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1.
Cardiovasc Res ; 2020 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-32170929

RESUMO

AIM: Cardiac dysfunction is a prevalent comorbidity of disrupted inflammatory homeostasis observed in conditions such as sepsis (acute) or obesity (chronic). Secreted and transmembrane protein 1a (Sectm1a) has previously been implicated to regulate inflammatory responses, yet its role in inflammation-associated cardiac dysfunction is virtually unknown. METHODS AND RESULTS: Using the CRISPR/Cas9 system, we generated a global Sectm1a-knockout (KO) mouse model and observed significantly increased mortality and cardiac injury after LPS injection, when compared to wild-type (WT) control. Further analysis revealed significantly increased accumulation of inflammatory macrophages in hearts of LPS-treated KO mice. Accordingly, ablation of Sectm1a remarkably increased inflammatory cytokines levels both in vitro [from bone marrow-derived macrophages (BMDMs)] and in vivo (in serum and myocardium) after LPS challenge. RNA-sequencing results and bioinformatics analyses showed that the most significantly downregulated genes in KO-BMDMs were modulated by LXRα, a nuclear receptor with robust anti-inflammatory activity in macrophages. Indeed, we identified that the nuclear translocation of LXRα was disrupted in KO-BMDMs when treated with GW3965 (LXR agonist), resulting in higher levels of inflammatory cytokines, compared to GW3965-treated WT-cells. Furthermore, using chronic inflammation model of high-fat diet (HFD) feeding, we observed that infiltration of inflammatory monocytes/macrophages into KO-hearts were greatly increased and accordingly, worsened cardiac function, compared to WT-HFD controls. CONCLUSION: This study defines Sectm1a as a new regulator of inflammatory-induced cardiac dysfunction through modulation of LXRα signaling in macrophages. Our data suggest that augmenting Sectm1a activity may be a potential therapeutic approach to resolve inflammation and associated cardiac dysfunction. TRANSLATIONAL PERSPECTIVE: Better understanding on the interaction between inflammatory responses and cardiac health is prominent for the development of safer and more efficacious therapies for heart failure patients. The present study, using both acute (LPS) and chronic (high-fat diet) inflammation models, reiterated the adverse effects of abnormal macrophages activation on cardiac function. Our Sectm1a knockout mouse model showed exacerbated cardiac and systemic inflammatory responses, resulting in further aggravation of contractile dysfunction on the heart after endotoxin challenge. We also demonstrated Sectm1a as a new regulator of macrophage function through LXRα pathway. These data suggest a novel approach to regulate macrophage-elicited inflammation.

2.
Redox Biol ; : 101453, 2020 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-32057709

RESUMO

Currently, most antioxidants do not show any favorable clinical outcomes in reducing myocardial ischemia-reperfusion (I/R) injury, suggesting an urgent need for exploring a new regulator of redox homeostasis in I/R hearts. Here, using heart-specific transgenic (TG) and knockdown (KD) mouse models, tumor susceptibility gene 101 (Tsg101) is defined as a novel cardiac-protector against I/R-triggered oxidative stress. RNA sequencing and bioinformatics data surprisingly reveal that most upregulated genes in Tsg101-TG hearts are transcribed by Nrf2. Accordingly, pharmacological inhibition of Nrf2 offsets Tsg101-elicited cardio-protection. Mechanistically, Tsg101 interacts with SQSTM1/p62 through its PRR domain, and promotes p62 aggregation, leading to recruitment of Keap1 for degradation by autophagosomes and release of Nrf2 to the nucleus. Furthermore, knockout of p62 abrogates Tsg101-induced cardio-protective effects during I/R. Hence, our findings uncover a previously unrecognized role of Tsg101 in the regulation of p62/Keap1/Nrf2 signaling cascades and provide a new strategy for the treatment of ischemic heart disease.

3.
Cells ; 9(1)2020 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-31947892

RESUMO

Macrophages are critical for regulation of inflammatory response during endotoxemia and septic shock. However, the mediators underlying their regulatory function remain obscure. Growth differentiation factor 3 (GDF3), a member of transforming growth factor beta (TGF-ß) superfamily, has been implicated in inflammatory response. Nonetheless, the role of GDF3 in macrophage-regulated endotoxemia/sepsis is unknown. Here, we show that serum GDF3 levels in septic patients are elevated and strongly correlate with severity of sepsis and 28-day mortality. Interestingly, macrophages treated with recombinant GDF3 protein (rGDF3) exhibit greatly reduced production of pro-inflammatory cytokines, comparing to controls upon endotoxin challenge. Moreover, acute administration of rGDF3 to endotoxin-treated mice suppresses macrophage infiltration to the heart, attenuates systemic and cardiac inflammation with less pro-inflammatory macrophages (M1) and more anti-inflammatory macrophages (M2), as well as prolongs mouse survival. Mechanistically, GDF3 is able to activate Smad2/Smad3 phosphorylation, and consequently inhibits the expression of nod-like receptor protein-3 (NLRP3) in macrophages. Accordingly, blockade of Smad2/Smad3 phosphorylation with SB431542 significantly offsets rGDF3-mediated anti-inflammatory effects. Taken together, this study uncovers that GDF3, as a novel sepsis-associated factor, may have a dual role in the pathophysiology of sepsis. Acute administration of rGDF3 into endotoxic shock mice could increase survival outcome and improve cardiac function through anti-inflammatory response by suppression of M1 macrophage phenotype. However, constitutive high levels of GDF3 in human sepsis patients are associated with lethality, suggesting that GDF3 may promote macrophage polarization toward M2 phenotype which could lead to immunosuppression.

4.
Clin Sci (Lond) ; 133(15): 1759-1777, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31383716

RESUMO

Macrophage polarization toward the M1 phenotype and its subsequent inflammatory response have been implicated in the progression of diabetic complications. Despite adverse consequences of autophagy impairment on macrophage inflammation, the regulation of macrophage autophagy under hyperglycemic conditions is incompletely understood. Here, we report that the autophagy-lysosome system and mitochondrial function are impaired in streptozotocin (STZ)-induced diabetic mice and high glucose (HG)-stimulated RAW 264.7 cells. Mitochondrial dysfunction promotes reactive oxygen species (ROS) production and blocks autophagic flux by impairing lysosome function in macrophages under hyperglycemic conditions. Conversely, inhibition of mitochondrial ROS by Mito-TEMPO prevents HG-induced M1 macrophage polarization, and its effect is offset by blocking autophagic flux. The role of mitochondrial ROS in lysosome dysfunction and M1 macrophage polarization is also demonstrated in mitochondrial complex I defective RAW 264.7 cells induced by silencing NADH:ubiquinone oxidoreductase subunit-S4 (Ndufs4). These findings prove that mitochondrial ROS plays a key role in promoting macrophage polarization to inflammatory phenotype by impairing autophagy-lysosome system, which might provide clue to a novel treatment for diabetic complications.

5.
J Transl Med ; 17(1): 279, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438970

RESUMO

BACKGROUND: Spaceflight or microgravity conditions cause myocardial atrophy and dysfunction, contributing to post-flight orthostatic intolerance. However, the underlying mechanisms remain incompletely understood and preventive approaches are limited. This study investigated whether and how losartan, a blocker of angiotensin-II receptor, preserved cardiomyocyte size and prevented myocardial dysfunction during microgravity. METHOD: Adult male mice were suspended with their tails to simulate microgravity. Echocardiography was performed to assess myocardial function. Heart weight and cardiomyocyte size were measured. NADPH oxidase activation was determined by analyzing membrane translocation of its cytosolic subunits including p47phox, p67phox and Rac1. Heart tissues were also assayed for oxidative stress, p47phox phosphorylation (Ser345), MuRF1 protein levels and angiotensin-II production. RESULTS: Tail-suspension for 28 days increased angiotensin-II production in hearts, decreased cardiomyocyte size and heart weight, and induced myocardial dysfunction. Administration of losartan preserved cardiomyocyte size and heart weight, and prevented myocardial dysfunction in tail-suspended mice. These cardioprotective effects of losartan were associated with inhibition of p47phox phosphorylation (Ser345), NADPH oxidase and oxidative stress in tail-suspended mouse hearts. Additionally, the NADPH oxidase inhibitor, apocynin, also reduced oxidative stress, preserved cardiomyocyte size and heart weight, and improved myocardial function in tail-suspended mice. Furthermore, losartan but not apocynin attenuated tail-suspension-induced up-regulation of MuRF1 protein in mouse hearts. CONCLUSIONS: Administration of losartan preserves cardiomyocyte size and prevents myocardial dysfunction under microgravity by blocking p47phox phosphorylation and NADPH oxidase activation, and by inhibiting MuRF1 expression. Thus, losartan may be a useful drug to prevent microgravity-induced myocardial abnormalities.

6.
Clin Sci (Lond) ; 133(13): 1505-1521, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31266854

RESUMO

Doxorubicin (DOX) is widely used as a first-line chemotherapeutic drug for various malignancies. However, DOX causes severe cardiotoxicity, which limits its clinical uses. Oxidative stress is one of major contributors to DOX-induced cardiotoxicity. While autophagic flux serves as an important defense mechanism against oxidative stress in cardiomyocytes, recent studies have demonstrated that DOX induces the blockage of autophagic flux, which contributes to DOX cardiotoxicity. The present study investigated whether nicotinamide riboside (NR), a precursor of nicotinamide adenine dinucleotide (NAD)+, prevents DOX cardiotoxicity by improving autophagic flux. We report that administration of NR elevated NAD+ levels, and reduced cardiac injury and myocardial dysfunction in DOX-injected mice. These protective effects of NR were recapitulated in cultured cardiomyocytes upon DOX treatment. Mechanistically, NR prevented the blockage of autophagic flux, accumulation of autolysosomes, and oxidative stress in DOX-treated cardiomyocytes, the effects of which were associated with restoration of lysosomal acidification. Furthermore, inhibition of lysosomal acidification or SIRT1 abrogated these protective effects of NR during DOX-induced cardiotoxicity. Collectively, our study shows that NR enhances autolysosome clearance via the NAD+/SIRT1 signaling, thereby preventing DOX-triggered cardiotoxicity.

7.
Antioxid Redox Signal ; 31(17): 1302-1319, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31218880

RESUMO

Aims: Clinical use of cisplatin (Cisp), one of the most widely used, common, and effective chemotherapeutic agents, is limited by its side effects, particularly tubular injury-associated nephrotoxicity. Previous studies suggest that hydrogen sulfide (H2S) alleviates Cisp-induced acute kidney injury (AKI). However, the underlying mechanism remains largely unclear. Results: A single intraperitoneal injection of Cisp is employed to induce AKI, and the mice exhibit severe kidney dysfunction and histological damage at day 4 after Cisp injection. Here, we reported that H2S alleviated Cisp-caused renal toxicity via SIRT3 activation and subsequent improvement of mitochondrial ATP production. Using a biotin-switch assay, we showed that H2S increased S-sulfhydration of SIRT3 and induced deacetylation of its target proteins (OPA1, ATP synthase ß, and superoxide dismutase 2). These effects of H2S were associated with a reduction of mitochondrial fragmentation, an increase in ATP generation, and less oxidative injury. Notably, the S-sulfhydration of SIRT3 induced by H2S was abrogated when Cys256, Cys259, Cys280, and Cys283 residues on SIRT3 (two zinc finger domains) were mutated. Innovation and Conclusion: Our data suggest that H2S attenuates Cisp-induced AKI by preventing mitochondrial dysfunction via SIRT3 sulfhydrylation. Antioxid. Redox Signal. 31, 1302-1319.

8.
J Biol Chem ; 294(27): 10438-10448, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31118273

RESUMO

The initiation and development of diabetes are mainly ascribed to the loss of functional ß-cells. Therapies designed to regenerate ß-cells provide great potential for controlling glucose levels and thereby preventing the devastating complications associated with diabetes. This requires detailed knowledge of the molecular events and underlying mechanisms in this disorder. Here, we report that expression of microRNA-223 (miR-223) is up-regulated in islets from diabetic mice and humans, as well as in murine Min6 ß-cells exposed to tumor necrosis factor α (TNFα) or high glucose. Interestingly, miR-223 knockout (KO) mice exhibit impaired glucose tolerance and insulin resistance. Further analysis reveals that miR-223 deficiency dramatically suppresses ß-cell proliferation and insulin secretion. Mechanistically, using luciferase reporter gene assays, histological analysis, and immunoblotting, we demonstrate that miR-223 inhibits both forkhead box O1 (FOXO1) and SRY-box 6 (SOX6) signaling, a unique bipartite mechanism that modulates expression of several ß-cell markers (pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1), and urocortin 3 (UCN3)) and cell cycle-related genes (cyclin D1, cyclin E1, and cyclin-dependent kinase inhibitor P27 (P27)). Importantly, miR-223 overexpression in ß-cells could promote ß-cell proliferation and improve ß-cell function. Taken together, our results suggest that miR-223 is a critical factor for maintaining functional ß-cell mass and adaptation during metabolic stress.

9.
Basic Res Cardiol ; 114(3): 17, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30874894

RESUMO

We and others have reported that calpain-1 was increased in myocardial mitochondria from various animal models of heart disease. This study investigated whether constitutive up-regulation of calpain-1 restricted to mitochondria induced myocardial injury and heart failure and, if so, whether these phenotypes could be rescued by selective inhibition of mitochondrial superoxide production. Transgenic mice with human CAPN1 up-regulation restricted to mitochondria in cardiomyocytes (Tg-mtCapn1/tTA) were generated and characterized with low and high over-expression of transgenic human CAPN1 restricted to mitochondria, respectively. Transgenic up-regulation of mitochondria-targeted CAPN1 dose-dependently induced cardiac cell death, adverse myocardial remodeling, heart failure, and early death in mice, the changes of which were associated with mitochondrial dysfunction and mitochondrial superoxide generation. Importantly, a daily injection of mitochondria-targeted superoxide dismutase mimetics mito-TEMPO for 1 month starting from age 2 months attenuated cardiac cell death, adverse myocardial remodeling and heart failure, and reduced mortality in Tg-mtCapn1/tTA mice. In contrast, administration of TEMPO did not achieve similar cardiac protection in transgenic mice. Furthermore, transgenic up-regulation of mitochondria-targeted CAPN1 induced a reduction of ATP5A1 protein and ATP synthase activity in hearts. In cultured cardiomyocytes, increased calpain-1 in mitochondria promoted mitochondrial permeability transition pore (mPTP) opening and induced cell death, which were prevented by over-expression of ATP5A1, mito-TEMPO or cyclosporin A, an inhibitor of mPTP opening. In conclusion, this study has provided direct evidence demonstrating that increased mitochondrial calpain-1 is an important mechanism contributing to myocardial injury and heart failure by disrupting ATP synthase, and promoting mitochondrial superoxide generation and mPTP opening.


Assuntos
Calpaína/metabolismo , Cardiomiopatia Dilatada/etiologia , Insuficiência Cardíaca/etiologia , Mitocôndrias/metabolismo , Animais , Cardiomiopatia Dilatada/metabolismo , Morte Celular , Óxidos N-Cíclicos , Modelos Animais de Doenças , Insuficiência Cardíaca/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Miócitos Cardíacos/metabolismo , Superóxidos/metabolismo
10.
FASEB J ; 33(6): 7451-7466, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30884248

RESUMO

Development of physiologic cardiac hypertrophy has primarily been ascribed to the IGF-1 and its receptor, IGF-1 receptor (IGF-1R), and subsequent activation of the protein kinase B (Akt) pathway. However, regulation of endosome-mediated recycling and degradation of IGF-1R during physiologic hypertrophy has not been investigated. In a physiologic hypertrophy model of treadmill-exercised mice, we observed that levels of tumor susceptibility gene 101 (Tsg101), a key member of the endosomal sorting complex required for transport, were dramatically elevated in the heart compared with sedentary controls. To determine the role of Tsg101 on physiologic hypertrophy, we generated a transgenic (TG) mouse model with cardiac-specific overexpression of Tsg101. These TG mice exhibited a physiologic-like cardiac hypertrophy phenotype at 8 wk evidenced by: 1) the absence of cardiac fibrosis, 2) significant improvement of cardiac function, and 3) increased total and plasma membrane levels of IGF-1R and increased phosphorylation of Akt. Mechanistically, we identified that Tsg101 interacted with family-interacting protein 3 (FIP3) and IGF-1R, thereby stabilizing FIP3 and enhancing recycling of IGF-1R. In vitro, adenovirus-mediated overexpression of Tsg101 in neonatal rat cardiomyocytes resulted in cell hypertrophy, which was blocked by addition of monensin, an inhibitor of endosome-mediated recycling, and by small interfering RNA-mediated knockdown (KD) of FIP3. Furthermore, cardiac-specific KD of Tsg101 showed a significant reduction in levels of endosomal recycling compartment members (Rab11a and FIP3), IGF-1R, and Akt phosphorylation. Most interestingly, Tsg101-KD mice failed to develop cardiac hypertrophy after intense treadmill training. Taken together, our data identify Tsg101 as a novel positive regulator of physiologic cardiac hypertrophy through facilitating the FIP3-mediated endosomal recycling of IGF-1R.-Essandoh, K., Deng, S., Wang, X., Jiang, M., Mu, X., Peng, J., Li, Y., Peng, T., Wagner, K.-U., Rubinstein, J., Fan, G.-C. Tsg101 positively regulates physiologic-like cardiac hypertrophy through FIP3-mediated endosomal recycling of IGF-1R.

11.
Diabetologia ; 62(5): 860-872, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30778623

RESUMO

AIMS/HYPOTHESIS: The role of non-cardiomyocytes in diabetic cardiomyopathy has not been fully addressed. This study investigated whether endothelial cell calpain plays a role in myocardial endothelial injury and microvascular rarefaction in diabetes, thereby contributing to diabetic cardiomyopathy. METHODS: Endothelial cell-specific Capns1-knockout (KO) mice were generated. Conditions mimicking prediabetes and type 1 and type 2 diabetes were induced in these KO mice and their wild-type littermates. Myocardial function and coronary flow reserve were assessed by echocardiography. Histological analyses were performed to determine capillary density, cardiomyocyte size and fibrosis in the heart. Isolated aortas were assayed for neovascularisation. Cultured cardiac microvascular endothelial cells were stimulated with high palmitate. Angiogenesis and apoptosis were analysed. RESULTS: Endothelial cell-specific deletion of Capns1 disrupted calpain 1 and calpain 2 in endothelial cells, reduced cardiac fibrosis and hypertrophy, and alleviated myocardial dysfunction in mouse models of diabetes without significantly affecting systemic metabolic variables. These protective effects of calpain disruption in endothelial cells were associated with an increase in myocardial capillary density (wild-type vs Capns1-KO 3646.14 ± 423.51 vs 4708.7 ± 417.93 capillary number/high-power field in prediabetes, 2999.36 ± 854.77 vs 4579.22 ± 672.56 capillary number/high-power field in type 2 diabetes and 2364.87 ± 249.57 vs 3014.63 ± 215.46 capillary number/high-power field in type 1 diabetes) and coronary flow reserve. Ex vivo analysis of neovascularisation revealed more endothelial cell sprouts from aortic rings of prediabetic and diabetic Capns1-KO mice compared with their wild-type littermates. In cultured cardiac microvascular endothelial cells, inhibition of calpain improved angiogenesis and prevented apoptosis under metabolic stress. Mechanistically, deletion of Capns1 elevated the protein levels of ß-catenin in endothelial cells of Capns1-KO mice and constitutive activity of calpain 2 suppressed ß-catenin protein expression in cultured endothelial cells. Upregulation of ß-catenin promoted angiogenesis and inhibited apoptosis whereas knockdown of ß-catenin offset the protective effects of calpain inhibition in endothelial cells under metabolic stress. CONCLUSIONS/INTERPRETATION: These results delineate a primary role of calpain in inducing cardiac endothelial cell injury and impairing neovascularisation via suppression of ß-catenin, thereby promoting diabetic cardiomyopathy, and indicate that calpain is a promising therapeutic target to prevent diabetic cardiac complications.


Assuntos
Calpaína/genética , Calpaína/fisiologia , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/terapia , Células Endoteliais/enzimologia , Neovascularização Patológica , Neovascularização Fisiológica , Animais , Apoptose , Diabetes Mellitus Tipo 2/metabolismo , Fibroblastos/metabolismo , Deleção de Genes , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Transdução de Sinais , beta Catenina/metabolismo
12.
Arch Toxicol ; 93(4): 1051-1065, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30810770

RESUMO

We recently reported that doxorubicin decreased the expression of calpain-1/2, while inhibition of calpain activity promoted doxorubicin-induced cardiac injury in mice. In this study, we investigated whether and how elevation of calpain-2 could affect doxorubicin-triggered cardiac injury. Transgenic mice with inducible cardiomyocyte-specific expression of calpain-2 were generated. An acute cardiotoxicity was induced in both transgenic mice and their relevant wild-type littermates by injection of a single dose of doxorubicin (20 mg/kg) and cardiac injury was analyzed 5 days after doxorubicin injection. Cardiomyocyte-specific up-regulation of calpain-2 did not induce any adverse cardiac phenotypes under physiological conditions by age 3 months, but significantly reduced myocardial injury and improved myocardial function in doxorubicin-treated mice. Cardiac protection of calpain-2 up-regulation was also observed in a mouse model of chronic doxorubicin cardiotoxicity. Up-regulation of calpain-2 increased the protein levels of mitogen activated protein kinase phosphatase-1 (MKP-1) in cultured mouse cardiomyocytes and heart tissues. Over-expression of MKP-1 prevented, whereas knockdown of MKP-1 augmented doxorubicin-induced apoptosis in cultured cardiomyocytes. Moreover, knockdown of MKP-1 offset calpain-2-elicited protective effects against doxorubicin-induced injury in cultured cardiomyocytes. Mechanistically, up-regulation of calpain-2 reduced the protein levels of phosphatase and tensin homolog and consequently promoted Akt activation, leading to increased MKP-1 protein steady-state levels by inhibiting its degradation. Collectively, this study reveals a new role of calpain-2 in promoting MKP-1 expression via phosphatase and tensin homolog/Akt signaling. This study also suggests that calpain-2/MKP-1 signaling may represent new therapeutic targets for doxorubicin-induced cardiac injury.

13.
J Cell Physiol ; 234(7): 10761-10770, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30417356

RESUMO

Cardiovascular dysfunction is a common complication among heatstroke patients, but its underlying mechanism is unclear. This study was designed to investigate the role of calpain-2 and its downstream signal pathway in heat stress-induced cardiomyocyte apoptosis and heart dysfunction. In cultured primary mouse neonatal cardiomyocytes (MNCs), heat stress (43°C for 2 hr) induced a heat-shock response, as indicated by upregulated heat-shock protein 27 (HSP27) expression and cellular apoptosis, as indicated by increased caspase-3 activity, DNA fragmentation and decreased cell viability. Meanwhile, heat stress decreased calpain activity, which was accompanied by downregulated calpain-2 expression and increased phosphorylation of p38, extraceIIuIar signaI-reguIated protein kinase (ERK1/2) and c-Jun N-terminaI kinase (JNK). Calpain-2 overexpression abrogated heat stress-induced apoptosis and phosphorylation of p38 and JNK, but not of ERK1/2. Blocking only p38 prevented heat stress-induced apoptosis in MNCs. In cardiac-specific calpain-2 overexpressing transgenic mice, p38 phosphorylation and cardiomyocyte apoptosis were decreased in the heart tissue of heatstroke mice, as revealed by western blot and terminal deoxynucleotidyl transferase dUTP nick end labelling assays, respectively. M-mode echocardiography also demonstrated that calpain-2 overexpression significantly improved heatstroke-induced decreases in ventricular end-diastolic volume and cardiac output. In conclusion, our study suggests that heat stress reduces calpain-2 expression, which then activates p38, leading to cardiomyocyte apoptosis and heart dysfunction.

14.
J Thorac Dis ; 10(9): 5283-5297, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30416776

RESUMO

Background: Ischemic heart injury activates calpains and endoplasmic reticulum (ER) stress in cardiomyocytes. This study investigated whether over-expression of calpastatin, an endogenous calpain inhibitor, protects the heart against myocardial infarction (MI) by inhibiting ER stress. Methods: Mice over-expressing calpastatin (Tg-CAST) and littermate wild type (WT) mice were divided into four groups: WT-sham, Tg-CAST-sham, WT-MI, and Tg-CAST-MI, respectively. WT-sham and Tg-CAST-sham mice showed similar cardiac function at baseline. MI for 7 days impaired cardiac function in WT-MI mice, which was ameliorated in Tg-CAST-MI mice. Results: Tg-CAST-MI mice exhibited significantly decreased diameter of the left ventricular cavity, scar area, and cardiac cell death compared to WT-MI mice. WT-MI mice had higher cardiac expression of C/EBP homologous protein (CHOP) and BIP, indicators of ER stress, compared to WT-sham mice, indicative of MI-induced ER stress. This increase was abolished in Tg-CAST-MI hearts. Furthermore, administration of tauroursodeoxycholic acid, an inhibitor of ER stress, reduced MI-induced expression of CHOP and BIP, scar area, and myocardial dysfunction. In an in vitro model of oxidative stress, H2O2 stimulation of H9c2 cardiomyoblasts induced calpain activation, CHOP expression, and cell death, all of which were prevented by the calpain inhibitor PD150606, as well as CHOP silencing. Conclusions: Over-expression of calpastatin ameliorates MI-induced myocardial injury in mice. These protective effects of calpastatin are partially achieved through suppression of the ER stress/CHOP pathway.

15.
Cell Rep ; 23(12): 3607-3620, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29925002

RESUMO

Exposure to cold temperature is well known to upregulate heat shock protein (Hsp) expression and recruit and/or activate brown adipose tissue and beige adipocytes in humans and animals. However, whether and how Hsps regulate adipocyte function for energy homeostatic responses is poorly understood. Here, we demonstrate a critical role of Hsp20 as a negative regulator of adipocyte function. Deletion of Hsp20 enhances non-shivering thermogenesis and suppresses inflammatory responses, leading to improvement of glucose and lipid metabolism under both chow diet and high-fat diet conditions. Mechanistically, Hsp20 controls adipocyte function by interacting with the subunit of the ubiquitin ligase complex, F-box only protein 4 (FBXO4), and regulating the ubiquitin-dependent degradation of peroxisome proliferation activated receptor gamma (PPARγ). Indeed, Hsp20 deficiency mimics and enhances the pharmacological effects of the PPARγ agonist rosiglitazone. Together, our findings suggest a role of Hsp20 in mediating adipocyte function by linking ß-adrenergic signaling to PPARγ activity.


Assuntos
Adipócitos/metabolismo , Proteínas F-Box/metabolismo , Proteínas de Choque Térmico HSP20/metabolismo , PPAR gama/metabolismo , Ubiquitinação , Adipócitos/efeitos dos fármacos , Tecido Adiposo Branco/metabolismo , Adiposidade/efeitos dos fármacos , Animais , Temperatura Baixa , Metabolismo Energético/efeitos dos fármacos , Glucose/metabolismo , Proteínas de Choque Térmico HSP20/deficiência , Proteínas de Choque Térmico HSP20/genética , Inflamação/patologia , Resistência à Insulina , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/patologia , Estabilidade Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rosiglitazona/farmacologia , Ubiquitinação/efeitos dos fármacos
16.
Free Radic Biol Med ; 123: 125-137, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29803807

RESUMO

AIMS: Sepsis-caused multiple organ failure remains the major cause of morbidity and mortality in intensive care units. Nicotinamide riboside (NR) is a precursor of nicotinamide adenine dinucleotide (NAD+), which is important in regulating oxidative stress. This study investigated whether administration of NR prevented oxidative stress and organ injury in sepsis. METHODS: Mouse sepsis models were induced by injection of lipopolysaccharides (LPS) or feces-injection-in-peritoneum. NR was given before sepsis onset. Cultured macrophages and endothelial cells were incubated with various agents. RESULTS: Administration of NR elevated the NAD+ levels, and elicited a reduction of oxidative stress, inflammation and caspase-3 activity in lung and heart tissues, which correlated with attenuation of pulmonary microvascular permeability and myocardial dysfunction, leading to less mortality in sepsis models. These protective effects of NR were associated with decreased levels of plasma high mobility group box-1 (HMGB1) in septic mice. Consistently, pre-treatment of macrophages with NR increased NAD+ content and reduced HMGB1 release upon LPS stimulation. NR also prevented reactive oxygen species (ROS) production and apoptosis in endothelial cells induced by a conditioned-medium collected from LPS-treated macrophages. Furthermore, inhibition of SIRT1 by EX527 offset the negative effects of NR on HMGB1 release in macrophages, and ROS and apoptosis in endothelial cells. CONCLUSIONS: Administration of NR prevents lung and heart injury, and improves the survival in sepsis, likely by inhibiting HMGB1 release and oxidative stress via the NAD+/SIRT1 signaling. Given NR has been used as a health supplement, it may be a useful agent to prevent organ injury in sepsis.


Assuntos
Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Insuficiência de Múltiplos Órgãos/prevenção & controle , Niacinamida/análogos & derivados , Estresse Oxidativo/efeitos dos fármacos , Sepse/complicações , Animais , Proteína HMGB1/metabolismo , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Insuficiência de Múltiplos Órgãos/etiologia , Insuficiência de Múltiplos Órgãos/metabolismo , Niacinamida/administração & dosagem , Niacinamida/farmacologia , Sepse/induzido quimicamente
17.
Shock ; 49(4): 429-441, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28650928

RESUMO

Septic shock increases vascular permeability, leading to multiple organ failure including cardiac dysfunction, a major contributor to septic death. Podosome, an actin-based dynamic membrane structure, plays critical roles in extracellular matrix degradation and angiogenesis. However, whether podosome contributes to endothelial barrier dysfunction during septic shock remains unknown. In this study, we found that the endothelial hyperpermeability, stimulated by phorbol 12-myristate 13-acetate and thrombin, was accompanied by increased formation of podosome clusters at the cell periphery, indicating a positive correlation between podosome clusters and endothelial leakage. Interestingly, we observed that circulating exosomes collected from septic mice were able to stimulate podosome cluster formation in cardiac endothelial cells, together with increased permeability in vitro/in vivo and cardiac dysfunction. Mechanistically, we identified that septic exosomes contained higher levels of reactive oxygen species (ROS) than normal ones, which were effectively transported to endothelial cells (ECs). Depletion of ROS in septic exosomes significantly reduced their capacity for promoting podosome cluster formation and thereby dampened vascular leakage. Finally, we elucidated that podosome cluster-induced endothelial hyperpermeability was associated with fragmentation/depletion of zonula occludens-1 (ZO-1) at the cell periphery. Our results demonstrate that septic exosomes were enriched with high amounts of ROS, which can be transported to ECs, leading to the generation of podosome clusters in target ECs and thereby, causing ZO-1 relocation, vascular leakage, and cardiac dysfunction.


Assuntos
Exossomos/metabolismo , Podossomos/metabolismo , Sepse/metabolismo , Animais , Western Blotting , Permeabilidade Capilar/fisiologia , Células Endoteliais/metabolismo , L-Lactato Desidrogenase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Espécies Reativas de Oxigênio/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
18.
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
19.
Biochim Biophys Acta ; 1862(11): 2023-2033, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27523632

RESUMO

Diabetes and obesity are prevalent in westernized countries. In both conditions, excessive fatty acid uptake by cardiomyocytes induces cardiac lipotoxicity, an important mechanism contributing to diabetic cardiomyopathy. This study investigated the effect of calpain disruption on cardiac lipotoxicity. Cardiac-specific capns1 knockout mice and their wild-type littermates (male, age of 4weeks) were fed a high fat diet (HFD) or normal diet for 20weeks. HFD increased body weight, altered blood lipid profiles and impaired glucose tolerance comparably in both capns1 knockout mice and their wild-type littermates. Calpain activity, cardiomyocyte cross-sectional areas, collagen deposition and triglyceride were significantly increased in HFD-fed mouse hearts, and these were accompanied by myocardial dysfunction and up-regulation of hypertrophic and fibrotic collagen genes as well as pro-inflammatory cytokines. These effects of HFD were attenuated by disruption of calpain in capns1 knockout mice. Mechanistically, deletion of capns1 in HFD-fed mouse hearts and disruption of calpain with calpain inhibitor-III, silencing of capn1, or deletion of capns1 in palmitate-stimulated cardiomyocytes prevented endoplasmic reticulum stress, apoptosis, cleavage of caspase-12 and junctophilin-2, and pro-inflammatory cytokine expression. Pharmacological inhibition of endoplasmic reticulum stress diminished palmitate-induced apoptosis and pro-inflammatory cytokine expression in cardiomyocytes. In summary, disruption of calpain prevents lipotoxicity-induced apoptosis in cardiomyocytes and cardiac injury in mice fed a HFD. The role of calpain is mediated, at least partially, through endoplasmic reticulum stress. Thus, calpain/endoplasmic reticulum stress may represent a new mechanism and potential therapeutic targets for cardiac lipotoxicity.

20.
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
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