Early discharge of patients with new-onset atrial fibrillation after cardiovascular surgery

The feasibility of monitoring intracellular sodium changes using Na triple quantum filtered NMR without a chemical shift reagent (SR) was investigated in an isolated rat heart during a variety of interventions for Na(i) loading. Perfusion with 1 mM ouabain or without K+ present in the perfusate for 30 min produced a rise of the Na TQF signal with a plateau of approximately 190% and approximately 228% relative to the preintervention level, respectively. Stop-flow ischemia for 30 min resulted in a TQF signal growth of approximately 147%. The maximal Na TQF signal increase of 460% was achieved by perfusion without K+/Ca2+, corresponding to an elimination of the Na transmembrane gradient. The observed values of Na NMR TQF growth in the physiological and pathological ranges are in agreement with reported data by other methods and have a linear correlation with intracellular sodium content as determined in this study by Co-EDTA method and by sucrose-histidine washout of the extracellular space. Our data indicate that the increase in Na TQF NMR signal is determined by the growth of Na(i), and the extracellular Na contribution to the total TQF signal is unchanged at approximately 64%. In conclusion, Na TQF NMR without using SR offers a unique and noninvasive opportunity to monitor alterations of intracellular sodium. It may provide valuable insights for developing cardioprotective strategies and for observing the effects of pharmaceutical treatments on sodium homeostasis.     

In situ remineralization of subsurface enamel lesion after the use of a fluoride chewing gum

The intracellular sodium of the perfused rat mandibular salivary gland was measured by double-quantum filtered 23Na-NMR spectroscopy at 2.34, 4.7 and 8.45 T. Biexponential relaxation of the intracellular 23Na signal was observed, and its intensity was increased by administration of acetylcholine with ouabain at 25 degrees C. The transverse and longitudinal relaxation rate constants were determined by the 'transverse experiment' (D-90 degrees-tau/2-180 degrees-tau/2-90 degrees-delta-90 degrees-acquire) and the 'longitudinal experiment' (D-180 degrees-tau-54.7 degrees-delta-90 degrees-acquire), respectively. From observed dependencies on B0 and temperature (5-37 degrees C), a possibility of exchange between two populations of intracellular Na+ was suggested. A small fraction of Na+ is in the slow-motion condition (with a quadrupole coupling constant of approx. 1.75 MHz and a correlation time of 6 x 10(-8) s). The major portion of intracellular Na+ is in the extreme narrowing condition with a transverse relaxation rate constant of approx. 100 s-1, which corresponds to a viscosity of approx. 5 cP.     

Ischemic preconditioning: effects on pH, Na and Ca in newborn rabbit hearts during Ischemia/Reperfusion

In adult hearts, ischemic preconditioning (PC) has been shown to decrease ischemia-induced changes in intracellular pH (pHi) and [Ca] ([Ca]i) and decrease associated injury. These results are consistent with the interpretation that PC decreases the stimulus for Na uptake via Na/H exchange, thereby decreasing intracellular Na (Nai) accumulation, and thus decreasing the change in force driving Na/Ca exchange, which otherwise contributes to ischemia-induced increases in [Ca]i. Given documented age-related differences in myocardial responses to ischemia, we tested the hypothesis that in newborn hearts, PC will diminish intracellular [H], Nai, and [Ca]i during ischemia/reperfusion. NMR was used to measure pHi, Nai, [Ca]i, ATP, and PCr in isolated newborn (4-7 days) rabbit hearts Langendorff-perfused with Krebs-Henseleit solution equilibrated with 95% O2/5% CO2 at 36+/-1 degrees C. Control hearts were perfused 30 min before initiating 40 min global ischemia followed by 40 min reperfusion. PC hearts were treated the same except four 5-min intervals of ischemia each followed by 10 min of perfusion which preceded global ischemia. At end ischemia, pHi was higher in PC than control hearts (6.31+/-0.03 v 5.83+/-0.05; P<0.05). Similarly, PC diminished Nai-accumulation during ischemia and reperfusion (P<0.05). Control Nai rose from 16.2+/-2.6 to 108.8+/-10.3 (mEq/kg dry weight) and recovered to 55.2+/-10.1 and the corresponding values for PC hearts were 25.6+/-6.2, 70.0+/-7.9 and 21.9+/-5.2. PC also improved [Ca]i recovery during reperfusion (P<0.05). Control [Ca]i rose from 418+/-43 to 1100+/-78 (nm/l) and recovered to 773+/-63, whereas in PC hearts the values were 382+/-40, 852+/-136 and 371+/-45, respectively. In addition, PC decreased coronary resistance during reperfusion (P<0.05) as reflected by lower perfusion pressures under constant flow conditions (65.9+/-1.5 v 56. 1+/-4.1 mmHg at end of reperfusion). Finally, PC improved recovery of left-ventricular developed pressure (LVDP-43.8+/-12.0 v 17.2+/-3. 0% of control; P<0.05) and diminished CK release (607+/-245 v 2432+/-639 IU/g dry weight; P<0.05) during reperfusion. The results are consistent with the hypothesis.     

Is a high glycogen content beneficial or detrimental to the ischemic rat heart? A controversy resolved

A high glycogen level may be beneficial to the ischemic heart by providing glycolytic ATP or detrimental by increasing intracellular lactate and protons. To determine the effect of high glycogen on the ischemic myocardium, the glycogen content of Langendorff-perfused rat hearts was either depleted or elevated before 32 minutes of low-flow (0.5 mL/min) ischemia with Krebs-Henseleit buffer with or without 11 mmol/L glucose, followed by 32 minutes of reperfusion with buffer containing 11 mmol/L glucose. 31P nuclear magnetic resonance spectra were acquired sequentially throughout. Further experiments involved early reperfusion or the addition of HOE 694, a Na+-H+ exchange inhibitor, during reperfusion. When glucose was supplied throughout ischemia, no ischemic contracture occurred, and postischemic recovery of contractile function was highest, at 88% of preischemic function. In the absence of glucose, normal-glycogen hearts underwent ischemic contracture at 5 minutes, had an end-ischemic pH of 6.87, and recovered to 54%, whereas in high-glycogen hearts, contracture was delayed to 13 minutes, the end-ischemic pH was 6.61, and functional recovery decreased to 13%. Contracture onset coincided with the decrease in glycolysis, which occurred as glycogen became fully depleted. Functional recovery in the high-glycogen hearts increased to 89% when reperfused before contracture and to 56% when reperfused in the presence of HOE 694. Thus, during brief ischemia in the high-glycogen hearts, ischemic glycogen depletion and contracture were avoided, and the hearts were protected from injury. In contrast, during prolonged ischemia in the high-glycogen hearts, glycogen became fully depleted, and myocardial injury occurred; the injury was exacerbated by the lower ischemia pH in these hearts, leading to increased Na+-H+ exchange during reperfusion. The contradictory findings of past studies concerning the effect of high glycogen on the ischemic myocardium may thus be due to differences in the extent of glycogen depletion during ischemia.     

Overexpression of the cardiac Na+/Ca2+ exchanger increases susceptibility to ischemia/reperfusion injury in male, but not female, transgenic mice

Influx of Ca2+ into myocytes via Na+/Ca2+ exchange may be stimulated by the high levels of intracellular Na+ and the changes in membrane potential known to occur during ischemia/reperfusion. This increased influx could, in turn, lead to Ca2+ overload and injury. Overexpression of the cardiac Na+/Ca2+ exchanger therefore may increase susceptibility to ischemia/reperfusion injury. To test this hypothesis, the hearts of male and female transgenic mice, overexpressing the Na+/Ca2+ exchange protein, and hearts of their wild-type littermates, were perfused with Krebs-Henseleit buffer and subjected to 20 minutes of ischemia and 40 minutes of reperfusion. Preischemic left ventricular developed pressures and +dP/dtmax, as well as -dP/dtmin, were higher in the male transgenic hearts compared with wild-type, implying a role for Na+/Ca2+ exchange in the contraction, as well as the relaxation, phases of the cardiac beat. Postischemic function was lower in male transgenic than in male wild-type hearts (7+/-2% versus 32+/-6% of preischemic function), but there was no difference between female transgenic and female wild-type hearts, both at approximately 30% of preischemic function. To assess whether this male/female difference was due to female-specific hormones such as estrogen, the hearts of bilaterally ovariectomized and sham-operated transgenic females were subjected to the same protocol. The functional recoveries of ovariectomized female transgenic hearts were lower (17+/-3% of preischemic function) than those of wild-type and sham-operated transgenic females. The lower postischemic functional recovery in the male transgenic and female ovariectomized transgenic hearts correlated with lower recoveries of the energy metabolites, ATP and phosphocreatine, as measured by 31P nuclear magnetic resonance spectroscopy. Alternans were observed during reperfusion in male transgenic and female ovariectomized transgenic hearts only, consistent with intracellular Ca2+ overload. Western analyses showed that alterations in the expression of the Na+/Ca2+ exchange or L-type Ca2+ channel proteins were not responsible for the protection observed in the female transgenic hearts. In conclusion, in males, overexpression of the Na+/Ca2+ exchanger reduced postischemic recovery of both contractile function and energy metabolites, indicating that the Na+/Ca2+ exchanger may play a role in ischemia/reperfusion injury. From the studies of females, however, it appears that this exacerbation of ischemia/reperfusion injury by overexpression of the Na+/Ca2+ exchanger can be overcome partially by female-specific hormones such as estrogen.     

Post-ischemic stimulation of 2-deoxyglucose uptake in rat myocardium: role of translocation of Glut-4

Myocardial ischemia elicits translocation of the insulin-sensitive glucose transporter GLUT-4 from intracellular membrane stores to the sarcolemma. Because glucose metabolism is of crucial importance for post-ischemic recovery of the heart, myocardial uptake of [3H]-labeled 2-deoxyglucose and subcellular localization of GLUT-4 were determined during reperfusion in isolated rat hearts perfused with medium containing 0.4 mm palmitate and 8 mm glucose. Hearts were subjected to 20 min of no-flow ischemia, followed by reperfusion for up to 60 min. Subcellular localization of GLUT-4 was determined by cell fractionation followed by immunoblotting. After 15 and 60 min of reperfusion uptake of 2-deoxyglucose was significantly higher (91+/-9 and 96+/-8 nmol/min/g wet weight, respectively) as compared to control values (65+/-1 nmol/min/g wet weight). Ischemia elicited translocation of GLUT-4 to the sarcolemma, which persisted after 15 min of reperfusion. However, after 60 min of reperfusion the subcellular distribution of GLUT-4 was similar to control hearts. In conclusion, reversal of ischemia-induced translocation of GLUT-4 to the sarcolemma is rather slow, possibly facilitating glucose uptake early during reperfusion. However, myocardial uptake and phosphorylation of 2-deoxyglucose remains enhanced late during reperfusion, when pre-ischemic distribution of GLUT-4 is almost completely restored, indicating that additional mechanisms are likely to be involved in post-ischemic stimulation of glucose uptake.     

Pathways of Rb+ influx and their relation to intracellular [Na+] in the perfused rat heart. A 87Rb and 23Na NMR study

The aims of this study were to characterize the routes of influx of the K+ congener, Rb+, into cardiac cells in the perfused rat heart and to evaluate their links to the intracellular Na+ concentration ([Na+]i) using 87Rb and 23Na nuclear magnetic resonance (NMR) spectroscopy. The rate constant for Rb+ equilibration in the extracellular space was 8.5 times higher than that for the intracellular space. The sensitivity of the rate of Rb+ accumulation in the intracellular space of the perfused rat heart to the inhibitors of the K+ and Na+ transport systems has been analyzed. The Rb+ influx rates were measured in both beating and arrested hearts: both procaine (5 mmol/L) and lidocaine (1 mmol/L) halved the Rb+ influx rate. In procaine-arrested hearts, the Na+,K(+)-ATPase inhibitor ouabain (0.6 mmol/L) decreased Rb+ influx by 76 +/- 24% relative to that observed in untreated but arrested hearts. Rb+ uptake was insensitive to the K+ channel blocker 4-aminopyridine (1 mmol/L). The inhibitor of Na+/K+/2 Cl- cotransport bumetanide (30 mumol/L) decreased Rb+ uptake only slightly (by 9 +/- 8%). Rb+ uptake was dependent on [Na+]i: it increased by 58 +/- 34% when [Na+]i was increased with the Na+ ionophore monensin (1 mumol/L) and decreased by 48 +/- 9% when [Na+]i was decreased by the Na+ channel blockers procaine and lidocaine. Dimethylamiloride (15 to 20 mumol/L), an inhibitor of the Na+/H+ exchanger, slightly reduced [Na+]i and Rb+ entry into the cardiomyocytes (by 15 +/- 5%). 31P NMR spectroscopy was used to monitor the energetic state and intracellular pH (pHi) in a parallel series of hearts. Treatment of the hearts with lidocaine, 4-aminopyridine, dimethylamiloride, or bumetanide for 15 to 20 minutes at the same concentrations as used for the Rb+ and Na+ experiments did not markedly affect the levels of the phosphate metabolites or pHi. These data show that under normal physiological conditions, Rb+ influx occurs mainly through Na+,K(+)-ATPase; the contribution of the Na+/K+/2 Cl- cotransporter and K+ channels to Rb+ influx is small. The correlation between Rb+ influx and [Na+bdi during infusion of drugs that affect [Na+]i indicates that, in rat hearts at 37 degrees C, Rb+ influx can serve as a measure of Na+ influx. We estimate that, at normothermia, at least 50% of the Na+ entry into beating cardiac cells is provided by the Na+ channels, with only minor contributions (< 15%) from the Na+/K+/2 Cl- cotransporter and the Na+/H+ exchanger.     

Importance of diabetes mellitus and systemic hypertension rather than completeness of revascularization in determining long-term outcome after coronary balloon angioplasty (the LDCMC registry). Lady Davis Carmel Medical Center

To characterize KC 12291 (1-(5-phenyl-1,2, 4-thiadiazol-3-yl-oxypropyl)-3-[N-methyl-N-[2-(3,4-dimethoxy phenyl) ethyl] amino] propane hydrochloride), a newly synthezised inhibitor of voltage-gated Na+ channels, the effects of the agent on Na+ current and ischemia-induced Na+ overload were investigated in isolated cardiomyocytes, atria and saline-perfused hearts. As measured by the patch clamp technique, KC 12291 (1 microM) significantly reduced peak Na+ current after activation of voltage-gated Na+ channels in rat cardiomyocytes. Partial depolarization enhanced the inhibitory effects during steady state conditions of the channel. In isolated guinea pig atria, 1 microM KC 12291 had no effect on contractility under basal conditions but effectively delayed the onset and reduced the extent of anoxic contracture. The concentration-response curve was clearly shifted to the left when atria were partially depolarized by increased extracellular K+. As measured by 23Na NMR spectroscopy in isolated perfused guinea pig hearts, intracellular Na+ rose more than four-fold in a linear fashion during 60 min of low-flow ischemia. KC 12291 (1 microM) prevented Na+ overload within the initial 12 min of ischemia; thereafter the slope of Na+ accumulation was identical to controls. Electrical excitability of hearts, evaluated by intracardial ECG, completely ceased within 15 min after the onset of ischemia. KC 12291 (1 microM) accelerated this process by more than 6 min. The data provide first evidence that KC 12291 reduces Na+ influx through voltage-gated Na+ channels during ischemia and thus delays Na+ overload by enhancing the inexcitability of the heart.     

Screening a peptidyl database for potential ligands to proteins with side-chain flexibility

OBJECTIVE: Microdialysis and 31P-NMR spectroscopy were used to test opposing hypotheses that ischemic preconditioning inhibits adenine nucleotide degradation and purine efflux, or that preconditioning activates cardiovascular adenosine formation to provide enhanced cardioprotection. METHODS: 31P-NMR spectra and matching interstitial fluid (ISF) or venous effluent samples were obtained from Langendorff perfused rat hearts. Control hearts (n = 9) underwent 30 min of global normothermic ischemia and 30 min reperfusion. Preconditioned hearts (n = 6) were subjected to a 5 min ischemic episode and 10 min reflow prior to 30 min ischemia and 30 min reperfusion. Effects of repetitive ischemia-reperfusion (3 x 5 min ischemic episodes) on adenosine levels and energy metabolism were also assessed (n = 8). RESULTS: Preconditioning improved post-ischemic recovery of heart rate x left ventricular developed pressure (71 +/- 5 vs 43 +/- 8%, P < 0.05) and end-diastolic pressure (14 +/- 3 vs 29 +/- 4 mmHg, P < 0.05) compared with control hearts, respectively. Preconditioning did not alter intracellular ATP, phosphocreatine (PCr), inorganic phosphate (Pi), H+ or free Mg2+ during global ischemia, but improved recoveries of PCr, Pi, and delta GATP on reperfusion. ISF adenosine increased more than 20-fold during 30 min ischemia. The 5 min preconditioning episode increased ISF adenosine 3-fold, and reduced ISF adenosine and inosine during subsequent prolonged ischemia by up to 75%. Venous purine levels during reperfusion were also reduced by preconditioning. Accumulation of adenosine in ISF and venous effluent during repetitive ischemia was progressively reduced despite comparable changes in substrate for adenosine formation via 5'-nucleotidase, (5'-AMP), and in allosteric modulators of this enzyme (Mg2+, H+, Pi, ADP, ATP). CONCLUSIONS: (i) Ischemic preconditioning reduces interstitial and vascular adenosine levels during ischemia-reperfusion, (ii) reduced ISF adenosine during ischemia is not due to reduced ischemic depletion of adenine nucleotides in preconditioned rat hearts, (iii) preconditioning may inhibit adenosine formation via 5'-nucleotidase in ischemic rat hearts, and (iv) improved functional recovery with preconditioning is unrelated to metabolic/bioenergetic changes during the ischemic insult, but may be related to improved post-ischemic recovery of [Pi] and delta GATP in this model.     

The blood contribution to early myocardial reperfusion injury is amplified in diabetes

Cardiovascular disease is excessive in diabetes, and blood cell function is altered. It is not clear, however, if alterations in the blood contribute to the excessive cardiovascular complications of this disease. In this study, we compared the contribution of nondiabetic and diabetic blood to myocardial reperfusion injury. The recovery of cardiac contractile function following no-flow ischemia was studied in isolated diabetic and nondiabetic rat hearts perfused with diabetic or nondiabetic diluted whole blood. Hearts were isolated from 10- to 12-week-old diabetic (streptozotocin, 65 mg/kg, i.v.) and nondiabetic rats and perfused with a Krebs-albumin-red cell solution (K2RBC, Hct 20%). After a 30-min pre-ischemic control period, during which cardiac pump function was evaluated, diabetic and nondiabetic hearts were perfused for 5 min with diluted whole blood (DWB; Hct 20%) collected from either diabetic or nondiabetic donor animals. Coronary flow was then stopped and the hearts subjected to 30 min of no-flow ischemia. Following ischemia, the hearts were reperfused with the K2RBC perfusate. Cardiac contractile function was evaluated throughout the 60-min reperfusion period. Six groups were studied: diabetic and nondiabetic hearts perfused before ischemia with either K2RBC, nondiabetic DWB (NDDWB), or diabetic DWB (DDWB). Perfusion with DWB prior to ischemia impaired the recovery of contractile function in all cases. The impairment to recovery was greater with DDWB than with NDDWB. Although diabetic hearts perfused with K2RBC throughout recovered quite well, the effect of DDWB perfusion in the diabetic hearts was dramatic. In an effort to determine why diabetic blood impaired functional recovery, measures of blood filterability and the generation of reactive oxygen species (ROS) were made. We found that diabetic blood was less filterable than nondiabetic blood; that is, the diabetic blood cells tended to plug the 5-microm filter pores more readily than the nondiabetic blood cells. Also, we found that the diabetic blood was capable of generating significantly greater ROS (oxygen free radicals) than nondiabetic blood (P < 0.05). These findings suggest that the blood contribution to myocardial reperfusion injury is amplified in diabetes. A tendency for diabetic blood cells to plug capillary-sized pores and show enhanced oxygen free radical production may account for the excessive contribution of diabetic blood to reperfusion injury in the heart.     

Potentiation of histamine-induced catecholamine secretion by ouabain in cultured bovine adrenal chromaffin cells is dependent on calcium and sodium influx

The effects of histamine on catecholamine secretion from cultured bovine adrenal chromaffin cells were studied in the presence of ouabain, an inhibitor of Na+-K+ ATPase. The purpose of this study was to determine whether Na+, as well as Ca2+, was involved in histamine receptor-mediated catecholamine secretion. Histamine (10(-8)-10(-5) M)-induced catecholamine secretion was markedly potentiated by addition of ouabain (10(-5) M) and was inhibited by a histamine-H1 receptor antagonist or incubation in a Ca2+-free medium. Histamine-induced 45Ca2+ influx was also potentiated by addition of ouabain. Ouabain alone or in the presence of histamine increased 22Na+ influx into the cells. In an additional set of experiments, cells were preincubated in the presence or absence of Na+ for 30 min (+/- histamine and ouabain), washed and then catecholamine secretion was measured following exposure to 2.2 mM Ca2+ for 15 min. Preincubation with histamine alone with or without Na+ had no effect of Ca2+-induced secretion of catecholamine. Preincubation with ouabain alone or with ouabain plus histamine produced a slight stimulation of catecholamine secretion in Na+-free medium and a large stimulation in Na+-containing medium. These results suggested that stimulation of the histamine-H1 receptor and inhibition of the Na+ pump both increase intracellular Na+ levels, resulting in increases in Ca2+ influx and catecholamine secretion.     

Generalized peroxisomal disorder in male twins: fatty acid composition of serum lipids and response to n-3 fatty acids

Ischemia leads to intracellular acidification which can be counteracted by the Na+/H+-exchange mechanism. A blockade of this exchanger has been hypothesized to cause stronger intracellular acidification in the course of ischemia thereby protecting the heart from ischemic damage. The aim of our study was to find out (1) whether in the course of ischemia areas become electrically silent, (2) whether this is enhanced by the Na+/H+-exchange inhibitor cariporide (4-Isopropyl-3-methylsulfonylbenzoyl-guanidine; Hoe 642) and whether cariporide has protective effects. Therefore, we submitted isolated rabbit hearts, perfused according to the Langendorff technique to regional ischemia (LAD occlusion) for 30 min followed by 30 min reperfusion with (n=7) or without (n=7) pre-treatment with 1 microM cariporide. Under these conditions 256-channel epicardial potential mapping was carried out. Under non-ischemic conditions cariporide did not alter any of the parameters under observation. We found that ischemia led to marked alterations of the activation pattern, to action potential shortening and a marked increase in the dispersion of refractoriness. In the ischemic region there was a significant ST deviation from the isoelectrical line (control 32+/-10; 30 min ischemia: 290+/-35 arbitrary units [a.u.]). This was markedly reduced by cariporide (control 39+/-10; 30 min ischemia: 170+/-25 a.u.). The increase in dispersion by ischemia (by 50+/-5 ms) was significantly counteracted by cariporide (increased dispersion by 20+/-4 ms). In a similar way the alteration of the activation pattern was antagonized. Under the influence of cariporide we found a lower increase in the left ventricular enddiastolic pressure, and a significantly slower recovery of the action potential duration. After 30 min of ischemia 24+/-5 (control series) 24.5+/-5 mm2 (cariporide) became electrically silent. In a second series of experiments the incidence of arrhythmia was assessed: we found ventricular fibrillation in 6/7 untreated control hearts and in 4/7 cariporide treated hearts. In a third series of experiments we determined the intracellular [ATP] after 30 min of LAD occlusion using a histochemical method. We observed a decrease in [ATP] in the ischemic region as compared to the non-ischemic right ventricular wall, which was less pronounced in cariporide-treated hearts. Thus, we conclude that (1) cariporide protects the heart from ischemic damage and (2) at least under these conditions an enlargement of the electrically silent area did not occur.     

Effects of glucose deprivation, chemical hypoxia, and simulated ischemia on Na+ homeostasis in rat spinal cord astrocytes

A steep inwardly directed Na+ gradient is essential for glial functions such as glutamate reuptake and regulation of intracellular ion concentrations. We investigated the effects of glucose deprivation, chemical hypoxia, and simulated ischemia on intracellular Na+ concentration ([Na+]i) in cultured spinal cord astrocytes using fluorescence ratio imaging with sodium-binding benzofuran isophthalate (SBFI) AM. Glucose removal or chemical hypoxia (induced by 10 mM NaN3) for 60 min increased [Na+]i from a baseline of 8.3 to 11 mM. Combined glycolytic and respiratory blockage by NaN3 and 0 glucose saline caused [Na+]i to increase by 20 mM, similar to the [Na+]i increases elicited by blocking the Na+/K+-ATPase with ouabain. Recovery from large [Na+]i increases (>15 mM) induced by the glutamatergic agonist kainate was attenuated during glucose deprivation or NaN3 application and was blocked in NaN3 and 0 glucose. To mimic in vivo ischemia, we exposed astrocytes to NaN3 and 0 glucose saline containing L-lactate and glutamate with increased [K+] and decreased [Na+], [Ca2+], and pH. This induced an [Na+]i decrease followed by an [Na+]i rise and a further [Na+]i increase after reperfusion with standard saline. Similar multiphasic [Na+]i changes were observed after NaN3 and 0 glucose saline with only reduced [Na+]e. Our results suggest that the ability to maintain a low [Na+]i enables spinal cord astrocytes to continue uptake of K+ and/or glutamate at the onset of energy failure. With prolonged energy failure, however, astrocytic [Na+]i rises; with loss of their steep transmembrane Na+ gradient, astrocytes may aggravate metabolic insults by carrier reversal and release of acid, K+, and/or glutamate into the extracellular space.     

Inhibition of Na+/K+ ATPase under hypertonic conditions in rat mast cells

Ionic fluxes that contribute to changes in membrane potential and variations of pHi (intracellular pH) are not well known in mast cells, although they can be important in the stimulus-secretion coupling. Cellular volume regulation implies changes in the concentration of intracellular ions, such as sodium and potassium and volume changes can be imposed varying the tonicity of the medium. We studied the physiology of sodium and examined the effect of ouabain on [22Na] entry in mast cells in isotonic and hypertonic media. We also recorded changes in membrane potential and pHi using the fluorescent dyes bis-oxonol (Bis-(1,3-diethylthiobarbituric acid) trimethineoxonol) a n d BCECF (2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester) in hypertonic conditions. The results show that [22Na] influx increases four fold in hypertonic solutions and it is mediated mainly by an amiloride-sensitive Na+/H+ exchanger. This transporter is involved in the shrinkage-activated cellular alkalinization and the pHi recovery is accelerated by inhibition of the Na+/K+ ATPase with ouabain in the absence of extracellular calcium. Under hypertonic conditions 22Na influx is apparently not increased by ouabain, while the Na+/K+ ATPase inhibitor clearly increases [22Na] uptake and also induces membrane depolarization in isotonic conditions. All together, these findings suggest that Na+/K+ ATPase is partially inhibited in hypertonic conditions.     

Cytokine and lipid mediator blood concentrations after coronary artery surgery

The redox state of the carriers of electron-transport chain of cardiac mitochondria was studied in the conditions of normal perfusion, global ischemia and reoxygenation of the myocardial tissue. Experiments were performed on isolated rat hearts perfused at 37 degrees C by the "working heart" procedure. The EPR spectra of the freeze-clamped hearts were measured at 6-30 K. An analysis of the main values of g-tensor, line-shape, line-width and relaxation parameters of the components of low-temperature EPR spectra allowed to distinguish the signals from Fe-S centers of NADH-CoQ reductase and succinate-CoQ reductase, and the signals from free radical species of coenzyme Q and flavin coenzymes. The EPR spectra of hearts that were fixed during control perfusion and reperfusion contained predominantly the signal of oxidized S3 center of succinate-CoQ reductase. The free radical signal in these conditions was mainly due to ubisemiquinones. Besides the intensive signal of S3 center, the low-temperature EPR spectra contained also signals from different Fe-S centers paramagnetic in reduced state. The global ischemia of cardiac muscle caused essential reduction of the Fe-S clusters of the mitochondrial electron-transport chain. In ischemic condition the free radical EPR signal was mainly due to flavosemiquinones. The changes of the redox state of carriers of the mitochondrial respiratory chain correlated with the changes of the physiological parameters of cardiac muscle.     

Relationship between Na+-Ca2+-exchanger protein levels and diastolic function of failing human myocardium

BACKGROUND: In the failing human heart, sarcoplasmic reticulum (SR) calcium handling is impaired, and therefore, calcium elimination and diastolic function may depend on the expression of sarcolemmal Na+-Ca2+ exchanger. METHODS AND RESULTS: Force-frequency relations were studied in ventricular muscle strip preparations from failing human hearts (n=29). Protein levels of Na+-Ca2+ exchanger and SR Ca2+-ATPase were measured in the same hearts. Hearts were divided into 3 groups by discriminant analysis according to the behavior of diastolic function when stimulation rate of muscle strips was increased from 30 to 180 min-1. At 180 compared with 30 min-1, diastolic force was increased by 160%, maximum rate of force decline was decreased by 46%, and relaxation time was unchanged in group III. In contrast, in group I, diastolic force and maximum rate of force decline did not change, and relaxation time decreased by 20%. Na+-Ca2+ exchanger was 66% higher in group I than in group III. Na+-Ca2+ exchanger was inversely correlated with the frequency-dependent rise of diastolic force when stimulation rate was increased (r=-0.74; P<0.001). Compared with nonfailing human hearts (n=6), SR Ca2+-ATPase was decreased and Na+-Ca2+ exchanger unchanged in group III, whereas Na+-Ca2+ exchanger was increased and SR Ca2+-ATPase unchanged in group I. Results with group II hearts were between those of group I and group III hearts. CONCLUSIONS: By discriminating failing human hearts according to their diastolic function, we identified different phenotypes. Disturbed diastolic function occurs in hearts with decreased SR Ca2+-ATPase and unchanged Na+-Ca2+ exchanger, whereas increased expression of the Na+-Ca2+ exchanger is associated with preserved diastolic function.     

Ability of activation recovery intervals to assess action potential duration during acute no-flow ischemia in the in situ porcine heart. Experimental Cardiology Group, University of North Carolina at Chapel Hill

INTRODUCTION: The ability to assess transmural changes in action potential duration during acute no-flow ischemia is essential to an understanding of the tachyarrhythmias that occur in this setting. The purpose of this study was to determine if activation recovery intervals determined from unipolar electrograms would provide this information. METHODS AND RESULTS: We recorded simultaneously transmembrane action potentials and unipolar electrograms from sites located as closely together as possible in the center and at the lateral margin of the ischemic zone during acute no-flow ischemia and correlated the changes in activation recovery intervals obtained from the unipolar electrograms to the changes in action potential duration. We found that the activation recovery intervals provided an accurate measure of the changes in action potential duration during acute no-flow ischemia provided the electrograms had a well-defined, single negative component to the QRS complex with a maximum negative dV/dt > 10 V/sec and a single positive component to the T wave having a maximum positive dV/dt > 1.6 V/sec. Electrograms meeting these criteria comprised 90% of the electrograms recorded at the margin of the ischemic zone throughout 60 minutes of no-flow ischemia. In the center of the ischemic zone, 75% of the recorded electrograms met these criteria for the first 20 minutes of no-flow ischemia. Thereafter, the percentage declined and after 40 minutes of no-flow ischemia, none of the electrograms recorded in the center of the ischemic zone met these criteria. CONCLUSION: Activation recovery intervals obtained from unipolar electrograms provide an accurate assessment of changes in action potential duration throughout the ischemic zone during acute no-flow ischemia, provided the characteristics of the electrograms meet specific predetermined criteria.     

Electrodeposited iridium oxide pH electrode for measurement of extracellular myocardial acidosis during acute ischemia

In the present paper, fabrication, characterization, and physiological applications of a solid-state pH electrode are described. The pH sensing layer was based on an anodic electrodeposited iridium oxide film (AEIROF). Sputtered platinum electrodes (1 mm diameter) fabricated on flexible Kapton films or platinum wires were used as planar or cylindrical supports. Each electrode site was coated with Nafion to attenuate the interference of anionic redox species and to protect the electrode surface during in vivo measurements. Performance of the AEIROF was evaluated, for the first time, as a pH electrode and proved to have a slightly super-Nernstian response with slope of -63.5 +/- 2.2 mV/pH unit for both wire and planar sputtered platinum electrodes. Linear pH responses were obtained in the pH range 2-10. The electrodes have a working lifetime of at least 1 month with accuracy of about 0.02 pH unit and fast response time. The electrodes showed very low sensitivities for different species, such as Na+, K+, Li+, NH4+, Ca2+, Mg2+, dissolved oxygen, lactate, ascorbate, and urate, which are important for physiological applications. The electrodes were applied in extracellular pH measurements during brief regional ischemia in a swine heart and no-flow ischemia in an isolated rabbit papillary muscle. A first report on extracellular pH, K+, and lactate simultaneous measurements during no-flow ischemia using the AEIROF pH electrode and the previously described K+ and lactate electrodes is presented as well.     

Aging impairs functional, metabolic and ionic recovery from ischemia-reperfusion and hypoxia-reoxygenation

Functional and metabolic responses to ischemia-reperfusion and hypoxia-reoxygenation were studied in Langendorff perfused hearts from mature (2-4 months) and aged (18-24 months) Wistar rats. Hearts were subjected to 20 min global ischemia or hypoxia followed by 30 min reperfusion or reoxygenation. Cellular metabolism was assessed by 31P-NMR spectroscopy. Normoxic function, phosphate metabolite levels, and cytosolic free energy state (delta GATP) were comparable in both age groups, although free [5'-AMP] and purine efflux were elevated in aged hearts. There were no aging-related differences in phosphate metabolite levels, pH or delta GATP during ischemia or hypoxia. Nevertheless, ischemic and hypoxic contracture tended to be higher in aged hearts. After reperfusion, heart rate x left-ventricular pressure recovered to 55% of pre-ischemia in mature hearts, and only 25% in aged hearts. After reoxygenation, function recovered to 75% in mature hearts and 55% in aged hearts. Recoveries of cellular [ATP], [phosphocreatine], [inorganic phosphate] and [Mg2+] were impaired, and delta GATP was consistently depressed in aged v mature hearts, Impaired recovery of delta GATP was associated with enhanced purine efflux in aged hearts. Post-ischemic Na+ and Ca2+ accumulation was also increased by 30-40% in aged hearts. Tissue damage assessed by post-ischemic creatine kinase efflux was modest in mature hearts (< 2% total tissue activity) and was 2.5-fold higher in aged hearts. The data show that: (i) aging reduces contractile recovery from ischemia/hypoxia; (ii) this is unrelated to the metabolic insult during ischemia/hypoxia, but parallels reduced recovery of delta GATP [inorganic phosphate], [Mg2+]i [Na+] and [Ca2+]; and (iii) increased purine catabolism may contribute to poor metabolic recovery in aged hearts.