|کد مقاله||کد نشریه||سال انتشار||مقاله انگلیسی||ترجمه فارسی||نسخه تمام متن|
|5533633||1550395||2017||9 صفحه PDF||سفارش دهید||دانلود رایگان|
RationaleQuantifying cellular proteins in ventricular myocytes (MCs) is challenging due to tissue heterogeneity and the variety of cell sizes in the heart. In post-weaning cardiac ontogeny, rod-shaped MCs make up the majority of the cardiac mass while remaining a minority of cardiac cells in number. Current biochemical analyses of cardiac proteins do not correlate well the content of MC-specific proteins to cell type or size in normally developing tissue.ObjectiveTo develop a new large-particle fluorescent-activated cell sorting (LP-FACS) strategy for the purification of adult rod-shaped MCs. This approach is developed to enable growth-scaled measurements per-cell of the MC proteome and sarcomeric proteins (i.e. myosin heavy chain (MyHC) and alpha-actin (Î±-actin)) content.Methods and resultsIndividual cardiac cells were isolated from 21 to 94Â days old mice. An LP-FACS jet-in-air system with a 200-Î¼m nozzle was defined for the first time to purify adult MCs. Cell-type specific immunophenotyping and sorting yielded â¥Â 95% purity of adult MCs independently of cell morphology and size. This approach excluded other cell types and tissue contaminants from further analysis. MC proteome, MyHC and Î±-actin proteins were measured in linear biochemical assays normalized to cell numbers. Using the allometric coefficient Î±, we scaled the MC-specific rate of protein accumulation to growth post-weaning. MC-specific volumes (Î±Â =Â 1.02) and global protein accumulation (Î±Â =Â 0.94) were proportional (i.e. isometric) to body mass. In contrast, MyHC and Î±-actin accumulated at a much greater rate (i.e. hyperallometric) than body mass (Î±Â =Â 1.79 and 2.19 respectively) and MC volumes (Î±Â =Â 1.76 and 1.45 respectively).ConclusionChanges in MC proteome and cell volumes measured in LP-FACS purified MCs are proportional to body mass post-weaning. Oppositely, MyHC and Î±-actin are concentrated more rapidly than what would be expected from MC proteome accumulation, cell enlargement, or animal growth alone. LP-FACS provides a new standard for adult MC purification and an approach to scale the biochemical content of specific proteins or group of proteins per cell in enlarging MCs.
Journal: Journal of Molecular and Cellular Cardiology - Volume 111, October 2017, Pages 114-122