Sheep Cardiac Muscle Research Articles

Elevated Homocysteine Levels Result in Carbonyl Formation on RyR2 and Enhanced Sensitivity of Sarcoplasmic Reticulum to Activation by Calcium

Elevated levels in blood serum of homocysteine (>10µmol/L) is strongly correlated with the incidence of heart failure (HF) in humans. We demonstrate that the cyclic thioester, homocysteine thiolactone (HTL), a metabolic product of homocysteine, at a concentration of 100 nM, results in the formation of carbonyls on the ryanodine receptor from sheep cardiac muscle (RyR2), and an enhancement of Ca2+ dependent activation of ryanodine binding from 129.7 ± 3.4 nM to 102.0 ± 2.7 nM. While both improper Ca2+ handling and elevated homocysteine levels have been considered bio-markers in HF, a direct connection between the two has not previously been made. We propose that HTL reacts with lysine residues on RyR2, generating an Ne-homocysteine-protein, which results in carbonyl formation and an enhancement in the Ca2+ sensitivity of RyR2. This is a new molecular mechanism linking elevated levels of Homocysteine, post-translational modification of RyR2, improper Ca2+ handling and heart failure. This work was supported by NIH 1 R41 HL105063-01 to J. Abramson and R. Strongin.

Quantification of calsequestrin 2 (CSQ2) in sheep cardiac muscle and Ca2+-binding protein changes in CSQ2 knockout mice

Calsequestrin 2 (CSQ2) is generally regarded as the primary Ca2+-buffering molecule present inside the sarcoplasmic reticulum (SR) in cardiac cells, but findings from CSQ2 knockout experiments raise major questions about its role and necessity. This study determined the absolute amount of CSQ2 present in cardiac ventricular muscle to gauge its likely influence on SR free Ca2+ concentration ([Ca2+]) and maximal Ca2+ capacity. Ventricular tissue from hearts of freshly killed sheep was examined by SDS-PAGE without any fractionation, and CSQ2 was detected by Western blotting; this method avoided the >90% loss of CSQ2 occurring with usual fractionation procedures. Band intensities were compared against those for purified CSQ2 run on the same blots. Fidelity of quantification was verified by demonstrating that CSQ2 added to homogenates was detected with equal efficacy as purified CSQ2 alone. Ventricular tissue from sheep (n=8) contained 24±2 μmol CSQ2/kg wet wt. Total Ca2+ content of the ventricular tissue, measured by atomic absorption spectroscopy, was 430±20 μmol/kg (with SR Ca2+ likely<250 μmol/kg) and displayed a linear correlation with CSQ2 content, with gradient of ∼10 Ca2+ per CSQ2. The large amount of CSQ2 bestows the SR with a high theoretical maximal Ca2+-binding capacity (∼1 mmol Ca2+/kg ventricular tissue, assuming a maximum of ∼40 Ca2+ per CSQ2) and would keep free [Ca2+] within the SR relatively low, energetically favoring Ca2+ uptake and reducing SR leak. In mice with CSQ2 ablated, histidine-rich Ca2+-binding protein was upregulated ∼35% in ventricular tissue, possibly in compensation.

Porin-type 1 proteins in sarcoplasmic reticulum and plasmalemma of striated muscle fibres.

The location of porin-type 1 proteins in mammalian striated muscle has been assessed using immunogold electron microscopy with an anti-porin 31HL monoclonal antibody as the primary antibody. Gold particles were found on the mitochondrial outer membrane, the sarcoplasmic reticulum and plasmalemma in longitudinal sections of rat and rabbit skeletal muscle and rabbit and sheep cardiac muscle. The relative densities of gold particles in the mitochondrial outer membrane, sarcoplasmic reticulum and plasmalemma were 7:3:1 in white sternomastoid muscle, for example. Skeletal and cardiac sarcoplasmic reticulum vesicles, which had been fractionated by discontinuous sucrose density centrifugation, were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting. The anti-porin 31HL monoclonal antibody detected a band of relative molecular mass (M(r)) 31,000 in all muscle sarcoplasmic reticulum vesicle fractions and also in liver mitochondria. The intensity of immunostaining of the sarcoplasmic reticulum fractions was 2.5-10% that of mitochondrial outer membranes per microgram of membrane protein blotted. Contamination of the sarcoplasmic reticulum fractions by mitochondrial outer membrane was < 0.75% as determined from the specific activity of monoamine oxidase. Thus, only a small part of the porin detected in sarcoplasmic reticulum vesicles can be attributed to mitochondrial contamination. These results show that porin-type1 immunoreactivity is not restricted to mitochondria but found in the sarcoplasmic reticulum and plasmalemma of both mammalian skeletal and cardiac muscle.

Ion selectivity of porcine skeletal muscle Ca2+ release channels is unaffected by the Arg615 to Cys615 mutation

The Arg615 to Cys615 mutation of the sarcoplasmic reticulum (SR) Ca2+ release channel of malignant hyperthermia susceptible (MHS) pigs results in a decreased sensitivity of the channel to inhibitory Ca2+ concentrations. To investigate whether this mutation also affects the ion selectivity filter of the channel, the monovalent cation conductances and ion permeability ratios of single Ca2+ release channels incorporated into planar lipid bilayers were compared. Monovalent cation conductances in symmetrical solutions were: Li+, 183 pS +/- 3 (n = 21); Na+, 474 pS +/- 6 (n = 29); K+, 771 pS +/- 7 (n = 29); Rb+, 502 pS +/- 10 (n = 22); and Cs+, 527 pS +/- 5 (n = 16). The single-channel conductances of MHS and normal Ca2+ release channel were not significantly different for any of the monovalent cations tested. Permeability ratios measured under biionic conditions had the permeability sequence Ca2+ >> Li+ > Na+ > K+ > or Rb+ > Cs+, with no significant difference noted between MHS and normal channels. This systematic examination of the conduction properties of the pig skeletal muscle Ca2+ release channel indicated a higher Ca2+ selectivity (PCa2+:Pk+ approximately 15.5) than the sixfold Ca2+ selectivity previously reported for rabbit skeletal (Smith et al., 1988) or sheep cardiac muscle (Tinker et al., 1992) Ca2+ release channels. These results also indicate that although Ca2+ regulation of Ca2+ release channel activity is altered, the Arg615 to Cys615 mutation of the porcine Ca2+ release channel does not affect the conductance or ion selectivity properties of the channel.

A model for ionic conduction in the ryanodine receptor channel of sheep cardiac muscle sarcoplasmic reticulum.

A model is developed for ionic conduction in the sheep cardiac sarcoplasmic reticulum ryanodine receptor channel based on Eyring rate theory. A simple scheme is proposed founded on single-ion occupancy and an energy profile with four barriers and three binding sites. The model is able to quantitatively predict a large number of conduction properties of the purified and native receptor with monovalent and divalent cations as permeant species. It suggests that discrimination between divalent and monovalent cations is due to a high affinity central binding site and a process that favors the passage of divalent cations between binding sites. Furthermore, differences in conductance among the group Ia cations and among the alkaline earths are largely explained by differing affinity at this putative central binding site.

Divalent cation conduction in the ryanodine receptor channel of sheep cardiac muscle sarcoplasmic reticulum.

The conduction properties of the alkaline earth divalent cations were determined in the purified sheep cardiac sarcoplasmic reticulum ryanodine receptor channel after reconstitution into planar phospholipid bilayers. Under bi-ionic conditions there was little difference in permeability among Ba2+, Ca2+, Sr2+, and Mg2+. However, there was a significant difference between the divalent cations and K+, with the divalent cations between 5.8- and 6.7-fold more permeant. Single-channel conductances were determined under symmetrical ionic conditions with 210 mM Ba2+ and Sr2+ and from the single-channel current-voltage relationship under bi-ionic conditions with 210 mM divalent cations and 210 mM K+. Single-channel conductance ranged from 202 pS for Ba2+ to 89 pS for Mg2+ and fell in the sequence Ba2+ greater than Sr2+ greater than Ca2+ greater than Mg2+. Near-maximal single-channel conductance is observed at concentrations as low as 2 mM Ba2+. Single-channel conductance and current measurements in mixtures of Ba(2+)-Mg2+ and Ba(2+)-Ca2+ reveal no anomalous behavior as the mole fraction of the ions is varied. The Ca(2+)-K+ reversal potential determined under bi-ionic conditions was independent of the absolute value of the ion concentrations. The data are compatible with the ryanodine receptor channel acting as a high conductance channel displaying moderate discrimination between divalent and monovalent cations. The channel behaves as though ion translocation occurs in single file with at most one ion able to occupy the conduction pathway at a time.

Large tetraalkyl ammonium cations produce a reduced conductance state in the sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel

The purified Ca(2+)-release/ryanodine receptor channel of the sheep cardiac muscle sarcoplasmic reticulum (SR) functions as a calcium-activated cation-selective channel under voltage clamp conditions following reconstitution into planar phospholipid bilayers. We have investigated the effect of large tetraalkyl ammonium (TAA) cations, (CnH2n+1)4N+ (n = 4 and 5) on monovalent cation conduction. These cations modify the conductance of the receptor channel at positive holding potentials from the cytosolic side of the channel. Under these conditions, openings are resolved as a mixture of normal full amplitude events and events of reduced conductance. The amplitude of the reduced conductance state is a fixed proportion of the normal open state. As a proportion of all open events, the occurrence of the tetrabutyl ammonium (TBA+) related subconductance state increases with concentration and increasingly positive holding potential. The TBA+ related subconductance state displays similar conduction properties to the unmodified channel; with a linear current-voltage relationship, a similar affinity for K+ and voltage-dependent block by TEA+. A method was used to quantify the voltage dependence of the occurrence of the TBA+ effect, which yielded an effective gating charge of 1.66. A second method based on kinetic analysis of the voltage dependence of transitions between the full open state and the TBA+ related subconductance state produced a similar value. In addition, this analysis revealed that the bulk of the voltage-dependence resided in the off rate. TBA+ related subconductance events, expressed as a proportion of all open events, saturated with increasing TBA+ concentration. Kinetic analysis revealed that this could be entirely accounted for by changes in the on rate. Tetrapentyl ammonium (TPeA+) causes a qualitatively similar effect with a subconductance state of lower amplitude. The voltage-dependence of the effect was comparable to that displayed by TBA+. These findings are interpreted as a form of partial block in which more than one large TAA cation binds at the extremity of the voltage drop to produce an electrostatic barrier for ion translocation.

Block of the sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel by tetra-alkyl ammonium cations.

The purified ryanodine receptor channel of the sheep cardiac muscle sarcoplasmic reticulum (SR) membrane functions as a calcium-activated cation-selective channel under voltage-clamp conditions following reconstitution into planar phospholipid bilayers. We have investigated the effects of the tetra-alkyl ammonium (TAA) cations, (CnH2n+1)4N+ and the trimethyl ammonium cations, ethyltrimethyl ammonium and propyltrimethyl ammonium, on potassium conductance through the receptor channel. Small TAA cations (n = 1-3) and the trimethyl ammonium derivatives act as asymmetric, voltage-dependent blockers of potassium current. Quantitative analysis of the voltage dependence of block indicates that the conduction pathway of the sheep cardiac SR ryanodine receptor channel contains two distinct sites for the interaction of these small organic cations. Sites are located at approximately 50% for tetramethyl ammonium (TMA+) and 90% for tetraethyl ammonium (TEA+) and tetrapropyl ammonium (TPrA+) of the voltage drop across the channel from the cytosolic face of the protein. The chemical substitution of an ethyl or propyl group for one of the methyl groups in TMA+ increases the voltage dependence of block to a level similar to that of TEA+ and TPrA+. The zero-voltage dissociation constant (Kb(0)) falls with the increasing number of methyl and methylene groups for those blockers acting 90% of the way across the voltage drop. This is interpreted as suggesting a hydrophobic binding site at this point in the conduction pathway. The degree of block increases as the concentration of small TAA cations is raised. The concentration dependence of tetraethyl ammonium block indicates that the cation interacts with a single site within the conduction pathway with a Km of 9.8 +/- 1.7 mM (mean +/- SD) at 40 mV. Larger TAA cations (n = 4-5) do not induce voltage-dependent block of potassium current of the form seen with the smaller TAA cations. These data support the contention that the sheep cardiac SR ryanodine receptor channel may be occupied by at most one ion at a time and suggest that a large proportion of the voltage drop falls over a relatively wide region of the conduction pathway.

Monovalent cation conductance in the ryanodine receptor‐channel of sheep cardiac muscle sarcoplasmic reticulum.

1. The ryanodine receptor protein of sheep cardiac muscle sarcoplasmic reticulum membranes functions as a ligand-regulated ion channel following solubilization with the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1- propane sulphonate); purification by density gradient centrifugation, reconstitution into proteo-liposomes and incorporation into planar phospholipid bilayers. 2. In the absence of divalent cations, measurable conductance is observed with the group 1a cations and with some larger organic cations. In symmetric 210 mM solutions the following conductance sequence was determined: K+ greater than Rb+ = NH4+ greater than Na+ = Cs+ greater than Li+ much greater than Tris+. 3. Other organic cations, e.g. TEA+, do not produce measurable current under these conditions. 4. Single-channel conductance saturates with increasing ionic activities of K+, Na+ and Li+. Saturation curves are described by Michaelis-Menten kinetic schemes with the following values of maximal conductance and apparent dissociation constant: K+ 900 pS, 19.9 mM; Na+ 516 pS, 17.8 mM; Li+ 248 pS, 9.1 mM. 5. The channel displays only minor differences in permeability amongst the group 1a cations. Relative permeability, monitored under bi-ionic conditions, yields the following sequence: Na+, 1.15 greater than K+, 1.00 = Li+, 0.99 greater than Rb+, 0.87 greater than Cs+, 0.61. Under similar conditions the permeability ratio of NH4+ to K+ was found to be 1.32 and that for Tris+ to K+ was 0.22. 6. The K+ conductance is reduced by low concentrations of the impermeant cation TEA+. Block appears as a smooth reduction in single-channel current amplitude and the degree of block is dependent upon applied voltage. These observations are consistent with a single-site blocking scheme in which TEA+ has access to a site within the voltage drop of the channel from only the cytosolic face of the channel protein and interacts with a site located approximately 90% of the electrical distance across the channel. The zero-voltage dissociation constant for TEA+ block is 50 mM. 7. Single-channel conductance measurements in mixtures of K(+)-Na+ and K(+)-Li+ reveal no anomalous behaviour as the mole fraction of the ions is varied. 8. With monovalent cations as permeant species, the sheep cardiac sarcoplasmic reticulum ryanodine receptor protein functions as a poorly selective, ligand-regulated channel. Under the conditions described here the channel functions as a single-ion pore. It is proposed that discrimination is largely dependent upon the strength of interaction of the permeant ion with a binding site in the conduction pathway.

Functional characterisation of the ryanodine receptor purified from sheep cardiac muscle sarcoplasmic reticulum

Sheep cardiac muscle sarcoplasmic reticulum ryanodine receptors have been isolated by density-gradient centrifugation following solubilisation with the zwitterionic detergent, CHAPS. The functional state of the receptor complex has been assessed by quantification of [ 3H]ryanodine binding and by characterisation of single-channel conductance and gating properties following reconstitution into unilamellar proteo-liposomes and incorporation into planar phospholipid bilayers. A method of solubilisation is described which yields a receptor displaying high-affinity [ 3H]ryanodine binding ( K d 2.8 nM, B max 352 pmol/mg protein) and which functions as a cation-selective, ligand-regulated channel under voltage clamp conditions. Previous reports of channel activity of purified rabbit skeletal and canine cardiac muscle ryanodine receptors describe a range of sub- or variable-conductance events. In contrast, the sheep cardiac ryanodine receptor-channels isolated using the optimal conditions described in this report consistently display a single open state conductance with either Ca 2+ or K + as the charge carrying species.

Sulmazole (AR-L 115BS) activates the sheep cardiac muscle sarcoplasmic reticulum calcium-release channel in the presence and absence of calcium

The properties of calcium-release channels of sheep cardiac muscle junctional sarcoplasmic reticulum (SR), have been investigated under voltage-clamp conditions following the fusion of isolated membrane vesicles with planar phospholipid bilayers. In the presence of activating calcium on the cytosolic side of the membrane, additions of the benzimidazole derivative sulmazole (AR-L 115BS) increased the open probability (Po) of the channel reaching saturating values of 1.0 at 3 mM sulmazole. The drug did not affect single-channel conductance and activation was readily reversible. Analysis of channel open and closed lifetimes suggested that low concentrations of sulmazole (0.1 mM) may sensitize the channel to activating calcium, while at higher concentrations (1 mM and above), calcium and sulmazole act synergistically to produce a unique gating scheme for the channel. Millimolar concentrations of sulmazole also stimulate a degree of channel opening at subactivating (60 pM) calcium concentrations. Openings occurring under these conditions show very different kinetics to those of the calcium-activated channel but have an identical single-channel conductance and are modified by ATP, magnesium, ruthenium red and ryanodine in a similar manner to the calcium-activated channel. The release of calcium from the SR following the activation of the calcium-release channel by sulmazole may contribute to the positive inotropic action of this drug on mammalian cardiac muscle.

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

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

The effects of membrane potential, extracellular potassium, and tetrodotoxin on the intracellular sodium ion activity of sheep cardiac muscle.

The intracellular sodium ion activity was measured using liquid ion-exchange microelectrodes with rapid response times in sheep Purkinje fibers and ventricular muscle under voltage control. The mean sodium ion activity in quiescent Purkinje fibers was 8.5 mM at a holding potential of -80 mV. With maintained hyperpolarizing (-110 mV) or depolarizing (-40 and 0 mV) voltage steps, sodium ion activity increased or decreased, respectively. At 0 mV, the mean steady state value for the sodium ion activity was 3.8 mM. Following a voltage step to 0 mV, or back to -80 mV, the time course of the sodium ion activity change could be fitted by single exponentials, with similar half-times. Increasing the extracellular potassium ion concentration from 5.4 to 15 mM did not alter the steady state value of the sodium ion activity at clamped voltages of -80 or 0 mV, which suggests that the external potassium ion activating site of the Na-K pump was saturated. With the extracellular potassium concentration 0 mM (holding potential -80 mV), the sodium ion activity increased. When maintained depolarizing steps to 0 mV were applied, the sodium ion activity decreased by up to 20 mM. This large fall in sodium ion activity is assumed to represent partial reactivation of the Na-K pump due to potassium ion accumulation in clefts. We also studied the stimulation-dependent change in sodium ion activity. Trains of action potentials or short duration depolarizing voltage clamp steps caused a frequency dependent rise in sodium ion activity. The magnitude of the rise of sodium ion activity was not altered by lengthening the duration of each voltage clamp step, but was inhibited by tetrodotoxin or by holding the membrane potential at -50 mV between depolarizing steps. These results show that sodium ion activity is a complex function of membrane voltage, depolarization frequency, and time. The rise in sodium ion activity with stimulation appears to depend on sodium ion entry regulated by the sodium channel, and may be important in the modulation of intracellular calcium and tension through the Na+-Ca++ exchange mechanism.

The interaction of ouabain and salicylate on sheep cardiac muscle.

1. The action potential duration in sheep ventricular fibres is rapidly diminished on exposure to 10(-6) M-ouabain. However, if 10--20 mM-sodium salicylate is added to the ouabain solution, glycoside-induced shortening is prevented, and a substantial increase in duration then occurs. Sodium salicylate also reverses the shortening effect of ouabain if applied after the glycoside has been allowed to act alone. 2. Sodium salicylate alone produces a much smaller prolongation than in the presence of ouabain. Alone and in the presence of ouabain it eventually increases the threshold and produces inexcitability. 3. Three other surface charge agents have been compared with salicylate: aminonaphthalene sulphonate, sodium dodecylsulphate and salicylamide, were unable to counter the actions of ouabain at the concentrations used. Since they also produced no changes in excitability it is likely that they did not bind significantly to the cell membrane. 4. In Purkinje fibres the reversal potential for the pacemaker current, iK2, is initially shifted in a negative direction in the presence of 10(-6) M-ouabain and 10 mM-salicylate instead of the positive shift expected with ouabain alone at this concentration. 5. In guinea-pig ventricle, salicylate alone reduces the duration of the action potential. This effect is rapidly reversible. Toxic levels of ouabain also reduce the action potential duration but this effect takes several hours to reverse. By contrast, the effects of salicylate and ouabain applied together are readily reversible. 6. It is suggested that the mechanism of these effects may depend on the ability of a surface negative charge agent like salicylate to increase the surface K+ concentration at the membrane and so protect the sodium-potassium pump from inhibition by large doses of ouabain.

High potassium and low sodium contractures in sheep cardiac muscle.

Contractures develop in sheep atrial trabeculae if Tyrode's solution is rapidly replaced by a solution containing elevated potassium, reduced sodium, or both. Two phases of the contracture can be identified on the basis of differences in physiological behavior: a rapid and transient phase that predominates during the first few seconds of the contracture, and a slowly developed phase that is responsible for the steady level of tension reached later in the contracture. The transient phase is particularly prominent if the muscle is stimulated rapidly before the contracture, and reduced or absent if the muscle is not stimulated or if calcium is not present before the contracture. Recovery of the transient phase after a contracture parallels the recovery of twitches. This transient phase appears to reflect the depolarization-induced release of activator (calcium) from an internal store, possibly the same store that is involved in the normal contraction. The slowly developed tension is dependent on the contracture solution used, and is decreased if the calcium concentration is reduced or if the sodium concentration is increased. It does not depend on conditions before the contracture and does not require time to recover. This phase of the contracture may be due to entry of calcium from the extracellular solution.

Relationship of the Sarcoplasmic Reticulum to Sarcolemma in Sheep Cardiac Muscle

SINCE Huxley and Taylor1 demonstrated that in striated muscle each myofibril segment probably receives an individual stimulus to contract, electron microscope studies have suggested that the most likely pathway for such stimuli from the sarcolemma to the myofibrils is the sarcoplasmic reticulum2, particularly3 its transverse tubular elements (also named the ‘transverse sarcotubular system’4). Theories about this have recently been elaborated and the evidence has been reviewed5,6. Such theories are necessarily based on the possibility that the membrane of the tubules is polarized and is capable of undergoing depolarization in the same way as the plasma membrane (inner layer of sarcolemma).

In vitro Studies of a Growth Inhibitory Fraction Obtained from Adult Sheep Cardiac Muscle.

In vitro Studies of a Growth Inhibitory Fraction Obtained from Adult Sheep Cardiac Muscle.