Animals and Reagents
All animal experiments were approved by the Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine (Shanghai, China). 8 ~ 10 weeks old male wild-type (WT) C57BL/6J mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. RCAN1 knockout mice were kindly provided by Dr. Jian Wu from Institute of Biomedical Sciences, Fudan University. Generation of RCAN1−/− mice has been described previously [11]. Exon 2 ~ 3 was removed from RCAN1 gene (ENSMUST00000060005) using CRISPR/Cas9 strategy. Mouse genotyping was performed by PCR and agarose gel electrophoresis. All the mice were raised alone in specific pathogen-free conditions. Mice were randomly divided into four groups: the wild-type (WT) control group, RCAN1−/− control group, WT mice with intraperitoneal LPS (10 mg/kg) injection (WT-LPS group) and RCAN1−/−-LPS group. In addition, mice from RCAN1−/−-LPS group were randomly selected for intraperitoneal injection of Mdivi-1 (10 mg/kg, twice per week, 4 weeks, S716201, Selleck.cn, USA); or for intraperitoneal injection of KN93 (5 mg/kg, twice per week, 4 weeks, S6787, Selleck.cn, USA).
Measurement of echocardiography
Cardiac function of mice was measured by a preclinical ultrasound system (Vevo 2100, FUJIFILM Visual Sonics, Canada) after LPS injection for 4 weeks. In brief, mice were anesthetized with inhaled 1.5% isoflurane and were placed on a heating pad to maintain normal body temperature and heart rate at about 500 beats/min. The intercept angle between the Doppler beam line and flow direction was controlled within 60°, and M-mode echocardiographic images were recorded. The left ventricular end systolic inner diameter (LVIDs) and left ventricular end diastolic inner diameter (LVIDd) were measured from the M-mode view; and left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were calculated and analyzed. All the measurements were double-blind.
Cell culture and isolation of cardiomyocytes
H9c2 cells, a cardiomyoblast cell line originally from the left ventricle of rat heart, was purchased from Shanghai Institute for Biological Sciences, Chinese Academy of Science (Shanghai, China). The cells were cultured in DMEM/F-12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a 37°C, 5% CO2 incubator.
Adult mouse (6 ~ 8 weeks) ventricular cardiomyocytes were isolated from RCAN1−/− and WT mice with C57BL/6 background using standard enzymatic method as previously described. The animal procedure was approved by the Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine (Shanghai, China). In brief, mice were anesthetized and hearts were excised. Then the hearts were transferred to a 6-cm dish containing fresh EDTA buffer. Cell digestion was completed by a sequential injection with EDTA buffer, perfusion buffer and collagenase buffer into the left ventricle. After stopping digestion and tissue dissociation, the isolated cardiomyocytes were stabilized in 1.0 mM Ca2+ Tyrode solution (126 mM NaCl, 5.4 mM KCl, 1.0 mM CaCl2, 1.0 mM MgCl2, 0.33 mM NaH2PO4, 10 mM HEPES, and 10 mM glucose) for 10 min, and then suspended in Dulbecco's modified Eagle medium Minimum Essential Medium (DMEM) with 10% FBS. Cells were dispersed in 3-cm dishes precoating with laminin (10 µg/ml) at the glass bottom, and cultured in 5% CO2 and 37°C incubator. After 1 hour and every 24 hours thereafter, the media was changed with fresh, prewarmed culture medium.
For laser confocal scanning, the isolated cardiomyocytes were cultured with FBS free medium for 2 hours and stimulated by LPS for 6 hours, then cells were fixed by 0.5% paraformaldehyde for 1 hour. After pretreating with 0.1 Triton X-100 for 10 min, cells were blocked with 1% BSA for 1 hour, and then were incubated with anti-p-Drp1Ser616(1:200, #3455, CST, US) and anti-p-CaMKIIThr286(1:200, ab171095, Abcam, US) at 4°C overnight. After incubating with the respective fluorescent labeled second antibodies, cells were visualized under laser confocal microscopy.
Serum LDH determination
Blood samples were collected by extracting the eyeball blood after mice were anesthetized with isoflurane. Then, all the samples were centrifugated at 3000 rpm for 5 min, serum supernatant was carefully collected and stored at − 80°C for subsequent analysis. Serum level of lactate dehydrogenase (LDH), one of the major cardiac injury markers, was determined using commercial kits (A020-1, Nanjing Jiancheng Biotechnology Institute, Nanjing, China) according to the manufacturer’s instructions.
Transmission electron microscope (TEM) analysis
Left ventricular tissues of mice were minced into 0.5 cm3 small pieces and were prefixed in 2.5% glutaraldehyde in PBS at 4°C overnight and post-fixed with 1% buffered osmium tetroxide for 2 h at 4°C. Then, the specimens went dehydrated with graded ethanol and two final 15min. rinses in 100% ethanol. After being embedded, the specimens were taken for ultrathin sections and were doubly stained with uranyl acetate and lead citrate. Ultrathin sections were examined using a transmission electron microscope (JEM-1010, JEOL, Japan).
Mitochondrial ROS and TMRE examination
Mouse hearts were rapidly removed and placed into ice-cold Krebs-Henseleit buffer containing 2.5mM CaCl2, 115mM NaCl, 25mM NaHCO3, 5.9mM KCl, 1.18mM MgC12, 1.23 mM NaH2PO4, 1.2 mM Na2SO4 and 10 mM glucose. Hearts were then perfused with Krebs-Henseleit medium for 10 min at 37°C, and the fluorescent probe, 2′,7′-Dichlorofluorescein diacetate (DCFH-DA) or tetramethylrhodamine ethyl ester (TMRE) was infused into the perfusion medium immediately prior to the perfusion at a final concentration of 50 µM or 5 µM. Then, mitochondria from the perfused heart tissues were isolated by a rapid tissue mitochondrial isolation kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Mitochondrial particles were washed, resuspended and ultrasonic crushed in 1.5 ml ice-old PBS buffer. After centrifuging at maximum speed 14000 g for 30 sec, the supernatant was collected and placed on ice, and fluorescence intensity was detected on a Biotek Synergy HT fluorometric plate reader (Biotek). DCF fluorescence was examined at an excitation wavelength of 488 nm and an emission wavelength of 525 nm; and TMRE fluorescence was examined at an excitation wavelength of 535 nm and an emission wavelength of 587 nm.
H&E and Masson’s trichrome staining
A thin midsection of heart was fixed in 4% paraformaldehyde, embedded in paraffin and cut into 5-µm-thick sections. The sections were then deparaffinized in xylene, dehydrated with graded concentrations of ethanol. The sections were stained with H&E for cardiomyocyte size determination and Masson's trichrome for collagen deposition. For each image, collagen volume fraction was calculated as the ratio of collagen surface area (aniline blue) with respect to myocardial surface area (red).
Western blot analysis
LV tissue or isolated cardiomyocytes were homogenized in protein lysis buffer with 1mM PMSF (Beyotime Biotechnology, Shanghai, China). Protein supernatants were extracted from the lysis buffer after centrifuging at maximum speed 14000 g for 15 min at 4°C and heated at 95°C for 3 min for denaturation. Proteins (20 µg) were separated on 8 ~ 12% SDS–PAGE gels and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). The membranes were blocked in PBS containing 1% skim milk powder for 1 h at room temperature and incubated with the primary antibodies at 4°C overnight, including anti-RCAN1(1:1000, ab140131, Abcam, US), anti-p-ERK1/2(1:1000, #4370, CST, US), anti-NFAT3(1:2000, A302-769A, Bethyl Laboratories, US), anti-Drp1(1:1000, #8570, CST, US), anti-p-Drp1Ser616(1:1000, #3455, CST, US), anti-p-CaMKIIThr286(1:1000, ab171095, Abcam, US), anti-β-actin (1:1000, #3700, CST), anti-GAPDH(1:1000, #2118, CST). Then, the membranes were washed with TBST (Tris-buffered saline plus 0.1% Tween-20) for 3 times and were incubated with the secondary antibodies (peroxidase-affinipure goat anti-rabbit IgG) for 0.5 hour at room temperature. The membranes were again washed with TBST for 3 times and were treated with enhanced ECL chemiluminescence reagents (Thermo Fisher Scientific). Blots from the membranes were visualized by using the ChemiDoc Imaging System (Bio-Rad).
Statistical analyses
Data were reported as means ± SEM. The comparisons were performed using unpaired Student’s T-test or one-way ANOVA followed by a Tukey multiple comparison posthoc test. A P value less than 0.05 was considered significant difference.