page 1 of 63 diabetes...jul 12, 2016 · 2 abstract micrornas have a fundamental role in diabetic...

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Lack of miR-133a decreases contractility in diabetic hearts: a role for novel cross-talk

between tyrosine aminotransferase and tyrosine hydroxylase

Shyam Sundar Nandi1, Hong Zheng

1, Neeru M. Sharma

1, Hamid R. Shahshahan

1, Kaushik P.

Patel1

and Paras K. Mishra1, 2

Affiliations:

1. Department of Cellular and Integrative Physiology, University of Nebraska Medical

Center, 985850 Nebraska Medical Center, Omaha, NE 68198, USA

2. Department of Anesthesiology, University of Nebraska Medical Center, 985850

Nebraska Medical Center, Omaha, NE 68198, USA.

Running title: MiR-133a regulates cardiac contractility

Corresponding author:

Paras Kumar Mishra

Department of Cellular and Integrative Physiology

University of Nebraska Medical Center

985850 Nebraska Medical Center

Omaha, NE-68198, USA

Phone: 402-559-8524

Fax: 402-559-4438

Email: [email protected]

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Diabetes Publish Ahead of Print, published online July 13, 2016

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Abstract

MicroRNAs have a fundamental role in diabetic heart failure. The cardioprotective microRNA-

133a (miR-133a) is downregulated, and contractility is decreased in diabetic hearts.

Norepinephrine (NE) is a key catecholamine that stimulates contractility by activating beta-

adrenergic receptors (β-AR). NE is synthesized from tyrosine by the rate-limiting enzyme,

tyrosine hydroxylase (TH), and tyrosine is catabolized by tyrosine aminotransferase (TAT).

However, the cross-talk/link between TAT and TH in the heart is unclear. To determine whether

miR-133a plays a role in the cross-talk between TH and TAT, and regulates contractility by

influencing NE biosynthesis and/or β-AR levels in diabetic hearts, Sprague-Dawley rats and

miR-133a transgenic (miR-133aTg) mice were injected with streptozotocin to induce diabetes.

The diabetic rats were then treated with miR-133a mimic or scrambled miRNA. Our results

revealed that miR-133a mimic treatment improved the contractility of the diabetic rat’s heart

concomitant with upregulation of TH, cardiac NE, β-AR, and downregulation of TAT and

plasma levels of NE. In miR-133aTg mice, cardiac specific miR-133a overexpression prevented

upregulation of TAT and suppression of TH in the heart after streptozotocin treatment.

Moreover, miR-133a overexpression in CATH.a neuronal cells suppressed TAT with

concomitant upregulation of TH, whereas knockdown and overexpression of TAT demonstrated

that TAT inhibited TH. Luciferase reporter assay confirmed that miR-133a targets TAT. In

conclusion, miR-133a controls the contractility of diabetic hearts by targeting TAT, regulating

NE biosynthesis, and consequently β-AR and cardiac function.

Key Words: microRNA, diabetes, heart failure, β-adrenergic receptors, norepinephrine

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INTRODUCTION

MicroRNAs (miRNAs) are non-coding, regulatory RNAs that play a crucial role in the

pathophysiology of several diseases including heart failure and diabetic cardiomyopathy (1). A

number of cardioprotective miRNAs are downregulated in the failing heart, which contributes to

pathological cardiac remodeling (2). MiRNA-133a (miR-133a) is one of the most abundant

miRNAs in the heart (3). It is shared between the central nervous system and the heart (4) and

has a multifaceted cardioprotective role (5). It is downregulated in failing hearts in humans and

mice (6). Downregulation of miR-133a is associated with upregulation of cardiac autophagy in

humans with diabetic heart failure (7). On the other hand, transgenic over-expression of miR-

133a in mice protects the diabetic heart from cardiac fibrosis (8). Albeit, the cardioprotective role

for miR-133a has been demonstrated at the myocardial level, its role in catecholamine

biosynthesis and action via adrenergic receptors that is required for neurohumoral stimulation of

cardiac contractility in diabetic hearts is poorly understood.

Diabetes mellitus (DM) is a complex disease caused due to insufficient insulin secretion

from pancreatic beta cells (T1D), and/or insulin resistance (T2D) that results in an increased

blood glucose level leading to morbidity and mortality (9). The number of DM patients are

increasing at an alarming rate in the world (10;11). However, the causes for increased prevalence

of DM and DM-mediated cardiomyopathy are poorly understood. Diabetes is a miRNA-

associated disease (12), which causes heart failure independent of coronary artery disease,

hypertension or valvular disease (13). In DM hearts, miR-133a is downregulated (7;8) and

contractility is decreased (14). Decreased contractility is caused primarily due to

inactivation/reduction of beta-adrenergic receptors (β-ARs) (15). β-ARs are G-protein-coupled

receptors, and β1-AR and β2-AR are the predominant subtypes in the heart. They are present in

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the ratio of 70:30 in the left ventricle, respectively, and increase contractility of the heart (16;17).

β-AR activation augments calcium uptake and increases sarco-endoplasmic reticulum activity by

upregulating sarco-endoplasmic reticulum ATPase-2a (SERCA-2a), which increases contractility

of the cardiomyocytes (18). In diabetic hearts, SERCA-2a is decreased (18), and β1-AR and β2-

AR are downregulated (19). The activation of β-AR depends on release of neuronal

norepinephrine (NE), a key catecholamine of sympathetic nervous system (20), into the synaptic

cleft, where it binds to β-AR on the cardiomyocyte membrane (21). Decreased contractility due

to β-AR inactivity/reduction may be a consequence of increased sympatho-excitation (22). The

biosynthesis of NE is achieved through a cascade of reactions beginning with the rate-limiting

enzyme tyrosine hydroxylase (TH), which converts tyrosine to dihydroxyphenylalanine, and TH

is decreased in diabetic hearts (23). Tyrosine, which is a precursor for NE biosynthesis (20), is

catabolized by the enzyme tyrosine aminotransferase (TAT). TAT catalyzes transamination of

tyrosine in the liver, and deficiency of this enzyme causes tyrosinemia (24). In addition to liver,

TAT is present in the heart, brain and kidney (25). However, the interaction between TH and

TAT for the regulation of NE biosynthesis in the heart under normal and diseased conditions

such as diabetes is unknown.

The purpose of the present study was to determine the role of miR-133a in the regulation

of TAT, cross-talk between TH and TAT, and contractility by influencing NE biosynthesis

and/or β-AR levels in diabetic hearts.

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RESEARCH DESIGN AND METHODS

Ethics statement

All animal studies were performed following the guidelines of the National Institutes of Health

and protocol approved by the Institutional Animal Care and Use Committee (IACUC) of the

University of Nebraska Medical Center.

Animal model and treatment

Male Sprague-Dawley rats were obtained from the Charles River Laboratory. Each rat was caged

individually in the animal care facility of the University of Nebraska Medical Center. Rats were

kept in an ambient environment with the temperature maintained at 22°C and humidity at 30–

40% with diurnal cycle of 12 hour dark and 12 hour light. Laboratory chow and water was made

available to the rats ad libitum. Eight week male rats (~225 g) were injected with streptozotocin

(STZ, 45 mg/kg i.v., cat # S0130, Sigma-Aldrich, Saint Louis, MO, USA) to induce diabetes.

Control animals were treated with citrate buffer in which streptozotocin was dissolved. Four

weeks after streptozotocin treatment, blood glucose was measured to ensure rats developed

diabetic phenotype (blood glucose >350 mg/dL). To assess the effect of miR-133a

overexpression on diabetic hearts, these diabetic rats were treated with lentivirus containing

either miR-133a mimic or scrambled miRNA and sacrificed at the age of fourteen weeks

(Supplementary Figure 1A).

The miR-133a transgenic (miR-133aTg) mouse was a kind gift from Dr. Scot Matkovich,

Washington University, St. Louis, USA. C57BL/6J (WT) mice were procured from the Jackson

Laboratory. Both strains of the mouse were maintained in the animal facility of University of

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Nebraska Medical Center. Eight weeks male WT and miR-133aTg mice were treated with

streptozotocin (65 mg/Kg, i. p.) following a previously published protocol (8) for five

consecutive days. At the tenth-week blood glucose level was measured to ensure diabetic

phenotype (blood glucose >300mg/dL). At the fourteenth-week hemodynamic measurements

were performed, mice were sacrificed, and heart tissue was collected (Supplementary Figure

1B). No exogenous insulin was administered at any time to either rats or mice.

Assessment of Cardiac Function: In vivo hemodynamics

Left ventricular pressure and rate of change of left ventricular pressure (±dP/dt) was evaluated in

the controls, diabetic rats treated with scrambled miRNA or miR-133a mimic. Rats were

anesthetized with α-chloralose (70 mg/kg, i. p.) and urethane (0.75 g/kg, i. p.), and a Millar

catheter (Millar Instruments, Houston, TX, USA) containing a pressure transducer was

introduced into the left ventricle via the right carotid artery. Another catheter was inserted via the

right femoral vein for administration of isoproterenol. Cardiac hemodynamic parameters were

measured in the anesthetized state. After assessing basal parameters, a bolus dose (0.05, 0.1, 0.25

µg/kg) of isoproterenol, a β-AR agonist was administered into the right femoral vein to assess the

responsiveness of the heart to β-AR stimulation. A Powerlab data-acquisition system (AD

Instruments, Colorado Springs, CO, USA) was used for acquiring data.

Plasmids and constructs

MiR-133a (cat # MmiR3445-MR03), scrambled miRNA (cat # CmiR0001-MR03), anti-miR-

133a (cat # MmiR-AN0880-AM04) and TAT 3/ UTR clones (WT 3

/ UTR: cat # RmiT048999-

MT01 and Mutant 3/

UTR: CS-RmiT048999-MT01-01) were purchased from GeneCopoeia,

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Rockville, MD. TAT mouse cDNA clone (Cat # MC204147) was purchased from OriGene

Technologies, Inc. Rockville, MD, USA. TAT siRNA and negative control siRNA

oligonucleotides (cat # 4390771 and cat # 4390843) were purchased from Life Technologies,

Carlsbad, CA, USA.

In vitro model, Cell culture and transfection

In vitro studies were performed on a CATH.a neuronal (dopaminergic) cell line using standard

protocol and medium (RPMI, cat # R8758 with 8% horse serum and 4% fetal bovine serum, Life

Technologies, Carlsbad, CA, USA). In brief, cells were cultured and differentiated by treating

N6, 2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate (DB-cAMP, cat # D0627, Sigma-

Aldrich, St. Louis, MO, USA) for 48 hours. Then they were transfected with plasmid or siRNA

oligonucleotides for 24 hours using Lipofectamine 2000 (cat # 11668-019, Life Technologies,

Carlsbad, CA, USA). Transfected cells were processed for immunocytochemistry or harvested

for protein isolation at 48 hour post transfection.

Lentiviral packaging

The 293T cells were cultured on 10 cm2 plate up to ~90% confluence. 20µg of the vector (miR-

133a or scrambled) and 10 µg of each of RSV-REV, VSVG, and pMDLg/p RRE (plasmids for

virus proteins packaging) were co-transfected by using Opti-MEM media and lipofectamine

2000 (cat # 11668-019 and cat # 31985-070, Life Technologies, Carlsbad, CA, USA). Viral

supernatant was collected at 48 and 72 hours after transfection, and precipitated with sterile PEG

solution (cat # 81280, Sigma-Aldrich, St. Louis, MO, USA; 1 volume PEG to 4 volume viral

supernatant collection). The virus pellets were collected by centrifuging the PEG-precipitated

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viral supernatant solution at 2300 rpm for 2 hours at 40C. The virus pellets were re-suspended in

PBS and aliquoted in PCR tubes and kept at -800C until used. The virus titer was calculated by

infecting virus aliquots (2, 4, 8, 16 µL) in a 6-well plate seeded with 293T cell. After two days,

GFP-tagged (miR-133a-GFP and scm-GFP positive) cells were counted at 40X magnification in

four different fields of view for each well. The average number of GFP positive cells per 40X

magnification view was counted. The titer was calculated with the formula: total number of virus

particle/µL(volume) = average number of GFP +ve cell in 40X field of view x 4900/the volume

of virus infected in the cell culture well.

MiR-133a assay

MiRNA was isolated from the heart tissue (left ventricle) using the mirVana™ miRNA isolation

kit (cat # AM1560, Life Technologies, Carlsbad, CA, USA). The purity of RNA was determined

by NanoDrop 2000c (Thermo Scientific Inc., Wilmington, DE, USA), and highly pure RNA

(ratio of 260/280 ≥ 1.8 and 260/230 ≥ 1.8) was used for the assays. Individual miR-133a assay

was performed using miRNA-133a primers (Assay ID: 002246, Life Technologies, Carlsbad,

CA, USA) specific for RT and Taqman qPCR. U6 SnRNA primer (Assay ID: 001973, Life

Technologies, Carlsbad, CA, USA) was used for endogenous control. MiRNA amplification was

performed following manufacturer’s instructions using TaqMan Universal PCR Master Mix (cat

# 4427788, Life Technologies, Carlsbad, CA, USA). RT-qPCR was performed in a Bio-Rad

CFX qPCR System and the results were analyzed by using BioRad CFX Manager3.0 software

(Bio-Rad Laboratories, Hercules, CA, USA).

Reverse Transcription (RT)

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High-quality RNA was used for reverse transcription polymerase chain reaction (RT-PCR). First

strand cDNA was synthesized from an aliquot of 1 µg of RNA using iScript™ cDNA synthesis

kit (cat # 170-8841, Bio-Rad Laboratories, Hercules, CA, USA). The reaction was performed

with priming at 25°C for 5 min, reverse transcription at 42°C for 5 min and RT inactivation at

85°C for 5 min in a thermal cycler (C1000 Touch, Bio-Rad Laboratories, Hercules, CA, USA).

Quantitative RT-polymerase chains reaction (qRT-PCR)

The qPCR was performed using gene-specific oligonucleotide primers (Supplementary Table 1).

The assay was performed using 2X iTaq Universal SYBR Green Supermix (cat #172-5121, Bio-

Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. In brief, the

PCR program was 95°C for 3 min and then 40 cycles of 95°C for 15 sec; 55°C for 30 sec and

72°C for 45 sec. The quantitative PCR reaction was performed in duplicate and it included: 4.5

µl, cDNA template diluted in nuclease free water (100 ng); 5 µl iTaq Universal SYBR Green

Supermix and 0.5 µl gene specific primer (10 pm). The forward and reverse primer sequences of

different genes are listed in Supplementary Table1. Bio-Rad CFX qPCR System was used for

RT-qPCR and data were analyzed using BioRad CFX Manager3.0 software (Bio-Rad

Laboratories, Hercules, CA, USA). Relative quantification in fold change (2-∆∆Ct

) was

normalized from expression of endogenous control 18s RNA.

Western blotting

The standard Western blotting protocol was followed after protein estimation by Pierce™ BCA

protein assay kit (cat # 23227, Pierce Biotechnology, Rockford, IL, USA). RIPA buffer (cat #

BP-115, Boston BioProducts, Worcester, MA, USA) supplemented with protease inhibitor

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cocktail (cat # MSSAFE, Sigma-Aldrich, St. Louis, MO, USA) was used for whole protein

extraction from rat hearts. For protein quantification, 40 µg of protein extracts were subjected to

10% SDS-PAGE and transferred onto nitrocellulose membrane (cat # 1620115, Bio-Rad

Laboratories, Hercules, CA, USA). Transferred membrane was blocked overnight with 5%

blocking solution (TBS with 5% non-fat dried milk). The primary antibodies used were β1-AR,

β2-AR , β-actin, β-tubulin (cat # sc-568, sc-570, sc-47778, and sc-23949, respectively from

Santa-Cruz Biotechnology, CA, USA), tyrosine hydroxylase (cat # 22941, ImmunoStar, Inc.

Hudson, WI, USA), tyrosine aminotransferase (cat # EPR6121, GeneTex Inc. Irvine, CA, USA)

for rat study, and cat # ab125000, Abcam, Cambridge, MA, USA) for mice study, SERCA-2a, β-

MHC and tyrosine hydroxylase antibodies were raised in rabbit and used for multiplex Western

blotting (cat # ab2861, ab172967, and ab112, respectively, Abcam, Cambridge, MA, USA),

GAPDH (cat # MAB374, Millipore, Temecula, CA, USA). Antibodies were diluted in the ratio

of 1:1000 and incubated for overnight at 4°C. Respective secondary antibodies with HRP

conjugates (anti-rabbit-HRP, cat # sc-2054, anti-mouse-HRP, cat # sc-2005, Santa-Cruz

Biotechnology, CA, USA) and fluorophore conjugates (anti-rabbit Alexa Fluor® 488, cat # A-

21441, anti-mouse Alexa Fluor® 594, cat # A-21201, Life Technologies, Carlsbad, CA, USA)

were diluted at 1:5000 and incubated at room temperature for 2 hours. The blots were developed

using ECL substrate (cat # 170-5061, Bio-Rad Laboratories, Hercules, CA, USA) or by

multichannel fluorescence imaging using Molecular Imager Chemi-DocTM

XRS with Image Lab

software, version 3.0 (Bio-Rad Laboratories, Hercules, CA, USA). The band intensity was

measured using the Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA).

Immunocytochemistry

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Immunocytochemistry staining was performed on CATH.a neuronal cells differentiated with

DB-cAMP. Cells were transfected with 2 µg of scrambled miRNA or miR-133a mimic plasmid.

After treatment and transfection, the medium was removed and cells were washed in 1x

phosphate buffered saline (1xPBS) pH-7.4, and then fixed in 4% paraformaldehyde (cat #

158127, Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes. After fixation, cells were washed

in 1xPBS for 3x5 minutes and then permeabilized in 0.02% Triton-X-100 (cat # 215682500,

Acros Organics, NJ, USA) in 1xPBS for 20 minutes. They were blocked in 1% BSA in 1xPBS

for 1 hour. They were then washed in 1xPBS for 3x5 minutes and incubated with diluted primary

antibodies in 1xPBS with 0.1% BSA at 4°C for overnight. The primary antibodies used were

1:400 dilution of anti-tyrosine hydroxylase (cat # 22941, ImmunoStar, Hudson, WI, USA) and

1:200 dilution of anti-tyrosine aminotransferase (cat # EPR6121, GeneTex, Irvine, CA, USA).

On next day, primary antibody was removed, and cells were washed in 1xPBS for 3x5 minutes

and incubated with anti-mouse AlexaFluor 594 (cat # A21201, Life Technologies, Carlsbad, CA,

USA) or anti-rabbit AlexaFluor 488 (cat # A21441, Life Technologies, Carlsbad, CA, USA) for

1 hour in dark. The secondary antibody was removed and cells were washed with 1xPBS for 3x5

minutes. They were then incubated with 1µg/ml DAPI in 1xPBS (cat # A1001, AppliChem, St.

Louis, MO, USA) for 20 minutes. After that, cells were washed twice in 1xPBS and mounted

with a coverslip using the Fluoromount-G mounting medium (Cat # 0100-01, Southern Biotech,

Birmingham, AL, USA). Images were captured by EVOS Cell Imaging Systems (Life

Technologies, Carlsbad, CA, USA) and analyzed by Image J software (NIH, USA).

Bioinformatics analyses of TAT 3/-UTR

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In silico analyses predicts TAT as a putative target of miR-133a. The search websites were

TargetScan (www.targetscan.org) and miRDB (www.mirdb.org).

Noradrenaline assay

Norepinephrine (NE) content was measured by Norepinephrine ELISA Assay Kit (cat # NOR31-

K01, Eagle Biosciences, Inc. Nashua, NH, USA). Blood plasma and left ventricular tissue lysate

from treated rats were used for norepinephrine assay. Plasma was isolated by collecting the

blood from renal vein in a 1 ml syringe with EDTA and separated from blood clot by

centrifuging in a 2 ml centrifuge tube at 2000 rpm. Cleared plasma after centrifugation was

stored at -80°C until use. For tissues, the dissected left ventricle of the heart was rinsed with ice-

cold saline and frozen immediately on dry ice and weighed. Norepinephrine was extracted from

the tissue by homogenization in 10 volumes of 0.1 mol/L HCl followed by centrifugation at

5000×g, for 20 min at 4°C.

Sucrose-Phosphate-Glyoxylic acid (SPG) chemifluorescence

The glyoxylic acid condensation reaction was used for fluorescence detection and distribution of

catecholaminergic nerves on 15 µm histological cryosections. The staining was followed as per

previously described protocol (26). In brief, frozen histological sections were adhered onto glass

slides and immersed promptly in sucrose-phosphate-glyoxylic acid solution (1% glyoxylic acid,

0.23 M monobasic KH2PO4, 0.2 M sucrose, pH 7.4) for 5 sec. Slides were dried entirely under a

cool stream of air and heated to 95°C for 2.5 min after applying a thin layer of mineral oil on

tissue surface. Slides were then cooled to room temperature and sealed with cover slip. Bluish-

white fluorescence of catecholamine on tissue section was captured by fluorescence microscopy

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with UV filter (Life Technologies, Carlsbad, CA, USA) and images were quantified by Image J

software (NIH, USA).

DAB-HRP Immunohistochemistry

Immunohistochemistry was performed on 5 µm transverse cryosections. In brief, the histological

section was dipped in 1xPBS to dissolve the freezing medium, processed for citrate antigen

retrieval (heating-cooling) and fixed in 4% paraformaldehyde for 30 min at room temperature.

Endogenous peroxidase activity was quenched by incubating slides in peroxidase suppressor

solution (3% H2O2). After quenching, slides were blocked in 1% serum (goat or horse) in 1xPBS

for 30 minutes. Slides were washed two times for 3 minutes with 1xPBS and incubated with

primary antibodies for 3 hours at 4°C. Primary antibodies used were tyrosine hydroxylase, 1:400

(cat # 22941, ImmunoStar, Hudson, WI, USA), tyrosine aminotransferase, 1:200 (cat #

EPR6121, GeneTex, Irvine, CA, USA), and anti-GFP horseradish peroxidase (HRP) conjugate,

1:500 (cat # A10260, Life Technologies, Carlsbad, CA, USA). Sections were washed in 1xPBS

for 3x5 minutes and incubated with respective HRP-conjugated secondary antibodies, anti-

rabbit-HRP (cat # sc-2054, Santa-Cruz Biotechnology, CA, USA) or anti-mouse-HRP (cat # sc-

2005, Santa-Cruz Biotechnology, CA, USA), as applicable, for 1 hour at room temperature in a

humidified chamber. Secondary antibody was removed and sections were washed with 1xPBS

for 3x5 minutes and incubated with fresh Sigma FAST DAB tablet solution (cat # D4293,

Sigma-Aldrich, St. Louis, MO, USA). Sections were then washed twice in 1xPBS and

counterstained with Harris modified Hematoxylin (Cat # 1859352, Pierce Biotechnology,

Rockford, IL, USA), and mounted with a coverslip using Permount Mounting Media (cat #

SP15-100, Thermo Scientific Inc., Waltham, MA, USA). Images were captured by bright field

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colored microscope (Leica Microsystems, Buffalo Grove, IL, USA) with image Pro 7.0 capture

tool.

Fluorescence Immunohistochemistry

Fluorescence immunohistochemistry was performed on 5 µm transverse cryosections following

standard protocol. Primary antibodies used were anti-sercomeric alpha actinin, 1:100 (cat #

ab9465, Abcam, Cambridge, MA, USA) and anti-GFP, 1:500 (cat # ab13970, Abcam,

Cambridge, MA, USA). Secondary antibodies used were anti-mouse AlexaFluor 488 (cat #

A21200, Life Technologies, Carlsbad, CA, USA) and anti-chicken AlexaFluor 594 (cat #

ab150176, Abcam, Cambridge, MA, USA).

Wheat Germ Agglutinin (WGA) staining

Wheat germ agglutinin staining was performed to stain cardiomyocyte cell boundaries and was

used for measurement of cell area and hypertrophy. Frozen histological sections of the heart

were kept in 1xPBS for 5 min to dissolve the freezing medium. Hydrated sections were fixed

with freshly prepared 4% formaldehyde for 15 min at 37°C and then washed 3×5 min with

1xPBS. Next, 100-200 µL of 5 µg/mL wheat germ agglutinin (cat # W834, Life Technologies,

Carlsbad, CA, USA) conjugate solution was applied onto the sections and incubated for 1 hour at

room temperature. The sections were counterstained with DAPI. Slides are mounted with a

coverslip and observed under microscope. The images were captured by EVOS Cell Imaging

Systems (Life Technologies, Carlsbad, CA, USA) and analyzed by Image J software (NIH,

USA).

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Luciferase reporter assay

To measure luciferase activity, CATH.a cells were plated (60% confluence) in incomplete RPMI

medium. Cells were co-transfected with 1µg of 3/UTR clone with 0 µg, 1 µg and 2 µg of

scrambled miRNA or miR-133a mimic plasmid. Luciferase reporter assay was performed after

48 hours of transfection using Dual-Glow luciferase assay kit (cat # E2920, Promega

Corporation, Madison, WI, USA) following the manufacturer’s instructions in a GloMax®-

Multi+ Detection System (Promega, Madison, WI, USA).

Statistical analyses

The statistical values were expressed as mean ± standard error of mean (SEM). The reverse

transcription, qPCR and Western blotting experiments were repeated at least thrice in three

independent samples unless otherwise represented. Statistical analysis was performed by paired

Student's t-test and one-way ANOVA was used to compare among groups. P <0.05 value are

considered as statistically significant.

RESULTS

Decreased contractility of the diabetic heart is normalized by miR-133a mimic treatment

Previous studies have shown that miR-133a has a role in the contractility of pressure overload

heart (27) and miR-133a is downregulated in diabetic hearts (28). To determine whether the lack

of miR-133a decreased the contractility of the diabetic heart, we overexpressed miR-133a in the

diabetic hearts by miR-133a mimic treatment and validated the upregulated miR-133a in the

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diabetic hearts by individual miR-133a assay (Supplementary Figures 2A-C). We measured left

ventricular (LV) pressure and rate of contraction and relaxation (±dP/dt) of the miR-133a mimic-

treated diabetic hearts at the basal level and after isoproterenol (a β-AR agonist) infusion using a

Millar catheter. Our results showed that miR-133a mimic treatment normalized the decreased LV

pressure and ±dP/dt in the diabetic hearts (Figures 1A-B), suggesting that reduced level of miR-

133a may contribute to decreased contractility of the diabetic hearts. Interestingly, the rate of

contraction (dP/dt) was significantly improved by miR-133a mimic after isoproterenol treatment

(Figure 1B), suggesting that β-AR may be an important player in miR-133a-mediated

improvement in the contractility of the diabetic hearts.

MiR-133a mimic treatment upregulates β-AR and SERCA-2a in diabetic hearts

To investigate if miR-133a influenced β-AR expression in diabetic hearts, we measured β1-AR

and β2-AR mRNA and protein levels in scrambled miRNA-, and miR-133a mimic- treated

diabetic hearts. The mRNA and protein levels of both β1-AR and β2-AR were increased in miR-

133a mimic-treated hearts (Figures 2A-D), suggesting that miR-133a overexpression has

upregulated β-AR in the diabetic hearts. β-AR activation normally upregulates calcium influx in

the cytoplasm, which triggers sarco-endoplastic reticulum activity for sarcomeric contraction that

increases the contractility of the heart. Decreased sarco-endoplasmic reticulum activity

contributes to decreased contractility of diabetic hearts (14;18). To determine whether miR-133a

improved the contractility of the diabetic heart by influencing sarco-endoplasmic reticulum

activity, we measured the protein level of SERCA-2a, a calcium handling enzyme in sarco-

endoplasmic reticulum, in the heart. Our results showed that miR-133a mimic treatment

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upregulated SERCA-2a in the diabetic hearts (Figure 2E). Overall, these results suggest that

miR-133a improved the contractility of the diabetic hearts by upregulating β-AR and SERCA-2a.

To support that miR-133a mimic treatment had a similar effect on the diabetic heart as

previously reported, we investigated cardiac hypertrophy in miR-133a mimic-treated diabetic

hearts because the anti-hypertrophy effect of miR-133a, possibly by suppression of GLUT4 via

targeting KLF15 (29), was documented in the non-diabetic (6) and diabetic (30) hearts. To

determine cardiac hypertrophy, we measured the level of β-myosin heavy chain (β-MHC), a

molecular marker for hypertrophy (Supplementary Figure 3A), and cross-sectional area of

cardiomyocytes in histological sections of the heart (Supplementary Figure 3B). Our results

showed that miR-133a mimic treatment mitigated cardiac hypertrophy in the diabetic hearts

(Supplementary Figure 3) demonstrating that miR-133a had a similar impact on the diabetic

hearts as previously reported.

MiR-133a upregulates norepinephrine in diabetic hearts

To determine the role of miR-133a on upstream signaling molecules that might have activated β-

AR in the diabetic heart, we measured NE levels because NE activates β-AR, and cardiac NE is

decreased in the failing heart (31). Notably, miRNAs are associated with reduced NE level in

chronic heart failure (32). Therefore, we sought to determine the impact of miR-133a

overexpression on NE level in diabetic hearts. Before measuring the cardiac NE level, we

measured the plasma NE level because it is documented that plasma NE level is increased in

most forms of heart failure (33). The plasma NE level was increased in the scrambled miRNA-

treated diabetic rats. However, it was decreased in miR-133a mimic-treated diabetic rats (Figure

3A), suggesting that miR-133a mimic treatment decreases the plasma NE level in diabetic rats.

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Then we measured the cardiac tissue level of NE in miR-133a mimic-treated diabetic hearts.

Contrary to the plasma NE level, the cardiac NE level was lower in the scrambled miRNA-

treated diabetic rats, and it was restored after miR-133a mimic treatment (Figure 3B). These

findings suggest that miR-133a has a crucial role in regulating the cardiac and plasma NE levels

in diabetics.

The endogenously released NE is stored in the nerve endings and intraneuronal storage of NE

is decreased in diabetic hearts (34). To determine the role of miR-133a in intraneuronal storage

of NE in diabetic hearts, we stained cryosections of the heart with sucrose-phosphate-glyoxylic

acid (SPG). The SPG binds to catecholamines and detects catecholamine storage in sympathetic

neuron terminals in the heart by chemifluorescence (Figure 3C (i)). Our results showed that miR-

133a mimic treatment increased cardiac NE storage in the diabetic hearts (Figure 3C (ii-iii)),

which further support that miR-133a overexpression upregulated cardiac NE in the diabetic

hearts. Overall, these findings demonstrated that miR-133a has a pivotal role in maintaining

plasma and cardiac NE levels in diabetic rats. However, it was unclear how miR-133a

normalized the NE levels in diabetics.

MiR-133a mimic treatment upregulates tyrosine hydroxylase in diabetic hearts

To understand the underlying mechanism of miR-133a-mediated regulation of NE levels, it was

imperative to investigate the biosynthesis of NE. Since NE is synthesized from tyrosine by TH,

we determined TH levels in miR-133a mimic-treated diabetic hearts. The messenger RNA and

protein levels of TH were upregulated in miR-133a mimic-treated diabetic hearts (Figures 4A-B)

suggesting that miR-133a induces TH gene expression. To determine the expression of neuronal

TH in diabetic hearts, we performed immunohistochemistry of TH in cryosections of the diabetic

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hearts. Our results showed increased intensity of neuronal TH in miR-133a mimic-treated

diabetic hearts (Figure 4C) corroborating that miR-133a mimic treatment increased the neuronal

TH in the diabetic hearts.

To validate that increase in the TH level was neuronal, we stained the heart sections with

TH and a neuronal marker, microtubule-associated protein 2 (MAP2). The imaging of the heart

sections showed co-localization of MAP2 with TH (Supplementary Figure 4A) demonstrating

that increases in the TH level in the miR-133a mimic-treated diabetic hearts was indeed

neuronal. Further, we used the same antibody that stains neuronal TH in the heart

(Supplementary Figure 4B) to assess the expression of TH in CATH.a cells, a validated

catecholaminergic neuronal cell line (Supplementary Figure 4D), which corroborated that TH

expressed in the heart was neuronal.

To determine the specific role of miR-133a in the regulation of neuronal TH level, we

treated CATH.a neuronal cells that express miR-133a (Supplementary Figure 4C) with

scrambled, miR-133a mimic and anti-miR-133a, and determined the protein level of TH in these

three groups. Our results demonstrated that miR-133a mimic treatment upregulated TH (Figures

4D-E). However, there was no change in the TH level in anti-miR-133a-treated neurons (Figure

4E) suggesting that miR-133a may not have a direct or causative role in the regulation of TH

level in neurons. Since miRNA mostly inhibits genes, we infer that miR-133a mimic treatment

might have upregulated TH by inhibiting another gene that normally suppresses TH.

Tyrosine aminotransferase inhibits tyrosine hydroxylase in neuronal cells

Since tyrosine is a substrate for TH and tyrosine is catabolized by TAT (24), we suspected that

TAT might have an influence on TH level. Although TAT has been reported to be present in the

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heart (25), its role in the heart remains unclear. To determine the specific effect of TAT on the

TH level, we first validated whether TAT was present in the neurons. For that, CATH.a neuronal

cells were stained with anti-TAT antibody and cellular level of TAT was observed. As

speculated, TAT was present in the neuronal cells (Supplementary Figure 4E). Then, we either

overexpressed or inhibited TAT in CATH.a neuronal cells (Supplementary Figures 4F-G) and

measured the levels of TH. Our results revealed that suppression of TAT upregulated TH

(Figures 5A-B), whereas overexpression of TAT downregulated TH in neuronal cells (Figure

5A, C). These findings suggest that TAT has an inhibitory effect on TH. Since TH is the rate-

limiting enzyme in NE biosynthesis, TAT might have an indirect influence on NE biosynthesis.

MiR-133a modulates tyrosine aminotransferase in diabetic hearts

Although our results demonstrated that miR-133a mimic treatment increased the level of TH in

diabetic hearts (Figure 4), it was unclear whether miR-133a upregulated TH by downregulating

TAT in the heart. To determine the role of miR-133a on TAT level, we measured mRNA and

protein levels of TAT in scrambled miRNA-, and miR-133a mimic- treated diabetic hearts. Our

results revealed that miR-133a mimic treatment decreased TAT expression in the diabetic hearts

(Figures 6A-B) suggesting that miR-133a suppresses TAT. Since TAT inhibited neuronal TH,

we sought to determine whether miR-133a decreased neuronal TAT in diabetic hearts. For that,

neuronal TAT was stained in diabetic heart sections and was observed under a microscope. Our

results showed that miR-133a mimic treatment reduced the levels of neuronal TAT in diabetic

hearts (Figure 6C).

To determine whether miR-133a had a direct role in inhibition of neuronal TAT, we

treated CATH.a neuronal cells with scrambled miRNA, miR-133a mimic, and anti-miR-133a,

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and then measured the levels of TAT. Our results demonstrated that miR-133a mimic treatment

downregulated TAT expression at cellular and protein levels (Figure 6D-E), whereas anti-miR-

133a treatment upregulated TAT expression (Figure 6E). These findings suggest that miR-133a

might have a direct role in regulating TAT expression in neuronal cells.

Cardiac-specific overexpression of miR-133a in mice prevents streptozotocin-induced

upregulation of TAT and downregulation of TH in the heart

To rule out the systemic effect of miR-133a mimic delivery and to validate the cross-species role

of miR-133a on TAT and TH, we used miR-133Tg mice and treated them with streptozotocin

(Supplementary Figure 1B). We also genotyped (Supplementary Figure 2D) and measured the

cardiac levels of miR-133a (Supplementary Figure 2E) in these mice before measuring the TAT

and TH levels in the heart. The comparison of TH levels in WT and miR-133a transgenic mice

(miRTg) treated with and without streptozotocin demonstrated that TH level was decreased in

streptozotocin-treated WT mice, but it remained upregulated in streptozotocin-treated miR-

133aTg mice (Figures 7A (i and ii)). On the contrary, protein levels of TAT were elevated in

streptozotocin-treated WT mice, but it remained decreased in streptozotocin-treated miR-133aTg

mice (Figures 7A (i and iii)). Based on these results we infer that cardiac specific overexpression

of miR-133a prevents diabetes-mediated upregulation of TAT and downregulation of TH in the

heart. Moreover, we also performed immunohistochemistry for TAT and TH in the heart sections

of WT and miR-133aTg mice treated with or without streptozotocin. We observed that

streptozotocin treatment decreased the number of neurons expressing TH in WT, but the number

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of TH-expressing neurons remained comparatively high in miR-133aTg mice after streptozotocin

treatment (Figure 7B (i)). Then we measured the levels of TAT-expressing neurons. In contrast

to TH, the number of TAT-expressing neurons were increased in streptozotocin-treated WT

hearts. However, it remained downregulated in streptozotocin-treated miR-133aTg hearts (Figure

7B (ii)). Overall, these results revealed that the inhibitory effect of miR-133a on TAT is common

in both mice and rat species, and overexpression of miR-133a suppresses TAT and induces TH

in diabetic hearts of mice and rats.

MiR-133a targets tyrosine aminotransferase

MiRNA modulates gene expression by targeting 3 prime untranslated region (3ÚTR) of mRNA.

Therefore, we performed in silico analyses for miR-133a predicted targets, and found that TAT

was a target for miR-133a (Figure 8A). The binding site for miR-133a on TAT 3ÚTR was

conserved in mouse and rat, however, mouse had three binding sites whereas rat had a single

binding site (Supplementary Figure 5). To determine whether the TAT was a direct target for

miR-133a, we used TAT 3ÚTR, and the miR-133a binding sequence mutant TAT 3ÚTR

(Figure 8B), and performed luciferase reporter assay on CATH.a neuronal cells. Our results

showed that miR-133a downregulated luciferase activity of 3ÚTR of TAT, which was nullified

in the mutant 3ÚTR of TAT (Figures 8C-D). These findings revealed that miR-133a targets

TAT.

Overall, our results demonstrated that miR-133a has a direct role in suppressing TAT. Since

TAT inhibited TH, miR-133a mimic might be indirectly upregulating TH and thus regulates NE

biosynthesis, which consequently leads to upregulation of β-AR and improved myocardial

contractility in diabetics.

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DISCUSSION

In the present study, we demonstrate that lack of miR-133a contributes to decreased contractility

of diabetic hearts and miR-133a mimic treatment can improve the contractility of diabetic hearts.

We propose that downregulation of miR-133a in the diabetic heart increases TAT level that in

turn suppresses TH and decreases cardiac NE level, which compromises β-AR activation and the

contractility of the diabetic hearts. MiR-133a mimic treatment normalizes TAT and restores the

levels of TH, NE, β-AR, and the contractility of the diabetic hearts (Figure 8E). In this study, we

reveal several novel regulatory mechanisms such as miR-133a acts as an inducer of β-AR by

regulating upstream activators of β-AR in diabetic hearts. Lack of miR-133a decreases the

contractility of diabetic hearts. TAT is present in the neurons of diabetic hearts and it inhibits

TH, which may influence NE biosynthesis and β-AR in diabetic hearts. MiR-133a directly

modulates TAT expression in diabetic hearts.

MiRNAs play a crucial role in regulating the contractility of the heart. Several miRNAs

decrease the contractility while others increase the contractility of the heart. In human heart

failure, upregulation of miR-765 decreases contractility by regulating protein phosphatase

inhibitor-1 (35). In mice and humans, SERCA-2a is regulated by miR-25. The inhibition of miR-

25 improves the contractility of the failing heart by upregulating SERCA-2a (36). In a rabbit

model of congestive heart failure, pacing improves contractility concomitant with upregulation

of SERCA-2a and miR-133a (37). In heart failure and diabetic cardiomyopathy, SERCA-2a is

downregulated (38). Our results demonstrate that miR-133a mimic treatment upregulates

SERCA-2a in diabetic hearts (Figure 2E) and improves contractility (Figures 1A-B), which

further support that miRNAs have a pivotal role in SERCA-2a regulation and cardiac

contractility. Calcium influx also plays an important role during contractility of cardiomyocytes

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and it is regulated by the sodium/calcium exchanger1 (Ncx1). MiR-214 regulates Ncx1 and

improves the contractility of the ischemic heart (39). In the pressure-overload model of heart

failure, miR-133a modulates β1-AR and its downstream signaling molecules that regulate the

contractility of the heart (27). In abdominal aortic constriction-induced pressure-overload model

in mice, deficiency of insulin-like growth factor-1 upregulates miR-133a to alleviate myocardial

contractile dysfunction (40). However, the role of miRNAs in regulation of the contractility of

diabetic hearts is poorly understood. In this study, we reveal that miR-133a, which is anti-

hypertrophy (30) and anti-fibrotic (8) in diabetic hearts, regulates contractility of the heart.

Although miR-133a has been reported to modulate β-AR and its downstream signaling

cascade in a pressure overload heart failure (27), the role of miRNAs in the regulation of

upstream activators of β-AR have not been elucidated. Our data show for the first time that miR-

133a has a crucial role in controlling the upstream activators of β-AR, especially NE

biosynthesis in diabetic hearts (Figure 3). Our results also support a previous report indicating

that miRNA expression is associated with catecholamine sensitivity (32). However, miR-133a-

mediated activation of β-AR in diabetic hearts (Figures 2A-D) differs from miR-133a-mediated

inhibition of β1-AR in the pressure overload heart (27). The different roles of miR-133a in β-AR

activation in the diabetic and pressure overload hearts suggest that miR-133a may normalize the

contractility of diabetic hearts by targeting other upstream signaling molecules that activate β-

AR in the diabetic hearts. Another reason could be that diabetic heart differs from other failing

hearts in the metabolic conditions (9). Further, we overexpressed miR-133a in rats by delivering

miR-133a mimic, whereas in the pressure overload model miR-133a transgenic mice were used

(27). In the miR-133a transgenic mice, there are discrepancies in the results on cardiac functions

depending on the α-myosin heavy chain (MHC) or β-MHC promoter being used (41;42). Our

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studies on miR-133a transgenic mice with α-MHC promoter revealed that cardiac specific

overexpression of miR-133a prevents downregulation of TH in diabetic condition (Figure 7),

which is similar to our results with diabetic rats (Figures 4A-C, 6A-C). Further studies on other

models of heart failure will provide insight on the miR-133a-mediated regulation of TAT, NE

and β-AR in the heart.

Increased plasma NE level is common in all forms of heart failure including diabetic

heart failure (33;34;43;44). Our results show that miR-133a mimic treatment decreases plasma

NE level (Figure 3A). There is a possibility that miR-133a either mitigates the conditions that

increase plasma NE level or stimulates the conditions that decrease plasma NE level or both.

Since miR-133a increases the cardiac NE storage in diabetic hearts (Figures 3B-C), it is a

possibility that miR-133a may decrease/influence NE release/spill over from nerve terminals in

the heart that reduce plasma NE level. Although these processes remain to be elucidated, it was

interesting to observe that miR-133a mimic treatment decreases plasma NE level in diabetic rats.

Cardiac NE level may not be same in the heart failure (45) and diabetic cardiomyopathy (22).

The level of cardiac NE depends on NE storage in the nerve endings and its release in the

myocardium. By using [3H] NE, it is reported that intraneuronal storage granules of NE in the

diabetic myocardium is defective (34) suggesting that storage of cardiac NE may be

compromised in diabetic hearts. SPG–induced histofluorescence has been used to visualize the

nerve profile in the heart and to demonstrate that loss of noradrenergic nerve terminal contributes

to right heart failure (45). We also used SPG to assess nerve profile in miR-133a mimic-treated

diabetic hearts. Our results reveal that miR-133a mimic treatment increases the number of NE

containing nerves in the diabetic heart (Figure 3C) suggesting that miR-133a mimic treatment

protects the diabetic heart from noradrenergic nerve loss. Moreover, our results are consistent

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with the previous report that nerve density is reduced in diabetic hearts (23). It also supports that

miRNAs are involved in catecholamine level in heart failure patients (32).

The biosynthesis of NE is increased by upregulation of TH (20). TH gene has a

tetranucleotide repeat in intron 1, which is associated with regulation of NE level and

hypertension (46). The cardiac level of TH is decreased in the diabetic (23;47) and failing hearts

(31). Our results show that miR-133a mimic treatment increased TH level in diabetic hearts

(Figure 4). However, the regulation of TH in the diabetic heart has not been completely

understood and the impact of TAT on NE biosynthesis was unknown. For the first time, we

elucidate that neuronal TH is regulated by TAT (Figure 5). Therefore, our results provide a novel

insight into a possibility of TAT-mediated indirect regulation of NE biosynthesis. It opens a new

window to understanding the regulation of TH in different disease conditions including

hypertension and heart failure.

TAT has a crucial role in tyrosinemia type II, hepatitis and hepatic carcinoma (24). It

regulates glucocorticoids in the liver of diabetic rodents (48). Although TAT is present in the

heart (49), its functional role in the heart is yet unclear. In the present study, we reveal that TAT

is a regulator of TH and elevated level of TAT downregulates TH in nerve terminals in the

diabetic heart (Figure 5). Interestingly, TAT is a target for miR-133a (Figures 8A-D) and miR-

133a mimic treatment decreases the level of TAT in nerve terminals (Figure 6D-E). The miR-

133a mimic-treated diabetic hearts also show decreased levels of TAT (Figures 6A-C) indicating

a novel regulatory role for miR-133a on TAT expression. Our results also open an avenue for

exploring the role of miR-133a in TAT-regulated diseases such as tyrosinemia type II, hepatitis

and hepatic carcinoma.

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Diabetic heart failure is a multifactorial disease that increases the risk of heart failure (50)

and ultimately causes multi-organ failure. Despite the advancement of medical research, the

prevalence of diabetes is increasing at an alarming rate (10;11), which warrants novel therapeutic

strategies to combat its complications. MicroRNA-based therapy offers a novel and advanced

approach for developing the treatment strategy for several diseases (51) including cancer, where

miR-34 is currently in clinical trial (ClinicalTrails.gov Identifier: NCT01829971). Therefore,

miRNA-based therapy could be a promising approach to normalizing diabetes-mediated

complications in the heart. Considering the multi-faceted cardioprotective role of miR-133a (5)

and the results of the present study, it is suggested that miR-133a could be a novel candidate for

exploring future therapeutic modality for diabetic heart failure.

In conclusion, we demonstrate a novel, cardioprotective role of miR-133a in the diabetic

heart. We show that miR-133a protects the heart of streptozotocin-treated diabetic rats/mice by

directly targeting TAT and TAT-TH crosstalk. TH and TAT enzymes are critically involved in

the biosynthesis of NE, a key catecholamine stimulating the contractility of the heart muscle. Our

results also demonstrate that miR-133a mimic treatment decreased plasma NE levels. These

findings by elucidating the cardio-neuronal cross-talk may help to understand the molecular

mechanisms underlying diabetic and non-diabetic forms of heart failure.

Limitations

The role of miR-133a in the pathophysiology of diabetic hearts and impact of hyperglycemia on

miR-133a functions are poorly understood. Further, the results obtained from mice or rat data

may vary from humans with diabetic heart failure. Our results in the present study are focused on

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T1D model using streptozotocin treatment that needs to be further validated with other models of

T1D as well as T2D.

Acknowledgments

We would like to thank Dr. Scot Matkovich, Washington University, St. Louis, USA for giving

us cardiac specific miR-133a transgenic mice, Dr. Howard S. Fox, University of Nebraska

Medical Center for his generous gift of SH-SY5Y neuronal cell line, and Xuefei Liu from our

Department for his technical support.

Funding

This work was supported, in parts, by the National Institutes of Health grants HL-113281 and

HL-116205 to Paras K. Mishra.

Duality of Interest

No conflict of interest relevant to this article was reported.

Author Contributions

SSN designed the study, performed experiments, analyzed results, and contributed to discussion

and writing of the manuscript, HZ, NMS, HRS contributed to experiments and data analyses,

KPP contributed to discussion and correcting the manuscript draft, PKM conceptualized the idea,

supervised the project, and wrote the manuscript. PKM is the guarantor for this study and has full

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access to the data in the study and takes responsibility for the integrity of the data and accuracy

of the data analyses.

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professionals from the American Heart Association. Circulation 100:1134-1146, 1999

51. van,RE, Purcell,AL, Levin,AA: Developing microRNA therapeutics. Circ Res 110:496-

507, 2012

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Figures Legends

Figure 1. MiR-133a mimic treatment improves contractility of the diabetic heart. A,

Pressure-volume loop study on control, scrambled miRNA treated diabetic (DM+scm), and miR-

133a mimic treated diabetic (DM+miR) rats. A(i), Representative left ventricular pressure

recordings of hearts from three groups. A(ii), Bar graph showing the mean values of left

ventricular pressure with standard error. B(i), Representative recordings of rates of left

ventricular pressure changes over time (± dp/dt) in hearts from the three groups at the basal level

and after treatment with 0.1µg/kg isoproterenol (a β-AR agonist). B(ii), Bar graph showing the

mean value of dp/dt with standard error of mean in the three groups at the basal level and with

two increasing doses of isoproterenol. N, Sham=5, DM+scm=6, DM+miR-133a=5.

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Figure 2. MiR-133a mimic treatment upregulates β1-AR, β2-AR, and SERCA-2a in the

diabetic heart. RT- qPCR and Western blot analyses was performed to assess the levels of β1-

AR, β2-AR, and SERCA-2a in diabetic hearts treated with scrambled miRNA (DM+scm) and

miR-133a mimic (DM+miR). A, Bar graph represents relative fold change of β1-AR mRNA.

18sRNA was used as an internal control. B, Top, representative Western blot bands of β1-AR and

actin (a loading control); Bottom, bar graph represents densitometric quantification of band

intensity in fold change. C, Bar graph represents relative fold change of β2-AR mRNA. 18sRNA

was used as an internal control. D, Top, representative Western blot bands of β2-AR and actin

(loading control); Bottom, bar graph represents densitometric quantification of band intensity in

fold change. E, Top, representative Western blot bands of SERCA-2a and tubulin (a loading

control); Bottom, bar graph represents densitometric quantification of band intensity in fold

change. The values are mean±SEM. n=6.

Figure 3. MiR-133a mimic treatment decreases plasma norepinephrine (NE) and increases

cardiac NE in diabetic rats. A, ELISA based quantification of NE level from plasma samples

and B, from left ventricle tissue sample. The bar diagram shows NE level in ng/ml of plasma or

ng/mg of tissue. The values are mean±SEM. N, control=3, DM+scm=6, DM+miR=6. C,

Visualization of nerve profile in the cryosections of the heart by sucrose-phosphate-glyoxylic

acid (SPG) histofluorescence. C (i), Representative images showing histofluorescence of SPG in

15 µm transverse sections of left ventricle in different color channels. The arrow points to white

regions where catecholamine are stored in sympathetic nerve terminals. The merged green and

red channels on right panel validate the purity and source of intense signal from innervated

sympathetic neuron terminals (arrows) and the level of background autofluorescence. Scale bar:

100 µm. C (ii), Representative SPG histofluorescence images of cryosections of the heart from

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DM+scm and DM+miR groups. The intense white color (arrows) represents SPG

chemifluorescence of catecholamine from sympathetic neuron terminals, image are transformed

from RGB to gray scale for better clarity. C (iii), Bar diagram shows quantification of SPG

fluorescence intensity and represented as fold change. The values are mean ±SEM, n=3. Scale

bar: 200 µm.

Figure 4. MiR-133a mimic treatment increases tyrosine hydroxylase in diabetic hearts. A,

Evaluation of tyrosine hydroxylase (TH) mRNA in diabetic hearts treated with scrambled

miRNA (DM+scm) and miR-133a mimic (DM+miR) by RT-qPCR. 18sRNA was used as an

endogenous control. The mRNA expression is presented as fold change. The values are mean

±SEM, n=3. B, Western blot analysis of TH protein in the hearts from the above two groups.

Top, representative bands of TH and gapdh (a loading control); Bottom, bar graph shows

densitometric analyses of relative TH protein in the heart and represented as fold change. The

values are mean ±SEM, n=6. C, left, Schematic drawing shows the area of cryosections of the

heart and sympathetic innervations in the heart. Transverse section of the heart is observed for

sympathetic innervations. LV, Left Ventricle and RV, Right Ventricle. Right, Representative

diaminobenzidine (DAB) immunohistochemical staining of TH and counterstaining of

hematoxylin in the heart sections of the two groups. Scale bar: 200µm. D, MiR-133a mimic

treatment induces TH in CATH.a neuron cell. Undifferentiated CATH. a neuron cells were

transfected with miR-133a mimic plasmid for 24 hour, and the cellular level of miR-133a (green,

shown by an arrow) and TH (red, shown by an arrowhead) was observed in the neuronal cells.

DAPI (blue) stains nucleus. The miR-133a overexpressing cells have high expression of TH. E,

CATH.a cells were treated with either scrambled miRNA (scm), miR-133a mimic (miR), or

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36

antimiR-133a (anti-miR), and proteins were extracted after 24 hour. Top panel, representative

Western blot of TH and gapdh (control) from the three groups. The bottom panel shows

densitometry analyses of bands and represented as fold change. The values are mean ±SEM.

n=3.

Figure 5. TAT inhibits TH in neurons. CATH.a cells were transfected with TAT plasmid or

TAT siRNA for 24 hours and proteins were extracted from treated cells for evaluation of TH

level by Western blotting. A, Representative Western blot bands of TH in TAT knockdown (si-

TAT) and overexpressing (OE-TAT) cells. Gapdh is a loading control. B, Densitometric analyses

of TH level in the TAT knockdown neuronal cells. The bar graph shows relative upregulation of

TH after knockdown of TAT. C, Densitometric analyses of TH level in the TAT overexpressing

cells. The bar graph shows relative downregulation of TH after TAT overexpression. Values are

mean ± SEM. N=3.

Figure 6. MiR-133a mimic treatment decreases tyrosine hydroxylase in the diabetic heart.

RT-qPCR and Western blotting was performed for measuring TAT mRNA and protein levels,

respectively in the diabetic hearts treated with scrambled miRNA (DM+scm) and miR-133a

mimic (DM+miR). A, The bar graph shows TAT mRNA level and represented as fold change.

18sRNA is an endogenous control. The values are mean ±SEM, n=3. B, Top, representative

Western blot bands of TAT and actin (loading control). Bottom, densitometric analyses of the

bands of TAT and the relative expression of TAT is represented as fold change. The values are

mean ±SEM, n=3. C, left, Schematics showing the area of heart section used for staining of

TAT. LV, Left Ventricle and RV, Right Ventricle. Right, Representative diaminobenzidine

(DAB) immunohistochemical staining of TAT in the heart sections of DM+scm and DM+miR.

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Sections were counterstained with Hematoxylin. Scale bar: 200 µm. D, MiR-133a mimic

treatment inhibits TAT in CATH. a neuron cells. Undifferentiated CATH.a neuron cells were

transfected with miR-133a mimic plasmid for 24 hour, and stained for TAT. Representative

immunofluorescence showing the expression of miR-133a (green, shown by an arrow) and TAT

(red, shown by an arrowhead) in the neuronal cells. DAPI (blue) stains nuclei. The miR-133a

overexpressing cells have less expression of TAT. E, CATH.a cells were treated with either

scrambled miRNA (scm), miR-133a mimic (miR), or antimiR-133a (anti-miR), and proteins

were extracted after 24 hour. Top panel, representative Western blot of TAT and tubulin (a

control) from the three groups. The bottom panel shows densitometry analyses of bands and

represented as fold change. The values are mean ±SEM. n=4.

Figure 7. Cardiac specific overexpression of miR-133a prevents diabetes-mediated

downregulation of TH and upregulation of TAT in the mouse heart. WT and miR-133aTg

(TG) mice were treated with or without STZ to induce diabetes. In the heart, protein levels of

TH and TAT was determined by Western blotting. A (i), Representative Western blot bands for

TH, TAT and actin (a loading control). A (ii), Bar graph showing densitometric analyses of band

intensity for TH in the four groups. N, WT= 6, WT+STZ=7, miRTg+STZ=5; miRTg=6. A (iii),

Bar graph showing densitometric analyses of band intensity of TAT in the four groups. N, WT=

5, WT+STZ=5, miRTg+STZ=4; miRTg=3. The values are mean ±SEM. B, Representative

diaminobenzidine (DAB) immunohistochemical staining of TH (i) and TAT (ii), and

counterstaining of hematoxylin in the heart sections of the four groups. Scale bar: 200µm.

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Figure 8. Regulatory role of miR-133a in diabetic hearts. A-D. MiR-133a targets 3/UTR of

TAT. A, The binding sequence of miR-133a with TAT 3/UTR in rat. B, The plasmid clone of

TAT 3/UTR used for luciferase reporter assay. The mutant plasmid is identical except the miR-

133a binding site is deleted. C, The luciferase reporter assay results with TAT 3/UTR and D,

mutant TAT 3/UTR. The relative luciferase activity is measured in CATH.a cells treated with

3/UTR and increasing doses of miR-133a. The values are mean ±SEM. n=6. E. MiR-133a

regulates contractility by targeting TAT in diabetic hearts. Schematic showing that reduced

level of miR-133a upregulates tyrosine aminotransferase (TAT) in diabetic hearts. Elevated TAT

inhibits tyrosine hydroxylase (TH) that decreases cardiac norepinephrine (c-NE) level. It results

into inactivation of β1-AR and β2-AR that decreases contractility in diabetic hearts. On the other

hand, treatment with miR-133a mimic increases miR-133a level in the diabetic heart that

decreases TAT by binding to its 3/UTR. Decreased TAT increases the level of TH, which

induces c-NE biosynthesis. The elevated level of c-NE induces β-AR and improves the

contractility of diabetic hearts.

of 63Diabetes

0 2.0 1.5 1.0 0.5 0

20

40

60

80

100

Time (Sec)

Control

0 2.0 1.5 1.0 0.5 0

20

40

60

80

100

Time (Sec)

DM+scm

0 2.0 1.5 1.0 0.5 0

20

40

60

80

100

Time (Sec)

DM+miR

Le

ft V

en

tric

ula

r

Pre

ssu

re (

mm

Hg)

Le

ft V

en

tric

ula

r

Pre

ssu

re (

mm

Hg)

Le

ft V

en

tric

ula

r

Pre

ssu

re (

mm

Hg)

A (i)

Figure 1

60

80

100

120

Le

ft V

en

tric

ula

r P

ressu

re (

mm

Hg)

Sham DM+scm DM+miR

P=0.09

P=0.09

A (ii)

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Basal

-4000

-12000

-8000

-4000

0 4000

8000

12000

16000

1.5 1.0 0.5 0 2.0

± d

p/d

t (m

mH

g/s

ec)

± d

p/d

t (m

mH

g/s

ec)

-4000

-12000

-8000

-4000

0 4000

8000

12000

16000

1.5 1.0 0.5 0 2.0

-12000

-8000

-4000

0 4000

8000

12000

16000

1.5 1.0 0.5 0 2.0

Control

DM+scm

DM+miR

Time (Sec) Time (Sec)



-12000

-8000

-4000

0 4000

8000

12000

16000

1.5 1.0 0.5 0 2.0

-12000

-8000

0 4000

8000

12000

16000

1.5 1.0 0.5 0 2.0

-12000

-8000

0 4000

8000

12000

16000

1.5 1.0 0.5 0 2.0

± d

p/d

t (m

mH

g/s

ec)

Figure 1

B (i)

Isoproterenol 0.1ug/kg

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B (ii)

Figure 1

0

4000

8000

12000

16000

20000

Basal 0.05ug/kg 0.1ug/kg

dp

/dt (m

mH

g/s

ec)

P=0.02

P=0.002

P=0.2

P=0.03

Control

DM+scm

DM+miR

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A

β1-A

R:1

8S

(F

old

ch

an

ge

)

0

1

2

3

4

5P=0.03

DM+scm DM+miR

B

C

0

1

2

3

4

5

β2-A

R:1

8S

(F

old

ch

an

ge

) P=0.04

D

DM+scm DM+miR

E

Figure 2

β1-AR

Actin 43 kDa

65 kDa

β1-A

R:a

ctin

(F

old

ch

an

ge

) P=0.01

DM+scm DM+miR

0

1

2

3

0

1

2

3

β2-AR

Actin 43 kDa

65 kDa

β2-A

R:a

ctin

(F

old

ch

an

ge

) P=0.002

DM+scm DM+miR

SE

RC

A-2

a:t

ubulin

(F

old

ch

an

ge

)

DM+scm DM+miR

Tubulin

Serca2a 110 kDa

55 kDa

P=0.04

0

1

2

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A C(i)

B C(ii)

P=0.04

Sham DM+scm DM+miR 0

0.09

0.18

0.27

0.36

0.45

0.54

P=0.02

p-N

E (

ng/m

l)

0

0.09

0.18

0.27 P=0.01 P=0.02

Sham DM+scm DM+miR

c-N

E (

ng/m

g t

issue

)

Figure 3

DM+scm DM+miR

Le

ft v

en

tric

le

0

0.5

1

1.5

2

2.5

SP

G f

luo

rescence

(Fo

ld c

ha

nge

)

P=0.03

DM+scm DM+miR

C(iii)

Le

ft v

en

tric

le

Blue channel Blue+green channel Blue+red channel

of 63 Diabetes

A B

Le

ft v

en

tric

le

TH

200µm 200µm

C C

Sym

pa

the

tic inn

erv

atio

ns

Heart

RV

LV

Transverse Section

(i) (ii)

DM+scm DM+miR Figure 4

0

3

6

9

TH

:18

S (

Fo

ld c

ha

nge

)

DM+scm DM+miR

P=0.01

0

1

2

3

4

Gapdh

TH 60 kDa

37 kDa

TH

:ga

pd

h (

Fold

ch

an

ge

)

DM+scm DM+miR

P=0.01

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miR-133a TH miR-133a + TH + DAPI

25µm 25µm 25µm

D

E

Figure 4

Gapdh

TH 60 kDa

37kDa

scm miR anti-miR

P=0.02

P=0.02

TH

:ga

pd

h (

Fold

ch

an

ge

)

0

0.5

1

1.5

2

of 63 Diabetes

0

0.5

1

1.5

2

TH

:ga

pd

h (F

old

ch

an

ge

)

P=0.04

Negative si-TAT 0

0.2

0.4

0.6

0.8

1

1.2

TH

:ga

pd

h (

Fold

ch

an

ge

)

P=0.005

Negative OE-TAT Figure 5

B C

TH

Gapdh

60kDa

si-T

AT

Nega

tive

OE

-TA

T

37kDa

Nega

tive

si-T

AT

Nega

tive

si-T

AT

Nega

tive

OE

-TA

T

OE

-TA

T

Nega

tive

Nega

tive

Ma

rker

A of 63Diabetes

0

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1

Actin

TAT 50 kDa

43 kDa

A B

TAT

Le

ft v

en

tric

le

200µm 200µm

C C

Sym

pa

the

tic inn

erv

atio

ns

Heart

RV

LV

Transverse Section

(i) (ii)

TA

T:1

8S

(F

old

ch

an

ge

) P=0.04

DM+scm DM+miR

0

0.5

1

TA

T:a

ctin (

Fold

ch

an

ge

)

P=0.03

DM+scm DM+miR

DM+scm DM+miR Figure 6

of 63 Diabetes

miR-133a TAT miR-133a + TAT + DAPI

25µm 25µm 25µm

D

E

Figure 6

Tubulin

TAT 50 kDa

55 kDa

scm miR anti-miR

P=0.004

P=0.00003

TA

T:t

ubu

lin (

Fold

ch

an

ge

)

0

0.5

1

1.5

of 63Diabetes

0

0.5

1

1.5

2

0

0.5

1

1.5

2

Figure 7

A

60 kDa TH

WT WT+STZ TG+STZ TG

TAT

43 kDa

50 kDa

Actin

TA

T: a

ctin

(F

old

ch

an

ge

) P=0.002 P=0.038 T

H: a

ctin

(F

old

ch

an

ge

)

P= 0.015

P= 0.009

WT WT miRTg miRTg

+STZ +STZ

WT WT miRTg miRTg

+STZ +STZ

of 63 Diabetes

Figure 7

WT+Vehicle WT+STZ

miRTg+Vehicle miRTg+STZ

200µm

WT+Vehicle WT+STZ

miRTg+Vehicle miRTg+STZ

200µm

B (i)

B (ii)

TAT

TH

of 63Diabetes

TAT 3/UTR 237: 5' auccugaggguaucaGACCAAu 3'

| | | | | |

rno-miR-133a 3' gucgaccaacuucccCUGGUUu 5' Rat TAT

A

0

0.2

0.4

0.6

0.8

1

1.2

miR

scm

P=0.001

Ga

ussia

:renill

a lucife

rase

activity

(Fo

ld c

ha

nge

)

miR-133a : 0μg 1μgg 2μg

TAT 3/-UTR : 1µg 1µg 1µg

TAT 3/UTR hLuc hRLuc SV40 CMV

miR-133a binding sites

TAT 3/UTR-pEZX clone

Figure 8

B

C

Ga

ussia

:renill

a lu

cife

rase

activity

(Fo

ld c

ha

nge

)

miR-133a : 0μg 1μgg 2μg

Mutant-TAT 3/-UTR : 1µg 1µg 1µg

0

0.2

0.4

0.6

0.8

1

1.2

D

of 63 Diabetes

Figure 8E

Contractility Diabetic heart

β1 & β2-ARs

MiR-133a Tyrosine

TAT

Homogentisate

Pathway

MiR-133a Tyrosine

TAT

Homogentisate

Pathway

miR-133a mimic-treated

diabetic heart

TH

c-NE

TH

c-NE

of 63Diabetes

1

SUPPLEMENTAL MATERIAL

MiR-133a regulates contractility of diabetic hearts: a novel role for cross-talk between

tyrosine aminotransferase and tyrosine hydroxylase

Shyam Sundar Nandi1, Hong Zheng

1, Neeru M. Sharma

1, Hamid R. Shahshahan

1, Kaushik P.

Patel1

and Paras K. Mishra1, 2

Affiliations:

1. Department of Cellular and Integrative Physiology, University of Nebraska Medical

Center, 985850 Nebraska Medical Center, Omaha, NE 68198, USA

2. Department of Anesthesiology, University of Nebraska Medical Center, 985850

Nebraska Medical Center, Omaha, NE 68198, USA.

Running title: MiR-133a regulates cardiac contractility

Corresponding author:

Paras Kumar Mishra

Department of Cellular and Integrative Physiology

University of Nebraska Medical Center

985850 Nebraska Medical Center

Omaha, NE-68198, USA

Phone: 402-559-8524

Fax: 402-559-4438

Email: [email protected]

of 63 Diabetes

2

SUPPLEMENTARY FIGURE LEGENDS

Supplementary Figure 1. Schematic illustration of the treatment protocol. Eight week

Sprague-Dawley male rats were treated with streptozotocin and after four weeks blood glucose

level was measured. Rats with more than 350 mg/dL glucose level were considered diabetic and

used for lentivirus treatment. Both miR-133a and scrambled plasmids were GFP tagged and were

packaged into lentivirus, and 10^6 lentivirus particles were injected into diabetic rats through tail

vein. After two weeks, physiological data were acquired and rats were sacrificed for tissue

collection.

Supplementary Figure 2. Treatment with miR-133a mimic upregulated cardiac miR-133a

in diabetic hearts. Lentivirus containing miR-133a mimic or scrambled miRNA (GFP-tagged)

were injected into diabetic rat through tail vein and the levels of cardiac miR-133a was

determined. A, Individual miR-133a assay was performed and relative expression of miR-133a

was measured by qRT-PCR in scrambled miRNA (DM+scm)-, and miR-133a mimic

(DM+miR)- treated diabetic hearts. U6 was used as an endogenous control. The values are

expressed as mean ±SEM, n=3. B, Immunohistochemistry was performed on cryosections of the

heart from DM+miR group. The expression of miR-133a (red, anti-GFP) was co-localized with

cardiomyocyte marker α-actinin (green). The merged images show the expression of miR-133a,

α-actinin and dapi (blue, nuclear stain). C, Anti-GFP-HRP antibody was used to stain the

cryosections from “B” group. The brown color in the left panel show the expression of miR-

133a. D, Genotyping for miR-133Tg mice was performed. MiR-133aTg showed a band at

670kb, which was absent in C57BL/6J (WT control) mice. E, Individual miR-133a assay was

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3

performed to validate that cardiac levels of miR-133a was increased in miR-133aTg mice. U6

was an endogenous control.

Supplementary Figure 3. MiR-133a mimic treatment decreases cardiac hypertrophy in

diabetic hearts. A, Western blot analyses of beta-myosin heavy chain (β-MHC) protein in the

heart tissue obtained from scrambled miRNA (DM+scm)-, and miR-133a mimic (DM+miR)-

treated diabetic hearts. Top, representative bands of β-MHC and tubulin (loading control);

Bottom, bar graph showing densitometric quantification of band intensity, which is represented

as fold change. The values are mean ±SEM, n=3. B, Wheat Germ Agglutinin staining of

histological cryosections the heart from the above two groups of the diabetic rats. Green color

demarcates cell boundaries and asterisks denote typical cell sizes. Scale bars: 100µm. C,

Quantification of cell size per unit cross sectional area of left ventricle. Bar graph represents cell

size per unit area in fold change, values are mean ±SEM, n=3.

Supplementary Figure 4. Tyrosine hydroxylase and tyrosine amino transferase are present

in the neuronal terminals of the diabetic heart. Validation of presence of neuronal tyrosine

hydroxylase (TH) in diabetic hearts and in CATH.a neuronal cell line. A, Histological sections of

diabetic hearts treated with miR-133a mimic (DM+miR) were stained for neuronal marker,

microtubule associated protein-2 (MAP2, green) and TH (red). The merged image were

magnified 400X to show that TH is localized in neuronal terminals in the diabetic heart. Scale

bar: 100µm. B, Anti-HRP antibody was used for staining TH and TAT in the cryosections of the

heart. C, Validation of presence of miR-133a in neuronal cells. Individual miR-133a assay was

performed on two different neuronal cells: CATH.a (mouse origin) and SH-SY5Y (human

of 63 Diabetes

4

origin) to validate that neuronal cells express miR-133a. U6 was an endogenous control. D,

Expression of TH in CATH.a neuronal cells. The neuronal cells were stained with anti-TH

antibody (red) and dapi (blue), and observed under a fluorescent microscope. Scale bar: 25µm.

E, Expression of TAT in CATH.a neuronal cells. The neuronal cells were stained with anti-TAT

antibody (green) and dapi (blue) and observed under a fluorescent microscope. F, Validation of

TAT siRNA in CATH.a cells. G, Validation of TAT overexpression in CATH.a cells.

Supplementary Figure 5. Tyrosine aminotransferase (TAT) is a predicted target for miR-133a.

A, The binding site for miR-133a on 3ÚTR of TAT in rat. B, The binding site for miR-133a on

3ÚTR of TAT in mouse. C, Conserved binding sequences and sites of miR-133a on 3ÚTR of

TAT in rat and mouse (sequence highlighted by colored box in “A” and “B”) and other species.

The highlighted color represents conserved nucleotides among different species.

of 63Diabetes

Streptozotocin (STZ)

injection

Sprague-Dawley rat

Blood glucose measurement

(>350mg/dL)

Sham Diabetic

+miR-133a

Diabetic

+scrambled

Treatments

miR-133a mimic or scrambled

injection by tail vein

(106 lentivirus/one time)

8 weeks 12th weeks 14th weeks

Diabetic

±dP/dt measurement,

sacrifice, and tissue

collection

Groups

Supplementary Figure 1A

Steptozotocin (STZ)

injection (65mg/Kg BW)

cardiac specific

miR-133a transgenic or

C57BL/6J mice

Blood glucose measurement

(>300mg/dL)

WT+Veh. miR-133aTg

Diabetic miR-133aTg+Veh.

8 weeks 10th week 14th week

Diabetic

±dP/dt measurement,

sacrifice, and tissue

collection

Groups

Treatments

0

Supplementary Figure 1B

of 63 Diabetes

Anti-α-actinin-488 Anti-GFP-594 Merged+DAPI

DM

+m

iR

B

50µm DM+scm DM+miR

miR

-13

3a:U

6 s

nR

NA

(Fold

ch

an

ge

)

A

P=0.001

0

1

2

3

4

Supplementary Figure 2

C Anti-GFP-HRP Negative control

100kb

200kb

300kb

400kb

500kb

850kb

miR-Tg WT

670 bp

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

WT miR-133aTg

P=0.008

miR

-13

3a:U

6S

nR

NA

(F

old

ch

an

ge

)

3 4

D E

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Ca

rdio

myo

cyte

siz

e/u

nit a

rea

(F

old

ch

an

ge

)

P=0.005

* * *

*

DM+scm DM+miR

Le

ft v

en

tric

le

WGA staining B A

DM+scm DM+miR

β-M

HC

: tu

bu

lin (

Fo

ld c

ha

nge

)

P=0.002

0

0.5

1

DM+scm DM+miR

C


223 KDa

Tubulin

β-MHC

55 KDa

DM+scm DM+miR

0

0.4

0.8

1.2

of 63 Diabetes

Anti-TH-594

CA

TH

.a C

ell

25µm


C

CA

TH

.a C

ell

Anti-TAT-488

A

B Anti-TH Anti-TAT

DM

+m

iR

D E

0

0.05

0.1

0.15

0.2

0.25

0.3

CATH.a SH-SY5Y

miR

-13

3a:U

6S

nR

NA

(Re

lative

qu

an

tity

)

DM

+m

iR

Anti-MAP2-488+Anti-TH-594

100µm

400X

of 63Diabetes

Actin

TAT 50 kDa

43 kDa

Si-TAT - +

0

0.2

0.4

0.6

0.8

1

1.2

Control siRNA

P=0.003

TA

T: a

ctin

(F

old

ch

an

ge

)

F G


Actin

TAT 50 kDa

43 kDa

OE-TAT - +

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Control OverEx

P=0.047

TA

T: a

ctin

(F

old

ch

an

ge

)

of 63 Diabetes

TAT 3/UTR 237: 5' auccugaggguaucaGACCAAu 3'

| | | | | |

rno-miR-133a 3' gucgaccaacuucccCUGGUUu 5' Rat TAT

TAT 3/UTR 290: 5' ugagGGUACCAGUUUACCAGa 3'

| | | | | | | | | | | | | |

mmu-miR-133a* 3' uaaaCCAAGGUAAAAUGGUCg 5' Mouse TAT

A

B

TA

T 3

' UT

R

C SITE1 SITE3 SITE2


of 63Diabetes

SUPPLEMENTAL TABLE

Table 1. The forward and reverse primers sequences used for RT-PCR and qPCR.

Gene Forward sequences (5′–3′) Reverse sequences (5′–3′)

TAT TACAGACCCTGAAGTTACCC CCTTGGAATGAGGATGTTT

TH CTTGTCTCGGGCTGTAAA CACTTTTCTTGGGAACCA

β1-AR GCCGATCTGGTCATGGGA GTTGTAGCAGCGGCGCG

β2-AR ACCTCCTCCTTGCCTATCCA TAGGTTTTCGAAGAAGACCG

18s GATACCGCAGCTAGGAATAA ATCGTTTATGGTCGGAACTA

of 63 Diabetes

page 1 of 63 diabetes...jul 12, 2016 · 2 abstract micrornas have a fundamental role in diabetic...

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