Myocardial aging as a T-cell-mediated phenomenon

Abstract

In recent years, the myocardium has been rediscovered under the lenses of immunology, and lymphocytes have been implicated in the pathogenesis of cardiomyopathies with different etiologies. Aging is an important risk factor for heart diseases, and it also has impact on the immune system. Thus, we sought to determine whether immunological activity would influence myocardial structure and function in elderly mice. Morphological, functional, and molecular analyses revealed that the age-related myocardial impairment occurs in parallel with shifts in the composition of tissue-resident leukocytes and with an accumulation of activated CD4⁺ Foxp3^- (forkhead box P3) IFN-γ⁺ T cells in the heart-draining lymph nodes. A comprehensive characterization of different aged immune-deficient mouse strains revealed that T cells significantly contribute to age-related myocardial inflammation and functional decline. Upon adoptive cell transfer, the T cells isolated from the mediastinal lymph node (med-LN) of aged animals exhibited increased cardiotropism, compared with cells purified from young donors or from other irrelevant sites. Nevertheless, these cells caused rather mild effects on cardiac functionality, indicating that myocardial aging might stem from a combination of intrinsic and extrinsic (immunological) factors. Taken together, the data herein presented indicate that heart-directed immune responses may spontaneously arise in the elderly, even in the absence of a clear tissue damage or concomitant infection. These observations might shed new light on the emerging role of T cells in myocardial diseases, which primarily affect the elderly population.

Keywords: T cells; aging; inflammaging; inflammation; myocardial.

Fig. 1.

Leukocyte populations found within the healthy myocardium. Cell suspensions obtained from perfused/digested hearts of 2- to 3-mo-old or 12- to 15-mo-old (A–C) animals were used for flow cytometry. The major leukocyte populations were defined as follows: monocytes/macrophages (Mono/Mac) (CD45⁺ CD11b⁺ Ly6G⁻), granulocytes (Gran.) (CD45⁺ CD11b⁺ Ly6G⁺), B cells (CD45⁺ CD11b⁻ Ly6G⁻ B220⁺), and T cells (CD45⁺ CD11b⁻ Ly6G⁻ CD3e⁺). (D) The same tissue digestion protocol was applied to cardiac and skeletal muscle samples under steady-state conditions, revealing a more abundant leukocyte presence within the healthy myocardium. The temporal fluctuations in the absolute cell counts of each subset are represented in E and F, where ■ represents monocyte/macrophages, ○ represents granulocytes, □ represents T cells, and ▲ represents B cells. (G) Entire hearts from healthy animals were stained with anti-CD45 antibodies and prepared for light-sheet fluorescence microscopy (LSFM). A Z-stack reconstruction spanning large myocardial areas in the longitudinal axis shows abundant resident leukocyte populations (Left, 150-μm Z axis, 5× magnification; Right, 1.5-mm z axis, 26× magnification). (Scale bars: 50 μm.) (H) The cardiac-resident leukocyte (CD45⁺) cell numbers detected either using a tissue digestion–flow cytometry protocol or directly using LSFM were compared. The graphs represent the mean ± SEM of four to eight animals. Statistical tests performed were as follows: t test (bar graphs) and one-way ANOVA, followed by Dunnett’s post hoc test comparing the same cell subset at different time points (line graphs). *P < 0.05; in E, ** shows that *P < 0.05 for both of the overlapping Mono/Mac and Gran lines. Data were pooled from at least two independent experiments. LV, left ventricle; RV, right ventricle.

Fig. S1.

Tissue distribution of the heart-associated leukocytes. (A) Staining intravascular and parenchymal leukocytes in the healthy myocardium. Excised hearts had the aorta cannulated and mounted in a perfusion apparatus, and the coronary circulation was perfused with anti-CD45 eF450. Afterward, hearts were enzymatically digested, and cell suspensions were stained with anti-CD45-FITC. Thus, intravascular leukocytes were defined as CD45 eF450⁺ whereas parenchymal leukocytes were CD45-FITC⁺ CD45-eF450⁻. (B) The parenchymal distribution of different cardiac leukocytes’ populations was further confirmed by immunofluorescence. Cardiomyocytes were stained with Phalloidin-Atto 488 (green); nuclei were stained using DAPI (blue), and different leukocytes subsets appear in red/magenta, as indicated by the white arrows. Macrophages were defined as CD68⁺ cells, T cells were CD3ε⁺, and B cells were CD45/B220⁺. (Magnification: CD68 and CD3ε, 200×; B220, 400×.)

Fig. S2.

Further phenotypic characterization of the cardiac leukocytes. (A and B) Monocytic cells (CD11b⁺Ly6g⁻) were further gated as F4/80⁺CD206⁻ (M1) or as F4/80⁺CD206⁺ (M2) macrophages. (C and D) The surface expression of CCR2 on cardiac macrophages was also monitored along aging. (E) No difference was observed between using antibodies targeting B220 or as CD19 as a B-cell lineage marker. (F) B cells were divided into two major subpopulations: a predominant IgD^highIgM^low subset, mainly consisting of mature, follicular B cells; and an IgD^lowIgM^high subset, including immature, marginal zone, and B1 cells. No fluctuations in these B-cell subsets were observed in the aged myocardium (G). (H and I) Frequencies of CD4⁺ and CD8⁺ T-cell subsets (among bulk T cells). The graphs represent the means ± SEM of four to six animals. The statistical tests performed were as follows: t test (B, D, and G) and one-way ANOVA followed by the Dunnett post hoc test for each T-cell subset (I). *P < 0.05.

Fig. 2.

Myocardial aging. Cardiac function, fibrosis, and hypertrophy were assessed over a 15-mo period. (A–E) Echocardiographic analysis revealed some age-related alterations in cardiac function and structure, including a mild reduction in fractional shortening (A), an increased end diastolic area (B), and an increase in end-diastolic anterior wall thickness (C). (D and E) Representative echocardiographic registers from young (2 to 3 mo old) and aged (12 to 15 mo old) mice, respectively. (F) The myocardial gene expression levels of Tgfb3. (G and H) Representative pictures of young and aged heart slices stained with Picrosirius red (PSR). (Scale bars: 50 μm.) (I) The ratio of the myocardial gene expression levels of myosin heavy chain isoforms 6 (alpha, cardiac) and 7 (beta, muscle). (J and K) Representative pictures of young and aged WGA-stained heart slices, respectively. The bar graphs represent the mean ± SEM of 5 to 19 animals (A) or 4 to 8 animals (F and I). The statistical test performed was as follows: one-way ANOVA followed by Dunnett’s post hoc test. *P < 0.05 in comparison with young controls. Data are pooled from at least two independent experiments.

Fig. S3.

Myocardial aging. (A) Hemodynamics analysis revealed a mild myocardial impairment with aging, including increased end diastolic volume (A) and reduced ejection fraction (B). Cardiac hypertrophy was further validated by measuring heart weight/tibia length (C) and by measuring cardiomyocyte cross-sectional area in H&E-stained histological slices (D). The bar graphs represent the means ± SEM of three to nine animals. The statistical tests performed were as follows: t test (A, B, and D), and one-way ANOVA followed by the Dunnett post hoc test. *P < 0.05.

Fig. 3.

Cardiac aging and inflammation. (A) Scatter plot comparing the normalized myocardial gene expression levels (Log2 relative gene expression) of naive WT young (2 to 3 mo old) versus aged (12 to 15 mo old) mice. A custom-made PCR array including 45 genes related to cardiomyocyte responses to cell stress (blue dots), inflammation (red dots), antiinflammation (green dots), and extracellular matrix (ECM) biology (yellow dots) was designed, and pooled myocardial samples were tested (n = 3 per group). Next, the most relevant genes related to inflammation (B–E) and cell stress (F and G) were further tested individually with additional biological and technical replicates. The bar graphs represent the mean ± SEM of three to six animals. The statistical test performed was as follows: one-way ANOVA followed by Dunnett’s post hoc test. *P < 0.05 in comparison with young controls. The qPCR-arrays were performed as a single experiment whereas the standard qPCR reactions were performed using samples from at least two independent experiments.

Fig. 4.

Analysis of the heart-draining lymph nodes during aging. (A) Absolute cell number per lymph node station. Flow cytometry analysis revealed an age-related accumulation of CD44^high CD62L^low CD4⁺ T cells (i.e., activated, effector/memory phenotype) in both the popliteal and mediastinal lymph nodes although the accumulation was more evident in the mediastinal lymph nodes (B and C). The frequency of Foxp3⁺ cells among the CD44^high CD62L^low CD4⁺ T cells was significantly reduced in mediastinal but not popliteal LN from aged animals (D and E). Upon in vitro stimulation with PMA plus ionomycin, the CD4⁺ T cells harvested from the aged mediastinal LN preferentially produced IFNγ (F and G) whereas the cells isolated from the popliteal LN (same animals) produced IL-10 (H and I). Alongside with the IFN-γ expression pattern, the surface expression of the chemokine receptor CXCR3 was preferentially up-regulated in the CD4⁺ T cells found in the med-LN of aged animals (J and K). The bar graphs represent the mean ± SEM of five to eight animals. The statistical test performed was as follows: two-way ANOVA followed by the Tukey post hoc test. *P < 0.05, as indicated in the graphs.

Fig. S4.

Further analysis of the med-LN and pop-LN of young and aged animals. (A and B) Frequencies of CD4⁺ and CD8⁺ cells among bulk T cells. (C and D) Frequencies of Tconv (Foxp3⁻) and Tregs (Foxp3⁺) among CD4⁺ T cells. (E and F) The frequency of CD44^high cells among either Tconv (CD4⁺ FoxP3⁻) or Tregs (CD4⁺ FoxP3⁺). The bar graphs represent the means ± SEM of four to eight animals. The statistical tests performed were as follows: two-way ANOVA followed by the Tukey post hoc test. *P < 0.05 between different ages.

Fig. S5.

Age-related shifts in the subiliac LNs. (A) Absolute cell number. (B) The frequency of CXCR3⁺ cells among CD4⁺T cells. (C) Frequency of CD4⁺ T cells exhibiting an activated/memory phenotype (CD44^highCD62^low). (D) The frequency of Tregs among activated CD4⁺T cells. The bar graphs represent the means ± SEM of four to six animals. The statistical test performed was as follows: t test. *P < 0.05. NS, Not statistically significant.

Fig. S6.

Analysis of the CD8⁺T-cell compartment. Upon in vitro stimulation with PMA plus ionomycin, the CD8⁺ T cells harvested from the aged mediastinal LN preferentially produced IFNγ (A and B) whereas the cells isolated from the popliteal LN (same animals) produced IL-10 (C and D). The bar graphs represent the means ± SEM of four to six animals. The statistical tests performed were as follows: two-way ANOVA followed by the Tukey post hoc test. *P < 0.05.

Fig. 5.

Spontaneous heart-directed autoreactivity arises with aging. Heart-specific autoantibodies were detected by means of incubating the plasma of young/aged animals with histological heart slices prepared from B-cell–deficient animals (thus, with no endogenous immunoglobulins). IgGs reacting against cardiac antigens are depicted in green (A, C, E, and G) whereas autoreactive IgMs appear in red (B, D, and F). (A and B) Control heart slides incubated with secondary antibodies only (no plasma). C and D show heart-specific autoreactivity found in young animals’ plasma whereas E and F show autoreactivity found in aged animals. (G) Higher magnification showing that most IgGs that spontaneously arise with aging target sarcomeric antigens. (H) Myosin-specific antibodies. The bar graph represents the mean ± SEM of six to eight animals. The statistical test performed was as follows: t test. *P < 0.05. (Magnification: A–F, 400×; G, 1,000×.)

Fig. 6.

Assessing the cardiac phenotype of different lymphocyte-deficient mouse strains. We performed a comprehensive cardiac phenotyping of 12- to 15-mo-old animals of the following mouse strains: CD4KO (CD4 deficiency), MHCIIKO (CD4 deficiency), OT-II (in which most of CD4+ T cells bear a transgenic TCR to an irrelevant pep-MHC), and μMT (B-cell deficiency). Echocardiographic analysis revealed that WT and B-cell–deficient mice, but not MHC-II–deficient mice, presented an age-related reduction in fractional shortening (A) and increase in end diastolic area (B). (C–F) A custom-made PCR array including three housekeeping genes and 45 genes related to cardiomyocyte responses to cell stress (blue dots), inflammation (red dots), antiinflammation (green dots), and extracellular matrix biology (yellow dots) was used to profile myocardial gene expression levels of different aged immunodeficient mice. Scatter plots of normalized gene expression levels (Log2 relative gene expression) comparing WT vs. CD4KO, WT vs. MHC-IIKO, WT vs. OT-II, and WT x μMT mouse strains are shown in C–F, respectively. PCR array data were further validated by performing qPCR reactions with individual samples for specific genes (G–I). The bar graphs represent the mean ± SEM of 5 to 19 animals (A and B) or 3 to 5 animals (G–I). The statistical test performed in A and B was as follows: two-way ANOVA followed by the Tukey post hoc test; ^#P < 0.05 compared with age-matched WT mice; *P < 0.05 compared with genotype-matched young controls. The statistical test performed in G–I was as follows: one-way ANOVA followed by Dunnett’s post hoc test; *P < 0.05 compared with age-matched WT mice. The experiments including aged immunodeficient mouse strains were performed once using littermate controls.

Fig. S7.

The distribution of lymphocyte populations after heterochronic cell transfer. Juvenile lymphocyte-deficient RagKO mice were adoptively transferred with med-LN cells from young WT (YY) or aged WT donors (OY-med-LN). Additional groups received cells purified from the med-LNs of aged T-cell–deficient donors (OY-med-LN-TCRβKO) or from the subiliac LN cells of aged WT donors (OY-si-LN). (A–C) The total leukocyte counts (CD45⁺ cells) in the spleen, med-LN, and si-LN of recipient mice. (D–F) The frequencies of CD8⁺ TCRβ⁺ T cells in the spleen, med-LN, or si-LN of recipient mice. (G–I) The frequencies of CD4⁺ TCRβ⁺ T cells in the spleen, med-LN, or si-LN of recipient mice. (J–L) The frequencies of effector CD4⁺ T cells (i.e., CD44^high CD62^low) in the spleen, med-LN, or si-LN of recipient mice. The graphs show individual values and the mean ± SEM of five to six mice. The statistical tests performed were as follows: one-way ANOVA followed by the Tukey post hoc test. *P < 0.05 compared with the YY group.

Fig. 7.

Heterochronic lymphocyte adoptive cell transfer. Juvenile lymphocyte-deficient RagKO mice were adoptively transferred with med-LN cells from WT young (YY) or aged donors (OY-med-LN). Additional groups received cells purified from the med-LNs of aged T-cell–deficient donors (OY-med-LN-TCRβKO) or from the subiliac LN cells of aged WT donors (OY-si-LN) (A). (B–D) The frequencies of major cardiac leukocyte populations were determined by flow cytometry at 4 mo after cell transfer. Macrophages (B) were defined as CD45⁺ CD11b⁺ Ly6G⁻; B cells (C) were defined as CD45⁺ CD11b⁻ B220⁺; and alpha beta T cells (D) were defined as CD45⁺ CD11b⁻ TCRβ⁺. (E and F) Echocardiographic analysis performed at 4 mo after cell transfer. The dashed lines in E and F indicate the upper 95% confidence interval (CI) of age-matched RagKO controls, and the dashed line in G indicates the lower 95% CI of age-matched RagKO controls. The graphs show the individual values obtained from each mouse and the mean ± SEM of 5 to 16 mice. The statistical test performed was as follows: one-way ANOVA followed by the Tukey post hoc test. The letters “a,” “b,” and “c” represent P < 0.05 when the OY-med-LN group was compared with YY, OY-si-LN, and OY-med-LN TCRβKO, respectively. The letter “d” represents P < 0.05 compared with naive Ragko controls.

Fig. S8.

Heterochronic cell transfer—further phenotyping. Juvenile lymphocyte-deficient RagKO mice were adoptively transferred with med-LN cells from young WT (YY) or aged WT donors (OY-med-LN). Additional groups received cells purified from the med-LNs of aged T-cell–deficient donors (OY-med-LN-TCRβKO) or from the subiliac LN cells of aged WT donors (OY-si-LN). (A–C) Relative myocardial mRNA expression levels of genes related to inflammatory, hypertrophic, and fibrotic processes. (D) Heart weight to body weight ratio. (E) The levels of circulating myosin-specific antibodies. (F) The splenocytes from the OY-med-LN-TCRβKO group were isolated, and their capacity to produce IFN-γ (baseline and after stimulation with anti-CD3) was tested in vitro. The graphs show individual values and the mean ± SEM of five to six mice. The statistical tests performed in panels A–E were as follows: one-way ANOVA followed by the Tukey post hoc test. The statistical tests performed in F were as follows: two-way ANOVA followed by the Tukey post hoc test. *P < 0.05 as indicated in the graph.