Cardiac Muscle Sarcoplasmic Reticulum Research Articles

Medicinal Importance, Pharmacological Activities, and Analytical Aspects of an Isoflavone Glycoside Tectoridin

Background: Polyphenols are a group of secondary plant metabolites produced in plants as a protective system against oxidative stress, UV radiation, pathogens, and predatorial attacks. Flavonoids are a major class of plant phenolics present in fruits, vegetables, tea, and red wine. Tectoridin, also called 40,5,7-thrihydroxy-6-methoxyisoflavone-7-Ob-D-glucopyranoside is an isoflavone glycoside present in the flower of Porites lobata. Methods: Present work focused on the biological importance, therapeutic potential, and pharmacological activities of tectoridin in medicine. Numerous scientific data have been collected from different literature databases such as Google Scholar, Science Direct, PubMed, and Scopus to know the health beneficial potential of tectoridin. Pharmacological data have been analyzed in the present work to know the biological effectiveness of tectoridin against human disorders. Analytical data of tectoridin have been collected and analyzed in the present work to know the importance of modern analytical methods in the isolation, separation, and identification of tectoridin. Results: Scientific data analysis revealed the biological importance and therapeutic benefit of tectoridin in medicine, signifying the therapeutic potential in healthcare systems. The biological activities of tectoridin are mainly due to its anti-inflammatory, anti-platelet, anti-angiogenic, hepatoprotective, anti-tumor, estrogenic, antioxidant, and hypoglycemic activity. However, this work has also presented the effectiveness of tectoridin against rat lens aldose reductase, nitric oxide, skeletal and cardiac muscle sarcoplasmic reticulum, and enzymes. Analytical data signified the importance of modern analytical techniques for separation, identification, and isolation of tectoridin. Conclusion: The present work signified the biological importance and therapeutic benefit of tectoridin in medicine and other allied health sectors.

P5025Structural insights into catecholaminergic polymorphic ventricular tachycardia-associated RyR2 mutant channels using a three-dimensional in silico model

Abstract Background The cardiac ryanodine receptors (RyR2) are large tetrameric calcium-permeant ion channels found in cardiac muscle sarcoplasmic reticulum, which play an important role in the control of intracellular Ca2+ release and cardiac contraction. Mutations in the RYR2 gene are associated with lethal arrhythmia diseases including catecholaminergic polymorphic ventricular tachycardia (CPVT) resulting from increased diastolic Ca2+ leak from mutant channels. RyR2 is a huge protein that each subunit of tetramer is comprised of 4967 amino acids, which hampers the detailed in vitro analysis of RyR2 mutant channels. Purpose We aimed to analyze the structural features of RyR2 mutant channels identified in our cohort with inherited arrhythmias using RyR2 three-dimensional (3D) in silico model to reveal the arrhythmogenic mechanisms. Methods A targeted next-generation sequencing panel for inherited arrhythmias was employed for genetic diagnosis of the patients. Then, we mapped the identified mutations on RyR2 3D structural model developed by cryo-EM images (PDB: 5go9, 5goa, Peng Science 2016) and investigated the relationship between the location of the mutations and specific functional sites. Results As a result of genetic analysis, we identified 93 RYR2 mutations from 112 probands with CPVT (n=93) or long-QT syndrome (LQTS) (n=19).64 of 93 (69%) RYR2 mutations are located in three “hot-spot” area (N-terminal (residues 77–466), central (2246–2534), and channel (3778–4959) hotspot. RyR2 3D in silico modeling revealed that the mutations are regionally distributed mainly in three parts: N-terminal, periphery, and channel part (Figure A). In N-terminal part (1–642 amino acid), 9 of 13 mutations alter the charges of the amino acids (Figure B). Especially, R169L, R169Q, and G172E are close to the interface between two neighboring subunits (∼20Å). These mutations which change the amino acid charge may cause a complete disruption of the ionic pair network and result in largest structural changes, which facilitates RyR2 channel opening. In periphery part (643–3528aa), 22 of 33 mutations are close to the two predicted binding sites of FKBP12.6, a stabilizer of RyR2 (∼5–40Å, Figure C). The mutations are supposed to disturb the binding affinity to the FKBP12.6 resulting in RyR2 channel instability. In channel part (3613–4968aa), 16 of 40 mutations are located near two interface. (FigureD) 12 mutations are close to the Ca2+ sensor and the other 4 mutations are adjacent to the pore-forming segment. Especially, V4821I is just located on this segment and strongly expected to impair the channel function. Above all, RyR2 3D in silico modeling revealed that 63 of all 93 (68%) identified mutations are supposed to be pathogenic. Location of RYR2 mutations in 3D model Conclusion 3D structural model of RyR2 is useful for the investigation of the pathogenic mechanisms of CPVT-related mutations. Further studies are needed to elucidate the relationship between the location of the mutations and clinical phenotypes.

S4-S5 Linker Regulates RyR2 Channel Gating through Multiple Interactions

Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel in the sarcoplasmic reticulum of cardiac muscle and plays an important role in excitation-contraction coupling. Although recent cryo-EM studies provided near-atomic structures of type 1 ryanodine receptor (RyR1), molecular mechanism of the channel opening remains largely unknown. The S4-S5 linker (S45L) is an α-helical structure connecting the S4 and S5 transmembrane segments and mediates signal transmission in a wide variety of channels. To address the role of S45L in the RyR2 channel gating, we systematically mutated residues in S45L (Thr4751-Asn4762) and neighboring transmembrane segments (S5 and S6). The mutant RyR2 was stably expressed in HEK293 cells, and the channel activity was investigated by ER luminal Ca2+ measurements and [3H]ryanodine binding. Using high sequence identity between RyR1 and RyR2 (65% in the whole molecule and 93% in the core channel domain after S4), we constructed structural model of RyR2 based on the structures of RyR1, and the phenotypes of mutants were interpreted by the model. Plausible interactions between amino acid residues in S45L and neighboring domains that may regulate channel gating will be discussed.

Calsequestrins in skeletal and cardiac muscle from adult Danio rerio.

Calsequestrin (Casq) is a high capacity, low affinity Ca(2+)-binding protein, critical for Ca(2+)-buffering in cardiac and skeletal muscle sarcoplasmic reticulum. All vertebrates have multiple genes encoding for different Casq isoforms. Increasing interest has been focused on mammalian and human Casq genes since mutations of both cardiac (Casq2) and skeletal muscle (Casq1) isoforms cause different, and sometime severe, human pathologies. Danio rerio (zebrafish) is a powerful model for studying function and mutations of human proteins. In this work, expression, biochemical properties cellular and sub-cellular localization of D. rerio native Casq isoforms are investigated. By quantitative PCR, three mRNAs were detected in skeletal muscle and heart with different abundances. Three zebrafish Casqs: Casq1a, Casq1b and Casq2 were identified by mass spectrometry (Data are available via ProteomeXchange with identifier PXD002455). Skeletal and cardiac zebrafish calsequestrins share properties with mammalian Casq1 and Casq2. Skeletal Casqs were found primarily, but not exclusively, at the sarcomere Z-line level where terminal cisternae of sarcoplasmic reticulum are located.

Specific molecular and morphological changes in cardiomyocytes of hibernating ground squirrels in different periods of annual cycle

Structural and molecular changes in cardiomyocytes of hibernating ground squirrels in different periods of the annual cycle were analyzed by means of electron microscopy and polymerase chain reaction. Morphological analysis showed an increase in relative area of sarcoplasmic reticulum in cardiac muscle of ground squirrels preparing to torpor compared to active summer animals. The size of sarcoplasmic reticulum in cardiomyocytes of torpid animals was reliably less than in any other condition of ground squirrels in the annual cycle. The results of molecular analysis showed the decrease in sarcoplasmic reticulum Ca(2+)-ATPase gene (SERCA2a) expression .at all stages of hibernation process and also in periods of autumn activity compared to control mRNA level in active summer animals. The revealed season changes in structure of sarcoplasmic reticulum and sarcoplasmic reticulum Ca(2+)-ATPase gene expression are discussed in regard to adaptation of ground squirrels to hibernation.

A significant role of sarcoplasmic reticulum in cardiac contraction of a basal vertebrate, the river lamprey (Lampetra fluviatilis)

Cardiac contraction and relaxation are mediated by rapidly changing calcium concentration around the myofibrils. In comparison with endotherms, ectothermic hearts are more strongly dependent on extracellular calcium for contraction suggesting this trait might represent the primitive vertebrate mode of cardiac activation. This study tests the hypothesis that intracellular calcium stores play a minor role in cardiac excitation-contraction coupling of a basal vertebrate, the lamprey (Lampetra fluviatilis). Contribution of sarcoplasmic reticulum to cardiac calcium management was examined by measuring the ryanodine sensitivity of contraction, determining the number of cardiac ryanodine receptors and their calcium sensitivity, assessing the size of sarcoplasmic reticulum calcium stores in enzymatically isolated cardiac myocytes and qualitative electron microscopic observations of sarcoplasmic reticulum in cardiac muscle. An extensive network of nonjunctional sarcoplasmic reticulum around myofibrils and numerous junctional sarcoplasmic reticulum couplings at the periphery of the myocytes was present in atrial and ventricular muscle of the lamprey heart. High numbers of ryanodine receptors were present in atrial (0.16 ± 0.04 nmol mg(-1) prot) and ventricular membranes (0.27 ± 0.03) (P < 0.01), and 10 μm ryanodine inhibited large part of contraction force in atrial (37.8 ± 5.7%) and ventricular (49.2 ± 6.5%) muscle. Sarcoplasmic reticulum is well developed in the lamprey heart and plays a significant role in cardiac calcium management. This suggests that in the common ancestor of vertebrates, cardiac excitation-contraction coupling could have been fairly strongly dependent on sarcoplasmic reticulum calcium stores. Functionally, this trait might be associated with high cardiac output and active predatory lifestyle of the lamprey.

Modulation of ryanodine receptor Ca2+ channels (Review).

Ryanodine-sensitive Ca2+ release channels (ryanodine receptors, RyRs) play a crucial role in the mobilization of Ca2+ from the sarcoplasmic reticulum (SR) during the excitation-contraction coupling of muscle cells. In skeletal muscle, depolarization of transverse tubules activates the RyR, whereas in cardiac muscle, a Ca2+ influx through an L-type Ca2+ channel activates the RyR. The RyR is also activated by caffeine, a low concentration (<10 µM) of ryanodine or cyclic ADP-ribose. RyR activity is inhibited by Mg2+, ruthenium red, or higher concentrations (≥100 µM) of ryanodine. The activity of RyR channels is modulated by phosphorylation and by associated proteins, including calmodulin (CaM), calsequestrin (CSQ) and FK506-binding proteins (FKBPs). In muscle cells, apoCaM (Ca2+-free CaM) activates the RyR channel, and Ca2+ CaM (Ca2+-bound CaM) inhibits the channel. CSQ can bind approximately 40 moles of Ca2+/mole of CSQ in the SR lumen of muscle cells, and interacts functionally with RyR protein. When the RyR is stimulated, Ca2+ released from the lumen is dissociated from the CSQ- Ca2+ complex. A 12-kDa or 12.6-kDa FK506-binding protein (FKBP12 or FKBP12.6, respectively) is associated with RyR protein. When FKBP12 or FKBP12.6 is dissociated from the FKBP-RyR complex, the RyR is modulated (activated). Phosphorylation of the RyR by cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase II modulates the channel. PKA phosphorylation of the RyR on the skeletal and cardiac muscle SR dissociates FKBP12 or FKBP12.6 from the RyR complex. This review deals with the modulation mechanisms of RyR proteins by associated proteins and phosphorylation.

Modulation of ryanodine receptor Ca2+ channels (Review)

Ryanodine-sensitive Ca2+ release channels (ryanodine receptors, RyRs) play a crucial role in the mobilization of Ca2+ from the sarcoplasmic reticulum (SR) during the excitation-contraction coupling of muscle cells. In skeletal muscle, depolarization of transverse tubules activates the RyR, whereas in cardiac muscle, a Ca2+ influx through an L-type Ca2+ channel activates the RyR. The RyR is also activated by caffeine, a low concentration (<10 µM) of ryanodine or cyclic ADP-ribose. RyR activity is inhibited by Mg2+, ruthenium red, or higher concentrations (≥100 µM) of ryanodine. The activity of RyR channels is modulated by phosphorylation and by associated proteins, including calmodulin (CaM), calsequestrin (CSQ) and FK506-binding proteins (FKBPs). In muscle cells, apoCaM (Ca2+-free CaM) activates the RyR channel, and Ca2+ CaM (Ca2+-bound CaM) inhibits the channel. CSQ can bind approximately 40 moles of Ca2+/mole of CSQ in the SR lumen of muscle cells, and interacts functionally with RyR protein. When the RyR is stimulated, Ca2+ released from the lumen is dissociated from the CSQ- Ca2+ complex. A 12-kDa or 12.6-kDa FK506-binding protein (FKBP12 or FKBP12.6, respectively) is associated with RyR protein. When FKBP12 or FKBP12.6 is dissociated from the FKBP-RyR complex, the RyR is modulated (activated). Phosphorylation of the RyR by cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase II modulates the channel. PKA phosphorylation of the RyR on the skeletal and cardiac muscle SR dissociates FKBP12 or FKBP12.6 from the RyR complex. This review deals with the modulation mechanisms of RyR proteins by associated proteins and phosphorylation.

Phospholabman Regulation of SERCA2a Kinetics, as Modulated by Nitroxyl

Activation of cardiac muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) by 1-agonists involves cAMP- and PKA-dependent phosphorylation of phospholamban (PLB). Activation increases both the apparent Ca2+ affinity and the Vmax of SERCA2a, requiring a sustained input of free energy during cycling. In collaborative work with Jeffrey Froehlich, MD, and others, we investigated this process by comparing the kinetic and spectroscopic properties of SERCA2a expressed with and without PLB in High Five insect cell microsomes to those of SERCA1 and SERCA2a in native skeletal and cardiac muscle SR. Our data suggests PLB modifies the state of oligomerization of SERCA2a, which in the presence of unphosphorylated, inhibitory PLB is predominantly monomeric. The absence of PLB allows SERCA2a molecules to reorient and interact conformationally, resulting in catalytic behavior characteristic of SERCA1 in skeletal SR, which has no PLB. We propose that intermolecular conformational coupling of SERCA2a units increases the catalytic efficiency of the Ca2+ pump (raises Vmax) and may contribute to activation of the cardiac SR Ca2+ pump induced by 1-adrenergic agonists. We have recently applied these insights to the mechanism of SERCA2a stimulation by nitroxyl, HNO, which we propose uncouples PLB from SERCA2a by modification of one or more PLB cysteine residues. Dr. Froehlich's kinetics studies indicated the HNO-treated SERCA2a +PLB sample has the same kinetic properties as SERCA2a in the absence of PLB. SERCA2a coexpressed with a null-cysteine PLB construct showed no change in kinetic properties following treatment with HNO. Our ongoing fluorescence and electron paramagnetic resonance spectroscopic experiments agree, showing that HNO-treated SERCA+PLB samples have physical properties similar to SERCA2a without PLB. Our results support the conclusion that HNO-treatment, at least in part, uncouples PLB from SERCA2a, leading to HNO-stimulated SERCA2a activation. Direct effects of HNO on SERCA2a are also under investigation.

Effects of monovalent cations on Ca 2+ uptake by skeletal and cardiac muscle sarcoplasmic reticulum

Ca 2+ transport by the sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase (SERCA) is sensitive to monovalent cations. Possible K + binding sites have been identified in both the cytoplasmic P-domain and the transmembrane transport-domain of the protein. We measured Ca 2+ transport into SR vesicles and SERCA ATPase activity in the presence of different monovalent cations. We found that the effects of monovalent cations on Ca 2+ transport correlated in most cases with their direct effects on SERCA. Choline +, however, inhibited uptake to a greater extent than could be accounted for by its direct effect on SERCA suggesting a possible effect of choline on compensatory charge movement during Ca 2+ transport. Of the monovalent cations tested, only Cs + significantly affected the Hill coefficient of Ca 2+ transport ( n H). An increase in n H from ∼2 in K + to ∼3 in Cs + was seen in all of the forms of SERCA examined. The effects of Cs + on the maximum velocity of Ca 2+ uptake were also different for different forms of SERCA but these differences could not be attributed to differences in the putative K + binding sites of the different forms of the protein.

The shy Angeli and his elusive creature: the HNO route to vasodilation

what we now call Angeli's salt (AS) was discovered more than a century ago by the Italian chemist Angelo Angeli (Fig. 1) . He found that “salts of nitroxylaminic acid are readily resolved into the corresponding nitrites and the unsaturated residue nitroxyl” (HNO), proposing their use as suppliers of HNO (3). However, by analogy with the genial but very reclusive personality of the discoverer (17), the physiological properties of HNO didn't draw too much attention until early 1990s. In their seminal work, Fukuto and colleagues (19) reported that HNO elicits vasorelaxation in both the rabbit aorta and bovine intrapulmonary artery by a soluble guanylate cyclase (sGC)- dependent pathway. Later, it was evident that the thiol-donating agent l-cysteine can discriminate the vasodilatative profile of HNO from that of nitric oxide (NO·) or nitrosothiols: AS/HNO action can be blocked by this agent, whereas the NO· effect is potentiated (36). In combination with the suggestion that NO· is the only species able to directly activate sGC (9), these data led to the conclusion that HNO-induced vasorelaxation is due to its intracellular conversion to NO· with subsequent activation of sGC. For several years, this idea has relegated HNO to a mere precursor of the presence and biological activity of NO·, de facto negating to HNO its own right to exist.

Regulation of the Cardiac Muscle Ryanodine Receptor by O2 Tension and S-Nitrosoglutathione

The cardiac and skeletal muscle sarcoplasmic reticulum ryanodine receptor Ca(2+) release channels contain thiols that are potential targets of endogenously produced reactive oxygen and nitrogen intermediates. Previously, we showed that the skeletal muscle ryanodine receptor (RyR1) has O(2)-sensitive thiols; only when these thiols are in the reduced state (pO(2) approximately 10 mmHg) can physiological concentrations of NO (nanomolar) activate RyR1. Here, we report that cardiac muscle ryanodine receptor (RyR2) activity also depends on pO(2), but unlike RyR1, RyR2 was not activated or S-nitrosylated directly by NO. Rather, activation and S-nitrosylation of RyR2 required S-nitrosoglutathione. The effects of peroxynitrite were indiscriminate on RyR1 and RyR2. Our results indicate that both RyR1 and RyR2 are pO(2)-responsive yet point to different mechanisms by which NO and S-nitrosoglutathione influence cardiac and skeletal muscle sarcoplasmic reticulum Ca(2+) release.

Phospholamban Thiols Play a Central Role in Activation of the Cardiac Muscle Sarcoplasmic Reticulum Calcium Pump by Nitroxyl

Nitroxyl (HNO) donated by Angeli's salt activates uptake of Ca(2+) by the cardiac SR Ca(2+) pump (SERCA2a). To determine whether HNO achieves this by a direct interaction with SERCA2a or its regulatory protein, phospholamban (PLN), we measured its effects on SERCA2a activation (as reflected in dephosphorylation) using insect cell microsomes expressing SERCA2a with or without PLN (wild-type and Cys --> Ala mutant). The results show that activation of SERCA2a dephosphorylation by HNO is PLN-dependent and that PLN thiols are targets for HNO. We conclude that HNO produces a disulfide bond that alters the conformation of PLN, relieving inhibition of the Ca(2+) pump.

Getting energy to where it is required is a problem in the failing heart

Getting energy to where it is required is a problem in the failing heart

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.

Effects of FK506 on Ca2+ Release Channels (Review)

Tacrolimus (FK506), which was isolated from the fermentation broth of Streptomyces tsukubaensis No. 9993, has an immunosuppressive effect. In T-lymphocytes, FK506 binds to the intracellular receptor, a 12-kDa FK506-binding protein (FKBP12). The FK506-FKBP12 complex binds to the phosphatase calcineurin (CN) and inhibits the activity of CN. By inhibition of the activity of CN, dephosphorylation of a nuclear factor of activated T-cells (NFAT) is inhibited, and translocation of the NFAT to the nucleus is suppressed. Thereby, the production of T-cell-derived mediators such as interleukin 2 (IL-2) is inhibited, and the proliferation of cytotoxic T-cells is suppressed. In muscle cells, FKBP12 and FKBP12.6 are associated with ryanodine-sensitive Ca(2+) release channels (ryanodine receptors: RyRs) on the skeletal and cardiac muscle sarcoplasmic reticulum (SR), respectively. FK506 modulates the RyR by dissociating FKBP12 or FKBP12.6 from the RyR complex. FKBP12 is also associated with inositol 1,4,5-trisphosphate (IP(3))-sensitive Ca(2+) release channels (IP(3) receptors: IP(3)Rs) on the endoplasmic reticulum (ER) of non-muscle cells. The IP(3)R-FKBP12 complex binds to CN, which dephosphorylates the protein kinase C (PKC) phosphorylation site on the receptor. When FKBP12 is dissociated from the IP(3)R complex by FK506, CN is also dissociated from the IP(3)R. Thereby, the IP(3)R is phosphorylated by PKC, and the receptor is modulated. Recently, it was found that FK506 itself induces Ca(2+) release through RyRs in some tissues.

Redox Sensitivity of the Ryanodine Receptor Interaction with FK506-binding Protein

The ryanodine receptor (RyR) calcium release channel functions as a redox sensor that is sensitive to channel modulators. The FK506-binding protein (FKBP) is an important regulator of channel activity, and disruption of the RyR2-FKBP12.6 association has been implicated in cardiac disease. In the present study, we investigated whether the RyR-FKBP association is redox-regulated. Using co-immunoprecipitation assays of solubilized native RyR2 from cardiac muscle sarcoplasmic reticulum (SR) with recombinant [(35)S]FKBP12.6, we found that the sulfydryl-oxidizing agents, H(2)O(2) and diamide, result in diminished RyR2-FKBP12.6 binding. Co-sedimentation experiments of cardiac SR vesicles with [(35)S]FKBP12.6 also demonstrated that oxidizing reagents decreased FKBP binding. Matching results were obtained with skeletal muscle SR. Notably, H(2)O(2) and diamide differentially affected the RyR2-FKBP12.6 interaction, decreasing binding to approximately 75 and approximately 50% of control, respectively. In addition, the effect of H(2)O(2) was negligible when the channel was in its closed state or when applied after FKBP binding had occurred, whereas diamide was always effective. A cysteine-null mutant FKBP12.6 retained redox-sensitive interaction with RyR2, suggesting that the effect of the redox reagents is exclusively via sites on the ryanodine receptor. K201 (or JTV519), a drug that has been proposed to prevent FKBP12.6 dissociation from the RyR2 channel complex, did not restore normal FKBP binding under oxidizing conditions. Our results indicate that the redox state of the RyR is intimately connected with FKBP binding affinity.

The role of phosphorylation on the structure and dynamics of phospholamban: A model from molecular simulations

Phospholamban (PLB) is a small membrane protein that regulates the activity of the calcium ATP-ase in the cardiac, slow-twitch, and smooth muscle sarcoplasmic reticulum through the reversible phosphorylation of Ser16. We present here a comparative molecular dynamics study of unmodified and phosphorylated PLB immersed in a phospholipid membrane. The study has been performed under different ionic strength conditions, using the NMR structures of two PLB variants determined in mixed organic solvent and dodecylphosphocholine micelles. The simulations indicate that all PLB forms studied display a highly dynamic behavior of the N-terminal cytoplasmic moiety, with a decrease of its helical content in the phosphorylated forms. The cytoplasmic domain undergoes large collective motions sampling conformations parallel as well as perpendicular to the membrane surface in all the simulations. The transmembrane domain retains a tightly folded helical conformation with a small tilt with respect to the membrane plane probably induced by the presence of Asn30 and Asn34 within the hydrophobic environment. Furthermore, the phosphoric group on Ser16 establishes transient electrostatic interactions with the phospholipid heads. We propose a model in which phosphorylation diminishes the probability of interactions of PLB with residues near Lys400 in the SERCA pump, thus relieving its inhibition.

Histidine-rich Ca binding protein: a regulator of sarcoplasmic reticulum calcium sequestration and cardiac function

Defects in the pathways that regulate cardiac sarcoplasmic reticulum (SR) calcium (Ca) cycling represent prime targets for driving the deterioration of function and progression to heart failure. We hypothesized that the histidine-rich Ca binding protein (HRC) in the SR may be involved in SR Ca cycling and that alterations in HRC levels would result in abnormal cardiac Ca homeostasis. In order to test this hypothesis, we generated transgenic mice with cardiac overexpression (3-fold) of HRC. Increased cardiac HRC levels were associated with impaired SR Ca uptake rates (35%) and attenuated cardiomyocyte Ca transient decay (38%), without alterations in peak Ca transients or SR Ca load. The depressed SR Ca sequestration was associated with attenuated rate of Ca extrusion via Na-Ca exchange. Triadin protein expression levels and L-type Ca channel current density were increased, while the channel inactivation kinetics were not altered. Impaired SR Ca uptake and delayed Ca decline rates triggered hypertrophy and compromised the heart's responses to increased stress by either hemodynamic overload or the aging process. By 18 months of age, cardiac remodeling deteriorated to congestive heart failure in transgenic mice. Collectively, these data suggest that HRC may be an integral regulatory protein involved in cardiac muscle SR Ca uptake and Ca homeostasis.

Effects of Phytoestrogens on Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase 2a and Ca2+Uptake into Cardiac Sarcoplasmic Reticulum

Phytoestrogens are naturally occurring estrogenic compounds found in plants and plant products. These compounds are also known to exert cellular effects independent of their interactions with estrogen receptors. We studied the effects of the phytoestrogens phloretin, phloridzin, genistein, and biochanin A on Ca(2+) uptake into the cardiac muscle sarcoplasmic reticulum (SR). Genistein and biochanin A did not affect SR Ca(2+) uptake. On the other hand, phloretin and phloridzin decreased the maximum velocity of SR Ca(2+) uptake but did not affect the Hill coefficient or the Ca(2+) sensitivity of uptake. Measurements of the ATPase activity of the cardiac SR Ca(2+) pump (SERCA2a) revealed direct inhibitory effects of phloretin and phloridzin on SERCA2a. Neither compound induced a detectable change in the permeability of the SR membrane to Ca(2+). These results indicate that phloretin and phloridzin inhibit cardiac SR Ca(2+) uptake by directly inhibiting SERCA2a.