Rhodamine 800 as a near-infrared fluorescent deposition flow tracer in rodent hearts.

We investigated the use of a near-infrared (NIR) fluorescent dye, Rhodamine 800 (Rhod800, λ(exc) = 693 nm, λ(em) > 720 nm) as a flow-dependent molecular tracer for NIR spectroscopy and high-resolution cardiac imaging. Rhod800 accumulates in isolated mitochondria in proportion to the mitochondrial membrane potential (ΔΨ). However, in the intact myocardium, Rhod800 binding is ΔΨ-independent. Rat hearts were perfused in a Langendorff mode with Krebs-Henseleit buffer containing 45-nM Rhod800 at normal (100%), increased (150%), or reduced (50%) baseline coronary flow (CF) per gram, for 30 to 60 min. In a different group of hearts, the left anterior descending artery (LAD) was occluded prior to Rhod800 infusion to create a flow deficit area. Rhod800 deposition was analyzed by: 1. absorbance spectroscopy kinetics in the Rhod800-perfused hearts, 2. Rhod800 absorbance and fluorescence imaging in the short-axis heart slices, and 3. dynamic epicardial/subepicardial fluorescence imaging of Rhod800 in KCl-arrested hearts, with a spatial resolution of ∼ 200 µm. Rhod800 deposition was proportional to the perfusate volume (CF and perfusion time) and there was no Rhod800 loss during the washout period. In the LAD-ligated hearts, Rhod800 fluorescence was missing from the no-flow, LAD-dependent endocardial and epicardial/subepicardial area. We concluded that Rhod800 can be used as a deposition flow tracer for dynamic cardiac imaging.

Defects in myoglobin oxygenation in K(ATP)-deficient mouse hearts under normal and stress conditions characterized by near infrared spectroscopy and imaging.

BACKGROUND: Disruption of ATP-sensitive potassium (K(ATP)) channel activity results in the development of dilated cardiomyopathy in response to different forms of stress, likely due to the underlying metabolic defects. To further understand the role of Kir6.2-containing channels in the development of cardiac disease, we analysed the left ventricular (LV) wall oxygenation and the physiologic responses induced by acute stress in non-dilated Kir6.2(-/-) hearts. METHODS: Control (C57BL6) and Kir6.2(-/-) mouse hearts were perfused in constant flow Langendorff mode with Krebs-Henseleit buffer. Myocardial oxygenation was evaluated using a newly developed technique, near infrared spectroscopic imaging (NIRSI) of the myoglobin (Mb) oxygen saturation parameter (OSP, ratio of oxy- to total Mb). RESULTS: 2,4-dinitrophenol (DNP, 50-µM) and isoproterenol (0.1-µM) failed to produce a transient vasodilatory response and caused a significant diastolic pressure increase in Kir6.2(-/-) hearts. DNP strongly suppressed contractile function in both groups and induced severe mean OSP decreases in Kir6.2(-/-) hearts. Isoproterenol-induced decreases in OSP were similar despite the lack of contractile function stimulation in the Kir6.2(-/-) group. The index of OSP spatial heterogeneity (relative dispersion, RD) was lower by 15% in the Kir6.2(-/-) group at the baseline conditions. Recovery after stress caused reduction of RD values by 20% (DNP) and 8% (isoproterenol) in controls; however, these values did not change in the Kir6.2(-/-) group. CONCLUSIONS: 1) NIRSI can be used to analyse 2-D dynamics of LV oxygenation in rodent models of cardiomyopathy; 2) Kir6.2-containing K(ATP) channels play an important role in maintaining myocardial oxygenation balance under acute stress conditions and in post-stress recovery.

Magnetic resonance imaging tracking of alginate beads used for drug delivery of growth factors at sites of cardiac damage.

Alginate-based beads labeled with contrast agent and loaded with vascular growth hormones were used for site-specific chronic delivery of hormones at the site of myocardial damage in a porcine model. Position of the beads within the pericardium could be monitored by MRI for optimal hormone delivery due to the presence of contrast agent. The beads facilitate the slow release of cytochrome c, myoglobin and methemoglobin used as protein models of growth factors. This application allows for site-specific delivery of hormones while the incorporated contrast agent in the beads provides a tool for MRI tracking in chronic studies.

Potassium fluxes, energy metabolism, and oxygenation in intact diabetic rat hearts under normal and stress conditions.

We evaluated the function of Na(+)/K(+) ATPase and sarcolemmal K(ATP) channels in diabetic rat hearts. Six weeks after streptozotocin (STZ) injection, unidirectional K(+) fluxes were assayed by using (87)rubidium ((87)Rb(+)) MRS. The hearts were loaded with Rb(+) by perfusion with Krebs-Henseleit buffer, in which 50% of K(+) was substituted with Rb(+). The rate constant of Rb(+) uptake via Na(+)/K(+) ATPase was reduced. K(ATP)-mediated Rb(+) efflux was activated metabolically with 2,4-dinitrophenol (DNP, 50 micromol.L(-1)) or pharmacologically with a K(ATP) channel opener, P-1075 (5 micromol.L(-1)). Cardiac energetics were monitored by using (31)P MRS and optical spectroscopy. DNP produced a smaller ATP decrease, yet similar Rb(+) efflux activation in STZ hearts. In K(+)-arrested hearts, P-1075 had no effect on high-energy phosphates and stimulated Rb(+) efflux by interaction with SUR2A subunit of K(ATP) channel; this stimulation was greater in STZ hearts. In normokalemic hearts, P-1075 caused cardiac arrest and ATP decline, and the stimulation of Rb(+) efflux was lower in normokalemic STZ hearts arrested by P-1075. Thus, the Rb(+)efflux stimulation in STZ hearts was altered depending on the mode of K(ATP) channel activation: pharmacologic stimulation (P-1075) was enhanced, whereas metabolic stimulation (DNP) was reduced. Both the basal concentration of phosphocreatine ([PCr]) and [PCr]/[ATP] were reduced; nevertheless, the STZ hearts were more or equally resistant to metabolic stress.

Detection of moderate regional ischemia in pig hearts in vivo by near-infrared and thermal imaging: effects of dipyridamole.

Effects of coronary vasodilator, dipyridamole, on epicardial oxygenation and flow were investigated under conditions of moderate coronary occlusion using near-infrared spectroscopic (NIRS) and thermal imaging. In anesthetized open chest pigs an inflatable occluder and flow probe were placed around the left anterior descending artery (LAD). In the ischemic group (n = 11) LAD occlusion (50% flow, 80 min) was followed by complete occlusion (10 min, n = 4), and reflow. Dipyridamole was infused (0.14 mg/min/kg/4 min) intravenously during 50% occlusion. In the control group (n = 6) LAD flow was temporarily increased (hyperemic response) by two 2-min periods of complete LAD occlusion applied 120 min apart, with a 4-min period of dipyridamole infusion between the two occlusions. NIRS and thermal images were acquired throughout the protocol. Maps of subepicardial oxygen saturation parameter (OSP), and epicardial temperature (T) were obtained. Partial occlusion reduced OSP and the temperature by 0.23 +/- 0.08 and 0.88 +/- 0.39 degrees C versus remote region, respectively. Dipyridamole decreased systolic blood pressure by 36%, which caused further decline in the LAD flow to 18% and OSP and T by 0.37 +/- 0.01 and 2.46 +/- 0.32 degrees C, respectively. Reflow restored OSP and T to their baseline levels. In control group dipyridamole and hyperemia increased LAD flow 2-4-fold associated with moderate increase in OSP and T. OSP and T showed linear dependence on the flow below 100%, which is leveled-off at flows above normal. Dipyridamole increases differences in the epicardial oxygenation and T between normal and moderately ischemic areas due to enhancement of disparity in perfusion of these areas.

Visualization and grading of regional ischemia in pigs in vivo using near-infrared and thermal imaging.

Reductions in regional coronary flow result in tissue deoxygenation and decrease in surface temperature, changes detectable by near-infrared spectroscopic (NIRS) and thermal imaging, respectively. In anesthetized open-chest pigs, an inflatable occluder and flow probe were placed around the left anterior descending artery. Gated NIRS and nongated thermal images were acquired at baseline, partial (17% and 50%), and complete occlusion and reflow. At each step, dobutamine was infused (10 microg.min(-1).kg(-1)) for 7-9 min to increase blood pressure and flow. Changes in the oxygen saturation parameter, rate of indocyanine green flow tracer passage, and the surface temperature were correlated with the measured left anterior descending artery flow. Location and sizes of the areas of reduced oxygenation, indocyanine green uptake, and temperature were similar. Decrease in the coronary flow to 50% and 17% of baseline resulted in progressive decrease in the above parameters, whereas increase in flow from 75% to approximately 250% achieved by dobutamine and reactive hyperemia did not significantly change them. Dobutamine increased total and epicardial flow in ischemic areas and increased subepicardial oxygenation. NIRS and thermal imaging provide epicardial maps of oxygen saturation and perfusion that reveal ischemic areas. Combination of these techniques may be useful in the coronary artery bypass graft (CABG) surgery setting.

Regional cardiac tissue oxygenation as a function of blood flow and pO2: A near-infrared spectroscopic imaging study.

Near-infrared spectroscopic imaging (NIRSI) is useful to assess cardiac tissue oxygenation in arrested and beating hearts, and it shows potential as an intraoperative gauge of the effectiveness of bypass grafting. The purpose of this study was to determine whether NIRSI can reliably differentiate among a range of cardiac oxygenation states, using ischemia and hypoxia models independently. An ischemia-reperfusion model was applied to isolated, beating, blood-perfused porcine hearts, in which the left anterior descending (LAD) artery was cannulated. LAD flow was decreased stepwise to approximately 50, 20, and 0% of normal flow and was completely restored between ischemic episodes. Upon completion of the ischemia-reperfusion protocol, the hearts were further subjected to periods of increasingly severe global hypoxia. Regional oxy- and deoxy-hemoglobin (myoglobin) levels were derived from spectroscopic images (650 to 1050 nm) acquired at each step. Oxygenation maps vividly highlighted the area at risk for all degrees of ischemia. Oxygenation values differed significantly for different LAD flow rates, regardless of whether intermediate reperfusion was applied, and oxygenation values during progressive hypoxia correlated well with blood oxygen saturation. These results suggest that NIRSI is well suited, not only to identify ischemic or hypoxic regions of cardiac tissue, but also to assess the severity of deoxygenation.

K+ transport and energetics in Kir6.2(-/-) mouse hearts assessed by 87Rb and 31P magnetic resonance and optical spectroscopy.

Cardiac sarcolemmal K(ATP) channels are crucial in adaptation to stress caused by metabolic inhibition and moderate exercise, which requires not only down-regulation of energy spending, but also up-regulation of mitochondrial ATP synthesis. To investigate sarcolemmal and mitochondrial effects of a Kir6.2 (K(+) ion-selective subunit of the channel) knockout, we used non-invasive techniques ((87)Rb, (31)P NMR and optical spectroscopy) to study (1) K(+) fluxes, (2) high-energy phosphates, (3) the cytochrome c oxidase redox state, (4) myoglobin deoxygenation, and (5) contractile function at the baseline and in response to metabolic uncoupling with 2,4-dintrophenol (DNP) and stimulation with isoproterenol in Langendorff-perfused mouse hearts. Comparison with control C57BL6 hearts demonstrated that the Kir6.2 knockout resulted in: (a) a lack of stimulation of the unidirectional potassium efflux from the hearts when K(ATP) channels were activated metabolically by DNP (50 muM, 20 min); (b) a decrease in ATP, but not phosphocreatine, at the baseline, that became even more pronounced when the hearts were subjected to stress due to metabolic inhibition or increased workload caused by isoproterenol infusion (0.1 microM, 20 min); (c) significantly higher reduction of cytochrome c oxidase in response to DNP uncoupling; (d) a blunted response to isoproterenol stimulation. Thus Kir6.2 knockout is associated with decreased tolerance of mouse hearts to metabolic inhibition and catecholamine stress.

Interaction of a mitochondrial membrane potential-sensitive dye, rhodamine 800, with rat mitochondria, cells, and perfused hearts.

Fluorescence, absorbance, and binding of a mitochondrial membrane potential-sensitive probe, rhodamine 800 (rhod800), were measured in isolated rat mitochondria, hepatocytes, cardiomyocytes, and hearts in the presence or absence of mitochondrial uncouplers. Excitation of rhod800 was achieved with laser diodes (690 or 670 nm) and resulted in a fluorescence peak at 720 nm. Greater than 99% of rhod800 (1 microM) was taken up from the buffer by energized mitochondria. This resulted in a fluorescence decrease by 77% (13% in de-energized mitochondria). Sixty-seven percent of rhod800 was taken up by cardiomyocytes and 75% by hepatocytes resulting in the fluorescence decrease by 16% and 37%, respectively, which were reversed by approximately 10% upon cell uncoupling. In hearts, binding, absorbance, and fluorescence were almost uncoupler-insensitive possibly due to rhod800 interaction outside of mitochondria. Fluorescence of the hearts perfused with 27.5 and 55 nM rhod800 was measured in orthogonal and reflection modes. The former provided deep tissue penetration (approximately a centimeter); however, nonlinearity between absorbance and fluorescence was evident. In the latter setting, depth of tissue penetration was approximately a millimeter, which eliminated an inner filter effect and restored linearity. We concluded that excessive hydrophobicity of rhod800 complicates detection of energy-dependent fluorescence changes in myocardium.

Assessment of optical path length in tissue using neodymium and water absorptions for application to near-infrared spectroscopy.

Quantitative analysis of blood oxygen saturation using near-IR spectroscopy is made difficult by uncertainties in both the absolute value and the wavelength dependence of the optical path length. We introduce a novel means of assessing the wavelength dependence of path length, exploiting the relative intensities of several absorptions exhibited by an exogenous contrast agent (neodymium). Combined with a previously described method that exploits endogenous water absorptions, the described technique estimates the absolute path length at several wavelengths throughout the visible/near-IR range of interest. Isolated rat hearts (n = 11) are perfused separately with Krebs-Henseleit buffer (KHB) and a KHB solution to which neodymium had been added, and visible/near-IR spectra are acquired using an optical probe made up of emission and collection fibers in concentric rings of diameters 1 and 3 mm, respectively. Relative optical path lengths at 520, 580, 679, 740, 800, 870, and 975 nm are 0.41+/-0.13, 0.49+/-0.21, 0.90+/-0.09, 0.94+/-0.01, 1.00, 0.84+/-0.01, and 0.78+/-0.08, respectively. The absolute path length at 975 nm is estimated to be 3.8+/-0.6 mm, based on the intensity of the water absorptions and the known tissue water concentration. These results are strictly valid only for the experimental geometry applied here.

Potassium transport in Langendorff-perfused mouse hearts assessed by 87Rb NMR spectroscopy.

We studied the fluxes of a potassium congener (Rb(+)) in mouse hearts by (87)Rb MRS at 8.4T. The hearts were loaded with Rb(+) by perfusion with Krebs-Henseleit buffer, in which 50% of K(+) was substituted with Rb(+). We initiated Rb(+) efflux by changing the perfusion medium to Rb(+)-free buffer. Spectra were acquired every 1.85 min, and the kinetics of Rb(+) transport were analyzed by means of monoexponential fits. The rate constants of Rb(+) uptake and efflux were 0.0680 +/- 0.0028 and 0.0510 +/- 0.0051 min(-1), respectively (approximately 30% faster than in the rat heart). The ATP-sensitive potassium channel opener, P-1075 (5 microM), and mitochondrial uncoupler, 2,4-dintrophenol (50 microM), activated Rb(+) efflux from mouse hearts by approximately 35%. The mechanisms responsible for the differences in Rb(+) uptake and efflux under baseline conditions and stimulation, in comparison with rat hearts, are discussed. These data provide a background for studies of cardiac potassium transport in transgenic mouse strains.

Rubidium-87 magnetic resonance spectroscopy and imaging for analysis of mammalian K+ transport.

This review summarizes results 87Rb MRS/I studies of K+ transport in mammalian cells, organs and in vivo. It provides a brief description of K+ transport systems, their interactions with Rb+ and evidence that Rb+ is a best K+ congener. 87Rb MR studies have focused mostly on isolated perfused rat and pig hearts and to a lesser extent on kidney, skeletal muscle, salivary gland and red blood cells. The method has been used for three purposes: measurements of kinetics of unidirectional Rb+ uptake and efflux and steady-state Rb+ levels. In cardiovascular studies Rb+ has been used in the absence of shift reagent taking advantage of the predominantly intracellular Rb+/K+ distribution (approximately 20:1). Pharmacological analysis of Rb+ uptake and efflux allowed assessment of the contributions of various transporters to the total Rb+ fluxes in rat hearts. It was confirmed that Na+/K+ ATPase is responsible for the majority of K+ influx since Rb+ uptake is 80% ouabain-sensitive and dependent on the intracellular [Na+]. Energy deprivation caused by low-flow ischemia or metabolic inhibition reduced Rb+ uptake rate. Under normal conditions, Rb+ efflux is mediated mainly by voltage-gated K+ channels with a small contribution from the K+/Na+/2Cl- cotransporter. Intracellular alkalosis and osmotic swelling stimulated Rb+ efflux by activation of the putative K+/H+ antiporter. Activity of ATP-sensitive K+ (K(ATP)) channels was revealed by metabolic (2,4-dinitrophenol, ischemia) or pharmacological (K(ATP) opener, P-1075) stimulation of Rb+ efflux, which was reversed by the K(ATP) blocker, glibenclamide. Mitochondrial K+ transport was evaluated in hearts with saponin-permeabilized myocytes and under hypothermic conditions.Three-dimensional (3-D) spectroscopic MRI of isolated beating pig hearts has been used to obtain time series of Rb+ maps of normal and ischemic/infarcted hearts, which showed lower image intensity in the damaged area. Kinetics of Rb+ uptake in the ischemic areas depended on both regional flow and metabolism. The adrenergic agonist dobutamine stimulated Rb+ uptake in normal areas and did not affect uptake in ischemic areas. Drugs that may affect passive Rb+ transport (bumetanide, pinacidil, glibenclamide) did not change Rb+ uptake either in the normal or ischemic zones. 87Rb-MRI was also able to localize ischemia and infarction in blood-perfused hearts. 87Rb MRS/I is an excellent non-invasive research tool for studies of K+ transport in isolated organs and in vivo.

Relative contributions of hemoglobin and myoglobin to near-infrared spectroscopic images of cardiac tissue.

Near-infrared (NIR) spectroscopic imaging is emerging as a unique tool for intra-operative assessment of myocardial oxygenation, but quantitative interpretation of the images is not straightforward. One confounding factor specific to muscle tissue (both skeletal and cardiac) is that the visible/NIR absorbance spectrum of myoglobin (Mb), an intracellular O(2) storage protein, is virtually identical to that of hemoglobin (Hb). As a consequence, the relative contributions of Mb and Hb to the NIR spectra measured in vivo for blood perfused muscle tissue cannot be determined from the measured spectra alone. To estimate the relative contributions of Mb and Hb to NIR spectra and spectroscopic images, isolated pig hearts were perfused first with a Hb-free blood substitute (Krebs-Henseleit buffer; KHB) and then with a 50/50 KHB/blood mixture, with spectroscopic images acquired at each step. Tissue Mb levels were estimated directly from the measurements during KHB perfusion, and total (Mb+Hb) levels were estimated from the images acquired during 50/50 blood/KHB perfusion. Myoglobin accounted for 63 +/- 11% of the total heme content during perfusion with the 50/50 mixture (implying that Mb would contribute 46% of the combined (Mb+Hb) NIR profile during whole blood perfusion), confirming that Mb contributes substantially to near-infrared absorbance spectra of blood perfused cardiac tissue.

Mapping regional oxygenation and flow in pig hearts in vivo using near-infrared spectroscopic imaging.

We used near-infrared spectroscopic (NIRS) imaging to assess the flow-dependence of both myocardial oxygenation and distribution of an intravascular tracer, indocyanine green (ICG). In open-chest pigs, nominal flow through the left anterior descending artery was reduced for 90 to 0 min (n = 6), 20 +/- 1 (n = 5) and 44% +/- 5% (n = 4) by variable occlusion and subsequently restored (to 219% +/- 71% at 45 min) for 120 min. Electrocardiogram-gated NIRS images of the heart were obtained using a CCD-array camera with a liquid crystal tunable filter, which acquired absorbance spectra in the range of 650-1050 nm for each of 256 x 256 pixels (0.4 x 0.4 mm each). Deoxy- and oxy-(hemoglobin (Hb) + myoglobin (Mb)) levels were determined independently by applying a spectral fitting algorithm to the spectra between 650 and 890 nm. Maps for oxygenation parameter (OP = oxy-(Hb + Mb)/deoxy-(Hb + Mb)) and oxygen saturation parameter (OSP) (oxy-(Hb + Mb)/total-(Hb + Mb)) were constructed. To visualize flow distribution, a bolus of ICG (8.3 mg/5 ml) was injected I.V. at each step of the protocol and gated images were acquired at 800 nm every second over 60 s period. The ratio of ICG wash-in velocity to equilibrium absorbance (V/DeltaA(tail)) was calculated. Changes in flow to 0%, 20%, 44%, 100% and 208% of baseline resulted in OP values of 1.46 +/- 0.25, 1.66 +/- 0.30, 2.22 +/- 0.45, 2.78 +/- 0.30 and 3.94 +/- 0.33 in the affected area. Flow rates of 20%, 44%, 100% and 220% of baseline corresponded to V/DeltaA(tail) values of 0.11 +/- 0.11, 0.39 +/- 0.13, 0.54 +/- 0.17 and 0.61 +/- 0.20, respectively. Thus, measurements of oxygenation and normalized ICG wash-in velocity correlated well with coronary flow, allowing for intraoperative optical assessment of the severity of regional ischemia.

Detection of myocardial cell damage in isolated rat hearts with near-infrared spectroscopy.

One hallmark of cell death resulting from prolonged ischemia is cell membrane disruption. We apply optical spectroscopy to gauge membrane disruption in isolated rat hearts by monitoring (1) the washout of myoglobin (Mb) and (2) the accumulation of an exogenous contrast agent in permeabilized cells. The contrast agent, a neodymium (Nd) chelate, has several absorptions in the visible and near-IR, and when present in the perfusate, it cannot penetrate cellular membranes. When membrane integrity is disrupted, however, it is expected to accumulate within the intracellular space; moreover, cellular Mb is expected to wash out. To test this hypothesis, rat hearts (n=12) are perfused with Krebs-Henseleit buffer (KHB), followed by perfusion with KHB in which a 5 mM Nd-DTPA solution is present. Membrane damage is then induced by infusion of digitonin into the Nd-KHB perfusate to provide a digitonin concentration of 2.5, 5, or 10 microg/mL. After 30 min of infusion, Mb levels fall to 46+/-14% of baseline levels and Nd-DTPA rises to 161+/-19% of predigitonin levels. No apparent dependence of total membrane disruption on digitonin concentration over the concentration range studied is found, although higher concentrations do lead to more rapid membrane disruption.

Sarcolemmal and mitochondrial effects of a KATP opener, P-1075, in "polarized" and "depolarized" Langendorff-perfused rat hearts.

We investigated consequences of cardiac arrest on sarcolemmal and mitochondrial effects of ATP-sensitive potassium channel (KATP) opener, P-1075, in Langendorff-perfused rat hearts. Depolarised cardiac arrest (24.7 mM KCl) did not affect glibenclamide-sensitive twofold activation of rubidium efflux by P-1075 (5 microM) from rubidium-loaded hearts, but eliminated uncoupling produced by P-1075 in beating hearts: 40% depletion of phosphocreatine and ATP, 50% increase in oxygen consumption, and reduction of cytochrome c oxidase. Depolarized cardiac arrest by calcium channel blocker, verapamil (5 microM), also prevented uncoupling. Lack of P-1075 mitochondrial effects in depolarized hearts was not due to changes in phosphorylation potential, because 2,4-dintrophenol (10 microM) reversed the [PCr]/[Cr] increase and Pi decrease, characteristic of KCl-arrest, but did not restore uncoupling. In agreement with this conclusion, pyruvate (5 mM) increased [PCr]/[Cr] and decreased Pi, but did not prevent uncoupling in beating hearts. A decrease in mean [Ca2+] in KCl-arrested hearts could not account for lack of P-1075 mitochondrial effects, because calcium channel opener, S-(-)-Bay K8644 (50 nM), and beta-agonist, isoproterenol (0.5 microM), did not facilitate uncoupling. In contrast, in adenosine (1 mM)-arrested hearts (polarized arrest), P-1075 caused 40% phosphocreatine and ATP depletion. In isolated rat liver mitochondria, P-1075 (20 microM) decreased mitochondrial membrane potential (DeltaPsi) by approximately 14 mV (demonstrated by redistribution of DeltaPsi-sensitive dye, rhodamine 800) in a glibenclamide-sensitive manner. We concluded that cell membrane depolarization does not prevent activation of sarcolemmal KATP by P-1075, but it plays a role in mitochondrial uncoupling effects of P-1075.

Cardioselective sulfonylthiourea HMR 1098 blocks mitochondrial uncoupling induced by a KATP channel opener, P-1075, in beating rat hearts.

We investigated effects of blockade of cardiac ATP-sensitive potassium channels (KATP) with a novel cardioselective sulfonylthiourea, HMR 1098, on metabolic uncoupling caused by a potent KATP opener, P-1075, in Langendorff-perfused rat hearts. We used (1) 87Rb-NMR to detect activation-deactivation of sarcolemmal KATP, (2) 31P-NMR to monitor high-energy phosphates, (3) oxygen uptake measurements to monitor cellular respiration, and (4) myocardial optical absorbance measurements at 603 nm to follow changes in cytochrome c oxidase redox state. Activation of sarcolemmal KATP by P-1075 (5 microM) and a mitochondrial uncoupler 2,4-dinitrophenol (DNP) (50 microM) stimulated Rb+ efflux from the hearts by 130% and 60%, respectively. HMR 1098 (5 and 30 microM) blocked activation of sarcolemmal KATP in situ. HMR 1098 also prevented cardiac arrest and mitochondrial uncoupling induced by P-1075, such as (a) depletion of phosphocreatine and ATP by 40%, (b) two-fold decrease in venous oxygen, and (c) reduction of cytochrome c oxidase (demonstrated by an increase in 603 nm optical absorbance). The metabolic effects of P-1075 can be readily explained by activation of putative mitochondrial KATP. We concluded that blockade of mitochondrial uncoupling by HMR 1098 included an inhibiting effect of HMR 1098 on sarcolemmal and mitochondrial KATP in beating rat hearts.

Hyposmotic shock: effects on rubidium/potassium efflux in normal and ischemic rat hearts, assessed by 87Rb and 31P NMR.

The study evaluated effects of hyposmotic shock on the rate of Rb(+)/K(+) efflux, intracellular pH and energetics in Langendorff-perfused rat hearts with the help of 87Rb- and 31P-NMR. Two models of hyposmotic shock were compared: (1) normosmotic hearts perfused with low [NaCl] (70 mM) buffer, (2) hyperosmotic hearts equilibrated with additional methyl alpha-D-glucopyranoside (Me-GPD, 90 or 33 mM) or urea (90 mM) perfused with normosmotic buffer. Four minutes after hyposmotic shock, Rb(+) efflux rate constant transiently increased approximately two-fold, while pH transiently decreased by 0.08 and 0.06 units, in the first and the second models, respectively, without significant changes in phosphocreatine and ATP. Hyposmotic shock (second model) did not change the rate of Rb(+)/K(+) uptake, indicating that the activity of Na(+)/K(+) ATPase was not affected. Dimethylamiloride (DMA) (10 microM) abolished activation of the Rb(+)/K(+) efflux in the second model; however, Na(+)/H(+) exchanger was not involved, because intracellular acidosis induced by the hyposmotic shock was not enhanced by DMA treatment. After 12 or 20 min of global ischemia, the rate of Rb(+)/K(+) efflux increased by 120%. Inhibitor of the ATP-sensitive potassium channels, glibenclamide (5 microM), partially (40%) decreased the rate constant; however, reperfusion with hyperosmolar buffer (90 mM Me-GPD) did not. We concluded that the shock-induced stimulation of Rb(+)/K(+) efflux occurred, at least partially, through the DMA-sensitive cation/H(+) exchanger and swelling-induced mechanisms did not considerably contribute to the ischemia-reperfusion-induced activation of Rb(+)/K(+) efflux.

Regional variations in myocardial tissue oxygenation mapped by near-infrared spectroscopic imaging.

Near-infrared fibre-optic single point spectroscopy has been widely exploited to provide information regarding blood volume and oxygenation in vivo, but it does not provide any information on regional differences in perfusion. We have combined the chemical sensitivity of spectroscopy with the spatial sensitivity of imaging to generate maps of regional cardiac oxygenation. Spectroscopic images were acquired for isolated, arrested, blood-perfused porcine hearts (n=4) over the wavelength range 650 and 1050 nm. Spectroscopic images were acquired during normal perfusion, regional ischemia (occlusion of left anterior descending artery) global ischemia, and reperfusion. Hemoglobin (Hb) and myoglobin (Mb) content and oxygenation were determined by reconstructing the tissue spectra measured at each pixel as weighted sums of water, oxy- and deoxy-Hb (and -Mb) absorptivity spectra. The spectroscopic images acquired during regional ischemia clearly revealed increased deoxy-(Hb+Mb) levels and decreased oxy-(Hb+Mb) levels in the ischaemic regions relative to the normally-perfused regions. Global ischemia produced a dramatic decrease in oxy-(Hb+Mb) levels and a moderate increase in deoxy-(Hb+Mb). These images confirm that blood oxygenation can be mapped in cardiac tissue by near-infrared spectroscopic imaging.

The effects of drugs modulating K(+) transport on Rb(+) uptake and distribution in pig hearts following regional ischemia: (87)Rb MRI study.

The effects of drugs that can modulate passive permeability of K(+) into cardiomyocytes in normal and reperfusion-damaged cardiac muscle were assessed. Rubidium ion (Rb(+)) was used as a K(+) tracer and (87)Rb-MRI as a detection method. The left anterior descending artery (LAD) of isolated pig hearts perfused with Krebs-Henseleit buffer (KHB) was occluded for 2 h and subsequently reperfused for 2 h with KHB containing 4.7 mM RbCl instead of KCl. The buffer contained either a blocker of ATP-sensitive K(+) channels (K(ATP)), glibenclamide (Glib, 3 micro M), a K(ATP) opener, pinacidil (Pin, 10 micro M), a K(+)/Na(+)/2Cl(-) co-transporter inhibitor, bumetanide (Bum, 10 micro M) or no drug (control). Upon reperfusion three-dimensional (87)Rb MR images were acquired to obtain kinetics of Rb(+) uptake and its distribution. Areas at risk (AAR) and areas of necrosis were determined by Evans Blue and triphenyl tetrazolium chloride staining, respectively. Rb(+) uptake kinetics in the remote posterior (Pos) wall were similar in all groups. The kinetics remained monoexponential in the affected anterior (Ant) wall and the uptake rates were 32, 36, 37 and 21% of that in the Pos wall in the control, Glib, Pin and Bum groups, respectively. Infarct sizes determined histologically as a percentage of total ventricular (left + right) mass (14-22%) corresponded to sizes of areas with 20-40% of maximal Rb image intensity [I(Rb)(max), 15-22%], except for the Pin group (12.5 vs 21%). The sizes of areas with 20-50% of I(Rb)(max) (30-36%) closely correlated with those of AAR determined histologically (31-33%). Lactate dehydrogenase release did not differ in all groups. We conclude that: (1) reperfusion damage quickly inhibits Rb(+) uptake; (2) Rb(+) uptake in normal and reperfused tissue does not significantly depend on K(ATP) or the K(+)/Na(+)/2Cl(-) cotransporter; (3) areas with 20-40% of maximal image intensity correspond to infarct areas.