ABSTRACT
The mitochondrial calcium (mCa2+) uniporter channel (mtCU) resides at the inner mitochondrial membrane and is required for Ca2+ to enter the mitochondrial matrix. The mtCU is essential for cellular function, as mCa2+ regulates metabolism, bioenergetics, signaling pathways and cell death. mCa2+ uptake is primarily regulated by the MICU family (MICU1, MICU2, MICU3), EF-hand-containing Ca2+-sensing proteins, which respond to cytosolic Ca2+ concentrations to modulate mtCU activity. Considering that mitochondrial function and Ca2+ signaling are ubiquitously disrupted in cardiovascular disease, mtCU function has been a hot area of investigation for the last decade. Here we provide an in-depth review of MICU-mediated regulation of mtCU structure and function, as well as potential mtCU-independent functions of these proteins. We detail their role in cardiac physiology and cardiovascular disease by highlighting the phenotypes of different mutant animal models, with an emphasis on therapeutic potential and targets of interest in this pathway.
Subject(s)
Calcium Channels , Cardiovascular Diseases , Humans , Animals , Cardiovascular Diseases/metabolism , Cardiovascular Diseases/pathology , Calcium Channels/metabolism , Calcium Channels/genetics , Mitochondria, Heart/metabolism , Calcium Signaling , Calcium/metabolism , Ion Channel Gating , Structure-Activity RelationshipABSTRACT
Differentiation of cardiac fibroblasts to myofibroblasts is necessary for matrix remodeling and fibrosis in heart failure. We previously reported that mitochondrial calcium signaling drives α-ketoglutarate-dependent histone demethylation, promoting myofibroblast formation. Here we investigate the role of ATP-citrate lyase (ACLY), a key enzyme for acetyl-CoA biosynthesis, in histone acetylation regulating myofibroblast fate and persistence in cardiac fibrosis. We show that inactivation of ACLY prevents myofibroblast differentiation and reverses myofibroblasts towards quiescence. Genetic deletion of Acly in post-activated myofibroblasts prevents fibrosis and preserves cardiac function in pressure-overload heart failure. TGFß stimulation enhances ACLY nuclear localization and ACLY-SMAD2/3 interaction, and increases H3K27ac at fibrotic gene loci. Pharmacological inhibition of ACLY or forced nuclear expression of a dominant-negative ACLY mutant prevents myofibroblast formation and H3K27ac. Our data indicate that nuclear ACLY activity is necessary for myofibroblast differentiation and persistence by maintaining histone acetylation at TGFß-induced myofibroblast genes. These findings provide targets to prevent and reverse pathological fibrosis.
Subject(s)
ATP Citrate (pro-S)-Lyase , Cell Differentiation , Fibrosis , Histones , Myofibroblasts , Smad2 Protein , Myofibroblasts/metabolism , Myofibroblasts/drug effects , ATP Citrate (pro-S)-Lyase/metabolism , ATP Citrate (pro-S)-Lyase/genetics , Animals , Fibrosis/metabolism , Cell Differentiation/drug effects , Histones/metabolism , Smad2 Protein/metabolism , Smad2 Protein/genetics , Acetylation/drug effects , Cell Nucleus/metabolism , Cell Nucleus/drug effects , Smad3 Protein/metabolism , Smad3 Protein/genetics , Cells, Cultured , Chromatin/metabolism , Mice, Knockout , Transforming Growth Factor beta/metabolism , Disease Models, Animal , Signal Transduction , Mice, Inbred C57BL , Male , Mice , Gene Expression Regulation/drug effectsABSTRACT
Activating Ca2+-sensitive enzymes of oxidative metabolism while preventing calcium overload that leads to mitochondrial and cellular injury requires dynamic control of mitochondrial Ca2+ uptake. This is ensured by the mitochondrial calcium uptake (MICU)1/2 proteins that gate the pore of the mitochondrial calcium uniporter (mtCU). MICU1 is relatively sparse in the heart, and recent studies claimed the mammalian heart lacks MICU1 gating of mtCU. However, genetic models have not been tested. We find that MICU1 is present in a complex with MCU in nonfailing human hearts. Furthermore, using murine genetic models and pharmacology, we show that MICU1 and MICU2 control cardiac mitochondrial Ca2+ influx, and that MICU1 deletion alters cardiomyocyte mitochondrial calcium signaling and energy metabolism. MICU1 loss causes substantial compensatory changes in the mtCU composition and abundance, increased turnover of essential MCU regulator (EMRE) early on and, later, of MCU, that limit mitochondrial Ca2+ uptake and allow cell survival. Thus, both the primary consequences of MICU1 loss and the ensuing robust compensation highlight MICU1's relevance in the beating heart.
Subject(s)
Calcium Signaling , Calcium-Binding Proteins , Calcium , Cation Transport Proteins , Mitochondrial Membrane Transport Proteins , Myocytes, Cardiac , Animals , Calcium-Binding Proteins/metabolism , Calcium-Binding Proteins/genetics , Mice , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membrane Transport Proteins/genetics , Humans , Cation Transport Proteins/metabolism , Cation Transport Proteins/genetics , Myocytes, Cardiac/metabolism , Calcium Signaling/physiology , Calcium/metabolism , Mitochondria, Heart/metabolism , Calcium Channels/metabolism , Calcium Channels/genetics , Mice, Knockout , Myocardium/metabolism , MaleABSTRACT
Vitamin A/Retinoic Acid (Vit A/RA) signaling is essential for heart development. In cardiac progenitor cells (CPCs), RA signaling induces the expression of atrial lineage genes while repressing ventricular genes, thereby promoting the acquisition of an atrial cardiomyocyte cell fate. To achieve this, RA coordinates a complex regulatory network of downstream effectors that is not fully identified. To address this gap, we applied a functional genomics approach (i.e scRNAseq and snATACseq) to untreated and RA-treated human embryonic stem cells (hESCs)-derived CPCs. Unbiased analysis revealed that the Hippo effectors YAP1 and TEAD4 are integrated with the atrial transcription factor enhancer network, and that YAP1 is necessary for activation of RA-enhancers in CPCs. Furthermore, in vivo analysis of control and conditionally YAP1 KO mouse embryos (Sox2-cre) revealed that the expression of atrial lineage genes, such as NR2F2, is compromised by YAP1 deletion in the CPCs of the second heart field. Accordingly, we found that YAP1 is required for the formation of an atrial chamber but is dispensable for the formation of a ventricle, in hESC-derived patterned cardiac organoids. Overall, our findings revealed that YAP1 is a non-canonical effector of RA signaling essential for the acquisition of atrial lineages during cardiogenesis.
ABSTRACT
IMPORTANCE: The yeast C. albicans exhibits metabolic flexibility for adaptability to host niches with varying availability of nutrients including essential metals like iron. For example, blood is iron deplete, while the oral cavity and the intestinal lumen are considered iron replete. We show here that C. albicans can tolerate very high levels of environmental iron, despite an increase in high iron-induced reactive oxygen species (ROS) that it mitigates with the help of a unique oxidase, known as alternative oxidase (AOX). High iron induces AOX1/2 that limits mitochondrial accumulation of ROS. Genetic elimination of AOX1/2 resulted in diminished virulence during oropharyngeal candidiasis in high iron mice. Since human mitochondria lack AOX protein, it represents a unique target for treatment of fungal infections.
Subject(s)
Candida albicans , Oxidoreductases , Humans , Animals , Mice , Candida albicans/genetics , Candida albicans/metabolism , Reactive Oxygen Species/metabolism , Oxidoreductases/genetics , Oxidoreductases/metabolism , Iron/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolismABSTRACT
The balance between mitochondrial calcium (mCa2+) uptake and efflux regulates ATP production, but if perturbed causes energy starvation or mCa2+ overload and cell death. The mitochondrial sodium-calcium exchanger, NCLX, is a critical route of mCa2+ efflux in excitable tissues, such as the heart and brain, and animal models support NCLX as a promising therapeutic target to limit pathogenic mCa2+ overload. However, the mechanisms that regulate NCLX activity remain largely unknown. We used proximity biotinylation proteomic screening to identify the NCLX interactome and define novel regulators of NCLX function. Here, we discover the mitochondrial inner membrane protein, TMEM65, as an NCLX-proximal protein that potently enhances sodium (Na+)-dependent mCa2+ efflux. Mechanistically, acute pharmacologic NCLX inhibition or genetic deletion of NCLX ablates the TMEM65-dependent increase in mCa2+ efflux. Further, loss-of-function studies show that TMEM65 is required for Na+-dependent mCa2+ efflux. Co-fractionation and in silico structural modeling of TMEM65 and NCLX suggest these two proteins exist in a common macromolecular complex in which TMEM65 directly stimulates NCLX function. In line with these findings, knockdown of Tmem65 in mice promotes mCa2+ overload in the heart and skeletal muscle and impairs both cardiac and neuromuscular function. We further demonstrate that TMEM65 deletion causes excessive mitochondrial permeability transition, whereas TMEM65 overexpression protects against necrotic cell death during cellular Ca2+ stress. Collectively, our results show that loss of TMEM65 function in excitable tissue disrupts NCLX-dependent mCa2+ efflux, causing pathogenic mCa2+ overload, cell death and organ-level dysfunction, and that gain of TMEM65 function mitigates these effects. These findings demonstrate the essential role of TMEM65 in regulating NCLX-dependent mCa2+ efflux and suggest modulation of TMEM65 as a novel strategy for the therapeutic control of mCa2+ homeostasis.
ABSTRACT
Alzheimer's disease (AD) is characterized by the extracellular deposition of amyloid beta, intracellular neurofibrillary tangles, synaptic dysfunction, and neuronal cell death. These phenotypes correlate with and are linked to elevated neuronal intracellular calcium ( i Ca 2+ ) levels. Recently, our group reported that mitochondrial calcium ( m Ca 2+ ) overload, due to loss of m Ca 2+ efflux capacity, contributes to AD development and progression. We also noted proteomic remodeling of the mitochondrial calcium uniporter channel (mtCU) in sporadic AD brain samples, suggestive of altered m Ca 2+ uptake in AD. Since the mtCU is the primary mechanism for Ca 2+ uptake into the mitochondrial matrix, inhibition of the mtCU has the potential to reduce or prevent m Ca 2+ overload in AD. Here, we report that neuronal-specific loss of mtCU-dependent m Ca 2+ uptake in the 3xTg-AD mouse model of AD reduced Aß and tau-pathology, synaptic dysfunction, and cognitive decline. Knockdown of Mcu in a cellular model of AD significantly decreased matrix Ca 2+ content, oxidative stress, and cell death. These results suggest that inhibition of neuronal m Ca 2+ uptake is a novel therapeutic target to impede AD progression.
ABSTRACT
At least seven cell death programs are activated during myocardial infarction (MI), but which are most important in causing heart damage is not understood. Two of these programs are mitochondrial-dependent necrosis and apoptosis. The canonical function of the pro-cell death BCL-2 family proteins BAX and BAK is to mediate permeabilization of the outer mitochondrial membrane during apoptosis allowing apoptogen release. BAX has also been shown to sensitize cells to mitochondrial-dependent necrosis, although the underlying mechanisms remain ill-defined. Genetic deletion of Bax or both Bax and Bak in mice reduces infarct size following reperfused myocardial infarction (MI/R), but the contribution of BAK itself to cardiomyocyte apoptosis and necrosis and infarction has not been investigated. In this study, we use Bak-deficient mice and isolated adult cardiomyocytes to delineate the role of BAK in the pathogenesis of infarct generation and post-infarct remodeling during MI/R and non-reperfused MI. Generalized homozygous deletion of Bak reduced infarct size â¼50% in MI/R in vivo, which was attributable primarily to decreases in necrosis. Protection from necrosis was also observed in BAK-deficient isolated cardiomyocytes suggesting that the cardioprotection from BAK loss in vivo is at least partially cardiomyocyte-autonomous. Interestingly, heterozygous Bak deletion, in which the heart still retains â¼28% of wild type BAK levels, reduced infarct size to a similar extent as complete BAK absence. In contrast to MI/R, homozygous Bak deletion did not attenuate acute infarct size or long-term scar size, post-infarct remodeling, cardiac dysfunction, or mortality in non-reperfused MI. We conclude that BAK contributes significantly to cardiomyocyte necrosis and infarct generation during MI/R, while its absence does not appear to impact the pathogenesis of non-reperfused MI. These observations suggest BAK may be a therapeutic target for MI/R and that even partial pharmacological antagonism may provide benefit.
Subject(s)
Myocardial Infarction , bcl-2 Homologous Antagonist-Killer Protein , Animals , Mice , Apoptosis/physiology , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolism , Homozygote , Myocardial Infarction/genetics , Myocardial Infarction/metabolism , Myocardial Infarction/pathology , Necrosis/genetics , Sequence Deletion , bcl-2 Homologous Antagonist-Killer Protein/metabolismABSTRACT
Mitochondrial calcium (mCa2+) is a critical regulator of neuronal cell death, bioenergetics, and signaling pathways. Although the regulatory machinery governing mCa2+ uptake via the mitochondrial calcium uniporter (mtCU) has been identified and functionally characterized, regulation of the mitochondrial Na+/Ca2+ exchanger (NCLX), the primary means of mCa2+ efflux, is poorly understood. Rozenfeld et al. report that inhibition of phosphodiesterase 2 (PDE2) enhances mCa2+efflux via increased NCLX phosphorylation by protein kinase A (PKA) [1]. The authors demonstrate that enhancing NCLX activity by pharmacologic inhibition of PDE2 improves neuronal survival in response to excitotoxic insult in vitro and enhances cognitive performance. Here we contextualize this discovery within existing literature and provide conjecture to add clarity to the proposed novel regulatory mechanism.
Subject(s)
Calcium , Sodium-Calcium Exchanger , Calcium/metabolism , Sodium-Calcium Exchanger/metabolism , Mitochondria/metabolism , Signal Transduction , Phosphorylation , Calcium Signaling/physiologyABSTRACT
Background: Mitochondrial calcium (mCa2+) uptake through the mitochondrial calcium uniporter channel (mtCU) stimulates metabolism to meet acute increases in cardiac energy demand. However, excessive mCa2+ uptake during stress, as in ischemia-reperfusion, initiates permeability transition and cell death. Despite these often-reported acute physiological and pathological effects, a major unresolved controversy is whether mtCU-dependent mCa2+ uptake and long-term elevation of cardiomyocyte mCa2+ contributes to the heart's adaptation during sustained increases in workload. Objective: We tested the hypothesis that mtCU-dependent mCa2+ uptake contributes to cardiac adaptation and ventricular remodeling during sustained catecholaminergic stress. Methods: Mice with tamoxifen-inducible, cardiomyocyte-specific gain (αMHC-MCM × flox-stop-MCU; MCU-Tg) or loss (αMHC-MCM × Mcufl/fl; Mcu-cKO) of mtCU function received 2-wk catecholamine infusion. Results: Cardiac contractility increased after 2d of isoproterenol in control, but not Mcu-cKO mice. Contractility declined and cardiac hypertrophy increased after 1-2-wk of isoproterenol in MCU-Tg mice. MCU-Tg cardiomyocytes displayed increased sensitivity to Ca2+- and isoproterenol-induced necrosis. However, loss of the mitochondrial permeability transition pore (mPTP) regulator cyclophilin D failed to attenuate contractile dysfunction and hypertrophic remodeling, and increased isoproterenol-induced cardiomyocyte death in MCU-Tg mice. Conclusions: mtCU mCa2+ uptake is required for early contractile responses to adrenergic signaling, even those occurring over several days. Under sustained adrenergic load excessive MCU-dependent mCa2+ uptake drives cardiomyocyte dropout, perhaps independent of classical mitochondrial permeability transition pore opening, and compromises contractile function. These findings suggest divergent consequences for acute versus sustained mCa2+ loading, and support distinct functional roles for the mPTP in settings of acute mCa2+ overload versus persistent mCa2+ stress.
ABSTRACT
MICU1 is a calcium (Ca2+)-binding protein that regulates the mitochondrial Ca2+ uniporter channel complex (mtCU) and mitochondrial Ca2+ uptake. MICU1 knockout mice display disorganized mitochondrial architecture, a phenotype that is distinct from that of mice with deficiencies in other mtCU subunits and, thus, is likely not explained by changes in mitochondrial matrix Ca2+ content. Using proteomic and cellular imaging techniques, we found that MICU1 localized to the mitochondrial contact site and cristae organizing system (MICOS) and directly interacted with the MICOS components MIC60 and CHCHD2 independently of the mtCU. We demonstrated that MICU1 was essential for MICOS complex formation and that MICU1 ablation resulted in altered cristae organization, mitochondrial ultrastructure, mitochondrial membrane dynamics, and cell death signaling. Together, our results suggest that MICU1 is an intermembrane space Ca2+ sensor that modulates mitochondrial membrane dynamics independently of matrix Ca2+ uptake. This system enables distinct Ca2+ signaling in the mitochondrial matrix and at the intermembrane space to modulate cellular energetics and cell death in a concerted manner.
Subject(s)
Mitochondrial Membranes , Proteomics , Mice , Animals , Mitochondrial Membranes/metabolism , Calcium Channels/genetics , Calcium Channels/metabolism , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondria/genetics , Mitochondria/metabolism , Mice, Knockout , Calcium/metabolism , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Mitochondrial Proteins/metabolismABSTRACT
Mitochondrial calcium overload contributes to neurodegenerative disease development and progression. We recently reported that loss of the mitochondrial sodium/calcium exchanger (NCLX), the primary mechanism of mCa2+ efflux, promotes mCa2+ overload, metabolic derangement, redox stress, and cognitive decline in models of Alzheimer's disease (AD). However, whether disrupted mCa2+ signaling contributes to neuronal pathology and cognitive decline independent of pre-existing amyloid or tau pathology remains unknown. Here, we generated mice with neuronal deletion of the mitochondrial sodium/calcium exchanger (NCLX, Slc8b1 gene), and evaluated age-associated changes in cognitive function and neuropathology. Neuronal loss of NCLX resulted in an age-dependent decline in spatial and cued recall memory, moderate amyloid deposition, mild tau pathology, synaptic remodeling, and indications of cell death. These results demonstrate that loss of NCLX-dependent mCa2+ efflux alone is sufficient to induce an Alzheimer's disease-like pathology and highlights the promise of therapies targeting mCa2+ exchange.
ABSTRACT
Heart failure (HF) with preserved ejection fraction (HFpEF) is defined as HF with an ejection fraction (EF) ≥ 50% and elevated cardiac diastolic filling pressures. The underlying causes of HFpEF are multifactorial and not well-defined. A transgenic mouse with low levels of cardiomyocyte (CM)-specific inducible Cavß2a expression (ß2a-Tg mice) showed increased cytosolic CM Ca2+, and modest levels of CM hypertrophy, and fibrosis. This study aimed to determine if ß2a-Tg mice develop an HFpEF phenotype when challenged with two additional stressors, high-fat diet (HFD) and Nω-nitro-l-arginine methyl ester (l-NAME, LN). Four-month-old wild-type (WT) and ß2a-Tg mice were given either normal chow (WT-N, ß2a-N) or HFD and/or l-NAME (WT-HFD, WT-LN, WT-HFD-LN, ß2a-HFD, ß2a-LN, and ß2a-HFD-LN). Some animals were treated with the histone deacetylase (HDAC) (hypertrophy regulators) inhibitor suberoylanilide hydroxamic acid (SAHA) (ß2a-HFD-LN-SAHA). Echocardiography was performed monthly. After 4 mo of treatment, terminal studies were performed including invasive hemodynamics and organs weight measurements. Cardiac tissue was collected. Four months of HFD plus l-NAME treatment did not induce a profound HFpEF phenotype in FVB WT mice. ß2a-HFD-LN (3-Hit) mice developed features of HFpEF, including increased atrial natriuretic peptide (ANP) levels, preserved EF, diastolic dysfunction, robust CM hypertrophy, increased M2-macrophage population, and myocardial fibrosis. SAHA reduced the HFpEF phenotype in the 3-Hit mouse model, by attenuating these effects. The 3-Hit mouse model induced a reliable HFpEF phenotype with CM hypertrophy, cardiac fibrosis, and increased M2-macrophage population. This model could be used for identifying and preclinical testing of novel therapeutic strategies.NEW & NOTEWORTHY Our study shows that three independent pathological stressors (increased Ca2+ influx, high-fat diet, and l-NAME) together produce a profound HFpEF phenotype. The primary mechanisms include HDAC-dependent-CM hypertrophy, necrosis, increased M2-macrophage population, fibroblast activation, and myocardial fibrosis. A role for HDAC activation in the HFpEF phenotype was shown in studies with SAHA treatment, which prevented the severe HFpEF phenotype. This "3-Hit" mouse model could be helpful in identifying novel therapeutic strategies to treat HFpEF.
Subject(s)
Cardiomyopathies , Heart Failure , Mice , Animals , Heart Failure/genetics , Heart Failure/drug therapy , Stroke Volume/physiology , NG-Nitroarginine Methyl Ester/pharmacology , Mice, Transgenic , Fibrosis , Phenotype , HypertrophyABSTRACT
Intracellular Ca2+ concentrations are strictly controlled by plasma membrane transporters, the endoplasmic reticulum, and mitochondria, in which Ca2+ uptake is mediated by the mitochondrial calcium uniporter complex (MCUc), while efflux occurs mainly through the mitochondrial Na+ /Ca2+ exchanger (NCLX). RNAseq database repository searches led us to identify the Nclx transcript as highly enriched in astrocytes when compared with neurons. To assess the role of NCLX in mouse primary culture astrocytes, we inhibited its function both pharmacologically or genetically. This resulted in re-shaping of cytosolic Ca2+ signaling and a metabolic shift that increased glycolytic flux and lactate secretion in a Ca2+ -dependent manner. Interestingly, in vivo genetic deletion of NCLX in hippocampal astrocytes improved cognitive performance in behavioral tasks, whereas hippocampal neuron-specific deletion of NCLX impaired cognitive performance. These results unveil a role for NCLX as a novel modulator of astrocytic glucose metabolism, impacting on cognition.
Subject(s)
Astrocytes , Calcium , Mice , Animals , Astrocytes/metabolism , Calcium/metabolism , Sodium-Calcium Exchanger/genetics , Mitochondria/metabolism , Glycolysis , Cognition , Sodium/metabolism , Calcium Signaling/physiologyABSTRACT
BACKGROUND: Hydrogen sulfide is a critical endogenous signaling molecule that exerts protective effects in the setting of heart failure. Cystathionine γ-lyase (CSE), 1 of 3 hydrogen-sulfide-producing enzyme, is predominantly localized in the vascular endothelium. The interaction between the endothelial CSE-hydrogen sulfide axis and endothelial-mesenchymal transition, an important pathological process contributing to the formation of fibrosis, has yet to be investigated. METHODS: Endothelial-cell-specific CSE knockout and Endothelial cell-CSE overexpressing mice were subjected to transverse aortic constriction to induce heart failure with reduced ejection fraction. Cardiac function, vascular reactivity, and treadmill exercise capacity were measured to determine the severity of heart failure. Histological and gene expression analyses were performed to investigate changes in cardiac fibrosis and the activation of endothelial-mesenchymal transition. RESULTS: Endothelial-cell-specific CSE knockout mice exhibited increased endothelial-mesenchymal transition and reduced nitric oxide bioavailability in the myocardium, which was associated with increased cardiac fibrosis, impaired cardiac and vascular function, and worsened exercise performance. In contrast, genetic overexpression of CSE in endothelial cells led to increased myocardial nitric oxide, decreased endothelial-mesenchymal transition and cardiac fibrosis, preserved cardiac and endothelial function, and improved exercise capacity. CONCLUSIONS: Our data demonstrate that endothelial CSE modulates endothelial-mesenchymal transition and ameliorate the severity of pressure-overload-induced heart failure, in part, through nitric oxide-related mechanisms. These data further suggest that endothelium-derived hydrogen sulfide is a potential therapeutic for the treatment of heart failure with reduced ejection fraction.
Subject(s)
Heart Failure , Hydrogen Sulfide , Ventricular Dysfunction, Left , Mice , Animals , Hydrogen Sulfide/metabolism , Endothelial Cells/metabolism , Nitric Oxide/metabolism , Mice, Knockout , Endothelium, Vascular/metabolism , FibrosisABSTRACT
The molecular mechanisms that link propionyl-CoA metabolism and epigenetic regulation of gene expression are unclear, as are the implications for heart function. Now, new insights into the modulation of chromatin acylation and transcription by aberrant oxidation of propionyl-CoA are revealed in the dysfunctional hearts of mice with propionic acidemia.
Subject(s)
Acyl Coenzyme A , Chromatin , Transcription, Genetic , Animals , Mice , Acyl Coenzyme A/metabolism , Acylation , Chromatin/metabolism , Chromatin/genetics , Epigenesis, Genetic , Myocardium/metabolism , Oxidation-Reduction , Propionic Acidemia/metabolism , Propionic Acidemia/geneticsABSTRACT
Pharmacologic activation of branched-chain amino acid (BCAA) catabolism is protective in models of heart failure (HF). How protection occurs remains unclear, although a causative block in cardiac BCAA oxidation is widely assumed. Here, we use in vivo isotope infusions to show that cardiac BCAA oxidation in fact increases, rather than decreases, in HF. Moreover, cardiac-specific activation of BCAA oxidation does not protect from HF even though systemic activation does. Lowering plasma and cardiac BCAAs also fails to confer significant protection, suggesting alternative mechanisms of protection. Surprisingly, activation of BCAA catabolism lowers blood pressure (BP), a known cardioprotective mechanism. BP lowering occurred independently of nitric oxide and reflected vascular resistance to adrenergic constriction. Mendelian randomization studies revealed that elevated plasma BCAAs portend higher BP in humans. Together, these data indicate that BCAA oxidation lowers vascular resistance, perhaps in part explaining cardioprotection in HF that is not mediated directly in cardiomyocytes.
Subject(s)
Amino Acids, Branched-Chain , Heart Failure , Humans , Blood Pressure , Amino Acids, Branched-Chain/metabolism , Heart , Heart Failure/metabolism , Energy MetabolismABSTRACT
BACKGROUND: Fibrosis and extracellular matrix remodeling are mediated by resident cardiac fibroblasts (CFs). In response to injury, fibroblasts activate, differentiating into specialized synthetic and contractile myofibroblasts producing copious extracellular matrix proteins (e.g., collagens). Myofibroblast persistence in chronic diseases, such as HF, leads to progressive cardiac dysfunction and maladaptive remodeling. We recently reported that an increase in αKG (alpha-ketoglutarate) bioavailability, which contributes to enhanced αKG-dependent lysine demethylase activity and chromatin remodeling, is required for myofibroblast formation. Therefore, we aimed to determine the substrates and metabolic pathways contributing to αKG biosynthesis and their requirement for myofibroblast formation. METHODS: Stable isotope metabolomics identified glutaminolysis as a key metabolic pathway required for αKG biosynthesis and myofibroblast formation, therefore we tested the effects of pharmacologic inhibition (CB-839) or genetic deletion of glutaminase (Gls1-/-) on myofibroblast formation in both murine and human cardiac fibroblasts. We employed immunofluorescence staining, functional gel contraction, western blotting, and bioenergetic assays to determine the myofibroblast phenotype. RESULTS: Carbon tracing indicated enhanced glutaminolysis mediating increased αKG abundance. Pharmacological and genetic inhibition of glutaminolysis prevented myofibroblast formation indicated by a reduction in αSMA+ cells, collagen gel contraction, collagen abundance, and the bioenergetic response. Inhibition of glutaminolysis also prevented TGFß-mediated histone demethylation and supplementation with cell-permeable αKG rescued the myofibroblast phenotype. Importantly, inhibition of glutaminolysis was sufficient to prevent myofibroblast formation in CFs isolated from the human failing heart. CONCLUSIONS: These results define glutaminolysis as necessary for myofibroblast formation and persistence, providing substantial rationale to evaluate several new therapeutic targets to treat cardiac fibrosis.
Subject(s)
Myofibroblasts , Humans , Mice , Animals , Myofibroblasts/metabolism , Glutamine/metabolism , Fibroblasts/metabolism , Collagen/metabolism , Cells, CulturedABSTRACT
The role of store-operated Ca2+ entry (SOCE) in melanoma metastasis is highly controversial. To address this, we here examined UV-dependent metastasis, revealing a critical role for SOCE suppression in melanoma progression. UV-induced cholesterol biosynthesis was critical for UV-induced SOCE suppression and subsequent metastasis, although SOCE suppression alone was both necessary and sufficient for metastasis to occur. Further, SOCE suppression was responsible for UV-dependent differences in gene expression associated with both increased invasion and reduced glucose metabolism. Functional analyses further established that increased glucose uptake leads to a metabolic shift towards biosynthetic pathways critical for melanoma metastasis. Finally, examination of fresh surgically isolated human melanoma explants revealed cholesterol biosynthesis-dependent reduced SOCE. Invasiveness could be reversed with either cholesterol biosynthesis inhibitors or pharmacological SOCE potentiation. Collectively, we provide evidence that, contrary to current thinking, Ca2+ signals can block invasive behavior, and suppression of these signals promotes invasion and metastasis.