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1.
J Ovarian Res ; 17(1): 79, 2024 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-38610028

RESUMEN

OBJECTIVE: IR emerges as a feature in the pathophysiology of PCOS, precipitating ovulatory anomalies and endometrial dysfunctions that contribute to the infertility challenges characteristic of this condition. Despite its clinical significance, a consensus on the precise mechanisms by which IR exacerbates PCOS is still lacking. This study aims to harness bioinformatics tools to unearth key IR-associated genes in PCOS patients, providing a platform for future therapeutic research and potential intervention strategies. METHODS: We retrieved 4 datasets detailing PCOS from the GEO, and sourced IRGs from the MSigDB. We applied WGCNA to identify gene modules linked to insulin resistance, utilizing IR scores as a phenotypic marker. Gene refinement was executed through the LASSO, SVM, and Boruta feature selection algorithms. qPCR was carried out on selected samples to confirm findings. We predicted both miRNA and lncRNA targets using the ENCORI database, which facilitated the construction of a ceRNA network. Lastly, a drug-target network was derived from the CTD. RESULTS: Thirteen genes related to insulin resistance in PCOS were identified via WGCNA analysis. LASSO, SVM, and Boruta algorithms further isolated CAPN2 as a notably upregulated gene, corroborated by biological verification. The ceRNA network involving lncRNA XIST and hsa-miR-433-3p indicated a possible regulatory link with CAPN2, supported by ENCORI database. Drug prediction analysis uncovered seven pharmacological agents, most being significant regulators of the endocrine system, as potential candidates for addressing insulin resistance in PCOS. CONCLUSIONS: This study highlights the pivotal role of CAPN2 in insulin resistance within the context of PCOS, emphasizing its importance as both a critical biomarker and a potential therapeutic target. By identifying CAPN2, our research contributes to the expanding evidence surrounding the CAPN family, particularly CAPN10, in insulin resistance studies beyond PCOS. This work enriches our understanding of the mechanisms underlying insulin resistance, offering insights that bridge gaps in the current scientific landscape.


Asunto(s)
Resistencia a la Insulina , MicroARNs , Síndrome del Ovario Poliquístico , ARN Largo no Codificante , Humanos , Femenino , Resistencia a la Insulina/genética , Síndrome del Ovario Poliquístico/genética , ARN Largo no Codificante/genética , Algoritmos , Biología Computacional , Calpaína/genética
2.
Orphanet J Rare Dis ; 19(1): 140, 2024 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-38561828

RESUMEN

BACKGROUND: Limb Girdle Muscular Dystrophy R1 (LGMDR1) is an autosomal recessive neuromuscular disease caused by mutations in the calpain-3 (CAPN3) gene. As clinical and pathological features may overlap with other types of LGMD, therefore definite molecular diagnosis is required to understand the progression of this debilitating disease. This study aims to identify novel variants of CAPN3 gene in LGMDR1 patients. RESULTS: Thirty-four patients with clinical and histopathological features suggestive of LGMD were studied. The muscle biopsy samples were evaluated using Enzyme histochemistry, Immunohistochemistry, followed by Western Blotting and Sanger sequencing. Out of 34 LGMD cases, 13 patients were diagnosed as LGMDR1 by immunoblot analysis, demonstrating reduced or absent calpain-3 protein as compared to controls. Variants of CAPN3 gene were also found and pathogenicity was predicted using in-silico prediction tools. The CAPN3 gene variants found in this study, included, two missense variants [CAPN3: c.1189T > C, CAPN3: c.2338G > C], one insertion-deletion [c.1688delinsTC], one splice site variant [c.2051-1G > T], and one nonsense variant [c.1939G > T; p.Glu647Ter]. CONCLUSIONS: We confirmed 6 patients as LGMDR1 (with CAPN3 variants) from our cohort and calpain-3 protein expression was significantly reduced by immunoblot analysis as compared to control. Besides the previously known variants, our study found two novel variants in CAPN3 gene by Sanger sequencing-based approach indicating that genetic variants in LGMDR1 patients may help to understand the etiology of the disease and future prognostication.


Asunto(s)
Calpaína , Distrofia Muscular de Cinturas , Humanos , Calpaína/genética , Distrofia Muscular de Cinturas/diagnóstico , Mutación/genética , Mutación Missense , Proteómica
3.
Commun Biol ; 7(1): 261, 2024 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-38438476

RESUMEN

Calpains are cysteine proteases that control cell fate transitions whose loss of function causes severe, pleiotropic phenotypes in eukaryotes. Although mainly considered as modulatory proteases, human calpain targets are directed to the N-end rule degradation pathway. Several such targets are transcription factors, hinting at a gene-regulatory role. Here, we analyze the gene-regulatory networks of the moss Physcomitrium patens and characterize the regulons that are misregulated in mutants of the calpain DEFECTIVE KERNEL1 (DEK1). Predicted cleavage patterns of the regulatory hierarchies in five DEK1-controlled subnetworks are consistent with a pleiotropic and regulatory role during cell fate transitions targeting multiple functions. Network structure suggests DEK1-gated sequential transitions between cell fates in 2D-to-3D development. Our method combines comprehensive phenotyping, transcriptomics and data science to dissect phenotypic traits, and our model explains the protease function as a switch gatekeeping cell fate transitions potentially also beyond plant development.


Asunto(s)
Bryopsida , Péptido Hidrolasas , Humanos , Calpaína/genética , Endopeptidasas , Diferenciación Celular/genética
4.
Muscle Nerve ; 69(4): 472-476, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38299438

RESUMEN

INTRODUCTION/AIMS: Limb-girdle muscular dystrophy R1 (LGMDR1) calpain 3-related usually presents as a recessively transmitted weakness of proximal limb-girdle muscles due to pathogenic variants in the CAPN3 gene. Pathogenic variants in this gene have also been found in patients with an autosomal dominantly inherited transmission pattern (LGMDD4). The mechanism underlying this difference in transmission patterns has not yet been elucidated. Camptocormia, progressive limb weakness, myalgia, back pain, and increased CK levels are common clinical features associated with dominant forms. The p.Lys254del pathogenic variant was associated with camptocormia in two LGMDD4 families. This study aimed to present carriers found in recessively transmitted LGMDR1 families bearing the p.Lys254del variant that do not show muscle weakness. METHODS: DNA sequencing was performed on exon 5 of CAPN3 in family members to establish the carrier status of the pathogenic variant. They were evaluated clinically and MRI was performed when available. RESULTS: Two families presented with the p.Lys254del pathogenic variant in a homozygous or compound heterozygous state. Family members carrying only the pathogenic variant in the heterozygous state did not demonstrate the myopathic characteristics described in dominant patients. Camptocormia and other severe clinical symptoms were not observed. DISCUSSION: We conclude that the p.Lys254del pathogenic variant per se cannot be solely responsible for camptocormia in dominant patients. Other undisclosed factors may regulate the phenotype associated with the dominant inheritance pattern in CAPN3 pathogenic variant carriers.


Asunto(s)
Calpaína , Atrofia Muscular Espinal , Distrofia Muscular de Cinturas , Curvaturas de la Columna Vertebral , Humanos , Calpaína/genética , Distrofia Muscular de Cinturas/patología , Debilidad Muscular , Familia , Paresia , Mutación/genética , Proteínas Musculares/genética
5.
J Cell Sci ; 137(4)2024 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-38305737

RESUMEN

Tight control over transcription factor activity is necessary for a sensible balance between cellular proliferation and differentiation in the embryo and during tissue homeostasis by adult stem cells, but mechanistic details have remained incomplete. The homeodomain transcription factor MEIS2 is an important regulator of neurogenesis in the ventricular-subventricular zone (V-SVZ) adult stem cell niche in mice. We here identify MEIS2 as direct target of the intracellular protease calpain-2 (composed of the catalytic subunit CAPN2 and the regulatory subunit CAPNS1). Phosphorylation at conserved serine and/or threonine residues, or dimerization with PBX1, reduced the sensitivity of MEIS2 towards cleavage by calpain-2. In the adult V-SVZ, calpain-2 activity is high in stem and progenitor cells, but rapidly declines during neuronal differentiation, which is accompanied by increased stability of MEIS2 full-length protein. In accordance with this, blocking calpain-2 activity in stem and progenitor cells, or overexpression of a cleavage-insensitive form of MEIS2, increased the production of neurons, whereas overexpression of a catalytically active CAPN2 reduced it. Collectively, our results support a key role for calpain-2 in controlling the output of adult V-SVZ neural stem and progenitor cells through cleavage of the neuronal fate determinant MEIS2.


Asunto(s)
Células-Madre Neurales , Factores de Transcripción , Animales , Ratones , Calpaína/genética , Calpaína/metabolismo , Diferenciación Celular , Proliferación Celular , Endopeptidasas/metabolismo , Ventrículos Laterales/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis/genética , Neuronas/metabolismo , Péptido Hidrolasas/metabolismo , Factores de Transcripción/metabolismo
6.
Phytomedicine ; 127: 155392, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38412575

RESUMEN

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.


Asunto(s)
Ferroptosis , Neoplasias Pancreáticas , Humanos , Animales , Ratones , Calpaína/genética , Calpaína/farmacología , Especies Reactivas de Oxígeno/metabolismo , Flavonoides/farmacología , Neoplasias Pancreáticas/patología , Hierro/metabolismo , Homeostasis
7.
FASEB J ; 38(2): e23404, 2024 01 31.
Artículo en Inglés | MEDLINE | ID: mdl-38197290

RESUMEN

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


Asunto(s)
Calpaína , Proteómica , Animales , Ratones , Ratones Endogámicos C57BL , Calpaína/genética , Transporte de Electrón , Complejo I de Transporte de Electrón , Complejo IV de Transporte de Electrones , Estrés del Retículo Endoplásmico , Mitocondrias Cardíacas
8.
Free Radic Biol Med ; 212: 80-93, 2024 02 20.
Artículo en Inglés | MEDLINE | ID: mdl-38151212

RESUMEN

Remote Ischemic Preconditioning (RIPC) can reduce myocardial ischemia-reperfusion injury, but its mechanism is not clear. In order to explore the mechanism of RIPC in myocardial protection, we collected myocardial specimens during cardiac surgery in children with tetralogy of Fallot for sequencing. Our study found RIPC reduces the expression of the calcium channel subunit cacna2d3, thereby impacting the function of calcium channels. As a result, calcium overload during ischemia-reperfusion is reduced, and the activation of calpain 1 is inhibited. This ultimately leads to a decrease in calpain 1 cleavage of Bax, consequently inhibiting increased mitochondrial permeability-mediated apoptosis. Notably, in both murine and human models of myocardial ischemia-reperfusion injury, RIPC inhibiting the expression of the calcium channel subunit cacna2d3 and the activation of calpain 1, improving cardiac function and histological outcomes. Overall, our findings put forth a proposed mechanism that elucidates how RIPC reduces myocardial ischemia-reperfusion injury, ultimately providing a solid theoretical foundation for the widespread clinic application of RIPC.


Asunto(s)
Precondicionamiento Isquémico Miocárdico , Precondicionamiento Isquémico , Daño por Reperfusión Miocárdica , Daño por Reperfusión , Niño , Humanos , Animales , Ratones , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/prevención & control , Daño por Reperfusión Miocárdica/metabolismo , Calpaína/genética , Calpaína/metabolismo , Apoptosis , Canales de Calcio , Daño por Reperfusión/patología
9.
Biochim Biophys Acta Gen Subj ; 1868(1): 130506, 2024 01.
Artículo en Inglés | MEDLINE | ID: mdl-37949151

RESUMEN

BACKGROUND: Ischemia and reperfusion (I/R) injury exacerbate the prognosis of ischemic diseases. The cause of this exacerbation is partly a mitochondrial cell death pathway. Mitochondrial calpain-5 is proteolyzed/autolyzed under endoplasmic reticulum stress, resulting in inflammatory caspase-4 activation. However, the role of calpain-5 in I/R injury remains unclear. We hypothesized that calpain-5 is involved in ischemic brain disease. METHODS: Mitochondria from C57BL/6J mice were extracted via centrifugation with/without proteinase K treatment. The expression and proteolysis/autolysis of calpain-5 were determined using western blotting. The mouse and human brains with I/R injury were analyzed using hematoxylin and eosin staining and immunohistochemistry. HT22 cells were treated with tunicamycin and CAPN5 siRNA. RESULTS: Calpain-5 was expressed in the mitochondria of mouse tissues. Mitochondrial calpain-5 in mouse brains was responsive to calcium earlier than cytosolic calpain-5 in vitro calcium assays and in vivo bilateral common carotid artery occlusion model mice. Immunohistochemistry revealed that neurons were positive for calpain-5 in the normal brains of mice and humans. The expression of calpain-5 was increased in reactive astrocytes at human infarction sites. The knockdown of calpain-5 suppressed of cleaved caspase-11. CONCLUSIONS: The neurons of human and mouse brains express calpain-5, which is proteolyzed/autolyzed in the mitochondria in the early stage of I/R injury and upregulated in reactive astrocytes in the end-stage. GENERAL SIGNIFICANCE: Our results provide a comprehensive understanding of the mechanisms underlying I/R injury. Targeting the expression or activity of mitochondrial calpain-5 may suppress the inflammation during I/R injuries such as cerebrovascular diseases.


Asunto(s)
Isquemia Encefálica , Daño por Reperfusión , Animales , Ratones , Humanos , Calpaína/genética , Calpaína/metabolismo , Calcio/metabolismo , Ratones Endogámicos C57BL , Isquemia Encefálica/genética , Caspasas
10.
Int J Mol Sci ; 24(23)2023 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-38069105

RESUMEN

Calpain is defined as a member of the superfamily of cysteine proteases possessing the CysPC motif within the gene. Calpain-1 and -2, which are categorized as conventional isozymes, execute limited proteolysis in a calcium-dependent fashion. Accordingly, the calpain system participates in physiological and pathological phenomena, including cell migration, apoptosis, and synaptic plasticity. Recent investigations have unveiled the contributions of both conventional and unconventional calpains to the pathogenesis of cardiometabolic disorders. In the context of atherosclerosis, overactivation of conventional calpain attenuates the barrier function of vascular endothelial cells and decreases the immunosuppressive effects attributed to lymphatic endothelial cells. In addition, calpain-6 induces aberrant mRNA splicing in macrophages, conferring atheroprone properties. In terms of diabetes, polymorphisms of the calpain-10 gene can modify insulin secretion and glucose disposal. Moreover, conventional calpain reportedly participates in amino acid production from vascular endothelial cells to induce alteration of amino acid composition in the liver microenvironment, thereby facilitating steatohepatitis. Such multifaceted functionality of calpain underscores its potential as a promising candidate for pharmaceutical targets for the treatment of cardiometabolic diseases. Consequently, the present review highlights the pivotal role of calpains in the complications of cardiometabolic diseases and embarks upon a characterization of calpains as molecular targets.


Asunto(s)
Aterosclerosis , Calpaína , Humanos , Calpaína/genética , Calpaína/metabolismo , Células Endoteliales/metabolismo , Proteolisis , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aminoácidos/metabolismo
11.
Acta Biochim Pol ; 70(4): 963-968, 2023 Dec 08.
Artículo en Inglés | MEDLINE | ID: mdl-38063852

RESUMEN

Calcium-dependent peptidases of the calpain family are widespread in eukaryotes but uncommon in prokaryotes. A few bacterial calpain homologs have been discovered but none of them have been characterized in detail. Here we present an in-depth substrate specificity analysis of the bacterial calpain-like peptidase Tpr from Porphyromonas gingivalis. Using the positional scanning hybrid combinatorial substrate library method, we found that the specificity of Tpr peptidase differs substantially from the papain family of cysteine proteases, showing a strong preference for proline residues at positions P2 and P3. Such a degree of specificity indicates that this P. gingivalis cell-surface peptidase has a more sophisticated role than indiscriminate protein degradation to generate peptide nutrients, and may fulfil virulence-related functions such as immune evasion.


Asunto(s)
Péptido Hidrolasas , Porphyromonas gingivalis , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/metabolismo , Péptido Hidrolasas/metabolismo , Calpaína/genética , Calpaína/metabolismo , Especificidad por Sustrato , Endopeptidasas/metabolismo
12.
Int J Mol Sci ; 24(24)2023 Dec 13.
Artículo en Inglés | MEDLINE | ID: mdl-38139275

RESUMEN

Zinc (Zn) and copper (Cu) have been shown to have the potential to improve glucose metabolism through interactions with cytokines and signaling events with multiple genes. miRNA-375 and the Calpin-10 gene are potential genetic biomarkers for the early prediction of diabetic nephropathy (DN). 128 healthy controls and 129 type 2 diabetic (T2DM) participants were matched for age and sex. Three subgroups were identified from the T2DM group: 39 patients had microalbuminuria, 41 had macroalbuminuria, and 49 patients had renal problems. Circulating miR-375 expression levels were measured via qPCR. Calpain-10 SNP 19 (rs3842570) genotyping was assessed with allele-specific PCR in all the included participants. Spectrophotometry was used to measure the concentrations of serum copper, zinc, and magnesium, while ELISA was used to measure the levels of TGF-ß and IL-17. There was significant up-regulation in the expression of miR-375 and serum levels of TGF-ß, IL-17, Cu, and the Cu/Zn ratio, whereas, in contrast to the control group, the Zn and Mg levels were lower in the T2DM group. The DN groups had significantly lower miR-375, TGF-ß, IL-17, Mg, and Zn levels compared with the T2DM without nephropathy group. Furthermore, between TGF-ß, IL-17, and miRNA-375, there were notable correlations. Calpain-10 SNP 19 genotype 22 and allele 2 were linked to a higher incidence of T2DM and DN. Significant TGF-ß, Cu, Cu/Zn ratio, HbAc1, and creatinine levels, but insignificant miRNA-375 levels, were associated with genotype 22 of Calpain-10 SNP 19. interactions between the Calpain-10 SNP 19 genotype 22 and IL-17, TGF-ß, mineral levels, and miRNA-375 might contribute to the aetiology of DN and T2DM and may have clinical implications for diagnosis and management.


Asunto(s)
Diabetes Mellitus Tipo 2 , Nefropatías Diabéticas , Interleucina-17 , MicroARNs , Factor de Crecimiento Transformador beta , Humanos , Calpaína/genética , Cobre , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/genética , Interleucina-17/metabolismo , MicroARNs/genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Zinc
13.
Endocr Regul ; 57(1): 221-234, 2023 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-37823570

RESUMEN

Objective. Nowadays, type 2 diabetes mellitus (T2DM) is the most common chronic endocrine disorder, affecting an estimated 5-10% of adults worldwide and this disease rapidly increases in the Kurdistan region population. This research aims to identify DNA methylation change in the CPAN10 gene as a predictive biomarker in T2DM and the association between DNA methylation status with lipid profile and kidney function test. Methods. The participants (113) were divided into three groups: diabetes group (47), prediabetes group (36), and control group (30). The study was carried out on patients who visited the private clinical sectors between August and December 2021 in the Koya city Kurdistan region of Iraq. To determine DNA methylation status, methylation-specific PCR (MPS) with paired primer for each methylated and unmethylated region was used. The Mann-Whitney U test and Spearman's correlation were performed for statistical analysis of data and a value of p<0.05 was considered significant. Results. The obtained results show that DNA hypermethylation was recorded in the promoter region in the samples of the diabetes and prediabetes groups compared to the healthy group (control). Various factors also affected the level of DNA methylation, such as HbA1c in prediabetes group and body mass index in the control group. Conclusion. These results indicate that DNA methylation changes in the CAPN10 gene promoter region may be used as a potential predictive biomarker to diagnose T2DM; however, this study requires further data to support this evidence.


Asunto(s)
Calpaína , Diabetes Mellitus Tipo 2 , Estado Prediabético , Adulto , Humanos , Biomarcadores , Glucemia/análisis , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/genética , Metilación de ADN/genética , Hemoglobina Glucada/genética , Pruebas de Función Renal , Lípidos , Estado Prediabético/epidemiología , Factores de Riesgo , Calpaína/genética
14.
Free Radic Biol Med ; 208: 700-707, 2023 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-37748718

RESUMEN

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.


Asunto(s)
Síndrome Metabólico , Isquemia Miocárdica , Porcinos , Animales , Miocardio/metabolismo , Calpaína/genética , Calpaína/metabolismo , Calpaína/farmacología , Síndrome Metabólico/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Carbonilación Proteica , Isquemia Miocárdica/tratamiento farmacológico , Isquemia Miocárdica/metabolismo , Estrés Oxidativo , Proteínas Mitocondriales/metabolismo , Modelos Animales de Enfermedad
15.
HGG Adv ; 4(4): 100232, 2023 10 12.
Artículo en Inglés | MEDLINE | ID: mdl-37663545

RESUMEN

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart defect (CHD) characterized by hypoplasia of the left ventricle and aorta along with stenosis or atresia of the aortic and mitral valves. HLHS represents only ∼4%-8% of all CHDs but accounts for ∼25% of deaths. HLHS is an isolated defect (i.e., iHLHS) in 70% of families, the vast majority of which are simplex. Despite intense investigation, the genetic basis of iHLHS remains largely unknown. We performed exome sequencing on 331 families with iHLHS aggregated from four independent cohorts. A Mendelian-model-based analysis demonstrated that iHLHS was not due to single, large-effect alleles in genes previously reported to underlie iHLHS or CHD in >90% of families in this cohort. Gene-based association testing identified increased risk for iHLHS associated with variation in CAPN2 (p = 1.8 × 10-5), encoding a protein involved in functional adhesion. Functional validation studies in a vertebrate animal model (Xenopus laevis) confirmed CAPN2 is essential for cardiac ventricle morphogenesis and that in vivo loss of calpain function causes hypoplastic ventricle phenotypes and suggest that human CAPN2707C>T and CAPN21112C>T variants, each found in multiple individuals with iHLHS, are hypomorphic alleles. Collectively, our findings show that iHLHS is typically not a Mendelian condition, demonstrate that CAPN2 variants increase risk of iHLHS, and identify a novel pathway involved in HLHS pathogenesis.


Asunto(s)
Síndrome del Corazón Izquierdo Hipoplásico , Animales , Humanos , Síndrome del Corazón Izquierdo Hipoplásico/genética , Alelos , Aorta , Calpaína/genética , Ventrículos Cerebrales
16.
Neurogenetics ; 24(4): 273-278, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37589857

RESUMEN

Both, recessive (LGMD R1) and dominant (LGMD D4) inheritance occur in calpain 3-related muscular dystrophy. We report a family with calpain-related muscular dystrophy caused by two known variants in the calpain 3 gene (CAPN3, NM_000070.3; (I) c.700G>A, p.Gly234Arg and (II) c.1746-20C>G, p.?). Three family members are compound heterozygous and exhibit a relatively homogeneous phenotype characterized by progressive proximal weakness starting in the third to fourth decade of life in the shoulder girdle and spreading to the legs. Two family members affected only by the p.Gly234Arg heterozygous missense variants show a different phenotype characterized by severe exertional myalgia without overt pareses. We conclude that in our family, the missense variant causes a severe myalgic phenotype without pareses that is aggravated by the second intronic variant and put these findings in the context of previous studies of the same variants.


Asunto(s)
Distrofia Muscular de Cinturas , Distrofias Musculares , Humanos , Calpaína/genética , Distrofia Muscular de Cinturas/genética , Proteínas Musculares , Mutación , Fenotipo , Paresia
17.
Behav Brain Res ; 454: 114635, 2023 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-37598906

RESUMEN

Calpain 15 (CAPN15) is an intracellular cysteine protease belonging to the non-classical small optic lobe (SOL) family of calpains, which has an important role in development. Loss of Capn15 in mice leads to developmental eye anomalies and volumetric changes in the brain. Human individuals with biallelic variants in CAPN15 have developmental delay, neurodevelopmental disorders, as well as congenital malformations. In Aplysia, a reductionist model to study learning and memory, SOL calpain is important for non-associative long-term facilitation, the cellular analog of sensitization behavior. However, how CAPN15 is involved in adult behavior or learning and memory in vertebrates is unknown. Here, using Capn15 conditional knockout mice, we show that loss of the CAPN15 protein in excitatory forebrain neurons reduces self-grooming and marble burying, decreases performance in the accelerated roto-rod and reduces pre-tone freezing after strong fear conditioning. Thus, CAPN15 plays a role in regulating behavior in the adult mouse.


Asunto(s)
Aplysia , Calpaína , Animales , Ratones , Calpaína/genética , Ratones Noqueados , Prosencéfalo
18.
ESC Heart Fail ; 10(5): 3077-3090, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37602925

RESUMEN

AIMS: Molecular hydrogen has been exhibited a protective function in heart diseases. Our previous study demonstrated that hydrogen-rich saline (HRS) could scavenge free radicals selectively and alleviate the inflammatory response in the myocardial ischaemia/reperfusion (I/R) injury, but the underlying mechanism has not been fully clarified. METHODS AND RESULTS: Adult (10 weeks) C57BL/6 male mice and neonatal rat cardiomyocytes were used to establish I/R and hypoxia/reoxygenation (H/R) injury models. I/R and H/R models were treated with HRS to classify the mechanisms of cardioproctective function. In this study, we found that miR-124-3p was significantly decreased in both I/R and H/R models, while it was partially ameliorated by HRS pretreatment. HRS treatment also alleviated ischaemia-induced apoptotic cell death and increased cell viability during I/R process, whereas silencing expression of miR-124-3p abolished this protective effect. In addition, we identified calpain1 as a direct target of miR-124-3p, and up-regulation of miR-124-3 produced both activity and expression of calpain1. It was also found that compared with the HRS group, overexpression of calpain1 increased caspase-3 activities, promoted cleaved-caspase3 and Bax protein expressions, and correspondingly decreased Bcl-2, further reducing cell viability. These results illustrated that calpain1 overexpression attenuated protective effect of HRS on cardiomyocytes in H/R model. CONCLUSIONS: The present study showed a protective effect of HRS on I/R injury, which may be associated with miR-124-3p-calpain1 signalling pathway.


Asunto(s)
Calpaína , MicroARNs , Daño por Reperfusión Miocárdica , Animales , Masculino , Ratones , Ratas , Apoptosis , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/metabolismo , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/metabolismo , Miocitos Cardíacos/metabolismo , Calpaína/genética , Calpaína/metabolismo
19.
Am J Med Genet A ; 191(11): 2757-2767, 2023 11.
Artículo en Inglés | MEDLINE | ID: mdl-37596828

RESUMEN

Oculogastrointestinal neurodevelopmental syndrome has been described in seven previously published individuals who harbor biallelic pathogenic variants in the CAPN15 gene. Biallelic missense variants have been reported to demonstrate a phenotype of eye abnormalities and developmental delay, while biallelic loss of function variants exhibit phenotypes including microcephaly and craniofacial abnormalities, cardiac and genitourinary malformations, and abnormal neurologic activity. We report six individuals from three unrelated families harboring biallelic deleterious variants in CAPN15 with phenotypes overlapping those previously described for this disorder. Of the individuals affected, four demonstrate radiographic evidence of the classical triad of Dandy-Walker malformation including hypoplastic vermis, fourth ventricle enlargement, and torcular elevation. Cerebellar anomalies have not been previously reported in association with CAPN15-related disease. Here, we present three unrelated families with findings consistent with oculogastrointestinal neurodevelopmental syndrome and cerebellar pathology including Dandy-Walker malformation. To corroborate these novel clinical findings, we present supporting data from the mouse model suggesting an important role for this protein in normal cerebellar development. Our findings add six molecularly confirmed cases to the literature and additionally establish a new association of Dandy-Walker malformation with biallelic CAPN15 variants, thereby expanding the neurologic spectrum among patients affected by CAPN15-related disease.


Asunto(s)
Vermis Cerebeloso , Síndrome de Dandy-Walker , Microcefalia , Animales , Ratones , Humanos , Síndrome de Dandy-Walker/diagnóstico , Síndrome de Dandy-Walker/genética , Cerebelo/anomalías , Microcefalia/complicaciones , Fenotipo , Calpaína/genética
20.
mBio ; 14(4): e0074923, 2023 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-37477430

RESUMEN

The human cytomegalovirus (HCMV) US12 gene family contributes to virus-host interactions by regulating the virus' cell tropism and its evasion of host innate immune responses. US21, one of the 10 US12 genes (US12-US21), is a descendant of a captured cellular transmembrane BAX inhibitor motif-containing gene. It encodes a 7TMD endoplasmic reticulum (ER)-resident viroporin (pUS21) capable of reducing the Ca2+ content of ER stores, which, in turn, protects cells against apoptosis. Since regulation of Ca2+ homeostasis affects a broad range of cellular responses, including cell motility, we investigated whether pUS21 might also interfere with this cytobiological consequence of Ca2+ signaling. Indeed, deletion of the US21 gene impaired the ability of HCMV-infected cells to migrate, whereas expression of US21 protein stimulated cell migration and adhesion, as well as focal adhesion (FA) dynamics, in a way that depended on its ability to manipulate ER Ca2+ content. Mechanistic studies revealed pUS21-mediated cell migration to involve calpain 2 activation since its inhibition prevented the viroporin's effects on cell motility. Pertinently, pUS21 expression stimulated a store-operated Ca2+ entry (SOCE) mechanism that may determine the activation of calpain 2 by promoting Ca2+ entry. Furthermore, pUS21 was observed to interact with talin-1, a calpain 2 substrate, and crucial protein component of FA complexes. A functional consequence of this interaction was confirmed by talin-1 knockdown, which abrogated the pUS21-mediated increase in cell migration. Together, these results indicate the US21-encoded viroporin to be a viral regulator of cell adhesion and migration in the context of HCMV infection. IMPORTANCE Human cytomegalovirus (HCMV) is an opportunistic pathogen that owes part of its success to the capture, duplication, and tuning of cellular genes to generate modern viral proteins which promote infection and persistence in the host by interfering with many cell biochemical and physiological pathways. The US21 viral protein provides an example of this evolutionary strategy: it is a cellular-derived calcium channel that manipulates intracellular calcium homeostasis to confer edges to HCMV replication. Here, we report on the characterization of a novel function of the US21 protein as a viral regulator of cell migration and adhesion through mechanisms involving its calcium channel activity. Characterization of HCMV multifunctional regulatory proteins, like US21, supports the better understanding of viral pathogenesis and may open avenues for the design of new antiviral strategies that exploit their functions.


Asunto(s)
Citomegalovirus , Proteínas Viroporinas , Humanos , Citomegalovirus/fisiología , Proteínas Viroporinas/metabolismo , Calpaína/genética , Calpaína/metabolismo , Talina/metabolismo , Proteínas Virales/metabolismo , Canales de Calcio/metabolismo , Movimiento Celular
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