Abstract 18296: A Novel Flow Cytometry Approach Shows That Cardiac Myocyte Size and Myosin Heavy Chain Protein Content Are Disassociated in Normal Post-natal Development

Introduction: We have reported previously that α-myosin heavy chain (α–MyHC) expressing myocytes (MCs), the predominant MC in adult mouse hearts, hypertrophy under pressure-overload without a proportionate increase in total MyHC protein (T-MyHC) content (Lopez et al, Circ. Res., 2011). It is not yet known if during normal physiological growth, these MCs increase their T-MyHC content in proportion to cell size. Hypothesis: During normal post-natal growth, an increase in T-MyHC content is proportionate to changes in cardiac mass and MC size. Methods: Individual cardiac cells were isolated by enzymatic digestion from male C57Bl/6 mice age in days 22+/-1 (young, n=4) and 88+/-6 (adult, n=4). Body and heart weights (wt), mean MC volumes by Coulter Multisizer (vol), and cell protein content-per-MC by BCA assay (TotProtMC) were used to measure cardiac growth. A new approach using large-particle fluorescent activated cell sorting (FACS, see Figure) was validated to isolate 15K Troponin-T expressing MCs (~97% α–MyHC+) per heart with 94+/-2% purity (n=5). Relative T-MyHC content was measured by immunoblotting equal numbers of sorted MCs (50 MCs/lane) that was validated to be in linear range for the assay. Results: Body wt (gm), heart wt (mg), MC vol (μm3, in ~7,000 MCs per heart) and TotProtMC (ng/MC) were 3, 2.3, 2.7 and 2.5 fold greater in adult than young mice, respectively. The relative T-MyHC content per adult MC was 7.5 fold greater than in young MCs (441+/-107 vs. 59+/- 4 A.U./MC, n=3 each, p≤0.01). Conclusions: Unexpectedly, the increase in T-MyHC protein per cell during post-natal growth is ~2.5 fold greater than predicted by changes in cardiac mass, MC size or cell protein content-per-MC. These data demonstrate that in normal physiological growth, MCs accumulate more T-MyHC protein per unit volume than predicted by changes in size alone. This form of growth, “normotrophy”, is in contrast to hypertrophy where T-MyHC protein accumulation is less or equal to changes in cell size. ![][1] [1]: /embed/graphic-1.gif