MuRF1 activity is present in cardiac mitochondria and regulates reactive oxygen species production in vivo

Erratum: https://link.springer.com/article/10.1007/s10863-014-9597-1MuRF1 is a previously reported ubiquitin-ligase found in striated muscle that targets troponin I and myosin heavy chain for degradation. While MuRF1 has been reported to interact with mitochondrial substrates in yeast two-hybrid studies, no studies have identified MuRF1’s role in regulating mitochondrial function to date. In the present study, we measured cardiac mitochondrial function from isolated permeabilized muscle fibers in previously phenotyped MuRF1 transgenic and MuRF1−/− mouse models to determine the role of MuRF1 in intermediate energy metabolism and ROS production. We identified a significant decrease in reactive oxygen species production in cardiac muscle fibers from MuRF1 transgenic mice with increased α-MHC driven MuRF1 expression. Increased MuRF1 expression in ex vivo and in vitro experiments revealed no alterations in the respiratory chain complex I and II function. Working perfusion experiments on MuRF1 transgenic hearts demonstrated significant changes in glucose oxidation. This is an factual error as written; however, total oxygen consumption was decreased. This data provides evidence for MuRF1 as a novel regulator of cardiac ROS, offering another mechanism by which increased MuRF1 expression may be cardioprotective in ischemia reperfusion injury, in addition to its inhibition of apoptosis via proteasome-mediate degradation of c-Jun. The lack of mitochondrial function phenotype identified in MuRF1−/− hearts may be due to the overlapping interactions of MuRF1 and MuRF2 with energy regulating proteins found by yeast two-hybrid studies reported here, implying a duplicity in MuRF1 and MuRF2’s regulation of mitochondrial function.Funding support from Medical Research Council, United Kingdom; National Institutes of Health, United States; British Heart Foundation, United Kingdo

Two dimensional crystallization of three solid Lipid A-Diphosphate Phases

Surface-tension-induced liquid-crystal growth of monomeric lipid A-diphosphate in aqueous dispersions is reported as a function of concentration, (c), and temperature, (T), and at low ionic strength (10–3 M). As the temperature was varied, a solid–liquid transition was revealed in the surface layer at a fixed lipid A-diphosphate bulk concentration. Here, the development of different two-dimensional (2-d) faceted crystal morphologies was observed and, as growth proceeded, these faceted 2-d crystals became unstable. Selected area electron microscopy diffraction (SAED) and X-ray diffraction (XRD) measurements of the faceted 2-d crystalline lipid A-diphosphate layers exhibited a pseudohexagonal molecular arrangement. The crystalline layer was a smectic F, SF, phase below the critical temperature, TC, and a smectic I, SI, phase above TC (15 °C). Both phases could be described in terms of the same C-centered monoclinic unit cell. The in-plane order extended for a limited distance although the layers were coupled. The analysis of the SAED patterns revealed short-range order in the SF phase (5–15 °C), but long-range order in the SI phase, for the temperature range 15–30 °C. The observed 2-d solid hexatic phase and the 2-d liquid hexatic phase had correlation lengths of 220 Å. This, the hexatic phase, displayed short-range in-plane positional order and quasi long-range, sixfold bond-orientational order. The SI phase showed long-range order characteristics of a hexatic 2-d crystal. The two-, four-, or six-layer crystalline lipid A-diphosphate films exhibited 2-d hexatic order and 6n-fold bond-orientational order. These films did not evolve into the SF phase, demonstrating that the two phases were thermodynamically distinct. A finite tilt angle of φ = 15° was calculated for the lipid A-diphosphate molecule; the tilt was toward the small side of the rectangular 2-d lattice. The constraint of six close-packed acyl chains in two distinct phases with the same symmetry suggests that the SF → SI transition was first-order

Solution and structural properties of colloidal charged lipid A (diphosphate) dispersions

It has been possible to prepare electrostatically stabilized aqueous dispersions of lipid A (diphosphate) particles of low polydispersity at low ionic strength (1-10 mM NaCl) over a range of volume fractions of 1.5 × 10-4 < < 5.75 × 10-4 (25 C). These suspensions have been characterized by transmission electron microscopy, light scattering, osmotic pressure measurements, and small-angle X-ray scattering experiments at 25 C. All four measurements yielded independent values for particle sizes, weighted-average molecular weights, number-average molecular weights, and particle surface charge. The mean values obtained are = 37.59 ± 0.75 nm, = 24.89 ± 0.88 nm, = (10.55 ± 0.78) × 106 g/mol, = (9.81 ± 0.90) × 106 g/mol, and the effective surface charge Z* = (756 ± 85). Very good experimental agreement is found for the directly measured osmotic pressure values and those determined from light scattering and small-angle X-ray scattering measurements as a function of volume fraction, , by applying liquid-state theory models. Using the particle parameters for the lipid A (diphosphate) system as determined, the scattering functions and the osmotic pressures can be compared as a function of volume fraction with no adjustable parameters. The ordering of lipid A in solution revealed a body-centered cubic (bcc) type lattice (a = 36.14 nm) at volume fractions of 3.75 × 10-4 < < 4.15 × 10-4, whereas at volume fractions of 4.15 × 10-4 < < 5.75 × 10-4 in the presence of 1.0 mM NaCl a face-centered cubic (fcc) lattice type (a = 57.25 nm) was observed. Small-angle X-ray scattering experiments also indicate the presence of long-ranged order at 1.0 mM or at 10.0 mM NaCl for lipid A dispersions of 3.75 × 10-4 < < 5.75 × 10-4

Structures of the 2-nitrophenol alkali complexes in solution and the solid state

The materials studied in this investigation were aqueous solutions (0.02-25.0 mM) of the salts of alkali metal ion (Me+) and 2-nitrophenol (2-NP). In the investigation, small-angle X-ray scattering, wide-angle X-ray scattering, and membrane-pressure osmometry were used to study the 2-NP-Me+ molecular salt structures and the onset of crystallization as a function of concentration and temperature. The experimental methods used to examine the 2-NP-Me+ molecular salt complexes provided corroborative evidence for the existence of spherical clusters with hydrodynamic diameters between ∼12 Å (Li) and 14 Å (Cs). Guinier plots of the zero-angle scattering peak were characteristic of the scattering from lamellae-like shapes with thicknesses of ∼290 Å. Tetramer and pentamer 2-NP-Me+ molecular clusters for Me+ = Li, Na, K, and Rb were assembled from four or five 2-NP molecules bound to a central alkali metal ion. The coordination symmetry around the six coordinated Li+, Na+, and K+ ions was that of a trigonal prism (D3h), with an octahedral arrangement (D2h). The Rb+ also revealed six-coordinate geometry and the central Rb+ ion adopted an octahedral arrangement (D2h). The eight-coordinated Cs+ ions with six 2-NP ligands were characteristic of a square antiprism (D4d). The square antiprism was the outcome of leaving two o-nitro groups and two phenolic oxygens being left intermolecularly uncoordinated to the Cs+ ion. The 2-NP residues were strictly planar and contained short non-bonded intramolecular distances. van der Waals forces were present between the adjacently stacked phenyl rings. No water molecules were involved as ligands for any of the 2-nitrophenol-Me+ complexes

The liquidlike ordering of lipid A-diphosphate colloidal crystals: the influence of Ca2+, Mg2+, Na+, and K+ on the ordering of colloidal suspensions of lipid A-diphosphate in aqueous solutions

A comprehensive study was performed on electrostatically stabilized aqueous dispersion of lipid A-diphosphate in the presence of bound Ca2+, Mg2+, K+, and Na+ ions at low ionic strength (0.10-10.0-mM NaCl, 25 °C) over a range of volume fraction of 1.0×10-4<=f<=4.95×10-4. These suspensions were characterized by light scattering (LS), quasielastic light scattering, small-angle x-ray scattering, transmission electron microscopy, scanning electron microscopy, conductivity measurements, and acid-base titrations. LS and electron microscopy yielded similar values for particle sizes, particle size distributions, and polydispersity. The measured static structure factor, S(Q), of lipid A-diphosphate was seen to be heavily dependent on the nature and concentration of the counterions, e.g., Ca2+ at 5.0 nM, Mg2+ at 15.0 µM, and K+ at 100.0 µM (25 °C). The magnitude and position of the S(Q) peaks depend not only on the divalent ion concentration (Ca2+ and Mg2+) but also on the order of addition of the counterions to the lipid A-diphosphate suspension in the presence of 0.1-µM NaCl. Significant changes in the rms radii of gyration (RG2)1/2 of the lipid A-diphosphate particles were observed in the presence of Ca2+ (24.8±0.8 nm), Mg2+ (28.5±0.7 nm), and K+ (25.2±0.6 nm), whereas the Na+ salt (29.1±0.8 nm) has a value similar to the one found for the de-ionized lipid A-diphosphate suspensions (29.2±0.8 nm). Effective particle charges were determined by fits of the integral equation calculations of the polydisperse static structure factor, S(Q), to the light-scattering data and they were found to be in the range of Z*=700-750 for the lipid A-diphosphate salts under investigation. The light-scattering data indicated that only a small fraction of the ionizable surface sites (phosphate) of the lipid A-diphosphate was partly dissociated (~30%). It was also discovered that a given amount of Ca2+ (1.0-5.0 nM) or K+ (100 µM) influenced the structure much more than Na+ (0.1-10.0-mM NaCl) or Mg2+ (50 µM). By comparing the heights and positions of the structure factor peaks S(Q) for lipid A-diphosphate-Na+ and lipid A-diphosphate-Ca2+, it was concluded that the structure factor does not depend simply on ionic strength but more importantly on the internal structural arrangements of the lipid A-diphosphate assembly in the presence of the bound cations. The liquidlike interactions revealed a considerable degree of ordering in solution accounting for the primary S(Q) peak and also the secondary minimum at large particle separation. The ordering of lipid A-diphosphate-Ca2+ colloidal crystals in suspension showed six to seven discrete diffraction peaks and revealed a face-centered-cubic (fcc) lattice type (a=56.3 nm) at a volume fraction of 3.2×10-4<=f<=3.9×10-4. The K+ salt also exhibited a fcc lattice (a=55.92 nm) at the same volume fractions, but reveals a different peak intensity distribution, as seen for the lipid A-diphosphate-Ca2+ salt. However, the Mg2+ and the Na+ salts of lipid A-diphosphate showed body-centered-cubic (bcc) lattices with a=45.50 nm and a=41.50 nm, respectively (3.2×10-4<=f<=3.9×10-4), displaying the same intensity distribution with the exception of the (220) diffraction peaks, which differ in intensity for both salts of lipid A-diphosphate

Liquid-like ordered colloidal suspensions of lipid A: the influence of lipid A particle concentration

Electrostatically stabilized aqueous dispersions of nm-sized free lipid A particles at low volume fractions (1.0×10-4<=θ<=3.5×10-4) in the presence of 1.0-10.0 mM NaCl (25 °C) have been characterized by static and quasielastic light scattering (QELS) techniques, electron microscopy (SEM and TEM), conductivity measurements, and acid-base titrations. QELS and electron microscopy (<overbar>ρTEM=8.0±0.6%) yield similar values for the particle size and particle size distribution (<overbar>ρQELS=10.9±0.75 %), whereas conductivity and acid-base titrations estimate surface chemical parameters (dissociation constant, ionizable sites, and Stern capacitance). Effective particle charges were determined by fits of the integral equation calculations of the polydisperse static structure factor, <overbar>S(Q), to the light scattering data. Using the particle properties as determined from these experiments, the polydisperse structure factor, <overbar>S(Q), was calculated as a function of volume fraction, θ, which was found to be consistent with a <overbar>S(Q) dependence on the number particle density. It can be concluded that, at low volume fractions and low ionic strength, the light scattering data are well represented by a Poisson-Boltzmann model (PBC) of fluid-like ordering of free lipid A in aqueous solution. We find that the light scattering data of this dispersion are best described by a model where only a small fraction of the ionizable phosphate groups is dissociated at neutral pH. Finally, light scattering studies of lipid A dispersions of volume fractions of 3.9×10-4<=θ<=4.9×10-4 indicate the presence of long-range order, resulting in distinct peaks which can be assigned either to a face-centered cubic (fcc) lattice (a=51.7 nm) or a body-centered cubic (bcc) lattice (a=41.5 nm), respectively

The formation of colloidal crystals of lipid A diphosphate: evidence for the formation of nanocrystals at low ionic strength

Dilute electrostatically stabilized aqueous solutions of hexa-acylated (C14) lipid A diphosphate from Escherichia coli form stable and regularly shaped colloidal crystals in a size range of approximately 50-1000 nm in width and 50-100 nm in thickness. The formation of these nanocrystals occurs over a range of volume fractions between 3.5 × 10-3 and 1.2 × 10-2 and at a low ionic strength, ~10-5. The shape of these crystals appears to be cubic or rhombohedral, and when exposed to the electron beam, these fragile nanocrystals are easily damaged. Electron diffraction patterns obtained from single particles reveal that they are orientated (001) crystals that conform to a trigonal or hexagonal unit cell (a = 3.65 ± 0.07 nm and c = 1.97 ± 0.04 nm), revealing crystal-like pore walls that exhibit structural periodicity with a spacing of 0.65 nm and are at least four times the size of the unit cell adopted by lipid A diphosphate