Enhanced calcium transport by sarcoplasmic reticulum in mild cardiac hypertrophy

Cardiac hypertrophy is associated with changes in sarcoplasmic reticulum function which may depend on the method of hypertrophy induction, its severity, acuteness, and duration. To assess the effects of mild, pressure-overload hypertrophy on Ca²⁺ transport capacity of cardiac sarcoplasmic reticulum, abdominal aortic constriction was produced in adult rats. This resulted in a mild degree of hypertrophy (22% increase in left ventricle/body weight ratio compared to sham-operated controls). The effect of hypertrophy on the sarcoplasmic reticulum was assessed 6 weeks after the operation. The yield of microsomal protein was increased in hypertrophied hearts (1.43 ± 0.2 mg/g vs. 1.1 ± 0.1 mg/g in controls) and was associated with enhanced Ca²⁺ uptake (107 ± 3.2 nmol Ca²⁺ mg^?1 min^?1 vs. 63 ± 1 in controls, P < 0.01) and Ca²⁺ ATPase activity (60 ± 1.8 nmol P_i mg^?1 min^?1 vs. 35 ± 1.2 in controls, P < 0.01). The steady-state levels of the phosphoprotein intermediate [EP] of the transport ATPase were higher in operated animals (1.61 ± 0.2 nmol/mg vs. 1.1 ± 0.1 nmol/mg) without a change in the Ca²⁺-, ATP-, or pH-dependency. The rate of [EP] dephosphorylation was, however, significantly faster in the hypertrophied animals. These results indicate that, in this model of cardiac hypertrophy, Ca²⁺ transport by the sarcoplasmic reticulum is enhanced and may be an important determinant of contractile performance.     

Effects of local anaesthetics on calcium transport by canine cardiac microsomes (fragmented sarcoplasmic reticulum).

Local anaesthetics inhibit calcium transport by cardiac sarcoplasmic reticulum. However, the concentrations of lidocaine and procaineamide needed to produce these effects are high. No significant differences were found between these agents that could account for reported differences in their electrophysiological effects.     

Effects of lanthanum ion on calcium transport by guinea-pig, rat and canine cardiac sarcoplasmic reticulum.

The trivalent earth metal lanthanum has been shown to inhibit calcium uptake and the calcium-activated ATPase activity of isolated guinea-pig and canine cardiac sarcoplasmic reticulum, at concentrations of lanthanum of 10^?5 and 10^?4m. Calcium uptake, in the presence of 2.5 to 5.0 mm K-oxalate, and the calcium activated ATPase activity of isolated rat cardiac sarcoplasmic reticulum is less sensitive to lanthanum than guinea-pig or canine sarcoplasmic reticulum.Calcium storage, in the absence of precipitating anions, by the cardiac sarcoplasmic reticulum of all species studied is not affected by lanthanum at concentrations of 10^?5m and 10^?4m.     

Doxorubicin-induced calcium release from cardiac sarcoplasmic reticulum vesicles

Doxorubicin, an anthracycline glycoside antibiotic which has been widely used for treatment of several types of cancer (Goormaghtigh and Ruysschaer, 1984), displays a clinically important cardiac toxicity (Young et al., 1981) that can be dissociated from the antitumor activity. Although the main sites of toxicity have been postulated to be on the muscle membranes (Goormaghtigh and Ruysschaer, 1984; Harris and Doroshow, 1985), no information is available for a direct doxorubicin effect on the Ca2+ fluxes in cardiac sarcoplasmic reticulum (SR). Previous studies have shown that micromolar doxorubicin triggers Ca2+ release from skeletal SR vesicles (Zorzato et al., 1985). The objective of this study was to examine the effect of doxorubicin or caffeine on Ca2+ fluxes in cardiac SR in the presence of various Ca2+ release inhibitors. Addition of either doxorubicin (C1/2 = 5 microM), or caffeine (C1/2 = 0.8 mM) triggered Ca2+ release from canine cardiac SR loaded with 45Ca2+ in the presence of 2 mM ATP. The maximal amount of Ca2+ release triggered by doxorubicin (38% of the total loaded Ca2+) was significantly higher than that released by caffeine (25%). Plots of the amount of Ca2+ release triggered by 20 microM doxorubicin or 2 mM caffeine vs. free Ca2+ concentration were a bell-shaped, with maximal Ca2+ release at 0.2 microM Ca2+. Ca2+ release triggered by either 20 microM doxorubicin or 2 mM caffeine was inhibited by ruthenium red (0.1 to 2 microM), ryanodine (1 to 100 microM) or tetracaine (0.1 to 1 mM), whereas 2 mM caffeine did not further activate Ca2+ release triggered by 50 microM doxorubicin, suggesting that the drugs may share the same Ca2+ release channel.     

Effect of hypothermic ischemia and reperfusion on calcium transport by myocardial sarcolemma and sarcoplasmic reticulum.

The effects of hypothermic ischemia and reperfusion on sarcolemma and sarcoplasmic reticulum Ca2+ transport were studied in vesicles isolated from rabbit hearts. Hypothermic global ischemia was produced by immersing hearts in saline at 4 degrees C for 3 h. Following hypothermic ischemia, reperfusion was carried out for 40 min using a Langendorff perfusion system for the working heart. Na+,K(+)-ATPase activity of sarcolemmal vesicles (SL), was not depressed by hypothermic ischemia nor by ischemia and reperfusion. The initial rate of Na(+)-Ca2+ exchange in SL vesicles was not depressed, but the maximum amount of Ca2+ uptake was increased both after hypothermic ischemia and after reperfusion. Ca2+ uptake activity of sarcoplasmic reticulum vesicles (SR) isolated from hearts subjected to hypothermic ischemia was slightly lower than that of control, and was further reduced following reperfusion. Ca(2+)-ATPase activity of SR was unaffected by hypothermic ischemia, while it was markedly lowered after reperfusion. Although the phosphoenzyme level in SR vesicles was slightly decreased, the turnover rate was reduced after reperfusion. Reperfusion injury thus took place mainly in SR while SL appeared to be tolerant to ischemia and reperfusion.     

Molecular mechanism of calcium uptake and release by cardiac sarcoplasmic reticulum

Cardiac sarcoplasmic reticulum (SR) plays a special role in controlling free calcium ions (Ca) in heart muscle cells. Ca stored in the SR is released through the Ca release channels when the sarcolemmal membrane is depolarized, thereby inducing contraction, while Ca is reaccumulated by the Ca pump to induce relaxation. In the latter process, the Ca pump of cardiac SR has a regulatory system by cAMP-dependent phosphorylation of a SR protein, phospholamban. Recently, significant progress has been achieved in understanding the molecular mechanisms of Ca release and uptake and its regulation. The structures of the Ca pump and phospholamban have been defined at molecular levels. A direct interaction between these two proteins was demonstrated. The Ca release channel was identified, and turned out to be the foot structure which in situ connects the SR to the sarcolemma/transverse tubule.     

Alterations in cardiac sarcoplasmic reticulum calcium transport in the postischemic "stunned" myocardium

This study examined the possibility that the postischemic mechanical depression observed in the "stunned" myocardium is a result of an alteration in the control of intracellular calcium. Regional myocardial stunning was produced in five open-chest dogs by eight to twelve 5-minute occlusions of the left anterior descending coronary artery, alternated with 10-minute reflow periods and followed by a final 60-minute period of reperfusion. Systolic segment shortening in the postischemic zone, measured by sonomicrometry, fell from 14.9% at baseline to -1.1% at the end of reperfusion. Sarcoplasmic reticulum isolated from stunned myocardium demonstrated a 17% reduction in oxalate-supported 45Ca2+ transport compared with sarcoplasmic reticulum from normal myocardium (0.93 vs. 1.12 mumol Ca2+/mg protein/min, p less than 0.005). There was also a 20% decrease in the maximal activation by Ca2+ of the sarcoplasmic reticulum Ca2+, Mg2+-ATPase (2.46 vs. 1.96 mumol Pi/mg protein/min, p less than 0.005), and a downward shift in the Ca2+-activation curve of the Ca2+, Mg2+-ATPase. These results indicate that myocardial stunning is associated with damage to the calcium-transport system of the sarcoplasmic reticulum. Altered intracellular control may contribute to the inability of the stunned heart to maintain normal mechanical function.     

Free radical mediation of the effects of acidosis on calcium transport by cardiac sarcoplasmic reticulum in whole heart homogenates

Generation of oxygen free radicals by xanthine acting on xanthine oxidase as a substrate significantly depressed calcium transport by sarcoplasmic reticulum in canine whole heart homogenates at 37 degrees C. At pH 7.0, this effect was completely inhibited by the addition of superoxide dismutase (SOD), a scavenger of the superoxide anion radical. At pH 6.4, SOD (5 to 20 micrograms X ml-1) was ineffective but catalase (20 micrograms X ml-1) was able to inhibit the effects of the xanthine-xanthine oxidase system. SOD + catalase (20 micrograms X ml-1) and SOD + mannitol, a scavenger of the hydroxyl free radical, inhibited the effects of the xanthine-xanthine oxidase system at pH 6.4. Preincubation at pH 6.4, in the absence of an exogenous free radical generating system, depressed calcium transport. This depression was more severe the longer the duration of incubation. However, return of the pH to 7.0 after preincubation at pH 6.4 partially restored calcium uptake velocity. The degree of reversibility was decreased the longer the period of incubation at pH 6.4. SOD reversed the effects of incubation at pH 6.4 for 5 min, but not those for incubations of 10 and 15 min. Mannitol alone was ineffective. The combinations of SOD and mannitol significantly reversed the effects of pH 6.4 up to 15 min. These results demonstrate that both exogenously generated and endogenously generated free oxygen radicals are capable of depressing calcium transport by cardiac sarcoplasmic reticulum in the whole heart homogenate in the presence of endogenous scavenging systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of linoleic and arachidonic acid derivatives on calcium transport in vesicles from cardiac sarcoplasmic reticulum

The effect of linoleic and arachidonic acid derivatives on ATP-dependent calcium transport was studied in the isolated vesicles from cardiac sarcoplasmic reticulum of guinea-pigs. Oxidation products of linoleic and arachidonic acids, obtained either by autoxidation or incubation with soybean lipoxygenase, effectively blocked in a dose-dependent manner, the net influx of calcium in the absence or presence of 5 mM of oxalate. Unoxidized fatty acids were much weaker at lower concentrations as compared to their oxidized counterparts, except the lipoxygenase-generated product of arachidonic acid which had only a marginal effect even at high concentrations. Autoxidation products of arachidonic acid were the most potent inhibitors of calcium transport. Likewise, autoxidation products of linoleic and arachidonic acids and lipoxygenase-generated products of linoleic acid induced a dose-dependent release of calcium from vesicles previously loaded with 45Ca, and release was further enhanced in the presence of 0.5 mM of EGTA. In contrast, lipoxygenase metabolites of arachidonic acid caused a transient increase in net calcium content. The effect of the fatty acid derivatives on calcium transport did not appear to be due either to the inhibition of Ca2+-ATPase activity or to a non-specific detergent-like action. The effects of oxidized fatty acids, on ATP-dependent calcium accumulation into and release from cardiac microsomal fraction were similar but less potent than those of classical calcium ionophores, X537A or A23187.     

Effect of pH on calcium ion dependence of dog cardiac sarcoplasmic reticulum adenosine triphosphatase activity

The effect of [Ca2+] on (Ca2+ + Mg2+)-ATPase activity from dog heart sarcoplasmic reticulum fragments was evaluated under three different pH values: 6.0, 6.8 and 7.6. Considering pH 6.8 as reference, under pH 7.6 an increase of the activity at saturating [Ca2+], an increase of pCa needed for half-maximal activation, and a decrease of the Hill coefficient, were observed. Opposite results were obtained under pH 6.0. The positively cooperative effect of Ca2+ was only observed at the lowest pH. The results are in line with previous suggestions regarding the existence of two enzymatic states with different Ca2+ affinities; this paper suggests as well, that equilibrium between these states is affected by pH. The results are compared with data obtained with skeletal sarcoplasmic reticulum fragments under the same experimental conditions.     

Altered calcium uptake by the sarcoplasmic reticulum following cardiac transplantation in humans

Abnormalities in the diastolic properties of the heart have been described following human cardiac transplantation and may reflect, at least in part, decreased Ca2+ uptake by the sarcoplasmic reticulum. This possibility was evaluated by obtaining serial myocardial biopsies in 13 patients who underwent cardiac transplantation for severe heart failure. Oxalate-supported Ca2+ uptake by the sarcoplasmic reticulum was measured in homogenates of 83 ventricular biopsies from transplanted hearts. Biopsies from seven subjects with normal cardiac function and morphology served as controls. In the transplanted hearts, there was a tendency for Ca2+ uptake rate to decline with time so that 4-5 months postoperatively, it was significantly lower (4.5 +/- 0.5 nmoles Ca2+/mg/min) compared to controls (5.6 +/- 0.5 nmoles Ca2+/mg/min, p less than 0.01). Plasma norepinephrine levels fell from the high preoperative values (689 +/- 50 pg/mL) towards normal (215 +/- 7 pg/mL) within 30 days after transplantation. Subsequently, however, there was a tendency for norepinephrine levels to increase (369 +/- 55 pg/mL at 4 months). In four patients for which serial observations were available, there was an inverse relationship between myocardial Ca2+ uptake and plasma norepinephrine levels. These results indicate the feasibility of obtaining reproducible serial measurements of Ca2+ uptake in human cardiac biopsies. The decline in sarcoplasmic reticulum function following cardiac transplantation may be, in part, the biochemical basis for the reported impairment in diastolic relaxation.     

The reversal of the calcium pump of cardiac sarcoplasmic reticulum

Summary The reversal of the calcium pump of cardiac sarcoplasmic reticulum (SR) prepared from dogs was investigated. Phosphorylation of the calcium transport ATPase by orthophosphate and ATP synthesis from ADP and orthophosphate by SR passively preloaded with calcium are demonstrated. The ADP-dependent calcium efflux from SR loaded with calcium in the presence of acetylphosphate is stoichiometrically coupled to ATP synthesis from ADP and orthophosphate.

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Die Umkehr der Calcium-Pumpe des myokardialen sarkoplasmatischen Retikulums

Zusammenfassung Die Umkehr der Calcium-Pumpe des myokardialen sarkoplasmatischen Retikulums (SR) wurde untersucht. SR-Vesikel, die passiv durch Präinkubation mit hohen Calciumkonzentrationen mit Calcium beladen wurden, synthetisieren ATP aus Orthophosphat und ADP. Die ATP-Synthese erfolgt über die Phosphoryllerung der Calcium-Transport-ATPase des SR durch Orthophosphat und eine Dephosphorylierung des EP durch ADP. Die Rate der Calcium-Freisetzung aus SR-Vesikel — die Calcium-Beladung erfolgte aktiv in Gegenwart von Acetylphosphat — wird durch ADP erhöht. Der ADP-abhängige Calcium-Efflux ist stöchiometrisch mit der ATP-Synthese aus Orthophosphat und ADP gekoppelt.

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Paper, presented at the Erwin Riesch Symposium, Tübingen, September 26–29, 1976

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With 3 figures and 1 table     

老年大鼠心肌肌浆网功能的改变

目的 探讨老年大鼠心肌肌浆网（ＳＲ）钙转运的改变及其在心脏收缩功能障碍中的作用。方法 测定老年和成年Ｗｉｓｔａｒ大鼠心功能。取左心室肌组织制备肌浆网膜,采用Ｍｉｌｌｉｐｏｒｅ滤过法测定心肌ＳＲＣａ＾２＋摄取、Ｃａ＾２＋释放和＾３Ｈ－Ｒｙａｎｏｄｉｎｅ受体结合,并测定心肌ＳＲＣａ＾２＋－ＡＴＰ酶活性。结果 与成年组比较,老年组大鼠左室舒张末压（ＬＶＥＤＰ）升高８２％（Ｐ〈０．０５）,左室内压变化速度     

Ablation of sarcolipin enhances sarcoplasmic reticulum calcium transport and atrial contractility

Sarcolipin is a novel regulator of cardiac sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) and is expressed abundantly in atria. In this study we investigated the physiological significance of sarcolipin in the heart by generating a mouse model deficient for sarcolipin. The sarcolipin-null mice do not show any developmental abnormalities or any cardiac pathology. The absence of sarcolipin does not modify the expression level of other Ca2+ handling proteins, in particular phospholamban, and its phosphorylation status. Calcium uptake studies revealed that, in the atria, ablation of sarcolipin resulted in an increase in the affinity of the SERCA pump for Ca2+ and the maximum velocity of Ca2+ uptake rates. An important finding is that ablation of sarcolipin resulted in an increase in atrial Ca2+ transient amplitudes, and this resulted in enhanced atrial contractility. Furthermore, atria from sarcolipin-null mice showed a blunted response to isoproterenol stimulation, implicating sarcolipin as a mediator of beta-adrenergic responses in atria. Our study documented that sarcolipin is a key regulator of SERCA2a in atria. Importantly, our data demonstrate the existence of distinct modulators for the SERCA pump in the atria and ventricles.     

Mitochondrial free calcium regulation during sarcoplasmic reticulum calcium release in rat cardiac myocytes

Cardiac mitochondria can take up Ca²⁺, competing with Ca²⁺ transporters like the sarcoplasmic reticulum (SR) Ca²⁺-ATPase. Rapid mitochondrial [Ca²⁺] transients have been reported to be synchronized with normal cytosolic [Ca²⁺]_i transients. However, most intra-mitochondrial free [Ca²⁺] ([Ca²⁺]_mito) measurements have been uncalibrated, and potentially contaminated by non-mitochondrial signals. Here we measured calibrated [Ca²⁺]_mito in single rat myocytes using the ratiometric Ca²⁺ indicator fura-2 AM and plasmalemmal permeabilization by saponin (to eliminate cytosolic fura-2). The steady-state [Ca²⁺]_mito dependence on [Ca²⁺]_i (with 5 mM EGTA) was sigmoid with [Ca²⁺]_mito < [Ca²⁺]_i for [Ca²⁺]_i below 475 nM. With low [EGTA] (50 μM) and 150 nM [Ca²⁺]_i (± 15 mM Na⁺) cyclical spontaneous SR Ca²⁺ release occurred (5–15/min). Changes in [Ca²⁺]_mito during individual [Ca²⁺]_i transients were small ( 2–10 nM/beat), but integrated gradually to steady-state. Inhibition SR Ca²⁺ handling by thapsigargin, 2 mM tetracaine or 10 mM caffeine all stopped the progressive rise in [Ca²⁺]_mito and spontaneous Ca²⁺ transients (confirming that SR Ca²⁺ releases caused the [Ca²⁺]_mito rise). Confocal imaging of local [Ca²⁺]_mito (using rhod-2) showed that [Ca²⁺]_mito rose rapidly with a delay after SR Ca²⁺ release (with amplitude up to 10 nM), but declined much more slowly than [Ca²⁺]_i (time constant 2.8 ± 0.7 s vs. 0.19 ± 0.06 s). Total Ca²⁺ uptake for larger [Ca²⁺]_mito transients was  0.5 μmol/L cytosol (assuming 100:1 mitochondrial Ca²⁺ buffering), consistent with prior indirect estimates from [Ca²⁺]_i measurements, and corresponds to  1% of the SR Ca²⁺ uptake during a normal Ca²⁺ transient. Thus small phasic [Ca²⁺]_mito transients and gradually integrating [Ca²⁺]_mito signals occur during repeating [Ca²⁺]_i transients.     

Effect of ischemia and reperfusion on sarcoplasmic reticulum calcium uptake.

To investigate the mechanism underlying postischemic cardiac dysfunction (myocardial stunning), contractility and adenine nucleotide metabolism were studied in three groups of isolated perfused rabbit hearts (control, ischemic, and reperfused), whereas Ca2+ uptake by the sarcoplasmic reticulum (SR) was measured in homogenates obtained from them. The hearts were Langendorff-perfused under constant pressure with Krebs-Henseleit solution at 37 degrees C. Global normothermic ischemia was produced by closing the perfusion line. In the reperfused group, after 15 minutes of ischemia, Krebs-Henseleit solution was perfused for 10 minutes. Developed left ventricular pressure (control, 104 +/- 6.3 mm Hg) and left ventricular dP/dt (2,063 +/- 256.6 mm Hg.sec-1) were significantly decreased in reperfused hearts (left ventricular pressure, 78 +/- 5.9 mm Hg; left ventricular dP/dt, 1,339 +/- 216.3 mm Hg.sec-1). Myocardial ATP content (control, 13.6 +/- 0.98 mumol/g dry wt) decreased during ischemia (4.5 +/- 1.23 mumol/g) but was restored to control level on reperfusion (11.8 +/- 0.68 mumol/g). Maximum velocity of Ca2+ uptake by the SR (Vmax) (control, 49.3 +/- 2.54 nmol.min-1 x mg-1) was significantly depressed by ischemia (36.3 +/- 1.94 nmol.min-1 x mg-1) but was restored to the control value after a 10-minute reperfusion (45.3 +/- 0.79 nmol.min-1 x mg-1). Apparent dissociation constant KCa and the Hill coefficient for Ca2+ uptake were not different between control, ischemia, and reperfusion. To test for the possible role of the SR Ca(2+)-release channel in the effect of ischemia and reperfusion, we measured Ca2+ uptake after incubation of homogenates with 610 microM ryanodine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium efflux from cardiac sarcoplasmic reticulum: effects of calcium and magnesium

The dependence of the rate of calcium efflux from cardiac sarcoplasmic reticulum on the concentration of ionized calcium in the medium has been investigated. A high concentration of ionized calcium outside the sarcoplasmic reticulum stimulates calcium efflux. Stimulation of calcium efflux from cardiac sarcoplasmic reticulum by high levels of ionized calcium outside the membranes is inhibited by magnesium. This inhibition can be overcome at very high concentrations of calcium in the medium. This resembles the magnesium inhibition of calcium triggered calcium release from skinned cardiac muscle fibres, which can also be overcome by increasing the concentration of calcium in the medium. Ouabain at 10^?6m and verapamil at 10^?5m have no effet on the stimulation of calcium efflux by calcium.