The role of macrophage migration inhibitory factor in anesthetic-induced myocardial preconditioning

Abstract

Introduction: Anesthetic-induced preconditioning (AIP) is known to elicit cardioprotective effects that are mediated at least in part by activation of the kinases AMPK and PKCε as well as by inhibition of JNK. Recent data demonstrated that the pleiotropic cytokine macrophage migration inhibitory factor (MIF) provides cardioprotection through activation and/or inhibition of kinases that are also known to mediate effects of AIP. Therefore, we hypothesized that MIF could play a key role in the AIP response.

Methods: Cardiomyocytes were isolated from rats and subjected to isoflurane preconditioning (4 h; 1.5 vol. %). Subsequently, MIF secretion and alterations in the activation levels of protective kinases were compared to a control group that was exposed to ambient air conditions. MIF secretion was quantified by ELISA and AIP-induced activation of protein kinases was assessed by Western blotting of cardiomyocyte lysates after isoflurane treatment.

Results: In cardiomyocytes, preconditioning with isoflurane resulted in a significantly elevated secretion of MIF that followed a biphasic behavior (30 min vs. baseline: p = 0.020; 24 h vs. baseline p = 0.000). Moreover, quantitative polymerase chain reaction demonstrated a significant increase in MIF mRNA expression 8 h after AIP. Of note, activation of AMPK and PKCε coincided with the observed peaks in MIF secretion and differed significantly from baseline.

Conclusions: These results suggest that the pleiotropic mediator MIF is involved in anesthetic-induced preconditioning of cardiomyocytes through stimulation of the protective kinases AMPK and PKCε.

Figure 1. Protocol for experimental studies.

All isolated cardiomyocytes were subjected to either preconditioning stimulus of isoflurane or room air for 4

Figure 2. Characterization of MIF receptors by confocal microscopy.

Rat cardiomyocytes were grown in an Ibidi μ-dish for 12 days. They were fixed with paraformaldehyde, permeabilised with Triton X-100 and stained with antibodies against CD74 (A) and CXCR4 (B) and fluorescently labeled secondary antibodies (Alexa 633). Nuclei were stained with Hoechst33342, a cell membrane permeable, DNA-binding fluorophore staining nuclei of cells with blue fluorescence.

Figure 3. Measurement of survival.

Cardiomyocytes from sham group and after preconditioning were subjected to hypoxia (5 hours) during the first and second window of protection in order to compare the survival between both groups and demonstrate the effect of preconditioning. Cell survival is given in percent of seeded cells. Whereas unpreconditioned cells were diminished to 48±2%, isoflurane preconditioned cells reached a survival ratio of 65±5% (n = 6 per group, p = 0.0061).

Figure 4. MIF secretion after anesthetic induced preconditioning.

Comparison of MIF secretion in the supernatant of isolated cardiomyocytes after isoflurane or control treatment. 12 days after isolation cardiomyocytes were placed in an incubation chamber and treated for 4% isoflurane or normal room air as control (sham group). At the indicated time points, cell-free supernatants were collected and MIF concentrations were measured using a modified mouse/human combination-ELISA . Data represented means ± SEM of at least 3 independent experiments. The shaded area indicates duration of preconditioning. *(**) = p<0.05(0.01) vs. baseline, §(§§) = p<0.05(0.01) vs. sham group.

Figure 5. Isoflurane preconditioning stimulates MIF mRNA expression in cardiomyocytes.

Cardiomyocytes were treated with isoflurane (4 hours; 1.5%) for preconditioning and consecutively compared to cardiomyocytes that were exhibited to room air in the incubation chamber. 8 hours after the treatment, mRNA was isolated and qPCR was performed. mRNA levels were normalized to GAPDH. Data represented means ± SEM of at least 3 independent experiments. *(**)  =  p<0.05 (0.01) vs. sham group.

Figure 6. Increased MIF levels are associated with activation of protective kinases PKCε and AMPK.

Relative activation of protein kinases was assessed at the indicated time points after preconditioning with 1.5% isoflurane for 4 h by western blotting. Band intensities were normalized to actin. Data shows a biphasic activation for PKCε (A) and AMPK (B) that conforms with the elevated MIF levels after preconditioning. No effect was measured for the activation of either JNK1 (C) or JNK2 (D). Data represented means ± SEM of at least 3 independent experiments. *(**) = p<0.05(0.01) vs. baseline; §(§§) = p<0.05(0.01) vs. sham group

Figure 7. Recombinant MIF activates AMPK and PKCε whereas it inhibits phosphorylation of JNK1/2.

Cardiomyocytes were incubated with 100/ml recombinant mouseMIF for the indicated time points. Relative activation PKCε (A) AMPK (B), JNK1 (C) and JNK2 (D) was assessed via western blotting. Band intensities were normalized to actin. Data represented means ± SEM of at least 3 independent experiments. *(**)  =  p<0.05 (0.01) vs. baseline.