Abstract 20940: PKA-Phosphorylation of Hsp20 Promotes Cardiac Fibrosis Through Upregulation of IL-6

Introduction: Heat shock protein 20 (Hsp20) is the only small heat shock protein that can be phosphorylated by PKA upon β-adrenergic stimulation. Interestingly, phosphorylation of Hsp20 is highly elevated following I/R injury and in human heart failure. However, the functional significance of this phosphorylation in vivo is currently unclear. Results: Transgenic lines with cardiac overexpression of constitutively phosphorylated Hsp20 (S16D) were generated and we chose one with 10-fold overexpression to mimic the levels observed in human failing hearts. Longitudinal assessment of cardiac function via echocardiography indicated significantly reduced LVEF in S16D mice starting at 3 months of age which progressed to heart failure and early lethality (50% dead by six months). Histological examination revealed interstitial fibrosis as early as 2 months with no evidence of apoptosis. To elucidate the mechanisms underlying fibrosis in S16D hearts, we performed cardiomyocyte-fibroblast crosstalk experiments. Adult mouse cardiomyocytes were infected with GFP, S16D or S16A-Hsp20, cultured for 48 hours, and their supernatant was then used to culture adult mouse fibroblasts. S16D-cultured fibroblasts exhibited enhanced proliferation as well as pro-fibrotic signaling (increased expression of α-SMA, TGF-β and collagen as well as cytokines IL-6 & IL-1β), while GFP or S16A-cultured fibroblasts showed no alterations. At the sub-cellular level, Stat3 phosphorylation and nuclear translocation was increased in S16D-cultured fibroblasts. These pro-fibrotic effects were associated with increased expression and secretion of IL-6 from cardiomyocytes, which were inhibited by inclusion of an IL-6 neutralizing antibody to the S16D-myocyte supernatant upon fibroblast exposure. Consistent with our ex-vivo results, the cardiac levels of IL-6 as well as Stat3 activation were upregulated in S16D hearts at two months, concurrent with increased IL-6 levels in the serum. Conclusion: Phosphorylation of Hsp20 (P-Hsp20) in cardiomyocytes appears to contribute to fibrosis and ventricular remodeling via upregulation of IL-6. Thus, targeting the observed increases in failing hearts may hold therapeutic potential.