Although many factors contribute to cellular differentiation, the role of mitochondria Ca
dynamics during development remains unexplored. Because mammalian embryonic epiblasts reside in a hypoxic ...environment, we intended to understand whether
Ca
and its transport machineries are regulated during hypoxia. Tissues from multiple organs of developing mouse embryo evidenced a suppression of MICU1 expression with nominal changes on other MCU complex components. As surrogate models, we here utilized human embryonic stem cells (hESCs)/induced pluripotent stem cells (hiPSCs) and primary neonatal myocytes to delineate the mechanisms that control
Ca
and bioenergetics during development. Analysis of MICU1 expression in hESCs/hiPSCs showed low abundance of MICU1 due to its direct repression by Foxd1. Experimentally, restoration of MICU1 established the periodic
Ca
oscillations and promoted cellular differentiation and maturation. These findings establish a role of
Ca
dynamics in regulation of cellular differentiation and reveal a molecular mechanism underlying this contribution through differential regulation of MICU1.
Members of the Bcl-2 protein family modulate outer mitochondrial membrane permeability to control apoptosis. However, these proteins also localize to the endoplasmic reticulum (ER), the functional ...significance of which is controversial. Here we provide evidence that anti-apoptotic Bcl-2 proteins regulate the inositol 1,4,5-trisphosphate receptor (InsP(3)R) ER Ca(2+) release channel resulting in increased cellular apoptotic resistance and enhanced mitochondrial bioenergetics. Anti-apoptotic Bcl-X(L) interacts with the carboxyl terminus of the InsP(3)R and sensitizes single InsP(3)R channels in ER membranes to low InsP(3), enhancing Ca(2+) and InsP(3)-dependent regulation of channel activity in vitro and in vivo, reducing ER Ca(2+) content and stimulating mitochondrial energetics. The pro-apoptotic proteins Bax and tBid antagonize this effect by blocking the biochemical interaction of Bcl-X(L) with the InsP(3)R. These data support a novel model in which Bcl-X(L) is a direct effector of the InsP(3)R, increasing its sensitivity to InsP(3) and enabling ER Ca(2+) release to be more sensitively coupled to extracellular signals. As a consequence, cells are protected against apoptosis by a more sensitive and dynamic coupling of ER to mitochondria through Ca(2+)-dependent signal transduction that enhances cellular bioenergetics and preserves survival.
Isoflurane induces cell apoptosis by an unknown mechanism. The authors hypothesized that isoflurane activates inositol 1,4,5-trisphosphate (IP3) receptors on the endoplasmic reticulum (ER) membrane, ...causing excessive calcium release, triggering apoptosis.
The authors determined isoflurane-induced cytotoxicity by measuring caspase-3 activity, lactate dehydrogenase release, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) reduction, and imaging analysis of cell damage markers (annexin V and propidium iodide staining) in different cell types. The authors used the chicken B lymphocyte with a total knock-out of IP3 receptors, PC12 cells with elevated IP3 receptor activity (transfected with L286V presenilin 1), striatal cells with a knock-in of Q111 Huntingtin, and each cell line's corresponding wild-type controls. The authors also measured the isoflurane-evoked changes of calcium concentration in cytosol and/or mitochondria in these cells.
Isoflurane induced apoptosis concentration- and time-dependently, and sequentially elevated cytosolic and then mitochondrial calcium in the chicken B-lymphocyte wild-type but not the IP3 receptor total knock-out cells. Thapsigargin, a calcium adenosine triphosphatase inhibitor on ER membranes, induced apoptosis and elevations of calcium in cytosol and mitochondria in both chicken B-lymphocyte wild-type and IP3 receptor total knock-out cells. Isoflurane induced significantly more neurotoxicity and greater calcium release from the ER in L286V PC12 and Q111 Huntingtin striatal cells than in their corresponding wild-type controls, both of which were significantly inhibited by the IP3 receptor antagonist xestospongin C.
These findings suggest that isoflurane activates the ER membrane IP3 receptor, producing excessive calcium release and triggering apoptosis. Neurons with enhanced IP3 receptor activity, as in certain cases of familial Alzheimer or Huntington disease, may be especially vulnerable to isoflurane cytotoxicity.
Insulin resistance and altered hepatic mitochondrial function are central features of type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD), but the etiological role of these processes ...in disease progression remains unclear. Here we investigated the molecular links between insulin resistance, mitochondrial remodeling, and hepatic lipid accumulation.
Hepatic insulin sensitivity, endogenous glucose production, and mitochondrial metabolic fluxes were determined in wild-type, obese (ob/ob) and pioglitazone-treatment obese mice using a combination of radiolabeled tracer and stable isotope NMR approaches. Mechanistic studies of pioglitazone action were performed in isolated primary hepatocytes, whilst molecular hepatic lipid species were profiled using shotgun lipidomics.
Livers from obese, insulin-resistant mice displayed augmented mitochondrial content and increased tricarboxylic acid cycle (TCA) cycle and pyruvate dehydrogenase (PDH) activities. Insulin sensitization with pioglitazone mitigated pyruvate-driven TCA cycle activity and PDH activation via both allosteric (intracellular pyruvate availability) and covalent (PDK4 and PDP2) mechanisms that were dependent on PPARγ activity in isolated primary hepatocytes. Improved mitochondrial function following pioglitazone treatment was entirely dissociated from changes in hepatic triglycerides, diacylglycerides, or fatty acids. Instead, we highlight a role for the mitochondrial phospholipid cardiolipin, which underwent pathological remodeling in livers from obese mice that was reversed by insulin sensitization.
Our findings identify targetable mitochondrial features of T2D and NAFLD and highlight the benefit of insulin sensitization in managing the clinical burden of obesity-associated disease.
•Hepatic pyruvate dehydrogenase is hyperactivated in insulin-resistant mice.•Pioglitazone lowers hepatic PDH through covalent and allosteric actions.•Induction of hepatocyte PDK4 by pioglitazone requires PPARγ.•Improved mitochondrial function with pioglitazone precedes changes in liver fat.•Mitochondrial lipid remodeling is a modifiable feature of liver insulin resistance.
Sepsis is the overwhelming systemic immune response to infection, which can result in multiple organ dysfunction and septic shock. Myocardial dysfunction during sepsis is associated with advanced ...disease and significantly increased in-hospital mortality. Our group has shown that energetic failure and excess reactive oxygen species (ROS) generation constitute major components of myocardial dysfunction in sepsis. Because ROS production is central to cellular metabolic health, we tested if the synthetic anti-oxidant lignan secoisolariciresinol diglucoside (SDG; LGM2605) would alleviate septic cardiac dysfunction and investigated the underlying mechanism. Using the cecal ligation and puncture (CLP) mouse model of peritonitis-induced sepsis, we observed impairment of cardiac function beginning at 4 h post-CLP surgery. Treatment of mice with LGM2605 (100 mg/kg body weight, i.p.) 6 h post-CLP surgery reduced cardiac ROS accumulation and restored cardiac function. Assessment of mitochondrial respiration (Seahorse XF) in primary cardiomyocytes obtained from adult C57BL/6 mice that had undergone CLP and treatment with LGM2605 showed restored basal and maximal respiration, as well as preserved oxygen consumption rate (OCR) associated with spare capacity. Further analyses aiming to identify the cellular mechanisms that may account for improved cardiac function showed that LGM2605 restored mitochondria abundance, increased mitochondrial calcium uptake and preserved mitochondrial membrane potential. In addition to protecting against cardiac dysfunction, daily treatment with LGM2605 and antibiotic ertapenem (70 mg/kg) protected against CLP-associated mortality and reversed hypothermia when compared against mice receiving ertapenem and saline. Therefore, treatment of septic mice with LGM2605 emerges as a novel pharmacological approach that reduces cardiac ROS accumulation, protects cardiac mitochondrial function, alleviates cardiac dysfunction, and improves survival.
•Sepsis results in progressive cardiac dysfunction associated with oxidative stress.•ROS formation underlies mitochondrial dysfunction in septic cardiomyopathy.•Antioxidant LGM2605 scavenges ROS and alleviates mitochondrial dysfunction.•LGM2605 treats septic cardiac dysfunction and improves survival.
Ferroptosis is an iron- and reactive oxygen species (ROS)-dependent form of regulated cell death, that has been implicated in Alzheimer's disease and Parkinson's disease. Inhibition of ...cystine/glutamate antiporter could lead to mitochondrial fragmentation, mitochondrial calcium (Ca
) overload, increased mitochondrial ROS production, disruption of the mitochondrial membrane potential (ΔΨ
), and ferroptotic cell death. The observation that mitochondrial dysfunction is a characteristic of ferroptosis makes preservation of mitochondrial function a potential therapeutic option for diseases associated with ferroptotic cell death. Mitochondrial calcium levels are controlled via the mitochondrial calcium uniporter (MCU), the main entry point of Ca
into the mitochondrial matrix. Therefore, we have hypothesized that negative modulation of MCU complex may confer protection against ferroptosis. Here we evaluated whether the known negative modulators of MCU complex, ruthenium red (RR), its derivative Ru265, mitoxantrone (MX), and MCU-i4 can prevent mitochondrial dysfunction and ferroptotic cell death. These compounds mediated protection in HT22 cells, in human dopaminergic neurons and mouse primary cortical neurons against ferroptotic cell death. Depletion of MICU1, a Ca
gatekeeper, demonstrated that MICU is protective against ferroptosis. Taken together, our results reveal that negative modulation of MCU complex represents a therapeutic option to prevent degenerative conditions, in which ferroptosis is central to the progression of these pathologies.
The Ca2+‐sensing stromal interaction molecule (STIM) proteins are crucial Ca2+ signal coordinators. Cre‐lox technology was used to generate smooth muscle (sm)‐targeted STIM1‐, STIM2‐, and double ...STIM1/STIM2‐knockout (KO) mouse models, which reveal the essential role of STIM proteins in Ca2+ homeostasis and their crucial role in controlling function, growth, and development of smooth muscle cells (SMCs). Compared to Cre+/– littermates, sm‐STIM1‐KO mice showed high mortality (50% by 30 d) and reduced bodyweight. While sm‐STIM2‐KO was without detectable phenotype, the STIM1/STIM double‐KO was perinatally lethal, revealing an essential role of STIM1 partially rescued by STIM2. Vascular and intestinal smooth muscle tissues from sm‐STIM1‐KO mice developed abnormally with distended, thinned morphology. While depolarization‐induced aortic contraction was unchanged in sm‐STIM1‐KO mice, α1‐adrenergic‐mediated contraction was 26% reduced, and store‐dependent contraction almost eliminated. Neointimal formation induced by carotid artery ligation was suppressed by 54%, and in vitro PDGF‐induced proliferation was greatly reduced (79%) in sm‐STIM1‐KO. Notably, the Ca2+ store‐refilling rate in STIM1‐KO SMCs was substantially reduced, and sustained PDGF‐induced Ca2+ entry was abolished. This defective Ca2+ homeostasis prevents PDGF‐induced NFAT activation in both contractile and proliferating SMCs. We conclude that STIM1‐regulated Ca2+ homeostasis is crucial for NFAT‐mediated transcriptional control required for induction of SMC proliferation, development, and growth responses to injury.—Mancarella, S., Potireddy, S., Wang, Y., Gao, H., Gandhirajan, K., Autieri, M., Scalia, R., Cheng, Z., Wang, H., Madesh, M., Houser, S. R., Gill, D. L. Targeted STIM deletion impairs calcium homeostasis, NFAT activation, and growth of smooth muscle. FASEB J. 27, 893–906 (2013). www.fasebj.org
Reactive oxygen species (ROS) have been implicated in both cell signaling and pathology. A major source of ROS in endothelial cells is NADPH oxidase, which generates superoxide (O(2)(.-)) on the ...extracellular side of the plasma membrane but can result in intracellular signaling. To study possible transmembrane flux of O(2)(.-), pulmonary microvascular endothelial cells were preloaded with the O(2)(.-)-sensitive fluorophore hydroethidine (HE). Application of an extracellular bolus of O(2)(.-) resulted in rapid and concentration-dependent transient HE oxidation that was followed by a progressive and nonreversible increase in nuclear HE fluorescence. These fluorescence changes were inhibited by superoxide dismutase (SOD), the anion channel blocker DIDS, and selective silencing of the chloride channel-3 (ClC-3) by treatment with siRNA. Extracellular O(2)(.-) triggered Ca(2+) release in turn triggered mitochondrial membrane potential alterations that were followed by mitochondrial O(2)(.-) production and cellular apoptosis. These "signaling" effects of O(2)(.-) were prevented by DIDS treatment, by depletion of intracellular Ca(2+) stores with thapsigargin and by chelation of intracellular Ca(2+). This study demonstrates that O(2)(.-) flux across the endothelial cell plasma membrane occurs through ClC-3 channels and induces intracellular Ca(2+) release, which activates mitochondrial O(2)(.-) generation.
Shear stress is known to stimulate an intracellular free calcium concentration (Ca(2+)i) response in vascular endothelial cells (ECs). Ca(2+)i is a key second messenger for signaling that leads to ...vasodilation and EC survival. Although it is accepted that the shear-induced Ca(2+)i response is, in part, due to Ca(2+) release from the endoplasmic reticulum (ER), the role of mitochondria (second largest Ca(2+) store) is unknown. We hypothesized that the mitochondria play a role in regulating Ca(2+)i in sheared ECs. Cultured ECs, loaded with a Ca(2+)-sensitive fluorophore, were exposed to physiological levels of shear stress. Shear stress elicited Ca(2+)i transients in a percentage of cells with a fraction of them displaying oscillations. Peak magnitudes, percentage of oscillating ECs, and oscillation frequencies depended on the shear level. Ca(2+)i transients/oscillations were present when experiments were conducted in Ca(2+)-free solution (plus lanthanum) but absent when ECs were treated with a phospholipase C inhibitor, suggesting that the ER inositol 1,4,5-trisphosphate receptor is responsible for the Ca(2+)i response. Either a mitochondrial uncoupler or an electron transport chain inhibitor, but not a mitochondrial ATP synthase inhibitor, prevented the occurrence of transients and especially inhibited the oscillations. Knockdown of the mitochondrial Ca(2+) uniporter also inhibited the shear-induced Ca(2+)i transients/oscillations compared with controls. Hence, EC mitochondria, through Ca(2+) uptake/release, regulate the temporal profile of shear-induced ER Ca(2+) release. Ca(2+)i oscillation frequencies detected were within the range for activation of mechanoresponsive kinases and transcription factors, suggesting that dysfunctional EC mitochondria may contribute to cardiovascular disease by deregulating the shear-induced Ca(2+)i response.
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