A new unique form of microRNA from human heart, microRNA-499c, promotes myofibril formation and rescues cardiac development in mutant axolotl embryos
© Kochegarov et al.; licensee BioMed Central Ltd. 2013
Received: 3 December 2012
Accepted: 18 March 2013
Published: 23 March 2013
A recessive mutation “c” in the Mexican axolotl, Ambystoma mexicanum, results in the failure of normal heart development. In homozygous recessive embryos, the hearts do not have organized myofibrils and fail to beat. In our previous studies, we identified a noncoding Myofibril-Inducing RNA (MIR) from axolotls which promotes myofibril formation and rescues heart development.
We randomly cloned RNAs from fetal human heart. RNA from clone #291 promoted myofibril formation and induced heart development of mutant axolotls in organ culture. This RNA induced expression of cardiac markers in mutant hearts: tropomyosin, troponin and α-syntrophin. This cloned RNA matches in partial sequence alignment to human microRNA-499a and b, although it differs in length. We have concluded that this cloned RNA is unique in its length, but is still related to the microRNA-499 family. We have named this unique RNA, microRNA-499c. Thus, we will refer to this RNA derived from clone #291 as microRNA-499c throughout the rest of the paper.
This new form, microRNA-499c, plays an important role in cardiac development.
The Mexican axolotl, Ambystoma mexicanum, is an exciting and useful animal model to study vertebrate heart development and cardiac myofibrillogenesis. It carries a lethal cardiac recessive mutation, designated by gene “c”, which, when homozygous (c/c), prevents normal heart development in axolotl embryos. Our previous studies  have shown that a non-coding RNA, Myofibril-Inducing RNA (MIR) from normal axolotl, is capable of promoting myofibrillogenesis and beating hearts in the mutant (c/c) axolotl embryos. This study demonstrated that the MIR gene is essential for tropomyosin expression in axolotl hearts during development. Real-Time PCR studies showed that mRNA expression of various tropomyosin isoforms in untreated mutant hearts is similar to normal hearts knocked down with double-stranded MIR (dsMIR). These results suggest that MIR is involved in controlling expression of various tropomyosin isoforms and subsequently in regulating cardiac contractility. The MIR was sequenced and found to be 166 nucleotides in length . This RNA is unique in that it does not show significant homology to any known sequences in the online NCBI database. Comparison between MIR sequences obtained from normal and mutant embryos demonstrated a single point mutation at base 93 of the mutant MIR nucleotide sequence . Genebee (Moscow State University), an online bioinformatics tool for the computation and modeling of secondary structures of RNAs, showed a conformational difference between the normal bioactive MIR and the mutant MIR, suggesting that the secondary structure of MIR might be important in the mutant rescue process . More recently we have found that total human fetal heart RNA also has the ability to promote normal myofibril formation and restore function of the mutant axolotl hearts, suggesting that a functional homologue of the axolotl MIR could be present in human fetal heart tissue . In the present study, through the random cloning of genes expressed in human heart, we have found a clone with the capacity to produce RNAs with the capability of promoting myofibrillogenesis and rescuing cardiac mutant axolotl hearts similarly to the axolotl MIR. We have designated this clone as a microRNA-499c. The role of microRNA-499 in heart development in human cardiomyocyte differentiation was previously described [4, 5]. Our identification of the human microRNA which promotes myofibrillogenesis helps us to understand the molecular mechanism of heart development and may have important implications for future treatment of myocardial infarcts, cardiomyopathies and other congenital or acquired myocardial diseases in humans.
For cloning, a total of 2 μg of human fetal heart RNA (Agilent Technologies, Inc #540165) was used for each reaction. The cloning kit used was the CloneMiner™ II cDNA Library Construction Kit (Invitrogen, #A11180). First and second DNA strands were synthesized from template RNAs and ligated into the pDONR222 vector. The pDONR222 vector contains the kanamycin resistance gene which allows selection of transfected bacteria and the ccdB gene which interferes with E. coli DNA gyrase allowing negative selection of the donor vector in E. coli following recombination and transformation. The ElectroMAX™ DH10B™ T1 Phage Resistant E. coli strain provided with the kit was transformed using the EC 1000 Electroporator (Thermo ES) at 2800 V. To each sterile cuvette, 50 μl ElectroMAX™ DH10B cells, 1.5 μl of (150 ng/μl) vector and 50 μl of dH2O were added. In case the sample arced at this voltage setting, 100 μl of dH2O, or more, was added to increase electrical resistance. After electroporation, the cells were added to 1 ml of S.O.C. medium and cultured in 15 ml snap-cap tubes for at least 1 hour at 37°C on a shaker at 225–250 rpm to allow expression of the kanamycin resistance marker. Serial dilutions of sample aliquots with S.O.C. medium at the ratios 1:10, 1:100 and 1:1000 were plated on LB agar plates containing 50ug/ml of kanamycin. The remaining cells were frozen at -80°C. Plated cells were incubated overnight at 37°C. Individual colonies were collected and transferred into snap-cap tubes with 2 ml of 2xYT medium containing 50ug/ml of kanamycin and incubated overnight. Plasmids with clones were extracted according to the standard Miniprep Plasmid DNA Isolation Protocol found in the online archive of the Institute of Bioinformatics and Applied Biotechnology.
Extracted plasmids (5 μl sample) were digested by 20U (1U/μl) of enzyme BsrGI in 1X NE Buffer with 0.1 mg/μl of BSA. The mixtures were incubated for 1 h at 37°C and analyzed by Gel electrophoresis on 1% agarose gels containing 0.5 μg/ml of ethidium bromide.
T7 RNA polymerase binding site TAATACGACTCACTATAGGG was added to the 5′ end of forward and reverse M13 primers.
Forward primer: 5′-TAATACGACTCACTATAGGGGTAAAACGACGGCCAG-3′.
Reverse primer 5′-TAATACGACTCACTATAGGGCAGGAAACAGCTATGAC-3′.
PCR was performed using a MyTaq™ Red Mix kit (Bioline, BIO-25043) according to the instruction manual for this kit: denaturation at 95°C during 15 sec followed by annealing at 55°C for 15 sec and elongation at 72°C for 15 sec for 30 cycles. The resulting DNA was purified by 5 M sodium chloride salt and isopropanol precipitation. Pellets were washed with 70% ethanol and re-suspended in 1X Tris-EDTA buffer.
The transcription reaction mixture was assembled from the MAXIscript® T7 Kit, Ambion # AM1314M. Then, we added 1 μg of DNA from the PCR product, 2 μL of 10X transcription buffer, 2 μL of T7 Enzyme Mix and 1 μL of each (10 mM) NTP; and adjusted the volume to 20 μL by nuclease-free water. The reaction mixture was incubated at 37°C for 2 hours. RNA was purified using ammonium acetate and ethanol precipitation and resuspended in nuclease-free water. The concentration of RNA was determined spectrophotometrycally at 260 nm.using a Synergy HT (Bio-Tek) platereader.
Cardiac mutant non-function carrier (+/c) adult axolotls were obtained from the Ambystoma Genetic Stock Center, University of Kentucky, Lexington. These heterozygous adult animals were mated (+/c x +/c) to produce mutant (c/c) and wildtype (+/+) embryos for our studies. Embryos were collected and allowed to develop to heart-beat stages 35-36, according to the Bordzilovskaya et al. staging system . For bioassays, only double recessive mutant c/c embryos were selected which do not have beating hearts. The embryos were anaesthetized by 0.7 mg/ml tricaine methanesulfonate or Ms-222 (Argeitt Chemicals Labs) in Holtfreter’s solution . Embryos were dissected under a binocular microscope in clay-lined Petri dishes in Holtfreter’s medium containing 1% antibiotic/antimycotic (Gibco #15240). Hearts were transferred into the Petri dishes on Parafilm substrate into 50 μl of Holtfreter’s solution (without antibiotic) containing 7 ng/μl of human fetal heart RNA from individual clones along with 0.1 mg/ml of lipofectamine. reagent (Invitrogen, Carlsbad CA). The Petri dishes with hearts were enclosed in a plastic container containing wet paper towels to maintain a saturated humidity environment at 17°C.
Fixation and staining procedure
All steps were performed at room temperature as previously described . Hearts were fixed in 4% paraformaldehyde for 30 min and rinsed twice in PBS for 3 min. Hearts were permeabilized in 0.05% Tween-20 and 3% BSA in PBS for 1 h. Hearts were incubated overnight with monoclonal anti-tropomyosin CG3 antibody (Developmental Studies Hybridoma Bank, University of Iowa) diluted to 1:75 in PBS, and then washed several times in PBS. Hearts were incubated in goat anti-mouse polyclonal secondary antibody (Abcam, # ab6669) at a 1:75 dilution for 1 h. The hearts were rinsed in several changes of PBS and mounted on slides in SlowFade® Gold antifade reagent (Invitrogen, #S36936). Three layers of fingernail polish were applied to the edges of glass coverslips to prevent damaging of the whole hearts. Antibodies conjugated with FITC were excited at 488 nm with an emission at 520 nm. The stained heart samples were scanned under a laser confocal microscope, Olympus Fluoview, equipped with a computer to record the images.
Primers used for genes in real time RT-PCR experiments
Gene of interest
Results and discussion
On the basis of our results we hypothesize that normal human fetal heart expresses an RNA, which is a functional homologue to the axolotl MIR, and which probably is required for human heart development and function. Our results have clearly shown that if we clone this RNA from human fetal heart and transfect it into mutant axolotl hearts, normal heart development is restored. In an earlier publication, our laboratory showed that RNA extracted from human fetal and adult hearts, but not from skeletal muscle, rescued the development of mutant axolotl hearts in organ culture . These earlier experiments suggest that total human heart RNAs, but not skeletal muscle, contain functional homologues of the MIR (myofibril-inducing RNA) derived from normal embryonic axolotl anterior endoderm .
Our results show that RNA cloned from human fetal heart has the capability of rescuing mutant axolotl hearts in organ culture bioassays. The rescue of mutant hearts was demonstrated by the development of beating in the hearts and expression of tropomyosin in organized myofibrils after incubation with the microRNA-499c. All three microRNA-499 precursors apparently originate from the same intron, which is initially the intron of the myosin heavy chain (MHC) genes.
The human microRNA-499 belongs to a family of micro RNAs encoded by the intron of the myosin heavy chain (MHC) genes, referred to as MyomiRs which also include microRNA-208a and microRNA-208b. MicroRNA-499 is highly conserved, being present in the genome of many vertebrate species (human, mouse, rat, bovine, Xenopus and zebrafish) . Micro-RNAs are non-coding RNAs which regulate the translation of genes by binding to untranslated sites (UTRs) in their targets. Nascent precursor microRNAs are usually about one hundred or more nucleotides long, and later, microRNAs are split by enzymes into a short active 22 bp fragment.
In animals, expression of microRNAs occurs in two stages. First, in the nucleus, pre-microRNAs are cleaved from an extended primary transcript of the gene which then is exported to the cytoplasm of the cell. In the cell cytoplasm, endoribonuclease, called Dicer, cleaves the double-stranded RNA (dsRNA) into a short 22 nucleotide micro-RNA. The mature microRNA is incorporated into the RNA-induced silencing complex (RISC) which includes Dicer and other proteins. The miRNA binds complementary mRNAs and directs them to degradation by endonucleases or prevent their translation by holding them in the RISC. In situ hybridization analysis of mouse heart, brain, spleen, liver, lung, quadriceps muscle, kidney, and gut tissues shows that the mature microRNA-499 is abundantly expressed in cardiac tissue and almost absent in other tissues, including skeletal muscle . Some of those predicted targets of microRNA-499 and microRNA-1 were shown to regulate cardiomyocyte differentiation. Transient transfection of microRNA-1 and -499 in human cardiomyocyte progenitor cells (CMPCs) reduced the proliferation rate and increased differentiation into cardiomyocytes. This effect most likely occurs by repression of histone deacetylase 4 or Sox6 because levels of these proteins are reduced . It has been found that microRNA-499 promotes ventricular specification in differentiating human embryonic stem cells . It was shown further that microRNA-499 expression increased in human stem cells differentiating into cardiomyocytes. Also, microRNA-499 transduction by the lentivirus of hESC-derived cardiovascular progenitors significantly increased the yield of stem cell-derived ventricular specified cardiomyocytes . Other results suggest that expression of microRNA-499 in human cardiac stem cells (hCSCs) represses the microRNA-499 target genes Sox6 and Rod1, enhancing cardiomyogenesis in vitro and after infarction in vivo. The level of microRNA-499 was 400 times higher in cardiomyocytes than in rat cortical stem cells. Cardiomyocytes derived from differentiation of hCSCs treated with microRNA-499 appeared to be larger, and their sarcomere striations were more evident . Expression of microRNA-499 was observed to be increased a few hundred times during differentiation of human embryonic stem cells into beating clusters . Also, overexpression of microRNA-499 enhanced expression of myocyte-specific enhancer factor 2C, the transcription factor which is involved in cardiac morphogenesis and myogenesis and vascular development . Thus, microRNA-499 appears to play an important role during heart development, although the molecular mechanisms and the microRNA-499 pathways are not well understood at this time.
Our results demonstrate clearly and unequivocally that human-derived microRNA-499c promotes the formation of cardiac myofibrils in cells of cardiac mutant salamander hearts and thus restores normal embryonic heart development in these lower vertebrate species. This observation is consistent with earlier publications showing that microRNA-499 plays an important role in cardiac differentiation and cardiogenesis during embryonic development, and it strongly suggests a ubiquitous and conserved mechanism of classic embryonic heart induction [12, 13] and myofibrillogenesis across the spectrum of vertebrate species.
This work is supported by an NIH grant (HL061246) and an American Heart Association grant (10GRNT4530001) to LFL and an NSF-RUI Award (1121151) to MH.
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