RNA-sequencing of human aortic valves identifies that miR-629-3p and TAGLN miRNA-mRNA pair involving in calcified aortic valve disease.

This study aimed to uncover the microRNA and messenger RNA (miRNA/mRNA) interactions in the pathophysiological process of calcified aortic valve disease (CAVD) of the human aortic valve. RNA sequencing of six selected samples (3 healthy control samples vs. 3 CAVD samples) was performed to obtain mRNA and miRNA sequences, and differential expression (DE) analysis of miRNA and mRNAs was performed. To build a CAVD-specific miRNA-mRNA interactome, the upregulated mRNAs and downregulated miRNAs were selected, followed by the establishment of inverse DE of mRNA-miRNA co-expression network based on Pearson's correlation coefficient using miRanda in the R language software. Subsequently, pathway enrichment analysis was performed to elucidate CAVD-related pathways that were likely mediated by miRNA regulatory mechanisms. In addition, miRNAs with an mRNA correlation greater than 0.9 in the co-expression network were selected for anti-calcification verification in a CAVD cellular model. We identified 216 mRNAs (99 downregulated and 117 upregulated) and 602 miRNAs (371 downregulated and 231 upregulated) that were differentially expressed between CAVD and healthy aortic valves. After applying Pearson's correlation toward miRNA-mRNA targets, a regulatory network of 67 miRNAs targeting 76 mRNAs was created. The subsequent pathway enrichment analysis of these targeted mRNAs elucidated that genes within the focal adhesion pathway are likely mediated by miRNA regulatory mechanisms. The selected hsa-miR-629-3p and TAGLN pair exhibited anti-calcification effects on osteogenic differentiation-induced human aortic valve interstitial cells (hVICs). On integrating the miRNA and mRNA sequencing data for healthy aortic valves and those with CAVD, the CAVD-associated miRNA-mRNA interactome and related pathways were elucidated. Additional cell function data demonstrated anti-calcification effects of the selected hsa-miR-629-3p targeting TAGLN, validating that it is a potential therapeutic target for inhibiting CAVD.

Endothelial cell-derived tetrahydrobiopterin prevents aortic valve calcification.

AIMS: Tetrahydrobiopterin (BH4) is a critical determinant of the biological function of endothelial nitric oxide synthase. The present study was to investigate the role of valvular endothelial cell (VEC)-derived BH4 in aortic valve calcification. METHODS AND RESULTS: Plasma and aortic valve BH4 concentrations and the BH4:BH2 ratio were significantly lower in calcific aortic valve disease patients than in controls. There was a significant decrease of the two key enzymes of BH4 biosynthesis, guanosine 5'-triphosphate cyclohydrolase I (GCH1) and dihydrofolate reductase (DHFR), in calcified aortic valves compared with the normal ones. Endothelial cell-specific deficiency of Gch1 in Apoe-/- (Apoe-/-Gch1fl/flTie2Cre) mice showed a marked increase in transvalvular peak jet velocity, calcium deposition, runt-related transcription factor 2 (Runx2), dihydroethidium (DHE), and 3-nitrotyrosine (3-NT) levels in aortic valve leaflets compared with Apoe-/-Gch1fl/fl mice after a 24-week western diet (WD) challenge. Oxidized LDL (ox-LDL) induced osteoblastic differentiation of valvular interstitial cells (VICs) co-cultured with either si-GCH1- or si-DHFR-transfected VECs, while the effects could be abolished by BH4 supplementation. Deficiency of BH4 in VECs caused peroxynitrite formation increase and 3-NT protein increase under ox-LDL stimulation in VICs. SIN-1, the peroxynitrite generator, significantly up-regulated alkaline phosphatase (ALP) and Runx2 expression in VICs via tyrosine nitration of dynamin-related protein 1 (DRP1) at Y628. Finally, folic acid (FA) significantly attenuated aortic valve calcification in WD-fed Apoe-/- mice through increasing DHFR and salvaging BH4 biosynthesis. CONCLUSION: The reduction in endothelial-dependent BH4 levels promoted peroxynitrite formation, which subsequently resulted in DRP1 tyrosine nitration and osteoblastic differentiation of VICs, thereby leading to aortic valve calcification. Supplementation of FA in diet attenuated hypercholesterolaemia-induced aortic valve calcification by salvaging BH4 bioavailability.

Klotho suppresses the inflammatory responses and ameliorates cardiac dysfunction in aging endotoxemic mice.

BACKGROUND: Aging augments endotoxemic cardiac dysfunction, but the mechanism remains unclear. Anti-aging protein Klotho has been found to modulate tissue inflammatory responses. We tested the hypothesis that a reduced Klotho level in aging heart plays a role in the augmented endotoxemic cardiac dysfunction. MATERIALS AND METHODS: Endotoxin (0.5 mg/kg, iv) was injected to adults (4-6 months) and aging (18-20 months) C57BL/6 mice. Recombinant Klotho (10 µg/kg, iv) was administered to a group of aging mice after endotoxin injection. Cardiac function was analyzed using a microcatheter at 24 and 48 h after endotoxin administration. Myocardial levels of Klotho and heat shock protein 70 (HSP70) were determined by immunoblotting, and plasma and myocardial cytokines were analyzed using ELISA. RESULTS: More severe cardiac dysfunction in aging mice were accompanied by greater cytokine levels in the plasma and myocardium. Klotho was detected in the myocardial tissue. Klotho levels were lower in aging hearts and were further reduced during endotoxemia. Myocardial HSP70 levels were correlated with Klotho levels. Recombinant Klotho increased myocardial HSP70, inhibited NF-κB activation, reduced cytokine levels, and improved cardiac function in aging endotoxemic mice. Delivery of HSP70 into cultured macrophages suppressed endotoxin-induced NF-κB activation. CONCLUSIONS: Aging-related augmentation of inflammatory responses and cardiac dysfunction is associated with relative Klotho deficiency. Post-treatment with recombinant Klotho suppresses the inflammatory responses and improves cardiac function in aging endotoxemic mice. Klotho modulates HSP70 levels and HSP70 appears to be involved in the anti-inflammatory mechanism of Klotho. Klotho may have therapeutic potential in amelioration of aging-related endotoxemic cardiac dysfunction.

Interleukin-37 suppresses the inflammatory response to protect cardiac function in old endotoxemic mice.

Myocardial inflammatory responses to endotoxemia are enhanced in old mice, which results in worse cardiac dysfunction. Anti-inflammatory cytokine interleukin (IL)-37 has a broad effect on innate immunoresponses. We hypothesized that IL-37 suppresses myocardial inflammatory responses to protect cardiac function during endotoxemia in old mice. Old (20-24month) wild-type (WT), and IL-37 transgenic (IL-37tg) mice were treated with lipopolysaccharide (LPS, 0.5mg/kg, iv) or normal saline (0.1ml/mouse, iv). Six hours later, left ventricle (LV) function was assessed using a pressure-volume microcatheter. Levels of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-6 in plasma and myocardial tissue, as well as mononuclear cell density in the myocardium, were examined. Cardiac microvascular endothelial cells isolated from WT and IL-37tg mice were treated with LPS (0.2µg/ml) for 0.5-24h. Nuclear factor-kappa B (NF-κB) p65 phosphorylation was examined by immunoblotting, and MCP-1 levels in cell culture supernatant was determined using enzyme-linked immunosorbent assay. LV dysfunction in old WT endotoxemic mice was accompanied by up-regulated MCP-1, myocardial accumulation of mononuclear cells and production of TNF-α, IL-1ß and IL-6. Expression of IL-37 suppressed myocardial inflammatory responses to endotoxemia in old mice, resulting in improved LV function. Treatment of old WT endotoxemic mice with recombinant IL-37 also improved LV function. In vitro experiments revealed that cardiac microvascular endothelial cells from IL-37tg mice had attenuated NF-κB activation and MCP-1 production following LPS stimulation. In conclusion, IL-37 is potent to suppress myocardial inflammation and protects against cardiac dysfunction during endotoxemia in old mice.

[Value of evaluating diastolic function with the single-beat E/(e'×s) obtained by dual doppler echocardiograph in coronary heart disease patients with preserved left ventricular systolic function].

OBJECTIVE: To assess the value of E/(e'×s) in estimating left ventricular diastolic dysfunction in patients with coronary heart disease by dual Doppler echocardiograph. METHODS: Seventy-seven consecutive coronary heart disease patients with preserved systolic function underwent echocardiographic study were included. The E, e'and s were obtained by the dual Doppler echocardiography and E/(e'×s), E/e' were calculated. All patients underwent left ventricular catheterization to measure left ventricular end diastolic pressure (LVEDP). The relationship between E/(e'×s), E/e' and LVEDP were analyzed. Patients were divided into normal diastolic function (LVEDP < 12 mmHg, 1 mmHg = 0.133 kPa) and diastolic dysfunction group (LVEDP ≥ 12 mmHg) . RESULTS: (1) Pearson correlation analysis showed that both E/(e'×s) and E/e' correlated well with LVEDP (r = 0.68 and r = 0.79, both P < 0.01). (2)Using receiver operating characteristic analysis, the optimal cut-off for E/(e'×s) was 1.2(sensitivity was 80%, specificity was 77%,AUC was 0.85) and for E/e' was 9.2(sensitivity was 74%, specificity was 81%,AUC was 0.87) to predict left ventricular diastolic dysfunction. When combined cut-offs of E/(e'×s) ≥ 1.2 and E/e' ≥ 9.2, the sensitivity and specificity of predicting left ventricular diastolic dysfunction were 83% and 71% respectively, and AUC was 0.87. CONCLUSIONS: E/(e'×s) can correctly reflect diastolic function status in patients with coronary artery disease. However, combined use of E/(e'×s)and E/e' does not add the prediction value on diastolic dysfunction in this patient cohort.

GRK 2 level in peripheral blood lymphocytes of elderly patients with acute myocardial infarction.

OBJECTIVE: To investigate the G protein-coupled receptor kinase 2 (GRK 2) level in peripheral blood lymphocytes with cardiac function in elderly patients with acute myocardial infarction. METHODS: This study enrolled 40 patients with acute ST-segment elevation myocardial infarction (STEMI) and 40 patients with unstable angina. All patients were 65 years or older. Cardiac function was evaluated by echocardiography, and the GRK 2 level in peripheral blood lymphocytes was measured. Patients with STEMI were followed up for 2 years. RESULTS: The GRK 2 level in peripheral blood lymphocytes was significantly higher in patients with STEMI than in patients with unstable angina, and was negatively correlated with left ventricular ejection fraction, cardiac output, stroke volume, and left ventricular fractional shortening. The GRK 2 level was significantly elevated in some patients with acute STEMI and poor cardiac function. CONCLUSIONS: Increased GRK 2 level in patients with acute STEMI may contribute to poor myocardial systolic function and myocardial remodeling. Measurement of the GRK 2 level in peripheral blood lymphocytes may assist in the evaluation of cardiac function and myocardial remodeling in elderly patients with acute STEMI.

ADAR1 regulates ARHGAP26 gene expression through RNA editing by disrupting miR-30b-3p and miR-573 binding.

Rho GTPase activating protein 26 (ARHGAP26) is a negative regulator of the Rho family that converts the small G proteins RhoA and Cdc42 to their inactive GDP-bound forms. It is essential for the CLIC/GEEC endocytic pathway, cell spreading, and muscle development. The present study shows that ARHGAP26 mRNA undergoes extensive A-to-I RNA editing in the 3' UTR that is specifically catalyzed by ADAR1. Furthermore, the mRNA and protein levels of ARHGAP26 were decreased in cells in which ADAR1 was knocked down. Conversely, ADAR1 overexpression increased the abundance of ARHGAP26 mRNA and protein. In addition, we found that both miR-30b-3p and miR-573 target the ARHGAP26 gene and that RNA editing of ARHGAP26 mediated by ADAR1 abolished the repression of its expression by miR-30b-3p or miR-573. When ADAR1 was overexpressed, the reduced abundance of ARHGAP26 protein mediated by miR-30b-3p or miR-573 was rescued. Importantly, we also found that knocking down ADAR1 elevated RhoA activity, which was consistent with the reduced level of ARHGAP26. Conversely, when ADAR1 was overexpressed, the amount of RhoA-GTP decreased. The similar expression patterns of ARHGAP26 and ADAR1 in human tissue samples further confirmed our findings. Taken together, our results suggest that ADAR1 regulates the expression of ARHGAP26 through A-to-I RNA editing by disrupting the binding of miR-30b-3p and miR-573 within the 3' UTR of ARHGAP26. This study provides a novel insight into the mechanism by which ADAR1 and its RNA editing function regulate microRNA-mediated modulation of target genes.

[Protective effect of amlodipine against contrast agent-induced renal injury in elderly patients with coronary heart disease].

OBJECTIVE: To evaluate the protective effect of amlodipine against contrast agent-induced renal injury in elderly patients with coronary heart disease. METHODS: A total of 189 elderly patients (>60 years) with coronary heart disease undergoing coronary artery angiography were randomly assigned into amlodipine group and control group to receive amlodipine or placebo, respectively, before and after administration of the contrast agent. At 24 h, 48 h and 5 days after contrast agent administration, the parameters of renal function were measured including serum cystatin C, urea nitrogen, creatinine, creatinine clearance rate, urine ß2-microglobulin, and urine N-acetyl-ß-glucosaminidase. RESULTS: In both groups, the contrast agents obviously affected the renal functions of the patients (P<0.05). At 24 h after contrast administration, the levels of serum cystatin C, urine ß2-microglobulin and urine NAG were significantly lower in amlodipine group than in the control group, but the other functional parameters showed no significant difference. At 48 h after contrast administration, the glomerular and tubular functional parameters were all superior in amlodipine group (P<0.05). At 5 days, the two groups showed significant differences in such glomerular and tubular functional parameters as urea nitrogen, creatinine, creatinine clearance rate, urine ß2-microglobulin, and urine NAG (P<0.05), but not in serum cystatin C level. The incidence of contrast agent-induced nephropathy was significantly lower in amlodipine group than in the control group (5/95 vs 10/94, P<0.05). CONCLUSIONS: Amlodipine offers protection against radiographic contrast agent-induced renal injury in elderly patients with coronary heart disease.

[Treatment of chronic heart failure by overexpressing sarcoplasmic reticulum calcium ATPase through gene therapy: an experiment with rats].

OBJECTIVE: To evaluate the value of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) in the gene therapy of congestive heart failure. METHODS: (1) The abdominal aortas of 51 male SD rats were isolated and ligated so as to establish models of heart failure caused by contraction of abdominal aortas. 20 rats undergoing isolation of the abdominal aorta without ligation were used as controls. 18 approximately 20 days after the operation heart failure occurred, then the rats with contraction of abdominal aorta and heart failure were randomly divided into 3 groups: rAAV-SERCA2a group (recombinant adeno-associated virus containing SERCA2a cDNA, rAAV-SERCA2a, of the concentration of 2 x 10(11) v.g was injected via diaphragm into the pericardia cavity), heart failure control group (without trentment) and rAAV2-EGFP group (the control virus rAAV2-EGFP of the concentration of 2 x 10(11) v.g was injected via diaphragm into the pericardial cavity). 10 and 30 days after virus injection, a catheter was inserted through the jugular vein into the left ventricle to record the left ventricle systole pressure (LVSP), left ventricle end diastole pressure (LVEDP), left ventricle pressure maximum increase speed (+dp/dt), and left ventricle pressure maximum decrease speed (-dp/dt), and heart rate (HR). Then all the rats were killed and their hearts were taken out to examine the expression of the SERCA2a protein. (2) The left coronary arteries of 25 male SD rats were ligated so as to establish the models of cardiac infarction. 9 rats underwent isolation of the left coronary arteries without ligation and were used as controls. Four weeks after the operation thoracotomy was performed on the rats with heart failure caused by heart infarction, rAV-SERCA2a or rAV2-EGFP were injected into the myocardium, and dilute solution was injected to the control rats. 21 days later all the rats were performed hemodynamic exams. RESULTS: (1) Thirty days after the transfection the LVSP, +dp/dt, and -dp/dt of the rAAV-SERCA2a group were significantly higher than those of the rAAV2-EGFP group by 57% (94 mm Hg vs 147 mm Hg), 110% (5350 mm Hg/s vs 11 225 mm Hg/s), and 99.8% (4198 mm Hg/s vs 8390 mm Hg/s) respectively, meanwhile the LVEDP was significantly lower by 60% (22 mm Hg vs 9 mm Hg). These homodynamic parameters of the rAAV-SERCA2a group were not significantly different from those of the control group. Thirty days after transfection the expression of SERCA2a protein of the SERCA2a group was significantly higher than those of the control heart failure and rAAV2-EGFP groups. (2) Twenty-one days after the transfection, the LVSP, +dp/dt, and -dp/dt of the SERCA2a group were significantly higher than those of the control group by 28% (86 mm Hg vs 110 mm Hg), 41% (4272 mm Hg/s vs 6026 mm Hg/s), and 71% (2789 mm Hg/s vs 4756 mm Hg/s) respectively, and the LVEDP was significantly lower by 70% (3.89 mm Hg vs -5.34 mm Hg), however, these homodynamic parameters of the rAV-SERCA2a group were all worse compared with the control false operation group. CONCLUSION: The recombinant viruses, rAAV-SERCA2a and rAV-SERCA2a, effectively deliver the SERCA2a gene and improve the homodynamic state.

[Adeno-associated viral gene transfer of SERCA2a improves heart function in chronic congestive heart failure rats].

OBJECTIVE: To study the therapy effect of adeno-associated viral gene transfer of sarcoplasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) on chronic congestive heart failure (HF) in 30 days, and the possible mechanism of the therapy effect. METHODS: The rats were divided into four groups: control group, HF group, Group HF + EGFP, and Group HF + SERCA2a. HF rats were obtained by creating descending aortic constriction. 0.9% sodium chloride solution, recombinant adeno-associated virus carrying enhanced green fluorescent protein gene (rAAV2.eGFP) and recombinant adeno-associated virus carrying SERCA2a gene (rAAV2.SERCA2a), were respectively delivered to pericardium of HF rats in different groups by intrapericardial injection with a trans-diaphragmatic approach. 30 days after gene transfer, hemodynamic parameters, SERCA2a protein expression and SERCA2a activity were analyzed. The proteome difference from rat hearts between Groups HF + SERCA2a and HF was detected by expression proteomics. Electrophoretic separation and quantitation of cardiac myosin heavy chain isoforms of hearts in different groups were performed at 30 days. RESULTS: At 30 days, left ventricular function improved significantly in HF rats infected with rAAV2.SERCA2a (LVSP 146.52 +/- 13.86 vs 97.91 +/- 12.13, LVEDP 7.88 +/- 2.88 vs 21.15 +/- 3.57, LV +dp/dt 11 206.16 +/- 1730.11 vs 5948.93 +/- 1283.43, LV -dp/dt -8249.54 +/- 1076.09 vs -4497.50 +/- 652.12; P < 0.05). The recovered cardiac function in Group HF + SERCA2a rats was comparable to control rats, and had lower LV-weight/Body-weight ratio (2.46 +/- 0.17 vs 2.71 +/- 0.24, P < 0.05). Overexpression of SERCA2a increased both the protein content (0.39 +/- 0.11 vs 1.11 +/- 0.18, P < 0.05) and activity (228.62 +/- 25.11 vs 82.55 +/- 14.13, P < 0.05) up to nonfailing levels. Expressions of some energy metabolic enzymes in hearts of Group HF + SERCA2a were much higher than those of HF group. They included creatine kinase-muscle, enolase beta, fructose-bisphosphate aldolase, mitochondrial H(+)-ATP synthase alpha subunit, electron transfer flavoprotein alpha-subunit, H(+)-transporting ATP synthase and heart fatty acid binding protein. Downregulation of alpha-MHC and upregulation of beta-MHC in failing hearts were observed. Gene transfer of SERCA2a could increase the expression of alpha-MHC [(74.48 +/- 3.74)% vs (53.57 +/- 2.30)%, P < 0.05], and decrease the expression of beta-MHC [(25.52 +/- 3.74)% vs (46.43 +/- 2.30)%, P < 0.05] in HF rats. The expression profiles of alpha-MHC and beta-MHC and the ratio of alpha-MHC/beta-MHC were similar to those in controls. CONCLUSIONS: Adeno-associated viral gene transfer of SERCA2a can enhance SERCA2a functions, maintain calcium homeostasis, improve cardiac energy metabolism, and normalize the expression of cardiac myosin heavy chain isoforms in HF rats. As a result, the ventricular systolic and diastolic functions can be improved significantly, and the hypertrophy of the heart may be reduced in clinic. Adeno-associated viral gene transfer of SERCA2a demonstrated good therapy effects on HF rats.

[The effects of exercise training on plasma tumor necrosis factor-alpha, blood leucocyte and its components in congestive heart failure patients].

OBJECTIVE: To evaluate six-minute walking test (6-MWT) in treatment of CHF and to investigate the effect of exercise training on blood leucocyte and its components and plasma TNF alpha in CHF patients. METHODS: 60 cases with NYHA II-III CHF patients underwent 6-MWT within 24 hours after hospitalization. The walking distance, heartbeat, plasma TNF alpha, and blood leucocyte and its components were investigated before and after walking test. These patients were then randomized into two groups, a training group (32 cases) and a control group (28 cases). Besides routine medication, 6-MWT was performed in the training group twice a day for 8 weeks. The same parameters were redetermined after 8 weeks of follow-up. 23 cases with NYHA IV CHF patients were investigated with the same parameters in the first day after hospitalization. RESULTS: Blood leucocyte count and percentage of granulocytes and mononuclears in cases with NYHA III-IV were significantly higher than those with NYHA II. There were significant reduction of heartbeat and prolongation of walking distance in CHF patients after exercise training. Blood leucocyte count and the percentage of granulocytes and mononuclear and plasma TNFalpha level were reduced more in the training group than the control group after 8 weeks the treatment. CONCLUSIONS: There is significantly higher leucocyte count and percentage of granulocytes and mononuclears in CHF with NYHA III-IV than those with NYHA II. 6-MWT, as an exercise training, is simple, safe, and not harmful. It can not only evaluate the severity of CHF but also serve as a therapeutic measure for CHF. Moreover the excessive activation of cytokines as TNFalpha could be reduced by 6-MWT training in CHF patients.