Andriy M. Babsky

Non-invasive magnetic resonance thermometry using thulium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (TmDOTA-)

Non-invasive thermometry is pivotal to the future advances of regional hyperthermia as a cancer treatment modality. Current magnetic resonance (MR) thermometry methods suffer from poor thermal resolution due to relatively weak dependence of chemical shift of the 1 H water signal on temperature. This study evaluated the feasibility of using thulium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (TmDOTA-) for MR thermometry. TmDOTA- is non-toxic and the gadolinium complex of DOTA4- is widely used as a MR contrast agent. The results demonstrate that the temperature dependence of the TmDOTA- proton shifts are about two orders of magnitudes higher than the water proton and, thus, provide excellent accuracy and resolution. In addition, TmDOTA - proton shifts are insensitive to the paramagnetic complex concentration, pH, Ca2+ or presence of plasma macromolecules and ions. Because hyperthermia is known to produce changes in tissue pH and other physiological parameters, these properties of TmDOTA- greatly simplify the procedures for using the lanthanide complex for MR thermometry. Application of TmDOTA- for measurement of temperature in a subcutaneously implanted human melanoma xenograft is demonstrated. Finally, the feasibility of imaging one of the 1H resonances of the lanthanide complex is demonstrated in phantom experiments. Overall, TmDOTA- appears to be a promising probe for MR thermometry in vivo.

Na+ Effects on Mitochondrial Respiration and Oxidative Phosphorylation in Diabetic Hearts

Intracellular Na+ is approximately two times higher in diabetic cardiomyocytes than in control. We hypothesized that the increase in Na+1 activates the mitochondrial membrane Na+/Ca2+ exchanger, which leads to loss of intramitochondrial Ca2+, with a subsequent alteration (generally depression) in bioenergetic function. To further evaluate this hypothesis, mitochondria were isolated from hearts of control and streptozotocin-induced (4 weeks) diabetic rats. Respiratory function and ATP synthesis were studied using routine polarography and 31P-NMR methods, respectively. While addition of Na+ (1–10 mM) decreased State 3 respiration and rate of oxidative phosphorylation in both diabetic and control mitochondria, the decreases were significantly greater for diabetic than for control. The Na+ effect was reversed by providing different levels of extramitochondrial Ca2+ (larger Ca2+ levels were needed to reverse the Na+ depressant effect in diabetes mellitus than in control) and by inhibiting the Na+/Ca2+exchanger function with diltiazem (a specific blocker of Na+/Ca2+ exchange that prevents Ca2+ from leaving the mitochondrial matrix). On the other hand, the Na+ depressant effect was enhanced by Ruthenium Red (RR, a blocker of mitochondrial Ca2+ uptake, which decreases intramitochondrial Ca2+). The RR effect on Na+ depression of mitochondrial bioenergetic function was larger in diabetic than control. These findings suggest that intramitochondrial Ca2+ levels could be lower in diabetic than control and that the Na+ depressant effect has some relation to lowered intramitochondrial Ca2+. Conjoint experiments with 31P-NMR in isolated superfused mitochondria embedded in agarose beads showed that Na+ (3–30 mM) led to significantly decreased ATP levels in diabetic rats, but produced smaller changes in control. These data support our hypothesis that in diabetic cardiomyocytes, increased Na+ leads to abnormalities of oxidative processes and subsequent decrease in ATP levels, and that these changes are related to Na+ induced depletion of intramitochondrial Ca2+.

Influence of ischemic preconditioning on intracellular sodium, pH, and cellular energy status in isolated perfused heart

The possible relationships between intracellular Na+ (Na1+), bioenergetic status and intracellular pH (pH1) in the mechanism for ischemic preconditioning were studied using 23Na and 31P magnetic resonance spectroscopy in isolated Langendorff perfused rat heart. The ischemic preconditioning (three 5-min ischemic episodes followed by two 5-min and one 10-min period of reperfusion) prior to prolonged ischemia (20 min stop-flow) resulted in a decrease in ischemic acidosis and faster and complete recovery of cardiac function (ventricular developed pressure and heart rate) after 30 min of reperfusion. The response of Na1 during ischemia in the preconditioned hearts was characterized by an increase in Na+1 at the end of preconditioning and an accelerated decrease during the first few minutes of reperfusion. During post-ischemic reperfusion, bioenergetic parameters (PCr/P1 and βATP/P, ratios) were partly recovered without any significant difference between control and preconditioned hearts. The reduced acidosis during prolonged ischemia and the accelerated decrease in Na1+ during reperfusion in the preconditioned hearts suggest activation of Na+/H+ exchanger and other ion transport systems during preconditioning, which may protect the heart from intracellular acidosis during prolonged ischemia, and result in better recovery of mechanical function (LVDP and heart rate) during post-ischemic reperfusion.

Simultaneous Measurement of Oxygen Consumption and 13C16O2 Production From 13C-Pyruvate in Diabetic Rat Heart Mitochondria

Several investigators have shown defects in energy production in hearts from animal models of diabetes mellitus (DM). These defects generally involve changes in oxidative phosphorylation (ox-phos) or in Krebs cycle functions. Abnormalities in ox-phos have been observed in heart mitochondria isolated from streptozotocin-diabetic (Pierce and Dhalla, 1985) and alloxan-diabetic (Puckett and Reddy, 1979) models as well as in genetic diabetic models (Kuo et al., 1983). In DM mitochondria, State 3 is decreased when a variety of substrates are used: Pyr plus malate and palmitylcarnitine plus malate (Kuo et al., 1983), a-ketoglutarate (Taegtmeyer et al, 1987), glutamate (Mokhtar et al., 1993), 3-hy-droxybutyrate and malate, and acetoacetate plus malate (Grinblat et al., 1986). The RCI also decreased in mitochondria from DM animals. The ADP/O ratio is depressed only in the early stages of chemically induced DM, returning to normal ranges later on, indicating that mitochondria from DM hearts have normal ability to transfer electrons from NADH to O2 or to couple oxidation to phosphorylation. This suggests that the deficiency of energy production is related to a decrease in substrate oxidation through the Krebs cycle. Two important cellular functions which may contribute to abnormal ox-phos are alterations in mi-tochondrial transporter systems (Kazmi et al., 1985; Paulson et al., 1984) and decrease in substrate supply. One possible mechanism of impaired substrate oxidation in the DM heart is abnormal mitochondrial Ca2+ uptake (Baba and Kako, 1991). It is well known that Ca2+ plays an important role in regulation of several matrix dehydrogenases (including PDH,

AMP promotes oxygen consumption and ATP synthesis in heart mitochondria through the adenylate kinase reaction: an NMR spectroscopy and polarography study.

Adenylate kinase plays an important role in cellular energy homeostasis by catalysing the interconversion of adenine nucleotides. The goal of present study was to evaluate the contribution of the adenylate kinase reaction to oxidative ATP synthesis by direct measurements of ATP using 31P NMR spectroscopy. Results show that AMP can stimulate ATP synthesis in the presence or absence of ADP. In particular, addition of 1 mM AMP to the 0.6 mM ADP superfusion system of isolated superfused mitochondria (contained and maintained in agarose beads) led to a 25% increase in ATP synthesis as measured by the increase in βATP signal. More importantly, we show that AMP can support ATP synthesis in the absence of ADP, demonstrated as follows. Superfusion of mitochondria without ADP led to the disappearance of ATP γ, α and β signals and the increase of Pi. Addition of AMP to the medium restored the production of ATP, as demonstrated by the reappearance of γ, α and β ATP signals, in conjunction with a decrease in Pi, which is being used for ATP synthesis. Polarographic studies showed Mg2+ dependence of this process, confirming the specificity of the adenylate kinase reaction. Furthermore, data obtained from this study demonstrate, for the first time, that different aspects of the adenylate kinase reaction can be evaluated with 31P NMR spectroscopy. Copyright

Metabolic Abnormalities and Differential Responses to Stress Associated with Hamster Cardiomyopathy

Abstract Metabolic differences between cardiomyopathic hamsters (CMHs), as they progress through various physiologic phases before reaching end-stage heart failure (HF), and healthy hamsters (HHs) are often difficult to demonstrate. We suggest that metabolic differences, magnified by application of chronic stress (S: cold immobilization 2 hr/day for 5 days) followed by acute stress (AS: 55 min global ischemia /30 min reperfusion), can be used to characterize different stages in this cardiomyopathic process. High performance liquid chromatography (HPLC) and 31P NMR methods were used to monitor the effects of acute stress applied to nonstressed (NS) and previously stressed CMHs (NS-2.5-month NS-5-month; S-2.5-month, S-5-month) and HHs (NS-HH, S-HH). Cardiac tissue extracts from nonstressed and stressed hamsters were analyzed for ATP and PCr at baseline and after completion of ischemia/reperfusion (AS) using HPLC. In nonstressed hamsters, ATP and PCr were 12% lower in CMHs (both NS-2.5-and NS-5-month) than in NS-HHs. After exposure to stress, ATP was 26% lower in CMHs (S-2.5-and S-5-month) compared to S-HHs, whereas there were minimal differences in PCr between the groups. 31P NMR monitoring of metabolism in the perfused beating heart during application of acute stress produced similar changes (%) in ATP and PCr in all groups (NS and S), whereas P, increase was less in NS-5-month (118%) compared to NS-2.5-month (179%) and NS-HHs (306.8%), P < 0.05; and in S-5-month (148%) compared to S-2.5-month (216%) and S-HHs (222%). The changes in myocardial pH were inversely related to changes in Pi: NS-5-month (-13.5%); NS-2.5-month (-9.7%); NS-HH (-17.7%). pH changes in stressed cardiomyopathic hamsters were similar to those of S-HHs. The postischemic recovery of ATP and Pi return closer to baseline values in cardiomyopathic hamsters (both NS and S) compared to healthy hamsters. The data suggest that cardiomyopathic hamsters have baseline metabolic abnormalities, and their responses to chronic cold immobilization stress, acute ischemia, and chronic cold immobilization stress plus acute ischemia are different from those in HHs. These responses may help to characterize specific stages of disease.