Correlation of ischemia-induced extracellular and intracellular ion changes to cell-to-cell electrical uncoupling in isolated blood-perfused rabbit hearts. Experimental Working Group.

BACKGROUND: The relationships between the metabolic, ionic, and electrical changes of acute ischemia have not been determined precisely because they have been studied under different experimental conditions. We used ion-selective electrodes, nuclear magnetic resonance spectroscopy, and the four-electrode method to perform four series of experiments in the isolated blood-perfused rabbit heart loaded with 5F-BAPTA during 30 to 35 minutes of no-flow ischemia. We sought to determine the relationship between changes in phosphocreatine (PCr), adenosine triphosphate (ATP), intracellular calcium ([CA2+]i), intracellular pH (pHi) extracellular potassium ([K+]e), extracellular pH (pHe), and whole-tissue resistance (rt). METHODS AND RESULTS: In the first 8 minutes of ischemia, [K+]e rose from 4.9 to 10.8 mmol/L, PCr fell by 90%, ATP decreased by 25%, and pHi and pHe decreased by 0.5 U, while [Ca2+]i and rt changed only slightly. Between 8 and 23 minutes, [K+]e changed only slightly; pHi, pHe, and ATP continued to fall, and [Ca2+]i rose. rt did not increase until >20 minutes of ischemia, when pHi was <6.0 and [Ca2+]i had increased more than three-fold. The increase in rt, indicating electrical uncoupling, coincided with the third phase of the [K+]e change. CONCLUSIONS: Our study suggests that cellular uncoupling occurs only after a significant rise in [Ca2+]i and fall in pHi and that these ionic and electrical changes can be identified by the change in [K+]e. Our study underscores the importance of using a common model while attempting to formulate an integrated picture of the ionic, metabolic, and electrical events that occur during acute ischemia.

Effects of adenosine antagonists on hexose uptake and preconditioning in perfused rat heart.

Preconditioning with brief intermittent periods of ischemia has been shown to lessen the detrimental effects of a subsequent sustained (30-60 min) period of ischemia. Because adenosine has been suggested to be the mediator of preconditioning, we were interested in investigating whether adenosine antagonists would block the effect of preconditioning on ionic changes during ischemia. We found that 10 microM of the adenosine antagonist BW-A1433U did not reverse the effect of preconditioning on intracellular pH (pHi). Hearts preconditioned with BW-A1433U had virtually no decrease in pHi during the 30-min sustained period of ischemia; after 30 min of ischemia, the pH in untreated hearts was 5.97 +/- 0.16 compared with 6.52 +/- 0.10 in preconditioned hearts and 6.90 +/- 0.08 in hearts preconditioned plus BW-A1433U. Because anaerobic glycolysis is largely responsible for the fall in pHi during ischemia, we examined the effect of BW-A1433U [and other adenosine antagonists, such as PD-115,199 and 8-cyclopentyl-1,3-dipropylxanthine (CPDPX)] on glucose uptake and phosphorylation during aerobic perfusion using 31P-nuclear magnetic resonance to monitor uptake and phosphorylation of 2-deoxyglucose (2-DG) to 2-deoxyglucose 6-phosphate (2-DG-6-P) when one-half of the glucose in the perfusate was replaced with 2-DG. Uptake of 2-DG-6-P after 15 min was reduced by 66% in the presence of BW-A1433U and 82% in the presence of PD-115,199 as compared with untreated hearts, but was not reduced in the presence of CPDPX. Thus CPDPX was the only adenosine antagonist tested that did not block accumulation of 2-DG-6-P. We also found that CPDPX did not block the beneficial effect of preconditioning on ionic alterations during a sustained 30-min period of ischemia or the improved recovery of function on reflow.

Role of increased cytosolic free calcium concentration in myocardial ischemic injury.

Increases in cytosolic free calcium concentration ([Ca2+]I) may play an important role in myocardial ischemic injury. An early effect of the rise in [Ca2+]I may be impaired postischemic contractile function if the ischemic myocardium is reperfused during the reversible phase of ischemic injury; furthermore, if the rise in [Ca2+]I is prolonged, a cascade of events may be initiated which ultimately results in lethal injury. With the development of methods for measuring [Ca2+]I, it has become possible to evaluate directly the role of increased [Ca2+]I in myocardial ischemic injury. Although it has been possible to show that inhibition of the transport processes which contribute to the early rise in [Ca2+]I attenuates stunning and the rise in [Ca2+]I concurrently, if increased [Ca2+]I plays an important role in ischemic injury, then it should be possible to show that interventions which alter the timecourse of ischemic injury also alter the timecourse of the rise in [Ca2+]I in a parallel manner. Recently, considerable effort has been expended to investigate the mechanisms underlying the preconditioning phenomenon, whereby repetitive brief periods of ischemia prior to a sustained period of ischemia protects the myocardium from injury during the sustained period of ischemia, and this has stimulated additional work to understand the possible involvement of adenosine as a mediator of preconditioning as well as to understand the protective effects of adenosine. Measurements of [Ca2+]I using 19F NMR of 5FBAPTA-loaded hearts have shown that preconditioning attenuates the rise in [Ca2+]I during 30 min of ischemia and reduces stunning during reflow. Adenosine pretreatment mimics the effects of preconditioning on the rise in [Ca2+]I and on stunning, but adenosine receptor antagonists do not eliminate the protective effects of preconditioning, although some adenosine antagonists also block hexose transport and under these conditions, the ability of preconditioning to attenuate the rise in [Ca2+]I is abolished and there is a corresponding loss of the protective effect of preconditioning on stunning. Although it has been suggested that the beneficial effect of preconditioning on infarct size can be eliminated by pretreatment with glibenclamide, in the isolated rat heart glibenclamide does not affect the attenuation of the rise in [Ca2+]I induced by preconditioning and does not affect stunning. All of these studies show a consistent relationship between the magnitude of the rise in [Ca2+]I during ischemia and the degree of stunning during reperfusion. The data suggest that increased [Ca2+]I plays a very important role in myocardial ischemic injury.

Glibenclamide does not abolish the protective effect of preconditioning on stunning in the isolated perfused rat heart.

OBJECTIVE: The aim was to determine if the beneficial effects of preconditioning would be affected by inhibiting ATP sensitive potassium (KATP) channels in the isolated, perfused rat heart. METHODS: The effects of inhibiting KATP channels with glibenclamide (10 microM) were evaluated on ionic alterations and recovery of function after 30 min ischaemia in non-preconditioned hearts and in hearts that had been preconditioned with four intermittent periods of 5 min ischaemia each separated by 5 min of reflow. [Ca2+]i, pHi, and high energy phosphate levels were measured using 19F and 31P nuclear magnetic resonance during the preconditioning periods of ischaemia, during 30 min of ischaemia, and during reflow, in the presence and absence of 10 microM glibenclamide. RESULTS: High energy phosphate contents were decreased during the preconditioning period to a greater extent in glibenclamide treated hearts and the onset of contracture was hastened during the subsequent 30 min period of sustained ischaemia. However, glibenclamide (10 microM) did not abolish the protective effects of preconditioning on ion accumulation during ischaemia or on postischaemic recovery of contractile function. Recovery of left ventricular developed pressure (as % of initial value) following 30 min of ischaemia was 74(SEM 5)% in the preconditioned hearts without drug and 62(4)% in the preconditioned hearts with glibenclamide, while recovery was 25(5)% in the non-preconditioned hearts without drug and 19(2)% in the non-preconditioned hearts with drug. The alterations in [Ca2+]i and pHi during ischaemia were similar in the glibenclamide treated and untreated preconditioned hearts and in both cases were less marked than in the non-preconditioned untreated hearts. CONCLUSIONS: Thus, although inhibition of KATP channels accelerates high energy phosphate depletion during the preconditioning period, this does not result in accentuation of the ionic derangements during a subsequent sustained period of ischaemia and does not abolish the protective effect of preconditioning on stunning in the isolated rat heart.

Protective effects of adenosine in the perfused rat heart: changes in metabolism and intracellular ion homeostasis.

Increased concentrations of intracellular H+, Na+, and Ca2+ have been observed during ischemia, and these ionic alterations have been correlated with several indexes of cell injury in a number of studies. Recently, adenosine was proposed to play a role in ischemic preconditioning, since adenosine antagonists block the protective effects of these brief intermittent periods of ischemia and reflow. In this study we evaluated the protective effects of adenosine (20 microM) on high-energy phosphate metabolism, H+ and Ca2+ accumulation, and glycolytic rate during 30 min of no-flow ischemia. Adenosine was observed to slow the onset of contracture (7.0 +/- 0.9 min) and to improve left ventricular developed pressure (62 +/- 7% of initial) during reperfusion compared with untreated hearts (5.0 +/- 0.6 min and 18 +/- 5%, respectively). Intracellular Ca accumulation at the end of 30 min of ischemia was higher in the untreated (2,835 +/- 465 nM) than in the adenosine-treated (2,064 +/- 533 nM) hearts, while intracellular pH fell more in the untreated (5.85 +/- 0.17) than in the adenosine-treated hearts (6.27 +/- 0.16). Glycolytic rate and the rate of ATP decline were significantly attenuated in the adenosine-treated hearts during ischemia. Thus adenosine treatment slowed the rate of metabolism and delayed the accumulation of H+ and Ca2+ during ischemia, resulting in better recovery of function upon reflow.

Metabolic substrates can alter postischemic recovery in preconditioned ischemic heart.

The mechanisms that contribute to myocardial cell injury are not well understood. Furthermore, the ability of reperfusion conditions to modify ischemic injury is unclear. Recent studies have indicated that glucose utilization may improve ionic homeostasis. Because considerable derangement of ion concentrations occurs during ischemia, glucose utilization may be beneficial when stimulated during the reperfusion period. The effects of glycolytic vs. mitochondrial substrates on postischemic contractile function, high-energy phosphates and ion balance (intracellular Ca2+ and pH) were determined. Reperfusion conditions were compared in the "preconditioned ischemic" heart where baseline contractile recovery during reperfusion with glucose as the sole exogenous substrate was 74 +/- 5% (n = 10). Contractile recovery was determined for reperfusion with pyruvate (14 +/- 2%, n = 10), pyruvate+glucose (23 +/- 4%, n = 10), deoxyglucose+acetate (25 +/- 4%, n = 10), and lactate+glucose (60 +/- 11%, n = 10). Contractile dysfunction could not be attributed to differences in high-energy phosphate contents. Elevated levels of intracellular Ca2+ during reperfusion were, however, correlated with poor contractile function. After 20 min of reperfusion, the mean time-averaged intracellular Ca2+ values, measured with 19F-nuclear magnetic resonance of 5-fluoro-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-loaded hearts, were 994 +/- 110 nM (glucose, n = 5), 2,270 +/- 494 nM (pyruvate, n = 5), 2,671 +/- 419 nM (pyruvate+glucose, n = 5), 2,382 +/- 480 nM (deoxyglucose+acetate, n = 5), and 1,019 +/- 33 nM (lactate+glucose, n = 5). These results are consistent with a beneficial role for glucose utilization during reperfusion, where enhanced recovery of contractile function and ionic homeostasis were observed.

Myocardial oxygenation in the isolated working rabbit heart as a function of work.

Myocardial O2 consumption (MVO2) was stimulated up to two-fold by either increasing afterload or beta-receptor stimulation in working normothermic isolated rabbit hearts while noninvasively monitoring the O2 delivery or phosphate compounds (total n = 48). Intracellular O2 delivery was estimated with the use of myocardial optical absorbance changes centered at 603.5 and 582 nm that correlate with cytochrome aa3 redox and myoglobin oxygenation states. Phosphate-containing metabolites (ATP, phosphocreatine, free ADP) were assessed using 31P nuclear magnetic resonance spectroscopy. Measurements were made both with intact autoregulation and after maximal vasodilation by 1 microM nitroprusside (NP). When afterload was used to increase MVO2, absorbance decreased at 603.5 nm and increased at 582 nm, consistent with a 10-15% increase in myocardial oxygenation, without an associated change in cardiac phosphate compounds. NP caused a further increase in myocardial oxygenation and venous PO2 consistent with an increase in the O2 supply-to-demand ratio. Increases in MVO2 due to beta-stimulation alone were not associated with changes in 603.5-nm absorbance or phosphate compounds, but in combination with NP were accompanied by increased oxygenation, venous PO2, and cardiac phosphocreatine. KCl arrest caused maximal increases in oxygenation and phosphocreatine. These findings suggest that neither cytochrome aa3 nor myoglobin in the isolated working rabbit heart is fully oxidized or oxygenated, respectively. Furthermore, the oxygenation state of the tissue varied both with afterload-induced changes in cardiac work and with changes in O2 supply/demand.

Effect of work on intracellular calcium of the intact heart.

Intracellular calcium has been proposed to play a key role in the orchestration of metabolic rate with contractile activity in the mammalian heart. Calcium is believed to accomplish this task by modulating the contractile apparatus as well as the metabolic process directly, and perhaps simultaneously, during alterations in cardiac work. The purpose of this study was to evaluate whether appropriate changes in intracellular calcium accompany alterations in cardiac work in the intact working rabbit heart. A range of myocardial oxygen consumption was obtained from 0.94 to 6.51 mumol.g LV wt-1.min-1 by changing afterload or beta-agonist addition. With the increase in work and associated increase in respiration, an increase in intracellular calcium was observed, on the basis of indo-1 fluorescence. These results indicate that intracellular calcium is a valid candidate as a cytosolic transducer contributing to the orchestration of myofibril adenosinetriphosphatase activity and oxidative phosphorylation in the intact heart.

Lipid bilayer and water proton magnetization transfer: effect of cholesterol.

Magnetization transfer between macromolecules and water can be a significant factor contributing to tissue water 1H relaxation. Using saturation transfer techniques, the degree of magnetization transfer between the macromolecular matrix and bulk water 1H can be directly measured and magnetization transfer contrast (MTC) can be generated in MR images. A significant degree of MTC has been observed in tissues with high plasma membrane content such as kidney and brain. The purpose of this study was to establish whether lipid bilayers, as models for cell membranes, could exchange magnetization with the water solvent and whether this effect could contribute to MTC observed in intact tissues. Magnetization transfer was measured in aqueous dispersions of egg phosphatidylcholine (EPC) in the presence and absence of cholesterol. It was found that neither EPC bilayers nor cholesterol by themselves significantly exchanged magnetization with bulk water 1H. However, as the concentration of cholesterol was increased, the pseudo-first-order magnetization exchange rate increased to a maximum value of approximately 1 s-1. The cholesterol-induced 1H magnetization exchange may be related either to longer correlation times of the lipid or to an increase in the number of water molecules associated with the bilayer. These results indicate that EPC-cholesterol bilayers exchange 1H magnetization with bulk water. These results are consistent with lipid bilayer contributions to bulk water relaxation and MTC in intact biological tissues.

Effects of tissue absorbance on NAD(P)H and Indo-1 fluorescence from perfused rabbit hearts.

The effects of tissue optical absorbance on intracellular NAD(P)H and Indo-1 fluorescence emission have been evaluated in the perfused rabbit heart. These results demonstrate that the tissue optical absorbance significantly modifies the emission characteristics of these fluorophores. This tissue 'inner filter' effect, observed with both probes, changed as a function of tissue oxygenation and redox state in a wavelength-dependent manner. Pathlength calculations from these results indicate that this inner filter effect could occur with a mean pathlength of 310 microns due to the extremely high extinction coefficient of heart tissue. It is concluded that tissue optical absorbance significantly affects the fluorescent emission characteristics of both intrinsic and extrinsic probes in the intact heart, under a variety of conditions. Several potential methods of correcting for these tissue inner filter effects are discussed.