John C. Docherty

High levels of fatty acids delay the recoveryof intracellular pH and cardiac efficiency inpost-ischemic hearts by inhibiting glucose oxidation

OBJECTIVES This study was designed to determine if the fatty acid-induced increase in H(+) production from glycolysis uncoupled from glucose oxidation delays the recovery of intracellular pH (pH(i)) during reperfusion of ischemic hearts. BACKGROUND High rates of fatty acid oxidation inhibit glucose oxidation and impair the recovery of mechanical function and cardiac efficiency during reperfusion of ischemic hearts. METHODS pH(i) was measured by 31P nuclear magnetic resonance spectroscopy in isolated working rat hearts perfused in the absence (5.5 mmol/l glucose) or presence of 1.2 mmol/l palmitate (glucose+palmitate). Glycolysis and glucose oxidation were measured using [5-3H/U-14C]glucose. RESULTS When glucose+palmitate hearts were subjected to 20 min of no-flow ischemia, recoveries of mechanical function and cardiac efficiency were significantly impaired compared with glucose hearts. Glucose oxidation rates were significantly lower in glucose+palmitate hearts during reperfusion compared with glucose hearts, whereas glycolysis rates were unchanged. This resulted in an increase in H(+) production from uncoupled glucose metabolism, and a decreased rate of recovery of pH(i) in glucose+palmitate hearts during reperfusion compared with glucose-perfused hearts. Dichloroacetate (3 mmol/l) given at reperfusion to glucose+palmitate hearts resulted in a 3.2-fold increase in glucose oxidation, a 35% +/- 3% decrease in H(+) production from glucose metabolism, a 1.7-fold increase in cardiac efficiency and a 2.2-fold increase in the rate of pH(i) recovery during reperfusion. CONCLUSIONS A high level of fatty acid delays the recovery of pH(i) during reperfusion of ischemic hearts because of an increased H(+) production from glycolysis uncoupled from glucose oxidation. Improving the coupling of glucose metabolism by stimulating glucose oxidation accelerates the recovery of pH(i) and improves both mechanical function and cardiac efficiency.

An inhibitor of poly (ADP‐ribose) synthetase activity reduces contractile dysfunction and preserves high energy phosphate levels during reperfusion of the ischaemic rat heart

The cardioprotective properties of inhibition of poly (ADP‐ribose) synthetase (PARS) were investigated in the isolated perfused heart of the rat. Hearts were perfused in the Langendorff mode and subjected to 23 min total global ischaemia and reperfused for 60 min. Left ventricular function was assessed by means of an intra‐ventricular balloon. High energy phosphates were measured by 31P‐NMR spectroscopy. Intracellular levels of NAD were measured by capillary electrophoresis of perchloric acid extracts of hearts at the end of reperfusion. Reperfusion in the presence of the PARS inhibitor 1,5 didroxyisoquinoline (ISO, 100 μM) attenuated the mechanical dysfunction observed following 1 h of reperfusion; 27±13 and 65±8% recovery of preischaemic rate pressure product for control and 100 μM ISO, respectively. This cardioprotection was accompanied by a preservation of intracellular high‐energy phosphates during reperfusion; 38±2 vs 58±4% (P<0.05) of preischaemic levels of phosphocreatine (PCr) for control and 100 μM ISO respectively and 23±1 vs 31±3% (P<0.05) of preischaemic levels of ATP for control and 100 μM ISO respectively. Cellular levels of NAD were higher in ISO treated hearts at the end of reperfusion; 2.56±0.45 vs 4.76±1.12 μmoles g−1 dry weight (P<0.05) for control and ISO treated. These results demonstrate that the cardioprotection afforded by inhibition of PARS activity with ISO is accompanied by a preservation of high‐energy phosphates and cellular NAD levels and suggest that the mechanism responsible for this cardioprotection may involve prevention of intracellular ATP depletion.

Capsaicin as a source for painful stimulation in functional MRI

Functional magnetic resonance imaging (fMRI) was used to examine the brain processing of capsaicin‐induced painful stimulation in the α‐chloralose anesthetized rat. Experiments were performed on a 9.4‐T magnet (Magnex, UK) with Avance console (Bruker, Germany) using a surface coil tuned to 400.5 MHz centred over the rat forebrain. Gradient‐echo images of two slices, with an echo time of 25 msec, repetition time of 70 msec, and 50 repetitions, were acquired per experiment. These images were analyzed using a fuzzy cluster analysis technique (EvIdent™). Activation of areas of the brain known to be associated with the processing of pain, namely the anterior cingulate (bilateral), frontal cortex (bilateral), and sensory motor cortex (contralateral), was found in all animals (N = 6) following injection of 25μL of capsaicin (128μg/mL in 7.5% dimethylsulfoxide [DMSO]) into the dorsal forepaw. It is possible to reproduce the pain response in a given animal several times throughout the course of an experiment, provided that sufficient time is allowed between capsaicin injections. This acute phase of capsaicin‐induced pain involving stimulation of C polymodal nociceptors was examined by functional imaging. There was a substantial initial increase in activation in regions of the brain associated with pain and there was a trend towards increasing activation with repeated stimulations. Treatment with morphine (3 mg/kg, intravenously) was found to substantially reduce, if not completely eliminate, the areas of functional activation associated with physiologic pain (anterior cingulate and frontal cortex) after C‐nociceptor stimulation with capsaicin (N = 6). FMRI involving capsaicin‐induced painful stimulation could prove to be an effective tool for the study of novel analgesics and the central nervous system processing of pain. J. Magn. Reson. Imaging 2001;14:341–347.

Na+-H+ exchange inhibition at reperfusion is cardioprotective during myocardial ischemia reperfusion; 31P NMR studies

To help resolve the controversy as to whether or not Na+-H+ exchange is functioning during reperfusion of the ischemic myocardium we assessed the effects of dimethylamiloride (DMA, an amiloride analogue possessing selectivity for inhibition of the Na+-H+ exchanger) on cardiac function and intracellular pH during ischemia-reperfusion. Studies were performed in the presence of bicarbonate (modified Krebs-Henseleit buffer) or in the nominal absence of bicarbonate (HEPES buffer) in order to determine if similar cardioprotection and effects on intracellular pH were observed in the presence and absence of bicarbonate dependent transport processes. Isovolumic rat hearts were perfused in the Langendorff mode at a constant pressure of 80 mm Hg and subjected to 28 min total global ischemia at 37°C. Intracellular pH was determined from the pH dependent shift of the inorganic phosphate peak in 31P nuclear magnetic resonance spectra. DMA (20 μM) was infused for either 2.5 min before ischemia, for the initial 5 min of reperfusion, or at both time intervals. DMA had no effect on the intracellular pH during ischemia. Intracellular pH returned to pre-ischemic levels within 2.5 min of reperfusion in bicarbonate buffer. This normalization of pH was slower in HEPES perfusate. In both bicarbonate and HEPES perfused hearts all drug dosing regimens caused a significant increase in the recovery of mechanical function after reperfusion and slowed the recovery of intracellular pH during reperfusion. These results suggest that the Na+-H+ exchanger is activated during reperfusion of the ischemic myocardium, that this activation of the exchanger contributes to ischemia-reperfusion induced cardiac dysfunction and that administration of an inhibitor of Na+-H+ exchange at reperfusion significantly attenuates the deleterious effects of exchanger activation. (Mol Cell Biochem 176: 257–264, 1997)

Perfused Organs Studied Using NMR Spectroscopy

NMR spectroscopy of perfused isolated organs is described. The main system studied has been the heart but also other organs such as the liver and kidney have been studied using mostly 31 P NMR. 19 F NMR has been used to investigate intracellular Ca 2+ levels, whilst 23 Na, 87 Rb and 7 Li NMR have all been employed for studies of sodium–potassium exchange.

FTIR/NIR Assessment of Ischemic Damage in the Rat Heart

The utility of combined near- and mid-infrared spectroscopic measurements to monitor short-term changes in the heart subjected to ischemia-reperfusion and the ability to assess long-term modifications to the extracellular matrix following an ischemic insult to the heart is presented.

Cardiac Sarcolemmal Na+/H+ Exchange after a Myocardial Infarction in the Rat

A growing number of studies have implicated the Na+/H+ exchanger in ischemic/reperfusion damage to the heart [1–7]. Other studies have now demonstrated accelerated ion flow through this exchanger in hypoxic/ reoxygenation insult to the heart as well [8–10]. While these are important pathological events, they are largely immediate reactions of Na+/H+ exchange activity in response to changes in the intracellular ionic environment in the heart. These reactions occur so quickly that they likely do not involve structural changes in the protein or alterations in protein synthesis. However, during low-flow ischemia of a relatively long duration, there are indications that these pathways are beginning to be activated. Changes in the Na+/H+ exchange mRNA message have been detected in hearts after three hours of low-flow ischemia [11]. These changes do not occur in global ischemia experiments, which employ considerably shorter ischemic periods (≤1 hour) [11,12]. Chronic alterations in the exchanger would be more likely to occur during conditions where an adaptive stimulus is placed upon the heart for a much longer period of time and/or where the tissue is allowed sufficient time to induce its adaptive mechanisms. A stimulus that frequently induces an adaptive response in the heart is myocardial disease.