ryanodine and Myocardial-Ischemia

Considerable data support the point of view that calcium antagonists, whether given before the onset of ischemia or exactly at the time of reperfusion, ameliorate stunning. Benefit after the onset of reperfusion is much more controversial. It is proposed that the mechanisms whereby calcium antagonists act vary between these situations. When given before or at the onset of ischemia, then an antiischemic effect is likely. According to the hypothesis that the severity of ischemic damage determines the severity of reperfusion damage, the calcium antagonists indirectly lessen reperfusion damage. When given exactly at the time of reperfusion, the proposal is that the calcium antagonists are specifically limiting the entry of calcium ions via the calcium channel and thereby diminishing pathogenic cytosolic calcium oscillations. The reported benefit of calcium antagonists when given postreperfusion to the heart in situ, in the presence of established stunning, is of unknown mechanism and controversial significance. The hypothesis of a two-stage model of stunning with calcium as a pathogen is in accord with most of the available evidence.

Topics: Calcium; Calcium Channel Blockers; Cytosol; Free Radicals; Humans; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardial Stunning; Phosphatidylinositols; Ryanodine; Sarcoplasmic Reticulum; Sodium-Hydrogen Exchangers; Time Factors

The impairment of intracellular calcium homeostasis plays an essential role during ischemia-reperfusion injury. Calcium release from sarcoplasmic reticulum which is triggered by myocardial ischemia is mainly mediated by ryanodine receptors. Dantrolene sodium is a ryanodine receptor antagonist. The objective of the present study was to evaluate the in-vivo impact of dantrolene sodium on myocardial ischemia-reperfusion injury in swine models. An in vivo, experimental trial comparing 10 experimental animals which received dantrolene sodium with 9 control swine models was conducted. Their left anterior descending coronary artery was temporarily occluded for 75 minutes via a vessel tourniquet, which was then released. Myocardial reperfusion was allowed for 24 hours. Dantrolene was administered at the onset of the reperfusion period and levels of troponin, creatine phosphokinase and creatine kinase myocardial band between the two groups were compared. Additionally, various other hemodynamic parameters and left ventricular morphology and function were examined. There were significantly lower values of troponin, creatine phosphokinase and creatine kinase myocardial band in the dantrolene group indicating less ischemia-reperfusion injury. Moreover, the postischemic cardiac index was also greater in the dantrolene group, whereas viable myocardium was also better preserved. In conclusion, the in vivo cardioprotective role of dantrolene sodium against ischemia-reperfusion injury in swine models was indicated in this study. Therefore, dantrolene sodium administration could be a promising treatment against ischemia-reperfusion injury in humans. However, large randomized clinical studies should be firstly carried out to prove this hypothesis.

Topics: Animals; Calcium; Creatine Kinase; Dantrolene; Homeostasis; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Ryanodine; Ryanodine Receptor Calcium Release Channel; Swine; Treatment Outcome; Troponin

To investigate the mechanism underlying T-wave alternans (TWA) and its hysteresis under ischemia conditions.. Transmembrane action potential (AP) from endocardial, M, and epicardial cells and monophasic AP (MAP) from four epicardial sites were recorded in ventricular wedge preparation and in isolated intact rabbit heart, respectively. The AP/MAP duration (APD/ MAPD), effective refractory period (ERP), activation time, and APD/MAPD restitution were determined under control and ischemia conditions. The effects of ryanodine (0.01 and 1 micromol x l(-1)) on TWA, and the effects of low extracellular Ca2+ and 4-aminopyridine on its hysteresis were studied.. Ischemia shortened the APD/MAPD and effective refractory period of all recording sites symmetrically, except the APD of M cells, which shortened markedly. In the ischemia group, TWA was induced within a cycle length (CL) range from 160 to 250 ms, which corresponded to a diastolic interval region of 0-70 ms. In this diastolic interval region, the repolarization restitution curve was the steepest (slope > 1.0). All TWA were accompanied by repolarization alternations. Low concentration ryanodine (0.01 micromol x l(-1)) facilitated TWA, high concentration (1 micromol x l(-1)) abolished it. Alternans of calcium transient were observed in myocytes purfused with ischemia solution during rapid stimulation. Ryanodine (0.1 micromol x l(-1)) abolished alternans of calcium transient, and ryanodine (0.01 micromol x l(-1)) facilitated them. After 60 min pacing at a CL of 200 ms, TWA persisted until the initial several beats at a CL of 300 ms at which a TWA was exceptional. The suppression of hysteresis by low extracellular Ca2+ and 4-aminopyridine indicated an underlying role of the intracellular Ca2+ overload and transient outward current (I(to)).. TWA is principally due to repolarization alternans, which is secondary to steep APD/MAPD restitution, and relates to intracellular calcium cycling. Hysteresis relates to intracellular Ca2+ overload and I(to).

Topics: 4-Aminopyridine; Animals; Arrhythmias, Cardiac; Calcium; Calcium Signaling; Electrocardiography; Endocardium; Heart Conduction System; Membrane Potentials; Myocardial Ischemia; Myocytes, Cardiac; Pericardium; Potassium Channel Blockers; Rabbits; Refractory Period, Electrophysiological; Ryanodine

We have previously demonstrated that brief episodes of tachycardia prior to a prolonged occlusion of a coronary artery, followed by reperfusion, substantially reduce the infarct size. Adenosine receptors and mitochondrial ATP-dependent K(+) channels mediate this effect. Since preconditioning can be induced or reverted by maneuvers that increase or decrease [Ca(2+)](i), respectively, and tachycardia increases [Ca(2+)](i), we studied the participation of sarcoplasmic reticulum and Ca(2+) in the preconditioning effect of tachycardia. We measured the effect of ischemia and tachycardia on Ca(2+) uptake and release by sarcoplasmic reticulum vesicles isolated from left ventricular canine myocardium. Myocardial ischemia increased Ca(2+)-release rate constants and decreased both the initial rates of Ca(2+) uptake and [(3)H]-ryanodine binding by sarcoplasmic reticulum. In addition, ischemia induced a decrease in the pentameric form of phospholamban and in the content of ryanodine-receptor Ca(2+)-release channel protein. All these effects were reverted in hearts preconditioned with tachycardia. Furthermore, tachycardia by itself increased [(3)H]-ryanodine binding, Ca(2+)-release rate constants and the protein levels of ryanodine-receptor Ca(2+)-release channels and the ATP-dependent Ca(2+) pump. These results suggest that tachycardia preserves the integrity of the sarcoplasmic reticulum preventing the excess of release and the decrease of uptake of Ca(2+) produced by ischemia, thereby avoiding cytosolic Ca(2+) overload. This sarcoplasmic reticulum protection could partly explain the preconditioning effect of tachycardia.

Topics: Animals; Blotting, Western; Calcium; Calcium-Binding Proteins; Dogs; Heart Ventricles; Ischemic Preconditioning, Myocardial; Kinetics; Myocardial Ischemia; Myocardium; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Tachycardia

There is recent evidence that Ca(2+) influx via reverse mode Na(+)/Ca(2+) exchange (NCX) at the time of reperfusion can contribute to cardiomyocyte hypercontracture. However, forward NCX is essential for normalization of [Ca(2+)](i) during reperfusion, and its inhibition may be detrimental. This study investigates the effect of NCX inhibition with KB-R7943 at the time of reperfusion on cell viability.. The effect of several concentrations of KB-R7943 added at reperfusion was studied in Fura-2 loaded quiescent cardiomyocytes submitted to 40 min of simulated ischemia (NaCN 2 mM, pH 6.4), and in rat hearts submitted to 60 min of ischemia. [Ca(2+)](i) and cell length were monitored in myocytes, and functional recovery and LDH release in isolated hearts. From these experiments an optimal concentration of KB-R7943 was identified and tested in pigs submitted to 48 min of coronary occlusion and 2 h of reperfusion.. In myocytes, KB-R7943 at concentrations up to 15 microM reduced [Ca(2+)](i) rise and the probability of hypercontracture during re-energization (P<0.01). Nevertheless, in rat hearts, the effects of KB-R7943 applied during reperfusion after 60 min of ischemia depended on concentration and timing of administration. During the first 5 min of reperfusion, KB-R7943 (0.3-30 microM) induced a dose-dependent reduction in LDH release (half-response concentration 0.29 microM). Beyond 6 min of re-flow, KB-R7943 had no effect on LDH release, except at concentrations > or = 15 microM, which increased LDH. KB-R7943 at 5 microM given during the first 10 min of reflow reduced contractile dysfunction (P=0.011), LDH release (P=0.019) and contraction band necrosis (P=0.014) during reperfusion. Intracoronary administration of this concentration during the first 10 min of reperfusion reduced infarct size by 34% (P=0.033) in pigs submitted to 48 min of coronary occlusion.. These results are consistent with the hypothesis that during initial reperfusion NCX activity results in net reverse mode operation contributing to Ca(2+) overload, hypercontracture and cell death, and that NCX inhibition during this phase is beneficial. Beyond this phase, NCX inhibition may impair forward mode-dependent Ca(2+) extrusion and be detrimental. These findings may help in the design of therapeutic strategies against lethal reperfusion injury, with NCX as the target.

Topics: Analysis of Variance; Animals; Calcium; Cell Death; Cell Size; Cells, Cultured; Dose-Response Relationship, Drug; Male; Models, Animal; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Perfusion; Random Allocation; Rats; Rats, Sprague-Dawley; Ryanodine; Sarcoplasmic Reticulum; Sodium-Calcium Exchanger; Swine; Thapsigargin; Thiourea; Time Factors

We investigated the effect of A(1) adenosine receptor overexpression, which has been reported to increase myocardial tolerance to ischemia-reperfusion injury, on sarcoplasmic reticulum (SR) Ca(2+) handling.. Transgenic mouse hearts (approximately 300-fold A(1) adenosine receptor overexpression) and wild-type mouse hearts were perfused in the Langendorff mode and subjected either to 80 min of aerobic perfusion or to 30 min of aerobic perfusion, 20 min of global ischemia and 30 min of reperfusion. The hearts were then homogenized and used to assay SR oxalate-supported 45Ca(2+) uptake and [3H]-ryanodine binding.. Transgenic hearts showed increased resistance to ischemia-reperfusion, as shown by lower diastolic tension (1.5 +/- 0.2 vs. 2.6 +/- 0.1 g, P<0.05) and higher recovery of developed tension (45 +/- 3 vs. 30 +/- 4% of the baseline, P<0.05) following ischemia-reperfusion. Under baseline conditions, oxalate-supported 45Ca(2+) uptake was lower in transgenic hearts, owing to reduced V(max) (10.6 +/- 2.0 vs. 17.8 +/- 2.7 nmol/min per mg of protein, P<0.05), and the difference was preserved after ischemia-reperfusion (10.0 +/- 1.0 vs. 15.7 +/- 2.5 nmol/min per mg of protein, P<0.05). No significant difference in [3H]-ryanodine binding was observed.. A(1) adenosine receptor overexpression is associated with a decreased rate of active Ca(2+) transport into the SR. We hypothesize that changes in SR function may cause a depletion of the SR Ca(2+) pool, which might protect from ischemic injury by delaying the development of cytosolic Ca(2+) overload during ischemia.

Topics: Analysis of Variance; Animals; Calcium; Female; Male; Mice; Mice, Transgenic; Myocardial Ischemia; Myocardium; Perfusion; Protein Binding; Rats; Receptors, Purinergic P1; Ryanodine; Sarcoplasmic Reticulum

Regular fish consumption is associated with low cardiovascular disease morbidity and mortality. Fish oils modify cardiac membrane phospholipid fatty acid composition with potent antiarrhythmic effects. We tested the effects of dietary fish oil on ventricular hemodynamics and myocardial oxygen consumption (MVO2).. Male Wistar rats were fed for 16 weeks on a reference diet rich in n-6 polyunsaturated fatty acids (PUFA), a diet rich in saturated animal fat (SAT), or a diet rich in n-3 PUFA from fish oil. Isolated working hearts were perfused with porcine erythrocytes (40% hematocrit) at 75 mm Hg afterload with variable preload (5 to 20 mm Hg) or with low coronary flow ischemia with maintained afterload, preload, and heart rate, then reperfused. MVO2 was low and coronary perfusion reserve high in n-3 PUFA hearts, and cardiac output increased with workload. The n-3 PUFA reduced ischemic markers-acidosis, K+, lactate, and creatine kinase-and increased contractile recovery during reperfusion. SAT hearts had high MVO2, low coronary perfusion reserve, and poor contractile function and recovery. Dietary differences in MVO2 were abolished by KCl arrest (basal metabolism) or ruthenium red (3.4 micromol/L) but not by ryanodine (1 nmol/L). Fish oil or ryanodine, but not ruthenium red, prevented ventricular fibrillation in reperfusion.. Dietary fish oil directly influenced heart function and improved cardiac responses to ischemia and reperfusion. The n-3 PUFA reduced oxygen consumption at any given work output and increased postischemic recovery. Thus, direct effects on myocardial function may contribute to the altered cardiovascular disease profile associated with fish consumption.

Topics: Animals; Blood Flow Velocity; Calcium; Cardiac Output; Cell Membrane; Coronary Circulation; Diet; Dietary Fats, Unsaturated; Fatty Acids; Fatty Acids, Omega-3; Fatty Acids, Omega-6; Fatty Acids, Unsaturated; Fish Oils; Hemodynamics; In Vitro Techniques; Intracellular Fluid; Male; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Oxygen Consumption; Rats; Rats, Wistar; Ruthenium Red; Ryanodine

Cytosolic Ca2+ overload plays a major role in the development of irreversible injury during myocardial ischemia. Such overload is due at least in part to the release of Ca2+ from the sarcoplasmic reticulum. Therefore, we investigated whether dantrolene, a blocker of the sarcoplasmic reticulum Ca2+ release channel, may protect from ischemic injury. In binding experiments, we determined the effect of dantrolene on [3H]-ryanodine binding in rat cardiac tissue. In perfusion experiments, isolated rat hearts were perfused for 20 min in the working mode, in the presence of 0-45 microM dantrolene. The hearts were then subjected to 30 min of global ischemia and 120 min of retrograde reperfusion. Tissue injury was evaluated on the basis of triphenyltetrazolium chloride (TTC) staining and LDH release. The binding experiments showed that dantrolene displaced 4 nM [3H]-ryanodine with IC50 of 34 microM. In the perfusion experiments, tissue necrosis (i.e., TTC-negative tissue) averaged 28.3 +/- 1.6% of the ventricular mass under control conditions. Dantrolene was protective at micromolar concentrations: tissue necrosis decreased to 21.4 +/- 1.0% and 8.4 +/- 1.4% with 1 microM and 45 microM dantrolene, respectively (P < 0.05 and P < 0.01). Similar results were obtained with regard to LDH release. At low concentrations (up to 4 microM), dantrolene did not produce any significant hemodynamic effect, except for a slight increase in coronary flow, whereas at higher concentration a negative inotropic effect was apparent. In conclusion, dantrolene reduced ischemic injury even at concentrations that did not affect contractile performance. Modulation of sarcoplasmic reticulum Ca2+ release might represent a new cardioprotective strategy.

Topics: Animals; Calcium; Dantrolene; Heart; Hemodynamics; Male; Myocardial Ischemia; Rats; Rats, Wistar; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Verapamil

Several studies have shown that myocardial ischemia leads to functional failure of endothelial cells (EC) whereby disturbance of Ca(2+) homeostasis may play an important role. The mechanisms leading to Ca(2+) disbalance in ischemic EC are not fully understood. The aim of this study was to test effects of different components of simulated ischemia (glucose deprivation, anoxia, low extracellular pH (pH(o)) and lactate) on Ca(2+) homeostasis in EC.. Cytosolic Ca(2+) (Ca(i)), cytosolic pH (pH(i)) and ATP content were measured in cultured rat coronary EC.. In normoxic cells 60 min glucose deprivation at pH(o) 7.4 had no effect on pH(i). It only slightly increased Ca(i) and decreased ATP content. Reduction of pH(o) to 6.5 under these conditions led to marked cytosolic acidosis and Ca(i) overload, but had no effect on ATP content. Anoxia at pH(o) 6.5 had no additional effect on Ca(i) overload, but significantly reduced cellular ATP. Addition of 20 mmol/l lactate to anoxia at pH(o) 6.5 accelerated Ca(i) overload due to faster cytosolic acidification. Acidosis-induced Ca(i) overload was prevented by inhibition of Ca(2+) release channels of endoplasmic reticulum (ER) with 3 micromol/l ryanodine or by pre-emptying the ER with thapsigargin. Re-normalisation of pH(o) for 30 min led to recovery of pH(i), but not of Ca(i).. The ischemic factors leading to cytosolic acidosis (low pH(o) and lactate) cause Ca(i) overload in endothelial cells, while anoxia and glucose deprivation play only a minor role. The ER is the main source for this Ca(i) rise. Ca(i) overload is not readily reversible.

Topics: Adenosine Triphosphate; Analysis of Variance; Animals; Calcium; Calcium Channel Blockers; Cell Size; Cells, Cultured; Coronary Vessels; Cytosol; Endoplasmic Reticulum; Endothelium, Vascular; Enzyme Inhibitors; Guanidines; Hydrogen-Ion Concentration; Lactic Acid; Male; Manganese; Myocardial Ischemia; Rats; Rats, Wistar; Ryanodine; Sodium-Hydrogen Exchangers; Sulfones; Thapsigargin

We investigated the effect of sulfhydryl and disulfide reagents on ischemic preconditioning and on sarcoplasmic reticulum Ca2+ release. Isolated working rat hearts were subjected to ischemic preconditioning (three 3-minute periods of global ischemia) or to control aerobic perfusion, which was followed by 30 minutes of global ischemia and 120 minutes of retrograde reperfusion. Necrosis was evaluated on the basis of lactate dehydrogenase release and triphenyltetrazolium chloride staining. In parallel experiments, sarcoplasmic reticulum Ca2+ release and [3H]-ryanodine binding were determined before the sustained ischemia. Ischemic preconditioning was associated with protection versus ischemic injury, decreased Ca2+ release and reduced [3H]-ryanodine binding. The disulfide reducing agent dithiothreitol (1 mmol/L) removed the protection provided by ischemic preconditioning, if added to the perfusion buffer either before or after the preconditioning procedure. In preconditioned hearts, dithiothreitol increased sarcoplasmic reticulum Ca2+ release and ryanodine binding, whereas in control hearts it had no effect on either tissue injury or sarcoplasmic reticulum function. Perfusion of control hearts with the sulfhydryl blocking agents 4,4'-dithiodipyridine (25 micromol/L) and N-ethylmaleimide (16 micromol/L) increased the resistance to ischemia and reduced sarcoplasmic reticulum Ca2+ release and [3H]-ryanodine binding. These effects were not additive with those induced by preconditioning. Sulfhydryl and disulfide reagents produced similar effects on Ca2+ release and [3H]-ryanodine binding if added in vitro to preparations obtained from control and preconditioned hearts. We conclude that ischemic preconditioning is associated with the oxidation of sulfhydryl groups involved in the modulation of sarcoplasmic reticulum Ca2+ release.

Topics: Animals; Calcium; Disease Susceptibility; Dithiothreitol; Ischemic Preconditioning; L-Lactate Dehydrogenase; Male; Myocardial Ischemia; Myocardium; Oxidation-Reduction; Rats; Rats, Wistar; Ryanodine; Sarcoplasmic Reticulum; Sulfhydryl Compounds

Abnormal intracellular calcium handling in cardiomyopathic human hearts has been associated with an impaired function of the sarcoplasmic reticulum, but previous reports on the gene expression of the ryanodine receptors (Ry2) are contradictory. We measured the mRNA levels, the protein levels and the number of high affinity [3H]ryanodine binding sites in the left ventricle of non-failing (n = 9) and failing human hearts [idiopathic dilated (IDCM n = 16), ischemic (ICM n = 7) or mixed (MCM n = 8) cardiomyopathies]. Ry2 mRNA levels were significantly reduced in IDCM (-30%) and unchanged in MCM and ICM and Ry2 protein levels were similar. In contrast, we observed a two-fold increase in the number of high affinity Ry2 (B(max) = 0.43 +/- 0.11 v 0.22 +/- 0.13 pmol/mg protein, respectively; P<0.01) and an unchanged K(d). Furthermore, levels of myosin heavy chain mRNA and protein per g of tissue were similar in failing and non-failing hearts, suggesting that the observed differences in Ry2 are not caused by the increase in fibrosis in failing heart. Therefore, the dissociation between the two-fold increase in the number of high affinity ryanodine receptors observed in all failing hearts and the slightly decreased mRNA level or unchanged protein level suggests that the ryanodine binding properties are affected in failing myocardium and that such modifications rather than a change in gene expression alter the channel activity and could contribute to abnormalities in intracellular Ca2+ handling.

Topics: Adult; Binding Sites; Blotting, Western; Calcium Channels; Cardiomyopathies; Cardiomyopathy, Dilated; Female; Humans; Male; Middle Aged; Muscle Proteins; Myocardial Ischemia; Myocardium; RNA Processing, Post-Transcriptional; RNA, Messenger; Ryanodine; Ryanodine Receptor Calcium Release Channel; Tritium

The effect of 15 min of global, normothermic ischemia on cardiac sarcoplasmic reticulum (SR) was investigated using the Ca2+ uptake rate and 3H-ryanodine binding of ventricular homogenates and isolated SR vesicles. Ischemia did not affect ryanodine binding in the homogenate, while it increased it in the isolated SR vesicles. Although ischemia decreased the homogenate oxalate-supported Ca2+ uptake rate, measured in the presence of high ryanodine to close the ryanodine-sensitive efflux pathway (+RY), its decrease of the Ca2+ uptake rate, measured in the absence of ryanodine (-RY), was more marked. This finding was also observed in the isolated SR. Although inhibition of the Ca-ATPase and its coupled Ca2+ uptake by thapsigargin proportionately decreased SR Ca2+ uptake -RY and +RY, ischemia decreased the Ca2+ uptake -RY proportionately more. This result suggested that there was a greater fraction of Ca2+ uptake activity in ryanodine-sensitive vesicles after ischemia. However, ischemia also reduced the yield of SR activity in the isolated SR fraction and the results could potentially be due to differential selection of ryanodine-sensitive and ryanodine-insensitive SR in the isolation procedure. We directly tested the hypothesis that ischemia changes the fraction of Ca2+ uptake activity in the ryanodine-sensitive vesicles by estimating the Ca-oxalate capacity measured +RY and -RY. Ischemia decreased the capacity -RY much more than +RY in the homogenate, indicating that more of the SR volume and Ca2+ uptake activity was in the ryanodine-sensitive vesicles after ischemia.

Topics: Animals; Calcium; Calcium Oxalate; Heart Ventricles; In Vitro Techniques; Male; Myocardial Ischemia; Rats; Rats, Sprague-Dawley; Ryanodine; Sarcoplasmic Reticulum

The effects of ischemia and reperfusion on sarcoplasmic reticulum (SR) calcium uptake were measured in crude heart homogenates of rats and were compared to published results for rabbit hearts. Isolated rat hearts (n = 5 in each group) were Langendorff-perfused at 37 degrees C and were either kept normally perfused (control group), or submitted to 15 min normothermic ischemia (ischemic group), or reperfused for 10 min after 15 min ischemia (reperfused group). Mechanical function recovered to 50-60% of control after 10 min reperfusion following ischemia. Ca uptake (control Vmax: 23.0 +/- 2.20 nmol.min.1.mg of protein-1) decreased during ischemia (Vmax: 15.7 +/- 1.60 nmol.min-1.mg-1) but recovered to control level on reperfusion (Vmax: 20.8 +/- 2.02 nmol.min-1.mg-1). An increased Ca uptake was obtained when the measurements were carried out in the presence of ryanodine (430 microM) to block Ca leakage through SR Ca-release channels. The relative magnitude of ryanodine effect in the ischemic myocardium (increase: 77.2 +/- 18.20%) was more marked than in control (32.0 +/- 8.22%) or reperfused myocardium (39.0 +/- 10.66%). This result is different from that of rabbit myocardium where similar ryanodine effect is present in all groups (56.7 +/- 13.76%, 50.0 +/- 13.56% and 54.2 +/- 6.88% in control, ischemic and reperfused hearts, respectively) and suggests that a component of cytosolic Ca overload via SR Ca-release channels is present during ischemia in rat, but not in rabbit myocardium.

Topics: Animals; Calcium; Heart; Male; Myocardial Ischemia; Myocardial Reperfusion; Myocardial Stunning; Rabbits; Rats; Rats, Sprague-Dawley; Ryanodine; Sarcoplasmic Reticulum

Although protein kinase C (PKC)-mediated cardioadaptation to ischemia-reperfusion (IR) is accompanied by increased intracellular Ca2+ concentration, it is unknown whether a preischemia sarcoplasmic reticulum (SR) Ca2+ release affects PKC-mediated post-IR functional protection. To study this, crystalloid-perfused (Langendorff) Sprague-Dawley rat hearts were used to assess the effects of a ryanodine (Ry)-induced preischemia Ca2+ load (Ry, 5 nM/2 min, retrograde coronary) 10 min before global IR (20 min). Ry was administered with and without each of two different PKC inhibitors (20 microM chelerythrine and 150 nM bisindolylmaleimide I-HCl). Ry improved myocardial functional recovery (developed pressure, end-diastolic pressure, coronary flow, and creatine kinase activity), which was eliminated after PKC inhibition. Immunohistochemical staining for PKC isoforms demonstrated that Ry induces specific PKC translocation of alpha-, delta-, and zeta-isoforms. We conclude that 1) a preischemia Ca2+ load from the SR results in post-IR myocardial functional protection 2) Ca(2+)-induced functional protection is PKC regulated via the translocation of specific isoforms, and 3) Ca(2+)-induced cardioadaptation to IR injury may have important therapeutic implications prior to planned ischemic events such as cardiac allograft preservation and cardiac bypass surgery.

Topics: Adaptation, Physiological; Alkaloids; Animals; Benzophenanthridines; Biological Transport; Calcium; Calcium Channels; Creatine Kinase; Enzyme Inhibitors; Heart; Hemodynamics; Indoles; Isoenzymes; Male; Maleimides; Muscle Proteins; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Phenanthridines; Protein Kinase C; Rats; Rats, Sprague-Dawley; Ryanodine; Ryanodine Receptor Calcium Release Channel

We have previously demonstrated deficiencies in myocardial cycling of Ca2+, and ATP turnover, in animals with heart failure (HF). The objective of this study was to determine the relevance of these changes to human HF.. We used the Ca2+ dye, indo-1, and the Ca(2+)-channel modulator ryanodine to examine Ca(2+)-cycling in homogenates containing 2.5% myocardium from 12 patients undergoing cardiac transplantations because of ischemic or idiopathic dilated cardiomyopathies (ISCM, DCM), and compared them to homogenates from 11 organ donors who died from noncardiac causes. Key enzymes of ATP production and utilization were also assayed.. In HF due to either ISCM or DCM, compared to nonfailing myocardium, rate constants (x 10(-3) s-1) for sarcoplasmic reticulum Ca(2+)-pumping (41.6 +/- 16.0 versus 15.1 +/- 5.9) and Ca(2+)-channel (25.1 +/- 8.3 versus 6.2 +/- 4.1) activities were decreased by 64 and 75%, respectively. These changes in rate constants were associated with a three-fold increase in ionized Ca2+ concentration. Compared to nonfailing myocardium, activities (IU/g) of ATP turnover were also decreased in ISCM and DCM HF by 39%, 30%, and 34%, respectively, for ATP production capacity of creatine kinase (1830 +/- 130 versus 1110 +/- 411) and oxidative phosphorylation (20.0 +/- 3.3 and 14.1 +/- 4.8), and for ATP utilization (28.2 +/- 18.7 versus 18.7 +/- 4.0). Myoglobin, a key component of oxidative phosphorylation, was approximately 50% lower with HF (1.72 +/- 0.30 versus 0.97 +/- 0.20 mg/g).. As in animal models, cycling of Ca2+ and ATP turnover were markedly impaired in human heart failure. There were no consistent biochemical differences attributable to difference in etiology, excepting that myoglobin deficiency was 33% greater in ISCM than DCM. We conclude that ATP and Ca2+ cycling are significantly impaired in human HF due to DCM and ISCM.

Topics: Adenosine Triphosphate; Calcium; Calcium Channels; Cardiomyopathy, Dilated; Creatine Kinase; Down-Regulation; Humans; Myocardial Ischemia; Myocardium; Myoglobin; Oxidative Phosphorylation; Ryanodine

We investigated the modifications of cardiac ryanodine receptors/sarcoplasmic reticulum Ca2+ release channels occurring in ischemic preconditioning. In an isolated rat heart model, the injury produced by 30 minutes of global ischemia was reduced by preexposure to three 3-minute periods of global ischemia (preconditioning ischemia). The protection was still present 120 minutes after preconditioning ischemia but disappeared after 240 minutes. Three 1-minute periods of global ischemia did not provide any protection. In the crude homogenate obtained from ventricular myocardium, the density of [3H]ryanodine binding sites averaged 372 +/- 18 fmol/mg of protein in the control condition, decreased 5 minutes after preconditioning ischemia (290 +/- 15 fmol/mg, P < .01), was still significantly reduced after 120 minutes (298 +/- 17 fmol/mg, P < .05), and recovered after 240 minutes (341 +/- 21 fmol/mg). Three 1-minute periods of ischemia did not produce any change in ryanodine binding. The Kd for ryanodine (1.5 +/- 0.3 nmol/L) was unchanged in all cases. In parallel experiments, the crude homogenate or a microsomal fraction was passively loaded with 45Ca, and Ca(2+)-induced Ca2+ release was studied by the quick filtration technique. In both preparations, the rate constant of Ca(2+)-induced Ca2+ release decreased 5 and 120 minutes after preconditioning ischemia (homogenate values: 19.7 +/- 1.4 and 18.9 +/- 0.9 s-1 vs a control value of 25.4 +/- 1.7 s-1, P < .05 in both cases) and recovered after 240 minutes (23.0 +/- 1.9 s-1). The Ca2+ dependence of Ca(2+)-induced Ca2+ release was not affected by preconditioning ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Analysis of Variance; Animals; Calcium Channels; Hemodynamics; In Vitro Techniques; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Rats; Rats, Sprague-Dawley; Ryanodine; Sarcoplasmic Reticulum; Time Factors

Isolated adult rat hearts in an isovolumic nonworking Langendorff preparation were loaded with the Ca2+ indicator aequorin to investigate the effects of ischemic reperfusion on free intracellular Ca2+ concentration ([Ca2+]i) homeostasis and left ventricular (LV) contractile function. In three groups (each n = 8) that underwent 10, 20, and 30 min of ischemia, recovery of developed pressure amounted to, respectively, 63% [77 +/- 3 (SE) mmHg], 48% (56 +/- 4 mmHg), and 34% (43 +/- 4 mmHg) of preischemic control (122 +/- 5 mmHg) after 60 min of reperfusion. Diastolic pressure remained elevated at 40 +/- 4, 55 +/- 3, and 65 +/- 6 mmHg, respectively (preischemic control, 12 mmHg). During early reperfusion (0-20 min), the light transient demonstrated a prolonged time to 90% decline from peak light (t90L), which was paralleled by a delayed relaxation on the LV pressure tracing in the 10- and 20-min ischemia groups. After 60 min of reperfusion, the prolongation of t90L persisted in all groups (10-min ischemia, 89 +/- 2 ms; 20 min, 95 +/- 3 ms; 30 min, 96 +/- 2 ms; control, 82 +/- 2 ms; P < 0.05). In contrast, the LV pressure tracing was abbreviated beyond the preischemic control, indicating altered myofibrillar Ca2+ responsiveness. Diastolic [Ca2+]i was elevated after 60 min of reperfusion (10-min ischemia, 0.40 +/- 0.06 microM; 20 min, 0.48 +/- 0.04 microM; 30 min, 0.51 +/- 0.06 microM; control, 0.32 +/- 0.01 microM) and had a significant positive correlation with LV diastolic pressure (r = 0.79; P < 0.001). A positive correlation was also found for the amplitude of the Ca2+ transient and LV developed pressure (r = 0.53; P < 0.05). These findings suggest that postischemic contractile dysfunction is related to altered Ca2+ modulation with impaired [Ca2+]i homeostasis following moderate to severe reperfusion injury in the rat.

Topics: Aequorin; Analysis of Variance; Animals; Blood Pressure; Calcium; Diastole; Homeostasis; In Vitro Techniques; Male; Myocardial Contraction; Myocardial Ischemia; Myocardial Reperfusion; Myofibrils; Rats; Rats, Wistar; Reference Values; Ryanodine; Systole; Time Factors; Ventricular Function, Left; Ventricular Pressure

Calcium-induced calcium release (CICR) from sarcoplasmic reticulum (SR) may contribute to calcium depletion of SR during the infusion of cardioplegic solution, which may protect the intracellular calcium overload observed during myocardial reperfusion. We have, therefore, investigated (1) the ability of ryanodine-containing cardioplegic solution to enhance myocardial protection and (2) the influence of diltiazem, L-type calcium channel blocker, on the ryanodine-enhanced cardioprotective effect in the isolated working rat heart. Hearts (n = 6-8/group) from male Wistar rats were aerobically (37 degrees C) perfused (20 min) with bicarbonate buffer (Ca2+ = 2.4 mM). This was followed by a 3 min infusion of St. Thomas' Hospital cardioplegic solution containing (1) 0 nmol/L of ryanodine or (2) 1.75 nmol/L of ryanodine combined with various concentrations of diltiazem (0, 0.13, 0.25 and 0.50 mumol/L). Hearts were then subjected to 40 min of normothermic (37 degrees C) global ischemia and 35 min of reperfusion (15 min Langendorff, 20 min working). (1) The recovery of aortic flow (%AF) was 52.2 +/- 3.5% in the ryanodine-free group. (2) %AF was 72.0 +/- 1.4%, 50.0 +/- 2.6*, 61.7 +/- 3.2* and 58.3 +/- 2.8*% in the 0, 0.13, 0.25 and 0.50 mumol/L diltiazem groups, respectively (*p < 0.05 vs the 0 mumol/L diltiazem group). Creatine kinase (CK) leakage during Langendorff reperfusion was less in the 0 mumol/L diltizaem (plus 1.75 nmol/L ryanodine) group than the ryanodine-free group.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Bicarbonates; Calcium; Calcium Channels; Calcium Chloride; Diltiazem; Heart Arrest, Induced; Magnesium; Male; Models, Cardiovascular; Myocardial Ischemia; Potassium Chloride; Rats; Rats, Wistar; Ryanodine; Sarcoplasmic Reticulum; Sodium Chloride

The effect of 15 min of global, normothermic ischemia on 3H-ryanodine binding and the oxalate-supported Ca2+ uptake of cardiac sarcoplasmic reticulum (SR) was investigated in parallel using ventricular homogenates of isolated perfused rat hearts. Ischemia increased the Ca2+ efflux under the uptake assay conditions, as demonstrated by the greater stimulation of Ca2+ uptake by high concentrations of ryanodine (+RY) to close the SR Ca2+ channel. This effect was partially reversed by reperfusion. Ischemia depressed Ca2+ uptake rate -RY at free [Ca2+] of 0.4 microM and above, while the depression + RY was significant only above 10 microM Ca2+. We tested the hypothesis that inhibition of the Ca-ATPase alone, by adding thapsigargin or cyclopiazonic acid, could reproduce the effects of ischemia on the homogenate Ca2+ uptake rate. Thapsigargin or cyclopiazonic acid proportionally depressed Ca2+ uptake rate +RY and -RY and produced distinctly different effects of ischemia. Ischemia did not change the Bmax or Kd for equilibrium 3H-ryanodine binding, or the Hill coefficient or KCa for the [Ca2+]-dependence of equilibrium 3H-ryanodine binding. The rate of ryanodine binding, measured under the uptake conditions, was increased by ischemia and further increased by reperfusion. The effect of ischemia on the rate and extent of equilibrium binding to the high-affinity ryanodine binding site were unrelated to the highly reproducible effects on SR Ca2+ uptake rates measured in the homogenate.

Topics: Animals; Calcium; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Radioligand Assay; Rats; Rats, Sprague-Dawley; Ryanodine; Sarcoplasmic Reticulum

Sarcoplasmic reticular (SR) calcium depletion may contribute to the myocardial protection against ischemia and reperfusion-induced injury. We have, therefore, investigated the effect of ryanodine-induced SR calcium depletion on the myocardial protection when given during Langendorff perfusion before ischemia in the isolated working rat heart. Hearts (n = 6/group) from male Wistar rats were aerobically (37 degrees C) perfused (20 min) with bicarbonate buffer (the first working perfusion), and then aerobically (37 degrees C) perfused with bicarbonate buffer containing 0, 1.75 or 7.00 nmol/L of ryanodine (a Langendorff perfusion). Hearts were then aerobically (37 degrees C) perfused (20 min) with ryanodine-free bicarbonate buffer (the second working perfusion). This was followed by a 3 min infusion of St. Thomas' Hospital cardioplegic solution, and then subjected to 38 min of normothermic (37 degrees C) global ischemia and 35 min of reperfusion (15 min Langendorff, 20 min working). After the ryanodine treatment, the percent reduction in aortic flow was significantly greater in the 7.00 nmol/L ryanodine group than in the control group. The post-ischemic recoveries of aortic flow were 39.1 +/- 3.3, 32.8 +/- 5.3 and 37.3 +/- 4.4% in the 0, 1.75 and 7.00 nmol/L ryanodine groups, respectively. There was no difference in the creatine kinase leakage during Langendorff reperfusion between the ryanodine-treated groups and the control group. Thus, ryanodine decreased the cardiac function after the ryanodine treatment, indicating a reduction in SR calcium content. Ryanodine, however, failed to improve the post-ischemic functional recoveries.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcium; Heart; Heart Arrest, Induced; In Vitro Techniques; Male; Myocardial Ischemia; Myocardium; Rats; Rats, Wistar; Ryanodine; Sarcoplasmic Reticulum

Calcium release from sarcoplasmic reticulum (SR) may contribute to the intracellular calcium overload observed during myocardial ischemia and reperfusion. We have therefore investigated the ability of ryanodine to enhance myocardial protection when given before ischemia or during reperfusion in the isolated working rat heart. Hearts (n = 6-9/group) from male Wistar rats were aerobically (37 degrees C) perfused (20 min) with bicarbonate buffer (Ca2+ = 2.4 mM). In the first series of studies, this was followed by a 3 min infusion of St Thomas' Hospital cardioplegic solution containing various concentrations of ryanodine. Hearts were then subjected to 38 min of normothermic (37 degrees C) global ischemia and 35 min of reperfusion (15 min Langendorff, 20 min working). The recoveries of aortic flow (%AF) were 50.3 +/- 2.5% in the ryanodine free controls versus 55.2 +/- 5.8, 72.0 +/- 1.3 (p < 0.05), 61.0 +/- 4.3, 51.8 +/- 5.1 and 32.1 +/- 5.0 (p < 0.05)% in the 0.88, 1.75, 2.13, 2.50 and 10.00 nM ryanodine groups, respectively. Creatine kinase (CK) leakage during Langendorff reperfusion was reduced in the 1.75 nM group but was similar to control in all other groups. In the second series of studies, 3 min of cardioplegia without ryanodine and 38 min of ischemia (37 degrees C) were followed by 15 min of Langendorff reperfusion with 0, 0.09, 0.18, 0.88 or 1.75 nM ryanodine, %AF was 59.3 +/- 3.3%, 54.7 +/- 3.3, 53.8 +/- 3.5, 38.4 +/- 8.9 (p < 0.05) and 33.3 +/- 5.8 (p < 0.05)% in the 0, 0.09, 0.18, 0.88 and 1.75 nM ryanodine groups, respectively. CK leakage tended to increase dose-dependently.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcium; Cardioplegic Solutions; Heart Arrest, Induced; In Vitro Techniques; Male; Models, Cardiovascular; Myocardial Ischemia; Myocardial Reperfusion Injury; Rats; Rats, Wistar; Ryanodine; Sarcoplasmic Reticulum

We investigated the effect of ischemia and reperfusion on the cardiac ryanodine receptor, which corresponds to the sarcoplasmic reticulum Ca2+ channel. Isolated working rat hearts were subjected to 10 to 30 minutes of global ischemia, followed or not by reperfusion. Ischemia produced significant reduction in the density of high-affinity 3H-ryanodine binding sites, determined either in whole-heart homogenate (Bmax, 220 +/- 22, 203 +/- 12, and 228 +/- 14 fmol/mg protein after 10, 20, and 30 minutes of ischemia versus 298 +/- 18 fmol/mg protein in the control condition; P < .01) or in a fraction enriched in sarcoplasmic reticulum (Bmax, 1.08 +/- 0.15 pmol/mg protein after 20 minutes of ischemia versus 1.69 +/- 0.08 pmol/mg protein in the control condition; P < .01). The Kd (1.5 +/- 0.1 nmol/L) and the Ca2+ dependence of high-affinity 3H-ryanodine binding were not affected by ischemia. The density of low-affinity 3H-ryanodine binding sites was also reduced after 20 minutes of ischemia (14.0 +/- 2.3 versus 34.0 +/- 8.2 pmol/mg protein in the sarcoplasmic reticulum fraction, P < .05), without significant changes in Kd (4.7 +/- 1.2 versus 2.4 +/- 1.0 mumol/L). All these changes persisted after 20 minutes of reperfusion. Analysis of tissue fractions showed that 55% of the ryanodine binding sites were retained in the pellet of a low-speed centrifugation ("nuclear pellet") and that the effects of ischemia concerned only the receptors released in the supernatant ("postnuclear supernatant"). In parallel experiments, we evaluated the effect of ryanodine on oxalate-supported Ca2+ uptake, which represents sarcoplasmic reticulum Ca2+ uptake. As expected, we found that high concentrations of ryanodine stimulated Ca2+ uptake, owing to channel blockade. The response to 900 mumol/L ryanodine was slightly reduced in crude homogenate and significantly reduced in postnuclear supernatant obtained from ischemic hearts. In conclusion, the number of ryanodine receptors is reduced after ischemia; this effect concerns a subpopulation of the receptors, persists after reperfusion, and might contribute to modify sarcoplasmic reticulum function.

Topics: Animals; Calcium; Calcium Channels; Hemodynamics; Muscle Proteins; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Rats; Rats, Sprague-Dawley; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum

Cocaine is a potent cardiac stimulant that can provoke lethal cardiac events, including ventricular fibrillation (VF). The cocaine-induced accumulation of intracellular calcium could contribute significantly to the development of these lethal arrhythmias. To test this hypothesis, VF was induced in 12 mongrel dogs by the combination of cocaine (1.0 mg/kg) and a 2-min coronary occlusion during exercise. This test without cocaine failed to induce arrhythmias. Pretreatment with the intracellular calcium-specific chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM; 1.0 mg/kg iv) prevented VF in 8 of 12 animals (P < 0.001) and delayed the onset of lethal arrhythmias in 3 of the remaining animals. Cocaine induced significant increases in left ventricular (LV) systolic pressure (control 154.7 +/- 8.7, cocaine 167.4 +/- 8.4 mmHg), heart rate (control 195.9 +/- 6.1, cocaine 222.3 +/- 10.6 beats/min), and LV maximum rate of pressure development (dP/dtmax; control 5,251 +/- 317.6, cocaine 6,016 +/- 435.1 mmHg/s). BAPTA-AM attenuated the increase in LV dP/dtmax (BAPTA-AM 4,591 +/- 479.3 mmHg/s) and LV systolic pressure (BAPTA-AM 154.5 +/- 6.8 mmHg). Because vascular muscle relaxation could contribute to the cardioprotection, the cocaine and exercise plus ischemia test was repeated after nitroprusside. The nitroprusside prevented cocaine-induced increases in LV systolic pressure but failed to prevent VF. These data suggest that BAPTA-AM may prevent cocaine-induced VF independently of its vascular actions.

Topics: Animals; Blood Pressure; Chelating Agents; Cocaine; Dogs; Egtazic Acid; Heart Rate; Hemodynamics; Myocardial Ischemia; Physical Exertion; Ryanodine; Ventricular Fibrillation; Ventricular Function, Left

Myocardial ischaemia can provoke a rise in cytosolic calcium which may in turn trigger malignant ventricular arrhythmias. Recently, inhibition of calcium entry has been shown to prevent these lethal arrhythmias. However, the contributions of calcium release from cytosolic stores to these disruptions in cardiac rhythm have not been investigated. This study examines the role of calcium release from the sarcoplasmic reticulum in the initiation of lethal ventricular arrhythmias.. Mongrel dogs were chronically instrumented to measure left ventricular pressure, coronary blood flow, and cardiac electrical activity (ventricular electrocardiogram). The left anterior descending coronary artery was ligated during the surgery to produce a myocardial infarction. In addition, a hydraulic occluder was placed around the left circumflex artery. The susceptibility to ventricular fibrillation was then evaluated by the combination of acute myocardial ischaemia and exercise.. Ventricular fibrillation was induced in 10 animals during the exercise plus ischaemia test. On a subsequent day the exercise plus ischaemia test was repeated after pretreatment with ryanodine (10 micrograms.kg-1, n = 10), a drug which impairs calcium efflux from the sarcoplasmic reticulum. Ryanodine failed to prevent ventricular fibrillation induced by ischaemia. Ryanodine significantly (p < 0.01) increased heart rate [control 115.3(SEM 6.3) v ryanodine 156.4(14.7) beats.min-1] but reduced left ventricular systolic pressure [control 141.8(4.9) v ryanodine 111.1(12.7) mm Hg] and positive left ventricular dP/dt [3312.9(217.4) v ryanodine 1462.9(226.3) mm Hg.s-1] both at rest and during exercise. In contrast, this drug abolished ventricular tachycardia induced by ouabain toxicity (n = 10, 40 micrograms.kg-1 bolus followed by 0.076 microgram.kg-1.min-1 for 1 h, then 20 micrograms.kg-1 bolus, intravenously).. These data suggest that calcium release from ryanodine sensitive channels in the sarcoplasmic reticulum may contribute significantly to the arrhythmias induced by ouabain toxicity but not to ventricular fibrillation provoked by ischaemia.

Topics: Animals; Blood Pressure; Dogs; Electrocardiography; Exercise Test; Heart Rate; Myocardial Ischemia; Ryanodine; Ventricular Fibrillation; Ventricular Pressure

To investigate mechanisms underlying the contractile dysfunction during myocardial "stunning," potentiated contractions were studied in Langendorff-perfused rabbit hearts paced at 2.5 Hz. Isovolumetric left ventricular pressure (LVP) and the first derivative of LVP (dP/dt) were measured via a balloon. Potentiated contractions, elicited after 3 s of rest (postrest potentiation, PRP) or with paired pulses (paired-pulse potentiation, PPP) were first characterized in nonischemic conditions. Exposure to 5 nM ryanodine changed PRP into postrest depression [control, 134 +/- 1.7% (SE); ryanodine, 65 +/- 3.4%; n = 5] but did not decrease PPP (control, 125 +/- 7.2%; ryanodine, 141 +/- 14.5%). When sarcolemmal Ca2+ influx was decreased by 0.2-2 microM verapamil, PRP increased (control, 136 +/- 3.7%; 1 microM verapamil, 214 +/- 23.8%; n = 5), whereas PPP was maintained (control, 134 +/- 8.0%; 1 microM verapamil, 154 +/- 11.5%). During ischemia, both PRP and PPP were increased above preischemic values (from 128 +/- 1.9 to 355 +/- 60.4% and from 122 +/- 5.4 to 313 +/- 37.4%, respectively, n = 5). Changes of potentiation of dP/dt were qualitatively similar to those of LVP. On reperfusion, rest potentiation transiently decreased (PRP of dP/dt: 127 +/- 6% preischemia vs. 112 +/- 3% at 2 min postischemia; n = 6). However, PPP increased during the first 20 min of reperfusion (PPP of dP/dt: 184 +/- 22% preischemia vs. 236 +/- 34% postischemia; n = 6). This transient depression of PRP during reperfusion suggests an impairment of sarcoplasmic reticulum function in stunned myocardium, at least during the early phase of reperfusion.

Topics: Animals; Calcium; Cardiac Pacing, Artificial; Heart; In Vitro Techniques; Myocardial Contraction; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Pressure; Rabbits; Ryanodine; Sarcoplasmic Reticulum; Ventricular Function, Left; Verapamil

In mammalian myocardium, muscle contraction is regulated by the rapid release of Ca2+ ions through ryanodine-sensitive Ca2+ release channels present in the intracellular membrane compartment, sarcoplasmic reticulum (SR). In this study, the effects of regional ischemia on intrinsic SR Ca2+ release channel function were determined by studying the Ca2+ transport and release, and [3H]ryanodine binding properties of whole muscle homogenates and SR-enriched membrane fractions from normal and ischemic myocardium. Measurement of oxalate-supported 45Ca(2+)-uptake rates before and after pretreatment with 1 mM ryanodine, indicated that the SR Ca2+ release channel retained its ability to be effectively closed by the channel-specific probe ryanodine after 15 and 60 min of ischemia. 45Ca2+ efflux from, and high-affinity [3H]ryanodine binding to SR-enriched vesicle fractions indicated retention of regulation of Ca2+ release channel activity by Ca2+, Mg2+ and adenine nucleotide in 15 and 60 min ischemic samples. Further, sodium dodecylsulfate polyacrylamide gel and immunoblot analysis revealed no proteolytic degradation of the M(r) 565,000 SR Ca2+ release channel polypeptide after 15 and 60 min of ischemia. These results suggested a minimal, if any, loss of intrinsic SR Ca2+ release channel function in ischemic hearts.

Topics: Animals; Calcium; Calcium Channels; Dogs; Muscle Contraction; Myocardial Ischemia; Ryanodine; Sarcoplasmic Reticulum