The effects of age and the expression of SPARC on extracellular matrix production by cardiac fibroblasts in 3-D cultures.
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.
Department of Craniofacial Biology, Medical University of South Carolina, Charleston, South Carolina, United States of America.
Jessica
J
Trombetta-eSilva
Erik P
EP
Eadie
Yuhua
Y
Zhang
Russell A
RA
Norris
Thomas K
TK
Borg
Amy D
AD
Bradshaw
D000375:Aging / Q000378:metabolism*; D000818:Animals; D018929:Cell Culture Techniques; D024042:Collagen Type I / Q000032:analysis / Q000737:chemistry; D005109:Extracellular Matrix / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D005786:Gene Expression Regulation; D051379:Mice; D009206:Myocardium / Q000166:cytology*; D015676:Osteonectin / Q000378:metabolism*; D011347:Procollagen / Q000737:chemistry / Q000378:metabolism; D012995:Solubility
D024042:Collagen Type I; D015676:Osteonectin; D011347:Procollagen; C578250:SPARC protein, mouse