Postconditioning with CpG-Containing TLR9 Ligand 1668-Thioate Attenuates Inflammatory Response and Remodeling Leading to Less Fibrosis and Better Left Ventricular Function in Murine Myocardial Infarction

Objectives: Development of ischemic cardiomyopathy has been associated with inflammation and toll-like receptor (TLR)-signaling. It has been shown that post-conditioning (PCon) is able to attenuate inflammation and fibrosis in myocardial infarction. Here, we investigated whether PCon with the synthetic CpG-containing TLR9 ligand 1668-thioate (CpG) can modulate the development of inflammation and remodeling in reperfused murine myocardium.

Methods: Thirty minutes of LAD-ligation followed by reperfusion was conducted in 12 weeks old male C57BL/6 mice. Mice where treated with CpG i.p. 5 minutes. before reperfusion. Control group received PBS; sham group did not undergo ischemia. After 3, 7, and 28 days M-mode echocardiography and Millar^® left ventricular (LV) pressure volume catheter measurements were performed. Hearts where excised and harvested for immunohistochemical analysis. Gene expression (Taqman^® RT-qPCR) was measured after 6 and 24 hour reperfusion.

Results: Apoptosis markers Caspase 3 and 8, and matrix metalloproteinase (MMP)9 were not induced in CpG PCon group compared with high induction in PBS PCon, indicating lesser degree of apoptosis and extracellular matrix degeneration. However, proinflammatory chemokines CCL2, CCL3 and CCL4, and cytokines TNF-α and IL-1β were significantly up-regulated in CpG PCon group compared with PBS PCon after 6 hour. Interestingly, this peak of inflammatory activation was accompanied by significant induction of anti-inflammatory IL-10. Further, after 3 and 7 days significantly lower macrophage density (stained with MAC-2) was observed in the ischemic myocardium of CpG PCon mice compared with PBS PCon, suggesting that anti-inflammatory and other mechanisms mitigate proinflammatory action. Total LV collagen area using picrosirius red planimetry was significantly attenuated in CpG PCon mice after 7 and 28 days compared with PBS PCon mice. CpG PCon mice showed significantly lower level of ventricular dysfunction than PBS PCon. TLR1 mRNA was induced in CpG PCon hearts, while TLR4 and 9 were not induced. The specific role of the early inflammatory peak, followed by less macrophage infiltration, has to be further elucidated.

Conclusion: Our study suggests a cardioprotective mechanism of CpG PCon in modulation of remodeling and subsequent development of LV dysfunction in a murine model of reperfused myocardial infarction. This mechanism seems to involve TLR-modulation being associated with early chemokine and cytokine action.