Effects of temperature and calcium availability on cardiac contractility in Synbranchus marmoratus, a neotropical teleost

An isometric muscle preparation was used to investigate the importance of the ventricular sarcoplasmic reticulum (SR) and extracellular Ca2+ (1.25 up to 11.25 mM) to force generation at 25 degrees C (acclimation temperature), 15 and 35 degrees C. The post-rest tension and force-frequency relationship were conducted with and without 10 microM ryanodine in the bathing medium. Increments in extracellular Ca2+ resulted in increases in twitch force development only at 35 degrees C. A significant post-rest potentiation was recorded for the control preparations at 25 degrees C (100% to 119.8+/-4.1%). However, this post-rest potentiation was inhibited by ryanodine only at 25 degrees C (100% to 97.6+/-1.5%). At 35 degrees C, force remained unchanged in the control preparations, but a significant post-rest decay was recorded in the presence of ryanodine (100% to 76.6+/-4.6%) while at 15 degrees C, ryanodine was not able to preventing the post-rest potentiation observed in the control preparations. The increases in the imposed contraction frequency caused a decline of the force at 25 and 35 degrees C and ryanodine decreased significantly peak tension at both temperatures. The findings suggest a high or medium calcium turnover, possibly related to the presence of a functional SR, whose functionality is diminished when temperature is decreased.     

Pyruvate potentiates inotropic effects of isoproterenol and Ca(2+) in rabbit cardiac muscle preparations

Catecholamines and elevated extracellular Ca(2+) concentration ([Ca(2+)](o)) augment contractile force by increased Ca(2+) influx and subsequent increased sarcoplasmic reticulum (SR) Ca(2+) release. We tested the hypothesis that pyruvate potentiates Ca(2+) release and inotropic response to isoproterenol and elevated [Ca(2+)](o), since this might be of potential importance in a clinical setting to circumvent deleterious effects on energy demand during application of catecholamines. Therefore, we investigated isometrically contracting myocardial preparations from rabbit hearts at 37 degrees C, pH 7.4, and a stimulation frequency of 1 Hz. At a [Ca(2+)](o) of 1.25 mM, pyruvate (10 mM) alone increased developed force (F(dev)) from 1.89 +/- 0.42 to 3.62 +/- 0.62 (SE) mN/mm(2) (n = 8, P < 0.05) and isoproterenol (10(-6) M) alone increased F(dev) from 2.06 +/- 0. 55 to 25.11 +/- 2.1 mN/mm(2) (P < 0.05), whereas the combination of isoproterenol and pyruvate increased F(dev) overproportionally from 1.89 +/- 0.42 to 33.31 +/- 3.18 mN/mm(2) (P < 0.05). In a separate series of experiments, we assessed SR Ca(2+) content by means of rapid cooling contractures and observed that, despite no further increase in F(dev) by increasing [Ca(2+)](o) from 8 to 16 mM, 10 mM pyruvate could still increase F(dev) from 26.4 +/- 6.8 to 29.7 +/- 7. 1 mN/mm(2) (P < 0.05, n = 9) as well as the Ca(2+) load of the SR. The results show that the positive inotropic effects of pyruvate potentiate the inotropic effects of isoproterenol or Ca(2+), because in the presence of pyruvate, Ca(2+) and isoproterenol induced larger increases in inotropy than can be calculated by mere addition of the individual effects.     

Contractile and Ca2+-handling properties of the right ventricular papillary muscle in the late-gestation sheep fetus.

The force-generating capacity of cardiomyocytes rapidly changes during gestation and early postnatal life coinciding with a transition in cardiomyocyte nucleation in both mice and rats. Changes in nucleation, in turn, appear to coincide with important changes in the excitation-contraction coupling architecture. However, it is not clear whether similar changes are observed in other mammals in which this transition occurs prenatally, such as sheep. Using small (70-300 microM diameter) chemically skinned cardiomyocyte bundles from the right ventricular papillary muscle of sheep fetuses at 126-132 and 137-140 days (d) gestational age (GA), we aimed to examine whether changes in cardiomyocyte nucleation during late gestation coincided with developmental changes in excitation-contraction coupling parameters (e.g., Ca(2+) uptake, Ca(2+) release, and force development). All experiments were conducted at room temperature (23 +/- 1 degrees C). We found that the proportion of mononucleate cardiomyocytes decreased significantly with GA (126-132 d, 45.7 +/- 4.7%, n = 7; 137-140 d, 32.8 +/- 1.6%, n = 6; P < 0.05). When we then examined force development between the two groups, there was no significant difference in either the maximal Ca(2+)-activated force (6.73 +/- 1.54 mN/mm(2), n = 14 vs. 6.55 +/- 1.25 mN/mm(2), n = 7, respectively) or the Ca(2+) sensitivity of the contractile apparatus (pCa at 50% maximum Ca(2+)-activated force: 126-132 d, 6.17 +/- 0.06, n = 14; 137-140 d, 6.24 +/- 0.08, n = 7). However, sarcoplasmic reticulum (SR) Ca(2+) uptake rates (but not Ca(2+) release) increased with GA (P < 0.05). These data reveal that during late gestation in sheep when there is a major transition in cardiomyocyte nucleation, SR Ca(2+) uptake rates increase, which would influence total SR Ca(2+) content and force production.     

Effects of temperature and calcium availability on cardiac contractility in Synbranchus marmoratus, a neotropical teleost

An isometric muscle preparation was used to investigate the importance of the ventricular sarcoplasmic reticulum (SR) and extracellular Ca²⁺ (1.25 up to 11.25 mM) to force generation at 25 °C (acclimation temperature), 15 and 35 °C. The post-rest tension and force–frequency relationship were conducted with and without 10 μM ryanodine in the bathing medium. Increments in extracellular Ca²⁺ resulted in increases in twitch force development only at 35 °C. A significant post-rest potentiation was recorded for the control preparations at 25 °C (100% to 119.8 ± 4.1%). However, this post-rest potentiation was inhibited by ryanodine only at 25 °C (100% to 97.6 ± 1.5%). At 35 °C, force remained unchanged in the control preparations, but a significant post-rest decay was recorded in the presence of ryanodine (100% to 76.6 ± 4.6%) while at 15 °C, ryanodine was not able to preventing the post-rest potentiation observed in the control preparations. The increases in the imposed contraction frequency caused a decline of the force at 25 and 35 °C and ryanodine decreased significantly peak tension at both temperatures. The findings suggest a high or medium calcium turnover, possibly related to the presence of a functional SR, whose functionality is diminished when temperature is decreased.     

Divergent effects of the malignant hyperthermia-susceptible Arg(615)-->Cys mutation on the Ca(2+) and Mg(2+) dependence of the RyR1

The sarcoplasmic reticulum (SR) Ca(2+) release channel (RyR1) from malignant hyperthermia-susceptible (MHS) porcine skeletal muscle has a decreased sensitivity to inhibition by Mg(2+). This diminished Mg(2+) inhibition has been attributed to a lower Mg(2+) affinity of the inhibition (I) site. To determine whether alterations in the Ca(2+) and Mg(2+) affinity of the activation (A) site contribute to the altered Mg(2+) inhibition, we estimated the Ca(2+) and Mg(2+) affinities of the A- and I-sites of normal and MHS RyR1. Compared with normal SR, MHS SR required less Ca(2+) to half-maximally activate [(3)H]ryanodine binding (K(A,Ca): MHS = 0.17 +/- 0.01 microM; normal = 0.29 +/- 0.02 microM) and more Ca(2+) to half-maximally inhibit ryanodine binding (K(I,Ca): MHS = 519.3 +/- 48.7 microM; normal = 293.3 +/- 24.2 microM). The apparent Mg(2+) affinity constants of the MHS RyR1 A- and I-sites were approximately twice those of the A- and I-sites of the normal RyR1 (K(A,Mg): MHS = 44.36 +/- 4.54 microM; normal = 21.59 +/- 1.66 microM; K(I,Mg): MHS = 660.8 +/- 53.0 microM; normal = 299.2 +/- 24.5 microM). Thus, the reduced Mg(2+) inhibition of the MHS RyR1 compared with the normal RyR1 is due to both an enhanced selectivity of the MHS RyR1 A-site for Ca(2+) over Mg(2+) and a reduced Mg(2+) affinity of the I-site.     

Defective Ca(2+) handling proteins regulation during heart failure

Abnormal release of Ca(2+) from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction in heart failure (HF). We previously demonstrated that RyR2 macromolecular complexes from HF rat were significantly more depleted of FK506 binding protein (FKBP12.6). Here we assessed expression of key Ca(2+) handling proteins and measured SR Ca(2+) content in control and HF rat myocytes. Direct measurements of SR Ca(2+) content in permeabilized cardiac myocytes demonstrated that SR luminal [Ca(2+)] is markedly lowered in HF (HF: DeltaF/F(0) = 26.4+/-1.8, n=12; control: DeltaF/F(0) = 49.2+/-2.9, n=10; P<0.01). Furthermore, we demonstrated that the expression of RyR2 associated proteins (including calmodulin, sorcin, calsequestrin, protein phosphatase 1, protein phosphatase 2A), Ca(2+) ATPase (SERCA2a), PLB phosphorylation at Ser16 (PLB-S16), PLB phosphorylation at Thr17 (PLB-T17), L-type Ca(2+) channel (Cav1.2) and Na(+)- Ca(2+) exchanger (NCX) were significantly reduced in rat HF. Our results suggest that systolic SR reduced Ca(2+) release and diastolic SR Ca(2+) leak (due to defective protein-protein interaction between RyR2 and its associated proteins) along with reduced SR Ca(2+) uptake (due to down-regulation of SERCA2a, PLB-S16 and PLB-T17), abnormal Ca(2+) extrusion (due to down-regulation of NCX) and defective Ca(2+) -induced Ca(2+) release (due to down-regulation of Cav1.2) could contribute to HF.     

Influence of sarcoplasmic reticulum calcium loading on mechanical and relaxation restitution

Mechanical and relaxation restitution represent the restoration of contractile force and relaxation, respectively, in premature beats having progressively longer extrasystolic intervals (ESI); these phenomena are related to intracellular activator Ca(2+) by poorly defined mechanisms. We tested the hypothesis that the level of phospholamban [which modulates the affinity of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase for Ca(2+), and thus the SR Ca(2+) load] may be an important determinant of both mechanical and relaxation restitution. Five mice with ablation of the phospholamban (PLB) gene (PLBKO), eight isogenic wild-type controls (129SvJ), eleven mice with PLB overexpression (PLBOE), and nine isogenic wild-type (FVB/N) controls were anesthetized and instrumented with a 1.4-Fr Millar catheter in the left ventricle and a 1-Fr pacemaker in the right atrium. At a cycle length of 200 ms, extrastimuli with increasing ESI were introduced, and the peak rates of left ventricular isovolumic contraction (+/-dP/dt(max)) were normalized and fit to monoexponential equations. In a subset, the protocols were repeated after ryanodine (4 ng/g) was administered to deplete SR Ca(2+) stores. The time constant of mechanical restitution in PLBKO was significantly shorter [6.3 +/- 1.2 (SE) vs. 47.7 +/- 7.6 ms] and began earlier (50 +/- 10 vs. 70 +/- 19 ms) than in 129SvJ. In contrast, the time constant of mechnical restitution was significantly longer (80.3 +/- 7.6 vs. 54.1 +/- 9.2 ms) in PLBOE than in FVB/N. The time constant of relaxation restitution was less in PLBKO than in 129SvJ (26.2 +/- 9.9 vs. 44.6 +/- 3.3, P < 0.05) but was similar in PLBOE and FVB/N (21.1 +/- 6.3 vs. 20.5 +/- 5.7 ms). Intravenous ryanodine decreased significantly the time constants of mechanical restitution in PLBOE, 129SvJ, and FVB/N but was lethal in PLBKO. In contrast, ryanodine increased the time constant of relaxation restitution. Thus 1) the phospholamban level is a critical determinant of mechanical restitution and (to a lesser extent) relaxation restitution in these transgenic models, and 2) ryanodine differentially affects mechanical and relaxation restitution. Furthermore, our data suggest a dissociation of processes within the SR that govern contraction and relaxation.     

Modification of sarcoplasmic reticulum (SR) Ca2+ release by FK506 induces defective excitation-contraction coupling only when SR Ca2+ recycling is disturbed

This study examined whether the effects of FK506-binding protein dissociation from sarcoplasmic reticulum (SR) Ca(2+) release channels on excitation-contraction (EC) coupling changed when SR Ca(2+) reuptake and (or) the trans-sarcolemmal Ca(2+) extrusion were altered. The steady-state twitch Ca(2+) transient (CaT), cell shortening, post-rest caffeine-induced CaT, and Ca(2+) sparks were measured in rat ventricular myocytes using laser-scanning confocal microscopy. In the normal condition, 50 micromol FK506/L significantly increased steady-state CaT, cell shortening, and post-rest caffeine-induced CaT. When the cells were solely perfused with thapsigargin, FK506 did not reduce any of the states, but when low [Ca(2+)](0) (0.1 mmol/L) was perfused additionally, FK506 reduced CaT and cell shortening, and accelerated the reduction of post-rest caffeine-induced CaT. FK506 significantly increased Ca(2+) spark frequency in the normal condition, whereas it mainly prolonged duration of individual Ca(2+) sparks under the combination of thapsigargin and low [Ca(2+)](0) perfusion. Modification of SR Ca(2+) release by FK506 impaired EC coupling only when released Ca(2+) could not be taken back into the SR and was readily extruded to the extracellular space. Our findings could partly explain the controversy regarding the contribution of FK506-binding protein dissociation to defective EC coupling.     

Low N-ethylmaleimide concentrations activate ryanodine receptors by a reversible interaction, not an alkylation of critical thiols

Previous studies proposed that N-ethylmaleimide (NEM) alkylates 3 classes of thiols on skeletal muscle ryanodine receptors (RyRs) producing 3 phases of channel modification, as function of time and concentration. NEM (5 mm) decreased, increased, and then decreased the open probability (P(o)) of the channel by thiol alkylation, a reaction not reversed by reducing agents. We now show that low NEM concentrations (20-200 microm) elicit Ca(2+) release from sarcoplasmic reticulum (SR) vesicles, but contrary to expectations, the effect was fully reversed by reducing agents or by washing SR vesicles. In bilayers, NEM (0.2 mm) increased P(o) of RyRs within seconds when added to the cis (not trans) side, and dithiothreitol (DTT; 1 mm) decreased P(o) in seconds. High (5 mm) NEM concentrations elicited SR Ca(2+) release that was not reversed by DTT, as expected for an alkylation reaction. A non-sulfhydryl reagent structurally related to NEM, N-ethylsuccinimide (0.1-0.5 mm), also elicited SR Ca(2+) release that was not reversed by DTT (1 mm). Other alkylating agents elicited SR Ca(2+) release, which was fully (N-methylmaleimide) or partially (iodoacetic acid) reversed by DTT and inhibited by ruthenium red. Nitric oxide (NO) donors at concentrations that did not activate RyRs inhibited NEM-induced Ca(2+) release, most likely by an interaction of NO with NEM rather than an inactivation of RyRs by NO. Thus, at low concentrations, NEM does not act as a selective thiol reagent and activates RyRs without alkylating critical thiols indicating that the multiple phases of ryanodine binding are unrelated to RyR activity or to NEM alkylation of RyRs.     

Role of sarcoplasmic reticulum in regulation of tonic contraction of rabbit basilar artery

Superficial sarcoplasmic reticulum (SR) regulates smooth muscle force development directly by Ca(2+) release and removal to and from the cytoplasm (Somlyo and Somlyo. J Cardiovasc Pharmacol 8, Suppl 8: S42-S47, 1986) by buffering Ca(2+) influx and contributing to Ca(2+) extrusion (Mueller and van Breemen. Nature 281: 682-683, 1979) and indirectly by releasing Ca(2+) near Ca(2+)-activated K(+) channels (K(Ca)) to hyperpolarize the plasma membrane (Bolton and Imaizumi. Cell Calcium 20: 141-152, 1996 and Nelson et al. Science 270: 633-637, 1995). In the rabbit basilar artery, relative contributions of direct effects and those mediated through activation of K(Ca) were evaluated by measuring force and intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to the SR-depleting agents thapsigargin and ryanodine and the large conductance K(Ca) (BK(Ca)) blockers iberiotoxin (IbTX) and tetraethylammonium ion (TEA). A large contraction was observed in response to K(Ca) blockade with either 3 mM TEA or 100 nM IbTX and also after addition of 10 microM ryanodine or 2 microM thapsigargin. When K(Ca) was blocked first with TEA or IbTX, subsequent addition of thapsigargin or ryanodine also increased force. Measurements of fura 2 fluorescence showed parallel increases in [Ca(2+)](i) in response to sequential blockade of sarco(endo)plasmic reticulum Ca(2+)-ATPase and K(Ca) regardless of the order of application. It appears that a significant fraction of K(Ca) remains activated in the absence of SR function and that SR contributes to relaxation after blockade of K(Ca). We found that depletion of SR before stimulating Ca(2+) influx through voltage-gated Ca(2+) channels markedly reduced force development rate and that thapsigargin abolished this effect. We conclude that the SR of rabbit cerebral arteries modulates constriction by direct and indirect mechanisms.     

cADP-ribose activates reconstituted ryanodine receptors from coronary arterial smooth muscle

The present study was designed to test the hypothesis that cADP-ribose (cADPR) increases Ca(2+) release through activation of ryanodine receptors (RYR) on the sarcoplasmic reticulum (SR) in coronary arterial smooth muscle cells (CASMCs). We reconstituted RYR from the SR of CASMCs into planar lipid bilayers and examined the effect of cADPR on the activity of these Ca(2+) release channels. In a symmetrical cesium methanesulfonate configuration, a 245 pS Cs(+) current was recorded. This current was characterized by the formation of a subconductance and increase in the open probability (NP(o)) of the channels in the presence of ryanodine (0.01-1 microM) and imperatoxin A (100 nM). A high concentration of ryanodine (50 microM) and ruthenium red (40-80 microM) substantially inhibited the activity of RYR/Ca(2+) release channels. Caffeine (0.5-5 mM) markedly increased the NP(o) of these Ca(2+) release channels of the SR, but D-myo-inositol 1,4,5-trisphospate and heparin were without effect. Cyclic ADPR significantly increased the NP(o) of these Ca(2+) release channels of SR in a concentration-dependent manner. Addition of cADPR (0.01 microM) into the cis bath solution produced a 2.9-fold increase in the NP(o) of these RYR/Ca(2+) release channels. An eightfold increase in the NP(o) of the RYR/Ca(2+) release channels (0.0056 +/- 0.001 vs. 0.048 +/- 0.017) was observed at a concentration of cADPR of 1 microM. The effect of cADPR was completely abolished by ryanodine (50 microM). In the presence of cADPR, Ca(2+)-induced activation of these channels was markedly enhanced. These results provide evidence that cADPR activates RYR/Ca(2+) release channels on the SR of CASMCs. It is concluded that cADPR stimulates Ca(2+) release through the activation of RYRs on the SR of these smooth mucle cells.     

Ca(2+) handling in isolated human atrial myocardium

Physiologically, human atrial and ventricular myocardium are coupled by an identical beating rate and rhythm. However, contractile behavior in atrial myocardium may be different from that in ventricular myocardium, and little is known about intracellular Ca(2+) handling in human atrium under physiological conditions. We used rapid cooling contractures (RCCs) to assess sarcoplasmic reticulum (SR) Ca(2+) content and the photoprotein aequorin to assess intracellular Ca(2+) transients in atrial and ventricular muscle strips isolated from nonfailing human hearts. In atrial myocardium (n = 19), isometric twitch force frequency dependently (0. 25-3 Hz) increased by 78 +/- 25% (at 3 Hz; P < 0.05). In parallel, aequorin light signals increased by 111 +/- 57% (P < 0.05) and RCC amplitudes by 49 +/- 13% (P < 0.05). Similar results were obtained in ventricular myocardium (n = 13). SR Ca(2+) uptake (relative to Na(+)/Ca(2+) exchange) frequency dependently increased in atrial and ventricular myocardium (P < 0.05). With increasing rest intervals (1-240 s), atrial myocardium (n = 7) exhibited a parallel decrease in postrest twitch force (at 240 s by 68 +/- 5%, P < 0.05) and RCCs (by 49 +/- 10%, P < 0.05). In contrast, postrest twitch force and RCCs significantly increased in ventricular myocardium (n = 6). We conclude that in human atrial and ventricular myocardium the positive force-frequency relation results from increased SR Ca(2+) turnover. In contrast, rest intervals in atrial myocardium are associated with depressed contractility and intracellular Ca(2+) handling, which may be due to rest-dependent SR Ca(2+) loss (Ca(2+) leak) and subsequent Ca(2+) extrusion via Na(+)/Ca(2+) exchange. Therefore, the influence of rate and rhythm on mechanical performance is not uniform in atrial and ventricular myocardium.     

Overexpression of Na+/Ca2+ exchanger gene attenuates postinfarction myocardial dysfunction

We monitored myocardial function in postinfarcted wild-type (WT) and transgenic (TG) mouse hearts with overexpression of the cardiac Na(+)/Ca(2+) exchanger. Five weeks after infarction, cardiac function was better maintained in TG than WT mice [left ventricular (LV) systolic pressure: WT, 41 +/- 2; TG, 58 +/- 3 mmHg; P < 0.05; maximum rising rate of LV pressure (+dP/dt(max)): WT, 3,750 +/- 346; TG, 5,075 +/- 334 mmHg/s; P < 0.05]. The isometric contractile response to beta-adrenergic stimulation was greater in papillary muscles from TG than WT mice (WT, 13.2 +/- 0.9; TG, 16.3 +/- 1.0 mN/mm(2) at 10(-4) M isoproterenol). The sarcoplasmic reticulum (SR) Ca(2+) content investigated by rapid cooling contractures in papillary muscles was greater in TG than WT mouse hearts. We conclude that myocardial function is better preserved in TG mice 5 wk after infarction, which results from enhanced SR Ca(2+) content via overexpression of the Na(+)/Ca(2+) exchanger.     

CaMKII-dependent reactivation of SR Ca(2+) uptake and contractile recovery during intracellular acidosis

In hearts, intracellular acidosis disturbs contractile performance by decreasing myofibrillar Ca(2+) response, but contraction recovers at prolonged acidosis. We examined the mechanism and physiological implication of the contractile recovery during acidosis in rat ventricular myocytes. During the initial 4 min of acidosis, the twitch cell shortening decreased from 2.3 +/- 0.3% of diastolic length to 0.2 +/- 0.1% (means +/- SE, P < 0.05, n = 14), but in nine of these cells, contractile function spontaneously recovered to 1.5 +/- 0.3% at 10 min (P < 0.05 vs. that at 4 min). During the depression phase, both the diastolic intracellular Ca(2+) concentration ([Ca(2+)](i)) and Ca(2+) transient (CaT) amplitude increased, and the twitch [Ca(2+)](i) decline prolonged significantly (P < 0.05). In the cells that recovered, a further increase in CaT amplitude and a reacceleration of twitch [Ca(2+)](i) decline were observed. The increase in diastolic [Ca(2+)](i) was less extensive than the increase in the cells that did not recover (n = 5). Blockade of sarcoplasmic reticulum (SR) function by ryanodine (10 microM) and thapsigargin (1 microM) or a selective inhibitor of Ca(2+)-calmodulin kinase II, 2-[N- (2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methyl benzylamine (1 microM) completely abolished the reacceleration of twitch [Ca(2+)](i) decline and almost eliminated the contractile recovery. We concluded that during prolonged acidosis, Ca(2+)-calmodulin kinase II-dependent reactivation of SR Ca(2+) uptake could increase SR Ca(2+) content and CaT amplitude. This recovery can compensate for the decreased myofibrillar Ca(2+) response, but may also cause Ca(2+) overload after returning to physiological pH(i).     

Postcontractile force depression in humans is associated with an impairment in SR Ca(2+) pump function

To investigate the hypothesis that intrinsic changes in sarcoplasmic reticulum (SR) Ca(2+)-sequestration function can be implicated in postcontractile depression (PCD) of force in humans, muscle tissue was obtained from the vastus lateralis and determinations of maximal Ca(2+) uptake and maximal Ca(2+)-ATPase activity were made on homogenates obtained before and after the induction of PCD. Eight untrained females, age 20.6+/-0.75 yr (mean +/- SE), performed a protocol consisting of 30 min of isometric exercise at 60% maximal voluntary contraction and at 50% duty cycle (5-s contraction and 5-s relaxation) to induce PCD. Muscle mechanical performance determined by evoked activation was measured before (0 min), during (15 and 30 min), and after (60 min) exercise. The fatiguing protocol resulted in a progressive reduction (P<0.05) in evoked force, which by 30 min amounted to 52% for low frequency (10 Hz) and 20% for high frequency (100 Hz). No force restoration occurred at either 10 or 100 Hz during a 60-min recovery period. Maximal SR Ca(2+)-ATPase activity (nmol x mg protein(-1) x min(-1)) and maximal SR Ca(2+) uptake (nmol. mg protein(-1) x min(-1)) were depressed (P<0.05) by 15 min of exercise [192+/-45 vs. 114+/-8.7 and 310+/-59 vs. 205+/-47, respectively; mean +/- SE] and remained depressed at 30 min of exercise. No recovery in either measure was observed during the 60-min recovery period. The coupling ratio between Ca(2+)-ATPase and Ca(2+) uptake was preserved throughout exercise and during recovery. These results illustrate that during PCD, Ca(2+) uptake is depressed and that the reduction in Ca(2+) uptake is due to intrinsic alterations in the Ca(2+) pump. The role of altered Ca(2+) sequestration in Ca(2) release, cytosolic-free calcium, and PCD remains to be determined.     

Complex effects of ryanodine on the sarcoplasmic reticulum Ca2+ levels in smooth muscle cells

We have studied the effects of ryanodine and inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) with thapsigargin, on both [Ca(2+)](i) and the sarcoplasmic reticulum (SR) Ca(2+) level during caffeine-induced Ca(2+) release in single smooth muscle cells. Incubation with 10 microM ryanodine did not inhibit the first caffeine-induced [Ca(2+)](i) response, although it abolished the [Ca(2+)](i) response to a second application of caffeine. To assess whether ryanodine was inducing a permanent depletion of the internal Ca(2+) stores, we measured the SR Ca(2+) level with Mag-Fura-2. The magnitude of the caffeine-induced reduction in the SR Ca(2+) level was not augmented by incubating cells with 1 microM ryanodine. Moreover, on removal of caffeine, the SR Ca(2+) levels partially recovered in 61% of the cells due to the activity of thapsigargin-sensitive SERCA pumps. Unexpectedly, 10 microM ryanodine instead of inducing complete depletion of SR Ca(2+) stores markedly reduced the caffeine-induced SR Ca(2+) response. It was necessary to previously inhibit SERCA pumps with thapsigargin for ryanodine to be able to induce caffeine-triggered permanent depletion of SR Ca(2+) stores. These data suggest that the effect of ryanodine on smooth muscle SR Ca(2+) stores was markedly affected by the activity of SERCA pumps. Our data highlight the importance of directly measuring SR Ca(2+) levels to determine the effect of ryanodine on the internal Ca(2+) stores.     

Serine phosphorylation of the sarcoplasmic reticulum Ca(2+)-ATPase in the intact beating rabbit heart.

Recent studies have demonstrated that Ca(2+)/calmodulin-dependent protein kinase phosphorylates the Ca(2+)-pumping ATPase of cardiac sarcoplasmic reticulum (SR) in vitro. Also, evidence from in vitro studies suggested that this phosphorylation, occurring at Ser(38), results in stimulation of Ca(2+) transport. In the present study, we investigated whether serine phosphorylation of the SR Ca(2+)-ATPase occurs in the intact functioning heart. Hearts removed from anesthetized rabbits were subjected to retrograde aortic perfusion of the coronary arteries with oxygenated mammalian Ringer solution containing (32)P(i) and contractions were monitored by recording systolic left ventricular pressure development. Following 45-50 min of (32)P perfusion, the hearts were freeze-clamped, SR isolated, and analyzed for protein phosphorylation. SDS-polyacrylamide gel electrophoresis and autoradiography showed phosphorylation of several peptides including the Ca(2+)-ATPase and Ca(2+) release channel (ryanodine receptor). The identity of Ca(2+)-ATPase as a phosphorylated substrate was confirmed by Western immunoblotting as well as immunoprecipitation using a cardiac SR Ca(2+)-ATPase-specific monoclonal antibody. The Ca(2+)-ATPase showed immunoreactivity with a phosphoserine monoclonal antibody indicating that the in situ phosphorylation occurred at the serine residue. Quantification of Ca(2+)-ATPase phosphorylation in situ yielded a value of 208 +/- 12 pmol (32)P/mg SR protein which corresponded to the phosphorylation of approximately 20% of the Ca(2+) pump units in the SR membrane. Since this phosphorylation occurred under basal conditions (i.e., in the absence of any inotropic intervention), a considerable steady-state pool of serine-phosphorylated Ca(2+)-ATPase likely exists in the normally beating heart. These findings demonstrate that serine phosphorylation of the Ca(2+)-ATPase is a physiological event which may be important in the regulation of SR function.     

Maintenance of resting tension in the american eel (Anguilla rostrata L.) heart is dependent upon exogenous fuel and the sarcoplasmic reticulum

The relationship between extracellular glucose and management of cell Ca(2+) in the heart of the American eel (Anguilla rostrata) was indirectly assessed by monitoring the performance of isolated ventricular strips at 20 degrees C. Twitch force increased in ventricular strips under specific conditions of 30 bpm pacing and an extracellular Ca(2+) challenge from 1.5 to 9.5 mM. The response was independent of any exogenous metabolic fuel in the medium. Resting tension was maintained when glucose was available, but in the absence of a metabolic fuel, resting tension increased in response to the increase in extracellular Ca(2+) level. When ventricular strips were treated with iodoacetate to inhibit glycolysis, a Ca(2+) challenge resulted in a decrease in twitch force in association with an approximately equivalent increase in resting tension even in the presence of exogenous glucose. However, when pyruvate (5 mM) was substituted as a metabolic fuel, twitch force increased as a function of extracellular Ca(2+), and resting tension was maintained in the presence of iodoacetate. Therefore, there is a need for an extracellular fuel but not a specific metabolic requirement for glucose to maintain the performance characteristics, which are presumably related to the management of intracellular Ca(2+) levels. Ventricular strips were treated with ryanodine to inhibit Ca(2+) release and uptake by the sarcoplasmic reticulum (SR). Ryanodine treatment impaired postrest potentiation at high extracellular Ca(2+) levels. In the presence of ryanodine, the protective effect of glucose on the increase in resting tension in the face of an extracellular Ca(2+) challenge was eliminated. Considered together, the results reveal that the heart of the American eel has a requirement for an extracellular fuel to manage intracellular Ca(2+) at high Ca(2+) loads, and that the SR plays a role in the beat-to-beat regulation of Ca(2+) at a frequency of 30 bpm, high Ca(2+) load, and 20 degrees C.     

Arg(615)Cys substitution in pig skeletal ryanodine receptors increases activation of single channels by a segment of the skeletal DHPR II-III loop

The effect of peptides, corresponding to sequences in the skeletal muscle dihydropyridine receptor II-III loop, on Ca(2+) release from sarcoplasmic reticulum (SR) and on ryanodine receptor (RyR) calcium release channels have been compared in preparations from normal and malignant hyperthermia (MH)-susceptible pigs. Peptide A (Thr(671)-Leu(690); 36 microM) enhanced the rate of Ca(2+) release from normal SR (SR(N)) and from SR of MH-susceptible muscle (SR(MH)) by 10 +/- 3.2 nmole/mg/min and 76 +/- 9.7 nmole/mg/min, respectively. Ca (2+) release from SR(N) or SR(MH) was not increased by control peptide NB (Gly(689)-Lys(708)). AS (scrambled A sequence; 36 microM) did not alter Ca (2+) release from SR(N), but increased release from SR(MH) by 29 +/- 4.9 nmoles/mg/min. RyR channels from MH-susceptible muscle (RyR(MH)) were up to about fourfold more strongly activated by peptide A (> or =1 nM) than normal RyR channels (RyR(N)) at -40 mV. Neither NB or AS activated RyR(N). RyR(MH) showed an approximately 1.8-fold increase in mean current with 30 microM AS. Inhibition at +40 mV was stronger in RyR(MH) and seen with peptide A (> or = 0.6 microM) and AS (> or = 0.6 microM), but not NB. These results show that the Arg(615)Cys substitution in RyR(MH) has multiple effects on RyRs. We speculate that enhanced DHPR activation of RyRs may contribute to increased Ca(2+) release from SR in MH-susceptible muscle.     

Positive inotropic effect of ryanodine on rabbit ventricular muscle: dependence on the intracellular calcium load

Two types of electrical and mechanical responses to 1 mumol/l ryanodine, depending on the intracellular calcium load, were observed in rabbit papillary muscles. In a normal calcium solution, ryanodine induced a transient decline followed by a stable increase in the developed force (by 20 +/- 5% of the pretreatment level; n = 30) and prolonged the action potential (AP). The positive ryanodine response showed an increased time-to-peak force and was completely suppressed by 2 mumol/l nifedipine, partially blocked by 50 mumol/l tetracaine (Ca2+ release blocker), but greatly potentiated by 20 mmol/l CsCl or (-) Bay R 5414 which prolonged the AP. The prolonged time-to-peak force of the positive ryanodine response was shortened by procedures raising the content of Ca2+ in the sarcoplasmic reticulum (SR). It is suggested that the initial decline in the force amplitude results from Ca2+ leakage from the SR which is further compensated for by an elevation of both the transmembrane Ca2+ entry and intracellular Ca2+ release. In calcium overloaded myocardium, 1 mumol/l ryanodine caused irreversible contracture and dramatic AP shortening, explained by a massive Ca2+ release from the overloaded SR into the cytoplasm. It is concluded that the calcium content in the SR is the main modulator of the electrical and mechanical effects of ryanodine in ventricular myocardium.