Neonatal Rat Cardiac Fibroblasts Research Articles

Assessment of ursolic acid effect on in vitro model of cardiac fibrosis

This study aimed to evaluate the effects of ursolic acid (UA) on Angiotensin II (Ang II)-treated neonatal rat cardiac fibroblasts (rCFs) as an in vitro model of cardiac fibrosis. The rCFs were isolated from two-day-old neonatal rats. An in vitro model of cardiac fibrosis was established using 500 nm Ang II treatment for 48 h. The cells were then treated with 5 and 10 μM of UA for 24 and 48 h. Masson's trichrome staining, hydroxyproline content assay, scratch assay, apoptosis assay, measurements of superoxide dismutase (SOD) and malondialdehyde (MDA) levels, real-time PCR, immunocytology and western blotting, were employed to assess the impact of UA. Ang II induced fibrosis in rCFs, as evidenced by the examination of various fibrotic markers. Upon treatment with 5 and 10 μM of UA, the amount of fibrosis in Ang II-treated rCFs was significantly decreased, so that the hydroxyproline concentration was reduced to 0.3 and 0.7 times, respectively. The RNA expression of the Col1a1, Col3a1, Tgfb1, Acta2 and Mmp2 genes had a decrease as well as Nrf2 and HO-1 had an increase after UA treatment. UA could lessen the harmful effects of cardiac fibrosis in a dose- and time-dependent manner, due to its antiapoptotic, antioxidant and cardioprotective properties. This suggests the potential of UA for treatment of cardiac fibrosis.

Supersulfide catabolism participates in maladaptive remodeling of cardiac cells

The atrophic myocardium resulting from mechanical unloading and nutritional deprivation is considered crucial as maladaptive remodeling directly associated with heart failure, as well as interstitial fibrosis. Conversely, myocardial hypertrophy resulting from hemodynamic loading is perceived as compensatory stress adaptation. We previously reported the abundant presence of highly redox-active polysulfide molecules, termed supersulfide, with two or more sulfur atoms catenated in normal hearts, and the supersulfide catabolism in pathologic hearts after myocardial infarction correlated with worsened prognosis of heart failure. However, the impact of supersulfide on myocardial remodeling remains unclear. Here, we investigated the involvement of supersulfide metabolism in cardiomyocyte remodeling, using a model of adenosine 5′-triphosphate (ATP) receptor-stimulated atrophy and endothelin-1 receptor-stimulated hypertrophy in neonatal rat cardiomyocytes. Results revealed contrasting changes in intracellular supersulfide and its catabolite, hydrogen sulfide (H2S), between cardiomyocyte atrophy and hypertrophy. Stimulation of cardiomyocytes with ATP decreased supersulfide activity, while H2S accumulation itself did not affect cardiomyocyte atrophy. This supersulfide catabolism was also involved in myofibroblast formation of neonatal rat cardiac fibroblasts. Thus, unraveling supersulfide metabolism during myocardial remodeling may lead to the development of novel therapeutic strategies to improve heart failure.

BAG3 localizes to mitochondria in cardiac fibroblasts and regulates mitophagy.

The co-chaperone Bcl2-associated athanogene 3 (BAG3) is a central node in protein quality control in the heart. In humans and animal models, decreased BAG3 expression is associated with cardiac dysfunction and dilated cardiomyopathy. Although previous studies focused on BAG3 in cardiomyocytes, cardiac fibroblasts are also critical drivers of pathologic remodeling. Yet, the role of BAG3 in cardiac fibroblasts is almost completely unexplored. Here, we show that BAG3 is expressed in primary rat neonatal cardiac fibroblasts and preferentially localizes to mitochondria. Knockdown of BAG3 reduces mitophagy and enhances fibroblast activation, which is associated with fibrotic remodeling. Heat shock protein 70 (Hsp70) is a critical binding partner for BAG3 and inhibiting this interaction in fibroblasts using the drug JG-98 decreased autophagy, decreased mitofusin-2 expression, and disrupted mitochondrial morphology. Together, these data indicate that BAG3 is expressed in cardiac fibroblasts, where it facilitates mitophagy and promotes fibroblast quiescence. This suggests that depressed BAG3 levels in heart failure may exacerbate fibrotic pathology, thus contributing to myocardial dysfunction through sarcomere-independent pathways.NEW & NOTEWORTHY We report BAG3's localization to mitochondria and its role in mitophagy for the first time in primary ventricular cardiac fibroblasts. We have also collected the first evidence showing that loss of BAG3 increases cardiac fibroblast activation into myofibroblasts, which are major drivers of cardiac fibrosis and pathological remodeling during heart disease.

Downregulation of salusins alleviates hypertrophic cardiomyopathy via attenuating oxidative stress and autophagy

IntroductionSalusins, which are translated from the alternatively spliced mRNA of torsin family 2 member A (TOR2A), play a vital role in regulation of various cardiovascular diseases. However, it remains unclear precisely regarding their roles in hypertrophic cardiomyopathy (HCM). Therefore, this study was conducted to explore therapeutic effect and the underlying mechanisms of salusins on HCM.Material and methodsIn vivo experiments, Sprague–Dawley rats were used to induce HCM model by angiotensin (Ang) II infusion for 4 weeks. The rats were randomly divided into four groups, namely, Saline + Control shRNA (n = 7), Ang II + Control shRNA (n = 8), Saline + TOR2A shRNA (n = 7), and Ang II + TOR2A shRNA groups (n = 8). After HCM induction, doppler echocardiography is recommended to evaluate heart function. In vitro experiments, primary neonatal rat cardiomyocytes (NRCMs) and cardiac fibroblasts (NRCFs) were obtained from newborn rats, and were treated with Ang II (10–6 M) for 24 h.ResultsAfter treatment with Ang II, levels of salusin-α and salusin-β were elevated in serum and cardiac tissues of rats and in the neonatal rat cardiomyocytes and cardiac fibroblasts. Downregulation of salusins alleviated the Ang II-induced cardiac hypertrophy by suppressing the increased atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and beta-myosin heavy chain (β-MHC) and cardiac fibrosis by blocking collagen I, collagen III and transforming growth factor-beta (TGF-β), and it also attenuated oxidative stress by suppressing the increased reactive oxygen species (ROS) and malondialdehyde (MDA) levels and reversing the decreased superoxide dismutase (SOD) activity and autophagy by inhibiting the increased microtubule-associated protein light chain 3B (LC3B), Beclin1, autophagy related gene (Atg) 3 and Atg5 in the cardiac tissues of Ang II-infused rats and in the Ang II-treated NRCMs.ConclusionsAll these findings suggest that the levels of salusins were elevated in the HCM, and targeting of salusins contributes to alleviation of cardiac hypertrophy and fibrosis probably via attenuating oxidative stress and autophagy. Accordingly, targeting of salusins may be a strategy for HCM therapy.

Development of photocrosslinkable bioinks with improved electromechanical properties for 3D bioprinting of cardiac BioRings

Three-dimensional (3D) bioprinting is an advanced fabrication technique to build biomimetic constructs for different biomedical applications. Here, we developed composite bioinks based on photocrosslinkable natural polymers, gelatin methacryloyl (GelMA) and alginate methacrylate (AlgMA), and electroconductive reduced graphene oxide (rGO) nanomaterials. The photocrosslinked hydrogels indicated interconnected porous microarchitecture with detectable rGO nanosheets on the pore walls. The addition of AlgMA and rGO to GelMA bioinks significantly enhanced tensile and compressive mechanical properties, improved the rheological properties and printability of inks, and controlled the degradation profile of the constructs. The optimization was done based on the suitable electromechanical properties for cardiac tissue engineering applications. The ring-shaped cardiac constructs were further 3D bioprinted with different cardiac cell types (neonatal rat cardiomyocytes, cardiac fibroblasts, and HL-1 cells). The optimized bioink supported a high level of cardiac cell viability, proliferation, spreading, elongation, and alignment with functional hallmarks (e.g., calcium transient and cellular spontaneous beating). Proof-of-concept validation was demonstrated for utilizing this platform as a 3D bioprinted heart-on-a-chip model with automated high-throughput in the 12-well plates. The developed bioprinted cardiac model could be used for different applications, including drug screening and disease modeling.

Biochanin A inhibits cardiac hypertrophy and fibrosis in vivo and in vitro

The heart undergoes pathological cardiac hypertrophy as an adaptive response to prolonged pathological stimulation, leading to cardiomyocyte hypertrophy, fibroblast proliferation, and an increase in extracellular matrix. Chinese medicine monomers are now receiving much attention for the treatment of cardiac hypertrophy and myocardial remodeling. Biochanin A (BCA) is a kind of flavonoid structural monomer, which has a certain therapeutic effect on bone thinning disease, aging syndrome, lung cancer, etc. Moreover, it exhibits hypoglycemic, anti-inflammatory, anti-oxidation, anti-bacteria and other pharmacological properties. It is still unknown whether BCA has an impact on the mechanism of TAC-induced cardiac hypertrophy. Here, cardiac remodeling was induced by TAC. BCA was injected intraperitoneally at 25 and 50 mg/kg/day one week in advance. Masson, WGA, DHE and other pathological staining and serum were used to detect the inhibitory effect of BCA on cardiac hypertrophy in mice. The anti-hypertrophic effect of BCA was demonstrated by studying the pathological manifestations of Neonatal rat cardiomyocytes (NRCMs) and cardiac fibroblasts (CFs) in vitro. The results showed that BCA significantly reduced TAC-induced fibrosis, inflammation, oxidative stress, and myocardial hypertrophy. BCA inhibited Ang II-induced cell hypertrophy and oxidative stress in NRCMs in vitro and Ang II-induced CF migration, proliferation, and collagen secretion. This suggests that BCA plays a key role in inhibiting the progression of myocardial remodeling, suggesting that BCA may be a promising agent for the treatment of myocardial hypertrophy and fibrosis.

CTRP3 inhibits myocardial fibrosis through the P2X7R-NLRP3 inflammasome pathway in SHR rats.

Reducing hypertensive myocardial fibrosis is the fundamental approach to preventing hypertensive ventricular remodelling. C1q/TNF-related protein-3 (CTRP3) is closely associated with hypertension. However, the role and mechanism of CTRP3 in hypertensive myocardial fibrosis are unclear. In this study, we aimed to explore the effect of CTRP3 on hypertensive myocardial fibrosis and the potential mechanism. WKY and SHR rats were employed, blood pressure, body weight, heart weight, H/BW were measured, and fibrotic-related proteins, CTRP3 and Collagen I were tested in myocardium at 12 and 20 weeks by immunohistochemical staining and Western blotting, respectively. The results showed that compared with the WKY, SBP, DBP, mean arterial pressure and heart rate (HR) were all significantly increased in SHR at 12 and 20 weeks, while heart weight and H/BW were only increased at 20 weeks. Meanwhile, CTRP3 decreased, while Collagen I increased significantly in the SHR rat myocardium at 20 weeks, which compared to the WKY. Moreover, the expression of α-SMA increased from 12 weeks, Collagen I/III and MMP2/9 increased and TIMP-2 decreased until 20 weeks. In order to explore the function and mechanism of CTRP3 in hypertensive fibrosis, Angiotensin II (Ang II) was used to induce hypertension in primary neonatal rat cardiac fibroblasts in vitro . CTRP3 significantly inhibited the Ang II induced activation of fibrotic proteins, purinergic 2X7 receptor (P2X7R)-NLRP3 inflammasome pathway. The P2X7R agonist BzATP significantly exacerbated Ang II-induced NLRP3 inflammasome activation, which was decreased by the P2X7R antagonists A43079, CTRP3 and MCC950. CTRP3 expression was decreased in the myocardium of SHR rats, and exogenous CTRP3 inhibited Ang II-induced fibrosis in cardiac fibroblasts by regulating the P2X7R-NLRP3 inflammasome pathway, suggesting that CTRP3 is a potential drug for alleviating myocardial fibrosis in hypertensive conditions.

Rivaroxaban, a direct inhibitor of coagulation factor Xa, attenuates adverse cardiac remodeling in rats by regulating the PAR-2 and TGF-β1 signaling pathways.

Factor Xa (FXa) not only plays an active role in the coagulation cascade but also exerts non-hemostatic signaling through the protease-activated receptors (PARs). This study aimed to investigate whether the FXa inhibitor, Rivaroxaban (RIV), attenuates adverse cardiac remodeling in rats with myocardial infarction (MI) and to identify the underlying molecular mechanisms it uses. An MI model was induced in eight-week-old, male Wistar rats, by permanent ligation of the left anterior descending coronary artery. MI rats were randomly assigned to receive RIV or protease-activated receptors 2-antagonist (PAR-2 antagonist, FSLLRY) treatment for four weeks. Histological staining, echocardiography and hemodynamics were used to assess the cardioprotective effects of RIV. Meanwhile, pharmacological approaches of agonist and inhibitor were used to observe the potential pathways in which RIV exerts antifibrotic effects in neonatal rat cardiac fibroblasts (CFs). In addition, real-time PCR and western blot analysis were performed to examine the associated signaling pathways. RIV presented favorable protection of left ventricular (LV) cardiac function in MI rats by significantly reducing myocardial infarct size, ameliorating myocardial pathological damage and improving left ventricular (LV) remodeling. Similar improvements in the PAR-2 antagonist FSLLRY and RIV groups suggested that RIV protects against cardiac dysfunction in MI rats by ameliorating PAR-2 activation. Furthermore, an in vitro model of fibrosis was then generated by applying angiotensin II (Ang II) to neonatal rat cardiac fibroblasts (CFs). Consistent with the findings of the animal experiments, RIV and FSLLRY inhibited the expression of fibrosis markers and suppressed the intracellular upregulation of transforming growth factor β1 (TGFβ1), as well as its downstream Smad2/3 phosphorylation effectors in Ang II-induced fibrosis, and PAR-2 agonist peptide (PAR-2 AP) reversed the inhibition effect of RIV. Our findings demonstrate that RIV attenuates MI-induced cardiac remodeling and improves heart function, partly by inhibiting the activation of the PAR-2 and TGF-β1 signaling pathways.

Calcineurin/NFATc3 pathway mediates myocardial fibrosis in diabetes by impairing enhancer of zeste homolog 2 of cardiac fibroblasts

BackgroundDiabetes is associated with myocardial fibrosis, while the underlying mechanisms remain elusive. The aim of this study is to investigate the underlying role of calcineurin/nuclear factor of activated T cell 3 (CaN/NFATc3) pathway and the Enhancer of zeste homolog 2 (EZH2) in diabetes-related myocardial fibrosis.MethodsStreptozotocin (STZ)-injected diabetic rats were randomized to two groups: the controlled glucose (Con) group and the diabetes mellitus (DM) group. Eight weeks later, transthoracic echocardiography was used for cardiac function evaluation, and myocardial fibrosis was visualized by Masson trichrome staining. The primary neonatal rat cardiac fibroblasts were cultured with high-glucose medium with or without cyclosporine A or GSK126. The expression of proteins involved in the pathway was examined by western blotting. The nuclear translocation of target proteins was assessed by immunofluorescence.ResultsThe results indicated that high glucose treatment increased the expression of CaN, NFATc3, EZH2 and trimethylates lysine 27 on histone 3 (H3K27me3) in vitro and in vivo. The inhibition of the CaN/NFATc3 pathway alleviated myocardial fibrosis. Notably, inhibition of CaN can inhibit the nuclear translocation of NFATc3, and the expression of EZH2 and H3K27me3 protein induced by high glucose. Moreover, treatment with GSK126 also ameliorated myocardial fibrosis.ConclusionDiabetes can possibly promote myocardial fibrosis by activating of CaN/NFATc3/EZH2 pathway.

Abstract P2012: Development Of An In Vitro Assay To Model Loss Of Neighbor At The Infarct Border Zone

The borderzone (BZ) of the infarcted heart separates poorly-perfused areas of cell death from well-perfused territories of comparatively normal myocardium. The BZ after myocardial infarction is of key importance for the remodeling process that drives heart failure and sudden cardiac death. We recently defined the transcriptional features of the infarct borderzone using single cell RNA-Seq and spatial transcriptomics which revealed prominent upregulation of mechanotransduction transcriptional programs. A similar transcriptional response was inducible by needle trauma alone. To explain these results, we proposed the “Loss of Neighbor” (LON) Hypothesis, where surviving cells neighboring the infarct experience mechanical instability due to the highly asymmetric loss of load, leading to cell signal activation. Here, we develop and validate an in vitro model of loss of neighbor using a mechanical scratch assay to model the biology of the infarct borderzone. We cultured neonatal rat cardiomyocytes and cardiac fibroblasts and subjected them to mechanical “scratch” injury. To evaluate how the mechanical wound altered gene expression, we performed qPCR for genes expressed by cardiomyocytes in the remote zone and the borderzone using RNA isolated from cells with or without exposure to mechanical scratch injury. Compared to non-scratched cultures, scratched cells significantly upregulated BZ-specific genes such as Ankrd1, Nppa, Flnc, and Xirp2 . In addition, scratched cells significantly upregulated expression of interferon-stimulated genes (ISGs) compared to non-scratched cells or scratched cells exposed to anti-IFNAR Ab. This suggests that the interferon-inducible cell (IFNIC) response observed at the infarct borderzone in vivo can be induced by in vitro cardiomyocyte LON. Taken together, our results suggest that the mechanical scratch assay in vitro captures key elements of the transcriptional borderzone in vivo . Overall, this model can guide further elucidation of the transcriptional program states during cardiac remodeling.

Celastrol relieves myocardial infarction-induced cardiac fibrosis by inhibiting NLRP3 inflammasomes in rats.

Myocardial infarction (MI) triggers a strong inflammatory response mediating by NLRP3 inflammasome which is associated with cardiac fibrosis. The key players in this response are Interleukin (IL)-1 and IL-18, which are regulated by NLRP3 inflammasomes. Celastrol, a traditional Chinese medicine with strong anti-inflammatory activity, has recently reported as a cardioprotective agent. However, the mechanisms by which celastrol is cardioprotective in MI remain elusive. We hypothesized that Celastrol could reduce IL-1β and IL-18 expression and ameliorate myocardial fibrosis after myocardial infarction in rats, improve poor heart remodeling, and preserve heart function. Myocardial infarction (MI) was caused by ligating the left anterior descending of male SD rats. Celastrol (1mg/kg) or saline was administered every other day for 4weeks. Heart function and fibrosis were assessed. Inflammatory and fibrotic markers in the myocardia were evaluated with immunohistochemistry, western blot, and ELISA. Molecular docking was employed to predict Celastrol's binding to NLRP3 protein. The effects of Celastrol on the expression of NLRP3 inflammasome and myocardial fibrosis genes were then examined in vitro. Celastrol maintained the left ventricular fractional shortening (FS) and ejection fraction (EF). Fibrosis was significantly reduced in animals treated with 1mg/kg Celastrol (15.17±1.82%) relative to controls (29.88±4.28%). Celastrol also significantly reduced the NLRP3, IL-18, and IL-1β levels, together with macrophage and neutrophil infiltration in the myocardium. Molecular docking predicted that NLRP3 would bind tightly to Celastrol [Docking energy: -8.9 (kcal/mol)]. In vitro experiments showed reduced NLRP3 inflammasome and myocardial fibrosis-associated proteins expression in neonatal rat cardiac fibroblasts treated with Celastrol. In post-MI rats, Celastrol, a naturally occurring active ingredient, was able to reduce myocardial fibrosis and improve cardiac function, according to our study. These effects may result from inhibiting the NLRP3 inflammasome and attenuating the early inflammatory storm after MI, suggesting that Celastrol may be useful in treating acute MI.

Protocatechuic acid prevents isoproterenol-induced heart failure in mice by downregulating kynurenine-3-monooxygenase.

Protocatechuic acid (3,4-dihydroxybenzoic acid) prevents oxidative stress, inflammation and cardiac hypertrophy. This study aimed to investigate the therapeutic effects of protocatechuic acid in an isoproterenol-induced heart failure mouse model and to identify the underlying mechanisms. To establish the heart failure model, C57BL/6NTac mice were given high-dose isoproterenol (80 mg/kg body weight) for 14 days. Echocardiography revealed that protocatechuic acid reversed the isoproterenol-induced downregulation of fractional shortening and ejection fraction. Protocatechuic acid attenuated cardiac hypertrophy as evidenced by the decreased heart-weight-to-body-weight ratio and the expression of Nppb. RNA sequencing analysis identified kynurenine-3-monooxygenase (Kmo) as a potential target of protocatechuic acid. Protocatechuic acid treatment or transfection with short-interfering RNA against Kmo ameliorated transforming growth factor β1-induced upregulation of Kmo, Col1a1, Col1a2 and Fn1 in vivo or in neonatal rat cardiac fibroblasts. Kmo knockdown attenuated the isoproterenol-induced increase in cardiomyocyte size, as well as Nppb and Col1a1 expression in H9c2 cells or primary neonatal rat cardiomyocytes. Moreover, protocatechuic acid attenuated Kmo overexpression-induced increases in Nppb mRNA levels. Protocatechuic acid or Kmo knockdown decreased isoproterenol-induced ROS generation in vivo and in vitro. Thus, protocatechuic acid prevents heart failure by downregulating Kmo. Therefore, protocatechuic acid and Kmo constitute a potential novel therapeutic agent and target, respectively, against heart failure.

Tissue kallikrein-related peptidase8 accentuates cardiac fibrosis after myocardial ischemia-reperfusion injury via regulation of cardiac fibroblasts

AimsTissue kallikrein-related peptidase8 (KLK8) has been found to mitigate acute myocardial ischemia-reperfusion (IR) injury. However, the effect of KLK8 on cardiac remodeling in response to IR injury has not been determined. Materials and methodsKLK8 overexpressing transgenic rat (KLK8-TG) was used as the animal model. IR injury was induced by ligating the left anterior descending coronary artery for 1 h and subsequent reperfusion. The functional and morphological changes of the heart were examined 14 days after the injury. Neonatal rat cardiac fibroblasts (CFs) were used to investigate the molecular mechanisms in vitro. Key findingsKLK8 overexpression enhanced cardiac diastolic dysfunction, fibrosis, and hypertrophy after IR injury, indicating that KLK8 accentuated cardiac remodeling in response to IR injury. Moreover, KLK8 overexpression increased epidermal growth factor (EGF) release and promoted the phosphorylation of EGF receptor (EGFR) and ERK1/2 in the heart after IR injury. It was interesting to find that both EGFR antagonist (AG 1478) and MEK inhibitor (PD98059) attenuated the KLK8-induced proliferation and activation of CFs in vitro, indicating that EGFR signaling might mediate the pro-fibrotic action of KLK8. SignificanceKLK8 plays a crucial role in cardiac remodeling after myocardial infarction. KLK8 accentuates cardiac fibrosis after IR injury, possibly mediated by EGFR signaling in CFs.

PFKFB3 Inhibitor 3PO Reduces Cardiac Remodeling after Myocardial Infarction by Regulating the TGF-β1/SMAD2/3 Pathway.

Adverse cardiac remodeling, including cardiac fibrosis, after myocardial infarction (MI) is a major cause of long-term heart failure. 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), an enzyme that regulates glucose metabolism, also plays an important role in various fibrotic and cardiovascular diseases. However, its effects on MI remain unknown. Here, PFKFB3 inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) and a permanent left anterior descending ligation mouse model were used to explore the functional role of PFKFB3 in MI. We showed that PFKFB3 expression increased significantly in the area of cardiac infarction during the early phase after MI, peaking on day 3. 3PO treatment markedly improved cardiac function, accompanied by decreased infarction size and collagen density in the infarct area. Meanwhile, 3PO attenuated cardiac fibrosis after MI by reducing the expression of collagen and fibronectin in murine hearts. Notably, 3PO reduced PFKFB3 expression and inhibited the transforming growth factor-beta 1/mothers against the decapentaplegic homolog 2/3 (TGF-β1/SMAD2/3) signaling pathway to inhibit cardiac fibrosis after MI. Moreover, PFKFB3 expression in neonatal rat cardiac fibroblasts (NRCFs) increased significantly after MI and under hypoxia, whereas 3PO alleviated the migratory capacity and activation of NRCFs induced by TGF-β1. In conclusion, 3PO effectively reduced fibrosis and improved adverse cardiac remodeling after MI, suggesting PFKFB3 inhibition as a novel therapeutic strategy to reduce the incidence of chronic heart failure following MI.

TRPA1 deficiency attenuates cardiac fibrosis via regulating GRK5/NFAT signaling in diabetic rats

BackgroundTransient receptor potential ankyrin 1 (TRPA1) has been linked to the development of various cardiovascular diseases, but its role in diabetic cardiomyopathy is not well understood. This study aimed to investigate the protective effects of TRPA1 deficiency on diabetic cardiomyopathy in rats with streptozotocin-induced diabetes and in neonatal rat cardiac fibroblasts (CFs) exposed to high glucose (HG). MethodsCardiac TRPA1 expression levels were measured in diabetic rats. Cardiac function, remodeling, and fibrosis were analyzed in Sprague-Dawley (SD) rats and TRPA1-deficient rats with diabetic cardiomyopathy. In vitro, fibrosis was measured in CFs exposed to HG. Additionally, 1,8-cineole, a natural inhibitor of TRPA1, was used to treat SD rats with diabetic cardiomyopathy. ResultsTRPA1 expression was increased in the heart tissue of diabetic rats and in CFs treated with HG. TRPA1 deficiency significantly improved cardiac function in diabetic rats, as evidenced by improved echocardiography and reduced cardiac hypertrophy and fibrosis. In vitro, TRPA1 deficiency suppressed the transformation of HG-induced CFs into myofibroblasts. The cardioprotective effect of TRPA1 deficiency was found to inhibit cardiac fibrosis by regulating GRK5/NFAT signaling. Furthermore, inhibition of GRK5/NFAT signaling abolished the promotion of CF transformation into myofibroblasts by TRPA1 activation. Inhibition of TRPA1 activation by 1,8-cineole reduced cardiac dysfunction and remodeling in diabetic rats by regulating GRK5/NFAT signaling. ConclusionsTRPA1 deficiency reduced cardiac fibrosis in diabetic rats and inhibited HG-induced CF activation in vitro by regulating GRK5/NFAT signaling. The TRPA1 inhibitor 1,8-cineole may serve as a novel therapeutic agent for the treatment of diabetic cardiomyopathy.

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.

Shexiang Tongxin Dropping Pill Allieviates Heart Failure via Extracellula Matrix-Receptor Interaction Pathways Based on RNA-Seq Transcriptomics and Experimental Studies.

To investigate the protective mechanisms of Chinese medicine Shexiang Tongxin Dropping Pills (STDP) on heart failure (HF). Isoproterenol (ISO)-induced HF rat model and angiotensin II (Ang II)-induced neonatal rat cardiac fibroblast (CFs) model were used in the present study. HF rats were treated with and without STDP (3 g/kg). RNA-seq was performed to identify differentially expressed genes (DEGs). Cardiac function was evaluated by echocardiography. Hematoxylin and eosin and Masson's stainings were taken to assess cardiac fibrosis. The levels of collagen I (Col I) and collagen III (Col III) were detected by immunohistochemical staining. CCK8 kit and transwell assay were implemented to test the CFs' proliferative and migratory activity, respectively. The protein expressions of α-smooth muscle actin (α-SMA), matrix metalloproteinase-2 (MMP-2), MMP-9, Col I, and Col III were detected by Western blotting. The results of RNA-seq analysis showed that STDP exerted its pharmacological effects on HF via multiple signaling pathways, such as the extracellular matrix (ECM)-receptor interaction, cell cycle, and B cell receptor interaction. Results from in vivo experiments demonstrated that STDP treatment reversed declines in cardiac function, inhibiting myocardial fibrosis, and reversing increases in Col I and Col III expression levels in the hearts of HF rats. Moreover, STDP (6, 9 mg/mL) inhibited the proliferation and migration of CFs exposed to Ang II in vitro (P<0.05). The activation of collagen synthesis and myofibroblast generation were markedly suppressed by STDP, also the synthesis of MMP-2 and MMP-9, as well as ECM components Col I, Col III, and α-SMA were decreased in Ang II-induced neonatal rats' CFs. STDP had anti-fibrotic effects in HF, which might be caused by the modulation of ECM-receptor interaction pathways. Through the management of cardiac fibrosis, STDP may be a compelling candidate for improving prognosis of HF.

Gβγ subunits colocalize with RNA polymerase II and regulate transcription in cardiac fibroblasts

Gβγ subunits mediate many different signaling processes in various compartments of the cell, including the nucleus. To gain insight into the functions of nuclear Gβγ signaling, we investigated the functional role of Gβγ signaling in the regulation of GPCR-mediated gene expression in primary rat neonatal cardiac fibroblasts. We identified a novel, negative, regulatory role for the Gβ1γ dimer in the fibrotic response. Depletion of Gβ1 led to derepression of the fibrotic response at the mRNA and protein levels under basal conditions and an enhanced fibrotic response after sustained stimulation of the angiotensin II type I receptor. Our genome-wide chromatin immunoprecipitation experiments revealed that Gβ1 colocalized and interacted with RNA polymerase II on fibrotic genes in an angiotensin II-dependent manner. Additionally, blocking transcription with inhibitors of Cdk9 prevented association of Gβγ with transcription complexes. Together, our findings suggest that Gβ1γ is a novel transcriptional regulator of the fibrotic response that may act to restrict fibrosis to conditions of sustained fibrotic signaling. Our work expands the role for Gβγ signaling in cardiac fibrosis and may have broad implications for the role of nuclear Gβγ signaling in other cell types.

Berberine Alleviates Doxorubicin-Induced Myocardial Injury and Fibrosis by Eliminating Oxidative Stress and Mitochondrial Damage via Promoting Nrf-2 Pathway Activation.

Doxorubicin (DOX)-related cardiotoxicity has been recognized as a serious complication of cancer chemotherapy. Effective targeted strategies for myocardial protection in addition to DOX treatment are urgently needed. The purpose of this paper was to determine the therapeutic effect of berberine (Ber) on DOX-triggered cardiomyopathy and explore the underlying mechanism. Our data showed that Ber markedly prevented cardiac diastolic dysfunction and fibrosis, reduced cardiac malondialdehyde (MDA) level and increased antioxidant superoxide dismutase (SOD) activity in DOX-treated rats. Moreover, Ber effectively rescued the DOX-induced production of reactive oxygen species (ROS) and MDA, mitochondrial morphological damage and membrane potential loss in neonatal rat cardiac myocytes and fibroblasts. This effect was mediated by increases in the nuclear accumulation of nuclear erythroid factor 2-related factor 2 (Nrf2) and levels of heme oxygenase-1 (HO-1) and mitochondrial transcription factor A (TFAM). We also found that Ber suppressed the differentiation of cardiac fibroblasts (CFs) into myofibroblasts, as indicated by decreased expression of α-smooth muscle actin (α-SMA), collagen I and collagen III in DOX-treated CFs. Pretreatment with Ber inhibited ROS and MDA production and increased SOD activity and the mitochondrial membrane potential in DOX-challenged CFs. Further investigation indicated that the Nrf2 inhibitor trigonelline reversed the protective effect of Ber on both cardiomyocytes and CFs after DOX stimulation. Taken together, these findings demonstrated that Ber effectively alleviated DOX-induced oxidative stress and mitochondrial damage by activating the Nrf2-mediated pathway, thereby leading to the prevention of myocardial injury and fibrosis. The current study suggests that Ber is a potential therapeutic agent for DOX-induced cardiotoxicity that exerts its effects by activating Nrf2.

Cryptotanshinone Attenuated Pathological Cardiac Remodeling In Vivo and In Vitro Experiments.

Cardiac remodeling has been demonstrated to be the early stage and common pathway for various types of cardiomyopathy, but no specific treatment has been suggested to prevent its development and progress. This study was aimed at assessing whether Cryptotanshinone (CTS) treatment could effectively attenuate cardiac remodeling in vivo and in vitro. Aortic banding (AB) surgery was performed to establish a pressure-overload-induced mouse cardiac remodeling model. Echocardiography and pressure-volume proof were used to examine mouse cardiac function. Hematoxylin and eosin (HE) and Picro-Sirius Red (PSR) staining were used to assess cardiac remodeling in vivo. Mouse hearts were collected to analysis signaling pathway and cardiac remodeling markers, respectively. Furthermore, neonatal rat cardiomyocyte (NRCMs) and cardiac fibroblast (CF) were isolated to investigate the roles and mechanisms of CTS treatment in vitro. CTS administration significantly alleviated pressure-overload-induced mouse cardiac dysfunction, inhibited cardiac hypertrophy, and reduced cardiac fibrosis. Mechanically, CTS treatment significantly inhibited the STAT3 and TGF-β/SMAD3 signaling pathways. In vitro experiments, CTS treatment markedly inhibited AngII-induced cardiomyocyte hypertrophy and TGF-β-induced myofibroblast activation via inhibiting STAT3 phosphorylation and its nuclear translocation. Finally, CTS treatment could not protect against pressure overload-induced mouse cardiac remodeling after adenovirus-associated virus (AAV)9-mediated STAT3 overexpression in mouse heart. CTS treatment might attenuate pathological cardiac remodeling via inhibiting STAT3-dependent pathway.