Olga Jilkina

Generation of antibodies specific for the RalA and RalB GTP-binding proteins and determination of their concentration and distribution in human platelets

Peptide specific polyclonal antibodies directed against C-termini of ras p21 related GTP-binding proteins, ralA and ralB, were generated. To assess antibody specificity, cDNAs coding for full length ralA and ralB were expressed in Escherichia coli as GST fusion proteins. Western blotting analysis using enhanced chemiluminescence technique confirmed that ralA and ralB antibodies were specific for their respective protein. To determine the concentration and distribution, varying amounts of GST-ralA and GST-ralB and, human platelet particulate and cytosolic proteins were loaded during Western blotting. The amount of ralA and ralB proteins in the platelet particulate fraction was determined to be 0.16 +/- 0.017 microgram/mg protein (n = 3) and 0.15 +/- 0.009 microgram/mg protein (n = 3) respectively. In the cytosol, only ralB protein was detected and its concentration was estimated to be 0.03 +/- 0.009 microgram/mg protein (n = 3). Both ralA and ralB proteins were isoprenylated in the presence of [3H] mevalonolactone plus rabbit reticulocyte lysate although radioactivity incorporated into ralA was three times higher than that associated with the ralB protein. Addition of geranylgeranyl pyrophosphate to the reaction mixture inhibited incorporation of radioactivity into ralA and ralB but not cH-ras suggesting that both ralA and ralB proteins are geranylgeranylated. Differential distribution of ralA and ralB GTP-binding proteins in human platelets suggests a distinct role for each of these proteins in platelet function.

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.

Three-dimensional 87Rb NMR imaging and spectroscopy of K+ fluxes in normal and postischemic pig hearts†

K+ uptake rates were measured in the anterior (An) and posterior (Pos) LV walls of pig hearts before and after regional ischemia and reperfusion using Rb+ as a K+ congener and 3D 87Rb NMR imaging and spectroscopy as detection methods. The hearts were perfused by the Langendorff method with Krebs‐Henseleit (KH) buffer and loaded with Rb+ (4.7 mM, Rb‐KH) after 120‐min ischemia and 60‐min reperfusion. A second protocol involved Rb+ loading prior to ischemia. Ischemia was produced by occlusion of the left anterior descending artery, which after 110 min of reperfusion resulted in infarction in the An wall (24 ± 6% of the LV mass) determined by triphenyltetrazolium chloride staining. At the end of reperfusion pressure‐rate product and oxygen consumption rate decreased to 58 ± 10 and 74 ± 4% of their preischemic values, respectively. Phosphocreatine, ATP, and intracellular pH (pHi), measured by 31P NMR spectroscopy in the infarcted area, decreased to 59 ± 17, 32 ± 6%, and 6.7 ± 0.36 (from 7.05 ± 0.13), respectively. Serial 87Rb images were acquired according to both protocols. Rate constants (k × 103, min−1), relative amount of intracellular Rb+ (A, %) and relative fluxes (F = kA, %/min) for the An and Pos walls were determined from the images. Before ischemia, F and k were comparable in the Pos and An walls. Ischemia + reperfusion decreased F in the An wall (from 4.4 ± 0.3 to 1.4 ± 0.85) due to a decrease in A (20 vs. 73) and increased F in Pos wall (from 3.2 ± 0.6 to 6.6 ± 0.23) due to an increase in k (from 42 ± 3 to 93 ± 6). The intensities of the Rb images correlated with the Rb+ content measured in tissue samples. Magn Reson Med 44:83–91, 2000. Published 2000 Wiley‐Liss, Inc.

Noninvasive assessment of cardiac ischemic injury using87Rb and23Na MR imaging,31P MR, and optical spectroscopy

The aim of the study was to compare and analyze different noninvasive indices of cell damage in the isolated pig heart model of regional ischemia. We used 23Na and 87Rb MR imaging to evaluate Na+/K+ balance, 31P MR spectroscopy to measure energetics, and optical spectroscopy to assess oxymyoglobin (MbO2). Hearts were subjected to 120‐min occlusion of the left anterior descending artery and were then reperfused for 120 min. Reperfusion resulted in an increase in 23Na (37 ± 18% of the posterior wall) and decrease in 87Rb (55 ± 15%) image intensities, partial recovery of PCr, ATP, the total phosphates, and MbO2 in the anterior wall. The above changes are consistent with the irreversible cell damage in the anterior wall, confirmed by lack of staining with triphenyltetrazolium chloride. Changes in Na+ and Rb+ in the infarct area inversely correlated and their ratio is a more sensitive index of cell injury than either of them alone. Magn Reson Med 44:899–908, 2000. Published 2000 Wiley‐Liss, Inc.

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.

MRI study of cryoinjury infarction in pig hearts: i. Effects of intrapericardial delivery of bFGF/VEGF embedded in alginate beads

The aim of the study was the testing of sustained intrapericardial delivery of vascular growth factors (GFs) from alginate beads on cryoinjury size and perfusion. In domestic pigs (15–20 kg, n = 21), the left ventricular (LV) anterolateral wall of exposed hearts was cryoinjured using an aluminum rod (25 mm o.d.) cooled in liquid nitrogen. Alginate beads (d = 3.2 ± 0.2 mm), containing human recombinant basic fibroblast GF (bFGF, 50 µg) and vascular endothelial GF (VEGF, 50 µg) + heparin (50 µg) or heparin alone (Con, n = 5), were sutured to the cryoinjured epicardium (GF, n = 5; Con, n = 3 ) or pericardium (GF, n = 3; Con, n = 2), or no beads were implanted (n = 4). Four pigs were sham‐operated. Cine and T1‐weighted MRI was performed in vivo at ~2.5 h and 1, 2, 3 and 4 weeks after injury in a 3T imager. A double bolus of GdDTPA was injected (0.05 and 0.15 mmol/kg) and first‐pass and late enhancement kinetics were monitored. After 4‐week cryoinjury, following the injection of 5x106 15‐µm NIR fluorescent microspheres (FMS, 645/680 nm), hearts were sliced and examined with fluorescence imaging. Triphenyltetrazolium chloride (TTC) staining was used to determine infarct areas. Epicardial GF‐containing beads were encapsulated within the hypointense 3‐4‐week infarct tissue. This tissue had a 75% higher LV thickening index, a lower distribution volume for GdDTPA (0.44 ± 0.12 vs 0.68 ± 0.05, p = 0.02), and 25% faster first‐pass Gd kinetics relative to control infarctions. TTC staining revealed TTC‐positive islands in the core of treated infarcts, which showed higher FMS fluorescence relative to surrounding infarct tissue (0.64 ± 0.14 vs. 0.31 ± 0.14; p < 0.0001) and to control infarcts (0.37 ± 0.09, p < 0.05). GF‐beads attached to the pericardium were not effective. We conclude that sustained intrapericardial release of bFGF + VEGF from alginate beads attached to the epicardium facilitated vascular growth in the cryoinjured area. Copyright

Effects of flow and energy metabolism on injury and Rb+ uptake in pig hearts: An 87Rb and 31P NMR study

In this work the roles of coronary flow (CF) and metabolism in Rb+ (K+ congener) uptake were studied. In isolated pig hearts the left anterior descending artery (LAD) was cannulated to maintain adequate perfusion of the LAD bed. Rb+ loading was initiated and the LAD flow was either completely stopped (no flow (NF)) or reduced to 12% (low flow (LF)), or buffer was deoxygenated without change in flow (hypoxic flow (HYP)) for 2 h. CF through the LAD was then restored to normal, or perfusion was switched to oxygenated buffer. Serial 87Rb MR images or localized 31P spectra were acquired to compare the Rb+ uptake and energetics in the left ventricular (LV) anterior (ischemic/hypoxic) and posterior (normal) walls. End‐ischemic/hypoxic 87Rb signal intensities in the anterior wall were higher and the fluxes were greater in the HYP and LF groups than in the NF group. Phosphocreatine and ATP decreased less significantly and recovered better in the HYP and LF groups. Upon reperfusion/reoxygenation, the HYP and LF groups showed higher 87Rb signal intensities and smaller or no infarctions in the anterior wall compared to those in the NF group. Ischemia reduces Rb+ uptake due to both flow limitations and metabolic inhibition of cellular transport. 87Rb MRI has a potential for distinguishing necrotic and reversibly damaged tissue. Magn Reson Med 46:963–973, 2001.

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.

Effects of ischemia on intracellular rubidium in pig and rat hearts: 87Rb NMR imaging and spectroscopic study

87Rb MR imaging and spectroscopy were used to study the effects of ischemia on the properties of K+ in cardiac tissue. Isolated pig and rat hearts perfused by the Langendorff method with Krebs‐Henseleit buffer were preloaded with Rb+. Ischemia (Isc) was induced by 120‐min occlusion of the left anterior descending artery in the pig hearts or by stopping perfusion for 33 min in the rat hearts. Serial 87Rb MR images or spectra from the anterior (An) LV wall of pig hearts were acquired continuously. The intensities of the Rb images of the An and posterior (Pos) walls were similar and stable during the first 45 min of ischemia. The intensity of signal from the An wall (Isc) then gradually increased by 60 ± 8% relative to the preischemic value (vs. 31 ± 5% increase in Pos wall) and necrosis (19 ± 5% of the LV wall mass) developed upon reperfusion. The Rb+ content was lower in the ischemic (An) than in the normal (Pos) area (22.3 ± 3 vs. 28.4 ± 1.3 mmol/g wet wt). A similar pattern was observed in the peak heights of 87Rb spectra from the An wall, which increased by 40 ± 16% (vs. 21 ± 11% in control) due to a 12% decrease in the apparent Rb linewidth (LW) and a 24 ± 14% increase in the peak area. The Rb peak comprised narrow (297 ± 21 Hz) and broad (1098 ± 40 Hz, 59 ± 3% of total area) Lorentzian components. The LW of the broad component decreased by 14%, while the narrow component did not change markedly. In the rat hearts ischemia caused a 33 ± 4% increase in the 87Rb peak height as a result of peak narrowing (13 ± 1%), and an increase in peak area (17 ± 5%). The decreases in LW and increases in Rb+ visibility can be explained by an increase in Rb+ mobility caused by displacement of Rb+ from anionic binding sites by H+ (ischemic acidosis) and changes in intracellular compartmentalization of Rb+. Magn Reson Med 44:193–200, 2000.

Defects in myoglobin oxygenation in KATP-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.