title: 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 globally distributed hereditary red blood cell (RBC) disorder. One of the hallmarks of SCD is the presence of circulating dense RBCs, which are important in SCD-related clinical manifestations. In human dense sickle cells, we found reduced calpastatin activity and protein expression compared to either healthy RBCs or unfractionated sickle cells, suggesting an imbalance between activator and inhibitor of calpain-1 in favor of activator in dense sickle cells. Calpain-1 is a nonlysosomal cysteine proteinase that modulates multiple cell functions through the selective cleavage of proteins. To investigate the relevance of this observation in vivo, we evaluated the effects of the orally active inhibitor of calpain-1, BDA-410 (30 mg/kg/d), on RBCs from SAD mice, a mouse model for SCD. In SAD mice, BDA-410 improved RBC morphology, reduced RBC density (D(20); from 1106 ± 0.001 to 1100 ± 0.001 g/ml; P<0.05) and increased RBC-K(+) content (from 364 ± 10 to 429 ± 12.3 mmol/kg Hb; P<0.05), markedly reduced the activity of the Ca(2+)-activated K(+)channel (Gardos channel), and decreased membrane association of peroxiredoxin-2. The inhibitory effect of calphostin C, a specific inhibitor of protein kinase C (PKC), on the Gardos channel was eliminated after BDA-410 treatment, which suggests that calpain-1 inhibition affects the PKC-dependent fraction of the Gardos channel. BDA-410 prevented hypoxia-induced RBC dehydration and K(+) loss in SAD mice. These data suggest a potential role of BDA-410 as a novel therapeutic agent for treatment of SCD.
authors.affiliation: Department of Medicine, University of Verona, Verona, Italy. lucia.defranceschi@univr.it
authors.forename: Lucia
authors.initials: L
authors.lastname: De Franceschi
authors.forename: Robert S
authors.initials: RS
authors.lastname: Franco
authors.forename: Mariarita
authors.initials: M
authors.lastname: Bertoldi
authors.forename: Carlo
authors.initials: C
authors.lastname: Brugnara
authors.forename: Alessandro
authors.initials: A
authors.lastname: Matté
authors.forename: Angela
authors.initials: A
authors.lastname: Siciliano
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Wieschhaus
authors.forename: Athar H
authors.initials: AH
authors.lastname: Chishti
authors.forename: Clinton H
authors.initials: CH
authors.lastname: Joiner
mesh_terms: D000755:Anemia, Sickle Cell / Q000097:blood* / Q000188:drug therapy* / Q000235:genetics; D000818:Animals; D002135:Calcium-Binding Proteins / Q000097:blood; D002154:Calpain / Q000037:antagonists & inhibitors* / Q000097:blood; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003681:Dehydration / Q000097:blood / Q000517:prevention & control; D004195:Disease Models, Animal; D004913:Erythrocytes, Abnormal / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000037:antagonists & inhibitors / Q000097:blood*; D051379:Mice; D008817:Mice, Mutant Strains; D008822:Mice, Transgenic; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C496923:KCNN4 protein, human; C496925:Kcnn4 protein, mouse; C572617:N-1-(hydroxy(3-oxo-2-phenyl-1-cyclopropen-1-yl)methyl)-2-methylpropyl-2-benzenesulfonylamino-4-methylpentanamide; D013449:Sulfonamides; C033668:calpastatin; D002154:Calpain; C112682:Capn1 protein, mouse