Inhibiting microRNA-144 abates oxidative stress and reduces apoptosis in hearts of streptozotocin-induced diabetic mice Manli Yu, Yu Liu, Bili Zhang, Yicheng Shi, Ling Cui, Xianxian Zhao PII: DOI: Reference:

S1054-8807(15)00077-0 doi: 10.1016/j.carpath.2015.06.003 CVP 6849

To appear in:

Cardiovascular Pathology

Received date: Revised date: Accepted date:

1 April 2015 16 June 2015 17 June 2015

Please cite this article as: Yu Manli, Liu Yu, Zhang Bili, Shi Yicheng, Cui Ling, Zhao Xianxian, Inhibiting microRNA-144 abates oxidative stress and reduces apoptosis in hearts of streptozotocin-induced diabetic mice, Cardiovascular Pathology (2015), doi: 10.1016/j.carpath.2015.06.003

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ACCEPTED MANUSCRIPT Inhibiting microRNA-144 abates oxidative stress and reduces apoptosis in hearts of

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streptozotocin-induced diabetic mice

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Manli Yu*, Yu Liu*, Bili Zhang, Yicheng Shi, Ling Cui, Xianxian Zhao

University, Shanghai, China

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Department of Cardiovasology, Changhai Hospital, Second Military Medical

*The first two authors contributed equally to the work.

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Correspondence to Xianxian Zhao, Department of Cardiovasology, Changhai Hospital,

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Second Military Medical University, Changhai Road 168, Shanghai, 200433, China Tel: +8602131161245; Fax: +8602131161263;

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E-mail: [email protected]

Summary:

Inhibiting microRNA-144 abated oxidative stress, reduced apoptosis, and improved cardiac function in STZ-induced diabetic mice, possibly via enhancing Nrf2 expression.

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ACCEPTED MANUSCRIPT ABSTRACT: Introduction: Hyperglycemia-induced reactive oxygen species (ROS) generation

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contributes to the development of diabetic cardiomyopathy. However, little is known about the role of microRNAs (miRNAs) in the regulation of ROS formation and

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myocardial apoptosis in streptozotocin (STZ)-induced diabetic mice. Methods and Results: It was observed that microRNA-144 (miR-144) level was

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lower in heart tissues of STZ-induced diabetic mice. High glucose exposure also reduced miR-144 levels in cultured cardiomyocytes. Moreover, miR-144 modulated high glucose-induced oxidative stress in cultured cardiomyocytes by directly targeting

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nuclear factor-erythroid 2-related factor 2 (Nrf2), which was a central regulator of

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cellular response to oxidative stress. The miR-144 mimics aggravated high glucose-induced ROS formation and apoptosis in cardiomyocytes, which could be

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attenuated by treatment with Dh404, an activator of Nrf2. Meanwhile, inhibition of

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miR-144 suppressed ROS formation and apoptosis induced by high-glucose in cultured cardiomyocytes. What was more important, reduced myocardial oxidative stress and apoptosis and improved cardiac function were identified in STZ-induced diabetic mice when treated with miR-144 antagomir. Conclusion: Although miR-144 cannot explain the increased oxidative stress in STZ, therapeutic interventions directed at decreasing miR-144 may help to decrease oxidative stress in these hearts. Inhibition of miR-144 might have clinical potential to abate oxidative stress as well as to reduce cardiomyocyte apoptosis, and improve cardiac function in diabetic cardiomyopathy.

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Keywords: streptozotocin, diabetic cardiomyopathy, microRNA-144, nuclear

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factor-erythroid 2-related factor 2, oxidative stress

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ACCEPTED MANUSCRIPT INTRODUCTION Diabetes mellitus has reached an epidemic level worldwide, with a prevalence of 4%

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in 1995 and an anticipated prevalence of 5.4% in 2025, corresponding to 365 million people suffered from diabetes in 2011 and this number is expected to rise up to 552

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million by 2030 [1]. Diabetic cardiomyopathy is responsible for higher incidence of

the diabetic population [2].

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sudden cardiac death and represents the leading cause of morbidity and mortality in

Apoptotic cell death is increased in the diabetic heart of patients and animal models [3, 4]. In diabetic cardiomyopathy, apoptosis as a comprehensive consequence of cardiac

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responses to various stresses causes a loss of contractile tissue, which initiates a

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cardiac remodeling and fibrosis; therefore, the cardiac apoptosis has been identified as a pivotal cause of various cardiomyopathies [5, 6]. Among apoptotic stimuli, cardiac

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ROS formation was closely related to apoptosis [7]. Oxidative stress occurred in

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diabetic hearts from both type 1 and type 2 diabetes [8, 9]. Treatment with antioxidants, such as vitamin E, N-acetyl-L-cysteine, and metallothionein, protected cardiomyocytes from apoptosis in high glucose conditions, and prevented diabetic cardiomyopathy and enhanced survival of diabetic animals [10-12]. MicroRNAs (miRNAs, miRs) are an evolutionarily conserved class of small noncoding RNAs of 22-24 nucleotides in length, that act as post-transcriptional regulators of gene expression by binding to the 3’-untranslated region (3’-UTR), finally induce mRNA degradation and/or translational repression in diverse biological processes [13]. The search for regulatory nucleotide sequences that have specific gene

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ACCEPTED MANUSCRIPT targets have put miRNAs at the forefront of development of therapeutics, and may serve as valuable diagnostic and/or therapeutic targets. Therefore, it is of crucial

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importance to gain insight into the role of miRNAs in diabetic cardiomyopathy which will help clarify the molecular mechanisms as well as lead to the development of

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novel and effective therapeutic interventions for diabetic cardiomyopathy. The miR-144 was found down-regulated in left ventricles of streptozotocin

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(STZ)-induced type I diabetic mice by RT-PCR analysis in this work. Using a combination of gain- and loss-of-function studies, we investigated the role of miR-144 on regulation of oxidative stress and apoptosis in cardiomyocytes exposed to

METHODS

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Ethical statement

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high glucose in vitro and diabetic cardiomyopathy induced by STZ in vivo.

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All the animals used in this work received humane care in compliance with institutional animal care guidelines, and were approved by the Local Institutional Committee. All protocols were conducted in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China.

Study design Experiment 1: Diabetes was induced in adult male mice by consecutive peritoneal injection of streptozotocin (two group, control and STZ group, n=9 in each group). 8

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ACCEPTED MANUSCRIPT weeks later, heart tissues were collected for determination of miR-144 expression. Mouse cardiomyocyte cells were incubated in either normal glucose (5 mmol/l) or

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high glucose (33 mmol/l). 48 hours later, cells were collected for determination of miR-144 expression.

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Experiment 2: Mouse cardiomyocyte cells were transfected with 40 nM miRNA mimic (miR-144) or with 60 nM miRNA inhibitor (anti-miR-144) (Dharmacon)

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utilizing RNAimax (Invitrogen). 48 hours later, the cells were then incubated in either normal glucose (5 mmol/l) or high glucose (33 mmol/l). 48 hours later, the Nrf2 protein expression was determined by Western blot analysis.

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Experiment 3: Mouse cardiomyocyte cells were transfected with 40 nM miRNA

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mimic (miR-144) utilizing RNAimax (Invitrogen). 48 hours later, the cells were then incubated in either normal glucose (5 mmol/l) or high glucose (33 mmol/l), and

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treated with Dh404 (an Nrf2 activator, 0.2μM). 48 hours later, intracellular ROS and

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apoptosis were assayed by DCFH-DA and TUNEL, respectively. Experiment 4: Mouse cardiomyocyte cells were transfected with 60 nM miRNA inhibitor (anti-miR-144). 48 hours later, the cells were then incubated in either normal glucose (5 mmol/l) or high glucose (33 mmol/l). 48 hours later, intracellular ROS and apoptosis were assayed by DCFH-DA and TUNEL, respectively. Experiment 5: Diabetes was induced in adult male mice by consecutive peritoneal injection of streptozotocin. 7 days later, mice were randomized into four groups (n=30-34 in each group). Control and miR-144 antagomir (anti-miR-144) and injected via the tail vein with either a scrambled anti-miR-144 or anti-miR-144 at a dose of 20

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ACCEPTED MANUSCRIPT mg/kg in 0.2 ml saline twice a week. 7 weeks later, cardiac function was measured

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and left ventricles were removed for determination of oxidative stress and apoptosis.

Animal model

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C57BL/6 mice (male, 2 months old) were purchased from the Sino-British SIPPR/BK Lab Animal Ltd (Shanghai, China) and housed two per cage under controlled

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temperature (23-25°C), humidity (50%) and lighting (12-hour light/dark cycle) with food and water provided ad libitum.

Mice were given streptozotocin (STZ) (150 mg/kg i.p.; Sigma-Aldrich, St. Louis, MO,

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USA) to induce diabetes, whereas control mice were injected with the same amount of

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citrate buffer. 3 days after the injection of STZ, blood was obtained from the tail-vein and glucose levels were measured using the OneTouch Ultra 2 blood glucose

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monitoring system (LifeScan, Milpitas, CA, USA). Mice were considered diabetic

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and used for the study only if they had hyperglycemia (≥16.7 mM). The control and diabetic mice both raised on standard food and water for the whole experimental period.

Cardiomyocyte isolation and culture Cardiomyocytes were cultured as described previously [14]. Briefly, hearts from adult C57BL/6 mice were excised under deep ether anesthesia and mounted on the cannula of a Langendorff perfusion system. Cardiomyocytes were isolated via perfusion with collagenase, followed by mincing, filtering and transfer to culture medium M199

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ACCEPTED MANUSCRIPT supplemented with carnitine (2 mM), creatine (5 mM) and taurine (5 mM). Normal glucose: 5 mmol/l, the osmolality were equal by adding 28 mmol/l of mannitol; high

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glucose: 33 mmol/l.

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Real-time RT-PCR quantification of microRNA expression

Total RNA was isolated from heart tissues or cultured cells using the miRNeasy Mini

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kit (Qiagen), according to the manufacturer’s protocol. The concentration of RNA was determined by a NanoDrop ND-1000 Spectrophotometer (NanoDrop Tech., Rockland, DE). To detect and quantify mature miR-144 expression, we used TaqMan miRNA

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assays (Applied Biosystems) according to manufacturer’s directions and as previously

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described [15]. Briefly, 500ng total RNA was used in each reaction and mixed with the RT primer (total 15 μl). The reverse transcription reaction was carried out and 1.33

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ul of cDNA was used for PCR reaction along with TaqMan primers (20ul total).

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Real-time PCR reactions, including no-template controls, were conducted in triplicate using the ABI 7500 real-time PCR system. Results were analyzed and expressed as CT (threshold cycle) values, and relative expression was calculated using the comparative CT method [16]. Unless otherwise specified, we compared relative levels of microRNA with probes specific for the indicated mature microRNA using total RNA and normalized by evaluating U6 expression.

Luciferase reporter assay The 3’untranslated region (UTR) of the Nrf2 was amplified using primers (forward:

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ACCEPTED MANUSCRIPT 5’-atttaggaggatttgacc-3’; reverse: 5’-tttttgccagagctaaacaattt-3’) and cloned into the XbaI site downstream of the firefly luciferase gene in the pGL3-control vector

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(Promega). The Nrf2 3’UTR mutant reporters were constructed with QuikChange II Site-Directed Mutagenesis (Stratagene). Expression constructs encoding miR-144

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were created by insertion into a cytomegalovirus-based pcDNA3 cloning vector (Invitrogen).

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For Nrf2-3′UTR assay, cardiomyocytes were cotransfected in 12-well plates using the DharmaFECT Duo Transfection Reagent according to the protocol of the manufacturer, with 0.4 μg of the Nrf2-3′UTR luciferase reporter vector and 0.08 μg of

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the control vector pMIR-REPORT (Ambion, Texas, USA). For each well, 100nM

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miR-144 or scrambled miR control was used. Cell lysates were prepared 72 hous later, and luciferase activity was measured, and expressed as relative light units using a

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luciferase assay kit (Promega, Madison, WI, USA). β-galactosidase activity was

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measured with a commercially available kit (Promega). 3′UTR activity of each construct was expressed as the ratio of luciferase/β-galactosidase activity. All transfections were performed in triplicate from three independent experiments.

ROS assay in vitro Cells were seeded in a 6-well plate. After treatment for 48 hours, cells were gently washed twice with PBS. Intracellular ROS were quantified by employing ROS-sensitive dye, DCFH-DA (10μM, Beyotime, Shanghai, China). Fluorescent images were acquired from a laser confocal microscope (Zeiss LSM 510 META), and

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ACCEPTED MANUSCRIPT the intensity on regions of interest was measured.

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Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Assay in vitro

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The in situ TUNEL cell death detection kit was used to detect apoptotic cells according to manufacturer’s instructions (Beyotime, Shanghai, China). Briefly, cells

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were fixed with 4% paraformaldehyde and permeabilized by 0.3% Triton X-100, and then washed twice with PBS. DNA breaks were labeled by incubation (1 hour, 37°C) with terminal deoxynucleotidyltransferase and nucleotide mixture containing

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fluorescein isothiocyanate-conjugated dUTP. Cells were nuclear stained with

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4’,6-diamidino-2-phenylindole fluorescent dye (DAPI), and the TUNEL positive and

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Japan).

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total nuclei were observed under a laser scanning confocal microscope (Nikon,

Western blotting

The cells were gently washed twice with iced PBS and harvested at 4℃ in lysis buffer containing 50 mM Tris (pH 7.5), 300 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1 mM PMSF and 2 μM leupeptin. The left ventricles were homogenized in ice cold buffer (0.15 M NaCl, 5 mM EDTA, 10 mM Tris-Cl, 1% Triton X-100 and protease inhibitors cocktail). The homogenates were centrifuged (12,000× g for 15 min at 4°C) and the supernatants were collected. The protein concentration was determined with bovine serum albumin as a standard

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ACCEPTED MANUSCRIPT by a Bradford assay. Equal amount of protein preparations were run on SDS-polyacrylamide gels, electrotransferred to polyvinylidine difluoride membranes,

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and blotted with a primary antibody against Nrf2 (Abcam, San Francisco, CA, USA), cleaved caspase-3 (Abcam, San Francisco, CA, USA), and Pro-caspase-3 (Abcam,

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San Francisco, CA, USA) to detect their protein levels, with HRP-conjugated monoclonal antibody against GAPDH (Sigma) serving as a control. Immunoreactive

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bands were detected by a chemiluminescent reaction (ECL kit, Amersham Pharmacia, USA).

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Measurement of cardiac total ROS formation

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Total ROS production was detected as the method described by Elks [17]. Briefly, the electron paramagnetic resonance (EPR) measurement was performed with a

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BenchTop EPR spectrophotometer e-scan R (Noxygen Science Transfer and

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Diagnostics, Elzach, Germany). The spin probe, 1-Hydroxy-3-methoxycarbonyl2,2,5,5-tetramethyl-pyrrolidine (CMH), was used for EPR studies. Small portions (15-20 mg each) of left ventricles from each mice were minced and placed into four wells of a 24-well tissue culture plate containing 20μM Krebs-HEPES buffer with defferoxamine and diethyldithiocarbamate (metal chelators). Tissue pieces were then washed twice with the same buffer to remove any trace contamination. Samples were incubated at 37℃ with 6.6 μl of CMH (200 mM, Enzo Life Sciences, San Diego, USA) for 30 min for ROS measurement.

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ACCEPTED MANUSCRIPT Measurement of malondialdehyde (MDA) MDA concentration is a presumptive marker of oxidant-mediated lipid peroxidation.

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Ventricular homogenates were used for the determination of MDA using a kit

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(Cayman, Ann Arbor, USA).

TUNEL assay in vivo

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Left ventricles were fixed in 4% formaldehyde and paraffin embedded, and 5-μm sections were prepared. TUNEL analysis was performed with a commercially available kit (Dead End Colorimetric TUNEL System) according to the

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manufacturer's instructions (ROCHE, Mannheim, DE, USA). The slides were

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counterstained with hematoxylin. Three midventricular sections (from the apex to the base) of each heart tissue were analyzed. Cardiomyocyte nuclei were quantified by

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randomly counting 10 fields/section. The apoptotic index (percentage of apoptotic

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nuclei) was calculated as apoptotic nuclei/total nuclei counted ×100. The evaluation was conducted by an investigator blinded to the study groups.

Cardiac function measurement Transthoracic echocardiography was performed noninvasively with a Vevo 770 high-resolution imaging system equipped with a 30-MHz transducer (RMV-707B; VisualSonics, Toronto, Canada). Mice were lightly anesthetized (0.3 mL of a cocktail containing 100 mg/ml ketamine and 10 mg/ml acepromazine given i.p.) for the duration of the recordings. The heart rate was monitored simultaneously by

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ACCEPTED MANUSCRIPT electrocardiography (ECG). Left ventricular (LV) end diastolic diameter (LVEDD) and end systolic diameter (LVESD) were used to calculate fractional shortening by

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the following formula: Fractional shortening (%) = [(LVEDD – LVESD)/LVEDD] ×100%. LV end diastolic volume (LVEDV) and end systolic volume (LVESV) were

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calculated as described previously [18]. Ejection fraction was calculated by the following formula: Ejection fraction (%) = [(LVEDV – LVESV)/LVEDV] ×100%. All

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echocardiographically derived measures were obtained by averaging the readings of three consecutive beats.

Hearts were isolated and perfused on a Langendorff-system. Maximal and minimal

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first derivatives of force (+dF/dtmax and –dF/dtmin) as the rate of contraction and

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relaxation were analyzed by PowerLab Chart program (ADInstruments) as described

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in previous study [19, 20].

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Statistical analysis

All the data are presented as mean ± standard deviations. Comparison between two groups was analyzed using paired Student’s t-test. Comparison among groups was analyzed using a two-way analysis of variance followed by Bonferroni t-test. P < 0.05 was considered statistically significant. Statistical analysis was performed using SPSS 11.0.0 software (SPSS Inc., Chicago, IL, USA).

RESULTS Expression of miR-144 in hearts of STZ-induced diabetic mice

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ACCEPTED MANUSCRIPT Quantitative RT-PCR results demonstrated significantly lower levels of miR-144 in

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hearts of STZ-induced diabetic mice (Fig. 1), when compared to control mice.

Nrf2 is a direct target of miR-144

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To understand the potential functional connection between miR-144 and oxidative stress in diabetic cardiomyopathy, we analyzed its predicted target gene. Recent

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studies have documented that Nrf2 was repressed by miR-144 in K562 cells [21] and cerebromicrovascular endothelial cells [22]. To validate whether miR-144 directly recognized the 3’-UTR of Nrf2, we generated serial constructs harboring the 3’UTR

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segment of Nrf2 and its mutant fused downstream to the luciferase coding sequence.

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Co-transfection with miR-144 strongly inhibited the luciferase activity; whereas no effect was observed in cells co-transfected with a construct containing a mutated

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segment of Nrf2 3’UTR (Fig. 2A).

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High glucose reduced miR-144 levels in cultured cardiomyocytes (Fig. 2B). High glucose down-regulated the expression of Nrf2 in cultured cardiomyocytes. More strikingly, protein levels of Nrf2 was further reduced when transfected with miR-144 mimic (Fig. 2C) and enhanced when transfected with miR-144 inhibitor (Fig. 2D). When exposed to normal glucose, transfection with miR-144 mimic or inhibitor had no significant effect on Nrf2 protein expression.

High glucose-induced oxidative stress and apoptosis Transfection with miR-144 mimic aggravated high glucose-induced ROS formation

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ACCEPTED MANUSCRIPT (Fig. 3A) and apoptosis (Fig. 3B, C) in cultured cardiomyocytes. Treatment with Nrf2 activator-Dh404 fully reversed the effect of miR-144 mimic. When exposed to normal

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glucose, neither miR-144 mimic nor Dh404 affected ROS formation or apoptosis. Transfection with miR-144 inhibitor attenuated ROS formation (Fig. 3D) and

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apoptosis (Fig. 3E) induced by high glucose in cultured cardiomyocytes.

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Oxidative stress

In STZ-induced diabetic mice, Nrf2 protein expression (Fig. 4A) was down-regulated, ROS formation (Fig. 4B) and MDA production (Fig. 4C) was enhanced in left

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ventricles.

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According to the results obtained from in vitro studies, miR-144 antagomir was employed for treatment of STZ-induced cardiomyopathy. Treatment with miR-144

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antagomir up-regulated Nrf2 protein expression, suppressed ROS formation, and

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reduced MDA content.

Apoptosis

In STZ-induced diabetic mice, caspase-3 (Fig. 5A) was activated in left ventricles when compared to control mice. TUNEL results (Fig. 5B) further validated that apoptosis was enhanced in left ventricles of STZ-induced diabetic mice than that in control mice. Treatment with miR-144 antagomir reduced caspase-3 activation and TUNEL positive cells in left ventricles of STZ-induced diabetic mice.

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ACCEPTED MANUSCRIPT Cardiac function As shown in Table 1, treatment with miR-144 antagomir had no significant effect on

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body weight or levels of fasting blood glucose. When compared to control mice, STZ-diabetic mice showed a significant reduction of +dF/dtmax, -dF/dtmin, fractional

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shortening, and ejection fraction. Importantly, myocardial function of diabetic mice

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was significantly improved when treated with miR-144 antagomir.

DISCUSSION

Diabetic hyperglycemia promotes the production of ROS to lead to oxidative stress

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and apoptosis responsible for progressive deterioration of the structure and function of

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organs [23, 24]. These findings indicated that factors and pathways regulating ROS production and the cellular redox state play a key role in the progression of diabetes

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and diabetes complications including cardiomyopathy [25]. Cells possess an array of

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antioxidant defense machinery to prevent or counterbalance damage caused by reactive radicals. One central regulator of antioxidant response is Nrf2, a basic leucine zipper transcription factor. Under oxidative stress, Nrf2 binds to the antioxidant response element (ARE) [26, 27], which is important for the coordinately inducible expression of antioxidant enzymes such as superoxide dismutase, catalase, GPX1, phase II detoxification enzymes such as NAD(P)H: quinone oxidoreductase (NQO1), and glutathione synthesis [28]. Nrf2 is critical in defense against oxidative damage induced by high glucose in cardiomyocytes [29]. STZ-induced diabetes developed more rapidly and severely in Nrf2 KO than WT mice and diabetes induced oxidative

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ACCEPTED MANUSCRIPT stress and apoptosis in hearts were aggravated in Nrf2 KO mice [30], which indicated the key role of Nrf2 in STZ-induced diabetic cardiomyopathy. It was found that Nrf2

observed in hearts of STZ-induced mice in this work.

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expression was down-regulated in human diabetic hearts [31], which was also

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Recent studies have documented that Nrf2 was repressed by miR-144 in K562 cells [21] and cerebromicrovascular endothelial cells [22]. In this work, it was validated

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that Nrf2 was a direct target of miR-144 in adult cardiomyocytes, and miR-144 inhibitor enhanced Nrf2 expression in cells exposed to high glucose in vitro and diabetic hearts in vivo. In addition, miR-144 mimic aggravated high glucose-induced

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oxidative stress and apoptosis, and the effect was reversed by treatment with Nrf2

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activator, indicating the key role of Nrf2 in the effect of miR-144. Importantly, treatment with miR-144 inhibitor abated oxidative stress, attenuated

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apoptosis, and improved cardiac function, which might lead to the development of

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novel and effective therapeutic interventions for type I diabetic cardiomyopathy. In diabetic mice, treatment with miR-144 inhibitor enhanced expression of Nrf2 in hearts, which might contribute to the beneficial effect of miR-144 inhibitor. However, Nrf2 was not the unique target of miR-144. It was reported that miR-144 impaired insulin signaling by inhibiting the expression of insulin receptor substrate 1 in type 2 diabetes mellitus [32]. An agomir of miR-144 accelerated plaque formation through impairing reverse cholesterol transport and promoting pro-inflammatory cytokine production by inhibiting expression of ATP binding cassette transporter A1 (ABCA1) [33]. Silencing miR-144 in mice increased hepatic ABCA1 protein, which in turn enhanced plasma

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ACCEPTED MANUSCRIPT HDL levels [34]. Thus, both of insulin receptor substrate 1 and ABCA1 could be the candidate for further investigation of effect of miR-144 in diabetic cardiomyopathy.

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In this work, although miR-144 modulated post-transcription of Nrf2, when exposed to normal glucose, transfection with miR-144 mimic or inhibitor had no significant

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effect on Nrf2 protein expression. The protein levels of Nrf2 were modulated exquisitely in cells [35, 36]. Under normal condition, the effect of miR-144 on Nrf2

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expression in cardiomyocytes might be neutralized through unknown mechanism. Although miR-144 cannot explain the occurred oxidative stress in STZ-induced diabetic hearts (because miR-144 is actually decreased in the diabetic heart),

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therapeutic interventions directed at decreasing miR-144 may help to decrease

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oxidative stress and apoptosis in the hearts, and pharmacological targeting miR-144 may represent a promising strategy in the management of type I diabetic

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cardiomyopathy.

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In conclusion, inhibiting microRNA-144 abated oxidative stress, reduced apoptosis, and improved cardiac function in STZ-induced diabetic mice, possibly via enhancing Nrf2 expression.

Conflict of Interest statement The authors declare that they have no conflict of interest.

Author contributions 1. Conception and design of the experiments: Manli Yu, Yu Liu, Xianxian Zhao

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ACCEPTED MANUSCRIPT 2. Collection, analysis and interpretation of data: Manli Yu, Yu Liu, Bili Zhang,

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3. Drafting the article: Manli Yu, Yu Liu, Xianxian Zhao

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Yicheng Shi, Ling Cui

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ACCEPTED MANUSCRIPT Table 1. Effect of treatment with miR144 antagomir (anti-miR144) on STZ-induced

Con+anti-miR144 STZ

Body weight (g)

32.6±4.8

31.7±5.2

Fasting blood glucose (mM)

5.3±1.2

5.1±1.0

+dF/dt max (g/s)

72.8±9.1

75.5±8.6

-dF/dt min (g/s)

55.2±6.5

54.1±6.2

FS (%)

57.9±5.2

EF (%)

83.5±5.8

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cardiac dysfunction in mice. STZ+anti-miR144 22.7±4.1 *

19.1±4.2 *

19.4±3.1 *

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21.6±3.6 *

64.7±8.0 #

26.0±4.8 *

51.9±5.7 #

59.5±6.6

40.6±3.5 *

52.8±4.5 #

84.3±6.0

67.9±5.2 *

80.2±4.9 #

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44.1±7.2 *

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shortening; EF, Ejection fraction; * P < 0.05 versus control group, # P < 0.05 versus

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STZ group.

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ACCEPTED MANUSCRIPT Figure legend Figure 1. Levels of miR-144 in heart tissues of STZ-induced diabetic mice. Mice

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were injected with STZ to induce diabetes. 8 weeks later, hearts were removed for determination of miR-144 levels by RT-PCR method. Values are means ± SD. n=9 in

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each group; STZ, streptozotocin; * P < 0.05 versus control group.

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Figure 2. miR-144 directly targeted Nrf2 gene. Dual luciferase activity assay of cardiomyocytes cotransfected with the plasmid containing the segment of Nrf2 3’UTR for miR-144 or a control oligoribonucleotide showed that miR-144 inhibited

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luciferase activity, compared with controls. Mutation of the miR-144 binding site

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abolished this effect (A). Levels of miR-144 (B) in cardiomyocytes exposed to high glucose. Western blot analysis of Nrf2 protein levels after transfection with miR-144

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mimic (C) or inhibitor (D) in cardiomyocytes exposed to high glucose. Nrf2, nuclear

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factor-erythroid 2-related factor 2; NG, normal glucose; HG, high glucose; * P < 0.05 versus NG group, # P < 0.05 versus HG group.

Figure 3. Effect of miR-144 on high glucose-induced ROS formation and apoptosis in cardiomyocytes. Transfection with miR-144 mimic increased ROS formation (A) and apoptosis (B, C) induced by HG exposure in cultured cells, which was reversed by treatment with Dh404 (0.2μM). Transfection with miR-144 inhibitor attenuated ROS formation (D) and apoptosis (E) induced by HG exposure in cultured cells. TUNEL assay for cardiomyocyte apoptosis. Sections were stained for DAPI to identify

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ACCEPTED MANUSCRIPT cardiomyocyte nuclei (blue signal, left panel) and for TUNEL activity to identify apoptotic cells (FITC-conjugated secondary antibody, green signal, middle panel).

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Apoptotic cardiomyocytes are identified in the merged image. ROS, reactive oxygen species; DAPI, 4’,6-diamidino-2-phenylindole fluorescent dye; TUNEL, terminal

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deoxynucleotidyl transferase dUTP nick end labeling; NG, normal glucose; HG, high glucose; * P < 0.05 versus NG group, # P < 0.05 versus HG group, ^ P < 0.05 versus

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HG group transfected with miR-144 mimic.

Figure 4. Effect of treatment with miR-144 antagomir on cardiac oxidative stress

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induced by STZ in mice. Mice were injected with STZ to induce diabetes, and treated

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with miR-144 antagomir. Left ventricles were removed for determination of Nrf2 protein expression (A), ROS formation (B), and MDA levels (C). Values are means ±

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SD. n=9-11 in each group; STZ, streptozotocin; Nrf2, nuclear factor-erythroid

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2-related factor 2; ROS, reactive oxygen species; MDA, malondialdehyde; * P < 0.05 versus control group, # P < 0.05 versus STZ group. Figure 5. Effect of treatment with miR-144 antagomir on apoptosis induced by STZ in mice. Mice were injected with STZ to induce diabetes, and treated with miR-144 antagomir. Left ventricles were removed for determination of caspase-3 activation and apoptosis, by Western blot and TUNEL, respectively. Caspase-3 activation was calculated by the ratio of cleaved caspase-3 to Pro-caspase-3. TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; STZ, streptozotocin; * P < 0.05 versus control group, # P < 0.05 versus STZ group.

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Funding: This study was supported by the National Natural Science Foundation of China (81170223, 81400287) and Natural Science Foundation of Shanghai (81170223). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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