adipose formation in the myocardium should be a more
common pathology than is currently recognized. A new
mechanism of arrhythmogenesis in ventricular tachycardia
proposes that intramyocardial adipose tissue hinders myocar-
dial conduction and worsens local electrophysiological proper-
ties, which in turn results in an increased propensity for
ventricular tachycardia.
The KM genes transduced into
CMP maintained high expression for about 1 week, resulting
in differentiation of the CMPs to lipid-laden adipocytes and
activation of the second wave of TFs for adipogenesis. In
contrast, cardiac IRI temporarily induced
Klf4
and
c-Myc
expression, which sharply fell and disappeared after only a
few days, resulting in failure to maintain the second wave and
generate adipocytes. During left ventricular (LV) remodeling
post MI, the renin
–
angiotensin
–
aldosterone system (RAAS) is
activated, which leads to AP1 activation
and might result in
c-Myc
induction.
Angiotensin II can induce
Klf4
expression in
cardiac fibroblasts including CMPs.
Although RAAS does not
induce a high expression of KM such as that which we observed
during
in vitro
adipogenesis in this study, the low level of KM
expression over a long period induced by RAAS might slowly
form adipogenic enhanceosomes at enhancer regions for late-
acting TFs in adipogenesis, such as
Ppar
γ
.
Inhibition of either
Klf4
or
c-Myc
induction might be a novel strategy to treat LV
remodeling post MI.
Global mRNA profiling of the myocardium after IRI has
revealed that
Klf
family members, including
Klf4
and
c-Myc
,
exhibit significantly increased expression following ischemia
and additional increases after reperfusion.
Ischemic events
generate interleukin 6 in the heart, activating STAT3,
which
is linked to
Klf4
expression.
However, ROS, which have
been characterized as negative factors in reperfusion injuries,
are involved in signal transduction in many biological
processes, including inflammation, stemness and differentia-
tion, cancer, and aging.
The thioredoxin family member
nucleoredoxin (NRX), which is a redox sensor regulated by
ROS, interacts with dishevelled (Dvl) under a reduction state.
The oxidized form of NRX liberates Dvl, which in turn stabilizes
β
-catenin, leading to the transcription of WNT target genes
including
c-Myc
Consistent with the aforementioned studies
on signal transduction, myocardial ischemia led to increased
expression of
Klf4
and reperfusion stimulated
c-Myc
expres-
sion. These results strongly suggest that the two TFs KLF4
and c-MYC in CMPs are causative factors for intracardiac
adipogenesis following myocardial reperfusion. The regional
assessment revealed that the expression levels of
Klf4
,
c-Myc
,
c-Fos
and C/Ebp
δ
in AON and AAR were raised more than
those in RA, suggesting that the adipogenic differentiation
process had already been launched in the area directly
affected by the insult of ischemia and reperfusion at 2 h.
MSCs can be isolated from various tissue types including
bone marrow, adipose tissue, heart and skeletal muscle.
However, the characteristics and epigenetic background of
these MSCs differ.
Transduction of CMPs with KM genes
was highly effective in inducing their differentiation into
adipocytes, whereas transducing the same genes into MSCs
derived from other tissues did not induce them to differentiate.
Furthermore, these phenomena might provide a basis for
ectopic fat formation in ischemic hearts. Understanding
CMP adipogenesis should shed light on post-MI and IRI
pathophysiology and facilitate the development of better
treatments for these disorders.
Materials and Methods
Materials.
Geltrex and basic fibroblast growth factor (bFGF) were purchased
from Life Technologies (Carlsbad, CA, USA). The CytoTune-iPS ver. 1.0 Sendai
Reprogramming Kit was purchased from DNAVEC (Ibaraki, Japan). Oil Red O
powder was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Percoll Plus was
purchased from GE Healthcare UK (Buckinghamshire, England). The adipogenic
stimulation cocktail ingredients insulin, IBMX, and dexamethasone were purchased
from Sigma-Aldrich (St. Louis, MO, USA).
Cell preparation.
Experimental procedures and protocols were approved by
the Animal Experiment Ethics Committee of the Kyoto Prefectural University of
Medicine. Murine CMPs were isolated from wild-type C57BL/6 mouse hearts
(10- to 16-week-old).
Briefly, the mice were killed by deep anesthesia with
pentobarbital. The hearts were excised, and atria were used in this study. The
minced tissue fragments were digested twice for 30 min at 37 °C with 0.2% (w/v)
type II collagenase and 0.01% (w/v) DNase I (Worthington Biochemical, Lakewood,
NJ, USA). After digestion, cells were passed through a 70-
μ
m filter to remove debris
and transferred to Dulbecco's modified Eagle
’
s medium (DMEM)/F12 supplemented
with 10% (v/v) fetal bovine serum (FBS) (Life Technologies). The cells were
collected and size fractionated on a 30
–
70% Percoll gradient to obtain CMPs
expressing the Sca-1 antigen. CMPs were seeded on 60-mm collagen I-coated
dishes (Asahi Glass, Tokyo, Japan) in DMEM/F12 supplemented with 10% (v/v)
FBS and 20 ng/ml bFGF. The medium was changed every 3 days.
Cell culture and adipocyte differentiation.
CMPs were cultured in
DMEM/F12 supplemented with 10% (v/v) FBS and 20 ng/ml bFGF in a humidified
atmosphere containing 5% CO
2
. NIH3T3 fibroblasts and MSCs derived from bone
marrow KUM5, KUM9 and KUSA-A1
were cultured in DMEM (Wako Chemical
Co., Osaka, Japan) supplemented with 10% (v/v) FBS in a humidified atmosphere
containing 5% CO
2
. The 3T3-L1 preadipocytes were cultured in minimum essential
media (MEM) (Life Technologies) supplemented with 10% (v/v) FBS in a humidified
atmosphere containing 5% CO
2
. For adipocyte differentiation, before viral
transduction, cells were seeded at 0.5 × 10
5
per well on Geltrex-coated six-well
plates (1:40, Life Technologies) in growth medium (day
–
2). On the next day
(day
–
1), cells were transduced using the CytoTune-iPS ver. 1.0 Sendai
Reprogramming Kit according to the manufacturer
’
s recommendations. At 24 h
after transduction (day 0), cells were transferred to reprogramming media, that is,
knockout DMEM (KO-DMEM) with 5% (v/v) knockout serum replacement, 15% (v/v)
FBS, 1% (v/v) GlutaMAX solution, 1% (v/v) nonessential amino acids solution and
0.1 mM
β
-mercaptoethanol (all components obtained from Life Technologies). Using
another conventional method for adipocyte differentiation, cells were exposed to
adipogenic differentiation cocktails containing dexamethasone (1
μ
M), IBMX
(0.5 mM), insulin (5
μ
g/ml) and 10% (v/v) FBS. The cells were maintained in
reprogramming medium for 8 days beginning at day 0, and the media was
exchanged every 48 h throughout all experiments (Figure 1a).
Total RNA extraction and qRT-PCR analysis.
Total RNAs from cells
were extracted using TRIzol (Life Technologies) and a Direct-zol RNA MiniPrep Kit
(Zymo Research, Irvine, CA, USA) with DNase I according to the manufacturer
’
s
recommendations. To perform the qRT-PCR assay, 400 ng of total RNAs was
reverse-transcribed using the PrimeScript RT Reagent Kit and SYBR Premix Ex Taq
(Takara Bio, Shiga, Japan) according to the manufacturer
’
s recommendations. qRT-
PCR was performed using a Thermal Cycler Dice Real Time System using the
default cycling program (Takara Bio). The primers used in this experiment are listed
in Supplementary Table 2. The relative gene expression levels of mouse total heart
RNAs (Takara Bio) or human iPSC RNAs were normalized to
Gapdh
expression.
Tissue preparation.
Ten- to 12-week-old C57BL/6 mice were anesthetized
and killed, and their hearts were removed at indicated time points. For total RNA
and proteins extraction, the walls of the LV were dissociated from the whole heart.
For total RNA isolation, the samples were cut into small pieces and homogenized
with TRIzol using Bio Masher II (Nippi, Tokyo, Japan).
To isolate whole proteins, the samples were cut into small pieces and
homogenized with lysis buffer (20 mM Tris-HCl (pH7.5), 137 mM NaCl, 10% glycerol
(vol/vol), 1% NP-40 (vol/vol) (Wako Chemical Co.)), subsequently the lysates were
Cardiac adiposity is regulated by
Klf4
and
c-Myc
D Kami
et al
10
Cell Death and Disease
