Mitochondrial calcium (sub.mCasup.2+) has a central role in both metabolic regulation and cell death signalling, however its role in homeostatic function and disease is controversial. Slc8b1 encodes ...the mitochondrial Nasup.+/Casup.2+ exchanger (NCLX), which is proposed to be the primary mechanism for sub.mCasup.2+ extrusion in excitable cells. Here we show that tamoxifen-induced deletion of Slc8b1 in adult mouse hearts causes sudden death, with less than 13% of affected mice surviving after 14 days. Lethality correlated with severe myocardial dysfunction and fulminant heart failure. Mechanistically, cardiac pathology was attributed to sub.mCasup.2+ overload driving increased generation of superoxide and necrotic cell death, which was rescued by genetic inhibition of mitochondrial permeability transition pore activation. Corroborating these findings, overexpression of NCLX in the mouse heart by conditional transgenesis had the beneficial effect of augmenting sub.mCasup.2+ clearance, preventing permeability transition and protecting against ischaemia-induced cardiomyocyte necrosis and heart failure. These results demonstrate the essential nature of sub.mCasup.2+ efflux in cellular function and suggest that augmenting sub.mCasup.2+ efflux may be a viable therapeutic strategy in disease.
RATIONALE:Cardiac myocyte–specific deletion of either glycogen synthase kinase (GSK)-3α and GSK-3β leads to cardiac protection after myocardial infarction, suggesting that deletion of both isoforms ...may provide synergistic protection. This is an important consideration because of the fact that all GSK-3–targeted drugs, including the drugs already in clinical trial target both isoforms of GSK-3, and none are isoform specific.
OBJECTIVE:To identify the consequences of combined deletion of cardiac myocyte GSK-3α and GSK-3β in heart function.
METHODS AND RESULTS:We generated tamoxifen-inducible cardiac myocyte–specific mice lacking both GSK-3 isoforms (double knockout). We unexpectedly found that cardiac myocyte GSK-3 is essential for cardiac homeostasis and overall survival. Serial echocardiographic analysis reveals that within 2 weeks of tamoxifen treatment, double-knockout hearts leads to excessive dilatative remodeling and ventricular dysfunction. Further experimentation with isolated adult cardiac myocytes and fibroblasts from double-knockout implicated cardiac myocytes intrinsic factors responsible for observed phenotype. Mechanistically, loss of GSK-3 in adult cardiac myocytes resulted in induction of mitotic catastrophe, a previously unreported event in cardiac myocytes. Double-knockout cardiac myocytes showed cell cycle progression resulting in increased DNA content and multinucleation. However, increased cell cycle activity was rivaled by marked activation of DNA damage, cell cycle checkpoint activation, and mitotic catastrophe–induced apoptotic cell death. Importantly, mitotic catastrophe was also confirmed in isolated adult cardiac myocytes.
CONCLUSIONS:Together, our findings suggest that cardiac myocyte GSK-3 is required to maintain normal cardiac homeostasis, and its loss is incompatible with life because of cell cycle dysregulation that ultimately results in a severe fatal dilated cardiomyopathy.
OBJECTIVE—Hyperlipidemia-induced endothelial cell (EC) activation is considered as an initial event responsible for monocyte recruitment in atherogenesis. However, it remains poorly defined what is ...the mechanism underlying hyperlipidemia-induced EC activation. Here, we tested a novel hypothesis that mitochondrial reactive oxygen species (mtROS) serve as signaling mediators for EC activation in early atherosclerosis.
APPROACH AND RESULTS—Metabolomics and transcriptomics analyses revealed that several lysophosphatidylcholine (LPC) species, such as 16:0, 18:0, and 18:1, and their processing enzymes, including Pla2g7 and Pla2g4c, were significantly induced in the aortas of apolipoprotein E knockout mice during early atherosclerosis. Using electron spin resonance and flow cytometry, we found that LPC 16:0, 18:0, and 18:1 induced mtROS in primary human aortic ECs, independently of the activities of nicotinamide adenine dinucleotide phosphate oxidase. Mechanistically, using confocal microscopy and Seahorse XF mitochondrial analyzer, we showed that LPC induced mtROS via unique calcium entry–mediated increase of proton leak and mitochondrial O2 reduction. In addition, we found that mtROS contributed to LPC-induced EC activation by regulating nuclear binding of activator protein-1 and inducing intercellular adhesion molecule-1 gene expression in vitro. Furthermore, we showed that mtROS inhibitor MitoTEMPO suppressed EC activation and aortic monocyte recruitment in apolipoprotein E knockout mice using intravital microscopy and flow cytometry methods.
CONCLUSIONS—ATP synthesis–uncoupled, but proton leak-coupled, mtROS increase mediates LPC-induced EC activation during early atherosclerosis. These results indicate that mitochondrial antioxidants are promising therapies for vascular inflammation and cardiovascular diseases.
Mg2+ is the most abundant divalent cation in metazoans and an essential cofactor for ATP, nucleic acids, and countless metabolic enzymes. To understand how the spatio-temporal dynamics of ...intracellular Mg2+ (iMg2+) are integrated into cellular signaling, we implemented a comprehensive screen to discover regulators of iMg2+ dynamics. Lactate emerged as an activator of rapid release of Mg2+ from endoplasmic reticulum (ER) stores, which facilitates mitochondrial Mg2+ (mMg2+) uptake in multiple cell types. We demonstrate that this process is remarkably temperature sensitive and mediated through intracellular but not extracellular signals. The ER-mitochondrial Mg2+ dynamics is selectively stimulated by L-lactate. Further, we show that lactate-mediated mMg2+ entry is facilitated by Mrs2, and point mutations in the intermembrane space loop limits mMg2+ uptake. Intriguingly, suppression of mMg2+ surge alleviates inflammation-induced multi-organ failure. Together, these findings reveal that lactate mobilizes iMg2+ and links the mMg2+ transport machinery with major metabolic feedback circuits and mitochondrial bioenergetics.
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•L-lactate triggers ER Mg2+ release that promotes mitochondrial Mg2+ uptake•Mg2+ is a second messenger for metabolic circuits•Limiting Mrs2-mediated Mg2+ uptake enhances mitochondrial bioenergetics•Inflammation-induced lactate contributes to organ failure via mMg2+ surge
Lactate operates as a signaling molecule to control Mg2+ handling between the ER and mitochondria.
Cardiac contractility is mediated by a variable flux in intracellular calcium (Ca2+), thought to be integrated into mitochondria via the mitochondrial calcium uniporter (MCU) channel to match ...energetic demand. Here, we examine a conditional, cardiomyocyte-specific, mutant mouse lacking Mcu, the pore-forming subunit of the MCU channel, in adulthood. Mcu−/− mice display no overt baseline phenotype and are protected against mCa2+ overload in an in vivo myocardial ischemia-reperfusion injury model by preventing the activation of the mitochondrial permeability transition pore, decreasing infarct size, and preserving cardiac function. In addition, we find that Mcu−/− mice lack contractile responsiveness to acute β-adrenergic receptor stimulation and in parallel are unable to activate mitochondrial dehydrogenases and display reduced bioenergetic reserve capacity. These results support the hypothesis that MCU may be dispensable for homeostatic cardiac function but required to modulate Ca2+-dependent metabolism during acute stress.
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•The MCU is dispensable for baseline homeostatic cardiac function•Deletion of Mcu protects against myocardial IR injury by reducing MPTP activation•The MCU is required to match energetics with contractile demand during stress•A slow MCU-independent uptake mechanism may maintain basal matrix mCa2+ content
Luongo et al. show, using a conditional knockout mouse model, that the mitochondrial Ca2+ uniporter (MCU), although dispensable for homeostatic function, is necessary for the cardiac “fight-or-flight” contractile response and a significant contributor to mitochondrial permeability transition during ischemia-reperfusion injury.
Reactive oxygen species (ROS) stimulate cytoplasmic Ca2+ (Ca2+c) signaling, but the exact role of the IP3 receptors (IP3R) in this process remains unclear. IP3Rs serve as a potential target of ROS ...produced by both ER and mitochondrial enzymes, which might locally expose IP3Rs at the ER-mitochondrial associations. Also, IP3Rs contain multiple reactive thiols, common molecular targets of ROS. Therefore, we have examined the effect of superoxide anion (O2⨪) on IP3R-mediated Ca2+ signaling. In human HepG2, rat RBL-2H3, and chicken DT40 cells, we observed Ca2+c spikes and frequency-modulated oscillations evoked by a O2⨪ donor, xanthine (X) + xanthine oxidase (XO), dose-dependently. The Ca2+c signal was mediated by ER Ca2+ mobilization. X+XO added to permeabilized cells promoted the Ca2+c rise evoked by submaximal doses of IP3, indicating that O2⨪ directly sensitizes IP3R-mediated Ca2+ release. In response to X+XO, DT40 cells lacking two of three IP3R isoforms (DKO) expressing either type 1 (DKO1) or type 2 IP3Rs (DKO2) showed a Ca2+c signal, whereas DKO expressing type 3 IP3R (DKO3) did not. By contrast, IgM that stimulates IP3 formation, elicited a Ca2+c signal in every DKO. X+XO also facilitated the Ca2+ release evoked by submaximal IP3 in permeabilized DKO1 and DKO2 but was ineffective in DKO3 or in DT40 lacking every IP3R (TKO). However, X+XO could also facilitate the effect of suboptimal IP3 in TKO transfected with rat IP3R3. Although in silico studies failed to identify a thiol missing in the chicken IP3R3, an X+XO-induced redox change was documented only in the rat IP3R3. Thus, ROS seem to specifically sensitize IP3Rs through a thiol group(s) within the IP3R, which is probably inaccessible in the chicken IP3R3.
Background: Reactive oxygen species (ROS) affect cytoplasmic calcium signaling.
Results: Superoxide anion causes oxidation of the IP3 receptor and sensitization of calcium release to promote cytoplasmic calcium oscillations and mitochondrial calcium uptake.
Conclusion: Physiologically relevant ROS controls cytoplasmic and mitochondrial calcium transport through IP3 receptors.
Significance: Mechanisms of calcium and ROS interactions are relevant for both physiological and pathophysiological signaling.
RATIONALE:Autologous bone marrow–derived or cardiac-derived stem cell therapy for heart disease has demonstrated safety and efficacy in clinical trials, but functional improvements have been limited. ...Finding the optimal stem cell type best suited for cardiac regeneration is the key toward improving clinical outcomes.
OBJECTIVE:To determine the mechanism by which novel bone-derived stem cells support the injured heart.
METHODS AND RESULTS:Cortical bone–derived stem cells (CBSCs) and cardiac-derived stem cells were isolated from enhanced green fluorescent protein (EGFP+) transgenic mice and were shown to express c-kit and Sca-1 as well as 8 paracrine factors involved in cardioprotection, angiogenesis, and stem cell function. Wild-type C57BL/6 mice underwent sham operation (n=21) or myocardial infarction with injection of CBSCs (n=67), cardiac-derived stem cells (n=36), or saline (n=60). Cardiac function was monitored using echocardiography. Only 2/8 paracrine factors were detected in EGFP+ CBSCs in vivo (basic fibroblast growth factor and vascular endothelial growth factor), and this expression was associated with increased neovascularization of the infarct border zone. CBSC therapy improved survival, cardiac function, regional strain, attenuated remodeling, and decreased infarct size relative to cardiac-derived stem cells– or saline-treated myocardial infarction controls. By 6 weeks, EGFP+ cardiomyocytes, vascular smooth muscle, and endothelial cells could be identified in CBSC-treated, but not in cardiac-derived stem cells–treated, animals. EGFP+ CBSC-derived isolated myocytes were smaller and more frequently mononucleated, but were functionally indistinguishable from EGFP− myocytes.
CONCLUSIONS:CBSCs improve survival, cardiac function, and attenuate remodeling through the following 2 mechanisms(1) secretion of proangiogenic factors that stimulate endogenous neovascularization, and (2) differentiation into functional adult myocytes and vascular cells.
Annexin V and Sytox Green are widely used markers to evaluate apoptosis in various cell types using flow cytometry and fluorescent microscopy. Recently, a novel fluoroprobe MitoSOX Red was introduced ...for selective detection of superoxide in the mitochondria of live cells and was validated for confocal microscopy and flow cytometry. This protocol describes simultaneous measurements of mitochondrial superoxide generation with apoptotic markers (Annexin V and Sytox Green) by both flow cytometry and confocal microscopy in endothelial cell lines. The advantages of the described flow cytometry method over other cell-based techniques are the tremendous speed (1-2 h), exquisite precision and the possibility of simultaneous quantitative measurements of mitochondrial superoxide generation and apoptotic (and other) markers, with maximal preservation of cellular functions. This method combined with fluorescent microscopy may be very useful to reveal important spatial-temporal changes in mitochondrial superoxide production and execution of programmed cell death in virtually any cell type.
Transient receptor potential melastatin channel subfamily member 2 (TRPM2) has an essential function in cell survival and is highly expressed in many cancers. Inhibition of TRPM2 in neuroblastoma by ...depletion with CRISPR technology or expression of dominant negative TRPM2-S has been shown to significantly reduce cell viability. Here, the role of proline-rich tyrosine kinase 2 (Pyk2) in TRPM2 modulation of neuroblastoma viability was explored. In TRPM2-depleted cells, phosphorylation and expression of Pyk2 and cAMP-responsive element-binding protein (CREB), a downstream target, were significantly reduced after application of the chemotherapeutic agent doxorubicin. Overexpression of wild-type Pyk2 rescued cell viability. Reduction of Pyk2 expression with shRNA decreased cell viability and CREB phosphorylation and expression, demonstrating Pyk2 modulates CREB activation. TRPM2 depletion impaired phosphorylation of Src, an activator of Pyk2, and this may be a mechanism to reduce Pyk2 phosphorylation. TRPM2 inhibition was previously demonstrated to decrease mitochondrial function. Here, CREB, Pyk2, and phosphorylated Src were reduced in mitochondria of TRPM2-depleted cells, consistent with their role in modulating expression and activation of mitochondrial proteins. Phosphorylated Src and phosphorylated and total CREB were reduced in TRPM2-depleted nuclei. Expression and function of mitochondrial calcium uniporter (MCU), a target of phosphorylated Pyk2 and CREB, were significantly reduced. Wild-type TRPM2 but not Ca
-impermeable mutant E960D reconstituted phosphorylation and expression of Pyk2 and CREB in TRPM2-depleted cells exposed to doxorubicin. Results demonstrate that TRPM2 expression protects the viability of neuroblastoma through Src, Pyk2, CREB, and MCU activation, which play key roles in maintaining mitochondrial function and cellular bioenergetics.
Mitochondrial permeability transition is a phenomenon in which the mitochondrial permeability transition pore (PTP) abruptly opens, resulting in mitochondrial membrane potential (ΔΨm) dissipation, ...loss of ATP production, and cell death. Several genetic candidates have been proposed to form the PTP complex, however, the core component is unknown. We identified a necessary and conserved role for spastic paraplegia 7 (SPG7) in Ca2+- and ROS-induced PTP opening using RNAi-based screening. Loss of SPG7 resulted in higher mitochondrial Ca2+ retention, similar to cyclophilin D (CypD, PPIF) knockdown with sustained ΔΨm during both Ca2+ and ROS stress. Biochemical analyses revealed that the PTP is a heterooligomeric complex composed of VDAC, SPG7, and CypD. Silencing or disruption of SPG7-CypD binding prevented Ca2+- and ROS-induced ΔΨm depolarization and cell death. This study identifies an ubiquitously expressed IMM integral protein, SPG7, as a core component of the PTP at the OMM and IMM contact site.
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•Mitochondrial SPG7 is essential for the PTP complex formation in multiple cell types•SPG7 interacts with CypD and VDAC at the IMM and OMM contact site•C terminus of SPG7 and CsA-binding region of CypD are necessary for PTP formation•Loss of SPG7 protects mitochondria from Ca2+- and ROS-induced PTP-dependent necrosis
Using an RNAi-based screen, Shanmughapriya et al. identify mitochondrial spastic paraplegia 7 as essential for the PTP complex formation. They show that SPG7 interacts with CypD and VDAC1 at the IMM and OMM contact site, and loss of SPG7 protects mitochondria from Ca2+- and ROS-induced PTP-dependent necrosis.
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