Calpain-2 mediates SARS-CoV-2 entry via regulating ACE2 levels.

Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, much effort has been dedicated to identifying effective antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A number of calpain inhibitors show excellent antiviral activities against SARS-CoV-2 by targeting the viral main protease (Mpro), which plays an essential role in processing viral polyproteins. In this study, we found that calpain inhibitors potently inhibited the infection of a chimeric vesicular stomatitis virus (VSV) encoding the SARS-CoV-2 spike protein but not Mpro. In contrast, calpain inhibitors did not exhibit antiviral activities toward the wild-type VSV with its native glycoprotein. Genetic knockout of calpain-2 by CRISPR/Cas9 conferred resistance of the host cells to the chimeric VSV-SARS-CoV-2 virus and a clinical isolate of wild-type SARS-CoV-2. Mechanistically, calpain-2 facilitates SARS-CoV-2 spike protein-mediated cell attachment by positively regulating the cell surface levels of ACE2. These results highlight an Mpro-independent pathway targeted by calpain inhibitors for efficient viral inhibition. We also identify calpain-2 as a novel host factor and a potential therapeutic target responsible for SARS-CoV-2 infection at the entry step. IMPORTANCE: Many efforts in small-molecule screens have been made to counter SARS-CoV-2 infection by targeting the viral main protease, the major element that processes viral proteins after translation. Here, we discovered that calpain inhibitors further block SARS-CoV-2 infection in a main protease-independent manner. We identified the host cysteine protease calpain-2 as an important positive regulator of the cell surface levels of SARS-CoV-2 cellular receptor ACE2 and, thus, a facilitator of viral infection. By either pharmacological inhibition or genetic knockout of calpain-2, the SARS-CoV-2 binding to host cells is blocked and viral infection is decreased. Our findings highlight a novel mechanism of ACE2 regulation, which presents a potential new therapeutic target. Since calpain inhibitors also potently interfere with the viral main protease, our data also provide a mechanistic understanding of the potential use of calpain inhibitors as dual inhibitors (entry and replication) in the clinical setting of COVID-19 diseases. Our findings bring mechanistic insights into the cellular process of SARS-CoV-2 entry and offer a novel explanation to the mechanism of activities of calpain inhibitors.

Tiliroside disrupted iron homeostasis and induced ferroptosis via directly targeting calpain-2 in pancreatic cancer cells.

BACKGROUND: Tiliroside (TIL) is a flavonoid compound that exists in a variety of edible plants. These dietary plants are widely used as food and medicine to treat various diseases. However, the effect of TIL on pancreatic cancer (PC) and its underlying mechanisms are unclear. PURPOSE: This study aims to reveal the anti-PC effect of TIL and clarify its mechanism. METHODS: The inhibitory effects of TIL on PC growth were studied both in vitro and in vivo. Flow cytometry, transmission electron microscopy, immunofluorescence, biochemical analyses, RT-qPCR, genetic ablation, and western blotting were employed to evaluate ferroptosis, autophagy, and iron regulation. Additionally, RNA sequencing (RNA-seq), biomolecular layer interferometry (BLI), and molecular simulation analysis were combined to identify TIL molecular targets. The clinicopathological significance of Calpain-2 (CAPN2) was determined through immunohistochemistry (IHC) on a PC tissue microarray. RESULTS: Herein, we showed that TIL was an effective anti-PC drug. CAPN2 was involved in the TIL - induced elevation of the labile iron pool (LIP) in PC cells. TIL directly bound to and inhibited CAPN2 activity, resulting in AKT deactivation and decreased expression of glucose transporters (GLUT1 and GLUT3) in PC cells. Consequently, TIL impaired ATP and NADPH generation, inducing autophagy and ROS production. The accumulation of TIL-induced ROS combined with LIP iron causes the Fenton reaction, leading to lipid peroxidation. Meanwhile, TIL-induced reduction of free iron ions promoted autophagic degradation of ferritin to regulate cellular iron homeostasis, which further exacerbated the death of PC cells by ferroptosis. As an extension of these in vitro findings, our murine xenograft study showed that TIL inhibited the growth of PANC-1 cells. Additionally, we showed that CAPN2 expression levels were related to clinical prognoses in PC patients. CONCLUSION: We identify TIL as a potent bioactive inhibitor of CAPN2 and an anti-PC candidate of natural origin. These findings also highlight CAPN2 as a potential target for PC treatment.

Shortwave radiation-induced reproductive organ damage in male rats by enhanced expression of molecules associated with the calpain/Cdk5 pathway and oxidative stress.

Shortwave radiation has been reported to have harmful effects on several organs in humans and animals. However, the biological effects of 27 MHz shortwave on the reproductive system are not clear. In this study, we investigated the effects of shortwave whole-body exposure at a frequency of 27 MHz on structural and functional changes in the testis. Male Wistar rats were exposed to 27 MHz continuous shortwaves at average power densities of 0, 5, 10, or 30 mW/cm2 for 6 min. The levels of insulin-like factor 3 (INSL3) and anti-sperm antibodies (AsAb) in the peripheral serum, sperm motility, sperm malformation rate, and testicular tissue structure of rats were analyzed. Furthermore, the activity of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) content, calpain, and Cdk5 expression were analyzed at 1, 7, 14, and 28 days after exposure. We observed that the rats after radiation had decreased serum INSL3 levels (p < 0.01), increased AsAb levels (p < 0.05), decreased percentage of class A+B sperm (p < 0.01 or p < 0.05), increased sperm malformation (p < 0.01 or p < 0.05), injured testicular tissue structure, decreased SOD and CAT activities (p < 0.01 or p < 0.05), increased MDA content (p < 0.01), and testicular tissue expressions of calpain1, calpain2, and Cdk5 were increased (p < 0.01 or p < 0.05). In conclusion, Shortwave radiation caused functional and structural damage to the reproductive organs of male rats. Furthermore, oxidative stress and key molecules in the calpain/Cdk5 pathway are likely involved in this process.

Shortwave radiation has been used in communications, medical and military applications, and its damaging effects on several organs of the human body have been reported in the literature. However, the biological effects of shortwave radiation on the male reproductive system are unknown. The present study, by constructing an animal model of short-wave radiation and analyzing the experimental results, revealed that shortwave radiation could cause functional and structural damage to the reproductive organs of male rats, and that oxidative stress and key molecules in the calpain/Cdk5 pathway might be involved in this process. It will provide organizational data for further studies on the mechanisms of male reproductive damage by shortwave radiation.

Calpain inhibition decreases oxidative stress via mitochondrial regulation in a swine model of chronic myocardial ischemia.

INTRODUCTION: Calpain overexpression is implicated in mitochondrial damage leading to tissue oxidative stress and myocardial ischemic injury. The aim of this study was to determine the effects of calpain inhibition (CI) on mitochondrial impairment and oxidative stress in a swine model of chronic myocardial ischemia and metabolic syndrome. METHODS: Yorkshire swine were fed a high-fat diet for 4 weeks to induce metabolic syndrome then underwent placement of an ameroid constrictor to the left circumflex artery. Three weeks later, animals received: no drug (control, "CON"; n= 7); a low-dose calpain inhibitor (0.12 mg/kg; "LCI", n= 7); or high-dose calpain inhibitor (0.25 mg/kg; "HCI", n=7). Treatment continued for 5 weeks, followed by tissue harvest. Cardiac tissue was assayed for protein carbonyl content, as well as antioxidant and mitochondrial protein expression. Reactive oxygen species (ROS) and mitochondrial respiration was measured in H9c2 cells following exposure to normoxia or hypoxia (1%) for 24 h with or without CI. RESULTS: In ischemic myocardial tissue, CI was associated with decreased total oxidative stress compared to control. CI was also associated with increased expression of mitochondrial proteins superoxide dismutase 1, SDHA, and pyruvate dehydrogenase compared to control. 100 nM of calpain inhibitor decreased ROS levels and respiration in both normoxic and hypoxic H9c2 cardiomyoblasts. CONCLUSIONS: In the setting of metabolic syndrome, CI improves oxidative stress in chronically ischemic myocardial tissue. Decreased oxidative stress may be via modulation of mitochondrial proteins involved in free radical scavenging and production.

Hydroxyapatite nanoparticles induced calcium overload-initiated cancer cell-specific apoptosis through inhibition of PMCA and activation of calpain.

Hydroxyapatite nanoparticles (HAPNs) have been reported to specifically induce apoptosis and sustained elevation of intracellular Ca2+ concentration ([Ca2+]i) in cancer cells. However, it remains unclear whether calcium overload, the abnormal intracellular accumulation of Ca2+, is the intrinsic cause of cell apoptosis, how HAPNs specifically evoke calcium overload in cancer cells, and which potential pathways were involved in apoptosis initiation in response to calcium overload. In this study, using various cancer and normal cells, we observed a positive correlation between the degree of increased [Ca2+]i and the specific toxicity of HAPNs. Moreover, chelating intracellular Ca2+ with BAPTA-AM inhibited HAPN-induced calcium overload and apoptosis, thus demonstrating that calcium overload was the main cause of HAPN-induced cytotoxicity in cancer cells. Notably, the dissolution of particles outside the cells did not affect cell viability or [Ca2+]i. In contrast, internalized HAPNs dissolved more readily in cancer cells than in normal cells and inhibited the activity of plasma membrane calcium-ATPase solely in cancer cells to prevent extrusion of excessive Ca2+, hence leading to calcium overload in tumor cells. Upon exposure to HAPNs, the Ca2+-sensitive cysteine protease calpain was activated and then cleaved the BH3-only protein Bid. Consequently, cytochrome c was released, and caspase-9 and -3 were activated, leading to mitochondrial apoptosis. However, these effects were alleviated by the calpain inhibitor calpeptin, confirming the involvement of calpain in HANP-induced apoptosis. Therefore, our results demonstrated that calcium overload induced by HAPNs caused cancer cell-specific apoptosis by inhibiting PMCA and activating calpain in tumor cells and thus may contribute to a more comprehensive understanding of biological effects of this nanomaterial and facilitate the development of calcium overload cancer therapy.

ATO Increases ROS Production and Apoptosis of Cells by Enhancing Calpain-Mediated Degradation of the Cancer Survival Protein TG2.

Transglutaminase 2 (TG2) is a critical cancer cell survival factor that activates several signalling pathways to foster drug resistance, cancer stem cell survival, metastasis, inflammation, epithelial-mesenchymal transition, and angiogenesis. All-trans retinoic acid (ATRA) and chemotherapy have been the standard treatments for acute promyelocytic leukaemia (APL), but clinical studies have shown that arsenic trioxide (ATO), alone or in combination with ATRA, can improve outcomes. ATO exerts cytotoxic effects in a variety of ways by inducing oxidative stress, genotoxicity, altered signal transduction, and/or epigenetic modification. In the present study, we showed that ATO increased ROS production and apoptosis ratios in ATRA-differentiated NB4 leukaemia cells, and that these responses were enhanced when TG2 was deleted. The combined ATRA + ATO treatment also increased the amount of nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor, an adaptive regulator of the cellular oxidative stress response, and calpain proteolytic activity, resulting in TG2 degradation and the reduced survival of WT leukaemia cells. We further showed that the induced TG2 protein expression was degraded in the MCF-7 epithelial cell line and primary peripheral blood mononuclear cells upon ATO treatment, thereby sensitising these cell types to apoptotic signals.

Water-soluble Yb3+, Er3+ codoped NaYF4 nanoparticles induced SGC-7901 cell apoptosis through mitochondrial dysfunction and ROS-mediated ER stress.

BACKGROUND: Nanoparticles are potential luminescent probes; among them, upconversion nanoparticles (UCNP) are currently being developed as fluorescent probes for biomedical applications. However, the molecular mechanisms of UCNP in human gastric cell lines remain poorly understood. Here, we aimed to examine UCNP cytotoxicity to SGC-7901 cells and explore its underlying mechanisms. METHODS: The effects of 50-400 µg/mL UCNP on human gastric adenocarcinoma (SGC-7901) cells were investigated. Flow cytometry was used to evaluate reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), intracellular Ca2+ levels, and apoptosis. Activated caspase-3 and nine activities were measured; meanwhile, cytochrome C (Cyt C) in the cytosol and B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), protein kinase B (Akt), phosphorylated-Akt (p-Akt), 78 kDa glucose-regulated protein (GRP78), 94 kDa glucose-regulated protein (GRP94), calpain-1, and calpain-2 protein levels were also detected. RESULTS: UCNP inhibited the viability of SGC-7901 cells in a concentration- and time-dependent manner and increased the proportion of cell apoptosis. Exposure to UCNP enhanced the ratio of Bax/Bcl-2, elevated the level of ROS, decreased ΔΨm, increased intracellular Ca2+ and Cyt C protein levels, decreased the levels of phosphorylated Akt, increased the activity of caspase-3 and caspase-9, and upregulated the protein expression of GRP-78, GRP-94, calpain-1 and calpain-2 in SGC-7901 cells. CONCLUSION: UCNP induced SGC-7901 cell apoptosis by promoting mitochondrial dysfunction and ROS-mediated endoplasmic reticulum (ER) stress, initiating the caspase-9/caspase-3 cascade.

Effects of heat stress on markers of skeletal muscle proteolysis in dairy cattle.

Heat stress (HS) markedly affects postabsorptive energetics and protein metabolism. Circulating urea nitrogen increases in multiple species during HS and it has been traditionally presumed to stem from increased skeletal muscle proteolysis; however, this has not been empirically established. We hypothesized HS would increase activation of the calpain and proteasome systems as well as increase degradation of autophagosomes in skeletal muscle. To test this hypothesis, lactating dairy cows (~139 d in milk; parity ~2.4) were exposed to thermal neutral (TN) or HS conditions for 7 d (8 cows/environment). To induce HS, cattle were fitted with electric blankets for the duration of the heating period and the semitendinosus was biopsied on d 7. Heat stress increased rectal temperature (1.3°C) and respiratory rate (38 breaths per minute) while it decreased dry matter intake (34%) and milk yield (32%). Plasma urea nitrogen (PUN) peaked following 3 d (46%) and milk urea nitrogen (MUN) peaked following 4 d of environmental treatment and while both decreased thereafter, PUN and MUN remained elevated compared with TN (PUN: 20%; MUN: 27%) on d 7 of HS. Contrary to expectations, calpain I and II abundance and activation and calpain activity were similar between groups. Likewise, relative protein abundance of E3 ligases, muscle atrophy F-box protein/atrogin-1 and muscle ring-finger protein-1, total ubiquitinated proteins, and proteasome activity were similar between environmental treatments. Finally, autophagosome degradation was also unaltered by HS. Counter to our hypothesis, these results suggest skeletal muscle proteolysis is not increased following 7 d of HS and call into question the presumed dogma that elevated skeletal muscle proteolysis, per se, drives increased AA mobilization.

Inhibition of Ca2+-calpain signaling is a new mechanism using Laminaria japonica polysaccharide to prevent macrophage foam cell formation and atherosclerosis.

The Ca2+-calpain signaling plays a pivotal role in regulating the upstream signaling pathway of cellular autophagy. The aim of the current work was to investigate the role of Ca2+-calpain signaling in the regulation of macrophage autophagy by a Laminaria japonica polysaccharide (LJP61A) in Ox-LDL induced macrophages and high fat diet fed atherosclerotic mice. Results revealed that the LJP61A markedly decreased the levels of intracellular Ca2+, calpain1, calpain2 and their downstream effectors (Gsα, cAMP and IP3), and simultaneously enhanced autophagy activity and lipid metabolism, thereby reducing lipid accumulation in the Ox-LDL stimulated macrophages and lipid-laden plaques in atherosclerotic mice. Moreover, BAPTA-AM (a Ca2+ chelator) and calpeptin (a calpain inhibitor) synergistically strengthened the beneficial effects of LJP61A on autophagy and lipid metabolism by decreasing the levels of intracellular Ca2+, calpain1, calpain2, and their downstream effectors (Gsα, cAMP and IP3) induced by Ox-LDL. These findings suggested that the LJP61A suppressed macrophage derived foam cell formation and atherosclerosis by modulating the Ca2+-calpain-mediated autophagy.

Protective Effect of Calpain Inhibition During Cold Ischemia on Ischemia-reperfusion Injury After Lung Transplantation.

BACKGROUND: Necroptosis, one of the types of regulated necrosis, causes ischemia-reperfusion (IR) lung injury. N-acetyl-leucyl-leucyl-norleucinal (ALLN), a calpain inhibitor, is known to attenuate necroptosis and apoptosis, and the purpose of this study was to evaluate the protective effect of ALLN during cold ischemia against IR injury in a rat lung transplant model. METHODS: Male Lewis rats (250-350 g) were divided into 3 groups: sham group (n = 4), nontransplantation; control group (n = 8), transplantation with IR lung injury; and ALLN group (n = 8), transplantation with IR lung injury/ALLN. Rats in the sham group underwent a simple thoracotomy, and the remaining 2 groups of rats underwent an orthotopic left lung transplant. Cold ischemic time was 15 h. After 2 h of reperfusion, physiological function, inflammatory cytokine expression, pathway activation, and the degrees of necroptosis and apoptosis were evaluated. RESULTS: Lung gas exchange (PaO 2 /FiO 2 ) was significantly better, and pulmonary edema was significantly improved in the ALLN group compared with the control group ( P = 0.0009, P = 0.0014). Plasma expression of interleukin-1ß was significantly lower in the ALLN group than in the control group ( P = 0.0313). The proportion of necroptotic and apoptotic cells was significantly lower in the ALLN group than in the control group ( P = 0.0009), whereas the proportion of apoptotic cells remained unchanged ( P = 0.372); therefore, the calpain inhibitor was thought to suppress necroptosis. CONCLUSIONS: The administration of ALLN during cold ischemia appears to improve IR lung injury in a lung transplant animal model via the inhibition of necroptosis.

Calpain inhibitors inhibit mitochondrial calpain activity to ameliorate apoptosis of cocultured myoblast.

Cancer cachexia is a fatal syndrome associated with muscle regeneration disability. Tumor factors induce the apoptosis of myoblasts to impair the regeneration of skeletal muscle. Cancer cachectic myoblast apoptosis is associated with mitochondria injury. It has been reported that activated mitochondrial calpain caused mitochondria injury in mouse cardiomyocytes and pulmonary smooth muscle. We wondered if mitochondrial calpains exist in skeletal myoblast and their potential role in myoblast apoptosis of cancer cachexia. We used a transwell to build a novel myoblast-carcinoma cell coculture model to simulate the cancer cachexia environment in vitro. Calpain inhibitors, calpastatin (CAST) and calpeptin (CAPT), were used during coculture. We found for the first time that two calpains (calpain-1 and calpain-2) and CAST were present in the mitochondria of myoblast. The activation of mitochondrial calpain decreased mitochondrial complex I activity, promoted mitochondrial permeability transition pore opening, and impaired mitochondrial membrane potential in myoblast during coculture, which induced myoblasts apoptosis. CAST and CAPT protected myoblasts from apoptosis by inhibiting mitochondrial calpain activity, which may attenuate or even reverse cancer cachectic muscle atrophy by improving muscle regeneration ability. Our study provides a new perspective for understanding the mechanism of cancer cachexia, and will further contribute to treat cancer cachexia by focusing on the mitochondrial calpain activity.

N-Acetylcysteine, an ROS Inhibitor, Alleviates the Pathophysiology of Hyperthyroidism-Induced Cardiomyopathy via the ROS/Ca2+ Pathway.

Hyperthyroidism is common and can induce cardiomyopathy, but there is no effective therapeutic strategy. The purpose of this study was to investigate the molecular mechanism of hyperthyroidism-induced cardiomyopathy (HTC) and the effect of N-acetylcysteine (NAC), an ROS inhibitor, on the pathophysiology of HTC in vivo and in vitro. Compared with those in the control groups in vivo and in vitro, TT3 and TT4 were significantly increased, the structure of myocardial cells was enlarged and disordered, and interstitial fibrosis and the apoptosis of myocardial cells were markedly increased in the L-Thy group. The ROS and inflammatory response were increased in the hyperthyroidism group. In the NAC group, the contents of TT3 and TT4 were decreased, the myocardial cell structure was slightly disturbed, fibrosis and apoptosis were significantly reduced, and the ROS level and inflammatory response were significantly reduced. Interestingly, L-Thy decreased the viability of fibroblasts and H9c2 cells, suggesting that L-Thy-induced fibrosis was not caused by the proliferation of fibroblasts. The molecular mechanism of HTC could be explained by the fact that L-Thy could cause cardiac hypertrophy, inflammation, and fibrosis by regulating the Ca2+/calpain/Rcan1-dependent signalling pathway, the Ca2+/Rcan1/NF-κB/p65-dependent signalling pathway, and the Ca2+/ROS/Bcl-2/caspase-3-dependent signalling pathway. In conclusion, NAC can alleviate the pathophysiology of hyperthyroidism-induced cardiomyopathy, probably by regulating the ROS/Ca2+-dependent pathway.

Inhibition of endoplasmic reticulum stress and the downstream pathways protects CD4+ T cells against apoptosis and immune dysregulation in sepsis.

Immunosuppression mediated by CD4+ T cell apoptosis and dysfunction is a key factor in promoting the progression of sepsis. Endoplasmic reticulum (ER) stress participates in the apoptosis and dysfunction of immune cells. We aimed to investigate the role of ER stress inhibition in CD4+ T cells in both in vitro and in vivo models of sepsis. In vitro model of sepsis was established with lipopolysaccharide (LPS) and the rat model of sepsis was established using cecal ligation and puncture (CLP). After the LPS treatment or CLP, ER stress inhibitors including 4-PBA, SNJ-1945, and SP600125 were used to treat cells or rats, and the CD4+ T cells were obtained by magnetic bead sorting. The effects of ER stress inhibitors on apoptosis and the function of CD4+ T cells were evaluated. After the LPS stimulation or CLP, the levels of ER stress and downstream markers (PERK, eIF2α, IRE-1α, ATF6, ATF4, XBP-1 s, GRP78, CHOP, and p-JNK) were increased in CD4+ T cells at the beginning of sepsis. Meanwhile, the number of apoptotic CD4+ T cells markedly increased. In addition, sepsis impaired the function of CD4+ T cells, manifested by the increased population of Th1, Th2, Th17, and Treg, as well as the production of TNF-α, interleukin (IL)-6, IL-4, and IL-10. However, inhibitors of ER stress, JNK, and calpain all decreased the induction of Th1 and Th17, enhanced the increase of Th2 and Treg, decreased the production of TNF-α and IL-6, and enhanced the production of IL-4 and IL-10. Our findings indicate that ER stress inhibitors may play a protective role by reducing CD4+ T cell apoptosis and maintaining CD4+ T cell function, which may be useful for enhancing the immune function and poor prognosis of patients with sepsis.

Inhibition of noncaspase proteases, calpain and proteasome, via ALLN and Bortezomib contributes to cell death through low degradation of pro-/anti-apoptotic proteins and apoptosis induction.

Dysfunction at any regulatory point along the apoptotic signaling pathway is closely related to many diseases including cancers. The apoptotic protein expression level is an important cause of cancer-related death, and the correct degradation of apoptotic proteins is involved in tumor development. Therefore, understanding of a regulatory point that underlying cancer-related death may help the development of new strategies to overcome the clinical challenges. Here, proteasome inhibitor Bortezomib and calpain inhibitor ALLN were examined on protein levels of caspase-3, caspase-9, XIAP, and E3-ligase PARC in HEK293T cells overexpressing XIAP and caspase-9. ATP depletion and caspase-3 activation were as a consequence of Bortezomib and ALLN function. Higher numbers of PI-stained cells provided evidence of cell death by both inhibitors. Western blotting analysis showed that both ALLN and Bortezomib equally inhibited degradation of XIAP, but only ALLN was effective at inhibiting caspase proteolytic degradation. Moreover, treatment of cells with both types of inhibitors significantly increased the level of E3-ligase PARC. Our findings showed that inhibition of proteasome and calpains enhanced the level of anti-apoptotic, XIAP and PARC, and pro-apoptotic, caspase-9 and 3 proteins, which totally promote cell death significantly.

TRPC1 contributes to endotoxemia-induced myocardial dysfunction via mediating myocardial apoptosis and autophagy.

Cardiac dysfunction is a vital complication of endotoxemia (ETM) with limited therapeutic options. Transient receptor potential canonical channel (TRPC)1 was involved in various heart diseases. While, the role of TRPC1 in ETM-induced cardiac dysfunction remains to be defined. In this study, we found that TRPC1 protein expression was significantly upregulated in hearts of lipopolysaccharide (LPS)-challenged mice. What's more, TRPC1 knockdown significantly alleviated LPS-induced cardiac dysfunction and injury. Further myocardial mRNA-sequencing analysis revealed that TRPC1 might participate in pathogenesis of ETM-induced cardiac dysfunction via mediating myocardial apoptosis and autophagy. Data showed that knockdown of TRPC1 significantly ameliorated LPS-induced myocardial apoptotic injury, cardiomyocytes autophagosome accumulation, and myocardial autophagic flux. Simultaneously, deletion of TRPC1 reversed LPS-induced molecular changes of apoptosis/autophagy signaling pathway in cardiomyocytes. Moreover, TRPC1 could promote LPS-triggered intracellular Ca2+ release, subsequent calpain activation and caveolin-1 degradation. Either blocking calpain by PD150606 or enhancing the amount of caveolin-1 scaffolding domain that interacts with TRPC1 by cell-permeable peptide cavtratin significantly alleviated the LPS-induced cardiac dysfunction and cardiomyocytes apoptosis/autophagy. Furthermore, cavtratin could inhibit LPS-induced calpain activation in cardiomyocytes. caveolin-1 could directly interact with calpain 2 both in vivo and in vitro. Importantly, cecal ligation and puncture-stimulated cardiac dysfunction and mortality were significantly alleviated in Trpc1-/- and cavtratin-treated mice, which further validated the contribution of TRPC1-caveolin-1 signaling axis in sepsis-induced pathological process. Overall, this study indicated that TRPC1 could promote LPS-triggered intracellular Ca2+ release, mediate caveolin-1 reduction, and in turn activates calpain to regulate myocardial apoptosis and autophagy, contributing to ETM-induced cardiac dysfunction of mice.

Calpain activation and progression of inflammatory cycles in Parkinson's disease.

Parkinson's disease (PD) is a progressive, neurodegenerative condition of the central nervous system (CNS) affecting 6.3 million people worldwide with no curative treatments. Current therapies aim to mitigate PD's effects and offer symptomatic relief for patients. Multiple pathways are involved in the pathogenesis of PD, leading to neuroinflammation and the destruction of dopaminergic neurons in the CNS. This review focuses on PD pathology and the role of calpain, a neutral protease, as a regulator of various immune cells such as T-cells, microglia and astrocytes which lead to persistent neuroinflammatory responses and neuronal loss in both the brain and spinal cord (SC). Calpain plays a significant role in the cleavage and aggregation of toxic α-synuclein (α-syn), a presynaptic neural protein, and other organelles, contributing to mitochondrial dysfunction and oxidative stress. α-Syn aggregation results in the formation of Lewy bodies (LB) that further contribute to neuronal damage through lipid bilayer penetration, calcium ion (Ca2+) influx, oxidative stress and damage to the blood brain barrier (BBB). Dysfunctional mitochondria destabilize cytosolic Ca2+ concentrations, raising intracellular Ca2+; this leads to excessive calpain activation and persistent inflammatory responses. α-Syn aggregation also results in the disruption of dopamine synthesis through phosphorylation of tyrosine hydroxylase (TH), a key enzyme involved in the conversion of tyrosine to levodopa (L-DOPA), the amino acid precursor to dopamine. Decreased dopamine levels result in altered dopamine receptor (DR) signaling, ultimately activating pro-inflammatory T-cells to further contribute to the inflammatory response. All of these processes, together, result in neuroinflammation, degeneration and ultimately neuronal death seen in PD. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP-a prodrug to the neurotoxin 1-methyl-4-phenylpyridinium (MPP+)), rotenone (an environmental neurotoxin), and 6-hydroxydopamine (6-OHDA - a neurotoxic synthetic organic compound) induce PD-like conditions when injected into rodents. All three agents work through similar mechanisms and lead to degeneration of dopaminergic neurons in the substantia nigra (SN) and more recently discovered in motor neurons of the spinal cord (SC). These neurotoxins also increase calpain activity, furthering the neuroinflammatory response. Hence, calpain inhibitors have been posited as potential therapeutics for PD to prevent calpain-related inflammation and neurodegenerative responses in not only the SN but the SC as well.

Effect of chronic alcohol consumption on myocardial apoptosis in the rat model of isoproterenol-induced myocardial injury and investigation on the cardioprotective role of calpain inhibitor 1.

We investigated the presence of myocardial apoptosis on isoproterenol (ISO)-induced myocardial injury (MI) after long-term high dose alcohol consumption and examined the antiapoptotic role of calpain inhibitor 1. Male Wistar Albino rats (n = 108) were divided into six groups: Control, alcohol (ethanol was given during 30 days for chronic alcohol consumption), MI (150 mg/kg ISO injection at last two days of alcohol consumption), alcohol + MI, alcohol + MI + calpain inhibitor 1 (10 mg/kg inhibitor was injected at 15 min before ISO injections) and Dimethyl Sulfoxide (DMSO) groups. Biochemical, histological, and morphometric methods determined apoptosis levels in the heart tissue of rats. Cytochrome c, caspase 3, and calpain levels were significantly high in alcohol, MI, and alcohol + MI groups. In contrast, mitochondrial cardiolipin content was found to be low in alcohol, MI, and alcohol + MI groups. These parameters were close to the control group in the therapy group. Histological and morphometric data have supported biochemical results. As a result of our biochemical data, myocardial apoptosis was seen in the alcohol, MI, and especially alcohol after MI groups. Calpain inhibitor 1 reduced apoptotic cell death and prevented myocardial tissue injury in these groups. The efficiency of calpain inhibitor was very marked in MI after long-term high dose alcohol consumption.

Inhibition of calpain reduces cell apoptosis by suppressing mitochondrial fission in acute viral myocarditis.

Cardiomyocyte apoptosis is critical for the development of viral myocarditis (VMC), which is one of the leading causes of cardiac sudden death in young adults. Our previous studies have demonstrated that elevated calpain activity is involved in the pathogenesis of VMC. This study aimed to further explore the underlying mechanisms. Neonatal rat cardiomyocytes (NRCMs) and transgenic mice overexpressing calpastatin were infected with coxsackievirus B3 (CVB3) to establish a VMC model. Apoptosis was detected with flow cytometry, TUNEL staining, and western blotting. Cardiac function was measured using echocardiography. Mitochondrial function was measured using ATP assays, JC-1, and MitoSOX. Mitochondrial morphology was observed using MitoTracker staining and transmission electron microscopy. Colocalization of dynamin-related protein 1 (Drp-1) in mitochondria was examined using immunofluorescence. Phosphorylation levels of Drp-1 at Ser637 site were determined using western blotting analysis. We found that CVB3 infection impaired mitochondrial function as evidenced by increased mitochondrial ROS production, decreased ATP production and mitochondrial membrane potential, induced myocardial apoptosis and damage, and decreased myocardial function. These effects of CVB3 infection were attenuated by inhibition of calpain both by PD150606 treatment and calpastatin overexpression. Furthermore, CVB3-induced mitochondrial dysfunction was associated with the accumulation of Drp-1 in the outer membrane of mitochondria and subsequent increase in mitochondrial fission. Mechanistically, calpain cleaved and activated calcineurin A, which dephosphorylated Drp-1 at Ser637 site and promoted its accumulation in the mitochondria, leading to mitochondrial fission and dysfunction. In summary, calpain inhibition attenuated CVB3-induced myocarditis by reducing mitochondrial fission, thereby inhibiting cardiomyocyte apoptosis. Calpain is activated by CVB3 infection. Activated calpain cleaves calcineurin A and converts it to active form which could dephosphorylate Drp-1 at Ser637 site. Then, the active Drp-1 translocates from the cytoplasm to mitochondria and triggers excessive mitochondrial fission. Eventually, the balance of mitochondrial dynamics is broken, and apoptosis occurs.

Effects of 6-(Methylsulfinyl)hexyl Isothiocyanate Ingestion on Muscle Damage after Eccentric Exercise in Healthy Males: A Pilot Placebo-Controlled Double-Blind Crossover Study.

An animal study demonstrated that 6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), a major bioactive compound in Japanese pungent spice wasabi, has an action of inhibiting the activation of calpain-1 (a protease). Increases in calpain activity can cause continual strength loss after eccentric exercise. It remains to be determined in humans whether 6-MSITC intake would modulate calpain and/or muscle damage responses after eccentric exercise. We performed a randomized, double-blind, crossover design study wherein eight healthy young males were randomly assigned to ingest 9 mg/day of 6-MSITC or placebo from 1 day before exercise to 4 days after exercise (30 maximal isokinetic eccentric contractions of the elbow flexors using an isokinetic dynamometer). Calpain-1 concentration, inflammatory and muscle damage markers (creatine kinase activity, urinary titin concentration, muscle strength, range of motion, muscle soreness and transverse relaxation time) were assessed. Plasma calpain-1 concentration after eccentric exercise was similar between the placebo- and 6-MSITC-treated conditions. All muscle damage and inflammatory markers were not affected by 6-MSITC relative to those in the placebo-treated condition. Our results suggest that 6-MSITC has no effect on plasma calpain-1 concentration and muscle damage and inflammatory markers measured after eccentric exercise.

Truncation-Driven Lateral Association of α-Synuclein Hinders Amyloid Clearance by the Hsp70-Based Disaggregase.

The aggregation of α-synuclein is the hallmark of a collective of neurodegenerative disorders known as synucleinopathies. The tendency to aggregate of this protein, the toxicity of its aggregation intermediates and the ability of the cellular protein quality control system to clear these intermediates seems to be regulated, among other factors, by post-translational modifications (PTMs). Among these modifications, we consider herein proteolysis at both the N- and C-terminal regions of α-synuclein as a factor that could modulate disassembly of toxic amyloids by the human disaggregase, a combination of the chaperones Hsc70, DnaJB1 and Apg2. We find that, in contrast to aggregates of the protein lacking the N-terminus, which can be solubilized as efficiently as those of the WT protein, the deletion of the C-terminal domain, either in a recombinant context or as a consequence of calpain treatment, impaired Hsc70-mediated amyloid disassembly. Progressive removal of the negative charges at the C-terminal region induces lateral association of fibrils and type B* oligomers, precluding chaperone action. We propose that truncation-driven aggregate clumping impairs the mechanical action of chaperones, which includes fast protofilament unzipping coupled to depolymerization. Inhibition of the chaperone-mediated clearance of C-truncated species could explain their exacerbated toxicity and higher propensity to deposit found in vivo.