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

Javier E Lopez, Janhavi Sharma, Jorge Avila, Taylor S. Wood, Jonathan E. VanDyke, Bridget McLaughlin, Craig K. Abbey, Andrew Wong, Bat Erdene Myagmar, Philip M. Swigart, Paul C. Simpson, Nipavan Chiamvimonvat

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)114-122
Number of pages9
JournalJournal of Molecular and Cellular Cardiology
Volume111
DOIs
StatePublished - Oct 1 2017

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Proteome
Myosins
Weaning
Cell Size
Muscle Cells
Actins
Myosin Heavy Chains
Proteins
Growth
Cell Count
Cell Enlargement
Immunophenotyping

Keywords

  • Cell size
  • FACS
  • Myosin heavy chain
  • Ontogenic allometry
  • Proteostasis
  • Single-cell analysis
  • α-actin

ASJC Scopus subject areas

  • Molecular Biology
  • Cardiology and Cardiovascular Medicine

Cite this

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. / Lopez, 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.

In: Journal of Molecular and Cellular Cardiology, Vol. 111, 01.10.2017, p. 114-122.

Research output: Contribution to journalArticle

Lopez, 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. / 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. In: Journal of Molecular and Cellular Cardiology. 2017 ; Vol. 111. pp. 114-122.
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title = "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 (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.",
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author = "Lopez, {Javier E} and Janhavi Sharma and Jorge Avila and Wood, {Taylor S.} and VanDyke, {Jonathan E.} and Bridget McLaughlin and Abbey, {Craig K.} and Andrew Wong and Myagmar, {Bat Erdene} and Swigart, {Philip M.} and Simpson, {Paul C.} and Nipavan Chiamvimonvat",
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T1 - 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

AU - Lopez, Javier E

AU - Sharma, Janhavi

AU - Avila, Jorge

AU - Wood, Taylor S.

AU - VanDyke, Jonathan E.

AU - McLaughlin, Bridget

AU - Abbey, Craig K.

AU - Wong, Andrew

AU - Myagmar, Bat Erdene

AU - Swigart, Philip M.

AU - Simpson, Paul C.

AU - Chiamvimonvat, Nipavan

PY - 2017/10/1

Y1 - 2017/10/1

N2 - 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.

AB - 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.

KW - Cell size

KW - FACS

KW - Myosin heavy chain

KW - Ontogenic allometry

KW - Proteostasis

KW - Single-cell analysis

KW - α-actin

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