Investigating the Role of Mitochondrial Fission in Cardiac Myocyte Hypoxia/Re-oxygenation-Induced Cell Death

Currently there is no effective pharmacological intervention to attenuate myocardialischemia reperfusion (IR) injury. Understanding the mechanisms that govern IR injury would benefit myocardial infarction, coronary bypass and organ transplantpatients. Recent studies suggest that cardiac mitochondria undergo excessive fragmentation during IR, a process that is called fission. Increased mitochondrial fission may lead to increased cell death during IR. Mitochondrial fission has been shown to be mediated by association of dynamin related protein-1(Drp1), a GTPasethat translocates from the cytosol to interact with outer mitochondrial membrane proteins, fission protein 1 and mitochondrial fission factor, to induce fission. We hypothesize that attenuating mitochondrial fission during IR may prove to be an effective strategy to mitigate IR injury. To test this hypothesis, we used a pharmacological inhibitor of Drp-1 GTPase activity, Mdivi-1(MW=353 g/mol). We subjected murine cardiac myocytes (HL-1 cells) to 12 hours of hypoxia in hypoxic buffer (pH 6.8, no glucose or pyruvate) and then 1 hour of re-oxygenation in normoxic media (pH 7.4), in the presence/absence of Mdivi-1 (5-25μM). We predicted that HL-1 cells subjected to simulated IR (SIR) would exhibit increased cell death in relation to their normoxic control counterparts, and that Mdivi-1 treated cells would have attenuated SIR-induced cell death. Cell death was determined by trypan blue (0.3%) staining. Preliminary data shows that control SIRcells (n=4) exhibited 25% cell death, by contrast Mdivi-1 treated cells only showed 7% (5μM, n=4) and 11% (25μM n=4) cell death. Normoxic untreated (n=3) and Mdivi-1 treated cells (5 and 25 μM; n=3) showed only 2% cell death in all study groups. The data suggests that inhibiting Drp-1 may mitigate cardiac cell death during SIR. Future studies will determine the role of Drp-1 in the regulation of mitochondrial dynamics during SIR

Nanomanufacturing for biological sensing applications

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2007."February 2007."Includes bibliographical references (leaves 219-226).Over the past 10-15 years, there have been tremendous research efforts in the synthesis of nanomaterials with unique electronic properties. Much less work, however, has focused on the incorporation of the nanomaterials into electronic devices. In order for nanomaterials to have a technological impact in electronic devices, nanomanufacturing techniques must be established for the reliable and reproducible creation of devices with nanomaterials as the active component. In this thesis, the incorporation of 3-20 nm diameter ligand coated gold nanoparticles into an electronic device is studied. Ligand coated nanoparticles provide great control over their solubility and electronic properties through the choice of protecting ligand molecule. The use of an isolated nanoparticle in electronic devices presents two major difficulties which are studied in detail in this work. In order to use the electrical properties of a single particle or a few particles, insulating gaps in metallic electrodes must be fabricated with dimensions of 5-50 nm. Several methods including direct patterning with electron beam lithography, physical methods of gap formation, and electrical methods of gap formation are described, studied and evaluated for use in nanomanufacturing.(cont.) A second major challenge is the specific assembly of nanoparticles into the nanogaps. The use of chemically directed assembly to pattern particles on templates generated by Dip Pen Nanolithography is described using several different surface chemistries. An electrical based method, dielectrophoresis, is found to be better suited for assembly of particles into the gaps and the forces which affect assembly are studied in detail. Electrical characterizations of networks of 10-200 nanoparticles are studied as a function of protecting ligand molecule. Preliminary results on the use of nanomanufactured devices consisting of gold nanoparticles-oglionucleotide conjugates bridging a nano-gap for DNA sensing are presented.by Robert J. Barsotti, Jr.Ph.D

Stretch-induced Calcium Release in Smooth Muscle

Smooth muscle cells undergo substantial increases in length, passively stretching during increases in intraluminal pressure in vessels and hollow organs. Active contractile responses to counteract increased transmural pressure were first described almost a century ago (Bayliss, 1902) and several mechanisms have been advanced to explain this phenomenon. We report here that elongation of smooth muscle cells results in ryanodine receptor–mediated Ca2+ release in individual myocytes. Mechanical elongation of isolated, single urinary bladder myocytes to ∼120% of slack length (ΔL = 20) evoked Ca2+ release from intracellular stores in the form of single Ca2+ sparks and propagated Ca2+ waves. Ca2+ release was not due to calcium-induced calcium release, as release was observed in Ca2+-free extracellular solution and when free Ca2+ ions in the cytosol were strongly buffered to prevent increases in [Ca2+]i. Stretch-induced calcium release (SICR) was not affected by inhibition of InsP3R-mediated Ca2+ release, but was completely blocked by ryanodine. Release occurred in the absence of previously reported stretch-activated currents; however, SICR evoked calcium-activated chloride currents in the form of transient inward currents, suggesting a regulatory mechanism for the generation of spontaneous currents in smooth muscle. SICR was also observed in individual myocytes during stretch of intact urinary bladder smooth muscle segments. Thus, longitudinal stretch of smooth muscle cells induces Ca2+ release through gating of RYR. SICR may be an important component of the physiological response to increases in luminal pressure in smooth muscle tissues

Effects of Mitochondria-Targeted Antioxidants on Real-time Blood Nitric Oxide and Hydrogen Peroxide Release in Hind Limb Ischemia and Reperfusion

In the body, reperfusion of ischemic tissue with blood causes the release of reactive oxygen species (ROS), in part, from damaged mitochondria leading to endothelial and organ dysfunction. Endothelial dysfunction occurs within 5 min of reperfusion, is common to all vascular beds, and is characterized by increased hydrogen peroxide (H2O2) and decreased nitric oxide (NO) levels in the blood that further exacerbate reperfusion injury. Previous studies have shown that promoting endothelial NO synthase coupling during reperfusion increases blood NO and decreases blood H2O2 levels in hind limb I/R and attenuates myocardial I/R injury (1). This study specifically examines the effects mitochondria-targeted antioxidants, mitoquinone (mitoQ; Fig. 1), a cell permeable coenzyme Q analogue or SS-31 ((D-Arg)-Dmt-Lys-Phe-Amide; Genemed Synthesis, San Antonio, TX) (Fig.1), a cell permeable peptide, on inhibiting H2O2 release and increasing NO bioavailability in hind limb I/R. MitoQ (2) and SS-31 (3,4) are able to concentrate into the inner mitochondrial membrane via an electrical potential gradient or selective diffusion respectively, where they attenuate superoxide and subsequent H2O2 production thus allowing a concurrent increase in NO bioavailability

Theology, News and Notes - Vol. 52, No. 02

Theology News & Notes was a theological journal published by Fuller Theological Seminary from 1954 through 2014.https://digitalcommons.fuller.edu/tnn/1153/thumbnail.jp

Myristoylated protein kinase C beta II peptide inhibitor exerts dose-dependent inhibition of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced leukocyte superoxide release

Protein kinase C (PKC) phosphorylation of leukocyte NADPH oxidase is essential to generatesuperoxide (SO) release. Inhibition of leukocyte SO release attenuates inflammation mediated vascular injury. However, the role of PKC isoforms mediating this response has not been fully elucidated. We hypothesize that PKC beta II (βII) isoform positively regulates leukocyte NADPH oxidase, and that a cell-permeable (myr)-PKC βII peptide inhibitor (N-myr-SLNPEWNET) would dose-dependently attenuate fMLP induced leukocyte SO release. fMLP is a leukocyte chemoattractant cell membrane receptor agonist. We isolated leukocytes by peritoneal lavage from male Sprague-Dawley rats using standard methods. fMLP (1 M)-induced leukocyte SO release was measured for 120 sec spectrophotometrically by reduction of ferricytochrome c in the presence/absence of myr-PKC βII peptide inhibitor (0.2 to 20 M) in 5 x 106 leukocytes. After each assay, cell viability was determined by 0.3% trypan blue exclusion. fMLP-induced leukocyte SO release increased peak absorbance to 0.18±0.03 in controls (n=20). This response was dose-dependently inhibited by myr-PKC βII peptide inhibitor at 0.17±0.05 (0.2 M; n=9), 0.14±0.05 (0.5 M; n=11), 0.1±0.05 (1 M; n=9), 0.05±0.03 (5 M; n=9), 0.04±0.03 (10 M; n=8) and 0.05±0.03 (20 µM; n=7) and was significantly attenuated in the 5 to 20 M range compared to controls (p\u3c0.05). Moreover, cell viability was \u3e 94±1% in all study groups. These results suggest that myr-PKC βII peptide inhibitor dose-dependently inhibits fMLP-induced leukocyte SO release in the 0.2 to 5 M dose-range and these effects are attributed to inhibition of PKC βII isoform

The role of endothelial nitric oxide synthase (eNOS) uncoupling in acute hyperglycemia – induced oxidative stress and vascular endothelial dysfunction by measuring blood nitric oxide and hydrogen peroxide in real-time

Acute hyperglycemia can impair vascular endothelial function in non-diabetic subjects in addition to diabetic patients. Decreased eNOS derived nitric oxide (NO) bioavailability and increased reactive oxygen species (ROS), such as superoxide (SO) and hydrogen peroxide (H2O2), are the major characteristics of vascular endothelial dysfunction. Furthermore, eNOS can change from coupled to an uncoupled status resulting in SO production instead of NO production. The role of eNOS uncoupling in acute hyperglycemia induced vascular dysfunction is unclear in vivo. In this study we hypothesized that acute hyperglycemia (200 mg/dL) would increase H2O2 and decrease NO release in blood relative to saline control. By contrast, 5,6,7,8-tetrahydrobiopterin (BH4, an essential cofactor of coupled eNOS) (MW=241.247 g/mol, 6.5 mg/kg) or L-arginine (the substrate of coupled eNOS) (MW=210.66 g/mol, 600 mg/kg) would attenuate acute hyperglycemia-induced blood NO/H2O2 change. However, 7,8-dihydrobiopterin (BH2, an oxidized form of BH4 and serves as a cofactor for uncoupled eNOS) (MW=239.231 g/mol, 4 mg/kg) will exacerbate acute hyperglycemia-induced blood NO/H2O2 change. Blood NO or H2O2 levels were measured simultaneously using calibrated NO or H2O2 microsensors (100 µm WPI Inc.) by placing them into the femoral veins of male Sprague-Dawley rats. The electrical traces were recorded at baseline and throughout 3 hours of infusion with saline or 20% D-glucose with or without a drug and converted into concentration based on the calibration curve. We found that acute hyperglycemia (200 mg/dL) significantly increased H2O2 (n=6) and reduced NO (n=6) blood levels compared to the saline group (n=7, p2 exacerbated hyperglycemia– induced increased H2O2 levels (n=7) and decreased NO levels (n=4) (p4 (n=6), significantly attenuated hyperglycemia– induced increased H2O2 levels and decreased NO levels (p2O2 (n=5) and NO (n=6) blood levels as BH4, showing significant reduction of blood H2O2 and enhancement of blood NO (p2O2 and reduced NO blood levels. Uncoupled eNOS serves as a significant source mediating acute hyperglycemia-induced vascular dysfunction. Therefore, promotion of eNOS coupling may be effective in protecting vascular endothelial function from hyperglycemic insult

Effects of Mitochondrial-Targeted Antioxidants on Real-Time Blood Nitric Oxide and Hydrogen Peroxide Release in Acute Hyperglycemia Rats

Acute hyperglycemia in non-diabetic subjects can impair vascularendothelial function, causing decreased endothelium-derived nitric oxide (NO) release and increased reactive oxygen species (ROS), such assuperoxide and hydrogen peroxide (H2O2). Hyperglycemia may induce mitochondrial dysfunction leading to ROS production and exacerbation of vascular endothelial dysfunction. We investigated whether mitochondrial-targeted antioxidants mitigate acute hyperglycemia-induced oxidative stress and reduced blood NO. To test this hypothesis, blood NO or H2O2 levels were measured simultaneously using NO or H2O2 microsensors (100 µm) which were placed into the femoral veins of anesthesized male Sprague-Dawley rats. Acute hyperglycemia was induced by infusion 20% D-glucose intravenously with or without mitochondria-targeted antioxidants (mitoquinone: mitoQ, MW=1714 g/mol, 2.3 mg/Kg; SS-31: (D-Arg)-Dmt-Lys-Phe-Amide, MW=640g/mol, 2.7 mg/Kg) for 3 hours. We found that acute hyperglycemia (200 mg/dL) significantly increased blood H2O2 by 3.0±0.5 M (n=7) and reduced blood NO by 68.0±13.5 nM (n=9) compared to the saline group at end of infusion (both p\u3c0.05). MitoQ significantly attenuated hyperglycemia– induced H2O2 levels by 2.5±0.2 M (n=7) and increased blood NO levels by 59.3±9.7 nM (n=5) (both p\u3c0.05 compared to hyperglycemia). Similarly, SS-31 significantly reduced hyperglycemia-induced blood H2O2 level by 4.0±0.6 M (n=5) and enhanced blood NO levels by 52.8±7.7 nM (n=6) at end of infusion (both p\u3c0.05 compared to hyperglycemia). In summary, acute hyperglycemia induces mitochondria-derived ROS which in turn contribute to vascular endothelial dysfunction. Therefore, mitochondria-targeted antioxidants are useful to attenuate acute hyperglycemia-induced vascular endothelial dysfunction and oxidative stress

Collagen Fibrils and Proteoglycans of Peripheral and Central Stroma of the Keratoconus Cornea - Ultrastructure and 3D Transmission Electron Tomography

Keratoconus (KC) is a progressive corneal disorder in which vision gradually deteriorates as a result of continuous conical protrusion and the consequent altered corneal curvature. While the majority of the literature focus on assessing the center of this diseased cornea, there is growing evidence of peripheral involvement in the disease process. Thus, we investigated the organization of collagen fibrils (CFs) and proteoglycans (PGs) in the periphery and center of KC corneal stroma. Three-dimensional transmission electron tomography on four KC corneas showed the degeneration of microfibrils within the CFs and disturbance in the attachment of the PGs. Within the KC corneas, the mean CF diameter of the central-anterior stroma was significantly (p ˂ 0.001) larger than the peripheral-anterior stroma. The interfibrillar distance of CF was significantly (p ˂ 0.001) smaller in the central stroma than in the peripheral stroma. PGs area and the density in the central KC stroma were larger than those in the peripheral stroma. Results of the current study revealed that in the pre- Descemet\u27s membrane stroma of the periphery, the degenerated CFs and PGs constitute biomechanically weak lamellae which are prone to disorganization and this suggests that the peripheral stroma plays an important role in the pathogenicity of the KC cornea

Determination of regional coronary flow utilizing microspheres in ex vivo myocardial ischemia/reperfusion (MI/R) injury

MI/R results in marked cardiac contractile dysfunction and cell death. We previously discovered that protein kinase C epsilon peptide inhibitor (PKC ε-) robustly restored post-reperfused cardiac function, and reduced infarct size, oxidative stress, and leukocyte endothelial interactions in coronary, hind limb, renal, and mesenteric vascular inflammation models. The mechanisms of these effects in part are due to attenuating uncoupled endothelial nitric oxide (NO) synthase activity and increase endothelial NO bioavailability. Therefore, we hypothesize that PKC ε- will increase regional flow during reperfusion. We determined regional flow to the right ventricle, left ventricle, and septum at baseline and after 10 and 45 min reperfusion using fluorescent microspheres in isolated rat perfused hearts subjected to global I(30min)/R(45min). A cell permeable PKC ε- (myr-EAVSLKPT, MW=1054 g/mol, 10μM, n=8) was given at beginning of reperfusion for 5 min. We found that final left ventricular developed pressure (LVDP) recovered to 70 ± 7%, maximal rate of left ventricular contraction, +dP/dtmax to 57 ± 7% and maximal rate of left ventricular relaxation, -dP/dtmin to 58 ± 6% of baseline values. These parameters were significantly improved compared to untreated I/R hearts (n=7) that only recovered to 30 ± 5% in LVDP, 21 ± 5% in dP/dtmax and 25 ± 5% in dP/dtmin relative to baseline values (all p \u3c 0.01). Moreover, our preliminary data suggest that PKC ε- treatment increased regional flow by 190 ± 40% in the right ventricle, 140 ± 30% in the left ventricle, and 120 ± 20% in the septum at 10 min. post reperfusion compared to non-treated control MI/R hearts. These values were maintained throughout the remaining 35 min of reperfusion. In summary, the data indicates that PKC ε- improves postreperfused cardiac function in part by restoration of regional flow