Thyroid hormone controls the timing of cochlear ribbon synapse maturation.

Ribbon synapses in the cochlear hair cells are subject to extensive pruning and maturation processes before hearing onset. Previous studies have highlighted the pivotal role of thyroid hormone (TH) in this developmental process, yet the detailed mechanisms are largely unknown. In this study, we found that the thyroid hormone receptor α (Thrα) is expressed in both sensory epithelium and spiral ganglion neurons in mice. Hypothyroidism, induced by Pax8 gene knockout, significantly delays the synaptic pruning during postnatal development in mice. Detailed spatiotemporal analysis of ribbon synapse distribution reveals that synaptic maturation involves not only ribbon pruning but also their migration, both of which are notably delayed in the cochlea of Pax8 knockout mice. Intriguingly, postnatal hyperthyroidism, induced by intraperitoneal injections of liothyronine sodium (T3), accelerates the pruning of ribbon synapses to the mature state without affecting the auditory functions. Our findings suggest that thyroid hormone does not play a deterministic role but rather controls the timing of cochlear ribbon synapse maturation.

Cardiac-specific overexpression of miR-122 induces mitochondria-dependent cardiomyocyte apoptosis and promotes heart failure by inhibiting Hand2.

MicroRNA-122 (miR-122) is one of several microRNAs elevated in heart failure patients. To investigate the potential role and mechanism of miR-122 in heart failure, we constructed a transgenic mouse overexpressing miR-122 in the heart. This mouse exhibited cardiac dysfunction (as assessed by transthoracic echocardiography), morphological abnormalities of the heart and cardiomyocyte apoptosis characteristic of heart failure. Mechanistically, we identified the Hand2 transcription factor as a direct target of miR-122 using a dual-luciferase reporter assay. In Tg-miR-122 mice and H9C2 cells with miR-122 mimics, we detected apoptosis and increased expression of dynamin-related protein-1 (Drp1). This effect was blocked with prior knockdown of Hand2 in vitro. Our work suggests that miR-122 causes cardiomyocyte apoptosis by inhibiting Hand2 and consequently increasing Drp1-mediated mitochondrial fission. Such a mechanism likely contributes to heart failure and so modulating this pathway could be therapeutically valuable against heart failure.

Preliminary assessment of video-based blood pressure measurement according to ANSI/AAMI/ISO81060-2: 2013 guideline accuracy criteria: Anura smartphone app with transdermal optimal imaging technology.

OBJECTIVE: A new smartphone app called Anura can measure blood pressure (BP) any time and any place without cuffs or special equipment from video of the face. This study assessed its accuracy in close conformity with the American National Standards Institute/Association for the Advancement of Medical Instrumentation/International Organization for Standardization (ANSI/AAMI/ISO) 81060-2:2013 standard for BP measurement devices. METHODS: We validated Anura in reference to auscultation using a mercury sphygmomanometer and then assessed accuracy against the two accuracy criteria described in the guideline (n = 85 subjects; three measurement pairs per subject). RESULTS: The mean difference between the Anura measurement and its paired auscultatory reference measurement across all 255 measurement pairs was -0.4 ± 6.7 mmHg for systolic blood pressure (SBP) and 1.2 ± 7.0 mmHg for diastolic blood pressure (DBP). Both are within the acceptable limit of 5 ± 8 mmHg and thus satisfy accuracy criterion 1. When mean differences are averaged for each subject, the mean across all 85 subjects is -0.4 ± 5.8 mmHg for SBP and 1.2 ± 6.7 mmHg for DBP. Both are within acceptable limits (based on the mean difference) and thus satisfy accuracy criterion 2. CONCLUSIONS: Anura meets ANSI/AAMI/ISO 81060-2:2013 standard with respect to BP measurement accuracy. As the ANSI/AAMI/ISO 81060-2:2013 standard has not been developed for cuffless devices, further research assessing additional accuracy issues specific to such devices is needed.

Potential Impact of the 2017 High Blood Pressure Guideline Beyond the United States: A Case Study of the People's Republic of China.

BACKGROUND: The 2017 American College of Cardiology (ACC)/American Heart Association (AHA) (US) Guideline for the Prevention, Detection, Evaluation and Management of High Blood Pressure in Adults expanded the definition of hypertension and now considers atherosclerotic cardiovascular disease (ASCVD) risk in determining treatment for people with hypertension. US guidelines are influential around the world and it is therefore justified to study their impact in other settings. Our study determined the impact of adopting the 2017 ACC/AHA guideline in China. METHODS: We analyzed the population impact of the 2017 ACC/AHA guideline using the 2011-2012 year of the China Health and Retirement Longitudinal Study (CHARLS), a nationally representative sample of Chinese adults 45-74 years of age (n = 11,822). Our analysis was unique because for the first time it used a population-appropriate equation to calculate ASCVD risk instead of the US Pooled Cohort Equation (the latter misrepresents risk in non-US populations). RESULTS: Adopting the 2017 ACC/AHA guideline in China would increase the prevalence of hypertension from 44.1% to 56.4% (12.3 percentage points) and increase the number of adults recommended for antihypertensive medication from 41.6% to 49.1% (7.5 percentage points) in the 45-74-year age range. According to Chinese (but not US) risk calculations, the 2017 ACC/AHA guideline more selectively assigns antihypertensive medication to patients at higher risk for ASCVD. CONCLUSIONS: The 2017 ACC/AHA guideline brings potential for risk reduction in China and selectively recommends medication for those who would benefit most. Realizing such benefits would ultimately depend on the acceptance, adherence, and feasibility of adopting this guideline.

Smartphone-Based Blood Pressure Measurement Using Transdermal Optical Imaging Technology.

BACKGROUND: Cuff-based blood pressure measurement lacks comfort and convenience. Here, we examined whether blood pressure can be determined in a contactless manner using a novel smartphone-based technology called transdermal optical imaging. This technology processes imperceptible facial blood flow changes from videos captured with a smartphone camera and uses advanced machine learning to determine blood pressure from the captured signal. METHODS: We enrolled 1328 normotensive adults in our study. We used an advanced machine learning algorithm to create computational models that predict reference systolic, diastolic, and pulse pressure from facial blood flow data. We used 70% of our data set to train these models and 15% of our data set to test them. The remaining 15% of the sample was used to validate model performance. RESULTS: We found that our models predicted blood pressure with a measurement bias±SD of 0.39±7.30 mm Hg for systolic pressure, -0.20±6.00 mm Hg for diastolic pressure, and 0.52±6.42 mm Hg for pulse pressure, respectively. CONCLUSIONS: Our results in normotensive adults fall within 5±8 mm Hg of reference measurements. Future work will determine whether these models meet the clinically accepted accuracy threshold of 5±8 mm Hg when tested on a full range of blood pressures according to international accuracy standards.

Increased SLC7A8 expression mediates L-DOPA uptake by renal tubular epithelial cells.

The kidney serves a central role in the control of blood pressure through the release of vasoactive substances and the urinary excretion of Na+. Patients with essential hypertension usually exhibit persistent high blood pressure accompanied by Na+ retention. L-dihydroxyphenylalanine (L­DOPA) is an amino acid, converted by the enzyme aromatic L­amino acid decarboxylase to dopamine. The uptake of L­DOPA by cells of the proximal tubular epithelium of the kidney is controlled by the L­type amino acid transporter 2 (LAT2). LAT2 belongs to the solute carrier family 7 (SLC7) of amino acid transporters and is coded by the SLC7A8 gene. SLC7A8 expression is increased in the second­order mesenteric arteries and kidneys of spontaneously hypertensive rats. The present study aimed to investigate the physiological role of the SLC7A8 gene in L­DOPA handling by kidney cells. Selective upregulation of SLC7A8 mRNA and protein levels was achieved by adenoviral transduction of NRK­52E cells, which retain several properties of proximal tubular epithelial cells. In addition, L­DOPA uptake was determined using high performance liquid chromatography; NRK­52E cells expressing SLC7A8 exhibited increased uptake of L­DOPA. The results of the present study suggested that SLC7A8 may serve a critical role in blood pressure control through regulating L­DOPA uptake in renal epithelial cells of the proximal tubule.

Pax8 plays a pivotal role in regulation of cardiomyocyte growth and senescence.

Congenital heart disease (CHD) is a worldwide health problem, particularly in young populations. In spite of the advancement and progress in medical research and technology, the underlying causative factors and mechanisms of CHD still remain unclear. Bone morphogenetic protein receptor IA (ALK3) mediates the development of ventricular septal defect (VSD). We have recently found that paired box gene 8 (Pax8) may be the downstream molecule of ALK3. Paired box gene 8 plays an essential role in VSD, and apoptosis and proliferation imbalance leads to septal dysplasia. Recent studies have also disclosed that cellular senescence also participates in embryonic development. Whether programmed senescence exists in cardiac organogenesis has not ever been reported. We hypothesized that together with various biological processes, such as apoptosis, enhanced cellular senescence may occur actively in the development of Pax8 null mice murine hearts. In H9C2 myogenic cells, Pax8 overexpression can rescue caspase-dependent apoptosis induced by ALK3 silencing. Senescent cells and senescence-associated mediators in Pax8 knockout hearts increased compared with the wild-type ones in an age-dependent manner. These results suggest that Pax8 maybe the downstream molecule of ALK3, it mediates the murine heart development perhaps via cellular senescence, which may serve as a mechanism that compensates for the cell loss via apoptosis in heart development.

Microarray expression profiling and gene ontology analysis of long non-coding RNAs in spontaneously hypertensive rats and their potential roles in the pathogenesis of hypertension.

Long non-coding RNAs (lncRNAs) have been demonstrated to be significant in numerous biological processes. Hypertension is a form of cardiovascular disease with at least one billion cases worldwide. The present study sought to compare the differential expression profiles of lncRNAs in the renal cortex of spontaneously hypertensive rats (SHRs) and normotensive Wistar­Kyoto (WKY) rats. The ipsilateral renal cortex was obtained from 15­week­old SHRs and WKY rats whose blood pressures had been monitored. Total RNA was extracted using TRIzol, and lncRNAs and messenger RNAs were profiled by microarray and validated using fluorescent quantitative reverse transcription­polymerase chain reaction. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to predict the function of differentially expressed genes. Microarray analysis demonstrated that 145 lncRNAs were differentially expressed between SHRs and WKY rats. GO and KEGG pathway analysis indicated that these lncRNAs are involved in numerous biological processes. Thus, lncRNAs may contribute to the pathogenesis of hypertension.

Quantitation of Mitochondrial DNA Deletions Via Restriction Digestion/Long-Range Single-Molecule PCR.

Quantification of deletions in mtDNA is a long-standing problem in mutational analysis. We describe here an approach that combines the power of single-molecule PCR of the entire mitochondrial genome with the enrichment of the deletions by restriction digestion. This approach is indispensable if information about wide range of deletion types in a sample is critical, such as in studies concerning distribution of deletion breakpoints (as opposed to approaches where fraction of a single deletion or a limited set of deletions is used as a proxy for total deletion load). Because deletions in a sample are quantified almost exhaustively, the other important application of this approach involves studies where only small amounts of tissue, such as biopsies, are available.

[Telmisartan reduces retina vessel endothelial cell apoptosis via upregulating retinal ACE2-Ang-(1-7)-Mas axis in spontaneous hypertensive rats].

OBJECTIVE: To investigate the effects of angiotensin II (Ang II) antagonist telmisartan on retina vessel endothelial cell apoptosis and its impact on the ACE2-Ang-(1-7)-Mas axis in spontaneous hypertensive rats (SHR). METHODS: Thirty-six SHR 16 week-old were randomly divided into 3 groups (n = 12 each): SHR, SHRT (telmisartan 10 mg · kg-1 · d-1 by gastric gavage) and SHRTA group (telmisartan 10 mg · kg-1 · d-1 by gastric gavage plus intravenous injection of A-779 0.5 mg · kg-1 · d-1), twelve WKY rats served as normotensive control group. Systolic blood pressure was measured at pre-treatment and 8 weeks later. After 8 weeks, rats were sacrificed, the expression of ACE2 and Mas in retina were analyzed by qRT-PCR, Western blot and Immunohistochemistry, the Ang-(1-7) concentration in serum was measured by ELISA. Specimens were obtained and stained by hematoxylin and eosin, and the morphology of retina vessel was observed. Apoptosis of vessel endothelial cells were determined by using terminal deoxynucleotidyl transferase mediated dUTP nick end labeling method. RESULTS: The systolic blood pressure of SHR, SHRT and SHRTA groups at baseline were significantly higher than age-matched WKY group (all P < 0.01). Eight weeks later, the systolic blood pressure group was significantly lower in SHRT group than in the SHR group (P < 0.01), this effect was partly reversed in SHRTA group. The retinal ACE2 mRNA and protein expression was significantly lower in SHR group than in WKY and SHRT groups (P < 0.01), which was similar between SHRT group and SHRTA group (P > 0.05). The retinal Mas mRNA and protein expression were significantly lower in SHR group compared to WKY and SHRT groups (all P < 0.01), which was significantly lower in SHRTA group than in the SHRT group (P < 0.05). ELISA results showed that serum Ang-(1-7) protein level was significantly lower in SHR group than in WKY group and SHRT group (both P < 0.05), which was lower in SHRTA group compared to SHRT group. Retinal vessel endothelial cell apoptosis was higher in SHR group than in WKY group, which could be reduced by cotreatment with telmisartan and this beneficial effect could be reversed by A-779. CONCLUSION: Telmisartan can reduce retinal vessel endothelial cell apoptosis via upregulating the ACE2-Ang-(1-7)-Mas axis.

Role of microRNA-29b in angiotensin II-induced epithelial-mesenchymal transition in renal tubular epithelial cells.

Angiotensin II (Ang II) has been proven to induce epithelial-mesenchymal transition (EMT). The aim of the present study was to determine the role of microRNA-29b (miR-29b) during Ang II-induced EMT. For this purpose, we used spontaneously hypertensive rats (SHRs) and age-matched Wistar-Kyoto (WKY) rats. The levels of Ang II and its receptor in the kidneys of the SHRs are significantly higher than those in the age-matched WKY rats. As shown by RT-qPCR, the expression of miR-29b in the renal cortex was lower in the SHRs than in the WKY rats. For in vitro experiments, NRK-52E renal tubular epithelial cells were treated with 10(-7) M Ang II; we found that the expression of miR-29b was decreased in the cells treated with Ang II. In addition, transfection of the NRK-52E cells with miR-29b inhibitor led to the downregulation of miR-29b in these cells, and increased the expression of transforming growth factor (TGF)-ß, α-smooth muscle actin (α-SMA) and collagen I (Col I). Similar results were observed with the induction of Ang II expression in the NRK-52E cells. By contrast, the upregulation of miR-29b by transfection with miR-29b mimics inhibited the overexpression of these genes induced by Ang II. These results suggest that miR-29b plays an important role in Ang II-induced EMT.

[Impact of Pax-8 gene interference on mitochondrial function and cardiomyocyte apoptosis].

OBJECTIVE: To observe the effects of paired box gene 8 (Pax-8) silencing by RNA interference on mitochondrial function and cardiomyocytes apoptosis. METHODS: The cultured H9C2 (2-1) myocytes were divided into 3 groups: short interference RNA targeting Pax-8 (Pax-8 siRNA) group, non-specific siRNA group as the negative control (NC siRNA), and blank control group (BC siRNA). Fluorescence spectrophotometry was used to detect the activity of caspase-3. RT-PCR was performed to detect mRNA expression of Bcl2 and Bax. The protein expression of Bcl2, Bax and cytoplasm of Cytochrome was examined by Western blot. Changes of ΔΨm were detected by flow cytometry.ΔΨm with JC-1 monomer/polymer ratio was calculated for measuring mitochondrial depolarization proportion. RESULTS: Compared to NC siRNA and BC siRNA group (0.075 ± 0.021, 0.072 ± 0.019), the activity of caspase-3 in Pax-8 siRNA group (0.167 ± 0.012) was significantly increased (P < 0.05); Bcl2 mRNA and protein expression in Pax-8 siRNA group (0.61 ± 0.06, 0.94 ± 0.11) were significantly downregulated compared with NC siRNA group (0.90 ± 0.070, 1.39 ± 0.15) and BC siRNA group (0.94 ± 0.087, 1.49 ± 0.20) (P < 0.05); Bax mRNA and protein expression in Pax-8 siRNA group (1.05 ± 0.10, 1.25 ± 0.12) were markedly upregulated compared with NC siRNA group (0.72 ± 0.03, 0.99 ± 0.12) and BC siRNA group (0.64 ± 0.03, 0.92 ± 0.06), P < 0.05; cytosolic cytochrome expression in Pax-8 siRNA group (0.75 ± 0.14) was significantly upregulated compared with NC siRNA group (0.51 ± 0.06) and BC siRNA group (0.48 ± 0.07) (P < 0.05); JC-1 monomer/polymer ratio in Pax-8 siRNA group (0.163 ± 0.011) was significantly increased compared with NC siRNA group (0.092 ± 0.015) and BC siRNA group (0.072 ± 0.025) (P < 0.05) indicating mitochondrial membrane potential was significantly reduced in Pax-8 siRNA group. Above parameters were similar between NC siRNA group and BC siRNA group (P > 0.05). CONCLUSION: Inhibiting Pax-8 results in enhanced cardiomyocytes apoptosis through the mitochondrial pathway.

Expression of microRNA-122 contributes to apoptosis in H9C2 myocytes.

The microRNAs (miRNAs) can post-transcriptionally regulate gene expression and heart development. The Pax-8 gene knockout mice have apparent heart abnormalities. This study investigated the role of miRNAs in regulation of cardiac apoptosis and development in the knockout mice. MicroRNA microarrays demonstrated differential expression of microRNAs between Pax-8(-/-) and Pax-8(+/-) mice, confirmed by real-time PCR. The miR-122 was up-regulated by 1.92 folds in Pax-8(-/-) mice. There were ventricular septum defects in Pax-8(-/-) mice, and increased numbers of apoptotic cells in the left ventricular wall and interventricular septum in Pax-8(-/-) mice. In H9C2 myocytes, treatment with miR-122 mimics or miR-122 inhibitor affects the expression of CCK-8 and activity of Caspase-3. The miR-122 is up-regulated in the myocytes of Pax-8(-/-) mice and may participate in the apoptotic gene expression and pathogenesis of heart development defect.

Reduced expression of FXYD domain containing ion transport regulator 5 in association with hypertension.

Experimental evidence indicates that hypertension is a multifactorial disorder and that the products of several genes may contribute to its development. The aim of this study was to investigate the expression of hypertension-related genes in spontaneous hypertensive rats (SHRs). A microarray screening for hypertension-related genes was conducted in SHRs and Wistar-Kyoto (WKY) rats using total-RNA extracted from second-order mesenteric arteries and kidneys. The FXYD5 mRNA expression in vascular smooth muscle cells (VSMCs) was silenced by RNA interference (RNAi). Meanwhile, the FXYD5 mRNA overexpression in renal tubular epithelial cells (RTECs) was induced by the recombinant plasmid pcDNA3.1(+)-FXYD5. The expression of FXYD5 mRNA was found to be 14.8-fold lower in SHR rats compared to that in WKY rats (P<0.01). The levels of FXYD5 mRNA expression were the highest in kidneys of SHR 13-week-old rats when the blood pressure reached the highest levels. The down-regulated FXYD5 mRNA expression inhibited the migration of smooth muscle cells (P<0.01) and cell membrane Na⁺-K⁺-ATPase activity (P<0.01). Up-regulated FXYD5 mRNA expression enhanced the renal tubular epithelial cell membrane Na⁺-K⁺-ATPase activity (P<0.05) and cell proliferation (P<0.05). FXYD5 is related to the migration of smooth muscle cells and cell membrane Na⁺-K⁺-ATPase activity in rodents. The results of the present study suggest that FXYD5 may have profound impact on the regulation of blood pressure, and that this gene may be a potential target for anti-hypertensive therapy.

The defects in development and apoptosis of cardiomyocytes in mice lacking the transcriptional factor Pax-8.

BACKGROUND: Cardiac-specific deletion of ALK3 is lethal in mid-gestation with ventricular septum malformations (VSM). This study was designed to define the Pax-8's role in heart development and cardiomyocyte apoptosis. METHODS: Pathologic changes in the hearts of Pax-8 or ALK3 knockout and wild type control mice were determined by light and electron microscopy. Analysis of cardiomyocyte apoptosis was performed by TUNEL. The effect of Pax-8 gene deficiency on caspase-3 activity was examined after transfecting Pax-8 siRNA into cultured myoblast cell line. RESULTS: Mice with ALK3 or Pax-8 gene knockout but not wild type control animals showed the development of VSM. Increased cardiomyocyte apoptosis was found in homozygotes. Echocardiography showed that Pax-8 homozygote mice developed malfunction of the heart. Furthermore, the caspase-3 activity was significantly higher in the cells treated with Pax-8 siRNA as compared to those treated with negative control siRNA in H9C2 (2-1) cell line. CONCLUSIONS: The Pax-8 gene may play a crucial role in heart development and regulating cardiocyte apoptosis. Knockout of Pax-8 may exert a similar effect on myocardial morphology and apoptosis as those seen in ALK3 knockouts. Furthermore, the ventricular septum malformations could be partially attributed to accelerated cardiomyocyte apoptosis.

[MicroRNA-144 over-expression induced myocytes apoptosis].

OBJECTIVE: To investigate the effects of microRNA-144 (miR-144) expression on H9C2 (2-1) myocytes. METHODS: MiR-144 was up-regulated in primary cultured H9C2 (2-1) myocytes through transfection. Cells transfected with Lipofectamine(TM) 2000 and its mixture with miRNA synthesized randomly as blank control and negative control respectively. The up-regulation of miR-144 was confirmed by real-time PCR. Cell apoptosis was evaluated by means of CCK-8, Caspase-3 and flow cytometry. RESULTS: Real-time PCR results showed that the miR-144 expression was obviously increased in miR-144 up-regulation group (2178.84 ± 838.52) compared with negative (2.06 ± 0.73) and blank (1.00 ± 0.00) control group (all P < 0.01). The proliferation was lower, the activity of Caspase-3 was elevated and the apoptosis rates were significantly increased in miR-144 up-regulation group compared with negative and blank control group, while no significant difference was found between the latter 2 groups. CONCLUSION: MiR-144 mimics may selectively up-regulate the expression of miR-144 in myocardial cells and consequently promote apoptosis and inhibit proliferation in myocardial cells.

[Ryanodine downregulates the expression of p-eNOS (Thr495) and improves the functions of rapamycin treated endothelial outgrowth cells].

OBJECTIVE: To observe the effects of ryanodine on rapamycin treated endothelial outgrowth cells (EOCs). METHODS: The mononuclear cells were harvested from umbilical cord blood by Ficoll density gradient centrifugation, then induced into EOCs and expanded in vitro. The endothelial characteristics of EOCs were identified by immunostaining and fluorescent staining. The EOCs were pretreated with or without ryanodine (10 µmol/L) for 1 h, and then treated with or without rapamycin (10 nmol/L) for 24 h. Proliferation was evaluated by CCK8 and migration was measured by Transwell. The protein expression of EOCs was evaluated by immunobloting technique with total eNOS antibody and phospho-eNOS (Thr495) antibody. RESULTS: Compared with control group, the proliferation and migration capacities of EOCs were significantly reduced while the phosphorylation of eNOS (Thr495) protein was significantly upregulated in rapamycin group (P < 0.05), expression of total eNOS was not affected by rapamycin (P > 0.05). Compared with rapamycin group, the proliferation and migration capacities of EOCs were significantly increased and the phosphorylation of eNOS (Thr495) protein was significantly downregulated in ryanodine + rapamycin group (P < 0.05). The proliferation and migration capacities, the phosphorylation of eNOS (Thr495) protein and the expression of total eNOS were not affected by ryanodine alone (P > 0.05). CONCLUSIONS: Rapamycin reduced proliferation and migration capacities while upregulated the phosphorylation of eNOS (Thr495) protein of EOCs and these effects could be partly reversed by cotreatment with ryanodine.

Mitochondrial DNA deletions in mice in men: substantia nigra is much less affected in the mouse.

Mitochondrial DNA (mtDNA) deletions have been reported to accumulate to high levels in substantia nigra of older humans, and these mutations are suspected of causing age-related degeneration in this area. We have compared levels of mtDNA deletions in humans and mice and report here that levels of deletions in the mouse are very significantly lower than in humans. While human mtDNA from substantia nigra contained more than 5% of deleted molecules, mouse substantia nigra contained less than 0.5%. These results imply that mtDNA deletions are unlikely to play any significant role in of murine substantia nigra aging and further call for caution in using mouse models in studies of the role of mtDNA deletions in aging and neurodegeneration. On a more general note, these results support the view that critical targets of the various aging processes may differ significantly between distant species.