The Goto Kakizaki rat: Impact of age upon changes in cardiac and renal structure, function.

BACKGROUND: Patients with diabetes are at a high risk for developing cardiac dysfunction in the absence of coronary artery disease or hypertension, a condition known as diabetic cardiomyopathy. Contributing to heart failure is the presence of diabetic kidney disease. The Goto-Kakizaki (GK) rat is a non-obese, non-hypertensive model of type 2 diabetes that, like humans, shares a susceptibility locus on chromosome 10. Herein, we perform a detailed analysis of cardio-renal remodeling and response to renin angiotensin system blockade in GK rats to ascertain the validity of this model for further insights into disease pathogenesis. METHODS: Study 1: Male GK rats along with age matched Wistar control animals underwent longitudinal assessment of cardiac and renal function for 32 weeks (total age 48 weeks). Animals underwent regular echocardiography every 4 weeks and at sacrifice, early (~24 weeks) and late (~48 weeks) timepoints, along with pressure volume loop analysis. Histological and molecular characteristics were determined using standard techniques. Study 2: the effect of renin angiotensin system (RAS) blockade upon cardiac and renal function was assessed in GK rats. Finally, proteomic studies were conducted in vivo and in vitro to identify novel pathways involved in remodeling responses. RESULTS: GK rats developed hyperglycaemia by 12 weeks of age (p<0.01 c/w Wistar controls). Echocardiographic assessment of cardiac function demonstrated preserved systolic function by 48 weeks of age. Invasive studies demonstrated left ventricular hypertrophy, pulmonary congestion and impaired diastolic function. Renal function was preserved with evidence of hyperfiltration. Cardiac histological analysis demonstrated myocyte hypertrophy (p<0.05) with evidence of significant interstitial fibrosis (p<0.05). RT qPCR demonstrated activation of the fetal gene program, consistent with cellular hypertrophy. RAS blockade resulted in a reduction blood pressure(P<0.05) cardiac interstitial fibrosis (p<0.05) and activation of fetal gene program. No significant change on either systolic or diastolic function was observed, along with minimal impact upon renal structure or function. Proteomic studies demonstrated significant changes in proteins involved in oxidative phosp4horylation, mitochondrial dysfunction, beta-oxidation, and PI3K/Akt signalling (all p<0.05). Further, similar changes were observed in both LV samples from GK rats and H9C2 cells incubated in high glucose media. CONCLUSION: By 48 weeks of age, the diabetic GK rat demonstrates evidence of preserved systolic function and impaired relaxation, along with cardiac hypertrophy, in the presence of hyperfiltration and elevated protein excretion. These findings suggest the GK rat demonstrates some, but not all features of diabetes induced "cardiorenal" syndrome. This has implications for the use of this model to assess preclinical strategies to treat cardiorenal disease.

Cardiac Fibrosis: Key Role of Integrins in Cardiac Homeostasis and Remodeling.

Cardiac fibrosis is a common finding that is associated with the progression of heart failure (HF) and impacts all chambers of the heart. Despite intense research, the treatment of HF has primarily focused upon strategies to prevent cardiomyocyte remodeling, and there are no targeted antifibrotic strategies available to reverse cardiac fibrosis. Cardiac fibrosis is defined as an accumulation of extracellular matrix (ECM) proteins which stiffen the myocardium resulting in the deterioration cardiac function. This occurs in response to a wide range of mechanical and biochemical signals. Integrins are transmembrane cell adhesion receptors, that integrate signaling between cardiac fibroblasts and cardiomyocytes with the ECM by the communication of mechanical stress signals. Integrins play an important role in the development of pathological ECM deposition. This review will discuss the role of integrins in mechano-transduced cardiac fibrosis in response to disease throughout the myocardium. This review will also demonstrate the important role of integrins as both initiators of the fibrotic response, and modulators of fibrosis through their effect on cardiac fibroblast physiology across the various heart chambers.

Bright Ferritin-a Reporter Gene Platform for On-Demand, Longitudinal Cell Tracking on MRI.

A major unresolved challenge in cell-based regenerative medicine is the absence of non-invasive technologies for tracking cell fate in deep tissue and with high spatial resolution over an extended interval. MRI is highly suited for this task, but current methods fail to provide longitudinal monitoring or high sensitivity, or both. In this study, we fill this technological gap with the first discovery and demonstration of in vivo cellular production of endogenous bright contrast via an MRI genetic reporter system that forms manganese-ferritin nanoparticles. We demonstrate this technology in human embryonic kidney cells genetically modified to stably overexpress ferritin and show that, in the presence of manganese, these cells produce far greater contrast than conventional ferritin overexpression with iron or manganese-permeable cells. In living mice, diffusely implanted bright-ferritin cells produce the highest and most sustained contrast in skeletal muscle. The bright-ferritin platform has potential for on-demand, longitudinal, and sensitive cell tracking in vivo.

Functional culture and in vitro genetic and small-molecule manipulation of adult mouse cardiomyocytes.

Primary adult cardiomyocyte (aCM) represent the mature form of myocytes found in the adult heart. However, culture of aCMs in particular is challenged by poor survival and loss of phenotype, rendering extended in vitro experiments unfeasible. Here, we establish murine aCM culture methods that enhance survival and maintain sarcomeric structure and Ca2+ cycling to enable physiologically relevant contractile force measurements. We also demonstrate genetic and small-molecule manipulations that probe mechanisms underlying myocyte functional performance. Together, these refinements to aCM culture present a toolbox with which to advance our understanding of myocardial physiology.

The low-resolution structure of nHDL reconstituted with DMPC with and without cholesterol reveals a mechanism for particle expansion.

Small-angle neutron scattering (SANS) with contrast variation was used to obtain the low-resolution structure of nascent HDL (nHDL) reconstituted with dimyristoyl phosphatidylcholine (DMPC) in the absence and presence of cholesterol, [apoA1:DMPC (1:80, mol:mol) and apoA1:DMPC:cholesterol (1:86:9, mol:mol:mol)]. The overall shape of both particles is discoidal with the low-resolution structure of apoA1 visualized as an open, contorted, and out of plane conformation with three arms in nascent HDL/dimyristoyl phosphatidylcholine without cholesterol (nHDL(DMPC)) and two arms in nascent HDL/dimyristoyl phosphatidylcholine with cholesterol (nHDL(DMPC+Chol)). The low-resolution shape of the lipid phase in both nHDL(DMPC) and nHDL(DMPC+Chol) were oblate ellipsoids, and fit well within their respective protein shapes. Modeling studies indicate that apoA1 is folded onto itself in nHDL(DMPC), making a large hairpin, which was also confirmed independently by both cross-linking mass spectrometry and hydrogen-deuterium exchange (HDX) mass spectrometry analyses. In nHDL(DMPC+Chol), the lipid was expanded and no hairpin was visible. Importantly, despite the overall discoidal shape of the whole particle in both nHDL(DMPC) and nHDL(DMPC+Chol), an open conformation (i.e., not a closed belt) of apoA1 is observed. Collectively, these data show that full length apoA1 retains an open architecture that is dictated by its lipid cargo. The lipid is likely predominantly organized as a bilayer with a micelle domain between the open apoA1 arms. The apoA1 configuration observed suggests a mechanism for accommodating changing lipid cargo by quantized expansion of hairpin structures.

Three-dimensional structure of a schistosome serpin revealing an unusual configuration of the helical subdomain.

Parasitic organisms are constantly challenged by the defence mechanisms of their respective hosts, which often depend on serine protease activities. Consequently, protease inhibitors such as those belonging to the serpin superfamily have emerged as protective elements that support the survival of the parasites. This report describes the crystal structure of ShSPI, a serpin from the trematode Schistosoma haematobium. The protein is exposed on the surface of invading cercaria as well as of adult worms, suggesting its involvement in the parasite-host interaction. While generally conforming to the well established serpin fold, the structure reveals several distinctive features, mostly concerning the helical subdomain of the protein. It is proposed that these peculiarities are related to the unique biological properties of a small serpin subfamily which is conserved among pathogenic schistosomes.

The low resolution structure of ApoA1 in spherical high density lipoprotein revealed by small angle neutron scattering.

Spherical high density lipoprotein (sHDL), a key player in reverse cholesterol transport and the most abundant form of HDL, is associated with cardiovascular diseases. Small angle neutron scattering with contrast variation was used to determine the solution structure of protein and lipid components of reconstituted sHDL. Apolipoprotein A1, the major protein of sHDL, forms a hollow structure that cradles a central compact lipid core. Three apoA1 chains are arranged within the low resolution structure of the protein component as one of three possible global architectures: (i) a helical dimer with a hairpin (HdHp), (ii) three hairpins (3Hp), or (iii) an integrated trimer (iT) in which the three apoA1 monomers mutually associate over a portion of the sHDL surface. Cross-linking and mass spectrometry analyses help to discriminate among the three molecular models and are most consistent with the HdHp overall architecture of apoA1 within sHDL.

Characterization of the secondary binding sites of Maclura pomifera agglutinin by glycan array and crystallographic analyses.

The Maclura pomifera agglutinin (MPA) recognizes the T-antigen disaccharide Galß1,3GalNAc mainly through interaction of the α-GalNAc moiety with its primary site, but the interactions of the two flanking subsites A and B with aglycones and substituents other than Gal, respectively, are not well understood. We therefore characterized the specificity of MPA in more detail by glycan microarray analysis and determined the crystal structures of MPA without ligand and in complexes with Galß1,3GalNAc and p-nitrophenyl α-GalNAc. In both sugar complexes, pairs of ligands created inter-tetramer hydrogen-bond bridging networks. While subsite A showed increased affinity for hydrophobic aglycones, it also accommodated several sugar substituents. Notably, a GalNAc-O-tripeptide, a Tn-antigen mimic, showed lower affinity than these compounds in surface plasmon resonance (SPR) experiments. The glycan array data that showed subsite B accepted compounds in which the O3 position of the GalNAc was substituted with various sugars other than Gal, but substitutions at O6 led to inactivity. Additions to the Gal moiety of the disaccharide also had only small effects on reactivity. These results are all compatible with the features seen in the crystal structures.

Congruency between biophysical data from multiple platforms and molecular dynamics simulation of the double-super helix model of nascent high-density lipoprotein.

The predicted structure and molecular trajectories from >80 ns molecular dynamics simulation of the solvated Double-Super Helix (DSH) model of nascent high-density lipoprotein (HDL) were determined and compared with experimental data on reconstituted nascent HDL obtained from multiple biophysical platforms, including small angle neutron scattering (SANS) with contrast variation, hydrogen-deuterium exchange tandem mass spectrometry (H/D-MS/MS), nuclear magnetic resonance spectroscopy (NMR), cross-linking tandem mass spectrometry (MS/MS), fluorescence resonance energy transfer (FRET), electron spin resonance spectroscopy (ESR), and electron microscopy. In general, biophysical constraints experimentally derived from the multiple platforms agree with the same quantities evaluated using the simulation trajectory. Notably, key structural features postulated for the recent DSH model of nascent HDL are retained during the simulation, including (1) the superhelical conformation of the antiparallel apolipoprotein A1 (apoA1) chains, (2) the lipid micellar-pseudolamellar organization, and (3) the solvent-exposed Solar Flare loops, proposed sites of interaction with LCAT (lecithin cholesteryl acyltransferase). Analysis of salt bridge persistence during simulation provides insights into structural features of apoA1 that forms the backbone of the lipoprotein. The combination of molecular dynamics simulation and experimental data from a broad range of biophysical platforms serves as a powerful approach to studying large macromolecular assemblies such as lipoproteins. This application to nascent HDL validates the DSH model proposed earlier and suggests new structural details of nascent HDL.

Double superhelix model of high density lipoprotein.

High density lipoprotein (HDL), the carrier of so-called "good" cholesterol, serves as the major athero-protective lipoprotein and has emerged as a key therapeutic target for cardiovascular disease. We applied small angle neutron scattering (SANS) with contrast variation and selective isotopic deuteration to the study of nascent HDL to obtain the low resolution structure in solution of the overall time-averaged conformation of apolipoprotein AI (apoA-I) versus the lipid (acyl chain) core of the particle. Remarkably, apoA-I is observed to possess an open helical shape that wraps around a central ellipsoidal lipid phase. Using the low resolution SANS shapes of the protein and lipid core as scaffolding, an all-atom computational model for the protein and lipid components of nascent HDL was developed by integrating complementary structural data from hydrogen/deuterium exchange mass spectrometry and previously published constraints from multiple biophysical techniques. Both SANS data and the new computational model, the double superhelix model, suggest an unexpected structural arrangement of protein and lipids of nascent HDL, an anti-parallel double superhelix wrapped around an ellipsoidal lipid phase. The protein and lipid organization in nascent HDL envisages a potential generalized mechanism for lipoprotein biogenesis and remodeling, biological processes critical to sterol and lipid transport, organismal energy metabolism, and innate immunity.

Dynamic flexibility of double-stranded RNA activated PKR in solution.

PKR, an interferon-induced double-stranded RNA activated serine-threonine kinase, is a component of signal transduction pathways mediating cell growth control and responses to stress and viral infection. Analysis of separate PKR functional domains by NMR and X-ray crystallography has revealed details of PKR RNA binding domains and kinase domain, respectively. Here, we report the structural characteristics, calculated from biochemical and neutron scattering data, of a native PKR fraction with a high level of autophosphorylation and constitutive kinase activity. The experiments reveal association of the protein monomer into dimers and tetramers, in the absence of double-stranded RNA or other activators. Low-resolution structures of the association states were obtained from the large angle neutron scattering data and reveal the relative orientation of all protein domains in the activated kinase dimer. Low-resolution structures were also obtained for a PKR tetramer-monoclonal antibody complex. Taken together, this information leads to a new model for the structure of the functioning unit of the enzyme, highlights the flexibility of PKR and sheds light on the mechanism of PKR activation. The results of this study emphasize the usefulness of low-resolution structural studies in solution on large flexible multiple domain proteins.

Biochemical analyses of multiple fractions of PKR purified from Escherichia coli.

PKR is a cellular protein kinase activated by double-stranded RNA (dsRNA) that phosphorylates eukaryotic initiation factor alpha (eIF2alpha) and inhibits protein translation. Activation of PKR is accompanied by Ser/Thr autophosphorylation on multiple sites. Because PKR negatively regulates cell growth, overexpression and purification of PKR are difficult to achieve. Here, we describe overexpression and purification of recombinant PKR protein from Escherichia coli under native conditions at the milligram level. Affinity, ion exchange, and gel filtration chromatographies revealed multiple fractions of PKR with distinctive biochemical characteristics. During gel filtration, a small amount of PKR was found in a high molecular weight (>300 kDa) fraction that also contained endogenous bacterial RNA. The PKR in this fraction has a constitutive substrate phosphorylation activity. The majority of PKR is found in fractions of lower molecular weight and is free of RNA but is differentially phosphorylated as examined by isoelectric focusing electrophoresis and can be further separated by gradient anion exchange chromatography. PKR eluted with low salt has a lower level of basal autophosphorylation, and its kinase activity can be induced by dsRNA. With an increasing NaCl gradient, the purified PKR exhibits an increased level of autophosphorylation and constitutive kinase activity but reduced dsRNA inducibility. The highest salt eluent of PKR exhibits little dsRNA-induced activation. The inducible activation of high salt eluent PKR by dsRNA can be partially restored by treatment with protein phosphatase 1. The production of multiple fractions of PKR with different biochemical properties in E. coli suggests that the spectrum of PKR activity and regulation in mammalian cells is likely to be similarly complex.