MicroRNA profiling in early hypertrophic growth of the left ventricle in rats
Introduction
Precise regulation of gene expression is vital for the normal development of living organisms. Gene regulation is achieved by multiple mechanisms operating at transcriptional, post-transcriptional and post-translational levels. The discovery of microRNAs by Ambros and coworkers [1] radically changed our understanding of gene regulation in eukaryotes. MicroRNAs (miRNAs) are highly conserved 21–23 nucleotides long non-coding RNAs that regulate gene expression at the post-transcriptional level. By annealing to partially complementary sequences in the target messenger RNAs, the microRNAs mostly mediate translational repression or degradation of the mRNAs, resulting in down-regulation of the protein level. However, miRNA are also able to stimulate gene expression [2]. More than 700 microRNAs have been identified in the human genome (miRBase release 14.0). Many studies concerning functional characterization of known microRNAs in different physiological scenarios have been published in the last years. The majority of microRNAs seem to act cooperatively with many target sites in one gene and they can also target several genes, which add high complexity to the gene regulatory networks [3]. MicroRNAs are important regulators of many biological processes, including development, differentiation and apoptosis [4]. Consequently aberrant expression of microRNAs has been associated with disease. For example, several miRNAs behave as oncogenes or tumour suppressor genes in many types of cancer (reviewed by [5]).
Cardiac hypertrophy or thickening of the heart muscle occurs in response to increased blood pressure that preserves myocardial wall stress, chamber size and contractile function. Cardiac hypertrophy, which is a risk factor for cardiovascular disease, can be induced by a number of stimuli, like long-term exposure to pressure-overload, and if untreated it can lead to cardiomyopathy (defined as weakness of the individual muscle fibres of the heart) and heart failure [6].
MicroRNAs are highly expressed in the heart and they have important regulatory roles during cardiomyocyte differentiation, cell cycle and during cardiac hypertrophy [4], [7]. In particular miR-133 and miR-1 are specifically expressed in cardiac and skeletal muscle and play important roles in muscle cell proliferation and differentiation [8]. However, in rats, hypertrophic growth of the left ventricle defined as weight gain relative to body weight ceases within 4 weeks of aortic banding [16] and miRNA expression in the heart has mainly been investigated after this time. It was therefore of interest to investigate whether the miRNAs expressed in late stage hypertrophy and heart failure are also involved in early hypertrophic growth.
Section snippets
Aortic Banding
Male Wistar rats weighing 70–90 g from Møllegaard Breeding Center (Lille Skensved, Denmark) were kept at Rigshospitalet with a 12:12-h light–dark cycle, and fed standard rat chow. During Hypnorm/Dormicum (0.2 ml/100 g sc; Boehringer Ingelheim, Ingelheim, Germany) and 1–1.5% isoflurane (Abbott Laboratories, Abbott Park, IL) anesthesia the ascending aorta was exposed by left-side thoracotomy, and a titanium clip with an inner diameter of 0.6 mm was placed 5 mm above the aortic valves with a
Physiological results
In animals with aortic banding, the left ventricle weight (LVW) relative to body weight (BW) increased 65% compared to the sham group after 14 days (P < 0.001) whereas there was no difference (P = 0.325) in the body weight of the two groups (Fig. 1, Table 1). The result indicates that there was hypertrophic growth of the left ventricle in the animals with aortic banding.
Expression of hypertrophic markers
The growth of the left ventricle was accompanied by upregulation of mRNA for the hypertrophic markers atrial natriuretic peptide
Discussion
In the present study, we used a rat model for pressure overload-induced hypertrophy that was previously established in our lab [10], [12], [16]. In agreement with our previous reports, the weight of the left ventricle of animals with aortic constriction was significantly higher than in sham-operated animals. This is in accordance with that the animals with aortic constriction develop pressure overload-induced left ventricle hypertrophy [16]. Furthermore, expression analysis showed that the mRNA
Role of the funding sources
None of the supporting sources had any role in study design; in the collection, analysis, and interpretation of data; in the writing of the report and in the decision to submit the paper for publication.
Acknowledgments
We thank Pernille Gundelach and Minna Jakobsen for excellent technical help, Tue E.H. Christoffersen for discussions on animal experiments and Frederikke Egerod for storage of samples.
This work was supported by grants from the Danish Heart Association, the Danish Medical Research Council, the Birthe and John Meyer Foundation, Villadsen Family Foundation, Kong Christian X’s Foundation, the Illum Foundation and Fru Asta Florida Bolding Foundation.
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