Dog Cardiac Sarcoplasmic Reticulum Research Articles

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

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

FK-binding Protein Is Associated with the Ryanodine Receptor of Skeletal Muscle in Vertebrate Animals

The ryanodine receptor/calcium release channel (RyR1) of sarcoplasmic reticulum from rabbit skeletal muscle terminal cisternae (TC) contains four tightly associated FK506-binding proteins (FKBP12). Dissociation and reconstitution studies have shown that RyR1 can be modulated by FKBP12, which helps to maintain the channel in the quiescent state. In this study, we found that the association of FKBP with RyR1 of skeletal muscle is common to each of the five classes of vertebrates. TC from skeletal muscle representing animals from different vertebrates, i.e. mammals (rabbit), birds (chicken), reptiles (turtle), fish (salmon and rainbow trout), and amphibians (frog), were isolated. For each, we find the following: 1) FKBP12 is localized to the TC (there are four FKBP binding sites/ryanodine receptor); 2) soluble FKBP exchanges with the bound form on RyR1 of TC; 3) release of FKBP from terminal cisternae by drug (FK590) treatment leads to a significant reduction in the net calcium loading rate, consistent with channel activation (the calcium loading rate is restored to the control value by reconstitution with FKBP12); and 4) RyR1 of skeletal muscle TC can bind to and exchange with either FKBP12 or FKBP12.6 (FKBP12.6 is the novel FKBP isoform found selectively associated with RyR2 of dog cardiac sarcoplasmic reticulum). We conclude that FKBP is an integral part of the RyR1 of skeletal muscle in each of the classes of vertebrate animals. The studies are consistent with a role for FKBP in skeletal muscle excitation-contraction coupling.

CADP-ribose releases Ca2+ from cardiac sarcoplasmic reticulum independently of ryanodine receptor.

Cyclic ADP-ribose (cADPR), an endogenous metabolite of beta-NAD+, activates Ca2+ release from endoplasmic reticulum in sea urchin eggs via the ryanodine receptor (RyR) pathway. A similar role has been proposed in cardiac sarcoplasmic reticulum (SR), although this remains controversial. We therefore investigated the ability of cADPR to induce Ca2+ release from canine cardiac SR microsomes using fluo 3 to monitor extravesicular Ca2+ concentration. We found that cADPR induced Ca2+ release in a concentration-dependent manner, whereas neither its precursor, NAD+, nor its metabolite, ADP-ribose, elicited a consistent effect. In addition, an additive effect on calcium release between cADPR and 9-Me-7-Br-eudistomin-D (MBED), an activator of RyR, was found as well as no cross-desensitization between cADPR and MBED. Specific blockers of the RyR did not abolish the cADPR-induced Ca2+ release. These results provide evidence for cADPR-induced Ca2+ release from dog cardiac SR via a novel mechanism which is independent of RyR activation.

Reversal of Phospholamban-Induced Inhibition of Cardiac Sarcoplasmic Reticulum Ca2+-ATPase by Tannin

The plant phenol tannin stimulated severalfold the Ca2+-dependent ATPase and Ca2+-uptake activities of dog cardiac sarcoplasmic reticulum (SR) with an EC50 value of 0.6 μM, The stimulation was due to a marked increase in the apparent affinity of the cardiac SR ATPase for Ca2+ ions while the Vmax was not affected. No stimulation of skeletal muscle SR preparations could be observed. The characteristics of stimulation were similar to those observed after phosphorylation of the regulatory protein phospholamban (PLN) by protein kinase A. The ability of protein kinase A to phosphorylate PLN was prevented by tannin with an IC50 of 3 μM. Phosphorylation of troponin I, another physiological substrate of protein kinase A, was resistant to tannin inhibition. The data show that submicromolar concentrations of tannin prevent PLN phosphorylation by interacting with the cytosolic portion of PLN. The specific binding of tannin reverses the inhibition that PLN exerts on cardiac SR ATPase.

Protein and lipid rotational dynamics in cardiac and skeletal sarcoplasmic reticulum detected by EPR and phosphorescence anisotropy.

We have used time-resolved phosphorescence anisotropy and electron paramagnetic resonance (EPR) spectroscopy to detect the rotational dynamics of the Ca-ATPase and its associated lipids in dog cardiac sarcoplasmic reticulum (DCSR), in comparison with rabbit skeletal SR (RSSR), in order to obtain insight into the physical bases for different activities and regulation in the two systems. Protein rotational motions were studied with time-resolved phosphorescence anisotropy (TPA) of erythrosin isothiocyanate (ERITC) and saturation-transfer EPR (ST-EPR) of a maleimide spin-label (MSL). Both labels were attached selectively and rigidly to the Ca-ATPase. Lipid rotational motions were studied with conventional EPR of stearic acid spin-labels. As in previous studies on RSSR, the phosphorescence anisotropy decays of both preparations at 4 degrees C were multiexponential, due to the presence of different oligomeric species. The rotational correlation times for the different rotating species were similar for the two preparations, but the total decay amplitude was substantially less for cardiac SR, indicating that more of the Ca-ATPase molecules are in large aggregates in DCSR. ST-EPR spectra confirmed that the Ca-ATPase is less rotationally mobile in DCSR than in RSSR. Lipid probe mobility and fatty acid composition were very similar in the two preparations, indicating that the large differences observed in protein mobility are not due to differences in lipid fluidity. We conclude that the higher restriction in protein mobility observed by both ST-EPR and TPA is due to more extensive protein-protein interactions in DCSR than in RSSR.

The effect of pH on the calcium dependence of calcium accumulation in dog cardiac muscle sarcoplasmic reticulum

Net Ca 2+ accumulation in vesicles of dog cardiac sarcoplasmic reticulum (CSR) was evaluated at three different pHs: 6.0, 6.8 and 7.6. The Ca 2+ sequestration by CSR depends on Ca 2+ concentration and on pH values. The curves that show the relationship between Ca 2+ accumulated by CSR and external Ca 2+ concentrations were shifted with pH changes, both in the absence and in the presence of potassium oxalate. Considering the curve at pH 6.8 as reference, a lower Ca concentration was needed to obtain the half-maximal value in Ca sequestration under pH 7.6 (0.04 ± 0.006 and 0.79 ± 0.09 μ m at pH 7.6 and 6.8, respectively). Opposite results were obtained under pH 6.0 (13.66 ± 1.29 μ m). Net calcium release during active accumulation of Ca 2+ and Ca 2+ efflux from passively 45Ca 2+ loaded CSR microsomes were significantly higher at alkaline pH than at acidic pH. The results suggest than in CSR alkaline pH would promote the increase in the rates of both, Ca 2+ release and active Ca 2+ accumulation, while opposite effects would be expected under acidic pH. Therefore, pH changes may regulate both, the Ca 2+ level upon which the SR Ca 2+ pump works (permeability effect) and the sequestration rate of the Ca 2+ pump (variation in the affinity for calcium).

Heterogeneous distribution of calmodulin- and cAMP-dependent regulation of Ca2+ uptake in cardiac sarcoplasmic reticulum subfractions.

The activity of the Ca2+-pumping ATPase of cardiac sarcoplasmic reticulum is controlled by the phosphorylation level of the intrinsic membrane protein phospholamban. Phospholamban monomers contain two distinct phosphorylation sites for either the cAMP-dependent or the calmodulin-dependent kinase. The two kinases, however, preferentially phosphorylate different populations of phospholamban molecules and double phosphorylation of the same subunit by their concerted action is a phenomenon that occurs only under particular experimental conditions. This study investigates the phosphorylation pattern of phospholamban in various subfractions derived from dog cardiac sarcoplasmic reticulum. The results show that the endogenous calmodulin-dependent kinase preferentially phosphorylates the phospholamban population found in association with the cisternal compartments of the reticulum. The differential phosphorylation occurs despite the presence of sufficient amounts of the kinase in all sarcoplasmic reticulum subfractions. On the other hand, phospholamban molecules localized on the longitudinal system are preferential substrates for the cAMP-dependent kinase. Possibly, the different lipid and/or protein microenvironment of phospholamban in the various sarcoplasmic reticulum domains is responsible for the apparent heterogeneity of phosphorylation. The present findings are compatible with the concept of additive and independent action of the cAMP-dependent and calmodulin-dependent kinases on cardiac sarcoplasmic reticulum. The imply, however, that different regions of the sarcoplasmic reticulum network are controlled by the two regulatory mechanisms.

Phosphoenzyme decomposition in dog cardiac sarcoplasmic reticulum Ca2+-ATPase.

A five-syringe quench-flow apparatus was used in the transient-state kinetic study of intermediary phosphoenzyme (EP) decomposition in a Triton X-100 purified dog cardiac sarcoplasmic reticulum (SR) Ca2+-ATPase at 20 degrees C. Phosphorylation of the enzyme by ATP in the presence of 100 mM K+ for 116 ms gave 32% ADP-sensitive E1P, 52% ADP- and K+-reactive E2P, and 16% unreactive residual EPr. The EP underwent a monomeric, sequential E1P 17 s-1----E2P 10.5 s-1----E2 + Pi transformation and decomposition in the ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid quenched Ca2+-devoid medium. The calculated rate constant for the total EP (i.e., E1P + E2P) dephosphorylation was 7.8 s-1. The E1P had an affinity for ADP with an apparent Kd congruent to 100 microM. When the EP was formed in the absence of K+ for 116 ms, no appreciable amount of the ADP-sensitive E1P was detected. The EP comprised about 80% ADP- and K+-reactive E2P and 20% residual EPr, suggesting a rapid E1P----E2P transformation. Both the E2P's formed in the presence and absence of K+ decomposed with a rate constant of about 19.5 s-1 in the presence of 80 mM K+ and 2 mM ADP, showing an ADP enhancement of the E2P decomposition. The results demonstrate mechanistic differences in monomeric EP transformation and decomposition between the Triton X-100 purified cardiac SR Ca2+-ATPase and deoxycholate-purified skeletal enzyme [Wang, T. (1986) J. Biol. Chem. 261, 6307-6319].

Effects of verapamil, diltiazem, nisoldipine and felodipine on sarcoplasmic reticulum

Verapamil, diltiazem, nisoldipine and felodipine, calcium antagonist drugs with different chemical structures, were studied for their effects on activities of sarcoplasmic reticulum (SR) isolated from dog cardiac and rabbit skeletal muscles. Nisoldipine and felodipine exerted biphasic actions on both cardiac and skeletal SR Ca2+-ATPase with maximum activation of 40–60% occurring at 20–40 μM for nisoldipine and 30–40% occurring at 15–30 μM for felodipine. At higher drug concentrations, Ca2+-ATPase was inhibited. In the presence of oxalate the maximum activation of the Ca2+ uptake rates at 5–20 μM nisoldipine were 30–50% for cardiac SR and 80–100 μM of the drug were 300–500% for skeletal SR. Felodipine inhibited the rate of Ca2+ uptake by dog cardiac SR, but activated Ca2+ uptake by rabbit skeletal SR with a maximum of 30–50% at 12–25 μM. At higher concentrations of the two drugs the rate of Ca2+ uptake was inhibited. In the absence of oxalate, i.e., limited tranport, nisoldipine shortened the duration of time that Ca2+ was bound to the cardiac and skeletal SR, while the rate of release of Ca2+ from skeletal SR was stimulated. Felodipine at low concentrations similarly caused a premature release of Ca2+ from skeletal SR at a rapid rate; at high concentrations both drugs did not alter Ca2+ binding but delayed Ca2+ release. Unlike nisoldipine and felodipine, verapamil and diltiazem inhibited the rates of Ca2+ transport both in cardiac and skeletal SR. The two drugs inhibited Ca2+-ATPase in cardiac SR but activated the enzyme in skeletal SR. Thus, these drugs caused complex and different effects on cardiac and skeletal SR, possibly resulting from perturbations of the lipid environment of the SR Ca2+-ATPase.

Studies on the Mechanism of the Cardiotonic Activity of MDL 19205

MDL 19205, 4-ethyl-1,3-dihydro-5-(4-pyridinyl-carbonyl)-2H-imidazol-2-one, is a new drug with cardiotonic properties. Its effects on several biochemical systems considered to be important in myocardial contraction were investigated. Cyclic nucleotide phosphodiesterases (PDEs) from dog hearts were separated into three isoenzymes, F I, F II, and F III, and effect of the drug on these enzymes was tested. MDL 19205 inhibited F III PDE specifically and produced little or no inhibition of F I and F II PDEs. The IC50 for inhibition of F III PDE was 8.6 microM when 0.5 microM cyclic AMP (cAMP) was used, whereas no more than 10% inhibition of F I and 18% of F II PDEs occurred at drug concentrations up to 200 microM when 1 microM cAMP was used. Concentrations of MDL 19205 up to 100 microM had no effect on Ca2+-adenosine triphosphatase (ATPase) or Ca2+ uptake by dog cardiac sarcoplasmic reticulum. At 100 microM, the drug produced a weak (18%) inhibition of Na+,K+-ATPase. It is suggested that inhibition of F III PDE may be the primary mechanism by which MDL 19205 produces its cardiotonic effect. Inhibition of Na+,K+-ATPase may also be involved at very high concentrations of this drug.

Correlation between calmodulin‐dependent increase in the rate of calcium transport and calmodulin‐dependent phosphorylation of cardiac sarcoplasmic reticulum

The aim of the present study was to prove a correlation between the calmodulin-dependent increase in the rate of calcium transport by dog cardiac sarcoplasmic reticulum and calmodulin-dependent phosphorylation. The dependence of phosphorylation on the total calmodulin concentration at 75 microM and 1 microM free calcium gave apparent calmodulin half-saturation constants Km (CaM) of 9.4 nM and 181 nM, respectively, whilst the apparent Km (CaM) for the rate of calmodulin-stimulated calcium transport carried out at 1 microM calcium, but phosphorylated prior to the calcium uptake at 75 microM or 1 microM calcium, were 12.5 nM and 127 nM, respectively. A positive correlation was obtained between calmodulin-dependent increase in the rate of calcium transport and hydroxylamine-insensitive phosphoester formed by the calcium/calmodulin-regulated, membrane-bound protein kinase. More than 90% of incorporated [32P]phosphate is confined to a 26-28-kDa or 9-11-kDa protein as determined by polyacrylamide gel electrophoresis following solubilization in sodium dodecyl sulfate at 37 degrees C and at 100 degrees C, respectively, similar to the results obtained by phosphorylation with cAMP-dependent protein kinase. The data indicate that calmodulin-dependent phosphorylation of the above protein(s) is causally related to the stimulation of the rate of calcium transport by cardiac sarcoplasmic reticulum, which is at least partially due to a shift in the calcium dependence of the rate of calcium transport to lower free calcium concentrations, K(Ca), of 1.25 microM and 0.61 microM in controls and calmodulin-dependent phosphorylation, respectively. Activation of calmodulin-dependent phosphorylation by free calcium at total calmodulin concentrations of 300 nM, 100 nM and 30 nM gave apparent K(Ca) values of 0.83 microM, 1.44 microM and 2.3 microM and Hill coefficients of 4.13, 3.76 and 3.79, respectively, indicating that all four calcium binding sites of calmodulin have to be saturated to obtain activation of the calcium/calmodulin-regulated protein kinase. The calmodulin-dependent modulation of calcium transport in vivo is, therefore, determined to great extent by the total calmodulin concentration present in the sarcoplasm.

Influence of monovalent cations on the Ca 2+-ATPase of sarcoplasmic reticulum isolated from rabbit skeletal and dog cardiac muscles. An interpretation of transient-state kinetic data

Transient-state kinetics of phosphorylation and dephosphorylation of the Ca 2+-ATPase of sarcoplasmic reticulum vesicles from rabbit skeletal and dog cardiac muscles were studied in the presence of varying concentrations of monovalent and divalent cations. Monovalent cations affect the two types of sarcoplasmic reticulum differently. When the rabbit skeletal sarcoplasmic reticulum was Ca 2+ deficient, preincubation with K + (as compared with preincubation with choline chloride) did not affect initial phosphorylation at various concentrations of Ca 2+, added with ATP to phosphorylate the enzyme. This is in contrast to preincubation with K + of the Ca 2+-deficient dog cardiac sarcoplasmic reticulum, which resulted in an increase in the phosphoenzyme level. When Ca 2+ was bound to the rabbit skeletal sarcoplasmic reticulum, K + inhibited E ~ P formation; but under the same conditions, E ~ P formation of dog cardiac sarcoplasmic reticulum was activated by K + at 12 μM Ca 2+ and inhibited at 0.33 and 1.3 μM Ca 2+. Li +, Na + and K + also have different effects on E ~ P decomposition of skeletal and cardiac sarcoplasmic reticulum. The latter responded less to these cations than the former. Studies with ADP revealed differences between the two types of sarcoplasmic reticulum. For rabbit skeletal sarcoplasmic reticulum, 40% of the phosphoenzyme formed was ‘ADP sensitive’, and the decay of the remaining E ~ P was enhanced by K + and ADP. Dog cardiac sarcoplasmic reticulum yielded about 40–48% ADP-sensitive E ~ P, but the decomposition rate of the remaining E ~ P was close to the rate measured in the absence of ADP. Thus, these studies showed certain qualitative differences in the transformation and decomposition of phosphoenzymes between skeletal and cardiac muscle which may have bearing on physiological differences between the two muscle types.

Temperature-dependency of the functional activities of dog cardiac sarcoplasmic reticulum: A comparison with sarcoplasmic reticulum from rabbit and lobster muscle

Temperature-dependency studies of the functional activities of sarcoplasmic reticulum vesicles from different muscle sources have been carried out. The energies of activation of transport and hydrolytic processes of microsomes from dog heart have been found to be higher than those obtained with rabbit and lobster skeletal muscle. This difference is reduced in the presence of membrane perturbing agents as the detergent Triton X-100 or the Ca 2+-ionophore A-23187, indicating that the different level of the functional ability of microsomal vesicles isolated from different tissues might be due mainly to differences in the lipid environment. Thermodynamic parameters ( ΔG ‡ , ΔH ‡ , ΔS ‡ of the Ca 2+-transport and Ca 2+-dependent ATPase reactions of cardiac microsomes pre-phosphorylated with cAMP-dependent protein kinase have been determined. The stimulatory effect of protein kinase can be interpreted as a change in the fluidity of the hydrophobic microenvironment of the ATPase molecules. The transport process if entropically more favourable in pre-phosphorylated than in control microsomes.

Effects of adenosine 3':5'-monophosphate-dependent protein kinase on sarcoplasmic reticulum isolated from cardiac and slow and fast contracting skeletal muscles.

Effects of cyclic adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase were studied in sarcoplasmic reticulum prepared from cardiac and slow and fast (white) skeletal muscle. Cyclic AMP-dependent protein kinase failed to catalyze phosphorylation of fast skeletal muscle microsomes as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cyclic AMP-dependent protein kinase was without effect on calcium uptake by these microsomes. Treatment of cardiac microsomes obtained from dog, cat, rabbit, and guinea pig with cyclic AMP-dependent protein kinase and ATP resulted in phosphorylation of a 22,000-dalton protein component in the amounts of 0.75, 0.25, 0.30, and 0.14 nmol of phosphorus/mg of microsomal protein, respectively. Calcium uptake by cardiac microsomes was stimulated 1.8- to 2.5-fold when microsomes were treated with cyclic AMP-dependent protein kinase. Protein kinases partially purified from bovine heart and rabbit skeletal muscle were both effective in mediating these effects on phosphorylation and calcium transport in dog cardiac sarcoplasmic reticulum. Slow skeletal muscle sarcoplasmic reticulum also contains a protein with a molecular weight of approximately 22,000 that can be phosphorylated by protein kinase. Phosphorylation of this component ranged from 0.005 to 0.016 nmol of phosphorous/mg of microsomal protein in dog biceps femoris. A statistically significant increase in calcium uptake by these membranes was produced by the protein kinase. Increases in protein kinase-catalyzed phosphorylation of a low molecular weight microsomal component and in calcium transport by sarcoplasmic reticulum of cardiac and slow skeletal muscle may be related to the relaxation-promoting effects of epinephrine seen in these types of muscle. Conversely, the absence of a relaxation-promoting effect of epinephrine in fast skeletal muscle may be associated with the lack of effect of cyclic AMP and protein kinase on calcium transport by the sarcoplasmic reticulum of this type of muscle.

Effect of ions on sarcoplasmic reticulum fragments

Fragments of cardiac sarcoplasmic reticulum (FSR) were prepared from homogenates of dog hearts and skeletal FSR from rabbit white skeletal muscle by differential centrifugation. The effect of substituting NO 3 −, I −, and SCN − for Cl on the ATPase and Ca uptake of fragmented dog cardiac sarcoplasmic reticulum (grana) was studied. Calcium uptake and ATPase were depressed in the order SCN − > I − > N0 3 −, the sequence found for skeletal FSR (2). The effects of the divalent cations Zn 2+ and UO 2 2+ were studied on both rabbit white skeletal muscle and dog cardiac FSR; 1 m m Zn 2+ and uranyl depressed calcium uptake of both preparations. The results are discussed in terms of the effects of these ions on cardiac and skeletal muscle contraction and current views of excitation-contraction coupling.