Cardiac Fibroblast-Dependent Extracellular Matrix Accumulation Is Associated with Diastolic Stiffness in Type 2 Diabetes

Cardiovascular complications are a leading cause of death in patients with type 2 diabetes mellitus (T2DM). Diastolic dysfunction is one of the earliest manifestations of diabetes-induced changes in left ventricular (LV) function, and results from a reduced rate of relaxation and increased stiffness. The mechanisms responsible for increased stiffness are not completely understood. Chronic hyperglycemia, advanced glycation endproducts (AGEs), and increased levels of proinflammatory and profibrotic cytokines are molecular pathways known to be involved in regulating extracellular matrix (ECM) synthesis and accumulation resulting in increased LV diastolic stiffness. Experiments were conducted using a genetically-induced mouse model of T2DM generated by a point mutation in the leptin receptor resulting in nonfunctional leptin receptors (db/db murine model). This study correlated changes in LV ECM and stiffness with alterations in basal activation of signaling cascades and expression of profibrotic markers within primary cultures of cardiac fibroblasts from diabetic (db/db) mice with nondiabetic (db/wt) littermates as controls. Primary cultures of cardiac fibrobroblasts were maintained in 25 mM glucose (hyperglycemic-HG; diabetic db/db) media or 5 mM glucose (normoglycemic-NG, nondiabetic db/wt) media. The cells then underwent a 24-hour exposure to their opposite (NG; diabetic db/db) media or 5 mM glucose (HG, nondiabetic db/wt) media. Protein analysis demonstrated significantly increased expression of type I collagen, TIMP-2, TGF-beta, PAI-1 and RAGE in diabetic db/db cells as compared to nondiabetic db/wt, independent of glucose media concentration. This pattern of protein expression was associated with increased LV collagen accumulation, myocardial stiffness and LV diastolic dysfunction. Isolated diabetic db/db fibroblasts were phenotypically distinct from nondiabetic db/wt fibroblasts and exhibited a profibrotic phenotype in normoglycemic conditions.