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Additional file 1: Figure S1. The results on the acute toxicity of nanoFe. A, Weight change statistics. B, Representative images of H&E staining of mouse heart, liver, and kidney tissues. Figure S2. Establishment of mouse CLP models A and aggravated CLP models B. The cecum was tightly ligated at 1/3 site from its end using 4–0 nylon suture, and double punctures of the cecal wall were performed with a 25 G needle. For the aggravated CLP model, the cecum was tightly ligated at 2/3 site from its end. Figure S3. Additional echocardiographic data of nanoFe treatment on septic mice. A, Left ventricular end-systolic posterior wall thickness (LVPWs), left ventricular end-diastolic posterior wall thickness (LVPWd), heart rate (HR), and corrected left ventricular mass (LV mass) in the long axis view calculated 8 h after CLP. B, LVPWs, LVPWd, HR, and corrected LV mass in the short axis view calculated 8 h after CLP. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Sham or vs. CLP; ns, non-significant. n = 6 for each group. Figure S4. A schematic illustration of the present study. Table S1. Peak area % mean value of nanoFe from XPS measurements. Table S2. Acute toxicity of nanoFe.

Adverse myocardial remodeling, manifesting pathologically as myocardial hypertrophy and fibrosis, often follows myocardial infarction (MI) and results in cardiac dysfunction. In this study, an obvious epicardial adipose tissue (EAT) was observed in the rat model of MI and the EAT weights were positively correlated with cardiomyocyte size and myocardial fibrosis areas in the MI 2- and 4-week groups. Then, rat cardiomyocyte cell line H9C2 and primary rat cardiac fibroblasts were cultured in conditioned media generated from EAT of rats in the MI 4-week group (EAT-CM). Functionally, EAT-CM enlarged the cell surface area of H9C2 cells and reinforced cardiac fibroblast activation into myofibroblasts by elevating intracellular reactive oxygen species (ROS) levels. Mechanistically, miR-134-5p was upregulated by EAT-CM in both H9C2 cells and primary rat cardiac fibroblasts. miR-134-5p knockdown promoted histone H3K14 acetylation of manganese superoxide dismutase and catalase by upregulating lysine acetyltransferase 7 expression, thereby decreasing ROS level. An in vivo study showed that miR-134-5p knockdown limited adverse myocardial remodeling in the rat model of MI, manifesting as alleviation of cardiomyocyte hypertrophy and fibrosis. In general, our study clarified a new pathological mechanism involving an EAT/miRNA axis that explains the adverse myocardial remodeling occurring after MI.

Objective To explore the underlying mechanisms of Hrd1/sema3a/IGF-1R on cardiomyocyte apoptosis. Methods AMI model was established by the left-anterior descending coronary artery (LAD) ligation. The expressions of Hrd1, sema3a and IGF-1R were examined by western blot. The activity of caspase-3 and caspase-8 was measured using the corresponding activity detection kit. Cardiomyocyte apoptosis was detected by flow cytometry assay. Co-immunoprecipitation and ubiquitination assay were used to test the relationship among Hrd1, sema3a and IGF-1R. Results Hrd1 expression and the activity of caspase-3 and caspase-8 were increased in cardiac tissues of AMI rats and hypoxia-induced cardiomyocytes, while IGF-1R expression was decreased. Hrd1 overexpression promoted IGF-1R degradation, whereas knockdown of sema3a suppressed this degradation. Moreover, knockdown of Hrd1 or sema3a could inhibit the decrease of IGF-1R expression induced by hypoxia, and reverse the enhanced activity of caspase-3 and caspase-8 and the increase of cardiomyocytes apoptosis induced by hypoxia, while si-IGF-1R countered these effects. In AMI rat experiments, interfering Hrd1 or sema3a reduced the infarct size and increased IGF-1R expression, but these could be abolished by si-IGF-1R. Conclusion Hrd1 might mediate the ubiquitination of IGF-1R through sema3a and then participate in the regulation of cardiomyocyte apoptosis.

Prokineticin 2 (PK2) was reported to be decreased in the hearts of end-state heart failure patients. Our study aimed to explore the effects of PK2 on hypoxia/reoxygenation (H/R) injury and the underlying mechanism. H9c2 cardiomyocytes were treated with 5 nM PK2 in the presence or absence of 5 mM dual phosphatidylinositol 3-kinase (PI3K)/the mammalian target of rapamycin (mTOR) inhibitor (BEZ235) for 24 h and then subjected to H/R treatment. Cell viability and lactate dehydrogenase (LDH) release were evaluated by CCK-8 and LDH release assays, respectively. Apoptosis was determined by flow cytometry analysis. Oxidative stress was assessed by measuring superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) content. Results showed that H/R treatment decreased PK2 expression and inactivated the Akt/mTOR pathway in H9c2 cardiomyocytes. PK2 treatment activated the Akt/mTOR pathway in H/R-exposed H9c2 cardiomyocytes. H/R stimulation suppressed cell viability, increased LDH release, induced apoptosis and oxidative stress in H9c2 cardiomyocytes, while these effects were neutralised by treatment with PK2. However, the inhibitory effects of PK2 on H/R-induced injury in H9c2 cardiomyocytes were abolished by the addition of BEZ235. In conclusion, PK2 relieved H/R-induced injury in H9c2 cardiomyocytes by activation of the Akt/mTOR pathway.

Several miRNAs have been demonstrated to be involved in endothelial dysfunction during atherosclerosis (AS). However, the detailed roles and underlying mechanisms of miR-34a in AS-associated endothelial cell apoptosis are far from being addressed. Apolipoprotein E-deficient (ApoE−/−) mice fed with high-fat diet (HFD) were used as in vivo model of AS. Oxidized low-density lipoprotein (ox-LDL)-treated human aortic endothelial cells (HAECs) were applied as in vitro model of AS. The effects of miR-34a on atherosclerotic lesions were evaluated by hematoxylin–eosin (HE) and Oil Red O staining. Pecam-1+ endothelial cells were isolated from the aortic arch with flow cytometry. qRT-PCR and western blot were employed to measure gene and protein expression. The effects of miR-34a on cell viability, cell cycle distribution, and apoptosis were assessed by Cell counting kit (CCK)-8 and flow cytometry analysis. The relationship between miR-34a and Bcl-2 was confirmed by online softwares, luciferase reporter assay, and RNA immunoprecipitation (RIP). miR-34a was upregulated in HFD-induced ApoE−/− mice and ox-LDL-treated HAECs. Anti-miR-34a decreased atherosclerotic lesions and inhibited Pecam-1+ endothelial cells apoptosis in HFD-induced ApoE−/− mice. Moreover, anti-miR-34a significantly promoted cell viability, alleviated cell cycle arrest, and restrained apoptosis in ox-LDL-treated HAECs. Furthermore, Bcl-2 was identified as a target of miR-34a, and miR-34a inhibited Bcl-2 expression via binding to its 3′UTR. Rescue experiments demonstrated that Bcl-2 overexpression dramatically reversed miR-34a-mediated inhibition of cell growth and promotion of apoptosis in ox-LDL-exposed HAECs. Depletion of miR-34a facilitated endothelial cell growth and blocked apoptosis in AS by upregulating Bcl-2, offering a promising avenue for AS therapy.

Objective To investigate the role of GAS5 on cardiomyocyte apoptosis. Methods Myocardial infarction (MI) model was established by the left-anterior descending coronary artery ligation. Norepinephrine (NE) was used to induce cardiomyocyte apoptosis. GAS5 levels and mRNA expressions of Semaphorin 3a (sema3a) were measured by qRT-PCR. Protein level of sema3a was detected by Western blotting. Cardiomyocyte apoptosis was detected by flow cytometry assay. RNA pull-down and RIP assay were used to verify the combination between GAS5 and sema3a. Infarct size was measured by TTC staining. Results GAS5 expression was increased in infarct boundary zone of MI group, while sema3a protein expression was decreased. Moreover, GAS5 expression in cardiomyocyte induced by NE was higher than control group, while sema3a protein expression was lower than control group. In addition, GAS5 could negatively regulate sema3a protein expression. pcDNA-GAS5 reversed cardiomyocyte apoptosis induced by NE, while pcDNA-sema3a countered the inhibitory effect. In animal experiment, overexpression of GAS5 decreased sema3a protein expression and reduced infarct size. Conclusion GAS5 could ameliorate cardiomyocyte apoptosis induced by MI via down-regulating sema3a.

Background/Aims: Cardiac fibrosis is a major cause of diverse cardiovascular diseases. MicroRNAs have recently been proven a novel class of regulators of cardiac fibrosis. In this study, we sought to investigate the role of miR-323a-3p and its mechanisms in regulating cardiac fibrosis. Methods: The transverse aortic constriction (TAC) mice model was induced and neonatal cardiac fibroblasts (CFs) were cultured. MTT (3- [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay was used to detect the cell viability. Echocardiography was used to evaluate cardiac function. Masson’s Trichrome stain was used to evaluate the development of fibrosis. Luciferase activity assay was performed to confirm the miRNA’s binding site. Real-time PCR and Western blot were used to evaluate the level of mRNA and protein. Results: MiR-323a-3p was found up-regulated in myocardial tissues subjected to TAC and in CFs cultured with Angiotensin Ⅱ (Ang Ⅱ). Overexpression of miR-323a-3p significantly increased the mRNA levels of collagen Ⅰ, collagen Ⅲ, MMP2 and MMP9, while inhibition of miR-323a-3p prevented the proliferation, collagen production and the protein level of transforming growth factor (TGF-β) in rat neonatal CFs. Strikingly, injection of antagomiR-323a-3p elevated cardiac function and inhibited the expression of TGF-β in the TAC mice. TIMP3 was a direct target of miR-323a-3p, as the overexpression of miR-323a-3p decreased the protein and mRNA levels of TIMP3. In the CFs with pre-treatment of Ang Ⅱ, siRNA-TIMP abolished the effects of AMO-323a-3p on the inhibition of the proliferation of CFs, the down-regulation of collagen Ⅰ and collagen Ⅲ, and the expression of TGF-β. Conclusion: Our findings provide evidence that miR-323a-3p promotes cardiac fibrosis via miR-323a-3p-TIMP3-TGF-β pathway. miR-323a-3p may be a new marker for cardiac fibrosis progression and that inhibition of miR-323a-3p may be a promising therapeutic target for the treatment of cardiac fibrosis.