S2). responses arise even in the absence of previous myocardial tissue damage. and and axis, 26 magnification). (Scale bars: 50 m.) (test (bar graphs) and one-way ANOVA, followed by Dunnetts post hoc test comparing the same cell subset at different time points (line graphs). * 0.05; in 0.05 for both of the shikonofuran A overlapping Mono/Mac and Gran lines. Data were pooled from at least two independent experiments. LV, left ventricle; RV, right ventricle. To assess whether these leukocytes were located in contact with the coronary circulation or in the myocardial parenchyma, we performed intravascular (in an isolatedCperfused heart system) and postdigestion staining using anti-CD45 antibodies coupled to different fluorochromes (Fig. S1and and Movie S1). shikonofuran A This approach enabled us to generate 3D reconstructions spanning large areas of intact myocardium and confirmed an abundant leukocyte distribution in hearts harvested from healthy, nonmanipulated animals (3,380 1,279 CD45+ cells per cubic millimeter of tissue). Myocardial Aging Is Accompanied by Important Shifts in Resident Leukocyte Composition. Next, we analyzed the myocardial tissues obtained from 2- to 3-mo-old mice (from here on, termed young animals), 6- to 8-mo-old mice (adult animals), and 12- to 15-mo-old mice (aged animals) and found that important changes in cardiac leukocyte composition occur over time (Fig. 1 and and Fig. S2). The macrophage population (primarily CD206+ cells) significantly decreased with aging, in parallel with an increase in the granulocyte infiltration (Fig. 1and and and and and test ( 0.05. The age-related shifts in the composition of heart-associated leukocyte populations occurred together with myocardial functional and structural alterations (Fig. 2). Echocardiographic studies revealed an age-related decrease in fractional shortening (FS) (young animals FS, 63.76 1.35% vs. aged animals, 51.24 2.62%, 0.05), an increase in end-diastolic area (EDA) (young animals EDA, 7.43 0.41 mm vs. aged animals, 9.93 0.34 mm, 0.05), and an increase in end-diastolic anterior wall thickness (EDWT) (young animals EDWT, 0.067 0.002 mm vs. aged animals, 0.086 0.004 mm, 0.05) shikonofuran A (Fig. 2 and and and and 0.05 in comparison with young controls. Data are pooled from at least two independent experiments. Open in a separate window Fig. S3. Myocardial aging. (test ( 0.05. Together with functional impairment, myocardial fibrosis and hypertrophy were observed in senescent hearts. Histological analysis revealed increased interstitial collagen deposition (Fig. 2 and and and Fig. S3= 3 per group). Next, the most relevant genes related to inflammation (and 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. In an attempt to delineate the sequence of events that lead to myocardial aging, we assessed the expression BRIP1 levels of key genes that are responsive to different stress contexts. Shifts in the expression of (GATA-binding protein 4), which is a cardiomyocyte transcription factor that is responsive to loading stress conditions, were the earliest alteration seen in the myocardium, occurring in animals aged 6 to 8 8 mo old. Only at later time points (12 to 15 mo) were the expression levels of genes related to redox stress ((hypoxia-inducible factor 1 alpha subunit) expression levels were observed with aging, suggesting that myocardial senescence is not mediated by hypoxic stress. Spontaneous CD4+ T-Cell shikonofuran A Activation Occurs in the Mediastinal Lymph Nodes of Aged Mice. The observation that age-related cardiac functional and structural impairment occurs in parallel with increased myocardial expression of genes related to the adaptive immune response prompted us to investigate whether lymphocyte activity plays a role in myocardial aging. Thus, we next characterized the heart-draining (mediastinal) lymph nodes (med-LNs) of young and aged mice under steady-state conditions. As controls, we used the popliteal lymph nodes (pop-LN) because they drain primarily the hind limb skeletal muscle (Fig. 4 and Figs. S4 and ?andS5S5). Open in a separate window Fig. 4. Analysis of the heart-draining lymph nodes during aging. (and and and and and 0.05, as indicated in the graphs. Open in a separate window Fig. S4. Further analysis of the med-LN and pop-LN of young and aged animals. (and and and 0.05 between different ages. Open in a separate window Fig. S5. Age-related shifts in the subiliac LNs. (test. * 0.05. NS, Not statistically significant. Surprisingly, we observed that aging was accompanied by an increased cellularity in the heart-draining LNs, but not in.