RESUMO
S-palmitoylation is a reversible lipid modification catalyzed by 23 S-acyltransferases with a conserved zinc finger aspartate-histidine-histidine-cysteine (zDHHC) domain that facilitates targeting of proteins to specific intracellular membranes. Here we performed a gain-of-function screen in the mouse and identified the Golgi-localized enzymes zDHHC3 and zDHHC7 as regulators of cardiac hypertrophy. Cardiomyocyte-specific transgenic mice overexpressing zDHHC3 show cardiac disease, and S-acyl proteomics identified the small GTPase Rac1 as a novel substrate of zDHHC3. Notably, cardiomyopathy and congestive heart failure in zDHHC3 transgenic mice is preceded by enhanced Rac1 S-palmitoylation, membrane localization, activity, downstream hypertrophic signaling, and concomitant induction of all Rho family small GTPases whereas mice overexpressing an enzymatically dead zDHHC3 mutant show no discernible effect. However, loss of Rac1 or other identified zDHHC3 targets Gαq/11 or galectin-1 does not diminish zDHHC3-induced cardiomyopathy, suggesting multiple effectors and pathways promoting decompensation with sustained zDHHC3 activity. Genetic deletion of Zdhhc3 in combination with Zdhhc7 reduces cardiac hypertrophy during the early response to pressure overload stimulation but not over longer time periods. Indeed, cardiac hypertrophy in response to 2 weeks of angiotensin-II infusion is not diminished by Zdhhc3/7 deletion, again suggesting other S-acyltransferases or signaling mechanisms compensate to promote hypertrophic signaling. Taken together, these data indicate that the activity of zDHHC3 and zDHHC7 at the cardiomyocyte Golgi promote Rac1 signaling and maladaptive cardiac remodeling, but redundant signaling effectors compensate to maintain cardiac hypertrophy with sustained pathological stimulation in the absence of zDHHC3/7.
Assuntos
Cardiomiopatias , Miócitos Cardíacos , Animais , Camundongos , Aciltransferases/genética , Aciltransferases/metabolismo , Cardiomegalia/metabolismo , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Histidina/metabolismo , Lipoilação , Camundongos Transgênicos , Miócitos Cardíacos/metabolismoRESUMO
Introduction: The ribosomal protein L3-like (RPL3L) is a heart and skeletal muscle-specific ribosomal protein and paralogue of the more ubiquitously expressed RPL3 protein. Mutations in the human RPL3L gene are linked to childhood cardiomyopathy and age-related atrial fibrillation, yet the function of RPL3L in the mammalian heart remains unknown. Methods and Results: Here, we observed that mouse cardiac ventricles express RPL3 at birth, where it is gradually replaced by RPL3L in adulthood but re-expressed with induction of hypertrophy in adults. Rpl3l gene-deleted mice were generated to examine the role of this gene in the heart, although Rpl3l -/- mice showed no overt changes in cardiac structure or function at baseline or after pressure overload hypertrophy, likely because RPL3 expression was upregulated and maintained in adulthood. mRNA expression analysis and ribosome profiling failed to show differences between the hearts of Rpl3l null and wild type mice in adulthood. Moreover, ribosomes lacking RPL3L showed no differences in localization within cardiomyocytes compared to wild type controls, nor was there an alteration in cardiac tissue ultrastructure or mitochondrial function in adult Rpl3l -/- mice. Similarly, overexpression of either RPL3 or RPL3L with adeno-associated virus -9 in the hearts of mice did not cause discernable pathology. However, by 18 months of age Rpl3l -/- null mice had significantly smaller hearts compared to wild type littermates. Conclusion: Thus, deletion of Rpl3l forces maintenance of RPL3 expression within the heart that appears to fully compensate for the loss of RPL3L, although older Rpl3l -/- mice showed a mild but significant reduction in heart weight.
RESUMO
Recent high-throughput approaches have revealed a vast number of transcripts with unknown functions. Many of these transcripts are long noncoding RNAs (lncRNAs), and intergenic region-derived lncRNAs are classified as long intergenic noncoding RNAs (lincRNAs). Although Myosin heavy chain 6 (Myh6) encoding primary contractile protein is down-regulated in stressed hearts, the underlying mechanisms are not fully clarified especially in terms of lincRNAs. Here, we screen upregulated lincRNAs in pressure overloaded hearts and identify a muscle-abundant lincRNA termed Lionheart. Compared with controls, deletion of the Lionheart in mice leads to decreased systolic function and a reduction in MYH6 protein levels following pressure overload. We reveal decreased MYH6 results from an interaction between Lionheart and Purine-rich element-binding protein A after pressure overload. Furthermore, human LIONHEART levels in left ventricular biopsy specimens positively correlate with cardiac systolic function. Our results demonstrate Lionheart plays a pivotal role in cardiac remodeling via regulation of MYH6.
Assuntos
Coração/fisiopatologia , Pressão , RNA Longo não Codificante/genética , Sístole/genética , Animais , Biópsia , Dependovirus/metabolismo , Ventrículos do Coração/ultraestrutura , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Regiões Promotoras Genéticas/genética , RNA Longo não Codificante/metabolismo , Ratos , Regulação para Cima/genéticaRESUMO
No effective treatment is yet available to reduce infarct size and improve clinical outcomes after acute myocardial infarction by enhancing early reperfusion therapy using primary percutaneous coronary intervention. The study showed that Kyoto University Substance 121 (KUS121) reduced endoplasmic reticulum stress, maintained adenosine triphosphate levels, and ameliorated the infarct size in a murine cardiac ischemia and reperfusion injury model. The study confirmed the cardioprotective effect of KUS121 in a porcine ischemia and reperfusion injury model. These findings confirmed that KUS121 is a promising novel therapeutic agent for myocardial infarction in conjunction with primary percutaneous coronary intervention.
RESUMO
Background Micro RNA (miR)-33 targets cholesterol transporter ATP -binding cassette protein A1 and other antiatherogenic targets and contributes to atherogenic progression. Its inhibition or deletion is known to result in the amelioration of atherosclerosis in mice. However, mice lack the other member of the miR-33 family, miR-33b, which exists in humans and other large mammals. Thus, precise evaluation and comparison of the responsibilities of these 2 miRs during the progression of atherosclerosis has not been reported, although they are essential. Methods and Results In this study, we performed a comprehensive analysis of the difference between the function of miR-33a and miR-33b using genetically modified mice. We generated 4 strains with or without miR-33a and miR-33b. Comparison between mice with only miR-33a (wild-type mice) and mice with only miR-33b (miR-33a-/-/miR-33b+/+) revealed the dominant expression of miR-33b in the liver. To evaluate the whole body atherogenic potency of miR-33a and miR-33b, we developed apolipoprotein E-deficient/miR-33a+/+/miR-33b-/- mice and apolipoprotein E-deficient/miR-33a-/-/miR-33b+/+ mice. With a high-fat and high-cholesterol diet, the apolipoprotein E-deficient/miR-33a-/-/miR-33b+/+ mice developed increased atherosclerotic plaque versus apolipoprotein E-deficient/miR-33a+/+/miR-33b-/- mice, in line with the predominant expression of miR-33b in the liver and worsened serum cholesterol profile. By contrast, a bone marrow transplantation study showed no significant difference, which was consistent with the relevant expression levels of miR-33a and miR-33b in bone marrow cells. Conclusions The miR-33 family exhibits differences in distribution and regulation and particularly in the progression of atherosclerosis; miR-33b would be more potent than miR-33a.
Assuntos
Aterosclerose/genética , Hepatócitos/metabolismo , Fígado/metabolismo , MicroRNAs/genética , Placa Aterosclerótica/genética , Animais , Apolipoproteínas B/metabolismo , Transplante de Medula Óssea , Colesterol/metabolismo , Colesterol na Dieta , Dieta Hiperlipídica , Progressão da Doença , Perfilação da Expressão Gênica , Técnicas de Introdução de Genes , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Knockout , Camundongos Knockout para ApoE , Camundongos Transgênicos , MicroRNAs/metabolismo , Triglicerídeos/metabolismoRESUMO
Acute cardiac rupture and adverse left ventricular (LV) remodeling causing heart failure are serious complications of acute myocardial infarction (MI). While cardio-hepatic interactions have been recognized, their role in MI remains unknown. We treated cultured cardiomyocytes with conditioned media from various cell types and analyzed the media by mass spectrometry to identify α1-microglobulin (AM) as an Akt-activating hepatokine. In mouse MI model, AM protein transiently distributed in the infarct and border zones during the acute phase, reflecting infiltration of AM-bound macrophages. AM stimulation activated Akt, NFκB, and ERK signaling and enhanced inflammation as well as macrophage migration and polarization, while inhibited fibrogenesis-related mRNA expression in cultured macrophages and cardiac fibroblasts. Intramyocardial AM administration exacerbated macrophage infiltration, inflammation, and matrix metalloproteinase 9 mRNA expression in the infarct and border zones, whereas disturbed fibrotic repair, then provoked acute cardiac rupture in MI. Shotgun proteomics and lipid pull-down analysis found that AM partly binds to phosphatidic acid (PA) for its signaling and function. Furthermore, systemic delivery of a selective inhibitor of diacylglycerol kinase α-mediated PA synthesis notably reduced macrophage infiltration, inflammation, matrix metalloproteinase activity, and adverse LV remodeling in MI. Therefore, targeting AM signaling could be a novel pharmacological option to mitigate adverse LV remodeling in MI.
Assuntos
alfa-Globulinas/metabolismo , Hormônios/metabolismo , Infarto do Miocárdio/patologia , Transdução de Sinais , Animais , Membrana Celular/metabolismo , Movimento Celular , Ativação Enzimática , Fibrose , Inflamação/metabolismo , Fígado/metabolismo , Macrófagos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ácidos Fosfatídicos/biossíntese , Proteínas Proto-Oncogênicas c-akt/metabolismo , Remodelação VentricularRESUMO
Objective- Atherosclerosis is a common disease caused by a variety of metabolic and inflammatory disturbances. MicroRNA (miR)-33a within SREBF2 (sterol regulatory element-binding factor 2) is a potent target for treatment of atherosclerosis through regulating both aspects; however, the involvement of miR-33b within SREBF1 remains largely unknown. Although their host genes difference could lead to functional divergence of miR-33a/b, we cannot dissect the roles of miR-33a/b in vivo because of lack of miR-33b sequences in mice, unlike human. Approach and Results- Here, we analyzed the development of atherosclerosis using miR-33b knock-in humanized mice under apolipoprotein E-deficient background. MiR-33b is prominent both in human and mice on atheroprone condition. MiR-33b reduced serum high-density lipoprotein cholesterol levels and systemic reverse cholesterol transport. MiR-33b knock-in macrophages showed less cholesterol efflux capacity and higher inflammatory state via regulating lipid rafts. Thus, miR-33b promotes vulnerable atherosclerotic plaque formation. Furthermore, bone marrow transplantation experiments strengthen proatherogenic roles of macrophage miR-33b. Conclusions- Our data demonstrated critical roles of SREBF1-miR-33b axis on both lipid profiles and macrophage phenotype remodeling and indicate that miR-33b is a promising target for treating atherosclerosis.
Assuntos
Aterosclerose/metabolismo , MicroRNAs/metabolismo , Placa Aterosclerótica , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose , Aterosclerose/genética , Aterosclerose/patologia , Transplante de Medula Óssea , Estudos de Casos e Controles , HDL-Colesterol/sangue , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Humanos , Absorção Intestinal , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Microdomínios da Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , MicroRNAs/genética , Pessoa de Meia-Idade , Fenótipo , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Triglicerídeos/sangueRESUMO
Recent evidence suggests that the accumulation of macrophages as a result of obesity-induced adipose tissue hypoxia is crucial for the regulation of tissue fibrosis, but the molecular mechanisms underlying adipose tissue fibrosis are still unknown. In this study, we revealed that periostin (Postn) is produced at extraordinary levels by adipose tissue after feeding with a high-fat diet (HFD). Postn was secreted at least from macrophages in visceral adipose tissue during the development of obesity, possibly due to hypoxia. Postn-/- mice had lower levels of crown-like structure formation and fibrosis in adipose tissue and were protected from liver steatosis. These mice also showed amelioration in systemic insulin resistance compared with HFD-fed WT littermates. Mice deficient in Postn in their hematopoietic compartment also had lower levels of inflammation in adipose tissue, in parallel with a reduction in ectopic lipid accumulation compared with the controls. Our data indicated that the regulation of Postn in visceral fat could be beneficial for the maintenance of healthy adipose tissue in obesity.
Assuntos
Moléculas de Adesão Celular/deficiência , Celulite (Flegmão)/metabolismo , Gorduras na Dieta/efeitos adversos , Resistência à Insulina , Gordura Intra-Abdominal/metabolismo , Obesidade/metabolismo , Animais , Celulite (Flegmão)/induzido quimicamente , Celulite (Flegmão)/genética , Celulite (Flegmão)/patologia , Gorduras na Dieta/farmacologia , Fibrose , Gordura Intra-Abdominal/patologia , Camundongos , Camundongos Knockout , Obesidade/induzido quimicamente , Obesidade/genética , Obesidade/patologiaRESUMO
MicroRNA 33 (miR-33) targets ATP-binding cassette transporter A1 (ABCA1), and its deficiency increases serum high-density lipoprotein (HDL)-cholesterol (HDL-C) and ameliorates atherosclerosis. Although we previously reported that miR-33 deficiency increased peripheral Ly6Chigh monocytes on an ApoE-deficient background, the effect of miR-33 on the monocyte population has not been fully elucidated, especially in a wild-type (WT) background. We found that Ly6Chigh monocytes in miR-33-/- mice were decreased in peripheral blood and increased in bone marrow (BM). Expansion of myeloid progenitors and decreased apoptosis in Lin- Sca1+ c-Kit+ (LSK) cells were observed in miR-33-/- mice. A BM transplantation study and competitive repopulation assay revealed that hematopoietic miR-33 deficiency caused myeloid expansion and increased peripheral Ly6Chigh monocytes and that nonhematopoietic miR-33 deficiency caused reduced peripheral Ly6Chigh monocytes. Expression of high-mobility group AT-hook 2 (HMGA2) targeted by miR-33 increased in miR-33-deficient LSK cells, and its knockdown abolished the reduction of apoptosis. Transduction of human apolipoprotein A1 and ABCA1 in WT mouse liver increased HDL-C and reduced peripheral Ly6Chigh monocytes. These data indicate that miR-33 deficiency affects distribution of inflammatory monocytes through dual pathways. One pathway involves the enhancement of Hmga2 expression in hematopoietic stem cells to increase Ly6Chigh monocytes, and the other involves the elevation of HDL-C to decrease peripheral Ly6Chigh monocytes.
Assuntos
Antígenos Ly/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Monócitos/metabolismo , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Apolipoproteínas E/metabolismo , Apoptose , Aterosclerose/genética , Aterosclerose/metabolismo , HDL-Colesterol/sangue , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Knockout para ApoE , Monócitos/classificação , Monócitos/citologia , Células Progenitoras Mieloides/citologia , Células Progenitoras Mieloides/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução GenéticaRESUMO
OBJECTIVES: The aim of this study was to evaluate the prognostic impact of left ventricular ejection fraction (LVEF) in patients with severe aortic stenosis (AS). BACKGROUND: The prognostic impact of LVEF in severe AS remains controversial. METHODS: Among 3,815 consecutive patients with severe AS enrolled in the CURRENT AS (Contemporary Outcomes After Surgery and Medical Treatment in Patients With Severe Aortic Stenosis) registry, the present study population consisted of 3,794 patients after excluding 21 patients without LVEF data. Patients were divided into 4 groups according to LVEF at index echocardiography (<50%, 50% to 59%, 60% to 69%, and ≥70%; conservative strategy: n = 388, n = 390, n = 1,025, and n = 800; initial aortic valve replacement strategy: n = 206, n = 170, n = 375, and n = 440). Echocardiographic data were site reported, and there was no echocardiography core laboratory. RESULTS: In the conservative group, the cumulative 5-year incidence of the primary outcome measure (a composite of aortic valve-related death or heart failure hospitalization) was significantly higher in patients with LVEFs <50% and 50% to 59% than in those with LVEFs 60% to 69% and ≥70% (72.3%, 58.4%, 38.7%, and 35.0%, respectively, p < 0.001), whereas in the initial aortic valve replacement group, the negative effect of low LVEF was markedly attenuated (20.2%, 20.3%, 17.7%, and 12.4%, respectively, p = 0.03). After adjusting for confounders, LVEF <50% (hazard ratio: 1.82; 95% confidence interval: 1.44 to 2.28; p < 0.001) and 50% to 59% (hazard ratio: 1.77; 95% confidence interval: 1.42 to 2.20; p < 0.001) but not 60% to 69% (hazard ratio: 1.14; 95% confidence interval: 0.94 to 1.39; p = 0.17) were independently associated with poorer outcomes compared with LVEF ≥70% (reference) in the conservative group. In the initial aortic valve replacement group, the adjusted risk for the primary outcome measure was not significantly different across the 4 LVEF groups. CONCLUSIONS: This study demonstrates that survival in patients with severe AS is impaired when LVEF is <60%, and these findings have implications for decision making with regard to the timing of surgical intervention.
Assuntos
Estenose da Valva Aórtica/fisiopatologia , Volume Sistólico , Função Ventricular Esquerda , Idoso , Idoso de 80 Anos ou mais , Estenose da Valva Aórtica/diagnóstico por imagem , Estenose da Valva Aórtica/mortalidade , Estenose da Valva Aórtica/cirurgia , Tratamento Conservador/efeitos adversos , Tratamento Conservador/mortalidade , Progressão da Doença , Ecocardiografia Doppler , Feminino , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/terapia , Implante de Prótese de Valva Cardíaca/efeitos adversos , Implante de Prótese de Valva Cardíaca/mortalidade , Hospitalização , Humanos , Japão , Masculino , Sistema de Registros , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Índice de Gravidade de Doença , Fatores de Tempo , Resultado do TratamentoRESUMO
OBJECTIVE: Abdominal aortic aneurysm (AAA) is an increasingly prevalent and ultimately fatal disease with no effective pharmacological treatment. Because matrix degradation induced by vascular inflammation is the major pathophysiology of AAA, attenuation of this inflammation may improve its outcome. Previous studies suggested that miR-33 (microRNA-33) inhibition and genetic ablation of miR-33 increased serum high-density lipoprotein cholesterol and attenuated atherosclerosis. APPROACH AND RESULTS: MiR-33a-5p expression in central zone of human AAA was higher than marginal zone. MiR-33 deletion attenuated AAA formation in both mouse models of angiotensin II- and calcium chloride-induced AAA. Reduced macrophage accumulation and monocyte chemotactic protein-1 expression were observed in calcium chloride-induced AAA walls in miR-33-/- mice. In vitro experiments revealed that peritoneal macrophages from miR-33-/- mice showed reduced matrix metalloproteinase 9 expression levels via c-Jun N-terminal kinase inactivation. Primary aortic vascular smooth muscle cells from miR-33-/- mice showed reduced monocyte chemotactic protein-1 expression by p38 mitogen-activated protein kinase attenuation. Both of the inactivation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were possibly because of the increase of ATP-binding cassette transporter A1 that is a well-known target of miR-33. Moreover, high-density lipoprotein cholesterol derived from miR-33-/- mice reduced expression of matrix metalloproteinase 9 in macrophages and monocyte chemotactic protein-1 in vascular smooth muscle cells. Bone marrow transplantation experiments indicated that miR-33-deficient bone marrow cells ameliorated AAA formation in wild-type recipients. MiR-33 deficiency in recipient mice was also shown to contribute the inhibition of AAA formation. CONCLUSIONS: These data strongly suggest that inhibition of miR-33 will be effective as a novel strategy for treating AAA.
Assuntos
Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/prevenção & controle , Aortite/prevenção & controle , Mediadores da Inflamação/metabolismo , MicroRNAs/metabolismo , Angiotensina II , Animais , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/metabolismo , Aortite/induzido quimicamente , Aortite/genética , Aortite/metabolismo , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Transplante de Medula Óssea , Cloreto de Cálcio , Linhagem Celular , Quimiocina CCL2/metabolismo , HDL-Colesterol/sangue , Dilatação Patológica , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Fenótipo , Transdução de Sinais , Fatores de Tempo , Transfecção , Remodelação Vascular , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Recently, it has been reported that specific microRNA (miRNA) levels are elevated in serum and can be used as biomarkers in patients with cardiovascular diseases. However, miRNAs expression profiles and their sources in pericardial fluid (PF) are unclear. METHODS AND RESULTS: The purpose of this study was to identify the levels of miRNAs in PF in relation to those in the serum in patients undergoing cardiac surgery. Serum (S) and PF from patients undergoing coronary artery bypass graft (CABG) due to stable angina pectoris (sAP) and unstable AP (uAP) and aortic valve replacement due to aortic stenosis (AS) were analyzed for the detection of miRNAs. We named these samples S-sAP, S-uAP, S-AS, PF-sAP, PF-uAP, and PF-AS, respectively. We first measured the levels of miR-423-5p, which was recognized previously as a biomarker for heart failure. miR-423-5p levels were significantly higher in PF than serum. Although there was no difference in miR-423-5p levels among the PF-AS, PF-sAP, and PF-uAP, its levels were significantly elevated in S-uAP compared with those in S-AS and S-sAP. In order to clarify the source of miR-423-5p in PF, we measured the levels of muscle-enriched miR-133a and vascular-enriched miR-126 and miR-92a in the same samples. miR-133a levels were significantly higher in serum than in PF, and it was elevated in S-uAP compared with S-AS. miR-126 level was significantly increased in serum compared with PF, and the level of miR-92a the similar tendency. miR-423-5p is located in the first intron of NSRP1. There is another miRNA, miR-3184, encoded in the opposite direction in the same region. In vitro experiments indicated that the duplex of miR-423-5p and miR-3184-3p was more resistant to RNase than the duplex of miR-423-5p and miR-133-3p, which may help to stabilize miR-423-5p in the PF. CONCLUSIONS: Our results suggested that miR-423-5p is enriched in PF, and serum miR-423-5p may be associate with uAP. Its expression pattern was different to that of muscle- and vascular-enriched miRNAs, miR-133a, miR-126, and miR-92a.
Assuntos
Angina Pectoris/genética , Estenose da Valva Aórtica/genética , MicroRNAs/sangue , MicroRNAs/genética , Líquido Pericárdico/metabolismo , Idoso , Idoso de 80 Anos ou mais , Angina Pectoris/sangue , Angina Pectoris/cirurgia , Estenose da Valva Aórtica/sangue , Estenose da Valva Aórtica/cirurgia , Sequência de Bases , Ponte de Artéria Coronária , Feminino , Perfilação da Expressão Gênica , Implante de Prótese de Valva Cardíaca , Humanos , Íntrons , Masculino , MicroRNAs/análise , Pessoa de Meia-IdadeRESUMO
BACKGROUND: The mechanism of cardiac energy production against sustained pressure overload remains to be elucidated. METHODS AND RESULTS: We generated cardiac-specific kinase-dead (kd) calcium/calmodulin-dependent protein kinase kinase-ß (CaMKKß) transgenic (α-MHC CaMKKßkd TG) mice using α-myosin heavy chain (α-MHC) promoter. Although CaMKKß activity was significantly reduced, these mice had normal cardiac function and morphology at baseline. Here, we show that transverse aortic binding (TAC) in α-MHC CaMKKßkd TG mice led to accelerated death and left ventricular (LV) dilatation and dysfunction, which was accompanied by significant clinical signs of heart failure. CaMKKß downstream signaling molecules, including adenosine monophosphate-activated protein kinase (AMPK), were also suppressed in α-MHC CaMKKßkd TG mice compared with wild-type (WT) mice. The expression levels of peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, which is a downstream target of both of CaMKKß and calcium/calmodulin kinases, were also significantly reduced in α-MHC CaMKKßkd TG mice compared with WT mice after TAC. In accordance with these findings, mitochondrial morphogenesis was damaged and creatine phosphate/ß-ATP ratios assessed by magnetic resonance spectroscopy were suppressed in α-MHC CaMKKßkd TG mice compared with WT mice after TAC. CONCLUSIONS: These data indicate that CaMKKß exerts protective effects on cardiac adaptive energy pooling against pressure-overload possibly through phosphorylation of AMPK and by upregulation of PGC-1α. Thus, CaMKKß may be a therapeutic target for the treatment of heart failure.
Assuntos
Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Insuficiência Cardíaca/etiologia , Remodelação Ventricular/genética , Trifosfato de Adenosina , Animais , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/mortalidade , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Cadeias Pesadas de Miosina/genética , Fosforilação , Regiões Promotoras Genéticas , Transdução de Sinais , Regulação para Cima , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports, including ours, indicated that miR-33a located within the intron of sterol regulatory element-binding protein (SREBP) 2 controls cholesterol homeostasis and can be a possible therapeutic target for treating atherosclerosis. Primates, but not rodents, express miR-33b from an intron of SREBF1. Therefore, humanized mice, in which a miR-33b transgene is inserted within a Srebf1 intron, are required to address its function in vivo. We successfully established miR-33b knock-in (KI) mice and found that protein levels of known miR-33a target genes, such as ABCA1, ABCG1, and SREBP-1, were reduced compared with those in wild-type mice. As a consequence, macrophages from the miR-33b KI mice had a reduced cholesterol efflux capacity via apoA-I and HDL-C. Moreover, HDL-C levels were reduced by almost 35% even in miR-33b KI hetero mice compared with the control mice. These results indicate that miR-33b may account for lower HDL-C levels in humans than those in mice and that miR-33b is possibly utilized for a feedback mechanism to regulate its host gene SREBF1. Our mice will also aid in elucidating the roles of miR-33a/b in different genetic disease models.
Assuntos
HDL-Colesterol/metabolismo , Íntrons/genética , MicroRNAs/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Animais , Sequência de Bases , Western Blotting , Células Cultivadas , Perfilação da Expressão Gênica , Células Hep G2 , Humanos , Macrófagos Peritoneais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismoRESUMO
BACKGROUND: Cholesterol efflux from cells to apolipoprotein A-I (apoA-I) acceptors via the ATP-binding cassette transporters ABCA1 and ABCG1 is thought to be central in the antiatherogenic mechanism. MicroRNA (miR)-33 is known to target ABCA1 and ABCG1 in vivo. METHODS AND RESULTS: We assessed the impact of the genetic loss of miR-33 in a mouse model of atherosclerosis. MiR-33 and apoE double-knockout mice (miR-33(-/-)Apoe(-/-)) showed an increase in circulating HDL-C levels with enhanced cholesterol efflux capacity compared with miR-33(+/+)Apoe(-/-) mice. Peritoneal macrophages from miR-33(-/-)Apoe(-/-) mice showed enhanced cholesterol efflux to apoA-I and HDL-C compared with miR-33(+/+)Apoe(-/-) macrophages. Consistent with these results, miR-33(-/-)Apoe(-/-) mice showed reductions in plaque size and lipid content. To elucidate the roles of miR-33 in blood cells, bone marrow transplantation was performed in these mice. Mice transplanted with miR-33(-/-)Apoe(-/-) bone marrow showed a significant reduction in lipid content in atherosclerotic plaque compared with mice transplanted with miR-33(+/+)Apoe(-/-) bone marrow, without an elevation of HDL-C. Some of the validated targets of miR-33 such as RIP140 (NRIP1) and CROT were upregulated in miR-33(-/-)Apoe(-/-) mice compared with miR-33(+/+)Apoe(-/-) mice, whereas CPT1a and AMPKα were not. CONCLUSIONS: These data demonstrate that miR-33 deficiency serves to raise HDL-C, increase cholesterol efflux from macrophages via ABCA1 and ABCG1, and prevent the progression of atherosclerosis. Many genes are altered in miR-33-deficient mice, and detailed experiments are required to establish miR-33 targeting therapy in humans.
Assuntos
Apolipoproteínas E/fisiologia , Aterosclerose/fisiopatologia , HDL-Colesterol/sangue , Progressão da Doença , MicroRNAs/fisiologia , Transportador 1 de Cassete de Ligação de ATP , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/fisiologia , Animais , Aterosclerose/sangue , Aterosclerose/genética , Western Blotting , Transplante de Medula Óssea , Células Cultivadas , Colesterol/metabolismo , Primers do DNA/química , Lipoproteínas/fisiologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Knockout , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Sterol regulatory element-binding protein 2 (SREBP-2) transcription factor has been identified as a key protein in cholesterol metabolism through the transactivation of the LDL receptor and cholesterol biosynthesis genes. Here, we generated mice lacking microRNA (miR)-33, encoded by an intron of the Srebp2, and showed that miR-33 repressed the expression of ATP-binding cassette transporter A1 (ABCA1) protein, a key regulator of HDL synthesis by mediating cholesterol efflux from cells to apolipoprotein A (apoA)-I. In fact, peritoneal macrophages derived from miR-33-deficient mice showed a marked increase in ABCA1 levels and higher apoA-I-dependent cholesterol efflux than those from WT mice. ABCA1 protein levels in liver were also higher in miR-33-deficient mice than in WT mice. Moreover, miR-33-deficient mice had significantly higher serum HDL cholesterol levels than WT mice. These data establish a critical role for miR-33 in the regulation of ABCA1 expression and HDL biogenesis in vivo.
Assuntos
HDL-Colesterol/metabolismo , Íntrons , MicroRNAs/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Sequência de Bases , Bovinos , Linhagem Celular , Galinhas , HDL-Colesterol/genética , Feminino , Humanos , Macrófagos/metabolismo , Camundongos , MicroRNAs/genética , Dados de Sequência Molecular , Alinhamento de Sequência , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismoRESUMO
AIMS: A significant increase in congestive heart failure (CHF) was reported when the anti-ErbB2 antibody trastuzumab was used in combination with the chemotherapy drug doxorubicin (Dox). The aim of the present study was to investigate the role(s) of miRNAs in acute Dox-induced cardiotoxicity. METHODS AND RESULTS: Neuregulin-1-ErbB signalling is essential for maintaining adult cardiac function. We found a significant reduction in ErbB4 expression in the hearts of mice after Dox treatment. Because the proteasome pathway was only partially involved in the reduction of ErbB4 expression, we examined the involvement of microRNAs (miRs) in the reduction of ErbB4 expression. miR-146a was shown to be up-regulated by Dox in neonatal rat cardiac myocytes. Using a luciferase reporter assay and overexpression of miR-146a, we confirmed that miR-146a targets the ErbB4 3'UTR. After Dox treatment, overexpression of miR-146a, as well as that of siRNA against ErbB4, induced cell death in cardiomyocytes. Re-expression of ErbB4 in miR-146a-overexpressing cardiomyocytes ameliorated Dox-induced cell death. To examine the loss of miR-146a function, we constructed 'decoy' genes that had tandem complementary sequences for miR-146a in the 3'UTR of a luciferase gene. When miR-146a 'decoy' genes were introduced into cardiomyocytes, ErbB4 expression was up-regulated and Dox-induced cell death was reduced. CONCLUSION: These findings suggested that the up-regulation of miR-146a after Dox treatment is involved in acute Dox-induced cardiotoxicity by targeting ErbB4. Inhibition of both ErbB2 and ErbB4 signalling may be one of the reasons why those patients who receive concurrent therapy with Dox and trastuzumab suffer from CHF.
Assuntos
Cardiomiopatias/metabolismo , Receptores ErbB/metabolismo , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Neurregulinas/metabolismo , Regiões 3' não Traduzidas , Animais , Apoptose , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/genética , Cardiomiopatias/patologia , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Doxorrubicina/toxicidade , Receptores ErbB/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Interferência de RNA , Ratos , Receptor ErbB-4 , Transdução de Sinais , Fatores de Tempo , Transfecção , Regulação para CimaRESUMO
MicroRNAs (miRNAs or miRs) are small, non-coding RNAs that modulate mRNA stability and post-transcriptional translation. A growing body of evidence indicates that specific miRNAs can affect the cellular function of cardiomyocytes. In the present study, miRNAs that are highly expressed in the heart were overexpressed in neonatal rat ventricular myocytes, and cellular ATP levels were assessed. As a result, miR-15b, -16, -195, and -424, which have the same seed sequence, the most critical determinant of miRNA targeting, decreased cellular ATP levels. These results suggest that these miRNAs could specifically down-regulate the same target genes and consequently decrease cellular ATP levels. Through a bioinformatics approach, ADP-ribosylation factor-like 2 (Arl2) was identified as a potential target of miR-15b. It has already been shown that Arl2 localizes to adenine nucleotide transporter 1, the exchanger of ADP/ATP in mitochondria. Overexpression of miR-15b, -16, -195, and -424 suppressed the activity of a luciferase reporter construct fused with the 3'-untranslated region of Arl2. In addition, miR-15b overexpression decreased Arl2 mRNA and protein expression levels. The effects of Arl2 siRNA on cellular ATP levels were the same as those of miR-15b, and the expression of Arl2 could restore ATP levels reduced by miR-15b. A loss-of-function study of miR-15b resulted in increased Arl2 protein and cellular ATP levels. Electron microscopic analysis revealed that mitochondria became degenerated in cardiomyocytes that had been transduced with miR-15b and Arl2 siRNA. The present results suggest that miR-15b may decrease mitochondrial integrity by targeting Arl2 in the heart.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Ligação ao GTP/metabolismo , MicroRNAs/fisiologia , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Sobrevivência Celular , Células Cultivadas , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , MicroRNAs/genética , Microscopia Eletrônica de Transmissão , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Miócitos Cardíacos/ultraestrutura , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Screening for cell surface proteins up-regulated under stress conditions may lead to the identification of new therapeutic targets. To search for genes whose expression was enhanced by treatment with oligomycin, a mitochondrial-F(0)F(1) ATP synthase inhibitor, signal sequence trapping was performed in H9C2 rat cardiac myoblasts. One of the genes identified was that for neural cell adhesion molecule (NCAM, CD56), a major regulator of development, cell survival, migration, and neurite outgrowth in the nervous system. Immunohistochemical analyses in a mouse myocardial infarction model revealed that NCAM was strongly expressed in residual cardiac myocytes in the infarcted region. Increased expression of NCAM was also found during the remodeling period in a rat model of hypertension-induced heart failure. Lentivirus-mediated knockdown of NCAM decreased the cell growth and survival following oligomycin treatment in H9C2 cells. In primary rat neonatal cardiac myocytes, NCAM was also found to be up-regulated and played a protective role following oligomycin treatment. Analyses of downstream signaling revealed that knockdown of NCAM significantly decreased the basal AKT phosphorylation level. In contrast, NCAM mimetic peptide P2d activated AKT and significantly reduced oligomycin-induced cardiomyocyte death, which was abolished by treatment with the PI3K inhibitor LY-294002 as well as overexpression of the dominant-negative AKT mutant. These findings demonstrate that NCAM is a cardioprotective factor up-regulated under metabolic stress in cardiomyocytes and augmentation of this signal improved survival.
Assuntos
Cardiotônicos/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Regulação para Cima , Animais , Membrana Celular/metabolismo , Proliferação de Células , Sobrevivência Celular , Inibidores Enzimáticos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Miócitos Cardíacos/citologia , Oligomicinas/farmacologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , RatosRESUMO
During the progression of heart failure (HF), phases of chronic compensation and acute decompensation alternate and the clinical status worsens during the acute phase. At the present time, few studies have focused their attention on the cycles of compensated and decompensated phases from the perspective of myocyte injury. We hypothesize that persistently increased serum cTn concentrations during chronic compensated HF identify patients likely to need multiple admissions to the hospital for management of acute cardiac decompensation, worsening their long-term prognosis by causing further myocyte injury during the acute phase. In patients with acute cardiac decompensation, myocyte injury observed within hours or days has a long-term predictive value, and the acute surge of myocyte injury occurring in the acutely decompensated phase might be an important therapeutic target from the perspective of myocyte preservation. Clinical trials that limit myocyte injury during acutely decompensated as well as during chronic compensated HF are warranted.