Tom J.C. Ruigrok

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.

Cytosolic Ca2+ concentration during Ca2+ depletion of isolated rat hearts.

Reperfusion of isolated mammalian hearts with a Ca2+-containing solution after a short Ca2+-free period at 37°: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.

An increase in intracellular [Na+] during Ca2+ depletion is not related to Ca2+ paradox damage in rat hearts

Ca2+paradox damage has been suggested to be determined by Na+ entry during Ca2+ depletion and exchange of Na+ for Ca2+ during Ca2+ repletion. With the use of23Na nuclear magnetic resonance, we previously observed a Ca2+ paradox without a prior Na+ increase. We have now demonstrated a Na+ increase during Ca2+ and Mg2+ depletion without the occurrence of the Ca2+ paradox during Ca2+ repletion. Isolated rat hearts were perfused for 20 min with a Ca2+-free or a Ca2+- and Mg2+-free (Ca2+/Mg2+-free) solution under hypothermic conditions (20 and 25°C). Intracellular Na+ concentration ([Na+]i) increased from 11.9 ± 1.2 to 26.9 ± 5.8 mM ( P < 0.001) during Ca2+/Mg2+-free perfusion at 20°C, whereas no significant change in [Na+]ioccurred during 20 min of Ca2+-free perfusion at 20°C. In addition, we confirmed that [Na+]idid not change significantly during 20 min of normothermic Ca2+-free perfusion. Creatine kinase release during normothermic Ca2+ repletion in the 20°C groups was ∼10% and in the 25°C groups 75% of the release in the normothermia group. Recovery of rate-pressure product was ∼50% in the 20°C groups versus 0% in the normothermia group. In conclusion, hypothermic Ca2+/Mg2+-free perfusion results in a significant increase of [Na+]i, which does not contribute to the extent of the Ca2+ paradox on normothermic Ca2+ repletion.

Heterotopic heart transplantation alters high-energy phosphate metabolism irrespective of cardiac allograft rejection

This study was undertaken to validate the potential of 31P magnetic resonance spectroscopy (MRS) as a noninvasive alternative for transvenous endomyocardial biopsy in detecting cardiac allograft rejection. Donor hearts from either Lewis rats (L) or Brown-Norway rats (BN) were transplanted into the neck of L rats resulting in a non-rejecting group L-L and a rejecting group L-BN. L-L and L-BN rats were serially studied by means of 31P MRS from postoperatine day 1–8. In addition, rejection was confirmed by histology. A similar, marked decrease in phosphocreative/β-adenosinetriphosphate (PCr/ATP) ratio from day 1–3 was observed in both L-L and L-BN hearts. This ratio levelled off on postoperative day 3 and remained depressed on subsequent postoperative days in both groups, although histology showed an increase in the severity of rejection in L-BN. However, the PCr signal/noise ratio in L-BN started to decrease after day 4, coinciding with the histologic evidence of severe rejection (score IV), whereas in L-L hearts (score 0) this ratio remained unaltered until day 8. Since high-energy phosphate metabolism is affected by the unloaded status of the heterotopically transplanted heart, irrespective of rejection, the PCr/ATP ratio appears not to be a specific marker for the detection of acute rejection in this model. In contrast, the PCr S/N ratio appears to be a specific and sensitive marker of acute rejection, but only in a late, severe stage.

Lidocaine does not prevent the calcium paradox in rat hearts: A laser microprobe mass analysis (LAMMA) study

The calcium paradox stands for the cell damage that occurs when isolated hearts are perfused with a Ca(2+)-free solution followed by perfusion with a Ca(2+)-containing solution. Although it is generally accepted that a massive Ca2+ influx during the Ca(2+)-repletion phase is responsible for the cell damage, there is no consensus about what makes the heart susceptible to the calcium paradox during the Ca(2+)-depletion phase. It has been suggested that the extent of the calcium paradox is primarily determined by accumulation of Na+ during Ca2+ depletion and a subsequent accumulation of Ca2+ via reverse Na(+)-Ca2+ exchange during Ca2+ repletion. According to another theory, weakening of intercalated disc junctions during Ca2+ depletion and contracture-mediated disruption of the cell membrane during Ca2+ repletion are responsible for the calcium paradox. In the present study we further investigated the possible role of Na+ in the development of the calcium paradox. During Ca2+ depletion, lidocaine was used to inhibit Na+ entry through the Na+ channels. Isolated rat hearts were perfused with Krebs Henseleit buffer (KH) containing 1.4 mM Ca2+ for 15 min, followed by 10 min of Ca(2+)-free perfusion and 10 min of reperfusion with Ca2+. In the treated group 0.1 mM lidocaine was present throughout the experiment. At the end of each experiment, Ca2+ cytochemistry was performed and the intracellular Ca2+ content was analyzed by laser microprobe mass analysis (LAMMA). The results show that during Ca2+ depletion, the intracellular Ca2+ content did not change significantly. Ca2+ repletion, however, gave rise to a full calcium paradox irrespective of the presence of lidocaine: massive cell damage and Ca2+ accumulation in the mitochondria. The results provide further evidence that intracellular Na+ accumulation during Ca2+ depletion is not involved in the occurrence of the calcium paradox.

Glycolytic ATP production is not essential for Na+-K+ ATPase function and contractile recovery during postischemic reperfusion in isolated rat hearts

Accumulation of intracellular sodium (Nai +) during ischemia is an important determinant of calcium overload upon reperfusion via Na + -Ca 2 + exchange. Postischemic reperfusion of ischemic myocardium results in an immediate decrease of Na + via (resumption) of Na + -K + ATPase activity [1]. Since energy from glycolysis may be preferentially used to fuel membrane functions [2], in the present study the requirement of glycolytic ATP for the postischemic decline of Nai + was evaluated.

The effect of hypothermia during the period of calcium repletion on the calcium paradox

SummaryReperfusion of an isolated heart with a calcium-containing solution after a short calcium-free perfusion may result in irreversible cell damage: the calcium paradox. In this investigation the effect of hypothermia during reperfusion with calcium-containing solution on the calcium paradox damage in the isolated rat heart was studied. In addition, the effect of pre-cooling the heart during the calcium-free period was investigated. Creatine kinase release was used to define cell damage. Normothermic (37°C) calcium-free perfusion followed by normothermic reperfusion with calcium-containing solution resulted in a massive release of CK. When the normothermic calcium-free perfusion was followed by hypothermic (10°C) calcium-containing reperfusion, CK release was reduced by 20% (P<0.005). This CK release during reperfusion was further reduced by 55% and 80% when the normothermic calcium-free perfusion was followed by 5 or 10 min respectively of hypothermic calcium-free perfusion prior to the hypothermic calcium-containing reperfusion. The results show that hypothermia during the period of calcium repletion retards the sequence of events which ultimately results in release of large amounts of intracellular components.

Pathophysiology of Acute or Short-Term Hibernation

Myocardial hibernation refers to a clinical state of chronic regional contractile dysfunction characterized by a reduced regional myocardial blood flow, either persistently at rest1-2 or repetitively during stress 3, that can be partially or completely restored to normal upon coronary revascularization. In hibernation, the observed reduction in function reflects preservation of viability rather than the occurrence of necrosis. Stress echocardiography using low-dose dobutamine infusion is at present the preferred initial approach for the selection of patients with hibernating or viable myocardium who would benefit from coronary revascularization 4. Additional techniques to assess viability include fluorine-18(18F) fluorodeoxyglucose positron emission tomography (FDG-PET), technetium- 99m (99mTc) sestamibi single photon emission tomography (SPECT), or thallium-201 (201TI) rest-redistribution SPECT imaging. Although the concept of chronic adaptive reduction of contractile function in response to reduction in myocardial blood flow is straightforward and simple, the mechanisms responsible for the development and maintenance of hibernation are unclear at present. This is mainly due to a large distance between the available experimental models of (acute or short-term) hibernation and the clinical scenario of (chronic or long-term) hibernation. In this chapter an experimental model of short-term hibernation will be discussed that is based on the observation that the majority of patients with hibernating myocardium has a history of an acute ischemic insult (either in the form of a transmural myocardial infarction or prolonged ischemic pain) followed by hypoperfusion5.

Recent advances of magnetic resonance spectroscopy in myocardial ischemia

The anatomical and functional information that can be obtained with nuclear magnetic resonance imaging (MRI) differs fundamentally from the biochemical information that can be provided in a unique, non-invasive way by nuclear magnetic resonance spectroscopy (MRS). Yet, the basic principles of MRI and MRS are quite similar. Nuclear magnetic resonance relies on the response of atomic nuclei, which are polarized by a strong magnetic field, to a radiofrequency (RF) pulse. Many atomic nuclei behave as magnetic dipoles due to a property called ‘spin’ and will orient themselves in a magnetic field, either parallel or antiparallel to the magnetic field direction. Since both orientations are not equally populated, a net magnetization results from the contributions of all nuclei. This net magnetization can be manipulated using RF-pulses, which induce transitions between the orientations of the tiny nuclear magnets and force them to rotate in phase. Macroscopically, the RF-pulses rotate the net magnetization vector from its equilibrium position along the magnetic field direction, which by convention is the z-axis, towards or into the xy-plane (90°pulse), or even further, resulting in inversion of the net magnetization (180°pulse). The extent of this rotation, the pulse angle, depends on the length of the pulse and the strength of the RF-field. Since the atomic nuclei are forced by the RF-field to rotate or spin in phase around the z-axis, so does the net magnetization vector, once it is displaced from the z-axis.