Enhanced Ca2+ transport and muscle relaxation in skeletal muscle from sarcolipin-null mice

Sarcolipin (SLN) inhibits sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pumps. To evaluate the physiological significance of SLN in skeletal muscle, we compared muscle contractility and SERCA activity between Sln-null and wild-type mice. SLN protein expression in wild-type mice was abundant in soleus and red gastrocnemius (RG), low in extensor digitorum longus (EDL), and absent from white gastrocnemius (WG). SERCA activity rates were increased in soleus and RG, but not in EDL or WG, from Sln-null muscles, compared with wild type. No differences were seen between wild-type and Sln-null EDL muscles in force-frequency curves or maximum rates of force development (+dF/dt). Maximum relaxation rates (-dF/dt) of EDL were higher in Sln-null than wild type across a range of submaximal stimulation frequencies, but not during a twitch or peak tetanic contraction. For soleus, no differences were seen between wild type and Sln-null in peak tetanic force or +dF/dt; however, force-frequency curves showed that peak force during a twitch and 10-Hz contraction was lower in Sln-null. Changes in the soleus force-frequency curve corresponded with faster rates of force relaxation at nearly all stimulation frequencies in Sln-null compared with wild type. Repeated tetanic stimulation of soleus caused increased (-dF/dt) in wild type, but not in Sln-null. No compensatory responses were detected in analysis of other Ca(2+) regulatory proteins using Western blotting and immunohistochemistry or myosin heavy chain expression using immunofluorescence. These results show that 1) SLN regulates Ca(2+)-ATPase activity thereby regulating contractile kinetics in at least some skeletal muscles, 2) the functional significance of SLN is graded to the endogenous SLN expression level, and 3) SLN inhibitory effects on SERCA function are relieved in response to repeated contractions thus enhancing relaxation rates.     

The relationship between contractile force and intracellular [Ca2+] in intact rat cardiac trabeculae

The control of force by [Ca2+] was investigated in rat cardiac trabeculae loaded with fura-2 salt. At sarcomere lengths of 2.1-2.3 microns, the steady state force-[Ca2+]i relationship during tetanization in the presence of ryanodine was half maximally activated at a [Ca2+]i of 0.65 +/- 0.19 microM with a Hill coefficient of 5.2 +/- 1.2 (mean +/- SD, n = 9), and the maximal stress produced at saturating [Ca2+]i equalled 121 +/- 35 mN/mm2 (n = 9). The dependence of steady state force on [Ca2+]i was identical in muscles tetanized in the presence of the Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA). The force-[Ca2+]i relationship during the relaxation of twitches in the presence of CPA coincided exactly to that measured at steady state during tetani, suggesting that CPA slows the decay rate of [Ca2+]i sufficiently to allow the force to come into a steady state with the [Ca2+]i. In contrast, the relationship of force to [Ca2+]i during the relaxation phase of control twitches was shifted leftward relative to the steady state relationship, establishing that relaxation is limited by the contractile system itself, not by Ca2+ removal from the cytosol. Under control conditions the force-[Ca2+]i relationship, quantified at the time of peak twitch force (i.e., dF/dt = 0), coincided fairly well with steady state measurements in some trabeculae (i.e., three of seven). However, the force-[Ca2+]i relationship at peak force did not correspond to the steady state measurements after the application of 5 mM 2,3-butanedione monoxime (BDM) (to accelerate cross-bridge kinetics) or 100 microM CPA (to slow the relaxation of the [Ca2+]i transient). Therefore, we conclude that the relationship of force to [Ca2+]i during physiological twitch contractions cannot be used to predict the steady state relationship.     

Inactivation of a Ca2+-induced Ca2+ release channel from skeletal muscle sarcoplasmic reticulum during active Ca2+ transport

ATP-dependent Ca2+ uptake by subfractions of skeletal muscle sarcoplasmic reticulum (SR) was studied with the Ca2+ indicator dye, antipyrylazo III. Ca2+ uptake by heavy SR showed two phases, a slow uptake phase and a fast uptake phase. By contrast, Ca2+ uptake by light SR exhibited a monophasic time course. In both fractions a steady state of Ca2+ uptake was observed when the concentration of free Ca2+ outside the vesicles was reduced to less than 0.1 microM. In the steady state, the addition of 5 microM Ca2+ to the external medium triggered rapid Ca2+ release from heavy SR but not from light SR, indicating that the heavy fraction contains a Ca2+-induced Ca2+ release channel. During Ca2+ uptake, heavy SR showed a constant Ca2+-dependent ATPase activity (1 mumol/mg protein X min) which was about 150 times higher than the rate of Ca2+ uptake in the slow uptake phase. Ruthenium red, an inhibitor of Ca2+-induced Ca2+ release, enhanced the rate of Ca2+ uptake during the slow phase without affecting Ca2+-dependent ATPase activity. Adenine nucleotides, activators of Ca2+ release, reduced the Ca2+ uptake rate. These results suggest that the rate of Ca2+ accumulation by heavy SR is not proportional to ATPase activity during the slow uptake phase due to the activation of the channel for Ca2+-induced Ca2+ release. In addition, they suggest that the release channel is inactivated during the fast Ca2+ uptake phase.     

Relaxation kinetics following sudden Ca(2+) reduction in single myofibrils from skeletal muscle

To investigate the roles of cross-bridge dissociation and cross-bridge-induced thin filament activation in the time course of muscle relaxation, we initiated force relaxation in single myofibrils from skeletal muscles by rapidly (approximately 10 ms) switching from high to low [Ca(2+)] solutions. Full force decay from maximal activation occurs in two phases: a slow one followed by a rapid one. The latter is initiated by sarcomere "give" and dominated by inter-sarcomere dynamics (see the companion paper, Stehle, R., M. Krueger, and G. Pfitzer. 2002. Biophys. J. 83:2152-2161), while the former occurs under nearly isometric conditions and is sensitive to mechanical perturbations. Decreasing the Ca(2+)-activated force preceding the start of relaxation does not increase the rate of the slow isometric phase, suggesting that cycling force-generating cross-bridges do not significantly sustain activation during relaxation. This conclusion is strengthened by the finding that the rate of isometric relaxation from maximum force to any given Ca(2+)-activated force level is similar to that of Ca(2+)-activation from rest to that given force. It is likely, therefore, that the slow rate of force decay in full relaxation simply reflects the rate at which cross-bridges leave force-generating states. Because increasing [P(i)] accelerates relaxation while increasing [MgADP] slows relaxation, both forward and backward transitions of cross-bridges from force-generating to non-force-generating states contribute to muscle relaxation.     

Interactions between intracellular calcium and phosphate in intact mouse muscle during fatigue

Fatigue was studied in intact tibialis anterior muscle of anesthetized mice. The distal tendon was detached and connected to a force transducer while blood flow continued normally. The muscle was stimulated with electrodes applied directly to the muscle surface and fatigued by repeated (1 per 4 s), brief (0.4 s), maximal (100-Hz stimulation frequency) tetani. Force declined monotonically to 49 ± 5% of the initial value with a half time of 36 ± 5 s and recovered to 86 ± 4% after 4 min. Intracellular phosphate concentration ([P(i)]) was measured by (31)P-NMR on perchloric acid extracts of muscles. [P(i)] increased during fatigue from 7.6 ± 1.7 to 16.0 ± 1.6 mmol/kg muscle wet wt and returned to control during recovery. Intracellular Ca(2+) was measured with cameleons whose plasmids had been transfected in the muscle 2 wk before the experiment. Yellow cameleon 2 was used to measure myoplasmic Ca(2+), and D1ER was used to measure sarcoplasmic reticulum (SR) Ca(2+). The myoplasmic Ca(2+) during tetani declined steadily during the period of fatigue and showed complete recovery over 4 min. The SR Ca(2+) also declined monotonically during fatigue and showed a partial recovery with rest. These results show that the initial phase of force decline is accompanied by a rise in [P(i)] and a reduction in the tetanic myoplasmic Ca(2+). We suggest that both changes contribute to the fatigue. A likely cause of the decline in tetanic myoplasmic Ca(2+) is precipitation of CaP(i) in the SR.     

Voltage-independent changes in L-type Ca(2+) current uncoupled from SR Ca(2+) release in cardiac myocytes

To determine the effect of voltage-independent alterations of L-type Ca(2+) current (I(Ca)) on the sarcoplasmic reticular (SR) Ca(2+) release in cardiac myocytes, we measured I(Ca) and cytosolic Ca(2+) transients (Ca(i)(2+); intracellular Ca(2+) concentration) in voltage-clamped rat ventricular myocytes during 1) an abrupt increase of extracellular [Ca(2+)] (Ca(o)(2+)) or 2) application of 1 microM FPL-64176, a Ca(2+) channel agonist, to selectively alter I(Ca) in the absence of changes in SR Ca(2+) loading. On the first depolarization in higher Ca(o)(2+), peak I(Ca) was increased by 46 +/- 6% (P < 0.001), but the increases in the maximal rate of rise of Ca(i)(2+) (dCa(i)(2+)/dt(max), where t is time; an index of SR Ca(2+) release flux) and the Ca(i)(2+) transient amplitude were not significant. Rapid exposure to FPL-64176 greatly slowed inactivation of I(Ca), increasing its time integral by 117 +/- 8% (P < 0.001) without significantly increasing peak I(Ca), dCa(i)(2+)/dt(max), or amplitude of the corresponding Ca(i)(2+) transient. Prolongation of exposure to higher Ca(o)(2+) or FPL-64176 did not further increase peak I(Ca) but greatly increased dCa(i)(2+)/dt(max), Ca(i)(2+) transient amplitude, and the gain of Ca(2+) release (dCa(i)(2+)/dt(max)/I(Ca)), evidently due to augmentation of the SR Ca(2+) loading. Also, the time to peak dCa(i)(2+)/dt(max) was significantly increased in the continuous presence of higher Ca(o)(2+) (by 37 +/- 5%, P < 0.001) or FPL-64176 (by 63 +/- 5%, P < 0.002). Our experiments provide the first evidence of a marked disparity between an increased peak I(Ca) and the corresponding SR Ca(2+) release. We attribute this to saturation of the SR Ca(2+) release flux as predicted by local control theory. Prolongation of the SR Ca(2+) release flux, caused by combined actions of a larger I(Ca) and maximally augmented SR Ca(2+) loading, might reflect additional Ca(2+) release from corbular SR.     

Impaired sarcoplasmic reticulum function leads to contractile dysfunction and cardiac hypertrophy

Sarcoplasmic reticulum (SR)-mediated Ca(2+) sequestration and release are important determinants of cardiac contractility. In end-stage heart failure SR dysfunction has been proposed to contribute to the impaired cardiac performance. In this study we tested the hypothesis that a targeted interference with SR function can be a primary cause of contractile impairment that in turn might alter cardiac gene expression and induce cardiac hypertrophy. To study this we developed a novel animal model in which ryanodine, a substance that alters SR Ca(2+) release, was added to the drinking water of mice. After 1 wk of treatment, in vivo hemodynamic measurements showed a 28% reduction in the maximum speed of contraction (+dP/dt(max)) and a 24% reduction in the maximum speed of relaxation (-dP/dt(max)). The slowing of cardiac relaxation was confirmed in isolated papillary muscles. The late phase of relaxation expressed as the time from 50% to 90% relaxation was prolonged by 22%. After 4 wk of ryanodine administration the animals had developed a significant cardiac hypertrophy that was most prominent in both atria (right atrium +115%, left atrium +100%, right ventricle +23%, and left ventricle +13%). This was accompanied by molecular changes including a threefold increase in atrial natriuretic factor mRNA and a sixfold increase in beta-myosin heavy chain mRNA. Sarcoplasmic endoplasmic reticulum Ca(2+) mRNA was reduced by 18%. These data suggest that selective impairment of SR function in vivo can induce changes in cardiac gene expression and promote cardiac growth.     

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.     

Repeated static contractions increase mitochondrial vulnerability toward oxidative stress in human skeletal muscle.

Repeated static contractions (RSC) induce large fluctuations in tissue oxygen tension and increase the generation of reactive oxygen species (ROS). This study investigated the effect of RSC on muscle contractility, mitochondrial respiratory function, and in vitro sarcoplasmic reticulum (SR) Ca(2+) kinetics in human muscle. Ten male subjects performed five bouts of static knee extension with 10-min rest in between. Each bout of RSC (target torque 66% of maximal voluntary contraction torque) was maintained to fatigue. Muscle biopsies were taken preexercise and 0.3 and 24 h postexercise from vastus lateralis. Mitochondria were isolated and respiratory function measured after incubation with H(2)O(2) (HPX) or control medium (Con). Mitochondrial function was not affected by RSC during Con. However, RSC exacerbated mitochondrial dysfunction during HPX, resulting in decreased respiratory control index, decreased mitochondrial efficiency (phosphorylated ADP-to-oxygen consumed ratio), and increased noncoupled respiration (HPX/Con post- vs. preexercise). SR Ca(2+) uptake rate was lower 0.3 vs. 24 h postexercise, whereas SR Ca(2+) release rate was unchanged. RSC resulted in long-lasting changes in muscle contractility, including reduced maximal torque, low-frequency fatigue, and faster torque relaxation. It is concluded that RSC increases mitochondrial vulnerability toward ROS, reduces SR Ca(2+) uptake rate, and causes low-frequency fatigue. Although conclusive evidence is lacking, we suggest that these changes are related to increased formation of ROS during RSC.     

Sarcolipin overexpression in rat slow twitch muscle inhibits sarcoplasmic reticulum Ca2+ uptake and impairs contractile function

Sarcolipin (SLN) is an inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs) in vitro, but its function in vivo has not been defined. NF-SLN cDNA (SLN tagged N-terminally with a FLAG epitope) was introduced into rat soleus muscle in one hindlimb by plasmid injection and electrotransfer. Western blotting showed expression and co-immunoprecipitation showed physical interaction between NF-SLN and SERCA2a. Contractile properties and SERCA2a function were assessed and compared with vector-injected contralateral soleus muscles. NF-SLN reduced both peak twitch force (P(t)) (123.9 +/- 12.5 versus 69.8 +/- 8.9 millinewtons) and tetanic force (P(o)) (562.3 +/- 51.0 versus 300.7 +/- 56.9 millinewtons) and reduced both twitch and tetanic rates of contraction (+dF/dt) and relaxation (-dF/dt) significantly. Repetitive stimulation (750-ms trains at 50 Hz once every 2 s for 3 min) showed that NF-SLN increased susceptibility to fatigue. These changes in contractile function were observed in the absence of endogenous phospholamban, and NF-SLN had no effect on either SERCA2a or SERCA1a expression levels. NF-SLN also decreased maximal Ca(2+) transport activity at pCa 5 by 31% with no significant change in apparent Ca(2+) affinity (6.36 +/- 0.07 versus 6.39 +/- 0.08 pCa units). These results show that NF-SLN expression impairs muscle contractile function by inhibiting SERCA function and diminishing sarcoplasmic reticulum Ca(2+) stores.     

Sex differences in expression of calcium-handling proteins and beta-adrenergic receptors in rat heart ventricle

Human studies reveal sex differences in myocardial function as well as in the incidence and manifestation of heart disease. Myocellular Ca(2+) cycling regulates normal contractile function; whereas cardiac dysfunction in heart failure has been associated with alterations in Ca(2+)-handling proteins. Beta-adrenergic receptor (beta-AR) signaling regulates activity of several Ca(2+)-handling proteins and alterations in beta-AR signaling are associated with heart disease. This study examines sex differences in expression of beta(1)-AR, beta(2)-AR, and Ca(2+)-handling proteins including: L-type calcium channel (Ca(v)1.2) , ryanodine calcium-release channels (RyR), sarcoplasmic reticular Ca(2+) ATPase (SERCA2), phospholamban (PLB) and Na(+)-Ca(2+) exchange protein (NCX) in healthy hearts from male and female Sprague-Dawley rats. Protein levels were examined using Western blot analysis. Abundance of mRNA was determined by real time RT-PCR normalized to abundance of GAPDH mRNA. Contraction parameters were measured in right ventricular papillary muscle in the presence and absence of isoproterenol. Results demonstrate that female ventricle has significantly higher levels of Ca(v)1.2, RyR, and NCX protein compared to males. Messenger RNA abundance for RyR, and NCX protein was significantly higher in females whereas Ca(v)1.2 mRNA was higher in males. No differences were detected in beta-ARs, SERCA2 or PLB. Female right papillary muscle had a faster maximal rate of force development and decline (+/- dF/dt). There were no sex differences in response to isoproterenol. Results show significant sex differences in expression of key ventricular Ca(2+)-handling proteins that are associated with small functional differences in +/- dF/dt. Further studies will determine whether differences in the abundance of these key proteins play a role in sex disparities in the incidence and manifestation of heart disease.     

4-Aminopyridine induces positive lusitropic effects and prevents the negative inotropic action of phenylephrine in the rat cardiac tissue subjected to ischaemia.

The effects of 4-aminopyridine (4-AP) at concentration of 1 mM on the contractility of rat isolated papillary muscle subjected to simulated ischaemia has been evaluated. Additionally, the effects of 4-AP on the phenylephrine inotropic action (a selective agonist of alpha1-adrenergic receptor) on rat isolated cardiac tissue underwent simulated ischaemia and reperfusion was studied. Experiments were performed on rat isolated papillary muscles obtained from left ventricle. The following parameters have been measured: force of contraction (Fc), velocity of contraction (+dF/dt), velocity of relaxation (-dF/dt) and the ratio between time to peak contraction (ttp) and relaxation time at the level of 10% of total contraction amplitude (tt10) as an index of lusitropic effects. Simulated ischaemia lasting 45 min was induced by replacement of standard normoxic solution by no-substrat one gassing with 95% N2/5%CO2. Although 4-AP exerted a slight, but significant positive inotropic action itself, pretreatment with 1 mM of this compound significantly depressed a recovery of Fc and +dF/dt, but improves recovery of -dF/dt in the rat papillary muscle during reperfusion as compared with control group of preparations. Moreover, the paradoxical negative inotropic action of phenylephrine observed in rat stunned papillary muscle was prevented in preparations previously treated by 4-AP. These findings suggest that an inhibition of outward K+ current (probably transient outward and rapid component of delayed rectifying currents at 1 mM of 4-AP) aggravates ischaemia-induced failure in contractility but prevents changes in alpha1-adrenergic receptor signaling pathway occuring during ischaemia.     

Effects of lindane (gamma-hexachlorocyclohexane) on rat heart muscle contraction

The effects of micromolar concentrations of lindane on the mechanical activity of cardiac left ventricular papillary muscles were studied in adult female rats. Lindane decreased the amplitude and duration of the contraction, and slowed down the time course of its ascending phase (i.e. decreased the maximum rate of rise of the initial phase (dC/dt(max))). Both amplitude and duration of the contraction, but not dC/dt(max), were restored by subsequent application of the rapid delayed outward K(+) current (I(Kr)) blocker E-4031 (10 nmol/l). Increasing the stimulation frequency from 1 to 3.3 Hz in the control solution produced a decrease in the amplitude of the first beat peak contraction while a slow recovery phase (srp) developed, as the result of the Na(+)-Ca(2+) exchanger activity. When the frequency was restored to 1 Hz, a post rest potentiation (prp) with a negative staircase (ns) developed due to the sarcoplasmic reticulum (SR) Ca(2+) refilling. Lindane increased the amplitude of both srp and prp, but did not affect ns, which indicates that SR Ca(2+) refilling was not altered by the pesticide. In conclusion, the results strongly suggest that some of the lindane-induced negative inotropic and chronotropic-like effects on the contraction are due to an increased I(Kr) while the decrease in dC/dt(max) (i.e. the rate of cross-bridge formation) results from lindane oxidative properties.     

Modeling short-term interval-force relations in cardiac muscle

This study employs two modeling approaches to investigate short-term interval-force relations. The first approach is to develop a low-order, discrete-time model of excitation-contraction coupling to determine which parameter combinations produce the degree of postextrasystolic potentiation seen experimentally. Potentiation is found to increase 1) for low recirculation fraction, 2) for high releasable fraction, i.e., the maximum fraction of Ca(2+) released from the sarcoplasmic reticulum (SR) given full restitution, and 3) for strong negative feedback of the SR release on sarcolemmal Ca(2+) influx. The second modeling approach is to develop a more detailed single ventricular cell model that simulates action potentials, Ca(2+)-handling mechanisms, and isometric force generation by the myofilaments. A slow transition from the adapted state of the ryanodine receptor produces a gradual recovery of the SR release and restitution behavior. For potentiation, a small extrasystolic release leaves more Ca(2+) in the SR but also increases the SR loading by two mechanisms: 1) less Ca(2+)-induced inactivation of L-type channels and 2) reduction of action potential height by residual activation of the time-dependent delayed rectifier K(+) current, which increases Ca(2+) influx. The cooperativity of the myofilaments amplifies the relatively small changes in the Ca(2+) transient amplitude to produce larger changes in isometric force. These findings suggest that short-term interval-force relations result mainly from the interplay of the ryanodine receptor adaptation and the SR Ca(2+) loading, with additional contributions from membrane currents and myofilament activation.     

Effects of fatigue on sarcoplasmic reticulum and myofibrillar properties of rat single muscle fibers.

Force decline during fatigue in skeletal muscle is attributed mainly to progressive alterations of the intracellular milieu. Metabolite changes and the decline in free myoplasmic calcium influence the activation and contractile processes. This study was aimed at evaluating whether fatigue also causes persistent modifications of key myofibrillar and sarcoplasmic reticulum (SR) proteins that contribute to tension reduction. The presence of such modifications was investigated in chemically skinned fibers, a procedure that replaces the fatigued cytoplasm from the muscle fiber with a normal medium. Myofibrillar Ca(2+) sensitivity was reduced in slow-twitch muscle (for example, the pCa value corresponding to 50% of maximum tension was 6.23 +/- 0.03 vs. 5.99 + 0.05, P < 0.01, in rested and fatigued fibers) and not modified in fast-twitch muscle. Phosphorylation of the regulatory myosin light chain isoform increased in fast-twitch muscle. The rate of SR Ca(2+) uptake was increased in slow-twitch muscle fibers (14.2 +/- 1.0 vs. 19.6 +/- 2. 5 nmol. min(-1). mg fiber protein(-1), P < 0.05) and not altered in fast-twitch fibers. No persistent modifications of SR Ca(2+) release properties were found. These results indicate that persistent modifications of myofibrillar and SR properties contribute to fatigue-induced muscle force decline only in slow fibers. These alterations may be either enhanced or counteracted, in vivo, by the metabolic changes that normally occur during fatigue development.     

Eugenol-induced contractions of saponin-skinned fibers are inhibited by heparin or by a ryanodine receptor blocker

The effects of eugenol on the sarcoplasmic reticulum (SR) and contractile apparatus of chemically skinned skeletal muscle fibers of the frog Rana catesbeiana were investigated. In saponin-skinned fibers, eugenol (5 mmol/L) induced muscle contractions, probably by releasing Ca(2+) from the SR. The Ca(2+)-induced Ca(2+) release blocker ruthenium red (10 micromol/L) inhibited both caffeine- and eugenol-induced muscle contractions. Ryanodine (200 micromol/L), a specific ryanodine receptor/Ca(2+) release channel blocker, promoted complete inhibition of the contractions induced by caffeine, but only partially blocked the contractions induced by eugenol. Heparin (2.5 mg/mL), an inositol 1,4,5-trisphosphate (InsP3) receptor blocker, strongly inhibited the contractions induced by eugenol but had only a small effect on the caffeine-induced contractions. Eugenol neither altered the Ca(2+) sensitivity nor the maximal force in Triton X-100 skinned muscle fibers. These data suggest that muscle contraction induced by eugenol involves at least 2 mechanisms of Ca(2+) release from the SR: one related to the activation of the ryanodine receptors and another through a heparin-sensitive pathway.     

Effects of 4-h ischemia and 1-h reperfusion on rat muscle sarcoplasmic reticulum function

To investigate the hypothesis that ischemia and reperfusion would impair sarcoplasmic reticulum (SR) Ca(2+) regulation in skeletal muscle, Sprague-Dawley rats (n = 20) weighing 290 +/- 3.5 g were randomly assigned to either a control control (CC) group, in which only the effects of anesthetization were studied, or to a group in which the muscles in one hindlimb were made ischemic for 4 h and allowed to recover for 1 h (I). The nonischemic, contralateral muscles served as control (C). Measurements of Ca(2+)-ATPase properties in homogenates and SR vesicles, in mixed gastrocnemius and tibialis anterior muscles, indicated no differences between groups on maximal activity, the Hill coefficient, and Ca(50), defined as the Ca(2+) concentration needed to elicit 50% of maximal activity. In homogenates, Ca(2+) uptake was lower (P < 0.05) by 20-25%, measured at 0.5 and 1.0 microM of free Ca(2+) ([Ca(2+)](f)) in C compared with CC. In SR vesicles, Ca(2+) uptake was lower (P < 0.05) by 30-38% in I compared with CC at [Ca(2+)](f) between 0.5 and 1.5 microM. Silver nitrate induced Ca(2+) release, assessed during both the initial, early rapid (phase 1), and slower, prolonged late (phase 2) phases, in homogenates and SR vesicles, indicated a higher (P < 0.05) release only in phase 1 in SR vesicles in I compared with CC. These results indicate that the alterations in SR Ca(2+) regulation, previously observed after prolonged ischemia by our group, are reversed within 1 h of reperfusion. However, the lower Ca(2+) uptake observed in long-term, nonischemic homogenates suggests that altered regulation may occur in the absence of ischemia.     

Pharmacological blockade of sarcoplasmic reticulum induces a negative lusitropic effect

The relaxation and the inter-beat mechanical tension are termed lusitropic functions. It is generally assumed that they are primarily determined by Ca(2+) homeostasis of cardiac cell and by interactions of Ca(2+) with the contractile machinery. In the present study we studied the effects of various pharmacological interventions on the excitation-contraction coupling in right ventricular papillary muscles of adult rabbits at various stimulation rates. The maximal force of isometric contraction (MG, a.u.), the time to peak of isometric contraction (TTP, ms), the maximal speed of relaxation (dF/dt(relax)), the diastolic tension (DT, a.u.) and the total tension (MG+DT, a.u.) were measured. To affect excitation-contraction coupling, caffeine (5 mmol x l(-1)), ryanodine (1 micromol x l(-1)) and dantrolene sodium (50 micromol x l(-1)) were used. Whereas caffeine and ryanodine elicited a pronounced negative lusitropic effect, the effect of dantrolene was less dramatic with preserved frequency dependence. The results indicate that the key element for affecting the lusitropic functions is the ryanodine receptor of the sarcoplasmic reticulum (SR). The lusitropic effects of dantrolene, that affects cardiac excitation-contraction coupling but only minimally the ryanodine receptors of SR, were considerably less pronounced. The findings agree with the assumption that the lusitropic disturbances are closely related to the defects of SR ryanodine receptors of cardiac myocytes.     

How source content determines intracellular Ca2+ release kinetics. Simultaneous measurement of [Ca2+] transients and [H+] displacement in skeletal muscle

In skeletal muscle, the waveform of Ca(2+) release under clamp depolarization exhibits an early peak. Its decay reflects an inactivation, which locally corresponds to the termination of Ca(2+) sparks, and is crucial for rapid control. In cardiac muscle, both the frequency of spontaneous sparks (i.e., their activation) and their termination appear to be strongly dependent on the Ca(2+) content in the sarcoplasmic reticulum (SR). In skeletal muscle, no such role is established. Seeking a robust measurement of Ca(2+) release and a way to reliably modify the SR content, we combined in the same cells the "EGTA/phenol red" method (Pape et al., 1995) to evaluate Ca(2+) release, with the "removal" method (Melzer et al., 1987) to evaluate release flux. The cytosol of voltage-clamped frog fibers was equilibrated with EGTA (36 mM), antipyrylazo III, and phenol red, and absorbance changes were monitored simultaneously at three wavelengths, affording largely independent evaluations of Delta[H(+)] and Delta[Ca(2+)] from which the amount of released Ca(2+) and the release flux were independently derived. Both methods yielded mutually consistent evaluations of flux. While the removal method gave a better kinetic picture of the release waveform, EGTA/phenol red provided continuous reproducible measures of calcium in the SR (Ca(SR)). Steady release permeability (P), reached at the end of a 120-ms pulse, increased as Ca(SR) was progressively reduced by a prior conditioning pulse, reaching 2.34-fold at 25% of resting Ca(SR) (four cells). Peak P, reached early during a pulse, increased proportionally much less with SR depletion, decreasing at very low Ca(SR). The increase in steady P upon depletion was associated with a slowing of the rate of decay of P after the peak (i.e., a slower inactivation of Ca(2+) release). These results are consistent with a major inhibitory effect of cytosolic (rather than intra-SR) Ca(2+) on the activity of Ca(2+) release channels.     

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.