Pharmacological inhibition of calpain-1 prevents red cell dehydration and reduces Gardos channel activity in a mouse model of sickle cell disease

ABSTRACT Sickle cell disease (SCD) is a globallydistributed hereditary red blood cell (RBC) disorder.One of the hallmarks of SCD is the presence ofcirculating dense RBCs, which are important in SCDrelatedclinical manifestations. In human dense sicklecells, we found reduced calpastatin activity and proteinexpression compared to either healthy RBCs or unfractionatedsickle cells, suggesting an imbalance betweenactivator and inhibitor of calpain-1 in favor of activatorin dense sickle cells. Calpain-1 is a nonlysosomal cysteineproteinase that modulates multiple cell functionsthrough the selective cleavage of proteins. To investigatethe relevance of this observation in vivo, weevaluated the effects of the orally active inhibitor ofcalpain-1, BDA-410 (30 mg/kg/d), on RBCs from SADmice, a mouse model for SCD. In SAD mice, BDA-410improved RBC morphology, reduced RBC density (D20;from 11060.001 to 11000.001 g/ml; P<0.05) andincreased RBC-K content (from 36410 to 42912.3mmol/kg Hb; P<0.05), markedly reduced the activityof the Ca2-activated Kchannel (Gardos channel), anddecreased membrane association of peroxiredoxin-2.The inhibitory effect of calphostin C, a specific inhibitorof protein kinase C (PKC), on the Gardos channelwas eliminated after BDA-410 treatment, which suggeststhat calpain-1 inhibition affects the PKC-dependentfraction of the Gardos channel. BDA-410 preventedhypoxia-induced RBC dehydration and K lossin SAD mice. These data suggest a potential role ofBDA-410 as a novel therapeutic agent for treatment ofSCD.