The muscle-specific microRNAs miR-1 and miR-133 produce opposing effects on apoptosis by targeting HSP60, HSP70 and caspase-9 in cardiomyocytes

MicroRNAs (miRNAs) are endogenous ∼22 nucleotide (nt) non-coding RNAs that anneal to inexactly complementary sequences in the 3′UTRs of target mRNAs of protein-coding genes to specify translational repression or/and mRNA cleavage (Ambros, 2004; Jackson and Standart, 2007; Meister and Tuschl, 2004; Lim et al., 2005). Having generated a tremendous amount of excitement about miRNAs in many areas of biology, research over the past five years has put miRNAs at centre stage. However, in spite of our ability to identify miRNAs, regulatory targets have not been established or even confidently predicted for any of the vertebrate miRNAs, which has hampered progress toward elucidating the functions of miRNAs. Our current understanding of the functions of miRNAs primarily relies on their tissue-specific or developmental stage-specific expression patterns as well as their evolutionary conservation and thus is limited to developmental regulation and oncogenesis (Ambros, 2004; Hwang and Mendell, 2006; Hammond, 2006), and much less is known about their role in other biological processes.

Among the known miRNAs, miR-1 and miR-133 are believed to be specifically expressed in adult cardiac and skeletal muscle tissues (Zhao et al., 2005; Chen et al., 2006; Rao et al., 2006; Kwon et al., 2005). Both of them have been found in most animal species, from Drosophila to human, indicating that they are evolutionally conserved. Increasing expression was found in neonatal hearts and skeletal muscle, and substantially higher levels are maintained in adult muscular tissues. Authors of a recent study (Chen et al., 2006) have proposed a model in which miR-1 and miR-133 regulate myogenesis by controlling distinct aspects of the differentiation process; miR-1 promotes myogenic differentiation and miR-133 enhances myoblast proliferation. One of the questions we asked is whether miR-1 and miR-133 are involved in apoptotic cell death under pathological conditions relevant to human cardiac disease. This study was designed to shed light on this issue.

H9c2 cells (rat embryonic ventricular cell line) were divided into five groups: control, wild-type (WT) miR-1, mutant (MT) miR-1, WT miR-133 and MT miR-133 groups, and they were all subjected to transfection procedures. Apoptotic cell death was verified by TUNEL of chromosomal cleavage (Fig. 1A) and enzyme-linked immunosorbent assay (ELISA) quantification of DNA fragmentation (by measuring optical density). Overexpression of miR-1 induced significant apoptotic cell death; DNA fragmentation increased by ∼15 fold (Fig. 1B). By comparison, overexpression of miR-133 did not cause H9c2 cell death. The apoptosis-inducing effect was not seen in cells transfected with MT miR-1. Noticeably, co-transfection of miR-1 and miR-133 largely prevented the apoptosis caused by miR-1 alone, indicating an anti-apoptotic effect of miR-133.

Incubation of H9c2 cells with H₂O₂ (ranging from 0 to 600 μM) produced concentration-dependent cell death, as determined by ELISA. The mean IC₅₀ value for DNA fragmentation induction by H₂O₂ was 65.5±5.2 μM (n=5 batches of cells) under our experimental conditions. Furthermore, overexpression of WT miR-1 promoted H₂O₂-induced apoptosis, as indicated by ∼60% reduction of IC₅₀ value (Fig. 1C). In sharp contrast, overexpression of WT miR-133 produced a cytoprotective effect against H₂O₂-induced apoptosis; the IC₅₀ was increased by ∼40%. Consistently, overexpression of miR-133 markedly increased cell viability, whereas that of miR-1 did the opposite, as determined by MTT assay (Fig. 1D). These effects were not seen when MT miR-1 or MT miR-133 was transfected. To further verify the opposing effect of miR-1 and miR-133, we performed reciprocal experiments wherein we transfected H9c2 cells with 2′-O-methyl antisense inhibitory oligonucleotides (AMOs) against miR-1 (AMO-1) or miR-133 (AMO-133) (Krutzfeldt et al., 2005; Cheng et al., 2005). AMO-1 reduced H₂O₂-induced apoptosis, whereas AMO-133 facilitated it (Fig. 1C,D). Most strikingly, co-transfection of WT miR-1 and WT miR-133 failed to alter H₂O₂-induced apoptosis, indicating that they counteract each other.

To confirm the above results obtained with the H9c2 cell line, the same experiments were conducted in neonatal rat ventricular myocytes, which are known to be terminally differentiated cardiomyocytes. Quantitatively the same results were obtained with these cells (Fig. 1E,F).

The above results suggest that miR-1 is pro-apoptotic whereas miR-133 is anti-apoptotic and these opposing actions are probably due to regulation of distinct apoptotic and survival factors by these miRNAs. To test this notion, we first used a computational and bioinformatics-based approach to predict the putative targets related to apoptosis versus survival using TargetScan hosted by the Wellcome Trust Sanger Institute (Griffiths-Jones, 2004). In this way, we identified two important candidate targets for miR-1: HSP60 and HSP70 (heat shock proteins). We also identified several targets sites for miR-133 in the caspase-9 (Casp9) gene by complementarity (Fig. 2).

To verify that HSP60, HSP70 and Casp9 are indeed the cognate targets of miR-1 and miR-133, respectively, for post-transcriptional repression, we took the following approaches. We first determined the effects of the two miRNAs on protein levels, by western blotting and caspase activity assays. miR-1 remarkably reduced the levels of HSP60 proteins by ∼73% in H9c2 cells both in the absence and presence of H₂O₂ (Fig. 3A). HSP70 protein level was similarly reduced, albeit to a less extent (∼53%; Fig. 3B). Co-application of miR-1 and AMO-1 almost abolished the effects. By comparison, miR-133 decreased total Casp9 protein level by ∼89% (Fig. 3C), an effect eliminated by co-transfection with AMO-133. When transfected with MT miR-1 and MT miR-133, decreases in HSP60, HSP70 and Casp9 were hardly seen. Since reduction of Casp9 protein level may well result in reduction of Casp9 activation, Casp9 activities were determined. As shown in Fig. 3D, miR-133 diminished basal Casp9 activity and prevented H₂O₂-induced increase in Casp9 activity. These effects of miR-133 were antagonized by AMO-1. Moreover, application of AMOs alone increased the levels of HSP60, HSP70 and Casp9 in H9c2 cells (Fig. 3E), indicating the roles of basal miR-1 and miR-133 in cardiac cells.

We subsequently investigated the effects of the miRNAs on mRNA levels of HSP60, HSP70 and Casp9 in H9c2 cells. The mRNA levels of HSP60, HSP70 were unaffected by miR-1, indicating that miR-1 and miR-133 do not affect their mRNA stability. Casp9 mRNA was reduced by miR-133 (Fig. 3F).

Casp3 genes do not contain any domains bearing putative target sites for miR-1 or miR-133. Thus, miR-1 or miR-133 is not expected to alter Casp3 protein levels. This was indeed verified by our experiments using western blot analysis with the antibody against the total Casp3 protein (Fig. 4A). However, miR-1 and miR-133 may indirectly affect Casp3 activities because these miRNAs repress HSP60, HSP70 and Casp9, which could in turn alter Casp3 activities (but not total protein levels). To test this notion, we determined Casp3 activities in H9c2 cells with or without H₂O₂ treatments, miR-1, miR-133 and their AMOs. Transfection of cells with miR-1 or challenge of cells with H₂O₂ both robustly increased Casp3 activities and the effects were abolished by co-application of AMO-1. By contrast, transfection of cells with miR-133 diminished Casp3 activity, an effect prevented by co-application of AMO-133. Moreover, H₂O₂ enhanced Casp3 activity and co-application with AMO-133 further increased Casp3 activity (Fig. 4B), in line with the notion that miR-133 diminishes Casp3 activation as a result of repression of Casp9.

We placed the 3′UTRs of HSP60 and HSP70, or the full-length cDNA of Casp9 into the 3′UTR of a luciferase reporter plasmid to construct chimeric vectors. Co-transfection of the chimeric constructs with miR-1 or miR-133 (Fig. 5) into HEK293 cells, consistently resulted in smaller luciferase activity relative to transfection of the chimeric plasmid alone. Co-application of miR-1 or miR-133 with their respective AMOs eliminated the silencing effects. A mutated target sequences of HSP60 or HSP70 fused to the 3′UTR of luciferase was not responsive to miR-1 or miR-133, suggesting specificity of the repression effect. Furthermore, MT miR-1 or MT miR-133 had no effect on the WT target sequences, but could efficiently repress luciferase activities with the constructs containing the MT 3′UTRs of HSP60 or HSP70 complementary to MT miR-1.

We then used miR-1 and miR-133 standards in which the complementary sequences of miR-1 and miR-133 were cloned downstream of the luciferase gene in the pMIR-REPORT plasmid (Chen et al., 2006; Krutzfeld et al., 2005). With these constructs, we were able to confirm the uptake and activities of transfected miRNAs. Real-time RT-PCR analyses on the effects of AMO-1 and AMO-133 on miR-1 and miR-133 levels in H9c2 cells, to verify the efficacy and specificities against the exogenous miRNAs, have been reported in our previous study (Yang et al., 2007; Luo et al., 2007).

Successful delivery of miR-1 or miR-133 and AMO-1 or AMO-133 into the cells was further verified by comparing the miR-1 or miR-133 levels before and 48 hours after transfection of the constructs in cultured neonatal ventricular myocytes. As shown in Fig. 6A, transfection resulted in approximately three- to fourfold increases in miR-1 and miR-133 levels. For control purpose, transfection of miR-1 or miR-133 did not significantly alter the level of miR-133 or miR-1. It should be mentioned that the miR-1 and miR-133 levels are dynamic with transfection. Our data were collected at a specific time, 48 hours after transfection (because all our measurements were performed at this time) and the levels do not apply to other times. Coincidently, incubation of cells with H₂O₂ (150 μM) caused an approx. threefold elevation of miR-1 (Fig. 6B). By comparison, H₂O₂ induced only 70% increase in miR-13. The results are consistent with the fact that H₂O₂ induces apoptosis.

The H9c2 rat ventricular cell line and the HEK293 human embryonic kidney cell line were used in our study for different specific objectives. The former was used for experiments involving endogenous miR-1 or miR-133, such as the experiments involving application of AMO-1 alone, and the latter was used for experiments involving only exogenously delivered miR-1 or miR-133 by transfection, such as the luciferase reporter gene experiments. We quantified the levels of miR-1 and miR-133 in these cell lines as well as in the A549 human lung cancer cell line. Our data confirmed that H9c2 expresses endogenous miR-1 and miR-133, which are known to be muscle-specific, whereas the non-muscle cells HEK293 and A549 express only minimal levels (∼1/100-1/1000 of H9c2) of endogenous miR-1 and miR-133 (Fig. 6C).

The study reported by Chen et al. (Chen et al., 2006) revealed that miR-1 and miR-133 are clustered on the same chromosomal loci and transcribed together as a single transcript which becomes two independent, mature miRNAs with distinct biological functions. One important characteristic of miR-1 and miR-133 regulation of cardiomyocyte apoptosis, revealed in this study, is that they produce opposing effects; miR-1 is pro-apoptotic whereas miR-133 is anti-apoptotic. This suggests that the relative levels of miR-1 and miR-133 is more important than their absolute levels, to determining the fate (apoptosis and survival) of cardiac cells. This notion is supported by the fact that co-transfection of miR-1 and miR-133 failed to induce apoptosis or to affect oxidative stress-induced apoptosis. In addition, in the presence of oxidative stress to induce apoptosis, both miR-1 and miR-133 levels were increased relative to those in cells under normal conditions, but the increase in miR-1 predominantly overweighed that in miR-133, favoring the occurrence of apoptosis. However, at this stage the notion is merely a speculation that needs further studies to verify. Moreover, it should also be noted that this study merely provided indirect evidence for the interactions between miR-1, miR-133 and their target genes, and more rigorous experimentation is required to fully establish the relationships.

Mitochondrial death pathway is one of the major mechanisms for apoptosis, which involves selective disruption of the outer membrane as a result of mitochondrial matrix hyperpolarization and/or matrix swelling, pore formation by proteins such as Bax and Bcl-xS, or rapid loss of ΔΨ following permeability transition (Latchman, 2001; Gupta and Knowlton, 2005). HSPs are expressed both constitutively (cognate proteins) and under stressful conditions (inducible forms), with constitutive expression being most prominent in mammalian tissues. HSPs are primarily anti-apoptotic and different HSPs have been shown to inhibit the mitochondrial death pathway at different points. HSP60 in the heart has key anti-apoptotic functions because of its ability to form complexes with Bax, Bak and Bcl-xS (Lin et al., 2001; Kirchhoff et al., 2002; Shan et al., 2003; Marber et al., 1995), but not with Bcl-2. Binding of HSP60 in the normal cardiac cells prevents Bax from oligomerizing and inserting into the mitochondrial membrane. Reduction of HSP60 is associated with an overall decrease in Bcl-2 along with an increase in Bax and Bak and is sufficient to precipitate apoptosis (Lin et al., 2001; Kirchhoff et al., 2002; Shan et al., 2003; Marber et al., 1995). HSP70 exerts its anti-apoptotic effect by preventing oligomerized Apaf-1 from recruiting pro-Casp9 (Latchman, 2001; Marber et al., 1995). HSP70 can also inhibit apoptosis in a caspase-independent manner by inhibiting the c-Jun N-terminal kinase (JNK kinase). However, Casp9 is a critical regulator of mitochondria-mediated apoptosis; it forms a multimeric complex with cytochrome c and Apaf-1 to activate downstream caspases such as caspase-3 leading to apoptotic cell death (Han et al., 2006; Bialik et al., 1999; Kannan and Jain, 2000). The data in the present study showing repression of HSP60 and HSP70 by miR-1 and Casp9 by miR-133 and the opposing actions of these two miRNAs on apoptosis are in line with these previous findings.

Our data demonstrated silencing of HSP60 and HSP70 by miR-1 only at the protein level, and knockdown of Casp9 by miR-133 at both protein and mRNA levels. Earlier, miRNAs were though to primarily repress their targets at the protein level without affecting mRNA stability (Meister and Tuschl, 2004; Lewis et al., 2003). Increasing evidence, however, indicate that miRNAs silence genes by multiple mechanisms including degrading their target mRNAs (Kannan and Jain, 2000; Nilsen, 2007; Pillai et al., 2007). Our observations seem to be in line with multiple mechanisms of the action. However, it is presently unclear what determines the exact mechanisms of miRNA actions.

Collectively, our study revealed a novel aspect of cellular functions of the muscle-specific miRNAs miR-1 and miR-133, i.e. regulation of apoptosis and survival in cardiomyocytes. A unique feature of this regulation is the opposing actions with miR-1 being pro-apoptotic and miR-133 being anti-apoptotic, suggesting a possible role of relative miR-1 and miR-133 levels in regulating the cell fate. Post-transcriptional repression of HSP60 and HSP70 by miR-1 and of Casp9 by miR-133 is probably one of the mechanisms underlying their regulation of apoptosis versus survival. Our present and previous studies revealed the pathological elevations of miR-1 levels in cardiomyocytes in conditions favoring apoptosis (ischemia and oxidative stress). However, how these conditions lead to overexpression of miR-1 remains unclear.

The cell lines used in this study were all purchased from American Type Culture Collection (ATCC, Manassas, VA). H9c2 (rat ventricular cell line) and HEK293 (human embryonic kidney cell line) were cultured in Dulbecco's Modified Eagle Medium (DMEM). The cultures were supplemented with 10% fetal bovine serum and 100 μg/ml penicillin/streptomycin.

miR-1 and miR-133 and their respective mutant constructs were synthesized by Integrated DNA Technologies (IDT) (1). The sequences of miR-1 and miR-133 inhibitors (AMOs; anti-miRNA oligonucleotides) used in our studies are the exact antisense copies of their respective mature miRNA sequences: 3′-AAUGUAUGAAGAAAUGUAAGGU-5′ for human miR-1 (GenBank acc. no.: HSM808714), 3′-AAUGUAUGAAGAAAUGUAAGGU-5′ for rat miR-1 (GenBank ac. no.: DQ066650), and 3′-UGUCGACCAACUUCCCCUGGUU-5′ for both human and rat miR-133 [the sequences of miR-133 are identical in human (HSM808714) and rat (RATNCRNAB)]. All the nucleotides in the AMOs (AMO-1 for miR-1 and AMO-133 for miR-133) contain 2′-O-methyl modifications at every base and a 3′ C3-containing amino linker. The antagomers were also synthesized by IDT.

To generate reporter vectors bearing miRNA-binding sites, we generated direct match miR-1 and miR-133 sites (synthesized by Invitrogen), respectively, and the sequences around the putative target sites for these miRNAs in the 3′ UTRs of HSP60 and HSP70, and the full-length Casp9 mRNA (1). These inserts were cloned into the multiple cloning sites in the pMIR-REPORT™ luciferase miRNA expression reporter vector (Ambion, Inc.). The sense and antisense strands of the oligonucleotides were annealed by adding 2 μg of each oligonucleotide to 46 μl of annealing solution (100 mM potassium acetate, 30 mM Hepes-KOH, pH 7.4 and 2 mM magnesium acetate) and incubating at 90°C for 5 minutes and then at 37°C for 1 hour. The annealed oligonucleotides were digested with HindIII and SpeI and used to ligate into HindIII and SpeI sites.

Nucleotide-substitution mutations (MT) were carried out using direct oligomer synthesis for miR-1 and miR-133, and PCR-based methods for the 3′ UTRs of HSP60 and HSP70 genes. The substitution nucleotides were so designed to avoid producing new binding sites for other miRNAs potentially existing in HEK293 cells. All constructs were sequencing verified. See Fig. S1 in supplementary material for details of the mutations.

After 24 hours starvation in serum-free medium, cells (1×10⁵ per well) were transfected with 1 μg miR-1, miR-133, or other constructs, with Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions.

For luciferase assay, cells were transfected with 1 μg PGL3-target DNA (firefly luciferase vector) and 0.1 μg PRL-TK (TK-driven Renilla luciferase expression vector) with Lipofectamine 2000. Luciferase activities were measured 48 hours after transfection with a dual luciferase reporter assay kit (Promega) on a luminometer (Lumat LB9507) (Yang et al., 2007; Luo et al., 2007).

For quantification of HSP60, HSP70 and Casp9 transcripts, conventional real-time RT-PCR was carried out with total RNA samples extracted from H9c2 cells and neonatal rat ventricular cells 48 hours after transfection. TaqMan quantitative assay was performed with the expression level of GAPDH as an internal control.

The mirVana™ qRT-PCR miRNA Detection Kit (Ambion) was used in conjunction with real-time PCR with SYBR Green I for quantification of miR-1 and miR-133 transcripts, as detailed elsewhere (Yang et al., 2007; Luo et al., 2007). Fold variations in expression of miR-1 and miR-133 between RNA samples were calculated after normalization to 5s rRNA.

The protein samples were extracted from H9c2 cells and cultured neonatal rat ventricular cells, with the procedures essentially the same as described in detail elsewhere (Han et al., 2001; Han et al., 2004a; Han et al., 2004b; Luo et al., 2007; Wang et al., 2002). Protein samples (∼50 μg) were fractionated by SDS-PAGE (7.5-10% polyacrylamide gels). The primary antibodies against HSP60 (Stressgen Bioreagents, Ann Arbor, MI; rabbit polyclonal), HSP70 (Cell Signaling; rabbit polyclonal) and total caspase-3 and total caspase-9 (Cell Signaling; rat specific, rabbit polyclonal) were used, with GAPDH (anti-GAPDH antibody from Research Diagnostics, Concord, MA) as an internal control.

The procedures were the same as previously described in detail (Han et al., 2001; Han et al., 2004a; Han et al., 2004b; Wang et al., 2002).

Cell Proliferation Kit I [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT); Roche Molecular Biochemicals, Laval, PQ, Canada] was used to quantify survival of cells from oxidative stress (Han et al., 2001; Han et al., 2004a; Han et al., 2004b; Wang et al., 2002).

The Cell Death Detection ELISA kit (Roche Molecular Biochemicals) was employed to quantify DNA fragmentation on the basis of antibody detection of free histone and fragmented DNA (Han et al., 2001; Han et al., 2004a; Han et al., 2004b; Wang et al., 2002).

DNA fragmentation of individual cells was detected in situ by TUNEL with the In Situ Cell Death Detection kit, Fluorescein (Roche Molecular Biochemicals) (Han et al., 2001; Han et al., 2003; Han et al., 2004a; Han et al., 2004b; Wang et al., 2002).

Group data are expressed as mean ± s.e.m. Statistical comparisons (performed using ANOVA followed by Dunnett's method) were carried out using Microsoft Excel. A two-tailed P<0.05 was taken to indicate a statistically significant difference.

The authors thank XiaoFan Yang for excellent technical support. This work was supported in part by the Natural Sciences and Engineering Research Council of Canada and Fonds de la Recherche de l'Institut de Cardiologie de Montreal, awarded to Z. Wang, and by the National Nature Science Foundation of China (30430780), the Foundation of National Department of Science and Technology of China (2004CCA06700), and National Basic Research Program of China (973 Program; 2007CB512000/2007CB512006) awarded to B. Yang. Z. Wang is a senior research scholar of the Fonds de Recherche en Sante de Quebec.