Acute endoplasmic reticulum stress-induced mitochondria respiratory chain damage: The role of activated calpains.

The induction of acute endoplasmic reticulum (ER) stress damages the electron transport chain (ETC) in cardiac mitochondria. Activation of mitochondria-localized calpain 1 (CPN1) and calpain 2 (CPN2) impairs the ETC in pathological conditions, including aging and ischemia-reperfusion in settings where ER stress is increased. We asked if the activation of calpains causes the damage to the ETC during ER stress. Control littermate and CPNS1 (calpain small regulatory subunit 1) deletion mice were used in the current study. CPNS1 is an essential subunit required to maintain CPN1 and CPN2 activities, and deletion of CPNS1 prevents their activation. Tunicamycin (TUNI, 0.4 mg/kg) was used to induce ER stress in C57BL/6 mice. Cardiac mitochondria were isolated after 72 h of TUNI treatment. ER stress was increased in both control littermate and CPNS1 deletion mice with TUNI treatment. The TUNI treatment activated both cytosolic and mitochondrial CPN1 and 2 (CPN1/2) in control but not in CPNS1 deletion mice. TUNI treatment led to decreased oxidative phosphorylation and complex I activity in control but not in CPNS1 deletion mice compared to vehicle. The contents of complex I subunits, including NDUFV2 and ND5, were decreased in control but not in CPNS1 deletion mice. TUNI treatment also led to decreased oxidation through cytochrome oxidase (COX) only in control mice. Proteomic study showed that subunit 2 of COX was decreased in control but not in CPNS1 deletion mice. Our results provide a direct link between activation of CPN1/2 and complex I and COX damage during acute ER stress.

Otud6b induces pulmonary arterial hypertension by mediating the Calpain-1/HIF-1α signaling pathway.

Pulmonary hypertension (PAH) is a cardiopulmonary disease in which pulmonary artery pressure continues to rise, leading to right heart failure and death. Otud6b is a member of the ubiquitin family and is involved in cell proliferation, apoptosis and inflammation. The aim of this study was to understand the role and mechanism of Otud6b in PAH. C57BL/6 and Calpain-1 knockout (KO) mice were exposed to a PAH model induced by 10% oxygen. Human pulmonary artery endothelial cells (HPACEs) and human pulmonary artery smooth muscle cells (HPASMCs) were exposed to 3% oxygen to establish an in vitro model. Proteomics was used to determine the role of Otud6b and its relationship to Calpain-1/HIF-1α signaling. The increased expression of Otud6b is associated with the progression of PAH. ROtud6b activates Otud6b, induces HIF-1α activation, increases the production of ET-1 and VEGF, and further aggravates endothelial injury. Reducing Otud6b expression by tracheal infusion of siOtud6b has the opposite effect, improving hemodynamic and cardiac response to PAH, reducing the release of Calpain-1 and HIF-1α, and eliminating the pro-inflammatory and apoptotic effects of Otud6b. At the same time, we also found that blocking Calpain-1 reduced the effect of Otud6b on HIF-1α, and inhibiting HIF-1α reduced the expression of Calpain-1 and Otud6b. Our study shows that increased Otud6b expression during hypoxia promotes the development of PAH models through a positive feedback loop between HIF-1α and Calpain-1. Therefore, we use Otud6b as a biomarker of PAH severity, and regulating Otud6b expression may be an effective target for the treatment of PAH.

Metformin Pre-Treatment as a Means of Mitigating Disuse-Induced Rat Soleus Muscle Wasting.

Currently, no ideal treatment exists to combat skeletal muscle disuse-induced atrophy and loss of strength. Because the activity of AMP-activated protein kinase (AMPK) in rat soleus muscle is suppressed at the early stages of disuse, we hypothesized that pre-treatment of rats with metformin (an AMPK activator) would exert beneficial effects on skeletal muscle during disuse. Muscle disuse was performed via hindlimb suspension (HS). Wistar rats were divided into four groups: (1) control (C), (2) control + metformin for 10 days (C+Met), (3) HS for 7 days (HS), (4) metformin treatment for 7 days before HS and during the first 3 days of 1-week HS (HS+Met). Anabolic and catabolic markers were assessed using WB and RT-PCR. Treatment with metformin partly prevented an HS-induced decrease in rat soleus weight and size of slow-twitch fibers. Metformin prevented HS-related slow-to-fast fiber transformation. Absolute soleus muscle force in the HS+Met group was increased vs. the HS group. GSK-3ß (Ser9) phosphorylation was significantly increased in the HS+Met group vs. the HS group. Metformin pre-treatment partly prevented HS-induced decrease in 18S+28S rRNA content and attenuated upregulation of calpain-1 and ubiquitin. Thus, pre-treatment of rats with metformin can ameliorate disuse-induced reductions in soleus muscle weight, the diameter of slow-type fibers, and absolute muscle strength.

An Activity-Based Nanosensor for Minimally-Invasive Measurement of Protease Activity in Traumatic Brain Injury.

Current screening and diagnostic tools for traumatic brain injury (TBI) have limitations in sensitivity and prognostication. Aberrant protease activity is a central process that drives disease progression in TBI and is associated with worsened prognosis; thus direct measurements of protease activity could provide more diagnostic information. In this study, a nanosensor is engineered to release a measurable signal into the blood and urine in response to activity from the TBI-associated protease calpain. Readouts from the nanosensor were designed to be compatible with ELISA and lateral flow assays, clinically-relevant assay modalities. In a mouse model of TBI, the nanosensor sensitivity is enhanced when ligands that target hyaluronic acid are added. In evaluation of mice with mild or severe injuries, the nanosensor identifies mild TBI with a higher sensitivity than the biomarker GFAP. This nanosensor technology allows for measurement of TBI-associated proteases without the need to directly access brain tissue, and has the potential to complement existing TBI diagnostic tools.

Changes in calpain-2 expression during glioblastoma progression predisposes tumor cells to temozolomide resistance by minimizing DNA damage and p53-dependent apoptosis.

BACKGROUND: Glioblastoma multiforme (GBM) is characterized by an unfavorable prognosis for patients affected. During standard-of-care chemotherapy using temozolomide (TMZ), tumors acquire resistance thereby causing tumor recurrence. Thus, deciphering essential molecular pathways causing TMZ resistance are of high therapeutic relevance. METHODS: Mass spectrometry based proteomics were used to study the GBM proteome. Immunohistochemistry staining of human GBM tissue for either calpain-1 or -2 was performed to locate expression of proteases. In vitro cell based assays were used to measure cell viability and survival of primary patient-derived GBM cells and established GBM cell lines after TMZ ± calpain inhibitor administration. shRNA expression knockdowns of either calpain-1 or calpain-2 were generated to study TMZ sensitivity of the specific subunits. The Comet assay and É£H2AX signal measurements were performed in order to assess the DNA damage amount and recognition. Finally, quantitative real-time PCR of target proteins was applied to differentiate between transcriptional and post-translational regulation. RESULTS: Calcium-dependent calpain proteases, in particular calpain-2, are more abundant in glioblastoma compared to normal brain and increased in patient-matched initial and recurrent glioblastomas. On the cellular level, pharmacological calpain inhibition increased the sensitivities of primary glioblastoma cells towards TMZ. A genetic knockdown of calpain-2 in U251 cells led to increased caspase-3 cleavage and sensitivity to neocarzinostatin, which rapidly induces DNA strand breakage. We hypothesize that calpain-2 causes desensitization of tumor cells against TMZ by preventing strong DNA damage and subsequent apoptosis via post-translational TP53 inhibition. Indeed, proteomic comparison of U251 control vs. U251 calpain-2 knockdown cells highlights perturbed levels of numerous proteins involved in DNA damage response and downstream pathways affecting TP53 and NF-κB signaling. TP53 showed increased protein abundance, but no transcriptional regulation. CONCLUSION: TMZ-induced cell death in the presence of calpain-2 expression appears to favor DNA repair and promote cell survival. We conclude from our experiments that calpain-2 expression represents a proteomic mode that is associated with higher resistance via "priming" GBM cells to TMZ chemotherapy. Thus, calpain-2 could serve as a prognostic factor for GBM outcome.

Hippocampal mitochondrial Ca++ in experimentally induced Alzheimer's disease, link to calpains and impact of vitamin D3 supplementation.

Vitamin D impact on hippocampal mitochondrial Ca++ and calpains was not previously investigated in Alzheimer's disease (AD). The current work aimed to assess the alteration in hippocampal mitochondrial Ca++, ATP & ADP and hippocampal calpains' level in (AlCl3)-induced AD model, and the effect of 2 regimens of vitamin D supplementation on these alterations. METHODS: Forty male Wistar rats were randomized into 4 groups; control, AD (AlCl3100 mg/kg, p.o. daily for 42 days), AD and vitamin D co-treated group (AlCl3 as in AD group with vitamin D3 400 IU/kg/day, p.o. for 42 days) and AD, followed by vitamin D3 group (AlCl3 was given as in AD group for 42 days, then vitamin D3 for two weeks). AD was assessed by hippocampal levels of Aß42, p-tau and spatial memory assessment in Morris water maze. Hippocampal mitochondrial Ca++, ATP and ADP levels besides to calpain-1 & 2 and cytochrome C were assessed in addition to CA1 histological examination. RESULTS: AD animals showed impaired mitochondrial function as denoted by high Ca++ and decreased ATP and ADP and elevated calpain-1 & 2 and cytochrome C. Hippocampal CA1 region showed increased degenerated neurons and reduced thickness of its pyramidal layer. Vitamin D administration minimized the hippocampal mitochondrial impairement induced by AD and mitigated histological alterations even when supplemented post AD establishment. CONCLUSION: Vitamin D administration to AD rats breaks the deleterious loop in the hippocampus that involves increased Ca++, calpain activation, mitochondrial failure, neuronal degeneration and AD disease progression.

Calpain-1 mediates vascular remodelling and fibrosis via HIF-1α in hypoxia-induced pulmonary hypertension.

Calpain-1, a calcium-activated neutral cysteine proteases, has been reported to be involved in the formation of pulmonary hypertension. HIF-1α, an oxygen-sensitive transcription factor, has been reported to activate genes involved in cell proliferation and extracellular matrix recombination. This study was designed to investigate the effect of calpain-1 in hypoxic pulmonary hypertension (HPH) and to explore whether there is a relationship between calpain-1 and HIF-1α in this disease. In the hypoxia-induced model of HPH, we found that hypoxia resulted in increased right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodelling and collagen deposition in lung tissues of mice. The levels of calpain-1 and HIF-1α were up-regulated in the lung tissues of hypoxia-treated mice and pulmonary arterial smooth muscle cells (PASMCs). Knock-out of calpain-1 restrained haemodynamic and histological changes induced by chronic hypoxia in mice, and inhibition of calpain-1 also repressed the abnormal proliferation and migration of PASMCs. Besides, knock-out or inhibition of calpain-1 suppressed hypoxia-induced expression of HIF-1α, VEGF, PCNA, TGF-ß1, MMP2 and collagen I in vivo and in vitro. While inhibition of HIF-1α abolished the above effects of calpain-1. Furthermore, we found that calpain-1 mediates the expression of HIF-1α through NF-κB (P65) under hypoxia conditions. In conclusion, our results suggest that calpain-1 plays a pivotal role in hypoxia-induced pulmonary vascular remodelling and fibrosis through HIF-1α, providing a better understanding of the pathogenesis of HPH.

LXR activation ameliorates high glucose stress-induced aberrant mitochondrial dynamics via downregulation of Calpain1 expression in H9c2 cardiomyoblasts.

Liver-X-receptor (LXR) has previously been shown to exert a cardioprotective effect against the development of diabetic cardiomyopathy (DCM) associated with a reduction in mitochondrial dysfunction. However, the underlying mechanism by which LXR activation attenuates the structural and functional mitochondrial impairments caused by high glucose (HG) stress remains unclear. We demonstrate here that LXR activation inhibits HG stress-induced mitochondrial dysfunction and ameliorates aberrant mitochondrial dynamics. Furthermore, LXR activation regulates mitochondrial dynamics by inhibiting HG stress-induced upregulation of Calpain1 expression. These data indicate that amelioration of Calpain1-mediated aberrant mitochondrial dynamics may be at least part of the mechanism underlying the cardioprotective effects of LXR against HG stress. Therefore, LXR is a potentially attractive molecular target for treating cardiac mitochondrial dysfunction in patients with diabetes.

Effect of enhanced muscle tone on the expression of atrogenes and cytoskeletal proteins during postural muscle unloading.

Skeletal muscle unloading leads to the decreased electrical activity and decline of muscle tone. AIMS: Current study evaluated the effect of muscle tone preservation achieved by tetanus toxin (TeNT) treatment on signaling pathways regulating atrophic processes during unloading. MAIN METHODS: Four groups of rats were used: non-treated control (C), control rats with TeNT administration (CT), 7 days of unloading/hindlimb suspension with placebo (HS), and 7 days of unloading with TeNT administration (HST). KEY FINDINGS: Absolute and relative force of tetanic contractions was decreased by 65% in soleus muscle of HS rats when compared with C. Treatment with TeNT significantly lessened force decline in soleus muscle of HST rats when compared with HS. TeNT administration increased myosin heavy chain I beta (MyHC Iß) expression in CT rats and prevented MyHC Iß loss in HST group when compared with C rats. Desmin content was lower by 31.4% (p < 0.05) in HS group when compared with HST. Calpain-1 expression was increased in HS group when compared with C, CT and HST. There was a decrease in p-p70S6K content (41%, p < 0,05) and an increase in p-eEF2 content (77%, p < 0,05) in HS group when compared with C, while there were no significant differences in the content of these proteins between HST, CT and C groups. SIGNIFICANCE: Treatment with TeNT significantly diminished unloading-induced decline of soleus muscle mass and mechanical properties and affected the regulation of MyHC Iß expression. These effects are mediated by signaling pathways regulating protein synthesis and degradation.

Baicalein-ameliorated cerebral ischemia-reperfusion injury dependent on calpain 1/AIF pathway.

Cerebral ischemia reperfusion (CIR) has become the leading cause of death and disability. Baicalein is a natural bioactive ingredient extracted from Scutellaria baicalensis Georgi and has neuroprotective activity. In our work, baicalein was found to reduce neurological deficits, brain water content, infarct area, and neuronal death of rats induced by middle cerebral artery occlusion/reperfusion. In vitro, oxygen-glucose deprivation/reperfusion induced inordinate ROS production and apoptosis that could be reversed by baicalein. Our study revealed for the first time that baicalein has the potential to bind and inhibit the activity of calpain 1, thereby inhibiting AIF nuclear translocation. These findings demonstrated that baicalein protected against CIR injury via inhibiting AIF nuclear translocation by inhibiting calpain 1 activity.

Calpain-2 specifically cleaves Junctophilin-2 at the same site as Calpain-1 but with less efficacy.

Calpain proteolysis contributes to the pathogenesis of heart failure but the calpain isoforms responsible and their substrate specificities have not been rigorously defined. One substrate, Junctophilin-2 (JP2), is essential for maintaining junctional cardiac dyads and excitation-contraction coupling. We previously demonstrated that mouse JP2 is cleaved by calpain-1 (CAPN1) between Arginine 565 (R565) and Threonine 566 (T566). Recently, calpain-2 (CAPN2) was reported to cleave JP2 at a novel site between Glycine 482 (G482) and Threonine 483 (T483). We aimed to directly compare the contributions of each calpain isoform, their Ca2+ sensitivity, and their cleavage site selection for JP2. We find CAPN1, CAPN2 and their requisite CAPNS1 regulatory subunit are induced by pressure overload stress that is concurrent with JP2 cleavage. Using in vitro calpain cleavage assays, we demonstrate that CAPN1 and CAPN2 cleave JP2 into similar 75 kD N-terminal (JP2NT) and 25 kD C-terminal fragments (JP2CT) with CAPNS1 co-expression enhancing proteolysis. Deletion mutagenesis shows both CAPN1 and CAPN2 require R565/T566 but not G482/T483. When heterologously expressed, the JP2CT peptide corresponding to R565/T566 cleavage approximates the 25 kD species found during cardiac stress while the C-terminal peptide from potential cleavage at G482/T483 produces a 35 kD product. Similar results were obtained for human JP2. Finally, we show that CAPN1 has higher Ca2+ sensitivity and cleavage efficacy than CAPN2 on JP2 and other cardiac substrates including cTnT, cTnI and ß2-spectrin. We conclude that CAPN2 cleaves JP2 at the same functionally conserved R565/T566 site as CAPN1 but with less efficacy and suggest heart failure may be targeted through specific inhibition of CAPN1.

Astragaloside IV attenuates inflammatory response mediated by NLRP-3/calpain-1 is involved in the development of pulmonary hypertension.

Inflammation eventually leads to pulmonary arterial hypertension (PAH). Astragaloside IV(AS-IV) has a protective effect on pulmonary hypertension, but the specific protective mechanism has been unclear until now. Therefore, in this study, our aim was to investigate the mechanisms underlying the effects of AS-IV on PAH. In vivo, male Sprague-Dawley (SD) rats were injected intraperitoneally with monocrotaline (MCT, 60 mg/kg) and treated with AS-IV (40 mg/kg, 80 mg/kg), MCC950 and MDL-28170. In vitro, human pulmonary artery endothelial cells (HPAECs) were treated with monocrotaline pyrrole (MCTP, 60 µg/mL). The protein expression levels of NLRP-3, caspase-1, ASC, IL-18, IL-1ß and calpain-1 were measured in vivo and/or in vitro. The results showed that AS-IV decreased the protein expression levels of NLRP-3, caspase-1, ASC, IL-18, IL-1ß and calpain-1 in vivo and/or vitro. In conclusion, in this study the results suggested that AS-IV could inhibit monocrotaline-induced pulmonary arterial hypertension via the NLRP-3/calpain-1 pathway.

Thioredoxin-1 regulates calcium homeostasis in MPP+ /MPTP-induced Parkinson's disease models.

Disturbance in calcium (Ca2+ ) homeostasis has been involved in a variety of neuropathological conditions including Parkinson's disease (PD). The Ca2+ channel, transient receptor potential channel 1 (TRPC1), plays a protective role in regulating entry of Ca2+ activated by store depletion of Ca2+ in endoplasmic reticulum (ER). We have showed that thioredoxin-1 (Trx-1) plays a role in suppressing ER stress in PD. However, whether Trx-1 regulates TRPC1 expression in PD is still unknown. In the present study, we demonstrated that treatment of 1-methyl-4-phenylpyridinum ion (MPP+ ) significantly reduced the expression of TRPC1 in PC12 cells, which was restored by Trx-1 overexpression, and further decreased significantly by Trx-1 siRNA. Moreover, we found that Ca2+ entered into the cells was decreased by MPP+ in PC 12 cells, which was restored by Trx-1 overexpression, and further decreased by Trx-1 siRNA. MPP+ significantly increased calcium-dependent cysteine protease calpain1 expression in PC12 cells, which was suppressed by Trx-1 overexpression. Calpain1 expression was increased by Trx-1 siRNA or SKF96365, an inhibitor of TRPC1. Moreover, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) decreased TRPC1 expression in the substantia nigra pars compacta region (SNpc), which was restored in mice overexpressing Trx-1, and further decreased in mice of knockdown Trx-1. Inversely, the expression of calpain1 was increased by MPTP, which was suppressed in mice overexpressing Trx-1, and further increased in mice of knockdown Trx-1. In conclusion, Trx-1 regulates the Ca2+ entry through regulating TRPC1 expression after treatment of MPP+ /MPTP.

Evidence for the involvement of interleukin-1α during development of experimental cytomegalovirus retinitis in immunosuppressed mice.

Interleukin-1α (IL-1α) is an alarmin involved in the recruitment of macrophages and neutrophils during tissue inflammation. IL-1α can undergo cleavage by proteases, such as calpain-1, that enhances IL-1α binding to its receptor, although proteolytic cleavage is not necessary for biological activity. Macrophages and neutrophils are involved in the retinal inflammation associated with development of AIDS-related human cytomegalovirus (HCMV) retinitis. We therefore performed studies to test the hypothesis that IL-1α gene expression is stimulated intraocularly during retinitis development using two mouse models of murine cytomegalovirus (MCMV) retinitis that differ in method of immunosuppression, one by retrovirus-induced immunosuppression (MAIDS) and the other by corticosteroid-induced immunosuppression. MCMV-infected eyes of groups of retinitis-susceptible mice with MAIDS of 10 weeks duration (MAIDS-10 mice) and retinitis-susceptible corticosteroid-treated mice showed significant stimulation of IL-1α mRNA. Western blot analysis confirmed IL-1α protein production within the MCMV-infected eyes of MAIDS-10 mice. Whereas significant intraocular calpain-1 mRNA and protein production were also observed within MCMV-infected eyes of MAIDS-10 mice, the MCMV-infected eyes of retinitis-susceptible corticosteroid-treated mice showed a pattern of mRNA synthesis equivalent to that found within the MCMV-infected eyes of healthy mice that fail to develop retinitis. Our findings suggest a role for the alarmin IL-1α in the pathogenesis of MCMV retinitis in immunosuppressed mice. These findings may extend to the pathogenesis of HCMV retinitis in patients with AIDS or other forms of immunosuppression.

Inhibition of the ERK1/2-ubiquitous calpains pathway attenuates experimental pulmonary fibrosis in vivo and in vitro.

Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease with poor prognosis. Epithelial-mesenchymal transition (EMT) has been reported to play an important role in IPF. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) cascade, which regulates EMT and oncogenesis, has been implicated in the pathogenesis of IPF. Calpains, Ca2+-dependent cysteine proteinases that mediate controlled proteolysis of many specific substrates including epithelial cell marker E-cadherin, participate in organ fibrosis. Calpain-1 and calpain-2 of calpain family are ubiquitous calpains. ERK1/2 signaling stimulates the ubiquitous calpains activity in cancer development, but whether ERK1/2 signaling mediates the ubiquitous calpains activity in pulmonary fibrosis is unknown. Here we investigated whether inhibition of ERK1/2 signaling and the ubiquitous calpains attenuated experimental pulmonary fibrosis and examined the potential mechanism. Our results showed that inhibition of ERK1/2 signaling and the ubiquitous calpains both attenuated bleomycin (BLM)-induced lung fibrosis in mice. Inhibition of ERK1/2 signaling downregulated the expression of calpain-1 and calpain-2 in vivo and in vitro. We detected decreased E-cadherin expression and increased calpain-1 expression in IPF patients. Inhibition of ERK1/2 signaling and the ubiquitous calpains both suppressed the development of EMT in vivo and in vitro. Our study indicated that inhibition of the ERK1/2-ubiquitous calpains pathway protected pulmonary fibrosis from BLM, possibly via inhibition of EMT. Therefore, targeting ubiquitous calpains may be a potential strategy to attenuate IPF.

Inhibition of calpain 1 restores plasma membrane stability to pharmacologically rescued Phe508del-CFTR variant.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a chloride channel normally expressed at the surface of epithelial cells. The most frequent mutation, resulting in Phe-508 deletion, causes CFTR misfolding and its premature degradation. Low temperature or pharmacological correctors can partly rescue the Phe508del-CFTR processing defect and enhance trafficking of this channel variant to the plasma membrane (PM). Nevertheless, the rescued channels have an increased endocytosis rate, being quickly removed from the PM by the peripheral protein quality-control pathway. We previously reported that rescued Phe508del-CFTR (rPhe508del) can be retained at the cell surface by stimulating signaling pathways that coax the adaptor molecule ezrin (EZR) to tether rPhe508del-Na+/H+-exchange regulatory factor-1 complexes to the actin cytoskeleton, thereby averting the rapid internalization of this channel variant. However, the molecular basis for why rPhe508del fails to recruit active EZR to the PM remains elusive. Here, using a proteomics approach, we characterized and compared the core components of wt-CFTR- or rPhe508del-containing macromolecular complexes at the surface of human bronchial epithelial cells. We identified calpain 1 (CAPN1) as an exclusive rPhe508del interactor that prevents active EZR recruitment, impairs rPhe508del anchoring to actin, and reduces its stability in the PM. We show that either CAPN1 down-regulation or its chemical inhibition dramatically improves the functional rescue of Phe508del-CFTR in airway cells. These observations suggest that CAPN1 constitutes an appealing target for pharmacological intervention, as part of CF combination therapies restoring Phe508del-CFTR function.

Impaired cerebellar plasticity and eye-blink conditioning in calpain-1 knock-out mice.

Calpain-1 and calpain-2 are involved in the regulation of several signaling pathways and neuronal functions in the brain. Our recent studies indicate that calpain-1 is required for hippocampal synaptic plasticity, including long-term depression (LTD) and long-term potentiation (LTP) in field CA1. However, little is known regarding the contributions of calpain-1 to cerebellar synaptic plasticity. Low frequency stimulation (LFS, 5 Hz, 5 min)-induced LTP at parallel fibers to Purkinje cell synapses was markedly impaired in cerebellar slices from calpain-1 knock-out (KO) mice. Application of a selective calpain-2 inhibitor enhanced LFS-induced LTP in both wild-type (WT) and calpain-1 KO mice. Three protocols were used to induce LTD at these synapses: LFS (1 Hz, 15 min), perfusion with high potassium and glutamate (K-Glu) or dihydroxyphenylglycine (DHPG), a mGluR1 agonist. All three forms of LTD were impaired in calpain-1 KO mice. DHPG application stimulated calpain-1 but not calpain-2 in cerebellar slices, and DHPG-induced LTD impairment was reversed by application of a protein phosphatase 2A (PP2A) inhibitor, okadaic acid. As in hippocampus, BDNF induced calpain-1 activation and PH domain and Leucine-rich repeat Protein Phosphatase 1/suprachiasmatic nucleus oscillatory protein (PHLPP1/SCOP) degradation followed by extracellular signal-regulated kinase (ERK) activation, as well as calpain-2 activation leading to degradation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) in cerebellar slices. The role of calpain-1 in associative learning was evaluated in the delay eyeblink conditioning (EBC). Calpain-1 KO mice exhibited significant learning impairment in EBC during the first 2 days of acquisition training. However, after 5 days of training, the percentage of conditioned responses (CRs) between calpain-1 KO and WT mice was identical. Both calpain-1 KO and WT mice exhibited typical extinction patterns. Our results indicate that calpain-1 plays critical roles in multiple forms of synaptic plasticity and associative learning in both hippocampus and cerebellum.

Taurine prevents cardiomyocyte apoptosis by inhibiting the calpain-1/cytochrome c pathway during RVH in broilers.

The calpain-1-activated apoptotic pathway plays a key role in right ventricular hypertrophy (RVH). Taurine has been shown to attenuate apoptosis by inhibiting calpain activity. This experiment aimed to determine whether taurine could prevent RVH by inhibiting the calpain-1/cytochrome c apoptotic pathway. The broilers were given 1% taurine dissolved in drinking water and were raised at 10 °C ~ 12 °C from day 21 to day 42. At 21 d, 28 d, 35 d and 42 d, the right ventricular (RV) tissues were collected. Increased RVH index, angiotensin II, norepinephrine and atrial natriuretic peptide mRNA expression were reduced by taurine in the broiler RVs. Taurine obviously inhibited cardiomyocyte apoptosis via maintaining the mitochondrial membrane potential and decreased the activation of caspase-9 and caspase-3 in the broiler RVs. The antioxidant assay demonstrated that taurine enhanced the activities of superoxide dismutase, total antioxidant capacity and glutathione peroxidase and the glutathione/glutathione disulfide ratio. Western blot results revealed that taurine also downregulated the expression of calpain-1 and cytosolic cytochrome c while upregulating the expression of Bcl-2/Bax and mitochondrial cytochrome c in broiler cardiomyocytes during RVH. In summary, we found that taurine could enhance cardiomyocyte antioxidant ability and further prevented cardiomyocyte apoptosis by inhibiting the calpain-1/cytochrome c pathway during RVH in broilers.

Taurine attenuates isoproterenol-induced H9c2 cardiomyocytes hypertrophy by improving antioxidative ability and inhibiting calpain-1-mediated apoptosis.

Pathological cardiac hypertrophy is ultimately accompanied by cardiomyocyte apoptosis. Apoptosis mainly related to calpain-1-mediated apoptotic pathways. Studies had proved that taurine can maintain heart health through antioxidation and antiapoptotic functions, but the effect of taurine on cardiac hypertrophy is still unclear. This study aimed to determine whether taurine could inhibit calpain-1-mediated mitochondria-dependent apoptotic pathways in isoproterenol (ISO)-induced hypertrophic cardiomyocytes. We found that taurine could inhibit the increase in cell surface area and reduce the protein expression levels of the hypertrophic markers atrial natriuretic peptide, brain natriuretic polypeptide, and ß-myosin heavy chain. Taurine also reduced ROS, intracellular Ca2+ overload and mitochondrial membrane potential. Moreover, taurine inhibited cardiomyocyte apoptosis by decreasing the protein expression of calpain-1, Bax, t-Bid, cytosolic cytochrome c, Apaf-1, cleaved caspase-9 and cleaved caspase-3 and by enhancing calpastatin and Bcl-2 protein expression. Calpain-1 small interfering RNA transfection results showed similar antiapoptotic effects as the taurine prevention group. However, compared with the two treatments, taurine inhibited the expression of cleaved caspase-9 more significantly. Therefore, we believe that taurine prevents ISO-induced H9c2 cardiomyocyte hypertrophy by inhibiting oxidative stress, intracellular Ca2+ overload, the calpain-1-mediated mitochondria-dependent apoptotic pathway and cleaved caspase-9 levels.

P38α-MAPK Signaling Inhibition Attenuates Soleus Atrophy during Early Stages of Muscle Unloading.

To test the hypothesis that p38α-MAPK plays a critical role in the regulation of E3 ligase expression and skeletal muscle atrophy during unloading, we used VX-745, a selective p38α inhibitor. Three groups of rats were used: non-treated control (C), 3 days of unloading/hindlimb suspension (HS), and 3 days HS with VX-745 inhibitor (HSVX; 10 mg/kg/day). Total weight of soleus muscle in HS group was reduced compared to C (72.3 ± 2.5 vs 83.0 ± 3 mg, respectively), whereas muscle weight in the HSVX group was maintained (84.2 ± 5 mg). The expression of muscle RING-finger protein-1 (MuRF1) mRNA was significantly increased in the HS group (165%), but not in the HSVX group (127%), when compared with the C group. The expression of muscle-specific E3 ubiquitin ligases muscle atrophy F-box (MAFbx) mRNA was increased in both HS and HSVX groups (294% and 271%, respectively) when compared with C group. The expression of ubiquitin mRNA was significantly higher in the HS (423%) than in the C and HSVX (200%) groups. VX-745 treatment blocked unloading-induced upregulation of calpain-1 mRNA expression (HS: 120%; HSVX: 107%). These results indicate that p38α-MAPK signaling regulates MuRF1 but not MAFbx E3 ligase expression and inhibits skeletal muscle atrophy during early stages of unloading.