Atrial fibrillation (AF) is a growing public health problem without adequate therapies. Understanding the underlying mechanisms involved in AF is fundamental in developing new therapeutic strategies. Recent research has suggested an important role for calcium-dysregulation in AF [1]. Lysosomal calcium signalling is a little-studied pathway in the heart which has been implicated in the adrenergic responses of both atrial [2] and ventricular myocytes [3]. Published work has suggested that inhibition of the lysosomal Ca2+ release pathway is protective against adrenergically-induced arrhythmias [4]. Studying the proteomic expression of variously sized acidic organelles relies on the suitable isolation of these organelles in separate density fractions. Here we describe a fractionation protocol which allows us to study organellar proteins in guinea-pig atria (experiments performed in accordance with Home Office Guidance on the Animals (Scientific Procedures) Act (1986)). Amongst the different density gradient fractions obtained, the three most relevant fractions, confirmed using Western Blot and MS/MS peptide analysis, were yielded from an adapted discontinuous Percoll, and sucrose differential density gradients. The first saved fraction consists of the whole tissue lysate; the second fraction (1.3 g/mL) contains fully mature lysosomes and some sarcoplasmic reticulum (SR) and mitochondrial contamination; the third fraction (1.04g/mL) consists of endo-lysosomes and exosomes with negligible contamination from SR or mitochondria. Western blots for LAMP2, V type H+ ATPase, COX IV and Phospholamban were performed to quantify the different organelles (lysosome, mitochondria and SR) in the respective fractions. The organelle purification protocol was first developed on mice and optimised in the guinea pig. MS/MS peptide analysis was performed on a QExec LumosTM mass spectrometer. The proteomic pathway analysis of the wild type guinea-pig atrial tissue fractions was performed using Panther Pathway. Atrial specific proteins such as Myosin heavy chain 7 (MYH7), peptidyl Glycine amidating monooxygenase (PAM) and atrial natriuretic peptide (NPPA), were detected in our whole cell tissue lysate. In the endo-lysosomal and exosome fraction, proteins as such Beta Arrestin – 1 (ARRB1, involved in intracellular trafficking pathway) and autophagy promoting protein BAG3 (intracellular transport chaperone protein which transport parts of the cytoskeleton to lysosomes during cellular stress) were detected. The molecular, biological, functional and protein networks information was extracted using GeneMANIA, UniProt, NCBI databases and protein-hits were cross-checked with the published research literature. This method allows us to explore quantitatively the protein interactions. This will help us to identify differences in protein expression, specifically related to lysosomes and globally which will inform future research.
Physiology 2019 (Aberdeen, UK) (2019) Proc Physiol Soc 43, PC030
Poster Communications: Cardiac Atrial Acidic Organelle Proteomics: A Method to Analyse Lysosomal Proteins
T. Ayagama1, R. Capel1, R. Fischer2, H. Kramer3, R. Burton1
1. Pharmacology, University of Oxford, Oxford, United Kingdom. 2. Target Discovery Institute, University of Oxford, Oxford, United Kingdom. 3. Biological Mass Spectrometry and Proteomics Facility, Imperial College London, London, United Kingdom.
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Where applicable, experiments conform with Society ethical requirements.