Protective mechanism of SIRT1 on Hcy‐induced atrial fibrosis mediated by TRPC3

Figure 1. High plasma levels of Hcy are closely related to the occurrence of cardiac fibrosis. A‐B, The expression levels of TRPC3 and the relevant proteins, such as TGF‐β and Collagen‐I in AF patients. C, The relative mRNA level. D1‐D2, The atrial nucleus in patients with AF is shown with haematoxylin and eosin (HE) staining, Masson's trichrome ‐stain and immunohistochemistry, as well as statistical results of positive areas and ratio to total area of the fibre (Magnification × 100, Scale bar = 200 μm; n = 4 per group). E, The ultra‐structure of atrial tissues was observed by electron microscopy: E1‐E3: SR patients, E4‐E6: AF patients. Error line indicates mean and standard deviation. *P < .01, **P < .001. Multiple t‐tests were performed using step‐down bootstrap sampling to control the familywise error rate at 0.05 for 1B, C, and unpaired t‐test was used for 1D. Fisher's exact test was used for analysing Table 1

Figure 2. Timeline for establishing mouse HFpHR model with a high Hcy diet and echocardiographic parameters for assessing systolic and diastolic function. A, Mice underwent TAC or sham surgery at 4 weeks of age in the HH group (n = 25), which were fed a high‐Hcy diet (4% methionine) from 4‐18 weeks of age. The NH group (n = 25) also underwent the above TAC operation but were fed a normal diet. Mice that underwent sham surgery and were fed a normal diet were considered the sham (SH) group (n = 10). Repeat echocardiogram (ECG) was performed at 16 and 18 weeks of age. We performed programmed ECG analysis, RT‐qPCR and Western blot in the three groups at 18 weeks of age. B1, The value of ejection fraction (EF) in SH, NH and HH mice. B2, The value of interventricular septum thickness (IVS‐S). B3, B4, Baseline E (early filling)/A (atrial filling) and E/E′ ratios amongst the three groups. B5, The value of left ventricular internal diastolic diameter (LVIDd). C, Representative images from pulsed‐wave Doppler showing E and A wave. D, Representative M‐mode images of the three groups. Error line indicates mean and standard deviation. *P < .01 and **P < .001. Mixed model regression with post hoc…

Figure 3. Programmed acetylcholine stimulation (PES) and ECG analyses in SH, NH and HH mice. A, Normal SR was typically observed in the ECGs of the SH group (n = 7/9), and the speed was 125 ms. B, ECGs indicating AF were frequently observed in the HH group (n = 10/17). C, Ventricular fibrillation (VF) was observed in the NH (n = 3/18) and HH (n = 4/17) groups. D, Increased susceptibility to AF in the HH group (N = 10/17) than in the NH group (N = 8/18), with the lowest susceptibility in the SH group (N = 2/9). E, The duration of AF in the three groups. F, The value of heart weight‐to‐body weight (HW/BW). G, The heart weight‐to‐tibia length ratio (HW/TL). H, Representative Masson staining showed transverse section in atria. I, Representative Masson staining images with magnified local images in atrial tissues. J, Survival rate of the three groups over different time periods. K, Results are represented as cardiac sizes in the three groups. *P < .05, **P < .01. Mixed model regression with post hoc testing (Tukey adjustment) was used in 3F, 3G, and Fisher's exact test was used in 3D

Figure 4. TRPC3‐KD or SIRT1‐OE in mice could effectively alleviate atrial fibrosis. A, C57B6 mice (n = 10) at 1 week of age were injected with purified lentivirus via the caudal vein. Mice underwent TAC and were fed with a high‐Hcy diet at 4 weeks of age, with relevant tissues obtained after 16 weeks. B, F, Heart weight‐to‐tibia length ratio (HW/TL). C, The Masson staining images with magnified local images in atrial tissues. D, The protein levels of TRPC3 and TGF‐β measured in the three groups. E, Ten C57B6 mice underwent TAC at 4 weeks of age and were fed with a high‐Hcy and folic acid mixed diet during 4‐16 weeks (HH+FC group). F, A subset of these animals were injected with Res (20 mg/kg/d) for 21 day (HH+Res group). G, The statistical total area of the fibre ratio. H, I, The protein and mRNA expression levels of SIRT1 and TRPC3. Error line indicates mean and standard deviation. *P < .05 and **P < .01. Mixed model regression with post hoc testing (Tukey adjustment) was used for 4B and 4F. Multiple t‐tests were performed using step‐down bootstrap sampling to control the familywise error rate at 0.05 for 4C, 4D and 4G‐4I

Figure 5. Hcy modulates the expression of TRPC3 protein and affects the activity of SIRT1. A, B, The expression levels of TRPC3, SIRT1, TGF‐β and Col‐I in mouse atrial fibroblast under different concentrations of Hcy or different treatments. C, D. The protein levels of TRPC3, SIRT1 and TGF‐β in TRPC3‐shRNA‐transfected stable cell lines with or without Hcy stimulation. E, F. The expression levels of TRPC3, SIRT1, TGF‐β and Col‐I in TRPC3‐OE or SIRT1‐OE stable cell lines. G, H, The promotion of fibroblast proliferation was detected by CCK‐8. Error line indicates mean and standard deviation. *P < .05 and **P < .01 vs scramble; # P < .05, ## P < .01 vs (500 μmol/L) Hcy group. Unpaired t‐test was used for 5A‐5F, and mixed model regression with post hoc testing (Tukey adjustment) was used for 5G, 5H

Figure 6. The interaction between TRPC3 and SIRT1. A, Co‐localization of TRPC3 and SIRT1. Cells are co‐stained with antibodies against TRPC3 (Alexa Fluor 488, green) and SIRT1 (Alexa Flour 564, red). Arrow indicates co‐localization. B, Interaction of TRPC3 with SIRT1 in mammalian cells. C, Endogenous TRPC3 binds to SIRT1 in MCFs. D, The interaction of endogenous TRPC3 with SIRT1 in MCFs. E, Schematic illustration of the domains of TRPC3 and its association with SIRT1. F, The C‐terminal tail (659‐836) of TRPC3 interacts with SIRT1. G, H, Analyse of the binding affinity between full‐length SIRT1 and TRPC3‐C (G) or TRPC3‐N (H) by bio‐layer interferometry (BLI). All experiments were performed in triplicate

Figure 7. Schematic representation of our working hypothesis. Increased Hcy levels promote the proliferation and differentiation of fibroblasts, which can bind to GPCRs, and increase the protein levels of TPRC3 and through interaction with SIRT1 promote atrial fibrosis. This may occur though the direct activation of TGF‐β to induce collagen‐I accumulation associated with the pathological mechanism of AF. However, the specific mechanism of AF requires further investigation