Molecular mechanisms of downregulated Kv channels in IPAH: Role of microRNA

项目总结/文摘

Project Summary/Abstract Idiopathic pulmonary arterial hypertension (IPAH) is a fatal and progressive disease that predominantly affects women. Pulmonary vascular remodeling, sustained vasoconstriction and in situ thrombosis are the major causes for the elevated pulmonary vascular resistance in IPAH patients. Pulmonary vascular remodeling is characterized in part by significant medial and intimal hypertrophy, due to increased pulmonary arterial smooth muscle cell (PASMC) proliferation and decreased PASMC apoptosis. A rise in cytosolic Ca ([Ca ]cyt) in PASMC is a 2+ 2+ major trigger for pulmonary vasoconstriction and an important stimulus for PASMC proliferation. Downregulation of voltage-gated K (Kv) channel expression and decrease in Kv currents (IK(V)) + contribute to a) increasing PASMC proliferation by raising [Ca ]cyt via membrane depolarization 2+ and b) decreasing PASMC apoptosis by inhibiting apoptotic volume decrease and maintaining sufficient K+ in the cytoplasm to inhibit caspases and nucleases. Our data showed that the expression and activity of Kv channels are reduced, while proliferation is enhanced and apoptosis is inhibited, in IPAH-PASMC compared to normal PASMC. MicroRNAs (miRNAs) are short RNAs that bind to complementary sequences in the 3'-untranslated regions (3'-UTR) of target mRNAs to inhibit mRNA translation and/or induce mRNA degradation, thereby inhibiting the expression of specific mRNA targets. miRNAs are thus posttranscriptional regulators that potentially regulate the level of Kv channel mRNAs in IPAH-PASMC. We recently identified several miRNAs (e.g., miR-29b, miR-138 and miR-222) that are highly expressed, whereas several Kv channels (e.g., KCNA1-7/10) are significantly downregulated, in IPAH-PASMC compared to normal PASMC. Overexpression of miR-29b, miR-138 or miR-222 in normal PASMC decreases whole-cell IK(V), whereas inhibition of miR-29b rescues (i.e., significantly enhances) IK(V) in IPAH-PASMC. These data suggest that miR-138, miR-222 and miR-29b are sufficient to decrease IK(V) in normal PASMC, while miR-29b is necessary for the decreased IK(V) in IPAH-PASMC. Based on these observations, we hypothesize that selectively upregulated miRNAs are involved in the posttranscriptional inhibition of Kv channels in IPAH-PASMC and the miRNA-mediated Kv channel inhibition contributes to increasing proliferation and decreasing apoptosis in IPAH-PASMC. Three Specific Aims are proposed to test this hypothesis: 1) To identify the miRNAs that are upregulated and the Kv channels that are downregulated in IPAH- PASMC, and to determine which Kv channel subunits are posttranscriptionally regulated by the upregulated miRNAs in IPAH-PASMC; 2) To determine whether upregulated miRNAs in IPAH- PASMC regulate the stability and translation of target Kv channel mRNAs; and 3) To determine the role of miRNA-mediated posttranscriptional inhibition of Kv channels in PASMC proliferation and apoptosis. The long-term goal of this study is to define the mechanism underlying the inhibition of Kv channels in IPAHPASMC and to explore the possibility to target miRNA for developing therapeutic approaches for IPAH.