Genetic inactivation of calpain-1 attenuates pain sensitivity in a humanized mouse model of sickle cell disease

Abstract

Calpain-1, a calcium-activated cysteine protease, is ubiquitously expressed in hematopoietic cells, and is known to play a functional role in a myriad of cellular processes by regulating limited cleavage of multiple substrates.1 Using a calpain-1 null model (CKO) previously generated in our laboratory, recent studies revealed a functional role of calpain-1 in IgE-dependent mast cell activation.2 Interestingly, in the Berkeley sickle mice, mast cell activation contributes to neurogenic inflammation, chronic pain, and hypoxia/reoxygenation (H/R)-evoked hyperalgesia, which were ameliorated upon treatment with mast cell inhibitor imatinib, and cannabinoids as well as nociception receptor ligand AT200. 3-5Therefore, we examined if calpain-1 contributes to chronic and/or hypoxia/reoxygenation (H/R)-evoked acute pain in sickle mice. We generated calpain-1 knockout Townes sickle (SSCKO) mice by cross-breeding HbSS-Townes sickle mice with calpain-1 KO mice. Systemic deletion of calpain-1 in Townes sickle mice 6 ameliorated chronic pain behaviors including mechanical, heat, cold, and deep tissue/musculoskeletal hyperalgesia.Calpains in mammals are encoded by 14 genes; however, two conventional calpains termed calpain-1 (m-calpain) and calpain-2 (m-calpain), with 61% amino acid identity, are highly expressed.1 Calpain-1 is activated at micromolar calcium, whereas active calpain-2 is detected at millimolar calcium concentration in vitro.1 Calpastatin, an endogenous inhibitor of both calpains, provides a regulatory mechanism for suppressing calpain activity under steady state conditions. 1 While both calpains are ubiquitously expressed, calpain-1 generally dominates in hematopoietic cells such as RBCs and platelets. In contrast, calpain-2 is more prominent in the nervous system. Sickle RBCs are known to exhibit high levels of intracellular calcium.7 Our previous study demonstrated that dense/dehydrated RBCs from patients with sickle cell disease show enhanced calpain-1 activity as measured by the calpastatin levels, and pharmacological inhibition of calpain-1 in the transgenic SAD mouse model of mild sickle cell disease reduced sickle RBC density/dehydration. 8Due to high sequence similarity between the two calpains, gene targeting and not the use of pharmacological inhibitors is preferable to elucidate the unique function of each calpain isoform in vivo. While the calpain-1 knockout mice are viable and fertile, 9 calpain-2 gene inactivation causes early embryonic lethality in mice. Following up on our recent investigation of the role of calpain-1 in the SAD mouse model of mild sickle cell disease (SCD), 8in the present study we utilized genetic inactivation of calpain-1 in the homozygous, HbSS-Townes mice with severe SCD to define the role of calpain-1 in pain sensitivity. A comprehensive breeding strategy was designed to generate viable calpain-1 knockout Townes sickle (SSCKO) mice ( Figure 1A and B), which express human but not mouse α-and b-globin genes. After 15 generations of breeding, the SSCKO mice were generated by ...

Calpain-1 regulates platelet function in a humanized mouse model of sickle cell disease

“…Townes sickle (SS) mice were obtained from the Jackson Laboratory and backcrossed with our calpain-1 knockout C57BL/6 (CKO) mice [17] to generate humanized calpain-1 knockout sickle (SSCKO) mice [26]. Humanized (AA) or wild type C57BL/6 mice were used as controls.…”

“…Townes Sickle mice offer several advantages such as: (i) it is a humanized mouse model of SCD that expresses exclusively human alpha, beta, and gamma globins, (ii) expresses 99% human sickle beta globin resulting in a severe disease phenotype with hemolytic anemia and multi-organ damage, and (iii) displays extensive RBC sickling at steady state without requiring hypoxia/reoxygenation treatment. Using established breeding and gene mapping techniques, we generated the first calpain-1 knockout Townes sickle (SSCKO) mice, which demonstrated reductions in all aspects of sickle chronic pain phenotype including mechanical hyperalgesia, sensitivity to heat and cold, and deep tissue hyperalgesia [26]. …”

“…In the present study, we tested our hypothesis that platelets from the novel SSCKO mouse model [26] would display reduced platelet activation and aggregation, consistent with our previous studies on washed platelets from calpain-1 knockout C57BL/6 (CKO) mice, which display impaired platelet aggregation and clot retraction, but normal bleeding times [17, 27]. We further hypothesized that compared to control Townes AA mice, Townes sickle (SS) mice would display thrombosis in major organs such as the liver and lungs, which might contribute to pulmonary arterial hypertension (PAH) consistent with elevated markers of platelet activation that correlate with incidence of PAH in SCD patients [28].…”

Non-Conventional Pain Management for Sickle Cell Disease

“…The chronic thermal and deep tissue hyperalgesia are completely ameliorated by the deletion of calpain-1 gene in SS mice. Thus, calpain-1 may contribute to the regulation of chronic hyperalgesia in SCD [101].…”

“…Systemic deletion of calpain-1 in Townes sickle mice ameliorated chronic pain behaviors including mechanical, heat, cold, and deep tissue/musculoskeletal hyperalgesia. The ultimate goal of any sickle cell therapy is the reduction of chronic pain and episodes of acute pain crises in patients with SCD [101]. In rat models of spinal nerve injury, inhibition of calpain-1 reduced neuropathic pain [102].…”