Animal models

The animal experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals in China and were approved by the Animal Ethics Committee of Nanchang University. Cardiomyocyte-specific Cdc42 conditional knockout (Cdc42CKO) mice were generated by crossing Cdc42loxP/loxP mice with MLC2v-Cre mice [16,17,18], and all the mice were backcrossed to the C57BL/6 background for at least 6 generations before the experiments. The mice were housed in individually ventilated cages under standard conditions with a 12-hour light/dark cycle and were given access to a rodent chow diet (Beijing Keao Xieli Feed Co., Ltd.) and autoclaved water ad libitum. The mice were euthanized via carbon dioxide (CO2) inhalation or by cervical dislocation after being anesthetized via the inhalant isofluorane as needed. Cardiac hypertrophy was induced in 2-month-old male mice by AngII (1500 ng/kg/min, Alzet, Cat. No. 2002) infusion with a 7-day osmotic mini-pump (Alzet, model 1007D, Cupertino, CA) or saline alone as described [19, 20]. Cdc42CKO and Cdc42loxP/loxP mice were divided into AngII–treated and saline groups, respectively. The systolic/diastolic blood pressure and pulse were measured in conscious mice by a tail-cuff system (BP-98 A; Softron, Tokyo, Japan) on day 0 and day 7 post-implanting minipump, and the average of ten consecutive measurements was recorded.

Minimally invasive transverse aortic constriction (mTAC) surgery without chest opening was performed according to a previous protocol with modifications in mice [21]. Briefly, mice were anesthetized via inhalation of 2–4% isoflurane mixed with air, fixed in the supine position on a warm anatomical plate, and maintained unconsciously by endotracheal intubation at 120–130 breaths/minute and a tidal volume of 1–3 ml with isoflurane by a rodent ventilator (ALC-VBS). mTAC surgery was performed around the area between the second and third ribs on the left side to expose the thymus, the thymus lubes were carefully separated from one another, and the aorta was visually exposed under a dissecting microscope. The aortic arch was slightly lifted with a blunt needle parallel to a 27-gauge cushion needle, and the transverse aorta at the site between the right and left common carotid artery was tightened with a 6 − 0 nonabsorptive silk thread. After the animals regained self-breathing and consciousness, they were monitored in individually ventilated cages for 48 h for postoperative signs. Echocardiography was used to evaluate the success of banding and cardiac morphology and function after 2 and 8 weeks.

Echocardiography

Echocardiography was performed on mice at AngII infusion day 7, on TAC week 2 or week 8 with a high-resolution Micro-Ultrasound system (Vevo2100 or Vevo3100; Visual Sonics, Toronto, Ontario, Canada), in which the mouse was anesthetized by inhalation of 1.5–2% isoflurane mixed with air during the entire echocardiography procedure. The values of intraventricular septal thickness (IVS), left ventricular internal dimension (LVID) and left ventricle posterior wall thickness (LVPWth) at the diastole and systole stages, LV fractional shortening (FS), and LV ejection fraction (EF) were collected and analyzed.

Histology

The hearts were isolated, fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. The cross sections were harvested at 5 μm and stained with hematoxylin and eosin (H&E, Solarbio, Cat. No. G1120) for histological analysis or Masson trichrome (Solarbio, Cat. No. G1340) for fibrous areas.

Isolation and culture of cardiomyocytes from adult mice

Cardiomyocytes were isolated from 8- to 10-week-old Cdc42CKO and Cdc42loxP/loxP mice as described previously [22, 23]. In brief, the mice were anesthetized by an intraperitoneal injection of an overdose of sodium pentobarbital (150 mg·kg−1) with heparin (8,000 U·kg−1), and the hearts were rapidly excised into ice-cold perfusion buffer and cannulated via the aorta before retrograde perfusion on a modified Langendorff system at 37 °C with filtered calcium-free perfusion buffer containing 113 mM NaCl, 4.7 mM KCl, 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 1.2 mM MgSO4, 10 mM Na-HEPES, 12 mM NaHCO3, 10 mM KHCO3, 0.032 mM phenol red, 30 mM taurine, 10 mM BDM, and 5.5 mM glucose at pH 7.0. The hearts were perfused with perfusion buffer at a flow rate of 4 mL/min for approximately 4–5 min until the effluents became clear and then switched to digestion buffer for 10–12 min, which consisted of 50 ml of perfusion buffer with 15,000 U of total type II collagenase (Worthington Biochemical Corporation, Lakewood, NJ, USA, Cat. No. LS0004176) and 50 µM CaCl2. When the heart became enlarged, pale, and soft, the heart was gently removed from the cannula, placed in a sterile 60-mm dish containing 2.5 ml of digestion buffer, and gently teased into 10–12 small pieces. The heart pieces and cells were gently pipetted with a Pasteur pipette and transferred into a new 15 ml polypropylene conical tube. The solution was diluted to 10 ml with stop buffer (perfusion buffer with 10% FBS) containing 12.5 µM CaCl2. The cells were centrifuged for 3 min at 20 × g, after which the supernatants were removed, and the cell pellets were resuspended in serial stop buffer supplemented with increasing concentrations of Ca2+ (100 µmol/L, 400 µmol/L, and 900 µmol/L). Approximately 80% of the cells were rod shaped. Finally, the cells were resuspended in plating medium (43 ml of MEM containing 2 mM L-glutamine, 1.26 mM CaCl2, 5 ml of FBS, 500 mM BDM and 10,000 U/ml penicillin/streptomycin) at 25,000 myocytes/ml. Cardiomyocytes were plated onto 6 cm prelaminated dishes (8 mL/dish) and incubated for 3 h in a 5% CO2 incubator at 37 °C to allow the myocytes to attach. Then, the culture medium (48 ml of MEM containing 2 mM L-glutamine, 1.26 mM CaCl2, 500 mM BDM and 10,000 U/ml penicillin/streptomycin) was changed to the sides. Cardiomyocytes were treated with AngII (1000 ng/ml) or saline for 15 min, respectively.

Isolation and culture of cardiomyocytes from newborn mice

Neonatal ventricular myocytes were isolated from the hearts of 1- to 3-day-old C57BL/6 mice. Briefly, neonatal hearts were digested with 0.08% trypsin (Gibco, 27250-018) and 0.05% collagenase II (Sigma, C6885) in a shaking water bath (Magnetic Stirrer) at 37 °C and 750 rpm for 10 min 4–5 times and neutralized with 10% FBS in DMEM. Cardiomyocytes were collected and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Cat. No. 11965092) supplemented with 10% FBS, penicillin (100 U/mL, Gibco, Cat. No. 15140122) and streptomycin (100 µg/mL, Gibco, Cat. No. 15140122). After preseeding, the isolated cells were placed in an uncoated dish at 37 °C for 90–180 min, and 5-bromo-2-deoxyuridine (BrdU, 100 µmol/L, Sigma, Cat. No. B5002) was added to prevent overgrowth of rapidly proliferating fibroblasts at 37 °C with 5% CO2.

Cell culture

H9c2 cells (ATCC, CRL-1446™) were cultured in DMEM (Thermo Fisher, Wal-tham, MA, USA) supplemented with 10% FBS and 100 µg/mL of each of penicillin and streptomycin (Thermo Fisher, Cat. No. 15140163) at 37 °C with 5% CO2. H9c2 cells were treated with AngII (1000 ng/ml) or saline for 15 min, respectively.

Western blot analysis

Total tissue or cellular proteins were extracted by RIPA buffer containing 25 mmol/L HEPES, pH 7.4, 1% NP40, 137 mmol/L NaCl, 10% glycerol, 50 mmol/L NaF and protease inhibitor cocktail (Roche, Mannheim, Germany). Serum proteins were enriched by precipitating mouse serum with methanol and chloroform [24]. The extracted proteins were separated by 10–15% denaturing SDS‒PAGE and then transferred to NC membranes (Bio-Rad), which were incubated with primary antibodies followed by secondary antibodies. Primary antibodies against Cdc42 (Santa Cruz, sc-8401), RhoA (Abcam, ab187027), ERK1/2 (CST, 4695 S), phospho-ERK1/2 (CST, 4370 S), JNK (Abcam, ab179461), phospho-JNK (Abcam, ab124956), NF-κB/p65 (Abcam, ab16502), phospho-NF-κB/p65 (Abcam, ab86299), CaMKII (Abcam, ab32422), phospho-CaMKII (Millipore, 05–574), NFAT-C4 (Abcam, ab23672), calcineurin (Abcam, ab18932), p38 (Abcam, ab170099), phospho-p38 (Abcam, ab4822), phospho-MKK3/MKK6 (CST, 9231 S), GSK3β (CST, 9315 S), phospho-GSK3β (CST, 9336 S), AKT1 (CST, 2967 S), phospho-AKT1 (CST, 4060 S), PI3 K (CST, 4257 S), phospho-PI3K (CST, 4228 S), IL6 (CST, 12912 S), TNFα (Proteintech, 60298-1-Ig), IL10 (Proteintech, 10008-1-AP), Col1 (Servicebio, GB11013), ANP (Santa Cruz, sc-515701), BNP (Abcam, ab19645), Col3 (Servicebio, GB11015) and GAPDH (Santa Cruz, sc − 47724) were used at 4 °C overnight. Blots were detected with HRP-conjugated goat anti–rabbit or rabbit anti-mouse secondary antibodies (Invitrogen, A16078 for goat anti - rabbit; A28175 for rabbit anti - mouse) for 1 h at room temperature. The luminescence was visualized using a Bio-Rad luminescent imaging system.

RNA extraction, transcriptome assembly and real-time PCR

Total RNA was extracted from mouse heart tissues by the TRIzol (TaKaRa, Code No. 9109) method. RNA was transcribed into cDNA using random hexamers and oligo (dT) with Superscript III Reverse Transcriptase (Invitrogen, 18080051). The expression levels of Cdc42, Cre and β-Actin were determined using semiquantitative RT‒PCR with the following primer pairs (displayed in order of forward primer, reverse primer and the length of cDNA product, Cdc42: (5′-CGG AGA AGC TGA GGA CAA GA-3′, 5′-TGG GTC CCA ACA AGC AAG AA-3′, 410 bp), Cre: (5′-GAC CAG GTT TCA CTC A-3′, 5′-ACC AGA GTC ATC CTT AGC G-3′, 400 bp) and β-Actin: (5′-ATG GAG GGG AAT ACA GCC C-3′, 5′-TTC TTT GCA GCT CCT TCG TT-3′, 149 bp). Cdc42 and Rac1 mRNA levels were quantified by SYBR Green Real-Time PCR. GAPDH served as an internal control. The reaction mixtures consisted of 10 µL of SYBR Green PCR Master Mix, 8.2 µL of water, 10 ng of DNA, and 0.4 µL of primers (10 mM) using the following primer pairs (forward, reverse): GAPDH (5’-CGT CCC GTA GAC AAA ATG GT-3’, 5’-TTG ATG GCA ACA ATC TCC AC-3’), Cdc42 (5’-GTT GGT GAT GGT GCT GTT-3’, 5’-GGA TAA CTT AGC GGT CGT-3’) and Rac1 (5’- TCT CCA GGA AAT GCA TTG GT-3’, 5’- AGA TGC AGG CCA TCA AGT GT-3’).

Approximately 3 µg of total RNA per mouse heart was used as input material for RNA high-throughput sequencing by the Novogene Experimental Department. Sequencing libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA, E7530L) following the manufacturer’s recommendations, and index codes were added to attribute the sequences. Briefly, twelve cDNA libraries were constructed with three samples per group. First-strand cDNA was synthesized using random hexamer primers and M-MuLV reverse transcriptase (RNase H-). Second-strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. After adenylation of the 3’ ends of cDNA fragments, NEBNext adaptors with hairpin loop structures were ligated to prepare for hybridization. To preferentially select cDNA fragments 150 ~ 200 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA, Cat. No. A63880) and selected with USER Enzyme (NEB, USA, Cat. No. M5505L) to obtain adaptor-ligated 150 ~ 200 bp cDNAs, which were subsequently amplified with Phusion High-Fidelity DNA polymerase, universal PCR primers and index (X) primers. Then, the PCR products were purified (AMPure XP system) and assessed on an Agilent Bioanalyzer 2100 system for quality. Clustering of the index-coded samples was performed on a cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina HiSeq platform, 125 bp/150 bp paired-end reads were generated, and approximately 6 Gb of clean data were obtained per sample. Differential expression analysis (DESeq) was performed after the reads were mapped to the mouse reference genome, followed by GO and KEGG enrichment analysis.

Immunofluorescence staining

About 5-µm paraffin tissue sections were dewaxed and heated in 1x citrate unmasking solution at 95–98 °C for 10 min, then immersed in 0.25% Triton X-100/PBS (PBST) solution and cooled at RT. The phospho-p38 primary antibody (CST #4511, 1:200) was applied on the sections in antibody dilution buffer and incubated overnight at 4 °C. H9c2 cells grown on glass coverslips were fixed in 4% PFA for 20 min and permeabilized in PBST. The primary antibody (mouse anti-IL-6, CST#12912, 1:100) was applied and incubated overnight at 4 °C. Later, the slides were applied with fluorochome-conjugated secondary antibodies (Proteintech, SA00013-4 or SA00013-1), respectively. Finally, the slides were mounted with anti-fluorescence quenching sealing solution containing DAPI (Santa Cruz, sc-24941). The fluorescent images were obtained under a Zeiss LSM 800 confocal microscope (version ZEN 3.3 software). The mean fluorescence intensity of the sections was analyzed by ImageJ software.

TUNEL assay

The apoptosis assay was performed according to the manufacturer’s instruction for One-step TUNEL In Situ Apoptosis Kit (Elabscience, E-CK-321). Briefly, the paraffin slides were dewaxed, then incubated in proteinase K 1 mg/ml in PBS solution for 20 min and washed with PBS 3 times. About 50 µL of TUNEL reaction mixture was added per sample and incubated in a dark and wet box at 37 °C for 1 h. After washing 3 times with PBS, an anti-fluorescence sealing solution with DAPI was applied on slide. The fluorescent images were taken under Zeiss LSM 800 confocal microscope.

Statistical analysis

The data are presented as the mean ± SD. Student’s t test was used for two-group analyses. One-way or two-way analysis of variance (ANOVA) was used to compare data among three or more groups. P < 0.05 was considered to indicate statistical significance.