Xiangxiang Shi

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.

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-8s 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.

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.

Induced cardiac myocytes apoptosis in vitro by microRNA-122 over expression

Objective The role of microRNA-122 (miR122) in myocardial cells is still unknown. Thus we investigated the involvement of miRNAs in the knockout Pax8 mice and study the function of miR122 during cardiac development. Methods The knockout Pax-8 mice model was established and the heart morphology of Pax-8 KO-/-, and Pax-8 KO+/- mice were detected. The total RNA of Pax-8 KO-/-, and Pax-8 KO+/- mice were extracted. MicroRNA microarray was used to investigate the differentially expressed microRNAs between Pax-8 KO-/-, and Pax-8 KO+/- mice, and the discovered microRNAs were further confirmed by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Primary cultured H9C2 (2-1) myocytes were transfected with extraneous miR-122 mimics to up-regulate the level of miR-122 expression and the transfection was confirmed by qRT-PCR. Myocytes apoptosis was determined by means of CCK-8, caspase-3 and flow cytometry. Results Ventricular septum defect in Pax-8-/- mice, and many apoptotic cells in left ventricular wall and interventricular septum in Pax-8 KO-/- mice were found. Differential expression profiles of miRNAs in Pax-8 KO-/- mice and Pax-8 KO+/- mice showed 10 microRNAs expressed differently between the two kinds of mice. miR-122 was up-regulated by 1.92 folds in Pax-8 KO-/-mice. Up-regulating the Level of miR-122 expression in primary cultured H9C2 (2-1) myocytes promoted cardiac myocytes apoptosis and inhibited myocytes proliferation. Taken together, these studies demonstrate that miR-122 is a critical regulator of heart development and ventricular septum defect pathogenesis.

Reduced expression of FXYD domain containing ion transport regulator 5 (FXYD5) in hypertension

l-nitroarginine methyl ester (L-NAME, 40 mg/kg, i.p.) significantly inhibited post exercise hypotension (25±11 and 5±3mm Hg, respectively; P<0.05). In addition, the superoxide anion generation was decreased, while the plasma nitrite production and serine phosphorylation of endothelial nitric oxide synthase were significantly elevated in spontaneously hypertensive rats at 30 min after the termination of exercise. Taken together, these data demonstrate that the increased phosphorylation of endothelial nitric oxide synthase plays a crucial role in the reduction of arterial pressure following a single bout of dynamic exercise in spontaneously hypertensive rats.

Protection of retinal vasculature by losartan against apoptosis and vasculopathy in rats with spontaneous hypertension

Methods Losartan potassium was administered to spontaneously hypertensive rats (SHR). Blood pressure was measured in SHR as well as normotensive Wistar– Kyoto rats (WKY). Eye fundus was examined in living animals and then tissue specimens were collected for histochemistry by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’triphosphate nick end labeling (TUNEL), immunohistochemistry and transmission electron microscopy.