Myocardial Sarcoplasmic Reticulum Research Articles

Haemodynamics and myocardial metabolism of phosphorus depleted dogs: effects of catecholamines and angiotensin II.

The responses of arterial pressure and myocardial contractile force (VPM) to infusion of angiotensin II, noradrenaline and orciprenaline were examined in twelve dogs during a control phase, after 30 days of dietary phosphorus deprivation and after 21 days of phosphorus repletion. In the phosphorus depletion period, animals had low skeletal and heart muscle Pi content, low magnesium, ATP and creatine phosphate in skeletal and heart muscle with no change of ADP, AMP or energy charge. In the basal state, VPM was diminished with no change of end-diastolic and systolic pressure. Infusion of angiotensin II caused a significantly smaller rise of arterial pressure (angiotensin II resistance), and the stimulatory effect of noradrenaline and orciprenaline on VPM was diminished (catecholamine resistance). These effects were reversible with Pi repletion. In phosphorus depletion, arterial concentrations were increased for lactate, unchanged for FFA and decreased for acetoacetate/beta-hydroxybutyrate. Unchanged myocardial extraction of lactate or beta-hydroxybutyrate and preserved cell Pi uptake for glycogenolysis were observed. The initial rate of uptake of calcium and concentrating ability of myocardial sarcoplasmic reticulum were unchanged.

Electron probe x-ray microanalysis and cryoultramicrotomy of unstained myocardial sarcoplasmic reticulum, in situ and fragmented

Myocardial sarcoplasmic reticulum of cats in situ and fragmented sarcoplasmic reticulum (FSR) were analysed using X-ray microanalysis, cryoultramicrotomy and scanning transmission electron microscopy. 2 types of FSR vesicles can be distinguished morphologically and by their different elemental composition especially by different Ca loading. The Ca content of the sarcoplasmic reticulum can also be detected in situ.

Quantitation of calcium in native myocardial and fractioned sarco-plasmic reticulum by electron probe X-ray microanalysis

The importance of the sarcoplasmic reticulum in the regulation of myocardial contraction is today still unclear. X-ray microanalysis of elements in the cell compartments involved in excitation-contraction coupling can contribute to the understanding of this question if the following prerequisites are met: treatment of the tissue should not alter the distribution of diffusible ions, and the method of detection should be sufficiently sensitive.In order to test whether electron probe microanalysis is suitable for myocardial Ca in sarcoplasmic reticulum, ultrathin cryosections were taken from native myocardial tissue and from isolated sarcoplasmic reticulum of the cat. This fragmented sarcoplasmic reticulum (FSR) was chosen for two reasons: first, if the x-ray microanalysis of isolated active SR vesicles maximally loaded with Ca did not show Ca, further experiments on myocardial tissue using this method would have been useless. Second, if Ca identification is possible using this method, the isolated FSR can be utilized as a standard for quantitation of Ca in native tissue since the amount of Ca taken up in the FSR is known before the analysis and can be arbitrarily varied.

Enhanced phosphorylation of myocardial sarcoplasmic reticulum in experimental hyperthyroidism

Calcium transport by cardiac sarcoplasmic reticulum (SR) was compared in hyperthyroid (HT) and euthyroid (ET) rats. Both Ca2+ uptake (97 +/- 3.1 nmol/mg per min in HT vs. 63 +/- 2.9 nmol/mg per min in ET, P less than 0.01) and CA2+ -stimulated ATPase activity (61 +/- 4.1 vs. 37 +/- 1.6 nmol Pi/mg per min, P less than 0.01) were higher in the thyroxine-treated animals. These changes were accompanied by enhanced cyclic AMP-dependent phosphorylation of cardiac SR in hyperthyroid rats (180 +/- 4.3 pmol Pi/mg per min vs. 117 +/- 4.2 pmol Pi/mg per min, P less than 0.01). SDS-polyacrylamide gel electrophoresis of cardiac SR showed that phosphorylation of a 22,000-dalton protein (phospholamban) primarily accounted for the differences between the two groups. There was no difference in the rate of SR dephosphorylation by endogenous phosphoprotein phosphatase between HT and ET rats. Differences in cyclic AMP-dependent phosphorylation between the two groups were blunted in the presence of excess exogenous cyclic AMP-dependent protein kinase. These results suggest that increased levels or activity of endogenous cyclic AMP-dependent protein kinases may partially explain enhanced calcium transport by the cardiac SR of hyperthyroid animals.

Cytochemical staining procedures selective for sarcotubular systems of muscle: Modifications and applications

The diaminobenzidine (DAB)-ferrocyanide cytochemical procedure for staining of myocardial sarcoplasmic reticulum (SR), first described by Waugh and his co-workers, has been subjected to further experimentation and modification. We have found, on the basis of studies utilizing inhibitory substances and conditions, that the staining reaction in the SR apparently is not attributable to endogenous peroxidase activity within the lumina of the SR tubules and saccules (nor, for that matter, to the activity of any species of enzyme). Perfusion of the vascular system with DAB-H2O2-Tris solution is unnecessary. In fact, exposure of tissues to DAB itself is superfluous, since mere postfixation in osmium—ferrocyanide solution serves to produce SR staining in muscle cells that have been fixed in an aldehyde solution containing calcium (or other divalent or trivalent cations), but lacking phosphate. This technique is applicable to both cardiac and skeletal muscle cells of mammals and lower vertebrates; it has been applied with success to mammalian vascular and nonvascular smooth muscle as well. Some degree of filling (or staining) of the system of extracellular spaces [ECS, including, in mammalian myocardium, the T-axial tubular system (TAx)] often occurs, which appears to be the result of colloid formation by the osmium ferrocyanide. SR staining in skeletal muscle usually is inseparable from T-system filling, although filling of T tubules may occur in the absence of SR staining. Choice of concentration or type of aldehyde(s) in the fixative solution does not appreciably alter the staining; however, exposure of tissues to aldehyde fixative which either does not contain calcium or contains phosphate brings about filling of the ECS-TAx without SR staining, thereby mimicking in effect the results obtained by more complicated tracer techniques.

The effect of taurine on cardiac sarcoplasmic reticulum

A recent communication reported that the rate of calcium uptake by sarcoplasmic reticulum (SR) isolated from rat skeletal muscle could be increased by the isolation of the SR in 15 mM taurine, and that exposure of the SR to taurine throughout the isolation procedure resulted in an increased yield of SR. Because of these results in rat skeletal muscle SR studies were carried out on dog myocardial SR. Sarcoplasmic reticulum isolated from adult dog cardiac muscle was not affected by taurine in concentrations as high as 15 millimolar. The addition of taurine to isolation media did not affect calcium transport, ATPase, binding, or release. Sarcoplasmic reticulum fragments were stored and re-examined over a period of a week without appreciable difference in stability of activity between those isolated in the presence of taurine and the control group. This lack of effect suggests that the role of taurine in cardiac muscle metabolism is not likely to be found in regulation of the sarcoplasmic reticulum.

The effect of gram negative endotoxin on the calcium uptake activity of sarcoplasmic reticulum isolated from canine myocardium

Gram negative endotoxin (Salmonella abortus equi) significantly inhibited the ATP dependent calcium uptake of canine myocardial sarcoplasmic reticulum (SR). The degree of inhibition was dependent upon the concentration of the endotoxin and its time of exposure to the SR. Significant inhibition was seen at endotoxin concentrations ranging between 0.024 mg/ml and 0.24 mg/ml. Inhibition, at a dose of 0.24 mg/ml, varied between 10.1 ± 0.1 percent with no preincubation to 61.0 ± 5.0 percent after fifteen minutes of preincubation. The relation between depression of the sarcotubular calcium pump and myocardial depression is discussed.

Factors affecting the uptake of calcium by canine myocardial sarcoplasmic reticulum.

1. Cardiac sarcoplasmic reticulum was prepared from canine ventricular muscle by differential centrifugation of a muscle homogenate and was purified in a surose gradient and the mean protein yield was 48.2 mg. per 100 gram myocardium. The uptake of45calcium was measured under various experimental conditions. 2. In the presence of 2×10−4 M calcium chloride the uptake of45Ca was 1 micromole per mg. protein after eight minutes although there was slight variation from experiment to experiment and according to the time interval following isolation of the sarcoplasmic reticulum. 3. Omission of magnesium from the incubation mixture inhibited45Ca uptake (81%), while parallel increase of both Mg and ATP concentrations produced an increased calcium uptake. When sodium or potassium were omitted from the incubation mixture activity was also reduced. 4. Potassium cyanide had no effect on calcium uptake, and neither did digoxin in concentrations 10−7 to 10−5 M even when the sarcoplasmic reticulum was exposed to digoxin for one hour prior to the addition of the45Ca. 5. Noradrenaline (10−3 and 10−4 M) and isoprenaline (10−3 and 10−4 M) both caused an increased accumulation of calcium to approximately the same extent, while lignocaine in a higher concentration caused a depression of calcium accumulation. 6. It is concluded that canine myocardial sarcoplasmic reticulum accumulates calcium actively by a process which requires ATP and magnesium for ATPase activation. It is also dependent on sodium and potassium and is not dependent on oxidative phosphorylation. Positive inotropic agents increase the rate of calcium accumulation wheras the negative inotropic drug, lignocaine, depresses the rate of calcium accumulation. The action of these agents on myocardial contractile force may be explained by this effect.

Relation between plasma lignocaine levels and induced haemodynamic changes.

Intravenous lignocaine (1 mg./kg. body weight) was found to produce insignificant haemodynamic changes, and in particular no reduction in myocardial contractility. A rate of 2 mg./minute infused intravenously is suggested for therapeutic purposes.In anaesthetized dogs an infusion of 13.5 mg./minute caused moderate haemodynamic depression and a maximum plasma level of 7 mug./ml. Massive injections of 200 and 400 mg. of lignocaine produced a maximum plasma level of 13.8 and 27.8 mug./ml., respectively, and in the latter failure of myocardial contraction in the presence of a normal E.C.G. ensued ("pump failure"). Lignocaine appears to alter the uptake of calcium by myocardial sarcoplasmic reticulum, and this may explain the negative inotropic effect of large doses.

Calcium uptake activity of canine myocardial sarcoplasmic reticulum in the presence of anesthetic agents.

The anesthetics chloroform, halothane, ethyl ether, and sodium pentobarbital, which have negative inotropic properties, were examined for effects on the calcium pump of the sarcoplasmic reticulum of the dog myocardium. Chloroform and sodium pentobarbital, at concentrations similar to those depressing cardiac contractility, inhibited calcium uptake. The concentration of halothane required to inhibit calcium uptake was ten times greater than that required to produce depression of contractility. Ether at concentrations eight times greater than those producing inhibition of contraction was completely without effect on calcium uptake. Chloroform and halothane depressed calcium uptake and calcium-stimulated ATPase activity in parallel, indicating that these agents do not uncouple transport from ATP hydrolysis. Sodium pentobarbital, in contrast, uncoupled transport from ATP hydrolysis.

Antero-Lateral Myocardial Infarction: Peri-Infarction Block

Pathological intracellular calcium handling has been proposed to underlie the alterations of contractile behavior in hypertrophied myocardium. However, the myocardial protein expression of intracellular calcium transport proteins in compensated human left ventricular hypertrophy has not yet been studied. We investigated septal myocardial specimens of patients suffering from hypertrophic obstructive cardiomyopathy (n=14) or from acquired aortic valve stenosis (n=11) undergoing myectomy or aortic valve replacement, respectively. For comparison, we studied non-hypertrophied myocardium of six non-failing hearts which could not be transplanted for technical reasons. The myocardial density of the calcium release channel of the sarcoplasmic reticulum (SR) was determined by3H-ryanodine binding. Myocardial contents of SR Ca2+-ATPase, phospholamban, calsequestrin and Na+/Ca2+-exchanger were analysed by Western blot analysis. The myocardial SR calcium release channel density was not significantly different in hypertrophied and non-failing human myocardium. In both hypertrophic obstructive cardiomyopathy and in aortic valve stenosis, SR Ca2+-ATPase expression was reduced by about 30% compared to non-failing myocardium (P<0.05), whereas the expression of phospholamban, calsequestrin, and the Na+/Ca2+-exchanger was unchanged. The decrease of SR Ca2+-ATPase expression was still observable when related to its regulatory protein phospholamban or to the myosin content of the homogenates (P<0.05). Furthermore, the SR Ca2+-ATPase expression was inversely correlated to the septum thickness assessed by echocardiography, but not to age, cardiac index or outflow tract gradient. In primary as well as in secondary hypertrophied human myocardium, the expression of SR Ca2+-ATPase is reduced and inversely related to the degree of the hypertrophy. The diminished SR Ca2+-ATPase expression might result in reduced Ca2+reuptake into the SR and might contribute to altered contractile behavior in hypertrophied human myocardium.

Depressant effect of ether on myocardium of the dog and its modification by reflex release of epinephrine and nor-epinephrine.

Depressant effect of ether on myocardium of the dog and its modification by reflex release of epinephrine and nor-epinephrine.