[II-AEPCYIA-3] Can regional differences in expression of cardiomyopathy-related proteins explain the clinical phenotype : a pilot study
Introduction
Recognised gene mutations poorly explain regional phenotypic differences in the myocardium of patients developing cardiomyopathy. Understanding the mechanisms driving these patterns, which often begin during childhood, may offer clues to innovate new treatment and diagnostic strategies. Previous proteomic studies have typically analysed single, small tissue samples obtained from a cardiac chamber or cell culture. Developing a novel approach, we aim to describe regional differences in the expression of important cardiomyopathy-associated proteins, with high resolution in different axes across each ventricular wall.
Methods
Continuous samples were obtained from 4-chamber cross-sections of bovine myocardium. Proteins from each were solubilised, extracted and digested, before analysis by mass spectrometry using a ‘hypothesis-free’ approach. Multivariate analysis was applied, to make unbiased comparisons between samples at whole-proteome level. Twenty-eight cardiomyopathy-associated proteins were selected and compared between samples by relative abundance. Multiple correlation analysis described variation from endocardium-toepicardium, apex-to-base and between each ventricular free-wall. Relative intensity maps were additionally generated.
Results
One-hundred and twenty-two samples of ventricular myocardium were analysed over 128 hours, generating 278 GB of data. 1,017 unique proteins were consistently detected among intra-sample repeats. Their relative expression conformed to three distinct regional patterns, varying predominantly from epicardial to endocardial layers. Regional variations in abundance were demonstrated across all selected proteins. Eleven disease-associated proteins, including Myomesin-1 and Actin alpha-1, were enriched within the ventricular septum (p<0.05). Likewise, eight proteins were specifically enriched within the right ventricular epicardial wall (p<0.05). Interestingly, some proteins were most abundant within regions associated with their corresponding cardiomyopathy. Mutations in the Desmoglein-2 gene, for example, are associated with a more left-ventricular dominant phenotype of arrhythmogenic cardiomyopathy (AVC). Unlike other AVC-related proteins, Desmoglein-2 was significantly more abundance within the left ventricular free-wall (figure).
Conclusions
This novel approach describes considerable and detailed variation in the regional abundance of 28 proteins implicated in three major cardiomyopathies. Such variation questions the interpretation of previous cardiac proteomic studies, which typically assume random tissue samples to be representative of the wider myocardium. Application of this approach to disease models at different stages, may offer new insights into development of a cardiomyopathy phenotype in populations of genotype-positive children and adolescents.
Recognised gene mutations poorly explain regional phenotypic differences in the myocardium of patients developing cardiomyopathy. Understanding the mechanisms driving these patterns, which often begin during childhood, may offer clues to innovate new treatment and diagnostic strategies. Previous proteomic studies have typically analysed single, small tissue samples obtained from a cardiac chamber or cell culture. Developing a novel approach, we aim to describe regional differences in the expression of important cardiomyopathy-associated proteins, with high resolution in different axes across each ventricular wall.
Methods
Continuous samples were obtained from 4-chamber cross-sections of bovine myocardium. Proteins from each were solubilised, extracted and digested, before analysis by mass spectrometry using a ‘hypothesis-free’ approach. Multivariate analysis was applied, to make unbiased comparisons between samples at whole-proteome level. Twenty-eight cardiomyopathy-associated proteins were selected and compared between samples by relative abundance. Multiple correlation analysis described variation from endocardium-toepicardium, apex-to-base and between each ventricular free-wall. Relative intensity maps were additionally generated.
Results
One-hundred and twenty-two samples of ventricular myocardium were analysed over 128 hours, generating 278 GB of data. 1,017 unique proteins were consistently detected among intra-sample repeats. Their relative expression conformed to three distinct regional patterns, varying predominantly from epicardial to endocardial layers. Regional variations in abundance were demonstrated across all selected proteins. Eleven disease-associated proteins, including Myomesin-1 and Actin alpha-1, were enriched within the ventricular septum (p<0.05). Likewise, eight proteins were specifically enriched within the right ventricular epicardial wall (p<0.05). Interestingly, some proteins were most abundant within regions associated with their corresponding cardiomyopathy. Mutations in the Desmoglein-2 gene, for example, are associated with a more left-ventricular dominant phenotype of arrhythmogenic cardiomyopathy (AVC). Unlike other AVC-related proteins, Desmoglein-2 was significantly more abundance within the left ventricular free-wall (figure).
Conclusions
This novel approach describes considerable and detailed variation in the regional abundance of 28 proteins implicated in three major cardiomyopathies. Such variation questions the interpretation of previous cardiac proteomic studies, which typically assume random tissue samples to be representative of the wider myocardium. Application of this approach to disease models at different stages, may offer new insights into development of a cardiomyopathy phenotype in populations of genotype-positive children and adolescents.