Early detection of cardiac fibrosis in diabetic mice by targeting myocardiopathy and matrix metalloproteinase 2

Tinglu Yixin granule inhibited fibroblast-myofibroblast transdifferentiation to ameliorate myocardial fibrosis in diabetic mice

Diabetic cardiomyopathy (DCM) constitutes a primary cause of mortality in diabetic patients. Histone deacetylase (HDAC) inhibition can alleviate diabetes-associated myocardial injury. This study investigated the mechanism of HDAC1 on myocardial fibrosis (MF) in DCM. A murine model of DCM was established by a high-fat diet and streptozotocin injection. The bodyweight, blood glucose, serum insulin, and cardiac function of mice were analyzed. Lentivirus-packaged sh-HDAC1 was injected into DCM mice and high glucose (HG)-induced cardiac fibroblasts (CFs). The pathological structure of the myocardium and the level of myocardial fibrosis were observed by histological staining. HDAC1 expression in mouse myocardial tissues and CFs was determined. Collagen I, collagen III, alpha-smooth muscle actin (α-SMA), and vimentin levels in CFs were detected, and CF proliferation was tested. HDAC activity and histone acetylation levels in tissues and cells were measured. Bone morphogenetic protein-7 (BMP-7) expression in myocardial tissues and CFs was determined. Functional rescue experiments were conducted to confirm the effects of histone acetylation and BMP-7 on myocardial fibrosis. DCM mice showed decreased bodyweight, elevated blood glucose and serum insulin, and cardiac dysfunction. Elevated HDAC1 and reduced BMP-7 expressions were detected in DCM mice and HG-induced CFs. HDAC1 repressed BMP-7 transcription through deacetylation. HDAC1 silencing alleviated MF, reduced CF proliferation and decreased collagen I, -III, α-SMA, and vimentin levels. However, reducing histone acetylation level or BMP-7 downregulation reversed the effects of HDAC1 silencing on CF fibrosis. HDAC1 repressed BMP-7 transcription by enhancing histone deacetylation, thereby promoting MF and aggravating DCM.

Diabetic cardiomyopathy (DCM) is a form of idiopathic heart disease, with signs including hypertrophy of myocardial cells, hypertension-independent fibrosis and coronary artery disease. Considering the involvement of dimethylarginine dimethylaminohydrolase 2 (DDAH2) in diabetes, it was hypothesized that DDAH2 may be beneficial to cardiac function and myocardial fibrosis during the progression of DCM with involvement of the DDAH/asymmetric NG, NGdimethyl-L-arginine (ADMA)/nitric oxide synthase (NOS)/nitric oxide (NO) signaling pathway. Following establishment of diabetic rat models, diabetes-related blood biochemical indices and cardiac function were measured in diabetic rats treated with lentivirus expressing DDAH2, short hairpin RNA against DDAH2, or L-NNA (inhibitor of NOS) to identify the roles of DDAH2 in DCM. The functional roles of DDAH2 in DCM were further determined through detection of the levels of collagen I, matrix metalloproteinase 2 (MMP2) and tissue inhibitor of metalloproteinase 2 (TIMP2). The H9C2 myocardial cell line was selected for in vitro experiments. The effects of DDAH2 on the migration of myocardial cells under high glucose conditions were also examined. To further investigate the underlying regulatory mechanism of DDAH2 in DCM, the contents of ADMA and NO, and the activities of DDAH and NOS were observed. The DCM model rats treated with DDAH2 exhibited reduced left ventricular end-diastolic pressure, and decreased blood glucose, total cholesterol, triglyceride, fasting blood glucose, and fasting insulin levels, but exhibited increased left ventricular systolic pressure and maximum rate of left ventricular pressure rise/fall levels in myocardial tissues. Myocardial cells under high glucose conditions treated with DDAH2 showed reductions in collagen I, MMP2 and TIMP2, indicating that DDAH2 reduced cell migration. Decreased levels of ADMA and NO but increased levels of DDAH and NOS were observed following treatment with DDAH2, indicating that the DDAH/ADMA/NOS/NO pathway was activated. These results reveal that the overexpression of DDAH2 attenuates myocardial fibrosis and protects against DCM through activation of the DDAH/ADMA/NOS/NO pathway in DCM rats. These results indicate that DDAH2 is a potential therapeutic candidate for the treatment of DCM.

Diabetic cardiomyopathy (DCM) is characterized by myocardial hypertrophy and fibrosis. This study aimed to investigate the effects of microRNA (miR)-34a on myocardial fibrosis in DCM and its potential mechanism of targeting Pin-1 signaling. Vimentin and Pin-1 proteins in mouse cardiac tissues were detected by immunohistochemical staining. Locked nucleic acid in situ hybridization was used to measure miR-34a expression in cardiac tissues. Primary mouse cardiac fibroblasts (CFs) were transfected with a mimics control/miR-34a mimics or Pin-1 plasmid and cultured in high-glucose (HG) Dulbecco's modified Eagle's medium. The miR-34a levels were measured by quantitative polymerase chain reaction. The apoptosis and viability of transfected cells were detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling and Cell Counting Kit-8 assays respectively. A cell migration experiment and dual-luciferase reporter assay were also performed. The body weight and fasting blood glucose of DCM mice were significantly higher than those in the control (CTL) group. In addition, DCM mice had decreased serum insulin levels and impaired cardiac function. The number of CFs in the DCM group was higher than in the CTL group and Pin-1 expression was upregulated. The expression level of miR-34a in the cardiac tissue of mice in the DCM group was obviously downregulated compared with the CTL group. The HG stimulation of CFs for 48 h significantly downregulated the expression level of miR-34a and was associated with increased Type I collagen expression, cell viability, and migration and decreased apoptosis. However, these effects could be reversed by overexpressing miR-34a in HG-induced CFs. Furthermore, we found that Pin-1 was a direct target of miR-34a. Our results suggest that miR-34a can attenuate myocardial fibrosis in DCM by reducing Type I collagen production, cell viability, and migration and increasing the apoptosis of CFs by targeting Pin-1 signaling.

Non-invasive Quantification of Myocardial Fibrosis in Diabetic Cardiomyopathy: T1 Mapping or Integrated Backscatter?

Hepatology Basic Science

A disintegrin and metalloprotease domain family protein 17 (ADAM17) is a new member of renin-angiotensin system (RAS) but its role in the pathogenesis of diabetic cardiomyopathy (DCM) is obscure. To test the hypothesis that ADAM17 knockdown mitigates while ADAM17 overexpression aggravates cardiac fibrosis via regulating ACE2 shedding and myofibroblast transformation in diabetic mice, ADAM17 gene was knocked down and overexpressed by means of adenovirus-mediated short-hairpin RNA (shRNA) and adenovirus vector carrying ADAM17 cDNA, respectively, in a mouse model of DCM. Two-dimensional and Doppler echocardiography, histopathology and immunohistochemistry were performed in all mice and in vitro experiments conducted in primary cardiofibroblasts. The results showed that ADAM17 knockdown ameliorated while ADAM17 overexpression worsened cardiac dysfunction and cardiac fibrosis in diabetic mice. In addition, ADAM17 knockdown increased ACE2 while reduced AT1R expression in diabetic hearts. Mechanistically, ADAM17 knockdown decreased while ADAM17 overexpression increased cardiac fibroblast-to-myofibroblast transformation through regulation of TGF-β1/Smad3 signaling pathway. In conclusion, ADAM17 knockdown attenuates while ADAM17 overexpression aggravates cardiac fibrosis via regulating ACE2 shedding and myofibroblast transformation through TGF-β1/Smad3 signaling pathway in diabetic mice. Targeting ADAM17 may provide a promising approach to the prevention and treatment of cardiac fibrosis in DCM.

To investigate the effects of exogenous hydrogen sulfide (H2S) on the hepatic fibrosis in diabetic mice and its mechanism. Twenty-four C57 male mice (weight 22±2 g) were randomly divided into three groups (n=8): ① Normal control group (Control): Mice were intraperitoneally injected equal amount of normal saline, the injection time was the same as that of the experimental groups; ② Diabetes model groups (HG): Streptozotocin (STZ) was injected intraperitoneally once according to body weight (150 mg/kg) to establish diabetes model; ③ NaHS treatment groups (HG + NaHS): Mice were intraperitoneally injected with NaHS (100 μmol/L·kg·d) once a day for 12 consecutive weeks. The hepatocyte injury was detected by HE staining; the hepatic fibrosis was observed through Masson staining; the protein expressions of cystathionine - β - synthetase (CBS), collagen-I (CoL-I), collagen-III (CoL-III) and matrix metalloproteinase-9 (MMP-9) were detected by Western blot. Compared with the control group, the damage and fibrosis of hepatocyte were significantly aggravated, the expression of CBS proteins was decreased (P＜0.01), and the expression levels of CoL-I, CoL-III and MMP-9 proteins were increased (P＜0.01) in the diabetic model group. Compared with the diabetic model group, the damage and fibrosis of hepatocyte were significantly lightened, the expression of CBS proteins was obviously increased (P＜0.01), and the expression levels of CoL-I, CoL-III and MMP-9 proteins were markedly decreased (P＜ 0.01). H2S inhibits the hepatic fibrosis in diabetic mice, and its mechanism is related to the decrease of collagen and matrix metalloproteinase-9.

Persistent hyperglycemia in diabetic patients has been associated with cardiac hypertrophy, myocardial fibrosis and cardiac dysfunction. However, the underlying mechanisms of this association have yet to be fully elucidated. The aim of this study was to investigate the expression and function of microRNAs (miRNAs) in diabetic cardiomyopathy. miRNA expression profiles were examined by miRNA microarray analysis in heart tissue from streptozotocin (STZ)-induced diabetic mice and non-diabetic mice. The targets of the altered miRNAs were predicted using the Sanger database. Then, the targets RASA1, RAC1, TGFB3 and COL1A1, related to cardiac hypertrophy or myocardial fibrosis, were selected to analyze the miRNA level by real-time reverse transcription (RT-PCR). Gene Ontology (GO) was further applied to describe the function of each miRNA target gene and to elucidate their combined effects in diabetic cardiomyopathy. Up-regulated (n=10) and down-regulated (n=6) miRNAs were identified in diabetic cardiomyopathy. Target genes (n=192) were pooled from the Sanger database. Among the 192 targets, the mRNA expression of RASA1, RAC1, TGFB3 and COL1A1 was increased in diabetic cardiomyopathy. Thirty one GO functions were enriched in diabetic cardiomyopathy. These results demonstrate that miRNAs may mediate cardiac hypertrophy and myocardial fibrosis in diabetic cardiomyopathy via their targets, and provide insights into the pathogenesis of diabetic cardiomyopathy.

Nox4 is the major isoform of NADPH oxidase found in the kidney and contributes to the pathogenesis of diabetic nephropathy. However, the molecular mechanisms of increased Nox4 expression induced by hyperglycaemia remain to be elucidated. Here, the role of the connexin32-Nox4 signalling axis in diabetic nephropathy and its related mechanisms were investigated. Diabetes was induced in mice by low-dose streptozotocin (STZ) combined with a high-fat diet. Effects of connexin32 on Nox4 expression and on renal function and fibrosis in STZ-induced diabetic mice were investigated using adenovirus-overexpressing connexin32 and connexin32-deficient mice. Interactions between connexin32 and Nox4 were analysed by co-immunoprecipitation and immunofluorescence assays. Connexin32 was down-regulated in the kidneys of STZ-induced diabetic mice. Overexpression of connexin32 reduced expression of Nox4 and improved renal function and fibrosis in diabetic mice, whereas connexin32 deficiency had opposite effects. Down-regulation of fibronectin expression by connexin32 was not dependent on gap junctional intercellular communication involving connexin32. Connexin32 interacted with Nox4 and reduced the generation of hydrogen peroxide, leading to the down-regulation of fibronectin expression. Mechanistically, connexin32 decreased Nox4 expression by promoting its K48-linked polyubiquitination. Interestingly, Smurf1 overexpression inhibited K48-linked polyubiquitination of Nox4. Furthermore, connexin32 interacted with Smurf1 and inhibited its expression. Connexin32 ameliorated renal fibrosis in diabetic mice by promoting K48-linked Nox4 polyubiquitination and degradation via inhibition of Smurf1 expression. Targeting the connexin32-Nox4 signalling axis may contribute to the development of novel treatments for diabetic nephropathy.

Cardiac fibrosis plays a key role in the progression of diabetic cardiomyopathy (DCM). Previous studies demonstrated the cardioprotective effects of natriuretic peptides. However, the effects of natriuretic peptide receptor C (NPRC) on cardiac fibrosis in DCM remains unknown. Here, we observed that myocardial NPRC expression was increased in mice and patients with DCM. NPRC−/− diabetic mice showed alleviated cardiac fibrosis, as well as improved cardiac function and remodeling. NPRC knockdown in both cardiac fibroblasts and cardiomyocytes decreased collagen synthesis and proliferation of cardiac fibroblasts. RNA sequencing identified that NPRC deletion up-regulated the expression of TGF-β–induced factor homeobox 1 (TGIF1), which inhibited the phosphorylation of Smad2/3. Furthermore, TGIF1 up-regulation was mediated by the activation of cAMP/PKA and cGMP/PKG signaling induced by NPRC deletion. These findings suggest that NPRC deletion attenuated cardiac fibrosis and improved cardiac remodeling and function in diabetic mice, providing a promising approach to the treatment of diabetic cardiac fibrosis.

Kidney fibrosis is the final common pathway of all progressive chronic kidney diseases, of which diabetic nephropathy is the leading cause. Endothelial-to-mesenchymal transition (EndMT) has emerged as one of the most important origins of matrix-producing fibroblasts. Dipeptidyl peptidase-4 (DPP-4) inhibitors have been introduced into the market as antidiabetes drugs. Here, we found that the DPP-4 inhibitor linagliptin ameliorated kidney fibrosis in diabetic mice without altering the blood glucose levels associated with the inhibition of EndMT and the restoration of microRNA 29s. Streptozotocin-induced diabetic CD-1 mice exhibited kidney fibrosis and strong immunoreactivity for DPP-4 by 24 weeks after the onset of diabetes. At 20 weeks after the onset of diabetes, mice were treated with linagliptin for 4 weeks. Linagliptin-treated diabetic mice exhibited a suppression of DPP-4 activity/protein expression and an amelioration of kidney fibrosis associated with the inhibition of EndMT. The therapeutic effects of linagliptin on diabetic kidneys were associated with the suppression of profibrotic programs, as assessed by mRNA microarray analysis. We found that the induction of DPP-4 observed in diabetic kidneys may be associated with suppressed levels of microRNA 29s in diabetic mice; linagliptin restored microRNA 29s and suppressed DPP-4 protein levels. Using cultured endothelial cells, we found that linagliptin inhibited TGF-β2-induced EndMT, and such anti-EndMT effects of linagliptin were mediated through microRNA 29 induction. These results indicate the possible novel pleiotropic action of linagliptin to restore normal kidney function in diabetic patients with renal impairment.

BackgroundSalvianolic acid B (Sal B), a water-soluble phenolic compound derived from Salvia miltiorrhiza Bunge, is commonly used in Traditional Chinese Medicine to treat cardiovascular disease. In our previous study, Sal B protected against myocardial fibrosis induced by diabetic cardiomyopathy (DCM). This study aimed to investigate the ameliorative effects and potential mechanisms of Sal B in mitigating myocardial fibrosis induced by DCM.MethodsVarious methods were used to investigate the effects of Sal B on myocardial fibrosis induced by DCM in vivo and in vitro. These methods included blood glucose measurement, echocardiography, HE staining, Masson’s trichrome staining, Sirius red staining, cell proliferation assessment, determination of hydroxyproline levels, immunohistochemical staining, evaluation of fibrosis-related protein expression (Collagen-I, Collagen-III, TGF-β1, p-Smad3, Smad3, Smad7, and α-smooth muscle actin), analysis of Smad7 gene expression, and analysis of Smad7 ubiquitin modification.ResultsThe animal test results indicated that Sal B significantly improved cardiac function, inhibited collagen deposition and phenotypic transformation, and ameliorated myocardial fibrosis in DCM by upregulating Smad7, thereby inhibiting the TGF-β1 signaling pathway. In addition, cell experiments demonstrated that Sal B significantly inhibited the proliferation, migration, phenotypic transformation, and collagen secretion of cardiac fibroblasts (CFs) induced by high glucose (HG). Sal B significantly decreased the ubiquitination of Smad7 and stabilized the protein expression of Smad7, thereby increasing the protein expression of Smad7 in CFs and inhibiting the TGF-β1 signaling pathway, which may be the potential mechanism by which Sal B mitigates myocardial fibrosis induced by DCM.ConclusionThis study revealed that Sal B can improve myocardial fibrosis in DCM by deubiquitinating Smad7, stabilizing the protein expression of Smad7, and blocking the TGF-β1 signaling pathway.

Diabetic cardiomyopathy (DCM) is a major complication of diabetes, and features myocardial fibrosis as its main pathological feature. Calcium sensing receptor (CaSR) is a G protein-coupled receptor, which involves in myocardial fibrosis by regulation of calcium homeostasis. Calhex231, the CaSR inhibitor, is not clear whether it regulates myocardial fibrosis in DCM. In the present study, type 1 diabetic (T1D) rats and primary neonatal rat cardiac fibroblasts were used to observe the role of Calhex231. In vivo experiments showed that in the T1D group, contractile dysfunction and the deposition of collagen I and III were obvious after 12 weeks. In vitro experiments, we found that high glucose (HG) could increase the expression of CaSR, α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1) collagen I/III, matrix metalloproteinase-2 (MMP-2), MMP9, along with cardiac fibroblast migration and proliferation. We further demonstrated that CaSR activation increased intracellular Ca2+ concentration and upregulated the expression of Itch (atrophin-1 interacting protein 4), which resulted in increasing the ubiquitination levels of Smad7 and upregulating the expression of p-Smad2, p-Smad3. However, treatment with Calhex231 clearly inhibited the above-mentioned changes. Collectively these results suggest that Calhex231 could inhibit Itch-ubiquitin proteasome and TGF-β1/Smads pathways, and then depress the proliferation of cardiac fibroblasts, along with the reduction deposition of collagen, alleviate glucose-induced myocardial fibrosis. Our findings indicate an important new mechanism for myocardial fibrosis, and suggest Calhex231 would be a new therapeutic agent for the treatment of DCM.

Myocardial fibrosis is a major complication of diabetic cardiomyopathy (DCM) that is primarily caused by cardiac fibroblasts that are highly activated by persistent hyperglycemic stimulation, resulting in excessive collagen deposition. Calcium sensing receptor (CaSR) is a member of the G protein-coupled receptor superfamily and regulates intracellular calcium concentrations, which are associated with numerous diseases, including myocardial infarction, tumors and pulmonary hypertension. However, whether CaSR participates in the pathological process of myocardial fibrosis in DCM remains unknown. The present study aimed to investigate the mechanism via which CaSR regulates high glucose (HG)-induced cardiac fibrosis in vitro. HG treated-cardiac fibroblast (CFs) were used and western blotting, immunoprecipitation, Cell Counting Kit-8 assay, ELISA and transfection technology were performed to examine the role of CaSR. In the HG group, treatment with HG increased CaSR, α-smooth muscle actin, collagen I/III and matrix metalloproteinase 2/9 expression and enhanced autophagosome generation and CF proliferation. Furthermore, CaSR activation upregulated the expression of Smad ubiquitin regulatory factor 2 (Smurf2), which led to increased intracellular Ca2+ concentrations, increased ubiquitination levels of SKI like proto-oncogene and Smad7 and autophagy activation. Furthermore, the CaSR agonist (R568) or the CaSR inhibitor (Calhex231) and Smurf2-small interfering RNA promoted or inhibited HG-induced alterations, including the enhanced and weakened effects, respectively. Taken together, the results from the present study suggested that increased CaSR expression in CFs activated the Smurf2-ubiquitin proteasome and autophagy, causing excessive CF proliferation and extensive collagen deposition, which resulted in HG-induced myocardial fibrosis. These findings indicated a novel pathogenesis of DCM and may provide a novel strategy for the diagnosis and treatment of DCM.