TRIM16 exerts protective function on myocardial ischemia/reperfusion injury through reducing pyroptosis and inflammation via NLRP3 signaling.

Myocardial infarction is still a leading cause of morbidity and mortality worldwide, but its pathogenesis has not been fully understood. In the study, we attempted to explore the effects of E3 ligase tripartite motif 16 (TRIM16) on myocardial ischemia-reperfusion (MI/R) injury in vivo and in vitro, and the underlying mechanisms. We identified that TRIM16 was indeed a potent regulator during MI/R progression in murine models and surprisingly showed a negative correlation with the concentrations of cardiac pro-inflammatory cytokines. Adenoviral vectors encoding GFP or TRIM16 (Ad-TRIM16) were subjected to mice through direct injection into the left ventricular (LV). We found that Ad-TRIM16 significantly reduced the infarct size, and improved the cardiac function and structure compared with the Ad-GFP mice after MI/R operation. More studies indicated that TRIM16 over-expression strongly meliorated nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome and associated inflammatory response in hearts of MI/R-induced mice, which were validated in hypoxia/reoxygenation (H/R)-exposed primary cardiomyocytes in vitro. In particular, MI/R operation led to cardiac pyroptosis by increasing the cleavage of Caspase-1 and Gasdermin D (GSDMD), while being considerably abrogated upon TRIM16 over-expression. Mechanistically, TRIM16 interacted with NLRP3 and promoted the K48-linked polyubiquitination of NLRP3, ultimately promoted its degradation. Together, we identified TRIM16 as a novel E3 ubiquitin ligase for NLRP3, which played an essential role in modulating its expression, and subsequently influenced inflammatory response and pyroptosis in MI/R murine model, confirming that TRIM16 may be a potential therapeutic target for myocardial infarction.

Recombinant high­mobility group box 1 induces cardiomyocyte hypertrophy by regulating the 14­3­3η, PI3K and nuclear factor of activated T cells signaling pathways.

High­mobility group box 1 (HMGB1) is released by necrotic cells and serves an important role in cardiovascular pathology. However, the effects of HMGB1 in cardiomyocyte hypertrophy remain unclear. Therefore, the aim of the present study was to investigate the potential role of HMGB1 in cardiomyocyte hypertrophy and the underlying mechanisms of its action. Neonatal mouse cardiomyocytes (NMCs) were co­cultured with recombinant HMGB1 (rHMGB1). Wortmannin was used to inhibit PI3K activity in cardiomyocytes. Subsequently, atrial natriuretic peptide (ANP), 14­3­3 and phosphorylated­Akt (p­Akt) protein levels were detected using western blot analysis. In addition, nuclear factor of activated T cells 3 (NFAT3) protein levels were measured by western blot analysis and observed in NMCs under a confocal microscope. The results revealed that rHMGB1 increased ANP and p­Akt, and decreased 14­3­3η protein levels. Furthermore, wortmannin abrogated the effects of rHMGB1 on ANP, 14­3­3η and p­Akt protein levels. In addition, rHMGB1 induced nuclear translocation of NFAT3, which was also inhibited by wortmannin pretreatment. The results of this study suggest that rHMGB1 induces cardiac hypertrophy by regulating the 14­3­3η/PI3K/Akt/NFAT3 signaling pathway.

MiR-223/NFAT5 signaling suppresses arterial smooth muscle cell proliferation and motility in vitro.

Aberrant proliferation and migration of vascular smooth muscle cells contributes to cardiovascular diseases (CVDs), including atherosclerosis. MicroRNA-223 (miR-223) protects against atherosclerotic CVDs. We investigated the contribution of miR-223 to platelet-derived growth factor-BB (PDGF-BB)-induced proliferation and migration of human aortic smooth muscle cells (HASMCs). We found that miR-223 was downregulated in PDGF-BB-treated HASMCs in a dose- and time-dependent manner, while nuclear factor of activated T cells 5 (NFAT5) was upregulated. Gain- and loss-of-function studies demonstrated that miR-223 treatment reduced PDGF-BB-induced HASMC proliferation and motility, whereas miR-223 inhibitor enhanced these processes. Moreover, NFAT5 was identified as a direct target of miR-223 in HASMC. The inhibitory effects of miR-223 on HASMC proliferation and migration were partly rescued by NFAT5 restoration. Overall, these findings suggest that miR-223 inhibits the PDGF-BB-induced proliferation and motility of HASMCs by targeting NFAT5 and that miR-223 and NFAT5 may be potential therapeutic targets for atherosclerosis.

Simvastatin Protects Heart from Pressure Overload Injury by Inhibiting Excessive Autophagy.

Cardiac hypertrophy is an independent predictor of cardiovascular morbidity and mortality. To identify the mechanisms by which simvastatin inhibits cardiac hypertrophy induced by pressure overload, we determined effects of simvastatin on 14-3-3 protein expression and autophagic activity. Simvastatin was administered intragastrically to Sprague-Dawley (SD) rats before abdominal aortic banding (AAB). Neonatal rat cardiomyocytes (NRCs) were treated with simvastatin before angiotensin II (AngII) stimulation. 14-3-3, LC3, and p62 protein levels were determined by western blot. Autophagy was also measured by the double-labeled red fluorescent protein-green fluorescent protein autophagy reporter system. Simvastatin alleviated excessive autophagy, characterized by a high LC3II/LC3I ratio and low level of p62, and blunted cardiac hypertrophy while increasing 14-3-3 protein expression in rats that had undergone AAB. In addition, it increased 14-3-3 expression and inhibited excessive autophagy in NRCs exposed to AngII. Our study demonstrated that simvastatin may inhibit excessive autophagy, increase 14-3-3 expression, and finally exert beneficial effects on cardioprotection against pressure overload.

Cilostazol suppresses angiotensin II-induced apoptosis in endothelial cells.

Patients with essential hypertension undergo endothelial dysfunction, particularly in the conduit arteries. Cilostazol, a type III phosphodiesterase inhibitor, serves a role in the inhibition of platelet aggregation and it is widely used in the treatment of peripheral vascular diseases. Previous studies have suggested that cilostazol suppresses endothelial dysfunction; however, it remains unknown whether cilostazol protects the endothelial function in essential hypertension. The aim of the present study was to investigate whether, and how, cilostazol suppresses angiotensin II (angII)­induced endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) and Sprague Dawley rats were exposed to angII and treated with cilostazol. Endothelial cell apoptosis and function, nitric oxide and superoxide production, phosphorylation (p) of Akt, and caspase­3 protein expression levels were investigated. AngII exposure resulted in the apoptosis of endothelial cells in vitro and in vivo. In vitro, cilostazol significantly suppressed the angII­induced apoptosis of HUVECs; however, this effect was reduced in the presence of LY294002, a phosphoinositide 3 kinase (PI3K) inhibitor. Furthermore, cilostazol suppressed the angII­induced p­Akt downregulation and cleaved caspase­3 upregulation. These effects were also alleviated by LY294002. In vivo, cilostazol suppressed the angII­induced endothelial cell apoptosis and dysfunction. Cilostazol was also demonstrated to partially reduced the angII­induced increase in superoxide production. The results of the present study suggested that cilostazol suppresses endothelial apoptosis and dysfunction by modulating the PI3K/Akt pathway.

High-mobility group box 1 induces calcineurin-mediated cell hypertrophy in neonatal rat ventricular myocytes.

Cardiac hypertrophy is an independent predictor of cardiovascular morbidity and mortality. In recent years, evidences suggest that high-mobility group box 1 (HMGB1) protein, an inflammatory cytokine, participates in cardiac remodeling; however, the involvement of HMGB1 in the pathogenesis of cardiac hypertrophy remains unknown. The aim of this study was to investigate whether HMGB1 is sufficient to induce cardiomyocyte hypertrophy and to identify the possible mechanisms underlying the hypertrophic response. Cardiomyocytes isolated from 1-day-old Sprague-Dawley rats were treated with recombinant HMGB1, at concentrations ranging from 50 ng/mL to 200 ng/mL. After 24 hours, cardiomyocytes were processed for the evaluation of atrial natriuretic peptide (ANP) and calcineurin A expression. Western blot and real-time RT-PCR was used to detect protein and mRNA expression levels, respectively. The activity of calcineurin was also evaluated using a biochemical enzyme assay. HMGB1 induced cardiomyocyte hypertrophy, characterized by enhanced expression of ANP, and increased protein synthesis. Meanwhile, increased calcineurin activity and calcineurin A protein expression were observed in cardiomyocytes preconditioned with HMGB1. Furthermore, cyclosporin A pretreatment partially inhibited the HMGB1-induced cardiomyocyte hypertrophy. Our findings suggest that HMGB1 leads to cardiac hypertrophy, at least in part through activating calcineurin.

Simvastatin modulates remodeling of Kv4.3 expression in rat hypertrophied cardiomyocytes.

OBJECTIVES: Hypertrophy has been shown to be associated with arrhythmias which can be caused by abnormal remodeling of the Kv4-family of transient potassium channels. Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) have recently been shown to exert pleiotropic protective effects in cardiovascular diseases, including anti-arrhythmias. It is hypothesized that remodeling of Kv4.3 occurs in rat hypertrophied cardiomyocytes and is regulated by simvastatin. METHODS: Male Sprague-Dawley rats and neonatal rat ventricular myocytes (NRVMs) underwent abdominal aortic banding (AAB) for 7 weeks and angiotensin II (AngII) treatment, respectively, to induce cardiac hypertrophy. Kv4.3 expression by NRVMs and myocardium (subepicardial and subendocardial) in the left ventricle was measured. The transient outward potassium current (I(to)) of NRVMs was recorded using a whole-cell patch-clamp method. RESULTS: Expression of the Kv4.3 transcript and protein was significantly reduced in myocardium (subepicardial and subendocardial) in the left ventricle and in NRVMs. Simvastatin partially prevented the reduction of Kv4.3 expression in NRVMs and subepicardial myocardium but not in the subendocardial myocardium. Hypertrophied NRVMs exhibited a significant reduction in the I(to) current and this effect was partially reversed by simvastatin. CONCLUSIONS: Simvastatin alleviated the reduction of Kv4.3 expression, I(to) currents in hypertrophied NRVMs and alleviated the reduced Kv4.3 expression in subepicardial myocardium from the hypertrophied left ventricle. It can be speculated that among the pleiotropic effects of simvastatin, the anti-arrhythmia effect is partly mediated by its effect on Kv4.3.