Effect of vagus nerve stimulation on tissue damage and function loss in a mouse myocardial ischemia-reperfusion model.

OBJECTIVES: In cardiac ischemia, acute inflammatory responses further increase the detrimental effect on myocardial tissue. Since vagus nerve stimulation (VS) attenuates inflammatory responsiveness this study examines the effect of VS on myocardial damage development in a cardiac ischemia-reperfusion (IR) mouse model. METHODS: 54 male C57Bl/6j mice were subjected to an IR procedure with or without prior VS. The effects on inflammatory responsiveness, infarct size, cardiac function, neutrophils, lymphocytes and vascular endothelial growth factor (VEGF) in the infarcted myocardium were measured at 48 h after intervention. Group results were compared with unpaired Mann-Whitney or Kruskall-Wallis test. RESULTS: A significant decrease in inflammatory responsiveness was not verified by decreased TNFα levels in blood from VS and IR treated mice. The percentage infarct size over area at risk was smaller in the group with VS + IR compared with IR (22.4 ±â€¯10.2% vs 37.6 ±â€¯9.0%, p = 0.003). The degree of the reduction in cardiac function was not different between the IR groups with or without VS and no group differences were found in amounts of neutrophils, CD3+ lymphocytes and VEGF in the reperfused mouse heart. CONCLUSION: The present study does not provide clear evidence of a reducing role for VS on cardiac function loss. This could mean that VS has a less inhibiting effect on myocardial inflammation than may be expected from the literature.

Brain death-related energetic failure of the donor heart becomes apparent only during storage and reperfusion: an ex vivo phosphorus-31 magnetic resonance spectroscopy study on the feline heart.

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.

Postischemic Na(+)-K(+)-ATPase reactivation is delayed in the absence of glycolytic ATP in isolated rat hearts.

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.

Bio-energetic response of the heart to dopamine following brain death-related reduced myocardial workload: a phosphorus-31 magnetic resonance spectroscopy study in the cat.

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.

Brain death-induced alterations in myocardial workload and high-energy phosphates: a phosphorus 31 magnetic resonance spectroscopy study in the cat.

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.

Experimentally induced stress in rheumatoid arthritis of recent onset: effects on peripheral blood lymphocytes.

OBJECTIVE: To examine the effects of experimentally-induced stress on the mobilization of peripheral blood lymphocytes (PBL) in patients with rheumatoid arthritis (RA) of recent onset. METHODS: Twenty-two (16 F, 6 M) patients (mean age 57.6 yrs.) and 23 (15 F, 8 M) healthy subjects (mean age 54.7 yrs.) were subjected to experimental stressors. The numbers of T-cells, B-cells, and NK-cells were determined before and after the completion of tasks inducing physical and mental effort. RESULTS: The change in PBL in response to stress was about equal for patients and healthy subjects (p > 0.75 in all PBL subsets). In patients as well as in healthy subjects, the correlations between PBL and cortisol changes in response to stress tended to be positive, while the correlations between PBL and cardiovascular changes were positive in healthy subjects, but zero or negative in patients. Moderate to high (0.32 < or = r < or = 0.55) correlations between PBL changes and pain were observed. CONCLUSION: Experimentally-induced changes in PBL (as well as cortisol) are normal in patients with early RA who are receiving long term medication, but correlations between these changes and autonomic nervous system responses are zero or negative. This apparent shift in the control of the change in PBL in response to stress is observed in particular in patients with more pain. The pathophysiological significance of these findings should be clarified in longitudinal studies.

Mathematical model of compartmentalized energy transfer: its use for analysis and interpretation of 31P-NMR studies of isolated heart of creatine kinase deficient mice.

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

31P NMR studies of creatine kinase flux in M-creatine kinase-deficient mouse heart.

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).

Fluxes through cytosolic and mitochondrial creatine kinase, measured by P-31 NMR.

The kinetic properties of the cytoplasmic and the mitochondrial iso-enzymes of creatine kinase from striated muscle were studied in vitro and in vivo. The creatine kinase (CK) iso-enzyme family has a multi-faceted role in cellular energy metabolism and is characterized by a complex pattern of tissue-specific expression and subcellular distribution. In mammalian tissues, there is always co-expression of at least two different CK isoforms. As a result, previous studies into the role of CK in energy metabolism have not been able to directly differentiate between the individual CK species. Here, we describe experiments which were directed at achieving this goal. First, we studied the kinetic properties of the muscle-specific cytoplasmic and mitochondrial CK isoforms in purified form under in vitro conditions, using a combination of P-31 NMR and spectrophotometry. Secondly, P-31 NMR measurements of the flux through the CK reaction were carried out on intact skeletal and heart muscle from wild-type mice and from transgenic mice, homozygous for a complete deficiency of the muscle-type cytoplasmic CK isoform. Skeletal muscle and heart were compared because they differ strongly in the relative abundance of the CK isoforms. The present data indicate that the kinetic properties of cytoplasmic and mitochondrial CK are substantially different, both in vitro and in vivo. This finding particularly has implications for the interpretation of in vivo studies with P-31 NMR.

Acute increase of myocardial workload, hemodynamic instability, and myocardial histological changes induced by brain death in the cat.

Brain death-related hemodynamic instability may preclude donor heart procurement. The relationships between the initial changes of myocardial workload, hemodynamic deterioration, and myocardial histological changes caused by acute induction of brain death are unclear. Cats (n = 15) were submitted to brain death by rapid inflation of an intracranial balloon. A further 12 cats served as a sham-operated control group. The changes in heart rate, mean arterial blood pressure, systolic and diastolic arterial blood pressure, left ventricular developed pressure, LV dP/dtmax, rate-pressure product (RPP), and circulating noradrenaline and adrenaline were studied during 240 min after the induction of brain death. Central venous pressure was kept constant. The hearts were histologically examined afterward. Electrocerebral activity disappeared within 30 sec after balloon inflation. At 3 min, noradrenaline and adrenaline levels had increased 75- and 40-fold, respectively, compared to pre-induction levels. The hemodynamic response was characterized by an early and rapid increase of hemodynamic variables at 2.9 +/- 0.2 min. This was followed by a second phase of normalization or deterioration. Two distinct subgroups (n = 9) became hemodynamically unstable (HDU), characterized by a systolic arterial blood pressure < 90 mm Hg, at 108 +/- 29 min, and progressively deteriorated to 67 +/- 8 mm Hg at 240 min after inflation of the balloon. The hemodynamic variables of the other, hemodynamically stable (HDS), subgroup (n = 6) normalized at 60 min after inflation. Hemodynamic deterioration of the HDU subgroup compared to the HDS subgroup was significant at 10 min after induction of brain death. The maximum values of RPP were similar in the two subgroups. Respiratory and metabolic variables at the end of the experiment were not different in both subgroups. Histological evidence of myocardial damage was present in 73% (11/15) of the brain dead cats and absent in the control group. The histological changes were identified both in hearts of HDU (6/9) and HDS (5/6) cats. In the cat, no relationships were demonstrated between the acute increase of myocardial workload, the occurrence of hemodynamic deterioration, and myocardial histological changes after rapid induction of brain death. These results may contribute to the discussion whether hemodynamic instability of the donor is an appropriate exclusion criterion for heart transplantation.

The role of the Na+ channel in the accumulation of intracellular Na+ during myocardial ischemia: consequences for post-ischemic recovery.

To further elucidate the role of the Na+ channel in the ischemic accumulation of intracellular Na+ (Na+i), 200 microM lidocaine was included in the perfusate for 5 min prior to 30 min of ischemia in isolated rat hearts paced at 5 Hz. Na+i and high-energy phosphates were measured, using 23Na-NMR with the shift reagent TmDOTP5- and 31P-NMR, respectively. Control values of phosphocreatine (PCr) and ATP were 14.1 +/- 1.5 mM and 7.7 +/- 0.7 mM, respectively (all data: mean +/- S.D.). During lidocaine perfusion the rate pressure product (RPP) decreased by approximately 50% and Na+i declined from 11.5 +/- 1.5 mM to 9.8 +/- 2.1 mM. During ischemia Na+i in lidocaine hearts rose to 17.9 +/- 2.5 mM v 28.4 +/- 1.7 mM in control hearts (P<0.05). In hearts in which extracellular Ca2+ was lowered prior to ischemia to reach a similar RPP decrease as in lidocaine hearts, Na+i rose to 26.3 +/- 3.0 mM during ischemia (P<0.05 v lidocaine, N.S. v control). Lidocaine did not affect the decline of PCr during ischemia (to 0.5 +/- 0.5 v 0.7 +/- 0.8 mM in lidocaine and control hearts, respectively) but significantly attenuated the initial decrease of pH(i) (6.06 +/- 0.07 v 5.76 +/- 0.04 after 20 min, P<0.01), attenuated the initial decline of ATP (3.3 +/- 1.3 v 1.5 +/- 0.9 mM after 20 min, P<0.05) and delayed the time to onset of contracture. However, at the end of ischemia pH(i) (5.73 +/- 0.04 and 5.78 +/- 0.05) and ATP (1.2 +/- 0.6 and 0.9 +/- 0.8 mM) were not significantly different. At 30 min of reperfusion Na+i was 14.9 +/- 2.6 mM in lidocaine hearts v 20.0 +/- 3.1 mM in controls. PCr (9.6 +/- 2.3 v 4.9 +/- 0.9 mM, P<0.05) and ATP (3.0 +/- 0.6 v 1.8 +/- 0.6 mM) recovered better in lidocaine hearts. Furthermore, developed and end-diastolic pressure recovered better in lidocaine hearts. In conclusion, Na+ influx during ischemia occurs, at least partly, via the Na+ channels, and blocking this channel during ischemia improves post-ischemic functional and metabolic recovery.

Abnormal myotonic dystrophy protein kinase levels produce only mild myopathy in mice.

Myotonic dystrophy (DM) is commonly associated with CTG repeat expansions within the gene for DM-protein kinase (DMPK). The effect of altered expression levels of DMPK, which is ubiquitously expressed in all muscle cell lineages during development, was examined by disrupting the endogenous Dmpk gene and overexpressing a normal human DMPK transgene in mice. Nullizygous (-/-) mice showed only inconsistent and minor size changes in head and neck muscle fibres at older age, animals with the highest DMPK transgene expression showed hypertrophic cardiomyopathy and enhanced neonatal mortality. However, both models lack other frequent DM symptoms including the fibre-type dependent atrophy, myotonia, cataract and male-infertility. These results strengthen the contention that simple loss- or gain-of-expression of DMPK is not the only crucial requirement for development of the disease.

Cardiac high-energy phosphates adapt faster than oxygen consumption to changes in heart rate.

To investigate the dynamic control of cardiac ATP synthesis, we simultaneously determined the time course of mitochondrial oxygen consumption with the time course of changes in high-energy phosphates following steps in cardiac energy demand. Isolated isovolumically contracting rabbit hearts were perfused with Tyrode's solution at 28 degrees C (n = 7) or at 37 degrees C (n = 7). Coronary arterial and venous oxygen tensions were monitored with fast-responding oxygen electrodes. A cyclic pacing protocol in which we applied 64 step changes between two different heart rates was used. This enabled nuclear magnetic resonance measurement of the phosphate metabolites with a time resolution of approximately 2 seconds. Oxygen consumption changed after heart-rate steps with time constants of 14 +/- 1 (mean +/- SEM) seconds at 28 degrees C and 11 +/- 1 seconds at 37 degrees C, which are already corrected for diffusion and vascular transport delays. Doubling of the heart rate resulted in a significant decrease in phosphocreatine (PCr) content (11% at 28 degrees C, 8% at 37 degrees C), which was matched by an increase in inorganic phosphate (P(i)) content, although oxygen supply was shown to be nonlimiting. The time constants for the change of both P(i) and PCr content, approximately 5 seconds at 28 degrees C and 2.5 seconds at 37 degrees C, are significantly smaller than the respective time constants for oxygen consumption.(ABSTRACT TRUNCATED AT 250 WORDS)