Purpose: Elderly patients often suffer from left ventricular hypertrophy and diastolic dysfunction with preserved systolic function resulting from ongoing cardiomyocyte loss and cardiac fibrosis. The protease activated receptor (PAR) 2 is known to be a pro-fibrotic mediator. In a mouse model of myocardial infarction PAR2 overexpression in cardiomyocytes led to the development of fibrosis. In this study we examine the role of PAR2 in the aged heart regarding fibrosis and hemodynamic function.
Methods: 8 weeks (wks) and 1 year (yr) old wild-type (wt) and PAR2 knockout (ko) mice underwent hemodynamic measurements with a 1.2F microconductance catheter and hearts were collected for histological and biochemical analysis. Collagen release and Smad2 phosphorylation were determined with western blots and the ROS activity was analysed with a DCF dependent immunfluorescence assay on adult cardiac fibroblasts. The PAR2 gene expression was determined in myocardial biopsies from HFPEF patients.
Results: 1 year old PAR2ko mice suffered from a left ventricular dysfunction with preserved systolic function, which was accompanied by an age dependent fibrosis. In hearts of 8 wks old wt and PAR2ko mice no differences in collagen expression were present. In contrast, 1 yr old PAR2ko mice showed collagen depositions in the heart and the collagen I / collagen III ratio revealed a fibrosis in PAR2ko mice but not in wt mice (p<0.05). Moreover, adult cardiac PAR2ko fibroblasts also showed an increased collagen I release into the supernatant compared to wt fibroblasts. Furthermore, the TGFβ-dependent Smad2 phosphorylation was stronger in PAR2ko fibroblasts compared to wt fibroblasts. Oxidative stress in the heart often triggers cardiac dysfunction. After treatment with H2O2, PAR2ko fibroblasts exhibited higher ROS levels than wt fibroblasts (wt vs PAR2ko: 4.26±1.78 vs. 6.42±5.35, p<0.05). The GSH / GSSG ratio in hearts of 1yr hearts pointed also to an increased oxidative stress in PAR2ko mice compared to wt mice (wt vs PAR2ko: 8.31±2.59 vs.4.80±1.53, p<0.05). These results indicate that the loss of PAR2 is associated with elevated oxidative stress, which leads to fibrosis and an impaired heart function. In HFPEF patients a decreased PAR2 expression was associated with severe diastolic dysfunction and vice versa.
Conclusion: The cardiac PAR2 expression is essential for the maintenance of the heart function in the aged heart. The loss of PAR2 results in increased oxidative stress, an age-dependent cardiac fibrosis and a left ventricular diastolic dysfunction.