Periconditioning with CpG-Containing TLR9 Ligand 1668-Thioate Ameliorates Left Ventricular Function via Modulation of Inflammation and Remodeling Resulting in Less Fibrosis in a Mouse Model of Reperfused Myocardial Infarction

Objectives: It has been shown that postconditioning (PCon) is able to attenuate inflammation and fibrosis in myocardial infarction. Aim of this study was to investigate 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-week-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. M-mode echocardiography and Millar left ventricular (LV) pressure volume catheter measurements were performed before hearts were excised and harvested for immunohistochemical, FACS, and gene expression analysis (Taqman RT-qPCR).

Results: Caspase 3 and 8 mRNA expression was not induced in CpG PCon group compared to high induction in PBS after 6 hours. Also, TUNEL and cleaved Caspase-3 staining showed significantly higher apoptosis in PBS animals. Abrogated Tenascin-C induction in CpG PCon mice further lessen degree of extracellular matrix remodeling, which is corroborated by fewer ASMAC+ myofibroblasts after 7 days. Total LV collagen area using picrosirius red planimetry was significantly smaller in CpG PCon mice after 7 and 28 days compared to PBS, resulting in significantly better LV function at the same time. The mRNA expression of proinflammatory chemokines CCL2, CCL3, and CCL4 and IL-1β protein levels (ELISA) were significantly upregulated in CpG PCon group. This was accompanied by significant induction of anti-inflammatory IL-10 mRNA. After 3 days, significantly lower number of M1 macrophage (FACS) was observed in CpG PCon mice, suggesting that anti-inflammatory mechanisms like IL-10 induction mitigate proinflammatory action. Although significantly more M2a macrophages were found in both groups after 3 days, and their number was significantly higher in PBS than in PCon mice. Interestingly, MCA771G staining revealed significantly higher number of neutrophils in PBS group compared to CpG PCon after 3 days. TLR1 mRNA was induced in CpG PCon hearts.

Conclusion: Our study suggests a cardioprotective mechanism of CpG PCon involving modulation of inflammatory reaction toward a more neutrophil-driven pathway and less cardiomyocyte apoptosis. This is followed by attenuated remodeling and LV dysfunction in a murine model of reperfused MI. This mechanism seems to involve TLR modulation.