Final Report Summary - CHONDRION (Chondrocyte ion channel function and regulation in health and disease)
Final report (October 2016)
Background and objectives
Osteoarthritis (OA) is one of the leading causes of disability and represents a major healthcare issue in the European Union. The aim of this fellowship was to investigate the expression, function and regulation of plasma membrane ion channels and calcium signalling pathways in cells of OA cartilage by using state of the art proteomic and electrophysiological techniques. As no effective treatment is available, there is an urgent need to understand the underlying cellular and molecular mechanisms and develop more targeted therapeutic strategies. Ion channels are important for chondrocyte function and survival; however, the full complement of chondrocyte membrane proteins (membranome) and ion channels (channelome), as well as their expression in OA has yet to be explored. Correlating altered ion channel expression and function during the development of OA may provide a better understanding of mechanisms controlling disease progression and may also lead to the discovery of novel biomarkers for OA.
Methodology
We have used the four different disciplines of transcriptomics, proteomics, bioinformatics and live cell electrophysiology to better characterise the plasma membrane proteins (membranome) of healthy and osteoarthritic progenitor cells that are involved in OA development, and thus potentially identify novel biomarkers.
Main results achieved so far
(1) Exploring the membranome and surfaceome of chondrocytes and chondroprogenitor cells
The first objective of the project was to optimise methodology to be able to explore the membrane protein complement (membranome), and more specifically, the cell surface protein complement (surfaceome) of chondrocytes and chondroprogenitors. We have made a good progress towards establishing methodology capable of enriching cell surface proteins in samples that were subsequently analysed using high throughput mass spectrometry. Whilst the Triton X-114 phase separation method (Fig. 1, left panel) and the Cell Surface Protein Isolation Kit (Sulfo-NHS-SS-Biotin; Fig. 1, middle panel) were only able to provide a ~30% enrichment, almost 80% of the identified proteins were localised to the cell membrane following the modified aminooxy-biotin labelling (Fig. 1, right panel). Data collection and analysis is still under way, and we hope to identify novel and yet unidentified biomarkers for chondroprogenitor cells and/or for OA disease development. We aim to publish at least one primary research article with these results.
(2) Identifying differentially expressed ion channels in healthy and OA cells
We applied Affymetrix microarrays to analyse differences in the global mRNA expression profile of osteoarthritic chondroprogenitor cells (CPCs) and bone marrow-derived mesenchymal stem cells (MSCs). Following a focussed analysis of the data, we have selected ~100 genes that were differentially expressed between the two cell types and used a TaqMan array plate to further narrow down the potentially important ion channel genes. Finally, we used SYBR Green-based qPCRs to verify changes in mRNA expression. The most obvious difference in expression was found in case of a specific calcium-dependent potassium channel, and we confirmed that the expression pattern was evident at the protein level as well. We are in the process now to finalise the experiments and publish the results in a primary research article.
(3) Functional analysis of ion channels using electrophysiology
In order to characterise the electrophysiological properties of OA chondroprogenitor cells and healthy mesenchymal stem cells, we employed two methodologies. Besides using conventional single cell patch clamp electrophysiology to determine the resting membrane potential, the capacitance and ionic currents across the cell membrane, we have also used an innovative method called 3D dielectrophoresis (3-DEP), which provides information on membrane and cytoplasm conductivity and capacitance at a population level. We found that although the resting membrane characteristics were quite similar between the two cell types, the cellular response and the detected changes in resting membrane potential were different following administration of the specific inhibitor and activator of the ion channel identified in the previous task. We will need to increase the number of cells studied to generate statistically robust results.
Final results and their potential impact and use
Current understanding of the chondrocyte channelome and membranome is very limited, despite the fact that cell surface proteins, including ion channels, are probably the first components of chondrocytes to respond to altered ECM composition in OA, and they are also the first molecules on which currently marketed drugs act. Therefore, there is a strong need to fully characterise the composition and function of the plasma membrane proteins in health and disease. Until now, the full complement of chondrocyte and chondroprogenitor plasma membrane ion channels (surfaceome and channelome) has not been investigated in full detail. The main outcome of the project is the identification of new plasma membrane biomarkers for the early detection and monitoring of inflammatory changes in OA. We have employed state-oftheart methodology and a truly innovative, novel approach in this field to characterise the surfaceome both in terms of composition and function using live cell measurements.
The outlined research has been successfully completed in line with a Personal Career Development Plan (PCDP) based on the Dr Matta’s specific training needs. The training programme, through the extensive network of the Scientist in charge, has provided Dr Matta with key practical skills to allow him to (1) perform patch clamp electrophysiological ion channel measurements, (2) learn state-of-the-art proteomics methodology, (3) evaluate and comprehend proteomics and bioinformatics results, and (4) be able to disseminate results at a professional level.
Background and objectives
Osteoarthritis (OA) is one of the leading causes of disability and represents a major healthcare issue in the European Union. The aim of this fellowship was to investigate the expression, function and regulation of plasma membrane ion channels and calcium signalling pathways in cells of OA cartilage by using state of the art proteomic and electrophysiological techniques. As no effective treatment is available, there is an urgent need to understand the underlying cellular and molecular mechanisms and develop more targeted therapeutic strategies. Ion channels are important for chondrocyte function and survival; however, the full complement of chondrocyte membrane proteins (membranome) and ion channels (channelome), as well as their expression in OA has yet to be explored. Correlating altered ion channel expression and function during the development of OA may provide a better understanding of mechanisms controlling disease progression and may also lead to the discovery of novel biomarkers for OA.
Methodology
We have used the four different disciplines of transcriptomics, proteomics, bioinformatics and live cell electrophysiology to better characterise the plasma membrane proteins (membranome) of healthy and osteoarthritic progenitor cells that are involved in OA development, and thus potentially identify novel biomarkers.
Main results achieved so far
(1) Exploring the membranome and surfaceome of chondrocytes and chondroprogenitor cells
The first objective of the project was to optimise methodology to be able to explore the membrane protein complement (membranome), and more specifically, the cell surface protein complement (surfaceome) of chondrocytes and chondroprogenitors. We have made a good progress towards establishing methodology capable of enriching cell surface proteins in samples that were subsequently analysed using high throughput mass spectrometry. Whilst the Triton X-114 phase separation method (Fig. 1, left panel) and the Cell Surface Protein Isolation Kit (Sulfo-NHS-SS-Biotin; Fig. 1, middle panel) were only able to provide a ~30% enrichment, almost 80% of the identified proteins were localised to the cell membrane following the modified aminooxy-biotin labelling (Fig. 1, right panel). Data collection and analysis is still under way, and we hope to identify novel and yet unidentified biomarkers for chondroprogenitor cells and/or for OA disease development. We aim to publish at least one primary research article with these results.
(2) Identifying differentially expressed ion channels in healthy and OA cells
We applied Affymetrix microarrays to analyse differences in the global mRNA expression profile of osteoarthritic chondroprogenitor cells (CPCs) and bone marrow-derived mesenchymal stem cells (MSCs). Following a focussed analysis of the data, we have selected ~100 genes that were differentially expressed between the two cell types and used a TaqMan array plate to further narrow down the potentially important ion channel genes. Finally, we used SYBR Green-based qPCRs to verify changes in mRNA expression. The most obvious difference in expression was found in case of a specific calcium-dependent potassium channel, and we confirmed that the expression pattern was evident at the protein level as well. We are in the process now to finalise the experiments and publish the results in a primary research article.
(3) Functional analysis of ion channels using electrophysiology
In order to characterise the electrophysiological properties of OA chondroprogenitor cells and healthy mesenchymal stem cells, we employed two methodologies. Besides using conventional single cell patch clamp electrophysiology to determine the resting membrane potential, the capacitance and ionic currents across the cell membrane, we have also used an innovative method called 3D dielectrophoresis (3-DEP), which provides information on membrane and cytoplasm conductivity and capacitance at a population level. We found that although the resting membrane characteristics were quite similar between the two cell types, the cellular response and the detected changes in resting membrane potential were different following administration of the specific inhibitor and activator of the ion channel identified in the previous task. We will need to increase the number of cells studied to generate statistically robust results.
Final results and their potential impact and use
Current understanding of the chondrocyte channelome and membranome is very limited, despite the fact that cell surface proteins, including ion channels, are probably the first components of chondrocytes to respond to altered ECM composition in OA, and they are also the first molecules on which currently marketed drugs act. Therefore, there is a strong need to fully characterise the composition and function of the plasma membrane proteins in health and disease. Until now, the full complement of chondrocyte and chondroprogenitor plasma membrane ion channels (surfaceome and channelome) has not been investigated in full detail. The main outcome of the project is the identification of new plasma membrane biomarkers for the early detection and monitoring of inflammatory changes in OA. We have employed state-oftheart methodology and a truly innovative, novel approach in this field to characterise the surfaceome both in terms of composition and function using live cell measurements.
The outlined research has been successfully completed in line with a Personal Career Development Plan (PCDP) based on the Dr Matta’s specific training needs. The training programme, through the extensive network of the Scientist in charge, has provided Dr Matta with key practical skills to allow him to (1) perform patch clamp electrophysiological ion channel measurements, (2) learn state-of-the-art proteomics methodology, (3) evaluate and comprehend proteomics and bioinformatics results, and (4) be able to disseminate results at a professional level.