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Single fast spin echo scans covering limited time frames are mostly used for contrast-enhanced MRI of atherosclerotic plaque biomarkers. Knowledge on inter-scan variability of the normalized enhancement ratio of plaque (NER(plaque)) and relation between NER(plaque) and gadolinium content for inversion-recovery fast spin echo is limited. Study aims were: evaluation of (1) timing of MRI after intravenous injection of cannabinoid-2 receptor (CB2-R) (expressed by human and mouse plaque macrophages) targeted micelles; (2) inter-scan variability of inversion-recovery fast spin echo and fast spin echo; (3) relation between NER(plaque) and gadolinium content for inversion-recovery fast spin echo and fast spin echo. Inversion-recovery fast spin echo/fast spin echo imaging was performed before and every 15 min up to 48 h after injection of CB2-R targeted or control micelles using several groups of mice measured in an interleaved fashion. NER(plaque) (determined on inversion-recovery fast spin echo images) remained high (∼2) until 48 h after injection of CB2-R targeted micelles, whereas NER(plaque) decreased after 36 h in the control group. The inter-scan variability and relation between NER(plaque) and gadolinium (assessed with inductively coupled plasma- mass spectrometry) were compared between inversion-recovery fast spin echo and fast spin echo. Inter-scan variability was higher for inversion-recovery fast spin echo than for fast spin echo. Although gadolinium and NER(plaque) correlated well for both techniques, the NER of plaque was higher for inversion-recovery fast spin echo than for fast spin echo. In mice injected with CB2-R targeted micelles, NER(plaque) can be best evaluated at 36-48 h post-injection. Because NER(plaque) was higher for inversion-recovery fast spin echo than for fast spin echo, but with high inter-scan variability, repeated inversion-recovery fast spin echo imaging and averaging of the obtained NER(plaque) values is recommended.

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AIMS/HYPOTHESIS: Mitochondrial dysfunction and increased intramyocellular lipid (IMCL) content have both been implicated in the development of insulin resistance and type 2 diabetes mellitus, but the relative contributions of these two factors in the aetiology of diabetes are unknown. As obesity is an independent determinant of IMCL content, we examined mitochondrial function and IMCL content in overweight type 2 diabetes patients and BMI-matched normoglycaemic controls. METHODS: In 12 overweight type 2 diabetes patients and nine controls with similar BMI (29.4 +/- 1 and 29.3 +/- 0.9 kg/m(2) respectively) in vivo mitochondrial function was determined by measuring phosphocreatine recovery half-time (PCr half-time) immediately after exercise, using phosphorus-31 magnetic resonance spectroscopy. IMCL content was determined by proton magnetic resonance spectroscopic imaging and insulin sensitivity was measured with a hyperinsulinaemic-euglycaemic clamp. RESULTS: The PCr half-time was 45% longer in diabetic patients compared with controls (27.3 +/- 3.5 vs 18.7 +/- 0.9 s, p < 0.05), whereas IMCL content was similar (1.37 +/- 0.30 vs 1.25 +/- 0.22% of the water resonance), and insulin sensitivity was reduced in type 2 diabetes patients (26.0 +/- 2.2 vs 18.9 +/- 2.3 mumol min(-1) kg(-1), p < 0.05 [all mean +/- SEM]). PCr half-time correlated positively with fasting plasma glucose (r (2) = 0.42, p < 0.01) and HbA(1c) (r (2) = 0.48, p < 0.05) in diabetic patients. CONCLUSIONS/INTERPRETATION: The finding that in vivo mitochondrial function is decreased in type 2 diabetes patients compared with controls whereas IMCL content is similar suggests that low mitochondrial function is more strongly associated with insulin resistance and type 2 diabetes than a high IMCL content per se. Whether low mitochondrial function is a cause or consequence of the disease remains to be investigated.

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BACKGROUND AND OBJECTIVE: Recently, we have shown, by using localized in vivo phosphorus-31 magnetic resonance spectroscopy (31P MRS) of the anterior left ventricular wall, that brain death (BD) is not associated with reduced myocardial energy status. In this study, we applied ex vivo 31P MRS of the entire heart to study the effects of BD on the energy status of the feline donor heart following explantation. METHODS: We used cats (6 BD and 6 controls [C]) in a 26-hour protocol. After 2 hours of preparation, we induced BD by filling an intracranial balloon at t = 0 hour. At t = 6 hours, the hearts were arrested with St. Thomas' Hospital cardioplegic solution, explanted, and stored in the same solution at 4 degrees C in a 4.7 Tesla magnet for 17 hours. Subsequently, the hearts were reperfused in the Langendorff mode at 38 degrees C for 1 hour. The first 5-minute 31P MRS spectrum was obtained 1 hour after crossclamping the aorta; we obtained subsequent spectra every hour during storage and every 5 minutes during reperfusion. At the end, the hearts were dried and weighed. Phosphocreatine (PCr), gamma-adenosine triphosphate (gamma-ATP), inorganic phosphate (Pi), and phosphomonoesters (PME), were expressed per g dry heart weight. The intracellular pH (pH(i)) and the PCr/ATP ratio were calculated. RESULTS: During storage, we identified a significant but similar decrease of pH(i), PCr/ATP ratio, and PCr in both groups. During reperfusion, pH(i) and PCr/ATP ratio recovered similarly in both groups, whereas the recovery of PCr in the BD group was significantly lower (p < 0.05). The Pi and PME increased in both groups during storage but to a lesser extent in the BD group (p < 0.05). This difference disappeared during reperfusion. The gamma-ATP was already significantly lower in the BD group at the onset of storage, and this remained so throughout storage and reperfusion (p < 0.05 vs C). Contractile capacity was lost in all hearts, except for 1 heart in the BD group. CONCLUSION: Brain death-related failure of the energetic integrity of the feline donor heart becomes apparent only when using 31P MRS during ischemic preservation and subsequent reperfusion.

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Normalization of intracellular sodium (Na) after postischemic reperfusion depends on reactivation of the sarcolemmal Na(+)-K(+)-ATPase. To evaluate the requirement of glycolytic ATP for Na(+)-K(+)-ATPase function during postischemic reperfusion, 5-s time-resolution 23Na NMR was performed in isolated perfused rat hearts. During 20 min of ischemia, Na increased approximately twofold. In glucose-reperfused hearts with or without prior preischemic glycogen depletion, Na decreased immediately upon postischemic reperfusion. In glycogen-depleted pyruvate-reperfused hearts, however, the decrease of Na was delayed by approximately 25 s, and application of the pyruvate dehydrogenase (PDH) activator dichloroacetate (DA) did not shorten this delay. After 30 min of reperfusion, Na had almost normalized in all groups and contractile recovery was highest in the DA-treated hearts. In conclusion, some degree of functional coupling of glycolytic ATP and Na(+)-K(+)-ATPase activity exists, but glycolysis is not essential for recovery of Na homeostasis and contractility after prolonged reperfusion. Furthermore, the delayed Na(+)-K(+)-ATPase reactivation observed in pyruvate-reperfused hearts is not due to inhibition of PDH.

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Reperfusion of isolated mammalian hearts with a Ca2+-containing solution after a short Ca2+-free period at 37 degrees C results in massive influx of Ca2+ into the cells and irreversible cell damage: the Ca2+ paradox. Information about the free intracellular, cytosolic [Ca2+] ([Ca2+]i) during Ca2+ depletion is essential to assess the possibility of Ca2+ influx through reversed Na+/Ca2+ exchange upon Ca2+ repletion. Furthermore, the increase in end-diastolic pressure often seen during Ca2+-free perfusion of intact hearts may be similar to that seen during ischemia and caused by liberation of Ca2+ from intracellular stores. Therefore, in this study, we measured [Ca2+]i during Ca2+-free perfusion of isolated rat hearts. To this end, the fluorescent indicator Indo-1 was loaded into isolated Langendorff-perfused hearts and Ca2+-transients were recorded. Ca2+-transients disappeared within 1 min of Ca2+ depletion. Systolic [Ca2+]i during control perfusion was 268 +/- 54 nM. Diastolic [Ca2+]i during control perfusion was 114 +/- 34 nM and decreased to 53 +/- 19 nM after 10 min of Ca2+ depletion. Left ventricular end-diastolic pressure (LVEDP) significantly increased from 13 +/- 4 mmHg during control perfusion after Indo-1 AM loading to 31 +/- 5 mmHg after 10 min Ca2+ depletion. Left ventricular developed pressure did not recover during Ca2+ repletion, indicating a full Ca2+ paradox. These results show that LVEDP increased during Ca2+ depletion despite a decrease in [Ca2+]i, and is therefore not comparable to the contracture seen during ischemia. Furthermore, calculation of the driving force for the Na+/Ca2+ exchanger showed that reversed Na+/Ca2+ exchange during Ca2+ repletion is not able to increase [Ca2+]i to cytotoxic levels.

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OBJECTIVE: Long-term exposure of the donor heart to high dosages of dopamine in the treatment of brain death-related hemodynamic deterioration has been shown to reduce myocardial phosphocreatine (PCr) and adenosine triphosphate (ATP) in myocardial biopsy specimens and may preclude heart donation for transplantation. Short-term exposure to the acute catecholamine release during the onset of brain death has shown an unchanged PCr/ATP ratio using in vivo phosphorus-31 magnetic resonance spectroscopy (31P MRS). In this study 31P MRS was used to evaluate in vivo myocardial energy metabolism during long-term dopamine treatment. METHODS: Twelve cats were studied in a 4.7 Tesla magnet for 360 minutes. At t = 0 minutes, brain death was induced (n = 6). At 210 minutes, when myocardial workload in the brain-death group was reduced significantly, dopamine was infused (n = 12) at 5 microg/kg/min and its dose was consecutively doubled every 30 minutes and was withheld during the last 30 minutes of the experiment. Phosphorus-31 magnetic resonance spectra were obtained from the left ventricular wall during 5-minute time frames, and PCr/ATP ratios were calculated. The hearts were histologically examined. RESULTS: Although significant changes in myocardial workload were observed after the induction of brain death and during support and withdrawal of dopamine in both groups, the initial PCr/ATP ratio of 2.00+/-0.12 and the contents of PCr and ATP did not vary significantly. Histologically identified sub-endocardial hemorrhage was observed in 3 of 6 of the brain-dead animals and in 1 of 6 of the control animals. CONCLUSIONS: High dosages of dopamine in the treatment of brain death-related reduced myocardial workload do not alter PCr/ATP ratios and the contents of PCr and ATP of the potential donor heart despite histologic damage.

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A reliable, sensitive, non-invasive alternative for transvenous endomyocardial biopsy in detecting cardiac allograft rejection is desirable for optimal management of heart transplant patients. To establish whether (31)P magnetic resonance spectroscopy can become a non-invasive tool for detecting cardiac allograft rejection, the cardiac high-energy phosphate metabolism of human heart transplants was serially examined in 13 patients by means of (31)P MRS from post-operative day 13 to day 294, and compared with histologic evaluation of endomyocardial biopsies. Biopsy scores of 2 or higher, according to the Working Formulation criteria of Billingham et al., were considered to indicate rejection. Logistic regression, which was corrected for differences between the individual patients and the time after transplantation, showed no significant correlation between the occurrence of histologically detected rejection and the PCr:ATP ratio. However, using an analysis of variance, the PCr:ATP ratios of non-rejecting cases obtained within 50 days after transplantation (mean: 27 +/- 11 days) appeared to be significantly different from those obtained after post-operative day 50 [0.95 +/- 0.17 (n = 25) vs 1.17 +/- 0.17 (n = 32), mean +/- SD; p < 0.01]. No significant difference was observed between the PCr:ATP ratios obtained 100 days after transplantation (mean: 162 +/- 52 days) and the PCr:ATP ratios in the hearts of healthy volunteers [1.18 +/- 0. 18 (n = 19) and 1.23 +/- 0.17 (n = 6), mean +/- SD, respectively; p = 0.55]. The PCr:ATP ratio in transplanted human hearts is not a sensitive indicator for the detection of early acute human cardiac allograft rejection. This may be due to a temporarily altered high-energy phosphate metabolism early after transplantation irrespective of rejection.

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BACKGROUND: Hemodynamic deterioration resulting from brain death-induced myocardial left ventricular dysfunction may preclude heart donation. A reduced myocardial high-energy phosphate content, assessed by biopsy specimens, has been suggested to be responsible for this phenomenon. By applying phosphorus 31 magnetic resonance spectroscopy, in vivo myocardial high-energy phosphate metabolism can be studied continuously. METHODS: Twelve cats were sedated, intubated, ventilated, and studied for 240 minutes. Heart rate, arterial blood pressure, and arterial blood gases were monitored. Central venous pressure was kept constant. Myocardial work was expressed as rate-pressure product (RPP=heart rate x systolic arterial blood pressure). After sternotomy a radio frequency surface coil was positioned onto the left ventricle. A parietal trephine hole was drilled, and an inflatable balloon was inserted. The animal was placed into a 4.7 T horizontal 40 cm bore magnet interfaced to a spectrometer. Brain death (n=6) was induced by rapid inflation of the balloon; the six other cats served as a sham-operated control group. 31P spectra were obtained in 30 seconds, with ventilation and arterial blood pressure curve triggering. The phosphocreatine/to/adenosine triphosphate ratio, as an estimator of energy metabolism, was calculated. RESULTS: Brain death was established within 30 seconds after inflation of the balloon. Changes in RPP were characterized by a triphasic profile with a maximum increase from 19.3+/-1.4 x 10(3) to 87.5+/-8.1 x 10(3) mm Hg x min(-1) (p < .0001 vs control group) at 2 minutes after inflation of the balloon. Subsequently, RPP decreased and was normalized at 15 minutes after inflation. The third phase was characterized by hemodynamic deterioration, which became significant at 180 minutes and resulted in mean arterial pressure of 71+/-12 mm Hg (p < .05 vs control group) at the end of the experimental period. RPP deteriorated to 14.6+/-2.0 x 10(3) mm Hg x min(-1) (p < .05 vs control group) at 240 minutes. Because the heart rate remained constant during the third phase, the decrease in RPP was caused by a decrease in systolic arterial blood pressure. The initial phosphocreatine/adenosine triphosphate ratio of 1.65+/-0.16 varied to 1.52+/-0.06 at 2 minutes, and to 1.73 +/-0.17 (all values NS vs control group and vs initial ratio) at 240 minutes. CONCLUSIONS: The energy status of the heart is not affected by brain death. Therefore brain death-induced hemodynamic deterioration is not caused by impaired myocardial high-energy phosphate metabolism.

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The effect of ischemia and reperfusion on transverse relaxation (T2) of intracellular Na+ (Na+i) was measured with 5-min time resolution in isolated rat hearts. Nai T2 relaxation was biexponential with 28+/-7% fast (T2f) and 72+/-7% slow (T2s) decay. This ratio was constant throughout the protocol. During 20 min of ischemia, Na(i) T(2s) increased from 18.9+/-2.7 ms to 26.4+/-1.1 ms (P < 0.001), whereas T2f did not change significantly (3.1+/-1.8 versus 2.3+/-1.6 ms during control), and Na+i increased from 9.0+/-1.0 to 19.5+/-1.0 mmol/liter (P < 0.001). T(2s) and Na(+)i declined again during reperfusion. Changes in T2s relaxation correlated significantly (r = 0.73, P < 0.001) with the time course of Na+i.

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P-31 nuclear magnetic resonance (NMR) is uniquely suited to measure the kinetics of the phosphoryl-exchange reaction catalyzed by creatine kinase in intact mammalian tissue, especially striated muscle. Recently developed transgenic mouse models of the creatine kinase iso-enzyme system open novel opportunities to assess the functional importance of the individual iso-enzymes and their relative contribution to the total in situ flux through the CK reaction. This chapter reviews the most recent findings from NMR flux measurements on such genetic models of CK function. Findings in intact mouse skeletal and cardiac muscle in vivo are compared to data from purified mitochondrial and cytosolic creatine kinase in vitro. The relevance of findings in transgenic animals for the function of CK in wild-type tissue is described and the perspectives of transgenic techniques in future quantitative studies on the creatine kinase iso-enzyme system are indicated.

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A mathematical model of the compartmentalized energy transfer in cardiac cells is described and used for interpretation of novel experimental data obtained by using phosphorus NMR for determination of the energy fluxes in the isolated hearts of transgenic mice with knocked out creatine kinase isoenzymes. These experiments were designed to study the meaning and importance of compartmentation of creatine kinase isoenzymes in the cells in vivo. The model was constructed to describe quantitatively the processes of energy production, transfer, utilization, and feedback between these processes. It describes the production of ATP in mitochondrial matrix space by ATP synthase, use of this ATP for phosphocreatine production in the mitochondrial creatine kinase reaction coupled to the adenine nucleotide translocation, diffusional exchange of metabolites in the cytoplasmic space, and use of phosphocreatine for resynthesis of ATP in the myoplasmic creatine kinase reaction. It accounts also for the recently discovered phenomenon of restricted diffusion of adenine nucleotides through mitochondrial outer membrane porin pores (VDAC). Practically all parameters of the model were determined experimentally. The analysis of energy fluxes between different cellular compartments shows that in all cellular compartments of working heart cells the creatine kinase reaction is far from equilibrium in the systolic phase of the contraction cycle and approaches equilibrium only in cytoplasm and only in the end-diastolic phase of the contraction cycle. Experimental determination of the relationship between energy fluxes by a 31P-NMR saturation transfer method and workload in isolated and perfused heart of transgenic mice deficient in MM isoenzyme of the creatine kinase, MM-/-showed that in the hearts from wild mice, containing all creatine kinase isoenzymes, the energy fluxes determined increased 3-4 times with elevation of the workload. By contrast, in the hearts in which only the mitochondrial creatine kinase was active, the energy fluxes became practically independent of the workload in spite of the preservation of 26% of normal creatine kinase activity. These results cannot be explained on the basis of the conventional near-equilibrium theory of creatine kinase in the cells, which excludes any difference between creatine kinase isoenzymes. However, these apparently paradoxical experimental results are quantitatively described by a mathematical model of the compartmentalized energy transfer based on the steady state kinetics of coupled creatine kinase reactions, compartmentation of creatine kinase isoenzymes in the cells, and the kinetics of ATP production and utilization reactions. The use of this model shows that: (1) in the wild type heart cells a major part of energy is transported out of mitochondria via phosphocreatine, which is used for complete regeneration of ATP locally in the myofibrils--this is the quantitative estimate for PCr pathway; (2) however, in the absence of MM-creatine kinase in the myofibrils in transgenic mice the contraction results in a very rapid rise of ADP in cytoplasmic space, that reverses the mitochondrial creatine kinase reaction in the direction of ATP production. In this way, because of increasing concentrations of cytoplasmic ADP, mitochondrial creatine kinase is switched off functionally due to the absence of its counterpart in PCr pathway, MM-creatine kinase. This may explain why the creatine kinase flux becomes practically independent from the workload in the hearts of transgenic mouse without MM-CK. Thus, the analysis of the results of studies of hearts of creatine kinase-deficient transgenic mice, based on the use of a mathematical model of compartmentalized energy transfer, show that in the PCr pathway of intracellular energy transport two isoenzymes of creatine kinase always function in a coordinated manner out of equilibrium, in the steady state, and disturbances in functioning of one of them inevitably result

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Although usually steady-state fluxes and metabolite levels are assessed for the study of metabolic regulation, much can be learned from studying the transient response during quick changes of an input to the system. To this end we study the transient response of O2 consumption in the heart during steps in heart rate. The time course is characterized by the mean response time of O2 consumption which is the first statistical moment of the impulse response function of the system (for mono-exponential responses equal to the time constant). The time course of O2 uptake during quick changes is measured with O2 electrodes in the arterial perfusate and venous effluent of the heart, but the venous signal is delayed with respect to O2 consumption in the mitochondria due to O2 diffusion and vascular transport. We correct for this transport delay by using the mass balance of O2, with all terms (e.g. O2 consumption and vascular O2 transport) taken as function of time. Integration of this mass balance over the duration of the response yields a relation between the mean transit time for O2 and changes in cardiac O2 content. Experimental data on the response times of venous [O2] during step changes in arterial [O2] or in perfusion flow are used to calculate the transport time between mitochondria and the venous O2 electrode. By subtracting the transport time from the response time measured in the venous outflow the mean response time of mitochondrial O2 consumption (tmito) to the step in heart rate is obtained. In isolated rabbit heart we found that tmito to heart rate steps is 4-12 s at 37 degrees C. This means that oxidative phosphorylation responds to changing ATP hydrolysis with some delay, so that the phosphocreatine levels in the heart must be decreased, at least in the early stages after an increase in cardiac ATP hydrolysis. Changes in ADP and inorganic phosphate (Pi) thus play a role in regulating the dynamic adaptation of oxidative phosphorylation, although most steady state NMR measurements in the heart had suggested that ADP and Pi do not change. Indeed, we found with 31P-NMR spectroscopy that phosphocreatine (PCr) and Pi change in the first seconds after a quick change in ATP hydrolysis, but remarkably they do this significantly faster (time constant approximately 2.5 s) than mitochondrial O2 consumption (time constant 12 s). Although it is quite likely that other factors besides ADP and Pi regulate cardiac oxidative phosphorylation, a fascinating alternative explanation is that the first changes in PCr measured with NMR spectroscopy took exclusively place in or near the myofibrils, and that a metabolic wave must then travel with some delay to the mitochondria to stimulate oxidative phosphorylation. The tmito slows with falling temperature, intracellular acidosis, and sometimes also during reperfusion following ischemia and with decreased mitochondrial aerobic capacity. In conclusion, the study of the dynamic adaptation of cardiac oxidative phosphorylation to demand using the mean response time of cardiac mitochondrial O2 consumption is a very valuable tool to investigate the regulation of cardiac mitochondrial energy metabolism in health and disease.

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Hearts of wild-type and cytosolic muscle creatine kinase (M-CK)-knockout mice were perfused with Krebs-Henseleit buffer containing 10 mM glucose and 5 mM pyruvate and studied during pacing at 400 and 600 beats/min and during K+ arrest. Phosphocreatine (PCr) and ATP concentrations in M-CK-deficient hearts were not significantly different from those in wild-type hearts. With the use of 31P NMR saturation transfer, the flux mediated predominantly by mitochondrial creatine kinase (Mi-CK) was clearly detected in M-CK-deficient hearts. Mi-CK flux was 4.8 +/- 0.6 and 4.5 +/- 0.6 mM/s during pacing at 400 and 600 beats/min, respectively, and was 3. 5 +/- 0.4 mM/s during cardiac arrest. In control hearts total CK flux was 7.8 +/- 1.1 and 6.6 +/- 1.3 mM/s during pacing at 400 and 600 beats/min, respectively, and decreased to 3.8 +/- 0.5 mM/s during arrest. It is suggested that the relative contribution of Mi-CK to the total NMR-measured CK flux in the wild-type heart is higher than that of the homodimeric M-CK isoform (MM-CK).

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Ca2+ paradox damage has been suggested to be determined by Na+ entry during Ca2+ depletion and exchange of Na+ for Ca2+ during Ca2+ repletion. Since previously a Ca2+ paradox without prior increase of total intracellular [Na+] ([Na+]i) has been observed, we investigated whether local accumulation of Na+ close to the inner side of the sarcolemma during Ca2+ depletion plays a role in the Ca2+ paradox by replacing all extracellular Na+ by Li+ 5 min before and during 10 min Ca2+-free perfusion (37°C) in isolated rat hearts (group I). Subsequently, hearts were perfused with a standard, Na+- and Ca2+-containing solution. Verapamil was used to prevent contracture due to the absence of Na+/Ca2+ exchange during Na+-free perfusion in the presence of Ca2+. In group II, the Ca2+-free period was omitted, and in group III normal extracellular [Na+] was used throughout. 23Na-NMR was used to monitor intra- and extracellular Na+ signals. Total creatine kinase release was 2,977±413, 36±24 and 3170±297 IU/g dry weight in groups I, II and III respectively, indicating a full Ca2+ paradox in groups I and III. [Na+]i decreased from 11.3±0.6 mM during control perfusion to 1.2±0.4 mM after 10 min Ca2+ depletion in group I, whereas in group III [Na+]i was 10.9±2.2 mM during control perfusion and did not change significantly after 10 min Ca2+-free perfusion. It is concluded that accumulation of Na+ close to the inner side of the sarcolemma during Ca2+ depletion is not a prerequisite for the Ca2+ paradox.

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Limited time resolution has hampered proper evaluation of changes in intracellular Na+ (Na+i) in whole hearts upon post-ischemic reperfusion. In isolated rat hearts perfused at 37 degrees C, we studied the contribution of the Na+-K+ ATPase and the Na+-H+ exchanger to control of Na+i during reperfusion using 23Na NMR and the shift reagent Tm(DOTP)5- with a time resolution of 5 s. To assess activities of the Na +-K+ ATPase and the Na+-H+ exchanger, 250 micro mol/l ouabain and/or 3 micro mol/l EIPA, respectively, was added to the perfusate during the first 5 min of reperfusion, following 20 min of ischemia. When used, ouabain was also present for 2 min prior to ischemia. Na+i increased during ouabain perfusion prior to ischemia (132+/-5 and 133+/-4% of the pre-ischemic control value after 2 min, in ouabain and ouabain+EIPA hearts, respectively; mean+/-s.e.m.; n=6 per group) resulting in higher end-ischemic values in ouabain and ouabain+EIPA hearts (249+/-9 and 267+/-17% of the pre-ischemic control value, respectively) than in control and EIPA hearts (207+/-21 and 199+/-10% of the pre-ischemic control value, respectively). In ouabain, hearts Na+i started to rise directly upon reperfusion and amounted to 117+/-6% of the end-ischemic value after 60 s of reperfusion. In control hearts, however, Na+i dropped immediately and was 87+/-5% of the end-ischemic value after 60 s, indicating that the Na+-K+ ATPase resumed function directly upon reperfusion. The initial steep increase of Na+i upon reperfusion in ouabain hearts, which diminished after approximately 40 s to the rate of increase observed during ischemia, was absent in ouabain + EIPA hearts. This indicates the existence, although masked by Na+-K+ ATPase activity, of a Na+-H + exchange mediated Na+ influx upon reperfusion. If only EIPA was present during reperfusion the initial decrease in Na+i was faster than in control hearts, corroborating this finding.

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