Table 1.
Morphological and Physiological Analysis.
Figure 1.
LV collagen volume fractions and collagen crosslinking was increased in diabetic (db/db) mouse hearts.
LV collagen volume fractions were determined from picric acid sirius red (PASR) stained mid ventricle for non-diabetic (db/wt) and diabetic (db/db) mice. Figure 1a shows representative photographs of PASR stained diabetic (db/db) mouse hearts under brightfield and polarized light microscopy. Figure 1b shows representative photographs of PASR stained of non-diabetic (db/wt) mouse hearts under brightfield and polarized light microscopy. Figure 1c depicts a significant increase in PASR stained collagen fibrils in db/db LVs (*p<0.05; n = 11 hearts per group). LV collagen crosslinks were determined from subsequent digestions of pepsin-acetic acid and HCl hydrolysis. Figure 1d depicts a significant increase in crosslinked collagen in db/db as measured by hydroxyproline concentrations. NG = normoglycemic media; HG = hyperglycemic media (*p<0.05; n = 11 hearts per group).
Figure 2.
Profibrotic markers were increased in isolated diabetic (db/db) cardiac fibroblasts.
Figure 2a shows type I collagen was increased in isolated fibroblasts from diabetic (db/db) protein secreted into conditioned media and in Figure 2b. type 1 collagen mRNA expression. Figure 2c shows an increased PAI-1 protein expression from cardiac fibroblast conditioned media of diabetic (db/db) fibroblasts. Figure 2d demonstrates an increase in TGF-β protein expression in diabetic (db/db) from cardiac fibroblast conditioned media as measured by commercial ELISA kit. Smooth muscle expression did not change between groups (data not shown). NG = normoglycemic media 5 mM glucose; HG = hyperglycemic media 25 mM glucose (*p<0.05; n = 4–5 independent experiments with 6 hearts per experiment).
Figure 3.
AGE accumulation was increased in diabetic (db/db) mouse hearts, and RAGE expression was increased in diabetic (db/db) isolated fibroblasts.
Figure 3a. demonstrates an increased AGE expression in diabetic (db/db) LV collagen extracts. Figure 3b. depicts an increase in RAGE protein expression in isolated db/db LV fibroblasts. NG = normoglycemic media 5 mM glucose; HG = hyperglycemic media 25 mM glucose (*p<0.05; n = 4 independent experiments with 6 hearts per experiment).
Figure 4.
Extracellular matrix protein regulators MMP-2 and TIMP-2 were increased in diabetic (db/db) mouse hearts and conditioned media from isolated diabetic (db/db) cardiac fibroblasts.
Figure 4a demonstrates increased MMP-2 gelatinase activity levels from conditioned media of isolated db/wt and db/db hearts as measured by gelatin zymography. MMP-2 activity levels were also significantly increased in db/db LV lysates as measured by gelatin zymography (data not shown). Figure 4b shows a dramatic increase in TIMP-2 protein levels for isolated db/db cardiac fibroblasts. NG = normoglycemic media 5 mM glucose; HG = hyperglycemic media 25 mM glucose (*p<0.05; n = 4 independent experiments with 6 hearts per experiment).