Impairments in neuronal intracellular calcium (
Ca
) handling may contribute to Alzheimer's disease (AD) development. Metabolic dysfunction and progressive neuronal loss are associated with AD ...progression, and mitochondrial calcium (
Ca
) signaling is a key regulator of both of these processes. Here, we report remodeling of the
Ca
exchange machinery in the prefrontal cortex of individuals with AD. In the 3xTg-AD mouse model impaired
Ca
efflux capacity precedes neuropathology. Neuronal deletion of the mitochondrial Na
/Ca
exchanger (NCLX, Slc8b1 gene) accelerated memory decline and increased amyloidosis and tau pathology. Further, genetic rescue of neuronal NCLX in 3xTg-AD mice is sufficient to impede AD-associated pathology and memory loss. We show that
Ca
overload contributes to AD progression by promoting superoxide generation, metabolic dysfunction and neuronal cell death. These results provide a link between the calcium dysregulation and metabolic dysfunction hypotheses of AD and suggest
Ca
exchange as potential therapeutic target in AD.
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.
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.
Since the identification of the mitochondrial calcium uniporter (MCU) in 2011, several studies utilizing genetic models have attempted to decipher the role of mitochondrial calcium uptake in cardiac ...physiology. Confounding results in various mutant mouse models have led to an ongoing debate regarding the function of MCU in the heart. In this review, we evaluate and discuss the totality of evidence for mitochondrial calcium uptake in the cardiac stress response and highlight recent reports that implicate MCU in the control of homeostatic cardiac metabolism and function. This review concludes with a discussion of current gaps in knowledge and remaining experiments to define how MCU contributes to contractile function, cell death, metabolic regulation, and heart failure progression.
•MCU is required to support increased cardiac contractility during sympathetic stimulation.•New data suggest MCU-dependent mCa2+ uptake also supports basal cardiac function.•Acute MCU disruption protects against cardiac I/R injury.•The heart has robust compensatory mechanisms to adapt to chronic MCU disruption.•MCU-independent pathways may mediate mCa2+ uptake during sustained stress.
Fibroblast to myofibroblast differentiation is crucial for the initial healing response but excessive myofibroblast activation leads to pathological fibrosis. Therefore, it is imperative to ...understand the mechanisms underlying myofibroblast formation. Here we report that mitochondrial calcium (
Ca
) signaling is a regulatory mechanism in myofibroblast differentiation and fibrosis. We demonstrate that fibrotic signaling alters gating of the mitochondrial calcium uniporter (mtCU) in a MICU1-dependent fashion to reduce
Ca
uptake and induce coordinated changes in metabolism, i.e., increased glycolysis feeding anabolic pathways and glutaminolysis yielding increased α-ketoglutarate (αKG) bioavailability.
Ca
-dependent metabolic reprogramming leads to the activation of αKG-dependent histone demethylases, enhancing chromatin accessibility in loci specific to the myofibroblast gene program, resulting in differentiation. Our results uncover an important role for the mtCU beyond metabolic regulation and cell death and demonstrate that
Ca
signaling regulates the epigenome to influence cellular differentiation.
Pharmacological agents targeting the mTOR complexes are used clinically as immunosuppressants and anticancer agents and can extend the lifespan of model organisms. An undesirable side effect of these ...drugs is hyperlipidemia. Although multiple roles have been described for mTOR complex 1 (mTORC1) in lipid metabolism, the etiology of hyperlipidemia remains incompletely understood. The objective of this study was to determine the influence of adipocyte mTORC1 signaling in systemic lipid homeostasis in vivo.
We characterized systemic lipid metabolism in mice lacking the mTORC1 subunit Raptor (RaptoraKO), the key lipolytic enzyme ATGL (ATGLaKO), or both (ATGL-RaptoraKO) in their adipocytes.
Mice lacking mTORC1 activity in their adipocytes failed to completely suppress lipolysis in the fed state and displayed prominent hypertriglyceridemia and hypercholesterolemia. Blocking lipolysis in their adipose tissue restored normal levels of triglycerides and cholesterol in the fed state as well as the ability to clear triglycerides in an oral fat tolerance test.
Unsuppressed adipose lipolysis in the fed state interferes with triglyceride clearance and contributes to hyperlipidemia. Adipose tissue mTORC1 activity is necessary for appropriate suppression of lipolysis and for the maintenance of systemic lipid homeostasis.
•Inhibition of adipose mTORC1 causes hypertriglyceridemia prior to lipodystrophy.•Genetically inhibiting lipolysis reverses the increase in plasma TG.•Acute pharmacological inhibition of lipolysis reverses the increase in plasma TG caused by rapamycin treatment.•Unrestrained lipolysis impairs LPL activity and decreases TG clearance.
GRK2, a G protein-coupled receptor kinase, plays a critical role in cardiac physiology. Adrenergic receptors are the primary target for GRK2 activity in the heart; phosphorylation by GRK2 leads to ...desensitization of these receptors. As such, levels of GRK2 activity in the heart directly correlate with cardiac contractile function. Furthermore, increased expression of GRK2 after cardiac insult exacerbates injury and speeds progression to heart failure. Despite the importance of this kinase in both the physiology and pathophysiology of the heart, relatively little is known about the role of GRK2 in skeletal muscle function and disease. In this study we generated a novel skeletal muscle-specific GRK2 knock-out (KO) mouse (MLC-Cre:GRK2
) to gain a better understanding of the role of GRK2 in skeletal muscle physiology. In isolated muscle mechanics testing, GRK2 ablation caused a significant decrease in the specific force of contraction of the fast-twitch extensor digitorum longus muscle yet had no effect on the slow-twitch soleus muscle. Despite these effects in isolated muscle, exercise capacity was not altered in MLC-Cre:GRK2
mice compared with wild-type controls. Skeletal muscle hypertrophy stimulated by clenbuterol, a β
-adrenergic receptor (β
AR) agonist, was significantly enhanced in MLC-Cre:GRK2
mice; mechanistically, this seems to be due to increased clenbuterol-stimulated pro-hypertrophic Akt signaling in the GRK2 KO skeletal muscle. In summary, our study provides the first insights into the role of GRK2 in skeletal muscle physiology and points to a role for GRK2 as a modulator of contractile properties in skeletal muscle as well as β
AR-induced hypertrophy.
BACKGROUND:The mitochondrial calcium uniporter (mtCU) is an ≈700-kD multisubunit channel residing in the inner mitochondrial membrane required for mitochondrial Ca (mCa) uptake. Here, we detail the ...contribution of MCUB, a paralog of the pore-forming subunit MCU, in mtCU regulation and function and for the first time investigate the relevance of MCUB to cardiac physiology.
METHODS:We created a stable MCUB knockout cell line (MCUB) using CRISPR-Cas9n technology and generated a cardiac-specific, tamoxifen-inducible MCUB mutant mouse (CAG-CAT-MCUB x MCM; MCUB-Tg) for in vivo assessment of cardiac physiology and response to ischemia/reperfusion injury. Live-cell imaging and high-resolution spectrofluorometery were used to determine intracellular Ca exchange and size-exclusion chromatography; blue native page and immunoprecipitation studies were used to determine the molecular function and impact of MCUB on the high-molecular-weight mtCU complex.
RESULTS:Using genetic gain- and loss-of-function approaches, we show that MCUB expression displaces MCU from the functional mtCU complex and thereby decreases the association of mitochondrial calcium uptake 1 and 2 (MICU1/2) to alter channel gating. These molecular changes decrease MICU1/2–dependent cooperative activation of the mtCU, thereby decreasing mCa uptake. Furthermore, we show that MCUB incorporation into the mtCU is a stress-responsive mechanism to limit mCa overload during cardiac injury. Indeed, overexpression of MCUB is sufficient to decrease infarct size after ischemia/reperfusion injury. However, MCUB incorporation into the mtCU does come at a cost; acute decreases in mCa uptake impair mitochondrial energetics and contractile function.
CONCLUSIONS:We detail a new regulatory mechanism to modulate mtCU function and mCa uptake. Our results suggest that MCUB-dependent changes in mtCU stoichiometry are a prominent regulatory mechanism to modulate mCa uptake and cellular physiology.
Mitochondria require nicotinamide adenine dinucleotide (NAD
) to carry out the fundamental processes that fuel respiration and mediate cellular energy transduction. Mitochondrial NAD
transporters ...have been identified in yeast and plants
, but their existence in mammals remains controversial
. Here we demonstrate that mammalian mitochondria can take up intact NAD
, and identify SLC25A51 (also known as MCART1)-an essential
mitochondrial protein of previously unknown function-as a mammalian mitochondrial NAD
transporter. Loss of SLC25A51 decreases mitochondrial-but not whole-cell-NAD
content, impairs mitochondrial respiration, and blocks the uptake of NAD
into isolated mitochondria. Conversely, overexpression of SLC25A51 or SLC25A52 (a nearly identical paralogue of SLC25A51) increases mitochondrial NAD
levels and restores NAD
uptake into yeast mitochondria lacking endogenous NAD
transporters. Together, these findings identify SLC25A51 as a mammalian transporter capable of importing NAD
into mitochondria.
The interface between the HIV-1 gp120 envelope glycoprotein and the CD4 receptor contains an unusual interfacial cavity, the “Phe43 cavity”, which CD4-mimetic miniproteins with nonnatural extensions ...can potentially utilize to enhance their neutralization of HIV-1. Here, we report cocrystal structures of HIV-1 gp120 with miniproteins M48U1 and M48U7, which insert cyclohexylmethoxy and 5-hydroxypentylmethoxy extensions, respectively, into the Phe43 cavity. Both inserts displayed flexibility and hydrophobic interactions, but the M48U1 insert showed better shape complementarity with the Phe43 cavity than the M48U7 insert. Subtle alteration in the gp120 conformation played a substantial role in optimizing fit. With M48U1, these translated into a YU2-gp120 affinity of 0.015 nM and neutralization of all 180 circulating HIV-1 strains tested, except clade-A/E isolates with noncanonical Phe43 cavities. Ligand chemistry, shape complementarity, surface burial, and gp120 conformation act in concert to modulate binding of ligands to the gp120-Phe43 cavity and, when optimized, can effect near-pan-neutralization of HIV-1.
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•M48U1 displays a gp120 affinity of 0.015 nM and near-pan-neutralization of HIV-1•Remarkable properties of M48U1 result from filling a hydrophobic interfacial cavity•Optimal cavity fit requires both specific chemistry and complementary shape•A Phe43-cavity mechanism for enhancing affinity of CD4-binding-site ligands
Acharya et al. establish that CD4-mimetic miniprotein M48U1 accesses an unusual interfacial cavity in HIV-1 gp120 envelope glycoprotein to achieve high-affinity binding. Ligand chemistry, shape complementary, surface burial, and gp120 conformation act in concert to modulate ligand binding.
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