Cell culture
Embryonic rat heart derived H9C2 cells, purchased from ATCC (CRL-1446, Manassas, VA), were maintained in the conditions as instructed by ATCC. H9C2 cells were exposed to palmitate at 62.5 µM for 15 h, with SDF-1β co-treatment at 100 nM, and some of these cultures were pre-treated with siRNAs based on experimental needs. Palmitate (Sigma Aldrich, St. Louis, MO) was dissolved in 50% ethanol, heated at 70 °C for 2 min, and then added to 2% fatty acid free bovine serum albumin (BSA, Sigma-Aldrich) in medium as stock solution (2.5 mM). Before use, the stock palmitate solution was gently rotated for 1 h at 37 °C and further diluted to the required concentrations for treatment. SDF-1β, prepared as in our previous study (7, 33), was dissolved in phosphate buffered saline (PBS) to the required concentrations.
siRNAs transfection
siRNAs specific for rat p38β (Silencer® Select siRNA ID: s236087), p38α (Silencer® Select siRNA ID: s135447), CXCR7 (Silencer® Select siRNA ID: s136443), CXCR4 (Silencer® Select siRNA ID: s133527) and KLF15 (Silencer® Select siRNA ID: s137039) along with their parallel Silencer® Select negative control siRNAs (Thermo Fisher Scientific) were transfected into H9C2 cells for 48 h by transfection reagent. Following which cells were co-treatment with SDF-1β at 100 nM and palmitate at 62.5 µM for 15 h. Transfection efficiency was assessed by ELISA kit and western blot analysis for the target genes or proteins.
Recombinant adenovirus infection
Rat KLF15 (Accession no. AAH89782.1) cloned into adenovirus vector (pAdenoMCMV-EGFP-P2A-3FLAG) were constructed by Obio Technology (Shanghai, China) (34). Adenoviral infection was performed on H9C2 cells at multiplicity of infection (MOI) of 50 for 48 hours (> 90% infection efficiency as assessed by GFP signal) (35).
Animal models
All animal protocols were approved by the Animal Ethics Committee of Jilin University. Eight-week-old male C57BL/6J mice (Weitonglihua, Beijing, China) and conditional knockout of cardiac specific Klf15 gene (Klf15-cKO) mice (VivoCure, Beijing, China) were acclimated in an air-conditioned room at 22 °C with a 12 h light / dark cycle and fed with standard rodent chow and tap water. Mice were fed with either high fat diet (HFD, 60%kcal from fat, No. 12492, Research Diets, New Brunswick, NJ) or normal diet (ND, 10% kcal from fat, No. 12450B, Research Diets) for 3 months to induce insulin resistance. These insulin resistant mice were injected with a single dose of STZ (freshly dissolved in 0.1 M sodium citrate (pH 4.5), Sigma Aldrich, St. Louis, MO) at 100 mg/kg body weight to induce partial insulin deficiency. Five days after STZ injection, mice with hyperglycemia (3 h fasting blood glucose levels ≥ 250 mg/dl) were defined as diabetic. After diabetes onset, mice were divided into eight groups: Control (Ctrl, n = 6), SDF-1β (n = 6), Type 2 Diabetes (T2D, n = 7), T2D plus SDF-1β (T2D/SDF, n = 7), Klf15-cKO (n = 6), Klf15-cKO plus T2D (Klf15-cKO/T2D, n = 7), Klf15-cKO plus T2D plus SDF-1β (Klf15-cKO/T2D/SDF-1β, n = 7), Klf15-cKO plus SDF-1β (Klf15-cKO/SDF-1β, n = 7). At baseline, Klf15-cKO mice are viable and have mild pathologic remodeling of the heart (mild LVH with preserved systolic function). However, after T2D onset, Klf15-cKO mice develop a severe cardiomyopathy characterized by LV dysfunction, cavity dilation with attenuated wall thickening. These pathologic changes were associated with augmented cardiac mass (Table 1). SDF-1β was given by tail vein at 5 mg/kg body-weight twice a week (Tuesday and Friday) for 3 months. The dose of SDF-1β used in the present study was based on our previous study. The control mice were given the same volume of vehicle (1% dimethyl sulfoxide diluted with PBS). All T2D and age-matched control mice continually received their HFD or ND for an additional 3 months. After 3 months of SDF-1β or vehicle treatment, their cardiac function was measured, after which animals were euthanized and heart tissues were collected.
Table 1
Klf15-cKO cardiac function data
|
Ctrl
|
T2D
|
T2D
/SDF-1β
|
SDF-1β
|
KLF15-cKO
|
KLF15-cKO
/T2D
|
KLF15-cKO
/T2D
/SDF-1β
|
KLF15-cKO
/SDF-1β
|
IVS; d (mm)
|
0.61 ± 0.02
|
0.57 ± 0.02*
|
0.60 ± 0.03
|
0.62 ± 0.02
|
0.58 ± 0.02*
|
0.55 ± 0.03#$
|
0.58 ± 0.03&
|
0.60 ± 0.02
|
LVID; d (mm)
|
3.79 ± 0.04
|
3.91 ± 0.05*
|
3.84 ± 0.06#
|
3.81 ± 0.06
|
3.89 ± 0.04*
|
4.03 ± 0.07#$
|
3.94 ± 0.07&
|
3.94 ± 0.05
|
LVPW; d (mm)
|
0.79 ± 0.02
|
0.70 ± 0.03*
|
0.75 ± 0.03*#
|
0.77 ± 0.03
|
0.72 ± 0.02*
|
0.63 ± 0.04#$
|
0.67 ± 0.04$
|
0.73 ± 0.02
|
IVS; s (mm)
|
1.07 ± 0.02
|
0.95 ± 0.03*
|
1.01 ± 0.03*#
|
1.06 ± 0.02
|
1.01 ± 0.03*#
|
0.84 ± 0.05#$
|
0.93 ± 0.04$&
|
1.03 ± 0.03
|
LVID; s (mm)
|
1.92 ± 0.09
|
2.39 ± 0.11*
|
2.03 ± 0.12#
|
1.94 ± 0.08
|
2.27 ± 0.13*
|
2.53 ± 0.14#$
|
2.41 ± 0.09$&
|
2.15 ± 0.09$
|
LVPW; s (mm)
|
1.33 ± 0.02
|
1.12 ± 0.03*
|
1.24 ± 0.03*#
|
1.31 ± 0.04
|
1.21 ± 0.03*
|
1.02 ± 0.05#$
|
1.11 ± 0.04$&
|
1.24 ± 0.05
|
LV Vol; d (mm)
|
61.82 ± 1.38
|
67.65 ± 1.74*
|
63.01 ± 1.53#
|
60.91 ± 1.64
|
65.91 ± 1.24*
|
72.11 ± 2.09#$
|
69.17 ± 2.01$
|
70.26 ± 1.65$
|
LV Vol; s (mm)
|
12.17 ± 1.25
|
21.12 ± 1.53*
|
15.77 ± 1.43*#
|
13.02 ± 1.39
|
18.37 ± 1.47*
|
28.12 ± 1.93#$
|
24.06 ± 1.75$&
|
17.14 ± 1.69
|
%EF
|
78.85 ± 2.93
|
63.22 ± 3.35*
|
69.46 ± 3.73*#
|
76.91 ± 3.09
|
68.27 ± 3.25*
|
51.57 ± 3.87#$
|
62.19 ± 3.54$&
|
73.27 ± 3.18$
|
% FS
|
49.06 ± 2.81
|
37.91 ± 2.86*
|
42.05 ± 3.01*#
|
50.51 ± 3.13
|
40.83 ± 3.26*
|
29.91 ± 3.54#$
|
35.53 ± 2.97$&
|
42.09 ± 3.83
|
LV Mass (mg)
|
84.23 ± 2.05
|
82.01 ± 3.41
|
82.87 ± 2.53
|
83.25 ± 2.43
|
84.05 ± 2.73
|
82.41 ± 3.27
|
82.93 ± 2.96
|
83.71 ± 3.03
|
Abbreviations: EF, ejection fraction; FS, fractional shortening; IVS, interventricular septum; LV mass, left ventricular mass; LVID;d, left ventricular internal diastolic diameter; LVID;s, left ventricular internal systolic diameter; LVPW, left ventricular posterior wall; LV vol;d, left ventricular end diastolic volume; LV vol;s, left ventricular end systolic volume. Data are presented as means ± SD (n = 6 at least in each group). *P<0.05 vs. Ctrl group; #P<0.05 vs. T2D group; $P<0.05 vs. Klf15-cKO group; &P<0.05 vs. Klf15-cKO/T2D group.
Echocardiography
Transthoracic echocardiography was measured by a Vevo 770 ultrasound system (Visualsonics, Toronto, Canada) equipped with a high frequency ultrasound probe (RMV-707B) as described previously (36). Mice were anesthetized with intraperitoneal injection of 1.2% Avertin (Sigma, St. Louis, MO) and placed in the supine position on a temperature-controlled platform. The chest hair was removed with a depilatory to reduce ultrasound attenuation. The images were recorded in parasternal long-axis and short-axis views. The LV wall thicknesses and dimensions were measured in parasternal short axis M-mode images. At the same time, the ejection fraction (EF), fractional shortening (FS), and LV mass were calculated using Vevo770 software. The data were averaged over 10 cardiac cycles.
Western blotting
The cardiac tissues were homogenized in lysis buffer and proteins were collected by centrifuging at 12,000 × g at 4 °C (37). Western blots were performed according to our previous studies (37). Briefly, the proteins were separated on 10% SDS-PAGE gels, and then were transferred to a nitrocellulose membrane. The membrane was blocked with a 5% non-fat dried milk for 1 h and incubated overnight at 4 °C with the following antibodies: anti-phospho-p38 MAPK (Thr180/Tyr182), anti-p38 MAPK and anti-p38α MAPK (1:1000, Cell Signaling, Beverly, MA, USA), anti-CTGF and anti-p38β MAPK (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-CXCR7, anti-TGF-β1 and anti-Fibronectin (1:1000, Proteintech, Rosemont, IL, USA), anti-KLF15, anti-Collagen I and anti-CXCR4 (1:1000, Abcam, Cambridge, MA, USA) respectively were prepared in 5% milk in Tris-buffered saline (pH 7.2) containing 0.05% Tween 20 (TBST) solution. After removal of unbound antibodies using TBST, membranes were incubated with the secondary antibody for 1 h at room temperature. Antigen-antibody complexes were visualized using an enhanced chemiluminescence detection kit (Thermo Scientific, Barrington, IL, USA). In order to determine loading, blots were stripped using stripping buffer (Sigma Aldrich, St. Louis, MO, USA) and reprobed for β-actin as loading control of total protein. Histone was used as loading control of nuclei proteins. Quantitative densitometry was performed on the identified bands using a computer-based measurement system, as employed in previous studies (37).
Statistical analysis
Data were collected from the repeated experiments at least three times for in vitro studies and six animals at least for in vivo study and were presented as mean ± SD. One-way analysis of variance (ANOVA) was used to determine whether differences exist and if so, a post hoc Tukey’s test was used for analysis of the difference between groups, with Origin 7.5 laboratory data analysis and graphing software. Statistical significance was considered as p < 0.05.