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1.
Proc Natl Acad Sci U S A ; 120(51): e2303713120, 2023 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-38091291

RESUMO

The mitochondrial permeability transition pore (mPTP) is a channel in the inner mitochondrial membrane whose sustained opening in response to elevated mitochondrial matrix Ca2+ concentrations triggers necrotic cell death. The molecular identity of mPTP is unknown. One proposed candidate is the mitochondrial ATP synthase, whose canonical function is to generate most ATP in multicellular organisms. Here, we present mitochondrial, cellular, and in vivo evidence that, rather than serving as mPTP, the mitochondrial ATP synthase inhibits this pore. Our studies confirm previous work showing persistence of mPTP in HAP1 cell lines lacking an assembled mitochondrial ATP synthase. Unexpectedly, however, we observe that Ca2+-induced pore opening is markedly sensitized by loss of the mitochondrial ATP synthase. Further, mPTP opening in cells lacking the mitochondrial ATP synthase is desensitized by pharmacological inhibition and genetic depletion of the mitochondrial cis-trans prolyl isomerase cyclophilin D as in wild-type cells, indicating that cyclophilin D can modulate mPTP through substrates other than subunits in the assembled mitochondrial ATP synthase. Mitoplast patch clamping studies showed that mPTP channel conductance was unaffected by loss of the mitochondrial ATP synthase but still blocked by cyclophilin D inhibition. Cardiac mitochondria from mice whose heart muscle cells we engineered deficient in the mitochondrial ATP synthase also demonstrate sensitization of Ca2+-induced mPTP opening and desensitization by cyclophilin D inhibition. Further, these mice exhibit strikingly larger myocardial infarctions when challenged with ischemia/reperfusion in vivo. We conclude that the mitochondrial ATP synthase does not function as mPTP and instead negatively regulates this pore.


Assuntos
Poro de Transição de Permeabilidade Mitocondrial , ATPases Mitocondriais Próton-Translocadoras , Camundongos , Animais , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Ciclofilinas/genética , Ciclofilinas/metabolismo , Peptidil-Prolil Isomerase F , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Cálcio/metabolismo
2.
J Mol Cell Cardiol ; 184: 1-12, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37709008

RESUMO

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 ∼50% 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 ∼28% 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.


Assuntos
Infarto do Miocárdio , Proteína Killer-Antagonista Homóloga a bcl-2 , Animais , Camundongos , Apoptose/fisiologia , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Homozigoto , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Necrose/genética , Deleção de Sequência , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo
3.
Biomed Res Int ; 2019: 1580982, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31341888

RESUMO

Cardiac hypertrophy mainly predicts heart failure and is highly linked with sudden loss of lives. MicroRNAs (miRNAs) play essential roles in the development of cardiac hypertrophy through binding to corresponding mRNA targets. In this study, in order to investigate the roles of two mature forms of miRNA-195, miR-195-3p, and miR-195-5p, in vitro and in vivo models of cardiac hypertrophy were established by applying angiotensin II (Ang II) to H9c2 cardiomyocytes and infusing chronic Ang II to mice, respectively. We found that miR-195-5p was evidently equally upregulated in the in vitro and in vivo studies of cardiac hypertrophy induced by Ang II. High expressed miR-195-5p could adequately promote hypertrophy, whereas the suppression of miR-195-5p prevented hypertrophy of H9c2 cardiomyocytes under Ang II treatment. Furthermore, the luciferase reporter system demonstrated that MFN2 and FBWX7 were target genes of miR-195-5p, which negatively regulated the expression of these two genes in H9c2 cells. By contrast, in both models, expression of miR-195-3p was only slightly changed without statistical significance. In addition, we observed a trend towards decreased expression of hypertrophic markers by overexpressing miR-195-3p in AngII-treated H9c2 cardiomyocytes in vitro. Taken together, our study indicates that miR-195-5p promotes cardiac hypertrophy via targeting MFN2 and FBXW7 and may provide promising therapeutic strategies for interfering cardiac hypertrophy.


Assuntos
Cardiomegalia/metabolismo , Proteína 7 com Repetições F-Box-WD/antagonistas & inibidores , GTP Fosfo-Hidrolases/biossíntese , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Angiotensina II/efeitos adversos , Angiotensina II/farmacologia , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/patologia , Masculino , Camundongos , Miócitos Cardíacos/patologia
4.
Shock ; 51(3): 372-380, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-29470359

RESUMO

This study tested the hypothesis that CD44 is involved in the development of cardiac fibrosis via angiotensin II (Ang II) AT1 receptor-stimulated TNFα/NFκB/IκB signaling pathways. Study was conducted in C57BL/6 wild type and CD44 knockout mice subjected to Ang II infusion (1,000 ng/kg/min) using osmotic minipumps up to 4 weeks or with gastric gavage administration of the AT1 receptor blocker, telmisartan at a dose of 10 mg/kg/d. Results indicated that Ang II enhances expression of the AT1 receptor, TNFα, NFκB, and CD44 as well as downregulates IκB. Further analyses revealed that Ang II increases macrophage migration, augments myofibroblast proliferation, and induces vascular/interstitial fibrosis. Relative to the Ang II group, treatment with telmisartan significantly reduced expression of the AT1 receptor and TNFα. These changes occurred in coincidence with decreased NFκB, increased IκB, and downregulated CD44 in the intracardiac vessels and intermyocardium. Furthermore, macrophage migration and myofibroblast proliferation were inhibited and fibrosis was attenuated. Knockout of CD44 did not affect Ang II-stimulated AT1 receptor and modulated TNFα/NFκB/IκB signaling, but significantly reduced macrophage/myofibroblast-mediated fibrosis as identified by less extensive collagen-rich area. These results suggest that the AT1 receptor is involved in the development of cardiac fibrosis by stimulating TNFα/NFκB/IκB-triggered CD44 signaling pathways. Knockout of CD44 blocked Ang II-induced cell migration/proliferation and cardiac fibrosis. Therefore, selective inhibition of CD44 may be considered as a potential therapeutic target for attenuating Ang II-induced deleterious cardiovascular effects.


Assuntos
Angiotensina II/efeitos adversos , Cardiopatias/prevenção & controle , Receptores de Hialuronatos/deficiência , Miocárdio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Angiotensina II/farmacologia , Animais , Feminino , Fibrose , Cardiopatias/induzido quimicamente , Cardiopatias/genética , Cardiopatias/metabolismo , Receptores de Hialuronatos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Miocárdio/patologia , NF-kappa B/genética , NF-kappa B/metabolismo , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
5.
Int J Nanomedicine ; 12: 1317-1328, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28243092

RESUMO

An easier method for constructing the hierarchical micro-/nano-structures on the surface of dental implants in the clinic is needed. In this study, three different titanium surfaces with microscale grooves (width 0.5-1, 1-1.5, and 1.5-2 µm) and nanoscale nanoparticles (diameter 20-30, 30-50, and 50-100 nm, respectively) were obtained by treatment with different concentrations of hydrofluoric acid (HF) and at different etching times (1%, 3 min; 0.5%, 12 min; and 1.5%, 12 min, respectively; denoted as groups HF1, HF2, and HF3). The biological response to the three different titanium surfaces was evaluated by in vitro human bone marrow-derived mesenchymal stem cell (hBMMSC) experiments and in vivo animal experiments. The results showed that cell adhesion, proliferation, alkaline phosphatase activity, and mineralization of hBMMSCs were increased in the HF3 group. After the different surface implants were inserted into the distal femurs of 40 rats, the bone-implant contact in groups HF1, HF2, and HF3 was 33.17%±2.2%, 33.82%±3.42%, and 41.04%±3.08%, respectively. Moreover, the maximal pullout force in groups HF1, HF2, and HF3 was 57.92±2.88, 57.83±4.09, and 67.44±6.14 N, respectively. The results showed that group HF3 with large micron grooves (1.5-2.0 µm) and large nanoparticles (50-100 nm) showed the best bio-functionality for the hBMMSC response and osseointegration in animal experiments compared with other groups.


Assuntos
Ácido Fluorídrico/farmacologia , Nanopartículas/química , Osteogênese/efeitos dos fármacos , Titânio/química , Titânio/farmacologia , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Células da Medula Óssea/citologia , Adesão Celular/efeitos dos fármacos , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Feminino , Fêmur/efeitos dos fármacos , Fêmur/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Implantes Experimentais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/ultraestrutura , Nanopartículas/ultraestrutura , Osseointegração/fisiologia , Osteogênese/genética , Ratos Sprague-Dawley , Espectrometria por Raios X , Propriedades de Superfície
6.
J Biol Chem ; 291(38): 20247-59, 2016 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-27502281

RESUMO

Recent studies have shown that myocardial ischemia/reperfusion (I/R)-induced necrosis can be controlled by multiple genes. In this study, we observed that both strands (5p and 3p) of miR-223 were remarkably dysregulated in mouse hearts upon I/R. Precursor miR-223 (pre-miR-223) transgenic mouse hearts exhibited better recovery of contractile performance over reperfusion period and lesser degree of myocardial necrosis than wild type hearts upon ex vivo and in vivo myocardial ischemia. Conversely, pre-miR-223 knock-out (KO) mouse hearts displayed opposite effects. Furthermore, we found that the RIP1/RIP3/MLKL necroptotic pathway and inflammatory response were suppressed in transgenic hearts, whereas they were activated in pre-miR-223 KO hearts upon I/R compared with wild type controls. Accordingly, treatment of pre-miR-223 KO mice with necrostatin-1s, a potent necroptosis inhibitor, significantly decreased I/R-triggered cardiac necroptosis, infarction size, and dysfunction. Mechanistically, we identified two critical cell death receptors, TNFR1 and DR6, as direct targets of miR-223-5p, whereas miR-223-3p directly suppressed the expression of NLRP3 and IκB kinase α, two important mediators known to be involved in I/R-induced inflammation and cell necroptosis. Our findings indicate that miR-223-5p/-3p duplex works together and cooperatively inhibits I/R-induced cardiac necroptosis at multiple layers. Thus, pre-miR-223 may constitute a new therapeutic agent for the treatment of ischemic heart disease.


Assuntos
MicroRNAs/biossíntese , Traumatismo por Reperfusão Miocárdica/metabolismo , Animais , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Imidazóis/farmacologia , Indóis/farmacologia , Camundongos , Camundongos Knockout , MicroRNAs/genética , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Necrose , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/biossíntese , Receptores Tipo I de Fatores de Necrose Tumoral/genética
7.
Diabetes ; 65(10): 3111-28, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27284111

RESUMO

Decreased heat shock protein (Hsp) expression in type 1 and type 2 diabetes has been implicated as a primary factor contributing to diabetes-induced organ damage. We recently showed that diabetic cardiomyocytes could release detrimental exosomes, which contain lower levels of Hsp20 than normal ones. To investigate whether such detrimental exosomes could be modified in cardiomyocytes by raising Hsp20 levels to become protective, we used a transgenic (TG) mouse model with cardiac-specific overexpression of Hsp20. TG and control wild-type (WT) mice were injected with streptozotocin (STZ) to induce diabetes. We observed that overexpression of Hsp20 significantly attenuated STZ-caused cardiac dysfunction, hypertrophy, apoptosis, fibrosis, and microvascular rarefaction. Moreover, Hsp20-TG cardiomyocytes exhibited an increased generation/secretion of exosomes by direct interaction of Hsp20 with Tsg101. Of importance, exosomes derived from TG cardiomyocytes encased higher levels of Hsp20, p-Akt, survivin, and SOD1 than WT exosomes and protected against in vitro hyperglycemia-triggered cell death, as well as in vivo STZ-induced cardiac adverse remodeling. Last, blockade of exosome generation by GW4869 remarkably offset Hsp20-mediated cardioprotection in diabetic mice. Our results indicate that elevation of Hsp20 in cardiomyocytes can offer protection in diabetic hearts through the release of instrumental exosomes. Thus, Hsp20-engineered exosomes might be a novel therapeutic agent for diabetic cardiomyopathy.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Exossomos/metabolismo , Proteínas de Choque Térmico HSP20/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Neovascularização Fisiológica/fisiologia , Compostos de Anilina/farmacologia , Animais , Compostos de Benzilideno/farmacologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Exossomos/efeitos dos fármacos , Proteínas de Choque Térmico HSP20/genética , Coração/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase-1/metabolismo
8.
J Biol Chem ; 291(30): 15700-13, 2016 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-27226563

RESUMO

MicroRNAs (miRNAs) have been extensively examined in pathological cardiac hypertrophy. However, few studies focused on profiling the miRNA alterations in physiological hypertrophic hearts. In this study we generated a transgenic mouse model with cardiac-specific overexpression of miR-223. Our results showed that elevation of miR-223 caused physiological cardiac hypertrophy with enhanced cardiac function but no fibrosis. Using the next generation RNA sequencing, we observed that most of dys-regulated genes (e.g. Atf3/5, Egr1/3, Sfrp2, Itgb1, Ndrg4, Akip1, Postn, Rxfp1, and Egln3) in miR-223-transgenic hearts were associated with cell growth, but they were not directly targeted by miR-223. Interestingly, these dys-regulated genes are known to regulate the Akt signaling pathway. We further identified that miR-223 directly interacted with 3'-UTRs of FBXW7 and Acvr2a, two negative regulators of the Akt signaling. However, we also validated that miR-223 directly inhibited the expression of IGF-1R and ß1-integrin, two positive regulators of the Akt signaling. Lastly, Western blotting did reveal that Akt was activated in miR-223-overexpressing hearts. Adenovirus-mediated overexpression of miR-223 in neonatal rat cardiomyocytes induced cell hypertrophy, which was blocked by the addition of MK2206, a specific inhibitor of Akt Taken together, these data represent the first piece of work showing that miR-223 tips the balance of promotion and inactivation of Akt signaling cascades toward activation of Akt, a key regulator of physiological cardiac hypertrophy. Thus, our study suggests that the ultimate phenotype outcome of a miRNA may be decided by the secondary net effects of the whole target network rather than by several primary direct targets in an organ/tissue.


Assuntos
Cardiomegalia/metabolismo , Regulação da Expressão Gênica , MicroRNAs/biossíntese , Transdução de Sinais , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Adenoviridae , Animais , Cardiomegalia/genética , Cardiomegalia/patologia , Modelos Animais de Doenças , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Proteína 7 com Repetições F-Box-WD , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução Genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
9.
Sci Rep ; 5: 13721, 2015 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-26348153

RESUMO

Mesenchymal stem cells (MSCs) have been shown to elicit cardio-protective effects in sepsis. However, the underlying mechanism remains obscure. While recent studies have indicated that miR-223 is highly enriched in MSC-derived exosomes, whether exosomal miR-223 contributes to MSC-mediated cardio-protection in sepsis is unknown. In this study, loss-of-function approach was utilized, and sepsis was induced by cecal ligation and puncture (CLP). We observed that injection of miR-223-KO MSCs at 1 h post-CLP did not confer protection against CLP-triggered cardiac dysfunction, apoptosis and inflammatory response. However, WT-MSCs were able to provide protection which was associated with exosome release. Next, treatment of CLP mice with exosomes released from miR-223-KO MSCs significantly exaggerated sepsis-induced injury. Conversely, WT-MSC-derived-exosomes displayed protective effects. Mechanistically, we identified that miR-223-KO exosomes contained higher levels of Sema3A and Stat3, two known targets of miR-223 (5p &3p), than WT-exosomes. Accordingly, these exosomal proteins were transferred to cardiomyocytes, leading to increased inflammation and cell death. By contrast, WT-exosomes encased higher levels of miR-223, which could be delivered to cardiomyocytes, resulting in down-regulation of Sema3A and Stat3. These data for the first time indicate that exosomal miR-223 plays an essential role for MSC-induced cardio-protection in sepsis.


Assuntos
Exossomos/genética , Cardiopatias/etiologia , Cardiopatias/prevenção & controle , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Sepse/complicações , Sepse/microbiologia , Animais , Morte Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Exossomos/metabolismo , Feminino , Cardiopatias/metabolismo , Cardiopatias/mortalidade , Cardiopatias/fisiopatologia , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Fator de Transcrição STAT3/metabolismo , Semaforina-3A/metabolismo , Sepse/mortalidade
10.
Biochim Biophys Acta ; 1852(11): 2362-71, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26300484

RESUMO

Sepsis is an infection-induced severe inflammatory disorder that leads to multiple organ failure. Amongst organs affected, myocardial depression is believed to be a major contributor to septic death. While it has been identified that large amounts of circulating pro-inflammatory cytokines are culprit for triggering cardiac dysfunction in sepsis, the underlying mechanisms remain obscure. Additionally, recent studies have shown that exosomes released from bacteria-infected macrophages are pro-inflammatory. Hence, we examined in this study whether blocking the generation of exosomes would be protective against sepsis-induced inflammatory response and cardiac dysfunction. To this end, we pre-treated RAW264.7 macrophages with GW4869, an inhibitor of exosome biogenesis/release, followed by endotoxin (LPS) challenge. In vivo, we injected wild-type (WT) mice with GW4869 for 1h prior to endotoxin treatment or cecal ligation/puncture (CLP) surgery. We observed that pre-treatment with GW4869 significantly impaired release of both exosomes and pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6) in RAW264.7 macrophages. At 12h after LPS treatment or CLP surgery, WT mice pre-treated with GW4869 displayed lower amounts of exosomes and pro-inflammatory cytokines in the serum than control PBS-injected mice. Accordingly, GW4869 treatment diminished the sepsis-induced cardiac inflammation, attenuated myocardial depression and prolonged survival. Together, our findings indicate that blockade of exosome generation in sepsis dampens the sepsis-triggered inflammatory response and thereby, improves cardiac function and survival.

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