Experimental animals

Syt1 heterozygous knockout (Syt1+/−) mice were constructed on the C57BL/6J background by deleting the exon 6 to exon 8 in Syt1 gene using the CRISPR/Cas9 approach. Syt1−/− mice were not used as they usually died after birth. WT littermates were used as controls. Genotype identification of Syt1+/− mice was performed based on PCR assay using genomic DNA extracted from mouse tails. Syt1+/− mice and WT littermates were housed in cages (4 − 5 mice per cage) under a 12:12 h light: dark cycle and had free access to regular chaw and water. Male mice of all the strains were used in the experiments. Experimental procedures involving the care and use of animals were approved by the Animal Care and Use Committee of Shanxi Medical University (Approval ID-SYDL2024007) and the experimental protocols conformed to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health.

Transverse aortic constriction (TAC)

TAC was performed to induce cardiac hypertrophy in 8–10 weeks old male mice. Mice of both strains (Syt1+/− and WT) were anesthetized with 2% isoflurane inhalation. Animal were then limb fixed in a supine position. A median thoracotomy was performed at the level of the first intercostal space. The aortic arch between the innominate and left common carotid arteries was isolated and transversely constricted using a 27 G cannula. The needle was immediately removed, leaving a stenotic lumen in the aortic arch. Sham mice underwent an identical surgical procedure without aortic banding. Finally, the chest walls were closed and animals were allowed to live for 8 weeks after TAC.

Echocardiography

Doppler echocardiography for small animals were performed to measure cardiac structure and function of the mice eight weeks after TAC or sham surgery. A Prospect 3.0 high-resolution in vivo imaging system (S-Sharp) with a scanhead (a 30–50 MHz transducer) was used for echocardiographic analysis. Mice were anesthetized with 2% isoflurane inhalation before analysis. M-mode images and two-dimensional images were obtained in a short-axis view. Echocardiographic parameters including LV diastolic and systolic internal dimensions (LVIDd, LVIDs), LV diastolic and systolic posterior wall thickness (LVPWd, LVPWs) and left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were then measured and analyzed.

Histology

Eight weeks after TAC or sham surgery, mice were deeply anaesthetized with isoflurane, hearts were transversely perfused via the aortae by ice-cold 1 × PBS, and then hearts were harvested. Heart tissues were fixed with 10% neutral buffered formalin for overnight, then embedded in paraffin, and cut to 5-μm sections. Tissue sections were deparaffinized and then stained with hematoxylin & eosin (H&E) or Sirius red to evaluate the microstructures including hypertrophy and fibrosis. To perform H&E staining, the sections were soaked with hematoxylin for 3 min and then washed with running tap water for 1 min. Then, the sections were blued with 0.1% ammonia water, followed by staining with 0.1% eosin for 2 min. The sections were dehydrated with ethanol and transparentized with xylene, and sealed with neutral resin. To perform Sirius red staining, tissue sections were stained with Sirius Red staining kit (G1472, Solarbio Life Science, Beijing China). The sections were incubated in 0.1% Sirius red solution for approximately 8 min and then rinsed with absolute alcohol, followed by xylene transparency and sealed with neutral gum. The stained sections were visualized and photographed under a microscope (Olympus, Tokyo, Japan) at a 20 × magnification. For fibrosis area quantification, at least ten random fields of each sample were analyzed. Briefly, the stained-red fibrotic areas were measured by adjusting the color-based threshold. The total fibrosis areas and the percentage of fibrosis area over the whole inspected areas in each slide were calculated [11] using the Image J software (National Institutes of Health).

RNA extraction and quantitative real-time PCR

The total RNA was isolated from the left ventricular (LV) tissues using Trizol reagent according to the manufacturer’s protocol. Extracted RNA was reversely transcribed to cDNA using PrimeScript RT reagent kit (RR064A, TaKaRa, Toyobo, Japan). Real-time quantitative PCR (qPCR) was performed by using SYBR premix Ex Taq (RR420, TaKaRa, Toyobo, Japan) on a QuantStudio real-time PCR system (Life Technologies, Thermo Fisher Scientific). PCR primer sequences used were listed below.

ANP:

forward: 5′-TCTTCCTCGTCTTGGCCTTT-3′

reverse: 5′-CCAGGTGGTCTAGCAGGTTC-3′

BNP:

forward: 5′-TGGGAGGTCACTCCTATCCT-3′

reverse: 5′-GGCCATTTCCTCCGACTTT-3′

β-MHC:

forward: 5′-ACTGTCAACACTAAGAGGGTCA-3′

reverse: 5′-TTGGATGATTTGATCTTCCAGGG-3′

Col1a1:

forward: 5′-CTGGCGGTTCAGGTCCAAT-3′

reverse: 5′-TTCCAGGCAATCCACGAGC-3′

Col3a1:

forward: 5′-TGAATGGTGGTTTTCAGTTCAG-3′

reverse: 5′-GATCCCATCAGCTTCAGAGACT-3′

GAPDH:

forward: 5′-AGAACATCATCCCTGCATCC -3′

reverse: 5′-AGTTGCTGTTGAAGTCGC-3′

Western blotting

Small pieces of mouse left ventricular tissues or H9C2 cells were homogenized in RIPA buffer (Beyotime, Shanghai, China) to extract total proteins. The protein concentrations in samples were measured using the BCA protein assay kit (Beyotime, Shanghai, China). Extracted proteins were separated with 10% SDS-PAGE and then were transferred onto PVDF membranes (Millipore, Billerica, MA, USA), followed by treatment with the blocking buffer. The membranes were incubated with respective primary antibodies at 4 °C for overnight. After four 5-min washes in TBST, the membranes were incubated with HRP-conjugated secondary antibody for 1 h. Then the positive blots in the membranes were visualized by autoradiography using ChemiDoc™ (Bio-Rad Laboratories, Hercules, CA, USA). β-actin was used as an internal control. The antibodies used were: anti-SYT1 (1:1000, ab254104, Abcam, Cambridge, UK), anti-B cell lymphoma-2 (Bcl-2) (1:1000, ab196495, Abcam, Cambridge, UK), anti-Bcl 2-associated X (Bax) (1:1000, ab32503, Abcam, Cambridge, UK), anti-p-p38 (Thr180/Tyr182) (D3F9) (1:1000, #4511, CST, Danvers, MA,USA), anti-p38 (D13E1) (1:1000, #8690, CST, Danvers, MA, USA), anti-p-JNK (Thr183/Tyr185) (81E11) (1:1000, #4668, CST, Danvers, MA, USA), anti-JNK (1:1000, #9252, CST, Danvers, MA, USA), anti-p-ERK1/2 (Thr202/Tyr204) (D13.14.4E) (1:1000, #4370, CST, Danvers, MA, USA), anti-ERK1/2 (137F5) (1:1000, #4695, CST, Danvers, MA, USA), and anti-β-actin (13E5) (1:1000, #4970, CST, Danvers, MA, USA). The secondary antibodies used were goat antimouse (1:1000, ZB-2305, Zhongshan Company, Beijing, China) and goat antirabbit (1:1000, ZB-2301, Zhongshan Company, Beijing, China).

Enzyme-linked immunosorbent assay (ELISA) analysis

Ang II levels in left ventricular tissues and serum were measured using Ang II ELISA kits (JL31138-96 T, JONLNBIO, Shanghai China) following the manufacturer’s instructions. Briefly, the microplate wells are precoated with mouse Ang II. Then the Ang II standards, samples and biotin-labeled antibody were added into the microplate and incubated at 37 °C for 1 h. After washing, 100 µL of HRP conjugated streptavidin was added to the microplate and incubated for 30 min and washing, followed by adding 100 µL of 3,3´,5,5´-tétraméthylbenzidine (TMB) for color development. The reaction was stopped with 50 µL/well of stopping solution, and the absorbance was measured at 450 nm. By applying a four-parameter fit to the standard curve, the concentrations of Ang-II in the samples were determined.

TUNEL staining

Myocardial apoptosis was examined using TUNEL reaction kit (Boster, Wuhan, China). Paraffin sections were incubated with protease K solution at 37 °C for 20 min and washed with TBS for 2 min. The sections were then stained with TdT labeling solution at 37 °C for 2 h, rinsed with TBS for three times, incubated with blocking buffer for 30 min and incubated with anti-DIG-biotin at 37 °C for 60 min, washed with TBS for 5 min, stained with DAPI solution for 5 min at room temperature. Positive TUNEL signals were observed and photographed under a fluorescence microscope.

Cell culture and treatment

The embryonic rat heart-derived H9C2 cells were purchased from SEVEN BIOTECH (AC601, Beijing, China). Cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (H-DMEM) (SEVEN, Beijing, China) supplemented with 10% fetal bovine serum (FBS) (Hyclone, USA), 100 units/ml penicillin and 100 μg/ml streptomycin at 37 °C and 5% CO2. Cells were stimulated with 1 μM Ang II (05–23-0101, Sigma, USA) to induce hypertrophy. Cells treated with equivalent saline were used as a control. To inhibit p-38 MAPK, cells were pretreated with 1 μM SB203580 (S8307, Sigma, USA) for 1 h. To knock down Syt1 expression in H9C2 cells, siRNA (Anbote, Jinan, China) and the negative control siRNA were mixed with Lipofectamine 2000 (Thermo Fisher Scientific, USA) for transfection according to the manufacturer’s instruction. Cells were then harvested 24 h after transfection for further analysis.

Immunofluorescent staining

Paraffin sections were incubated with 0.1% Triton X-100 for 20 min and then rinsed with PBS for 15 min. Following incubation in 5% goat serum at 37 °C for 1 h, sections were incubated overnight with primary antibodies against α-SMA (1:500, ab179467, Abcam, Cambridge, USA) at 4 °C. The sections were washed and then incubated with fluorescence-labeled secondary antibody (1:500, P11047, Thermo Scientific, Waltham, MA, USA) for 1 h at room temperature. Afterwards, sections were counterstained with DAPI solution for 2 min at room temperature in a humidifying box and photographed under a luminescence microscope.

Statistical analysis

The results were presented as mean ± standard error (SEM). The data were analyzed and graphed using GraphPad Prism 7.0 (GraphPad Software, In., San Diego, CA, USA). Statistical differences were analyzed by grouped t test for two groups or one-way ANOVA followed by Turkey’s multiple comparison for three or more groups. The Kaplan–Meier method with log-rank test was used for survival analysis. P < 0.05 were considered statistically significant.