. 2003; 278:23480-23486; Balshaw et al., J Biol Chem. 2001; 276:20144-20153; Yang et al., Circ Res. 2014; 114:295-306); Yamaguchi et al., J Clin Invest. 2007; 117:1344-1353). We measured the effect of H.sub.2O.sub.2 on CaM-RyR2 binding affinity using F-CaM as in our previous reports (Yang et al., Circ Res. 2014; 114:295-306; Cornea et al., Proc Natl Acad Sci USA. 2009; 106:6128-6133). Both 10 and 50 mol/L H.sub.2O.sub.2 pretreatment of myocytes reduced the F-CaM binding at the Z-line significantly (using [F-CaM]=20 nmol/L, near its K.sub.d (Yang et al., Circ Res. 2014; 114:295-306)), suggesting a decreased affinity of F-CaM/RyR2 binding (
(189) To test whether CaM oxidation is likely to explain the reduced RyR2 binding of H.sub.2O.sub.2-treated myocytes, we first incubated [F-CaM] (50 mol/L) in media containing 50 mol/L H.sub.2O.sub.2 (the same concentration used in myocyte pre-incubation experiments). This allowed F-CaM oxidation (and call this F-CaM.sup.Ox). This was then diluted 2500-fold to 20 nmol/L F-CaM.sup.Ox and no H.sub.2O.sub.2 was present in the myocyte bath. As shown in
(190) As a further test of whether RyR2 oxidation alone inhibits subsequent CaM affinity, we removed H.sub.2O.sub.2 from the myocytes by repeated washing, prior to the addition of F-CaM.
(191) Since CaM binding to RyR2 in myocytes is known to suppress Ca.sup.2+ sparks (Yang et al., Circ Res. 2014; 114:295-306; Guo et al., Circ Res. 2006; 99: 398-406) the H.sub.2O.sub.2-induced increase in SR Ca.sup.2+ leak (measured as Ca.sup.2+ sparks in
(192) Effects of Dantrolene or H.sub.2O.sub.2 on Wash-In Kinetics of HF647-DPc10
(193) We previously reported that the wash-in kinetics of DPc10 labeled with HYLITE FLUOR 647 (HF647-DPc10) were greatly slowed when CaM was bound to the RyR2, which also prevented RyR2 activation by DPc10 exposure (Oda et al., Circ Res. 2013;112:487-497). Our working model was that DPc10 access to its binding site was sterically blocked when CaM was bound. Here, we first tested whether dantrolene has the same effect as CaM in preventing DPc10 access.
(194)
(195) Next we tested whether RyR2 oxidation would increase HF647-DPc10 access. Here, we used FRET between F-FKBP as a donor and HF647-DPc10 as an acceptor, to detect DPc10 that specifically binds at RyR2 (Oda et al., Circ Res. 2013; 112:487-497). Note that AF488-FKBP12.6 binds specifically at RyR2 with 1 nM affinity, and its binding is not influenced by CaM, H.sub.2O.sub.2 or dantrolene (Guo et al., Circ Res. 2010; 106:1743-1752 and
(196) Discussion
(197) Novel findings of this study are the following: In the relatively intact ventricular myocyte environment, (1) H.sub.2O.sub.2 treatment increases both CaSpF and the occurrence of SCW, but dantrolene prevents this elevated Ca.sup.2+ leak; (2) RyR2 oxidation by H.sub.2O.sub.2 decreases its binding affinity for CaM, but does not alter its FKBP12.6 affinity; (3) RyR2 oxidation leads to domain unzipping; and (4) dantrolene corrects domain unzipping, restores the CaM-RyR2 binding affinity, and inhibits pathological RyR2 channel gating. Our working model is that modest H.sub.2O.sub.2 (or ROS) levels causes a similar pathological change in RyR2 conformation as seen in HF, in which CaM affinity is reduced, DPc10 access is increased and SR Ca leak is elevated.
(198) RyR2 Function is Activated by H.sub.2O.sub.2
(199) Increased ROS production has been associated with pathological states, such as HF (McMurray et al., Eur Heart J. 1993; 14:1493-1498), and RyR2 activity in pathological states is increased by thiol oxidation (Boraso et al., Am J Physiol. 1994; 267:H1010-H1016; Donoso et al., J Mol Cell Cardiol. 2014; 68:38-46). H.sub.2O.sub.2 can activate RyR2 function, but it also alters function of other important Ca.sup.2+-handing proteins, including SERCA and Na.sup.+Ca.sup.2+ exchange (NCX). Yan et al. (Yan et al., Cardiovasc Res. 2008; 77:432-41) showed that CaSpF increased during 10 min of treatment with 50 mol/L H.sub.2O.sub.2 in intact myocytes. On the other hand, higher [H.sub.2O.sub.2] (200 mol/L) can reduce Ca.sup.2+ transient amplitude, CaSpF, and SR Ca.sup.2+ content, consistent with H.sub.2O.sub.2-dependent inhibition of SERCA activity (Greensmith et al., Cell Calcium. 2010; 48:341-51). To assess whether H.sub.2O.sub.2 activates the RyR2 function, we measured CaSpF under conditions where both amplitude and the rate of Ca.sup.2+ transient decline (reflecting SERCA activity), and the decay of caffeine-induced Ca.sup.2+ transient (reflecting NCX activity), were similar. This indicates that both SERCA and NCX function were not appreciably altered under our conditions. Thus, RyR2 function may be more sensitive to H.sub.2O.sub.2-induced modulation than are SERCA or NCX. We also found a significant increase in the occurrence of arrhythmogenic SCW in H.sub.2O.sub.2-treated myocytes, when pacing rate increased from 1 to 5 Hz, indicating that hyperactivity of RyR2 by oxidation may contribute to triggering lethal arrhythmias.
(200) Dantrolene Restores H.sub.2O.sub.2-Induced RyR2 Activation without Altering Cellular and RyR2 Oxidation
(201) Dantrolene has been shown to bind to amino acids 601-620 of RyR2 (Paul-Pletzer et al., Biochem J. 2005; 387:905-909) and stabilize the RyR2 channel gating in pathological states, such as HF (Maxwell et al., Am J Physiol Heart Circ Physiol. 2012; 302:H953-63) or CPVT (Jung et al., EMBO Mol Med. 2012; 4:180-191). However, dantrolene has no effect on Ca.sup.2+ signaling under control condition (Maxwell et al., Am J Physiol Heart Circ Physiol. 2012; 302:H953-63; Diaz-Sylvester et al., Am J Physiol Cell Physiol. 2008; 294:C1103-C1112). These are consistent with our observation that dantrolene significantly reduced the CaSpF and prevented potentially deleterious spontaneous arrhythmogenic Ca.sup.2+ waves in H.sub.2O.sub.2-treated myocytes, but did not alter the frequency of Ca.sup.2+ sparks and SCW under control condition. To exclude the possibility that dantrolene attenuated the oxidation level to achieve this effect, we measured intracellular ROS production and RyR2 free thiol content in dantrolene-treated myocytes. Dantrolene influenced neither cellular nor RyR2 oxidation level, suggesting that dantrolene directly stabilized RyR2, possibly by inhibiting domain unzipping (Krause et al., Anaesthesia. 2004; 59:364-373).
(202) H.sub.2O.sub.2 Reduced CaM, but not FKBP12.6 Binding to RyR2
(203) There are two main possible explanations for H.sub.2O.sub.2-induced defective CaM binding to RyR2: (1) Oxidation of CaM inhibits the productive association of CaM with RyR2 or (2) Oxidation of RyR2 prevents the CaM binding to its binding site. Balog et al. (Balog et al., Am J Physiol Heart Circ Physiol. 2006; 290:H794-H799) reported that extensive in vitro oxidation of CaM abolishes the functional interaction between CaM and RyR2. That is consistent with our in situ observation that our much milder exposure to H.sub.2O.sub.2 caused some CaM oxidation and reduction of binding to RyR2 (
(204) FKBP12.6 binds to RyR2 with high affinity and can also influence RyR2 gating (Guo et al., Circ Res. 2010; 106:1743-1752; Prestle et al., Circ Res. 2001; 88:188-194; Marx et al., Circ Res. 2001; 88:1151-1158) and has been proposed to play an important role in stabilizing RyR2 function (Prestle et al., Circ Res. 2001; 88:188-194; Marx et al., Circ Res. 2001; 88:1151-1158; Shan et al., J Cin Invest. 2010; 120:4375-4387), although this issue is controversial (Guo et al., Circ Res. 2010; 106:1743-1752; Greensmith et al., Cell Calcium. 2010; 48:341-51; Stange et al., J Biol Chem. 2003; 278:51693-51702; Houser et al., Circ Res. 2014 11;114:1320-1327). Shan et al. reported (Shan et al., J Cin Invest. 2010; 120:4375-4387) that 1 mmol/L H.sub.2O.sub.2 combined with phosphorylation of Ser2808 by PKA could reduce FKBP12.6 binding to RyR2 by 70%. In contrast, we find that neither PKA-dependent phosphorylation (Guo et al., Circ Res. 2010; 106:1743-1752), DPc10-induced unzipping (Oda et al., Circ Res. 2013; 112:487-497) nor the more moderate levels of H.sub.2O.sub.2 used here had any effect on FKBP12.6 binding to RyR2 in myocytes (
(205) Dantrolene Corrects RyR2 Conformation Caused by Either H.sub.2O.sub.2 or DPc10
(206) We previously demonstrated that monitoring F-DPc10 binding kinetics is a powerful tool to evaluate functionally important RyR2 conformational changes, likely related to an interaction between the N-terminal and central domains of RyR2. Using this method, we now show that H.sub.2O.sub.2 significantly accelerates F-DPc10 association rate in situ, indicating that H.sub.2O.sub.2 causes domain unzipping (
(207) CaMKII can also be activated by oxidation at methionine 281/282 (Erickson et al., Cell. 2008; 133:462-474) and can also phosphorylate and activate RyR2 (Guo et al., Circ Res. 2006; 99: 398-406; Erickson et al., Cell. 2008; 133:462-474) and this CaMKII pathway would be expected to exacerbate the direct RyR2 and CaM effects on RyR2 gating that has been the focus here. Moreover, here we used the CaMKII inhibitors AIP (CaM binding experiments) and KN-93 (SCW experiments) specifically to assess CaMKII-independent effects of H.sub.2O.sub.2 on Ca.sup.2+ waves, CaM binding to RyR2. Wagner et al. (Wagner et al., Circ Res. 2011; 108:555-65) have shown that the increase in CaSpF and SR Ca leak observed with 200 mol/L H.sub.2O.sub.2 was not prevented by KN-93, consistent with a CaMKII-independent effect of H.sub.2O.sub.2 on RyR2 dysfunction.
(208) These results indicate that abnormal oxidative modification of RyR2 by H.sub.2O.sub.2 causes reduced CaM affinity of RyR2 (by oxidation of sites on both CaM and RyR2) and RyR2 conformation changes (domain unzipping) that lead to untimely and potentially arrhythmogenic RyR2 channel opening. Dantrolene restores normal CaM binding, conformational state and quiets pathological RyR2 channel gating. This H.sub.2O.sub.2-induced structural unzipping, reduced CaM binding and more active RyR2 may represent a functionally integrated common pathological RyR2 state that is relevant for HF, oxidative stress and even CPVT-linked genetic mutations.
EXAMPLE 3
(209) Using the method illustrated in
(210) The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. Supplementary materials referenced in publications (such as supplementary tables, supplementary figures, supplementary materials and methods, and/or supplementary experimental data) are likewise incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
(211) Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term about. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
(212) Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.
(213) All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.