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. (October 2017)

• Record Type: Journal Article
• 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
• Is Part Of: Journal of molecular and cellular cardiology. Volume 111(2017)
• Page Start: 114
• Page End: 122
• Publication Date: 2017-10
• Subjects: Single-cell analysis -- Myosin heavy chain -- α-actin -- Cell size -- Proteostasis -- FACS -- Ontogenic allometry; Ab antibody -- α-actin alpha-actin -- APC allophycocyanin -- BDM 2, 3-butanedione monoxime -- cTnT cardiac troponin T -- DAPI 4′, 6-diamidino-2-phenylindole -- DSHB Developmental Studies Hybridoma Bank -- ECM extra cellular matrix -- FCM Flow cytometric analysis -- H/BM heart to body mass ratio -- HRP horseradish peroxidase -- HT hypertrophy -- LP-FACS large-particle fluorescent-activated cell sorting -- mAb monoclonal antibody -- MC myocyte -- MF20 anti-myosin heavy chain monoclonal antibody -- MyHC myosin heavy chain -- NMC non-muscle cell -- PI propidium iodide -- PFA paraformaldehyde; Cardiology -- Periodicals; Heart Diseases -- Periodicals; Molecular Biology -- Periodicals; Cardiologie -- Périodiques; Cardiology; Electronic journals; Periodicals; 616.12
• DOI: 10.1016/j.yjmcc.2017.07.012
• Languages: English
• ISSNs: 0022-2828

Abstract

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 (α-actin)) content. Methods and results: Individual cardiac cells were isolated from 21 to 94 days 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). Conclusion: 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.