David Piwnica-Worms

Uptake and retention of hexakis (2-methoxyisobutyl isonitrile) technetium(I) in cultured chick myocardial cells. Mitochondrial and plasma membrane potential dependence.

The fundamental myocellular uptake and retention mechanisms of hexakis (2-methoxyisobutyl isonitrile) technetium(I) (Tc-MIBI), a technetium-99m-based myocardial perfusion imaging agent, are unresolved. Because of the lipophilic cationic nature of Tc-MIBI, it may be distributed across biological membranes in response to transmembrane potential. To test this hypothesis, net uptake and retention of Tc-MIBI in cultured chick embryo ventricular myocytes were determined under conditions known to alter mitochondrial and plasma membrane potentials. Isovolumic depolarization of plasma membrane potentials in 130 mM extracellular K (Ko) 20 mM extracellular Cl buffer reduced net accumulation of Tc-MIBI from 171 +/- 16 (control) to 29 +/- 3.3 fmol intracellular Tc-MIBI/mg protein.nM extracellular Tc-MIBI. Unidirectional influx of Tc-MIBI in cells depolarized in 30 mM Ko buffer was also reduced; a resting plasma membrane potential of -87 +/- 6 mV was calculated from the Goldman flux equation using normal Ko/high Ko Tc-MIBI influx ratios. Addition of the potassium ionophore valinomycin to cells incubated in 130 mM Ko buffer to additionally depolarize mitochondrial membrane potentials further reduced net uptake of Tc-MIBI to levels comparable to that found in nonviable freeze-thawed preparations ([Tc-MIBI]i/[Tc-MIBI]o = 1). By depolarizing mitochondrial (and in part plasma membrane) potentials with the protonophores 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone (CCCP) Tc-MIBI was rapidly depleted from 181 +/- 16 (control) to 16 +/- 2.6 and 31 +/- 4.2 fmol/mg protein.nMo, respectively, with kinetics that did not correlate with loss of cellular ATP content. CCCP alone inhibited 90 +/- 3% of net accumulation or 66 +/- 3% of unidirectional influx of Tc-MIBI in a concentration-dependent manner. By hyperpolarizing mitochondrial membrane potentials with the K+/H+ ionophore nigericin or the ATP synthase inhibitor oligomycin, net uptake and retention of Tc-MIBI were increased by 60 +/- 9% and 375 +/- 20%, respectively. Caffeine, as well as the respiratory chain electron transport inhibitor rotenone, did not significantly alter net cell uptake (p greater than 0.2). These data indicate that the fundamental myocellular uptake mechanism of Tc-MIBI involves passive distribution across plasma and mitochondrial membranes and that at equilibrium Tc-MIBI is sequestered within mitochondria by the large negative transmembrane potentials.

Ovarian vein thrombosis

We report five cases of OVT and review the literature on this disease. Among our patients OVT was not suspected clinically and was diagnosed by CT scanning or MRI. Ultrasound imaging, utilized in three cases, failed to detect OVT. Although this disease usually occurs after delivery, in two cases it was diagnosed before delivery. Of the two postpartum cases, one had a typical presentation and the other was asymptomatic. The final case was diagnosed incidentally 5 months after removal of an ovarian carcinoma. Our experience suggests that this disease may be underdiagnosed. More widespread use of CT scanning and MRI may lead to more frequent diagnosis of OVT. The role that clinically silent OVT may play in peripartum pulmonary embolism should be clarified.

Mitochondrial localization and characterization of 99Tc-sestamibi in heart cells by electron probe x-ray microanalysis and 99Tc-NMR spectroscopy

As the development of targeted intracellular magnetic resonance contrast agents proceeds, techniques for the quantitative analysis of the subcellular compartmentation and characterization of metallopharmaceuticals must also advance. To this end, the subcellular distribution and chemical state of hexakis (2-methoxyisobutyl isonitrile) technetium-99 (99Tc-SESTAMIBI), the ground state of the organotechnetium radiopharmaceutical used for the noninvasive evaluation of myocardial perfusion and viability by scintigraphy, has been determined by a novel application of electron probe X-ray microanalysis (EPXMA) and 99Tc-NMR spectroscopy. In cryopreserved cultured chick heart cells equilibrated in 36 microM 99Tc-SESTAMIBI, EPXMA imaging of mitochondria yielded a respiratory uncoupler-sensitive characteristic 99Tc X-ray peak representing 32.0 +/- 2.9 nmoles Tc/mg dry weight, while EPXMA of cytoplasm or nucleus showed no peak significantly greater than the threshold detectability limit of approximately 1 nmole/mg dry weight. Technetium-99 NMR spectroscopy of heart cells equilibrated with 99Tc-SESTAMIBI showed a single peak at -45.5 ppm with no evidence of significant line broadening or chemical shift compared to aqueous chemical standards, indicating that the majority of the complex exists unbound within the mitochondrial matrix. These data quantitatively demonstrate the localization of this lipophilic cationic organometallic complex within mitochondria in situ, consistent with a sequestration mechanism dependent on membrane potentials. Furthermore, this study establishes the general feasibility of combined EPXMA and NMR spectroscopy for the direct subcellular localization and characterization of metallopharmaceuticals, techniques that are readily applicable to MR contrast agents.

Aortic flow velocity patterns in chronic aortic regurgitation: implications for Doppler echocardiography.

Aortic regurgitation is associated with retrograde diastolic flow in the aorta. Echocardiographic quantitative analysis of the magnitude of the flow reversal is believed to provide an estimate of severity of regurgitant disease despite variations in flow profiles. The purpose of this study was to evaluate the uniformity of flow patterns in the aorta of patients with aortic regurgitation and to investigate the relationship between these profiles and the echocardiographic estimates of flow reversal. Seventeen patients with chronic aortic regurgitation underwent cine-phase magnetic resonance imaging in an axial section through the ascending and descending aorta. The regurgitant fraction in the ascending aorta 4 cm above the aortic valve and the descending aorta were calculated from the velocity maps. These results were compared with data from nine individual sample volumes in the ascending and descending aorta. The magnetic resonance ascending aortic regurgitant fraction was compared with Doppler echocardiographic descending aortic flow velocity patterns. The descending aortic regurgitant fraction correlated only weakly with the ascending aortic regurgitant fraction (descending aortic regurgitant fraction = 0.62% ascending aortic regurgitant fraction + 0.04%; r = 0.75; p < 0.001). Regurgitant proportions in all sample volumes in the descending aorta, but not in the ascending aorta, were significantly related to the ascending aortic regurgitant fraction. The best descending aortic Doppler echocardiographic parameter for predicting ascending aortic regurgitant fraction was the end-diastolic velocity (end-diastolic velocity = 32.2 cm/sec. ascending aortic regurgitant fraction + 1.4 cm/sec; r = 0.94; p < 0.001). Pulsedwave Doppler sampling of descending aortic flow reflects severity of aortic regurgitant disease, in part the result of more uniform blood-velocity profiles in the descending aorta compared with the ascending aorta. The Doppler end-diastolic velocity in the descending aorta is a useful parameter of severity of aortic regurgitation.

Membrane potential determination in large unilamellar vesicles with hexakis(2-methoxyisobutylisonitrile)technetium(I)

The accumulation of the lipophilic cation hexakis (2-methoxyisobutylisonitrile)technetium (99mTc-MIBI) within large unilamellar vesicles made from egg phosphatidylcholine was examined as a function of time and membrane potential (Em). Equilibrium distribution occurred within minutes at 30 degrees C. The transmembrane distribution of Tc-MIBI was measured at Em = 0 mV and at a series of negative membrane potentials. The distribution of Tc-MIBI was in close agreement with the Nernst equation for passive distribution of a permeant ion across a bilayer, permitting the membrane potential to be predicted from Tc-MIBI distribution. In this respect, Tc-MIBI behaves similarly to other radioprobes of membrane potential, but with unique properties including high specific activity (10(9) Ci/mol), rapid kinetics of distribution, low potential-independent binding, and short half-life (6.02 h). The results indicate a mechanism for tissue accumulation of Tc-MIBI in vivo that may in part account for its utility in clinical imaging of ischemic myocardium.

Comparison of neutral and cationic myocardial perfusion agents: Characteristics of accumulation in cultured cells

Uptake and washout kinetics of two new neutral lipophilic technetium-99m-labeled boronic acid adducts of technetium tris(dioxime) (BATO complexes) were studied in monolayers of contractile chick heart cells and compared to the cationic myocardial perfusion agents, 99mTc(CNCH2C(CH3)2OCH3)6+ (Tc-MIBI) and 201Tl+. 99mTcCl(CDOH)2(CDO)(BCH3), where CDO = cyclohexanedione dioxime (CDO-MeB), had a 7-fold greater net accumulation than Tc-MIBI and the most rapid unidirectional washout with a fast initial phase and a slower secondary component. Incubation with cationic membrane transport inhibitors or metabolic inhibitors had little or modest influence, respectively, on uptake of these BATO complexes. Studies with NIH 3T3 fibroblasts indicated that the neutral complexes did not show myocyte specific accumulation.

Dynamic nature of the aortic regurgitant orifice area during diastole in patients with chronic aortic regurgitation.

BACKGROUND The effective aortic regurgitant orifice area varies with aortic pressure in animal models of acute aortic regurgitation. The purpose of this study was to determine whether the aortic regurgitant orifice area changes during diastole in patients with chronic aortic regurgitation. METHODS AND RESULTS Two-dimensional and Doppler echocardiography were performed immediately before and after magnetic resonance velocity mapping using a cine phase contrast sequence in 17 patients with chronic aortic regurgitation. ECG-gated continuous-wave Doppler velocity time integrals and magnetic resonance flow rates were measured 16 times per cardiac cycle. The mean aortic regurgitant orifice area (centimeters squared) was calculated by the continuity equation. The regurgitant orifice area was also determined for each diastolic acquisition interval. Changes in the regurgitant orifice area during diastole were modeled using an asymptotic exponential decay model to determine the static and dynamic components of the orifice. The regurgitant orifice area increased directly with regurgitant fraction (y[cm2] = 0.0072[cm2/%]*x[%]-0.0409[cm2]; r = .86, P < .0001). In 15 of 17 (88%) patients, the regurgitant orifice area decreased during diastole. The dynamic component of the regurgitant orifice area decreased with increasing regurgitant fraction (y[%] = -0.98x[%]+96.9[%]; r = -.90, P < .0001). There were no significant differences in heart rate, systolic or diastolic blood pressures, or continuous-wave Doppler velocity time integrals measured before or after the magnetic resonance examination. CONCLUSIONS The effective regurgitant orifice area decreases during diastole in patients with chronic aortic regurgitation. This phenomenon should be considered when evaluating aortic regurgitant severity.

Detection of Adriamycin-induced cardiotoxicity in cultured heart cells with technetium 99m-SESTAMIBI

Adriamycin, a broad-spectrum cytotoxic agent useful in cancer chemotherapy, is limited by a dose-dependent cardiomyopathy mediated in part by disruption of mitochondrial energetics. Hexakis(2-methoxyisobutyl isonitrile)technetium(I)(99mTc-SESTAMIBI) is a gammaemitting radiopharmaceutical with myocellular accumulation properties dependent on mitochondrial membrane potential. To test the hypothesis that99mTc-SESTAMIBI could monitor Adriamycin-induced alterations in cardiac energetics, cultured chick heart cells were treated with Adriamycin and99mTc-SESTAMIBI tracer kinetics were determined. Concentration- and time-dependent depression of99mTc-SESTAMIBI accumulation was evident within 60 min of treatment. The apparentKi for acute Adriamycin inhibition of tracer accumulation was 82 μM. After 24 h of treatment, Adriamycin concentrations as low as 0.1 μM demonstrated detectable inhibitory effects. The apparentKi for this subchronic Adriamycin inhibition of99mTc-SESTAMIBI accumulation was 18 μM. Subchronic concentration-dependent increases in adriamycin-induced myocellular injury as reflected by lactate dehydrogenase (LDH) release correlated inversely with decreases in99mTc-SESTAMIBI accumulation. These data further support a contribution from altered mitochondrial energetics to Adriamycin-induced injury and establish a pharmacological foundation for pursuing the possibility of noninvasive imaging of chronic Adriamycin cardiotoxicity in cancer patients using99mTc-SESTAMIBI.

Interaction of (Na + K + 2 Cl) cotransport and the Na/K pump in cultured chick cardiac myocytes

We have recently reported the presence of an electroneutral (Na + K + 2 Cl) cotransport mechanism that is bumetanide-sensitive and maintains Cli above its electrochemical equilibrium in cultured chick heart cells. In steady state, (Na + K + 2 Cl) cotransport is inwardly directed and so contributes to the Na influx that must be counterbalanced by the activity of the Na/K pump to maintain Nai homeostasis. We now show that manipulating (Na + K + 2 Cl) cotransport by restoring Clo to a Cl-free solution indirectly influences Na/K pump activity because the bumetanide-sensitive recovery of ainfNasupito its control level and the accompanying hyperpolarization could be blocked by 10−4M ouabain. In another protocol, when the Na/K pump was reactivated by restoring Ko (from 0.5 mM to 5.4 mM) and removing ouabain, the recovery of aNa was attenuated by 10−4M bumetanide. The relatively slow rate of ouabain dissociation coupled with the activation of Na influx by (Na + K + 2 Cl) cotransport clearly establishes the interaction of these transport mechanisms in regulating Nai. Although (Na + K + 2 Cl) cotransport is electroneutral, secondary consequences of its activity can indirectly affect the electrophysiological properties of cardiac cells.