Near-infrared fluorescence imaging of mouse myocardial microvascular endothelium using Cy5.5-lectin conjugate.

Cy5.5-lectin, a non-toxic conjugate, combines the benefits of near-infrared (NIR) imaging, such as significant reduction of background fluorescence and increased tissue depth penetration, with its affinity for vascular endothelial cells. When compared to endothelial staining methods using FITC-lectin and ICAM2 antibodies, Cy5.5-lectin was confirmed to specifically bind endothelial cells and produce a fluorescence signal both in real-time and post-infusion. Ex-vivo experiments with isolated hearts demonstrated that binding was limited to perfused areas of the myocardium. With mouse in-vivo tail-vein injections, other organs such as the liver, spleen, and kidney were also stained and yielded similar quality images of the heart.

NIR spectroscopic imaging to map hemoglobin + myoglobin oxygenation, their concentration and optical pathlength across a beating pig heart during surgery.

The purpose of this paper is to demonstrate that near-infrared (NIR) spectroscopic imaging can provide spatial distribution (maps) of the absolute concentration of hemoglobin + myoglobin, oxygen saturation parameter and optical pathlength, reporting on the biochemico-physiological status of a beating heart in vivo. The method is based on processing the NIR spectroscopic images employing a first-derivative approach. Blood-pressure-controlled gating compensated the effect of heart motion on the imaging. All the maps are available simultaneously and noninvasively at a spatial resolution in the submillimeter range and can be obtained in a couple of minutes. The equipment has no mechanical contact with the tissue, thereby leaving the heart unaffected during the measurement.

Dynamic manganese-enhanced magnetic resonance imaging can detect chronic cryoinjury-induced infarction in pig hearts in vivo.

The purpose was to investigate whether MnCl(2) can serve as an MRI contrast agent to detect chronic cryoinjury infarction in pigs in vivo and whether MnCl(2) causes significant hemodynamic disturbances. Hearts were subjected to a topical 2 min cryothermia to establish myocardial infarction (MI). Thereafter GdDTPA-enhanced MRI was performed at 0, 1, 2 and 3 weeks using a 3 T scanner. Four weeks post-cryoinjury the pigs underwent in vivo Mn-enhanced magnetic resonance imaging (MEMRI). MnCl(2) (70 µmol/kg, 14 min) was infused i.v. intermittently (n = 4) or continuously (n = 5) and T(1)-weighted images were acquired every 2 min simultaneously recording heart rate and arterial blood pressure. Either infusion scheme led to an immediate increment in MR signal intensity (SI) within the left ventricular (LV) blood pool and LV normal and cryoinjured myocardium, which reached a maximum at the end of infusion. No significant difference was observed between the normal and cryoinjured myocardium. After infusion termination, SI decreased faster within the LV blood pool and the MI, as compared with the normal myocardium in either group, resulting in significant contrast between the MI and normal tissue (intermittent: 18 ± 7 vs 49 ± 13%, p = 0.002; continuous: 19 ± 8 vs 36 ± 9%, p = 0.004). Infarction sizes were similar in Mn(2+)- and GdDTPA-enhanced images at 4 and 3 weeks post injury, respectively. Thus, in vivo MEMRI differentiated infarcted from normal myocardium in pig hearts subjected to 4-week cryoinjury. Compared with intermittent infusion, continuous infusion minimized hemodynamic fluctuations.

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.

Fluorescence imaging to quantify the fluorescent microspheres in cardiac tissue.

To quantify the fluorescent microsphere (FM) content in cardiac tissue, which is an indicative of blood flow, fluorescence imaging of both sides of the pig heart slice was employed. Despite the light scattering inside the tissue and contributions from multiple tissue layers to the total emission, it is shown that the fluorescence intensity at any pixel is proportional to the FM content and the fluorescence image may be transformed to the image of the FM concentration. A convenient standard for the emission-FM concentration transformation is proposed. The approach has several advantages in comparison with the traditional "digestion & extraction" method such as: non-destructiveness, high spatial resolution, high throughput, repeatability and simplicity of operation.

Mapping the myoglobin concentration, oxygenation, and optical pathlength in heart ex vivo using near-infrared imaging.

A method that provides maps of absolute concentrations of oxygenated and deoxygenated myoglobin (Mb), its oxygenation, and its near-infrared (NIR) optical pathlength in cardiac tissue was developed. These parameters are available simultaneously. The method is based on NIR diffuse reflectance spectroscopic imaging and specific processing of the NIR images, which included a first derivative of the diffuse reflectance spectrum. Mb oxygenation, total Mb concentration, and NIR light pathlength were found to be in the range of 92%, 0.3 mM, and 12.5 mm, respectively, in beating isolated buffer-perfused and arrested pig hearts. The charge-coupled device camera enables sub-millimeter spatial resolution and spectroscopic imaging in 1.5 to 2.0 min. The technique is noninvasive and nondestructive. The equipment has no mechanical contact with the tissue of interest, leaving it undisturbed.

Characterization of cryoinjury-induced infarction with manganese-and gadolinium-enhanced MRI and optical spectroscopy in pig hearts.

PURPOSE: To investigate progression of cryoinjury in pigs using contrast-enhanced magnetic resonance imaging (MRI) as well as optical spectroscopy and imaging. METHODS: Cryoinjury was produced in 16 pigs in vivo and investigated using Gd-and Mn-enhanced MRI, optical imaging/spectroscopy and histology in acute and chronic setting up to 4 weeks after the injury. RESULTS: (1) Acute cryoinjury resulted in formation of a lesion with a severely reduced rate of sub-epicardial indocyanine green (intravascular optical flow tracer) passage. In vivo late Gd-enhanced MRI showed a approximately 10 mm deep hypointense area that was surrounded by a hyperintense rim while ex vivo Mn-enhanced MRI (MEMRI) detected a homogenous hypointense zone. Histological and spectroscopic examination revealed embolic erythrocytes blockages within the cryolesion with a thin necrotic rim neighboring the normal myocardium. (2) Chronic 4-week cryoinjury was characterized by uniform Gd-enhancement, whereas MEMRI revealed reduced Mn(2+)enhancement. Histological examination showed replacement of the cryoinjured myocardium by scar tissue. CONCLUSIONS: Acute cryoinjury resulted in formation of a no-reflow core embolized by erythrocytes and surrounded by a rim of necrotic tissue. Upon injury progression, the no-reflow zone shrunk and was completely replaced with scar tissue by 4 weeks after injury.

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.

Hemoglobin plus myoglobin concentrations and near infrared light pathlength in phantom and pig hearts determined by diffuse reflectance spectroscopy.

To noninvasively determine absolute concentrations of hemoglobin (Hb) plus myoglobin (Mb) in cardiac tissue by means of regular near infrared (NIR) light diffuse reflectance measurements, a first derivative approach was applied. The method was developed to separately calculate oxygenated and deoxygenated [Hb+Mb] as well as an effective pathlength, which NIR light passes through in the tissue between optodes. Applying a cotton wool-based phantom, which mimics muscle tissue, it was shown that the intensity of the pseudo-optical density first derivative depends linearly on both oxygenated and deoxygenated Hb concentration, thereby validating the Lambert-Beer law in the range of 0 to 0.25 mM tetrameric Hb. A high correlation (R=0.995) was found between concentrations of Hb loaded onto the phantom and those determined spectrophotometrically, thereby verifying the first derivative method validity. The efficiency of the method was tested using in vivo pig hearts prior to and after ischemia initiated experimentally by left anterior descending artery branches occlusion. The results showed that the total [Hb+Mb] was 0.9-1.2 mM heme, the average tissue oxygen saturation was approximately 70% (which reduced to nearly 0% after occlusion), and the NIR (700-965 nm) light pathlength was 2.3 mm (differential pathlength factor [DPF]=2.7-2.8) in a living heart tissue.

Assessment of near-infrared path length in fibrous phantom and muscle tissue.

The first derivative of the pseudo-absorption spectrum of a water-loaded cotton wool (water-CW) phantom, which mimics muscle tissues, was used to determine the light path length in the near-infrared (NIR) region. The light path length increased as the density of the turbid medium decreased. It is independent of both water content in the range of 75-85% (by weight) and the diffuse reflecting reference used to determine the pseudo-absorbance. The path length determination procedure was verified by measurements of diffuse reflectance in chicken breast tissue for which the path length of 1.8 mm (differential path length factor, DPF = 2.1) was found to be similar to the path length of NIR light of 1.5-2.2 mm (DPF = 1.8-2.6) in a water-CW phantom of density similar to chicken breast. We conclude that the NIR light path length can serve as a characteristic of muscle tissue density.

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.