Construction of C3G Knockout Recombinant Lentiviruses
The candidate sequences knockouting specifically all 13 variant mRNA transcripts of rat C3G gene were designed (http://www.e-crisp.org/E-CRISP/)  and cloned respectively into pLenti-Cas-Guide lentiviral vector (Origene Co., Ltd, Rockville MD, USA). RT-PCR (reverse transcription polymerase chain reaction) and protein immunoblot screening experiments revealed that the recombinant vector containing the targeting sequence was the most effective one to knockout C3G gene in the rat-derived H9C2 cardiac myoblastic cell line (purchased from American Type Culture Collection, Manassas, VA, USA), and was used in subsequent experiments to knockout rat endogenous C3G mRNA (Table 1). The non-target (NT) was also cloned into the pLenti-Cas-Guide lentiviral vector, and formed a NT recombinant lentiviral vector, which was used for negative control (Table 1).
The C3G knockout (KO) and NT recombinant vectors were packaged into CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 recombinant lentiviruses (x 107 TU/ml) respectively in 293T cells through using Lenti-vpak Lentiviral Packaging Kit, and selected respectively. DNA sequencing experiment revealed that the sequence knocking out C3G gene was successfully inserted into the recombinant lentivirus.
Cell Culture in Vitro and Treatment with C3G Knockout Lentivirus and Hypoxia
Primary cardiac fibroblastic cell line (ScienCell Research Laboratories, Carlsbad, CA, USA) and H9C2 cardiac myoblastic cell line were cultured as described previously [7, 9]. An equal number of the cells (1 x 105) were infected with C3G KO and NT recombinant lentiviruses respectively. The cells were divided into C3G KO and NT groups, and subjected to treatment with hypoxia [7, 9]. The cells were finally randomly divided into NT, C3G KO, NT + hypoxia and C3G KO + hypoxia groups. The cells were washed with PBS, and harvested at indicated time points respectively for experimental analysis by RT-PCR (reverse transcription-polymerase chain reaction), Western immunoblot, MTT [3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide] assay and flow cytometry.
RT-PCR Analysis for C3G mRNA
Total RNA was purified, quantified and reversely transcribed into cDNA as described previously [7, 9]. The generated cDNA was amplified using specific primers of rat C3G and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) genes respectively (Table 1). RT-PCR products were then analyzed.
Western Immunoblot Experimental Analysis for C3G, p-ERK1/2, Bcl-2 and Bax Proteins
Protein isolation and its concentration measurement, electrophoretic separation and membrane transferring were similar as previously described [7, 8]. After adding anti-rat C3G, p-ERK1/2, Bcl-2, Bax, GAPDH rabbit or mouse primary antibodies respectively, the membrane was incubated overnight, and then incubated with anti-rabbit or anti-mouse second antibody. The intensity of immunoreactive band was then determined [7, 8]. The C3G / GAPDH, p-ERK1/2 / GAPDH, Bcl-2 / GAPDH and Bax / GAPDH represented C3G, p-ERK1/2, Bcl-2 and Bax protein expression levels respectively.
MTT Assay for Cell Viability in Vitro
At the designated time points (24 h, 72 h) after infection, MTT working solution was added to cells in each well plate. Then subsequent performances were similar as described previously[7, 9]. The cell proliferative rate was counted as (absorbance at 72 h – absorbance at 24 h) / absorbance at 24 h, and expressed as a percentage.
Flow Cytometry Analysis for Cell Apoptosis in Vitro
Seventy-two hours after infection, the apoptotic and necrotic cells were labeled respectively with annexin V fluorescein isothiocyanate (FITC) and propidium iodide (PI). The labeled cells were then sorted by ﬂow cytometry as described previously [7, 9]. The cell apoptotic rates were calculated as the percentage of the number of the annexin V FITC positive and PI negative cells over the number of the total cells.
Myocardial Infarction Model and Experimental Protocol
Sixty Sprague-Dawley adult male rats (body weight (BW), 267.1 ± 19.2 g) were divided randomly into sham-operation (sham) + NT (n = 15), Sham + C3G KO (n = 15), MI + NT (n = 15) and MI + C3G KO (n = 15) groups respectively. MI model and sham-operation were established as described previously . After establishment of MI model and sham-operation, surviving rats were as follows: in sham + NT CRISPR/Cas9 (n = 14), sham + C3G CRISPR/Cas9 (n = 13), MI + NT CRISPR/Cas9 (n = 11) and MI + C3G CRISPR/Cas9 (n = 10) groups. For each injection point, C3G KO or NT lentiviruses (25 μl) were then injected to myocardium at the following points; 3 points located in the infarcted central zones, and another 8 points in the border zones in each rat with MI or at the corresponding injection points in each reference rat in the above 4 groups respectively (Fig. 3(d)). The chest was then closed, and the rats were treated as described previously . The animals were supplied by the Laboratory Animal Center of Chongqing Medical University, Chongqing, China. All the animal experiments were approved by our institute laboratory animal committee.
Transthoracic doppler echocardiographic study was performed as described previously . One week and 12 weeks after treatment with C3G KO or NT lentiviruses, the number of surviving rats were shown in each group (Table 2). Interventricular septal thickness (IVST), left ventricle (LV) posterior wall thickness (LVPWT), end-diastolic diameter (LVEDD) and ejection fraction (LVEF) were measured from the M-mode tracings of echocardiography Vivid 7 with a 3.5-MHz sector scan probe (General Electric Medical Systems, Milwaukee, WI, USA). The sonographer, who collected the data, was kept unaware of which treatments the rats were assigned to.
Histology and Collagen Volume Fraction Examination
Twelve weeks after sham-operation and MI, BW was measured again; all rats were then euthanized after the echocardiography. The heart was excised rapidly, and the LV was separated from other parts of the heart and weighed. The LV/BW ratio was calculated. Histological slice was obtained and stained with hematoxylin and eosin and picrosirius red respectively. Then infarction size and collagen volume fraction (CVF) were measured as described previously . The myocardium in remote zone around the infarcted lesion scar in LV was frozen in liquid nitrogen, and stored in the -80°C freezer for Western immunoblot examination.
Detection of Cardiomyocytic Apoptosis in Myocardium
According to the instruction of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) in situ cell death detection kit (No. 11684817910, F. Hoffmann-La Roche Co. Ltd., CH-4070 Basel, Switzerland), the slice was incubated in TUNEL reaction mixture and converter-peroxidase. After the incubation period, the slice was added with diaminobenzidine substrate. The cardiomyocyte with brown nucleus was defined as apoptotic cell, while the cell with blue nucleus was defined as normal cell. The apoptotic rate was calculated as the ratio of apoptotic to total cell count of cardiomyocytes in each field, and an averaged apoptotic rate was calculated .
Data is presented as mean ± standard deviation (SD). All analyses were performed using the SPSS22.0 statistical software. The statistical significance between the groups was evaluated by one-way ANOVA, then, in case of significance, by a two-side Tukey test for multiple comparisons. A value of p < 0.05 was considered statistically significant.