We spent much of our grant year optimizing the C2A domain constructs for use in our adeno-associated virus (AAV) experiments. We had previously found that placing the C2A domain of dysferlin with the adjacent Romeo epitope in tandem with C2 domains from another muscle protein allowed our newly created hybrid protein to concentrate at the triad junctions of dysferlin-null skeletal muscle, where dysferlin would normally be concentrated. This protein had the two key activities of native dysferlin that we have been studying: proper regulation of Ca2+ signaling, and good support of membrane repair. The problem we faced was with the controls, for which we had originally planned to use inactive variants of the C2A domain, such as the V67D or the A84R mutants. When placed next to the same muscle protein C2 domains, neither was expressed well in dysferlin-null muscle – not nearly as well as the wild type. As the amount of the virally expressed control protein should be approximately the same as our experimental one containing C2A and Romeo, we decided to use virus expressing the other C2 domains alone. An added advantage of this approach is that this hybrid protein also concentrates at the triad junctions but it is inactive both in stabilizing Ca2+ signaling and in promoting membrane repair (the latter was assayed by Noah Weisleder and his colleagues at OSU in Columbus, OH). We can now express the experimental and control proteins as mCrimson fusion proteins and have provided the plasmid constructs to a commercial facility for production of the AAV.
In a separate series of experiments, we began to investigate the relationship between the abnormal Ca2+ signaling that occurs in dysferlin-null muscle after a mild injury, which is mediated by a process call Ca2+-induced Ca2+ release, or CICR, and the normal signaling seen in healthy muscle, even after injury, which is mediated by what is called voltage-induced Ca2+ release, or VICR. CICR is a phenomenon that underlies many diseases of muscle and it is generally thought that suppressing it, when it occurs, can improve muscle health. Our results so far suggest that VICR and CICR may be interconvertible and that their interconversion may be mediated by enzymes that add or remove phosphate residues from a protein or proteins at the triad junction. We are trying to identify these enzymes now, as drugs that affect their activity may be useful in suppressing some of the abnormal Ca2+ signaling that is typical of dysferlinopathic muscle and thereby improve the length of time over which dysferlinopathic muscle can remain functional. We are currently seeking separate funding for this aspect of our research.