Apoptosis Of Cardiac Fibroblasts Research Articles

Regulation of cardiac fibroblast cell death by unfolded protein response signaling.

The endoplasmic reticulum (ER) is a tightly regulated organelle that requires specific environmental properties to efficiently carry out its function as a major site of protein synthesis and folding. Embedded in the ER membrane, ER stress sensors inositol-requiring enzyme 1 (IRE1), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) serve as a sensitive quality control system collectively known as the unfolded protein response (UPR). In response to an accumulation of misfolded proteins, the UPR signals for protective mechanisms to cope with the cellular stress. Under prolonged unstable conditions and an inability to regain homeostasis, the UPR can shift from its original adaptive response to mechanisms leading to UPR-induced apoptosis. These UPR signaling pathways have been implicated as an important feature in the development of cardiac fibrosis, but identifying effective treatments has been difficult. Therefore, the apoptotic mechanisms of UPR signaling in cardiac fibroblasts (CFs) are important to our understanding of chronic fibrosis in the heart. Here, we summarize the maladaptive side of the UPR, activated downstream pathways associated with cell death, and agents that have been used to modify UPR-induced apoptosis in CFs.

MiR-214 could promote myocardial fibrosis and cardiac mesenchymal transition in VMC mice through regulation of the p53 or PTEN-PI3K-Akt signali pathway, promoting CF proliferation and inhibiting its ng pathway

IntroductionThis study aimed to investigate the role of miR-214 in the bidirectional regulation of p53 and PTEN and its influence on myocardial fibrosis and cardiac mesenchymal transformation in mice with viral myocarditis (VMC). MethodsThe study established a VMC model in BALB/c mice by injecting them with the CVB3 virus intraperitoneally. Techniques such as ELISA, H&E staining, Masson staining, immunohistochemical staining, RT-qPCR, western blot, and dual-luciferase reporter gene assay were used to detect the expression levels of relevant factors in tissues and cells. Isolation and culture of cardiac fibroblasts (CFs) were also conducted. ResultsThe study found that miR-214 bidirectional regulation of p53 and PTEN promotes myocardial fibrosis and cardiac mesenchymal transformation in mice with VMC. The expression levels of collagen-related peptides, inflammatory-related factors, miR-214, mesenchymal transformation-related factors, and fibrosis-related factors were significantly increased, while the expression levels of p53, PTEN, and epithelial/endothelial cell phenotype marker factors were significantly decreased. Downregulation of miR-214 or upregulation of p53 and PTEN expression inhibited inflammatory cell and fibroblast infiltration in VMC mouse myocardial tissue. It reduced the proliferation ability while increasing the apoptosis of cardiac fibroblasts. ConclusionmiR-214 plays a significant role in the bidirectional inhibition of p53 and PTEN, which leads to myocardial fibrosis and cardiac mesenchymal transformation in mice with VMC. Downregulation of miR-214 or upregulation of p53 and PTEN expression may provide potential therapeutic targets for treating VMC-induced cardiac fibrosis and mesenchymal transformation.

The p53/miR-29a-3p axis mediates the antifibrotic effect of leonurine on angiotensin II-stimulated rat cardiac fibroblasts.

Overactivation of cardiac fibroblasts (CFs) is one of the main causes of myocardial fibrosis (MF), and inhibition of CF activation is a crucial strategy for MF therapy. A previous study by our group demonstrated that leonurine (LE) effectively inhibits collagen synthesis and myofibroblast generation originated from CFs, and eventually mitigates the progression of MF (where miR-29a-3p is likely to be a vital mediator). However, the underlying mechanisms involved in this process remain unknown. Thus, the present study aimed to investigate the precise role of miR-29a-3p in LE-treated CFs, and to elucidate the pharmacological effects of LE on MF. Neonatal rat CFs were isolated and stimulated by angiotensin II (Ang II) to mimic the pathological process of MF in vitro. The results show that LE distinctly inhibits collagen synthesis, as well as the proliferation, differentiation and migration of CFs, all of which could be induced by Ang II. In addition, LE promotes apoptosis in CFs under Ang II stimulation. During this process, the down-regulated expressions of miR-29a-3p and p53 are partly restored by LE. Either knockdown of miR-29a-3p or inhibition of p53 by PFT-α (a p53 inhibitor) blocks the antifibrotic effect of LE. Notably, PFT-α suppresses miR-29a-3p levels in CFs under both normal and Ang II-treated conditions. Furthermore, ChIP analysis confirmed that p53 is bound to the promoter region of miR-29a-3p, and directly regulates its expression. Overall, our study demonstrates that LE upregulates p53 and miR-29a-3p expression, and subsequently inhibits CF overactivation, suggesting that the p53/miR-29a-3p axis may play a crucial role in mediating the antifibrotic effect of LE against MF.

Silencing RIPK1/mTORC1 signalling attenuated the inflammation and oxidative stress in diabetic cardiomyopathy

BackgroundDiabetes cardiomyopathy (DCM) is one of the major risk factors for the heart failure of the diabetic patients. RIPK1 maybe participate in the regulation of the oxidative stress and inflammation during DCM. MethodsH&E and Masson staining were utilized to assess the inflammation and fibrosis in myocardial tissues. CCK-8 and TUNEL staining were utilized to analyze cell viability and apoptosis, respectively. SOD activity and MDA content were detected utilizing the kits. The formation of autophagosomes was measured by immunofluorescence assay. ResultsRIPK1 and RPTOR (a component of mTORC1) expression and oxidative stress level were upregulated, but autophagy was decreased in the myocardial tissues of DCM rat characterized by the high body weight and blood glucose, abnormal cardiac function, myocardial inflammation and fibrosis. High glucose (HG) treatment resulted in cell viability and autophagy level decreasing, inflammatory cytokines expression increasing and oxidative stress increasing in cardiac fibroblasts (CFs). Meanwhile, RIPK1 and RPTOR expression also was increased in HG-treated cells. HG-induced CFs apoptosis, inflammation, oxidative stress and the inhibition of HG to cell viability and autophagy was partly reversed by the inhibitor of RIPK1 and mTORC1. ConclusionOverall, RIPK1/mTORC1 signalling suppression improved HG-induced apoptosis, inflammation and oxidative stress through activation autophagy. These data provided a reliable evidence that RIPK1 may be a potential target for DCM therapeutic.

Dihydro-artemisinin inhibits angiotensin II-induced cardiac fibroblasts proliferation and induces apoptosis through phosphoinositide 3-kinase/akt pathway

Cardiac fibroblasts (CFs) abnormal proliferation can trigger myocardial fibrosis, which is closely related to a variety of cardiovascular diseases. This study investigated the effect of dihydroartemisinin (DHA) on CF growth through PI3K/AKT with angiotensin II (AngII). CCK-8 method and EdU were used detect cell proliferation, and flow cytometry assessed apoptosis of CFs. The PI3K and AKT expressions were determined, whilst immunofluorescence staining and RT-qPCR measured α-smooth muscle actin (α-SMA) level. With rat CFs exhibiting typical fibroblast morphology and nuclei in spindle, oval or polygonal shape, these cells were positive for the fibroblast marker vimentin. Treatment with AngII resulted in increased proliferation of CFs, but the proliferation declined in the presence of DHA (p < 0.05). Meanwhile, the AngII group had lower apoptosis of CFs than blank and DHA groups with highest apoptosis observed in the DHA group. α-SMA increased at the mRNA and protein levels after Ang II treatment and decreased after DHA treatment (p < 0.05). Importantly, the Ang II upregulated PI3K and AKT in CFs (p < 0.05) and the increase was abrogated by DHA administration with lowest levels (p < 0.05). Ang II activates PI3K/Akt in normal CFs, as administration of DHA can inhibit the effect of Ang II, thereby inhibiting proliferation of CFs induced by AngII and inducing apoptosis.

Transcription pattern of key molecular chaperones in heat shocked caprine cardiac fibroblasts

The present study was attempted to unveil the impact of heat stress on transcription pattern of major heat shock response genes in caprine cardiac fibroblasts. Cardiac tissues (n = 6) were collected and primary cardiac cell culture was done. Cultured cardiac fibroblasts were kept in an atmosphere of 5% CO2 and 95% air at 38.5 °C. Cardiac cells achieved 70-75% confluence after 72 hours of incubation. Heat stress was induced on confluent cardiac fibroblasts at 42 °C for 0 (control), 20, 60, 100 and 200 min. Quantitative RT-PCR for β2m (internal control), HSP60, HSP70, HSP90, and HSP110 was done and their transcription pattern was assessed by Pfaffl method. HSP60, HSP90, and HSP110 transcription did not differ at 20 min, up-regulated (p < 0.05) from 60 to 200 min and registered highest at 200 min of heat exposure. HSP70 transcription was gradually escalated (p < 0.05) time dependently from 20 to 200 min and reached zenith at 200 min of heat exposure. Differential induction in transcription of key molecular chaperones at various durations of heat exposure might reduce cardiac fibroblasts apoptosis and thus could maintain cardiac tissue function during heat stress.

Nrg1/ErbB signaling-mediated regulation of fibrosis after myocardial infarction.

Appropriate fibrotic tissue formation after myocardial infarction (MI) is crucial to the maintenance of the heart's structure. M2-like macrophages play a vital role in post-MI fibrosis by activating cardiac fibroblasts. Because the mechanism by which post-MI cardiac fibrosis is regulated is not fully understood, we investigated, in vitro and in vivo, the cellular and molecular mechanisms of post-MI fibrotic tissue formation, especially those related to the regulation of cellular senescence and apoptosis. CD206+ F4/80+ CD11b+ M2-like macrophages collected from mouse hearts on post-MI day 7showed increased expression of neuregulin 1 (Nrg1). Nrg1 receptor epidermal growth factor receptors ErbB2 and ErbB4 were expressed on cardiac fibroblasts in the infarct area. M2-like macrophage-derived Nrg1suppressed both hydrogen peroxide-induced senescence and apoptosis of fibroblasts, whereas blockade of ErbB function significantly accelerated both processes. M2-like macrophage-derived Nrg1/ErbB/PI3K/Akt signaling, shown to be related to anti-senescence, was activated in damaged cardiac fibroblasts. Interestingly, systemic blockade of ErbB function in MI model mice enhanced senescence and apoptosis of cardiac fibroblasts and exacerbated inflammation. Further, increased accumulation of M2-like macrophages resulted in excessive post-MI progression of fibrosis in mice hearts. The molecular mechanism underlying the regulation of fibrotic tissue formation in the infarcted myocardium was shown in part to be attenuation of apoptosis and senescence of cardiac fibroblasts by the activation of Nrg1/ErbB/PI3K/Akt signaling. M2-like macrophage-mediated regulation of Nrg1/ErbB signaling has a substantial effect on fibrotic tissue formation in the infarcted adult mouse heart and is critical for suppressing the progression of senescence and apoptosis of cardiac fibroblasts.

Homocysteine promotes cardiac fibrosis by regulating the Akt/FoxO3 pathway

BackgroundEvaluated plasma homocysteine (Hcy) is an independent risk factor for cardiac fibrosis which is a common feature of cardiovascular disease, although the mechanisms are still unclear. This study aims to explore the mechanism of Hcy-induced cardiac fibrosis.MethodsThe mRNA and protein levels of Forkhead box O3 (FoxO3) and differentiation markers were detected in primary cardiac fibroblasts (CFs) after 300 µM Hcy treatment. Scratch and transwell migration assay were used to determine the effect of Hcy on proliferation and migration in CFs. The protein levels involved in the fibrotic processes in mice fed with high methionine diet (HMD) for 4 or 8 weeks were investigated by western blot. CFs were infected with FoxO3 recombinant adenovirus to explore the potential role of FoxO3 in Hcy-induced cardiac dysfunction.ResultsHcy treatment significantly promoted the differentiation, proliferation and migration of CFs, while FoxO3 activity were decreased in CFs. In HMD hearts, the protein levels of TIMP1, Fibronectin and α-SMA were increased after 4 or 8 weeks, but the FoxO3 activity was decreased. Moreover, the HMD hearts had a higher level of Bcl2 but lower of Bax and LC3II protein. In addition, FoxO3 overexpression attenuates Hcy-induced dysfunction in CFs.ConclusionsHcy promotes myofibroblast activation and resistance to autophagy and apoptosis in CFs, and eventually results in cardiac fibrosis by regulating the Akt/FoxO3 pathway. Thus, FoxO3 is a promising therapeutic target to prevent cardiac remodeling.

Exercise Training Alleviates Cardiac Fibrosis through Increasing Fibroblast Growth Factor 21 and Regulating TGF-β1-Smad2/3-MMP2/9 Signaling in Mice with Myocardial Infarction.

Exercise training has been reported to alleviate cardiac fibrosis and ameliorate heart dysfunction after myocardial infarction (MI), but the molecular mechanism is still not fully clarified. Fibroblast growth factor 21 (FGF21) exerts a protective effect on the infarcted heart. This study investigates whether exercise training could increase FGF21 protein expression and regulate the transforming growth factor-β1 (TGF-β1)-Smad2/3-MMP2/9 signaling pathway to alleviate cardiac fibrosis following MI. Male wild type (WT) C57BL/6J mice and Fgf21 knockout (Fgf21 KO) mice were used to establish the MI model and subjected to five weeks of different types of exercise training. Both aerobic exercise training (AET) and resistance exercise training (RET) significantly alleviated cardiac dysfunction and fibrosis, up-regulated FGF21 protein expression, inhibited the activation of TGF-β1-Smad2/3-MMP2/9 signaling pathway and collagen production, and meanwhile, enhanced antioxidant capacity and reduced cell apoptosis in the infarcted heart. In contrast, knockout of Fgf21 weakened the cardioprotective effects of AET after MI. In vitro, cardiac fibroblasts (CFs) were isolated from neonatal mice hearts and treated with H2O2 (100 μM, 6 h). Recombinant human FGF21 (rhFGF21, 100 ng/mL, 15 h) and/or 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR, 1 mM, 15 h) inhibited H2O2-induced activation of the TGF-β1-Smad2/3-MMP2/9 signaling pathway, promoted CFs apoptosis and reduced collagen production. In conclusion, exercise training increases FGF21 protein expression, inactivates the TGF-β1-Smad2/3-MMP2/9 signaling pathway, alleviates cardiac fibrosis, oxidative stress, and cell apoptosis, and finally improves cardiac function in mice with MI. FGF21 plays an important role in the anti-fibrosis effect of exercise training.

High Glucose Activated Cardiac Fibroblasts by a Disruption of Mitochondria-Associated Membranes.

Cardiac fibrosis is evident even in the situation without a significant cardiomyocyte loss in diabetic cardiomyopathy and a high glucose (HG) level independently activates the cardiac fibroblasts (CFs) and promotes cell proliferation. Mitochondrial respiration and glycolysis, which are key for cell proliferation and the mitochondria-associated membranes (MAMs), are critically involved in this process. However, the roles and the underlying mechanism of MAMs in the proliferation of HG-induced CFs are largely unknown. The proliferation and apoptosis of CFs responding to HG treatment were evaluated. The MAMs were quantified, and the mitochondrial respiration and cellular glycolytic levels were determined using the Seahorse XF analyzer. The changes of signal transducer and activator of transcription 3 (STAT3) and mitofusin-2 (MFN2) in responding to HG were also determined, the effects of which on cell proliferation, MAMs, and mitochondrial respiration were assessed. The effects of STAT3 on MFN2 transcription was determined by the dual-luciferase reporter assay (DLRA) and chromatin immunoprecipitation (CHIP). HG-induced CFs proliferation increased the glycolytic levels and adenosine triphosphate (ATP) production, while mitochondrial respiration was inhibited. The MAMs and MFN2 expressions were significantly reduced on the HG treatment, and the restoration of MFN2 expression counteracted the effects of HG on cell proliferation, mitochondrial respiration of the MAMs, glycolytic levels, and ATP production. The mitochondrial STAT3 contents were not changed by HG, but the levels of phosphorylated STAT3 and nuclear STAT3 were increased. The inhibition of STAT3 reversed the reduction of MFN2 levels induced by HG. The DLRA and CHIP directly demonstrated the negative regulation of MFN2 by STAT3 at the transcription levels via interacting with the sequences in the MFN2 promoter region locating at about −400 bp counting from the start site of transcription. The present study demonstrated that the HG independently induced CFs proliferation via promoting STAT3 translocation to the nucleus, which switched the mitochondrial respiration to glycolysis to produce ATP by inhibiting MAMs in an MFN2-depression manner.

Latent-transforming growth factor β-binding protein 2 accelerates cardiac fibroblast apoptosis by regulating the expression and activity of caspase-3.

Cardiac fibrosis is a core process in the development of heart failure. However, the underlying mechanism of cardiac fibrosis remains unclear. Recently, a study found that in an isoproterenol (ISO)-induced cardiac fibrosis animal model, there is high expression of latent-transforming growth factor β-binding protein 2 (LTBP2) in cardiac fibroblasts. Whether LTBP2 serves a role in cardiac fibrosis is currently unknown. In the present study, mouse cardiac fibroblasts (MCFs) were treated with 100 µM/l ISO for 24, 48, or 72 h, and small interfering RNAs (siRNAs) were used to knockdown LTBP2. Reverse transcription-quantitative PCR and western blotting were used to determine gene and protein expression levels, respectively. Caspase-3 serves a key role in cell apoptosis and is related to cardiac fibrosis-induced heart failure. Caspase-3 activity was therefore determined using a caspase-3 assay kit, CCK8 was used to determine the rate of cell proliferation and apoptosis rates were quantified using a cell death detection ELISA kit. The present study demonstrated that cell apoptosis and LTBP2 expression increased in MCFs treated with 100 µM/l ISO in a time-dependent manner. Expression and activity of caspase-3 also increased in MCFs treated with 100 µM/l ISO for 48 h compared with the control group. In addition, ISO stimulation-induced MCF apoptosis, along with the increased expression of caspase-3 were partly abolished when LTBP2 was knocked down. In conclusion, LTBP2 expression increased in ISO-treated MCFs and accelerated mouse cardiac fibroblast apoptosis by enhancing the expression and activity of caspase-3. LTBP2 may therefore be a potential therapeutic target for treating patients with cardiac fibrosis.

Ultrafiltration Extract of Radix Angelica Sinensis and Radix Hedysari Attenuates Risk of Low-Dose X-Ray Radiation-Induced Myocardial Fibrosis In Vitro.

The risk of radiation-induced heart damage (RIHD) is a growing concern since recent advances in radiation therapy (RT) for cancer treatments have significantly improved the number of survivors. Radiation-induced myocardial fibrosis (RIMF) is the final pathological condition of RIHD and main change leading to serious cardiovascular complications following RT. The aim of this study was to investigate the effect of ultrafiltration extract of Radix Angelica Sinensis and Radix Hedysari (RAS-RH) on the proliferation, apoptosis, and reactive oxygen species (ROS) of cardiac fibroblasts after X-irradiation in vitro. The RAS-RH extract was from the Danggui Buxue decoction (DBD) in TCM. Primary cardiac fibroblasts were irradiated with 1 Gy X-ray to evaluate the effect of RAS-RH on the expression levels of cell proliferation, apoptosis, ROS, and fibrotic molecules. Our data demonstrated that X-irradiation at 1 Gy resulted in the proliferation of cardiac fibroblasts; RAS-RH attenuated the myocardial fibrosis. Furthermore, X-ray radiation reduced the apoptosis of cardiac fibroblasts; RAS-RH accelerated the apoptosis of these cells after irradiation. In addition, the damage driven by ROS in primary cardiac fibroblasts after irradiation was weakened by RAS-RH and the expression of TGF-β1, Col1, and α-SMA increased after irradiation; RAS-RH decreased the expression of these makers. Overall, these data indicate that low-dose X-ray irradiation boosts myocardial fibrosis, and the effect of RAS-RH protects against fibrosis via attenuating the proliferation and accelerating the apoptosis of myocardial fibroblasts after X-irradiation.

Long Noncoding RNA Small Nuclear RNA Host Gene 7 Knockdown Protects Mouse Cardiac Fibroblasts Against Myocardial Infarction by Regulating miR-455-3p/Platelet-Activating Factor Receptor Axis.

Myocardial infarction (MI) is a leading cause of heart failure all over the world. Long noncoding RNAs have been reported to be associated with the development of MI. In this article, we aimed to explore the effects of long noncoding RNA small nuclear RNA host gene 7 (SNHG7) on MI and the possible mechanism. In this study, an MI model was established by ligating the left anterior descending coronary artery of mice. Cardiac fibroblasts (CFs) derived from neonatal mice were activated by angiotensin II (Ang-II) treatment. The expression of SNHG7 and miR-455-3p was examined by quantitative real-time polymerase chain reaction, and protein levels of platelet-activating factor receptor (PTAFR) and fibrosis-related proteins were analyzed by western blot assay. Cell apoptosis of CFs was monitored by flow cytometry. Enzyme-linked immunosorbent assay was performed to evaluate inflammatory responses in CFs. Moreover, dual-luciferase reporter assay was used to confirm the target relationship between miR-455-3p and SNHG7 or PTAFR. LncRNA SNHG7 and PTAFR were upregulated, whereas miR-455-3p was downregulated in cardiac tissues of mice with MI and Ang-II-induced CFs. SNHG7 depletion or miR-455-3p overexpression attenuated Ang-II-induced apoptosis, fibrosis, and inflammation in CFs, which was severally weakened by miR-455-3p inhibition or PTAFR upregulation. LncRNA SNHG7 targeted miR-455-3p, and PTAFR was a target of miR-455-3p. LncRNA SNHG7 depletion exerted protective roles in apoptosis, fibrosis, and inflammation in Ang-II-induced CFs by regulating miR-455-3p/PTAFR axis, providing a potential molecular target for MI therapy.

Cyclic stretching boosts microRNA-499 to regulate Bcl-2 via microRNA-208a in atrial fibroblasts.

MicroRNAs that modulate transcription can regulate other microRNAs and are also up‐regulated under pathological stress. MicroRNA‐499 (miR‐499), microRNA‐208a (miR‐208a) and B‐cell lymphoma 2 (Bcl‐2) play roles in cardiovascular diseases, such as direct reprogramming of cardiac fibroblast into cardiomyocyte and cardiomyocyte apoptosis. Whether miR208a, miR499 and Bcl‐2 were critical regulators in cardiac fibroblast apoptosis under mechanical stretching conditions in human cardiac fibroblasts‐adult atrial (HCF‐aa) was investigated. Using negative pressure, HCF‐aa grown on a flexible membrane base were cyclically stretched to 20% of their maximum elongation. In adult rats, an aortocaval shunt was used to create an in vivo model of volume overload. MiR208a was up‐regulated early by stretching and returned to normal levels with longer stretching cycles, whereas the expression of miR499 and Bcl‐2 was up‐regulated by longer stretching times. Pre‐treatment with antagomir‐499 reversed the miR‐208a down‐regulation, whereas Bcl‐2 expression could be suppressed by miR‐208a overexpression. In the HCF‐aa under stretching for 1 h, miR‐499 overexpression decreased pri‐miR‐208a luciferase activity; this inhibition of pri‐miR‐208a luciferase activity with stretching was reversed when the miR‐499‐5p binding site in pri‐miR‐208a was mutated. The addition of antagomir‐208a reversed the Bcl‐2‐3′UTR suppression from stretching for 1 h. Flow cytometric analysis revealed that pre‐treatment with miR‐499 or antagomir‐208a inhibited cellular apoptosis in stretched HCF‐aa. In hearts with volume overload, miR‐499 overexpression inhibited myocardial miR‐208a expression, whereas Bcl‐2 expression could be suppressed by the addition of miR‐208a. In conclusion, miR‐208a mediated the regulation of miR‐499 on Bcl‐2 expression in stretched HCF‐aa and hearts with volume overload.

Mitochondrial Fission and Mitophagy Reciprocally Orchestrate Cardiac Fibroblasts Activation.

Although mitochondrial fission has been reported to increase proliferative capacity and collagen production, it can also contribute to mitochondrial impairment, which is detrimental to cell survival. The aim of the present study was to investigate the role of mitochondrial fission in cardiac fibroblasts (CF) activation and explore the mechanisms involved in the maintenance of mitochondrial health under this condition. For this, changes in the levels of mitochondrial fission/fusion-related proteins were assessed in transforming growth factor beta 1 (TGF-β1)-activated CF, whereas the role of mitochondrial fission during this process was also elucidated, as were the underlying mechanisms. The interaction between mitochondrial fission and mitophagy, the main defense mechanism against mitochondrial impairment, was also explored. The results showed that the mitochondria in TGF-β1-treated CF were noticeably more fragmented than those of controls. The expression of several mitochondrial fission-related proteins was markedly upregulated, and the levels of fusion-related proteins were also altered, but to a lesser extent. Inhibiting mitochondrial fission resulted in a marked attenuation of TGF-β1-induced CF activation. The TGF-β1-induced increase in glycolysis was greatly suppressed in the presence of a mitochondrial inhibitor, whereas a glycolysis-specific antagonist exerted little additional antifibrotic effects. TGF-β1 treatment increased cellular levels of reactive oxygen species (ROS) and triggered mitophagy, but this effect was reversed following the application of ROS scavengers. For the signals mediating mitophagy, the expression of Pink1, but not Bnip3l/Nix or Fundc1, exhibited the most significant changes, which could be counteracted by treatment with a mitochondrial fission inhibitor. Pink1 knockdown suppressed CF activation and mitochondrial fission, which was accompanied by increased CF apoptosis. In conclusion, mitochondrial fission resulted in increased glycolysis and played a crucial role in CF activation. Moreover, mitochondrial fission promoted reactive oxygen species (ROS) production, leading to mitophagy and the consequent degradation of the impaired mitochondria, thus promoting CF survival and maintaining their activation.

MiR-34a attenuates myocardial fibrosis in diabetic cardiomyopathy mice via targeting Pin-1.

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.

TGF-β1 decreases CHOP expression and prevents cardiac fibroblast apoptosis induced by endoplasmic reticulum stress

Transforming growth factor-beta 1 (TGF-β1) is a cytokine with marked pro-fibrotic action on cardiac fibroblasts (CF). TGF-β1 induces CF-to-cardiac myofibroblast (CMF) differentiation, defined by an increase in α-smooth muscle cells (α-SMA), collagen secretion and it has a cytoprotective effect against stimuli that induce apoptosis. In the Endoplasmic Reticulum (ER) lumen, misfolded protein accumulation triggers ER stress and induces apoptosis, and this process plays a critical role in cell death mediated by Ischemia/Reperfusion (I/R) injury and by ER stress inducers, such as Tunicamycin (Tn). Here, we studied the regulation of CHOP, a proapoptotic ER-stress-related transcription factor in CF under simulated I/R (sI/R) or exposed to Tn. Even though TGF-β1 has been shown to participate in ER stress, its regulatory effect on CF apoptosis and ER stress-induced by sI/R or TN has not been evaluated yet.CF from neonatal rats were exposed to sI/R, and cell death was evaluated by cell count and apoptosis by flow cytometry. ER stress was assessed by western blot against CHOP. Our results evidenced that sI/R (8/24) h or Tn triggers CF apoptosis and an increase in CHOP protein levels. TGF-β1 pre-treatment partially prevented apoptosis induced by sI/R or Tn. Furthermore, TGF-β1 pre-treatment completely prevented CHOP increase by sI/R or Tn. Additionally, we found a decrease in α-SMA expression induced by sI/R and in collagen secretion induced by Tn, which were not prevented by TGF-β1 treatment.In conclusion, TGF-β1 partially protects CF apoptosis induced by sI/R or Tn, through a mechanism that would involve ER stress.

MiR-145 attenuates cardiac fibrosis through the AKT/GSK-3β/β-catenin signaling pathway by directly targeting SOX9 in fibroblasts.

Myocardial infarction (MI) will inevitably result in cardiac fibrosis. In this study, we investigated the effect of microRNA-145 (miR-145) and transcription factor sex-determining region Y box 9 (SOX9) in the production of cardiac fibrosis induced by MI. MI rat models were established by left anterior descending coronary artery (LAD) occlusion. Four weeks after LAD, the cardiac fibrosis level was assessed by Masson's trichrome staining. Cardiac fibroblasts (CFs) exposed to hypoxia were used to simulate MI-induced fibrosis. Flow cytometry, cell counting kit-8, and transwell assays were used to examine changes in CF apoptosis, proliferation, and migration, respectively. miR-145 expression was measured by quantitative real-time polymerase chain reaction. Immunofluorescence and Western blot analysis were performed to determine the relative expression of proteins. In comparison to the sham-operated group, the expression of miR-145 was significantly downregulated in the infarction peripheral area, whereas, SOX9 was upregulated. In the infarcted heart, the overexpression of miR-145 significantly ameliorated cardiac fibrosis and cardiac function, and there was a negative correlation between miR-145 and SOX9 expressions in hypoxic CFs in vitro. In addition, SOX9 was verified to be a functional target of miR-145. Overexpression of miR-145 or inhibition of SOX9 decreased CF proliferation, migration, and fibrosis, but augmented their apoptotic rate. Moreover, the upregulation of miR-145 or suppression of SOX9 inhibited AKT and β-catenin signaling in hypoxic CFs. Taken together, this study highlights a potential treatment for cardiac fibrosis through the targeted regulation of SOX9 by miR-145, and our findings indicate that miR-145 exerts anti-fibrotic effects in MI via the negative regulation of SOX9 and its downstream AKT/GSK-3β/β-catenin pathways.

Notch3 Modulates Cardiac Fibroblast Proliferation, Apoptosis, and Fibroblast to Myofibroblast Transition via Negative Regulation of the RhoA/ROCK/Hif1α Axis.

Cardiac fibrosis is a common pathological process in multiple cardiovascular diseases, including myocardial infarction (MI). Abnormal cardiac fibroblast (CF) activity is a key event in cardiac fibrosis. Although the Notch signaling pathway has been reported to play a vital role in protection from cardiac fibrosis, the exact mechanisms underlying cardiac fibrosis and protection from it have not yet been elucidated. Similarly, Hif1α and the RhoA/ROCK signaling pathway have been shown to participate in cardiac fibrosis. The RhoA/ROCK signaling pathway has been reported to be an upstream pathway of Hif1α in several pathophysiological processes. In the present study, we aimed to determine the effects of notch3 on CF activity and its relationship with the RhoA/ROCK/Hif1α signaling pathway. Using in vitro experiments, we demonstrated that notch3 inhibited CF proliferation and fibroblast to myofibroblast transition (FMT) and promoted CF apoptosis. A knockdown of notch3 using siRNAs had the exact opposite effect. Next, we found that notch3 regulated CF activity by negative regulation of the RhoA/ROCK/Hif1α signaling pathway. Extending CF-based studies to an in vivo rat MI model, we showed that overexpression of notch3 by the Ad-N3ICD injection attenuated the increase of RhoA, ROCK1, ROCK2, and Hif1α levels following MI and further prevented MI-induced cardiac fibrosis. On the basis of these results, we conclude that notch3 is involved in the regulation of several aspects of CF activity, including proliferation, FMT, and apoptosis, by inhibiting the RhoA/ROCK/Hif1α signaling pathway. These findings are significant to further our understanding of the pathogenesis of cardiac fibrosis and to ultimately identify new therapeutic targets for cardiac fibrosis, potentially based on the RhoA/ROCK/Hif1α signaling pathway.

Sodium tanshinone IIA sulfonate prevents radiation-induced damage in primary rat cardiac fibroblasts.

This study investigated the effects of X-ray irradiation on primary rat cardiac fibroblasts (CFs) and its potential mechanism, as well as whether sodium tanshinone IIA sulfonate (STS) has protective effect on CFs and its possible mechanism. Our data demonstrated that X-rays inhibited cell growth and increased oxidative stress in CFs, and STS mitigated X-ray-induced injury. Enzyme-linked immuno-sorbent assay showed that X-rays increased the levels of secreted angiotensin II (Ang II) and brain natriuretic peptide (BNP). STS inhibited the X-ray-induced increases in Ang II and BNP release. Apoptosis and cell cycle of CFs were analyzed using flow cytometry. X-rays induced apoptosis in CFs, whereas STS inhibited apoptosis in CFs after X-ray irradiation. X-rays induced S-phase cell cycle arrest in CFs, which could be reversed by STS. X-rays increased the expression of phosphorylated-P38/P38, cleaved caspase-3 and caspase-3 as well as decreased the expression of phosphorylated extracellular signal-regulated kinase 1/2 (ERK 1/2)/ERK 1/2 and B cell lymphoma 2 (Bcl-2)/Bcl-2 associated X protein (BAX) in CFs, as shown by Western blotting. STS mitigated the X-ray radiation-induced expression changes of these proteins. In conclusion, our results demonstrated that STS may potentially be developed as a medical countermeasure to mitigate radiation-induced cardiac damage.