Novel large-particle FACS purification of adult ventricular myocytes reveals accumulation of myosin and actin disproportionate to cell size and proteome in normal post-weaning development.

ABSTRACT: RATIONALE:Quantifying 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. OBJECTIVE:To 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 (?-actin)) content. METHODS AND RESULTS:Individual cardiac cells were isolated from 21 to 94days old mice. An LP-FACS jet-in-air system with a 200-?m nozzle was defined for the first time to purify adult MCs. Cell-type specific immunophenotyping and sorting yielded ?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 ?-actin proteins were measured in linear biochemical assays normalized to cell numbers. Using the allometric coefficient ?, we scaled the MC-specific rate of protein accumulation to growth post-weaning. MC-specific volumes (?=1.02) and global protein accumulation (?=0.94) were proportional (i.e. isometric) to body mass. In contrast, MyHC and ?-actin accumulated at a much greater rate (i.e. hyperallometric) than body mass (?=1.79 and 2.19 respectively) and MC volumes (?=1.76 and 1.45 respectively). CONCLUSION:Changes in MC proteome and cell volumes measured in LP-FACS purified MCs are proportional to body mass post-weaning. Oppositely, MyHC and ?-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.

SUBMITTER: Lopez JE 

PROVIDER: S-EPMC5791888 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature