Cardiac Sarcoplasmic Reticulum Research Articles

Mechanisms for cardiac calcium pump activation by its substrate and a synthetic allosteric modulator using fluorescence lifetime imaging.

The discovery of allosteric modulators is an emerging paradigm in drug discovery, and signal transduction is a subtle and dynamic process that is challenging to characterize. We developed a time-correlated single photon-counting imaging approach to investigate the structural mechanisms for small-molecule activation of the cardiac sarcoplasmic reticulum Ca2+-ATPase, a pharmacologically important pump that transports Ca2+ at the expense of adenosine triphosphate (ATP) hydrolysis. We first tested whether the dissociation of sarcoplasmic reticulum Ca2+-ATPase from its regulatory protein phospholamban is required for small-molecule activation. We found that CDN1163, a validated sarcoplasmic reticulum Ca2+-ATPase activator, does not have significant effects on the stability of the sarcoplasmic reticulum Ca2+-ATPase-phospholamban complex. Time-correlated single photon-counting imaging experiments using the nonhydrolyzable ATP analog β,γ-Methyleneadenosine 5'-triphosphate (AMP-PCP) showed ATP is an allosteric modulator of sarcoplasmic reticulum Ca2+-ATPase, increasing the fraction of catalytically competent structures at physiologically relevant Ca2+ concentrations. Unlike ATP, CDN1163 alone has no significant effects on the Ca2+-dependent shifts in the structural populations of sarcoplasmic reticulum Ca2+-ATPase, and it does not increase the pump's affinity for Ca2+ ions. However, we found that CDN1163 enhances the ATP-mediated modulatory effects to increase the population of catalytically competent sarcoplasmic reticulum Ca2+-ATPase structures. Importantly, this structural shift occurs within the physiological window of Ca2+ concentrations at which sarcoplasmic reticulum Ca2+-ATPase operates. We demonstrated that ATP is both a substrate and modulator of sarcoplasmic reticulum Ca2+-ATPase and showed that CDN1163 and ATP act synergistically to populate sarcoplasmic reticulum Ca2+-ATPase structures that are primed for phosphorylation. This study provides novel insights into the structural mechanisms for sarcoplasmic reticulum Ca2+-ATPase activation by its substrate and a synthetic allosteric modulator.

Calcium Signaling Consequences of RyR2-S4938F Mutation Expressed in Human iPSC-Derived Cardiomyocytes.

Type-2 ryanodine receptor (RyR2) is the major Ca2+ release channel of the cardiac sarcoplasmic reticulum (SR) that controls the rhythm and strength of the heartbeat, but its malfunction may generate severe arrhythmia leading to sudden cardiac death or heart failure. S4938F-RyR2 mutation in the carboxyl-terminal was expressed in human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) using CRISPR/Cas9 gene-editing technique. Ca2+ signaling and electrophysiological properties of beating cardiomyocytes carrying the mutation were studied using total internal reflection fluorescence microscopy (TIRF) and patch clamp technique. In mutant cells, L-type Ca2+ currents (ICa), measured either by depolarizations to zero mV or repolarizations from +100 mV to -50 mV, and their activated Ca2+ transients were significantly smaller, despite their larger caffeine-triggered Ca2+ release signals compared to wild type (WT) cells, suggesting ICa-induced Ca2+ release (CICR) was compromised. The larger SR Ca2+ content of S4938F-RyR2 cells may underlie the higher frequency of spontaneously occurring Ca2+ sparks and Ca2+ transients and their arrhythmogenic phenotype.

RyR2 Binding of an Antiarrhythmic Cyclic Depsipeptide Mapped Using Confocal Fluorescence Lifetime Detection of FRET.

Hyperactivity of cardiac sarcoplasmic reticulum (SR) ryanodine receptor (RyR2) Ca2+-release channels contributes to heart failure and arrhythmias. Reducing the RyR2 activity, particularly during cardiac relaxation (diastole), is a desirable therapeutic goal. We previously reported that the unnatural enantiomer (ent) of an insect-RyR activator, verticilide, inhibits porcine and mouse RyR2 at diastolic (nanomolar) Ca2+ and has in vivo efficacy against atrial and ventricular arrhythmia. To determine the ent-verticilide structural mode of action on RyR2 and guide its further development via medicinal chemistry structure-activity relationship studies, here, we used fluorescence lifetime (FLT)-measurements of Förster resonance energy transfer (FRET) in HEK293 cells expressing human RyR2. For these studies, we used an RyR-specific FRET molecular-toolkit and computational methods for trilateration (i.e., using distances to locate a point of interest). Multiexponential analysis of FLT-FRET measurements between four donor-labeled FKBP12.6 variants and acceptor-labeled ent-verticilide yielded distance relationships placing the acceptor probe at two candidate loci within the RyR2 cryo-EM map. One locus is within the Ry12 domain (at the corner periphery of the RyR2 tetrameric complex). The other locus is sandwiched at the interface between helical domain 1 and the SPRY3 domain. These findings document RyR2-target engagement by ent-verticilide, reveal new insight into the mechanism of action of this new class of RyR2-targeting drug candidate, and can serve as input in future computational determinations of the ent-verticilide binding site on RyR2 that will inform structure-activity studies for lead optimization.

PO-01-244 BMAL1 REGULATES THE CIRCADIAN RHYTHM OF RYR2 AND CONTRIBUTES TO CIRCADIAN VENTRICULAR ARRHYTHMIA IN CHRONIC HEART FAILURE AFTER MYOCARDIAL INFARCTION

Ventricular arrhythmia (VAs) is the major cause of sudden cardiac death in chronic heart failure (CHF) after myocardial infarction (MI), and previous studies have reported that VAs exist circadian oscillation in CHF. Peripheral circadian clock can regulate expression and function of ion channels in cardiomyocytes, which is associated with circadian rhythm of VAs in CHF. However, the regulation of peripheral circadian clock on Ryanodine Receptor 2 (RYR2), a calcium channel on cardiac sarcoplasmic reticulum, remain to be fully understood. We aimed to explore the regulation of peripheral circadian clock on RYR2 and VA events in CHF. CHF was induced by ligation of coronary artery of mice, and RT-PCR were performed to investigate the circadian rhythm of RYR2 in CHF myocardial tissue. Amplitude of calcium transient of ISO-induced cardiomyocytes at different time was measured to study the circadian rhythm of RYR2 function. To confirm the circadian oscillation of VAs, VAs incidences at different time of CHF wild type mice and cardiomyocyte-specific Bmal1 knockout (CKO) mice were investigated by programmed electrical stimulation. Infections of Ad-CLOCK and/or Ad-BMAL1 were applied to overexpress CLOCK, BMAL1 and CLOCK-BMAL1 in cardiomyocytes, and expression of RYR2 was measured by Western blot. Chromatin immunoprecipitation assay (ChIP) and luciferase (LUC) were applied to determine whether CLOCK-BMAL1 transcriptionally regulate RYR2 expression. CLOCK, BMAL1, and RYR2 exhibited notable circadian rhythm in CHF mice (Figure A, P<0.05). Amplitude of calcium transient at CT36 was lower than CT24 (Figure B, P<0.05). Compared to CT36, VAs had higher incidence at CT24(Figure C, P<0.05), and incidences of VAs had no significant difference between CT24 and CT36 in CKO Mice or using Dantrolene, an antagonist of RYR2. Ad-CLOCK and Ad-BMAL1 co-infection resulted in overexpression of RYR2 (Figure D, P<0.05), and BMAL1 bound to the enhancer of RYR2 gene and upregulated RYR2 expression transcriptionally (Figure E). In conclusion, BMAL1, as peripheral circadian clock, could contribute to circadian oscillation of VAs in CHF by regulating the circadian rhythm of RYR2.

Reduced O-GlcNAcylation diminishes cardiomyocyte Ca2+ dependent facilitation and frequency dependent acceleration of relaxation

Ca2+ dependent facilitation (CDF) and frequency dependent acceleration of relaxation (FDAR) are regulatory mechanisms that potentiate cardiomyocyte Ca2+ channel function and increase the rate of Ca2+ sequestration following a Ca2+-release event, respectively, when depolarization frequency increases. CDF and FDAR likely evolved to maintain EC coupling at increased heart rates. Ca2+/calmodulin-dependent kinase II (CaMKII) was shown to be indispensable to both; however, the mechanisms remain to be completely elucidated. CaMKII activity can be modulated by post-translational modifications but if and how these modifications impact CDF and FDAR is unknown. Intracellular O-linked glycosylation (O-GlcNAcylation) is a post-translational modification that acts as a signaling molecule and metabolic sensor. In hyperglycemic conditions, CaMKII was shown to be O-GlcNAcylated resulting in pathologic activity. Here we sought to investigate whether O-GlcNAcylation impacts CDF and FDAR through modulation of CaMKII activity in a pseudo-physiologic setting. Using voltage-clamp and Ca2+ photometry we show that cardiomyocyte CDF and FDAR are significantly diminished in conditions of reduced O-GlcNAcylation. Immunoblot showed that CaMKIIδ and calmodulin expression are increased but the autophosphorylation of CaMKIIδ and the muscle cell-specific CaMKIIβ isoform are reduced by 75% or more when O-GlcNAcylation is inhibited. We also show that the enzyme responsible for O-GlcNAcylation (OGT) can likely be localized in the dyad space and/or at the cardiac sarcoplasmic reticulum and is precipitated by calmodulin in a Ca2+ dependent manner. These findings will have important implications for our understanding of how CaMKII and OGT interact to impact cardiomyocyte EC coupling in normal physiologic settings as well as in disease states where CaMKII and OGT may be aberrantly regulated.

Activation of Ca2+ transport in cardiac microsomes enriches functional sets of ER and SR proteins

The importance of sarcoplasmic reticulum (SR) Ca2+-handling in heart has led to detailed understanding of Ca2+-release and re-uptake protein complexes, while less is known about other endoplasmic reticulum (ER) functions in the heart. To more fully understand cardiac SR and ER functions, we analyzed cardiac microsomes based on their increased density through the actions of the SR Ca2+-ATPase (SERCA) and the ryanodine receptor that are highly active in cardiomyocytes. Crude cardiac microsomal vesicles loaded with Ca oxalate produced two higher density subfractions, MedSR and HighSR. Proteins from 20.0 μg of MV, MedSR, and HighSR protein were fractionated using SDS-PAGE, then trypsinized from 20 separate gel pieces, and analyzed by LC–MS/MS to determine protein content. From 62,000 individual peptide spectra obtained, we identified 1105 different proteins, of which 354 were enriched ≥ 2.0-fold in SR fractions compared to the crude membrane preparation. Previously studied SR proteins were all enriched, as were proteins associated with canonical ER functions. Contractile, mitochondrial, and sarcolemmal proteins were not enriched. Comparing the levels of SERCA-positive SR proteins in MedSR versus HighSR vesicles produced a range of SR subfraction enrichments signifying differing levels of Ca2+ leak co-localized in the same membrane patch. All known junctional SR proteins were more enriched in MedSR, while canonical ER proteins were more enriched in HighSR membrane. Proteins constituting other putative ER/SR subdomains also exhibited average Esub enrichment values (mean ± S.D.) that spanned the range of possible Esub values, suggesting that functional sets of proteins are localized to the same areas of the ER/SR membrane. We conclude that active Ca2+ loading of cardiac microsomes, reflecting the combined activities of Ca2+ uptake by SERCA, and Ca2+ leak by RyR, permits evaluation of multiple functional ER/SR subdomains. Sets of proteins from these subdomains exhibited similar enrichment patterns across membrane subfractions, reflecting the relative levels of SERCA and RyR present within individual patches of cardiac ER and SR.

Calcium-dependent shifts between phospholamban (PLB) monomers and homo-pentamers reveal the interactions between PLB and SERCA in native cardiac membranes.

Phospholamban monomer (PLB1) binds to the cardiac calcium pump (SERCA2a) and inhibits the enzyme. PLB homopentamers (PLB5) are inactive species, but stable in SDS that correlate well to that in the native membranes. In contrast, SDS separates PLB1 from SERCA2a. Using this feature, we developed assays to probe the constant exchanging PLB1 with SERCA2a, and with PLB5, in native cardiac sarcoplasmic reticulum (SR) membrane vesicles. Dog cardiac SR was pre-incubated at 37°C in 1mM EGTA, allowing PLB1 binding to SERCA2a. Then, 8 µg samples were incubated for 1 min with control buffer, 1mM Ca (free [Ca] = 16.4 µM), 1 mM Ca first and then 5 mM EGTA (free [Ca] = 0.06 µM), or 10µM SERCA2a inhibitor thapsigargin. These reactions were stopped by 1% SDS before SDS-PAGE and immunoblotting to measure PLB5/PLB1 ratios. Addition of Ca significantly increased PLB5/PLB1 to 2.91 ± 0.38 from 1.22 ± 0.08 in the control, Ca-free condition, suggesting that Ca activation of the pump dissociates the whole PLB monomer from inhibitory site on SERCA2a in cardiac membranes. Furthermore, subsequent addition of EGTA reversed Ca-induced increased in PLB5/PLB1 back to 1.38 ± 0.15. Meanwhile, thapsigargin virtually eliminated PLB1 binding to SERCA2a, increasing PLB5/PLB1 to 3.13 ± 0.40. In control experiments, none of these factors affected PLB5/PLB1 of WT-PLB expressed alone in insect cells. Therefore, the detected changes in PLB5/PLB1 must occur in the native SR membranes, reflecting fully reversible processes of Ca-dependent PLB1 association and dissociation from the inhibitory site on SERCA2a, followed by disassembling and assemblage into PLB5, respectively. Such assays will allow further studies to determine the effect of allosteric factors and proteins phosphorylation on the interactions between PLB and SERCA2a directly in native cardiac SR membranes.

Calcium signaling consequences of RyR2-S4938F mutation expressed in hiPSC-CMs.

Ryanodine receptor 2 (RyR2) is the major Ca2+ release channel of cardiac sarcoplasmic reticulum (SR) that regulates the rhythm and strength of the heartbeat, but its malfunction generates arrhythmia leading to heart failure. Here we report on a catecholaminergic polymorphic ventricular tachycardia (CPVT1)-associated RyR2 mutation (S4938F) located in RyR2 carboxyl-terminal that was expressed in human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) using CRISPR/Cas9 gene-editing. Ca2+ signaling and electrophysiological properties of beating cardiomyocytes carrying S4938F mutation were studied using total internal fluorescence microscopy (TIRF) and patch clamp technique. ICa densities measured either by depolarizations to zero mV or repolarizations from +100 mV to −50 mV in mutant hiPSC-CMs were comparable to WT cells, but their accompanying Ca2+-transients detected by Fura-2 (cytosolic Ca2+) or ER-GCaMP6 (SR Ca2+ release) were significantly suppressed, suggesting ICa induced Ca2+ release (CICR) was compromised. Even though 5mM caffeine triggered Fura-2 or ER-GCaMP6 signals and the frequency of spontaneously generated calcium transients of mutant cells were comparable to those of WT cells, the 4-CmC triggered cytosolic or SR Ca2+ signals and magnitude of spontaneously occurring Ca2+-transients were significantly suppressed. Spontaneously occurring Ca2+ sparks in S4938F hiPSC-CMs had longer durations but their frequency was comparable to those of WT cells. Isoprenaline increased the beating frequency that led to arrhythmic beating and cessation of excitability. Our data suggests that S4938F-RyR2 mutation does not significantly affect Ca2+content of SR, but decreases its Ca2+ sensitivity resulting in suppressed ICa-gated and spontaneous Ca2+ release from SR. Supported by NIH grant R01HL153504.

Junctophilin-2 allosterically interacts with ryanodine receptor type 2 to regulate calcium release units in mouse cardiomyocytes.

The interaction between junctophilin-2 (JPH2) and ryanodine receptor type 2 (RyR2) regulated Ca2+ signaling in mouse cardiomyocytes. However, their exact interaction remains unclear. This study elucidates the interaction between JPH2 with RyR2 using co-immunoprecipitation of cardiac sarcoplasmic reticulum vesicles. Additionally, a glutathione S-transferase (GST) pull-down analysis was performed to investigate the physical interaction between RyR2 and JPH2 fragments. JPH2 interacted with RyR2 and the C terminus of the JPH2 protein can pull-down RyR2 receptors. Confocal immunofluorescence imaging indicated that the majority of JPH2 and RyR2 proteins were colocalized near Z-lines in isolated mouse cardiomyocytes. Knockdown of JPH2 reduced the amplitude of Ca2+ transients and disrupted its interaction with RyR2. Therefore, the C-terminus domain of JPH2 is required for interactions with RyR2 in mouse cardiomyocytes, which provides a molecular mechanism for seeking Ca2+-related disease prevention strategies.

A Large-Scale High-Throughput Screen for Modulators of SERCA Activity

The sarco/endoplasmic reticulum Ca-ATPase (SERCA) is a P-type ion pump that transports Ca2+ from the cytosol into the endoplasmic/sarcoplasmic reticulum (ER/SR) in most mammalian cells. It is critically important in muscle, facilitating relaxation and enabling subsequent contraction. Increasing SERCA expression or specific activity can alleviate muscle dysfunction, most notably in the heart, and we seek to develop small-molecule drug candidates that activate SERCA. Therefore, we adapted an NADH-coupled assay, measuring Ca-dependent ATPase activity of SERCA, to high-throughput screening (HTS) format, and screened a 46,000-compound library of diverse chemical scaffolds. This HTS platform yielded numerous hits that reproducibly alter SERCA Ca-ATPase activity, with few false positives. The top 19 activating hits were further tested for effects on both Ca-ATPase and Ca2+ transport, in both cardiac and skeletal SR. Nearly all hits increased Ca2+ uptake in both cardiac and skeletal SR, with some showing isoform specificity. Furthermore, dual analysis of both activities identified compounds with a range of effects on Ca2+-uptake and ATPase, which fit into distinct classifications. Further study will be needed to identify which classifications are best suited for therapeutic use. These results reinforce the need for robust secondary assays and criteria for selection of lead compounds, before undergoing HTS on a larger scale.

Abstract 9703: Cardiac β 3 -Adrenergic Receptor Deficiency Mice Protected from Cardiac Aging: Insights into the Cellular and Molecular Mechanism of Myocardial Aging

Background: We have shown previously that aging-caused declines in cardiac function and β-adrenergic reserve were prevented in β 3 -adrenergic receptor (AR) knockout (β 3 KO) aged mice. The cellular and molecular mechanisms are unclear. We hypothesize that reversal of aging-induced downregulations of cardiac SR Ca 2+ -ATPase (SERCA2a) and β 1 -AR may lead to normalization of myocyte [Ca 2+ ] i regulation, β-AR regulation, and preserving normal myocyte function, thereby preventing aging-induced cardiac dysfunction and eliminating myocardial aging. Methods: We compared protein levels of β 1 - and β 3 -AR, and SERCA2a, myocyte contractile, [Ca 2+ ] i transient ([Ca 2+ ] iT ) and calcium current (I Ca,L ) responses to isoproterenol (ISO, 10 -8 M) in LV myocytes obtained from 2 young (Y) (~6 m) and 2 aged (A) (~28 m) groups (7/group) of wild-type (WT) and β 3 KO mice, respectively. Results: Compared with YWT, AWT myocytes had significantly decreased protein levels of SERCA2a (AWT: 0.24 vs YWT: 0.67), and β 1 -AR (0.35 vs 0.59), but increased β 3 -AR (0.36 vs 0.16) with reduced basal cell contraction (dL/dt max ) (85.3 vs 126.8μm/s), relaxation (dR/dt max ) (66.7 vs 102.1 μm/s), [Ca 2+ ] iT (0.18 vs 0.24), and I Ca,L (4.3 vs 7.6 pA/pF). These changes were accompanied by diminished ISO-stimulated inotropic response. In AWT myocytes, ISO caused significantly less increases in dL/dt max (34% vs 82%), dR/dt max (26% vs 64%), [Ca 2+ ] iT (16% vs 38%) and I Ca,L (15% vs 34%). Versus YWT, Yβ 3 KO did not alter basal myocyte contraction and relaxation and response to ISO stimulation, but had significantly increased protein levels of SERCA2a (1.41 vs 0.61) and unchanged β 1 -AR (0.65 vs 0.62). Aβ 3 KO mice had similar alterations. Importantly, in contrast to AWT, in Aβ 3 KO myocytes, there was increased SERCA2a (1.3) associated with normal basal cell contraction and relaxation, and preserved ISO-stimulated inotropic response. ISO caused similar increases in dL/dt max (Aβ 3 KO: 81% vs Yβ 3 KO: 83%), dR/dt max (68% vs 62%), [Ca 2+ ] iT (31% vs 32%) and I Ca,L (30% vs 32%). Conclusions: β 3 KO prevents aging-caused downregulations of cardiac β 1 -ARs and SERCA2a, leading to the preservation of myocyte function, [Ca 2+ ] iT , I Ca,L and β-adrenergic reserve in aged hearts. Thus, β 3 -AR may provide a new target for cardiac aging.

Synergistic FRET assays for drug discovery targeting RyR2 channels

A key therapeutic target for heart failure and arrhythmia is the deleterious leak through sarcoplasmic reticulum (SR) ryanodine receptor 2 (RyR2) calcium release channels. We have previously developed methods to detect the pathologically leaky state of RyR2 in adult cardiomyocytes by monitoring RyR2 binding to either calmodulin (CaM) or a biosensor peptide (DPc10). Here, we test whether these complementary binding measurements are effective as high-throughput screening (HTS) assays to discover small molecules that target leaky RyR2. Using FRET, we developed and validated HTS procedures under conditions that mimic a pathological state, to screen the library of 1280 pharmaceutically active compounds (LOPAC) for modulators of RyR2 in cardiac SR membrane preparations. Complementary FRET assays with acceptor-labeled CaM and DPc10 were used for Hit prioritization based on the opposing binding properties of CaM vs. DPc10. This approach narrowed the Hit list to one compound, Ro 90–7501, which altered FRET to suggest increased RyR2-CaM binding and decreased DPc10 binding. Follow-up studies revealed that Ro 90–7501 does not detrimentally affect myocyte Ca2+ transients. Moreover, Ro 90–7501 partially inhibits overall Ca2+ leak, as assessed by Ca2+ sparks in permeabilized rat cardiomyocytes. Together, these results demonstrate (1) the effectiveness of our HTS approach where two complementary assays synergize for Hit ranking and (2) a drug discovery process that combines high-throughput, high-precision in vitro structural assays with in situ myocyte assays of the pathologic RyR2 leak. These provide a drug discovery platform compatible with large-scale HTS campaigns, to identify agents that inhibit RyR2 for therapeutic development.

The Cardiac Ryanodine Receptor Provides a Suitable Pathway for the Rapid Transport of Zinc (Zn2+).

The sarcoplasmic reticulum (SR) in cardiac muscle is suggested to act as a dynamic storage for Zn2+ release and reuptake, albeit it is primarily implicated in the Ca2+ signaling required for the cardiac cycle. A large Ca2+ release from the SR is mediated by the cardiac ryanodine receptor (RYR2), and while this has a prominent conductance for Ca2+ in vivo, it also conducts other divalent cations in vitro. Since Zn2+ and permeant Mg2+ have similar physical properties, we tested if the RYR2 channel also conducts Zn2+. Using the method of planar lipid membranes, we evidenced that the RYR2 channel is permeable to Zn2+ with a considerable conductance of 81.1 ± 2.4 pS, which was significantly lower than the values for Ca2+ (127.5 ± 1.8 pS) and Mg2+ (95.3 ± 1.4 pS), obtained under the same asymmetric conditions. Despite similar physical properties, the intrinsic Zn2+ permeability (PCa/PZn = 2.65 ± 0.19) was found to be ~2.3-fold lower than that of Mg2+ (PCa/PMg = 1.146 ± 0.071). Further, we assessed whether the channel itself could be a direct target of the Zn2+ current, having the Zn2+ finger extended into the cytosolic vestibular portion of the permeation pathway. We attempted to displace Zn2+ from the RYR2 Zn2+ finger to induce its structural defects, which are associated with RYR2 dysfunction. Zn2+ chelators were added to the channel cytosolic side or strongly competing cadmium cations (Cd2+) were allowed to permeate the RYR2 channel. Only the Cd2+ current was able to cause the decay of channel activity, presumably as a result of Zn2+ to Cd2+ replacement. Our findings suggest that the RYR2 channel can provide a suitable pathway for rapid Zn2+ escape from the cardiac SR; thus, the channel may play a role in local and/or global Zn2+ signaling in cardiomyocytes.

Effect of hyperhomocysteinemia on rat cardiac sarcoplasmic reticulum.

Increased concentration of plasma homocysteine (Hcy) is an independent risk factor of cardiovascular disease, yet the mechanism by which hyperhomocysteinemia (HHcy) causes cardiac dysfunction is largely unknown. The aim of present study was to investigate the contribution of sarcoplasmic reticulum to impaired cardiac contractile function in HHCy. HHcy-induced by subcutaneous injection of Hcy (0.45μmol/g of body weight) twice a day for a period of 2weeks resulted in significant decrease in developed left ventricular pressure and maximum rate of ventricular relaxation. Our results show that abundances of SR Ca2+-handling proteins, Ca2+-ATPase (SERCA2), calsequestrin and histidine-rich calcium-binding protein are significantly reduced while the content of phospholamban is unchanged. Moreover, we found that increased PLN:SERCA2 ratio results in the inhibition of SERCA2 activity at low free Ca2+ concentrations. We further discovered that HHcy is not associated with increased oxidative stress in SR. Taken together, these findings suggest that disturbances in SR Ca2+ handling, caused by altered protein contents but not oxidative damage, may contribute to impaired cardiac contractility in HHcy.

Stabilizing Ryanodine Receptors Improves Left Ventricular Function in Juvenile Dogs With Duchenne Muscular Dystrophy

BackgroundDuchenne muscular dystrophy is associated with progressive deterioration in left ventricular (LV) function. The golden retriever muscular dystrophy (GRMD) dog model recapitulates the pathology and clinical manifestations of Duchenne muscular dystrophy. Importantly, they develop progressive LV dysfunction starting at early age. ObjectivesThe authors tested the cardioprotective effect of chronic administration of the ARM036, a small molecule that stabilizes the closed conformation of the cardiac sarcoplasmic reticulum ryanodine receptor/calcium release channel (RyR2) in young GRMD-dogs. MethodsTwo-month-old GRMD-dogs were treated with ARM036 or placebo for 4 months. Healthy-dogs of the same genetic background served as controls. Cardiac function was evaluated by conventional and 2-dimensional speckle-tracking echocardiography. Cardiac cellular and molecular analyses were performed at 6 months old. ResultsConventional echocardiography showed normal LV dimensions and ejection fraction in 6-month-old GRMD dogs. Interestingly, 2-dimensional speckle-tracking echocardiography revealed decreased global longitudinal strain and the presence of hypokinetic segments in placebo-treated GRMD dogs. Single-channel measurements revealed higher RyR2 open probability at low resting Ca2+ in GRMD cardiomyocytes than in controls. ARM036 prevented those in vivo and in vitro dysfunctions in GRMD dogs. Myofilament Ca2+-sensitivity was increased in permeabilized GRMD cardiomyocytes at short sarcomere length. ARM036 had no effect on this parameter. Cross-bridge cycling kinetics were altered in GRMD myocytes and recovered with ARM036 treatment, which coincided with the level of myosin binding protein-C-S glutathionylation. ConclusionsGRMD-dogs exhibit early LV dysfunction associated with altered myofilament contractile properties. These abnormalities were prevented pharmacologically by stabilizing RyR2 with ARM036.

Abstract 9024: Molecular Mechanism of Chronic Beta3-Adrenergic Receptor Antagonist Caused Regression of Diabetic Cardiomyopathy in a Murine Model With Type 2 Diabetes

Background: Diabetic cardiomyopathy (DCM) is a serious complication of diabetes (DM). As yet, no effective therapeutic strategies exist. Recently, we found that DM-induced LV dysfunction was prevented by chronic treatment with a β 3 -adrenergic receptor (AR) antagonist (β 3 ANT). However, the molecular mechanism is unclear. We hypothesized that reversal of DM-induced detrimental alterations on the expressions of cardiac SR Ca 2+ -ATPase (SERCA2a), β-ARs, nitric oxide synthase (NOS) by β 3 ANT may play a key role for its salutary role in DCM. Methods: The protein levels of myocyte β 1 - and β 3 - AR, SERCA2a, NOS and myocyte functional responses were determined in 3 groups (7/group) wild-type female mice over 14 weeks (W): Type 2 DM ( T2 ), 14 W fed a high-fat diet (HFD), but after HFD for 4 W receiving streptozotocin (STZ, 40 mg/kg/day, i.p. 5 days); T2β 3 ANT , T2 mice at 10 W received L-748,337, a selective β 3 ANT (10 -7 M/kg/day, mini-pump) for 4 W; and Vehicle controls (C). Results: Versus C, T2DM was induced in mice received HFD and low dose STZ with significantly elevated blood glucose (~340 to 380 mg/dl). Only T2 mice had significantly decreased protein levels of LV myocyte β 1 -AR (T2: 0.35 vs C: 0.49) and SERCA2a (0.19 vs 0.29), but increased β 3 -AR (0.25 vs 0.14) and iNOS (0.25 vs 0.15) without significant changes in eNOS (0.14 vs 0.13) and nNOS. These changes were followed by significantly reduced basal cell contraction (dL/dt max , 75.4 vs 133.7 μm/s), relaxation (dR/dt max , 59.6 vs 113.8 μ m/s) and [Ca 2+ ] iT (0.16 vs 0.21). The isoproterenol (ISO, 10 -8 M)-stimulated increases in dL/dt max (38% vs 58%), dR/dt max (34% vs 55%) and [Ca 2+ ] iT (19% vs 30%) were also significantly reduced. By contrary, versus C, T2β 3 ANT myocytes had similar protein levels of β 1 -AR (0.52), SERCA2a (0.75) and iNOS (0.36) with preserved normal basal cell contractility (127.8 μm/s), relaxation (109.4 μm/s) and [Ca 2+ ] iT (0.21). ISO caused increases in dL/dt max (57%), dL/dt max (52%), and [Ca 2+ ] iT (30%) were also closed to control values. Conclusions: Chronic β 3 ANT prevents DCM-caused downregulations of cardiac β 1 -ARs and SERCA2a, and normalized upregulations of iNOS and β 3 -AR, thus leading to the preservation of normal myocyte contractile function, [Ca 2+ ] iT , and β-adrenergic reserve in a mouse model of T2DM.

Simulating cardiac Ca2+ release units: effects of RyR cluster size and Ca2+ buffers on diastolic Ca2+ leak.

Leak of Ca2+ out of the cardiac sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) during diastole is vital to regulate SR Ca2+ levels. This leak can become deleterious when large spontaneous RyR-mediated Ca2+ release events evoke proarrhythmic Ca2+ waves that can lead to delayed after-depolarizations. Here, we model diastolic SR Ca2+ leak at individual SR Ca2+ release sites using computer simulations of RyR arrays like those in the dyadic cleft. The results show that RyR arrays size has a significant effect on SR Ca2+ leak, with bigger arrays producing larger and more frequent Ca2+ release events. Moreover, big RyR arrays are more susceptible to small changes in the levels of dyadic Ca2+ buffers. Such changes in buffering shift Ca2+ leak from small Ca2+ release events (involving few open RyRs) to larger events (with many open RyRs). Moreover, by analyzing a large parameter space of possible buffering and SR Ca2+ loads, we find further evidence for the hypothesis that SR Ca2+ leak by RyR arrays can undergo a sudden phase transition.

High-Throughput Screening and Drug Development to Fix RyR Pathology

Using fluorescence lifetime FRET-based high-throughput screening (HTS) assays, we identified compounds that modulate the sarcoplasmic reticulum's (SR) Ca2+ gatekeeper, the ryanodine receptor (RyR) channel. Intracellular Ca2+ regulation is critical for striated muscle function, and SR Ca2+ release via opening of RyR is essential for triggering muscle contraction. Under cellular rest, increased propensity of channel opening due to RyR dysregulation is associated with severe cardiac and skeletal myopathies, diabetes and neurological disorders. Thus, this leaky state of the RyR is a high-value pharmacological target for treating such pathologies. Our FRET-based HTS detects the RyR leaky state by monitoring binding of the accessory proteins calmodulin, FKBP12.0, FKBP12.6, and DPc10. Under conditions that mimic a pathological state, we carried out screens of a 50,000-compound library, to identify modulators of RyR in either skeletal or cardiac SR membrane preparations. These screens yielded >60 compounds (Hits) that reproducibly altered FRET. Following functional validation, the resulting 37 Hits fall within 10 chemical scaffold groups that are specific for either RyR1 (skeletal isoform) or RyR2 (cardiac isoform), or that act on both RyR1 and RyR2. With the first round of Hit analogue testing, we are pursuing >80 compounds to further inform medicinal chemistry synthesis of more potent RyR stabilizers. Good candidate compounds for treatment of striated muscle myopathies will progress to testing on RyR function in skeletal muscle fibers and adult ventricular myocytes. This work is supported by NIH grants R01HL092097 and R01HL138539 (RLC/DMB) and R37AG026160 (DDT).

Nano-scale morphology of cardiomyocyte t-tubule/sarcoplasmic reticulum junctions revealed by ultra-rapid high-pressure freezing and electron tomography

Detailed knowledge of the ultrastructure of intracellular compartments is a prerequisite for our understanding of how cells function. In cardiac muscle cells, close apposition of transverse (t)-tubule (TT) and sarcoplasmic reticulum (SR) membranes supports stable high-gain excitation-contraction coupling. Here, the fine structure of this key intracellular element is examined in rabbit and mouse ventricular cardiomyocytes, using ultra-rapid high-pressure freezing (HPF, omitting aldehyde fixation) and electron microscopy. 3D electron tomograms were used to quantify the dimensions of TT, terminal cisternae of the SR, and the space between SR and TT membranes (dyadic cleft). In comparison to conventional aldehyde-based chemical sample fixation, HPF-preserved samples of both species show considerably more voluminous SR terminal cisternae, both in absolute dimensions and in terms of junctional SR to TT volume ratio. In rabbit cardiomyocytes, the average dyadic cleft surface area of HPF and chemically fixed myocytes did not differ, but cleft volume was significantly smaller in HPF samples than in conventionally fixed tissue; in murine cardiomyocytes, the dyadic cleft surface area was higher in HPF samples with no difference in cleft volume. In both species, the apposition of the TT and SR membranes in the dyad was more likely to be closer than 10 nm in HPF samples compared to CFD, presumably resulting from avoidance of sample shrinkage associated with conventional fixation techniques. Overall, we provide a note of caution regarding quantitative interpretation of chemically-fixed ultrastructures, and offer novel insight into cardiac TT and SR ultrastructure with relevance for our understanding of cardiac physiology.

Abstract 13385: Protective Effect of Beta3-Adrenergic Receptor Deficiency on Diabetic Cardiomyopathy: Reversal of Type 2 Diabetes-Induced Contrast Changes of Cardiac SERCA 2a and Inducible Nitric Oxide Synthase

Background: We have shown that diabetes (DM)-induced cardiac dysfunction and β-adrenergic desensitization were prevented in β 3 -adrenergic receptor (AR) knockout (β 3 KO) mice. However, the molecular mechanism is unclear. We hypothesize that reversal of type 2 (T2) DM-induced alterations of cardiac SR Ca 2+ -ATPase (SERCA2a) and inducible nitric oxide synthase (iNOS) by β 3 KO may play a key role for the protective effect. Methods: LV myocyte protein levels of β 1 - and β 3 -AR, SERCA2a and iNOS, myocyte contractile and [Ca 2+ ] i transient ([Ca 2+ ] iT ) responses to isoproterenol (ISO, 10 -8 M) were compared in 4 groups (n=6) female mice: 2 Vehicle controls wild-type (CWT) and Cβ 3 KO; and 2 T2DM of T2WT and T2β 3 KO.T2 was induced by fed high-fat diet (HFD) for 14 weeks (W), but after HFD for 4 W receiving streptozotocin (STZ, 40 mg/kg/day, i.p. for 5 days). Results: Mice-treated with HFD plus STZ had high blood glucose (~350 to 380 mg/dl). Versus CWT, T2WT myocytes had significantly decreased protein levels of SERCA2a (0.18 vs 0.29), and β 1 -AR (0.35 vs 0.49), but increased iNOS (0.25 vs 0.15) and β 3 -AR (0.25 vs 0.14) with reduced cell contraction (dL/dt max ) (72.7 vs 140.9 μm/s), relaxation (dR/dt max ) (50.9 vs 117.3 μm/s) and [Ca 2+ ] iT (0.16 vs 0.22). Moreover, in T2WT myocytes, ISO caused significantly less increases in dL/dt max (37% vs 59%), dR/dt max (30% vs 53%) and [Ca 2+ ] iT (19% vs 30%). Versus CWT, T2β 3 KO did not alter basal myocyte contraction and relaxation and response to ISO stimulation, but had significantly increased protein levels of SERCA2a (0.38 vs 0.29). T2β 3 KO prevented DM-caused increase in iNOS (T2β 3 KO: 0.16 vs CWT: 0.15) and decrease in β 1 -AR (0.46 vs 0.49). Versus T2β 3 KO, Cβ 3 KO mice had similar alterations. Importantly, in contrast to T2WT, in T2β 3 KO myocytes, the increased SERCA2a and reduced iNOS correlated with normal basal cell contraction and relaxation with preserved ISO-stimulated inotropic response. ISO caused similar increases in dL/dt max (60% vs 59%), dR/dt max (51% vs 53%) and [Ca 2+ ] iT (32% vs 30%). Conclusions: β 3 KO prevents T2DM-caused downregulation of cardiac β 1 -ARs and reverses increased iNOS and decreased SERCA2a, leading to the preservation of myocyte function, [Ca 2+ ] iT , and β-adrenergic reserve. Thus, β 3 -AR may provide a new target for DCM.