William E. Jacobus

Improvement of postischemic myocardial function and metabolism induced by administration of deferoxamine at the time of reflow: the role of iron in the pathogenesis of reperfusion injury.

Reperfusion of ischemic myocardium has been postulated to result in a specific oxygen radical-mediated component of tissue injury. In a previous study we demonstrated improved recovery of ventricular function and metabolism when the superoxide radical scavenger superoxide dismutase was administered at the time of postischemic reflow. Studies in vitro, have suggested that superoxide toxicity might be mediated via the generation of more reactive hydroxyl radicals in an iron-catalyzed reaction. The present study was designed to test the hypothesis that myocardial reperfusion injury might be reduced by administration of the iron chelator deferoxamine at the time of reflow, most likely by preventing hydroxyl radical formation. Sixteen isolated Langendorff rabbit hearts, perfused within the bore of a superconducting magnet, were subjected to 30 min of normothermic (37 degrees C) total global ischemia followed by 45 min of reperfusion. At reflow eight treated hearts received a 10 ml bolus containing 50 mumol of deferoxamine followed by an infusion of 11 mumol/min for the first 15 min of reflow. The hearts were then perfused with standard perfusate for an additional 30 min. Eight untreated control hearts received a similar bolus of perfusate followed by 45 min of standard reperfusion. Serial 5 min 31P nuclear magnetic resonance spectra were recorded. Myocardial phosphocreatine (PCr) content fell to 5% to 7% of control during ischemia in both groups of hearts. Deferoxamine-treated hearts recovered 99 +/- 10% of control PCr content, while untreated hearts recovered 60 +/- 16% (p less than .05). Intracellular pH fell to 5.9 during ischemia in both groups, before showing more rapid and complete recovery in treated hearts (p less than .01). Recovery of developed pressure reached 70 +/- 6% of control in treated hearts compared with 35 +/- 10% in untreated hearts (p less than .05). Iron content of the perfusate was 7 microM, and by electron paramagnetic resonance spectroscopy was in the form of Fe3+-EDTA complexes. In the effluent of treated hearts iron was in the form of Fe3+-deferoxamine chelates. In summary, administration of the iron chelator deferoxamine at the time of postischemic reflow results in greater recovery of myocardial function and energy metabolism, which supports the hypothesis that iron plays an important role in the pathogenesis of reperfusion injury.

Mechanisms of ischemic myocardial cell damage assessed by phosphorus-31 nuclear magnetic resonance.

Phosphorus-31 nuclear magnetic resonance (91P NMR) can estimate tissue intracellular pH as well as the content of high-energy phosphate metabolites in isolated perfused hearts. We used 3P NMR to examine mechanisms associated with the recovery of ventricular function in hearts subjected to global ischemia and reperfusion, with special emphasis on intracellular pH, a previously unreported variable. Single-dose and multiple-dose administration of a hyperkalemic cardioplegic solution were compared with hypothermia alone in 18 isolated perfused rabbit hearts. Hearts in group 1 were subjected to 24°C hypothermia during 60 minutes of global ischemia; group 2 hearts received a single injection of 37-mM KCL cardioplegic solution at 10°C at the onset of ischemia; and group 3 hearts received a similar initial cardioplegic injection followed by two subsequent 24°C injections at 20-minute intervals during the ischemic period. Using an intraventricular balloon, maximal dP/dt provided a quantitative index of left ventricular performance before and after ischemia. Return of ventricular function expressed as a percentage of control was 54 ± 11% for group 1, 84 ± 6% for group 2, and 101 ± 18% for group 3. Differences in the rate of development of intracellular acidosis were noted during the 60-minute ischemic period. Intracellular pH fell to 6.09 ± 0.12 in group 1, 6.31 ± 0.09 in group 2, and 6.79 ± 0.03 in group 3. In all three groups intracellular pH returned to control (pH 7.20) within 10 minutes of reflow. The metabolic correlates of functional recovery appeared to be the tissue content of ATP at the end of ischemia and after reflow. ATP content at the end of ischemia was 22 ± 2% of control in group 1 hearts, 31 ± 4% in group 2 and 64 + 2% in group 3. After 45 minutes of reperfusion, ATP levels recovered to 33 ± 9% of control in group 1, to 71 ± 9% in group 2 and to 86 ± 6% in group 3. Although there were no differences between groups in the content of creatine phosphate after 60 minutes of ischemia, the rates of creatine phosphate decline were dissimilar. Further, during the early reflow period, a marked overshoot in tissue creatine phosphate was detected, especially in groups 1 and 2. Histologic damage assessed by light microscopy correlated with the metabolic data, confirming that multidose cardioplegia provided the best preservation of cellular morphology. These results demonstrate that the magnitude of intracellular acidosis and the associated increase in inorganic phosphate correlate inversely with recovery of postischemic ventricular structure and function. ATP, but not creatine phosphate, content correlates with return of contractile performance after reperfusion. The overshoot in creatine phosphate during early reperfusion might impede optimal restoration of ATP content and, as a result, optimal recovery of cell functions.

Creatine kinase of heart mitochondria: Changes in its kinetic properties induced by coupling to oxidative phosphorylation

Abstract A complete kinetic analysis of the forward mitochondrial creatine kinase reaction was conducted to define the mechanism for its rate enhancement when coupled to oxidative phosphorylation. Two experimental systems were employed. In the first, ATP was produced by oxidative phosphorylation. In the second, heart mitochondria were pretreated with rotenone and oligomycin, and ATP was regenerated by a phosphoenolpyruvate-pyruvate kinase system. Product inhibition studies showed that oxidative phosphorylation did not effect the binding of creatine phosphate to the enzyme. Creatine phosphate interacted competitively with both ATP and creatine, and the E · MgATP · CrP dead-end complex was not readily detected. In a similar manner, the dissociation constants for creatine were not influenced by the source of ATP: K ib = 29 mm; K b = 5.3 mM, and the maximum velocity of the reaction was unchanged: V 1 = 1 μmol/ min/mg. Slight differences were noted for the dissociation constant ( K ia ) of MgATP from the binary enzyme complex, E · MgATP. The values were 0.75 and 0.29 m m in the absence and presence of respiration. However, a 10-fold decrease in the steady-state dissociation constant ( K a ) of MgATP from the ternary complex, E · MgATP · creatine, was documented: 0.15 m m with exogenous ATP and 0.014 m m with oxidative phosphorylation. Since K ia × K b does not equal K a × K ib under respiring conditions, the enzyme appears to be altered from its normal rapid-equilibrium random binding kinetics to some other mechanism by its coupling to oxidative phosphorylation.

Preserved high energy phosphate metabolic reserve in globally “stunned” hearts despite reduction of basal ATP content and contractility+

Impaired energy production has been proposed as one mechanism to explain the contractile abnormality in post-ischemic stunned myocardium. If energy production were impaired, administration of inotropic agents should result in a deterioration of cellular energy stores because of an inability of ATP synthesis to match the rate of increased utilization. In this study we correlated changes in myocardial high energy phosphates, measured by 31P-NMR spectroscopy, with changes in left ventricular function and energy requirement in buffer perfused rabbit hearts following ischemia and reperfusion, and during stimulation with isoproterenol. Hearts were stunned by 20 min of zero flow global ischemia at room temperature. After reperfusion, isovolumic developed pressure returned to 77.8 +/- 2.2% of baseline and ATP content was reduced to 80.9 +/- 4.1% of baseline. Isoproterenol (5 x 10(-8) M for 10 min) caused increases in developed pressure and rate-pressure product (to 134.1 +/- 12.6% and 195.0 +/- 21.4% of baseline, respectively) without a decrease in ATP or phosphocreatine (PCr) content (80.0 +/- 7.1% and 103.0 +/- 3.8% of preischemia, respectively), and without functional or metabolic deterioration of the hearts after discontinuation of the drug. Control hearts not subjected to ischemia showed similar functional and metabolic responses to isoproterenol. The phosphocreatine/inorganic phosphate (PCr/Pi) ratio, an index of the balance between energy production and utilization, was higher (not lower) than baseline in stunned hearts, thus confirming that energy production was not intrinsically impaired. Together these data indicate that despite reduced myocardial ATP content, mitochondrial function in stunned hearts is capable of sustaining a large increase in function and energetic requirements.

Perfusate sodium during ischemia modifies post-ischemic functional and metabolic recovery in the rabbit heart

Metabolic and functional recovery following 60 minutes of low flow (0.1 ml/min) ischemia were compared in rabbit hearts perfused with normal sodium and potassium, low sodium (120 mM NaCl replaced by 120 mM LiCl), or zero potassium perfusate during ischemia. During the control, pre-ischemic, and reperfusion periods, all hearts were perfused identically with normal sodium and potassium. 31P NMR was used to monitor intracellular pH (pHi), ATP, and phosphocreatine (PGr). Developed pressure, end diastolic pressure, pHi, and the integrated areas of ATP and PCr were equivalent in the three groups in the pre-ischemic period. The fall in pHi, PCr, ATP, and developed pressure and the rise in end diastolic pressure during 60 min ischemia also did not differ among the three groups. In contrast to the lack of an effect of perfusate sodium and potassium on the decline in parameters of metabolism and function during ischemia, there was a marked difference in the recovery of these indices during reperfusion. Hearts perfused with low sodium during ischemia exhibited the best recovery (expressed as percent of control) of developed pressure (95 +/- 4%), PCr (106 +/- 6%), and ATP (51 +/- 2%) and the smallest rise in end diastolic pressure (229 +/- 50%); hearts perfused with normal sodium and potassium during ischemia had intermediate recovery values for developed pressure (53 +/- 10%), PCr (78 +/- 9%), ATP (45 +/- 4%) and end diastolic pressure (487 +/- 73%) and the hearts perfused with zero potassium solution during ischemia exhibited the poorest recovery of developed pressure (23 +/- 6%), PCr (49 +/- 6%), ATP (39 +/- 5%) and end diastolic pressure (968 +/- 185%).(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular acidosis and contractility in the normal and ischemic heart as examined by 31P NMR.

Abstract 31P was used to investigate correlations between intracellular pH (pHi) and myocardial contractility in the normal and ischemic isolated, perfused isovolumic rabbit heart. Intracellular pH was calculated from the chemical shift of Pi in hearts perfused with phosphate-free buffer. Normal intracellular pH was 7.22 ± 0.02 (n = 15). To calibrate the relationship between pHi and left ventricular developed pressure (LVDP), respiratory acidosis was induced by mixing 65% O2: 30% N2: 5% CO2 with 65% O2: 35% CO2 using Krebs buffer containing 24 m m HCO3−. The results show that a 0.22 pH unit acidification correlates with a 50% reduction in LVDP. The correlation between pHi and LVDP was also studied in two models of ischemia: total global ischemia and steady state partial ischemia (50% reduction of LVDP). In both ischemic conditions, a 50% lowering in LVDP correlated with only a 0.09 pH unit acidification. Thus, while intracellular acidosis may account for 40 to 50% of the depression of LVDP oberved during the early phases of ischemia, other factor must also play a role. Mass spectrometry was used to examine the potential regulatory role of tissue oxygen (PmO2). The model of steady-state partial ischemia was employed. Changes in LVDP and MVO2 correlated quite closely with reductions in coronary flow. However, up to a 50% reduction in flow, pHi remained near normal, and tissue PmO2 was normal or slightly elevated. These latter results suggest that an efficient autoregulatory mechanism controls both function and MVO2 in close parallel to changes in flow. As a result, the metabolic supply/demand balance is maintained. However, beyond a 50% reduction in flow, this mechanism fails and metabolic indicies of ischemia are expressed.

Phosphorus nuclear magnetic resonance studies of heart physiology

Abstract 31 P NMR at 72.9 MHz using a 25-mm-diameter phosphorus probe has been used to study correlations among cardiac metabolism, tissue pH, and contractile performance. In all studies, isovolumic left ventricular pressure (LVP) was measured in the paced hearts. Under these conditions, excellent spectra were collected in 5 min, on 6-g rabbit hearts. Good spectra were obtained in 30-sec intervals. Rapid sequential spectra illustrate the metabolic and pH events associated with the onset and recovery of total, global ischemia. Tissue pH was stable for the first minute, but then fell from 7.4 to 6.9 by 6 min, and progressed to a value of 6.4 after 40 min. During the first minute, LVP fell 80%. Metabolites and tissue pH recovered within 6 min of reperfusion, a time when ventricular pressure remained depressed by 50%. These results suggest that both the early fall and initial postischernic recovery of ventricular pressure may not be exclusively regulated by tissue pH, as estimated by NMR. We also used NMR to investigate the metabolic changes associated with regional ischemia. The 31 P NMR spectrum of a regionally ischemic, perfused rabbit heart showed two inorganic phosphate (P i ) peaks. Before ligation there was only a single P i signal at a position corresponding to one of the peaks noted during regional ischemia. Since the resonance frequency of the P i peak is determined by pH and since ischemia causes acidosis the two signals in the regionally ischemic heart result from P i at different intracellular pH values in the normal (pH 7.4) and ischemic zones (pH 6.4). And finally, we compared the status of KCl-arrested, ischemic rabbit hearts and non-KCl-treated hearts and correlated the 31 P NMR spectra with the ability of the heart to return to normal function following a period of ischemia. A rabbit heart was arrested by perfusing it with 30 m M KCl and was then made globally ischemic; a second heart was made ischemic without KCl arrest. After 40 min of global ischemia the KCl-arrested heart showed a near-normal level of ATP, low P i and a pH of 7.0. The nonarrested heart, on the other hand, showed low ATP, high P i , and a pH of 6.4. On reperfusion, the KCl-arrested heart recovered 100% of control function within 5 min but the control heart recovered only 70% of control function after 30 min. The 31 P NMR confirms that KCl arrest preserves ischemic myocardial metabolites and suggests that it can be used to test currently untried treatments for functional protection.

Theoretical support for the heart phosphocreatine energy transport shuttle based on the intracellular diffusion limited mobility of ADP

Flux rates for phosphate metabolites were calculated using the equation for radial diffusion, assuming heart intracellular conditions and a 5% concentration gradient. The data show that while the flux of phosphocreatine is about 3 times faster than ATP, both are more than two orders of magnitude greater than the known maximum rate of ATP utilization. In contrast, since the concentration of free ADP is very low, its flux is below the maximum rate of ATP turnover, while the flux of creatine is almost 3 orders of magnitude greater than ADP. The data suggest that the rate of high-energy phosphate production could be limited by ADP diffusion, with creatine thus substituting as the primary cytoplasmic-mitochondrial phosphate acceptor.

Reduced aerobic metabolic efficiency in globally “stunned” myocardium

Post-ischemic stunned myocardium appears to be metabolically inefficient, since oxygen consumption is preserved, while mechanical work is depressed. The present study investigated whether this metabolic inefficiency represents a basal functional abnormality present in the quiescent myocardium (e.g. abnormal mitochondrial coupling) or is specifically related to muscle contraction. Isolated perfused rabbit hearts (n = 7) were exposed to 20 min zero-flow ischemia to produce post-ischemic myocardial stunning. After 10 min of reperfusion, mean rate-pressure product (mmHg/min), was reduced to 56.1% of baseline in stunned hearts, while mean oxygen consumption (mumol O2/min/g LV) was reduced to only 71.8% of baseline. The ratio of oxygen consumption to rate-pressure product remained significantly elevated throughout 40 min of reperfusion when compared with non-ischemic controls (P less than 0.01). Despite inappropriately high oxygen consumption in the beating stunned heart, basal oxygen consumption measured after KCl arrest was not significantly different from controls (1.07 +/- 0.07 vs. 1.03 +/- 0.04, respectively). These results indicate that the metabolic inefficiency found in stunned myocardium is not a basal abnormality, but rather is related specifically to abnormalities in contraction or electromechanical coupling.

Control of heart oxidative phosphorylation by creatine kinase in mitochondrial membranes.

Three important points must be emphasized in summary. First is the idea that a cellular microcompartment need not be limited by a semi-permeable membrane. We recognize microcompartments in multi-enzyme complexes where substrates are covalently transported from subunit to subunit. An example of this is the lipoic acid moiety of the pyruvate dehydrogenase complex. However, to act as a kinetic microcompartment, covalent transfer is not an obligatory requirement. Proximity effects may be sufficient for substantial rate enhancement. Our data clearly show that the kinetics of ADP translocation are influenced by the site of ADP formation. We contend that this represents a newly recognized and important form of cellular microcompartmentation. The second point is that we do not want our results misinterpreted as an overextension of the known data concerning tissue respiration. We believe that the primary parameter controlling heart mitochondrial oxygen consumption is the availability of ADP at the adenine nucleotide translocase. Our data show, however, that this is not a simple process. Secondary control is exerted by the localization of ADP formation, i.e. microcompartmentation. As a result of the kinetic data (Table 3), we conclude that the forward rate of mitochondrial creatine kinase is the preferential reaction controlling ADP delivery to the translocase. We are left, nonetheless, with questions concerning the secondary regulation of this enzyme in vivo by substrate (ATP and creatine) and inhibition by product (phosphocreatine). The nature of this control awaits further experimental data. Finally, the results are consistent with the creatine kinase energy transport hypothesis. Overall, the rate of tissue oxygen consumption reflects the metabolic activity of the organ, determined by the rate of ATP utilization (see right side of Figure 1). This results in the cytoplasmic production of ADP. In heart, this is coupled via the bound cytoplasmic isozymes of creatine kinase to the local rephosphorylation of ADP to ATP and the simultaneous production of creatine.(ABSTRACT TRUNCATED AT 400 WORDS)