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

• Published in: Journal of molecular and cellular cardiology Vol. 111; pp. 114 - 122
• Main Authors: López, Javier E., Sharma, Janhavi, Avila, Jorge, Wood, Taylor S., VanDyke, Jonathan E., McLaughlin, Bridget, Abbey, Craig K., Wong, Andrew, Myagmar, Bat-Erdene, Swigart, Philip M., Simpson, Paul C., Chiamvimonvat, Nipavan
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.10.2017
• Subjects: Actins - metabolism; Aging - metabolism; Animals; Animals, Newborn; Body Weight; Cell Separation - methods; Cell Size; FACS; Flow Cytometry - methods; Heart Ventricles - cytology; Immunophenotyping; Mice, Inbred C57BL; Myocytes, Cardiac - cytology; Myocytes, Cardiac - metabolism; Myosin heavy chain; Myosins - metabolism; Ontogenic allometry; Organ Size; Particle Size; Proteome - metabolism; Proteostasis; Sarcomeres - metabolism; Single-cell analysis; Weaning; α-actin; Ab; Myosin heavy chain; Proteostasis; Single-cell analysis; HRP; BDM; PFA; FACS; HT; H/BM; MC; cTnT; MyHC; NMC; DAPI; DSHB; α-actin; LP-FACS; ECM; mAb; FCM; MF20; APC; Ontogenic allometry; PI; Cell size
• ISSN: 0022-2828; 1095-8584
• DOI: 10.1016/j.yjmcc.2017.07.012

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. 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 (α-actin)) content. Individual cardiac cells were isolated from 21 to 94days old mice. An LP-FACS jet-in-air system with a 200-μm 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 α-actin proteins were measured in linear biochemical assays normalized to cell numbers. Using the allometric coefficient α, we scaled the MC-specific rate of protein accumulation to growth post-weaning. MC-specific volumes (α=1.02) and global protein accumulation (α=0.94) were proportional (i.e. isometric) to body mass. In contrast, MyHC and α-actin accumulated at a much greater rate (i.e. hyperallometric) than body mass (α=1.79 and 2.19 respectively) and MC volumes (α=1.76 and 1.45 respectively). Changes in MC proteome and cell volumes measured in LP-FACS purified MCs are proportional to body mass post-weaning. Oppositely, MyHC and α-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.