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. 2020 Sep 8;15(9):e0238857. doi: 10.1371/journal.pone.0238857

Propofol induces mitochondrial-associated protein LRPPRC and protects mitochondria against hypoxia in cardiac cells

Qianlu Zhang 1, Shiwei Cai 2, Liping Guo 3, Guojun Zhao 3,*
Editor: Partha Mukhopadhyay4
PMCID: PMC7478836  PMID: 32898195

Abstract

Background

Hypoxia-induced oxidative stress is one of the main mechanisms of myocardial injury, which frequently results in cardiomyocyte death and precipitates life-threatening heart failure. Propofol (2,6-diisopropylphenol), which is used to sedate patients during surgery, was shown to strongly affect the regulation of physiological processes, including hypoxia-induced oxidative stress. However, the exact mechanism is still unclear.

Methods

Expression of LRPPRC, SLIRP, and Bcl-2 after propofol treatment was measured by RT-qPCR and western blot analyses. The effects of propofol under hypoxia were determine by assessing mitochondrial homeostasis and mitochondrial function, including the ATP level and mitochondrial mass. Autophagy/mitophagy was measured by detecting the presence of LC3B, and autophagosomes were observed by transmission microscopy

Results

Propofol treatment inhibited cleaved caspase-9 and caspase-3, indicating its inhibitory roles in mitochondrial-related apoptosis. Propofol treatment also transcriptionally activated LRPPRC, a mitochondrial-associated protein that exerts multiple functions by maintaining mitochondrial homeostasis, in a manner dependent on the presence of hypoxia-induced factor (HIF)-1α transcriptional activity in H9C2 and primary rat cardiomyocytes. LRPPRC induced by propofol maintained the mitochondrial membrane potential (MMP) and promoted mitochondrial function, including ATP synthesis and transcriptional activity. Furthermore, LRPPRC induced by propofol contributes, at least partially, to the inhibition of apoptotic cell death induced by hypoxia.

Conclusion

Taken together, our results indicate that LRPPRC may have a protective antioxidant effect by maintaining mitochondrial homoeostasis induced by propofol and provide new insight into the protective mechanism of propofol against oxidative stress.

Introduction

Under hypoxic conditions, cardiomyocyte hypertrophy is a process that meets the increased oxygen demand and maintains homeostasis [1]. Increased oxygen consumption induces oxidative stress and might induce physiological and even pathological events [2]. Thus, myocardial hypoxia is considered a major factor in cardiac ischemia and myocardial infarction and may cause microvascular disease [3] and heart failure [4]. Since cardiomyocytes are terminally differentiated cells that are not believed to regenerate, preventing the cardiomyocyte injury and loss induced by hypoxia is a vital therapeutic strategy. Thus, targeting hypoxic signaling is considered a promising strategy to protect against heart injury [5].

Propofol is a widely employed intravenous anesthetic that has also been found to display protective effects on oxidative stress in different types of cells, including cardiomyocytes [6], hippocampal neurons [7], and kidney cells [8]. It has been reported that in rat cardiac H9c2 cells, propofol transcriptionally activates antioxidant enzymes to abolish oxidative stress induced by H2O2 [6]. Li and colleagues found that hypoxia-induced oxidative stress was decreased after inhibition of calcineurin-induced calcium overload and activation of the subsequent YAP signaling pathway in hippocampal neurons [6]. Propofol treatment was also proven to exert a protective effect on kidney damage after sepsis-induced AKI by inhibiting oxidative stress [9]. However, given that oxidative stress is one of the most critical factors of mitochondrial damage [10], the exact effect of propofol on oxidative stress-induced mitochondrial damage is still largely unknown.

Leucine-rich pentatricopeptide repeat-containing (LRPPRC), also known as LRP130, is a member of the pentatricopeptide repeat protein family and has multiple functions in various processes, including homeostasis, microtubule alterations, RNA stability, DNA/RNA binding, transcriptional activity in the mitochondria, metabolic processes, RNA nuclear export, tumorigenesis and progression [11–15]. LRPPRC is essential for maintaining mitochondrial homeostasis by protecting the mitochondrial membrane through interactions with Beclin 1 and Bcl-2 and formation of a ternary complex to maintain Bcl-2 stability [16,17,18]. This protein was also found in the mitochondrial matrix and binds to single-stranded RNA to post-transcriptionally regulate mitochondrial genes [19]. LRPPRC acts as an inhibitor of autophagy/mitophagy by maintaining MMP and thus promoting mitochondrial function [17].

SRA stem-loop interacting RNA binding protein (SLIRP) was initially isolated as a protein that binds to the stem-loop structure of the RNA molecule SRA [20]. An RNA recognition motif (RRM) domain of SLIRP was shown to physically bind to pentatricopeptide repeat (PPR) domains on LRPPRC and thus regulates mitochondrial protein synthesis by protecting LRPPRC from degradation [21–23]. Interestingly, SLIRP was also found to be a novel protein that interacted with Bcl-2 [23]. Although SLIRP was not found to mediate the ability of Bcl-2 to protect against apoptosis and oxidative damage, the binding of Bcl-2 to SLIRP stabilizes the SLIRP protein and regulates mitochondrial mRNA levels. This finding raised the question of whether LRPPRC, SLIRP and Bcl-2 form heterotrimers and function synergistically. These results could provide new insight into the regulation of these proteins on cell physiological processes and mitochondrial function.

In this study, we established a model of hypoxia in H9c2 cells and primary rat cardiomyocytes to investigate the effects of propofol on hypoxia-induced myocardial damage. We found that propofol exerts protective effects against hypoxia-induced apoptosis potentially by upregulating LRPPRC. LRPPRC upregulation attenuated the hypoxia-induced cardiomyocyte injury by maintaining mitochondrial homeostasis and function, suggesting a novel protective mechanism of propofol against hypoxia-related heart diseases.

Material and methods

Cell culture and treatment

H9C2 cell line was originally derived from the embryonic rat ventricle and was bought from American Type Culture Collection (ATCC Manassas, VA, USA). Cells were stored in liquid nitrogen and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% Fetal bovine serum (FBS, Life Technologies, Grand Island, NY, USA), 100 U/mL penicillin and 100 g/mL streptomycin in a humidified atmosphere of 5% CO2 and 95% air at 37°C.

Hypoxic cultures were carried out in a humidified hypoxia workstation invivo 400 model (Ruskin Technology Ltd, Bridgend, UK). Briefly, medium was pre-exposed to 5% CO2, 20% (Normoxia) or 1%O2 (Hypoxia) balanced with nitrogen. Then, cells were cultured in pre-treated medium and kept in corresponding conditions for 24 or 48 h.

Animals and isolation of cardiac cells

2-day-old Wistar rats were used for isolating primary rat cardiac myocytes (RCM). All of the experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the First Affiliated Hospital of the University of South China ethics committee.

The hearts were dissociated into single suspended cells by using SoniConvert® Sonicator (UTL: http://www.doc-sense.com/index.html, DocSense, Chengdu, China) following the manufacturer’s instruction. Briefly, the hearts were rapidly excised and rinsed in ice-cold PBS. The hearts were minced and incubated in DS buffer supplied by DocSense (Chengdu, China), and incubated at 37°C for 5 min with several times of rotation. Then Sonicator was employed to dissociate tissue into single cells with the procedure: 3 s under 10% power. After a brief centrifugation at 400 g, 4°C for 5 min, cell pellet was collected for further culturing and analysis. For culturing, cells were transferred to serum-free Cardiac Myocyte Medium (ScienCell Research Laboratories, cat. No.: 6101, Carlsbad, CA). Cells were maintained in a CO2 incubator at 37°C in humid air containing 5% CO2. The medium was refreshed every two days.

Annexin V/PI double staining

Cultured cells were suspended using 0.25% Trypsin and washed with ice-cold PBS twice to remove trypsin and EDTA. Then cells were suspended in ice-cold PBS and adjust the cell concentration to 1×106 cells per 1mL. The cells were then stained with Annexin V-FITC (green fluorescence) and PI (red fluorescence), which allowed the identification of intact cells (FITC-/PI-), early stage of apoptotic cells (FITC+/PI), late stage of apoptotic cells (FITC+/PI+) and unapoptotic cells (FITC-/PI+). Then stained cells were analyzed by 3 laser Navios flow cytometers (Beckman Coulter, Brea, CA, USA). The data files was analyzed using FlowJo 9.7.6 (FlowJo LLC, Ashland, OR USA).

JC-1 staining

JC-1 probe was employed to investigate the mitochondrial membrane potential to determine the mitochondrial homeostasis. JC-1 (20 μg/mL, Life Technologies, Grand Island, NY, USA) was added and incubated at 37°C for 20 min and then washed twice with ice-cold PBS to remove remained dye. Images were taken under a X71 (U-RFL-T) fluorescence microscope (Olympus, Melville, NY). The red fluorescence emission maximum at 590 nm represents JC-1 aggregates, and green fluorescence emission maximum at 529 nm represents JC-1 monomers.

siRNA transfection

Knockdown of mRNA level of LRPPRC was achieved by transient transfection of cells with siRNA duplexes (Thermo Scientific, Waltham, MA, USA), specific to the mRNAs of LRPPRC. The relevant siRNA sequences were: 5′-GCCUGCCGAUUGAACCAAATT-3`and 5′-UUUGGUU CAAUCGGCAGGCAA-3′; negative control (NC) sense 5’-GUUCAAUAUUAUCAAGCGGUU-3’ and antisense 5’-CCGCUUGAUAAUAUUGAACUU-3’. According to author’s instruction, 2×105 cells were grown in 2 ml of serum-free medium. A siRNA/transfection reagent complex was formed at room temperature by combining siRNA oligomer (50 nM) with 5 μl (2 μg/ml) Lipofectamine™ 2000 transfection reagent (Thermo Scientific, Waltham, MA, USA) in 0.5 ml OptiMEM medium (Thermo Scientific, Waltham, MA, USA), and this was applied to cells for 48 h until they were harvested. Cells transfected with NC siRNA was considered as Control cells.

Real-time quantitative PCR (RT-qPCR)

To detect mitochondrial transcriptional activity, the transcriptional levels of mitochondrial-coded genes, including COX I and ND1, were analyzed by RT-qPCR. 1×106 cells were collected by trypsin and pelleted by centrifugation at 1000g for 5 min. 1 ml of TRIzol reagent (Life Technologies, Grand Island, NY, US) was added and pellet was sonicated by SoniConvertTM system (DocSense, Chengdu, China) and total RNA was extracted according to the manual. Then isolated RNA was reverse transcripted using a Reverse Transcriptase Kit (RIBOBIO, Guangzhou, China). The PCR was performed using Power SYBRTM Green PCR Master Mix Kit (Applied Biosystems, Foster City, CA, USA). The qPCR was performed in a ABI7500 system (Applied Biosystems, Foster City, CA, USA) under the following conditions: 95°C 5 min, 35 cycles of 95°C 10s, and 60°C 1min. The specific primers used were followed: LRPPRC, 5’-CTGCACTGTGCTCTTCAAGC-3’ and 5’-GACTGCACACTACCGAAGCA-3’; COX I forward: 5’- GGAGCAGTATTCGCCATCAT-3’; reverse: 5’-GAGCACTTCTCGTTTTGATGC-3’; ND1 forward: 5’- CACCCCCTTATCAACCTCAA-3’; reverse: 5’-ATTTGTTTCTGCGAGGGTTG-3’; GAPDH forward: 5’-CCTTCATTGACCTCAACTACAT-3’; reverse: 5’-CCAAAGTTGTCATGGATGACC-3’.

To detect mitochondrial DNA contents, total DNA was isolated using Genome DNA isolation kit (DocSense, Chengdu, China) and 30 ng of DNA was used as template for each reaction. The same reacting system and running condition was the same with RT-qPCR. The specific primers used were followed: 12S rDNA forward: 5’- ACCGCGGTCATACGATTAAC-3’; reverse: 5’- AGTACCGCCAAGTCCTTTGA-3’; 18S rDNA forward: 5’-TCAATCTCGGGTGGCTGAACG-3’; reverse: 5’-GGACCAGAGCGAAAGCATTTG-3’.

Western blot

The primary antibodies used was listed as followed: Rabbit monoclonal anti-LRPPRC antibody (1: 2000, #ab97505); Rabbit monoclonal anti- Bcl-2 antibody (1:1000, #abab32124); Rabbit monoclonal anti- Beclin 1 antibody (1:1000, #ab208612); rabbit monoclonal anti-cleaved PARP1 antibody (1:2000, #ab32561); rabbit monoclonal anti-pro-caspase-3 antibody (1:2000, #ab32150); rabbit monoclonal anti-cleaved-caspase-3 antibody (1:1000, #ab49822); rabbit monoclonal anti-pro caspase-8 antibody (1:1000, #ab108333); mouse monoclonal anti-cleaved-caspase-8 antibody (1:1000, #9496S, CST); mouse monoclonal anti-caspase-9 antibody (1:1000, #9508T, CST). Rabbit monoclonal anti-β-actin antibody (1:5000, #ab8227). Goat anti-rabbit IgG H&L antibody (HRP ladled, 1:10000, #ab7090) was used as secondary antibody. Blot bands were quantified via densitometry with Image J software (National Institutes of Health Baltimore, MD, USA). β-actin was used as an internal reference.

Immunoprecipitation

For immunoprecipitation assay, 5×106 cells were lyzed using SoniConvert® sonicator (DocSense, Chengdu, Sichuan, China. URL: http://www.doc-sense.com/index.html) according to the manufacturer’s instruction. 200 μl of supernatant was incubated with 10 μg of Anti-Flag antibody, Anti-HA antibody or rabbit IgG (negative control) followed by immunoprecipitation with 50 ul of protein A agarose beads during and overnight incubation at 4°C with rotation. Product was denatured in 200 μl of 1×SDS loading buffer and immunoblotting was performed as described before.

Mitochondrial ATP synthesis

To measure ATP synthesis, cells were suspended in solution containing 0.22M sucrose, 0.12 M mannitol, 40 mM Tricine, pH7.5, and 1mM EDTA and lysed using SoniConvertTM system and analyzed using Optocomp I BG-1 luminometer (GEM Biomedical Inc.) using the ATP Bioluminescent Assay kit (Sigma).

Statistical analysis

All data were analyzed for statistical significance using SPSS 13.0 software (SPSS, Chicago, IL, USA) and presented as means±SD from at least 3 independent experiments performed in duplicate. Statistical comparisons of the results were made using analysis of One-way ANOVA. P<0.05 was considered statistically significant.

Results

Propofol exerts a protective effect against hypoxia-induced cell injury

To determine the effect of propofol on hypoxia-induced cell injury, we subjected the cells to hypoxic conditions for 0, 24 and 48 h with different concentrations of propofol ranging from 10 to 100 mM, and cell viability was assayed using the CCK-8 assay. As shown in Fig 1A, 25, 50 and 100 mM propofol inhibited the decrease in viability after 48 h of treatment normalized to normoxia group (P<0.05). Treatment with 50 mM propofol was further used to detect hypoxia-induced apoptotic cell death after 48 h. As expected, both early- and late-stage apoptosis induced by hypoxia decreased significantly after propofol treatment (P<0.05, Fig 1B). We then investigated PARP, pro-caspase 3, cleaved-caspase 3, pro-caspase-9, cleaved-caspase 9 and pro-caspase 8 and observed increased protein levels of cleaved-caspase-3, cleaved-caspase 9, and cleaved-PARP after hypoxia exposure; these changes were significantly reversed by the addition of propofol (P<0.05, Fig 1C). Consistently, propofol also decreased the caspase 3 and caspase 9 activities, which were stimulated by hypoxia exposure (P<0.05, Fig 1D).

Fig 1. Propofol exerts protective effect against hypoxia-induced cell injury via inhibiting mitochondria-related apoptosis.

Fig 1

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(A) The cell viability was assayed using CCK-8 when H9c2 cardiomyocytes were incubated with 1% O2 after 0, 24 and 48 h. (B) Cell apoptosis was detected using Annexin V-FITC/PI double staining after being incubated with 1% O2 with or without 50 mM of propofol for 48 h. (C) the protein level of PARP, pro-caspase 3, pro-caspase 8, pro-caspase 9, cleaved-caspase 3 and cleaved-caspase 9 were detected using western blot. *P<0.05, vs. Normoxia group; #P<0.05, Hypoxia/Mock group. (D) caspase 3 and caspase 9 activity was measured. *P<0.05, vs. Normoxia group; #P<0.05, Hypoxia/Mock group.

Propofol transcriptionally activates LRPPRC under hypoxic but not normoxic conditions

To determine whether LRPPRC and its related proteins are involved in the effects of propofol, we detected LRPPRC, Bcl-2 and Beclin 1. As expected, LRPPRC increased after 24 h and 48 h of incubation with propofol under hypoxia (P<0.05, Fig 2A). Furthermore, Bcl-2 showed a similar trend as LRPPRC. RT-qPCR analysis further confirmed the transcriptional activation of LRPPRC (P<0.05, Fig 2B). We then performed immunofluorescence staining to localize LRPPRC and found that propofol-induced LRPPRC localized around the nucleus (Fig 2C). Notably, propofol treatment under normoxic conditions failed to affect both the mRNA and protein levels of LRPPRC (Fig 2D), indicating that propofol regulates LRPPRC in the context of hypoxia biology.

Fig 2. Propofol transcriptionally induced LRPPRC under hypoxic condition.

Fig 2

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(A) western blot was performed to detect the effects of propofol on LRPPRC, Bcl-2 and Beclin 1 protein levels under hypoxic condition. *P<0.05, vs. Hypoxia/24 h group. #P<0.05, Hypoxia/48 h group. (B) RT-qPCR assay was performed to detect the mRNA level of LRPPRC after propofol exposure. (C) immunofluorescent staining was performed to localize LRPPRC in H9c2 cells. (D) RT-qPCR assay was performed to detect the effect of propofol under normoxic condition.

Propofol transcriptionally activates LRPPRC depending on the transcriptional activity of HIF-1α

To detect the possible mechanism of upregulation of LRPPRC by propofol, we used hypoxia, the hypoxia mimic DFO and CoCl2 for treatment of H9c2 cells. As shown in Fig 3A, hypoxia, DFO and CoCl2 cotreatment with propofol all significantly increased LRPPRC, and this change was reversed by the addition of a transcriptional inhibitor of HIF-1α, 1 ng/ml echinomycin. RT-qPCR showed that propofol transcriptionally activated LRPPRC under oxidative stress (Fig 3B). All these results demonstrated that propofol transcriptionally activated LRPPRC in the presence of HIF-1α transcriptional activity. Analysis of apoptotic death demonstrated consistent results, showing that the antiapoptotic effect of propofol was reversed by the addition of echinomycin (Fig 3C).

Fig 3. Propofol transcriptionally activated LRPPRC.

Fig 3

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(A) Western blot was employed to detect LRPPRC protein after hypoxia, DFO and CoCl2 treatment. (B) RT-qPCR assay was employed to detect mRNA level of LRPPRC. (C) Cell treated with propofol under hypoxia was analyzed for apoptotic death rate.

To further confirm whether propofol regulates LRPPRC in primary cardiomyocytes, neonate cardiomyocytes were chosen for further confirmation, instead of adult cardiomyocytes for the poor survival period and low transfection efficacy. Firstly, we isolated RCM from 2-day-old rat and cultured in 6-well plate (Fig 4A). After normoxia, hypoxia and hypoxa/propofol treatment for 24 h, consistently, LRPPRC was immune-stained and presented to be uprelated in hypxia/propofol co-treated group (Fig 4B). Hypoxia exposure, DFO and CoCl2 treatment obviously upregulated HIF-1α protein (Fig 4C), and upregulated LRPPRC protein with the presence of propofol, which were reversed by inhibiting HIF-1α transcriptional activity, indicated the consistent finding which was observed in H9C2 cells. We then also detect the effect of propofol under hypoxia on apoptosis in primary cardiomyocytes. As it is shown in Fig 4D, addition of propofol inhibited hypoxia-induced apoptosis, which was reversed by inhibiting HIF-1α transcriptional activity. All these results suggested that, propofol exerts protective effect against hypoxia-induced apoptosis and upregulated LRPPRC via HIF-1α transcriptional activity.

Fig 4. Propofol exerts protective effect against hypoxia-induced cell apoptosis in RCM.

Fig 4

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(A) Isolated RCM was cultured. (B) immunofluorescent staining was performed to localize LRPPRC in RCM. (C) The LRPPRC and HIF-1α protein levels were detected by western blot. (D) Cell treated with propofol under hypoxia was analyzed for apoptotic death rate.

Propofol maintains the mitochondrial membrane potential and mitochondrial functions under hypoxic conditions depending on the activation of LRPPRC

After exposure to hypoxia for 24 h with or without propofol, JC-1 staining was performed, and we observed that the decrease in aggregates induced by hypoxia exposure was reversed by the addition of propofol, and this change was abolished by inhibition of HIF-1α transcriptional activity (Fig 5A). As expected, propofol also promoted ATP synthesis (Fig 5B), mitochondrial DNA content (Fig 5C) and transcriptional activity (Fig 5D), demonstrating that propofol protects mitochondria from hypoxia-induced mitochondrial dysfunction. Furthermore, we detected the status of autophagy after hypoxic exposure. As shown in Fig 5E, the LC3-II level was decreased at both 12 and 24 h after the addition of propofol, indicating that propofol also potentially inhibited hypoxia-induced autophagy and thus inhibited mitochondria degradation induced by membrane damage. We then = observed autophagic vacuoles affected by LRRPRC. As shown in Fig 4F, in the presence of the lysosomal inhibitor bafilomycin A1, propofol treatment decreased the number of autophagic vacuoles, which was reversed by the addition of echinomycin (Fig 5F).

Fig 5. Propofol contributes to maintenance of mitochondrial homeostasis and mitochondrial functions.

Fig 5

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(A) H9c2 cells exposed to hypoxia with or without propofol were stained with JC-1 to identify mitochondrial membrane potential. (Red, JC-1 aggregates; green, JC-1 monomer). The effects of propofol on regulating ATP synthesis (B), mitochondrial DNA mass (C), and transcriptional activity (D) were measured. (E) western blot was performed to detect LC-3 level. (F) Transmission electron microscopy imaging of cells exposed hypoxia with or without propofol. *, autophagic vacuoles.

We further determined whether propofol-induced LRPPRC is critical for propofol’s protective effect against hypoxia. siRNA targeting LRPPRC mRNA (siLRPPRC) was introduced before hypoxia exposure, and western blot analysis showed that siLRPPRC efficiently knocked down endogenous LRPPRC under normoxia and upregulated LRPPRC under hypoxia (Fig 6A). After LRPPRC knockdown, the ratio of LC3B-II/LC3B-I increased compared to that of the control group (siNC transfected), indicating that endogenous LRPPRC inhibited the basal level of mitophagy/autophagy (Fig 6A). As expected, under hypoxic conditions, propofol reversed the increased ratio of LC3B-II/LC3B-I, which was abolished by LRPPRC knockdown (Fig 6A). We detected ATP synthesis and mitochondrial DNA mass and consistently observed that knockdown of LRPPRC under both normoxia and hypoxia decreased ATP synthesis and mitochondrial DNA mass, demonstrating that LRPPRC may be critical for maintaining mitochondrial function (Fig 6B&6C).

Fig 6. Propofol potentially inhibited autophagy/mitophagy and maintained mitochondrial function via upregulated LRPPRC.

Fig 6

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(A) After efficiently knockdown of LRPPRC, LC3B-I/II level was detected by western blot. *P<0.05, vs. hypoxia/mock group. #P<0.05, vs. hypoxia/siNC group. After hypoxia exposure with or without LRPPRC knockdown, ATP synthesis (B) and mitochondrial DNA mass (C) were measured. *P<0.05, vs. hypoxia/mock group. #P<0.05, vs. hypoxia/siNC group.

Hypoxia-induced LRPPRC stabilizes Bcl-2 potentially by binding to it

To confirm whether hypoxia regulates Bcl-2 via LRPPRC induction, we treated the hypoxia-exposed cells with propofol after LRPPRC knockdown. As expected, propofol treatment obviously increased LRPPRC under hypoxia exposure, and LRPPRC knockdown obviously decreased the Bcl-2 protein level compared to that in the hypoxia/mock group (Fig 7A). Moreover, Beclin 1 was not obviously affected by upregulated LRPPRC. As LRPPRC tightly regulates the Bcl-2 protein stability by binding directly to it, we wanted to determine whether propofol-induced LRPPRC protein enhances binding to Bcl-2 [16–18]. After immunoprecipitation, the obtained products were detected for the existence of LRPPRC, Bcl-2, Beclin 1 and β-actin. The intensive signal of LRPPRC and undetectable β-actin demonstrated the specific and efficient precipitation of LRPPRC by an anti-LRPPRC antibody (Fig 7B). Notably, the most intensive signals of LRPPRC, Bcl-2 and Beclin 1 were observed after propofol treatment, and LRPPRC knockdown decreased Bcl-2 and Beclin 1 in the IP product. These results indicated that propofol-upregulated LRPPRC binds more strongly to Bcl-2 and Beclin 1 and may affect the stability of Bcl-2 but not Beclin 1.

Fig 7. Propofol-induced LRPPRC physically interacted with Bcl-2 and Beclin 1.

Fig 7

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(A) LRPPRC knockdown using siRNA transfection obviously decreased Bcl-2 protein. (B) Immunoprecipitation was carried out to detect the physical interaction between LRPPRC, Bcl-2 and Beclin 1.

Hypoxia-induced LRPPRC binds the SLIRP protein

Given that SLIRP was reported to bind to and prevent the degradation of LRPPRC and also interact with Bcl-2 [21–23], we assessed whether SLIRP is affected under hypoxic conditions. Western blotting showed that SLIRP had a similar trend to LRPPRC at the protein level (Fig 8A), without being affected transcriptionally. The direct binding of SLIRP to LRPPRC or Bcl-2 is necessary for its stabilizing effects on LRPPRC or Bcl-2; thus, we tried to determine whether SLIRP, LRPPRC and Bcl-2 form heterotrimers. We tried to precipitate endogenous SLIRP or LRPPRC. However, SLIRP and LRPPRC interactions were not observed, which may be due to low endogenous protein levels. Thus, we overexpressed Flag-tagged LRPPRC and HA-tagged SLIRP in H9C2 cells and reperformed the immunoprecipitation analyses. As presented in Fig 8C (right panel), Flag-LRPPRC and HA-SLIRP were intensively expressed after enrichment. Flag-LRPPRC and HA-SLIRP were successfully precipitated, and in separate IP products, the LRPPRC, SLIRP and Bcl-2 proteins were all detected (Fig 8C), indicating that LRPPRC, SLIRP and Bcl-2 potentially form heterotrimers and thus function synergistically.

Fig 8. Propofol-induced LRPPRC interacted with SLIRP.

Fig 8

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(A) Propofol treatment affected SLIRP protein level under hypoxia. (B) SLIRP mRNA level was measured by RT-qPCR after propofol treatment under hypoxia. (C) immunoprecipitation was carried out to detect the potential formation of heterotrimer of LRPPRC, SLIRP and Bcl-2.

Discussion

In this study, we demonstrated the protective effects of propofol on cardiomyocyte injury induced by hypoxia. Propofol maintained mitochondrial homeostasis and promoted mitochondrial function. In rat H9C2 cells, propofol reversed apoptotic cell death induced by oxidative stress, indicating its potential protective mechanism by protecting mitochondria.

Propofol has critical antioxidant and antiapoptotic effects [24]. In human umbilical vein endothelial cells (HUVECs) exposed to H2O2-induced oxidative stress, propofol abolished reactive oxygen species and thus inhibited apoptotic cell death. Propofol attenuated the cleavage of caspase-3 and the expression of Bcl-2 and Bax in a dose-dependent manner. This drug also ameliorated H2O2-induced phosphorylation of the p38 MAPK, JNK, and Akt signaling pathways, which are critical for initiating or promoting apoptosis [25]. Here, we revealed that propofol treatment protected H9c2 cells from hypoxia-induced cell apoptosis. We further found that propofol treatment substantially increased mitochondrial function, including increasing ATP synthesis, mitochondrial DNA content and transcriptional activity. Mitochondria play key roles in inducing apoptotic cell death via ROS-inducing pathways [26], including regulating calcium levels and opening of the mitochondrial permeability transition pore followed by release of cytochrome c. Sumi and colleagues reported that propofol treatment targets mitochondrial complexes I, II, and III and induces a cellular metabolic switch from oxidative phosphorylation to glycolysis [26]. Subsequently, accumulated ROS activated caspase 9 and 3/7 and induced mitochondrial-dependent apoptosis. According to our data, propofol treatment decreased mitochondrial function, which may cause the decrease in ROS, and maintained mitochondrial homeostasis under oxidative stress.

LRPPRC tightly regulates the promotion of cell cycle phases probably by regulating mitochondrial function and mitophagy in physiological and pathological processes. Previous studies have shown that in cancer progression, LRPPRC interacts with Beclin 1 and Bcl-2 and forms a ternary complex to maintain Bcl-2 stability [27,28], resulting in the maintenance of mitochondrial homeostasis and mitochondrial function. By considering that the relative high level of LRPPRC, we employed siRNA targeting to LRPPRC mRNA to modify LRPPRC protein. As expected, knockdown of LRPPRC causes a decrease in Bcl-2, followed by Beclin 1 release to form complexes with PI3KCIII to activate basal levels of autophagy [29]. In our study, we found that under hypoxic conditions, propofol treatment transcriptionally upregulated LRPPRC. Induced HIF-1α transcriptional activity or propofol treatment under normoxic conditions failed to affect LRPPRC, indicating that instead of directly regulating LRPPRC, propofol may exert synergistic effects with a downstream target gene of HIF-1α, which was not detected in this study.

LRPPRC was reported to physically bind to SLIRP and thus was stabilized. Loss of SLIRP impacts mitochondrial gene expression with a slight decrease in mitochondrial DNA mass [23] due to the decrease in LRPPRC resulting in a decrease in mitochondrial membrane potential and mitochondrial dysfunction. By performing immunoprecipitation, we observed that three of these proteins, LRPPRC, SLIRP and Bcl-2, were all observed in both LRPPRC and SLIRP immunoprecipitated products. This indicated that LRPPRC, SLIRP and Bcl-2 may form allotrimer, and potentially regulate stability mutually. These results indicated that LRPPRC, SLIRP and Bcl-2 may form heterotrimers and synergistically promote stabilization and regulate mitochondrial gene expression and mitochondrial function.

In summary, propofol promotes mitochondrial function, maintains mitochondrial homeostasis, inhibits ROS accumulation, and exerts a protective effect against hypoxia-induced oxidative stress, potentially by upregulating LRPPRC. These results provide new insight into the regulatory effects of propofol in mitochondria under oxidative stress.

Supporting information

S1 File

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Acknowledgments

The authors apologize to those whose work has not been cited due to space limitations.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported in part by research grants from the National Natural Science Foundation of China [81601103].

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Decision Letter 0

Partha Mukhopadhyay

4 Jun 2020

PONE-D-20-10919

Mitochondrial-associated protein LRPPRC is critical in propofol-induced protective effect against oxidative stress in cardiac cells

PLOS ONE

Dear Dr. Zhao,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewer #1: In the current study “Mitochondrial-associated protein LRPPRC is critical in propofol-induced protective effect against oxidative stress in cardiac cells” the authors discussed the involvement of LRPPRC in propoofl-induced cardiac cell protection in hypoxia. Here are some major and minor concerns:

Major:

1. In Figure 1A, why would 48h normoxia group drop in cell viability comparable to the hypoxia group? Does it mean hypoxia is not the reason of cell death?

2. In Figure 1C, pro-caspase-9 and cleaved caspase-9 image is not consistent with statistics.

3. In Figure 2A, without propofol, 24h hypoxia reduces LRPPRC protein expression compared with normoxia group. However, this trend does not show in Figure 2D and many other subsequent figures. The authors need to explain the discrepancy.

4. In Figure 6A, why would hypoxia mock treatment be so different from hypoxia siNC treatment? The increase of LC3-II in hypoxia condition compared to normoxia in Figure 5E is also absent in Figure 6A.

5. The authors found a direct binding between LRPPRC and Bcl-2, and the two protein expression is the same trend. However, it is not sufficient enough to claim that LRPPRC stabilizes Bcl-2. At least the authors need to detect the mRNA Bcl-2, and use translational inhibitors as well as proteasome inhibitors combined with the knockdown of LRPPRC to make a conclusion.

6. Figure 8C right, why would LRPPRC and SLIRP blots not detect the overexpression of these genes?

7. In Figure 8, results claim LRPPRC stabilizes SLIRP, instead of SLIRP stabilizes LRPPRC. This is the opposite of regulation mechanism to the published articles. The authors need to point it out and give a thorough discussion.

8. It is best the authors detect the endogenous SLIRP and LRPPRC binding, because the overexpression system sometimes introduce artificial results due to the expression abundance and overexpression techniques.

Minor:

9. There is no evidence to “Bcl-2 binds and stabilizes the SLIRP protein and thus enhances its function” in the discussion. The authors should be careful in conclusions.

10. Legend of Figure 4 is missing.

11. Instead of LRPPRC protects the mitochondria from autophagy, it is more likely that with LRPPRC, mitochondria are not damaged so that they do not have to be eliminated by autophagy (membrane potential and function is rescued). The authors need to revise the manuscript.

12. It is best the authors put the same genes into groups and distinguish with experimental conditions in the group graphs.

13. The authors never discussed oxidative stress by analyzing ROS or the ROS eliminating mechanisms. The model used is hypoxia and there may be a lot of mechanisms in cytotoxicity. Plus, oxidative stress is best manifested in the reperfusion phase which the authors did not study. So it is best the authors correct the title and related areas from oxidative stress by focusing on hypoxia.

Reviewer #2: The manuscript entitled “Mitochondrial-associated protein LRPPRC is critical in propofol-induced protective effect against oxidative stress in cardiac cells” suggested the protective effects of propofol on hypoxia induced cell injury on H9C2 cells and rat cardiac myocytes via LRPPRC-SLIRP-Bcl2 regulation. In detail, propofol treatment inhibited hypoxia induced apoptosis and autophagy in H9C2 cells. Authors further demonstrated that LRPPRC, SLIRP and Bcl-2 potentially form heterotrimers using immunoprecipitation. Based on accumulated results, the authors clearly described the mechanism of propofol on regulation of mitochondrial homeostasis under hypoxia conditions. However, there are several concerns must be addressed.

1) To support authors’ idea that anti-oxidative stress effects of propofol, oxidative stress markers such as 4HNE or MDA should be checked.

2) In Fig1A, cell viability of normoxia condition at 48h is significantly decreased similar to that of hypoxia condition. Please check the data and if that is correct please discuss it whether that result is related to unchanged expression of LRPPRC with hypoxia induction compared to normoxia group (Fig 2 B, C).

3) For the western blot analysis, please include quantification results. For Fig 1C and 2A, what do the apoptotic death rate graphs mean?

4) For Fig 2A & B, please include normoxia 48h group for control.

5) For Fig 3A, please include HIF-1a western results. The asterisk (*) on results of DFO and CoCl2 treatment groups seems be missing, considering the mention on p16.

6) For Fig 4C, please describe the reason of unchanged HIF-1a protein expression with echinomycin treatment. Please consider for checking HIF-1a expression from nuclear fraction.

7) For Fig 6, data from mock group is confusing. It should be similar to that of siNC.

8) For Fig 6, it should be checked whether maintaining mitochondrial homeostasis effects of propofol against hypoxic conditions was blocked with siLRPPRC transfection.

9) In general, there are some errors/mistakes in the figures. Please re-check them carefully and thoroughly. Some identified was shown below.

- For AnnexinV/PI assay results, indications seem to be incorrect.

Fig 1B & 3C: horizontal: PI/ vertical: Annexin V-> horizontal: Annenxin V/ vertical: PI

Fig4D: missing-> horizontal :PI , vertical : PI

- Fig 4C, please check the indication for propofol treatment +/-.

10) Materials and methods part contains less information.

- Please describe how hypoxia condition was induced on cell cultures.

- Please include methods for siLRPPRC transfection.

- Please add methods for caspase3/9 activity measurements and immunoprecipitation.

**********

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Reviewer #2: No

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PLoS One. 2020 Sep 8;15(9):e0238857. doi: 10.1371/journal.pone.0238857.r002

Author response to Decision Letter 0

28 Jun 2020

Reviewer #1: In the current study “Mitochondrial-associated protein LRPPRC is critical in propofol-induced protective effect against oxidative stress in cardiac cells” the authors discussed the involvement of LRPPRC in propoofl-induced cardiac cell protection in hypoxia. Here are some major and minor concerns:

Major:

1. In Figure 1A, why would 48h normoxia group drop in cell viability comparable to the hypoxia group? Does it mean hypoxia is not the reason of cell death?

Answer： Dear editor, thanks for the notification. In this section, Normoxia condition with supplemented CoCl2, which is a hypoxia mimic, was considered as a control of hypoxia. By comparing to normoixa+CoCl2 group, the decrease in cell viability by hypoxia exposure was considered to be induced by upregulation of HIF-1a. The figure has been modified.

2. In Figure 1C, pro-caspase-9 and cleaved caspase-9 image is not consistent with statistics.

Answer： Sorry for the mistake. In figure 1C, we incorrectly present the statistical data of image.

3. In Figure 2A, without propofol, 24h hypoxia reduces LRPPRC protein expression compared with normoxia group. However, this trend does not show in Figure 2D and many other subsequent figures. The authors need to explain the discrepancy.

Answer： Thanks for the precious suggestion. According to our results, in figure 2A and C, 24-hour exposure to hypoxia decreased LRPPRC. In figure 2D, 48-hour exposure to hypoxia decreased LRPPRC, but not at 24-hour time point. To figure out the discrepancy, we detected LRPPRC protein level at different time points, including 12, 16, 20, 24, 28, 32, 36, 40, 44, 48h in normoxia and hypoxia group, repeatedly. As shown in our supplemented results (Figure 1), LRPPRC was obviously decreased at 28, 32, 36, 40, and 48 h. However, at 24-hour time point, LRPPRC was not obviously decreased. This instability may explain at 24-h time point after hypoxia exposure, the observation of decrease in LRPPRC was not consistent. That’s why we mainly focused on the 48-hour time point.

Figure 1. After 12, 16, 20, 24, 28, 32, 36, 40, 44 and 48-hour hypoxia exposure,

4. In Figure 6A, why would hypoxia mock treatment be so different from hypoxia siNC treatment? The increase of LC3-II in hypoxia condition compared to normoxia in Figure 5E is also absent in Figure 6A.

Answer：Dear editor, we found that transfection procedure obviously upregulated LRPPRC. This may due to a technical character. If you think it is unacceptable, we’d like to change another technic to introduce siRNA.

5. The authors found a direct binding between LRPPRC and Bcl-2, and the two protein expression is the same trend. However, it is not sufficient enough to claim that LRPPRC stabilizes Bcl-2. At least the authors need to detect the mRNA Bcl-2, and use translational inhibitors as well as proteasome inhibitors combined with the knockdown of LRPPRC to make a conclusion.

Answer： The stabilizing activity of binding of LRRPRC to Bcl-2 was reported previously (1). We would like to claim that upregulated LRPPRC may function via stabilizes Bcl-2.

Zou J, Yue F, Jiang X, Li W, Yi J, et al. (2013) Mitochondrion-associated protein LRPPRC suppresses the initiation of basal levels of autophagy via enhancing Bcl-2 stability. Biochem J 454: 447-457.

6. Figure 8C right, why would LRPPRC and SLIRP blots not detect the overexpression of these genes?

Answer：Dear editor, in mammal cells, the overexpression of introduced coding sequence is usually not so obvious because of relative low introducing efficacy. The flag- or HA-tagged protein was usually obvious in IP product because of immune enrichment.

7. In Figure 8, results claim LRPPRC stabilizes SLIRP, instead of SLIRP stabilizes LRPPRC. This is the opposite of regulation mechanism to the published articles. The authors need to point it out and give a thorough discussion.

Answer： Thanks for the suggestion, we also agree with that it is not sufficient to clain LRPPRC stabilizes SLIRP. According to our results in figure 8, we presented the evidence showing that LRPPRC binds to SLIRP only. So we modified the manuscript.

8. It is best the authors detect the endogenous SLIRP and LRPPRC binding, because the overexpression system sometimes introduce artificial results due to the expression abundance and overexpression techniques.

Answer：We previously detected endogenous LRPPRC and SLIRP. However, we failed to intensively detect the binding between endogenous LRPPRC and SLIRP. The potential reasons are listed as followed: (1) antibody target to endogenous LRPPRC and SLIRP are not suitable for immunoprecipitation analysis because of low efficacy. (2) The low endogenous protein level of LRPPRC or SLIRP.

After several failure, we have to decide to introduce exogenous LRPPRC and SLIRP with Flag or HA tag.

Minor:

9. There is no evidence to “Bcl-2 binds and stabilizes the SLIRP protein and thus enhances its function” in the discussion. The authors should be careful in conclusions.

Answer： Thanks for the suggestion, it has been modified in 4 th paragraph of discussion section.

10. Legend of Figure 4 is missing.

Answer： Sorry for the mistake!

11. Instead of LRPPRC protects the mitochondria from autophagy, it is more likely that with LRPPRC, mitochondria are not damaged so that they do not have to be eliminated by autophagy (membrane potential and function is rescued). The authors need to revise the manuscript.

Answer： Thanks for the suggestion. We agree with this precious suggestion.

12. It is best the authors put the same genes into groups and distinguish with experimental conditions in the group graphs.

Answer： Do you mean the figure 2A, protein levels?

13. The authors never discussed oxidative stress by analyzing ROS or the ROS eliminating mechanisms. The model used is hypoxia and there may be a lot of mechanisms in cytotoxicity. Plus, oxidative stress is best manifested in the reperfusion phase which the authors did not study. So it is best the authors correct the title and related areas from oxidative stress by focusing on hypoxia.

Answer： Thanks for the suggestion. We modified the title and manuscript accordingly.

Reviewer #2: The manuscript entitled “Mitochondrial-associated protein LRPPRC is critical in propofol-induced protective effect against oxidative stress in cardiac cells” suggested the protective effects of propofol on hypoxia induced cell injury on H9C2 cells and rat cardiac myocytes via LRPPRC-SLIRP-Bcl2 regulation. In detail, propofol treatment inhibited hypoxia induced apoptosis and autophagy in H9C2 cells. Authors further demonstrated that LRPPRC, SLIRP and Bcl-2 potentially form heterotrimers using immunoprecipitation. Based on accumulated results, the authors clearly described the mechanism of propofol on regulation of mitochondrial homeostasis under hypoxia conditions. However, there are several concerns must be addressed.

1) To support authors’ idea that anti-oxidative stress effects of propofol, oxidative stress markers such as 4HNE or MDA should be checked.

Answer： Thanks for the precious suggestion. Instead of 4HNE or MDA, we measured the ROS accumulation, which is also considered as a major cause of oxidative stress. Moreover, we analyzed cell viability and apoptosis as a presentation of oxidative stress-related cell injury. 4HNE and MDA were often employed in patient serum who is suffering oxidative stress-related disease.

2) In Fig1A, cell viability of normoxia condition at 48h is significantly decreased similar to that of hypoxia condition. Please check the data and if that is correct please discuss it whether that result is related to unchanged expression of LRPPRC with hypoxia induction compared to normoxia group (Fig 2 B, C).

Answer： Dear editor, thanks for the notification. In this section, Normoxia condition with supplemented CoCl2, which is a hypoxia mimic, was considered as a control of hypoxia. By comparing to normoixa+CoCl2 group, the decerase in cell viability by hypoxia exposure was considered to be induced by upregulation of HIF-1a. The figure has been modified.

3) For the western blot analysis, please include quantification results. For Fig 1C and 2A, what do the apoptotic death rate graphs mean?

Answer： Sorry for the incorrect labeling. They have been modified.

4) For Fig 2A & B, please include normoxia 48h group for control.

Answer： The control group was originally included.

5) For Fig 3A, please include HIF-1a western results. The asterisk (*) on results of DFO and CoCl2 treatment groups seems be missing, considering the mention on p16.

Answer： Thanks for the remaindering. The missing part has been added.

6) For Fig 4C, please describe the reason of unchanged HIF-1a protein expression with echinomycin treatment. Please consider for checking HIF-1a expression from nuclear fraction.

Answer： Echinomycin was considered as a inhibitor of HIF-1a’s transcriptional activity. It was proved that echinomycin do not affect HIF-1a protein level.

7) For Fig 6, data from mock group is confusing. It should be similar to that of siNC.

Dear editor, we found that transfection procedure obviously upregulated LRPPRC. This may due to a technical character. If you think it is unacceptable, we’d like to change another technic to introduce siRNA.

8) For Fig 6, it should be checked whether maintaining mitochondrial homeostasis effects of propofol against hypoxic conditions was blocked with siLRPPRC transfection.

Answer： Introduction of siNC obviously upregulated LRPRC, which is unexpected. Knockdown of LRPPRC by siLRPPRC introduction obviously decreased mitochondrial mass.

9) In general, there are some errors/mistakes in the figures. Please re-check them carefully and thoroughly. Some identified was shown below.

- For AnnexinV/PI assay results, indications seem to be incorrect.

Fig 1B & 3C: horizontal: PI/ vertical: Annexin V-> horizontal: Annenxin V/ vertical: PI

Fig4D: missing-> horizontal :PI , vertical : PI

- Fig 4C, please check the indication for propofol treatment +/-.

Answer： Thanks for these suggestions. All figures have been re-checked.

10) Materials and methods part contains less information.

- Please describe how hypoxia condition was induced on cell cultures.

- Please include methods for siLRPPRC transfection.

- Please add methods for caspase3/9 activity measurements and immunoprecipitation.

Answer： Thanks for these suggestions. It has been re-checked.

Attachment

Submitted filename: respond to reviewers.docx

Click here for additional data file. (92KB, docx)

Decision Letter 1

Partha Mukhopadhyay

28 Jul 2020

PONE-D-20-10919R1

Propofol induces Mitochondrial-associated protein LRPPRC and protectives mitochondria against oxidative stress in cardiac cells

PLOS ONE

Dear Dr. Zhao,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Your manuscript was reviewed by same reviewers and one reviewer raised some suggestions to improve the quality of the manuscript.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In the present version of “Propofol induces Mitochondrial-associated protein LRPPRC and protectives mitochondria against oxidative stress in cardiac cells”, the authors made a lot of explanation, correction and improvement. Here are some more minor concerns to be addressed:

1. If Figure 1A is the only experiment to supplement CoCl2 to mimic hypoxia but called control or normoxia, it is best not to show the CoCl2 treatment results. Instead, a non-treated group should be shown to make the whole study consistent. CoCl2 does not provide any more information, but confusion to the study.

2. If LRPPRC expression is modified by siRNA transfection, the authors should mention it in the result or discussion parts to reduce confusion.

3. Although the authors changed the title, oxidative stress is still in it. If the authors insist on claiming oxidative stress, ROS detection should be added and the reperfusion phase should be studied. The source of ROS and ROS elimination machinery should also be analyzed. The easier way is to correct oxidative into hypoxia, which is what the authors studied in the present research project. Also, is it "protectives" or "protects"?

4. For Figure 1C, Figure 2A, the authors should put genes in groups and assign different colors to different experimental condition, the same way of Figure 6D.

5. The authors need to double check the Annexin V/PI staining original figures. It seems in Figure 1 and 3, all the axes for Annexin V is labeled PI and vice versa.

Reviewer #2: (No Response)

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Sep 8;15(9):e0238857. doi: 10.1371/journal.pone.0238857.r004

Author response to Decision Letter 1

31 Jul 2020

Reviewer #1: In the present version of “Propofol induces Mitochondrial-associated protein LRPPRC and protectives mitochondria against oxidative stress in cardiac cells”, the authors made a lot of explanation, correction and improvement. Here are some more minor concerns to be addressed:

1. If Figure 1A is the only experiment to supplement CoCl2 to mimic hypoxia but called control or normoxia, it is best not to show the CoCl2 treatment results. Instead, a non-treated group should be shown to make the whole study consistent. CoCl2 does not provide any more information, but confusion to the study.

Answer: Thanks for the suggestion. Originally, we already performed normoxia group without cocl2. We’d like to replace normoxia+cocl2 group with normoxia group.

2. If LRPPRC expression is modified by siRNA transfection, the authors should mention it in the result or discussion parts to reduce confusion.

Answer: It has been mentioned in discussion parts.

3. Although the authors changed the title, oxidative stress is still in it. If the authors insist on claiming oxidative stress, ROS detection should be added and the reperfusion phase should be studied. The source of ROS and ROS elimination machinery should also be analyzed. The easier way is to correct oxidative into hypoxia, which is what the authors studied in the present research project. Also, is it "protectives" or "protects"?

Answer: Thanks for the suggestion. We further modified the title.

4. For Figure 1C, Figure 2A, the authors should put genes in groups and assign different colors to different experimental condition, the same way of Figure 6D.

Answer: Thanks for the suggestion. We further modified the figures.

5. The authors need to double check the Annexin V/PI staining original figures. It seems in Figure 1 and 3, all the axes for Annexin V is labeled PI and vice versa.

Answer: Thanks for the suggestion. We further modified the figures.

Attachment

Submitted filename: response to reviewers.docx

Click here for additional data file. (14.9KB, docx)

Decision Letter 2

Partha Mukhopadhyay

26 Aug 2020

Propofol induces Mitochondrial-associated protein LRPPRC and protects mitochondria against hypoxia in cardiac cells

PONE-D-20-10919R2

Dear Dr. Zhao,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Partha Mukhopadhyay, Ph.D.

Section Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Acceptance letter

Partha Mukhopadhyay

28 Aug 2020

PONE-D-20-10919R2

Propofol induces Mitochondrial-associated protein LRPPRC and protects mitochondria against hypoxia in cardiac cells

Dear Dr. Zhao:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

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PLOS ONE Editorial Office Staff

on behalf of

Dr. Partha Mukhopadhyay

Section Editor

PLOS ONE

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    Click here for additional data file. (92KB, docx)
    Attachment

    Submitted filename: response to reviewers.docx

    Click here for additional data file. (14.9KB, docx)

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