Calpain-dependent degradation of cytoskeletal proteins as a key mechanism for a reduction in intrinsic passive stiffness of unloaded rat postural muscle.

In mammals, prolonged mechanical unloading results in a significant decrease in passive stiffness of postural muscles. The nature of this phenomenon remains unclear. The aim of the present study was to investigate possible causes for a reduction in rat soleus passive stiffness after 7 and 14 days of unloading (hindlimb suspension, HS). We hypothesized that HS-induced decrease in passive stiffness would be associated with calpain-dependent degradation of cytoskeletal proteins or a decrease in actomyosin interaction. Wistar rats were subjected to HS for 7 and 14 days with or without PD150606 (calpain inhibitor) treatment. Soleus muscles were subjected to biochemical analysis and ex vivo measurements of passive tension with or without blebbistatin treatment (an inhibitor of actomyosin interactions). Passive tension of isolated soleus muscle was significantly reduced after 7- and 14-day HS compared to the control values. PD150606 treatment during 7- and 14-day HS induced an increase in alpha-actinin-2 and -3, desmin contents compared to control, partly prevented a decrease in intact titin (T1) content, and prevented a decrease in soleus passive tension. Incubation of soleus muscle with blebbistatin did not affect HS-induced reductions in specific passive tension in soleus muscle. Our study suggests that calpain-dependent breakdown of cytoskeletal proteins, but not a change in actomyosin interaction, significantly contributes to unloading-induced reductions in intrinsic passive stiffness of rat soleus muscle.

Simvastatin alleviates liver function injury in ApoE knockout mice by inhibiting calpain activity / 中国药理学通报

Aim To study the protective effect of simvastatin(Sim)on liver function injury in apolipoprotein E gene knockout(ApoE KO)mice fed with high-fat diet and the underlying mechanism.Methods Twenty-four 8-week-old male ApoE KO mice were randomly divided into ApoE KO group,ApoE KO+Sim group and ApoE KO+PD150606 group.The contents of total cholesterol(TC)and triglyceride(TG)in serum and liver,and the activities of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)in serum were measured.The contents of malondialdehyde(MDA)and reactive oxygen species(ROS)and the activity of superoxide dismutase(SOD)in liver were determined.The contents of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)and the activity of calpain in liver were examined.Results Compared with C57 group,ApoE KO group showed significant increase in the contents of TC and TG in both serum and liver.In addition,the activities of AST and ALT in serum and the contents of MDA and ROS in liver significantly increased,while SOD activity in liver decreased in ApoE KO group.The contents of TNF-α and IL-6 and the activity of calpain in liver significantly increased.Compared with ApoE KO group,Sim group had no significant effects on TC and TG,while reduced the activities of AST and ALT,decreased the contents of MDA and ROS,increased the activity of SOD and decreased the contents of TNF-α and IL-6 as well as the activity of calpain in liver.PD,the calpain inhibitor,had the similar effects with Sim regarding the above mentioned parameters.Conclusions Sim improved the liver function injury of ApoE KO mice,which might be related to the inhibition of calpain activity,subsequently increasing the antioxidant capacity and reducing the inflammatory response.

Calpain Activation Is the Major Cause of Cell Death in Photoreceptors Expressing a Rhodopsin Misfolding Mutation.

The majority of mutations in rhodopsin (RHO) cause misfolding of the protein and has been linked to degeneration of photoreceptor cells in the retina. A lot of attention has been set on targeting ER stress for the development of new therapies for inherited retinal degeneration caused by mutations in the RHO gene. Nevertheless, the cell death pathway activated by RHO misfolded protein is still debated. In this study, we analyzed the retina of the knock-in mouse expressing the P23H misfolded mutant RHO. We found persistent unfolded protein response (UPR) during degeneration. Interestingly, long-term stimulation of the PERK branch of ER stress had a protective effect by phosphorylating nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor, associated with antioxidant responses. Otherwise, we provide evidence that increased intracellular calcium and activation of calpains strongly correlated with rod photoreceptor cell death. By blocking calpain activity, we significantly decreased the activation of caspase-7 and apoptosis-inducing factor (AIF), two cell death effectors, and cell demise, and effectively protected the retina from degeneration caused by the P23H dominant mutation in RHO.

Calpain modulates fear memory consolidation, retrieval and reconsolidation in the hippocampus.

It has been proposed that long-lasting changes in dendritic spines provide a physical correlate for memory formation and maintenance. Spine size and shape are highly plastic, controlled by actin polymerization/depolymerization cycles. This actin dynamics are regulated by proteins such as calpain, a calcium-dependent cysteine protease that cleaves the structural cytoskeleton proteins and other targets involved in synaptic plasticity. Here, we tested whether the pharmacological inhibition of calpain in the dorsal hippocampus affects memory consolidation, retrieval and reconsolidation in rats trained in contextual fear conditioning. We first found that post-training infusion of the calpain inhibitor PD150606 impaired long-term memory consolidation, but not short-term memory. Next, we showed that pre-test infusion of the calpain inhibitor hindered memory retrieval. Finally, blocking calpain activity after memory reactivation disrupted reconsolidation. Taken together, our results show that calpain play an essential role in the hippocampus by enabling memory formation, expression and reconsolidation.

PD150606 protects against ischemia/reperfusion injury by preventing µ-calpain-induced mitochondrial apoptosis.

Calpain plays an important role in myocardial ischemia/reperfusion (I/R) injury. PD150606, a nonpeptide, cell-permeable and noncompetitive calpain inhibitor, has been shown to have protective properties in ischemic disease. The aims of the present study were to investigate whether PD150606 could alleviate myocardial I/R injury and to examine the possible mechanisms involved. The I/R model was established in vivo in C57BL/6 mice and in vitro using neonatal mouse cardiomyocytes, respectively. To evaluate the protective effects of PD150606 on I/R injury, we measured the myocardial infarct area, apoptosis, and expression of cleaved caspase-3. We also investigated the underlying mechanisms by examining mitochondrial function as reflected by the ATP concentration, translocation of cytochrome c, dynamics of mPTP opening, and membrane potential (ΔΨm), coupled with calpain activity. Pretreatment with PD150606 significantly reduced the infarct area and apoptosis caused by I/R. PD150606 pretreatment also reduced mitochondrial dysfunction by inhibiting calpain activation. Moreover, we found that µ-calpain is the main contributor to I/R-induced calpain activation. Knockdown of µ-calpain with siRNA significantly reversed calpain activation, mitochondrial dysfunction, and cardiomyocyte apoptosis caused by I/R in vitro. Our results suggest that PD150606 may protect against I/R injury via preventing µ-calpain-induced mitochondrial apoptosis.

Allosteric inhibitors of calpains: Reevaluating inhibition by PD150606 and LSEAL.

BACKGROUND: The mercaptoacrylate calpain inhibitor, PD150606, has been shown by X-ray crystallography to bind to a hydrophobic groove in the enzyme's penta-EF-hand domains far away from the catalytic cleft and has been previously described as an uncompetitive inhibitor of calpains. The penta-peptide LSEAL has been reported to be an inhibitor of calpain and was predicted to bind in the same hydrophobic groove. The X-ray crystal structure of calpain-2 bound to its endogenous calpain inhibitor, calpastatin, shows that calpastatin also binds to the hydrophobic grooves in the two penta-EF-hand domains, but its inhibitory domain binds to the protease core domains and blocks the active site cleft directly. METHODS: The mechanisms of inhibition by PD150606 and LSEAL were investigated using steady-state kinetics of cleavage of a fluorogenic substrate by calpain-2 and the protease core of calpain1, as well as by examining the inhibition of casein hydrolysis by calpain and the autoproteolysis of calpain. RESULTS: PD150606 inhibits both full-length calpain-2 and the protease core of calpain-1 with an apparent noncompetitive kinetic model. The penta-peptide LSEAL failed to inhibit either whole calpain or its protease core in vitro. CONCLUSIONS: PD150606 cannot inhibit cleavage by calpain-2 of small substrates via binding to the penta-EF-hand domain. GENERAL SIGNIFICANCE: PD150606 is often described as a calpain-specific inhibitor due to its ability to target the penta-EF-hand domains of calpain, but we show that it must be acting at a site on the protease core domain instead.