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1Department of Pathophysiology, PLA General Hospital, and 2Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
Submitted 15 March 2005 ; accepted in final form 4 August 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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RNA interference; left ventricle; cardiac hypertrophy; cardiac remodeling
Recently, we identified a novel homologous gene, myofibrillogenesis regulator-1 (MR-1), from a human skeletal muscle cDNA library (10). The MR-1 gene (GenBank accession no. AF417001) is located on human chromosome 2q35 (GenBank accession no. AC021016) and spans 
2,887 bp of contiguous DNA. The MR-1 gene is composed of three distinct exons between bp 169 and 713. The 5'- and 3'-acceptor splice sites in each of the introns follow the GT-AG consensus sequence for eukaryotic genes. Exon 1 (212 bp) encodes the whole 5'-untranslated region plus the first 22 amino acids. Exon 2 (169 bp) encodes the next 56 amino acids, whereas exon 3 (713 bp) contains the final 64 codons and an extensive 3'-untranslated region of 182 bp. This gene encodes a 142-amino acid protein with a hydrophobic transmembrane structure between amino acids 75 and 92. Bioinformatic analysis revealed that MR-1 has no homologous gene in the known human gene database. A computer search of an expressed sequence tag database with the MR-1 amino acid sequence identified MR-1 orthologs in mammals such as mice, rats, cows, and pigs, but no detectable homologs were present in invertebrates such as fugu, zebrafish, Drosophila, Caenorhabditis elegans, zea, yeast, fungi, Plasmodium falciparum, and all 278 known microbial genomes. These findings support the hypothesis that the MR-1 gene exists only in mammals. Further immunohistochemical staining revealed that the MR-1 protein is located mainly on the cytoplasm in fibroblast cells, as well as on the nucleic membrane in the human HeLa cell (11, 12). Northern blot and serial analysis of gene expression revealed that the transcription level of MR-1 in human tissues is especially high in skeletal muscle and myocardium (10).
Muscle consists of myocytes full of strands called myofibrils, which are in turn made up of contractile units called sarcomeres. Sarcomeres mainly consist of two kinds of filamentary protein: actin and myosin. These protein molecules slide over one another telescopically as the sarcomere contracts and relaxes. Yeast two-hybrid screening and in vitro GST pulldown assay revealed that MR-1 interacts with three proteins involved in muscle contraction (myosin regulatory light chain, myomesin, and 
-enolase) and several cell signal transduction-related proteins (10). The interaction of MR-1 with sarcomeric structural proteins involved in muscle contraction and its presence in human myocardial myofibrils indicate that MR-1 is involved in the regulation of contractile proteins in the myocardium and might be associated with cardiac hypertrophy. To test this hypothesis, MR-1 expression in hypertrophied myocardium and cardiomyocytes was compared with that in sham-operated (SO) myocardium and control cardiomyocytes of rats. Also the effects of rat MR-1 (rMR-1) RNA interference (RNAi) on angiotensin II (ANG II)-induced hypertrophy were studied in cultured cardiomyocytes from neonatal rats.
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METHODS |
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Male Sprague-Dawley rats (90–100 g body wt) were housed four per cage at 23°C with a 12:12-h light-dark cycle, fed Purina rat ration, and allowed free access to water. All studies were approved by the Council for Animal Research, Health Center, Peking University. All animals were obtained from the Experimental Animal Center at PLA General Hospital.
Rats underwent abdominal aortic stenosis (AAS, n = 12) or SO (n = 12) as described by Woodiwiss et al. (20). Tail systolic blood pressure (SBP) was measured 3, 7, 14, 21, and 28 days after AAS with a tail-cuff blood pressure analyzer (Manoreter-Tachometer KN-210). At 28 days after AAS, mean arterial pressure (MAP) and heart rate (HR) were measured for 3 consecutive minutes in anesthetized rats. For measurement of these parameters, the right carotid artery was cannulated and connected to a pressure transducer in-line with a Grass polygraph.
At 28 days after AAS, anesthetized rats were weighed, and plasma angiotensin peptide concentration was assessed by the method of Cassis et al. (2). Then the rats underwent median thoracotomy for removal of the heart, and the LV was dissected and weighed for detection of LV hypertrophy assessed by the ratio of LV wet weight to whole heart weight (LV/HW) or LV wet weight to body weight (LV/BW). A longitudinal slice of the anterior LV wall was cut and immersed in neutral and tamponade formalin (10%). The tissue was washed, dehydrated, and embedded in paraffin. Serial sections (6 µm) were incubated at 4°C overnight with a 1:500 dilution of rabbit anti-rMR-1 polyclonal antibody and then incubated for 1 h at 37°C with a 1:500 dilution of goat anti-rabbit immunoglobulin (10). Antibody-biotin conjugate was detected with a 3,3'-diaminobenzidine kit according to the manufacturer's instructions. The immunohistochemically stained slides were viewed with a light microscope, and digital gray-scale images were acquired with a charge coupled device camera with constant settings. Images were collected and analyzed with Microcomputer Imaging Device software (Imaging Research). Gray-scale values within the regions of interest were plotted as histograms, and minimum, maximum, and mean pixel intensity values were calculated with conventional software. Data are expressed as intensity units compared with values for which IgG was used as the primary antibody and as a control for comparison.
In Vitro Studies
For each experiment, primary cultures of cardiomyocytes were prepared from three litters of neonatal Sprague-Dawley rats and pooled as described previously (14). After 24 h of culture, cells were transferred to maintenance medium (DMEM containing 1% penicillin-streptomycin without serum) for 24 h before the experiment. Lipofectamine 2000 (Life Technologies, Carlsbad, CA) was used for transfection of RNAi plasmids for targeting endogenous genes.
Plasmid Construction
With the use of bioinformatics analysis, two pairs of primers were designed and synthesized according to human MR-1 homologous sequences on rat genome chromosome 9. GenBank accession no. NW_047816.1 Olig1 and Olig2 (5'-ATGGCGGCGGTGGTAGCTGCT-3' and 5'-TCAGGTCTGTACTCCAGACCCAAC-3', respectively) were used for the first RT-PCR; Olig3 and Olig4 [5'-ATTAGAATTCATGGCGGCGGTGGTAGCTGCT-3' (Olig3), with the EcoR I site underlined, and 5'-ATATAAGCTTTCAGGTCTGTACTCCAGACCCAAC-3' (Olig4), with the Hind III site underlined] were used for the second PCR. A 0.65-kb RT-PCR fragment was amplified from the total RNA of cardiomyocytes. A total of 0.1 µl of this product was used as a template for the second PCR. The RT-PCR was carried out in a volume of 25 µl containing 2.4 mM MgSO4, incubated for 30 min at 50°C, and then subjected to 40 cycles of 15 s at 94°C, 30 s at 55°C, and 1 min at 72°C on a thermal cycler (model PTC-200, MJ Research, Waltham, MA). The PCR was conducted at 94°C for 4 min followed by 30 cycles at 94°C for 30 s, 55°C for 50 s, and 72°C for 1 min, with a final extension at 72°C for 5 min. Plasmids were constructed using standard techniques (6). Each constructed plasmid was sequenced for verification of accuracy. The prediction of rMR-1 mRNA secondary structures from the sequence we cloned was based on the principle of minimizing free energy using the RNA structure 3.71 program. Figure 1 shows the structure with the minimum thermodynamic free energy calculated: –176.5 kcal/mol. The red regions represent the three double-stranded RNA (dsRNA) delivering plasmids pSi-1, pSi-2, and pSi-3 targets (Fig. 1). With the use of these recombinant plasmids, three RNAi were constructed from the pSilencer 3.0-H1 (Ambion, Austin, TX), each targeting different regions of rMR-1 (Table 1).
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Total RNA was extracted using Trizol reagent. After transfection of the cultured cardiomyocytes with the RNAi plasmids for 24 h, a semiquantitative RT-PCR was performed for detection of trigger-silencing efficiency. This method enables quantitation on the basis of the comparison of the target gene with the -actin housekeeping gene, a commonly used internal standard in hypertrophy study (4). Two pairs of competitive primers were synthesized: P1 (5'-TCCTCCGGGGGATTCTCGCAG-3') and P2 (5'-CGATGGCCCAGGCCAGTCCCACA-3'), corresponding to 203- to 388-nt positions of the rMR-1 open reading frame (ORF) sequence, respectively. P3 (5'-GCCCTAGGCACCAGGGTGT-3') and P4 (5'-GCCACAGGATTCCATACCC-3') corresponded to nt 110–819 of rat -actin (GenBank accession no. NM_031144). The RT-PCR is the same as that described above. Amplification yielded a 186-bp rMR-1 fragment and a 710-bp -actin fragment. The products were analyzed on 1.5% agarose gel stained with ethidium bromide. The gels were scanned by an Alpha 5500 scanner, and the band intensities were evaluated using AlphaEase FC3.1.2 software. The relative level of rMR-1 mRNA was obtained from the ratio of its individual band intensity to internal standard band (i.e., -actin) intensity.
Incorporation of [3H]Leu
Neonatal cardiomyocytes were plated on 24-well culture plates at a density of 1 x 106 cells per well with maintenance medium for 24 h. Cells were randomly distributed into the following groups: 1) control group, i.e., untransfected cardiomyocytes incubated in a cell incubator for 50.5 h; 2) ANG II group, i.e., untransfected cardiomyocytes cultured for 36 h and then incubated for 24 h with ANG II (10–7 mol/l); 3) losartan + ANG II group, i.e., untransfected cardiomyocytes cultured for 35.5 h and preincubated with losartan (10–5 mol/l) for 30 min before 24 h of ANG II treatment; 4) MR-1 RNAi + ANG II group, i.e., pSi-1-transfected cardiomyocytes cultured for 36 h before 24 h of ANG II treatment; 5) MR-1 RNAi group, i.e., pSi-1-transfected cardiomyocytes cultured for 50 h; 6) pSilencer 3.0-H1 transfection group, i.e., cardiomyocytes transfected with pSilencer 3.0-H1 and cultured for 50 h; and 7) pSilencer 3.0-H1 transfection + ANG II group, i.e., cardiomyocytes transfected with pSilencer 3.0-H1 and cultured for 36 h before 24 h of ANG II treatment. Then the cardiomyocytes were incubated in DMEM containing 12.5 µCi/ml [3H]Leu for 24 h, and [3H]Leu uptake was determined according to the methods described by Liu et al. (13).
Calculation of Cardiomyocyte Size
Neonatal cardiomyocytes were plated on 24-well culture plates at a density of 1 x 104 cells per well with maintenance medium for 24 h. Serum-starved cardiomyocytes were randomly distributed into the seven groups described above, but the ANG II incubation was 48 h, instead of 24 h as in the incorporation experiment. The surface area of cardiomyocytes was assessed by the method of Kodama et al. (9).
Preparation of Whole Cell Extracts From Cardiomyocytes and Western Blotting Analysis
The proteins were extracted from cardiomyocytes or myocardium as previously described (15). Protein concentration in the detergent-soluble supernatant was determined with a bicinchoninic acid protein assay kit (Pierce, Rockford, IL) according to the manufacturer's protocol. The proteins in the supernatant (50 µg) were separated by SDS-PAGE (10% separate gel) and transferred to a polyvinylidene difluoride filter membrane blocked with 10% nonfat dry milk in Tris-buffered saline + Tween 20 (TBST) containing 20 mmol/l Tris·HCl (pH 7.6), 137 mmol/l NaCl, and 0.1% Tween 20. The blots were incubated at 4°C overnight with rabbit anti-rMR-1 polyclonal antibody at 1:1,000 dilution in TBST and then for 1 h at 37°C with a 1:500 dilution of goat anti-rabbit IgG conjugated to horseradish peroxidase in TBST. Antibody-antigen complexes were visualized by chemiluminescence. Membranes were exposed to X-ray film for 0.5–2 min. The staining was quantified by scanning the films, and the band density was determined with Image-Pro software.
Chemicals
DNA restriction endonucleases, T4 ligase, and the LA-Taq PCR amplification kit were purchased from TakaRa (Otsu, Shiga, Japan), the SV total RNA isolation kit from Promega (Madison, WI), Lipofectamine 2000, Trizol reagent, and the Platinum Quantitative RT-PCR ThermoScript One-Step System from Invitrogen (Carlsbad, CA), and fetal bovine serum from HyClone (Logan, UT). Rabbit anti-rat MR-1 (rMR-1) polyclonal antibody was generated in our laboratory (11). Other monoclonal antibodies, enhanced chemiluminescence immunodetection kits, and 3,3'-diaminobenzidine kits were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Prestained SDS-PAGE protein standards (broad range) were purchased from Bio-Rad Laboratories (Hercules, CA) and L-[3,4,5-3H]Leu from Amersham (Buckinghamshire, UK). Other chemicals were purchased from Sigma (St. Louis, MO), unless otherwise noted.
Statistical Analysis
Values are means ± SD from at least three independent experiments. Each treatment in each experiment was performed in triplicate. In each experiment, cardiomyocytes were pooled from three different litters, and data from the three experiments were pooled (n = 3). Differences in means between groups were tested by one-way ANOVA. P < 0.05 was considered statistically significant.
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RESULTS |
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Magnitude of pressure overload. The time course for alterations in tail SBP and HR after 3, 7, 14, 21, and 28 days of pressure overload is presented in Table 2. AAS resulted in a significant increase in tail SBP (P < 0.01) and HR (P < 0.01). Tail SBP increased by 16%, 19%, 29%, 39%, and 40% and HR by 19%, 37%, 49%, 69%, and 107% in AAS-induced rats at 3, 7, 14, 21, and 28 days, respectively, compared with SO rats.
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Protein expression of rMR-1 in myocardium. Although only faint immunoreactivity of rMR-1 was detected in myocardium of SO rats, strong signals were seen in the AAS rats (Fig. 2). Quantitative image analysis indicated a 110% increase in the area of rMR-1 staining in AAS-induced hypertrophied myocardium (P < 0.05 vs. SO rats).
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Figure 4 shows mRNA expression of rMR-1 24 h after transfection of cardiomyocytes with plasmids targeting different regions of rMR-1. pSi-1, targeting the rMR-1 ORF 91–111 sequence, gave the best target gene-silencing effect and was chosen for the following experiments.
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Surface area of cells. Measurement of the surface area of cardiomyocytes revealed that ANG II caused a 66% increase in cell size compared with controls (2,645.8 ± 129.3 vs. 1,589.3 ± 135.2 µm2, P < 0.01). The ANG II-induced increase in surface area of cells was prevented by losartan, an angiotensin (AT1) receptor inhibitor (1,628.4 ± 173.2 µm2 in losartan + ANG II, P < 0.01 vs. ANG II group) and transfection with the plasmid pSi-1. The surface area of cells in the rMR-1 RNAi + ANG II group was decreased by 36% compared with the ANG II group (1,701.6 ± 145.6 µm2 in MR-1 RNAi + ANG II, P < 0.01 vs. ANG II group). Transfection with an empty vector did not influence ANG II-induced hypertrophy (Fig. 5).
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Protein expression of rMR-1 in cardiomyocytes. Alteration of protein expression of rMR-1 in cardiomyocytes was detected by Western blotting analysis. Blotting of whole cell extracts from ANG II-induced hypertrophied cardiomyocytes resulted in a 66% increase of rMR-1 expression compared with that in whole cell extracts from controls (Fig. 6). The upregulation of rMR-1 expression was abolished in losartan-pretreated and pSi-1-transfected cardiomyocytes. Transfection with a vector had no effect on rMR-1 protein expression in control and ANG II-stimulated cardiomyocytes (Fig. 6).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Several experimental models have been proposed for the study of LV hypertrophy due to pressure overload. One of these is induction of AAS in rats, which leads to systemic hypertension, LV hypertrophy, and activation of the renin-angiotensin-aldosterone system (1, 3). Using a model of AAS-induced cardiac hypertrophy, we assessed cardiac function, blood pressure, and LV hypertrophy in adult Sprague-Dawley rats. In this study, pressure overload induced by suprarenal AAS increased MAP proximal to the site of aortic constriction. The magnitude of the pressure increase was sufficient to produce sustained LV hypertrophy, demonstrated as increased LV/HW and LV/BW, within 28 days. Our results also demonstrate that the systemic renin-angiotensin system is activated in response to pressure overload and coincides with the appearance of LV hypertrophy in AAS rats. The results agree with findings from previous studies examining the time course for cardiac hypertrophy and function in response to pressure overload (20).
The mechanisms governing the development of cardiac hypertrophy have been extensively studied; however, they are incompletely understood. Investigation of alterations in myocardial contractile protein-related genes during hypertrophy helps in our understanding of the mechanisms and provides a new pathway for treatment of hypertrophy and heart failure. The MR-1 gene encodes a protein that shows interaction with three proteins involved in muscle contraction and several cell signal transduction-related proteins (10). However, the role of MR-1 in cardiac hypertrophy remains unknown. In the present study, imunohistochemistry and Western blot analyses demonstrated that MR-1 protein expression is upregulated in myocardium subjected to AAS-induced cardiac hypertrophy in rats. To our knowledge, this is the first direct evidence that MR-1 is involved in the hypertrophic response of the heart.
Cardiomyoyctes are terminally differentiated cells (17). In response to various extracellular stimuli, cardiomyoyctes grow in a hypertrophic manner, characterized by enlarged individual cell size and increased content of contractile proteins (18). ANG II is a major determinant of arterial pressure and volume homeostasis. Moreover, ANG II has been postulated to be the humoral mediator of mechanical stretch-induced cardiac hypertrophy. In this study, we showed that ANG II induced an increase in [3H]Leu uptake and cell area in cardiomyocytes. The results also confirmed that ANG II augmented MR-1 protein synthesis in cardiomyocytes, which coincides with results of our in vivo experiments. Pretreatment with losartan, an AT1 receptor inhibitor, prevented the hypertrophic events and the increased MR-1 protein secretion, which indicated that the upregulation of MR-1 protein expression is associated with ANG II-induced cardiac hypertrophy.
RNAi is a process of sequence-specific gene silencing in animals and plants; it is initiated by dsRNA that is homologous in sequence to the targeted genes (7). The specific messenger RNA degradation is mediated by 21- or 22-nt small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs (5). In the present study, RNAi was used to further study the relation between MR-1 expression and cardiac hypertrophy. After transfection of neonatal cardiomyocytes with a pSi-1 targeting the rMR-1 ORF 91–111 sequence for 24 h, quantitative RT-PCR results showed optimal trigger-silencing efficiency by downregulation of the rMR-1 mRNA and protein expression. Furthermore, results of [3H]Leu incorporation and cell size measurement in cardiomyocytes clearly demonstrated that the silencing of rMR-1 prevents cardiomyocyte hypertrophy induced by ANG II. The effect of RNAi is similar to that of AT1 receptor inhibitor treatment. Our observations indicate that MR-1 is required for hypertrophy of cardiomyocytes. Additional studies are required to elucidate the interconnectivity between MR-1 and other intracellular signaling pathways to gain a greater understanding of the coordinate regulation of cardiac hypertrophy.
In summary, our results demonstrate for the first time, we believe, the functional role and direct effect of MR-1 in cardiac hypertrophy. The findings may provide a better understanding of the mechanism(s) of cardiac remodeling and a new insight into the development of novel therapeutic strategies in cardiac hypertrophy.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* Both authors contributed equally to this work. 
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REFERENCES |
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induced by p42/p44 mitogen-activated protein kinases is involved. Pathophysiology 9: 201–205, 2003.[CrossRef][Medline]1 mediates the hypertrophic cardiomyocyte growth induced by angiotensin II. J Clin Invest 109: 787–796, 2002.[CrossRef][Web of Science][Medline]This article has been cited by other articles:
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  H.-L. Li, Z.-G. She, T.-B. Li, A.-B. Wang, Q. Yang, Y.-S. Wei, Y.-G. Wang, and D.-P. Liu Overexpression of Myofibrillogenesis Regulator-1 Aggravates Cardiac Hypertrophy Induced by Angiotensin II in Mice Hypertension, June 1, 2007; 49(6): 1399 - 1408. [Abstract] [Full Text] [PDF]
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