Trimethylamine N-oxide induces osteogenic responses in human aortic valve interstitial cells in vitro and aggravates aortic valve lesions in mice.

AIMS: Recent studies have shown that the choline-derived metabolite trimethylamine N-oxide (TMAO) is a biomarker that promotes cardiovascular disease through the induction of inflammation and stress. Inflammatory responses and stress are involved in the progression of calcified aortic valve disease (CAVD). Here, we examined whether TMAO induces the osteogenic differentiation of aortic valve interstitial cells (AVICs) through endoplasmic reticulum (ER) and mitochondrial stress pathways in vitro and in vivo. METHODS AND RESULTS: Plasma TMAO levels were higher in patients with CAVD (n = 69) than in humans without CAVD (n = 263), as examined by liquid chromatography-tandem mass spectrometry. Western blot and staining probes showed that TMAO-induced an osteogenic response in human AVICs. Moreover, TMAO promoted ER stress, mitochondrial stress, and nuclear factor-κB (NF-κB) activation in vitro. Notably, the TMAO-mediated effects were reversed by the use of ER stress, mitochondrial stress, and NF-κB activation inhibitors and small interfering RNA. Mice treated with supplemental choline in a high-fat diet had markedly increased TMAO levels and aortic valve thicknesses, which were reduced by 3,3-dimethyl-1-butanol (an inhibitor of trimethylamine formation) treatment. CONCLUSIONS: Choline-derived TMAO promotes osteogenic differentiation through ER and mitochondrial stress pathways in vitro and aortic valve lesions in vivo.

[cGAS/STING signaling pathways induces the secretion of type â interferon in porcine alveolar macrophages infected with porcine circovirus type 2].

In order to study the signal pathway secreting type Ⅰ interferon in porcine alveolar macrophages (PAMs) infected with porcine circovirus type 2 (PCV2), the protein and the mRNA expression levels of cGAS/STING pathways were analyzed by ELISA, Western blotting and quantitative reverse transcriptase PCR in PAMs infected with PCV2. In addition, the roles of cGAS, STING, TBK1 and NF-κB/P65 in the generation of type I interferon (IFN-I) from PAMs were analyzed by using the cGAS and STING specific siRNA, inhibitors BX795 and BAY 11-7082. The results showed that the expression levels of IFN-I increased significantly at 48 h after infection with PCV2 (P<0.05), the mRNA expression levels of cGAS increased significantly at 48 h and 72 h after infection (P<0.01), the mRNA expression levels of STING increased significantly at 72 h after infection (P<0.01), and the mRNA expression levels of TBK1 and IRF3 increased at 48 h after infection (P<0.01). The protein expression levels of STING, TBK1 and IRF3 in PAMs infected with PCV2 were increased, the content of NF-κB/p65 was decreased, and the nuclear entry of NF-κB/p65 and IRF3 was promoted. After knocking down cGAS or STING expression by siRNA, the expression level of IFN-I was significantly decreased after PCV2 infection for 48 h (P<0.01). BX795 and BAY 11-7082 inhibitors were used to inhibit the expression of IRF3 and NF-κB, the concentration of IFN-I in BX795-treated group was significantly reduced than that of the PCV2 group (P<0.01), while no significant difference was observed between the BAY 11-7028 group and the PCV2 group. The results showed that PAMs infected with PCV2 induced IFN-I secretion through the cGAS/STING/TBK1/IRF3 signaling pathway.

Iron deficiency is an independent risk factor of increased myocardial energy expenditure in chronic heart failure patients.

BACKGROUND: Chronic heart failure (CHF) is a major public health burden and is associated with high morbidity, mortality, and cost. Recent studies demonstrated iron metabolism and myocardial energy metabolism were altered in CHF patients. In this study, we aimed to analyze the effects and correlations of iron metabolism on myocardial energy metabolism in CHF. METHODS: One hundred and thirty patients with CHF [age: 66.2±11.5 years, males: 58.5% and New York Heart Association (NYHA) class (II/III/IV): 67/43/20] were included. Serum concentrations of ferritin, transferrin saturation (Tsat), and soluble transferrin receptor (sTfR) were quantified as the indexes of iron metabolism, and echocardiography was used to assess myocardial energy expenditure (MEE) levels. Iron deficiency (ID) was defined as ferritin <100 or 100-300 µg/L with Tsat <20%. RESULTS: Patients with CHF were divided into two groups based on iron status. The prevalence of ID in CHF was 36.9%, and increased with the severity of CHF, reaching 80.0% in those with NYHA class IV (NYHA class II/III/IV: 17.9% vs. 46.5% vs. 80.0%, P=0.000). The demographic characteristics [age, sex, body mass index (BMI), blood pressure, and heart rate] and hemoglobin (HGB) concentrations in two groups were similar (all P>0.05). MEE was significantly higher in the ID group (92.7±23.0 vs. 65.6±20.8 cal/min, P=0.000), while NYHA classes II and III was significantly higher in the ID group (71.6±16.4 vs. 60.3±14.8 cal/min, P=0.022; 88.9±10.4 vs. 69.1±20.1 cal/min, P=0.000). In univariable linear regression models, the presence of ID, higher NYHA class, increased N-terminal pro-B-type natriuretic peptide (NT-proBNP), sTfR, left ventricular internal diastolic diameter (LVIDd), as well as reduced ferritin, Tsat levels, and lower left ventricular ejection fraction (LVEF) were associated with elevated MEE levels (all P<0.05). In multivariable regression models, the presence of ID, reduced Tsat. and increased sTfR remained independent predictors of elevated MEE levels after adjustment for all variables that showed a significant association with MEE (all P<0.05). CONCLUSIONS: The prevalence of ID is high in CHF and is associated with the severity of cardiac dysfunction. The presence of ID as well as reduced Tsat and increased sTfR concentrations are associated with elevated MEE levels in CHF.

A genetic variation in CHI3L1 is associated with bronchial asthma.

BACKGROUND: This study was aimed to investigate the associations among Chitinase 3-like 1 (CHI3L1) polymorphisms, asthma and plasma YKL-40 levels in Chinese population. MATERIAL AND METHODS: Four CHI3L1 single nucleotide polymorphisms (SNPs) were genotyped. The YKL-40 level in plasma and eosinophil percentage in peripheral blood were quantified. RESULTS: A SNP (rs4950928) in the CHI3L1 promoter was associated with elevated plasma YKL-40 levels (p = .02), asthma (p = .042) and lung function (p = .029 to .002) in this Chinese population. Plasma YKL-40 levels were significantly elevated in patients with asthma compared to those in control subjects (p < .05). Plasma YKL-40 levels were significantly correlated with forced expiratory volume per cent (FEV1%) measurements (p < .05). Although plasma YKL-40 levels were decreased after treatment, the correlation with FEV1% still existed. CONCLUSIONS: CHI3L1 locus is a risk factor for asthma in Chinese population.

Association of Urine Albumin/Creatinine Ratio below 30 mg/g and Left Ventricular Hypertrophy in Patients with Type 2 Diabetes.

Several studies show that even a level of urine albumin/creatinine ratio (UACR) within the normal range (below 30 mg/g) increases the risk of cardiovascular diseases. We speculate that mildly increased UACR is related to left ventricular hypertrophy (LVH) in patients with type 2 diabetes mellitus (T2DM). In this retrospective study, 317 patients with diabetes with normal UACR, of whom 62 had LVH, were included. The associations between UACR and laboratory indicators, as well as LVH, were examined using multivariate linear regression and logistic regression, respectively. The diagnostic efficiency and the optimal cutoff point of UACR for LVH were evaluated using the area under the receiver operating characteristic curve (AUC) and Youden index. Our results showed that patients with LVH had significantly higher UACR than those without LVH (P < 0.001). The prevalence of LVH presented an upward trend with the elevation of UACR. UACR was independently and positively associated with hemoglobin A1c (P < 0.001). UACR can differentiate LVH (AUC = 0.682, 95% CI (0.602-0.760), P < 0.001). The optimal cutoff point determined with the Youden index was UACR = 10.2 mg/g. When categorized by this cutoff point, the odds ratio (OR) for LVH in patients in the higher UACR group (10.2-30 mg/g) was 3.104 (95% CI: 1.557-6.188, P=0.001) compared with patients in the lower UACR group (<10.2 mg/g). When UACR was analyzed as a continuous variable, every double of increased UACR, the OR for LVH was 1.511 (95% CI: 1.047-2.180, P=0.028). Overall, UACR below 30 mg/g is associated with LVH in patients with T2DM. The optimal cutoff value of UACR for identifying LVH in diabetes is 10 mg/g.

SULT2B1b inhibits reverse cholesterol transport and promotes cholesterol accumulation and inflammation in lymphocytes from AMI patients with low LDL-C levels.

The current main treatment for coronary artery disease (CAD) is to reduce low-density lipoprotein cholesterol (LDL-C) by statins, which could decrease the incidence of major adverse cardiovascular events (MACEs) by 30%. However, many residual risks still remain. To clarify the mechanism involved, we studied patients with acute myocardial infarction (AMI) with low LDL-C levels. Lymphocytes were isolated, and it was found that despite no difference in plasma LDL-C level, the lymphocyte cholesterol content was higher in AMI patient than those in non-CAD patients; thus, the decrease in intracellular cholesterol content was inconsistent with that in the plasma. Additionally, [3H]-cholesterol efflux rates were lower and mRNA levels of the inflammatory factors tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) higher in AMI lymphocytes. It was found that sulphotransferase 2B1b (SULT2B1b) expression was higher in AMI lymphocytes. Further research using Jurkat T lymphocytes confirmed that SULT2B1b knockdown increased cholesterol efflux capacity and decreased mRNA levels of TNF-α and IFN-γ by increasing liver X receptor (LXR)-ß levels. Furthermore, the degree of CpG island methylation in the SULT2B1b promoter was reduced in cells from AMI patients. In conclusion, SULT2B1b up-regulation due to hypomethylation of its promoter promotes cholesterol accumulation and inflammation by inhibiting LXR-ß in lymphocytes of AMI patients with low LDL-C levels. Therefore, reducing intracellular cholesterol is also important as plasma cholesterol levels. Therapeutic approaches to decrease SULT2B1b expression might be potentially beneficial for CAD prevention by decreasing intracellular cholesterol.

LCK inhibitor attenuates atherosclerosis in ApoE-/- mice via regulating T cell differentiation and reverse cholesterol transport.

Lots of studies demonstrated that CD4+ T cells regulate the development of atherosclerosis (AS). Previously, we reported that LCK, a key molecule in activation of T cell receptor (TCR) signalling and T cells, adversely affects reverse cholesterol transport (RCT), which ameliorates AS in vitro. To investigate the effect of LCK on AS in vivo, we injected the LCK inhibitor, PP2, into ApoE-/- mice fed a chow diet or a high-fat diet (HFD). Although, AS plaques were not affected by PP2 in chow diet-fed mice, PP2 significantly reduced the lesion percentage and necrotic core areas in HFD-fed mice. We further analysed the plaque contents and found that the accumulation of lipids and macrophages were decreased, while the contents of collagen and smooth muscle cells were increased by the LCK inhibitor. Thus, inhibiting LCK enhanced the plaque stability. We also found the LCK inhibitor improved cholesterol efflux capacity of HDL and up-regulated RCT regulatory proteins in the spleen. Moreover, inhibiting LCK regulated differentiation of T cells by increasing regulatory T (Treg) cells and decreasing the number of T helper 1 (Th1) cells in the aorta, thymus and spleen. Consistent with these results, infiltration of CD4+ T cells in plaques, secretion of pro-atherosclerotic cytokines, INF-γ and TNF-α synthesized mostly by Th1 cells, and the activation of PI3K/AKT/mTOR signalling were inhibited by the LCK inhibitor. Moreover, the effect of LCK inhibitor on the ratio of Th1 to Treg cells were compromised by activation of mTOR. Together, these data indicate that inhibiting LCK in TCR signalling attenuated the development of AS and promoted plaque stability. Improving RCT by upregulating RCT regulatory proteins and decreasing the Th1/Treg ratio by inhibiting PI3K/AKT/mTOR signalling may contribute to the anti-atherosclerotic effects of LCK inhibition.

Ferulic acid increases intestinal Lactobacillus and improves cardiac function in TAC mice.

Ferulic acid, a main ingredient of Ligusticum, exhibits anti-oxidant and anti-inflammation effects in heart diseases. Some studies indicate that gut microbiome is associated with the generation of ferulic acid. Whether the beneficial effect of ferulic is raised by the alteration of gut microbiota is still unknown. This study examined the effect of sodium ferulate on gut microbiome and cardiac function in TAC mice. Cell Counting Kit-8 (CCK8) assay verified that ferulic acid has low toxicity in vitro and that ferulic acid inhibited the up-regulation of ß-MHC and ANP induced by Angiotensin II. In addition, daily supplement of 50 mg/kg sodium ferulate improved the ejection fraction and changed the gut microbiota composition of TAC mice. Relative abundance of Lactobacillus and Parabacteroides are increased in TAC mice gavaged with sodium ferulate. In addition, Lactobacillus is negatively correlated with HW/BW and LW/BW ratio. These results suggest that the beneficial effect of ferulic in TAC mice is probably through the regulation of gut microbiota.

A CRM1 Inhibitor Alleviates Cardiac Hypertrophy and Increases the Nuclear Distribution of NT-PGC-1α in NRVMs.

Chromosomal maintenance 1 (CRM1) inhibitors display antihypertrophic effects and control protein trafficking between the nucleus and the cytoplasm. PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1alpha) is a type of transcriptional coactivator that predominantly resides in the nucleus and is downregulated during heart failure. NT-PGC-1α is an alternative splicing variant of PGC-1α that is primarily distributed in the cytoplasm. We hypothesized that the use of a CRM1 inhibitor could shuttle NT-PGC-1α into the nucleus and activate PGC-1α target genes to potentially improve cardiac function in a mouse model of myocardial infarction (MI). We showed that PGC-1α and NT-PGC-1α were decreased in MI-induced heart failure mice. Phenylephrine and angiotensin II were applied to induce hypertrophy in neonatal rat ventricular myocytes (NRVMs). The antihypertrophic effects of the CRM1-inhibitor Selinexor was verified through profiling the expression of ß-MHC and through visualizing the cell cross-sectional area. NRVMs were transfected with adenovirus-NT-PGC-1α or adenovirus-NLS (nucleus localization sequence)-NT-PGC-1α and then exposed to Selinexor. Confocal microscopy was then used to observe the shuttling of NT-PGC-1α. After NT-PGC-1α was shuttled into the nucleus, there was increased expression of its related genes, including PPAR-α, Tfam, ERR-γ, CPT1b, PDK4, and Nrf2. The effects of Selinexor on post-MI C57BL/6j mice were determined by echocardiography and qPCR. We found that Selinexor showed antihypertrophic effects but did not influence the ejection fraction of MI-mice. Interestingly, the antihypertrophic effects of Selinexor might be independent of NT-PGC-1α transportation.

Mitofusin 2 Participates in Mitophagy and Mitochondrial Fusion Against Angiotensin II-Induced Cardiomyocyte Injury.

BACKGROUND: Mitochondrial dynamics play a critical role in mitochondrial function. The mitofusin 2 (MFN2) gene encodes a mitochondrial membrane protein that participates in mitochondrial fusion to maintain and operate the mitochondrial network. Moreover, MFN2 is essential for mitophagy. In Ang II-induced cardiac remodeling, the combined effects of MFN2-mediated mitochondrial fusion and mitophagy are unclear. This study was designed to explore a novel strategy for preventing cardiomyocyte injury via modulation of mitochondrial dynamics. METHODS: We studied the function of MFN2 in mitochondrial fusion and mitophagy in Ang II-stimulated cardiomyocyte injury. Cardiomyocyte injury experiments, including reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), and apoptosis rate of cardiomyocytes were performed. The mitochondrial morphology in cardiomyocytes was examined via transmission electron microscopy (TEM) and confocal microscopy. Autophagic levels in response to Ang II were examined by immunoblotting of autophagy-related proteins. Moreover, PINK1/MFN2/Parkin pathway-related proteins were examined. RESULTS: With stimulation by Ang II, MFN2 expression was progressively reduced. MFN2 deficiency impaired mitochondrial quality, resulting in exacerbated mitochondrial damage induced by Ang II. The Ang II-induced increases in ROS production and apoptosis rate were alleviated by MFN2 overexpression. Moreover, MFN2 alleviated the Ang II-induced reduction in MMP. MFN2 promoted mitochondrial fusion, and MFN2 promoted Parkin translocation and phosphorylation, leading to mitochondrial autophagy. The effects of MFN2 overexpression were reversed by autophagy inhibitors. CONCLUSION: Mitofusin 2 promotes Parkin translocation and phosphorylation, leading to mitophagy to clear damaged mitochondria. However, the beneficial effects of MFN2 were reversed by autophagy inhibitors. Additionally, MFN2 participates in mitochondrial fusion to maintain mitochondrial quality. Thus, MFN2 participated in mitophagy and mitochondrial fusion against Ang II-induced cardiomyocyte injury.

Association between admission plasma 2-oxoglutarate levels and short-term outcomes in patients with acute heart failure: a prospective cohort study.

BACKGROUND: 2-oxoglutarate (2OG), an intermediate metabolite in the tricarboxylic acid cycle, has been found to associate with chronic heart failure (HF), but its effect on short-term adverse outcomes in patients with acute HF (AHF) is uncertain. METHODS: This prospective cohort study included 411 consecutive hospitalized patients with AHF. During hospitalization, fasting plasma samples were collected within the first 24 h of admission. Plasma 2OG levels were measured by hydrophilic interaction liquid chromatography-liquid chromatography tandem mass spectrometry (HILIC-LC/MS/MS). All participants were followed up for six months. Multiple logistic regression was used to determine the odds ratio (OR) and 95% confidence interval (CI) for primary outcomes. RESULTS: The AHF cohort consisted of HF with preserved ejection fraction (EF) (64.7%), mid-range EF (16.1%), and reduced EF (19.2%), the mean age was 65 (±13) years, and 65.2% were male. Participants were divided into two groups based on median 2OG levels (µg/ml): low group (< 6.0, n = 205) and high group (≥6.0, n = 206). There was a relatively modest correlation between 2OG and N-terminal pro B-type natriuretic peptide (NT-proBNP) levels (r = 0.25; p < 0.001). After adjusting for age, sex, and body mass index, we found that the progression of the NYHA classification was associated with a gradual increase in plasma 2OG levels (p for trend< 0.001). After six months of follow-up, 76 (18.5%) events were identified. A high baseline 2OG level was positively associated with a short-term rehospitalization and all-cause mortality (OR: 2.2, 95% CI 1.3-3.7, p = 0.003), even after adjusting for NT-proBNP and estimated glomerular filtration rate (eGFR) (OR: 1.9, 95% CI 1.1-3.4, p = 0.032). After a similar multivariable adjustment, the OR was 1.4 (95% CI 1.1-1.7, p = 0.018) for a per-SD increase in 2OG level. CONCLUSIONS: High baseline 2OG levels are associated with adverse short-term outcomes in patients with AHF independent of NT-proBNP and eGFR. Hence plasma 2OG measurements may be helpful for risk stratification and treatment monitoring in AHF. TRIAL REGISTRATION: ChiCTR-ROC-17011240 . Registered 25 April 2017.

The intestinal microbiota associated with cardiac valve calcification differs from that of coronary artery disease.

BACKGROUND AND AIMS: Although most risk factors for cardiac valve calcification (VC) are similar to those for coronary artery disease (CAD), they differ regarding lesions and clinical symptoms. Recently, increasing evidence suggests that intestinal bacteria play essential roles in cardiovascular disease (CVD). It is plausible that the gut microbiota is linked to the occurrence of different CVDs under similar risk factors. Thus, we aimed to explore the gut microbiomes in patients with VC or CAD and determine their underlying connections. METHODS: We collected samples from 119 subjects and performed 16S rRNA gene sequencing to analyze the gut microbiomes in VC and CAD patients and in control volunteers. RESULTS: The gut microbiomes of VC and CAD patients were significantly different in terms of beta-diversity. Bacteria from Veillonella dispar, Bacteroides plebeius and Fusobacterium were enriched in the VC group, while members of Collinsella aerofaciens, Megamonas, Enterococcus, Megasphaera, Dorea and Blautia were decreased. According to the association with dyslipidemia, seven operational taxonomic units (OTUs), including Parabacteroides distasonis, Megamonas, Fusobacterium, Bacteroides sp., Bacteroides plebeius, Lactobacillus and Prevotella copri, were regarded as potential pathogens for CVDs. Additionally, Prevotella copri might be a keystone of CVDs, especially in VC patients, while Collinsella aerofaciens is a possible keystone of CAD, based on the multi-correlations of these bacteria with other OTUs in microbial communities. CONCLUSIONS: Patients with VC and CAD suffer from different gut microbial dysbiosis. The gut microbiomes are associated with the clinical characteristics in these diseases and might be potential therapeutic targets.

ZAP70 deficiency promotes reverse cholesterol transport through MAPK/ERK pathway in Jurkat cell.

BACKGROUND: Lots of studies have demonstrated that immune cells could regulate reverse cholesterol transport (RCT). However, neither T cell receptor (TCR) signalling nor Zeta-chain associated protein 70 (ZAP70) have been demonstrated to be associated with RCT. To investigate this association, we used a ZAP70-deficient Jurkat-derived mutant, P116 cell line, to detect the effect of ZAP70 on RCT and inflammatory response. ZAP70 deficiency improved cholesterol efflux capacity by 14%. Meanwhile, mRNA and proteins expression of RCT regulatory proteins such as ABCA1, ABCG1 and SR-BI were increased in P116 cells. ZAP70-deficiency had no influence on LXR-α and PPAR-γ. Regarding the inflammatory response, the mRNA expression and secretion of pro-atherosclerotic cytokines, TNF-α, IFN-γ, IL-2 and IL-6, were significantly decreased in the ZAP70-deficient cell line. Activation of MAP kinases cascades, as determined by of ERK, JNK and p38 MAPK phosphorylation, were found to be inhibited in the absence of ZAP70. Specific inhibition of ERK, JNK and p38 MAPK activity was also found to decreased TNF-α, IFN-γ, and IL-6 secretion. However, only the ERK inhibition was observed to reduce IL-2 secretion, improve cholesterol efflux capacity and increase expression of ABCA1, ABCG1 and SR-BI without increasing LXR-α and PPAR-γ. Using ChIP assay to detect the binding of LXR-α to LXRE, which promotes the expression of ABCG1, we found that inhibiting ERK improved binding without increasing LXR-α levels. Thus, we speculate that ZAP70-deficiency may improve RCT and decrease the inflammatory response of T cells. Furthermore, these effects are probably achieved via ERK signalling pathway.

Serum Peroxisome Proliferator-activated Receptor Gamma Coactivator-1α Related to Myocardial Energy Expenditure in Patients With Chronic Heart Failure.

BACKGROUND: Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) plays key roles in controlling cardiac metabolism and function. Myocardial energy expenditure (MEE) can reflect myocardial energy metabolism and cardiac function. Whether the variation of PGC-1α can influence MEE levels in chronic heart failure (CHF) is unclear. Therefore, we investigated the relationship between PGC-1α and MEE. MATERIAL AND METHODS: We studied 219 patients with CHF and 66 healthy controls. MEE was measured according to echocardiographic parameters. Serum PGC-1α, N-terminal pro-B-type natriuretic peptide and other parameters were detected. Patients with CHF were divided into different groups according to the left ventricular ejection fraction (LVEF) and the tertile range of MEE. RESULTS: Serum PGC-1α was lower in the MEE 2 and 3 groups compared with controls (both P < 0.05). Patients in the MEE 2 (1.73 ± 0.83 versus 2.16 ± 0.82 ng/mL, P = 0.001) and 3 groups (1.65 ± 0.73 versus 2.16 ± 0.82 ng/mL, P < 0.001) possessed lower levels of PGC-1α than those in the MEE 1 group. Compared with high LVEF, patients with low LVEF had higher MEE (median, 167 versus 73 cal/minute, P < 0.05) and lower PGC-1α (1.71 ± 0.65 versus 1.95 ± 0.91 ng/mL, P = 0.032). Multivariate logistic regression analysis showed that MEE (OR = 0.517, 95% CI = 0.267-0.998, P = 0.049) and creatinine (OR = 2.704, 95% CI = 1.144-6.391, P = 0.023) were independently associated with increased PGC-1α. CONCLUSIONS: Serum PGC-1α was related to MEE and LVEF in patients with CHF and can reflect the degree of MEE and the systolic function of the left ventricle.

Metformin Increases Cardiac Rupture After Myocardial Infarction via the AMPK-MTOR/PGC-1α Signaling Pathway in Rats with Acute Myocardial Infarction.

BACKGROUND Cardiac rupture often occurs after acute myocardial infarction due to complex and unclear pathogenesis. This study investigated whether metformin increases the incidence of cardiac rupture after myocardial infarction through the AMPK-MTOR/PGC-1α signaling pathway. MATERIAL AND METHODS An acute myocardial infarction (MI) mouse model was established. A series of experiments involving RT-qPCR, Western blot, TUNEL staining, and Masson staining were performed in this study. RESULTS Myocardial infarction occurred, resulting in the cardiac rupture, and the expression level of PGC-1α increased in the cardiac myocardium. Meanwhile, the proportion of myocardial NT-PGC-1α/PGC-1α decreased. The expression level of myocardial PGC-1α in MI mice with cardiac rupture after MI was significantly higher than that in the mice without cardiac rupture, and the ratio of myocardial NT-PGC-1α/PGC-1α was low. In addition, increasing the dose of metformin significantly increased the incidence of cardiac rupture after myocardial infarction in MI mice. High-dose metformin caused cardiac rupture in MI mice. Moreover, high-dose metformin (Met 2.0 nM) reduces the proportion of NT-PGC-1α/PGC-1α in primary cardiomyocytes of SD mice (SD-NRVCs [Neonatal rat ventricular cardiomyocytes]), and its effect was inhibited by Compound C (AMPK inhibitor). Further, after 3 days of treatment with high-dose metformin in MI mice, myocardial fibrin synthesis decreased and fibrosis was significantly inhibited. Meanwhile, cardiomyocyte apoptosis increased significantly. With the increase in metformin concentration, the expression level of myocardial LC3b gradually increased in MI mice, suggesting that metformin enhances the autophagy of cardiomyocytes. CONCLUSIONS These results suggest that metformin increases cardiac rupture after myocardial infarction through the AMPK-MTOR/PGC-1α signaling pathway.

N­terminal truncated peroxisome proliferator­activated receptor­Î³ coactivator­1α alleviates phenylephrine­induced mitochondrial dysfunction and decreases lipid droplet accumulation in neonatal rat cardiomyocytes.

N­terminal truncated peroxisome proliferator­activated receptor­Î³ coactivator­1α (NT­PGC­1α) is an alternative splice variant of PGC­1α. NT­PGC­1α exhibits stronger anti­obesity effects in adipose tissue than PGC­1α; however, NT­PGC­1α has not yet been investigated in neonatal rat cardiomyocytes (NRCMs). The present study aimed to investigate the role of NT­PGC­1α in mitochondrial fatty acid metabolism and its possible regulatory mechanism in NRCMs. NRCMs were exposed to phenylephrine (PE) or angiotensin II (Ang II) to induce cardiac hypertrophy. Following this, NRCMs were infected with adenovirus expressing NT­PGC­1α, and adenosine 5'­triphsophate (ATP) levels, reactive oxygen species (ROS) generation and mitochondrial membrane potential were subsequently detected. In addition, western blotting, lipid droplet staining and oxygen consumption assays were performed to examine the function of NT­PGC­1α in fatty acid metabolism. NT­PGC­1α was demonstrated to be primarily expressed in the cytoplasm, which differed from full­length PGC­1α, which was predominantly expressed in the nucleus. NT­PGC­1α overexpression alleviated mitochondrial function impairment, including ATP generation, ROS production and mitochondrial membrane potential integrity. Furthermore, NT­PGC­1α overexpression alleviated the PE­induced suppression of fatty acid metabolism­associated protein expression, increased extracellular oxygen consumption and decreased lipid droplet accumulation in NRCMs. Taken together, the present study demonstrated that NT­PGC­1α alleviated PE­induced mitochondrial impairment and decreased lipid droplet accumulation in NRCMs, indicating that NT­PGC­1α may have ameliorated mitochondrial energy defects in NRCMs, and may be considered as a potential target for the treatment of heart failure.

Serum amyloid P component therapeutically attenuates atherosclerosis in mice via its effects on macrophages.

Background: A hallmark of atherosclerosis is the formation of macrophage-derived foam cells. Serum amyloid P component (SAP), a member of the pentraxin family of proteins, is known to affect macrophage activation. However, the role of SAP in atherosclerosis is still unclear. Methods: Apolipoprotein E-deficient (Apoe-/-) mice fed a high-fat diet were given intraperitoneal injections of SAP (6 mg/kg) every other day for a total of 2 weeks to characterize atherosclerosis development. Results: We showed that intraperitoneal injection of SAP attenuated atherosclerosis in Apoe-/- mice. Immunostaining of aortic roots indicated that SAP was up-taken by the lesion area. In SAP-treated mice, serum paraoxonase1 (PON1) activity was increased whereas high-density lipoprotein inflammatory index (HII) was reduced. The cholesterol efflux rate in macrophages was elevated along with the expression of cholesterol efflux proteins. Through bioinformatics analysis followed by experimental validation, we found that proline/serine-rich coiled-coil protein 1 (Psrc1) was an important downstream effector of SAP in macrophages. Conclusions: Our findings reveal an anti-atherosclerotic role of SAP and extend the current knowledge regarding this molecule as a marker for atherosclerosis.

PTEN induced putative kinase 1 (PINK1) alleviates angiotensin II-induced cardiac injury by ameliorating mitochondrial dysfunction.

BACKGROUND: Mitochondrial quality control is crucial to the development of angiotensin II (AngII)-induced cardiac hypertrophy. PTEN induced putative kinase 1 (PINK1) is rapidly degraded in normal mitochondria but accumulates in damaged mitochondria, triggering autophagy to protect cells. PINK1 mediates mitophagy in general, but whether PINK1 mediates AngII-induced mitophagy and the effects of PINK1 on AngII-induced injury are unknown. This study was designed to investigate the function of PINK1 in an AngII stimulation model and its regulation of AngII-induced mitophagy. METHODS: We studied the function of PINK1 in mitochondrial homeostasis in AngII-stimulated cardiomyocytes via RNA interference-mediated knockdown and adenovirus-mediated overexpression of the PINK1 protein. Mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production, adenosine triphosphate (ATP) content, cell apoptosis rates and cardiomyocyte hypertrophy were measured. The expression of LC3B, Beclin1 and p62 was measured. Mitochondrial morphology was examined via electron microscopy. Mitophagy was detected by confocal microscopy based on the co-localization of lysosomes and mitochondria. Additionally, endogenous PINK1, phosphorylated PINK1, mito-PINK1, total Parkin, cyto-Parkin, mito-Parkin and phosphorylated Parkin protein levels were measured. RESULTS: Cardiomyocytes untreated by AngII had very low levels of total and phosphorylated PINK1. However, in the AngII stimulation model, the MMP was decreased, and the levels of total and phosphorylated PINK1 were increased. After PINK1 was knocked down, Parkin translocation to the mitochondria was inhibited. Moreover, levels of phosphorylated Parkin were reduced, and autophagy markers were downregulated. MMP and ATP contents were further reduced, ROS production and the apoptotic rate were further increased, and myocardial hypertrophy was further aggravated compared with those in the AngII group. However, PINK1 overexpression promoted Parkin translocation and phosphorylation, autophagy markers were upregulated, and myocardial injury was reduced. In addition, the effects of PINK1 overexpression were reversed by autophagy inhibitors. CONCLUSION: Decreased MMP induced by AngII maintains the stability of PINK1, causing PINK1 autophosphorylation. PINK1 activation promotes Parkin translocation and phosphorylation and increases autophagy to clear damaged mitochondria. Thus, PINK1/Parkin-mediated mitophagy has a compensatory, protective role in AngII-induced cytotoxicity.

Contraction-dependent TGF-ß1 activation is required for thrombin-induced remodeling in human airway smooth muscle cells.

AIMS: Thrombin is a serine proteinase that is not only involved in coagulation cascade, but also mediates a number of biological responses relevant to tissues repair, and induces bronchoconstriction. TGF-ß plays a pivotal role in airway remodeling due to its effects on airway smooth muscle proliferation and extracellular matrix (ECM) deposition. Recently, bronchoconstriction itself is found to constitute a form of strain and is highly relevant to asthmatic airway remodeling. However, the underlying mechanisms remain unknown. Here, we investigated the role of contraction- dependent TGF-ß activation in thrombin-induced remodeling in human airway smooth muscle (HASM) cells. MATERIALS AND METHODS: Primary HASM cells were treated with or without thrombin in the absence or presence of anti-TGF-ß antibody, cytochalasin D and formoterol. CFSE labeling index or CCK-8 assay were performed to test cell proliferation. RT-PCR and Western blotting were used to examined ECM mRNA level and collagen Iα1, α-actin protein expression, respectively. Immunofluorescence was also used to confirm contraction induced by thrombin in HASM cells. KEY FINDING: Thrombin stimulation enhanced HASM cells proliferation and activated TGF-ß signaling. Thrombin induced ECM mRNA and collagen Iα1 protein expression, and these effects are mediated by TGF-ß. Abrogation of TGF-ß activation by contraction inhibitors cytochalasin D and formoterol prevents the thrombin-induced effects. SIGNIFICANCE: These findings suggest that contraction-dependent TGF-ß activation could be a mechanism by which thrombin leads to the development of asthmatic airway remodeling. Blocking physical forces with bronchodilator would be an intriguing way in reducing airway remodeling in asthma.

PSRC1 overexpression attenuates atherosclerosis progression in apoE-/- mice by modulating cholesterol transportation and inflammation.

AIMS: Human genome-wide association studies (GWAS) have found that proline/serine-rich coiled-coil 1 (PSRC1) encodes a protein that is associated with serum lipid levels and coronary artery disease. In addition, our previous study showed that the cholesterol efflux capacity is decreased in macrophages following a treatment silencing Psrc1, indicating that PSRC1 has anti-atherosclerotic effects. However, the role of PSRC1 in the development of atherosclerosis is unknown. This study aims to explore the effect of PSRC1 on atherosclerosis and its underlying mechanisms. METHOD AND RESULTS: A recombinant adenovirus expressing Psrc1 (Ad-PSRC1) was constructed and transfected in RAW264.7 cells as well as injected intravenously into apoE-/- mice. The in vitro study showed that PSRC1 overexpression reduced the cellular cholesterol content, increased the cholesterol efflux capacity and inhibited foam cell formation by upregulating the expression of peroxisome proliferator-activated receptor γ (PPAR-γ) and liver X receptor α (LXR-α), which are key cholesterol transportation-related proteins. Infecting apoE-/- mice with Ad-PSRC1 inhibited the development of atherosclerotic lesions and enhanced atherosclerotic plaque stability. Consistent with these results, PSRC1 overexpression in apoE-/- mice decreased the plasma levels of TC, TG, LDL-C, TNF-α, IL-1ß and IL-6, increased the plasma HDL-C levels and improved HDL function. Similarly, the PPAR-γ and LXR-α expression levels were upregulated in the liver and in peritoneal macrophages of PSRC1-overexpressing apoE-/- mice. Finally, the liver and peritoneal macrophages of apoE-/- mice displayed elevated expression of ß-catenin, which is a direct downstream gene of PSRC1 and an upstream gene of PPAR-γ and LXR-α, but decreased activity of nuclear transcription factor (NF-κB), which acts as a key gene in the regulation of inflammation. CONCLUSIONS: PSRC1 protects against the development of atherosclerosis and enhances the stability of plaques by modulating cholesterol transportation and inflammation in macrophages and the liver of apoE-/- mice.