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1.
Circ Res ; 127(3): 379-390, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32299299

RESUMO

RATIONALE: Mitochondrial Ca2+ loading augments oxidative metabolism to match functional demands during times of increased work or injury. However, mitochondrial Ca2+ overload also directly causes mitochondrial rupture and cardiomyocyte death during ischemia-reperfusion injury by inducing mitochondrial permeability transition pore opening. The MCU (mitochondrial Ca2+ uniporter) mediates mitochondrial Ca2+ influx, and its activity is modulated by partner proteins in its molecular complex, including the MCUb subunit. OBJECTIVE: Here, we sought to examine the function of the MCUb subunit of the MCU-complex in regulating mitochondria Ca2+ influx dynamics, acute cardiac injury, and long-term adaptation after ischemic injury. METHODS AND RESULTS: Cardiomyocyte-specific MCUb overexpressing transgenic mice and Mcub gene-deleted (Mcub-/-) mice were generated to dissect the molecular function of this protein in the heart. We observed that MCUb protein is undetectable in the adult mouse heart at baseline, but mRNA and protein are induced after ischemia-reperfusion injury. MCUb overexpressing mice demonstrated inhibited mitochondrial Ca2+ uptake in cardiomyocytes and partial protection from ischemia-reperfusion injury by reducing mitochondrial permeability transition pore opening. Antithetically, deletion of the Mcub gene exacerbated pathological cardiac remodeling and infarct expansion after ischemic injury in association with greater mitochondrial Ca2+ uptake. Furthermore, hindlimb remote ischemic preconditioning induced MCUb expression in the heart, which was associated with decreased mitochondrial Ca2+ uptake, collectively suggesting that induction of MCUb protein in the heart is protective. Similarly, mouse embryonic fibroblasts from Mcub-/- mice were more sensitive to Ca2+ overload. CONCLUSIONS: Our studies suggest that Mcub is a protective cardiac inducible gene that reduces mitochondrial Ca2+ influx and permeability transition pore opening after ischemic injury to reduce ongoing pathological remodeling.


Assuntos
Cálcio/metabolismo , Membro Posterior/irrigação sanguínea , Proteínas de Membrana/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Infarto do Miocárdio/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Remodelação Ventricular , Animais , Sinalização do Cálcio , Morte Celular , Linhagem Celular , Modelos Animais de Doenças , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Precondicionamento Isquêmico , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/patologia , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Proteínas Mitocondriais/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/patologia
2.
J Mol Cell Cardiol ; 144: A3-A13, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32454061

RESUMO

The adenosine nucleotide translocase (ANT) family of proteins are inner mitochondrial membrane proteins involved in energy homeostasis and cell death. The primary function of ANT proteins is to exchange cytosolic ADP with matrix ATP, facilitating the export of newly synthesized ATP to the cell while providing new ADP substrate to the mitochondria. As such, the ANT proteins are central to maintaining energy homeostasis in all eukaryotic cells. Evidence also suggests that the ANTs constitute a pore-forming component of the mitochondrial permeability transition pore (MPTP), a structure that forms in the inner mitochondrial membrane that is thought to underlie regulated necrotic cell death. Additionally, emerging studies suggest that ANT proteins are also critical for mitochondrial uncoupling and for promoting mitophagy. Thus, the ANTs are multifunctional proteins that are poised to participate in several aspects of mitochondrial biology and the greater regulation of cell death, which will be discussed here.


Assuntos
Mitocôndrias/genética , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/genética , Translocases Mitocondriais de ADP e ATP/metabolismo , Necroptose , Animais , Regulação da Expressão Gênica , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Modelos Biológicos , Família Multigênica , Necroptose/genética , Fosforilação Oxidativa
3.
Pharmacol Res ; 151: 104548, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31759087

RESUMO

Ischemia/reperfusion (I/R) injury is mediated in large part by opening of the mitochondrial permeability transition pore (PTP). Consequently, inhibitors of the PTP hold great promise for the treatment of a variety of cardiovascular disorders. At present, PTP inhibition is obtained only through the use of drugs (e.g. cyclosporine A, CsA) targeting cyclophilin D (CyPD) which is a key modulator, but not a structural component of the PTP. This limitation might explain controversial findings in clinical studies. Therefore, we investigated the protective effects against I/R injury of small-molecule inhibitors of the PTP (63 and TR002) that do not target CyPD. Both compounds exhibited a dose-dependent inhibition of PTP opening in isolated mitochondria and were more potent than CsA. Notably, PTP inhibition was observed also in mitochondria devoid of CyPD. Compounds 63 and TR002 prevented PTP opening and mitochondrial depolarization induced by Ca2+ overload and by reactive oxygen species in neonatal rat ventricular myocytes (NRVMs). Remarkably, both compounds prevented cell death, contractile dysfunction and sarcomeric derangement induced by anoxia/reoxygenation injury in NRVMs at sub-micromolar concentrations, and were more potent than CsA. Cardioprotection was observed also in adult mouse ventricular myocytes and human iPSc-derived cardiomyocytes, as well as ex vivo in perfused hearts. Thus, this study demonstrates that 63 and TR002 represent novel cardioprotective agents that inhibit PTP opening independent of CyPD targeting.


Assuntos
Cardiotônicos/uso terapêutico , Poro de Transição de Permeabilidade Mitocondrial/antagonistas & inibidores , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/uso terapêutico , Animais , Cardiotônicos/farmacologia , Linhagem Celular , Células Cultivadas , Humanos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/efeitos dos fármacos , Ratos Sprague-Dawley , Ratos Wistar , Bibliotecas de Moléculas Pequenas/farmacologia
4.
J Biol Chem ; 291(45): 23490-23505, 2016 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-27621312

RESUMO

Cardiac ryanodine receptor (Ryr2) Ca2+ release channels and cellular metabolism are both disrupted in heart disease. Recently, we demonstrated that total loss of Ryr2 leads to cardiomyocyte contractile dysfunction, arrhythmia, and reduced heart rate. Acute total Ryr2 ablation also impaired metabolism, but it was not clear whether this was a cause or consequence of heart failure. Previous in vitro studies revealed that Ca2+ flux into the mitochondria helps pace oxidative metabolism, but there is limited in vivo evidence supporting this concept. Here, we studied heart-specific, inducible Ryr2 haploinsufficient (cRyr2Δ50) mice with a stable 50% reduction in Ryr2 protein. This manipulation decreased the amplitude and frequency of cytosolic and mitochondrial Ca2+ signals in isolated cardiomyocytes, without changes in cardiomyocyte contraction. Remarkably, in the context of well preserved contractile function in perfused hearts, we observed decreased glucose oxidation, but not fat oxidation, with increased glycolysis. cRyr2Δ50 hearts exhibited hyperphosphorylation and inhibition of pyruvate dehydrogenase, the key Ca2+-sensitive gatekeeper to glucose oxidation. Metabolomic, proteomic, and transcriptomic analyses revealed additional functional networks associated with altered metabolism in this model. These results demonstrate that Ryr2 controls mitochondrial Ca2+ dynamics and plays a specific, critical role in promoting glucose oxidation in cardiomyocytes. Our findings indicate that partial RYR2 loss is sufficient to cause metabolic abnormalities seen in heart disease.


Assuntos
Sinalização do Cálcio , Glucose/metabolismo , Contração Miocárdica , Miocárdio/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Cálcio/metabolismo , Deleção de Genes , Metaboloma , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/citologia , Miocárdio/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Oxirredução , Proteoma , Piruvatos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética
5.
J Biol Chem ; 288(26): 18975-86, 2013 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-23678000

RESUMO

Ca(2+) fluxes between adjacent organelles are thought to control many cellular processes, including metabolism and cell survival. In vitro evidence has been presented that constitutive Ca(2+) flux from intracellular stores into mitochondria is required for basal cellular metabolism, but these observations have not been made in vivo. We report that controlled in vivo depletion of cardiac RYR2, using a conditional gene knock-out strategy (cRyr2KO mice), is sufficient to reduce mitochondrial Ca(2+) and oxidative metabolism, and to establish a pseudohypoxic state with increased autophagy. Dramatic metabolic reprogramming was evident at the transcriptional level via Sirt1/Foxo1/Pgc1α, Atf3, and Klf15 gene networks. Ryr2 loss also induced a non-apoptotic form of programmed cell death associated with increased calpain-10 but not caspase-3 activation or endoplasmic reticulum stress. Remarkably, cRyr2KO mice rapidly exhibited many of the structural, metabolic, and molecular characteristics of heart failure at a time when RYR2 protein was reduced 50%, a similar degree to that which has been reported in heart failure. RYR2-mediated Ca(2+) fluxes are therefore proximal controllers of mitochondrial Ca(2+), ATP levels, and a cascade of transcription factors controlling metabolism and survival.


Assuntos
Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Alelos , Animais , Apoptose , Autofagia , Morte Celular , Sobrevivência Celular , Retículo Endoplasmático/metabolismo , Hipóxia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxigênio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Transcrição Gênica
6.
Arterioscler Thromb Vasc Biol ; 33(12): 2830-8, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24115032

RESUMO

OBJECTIVE: During diabetes mellitus, coronary lipoprotein lipase increases to promote the predominant use of fatty acids. We have reported that high glucose stimulates active heparanase secretion from endothelial cells to cleave cardiomyocyte heparan sulfate and release bound lipoprotein lipase for transfer to the vascular lumen. In the current study, we examined whether heparanase also has a function to release cardiomyocyte vascular endothelial growth factor (VEGF), and whether this growth factor influences cardiomyocyte fatty acid delivery in an autocrine manner. APPROACH AND RESULTS: Acute, reversible hyperglycemia was induced in rats, and a modified Langendorff heart perfusion was used to separate the coronary perfusate from the interstitial effluent. Coronary artery endothelial cells were exposed to high glucose to generate conditioned medium, and VEGF release from isolated cardiomyocytes was tested using endothelial cell conditioned medium or purified active and latent heparanase. Autocrine signaling of myocyte-derived VEGF on cardiac metabolism was studied. High glucose promoted latent and active heparanase secretion into endothelial cell conditioned medium, an effective stimulus for releasing cardiomyocyte VEGF. Intriguingly, latent heparanase was more efficient than active heparanase in releasing VEGF from a unique cell surface pool. VEGF augmented cardiomyocyte intracellular calcium and AMP-activated protein kinase phosphorylation and increased heparin-releasable lipoprotein lipase. CONCLUSIONS: Our data suggest that the heparanase-lipoprotein lipase-VEGF axis amplifies fatty acid delivery, a rapid and adaptive mechanism that is geared to overcome the loss of glucose consumption by the diabetic heart. If prolonged, the resultant lipotoxicity could lead to cardiovascular disease in humans.


Assuntos
Comunicação Autócrina , Vasos Coronários/enzimologia , Células Endoteliais/enzimologia , Glucuronidase/metabolismo , Hiperglicemia/enzimologia , Lipase Lipoproteica/metabolismo , Miócitos Cardíacos/enzimologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Glicemia/metabolismo , Cálcio/metabolismo , Meios de Cultivo Condicionados/metabolismo , Diazóxido , Modelos Animais de Doenças , Metabolismo Energético , Ativação Enzimática , Ácidos Graxos/metabolismo , Hiperglicemia/sangue , Hiperglicemia/induzido quimicamente , Fosforilação , Ratos , Transdução de Sinais , Fatores de Tempo
7.
Sci Rep ; 14(1): 6751, 2024 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-38514795

RESUMO

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.


Assuntos
Distrofia Muscular de Duchenne , Animais , Humanos , Camundongos , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Morte Celular , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Distrofia Muscular de Duchenne/patologia , Necrose/metabolismo
8.
Cell Rep ; 43(5): 114149, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38678560

RESUMO

Loss of muscle mass is a feature of chronic illness and aging. Here, we report that skeletal muscle-specific thrombospondin-1 transgenic mice (Thbs1 Tg) have profound muscle atrophy with age-dependent decreases in exercise capacity and premature lethality. Mechanistically, Thbs1 activates transforming growth factor ß (TGFß)-Smad2/3 signaling, which also induces activating transcription factor 4 (ATF4) expression that together modulates the autophagy-lysosomal pathway (ALP) and ubiquitin-proteasome system (UPS) to facilitate muscle atrophy. Indeed, myofiber-specific inhibition of TGFß-receptor signaling represses the induction of ATF4, normalizes ALP and UPS, and partially restores muscle mass in Thbs1 Tg mice. Similarly, myofiber-specific deletion of Smad2 and Smad3 or the Atf4 gene antagonizes Thbs1-induced muscle atrophy. More importantly, Thbs1-/- mice show significantly reduced levels of denervation- and caloric restriction-mediated muscle atrophy, along with blunted TGFß-Smad3-ATF4 signaling. Thus, Thbs1-mediated TGFß-Smad3-ATF4 signaling in skeletal muscle regulates tissue rarefaction, suggesting a target for atrophy-based muscle diseases and sarcopenia with aging.


Assuntos
Fator 4 Ativador da Transcrição , Músculo Esquelético , Atrofia Muscular , Transdução de Sinais , Proteína Smad2 , Proteína Smad3 , Trombospondina 1 , Fator de Crescimento Transformador beta , Animais , Masculino , Camundongos , Fator 4 Ativador da Transcrição/metabolismo , Autofagia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Trombospondina 1/metabolismo , Trombospondina 1/genética , Fator de Crescimento Transformador beta/metabolismo
9.
Sci Adv ; 9(34): eadi2767, 2023 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-37624892

RESUMO

Mitochondrial permeability transition pore (MPTP) formation contributes to ischemia-reperfusion injury in the heart and several degenerative diseases, including muscular dystrophy (MD). MD is a family of genetic disorders characterized by progressive muscle necrosis and premature death. It has been proposed that the MPTP has two molecular components, the adenine nucleotide translocase (ANT) family of proteins and an unknown component that requires the chaperone cyclophilin D (CypD) to activate. This model was examined in vivo by deleting the gene encoding ANT1 (Slc25a4) or CypD (Ppif) in a δ-sarcoglycan (Sgcd) gene-deleted mouse model of MD, revealing that dystrophic mice lacking Slc25a4 were partially protected from cell death and MD pathology. Dystrophic mice lacking both Slc25a4 and Ppif together were almost completely protected from necrotic cell death and MD disease. This study provides direct evidence that ANT1 and CypD are required MPTP components governing in vivo cell death, suggesting a previously unrecognized therapeutic approach in MD and other necrotic diseases.


Assuntos
Distrofias Musculares , Animais , Camundongos , Necrose , Morte Celular , Peptidil-Prolil Isomerase F , Modelos Animais de Doenças
10.
Cell Rep ; 42(11): 113465, 2023 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-37976157

RESUMO

Mitochondria use the electron transport chain to generate high-energy phosphate from oxidative phosphorylation, a process also regulated by the mitochondrial Ca2+ uniporter (MCU) and Ca2+ levels. Here, we show that MCUb, an inhibitor of MCU-mediated Ca2+ influx, is induced by caloric restriction, where it increases mitochondrial fatty acid utilization. To mimic the fasted state with reduced mitochondrial Ca2+ influx, we generated genetically altered mice with skeletal muscle-specific MCUb expression that showed greater fatty acid usage, less fat accumulation, and lower body weight. In contrast, mice lacking Mcub in skeletal muscle showed increased pyruvate dehydrogenase activity, increased muscle malonyl coenzyme A (CoA), reduced fatty acid utilization, glucose intolerance, and increased adiposity. Mechanistically, pyruvate dehydrogenase kinase 4 (PDK4) overexpression in muscle of Mcub-deleted mice abolished altered substrate preference. Thus, MCUb is an inducible control point in regulating skeletal muscle mitochondrial Ca2+ levels and substrate utilization that impacts total metabolic balance.


Assuntos
Cálcio , Mitocôndrias , Animais , Camundongos , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Ácidos Graxos/metabolismo , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo
11.
Sci Adv ; 5(8): eaaw4597, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31489369

RESUMO

The mitochondrial permeability transition pore (MPTP) has resisted molecular identification. The original model of the MPTP that proposed the adenine nucleotide translocator (ANT) as the inner membrane pore-forming component was challenged when mitochondria from Ant1/2 double null mouse liver still had MPTP activity. Because mice express three Ant genes, we reinvestigated whether the ANTs comprise the MPTP. Liver mitochondria from Ant1, Ant2, and Ant4 deficient mice were highly refractory to Ca2+-induced MPTP formation, and when also given cyclosporine A (CsA), the MPTP was completely inhibited. Moreover, liver mitochondria from mice with quadruple deletion of Ant1, Ant2, Ant4, and Ppif (cyclophilin D, target of CsA) lacked Ca2+-induced MPTP formation. Inner-membrane patch clamping in mitochondria from Ant1, Ant2, and Ant4 triple null mouse embryonic fibroblasts showed a loss of MPTP activity. Our findings suggest a model for the MPTP consisting of two distinct molecular components: The ANTs and an unknown species requiring CypD.


Assuntos
Nucleotídeos de Adenina/genética , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Necrose Dirigida por Permeabilidade Transmembrânica da Mitocôndria/genética , Peptidil-Prolil Isomerase F/genética , Deleção de Sequência/genética , Animais , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Knockout , Poro de Transição de Permeabilidade Mitocondrial
12.
JCI Insight ; 3(22)2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30429366

RESUMO

The mitochondrial Ca2+ uniporter (MCU) complex mediates acute mitochondrial Ca2+ influx. In skeletal muscle, MCU links Ca2+ signaling to energy production by directly enhancing the activity of key metabolic enzymes in the mitochondria. Here, we examined the role of MCU in skeletal muscle development and metabolic function by generating mouse models for the targeted deletion of Mcu in embryonic, postnatal, and adult skeletal muscle. Loss of Mcu did not affect muscle growth and maturation or otherwise cause pathology. Skeletal muscle-specific deletion of Mcu in mice also did not affect myofiber intracellular Ca2+ handling, but it did inhibit acute mitochondrial Ca2+ influx and mitochondrial respiration stimulated by Ca2+, resulting in reduced acute exercise performance in mice. However, loss of Mcu also resulted in enhanced muscle performance under conditions of fatigue, with a preferential shift toward fatty acid metabolism, resulting in reduced body fat with aging. Together, these results demonstrate that MCU-mediated mitochondrial Ca2+ regulation underlies skeletal muscle fuel selection at baseline and under enhanced physiological demands, which affects total homeostatic metabolism.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Músculo Esquelético/metabolismo , Animais , Canais de Cálcio/genética , Sinalização do Cálcio , Metabolismo Energético , Feminino , Marcação de Genes , Masculino , Camundongos , Camundongos Transgênicos , Músculo Esquelético/crescimento & desenvolvimento
13.
Cell Metab ; 23(1): 179-93, 2016 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-26626461

RESUMO

Pancreatic ß cells are mostly post-mitotic, but it is unclear what locks them in this state. Perturbations including uncontrolled hyperglycemia can drive ß cells into more pliable states with reduced cellular insulin levels, increased ß cell proliferation, and hormone mis-expression, but it is unknown whether reduced insulin production itself plays a role. Here, we define the effects of ∼50% reduced insulin production in Ins1(-/-):Ins2(f/f):Pdx1Cre(ERT):mTmG mice prior to robust hyperglycemia. Transcriptome, proteome, and network analysis revealed alleviation of chronic endoplasmic reticulum (ER) stress, indicated by reduced Ddit3, Trib3, and Atf4 expression; reduced Xbp1 splicing; and reduced phospho-eIF2α. This state was associated with hyper-phosphorylation of Akt, which is negatively regulated by Trib3, and with cyclinD1 upregulation. Remarkably, ß cell proliferation was increased 2-fold after reduced insulin production independently of hyperglycemia. Eventually, recombined cells mis-expressed glucagon in the hyperglycemic state. We conclude that the normally high rate of insulin production suppresses ß cell proliferation in a cell-autonomous manner.


Assuntos
Proliferação de Células , Estresse do Retículo Endoplasmático , Células Secretoras de Insulina/fisiologia , Insulina/biossíntese , Animais , Células Cultivadas , Metaboloma , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Mapas de Interação de Proteínas , Proteoma/metabolismo , Transdução de Sinais , Transcriptoma
14.
PLoS One ; 9(4): e95615, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24743769

RESUMO

A large genomic deletion in human cardiac ryanodine receptor (RYR2) gene has been detected in a number of unrelated families with various clinical phenotypes, including catecholaminergic polymorphic ventricular tachycardia (CPVT). This genomic deletion results in an in-frame deletion of exon-3 (Ex3-del). To understand the underlying disease mechanism of the RyR2 Ex3-del mutation, we generated a mouse model in which the RyR2 exon-3 sequence plus 15-bp intron sequences flanking exon-3 were deleted. Heterozygous Ex3-del mice (Ex3-del+/-) survived, but no homozygous Ex3-del mice were born. Unexpectedly, the Ex3-del+/- mice are not susceptible to CPVT. Ex3-del+/- cardiomyocytes exhibited similar amplitude but altered dynamics of depolarization-induced Ca2+ transients compared to wild type (WT) cells. Immunoblotting analysis revealed markedly reduced expression of RyR2 protein in the Ex3-del+/- mutant heart, indicating that Ex3-del has a major impact on RyR2 protein expression in mice. Cardiac specific, conditional knockout of the WT RyR2 allele in Ex3-del+/- mice led to bradycardia and death. Thus, the absence of CPVT and other phenotypes in Ex3-del+/- mice may be attributable to the predominant expression of the WT RyR2 allele as a result of the markedly reduced expression of the Ex3-del mutant allele. The effect of Ex3-del on RyR2 protein expression is discussed in relation to the phenotypic variability in individuals with the RyR2 exon-3 deletion.


Assuntos
Modelos Animais de Doenças , Éxons/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Animais , Camundongos , Taquicardia Ventricular/genética
15.
Protoplasma ; 249 Suppl 1: S49-58, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22105567

RESUMO

Nanometre-scale spaces between organelles represent focused nodes for signal transduction and the control of cellular decisions. The endoplasmic reticulum (ER) and the mitochondria form dynamic quasi-synaptic interaction nanodomains in all cell types examined, but the functional role of these junctions in cellular metabolism and cell survival remains to be fully understood. In this paper, we review recent evidence that ER Ca(2+) channels, such as the RyR and IP(3)R, can signal specifically across this nanodomain to the adjacent mitochondria to pace basal metabolism, with focus on the pancreatic ß-cell. Blocking these signals in the basal state leads to a form of programmed cell death associated with reduced ATP and the induction of calpain-10 and hypoxia-inducible factors. On the other hand, the hyperactivity of this signalling domain plays a deleterious role during classical forms of apoptosis. Thus, the nanospace between ER and mitochondria represents a critical rheostat controlling both metabolism and programmed cell death. Many aspects of the mechanisms underlying this control system remain to be uncovered, and new nanotechnologies are required understand these domains at a molecular level.


Assuntos
Sinalização do Cálcio , Retículo Endoplasmático/metabolismo , Células Secretoras de Insulina/metabolismo , Mitocôndrias/metabolismo , Animais , Apoptose , Cálcio/metabolismo , Sobrevivência Celular , Diabetes Mellitus/metabolismo , Retículo Endoplasmático/ultraestrutura , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , Mitocôndrias/ultraestrutura , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Transdução de Sinais
16.
Cardiovasc Res ; 96(3): 372-80, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-22869620

RESUMO

AIMS: The molecular mechanisms controlling heart function and rhythmicity are incompletely understood. While it is widely accepted that the type 2 ryanodine receptor (Ryr2) is the major Ca(2+) release channel in excitation-contraction coupling, the role of these channels in setting a consistent beating rate remains controversial. Gain-of-function RYR2 mutations in humans and genetically engineered mouse models are known to cause Ca(2+) leak, arrhythmias, and sudden cardiac death. Embryonic stem-cell derived cardiomyocytes lacking Ryr2 display slower beating rates, but no supporting in vivo evidence has been presented. The aim of the present study was to test the hypothesis that RYR2 loss-of-function would reduce heart rate and rhythmicity in vivo. METHODS AND RESULTS: We generated inducible, tissue-specific Ryr2 knockout mice with acute ∼50% loss of RYR2 protein in the heart but not in other tissues. Echocardiography, working heart perfusion, and in vivo ECG telemetry demonstrated that deletion of Ryr2 was sufficient to cause bradycardia and arrhythmia. Our results also show that cardiac Ryr2 knockout mice exhibit functional and structural hallmarks of heart failure, including sudden cardiac death. CONCLUSION: These results illustrate that the RYR2 channel plays an essential role in pacing heart rate. Moreover, we find that RYR2 loss-of-function can lead to fatal arrhythmias typically associated with gain-of-function mutations. Given that RYR2 levels can be reduced in pathological conditions, including heart failure and diabetic cardiomyopathy, we predict that RYR2 loss contributes to disease-associated bradycardia, arrhythmia, and sudden death.


Assuntos
Arritmias Cardíacas/metabolismo , Relógios Biológicos , Frequência Cardíaca , Miocárdio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Arritmias Cardíacas/diagnóstico por imagem , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Bradicardia/genética , Bradicardia/metabolismo , Bradicardia/fisiopatologia , Débito Cardíaco , Morte Súbita Cardíaca/etiologia , Regulação para Baixo , Eletrocardiografia Ambulatorial/métodos , Acoplamento Excitação-Contração , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Miocárdica , RNA Mensageiro/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/deficiência , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Telemetria , Fatores de Tempo , Ultrassonografia , Função Ventricular
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