Cardiac Muscle Sarcoplasmic Reticulum Research Articles

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).

Molecular cloning of cDNAs from human kidney coding for two alternatively spliced products of the cardiac Ca2+-ATPase gene.

Ca2+-ATPase molecules present in the microsomal fraction from non-muscle cells were examined immunologically. Rabbit whole brain, cerebellum, liver, kidney, and COS-1 cell microsomes all displayed a polypeptide of about 110 kDa which was immunoreactive with a polyclonal antiserum against the cardiac muscle sarcoplasmic reticulum Ca2+-ATPase molecule, but was not immunoreactive with a monoclonal antibody specific for the fast-twitch muscle Ca2+-ATPase. cDNAs encoding the full length of two Ca2+-ATPase molecules were isolated from a human kidney library using a mixture of nucleotide probes derived from both rabbit fast-twitch and cardiac muscle Ca2+-ATPase cDNAs. The human kidney cDNAs, HK1 and HK2, are the products of alternative splicing. HK2 codes for a protein identical to rabbit cardiac muscle Ca2+-ATPase, with the exception of 6 scattered amino acid replacements, whereas HK1 codes for a protein identical to that encoded by HK2, but with the carboxyl-terminal 4 amino acids replaced by an extended sequence of 49 amino acids. cDNAs of the HK1 type are by far the most abundant in the library. The partial structure of a 40-kilobase genomic DNA encoding all but the 5' end of the human cardiac Ca2+-ATPase is described. The exons which give rise to the alternatively spliced products were located by Southern blotting and sequencing, and the alternative splicing patterns were determined.

Inhibition of rapid Ca-release from isolated skeletal and cardiac sarcoplasmic reticulum (SR) membranes

Rapid Ca-release from the cisternal compartments isolated from skeletal and cardiac muscle SR was characterized by the use of inhibitors. Ruthenium red (RR) completely blocked (IC 50 = 90 nM) the Ca-channels of skeletal SR. Its effect on the rapid Ca-release from cardiac SR was marginal but became optimal (IC 50 = 200 nM) in the presence of FLA 365 ([2,6-dichloro-4-dimethyl-aminophenyl] isopropylamine) which by itself had no measurable effect. The antibiotic neomycin mimicked the properties of RR. The strong synergistic effect of RR or neomycin and FLA 365 indicates that either cardiac cisternae contain two distinct isoforms of Ca-release channel, or that different drugs are needed to effectively block the same channel.

Ryanodine binding to sarcoplasmic reticulum membrane; comparison between cardiac and skeletal muscle

[ 3H]Ryanodine binding to skeletal muscle and cardic sarcoplasmic reticulum (SR) vesicles was compared under experimental conditions known to inhibit or stimulate Ca 2+ release. In the skeletal muscle SR, ryanodine binds to a single class of high-affinity sites ( K d of 11.3 nM). In cardiac SR vesicles, more than one class of binding sites is observed ( d values of 3.6 and 28.1 nM). Ryanodine binding to skeletal muscle SR vesicles requires high concentrations of NaCl, whereas binding of the drug to cardiac SR is only slightly influenced by ionic strength. In the presence of 5′-adenylyl imidodiphosphate (p[NH]ppA), increased pH, and micromolar concentration of Ca 2+ (which all induce Ca 2+ release from SR) binding of ryanodine to SR is significantly increased in skeletal muscle, while being unchanged in cardiac muscle. Ryanodine binding to skeletal but not to cardiac muscle SR is inhibited in the presence of high Ca 2+ or Mg 2+ concentrations (all known to inhibit Ca 2+ release from skeletal muscle SR). Ruthenium red or dicyclohexylcarbodiimide modification of cardiac and skeletal muscle SR inhibit Ca 2+ release and ryanodine binding in both skeletal and cardiac membranes. These results indicate that significant differences exist in the properties of ryanodine binding to skeletal or cardiac muscle SR. Our data suggest that ryanodine binds preferably to site(s) which are accessible only when the Ca 2+ release channel is in the open state.

Biochemical characterization of the Ca2+ release channel of skeletal and cardiac sarcoplasmic reticulum

Rapid mixing-vesicle ion flux and planar lipid bilayer-single channel measurements have shown that a high-conductance, ligand-gated Ca2+ release channel is present in 'heavy', junctional-derived membrane fractions of skeletal and cardiac muscle sarcoplasmic reticulum. Using the release channel-specific probe, ryanodine, a 30S protein complex composed of polypeptides of Mr approximately 400,000 has been isolated from cardiac and skeletal muscle. Reconstitution of the complex into planar lipid bilayers has revealed a Ca2+ conductance with properties characteristic of the native Ca2+ release channel.

Lysophospholipid-mediated alterations in the calcium transport systems of skeletal and cardiac muscle sarcoplasmic reticulum.

The effects of various lysophospholipids on the calcium transport activity of sarcoplasmic reticulum (SR) from rabbit skeletal and canine cardiac muscles were examined. The lipids decreased calcium transport activity in both membrane types; the effectiveness being in the order lysoPC greater than lsyoPS, lysoPG greater than lysoPE. The maximum inhibition induced by lysoPC, lysoPG and lysoPS was greater than 85% of the normal Ca2+-transport rate. In cardiac SR lysoPE had a maximal inhibition of about 50%. Half maximal inhibition of calcium transport by lysoPC was achieved at 110 nmoles lysoPC/mg SR. At this concentration of lysoPC, the (Ca2+ + Mg2+)-ATPase and Ca2+-uptake activities were inhibited to the same extent (about 60%) in skeletal sarcoplasmic reticulum, while in cardiac sarcoplasmic reticulum, there was less than 20% inhibition of the Ca2+ + Mg2+-ATPase activity. Studies with EGTA-induced passive calcium efflux showed that up to 200 nmoles lysoPC/mg SR did not alter calcium permeability significantly in cardiac sarcoplasmic reticulum. In skeletal muscle membranes the lysophospholipid mediated decrease in calcium uptake correlated well with the increase in passive calcium efflux due to lysophosphatidylcholine. The difference in the lysophospholipid-induced effects on the sarcoplasmic reticulum from the two muscle types probably reflects variations in protein and other membrane components related to the respective calcium transport systems.

Phospholamban stoichiometry in canine cardiac muscle sarcoplasmic reticulum.

Treatment of cardiac sarcoplasmic reticulum with the crosslinking reagent dithiobis (succinimidyl propionate) in the presence of 125I-calmodulin, resulted in the formation of a 40,000-dalton affinity labeled component, consisting of a 1:1, phospholamban: 125I-calmodulin complex. In parallel experiments, sarcoplasmic reticulum was phosphorylated in the presence of calmodulin and [gamma-32P]ATP, and then treated with the crosslinking reagent to produce an affinity labeled component consisting of a 1:1, calmodulin: 32P-phospholamban complex. These experiments permitted determination of the amount of 125I and 32P incorporated into the 40,000-dalton complexes, as well as the amount of 32P incorporated into the 23,000-dalton form of phospholamban. If 1 mol of Ca2+-dependent ATPase phosphoprotein represents 1 mol of 100,000-dalton Ca2+-dependent ATPase monomer, then there are 4.88 +/- 1.33 mol Ca2+-dependent ATPase/mol of phospholamban. If there are 2 mol of Ca2+-dependent ATPase phosphoprotein/mol of 100,000-dalton Ca2+-dependent ATPase monomer, then there are 9.76 +/- 2.66 mol Ca2+-dependent ATPase/mol phospholamban.

Immunoelectron microscopical localization of phospholamban in adult canine ventricular muscle.

The subcellular distribution of phospholamban in adult canine ventricular myocardial cells was determined by the indirect immunogold-labeling technique. The results presented suggest that phospholamban, like the Ca2+-ATPase, is uniformly distributed in the network sarcoplasmic reticulum but absent from the junctional portion of the junctional sarcoplasmic reticulum. Unlike the Ca2+-ATPase, but like cardiac calsequestrin, phospholamban also appears to be present in the corbular sarcoplasmic reticulum. Comparison of the relative distribution of phospholamban immunolabeling in the sarcoplasmic reticulum with that of the sarcolemma showed that the density of phospholamban in the network sarcoplasmic reticulum was approximately 35-fold higher than that of the cytoplasmic side of the sarcolemma, which in turn was found to be three- to fourfold higher than the density of the background labeling. However, a majority of the specific phospholamban labeling within 30 nm of the cytoplasmic side of the sarcolemma was clustered and present over the sarcoplasmic reticulum in the subsarcolemmal region of the myocardial cells, suggesting that phospholamban is confined to the junctional regions between the sarcolemma and the sarcoplasmic reticulum, but absent from the nonjunctional portion of the sarcolemma. Although the resolution of the immunogold-labeling technique used (60 nm) does not permit one to determine whether the specific labeling within 30 nm of the cytoplasmic side of the sarcolemma is associated with the sarcolemma and/or the junctional sarcoplasmic reticulum, it is likely that the low amount of labeling in this region represents phospholamban associated with sarcoplasmic reticulum. These results suggest that phospholamban is absent from the sarcolemma and confined to the sarcoplasmic reticulum in cardiac muscle.

The mechanism of nucleotide induced calcium translocation across sarcoplasmic reticulum membranes: Evidence for a non-translocated intermediate pool of calcium

Our previous data suggested that, in cardiac muscle sarcoplasmic reticulum fragments, GTP hydrolysis occurs by an alternative enzyme cycle of the Ca2+ ATPase which is insensitive to (Ca2+) and does not involve an acyl phosphate intermediate. Despite this, GTP induces the incorporation of calcium into a membrane pool that is not translocated to the vesicular lumen. The present study suggests that this GTP-induced intermediate calcium pool is identical to a modulable component of the calcium translocation process in that: it has an identical pH sensitivity; the initial incorporation of calcium in response to GTP eliminates the initial rapid burst and lag component of the typical ATP-induced calcium uptake curve when ATP is added during GTP-induced calcium accumulation. Instead, the addition of ATP during GTP-induced calcium accumulation results in the prompt onset of the linear phase of calcium translocation; GTP-induced calcium accumulation directly affects the pH sensitivity of subsequent ATP-induced calcium accumulation. We suggest that the intermediate calcium pool is in series with calcium translocation and is the site of the pH sensitivity observed in calcium flux.

Thyroidectomy of SHR: effects on ventricular relaxation and on SR calcium uptake activity.

Ventricular hypertrophy and hypothyroidism are each characterized by impaired cardiac muscle relaxation and sarcoplasmic reticulum (SR) calcium uptake activity. A previous report also showed that hypothyroidism does not reverse ventricular hypertrophy (left-to-right ventricular weight ratios) of spontaneously hypertensive rats (SHR). We characterized the effects of thyroidectomy of 8 wk duration on relaxation of ejecting hearts and on SR calcium uptake activity from SHR and nonhypertrophic Wistar-Kyoto rat (WKY) controls. Relaxation was quantified by plotting maximum left ventricular pulse pressure (Pmax) vs. the area under the falling phase of the left ventricular pressure wave at three different pressure loads. Ventricles of euthyroid SHR were characterized by impaired relaxation and depressed SR calcium uptake activity compared with those of euthyroid WKY, confirming earlier studies. Thyroidectomy reduced ventricular relaxation and SR calcium uptake activities to about the same extent in SHR and WKY strains so that these measurements were most depressed in the SHR hypothyroid group. When all groups were considered, the extent of mechanical relaxation ex vivo and the rate of SR calcium uptake in vitro were well correlated.

Ca-induced Ca release in the sarcoplasmic reticulum of cardiac muscle of the guinea pig

Ca-induced Ca release in the sarcoplasmic reticulum of cardiac muscle of the guinea pig

A monovalent cation-selective channel of sheep cardiac muscle sarcoplasmic reticulum (SR)

A monovalent cation-selective channel of sheep cardiac muscle sarcoplasmic reticulum (SR)

Asymmetric block of a monovalent cation-selective channel of rabbit cardiac sarcoplasmic reticulum by succinyl choline

We have investigated the effect of the skeletal muscle relaxant succinyl choline (SC) on the conduction of potassium ions through a monovalent cation-selective channel present in the cardiac muscle sarcoplasmic reticulum membrane (CSR). This channel has been studied under voltage-clamp conditions following the fusion of purified CSR membrane vesicles with preformed planar phospholipid bilayers. The channel assumes a fixed orientation in the bilayer and displays two conducting states (B. Tomlins, A.J. Williams & R.A.P. Montgomery, 1984, J. Membrane Biol. 80:191-199). SC blocks potassium conductance through the channel in a voltage-dependent manner. Block occurs from both sides of the channel, in both conducting states and is resolved as discrete "flickering" events. Although SC is capable of blocking potassium conductance from both sides of the membrane, block is asymmetric. The zero-voltage dissociation constant for block from the cis side of the membrane is approximately threefold lower than that from the trans side. Block from the cis side displays a linear dependence on SC concentration for both open states and is competitive with potassium ions at saturating potassium activities, consistent with a single-site blocking model. The degree of SC-induced block is also influenced by membrane surface charge. SC block differs from that previously described for bis quaternary ammonium (bis Qn) compounds such as decamethonium in that SC blocks preferentially from the cis side of the channel.

Skeletal muscle relaxing agents as probes of a monovalent cation selective channel of cardiac muscle sarcoplasmic reticulum (SR)

Skeletal muscle relaxing agents as probes of a monovalent cation selective channel of cardiac muscle sarcoplasmic reticulum (SR)

Rapid purification of calsequestrin from cardiac and skeletal muscle sarcoplasmic reticulum vesicles by Ca2+-dependent elution from phenyl-sepharose.

Treatment of cardiac or skeletal muscle sarcoplasmic reticulum vesicles with 0.1 M sodium carbonate selectively extracts both the Ca2+-binding protein calsequestrin and the two "intrinsic glycoproteins," while leaving the Ca2+-dependent ATPase membrane bound. Phenyl-Sepharose chromatography in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and high salt (0.5 M NaCl) readily fractionates these solubilized proteins into a Ca2+-elutable fraction, which contains purified calsequestrin, and a low ionic strength elutable fraction, which contains one of the two intrinsic glycoproteins. Elution of calsequestrin from phenyl-Sepharose occurs near 1 mM Ca2+. Copurifying with calsequestrin are an homologous set of high molecular weight proteins, which like calsequestrin stain blue with Stains-All. These proteins are present in trace amounts and do not correspond to any sarcoplasmic reticulum proteins previously identified. Elution of calsequestrin from phenyl-Sepharose is consistent with the Ca2+-binding protein losing its hydrophobic character in the presence of millimolar Ca2+. This behavior is converse to that observed for several calmodulin-like proteins, which are eluted from hydrophobic gels in the presence of EGTA. The high yield and purity of calsequestrin prepared by this method makes possible a unique system for studying what may be a distinct class of Ca2+-binding proteins.

Nucleotide triphosphate utilization by cardiac and skeletal muscle sarcoplasmic reticulum. Further evidence for an alternative substrate hydrolysis cycle and the effect of calcium NTPase purification.

It was previously observed that the hydrolysis of GTP by cardiac sarcoplasmic reticulum (SR) (in contrast to skeletal muscle SR: (a) was identical in rate with total ATP hydrolysis; (b) gave a similar nonlinear substrate response; (c) was not Ca2+ dependent; and (d) stimulated Ca2+ accumulation but not Ca2+ translocation. Evidence was presented that both the ATPase and GTPase are effected by the same enzyme and represent different hydrolysis cycles (Van Winkle, W. B., Tate, C. A., Bick, R. J., and Entman, M. L. (1981) J. Biol. Chem. 256, 2268-2274). In the present paper, we report that purification of the NTPase from both muscle sources resulted in an alteration in the NTP concentration response compatible with a single high affinity binding site for ATP only in cardiac SR and for both substrates in skeletal muscle SR. As is the case with native skeletal muscle SR, purified skeletal muscle NTPase hydrolyzed GTP in a manner qualitatively similar to ATP (but with no Ca2+-independent NTPase) but with reduced velocity. In contrast, there was no GTPase activity or Ca2+-independent "basic" ATPase activity in the purified cardiac NTPase. Inclusion of oxalate or the ionophore, A23187, in assays with cardiac SR and ATP as the substrate increased the total ATPase activity but had no effect on GTPase activity. Furthermore, the nucleotide-dependent uptake of oxalate by cardiac SR was only apparent with ATP and not with GTP. In the presence of Ca2+, ATP was a potent inhibitor (noncompetitive, Ki of 2-5 microM) of GTPase activity, whereas it was a weaker competitive inhibitor in the absence of Ca2+. We suggest that GTPase and basic ATPase represent similar alternative enzyme cycles for the CaATPase enzyme that are inhibited by the presence of ATP plus Ca2+ but are rendered inactive during the purification of cardiac NTPase.

Ryanodine modification of cardiac muscle responses to potassium-free solutions. Evidence for inhibition of sarcoplasmic reticulum calcium release.

To test whether ryanodine blocks the release of calcium from the sarcoplasmic reticulum in cardiac muscle, we examined its effects on the aftercontractions and transient depolarizations or transient inward currents developed by guinea pig papillary muscles and voltage-clamped calf cardiac Purkinje fibers in potassium-free solutions. Ryanodine (0.1-1.0 microM) abolished or prevented aftercontractions and transient depolarizations by the papillary muscles without affecting any of the other sequelae of potassium removal. In the presence of 4.7 mM potassium and at a stimulation rate of 1 Hz, ryanodine had only a small variable effect on papillary muscle force development and action potential characteristics. In calf Purkinje fibers, ryanodine (1 nM-1 microM) completely blocked the aftercontractions and transient inward currents without altering the steady state current-voltage relationship. Ryanodine also abolished the twitch in potassium-free solutions, but it enhanced the tonic force during depolarizing voltage-clamp steps. This latter effect was dependent on the combination of ryanodine and potassium-free solutions. The slow inward current was not blocked by 1 microM ryanodine, but ryanodine did appear to abolish an outward current that remained in the presence of 0.5 mM 4-aminopyridine. Our observations are consistent with the hypothesis that ryanodine, by inhibiting the release of calcium from the sarcoplasmic reticulum, prevents the oscillations in intracellular calcium that activate the transient inward currents and aftercontractions associated with calcium overload states.

Factors that modify the molecular size of phospholamban, the 23,000-dalton cardiac sarcoplasmic reticulum phosphoprotein.

Phospholamban, a 23,000-dalton phosphoprotein present in cardiac muscle sarcoplasmic reticulum vesicles is quantitatively dissociated into three smaller sized phosphorylated components of 11,000, 15,000, and 20,000 daltons when sarcoplasmic reticulum, solubilized in 1% (w/v) sodium dodecyl sulfate and 1 mM MgCl2, is heated at 71 degrees C or greater for 2 min. This dissociation is inhibited by Mg2+ (50% at approximately 5 mM). The 23,000-dalton phosphoprotein reformed from the 11,000-, 15,000-, and 20,000-dalton phosphorylated components when phosphorylated sarcoplasmic reticulum that had been boiled in 1% sodium dodecyl sulfate and 1 MM MgCl2 was stored for 1 week at -70 degrees C. We propose that the 23,000-dalton phosphorylated protein is a trimer, composed of three subunits with molecular mass of 11,000, 8,000, and 4,000 daltons. In this model, only the 11,000-dalton subunit would be phosphorylated. The partial dissociation of the 23,000-dalton phosphorylated protein would result in the formation of the 19,000 (11,000 + 8,000)-dalton or the 15,000 (11,000 + 4,000)- dalton phosphorylated components. Full dissociation of the 23,000-dalton phosphorylated protein would result in the formation of the 11,000-dalton phosphorylated component.

Characterization of calmodulin-mediated phosphorylation of cardiac muscle sarcoplasmic reticulum

Sarcoplasmic reticulum, isolated from canine cardiac muscle, was phosphorylated in the presence of exogenous cAMP-dependent protein kinase or calmodulin. This phosphorylation has been shown previously to activate sarcoplasmic reticulum calcium uptake (LePeuch et al. (1979) Biochemistry 18, 5150–5157). Calmodulin appeared to activate an endogenous protein kinase present in sarcoplasmic reticulum membranes. The incorporation of phosphate increased with time. However, once all the ATP was consumed, the level of phosphorylated protein started to decrease due to the action of an endogenous protein phosphatase. Dephosphorylation occurred even when the level of phosphorylated sarcoplasmic reticulum remained constant at high ATP concentrations. The phosphorylation of sarcoplasmic reticulum in the presence of calmodulin, increased as the pH was increased from pH 5.5 to 8.5. This phosphorylation was only inhibited by KCl concentrations greater than 100 m m. The apparent K m of cAMP-dependent protein kinase for ATP was 5.2 ± 0.2 × 10 −5 m, and of the calmodulin-dependent protein kinase for ATP was 3.67 ± 0.29 × 10 −5 m. Phosphorylation was maximally activated by 5–10 m m MgCl 2; higher MgCl 2 concentrations inhibited this phosphorylation. Thus the calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum could be maximally activated at sarcoplasmic concentrations of K +, Mg 2+, and ATP. The calmodulindependent phosphorylation was half-maximally activated at Ca 2+ concentrations that were significantly greater than those required to promote the formation of the sarcoplasmic reticulum Ca-activated ATPase phosphoprotein intermediate. Thus at sarcoplasmic Ca 2+ concentrations that might be expected during systole, the sarcoplasmic reticulum calcium pump would be fully activated before any significant calmodul-independent sarcoplasmic reticulum phosphorylation occurred. However, under certain pathological conditions when the sarcoplasmic Ca 2+ becomes elevated (e.g., in ischemia) the kinase could be activated so that the sarcoplasmic reticulum would be phosphorylated and calcium uptake augmented. Thus, the calmodulin-dependent protein kinase may only function when the heart needs to rescue itself from a possibly fatal calcium overload.

Permeability of canine cardiac sarcoplasmic reticulum vesicles to K+, Na+, H+, and Cl-.

Cardiac muscle sarcoplasmic reticulum appears to contain channel-like structures that render the membrane permeable to small univalent ions. Canine heart microsomes fractionated according to buoyant density were examined by Millipore filtration, light scattering, and membrane potential m easurements. Enzymatic analysis and measurement of D-glucose permeation and Na/Ca exchange systems indicated two membrane fractions suitable for the permeability studies, one enriched in surface membranes with a buoyant density of 1.04-1.11 (10-25% sucrose) and one enriched in sarcoplasmic reticulum with a buoyant density of 1.13-1.15 (30-34% sucrose). Surface membrane vesicles impermeable to [3H]sucrose were largely impermeable to K+, Na+, and Cl-, while sarcoplasmic reticulum vesicles impermeable to [3H]sucrose were readily permeable to K+, Na+, H+, and Cl-. Sarcoplasmic reticulum vesicles were essentially impermeable to Ca2+, Mg2+, choline+, gluconate-, 1,4-piperazinediethanesulfonic acid (Pipes-), and D-glucose. These results suggest that cardiac muscle sarcoplasmic reticulum contains structures that facilitate the movement of small univalent ions. A possible function of these putative ion-conducting structures may be to allow rapid ion fluxes to counter electrogenic Ca2+ fluxes across sarcoplasmic reticulum during cardiac muscle contraction and relaxation.