ORIGINAL ARTICLE
Regulator of Calcineurin 1-1L Protects Cardiomyocytes Against Hypoxia-induced Apoptosis via Mitophagy Lijie Yan, PhD, Haitao Yang, PhD, Yongqiang Li, PhD, Hongyan Duan, PhD, Jintao Wu, MD, Peng Qian, PhD, Bing Li, MD, and Shanling Wang, MD
Abstract: Mitochondrial dysfunction induced by myocardial ischemia is the primary cause of cardiac cell death. Specific removal of damaged mitochondria through mitophagy may be beneficial for cardiomyocyte protection against ischemia. Regulator of calcineurin 1-1L (Rcan1-1L) has been implicated in mitophagy induction in neurons. However, whether or not Rcan1-1L can evoke mitophagy in cardiomyocytes during hypoxia remains unknown. This study aims to investigate the effect of Rcan1-1L overexpression on cardiomyocytes during hypoxia and the possible underlying mechanism. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that Rcan1-1L overexpression inhibited cell growth under normoxic conditions, whereas Rcan1-1L overexpression significantly reversed the growth inhibition induced by hypoxia. The results of terminal deoxynucleotidyl transferase biotin-dUTP nick end-labeling assay showed that cell apoptosis induced by hypoxia was markedly reduced by Rcan1-1L overexpression. In addition, Rcan1-1L overexpression inhibited the expression of the proapoptotic protein Bcl-2– associated death promoter and increased that of the antiapoptotic protein Bcl-2. Rcan1-1L overexpression opened the mitochondrial permeability transition pore and decreased mitochondrial mass. Meanwhile, the release of reactive oxygen species from mitochondria was suppressed by Rcan1-1L. Autophagy flow activation represented by mammalian target of rapamycin inhibition and microtubule-associated protein light chain 3 (LC3) upregulation was also demonstrated. Compared with endoplasmic reticulum and Golgi apparatus protein markers, the mitochondrial protein marker translocase of outer mitochondrial membranes 20 (TOM20) was downregulated by Rcan1-1L overexpression. Moreover, Rcan1-1L increased mitophagy receptor Parkin translocation into mitochondria from cytosol. Additionally, the effect of Rcan1-1L on cell growth, cell apoptosis and mitochondria mass was blocked by Parkin expression silencing. Overall, these data suggest that Rcan1-1L protects cardiomyocytes from hypoxia-induced apoptosis by inducing mitophagy partly through Parkin. This study provided novel insights into the prevention and treatment of ischemic heart disease. Key Words: myocardial ischemia, hypoxia, apoptosis, mitophagy (J Cardiovasc Pharmacol 2014;64:310–317)
Received for publication February 10, 2014; accepted April 23, 2014. From the Department of Cardiology, Henan Provincial People’s Hospital, Zhengzhou, China. Lijie Yan, PhD, and Haitao Yang, PhD, contributed equally to this manuscript. The authors report no conflicts of interest. Reprints: Shanling Wang, MD, Department of Cardiology, Henan Provincial People’s Hospital, Zhengzhou, China 450003 (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
310
| www.jcvp.org
INTRODUCTION Coronary heart disease with high morbidity and mortality is a leading cause of death worldwide.1 Reperfusion therapy can attenuate myocardial infarction and prevent heart failure; however, this treatment often causes myocardial ischemia/reperfusion, which leads to myocardial damage and pathological remodeling.2–4 Thus, development of effective strategies to protect cardiomyocytes from hypoxia-induced damage may provide novel insights into the prevention of myocardial damage during cardiac surgery. Mitochondria, the most abundant organelles in cardiomyocytes, supply energy for myocardial contraction that is vulnerable under hypoxic conditions. Cell death pathways activated by dysfunctional mitochondria lead to excessive reactive oxygen species production and insufficient ATP production, which promote infarct expansion and heart failure progression after ischemic injury progression.5–7 Efficient and specific clearance of dysfunctional mitochondria is beneficial for cardiomyocyte protection under hypoxic conditions. Recent studies have demonstrated that mitochondria can be selectively removed by autophagy (termed as mitophagy), which is essential for cellular homeostasis especially for cardiomyocytes.8 Mitophagy is controlled by PTEN-induced kinase protein 1 (PINK1) and Parkin. Parkin, which is usually located in the cytoplasm under normal conditions, can be quickly recruited to mitochondria by the accumulated PINK1 upon mitochondrial membrane depolarization; the recruited Parkin then ubiquitylates mitochondrial proteins by its E3 ubiquitin ligase activity and causes mitophagy.9 BNIP3 and NIX are mitophagy regulators that bind with the autophagy protein, microtubule-associated protein-light chain 3 (LC3) to form autophagy lysosomal membrane.10,11 Fundc1 has been recently identified to be a new receptor for mitophagy in mammalian cells; this receptor interacts with LC3 to form autophagy lysosomal membrane.12 These studies have demonstrated that mitochondria can be selectively cleared by mitophagy. Therefore, analyzing mitophagy may provide insights into the pathogenesis and treatment of disease therapy. Regulator of calcineurin 1 (Rcan1), also named Adapt78, DSCR1, and MCIP1, is an adaptive protein that is induced during cellular stress.13–16 The Rcan1 gene is located in the q22.12 region of chromosome 21; this gene consists of 7 exons that can produce different mRNA and protein isoforms.17,18 Rcan1 has 3 isoforms identified so far: RCAN11L, RCAN1-1S, and RCAN1-4.14,19,20 The original function J Cardiovasc Pharmacol Volume 64, Number 4, October 2014
J Cardiovasc Pharmacol Volume 64, Number 4, October 2014
of Rcan1 is to regulate calcineurin activity.21–23 Current studies have shown that Rcan1 has other functions. Rcan1 interacts with mitochondrial adenine nucleotide translocator and regulates ADP/ATP exchange in Drosophila.24 Rcan1 also modulates cell apoptosis.25–28 RCAN1-1L has been recently demonstrated to be a regulator of mitophagy that efficiently causes dramatic degradation of mitochondria.29 Whether Rcan1-1L can protect cardiomyocytes from hypoxia by inducing mitophagy remains unknown. In the present study, we investigated the effect of Rcan1-1L on hypoxia-treated cardiomyocytes for the first time. We found that Rcan1-1L overexpression in cardiomyocytes during hypoxia elicits cytoprotective effects by activating mitophagy. The results of this study suggest Rcan1-1L as a novel target for the treatment of ischemic heart disease.
Hypoxia-induced Apoptosis via Mitophagy
Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End-labeling Staining
After indicated treatments, ells were fixed with 4% paraformaldehyde solution for 10 minutes at room temperature followed by washing with PBS. Then, the cells were permeabilized with 0.1% sodium citrate and 0.1% Triton X-100 for 10 minutes. After washing with PBS, cell apoptosis were detected using a terminal deoxynucleotidyl transferase biotin-dUTP nick end-labeling (TUNEL) kit (Beyotime, Beijing, China) according to the supplier’s instructions. Cells were observed and photographed by fluorescence microscope (Olympus, Tokyo, Japan). Quantitative analysis was presented as TUNEL-positive cells per field counted in 5 random fields, and the counts were averaged.
Caspase-3 Activity Assay MATERIALS AND METHODS Cell Culture Human adult cardiomyocytes obtained from Celprogen (California) were maintained in human cardiomyocyte cell culture complete media with serum and antibiotics. These cells were cultured at 378C with 5% CO2 in an incubator (Life Technologies, Baltimore, MD).
Cell Treatment and Transfection For plasmid transfection, plasmid pcDNA-Rcan1-1L (1 mg) diluted in 500 mL of cardiomyocyte cell culture complete media with 5 mL lipofectamine (Invitrogen, Carlsbad, CA) were added to the cells and cultured for indicated times: 12, 24, and 48 hours. Cells transfected null vectors and phosphate-buffered saline (PBS) were taken as control. Cells were cultured under normoxic or hypoxia conditions. Hypoxia was achieved by placing the cells in a hypoxia chamber filled with 5% CO2 and 95% N2 at 378C. For small-interfering RNAs (siRNAs) transfection, 0.1 mM siRNA targeting Parkin (sc-42158; Santa Cruz, CA) diluted in 500 mL of medium with 5 mL lipofectamine (Invitrogen) were added to cells and incubated for 24 hours under hypoxic conditions. Cells transfected with nonspecific siRNA (sc-37007) were taken as control.
3-(4,5-Dimethylthiazol-2-yl)-2,5Diphenyltetrazolium Bromide Assay For the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the cells were plated in 96-well plates (5 · 104 cells per well) and cultured under regular conditions until 80% confluence was reached. After the cells transfected with plasmid DNA or siRNAs and incubated for 12, 24 and 48 hours, the culture medium was replaced with 200 mL fresh medium that contains MTT (5 mg/mL in PBS; Sangon, Shanghai, China) and then incubated with cells for an additional 4 hours. Dimethyl sulfoxide (150 mL/well) was added to dissolve formazan for 10 minutes, and the absorbance at 490 nm was determined using an ELISA reader (Bio-tek Instruments, Winooski, VT). The experiment was performed in quadruplicate and was repeated thrice. 2014 Lippincott Williams & Wilkins
Cells were harvested and homogenized in ice-cold Trisbuffered saline (TBS) and centrifuged at 12,000 rpm at 48C for 20 minutes. The supernatants were collected and incubated with 5 mM Ac-DEVD-MCA at 378C for 5 minutes. The release of 7-amino-4-methylcoumarin was measured using a spectrofluorometer (Hitachi F-2000; Tokyo, Japan) at 380 nm excitation wavelength and 460 nm emission wavelength.
Fluorescence-activated Cell Sorting Analysis For mitochondrial mass analysis, the cells were resuspended in 380 mL PBS that contains 200 nM Mitotracker Green FM (Invitrogen). Afterward, the mixture was incubated at 378C and then shaken gently for 15 minutes. Finally, fluorescence was assessed using a fluorescence-activated cell sorting (FACS)calibur analyzer flow cytometer at 530 nm excitation wavelength (FL-1). mPTP opening was determined as previously described.30 Briefly, the cells were incubated in culture that contains 1 mM calcein-AM (Molecular Probes) at 378C in the dark for 30 minutes. Afterward, 1 mM CoCl2 (Sigma, St Louis, MO) was added and incubated for another 10 minutes in the dark at 378C. Thereafter, the fluorescence signal of mitochondria-trapped calcein was analyzed using a FACSCalibur flow cytometer at 530 nm excitation wavelength (FL-1). For each experiment, calcein fluorescence from 30,000 cells was acquired using FACSCalibur flow cytometer, and median value analysis was conducted using FlowJo software.
Intracellular Reactive Oxygen Species Measurement Cells transfected with plasmid DNA or siRNAs were cultured under hypoxic conditions for 24 hours. After cells were harvested and washed with PBS, cells were incubated with 50 mM 20 ,70 -dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma) at 378C for 45 minutes in the dark. Then, the fluorescence intensity was measured by using Multi-Detection microplate reader (Bio-tek Instruments) at 485 nm excitation wavelength and 530 nm emission wavelength. The experiment was performed in quadruplicate and was repeated thrice.
Mitochondrial Protein Extraction Mitochondrial and cytosolic fractions were isolated using a mitochondria isolation kit (Pierce, Rockford) according to the manufacturer’s instructions. Briefly, the cells were www.jcvp.org |
311
Yan et al
collected and homogenized in ice-cold lysis buffer A followed by centrifugation at 800g for 10 minutes at 48C. After the supernatants were collected, a total of 500 mL mitochondria isolation reagent buffer were added in followed by centrifugation at 15,000g for 20 minutes at 48C. Then, the supernatant containing the cytosolic fraction was collected and stored at 2208C for further analysis. The pellet enriched in mitochondria were collected and washed by he rinsing buffer followed by centrifugation at 15,000g for 10 minutes at 48C. The pellet was collected and resuspended in ice-cold lysis buffer B followed by centrifugation at 12,000 rpm for 15 minutes at 48C. Finally, the supernatant containing mitochondrial protein were collected for further analysis.
J Cardiovasc Pharmacol Volume 64, Number 4, October 2014
antibodies (Boster Corporation, Wuhan, China) diluted in the blocking buffer for 1 hour. Finally, the membrane was washed, and 4-chloro-1-naphthol (4-CN) (1 mL) with TBS (9 mL) containing 6 mL of H2O2 was added for protein visualization.
Statistical Analysis All assays were performed in triplicate, and data are presented as mean 6 SD. Comparisons between 2 groups were determined by Student t test, and among groups were tested using 1-way analysis of variance followed by Bonferroni post hoc. Statistical significance was considered at P , 0.05. All statistical analyses were performed using SPSS version 11.5 (SPSS Inc, Chicago, IL).
Western Blot Analysis
Approximately 50 mg of protein was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by electroblotting onto a nitrocellulose membrane (Amersham, Little Chalfont, United Kingdom). The membrane was incubated in TBS that contains 2% nonfat dry milk to block nonspecific binding at room temperature for 1 hour. Afterward, the membrane was washed by TBS and incubated with primary antibodies in the blocking buffer overnight at 48C. Anti-Rcan1-1 antibodies (D6694) diluted in 1:1000 were purchased from Sigma. Anti-mTOR antibody (sc-1549) diluted in 1:200, anti-p70 S6 kinase a antibody (sc-393967) diluted in 1:200, anti-Bcl-2 antibody (sc-492) diluted in 1:500, anti-Bad antibody (sc-943) diluted in 1:200, anti-TOM20 (sc-17764) diluted in 1:500, anti-GM130 (sc-55590) diluted in (1:800) and anti-Calnexin (sc-11397) diluted in 1:800 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-LC3B antibody (ab63817) diluted in 1:400, anti-Parkin antibody (ab77924) diluted in 1:200 and anti-PINK1 antibody (ab23707) diluted in 1:600 were purchased from Abcam (Cambridge, United Kingdom). Anti-GAPDH antibody (bs-2188R) and anti-a-tubulin antibody (bs-4511R) were purchased from Bioss (Beijing, China). Subsequently, the membrane was washed thrice with TBS–Tween. The membrane was then incubated in horseradish peroxidase–conjugated secondary
RESULTS Rcan1-1L Improves Cardiomyocytes Under Hypoxic Conditions To investigate the role of Rcan1-1L on cell growth, we induced Rcan1-1L overexpression in cultured cardiomyocytes through transfection with pcDNA3.0-Rcan1-1L expression vectors under normoxic conditions. Western blot analysis showed that Rcan1-1L was overexpressed for 12, 24, and 48 hours compared with the control or null vector transfection (Fig. 1A). Using MTT assay, we found that cell growth was inhibited by Rcan1-1L (Fig. 1B). We next explored the effect of Rcan1-1L on cardiomyocytes under hypoxic conditions. The results showed that Rcan1-1L overexpression promoted cell growth and viability compared with the cells transfected with PBS or null vector under hypoxic conditions (Figs. 2C, D). These data indicated that Rcan1-1L overexpression under hypoxic conditions promoted the survival of cardiomyocytes.
Rcan1-1L Resists Hypoxia-induced Cell Apoptosis in Cardiomyocytes Hypoxia treatment generally induces cell apoptosis in cardiomyocytes. We conducted TUNEL assay to analyze cell apoptosis and explore the effect of Rcan1-1L on cell
FIGURE 1. Rcan1-1L expression in cardiomyocytes and its effect on cell growth. A, Western blot analysis was performed to analyze the protein expression of Rcan1-1L in normoxictreated cells. PBS, cells treated with PBS were taken as control. Vector, cells were treated with null vector without Rcan1-1L. Rcan1-1L, cells were treated with Rcan1-1L expression vectors. Cells were cultured under normoxic conditions for 12, 24, and 48 hours. B, Effect of Rcan11L on cell growth under normoxic conditions was measured by MTT assay. †P , 0.05 versus PBS group; *P , 0.05 versus null vector group. C, Western blot analysis of Rcan1-1L expression in hypoxia-treated cells. D, Effect of Rcan1-1L on cell growth under hypoxic conditions was measured by MTT assay. ††P , 0.01 versus PBS group; **P , 0.01 versus null vector group.
312
| www.jcvp.org
2014 Lippincott Williams & Wilkins
J Cardiovasc Pharmacol Volume 64, Number 4, October 2014
Hypoxia-induced Apoptosis via Mitophagy
FIGURE 2. Effect of Rcan1-1L on cardiomyocyte apoptosis under hypoxic conditions. A, Cell apoptosis was tested by TUNEL assay. Cells treated with Rcan1-1L expression vectors, null vectors or PBS for 24 hours under hypoxia for 24 hours. B, Quantitative analysis of TUNEL-positive cells. ††P , 0.01 versus PBS group; **P , 0.01 versus null vector group. C, Caspase-3 activity measurement. Protein lysate of cardiomyocytes was examined by incubation with Ac-DEVD-MCA for 5 minutes at 378C. The release of 7amino-4-methylcoumarin was measured using a spectrofluorometer. ††P , 0.01 versus PBS group; **P , 0.01 versus null vector group. D, Western blot analysis was performed to examine the protein expression levels of Bcl-2 and Bad in cardiomyocytes.
apoptosis during hypoxia treatment. Compared with the cells under normoxic conditions, the cells transfected with PBS or null vectors showed a significant increase in cell apoptotic rate; this increase was reversed by Rcan1-1L expression (Figs. 2A, B). To further confirm the results, we detected the activity of caspase-3, a key downstream effector protein of apoptosis. The activity of caspase-3 in the Rcan1-1L expression group was significantly downregulated compared with that in the cells transfected with PBS or null vector (Fig. 2C). We also monitored apoptosis-related proteins, including the antiapoptotic protein Bcl-2 and the proapoptotic protein Bcl2–associated death promoter (Bad) by western blot. The protein expression level of Bcl-2 that decreased during hypoxia was upregulated by Rcan1-1L. By contrast, the protein expression level of Bad that increased during hypoxia was downregulated by Rcan1-1L (Fig. 2D).
Rcan1-1L Overexpression Leads to Mitochondrial Mass Reduction and mPTP Opening We determined the effect of Rcan1-1L on mitochondria to further explore the mechanism by which Rcan1-1L regulates cell apoptosis. In the present study, mitochondrial mass was measured using FACS analysis. The cells were labeled with MitoTracker Green, and the total signals in each cell were measured. Rcan1-1L overexpression significantly decreased mitochondrial mass (Fig. 3A). We also determined the effects of Rcan1-1L on mPTP. The results showed that 2014 Lippincott Williams & Wilkins
mPTP opening peaked at 24 hours and then decreased; despite this decrease, this opening was still higher than that at 0 hours (Fig. 3B). Moreover, the release of ROS from mitochondria was significantly downregulated by Rcan1-1L (Fig. 3C).
Rcan1-1L Overexpression Stimulates Mitophagy Mitophagy is the most common mechanism of mitochondrial mass reduction. A previous study implied that Rcan1-1L can evoke mitophagy.29 In the present study, we determined whether Rcan1-1L expression in cardiomyocytes under hypoxic conditions can induce mitophagy. Therefore, we first analyzed the autophagy flow in the cells. The main mediator of autophagy is the kinase mTOR, which can inhibit the initiation of autophagy.31 LC3-I/II has an important function in forming autophagosomal membranes, and LC3-II is a validated marker of autophagic compartments.32 In the present study, mTOR and its downstream gene S6K were inhibited in the Rcan1-1L–transfected cells compared with the control groups. Furthermore, the protein expression level of LC3-II increased, implying that autophagy flow was activated by Rcan1-1L expression (Fig. 4A). We analyzed whether other organelles were altered to confirm that the autophagy flow was mitochondrion specific. The results showed that the protein expression level of the mitochondrial marker TOM20 was downregulated in the Rcan1-1L–transfected cells, whereas those of the Golgi body marker GM130 and endoplasmic reticulum marker calnexin remained unchanged www.jcvp.org |
313
Yan et al
J Cardiovasc Pharmacol Volume 64, Number 4, October 2014
FIGURE 4. Effect of Rcan1-1L on mitophagy in hypoxia-treated cardiomyocytes. Western blot analysis was performed to determine the protein expression levels of autophagy markers, mTOR, S6K, and LC3-I/II (A) mitochondria marker, TOM20, Golgi body marker GM130 and endoplasmic reticulum marker calnexin (B) in cardiomyocytes. GAPDH was used as control. C, Western blot analysis was conducted to determine the protein expression levels of Parkin and PINK1 in cytosol and mitochondria fractions. a-Tubulin was used as cytosol protein control and VDAC was used as mitochondria protein control. FIGURE 3. Effect of Rcan1-1L on mitochondria. A, Rcan1-1L overexpression reduced mitochondrial mass. Cells were transfected with vectors that contain Rcan1-1L or null vectors. Mitochondria were labeled with MitoTracker Green, and signals were measured using the FACS analyzer. Mitochondrial mass was evaluated by comparing fluorescence signals. †P , 0.05 versus PBS group; *P , 0.05 versus null vector group. B, Rcan1-IL overexpression led to mPTP opening. mPTP detection using calcein fluorescence dye. Mean fluorescence values were converted to arbitrary units (AU). Fluorescence values in null vector–transfected cells were set as 1.0. *P , 0.05 versus null vector group. C, Rcan1-1L overexpression decreased reactive oxygen species release. Reactive oxygen species levels were determined using DCFH-DA assay after cells were cultured under hypoxia for 24 hours. †P , 0.05 versus PBS group; *P , 0.05 versus null vector group.
(Fig. 4B). This result implied that the autophagy was mitophagy specific. To further verify whether the mitophagy pathway was activated by Rcan1-1L, we detected the protein levels of mitophagy receptor Parkin and PINK1. The results showed that Rcan1-1L promoted Parkin translocation from cytosol into mitochondria. The expression of PINK1, which was hardly detected in cytosol, was highly upregulated by Rcan1-1L in mitochondria (Fig. 4C).
Knockdown of Parkin-mediated Mitophagy Pathway Blocks the Effect of Rcan1-1L Overexpression To further investigate whether Rcan1-1L regulated mitophagy specifically to remit the hypoxia-induced growth
314
| www.jcvp.org
inhibition on cardiomyocytes, we silenced the expression of Parkin and then determined whether Rcan1-1L overexpression still promoted cell survival or reduced mitochondrial mass under hypoxic conditions (Fig. 5A). Results showed that knockdown of Parkin disturbed the effect of Rcan1-1L that promoted cell growth and survival under hypoxic conditions (Figs. 5B, C). Furthermore, Rcan1-1L–induced mitochondrial mass reduction was also blocked by silencing the expression of Parkin (Fig. 5D). These data implied that Rcan1-1L promoted cell survival through Parkin-mediated mitophagy pathway.
DISCUSSION Rcan1 has critical functions in inflammation and angiogenesis, which are linked to cardiac hypertrophy, cardiac valve development, and cancer.33 Particularly, Rcan1 is dysregulated in neurodegeneration in Down syndrome and Alzheimer disease.34–36 Rcan1-1 isoforms, such as Rcan1-1L and Rcan1-1S, are widely distributed in tissues from the heart, skeletal muscles, and nervous system.37,38 Evidence suggests that Rcan1 expression is involved in cell survival and death protection. Rcan1 reportedly protects cells from calcium overload–induced cell death.25 Rcan1 deficiency promotes T-helper type I cell apoptosis and increases cell susceptibility to factor associated suicide-mediated and etoposide-induced apoptosis.26,27 Similarly, knockdown of Rcan1-1 and Rcan1-4 isoforms triggers apoptosis in endothelial cells.28 Kim et al39 have recently demonstrated that Rcan1-1 can protect neurons from hydrogen peroxide–induced cell death by inducing 2014 Lippincott Williams & Wilkins
J Cardiovasc Pharmacol Volume 64, Number 4, October 2014
FIGURE 5. Parkin siRNA disturbs the effect of Rcan1-1L overexpression. A, Western blot analysis was performed to detect the protein expression of Parkin and Rcan1-1L. B, Cell growth and viability was assessed by MTT assay. *P , 0.05 denotes null vector group versus Rcan1-1L group or Rcan1-1L + nonsiRNA transfected group under hypoxia. †P , 0.05 denotes Rcan1-1L + Parkin siRNA transfected group versus Rcan1-1L group or Rcan1-1L + non-siRNA transfected group under hypoxia. C, Cell apoptosis was tested by TUNEL assay. **P , 0.01 denote null vector group versus Rcan1-1L group or Rcan1-1L + non-siRNA transfected group under hypoxia. ††P , 0.01 denote Rcan1-1L + Parkin siRNA transfected group versus Rcan1-1L group or Rcan1-1L + non-siRNA transfected group under hypoxia. D, Mitochondria were labeled with MitoTracker Green, and signals were measured using the FACS analyzer. Cells without treatment cultured under normoxic conditions were taken as control. *P , 0.05 denotes null vector group versus Rcan1-1L group or Rcan1-1L + non-siRNA transfected group under hypoxia. †P , 0.05 denotes Rcan1-1L + Parkin siRNA transfected group versus Rcan1-1L group or Rcan1-1L + non-siRNA transfected group under hypoxia. Cells under hypoxic conditions were divided into 4 groups without different treatments. Vector, cells were treated with null vector without Rcan1-1L. Rcan1-1L, cells were treated with Rcan1-1L expression vectors. Rcan1-1L + Non-siRNA, cells were 2014 Lippincott Williams & Wilkins
Hypoxia-induced Apoptosis via Mitophagy
cAMP response element-binding protein and its target gene Bcl-2. Short-term expression of Rcan1-1L protects neurons from oxidative stress-induced apoptosis by inhibiting caspase-3.40 Consistent with these findings, we found that Rcan1-1L overexpression protects cardiomyocytes from hypoxia-induced cell death. In addition, Rcan1-1L overexpression promoted the expression of the antiapoptotic protein Bcl-2 and inhibited that of the proapoptotic proteins caspase-3 and Bad. However, several inconsistent reports were also found. For example, glucocorticoid hormones specifically evoke the upregulation of Rcan1-1 that makes human leukemic cells susceptible to apoptosis.41 In addition, dexamethasone enhances neuronal apoptosis by elevating Rcan1 expression.33 These apparent discrepancies imply that Rcan1-1 has a complex function for cell survival or death under different stress and stimulus conditions. In the present study, we found that Rcan1-1L overexpression promoted mitochondria degradation through mitophagy. This result is consistent with the finding of Ermak et al29 We also demonstrated that Rcan1-1L overexpression decreased mitochondrial mass reduction and mtPTP opening. Previous studies suggested that mtPTP opening is involved in mitophagy induction.42,43 We identified that Rcan1-1L overexpression increased autophagy flow. mTOR, which inhibits autophagy during normal conditions, was downregulated. LC3, a critical component in autophagosomal membrane formation, was markedly upregulated. The mitochondrial protein marker TOM20 was significantly decreased, whereas Golgi body and endoplasmic reticulum remained unchanged. Thus, we speculated that Rcan1-1L overexpression specifically induces mitophagy in cardiomyocytes during hypoxia. However, we observed that Rcan1-1L overexpression inhibited cell growth under normoxic conditions. It is apparent that decreased mitochondrial impair cell growth under normal conditions. Under hypoxia, damaged mitochondria are increased, which is detrimental for cell growth and survival. Therefore, accelerating the degradation of damaged mitochondria by Rcan1-1L is beneficial for cell growth and survival under hypoxic stress. Mitophagy has been recently proven to have unique functions in ischemic heart disease. Integrated mitochondria are essential for cell survival. Damaged mitochondria caused by hypoxic treatment in cardiomyocytes generate excessive reactive oxygen species and activate cell death, which trigger myocardial damage.5–7 Therefore, efficient and specific clearance of dysfunctional mitochondria is beneficial for cardiomyocyte protection under hypoxic conditions. Knockdown of autophagy-related gene ATG5 causes mitochondrial dysfunction, which leads to rapid myocardial failure in mouse model.44 Parkin-deficient mice demonstrate enhanced accumulation of damaged mitochondria, which cause myocardial dysfunction
cotransfected with Rcan1-1L expression vectors and nonspecific siRNA. Rcan1-1L + Parkin siRNA, cells were cotransfected with Rcan1-1L expression vectors and Parkinspecific siRNA. Cells with different treatments were cultured for 24 hours under hypoxic conditions and then harvested for analysis. www.jcvp.org |
315
J Cardiovasc Pharmacol Volume 64, Number 4, October 2014
Yan et al
and decrease survival rate.45 Similarly, BNIP3 and/or Nix knockdown causes the accumulation of damaged mitochondria and cardiac dysfunction.46 A recent study has demonstrated that p53 knockdown stimulates mitophagy in cardiomyocytes, which inhibits myocardial damage induced by ischemia.47 In the present study, we found that Rcan1-1L overexpression induced mitophagy receptor Parkin and PINK1 in cardiomyocytes during hypoxia. Knockdown of Parkin apparently disturbed the effect of Rcan1-1L overexpression on cell survival and mitochondrial mass implying that Rcan1-1L might evoke mitophagy through Parkin-mediated mitophagy pathway. In conclusion, our data revealed that targeting Rcan1-1L to specifically remove damaged mitochondria has a potential application in the treatment of myocardial ischemia. Rcan1-1L gene expression strategies in animal models of myocardial ischemia should be performed to further confirm and verify the cytoprotective effects of Rcan1-1L. However, 1 major concern that should be considered is that excessive mitophagy can enhance cell death. Thus, more investigation should be performed to explore methods for monitoring and controlling appropriate mitophagy induced by Rcan1-1L. For instance, hypoxia response element–mediated Rcan1-1L expression in genetic therapy delivery agent such as recombinant adenoassociated virus could be used to monitoring Rcan1-1L– induced mitophagy, in which Rcan1-1L is only expressed under myocardial ischemia. Once ischemia condition is relieved, the Rcan1-1L expression is stopped that will avoid excessive mitophagy. However, certain limitations of this study should be noted. In the present study, we detected the mitochondrial mass alterations, and the autophagy flow represented by mTOR and LC3 protein levels to claim the conclusion that Rcan1-1L specifically initiated mitophagy. To a certain degree, the mitophagy receptor Parkin knockdown experiments confirmed the Rcan1-1L–induced mitophagy. However, there is still lack of direct evidences for Rcan1-1L–induced mitophagy such as a direct observation of mitochondria and the mitochondria-related autolysosome alterations by electron microscope scanning. Moreover, the effect of Rcan1-1L– induced on the phenotype of the cardiac cells should be concerned. However, studies of Rcan1-1L–induced mitophagy should be further conducted in our laboratory and the treatment of myocardial ischemia by Rcan1-1L–induced mitophagy will also be tested in animal models in our laboratory. REFERENCES 1. Go AS, Mozaffarian D, Roger VL, et al. Heart disease and stroke statistics–2013 update: a report from the American Heart Association. Circulation. 2013;127:e6–e245. 2. Fox KA, Steg PG, Eagle KA, et al. Decline in rates of death and heart failure in acute coronary syndromes, 1999-2006. JAMA. 2007;297:1892– 1900. 3. Simoons ML, Windecker S. Controversies in cardiovascular medicine: chronic stable coronary artery disease: drugs vs. revascularization. Eur Heart J. 2010;31:530–541. 4. Verma S, Fedak PW, Weisel RD, et al. Fundamentals of reperfusion injury for the clinical cardiologist. Circulation. 2002;105:2332–2336. 5. Chatterjee S, Stewart AS, Bish LT, et al. Viral gene transfer of the antiapoptotic factor Bcl-2 protects against chronic postischemic heart failure. Circulation. 2002;106:I212–I217. 6. Nakagawa T, Shimizu S, Watanabe T, et al. Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death. Nature. 2005;434:652–658.
316
| www.jcvp.org
7. Neubauer S. The failing heart–an engine out of fuel. N Engl J Med. 2007; 356:1140–1151. 8. Jimenez RE, Kubli DA, Gustafsson AB. Autophagy and mitophagy in the Myocardium: therapeutic potential and concerns. Br J Pharmacol. 2014; 171:1907–1916. 9. Youle RJ, Narendra DP. Mechanisms of mitophagy. Nat Rev Mol Cell Biol. 2011;12:9–14. 10. Hamacher-Brady A, Brady NR, Logue SE, et al. Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy. Cell Death Differ. 2007;14:146–157. 11. Zhang J, Ney PA. Role of BNIP3 and NIX in cell death, autophagy, and mitophagy. Cell Death Differ. 2009;16:939–946. 12. Liu L, Feng D, Chen G, et al. Mitochondrial outer-membrane protein FUNDC1 mediates hypoxia-induced mitophagy in mammalian cells. Nat Cell Biol. 2012;14:177–185. 13. Crawford DR, Leahy KP, Abramova N, et al. Hamster adapt78 mRNA is a Down syndrome critical region homologue that is inducible by oxidative stress. Arch Biochem Biophys. 1997;342:6–12. 14. Davies KJ, Ermak G, Rothermel BA, et al. Renaming the DSCR1/ Adapt78 gene family as RCAN: regulators of calcineurin. FASEB J. 2007;21:3023–3028. 15. Wang Y, De Keulenaer GW, Weinberg EO, et al. Direct biomechanical induction of endogenous calcineurin inhibitor Down Syndrome Critical Region-1 in cardiac myocytes. Am J Physiol Heart Circ Physiol. 2002; 283:H533–H539. 16. Ermak G, Pritchard MA, Dronjak S, et al. Do RCAN1 proteins link chronic stress with neurodegeneration? FASEB J. 2011;25:3306–3311. 17. Hattori M, Fujiyama A, Taylor TD, et al. The DNA sequence of human chromosome 21. Nature. 2000;405:311–319. 18. Qi YM, Lei T, Zhou L, et al. Genomic organization, alternative splicing and tissues expression of porcine CREB3L4 gene. Mol Biol Rep. 2009; 36:1881–1888. 19. Ermak G, Harris CD, Davies KJ. The DSCR1 (Adapt78) isoform 1 protein calcipressin 1 inhibits calcineurin and protects against acute calciummediated stress damage, including transient oxidative stress. FASEB J. 2002;16:814–824. 20. Ermak G, Harris CD, Battocchio D, Davies KJ. RCAN1 (DSCR1 or Adapt78) stimulates expression of GSK-3beta. FEBS J. 2006;273:2100–2109. 21. Gorlach J, Fox DS, Cutler NS, et al. Identification and characterization of a highly conserved calcineurin binding protein, CBP1/calcipressin, in Cryptococcus neoformans. EMBO J. 2000;19:3618–3629. 22. Kingsbury TJ, Cunningham KW. A conserved family of calcineurin regulators. Genes Dev. 2000;14:1595–1604. 23. Rothermel B, Vega RB, Yang J, et al. A protein encoded within the Down syndrome critical region is enriched in striated muscles and inhibits calcineurin signaling. J Biol Chem. 2000;275:8719–8725. 24. Chang KT, Min KT. Drosophila melanogaster homolog of Down syndrome critical region 1 is critical for mitochondrial function. Nat Neurosci. 2005;8:1577–1585. 25. Zhao P, Xiao X, Kim AS, et al. c-Jun inhibits thapsigargin-induced ER stress through up-regulation of DSCR1/Adapt78. Exp Biol Med (Maywood). 2008;233:1289–1300. 26. Ryeom S, Greenwald RJ, Sharpe AH, McKeon F. The threshold pattern of calcineurin-dependent gene expression is altered by loss of the endogenous inhibitor calcipressin. Nat Immunol. 2003;4:874–881. 27. Kim YS, Lee HJ, Jang C, et al. Knockdown of RCAN1.4 Increases Susceptibility to FAS-mediated and DNA-damage-induced Apoptosis by Upregulation of p53 Expression. Korean J Physiol Pharmacol. 2009;13:483–489. 28. Ryeom S, Baek KH, Rioth MJ, et al. Targeted deletion of the calcineurin inhibitor DSCR1 suppresses tumor growth. Cancer Cell. 2008;13:420–431. 29. Ermak G, Sojitra S, Yin F, et al. Chronic expression of RCAN1-1L protein induces mitochondrial autophagy and metabolic shift from oxidative phosphorylation to glycolysis in neuronal cells. J Biol Chem. 2012;287:14088–14098. 30. Petronilli V, Miotto G, Canton M, et al. Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys J. 1999;76:725–734. 31. Blommaart EF, Luiken JJ, Blommaart PJ, et al. Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes. J Biol Chem. 1995;270:2320–2326.
2014 Lippincott Williams & Wilkins
J Cardiovasc Pharmacol Volume 64, Number 4, October 2014 32. Klionsky DJ, Abdalla FC, Abeliovich H, et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy. 2012;8: 445–544. 33. Sun X, Wu Y, Chen B, et al. Regulator of calcineurin 1 (RCAN1) facilitates neuronal apoptosis through caspase-3 activation. J Biol Chem. 2011;286:9049–9062. 34. Harris CD, Ermak G, Davies KJ. RCAN1-1L is overexpressed in neurons of Alzheimer’s disease patients. FEBS J. 2007;274:1715–1724. 35. Ermak G, Morgan TE, Davies KJ. Chronic overexpression of the calcineurin inhibitory gene DSCR1 (Adapt78) is associated with Alzheimer’s disease. J Biol Chem. 2001;276:38787–38794. 36. Keating DJ, Dubach D, Zanin MP, et al. DSCR1/RCAN1 regulates vesicle exocytosis and fusion pore kinetics: implications for Down syndrome and Alzheimer’s disease. Hum Mol Genet. 2008;17:1020–1030. 37. Fuentes JJ, Pritchard MA, Planas AM, et al. A new human gene from the Down syndrome critical region encodes a proline-rich protein highly expressed in fetal brain and heart. Hum Mol Genet. 1995;4:1935–1944. 38. Fuentes JJ, Pritchard MA, Estivill X. Genomic organization, alternative splicing, and expression patterns of the DSCR1 (Down syndrome candidate region 1) gene. Genomics. 1997;44:358–361. 39. Kim SS, Jang SA, Seo SR. CREB-mediated Bcl-2 expression contributes to RCAN1 protection from hydrogen peroxide-induced neuronal death. J Cell Biochem. 2013;114:1115–1123.
2014 Lippincott Williams & Wilkins
Hypoxia-induced Apoptosis via Mitophagy
40. Wu Y, Song W. Regulation of RCAN1 translation and its role in oxidative stress-induced apoptosis. FASEB J. 2013;27:208–221. 41. Hirakawa Y, Nary LJ, Medh RD. Glucocorticoid evoked upregulation of RCAN1-1 in human leukemic CEM cells susceptible to apoptosis. J Mol Signal. 2009;4:6. 42. Rodriguez-Enriquez S, He L, Lemasters JJ. Role of mitochondrial permeability transition pores in mitochondrial autophagy. Int J Biochem Cell Biol. 2004;36:2463–2472. 43. Rodriguez-Enriquez S, Kai Y, Maldonado E, et al. Roles of mitophagy and the mitochondrial permeability transition in remodeling of cultured rat hepatocytes. Autophagy. 2009;5:1099–1106. 44. Nakai A, Yamaguchi O, Takeda T, et al. The role of autophagy in cardiomyocytes in the basal state and in response to hemodynamic stress. Nat Med. 2007;13:619–624. 45. Kubli DA, Zhang X, Lee Y, et al. Parkin protein deficiency exacerbates cardiac injury and reduces survival following myocardial infarction. J Biol Chem. 2013;288:915–926. 46. Dorn GW 2nd. Mitochondrial pruning by Nix and BNip3: an essential function for cardiac-expressed death factors. J Cardiovasc Transl Res. 2010;3:374–383. 47. Hoshino A, Matoba S, Iwai-Kanai E, et al. p53-TIGAR axis attenuates mitophagy to exacerbate cardiac damage after ischemia. J Mol Cell Cardiol. 2012;52:175–184.
www.jcvp.org |
317
Regulator of Calcineurin 1-1L Protects Cardiomyocytes Against Hypoxia-induced Apoptosis via Mitophagy Lijie Yan, PhD, Haitao Yang, PhD, Yongqiang Li, PhD, Hongyan Duan, PhD, Jintao Wu, MD, Peng Qian, PhD, Bing Li, MD, and Shanling Wang, MD
Abstract: Mitochondrial dysfunction induced by myocardial ischemia is the primary cause of cardiac cell death. Specific removal of damaged mitochondria through mitophagy may be beneficial for cardiomyocyte protection against ischemia. Regulator of calcineurin 1-1L (Rcan1-1L) has been implicated in mitophagy induction in neurons. However, whether or not Rcan1-1L can evoke mitophagy in cardiomyocytes during hypoxia remains unknown. This study aims to investigate the effect of Rcan1-1L overexpression on cardiomyocytes during hypoxia and the possible underlying mechanism. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that Rcan1-1L overexpression inhibited cell growth under normoxic conditions, whereas Rcan1-1L overexpression significantly reversed the growth inhibition induced by hypoxia. The results of terminal deoxynucleotidyl transferase biotin-dUTP nick end-labeling assay showed that cell apoptosis induced by hypoxia was markedly reduced by Rcan1-1L overexpression. In addition, Rcan1-1L overexpression inhibited the expression of the proapoptotic protein Bcl-2– associated death promoter and increased that of the antiapoptotic protein Bcl-2. Rcan1-1L overexpression opened the mitochondrial permeability transition pore and decreased mitochondrial mass. Meanwhile, the release of reactive oxygen species from mitochondria was suppressed by Rcan1-1L. Autophagy flow activation represented by mammalian target of rapamycin inhibition and microtubule-associated protein light chain 3 (LC3) upregulation was also demonstrated. Compared with endoplasmic reticulum and Golgi apparatus protein markers, the mitochondrial protein marker translocase of outer mitochondrial membranes 20 (TOM20) was downregulated by Rcan1-1L overexpression. Moreover, Rcan1-1L increased mitophagy receptor Parkin translocation into mitochondria from cytosol. Additionally, the effect of Rcan1-1L on cell growth, cell apoptosis and mitochondria mass was blocked by Parkin expression silencing. Overall, these data suggest that Rcan1-1L protects cardiomyocytes from hypoxia-induced apoptosis by inducing mitophagy partly through Parkin. This study provided novel insights into the prevention and treatment of ischemic heart disease. Key Words: myocardial ischemia, hypoxia, apoptosis, mitophagy (J Cardiovasc Pharmacol 2014;64:310–317)
Received for publication February 10, 2014; accepted April 23, 2014. From the Department of Cardiology, Henan Provincial People’s Hospital, Zhengzhou, China. Lijie Yan, PhD, and Haitao Yang, PhD, contributed equally to this manuscript. The authors report no conflicts of interest. Reprints: Shanling Wang, MD, Department of Cardiology, Henan Provincial People’s Hospital, Zhengzhou, China 450003 (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
310
| www.jcvp.org
INTRODUCTION Coronary heart disease with high morbidity and mortality is a leading cause of death worldwide.1 Reperfusion therapy can attenuate myocardial infarction and prevent heart failure; however, this treatment often causes myocardial ischemia/reperfusion, which leads to myocardial damage and pathological remodeling.2–4 Thus, development of effective strategies to protect cardiomyocytes from hypoxia-induced damage may provide novel insights into the prevention of myocardial damage during cardiac surgery. Mitochondria, the most abundant organelles in cardiomyocytes, supply energy for myocardial contraction that is vulnerable under hypoxic conditions. Cell death pathways activated by dysfunctional mitochondria lead to excessive reactive oxygen species production and insufficient ATP production, which promote infarct expansion and heart failure progression after ischemic injury progression.5–7 Efficient and specific clearance of dysfunctional mitochondria is beneficial for cardiomyocyte protection under hypoxic conditions. Recent studies have demonstrated that mitochondria can be selectively removed by autophagy (termed as mitophagy), which is essential for cellular homeostasis especially for cardiomyocytes.8 Mitophagy is controlled by PTEN-induced kinase protein 1 (PINK1) and Parkin. Parkin, which is usually located in the cytoplasm under normal conditions, can be quickly recruited to mitochondria by the accumulated PINK1 upon mitochondrial membrane depolarization; the recruited Parkin then ubiquitylates mitochondrial proteins by its E3 ubiquitin ligase activity and causes mitophagy.9 BNIP3 and NIX are mitophagy regulators that bind with the autophagy protein, microtubule-associated protein-light chain 3 (LC3) to form autophagy lysosomal membrane.10,11 Fundc1 has been recently identified to be a new receptor for mitophagy in mammalian cells; this receptor interacts with LC3 to form autophagy lysosomal membrane.12 These studies have demonstrated that mitochondria can be selectively cleared by mitophagy. Therefore, analyzing mitophagy may provide insights into the pathogenesis and treatment of disease therapy. Regulator of calcineurin 1 (Rcan1), also named Adapt78, DSCR1, and MCIP1, is an adaptive protein that is induced during cellular stress.13–16 The Rcan1 gene is located in the q22.12 region of chromosome 21; this gene consists of 7 exons that can produce different mRNA and protein isoforms.17,18 Rcan1 has 3 isoforms identified so far: RCAN11L, RCAN1-1S, and RCAN1-4.14,19,20 The original function J Cardiovasc Pharmacol Volume 64, Number 4, October 2014
J Cardiovasc Pharmacol Volume 64, Number 4, October 2014
of Rcan1 is to regulate calcineurin activity.21–23 Current studies have shown that Rcan1 has other functions. Rcan1 interacts with mitochondrial adenine nucleotide translocator and regulates ADP/ATP exchange in Drosophila.24 Rcan1 also modulates cell apoptosis.25–28 RCAN1-1L has been recently demonstrated to be a regulator of mitophagy that efficiently causes dramatic degradation of mitochondria.29 Whether Rcan1-1L can protect cardiomyocytes from hypoxia by inducing mitophagy remains unknown. In the present study, we investigated the effect of Rcan1-1L on hypoxia-treated cardiomyocytes for the first time. We found that Rcan1-1L overexpression in cardiomyocytes during hypoxia elicits cytoprotective effects by activating mitophagy. The results of this study suggest Rcan1-1L as a novel target for the treatment of ischemic heart disease.
Hypoxia-induced Apoptosis via Mitophagy
Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End-labeling Staining
After indicated treatments, ells were fixed with 4% paraformaldehyde solution for 10 minutes at room temperature followed by washing with PBS. Then, the cells were permeabilized with 0.1% sodium citrate and 0.1% Triton X-100 for 10 minutes. After washing with PBS, cell apoptosis were detected using a terminal deoxynucleotidyl transferase biotin-dUTP nick end-labeling (TUNEL) kit (Beyotime, Beijing, China) according to the supplier’s instructions. Cells were observed and photographed by fluorescence microscope (Olympus, Tokyo, Japan). Quantitative analysis was presented as TUNEL-positive cells per field counted in 5 random fields, and the counts were averaged.
Caspase-3 Activity Assay MATERIALS AND METHODS Cell Culture Human adult cardiomyocytes obtained from Celprogen (California) were maintained in human cardiomyocyte cell culture complete media with serum and antibiotics. These cells were cultured at 378C with 5% CO2 in an incubator (Life Technologies, Baltimore, MD).
Cell Treatment and Transfection For plasmid transfection, plasmid pcDNA-Rcan1-1L (1 mg) diluted in 500 mL of cardiomyocyte cell culture complete media with 5 mL lipofectamine (Invitrogen, Carlsbad, CA) were added to the cells and cultured for indicated times: 12, 24, and 48 hours. Cells transfected null vectors and phosphate-buffered saline (PBS) were taken as control. Cells were cultured under normoxic or hypoxia conditions. Hypoxia was achieved by placing the cells in a hypoxia chamber filled with 5% CO2 and 95% N2 at 378C. For small-interfering RNAs (siRNAs) transfection, 0.1 mM siRNA targeting Parkin (sc-42158; Santa Cruz, CA) diluted in 500 mL of medium with 5 mL lipofectamine (Invitrogen) were added to cells and incubated for 24 hours under hypoxic conditions. Cells transfected with nonspecific siRNA (sc-37007) were taken as control.
3-(4,5-Dimethylthiazol-2-yl)-2,5Diphenyltetrazolium Bromide Assay For the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the cells were plated in 96-well plates (5 · 104 cells per well) and cultured under regular conditions until 80% confluence was reached. After the cells transfected with plasmid DNA or siRNAs and incubated for 12, 24 and 48 hours, the culture medium was replaced with 200 mL fresh medium that contains MTT (5 mg/mL in PBS; Sangon, Shanghai, China) and then incubated with cells for an additional 4 hours. Dimethyl sulfoxide (150 mL/well) was added to dissolve formazan for 10 minutes, and the absorbance at 490 nm was determined using an ELISA reader (Bio-tek Instruments, Winooski, VT). The experiment was performed in quadruplicate and was repeated thrice. 2014 Lippincott Williams & Wilkins
Cells were harvested and homogenized in ice-cold Trisbuffered saline (TBS) and centrifuged at 12,000 rpm at 48C for 20 minutes. The supernatants were collected and incubated with 5 mM Ac-DEVD-MCA at 378C for 5 minutes. The release of 7-amino-4-methylcoumarin was measured using a spectrofluorometer (Hitachi F-2000; Tokyo, Japan) at 380 nm excitation wavelength and 460 nm emission wavelength.
Fluorescence-activated Cell Sorting Analysis For mitochondrial mass analysis, the cells were resuspended in 380 mL PBS that contains 200 nM Mitotracker Green FM (Invitrogen). Afterward, the mixture was incubated at 378C and then shaken gently for 15 minutes. Finally, fluorescence was assessed using a fluorescence-activated cell sorting (FACS)calibur analyzer flow cytometer at 530 nm excitation wavelength (FL-1). mPTP opening was determined as previously described.30 Briefly, the cells were incubated in culture that contains 1 mM calcein-AM (Molecular Probes) at 378C in the dark for 30 minutes. Afterward, 1 mM CoCl2 (Sigma, St Louis, MO) was added and incubated for another 10 minutes in the dark at 378C. Thereafter, the fluorescence signal of mitochondria-trapped calcein was analyzed using a FACSCalibur flow cytometer at 530 nm excitation wavelength (FL-1). For each experiment, calcein fluorescence from 30,000 cells was acquired using FACSCalibur flow cytometer, and median value analysis was conducted using FlowJo software.
Intracellular Reactive Oxygen Species Measurement Cells transfected with plasmid DNA or siRNAs were cultured under hypoxic conditions for 24 hours. After cells were harvested and washed with PBS, cells were incubated with 50 mM 20 ,70 -dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma) at 378C for 45 minutes in the dark. Then, the fluorescence intensity was measured by using Multi-Detection microplate reader (Bio-tek Instruments) at 485 nm excitation wavelength and 530 nm emission wavelength. The experiment was performed in quadruplicate and was repeated thrice.
Mitochondrial Protein Extraction Mitochondrial and cytosolic fractions were isolated using a mitochondria isolation kit (Pierce, Rockford) according to the manufacturer’s instructions. Briefly, the cells were www.jcvp.org |
311
Yan et al
collected and homogenized in ice-cold lysis buffer A followed by centrifugation at 800g for 10 minutes at 48C. After the supernatants were collected, a total of 500 mL mitochondria isolation reagent buffer were added in followed by centrifugation at 15,000g for 20 minutes at 48C. Then, the supernatant containing the cytosolic fraction was collected and stored at 2208C for further analysis. The pellet enriched in mitochondria were collected and washed by he rinsing buffer followed by centrifugation at 15,000g for 10 minutes at 48C. The pellet was collected and resuspended in ice-cold lysis buffer B followed by centrifugation at 12,000 rpm for 15 minutes at 48C. Finally, the supernatant containing mitochondrial protein were collected for further analysis.
J Cardiovasc Pharmacol Volume 64, Number 4, October 2014
antibodies (Boster Corporation, Wuhan, China) diluted in the blocking buffer for 1 hour. Finally, the membrane was washed, and 4-chloro-1-naphthol (4-CN) (1 mL) with TBS (9 mL) containing 6 mL of H2O2 was added for protein visualization.
Statistical Analysis All assays were performed in triplicate, and data are presented as mean 6 SD. Comparisons between 2 groups were determined by Student t test, and among groups were tested using 1-way analysis of variance followed by Bonferroni post hoc. Statistical significance was considered at P , 0.05. All statistical analyses were performed using SPSS version 11.5 (SPSS Inc, Chicago, IL).
Western Blot Analysis
Approximately 50 mg of protein was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by electroblotting onto a nitrocellulose membrane (Amersham, Little Chalfont, United Kingdom). The membrane was incubated in TBS that contains 2% nonfat dry milk to block nonspecific binding at room temperature for 1 hour. Afterward, the membrane was washed by TBS and incubated with primary antibodies in the blocking buffer overnight at 48C. Anti-Rcan1-1 antibodies (D6694) diluted in 1:1000 were purchased from Sigma. Anti-mTOR antibody (sc-1549) diluted in 1:200, anti-p70 S6 kinase a antibody (sc-393967) diluted in 1:200, anti-Bcl-2 antibody (sc-492) diluted in 1:500, anti-Bad antibody (sc-943) diluted in 1:200, anti-TOM20 (sc-17764) diluted in 1:500, anti-GM130 (sc-55590) diluted in (1:800) and anti-Calnexin (sc-11397) diluted in 1:800 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-LC3B antibody (ab63817) diluted in 1:400, anti-Parkin antibody (ab77924) diluted in 1:200 and anti-PINK1 antibody (ab23707) diluted in 1:600 were purchased from Abcam (Cambridge, United Kingdom). Anti-GAPDH antibody (bs-2188R) and anti-a-tubulin antibody (bs-4511R) were purchased from Bioss (Beijing, China). Subsequently, the membrane was washed thrice with TBS–Tween. The membrane was then incubated in horseradish peroxidase–conjugated secondary
RESULTS Rcan1-1L Improves Cardiomyocytes Under Hypoxic Conditions To investigate the role of Rcan1-1L on cell growth, we induced Rcan1-1L overexpression in cultured cardiomyocytes through transfection with pcDNA3.0-Rcan1-1L expression vectors under normoxic conditions. Western blot analysis showed that Rcan1-1L was overexpressed for 12, 24, and 48 hours compared with the control or null vector transfection (Fig. 1A). Using MTT assay, we found that cell growth was inhibited by Rcan1-1L (Fig. 1B). We next explored the effect of Rcan1-1L on cardiomyocytes under hypoxic conditions. The results showed that Rcan1-1L overexpression promoted cell growth and viability compared with the cells transfected with PBS or null vector under hypoxic conditions (Figs. 2C, D). These data indicated that Rcan1-1L overexpression under hypoxic conditions promoted the survival of cardiomyocytes.
Rcan1-1L Resists Hypoxia-induced Cell Apoptosis in Cardiomyocytes Hypoxia treatment generally induces cell apoptosis in cardiomyocytes. We conducted TUNEL assay to analyze cell apoptosis and explore the effect of Rcan1-1L on cell
FIGURE 1. Rcan1-1L expression in cardiomyocytes and its effect on cell growth. A, Western blot analysis was performed to analyze the protein expression of Rcan1-1L in normoxictreated cells. PBS, cells treated with PBS were taken as control. Vector, cells were treated with null vector without Rcan1-1L. Rcan1-1L, cells were treated with Rcan1-1L expression vectors. Cells were cultured under normoxic conditions for 12, 24, and 48 hours. B, Effect of Rcan11L on cell growth under normoxic conditions was measured by MTT assay. †P , 0.05 versus PBS group; *P , 0.05 versus null vector group. C, Western blot analysis of Rcan1-1L expression in hypoxia-treated cells. D, Effect of Rcan1-1L on cell growth under hypoxic conditions was measured by MTT assay. ††P , 0.01 versus PBS group; **P , 0.01 versus null vector group.
312
| www.jcvp.org
2014 Lippincott Williams & Wilkins
J Cardiovasc Pharmacol Volume 64, Number 4, October 2014
Hypoxia-induced Apoptosis via Mitophagy
FIGURE 2. Effect of Rcan1-1L on cardiomyocyte apoptosis under hypoxic conditions. A, Cell apoptosis was tested by TUNEL assay. Cells treated with Rcan1-1L expression vectors, null vectors or PBS for 24 hours under hypoxia for 24 hours. B, Quantitative analysis of TUNEL-positive cells. ††P , 0.01 versus PBS group; **P , 0.01 versus null vector group. C, Caspase-3 activity measurement. Protein lysate of cardiomyocytes was examined by incubation with Ac-DEVD-MCA for 5 minutes at 378C. The release of 7amino-4-methylcoumarin was measured using a spectrofluorometer. ††P , 0.01 versus PBS group; **P , 0.01 versus null vector group. D, Western blot analysis was performed to examine the protein expression levels of Bcl-2 and Bad in cardiomyocytes.
apoptosis during hypoxia treatment. Compared with the cells under normoxic conditions, the cells transfected with PBS or null vectors showed a significant increase in cell apoptotic rate; this increase was reversed by Rcan1-1L expression (Figs. 2A, B). To further confirm the results, we detected the activity of caspase-3, a key downstream effector protein of apoptosis. The activity of caspase-3 in the Rcan1-1L expression group was significantly downregulated compared with that in the cells transfected with PBS or null vector (Fig. 2C). We also monitored apoptosis-related proteins, including the antiapoptotic protein Bcl-2 and the proapoptotic protein Bcl2–associated death promoter (Bad) by western blot. The protein expression level of Bcl-2 that decreased during hypoxia was upregulated by Rcan1-1L. By contrast, the protein expression level of Bad that increased during hypoxia was downregulated by Rcan1-1L (Fig. 2D).
Rcan1-1L Overexpression Leads to Mitochondrial Mass Reduction and mPTP Opening We determined the effect of Rcan1-1L on mitochondria to further explore the mechanism by which Rcan1-1L regulates cell apoptosis. In the present study, mitochondrial mass was measured using FACS analysis. The cells were labeled with MitoTracker Green, and the total signals in each cell were measured. Rcan1-1L overexpression significantly decreased mitochondrial mass (Fig. 3A). We also determined the effects of Rcan1-1L on mPTP. The results showed that 2014 Lippincott Williams & Wilkins
mPTP opening peaked at 24 hours and then decreased; despite this decrease, this opening was still higher than that at 0 hours (Fig. 3B). Moreover, the release of ROS from mitochondria was significantly downregulated by Rcan1-1L (Fig. 3C).
Rcan1-1L Overexpression Stimulates Mitophagy Mitophagy is the most common mechanism of mitochondrial mass reduction. A previous study implied that Rcan1-1L can evoke mitophagy.29 In the present study, we determined whether Rcan1-1L expression in cardiomyocytes under hypoxic conditions can induce mitophagy. Therefore, we first analyzed the autophagy flow in the cells. The main mediator of autophagy is the kinase mTOR, which can inhibit the initiation of autophagy.31 LC3-I/II has an important function in forming autophagosomal membranes, and LC3-II is a validated marker of autophagic compartments.32 In the present study, mTOR and its downstream gene S6K were inhibited in the Rcan1-1L–transfected cells compared with the control groups. Furthermore, the protein expression level of LC3-II increased, implying that autophagy flow was activated by Rcan1-1L expression (Fig. 4A). We analyzed whether other organelles were altered to confirm that the autophagy flow was mitochondrion specific. The results showed that the protein expression level of the mitochondrial marker TOM20 was downregulated in the Rcan1-1L–transfected cells, whereas those of the Golgi body marker GM130 and endoplasmic reticulum marker calnexin remained unchanged www.jcvp.org |
313
Yan et al
J Cardiovasc Pharmacol Volume 64, Number 4, October 2014
FIGURE 4. Effect of Rcan1-1L on mitophagy in hypoxia-treated cardiomyocytes. Western blot analysis was performed to determine the protein expression levels of autophagy markers, mTOR, S6K, and LC3-I/II (A) mitochondria marker, TOM20, Golgi body marker GM130 and endoplasmic reticulum marker calnexin (B) in cardiomyocytes. GAPDH was used as control. C, Western blot analysis was conducted to determine the protein expression levels of Parkin and PINK1 in cytosol and mitochondria fractions. a-Tubulin was used as cytosol protein control and VDAC was used as mitochondria protein control. FIGURE 3. Effect of Rcan1-1L on mitochondria. A, Rcan1-1L overexpression reduced mitochondrial mass. Cells were transfected with vectors that contain Rcan1-1L or null vectors. Mitochondria were labeled with MitoTracker Green, and signals were measured using the FACS analyzer. Mitochondrial mass was evaluated by comparing fluorescence signals. †P , 0.05 versus PBS group; *P , 0.05 versus null vector group. B, Rcan1-IL overexpression led to mPTP opening. mPTP detection using calcein fluorescence dye. Mean fluorescence values were converted to arbitrary units (AU). Fluorescence values in null vector–transfected cells were set as 1.0. *P , 0.05 versus null vector group. C, Rcan1-1L overexpression decreased reactive oxygen species release. Reactive oxygen species levels were determined using DCFH-DA assay after cells were cultured under hypoxia for 24 hours. †P , 0.05 versus PBS group; *P , 0.05 versus null vector group.
(Fig. 4B). This result implied that the autophagy was mitophagy specific. To further verify whether the mitophagy pathway was activated by Rcan1-1L, we detected the protein levels of mitophagy receptor Parkin and PINK1. The results showed that Rcan1-1L promoted Parkin translocation from cytosol into mitochondria. The expression of PINK1, which was hardly detected in cytosol, was highly upregulated by Rcan1-1L in mitochondria (Fig. 4C).
Knockdown of Parkin-mediated Mitophagy Pathway Blocks the Effect of Rcan1-1L Overexpression To further investigate whether Rcan1-1L regulated mitophagy specifically to remit the hypoxia-induced growth
314
| www.jcvp.org
inhibition on cardiomyocytes, we silenced the expression of Parkin and then determined whether Rcan1-1L overexpression still promoted cell survival or reduced mitochondrial mass under hypoxic conditions (Fig. 5A). Results showed that knockdown of Parkin disturbed the effect of Rcan1-1L that promoted cell growth and survival under hypoxic conditions (Figs. 5B, C). Furthermore, Rcan1-1L–induced mitochondrial mass reduction was also blocked by silencing the expression of Parkin (Fig. 5D). These data implied that Rcan1-1L promoted cell survival through Parkin-mediated mitophagy pathway.
DISCUSSION Rcan1 has critical functions in inflammation and angiogenesis, which are linked to cardiac hypertrophy, cardiac valve development, and cancer.33 Particularly, Rcan1 is dysregulated in neurodegeneration in Down syndrome and Alzheimer disease.34–36 Rcan1-1 isoforms, such as Rcan1-1L and Rcan1-1S, are widely distributed in tissues from the heart, skeletal muscles, and nervous system.37,38 Evidence suggests that Rcan1 expression is involved in cell survival and death protection. Rcan1 reportedly protects cells from calcium overload–induced cell death.25 Rcan1 deficiency promotes T-helper type I cell apoptosis and increases cell susceptibility to factor associated suicide-mediated and etoposide-induced apoptosis.26,27 Similarly, knockdown of Rcan1-1 and Rcan1-4 isoforms triggers apoptosis in endothelial cells.28 Kim et al39 have recently demonstrated that Rcan1-1 can protect neurons from hydrogen peroxide–induced cell death by inducing 2014 Lippincott Williams & Wilkins
J Cardiovasc Pharmacol Volume 64, Number 4, October 2014
FIGURE 5. Parkin siRNA disturbs the effect of Rcan1-1L overexpression. A, Western blot analysis was performed to detect the protein expression of Parkin and Rcan1-1L. B, Cell growth and viability was assessed by MTT assay. *P , 0.05 denotes null vector group versus Rcan1-1L group or Rcan1-1L + nonsiRNA transfected group under hypoxia. †P , 0.05 denotes Rcan1-1L + Parkin siRNA transfected group versus Rcan1-1L group or Rcan1-1L + non-siRNA transfected group under hypoxia. C, Cell apoptosis was tested by TUNEL assay. **P , 0.01 denote null vector group versus Rcan1-1L group or Rcan1-1L + non-siRNA transfected group under hypoxia. ††P , 0.01 denote Rcan1-1L + Parkin siRNA transfected group versus Rcan1-1L group or Rcan1-1L + non-siRNA transfected group under hypoxia. D, Mitochondria were labeled with MitoTracker Green, and signals were measured using the FACS analyzer. Cells without treatment cultured under normoxic conditions were taken as control. *P , 0.05 denotes null vector group versus Rcan1-1L group or Rcan1-1L + non-siRNA transfected group under hypoxia. †P , 0.05 denotes Rcan1-1L + Parkin siRNA transfected group versus Rcan1-1L group or Rcan1-1L + non-siRNA transfected group under hypoxia. Cells under hypoxic conditions were divided into 4 groups without different treatments. Vector, cells were treated with null vector without Rcan1-1L. Rcan1-1L, cells were treated with Rcan1-1L expression vectors. Rcan1-1L + Non-siRNA, cells were 2014 Lippincott Williams & Wilkins
Hypoxia-induced Apoptosis via Mitophagy
cAMP response element-binding protein and its target gene Bcl-2. Short-term expression of Rcan1-1L protects neurons from oxidative stress-induced apoptosis by inhibiting caspase-3.40 Consistent with these findings, we found that Rcan1-1L overexpression protects cardiomyocytes from hypoxia-induced cell death. In addition, Rcan1-1L overexpression promoted the expression of the antiapoptotic protein Bcl-2 and inhibited that of the proapoptotic proteins caspase-3 and Bad. However, several inconsistent reports were also found. For example, glucocorticoid hormones specifically evoke the upregulation of Rcan1-1 that makes human leukemic cells susceptible to apoptosis.41 In addition, dexamethasone enhances neuronal apoptosis by elevating Rcan1 expression.33 These apparent discrepancies imply that Rcan1-1 has a complex function for cell survival or death under different stress and stimulus conditions. In the present study, we found that Rcan1-1L overexpression promoted mitochondria degradation through mitophagy. This result is consistent with the finding of Ermak et al29 We also demonstrated that Rcan1-1L overexpression decreased mitochondrial mass reduction and mtPTP opening. Previous studies suggested that mtPTP opening is involved in mitophagy induction.42,43 We identified that Rcan1-1L overexpression increased autophagy flow. mTOR, which inhibits autophagy during normal conditions, was downregulated. LC3, a critical component in autophagosomal membrane formation, was markedly upregulated. The mitochondrial protein marker TOM20 was significantly decreased, whereas Golgi body and endoplasmic reticulum remained unchanged. Thus, we speculated that Rcan1-1L overexpression specifically induces mitophagy in cardiomyocytes during hypoxia. However, we observed that Rcan1-1L overexpression inhibited cell growth under normoxic conditions. It is apparent that decreased mitochondrial impair cell growth under normal conditions. Under hypoxia, damaged mitochondria are increased, which is detrimental for cell growth and survival. Therefore, accelerating the degradation of damaged mitochondria by Rcan1-1L is beneficial for cell growth and survival under hypoxic stress. Mitophagy has been recently proven to have unique functions in ischemic heart disease. Integrated mitochondria are essential for cell survival. Damaged mitochondria caused by hypoxic treatment in cardiomyocytes generate excessive reactive oxygen species and activate cell death, which trigger myocardial damage.5–7 Therefore, efficient and specific clearance of dysfunctional mitochondria is beneficial for cardiomyocyte protection under hypoxic conditions. Knockdown of autophagy-related gene ATG5 causes mitochondrial dysfunction, which leads to rapid myocardial failure in mouse model.44 Parkin-deficient mice demonstrate enhanced accumulation of damaged mitochondria, which cause myocardial dysfunction
cotransfected with Rcan1-1L expression vectors and nonspecific siRNA. Rcan1-1L + Parkin siRNA, cells were cotransfected with Rcan1-1L expression vectors and Parkinspecific siRNA. Cells with different treatments were cultured for 24 hours under hypoxic conditions and then harvested for analysis. www.jcvp.org |
315
J Cardiovasc Pharmacol Volume 64, Number 4, October 2014
Yan et al
and decrease survival rate.45 Similarly, BNIP3 and/or Nix knockdown causes the accumulation of damaged mitochondria and cardiac dysfunction.46 A recent study has demonstrated that p53 knockdown stimulates mitophagy in cardiomyocytes, which inhibits myocardial damage induced by ischemia.47 In the present study, we found that Rcan1-1L overexpression induced mitophagy receptor Parkin and PINK1 in cardiomyocytes during hypoxia. Knockdown of Parkin apparently disturbed the effect of Rcan1-1L overexpression on cell survival and mitochondrial mass implying that Rcan1-1L might evoke mitophagy through Parkin-mediated mitophagy pathway. In conclusion, our data revealed that targeting Rcan1-1L to specifically remove damaged mitochondria has a potential application in the treatment of myocardial ischemia. Rcan1-1L gene expression strategies in animal models of myocardial ischemia should be performed to further confirm and verify the cytoprotective effects of Rcan1-1L. However, 1 major concern that should be considered is that excessive mitophagy can enhance cell death. Thus, more investigation should be performed to explore methods for monitoring and controlling appropriate mitophagy induced by Rcan1-1L. For instance, hypoxia response element–mediated Rcan1-1L expression in genetic therapy delivery agent such as recombinant adenoassociated virus could be used to monitoring Rcan1-1L– induced mitophagy, in which Rcan1-1L is only expressed under myocardial ischemia. Once ischemia condition is relieved, the Rcan1-1L expression is stopped that will avoid excessive mitophagy. However, certain limitations of this study should be noted. In the present study, we detected the mitochondrial mass alterations, and the autophagy flow represented by mTOR and LC3 protein levels to claim the conclusion that Rcan1-1L specifically initiated mitophagy. To a certain degree, the mitophagy receptor Parkin knockdown experiments confirmed the Rcan1-1L–induced mitophagy. However, there is still lack of direct evidences for Rcan1-1L–induced mitophagy such as a direct observation of mitochondria and the mitochondria-related autolysosome alterations by electron microscope scanning. Moreover, the effect of Rcan1-1L– induced on the phenotype of the cardiac cells should be concerned. However, studies of Rcan1-1L–induced mitophagy should be further conducted in our laboratory and the treatment of myocardial ischemia by Rcan1-1L–induced mitophagy will also be tested in animal models in our laboratory. REFERENCES 1. Go AS, Mozaffarian D, Roger VL, et al. Heart disease and stroke statistics–2013 update: a report from the American Heart Association. Circulation. 2013;127:e6–e245. 2. Fox KA, Steg PG, Eagle KA, et al. Decline in rates of death and heart failure in acute coronary syndromes, 1999-2006. JAMA. 2007;297:1892– 1900. 3. Simoons ML, Windecker S. Controversies in cardiovascular medicine: chronic stable coronary artery disease: drugs vs. revascularization. Eur Heart J. 2010;31:530–541. 4. Verma S, Fedak PW, Weisel RD, et al. Fundamentals of reperfusion injury for the clinical cardiologist. Circulation. 2002;105:2332–2336. 5. Chatterjee S, Stewart AS, Bish LT, et al. Viral gene transfer of the antiapoptotic factor Bcl-2 protects against chronic postischemic heart failure. Circulation. 2002;106:I212–I217. 6. Nakagawa T, Shimizu S, Watanabe T, et al. Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death. Nature. 2005;434:652–658.
316
| www.jcvp.org
7. Neubauer S. The failing heart–an engine out of fuel. N Engl J Med. 2007; 356:1140–1151. 8. Jimenez RE, Kubli DA, Gustafsson AB. Autophagy and mitophagy in the Myocardium: therapeutic potential and concerns. Br J Pharmacol. 2014; 171:1907–1916. 9. Youle RJ, Narendra DP. Mechanisms of mitophagy. Nat Rev Mol Cell Biol. 2011;12:9–14. 10. Hamacher-Brady A, Brady NR, Logue SE, et al. Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy. Cell Death Differ. 2007;14:146–157. 11. Zhang J, Ney PA. Role of BNIP3 and NIX in cell death, autophagy, and mitophagy. Cell Death Differ. 2009;16:939–946. 12. Liu L, Feng D, Chen G, et al. Mitochondrial outer-membrane protein FUNDC1 mediates hypoxia-induced mitophagy in mammalian cells. Nat Cell Biol. 2012;14:177–185. 13. Crawford DR, Leahy KP, Abramova N, et al. Hamster adapt78 mRNA is a Down syndrome critical region homologue that is inducible by oxidative stress. Arch Biochem Biophys. 1997;342:6–12. 14. Davies KJ, Ermak G, Rothermel BA, et al. Renaming the DSCR1/ Adapt78 gene family as RCAN: regulators of calcineurin. FASEB J. 2007;21:3023–3028. 15. Wang Y, De Keulenaer GW, Weinberg EO, et al. Direct biomechanical induction of endogenous calcineurin inhibitor Down Syndrome Critical Region-1 in cardiac myocytes. Am J Physiol Heart Circ Physiol. 2002; 283:H533–H539. 16. Ermak G, Pritchard MA, Dronjak S, et al. Do RCAN1 proteins link chronic stress with neurodegeneration? FASEB J. 2011;25:3306–3311. 17. Hattori M, Fujiyama A, Taylor TD, et al. The DNA sequence of human chromosome 21. Nature. 2000;405:311–319. 18. Qi YM, Lei T, Zhou L, et al. Genomic organization, alternative splicing and tissues expression of porcine CREB3L4 gene. Mol Biol Rep. 2009; 36:1881–1888. 19. Ermak G, Harris CD, Davies KJ. The DSCR1 (Adapt78) isoform 1 protein calcipressin 1 inhibits calcineurin and protects against acute calciummediated stress damage, including transient oxidative stress. FASEB J. 2002;16:814–824. 20. Ermak G, Harris CD, Battocchio D, Davies KJ. RCAN1 (DSCR1 or Adapt78) stimulates expression of GSK-3beta. FEBS J. 2006;273:2100–2109. 21. Gorlach J, Fox DS, Cutler NS, et al. Identification and characterization of a highly conserved calcineurin binding protein, CBP1/calcipressin, in Cryptococcus neoformans. EMBO J. 2000;19:3618–3629. 22. Kingsbury TJ, Cunningham KW. A conserved family of calcineurin regulators. Genes Dev. 2000;14:1595–1604. 23. Rothermel B, Vega RB, Yang J, et al. A protein encoded within the Down syndrome critical region is enriched in striated muscles and inhibits calcineurin signaling. J Biol Chem. 2000;275:8719–8725. 24. Chang KT, Min KT. Drosophila melanogaster homolog of Down syndrome critical region 1 is critical for mitochondrial function. Nat Neurosci. 2005;8:1577–1585. 25. Zhao P, Xiao X, Kim AS, et al. c-Jun inhibits thapsigargin-induced ER stress through up-regulation of DSCR1/Adapt78. Exp Biol Med (Maywood). 2008;233:1289–1300. 26. Ryeom S, Greenwald RJ, Sharpe AH, McKeon F. The threshold pattern of calcineurin-dependent gene expression is altered by loss of the endogenous inhibitor calcipressin. Nat Immunol. 2003;4:874–881. 27. Kim YS, Lee HJ, Jang C, et al. Knockdown of RCAN1.4 Increases Susceptibility to FAS-mediated and DNA-damage-induced Apoptosis by Upregulation of p53 Expression. Korean J Physiol Pharmacol. 2009;13:483–489. 28. Ryeom S, Baek KH, Rioth MJ, et al. Targeted deletion of the calcineurin inhibitor DSCR1 suppresses tumor growth. Cancer Cell. 2008;13:420–431. 29. Ermak G, Sojitra S, Yin F, et al. Chronic expression of RCAN1-1L protein induces mitochondrial autophagy and metabolic shift from oxidative phosphorylation to glycolysis in neuronal cells. J Biol Chem. 2012;287:14088–14098. 30. Petronilli V, Miotto G, Canton M, et al. Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys J. 1999;76:725–734. 31. Blommaart EF, Luiken JJ, Blommaart PJ, et al. Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes. J Biol Chem. 1995;270:2320–2326.
2014 Lippincott Williams & Wilkins
J Cardiovasc Pharmacol Volume 64, Number 4, October 2014 32. Klionsky DJ, Abdalla FC, Abeliovich H, et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy. 2012;8: 445–544. 33. Sun X, Wu Y, Chen B, et al. Regulator of calcineurin 1 (RCAN1) facilitates neuronal apoptosis through caspase-3 activation. J Biol Chem. 2011;286:9049–9062. 34. Harris CD, Ermak G, Davies KJ. RCAN1-1L is overexpressed in neurons of Alzheimer’s disease patients. FEBS J. 2007;274:1715–1724. 35. Ermak G, Morgan TE, Davies KJ. Chronic overexpression of the calcineurin inhibitory gene DSCR1 (Adapt78) is associated with Alzheimer’s disease. J Biol Chem. 2001;276:38787–38794. 36. Keating DJ, Dubach D, Zanin MP, et al. DSCR1/RCAN1 regulates vesicle exocytosis and fusion pore kinetics: implications for Down syndrome and Alzheimer’s disease. Hum Mol Genet. 2008;17:1020–1030. 37. Fuentes JJ, Pritchard MA, Planas AM, et al. A new human gene from the Down syndrome critical region encodes a proline-rich protein highly expressed in fetal brain and heart. Hum Mol Genet. 1995;4:1935–1944. 38. Fuentes JJ, Pritchard MA, Estivill X. Genomic organization, alternative splicing, and expression patterns of the DSCR1 (Down syndrome candidate region 1) gene. Genomics. 1997;44:358–361. 39. Kim SS, Jang SA, Seo SR. CREB-mediated Bcl-2 expression contributes to RCAN1 protection from hydrogen peroxide-induced neuronal death. J Cell Biochem. 2013;114:1115–1123.
2014 Lippincott Williams & Wilkins
Hypoxia-induced Apoptosis via Mitophagy
40. Wu Y, Song W. Regulation of RCAN1 translation and its role in oxidative stress-induced apoptosis. FASEB J. 2013;27:208–221. 41. Hirakawa Y, Nary LJ, Medh RD. Glucocorticoid evoked upregulation of RCAN1-1 in human leukemic CEM cells susceptible to apoptosis. J Mol Signal. 2009;4:6. 42. Rodriguez-Enriquez S, He L, Lemasters JJ. Role of mitochondrial permeability transition pores in mitochondrial autophagy. Int J Biochem Cell Biol. 2004;36:2463–2472. 43. Rodriguez-Enriquez S, Kai Y, Maldonado E, et al. Roles of mitophagy and the mitochondrial permeability transition in remodeling of cultured rat hepatocytes. Autophagy. 2009;5:1099–1106. 44. Nakai A, Yamaguchi O, Takeda T, et al. The role of autophagy in cardiomyocytes in the basal state and in response to hemodynamic stress. Nat Med. 2007;13:619–624. 45. Kubli DA, Zhang X, Lee Y, et al. Parkin protein deficiency exacerbates cardiac injury and reduces survival following myocardial infarction. J Biol Chem. 2013;288:915–926. 46. Dorn GW 2nd. Mitochondrial pruning by Nix and BNip3: an essential function for cardiac-expressed death factors. J Cardiovasc Transl Res. 2010;3:374–383. 47. Hoshino A, Matoba S, Iwai-Kanai E, et al. p53-TIGAR axis attenuates mitophagy to exacerbate cardiac damage after ischemia. J Mol Cell Cardiol. 2012;52:175–184.
www.jcvp.org |
317
