Calpain Inhibitor Research Articles

Podocyte Injury in Diabetic Kidney Disease in Mouse Models Involves TRPC6-mediated Calpain Activation Impairing Autophagy.

Autophagy protects podocytes from injury in diabetic kidney disease (DKD). Restoring glomerular autophagy is a promising approach to limit DKD. This study demonstrates a novel regulatory mechanism of autophagy that blocks this critical protection of the glomerular filtration barrier. We demonstrated that TRPC6 induced in podocytes in mouse models of diabetes mediates calpain activation, thereby impairing podocyte autophagy, causing injury and accelerating DKD. Furthermore, this study provides proof of principle for druggable targets for DKD because restoration of podocyte autophagy by calpain inhibitors effectively limits glomerular destruction. Diabetic kidney disease is associated with impaired podocyte autophagy and subsequent podocyte injury. The regulation of podocyte autophagy is unique because it minimally uses the mTOR and AMPK pathways. Thus, the molecular mechanisms underlying the impaired autophagy in podocytes in diabetic kidney disease remain largely elusive. This study investigated how the calcium channel TRPC6 and the cysteine protease calpains deleteriously affect podocyte autophagy in diabetic kidney disease in mice. We demonstrated that TRPC6 knockdown in podocytes increased the autophagic flux because of decreased cysteine protease calpain activity. Diabetic kidney disease was induced in vivo using streptozotocin with unilateral nephrectomy and the BTBR ob/ob mouse models. Diabetes increased TRPC6 expression in podocytes in vivo with decreased podocyte autophagic flux. Transgenic overexpression of the endogenous calpain inhibitor calpastatin, as well as pharmacologic inhibition of calpain activity, normalized podocyte autophagic flux, reduced nephrin loss, and prevented the development of albuminuria in diabetic mice. In kidney biopsies from patients with diabetes, we further confirmed that TRPC6 overexpression in podocytes correlates with decreased calpastatin expression, autophagy blockade, and podocyte injury. Overall, we discovered a new mechanism that connects TRPC6 and calpain activity to impaired podocyte autophagy, increased podocyte injury, and development of proteinuria in the context of diabetic kidney disease. Therefore, targeting TRPC6 and/or calpain to restore podocyte autophagy might be a promising therapeutic strategy for diabetic kidney disease.

Calpain signaling: from biology to therapeutic opportunities in neurodegenerative disorders.

Neurodegenerative disorders represent a major and growing healthcare challenge globally. Among the numerous molecular pathways implicated in their pathogenesis, calpain signaling has emerged as a crucial player in neuronal dysfunction and cell death. Calpain is a family of calcium-dependent cysteine proteases that is involved in many biological processes, such as signal transduction, cytoskeleton remodeling, and protein turnover. Dysregulation of calpain activation and activity has been associated with several neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases. Understanding the intricate structure of calpains is crucial for unraveling their roles in cellular physiology and their implications in pathology. In addition, the identification of diverse abnormalities in both humans and other animal models with deficiencies in calpain highlights the significant progress made in understanding calpain biology. In this comprehensive review, we delve into the recent roles attributed to calpains and provide an overview of the mechanisms that govern their activity during the progression of neurodegenerative diseases. The possibility of utilizing calpain inhibition as a potential therapeutic approach for treating neuronal dysfunctions in neurodegenerative disorders would be an area of interest in future calpain research.

Downregulation of NHE1 expression attenuates apoptosis of primary hippocampal neurons of an epilepsy model through the calpain-1 pathway

ObjectiveNa(+)/H(+) exchanger isoform 1 (NHE1), a membrane protein that regulates intracellular pH, is abundantly expressed in brain tissues. It is associated with pathophysiologies in several brain diseases. The present study aimed to investigate the effects of NHE1 on the apoptosis of primary neurons of an epilepsy model. MethodsPrimary hippocampal neurons were cultured in an Mg2+-free medium to establish an epilepsy cell model. Designed shNHE1 lentivirus was used to silence NHE1 level in primary neurons. Nonselective pharmacological inhibitor MDL-28170 (20 μmol/L) was used to inhibit calpain-1 protein in neurons treated with Mg2+-free medium. The expression levels of NHE1 and calpain-1, intracellular Ca2+ (Ca2+i) and H+ (H+i) levels, and the expression levels of apoptosis-related proteins Bcl-2 and Bax were detected in neurons. TUNEL staining was performed to determine apoptosis in different groups. ResultsNHE1 expression was increased in primary neurons treated with an Mg2+-free medium, and it was correlated with increased expression of calpain-1 and cell apoptosis. Neurons from the in vitro epilepsy model showed significantly decreased Bcl-2 protein expression and significantly increased Bax protein expression. In the presence of LV-shNHE1 and the calpain-1 inhibitor MDL-28170, the changes in the expression of apoptosis-related proteins Bcl-2 and Bax were blocked in the epileptic model, and the percentage of apoptotic neurons among neurons from the in vitro epilepsy model was significantly decreased. The increase in calpain-1 expression was suppressed by LV-shNHE1; however, the inhibition of calpain-1 did not affect NHE1 expression. ConclusionThese results demonstrate that NHE1 participates in the promotion of neuronal apoptosis of epilepsy model in vitro through the calpain-1 pathway. Downregulation of NHE1 expression could exert a neuroprotective effect on epilepsy.

Calpain as a Therapeutic Target for Hypoxic-Ischemic Encephalopathy.

Hypoxic-ischemic encephalopathy (HIE) is a complex pathophysiological process with multiple links and factors. It involves the interaction of inflammation, oxidative stress, and glucose metabolism, and results in acute and even long-term brain damage and impairment of brain function. Calpain is a family of Ca2+-dependent cysteine proteases that regulate cellular function. Calpain activation is involved in cerebral ischemic injury, and this involvement is achieved by the interaction among Ca2+, substrates, organelles, and multiple proteases in the neuronal necrosis and apoptosis pathways after cerebral ischemia. Many calpain inhibitors have been developed and tested in the biochemical and biomedical fields. This study reviewed the potential role of calpain in the treatment of HIE and related mechanism, providing new insights for future research on HIE.

Calpain Promotes LPS-induced Lung Endothelial Barrier Dysfunction via Cleavage of Talin.

Acute lung injury (ALI) is characterized by lung vascular endothelial cell (EC) barrier compromise resulting in increased endothelial permeability and pulmonary edema. The infection of gram-negative bacteria that produce toxins like LPS is one of the major causes of ALI. LPS activates Toll-like receptor 4, leading to cytoskeleton reorganization, resulting in lung endothelial barrier disruption and pulmonary edema in ALI. However, the signaling pathways that lead to the cytoskeleton reorganization and lung microvascular EC barrier disruption remain largely unexplored. Here we show that LPS induces calpain activation and talin cleavage into head and rod domains and that inhibition of calpain attenuates talin cleavage, RhoA activation, and pulmonary EC barrier disruption in LPS-treated human lung microvascular ECs invitro and lung EC barrier disruption and pulmonary edema induced by LPS in ALI invivo. Moreover, overexpression of calpain causes talin cleavage and RhoA activation, myosin light chain (MLC) phosphorylation, and increases in actin stress fiber formation. Furthermore, knockdown of talin attenuates LPS-induced RhoA activation and MLC phosphorylation and increased stress fiber formation and mitigates LPS-induced lung microvascular endothelial barrier disruption. Additionally, overexpression of talin head and rod domains increases RhoA activation, MLC phosphorylation, and stress fiber formation and enhances lung endothelial barrier disruption. Finally, overexpression of cleavage-resistant talin mutant reduces LPS-induced increases in MLC phosphorylation in human lung microvascular ECs and attenuates LPS-induced lung microvascular endothelial barrier disruption. These results provide the first evidence that calpain mediates LPS-induced lung microvascular endothelial barrier disruption in ALI via cleavage of talin.

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

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. 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. 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-1β 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. The administration of ALLN during cold ischemia appears to improve IR lung injury in a lung transplant animal model via the inhibition of necroptosis.

Abstract P1023: Nonsense Mutations In Atrial Myofilament Regulatory Protein MyBP-HL Are Subject To Proteolytic Degradation And Loss Of Functional Protein

Background: Myosin binding protein-H like (MyBP-HL) is a recently identified atrial myofilament protein encoded by the gene MYBPHL. The Arg255Stop mutant has been linked to atrial enlargement, atrial fibrillation, and dilated cardiomyopathy. Deletion of MYBPHL in mice recapitulates this human phenotype. MyBP-HL is composed of two Immunoglobulin domains (Ig) and one Fibronectin domain (FnIII), like the structure and sequence of the last three C-terminal domains (C8-C9-C10) of cardiac myosin binding protein-C (cMyBP-C). cMyBP-C nonsense mutations fail in myofilament incorporation and result in a rapid degradation of the truncated protein and are a major cause of hypertrophic cardiomyopathy. There are 9 premature stop mutations in MYBPHL identified in humans (Gln29, Trp54, Arg113, Tyr123, Trp158, Trp192, Lys250, Arg255, and Tyr307). Hypothesis: We hypothesize that MYBPHL nonsense mutations will share a common mechanism that leads to MyBP-HL failing to integrate into the sarcomere, resulting in truncated MyBP-HL degradation and loss myofilament incorporation. Results: We demonstrate heterologous expression of MyBP-HL in neonatal rat cardiomyocytes can incorporate in a similar sarcomere pattern as cMyBP-C. Expression of the nine nonsense mutations do not localize into the sarcomere. Neonatal rat cardiomyocytes transfected with MyBP-HL constructs were treated with proteasome and/or calpain inhibitors. We observed that wild-type MyBP-HL and MyBP-HL truncating mutations are degraded mainly by the proteasome, although we also identified a significant calpain involvement in MyBP-HL degradation. Shorter MyBP-HL truncated peptides are degraded as a poisoning peptides, but longer peptides are not degraded. Conclusion: Based on these data, we demonstrated that MYBPHL nonsense mutations prevent MyBP-HL incorporation into the thick filament as are subject to degradation. The differential expression of short vs. long truncating MyBP-HL mutants suggest a differential pathogenic mechanism for subsets of these mutations.

Altered Caveolin-1 Dynamics Result in Divergent Mineralization Responses in Bone and Vascular Calcification.

Though vascular smooth muscle cells adopt an osteogenic phenotype during pathological vascular calcification, clinical studies note an inverse correlation between bone mineral density and arterial mineral-also known as the calcification paradox. Both processes are mediated by extracellular vesicles (EVs) that sequester calcium and phosphate. Calcifying EV formation in the vasculature requires caveolin-1 (CAV1), a membrane scaffolding protein that resides in membrane invaginations (caveolae). Of note, caveolin-1-deficient mice, however, have increased bone mineral density. We hypothesized that caveolin-1 may play divergent roles in calcifying EV formation from vascular smooth muscle cells (VSMCs) and osteoblasts (HOBs). Primary human coronary artery VSMCs and osteoblasts were cultured for up to 28days in an osteogenic media. CAV1 expression was knocked down using siRNA. Methyl β-cyclodextrin (MβCD) and a calpain inhibitor were used, respectively, to disrupt and stabilize the caveolar domains in VSMCs and HOBs. CAV1 genetic variation demonstrates significant inverse relationships between bone-mineral density (BMD) and coronary artery calcification (CAC) across two independent epidemiological cohorts. Culture in osteogenic (OS) media increased calcification in HOBs and VSMCs. siRNA knockdown of CAV1 abrogated VSMC calcification with no effect on osteoblast mineralization. MβCD-mediated caveolae disruption led to a 3-fold increase of calcification in VSMCs treated with osteogenic media (p < 0.05) but hindered osteoblast mineralization (p < 0.01). Conversely, stabilizing caveolae by calpain inhibition prevented VSMC calcification (p < 0.05) without affecting osteoblast mineralization. There was no significant difference in CAV1 content between lipid domains from HOBs cultured in OS and control media. Our data indicate fundamental cellular-level differences in physiological and pathophysiological mineralization mediated by CAV1 dynamics. This is the first study to suggest that divergent mechanisms in calcifying EV formation may play a role in the calcification paradox. The online version contains supplementary material available at 10.1007/s12195-023-00779-7.

Enriched environment ameliorates fear memory impairments induced by sleep deprivation via inhibiting PIEZO1/calpain/autophagy signaling pathway in the basal forebrain.

To verify the hypothesis that an enriched environment (EE) alleviates sleep deprivation-induced fear memory impairment by modulating the basal forebrain (BF) PIEZO1/calpain/autophagy pathway. Eight-week-old male mice were housed in a closed, isolated environment (CE) or an EE, before 6-h total sleep deprivation. Changes in fear memory after sleep deprivation were observed using an inhibitory avoidance test. Alterations in BF PIEZO1/calpain/autophagy signaling were detected. The PIEZO1 agonist Yoda1 or inhibitor GsMTx4, the calpain inhibitor PD151746, and the autophagy inducer rapamycin or inhibitor 3-MA were injected into the bilateral BF to investigate the pathways involved in the memory-maintaining role of EE in sleep-deprived mice. Mice housed in EE performed better than CE mice in short- and long-term fear memory tests after sleep deprivation. Sleep deprivation resulted in increased PIEZO1 expression, full-length tropomyosin receptor kinase B (TrkB-FL) degradation, and autophagy, as reflected by increased LC3 II/I ratio, enhanced p62 degradation, increased TFEB expression and nuclear translocation, and decreased TFEB phosphorylation. These molecular changes were partially reversed by EE treatment. Microinjection of Yoda1 or rapamycin into the bilateral basal forebrain induced excessive autophagy and eliminated the cognition-protective effects of EE. Bilateral basal forebrain microinjection of GsMTx4, PD151746, or 3-MA mimicked the cognitive protective and autophagy inhibitory effects of EE in sleep-deprived mice. EE combats sleep deprivation-induced fear memory impairments by inhibiting the BF PIEZO1/calpain/autophagy pathway.

Ginsenoside Rg1 attenuates diabetic vascular endothelial dysfunction by inhibiting the calpain-1/ROS/PKC-β axis

AimsVascular endothelial dysfunction (VED) is the onset event of cardiovascular complications in type 2 diabetes mellitus. Ginsenoside Rg1 (Rg1) can improve the cardiovascular system, but its mechanism in diabetic vascular endothelial dysfunction has received little attention. Main methodsMale calpain-1-knockout and wild-type C57BL/6 J mice were intraperitoneally injected with streptozotocin and treated with Rg1 (10 and 20 mg/kg) for 8 weeks. Human aortic endothelial cells (HAECs) were incubated with high glucose (HG) and were pretreated with Rg1 (10, 20 μM), MDL-28170 (calpain-1 inhibitor), LY-333531 (PKC-β inhibitor), NAC (ROS inhibitor) and calpain-1 overexpression. Then, factors related to mitochondrial dysfunction, oxidative stress and VED were measured. Key findingsThe administration of Rg1 and calpain-1 knockout ameliorated diabetic mitochondrial dysfunction, oxidative stress and VED and inhibited the calpain-1/ROS/PKC-β axis. LY-333531 and NAC treatment restored destructive endothelium-dependent vasodilation in mice with diabetes, while pyrogallol (ROS agonist), PMA (PKC-β agonist) or L-NAME (eNOS inhibitor) treatment abrogated the protective effect of Rg1 against diabetic endothelial dysfunction. The administration of Rg1, MDL-28170, LY-333531 and NAC improved mitochondrial dysfunction, oxidative stress and VED, whereas the overexpression of calpain-1 amplified mitochondrial dysfunction, oxidative stress and VED and further upregulated the expression of PKC-β in HAECs exposed to HG. Overexpression of calpain-1 abrogated the protective effect of Rg1 against HG-induced oxidative stress and VED. SignificanceThese findings reveal that Rg1 can protect against VED by suppressing the calpain-1/ROS/PKC-β axis and alleviating the development of mitochondrial dysfunction and oxidative stress.

Prevention of noise-induced hearing loss by calpain inhibitor MDL-28170 is associated with upregulation of PI3K/Akt survival signaling pathway.

Noise-induced calcium overload in sensory hair cells has been well documented as an early step in the pathogenesis of noise-induced hearing loss (NIHL). Alterations in cellular calcium homeostasis mediate a series of cellular events, including activation of calcium-dependent protein kinases and phosphatases. Using cell-membrane- and blood-brain-barrier-permeable calpain-1 (μ-calpain) and calpain-2 (m-calpain) inhibitor MDL-28170, we tested the involvement of calpains, a family of calcium-dependent cysteine proteases, and the potential of MDL-28170 in preventing NIHL. CBA/J mice at the age of 12 weeks were exposed to broadband noise with a frequency spectrum from 2-20 kHz for 2 h at 101 dB sound pressure level to induce permanent hearing loss as measured by auditory brainstem response and distortion product otoacoustic emissions. Morphological damage was assessed by quantification of remaining sensory hair cells and inner hair cell synapses 2 weeks after the exposure. MDL-28170 treatment by intraperitoneal injection significantly attenuated noise-induced functional deficits and cochlear pathologies. MDL-28170 treatment also prevented noise-induced cleavage of alpha-fodrin, a substrate for calpain-1. Furthermore, MDL-28170 treatment prevented reduction of PI3K/Akt signaling after exposure to noise and upregulated p85α and p-Akt (S473) in outer hair cells. These results indicate that noise-induced calpain activation negatively regulates PI3K/Akt downstream signaling, and that prevention of NIHL by treatment with MDL-28170 is associated with upregulation of PI3K/Akt survival signaling pathways.

Increased IP3R-3 degradation induced by acrylamide promoted Ca2+-dependent calpain activation and axon damage in rats

Occupational and environmental exposure to acrylamide (ACR) can cause selective peripheral and central nerve fiber degeneration. IP3R-3 is an important transmembrane Ca2+ channel on the endoplasmic reticulum (ER), previous studies have found that ACR could induce Ca2+-dependent calpain activation and axon injury, but the exact role of IP3R-3 in ACR neuropathy is still unclear. Here we show that ACR exposure (40 mg/kg) markedly increased the ubiquitination of IP3R-3 in rat spinal cords, and promoted the degradation of IP3R-3 through the ubiquitin-proteasome pathway. Furthermore, the normal structure of ER, especially the mitochondrial associated membranes (MAMs) component, was significantly impaired in ACR neuropathy, and the ER stress pathway was activated, which indicated that the aberrant increase of cytoplasmic Ca2+ could be attributed the destruction of IP3R-3. Further investigation demonstrated that the proteasome inhibitor MG-132 effectively rescued the IP3R-3 loss, attenuated the intracellular Ca2+ increase, and reduced the axon loss of Neuron 2a (N2a) cells following ACR exposure. Moreover, the calpain inhibitor ALLN also reduced the loss of IP3R-3 and axon injury in N2a cells, but did not alleviate the Ca2+ increase in cytosol, supporting that the abnormal ubiquitination of IP3R-3 was the upstream of the cellular Ca2+ rise and axon damage in ACR neuropathy. Taken together, our results suggested that the aberrant IP3R-3 degradation played an important role in the disturbance of Ca2+ homeostasis and the downstream axon loss in ACR neuropathy, thus providing a potential therapeutic target for ACR neurotoxicity.

#3680 CALPAIN INHIBITION PREVENTS FIBROSIS IN PROGRESSIVE CHRONIC KIDNEY DISEASE

Abstract Background and Aims Kidney fibrosis is one of the main pathological processes of chronic kidney disease (CKD), although the pathogenesis of renal scar formation is not fully understood. Calpains (CAPN) are intracellular cysteine ​​proteases that play a key role in multiple biological processes linked to tissue damage and repair mechanisms, such as fibrosis and epithelial-mesenchymal transition. However, CAPN contribution in CKD genesis and progression remains to be fully elucidated. The aim of this study was to investigate the possible role of CAPN in the development of CKD and renal fibrosis in an experimental model of CKD induced by adenine and in human kidney proximal tubular cells (HK-2). Method C57BL/6 wild-type mice were fed a 0.2% adenine-rich diet (WTA) for 5 days and 2 weeks to induce tubulointerstitial damage resembling what occurs in human CKD. Mice with a standard diet were used as controls (WT). WT and 5 days WTA mice were injected intraperitoneally for 5 consecutive days with a dose of calpain inhibitor III (20 mg/kg). In vitro experiments were performed in HK-2 cells in the presence or absence of TGF-β (1 ng/ml, different times) and calpain inhibitor III (50 µM). Mice renal function was assessed by measuring plasma BUN and creatinine. Interstitial fibrosis was evaluated in kidney samples embedded in paraffin by Sirius red staining of the collagen renal cortex content. Fibrosis markers were determined by RT-qPCR. CAPN localization in the kidney was assessed by immunofluorescence. CAPN 2 and 5 protein content were analyzed by western blot. CAPN activity was determined by fluorescence assays. All data were analyzed using non-parametric statistics for comparisons, applying the Kruskal-Wallis with Mann-Whitney post-test. A value of p&amp;lt;0.05 was considered statistically significant. Results Our results show a progressive worsening of renal function and structure in adenine-fed mice, with increased BUN, creatinine and COL I mRNA expression being significantly higher at 2 weeks of treatment. CAPN 2 and 5 protein content was significantly higher in animals that received adenine for 2 weeks compared to control, with a statistically significant increased renal CAPN activity. Our results point out that CAPN 2 and 5 content in renal tissue increases as CKD and fibrosis progress. To verify this potential relationship, we treated kidney tubular cells, where we observed a greater calpain expression compared to other kidney cells, with TGF-β to induce fibrosis. This treatment increased COL I mRNA expression, as well as CAPN 2 and 5 levels in HK-2 cells. However, calpain inhibitor III administration prevents increased COL I expression, both in vitro and in vivo. Conclusion We suggest a direct relationship between the cellular content of CAPN and the renal fibrosis observed in CKD, being involved in the genesis or progression of kidney disease. Thus, effective CAPN blockade or downregulation could be helpful as a therapeutic strategy to prevent CKD.

SEVERE BURN-INDUCED MITOCHONDRIAL RECRUITMENT OF CALPAIN CAUSES ABERRANT MITOCHONDRIAL DYNAMICS AND HEART DYSFUNCTION.

Mitochondrial damage is an important cause of heart dysfunction after severe burn injury. However, the pathophysiological process remains unclear. This study aims to examine the mitochondrial dynamics in the heart and the role of μ-calpain, a cysteine protease, in this scenario. Rats were subjected to severe burn injury treatment, and the calpain inhibitor MDL28170 was administered intravenously 1 h before or after burn injury. Rats in the burn group displayed weakened heart performance and decreased mean arterial pressure, which was accompanied by a diminishment of mitochondrial function. The animals also exhibited higher levels of calpain in mitochondria, as reflected by immunofluorescence staining and activity tests. In contrast, treatment with MDL28170 before any severe burn diminished these responses to a severe burn. Burn injury decreased the abundance of mitochondria and resulted in a lower percentage of small mitochondria and a higher percentage of large mitochondria. Furthermore, burn injury caused an increase in the fission protein DRP1 in the mitochondria and a decrease in the inner membrane fusion protein OPA1. Similarly, these alterations were also blocked by MDL28170. Of note, inhibition of calpain yielded the emergence of more elongated mitochondria along with membrane invagination in the middle of the longitude, which is an indicator of the fission process. Finally, MDL28170, administered 1 h after burn injury, preserved mitochondrial function and heart performance, and increased the survival rate. Overall, these results provided the first evidence that mitochondrial recruitment of calpain confers heart dysfunction after severe burn injury, which involves aberrant mitochondrial dynamics.

Abstract 683: Improved Myocardial Blood Flow And Arteriogenesis With Calpain Inhibition May Be Mediated By Vascular Endothelial Growth Factor And Fibroblast Growth Factor Angiogenic Pathways In A Large-animal Model Of Metabolic Syndrome And Chronic Myocardial Ischemia

Objective: Ischemic heart disease is the leading cause of death worldwide. Under pathological conditions like myocardial ischemia, endothelial calpain proteases are activated by hypoxia, leading to aberrant angiogenesis. Our group has shown calpain inhibition increases myocardial blood flow and vascular density, microvascular vasodilation, and expression of proangiogenic vascular endothelial growth factor (VEGF) and its receptors (VEGF-R1, VEGF-R2) in a porcine model of chronic myocardial ischemia and diet-induced metabolic syndrome. We aim to assess the role of downstream factors in angiogenic pathways. Methods: Yorkshire swine were fed a high cholesterol diet for four weeks, and then underwent placement of an ameroid constrictor to the left circumflex artery. After three weeks, pigs received either daily calpain inhibitor MDL28170 (0.25mg/kg, oral) for five weeks (n = 8) or no drug (n = 7), followed by tissue harvest of ischemic myocardium. Results: Treatment was associated with a significant increase in expression of proangiogenic mediators, fibroblast growth factor (FGF) receptor 1 (FGFR1, p &lt;0.001) and matrix metalloprotease-2 (MMP-2, p &lt;0.001). Cathepsins D and L have been implicated in both pro- and antiangiogenic actions. Cathepsin D was significantly increased (p = 0.004), but Cathepsin L was unchanged (p &gt;0.99). Cathepsin L proenzyme, procathepsin L, was significantly decreased (p = 0.04), while there was a strong trend toward decreased procathepsin L:cathepsin L ratio (p = 0.09). Proangiogenic Tie-2 (p = 0.28), and antiangiogenic collagen 18 (p = 0.54) and angiostatin (p = 0.24) were unchanged. Conclusions: In metabolic syndrome, calpain inhibition led to increased proangiogenic signaling in chronically ischemic myocardium, suggesting angiogenic VEGF- and FGF-mediated pathways may work in concert with improved microvascular vasodilation to explain the observed increase in vascular density and collateral-dependent coronary perfusion.

Allergen-stimulated secretion of DNA fragments by the airway epithelium enhances type 2 immune responses

Exposure of human bronchial epithelial cells (hBECs) and mouse airways to allergens from the fungus Alternaria alternata triggers the release of alarmin signaling molecules including ATP, IL-33 and DNA fragments. The goal of this study was to determine the mechanisms that regulate release of genomic DNA fragments and how extracellular DNA (eDNA) contributes to type 2 immunity. Treatment of hBECs with Alternaria extract induces oxidative stress, which leads to ATP release and uptake of Ca 2+ following activation of P2X receptors. DNA release was dependent on a sustained increase in intracellular [Ca 2+ ], which stimulates cleavage of caspase 3 by the proprotein convertase enzyme furin, resulting in activation of caspase 3 and ultimately nuclear DNA fragmentation as shown in comet assays. Some of the fragmented DNA was released into the media under conditions where barrier function of the epithelium was maintained, and nuclear and plasma membranes remained intact. Alternaria-induced DNA release was detected within 30 minutes following exposure, reached its peak at 4 hours and ceased after 12 hours. Pretreatment of hBECs with a calpain inhibitor resulted in continued DNA release and loss of cell viability by 24 hours. This result suggested that calpain activity contributed to termination of Alternaria-evoked DNA release. In mice, intranasal administration of Alternaria extract produced release of DNA fragments into bronchoalveolar lavage (BAL) fluid over a similar time course as observed in hBECs. In caspase 3 deficient mice however, DNA release into BAL fluid was significantly reduced. Furthermore, Alternaria-induced type 2 immune responses were suppressed following pretreatment with a DNA scavenger. Conversely, intranasal administration of mouse genomic DNA with Alternaria amplified IL-5 and IL-13 secretion into BAL fluid while DNA alone had no effect. These findings indicate that DNA release from the airway epithelium is a regulated process involving noncanonical activation of caspase 3 to produce DNA fragmentation and calpain activity, which appears to be essential for the termination of DNA release. Moreover in airways, eDNA appears to function as a secondary alarmin that acts on immune cells to enhance type 2 immunity. This work was supported by grants from the NIH (AI128729 and AI169530) to HK and SMO. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Calpain activity is negatively regulated by a KCTD7–Cullin-3 complex via non-degradative ubiquitination

Calpains are a class of non-lysosomal cysteine proteases that exert their regulatory functions via limited proteolysis of their substrates. Similar to the lysosomal and proteasomal systems, calpain dysregulation is implicated in the pathogenesis of neurodegenerative disease and cancer. Despite intensive efforts placed on the identification of mechanisms that regulate calpains, however, calpain protein modifications that regulate calpain activity are incompletely understood. Here we show that calpains are regulated by KCTD7, a cytosolic protein of previously uncharacterized function whose pathogenic mutations result in epilepsy, progressive ataxia, and severe neurocognitive deterioration. We show that KCTD7 works in complex with Cullin-3 and Rbx1 to execute atypical, non-degradative ubiquitination of calpains at specific sites (K398 of calpain 1, and K280 and K674 of calpain 2). Experiments based on single-lysine mutants of ubiquitin determined that KCTD7 mediates ubiquitination of calpain 1 via K6-, K27-, K29-, and K63-linked chains, whereas it uses K6-mediated ubiquitination to modify calpain 2. Loss of KCTD7-mediated ubiquitination of calpains led to calpain hyperactivation, aberrant cleavage of downstream targets, and caspase-3 activation. CRISPR/Cas9-mediated knockout of Kctd7 in mice phenotypically recapitulated human KCTD7 deficiency and resulted in calpain hyperactivation, behavioral impairments, and neurodegeneration. These phenotypes were largely prevented by pharmacological inhibition of calpains, thus demonstrating a major role of calpain dysregulation in KCTD7-associated disease. Finally, we determined that Cullin-3–KCTD7 mediates ubiquitination of all ubiquitous calpains. These results unveil a novel mechanism and potential target to restrain calpain activity in human disease and shed light on the molecular pathogenesis of KCTD7-associated disease.

Inherited Retinal Degeneration: Towards the Development of a Combination Therapy Targeting Histone Deacetylase, Poly (ADP-Ribose) Polymerase, and Calpain

Inherited retinal degeneration (IRD) represents a diverse group of gene mutation-induced blinding diseases. In IRD, the loss of photoreceptors is often connected to excessive activation of histone-deacetylase (HDAC), poly-ADP-ribose-polymerase (PARP), and calpain-type proteases (calpain). Moreover, the inhibition of either HDACs, PARPs, or calpains has previously shown promise in preventing photoreceptor cell death, although the relationship between these enzyme groups remains unclear. To explore this further, organotypic retinal explant cultures derived from wild-type mice and rd1 mice as a model for IRD were treated with different combinations of inhibitors specific for HDAC, PARP, and calpain. The outcomes were assessed using in situ activity assays for HDAC, PARP, and calpain, immunostaining for activated calpain-2, and the TUNEL assay for cell death detection. We confirmed that inhibition of either HDAC, PARP, or calpain reduced rd1 mouse photoreceptor degeneration, with the HDAC inhibitor Vorinostat (SAHA) being most effective. Calpain activity was reduced by inhibition of both HDAC and PARP whereas PARP activity was only reduced by HDAC inhibition. Unexpectedly, combined treatment with either PARP and calpain inhibitors or HDAC and calpain inhibitors did not produce synergistic rescue of photoreceptors. Together, these results indicate that in rd1 photoreceptors, HDAC, PARP, and calpain are part of the same degenerative pathway and are activated in a sequence that begins with HDAC and ends 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.

Tunicamycin induced inhibition of calpain 1 and 2 enzyme activity in ovarian cancer cells

Background: Tunicamycin (TN) is an antitumor agent and induced intracellular calcium levels in many cells, however its molecular mechanism is still needed to be explored. Calpeptin (Calp) is an inhibitor of both calpain 1 and 2 (CAPN-1/2) enzymes, and plays a fundamental role in tumor mechanism. In this study, the effects of TN and Calp were investigated on CAPN-1/2 enzyme activity in normal and ovarian cancer cells adhered to fibronectin. Methods: 24uM TN, 50uM Calp, and combined TN and Calp (TN+Calp) were applied for 1 and 12 hours to FN-bound (FN+) and non-FN-bound (FN-) normal human ovarian epithelial (IHOSE) and ovarian cancer (SKOV-3) cells. The activation of CAPN-1/2 was measured by the luminescent method and the significance of the results was analyzed with the t-test. Results: CAPN-1/2 enzyme activity (at 12 hour) was present in both cell lines, but the level of enzyme is higher in IHOSE cells compared to SKOV-3 cells. The results showed that 1 hour TN and TN+Calp applications stimulated CAPN-1/2 enzyme activity in IHOSE cells but did not show any stimulating effect in SKOV-3 cells. After 12-hour of treatment, the cells with TN, Calp or TN+Calp showed an inhibitory effect on CAPN-1/2 enzyme in both FN+IHOSE and SKOV-3 cells. At 12-hour TN+Calp administration was determined to be the most effective inhibitor in FN+ SKOV-3 since it inhibited CAPN1/2 activity statistically significantly more than both Calp and TN administrations. Conclusions: The effects of TN, Calp and TN+Calp applications on the CAPN-1/2 enzyme varied according to the cell type, normal or cancer cells, and whether the cell was bound to FN and the incubation period. 12 h administrations of TN, Calp or TN+Calp inhibited the CAPN-1/2 enzyme in both FN+ IHOSE and SKOV-3 cells.