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Myocardial fibrosis, a common pathological manifestation of cardiac remodeling (CR), often leads to heart failure (HF) and even death. The underlying molecular mechanism of the role of TRIM33 in Ang II-induced myocardial fibrosis is not fully understood. We found that TRIM33 was specifically upregulated in CFs and myocardial tissue after Ang II stimulation. Adult mice induced by Ang II were used as

Sheng Zhang,1,2 Peng Wu,1 Jiabao Liu,1 Yingqiang Du,1 Zhijian Yang1 1Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, People’s Republic of China; 2Department of Cardiology, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu 213004, People’s Republic of ChinaCorrespondence: Zhijian YangDepartment of Cardiology, The First Affiliated Hospital of Nanjing Medical University, No. 300, Guangzhou Road, Gulou District, Nanjing, Jiangsu, 210029, People’s Republic of ChinaTel/Fax +86-25-52271000Email yangzhijian173@163.comBackground and Purpose: Cardiotoxicity is an important side effect of the treatment of a malignant tumor with Doxorubicin. Currently, decreasing the dosage of Doxorubicin to alleviate the side effects on cardiac function is the common method to deal with the cardiotoxicity induced by Doxorubicin. The present study aims to investigate the therapeutic effects of Roflumilast on Doxorubicin-induced inflammation and cellular senescence, as well as the potential mechanism in H9c2 myocardial cells.Methods: The injured cardiac cell model was established by incubation with 5 μmol/L Doxorubicin. MTT was used to evaluate the cell viability of treated H9c2 cardiac cells. The expression of 4-HNE was determined using an immunofluorescence assay. The gene expression levels of IL-17, IL-6, TNF-α, IL-4, PAI-1, p21, and SIRT1 were evaluated using qRT-PCR and the protein levels of Gpx4, PAI-1, p21, and SIRT1 were determined using Western blot analysis. Secretions of IL-17, IL-6, TNF-α, IL-4, CK-MB, and cTnI were measured using ELISA. Cellular senescence was assessed using SA-β-Gal staining. Si-RNA technology was used to knockdown the expression of SIRT1 in H9c2 cardiac cells.Results: Cell viability of H9c2 cardiac cells was significantly inhibited by Doxorubicin but rescued by Roflumilast. The upregulated 4-HNE and downregulated Gpx4 were reversed by Roflumilast. The secretions of IL-6 and IL-17 were promoted by Doxorubicin and suppressed by Roflumilast. The increased SA-β-Gal staining induced by Doxorubicin was inhibited by Roflumilast. P21 and PAI-1 were significantly upregulated and SIRT1 was greatly downregulated by Doxorubicin, all of which were reversed by Roflumilast. The anti-senescent effect of Roflumilast was abolished by knocking down SIRT1.Conclusion: Roflumilast might attenuate Doxorubicin-induced inflammation and cellular senescence in cardiomyocytes by upregulating SIRT1.Keywords: roflumilast, doxorubicin, SIRT1, cellular senescence, chronic heart failure

Macrophage polarization determines the transition from the inflammation phase to the inflammation resolution phase after myocardial infarction (MI). The aim of the present study was to investigate whether resveratrol (RSV) could inhibit the inflammatory mediators associated with the regulation of macrophage phenotypes and functions in MI mice. We initially discovered that RSV significantly improved cardiac function and suppressed the expression of fibrosis markers, such as collagen-I, collagen-III, and fibronectin, and pro-inflammatory cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α). RSV inhibited the expression of M1-like macrophage-related biomarkers (e.g., TNF-α and MCP-1) when bone marrow-derived macrophages (BMDMs) were stimulated with lipopolysaccharide (LPS) and interferon-γ (INF-γ). In contrast, it upregulated M2-like macrophage-related biomarkers (e.g., CD163 and Arg-1) when BMDMs were stimulated with interleukin-4 (IL-4) and interleukin-10 (IL-10). In addition, we found that RSV promoted M2-like macrophage polarization under anoxic conditions, which could be related to JAK2-SATA3 phosphorylation. In summary, RSV might promote anti-inflammatory M2-like polarization of macrophages after MI to improve cardiac function via the regulation of JAK2-SATA3 phosphorylation.

Muscone is the main active monomer of traditional Chinese medicine musk. Previous studies have reported a variety of beneficial effects of muscone. However, the effects of muscone on chronic inflammation after myocardial infarction (MI) are rarely reported. This study evaluated the anti-inflammatory effects of muscone on myocardial infarction by establishing a MI model in mice. We found that muscone remarkably decreased the levels of inflammatory cytokines (IL-1β, TNF-α and IL-6), and ultimately improved cardiac function and survival rate. Furthermore, the main anti-inflammatory effect of muscone was alleviating cardiac macrophage-mediated inflammatory response in heart tissues after MI. Bone marrow-derived macrophages (BMDMs) induced with lipopolysaccharide (LPS) were used as an

It is well recognized that the incidence of heart failure and the risk of death is high in diabetic patients after myocardial infarction (MI). Accumulating evidence showed that puerarin (PUE) has protecting function on both cardiovascular disease and diabetes. The aim of this study is to explore whether puerarin could improve cardiac function in diabetic mice after MI and the underlying mechanism. The left anterior of Streptozotocin (STZ)-Nicotinamide (NA) induced diabetic mice were ligated permanently except for the Shame group. Then the operated mice were randomly treated with PUE or saline. Cardiac function was evaluated by echocardiograph before and at 1, 2, 4 weeks after MI. GLUT4/CD36/p-Akt/PPAR α of the heart was examined after treatment for 4 weeks. The results indicated that PUE significantly increased survival rate, improved cardiac function compared with MI group. Moreover, PUE increased expression and translocation of GLUT4 while attenuated expression and translocation of CD36. Western blot analysis showed that PUE enhanced phosphorylation of Akt and decreased PPAR α. This study demonstrated that PUE improved cardiac function after MI in diabetic mice through regulation of energy metabolism, the possible mechanism responsible for the effect of PUE was increasing the expression and translocation of GLUT4 while attenuating the expression and translocation of CD36.

Objective: This study is a long-term follow-up of our previous phase I clinical trial and aims at evaluating the long-term safety and efficacy of intracoronary Ad-HGF administration for treating coronary disease. Methods: This study includes 22 patients (11 in the experiment group and 11 in the control group) with diffused and severe coronary disease who had received the optimal standardized medication therapy and was not amenable to revascularization. Intracoronary Ad-HGF gene transfer was administered to the distal part of the accessible artery by over-the wire balloon or at the ostium of the target vessels by diagnostic coronary catheter in the experiment group. Safety parameters were measured and compared between baseline and follow-ups (5-week; 12-month; 36- month) only in the experiment group. The changes of efficacy parameters (ejection fraction, EF) from baseline to 36- month follow-up (δEF) were measured in both groups and compared with each other. Results: This study confirmed the long-term safety of intracoronary Ad-HGF administration for treating severe diffuse coronary disease. All the eleven patients of the experiment group were alive after 36-months follow-up. During the follow-up, no new-onset arrhythmia was recorded; no malignant tumor was diagnosed; no paroxysmal or long-term fever was recorded; no retinal vascular anomaly was diagnosed. There were no statistically significant differences between the follow-ups and baseline in regard to blood parameters, including WBC, Hb, ALT, AST, BUN, Cr, CEA and AFP. In addition, intracoronary Ad-HGF efficiently improved echocardiographic EF at the 36-month follow-up compared to baseline (F=4.4, p=0.024) and with the control group (δEF: 3.5 ± 1.1 vs. -4.5 ± 1.3, MD: 8, p=0.0001). The medium-high dose subgroup also showed higher ECT-EF at the 36-month follow-up than baseline (MD: 4.8, p=0.017, n=8) and higher improvement of ECT-EF than the control group (δEF: 4.8 ± 1.5 vs. 0.3 ± 1.7, MD: 4.5, p=0.08). Conclusion: Intracoronary Ad-HGF administration is safe and potentially efficient in improving EF of patients with severe diffuse coronary disease in 3-year follow-up.

Background/Aims: The discovery of c-kit+ cardiac stem cells (CSCs) provided us with new therapeutic targets to repair the damaged heart. However, the precise mechanisms regulating CSC proliferation and differentiation in the aged heart remained elusive. Necroptosis, a type of regulated cell death, has recently been shown to occur following myocardial infarction (MI); however, its effect on c-kit+ CSCs remains unknown. We investigated the effects of hepatocyte growth factor (HGF) and necroptosis on the proliferation and differentiation of endogenous c-kit+ CSCs in aged rat hearts following MI. Methods: The c-kit+ CSCs and HGF/p-Met expression levels in neonatal, adult and aged rats were compared using immunofluorescence and Western blotting. Immediately after MI, adenovirus carrying the HGF gene (Ad-HGF) was injected into the left ventricular wall surrounding the infarct areas of the aged rat heart. The proliferation and differentiation of the endogenous c-kit+ CSCs were studied using immunofluorescence. The signalling pathways were analysed via Western blotting and ELISA. Results: HGF/p-Met expression levels and c-kit+ CSC abundance gradually decreased with age. Ad-HGF promoted c-kit+ CSC differentiation into precursor cells of cardiomyocyte, endothelial and smooth muscle cell lineages and enhanced cardiomyocyte proliferation and angiogenesis in aged rats; these effects were reversed by the inhibition of necroptosis. Ad-HGF administration induced necroptosis by increasing the expression of receptor interacting protein kinase (RIP) 1 and receptor interacting protein kinase (RIP) 3 proteins in the infarcted heart. Moreover, Ad-HGF-induced necroptosis increased high-mobility group box 1 protein (HMGB1) levels and enhanced the abundance of c-kit+ cells in the bone marrow, which may partly account for the beneficial effect of necroptosis on the c-kit+ CSCs. Conclusion: Ad-HGF-induced necroptosis facilitated aged heart repair after MI by promoting c-kit+ CSC proliferation and differentiation. These findings may lead to the development of new methods for the treatment of ischaemic heart disease in aged populations.

Cell death in MI is the most critical determinant of subsequent left ventricular remodeling and heart failure. Besides apoptosis, autophagy and necroptosis have been recently found to be another two regulated cell death styles. HGF has been reported to have a protective role in MI, but its impact on the three death styles remains unclear. Thus, our study was performed to investigate the distribution of autophagy, apoptosis and necroptosis in cardiac tissues after MI and explore the role and mechanism of Ad-HGF on cardiac remodeling by regulating the three death styles. We firstly showed the distribution of autophagy, apoptosis and necroptosis differs in temporal and spatial context after MI using immunofluorescence. Notably, Ad-HGF treatment improves the cardiac remodeling of SD rats following MI by preserving the heart function, reducing the scar size and aggresomes. Further mechanism study reveals Ad-HGF promotes autophagy and necroptosis and inhibits apoptosis in vivo and in vitro. Co-immunoprecipitation assays showed Ad-HGF treatment significantly decreased the binding of Bcl-2 to Beclin1 but enhanced Bcl-2 binding to Bax in H9c2 cells under hypoxia. Moreover, HGF-induced sequestration of Bax by Bcl-2 allows Bax to become inactive, thereby inhibiting apoptosis. In addition, Ad-HGF markedly increased the formation of Beclin1-Vps34-Atg14L complex, which accounted for promoting autophagy. Both the western blot and activity assay showed Ad-HGF significantly decreased the caspase 8 protein and activity levels, which obligated the cell to undergo necroptosis under hypoxia and block apoptosis. Thus, our findings offer new evidence and strategies for the treatment of MI and post-MI cardiac remodeling.

Background: Hepatocyte growth factor (HGF) is widely known as a protective factor in ischemic myocardium, however HGF sensitive cellular mechanism remained ill-defined. Autophagy at early stage of hypoxia has been demonstrated to play a role in protecting myocardium both in vivo and vitro. We performed this study to investigate the association between the protective effect of HGF and autophagy. Methods: Ventricular myocytes were isolated from neonatal rat heart (NRVMs). We evaluated cardiomyocytes apoptosis by Hoechst staining and flow cytometry. Autophagy was assessed by transmission electron microscope and mRFP-GFP-LC3 adenovirus infection. Mitochondrial membrane potential was estimated by JC-1 staining. Western blotting and ELISA assay were used to quantify protein concentrations. Results: We found that autophagy in NRVMs increased at early stage after hypoxia and HGF release was consistent with the change of autophagy. Exogenous HGF enhanced autophagy and decreased apoptosis, while neutralizing HGF yielded opposite effects. Besides, inhibition of autophagy increased apoptosis of myocytes. Furthermore, exogenous HGF induced Parkin, the marker of mitochondrial autophagy, indicating increased clearance of injured mitochondria. Conclusions: Our results revealed a potential mechanism in which exogenous HGF prevented NRVMs from apoptosis after hypoxia. Upregulation of Parkin through administration of exogenous HGF may be a potential therapeutic strategy ptotecting myocytes during ischemia.