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1.
Physiol Genomics ; 52(8): 358-368, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32716698

RESUMO

MicroRNAs (miRNAs) are powerful regulators of protein expression. Many play important roles in cardiac development and disease. While several miRNAs and targets have been well characterized, the abundance of miRNAs and the numerous potential targets for each suggest that the vast majority of these interactions have yet to be described. The goal of this study was to characterize miRNA expression in the mouse heart after coronary artery ligation (LIG) and identify novel mRNA targets altered during the initial response to ischemic stress. We performed small RNA sequencing (RNA-Seq) of ischemic heart tissue 1 day and 3 days after ligation and identified 182 differentially expressed miRNAs. We then selected relevant mRNA targets from all potential targets by correlating miRNA and mRNA expression from a corresponding RNA-Seq data set. From this analysis we chose to focus, as proof of principle, on two miRNAs from the miR-125 family, miR-125a and miR-351, and two of their potential mRNA targets, Xin actin-binding repeat-containing protein 1 (XIRP1) and factor inhibiting hypoxia-inducible factor (FIH). We found miR-125a to be less abundant and XIRP1 more abundant after ligation. In contrast, the related murine miRNA miR-351 was substantially upregulated in response to ischemic injury, and FIH expression correspondingly decreased. Luciferase reporter assays confirmed direct interactions between these miRNAs and targets. In summary, we utilized a correlative analysis strategy combining miRNA and mRNA expression data to identify functional miRNA-mRNA relationships in the heart after ligation. These findings provide insight into the response to ischemic injury and suggest future therapeutic targets.


Assuntos
Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/genética , MicroRNAs/genética , Oxigenases de Função Mista/genética , Infarto do Miocárdio/genética , Regulação para Cima/genética , Animais , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Oxigenases de Função Mista/metabolismo , Infarto do Miocárdio/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA-Seq , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética
2.
Am J Physiol Heart Circ Physiol ; 316(3): H554-H565, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30575439

RESUMO

The principal regulator of cellular response to low oxygen is hypoxia-inducible factor (HIF)-1, which is stabilized in several forms of heart failure. Our laboratory developed a mouse strain in which a stable form of HIF-1 can be inducibly expressed in cardiomyocytes. Strikingly, these mice show a rapid decrease in cardiac contractility and a rapid loss of SERCA2 protein, which is also seen in heart failure. Interestingly, while the SERCA2 transcript decreased, it did not fully account for the observed decrease in protein. We therefore investigated whether HIF-1-regulated microRNA could impair SERCA translation. Multiple screening analyses identified the microRNA miR-29c to be substantially upregulated upon HIF-1 induction and to have complementarity to SERCA, and therefore be a potential regulator of SERCA2 expression in hypoxia. Subsequent evaluation confirmed that miR-29c reduced SERCA2 expression and Ca2+ reuptake. Additionally, administration of an antagonist sequence (antimir) improved cardiac contractility and SERCA2 expression in HIF transgenic mice. To extend the significance of these findings, we examined miR-29c expression in physiological hypoxia. Surprisingly, miR-29c decreased in these settings. We also treated mice with antimir before infarction to see if further suppression of miR-29c could improve cardiac function. While no improvement in contractility or SERCA2 was observed, reduction of heart size after infarction indicated that the antimir could modulate cardiac physiology. These results demonstrate that while a HIF-1-regulated microRNA, miR-29c, can reduce SERCA2 expression and contractility, additional factors in the ischemic milieu may limit these effects. Efforts to develop miRNA-based therapies will need to explore and account for these additional countervailing effects. NEW & NOTEWORTHY Our study demonstrated hypoxia-inducible factor-1-dependent upregulation of miR-29c, which, in turn, inhibited SERCA2 expression and reduced cardiac contractility in a transgenic overexpression system. Interestingly, these results were not recapitulated in a murine myocardial infarction model. These results underscore the complexity of the pathological environment and highlight the need for therapeutic target validation in physiologically relevant models.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , MicroRNAs/metabolismo , Contração Miocárdica , Miócitos Cardíacos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Animais , Cálcio/metabolismo , Hipóxia Celular , Células HEK293 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Miócitos Cardíacos/fisiologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
3.
Physiol Genomics ; 50(7): 479-494, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29652636

RESUMO

Alternative splicing of RNA is an underexplored area of transcriptional response. We expect that early changes in alternatively spliced genes may be important for responses to cardiac injury. Hypoxia inducible factor 1 (HIF1) is a key transcription factor that rapidly responds to loss of oxygen through alteration of metabolism and angiogenesis. The goal of this study was to investigate the transcriptional response after myocardial infarction (MI) and to identify novel, hypoxia-driven changes, including alternative splicing. After ligation of the left anterior descending artery in mice, we observed an abrupt loss of cardiac contractility and upregulation of hypoxic signaling. We then performed RNA sequencing on ischemic heart tissue 1 and 3 days after infarct to assess early transcriptional changes and identified 89 transcripts with altered splicing. Of particular interest was the switch in Pkm isoform expression (pyruvate kinase, muscle). The usually predominant Pkm1 isoform was less abundant in ischemic hearts, while Pkm2 and associated splicing factors (hnRNPA1, hnRNPA2B1, Ptbp1) rapidly increased. Despite increased Pkm2 expression, total pyruvate kinase activity remained reduced in ischemic myocardial tissue. We also demonstrated HIF1 binding to PKM by chromatin immunoprecipitation, indicating a direct role for HIF1 in mediating this isoform switch. Our study provides a new, detailed characterization of the early transcriptome after MI. From this analysis, we identified an HIF1-mediated alternative splicing event in the PKM gene. Pkm1 and Pkm2 play distinct roles in glycolytic metabolism and the upregulation of Pkm2 is likely to have important consequences for ATP synthesis in infarcted cardiac muscle.


Assuntos
Perfilação da Expressão Gênica , Fator 1 Induzível por Hipóxia/genética , Infarto do Miocárdio/genética , Piruvato Quinase/genética , Processamento Alternativo , Animais , Glicólise/genética , Humanos , Hipóxia , Fator 1 Induzível por Hipóxia/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Piruvato Quinase/metabolismo
4.
Kidney Int ; 91(6): 1386-1397, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28139294

RESUMO

C3 glomerulopathy is a potentially life-threatening disease of the kidney caused by dysregulated alternative pathway complement activation. The specific complement mediator(s) responsible for kidney injury in C3 glomerulopathy are yet to be defined and no specific therapy is currently available. We previously developed a mouse model of lethal C3 glomerulopathy with factor H and properdin gene double mutations. Therefore, we used this model to examine the role of C5 and C5a receptor (C5aR) in the pathogenesis of the disease. Disease severity in these factor H/properdin double-mutant mice was found to be correlated with plasma C5 levels, and prophylactic anti-C5 mAb therapy was effective in preventing lethal C3 glomerulopathy. When given to these double-mutant mice that had already developed active disease with severe proteinuria, anti-C5 mAb treatment also prevented death in half of the mice. Deficiency of C5aR significantly reduced disease severity, suggesting that C5aR-mediated inflammation contributed to C3 glomerulopathy. Thus, C5 and C5aR have a critical role in C3 glomerulopathy. Hence, early intervention targeting these pathways may be an effective therapeutic strategy for patients with C3 glomerulopathy.


Assuntos
Anticorpos Monoclonais/farmacologia , Complemento C3/metabolismo , Complemento C5/antagonistas & inibidores , Inativadores do Complemento/farmacologia , Via Alternativa do Complemento/efeitos dos fármacos , Glomerulonefrite/prevenção & controle , Rim/efeitos dos fármacos , Insuficiência Renal/prevenção & controle , Animais , Complemento C3/imunologia , Complemento C5/imunologia , Complemento C5/metabolismo , Fator H do Complemento/deficiência , Fator H do Complemento/genética , Modelos Animais de Doenças , Predisposição Genética para Doença , Glomerulonefrite/genética , Glomerulonefrite/imunologia , Glomerulonefrite/metabolismo , Rim/imunologia , Rim/metabolismo , Rim/patologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Properdina/deficiência , Properdina/genética , Proteinúria/imunologia , Proteinúria/metabolismo , Proteinúria/prevenção & controle , Receptor da Anafilatoxina C5a/deficiência , Receptor da Anafilatoxina C5a/genética , Insuficiência Renal/genética , Insuficiência Renal/imunologia , Insuficiência Renal/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo
5.
Sci Rep ; 11(1): 13116, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162925

RESUMO

Ischemic heart disease is a leading cause of heart failure and hypoxia inducible factor 1 (HIF1) is a key transcription factor in the response to hypoxic injury. Our lab has developed a mouse model in which a mutated, oxygen-stable form of HIF1α (HIF-PPN) can be inducibly expressed in cardiomyocytes. We observed rapid cardiac dilation and loss of contractility in these mice due to lower expression of excitation-contraction coupling genes and reduced calcium flux. As alternative splicing plays an underappreciated role in transcriptional regulation, we used RNA sequencing to search for splicing changes in calcium-handling genes of HIF-PPN hearts and compared them to previous sequencing data from a model of myocardial infarction (MI) to select for transcripts that are modified in a pathological setting. We found overlap between genes differentially expressed in HIF-PPN and post-MI mice (54/131 genes upregulated in HIF-PPN hearts at 1 day and/or 3 days post-MI, and 45/78 downregulated), as well as changes in alternative splicing. Interestingly, calcium/calmodulin dependent protein kinase II, gamma (CAMK2G) was alternatively spliced in both settings, with variant 1 (v1) substantially decreased compared to variants 2 (v2) and 3 (v3). These findings were also replicated in vitro when cells were transfected with HIF-PPN or exposed to hypoxia. Further analysis of CAMK2γ protein abundance revealed only v1 was detectable and substantially decreased up to 7 days post-MI. Rbfox1, a splicing factor of CAMK2G, was also decreased in HIF-PPN and post-MI hearts. Subcellular fractionation showed CAMK2γ v1 was found in the nuclear and cytoplasmic fractions, and abundance decreased in both fractions post-MI. Chromatin immunoprecipitation analysis of HIF1 in post-MI hearts also demonstrated direct HIF1 binding to CAMK2G. CaMK2 is a key transducer of calcium signals in both physiological and pathological settings. The predominantly expressed isoform in the heart, CaMK2δ, has been extensively studied in cardiac injury, but the specific role of CaMK2γ is not well defined. Our data suggest that loss of CaMK2γ after MI is HIF1-dependent and may play an important role in the heart's calcium signaling and transcriptional response to hypoxia.


Assuntos
Processamento Alternativo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Isquemia Miocárdica/metabolismo , Animais , Western Blotting , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
6.
Invest Ophthalmol Vis Sci ; 59(8): 3405-3415, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-30025090

RESUMO

Purpose: Dense deposit disease (DDD) is caused by dysregulation of the alternative pathway of the complement cascade and characterized by electron-dense deposits in the kidney glomerular basement membrane (GBM) and drusen in Bruch's membrane (BrM). Complement factor H (fH) and factor properdin (fP) regulate complement activation; fH inhibits alternative pathway (AP) activation, whereas fP promotes it. We report pathologic changes in eyes of an fH and fP double-mutant mouse, which we previously showed have dense deposits in the GBM and early mortality from nephropathy. Methods: fHm/m, fP-/-, and fHm/m/fP-/- mice were generated on a C57BL/6-129J background. Fundus imaging at 8 weeks of age was followed by analysis via light and electron microscopy. Retinal function was assessed by electroretinography (ERG). Complement levels and localization were tested by immunohistochemistry and ELISA. Retinas of fHm/m/fP-/- mice treated with intraperitoneal injections of an anti-C5 antibody were compared to those of age- and genotype-matched mice injected with an isotype control antibody. Results: fHm/m/fP-/- mice suffered early-onset retinal hypopigmented spots detected using in vivo retinal photography, and histologic examination showed basal laminar deposits (BLamD), degeneration of the photoreceptors, and RPE vacuolization. ERG showed diminished retinal function. The anti-C5 antibody was retina-protective. Conclusions: This unique mouse represents a new model of complement-mediated rapid-onset DDD, and could be useful in exploring the pathologic changes associated with BLamD in age-related macular degeneration.


Assuntos
Lâmina Basilar da Corioide/patologia , Fator H do Complemento/metabolismo , Modelos Animais de Doenças , Glomerulonefrite Membranoproliferativa/patologia , Properdina/metabolismo , Doenças Retinianas/patologia , Epitélio Pigmentado da Retina/patologia , Animais , Eletrorretinografia , Ensaio de Imunoadsorção Enzimática , Glomerulonefrite Membranoproliferativa/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Doenças Retinianas/metabolismo
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