Caffeine-induced Tension Transients Research Articles

Energetic state is a strong regulator of sarcoplasmic reticulum Ca2+ loss in cardiac muscle: different efficiencies of different energy sources

Increased diastolic sarcoplasmic reticulum (SR) Ca(2+) loss could depress contractility in heart failure. Since the failing myocardium has impaired energetics, we investigated whether Ca(2+) loss is linked to changes in energetic pathways. Leakage from SR in mouse permeabilized preparations was assessed using exogenous ATP, ATP + phosphocreatine (activation of bound creatine kinase, CK), ATP + mitochondrial substrates (mitochondrial activation), or with all of these together (optimal energetic conditions) in Ca(2+)-free solution. In ventricular fibres caffeine-induced tension transients under optimal energetic conditions were used to estimate SR [Ca(2+)]. In cardiomyocytes, intra-SR Ca(2+) was monitored by use of the fluorescent marker Mag-fluo 4. In fibres, SR Ca(2+) content after 5 min incubation strongly depended on energy supply (100%-optimal energetic conditions; 27 +/- 5%-exogenous ATP only, 52 +/- 5%-endogenous CK activation; 88 +/- 8%-mitochondrial activation, P < 0.01 vs. CK system). The significant loss with only exogenous ATP was not inhibited by the ryanodine receptor blockers tetracaine or ruthenium red. However, the SR Ca(2+)-ATPase (SERCA) inhibitors cyclopiazonic acid or 2,5-di(tert-butyl)-1,4-benzohydroquinone significantly decreased Ca(2+) loss. At 100 nM external [Ca(2+)], the SR Ca(2+) loss was also energy dependent and was not significantly inhibited by tetracaine. In cardiomyocytes, the decline in SR [Ca(2+)] at zero external [Ca(2+)] was almost two times slower under optimal energetic conditions than in the presence of exogenous ATP only. At low extra-reticular [Ca(2+)], the main leak pathway is an energy-sensitive backward Ca(2+) pump, and direct mitochondrial-SERCA ATP channelling is more effective in leak prevention than local ATP generation by bound CK.

Altered energy transfer from mitochondria to sarcoplasmic reticulum after cytoarchitectural perturbations in mice hearts

Sarcoplasmic reticulum (SR) calcium pump function requires a high local ATP/ADP ratio, which can be maintained by direct nucleotide channelling from mitochondria, and by SR-bound creatine kinase (CK)-catalysed phosphate-transfer from phosphocreatine. We hypothesized that SR calcium uptake supported by mitochondrial direct nucleotide channelling, but not bound CK, depends on the juxtaposition of these organelles. To test this, we studied a well-described model of cytoarchitectural disorganization, the muscle LIM protein (MLP)-null mouse heart. Subcellular organization was characterized using electron microscopy, and mitochondrial, SR and myofibrillar function were assessed in saponin-permeabilized fibres by measuring respiration rates and caffeine-induced tension transients. MLP-null hearts had fewer, less-tightly packed intermyofibrillar mitochondria, and more subsarcolemmal mitochondria. The apparent mitochondrial Km for ADP was significantly lower in the MLP-null heart than in control (175 +/- 15 and 270 +/- 33 microM, respectively), indicating greater ADP accessibility, although maximal respiration rate, mitochondrial content and total CK activity were unaltered. Active tension in the myofibres of MLP-null mice was 54% lower than in controls (39 +/- 3 and 18 +/- 1 mN mm(-2), respectively), consistent with cytoarchitectural disorganization. SR calcium loading in the myofibres of MLP-null mice was similar to that in control myofibres when energy support was provided via Bound CK, but approximately 36% lower than controls when energy support was provided by mitochondrial (P < 0.05). Mitochondrial support for SR calcium uptake was also specifically decreased in the desmin-null heart, which is another model of cytoarchitectural perturbation. Thus, despite normal oxidative capacity, direct nucleotide channelling to the SR was impaired in MLP deficiency, concomitant with looser mitochondrial packing and increased nucleotide accessibility to this organelle. Changes in cytoarchitecture may therefore impair subcellular energy transfer and contribute to energetic and contractile dysfunction.

Energetic crosstalk between organelles: architectural integration of energy production and utilization.

Cells with high and fluctuating energy demands such as cardiomyocytes need efficient systems to link energy production to energy utilization. This is achieved in part by compartmentalized energy transfer enzymes such as creatine kinase (CK). However, hearts from CK-deficient mice develop normal cardiac function under conditions of moderate workload. We have therefore investigated whether a direct functional interplay exists between mitochondria and sarcoplasmic reticulum or between mitochondria and myofilaments in cardiac cells that catalyzes direct energy and signal channeling between organelles. We used the selective permeabilization of sarcolemmal membranes with saponin to study the functional interactions between organelles within the cellular architecture. We measured contractile kinetics, oxygen consumption, and caffeine-induced tension transients. The results show that in hearts of normal mice, ATP produced by mitochondria (supplied with substrates, oxygen, and adenine nucleotides) was able to sustain calcium uptake and contractile speed. Moreover, direct mitochondrially supplied ATP was nearly as effective as CK-supplied ATP and much more effective than externally supplied ATP, suggesting that a direct ATP/ADP channeling exists between the sites of energy production (mitochondria) and energy utilization (sarcoplasmic reticulum and myofilaments). On the other hand, in cardiac cells of mice deficient in mitochondrial and cytosolic CK, marked cytoarchitectural modifications were observed, and direct adenine nucleotide channeling between mitochondria and organelles was still effective for sarcoplasmic reticulum and myofilaments. Such direct crosstalk between organelles may explain the preserved cardiac function of CK-deficient mice under moderate workloads.

Glycolysis Supports Calcium Uptake by the Sarcoplasmic Reticulum in Skinned Ventricular Fibres of Mice Deficient in Mitochondrial and Cytosolic Creatine Kinase

E. Boehm, R. Ventura-Clapier, P. Mateo, P. Lechène and V. Veksler. Glycolysis Supports Calcium Uptake by the Sarcoplasmic Reticulum in Skinned Ventricular Fibres of Mice Deficient in Mitochondrial and Cytosolic Creatine Kinase. Journal of Molecular and Cellular Cardiology (2000) 32, 891–902. Several works have shown the importance of the creatine kinase (CK) system for cardiac energetics and Ca2+homeostasis. Nevertheless, CK-deficient mice have cardiac function close to normal, at least under conditions of low or moderate workload. To characterize possible adaptive changes of the sarcoplasmic reticulum (SR) and potential role of glycolytic support in cardiac contractility we used the skinned fibre technique to study properties of the SR and myofibrils, in control and muscle-type homodimer (MM-/mitochondrial-CK)-deficient mice. In control fibres, SR Ca2+loading with ATP and phosphocreatine (solution PL) was significantly better than loading with ATP alone (solution AL), as determined by analysis of caffeine-induced tension transients. Loading in the presence of ATP and glycolytic intermediates (solution GL) was not significantly different from solution PL. These data indicate that Ca2+uptake by the SR in situ depends on a local ATP:ADP ratio that is controlled by both CK and glycolytic enzymes. In CK-deficient mice, Ca2+loading was impaired in solution PLdue to the absence of CK. In solution GL, loading was significantly increased, such that calculated Ca2+release parameters were normalized to those in control fibres in solution PL. In CK-deficient mice, fibre kinetic parameters of tension recovery were impaired after quick stretch in solution PLand were not improved in solution GL. These results show that in CK-deficient mice, at least under basal conditions, glycolysis can replace the CK system in fueling the SR Ca2+ATPase, but not the myosin ATPase, and may in part explain the limited phenotypic alterations seen in the hearts of these mice.

Biphasic nature of inotropic action of nitric oxide donor NOC7 in guinea-pig ventricular trabeculae.

The effects of nitric oxide (NO) donor on the contractility of guinea-pig ventricular trabeculae were explored to clarify whether NO affects the function of sarcoplasmic reticulum (SR) and the contractile elements. NO donor, 3-(2-hydroxy-1-methyl-2-nitroso-hydrazino)-N-methyl-1-propanamine (NOC7), increased monotonically the amplitude of the twitch tension induced by electrical stimulation at a concentration of 20 microM. A higher concentration of NOC7 (200 microM) caused a biphasic response: transiently increased the amplitude of twitch and then decreased it. On wash-off of the higher concentration of NOC7, a rebound increase of the twitch amplitude was observed. An inhibitor of NO-sensitive guanylyl cyclase, 1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ), abolished the mono-tonic increase and rebound increase in the amplitude of tension but did not affect the decrease in the amplitude of tension at the higher concentration of NOC7. Oscillatory contractions developed by beta-escin-skinned muscle fibers were not changed by NOC7 at either concentration. Caffeine-induced tension transients indicating the Ca(2+)-accumulating and -releasing functions of intracellular Ca(2+) stores were not affected by NOC7. NOC7 did not change the steady tension developed in 1.6 microM Ca(2+) containing solution with and without ODQ. These results suggest that the biphasic inotropic effects by NOC7 were not caused by modifying the function of SR and the Ca(2+) sensitivity of myofilaments of the guinea-pig ventricular trabecula, but at least the positive inotropic effect was mediated through cGMP-dependent mechanisms.

Mechanisms of action of isoflurane on contraction of rabbit conduit artery.

Isoflurane may cause differential effects on different vascular beds of the same animal species. The mechanisms of this action have not been elucidated. Accordingly, we compared in rabbit aorta and femoral artery the effects of isoflurane (1-3.3%) in isolated rings (endothelium denuded) activated by norepinephrine, and isoflurane effects on Ca2+ fluxes from the sarcoplasmic reticulum in skinned strips. When < 30 nM norepinephrine was used to cause ring contraction, isoflurane increased the force of contraction in aortic rings, but decreased force in femoral arterial rings. At 30 nM norepinephrine stimulation, 3.3% isoflurane decreased the force and, in the presence of verapamil, isoflurane actually increased the force in both arterial types. In skinned strips of both arterial types, isoflurane present during Ca2+ uptake decreased the caffeine-induced tension transients, whereas isoflurane present during Ca2+ release enhanced the transients. Isoflurane potentiated the depression of the tension transients by ryanodine. Isoflurane directly caused contracture even in the absence of caffeine. Thus, isoflurane has similar cellular mechanisms of action in the aortic and femoral arterial smooth muscle: inhibiting Ca2+ influx through the sarcolemma, decreasing Ca2+ uptake by the sarcoplasmic reticulum, and enhancing caffeine-induced Ca2+ release from the sarcoplasmic reticulum.

Modulation of the ryanodine receptor sarcoplasmic reticular Ca2+ channel in skinned fibers of fast- and slow-twitch skeletal muscles from rabbits

This study was performed to compare skinned fibers from rabbit adductor magnus (AM) and soleus (SL) muscles with regard to the influence of caffeine, Ca2+ and Mg2+ on the depressive effects of ryanodine (RYA) on the caffeine-induced tension transients. Single skinned fibers were immersed in solutions to load Ca2+ into, and release Ca2+ from the SR (a load-release cycle). Three cycles were sequentially performed in each skinned fiber: (1) a control (no RYA), (2) a conditioning period in which activation was carried out in the presence of ryanodine plus various concentrations of the modulators, i.e. caffeine, Ca2+ or Mg2+, and (3) a test (no RYA) which monitored the release activity retained after the conditioning cycle. The depressive effect of RYA was found to be a function of [ryanodine], [caffeine], or [Ca2+], and an inverse function of [Mg2+], where [] denotes concentration. The half-maximal effects of RYA in AM (5 microM RYA) and SL (10 microM RYA), respectively, occurred at a pCa50 of 5.32 versus 5.43 without caffeine, or pCa50 of 7.24 versus 6.88 and pMg50 of 3.29 versus 3.61 with 25 mM caffeine, at a [caffeine] of 4.96 versus 7.29 mM, and at a [ryanodine] of 31.0 versus 101.6 microM. Thus, the RYA depression in skinned muscle fibers is modulated by caffeine, Ca2+, and Mg2+ in both muscle types, and AM is at least two- to fourfold more sensitive than SL.

Effects of ryanodine on SR Ca2+ release channels demonstrated with caffeine-induced tension transients in skinned striated and vascular smooth muscle.

Ryanodine has been shown to cause cardiac arrest and contracture of skeletal muscle and to bind to a receptor with high specificity and affinity in isolated sarcoplasmic reticulum (SR).1 We tested the hypothesis that the ryanodine binding itself, to the receptor is the mechanism of ryanodine’s pharmacological effects. We used a skinned muscle fiber preparation2,3 which is unique in that the ryanodine effect on the SR can be monitored by tension development. This study shows that: (i) ryanodine, in skeletal muscle, enhances the caffeine-induced tension transient, and in striated and vascular smooth muscle, decreases the caffeine-induced tension transient of the subsequent control (C2 depression); (ii) C2 depression is dependent on the duration of the open state of the SR Ca2+ release channels; and (iii) C2 depression is a direct function of [Ca2+] and [caffeine], and is an inverse function of [Mg2+].

Intracellular Mechanisms of Halothane's Effect on Isolated Aortic Strips of the Rabbit

The intracellular mechanisms of halothane action were examined in vascular smooth muscle from the aorta. Medial layers of the aorta from rabbits were mounted on photodiode tension transducers, stretched to 20 mg resting tension, and "skinned" with saponin. The skinned fiber preparations were then immersed in bathing solutions to study the effects of halothane (0.5-2%) on Ca2+ activation of the contractile proteins, and Ca2+ uptake and release from the sarcoplasmic reticulum (SR) using caffeine-induced tension transients. For comparison, isolated intact aortic rings were mounted on Blinks' dual tissue bath and attached to force transducers. The preparations were contracted with either 40 mM KCl, or norepinephrine (NE) followed by acetylcholine (ACh)- or sodium nitroprusside (SNP)-induced relaxation. At steady state contraction or relaxation, the effects of halothane (1-3%) were studied. The steady state tension during halothane was expressed as a percentage of the steady state tension before administration of halothane. In the isolated intact aortic rings, halothane (1-3%) produced biphasic effects on KCl-induced tension, i.e., an initially slight increase followed by decreases, independent of endothelium. Halothane markedly increased tension in the ACh- or SNP-relaxed state. The effects were dose-dependent. In the skinned aortic strips, halothane slightly decreased maximum Ca2+-activated tension development of the contractile proteins. Halothane decreased Ca2+ accumulation in the SR and increased Ca2+ release from the SR in a dose-dependent manner. The halothane-induced increases in Ca2+ release from the SR were blocked by ryanodine, an SR Ca2+ release channel blocker. It is concluded that halothane directly causes vascular contraction or relaxation, depending on the condition, and that halothane's effects on the SR may play a role.

Intracellular mechanisms of verapamil and diltiazem action on striated muscle of the rabbit.

Skinned fibers from striated muscle were used to study the intracellular mechanisms (contractile proteins and sarcoplasmic reticulum [SR]) of action of diltiazem (DT) and verapamil (VP) on muscle contraction. Rabbit papillary muscle (PM), and the skeletal muscles adductor magnus (AM, fast-twitch) and soleus (SL, slow-twitch) were used. The muscles were skinned by homogenization and fibre bundles for PM and single fibres for AM and SL were dissected from the homogenate and mounted on photodiode force transducers. VP (0.1-3.0 mmol/l) (and to a lesser degree DT) increased Ca2+-activated tension development of the contractile protains in PM and SL and decreased it in AM (+[4-20]%, +4%, -[14-28]%, respectively). Both drugs increased the submaximal Ca2+-activated tension development at the order of PM = SL greater than AM in a dose-dependent manner. The changes of half-maximal pCa50 at 1 mmol/l VP were 0.25, 0.25, and 0.15, respectively. For Ca2+ uptake and release from the SR, VP as well as DT (0.1-3.0 mmol/l) in the uptake phase decreased caffeine-induced tension transients in a dose-dependent fashion. At 0.01-3.0 mmol/l, the drugs directly induced Ca2+ release from the SR or enhanced caffeine-induced tension transients with the exception that in PM, DT attenuated caffeine-induced tension transients. Thus, VP and DT have similar intracellular mechanisms of action in striated muscle. Both drugs induced calcium release from the SR and increase Ca2+ sensitivity of the contractile proteins, and thus could be the underlying mechanisms for potentiating twitch tension, and inducing contracture in skeletal muscle.

Mechanisms of ryanodine-induced depression of caffeine-induced tension transients in skinned striated rabbit muscle fibers

Evidence suggests that ryanodine affects ligand-gated calcium channels in the sarcoplasmic reticulum (SR) resulting in depressed muscle contraction. In skinned fibers from striated muscle the effects of ryanodine were examined (1) on Ca2+ uptake and on Ca2+ release to differentiate whether the effects are on the pump or channel, and (2) during the tension transient, with ryanodine exposure at various times either simultaneous with or directly after exposure to caffeine. Of total calcium content in the SR, 25 mM caffeine released greater than 90% in papillary muscle (PM), approximately equal to 25% in soleus (SL), and approximately equal to 20% in adductor magnus (AM). Ryanodine (100 microM for 1-3 s for AM and SL; 1 microM for 7-10 s for PM), in the initial loading phase, did not significantly change, and in the initial release phase, markedly depressed the subsequent control caffeine-induced tension transients (C2) in all three muscle types. The depression increased with increasing time of exposure to ryanodine (10 microM) in the order of PM greater than AM greater than SL. Upon introduction of ryanodine after caffeine-induced tension transients, maximal depression was observed at half-maximum rise of the tension transient, followed by recovery of depression to completion in SL, and only partially in AM and PM at steady state of relaxation. The extent of recovery was in the order of SL greater than AM greater than PM. The data suggest that ryanodine affects Ca2+ releasing channel as a result of its binding to open channels.

Effects of ryanodine on skinned myocardial fibers of the rabbit.

Skinned fiber bundles from papillary muscle of rabbits were used to study the effects of ryanodine (1) on direct Ca2+ activation of the contractile proteins, and (2) on direct Ca2+ uptake and release from the sarcoplasmic reticulum (SR). Caffeine (25 mM) was used to release Ca2+ from the SR and to generate a tension transient. Each tension transient occurred after sequential immersion of the fiber bundles into five solutions: loading (uptake phase, [U]) and releasing (release phase, [R]). The height of free Ca2+-activated tension development of the contractile proteins, and the area of the tension transient generated by caffeine were assessed. (1) The direct free Ca2+-activated tension development of the contractile proteins was not significantly affected by ryanodine up to 0.1 mM, either at the submaximal or maximal free Ca2+ concentrations. (2) Ryanodine (1 nM-1 microM), in U, R, or in U and R, did not significantly change the immediate caffeine-induced tension transients. In the same preparation after ryanodine treatments, the second control caffeine-induced tension transients (C2, no ryanodine) were decreased in a dose-dependent manner (IC50 = 50 nM, 10 nM, 10 nM for R, U, and U and R, respectively). The depression caused by ryanodine on the SR was "activity"-dependent and not readily reversible. Total calcium content in the SR of C2 was not significantly changed by small quantities of ryanodine (less than 0.1 microM) and was decreased with greater amounts of ryanodine (greater than or equal to 0.1 microM). Thus, at low concentrations of ryanodine, the negative inotropic action is due to decrease Ca2+ release from the SR; at high concentration of ryanodine, it is due to decrease in calcium accumulation in the SR.

Effects of ryanodine on skinned skeletal muscle fibers of the rabbit

The mechanism(s) of ryanodine-induced contracture of skeletal muscle were studied in skinned fibers from soleus (SL) and adductor magnus (AM) (slow- and fast-twitch skeletal muscles) of rabbits. Pieces of SL or AM were homogenized (sarcolemma disrupted). Single fibers were dissected from the homogenate and mounted on photodiode force transducers. At concentrations 1-50 microM, ryanodine slightly but significantly increased the submaximal Ca2+-activated tension development of the contractile proteins in skinned fibers of AM but not of SL. Ryanodine in uptake phase or release phase increased caffeine-induced tension transients in the SR of both muscle types; however, no dose-response relation was found. Ryanodine greater than or equal to 1 microM decreased, however, the second control tension transients in a dose-dependent manner. The depression was nearly irreversible and "activity"-dependent. The concentrations of ryanodine that inhibited the second control tension transients by 50% were 10 microM and 5 microM for SL and AM, respectively, following ryanodine administration in the release phase, and 100 microM and 30 microM, respectively, for these preparations after the drug was present in the uptake phase. The quantity of calcium released from the SR by Triton X-100 and caffeine in the second control tension transient was unchanged by ryanodine at all concentrations tested when compared with that of the absence of ryanodine. The present findings suggest that the ability of ryanodine to increase immediate calcium release from the SR, and in AM but not SL, to increase the sensitivity of the contractile proteins to Ca2+ underlies the contracture caused by this agent in intact skeletal muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of adenosine 3',5'-monophosphate (cAMP)-induced increase in Ca2+ uptake by the sarcoplasmic reticulum in functionally skinned myocardial fibers.

The increased rate of Ca2+ uptake and ATPase activity in isolated cardiac sarcoplasmic reticulum (SR) by adenosine 3',5'-monophosphate (cAMP) has been shown to be activated by a cAMP-dependent protein kinase (cAMP kinase). Functionally skinned myocardial fiber preparations were used to study the mechanisms of cAMP action on the SR at the same time that tension was monitored. cAMP effects were studied on Ca2+ -activated tension of the contractile proteins, and on Ca2+ uptake and release from the SR using caffeine-induced tension transients. Neither cyclic AMP (0.1-5 microM) nor the catalytic subunit of cAMP kinase (0.1-1 microM) (PK-C) significantly changed either the maximal or the submaximal Ca2+ -activated tension. The areas of the tension transients were unchanged when cAMP was present in the releasing solution (release phase), and were significantly increased up to a mean of about 80% when cAMP or PK-C was present in the Ca2+ loading solutions (uptake phase). The increased tension transient was blocked by heat-stable inhibitor of cAMP kinase. We conclude that cAMP-induced increases in Ca2+ uptake by the SR could play an important role in the positive inotropic effect. cAMP kinase could thus play a crucial role in the regulation of myocardial contractility.

Effects of halothane on functionally skinned rabbit soleus muscle fibers: a correlation between tension transient and 45Ca release.

The mechanism(s) involved in the halothane-induced increase in skeletal muscle contraction was studied using functionally skinned soleus muscle fibers from rabbits: For the tension study, single functionally skinned fibers were individually mounted on two pairs of forceps, with one end attached to a photodiode tension transducer. Ca2+-activated tension development of the contractile proteins, and Ca2+ uptake and release from the sarcoplasmic reticulum (SR) using caffeine-induced tension transients were studied. To measure the amount of calcium, skinned fibers at 0.1 g/ml were used and 0.075 μCi45Ca/ml was spiked in the solution 3 (pCa 6.5 and 1 mM [EGTA]) which promoted rapid loading of Ca2+. Halothane (1–3%) did not change the [Ca2+]-tension relationship; 2 and 3% halothane reduced the maximum Ca2+-activated tension by 6–7%. Halothane (1–3%) added to the solution 3, reduced45Ca uptake by 3, 22 and 23%; however, the subsequent caffeine-induced tension transient and45Ca release were increased by 10–40%. During the release phase only halothane increased both caffeine-induced tension transient and45Ca release by 20–60%. The effects of halothane on the tension transient and on the45Ca release were comparable. There was no dose-response relationship to the effects of halothane on the above parameters. It is concluded that halothane affects the SR by increasing its membrane permeability to Ca2+, resulting in an increase in myoplasmic [Ca2+] and thus in the twitch tension in skeletal muscle.

Effects of halothane on caffeine-induced tension transients in functionally skinned myocardial fibers.

The effects of halothane on caffeine-induced tension transients in functionally skinned myocardial fibers were investigated. Fiber bundles from mechanically disrupted rabbit right ventricular papillary muscles were mounted on a tension transducer. The fiber preparation was loaded with Ca2+; Ca2+ was then released by the use of caffeine (25 mM); and the area of the resulting tension transient was measured. Each preparation was sequentially transferred from control to test to control solution. The control solutions were equilibrated with 100% N2, and the test solutions with a mixture of N2 and various halothane concentrations. The preparation was exposed to halothane during the Ca2+ uptake or the release phase only, or during both Ca2+ uptake and release phases. The areas of the test tension transients were compared with those of the two control tension transients. It was found that halothane depressed the caffeine-induced tension transient either during the uptake phase or the combined-uptake-and-release phase but not during the release phase. The halothane-induced depression was dose-dependent, reversible, and comparable to the depression observed in intact isolated papillary muscles. We conclude that halothane could induce myocardial depression by inhibiting Ca2+ uptake by the sarcoplasmic reticulum.