ANKRD1 specifically binds CASQ2 in heart extracts and both proteins are co-enriched in piglet cardiac Purkinje cells

It has been suggested that the cardiac ankyrin repeat domain 1 protein (ANKRD1), also known as CARP, can play a pathophysiological role in the contractile responsiveness of myocardium. Here, we study the potential functional roles of ANKRD1 by searching for endogenous cardiac proteins that interact preferentially with ANKRD1 in the heart-tissue extract from neonatal piglets, using non-biased pull-down approaches. These approaches identified, for the first time, a selective interaction between ANKRD1 and endogenous cardiac calsequestrin-2 (CASQ2) that is important for Ca2+ release and excitation-contraction coupling. Blot-overlay and co-immunoprecipitation assays provided further confirmation of the direct and specific interaction between the two proteins. Mapping of the peptides involved in the interaction revealed five non-overlapping binding sequences for CASQ2 on ANKRD1, as well as, three binding peptides for ANKRD1 in CASQ2. For the first time, we show by immunohistochemistry that endogenous ANKRD1 and CASQ2 are co-enriched in piglet cardiac Purkinje cells. Collectively, the results provide the first sing of a possible functional interaction between ANKRD1 and CASQ2 and suggest a potentially novel role for both proteins in cardiac Purkinje fibers.