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 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). 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.

中文翻译：

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成人心室肌细胞的新型大颗粒FACS纯化揭示了正常断奶后发育中肌球蛋白和肌动蛋白的积累与细胞大小和蛋白质组不成比例。

原理由于组织异质性和心脏细胞大小的多样性，对心室肌细胞（MCs）中的细胞蛋白进行定量分析具有挑战性。在断奶后心脏发育中，杆状MC构成了心脏的大部分，而心脏细胞的数量却很少。当前心脏蛋白的生化分析与正常发育组织中MC特异性蛋白的含量与细胞类型或大小没有很好的相关性。目的开发新的大颗粒荧光激活细胞分选（LP-FACS）策略，用于纯化成人杆状MC。开发这种方法是为了能够按细胞按生长尺度测量MC蛋白质组和肌节蛋白（即肌球蛋白重链（MyHC）和α-肌动蛋白（α-肌动蛋白））的含量。方法和结果从21至94日龄的小鼠中分离出单个心脏细胞。首次定义了带有200μm喷嘴的LP-FACS空中喷射系统，以纯化成年MC。与细胞形态和大小无关，细胞类型特异性免疫表型和分选产生的成年MC纯度≥95％。这种方法排除了其他细胞类型和组织污染物的进一步分析。MC蛋白质组，MyHC和α-肌动蛋白在线性生化分析中根据细胞数进行了测量。使用异速系数α，我们对断奶后生长的蛋白质的MC积累速率进行了缩放。MC特异体积（α= 1.02）和总体蛋白质积累（α= 0.94）与体重成比例（即等轴测）。相比之下，MyHC和α-肌动蛋白的累积速率（即超称量性）比体重（α= 1）高得多。分别为79和2.19）和MC体积（分别为α= 1.76和1.45）。结论LP-FACS纯化的MC中MC蛋白质组和细胞体积的变化与断奶后体重成正比。相反，MyHC和α-肌动蛋白的浓缩速度比MC蛋白组堆积，细胞扩增或仅动物生长所预期的速度要快。LP-FACS为成人MC纯化提供了新的标准，并且为扩大MC中每个细胞的特定蛋白质或一组蛋白质的生化含量提供了一种方法。细胞增大或仅动物生长。LP-FACS为成人MC纯化提供了新标准，并为扩大MC中每个细胞的特定蛋白质或一组蛋白质的生化含量提供了一种方法。细胞增大或仅动物生长。LP-FACS为成人MC纯化提供了新标准，并为扩大MC中每个细胞的特定蛋白质或一组蛋白质的生化含量提供了一种方法。