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reoxygenation-induced injury by repairing microfilaments in neonatal rat cardiomyocytes
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Research Article
Myofibrillogenesis regulator-1 attenuates hypoxia/reoxygenation-induced injury by repairing microfilaments in neonatal rat cardiomyocytes Tianqi Tao, Xiaoreng Wang, Mi Liu, Xiuhua Liu n Department of Pathophysiology, Chinese PLA General Hospital, 100853 Beijing, China
art ic l e i nf o
a b s t r a c t
Article history: Received 1 March 2015 Received in revised form 20 May 2015 Accepted 30 May 2015
Hypoxia/reoxygenation (H/R) injury is characterized by microfilament reorganization in cardiomyocytes. Previous studies have shown that myofibrillogenesis regulator-1 (MR-1) is expressed in the myocardium and promotes actin organization in cardiomyocytes. The purpose of this study was to investigate the role of MR-1 in attenuating hypoxia/reoxygenation injury in cardiomyocytes through promoting restoration of the microfilament. To address this aim, an H/R model of cultured neonatal cardiomyocytes was used to assess filamentous actin (F-actin) and α-actinin organization through immunofluorescence microscopy analysis. RT-PCR was used to detect mRNA levels of MR-1 and myosin regulatory light chain-2 (MLC-2). Western blot analysis was used to detect protein levels of MR-1 and filamentous actin/globular actin (F-/ G-actin) as well as MLC-2 and myosin light chain kinase (MLCK) phosphorylation and protein expression. We also explored the effects of overexpressing or knocking down MR-1 on H/R injury and the MLCK/ MLC-2/F-actin pathway. We found that H/R induced cardiomyocyte injury and disruption of F-actin and α-actinin with a decrease in the F-/G-actin ratio compared with controls. Compared with the H/R group, MR-1 overexpression attenuated H/R-induced injury and disruption of F-actin and α-actinin in cardiomyocytes with an increase in the F-/G-actin ratio. MR-1 overexpression also up-regulated H/R-induced MLCK and MLC-2 phosphorylation. However, MR-1 knockdown aggravated H/R injury by further disrupting F-actin and α-actinin, as well as decreasing the F-/G-actin ratio. MR-1 knockdown also downregulated MLCK and MLC-2 phosphorylation induced by H/R injury. These findings suggest that MR-1 attenuates H/R-induced cardiomyocyte injury by promoting microfilament reorganization through the activation of the MLCK/MLC-2 pathway. & 2015 Elsevier Inc. All rights reserved.
Keywords: Hypoxia/reoxygenation Cardiomyocyte Myofibrillogenesis regulator-1 Microfilament
1. Introduction Reperfusion therapy can salvage the myocardium from ischemia injury induced by acute myocardial infarction. Restoration of blood flow to the ischemic myocardium may lead to further irreversible myocardium damage, termed myocardial ischemia/ reperfusion (I/R) injury [1]. Currently, substantial efforts are underway to improve strategies for the prevention of I/R injury. The myofibrils, comprised of thick and thin filaments, are the molecular motor for cardiac contraction and responsible for the shape and survival of cardiomyocytes [2–4]. The thin filament comprises two helical strands of actin filament, the elongated tropomyosin, and troponin complexes. The thick filament comprises two myosin heavy chain (MHC) molecules combined with two myosin regulatory light chain (MLC)-1 molecules and two MLC-2 molecules [5,6]. MLC-2 is an important molecule for regulating actin–myosin n
interaction, and driving actin–myosin contraction through the activation of ATPase [7]. MLC-2 also promotes globular actin (G-actin) polymerization to form filamentous actin (F-actin) in cardiomyocytes [8]. Studies of the molecular consequences of myocardial I/R injury suggest that myofibril rupture is a hallmark of cardiomyocyte necrosis [5,9]. The mechanism whereby I/R induces myofibril rupture remains unclear, but a putative mechanism is disturbance of F-actin, the backbone of the thin filament in cardiomyocytes. Hein et al. reported that global ischemia induced disturbances of the localization pattern of actin in 10 min, which fully completed at 20 min after the onset of ischemia in the myocardium from human left ventricles [10]. An actin-binding protein, α-actinin, cross-links actin and forms much of the dense component of the Z band [2,11]. Ischemia caused α-actinin disruption in the myocardium of the Langendorff perfused rat heart [5]. This infers that actin filaments and its binding proteins are
Corresponding author. E-mail addresses: [email protected] (T. Tao), [email protected] (X. Wang), [email protected] (M. Liu), [email protected] (X. Liu).
http://dx.doi.org/10.1016/j.yexcr.2015.05.026 0014-4827/& 2015 Elsevier Inc. All rights reserved.
Please cite this article as: T. Tao, et al., Myofibrillogenesis regulator-1 attenuates hypoxia/reoxygenation-induced injury by repairing microfilaments in neonatal rat cardiomyocytes, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.026i
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potential targets for cardioprotection during I/R injury. Myofibrillogenesis regulator-1 (MR-1), a human gene (AF417001) cloned from a human skeletal muscle cDNA library, is highly expressed in the myocardium and located in the myofibrils. It is a 755-bp length gene which is located on the human chromosome 2q35 and encodes a 142-amino-acid protein [8,12]. Our previous studies showed that MR-1 interacted directly with MLC-2 and induced cardiomyocyte hypertrophy via sarcomere organization [8]. MR-1 overexpression promoted actin polarity through the MLC-2-mediated reorganization of actin filaments and protected HK-2 cells from H/R injury [13]. This further suggests that MR-1 might protect cells from H/R injury through attenuating H/R-induced damage of the actin filament. Myosin light chain kinase (MLCK) is the predominant kinase for MLC-2 and important for normal cardiac contraction in vivo [6]. MR-1 overexpression up-regulated MLC-2 [8,13] while MR-1 knockdown down-regulated MLC-2 phosphorylation [13,14]. I/R and hypoxia injury also induced MLCK protein expression and MLC2 phosphorylation as well as aggravated actin polymerization and depolymerization in the myocardium [10,15,16]. We hypothesized that MR-1 attenuated H/R-induced cardiomyocyte injury by promoting actin filament reorganization through the MLCK/MLC-2 pathway. The present investigation was conducted to elucidate this possibility. We studied the effects of MR-1 on H/Rinduced F-actin and α-actinin organization, as well as the expression and phosphorylation of MLC-2 and MLCK in neonatal rat cardiomyocytes. We found that MR-1 overexpression attenuated H/Rinduced disruption of F-actin and α-actinin in cardiomyocytes along with an increase of the F-/G-actin ratio. MR-1 overexpression upregulated while MR-1 knockdown down-regulated MLCK and MLC2 phosphorylation. It suggested that MR-1 protected cardiomyocytes from H/R injury by promoting the formation of microfilaments through the up-regulation of the MLCK/MLC-2 pathway.
2. Materials and methods 2.1. Antibodies and reagents Dulbecco modified Eagle medium (DMEM) was purchased from Gibco (Grand Island, NY, USA), newborn calf serum (NCS) was purchased from PAA (Pasching, Austria), and trypsin was purchased from Amresco (Solon, OH, USA). The protease inhibitor, penicillin/streptomycin, Triton X-100, phalloidin-FITC and mouse monoclonal antibody against α-actinin were purchased from Sigma (St. Louis, MO, USA). A rabbit anti-MR-1 polyclonal antibody was obtained from polypeptide-immunized New Zealand rabbits [8]. The recombinant adenovirus overexpressing full-length rat MR-1, the stable stealth RNA targeted to rat MR-1, non-target stealth RNA and TRIzol reagents were purchased from Invitrogen (Foster, CA, USA). Phosphatase inhibitor and bovine serum albumin (BSA) were purchased from Merck (Rahway, NJ, USA). The lactate dehydrogenase (LDH) detection kit was purchased from Nanjing Jiancheng Biological Engineering Institute (Nanjing, China), and the Cell Counting Kit-8 (CCK-8) detection kit was from Kaiji Biological Engineering Company (Nanjing, China). Rabbit monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and rabbit monoclonal antibody against Panactin were purchased from Cell Signaling Technology (Danvers, MA, USA). Goat monoclonal antibody against phosphorylated MLCK (p-MLCK), daylight 488 green-conjugated donkey antimouse secondary antibody, and the enhanced chemiluminescence kit were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit monoclonal antibodies against MLC-2, phosphorylated MLC-2 (p-MLC-2), MLCK and Horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) and
rabbit anti-goat IgG were purchased from Epitomics (Burlingame, CA, USA). The mounting media was from Vector Laboratories (Burlingame, CA, USA). The EasyScript First-Strand cDNA Synthesis SuperMix Kit was from TransGen Company (Beijing, China) and 2 Taq mix was from TianGen Technology (Beijing, China). 2.2. Animals Sprague-Dawley (SD) neonatal rats were used for the cardiomyocyte cultures and were purchased from the Experimental Animal Center of Military Medical Sciences. The investigation conformed to the Guide for the Care and Use of Laboratory Animals, published by the United States National Institutes of Health (NIH publication no. 85-23, revised 1996). All animal studies were also approved by the Institutional Animal Care and Use Committee of the Chinese PLA General Hospital. 2.3. Cell culture and experiment protocol Methods for culturing neonatal rat ventricular cardiomyocytes were described in our previous publication [17]. In brief, cardiac ventricles were obtained from 1-day-old SD rats and enzymatically dissociated using a 0.15% trypsin solution in a shaker at 37 °C. Cells were collected by centrifugation to remove noncardiomyocytes and were pre-plated for 1.5 h in DMEM with 10% NCS and 1% penicillin/streptomycin (100 U/mL). Unattached cells were removed and placed into the T-75 flasks with the same medium, and incubated at 37 °C, 5% CO2. Following 24 h, the cells were transferred to serum-free maintenance media (DMEM containing 1% penicillin/streptomycin) for 24 h before experimentation. In order to assess for the protective effects of MR-1 against H/ R injury, cardiomyocytes were divided into seven groups: (1) control group: cells cultured in a 5% CO2 incubator at 37 °C for the duration of the experiment; (2) H/R group: cardiomyocytes were transferred to an incubator (Thermo Fisher Scientific, Waltham, MA, USA) filled with a gas mixture of 90% N2, 5% O2 and 5% CO2 for 8 h of hypoxia and then placed back in the normoxic CO2 incubator at 37°C for 16 h of reoxygenation; (3) Ad-MR-1 group: cardiomyocytes were infected with recombinant adenovirus overexpressing full-length rat MR-1 (MOI ¼50 ifu/cell); (4) st-MR1 group: cardiomyocytes were transfected with stable stealth RNA targeted to rat MR-1. The sequence of the stable stealth RNA against rat MR-1 was 5′-CGACAGCUAACAAGGCUUCCCAGAA-3′ [8]; (5) Ad-MR-1þH/R group: cardiomyocytes were infected with MR1-adenovirus for 24 h and then treated with H/R as described above; (6) st-MR-1þH/R group: cardiomyocytes were transfected with MR-1-RNAi for 24 h, and then treated with H/R as described above; and (7) Mock group: cardiomyocytes were exposed to nontarget 25-base pair silencing oligonucleotides. 2.4. Detection of cell injury and viability LDH activity was assessed in culture medium using an LDH assay kit to estimate LDH leakage under various treatment conditions. CCK-8 detection was an alternative method used for estimating cell viability. This measures metabolic activity through the ability of the cells to convert tetrazolium dye to its insoluble form, formazan [18]. Cells were seeded and plated in 96-well plates and then incubated with CCK-8 for 4 h. Samples were measured using a microplate reader at 450 nm wave-length (Tecan Infinite f200 Pro; Tecan Group Ltd., Männedorf, Switzerland). 2.5. RT-PCR Cardiomyocyte total RNA was extracted using TRIzol reagent, and quantified at 260 nm. The EasyScript First-Strand cDNA
Please cite this article as: T. Tao, et al., Myofibrillogenesis regulator-1 attenuates hypoxia/reoxygenation-induced injury by repairing microfilaments in neonatal rat cardiomyocytes, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.026i
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Synthesis SuperMix Kit was used to perform reverse transcription to obtain cDNA in each group. The cDNA was further amplified using Superscript One-Step polymerase chain reaction with 2 Taq mix. PCR primers (Table 1) were derived from sequences in the GenBank human database. PCR products were separated on 1.0% agarose gels and photographed for analysis using Image-Pro Plus software (Roper Industries, New York, NY, USA). 2.6. Western blot analysis
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Table 1 Primer sequences used for PCR. Gene
Forward primer
Reverse primer
r18s rMR-1
5′-CCAACCCGGTCAGCCCCCTC-3′ 5′-CCAGAACAGG ACCAGAGCA-3′
5′-TACCTCCCCGGGTCGGGAGT-3′ 5′-GGTGAGGACG AAGAAGAGGATA-3′ 5′-AAAGAGGAAA GGCTGTGAAAC3′
rMLC-2 5′-GCGAAAGACA AAGATGACTGA-3′ r¼ rat.
Cardiomyocyte protein was extracted as previously described [8,19]. In brief, cardiomyocytes were plated in T-25 flasks at a cell density of 5 105 cells per flask. The concentration of protein extracted from the cardiomyocytes was detected using Bradford Assay. Proteins were resolved on 10% sodium dodecyl sulfatepolyacrylamide gels using an equal amount of protein (80 μg/ lane). Following electrophoresis, proteins were electrophoretically transferred to nitrocellulose membranes and then blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) at room temperature for 1 h. Then membranes were probed with primary antibodies against MR-1, Pan-actin, MLC-2, p-MLC-2, MLCK, p-MLCK and GAPDH (all 1:500 diluted) at 4 °C overnight. The antibody-tagged membranes were incubated with a secondary antibody solution consisting of either a 1:1000 dilution of HRPconjugated goat anti-rabbit IgG (for MR-1, Pan-actin, MLC-2, p-MLC-2, MLCK and GAPDH) or a 1:1000 dilution of HRP-conjugated rabbit anti-goat IgG (for p-MLCK). An enhanced chemiluminescence detection system was used for immunoblot protein detection. Optical densities of the bands were analyzed using Image-Pro Plus software, and the densitometry results were normalized to GAPDH. 2.7. Immunofluorescence staining Cardiomyocytes were grown on coverslips and fixed using 4% paraformaldehyde at room temperature for 25 min. They were then blocked in phosphate-buffered saline containing 10% donkey serum, 1% BSA and 0.1% Triton X-100 for 50 min. Cardiomyocytes were identified by indirect immunofluorescence staining using anti-α-actinin mouse monoclonal antibody for 2 h at room temperature, followed by FITC green-conjugated donkey anti-mouse secondary antibody (1:400) for 1 h in the dark at room temperature. For F-actin detection, cardiomyocytes were stained in the dark at room temperature for 1 h with phalloidin-FITC at a final concentration of 0.33 μmol/L [8,13]. The coverslips were mounted on glass slides with mounting medium. Images were acquired using a confocal scanning microscope (Zeiss LSM-510 Meta, Jena, Germany), and a 63 oil immersion objective with a numerical aperture of 1.4 was used. 2.8. Statistical analysis The SPSS v13.0 program (Chicago, IL, USA) was used for statistical analysis. For multiple-group comparisons, one-way analysis of variance followed by Newman–Keuls post hoc analysis was performed. Values are presented as mean 7SD. Pearson bivariate correlation analysis was applied to determine the correlation between variables. Po 0.05 was considered to be statistically significant.
3. Results 3.1. Expression of MR-1 in H/R-induced cardiomyocytes To assess the effects of MR-1 on cardiomyocytes with or
without H/R injury, we overexpressed MR-1 using recombinant adenovirus and down-regulated MR-1 using siRNA. Alterations in mRNA expression of MR-1 in cardiomyocytes were detected by RTPCR (Fig. 1A and C). We found that MR-1 mRNA expression decreased by 27.5% in H/R-treated cardiomyocytes compared with controls (Po0.01). An infection of cardiomyocytes with recombinant adenovirus overexpressing MR-1 resulted in a significant increase in MR-1 mRNA expression following H/R treatment (Po0.01). MR-1 mRNA expression in cardiomyocytes increased by 442% in the Ad-MR-1 group (Po 0.01 vs. control) and 508% in the Ad-MR-1þ H/R group (P o0.01 vs. H/R), respectively. MR-1 siRNA treatment down-regulated MR-1 mRNA expression in cardiomyocytes. MR-1 mRNA expression decreased by 66.8% in cardiomyocytes from the st-MR-1 group (P o0.01 vs. Mock) and 58.7% in cardiomyocytes from the st-MR-1 þH/R group (P o0.01 vs. H/R), respectively. Alterations in MR-1 protein expression in cardiomyocytes were assessed using western blot analysis (Fig. 1B and C). H/R-treatment decreased MR-1 protein expression by 33.5% compared with control cardiomyocytes. MR-1 protein expression in cardiomyocytes increased by 610% in the Ad-MR-1 group (Po 0.01 vs. control) and 786% in the Ad-MR-1 þH/R group (Po 0.01 vs. H/R), respectively. MR-1 protein expression decreased by 53.1% in cardiomyocytes from the st-MR-1 group (P o0.01 vs. Mock) and 20.6% in cardiomyocytes from the st-MR-1 þH/R group (P o0.01 vs. H/R), respectively. These data indicate that H/R decreases MR-1 expression, and treatment with recombinant adenovirus or knockdown by siRNA is sufficient to either up-regulate or down-regulate MR-1 expression, respectively. 3.2. Effects of MR-1 on H/R injury in cardiomyocytes 3.2.1. LDH activity LDH activity in the culture medium was measured to estimate the amount of LDH leakage from cardiomyocytes, indicating cell injury (Fig. 2A). LDH activity in the culture medium was 174 712.3 U/L in control cardiomyocytes. H/R induced an increase in LDH leakage compared with controls, and LDH activity in the culture medium increased by 573% in the H/R group compared with control. MR-1 overexpression prior to H/R attenuated H/Rinduced LDH leakage from cardiomyocytes. Compared with the H/ R group, LDH activity in the culture medium decreased by 64.2% in the Ad-MR-1þH/R group (P o0.01), although no significant difference in LDH leakage was detected in cardiomyocytes from the Ad-MR-1 group compared with control (P 40.05). MR-1 knockdown increased LDH leakage from cardiomyocytes, and LDH activity in the culture medium increased by 331% in the st-MR-1 group (P o0.01 vs. Mock). MR-1 knockdown prior to H/R aggravated H/R-induced LDH leakage from cardiomyocytes, and LDH activity in the culture medium increased by 19.0% in the st-MR1 þH/R group (P o0.05 vs. H/R) and by 40.0% compared with the st-MR-1 group (P o0.01).
Please cite this article as: T. Tao, et al., Myofibrillogenesis regulator-1 attenuates hypoxia/reoxygenation-induced injury by repairing microfilaments in neonatal rat cardiomyocytes, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.026i
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3.2.2. Cell viability Cellular viability detected by CCK-8 is shown in Fig. 2B. H/Rtreatment induced a 46.9% decrease in cell viability compared with control (P o0.01). MR-1 overexpression prior to H/R attenuated H/ R-induced cardiomyocyte injury, increasing cell viability by 36.0% in the Ad-MR-1þ H/R group compared with the H/R group (P o0.01), although there was no significant difference in the AdMR-1 group (P4 0.05 vs. control). MR-1 knockdown induced a 20.3% decrease in cell viability (P o0.01 vs. Mock). MR-1 knockdown prior to H/R aggravated H/R-induced cardiomyocyte injury. Cell viability decreased by 8.5% in the st-MR-1 þH/R group (P o0.01 vs. H/R) and by 38.7% compared with the st-MR-1 group (P o0.01). 3.3. Effects of MR-1 on F-actin distribution 3.3.1. F-actin distribution We examined F-actin by staining the cardiomyocytes with phalloidin-FITC, which bound only to F-actin. The fluorescence microphotographs (Fig. 3A) revealed that in control cardiomyocytes, F-actin predominantly appeared in striated like and stress fibers. In H/R-treated cardiomyocytes, F-actin appeared predominantly disorganized with scattered ruptures. F-actin distribution in cardiomyocytes from the Ad-MR-1 group (MR-1 overexpression alone) was similar to controls with increased green fluorescence. MR-1 overexpression prior to H/R attenuated H/Rinduced F-actin fragility, and exhibited a uniform distribution of F-actin in the cytosol in cardiomyocytes from the Ad-MR-1 þH/R group, similar to control cardiomyocytes. MR-1 knockdown caused disorganization of F-actin similar to that in the H/R group. MR-1
Fig. 2. Myofibrillogenesis regulator-1 (MR-1) reduced cardiomyocyte injury and increased cardiomyocyte viability after H/R treatment in vitro. (A) LDH activity in cultured medium. (B) Cell viability by the Cell Counting Kit-8 assay. x¯ 7 s. n ¼3. * P o0.05 vs. control; #Po 0.05 vs. H/R; &P o0.05 vs. Mock.
knockdown prior to H/R aggravated H/R-induced F-actin fragility, showing much more disorganized and scattered rupture of F-actin than that in the H/R group.
Fig. 1. Relative amounts of myofibrillogenesis regulator-1 (MR-1) expression levels in hypoxia/reoxygenation (H/R)-treated cardiomyocytes infected and transfected with MR-1. (A) The level of MR-1 mRNA was examined using RT-PCR. 18s was used as a loading control and for normalization. (B) The level of MR-1 protein was examined using Western blot analysis. GAPDH was used as a loading control and for normalization of data. (C) Bar chart of densitometry of the bands shown in A and B. x¯ 7 s. n ¼3. *Po 0.05 vs. control; #Po 0.05 vs. H/R; &P o0.05 vs. Mock.
3.3.2. F-/G-actin ratio Actin polymerization is critical for cell shape and many regulatory responses including contraction and motility. Therefore, we examined the F-/G-actin ratio by Western blotting analysis to assess polymerization/depolymerization of actin in cardiomyocytes (Fig. 3B). We found that cardiomyocytes in the H/R group showed a 9.0% decrease in the F-/G-actin ratio compared with the control (P o0.01), suggesting that H/R induced depolymerization of actin. MR-1 overexpression increased the F-/G-actin ratio by 11.5% (P o0.01, Ad-MR-1 vs. control). MR-1 overexpression prior to H/R attenuated H/R-induced F-actin depolymerization, exhibiting a 36.2% increase in the F-/G-actin ratio compared with H/R alone (P o0.01). It suggests that MR-1 overexpression induces the polymerization of actin. Although there was no difference between control and Mock-transfected cells, knocking down MR-1 decreased the F-/G-actin ratio by 12.1% compared with the Mock group (P o0.01). Knockdown of MR-1 prior to H/R aggravated H/Rinduced F-actin depolymerization, and the F-/G-actin ratio decreased by 10.6% in the st-MR-1 þH/R group, compared with H/Rtreated cardiomyocytes (P o0.01). These data indicate that MR-1 promotes polymerization of actin monomers and plays an important role in organizing actin filament assembly.
Please cite this article as: T. Tao, et al., Myofibrillogenesis regulator-1 attenuates hypoxia/reoxygenation-induced injury by repairing microfilaments in neonatal rat cardiomyocytes, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.026i
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3.4. Effects of MR-1 on
α-actinin distribution
α-actinin is an actin-binding protein that cross-links actin and forms much of the dense component of both the Z band and fascia adherens plaque. Therefore, we examined the distribution of αactinin in cardiomyocytes by immunofluorescence microscopy (Fig. 4). The fluorescence microphotographs revealed that α-actinin appeared orderly distributed in control cardiomyocytes. In H/ R-treated cardiomyocytes, α-actinin showed a diffuse disorganization. The α-actinin distribution in cardiomyocytes from the Ad-MR-1 group (MR-1 overexpression alone) was similar to controls. MR-1 overexpression prior to H/R attenuated H/R-induced α-actinin disorganization, exhibiting a uniform distribution of α-actinin in the cytosol in cardiomyocytes from the Ad-MR1 þH/R group, which was similar to control cardiomyocytes.
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Although there was no difference between control and Mocktransfected cells, knocking down MR-1 itself induced α-actinin disorganization. MR-1 knockdown prior to H/R aggravated H/Rinduced α-actinin disorganization, and predominantly presented the disorganization of α-actinin in the st-MR-1þH/R group compared with H/R-treated cardiomyocytes.
3.5. Effects of MR-1 on MLC-2 expression and phosphorylation MR-1 interacts directly with MLC-2 and phosphorylation of MLC-2 promotes G-actin polymerization to form F-actin. Therefore, we examined MLC-2 expression and phosphorylation to investigate the regulating responses induced by MR-1.
Fig. 3. Effect of myofibrillogenesis regulator-1 (MR-1) on the localization and organization of F-actin and the F-/G-actin ratio influenced by H/R treatment in vitro. (A) Under laser scanning confocal microscope, F-actin in cardiomyocytes was labeled with phalloidin-FITC (600 , bar¼ 20 μm). The images are representative of the majority of the cells. More than 100 cells were investigated in total by IF microscopy, and more than 70% of the total number of investigated cells were the morphological changes of the actin cytoskeleton observed. The ratio in each group was as follows: 72.4% in control group, 70.8% in H/R group, 70.4% in Ad-MR-1 group, 76.5% in st-MR-1 group, 71.2% in AdMR-1þ H/R group, 71.3% in st-MR-1 þ H/R group and 76.1% in Mock group, respectively. (B) The levels of F-actin and G-actin were examined using Western blot analysis. Bar chart of the F-/G-actin ratio calculated by densitometry of the bands. x¯ 7 s. n¼ 3. *Po 0.05 vs. control; #P o 0.05 vs. H/R; &Po 0.05 vs. Mock.
Please cite this article as: T. Tao, et al., Myofibrillogenesis regulator-1 attenuates hypoxia/reoxygenation-induced injury by repairing microfilaments in neonatal rat cardiomyocytes, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.026i
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3.5.1. MLC-2 expression MLC-2 mRNA and protein expression was detected by RT-PCR and Western blotting analysis, respectively (Fig. 5A–D). Compared with control, the MLC-2 expression increased by 26.7% (mRNA) and 50.5% (protein) in H/R-treated cardiomyocytes, respectively (Po0.01). MR-1 overexpression increased MLC-2 expression by 92.6% (mRNA) and 112% (protein), respectively (Po0.01 vs. control). MR-1 overexpression prior to H/R increased H/R-induced MLC-2 expression, exhibiting a 26.8% (mRNA) and a 24.8% (protein) increase in MLC-2 expression compared with H/R alone, respectively (Po0.01). Although there was no difference between control and Mock-transfected cells, knocking down MR-1 decreased MLC-2 mRNA and protein expression by 31.5% and 23.2% compared with the Mock group, respectively (Po0.01). Knockdown of MR-1 prior to H/R decreased MLC-2 mRNA and protein expression by 18.8% and 55.9% as compared with H/R-treated cardiomyocytes, respectively (Po0.01). Our data suggest that MR-1 is involved in the regulation of transcription and translation of MLC-2. 3.5.2. MLC-2 phosphorylation MLC-2 phosphorylation was detected by using Western blotting analysis (Fig. 5C and D). Compared with control, the MLC-2 phosphorylation increased by 53.2% in H/R-treated cardiomyocytes (P o0.01). MR-1 overexpression increased MLC-2 phosphorylation by 70.8% (Po 0.01, Ad-MR-1 vs. control). MR-1 overexpression prior to H/R increased H/R-induced MLC-2 phosphorylation, exhibiting a 29.6% increase in MLC-2 phosphorylation compared with H/R alone (P o0.01). Although there was no difference between control and Mock-transfected cells, knocking down MR-1 decreased MLC-2 phosphorylation by 23.3% compared with the Mock group (P o0.01). Knockdown of MR-1 prior to H/R decreased MLC-2 phosphorylation by 40.6%, compared with H/Rtreated cardiomyocytes (Po 0.01). These data provide further evidence that MR-1 is also involved in the regulation of MLC-2 phosphorylation. 3.6. Effects of MR-1 on MLCK expression and phosphorylation MLCK is the predominant kinase for MLC-2 phosphorylation and MR-1 knockdown down-regulated MLC-2 phosphorylation.
Therefore, we examined the MLCK expression and phosphorylation to investigate the effects of MR-1 on the MLCK/MLC-2 pathway in this study. Alterations in MLCK protein expression and phosphorylation in cardiomyocytes were detected using Western blot analysis. As shown in Fig. 5E and F, cardiomyocytes from the H/R group showed a 14.7% and a 37.2% increase in MLCK expression and phosphorylation compared with controls, respectively (P o0.01). MR-1 overexpression increased MLCK expression and phosphorylation by 40.8% and 30.4%, respectively (P o0.01 vs. control). MR1 overexpression prior to H/R increased H/R-induced MLCK expression and phosphorylation, exhibiting a 17.7% and a 19.1% increase in MLCK expression and phosphorylation compared with H/ R alone, respectively (P o0.01). There were no differences in MLCK expression and phosphorylation between the control and Mock-transfected cells. Knocking down MR-1 decreased MLCK expression by 13.3% without the effect of MLCK phosphorylation, compared with the Mock group (P o0.01). Knockdown of MR-1 prior to H/R down-regulated H/R-induced MLCK expression and phosphorylation. MLCK expression and phosphorylation decreased by 17.9% and 13.1% in the st-MR-1þ H/R group, compared with H/ R-treated cardiomyocytes, respectively (P o0.01). These data indicate that MR-1 is also involved in the regulation of transcription and translation of MLCK. In addition, correlation analysis showed a significant positive correlation between the phosphorylation levels of MLC-2 and MLCK (r ¼0.829, Po 0.01). It provides further evidence to the suggestion that MR-1 protects cardiomyocytes from H/R injury by promoting F-actin organization through MLCK/MLC2 pathway.
4. Discussion Potential mechanisms underlying the pathogenesis and treatment of myocardial ischemia/reperfusion (I/R) injury are largely unknown. Studies of the molecular consequences of myocardial I/R injury suggest that the actin filaments and their binding proteins are potential targets for the prevention and treatment of I/R injury [5,9,10]. The rat neonatal cardiomyocyte model provides an efficient in vitro model for I/R as it assesses the profile of contractile
Fig. 4. Effect of myofibrillogenesis regulator-1 (MR-1) on the localization and organization of α-actinin influenced by H/R treatment in vitro. Under a laser scanning confocal microscope, α-actinin in cardiomyocytes was observed using immunofluorescence microscopy (600 , bar ¼ 20 μm). The images are representative of the majority of the cells. More than 100 cells were investigated in total by IF microscopy, and more than 70% of the total number of investigated cells were the morphological changes of the αactinin observed. The ratio in each group was as follows: 80.0% in control group, 73.7% in H/R group, 82.4% in Ad-MR-1 group, 86.3% in st-MR-1 group, 75.9% in Ad-MR-1 þH/ R group, 78.6% in st-MR-1 þH/R group and 84.0% in Mock group, respectively.
Please cite this article as: T. Tao, et al., Myofibrillogenesis regulator-1 attenuates hypoxia/reoxygenation-induced injury by repairing microfilaments in neonatal rat cardiomyocytes, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.026i
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Fig. 5. Effect of myofibrillogenesis regulator-1 (MR-1) on the expression and phosphorylation of MLC-2 and MLCK in H/R-treated cardiomyocytes. (A, B) MLC-2 mRNA level was examined by RT-PCR with 18s used as a loading control and for normalization of data. (C, D) MLC-2 protein expression and phosphorylation were examined using Western blot analysis. GAPDH was used as a loading control and for normalization of data. (E, F) MLCK protein expression and phosphorylation were examined using Western blot analysis. GAPDH was used as a loading control and for normalization of data. x¯ 7 s. n¼3. *P o0.05 vs. control; #Po 0.05 vs. H/R; &P o 0.05 vs. Mock.
proteins during early stages of H/R, which is compatible with myocardial injury during I/R [20]. MR-1 is a functional gene that is highly expressed in the myocardium, skeletal muscle, kidney and liver [8,12,13,21]. Our previous studies showed that overexpressing MR-1 attenuated H/R-induced apoptosis in cardiomyocytes [22]. In this study, we demonstrated that hypoxia/reoxygenation (H/R) down-regulated endogenous expression of MR1, induced F-actin disruption and depolymerization and α-actinin disorganization, and caused an increase in LDH leakage and a decrease in cell viability in cardiomyocytes. These results suggest that MR-1 down-regulation is involved in the damage of actin filaments in cardiomyocytes during H/R injury. We then examined the above possibility mechanistically in MR-1-overexpressing and MR-1-knockdown cardiomyocytes. We found that MR-1 overexpression attenuated, while MR-1 knockdown aggravated H/Rinduced F-actin disruption and depolymerization and α-actinin disorganization. MR-1 overexpression also decreased H/R-induced
LDH leakage and presented an increase in cell viability in cardiomyocytes subjected to H/R. Our data indicate that MR-1 promotes polymerization of actin monomers and plays an important role in repairing actin filament during H/R. Finally, we found that this process involved the increased expression and phosphorylation of MLC-2 and MLCK. MR-1 overexpression up-regulated while MR-1 knockdown down-regulated MLCK expression and phosphorylation, which both activated MLC-2. In addition, MR-1 also induced MLC-2 expression, which further increased MLC-2 total phosphorylation. When combined with our previous study that showed MR-1 interacted directly with MLC-2, and MLC-2 was critical for actin polymerization to form F-actin [23] and regulated by MLCK [13], these data demonstrate that MR-1 protects cardiomyocytes from H/R injury by attenuating damage of actin filaments and activating the MLCK/MLC-2 pathway. The cytoskeleton, comprised of actin filaments, microtubules, intermediate filaments, and their associated binding and
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attachment proteins, is responsible for contraction, survival and shapes of cardiomyocytes [2–6]. In cardiomyocytes, the actin filaments form fine filaments and attachments to the cytoplasmic membrane at the fascia adherens junctions of the intercalated disk, and at the lateral costamere junctions. Actin polymerization is critical for cell shape and many regulatory responses including contraction and motility [4]. I/R induced actin depolymerization and disorganization in cardiomyocytes [24]. Furthermore, Actin cytoskeleton disorganization causes the disappearance of central stress fibers and leads to an early step in most cells undergoing apoptosis by a loss of survival signals [25]. The disorganization of F-actin further induced apoptosis in mouse embryonic fibroblast cells, with the decrease of cell viability [26]. In this study, we used the F-/G-actin ratio to reflect the polymerization/depolymerization rate of actin. We found that H/R induced F-actin disorganization, causing depolymerization and re-arrangement, suggesting that H/ R induced disorganization and depolymerization of actin, which coincided with F-actin disassembly as a consequence of sustained myocardial I/R injury [24]. MR-1 overexpression attenuated, while MR-1 knockdown aggravated H/R-induced F-actin disorganization and depolymerization, indicating that MR-1 promoted polymerization of actin monomers and organizing actin filament assembly. Our data are consistent with the findings that MR-1 overexpression promotes actin polarity in H/R-treated HK-2 cells [13]. α-Actinin is one of the actin-binding proteins that ensures F-actin connects together to form bundles [2]. The damaged αactinin aggravates F-actin derangement. Ganote et al. indicated a loss of α-actinin from 90-min anoxic hearts when examined under high-voltage electron-microscopy [27]. Our study further confirmed that H/R induced α-actinin destruction. Furthermore, MR-1 overexpression attenuated while MR-1 knockdown aggravated H/ R-induced α-actinin disorganization, suggesting that MR-1 protects α-actinin from H/R injury. MR-1, located in the myofibrils, interacted directly with MLC-2 and induced cardiomyocyte hypertrophy via sarcomere organization [8]. MLC-2 is the predominant substrate of MLCK [6], which phosphorylated MLC-2 at two sites (Ser19 and Thr18) [7,28]. Studies verified that MLCK overexpression also induced MLC-2 activation [6]. MLC-2 phosphorylation is critical for actin polymerization to form F-actin. Tu et al. found that I/R induced rupture of myocardial fibers and degradation of F-actin as well as MLC-2 phosphorylation in the myocardial tissue [24]. Kim et al. observed that I/R increased expression of genes encoding MLC-2 in rat hearts, which compensated for disorganization of sarcomeric myofibrillar proteins [29,30]. In this study, we found that H/R down-regulated MR-1 expression and induced disruption of F-actin and α-actinin disorganization with cardiomyocyte injury, as well as the up-regulation of MLC-2 expression and phosphorylation. MLC-2 up-regulation may compensate for disorganization of F-actin, which is consistent with Kim's results. In addition, MR-1 overexpression increased, while MR-1 knockdown decreased MLCK expression and phosphorylation, which both activated MLC2. MR-1 also up-regulated MLC-2 expression and further increased total MLC-2 phosphorylation. MLC-2 phosphorylation accelerated actin polymerization [31]. It suggest that the MLCK/MLC-2 pathway is involved in MR-1-mediated F-actin organization in the setting of H/R injury, hence cardioprotection during H/R. Moreover, the mechanism of MR-1-mediated cardiac hypertrophy and microfilament reorganization is similar. MR-1 overexpression induced cardiomyocyte hypertrophy by promoting sarcomere rapid reorganization with the increase of MLC-2 expression [8]. Our data illustrated that MR-1 also up-regulated MLC-2 expression and phosphorylation, which attenuated H/Rinduced microfilament destruction by promoting microfilament reorganization. However, previous studies showed that 24 h after Ad-MR-1 overexpression promotes rapid organization of
Fig. 6. Proposed pathway of MR-1-mediated regulation of microfilament reorganization during H/R. H/R induced microfilament disorganization and depolymerization, and MR-1 regulated microfilament reorganization through MLCK/MLC2 pathway. MR-1 up-regulated expression and phosphorylation of MLCK, which both activated MLC-2. In addition, MR-1 also induced MLC-2 expression, which further increased MLC-2 total phosphorylation. MLC-2 phosphorylation promoted microfilament reorganization in cardiomyocytes. (H/R: hypoxia/reoxygenation, MR1: myofibrillogenesis regulator-1, MLCK: myosin light chain kinase, MLC-2: myosin regulatory light chain-2.)
sarcomeres while did not induce cardiomyocyte hypertrophy until 48 h after MR-1 overexpression. Hypertrophic hallmarks such as cell size, [3H]-leucine incorporation, and expression of ANF and BNP were significantly increased at 48 h after MR-1 overexpression. However, in this study, H/R was induced at 24 h after MR-1 overexpression, when cardiomyocyte hypertrophy was unhappened. It indicates that MR-1 overexpression protect disorganization of microfilaments against H/R injury, without involving MR-1-mediated hypertrophy at that time point. In summary, MR-1 promotes microfilament organization and protects cardiomyocytes from H/R injury. The underlying mechanism might involve the correct organization of F-actin through the MLCK/MLC-2 pathway. The proposed pathway of MR-1mediated regulation of microfilament reorganization was summarized in Fig. 6. Our studies may help uncover therapeutic targets for the prevention and treatment of I/R injury.
Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant nos. 81170140, 31471094 and 81070130 (XH Liu)).
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Research Article
Myofibrillogenesis regulator-1 attenuates hypoxia/reoxygenation-induced injury by repairing microfilaments in neonatal rat cardiomyocytes Tianqi Tao, Xiaoreng Wang, Mi Liu, Xiuhua Liu n Department of Pathophysiology, Chinese PLA General Hospital, 100853 Beijing, China
art ic l e i nf o
a b s t r a c t
Article history: Received 1 March 2015 Received in revised form 20 May 2015 Accepted 30 May 2015
Hypoxia/reoxygenation (H/R) injury is characterized by microfilament reorganization in cardiomyocytes. Previous studies have shown that myofibrillogenesis regulator-1 (MR-1) is expressed in the myocardium and promotes actin organization in cardiomyocytes. The purpose of this study was to investigate the role of MR-1 in attenuating hypoxia/reoxygenation injury in cardiomyocytes through promoting restoration of the microfilament. To address this aim, an H/R model of cultured neonatal cardiomyocytes was used to assess filamentous actin (F-actin) and α-actinin organization through immunofluorescence microscopy analysis. RT-PCR was used to detect mRNA levels of MR-1 and myosin regulatory light chain-2 (MLC-2). Western blot analysis was used to detect protein levels of MR-1 and filamentous actin/globular actin (F-/ G-actin) as well as MLC-2 and myosin light chain kinase (MLCK) phosphorylation and protein expression. We also explored the effects of overexpressing or knocking down MR-1 on H/R injury and the MLCK/ MLC-2/F-actin pathway. We found that H/R induced cardiomyocyte injury and disruption of F-actin and α-actinin with a decrease in the F-/G-actin ratio compared with controls. Compared with the H/R group, MR-1 overexpression attenuated H/R-induced injury and disruption of F-actin and α-actinin in cardiomyocytes with an increase in the F-/G-actin ratio. MR-1 overexpression also up-regulated H/R-induced MLCK and MLC-2 phosphorylation. However, MR-1 knockdown aggravated H/R injury by further disrupting F-actin and α-actinin, as well as decreasing the F-/G-actin ratio. MR-1 knockdown also downregulated MLCK and MLC-2 phosphorylation induced by H/R injury. These findings suggest that MR-1 attenuates H/R-induced cardiomyocyte injury by promoting microfilament reorganization through the activation of the MLCK/MLC-2 pathway. & 2015 Elsevier Inc. All rights reserved.
Keywords: Hypoxia/reoxygenation Cardiomyocyte Myofibrillogenesis regulator-1 Microfilament
1. Introduction Reperfusion therapy can salvage the myocardium from ischemia injury induced by acute myocardial infarction. Restoration of blood flow to the ischemic myocardium may lead to further irreversible myocardium damage, termed myocardial ischemia/ reperfusion (I/R) injury [1]. Currently, substantial efforts are underway to improve strategies for the prevention of I/R injury. The myofibrils, comprised of thick and thin filaments, are the molecular motor for cardiac contraction and responsible for the shape and survival of cardiomyocytes [2–4]. The thin filament comprises two helical strands of actin filament, the elongated tropomyosin, and troponin complexes. The thick filament comprises two myosin heavy chain (MHC) molecules combined with two myosin regulatory light chain (MLC)-1 molecules and two MLC-2 molecules [5,6]. MLC-2 is an important molecule for regulating actin–myosin n
interaction, and driving actin–myosin contraction through the activation of ATPase [7]. MLC-2 also promotes globular actin (G-actin) polymerization to form filamentous actin (F-actin) in cardiomyocytes [8]. Studies of the molecular consequences of myocardial I/R injury suggest that myofibril rupture is a hallmark of cardiomyocyte necrosis [5,9]. The mechanism whereby I/R induces myofibril rupture remains unclear, but a putative mechanism is disturbance of F-actin, the backbone of the thin filament in cardiomyocytes. Hein et al. reported that global ischemia induced disturbances of the localization pattern of actin in 10 min, which fully completed at 20 min after the onset of ischemia in the myocardium from human left ventricles [10]. An actin-binding protein, α-actinin, cross-links actin and forms much of the dense component of the Z band [2,11]. Ischemia caused α-actinin disruption in the myocardium of the Langendorff perfused rat heart [5]. This infers that actin filaments and its binding proteins are
Corresponding author. E-mail addresses: [email protected] (T. Tao), [email protected] (X. Wang), [email protected] (M. Liu), [email protected] (X. Liu).
http://dx.doi.org/10.1016/j.yexcr.2015.05.026 0014-4827/& 2015 Elsevier Inc. All rights reserved.
Please cite this article as: T. Tao, et al., Myofibrillogenesis regulator-1 attenuates hypoxia/reoxygenation-induced injury by repairing microfilaments in neonatal rat cardiomyocytes, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.026i
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potential targets for cardioprotection during I/R injury. Myofibrillogenesis regulator-1 (MR-1), a human gene (AF417001) cloned from a human skeletal muscle cDNA library, is highly expressed in the myocardium and located in the myofibrils. It is a 755-bp length gene which is located on the human chromosome 2q35 and encodes a 142-amino-acid protein [8,12]. Our previous studies showed that MR-1 interacted directly with MLC-2 and induced cardiomyocyte hypertrophy via sarcomere organization [8]. MR-1 overexpression promoted actin polarity through the MLC-2-mediated reorganization of actin filaments and protected HK-2 cells from H/R injury [13]. This further suggests that MR-1 might protect cells from H/R injury through attenuating H/R-induced damage of the actin filament. Myosin light chain kinase (MLCK) is the predominant kinase for MLC-2 and important for normal cardiac contraction in vivo [6]. MR-1 overexpression up-regulated MLC-2 [8,13] while MR-1 knockdown down-regulated MLC-2 phosphorylation [13,14]. I/R and hypoxia injury also induced MLCK protein expression and MLC2 phosphorylation as well as aggravated actin polymerization and depolymerization in the myocardium [10,15,16]. We hypothesized that MR-1 attenuated H/R-induced cardiomyocyte injury by promoting actin filament reorganization through the MLCK/MLC-2 pathway. The present investigation was conducted to elucidate this possibility. We studied the effects of MR-1 on H/Rinduced F-actin and α-actinin organization, as well as the expression and phosphorylation of MLC-2 and MLCK in neonatal rat cardiomyocytes. We found that MR-1 overexpression attenuated H/Rinduced disruption of F-actin and α-actinin in cardiomyocytes along with an increase of the F-/G-actin ratio. MR-1 overexpression upregulated while MR-1 knockdown down-regulated MLCK and MLC2 phosphorylation. It suggested that MR-1 protected cardiomyocytes from H/R injury by promoting the formation of microfilaments through the up-regulation of the MLCK/MLC-2 pathway.
2. Materials and methods 2.1. Antibodies and reagents Dulbecco modified Eagle medium (DMEM) was purchased from Gibco (Grand Island, NY, USA), newborn calf serum (NCS) was purchased from PAA (Pasching, Austria), and trypsin was purchased from Amresco (Solon, OH, USA). The protease inhibitor, penicillin/streptomycin, Triton X-100, phalloidin-FITC and mouse monoclonal antibody against α-actinin were purchased from Sigma (St. Louis, MO, USA). A rabbit anti-MR-1 polyclonal antibody was obtained from polypeptide-immunized New Zealand rabbits [8]. The recombinant adenovirus overexpressing full-length rat MR-1, the stable stealth RNA targeted to rat MR-1, non-target stealth RNA and TRIzol reagents were purchased from Invitrogen (Foster, CA, USA). Phosphatase inhibitor and bovine serum albumin (BSA) were purchased from Merck (Rahway, NJ, USA). The lactate dehydrogenase (LDH) detection kit was purchased from Nanjing Jiancheng Biological Engineering Institute (Nanjing, China), and the Cell Counting Kit-8 (CCK-8) detection kit was from Kaiji Biological Engineering Company (Nanjing, China). Rabbit monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and rabbit monoclonal antibody against Panactin were purchased from Cell Signaling Technology (Danvers, MA, USA). Goat monoclonal antibody against phosphorylated MLCK (p-MLCK), daylight 488 green-conjugated donkey antimouse secondary antibody, and the enhanced chemiluminescence kit were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit monoclonal antibodies against MLC-2, phosphorylated MLC-2 (p-MLC-2), MLCK and Horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) and
rabbit anti-goat IgG were purchased from Epitomics (Burlingame, CA, USA). The mounting media was from Vector Laboratories (Burlingame, CA, USA). The EasyScript First-Strand cDNA Synthesis SuperMix Kit was from TransGen Company (Beijing, China) and 2 Taq mix was from TianGen Technology (Beijing, China). 2.2. Animals Sprague-Dawley (SD) neonatal rats were used for the cardiomyocyte cultures and were purchased from the Experimental Animal Center of Military Medical Sciences. The investigation conformed to the Guide for the Care and Use of Laboratory Animals, published by the United States National Institutes of Health (NIH publication no. 85-23, revised 1996). All animal studies were also approved by the Institutional Animal Care and Use Committee of the Chinese PLA General Hospital. 2.3. Cell culture and experiment protocol Methods for culturing neonatal rat ventricular cardiomyocytes were described in our previous publication [17]. In brief, cardiac ventricles were obtained from 1-day-old SD rats and enzymatically dissociated using a 0.15% trypsin solution in a shaker at 37 °C. Cells were collected by centrifugation to remove noncardiomyocytes and were pre-plated for 1.5 h in DMEM with 10% NCS and 1% penicillin/streptomycin (100 U/mL). Unattached cells were removed and placed into the T-75 flasks with the same medium, and incubated at 37 °C, 5% CO2. Following 24 h, the cells were transferred to serum-free maintenance media (DMEM containing 1% penicillin/streptomycin) for 24 h before experimentation. In order to assess for the protective effects of MR-1 against H/ R injury, cardiomyocytes were divided into seven groups: (1) control group: cells cultured in a 5% CO2 incubator at 37 °C for the duration of the experiment; (2) H/R group: cardiomyocytes were transferred to an incubator (Thermo Fisher Scientific, Waltham, MA, USA) filled with a gas mixture of 90% N2, 5% O2 and 5% CO2 for 8 h of hypoxia and then placed back in the normoxic CO2 incubator at 37°C for 16 h of reoxygenation; (3) Ad-MR-1 group: cardiomyocytes were infected with recombinant adenovirus overexpressing full-length rat MR-1 (MOI ¼50 ifu/cell); (4) st-MR1 group: cardiomyocytes were transfected with stable stealth RNA targeted to rat MR-1. The sequence of the stable stealth RNA against rat MR-1 was 5′-CGACAGCUAACAAGGCUUCCCAGAA-3′ [8]; (5) Ad-MR-1þH/R group: cardiomyocytes were infected with MR1-adenovirus for 24 h and then treated with H/R as described above; (6) st-MR-1þH/R group: cardiomyocytes were transfected with MR-1-RNAi for 24 h, and then treated with H/R as described above; and (7) Mock group: cardiomyocytes were exposed to nontarget 25-base pair silencing oligonucleotides. 2.4. Detection of cell injury and viability LDH activity was assessed in culture medium using an LDH assay kit to estimate LDH leakage under various treatment conditions. CCK-8 detection was an alternative method used for estimating cell viability. This measures metabolic activity through the ability of the cells to convert tetrazolium dye to its insoluble form, formazan [18]. Cells were seeded and plated in 96-well plates and then incubated with CCK-8 for 4 h. Samples were measured using a microplate reader at 450 nm wave-length (Tecan Infinite f200 Pro; Tecan Group Ltd., Männedorf, Switzerland). 2.5. RT-PCR Cardiomyocyte total RNA was extracted using TRIzol reagent, and quantified at 260 nm. The EasyScript First-Strand cDNA
Please cite this article as: T. Tao, et al., Myofibrillogenesis regulator-1 attenuates hypoxia/reoxygenation-induced injury by repairing microfilaments in neonatal rat cardiomyocytes, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.026i
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Synthesis SuperMix Kit was used to perform reverse transcription to obtain cDNA in each group. The cDNA was further amplified using Superscript One-Step polymerase chain reaction with 2 Taq mix. PCR primers (Table 1) were derived from sequences in the GenBank human database. PCR products were separated on 1.0% agarose gels and photographed for analysis using Image-Pro Plus software (Roper Industries, New York, NY, USA). 2.6. Western blot analysis
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Table 1 Primer sequences used for PCR. Gene
Forward primer
Reverse primer
r18s rMR-1
5′-CCAACCCGGTCAGCCCCCTC-3′ 5′-CCAGAACAGG ACCAGAGCA-3′
5′-TACCTCCCCGGGTCGGGAGT-3′ 5′-GGTGAGGACG AAGAAGAGGATA-3′ 5′-AAAGAGGAAA GGCTGTGAAAC3′
rMLC-2 5′-GCGAAAGACA AAGATGACTGA-3′ r¼ rat.
Cardiomyocyte protein was extracted as previously described [8,19]. In brief, cardiomyocytes were plated in T-25 flasks at a cell density of 5 105 cells per flask. The concentration of protein extracted from the cardiomyocytes was detected using Bradford Assay. Proteins were resolved on 10% sodium dodecyl sulfatepolyacrylamide gels using an equal amount of protein (80 μg/ lane). Following electrophoresis, proteins were electrophoretically transferred to nitrocellulose membranes and then blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) at room temperature for 1 h. Then membranes were probed with primary antibodies against MR-1, Pan-actin, MLC-2, p-MLC-2, MLCK, p-MLCK and GAPDH (all 1:500 diluted) at 4 °C overnight. The antibody-tagged membranes were incubated with a secondary antibody solution consisting of either a 1:1000 dilution of HRPconjugated goat anti-rabbit IgG (for MR-1, Pan-actin, MLC-2, p-MLC-2, MLCK and GAPDH) or a 1:1000 dilution of HRP-conjugated rabbit anti-goat IgG (for p-MLCK). An enhanced chemiluminescence detection system was used for immunoblot protein detection. Optical densities of the bands were analyzed using Image-Pro Plus software, and the densitometry results were normalized to GAPDH. 2.7. Immunofluorescence staining Cardiomyocytes were grown on coverslips and fixed using 4% paraformaldehyde at room temperature for 25 min. They were then blocked in phosphate-buffered saline containing 10% donkey serum, 1% BSA and 0.1% Triton X-100 for 50 min. Cardiomyocytes were identified by indirect immunofluorescence staining using anti-α-actinin mouse monoclonal antibody for 2 h at room temperature, followed by FITC green-conjugated donkey anti-mouse secondary antibody (1:400) for 1 h in the dark at room temperature. For F-actin detection, cardiomyocytes were stained in the dark at room temperature for 1 h with phalloidin-FITC at a final concentration of 0.33 μmol/L [8,13]. The coverslips were mounted on glass slides with mounting medium. Images were acquired using a confocal scanning microscope (Zeiss LSM-510 Meta, Jena, Germany), and a 63 oil immersion objective with a numerical aperture of 1.4 was used. 2.8. Statistical analysis The SPSS v13.0 program (Chicago, IL, USA) was used for statistical analysis. For multiple-group comparisons, one-way analysis of variance followed by Newman–Keuls post hoc analysis was performed. Values are presented as mean 7SD. Pearson bivariate correlation analysis was applied to determine the correlation between variables. Po 0.05 was considered to be statistically significant.
3. Results 3.1. Expression of MR-1 in H/R-induced cardiomyocytes To assess the effects of MR-1 on cardiomyocytes with or
without H/R injury, we overexpressed MR-1 using recombinant adenovirus and down-regulated MR-1 using siRNA. Alterations in mRNA expression of MR-1 in cardiomyocytes were detected by RTPCR (Fig. 1A and C). We found that MR-1 mRNA expression decreased by 27.5% in H/R-treated cardiomyocytes compared with controls (Po0.01). An infection of cardiomyocytes with recombinant adenovirus overexpressing MR-1 resulted in a significant increase in MR-1 mRNA expression following H/R treatment (Po0.01). MR-1 mRNA expression in cardiomyocytes increased by 442% in the Ad-MR-1 group (Po 0.01 vs. control) and 508% in the Ad-MR-1þ H/R group (P o0.01 vs. H/R), respectively. MR-1 siRNA treatment down-regulated MR-1 mRNA expression in cardiomyocytes. MR-1 mRNA expression decreased by 66.8% in cardiomyocytes from the st-MR-1 group (P o0.01 vs. Mock) and 58.7% in cardiomyocytes from the st-MR-1 þH/R group (P o0.01 vs. H/R), respectively. Alterations in MR-1 protein expression in cardiomyocytes were assessed using western blot analysis (Fig. 1B and C). H/R-treatment decreased MR-1 protein expression by 33.5% compared with control cardiomyocytes. MR-1 protein expression in cardiomyocytes increased by 610% in the Ad-MR-1 group (Po 0.01 vs. control) and 786% in the Ad-MR-1 þH/R group (Po 0.01 vs. H/R), respectively. MR-1 protein expression decreased by 53.1% in cardiomyocytes from the st-MR-1 group (P o0.01 vs. Mock) and 20.6% in cardiomyocytes from the st-MR-1 þH/R group (P o0.01 vs. H/R), respectively. These data indicate that H/R decreases MR-1 expression, and treatment with recombinant adenovirus or knockdown by siRNA is sufficient to either up-regulate or down-regulate MR-1 expression, respectively. 3.2. Effects of MR-1 on H/R injury in cardiomyocytes 3.2.1. LDH activity LDH activity in the culture medium was measured to estimate the amount of LDH leakage from cardiomyocytes, indicating cell injury (Fig. 2A). LDH activity in the culture medium was 174 712.3 U/L in control cardiomyocytes. H/R induced an increase in LDH leakage compared with controls, and LDH activity in the culture medium increased by 573% in the H/R group compared with control. MR-1 overexpression prior to H/R attenuated H/Rinduced LDH leakage from cardiomyocytes. Compared with the H/ R group, LDH activity in the culture medium decreased by 64.2% in the Ad-MR-1þH/R group (P o0.01), although no significant difference in LDH leakage was detected in cardiomyocytes from the Ad-MR-1 group compared with control (P 40.05). MR-1 knockdown increased LDH leakage from cardiomyocytes, and LDH activity in the culture medium increased by 331% in the st-MR-1 group (P o0.01 vs. Mock). MR-1 knockdown prior to H/R aggravated H/R-induced LDH leakage from cardiomyocytes, and LDH activity in the culture medium increased by 19.0% in the st-MR1 þH/R group (P o0.05 vs. H/R) and by 40.0% compared with the st-MR-1 group (P o0.01).
Please cite this article as: T. Tao, et al., Myofibrillogenesis regulator-1 attenuates hypoxia/reoxygenation-induced injury by repairing microfilaments in neonatal rat cardiomyocytes, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.026i
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3.2.2. Cell viability Cellular viability detected by CCK-8 is shown in Fig. 2B. H/Rtreatment induced a 46.9% decrease in cell viability compared with control (P o0.01). MR-1 overexpression prior to H/R attenuated H/ R-induced cardiomyocyte injury, increasing cell viability by 36.0% in the Ad-MR-1þ H/R group compared with the H/R group (P o0.01), although there was no significant difference in the AdMR-1 group (P4 0.05 vs. control). MR-1 knockdown induced a 20.3% decrease in cell viability (P o0.01 vs. Mock). MR-1 knockdown prior to H/R aggravated H/R-induced cardiomyocyte injury. Cell viability decreased by 8.5% in the st-MR-1 þH/R group (P o0.01 vs. H/R) and by 38.7% compared with the st-MR-1 group (P o0.01). 3.3. Effects of MR-1 on F-actin distribution 3.3.1. F-actin distribution We examined F-actin by staining the cardiomyocytes with phalloidin-FITC, which bound only to F-actin. The fluorescence microphotographs (Fig. 3A) revealed that in control cardiomyocytes, F-actin predominantly appeared in striated like and stress fibers. In H/R-treated cardiomyocytes, F-actin appeared predominantly disorganized with scattered ruptures. F-actin distribution in cardiomyocytes from the Ad-MR-1 group (MR-1 overexpression alone) was similar to controls with increased green fluorescence. MR-1 overexpression prior to H/R attenuated H/Rinduced F-actin fragility, and exhibited a uniform distribution of F-actin in the cytosol in cardiomyocytes from the Ad-MR-1 þH/R group, similar to control cardiomyocytes. MR-1 knockdown caused disorganization of F-actin similar to that in the H/R group. MR-1
Fig. 2. Myofibrillogenesis regulator-1 (MR-1) reduced cardiomyocyte injury and increased cardiomyocyte viability after H/R treatment in vitro. (A) LDH activity in cultured medium. (B) Cell viability by the Cell Counting Kit-8 assay. x¯ 7 s. n ¼3. * P o0.05 vs. control; #Po 0.05 vs. H/R; &P o0.05 vs. Mock.
knockdown prior to H/R aggravated H/R-induced F-actin fragility, showing much more disorganized and scattered rupture of F-actin than that in the H/R group.
Fig. 1. Relative amounts of myofibrillogenesis regulator-1 (MR-1) expression levels in hypoxia/reoxygenation (H/R)-treated cardiomyocytes infected and transfected with MR-1. (A) The level of MR-1 mRNA was examined using RT-PCR. 18s was used as a loading control and for normalization. (B) The level of MR-1 protein was examined using Western blot analysis. GAPDH was used as a loading control and for normalization of data. (C) Bar chart of densitometry of the bands shown in A and B. x¯ 7 s. n ¼3. *Po 0.05 vs. control; #Po 0.05 vs. H/R; &P o0.05 vs. Mock.
3.3.2. F-/G-actin ratio Actin polymerization is critical for cell shape and many regulatory responses including contraction and motility. Therefore, we examined the F-/G-actin ratio by Western blotting analysis to assess polymerization/depolymerization of actin in cardiomyocytes (Fig. 3B). We found that cardiomyocytes in the H/R group showed a 9.0% decrease in the F-/G-actin ratio compared with the control (P o0.01), suggesting that H/R induced depolymerization of actin. MR-1 overexpression increased the F-/G-actin ratio by 11.5% (P o0.01, Ad-MR-1 vs. control). MR-1 overexpression prior to H/R attenuated H/R-induced F-actin depolymerization, exhibiting a 36.2% increase in the F-/G-actin ratio compared with H/R alone (P o0.01). It suggests that MR-1 overexpression induces the polymerization of actin. Although there was no difference between control and Mock-transfected cells, knocking down MR-1 decreased the F-/G-actin ratio by 12.1% compared with the Mock group (P o0.01). Knockdown of MR-1 prior to H/R aggravated H/Rinduced F-actin depolymerization, and the F-/G-actin ratio decreased by 10.6% in the st-MR-1 þH/R group, compared with H/Rtreated cardiomyocytes (P o0.01). These data indicate that MR-1 promotes polymerization of actin monomers and plays an important role in organizing actin filament assembly.
Please cite this article as: T. Tao, et al., Myofibrillogenesis regulator-1 attenuates hypoxia/reoxygenation-induced injury by repairing microfilaments in neonatal rat cardiomyocytes, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.026i
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3.4. Effects of MR-1 on
α-actinin distribution
α-actinin is an actin-binding protein that cross-links actin and forms much of the dense component of both the Z band and fascia adherens plaque. Therefore, we examined the distribution of αactinin in cardiomyocytes by immunofluorescence microscopy (Fig. 4). The fluorescence microphotographs revealed that α-actinin appeared orderly distributed in control cardiomyocytes. In H/ R-treated cardiomyocytes, α-actinin showed a diffuse disorganization. The α-actinin distribution in cardiomyocytes from the Ad-MR-1 group (MR-1 overexpression alone) was similar to controls. MR-1 overexpression prior to H/R attenuated H/R-induced α-actinin disorganization, exhibiting a uniform distribution of α-actinin in the cytosol in cardiomyocytes from the Ad-MR1 þH/R group, which was similar to control cardiomyocytes.
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Although there was no difference between control and Mocktransfected cells, knocking down MR-1 itself induced α-actinin disorganization. MR-1 knockdown prior to H/R aggravated H/Rinduced α-actinin disorganization, and predominantly presented the disorganization of α-actinin in the st-MR-1þH/R group compared with H/R-treated cardiomyocytes.
3.5. Effects of MR-1 on MLC-2 expression and phosphorylation MR-1 interacts directly with MLC-2 and phosphorylation of MLC-2 promotes G-actin polymerization to form F-actin. Therefore, we examined MLC-2 expression and phosphorylation to investigate the regulating responses induced by MR-1.
Fig. 3. Effect of myofibrillogenesis regulator-1 (MR-1) on the localization and organization of F-actin and the F-/G-actin ratio influenced by H/R treatment in vitro. (A) Under laser scanning confocal microscope, F-actin in cardiomyocytes was labeled with phalloidin-FITC (600 , bar¼ 20 μm). The images are representative of the majority of the cells. More than 100 cells were investigated in total by IF microscopy, and more than 70% of the total number of investigated cells were the morphological changes of the actin cytoskeleton observed. The ratio in each group was as follows: 72.4% in control group, 70.8% in H/R group, 70.4% in Ad-MR-1 group, 76.5% in st-MR-1 group, 71.2% in AdMR-1þ H/R group, 71.3% in st-MR-1 þ H/R group and 76.1% in Mock group, respectively. (B) The levels of F-actin and G-actin were examined using Western blot analysis. Bar chart of the F-/G-actin ratio calculated by densitometry of the bands. x¯ 7 s. n¼ 3. *Po 0.05 vs. control; #P o 0.05 vs. H/R; &Po 0.05 vs. Mock.
Please cite this article as: T. Tao, et al., Myofibrillogenesis regulator-1 attenuates hypoxia/reoxygenation-induced injury by repairing microfilaments in neonatal rat cardiomyocytes, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.026i
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3.5.1. MLC-2 expression MLC-2 mRNA and protein expression was detected by RT-PCR and Western blotting analysis, respectively (Fig. 5A–D). Compared with control, the MLC-2 expression increased by 26.7% (mRNA) and 50.5% (protein) in H/R-treated cardiomyocytes, respectively (Po0.01). MR-1 overexpression increased MLC-2 expression by 92.6% (mRNA) and 112% (protein), respectively (Po0.01 vs. control). MR-1 overexpression prior to H/R increased H/R-induced MLC-2 expression, exhibiting a 26.8% (mRNA) and a 24.8% (protein) increase in MLC-2 expression compared with H/R alone, respectively (Po0.01). Although there was no difference between control and Mock-transfected cells, knocking down MR-1 decreased MLC-2 mRNA and protein expression by 31.5% and 23.2% compared with the Mock group, respectively (Po0.01). Knockdown of MR-1 prior to H/R decreased MLC-2 mRNA and protein expression by 18.8% and 55.9% as compared with H/R-treated cardiomyocytes, respectively (Po0.01). Our data suggest that MR-1 is involved in the regulation of transcription and translation of MLC-2. 3.5.2. MLC-2 phosphorylation MLC-2 phosphorylation was detected by using Western blotting analysis (Fig. 5C and D). Compared with control, the MLC-2 phosphorylation increased by 53.2% in H/R-treated cardiomyocytes (P o0.01). MR-1 overexpression increased MLC-2 phosphorylation by 70.8% (Po 0.01, Ad-MR-1 vs. control). MR-1 overexpression prior to H/R increased H/R-induced MLC-2 phosphorylation, exhibiting a 29.6% increase in MLC-2 phosphorylation compared with H/R alone (P o0.01). Although there was no difference between control and Mock-transfected cells, knocking down MR-1 decreased MLC-2 phosphorylation by 23.3% compared with the Mock group (P o0.01). Knockdown of MR-1 prior to H/R decreased MLC-2 phosphorylation by 40.6%, compared with H/Rtreated cardiomyocytes (Po 0.01). These data provide further evidence that MR-1 is also involved in the regulation of MLC-2 phosphorylation. 3.6. Effects of MR-1 on MLCK expression and phosphorylation MLCK is the predominant kinase for MLC-2 phosphorylation and MR-1 knockdown down-regulated MLC-2 phosphorylation.
Therefore, we examined the MLCK expression and phosphorylation to investigate the effects of MR-1 on the MLCK/MLC-2 pathway in this study. Alterations in MLCK protein expression and phosphorylation in cardiomyocytes were detected using Western blot analysis. As shown in Fig. 5E and F, cardiomyocytes from the H/R group showed a 14.7% and a 37.2% increase in MLCK expression and phosphorylation compared with controls, respectively (P o0.01). MR-1 overexpression increased MLCK expression and phosphorylation by 40.8% and 30.4%, respectively (P o0.01 vs. control). MR1 overexpression prior to H/R increased H/R-induced MLCK expression and phosphorylation, exhibiting a 17.7% and a 19.1% increase in MLCK expression and phosphorylation compared with H/ R alone, respectively (P o0.01). There were no differences in MLCK expression and phosphorylation between the control and Mock-transfected cells. Knocking down MR-1 decreased MLCK expression by 13.3% without the effect of MLCK phosphorylation, compared with the Mock group (P o0.01). Knockdown of MR-1 prior to H/R down-regulated H/R-induced MLCK expression and phosphorylation. MLCK expression and phosphorylation decreased by 17.9% and 13.1% in the st-MR-1þ H/R group, compared with H/ R-treated cardiomyocytes, respectively (P o0.01). These data indicate that MR-1 is also involved in the regulation of transcription and translation of MLCK. In addition, correlation analysis showed a significant positive correlation between the phosphorylation levels of MLC-2 and MLCK (r ¼0.829, Po 0.01). It provides further evidence to the suggestion that MR-1 protects cardiomyocytes from H/R injury by promoting F-actin organization through MLCK/MLC2 pathway.
4. Discussion Potential mechanisms underlying the pathogenesis and treatment of myocardial ischemia/reperfusion (I/R) injury are largely unknown. Studies of the molecular consequences of myocardial I/R injury suggest that the actin filaments and their binding proteins are potential targets for the prevention and treatment of I/R injury [5,9,10]. The rat neonatal cardiomyocyte model provides an efficient in vitro model for I/R as it assesses the profile of contractile
Fig. 4. Effect of myofibrillogenesis regulator-1 (MR-1) on the localization and organization of α-actinin influenced by H/R treatment in vitro. Under a laser scanning confocal microscope, α-actinin in cardiomyocytes was observed using immunofluorescence microscopy (600 , bar ¼ 20 μm). The images are representative of the majority of the cells. More than 100 cells were investigated in total by IF microscopy, and more than 70% of the total number of investigated cells were the morphological changes of the αactinin observed. The ratio in each group was as follows: 80.0% in control group, 73.7% in H/R group, 82.4% in Ad-MR-1 group, 86.3% in st-MR-1 group, 75.9% in Ad-MR-1 þH/ R group, 78.6% in st-MR-1 þH/R group and 84.0% in Mock group, respectively.
Please cite this article as: T. Tao, et al., Myofibrillogenesis regulator-1 attenuates hypoxia/reoxygenation-induced injury by repairing microfilaments in neonatal rat cardiomyocytes, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.026i
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Fig. 5. Effect of myofibrillogenesis regulator-1 (MR-1) on the expression and phosphorylation of MLC-2 and MLCK in H/R-treated cardiomyocytes. (A, B) MLC-2 mRNA level was examined by RT-PCR with 18s used as a loading control and for normalization of data. (C, D) MLC-2 protein expression and phosphorylation were examined using Western blot analysis. GAPDH was used as a loading control and for normalization of data. (E, F) MLCK protein expression and phosphorylation were examined using Western blot analysis. GAPDH was used as a loading control and for normalization of data. x¯ 7 s. n¼3. *P o0.05 vs. control; #Po 0.05 vs. H/R; &P o 0.05 vs. Mock.
proteins during early stages of H/R, which is compatible with myocardial injury during I/R [20]. MR-1 is a functional gene that is highly expressed in the myocardium, skeletal muscle, kidney and liver [8,12,13,21]. Our previous studies showed that overexpressing MR-1 attenuated H/R-induced apoptosis in cardiomyocytes [22]. In this study, we demonstrated that hypoxia/reoxygenation (H/R) down-regulated endogenous expression of MR1, induced F-actin disruption and depolymerization and α-actinin disorganization, and caused an increase in LDH leakage and a decrease in cell viability in cardiomyocytes. These results suggest that MR-1 down-regulation is involved in the damage of actin filaments in cardiomyocytes during H/R injury. We then examined the above possibility mechanistically in MR-1-overexpressing and MR-1-knockdown cardiomyocytes. We found that MR-1 overexpression attenuated, while MR-1 knockdown aggravated H/Rinduced F-actin disruption and depolymerization and α-actinin disorganization. MR-1 overexpression also decreased H/R-induced
LDH leakage and presented an increase in cell viability in cardiomyocytes subjected to H/R. Our data indicate that MR-1 promotes polymerization of actin monomers and plays an important role in repairing actin filament during H/R. Finally, we found that this process involved the increased expression and phosphorylation of MLC-2 and MLCK. MR-1 overexpression up-regulated while MR-1 knockdown down-regulated MLCK expression and phosphorylation, which both activated MLC-2. In addition, MR-1 also induced MLC-2 expression, which further increased MLC-2 total phosphorylation. When combined with our previous study that showed MR-1 interacted directly with MLC-2, and MLC-2 was critical for actin polymerization to form F-actin [23] and regulated by MLCK [13], these data demonstrate that MR-1 protects cardiomyocytes from H/R injury by attenuating damage of actin filaments and activating the MLCK/MLC-2 pathway. The cytoskeleton, comprised of actin filaments, microtubules, intermediate filaments, and their associated binding and
Please cite this article as: T. Tao, et al., Myofibrillogenesis regulator-1 attenuates hypoxia/reoxygenation-induced injury by repairing microfilaments in neonatal rat cardiomyocytes, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.026i
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attachment proteins, is responsible for contraction, survival and shapes of cardiomyocytes [2–6]. In cardiomyocytes, the actin filaments form fine filaments and attachments to the cytoplasmic membrane at the fascia adherens junctions of the intercalated disk, and at the lateral costamere junctions. Actin polymerization is critical for cell shape and many regulatory responses including contraction and motility [4]. I/R induced actin depolymerization and disorganization in cardiomyocytes [24]. Furthermore, Actin cytoskeleton disorganization causes the disappearance of central stress fibers and leads to an early step in most cells undergoing apoptosis by a loss of survival signals [25]. The disorganization of F-actin further induced apoptosis in mouse embryonic fibroblast cells, with the decrease of cell viability [26]. In this study, we used the F-/G-actin ratio to reflect the polymerization/depolymerization rate of actin. We found that H/R induced F-actin disorganization, causing depolymerization and re-arrangement, suggesting that H/ R induced disorganization and depolymerization of actin, which coincided with F-actin disassembly as a consequence of sustained myocardial I/R injury [24]. MR-1 overexpression attenuated, while MR-1 knockdown aggravated H/R-induced F-actin disorganization and depolymerization, indicating that MR-1 promoted polymerization of actin monomers and organizing actin filament assembly. Our data are consistent with the findings that MR-1 overexpression promotes actin polarity in H/R-treated HK-2 cells [13]. α-Actinin is one of the actin-binding proteins that ensures F-actin connects together to form bundles [2]. The damaged αactinin aggravates F-actin derangement. Ganote et al. indicated a loss of α-actinin from 90-min anoxic hearts when examined under high-voltage electron-microscopy [27]. Our study further confirmed that H/R induced α-actinin destruction. Furthermore, MR-1 overexpression attenuated while MR-1 knockdown aggravated H/ R-induced α-actinin disorganization, suggesting that MR-1 protects α-actinin from H/R injury. MR-1, located in the myofibrils, interacted directly with MLC-2 and induced cardiomyocyte hypertrophy via sarcomere organization [8]. MLC-2 is the predominant substrate of MLCK [6], which phosphorylated MLC-2 at two sites (Ser19 and Thr18) [7,28]. Studies verified that MLCK overexpression also induced MLC-2 activation [6]. MLC-2 phosphorylation is critical for actin polymerization to form F-actin. Tu et al. found that I/R induced rupture of myocardial fibers and degradation of F-actin as well as MLC-2 phosphorylation in the myocardial tissue [24]. Kim et al. observed that I/R increased expression of genes encoding MLC-2 in rat hearts, which compensated for disorganization of sarcomeric myofibrillar proteins [29,30]. In this study, we found that H/R down-regulated MR-1 expression and induced disruption of F-actin and α-actinin disorganization with cardiomyocyte injury, as well as the up-regulation of MLC-2 expression and phosphorylation. MLC-2 up-regulation may compensate for disorganization of F-actin, which is consistent with Kim's results. In addition, MR-1 overexpression increased, while MR-1 knockdown decreased MLCK expression and phosphorylation, which both activated MLC2. MR-1 also up-regulated MLC-2 expression and further increased total MLC-2 phosphorylation. MLC-2 phosphorylation accelerated actin polymerization [31]. It suggest that the MLCK/MLC-2 pathway is involved in MR-1-mediated F-actin organization in the setting of H/R injury, hence cardioprotection during H/R. Moreover, the mechanism of MR-1-mediated cardiac hypertrophy and microfilament reorganization is similar. MR-1 overexpression induced cardiomyocyte hypertrophy by promoting sarcomere rapid reorganization with the increase of MLC-2 expression [8]. Our data illustrated that MR-1 also up-regulated MLC-2 expression and phosphorylation, which attenuated H/Rinduced microfilament destruction by promoting microfilament reorganization. However, previous studies showed that 24 h after Ad-MR-1 overexpression promotes rapid organization of
Fig. 6. Proposed pathway of MR-1-mediated regulation of microfilament reorganization during H/R. H/R induced microfilament disorganization and depolymerization, and MR-1 regulated microfilament reorganization through MLCK/MLC2 pathway. MR-1 up-regulated expression and phosphorylation of MLCK, which both activated MLC-2. In addition, MR-1 also induced MLC-2 expression, which further increased MLC-2 total phosphorylation. MLC-2 phosphorylation promoted microfilament reorganization in cardiomyocytes. (H/R: hypoxia/reoxygenation, MR1: myofibrillogenesis regulator-1, MLCK: myosin light chain kinase, MLC-2: myosin regulatory light chain-2.)
sarcomeres while did not induce cardiomyocyte hypertrophy until 48 h after MR-1 overexpression. Hypertrophic hallmarks such as cell size, [3H]-leucine incorporation, and expression of ANF and BNP were significantly increased at 48 h after MR-1 overexpression. However, in this study, H/R was induced at 24 h after MR-1 overexpression, when cardiomyocyte hypertrophy was unhappened. It indicates that MR-1 overexpression protect disorganization of microfilaments against H/R injury, without involving MR-1-mediated hypertrophy at that time point. In summary, MR-1 promotes microfilament organization and protects cardiomyocytes from H/R injury. The underlying mechanism might involve the correct organization of F-actin through the MLCK/MLC-2 pathway. The proposed pathway of MR-1mediated regulation of microfilament reorganization was summarized in Fig. 6. Our studies may help uncover therapeutic targets for the prevention and treatment of I/R injury.
Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant nos. 81170140, 31471094 and 81070130 (XH Liu)).
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Please cite this article as: T. Tao, et al., Myofibrillogenesis regulator-1 attenuates hypoxia/reoxygenation-induced injury by repairing microfilaments in neonatal rat cardiomyocytes, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.026i
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Please cite this article as: T. Tao, et al., Myofibrillogenesis regulator-1 attenuates hypoxia/reoxygenation-induced injury by repairing microfilaments in neonatal rat cardiomyocytes, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.026i