| Background:Cardiac hypertrophy is a pathological feature common to numerous forms of heart diseases such as hypertension, ischemic myocardial injury, diabetic cardiomyopathy, valvular dysfunction and aortic stenosis, etc. Persistence of hypertrophy can ultimately lead to ventricular dilatation, arrhythmia, fibrotic diseases, heart failure and even sudden death. Cardiac hypertrophy is a major risk factor in the development of heart failure, and its therapeutic reversal is associated with improved mortality.Recent studies have indicated that a key role of microRNAs (miRNAs) in biological processes including differentiation, apoptosis, proliferation, and development, and their dysregulation have been linked to several human diseases, including cardiovascular disease. MicroRNAs (miRNAs) are a class of highly conserved, small noncoding RNAs (-23 nucleotides) that regulate gene expression posttranscriptionally. They have been found in various organisms and regarded as powerful regulators of gene expression and cellular phenotype. Moreover, their roles in cardiovascular biology and diseases have been an area of intense investigation. Recently, studies have identified expression patterns of miRNAs associated with cardiovascular diseases. For example, miRNA-21, miRNA-23a, miRNA-24, miRNA-133, miRNA-208/miRNA-195 and miRNA-199 are involved in cardiac hypertrophy, miRNA-1 in arrhythmia, miRNA-29 and-21 in cardiac flbrosis, miRNA-210 and miRNA-494 in ischemic heart disease, and miRNA-129 in heart failure. But there is no research on the relationship between miRNA-455(miR-455) and cardiac hypertrophy. Very few studies have been published on miR-455, and, in fact, a PubMed search coupled with a search of PhenomiR, a knowledge base for miRNAs expression and function, reveals only 4 research papers on miR-455, none of which is related to the relationship between miR-455 and cardiac hypertrophy.We made a model of hypertrophy in mice by transverse aorta constriction (TAC), and aimed to investigate the role of an aberrantly expressed miR-455 in cardiac hypertrophy and its potential cellular and molecular mechanisms.Part 1 miR-455 promotes cardiac hypertrophy induced by short-term pressure overload by target calreticulinObjective:Recent studies have shown that microRNAs associated with a variety of cardiovascular disease, but so far there is no study about miRNA-455 (miR-455) and myocardial hypertrophy.We made a model of hypertrophy in mice by transverse aorta constriction (TAC) after 2 weeks, and aimed to investigate the role of an aberrantly expressed miR-455 in cardiac hypertrophy and its potential cellular and molecular mechanisms.Methods:Four-week-old kunming male mice were randomly divided into 3 groups,TAC mice with miR-455(TAC.miR-455)group,TAC mice with GFP(TAC.GFP)group,and sham operation mice with GFP(sham)group. TAC or sham operation was carried out.Hemodynamic and echocardiographic data was measured in mice after two weeks.HE and Masson staining was carried out on the myocardial tissue to observe the histopathological changes. Quantitative reverse transcription-polymerase chain reaction(qRT-PCR) was applied to detect hypertrophy gene and fibrosis.Western blot analysis was applied to detect apoptosis protein.qRT-PCR and Western blot analysis was applied to detect miR-455 target genes and proteins.Results:1 In vivo restoration of mature miR-455 expression.Significant decrease of miR-455 gene in the GFP-treated hearts of TAC, and significant increase of miR-455 gene in the miR-455-treated hearts of TAC were observed. Ad-mediated miR-455 gene transfer in vivo in the setting of pressure-overload hypertrophy restored the expression of the mature miR-455 in the hypertrophic hears,all P<0.01.2 The hemodynamic and echocardiographic effects of miR-455 on cardiac hypertrophy at 2 weeks after pressure overload.First, at this time point, all the experimental mice survived and some of them received hemodynamic and echocardiographic examination. BP and LVESP were similarly elevated, whereas LVEDP was not changed in all examined TAC mice. TAC induced significant cardiac hypertrophy characterized by increases in LV wall thicknesses, decreases of LV cavity, and increases in the ratio of heart weight to body weight, left ventricular ejection fraction and the cross-sectional area of cardiomyocytes in GFP-treated mice,all P<0.05. All these characteristics were significantly aggravated by the treatment with miR-455,all P<0.05,but left ventricular ejection fraction was similar,P>0.05.3 The effects of miR-455 on myocardial hypertrophy geneWe assessed the expression of the hypertrophic fetal genes, Anf, Actal as well as Myh7, at 2 weeks after gene transfer. Pressure-overload-induced hypertrophy in the GFP-treated group was associated with the reinduction of the "fetal gene program" characterized by a significant increase in the mRNA expression of Anf, Myh7 and Actal compared with shamoperated animals,all P<0.01. Furthermore, we observed an significant increase of three genes in the miR-455-treated hearts compared with the GFP-treated hearts. That is, the expression of hypertrophic genes such as Anf, Myh7 and Actal were significantly upregulated by the pressure overload in the hearts of GFP-treated mice,all P<0.01, and these hypertrophic responses were significantly aggravated after gene transfer.4 The effects of miR-455 on myocardial fibrosisHistological examination of LV sections by Masson staining and subsequent quantification of the fibrotic area revealed that TAC induced a profound increase in interstitial fibrosis in the GFP-treated hearts and miR-455 treatment compared with sham-operated hearts. But there was no obvious difference between the two groups.We observed a significant increase in mRNA expression of the profibrotic genes, the transforming growth factor beta-1 (Tgf?1), and the connective tissue growth factor (Ctgf), in the GFP-treated hearts and miR-455 treatment compared with sham,P<0.05. But there was no obvious difference between the two groups.5 The effects of miR-455 on myocardial cell apoptosisAntiapoptotic protein decreased and promoting apoptosis protein increased significantly in the GFP-treated hearts of TAC,P<0.05.Antiapoptotic protein in the miR-455-treated hearts of TAC was similar compared with GFP-treated hearts of TAC,P>0.05,even if it decreased.6 Calr is the target gene of miR-455 in the setting of pressure-overload-induced cardiac hypertrophyTo gain additional insights into the relation of the miR-455 and Calr in the setting of pressure-overload-induced cardiac hypertrophy, we evaluated whether Calr and GRP78 expression was regulated by miR-455 in vivo in the setting of pressure-overload-induced hypertrophy. Western blotting analysis demonstrated that at the protein level, the expression of both Calr and GRP78 was significantly increased in the GFP-treated LV tissue compared with sham animals. However, there was downregulation of Calr along with upregulation of miR-455 in the miR455-treated LV tissue, but GRP78 was still in a state of increase.That is to say, Calr is the target of miR-455, but GRP78 is not. In addition to translational repression, miRNAs can lead to mRNA degradation of their targets. A significant decrease in the mRNA expression levels of Calr in the miR-455 animals compared with GFP-treated ones was observed, as assessed by qRT-PCR. But, there is no change of mRNA expression levels of GRP78 in the miR-455 animals compared with GFP-treated mice.Conclusion:TAC induces significant cardiac hypertrophy at two weeks by increasing in LV wall thicknesses, decreasing of LV cavity, and increasing the ratio of heart weight to body weight, left ventricular ejection fraction and the cross-sectional area of cardiomyocytes. miR-455 aggravates the hypertrophic phenotype. miR-455 promotes cardiac hypertrophy induced by short-term pressure overload by targeting calreticulin.Part 2 miR-455 reduces myocardial fibrosis and myocardial cell apoptosispromotes cardiac hypertrophy induced by long-term pressure overload by target calreticulinObjective:Considering that cardiac fibrosis and apoptosis are prominent features in the transition from compensatory hypertrophy to heart failure, we investigated the potential involvement of miR-455 restoration in the regulation of cardiac ECM remodeling and apoptosis. Fibrosis is a pathological feature of cardiac adaptation to stress, where the proliferation of fibroblasts and the increased deposition of ECM components result in myocardial stiffness and diastolic dysfunction. We made a model of hypertrophy in mice by transverse aorta constriction (TAC) after 4 weeks, and aimed to investigate the role of an aberrantly expressed miR-455 in cardiac hypertrophy and its potential cellular and molecular mechanisms.Methods:Four-week-old kunming male mice were randomly divided into 3 groups,TAC mice with miR-455(TAC.miR-455)group,TAC mice with GFP(TAC.GFP)group,and sham operation mice with GFP(sham)group. TAC or sham operation was carried out.Hemodynamic and echocardiographic data was measured in mice after four weeks.HE and Masson staining was carried out on the myocardial tissue to observe the histopathological changes. Quantitative reverse transcription-polymerase chain reaction(qRT-PCR) was applied to detect hypertrophy gene and fibrosis.Western blot analysis was applied to detect apoptosis protein.qRT-PCR and Western blot analysis was applied to detect miR-455 target genes and proteins.Results:1 In vivo restoration of mature miR-455 expression.Significant decrease of miR-455 gene in the GFP-treated hearts of TAC, and significant increase of miR-455 gene in the miR-455-treated hearts of TAC were observed. Ad-mediated miR-455 gene transfer in vivo in the setting of pressure-overload hypertrophy restored the expression of the mature miR-455 in the hypertrophic hears,all P<0.01.2.The hemodynamic and echocardiographic effects of miR-455 on cardiac hypertrophy at 4 weeks after pressure overload.At 4 weeks after TAC, in the GFP-treated mice, although BP, LVESP, the ratio of heart weight to body weight, and the cardiomyocyte cross-sectional area did not change as compared with those at 2 weeks, an elevated LVEDP, a thinned LV wall thickness, an enlarged LV cavity, and a lowered LV contractility were observed,P<0.05, suggesting significant cardiac remodeling with the impaired cardiac function at that time in these mice. However, LVAWd was thinning in the miR-455-treated mice,P<0.05, but an enlarged LV cavity and a lowered LV contractility were not observed,P>0.05. That is to say, the miR-455-treated hearts still maintained a state of myocardial hypertrophy. These results suggest that miR-455 gene-transference are effective in the prevention of cardiac remodeling and dysfunction during a 4-week period of pressure overload.3 The effects of miR-455 on myocardial hypertrophy geneWe assessed the expression of the hypertrophic fetal genes, Anf, Actal as well as Myh7, at 4 weeks after gene transfer. Pressure-overload-induced hypertrophy in the GFP-treated group was associated with the reinduction of the "fetal gene program" characterized by a significant increase in the mRNA expression of Anf, Myh7 and Actal compared with shamoperated animals,all P<0.05. Furthermore, we observed an significant increase of two genes (Anf, Actal)in the miR-455-treated hearts compared with the GFP-treated hearts,all P<0.01. But there was a phenomenon worthy of attention, that was, the expression of Myh7 in GFP-treated mice was more than that in miR-455-treated mice at 4 weeks,P<0.01.4 The effects of miR-455 on myocardial fibrosisHistological examination of LV sections by Masson staining and subsequent quantification of the fibrotic area revealed that TAC induced a profound increase in interstitial fibrosis in the GFP-treated hearts compared with sham-operated hearts. In contrast, miR-455 treatment significantly decreased fibrosis.We observed a significant increase in mRNA expression of the profibrotic genes, the transforming growth factor beta-1 (Tgf?1), and the connective tissue growth factor (Ctgf), in the GFP-treated hearts-compared with sham,all P<0.01. However, these accelerated myocardial fibrosis genes after pressure overload were all significantly alleviated in the mice treated with miR-455,all P<0.01.5 The effects of miR-455 on myocardial cell apoptosisIn response to pressure overload, cardiomyocyte apoptosis may further contribute to the transition from LV hypertrophy to heart failure. The apoptotic cells by TUNEL staining decreased significantly in miR-455, P<0.01,compared with GFP-treated hearts.By Western blot analysis, we quantified the protein expression of the antiapoptotic gene, Bcl-2, and the proapoptotic gene, Bax, which are involved in the apoptotic pathway. Results showed that there was a significant increase in Bcl-2 and a decrease in Bax expression in miR-455, all P<0.01 compared with GFP-treated hearts. Consequently, the Bcl-2/Bax ratio, an important marker of myocardial cell survival probability, was significantly increased in the miR-455, compared with GFP-treated hearts.6 Calr is the target gene of miR-455 in the setting of pressure-overload-induced cardiac hypertrophyTo gain additional insights into the relation of the miR-455 and Calr in the setting of pressure-overload-induced cardiac hypertrophy, we evaluated whether Calr and GRP78 expression was regulated by miR-455 in vivo in the setting of pressure-overload-induced hypertrophy. Western blotting analysis demonstrated that at the protein level, the expression of both Calr and GRP78 was significantly increased in the GFP-treated LV tissue compared with sham animals. However, there was downregulation of Calr along with upregulation of miR-455 in the miR455-treated LV tissue, but GRP78 was still in a state of increase.That is to say, Calr is the target of miR-455, but GRP78 is not. In addition to translational repression, miRNAs can lead to mRNA degradation of their targets. A significant decrease in the mRNA expression levels of Calr in the miR-455 animals compared with GFP-treated ones was observed, as assessed by qRT-PCR. But, there is no change of mRNA expression levels of GRP78 in the miR-455 animals compared with GFP-treated mice.Conclusion:TAC induces significant heart failure at four weeks by decreasing in LV wall thicknesses, increasing of LV cavity, and decreasing left ventricular ejection fraction.A significant myocardial fibrosis and myocardial cell apoptosis are observed with changes of fibrosis gene and apoptosis protein.miR-455 treatment significantly decreases fibrosis and myocardial cell apoptosis,so retardes the progress of heart failure.miR-455 retardes ventricular remodeling induced by long-term pressure overload by target calreticulin.Conclusion:In conclusion, Ad-13-mediated normalization of miR-455 expression aggravates the hypertrophic phenotype but attenuates the progressive deterioration of LV function. Restoration or downregulation of miR-455 levels at different timing, therefore, may lead to a novel therapeutic strategy to reverse cardiac hypertrophy and alleviate function deterioration. |
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