Advertisement

MicroRNA-19 upregulation attenuates cardiac fibrosis via targeting connective tissue growth factor

Published:December 16, 2022DOI:https://doi.org/10.1016/j.amjms.2022.12.010

      Abstract

      Background

      Previous studies have shown the role of microRNA (miR)-19 in aging-related heart failure. The present study aimed to verify the effects of miR-19 on cardiac fibrosis and its target.

      Methods

      Cardiac fibrosis was induced by myocardial infarction (MI)-induced heart failure and angiotensin (Ang) II-treated rats in vivo, and was induced in Ang II-treated cardiac fibroblasts (CFs) in vitro.

      Results

      The expression of miR-19 was reduced in the heart tissue of MI and Ang II-treated rats, and Ang II-treated CFs. The impaired cardiac function in rats was repaired after miR-19 administration. The levels of collagen I, collagen III and transforming growth factor-beta (TGF-β) increased in the heart tissue of MI and Ang II-treated rats, and Ang II-treated CFs. These increases were reversed by miR-19 agomiR. Moreover, the bioinformatic analysis and luciferase reporter assays demonstrated that connective tissue growth factor (CTGF) was a direct target of miR-19. MiR-19 treatment inhibited CTGF expression in CFs, while CTGF overexpression inhibited miR-19 agomiR to attenuate the Ang II-induced increases of collagen I and collagen III in CFs. The increases of p-ERK, p-JNK and p-p38 in the CFs induced by Ang II were repressed by miR-19 agomiR.

      Conclusions

      Upregulating miR-19 can improve cardiac function and attenuate cardiac fibrosis by inhibiting the CTGF and MAPK pathways.

      Key Indexing Terms

      Introduction

      Heart failure is caused by multiple factors, and some of them, such as obesity, diabetes and hypertension, are interconnected.
      • Borlaug B.A.
      Evaluation and management of heart failure with preserved ejection fraction.
      In the case of heart failure, electrical conduction, energy metabolism and cardiac contractility are disrupted, all disabling the heart to fulfill its circulatory roles.
      • Stanley W.C.
      • Recchia F.A.
      • Lopaschuk G.D.
      Myocardial substrate metabolism in the normal and failing heart.
      ,
      • Jessup M.
      • Brozena S.
      Heart failure.
      Heart failure is preceded by adverse left ventricle (LV) remodeling, as manifested by hypertrophy or dilatation.
      • Sygitowicz G.
      • Maciejak-Jastrzebska A.
      • Sitkiewicz D.
      MicroRNAs in the development of left ventricular remodeling and postmyocardial infarction heart failure.
      ,
      • Luczak E.D.
      • Wu Y.
      • Granger J.M.
      • et al.
      Mitochondrial CaMKII causes adverse metabolic reprogramming and dilated cardiomyopathy.
      Cardiac fibrosis is a key driver of chronic heart failure.
      • Tarone G.
      • Balligand J.L.
      • Bauersachs J.
      • et al.
      Targeting myocardial remodelling to develop novel therapies for heart failure: a position paper from the Working Group on Myocardial Function of the European Society of Cardiology.
      ,
      • Cheng X.
      • Wang L.
      • Wen X.
      • et al.
      TNAP is a novel regulator of cardiac fibrosis after myocardial infarction by mediating TGF-beta/Smads and ERK1/2 signaling pathways.
      In addition, excessive cardiac fibrosis causes large infarction that impair cardiac function.
      • Kong P.
      • Christia P.
      • Frangogiannis N.G.
      The pathogenesis of cardiac fibrosis.
      ,
      • Heineke J.
      • Molkentin J.D.
      Regulation of cardiac hypertrophy by intracellular signalling pathways.
      Cardiac fibrosis is characterized by the accumulation of extracellular matrix in the myocardium.
      • Berk B.C.
      • Fujiwara K.
      • Lehoux S.
      ECM remodeling in hypertensive heart disease.
      Cardiac fibroblasts (CFs) are major participators in fibrosis,
      • Cheng X.
      • Wang L.
      • Wen X.
      • et al.
      TNAP is a novel regulator of cardiac fibrosis after myocardial infarction by mediating TGF-beta/Smads and ERK1/2 signaling pathways.
      ,
      • Tallquist M.D.
      • Molkentin J.D.
      Redefining the identity of cardiac fibroblasts.
      but their mechanisms remain poorly understood.
      MicroRNAs (miRs), a group of small and non-coding RNAs, can bind to the complementary 3’-untranslated region to inhibit messenger RNA (mRNA) translation, thus negatively regulating gene expression at the post-transcriptional level.
      • Bartel D.P.
      MicroRNAs: genomics, biogenesis, mechanism, and function.
      • Javadian M.
      • Gharibi T.
      • Shekari N.
      • et al.
      The role of microRNAs regulating the expression of matrix metalloproteinases (MMPs) in breast cancer development, progression, and metastasis.
      • van Rooij E.
      The art of microRNA research.
      • Mohr A.M.
      • Mott J.L.
      Overview of microRNA biology.
      Many miRNAs play pathogenic roles in processes of heart failure, such as apoptosis, remodeling, hypoxia, or hypertrophy.
      • Melman Y.F.
      • Shah R.
      • Das S.
      MicroRNAs in heart failure: is the picture becoming less miRky?.
      ,
      • Tijsen A.J.
      • Pinto Y.M.
      • Creemers E.E.
      Non-cardiomyocyte microRNAs in heart failure.
      In aging-associated heart failure, the expression level of miR-19 decreased, but in heart failure-resistant mice, this level increased.
      • van Almen G.C.
      • Verhesen W.
      • van Leeuwen R.E.
      • et al.
      MicroRNA-18 and microRNA-19 regulate CTGF and TSP-1 expression in age-related heart failure.
      The effects of miR-19 on cardiac fibrosis and associated mechanisms are still unclear.
      Connective tissue growth factor (CTGF) is a cysteine-rich matricellular protein involved in various biological processes, such as cell adhesion, angiogenesis, proliferation and differentiation, as well as tissue fibrosis.
      • Ramazani Y.
      • Knops N.
      • Elmonem M.A.
      • et al.
      Connective tissue growth factor (CTGF) from basics to clinics.
      CTGF monoclonal antibody can repair the infarcted tissue and attenuate LV hypertrophy and fibrosis to enhance survival and LV function.
      • Vainio L.E.
      • Szabo Z.
      • Lin R.
      • et al.
      Connective tissue growth factor inhibition enhances cardiac repair and limits fibrosis after myocardial infarction.
      MiR-19 can modulate the expression of extracellular matrix proteins and CTGF in cardiomyocytes.
      • van Almen G.C.
      • Verhesen W.
      • van Leeuwen R.E.
      • et al.
      MicroRNA-18 and microRNA-19 regulate CTGF and TSP-1 expression in age-related heart failure.
      The purpose of this study was to explore the cardioprotective effects of miR-19 in rats with MI-induced heart failure and associated regulatory mechanisms.

      Materials and methods

      Ethical approval

      The experiments were performed at the Animal Core Facility of Nanjing Medical University using male Sprague-Dawley (SD) rats weighing 180–220 g (Vital River Biological Co., Ltd, Beijing, China). All the animals were housed in an environment at a temperature of 22 ± 1 °C, a relative humidity of 50 ± 1%, and a light/dark cycle of 12/12 h, with free access to standard chow and tap water. All procedures were in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, revised 1996).

      Myocardial infarction model

      Myocardial infarction (MI) was induced in the rats by coronary artery ligation (CAL) with sterile techniques as previously reported.
      • Gan X.B.
      • Duan Y.C.
      • Xiong X.Q.
      • et al.
      Inhibition of cardiac sympathetic afferent reflex and sympathetic activity by baroreceptor and vagal afferent inputs in chronic heart failure.
      Briefly, the rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and randomly subjected to the ligation of the left anterior descending (LAD) coronary artery (MI group) or sham operation (Sham group). The heart was exposed through left intercostal thoracotomy, and the left coronary artery was looped by a 7-0 single nylon suture. Afterward, the heart was quickly repositioned into the chest. The Sham rats were treated in the same way as the CAL rats, except that their coronary arteries were not ligated.

      Ang II pump

      The rats were anesthetized with 3.5% isoflurane induction and 2.5% isoflurane maintenance. After a small incision was made in the back of the neck, the rats were subjected to a 4-week infusion of Ang II (200 ng/kg/min, Sigma, MO, USA) or saline delivered by mini-osmotic pumps (ALZET Osmotic Pumps, CA, USA) that were surgically inserted below the neck.

      MiR-19 agomiR treatment in rats

      The rats were injected with miR-19 agomiR (a 2′OME  +  5′chol modified miR-19 agonist, 40 mg/kg/day, once every three days) or negative control (NC) agomiR (40 mg/kg/day) via the tail vein at seven days after MI or Ang II infusion. The miR-19 agomiR was obtained from RIBOBIO (Guangzhou, China). After three weeks of injection, the rats were sacrificed with an overdose of sodium pentobarbital (100 mg/kg, i.p.).

      Echocardiography

      Transthoracic echocardiography was performed using an ultrasound system (VisualSonics, Toronto, Canada) and a 21-MHz probe in the rats under isoflurane (2.0-2.5%) anesthesia. Measurements over three consecutive cardiac cycles were averaged. The left ventricular (LV) ejection fraction (EF) and fractional shortening (FS), LV end-systolic diameter (LVESD), LV end-diastolic diameter (LVEDD), LV volumes in systole (LVVS), and LV volumes in diastole (LVVD) were measured.

      Hemodynamic monitoring

      The rats were anesthetized with isoflurane (2.0-2.5%). A conductance micromanometer catheter (1.4F, Millar Instruments, TX, USA) was inserted into the LV chamber via the right carotid artery for hemodynamic monitoring. The maximum first derivative of LV pressure (LV  + dp/dtmax), LV systolic pressure (LVSP) and LV end-diastolic pressure (LVEDP) were obtained using a PowerLab data acquisition system (AD Instruments, Sydney, Australia).

      Masson's trichrome staining

      Cardiac sections (5 µm) were examined by Masson's Trichrome Staining (Service Biological Technology Co., Ltd, Wuhan, China). Three to five random fields were selected from each of three sections from one rat, and analyzed under a light microscope (Carl Zeiss GmbH, Oberkochen, Germany). The images were analyzed using Image-Pro Plus software (Media Cybernetics, Inc., MD, USA).

      Quantitative real time-PCR (qRT-PCR)

      The rats were sacrificed with an overdose of pentobarbital (100 mg/kg, i.p.), and the hearts were sampled. The total RNA in the samples was extracted with TRIzol (Ambion, TX, USA). The cDNA was extracted from the RNA with reverse transcription using random primers in a total volume of 10 μL according to the instructions of the PrimeScript™ RT Master Mix (TaKaRa Biomedical Technology, Dalian, China). The cDNA was stored at − 80°C before use. mRNA was determined with SYBR Green I fluorescence. All samples were amplified in triplicates for 40 cycles in 384-well plates. The relative gene expression was determined by calculating the values of Δcycle threshold (ΔCt) as a relative quantity to the endogenous control. The primers (Genscript, Nanjing, China) are shown in Table 1.
      TABLE 1Primers used for qRT-PCR.
      GeneSpeciesForward primerReverse primer
      miR-19RatACCTGTGCAAATCCATGTGCGTGTCGTGGAGTC
      Collagen IRatTCAAGATGGTGGCCGTTACCTGCGGATGTTCTCAATCTG
      Collagen IIIRatCGAGATTAAAGCAAGAGGAAGAGGCTTCTTTACATACCAC
      TGF-βRatCAGGGAGTAAGGGACACGAACAGCAGTTAGGAACCCAGAT
      U6RatGCTTCGGCAGCACATATACTAAAATCGCTTCACGAATTTGCGTGTCAT
      GAPDHRatGGCACAGTCAAGGCTGAGAATGATGGTGGTGAAGACGCCAGTA
      miR-19, microRNA-19; TGF-β, transforming growth factor-beta; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

      Western blotting

      Heart tissue or CFs samples were sonicated in RIPA lysis buffer (Beyotime Biotechnology Shanghai, China) and homogenized. The debris was removed and the supernatant was obtained through centrifugation at 12,000 g for 10 min at 4°C. Then, the protein was separated by gel electrophoresis, transferred to PVDF membrane, and probed with primary antibodies against collagen I (Abcam, MA, USA), collagen III (Abcam), TGF-β (Abcam), p-ERK (Cell Signaling Technology, MA, USA), ERK (Cell Signaling Technology), p-JNK (Cell Signaling Technology), JNK (Cell Signaling Technology), p-p38 (Cell Signaling Technology), p38 (Cell Signaling Technology); and GAPDH (Abcam) as an internal control. Images were analyzed using Image-Pro Plus software.

      Isolation and culture of CFs

      Rat CFs were isolated from SD rats (1-3 days). Briefly, CFs were separated from cardiomyocytes by gravity separation and allowed to grow on 10-cm cell culture dishes coated with DMEM medium, containing 10% FBS, 1% penicillin and 1% streptomycin (Gibco), in humidified air with 5% CO2 and 95% O2 at 37 °C. The P2 CFs were used in the experiments. CFs were incubated with 10−6 M
      • Yang Z.
      • Zhang X.
      • Guo N.
      • Li B.
      • Zhao S.
      Substance P inhibits the collagen synthesis of rat myocardial fibroblasts induced by Ang II.
      ,
      • Yang X.
      • Wang Y.
      • Yan S.
      • et al.
      Effect of testosterone on the proliferation and collagen synthesis of cardiac fibroblasts induced by angiotensin II in neonatal rat.
      Ang II (Sigma, MO, USA) for 24 h to induce induce fibrosis, and treated with miR-19 agomiR according to the manufacturers’ instructions.

      CTGF overexpression

      The CFs were transfected with serum-free medium containing recombinant adenovirus (Ad)-CTGF (1e + 7Tu/ml; GeneChem Co., Shanghai, China) for 6-8 h, till a confluence of 70-80% was detected.

      Bioinformatics analysis and dual-luciferase reporter gene assay

      Briefly, endonuclease sites (SpeI and HindIII) were used to insert CTGF into the pMIR-reporter vector, and the mutation sites of complementary sequences of seed sequences were designed on wild-type CTGF (CTGF-WT). After the sequences were cut with restriction enzyme, the T4 DNA ligase was used to insert the target fragment into the pMIR‐reporter vector. The WT and mutant type (MUT) luciferase reporter plasmids were co-transfected into HEK-293T with miR-19, respectively. After 48 h of transfection, the cells were collected and centrifuged for 5 min to collect the supernatant. The luciferase kit (Beyotime Biotech Co, Ltd., Shanghai, China) was used to determine the relative light unit (RLU) according to manufacturer's instructions

      Statistical analyses

      Data were presented as mean ± standard error of the mean (SEM). Using GraphPad Prism 9.0 (GraphPad Software Inc., CA, USA), statistical significance was evaluated by unpaired t-test between two groups, or one-way analysis of variance (ANOVA) with the Bonferroni post-hoc test among multiple groups. A two-tailed P-value <0.05 was considered statistically significant.

      Results

      Expression of miR-19

      The level of miR-19 in the heart tissue of MI rats was lower than that in the Sham rats (Fig. 1A). The expression level of miR-19 was reduced in the heart tissue of Ang II pump rats, compared with that in the saline pump rats (Fig. 1B). The expression level of miR-19 in the CFs was reduced after Ang II treatment (Fig. 1C). The expression level of miR-19 was increased in the heart tissue of rats after miR-19 agomiR administration (Fig. 1D).
      Fig 1
      Figure 1The expression of miR-19. A, the expression of miR-19 was decreased in the heart of Ang II-treated rats. B, the expression of miR-19 was decreased in the heart of MI rats. C, the expression of miR-19 was decreased in the CFs treated with Ang II. D, the expression of miR-19 was increased in the heart of rats after miR-19 agomiR administration. The results are expressed as mean ± SEM. N = 8 for each group. *p < 0.05 vs. Sham (A and D), Saline (B) or PBS group (C); #p < 0.05 versus the NC agomiR group. Ang, angiotensin; CFs, cardiac fibroblasts; MI, myocardial infarction; miR, microRNA; NC, negative control; SEM, standard error of the mean.

      Effects of miR-19 agomiR on cardiac dysfunction in MI rats

      The EF and FS of LV in MI rats decreased, and these decreases were enhanced after miR-19 agomiR administration. LVESD, LVEDD, LVVS and LVVD increased in MI rats, but subsequently dropped after miR-19 agomiR administration (Fig. 2A). LV  + dp/dtmax and LVSP lowered in MI rats. miR-19 agomiR significantly enhanced the decreases of LV  + dp/dtmax and LVSP in MI rats. LVEDP increased in MI rats, and this increase was inhibited after miR-19 agomiR treatment (Fig. 2B).
      Fig 2
      Figure 2Effects of miR-19 agomiR on cardiac function in MI-induced heart failure rats. A, the EF and FS of LV were reduced, and LVESD, LVEDD, LVVS and LVVD were increased in MI-induced heart failure rats, and these changes were reversed by miR-19 agomiR treatment. B, the decreases of LV  +  dp/dtmax and LVSP, and the increase of LVEDP in the MI rats were reversed by miR-19 agomiR treatment. The results are expressed as mean ± SEM. N = 8 for each group. *p < 0.05 versus the Sham  +  NC agomiR group; #p < 0.05 versus the MI  +  NC agomiR group. Ang, angiotensin; CFs, cardiac fibroblasts; EF, ejection fraction; FS, fractional shortening; LV, left ventricular; LV ± dp/dtmax, the maximum first derivative of LV pressure; LVEDD, LV end-diastolic diameter LVESD, LV end-systolic diameter; LVSP, LV systolic pressure; LVVD, LV volumes in diastole; LVVS, LV volumes in systole; MI, myocardial infarction; miR, microRNA; NC, negative control; SEM, standard error of the mean.

      Effects of miR-19 agomiR on cardiac fibrosis in MI rats

      The LV fibrosis in MI rats increased markedly, and this increase was reduced by miR-19 agomiR (Fig. 3A). The expression levels of collagen I, collagen III and TGF-β mRNA in the heart increased in the rats with MI-induced heart failure, and these increases were blocked by miR-19 agomiR (Fig. 3B). In addition, miR-19 agomiR treatment inhibited the increases of collagen I, collagen III and TGF-β proteins in the heart of MI rats (Fig. 3C).
      Fig 3
      Figure 3Effects of miR-19 agomiR on cardiac fibrosis in MI rats. A, miR-19 agomiR inhibited cardiac fibrosis of MI rats. B, miR-19 agomiR inhibited the increases of collagen I, collagen III and TGF-β mRNA in the heart of MI rats. C, miR-19 agomiR inhibited the increases of collagen I, collagen III and TGF-β proteins in the heart of MI rats. The results are expressed as mean ± SEM. N = 8 for each group. *p < 0.05 versus the Sham  +  NC agomiR group; #p < 0.05 versus the MI  +  NC agomiR group. MI, myocardial infarction; miR, microRNA; NC, negative control; SEM, standard error of the mean; TGF-β, transforming growth factor-beta.

      Effects of miR-19 agomiR on cardiac fibrosis in Ang II-treated rats

      Ang II infusion aggravated the fibrosis of the rat heart, and this aggravation was inhibited by miR-19 agomiR (Fig. 4A). The expression levels of collagen I, collagen III and TGF-β mRNA in the heart increased in Ang II-treated rats, and these increases were blocked by miR-19 agomiR (Fig. 4B).
      Fig 4
      Figure 4Effects of miR-19 agomiR on cardiac fibrosis in rats treated with Ang II. A, miR-19 agomiR inhibited cardiac fibrosis of Ang II-treated rats. B, miR-19 agomiR inhibited the increases of collagen I, collagen III and TGF-β mRNA in the heart of Ang II-treated rats. The results are expressed as mean ± SEM. N = 8 for each group. *p < 0.05 versus the Saline  +  NC agomiR group; #p < 0.05 versus the Ang II  +  NC agomiR group. Ang, angiotensin; miR, microRNA; NC, negative control; SEM, standard error of the mean; TGF-β, transforming growth factor-beta.

      Effects of miR-19 agomiR on CFs fibrosis induced by Ang II

      The increased expression of collagen I, collagen III and TGF-β mRNA induced by Ang II- in CFs was inhibited by miR-19 agomiR (Fig. 5A). The protein levels of collagen I, collagen III and TGF-β elevated in CFs treated with Ang II, and these elevations were attenuated by miR-19 agomiR (Fig. 5B).
      Fig 5
      Figure 5Effects of miR-19 agomiR on the fibrosis of CFs induced by Ang II. A, miR-19 agomiR inhibited the Ang II-induced increases of collagen I, collagen III and TGF-β mRNA in the CFs. B, miR-19 agomiR attenuated the Ang II-induced increases of collagen I, collagen III and TGF-β proteins in the CFs. The results are expressed as mean ± SEM. N = 6 for each group. *p < 0.05 versus the PBS  +  NC agomiR group; #p < 0.05 versus the Ang II  +  NC agomiR group. Ang, angiotensin; CFs, cardiac fibroblasts; miR, microRNA; NC, negative control; SEM, standard error of the mean; TGF-β, transforming growth factor-beta.

      CTGF was a target of miR-19

      A bioinformatic analysis was performed to predict the interactions between miR-19 and the target mRNAs. We found that CTGF was a potential target of miR-19. The scheme showed the difference between the 3’UTR regions of CTGF gene and miR-19 (Fig. 6A). CTGF mRNA expression level in CFs was reduced after miR-19 agomiR treatment (Fig. 6B). miR-19 agomiR significantly weakened the luciferase activities of the vectors with the wt CTGF 3’UTR, but not those of mt CTGF 3’UTR (Fig. 6C). CTGF level increased in the heart of MI rats (Fig. 6D). CTGF expression level increased in the heart of rats treated with Ang II (Fig. 6E).
      Fig 6
      Figure 6miR-19 directly targeted CTGF. A, Predicted binding site of miR-19 in the 3’ UTR region of CTGF. B, CTGF mRNA expression level was reduced after miR-19 agomiR administration in CFs. C, MiR-19 overexpression decreased the luciferase activities of the vector with wt CTGF 3’UTR, but not of those with mt CTGF 3’UTR. D, CTGF expression level was increased in the heart of MI rats. E, CTGF expression level was increased in the heart of rats treated with Ang II. The results are expressed as mean ± SEM. N = 8 for each group. *p < 0.05 versus the NC agomiR (B and C) or Sham (D) or Saline (E), #p < 0.05 versus the PBS. Ang, angiotensin; CFs, cardiac fibroblasts; CTGF, connective tissue growth factor; MI, myocardial infarction; miR, microRNA; NC, negative control; SEM, standard error of the mean; TGF-β, transforming growth factor-beta.

      CTGF overexpression reversed the effects of miR-19

      CTGF overexpression reversed the inhibitory effects of miR-19 on the Ang II-induced increases of collagen I and collagen III mRNA levels in CFs (Fig. 7A). In addition, CTGF overexpression reversed the attenuative effects of miR-19 on the increases of collagen I and collagen III protein levels in CFs induced by Ang II (Fig. 7B).
      Fig 7
      Figure 7CTGF overexpression reversed the effects of miR-19. A, CTGF overexpression reversed miR-19 agomiR to inhibit the Ang II-induced increases of collagen I and collagen III mRNA in CFs. B, CTGF overexpression reversed miR-19 agomiR to inhibit the Ang II-induced increases of collagen I and collagen III protein in CFs. The results are expressed as mean ± SEM. N = 8 for each group. *p < 0.05 versus the PBS + NC agomiR + Ad-GFP, #p < 0.05 versus the Ang II + NC agomiR + Ad-GFP. &p < 0.05 versus the Ang II + miR-19 agomiR + Ad-GFP group. Ang, angiotensin; CFs, cardiac fibroblasts; CTGF, connective tissue growth factor; GFP, green fluorescent protein; miR, microRNA; NC, negative control; SEM, standard error of the mean.

      Effects of miR-19 agomiR on MAPKs pathway

      The levels of p-ERK, p-JNK and p-p38 increased in the CFs treated with Ang II. Treatment with miR-19 agomiR inhibited the increases of p-ERK, p-JNK and p-p38 in the CFs induced by Ang II (Fig. 8).
      Fig 8
      Figure 8Effects of miR-19 agomiR on MAPKs pathway. The increases of p-ERK, p-JNK and p-p38 in the CFs induced by angiotensin (Ang) II were attenuated by miR-19 agomiR. The results are expressed as mean ± SEM. N = 4 for each group. *p < 0.05 versus the PBS + NC agomiR group; #p < 0.05 versus the Ang II + NC agomiR group. Ang, angiotensin; MAPKs, mitogen-activated protein kinases; miR, microRNA; NC, negative control; SEM, standard error of the mean.

      Discussion

      In the present study, we found that miR-19 upregulation significantly improved cardiac function in MI rats. miR-19 agomiR could attenuate CF fibrosis induced by Ang II via targeting CTGF.
      MiR-19 has shown to be downregulated in the vascular epithelial tissues of ischemia-reperfusion injury (IRI) rats.
      • Antoniak S.
      • Sparkenbaugh E.
      • Pawlinski R.
      Tissue factor, protease activated receptors and pathologic heart remodelling.
      The serum level of miR-19 is lower in Crohn's disease (CD) patients with a stricture phenotype than in control patients.
      • Lewis A.
      • Mehta S.
      • Hanna L.N.
      • et al.
      Low serum levels of MicroRNA-19 are associated with a stricturing crohn's disease phenotype.
      In addition, low miR-19 expression is associated with severe heart failure in old-age mice.
      • van Almen G.C.
      • Verhesen W.
      • van Leeuwen R.E.
      • et al.
      MicroRNA-18 and microRNA-19 regulate CTGF and TSP-1 expression in age-related heart failure.
      In the current study, we found that miR-19 expression level was reduced in the heart tissue of MI and Ang II-treated rats and the CFs treated with Ang II, suggesting that miR-19 may be involved in the pathogenesis of heart failure.
      The cardiac function and hemodynamic profiles are significantly changed in heart failure induced by MI.
      • Yu Y.
      • Sun J.
      • Liu J.
      • Wang P.
      • Wang C.
      Ginsenoside Re preserves cardiac function and ameliorates left ventricular remodeling in a rat model of myocardial infarction.
      ,
      • Wu Y.
      • Hu Z.
      • Wang D.
      • Lv K.
      • Hu N.
      Resiniferatoxin reduces ventricular arrhythmias in heart failure via selectively blunting cardiac sympathetic afferent projection into spinal cord in rats.
      LV remodeling is manifested by the decreases in EF and FS, and the increases in LVVS, LVVD, LVESD and LVEDD.
      • Fei L.
      • Zhang J.
      • Niu H.
      • Yuan C.
      • Ma X.
      Effects of rosuvastatin and MiR-126 on myocardial injury induced by acute myocardial infarction in rats: role of vascular endothelial growth factor a (VEGF-A).
      ,
      • Zhang D.Y.
      • Wang B.J.
      • Ma M.
      • Yu K.
      • Zhang Q.
      • Zhang X.W.
      Correction to: MicroRNA-325-3p protects the heart after myocardial infarction by inhibiting RIPK3 and programmed necrosis in mice.
      In the current study, we found that miR-19 agomiR enhanced the decreases of EF, FS, LV ±dp/dt max and LVSP in MI rats. In addition, miR-19 agomiR reversed the increases in LVVS, LVVD, LVESD, LVEDD and LVEDP in MI-induced heart failure rats. These results demonstrated that the upregulation of miR-19 could ameliorate cardiac dysfunction in rats with MI-induced heart failure.
      Myocardial interstitial fibrosis contributes to LV dysfunction and even heart failure. CFs activation leads to excessive deposition of ECM proteins, such as fibronectin and collagen type I, during the progression of heart failure.
      • Moore-Morris T.
      • Guimaraes-Camboa N.
      • Banerjee I.
      • et al.
      Resident fibroblast lineages mediate pressure overload-induced cardiac fibrosis.
      A variety of miRs are involved in cardiac fibrosis.
      • Medzikovic L.
      • Aryan L.
      • Eghbali M.
      Connecting sex differences, estrogen signaling, and microRNAs in cardiac fibrosis.
      ,
      • Kura B.
      • Szeiffova Bacova B.
      • Kalocayova B.
      • Sykora M.
      • Slezak J.
      Oxidative stress-responsive MicroRNAs in heart injury.
      We found that cardiac fibrosis emerged in the heart of the MI rats, but was inhibited by miR-19 agomiR. The expression levels of fibrosis markers increased in the heart tissue of the MI and Ang II-treated rats, and these elevations were reversed by miR-19 agomiR. In addition, Ang II significantly elevated the levels of fibrosis markers in CFs, and these increases were attenuated by miR-19 agomiR. These results indicated that miR-19 upregulation could attenuate cardiac fibrosis to improve cardiac dysfunction in heart failure.
      CTGF, a cysteine-rich growth factor, is implicated in ECM protein synthesis induced by TGF-β.
      • Zhang D.Y.
      • Wang B.J.
      • Ma M.
      • Yu K.
      • Zhang Q.
      • Zhang X.W.
      Correction to: MicroRNA-325-3p protects the heart after myocardial infarction by inhibiting RIPK3 and programmed necrosis in mice.
      Fibroblast-derived CTGF is pro-fibrotic, while cardiomyocyte-derived CTGF is not.
      • Dorn L.E.
      • Petrosino J.M.
      • Wright P.
      • Accornero F.
      CTGF/CCN2 is an autocrine regulator of cardiac fibrosis.
      In our current study, the results showed that CTGF expression level was reduced after miR-19 agomiR administration in CFs. The overexpression of miR-19 significantly decreased the luciferase activities of vectors with wt CTGF 3’UTR. CTGF overexpression reversed the inhibitory effects of miR-19 agomiR on the Ang II-induced increases of collagen I and collagen III in CFs. Our results indicate that miR-19 could inhibite Ang II-induced fibrosis of CFs via targeting CTGF.
      MAPKs are central regulators during the transition of fibroblasts to myofibroblasts that is activated by chemical and mechanical stress signals.
      • Bretherton R.
      • Bugg D.
      • Olszewski E.
      • Davis J.
      Regulators of cardiac fibroblast cell state.
      The phosphorylation of p38 MAPK was enhanced in the mice with cardiac fibrosis induced by isoproterenol.
      • Zhao T.
      • Kee H.J.
      • Bai L.
      • Kim M.K.
      • Kee S.J.
      • Jeong M.H.
      Selective HDAC8 inhibition attenuates isoproterenol-induced cardiac hypertrophy and fibrosis via p38 MAPK pathway.
      ERK and JNK were activated in acute myocardial infarction model.
      • Liu Z.
      • Gao Z.
      • Zeng L.
      • Liang Z.
      • Zheng D.
      • Wu X.
      Nobiletin ameliorates cardiac impairment and alleviates cardiac remodeling after acute myocardial infarction in rats via JNK regulation.
      We found that ERK, JNK and p38 were activated in the CFs induced by Ang II, and these activations were inhibited by miR-19 agomiR. These results indicated that miR-19 could alleviate fibrosis of CFs via attenuating the MAPKs pathway.

      Conclusions

      In conclusion, miR-19 upregulation can inhibit CFs fibrosis by targeting CTGF. Upregulation of miR-19 alleviates cardiac fibrosis via attenuating the MAPKs pathway. Thus, miR-19 may be targeted to design a therapeutic strategy for heart failure.

      Funding

      This study was supported by Shandong Province Medical and Health Technology Development Plan (2019WS043).

      Authors Contributions statement

      XZ.S: Conceptualization, Investigation, Methodology and Project administration. YQ.C: Data curation and Formal analysis. T.Z: Conceptualization, Funding acquisition, Resources, Supervision, Writing - original draft and Writing - review & editing.

      Declaration of Competing Interests

      The author has no financial or other conflicts of interest to disclose.

      References

        • Borlaug B.A.
        Evaluation and management of heart failure with preserved ejection fraction.
        Nat Rev Cardiol. 2020; 17: 559-573
        • Stanley W.C.
        • Recchia F.A.
        • Lopaschuk G.D.
        Myocardial substrate metabolism in the normal and failing heart.
        Physiol Rev. 2005; 85: 1093-1129
        • Jessup M.
        • Brozena S.
        Heart failure.
        N Engl J Med. 2003; 348: 2007-2018
        • Sygitowicz G.
        • Maciejak-Jastrzebska A.
        • Sitkiewicz D.
        MicroRNAs in the development of left ventricular remodeling and postmyocardial infarction heart failure.
        Pol Arch Intern Med. 2020; 130: 59-65
        • Luczak E.D.
        • Wu Y.
        • Granger J.M.
        • et al.
        Mitochondrial CaMKII causes adverse metabolic reprogramming and dilated cardiomyopathy.
        Nat Commun. 2020; 11: 4416
        • Tarone G.
        • Balligand J.L.
        • Bauersachs J.
        • et al.
        Targeting myocardial remodelling to develop novel therapies for heart failure: a position paper from the Working Group on Myocardial Function of the European Society of Cardiology.
        Eur J Heart Fail. 2014; 16: 494-508
        • Cheng X.
        • Wang L.
        • Wen X.
        • et al.
        TNAP is a novel regulator of cardiac fibrosis after myocardial infarction by mediating TGF-beta/Smads and ERK1/2 signaling pathways.
        EBioMedicine. 2021; 67103370
        • Kong P.
        • Christia P.
        • Frangogiannis N.G.
        The pathogenesis of cardiac fibrosis.
        Cell Mol Life Sci. 2014; 71: 549-574
        • Heineke J.
        • Molkentin J.D.
        Regulation of cardiac hypertrophy by intracellular signalling pathways.
        Nat Rev Mol Cell Biol. 2006; 7: 589-600
        • Berk B.C.
        • Fujiwara K.
        • Lehoux S.
        ECM remodeling in hypertensive heart disease.
        J Clin Invest. 2007; 117: 568-575
        • Tallquist M.D.
        • Molkentin J.D.
        Redefining the identity of cardiac fibroblasts.
        Nat Rev Cardiol. 2017; 14: 484-491
        • Bartel D.P.
        MicroRNAs: genomics, biogenesis, mechanism, and function.
        Cell. 2004; 116: 281-297
        • Javadian M.
        • Gharibi T.
        • Shekari N.
        • et al.
        The role of microRNAs regulating the expression of matrix metalloproteinases (MMPs) in breast cancer development, progression, and metastasis.
        J Cell Physiol. 2018; 234: 5399-5412
        • van Rooij E.
        The art of microRNA research.
        Circ Res. 2011; 108: 219-234
        • Mohr A.M.
        • Mott J.L.
        Overview of microRNA biology.
        Semin Liver Dis. 2015; 35: 3-11
        • Melman Y.F.
        • Shah R.
        • Das S.
        MicroRNAs in heart failure: is the picture becoming less miRky?.
        Circ Heart Fail. 2014; 7: 203-214
        • Tijsen A.J.
        • Pinto Y.M.
        • Creemers E.E.
        Non-cardiomyocyte microRNAs in heart failure.
        Cardiovasc Res. 2012; 93: 573-582
        • van Almen G.C.
        • Verhesen W.
        • van Leeuwen R.E.
        • et al.
        MicroRNA-18 and microRNA-19 regulate CTGF and TSP-1 expression in age-related heart failure.
        Aging Cell. 2011; 10: 769-779
        • Ramazani Y.
        • Knops N.
        • Elmonem M.A.
        • et al.
        Connective tissue growth factor (CTGF) from basics to clinics.
        Matrix Biol. 2018; 68-69: 44-66
        • Vainio L.E.
        • Szabo Z.
        • Lin R.
        • et al.
        Connective tissue growth factor inhibition enhances cardiac repair and limits fibrosis after myocardial infarction.
        JACC Basic Transl Sci. 2019; 4: 83-94
        • Gan X.B.
        • Duan Y.C.
        • Xiong X.Q.
        • et al.
        Inhibition of cardiac sympathetic afferent reflex and sympathetic activity by baroreceptor and vagal afferent inputs in chronic heart failure.
        PLoS One. 2011; 6: e25784
        • Yang Z.
        • Zhang X.
        • Guo N.
        • Li B.
        • Zhao S.
        Substance P inhibits the collagen synthesis of rat myocardial fibroblasts induced by Ang II.
        Med Sci Monit. 2016; 22: 4937-4946
        • Yang X.
        • Wang Y.
        • Yan S.
        • et al.
        Effect of testosterone on the proliferation and collagen synthesis of cardiac fibroblasts induced by angiotensin II in neonatal rat.
        Bioengineered. 2017; 8: 14-20
        • Antoniak S.
        • Sparkenbaugh E.
        • Pawlinski R.
        Tissue factor, protease activated receptors and pathologic heart remodelling.
        Thromb Haemost. 2014; 112: 893-900
        • Lewis A.
        • Mehta S.
        • Hanna L.N.
        • et al.
        Low serum levels of MicroRNA-19 are associated with a stricturing crohn's disease phenotype.
        Inflamm Bowel Dis. 2015; 21: 1926-1934
        • Yu Y.
        • Sun J.
        • Liu J.
        • Wang P.
        • Wang C.
        Ginsenoside Re preserves cardiac function and ameliorates left ventricular remodeling in a rat model of myocardial infarction.
        J Cardiovasc Pharmacol. 2020; 75: 91-97
        • Wu Y.
        • Hu Z.
        • Wang D.
        • Lv K.
        • Hu N.
        Resiniferatoxin reduces ventricular arrhythmias in heart failure via selectively blunting cardiac sympathetic afferent projection into spinal cord in rats.
        Eur J Pharmacol. 2020; 867172836
        • Fei L.
        • Zhang J.
        • Niu H.
        • Yuan C.
        • Ma X.
        Effects of rosuvastatin and MiR-126 on myocardial injury induced by acute myocardial infarction in rats: role of vascular endothelial growth factor a (VEGF-A).
        Med Sci Monit. 2016; 22: 2324-2334
        • Zhang D.Y.
        • Wang B.J.
        • Ma M.
        • Yu K.
        • Zhang Q.
        • Zhang X.W.
        Correction to: MicroRNA-325-3p protects the heart after myocardial infarction by inhibiting RIPK3 and programmed necrosis in mice.
        BMC Mol Biol. 2019; 20: 18
        • Moore-Morris T.
        • Guimaraes-Camboa N.
        • Banerjee I.
        • et al.
        Resident fibroblast lineages mediate pressure overload-induced cardiac fibrosis.
        J Clin Invest. 2014; 124: 2921-2934
        • Medzikovic L.
        • Aryan L.
        • Eghbali M.
        Connecting sex differences, estrogen signaling, and microRNAs in cardiac fibrosis.
        J Mol Med (Berl). 2019; 97: 1385-1398
        • Kura B.
        • Szeiffova Bacova B.
        • Kalocayova B.
        • Sykora M.
        • Slezak J.
        Oxidative stress-responsive MicroRNAs in heart injury.
        Int J Mol Sci. 2020; 21: 358
        • Dorn L.E.
        • Petrosino J.M.
        • Wright P.
        • Accornero F.
        CTGF/CCN2 is an autocrine regulator of cardiac fibrosis.
        J Mol Cell Cardiol. 2018; 121: 205-211
        • Bretherton R.
        • Bugg D.
        • Olszewski E.
        • Davis J.
        Regulators of cardiac fibroblast cell state.
        Matrix Biol. 2020; 91-92: 117-135
        • Zhao T.
        • Kee H.J.
        • Bai L.
        • Kim M.K.
        • Kee S.J.
        • Jeong M.H.
        Selective HDAC8 inhibition attenuates isoproterenol-induced cardiac hypertrophy and fibrosis via p38 MAPK pathway.
        Front Pharmacol. 2021; 12677757
        • Liu Z.
        • Gao Z.
        • Zeng L.
        • Liang Z.
        • Zheng D.
        • Wu X.
        Nobiletin ameliorates cardiac impairment and alleviates cardiac remodeling after acute myocardial infarction in rats via JNK regulation.
        Pharmacol Res Perspect. 2021; 9: e00728