2.1 Mouse preparation
In this study, male SD rats of 8–10 g weight and 1–3 days of age were raised in the Animal Breeding Center of Beijing Viewsolid Biotech Co., Ltd. [SYXK (Beijing) 2016-0039., SPF-level management of the Experimental Animal Center. The experimental animals were provided by Beijing Viewsolid Biotech Co., Ltd [SCXK (Beijing) 2016-0009.. All experimental mice were cared for in accordance with the 3R principle. The approval number of the Experimental Animal Ethics Committee is 20200037.
2.2 Primary cardiomyocyte culture
After the suckling mice were immersed in 75% alcohol for disinfection, their hearts were immediately opened in an ultra-clean workbench in order to be extracted and washed twice in a glass dish filled with ice PBS solution (4 °C PBS). Following this, the hearts were transferred to a 10 ml sterile centrifuge tube, and digestive enzyme (containing 0.1% trypsin) prepared with PBS solution for digestion was added. The entire digestion process was carried out under constant temperature and stirring at 37 ℃, 130 rpm/min. After every 8 min of digestion, the supernatant was sucked in the centrifuge tube, it was added to the same amount of DMEM-F12 culture medium containing 10% FBS and a pipette was used to evenly mix it. This process was repeated approximately 7–8 times until the tissue block was completely digested, the collected tubes were centrifuged at 1000 rpm for 6 min at room temperature, the supernatant was discarded, the cells were resuspended in a DMEM medium containing 10% FBS the cardiomyocytes obtained each time were combined in a petri dish and statically incubated in an incubator containing 5% CO2 at 37 °C. The morphology, beating frequency, rhythm and intensity of the cardiomyocytes were observed under an electron microscope following the culturing in an incubator containing 5% CO2 at 37°C for 24 hours, and their growth status was gauged.
2.3 HL1 cardiomyocyte culture
The cells were cultured in a DMEM-F12 medium containing 10% fetal bovine serum. When the myocardial cell density grew to 70–80%, the cells were transferred out of the culture according to 1:4.
2.4 Construction of MH cell model
When the myocardial cell density grows to 60–70%, the cell culture medium is changed to a serum-free DMEM-F12 one, starved for 12 h. 1x10-^6 mmol angiotensin (AII) is added, and the stimulation time is set to 0 h, 6 h, 12 h, 24 h and 48 h. The mRNA expressions of ANP, BNP and PACSIN2 were detected to confirm the success of the cell model.
2.5 Cellular drug treatment
In order to achieve high expression of PKC in cardiomyocytes, the PKC agonist drug PMA is used. The concentration of the drug added to the cells is based on the amount provided in the instructions. The expression of 12 h ANP, BNP and PACSIN2 mRNA and the expression of PKC, PACSIN2 and RCAN1.4 protein are detected.
2.6 Plasmid construction and transfection
A PACSIN2 shRNA (sh-PACSIN2) plasmid was constructed, and the design and construction were completed by Beijing Viewsolid Biotech Co., Ltd. The purpose of constructing siRNA was to downregulate the expression of PACSIN2. According to the manufacturer's instructions, polybrene (viral transfer aid) is diluted at the ratio of transfer aid: virus = 1:500, and 500 μl medium is added 2 h after adding the cells.
2.7 Cellular immunofluorescence histochemistry
The cells were diluted in multiples in a 96-well plate (2000 is the most effective). They were fixed with cold 4% paraformaldehyde for 30 min, washed 3 times with PBS for 5 min each time, permeated with 0.3% Trition for 5 min (diluted with PBS), washed 3 times with PBS for 5 min each time. They were blocked with 5% goat serum (PBS) on a room temperature shaker for 1 h; the primary antibody was incubated overnight at 4 °C: α-actin (1:50); then, the primary antibody was recovered and washed 3 times with PBS for 5 min each; the PBS (1:1000) Dilution was used; the absorbance of the rabbit secondary antibody was 488 nm. This was incubated in an oven at 37 °C in the dark for 1 h; it was washed with PBS 3 times, for 5 min each time, and was observed under a microscope.
2.8 Quantitative reverse transcription polymerase chain reaction
The total RNA from primary cardiomyocytes and HL1 cells were extracted using Trizol reagent (Invitrogen) according to the manufacturer’s protocol for α-MHC and β-MHC mRNA expression; 1 mg of total RNA was reverse-transcribed into cDNA using PrimeScript ™ RTMaster Mix (TaKaRa, Dalian, China). Then, real-time PCR was performed using a SYBR ® Premix Ex TaqTM II kit (TakaRa) on the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).
2.9 West Blotting
The total protein was extracted using a RIPA lysis buffer (lifetime) for 20 min on ice. Its concentration was determined with a BCA protein assay kit (general, Shanghai, China). Equal amounts of protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to PVDF membranes. After being blocked with TBST buffer containing 5% nonfat skim milk for 1 h, the membranes were incubated overnight with primary antibodies against proliferating cell nuclear antigen (PACSIN2, 1:5000; Abcam, Cambridge, MA, USA), PKC (1:1000; Abcam), RCAN1.4 (1:1000), RCAN1.1 (1:1000) and GAPDH (1:10000; Cell signaling technology, Danvers, MA, USA) at 4 ℃. The membranes were then washed with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody. The blots were developed using an Enhanced Chem luminescence Detection System (Thermo Scientific, Carlsbad, CA, USA)
2.10 Statistical analysis
All data was presented as mean ± standard deviation (SD) and analyzed with the help of GraphPad Prism 7.0. The comparisons were performed with one-way ANOVA followed by Bonferroni post-hoc test. The statistical significance was defined as P < 0.05.