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RationaleQuantifying 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.ObjectiveTo 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 resultsIndividual 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).ConclusionChanges 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.

Previous work identified two common ethnically-dependent allelic variants of human flavin-containing monooxygenase 2 (FMO2) that encode protein with monooxygenase activity that could occur in combination with the functional FM02*1 allele (Q472 protein); the encoded proteins were S195L and N413K. To understand how these variants alter enzyme activity, we performed assays with the FMO substrate, methyl p-tolyl sulfide (MTS) under a range of conditions with S195L, N413K and Q472 produced by over-expression in Sf9 insect cells. MTS sulfoxidation was assayed by HPLC. All protein variants demonstrated the same pattern of response to the presence of magnesium (increased activity) and to heat (decreased activity). Increased activity was observed with Q472 and N413K in response to increasing pH and to cholate, while S195L had diminished activity. The major effect of the N413K substitution was to increase velocity compared with Q472, while S195L had reduced activity and altered patterns of response to incubation conditions. In contrast to Q472 and N413K, S195L activity was negligible unless NADPH was added before MTS. Also, the K,, for S195L is at least 10-fold higher than for Q472 and N413K. Although these allelic variants are expected to occur infrequently as mutations to the FM02*1 allele, they contribute to our overall understanding of mammalian FMO structure and function. [ABSTRACT FROM AUTHOR]