Nucleoporin 107 facilitates the nuclear export of Scn5a mRNA to regulate cardiac bioelectricity

Figures

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  Figure 1. Overexpression of Nup107 increases Nav1.5 protein expression in cardiomyocytes. (A) Western blotting examination of Nup107 overexpression on Nav1.5 protein expression. NRVMs were treated with either control (Ad‐GFP) or Ad‐Nup107 for 48 h, followed by the detection of Nav1.5 protein expression. (B) Data were pooled from three independent experiments. (C) Nup107 overexpression did not affect the mRNA levels of major ion channels. NRVMs were infected with Ad‐GFP or Ad‐Nup107 for 48 h, followed by the RT‐PCR analysis of sodium channel (Scn5a), calcium channel (Cacna1c), and potassium channels (Kcnd2, Kcnd3, Kcnj2, and Kcna4). (D)Western blotting examination of Nup107 overexpression on protein expression of other ion channels. NRVMs were treated with either Ad‐GFP or Ad‐Nup107 for 48 h, and then subjected to the detection of calcium channel (Cav1.2), and potassium channel associated proteins (Kv4.3, Kv1.4, and Kir2.1)

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  Figure 2. Overexpression of Nup107 changes the distribution of Scn5a mRNA between the cytoplasm and nucleus in NRVMs. (A) Schematic diagram of amplification regions of mature and nascent Scn5a mRNA. Junc, junction region; int, intron region. (B) Effects of Nup107 overexpression on the expression of mature and nascent Scn5a mRNA. The expression of Scn5a mRNA was normalized to multiple internal reference Gapdh, 18s, β‐actin, and β‐tubulin. Data were pooled from three independent experiments. (C) Overexpression of Nup107 altered the cytoplasmic‐nuclear distribution of Scn5a mRNA in NRVMs. The copies of Scn5a, Gapdh, β‐actin, and actinin mRNA in cytoplasmic and nuclear fractions were measured using absolute qualification. Data were expressed as the ratio of cytoplasmic and total mRNAs, and pooled from three independent experiments. *P < 0.01. (D) The half‐life of Nav1.5 was not affected in NRVMs, as determined by a cycloheximide (CHX) chase assay. NRVMs were infected with either control Ad‐GFP or Ad‐Nup107 for 24 h, followed by the addition of CHX (100 μg/mL) for the indicated time. Then the cells were harvested and immunoblotted. Left, typical blots of Nav1.5 expression in Nup107…

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  Figure 3. Identification of interactive sites for Nup107 and Scn5a mRNA. (A) Schematic diagram of primer pairs specific for different parts of Scna5a mRNA. UTR, untranslated region; CDS, coding sequences. (B) RNA‐IP assay for detecting the interaction between Nup107 protein and Scn5a mRNA. NRVMs were treated with Ad‐Nup107 or Ad‐GFP for 48 h, followed by RNA‐IP assay. The RIP‐PCR analysis showed that Nup107, but not GFP selectively regulated Scn5a mRNA. *P < 0.01. (C) Association of Nup107 with different regions of Scn5a mRNA in NRVMs as measured by RIP‐PCR analysis. Values are the means ± SD (n = 3). *P < 0.01 compared with GFP control group. (D) The interactive regions of Scn5a mRNA associated with Nup107 protein, verified by the luciferase reporter assay. Top, schematic diagram of the luciferase reporter vectors containing the 5′‐UTR, CDS, or 3′‐UTR of Scn5a mRNA; bottom, the luminescence intensity under the overexpression of Nup107 and control vector transfected with the 5′‐UTR, CDS, 3′‐UTR reporters, or their corresponding subcloning reporters in HEK293 cells. HEK293 cells overexpressed with Nup107 were cotransfected with the luminescence vectors containing the 5′‐UTR, CDS, or 3′‐UTR…

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  Figure 4. Nup107 regulates cardiac INa currents and Nup107‐mediated regulation of Nav1.5 is associated with ischaemic cardiac injury both in vitro and in vivo. (A) The representative Na+ currents recorded from NRVMs infected with Ad‐Nup107 or Ad‐GFP. The currents were recorded with the whole‐cell patch‐clamp technique. Cells were held at −120 mV and INa was elicited by a family of voltage steps to potentials ranging from −70 to +40 mV with 10 mV increments. (B) Current‐voltage relationship of INa in NRVMs infected with Ad‐Nup107 (n = 20) or Ad‐GFP (n = 12). Currents were normalized to cell capacity. *P < 0.01. (C) Protein expression levels of Nup107 and Nav1.5 in cultured NVRMs subjected to hypoxia condition (1% O2) for 48 h. Data are presented as mean ± SD of three independent experiments. *P < 0.01. (D) The protein expression of Nup107 and Nav1.5 in ischaemic ventricular tissues from the rat model of acute myocardial infarction. Left: representative blots; right: pooled data. *P < 0.01