Effects of elevated physiological temperatures on sarcoplasmic reticulum function in mechanically skinned muscle fibers of the rat

Properties of the sarcoplasmic reticulum (SR) with respect to Ca²⁺ loading and release were measured in mechanically skinned fiber preparations from isolated extensor digitorum longus (EDL) muscles of the rat that were either kept at room temperature (23°C) or exposed to temperatures in the upper physiological range for mammalian skeletal muscle (30 min at 40 or 43°C). The ability of the SR to accumulate Ca²⁺ was significantly reduced by a factor of 1.9–2.1 after the temperature treatments due to a marked increase in SR Ca²⁺ leak, which persisted for at least 3 h after treatment. Results with blockers of Ca²⁺ release channels (ruthenium red) and SR Ca²⁺ pumps [2,5-di(tert-butyl)-1,4-hydroquinone] indicate that the increased Ca²⁺ leak was not through the SR Ca²⁺ release channel or the SR Ca²⁺ pump, although it is possible that the leak pathway was via oligomerized Ca²⁺ pump molecules. No significant change in the maximum SR Ca²⁺-ATPase activity was observed after the temperature treatment, although there was a tendency for a decrease in the SR Ca²⁺-ATPase. The observed changes in SR properties were fully prevented by the superoxide (O₂^–) scavenger Tiron (20 mM), indicating that the production of O₂^– at elevated temperatures is responsible for the increase in SR Ca²⁺ leak. Results show that physiologically relevant elevated temperatures 1) induce lasting changes in SR properties with respect to Ca²⁺ handling that contribute to a marked increase in the SR Ca²⁺ leak and, consequently, to the reduction in the average coupling ratio between Ca²⁺ transport and SR Ca²⁺-ATPase and muscle performance, and 2) that these changes are mediated by temperature-induced O₂^– production. skeletal muscle; calcium ion leak; superoxide; skinned fibers     

Ca2+ sensitization of smooth muscle contractility induced by ruthenium red

The effects ofruthenium red (RuR) on contractility were examined in skinned fibers ofguinea pig smooth muscles, where sarcoplasmic reticulum function wasdestroyed by treatment with A-23187. Contractions of skinned fibers ofthe urinary bladder were enhanced by RuR in a concentration-dependentmanner (EC₅₀ = 60 µM at pCa6.0). The magnitude of contraction at pCa 6.0 was increased to 320% ofcontrol by 100 µM RuR. Qualitatively, the same results were obtainedin skinned fibers prepared from the ileal longitudinal smooth musclelayer and mesenteric artery. The maximal contraction induced by pCa 4.5 was not affected significantly by RuR. The enhanced contraction by RuRwas not reversed by the addition of guanosine5'-O-(2-thiodiphosphate) or a peptideinhibitor of protein kinase C [PKC-(1931)]. Theapplication of microcystin, a potent protein phosphatase inhibitor,induced a tonic contraction of skinned smooth muscle at lowCa²⁺ concentration([Ca²⁺]; pCa > 8.0).RuR had a dual effect on the microcystin-induced contraction-to-enhancement ratio at low concentrations and suppression at highconcentrations. The relaxation following the decrease in[Ca²⁺] from pCa 5.0 to>8.0 was significantly slowed down by an addition of RuR.Phosphorylation of the myosin light chain at pCa 6.3 was significantlyincreased by RuR in skinned fibers of the guinea pig ileum. Theseresults indicate that RuR markedly increases theCa²⁺ sensitivity of thecontractile system, at least in part via inhibition of myosin lightchain phosphatase.     

Denervation produces different single fiber phenotypes in fast- and slow-twitch hindlimb muscles of the rat

Using a single, mechanically skinned fiber approach, we tested the hypothesis that denervation (0 to 50 days) of skeletal muscles that do not overlap in fiber type composition [extensor digitorum longus (EDL) and soleus (SOL) muscles of Long-Evans hooded rats] leads to development of different fiber phenotypes. Denervation (50 day) was accompanied by 1) a marked increase in the proportion of hybrid IIB/D fibers (EDL) and I/IIA fibers (SOL) from 30% to >75% in both muscles, and a corresponding decrease in the proportion of pure fibers expressing only one myosin heavy chain (MHC) isoform; 2) complex muscle- and fiber-type specific changes in sarcoplasmic reticulum Ca²⁺-loading level at physiological pCa 7.1, with EDL fibers displaying more consistent changes than SOL fibers; 3) decrease by 50% in specific force of all fiber types; 4) decrease in sensitivity to Ca²⁺, particularly for SOL fibers (by 40%); 5) decrease in the maximum steepness of the force-pCa curves, particularly for the hybrid I/IIA SOL fibers (by 35%); and 6) increased occurrence of biphasic behavior with respect to Sr²⁺ activation in SOL fibers, indicating the presence of both slow and fast troponin C isoforms. No fiber types common to the two muscles were detected at any time points (day 7, 21, and 50) after denervation. The results provide strong evidence that not only neural factors, but also the intrinsic properties of a muscle fiber, influence the structural and functional properties of a particular muscle cell and explain important functional changes induced by denervation at both whole muscle and single cell levels. mechanically skinned fibers; myosin heavy chain isoforms; lineage; sarcoplasmic reticulum; Ca²⁺; Sr²⁺ sensitivity; Long-Evans hooded rat     

Ca2+-Activated Force Production and Calcium Handling by the Sarcoplasmic Reticulum of Crustacean Muscles During Molt-Induced Atrophy

Growth in crustaceans is an intermittent process centered aroundthe principal event of ecdysis. A major problem facing decapodcrustaceans at the time of ecdysis is the withdrawal of thelarge muscle mass of the chelae through the narrow basi-ischialjoints. To overcome this problem the muscle undergoes an atrophytriggered by the molt, which reduces the muscle mass. Once theanimal is freed from the old exoskeleton, the muscle fibers,must elongate to accommodate the new larger exoskeleton. Despitethis major myofibrillar remodification, the muscles are thoughtto remain functional over the molt cycle. Studies using skinnedmuscle fibers have shown that long-sarcomere fibers maintaintheir function over the molt cycle while the contractile propertiesof the short-sarcomere fibers are modified, as fibers couldnot withstand maximal activation with Ca²⁺ during the premoltstage. In this study the maximum Ca²⁺-activated force productionand the ability of the sarcoplasmic reticulum (SR) to releaseaccumulated Ca²⁺ has been investigated in the two major fibertypes in the claw muscle of Cherax destructor, in the stagesjust prior to ecdysis and during inter molt. In both long- andshort-sarcomere fibers, the amount of Ca²⁺ released by the SRwas not different in premolt and intermolt stages. However,the maximum releasing capacity of the SR was reached in a shortertime during the premolt suggesting that Ca²⁺ is being accumulatedat a faster rate. The force production was greatly reduced andwas graded during the premolt in both fiber types. This modulationof force appears to be the most likely candidate regulatingthe magnitude of the force development in the periods when fibersare undergoing myofibrillar remodification and thus may serveto prevent fiber damage.     

Mechanisms of P(i) regulation of the skeletal muscle SR Ca(2+) release channel

Inorganic phosphate(P_i) accumulates in the fibers of actively working musclewhere it acts at various sites to modulate contraction. To characterizethe role of P_i as a regulator of the sarcoplasmic reticulum(SR) calcium (Ca²⁺) release channel, we examined the actionof P_i on purified SR Ca²⁺ release channels,isolated SR vesicles, and skinned skeletal muscle fibers. In singlechannel studies, addition of P_i to the cis chamberincreased single channel open probability (P_o;0.079 ± 0.020 in 0 P_i, 0.157 ± 0.034 in 20 mMP_i) by decreasing mean channel closed time; mean channelopen times were unaffected. In contrast, the ATP analog,,-methyleneadenosine 5'-triphosphate (AMP-PCP), enhancedP_o by increasing single channel open time anddecreasing channel closed time. P_i stimulation of[³H]ryanodine binding by SR vesicles wassimilar at all concentrations of AMP-PCP, suggesting P_i andadenine nucleotides act via independent sites. In skinned musclefibers, 40 mM P_i enhanced Ca²⁺-inducedCa²⁺ release, suggesting an in situ stimulation ofthe release channel by high concentrations of P_i. Ourresults support the hypothesis that P_i may be an importantendogenous modulator of the skeletal muscle SR Ca²⁺ releasechannel under fatiguing conditions in vivo, acting via a mechanismdistinct from adenine nucleotides.

     

Contractile apparatus and sarcoplasmic reticulum function: effects of fatigue, recovery, and elevated Ca2+

Williams, Jay H. Contractile apparatus and sarcoplasmicreticulum function: effects of fatigue, recovery, and elevated Ca²⁺. J. Appl.Physiol. 83(2): 444-450, 1997.This investigationtested the notion that fatiguing stimulation induces intrinsic changes in the contractile apparatus and sarcoplasmic reticulum (SR) and thatthese changes are initiated by elevated intracellularCa²⁺ concentration([Ca²⁺]_i).Immediately after stimulation of frog semitendinosus muscle, contractile apparatus and SR function were measured. Despite a largedecline in tetanic force (P_o),maximal Ca²⁺-activated force(F_max) of the contractileapparatus was not significantly altered. However,Ca²⁺ sensitivity was increased. Inconjunction, the rate constant ofCa²⁺ uptake by the SR wasdiminished, and the caffeine sensitivity ofCa²⁺ release was decreased. Duringrecovery, P_o, contractileapparatus, and SR function each returned to near-initial levels.Exposure of skinned fibers to 0.5 µM freeCa²⁺ for 5 min depressed bothF_max andCa²⁺ sensitivity of thecontractile apparatus. In addition, caffeine sensitivity ofCa²⁺ release was diminished.Results suggest that fatigue induces intrinsic alterations incontractile apparatus and SR function. Changes in contractile apparatusfunction do not appear to be mediated by increased[Ca²⁺]_i.However, a portion of the change in SRCa²⁺ release seems to be due toelevated[Ca²⁺]_i.

     

Effects of MCC-135 on Ca2+ uptake by sarcoplasmic reticulum and myofilament sensitivity to Ca2+ in isolated ventricular muscles of rats with diabetic cardiomyopathy

Diabetic cardiomyopathy is characterized by delayed cardiac relaxation. Delayed relaxation is suggested to be associated with sarcoplasmic reticulum (SR) dysfunction and/or increase in myofilament sensitivity to Ca²⁺. Although MCC-135, an intracellular Ca²⁺-handling modulator, accelerates the delayed relaxation without inotropic effect in the ventricular muscle isolated from rats with diabetic cardiomyopathy, the underlying mechanism has not been fully understood. We tested the hypotheses that MCC-135 modulates Ca²⁺ uptake by SR and myofilament sensitivity to Ca²⁺. Wistar rats were made diabetic by a single injection of streptozotocin (40 mg/kg i.v.). Seven months later, the left ventricular papillary muscle was isolated and skinned fibers with and without functional SR were prepared by treatment of the papillary muscle with saponin to study SR Ca²⁺ uptake and myofilament sensitivity to Ca²⁺, respectively. In diabetic rats, SR Ca²⁺ uptake was decreased, which was related to decrease in protein level of SR Ca²⁺-ATPase determined by western blot analysis. MCC-135 enhanced SR Ca²⁺ uptake in diabetic rats, but not in normal rats. In diabetic rats, maximum force was decreased but force at diastolic level of Ca²⁺ was increased, without significant change in myofilament sensitivity to Ca²⁺ compared with normal rats. MCC-135 decreased force at any pCa tested (pCa 7.0-4.4), but had no significant effect on myofilament sensitivity to Ca²⁺ in diabetic rats. These results suggest that MCC-135 enhances SR Ca²⁺ uptake and shifts force-pCa curve downward without modulating myofilament sensitivity to Ca²⁺. These effects may contribute to positive lusitropic effect without inotropic effect of MCC-135 observed in the ventricular muscle of diabetic cardiomyopathy.     

Functional aspects of skeletal muscle contractile apparatus and sarcoplasmic reticulum after fatigue

This study examined the effects of fatigue on the functionalaspects of the contractile apparatus and sarcoplasmic reticulum (SR).Frog semitendinosus muscles were stimulated to fatigue, and skinnedfibers or a homogenate fraction was prepared from both fatigued andrested contralateral muscles. In fatigued fibers, maximalCa²⁺-activated force of thecontractile apparatus was unaltered, whereas maximal actomyosin-ATPaseactivity was depressed by 20%. TheCa²⁺ sensitivity of force wasincreased, whereas that of actomyosin-ATPase was not altered. Also, therate constant for tension redevelopment was decreased at submaximalCa²⁺ concentration. These latterfindings suggest that fatigue slows the dissociation offorce-generating myosin cross bridges.Ca²⁺ uptake andCa²⁺-ATPase activity of the SRwere depressed by 46 and 21%, respectively, in the fatigued muscles.Fatigue also reduced the rates of SR Ca²⁺ release evoked byAgNO₃ and4-chloro-m-cresol by 38 and 45%, respectively. During fatigue, the contractile apparatus and SR undergointrinsic functional alterations. These changes likely result inaltered force production and energy consumption by the intact muscle.

     

Effect of clenbuterol on sarcoplasmic reticulum function in single skinned mammalian skeletal muscle fibers

We examined the effect of the₂-agonist clenbuterol (50 µM)on depolarization-induced force responses and sarcoplasmic reticulum (SR) function in muscle fibers of the rat (Rattusnorvegicus; killed by halothane overdose) that had beenmechanically skinned, rendering the₂-agonist pathway inoperable.Clenbuterol decreased the peak of depolarization-induced forceresponses in the extensor digitorum longus (EDL) and soleus fibers to77.2 ± 9.0 and 55.6 ± 5.4%, respectively, ofcontrols. The soleus fibers did not recover. Clenbuterol significantlyand reversibly reduced SR Ca²⁺loading in EDL and soleus fibers to 81.5 ± 2.8 and 78.7 ± 4.0%, respectively, of controls. Clenbuterol also producedan ~25% increase in passive leak ofCa²⁺ from the SR of the EDL andsoleus fibers. These results indicate that clenbuterol has directeffects on fast- and slow-twitch skeletal muscle, in the absence of the₂-agonist pathway. Theincreased Ca²⁺ leak in the triadregion may lead to excitation-contraction coupling damage in the soleusfibers and could also contribute to the anabolic effect of clenbuterolin vivo.

     

Calcium phosphate precipitation in the sarcoplasmic reticulum reduces action potential-mediated Ca2+ release in mammalian skeletal muscle

During vigorous exercise, P_i concentration levels within the cytoplasm of fast-twitch muscle fibers may reach 30 mM. Cytoplasmic P_i may enter the sarcoplasmic reticulum (SR) and bind to Ca²⁺ to form a precipitate (CaP_i), thus reducing the amount of releasable Ca²⁺. Using mechanically skinned rat fast-twitch muscle fibers, which retain the normal action potential-mediated Ca²⁺ release mechanism, we investigated the consequences of P_i exposure on normal excitation-contraction coupling. The total amount of Ca²⁺ released from the SR by a combined caffeine/low-Mg²⁺ concentration stimulus was reduced by 20%, and the initial rate of force development slowed after 2-min exposure to 30 mM P_i (with or without the presence creatine phosphate). Peak (50 Hz) tetanic force was also reduced (by 25% and 45% after 10 and 30 mM P_i exposure, respectively). Tetanic force responses produced after 30 mM P_i exposure were nearly identical to those observed in the same fiber after depletion of total SR Ca²⁺ by 35%. Ca²⁺ content assays revealed that the total amount of Ca²⁺ in the SR was not detectably changed by exposure to 30 mM P_i, indicating that Ca²⁺ had not leaked from the SR but instead formed a precipitate with the P_i, reducing the amount of available Ca²⁺ for rapid release. These results suggest that CaP_i precipitation that occurs within the SR could contribute to the failure of Ca²⁺ release observed in the later stages of metabolic muscle fatigue. They also demonstrate that the total amount of Ca²⁺ stored in the SR cannot drop substantially below the normal endogenous level without reducing tetanic force responses. muscle fatigue; excitation-contraction coupling     

Characteristics of phosphate-induced Ca2+ efflux from the SR in mechanically skinned rat skeletal muscle fibers

The effects of P_i onsarcoplasmic reticulum (SR) Ca²⁺ regulation were studied inmechanically skinned rat skeletal muscle fibers. Brief application ofcaffeine was used to assess the SR Ca²⁺ content, andchanges in concentration of Ca²⁺([Ca²⁺]) within the cytosol were detected withfura 2 fluorescence. Introduction of P_i (1-40 mM)induced a concentration-dependent Ca²⁺ efflux from the SR.In solutions lacking creatine phosphate (CP), the amplitude of theP_i-induced Ca²⁺ transient approximatelydoubled. A similar potentiation of P_i-induced Ca²⁺ release occurred after inhibition of creatine kinase(CK) with 2,4-dinitrofluorobenzene. In the presence of ruthenium red or ryanodine, caffeine-induced Ca²⁺ release was almostabolished, whereas P_i-induced Ca²⁺ release wasunaffected. However, introduction of the SR Ca²⁺ ATPaseinhibitor cyclopiazonic acid effectively abolishedP_i-induced Ca²⁺ release. These data suggestthat P_i induces Ca²⁺ release from the SR byreversal of the SR Ca²⁺ pump but not via the SRCa²⁺ channel under these conditions. If this occurs inintact skeletal muscle during fatigue, activation of a Ca²⁺efflux pathway by P_i may contribute to the reporteddecrease in net Ca²⁺ uptake and increase in resting[Ca²⁺].

     

Coordinate downregulation of CaM kinase II and phospholamban accompanies contractile phenotype transition in the hyperthyroid rabbit soleus

This study investigated the effects of L-thyroxine-induced hyperthyroidism on Ca²⁺/calmodulin (CaM)-dependent protein kinase (CaM kinase II)-mediated sarcoplasmic reticulum (SR) protein phosphorylation, SR Ca²⁺ pump (Ca²⁺-ATPase) activity, and contraction duration in slow-twitch soleus muscle of the rabbit. Phosphorylation of Ca²⁺-ATPase and phospholamban (PLN) by endogenous CaM kinase II was found to be significantly lower (30–50%) in soleus of the hyperthyroid compared with euthyroid rabbit. Western blotting analysis revealed higher levels of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) 1 (150%) Ca²⁺ pump isoform, unaltered levels of SERCA2 Ca²⁺ pump isoform, and lower levels of PLN (50%) and -, -, and -CaM kinase II (40 70%) in soleus of the hyperthyroid rabbit. SR vesicles from hyperthyroid rabbit soleus displayed approximately twofold higher ATP-energized Ca²⁺ uptake and Ca²⁺-stimulated ATPase activities compared with that from euthyroid control. The V_max of Ca²⁺ uptake (in nmol Ca²⁺·mg SR protein^–1·min^–1: euthyroid, 818 ± 73; hyperthyroid, 1,649 ± 90) but not the apparent affinity of the Ca²⁺-ATPase for Ca²⁺ (euthyroid, 0.97 ± 0.02 µM, hyperthyroid, 1.09 ± 0.04 µM) differed significantly between the two groups. CaM kinase II-mediated stimulation of Ca²⁺ uptake by soleus muscle SR was 60% lower in the hyperthyroid compared with euthyroid. Isometric twitch force of soleus measured in situ was significantly greater (36%), and the time to peak force and relaxation time were significantly lower (30–40%), in the hyperthyroid. These results demonstrate that thyroid hormone-induced transition in contractile properties of the rabbit soleus is associated with coordinate downregulation of the expression and function of PLN and CaM kinase II and selective upregulation of the expression and function of SERCA1, but not SERCA2, isoform of the SR Ca²⁺ pump. calmodulin kinase II; phospholamban ; calcium ion-adenosinetriphosphatase; sarcoplasmic reticulum     

Milrinone inhibits contractility in skinned skeletal muscle fibers

Direct action of the cardiotonic bipyridine milrinone on thecross bridges of single fibers of skinned rabbit skeletal muscle wasinvestigated. At 10°C and pH 7.0, milrinone reduced isometric tension in a logarithmically concentration-dependent manner, with a55% reduction in force at 0.6 mM. Milrinone also reducedCa²⁺ sensitivity of skinned fibersin terms of force production; the shift in the force-pCa curveindicated a change in the pCa value at 50% maximal force from 6.10 to5.94. The unloaded velocity of shortening was reduced by 18% in thepresence of 0.6 mM milrinone. Parts of the transient tension responseto step change in length were altered by milrinone, so that the testand control transients could not be superimposed. The results indicatethat milrinone interferes with the cross-bridge cycle and possiblydetains cross bridges in low-force states. The results also suggestthat the positive inotropic effect of milrinone on cardiac muscle isprobably not due to the drug's direct action on the muscle crossbridges. The specific and reversible action of the bipyridine on muscle cross bridges makes it a potentially useful tool for probing the chemomechanical cross-bridge cycle.

     

Mechanical signals and mechanosensitive modulation of intracellular [Ca(2+)] in smooth muscle

We testedthe hypothesis that strain is the primary mechanical signal in themechanosensitive modulation of intracellular Ca²⁺concentration ([Ca²⁺]_i) in airway smoothmuscle. We found that [Ca²⁺]_i wassignificantly correlated with muscle length during isotonic shorteningagainst 20% isometric force (F_iso). When the isotonic loadwas changed to 50% F_iso, data points from the 20 and 50% F_iso experiments overlapped in thelength-[Ca²⁺]_i relationship. Similarly, datapoints from the 80% F_iso experiments clustered near thosefrom the 50% F_iso experiments. Therefore, despite 2.5- and4-fold differences in external load, [Ca²⁺]_idid not deviate much from the length-[Ca²⁺]_irelation that fitted the 20% F_iso data. Maximal inhibition of sarcoplasmic reticular (SR) Ca²⁺ uptake by 10 µMcyclopiazonic acid (CPA) did not significantly change[Ca²⁺]_i in carbachol-induced isometriccontractions and isotonic shortening. CPA also did not significantlychange myosin light-chain phosphorylation or force redevelopment whencarbachol-activated muscle strips were quickly released from optimallength (L_o) to 0.5 L_o. These results are consistent with thehypothesis and suggest that SR Ca²⁺ uptake is not theunderlying mechanism.

     

Skeletal muscle sarcoplasmic reticulum glycogen status influences Ca2+ uptake supported by endogenously synthesized ATP

The purpose of this investigation was to determine whether there is a link between sarcoplasmic reticulum (SR) glycogen status and SR Ca²⁺ handling. In this investigation, skeletal muscle SR was purified from female Sprague-Dawley rats (200–250 g). Glycogen was extracted from the SR purified from one hindlimb, whereas the SR purified from the contralateral limb served as control. Before removal of the tissue, the animals were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). Both -amylase treatment (AM) and removal of EDTA from the homogenization and storage buffers reduced the amount of glycogen associated with the SR (P < 0.05). AM treatment reduced the glycogen phosphorylase content of SR (P < 0.05). In contrast, creatine kinase (CK) and pyruvate kinase (PK) contents were increased after both glycogen extraction protocols (P < 0.05). Under exogenous ATP conditions, both AM and EDTA-free (EF) treatments resulted in an increase in Ca²⁺-stimulated ATPase activity when normalized to sarco(endo)plasmic reticulum calcium-ATPase (SERCA) content (P < 0.05). CK and PK-supported SR Ca²⁺ uptake was decreased (P < 0.05) in the AM group when normalized to SERCA and CK or SERCA and PK content, respectively. AM was more effective than the EF for extracting glycogen associated with purified SR. Glycogen extraction alters the yield of purified SR proteins and must be taken into account when investigating SR calcium handling. Removal of glycogen from purified SR causes a change in Ca²⁺-handling properties as measured by ATPase and uptake activities. glycogen extraction; fatigue; SERCA     

Ca2+ dependence of tension and ADP production in segments of chemically skinned muscle fibers

Both ADP production and tension have been measured in segments of chemically skinned fibers contracting at different Ca²⁺ concentrations. Full mechanical activation occurred between pCa 7.00 and pCa 6.50. The total ATPase was due to both actomyosin and non-actomyosin ATPase. Actomyosin ATPase was observed at pCa 7.09 without accompanying tension. The Ca²⁺ dependence of tension was steeper than actomyosin ATPase. This finding implies some rate constants of the mechanochemical cycle are Ca²⁺ dependent. Non-actomyosin ATPase was measured in fibers stretched beyond overlap of the thick and thin filaments. Sarcoplasmic reticulum was isolated and sarcoplasmic reticulum activity was measured in vitro under the same conditions as the single-fiber experiments. Non-actomyosin ATPase in the single fibers was found to be small compared to maximally activated actomyosin ATPase but larger than the ATPase that could be attributed to sarcoplasmic reticulum activity.     

Calcium-gated calcium channels in sarcoplasmic reticulum of rabbit skinned skeletal muscle fibers

The action of ruthenium red (RR) on Ca2+ loading by and Ca2+ release from the sarcoplasmic reticulum (SR) of chemically skinned skeletal muscle fibers of the rabbit was investigated. Ca2+ loading, in the presence of the precipitating anion pyrophosphate, was monitored by a light-scattering method. Ca2+ release was indirectly measured by following tension development evoked by caffeine. Stimulation of the Ca2+ loading rate by 5 microM RR was dependent on free Ca2+, being maximal at pCa 5.56. Isometric force development induced by 5 mM caffeine was reversibly antagonized by RR. IC50 for the rate of tension rise was 0.5 microM; that for the extent of tension was 4 microM. RR slightly shifted the steady state isometric force/pCa curve toward lower pCa values. At 5 microM RR, the pCa required for half-maximal force was 0.2 log units lower than that of the control, and maximal force was depressed by approximately 16%. These results suggest that RR inhibited Ca2+ release from the SR and stimulated Ca2+ loading into the SR by closing Ca2+-gated Ca2+ channels. Previous studies on isolated SR have indicated the selective presence of such channels in junctional terminal cisternae.     

Na+/Ca2+ exchange activity in neonatal rabbit ventricular myocytes

Much less is known about the contributions of the Na⁺/Ca²⁺ exchanger (NCX) and sarcoplasmic reticulum (SR) Ca²⁺ pump to cell relaxation in neonatal compared with adult mammalian ventricular myocytes. Based on both biochemical and molecular studies, there is evidence of a much higher density of NCX at birth that subsequently decreases during the next 2 wk of development. It has been hypothesized, therefore, that NCX plays a relatively more important role for cytosolic Ca²⁺ decline in neonates as well as, perhaps, a role in excitation-contraction coupling in reverse mode. We isolated neonatal ventricular myocytes from rabbits in four different age groups: 3, 6, 10, and 20 days of age. Using an amphotericin-perforated patch-clamp technique in fluo-3-loaded myocytes, we measured the caffeine-induced inward NCX current (I_NCX) and the Ca²⁺ transient. We found that the integral of I_NCX, an indicator of SR Ca²⁺ content, was greatest in myocytes from younger age groups when normalized by cell surface area and that it decreased with age. The velocity of Ca²⁺ extrusion by NCX (V_NCX) was linear with [Ca²⁺] and did not indicate saturation kinetics until [Ca²⁺] reached 1–3 µM for each age group. There was a significantly greater time delay between the peaks of I_NCX and the Ca²⁺ transient in myocytes from the youngest age groups. This observation could be related to structural differences in the subsarcolemmal microdomains as a function of age. ontogeny of cardiac excitation-contraction coupling; sodium/calcium exchanger; cytosolic calcium concentration; subsarcolemmal calcium concentration; sarcoplasmic reticulum calcium content     

Effects of ATP, Mg2+, and redox agents on the Ca2+ dependence of RyR channels from rat brain cortex

Despite their relevance for neuronal Ca²⁺-induced Ca²⁺ release (CICR), activation by Ca²⁺ of ryanodine receptor (RyR) channels of brain endoplasmic reticulum at the [ATP], [Mg²⁺], and redox conditions present in neurons has not been reported. Here, we studied the effects of varying cis-(cytoplasmic) free ATP concentration ([ATP]), [Mg²⁺], and RyR redox state on the Ca²⁺ dependence of endoplasmic reticulum RyR channels from rat brain cortex. At pCa 4.9 and 0.5 mM adenylylimidodiphosphate (AMP-PNP), increasing free [Mg²⁺] up to 1 mM inhibited vesicular [³H]ryanodine binding; incubation with thimerosal or dithiothreitol decreased or enhanced Mg²⁺ inhibition, respectively. Single RyR channels incorporated into lipid bilayers displayed three different Ca²⁺ dependencies, defined by low, moderate, or high maximal fractional open time (P_o), that depend on RyR redox state, as we have previously reported. In all cases, cis-ATP addition (3 mM) decreased threshold [Ca²⁺] for activation, increased maximal P_o, and shifted channel inhibition to higher [Ca²⁺]. Conversely, at pCa 4.5 and 3 mM ATP, increasing cis-[Mg²⁺] up to 1 mM inhibited low activity channels more than moderate activity channels but barely modified high activity channels. Addition of 0.5 mM free [ATP] plus 0.8 mM free [Mg²⁺] induced a right shift in Ca²⁺ dependence for all channels so that [Ca²⁺] <30 µM activated only high activity channels. These results strongly suggest that channel redox state determines RyR activation by Ca²⁺ at physiological [ATP] and [Mg²⁺]. If RyR behave similarly in living neurons, cellular redox state should affect RyR-mediated CICR. Ca²⁺-induced Ca²⁺ release; Ca²⁺ release channels; endoplasmic reticulum; thimerosal; 2,4-dithiothreitol; ryanodine receptor     

A probable role of dihydropyridine receptors in repression of Ca2+ sparks demonstrated in cultured mammalian muscle

To activate skeletal muscle contraction, action potentials must be sensed by dihydropyridine receptors (DHPRs) in the T tubule, which signal the Ca²⁺ release channels or ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) to open. We demonstrate here an inhibitory effect of the T tubule on the production of sparks of Ca²⁺ release. Murine primary cultures were confocally imaged for Ca²⁺ detection and T tubule visualization. After 72 h of differentiation, T tubules extended from the periphery for less than one-third of the myotube radius. Spontaneous Ca²⁺ sparks were found away from the region of cells where tubules were found. Immunostaining showed RyR1 and RyR3 isoforms in all areas, implying inhibition of both isoforms by a T tubule component. To test for a role of DHPRs in this inhibition, we imaged myotubes from dysgenic mice (mdg) that lack DHPRs. These exhibited T tubule development similar to that of normal myotubes, but produced few sparks, even in regions where tubules were absent. To increase spark frequency, a high-Ca²⁺ saline with 1 mM caffeine was used. Wild-type cells in this saline plus 50 µM nifedipine retained the topographic suppression pattern of sparks, but dysgenic cells in high-Ca²⁺ saline did not. Shifted excitation and emission ratios of indo-1 in the cytosol or mag-indo-1 in the SR were used to image [Ca²⁺] in these compartments. Under the conditions of interest, wild-type and mdg cells had similar levels of free [Ca²⁺] in cytosol and SR. These data suggest that DHPRs play a critical role in reducing the rate of spontaneous opening of Ca²⁺ release channels and/or their susceptibility to Ca²⁺-induced activation, thereby suppressing the production of Ca²⁺ sparks. excitation-contraction coupling; sarcoplasmic reticulum; ryanodine receptors; Ca²⁺ imaging