Quantitative analysis of myocardial inflammation by flow cytometry in murine autoimmune myocarditis: correlation with cardiac function

Abstract

Inflammation has been increasingly recognized as an important pathological component of heart failure. Existing methods of assessing myocardial infiltrate are labor-intensive and provide data that are difficult to quantify and not representative of the whole heart. As a result, little effort has been made to systematically assess the components of myocardial inflammation. We established an alternative method of quantitative assessment of myocardial inflammation by flow cytometry after enzymatic digestion of hearts to characterize the infiltrate and study the association between inflammation and cardiac function in murine experimental autoimmune myocarditis. The severity of acute myocarditis uniquely correlated with the proportion of neutrophils, but not T cells, B cells, or macrophages. Both acute and chronic phases were characterized by the presence of CD44high (activated) T cells in the heart, whereas T cells trafficking through normal hearts exhibited CD44low phenotype. During the chronic phase, the proportion of CD4+ T cells was associated with increased left-ventricular volumes and deterioration of systolic function, the hallmarks of dilated cardiomyopathy. We conclude that flow cytometry on uniformly digested mouse hearts provides sensitive and reproducible assessment of myocardial infiltrate and can be used to dissect out the specific role of individual immune components from the overall inflammatory response in the heart.

Figure 1

Flow cytometric assessment of heart-infiltrating leukocytes. A: Representative forward scatter/side scatter profile and a live cell gate. B: CD45/side scatter plot was used to obtain percentages of CD45⁺ leukocytes; cells were derived from the live cell gate shown in A. C: CD4/CD45 plot; cells were gated as shown in A to derive percentage of CD4⁺ T cells of total live cells. D: CD3/MHC class II plot was used to derive percentages of CD3⁺ T cells or MHC class II⁺ cells of CD45⁺ leukocytes. Cells were first gated as shown in A and then gated as shown in B. E and F: Histograms showing CD45⁺ leukocytes (the second peak) from unimmunized and immunized hearts, respectively; cells were gated as illustrated in A. Numbers are percentages of leukocytes of total live cells. Data in A to D and F are from day 30-immunized A/J mice and data in E are from an unimmunized A/J mouse. The arrows indicate the gating strategy.

Figure 2

Severity of myocarditis in A/J and BALB/c mice. A and B: Gross scores in A/J (A) and BALB/c (B) mice. C and D: Flow cytometry-derived percentages of infiltrating leukocytes (CD45⁺ cells) of total live cells in the heart in A/J (C) and BALB/c (D) mice. E and F: Relationship between gross scores and percentages of leukocytes in the heart in A/J (E) and BALB/c (F) mice. P values were calculated using the Mann-Whitney U (gross scores) or the Student’s t (leukocytes)-test. Day 0 represents unimmunized mice. The gross scores and flow cytometry data are from the same mice and each triangle or dot represents an individual mouse. In E and F the data are pooled from different time points after immunization.

Figure 3

Increased activation state of heart-infiltrating T cells in EAM. Flow cytometric events were gated on live CD45⁺CD3⁺ cells. A: Percentages of cells expressing high levels of CD44 of total CD3⁺ T cells. Data are from BALB/c mice and shown as mean ± SD, n ≥ 5 per group. *, P < 0.05 compared to unimmunized controls (day 0), Student’s t-test. Day 0 corresponds to unimmunized age-matched BALB/c control mice. B: Overlay histogram shows CD44 expression on CD3⁺ T cells from a representative unimmunized mouse (thin line, shaded) and an immunized mouse on day 23 after immunization (thick line, unshaded).

Figure 4

Severity of acute inflammation is associated with the proportion of neutrophils. Percentages of leukocytes (CD45⁺ cells) of total live cells correlate with percentages of neutrophils of total live cells in BALB/c mice (A) and with the proportion of neutrophils of leukocytes in BALB/c mice (B) and in A/J mice (C). D: Side scatter/CD45 dot plot of heart cells represents severe myocarditis in A/J mice. The rectangular and oval gates indicate granulocytes and lymphocytes, respectively. These gates were obtained by backgating Gr1⁺ and CD3⁺ populations. Representative CD45/Gr1 dot plots from A/J mice with mild (E) or severe (F) myocarditis. Neutrophils are CD45⁺Gr1^high cells (upper right quadrants). Numbers are percentages of neutrophils of total live cells. Data were obtained using the live cell gate from individual hearts on days 22 to 24 in BALB/c mice and on day 30 after immunization in A/J mice. R² represents a regression coefficient. P values indicate the significance of the slope of the repression line (the strength of the linear association).

Figure 5

Proportion of CD4⁺ T cells is associated with cardiac dysfunction during the chronic phase of EAM. The proportions of CD4⁺ T cells of leukocytes are associated with increased EDV (A), reduced ejection fraction (EF) (B), reduced preload-adjusted maximal power (PWR_max/EDV) (C), and reduced PRSW (D). Each dot represents an individual mouse. Pressure-volume loops and a flow cytometric plot are from the same mouse with the lowest (2.8%) (E) and highest (8.7%) proportion of CD4⁺ T cells of leukocytes (F). R² represents the regression coefficient. P values indicate the significance of the slope of the repression line (the strength of the linear association).