Immune-inflammatory dysregulation modulates the incidence of progressive fibrosis and diastolic stiffness in the aging heart

Abstract

Diastolic dysfunction in the aging heart is a grave condition that challenges the life and lifestyle of a growing segment of our population. This report seeks to examine the role and interrelationship of inflammatory dysregulation in interstitial myocardial fibrosis and progressive diastolic dysfunction in aging mice. We studied a population of C57BL/6 mice that developed progressive diastolic dysfunction over 30 months of life. This progressive dysfunction was associated with increasing infiltration of CD45(+) fibroblasts of myeloid origin. In addition, increased rates of collagen expression as measured by cellular procollagen were apparent in the heart as a function of age. These cellular and functional changes were associated with progressive increases in mRNA for MCP-1 and IL-13, which correlated both temporally and quantitatively with changes in fibrosis and cellular procollagen levels. MCP-1 protein was also increased and found to be primarily in the venular endothelium. Protein assays also demonstrated elevation of IL-4 and IL-13 suggesting a shift to a Th2 phenotype in the aging heart. In vitro studies demonstrated that IL-13 markedly enhanced monocyte-fibroblast transformation. Our results indicate that immunoinflammatory dysregulation in the aging heart induces progressive MCP-1 production and an increased shift to a Th2 phenotype paralleled by an associated increase in myocardial interstitial fibrosis, cellular collagen synthesis, and increased numbers of CD45(+) myeloid-derived fibroblasts that contain procollagen. The temporal association and functional correlations suggest a causative relationship between age-dependent immunoinflammatory dysfunction, fibrosis and diastolic dysfunction.

Figure 1

Noninvasive measurement of diastolic function in mice of different ages. The transmitral Peak E filling velocity decreased with age from three months to twenty four months, but the values increased in thirty month old mice (A). The isovolumic relaxation time was prolonged in the 24 month old mice, but was shorter in the 30 month old mice (B). The left atrial volume increased monotonically with age (C).

Figure 2

Immunohistochemistry of collagen type I in hearts from mice of different ages: 3 (A), 14 (B), 24 (C), and 30 month (D) at 20× magnification, and 3 (E), 14 (F), 24 (G) and 30 month (H) at 40× magnification.

Figure 3

Expression of collagen type I in CD45⁺ cells. Nonmyocytes were isolated from hearts of different ages. (A) Flow cytometry of those cells demonstrated that a proportion of the nonmyocytes expressed both CD45 (hematopoietic marker) and collagen type I (fibroblast marker) as seen in the upper right quadrant (cells isolated from a 14 month old animal). (B) The proportion of the nonmyocytes that were double positives (CD45 and collagen type I) increased with age. Linear regression R2= 0.66 and p=0.0001 (n=7, 4, 4, and 9 for 3, 14, 24, and 30 months). Immunofluorescence in paraffin sections of hearts with antibodies to CD45 (red) was negative in 3 month old hearts (C) and positive with a random distribution in 14 month old hearts (D). (E) Some CD45⁺ cells were also positive for procollagen type I (green). Cell nuclei are DAPI stained (blue).

Figure 4

Expression of procollagen type I in the aging heart. Protein extracts from whole hearts of different ages were analyzed for the presence of procollagen to indicate active collagen synthesis. (A) is an immunoblot of 3 month-old versus 30 month old hearts, (B) is the Ponceau stain of the membrane to ensure equal protein loading of the gel, and (C) is the densitometric analysis of multiple blots from hearts of the indicated ages (n=4). Immunofluorescence of paraffin sections of hearts of different ages indicates that there is no visible procollagen type I (green) in young hearts (D). Procollagen type I is visible at 14 months (E) and 24 months (F) in fibroblasts, and in blood vessel walls at 24 months and 30 months (G). Cell nuclei are DAPI stained (blue).

Figure 5

Measurement of MCP-1 mRNA and protein in hearts from mice of different ages. (A) mRNA expression of MCP-1 by quantitative PCR, with a linear regression R2=0.56 and p=0.0004; n=5, 3, 5, and 5 for 3, 14, 24, and 30 month old animals. MCP-1 protein levels in 3 month old hearts were compared to that in 30 month old hearts by protein array (B)(n=3). Immunofluorescence of an antibody against MCP-1 (green) in hearts from different ages showed no visible staining in 3 month old hearts (C), whereas there was staining in the blood vessels of all other ages (D, 14 month old heart). Cell nuclei are DAPI stained (blue).

Figure 6

Monocyte-to-fibroblast maturation after TEM in response to MCP-1. Human mononuclear leukocytes from four donors were allowed to migrate through human cardiac microvascular endothelial cells in response to 650 ng of MCP-1. The total number of fibroblasts in each well of triplicates was counted after two days of migration and two further days of maturation. (A) Data from wells treated with 10 ng/ml of either IL-13 or IL-12 were normalized to control wells with no further treatment. (B) All wells were treated with 10 ng/ml IL-13; some of those were also treated with 1 μg/ml of either an IL-13 Rα2/Fc chimera (IL13RFc) or the control construct IL-11 Rα/Fc chimera (IL11RFc) and the data normalized to the counts from the IL-13 only treated wells. An asterisk indicates which groups are statistically different from the control (p=0.01, Student's t test).

Figure 7

Measurement of cytokine mRNA and protein in hearts from mice of different ages. (A) Th2 (IL-4 and IL-13) and Th1 (IFN-γ) cytokines were measured by protein array in 3 month old versus 30 month old hearts (n=3). (B) mRNA expression of IL-13 by quantitative PCR, with a linear regression R2=0.43 and p=0.006; n=4, 3, 5, and 4 for 3, 14, 24, and 30 month old animals.