Transcriptome

analysis

of

the

response

to

chronic

constant

hypoxia

in

zebrafish

hearts

Insufficient

blood

supply

during

acute

infarction

and

chronic

ischemia

leads

to

tissue

hypoxia

which

can

significantly

alter

gene

expression

patterns

in

the

heart

In

contrast

to

most

mammals

some

teleost

fishes

are

able

to

adapt

to

extremely

low

oxygen

levels

We

describe

here

that

chronic

constant

hypoxia

-LRB-

CCH

-RRB-

leads

to

a

smaller

ventricular

outflow

tract

reduced

lacunae

within

the

central

ventricular

cavity

and

around

the

trabeculae

and

an

increase

in

the

number

of

cardiac

myocyte

nuclei

per

area

in

the

hearts

of

two

teleost

species

zebrafish

-LRB-

Danio

rerio

-RRB-

and

cichlids

-LRB-

Haplochromis

piceatus

-RRB-

In

order

to

identify

the

molecular

basis

for

the

adaptations

to

CCH

we

profiled

the

gene

expression

changes

in

the

hearts

of

adult

zebrafish

We

have

analyzed

over

different

transcripts

and

found

differentially

regulated

genes

of

which

genes

showed

increased

and

genes

decreased

expression

levels

Two

notch

receptors

-LRB-

and

-RRB-

as

well

as

regulatory

genes

linked

to

cell

proliferation

were

transcriptionally

upregulated

in

hypoxic

hearts

We

observed

a

simultaneous

increase

in

expression

of

and

and

upregulation

of

several

genes

important

for

the

protection

against

reactive

oxygen

species

-LRB-

ROS

-RRB-

We

have

identified

here

many

novel

genes

involved

in

the

response

to

CCH

in

the

heart

which

may

have

potential

clinical

implications

in

the

future

Introduction

Low

oxygen

levels

-LRB-

hypoxia

-RRB-

play

important

roles

in

clinical

conditions

such

as

stroke

and

heart

failure

Insufficient

blood

supply

leads

to

tissue

hypoxia

in

the

heart

during

acute

infarction

and

chronic

ischemia

-LRB-

Semenza

-RRB-

Effective

protection

of

the

heart

against

ischemia/reperfusion

injury

is

one

of

the

most

important

goals

of

experimental

and

clinical

research

in

cardiology

Besides

ischemic

preconditioning

as

a

powerful

temporal

protective

phenomenon

adaptation

to

chronic

hypoxia

also

increases

cardiac

tolerance

to

all

major

deleterious

consequences

of

acute

oxygen

deprivation

such

as

myocardial

infarction

contractile

dysfunction

and

ventricular

arrhythmias

-LRB-

Kolar

and

Ostadal

-RRB-

Although

many

factors

have

been

proposed

to

play

potential

roles

the

detailed

mechanism

of

this

long-term

protection

remains

poorly

understood

Some

of

the

molecular

mechanisms

of

cardiac

protection

by

adaptation

to

chronic

hypoxia

and

chronic

high-altitude

hypoxia

have

recently

been

reviewed

-LRB-

Kolar

and

Ostadal

Ostadal

and

Kolar

-RRB-

KATP

channels

PKCδ

as

well

as

the

different

MAPK

pathways

were

shown

to

be

involved

in

the

mechanism

of

increased

tolerance

of

chronically

hypoxic

hearts

and

further

the

controversial

role

of

ROS

in

hypoxia

tolerance

is

discussed

-LRB-

Kolar

and

Ostadal

-RRB-

Furthermore

a

recent

study

has

profiled

the

gene

expression

changes

induced

by

chronic

constant

hypoxia

-LRB-

CCH

-RRB-

and

chronic

intermittent

hypoxia

-LRB-

CIH

-RRB-

in

newborn

mice

-LRB-

Fan

et

al.

-RRB-

In

contrast

to

most

mammals

-LRB-

with

the

exception

of

some

marine

mammals

-RRB-

some

teleosts

have

developed

the

ability

to

withstand

extreme

chronic

hypoxia

-LRB-

Stecyk

et

al.

-RRB-

It

is

well

assumed

that

these

vertebrate

species

possess

unique

adaptations

in

order

to

survive

short

and

long

term

oxygen

deprivation

However

the

molecular

basis

of

these

adaptations

in

fish

has

so

far

not

been

extensively

investigated

Several

studies

have

profiled

gene

expression

changes

in

teleosts

exposed

to

hypoxia

Gracey

et

al.

showed

in

adults

of

the

euryoxic

gobiid

fish

Gillichthys

mirabilis

-LRB-

the

long-jawed

mudsucker

-RRB-

that

days

of

hypoxia

induced

a

complex

transcriptional

response

including

a

shut

down

of

energy

requiring

pathways

like

protein

synthesis

and

locomotion

and

an

induction

of

genes

needed

for

anaerobic

ATP

production

in

different

tissues

-LRB-

Gracey

et

al.

-RRB-

Recently

we

described

phenotypic

and

behavioral

adaptations

to

long-term

hypoxia

and

described

the

gene

expression

changes

induced

by

chronic

constant

hypoxia

in

the

gills

of

adult

zebrafish

-LRB-

van

der

Meer

et

al.

-RRB-

Ton

et

al.

identified

global

gene

expression

changes

in

zebrafish

embryos

Zebrafish

embryos

at

h

post

fertilization

were

exposed

to

water

with

oxygen

content

for

h

The

authors

identified

genes

responsive

to

short-term

hypoxia

and

could

also

show

that

transcriptional

changes

indicated

metabolic

depression

a

switch

from

aerobic

to

anaerobic

metabolism

and

energy

conservation

-LRB-

Ton

et

al.

-RRB-

In

this

study

we

have

identified

CCH-induced

gene

expression

changes

in

the

zebrafish

heart

by

looking

at

over

half

of

the

zebrafish

genome

We

have

compared

several

of

these

novel

changes

described

in

other

species

and

tissues

We

have

here

identified

the

heart-specific

molecular

adaptations

to

CCH

Future

functional

experiments

are

warranted

to

determine

whether

some

of

the

findings

can

be

used

to

better

adapt

mammalian

hearts

to

CCH

Material

and

methods

Animal

handling

Adult

wild-type

zebrafish

-LRB-

Danio

rerio

-RRB-

around

month

of

age

were

obtained

from

a

local

pet

store

Cichlids

-LRB-

Haplochromis

piceatus

-RRB-

have

been

collected

in

the

Mwanza

Gulf

of

Lake

Victoria

in

and

were

bred

in

our

laboratory

for

about

generations

All

animals

were

handled

in

compliance

with

animal

care

regulations

Our

animal

protocols

were

approved

by

the

review

board

of

Leiden

University

in

accordance

with

the

requirements

of

the

Dutch

government

Zebrafish

were

kept

at

°C

in

aquaria

with

day/night

light

cycles

-LRB-

h

dark

vs.

h

light

-RRB-

Cichlids

were

kept

at

°C

with

the

same

day/night

light

cycles

Hypoxia

treatment

For

gradual

hypoxia

treatment

oxygen

levels

were

gradually

decreased

in

days

from

air

saturated

water

to

-LRB-

day

-RRB-

-LRB-

day

-RRB-

-LRB-

day

-RRB-

and

the

final

air

saturation

-LRB-

day

-RRB-

After

day

the

fish

were

kept

for

an

additional

days

at

air

saturation

-LRB-

at

air

saturation

and

°C

the

concentration

is

mg/l

and

is

Torr

-RRB-

In

parallel

a

control

group

was

kept

at

air

saturated

water

Both

groups

were

kept

in

identical

aquaria

of

l

The

oxygen

level

on

the

hypoxia

group

was

kept

constant

by

a

controller

-LRB-

Applikon

Biotechnology

The

Netherlands

-RRB-

connected

to

an

and

solenoid

valve

in

line

with

an

air

diffuser

The

oxygen

level

in

the

tank

was

kept

constant

by

adding

oxygen

via

the

diffuser

and

thereby

compensating

the

oxygen

consumption

of

the

fish

In

case

of

immediate

hypoxia

exposure

tanks

were

pre-equilibrated

to

the

respective

concentration

and

fish

were

then

directly

set

in

the

equilibrated

aquaria

Perfusion

of

cichlid

hearts

In

order

to

minimize

blood

clotting

a

perfusion

protocol

was

developed

in

which

the

whole

blood

volume

of

clinically

dead

animals

was

initially

replaced

with

isotonic

buffer

and

with

a

fixative

solution

secondarily

Heart

dissection

The

fish

were

killed

with

an

overdose

of

anesthetic

-LRB-

Tricaine

Methanesulfonate

from

Argent

Chemical

Laboratories

USA

-RRB-

Hearts

were

dissected

from

the

fish

immediately

after

the

anesthetic

worked

For

RNA

preparation

the

hearts

were

immediately

shock-freezed

in

liquid

nitrogen

For

histology

and

microscopy

the

hearts

were

left

intact

and

fixed

immediately

in

Karnovsky

fixative

-LRB-

paraformaldehyde

-LRB-

PFA

-RRB-

and

glutaraldehyde

in

M

phosphate

buffer

pH

-RRB-

for

h

at

°C

After

three

washes

in

M

phosphate

buffer

pH

they

were

transferred

to

ethanol

Histology

of

adult

fish

hearts

statistical

analysis

and

scanning

electron

microscopy

Hearts

from

zebrafish

and

cichlids

raised

either

under

normoxic

or

hypoxic

conditions

were

dissected

from

the

fish

and

fixed

for

h

in

PFA

in

PBS

After

fixation

hearts

were

washed

with

PBS

cut

in

halfs

-LRB-

sagittal

through

the

midline

-RRB-

and

then

dehydrated

through

ethanol

series

starting

at

ethanol

followed

by

and

ethanol

each

step

was

done

once

for

h

except

the

last

one

which

was

done

twice

After

dehydration

the

hearts

were

embedded

in

increasing

gradients

of

Historesin

-LRB-

Technovit

Heraus

Kulzer

Germany

-RRB-

-LRB-

and

Historesin

in

ethanol

for

h

at

room

temperature

Historesin

h

at

°C

-RRB-

Afterwards

the

plastic

with

the

hearts

was

polymerized

at

°C

-LRB-

overnight

-RRB-

A

μm

sagittal

sections

were

made

using

the

Ultramicrotome

-LRB-

Reichter-Jung

-RRB-

and

a

glass

knife

Approximately

sections

on

each

side

of

the

midline

were

visually

analyzed

per

heart

-LRB-

sections

in

total

per

heart

-RRB-

Sections

were

left

to

dry

and

later

stained

with

hematoxylin

eosin

staining

Pictures

were

taken

with

Axioplan

imaging

-LRB-

Carl

Zeiss

Jena

-RRB-

Azan

staining

of

histological

sections

were

done

as

follows

Paraffin

sections

of

the

hearts

were

prepared

as

described

before

-LRB-

van

der

Meer

et

al.

-RRB-

Sections

were

incubated

with

Azokarmine

solution

for

min

at

°C

Afterwards

they

were

washed

in

water

and

differentiated

in

Anilin

alcohol

They

were

then

rinsed

in

acetic

acid

in

alcohol

followed

by

min

incubation

period

in

phosphotungstic

acid

After

that

sections

were

rinsed

in

distilled

water

and

incubated

for

min

with

aniline

blue

Finally

they

were

rinsed

with

distilled

water

differentiated

and

dehydrated

in

alcohol

followed

by

absolute

cleared

in

xylene

and

mounted

in

Entallan

The

protocols

used

here

for

SEM

had

been

described

before

-LRB-

van

der

Meer

et

al.

-RRB-

The

statistical

analysis

of

cardiac

myocyte

nuclei

per

section

was

done

using

Statistica

by

performing

an

independent

t

test

A

P

value

of

less

than

was

considered

significant

Immunohistochemistry

and

statistical

analysis

Zebrafish

raised

either

under

normoxic

or

hypoxic

conditions

were

killed

with

an

overdose

of

anesthetic

and

frozen

in

liquid

nitrogen

Subsequently

transversal

cross-sections

-LRB-

μm

thick

-RRB-

of

the

body

were

cut

using

a

cryostat

at

°C

and

mounted

on

glass

slides

coated

with

Vectabond

-LRB-

Vector

Laboratories

Burlingame

USA

-RRB-

Sections

were

fixed

in

formaldehyde

in

Tris-buffered

saline

-LRB-

TBS

mM

Tris

and

mM

NaCl

pH

-RRB-

for

min

and

subsequently

washed

in

TBS

with

-LRB-

TBST

-RRB-

-LRB-

Sigma-Aldrich

Zwijndrecht

The

Netherlands

-RRB-

Subsequently

sections

were

incubated

for

min

with

normal

swine

serum

-LRB-

Vector

laboratories

-RRB-

in

TBST

after

which

sections

were

incubated

for

h

at

°C

with

anti-phospho

Akt

polyclonal

antibody

-LRB-

Santa-Cruz

Biotechnology

USA

-RRB-

diluted

in

TBST

After

incubation

with

primary

antibody

the

slides

were

washed

in

TBST

and

subsequently

placed

in

-LRB-

v/v

-RRB-

acetic

anhydride

in

M

triethanolamine

buffer

-LRB-

pH

-RRB-

for

min

followed

by

rinsing

in

TBST

After

this

sections

were

incubated

for

min

at

°C

with

secondary

anti-rabbit

immunoglobulin

G

-LRB-

IgG

-RRB-

antibody

covalently

coupled

to

alkaline

phosphatase

-LRB-

Vector

Laboaratories

-RRB-

diluted

and

washed

in

TBST

After

this

sections

were

incubated

for

min

with

alkaline

phosphatase

-LRB-

AP

-RRB-

buffer

-LRB-

M

NaCl

M

Tris

mM

and

pH

-RRB-

followed

by

incubation

with

BM

Purple

AP

substrate

-LRB-

Roche

Applied

Sciences

Almere

The

Netherlands

-RRB-

for

or

min

which

was

followed

by

rinsing

in

TBST

All

sections

were

mounted

in

glycerine-gelatin

and

stored

at

°C

in

the

dark

until

staining

intensity

was

measured

The

absorbance

values

of

the

BM

Purple

in

the

sections

were

determined

using

a

Leica

DMRB

microscope

-LRB-

Wetzlar

Germany

-RRB-

fitted

with

calibrated

gray

filters

using

different

interference

filters

Absorbances

for

BM

Purple

were

determined

at

nm

Images

were

recorded

with

a

objective

and

a

Sony

camera

-LRB-

Towada

Japan

-RRB-

connected

to

a

frame

grabber

-LRB-

Scion

Frederick

MD

-RRB-

in

an

Apple

Power

Macintosh

computer

Recorded

images

were

analysed

with

the

public

domain

program

NIH-Image

-LRB-

US

National

Institutes

of

Health

available

at

-RRB-

Gray

values

were

converted

to

absorbance

values

per

pixel

using

the

gray

filters

and

a

third-degree

polynomial

fit

in

the

calibrate

option

of

NIH-image

programme

Morphometry

was

calibrated

using

a

slide

micrometer

and

the

set

scale

option

in

NIH-image

taking

the

pixel-aspect

ration

into

account

An

independent

t

test

was

used

to

test

for

differences

in

phospho-Akt

levels

in

cardiac

myocytes

of

normoxic

and

hypoxic

fish

A

P

value

of

less

than

was

considered

significant

Values

are

means

S.E.M.

RNA

preparation

and

biological

sampling

After

dissection

hearts

were

homogenized

in

a

Dounce

homogenizer

using

ml

Trizol

solution

-LRB-

GibcoBrl

Life

technologies

-RRB-

The

whole

heart

was

used

and

for

each

biological

sample

hearts

were

pooled

from

five

different

animals

After

Trizol

extraction

total

RNA

was

further

purified

using

RNAeasy

columns

-LRB-

Qiagen

-RRB-

RNA

samples

were

analyzed

for

quality

control

by

Lab-on-a-chip

analysis

-LRB-

Agilent

-RRB-

and

on

agarose

gels

For

the

array

experiment

five

arrays

were

done

for

normoxic

and

arrays

for

the

hypoxic

condition

Biological

samples

-LRB-

BS

-RRB-

came

from

two

independent

experiments

and

one

technical

replicate

-LRB-

TR

-RRB-

was

included

-LRB-

TR

for

normoxia

and

TR

for

hypoxia

-RRB-

As

mentioned

above

for

each

BS

hearts

from

five

different

animals

were

pooled

Microarray

analysis

The

Affymetrix

GeneChip

Zebrafish

Genome

Arrays

containing

Danio

rerio

gene

transcripts

were

used

Probe

sets

on

the

arrays

were

designed

with

oligonucleotide

pairs

to

detect

each

transcript

and

procedures

were

in

full

support

of

MIAME

standards

Labeling

and

microarray

hybridization

were

performed

by

ServiceXS

-LRB-

Leiden

The

Netherlands

-RRB-

including

prior

a

standard

round

of

RNA

amplification

according

to

standard

Affymetrix

protocols

The

criteria

used

for

differential

expression

were

greater

or

equal

than

induced

or

reduced

and

P

Data

analysis

was

done

using

Rosetta

Resolver

All

expression

data

was

submitted

to

the

NCBI

Gene

Expression

Omnibus

-LRB-

-RRB-

The

series

entry

number

is

and

the

following

accession

numbers

were

assigned

and

A

complete

list

of

differential

regulated

genes

is

shown

in

Supplemental

Table

Table

analysis

of

histological

sections

-LRB-

μm

-RRB-

from

zebrafish

-LRB-

Danio

rerio

-RRB-

and

cichlid

-LRB-

Haplochromis

piceatus

-RRB-

heartsNumber

of

cardiomyocyte

nuclei

per

sectionZebrafish

-LRB-

nuclei

per

-RRB-

Cichlid

-LRB-

nuclei

per

-RRB-

P

sections

of

zebrafish

and

cichlids

raised

under

normoxic

and

hypoxic

conditions

were

chosen

and

subdivided

in

smaller

areas

In

the

case

of

D.

rerio

each

subarea

of

the

section

was

whereas

in

the

case

of

the

H.

piceatus

it

was

Then

subareas

were

randomly

picked

and

the

amount

of

cardiac

myocyte

nuclei

were

counted

The

vast

majority

of

cardiac

myocytes

were

mononucleated

cells

Hundred

subareas

per

section

were

counted

per

specimen

Three

sections

per

heart

were

visually

analyzed

-LRB-

with

a

magnification

of

-RRB-

In

total

different

zebrafish

hearts

-LRB-

from

three

independent

experiments

-RRB-

were

investigated

for

hypoxic

conditions

and

for

normoxic

condition

In

total

subareas

were

quantified

per

heart

and

condition

used

The

same

was

done

for

the

cichlids

-LRB-

for

which

bigger

subareas

were

counted

-RRB-

A

two-tailed

t

test

was

applied

and

a

significant

difference

-LRB-

P

-RRB-

in

the

amount

of

nuclei

present

in

the

hearts

of

normoxia

versus

hypoxia

groups

was

observed

for

both

species

Gene

ontology

analysis

The

Gene

ontology

analysis

was

performed

using

eGOn

and

the

database

of

the

Norwegian

Microarray

Consortium

-LRB-

-RRB-

and

the

most

recent

updated

Unigene

numbers

for

zebrafish

-LRB-

May

-RRB-

Unigene

numbers

were

annotated

to

the

Affymetrix

gene

chip

used

eGOn

Enrichment

analysis

was

used

for

the

entire

Affymetrix

set

versus

the

identified

gene

set

based

on

the

new

Unigene

numbers

A

master

target

test

was

performed

-LRB-

using

Fishers

exact

test

-RRB-

and

the

cut

off

for

positives

was

set

to

P

The

available

three

categories

molecular

function

biological

process

and

cellular

component

were

all

tested

and

are

shown

in

this

dataset

-LRB-

Supplemental

Data

-RRB-

The

fold

enrichment

was

based

on

the

percentage

found

within

the

differentially

expressed

genes

divided

by

the

percentage

on

the

total

chip

A

list

of

all

Unigene

numbers

found

in

the

different

categories

is

given

in

Supplemental

Data

Real-time

quantitative

RT-PCR

For

verification

of

gene

expression

data

we

used

quantitative

real-time

RT-PCR

Biological

RNA

samples

were

obtained

from

a

third

independent

experiment

and

as

mentioned

above

for

the

microarrays

for

each

BS

hearts

from

five

different

animals

were

pooled

The

Roche

Master

SYBR

Green

kit

was

used

for

the

RT-PCR

reactions

The

annealing

and

synthesis

temperature

was

°C

alternating

with

°C

for

cycles

Dissociation

protocols

were

used

to

measure

melting

curves

and

control

for

unspecific

signals

from

the

primers

A

measure

of

ng

of

total

RNA

was

used

per

reaction

A

standard

curve

for

β-actin

using

and

ng

of

total

RNA

was

used

for

normalization

Samples

were

measured

in

the

Roche

LightCycler

The

software

-LRB-

-RRB-

was

used

to

design

primers

for

short

amplicons

between

and

bases

The

primers

used

are

shown

in

Supplemental

Table

Results

We

describe

here

the

survival

rates

of

adult

zebrafish

upon

immediate

and

gradual

exposure

to

different

concentrations

Immediate

exposure

to

of

Torr

-LRB-

concentration

of

mg/l

or

air

saturated

water

-RRB-

was

lethal

for

the

adult

zebrafish

and

survived

for

longer

than

h

-LRB-

Fig.

-RRB-

If

lowered

gradually

-LRB-

see

experimental

procedures

for

regimen

in

Sect

Hypoxia

treatment

-RRB-

zebrafish

were

able

to

grow

and

gain

weight

at

levels

of

air

saturation

The

zebrafish

were

able

to

survive

for

longer

than

months

and

no

mortality

was

observed

demonstrating

that

they

can

well

adapt

to

these

conditions

-LRB-

data

not

shown

-RRB-

For

the

experiments

with

zebrafish

and

cichlids

the

concentration

was

gradually

lowered

to

air

saturation

-LRB-

mg/l

-RRB-

-LRB-

see

Material

and

methods

-RRB-

and

fishes

were

kept

under

air

saturation

for

weeks

Control

groups

were

always

kept

in

parallel

under

normoxic

conditions

Fig.

of

zebrafish

embryos

after

immediate

exposure

to

hypoxia

Zebrafish

were

immediately

exposed

to

hypoxia

-LRB-

Torr

mg/ml

air

saturated

water

-RRB-

Results

are

derived

from

three

independent

experiments

with

n

in

each

experiment

-LRB-

adding

to

a

total

of

fish

tested

-RRB-

After

and

h

dead

and

alive

fish

were

counted

Shown

here

is

the

percentage

of

dead

fish

at

the

respective

time

points

and

the

standard

deviation

At

h

-LRB-

-RRB-

of

fish

were

dead

at

hours

-LRB-

-RRB-

and

at

hours

-LRB-

-RRB-

Control

groups

-LRB-

n

-RRB-

which

were

in

parallel

exposed

to

normoxic

conditions

showed

no

mortality

-LRB-

not

shown

-RRB-

of

the

zebrafish

survived

the

immediate

exposure

to

an

concentration

of

mg/l

-LRB-

air

saturation

-RRB-

over

the

period

In

contrast

fishes

gradually

exposed

to

hypoxia

showed

no

induced

mortality

for

even

the

day

time

periods

used

in

our

experiments

Phenotypic

changes

in

the

heart

of

adult

teleosts

under

chronic

constant

hypoxia

-LRB-

CCH

-RRB-

In

comparison

to

the

normoxic

control

groups

we

observed

a

significantly

smaller

ventricular

outflow

tract

and

reduced

lacunae

within

the

central

ventricular

cavity

and

reduced

lacunae

around

the

trabeculae

in

midline

sections

of

hearts

of

both

zebrafish

-LRB-

Danio

rerio

-RRB-

-LRB-

Fig.

-RRB-

and

cichlids

-LRB-

Haplochromis

piceatus

-RRB-

-LRB-

Fig.

-RRB-

exposed

to

CCH

In

addition

to

the

midline

sections

of

the

sections

investigated

from

hypoxia

treated

fishes

showed

a

ventricular

outflow

tract

in

comparable

size

to

the

normoxic

controls

-LRB-

data

not

shown

-RRB-

In

addition

to

the

midline

sections

of

the

lateral

sections

-LRB-

to

both

sides

of

the

midline

-RRB-

investigated

from

hypoxia

treated

fishes

showed

a

ventricular

outflow

tract

as

well

as

lacunae

in

a

comparable

size

to

the

normoxic

controls

-LRB-

data

not

shown

-RRB-

The

larger

cichlid

hearts

were

also

perfused

and

midline

sectioned

and

showed

similar

results

with

a

smaller

ventricular

outflow

tract

and

reduced

lacunae

-LRB-

Fig.

-RRB-

This

might

represent

ventricular

hypertrophy

or

hyperplasia

in

both

walls

and

trabeculae

which

could

lead

to

the

observed

cavity

obliteration

in

these

sections

We

quantified

the

number

of

cardiac

myocyte

nuclei

per

area

in

the

midline

sections

of

both

zebrafish

and

cichlids

under

normoxic

as

well

as

hypoxic

conditions

A

significant

difference

in

both

species

was

observed

and

showed

that

hypoxia

led

to

a

and

increase

in

the

number

of

cardiac

myocyte

nuclei

per

area

in

zebrafish

and

cichlid

hearts

respectively

-LRB-

Table

-RRB-

Cardiac

myocyte

nuclei

in

sections

were

clearly

distinguishable

from

nuclei

of

other

cells

like

erythrocytes

and

fibroblasts

and

only

centralized

nuclei

in

cardiac

myocytes

-LRB-

which

are

more

elongated

than

nuclei

from

erythrocytes

-RRB-

were

counted

Furthermore

scanning

electron

microscopy

-LRB-

SEM

-RRB-

was

used

to

confirm

these

findings

in

the

smaller

zebrafish

hearts

-LRB-

Fig.

-RRB-

Future

research

is

warranted

in

order

to

assess

further

how

the

cardiac

myocytes

adapt

to

CCH

in

the

fish

heart

Fig.

changes

of

zebrafish

and

cichlid

hearts

after

exposure

to

chronic

constant

hypoxia

a

shows

zebrafish

hearts

that

were

dissected

sectioned

and

stained

with

a

hematoxylin

eosin

staining

with

A

B

and

E

representing

normoxic

and

C

D

and

F

hypoxic

conditions

Cell

nuclei

are

seen

in

dark

-LRB-

dark

blue

in

online

version

-RRB-

and

cell

cytoplasm

in

light

-LRB-

pink

in

online

version

-RRB-

Pictures

A

C

E

and

F

have

the

same

magnification

-LRB-

-RRB-

Images

B

and

D

represent

a

magnification

of

cardiac

muscle

-LRB-

D

-RRB-

Abbreviations

used

are

a

atrium

v

ventricle

vo

ventricular

outflow

tract

and

ca

conus

arteriosus

b

-LRB-

A

F

-RRB-

corresponds

to

sections

of

cichlid

hearts

which

were

treated

the

same

way

as

the

ones

above

from

zebrafish

and

G

and

H

show

cichlid

hearts

which

have

been

perfused

prior

to

dissection

and

were

stained

with

either

hematoxylin

eosin

-LRB-

A

F

-RRB-

or

Azan

blue

-LRB-

G

H

-RRB-

In

A

B

C

and

G

pictures

of

normoxic

conditions

are

shown

and

D

E

F

and

H

represent

the

corresponding

hypoxic

conditions

Similar

results

for

both

the

zebrafish

and

the

cichlid

hearts

were

observed

in

three

independent

experimentsFig

changes

of

zebrafish

and

cichlid

hearts

after

exposure

to

chronic

constant

hypoxia

Scanning

electron

microscopy

pictures

of

hearts

from

normoxia

control

zebrafish

-LRB-

a

c

and

d

-RRB-

and

hypoxia-treated

zebrafish

-LRB-

b

e

and

f

-RRB-

The

bigger

images

show

half

of

a

heart

ventricle

sectioned

longitudinally

The

smaller

images

represent

a

higher

magnification

of

cardiac

muscle

The

scale

is

given

at

the

bottom

of

each

picture

Abbreviations

used

are

v

ventricle

and

vo

ventricular

outflow

tract

Gene

expression

changes

in

the

heart

of

adult

zebrafish

under

chronic

constant

hypoxia

-LRB-

CCH

-RRB-

In

this

study

we

used

microarrays

for

the

transcriptional

profiling

of

up

and

downregulated

genes

in

response

to

hypoxia

in

the

zebrafish

heart

We

identified

genes

that

were

differentially

expressed

under

hypoxic

conditions

out

of

which

genes

showed

a

decrease

in

gene

expression

-LRB-

-RRB-

in

comparison

to

genes

which

showed

increased

expression

levels

-LRB-

-RRB-

All

differentially

expressed

genes

including

the

ones

with

oligo

sequences

which

could

not

be

annotated

so

far

-LRB-

referred

therein

either

as

transcribed

locus

or

zebrafish

clone

-RRB-

are

shown

in

the

complete

file

-LRB-

Supplemental

Table

-RRB-

Functional

groups

are

color

coded

and

if

possible

gene

functions

are

briefly

summarized

and

OMIM

links

given

Functional

groups

of

differentially

expressed

genes

in

the

heart

We

have

clustered

the

differential

expressed

genes

according

to

known

functions

-LRB-

Table

-RRB-

Genes

can

have

more

than

one

particular

function

assigned

so

some

genes

can

appear

in

more

than

one

group

In

addition

a

gene

ontology

analysis

was

performed

using

eGOn

to

determine

gene

enrichment

and

overrepresentation

in

the

three

categories

of

molecular

function

biological

processes

and

cellular

components

-LRB-

Supplemental

Data

and

-RRB-

Table

groups

of

differentially

expressed

genesUniGeneGeneBankFoldGene

nameUpregulated

genesAngiogenesisDr

T-cell

acute

lymphocytic

leukemia

Fibrinogen

alpha/alpha-E

chain

Dr.

Fibrinogen

gamma

polypeptideApoptosisDr

interacting

Death

receptorDr

Apoptosis

inhibitor

BAX

inhibitor

adhesionDr

Cadherin

calcium

signal

transducer

Dr.

antigenDr.25140BQ2628027.7Tumor-associated

calcium

signal

transducer

glycoproteinDevelopmentDr

T-cell

acute

lymphocyte

leukemia

-LRB-

tal

-RRB-

Dr.

Bone

morphogenetic

protein

-LRB-

bmp

-RRB-

SRY-box

proteinDr

Notch

homolog

-LRB-

Drosophila

-RRB-

Dr.

Chemokine

receptor

NoelinDr

notch

relatedDr

Eparin

cofactor

IIDr

hydroxyphenylpyruvate

dioxygenase

HPDDr

Gata

binding

protein

-LRB-

-RRB-

Dr.

Prion

protein

-LRB-

prp

-RRB-

geneGrowth

regulationDr

Insulin

like

growth

factor

-LRB-

-RRB-

Dr.

Prolactin

receptorDr

Mad

homolog

Dr.

Spint

Spint

Tetraspan

membrane

protein

Insulin-like

growth

factor

binding

protein

Dr.

Cyclin

growth

factor

binding

protein

relatedDr

Lectin

galactoside-binding

soluble

-LRB-

-RRB-

Dr.

HaptoglobinDr

Angiotensinogen

Dr.

Complement

component

Dr.

Uridine

macro-globulin

Complement

Small

inducible

cytokine

A

-LRB-

scyba

-RRB-

Dr.

Kruppel-like

factor

Complement

component

gamma

polypeptideDr

complement

component

Complement

component

b

fbDr

Complement

Complement

and

Complement

component

Complement

component

complement

control

protein

factor

I-B

Dr.

Complement

component

Complement

Complement

component

factor

B

prostaglandin

D

synthase-like

proteinMetabolismDr

Sulfide

dehydrogenase

likeDr

Hypoxanthine

hosphor-ribosyltransferase

Angiopoietin

Dr.

Alcohol

dehydrogenase

bDr

nucleotidaseDr

OxidoreductaseDr

Pyruvate

kinaseDr.5504BI8795503.2Cystathionine-beta-synthaseDr.1202

Apolipoprotein

Urate

oxidaseDr

Aldolase

bDr

Fatty

acid

desaturase

ELOVL

family

member

Dr.

Apolipoprotein

A-IV

Muscle

relatedDr

AngiotensinogenDr

Complement

component

Trypsin

precursorDr.3025BG7382042.7Alpha-2-macroglobulin

Dr.22139AW0189653.0Alpha-1-antitrypsinDr.25331

AI6580724.1Alpha-2-macroglobulin-2Dr.12602

LysozymeDr

Protease

inhibitor

inhibitor

heavy

chain

Dr.

Serine

protease

inhibitor

alpha

1Dr.26371AI6676765.4ProstasinDr.3025BM5304275.6Alpha-2-macroglobulin-1Dr.3025

BM3168676.5Alpha-2-macroglobulin-2Dr.2960

pre-alpha

-LRB-

globulin

-RRB-

inhibitor

polypeptideDr.25379BI3267836.7Alpha-2-macroglobulinDr.4797

kD-Proteinase

proteinROS

protectionDr

Metallothionein

-LRB-

mt

-RRB-

Dr.

Biliverdin

I

Beta

ReductaseDr

Glutathione

S-transferase

theta

Metallothionein

CerulopasminDr

Uncoupling

protein

transductionDr

CAM

kinase

Rho

guanine

nucleotide

exchange

factor

Rho

guanine

nucleotide

exchange

factor

Phospholipase

C

deltaDr

RRAGC

Rag

C

-LRB-

Ras-related

GTP

binding

C

-RRB-

Dr.

Protein

phosphatase

Dr.

Phosphoprotein

phosphataseTranslationDr

Ribonuclease

P

Dr.

NucleolinDr

RNA

terminal

phosphate

cyclase-like

protein

-LRB-

-RRB-

Dr.

Methionyl

aminopeptidase

Eukaryotic

translation

initiation

factor

Dr.

S

ribosomal

protein

Ribosomal

protein

Dr.

Ribosomal

protein

Dr.

Heterogeneous

nuclear

ribonucleoprotein

K

Dr.

Heterogeneous

nuclear

ribonucleoprotein

KDr

Heterogeneous

nuclear

ribonucleoprotein

K

Dr.

Heterogeneous

nuclear

ribonucleoprotein

K

Dr.

Heterogeneous

nuclear

ribonucleoprotein

KDr

Heterogeneous

nuclear

ribonucleoprotein

KTransportDr

Clathrin

coat

assembly

protein

APOB

apolipoprotein

B

-LRB-

including

Ag

-LRB-

x

-RRB-

antigen

-RRB-

Dr.

Solute

carrier

family

Embryonic

globin

beta

Uroporphyrinogen

III

synthaseDr

K

transporting

alpha

polypeptideDr

HemopexinDownregulated

genesAngiogenesisDr

Fast

muscle

troponin

IDr

Similar

to

HUMAN

protein

precursor

Insulin-like

growth

factor-binding

protein

adhesionDr

Cadherin

relatedDr

GTP-binding

protein

Ataxin

proteinDr

FibrillarinDr

netrin

regulationDr

-LRB-

-RRB-

Dr.

Sprouty

homolog

Dr.

Suppressors

of

cytokine

signaling

translocation

gene

Dual

specificity

phosphatase

Epidermal

growth

factor

receptor

kinase

substrate

proliferation-associated

kDa

proteinDr

early

growth

response

protein

relatedDr

ATPase

Ca

transporting

cardiac

muscle

-LRB-

-RRB-

Dr.

Fast

skeletal

myosin

light

chain

TitinMetabolismDr

Creatine

kinase

Pyruvate

dehydrogenase

kinaseDr

Carnitine

O-palmitoyltransferase

IIDr

acyl-CoA

dehydrogenase

Dr.

FabG

beta-ketoacyl

reductaseDr

Elongation

of

very

long

chain

fatty

acids

-LRB-

-RRB-

Dr.

transport

protein

A

delta

isomerase

Dr.

Carnitine

ligaseDr

Creatine

kinase

mitochondrial

Muscle

relatedDr

Myosin

binding

protein

CDr

Myosin

light

polypeptide

Myosin

light

polypeptide

TitinDr

Caveolin

Carbonic

anhydrase

IIDr

Troponin

IProteolysisDr

Chymotrypsinogen

Dr.

Chymotrypsinogen

transductionDr

Serum

deprivation

response

protein

-LRB-

SDPR

-RRB-

TranslationDr

Mitochondrial

elongation

factor

Mitochondrial

ribosomal

protein

Mitochondrial

S

ribosomal

protein

TransportDr

ADP

ATP

carrier

proteinDr

mitochondrial

carrier

protein

-LRB-

Citrin

-RRB-

Dr.

Solute

carrier

family

Dr.

Synaptotagmin

IDr

Synaptotagmin

I

Dr.

protein

Dr.

ATPase

-LRB-

Ca

transporting

plasma

membrane

-RRB-

Proteinbiosynthesis

-LRB-

Translation

-RRB-

In

the

group

linked

to

proteinbiosynthesis

only

three

genes

showed

decreased

expression

under

hypoxic

conditions

All

three

were

found

to

be

part

of

the

mitochondrial

translational

machinery

In

contrast

non

mitochondrial

genes

linked

to

proteinbiosynthesis

showed

all

increased

expression

-LRB-

Table

-RRB-

Metabolism

The

group

with

metabolic

genes

contains

repressed

and

genes

with

enhanced

expression

-LRB-

Table

-RRB-

The

repression

majorly

involves

metabolic

genes

linked

to

β-oxidation

and

lipid

metabolism

Among

the

metabolic

genes

with

enhanced

expression

are

pyruvate

kinase

and

aldolase

which

both

are

key

enzymes

for

glycolysis

indicating

a

shift

from

aerobic

to

anaerobic

metabolism

induced

by

hypoxia

Protection

against

reactive

oxygen

species

-LRB-

ROS

-RRB-

The

group

of

genes

important

for

protection

against

ROS

contains

genes

which

are

all

enhanced

under

hypoxia

Apoptosis

We

found

four

genes

linked

to

programmed

cell

death

to

be

enhanced

by

hypoxia

Two

of

these

the

death

receptor

-LRB-

-RRB-

and

the

homologue

are

considered

to

be

pro-apoptotic

whereas

apoptosis

inhibitor

and

Bax

inhibitor

have

been

shown

to

have

anti-apoptotic

properties

-LRB-

Tewari

et

al.

Xu

and

Reed

-RRB-

Growth

regulation

In

the

group

of

genes

linked

to

growth

regulation

we

found

genes

to

be

repressed

and

genes

with

enhanced

expression

Within

the

group

of

repressed

genes

we

found

antiproliferative

genes

and

dual

specificity

phosphatase

both

inhibit

mitogen-activated

kinases

-LRB-

MAPK

-RRB-

binds

and

inhibits

Janus

kinases

-LRB-

JAK

-RRB-

and

thereby

prevents

activation

which

is

important

in

the

transition

and

is

a

transcriptional

repressor

with

antiproliferative

functions

Although

some

genes

involved

in

cell

proliferation

like

the

transcription

factor

c-fos

were

repressed

by

hypoxia

-LRB-

see

also

Discussion

-RRB-

the

regulation

of

the

vast

majority

-LRB-

of

-RRB-

of

identified

genes

in

this

group

suggests

stimulation

of

proliferation

-LRB-

Table

-RRB-

Inflammation

genes

involved

in

the

inflammatory

response

were

identified

to

be

differentially

regulated

and

all

showed

increased

expression

upon

hypoxia

treatment

Heart-related

function

Several

genes

linked

to

cardiac

hypertrophy

cardiomyopathy

-LRB-

disease

of

the

heart

muscle

-RRB-

and

cardiac

infarction

were

identified

in

this

study

-LRB-

see

Table

and

Discussion

-RRB-

Muscle-related

function

Several

sarcomeric

genes

linked

to

hypertrophy

showed

decreased

expression

under

hypoxic

conditions

-LRB-

Table

and

Discussion

-RRB-

Development

The

genes

in

this

group

were

all

found

to

be

upregulated

by

hypoxia

among

them

the

gene

for

and

Notch

receptors

are

transmembrane

receptors

with

essential

roles

in

development

including

heart

development

Transport

-LRB-

cellular

and

vascular

-RRB-

A

heterologous

group

containing

the

gene

for

embryonic

hemoglobin

beta

important

for

oxygen

transport

and

hemopexin

which

is

important

for

heme

and

iron

transport

and

was

found

to

be

upregulated

Angiogenesis

It

is

well

known

that

hypoxia

via

the

hypoxia

inducible

factor

1α

-LRB-

-RRB-

pathway

leads

to

angiogenesis

In

the

zebrafish

heart

under

CCH

we

observed

increased

expression

of

as

well

as

fibrinogen-α

and

γ

Fibrinogen-α

has

been

shown

to

stimulate

and

VEGF

expression

and

thereby

induces

angiogenesis

-LRB-

Shiose

et

al.

-RRB-

Expression

changes

of

known

hypoxia

responsive

genes

Examples

for

regulation

of

known

hypoxia

responsive

genes

are

insulin

growth

-LRB-

-RRB-

insulin

growth

factor

binding

protein

-LRB-

-RRB-

and

caveolin

The

transcription

factor

is

pivotal

in

the

cellular

response

to

hypoxic

stress

-LRB-

Semenza

-RRB-

We

further

observed

increased

expression

of

the

egl

nine

homolog

a

gene

which

was

shown

to

be

induced

by

hypoxia

through

the

pathway

-LRB-

Pescador

et

al.

-RRB-

gene

and

protein

expression

had

been

shown

to

be

upregulated

by

hypoxia

-LRB-

Beilharz

et

al.

-RRB-

It

was

shown

that

also

is

a

hypoxia-inducible

gene

in

zebrafish

embryos

and

it

mediates

hypoxia-induced

embryonic

growth-inhibition

and

developmental-retardation

-LRB-

Kajimura

et

al.

-RRB-

Both

and

were

found

upregulated

in

our

study

We

observed

decreased

expression

of

caveolin

in

the

hearts

of

zebrafish

exposed

to

CCH

earlier

findings

showed

that

chronic

myocardial

hypoxia

led

to

decreased

protein

expression

in

rabbit

hearts

-LRB-

Shi

et

al.

-RRB-

These

findings

indicate

that

the

hypoxic

conditions

used

lead

to

hypoxic

stress

in

the

fish

heart

Evaluation

of

microarray

results

by

quantitative

real-time

RT-PCR

To

further

verify

our

results

we

used

quantitative

real-time

PCR

for

of

the

transcripts

We

confirmed

the

gene

expression

changes

found

in

the

microarray

studies

for

these

up

and

downregulated

transcripts

by

this

independent

method

-LRB-

Fig.

-RRB-

The

downregulated

zebrafish

genes

tested

were

c-fos

creatine

kinase

-LRB-

-RRB-

nebulin

titin

and

the

upregulated

genes

tested

were

metallothionein

pyruvate

kinase

apoptosis

inhibitor

and

The

fold

induction

values

were

not

always

directly

comparable

to

the

array

data

but

in

all

cases

induction

or

reduction

was

confirmed

Quantitative

differences

between

array

data

and

qPCR

results

have

been

reported

before

-LRB-

Meijer

et

al.

Ton

et

al.

van

der

Meer

et

al.

-RRB-

Fig.

of

gene

expression

changes

by

quantitative

real-time

PCR

selected

genes

which

were

found

to

be

differentially

expressed

on

the

microarrays

were

further

analyzed

by

quantitative

real-time

RT-PCR

Relative

expression

is

given

based

on

normalization

to

β-actin

A

standard

curve

for

β-actin

was

included

in

each

experiment

and

data

represents

three

independent

experiments

each

done

in

triplicates

The

primers

used

are

given

in

Supplemental

Table

Assessment

of

microarray

results

for

the

pathway

by

comparing

phospho-Akt

levels

in

cardiac

myocytes

of

normoxic

versus

hypoxic

zebrafish

hearts

The

pathway

is

activated

by

IGFs

which

are

antagonized

by

the

To

test

the

effects

of

the

upregulation

of

both

and

we

assayed

phospo-Akt

levels

in

cardiac

myocytes

and

showed

that

phospho-Akt

levels

were

not

different

between

normoxic

and

hypoxic

cells

Figure

shows

cytoplasmic

immunohistochemical

staining

of

phospho-Akt

The

antibody

recognizes

phosphorylated

and

detects

the

forms

The

incubation

times

for

the

primary

and

secondary

antibody

as

well

as

the

BM

Purple

were

optimized

to

obtain

a

good

signal

to

noise

ratio

Absorbances

were

linearly

related

to

the

time

of

incubation

with

the

primary

and

secondary

antibodies

as

well

as

that

of

BM

Purple

Figure

shows

for

normoxic

fish

the

absorbance

values

of

the

phospho-Akt

staining

in

cardiac

myocytes

as

well

as

skeletal

muscle

fibers

from

the

tail

as

a

function

of

the

incubation

time

with

BM

Purple

AP

substrate

The

absorbance

values

for

the

heart

are

considerably

higher

than

for

the

skeletal

muscle

fibers

However

for

both

cardiac

and

skeletal

muscle

the

absorbances

are

linearly

related

with

the

incubation

time

with

BM

Purple

AP

substrate

and

increase

at

the

same

relative

rate

This

implicates

that

the

absorbance

of

BM

Purple

after

min

incubation

with

BM

Purple

AP

substrate

did

not

reach

saturation

and

therefore

provides

a

semi-quantitative

estimate

of

the

phospho-Akt

content

in

the

cardiac

myocytes

Absorbances

of

staining

for

phospho-Akt

in

normoxic

and

hypoxic

cardiac

myocytes

were

not

shown

to

be

significantly

different

-LRB-

Fig.

P

-RRB-

which

indicates

that

hypoxia

did

not

change

the

activation

of

the

Akt

pathway

in

the

cardiac

myocytes

Fig.

of

chronic

constant

hypoxia

of

the

activation

of

Akt

in

zebrafish

cardiac

myocytes

Immunohistochemical

staining

of

phospho-Akt

in

cardiac

myocytes

of

zebrafish

raised

under

hypoxic

a

or

normoxic

b

conditions

Specificity

is

shown

by

the

control

sections

obtained

from

normoxic

fish

which

were

not

incubated

with

primary

antibody

against

phospho-Akt

c.

For

both

cardiac

myocytes

and

skeletal

muscle

fibers

the

absorbance

of

the

BM

Purple

is

linearly

increasing

at

the

same

relative

rate

with

the

incubation

time

with

BM

Purple

AP

substrate

at

the

same

relative

rate

d.

Mean

absorbances

of

phospho-Akt

staining

-LRB-

S.E.M.

-RRB-

from

normoxic

and

hypoxic

cardiac

myocytes

was

not

different

e

-RRB-

Discussion

In

the

aquatic

environment

oxygen

concentrations

can

often

vary

and

being

able

to

adapt

to

changes

in

oxygen

levels

can

be

advantageous

for

the

survival

of

aquatic

animals

This

might

be

in

part

the

reason

why

some

teleosts

have

developed

the

ability

to

withstand

extreme

hypoxic

conditions

In

this

study

we

have

focused

on

the

long-term

response

to

hypoxia

in

the

fish

heart

The

hypothesis

is

that

the

zebrafish

heart

in

contrast

to

most

mammalian

hearts

which

are

characterized

by

relative

intolerance

to

injury

or

the

lack

of

oxygen

are

able

to

adapt

to

extreme

hypoxic

conditions

We

showed

that

chronic

hypoxia

of

zebrafish

caused

a

smaller

ventricular

outflow

tract

reduced

lacunae

and

increased

cardiac

myocyte

densities

in

the

heart

These

findings

suggest

that

hypoxia

induced

an

increase

of

the

cardiac

myocyte

volume

or

at

least

did

not

result

in

a

loss

of

cardiac

myocytes

This

is

in

contrast

to

mammals

where

tissue

hypoxia

in

chronic

heart

failure

leads

to

apoptosis

and

considerable

losses

of

cardiac

myocytes

-LRB-

see

for

review

Sabbah

et

al.

-RRB-

In

mammals

compensation

for

this

loss

of

cardiac

myocytes

occurs

mainly

by

hypertrophy

of

the

remaining

cardiac

myocytes

-LRB-

Ostadal

and

Kolar

-RRB-

although

regeneration

of

myocardium

by

proliferation

of

cardiac

myocytes

may

occur

also

but

to

a

limited

extend

-LRB-

Beltrami

et

al.

-RRB-

Our

assay

for

phosho-Akt

did

not

show

any

enhancement

of

Akt

activity

in

response

to

the

CCH

suggesting

a

lack

of

hypertrophic

signaling

via

the

phosphotadilinositol

kinase

pathway

However

the

density

of

cardiac

myocyte

nuclei

increased

by

during

CCH

which

indicates

substantial

proliferation

of

cardiac

myocytes

and/or

nuclear

hyperplasia

Recently

it

has

been

shown

that

the

zebrafish

heart

has

the

ability

to

regenerate

from

mechanical

cardiac

injury

by

proliferation

of

cardiac

myocytes

-LRB-

Poss

et

al.

-RRB-

If

the

zebrafish

heart

responds

to

chronic

hypoxia

in

a

similar

way

as

to

mechanical

dissection

this

will

be

beneficial

in

preventing

apoptosis

of

cardiac

myocytes

as

the

diffusion

distance

for

oxygen

are

not

increased

as

during

hypertrophy

which

will

help

to

prevent

the

development

of

anoxic

cores

in

the

cardiac

myocytes

-LRB-

Des

Tombe

et

al.

van

der

Laarse

et

al.

-RRB-

In

the

case

that

the

increase

in

cardiac

myocyte

nuclear

density

during

chronic

hypoxia

was

due

to

nuclear

hyperplasia

and

nuclear

ploidy

the

observed

smaller

ventricular

outflow

tract

and

reduced

lacunae

may

have

been

the

result

of

hypertrophy

of

the

existing

cardiac

myocytes

The

histological

assay

in

this

study

does

not

clearly

distinguish

between

hyperplasia

and

hypertrophy

of

cardiac

myocytes

Future

investigation

of

the

mechanisms

underlying

the

general

morphological

adaptations

of

teleost

fish

heart

in

response

to

CCH

requires

a

nuclear

stain

in

combination

with

a

clear

membrane

stain

which

allows

determination

of

cell

sizes

in

addition

to

counts

of

cardiac

myocyte

nuclei

We

were

interested

in

the

underlying

gene

expression

changes

of

these

adaptations

We

found

gene

regulations

in

a

transcriptional

network

of

the

serum

response

element

-LRB-

SRE

-RRB-

which

are

opposed

to

the

ones

described

in

mammals

In

the

zebrafish

heart

hypoxia

repressed

c-fos

and

expression

Both

genes

are

important

in

the

same

transcriptional

network

can

transduce

serum-responsive

transcriptional

activity

to

the

c-fos

-LRB-

SRE

-RRB-

by

interacting

with

serum

response

factor

-LRB-

SRF

-RRB-

-LRB-

Simon

et

al.

-RRB-

In

mammals

many

studies

showed

increased

expression

of

c-fos

by

hypoxia

e.g.

in

rats

hypoxia

induces

c-fos

expression

in

the

LV

and

RV

-LRB-

Deindl

et

al.

-RRB-

and

the

same

held

true

in

tissue

culture

cells

-LRB-

Webster

et

al.

-RRB-

Interestingly

repression

of

c-fos

by

hypoxia

has

also

been

shown

for

the

short-term

response

to

anoxia

in

anoxia

tolerant

turtles

-LRB-

Greenway

and

Storey

-RRB-

Is

it

possible

that

repression

of

c-fos

and

are

important

adaptations

in

hypoxia

tolerant

animals

Several

novel

gene

expression

changes

induced

by

CCH

have

been

identified

in

this

study

-LRB-

Table

and

Supplemental

Table

-RRB-

An

example

is

the

two

Notch

receptors

and

whose

expression

were

induced

by

CCH

Notch

receptors

have

been

shown

to

be

important

for

heart

patterning

and

differentiation

-LRB-

Armstrong

and

Bischoff

-RRB-

cell

fate

determination

and

self

renewal

of

stem

cells

-LRB-

Androutsellis-Theotokis

et

al.

Silvia

Bianchi

-RRB-

but

so

far

have

not

been

linked

to

the

hypoxic

response

Several

genes

with

links

to

human

heart

pathology

were

found

to

be

upregulated

in

this

study

Among

these

were

two

markers

for

myocardial

infarction

complement

component

and

haptoglobin

is

used

as

a

marker

for

myocardial

infarction

-LRB-

Doran

et

al.

-RRB-

and

a

polymorphism

for

haptoglobin

predicts

mortality

and

heart

failure

in

patients

with

diabetes

and

acute

myocardial

infarction

-LRB-

Levy

et

al.

-RRB-

We

also

observed

increased

expression

of

fetuin-α

a

circulating

calcium-regulatory

glycoprotein

that

inhibits

vascular

calcification

Low

fetuin-α

levels

have

been

associated

with

heart

failure

in

mice

-LRB-

Merx

et

al.

-RRB-

Upregulation

of

fetuin-α

in

the

zebrafish

heart

could

help

to

better

tolerate

CCH

Selenoprotein

P

-LRB-

-RRB-

is

a

heparin-binding

protein

that

appears

to

be

associated

with

endothelial

cells

and

has

been

implicated

as

an

oxidant

defense

in

the

extracellular

space

Human

populations

that

are

selenium

deficient

are

susceptible

to

the

development

of

Keshan

disease

a

potentially

fatal

form

of

cardiomyopathy

-LRB-

Nezelof

et

al.

-RRB-

expression

was

found

to

be

increased

in

our

study

indicating

a

potential

protective

mechanism

against

oxidative

stress

in

the

heart

This

was

further

supported

by

the

increased

expression

observed

for

several

genes

important

for

the

protection

against

reactive

oxygen

species

-LRB-

ROS

-RRB-

-LRB-

Table

-RRB-

Among

them

were

metallothionein

and

glutathione

S-transferase

which

are

both

known

ROS

scavengers

Metallothionein

has

further

been

described

to

be

protective

against

hypoxia-induced

apoptosis

when

overexpressed

-LRB-

Wang

et

al.

-RRB-

Our

findings

suggest

ROS

protection

as

an

important

adaptation

to

CCH

in

the

fish

heart

Furthermore

our

data

show

for

the

first

time

that

CCH

simultaneously

induced

upregulation

of

and

the

insulin-like

growth

factor

binding

protein

-LRB-

-RRB-

In

isolated

cardiomyoblasts

angiotensin

II

stimulates

expression

which

is

involved

in

the

induction

of

apoptotic

signaling

in

rat

hearts

-LRB-

Lee

et

al.

-RRB-

We

have

observed

here

both

angiotensin

and

upregulation

-LRB-

Table

-RRB-

The

upregulation

of

seems

to

be

a

general

response

to

hypoxia

in

zebrafish

embryos

where

it

mediates

hypoxia-induced

embryonic

growth

retardation

and

developmental

delay

-LRB-

Kajimura

et

al.

-RRB-

is

a

secreted

protein

which

binds

to

IGFs

in

the

extracellular

environment

and

prevents

receptor

activation

-LRB-

Florini

et

al.

Stewart

and

Rotwein

-RRB-

Here

by

binding

may

have

prevented

both

cardioprotective

as

well

as

apoptotic

effects

of

enhanced

expression

To

test

the

effects

of

the

upregulation

of

both

and

we

assayed

phospo-Akt

levels

in

cardiac

myocytes

and

showed

that

phospho-Akt

levels

were

not

different

between

normoxic

and

hypoxic

cells

This

suggests

that

either

hypoxia-induced

changes

in

mRNA

expression

did

not

occur

at

the

protein

level

or

the

effects

of

increased

expression

on

the

receptor

-LRB-

which

can

be

activated

by

-RRB-

were

blunted

by

the

upregulation

of

the

The

fact

that

we

did

not

observe

increased

phospho-Akt

levels

suggests

a

lack

of

hypertrophic

signaling

as

enhanced

phospho-Akt

is

required

for

cardiac

hypertrophy

-LRB-

DeBosch

et

al.

-RRB-

This

may

be

beneficial

for

the

heart

as

hypertrophy

of

cardiac

myocytes

implicates

an

increase

in

the

diffusion

distance

for

oxygen

which

may

cause

the

development

of

anoxic

cores

-LRB-

Des

Tombe

et

al.

-RRB-

release

of

cytochrome

c

-LRB-

van

Beek-Harmsen

and

van

der

Laarse

-RRB-

and

production

of

ROS

-LRB-

Lee

et

al.

Powers

et

al.

-RRB-

and

eventually

causing

apoptosis

of

cardiac

myocytes

The

importance

of

the

signaling

in

the

protection

of

the

zebrafish

heart

and

its

underlying

mechanisms

remain

to

be

determined

The

gene

expression

changes

we

observed

for

the

specific

response

to

CCH

in

the

fish

heart

were

very

different

from

the

responses

we

observed

in

an

earlier

study

in

the

gills

-LRB-

van

der

Meer

et

al.

-RRB-

Under

the

same

criteria

as

used

in

this

study

we

found

that

the

majority

of

differentially

regulated

genes

in

the

gills

showed

a

decrease

in

gene

expression

-LRB-

or

genes

in

total

-RRB-

in

comparison

to

genes

which

showed

increased

expression

levels

-LRB-

or

genes

in

total

-RRB-

This

is

opposed

to

the

heart

where

of

differentially

regulated

genes

-LRB-

genes

in

total

-RRB-

showed

increased

and

-LRB-

in

total

-RRB-

decreased

expression

levels

Many

genes

linked

to

protein

synthesis

showed

a

similar

trend

and

were

downregulated

in

the

gills

-LRB-

van

der

Meer

et

al.

-RRB-

and

upregulated

in

the

heart

-LRB-

Table

-RRB-

The

major

differences

observed

in

gene

regulation

between

heart

and

gills

point

to

very

tissue

specific

responses

to

CCH

A

list

of

genes

identified

in

both

studies

as

identified

by

a

direct

comparison

based

on

Accession

numbers

and

old

Unigenes

numbers

are

shown

in

Supplemental

Data

Teleosts

have

developed

specific

phenotypic

adaptations

to

low

oxygen

due

to

the

natural

occurrence

of

hypoxia

in

the

water

environment

Here

we

identified

the

changes

in

gene

expression

as

well

as

associated

morphological

changes

of

the

fish

heart

to

hypoxia

We

observed

repression

of

c-fos

which

differs

compared

to

the

described

increase

in

expression

in

mammals

-LRB-

Deindl

et

al.

-RRB-

Other

changes

found

like

upregulation

of

the

two

Notch

receptors

have

not

been

described

before

to

the

best

of

our

knowledge

Similarly

the

simultaneous

increase

in

expression

of

and

has

not

been

shown

The

changes

identified

here

can

contribute

to

the

ability

of

teleosts

to

adapt

to

severe

hypoxia

-LRB-

for

example

the

upregulation

of

fetuin-α

and

levels

-RRB-

Future

functional

studies

are

warranted

to

validate

the

role

of

the

identified

genes

in

cardiac

protection

to

hypoxia

Electronic

supplementary

material

Below

is

the

link

to

the

electronic

supplementary

material

-LRB-

XLS

kb

-RRB-

-LRB-

DOC

kb

-RRB-

-LRB-

DOC

kb

-RRB-

-LRB-

XLS

kb

-RRB-

O O O O O O O O B O B O O O O O O O O O O O O O B O O O O B I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O B O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B I O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B I O O O O O B O O O O O B O O O O O O O O O O O O O O B I O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B O O B O O O B O O O O O O O O O O O O O O O O O O O O O O B O O B B O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O B B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O B I O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B I O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

hypoxia
zebrafish
heart
hyperplasia
gene expression