Genetic Deletion of Calpain-1 Improves Reticulocyte Deformability in a Mouse Model of Severe Sickle Cell Disease

Introduction: Sickle cell disease (SCD) is caused by the substitution of a valine residue for glutamic acid in the β-globin chain of hemoglobin. The resulting sickle hemoglobin rapidly polymerizes upon deoxygenation, forming sickle RBCs (sRBCs) with reduced deformability. Less deformable sRBCs have an impaired ability to navigate the microvasculature, thereby potentiating vaso-occlusive crises, the hallmark of SCD. Deoxygenation-mediated sickling also induces influx of calcium leading to calpain-1 mediated RBC dehydration via the Gardos channel. Calpains are calcium-activated cysteine proteases involved in numerous cellular processes. In mammals, calpain-1 and calpain-2 are the dominantly expressed isoforms, along with the endogenous inhibitor, calpastatin. Previously, we have shown that genetic deletion of calpain-1 improves RBC deformability in C57BL/6 mice (Wieschhaus et al, Biochem J 2012). Similarly, pharmacological inhibition of calpain-1 prevents dehydration of sRBCs in the SAD model of mild SCD (De Franceschi, et al, FASEB J 2013). Here, our objective is to determine whether genetic deletion of calpain-1 in the HbSS-Townes mice – a severe SCD mouse model, improves RBC deformability in both normoxic and hypoxic conditions.

Methods: Transgenic SCD mice (HbSS-Townes) expressing >99% human HbS were backcrossed with calpain-1 knockout (CKO) mice to obtain sickle mice lacking calpain-1 (HbSS-Townes-CKO). Casein zymography was performed to confirm the complete loss of calpain-1 activity in the RBCs of the HbSS-Townes-CKO mice. Thiazole orange-based FACS revealed that HbSS-Townes and HbSS-Townes-CKO mice express ~60% reticulocytes. The elevated reticulocyte count prompted us to evaluate the effect of calpain-1 deficiency on reticulocyte deformability. We utilized a microfluidic-based flow cytometer to assess single reticulocyte deformability following thiazole orange staining upon treatment with calcium ionophore (A23187) and CaCl₂. We further probed the molecular mechanisms of RBC deformability by assessing calcium-induced proteolysis of RBC cytoskeletal proteins. RBCs were harvested from HbSS-Townes and HbSS-Townes-CKO mice, treated with A23187 and CaCl₂ to activate calpain-1, and subjected to RBC ghosts preparation and gel electrophoresis. Cytoskeletal proteins were visualized and quantified. Guided by our previous findings that calpain-1 knockout (CKO) mice exhibit a clot retraction defect (Kuchay, et al, MCB 2007), we harvested un-coagulated blood from HbSS-Townes and HbSS-Townes-CKO mice, and assessed their clot formation phenotype in calcium-free RPMI medium at different time intervals.

Results: Casein zymography revealed a complete loss of calpain-1 enzyme activity (indicated by lack of casein cleavage) in the HbSS-Townes-CKO mice. Furthermore, reticulocytes from HbSS-Townes-CKO mice are more deformable than their HbSS-Townes counterparts under steady state as well as upon treatment with calcium ionophore and CaCl₂. An improvement in sickle reticulocyte deformability has significant potential to prevent vaso-occlusive crisis under situations of hypoxia-induced ischemia reperfusion injury – a hypothesis that is currently being tested. In addition, our preliminary results show that RBC ghosts from HbSS-Townes mice exhibit a transglutaminase-mediated cross-linking defect in the cytoskeleton that is restored in the HbSS-Townes-CKO mice. This finding has significant implications for an increased in vivo RBC life span of HbSS-Townes-CKO mice. Finally, we found that HbSS-Townes-CKO mice display a whole blood clot retraction defect, similar to our previous findings on platelets harvested from calpain-1 knockout (CKO) mice. Our findings showing improved sickle cell deformability and clot retraction phenotype are consistent with recently published findings demonstrating an interaction between RBCs and platelets in clot formation and in vivo thrombosis. To test this intriguing hypothesis, we are currently evaluating whether HbSS-Townes-CKO mice exhibit reduced thrombus formation upon laser-induced in vivo thrombosis. Together, our results report the first mouse model of severe sickle cell disease lacking calpain-1 activity, and unveil a new role for calpain-1 in the multi-step process regulating SCD pathogenesis. These findings might serve as a basis for exploring calpain-1 inhibition as a therapeutic target for SCD.