Sham Surgery and Inter-Individual Heterogeneity Are Major Determinants of Monocyte Subset Kinetics in a Mouse Model of Myocardial Infarction

<div><p>Aims</p><p>Mouse models of myocardial infarction (MI) are commonly used to explore the pathophysiological role of the monocytic response in myocardial injury and to develop translational strategies. However, no study thus far has examined the potential impact of inter-individual variability and sham surgical procedures on monocyte subset kinetics after experimental MI in mice. Our goal was to investigate determinants of systemic myeloid cell subset shifts in C57BL/6 mice following MI by developing a protocol for sequential extensive flow cytometry (FCM).</p><p>Methods and Results</p><p>Following cross-sectional multiplex FCM analysis we provide for the first time a detailed description of absolute quantities, relative subset composition, and biological variability of circulating classical, intermediate, and non-classical monocyte subsets in C57BL/6 mice. By using intra-individual longitudinal measurements after MI induction, a time course of classical and non-classical monocytosis was recorded. This approach disclosed a significant reduction of monocyte subset dispersion across all investigated time points following MI. We found that in the current invasive model of chronic MI the global pattern of systemic monocyte kinetics is mainly determined by a nonspecific inflammatory response to sham surgery and not by the extent of myocardial injury.</p><p>Conclusions</p><p>Application of sequential multiplexed FCM may help to reduce the impact of biological variability in C57BL/6 mice. Furthermore, the confounding influence of sham surgical procedures should always be considered when measuring monocyte subset kinetics in a murine model of MI.</p></div

Sequential FCM may reduce the impact of inter-individual variability.

<p>Time course of the coefficient of variation for monocyte subset absolute cell numbers following MI within (<b>A</b>) inter-group (independently operated mice, n = 18–23/group) and (<b>B</b>) intra-group (sequential analysis, n = 8) experimental setups.</p

Sham surgery determines monocyte subset kinetics in a mouse model of MI.

<p>(<b>A</b>) Flow chart of the time-course study. (<b>B</b>) Mean ejection fraction (EF) and end-diastolic volume (EDV) obtained by sequential magnetic resonance imaging (MRI) in C57BL/6 mice (n = 15). Significance between time points was calculated by one-way ANOVA with Tukeys' post-hoc test: * p<0.05, ** p<0.01, *** p<0.001. (<b>C</b>) Sham-operated (n = 7) and MI mice displayed a similar intra-individual time course of circulating monocyte subsets. Calculated individual cell-delta data were used to display the intra-individual leukocyte subset kinetics and differences between MI, sham-operated and control (n = 4) groups. Significance between groups calculated by two-way ANOVA with Benferroni post-hoc test (MI vs. SHAM, ns - not significant). (<b>D</b>) Subset composition changes within the circulating MHCII^neg monocyte compartment following MI. (<b>E</b>) Example of MRI analysis with a short axis of hearts with mild and severe MI. EDV: end-diastolic volume, ESV: end-systolic volume. (<b>F</b>) Selection of mild versus severe MI based on the chronic impairment of the left ventricle ejection fraction (LVEF<35%) and increased LV dilatation (EDV) at day 21. (<b>G</b>) Monocyte time-course kinetics for both MI groups shows no correlation between development of blood monocytosis and the extent of myocardial injury.</p

Inter-individual variability of circulating leukocyte subsets in C57BL/6 mice.

<p>(<b>A</b>) Cross-sectional cell frequency analysis of 180 gender- and age-matched wild-type (WT) mice. Box-and-whiskers plots of the absolute cell numbers. The box shows the 25^th to 75^th percentiles, and the line in the box indicates the median value. Horizontal bars outside the box indicate 10^th to 90^th percentiles and the circles indicate 1^st to 99^th percentiles. (CV – standard deviation/mean). (<b>B</b>) Frequencies of main monocyte subsets in WT mice as based on Ly6C/CD43 classification are displayed. (<b>C</b>) Occurrence of monocytosis in WT animals depends on shifts towards classical (Ly6C^hiCD43^low) phenotype. (<b>D</b>) Analysis of MHC-II-pos compartment reveals predominance of “non-classical” (Ly6C^lowCD43^high) and “intermediate” phenotype. (<b>D</b>) Mean fluorescence intensity (MFI) for F4/80 in the major monocyte subsets in WT mice.</p

Polychromatic FCM is adaptable for low-volume measurements in mice.

<p>(<b>A</b>) Basic FCM gating strategy and phenotypic definitions for granulocyte and monocyte subsets as used in the current study. Monocytes (Lin<b>⁻</b>CD11b<b>⁻</b>Iab<b>⁻</b>) and MHC(Iab)⁺ cells were separated into main subsets based on the Ly6C/CD43 expression properties. (<b>B</b>) FCM immunobead-based assay for rapid leukocyte quantification in mice. (<b>C</b>) Comparison of assay-standardized (50 µl) and volume-reduced (20 µl) blood samples (n = 10 mice) show high correlation across the entire cell frequency range for all measured myeloid subsets.</p