Extracellular matrix remodeling in early cardiac fibrosis and diastolic dysfunction

Abstract

Background: Half of the patients with congestive heart failure suffer from diastolic dysfunction with persistent left ventricular function (HFpEF). Until today different explanations exist, but the exact mechanism of HFpEF is still unclear. From human biopsies and different animal models it is known that in HFpEF the extracellular matrix underlies remodeling processes with increased fibrosis and disturbance of cardiac architecture. Our study aimed to characterize changes of the extracellular matrix in early cardiac fibrosis and diastolic dysfunction. Methods: In C57Bl/6-mice, osmotic minipumps were implanted with a continuous infusion of Angiotensin II (Ang, 0.5μg/kg/min) or NaCl (Sham). After assessment of systolic and diastolic parameters using echocardiography at day 7 or 28, mice were euthanized (Ang7d, n=22/Ang28d, n=27/Sham, n=20). Cardiac tissue was investigated with micro array technology, western blot, qRT-PCR and immunohistochemistry. Results: Echocardiography revealed a reliable induction of diastolic dysfunction with persistent LVEF in the Ang groups (E/E': Sham 25.9±1.0 vs. Ang28d 38.5±4.4; p<0.01). BNP expression was significantly increased (Sham 0.7±0.1 vs. Ang28d 1.9±0.4; p<0.05). Histochemical examination showed a significantly elevated interstitial fibrosis with significant increase of Vimentin positive cardiac fibroblasts. Expressions of Collagen 1, 3, 4, 6 and 14 were increased. By using mRNA micro array technology different targets of the extracellular remodeling process were identified and validated by qRT-PCR. The expressions of the collagen maturation enzyme LOX and of the fibrillogenesis promoting proteins Sparc were significantly increased. Fibromodulin, which is known to regulate collagen fibril diameters, was also elevated (Sham 2.3±0.6 vs. Ang28d 14.5±3.9; p<0.05). Expressions of the Small leucine-rich repeat proteoglycan Decorin and Osteoglycin were reduced after 7 days, while Osteopontin showed significantly elevated expression. Lumican and Asporin were unchanged. The expression of adhesion permitting protein Tenascin C was significantly elevated. Interestingly, expression of the Cartilage Intermediate Layer Protein (CILP), which is only known to take part in the cartilage remodeling process, was clearly increased (Sham 1.9±0.3 vs. Ang28d 8.3±1.9; p<0.01). Conclusion: The Angiotensin induced cardiac fibrosis was composed by an intensified fibrillogenesis and a changed composition of SLRPs. Furthermore, the significantly elevated expression of CILP suggests a contribution of this extracellular protein to the process of cardiac fibrosis and diastolic dysfunction.