Animal model and treatment protocols
Our animal experimental protocol was approved by the Animal Care and Use Committee of Tianjin Union Medical Center. A total of 70 male adult Sprague-Dawley (SD) rats (weight, 200-250 g) were used in this study, which were provided by the Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animals were adapted to the new environment for 1 week prior to experiments, and they had free access to foods and water under laboratory conditions. All animals were randomized into two groups, namely, the ISO-induced cardiomyopathy group (n=60) and the control group (n=10). Typically, the ISO-induced cardiomyopathy group was given subcutaneous injection of isoproterenol (ISO) (Purty: 98%, Beijing Solarbio Science & Technology Co., Ltd, China) dissolved in normal saline (NS) at 10 mg/kg/d for once daily for the initial 2 consecutive weeks. The control group was given injection of NS at identical dose for the initial two weeks consecutively. Afterwards, 50 surviving animals from ISO-induced cardiomyopathy group were further classified into 5 groups, namely, low-dose TMYXP was given TMYXP (0.5 g/kg) administration for 6 weeks,,middle-dose TMYXP was given TMYXP (1 g/kg) administration for 6 weeks, high-dose TMYXP was given TMYXP (2 g/kg) administration for 6 weeks and The BIS group was given bisoprolol (BIS, 0.5 mg/kg) for 6 weeks as a positive control group, while those in ISO and control groups were given NS gavage at identical volume once a day for six consecutive weeks.
At the end of week 8, animals were euthanized, and blood samples were collected into tubes. At the same time, fresh cardiac tissues were rinsed with cold saline, of them, a fraction was utilized for mitochondrial protein extraction, while another part was used for monoplast suspension preparation to determine ROS and MMP, and the rest tissues were preserved at -80 °C for subsequent use. Moreover, the intact heart had been fixed using 4% paraformaldehyde, followed by paraffin embedding prior to pathological and TUNEL staining.
Echocardiography
At weeks 2 and 8, all animals were injected with 3% sodium pentobarbital solution at 10.0 mg/kg for anesthesia before echocardiographic measurements. Typically, data on the left ventricular end diastolic diameter (LVESD), left ventricular end diastolic diameter (LVEDD), left ventricular fractional shortening (LVFS%), and left ventricular ejection fraction (LVEF%), were determined based on short-axis M-mode echocardiography using the Vevo2100 imaging system (VisualSonics, Toronto, ON, Canada).
HWI assessment
The excised cardiac tissues were washed with ice-cold PBS; later, the adipose tissue, aorta and atria were separated, and the heart weight (HW, mg) was measured. The heart weight index (HWI, mg/g) was defined as the ratio of HW-to-body weight.
Histopathological analysis
Ventricular tissues were fixed with 4% paraformaldehyde, followed by paraffin embedding, sectioning (5 μm in thickness), and staining using Masson’s trichrome as well as hematoxylin-eosin (H&E) solution. Finally, the collagen contents and morphological changes of cardiac tissues were assessed using the light microscope (DS-Ri2, Nikon, Japan).
Determination of serum BNP
After standing for 30 min, blood samples were centrifuged for 15 min at 3000 g to separate the serum. Thereafter, the serum levels of B-type natriuretic peptide (BNP) were determined using the ELISA kits (Elabscience Biotechnology Co., Ltd, Wuhan, China).
Assessment of oxidase/antioxidase activities
Cardiac tissues (10% w/v) were homogenized using cold saline, followed by 10 min of centrifugation at 3000 g to collect the supernatant. Then, the contents of malondialdehyde (MDA), glutathione peroxidase (GSH-Px) and manganese superoxide dismutase (Mn-SOD) were measured in accordance with reagent protocols (Nanjing Jiancheng Bioengineering Institute, China)
Determination of the mitochondrial complexes I, II, III and IV activities
Myocardial mitochondria were extracted, and the mitochondrial respiratory chain complexes I, II, III and IV activities were measured using the Mitochondrial respiratory chain complexes I, II, III and IV Activity Assay kits (Beijing Solarbio Science & Technology Co., Ltd, China) following the manufacturer protocols.
Determination of ATP contents
Cardiac tissues (10% w/v) were homogenized using boiling double distilled water: The samples were then placed in a boiling water bath for 10 min, followed by 10 min of centrifuged at 3500 g to collect the supernatant. Thereafter, the ATP level was measured according to the reagent protocols (Nanjing Jiancheng Bioengineering Institute, China).
2.10 Flow cytometry
Cardiac tissues were cut into 1 mm3 pieces by ophthalmology, digested with trypsin in the constant temperature water bath (37 ° C) for 30 min, and filtered with the 300-mesh nylon filter. Afterwards, the filtrate was centrifuged at 600×g for 5 min at 4 °C, the supernatant was discarded, and the sediment was re-suspended with cold PBS for several times. The above-mentioned steps were repeated for three times, and finally the sediment was suspended with cold PBS. Then, the monoplast suspension was loaded using the DCFH-DA probes and JC-1 probes in accordance with the active oxygen detection kit (Nanjing Jiancheng Bioengineering Institute, China) and the Mitochondrial membrane potential assay kit with JC-1(Beyotime Biotechnology, Shanghai, China). The monoplast was washed with PBS for three times after incubation at 37 °C; finally, the DCF and JC-1 fluorescence intensity were determined through FC using the Guava EasyCyte flow cytometer (EMD Millipore Corporation, Hayward, Calif).
TUNEL assay
The apoptosis level was determined according to the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay performed using the One Step TUNEL Apoptosis Assay Kit (Dalian Meilun Biotechnology Co., LTD, China) following the manufacturer protocol. Cells with bright blue nuclei were deemed as positive for apoptosis. Five representative fields were randomly selected at ×200 magnification.
Protein extraction and Western blotting
Myocardial tissues were cut into pieces and homogenized within the lysis buffer containing 100×phosphatase inhibitor, 100 mM PMSF and 1000× protease inhibitor (KeyGen Biotech. Inc, Nanjing, China). Later, the homogenization solution was transferred into the ep tube to centrifuge at 12000 g for 5 min at 4 °C. Concentration of the supernatant was determined using the BCA protein assay kit (Beijing Solarbio Science & Technology Co., Ltd, China). Moreover, cytoplasmic protein was extracted according to operation procedure from KeyGen Biotech. Inc (Nanjing, China). In brief, tissues were homogenized, placed on ice for 30 min, and centrifuged at 3000 g for 10 at 4 °C, and the supernatant was cytoplasmic protein. To carry out immunoblotting, proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE), followed by transfer onto the polyvinylidene difluoride (PVDF) membranes. Then, the PVDF membranes were blocked with 5% defatted milk for 2 h at room temperature, and incubated with primary antibodies diluted by TBST overnight at 4 °C. The membranes were incubated with the following primary antibodies: SIRT3 (1:1000, Proteintech Group, Inc, # 10099-1-AP), PGC-1α (1:1000, Proteintech Group, Inc, #66369-1-Ig), NRF1 (1:500, BOSTER, #AO1129-2), TFAM (1:500, BOSTER, #PB0413), Cleaved Caspase-3 (1:1000, Affinity, # AF7022), Cyt C (1:800, Proteintech Group, Inc, # 10993-1-AP), GAPDH (1:1000, Cell Signaling Technology, #5174). After washing with TBST for 3 times, the membranes were incubated with peroxidase (HRP)-conjugated secondary antibody, and visualized using the luminol reagent (Engreen Biosystem Co, Ltd., Beijing, China).
Statistical analysis
All experimental data were expressed as means±S.D. The SPSS 19.0 (Lead Technologies, Chicago, Illinois) was utilized for all statistical analyses. Significance was determined by one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. A difference of P < 0.05 was considered as statistically significant.