Changes In Sarcoplasmic Reticulum Function Research Articles

Trypanosoma brucei cathepsin-L increases arrhythmogenic sarcoplasmic reticulum-mediated calcium release in rat cardiomyocytes

AimsAfrican trypanosomiasis, caused by Trypanosoma brucei species, leads to both neurological and cardiac dysfunction and can be fatal if untreated. While the neurological-related pathogenesis is well studied, the cardiac pathogenesis remains unknown. The current study exposed isolated ventricular cardiomyocytes and adult rat hearts to T. brucei to test whether trypanosomes can alter cardiac function independent of a systemic inflammatory/immune response.Methods and resultsUsing confocal imaging, T. brucei and T. brucei culture media (supernatant) caused an increased frequency of arrhythmogenic spontaneous diastolic sarcoplasmic reticulum (SR)-mediated Ca2+ release (Ca2+ waves) in isolated adult rat ventricular cardiomyocytes. Studies utilising inhibitors, recombinant protein and RNAi all demonstrated that this altered SR function was due to T. brucei cathepsin-L (TbCatL). Separate experiments revealed that TbCatL induced a 10–15% increase of SERCA activity but reduced SR Ca2+ content, suggesting a concomitant increased SR-mediated Ca2+ leak. This conclusion was supported by data demonstrating that TbCatL increased Ca2+ wave frequency. These effects were abolished by autocamtide-2-related inhibitory peptide, highlighting a role for CaMKII in the TbCatL action on SR function. Isolated Langendorff perfused whole heart experiments confirmed that supernatant caused an increased number of arrhythmic events.ConclusionThese data demonstrate for the first time that African trypanosomes alter cardiac function independent of a systemic immune response, via a mechanism involving extracellular cathepsin-L-mediated changes in SR function.

T.P.10 The acute effects of curcumin exposure on skeletal muscle contractile function

Curcumin is a component in the Asian spice turmeric which has demonstrated anti-inflammatory, anti-oxidant and anti-cancer effects in animal models of human disease. It has also been shown to alleviate the dystropathology in mdx mice. However, in vitro studies on isolated sarcoplasmic reticulum (SR) vesicles indicate that curcumin has inhibitory effects on SR function which may adversely affect muscle contraction. The aim of this study was to investigate the specific effects of curcumin on skeletal muscle contractile function. These experiments, conducted on isolated extensor digitorum longus (EDL) muscles from 8week old ARC mice, were approved by the animal ethics committee of the University of Western Australia. Mice were anaesthetized (pentobarbitone, 40mg/kg, IP) and the muscles surgically removed and mounted in an in vitro muscle test system containing mammalian Ringer solution bubbled with carbogen. Measures of contractile function were performed before and after 60min exposure to either 100μM curcumin (dissolved in DMSO) or control solution containing the equivalent DMSO concentration (0.05%). The effect of curcumin on the susceptibility to and recovery from fatigue was also assessed. Curcumin exposure significantly decreased maximum specific force in EDL muscles by 14% compared to control muscles exposed to DMSO alone (curcumin: 19.7±0.7N/cm2, n=6; control: 23.0±1.0N/cm2, n=6, P<0.05). Curcumin had no significant effect on peak twitch force, the time to peak or the 1/2 relaxation time of twitch contractions. The rate of muscle fatigue was significantly reduced after curcumin exposure compared to controls (P<0.01, n=6). This study shows for the first time that (100μM) curcumin exposure significantly affects skeletal muscle contractile function. As the twitch contraction and relaxation times are sensitive to changes in SR function, these results are not consistent with previous findings that curcumin inhibits SR Ca2+ handling in SR vesicles.

Upregulation Of Cam Kinase IIδ Modulates Spontaneous Ca2+ Wave Properties In A Rabbit Model Of Heart Failure

In ventricular cardiomyocytes, CaMKIIδ (calcium/calmodulin-dependent protein kinase IIδ) is known to modulate Ca2+-handling proteins of the sarcoplasmic reticulum (SR). Dysregulation of Ca2+-handling is evident in cardiomyopathy and may be mediated by changes in CaMKIIδ expression and/or activity. The present study assesses CaMKIIδ expression and activity in rabbit left ventricular(LV) whole homogenates one week after coronary artery ligation(CAL). Changes in CaMKIIδ are translated to changes in SR function in isolated LV cardiomyocytes.Quantitative immunoblotting of CaMKIIδ protein revealed expression was increased by ∼2-fold in CAL (0.096±0.01(sham)vs.0.214±0.042(CAL) normalised mean ratio, (n=7). Similarly, CaMKIIδ activity was increased by ∼1.5-fold in CAL (0.168±0.022 (sham) vs. 0.247±0.028 (CAL) pmolPO4−inc/min/μg protein, n=7). In isolated permeabilised cardiomyocytes, spontaneous Ca2+ waves were studied to assess changes in function associated with upregulation of CaMKIIδ. This was accomplished by measuring changes in Ca2+ wave properties in the presence and absence of AIP (autocamtide-2-related inhibitory peptide). Cells were incubated with a modest concentration (300nM) of AIP for >30 min before use; these were then loaded with fluo-3 and fluorescence was monitored by confocal linescan microscopy with subsequent conversion to Ca2+. In cells from sham animals, no changes in Ca2+ waves were observed in the presence of AIP. In cells from LVD animals AIP caused Ca2+ waves to be reduced in frequency (-20.8±3.4%), increased minimum Ca2+ (+17.4±5.0%) as well as increased rate of decline (+15.8±2.0%). These changes are consistent with CaMKIIδ playing an increased role in SR Ca2+ handling following CAL. Since CaMKIIδ expression and activity are both significantly increased in this model, increased SR Ca2+ handling could occur via CaMKII-mediated effects on SERCA activity producing increased SR accumulation of Ca2+. This would result in an elevated sensitivity to β-adrenergic stimulation that could be arrhythmogenic.

Hydrogen peroxide increases depolarization-induced contraction of mechanically skinned slow twitch fibres from rat skeletal muscles.

The effect of exogenous hydrogen peroxide (H(2)O(2)) on excitation-contraction (E-C) coupling and sarcoplasmic reticulum (SR) function was compared in mechanically skinned slow twitch fibres (prepared from the soleus muscles) and fast twitch fibres (prepared from the extensor digitorum longus; EDL muscles) of adult rats. Equilibration (5 min) with 1 mM H(2)O(2) diminished the ability of the Ca(2+)-depleted SR to reload Ca(2+) in both slow (P < 0.01) and fast twitch fibres (P < 0.05) compared to control. Under conditions when all Ca(2+) uptake was prevented, 1 mM H(2)O(2) increased SR Ca(2+) "leak" in fast twitch fibres by 24 +/- 5 % (P < 0.05), but leak was not altered in slow twitch fibres. Treatment with 1 mM H(2)O(2) also increased the peak force of low [caffeine] contracture by approximately 45% in both fibre types compared to control (P < 0.01), which could be partly reversed following treatment with 10 mM dithiothreitol (DTT). The changes in SR function caused by 1 mM H(2)O(2) were associated with an approximately 65% increase in the peak height of depolarization-induced contractile response (DICR) in slow twitch fibres, compared to control (no H(2)O(2); P < 0.05). In contrast, peak contractile force of fast twitch fibres was not altered by 1 mM H(2)O(2) treatment. Equilibration with 5 mM H(2)O(2) induced a spontaneous force response in both slow and fast twitch fibres, which could be partly reversed by 2 min treatment with 10 mM DTT. Peak DICR was also increased approximately 40% by 5 mM H(2)O(2) in slow twitch fibres compared to control (no H(2)O(2); P < 0.05). Our results indicate that exogenous H(2)O(2) increases depolarization-induced contraction of mechanically skinned slow but not fast twitch fibres. The increase in depolarization-induced contraction in slow twitch fibres might be mediated by an increased SR Ca(2+) release during contraction and/or an increase in Ca(2+) sensitivity.

Effect of cardiac A(1) adenosine receptor overexpression on sarcoplasmic reticulum function.

We investigated the effect of A(1) adenosine receptor overexpression, which has been reported to increase myocardial tolerance to ischemia-reperfusion injury, on sarcoplasmic reticulum (SR) Ca(2+) handling. Transgenic mouse hearts (approximately 300-fold A(1) adenosine receptor overexpression) and wild-type mouse hearts were perfused in the Langendorff mode and subjected either to 80 min of aerobic perfusion or to 30 min of aerobic perfusion, 20 min of global ischemia and 30 min of reperfusion. The hearts were then homogenized and used to assay SR oxalate-supported 45Ca(2+) uptake and [3H]-ryanodine binding. Transgenic hearts showed increased resistance to ischemia-reperfusion, as shown by lower diastolic tension (1.5 +/- 0.2 vs. 2.6 +/- 0.1 g, P<0.05) and higher recovery of developed tension (45 +/- 3 vs. 30 +/- 4% of the baseline, P<0.05) following ischemia-reperfusion. Under baseline conditions, oxalate-supported 45Ca(2+) uptake was lower in transgenic hearts, owing to reduced V(max) (10.6 +/- 2.0 vs. 17.8 +/- 2.7 nmol/min per mg of protein, P<0.05), and the difference was preserved after ischemia-reperfusion (10.0 +/- 1.0 vs. 15.7 +/- 2.5 nmol/min per mg of protein, P<0.05). No significant difference in [3H]-ryanodine binding was observed. A(1) adenosine receptor overexpression is associated with a decreased rate of active Ca(2+) transport into the SR. We hypothesize that changes in SR function may cause a depletion of the SR Ca(2+) pool, which might protect from ischemic injury by delaying the development of cytosolic Ca(2+) overload during ischemia.

Effects of cytosolic ATP on Ca(2+) sparks and SR Ca(2+) content in permeabilized cardiac myocytes.

Confocal imaging was used to study the influence of cytosolic ATP on the properties of spontaneous Ca(2+) sparks in permeabilized ventricular myocytes. Cells were perfused with mock intracellular solutions containing fluo 3. Reducing [ATP] to <0.5 mmol/L decreased the frequency but increased the amplitude of spontaneous Ca(2+) sparks. In the presence of 20 micromol/L ATP, the amplitude increased by 48.7+/-10.9%, and the frequency decreased by 77.07+/-3.8%, relative to control responses obtained at 5 mmol/L ATP. After exposure to a solution containing zero ATP, the frequency of Ca(2+) sparks decreased progressively and approached zero within 90 seconds. As ATP washed out of the cell, the sarcoplasmic reticulum (SR) Ca(2+) content increased, until reaching a maximum after 3 minutes. Subsequent introduction of adenylyl imidodiphosphate precipitated a burst of large-amplitude Ca(2+) sparks. This was accompanied by a rapid decrease in SR Ca(2+) content to 80% to 90% of the steady-state value obtained in the presence of 5 mmol/L ATP. Thereafter, the SR Ca(2+) content declined much more slowly over 5 to 10 minutes. The effects of ATP withdrawal on Ca(2+) sparks may reflect reduced occupancy of the adenine nucleotide site on the SR Ca(2+) channel. These effects may contribute to previously reported changes in SR function during myocardial ischemia and reperfusion, in which ATP depletion and Ca(2+) overload occur.

Dynamic changes in sarcoplasmic reticulum function in cardiac hypertrophy and failure

Dynamic changes in sarcoplasmic reticulum function in cardiac hypertrophy and failure

Dynamic changes in sarcoplasmic reticulum function in cardiac hypertrophy and failure.

Previous studies have demonstrated that cardiac function changes with development of pressure overload-induced hypertrophy. The present study was undertaken to discover the basis for the changes in sarcoplasmic reticulum (SR) functions: uptake, (as related to the SR Ca2+ pump properties) and release in isolated, perfused hypertrophied rat hearts. Our results demonstrated significant prolongation of the time-to-90%-relaxation, both during the period of compensation (8 weeks after banding the ascending aorta, group HR1), when systolic function was preserved, and later with progressive hypertrophy (20 weeks after banding, group HR2) and contractile failure (20-22 weeks after banding, group F). The initial rates of the oxalate-supported SR Ca2+ uptake and the maximum transport rate (Vmax) of the SR Ca2+ pump, measured in the left ventricular homogenates, during blockade of the SR Ca2+ release channels with ruthenium red, were preserved in group HR1. To correlate early relaxation abnormalities with SR function, the [Ca2+] required for half-maximal pump activation (EC50) was examined and increased significantly in HRI vs. Sham1 (0.95+/-0.06 vs. 0.81+/-0.04 microM, P<0.05) indicating that the affinity of the SR Ca2+ pump for Ca2+ was reduced. The same tendency was demonstrated in groups HR2 (0.94+/-0.06 vs. 0.79+/-0.05) and F (0.89+/-0.05 vs. 0.78+/-0.05). In addition, with progression of hypertrophy we observed a significant decline in the amount of SR Ca2+ pump, as assessed by the Vmax, from 31.22+/-1.20 (Sham2) to 26.47+/-1.58 HR2) nmol/mg protein per min (P<0.05), and from 33.81+/-1.23 (Sham3) to 25.15+/-1.57 (F) nmol/mg protein per min, (P<0.01). This decrease was accompanied by a parallel reduction in the number of SR Ca2+ release channels by 14% (HR2) and 23% (F), as determined by maximum [3H] ryanodine binding (Bmax). These results suggest that pressure overload-induced changes in SR Ca2+ uptake (as reflected by Vmax and EC50) and SR Ca2+ release (as reflected by Bmax), both leading to diminished Ca2+ sequestration, may contribute to impaired cardiac relaxation with compensatory hypertrophy and failure.

Effects of varied fatigue protocols on sarcoplasmic reticulum calcium uptake and release rates

The purpose of this investigation was to examine changes in sarcoplasmic reticulum (SR) function in muscles subjected to different patterns of muscle activity. Frog sartorius muscles were stimulated with tetanic trains (100 ms, 100 Hz) delivered at rates of 2.0, 0.5, and 0.2 trains/s. In one set of experiments, stimulation was continued until force had declined to approximately 17% of initial (constant fatigue), whereas in the other set, stimulation was continued for 1 min (constant duration). In the constant-fatigue experiments, Ca2+ uptake (1 mM MgATP) and release rates (25 microM AgNO3, 5 mM 4-chloro-m-cresol) were depressed by similar extents following each protocol. This occurred despite 1, 4, and 17 min of stimulation, respectively, used to induce fatigue. In the constant-duration experiments, larger reductions in SR function occurred following the highest frequency stimulation protocol. These data suggest that when muscles are fatigued to similar extents, depressions in SR function are independent of the activity protocol. On the other hand, when a constant duration of activity is imposed, changes in SR function are closely linked to the extent of force reduction.

Changes in skeletal muscle sarcoplasmic reticulum function and force production following myocardial infarction in rats.

In patients following myocardial infarction (MI), exercise tolerance and muscular strength are often markedly reduced. The purpose of this investigation was to determine if calcium (Ca2+) uptake and release by skeletal muscle sarcoplasmic reticulum (SR) are altered following MI and if such changes are associated with diminished muscular performance. SR vesicles were isolated from rat gastrocnemius muscle at 6 and 12 weeks following MI (via left coronary artery ligation) or sham surgery. At both post-surgery intervals, the rates of Ca2+ uptake (measured using fura-2) were 35% greater in the MI group. In addition, the rates of Ca2+ release were increased in the MI group by 10 and 30% at 6 and 12 weeks, respectively. At 12 weeks post-surgery, animals of the MI group showed significant reduction in in situ twitch and tetanic forces and significant elevations in the rates of tension increase and decrease. These data indicate that SR Ca2+ exchange is altered following MI. In addition, changes in SR function are associated with changes in force production by the whole muscle.

Phospholamban gene dosage effects in the mammalian heart.

Phospholamban ablation has been shown to result in significant increases in cardiac contractile parameters and loss of beta-adrenergic stimulation. To determine whether partial reduction in phospholamban levels is also associated with enhancement of cardiac performance and to further examine the sensitivity of the contractile system to alterations in phospholamban levels, hearts from wild-type, phospholamban-heterozygous, and phospholamban-deficient mice were studied in parallel at the subcellular, cellular, and organ levels. The phospholamban-heterozygous mice expressed reduced cardiac phospholamban mRNA and protein levels (40 +/- 5%) compared with wild type mice. The reduced phospholamban levels were associated with significant decreases in the EC50 of the sarcoplasmic reticulum Ca2+ pump for CA2+ and increases in the contractile parameters of isolated myocytes and beating hearts. The relative phospholamban levels among wild-type, phospholamban-heterozygous, and phospholamban-deficient mouse hearts correlated well with the (1) EC50 of the Ca(2+)-ATPase for Ca2+ in sarcoplasmic reticulum, (2) rates of relaxation and contraction in isolated cardiac myocytes, and (3) rates of relaxation and intact beating hearts. These findings suggest that physiological and pathological changes in the levels of phospholamban will result in parallel changes in sarcoplasmic reticulum function and cardiac contraction.

Sarcoplasmic reticulum-related changes in cytosolic calcium in pressure-overload-induced feline LV hypertrophy.

Alterations in Ca2+ homeostasis that involve the sarcoplasmic reticulum (SR) were studied in feline left ventricular (LV) myocytes isolated from hearts with LV hypertrophy induced by slow, progressive pressure overload. At death, severe hypertrophy was evidenced by increased heart weight-to-body weight ratio (8.4 +/- 0.6 vs. 4.2 +/- 0.2 g/kg in controls). Steady-state Ca2+ transients (measured as. indo 1 fluorescence at 410 nm/480 nm; I410/I480) in LV hypertrophy (LVH) myocytes had diminished peak amplitudes (I410/I480 2.28 +/- 0.07 vs. 2.53 +/- 0.07 in controls) and prolonged durations (0.75 +/- 0.03 vs. 0.59 +/- 0.02 s in controls). The magnitude of shortening was reduced and the contractile duration was prolonged in LVH myocytes. The idea that changes in SR function are responsible for these alterations in the Ca2+ transient was tested by studying two aspects of SR-related Ca2+ homeostasis. Restitution of releasable SR Ca2+ was studied by measuring indo 1 transients and contractions during premature beats. The time course of restitution of both the indo 1 transient and contraction of hypertrophy myocytes was significantly slower than in controls. These data suggest that restitution of releasable SR Ca2+ is slowed in hypertrophy myocytes. The reduction of the indo 1 transient and contraction in beats following long rest periods (rest decay) was measured to determine the rate of Ca2+ loss from the SR. Rest decay was significantly (P < 0.05) more pronounced in hypertrophy myocytes, suggesting that Ca2+ loss from the SR is accelerated in these myocytes. (ABSTRACT TRUNCATED AT 250 WORDS)

Altered sarcoplasmic reticulum function after high-intensity exercise

This study examined the effects of acute high-intensity exercise on the rate and capacity of Ca2+ uptake and Ca2+-stimulated adenosinetriphosphatase (ATPase) activity of the sarcoplasmic reticulum and the reversibility of these effects. Thoroughbred horses were run at maximal O2 uptake on a high-speed treadmill until fatigued. Muscle temperatures and biopsy samples were collected at rest, immediately after exercise, and 30 and 60 min after exercise. Blood samples were collected at rest and 5 min after exercise. Muscle and blood (lactate concentration) were three- and fivefold greater than pre-exercise values. Muscle temperature and pH immediately after post-exercise were 43 degrees C and 6.55, respectively, but approached rest values by 60 min after exercise. The initial rate and maximal capacity of Ca2+ uptake of muscle homogenates and isolated sarcoplasmic reticulum were significantly depressed immediately after exercise. This depression was paralleled by decreased activity of the Ca2+-stimulated ATPase. However, both Ca2+ uptake (rate and capacity) and Ca2+4-ATPase activity had returned to normal by 60 min after exercise. These findings demonstrate that changes in sarcoplasmic reticulum function after high-intensity exercise may be induced but not sustained by local changes in muscle pH and/or temperature.

Maturation of calcium transport in cardiac sarcoplasmic reticulum.

Developmental changes in myocardial function have been described by a number of investigators. To further define the cellular basis for these changes, the present study was designed to characterize age-related changes in Ca2+ transport in sarcoplasmic reticulum (SR) vesicles isolated from six groups of sheep: group (Sp) I (100-105 days gestation), Gp II (128-132 days gestation), Gp III (postnatal, 0-3 days), Gp IV (4 wk), Gp V (8 wk), and Gp VI (maternal sheep). The maximal Ca2+ uptake for vesicles isolated from Gp I-V was significantly (p less than 0.01) decreased as compared to that measured for Gp VI (maternal) vesicles. However, Ca2+-dependent ATP hydrolysis was decreased only in the fetal SR vesicles (Gp I-II). Thus, decreased ATP hydrolysis only partially explained the decreased Ca2+ uptake. In contrast, calculation of apparent Ca2+ pump coupling ratios (mol of Ca2+ transported/mol of ATP hydrolyzed) showed that there was a marked increase in the coupling of Ca2+ transport to ATP hydrolysis during maturation of the heart. Inasmuch as the contractile state of the heart depends on precise regulation of Ca2+ concentration by the SR, these age-related changes in SR function may contribute to developmental changes in myocardial function.

Cellular compartmentation in ischemic myocardium: indirect analysis by electron probe.

Electron probe microanalysis (EPMA) was carried out directly on myocardial cells and on the myofibrils and the mitochondria within them. A third subcellular compartment, which contains sarcoplasmic reticulum (SR), was measured indirectly. The percent of the total cell calcium content that resides within this "hidden" compartment was calculated from cell data minus weighted myofibril and mitochondria data. This approach was applied to control, ischemic, and reperfused myocardium, and other elements were also quantified. We found that the calcium content of this third compartment is little changed during global ischemia but is markedly depleted after 5 min reperfusion. We conclude that these changes are ascribable to changes in SR function.

SEPARATION OF CARDIAC SARCOPLASMIC RETICULUM FROM FETAL AND MATERNAL SHEEP INTO FREE AND JUNCTIONAL SUBPOPULATIONS

We have previously shown decreased Ca accumulation in crude cardiac sarcoplasmic reticulum (SR) vesicles isolated from fetal sheep. However, recent data indicate that the cardiac SR is subspecialized and that these crude preparations are quite heterogeneous. To further define developmental changes in SR Ca transport, SR vesicles from fetal and maternal sheep hearts were seperated by sucrose density gradient centrifugation into free (F) and junctional (J) fractions. The FSR vesicles displayed a high density of Ca pumps and high Ca uptake that was insensitive to ryanodine. In contrast, although Ca uptake was decreased in JSR vesicles, uptake increased markedly in the presence of ryanodine. Gel electrophoresis showed selective enrichment of JSR vesicles with the Ca binding protein, calsequestrin. CaATPase activity and maximal Ca uptake were decreased 30-50% in fetal FSR and JSR (p<0.05). These data indicate that previously described differences in Ca transport in cardiac SR vesicles isolated from fetal and maternal sheep persist in our highly purified preparations of FSR and JSR. Further characterization of these SR subpopulations should enable us to better define developmental changes in SR function. *mean±SD, n=3.

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 Ca 2+ 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 Ca 2+ uptake (107 ± 3.2 nmol Ca 2+ mg −1 min −1 vs. 63 ± 1 in controls, P < 0.01) and Ca 2+ 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 Ca 2+-, 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, Ca 2+ transport by the sarcoplasmic reticulum is enhanced and may be an important determinant of contractile performance.