Sarcoplasmic Reticulum Microsomes Research Articles

SERCA Inhibition as a Potential Therapeutic Target for Breast Cancer

Chemotherapy is both a palliative treatment for many cancers and adjuvant treatment before, during, or after local treatment (surgery and/or radiation therapy) in single or combination regimens. Despite current treatments, approximately 1 in 39 women (3%) still die from breast cancer. A major complication with systemic therapy is drug resistance which limits the efficacy of clinical chemotherapy. Hence, current research aims to uncover novel targets for breast cancer therapy. Various studies have shown altered calcium signaling in breast cancer cells, therefore, calcium modulators may be potential therapeutic targets. Studies have also shown that Thapsigargin (TG) and cyclopiazonic acid (CPZ), two potent and specific inhibitors of the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA), can impair cancer cell growth. However, TG and CPZ also impair non-malignant cells, which results in significant toxicity, thus preventing use in chemotherapy. Using spectrometry UV-visible measurements to assess calcium loading and ATPase activity in SR microsomes, our lab has found that various drugs which display cell protective effects in ischemia also inhibit SERCA. They include novel benzothiazepines (BZTs; K201, CGP37157, PH00095) and have shown to inhibit SERCA in a calcium dependent mechanism. They decreased SR uptake when cytosolic Ca2+ levels ([Ca2+]c) are low, similar to competitive inhibition of Ca2+ uptake, which produces mild store depletion. Contrarily, TG and CPZ block Ca2+ uptake at all [Ca2+]c and largely deplete SR stores. Using various SERCA inhibitors, we sought out to determine their effects on breast cancer cell lines of different subtypes. MTS assay studies show that TG and CPZ significantly reduce cell viability in MCF7 and MCF10A cell lines while having no significant effects on triple negative breast cancer (TNBC) cell line MDAMB231. Preliminary studies also show no significant effect by TG on BT20 cells, another TNBC cell line. BZT K201 also significantly reduced cell viability in MCF7 cells but not MDAMB231. Immunoblot shows MCF7 cells have significantly higher SERCA2 expression than MDAMB231. We observe no significant differences in plasma membrane calcium channel ATPase (PMCA) protein expression. Preliminary RT-PCR studies estimate MCF7 cells have a higher fold change in SERCA genes. Thus, targeting SERCA may serve as a potential therapeutic target for breast cancer. Here, we find that SERCA inhibitors TG, CPZ, and BZT K201 reduce cell viability in MCF7 cells. Still, these studies indicate that SERCA inhibition may only be efficacious in certain breast cancer cell types which do not express a TNBC phenotype. TNBC cell lines (MDAMB231, BT20) are not significantly affected by SERCA inhibition, which may be due to higher dependence for signaling on plasmalemma calcium channels and transporters. Additional studies are required to confirm the expression profiles of calcium transporters among cell lines, including those in the plasmalemma (PMCA, calcium channels), which may help to understand if alternative sources of Ca2+ for intracellular signaling make TNBC cell lines more resistant to SERCA inhibitors.

Orthograde signal of dihydropyridine receptor increases Ca2+ leakage after repeated contractions in rat fast-twitch muscles in vivo.

The purpose of this study was to investigate the mechanism underlying sarcoplasmic reticulum (SR) Ca2+ leakage after in vivo contractions. Rat gastrocnemius muscles were electrically stimulated in vivo, and then mechanically skinned fibers and SR microsomes were prepared from the muscles excised 30 min after repeated high-intensity contractions. The mechanically skinned fibers maintained the interaction between dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs), whereas the SR microsomes did not. Interestingly, skinned fibers from the stimulated muscles showed increased SR Ca2+ leakage, whereas Ca2+ leakage decreased in SR microsomes from the stimulated muscles. To enhance the orthograde signal of DHPRs, SR Ca2+ leakage in the skinned fiber was measured 1) under a continuously depolarized condition and 2) in the presence of nifedipine. As a result, in either of the two conditions, SR Ca2+ leakage in the rested fibers reached a level similar to that in the stimulated fibers. Furthermore, the increased SR Ca2+ leakage from the stimulated fibers was alleviated by treatment with 1 mM tetracaine (Tet) but not by treatment with 3 mM free Mg2+ (3 Mg). Tet exerted a greater inhibitory effect on the DHPR signal to RyR than 3 Mg, although their inhibitory effects on RyR were almost similar. These results suggest that the increased Ca2+ leakage after muscle contractions is mainly caused by the orthograde signal of DHPRs to RyRs.

Pharmacological Targeting of Serca May Have Potential for Cellular Protection

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

HAX-1 regulates SERCA2a oxidation and degradation

Ischemia/reperfusion injury is associated with contractile dysfunction and increased cardiomyocyte death. Overexpression of the hematopoietic lineage substrate-1-associated protein X-1 (HAX-1) has been shown to protect from cellular injury but the function of endogenous HAX-1 remains obscure due to early lethality of the knockout mouse. Herein we generated a cardiac-specific and inducible HAX-1 deficient model, which uncovered an unexpected role of HAX-1 in regulation of sarco/endoplasmic reticulum Ca-ATPase (SERCA2a) in ischemia/reperfusion injury. Although ablation of HAX-1 in the adult heart elicited no morphological alterations under non-stress conditions, it diminished contractile recovery and increased infarct size upon ischemia/reperfusion injury. These detrimental effects were associated with increased loss of SERCA2a. Enhanced SERCA2a degradation was not due to alterations in calpain and calpastatin levels or calpain activity. Conversely, HAX-1 overexpression improved contractile recovery and maintained SERCA2a levels. The regulatory effects of HAX-1 on SERCA2a degradation were observed at multiple levels, including intact hearts, isolated cardiomyocytes and sarcoplasmic reticulum microsomes. Mechanistically, HAX-1 ablation elicited increased production of reactive oxygen species at the sarco/endoplasic reticulum compartment, resulting in SERCA2a oxidation and a predisposition to its proteolysis. This effect may be mediated by NAPDH oxidase 4 (NOX4), a novel binding partner of HAX-1. Accordingly, NOX inhibition with apocynin abrogated the effects of HAX-1 ablation in hearts subjected to ischemia/reperfusion injury. Taken together, our findings reveal a role of HAX-1 in the regulation of oxidative stress and SERCA2a degradation, implicating its importance in calcium homeostasis and cell survival pathways.

K201 (JTV519) is a Ca2+-Dependent Blocker of SERCA and a Partial Agonist of Ryanodine Receptors in Striated Muscle.

K201 (JTV-519) may prevent abnormal Ca(2+) leak from the sarcoplasmic reticulum (SR) in the ischemic heart and skeletal muscle (SkM) by stabilizing the ryanodine receptors (RyRs; RyR1 and RyR2, respectively). We tested direct modulation of the SR Ca(2+)-stimulated ATPase (SERCA) and RyRs by K201. In isolated cardiac and SkM SR microsomes, K201 slowed the rate of SR Ca(2+) loading, suggesting potential SERCA block and/or RyR agonism. K201 displayed Ca(2+)-dependent inhibition of SERCA-dependent ATPase activity, which was measured in microsomes incubated with 200, 2, and 0.25 µM Ca(2+) and with the half-maximal K201 inhibitory doses (IC50) estimated at 130, 19, and 9 µM (cardiac muscle) and 104, 13, and 5 µM (SkM SR). K201 (≥5 µM) increased RyR1-mediated Ca(2+) release from SkM microsomes. Maximal K201 doses at 80 µM produced ∼37% of the increase in SkM SR Ca(2+) release observed with the RyR agonist caffeine. K201 (≥5 µM) increased the open probability (Po) of very active ("high-activity") RyR1 of SkM reconstituted into bilayers, but it had no effect on "low-activity" channels. Likewise, K201 activated cardiac RyR2 under systolic Ca(2+) conditions (∼5 µM; channels at Po ∼0.3) but not under diastolic Ca(2+) conditions (∼100 nM; Po < 0.01). Thus, K201-induced the inhibition of SR Ca(2+) leak found in cell-system studies may relate to potentially potent SERCA block under resting Ca(2+) conditions. SERCA block likely produces mild SR depletion in normal conditions but could prevent SR Ca(2+) overload under pathologic conditions, thus precluding abnormal RyR-mediated Ca(2+) release.

DHBP Block of Ryanodine Receptor Channels

1,1′-diheptyl-4,4′-bipyridinium dibromide (DHBP), a divalent organic cation, was reported to inhibit muscle contraction, which was attributed to inhibition of ryanodine receptor (RyR) mediated Ca2+ release from the sarcoplasmic reticulum (SR). Still, single RyR channel studies are lacking and the mechanism by which DHBP blocks RyRs is unknown. We reconstituted RyRs from skeletal muscle and heart SR microsomes into planar bilayers. We observed with DHBP addition to the cytosolic surface of RyRs (clamped at Vm = 0 mV) decreased channel conductance (IC50 ∼ 1 mM). The percentage block in channel conductance elicited by DHBP was invariable in the range −30 to +30 mV. DHBP did not affect RyR gating characteristics, which was measured under Ca2+ conditions that produced low, moderate or high Po and in the presence/absence of physiological modulators (ATP, Mg2+). DHBP added to luminal and cytosolic sides of the RyRs, resulted in additive effects on channel conductance. DHBP also induced inhibition of RyR-mediated SR Ca2+ release from skeletal SR microsomes. A similar block of RyRs conductance was observed with other bipyridinum derivatives, none of which affected SERCA ATPase activity. Previous reports in the literature indicated block of RyRs by DHBP in cells required lower drug concentrations, suggesting a possible cellular mechanism of DHBP accumulation. In summary, DHBP blocks RyRs Ca2+ conductance resembling the action of polyamines and organic cations. DHBP properties make it an attractive experimental probe for understanding the role of RyR conductance in Ca2+ release from intracellular Ca2+ stores (Supported by American Heart Association and Eskridge Foundation).

Eudistomin D and penaresin derivatives as modulators of ryanodine receptor channels and sarcoplasmic reticulum Ca2+ ATPase in striated muscle.

Eudistomin D (EuD) and penaresin (Pen) derivatives are bioactive alkaloids from marine sponges found to induce Ca(2+) release from striated muscle sarcoplasmic reticulum (SR). Although these alkaloids are believed to affect ryanodine receptor (RyR) gating in a "caffeine-like" manner, no single-channel study confirmed this assumption. Here, EuD and MBED (9-methyl-7-bromoeudistomin D) were contrasted against caffeine on their ability to modulate the SR Ca(2+) loading/leak from cardiac and skeletal muscle SR microsomes as well as the function of RyRs in planar bilayers. The effects of these alkaloids on [(3)H]ryanodine binding and SR Ca(2+) ATPase (SERCA) activity were also tested. MBED (1-5 μM) fully mimicked maximal activating effects of caffeine (20 mM) on SR Ca(2+) leak. At the single-channel level, MBED mimicked the agonistic action of caffeine on cardiac RyR gating (i.e., stabilized long openings characteristic of "high-open-probability" mode). EuD was a partial agonist at the maximal doses tested. The tested Pen derivatives displayed mild to no agonism on RyRs, SR Ca(2+) leak, or [(3)H]ryanodine binding studies. Unlike caffeine, EuD and some Pen derivatives significantly inhibited SERCA at concentrations required to modulate RyRs. Instead, MBED's affinity for RyRs (EC50 ∼ 0.5 μM) was much larger than for SERCA (IC50 > 285 μM). In conclusion, MBED is a potent RyR agonist and, potentially, a better choice than caffeine for microsomal and cell studies due to its reported lack of effects on adenosine receptors and phosphodiesterases. As a high-affinity caffeine-like probe, MBED could also help identify the caffeine-binding site in RyRs.

Gingerol discharges NG115–401L neural cell Ca2+ stores, activates Ca2+ influx and promotes cell death

Gingerol's effect on sarco/endoplasmic reticulum Ca2+‐ ATPase (SERCA) and cytosolic free Ca2+ ([Ca2+]i) was tested on sarcoplasmic reticulum (SR) microsomes and NG115–401L, a neural cell line with diminished level of stromal interacting molecule 1 (STIM1) and deficient endoplasmic reticulum (ER)− regulated Ca2+ signaling pathways. Cells were loaded with the Ca2+ probe fura‐2 to detect [Ca2+]i changes and SR microsomes were prepared to measure Ca2+‐ATPase activity. Gingerol stimulates an approximately two‐fold increase in ATP hydrolysis in skeletal muscle SR microsomes. Moreover, gingerol incubation nearly completely reverses SERCA enzymatic function against SERCA blocker cyclopiazonic acid (CPA). However, in 401L cells, gingerol fails to enhance ER Ca2+ uptake, but induces a transient [Ca2+]i elevation in the absence of external Ca2+ much like thapsigargin (TG) and CPA. Additionally, gingerol is unable to protect 401L cells against CPA‐induced apoptosis, confirming that the compound has no ER Ca2+ enhancing effect in 401L cells. Surprisingly, gingerol's apparent ER Ca2+ depleting action couples to long‐lived Ca2+ influx pathways, whereas those of TG and CPA do not. These results argue for a finer regulatory control on SERCA function with gingerol actions revealing potentially novel routes of coupling altered pump regulation to the assembly of functional Ca2+ influx units. Research supported by University of the Pacific

Benzodiazepines and Benzothiazepines as Modulators of the Sarcoplasmic Reticulum Calcium ATP-ase and Ryanodine Receptors in Striated Muscle

We have reported that CGP-37157, a benzothiazepine (BZT) derivative of clonazepam utilized as a blocker of the mitochondrial Na+/Ca2+ exchanger, also activates ryanodine receptors (RyRs) and inhibits the sarcoplasmic reticulum (SR) Ca2+-stimulated ATPase (SERCA). We extended the studies to other BZT (e.g., clonazepan, diltiazem) as well as to benzodiazepines (BZD; e.g. diazepan, lorazepan). We aimed to determine if these drug classes have as a common trait the ability to modulate RyRs and/or SERCA. The effects of BZD and BZT on RyRs activity were tested in SR microsomes with a Ca2+ leak assay as well as after reconstituting RyRs into lipid bilayers. The agents tested had variable potency to increase RyR-mediated Ca2+ leak from skeletal SR microsomes. As an example, while diazepam significantly increased RyR activity, most of the others (clonazepam, lorazepam) had minor effects at high doses. In contrast, diltiazem produced moderate inhibition. Planar bilayer studies confirmed the leak observations with both cardiac and skeletal RyR. In the presence of ruthenium red, most agents decreased the rate of loading which indicates inhibitory effects on SERCA activity. The effects of BZT and BZD on loading correlated with a decrease in ATPase activity of SERCA-enriched skeletal SR fractions. In summary, most BZT and BZD utilized in therapeutics or as pharmacological tools have an inhibitory action on SERCA. In contrast, they show a variety of effects on RyRs ranging from inhibition to activation. Hence, the pharmacological action of BZT and BZD on cellular Ca2+ homeostasis reported in the literature of cardiac and skeletal muscle as well as other non-muscle systems may require taking into consideration the contributions of all drug-sensitive intracellular Ca2+ transporters. (Supported by NIH-GM078665 and AHA-MWA 12180038).

Cross-Reactivity of Ryanodine Receptors with Plasma Membrane Ion Channel Modulators

Various pharmacological agents designed to modulate plasma membrane ion channels seem to significantly affect intracellular Ca²⁺ signaling when acting on their target receptor. Some agents could also cross-react (modulate channels or receptors beyond their putative target) with intracellular Ca²⁺ transporters. This study investigated the potential of thirty putative modulators of either plasma membrane K⁺, Na⁺, or transient receptor potential (TRP) channels to cross-react with intracellular Ca²⁺ release channels [i.e., ryanodine receptors (RyRs)] from skeletal muscle sarcoplasmic reticulum (SR). Screening for cross-reactivity of these various agents was performed by measuring the rate of spontaneous Ca²⁺ leak or caffeine-induced Ca²⁺ release from SR microsomes. Four of the agents displayed a strong cross-reactivity and were further evaluated with skeletal RyR (RyR1) reconstituted into planar bilayers. 6,12,19,20,25,26-Hexahydro-5,27:13,18:21,24-trietheno-11,7-metheno-7H-dibenzo [b,n][1,5,12,16]tetraazacyclotricosine-5, 13-diium dibromide (UCL 1684; K⁺ channel antagonist) and lamotrigine (Na⁺ channel antagonist) were found to significantly inhibit the RyR1-mediated caffeine-induced Ca²⁺ release. TRP channel agonists anandamide and (-)menthol were found to inhibit and activate RyR1, respectively. High concentrations of nine other agents produced partial inhibition of RyR1-mediated Ca²⁺ release from SR microsomes. Various pharmacological agents, especially TRP modulators, also inhibited a minor RyR1-independent component of the SR Ca²⁺ leak. Overall, ∼43% of the agents selected cross-reacted with RyR1-mediated and/or RyR1-independent Ca²⁺ leak from intracellular stores. Thus, cross-reactivity should be considered when using these classes of pharmacological agents to determine the role of plasmalemmal channels in Ca²⁺ homeostasis.

CGP37157 is an Inhibitor of the Sarcoplasmic Reticulum Calcium ATPase and an Activator of Ryanodine Receptors in Striated Muscle

CGP-37157 (CGP), a benzothiazepine derivative of clonazepam, is commonly utilized as a blocker of the mitochondrial Na+/Ca2+ exchanger. Yet, evidence suggests that CGP could also affect other targets, such as L-type Ca2+ channels and plasmalemma Na+/Ca2+ exchanger. Here, we tested the possibility of a direct modulation of ryanodine receptors (RyR) and/or sarcoplasmic reticulum (SR) Ca2+ stimulated ATPase (SERCA) by CGP. SERCA activity was measured in SR microsomes with a ATP-ase activity assay or Ca2+ loading in presence of ruthenium red (RyR blocker). The effects of CGP on RyR activity were performed in SR microsomes with a Ca2+ leak assay or after reconstitution of the channels into planar lipid bilayers. CGP inhibited SERCA-mediated Ca2+ uptake of cardiac and skeletal SR microsomes (IC50's = 6.6 and 9.9 µM, respectively). The CGP effects on SERCA activity correlated with a decreased Vmax of ATPase activity of SERCA-enriched skeletal SR fractions without an apparent change in Km. CGP (≥ 5 µM) also increased RyR-mediated Ca2+ leak from skeletal SR microsomes. Planar bilayer studies confirmed that both cardiac and skeletal RyR are directly activated by CGP (EC50's = 9.4 and 12.0 μM, respectively). In summary, we found that CPG inhibits SERCA and activates RyR channels. Hence, the action of CGP on cellular Ca2+ homeostasis reported in the literature of cardiac, skeletal muscle as well as other non muscle systems requires further analysis to take into account the contribution of all CGP-sensitive Ca2+ transporters. (Supported by NIH R01 GM078665).

Functional Properties of SR Cl- and K+ Channels during Postnatal Development of Cardiac Muscle

In adult (AD) heart, the sarcoplasmic reticulum (SR) contains Cl- & K+ channels presumably involved in controlling RyR-mediated SR Ca2+ release. These channels provide a countercurrent mechanism that attenuates the drop in Ca2+ driving force across the SR membrane, thereby preventing early termination of Ca2+ release. We showed that in newborn (NB), Ca2+ sparks occur with similar frequency than in AD but have shorter duration and smaller amplitude, implying an early termination of Ca2+ release. Although the functional properties of SR Cl- & K+ channels have been thoroughly described in AD, little is known about their presence and their role in NB. Consequently, we first tested the hypothesis that the early termination of Ca2+ release in NB coincides with absence/low density of SR Cl- & K+ channels at this stage. To this end, the heavy microsomal fraction was obtained from 5-days-old NB and AD rat hearts and SR Cl- & K+ channels were reconstituted into artificial planar lipid bilayers. Our results indicate that Cl- & K+ channels can be reconstituted from NB heavy SR microsomes with a similar success rate (number of SR channel incorporations / total number of bilayers) than in AD (∼0.2 for Cl- channels & ∼0.1 for K+ channels). Thus, an alternative mechanism would imply that in NB, smaller counterion fluxes result from different functional properties of SR Cl- & K+ channels. This assumption was tested by measuring their unitary conductance, open probability, and voltage dependence. The results in NB channels revealed no significant differences in any of these parameters in comparison to AD. Thus, we concluded that SR Cl- & K+ channels do not contribute to the developmental changes of Ca2+ release in NB cardiomyocytes. Supported by AHA-0655656Z to RMA.

Voltage-Dependent Modulation of Cardiac Ryanodine Receptors (RyR2) by Protamine

It has been reported that protamine (>10 µg/ml) blocks single skeletal RyR1 channels and inhibits RyR1-mediated Ca2+ release from sarcoplasmic reticulum microsomes. We extended these studies to cardiac RyR2 reconstituted into planar lipid bilayers. We found that protamine (0.02–20 µg/ml) added to the cytosolic surface of fully activated RyR2 affected channel activity in a voltage-dependent manner. At membrane voltage (Vm; SR lumen - cytosol) = 0 mV, protamine induced conductance transitions to several intermediate states (substates) as well as full block of RyR2. At Vm>10 mV, the substate with the highest level of conductance was predominant. Increasing Vm from 0 to +80 mV, decreased the number of transitions and residence of the channel in this substate. The drop in current amplitude (full opening to substate) had the same magnitude at 0 and +80 mV despite the ∼3-fold increase in amplitude of the full opening. This is more similar to rectification of channel conductance induced by other polycations than to the action of selective conductance modifiers (ryanoids, imperatoxin). A distinctive effect of protamine (which might be shared with polylysines and histones but not with non-peptidic polycations) is the activation of RyR2 in the presence of nanomolar cytosolic Ca2+ and millimolar Mg2+ levels. Our results suggest that RyRs would be subject to dual modulation (activation and block) by polycationic domains of neighboring proteins via electrostatic interactions. Understanding these interactions could be important as such anomalies may be associated with the increased RyR2-mediated Ca2+ leak observed in cardiac diseases.

Dissociation of FKBP12.6 from ryanodine receptor type 2 is regulated by cyclic ADP-ribose but not β-adrenergic stimulation in mouse cardiomyocytes

Beta-adrenergic augmentation of Ca(2+) sparks and cardiac contractility has been functionally linked to phosphorylation-dependent dissociation of FK506 binding protein 12.6 (FKBP12.6) regulatory proteins from ryanodine receptors subtype 2 (RYR2). We used FKBP12.6 null mice to test the extent to which the dissociation of FKBP12.6 affects Ca(2+) sparks and mediates the inotropic action of isoproterenol (ISO), and to investigate the underlying mechanisms of cyclic ADP-ribose (cADPR) regulation of Ca(2+) sparks. Ca(2+) sparks and contractility were measured in cardiomyocytes and papillary muscle segments from FKBP12.6 null mice, and western blot analysis was carried out on sarcoplasmic reticulum microsomes prepared from mouse heart. Exposure to ISO resulted in a three- and two-fold increase in Ca(2+) spark frequency in wild-type (WT) and FKBP12.6 knockout (KO) myocytes, respectively, and Ca(2+) spark kinetics were also significantly altered in both types of cells. The effects of ISO on Ca(2+) spark properties in KO cells were inhibited by pre-treatment with thapsigargin or phospholamban inhibitory antibody, 2D12. Moreover, twitch force magnitude and the rate of force development were not significantly different in papillary muscles from WT and KO mice. Unlike beta-adrenergic stimulation, cADPR stimulation increased Ca(2+) spark frequency (2.8-fold) and altered spark kinetics only in WT but not in KO mice. The effect of cADPR on spark properties was not entirely blocked by pre-treatment with thapsigargin or 2D12. In voltage-clamped cells, cADPR increased the peak Ca(2+) of the spark without altering the decay time. We also noticed that basal Ca(2+) spark properties in KO mice were markedly altered compared with those in WT mice. Our data demonstrate that dissociation of FKBP12.6 from the RYR2 complex does not play a significant role in beta-adrenergic-stimulated Ca(2+) release in heart cells, whereas this mechanism does underlie the action of cADPR.

Tricyclic antidepressant amitriptyline alters sarcoplasmic reticulum calcium handling in ventricular myocytes

Tricyclic antidepressants such as amitriptyline (AMT) have been reported to have adverse side effects on cardiac performance. AMT effects on Ca handling in ventricular myocytes, however, are not well understood. Therefore, we investigated AMT action on sarcoplasmic reticulum (SR) Ca release in ventricular myocytes, ryanodine receptor (RyR) activity, and Ca uptake by SR microsomes. In permeabilized myocytes, AMT transiently increased free luminal Ca concentration ([Ca]) followed by marked depletion. AMT (10 microM) caused a rapid and a transient increase of Ca spark frequency, followed by a significant suppression of spark activity. The latter was associated with a decrease of Ca spark amplitude and SR Ca load to 87 and 60%, respectively. AMT (10 microM) completely abolished propagation of spontaneous Ca waves. Higher concentrations of AMT (0.1-1 mM) evoked SR Ca release reminiscent of the effect of caffeine (20 mM) and caused almost complete depletion of SR Ca content. Studies on single calsequestrin-free RyR channels revealed that AMT increased the mean open time and open probability (Po) in a dose-dependent fashion (dissociation constant = 4.2 microM). High concentrations of AMT (> 25 microM) evoked frequent long openings with Po reaching very high levels (> 0.70). In studies with cardiac SR microsomes, AMT slowed the rate of ATP-dependent Ca uptake. We conclude that AMT affects SR Ca handling in ventricular myocytes by multiple mechanisms, including direct stimulation of RyRs and inhibition of SR Ca uptake. These effects could contribute to AMT cardiotoxicity.

Reduced Threshold for Luminal Ca2+ Activation of RyR1 Underlies a Causal Mechanism of Porcine Malignant Hyperthermia

Naturally occurring mutations in the skeletal muscle Ca(2+) release channel/ryanodine receptor RyR1 are linked to malignant hyperthermia (MH), a life-threatening complication of general anesthesia. Although it has long been recognized that MH results from uncontrolled or spontaneous Ca(2+) release from the sarcoplasmic reticulum, how MH RyR1 mutations render the sarcoplasmic reticulum susceptible to volatile anesthetic-induced spontaneous Ca(2+) release is unclear. Here we investigated the impact of the porcine MH mutation, R615C, the human equivalent of which also causes MH, on the intrinsic properties of the RyR1 channel and the propensity for spontaneous Ca(2+) release during store Ca(2+) overload, a process we refer to as store overload-induced Ca(2+) release (SOICR). Single channel analyses revealed that the R615C mutation markedly enhanced the luminal Ca(2+) activation of RyR1. Moreover, HEK293 cells expressing the R615C mutant displayed a reduced threshold for SOICR compared with cells expressing wild type RyR1. Furthermore, the MH-triggering agent, halothane, potentiated the response of RyR1 to luminal Ca(2+) and SOICR. Conversely, dantrolene, an effective treatment for MH, suppressed SOICR in HEK293 cells expressing the R615C mutant, but not in cells expressing an RyR2 mutant. These data suggest that the R615C mutation confers MH susceptibility by reducing the threshold for luminal Ca(2+) activation and SOICR, whereas volatile anesthetics trigger MH by further reducing the threshold, and dantrolene suppresses MH by increasing the SOICR threshold. Together, our data support a view in which altered luminal Ca(2+) regulation of RyR1 represents a primary causal mechanism of MH.

Ginsenoside Re suppresses electromechanical alternans in cat and human cardiomyocytes

Ginseng botanicals are increasingly used as complementary or alternative medicines for a variety of cardiovascular diseases, yet little is known about their cellular actions in cardiac muscle. Electromechanical alternans (EMA) is a proarrhythmic cardiac abnormality that results from disturbances of intracellular Ca(2+) homeostasis. This study sought to determine whether a purified ginsenoside extract of ginseng, Re, exerts effects to suppress EMA and to gain insight into its mechanism of action. Alternans was induced by electrically pacing cardiomyocytes at room temperature. Re (> or = 10 nM) reversibly suppressed EMA recorded from cat ventricular and atrial myocytes and Langendorff-perfused cat hearts. In cat ventricular myocytes, Re reversibly suppressed intracellular Ca(2+) concentration ([Ca(2+)](i)) transient alternans. Re exerted no significant effects on baseline action potential configuration or sarcolemmal L-type Ca(2+) current (I(Ca,L)), Na(+) current, or total K(+) conductance. In human atrial myocytes, Re suppressed mechanical alternans and exerted no effect on I(Ca,L). In cat ventricular myocytes, Re increased [Ca(2+)](i) transient amplitude and decreased sarcoplasmic reticulum (SR) Ca(2+) content, resulting in an increase in fractional SR Ca(2+) release. In SR microsomes isolated from cat ventricles, Re had no effect on SR Ca(2+) uptake. Re increased the open probability of ryanodine receptors (RyRs), i.e., SR Ca(2+)-release channels, isolated from cat ventricles and incorporated into planar lipid bilayers. We concluded that ginsenoside Re suppresses EMA in cat atrial and ventricular myocytes, cat ventricular muscle, and human atrial myocytes. The effects of Re are not mediated via actions on sarcolemmal ion channels or action potential configuration. Re acts via a subcellular mechanism to enhance the opening of RyRs and thereby overcome the impaired SR Ca(2+) release underlying EMA.

Halothane modulation of skeletal muscle ryanodine receptors: dependence on Ca2+, Mg2+, and ATP

Malignant hyperthermia (MH) susceptibility is a genetic disorder of skeletal muscle associated with mutations in the ryanodine receptor isoform 1 (RyR1) of sarcoplasmic reticulum (SR). In MH-susceptible skeletal fibers, RyR1-mediated Ca(2+) release is highly sensitive to activation by the volatile anesthetic halothane. Indeed, studies with isolated RyR1 channels (using simple Cs(+) solutions) found that halothane selectively affects mutated but not wild-type RyR1 function. However, studies in skeletal fibers indicate that halothane can also activate wild-type RyR1-mediated Ca(2+) release. We hypothesized that endogenous RyR1 agonists (ATP, lumenal Ca(2+)) may increase RyR1 sensitivity to halothane. Consequently, we studied how these agonists affect halothane action on rabbit skeletal RyR1 reconstituted into planar lipid bilayers. We found that cytosolic ATP is required for halothane-induced activation of the skeletal RyR1. Unlike RyR1, cardiac RyR2 (much less sensitive to ATP) responded to halothane even in the absence of this agonist. ATP-dependent halothane activation of RyR1 was enhanced by cytosolic Ca(2+) (channel agonist) and counteracted by Mg(2+) (channel inhibitor). Dantrolene, a muscle relaxant used to treat MH episodes, did not affect RyR1 or RyR2 basal activity and did not interfere with halothane-induced activation. Studies with skeletal SR microsomes confirmed that halothane-induced RyR1-mediated SR Ca(2+) release is enhanced by high ATP-low Mg(2+) in the cytosol and by increased SR Ca(2+) load. Thus, physiological or pathological processes that induce changes in cellular levels of these modulators could affect RyR1 sensitivity to halothane in skeletal fibers, including the outcome of halothane-induced contracture tests used to diagnose MH susceptibility.

Ca2+ entry-independent effects of L-type Ca2+ channel modulators on Ca2+ sparks in ventricular myocytes

During the cardiac action potential, Ca(2+) entry through dyhidropyridine receptor L-type Ca(2+) channels (DHPRs) activates ryanodine receptors (RyRs) Ca(2+)-release channels, resulting in massive Ca(2+) mobilization from the sarcoplasmic reticulum (SR). This global Ca(2+) release arises from spatiotemporal summation of many localized elementary Ca(2+)-release events, Ca(2+) sparks. We tested whether DHPRs modulate Ca(2+)sparks in a Ca(2+) entry-independent manner. Negative modulation by DHPR of RyRs via physical interactions is accepted in resting skeletal muscle but remains controversial in the heart. Ca(2+) sparks were studied in cat cardiac myocytes permeabilized with saponin or internally perfused via a patch pipette. Bathing and pipette solutions contained low Ca(2+) (100 nM). Under these conditions, Ca(2+) sparks were detected with a stable frequency of 3-5 sparks.s(-1).100 microm(-1). The DHPR blockers nifedipine, nimodipine, FS-2, and calciseptine decreased spark frequency, whereas the DHPR agonists Bay-K8644 and FPL-64176 increased it. None of these agents altered the spatiotemporal characteristics of Ca(2+) sparks. The DHPR modulators were also without effect on SR Ca(2+) load (caffeine-induced Ca(2+) transients) or sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) activity (Ca(2+) loading rates of isolated SR microsomes) and did not change cardiac RyR channel gating (planar lipid bilayer experiments). In summary, DHPR modulators affected spark frequency in the absence of DHPR-mediated Ca(2+) entry. This action could not be attributed to a direct action of DHPR modulators on SERCA or RyRs. Our results suggest that the activity of RyR Ca(2+)-release units in ventricular myocytes is modulated by Ca(2+) entry-independent conformational changes in neighboring DHPRs.

Istaroxime, a Stimulator of Sarcoplasmic Reticulum Calcium Adenosine Triphosphatase Isoform 2a Activity, as a Novel Therapeutic Approach to Heart Failure

Interventions involving calcium cycling may represent a promising approach to heart failure (HF) therapy because calcium handling is known to be deranged in human and experimental HF. Istaroxime is a sodium-potassium adenosine triphosphatase (ATPase) inhibitor with the unique property of increasing sarcoplasmic reticulum calcium ATPase (SERCA) isoform 2a (SERCA2a) activity. Because this was demonstrated in normal experimental models, we investigated whether istaroxime is able to improve global cardiac function and stimulate SERCA in failing hearts. In guinea pigs with 3-month aortic banding (AoB), echocardiographic results showed that istaroxime intravenous infusion (0.11 mg/kg per min) significantly increased both indices of contraction and relaxation (fractional shortening, +18+/-3.7%; aortic flow rate, +19+/-2.9%; peak myocardial systolic velocity, +36+/-7%; circumferential fiber shortening, +24+/-4.1%; peak atrial flow velocity, +69+/-8.6%; isovolumic relaxation time, +19+/-6.9%; and peak myocardial early diastolic velocity, +42+/-12%). In left ventricular sarcoplasmic reticulum microsomes from AoB animals, 100 nmol/L istaroxime normalized the depressed (-32%) SERCA2a maximum velocity and increased SERCA activity (+17%). In muscle strips from hearts from patients undergoing cardiac transplantation, istaroxime (0.1-1.0 micromol/L) increased (in a concentration-dependent manner) developed tension, the maximum and minimum first derivative of tension, and absolute velocity of contraction, while stimulating SERCA activity in sarcoplasmic reticulum microsomes at physiologic free calcium concentrations. In conclusion, istaroxime is presently the only available compound that stimulates SERCA2a activity and produces a luso-inotropic effect in HF.