Calpain-1 Levels Research Articles

Ginsenoside Rg1 alleviates vascular remodeling in hypoxia-induced pulmonary hypertension mice through the calpain-1/STAT3 signaling pathway

BackgroundHypoxic pulmonary hypertension (HPH) is the main pathological change in vascular remodeling, a complex cardiopulmonary disease caused by hypoxia. Some research results have shown that ginsenoside Rg1 (Rg1) can improve vascular remodeling, but the effect and mechanism of Rg1 on hypoxia-induced pulmonary hypertension are not clear. The purpose of this study was to discuss the potential mechanism of action of Rg1 on HPH. MethodsC57BL/6 mice, calpain-1 knockout mice and Pulmonary artery smooth muscle cells (PASMCs) were exposed to a low oxygen environment with or without different treatments. The effect of Rg1 and calpain-1 silencing on inflammation, fibrosis, proliferation and the protein expression levels of calpain-1, STAT3 and p-STAT3 were determined at the animal and cellular levels. ResultsAt the mouse and cellular levels, hypoxia promotes inflammation, fibrosis, and cell proliferation, and the expression of calpain-1 and p-STAT3 is also increased. Ginsenoside Rg1 administration and calpain-1 knockdown, MDL-28170, and HY-13818 treatment showed protective effects on hypoxia-induced inflammation, fibrosis, and cell proliferation, which may be associated with the downregulation of calpain-1 and p-STAT3 expression in mice and cells. In addition, overexpression of calpain 1 increased p-STAT3 expression, accelerating the onset of inflammation, fibrosis and cell proliferation in hypoxic PASMCs. ConclusionGinsenoside Rg1 may ameliorate hypoxia-induced pulmonary vascular remodeling by suppressing the calpain-1/STAT3 signaling pathway.

Calpain Inhibitor Calpeptin Improves Pancreatic Fibrosis in Mice with Chronic Pancreatitis by Inhibiting the Activation of Pancreatic Stellate Cells.

Pancreatic fibrosis is a hallmark feature of chronic pancreatitis (CP), resulting in persistent damage to the pancreas. The sustained activation of pancreatic stellate cells (PSCs) plays a pivotal role in the progression of pancreatic fibrosis and is a major source of extracellular matrix (ECM) deposition during pancreatic injury. Calpain is a calcium-independent lysosomal neutral cysteine endopeptidase and was found to be correlated to various fibrotic diseases. Studies have revealed that calpeptin, a calpain inhibitor, can improve the fibrosis process of multiple organs. This study investigated the effect of the calpain inhibitor, calpeptin, on fibrosis in experimental CP and activation of cultured PSCs in mice. CP was induced in mice by repeated injections of cerulein for four weeks in vivo, and the activation process of mouse PSCs was isolated and cultured in vitro. Then, the inhibitory effect of calpeptin on pancreatic fibrosis was confirmed based on the histological damage of CP, the expression of α-smooth muscle actin (α-SMA) and collagen-Iα1(Col1α1), and the decrease in mRNA levels of calpain-1 and calpain-2. In addition, it was revealed that calpeptin can inhibit the activation process of PSCs and induce significant PSCs apoptosis by downregulating the expression of calpain-1, calpain-2 and TGF-β1, and the expression and phosphorylation of smad3 in vitro. These results suggest that the calpain inhibitor, calpeptin, plays a key role in the regulation of PSC activation by inhibiting the TGF-β1/smad3 signaling pathway, which supports the potential of calpeptin as an inhibitor of pancreatic fibrosis in mice by interfering with calpain.

Calpain inhibitor MDL28170 alleviates cerebral ischemia‑reperfusion injury by suppressing inflammation and autophagy in a rat model of cardiac arrest.

Cerebral ischemia-reperfusion injury (CIRI) is associated with a poor neurological prognosis in patients who have experienced cardiac arrest (CA) and cardiopulmonary resuscitation (CPR). The aim of the current study was to investigate the potential role of a calpain inhibitor in CIRI using a rat model of CA. CA was induced in adult male Sprague-Dawley rats, and MDL28170 (a calpain inhibitor) was administered to the rats within 30 min after the return of spontaneous circulation. Differences between groups were evaluated by measuring survival rate, CPR duration and neurological deficit score. Hematoxylin-eosin staining and Nissl staining were performed to assess cerebral injury, and microstructure and autophagy were assessed by transmission electron microscopy. The levels of calpain-1, calpain-2, calpastatin, interleukin (IL)-1β, tumor necrosis factor (TNF)-α, P62, beclin-1 and LC3 in the brain tissues were determined using western blotting and double immunofluorescence staining. There was no significant difference in CPR duration or survival rate among the groups. At 24 h after CPR, the CA group demonstrated damaged tissue morphology; decreased neurological deficit scores, and P62 expression; and upregulated calpain-2, IL-1βp17, TNF-α, beclin-1 and LC3 levels in the cortex. However, MDL28170 improved neuronal function and suppressed inflammation and autophagy by inhibiting calpain-2 level, but there were no differences in the calpain-1 and calpastatin levels. These results suggest that calpain-2, inflammation and autophagy are involved in CA-induced CIRI. MDL28170 inhibited calpain-2 expression, inflammation and autophagy, which suggests its potential efficacy in treating post-CA nerve damage.

Exercise mitigates calpain induced Purkinje cell loss in diabetes

Calpain-1 is a ubiquitous calcium dependent cysteine protease and found in cytoplasm as well as mitochondria. We have earlier reported that active calpain-1 is translocated from cytosol to mitochondria and activates MMP9. Calpain-1 activation is detrimental to the heart in several different ways, but there is little evidence that it can degrade Purkinje cell protein (PCP-4) and impair contractility in diabetes. Our hypothesis is that in diabetes, PCP-4 is degraded by calpain-1, causing contractile dysfunction that can be mitigated by exercise. To test this hypothesis, we recruited four groups of mice, 1) db/+ control, 2) db/+ with exercise, 3) db/db, 4) db/db with exercise. The mice were exercised on treadmill for 8 weeks as per American Veterinary Research Guidelines. Adding calcium to isolated cardiomyocytes caused them to lose shape and die. Compared with live myocytes, we observed high calpain-1 levels as well as significantly lower levels of PCP-4 and increased levels of calmodulin and calmodulin kinase II (CaMKII) in dead myocytes. We used the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) plasmid to knock down calpain-1 in HL-1 myocytes which restored the levels of PCP-4 along with calmodulin and CaMKII. In vivo, we found upregulated levels of calpain-1 in db/db mice (diabetic) as compared to db/+ which were mitigated in the exercised mice. Conclusively our data strongly suggests that in diabetes there is high induction of calpain-1 with degrades PCP-4, a protein important for contractility and exercise can mitigate this.

Ginsenoside Rg1 ameliorates chronic intermittent hypoxia-induced vascular endothelial dysfunction by suppressing the formation of mitochondrial reactive oxygen species through the calpain-1 pathway

BackgroundAs the major pathophysiological feature of obstructive sleep apnea (OSA), chronic intermittent hypoxia (CIH) is vital for the occurrence of cardiovascular complications. The activation of calpain-1 mediates the production of endothelial reactive oxygen species (ROS) and impairs nitric oxide (NO) bioavailability, resulting in vascular endothelial dysfunction (VED). Ginsenoside Rg1 is thought to against endothelial cell dysfunction, but the potential mechanism of CIH-induced VED remains unclear. MethodsC57BL/6 mice and human coronary artery endothelial cells (HCAECs) were exposed to CIH following knockout or overexpression of calpain-1. The effect of ginsenoside Rg1 on VED, oxidative stress, mitochondrial dysfunction, and the expression levels of calpain-1, PP2A and p-eNOS were detected both in vivo and in vitro. ResultsCIH promoted VED, oxidative stress and mitochondrial dysfunction accompanied by enhanced levels of calpain-1 and PP2A and reduced levels of p-eNOS in mice and cellular levels. Ginsenoside Rg1, calpain-1 knockout, OKA, NAC and TEMPOL treatment protected against CIH-induced VED, oxidative stress and mitochondrial dysfunction, which is likely concomitant with the downregulated protein expression of calpain-1 and PP2A and the upregulation of p-eNOS in mice and cellular levels. Calpain-1 overexpression increased the expression of PP2A, reduced the level of p-eNOS, and accelerated the occurrence and development of VED, oxidative stress and mitochondrial dysfunction in HCAECs exposed to CIH. Moreover, scavengers of O2•−, H2O2, complex Ⅰ or mitoKATP abolished CIH-induced impairment in endothelial-dependent relaxation. ConclusionGinsenoside Rg1 may alleviate CIH-induced vascular endothelial dysfunction by suppressing the formation of mitochondrial reactive oxygen species through the calpain-1 pathway.

Polysaccharides from Polygonatum cyrtonema Hua Reduce Depression-Like Behavior in Mice by Inhibiting Oxidative Stress-Calpain-1-NLRP3 Signaling Axis.

Polysaccharides from Polygonatum cyrtonema Hua (PSP) exert antioxidant, anti-inflammatory, and antidepressant effects. Production of reactive oxygen species (ROS) and activation of the calpain system and the NOD-like receptor protein 3 (NLRP3) inflammasome are closely related to the pathogenesis of depression. However, the relationships among those pathways and the protective effects of PSP have not been characterized. In this study, lipopolysaccharide (LPS) and chronic unpredictable mild stress- (CUMS-) induced depression models were used to evaluate the protective mechanisms of PSP against depression. ROS levels were measured in HT-22 cells using flow cytometry. Brain tissues were collected to determine the levels of oxidation-related indicators and inflammatory cytokines. The protein levels of calpain-1, calpain-2, calpastatin, phosphatase and Tensin Homolog deleted on Chromosome 10 (PTEN), suprachiasmatic nucleus circadian oscillatory protein (SCOP), nuclear factor-erythroid factor 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), NLRP3, apoptosis-associated speck-like protein (ASC), caspase-1, cleaved-caspase-1, ionized calcium binding adapter molecule 1 (Iba1), phosphorylation of extracellular signal-regulated kinase (p-ERK), nuclear factor-kappa B (NF-κB), interleukin-1β (IL-1β), and glial fibrillary acidic protein (GFAP) were measured using western blotting or immunofluorescence. In cellular experiments, we showed that PSP attenuated LPS-induced production of ROS in HT-22 cells. In animal experiments, we found that LPS increased the expression of calpain-1, NLRP3, ASC, caspase-1, cleaved-caspase-1, Iba1, p-ERK, NF-κB, and GFAP and reduced the expression of calpastatin, PTEN, SCOP, and Nrf2. Administration of PSP reversed these changes. N-Acetyl-L-cysteine (NAC) administration also inhibited oxidative stress and activation of the calpain system and the NLRP3 inflammasome. Furthermore, PSP, calpeptin, MCC950 (a selective NLRP3 inflammasome inhibitor), and NAC reduced LPS-induced proinflammatory cytokine release. We also showed that PSP prevented CUMS-induced changes in the calpain system and the Nrf2 and NLRP3 signaling pathways and reduced depression-like behavior. These results indicate that PSP exerts antidepressant effects through regulation of the oxidative stress-calpain-1-NLRP3 signaling axis.

Effects of electroacupuncture on motor function and calpain expression in rats with acute spinal cord injury

To observe the effect of electroacupuncture (EA) on motor function, calpain and calpastatin expression in rats with spinal cord injury, so as to explore the mechanism of EA underlying improvement of acute spinal cord injury. Thirty male SD rats were randomly divided into sham operation group, model group and EA group, with 10 rats in each group. The acute moderate spinal cord injury model was established by using a NYU spinal cord impactor. EA was applied to "Jizhong"(GV6) and "Mingmen" (GV4) for 30 min, once daily for 28 d. The Basso, Beattie and Bresnahan (BBB) rating scale (0 to 21 points) was used to assess changes of locomotor function. Histopathological changes of the injured spinal cord were observed after sectioning and Nissl staining, and the expression levels of calpain1, calpain2 and calpastatin mRNA and protein in the spinal cord tissues were detected by using quantitative real-time PCR and Western blot, respectively. The BBB score of the model group was significantly lower than that of the sham operation group (P<0.01), and was significantly higher in the EA group than that of the model group on 14th and 28th day (P<0.01). Compared with the sham operation group, the number of neurons in the model group decreased, and Nissl body stained cells decreased or even disappeared, which was evidently milder in the EA group. Compared with the sham operation group, the expression levels of calpain1 mRNA and protein in the spinal cord of the model group were significantly increased (P<0.01), while the expression levels of calpastatin mRNA and protein were significantly reduced (P<0.01). Following EA intervention, in contrast to the model group, the expression levels of calpain1 mRNA and protein in the EA group were significant down-regulated (P<0.01), calpastatin mRNA and protein expression levels were significantly up-regulated(P<0.01). There was no significant difference in calpain2 mRNA and protein expression among the 3 groups(P>0.05). EA can improve the locomotor function of rats with spinal cord injury, which may be related to its effect in inhibiting the activity of calpain in the injured spinal cord.

Verapamil decreases calpain-1 and matrix metalloproteinase-2 activities and improves hypertension-induced hypertrophic cardiac remodeling in rats

AimsIncreased activity of calpain-1 and matrix metalloproteinase (MMP)-2 was observed in different models of arterial hypertension and contribute to thicken the left ventricle (LV) walls and to hypertrophy cardiac myocytes. MMP-2 activity may be regulated by calpain-1 via bioactive molecules activation such as transforming growth factor (TGF)-β in cardiovascular diseases. This study analyzed whether calpain-1 causes cardiac hypertrophy and dysfunction by modulating the expression and activity of MMP-2 in renovascular hypertension. Main methodsMale Wistar rats were submitted to two kidneys, one clip (2K1C) model of hypertension or sham surgery and were treated with verapamil (VRP, 8 mg/kg/bid) by gavage from the second to tenth week post-surgery. Systolic blood pressure (SBP) was weekly assessed by tail-cuff plethysmography and morphological and functional parameters of LV were analyzed by echocardiography. MMP-2 activity was analyzed by in situ and gelatin zymography, while calpain-1 activity by caseinolytic assay. MMP-2, calpain-1, TGF-β and MMP-14/TIMP-2 levels were identified in the LV by western blots. Fluorescence assays were performed to evaluate oxidative stress, MMP-2 and calpain-1 levels. Key findingsSBP increased in 2K1C rats and was unaltered by VRP. However, VRP notably ameliorated hypertension-induced increase in LV thickness. VRP decreased hypertension-induced enhances in calpain-1 and MMP-2 activities, oxidative stress and mature TGF-β levels. Treatment with VRP also decreased the accentuated MMP-14/TIMP-2 levels in 2K1C. SignificanceTreatment with VRP decreases calpain-1 and MMP-2 activities and also reduces TGF-β and MMP-14/TIMP-2 levels in the LV of hypertensive rats, thus contributing to ameliorate cardiac hypertrophy.

Use of a Mouse Model and Human Umbilical Vein Endothelial Cells to Investigate the Effect of Arsenic Exposure on Vascular Endothelial Function and the Associated Role of Calpains.

Background:Arsenic (As) is a well-known environmental contaminant. Chronic exposure to As is known to increase the risk of cardiovascular diseases, including atherosclerosis, hypertension, diabetes, and stroke. However, the detailed mechanisms by which As causes vascular dysfunction involving endothelial integrity and permeability is unclear.Objectives:Our goal was to investigate how exposure to As leads to endothelial dysfunction.Methods:Arsenic trioxide (ATO) was used to investigate the effects and mechanisms by which exposure to As leads to endothelial dysfunction using a mouse model and cultured endothelial cell monolayers.Results:Compared with the controls, mice exposed chronically to As (10 ppb in drinking water supplied by ATO) exhibited greater vascular permeability to Evans blue dye and fluorescein isothiocyanate–labeled bovine serum albumin (FITC-BSA). In addition, endothelial monolayers treated with ATO ( As) exhibited greater intracellular gaps and permeability to low-density lipoprotein or transmigrating THP-1 cells. Furthermore, activity and protein levels of calpain-1 (CAPN-1) were significantly higher in aortas and human umbilical vein endothelial cells (HUVECs) treated with ATO. These results were consistent with effects of ATO treatment and included a rapid increase of intracellular calcium () and higher levels of CAPN-1 in the plasma membrane. Endothelial cell dysfunction and the proteolytic disorganization of vascular endothelial cadherin (VE-cadherin) in HUVECs in response to ATO were partially mitigated by treatment with a CAPN-1 inhibitor (ALLM) but not a CAPN-2 inhibitor (Z-LLY-FMK).Conclusions:This study found that in mice and HUVEC models, exposure to ATO led to CAPN-1 activation by increasing and CAPN-1 translocation to the plasma membrane. The study also suggested that inhibitor treatment may have a role in preventing the vascular endothelial dysfunction associated with As exposure. The findings presented herein suggest that As-induced endothelial dysfunction involves the hyperactivation of the CAPN proteolytic system. https://doi.org/10.1289/EHP4538

High calpain-1 expression predicts a poor clinical outcome and contributes to tumor progression in pancreatic cancer patients.

Pancreatic cancer (PC) is a highly aggressive and metastatic disease, with an elevated mortality rate. It is, therefore, crucial to assess factors affecting the prognosis of PC patients. Meanwhile, calpain-1 is associated with malignant tumor progression and metastasis. Thus, it is meaningful to evaluate the relationship between calpain-1 and PC. Calpain-1 protein expression was assessed by immunohistochemistry in 96 pancreatic cancer samples and paired adjacent non-cancerous specimens. In addition, calpain-1 protein levels were assessed in six PC cell lines by western blot (WB). Next, PC cells were transfected with calpain-1 siRNA, and silencing was confirmed by WB. Finally, cell proliferation, colony formation, migration and invasion assays, and cell apoptosis analysis were performed to examine the effects of calpain-1 knockdown on proliferation, growth, apoptosis, migration, and invasion in PC cells. The results showed that calpain-1 was overexpressed in PC tissues and cells. Meanwhile, calpain-1 overexpression was associated with tumor site (P = 0.029), metastasis (P = 0.000), and TNM stage (P = 0.000), but showed no associations with histological grade (P = 0.396), age (P = 0.809), sex (P = 1.000), and lesion size (P = 0.679). The Kaplan-Meier method demonstrated that the low calpain-1 expression group had increased overall survival (OS) compared with patients expressing high calpain-1 levels (28.7 ± 4.1 vs. 17.0 ± 2.3months) (P = 0.005). Besides, calpain-1 in PC cells was successfully silenced by liposome-mediated RNA interference, resulting in reduced cell growth, invasion, and metastasis in PC cells, with no effect on apoptosis. The above findings suggest that calpain-1 should be considered a potential biomarker for PC prognosis and therapy.

Exploring the putative role of kallikrein-6, calpain-1 and cathepsin-D in the proteolytic degradation of α-synuclein in multiple system atrophy.

There is evidence that accumulation of α-synuclein (α-syn) in Parkinson's disease (PD) and dementia with Lewy bodies (DLB) results from impaired removal of α-syn rather than its overproduction. Kallikrein-6 (KLK6), calpain-1 (CAPN1) and cathepsin-D (CTSD) are among a small number of proteases that cleave α-syn and are dysregulated in PD and DLB. Our aim in this study was to determine whether protease activity is altered in another α-synucleinopathy, multiple system atrophy (MSA), and might thereby modulate the regional distribution of α-syn accumulation. mRNA and protein level and/or activity of KLK6, CAPN1 and CTSD were measured and assessed in relation to α-syn load in multiple brain regions (posterior frontal cortex, caudate nucleus, putamen, occipital cortex, pontine base and cerebellar white matter), in MSA (n=20) and age-matched postmortem control tissue (n=20). CTSD activity was elevated in MSA in the pontine base and cerebellar white matter. KLK6 and CAPN1 levels were elevated in MSA in the putamen and cerebellar white matter. However, the activity or level of these proteolytic enzymes did not correlate with the regional distribution of α-syn. Accumulation of α-syn in MSA is not due to reduced activity of the proteases we have studied. We suggest that their upregulation is likely to be a compensatory response to increased α-syn in MSA.

Muscle-specific activation of calpain system in hindlimb unloading rats and hibernating Daurian ground squirrels: a comparison between artificial and natural disuse.

To determine whether the regulation of calpain system is involved in non-hibernators and hibernators in disused condition, the soleus (SOL) and extensor digitorum longus (EDL) muscles were used for investigating the muscle mass, the ratio of muscle wet weight/body weight (MWW/BW), fiber-type distribution, fiber cross-sectional area (CSA), and the protein expression of MuRF1, calpain-1, calpain-2, calpastatin, desmin, troponin T, and troponin C in hindlimb unloading rats and hibernating Daurian ground squirrels. The muscle mass, MWW/BW, and fiber CSA were found significantly decreased in SOL and EDL of hindlimb unloading rats, but unchanged in hibernating ground squirrels. The MuRF1 expression was increased in both SOL and EDL of unloading rats, while it was only increased in SOL, but maintained in EDL of hibernating ground squirrels. The expression levels of calpain-1 and calpain-2 were increased in different degrees in unloaded SOL and EDL in rats, while they were maintained in EDL and even reduced in SOL of hibernating ground squirrels. Besides, the expression of calpastatin was decreased in unloaded rats, but increased in hibernating ground squirrels. The desmin expression was decreased in unloaded rats, but maintained in hibernating squirrels. Interestingly, the levels of troponin T and troponin C were decreased in both SOL and EDL of unloaded rats, but increased in hibernating ground squirrels with muscle-type specificity. In conclusion, differential calpain activation and substrate-selective degradation in slow and fast muscles are involved in the mechanisms of muscle atrophy of unloaded rats and remarkable ability of muscle maintenance of hibernating ground squirrels.

EphrinB/EphB signaling contributes to spinal nociceptive processing via calpain‑1 and caspase‑3.

Previous studies have indicated that an important subfamily of receptor tyrosine kinases, ephrins and their receptors, are important in pain signaling, particularly in spinal nociceptive processing. In the present study, the role of the ephrin/Eph signaling pathway was confirmed, and it was shown that this signaling was also involved in spinal nociceptive processing through the actions of calpain-1 and caspase-3. First, the ephrinB ligands, ephrinB1-Fc or ephrinB2-Fc, were introduced into experimental mice via intrathecal injection, and it was found that this injection induced marked time- and dose-dependent mechanical allodynia and thermal hyperalgesia, accompanied by increased levels of calpain-1 and caspase-3 in the spinal cord. MDL28170, an inhibitor of calpain-1, reversed the behavioral effects and ameliorated the increases in calpain-1 and caspase-3. Second, it was found that the administration of EphB1 between L5 and L6 in mice inhibited the mechanical allodynia and thermal hyperalgesia induced by chronic constrictive injury. In addition, to demonstrate the cell phenotypes responsible for the increased levels of calpain-1 and caspase-3 in the spinal cord following injection with ephrinB2-Fc, double immunofluorescent labeling was performed, which indicated that calpain-1 and caspase-3 were localized in neurons, but not in astrocytes or microglial cells. In conclusion, the present study suggested that ephrinB/EphB signaling contributes to spinal nociceptive processing via the actions of calpain-1 and caspase-3.

Increased Glycolysis Contributes to Pulmonary Vascular Remodeling via ERK‐dependent Calpain Activation in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a life‐threatening lung disease which is characterized by pulmonary vascular remodeling due to the abnormal proliferation of vascular smooth muscle cell (SMCs) and overproduction of extracellular matrix (ECM) proteins including collagen. It has been shown that a number of growth factors such as platelet‐derived growth factor (PDGF) as well as the metabolic shift in energy production from oxidative phosphorylation to glycolysis participate in PAH in animal models and patients. We recently reported that calpain mediates collagen synthesis and proliferation of pulmonary arterial SMCs (PASMCs) contributing to the pathogenesis of PAH. The aim of this study is to investigate the mechanisms by which glycolytic shift induces calpain activation in PAH. PASMCs were incubated with PDGF‐BB (10 ng/ml) for 24 h, after which protein content of 6‐Phosphofructo‐2‐kinase/fructose‐2, 6‐bisphosphatase 3 (PFKFB3) and lactate concentration in the cell lysates and in the culture medium were measured. We found that PDGF‐BB significantly increases the protein levels of PFKFB3 and the lactate production. Our results also showed that inhibition of PFKFB3 attenuates PDGF‐induced calpain activity, collagen synthesis and proliferation of PASMCs. Treatment of PASMCs with lactate markedly increased the activities of calpain and extracellular signal–regulated kinase (ERK) without affecting the protein levels of calpain‐1, calpain‐2 and calpastatin in PASMCs suggesting that it may regulate the calpain activity via posttranslational mechanism. Moreover, inhibition of calpain‐2, but not calpain‐1, attenuates the lactate‐induced collagen synthesis and proliferation of PASMCs. In addition, pre‐treatment of the PASMCs with ERK 1/2 inhibitor PD98059 diminishes increases in calpain‐2 phosphorylation at serine 50, collagen synthesis and proliferation of PASMCs induced by lactate. More importantly, pharmacological inhibition of ERK 1/2 by PD98059 attenuates the progression of Sugen/Hypoxia‐induced pulmonary hypertension and pulmonary vascular remodeling in rats. In summary, we have demonstrated that ERK‐stimulated calpain signaling is a critical pathway in PAH which mediates cell proliferation and collagen synthesis in pulmonary vascular smooth muscle cells via enhanced glycolysis.Support or Funding InformationSupported by NIH (YS), Department of Veterans Affairs (YS), FAMRI (YS) and AHA (LK).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Calpain and Spectrin Breakdown Products (SBDP) as Potential Biomarkers in Tuberculous Pleural Effusion

SESSION TITLE: Tuberculosis SESSION TYPE: Original Investigation Poster PRESENTED ON: Wednesday, November 1, 2017 at 01:30 PM - 02:30 PM PURPOSE: Early diagnosis of tuberculous pleural effusion remains difficult. Calpain is a family of calcium-dependent endopeptidase that plays an important role in ECM remodeling and collagen synthesis. The aim of this study was to explore the diagnostic value of pleural fluid angiotensin-converting enzyme (ACE), Calpain-1, Spectrin breakdown products (SBDP) and matrix metalloproteinase-1 (MMP-1) measurement in tuberculous pleural effusion (TPE) and malignant pleural effusion (MP). METHODS: The study included 47 patients with TP, 28 patients with MP and 10 patients with transudate of non-tuberculous and non-malignant origin as controls. Calpain, ACE, SBDP and MMP-1 levels in pleural fluid were measured by ELISA method. RESULTS: ACE level, Calpain-1 level, SBDP level and MMP-1 level were higher in TPE than MPE and transudate (all, P value<0.05). By logistic regression analysis, ACE > 47.16 ng/mL, Calpain-1> 787 ng/mL, SBDP > 2.745 ng/mL and MMP-1 >7229 pg/mL were independent factors associated with TPE. The predicted probability based on the four predictors had an area under the ROC curve of 0.935, with 89.3% sensitivity and 89.4% specificity under the cut-off value 0.429. CONCLUSIONS: Our data suggest that ACE, Calpain-1, SBDP and MMP-1 levels could be useful biomarkers for diagnosing TPE. CLINICAL IMPLICATIONS: Early diagnpsos of tuberculous pleural effusion remains difficult. Measuring pleural effusion ACE, Calpain-1, SBDP and MMP-1 levels can improve the diagnosis and may give information about pleural fibrosis. DISCLOSURE: The following authors have nothing to disclose: Ji Young Hong, Myung Goo Lee, Chang Youl Lee No Product/Research Disclosure Information

Association between myocardial cell apoptosis and calpain-1/caspase-3 expression in rats with hypoxic-ischemic brain damage.

The present study aimed to investigate the association between myocardial cell apoptosis and calpain-1/caspase-3 expression in a rat model of hypoxic-ischemic brain damage (HIBD). A total of 64 newborn rats were divided into control (n=8; sacrificed on day 7) and HIBD groups (n=56). HIBD group rats were sacrificed 2, 12 or 24h, or 2, 3, 5 or 7days following HIBD (n=8/group). A terminal deoxynucleotidyl transferase dUTP nick-end labeling assay was performed to detect myocardial apoptotic cells and calculate the apoptosis index (AI), reverse transcription-polymerase chain reaction was performed to detect myocardial calpain-1/caspase-3 mRNA expression levels and a western blot analysis was conducted to detect calpain‑1 protein expression levels. The correlations between calpain‑1 and caspase‑3 expression levels and AI were analyzed. The results demonstrated that apoptotic myocardial cells in the HIBD groups were markedly increased compared with the control group, with AI peaking in the day 3 group. Caspase‑3 and calpain‑1 mRNA expression levels were increased from 2 and 12h following HIBD, respectively, with the most elevated levels in the day 2 group. Compared with the control group, calpain‑1 protein expression levels were increased from 2h, with the greatest expression levels in the day 3 group (P<0.05). Calpain‑1 mRNA and protein (76/80kDa) expression levels demonstrated positive linear correlations with AI (r=0.786, P=0.001; and r=0.853, P=0.001, respectively) Caspase-3 mRNA expression levels were positively correlated with AI (r=0.894; P=0.001). In conclusion, the present study demonstrated that in rats with HIBD, there is a positive correlation between increased apoptosis of myocardial cells and expression levels of calpain-1 and caspase-3.

Activation of Both the Calpain and Ubiquitin-Proteasome Systems Contributes to Septic Cardiomyopathy through Dystrophin Loss/Disruption and mTOR Inhibition.

Cardiac dysfunction caused by the impairment of myocardial contractility has been recognized as an important factor contributing to the high mortality in sepsis. Calpain activation in the heart takes place in response to increased intracellular calcium influx resulting in proteolysis of structural and contractile proteins with subsequent myocardial dysfunction. The purpose of the present study was to test the hypothesis that increased levels of calpain in the septic heart leads to disruption of structural and contractile proteins and that administration of calpain inhibitor-1 (N-acetyl-leucinyl-leucinyl-norleucinal (ALLN)) after sepsis induced by cecal ligation and puncture prevents cardiac protein degradation. We also tested the hypothesis that calpain plays a role in the modulation of protein synthesis/degradation through the activation of proteasome-dependent proteolysis and inhibition of the mTOR pathway. Severe sepsis significantly increased heart calpain-1 levels and promoted ubiquitin and Pa28β over-expression with a reduction in the mTOR levels. In addition, sepsis reduced the expression of structural proteins dystrophin and β-dystroglycan as well as the contractile proteins actin and myosin. ALLN administration prevented sepsis-induced increases in calpain and ubiquitin levels in the heart, which resulted in decreased of structural and contractile proteins degradation and basal mTOR expression levels were re-established. Our results support the concept that increased calpain concentrations may be part of an important mechanism of sepsis-induced cardiac muscle proteolysis.

Calpain mediated pulmonary vascular remodeling in hypoxia induced pulmonary hypertension

To explore the role of calpain in pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension and the underlying mechanisms. Sprague-Dawley rats were randomly divided into the hypoxia group and the normoxia control group. Right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) were monitored by a method with right external jugular vein cannula. Right ventricular hypertrophy index was presented as the ratio of right ventricular weight to left ventricular weight (left ventricle plus septum weight). Levels of calpain-1, -2 and -4 mRNA in pulmonary artery were determined by real-time PCR. Levels of calpain-1, -2 and -4 protein were determined by Western blot. Primary rat pulmonary arterial smooth muscle cells (PASMCs) were divided into 4 groups: a normoxia control group, a normoxia+MDL28170 group, a hypoxia group and a hypoxia+MDL28170 group. Cell proliferation was detected by MTS and flow cytometry. Levels of Ki-67 and proliferating cell nuclear antigen (PCNA) mRNA were determined by real-time PCR. RVSP, mPAP and right ventricular remodeling index were significantly elevated in the hypoxia group compared to those in the normoxia group. In the hypoxia group, pulmonary vascular remodeling was significantly developed, accompanied by up-regulation of calpain-1, -2 and -4. MDL28170 significantly inhibited hypoxia-induced proliferation of PASMCs concomitant with the suppression of Ki-67 and PCNA mRNA expression. Calpain mediates vascular remodeling via promoting proliferation of PASMCs in hypoxia-induced pulmonary hypertension.

Regulation of gene expression by NFAT transcription factors in hibernating ground squirrels is dependent on the cellular environment.

Calcineurin is a calmodulin-stimulated phosphatase that regulates the nuclear translocation of nuclear factor of activated T cell (NFAT) c1-4 through dephosphorylation. We believe that this mechanism plays various roles in the remodeling and maintenance of Ictidomys tridecemlineatus skeletal muscle. During hibernation, bouts of torpor and arousal take place, and squirrels do not lose muscle mass despite being inactive. Protein expression of Ca(2+) signaling proteins were studied using immunoblotting. A DNA-protein interaction ELISA technique was created to test the binding of NFATs in the nucleus to DNA probes containing the NFAT response element under environmental conditions reflective of those during hibernation. Calcineurin protein levels increased by 3.08-fold during torpor (compared to euthermic control), whereas calpain1 levels also rose by 3.66-fold during torpor. Calmodulin levels were elevated upon entering torpor. NFATc4 binding to DNA showed a 1.4-fold increase during torpor, and we found that this binding was further enhanced when 600nM of Ca(2+) was supplemented. We also found that decreasing the temperature of ELISAs resulted in progressive decreases in the binding of NFATs c1, c3, and c4 to DNA. In summary, calmodulin and calpain1 appear to activate calcineurin and NFATc4 during torpor. NFAT binding to target promoters is affected by intranuclear [Ca(2+)] and environmental temperatures. Therefore, Ca(2+) signaling and temperature changes play key roles in regulation of the NFAT-calcineurin pathway in skeletal muscle of hibernating 13-lined ground squirrels over the torpor-arousal cycle, and they may contribute to the avoidance of disuse-induced muscle atrophy that occurs naturally in these animals.

Heat stress prevents lipopolysaccharide-induced apoptosis in pulmonary microvascular endothelial cells by blocking calpain/p38 MAPK signalling.

Pulmonary microvascular endothelial cells (PMECs) injury including apoptosis plays an important role in the pathogenesis of acute lung injury during sepsis. Our recent study has demonstrated that calpain activation contributes to apoptosis in PMECs under septic conditions. This study investigated how calpain activation mediated apoptosis and whether heat stress regulated calpain activation in lipopolysaccharides (LPS)-stimulated PMECs. In cultured mouse primary PMECs, incubation with LPS (1μg/ml, 24h) increased active caspase-3 fragments and DNA fragmentation, indicative of apoptosis. These effects of LPS were abrogated by pre-treatment with heat stress (43°C for 2h). LPS also induced calpain activation and increased phosphorylation of p38 MAPK. Inhibition of calpain and p38 MAPK prevented apoptosis induced by LPS. Furthermore, inhibition of calpain blocked p38 MAPK phosphorylation in LPS-stimulated PMECs. Notably, heat stress decreased the protein levels of calpain-1/2 and calpain activities, and blocked p38 MAPK phosphorylation in response to LPS. Additionally, forced up-regulation of calpain-1 or calpain-2 sufficiently induced p38 MAPK phosphorylation and apoptosis in PMECs, both of which were inhibited by heat stress. In conclusion, heat stress prevents LPS-induced apoptosis in PMECs. This effect of heat stress is associated with down-regulation of calpain expression and activation, and subsequent blockage of p38 MAPK activation in response to LPS. Thus, blocking calpain/p38 MAPK pathway may be a novel mechanism underlying heat stress-mediated inhibition of apoptosis in LPS-stimulated endothelial cells.