2.1. Reagents
Streptozocin (STZ), allyl-isothiocyanate (AITC), VIVIT and 1, 8-cineole were acquired from Sigma (St. Louis, MO). amlexanox and trifluoperazine were purchased from MedChemExpress (Shanghai, China). Anti-NOX2 (cat no. ab129068) and anti-α-SMA (cat no. ab5694) were purchased from Abcam Cambridge (Abcam, MA, USA). Anti-COL1 (cat no. 66761-1-Ig), anti-COL3 (cat no. 22734-1-AP) and anti-MMP2 (cat no. 10373-2-AP) were acquired from Proteintech Group (Proteintech, USA). Anti-TRPA1 (cat no. NB110-40763) and anti-NFAT1 (cat no. NB300-504) were purchased from Novus Biologicals (Novus, USA). Anti-GRK5 (cat no. GTX64841) was acquired from GeneTex (Hsinchu, Taiwan). Anti-NOX4 (cat no. GB11347) was purchased from Servicebio (Wuhan, China). Anti-TGFβ (cat no. sc-130348) was acquired from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-p-SMAD2/3 (cat no. 8828), anti-SMAD2/3 (cat no. 8685), anti-SMAD4 (cat no. 46535), anti-GAPDH (cat no. 5174) and anti-PCNA (cat no. 13110) were acquired from Cell Signaling Technology (CST, USA).
2.2. Bioinformatic tools
All expression profiling data analyzed in this study were downloaded from GENE EXPRESSION OMNIBUS (GEO, http://www.ncbi.nlm.nih.gov/geo). The genes differentially expressed in the cardiac tissue of diabetic patients and healthy controls were searched from the data GSE26887 from the GEO series.
2.3. Animal care and preparation
Male Sprague-Dawley (SD) rats (Shulaibao Biotechnology, Wuhan, China) and TRPA1 KO rats (Cyagen Biosciences, Wuhan, China) aged 8 weeks were used in this study. All animals were housed at constant room temperature on a 12:12 h light-dark cycle at the Animal Center of the Wuhan University Renmin Hospital and fed with standard rodent diet and water. All animals received humane care according to NIH (USA) guidelines. All animal care and experimental procedures were approved by the Animal Policy and Welfare Committee of Wuhan University Renmin Hospital (WDRM20210607A).
To model the cardiac injury in type 1 diabetes, SD and TRPA1KO rats were treated with single-dose intraperitoneal injection of STZ (60 mg/kg in citrate buffer, pH 4.5). This dose of STZ has been shown to reliably induce diabetes in SD rats [14]. Vehicle control mice received the same volume of citrate buffer. Use a blood glucose meter to measure fasting blood glucose levels from day 3. Mice showing fasting blood glucose levels > 12 mM (~ 216 mg/dL) for 3 consecutive days were considered diabetic. Body weight and fasting blood glucose levels of all mice were measured weekly during the study period. The experimental groupings were as follows: non-diabetic SD controls (SD, n = 6), non-diabetic TRPA1KO controls (KO, n = 6), STZ-induced SD diabetic rats (SD-STZ, n = 6) and STZ-induced TRPA1KO diabetic rats (KO-STZ, n = 6). All rats were euthanized 12 weeks after the onset of diabetes. Euthanasia was performed under pentobarbital sodium anesthesia, and blood was collected for subsequent analysis. Heart tissue was collected and fixed in 4% paraformaldehyde for pathological analysis and/or snap-frozen in liquid nitrogen for gene and protein expression analysis.
To examine the therapeutic effect of 1,8-cineole (CIN) in diabetic rats, we randomly divided SD rats into 4 groups (n = 6): no-diabetic controls (SHAM), SHAM + CIN (60 mg/ kg/d, i.g.12 weeks)[15], STZ-induced diabetic rats (diabetic cardiomyopathy) and diabetic cardiomyopathy + CIN (60 mg/kg/d, i.g.12 weeks). Rats were then euthanized under sodium pentobarbital anesthesia. Cardiac tissue was collected for subsequent analysis.
2.4. Echocardiography
Systolic and diastolic cardiac function in anesthetized rats was measured noninvasively by transthoracic echocardiography 1 day before sacrifice as described [16]. Rats were anesthetized with isoflurane using 1.5% isoflurane, and ventricular septum, chamber dimensions, systolic and diastolic function, and ejection fraction were recorded by MyLab 30 CV ultrasound system (Esaote S.P.A, Genoa, Italy).
2.5. Histology and tissue staining
Heart tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 µm thickness. After dehydration, sections were stained with hematoxylin and eosin (H&E). Stained sections were assessed for general histopathological damage using light microscopy. Cardiomyocyte size was assessed in cross-sections of cardiac tissue. Additional paraffin sections were stained with Masson's Trichrome to assess cardiac fibrosis. Stained sections were observed under a light microscope. Digital images of stained sections were captured and then measured using a digital image analysis program (Image-Pro Plus, version 6.0).
For immunofluorescence staining, harvested rat hearts were embedded in paraffin and cut into 5-micron thick sections. Sections were incubated with primary antibodies against GRK5 and NFAT overnight in a humidified chamber at 4°C. Sections were then incubated with secondary antibody for 1 hour in the dark. Sections were evaluated using a fluorescence microscope (Olympus DX51, Tokyo, Japan) and further analyzed using Image-Pro Plus software (version 6.0).
2.6. Cell isolation culture and treatments
Neonatal rat cardiac fibroblasts (CFs) were isolated from SD and TRPA1 KO rats. Briefly, the minced neonatal rat (1–2 days old) hearts were placed in 0.125% trypsin and digested at 150 r/min in a 37-degree thermostatic mixer. The digested fluid was collected and centrifuged after 1 hour of digestion. The supernatant after centrifugation was discarded and the cells were resuspended in culture medium. After the cells were seeded in 100 mm dishes and cultured for 90 min, the medium was changed to culture CFs. The medium consists of 10% FBS, 1% penicillin/streptomycin, and DMEM-F12. CFs were maintained at a density of 3x105 cells per 60 mm.
To promote CFs activation in vitro, cells were exposed to high glucose (HG, 33 mM) for 24 h [17]. To explore the mechanism by which TRPA1 regulates the activation of CFs, we treated CFs with amlexanox (35uM) [18], VIVIT (100uM) [19] or trifluoperazine (30uM)[20] for 12 hours in serum-free medium. Then, CFs were treated with AITC (200uM) [18] and the same dose of drugs above for 24 hours. CFs were fixed with 4% paraformaldehyde to evaluate the levels of activation and transdifferentiation by immunofluorescence staining for α-SMA. Images were captured and evaluated using a fluorescence microscope (Olympus DX51, Tokyo, Japan) and further analyzed using Image-Pro Plus software (version 6.0).
2.7. Cardiac fibroblast immunofluorescence analysis
Transdifferentiation of CFs, the expression and localization of NFAT and GRK5 were assessed by immunofluorescence staining. Briefly, after three washes with PBS, cells were fixed with 4% formaldehyde, permeabilized with 0.2% Triton, and diluted 1:100 in 1% with anti-α-SMA, anti-NFAT, and anti-GRK5 at 4°C goat serum (GTX27481, GeneTex, Sanantonio, TX, USA) overnight. The next day, cells were incubated with Alexa Fluor 488 (green fluorescent dye) or Alexa Fluor 568 (red) secondary antibody at a 1:200 dilution for 1 hour at 37°C. Nuclear were then counterstained with DAPI (S36939, Thermo Fisher Scientific, Eugene, OR, USA) for visualization. Finally, images were captured and evaluated using a fluorescence microscope (Olympus DX51, Tokyo, Japan) and further analyzed using Image-Pro Plus software (version 6.0).
2.8. GRK5 silencing
GRK5 was silenced in CFs by siRNA. Rat siGRK5 (5’-GACGAGTTAAGATGCTAAAATAT-3’) was purchased from Sangon biotech (Shanghai, China). Negative control transfections included scrambled siRNA sequences. All transfections were performed using LipofectAMINE™ 2000 (Invitrogen, Carlsbad, California). Knockdown of the gene in transfected cells was confirmed by western blot and immunofluorescence analysis.
2.9. Reactive oxygen species detection
Reactive oxygen species (ROS) production was assessed in vitro by DHE staining. Briefly, fresh heart samples or CFs were stained with DHE (5 µmol/L) for 30 min at 37°C in the dark and then observed blindly under an Olympus DX51 fluorescence microscope.
2.10. Western blot
Western blots were performed to assess protein expression levels. Lysed cells or homogenized cardiac tissue were collected for total protein extraction. Protein concentration was assessed using the BCA protein assay kit (23227, Thermo Fisher Scientific, Waltham, MA, USA) and normalized before western blotting. Protein samples (50 µg) were separated on 10% SDS-PAGE and subsequently transferred to PVDF membranes (FL00010, Millipore, Billerica, MA, USA). PVDF membranes were blocked with 5% nonfat milk for 1 h at room temperature to block non-specific binding sites, then incubated with primary antibody overnight at 4°C. The protein expression level of GAPDH was used as an internal standard. The next day, the western blot was incubated with the secondary antibody for 1 hour at room temperature. Nuclear protein fractions were separated by a commercial kit and were normalized to PCNA. Finally, blots were obtained and scanned by Image Lab software (Bio-Rad Laboratories, Inc., Hercules, CA, USA) to assess protein expression.
2.11 Real-time quantitative PCR
To detect mRNA expression of markers associated with cardiac hypertrophy and fibrosis, cells or cardiac tissues were homogenized in TRIZOL (15596-026, Invitrogen Corporation, Waltham, MA, USA), followed by a cDNA synthesis kit (489703001, Basel, Switzerland) reverse transcription of mRNA into complementary DNA (cDNA). The LightCycler 480 system (04896866001, Roche) was used for qPCR analysis. Primers for target genes are listed in Supplementary Table 1. The amount of each gene was determined and normalized to the amount of GAPDH.
2.12 Statistical analysis
All experiments were randomized and blinded. In vitro experiments were repeated at least three times. All statistical analyses in this study were performed using SPSS version 25.0 software and expressed as mean ± standard deviation (SD). Differences between groups were assessed by one-way ANOVA followed by post hoc Tukey test. Two groups were analyzed using an unpaired Student's t-test. P < 0.05 was considered statistically significant.