healthmedicinet

Animals

Two-month old ICR male mice were purchased from Charles River Laboratories (Wilmington,
MA, USA). All animal experiments were conducted under a protocol approved by the Institutional
Animal Care and Use Committee of Institute, which conforms to the Guide for the Care
and Use of Laboratory Animals published by the US National Institutes of Health (NIH
Publication No. 85-23, revised 1996).

Experiment protocols

Two month-old mice were made diabetic by intraperitoneal injection of a single dose
of freshly prepared STZ solution (200 mg/kg body wt dissolved in citrate buffer, pH
4.5) following overnight fasting 30]. Diabetic status was determined by measuring blood glucose concentration. Another
group of mice was injected with vehicle (0.1 mol/l citrate buffer, pH 4.5) to serve
as a control. Mice were randomly divided into four groups: Control group: mice only received an injection of vehicle (citrate buffer); Control + NaBu group: mice received sodium butyrate (1%), a specific HDAC inhibitor, in drinking
water on a daily basis; STZ-treated group: mice received intraperitoneal injection of STZ; STZ + NaBu group: mice received intraperitoneal injection of STZ injection followed by sodium
butyrate (1%), in drinking water on a daily basis. Tail vein blood glucose samples
were measured using One Touch II Glucometer (Lifescan, Inc., Milpitas, CA, USA) to
confirm the induction of diabetes. All animals were euthanized at 21 weeks after injection
of STZ.

Echocardiography

Echocardiographic parameters were accessed before and 7, 14, and 21 weeks after the
sodium butyrate treatments. Echocardiography was performed to evaluate left ventricular
(LV) functions using an Acuson Sequoia C512 system (Siemens Helathcare, PA, USA) equipped
with a 15L8 linear array transducer. Mice were placed in supine position on a heating
pad after being anesthetized with 1.5% isoflurane mixed with oxygen. Pre-warmed ultrasound
gel was applied on the chest throughout the measurements. At the signal depth of 25 mm,
2-D B-mode and M-mode images were recorded on short axis views at the level of the
papillary muscles. The following parameters were measured on the M-mode tracings and
averaged from 3 to 6 cardiac cycles: left ventricular internal dimension-diastole
(LVID;d), left ventricular internal dimension-systole (LVID;s), ejection fraction
(EF), fraction shortening (FS), left ventricular posterior wall thickness in end-diastole
(LVPW;d), and end-systole (LVPW;s). Data were calculated with accompanying software.

Immunohistochemistry

Tissue sections were de-paraffinized for 30 min at 70°C and subsequently immersed
in xylene and ethanol at decreasing concentration. Immunostaining was performed as
described previously 27]. All de-paraffinized tissue sections went through antigen retrieval by boiling of
slides at 100°C for 1 h. Wheat germ agglutinin (WGA) staining was carried out to measure
cell size. The outline of myocytes was traced in the LV of each animal, using NIH
Image J software to determine myocyte cross-sectional area. A value from each heart
was calculated by the measurements of approximately 400–600 cells in a remote area
from 5 randomly selected image areas in an individual heart. For evaluation of interstitial
fibrosis, Picrosirius red staining of cardiac sections was conducted. Interstitial
collagen was examined from five randomly selected regions from each tissue section
using an Olympus BX51 microscope (Olympus, Center Valley, PA, USA). For ?-smooth muscle
actin (?-SMA) and cluster of differentiated CD31 immunostaining, cardiac sections
were incubated with primary antibodies including CD31 (Millipore, Billerica, MA, USA)
and anti-?-SMA (Sigma, St. Louis, MO, USA) overnight at 4°C. Signals were visualized
by incubation with the corresponding secondary antibodies including goat-anti-rat-Cy3
and goat-anti-mouse-Cy3 (Life Technologies, Carlsbad, CA, USA) at room temperature
for 1 h. Nuclei were stained with 4?, 6-diamidino-2-phenylindole (DAPI).

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

De-paraffinized sections were processed for a TUNEL assay using a TACS
^®
TdT In Situ Apoptosis Detection Kits (Trevigen, Gaithersburg, MD, USA) following the
manufacturer’s instructions. TUNEL positive cells were observed using confocal laser
scanning microscopy LSM 700 (Carl Zeiss). The numbers of TUNEL positive cells were
determined and were normalized to the tissue area. DAPI was used to counter stain
for nuclei.

Immunoblotting and immunoprecipitation

Heart tissues were homogenized on ice in RIPA buffer with Halt protease and phosphatase
inhibitor cocktail. Proteins (50 µg/lane) were resolved by SDS gel and transferred
onto PVDF membranes. The membranes were then blocked with 5% powdered milk in TBST
for 1 h and were subsequently probed with primary antibodies overnight at 4°C. The
following primary antibodies were used in this study: HDAC4, acetylated-Lysine, phosphorylated
p38, and active casepase-3 from Cell Signaling (Danvers, MA, USA); GLUT 1, GLUT4,
p38, SOD1, and ?-actin were from Santa Cruz Biotechnology (Dallas, TX, USA). Horseradish
peroxidase-conjugated monoclonal antibodies (1: 2,000) were used for chemiluminescence
detection.

For immunoprecipitation, samples were pre-cleared prior to immunoprecipitation to
reduce the amount of non-specific contaminants. The EZView red protein A affinity
gel (Sigma-Aldrich, St.Louis, MO, USA) was incubated with myocardial lysates for 60 min
at 4°C. Samples were centrifuged, and supernatants were obtained. Proteins were incubated
with the indicated primary antibodies at 4°C overnight. Beads were added to lysate
plus antibody mix, and proteins were further incubated for 2 h at 4°C. In addition,
IgG was also used as immunoprecipitation control and non-immunoprecipitated lysate
was used as a control for detected molecular weight. After incubation, samples were
washed with RIPA buffer five times, and proteins were eluted with 4× loading buffer
by boiling and subjected to SDS-PAGE.

HDAC activity measurement

Myocardial HDAC activity was measured as described preciously in detail 25].

Statistical analysis

All data are expressed as mean ± SEM. Differences among multigroups were analyzed
by one-way analysis of variance (ANOVA), followed by Bonferroni correction. A probability
of p  0.05 was considered to be a significant difference.