ryanodine and Acidosis

Returning to normal pH after acidosis, similar to reperfusion after ischemia, is prone to arrhythmias. The type and mechanisms of these arrhythmias have never been explored and were the aim of the present work. Langendorff-perfused rat/mice hearts and rat-isolated myocytes were subjected to respiratory acidosis and then returned to normal pH. Monophasic action potentials and left ventricular developed pressure were recorded. The removal of acidosis provoked ectopic beats that were blunted by 1 muM of the CaMKII inhibitor KN-93, 1 muM thapsigargin, to inhibit sarcoplasmic reticulum (SR) Ca(2+) uptake, and 30 nM ryanodine or 45 muM dantrolene, to inhibit SR Ca(2+) release and were not observed in a transgenic mouse model with inhibition of CaMKII targeted to the SR. Acidosis increased the phosphorylation of Thr(17) site of phospholamban (PT-PLN) and SR Ca(2+) load. Both effects were precluded by KN-93. The return to normal pH was associated with an increase in SR Ca(2+) leak, when compared with that of control or with acidosis at the same SR Ca(2+) content. Ca(2+) leak occurred without changes in the phosphorylation of ryanodine receptors type 2 (RyR2) and was blunted by KN-93. Experiments in planar lipid bilayers confirmed the reversible inhibitory effect of acidosis on RyR2. Ectopic activity was triggered by membrane depolarizations (delayed afterdepolarizations), primarily occurring in epicardium and were prevented by KN-93. The results reveal that arrhythmias after acidosis are dependent on CaMKII activation and are associated with an increase in SR Ca(2+) load, which appears to be mainly due to the increase in PT-PLN.

Topics: Acidosis; Action Potentials; Animals; Arrhythmias, Cardiac; Benzylamines; Calcium; Calcium-Binding Proteins; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Dantrolene; Disease Models, Animal; Enzyme Inhibitors; Hydrogen-Ion Concentration; Male; Mice; Mice, Transgenic; Myocytes, Cardiac; Peptides; Phosphorylation; Rats; Rats, Wistar; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sulfonamides; Thapsigargin; Time Factors; Ventricular Function, Left; Ventricular Pressure

This study was conducted to investigate the mechanism of acidic pH-induced contraction (APIC) with regard to Ca2+ handling using isometric tension recording experiments.. Decreasing extracellular pH from 7.4 to 6.5 produced a marked and sustained contraction of spontaneously hypertensive rat (SHR) aorta, that was 128.7 +/- 2.0% of the 64.8 mm KCl-induced contraction. Verapamil, an inhibitor of voltage-dependent Ca2+ channels (VDCC) significantly inhibited the APIC. In Ca2+-deficient solution, sustained contraction induced by acidic pH was abolished completely, while a transient contraction was still observed suggesting the release of Ca2+ from intracellular site. Ryanodine (1 microm), a ryanodine receptor blocker, and 10 microm cyclopiazonic acid (CPA; a sarco/endoplasmic reticulum Ca2+ ATPase inhibitor) abolished the transient contraction induced by acidosis. In normal Ca2+-containing solution, ryanodine significantly decreased the rate of rise as well as maximum level of APIC. Interestingly, ryanodine and CPA showed an additive inhibitory effect with verapamil and the combined treatment of ryanodine or CPA with verapamil nearly abolished the APIC.. It is concluded that acidic pH induces Ca2+ release from ryanodine/CPA-sensitive store of sarcoplasmic reticulum in SHR aorta. This Ca2+ plays an important role in the facilitation of the rate of rise of APIC, as well as contributing to the sustained contraction via a mechanism which is independent of Ca2+ influx through VDCC.

Topics: Acidosis; Animals; Aorta, Thoracic; Calcium; Calcium Channel Blockers; Calcium-Transporting ATPases; Chelating Agents; Egtazic Acid; Enzyme Inhibitors; Hydrogen-Ion Concentration; Indoles; Male; Muscle Contraction; Muscle, Smooth, Vascular; Rats; Rats, Inbred SHR; Ryanodine; Sarcoplasmic Reticulum; Verapamil

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

Topics: Acidosis; Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Carbon Dioxide; Cell Separation; Enzyme Activation; Enzyme Inhibitors; Heart Ventricles; Hydrogen-Ion Concentration; In Vitro Techniques; Intracellular Fluid; Male; Myocardial Contraction; Myocardium; Propionates; Rats; Ryanodine; Sarcoplasmic Reticulum; Thapsigargin

ortho-Substituted PCBs mobilize Ca2+ from isolated brain microsomes by interaction with FKBP12/RyR complexes. Investigation into the cellular importance of this mechanism was undertaken using PC12 cells by fluoroimaging the actions of specific PCB congeners on [Ca2+]i and pH. RyR and IP3R share a common intracellular Ca2+ store in PC12 cells. Perfusion of nM to low microM PCB95 caused a transient rise of [Ca2+]i that was not completely dependent on extracellular Ca2+. Pre-incubation of the cells with ryanodine or FK506 completely eliminated PCB95 responses, suggesting a primary action on the FKPP12/RyR-sensitive store. PCB95, but not PCB126, induced a gradual decrease in cytosolic pH that could be completely eliminated by FK506 pre-incubation of the cells. Direct respiration measurement using isolated brain mitochondria demonstrated that neither of the PCBs directly altered any stage of mitochondrial respiration. These results revealed that PCB95 disrupts intracellular Ca2+ signaling in PC12 cells by interaction with the FKBP12/RyR complex that in turn accelerated cellular metabolism, possibly affecting signaling between ER and mitochondria. Since ortho-substituted PCBs have been shown to be neurotoxic and may affect neurodevelopment, studies on the molecular mechanism by which they alter cellular signaling may provide valuable information on the physiological roles of FKPB12 and RyR on neuronal functions.

Topics: Acidosis; Animals; Bradykinin; Brain Chemistry; Calcium; Calcium Signaling; Cell Respiration; Enzyme Inhibitors; Estrogen Antagonists; Hydrogen-Ion Concentration; Immunophilins; Inositol Phosphates; Intracellular Fluid; Male; Mitochondria; PC12 Cells; Pheochromocytoma; Polychlorinated Biphenyls; Rats; Rats, Sprague-Dawley; Ryanodine; Structure-Activity Relationship; Tacrolimus

We investigated the effects of acidosis on the intracellular Ca2+ concentration ([Ca2+]i) and contractile properties of intact mammalian cardiac muscle during tetanic and twitch contractions. Aequorin was injected into ferret papillary muscles, and the [Ca2+]i and tension were simultaneously measured. Acidosis was attained by increasing the CO2 concentration in the bicarbonate (20 mM)-buffered Tyrode solution from 5% (pH 7.35, control) to 15% (pH 6.89, acidosis). Tetanic contraction was produced by repetitive stimulation of the preparation following treatment with 5 microM ryanodine. The relationship between [Ca2+]i and tension was measured 6 s after the onset of the stimulation and was fitted using the Hill equation. Acidosis decreased the maximal tension to 81 +/- 2% of the control and shifted the [Ca2+]i-tension relationship to the right by 0.18 +/- 0.01 pCa units. During twitch contraction, a quick shortening of muscle length from the length at which developed tension became maximal (Lmax) to 92% Lmax produced a transient change in the [Ca2+]i (extra Ca2+). The magnitude of the extra Ca2+ was dependent on the [Ca2+]i immediately before the length change, suggesting that the extra Ca2+ is related to the amount of troponin-Ca complex. Acidosis decreased the normalized extra Ca2+ to [Ca2+]i immediately before the length change, which indicates that the amount of Ca2+ bound to troponin C is less when [Ca2+]i is the same as in the control. The decrease in the Ca2+ binding to troponin C explains the decrease in tetanic and twitch contraction, and mechanical stress applied to the preparation induced less [Ca2+]i change in acidosis.

Topics: Acidosis; Aequorin; Animals; Calcium; Electric Stimulation; Ferrets; In Vitro Techniques; Myocardial Contraction; Papillary Muscles; Ryanodine; Time Factors

The effect of stretch on cardiac muscle contraction and the Ca2+ transient was studied during hypoxia and acidosis in isolated ferret ventricular muscles. In control conditions, a maintained stretch produced an immediate increase in tension followed by a slow increase in tension and the Ca2+ transient. A stretch between contractions (diastolic stretch) caused only a slow increase in tension and the Ca2+ transient, whereas a stretch during the period of contraction (systolic stretch) produced an immediate increase in tension followed by a small slow increase in tension and the Ca2+ transient. In hypoxia, the immediate percent increase in tension was the same as in control. However, the slow increase was smaller during all three types of stretch. In acidosis, the immediate percent increase in tension was larger than in control. The slow change was the same during maintained stretch. However, the slow increase in tension was smaller during diastolic stretch and larger during systolic stretch. Thus the stretch-dependent increase in contraction is inhibited during hypoxia and modulated by acidosis.

Topics: Acidosis; Aequorin; Animals; Calcium; Female; Ferrets; Hypoxia; Male; Muscle Contraction; Papillary Muscles; Physical Stimulation; Ryanodine; Ventricular Function

Acidosis increases resting cytosolic [Ca2+], (Cai) of myocardial preparations; however, neither the Ca2+ sources for the increase in Cai nor the effect of acidosis on mitochondrial free [Ca2+], (Cam) have been characterized. In this study cytosolic pH (pHi) was monitored in adult rat left ventricular myocytes loaded with the acetoxymethyl ester (AM form) of SNARF-1. A stable decrease in the pHi of 0.52 +/- 0.05 U (n = 16) was obtained by switching from a bicarbonate buffer equilibrated with 5% CO2 to a buffer equilibrated with 20% CO2. Electrical stimulation at either 0.5 or 1.5 Hz had no effect on pHi in 5% CO2, nor did it affect the magnitude of pHi decrease in response to hypercarbic acidosis. Cai was measured in myocytes loaded with indo-1/free acid and Cam was monitored in cells loaded with indo-1/AM after quenching cytosolic indo-1 fluorescence with MnCl2. In quiescent intact myocytes bathed in 1.5 mM [Ca2+], hypercarbia increased Cai from 130 +/- 5 to 221 +/- 13 nM. However, when acidosis was effected in electrically stimulated myocytes, diastolic Cai increased more than resting Cai in quiescent myocytes, and during pacing at 1.5 Hz diastolic Cai was higher (285 +/- 17 nM) than at 0.5 Hz (245 +/- 18 nM; P < 0.05). The magnitude of Cai increase in quiescent myocytes was not affected either by sarcoplasmic reticulum (SR) Ca2+ depletion with ryanodine or by SR Ca2+ depletion and concomitant superfusion with a Ca(2+)-free buffer. In unstimulated intact myocytes hypercarbia increased Cam from 95 +/- 12 to 147 +/- 19 nM and this response was not modified either by ryanodine and a Ca(2+)-free buffer or by 50 microM ruthenium red in order to block the mitochondrial uniporter. In mitochondrial suspensions loaded either with BCECF/AM or indo-1/AM, acidosis produced by lactic acid addition decreased both intra- and extramitochondrial pH and increased Cam. Studies of mitochondrial suspensions bathed in indo-1/free acid-containing solution showed an increase in extramitochondrial Ca2+ after the addition of lactic acid. Thus, in quiescent myocytes, cytoplasmic and intramitochondrial buffers, rather than transsarcolemmal Ca2+ influx or SR Ca2+ release, are the likely Ca2+ sources for the increase in Cai and Cam, respectively; additionally, Ca2+ efflux from the mitochondria may contribute to the raise in Cai. In contrast, in response to acidosis, diastolic Cai in electrically stimulated myocytes increases more than resting Cai in quiescent cells; this suggests tha

Topics: Acidosis; Animals; Calcium; Carbon Dioxide; Cytosol; Electric Stimulation; Heart; Hydrogen-Ion Concentration; In Vitro Techniques; Male; Mitochondria, Heart; Myocardium; Rats; Rats, Wistar; Ryanodine; Sarcoplasmic Reticulum

We have investigated the effect of a CO2-induced (respiratory) acidosis on contraction and on intracellular Ca2+, Na+, and pH (measured using the fluorescent dyes fura-2, sodium-binding benzofuran isophthalate, and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, respectively) in ventricular myocytes isolated from rat hearts. Initial exposure to acidosis led to a rapid decrease in intracellular pH that was accompanied by an abrupt decline in contractility. There were no consistent changes of intracellular Na+ or Ca2+ during this period. The rapid decline of contractility was followed by a slower partial recovery, which was accompanied by increases in intracellular Na+, systolic and diastolic Ca2+, and an increase in the Ca2+ content of the sarcoplasmic reticulum (estimated using caffeine). Intracellular pH did not change during this slow recovery. The slow rise of intracellular Na+ and the recovery of the twitch were blocked by the Na(+)-H+ exchange inhibitor amiloride. The sarcoplasmic reticulum inhibitor ryanodine blocked the recovery of the twitch but had no effect on the rise of intracellular Na+ induced during acidosis. It is concluded that a major cause of the initial decline of the twitch during acidosis is a decrease in the response of the contractile proteins to Ca2+ due to the decrease of intracellular pH. The subsequent slow recovery of the twitch is due to the decrease of intracellular pH activating the Na(+)-H+ exchange mechanism. This elevates intracellular Na+ and presumably, via the Na(+)-Ca2+ exchange mechanism, intracellular Ca2+. This in turn may lead to increased Ca2+ loading of, and hence release from, the sarcoplasmic reticulum, and it is this that underlies the partial recovery of contraction during acidosis in this preparation.

Topics: Acidosis; Acidosis, Respiratory; Amiloride; Animals; Calcium; Heart Ventricles; Hydrogen; Hydrogen-Ion Concentration; Intracellular Membranes; Ions; Myocardial Contraction; Myocardium; Rats; Ryanodine; Sarcoplasmic Reticulum; Sodium

1. The photoprotein aequorin was micro-injected into papillary muscles from the right ventricle of ferrets and rats. Tension and aequorin light (a function of intracellular [Ca2+]) were monitored. 2. In stimulated ferret papillary muscles, increasing the [CO2] of the bicarbonate-buffered superfusate from 5% (pH 7.35) to 20% (pH 6.8) led to a rapid decrease of developed tension, with no significant change in the size of the intracellular Ca2+ transient which accompanies contraction. There was then a small brief recovery of tension which was accompanied by a large brief increase in the size of the Ca2+ transient. Tension then declined again before recovering more slowly, with no significant change in the size of the Ca2+ transient. 3. The time course of the Ca2+ transient was prolonged on exposure to the acid solution, but shortened on continued exposure to the acid solution. Relaxation of twitch tension became faster on exposure to the acid solution, but slowed again on continued exposure to the acid solution. 4. In the presence of 10 mM-caffeine the size of the Ca2+ transient increased during the initial decline of developed tension, the short-lived recovery of tension was abolished, and the Ca2+ transient became smaller during the slower recovery of developed tension. 2 microM-ryanodine had similar effects on developed tension. 5. Addition of 10 mM-lactic acid to the superfusate produced changes similar to those described in 2 and 3 above. 6. An intracellular acidosis, produced by the addition and subsequent withdrawal of 20 mM-NH4Cl from the superfusate also caused changes similar to those described above. In the presence of caffeine, withdrawal of NH4Cl produced changes similar to those described in 4 above. 7. In unstimulated ferret papillary muscles, increasing superfusate [CO2] produced an increase of aequorin light when the bathing [Ca2+] was increased or in the presence of ouabain (10 microM). This increase was not inhibited by verapamil (5 microM), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (1 microM) and oligomycin (2.5 microM), but was reduced by ryanodine (2 microM). 8. Rat papillary muscles showed responses which were quantitatively different from those observed in ferret papillary muscles: the initial recovery of tension developed more slowly, and sarcoplasmic reticulum (s.r.) inhibitors had a greater inhibitory effect on the recovery of tension. 9. It is concluded that the early decline of developed tension observed during acidosis

Topics: Acidosis; Aequorin; Aging; Ammonium Chloride; Animals; Caffeine; Calcium; Ferrets; Hydrogen-Ion Concentration; In Vitro Techniques; Lactates; Lactic Acid; Muscle Contraction; Papillary Muscles; Rats; Ryanodine; Sarcoplasmic Reticulum