scholarly journals Cardiac Atrial Compartmentalisation Proteomics: A Modified Density Gradient Method to Analyse Endo-lysosomal Proteins

2021 ◽  
Author(s):  
Thamali Ayagama ◽  
Samuel J Bose ◽  
Rebecca A Capel ◽  
David A Priestman ◽  
Georgina Berridge ◽  
...  
Keyword(s):  
Density Gradient ◽  
Cardiac Tissue ◽  
Calcium Signalling ◽  
Rab Proteins ◽  
Label Free ◽  
Enzyme Activity Assay ◽  
Differential Density ◽  
Tissue Lysate ◽  
Good Coverage ◽  
Lysosomal Proteins

SummaryThe importance of lysosomes in cardiac physiology and pathology are well established, and evidence for roles in calcium signalling are emerging. We describe a label-free proteomics method suitable for small cardiac tissue biopsies based on density-separated fractionation, which allows study of endo-lysosomal (EL) proteins.Density gradient fractions corresponding to tissue lysate; sarcoplasmic reticulum (SR), mitochondria (Mito) (1.3 g/ml); and EL with negligible contamination from SR or Mito (1.04 g/ml), were analysed using Western Blot, enzyme activity assay and LC-MS/MS analysis (adapted discontinuous Percoll, and sucrose differential density gradient).Kyoto Encyclopedia of Genes and Genomes, Reactome, Panther and Gene Ontology pathway analysis showed good coverage of RAB proteins and lysosomal cathepsins (including cardiac-specific cathepsin D) in the purified EL fraction. Significant EL proteins recovered included catalytic activity proteins. We thus present a comprehensive protocol and dataset of guinea-pig atrial EL organelle proteomics using techniques also applicable for non-cardiac tissue.

A label-free method based on MALDI-TOF mass spectrometry for the absolute quantitation of troponin T in mouse cardiac tissue

2008 ◽  
Vol 391 (5) ◽  
pp. 1969-1976 ◽  
Author(s):  
Mariano Bizzarri ◽  
Chiara Cavaliere ◽  
Patrizia Foglia ◽  
Chiara Guarino ◽  
Roberto Samperi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cardiac Tissue ◽  
Troponin T ◽  
Label Free ◽  
Absolute Quantitation ◽  
Maldi Tof Mass Spectrometry ◽  
Maldi Tof ◽  
The Absolute

scholarly journals Detection and Quantification of Myocardial Fibrosis Using Stain-Free Infrared Spectroscopic Imaging

2021 ◽  
Author(s):  
Eric Zimmermann ◽  
Sudipta S. Mukherjee ◽  
Kianoush Falahkheirkhah ◽  
Mark C. Gryka ◽  
Andre Kajdacsy-Balla ◽  
...  
Keyword(s):  
Myocardial Fibrosis ◽  
Cardiac Tissue ◽  
Infrared Imaging ◽  
Characteristic Curve ◽  
Area Under The Curve ◽  
Spectroscopic Imaging ◽  
Quantitative Agreement ◽  
Label Free ◽  
Infrared Spectroscopic ◽  
Infrared Spectroscopic Imaging

Context.— Myocardial fibrosis underpins a number of cardiovascular conditions and is difficult to identify with standard histologic techniques. Challenges include imaging, defining an objective threshold for classifying fibrosis as mild or severe, as well as understanding the molecular basis for these changes. Objective.— To develop a novel, rapid, label-free approach to accurately measure and quantify the extent of fibrosis in cardiac tissue using infrared spectroscopic imaging. Design.— We performed infrared spectroscopic imaging and combined that with advanced machine learning–based algorithms to assess fibrosis in 15 samples from patients belonging to the following 3 classes: (1) nonpathologic (control) donor hearts; (2) patients receiving transplant; and (3) tissue from patients undergoing implantation of ventricular assist device. Results.— Our results show excellent sensitivity and accuracy for detecting myocardial fibrosis as demonstrated by high area under the curve of 0.998 in the receiver-operating characteristic curve measured from infrared imaging. Fibrosis of various morphologic subtypes are then demonstrated with virtually generated picrosirius red images, which show good visual and quantitative agreement (correlation coefficient = 0.92, ρ = 7.76 × 10−15) with stained images of the same sections. Underlying molecular composition of the different subtypes were investigated with infrared spectra showing reproducible differences presumably arising from differences in collagen subtypes and/or crosslinking. Conclusions.— Infrared imaging can be a powerful tool in studying myocardial fibrosis and gleaning insights into the underlying chemical changes that accompany it. Emerging methods suggest that the proposed approach is compatible with conventional optical microscopy and its consistency makes it translatable to the clinical setting for real-time diagnoses as well as for objective and quantitative research.

Muscular Thin Films for Label-Free Mapping of Excitation Propagation in Cardiac Tissue

2020 ◽  
Vol 48 (10) ◽  
pp. 2425-2437
Author(s):  
Viktor A. Balashov ◽  
Vasily S. Gorbunov ◽  
Konstantin G. Guria ◽  
Konstantin I. Agladze
Keyword(s):  
Thin Films ◽  
Cardiac Tissue ◽  
Label Free ◽  
Excitation Propagation

scholarly journals Chronic Occupational Exposure to Ionizing Radiation Induces Alterations in the Structure and Metabolism of the Heart: A Proteomic Analysis of Human Formalin-Fixed Paraffin-Embedded (FFPE) Cardiac Tissue

2020 ◽  
Vol 21 (18) ◽  
pp. 6832
Author(s):  
Omid Azimzadeh ◽  
Tamara Azizova ◽  
Juliane Merl-Pham ◽  
Andreas Blutke ◽  
Maria Moseeva ◽  
...  
Keyword(s):  
Cardiac Tissue ◽  
Gamma Ray ◽  
Confounding Factor ◽  
Control Group ◽  
Histopathological Analysis ◽  
Label Free ◽  
Proteomics Analysis ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Epidemiological studies on workers employed at the Mayak plutonium enrichment plant have demonstrated an association between external gamma ray exposure and an elevated risk of ischemic heart disease (IHD). In a previous study using fresh-frozen post mortem samples of the cardiac left ventricle of Mayak workers and non-irradiated controls, we observed radiation-induced alterations in the heart proteome, mainly downregulation of mitochondrial and structural proteins. As the control group available at that time was younger than the irradiated group, we could not exclude age as a confounding factor. To address this issue, we have now expanded our study to investigate additional samples using archival formalin-fixed paraffin-embedded (FFPE) tissue. Importantly, the control group studied here is older than the occupationally exposed (>500 mGy) group. Label-free quantitative proteomics analysis showed that proteins involved in the lipid metabolism, sirtuin signaling, mitochondrial function, cytoskeletal organization, and antioxidant defense were the most affected. A histopathological analysis elucidated large foci of fibrotic tissue, myocardial lipomatosis and lymphocytic infiltrations in the irradiated samples. These data highlight the suitability of FFPE material for proteomics analysis. The study confirms the previous results emphasizing the role of adverse metabolic changes in the radiation-associated IHD. Most importantly, it excludes age at the time of death as a confounding factor.

scholarly journals X-ray diffraction and second harmonic imaging reveal new insights into structural alterations caused by pressure-overload in murine hearts

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jan-David Nicolas ◽  
Amara Khan ◽  
Andrea Markus ◽  
Belal A. Mohamed ◽  
Karl Toischer ◽  
...  
Keyword(s):  
Cardiac Remodeling ◽  
Cardiac Tissue ◽  
Immune Cell ◽  
Second Harmonic ◽  
Pressure Overload ◽  
Automated Analysis ◽  
Myosin Filament ◽  
Label Free ◽  
X Ray Diffraction ◽  
X Ray

Abstract We demonstrate a label-free imaging approach to study cardiac remodeling of fibrotic and hypertrophic hearts, bridging scales from the whole organ down to the molecular level. To this end, we have used mice subjected to transverse aortic constriction and imaged adjacent cardiac tissue sections by microfocus X-ray diffraction and second harmonic generation (SHG) imaging. In this way, the acto-myosin structure was probed in a spatially resolved manner for entire heart sections. From the recorded diffraction data, spatial maps of diffraction intensity, anisotropy and orientation were obtained, and fully automated analysis depicted the acto-myosin filament spacing and direction. X-ray diffraction presented an overview of entire heart sections and revealed that in regions of severe cardiac remodeling the muscle mass is partly replaced by connective tissue and the acto-myosin lattice spacing is increased at these regions. SHG imaging revealed sub-cellular structure of cardiac tissue and complemented the findings from X-ray diffraction by revealing micro-level distortion of myofibrils, immune cell infiltration at regions of cardiac remodeling and the development of fibrosis down to the scale of a single collagen fibril. Overall, our results show that both X-ray diffraction and SHG imaging can be used for label-free and high-resolution visualization of cardiac remodeling and fibrosis progression at different stages in a cardiac pressure-overload mouse model that cannot be achieved by conventional histology.

scholarly journals A Modified Density Gradient Proteomic-Based Method to Analyse Endolysosomal Proteins in Cardiac Tissue

iScience ◽  
2021 ◽  
pp. 102949
Author(s):  
Thamali Ayagama ◽  
Samuel J. Bose ◽  
Rebecca A. Capel ◽  
David A. Priestman ◽  
Georgina Berridge ◽  
...  
Keyword(s):  
Density Gradient ◽  
Cardiac Tissue
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