ObjectiveTo study the regulatory effect of the partial sequence within the 3’ untranslated region （3’UTR） of slit guidance ligand 1 （Slit1） （Slit1-3’UTR） on the fibrotic phenotypes of cardiac fibroblasts （CFs） and its potential mechanism. MethodsThe adenovirus vector was used to overexpress the 1526nt sequence of Slit1-3’UTR in ICR neonatal mouse CFs （mCFs）. The expression of fibrosis-related genes in mCFs， such as collagen type 1 alpha1（COL1A1）， collagen type 3 alpha3 （COL3A1） and alpha smooth muscle actin （α-SMA） were detected by Western blot assay. The effect of Slit1-3’UTR 1526nt on the proliferation and migration of mCFs was assessed by EdU staining and Trans-well assays. Angiotensin Ⅱ （Ang Ⅱ） was used to treat mCFs， and the impact of Slit1-3’UTR 1526nt on the fibrotic phenotypes of Ang Ⅱ-induced mCFs was evaluated. After overexpression of Slit1-3’UTR 1526nt， miR-34a-5p mimic was transfected into mCFs， followed by actinomycin D treatment to detect the mRNA stability of Slit1-3’UTR 1526nt， and the levels of miR-34a-5p and its target gene SIRT1（si-SIRT1） in mCFs were determined. The effects of miR-34a-5p and small interfering RNA targeting SIRT1 on the Slit1-3’UTR 1526nt-mediated regulation of fibrotic phenotypes were also determined. ResultsAdenovirus-mediated overexpression of Slit 1-3’UTR 1526nt was achieved in mCFs. Overexpression of Slit 1-3’UTR 1526nt markedly inhibited the expression of the fibrosis-related genes， proliferation and migration of mCFs and fibrotic phenotypes of Ang Ⅱ. The results of actinomycin D assay showed that miR-34a-5p inhibited the stability of Slit1-3’UTR 1526nt in mCFs， while the level of miR-34a-5p was reduced in mCFs with overexpression of Slit1-3’UTR 1526nt. Transfection of miR-34a-5p promoted the fibrotic phenotypes， and reversed the inhibitory effect of Slit1-3’UTR 1526nt on the fibrotic phenotypes of mCFs. Overexpression of Slit1-3’UTR 1526nt significantly increased the level of miR-34a-5p target gene SIRT1 in mCFs. Transfection of miR-34a-5p and si-SIRT1 consistently reversed the inhibitory effects of Slit1-3’UTR 1526nt on the fibrotic phenotypes of mCFs. ConclusionSlit1-3’UTR1526nt inhibits the fibrotic phenotypes of mCFs by binding to miR-34a-5p and increasing the expression of its target gene of SIRT1.

ObjectiveTo investigate the regulatory effect and potential mechanisms of isocitrate dehydrogenase 3A （IDH3A） on cardiomyocyte hypertrophy. MethodsThe expression of IDH3A in the myocardium of healthy volunteers （n=10） and patients with heart failure （HF） （n=10）， and in the myocardium of mice subjected to transverse aortic constriction （TAC） surgery and sham operation， as well as in phenylephrine （PE）-induced neonatal rat ventricular cardiomyocytes （NRVCs）， was assessed by real-time quantitative polymerase chain reaction （RT-qPCR） and Western blot assay. The effect of adenovirus-mediated overexpression of IDH3A on the expression of hypertrophy-related genes in PE-induced NRVCs was also evaluated. The effect of IDH3A on NRVCs area was examined by phalloidin staining assay. A mutant of IDH3A with abolished enzymatic activity， IDH3A_D208A， was generated through site-directed mutagenesis. The impact of this IDH3A mutant on the hypertrophic phenotype， ATP and ROS levels in NRVCs was evaluated to investigate whether the regulatory role of IDH3A in cardiomyocyte hypertrophy was dependent on its enzymatic activity. The effect of exogenous α-ketoglutaric acid （AKG） on cardiomyocyte hypertrophy was also detected by Western blot and phalloidin staining assay， respectively. ResultsIDH3A was significantly decreased in the myocardium of HF patients， in the myocardium of TAC-operated mice， and in PE-induced NRVCs （P = 0.005 2，P = 0.026 6，P = 0.041 3 and P = 0.006 6， respectively）. Overexpression of IDH3A markedly suppressed the expression of hypertrophy-related genes and the increase of cell size of PE-induced NRVCs （P < 0.000 1， P = 0.000 1 and P = 0.000 2， respectively）. The ATP and ROS analysis indicated that IDH3A inhibited the increases of ATP and ROS levels in PE-induced NRVCs （P = 0.001 2 and P<0.000 1， respectively）， whereas the enzymatically inactive IDH3A mutant lacked this effect. Exogenous AKG provision could， but overexpression of IDH3A mutant failed to suppress PE-induced NRVCs hypertrophy. ConclusionIDH3A inhibits cardiomyocyte hypertrophy via elevating AKG level， providing scientific evidence for study on IDH3A-based treatment of cardiac hypertrophy.

ObjectiveTo investigate the regulatory effect of circular RNA circ_0120051 on the fibrotic phenotype of cardiac fibroblasts and the potential mechanism involved. MethodsThe expression of circ_0120051 and its host gene of solute carrier family 8 member A1（SLC8A1） mRNA in the myocardium of healthy organ donors （n=24） and heart failure （HF） patients （n=21） were assessed by real-time quantitative polymerase chain reaction （RT-qPCR） assay. RNA stability of circ_0120051 was identified by RNase R exonuclease digestion assay. The cytoplasmic and nuclear distribution of circ_0120051 in human cardiomyocyte AC16 was detected by RT-qPCR assay. The expression of fibrosis-related genes in mouse cardiac fibroblasts （mCFs） with adenovirus-mediated overexpression of circ_0120051 was detected by RT-qPCR and Western blot assay， respectively. The effect of overexpression of circ_0120051 on the migration activity of mCFs was evaluated by wound-healing assay. RNA co-immunoprecipitation （RIP） was conducted to detect the interaction between circ_0120051 and miR-144-3p. The binding site of miR-144-3p in the 3'-UTR of isocitrate dehydrogenase 2 （Idh2） mRNA was identified by the dual luciferase reporter gene assay. ResultsCirc_0120051 was significantly up-regulated in the myocardium of HF patients， while the mRNA expression of its host gene SLC8A1 was not changed. Circ_0120051 was mainly located in the cytoplasm of human AC16 cells. Results of RNase R exonuclease digestion revealed that circ_0120051 possesses the characteristic stability of circular RNA compared to the linear SLC8A1 mRNA. Overexpression of circ_0120051 could inhibit the expression of fibrosis-related gene in mCFs and mCFs migration. RIP assay confirmed the specific interaction between circ_0120051 and miR-144-3p. Transfection of miR-144-3p mimic could efficiently promote the expression of fibrosis-related genes in mCFs and reverse the inhibitory effect of circ_0120051 on the fibrotic phenotype of mCFs. Results of the dual luciferase reporter gene assay confirmed the interaction between miR-144-3p and the 3'-UTR of Idh2. Transfection of miR-144-3p transcriptionally inhibited Idh2 expression， and overexpression of circ_0120051 enhanced IDH2 expression in mCFs. MiR-144-3p mimic and Idh2 small interfering RNA （siRNA） could consistently reverse the inhibitory effects of circ_0120051 on fibrosis-related genes expression in mCFs and mCFs migration. ConclusionsCirc_0120051 inhibits the fibrotic phenotype of cardiac fibroblasts via sponging miR-144-3p to enhance the target gene of IDH2 expression.

ObjectiveTo investigate the effect of circSLC8A1_005 on the fibrotic phenotype of cardiac fibroblasts and the potential mechanism involved. MethodsThe effect of adenovirus-mediated overexpression of circSLC8A1_005 on the expression of fibrosis-related genes， collagen type I alpha 1 chain （Col1a1）， collagen type Ⅲ alpha 1 chain （Col3a1） and smooth muscle actin alpha 2 （Acta2）， in mouse cardiac fibroblasts （mCFs） were detected. The proliferation and migration activities of mCFs were detected by EdU and wound-healing assay， respectively. Dual luciferase reporter gene assay was performed to detect the activity of potential internal ribozyme entry site （IRES） in circSLC8A1_005. CircSLC8A1_005-translated protein， SLC8A1-605aa， and its intracellular distribution was identified by Western blot assay. The effect of SLC8A1-605aa protein on transcription activity of Sod2 gene was detected by the dual luciferase reporter gene assay. RNA binding protein immunoprecipitation （RIP） was utilized to verify the interaction between SLC8A1-605aa and superoxide dismutase 2 （Sod2） mRNA. Actinomycin D treatment was used to detect the effect of SLC8A1-605aa on Sod2 mRNA stability in mCFs. ResultsAn efficient adenovirus-mediated overexpression of circSLC8A1_005 was achieved in mCFs. The enforced expression of circSLC8A1_005 suppressed proliferation and migration of mCFs， and inhibited the expression of fibrosis-related genes in mCFs. The dual luciferase reporter gene assay revealed the activities of 2 IRES in circSLC8A1_005. Results of Western blot assay showed that circSLC8A1_005 could translate protein SLC8A1-605aa with the prospected molecular weight of 70 ku， which is predominantly distributed in the nucleus. Overexpression of the circSLC8A1_005 and SLC8A1-605aa could consistently inhibit the fibrotic phenotype of mCFs. SLC8A1-605aa could up-regulate superoxide dismutase 2 （Sod2） expression， but not at the transcriptional level. RIP assay indicated that SLC8A1-605aa could specifically interact with Sod2 mRNA， and the results of actinomycin D assay showed that SLC8A1-605aa could enhance the stability of Sod2 mRNA in mCFs. ConclusionCircSLC8A1_005 inhibits the fibrotic phenotype of cardiac fibroblasts via translating SLC8A1-605aa protein， and SLC8A1-605aa may be a potential target for the treatment of myocardial fibrosis.

AIM:To investigate the effect of circular RNA MYO9A-006(circMYO9A_006)on hypertrophic phenotype of cardiomyocytes and the underlying mechanism.METHODS:The effect of adenovirus-mediated overexpres-sion of circMYO9A_006 on the expression of hypertrophy-related proteins,including β-myosin heavy chain(β-MHC),skeletal muscle actin alpha 1(ACTA1)and atrial natriuretic peptide(ANP),was evaluated in neonatal mouse ventricular cardiomyocytes(NMVCs).Moreover,a neonatal rat ventricular cardiomyocyte(NRVC)model of phenylephrine(PE)-in-duced hypertrophy was established.The effect of circMYO9A_006 overexpression on NRVC size was ascertained using Phalloidin-iFluor 647 staining method.Dual-luciferase reporter assay was employed to measure the activity of potential in-ternal ribosome entry sites(IRES)in circMYO9A_006.The translation and intracellular location of the circMYO9A_006-translated protein,MYO9A-208aa,were verified using Western blot.To investigate the role of MYO9A-208aa in the ef-fect of circMYO9A_006 on the cardiomyocyte hypertrophic phenotype,we prepared and used the following adenoviruses:the recombinant circMYO9A_006-ORF adenovirus to express MYO9A-208aa,the recombinant circMYO9A_006-ATG-mut adenovirus that does not express MYO9A-208aa,the recombinant circMYO9A_006 adenovirus,and the adenovirus vector control.These were then employed to infect NRVCs.RESULTS:Successful adenovirus-mediated overexpression of circMYO9A_006 was observed in NMVCs.The increased expression of circMYO9A_006 notably reduced the expres-sion of hypertrophy-related proteins in NMVCs(P<0.01).Concurrently,overexpression of circMYO9A_006 substantially reduced the expression of hypertrophy-associated proteins and diminished the size of PE-induced NRVCs(P<0.05).Dual-luciferase reporter assay identified the activity of 2 IRES in circMYO9A_006.Western blot results indicated that circ-MYO9A_006 could produce the MYO9A-208aa protein with an anticipated molecular weight of 28 kD in NRVCs,primari-ly found in the cytoplasm.Elevated expression of both circMYO9A_006 and MYO9A-208aa consistently reduced the ex-pression of hypertrophy-associated proteins(P<0.01),and counteracted the enlarged size of PE-induced NRVCs(P<0.05).However,increased expression of circMYO9A_006-ATG-mut did not counteract the PE-induced hypertrophic phe-notype of NRVCs.CONCLUSION:circMYO9A_006 attenuates the hypertrophic phenotype of cardiomyocytes by synthe-sizing the MYO9A-208aa protein.

Transthyretin-related amyloid cardiomyopathy (ATTR-CM) is a disease caused by the depo-sition of insoluble amyloid fibers formed by the misfolding of transthyretin precursor protein in the intercellular space of cardiomyocytes. This lesion may lead to myocardial dysfunction, cogestive heart failure, and death.When diagnosed earlier, the patient can be treated with drugs as soon as possible to intervene in the progress of the disease, so as to effectively improve the patient's prognosis.^99mtechnetium-pyrophosphate (⁹⁹Tc^m-PYP)single-photon emission computed tomography (SPECT) has been widely used in the imaging examination of cardiac amyloidosis (CA) in recent years. While achieving early non-invasive diagnosis, accurate pathological classification can be obtained through Perugini visual score analysis, semi-quantitative analysis of heart to contralateral lung (H/CL) ratio, and SPECT image analysis. This article presents the application, methods, and the precautions of ⁹⁹Tc^m-PYPSPECT in the diagnosis of ATTR-CM, aiming to provide clinical reference for the application of this technology.

Objective:To investigate the application of different imaging methods of 99Tc m-pyrophosphate (PYP) in the diagnosis and pathological classification of cardiac amyloidosis (CA). Methods:A total of 31 patients (22 males, 9 females, age 21-81(57.2±13.4) years) with suspected CA who underwent 99Tc m-PYP dual-phase scintigraphy (early-phase: 1 h, delay-phase: 2-3 h) and SPECT/CT (1 h) between December 2018 and December 2019 in Peking Union Medical College Hospital were retrospectively included. Taking clinical diagnosis as the standard, the results of visual score (≥2, positive) and semi-quantitative values (heart to contralateral lung (H/CL)≥1.5, positive) of 99Tc m-PYP uptake in dual-phase scintigraphy and SPECT/CT imaging were analyzed. One-way analysis of variance and Bonferroni test were used to analyze the data. Results:Among 31 patients with suspected CA, 15 were clinically diagnosed as CA (5 patients with transthyretin-related CA (ATTR-CA) and 10 patients with light chain CA (AL-CA)) and 16 were diagnosed as non-CA. All 5 patients with ATTR-CA had positive dual-phase scintigraphy and SPECT/CT imaging results. Three out of 10 patients with AL-CA had positive early-phase scintigraphy whereas negative delay-phase scintigraphy and SPECT/CT imaging results. Sixteen patients who were clinically diagnosed as non-CA had negative dual-phase scintigraphy and SPECT/CT imaging results. The sensitivity (5/5), specificity (10/10), positive predictive value (5/5), negative predictive value (10/10) and accuracy (15/15) of delay-phase scintigraphy and SPECT/CT imaging were the same. Among 31 patients, 16 patients carried transthyretin-related (TTR) gene mutation, and 4 of them who clinically diagnosed as variant ATTR (ATTRv) had positive image findings while 12 of them who not clinically diagnosed as CA had negative image findings. There were significant differences in H/CL between ATTR-CA group and AL-CA group in early-phase (2.11±0.24 vs 1.31±0.07) and delay-phase (2.02±0.19 vs 1.30±0.05; F values: 75.41 and 87.15, Bonferroni test, both P<0.01). Conclusions:99Tc m-PYP delay-phase scintigraphy and SPECT/CT have high diagnostic efficiencies in ATTR-CA, helping to determine the pathological classification of CA; while early-phase scintigraphy has false positive results. Moreover, 99Tc m-PYP imaging is helpful to detect CA in patients with TTR gene mutation.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with unknown etiology and limited therapeutic options. Activation of fibroblasts is a prominent feature of pulmonary fibrosis. Here we report that lncRNA DACH1 (dachshund homolog 1) is downregulated in the lungs of IPF patients and in an experimental mouse model of lung fibrosis. LncDACH1 knockout mice develop spontaneous pulmonary fibrosis, whereas overexpression of LncDACH1 attenuated TGF-β1-induced aberrant activation, collagen deposition and differentiation of mouse lung fibroblasts. Similarly, forced expression of LncDACH1 not only prevented bleomycin (BLM)-induced lung fibrosis, but also reversed established lung fibrosis in a BLM model. Mechanistically, LncDACH1 binding to the serine/arginine-rich splicing factor 1 (SRSF1) protein decreases its activity and inhibits the accumulation of Ctnnb1. Enhanced expression of SRSF1 blocked the anti-fibrotic effect of LncDACH1 in lung fibroblasts. Furthermore, loss of LncDACH1 promoted proliferation, differentiation, and extracellular matrix (ECM) deposition in mouse lung fibroblasts, whereas such effects were abolished by silencing of Ctnnb1. In addition, a conserved fragment of LncDACH1 alleviated hyperproliferation, ECM deposition and differentiation of MRC-5 cells driven by TGF-β1. Collectively, LncDACH1 inhibits lung fibrosis by interacting with SRSF1 to suppress CTNNB1 accumulation, suggesting that LncDACH1 might be a potential therapeutic target for pulmonary fibrosis.

Endoplasmic reticulum (ER) stress is mediated by disturbance of Ca²⁺ homeostasis. The store-operated calcium (SOC) channel is the primary Ca²⁺ channel in non-excitable cells, but its participation in agent-induced ER stress is not clear. In this study, the effects of tunicamycin on Ca²⁺ influx in human umbilical vein endothelial cells (HUVECs) were observed with the fluorescent probe Fluo-4 AM. The effect of tunicamycin on the expression of the unfolded protein response (UPR)-related proteins BiP and CHOP was assayed by western blotting with or without inhibition of Orai1. Tunicamycin induced endothelial dysfunction by activating ER stress. Orai1 expression and the influx of extracellular Ca²⁺ in HUVECs were both upregulated during ER stress. The SOC channel inhibitor SKF96365 reversed tunicamycin-induced endothelial cell dysfunction by inhibiting ER stress. Regulation of tunicamycin-induced ER stress by Orai1 indicates that modification of Orai1 activity may have therapeutic value for conditions with ER stress-induced endothelial dysfunction.
Blotting, Western ; Calcium ; Endoplasmic Reticulum ; Endoplasmic Reticulum Stress ; Endothelial Cells ; Homeostasis ; Human Umbilical Vein Endothelial Cells ; Tunicamycin ; Unfolded Protein Response

OBJECTIVETo investigate the role of miR-199a-3p in cardiac fibrosis and the potential target of miR-199a-3p.

METHODSCardiac fibroblasts were isolated from C57BL/6 mice and cultured. The miR-199a-3p mimic and Smad1 siRNA were transiently transfected into the cardiac fibroblasts via liposome. Dual luciferase reporter assay was performed to confirm the interaction between miR-199a-3p and the 3'-UTR of Smad1. The expressions of Smad1 and fibrosis-related genes at the mRNA and protein levels in the cells after miR-199a-3p mimic transfection were determined using RT-qPCR and Western blotting, respectively. The expressions of Smad1, Smad3 and fibrosis-related genes at the protein level in cells transfected with miR-199a-3p mimic and Smad1 siRNA were detected using Western blotting.

RESULTSOver-expression of miR-199a-3p significantly increased the expression of cardiac fibrosis-related genes in cultured mouse cardiac fibroblasts. Dual luciferase reporter assay revealed the interaction of miR-199a-3p with the 3'-UTR of Smad1. The results of RT-qPCR and Western blotting confirmed that miR-199a-3p inhibited Smad1 expression at the post- transcriptional level. Transfection with miR-199a-3p mimic and siRNA-mediated Smad1 silencing consistently activated the Smad3 signaling pathway and enhanced the expressions of cardiac fibrosis-related genes in the cardiac fibroblasts.

CONCLUSIONSAs the target gene of miR-199a-3p, Smad1 mediates the pro-fibrotic effect of miR-199a-3p by activating the Smad3 signaling in cultured mouse cardiac fibroblasts.