RAD51-Mediated DNA Homologous Recombination Is Independent of PTEN Mutational Status.

PTEN mutation occurs in a variety of aggressive cancers and is associated with poor patient outcomes. Recent studies have linked mutational loss of PTEN to reduced RAD51 expression and function, a key factor involved in the homologous recombination (HR) pathway. However, these studies remain controversial, as they fail to establish a definitive causal link to RAD51 expression that is PTEN-dependent, while other studies have not been able to recapitulate the relationship between the PTEN expression and the RAD51/HR function. Resolution of this apparent conundrum is essential due to the clinically-significant implication that PTEN-deficient tumors may be sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) commonly used in the clinical management of BRCA-mutated and other HR-deficient (HRD) tumors. METHODS: Primary Pten-deficient (and corresponding wild-type) mouse embryonic fibroblasts (MEFs) and astrocytes and PTEN-null human tumor cell lines and primary cells were assessed for RAD51 expression (via the Western blot analysis) and DNA damage repair analyses (via alkali comet and γH2AX foci assays). RAD51 foci analysis was used to measure HR-dependent DNA repair. Xrcc2-deficient MEFs served as an HR-deficient control, while the stable knockdown of RAD51 (shRAD51) served to control for the relative RAD51/HR-mediated repair and the phospho-53BP1 foci analysis served to confirm and measure non-homologous end joining (NHEJ) activity in PTEN-deficient and shRAD51-expressing (HRD) lines. Cell proliferation studies were used to measure any potential added sensitivity of PTEN-null cells to the clinically-relevant PARPi, olaparib. RAD51 levels and DNA damage response signaling were assessed in PTEN-mutant brain tumor initiating cells (BTICs) derived from primary and recurrent glioblastoma multiforme (GBM) patients, while expression of RAD51 and its paralogs were examined as a function of the PTEN status in the RNA expression datasets isolated from primary GBM tumor specimens and BTICs. RESULTS: Pten knockout primary murine cells display unaltered RAD51 expression, endogenous and DNA strand break-induced RAD51 foci and robust DNA repair activity. Defective HR was only observed in the cells lacking Xrcc2. Likewise, human glioblastoma multiforme (GBM) cell lines with known PTEN deficiency (U87, PTEN-mutated; U251 and U373, PTEN-null) show apparent expression of RAD51 and display efficient DNA repair activity. Only GBM cells stably expressing shRNAs against RAD51 (shRAD51) display dysfunctional DNA repair activity and reduced proliferative capacity, which is exacerbated by PARPi treatment. Furthermore, GBM patient-derived BTICs displayed robust RAD51 expression and intact DNA damage response signaling in spite of PTEN-inactivating mutations. RNA expression analysis of primary GBM tissue specimens and BTICs demonstrate stable levels of RAD51 and its paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and DMC1), regardless of the PTEN mutational status. CONCLUSIONS: Our findings demonstrate definitively that PTEN loss does not alter the RAD51 expression, its paralogs, or the HR activity. Furthermore, deficiency in PTEN alone is not sufficient to impart enhanced sensitivity to PARPi associated with HRD. This study is the first to unequivocally demonstrate that PTEN deficiency is not linked to the RAD51 expression or the HR activity amongst primary neural and non-neural Pten-null cells, PTEN-deficient tumor cell lines, and primary PTEN-mutant GBM patient-derived tissue specimens and BTICs.

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.

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.

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.

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.

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.

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.

Effects of K(ATP) channel openers, P-1075, pinacidil, and diazoxide, on energetics and contractile function in isolated rat hearts.

We investigated the metabolic effects of a potent opener of ATP-sensitive K(+) (K(ATP)) channels, P-1075, in perfused rat hearts with the help of(31)P NMR spectroscopy. A 20 min infusion of 5 microm P-1075 depleted phosphocreatine and ATP by approximately 40%, concomitantly with a two-fold increase in inorganic phosphate, while oxygen consumption by the hearts increased by 50%. P-1075 induced a cardiac contracture (left ventricular end diastolic pressure increased from 6 to 60 mmHg) and a cardiac arrest after an infusion of approximately 9 min. The effects were fully reversed by glibenclamide (5 microm), but not by sodium 5-hydroxydecanoate (0.4 m m). A P-1075-related K(ATP) opener, pinacidil (0.3 m m), partially reversed the effects of P-1075, but a structurally unrelated opener, diazoxide (0.5 m m), had no effect. Pinacidil and diazoxide alone did not significantly affect PCr and ATP levels. Bioenergetic and functional effects similar to those of P-1075 were induced by infusion of a classic mitochondrial uncoupler, 2,4-dinitrophenol (50 microm); however, they were not abolished by glibenclamide. In addition, it was shown, using(87)Rb NMR, that both agents, P-1075 and 2,4-dinitrophenol, resulted in a stimulation of Rb(+) efflux from the Rb(+) loaded rat hearts by approximately 130 and 65%, respectively, in a glibenclamide-sensitive manner. An inhibitory effect of P-1075 on ATP synthesis cannot be explained by its well-known action on sarcolemmal K(ATP) channels. We concluded that, unlike an uncoupling effect of 2,4-dinitrophenol, an inhibitory effect of P-1075 is produced by uncoupling of oxidative phosphorylation through the activation of mitochondrial K(ATP) channels.