ATR prevents Ca2+ overload-induced necrotic cell death through phosphorylation-mediated inactivation of PARP1 without DNA damage signaling.
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ABSTRACT:
SUBMITTER: Li Z
PROVIDER: S-EPMC8252533 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mitochondrial Ca2+ overload can mediate mitochondria-dependent cell death, a major contributor to several human diseases. Indeed, Duchenne muscular dystrophy (MD) is driven by dysfunctional Ca2+ influx across the sarcolemma that causes mitochondrial Ca2+ overload, organelle rupture, and muscle necrosis. The mitochondrial Ca2+ uniporter (MCU) complex is the primary characterized mechanism for acute mitochondrial Ca2+ uptake. One strategy for preventing mitochondrial Ca2+ overload is deletion of the Mcu gene, the pore forming subunit of the MCU-complex. Conversely, enhanced MCU-complex Ca2+ uptake is achieved by deleting the inhibitory Mcub gene. Here we show that myofiber-specific Mcu deletion was not protective in a mouse model of Duchenne MD. Specifically, Mcu gene deletion did not reduce muscle histopathology, did not improve muscle function, and did not prevent mitochondrial Ca2+ overload. Moreover, myofiber specific Mcub gene deletion did not augment Duchenne MD muscle pathology. Interestingly, we observed MCU-independent Ca2+ uptake in dystrophic mitochondria that was sufficient to drive mitochondrial permeability transition pore (MPTP) activation and skeletal muscle necrosis, and this same type of activity was observed in heart, liver, and brain mitochondria. These results demonstrate that mitochondria possess an uncharacterized MCU-independent Ca2+ uptake mechanism that is sufficient to drive MPTP-dependent necrosis in MD in vivo.
| S-EPMC10957967 | biostudies-literature
Project description:In budding yeast, a single DNA double-strand break (DSB) triggers the activation of Mec1ATR-dependent DNA damage checkpoint. After about 12 h, cells turn off the checkpoint signaling and adapt despite the persistence of the DSB. We report that the adaptation involves the autophosphorylation of Mec1 at site S1964. A non-phosphorylatable mec1-S1964A mutant causes cells to arrest permanently in response to a single DSB without affecting the initial kinase activity of Mec1. Autophosphorylation of S1964 is dependent on Ddc1Rad9 and Dpb11TopBP1, and it correlates with the timing of adaptation. We also report that Mec1's binding partner, Ddc2ATRIP, is an inherently stable protein that is degraded specifically upon DNA damage. Ddc2 is regulated extensively through phosphorylation, which, in turn, regulates the localization of the Mec1-Ddc2 complex to DNA lesions. Taken together, these results suggest that checkpoint response is regulated through the autophosphorylation of Mec1 kinase and through the changes in Ddc2 abundance and phosphorylation.
| S-EPMC7218798 | biostudies-literature
Project description:Disuse atrophy of skeletal muscle is associated with a severe imbalance in cellular Ca2+ homeostasis and marked increase in nuclear apoptosis. Nuclear Ca2+ is involved in the regulation of cellular Ca2+ homeostasis. However, it remains unclear whether nuclear Ca2+ levels change under skeletal muscle disuse conditions, and whether changes in nuclear Ca2+ levels are associated with nuclear apoptosis. In this study, changes in Ca2+ levels, Ca2+ transporters, and regulatory factors in the nucleus of hindlimb unloaded rat soleus muscle were examined to investigate the effects of disuse on nuclear Ca2+ homeostasis and apoptosis. Results showed that, after hindlimb unloading, the nuclear envelope Ca2+ levels ([Ca2+]NE) and nucleocytoplasmic Ca2+ levels ([Ca2+]NC) increased by 78% (p < 0.01) and 106% (p < 0.01), respectively. The levels of Ca2+-ATPase type 2 (Ca2+-ATPase2), Ryanodine receptor 1 (RyR1), Inositol 1,4,5-tetrakisphosphate receptor 1 (IP3R1), Cyclic ADP ribose hydrolase (CD38) and Inositol 1,4,5-tetrakisphosphate (IP3) increased by 470% (p < 0.001), 94% (p < 0.05), 170% (p < 0.001), 640% (p < 0.001) and 12% (p < 0.05), respectively, and the levels of Na+/Ca2+ exchanger 3 (NCX3), Ca2+/calmodulin dependent protein kinase II (CaMK II) and Protein kinase A (PKA) decreased by 54% (p < 0.001), 33% (p < 0.05) and 5% (p > 0.05), respectively. In addition, DNase X is mainly localized in the myonucleus and its activity is elevated after hindlimb unloading. Overall, our results suggest that enhanced Ca2+ uptake from cytoplasm is involved in the increase in [Ca2+]NE after hindlimb unloading. Moreover, the increase in [Ca2+]NC is attributed to increased Ca2+ release into nucleocytoplasm and weakened Ca2+ uptake from nucleocytoplasm. DNase X is activated due to elevated [Ca2+]NC, leading to DNA fragmentation in myonucleus, ultimately initiating myonuclear apoptosis. Nucleocytoplasmic Ca2+ overload may contribute to the increased incidence of myonuclear apoptosis in disused skeletal muscle.
| S-EPMC10093444 | biostudies-literature
Project description:BackgroundCardiac hypertrophy and heart failure are characterized by increased late sodium current and abnormal Ca2+ handling. Ranolazine, a selective inhibitor of the late sodium current, can reduce sodium accumulation and Ca 2+ overload. In this study, we investigated the effects of ranolazine on pressure overload-induced cardiac hypertrophy and heart failure in mice.Methods and resultsInhibition of late sodium current with the selective inhibitor ranolazine suppressed cardiac hypertrophy and fibrosis and improved heart function assessed by echocardiography, hemodynamics, and histological analysis in mice exposed to chronic pressure overload induced by transverse aortic constriction (TAC). Ca2+ imaging of ventricular myocytes from TAC mice revealed both abnormal SR Ca 2+ release and increased SR Ca 2+ leak. Ranolazine restored aberrant SR Ca 2+ handling induced by pressure overload. Ranolazine also suppressed Na + overload induced in the failing heart, and restored Na + -induced Ca 2+ overload in an sodium-calcium exchanger (NCX)-dependent manner. Ranolazine suppressed the Ca 2+ -dependent calmodulin (CaM)/CaMKII/myocyte enhancer factor-2 (MEF2) and CaM/CaMKII/calcineurin/nuclear factor of activated T-cells (NFAT) hypertrophy signaling pathways triggered by pressure overload. Pressure overload also prolonged endoplasmic reticulum (ER) stress leading to ER-initiated apoptosis, while inhibition of late sodium current or NCX relieved ER stress and ER-initiated cardiomyocyte apoptosis.ConclusionsOur study demonstrates that inhibition of late sodium current with ranolazine improves pressure overload-induced cardiac hypertrophy and systolic and diastolic function by restoring Na+ and Ca 2+ handling, inhibiting the downstream hypertrophic pathways and ER stress. Inhibition of late sodium current may provide a new treatment strategy for cardiac hypertrophy and heart failure.
| S-EPMC6515545 | biostudies-literature
Project description:BackgroundSERPINA3K, an extracellular serine proteinase inhibitor (serpin), has been shown to have decreased levels in the retinas of diabetic rats, which may contribute to diabetic retinopathy. The function of SERPINA3K in the retina has not been investigated.Methodology/principal findingsThe present study identified a novel function of SERPINA3K, i.e. it protects retinal cells against oxidative stress-induced cell death including retinal neuronal cells and Müller cells. Flow-cytometry showed that the protective effect of SERPINA3K on Müller cells is via reducing oxidation-induced necrosis. Measurements of intracellular calcium concentration showed that SERPINA3K prevented the intracellular calcium overload induced by H(2)O(2). A similar protective effect was observed using a calcium chelator (BAPTA/AM). Further, SERPINA3K inhibited the phosphorylation of phospholipase C (PLC)-gamma1 induced by H(2)O(2). Likewise, a specific PLC inhibitor showed similar protective effects on Müller cells exposed to H(2)O(2). Furthermore, the protective effect of SERPINA3K was attenuated by a specific PLC activator (m-3M3FBS). Finally, in a binding assay, SERPINA3K displayed saturable and specific binding on Müller cells.Conclusion/significanceThese results for the first time demonstrate that SERPINA3K is an endogenous serpin which protects cells from oxidative stress-induced cells death, and its protective effect is via blocking the calcium overload through the PLC pathway. The decreased retinal levels of SERPINA3K may represent a new pathogenic mechanism for the retinal Müller cell dysfunction and neuron loss in diabetes.
| S-EPMC2605247 | biostudies-literature
Project description:Overproduction of reactive oxygen species (ROS), metal ion accumulation, and tricarboxylic acid cycle collapse are crucial factors in mitochondria-mediated cell death. However, the highly adaptive nature and damage-repair capabilities of malignant tumors strongly limit the efficacy of treatments based on a single treatment mode. To address this challenge, a self-reinforced bimetallic Mito-Jammer is developed by incorporating doxorubicin (DOX) and calcium peroxide (CaO2) into hyaluronic acid (HA) -modified metal-organic frameworks (MOF). After cellular, Mito-Jammer dissociates into CaO2 and Cu2+ in the tumor microenvironment. The exposed CaO2 further yields hydrogen peroxide (H2O2) and Ca2+ in a weakly acidic environment to strengthen the Cu2+-based Fenton-like reaction. Furthermore, the combination of chemodynamic therapy and Ca2+ overload exacerbates ROS storms and mitochondrial damage, resulting in the downregulation of intracellular adenosine triphosphate (ATP) levels and blocking of Cu-ATPase to sensitize cuproptosis. This multilevel interaction strategy also activates robust immunogenic cell death and suppresses tumor metastasis simultaneously. This study presents a multivariate model for revolutionizing mitochondria damage, relying on the continuous retention of bimetallic ions to boost cuproptosis/immunotherapy in cancer.
| S-EPMC11022715 | biostudies-literature
Project description:Mitochondrial dysfunction impairs muscle health and causes subsequent muscle wasting. This study explores the role of mitochondrial dysfunction as an intramuscular signal for the extracellular matrix (ECM)-based proteolysis and, consequentially, muscle cell dystrophy. We found that inhibition of the mitochondrial electron transport chain causes paralysis as well as muscle structural damage in the nematode Caenorhabditis elegans. This was associated with a significant decline in collagen content. Both paralysis and muscle damage could be rescued with collagen IV overexpression, matrix metalloproteinase (MMP), and Furin inhibitors in Antimycin A-treated animal as well as in the C. elegans Duchenne muscular dystrophy model. Additionally, muscle cytosolic calcium increased in the Antimycin A-treated worms, and its down-regulation rescued the muscle damage, suggesting that calcium overload acts as one of the early triggers and activates Furin and MMPs for collagen degradation. In conclusion, we have established ECM degradation as an important pathway of muscle damage.-Sudevan, S., Takiura, M., Kubota, Y., Higashitani, N., Cooke, M., Ellwood, R. A., Etheridge, T., Szewczyk, N. J., Higashitani, A. Mitochondrial dysfunction causes Ca2+ overload and ECM degradation-mediated muscle damage in C. elegans.
| S-EPMC6662967 | biostudies-literature
Project description:Augmented Na(+) /Ca(2+) exchanger (NCX) activity may play a crucial role in cardiac arrhythmogenesis; however, data regarding the anti-arrhythmic efficacy of NCX inhibition are debatable. Feasible explanations could be the unsatisfactory selectivity of NCX inhibitors and/or the dependence of the experimental model on the degree of Ca(2+) i overload. Hence, we used NCX inhibitors SEA0400 and the more selective ORM10103 to evaluate the efficacy of NCX inhibition against arrhythmogenic Ca(2+) i rise in conditions when [Ca(2+) ]i was augmented via activation of the late sodium current (INaL ) or inhibition of the Na(+) /K(+) pump.Action potentials (APs) were recorded from canine papillary muscles and Purkinje fibres by microelectrodes. NCX current (INCX ) was determined in ventricular cardiomyocytes utilizing the whole-cell patch clamp technique. Ca(2+) i transients (CaTs) were monitored with a Ca(2+) -sensitive fluorescent dye, Fluo-4.Enhanced INaL increased the Ca(2+) load and AP duration (APD). SEA0400 and ORM10103 suppressed INCX and prevented/reversed the anemone toxin II (ATX-II)-induced [Ca(2+) ]i rise without influencing APD, CaT or cell shortening, or affecting the ATX-II-induced increased APD. ORM10103 significantly decreased the number of strophanthidin-induced spontaneous diastolic Ca(2+) release events; however, SEA0400 failed to restrict the veratridine-induced augmentation in Purkinje-ventricle APD dispersion.Selective NCX inhibition - presumably by blocking rev INCX (reverse mode NCX current) - is effective against arrhythmogenesis caused by [Na(+) ]i -induced [Ca(2+) ]i elevation, without influencing the AP waveform. Therefore, selective INCX inhibition, by significantly reducing the arrhythmogenic trigger activity caused by the perturbed Ca(2+) i handling, should be considered as a promising anti-arrhythmic therapeutic strategy.
| S-EPMC4290709 | biostudies-literature
Project description:BackgroundSepsis is a systemic inflammatory response to a local severe infection that may lead to multiple organ failure and death. Previous studies have shown that 40-50% of patients with sepsis have diverse myocardial injuries and 70 to 90% mortality rates compared to 20% mortality in patients with sepsis without myocardial injury. Therefore, uncovering the mechanism of sepsis-induced myocardial injury and finding a target-based treatment are immensely important.ObjectiveThe present study elucidated the mechanism of sepsis-induced myocardial injury and examined the value of human umbilical cord mesenchymal stem cells (huMSCs) for protecting cardiac function in sepsis.MethodsWe used cecal ligation and puncture (CLP) to induce sepsis in mice and detect myocardial injury and cardiac function using serological markers and echocardiography. Cardiomyocyte apoptosis and heart tissue ultrastructure were detected using TdT-mediated dUTP Nick-End Labeling (TUNEL) and transmission electron microscopy (TEM), respectively. Fura-2 AM was used to monitor Ca2+ uptake and efflux in mitochondria. FQ-PCR and Western blotting detected expression of mitochondrial Ca2+ distribution regulators and PTEN-induced putative kinase 1 (PINK1). JC-1 was used to detect the mitochondrial membrane potential (Δψm) of cardiomyocytes.ResultsWe found that expression of PINK1 decreased in mouse hearts during sepsis, which caused cardiomyocyte mitochondrial Ca2+ efflux disorder, mitochondrial calcium overload, and cardiomyocyte injury. In contrast, we found that exosomes isolated from huMSCs (huMSC-exo) carried Pink1 mRNA, which could be transferred to recipient cardiomyocytes to increase PINK1 expression. The reduction in cardiomyocyte mitochondrial calcium efflux was reversed, and cardiomyocytes recovered from injury. We confirmed the effect of the PINK1-PKA-NCLX axis on mitochondrial calcium homeostasis in cardiomyocytes during sepsis.ConclusionThe PINK1-PKA-NCLX axis plays an important role in mitochondrial calcium efflux in cardiomyocytes. Therefore, PINK1 may be a therapeutic target to protect cardiomyocyte mitochondria, and the application of huMSC-exo is a promising strategy against sepsis-induced heart dysfunction.
| S-EPMC8101124 | biostudies-literature
Project description:Stac protein (named for its SH3- and cysteine-rich domains) was first identified in brain 20 years ago and is currently known to have three isoforms. Stac2, Stac1, and Stac3 transcripts are found at high, modest, and very low levels, respectively, in the cerebellum and forebrain, but their neuronal functions have been little investigated. Here, we tested the effects of Stac proteins on neuronal, high-voltage-activated Ca2+ channels. Overexpression of the three Stac isoforms eliminated Ca2+-dependent inactivation (CDI) of l-type current in rat neonatal hippocampal neurons (sex unknown), but not CDI of non-l-type current. Using heterologous expression in tsA201 cells (together with β and α2-δ1 auxiliary subunits), we found that CDI for CaV1.2 and CaV1.3 (the predominant, neuronal l-type Ca2+ channels) was suppressed by all three Stac isoforms, whereas CDI for the P/Q channel, CaV2.1, was not. For CaV1.2, the inhibition of CDI by the Stac proteins appeared to involve their direct interaction with the channel's C terminus. Within the Stac proteins, a weakly conserved segment containing ∼100 residues and linking the structurally conserved PKC C1 and SH3_1 domains was sufficient to fully suppress CDI. The presence of CDI for l-type current in control neonatal neurons raised the possibility that endogenous Stac levels are low in these neurons and Western blotting indicated that the expression of Stac2 was substantially increased in adult forebrain and cerebellum compared with neonate. Together, our results indicate that one likely function of neuronal Stac proteins is to tune Ca2+ entry via neuronal l-type channels.SIGNIFICANCE STATEMENT Stac protein, first identified 20 years ago in brain, has recently been found to be essential for proper trafficking and function of the skeletal muscle l-type Ca2+ channel and is the site of mutations causing a severe, inherited human myopathy. In neurons, however, functions for Stac protein have remained unexplored. Here, we report that one likely function of neuronal Stac proteins is tuning Ca2+ entry via l-type, but not that via non-l-type, Ca2+ channels. Moreover, there is a large postnatal increase in protein levels of the major neuronal isoform (Stac2) in forebrain and cerebellum, which could provide developmental regulation of l-type channel Ca2+ signaling in these brain regions.
| S-EPMC6199411 | biostudies-literature