Abstract
Fibrillar collagen is the primary component of the cardiac interstitial extracellular matrix. This extracellular matrix undergoes dramatic changes from birth to adulthood and then into advanced age. As evidence, fibrillar collagen content was compared in sections from neonates, adult, and old hearts and was found to increase at each respective age. Cardiac fibroblasts are the principle cell type that produce and control fibrillar collagen content. To determine whether fibroblast production, processing, and deposition of collagen differed with age, primary cardiac fibroblasts from neonate, adult, and old mice were isolated and cultured in 3-dimensional (3D) fibrin gels. Fibroblasts from each age aligned in fibrin gels along points of tension and deposited extracellular matrix. By confocal microscopy, wild-type neonate fibroblasts appeared to deposit less collagen into fibrillar structures than fibroblasts from adults. However, by immunoblot analysis, differences in procollagen production and processing of collagen I were not detected in neonate versus adult fibroblasts. In contrast, fibroblasts from old mice demonstrated increased efficiency of procollagen processing coupled with decreased production of total collagen. SPARC is a collagen-binding protein previously shown to affect cardiac collagen deposition. Accordingly, in the absence of SPARC, less collagen appeared to be associated with fibroblasts of each age grown in fibrin gels. In addition, the increased efficiency of procollagen alpha 1(I) processing in old wild-type fibroblasts was not detected in old SPARC-null fibroblasts. Increased levels of fibronectin were detected in wild-type neonate fibroblasts over that of adult and old fibroblasts but not in SPARC-null neonate fibroblasts versus older ages. Immunostaining of SPARC overlapped with that of collagen I but not to that of fibronectin in 3D cultures. Hence, whereas increases in procollagen processing, influenced by SPARC expression, plausibly contribute to increased collagen deposition in old hearts, other cellular mechanisms likely affect differential collagen deposition by neonate fibroblasts.
Highlights
Fibrillar collagen is the primary component of the interstitial extracellular matrix (ECM) of the heart
Primary cardiac fibroblasts were isolated from neonate, adult and old mice and cultured in 3D fibrin gels with points of tension provided by insect pins anchored in Sylguard beneath the gels (Figure 1)
We sought to determine whether primary cardiac fibroblasts isolated from mice of different ages exhibited variations in collagen production and procollagen processing that might contribute to age-dependent changes in the cardiac interstitium
Summary
Fibrillar collagen is the primary component of the interstitial extracellular matrix (ECM) of the heart. Of the ECM components present in the cardiac interstitium, amounts of collagen type I are highest with lesser amounts of collagen III and V represented [2]. These 3 collagen types are the primary components of the collagen fibers in the heart and take on representative structures in the forms of weaves, struts, and coils [3]. As the murine heart develops from neonatal stages to those of adult, the cardiac interstitium undergoes notable changes [5]. We sought to determine whether changes in processing in the absence of SPARC expression were apparent in cardiac fibroblasts from neonate, adult and old myocardium
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