Attenuation of skeletal muscle reperfusion injury with intravenous 12 amino acid peptides that bind to pathogenic IgM

Background. The injury sustained by reperfused skeletal muscle is inflammatory and is initiated by binding of pre-formed IgM to involved tissue, followed by local complement activation and further inflammation. A clone of natural IgM has been described that initiates this injury, suggesting that specific antigens are exposed on ischemic tissues that act as ligands for this pathogenic antibody. In these experiments, we examine the Properties of short peptide sequences, and their homologues, that bind to the antigen-combining site of this pathogenic IgM clone. Methods. A 12-mer phage display library was biopanned with the pathogenic IgM clone and then negatively selected against an inactive natural IgM clone. All 8 clones that bound specifically to the pathogenic IgM had closely related amino acid sequences. P8 is the clone that bound most avidly. Tissue lysates from ischemic tissue were reacted with pathogenic IgM, and immune complexes isolated and analyzed on SDS-PAGE. Bands were excised and sequenced, identifying non-muscle myosin as the protein reacting with pathogenic antibody in ischemic gut and glycogen phosphorylase as the counterpart in ischemic skeletal muscle. Both proteins contain sequence homologous to P8; N2 and GP1 are the natural 12-mers homologues that are contained within non-muscle myosin and glycogen phosphorylase, respectively. Wild-type C57/B16 mice, divided into groups receiving saline, P8, N2, GP1, or a random peptide at the start of the experiment, were subjected to 2 hours of tourniquet induced hind limb ischemia and 3 hours of reperfusion. Muscle was assessed for injury with histology and for immune activation with histochemistry. Results. Intravenous administration of P8, N2, and GP1 led to significant attenuation of muscle injury (13 +/- 1.8 injuredfibers/50 counted, 12 +/- 0.81, 8.0 +/- 0.73 respectively) after reperfusion injury compared to animals receiving saline (26 +/- 2.3) or the same mass of a random peptide (22 2.3), P less than .05. This level of protection from injury is comparable to that seen in the absence of antibody altogether. As well, P8-treated animals exhibited a marked decrease in deposition of IgM (as well as C3) in comparison to saline treated controls. Conclusions. Specific peptide blockade of an injury-inducing IgM clone decreased the local consequences, of skeletal muscle ischemia/reperfusion injury in wild-type animals that have the full repertoire of IgM specificities. This indicates that the antibodies that initiate reperfusion injury have specificity only for P8-related antigens. This could also indicate that the variety of relevant ischemic antigens is quite restricted.