MiR-150 Attenuates Maladaptive Cardiac Remodeling Mediated by Long Noncoding RNA MIAT and Directly Represses Profibrotic Hoxa4

Background: MicroRNA-150 (miR-150) plays a protective role in heart failure (HF). Long noncoding RNA, myocardial infarction–associated transcript (MIAT) regulates miR-150 function in vitro by direct interaction. Concurrent with miR-150 downregulation, MIAT is upregulated in failing hearts, and gain-of-function single-nucleotide polymorphisms in MIAT are associated with increased risk of myocardial infarction (MI) in humans. Despite the correlative relationship between MIAT and miR-150 in HF, their in vivo functional relationship has never been established, and molecular mechanisms by which these 2 noncoding RNAs regulate cardiac protection remain elusive. Methods: We use MIAT KO (knockout), Hoxa4 (homeobox a4) KO, MIAT TG (transgenic), and miR-150 TG mice. We also develop DTG (double TG) mice overexpressing MIAT and miR-150. We then use a mouse model of MI followed by cardiac functional, structural, and mechanistic studies by echocardiography, immunohistochemistry, transcriptome profiling, Western blotting, and quantitative real-time reverse transcription-polymerase chain reaction. Moreover, we perform expression analyses in hearts from patients with HF. Lastly, we investigate cardiac fibroblast activation using primary adult human cardiac fibroblasts and in vitro assays to define the conserved MIAT/miR-150/HOXA4 axis. Results: Using novel mouse models, we demonstrate that genetic overexpression of MIAT worsens cardiac remodeling, while genetic deletion of MIAT protects hearts against MI. Importantly, miR-150 overexpression attenuates the detrimental post-MI effects caused by MIAT. Genome-wide transcriptomic analysis of MIAT null mouse hearts identifies Hoxa4 as a novel downstream target of the MIAT/miR-150 axis. Hoxa4 is upregulated in cardiac fibroblasts isolated from ischemic myocardium and subjected to hypoxia/reoxygenation. HOXA4 is also upregulated in patients with HF. Moreover, Hoxa4 deficiency in mice protects the heart from MI. Lastly, protective actions of cardiac fibroblast miR-150 are partially attributed to the direct and functional repression of profibrotic Hoxa4 . Conclusions: Our findings delineate a pivotal functional interaction among MIAT, miR-150, and Hoxa4 as a novel regulatory mechanism pertinent to ischemic HF.

Cardiac fibroblast activation detected by 68Gallium-FAPI-46 PET/MRI as a sign of chronic activity in cardiac sarcoidosis

In a patient with cardiac sarcoidosis, follow-up FDG-PET suggested complete remission under immunosuppression whereas FAPI-PET demonstrated ongoing fibroblast activation pointing at its potential to monitor chronic activity in sarcoidosis.

Fibroblast Growth Factor 23 Stimulates Cardiac Fibroblast Activity through Phospholipase C-Mediated Calcium Signaling

Fibroblast growth factor (FGF)-23 induces hypertrophy and calcium (Ca2+) dysregulation in cardiomyocytes, leading to cardiac arrhythmia and heart failure. However, knowledge regarding the effects of FGF-23 on cardiac fibrogenesis remains limited. This study investigated whether FGF-23 modulates cardiac fibroblast activity and explored its underlying mechanisms. We performed MTS analysis, 5-ethynyl-2’-deoxyuridine assay, and wound-healing assay in cultured human atrial fibroblasts without and with FGF-23 (1, 5 and 25 ng/mL for 48 h) to analyze cell proliferation and migration. We found that FGF-23 (25 ng/mL, but not 1 or 5 ng/mL) increased proliferative and migratory abilities of human atrial fibroblasts. Compared to control cells, FGF-23 (25 ng/mL)-treated fibroblasts had a significantly higher Ca2+ entry and intracellular inositol 1,4,5-trisphosphate (IP3) level (assessed by fura-2 ratiometric Ca2+ imaging and enzyme-linked immunosorbent assay). Western blot analysis showed that FGF-23 (25 ng/mL)-treated cardiac fibroblasts had higher expression levels of calcium release-activated calcium channel protein 1 (Orai1) and transient receptor potential canonical (TRPC) 1 channel, but similar expression levels of α-smooth muscle actin, collagen type IA1, collagen type Ⅲ, stromal interaction molecule 1, TRPC 3, TRPC6 and phosphorylated-calcium/calmodulin-dependent protein kinase II when compared with control fibroblasts. In the presence of ethylene glycol tetra-acetic acid (a free Ca2+ chelator, 1 mM) or U73122 (an inhibitor of phospholipase C, 1 μM), control and FGF-23-treated fibroblasts exhibited similar proliferative and migratory abilities. Moreover, polymerase chain reaction analysis revealed that atrial fibroblasts abundantly expressed FGF receptor 1 but lacked expressions of FGF receptors 2-4. FGF-23 significantly increased the phosphorylation of FGF receptor 1. Treatment with PD166866 (an antagonist of FGF receptor 1, 1 μM) attenuated the effects of FGF-23 on cardiac fibroblast activity. In conclusion, FGF-23 may activate FGF receptor 1 and subsequently phospholipase C/IP3 signaling pathway, leading to an upregulation of Orai1 and/or TRPC1-mediated Ca2+ entry and thus enhancing human atrial fibroblast activity.

Inhibition of SGLT1 alleviates the glycemic variability-induced cardiac fibrosisvia inhibiting activations of macrophage and cardiac fibroblast

Glycemic variability has been considered as one of the predictors of diabetes complications in patients with diabetes mellitus (DM). In this work, we evaluated whether glycemic variability induces cardiac fibrosis through regulating cardiac fibroblast activation and macrophage polarization. Moreover, we determined whether glucose transporter sodium-glucose cotransporter 1 (SGLT1) plays an important role in this process. Glycemic variability-induced mice were established using DM mice (GVDM mice) and intermittent high glucose (IHG) treatment was used to simulate glycemic variability in RAW264.7 macrophages and cardiac fibroblasts. The short hairpin RNA for SGLT1 was used to knock down SGLT1. The results showed that glycemic variability aggravated the cardiac fibrosis in GVDM mice. Additionally, glycemic variability promoted the expressions of fibrogenic cytokine and the extracellular matrix proteins in left ventricular tissues and cardiac fibroblasts. GVDM mice showed a higher incidence of macrophage infiltration and M1 polarization in left ventricular tissues. Moreover, IHG promoted RAW264.7 macrophages tended to differentiate to M1 phenotype. SGLT1 knockdown alleviated cardiac fibrosis in GVDM mice and inhibited activations of cardiac fibroblasts and macrophages M1 polarization. Our results indicated that glycemic variability aggravates cardiac fibrosis through activating cardiac fibroblast and macrophage M1 polarization, which could be partially inhibited by SGLT1 knockdown.

Epigenetic Repression of Chloride Channel Accessory 2 Transcription in Cardiac Fibroblast: Implication in Cardiac Fibrosis

Cardiac fibrosis is a key pathophysiological process that contributes to heart failure. Cardiac resident fibroblasts, exposed to various stimuli, are able to trans-differentiate into myofibroblasts and mediate the pro-fibrogenic response in the heart. The present study aims to investigate the mechanism whereby transcription of chloride channel accessory 2 (Clca2) is regulated in cardiac fibroblast and its potential implication in fibroblast-myofibroblast transition (FMyT). We report that Clca2 expression was down-regulated in activated cardiac fibroblasts (myofibroblasts) compared to quiescent cardiac fibroblasts in two different animal models of cardiac fibrosis. Clca2 expression was also down-regulated by TGF-β, a potent inducer of FMyT. TGF-β repressed Clca2 expression at the transcriptional level likely via the E-box element between −516 and −224 of the Clca2 promoter. Further analysis revealed that Twist1 bound directly to the E-box element whereas Twist1 depletion abrogated TGF-β induced Clca2 trans-repression. Twist1-mediated Clca2 repression was accompanied by erasure of histone H3/H4 acetylation from the Clca2 promoter. Mechanistically Twist1 interacted with HDAC1 and recruited HDAC1 to the Clca2 promoter to repress Clca2 transcription. Finally, it was observed that Clca2 over-expression attenuated whereas Clca2 knockdown enhanced FMyT. In conclusion, our data demonstrate that a Twist1-HDAC1 complex represses Clca2 transcription in cardiac fibroblasts, which may contribute to FMyT and cardiac fibrosis.

TGF-β1-containing exosomes from cardiac microvascular endothelial cells mediate cardiac fibroblast activation under high glucose conditions

Cardiac fibroblast (CF)-mediated extracellular matrix (ECM) remodeling is the key pathological basis for the occurrence and development of diabetic cardiomyopathy (DCM); its specific regulatory mechanisms have been widely studied but remain unclear. Exosomes are a type of stable signal transmission medium, and exosome-mediated cell-cell interactions play an important role in DCM. Endothelial cells form an important barrier between circulation and cardiomyocytes, in addition to being an important endocrine organ of the heart and an initial target for hyperglycemia, a key aspect in the development of DCM. We previously showed that exosomes derived from cardiac microvascular endothelial cells (CMECs) under high glucose conditions can be taken up by cardiomyocytes and regulate autophagy, apoptosis, and glucose metabolism. Consequently, in the present study, we focused on how exosomes mediate the interaction between CMECs and CFs. Surprisingly, exosomes derived from CMECs under high glucose were rich in TGF-β1 mRNA, which significantly promoted the activation of CFs. Additionally, exosomes derived from CMECs under high glucose conditions aggravated perivascular and interstitial fibrosis in mice treated with streptozotocin. Herein, we demonstrated for the first time the capacity of exosomes, released by CMECs under high glucose, to mediate fibroblast activation through TGF-β1 mRNA, which may be potentially beneficial in the development of exosome-targeted therapies to control DCM.

Necroptosis Inhibition by Hydrogen Sulfide Alleviated Hypoxia-Induced Cardiac Fibroblasts Proliferation via Sirtuin 3

Myocardial ischemia or hypoxia can induce myocardial fibroblast proliferation and myocardial fibrosis. Hydrogen sulfide (H2S) is a gasotransmitter with multiple physiological functions. In our present study, primary cardiac fibroblasts were incubated with H2S donor sodium hydrosulfide (NaHS, 50 μM) for 4 h followed by hypoxia stimulation (containing 5% CO2 and 1% O2) for 4 h. Then, the preventive effects on cardiac fibroblast proliferation and the possible mechanisms were investigated. Our results showed that NaHS reduced the cardiac fibroblast number, decreased the hydroxyproline content; inhibited the EdU positive ratio; and down-regulated the expressions of α-smooth muscle actin (α-SMA), the antigen identified by monoclonal antibody Ki67 (Ki67), proliferating cell nuclear antigen (PCNA), collagen I, and collagen III, suggesting that hypoxia-induced cardiac fibroblasts proliferation was suppressed by NaHS. NaHS improved the mitochondrial membrane potential and attenuated oxidative stress, and inhibited dynamin-related protein 1 (DRP1), but enhanced optic atrophy protein 1 (OPA1) expression. NaHS down-regulated receptor interacting protein kinase 1 (RIPK1) and RIPK3 expression, suggesting that necroptosis was alleviated. NaHS increased the sirtuin 3 (SIRT3) expressions in hypoxia-induced cardiac fibroblasts. Moreover, after SIRT3 siRNA transfection, the inhibitory effects on cardiac fibroblast proliferation, oxidative stress, and necroptosis were weakened. In summary, necroptosis inhibition by exogenous H2S alleviated hypoxia-induced cardiac fibroblast proliferation via SIRT3.

Increased release of serotonin from rat primary isolated adult cardiac myofibroblasts

Elevated blood serotonin levels have been observed in patients with heart failure and serotonin has a role in pathological cardiac function. The serotonin receptor system was examined in adult rat isolated cardiac fibroblast and myofibroblast cells. This is one of the first studies that has investigated serotonin receptors and other proteins involved in the serotonin receptor system in rat cardiac fibroblast and myofibroblast cells. Rat primary cardiac fibroblasts were isolated and transformed into myofibroblasts using 5 ng/ml TGF-β1. Transformation of cells to myofibroblasts was confirmed with the presence of α-smooth muscle actin using Western blot. Serotonin metabolism and receptor protein expression was assessed using Western blot techniques and serotonin levels measured using ELISA. The 5-HT1A, 5-HT2A and 5-HT2B receptors were found to be present in both rat cardiac fibroblasts and myofibroblast cells, however no significance in protein expression between the two cell types was found (P > 0.05). In this study a significant increase in the serotonin transporter (SERT), tryptophan hydroxylase 1 and extracellular serotonin levels was observed in rat cardiac myofibroblasts when compared to fibroblasts (P < 0.05). These results suggest that serotonin levels may rise in parallel with cardiac myofibroblast populations and contribute to the pathogenesis of heart failure via serotonin receptors.