Mechanism of hypoxic preconditionin in guinea pig papillary muscles

Brief ischemia or hypoxia has been found to protect the heart against susbsequent long-lasting ischemia and to improve contractile dysfunction as well to reduce cell necrosis and the incidence of lethal arrhythmias. This phenomenon, termed preconditioning (PC) has been demonstrated in different species. However, little is known about PC in guinea pigs. Moreover, electrophysiological changes underlying protection have not been studied so far in conjuntion with force recovery in a setting of PC. The aim of the study was to study PC in a guinea pig papillary muscle, using recovery of contractility after long hypoxic challenge as the main end-point of protection, and to investigate concominant electrophysiological alterations. In guinea pig papillary muscle preparations contracting isometrically (paced at 2 Hz), transmembrane action potentials (AP) and developed force (DF) were recorded by conventional microelectrode technique and a force tranducer. In addition, effective refractory periods (ERP) were determined. Hypoxia was induced by superfusion with 100% N₂ (pO₂ < 5 kPa) and pacing at 3,3 Hz. In the control group, long hypoxia lasted for 45 min and was followed by 30 min reoxygenation. In the PC group, muscles were subjected to 5 min hypoxia followed by 10 min recovery prior to sustained hypoxia/reoxygenation. Results: Long hypoxia induced a similar depression of DF in both, PC and control groups. However, a loss of contractile activity occured earlier in the PC group. AP duration and ERP decreased faster and were significantly shorter after PC. Upon reoxygenation, preconditioned muscles showed significantly better recovery of function (DF 86% of prehypoxic value vs. 36% in controls; p < 0,05). AP and ERP were completely restored in both, PC and control groups. Guinea pig papillary muscle can be preconditioned with a brief hypoxic challenge against contractile dysfunction upon long-lasting hypoxia/reoxygenation. Shortening of AP and loss of contractility occured more quickly during hypoxia and may participate in the protective effect of preconditioning. Possible mechanisms might involve facilitated opening of K_ATP-dependent channels.     

Development of an in vitro model for study of the efficacy of ischemic preconditioning in human skeletal muscle against ischemia-reperfusion injury.

Ischemia-reperfusion (I/R) injury causes skeletal muscle infarction and ischemic preconditioning (IPC) augments ischemic tolerance in animal models. To date, this has not been demonstrated in human skeletal muscle. This study aimed to develop an in vitro model to investigate the efficacy of simulated IPC in human skeletal muscle. Human skeletal muscle strips were equilibrated in oxygenated Krebs-Henseleit-HEPES buffer (37 degrees C). Aerobic and reperfusion phases were simulated by normoxic incubation and reoxygenation, respectively. Ischemia was simulated by hypoxic incubation. Energy store, cell viability, and cellular injury were assessed using ATP, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and lactate dehydrogenase (LDH) assays, respectively. Morphological integrity was assessed using electron microscopy. Studies were designed to test stability of the preparation (n = 5-11) under normoxic incubation over 24 h; the effect of 1, 2, 3, 4, or 6 h hypoxia followed by 2 h of reoxygenation; and the protective effect of hypoxic preconditioning (HPC; 5 min of hypoxia/5 min of reoxygenation) before 3 h of hypoxia/2 h of reoxygenation. Over 24 h of normoxic incubation, muscle strips remained physiologically intact as assessed by MTT, ATP, and LDH assays. After 3 h of hypoxia/2 h of reoxygenation, MTT reduction levels declined to 50.1 +/- 5.5% (P < 0.05). MTT reduction levels in HPC (82.3 +/- 10.8%) and normoxic control (81.3 +/- 10.2%) groups were similar and higher (P < 0.05) than the 3 h of hypoxia/2 h of reoxygenation group (45.2 +/- 5.8%). Ultrastructural morphology was preserved in normoxic and HPC groups but not in the hypoxia/reoxygenation group. This is the first study to characterize a stable in vitro model of human skeletal muscle and to demonstrate a protective effect of HPC in human skeletal muscle against hypoxia/reoxygenation-induced injury.     

Hypoxia-induced skeletal muscle fiber dysfunction: role for reactive nitrogen species

Hypoxia impairs skeletal muscle function, but the precise mechanisms are incompletely understood. In hypoxic rat diaphragm muscle, generation of peroxynitrite is elevated. Peroxynitrite and other reactive nitrogen species have been shown to impair contractility of skinned muscle fibers, reflecting contractile protein dysfunction. We hypothesized that hypoxia induces contractile protein dysfunction and that reactive nitrogen species are involved. In addition, we hypothesized that muscle reoxygenation reverses contractile protein dysfunction. In vitro contractility of rat soleus muscle bundles was studied after 30 min of hyperoxia (Po2 approximately 90 kPa), hypoxia (Po2 approximately 5 kPa), hypoxia + 30 microM N(G)-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor), hyperoxia + 30 microM L-NMMA, and hypoxia (30 min) + reoxygenation (15 min). One part of the muscle bundle was used for single fiber contractile measurements and the other part for nitrotyrosine detection. In skinned single fibers, maximal Ca2+-activated specific force (Fmax), fraction of strongly attached cross bridges (alphafs), rate constant of force redevelopment (ktr), and myofibrillar Ca2+ sensitivity were determined. Thirty minutes of hypoxia reduced muscle bundle contractility. In the hypoxic group, single fiber Fmax, alphafs, and ktr were significantly reduced compared with hyperoxic, L-NMMA, and reoxygenation groups. Myofibrillar Ca2+ sensitivity was not different between groups. Nitrotyrosine levels were increased in hypoxia compared with all other groups. We concluded that acute hypoxia induces dysfunction of skinned muscle fibers, reflecting contractile protein dysfunction. In addition, our data indicate that reactive nitrogen species play a role in hypoxia-induced contractile protein dysfunction. Reoxygenation of the muscle bundle partially restores bundle contractility but completely reverses contractile protein dysfunction.     

Increased [Mg2+]o reduces Ca2+ influx and disruption of mitochondrial membrane potential during reoxygenation

Increase in extracellular Mg2+ concentration ([Mg2+]o) reduces Ca2+ accumulation during reoxygenation of hypoxic cardiomyocytes and exerts protective effects. The aims of the present study were to investigate the effect of increased [Mg(2+)](o) on Ca2+ influx and efflux, free cytosolic Ca2+ ([Ca2+]i) and Mg2+ concentrations ([Mg2+]i), Ca2+ accumulation in the presence of inhibitors of mitochondrial or sarcoplasmatic reticulum Ca2+ transport, and finally mitochondrial membrane potential (Delta(psi)m). Isolated adult rat cardiomyocytes were exposed to 1 h of hypoxia and subsequent reoxygenation. Cell Ca2+ was determined by 45Ca2+ uptake, and the levels of [Mg2+]i and [Ca2+]i were determined by flow cytometry as the fluorescence of magnesium green and fluo 3, respectively. Ca2+ influx rate was significantly reduced by approximately 40%, whereas Ca2+ efflux was not affected by increased [Mg2+]o (5 mM) during reoxygenation. [Ca2+]i and [Mg2+]i were increased at the end of hypoxia, fell after reoxygenation, and were unaffected by increased [Mg2+]o. Clonazepam, a selective mitochondrial Na+/Ca2+ exchange inhibitor (100 microM), significantly reduced Ca2+ accumulation by 70% and in combination with increased [Mg2+]o by 90%. Increased [Mg2+]o, clonazepam, and the combination of both attenuated the hypoxia-reoxygenation-induced reduction in Delta(psi)m, determined with the cationic dye JC-1 by flow cytometry. A significant inverse correlation was observed between Delta(psi)m and cell Ca2+ in reoxygenated cells treated with increased [Mg2+]o and clonazepam. In conclusion, increased [Mg2+]o (5 mM) inhibits Ca2+ accumulation by reducing Ca2+ influx and preserves Delta(psi)m without affecting [Ca2+]i and [Mg2+]i during reoxygenation. Preservation of mitochondria may be an important effect whereby increased [Mg2+]o protects the postischemic heart.     

Recovery of force during postcontractile depression in single Xenopus muscle fibers

This study examined the relationship between force and cytosolic free calcium concentration ([Ca2+]c) in different fiber types from Xenopus before, during, and after cells underwent postcontractile depression (PCD). During a standardized fatigue run, force in the two fast fatiguing (FF) fiber types (types 1 and 2, n = 10) fell more quickly (5.8 vs. 8.1 min) and to a greater degree [0.36 vs. 0.51 of initial (P(o))] than in the slow fatiguing (SF) fiber type (type 3, n = 11). After the initial fatigue run, both FF and SF experienced a drop in force to <15% P(o) (PCD) at a similar time (20.6 vs. 21.4 min). A second stimulation period, undertaken during PCD, produced significant recovery of force in both groups, but significantly more so in SF than FF (64 +/- 7 vs. 29 +/- 2% P(o)). This force recovery during PCD was accompanied by a significant increase in peak [Ca2+]c, particularly in SF. However, despite the significant recovery of force during stimulation while in PCD, the amount of force produced for a given peak [Ca2+]c was significantly lower in both groups during PCD than at any other point in the experiment. A final stimulation period, initiated when all fibers had recovered from PCD, demonstrated a recovery of both force and peak [Ca2+]c in both groups, but this recovery was significantly greater in SF vs. FF. These data demonstrate that with continuous electrical stimulation, it is possible to produce a significant recovery of force production during the normally quiescent period of PCD, but that it occurs with a decreased muscle force production for a given peak [Ca2+]c. This suggests that factors other than structural alterations of the sarcoplasmic reticulum are likely the cause of PCD in these fibers.     

Cytosolic Free Calcium and Gene Expression During Chemical Hypoxia

Understanding the cellular response to hypoxia may help elucidate the role of altered oxidation in neuronal death or abnormal cell function. In PC12 cells, 30 min of chemical hypoxia (i.e., KCN) reduced ATP concentrations by 92%, but diminished viability by only 10%. Ten minutes of hypoxia increased cytosolic free calcium ([Ca2+]i) 2.5-fold above control, but after 30 min of hypoxia, [Ca2+]i was slightly below that of nonhypoxic cells. Short periods of hypoxia also exaggerated the K(+)-induced elevation of [Ca2+]i, but by 30 min these ATP-depleted cells reestablished a calcium gradient that was equal to nonhypoxic, K(+)-depolarized cells. Thus, 30 min of severe ATP depletion left [Ca2+]i and viability relatively unaffected. Nerve growth factor caused slight, but significant, improvements in ATP and viability of hypoxic cells, but had no effect on [Ca2+]i. Although [Ca2+]i was equivalent in control and hypoxic cells after 30 or 60 min, hypoxia abolished the K(+)-stimulated elevation of [Ca2+]i. The nerve growth factor induction of c-fos, an indicator of the genomic response, was diminished by approximately 80%. Thus, hypoxic PC12 cells with greatly reduced ATP stores maintained normal [Ca2+]i, but their ability to respond to external stimulation was impaired. Further, the reduced oxidation that occurs in the brain in a variety of pathological conditions may interfere with the cellular response to stimulation and growth factors.     

Effect of hypoxia upon intracellular calcium concentration of human endothelial cells.

Ischemia is a situation occurring in several diseases including myocardial infarction and organ transplantation in which oxygenated blood supply is impaired. Ischemia leads to many cellular and tissue modifications, the most important one being cell death. Several explanations have been proposed to account for these modifications and cell death; among them is calcium overload. However, the influence of calcium concentration on the alteration of endothelial cell functions or viability during ischemia are still unknown. We developed here an in vitro model where human endothelial cell monolayers were submitted to hypoxia with or without reoxygenation and variation in calcium concentration was followed using a specific intracellular probe Fura 2. We observed a significant increase of [Ca2+]i during 2 h hypoxia reaching values similar to those observed during agonist stimulation of endothelial cells but far lower than values toxic for the cells. This increase was constant during the hypoxic incubation and was due mainly to an influx of extracellular calcium. Viability was also followed during hypoxia and using calcium channel blockers, we could show that there was no correlation between viability and the rise in calcium concentration. During the reoxygenation period, [Ca2+]i decreased to reach the normal value of resting cells after 45 min, suggesting that cells were still able to recover their calcium homeostasis. The use of a ketone body (beta-hydroxybutyrate) indicated that an energy deficiency was responsible for the hypoxia-induced increase in [Ca2+]i. We actually observed a 43% decrease in ATP concentration after 2 h hypoxia. This decrease was already significant after 30 min which thus precedes the changes in [Ca2+]i. These results show that during hypoxia, energy deficiency led to an increase in [Ca2+]i which is, however, too low to account for the loss of viability but which is within the range of concentrations observed during stimulation of endothelial cells. We propose that such increased intracellular calcium concentrations could play a role in the synthesis of mediators leading to the development of local inflammation.     

The use of Fura-2 to estimate myoplasmic [Ca2+] in human skeletal muscle

Fura-2 was used to estimate myoplasmic [Ca2+] in intact fibers and fiber segments from normal and diseased human muscles. Small muscle bundles (20-50 fibers) were loaded with the membrane-permeant form of the dye (Fura-2 AM). High-performance liquid chromatography was utilized to study the ability of these cells to hydrolyze Fura-2 AM. Immediately after the 30 min loading period, Fura-2 (the Ca2+ indicator) was the predominant form of the dye in all preparations and the concentration within these fibers remained stable for over 4 1/2 hours. In addition, the resting myoplasmic [Ca2+] in fiber segments from normal subjects and those susceptible to malignant hyperthermia were the same. However, halothane administration (1.5%) induced correlated increases in myoplasmic [Ca2+] and force only in fibers from the susceptible patients. In contrast, caffeine administration causes correlated increases in myoplasmic [Ca2+] and force in both types of muscle, but lower concentrations were needed to do so in the fibers from the susceptible patients. The effects of halothane and caffeine were reversible. We conclude that Fura-2 can be used successfully to estimate resting levels and changes in myoplasmic [Ca2+] in human skeletal muscle.     

Dynamics of lipid peroxidation of membranes in cells and mitochondrial fraction of neocortex in non- and preconditioned rats after severe hypobaric hypoxia

There was studied effect of severe hypobaric hypoxia and subsequent reoxygenation on level and dynamics of lipid peroxidation in membranes of neocortex cells and in mitochondriaenriched neocortex fraction of non-preconditioned rats and of rats preconditioned thrice with a moderate hypobaric hypoxia. The threefold hypoxic preconditioning increasing brain resistance has been shown to significantly prevent disturbance of lipid peroxidation processes in neocortex—one of the most hypoxia-sensitive brain structures—and to modify development of these processes in mitochondria.     

BHK cells transfected with NCX3 are more resistant to hypoxia followed by reoxygenation than those transfected with NCX1 and NCX2: Possible relationship with mitochondrial membrane potential

The specific role played by NCX1, NCX2, and NCX3, the three isoforms of the Na+/Ca2+ exchanger (NCX), has been explored during hypoxic conditions in BHK cells stably transfected with each of these isoforms. Six major findings emerged from the present study: (1) all the three isoforms were highly expressed on the plasma membranes of BHK cells; (2) under physiological conditions, the three NCX isoforms showed similar functional activity; (3) hypoxia plus reoxygenation induced a lower increase of [Ca2+]i in BHK-NCX3-transfected cells than in BHK-NCX1- and BHK-NCX2-transfected cells; (4) NCX3-transfected cells were more resistant to chemical hypoxia plus reoxygenation than NCX1- and NCX2-transfected cells. Interestingly, such augmented resistance was eliminated by CBDMD (10 microM), an inhibitor of NCX and by the specific silencing of the NCX3 isoform; (5) chemical hypoxia plus reoxygenation produced a loss of mitochondrial membrane potential in NCX1- and NCX2-transfected cells, but not in NCX3-transfected cells; (6) the forward mode of operation in NCX3-transfected cells was not affected by ATP depletion, as it occurred in NCX1- and NCX2-transfected cells. Altogether, these results indicate that the brain specifically expressed NCX3 isoform more significantly contributes to the maintenance of [Ca2+]i homeostasis during experimental conditions mimicking ischemia, thereby preventing mitochondrial delta psi collapses and cell death.     

缺氧诱导因子-1α在缺氧预处理预防心肌细胞损伤中的作用

本工作旨在研究缺氧预处理(hypoxic preconditioning,HPC)对于心肌细胞外信号调节激酶(extracellular signal-regulated proteinkinases,ERK)活性、缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)表达的影响,及其在缺氧复氧诱导心肌细胞损伤中的作用。通过在培养的SD乳鼠心肌细胞缺氧／复氧(H／R)模型上,观察HPC对于24h后H／R诱导心肌细胞损伤的影响,以台盼蓝排斥实验检测心肌细胞存活率、以TUNEL法检测细胞凋亡、并用荧光素染料Hoechst33258测定心肌细胞凋亡率：制备心肌细胞蛋白提取物,以磷酸化的ERK1／2抗体测定ERK1／2活性,以抗HIF-1α抗体检测HIF-1α的表达,并观察ERKs的上游激酶(MEK1／2)抑制剂PD98059对于HPC诱导的ERKs磷酸化、HIF-1α表达以及心肌细胞保护作用的影响,并分析细胞损伤与ERK1／2活性、HIF-1α表达量之间的相互关系。结果 显示缺氧复氧造成心肌细胞损伤,HPC可以增加心肌细胞H／R后存活率,降低凋亡率,并激活ERKll2,诱导HIF-1α表达：细胞凋亡与ERKs活性、HIF-1α表达量之间存在负相关,即ERKs活化、HIF-1α表达与预防细胞损伤有关：而ERKs活性与HIF-1α表达量之间存在正相关,ERKs的上游激酶MEK抑制剂PD98059可以消除HPC诱导的ERKs磷酸化、HIF-1α表达和心肌细胞保护作用。由此得出的结论是HPC可以提高乳鼠心肌细胞对于H／R的耐受性,其机制涉及ERKs介导的HIF-1α表达。     

Characterization of hypoxia-induced [Ca2+]i rise in rabbit pulmonary arterial smooth muscle cells

We have investigated the effects of hypoxia on the intracellular Ca2+ concentration ([Ca2+]i) in rabbit pulmonary (PASMCs) and coronary arterial smooth muscle cells with fura-2. Perfusion of a glucose-free and hypoxic (PO2<50 mmHg) external solution increased [Ca2+]i in cultured as well as freshly isolated PASMCs. However it had no effect on [Ca2+]i in freshly isolated coronary arterial myocytes. In the absence of extracellular Ca2+, hypoxic stimulation elicited a transient [Ca2+]i increase in cultured PASMCs which was abolished by the simultaneous application of cyclopiazonic acid and ryanodine, suggesting the involvement of sarcoplasmic reticulum (SR) Ca2+ store. Pretreatment with the mitochondrial protonophore, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) enhanced the [Ca2+]i rise in response to hypoxia. A short application of caffeine gave a transient [Ca2+]i rise which was prolonged by CCCP. Decay of the caffeine-induced [Ca2+]i transients was significantly slowed by treatment of CCCP or rotenone. After full development of the hypoxia-induced [Ca2+]i rise, nifedipine did not decrease [Ca2+]i. These data suggest that the [Ca2+]i increase in response to hypoxia may be ascribed to both Ca2+ release from the SR and the subsequent activation of nifedipine-insensitive capacitative Ca2+ entry. Mitochondria appear to modulate hypoxia induced Ca2+ release from the SR.     

Impairment of Ca(2+) release in single Xenopus muscle fibers fatigued at varied extracellular PO(2).

We tested the hypothesis that the mechanisms involved in the more rapid onset of fatigue when O(2) availability is reduced in contracting skeletal muscle are similar to those when O(2) availability is more sufficient. Two series of experiments were performed in isolated, single skeletal muscle fibers from Xenopus laevis. First, relative force and free cytosolic Ca(2+) concentrations ([Ca(2+)](c)) were measured simultaneously in single fibers (n = 6) stimulated at increasing frequencies (0.25, 0.33, 0.5, and 1 Hz) at an extracellular PO(2) of either 22 or 159 Torr. Muscle fatigue (force = 50% of initial peak tension) occurred significantly sooner (P < 0.05) during the low- (237 +/- 40 s) vs. high-PO(2) treatments (280 +/- 38 s). Relative [Ca(2+)](c) was significantly decreased from maximal values at the fatigue time point during both the high- (72 +/- 4%) and low-PO(2) conditions (78 +/- 4%), but no significant difference was observed between the treatments. In the second series of experiments, using the same stimulation regime as the first, fibers (n = 6) exposed to 5 mM caffeine immediately after fatigue demonstrated an immediate but incomplete relative force recovery during both the low- (89 +/- 4%) and high-PO(2) treatments (82 +/- 3%), with no significant difference between treatments. Additionally, there was no significant difference in relative [Ca(2+)](c) between the high- (100 +/- 12% of prefatigue values) and low-PO(2) treatments (108 +/- 12%) on application of caffeine. These results suggest that in isolated, single skeletal muscle fibers, the earlier onset of fatigue that occurred during the low-extracellular PO(2) condition was modulated through similar pathways as the fatigue process during the high and involved a decrease in relative peak [Ca(2+)](c).     

Contractile response of skeletal muscle to low peroxide concentrations: myofibrillar calcium sensitivity as a likely target for redox-modulation.

Endogenous peroxides and related reactive oxygen species may influence various steps in the contractile process. Single mouse skeletal muscle fibers were used to study the effects of hydrogen peroxide (H2O2) and t-butyl hydroperoxide (t-BOOH) on force and myoplasmic Ca2+ concentration ([Ca2+]i). Both peroxides (1010 to 105 M) decreased tetanic [Ca2+]i and increased force during submaximal tetani. Catalase (1 kU/ml) blocked the effect of H2O2, but not of t-BOOH. The decrease in tetanic [Ca2+]i was constant, while the effect on force was biphasic: A transitory increase was followed by a steady decline to the initial level. Myofibrillar Ca2+ sensitivity remained increased during incubation with either peroxide. Only the highest peroxide concentration (10 mM) increased resting [Ca2+]i and slowed the return of [Ca2+]i to its resting level after a contraction, evidence of impaired sarcoplasmic reticulum Ca2+ re-uptake. The peroxides increased maximal force production and the rate of force redevelopment, and decreased maximum shortening velocity. N-ethylmaleimide (25 mM, thiol-alkylating agent) prevented the response to 1 mM H2O2. These results show that myofibrillar Ca2+ sensitivity and cross-bridge kinetics are influenced by H2O2 and t-BOOH concentrations that approach those found physiologically, and these findings indicate a role for endogenous oxidants in the regulation of skeletal muscle function.     

Effects of Hypoxia on the Catecholamine Release, Ca2+ Uptake, and Cytosolic Free Ca2+ Concentration in Cultured Bovine Adrenal Chromaffin Cells

The purpose of the present study is to clarify the effects of hypoxia on catecholamine release and its mechanism of action. For this purpose, using cultured bovine adrenal chromaffin cells, we examined the effects of hypoxia on high (55 mM) K(+)-induced increases in catecholamine release, in cytosolic free Ca2+ concentration ([Ca2+]i), and in 45Ca2+ uptake. Experiments were carried out in media preequilibrated with a gas mixture of either 21% O2/79% N2 (control) or 100% N2 (hypoxia). High K(+)-induced catecholamine release was inhibited by hypoxia to approximately 40% of the control value, but on reoxygenation the release returned to control levels. Hypoxia had little effect on ATP concentrations in the cells. In the hypoxic medium, [Ca2+]i (measured using fura-2) gradually increased and reached a plateau of approximately 1.0 microM at 30 min, whereas the level was constant in the control medium (approximately 200 nM). High K(+)-induced increases in [Ca2+]i were inhibited by hypoxia to approximately 30% of the control value. In the cells permeabilized by digitonin, catecholamine release induced by Ca2+ was unaffected by hypoxia. Hypoxia had little effect on basal 45Ca2+ uptake into the cells, but high K(+)-induced 45Ca2+ uptake was inhibited by hypoxia. These results suggest that hypoxia inhibits high K(+)-induced catecholamine release and that this inhibition is mainly the result of the inhibition of high K(+)-induced increases in [Ca2+]i subsequent to the inhibition of Ca2+ influx through voltage-dependent Ca2+ channels.     

Prolonged force increase following a high-frequency burst is not due to a sustained elevation of [Ca2+]i

A brief high-frequency burst of action potentials results in a sustained force increase in skeletal muscle. The present study investigates whether this force potentiation is the result of a sustained increase of the free myoplasmic [Ca2+] ([Ca2+]i). Single fibers from mouse flexor brevis muscles were stimulated with three impulses at 150 Hz (triplet) at the start of a 350-ms tetanus or in the middle of a 700-ms tetanus; the stimulation frequency of the rest of the tetanus ranged from 20 to 60 Hz. After the triplet, force was significantly (P < 0.05) increased between 17 and 20% when the triplet was given at the start of the tetanus and between 5 and 18% when the triplet was given in the middle (n = 7). However, during this potentiation, [Ca2+]i was not consistently increased. Hence, the increased force following a high-frequency burst is likely due to changes in the myofibrillar properties.     

Role of mitochondrial reactive oxygen species in hypoxia-dependent increase in intracellular calcium in pulmonary artery myocytes

Previous studies examining the role of mitochondria-derived reactive oxygen species (ROS) in hypoxic responses have been mainly conducted in isolated lungs and cultured pulmonary artery smooth muscle cells (PASMCs) using mitochondrial inhibitors, and yielded largely conflicting results. Here we report that in freshly isolated mouse PASMCs, which are devoid of the mixed responses from multi-types of cells in lungs and significant changes in gene expression in cultured cells, the mitochondrial electron transport chain (ETC) complex I, II, or III inhibitors blocked hypoxia-induced increases in intracellular ROS and Ca2+ concentration ([ROS]i and [Ca2+]i) without effects on their resting levels. Inhibition of the complex I plus II and/or III did not produce an additive effect. Glutathione peroxidase-1 (Gpx1) or catalase gene overexpression to enhance H2O2 removal remarkably reduced hypoxic increases in [ROS]i and [Ca2+]i, whereas Gpx1 gene deletion had the opposite effect. None of these genetic modifications changed the resting [ROS]i and [Ca2+]i. H2O2 at 51 microM caused a similar increase in DCF fluorescence ([ROS]i) as that by hypoxia, but only induced 33% of hypoxic increase in [Ca2+]i. Moreover, H2O2 (5.1 microM) reversed the inhibition of the hypoxia-induced increase in [Ca2+]i by rotenone. Collectively, our study using various mitochondrial inhibitors and genetic approaches demonstrates that in response to acute hypoxia, the mitochondrial ETC molecules prior to the complex III ubisemiquinone site act as a functional unit to increase the generation of ROS, particularly H2O2, which is important for, but may not fully cause, the hypoxic increase in [Ca2+]i in freshly isolated PASMCs.     

NHE and ICAM-1 expression in hypoxic/reoxygenated coronary microvascular endothelial cells

Although Na+/H+ exchange (NHE) has been implicated in myocardial reperfusion injury, participation of coronary microvascular endothelial cells (CMECs) in this pathogenesis has been poorly understood. NHE-induced intracellular Ca2+ concentration ([Ca2+]i) overload in CMECs may increase the synthesis of intercellular adhesion molecules (ICAM), which is potentially involved in myocardial reperfusion injury. The present study tested the hypothesis that NHE plays a crucial role in [Ca2+]i overload and ICAM-1 synthesis in CMECs. Primary cultures of CMECs isolated from adult rat hearts were subjected to acidic hypoxia for 30 min followed by reoxygenation. Two structurally distinct NHE inhibitors, cariporide and 5-(N-N-dimethyl)-amiloride (DMA), had no significant effect on the acidic hypoxia-induced decrease in intracellular pH (pH(i)) of CMECs but significantly retarded pH(i) recovery after reoxygenation. These NHE inhibitors abolished the hypoxia- and reoxygenation-induced increase in [Ca2+]i. Expression of ICAM-1 mRNA was markedly increased in the vehicle-treated CMECs 3 h after reoxygenation, and this was significantly inhibited by treatment with cariporide, DMA, or Ca2+-free buffer. In addition, enhanced ICAM-I protein expression on the cell surface of CMECs 8 h after reoxygenation was attenuated by treatment with cariporide, DMA, or Ca2+-free buffer. These results suggest that NHE plays a crucial role in the rise of [Ca2+]i and ICAM-1 expression during acidic hypoxia/reoxygenation in CMECs. We propose that inhibition of ICAM-1 expression in CMECs may represent a novel mechanism of action of NHE inhibitors against ischemia-reperfusion injury.     

Investigation of factors affecting fluorometric quantitation of cytosolic [Ca2+] in perfused hearts.

The goal of these studies was to examine the effects of several factors that may artifactually influence quantitation of cytosolic [Ca2+], [Ca2+]c, while using the fluorescent calcium indicator Indo-1. The following factors were investigated: 1) a possible fluorescence contribution from unhydrolized Indo-1/AM (by Mn2+ quenching), 2) Ca2+ buffering by Indo-1 (by varying [Indo-1]), 3) endothelial and mitochondrial Indo-1 loading (by bradykinin stimulation and calculations), and 4) effects of changing tissue fluorescence (predominantly NAD(P)H) on calculated [Ca2+]c during hypoxia (by a new method which allowed simultaneous determination of [Ca2+]c and changes in [NAD(P)H]). No significant contribution of Indo-1/AM was found. With increasing [Indo-1], calculated systolic [Ca2+]c fell significantly. Indo-1 incorporation (< 18%) into endothelial cells, caused a slight underestimation of systolic [Ca2+]c, while mitochondrial Indo-1 loading may cause overestimation of [Ca2+]c. With increased tissue fluorescence, during hypoxia, systolic [Ca2+]c may be underestimated by approximately 27% (for Indo-1 loading factors three to five times original tissue fluorescence). These studies suggest conditions in which experimental artifacts could be minimized to allow reliable quantitation of [Ca2+]c in intact perfused hearts using Indo-1 fluorometry. The major problem of obtaining reliable results depended on the ability to correct for changing NAD(P)H fluorescence while keeping [Indo-1] low.