Glycosylation of skeletal calsequestrin: implications for its function

Calsequestrin (CASQ) serves as a major Ca(2+) storage/buffer protein in the sarcoplasmic reticulum (SR). When purified from skeletal muscle, CASQ1 is obtained in its glycosylated form. Here, we have confirmed the specific site and degree of glycosylation of native rabbit CASQ1 and have investigated its effect on critical properties of CASQ by comparison with the non-glycosylated recombinant form. Based on our comparative approach utilizing crystal structures, Ca(2+) binding capacities, analytical ultracentrifugation, and light-scattering profiles of the native and recombinant rabbit CASQ1, we propose a novel and dynamic role for glycosylation in CASQ. CASQ undergoes a unique degree of mannose trimming as it is trafficked from the proximal endoplasmic reticulum to the SR. The major glycoform of CASQ (GlcNAc(2)Man(9)) found in the proximal endoplasmic reticulum can severely hinder formation of the back-to-back interface, potentially preventing premature Ca(2+)-dependent polymerization of CASQ and ensuring its continuous mobility to the SR. Only trimmed glycans can stabilize both front-to-front and the back-to-back interfaces of CASQ through extensive hydrogen bonding and electrostatic interactions. Therefore, the mature glycoform of CASQ (GlcNAc(2)Man(1-4)) within the SR can be retained upon establishing a functional high capacity Ca(2+) binding polymer. In addition, based on the high resolution structures, we propose a molecular mechanism for the catecholaminergic polymorphic ventricular tachycardia (CPVT2) mutation, K206N.     

Postulated role of calsequestrin in the regulation of calcium release from sarcoplasmic reticulum

Ca2+ release from heavy sarcoplasmic reticulum (SR) vesicles was induced by 2 mM caffeine, and the amount (A) and the rate constant (k) of Ca2+ release were investigated as a function of the extent of Ca2+ loading. Under both passive and active loading conditions, the A value increased monotonically in parallel to Ca2+ loading. On the other hand, k sharply increased at partial Ca2+ loading, and upon further loading, it decreased to a lower level. Since most of the intravesicular calcium appears to be bound to calsequestrin both under passive and under active loading conditions, these results suggest that the kinetic properties of induced Ca2+ release show significant variation depending upon how much calcium has been bound to calsequestrin at the time of the induction of Ca2+ release. An SR membrane segment consisting of the junctional face membrane (jfm) and attached calsequestrin (jfm-calsequestrin complex) was prepared. The covalently reacting thiol-specific conformational probe N-[7-(dimethylamino)-4-methyl-3-coumarinyl]maleimide (DACM) was incorporated into several proteins of the jfm, but not into calsequestrin. The fluorescence intensity of DACM increased with Ca2+. Upon dissociation of calsequestrin from the jfm by salt treatment, the DACM fluorescence change was abolished, while upon reassociation of calsequestrin by dilution of the salt it was partially restored. These results suggest that the events occurring in the jfm proteins are mediated via the attached calsequestrin rather than by a direct effect of Ca2+ on the jfm proteins. We propose that the [Ca2+]-dependent conformational changes of calsequestrin affect the jfm proteins and in turn regulate the Ca2+ channel functions.     

Phosphorylation of chicken gizzard myosin: Myosin filament hypothesis of calcium regulation

Myosin from chicken gizzard smooth muscle was found to be characteristically different from rabbit skeletal striated myosin: i) ATP induced a profound change in the conformation of gizzard myosin molecules. ii) ATP also induced disassembling of gizzard myosin filaments. iii) Enzymic phosphorylation of gizzard myosin light chains rendered both the myosin conformation and the myosin filaments resistant to the actions of ATP. iv) Very high concentrations of magnesium were required for formation of the ATP-resistant filaments as well as for superprecipitation (a model contraction) of actomyosin suspensions. v) ITP was a very poor substrate for MLCK, and was accordingly incapable of inducing “Ca-tension” in glycerinated fibers of gizzard muscle, but it did induce “Mg-tension.” Primarily from these findings, it was proposed that tje mechanism of gizzard muscle contraction involves ATP-induced changes in the morphology of myosin filaments which are reversibly altered by enzymic phosphorylation and dephosphorylation of myosin light chains in the presence of relatively high concentrations of magnesium.     

Fast kinetics of calcium dissociation from calsequestrin

We measured the kinetics of calcium dissociation from calsequestrin in solution or forming part of isolated junctional sarcoplasmic reticulum membranes by mixing calsequestrin equilibrated with calcium with calcium-free solutions in a stopped-flow system. In parallel, we measured the kinetics of the intrinsic fluorescence changes that take place following calcium dissociation from calsequestrin. We found that at 25 degrees C calcium dissociation was 10-fold faster for calsequestrin attached to junctional membranes (k = 109 s(-1)) than in solution. These results imply that calcium dissociation from calsequestrin in vivo is not rate limiting during excitation-contraction coupling. In addition, we found that the intrinsic fluorescence decrease for calsequestrin in solution or forming part of junctional membranes was significantly slower than the rates of calcium dissociation. The kinetics of intrinsic fluorescence changes had two components for calsequestrin associated to junctional membranes and only one for calsequestrin in solution; the faster component was 8-fold faster (k = 54.1 s(-1)) than the slower component (k = 6.9 s(-1)), which had the same k value as for calsequestrin in solution. These combined results suggest that the presence of calsequestrin at high concentrations in a restricted space, such as when bound to the junctional membrane, accelerates calcium dissociation and the resulting structural changes, presumably as a result of cooperative molecular interactions.     

Polymerization of calsequestrin. Implications for Ca2+ regulation

Two distinct dimerization contacts in calsequestrin crystals suggested a mechanism for Ca(2+) regulation resulting from the occurrence of coupled Ca(2+) binding and protein polymerization. Ca(2+)-induced formation of one contact was proposed to lead to dimerization followed by Ca(2+)-induced formation of the second contact to bring about polymerization (). To test this mechanism, we compared canine cardiac calsequestrin and four truncation mutants with regard to their folding properties, structures, and Ca(2+)-induced polymerization. The wild-type calsequestrin and truncation mutants exhibited similar K(+)-induced folding and end-point structures as indicated by intrinsic fluorescence and circular dichroism, respectively, whereas the polymerization tendencies of the wild-type calsequestrin differed markedly from the polymerization tendencies of the truncation mutants. Static laser light scattering and 3,3'-dithiobis sulfosuccinimidyl-propionate cross-linking indicated that wild-type protein exhibited an initial Ca(2+)-induced dimerization, followed by additional oligomerization as the Ca(2+) concentration was raised or as the K(+) concentration was lowered. None of the truncation mutants exhibited clear stepwise oligomerization that depended on increasing Ca(2+) concentration. Comparison of the three-dimensional structure of rabbit skeletal calsequestrin with a homology model of canine cardiac calsequestrin from the point of view of our coupled Ca(2+) binding and polymerization mechanism leads to a possible explanation for the 2-fold reduced Ca(2+) binding capacity of cardiac calsequestrin despite very similar overall net negative charge for the two proteins.     

Paradoxical buffering of calcium by calsequestrin demonstrated for the calcium store of skeletal muscle

Contractile activation in striated muscles requires a Ca²⁺ reservoir of large capacity inside the sarcoplasmic reticulum (SR), presumably the protein calsequestrin. The buffering power of calsequestrin in vitro has a paradoxical dependence on [Ca²⁺] that should be valuable for function. Here, we demonstrate that this dependence is present in living cells. Ca²⁺ signals elicited by membrane depolarization under voltage clamp were compared in single skeletal fibers of wild-type (WT) and double (d) Casq-null mice, which lack both calsequestrin isoforms. In nulls, Ca²⁺ release started normally, but the store depleted much more rapidly than in the WT. This deficit was reflected in the evolution of SR evacuability, E, which is directly proportional to SR Ca²⁺ permeability and inversely to its Ca²⁺ buffering power, B. In WT mice E starts low and increases progressively as the SR is depleted. In dCasq-nulls, E started high and decreased upon Ca²⁺ depletion. An elevated E in nulls is consistent with the decrease in B expected upon deletion of calsequestrin. The different value and time course of E in cells without calsequestrin indicate that the normal evolution of E reflects loss of B upon SR Ca²⁺ depletion. Decrement of B upon SR depletion was supported further. When SR calcium was reduced by exposure to low extracellular [Ca²⁺], release kinetics in the WT became similar to that in the dCasq-null. E became much higher, similar to that of null cells. These results indicate that calsequestrin not only stores Ca²⁺, but also varies its affinity in ways that progressively increase the ability of the store to deliver Ca²⁺ as it becomes depleted, a novel feedback mechanism of potentially valuable functional implications. The study revealed a surprisingly modest loss of Ca²⁺ storage capacity in null cells, which may reflect concurrent changes, rather than detract from the physiological importance of calsequestrin.     

Phosphorylation and dephosphorylation of calsequestrin on CK2-sensitive sites in heart

Calsequestrin (CSQ) concentrates in junctional sarcoplasmic reticulum (SR) where it functions in regulation of Ca2+ release. When purified from heart tissue, cardiac CSQ contains phosphate on a cluster of C-terminal serine residues, but little is known about the cellular site of kinase action, and the identity of the kinase remains uncertain. To determine basic features of the phosphorylation, we examined the reaction in canine heart preparations. CSQ phosphorylation was observed in [32P]metabolically-labeled heart cells after adenoviral overexpression, and its constitutive phosphorylation was limited to a CK2-sensitive C-terminal serine cluster. The CSQ kinase was oriented intralumenally, as was CSQ, inside membrane vesicles, such that exposure to each required detergent permeabilization. Yet even after detergent permeabilization, CSQ was phosphorylated much less efficiently by protein kinase CK2 in cardiac microsomes than was purified CSQ. Reduced phosphorylation was strongly dependent upon protein concentration, and phosphorylation time courses revealed a phosphatase activity that occurred constitutively as phosphorylated substrate accumulates. Evidence of selective dephosphorylation of CSQ glycoforms in heart homogenates was also seen by mass spectrometry analysis. Molecules with greater mannose content, a feature of early secretory pathway compartments, were more highly phosphorylated, while greater dephosphorylation was apparent in more distal compartments. Taken together, the analyses of CSQ phosphorylation in heart suggest that a constitutive process of phosphate turnover occurs for cardiac CSQ perhaps associated with its intracellular transport.     

Purinergic-independent calcium signaling mediates recovery from hepatocellular swelling: implications for volume regulation.

Swelling of hepatocytes and other epithelia activates volume-sensitive ion channels that facilitate fluid and electrolyte efflux to restore cell volume, but the responsible signaling pathways are incompletely defined. Previous work in model HTC rat hepatoma cells has indicated that swelling elicits ATP release, which stimulates P2 receptors and activates Cl(-) channels, and that this mechanism is essential for hepatocellular volume recovery. Since P2 receptors are generally coupled to Ca(2+) signaling pathways, we determined whether hepatocellular swelling affected cytosolic [Ca(2+)], and if this involved a purinergic mechanism. Exposure of HTC cells to hypotonic media evoked an increase in cytosolic [Ca(2+)], which was followed by activation of K(+) and Cl(-) currents. Maneuvers that interfered with swelling-induced increases in cytosolic [Ca(2+)], including extracellular Ca(2+) removal and intracellular Ca(2+) store depletion with thapsigargin, inhibited activation of membrane currents and volume recovery. However, the swelling-induced increases in cytosolic [Ca(2+)] were unaffected by either extracellular ATP depletion with apyrase or blockade of P2 receptors with suramin. These findings indicate that swelling elicits an increase in hepatocellular Ca(2+), which is essential for ion channel activation and volume recovery, but that this increase does not stem from activation of volume-sensitive P2 receptors. Collectively, these observations imply that regulatory responses to hepatocellular swelling involve a dual requirement for a purinergic-independent Ca(2+) signaling cascade and a Ca(2+)-independent purinergic signaling pathway.     

A calcium-induced calcium release mechanism mediated by calsequestrin

Calcium (Ca²⁺)-induced Ca²⁺ release (CICR) is widely accepted as the principal mechanism linking electrical excitation and mechanical contraction in cardiac cells. The CICR mechanism has been understood mainly based on binding of cytosolic Ca²⁺ with ryanodine receptors (RyRs) and inducing Ca²⁺ release from the sarcoplasmic reticulum (SR). However, recent experiments suggest that SR lumenal Ca²⁺ may also participate in regulating RyR gating through calsequestrin (CSQ), the SR lumenal Ca²⁺ buffer. We investigate how SR Ca²⁺ release via RyR is regulated by Ca²⁺ and calsequestrin (CSQ). First, a mathematical model of RyR kinetics is derived based on experimental evidence. We assume that the RyR has three binding sites, two cytosolic sites for Ca²⁺ activation and inactivation, and one SR lumenal site for CSQ binding. The open probability (P_o) of the RyR is found by simulation under controlled cytosolic and SR lumenal Ca²⁺. Both peak and steady-state P_o effectively increase as SR lumenal Ca²⁺ increases. Second, we incorporate the RyR model into a CICR model that has both a diadic space and the junctional SR (jSR). At low jSR Ca²⁺ loads, CSQs are more likely to bind with the RyR and act to inhibit jSR Ca²⁺ release, while at high SR loads CSQs are more likely to detach from the RyR, thereby increasing jSR Ca²⁺ release. Furthermore, this CICR model produces a nonlinear relationship between fractional jSR Ca²⁺ release and jSR load. These findings agree with experimental observations in lipid bilayers and cardiac myocytes.     

Modulation of sarcoplasmic reticulum calcium release by calsequestrin in cardiac myocytes

Calsequestrin (CASQ2) is a high capacity Ca-binding protein expressed inside the sarcoplasmic reticulum (SR). Mutations in the cardiac calsequestrin gene (CASQ2) have been linked to arrhythmias and sudden death induced by exercise and emotional stress. We have studied the function of CASQ2 and the consequences of arrhythmogenic CASQ2 mutations on intracellular Ca signalling using a combination of approaches of reverse genetics and cellular physiology in adult cardiac myocytes. We have found that CASQ2 is an essential determinant of the ability of the SR to store and release Ca2+ in cardiac muscle. CASQ2 serves as a reservoir for Ca2+ that is readily accessible for Ca(2+)-induced Ca2+ release (CICR) and also as an active Ca2+ buffer that modulates the local luminal Ca-dependent closure of the SR Ca2+ release channels. At the same time, CASQ2 stabilizes the CICR process by slowing the functional recharging of SR Ca2+ stores. Abnormal restitution of the Ca2+ release channels from a luminal Ca-dependent refractory state could account for ventricular arrhythmias associated with mutations in the CASQ2 gene.     

Comparing skeletal and cardiac calsequestrin structures and their calcium binding: a proposed mechanism for coupled calcium binding and protein polymerization

Calsequestrin, the major calcium storage protein of both cardiac and skeletal muscle, binds and releases large numbers of Ca(2+) ions for each contraction and relaxation cycle. Here we show that two crystal structures for skeletal and cardiac calsequestrin are nearly superimposable not only for their subunits but also their front-to-front-type dimers. Ca(2+) binding curves were measured using atomic absorption spectroscopy. This method enables highly accurate measurements even for Ca(2+) bound to polymerized protein. The binding curves for both skeletal and cardiac calsequestrin were complex, with binding increases that correlated with protein dimerization, tetramerization, and oligomerization. The Ca(2+) binding capacities of skeletal and cardiac calsequestrin are directly compared for the first time, with approximately 80 Ca(2+) ions bound per skeletal calsequestrin and approximately 60 Ca(2+) ions per cardiac calsequestrin, as compared with net charges for these molecules of -80 and -69, respectively. Deleting the negatively charged and disordered C-terminal 27 amino acids of cardiac calsequestrin results in a 50% reduction of its calcium binding capacity and a loss of Ca(2+)-dependent tetramer formation. Based on the crystal structures of rabbit skeletal muscle calsequestrin and canine cardiac calsequestrin, Ca(2+) binding capacity data, and previous light-scattering data, a mechanism of Ca(2+) binding coupled with polymerization is proposed.     

Hydrogen peroxide in human urine: implications for antioxidant defense and redox regulation.

The presence of hydrogen peroxide, at levels sometimes exceeding 100 microM, in human urine samples was established by three different assay methods: 2-oxoglutarate decarboxylation and the ferrous oxidation-xylenol orange (FOX) assay and an oxygen electrode. Detected levels of H(2)O(2) were decreased by addition of superoxide dismutase. We conclude that urine contains autooxidizable molecules that, upon exposure to 21% O(2), undergo rapid superoxide-dependent autooxidation reactions to generate H(2)O(2). The exposure of human tissues to hydrogen peroxide may be greater than is commonly supposed, which has implications in relation to the proposed role of this species in cell signaling.     

Increased calcium leak associated with reduced calsequestrin expression in hyperthyroid cardiomyocytes

IntroductionCalcium (Ca²⁺) leak during cardiac diastole is chiefly mediated by intracellular Ca²⁺ channel/Ryanodine Receptors. Increased diastolic Ca²⁺ leak has been proposed as the mechanism underlying the appearance of hereditary arrhythmias. However, little is known about alterations in diastolic Ca²⁺ leak and the specific roles played by key intracellular Ca²⁺-handling proteins in hyperthyroidism, a known arrhythmogenic condition.AimWe sought to determine whether there were modifications in diastolic Ca²⁺ leak, based on the recording of Ca²⁺ sparks and Ca²⁺ waves; we also investigated changes in the expression and activity of key Ca²⁺ handling proteins, including ryanodine receptors, Sarco-Endoplasmic Reticulum Ca²⁺ ATPase pump and calsequestrin in isolated left-ventricular cardiomyocytes isolated from hyperthyroid rats.Materials and methodsElectrocardiography (ECG) recordings were performed in control and hyperthyroid rats. Ca²⁺ sparks, Ca²⁺ waves, and electrically-stimulated Ca²⁺ transients were recorded in Fluo-3-loaded cardiomyocytes from both experimental groups using confocal microscopy. In addition, left-ventricular homogenates and Ryanodine Receptor-enriched membrane fractions were prepared for assessing [³H]-ryanodine binding, hydrolytic ATPase activity of SERCA pump and expression levels of key proteins by Western blot, and cDNA for real-time qPCR.Results and conclusionsExtrasystoles were observed in hearts of hyperthyroid rats by ECG recordings. Arrhythmogenic activity, high incidence of Ca²⁺ waves, and de novo Ca²⁺ wavelets −in the absence of sarcoplasmic reticulum Ca²⁺ overload- were recorded in these cardiomyocytes. The exacerbated diastolic Ca²⁺ leak and arrhythmogenic activities were related to a diminished expression of calsequestrin along with increased SERCA pump activity, which, in effect, promoted a gain-of-function in RyRs without alterations in SR Ca²⁺ load, RyR expression or its Ca²⁺ sensitivity.     

Phosphorylation of erythrocyte membrane liberates calcium

Washed and permeabilized human erythrocyte ghosts were found to discharge calcium on treatment with ATP. Concomitantly, there was a decrease in phosphatidylinositol (PI) and an increase in phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2). These results support the hypothesis that an inositide shuttle, PI in equilibrium PIP in equilibrium PIP2, operates to maintain intracellular Ca2+ levels. The cation is thought to be sequestered in a cage formed by the head groups of two acidic phospholipid molecules, e.g., phosphatidylserine and phosphatidylinositol, with participation of both PO and fatty acid ester CO groups. These cages are stabilized by inter-headgroup hydrogen bonding. When the inositol group is phosphorylated in positions 4 and 5, inter-lipid hydrogen bonding is disrupted and the cage opens to release its Ca2+.     

Phosphorylation of cardiac and skeletal muscle calsequestrin isoforms by casein kinase II. Demonstration of a cluster of unique rapidly phosphorylated sites in cardiac calsequestrin

Calsequestrin is an acidic Ca2(+)-binding protein of sarcoplasmic reticulum existing as different gene products in cardiac muscle and skeletal muscle. A unique feature of cardiac calsequestrin is a 31-amino acid-long COOH-terminal tail (Scott, B. T., Simmerman, H. K. B., Collins, J. H., Nadal-Ginard, B., and Jones, L. R. (1988) J. Biol. Chem. 263, 8958-8964), which is highly acidic and contains several consensus phosphorylation sites for casein kinase II. In the work described here, we tested whether this cardiac-specific sequence is a substrate for casein kinase II. Both cardiac and skeletal muscle calsequestrins were phosphorylated by casein kinase II, but cardiac calsequestrin was phosphorylated to a higher stoichiometry and at least 50 times more rapidly. The site of rapid phosphorylation of cardiac calsequestrin was localized to the distinct COOH terminus, where a cluster of three closely spaced serine residues are found (S378DEESN-DDSDDDDE-COOH). The slower phosphorylation of skeletal muscle calsequestrin occurred at its truncated COOH terminus, at threonine residue 363 (I351NTEDDDDDE-COOH). The similar sequence in cardiac calsequestrin (I351NTEDDDNEE) was not phosphorylated. Cardiac calsequestrin, as isolated, already contained 1.2 mol of Pi/mol of protein, whereas skeletal muscle calsequestrin contained only trace levels of Pi. The endogenous Pi of cardiac calsequestrin was also localized to the distinct COOH terminus. Our results indicate that the cardiac isoform of calsequestrin is the preferred substrate for casein kinase II both in vivo and in vitro.     

Volume regulation by human lymphocytes. Role of calcium

Human peripheral blood lymphocytes regulate their volumes in hypotonic solutions. In hypotonic media in which Na+ is the predominant cation, an initial swelling phase is followed by a regulatory volume decrease (RVD) associated with a net loss of cellular K+. In media in which K+ is the predominant cation, the rapid initial swelling is followed by a slower second swelling phase. 86Rb+ fluxes increased during RVD and returned to normal when the original volume was approximately regained. Effects similar to those induced by hypotonic stress could also be produced by raising the intracellular Ca++ level. In isotonic, Ca++- containing media cells were found to shrink upon addition of the Ca++ ionophore A23187 in K+-free media, but to swell in K+-rich media. Exposure to Ca++ plus A23187 also increased 86Rb+ fluxes. Quinine (75 microM), an inhibitor of the Ca++-activated K+ pathway in other systems blocked RVD, the associated K+ loss, and the increase in 86Rb+ efflux. Quinine also inhibited the volume changes and the increased 86Rb fluxes induced by Ca++ plus ionophore. The calmodulin inhibitors trifluoperazine, pimozide and chlorpromazine blocked RVD as well as Ca++ plus A23187-induced volume changes. Trifluoperazine also prevented the increase in 86Rb+ fluxes and K+ loss induced by hypotonicity. Chlorpromazine sulfoxide, a relatively ineffective calmodulin antagonist, was considerably less potent as an inhibitor of RVD than chlorpromazine. It is suggested than an elevation in cytoplasmic [Ca++], triggered by cell swelling, increases the plasma membrane permeability to K+, the ensuing increased efflux of K+, associated anions, and osmotically obliged water, leading to cell shrinking (RVD).     

Phosphorylation of the cardiac isoform of calsequestrin in cultured rat myotubes and rat skeletal muscle.

Calsequestrin is a high-capacity Ca(2+)-binding protein and a major constituent of the sarcoplasmic reticulum (SR) of both skeletal and cardiac muscle. Two isoforms of calsequestrin, cardiac and skeletal muscle forms, have been described which are products of separate genes. Purified forms of the two prototypical calsequestrin isoforms, dog cardiac and rabbit fast-twitch skeletal muscle calsequestrins, serve as excellent substrates for casein kinase II and are phosphorylated on distinct sites (Cala, S.E. and Jones, L.R. (1991) J. Biol. Chem 266, 391-398). Dog cardiac calsequestrin is phosphorylated at a 50 to 100-fold greater rate than is rabbit skeletal muscle calsequestrin, and only the dog cardiac isoform contains endogenous Pi on casein kinase II phosphorylation sites. In this study, we identified and examined both calsequestrin isoforms in rat muscle cultures and homogenates to demonstrate that the cardiac isoform of calsequestrin in rat skeletal muscle was phosphorylated in vivo on sites which are phosphorylated by casein kinase II in vitro. Phosphorylation of rat skeletal muscle calsequestrin was not detected. In tissue homogenates, cardiac and skeletal muscle calsequestrin isoforms were both found to be prominent substrates for endogenous casein kinase II activity with cardiac calsequestrin the preferred substrate. In addition, these studies revealed that the cardiac isoform of calsequestrin was the predominant form expressed in skeletal muscle of fetal rats and cultured myotubes.     

Altered spatial calcium regulation enhances electrical heterogeneity in the failing canine left ventricle: implications for electrical instability

Myocytes across the left ventricular (LV) wall of the mammalian heart are known to exhibit heterogeneity of electrophysiological properties; however, the transmural variation of cellular electrophysiology and Ca(2+) homeostasis in the failing LV is incompletely understood. We studied action potentials (APs), the L-type calcium (Ca(2+)) current (I(Ca,L)), and intracellular Ca(2+) transients ([Ca(2+)](i)) of subendocardial (Endo), midmyocardial (Mid), and subepicardial (Epi) tissue layers in the canine normal and tachycardia pacing-induced failing left ventricles. Heart failure (HF) was associated with significant prolongation of the AP duration in Mid myocytes. There were no differences in I(Ca,L) density in normal Endo, Mid, and Epi myocytes, whereas in the failing heart, I(Ca,L) density was downregulated by 45% and 26% (at +10 mV) in Endo and Mid myocytes, respectively. The rates of sarcoplasmic reticulum (SR) Ca(2+) release and decay of the [Ca(2+)](i) were slowed, and the amplitude of the [Ca(2+)](i) was depressed in Endo and Epi myocytes isolated from failing, compared with normal, hearts. Experiments in sodium (Na(+))-free solutions showed that Epi and Mid myocytes of the failing ventricle exhibit a greater reliance on the Na(+)-Ca(2+) exchanger to remove cytosolic Ca(2+) than myocytes isolated from normal hearts. Simulation studies in Endo, Mid, and Epi canine myocytes demonstrate the importance of L-type current density and SR Ca(2+) uptake in modulating the potentially arrhythmogenic repolarization in HF. In conclusion, these results demonstrate that spatially heterogeneous decreases in I(Ca,L) and defective cytosolic Ca(2+) removal contribute to the altered [Ca(2+)](i) and AP profiles across the canine failing LV. These distinct electrophysiological features in myocytes from a failing heart contribute to a characteristic electrogram arising from increased dispersion of refractoriness across the LV, which may result in significant arrhythmogenic sequellae.     

Catecholaminergic polymorphic ventricular tachycardia-related mutations R33Q and L167H alter calcium sensitivity of human cardiac calsequestrin

Two missense mutations, R33Q and L167H, of hCASQ2 (human cardiac calsequestrin), a protein segregated to the lumen of the sarcoplasmic reticulum, are linked to the autosomal recessive form of CPVT (catecholaminergic polymorphic ventricular tachycardia). The effects of these mutations on the conformational, stability and Ca(2+) sensitivity properties of hCASQ2, were investigated. Recombinant WT (wild-type) and mutant CASQ2s were purified to homogeneity and characterized by spectroscopic (CD and fluorescence) and biochemical (size-exclusion chromatography and limited proteolysis) methods at 500 and 100 mM KCl, with or without Ca(2+) at a physiological intraluminal concentration of 1 mM; Ca(2+)-induced polymerization properties were studied by turbidimetry. In the absence of Ca(2+), mutations did not alter the conformation of monomeric CASQ2. For L167H only, at 100 mM KCl, emission fluorescence changes suggested tertiary structure alterations. Limited proteolysis showed that amino acid substitutions enhanced the conformational flexibility of CASQ2 mutants, which became more susceptible to tryptic cleavage, in the order L167H>R33Q>WT. Ca(2+) at a concentration of 1 mM amplified such differences: Ca(2+) stabilized WT CASQ2 against urea denaturation and tryptic cleavage, whereas this effect was reduced in R33Q and absent in L167H. Increasing [Ca(2+)] induced polymerization and precipitation of R33Q, but not that of L167H, which was insensitive to Ca(2+). Based on CASQ2 models, we propose that the Arg(33)-->Gln exchange made the Ca(2+)-dependent formation of front-to-front dimers more difficult, whereas the Leu(167)-->His replacement almost completely inhibited back-to-back dimer interactions. Initial molecular events of CPVT pathogenesis begin to unveil and appear to be different depending upon the specific CASQ2 mutation.