Abundance of calpain and aggrecan-cleavage products of calpain in degenerated human intervertebral discs

Objective

To assess the expression of calpains and calpain-induced aggrecan fragmentation in early and advanced stages of degeneration of human intervertebral discs (IVDs).

Design

Disc tissue samples of 55 patients (mean age, 51.2 ± 22.3 years) who underwent intervertebral fusion were divided into groups with early and advanced degeneration based on the Thompson magnetic resonance imaging (MRI) scale. In advanced degeneration group, five patients (mean age, 35.5 ± 11.4 years) of lumbar disc herniation (LDH) were included. Protein levels of m- and μ-calpains and their inhibitor calpastatin were assayed, and immunohistochemical techniques were used to localize and quantify the production of the enzymes. To investigate calpain activity, we assayed purified aggrecan fragmentation in disc tissue by Western blotting and immunohistochemistry with VPGVA antibody, which recognizes the m-calpain generated neo-epitope GVA.

Results

Discs at early stages of degeneration expressed low levels of m- and μ-calpains and calpastatin, and few cells expressed degenerative enzymes. At more advanced stages of degeneration, the expression and number of cells immunopositive for m-calpain, μ-calpain and calpastatin were significantly higher. Further finding showed that anti-GVA-reactive aggrecan fragments were significantly higher in discs at advanced compared with early stages of degeneration. Herniated disc samples showed stronger expression and more cells immunopositive for calpains, calpastatin and GVA in the nucleus pulposus than in the annulus fibrous.

Conclusions

The expression of calpains, together with m-calpain-induced degradation products of extracellular matrix, was correlated with the degree of disc degeneration in human IVD tissue. These findings suggest that calpains may be involved in IVD degeneration via proteoglycan (PG) cleavage.     

Run-down of the cardiac L-type Ca2+ channel: partial restoration of channel activity in cell-free patches by calpastatin

 We have found previously that run-down of cardiac Ca²⁺ channels in cell-free patches is reversed by cytoplasm plus adenosine triphosphate (ATP). Characterization of the factor in cytoplasm revealed that it is likely to be calpastatin (CS), an endogenous inhibitor of calpain (Ca²⁺-activated neutral protease). We therefore investigated the possible restoring effect of CS obtained from various tissues (activity 1.3–23 U/ml) on Ca²⁺ channel activity after run-down in inside-out patches. Although CS from porcine erythrocytes (plus 3 mM ATP) had only a minimal effect in restoring channel activity (to 4% of the control level recorded before the run-down), CS from porcine heart restored channel activity to 19% of control. The product of recombinant complementary deoxyribonucleic acid (cDNA) of human heart CS, a membrane-bound CS partially purified from bovine heart and CS from rabbit skeletal muscle (Sigma) restored channel activity to 28%, 23% and 10% of control levels, respectively. These results suggest that tissue-type CS, but not erythrocyte-type (truncated) CS, seems to have an effect on the cardiac Ca²⁺ channel to maintain its activity. Purified CS had relatively small effects compared to that of crude cytoplasm, implying that some other factor(s) might contribute also to the regulation of Ca²⁺ channel activity. Received: 26 March 1997 / Received after revision: 8 August 1997 / Accepted: 5 September 1997     

Calpastatin and nucleotides stabilize cardiac calcium channel activity in excised patches

The activity of single L-type Ca²⁺ channels is rapidly lost (run-down) when contact between the membrane and cytosol is interrupted. We have now achieved the stabilization of cardiac Ca²⁺ channel activity of guinea-pig ventricular myocytes by using either cytosol or defined components added to excised patches. The endogenous protease inhibitor, calpastatin, together with nucleotides, ATP + GTP, was found to prevent rundown as effectively as cardiac cytosolic solution. These results suggest the involvement of proteolysis by calpain in run-down of channel activity and enable the study of cardiac Ca²⁺ channel regulation with free access to both sides of the membrane.     

Calpain in the pathophysiology of spinal cord injury: neuroprotection with calpain inhibitors

Spinal cord injury (SCI) evokes an increase in intracellular free Ca²⁺ level resulting in activation of calpain, a Ca²⁺-dependent cysteine protease, which cleaves many cytoskeletal and myelin proteins. Calpain is widely expressed in the central nervous system (CNS) and regulated by calpastatin, an endogenous calpain-specific inhibitor. Calpastatin degraded by overactivation of calpain after SCI may lose its regulatory efficiency. Evidence accumulated over the years indicates that uncontrolled calpain activity mediates the degradation of many cytoskeletal and membrane proteins in the course of neuronal death and contributes to the pathophysiology of SCI. Cleavage of the key cytoskeletal and membrane proteins by calpain is an irreversible process that perturbs the integrity and stability of CNS cells leading to cell death. Calpain in conjunction with caspases, most notably caspase-3, can cause apoptosis of the CNS cells following trauma. Aberrant Ca²⁺ homeostasis following SCI inevitably activates calpain, which has been shown to play a crucial role in the pathophysiology of SCI. Therefore, calpain appears to be a potential therapeutic target in SCI. Substantial research effort has been focused upon the development of highly specific inhibitors of calpain and caspase-3 for therapeutic applications. Administration of cell permeable and specific inhibitors of calpain and caspase-3 in experimental animal models of SCI has provided significant neuroprotection, raising the hope that humans suffering from SCI may be treated with these inhibitors in the near future.     

Relatively low levels of calpain expression in juvenile rat correlate with less neuronal apoptosis after spinal cord injury

Approximately 5% of spinal cord injuries in the US occur in patients younger than 16 years. These young patients have an increased mortality within the 24 h after trauma but have a greater capacity for functional recovery than adults, suggesting age-related differences in injury tolerance. Unfortunately, the response of the developing cord to secondary injury has not been thoroughly investigated. Calpain, a Ca(2+)-dependent protease, has been implicated in the pathogenesis of spinal cord injury (SCI) in rats. Our current investigation revealed that following SCI, calpain upregulation was qualitatively less in the 21-day-old rats than in adult rats, as shown by immunofluorescent labeling. Decreased levels of TUNEL+ neurons were also noted in juvenile rat spinal cord, indicating that the developing cord may have an increased resistance to injury.     

钙蛋白酶抑制蛋白转基因小鼠的构建和鉴定

目的 将人的钙蛋白酶抑制蛋白（calpastatin,CAST）外源基因整合到C57BL/6J小鼠中,构建高表达CAST的转基因小鼠模型。方法 利用Gateway技术构建pRP.EX3d-EF1A-CAST-IRES-eGFP载体,回收片段后通过显微注射法将目的基因片段注入到C57BL/6J小鼠受精卵中,将其胚胎移植至同期发情的假孕受体母鼠输卵管内获得子代小鼠。采用PCR方法鉴定出阳性的转基因小鼠,确定首建鼠,通过与C57BL/6J小鼠回交后互交数代建系。利用RT-PCR和Western blotting方法检测CAST基因和蛋白在各组织中的表达情况。结果 将90枚注射受精卵移植到3只假孕鼠中,3只均怀孕,移植成功率100%,产下23只子鼠,经PCR鉴定得到2只转基因阳性首建鼠,阳性率为9%。子代小鼠进行RT-PCR检查显示,CAST基因在转基因小鼠的心、肝、脾、肺、肾、脑和骨骼肌中均有表达;Western blotting检查显示,CAST蛋白表达在转基因小鼠中显著高于同窝阴性小鼠。结论 通过显微注射法成功构建CAST高表达的转基因小鼠,为进一步研究CAST奠定了良好的模型基础。     

Calpain inhibition attenuates right ventricular contractile dysfunction after acute pressure overload

Right ventricular contractile failure from acute RV pressure overload is an important cause of morbidity and mortality, but the mechanism of RV failure in this setting is incompletely defined. We hypothesized that RV dysfunction from acute RV pressure overload is, in part, due to activation of calpain, and that calpain inhibition would therefore attenuate RV dysfunction. Anesthetized, open chest pigs were treated with the calpain inhibitor MDL-28170 or with inactive vehicle, and then subjected to acute RV pressure overload for 90 min. RV contractile function was assessed by the regional Frank-Starling relation. RV myocardial tissue was analyzed for evidence of calpain activation and calpain-mediated proteolysis. RV pressure overload caused severe contractile dysfunction, along with significant alterations in the endogenous calpain inhibitor calpastatin typical of calpain activation. MDL-28170 attenuated RV free wall dysfunction by more than 50%. However, there were no differences in degradation of spectrin, desmin, troponin-I or SERCA2 between SHAM operated pigs and pigs subjected to acute RV pressure overload, or between vehicle and MDL-28170 treated pigs. Acute RV pressure overload causes calpain activation, and RV contractile dysfunction from acute RV pressure overload is attenuated by the calpain inhibitor MDL-28170; however, the effect is not explained by inhibition of calpain-mediated degradation of spectrin, desmin, troponin-I or SERCA2. Because this is the first report of any agent that can directly attenuate RV contractile dysfunction in acute RV pressure overload, further investigation of the mechanism of action of MDL-28170 in this setting is warranted.