The Role Of Kv1.5Channel In Hypoxic Pulmonary Arterial Hypertension Following Intrauterine Growth Retardation And Its Epigenetic Mechanism

Intrauterine growth retardation (IUGR) is defined as having a birth weight below the10th percentile of the corresponding gestational age caused by adverse intrauterine environment. Adverse intrauterine environment can affect fetal development and cause IUGR. A large number of epidemiological and laboratory evidence show that neonates with IUGR or low birth weight are being at increased risk of adult diseases, such as type2diabetes and cardiovascular diseases. Meanwhile, this phenomenon can be transmitted to the next generation and known as the "Developmental Origins of Adult Disease Hypothesis". There is accumulating evidence reveals an epigenetic regulatory mechanisms play an important role in the fetal origin of adult disease. Epigenetics is the study of heritable changes in gene activity that not caused by changes in the DNA sequence. miRNA is the main executor RNA interference and plays a role in epigenetic regulation. After binding with the RNA-induced silencing complex, miRNA is specifically complemented with target mRNA3’UTRs and then regulates gene expression leading to inhibition of translation or to destabilize mRNA.Recent research has found that IUGR could cause exaggerated CH-PAH at adult via endothelial mechanism. The latest evidence showed miRNA-1may be related with cardiovascular muscle cell in CH-PAH. miRNA-1family includes miRNA-1, miRNA- 206, and miRNA-133a/b. Whether miRNA-1family can regulate expression of Kv channel directly and its mechanism has not been totally identified.So, we established IUGR rat model and exposed them to2-week hypoxia, and then screen related Kv channel and its role in this model. miRNA array assay was used to screen related miRNA which can modulate Kv1.5in CH-PAH following IUGR. The process and mechanism was investigated thoroughly. Part I Kvl.5in intrauterine growth retardation rats with exaggerated pulmonary arterial hypertensionObjective:1. Assess pulmonary artery hemodynamics and morphometry in pulmonary arterial hypertension following IUGR.2. Assess changes in expression and function of related Kv channel of rat with hypoxic pulmonary arterial hypertension induced by IUGR.3. Establish primary PASMCs of IUGR rat, observe the different time points of hypoxia for proliferation of PASMCs and expression and function changes of related Kv channel, and then assess the impact of Kv channel on proliferation induced by hypoxia.Method:1. Animal model and groups:Control:Rats were born from mothers fed with standard chow ad libitum during the entire pregnancy.IUGR:Rats were born from mothers fed with standard chow50%of the ad libitum amount (determined from the amount of chow consumed by the mothers of control group) during the entire pregnancy.Control-hypoxia:Rats from control group were exposed to hypoxia for2weeks at12-week-old.IUGR-hypoxia:Rats from IUGR group were exposed to hypoxia for2weeks at12-week-old.2. Assessment of pulmonary hypertension:The right ventricular systolic pressure (RVSP) was detected after2weeks’ hypoxic exposure. The degree of muscularization of pulmonary vessels was assessed by immunohistochemistry staining and western blotting of a-SMA. The right ventricular (RV) hypertrophy was accessed by the ratio of the RV to the LV+Sep weight (RVHI).3. A vascular reactivity test was performed with K+channel inhibitors:4-AP, TEA, and BaC12.4. PASMCs were isolated using a modification of technique. The whole-cell Kv current and membrane potential were detected by patch clamp.5. The expression of Kv channel in rats’ pulmonary artery was detected by Western blot analysis.6. Proliferation was determined by MTT assays. The expression of Kv channel on the membrane of PASMCs was detected by immunofluorescence staining.7. Observe changes in proliferation of PASMCs when related subtypes of Kv channels are overexpressed.Result:1. RVSP and RVHI values were similar between the control and IUGR groups (RVSP:22.88±0.38mmHg vs.23.76±0.28mmHg; RVHI:0.26±0.01vs.0.28±0.01). After2weeks of hypoxia exposure, RVSP and RVHI values increased. The increase was more significant in the IUGR-hypoxia group compared with its age-matched control-hypoxia group (RVSP:32.50±0.30mmHg vs.37.80±0.63mmHg, P<0.05; RVHI:0.32±0.01vs.0.40±0.02, P<0.05).The percentage of pulmonary artery medial thickness was greater in IUGR-hypoxia group than that in the age-matched control-hypoxia group, as well as a-SMA protein expression.2. Microvascular tension determination showed that significant constriction in microvascular was induced by4-AP while it is weak by BaCl2and litter by TEA. After2weeks of hypoxia, the constriction to4-AP (10mM) was inhibited, and the inhibition was more severe in the IUGR-hypoxia group.3. After2weeks of hypoxia, the expression of Kv1.5was also decreased more severely in IUGR-hypoxia groups (p<0.05). There was no difference between two groups in Kv1.2, Kv2.1, Kv3.1, and Kv9.3channels. Kv current in single cell of PASMC was significant decreased in IUGR-hypoxia compared with that in control-hypoxia. No difference between groups in membrane potential.4. More than ninety-five percent of cells were smooth muscle cells judging from cell morphology and immunofluorescence staining of a-SMA.5. Overexpress Kv1.5was found after being transfected with PASMC by using immunofluorescence test. MTT was used to test cell proliferation. No significant difference in OD was found between IUGR-hypoxia group and IUGR-hypoxia control groupConclusions:1. IUGR rats developed exaggerated CH-PHT at adult when exposed to chronic hypoxia.2. Decreased expression and function of Kv1.5play an important role in the mechanism of IUGR-exaggerated CH-PHT.3. Overexpression of Kv1.5could inhibit primary PASMCs of IUGR proliferation induced by hypoxia. Part II miRNA-206regulates the effect of hypoxia on PASMCs of intrauterine growth retardation ratsObjective:1. To screen and verify multiplexed profiling of candidate miRNA for pulmonary artery smooth muscle tissues of IUGR in rats.2. To observe the changes of Kvl.5protein expression and cell proliferation after miRNA-206mimic intervention in PASMCs.Methods:1. We used miRNA3.0array assay combined database to screen multiplexed profiling of candidate miRNA for pulmonary artery smooth muscle tissues of IUGR in rats.2. We verified the result of miRNA3.0array assay by real-time PCR combine Taqman technique.3. We used miRNA mimic to interfere primary PASMCs of IUGR rats before hypoxia (CoCl2).4. After administration of miRNA mimic, Kvl.5expression was verified by Western blot analysis. EdU test was used to detect cell proliferation.Results:1. miRNA-206and miRNA-184were significantly up-regulated with a threshold of>2and q value<0.05in IUGR group when it was compared with control group.2. TargetScan database predicted that KCNA5is a target of miRNA-206.3. After miRNA-206mimic intervention of PASMCs, Kvl.5protein expression was down-regulated in control-hypoxia group, and there was no difference between control-hypoxia group and IUGR-hypoxia group.4. After miRNA-206mimic intervention of PASMCs, EdU test showed that proliferation rate of control-hypoxia group was accelerated as same as IUGR-hypoxia group. Conclusions:1. miRNA-206is important factor in IUGR rats with hypoxic pulmonary arterial hypertension at adult.2. miRNA-206can affect proliferation of PASMCs via inhibiting the role of hypoxia on Kvl.5protein expression.