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. 2011 Sep 6;124(10):1124-31.
doi: 10.1161/CIRCULATIONAHA.111.044495. Epub 2011 Aug 22.

Role of RBM25/LUC7L3 in abnormal cardiac sodium channel splicing regulation in human heart failure

Ge Gao  1 , An Xie, Shu-Ching Huang, Anyu Zhou, Jianhua Zhang, Amanda M Herman, Sassan Ghassemzadeh, Euy-Myoung Jeong, Srinivasan Kasturirangan, Mihai Raicu, Michael A Sobieski 2nd, Geetha Bhat, Antone Tatooles, Edward J Benz Jr, Timothy J Kamp, Samuel C Dudley Jr
Affiliations

Role of RBM25/LUC7L3 in abnormal cardiac sodium channel splicing regulation in human heart failure

Ge Gao et al. Circulation. .

Abstract

Background: Human heart failure is associated with decreased cardiac voltage-gated Na+ channel current (encoded by SCN5A), and the changes have been implicated in the increased risk of sudden death in heart failure. Nevertheless, the mechanism of SCN5A downregulation is unclear. A number of human diseases are associated with alternative mRNA splicing, which has received comparatively little attention in the study of cardiac disease. Splicing factor expression profiles during human heart failure and a specific splicing pathway for SCN5A regulation were explored in this study.

Methods and results: Gene array comparisons between normal human and heart failure tissues demonstrated that 17 splicing factors, associated with all major spliceosome components, were upregulated. Two of these splicing factors, RBM25 and LUC7L3, were elevated in human heart failure tissue and mediated truncation of SCN5A mRNA in both Jurkat cells and human embryonic stem cell-derived cardiomyocytes. RBM25/LUC7L3-mediated abnormal SCN5A mRNA splicing reduced Na+ channel current 91.1±9.3% to a range known to cause sudden death. Overexpression of either splicing factor resulted in an increase in truncated mRNA and a concomitant decrease in the full-length SCN5A transcript.

Conclusions: Of the 17 mRNA splicing factors upregulated in heart failure, RBM25 and LUC7L3 were sufficient to explain the increase in truncated forms and the reduction in full-length Na+ channel transcript. Because the reduction in channels was in the range known to be associated with sudden death, interruption of this abnormal mRNA processing may reduce arrhythmic risk in heart failure.

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Figures

Figure 1
Figure 1
Expressions of RBM25 and LUC7L3 in human HF tissue. (A) qPCR demonstrates the upregulation of RBM25 and LUC7L3 in human HF tissue. The relative expression changes of RBM25 and LUC7L3 in both normal control (white bars) and failing heart tissue (black bars) are shown. All mRNA abundances are normalized by β-actin. * P < 0.05 when compared with control. (B) Western blots quantification confirms the upregulation of RBM25 and LUC7L3 in human HF tissue. Control represents the mixture of 4 normal human heart tissue samples. HF1, HF2, HF3 and HF4 represent the HF tissue sample 1, sample 2, sample 3 and sample 4, respectively. Quantification is based on three replications for each sample. All protein levels are normalized by GAPDH. * P < 0.05 when compared with control.
Figure 2
Figure 2
Illustration of the C-terminal structure of SCN5A and the variants E28C and E28D. (A) * indicates the RBM25 binding site CGGGCA in exon 28 (982bp–987bp). Gel mobility shift assays are performed using the biotinylated wild type (WT) probe (B) or the mutant (Mu) probe (C) and purified RBM25 protein. For loading samples from left to right, the amount of RBM25 in each binding reaction is increased by a fold. For the competition assay (D), 0, 1-, 5-, or 20-fold molar excesses of unlabeled WT or Mu probes are added in each binding reaction.
Figure 3
Figure 3
RBM25 and LUC7L3 are involved in SCN5A regulation in Jurkat cells. (A) qPCR demonstrates the upregulation of RBM25 and LUC7L3. The expression changes of RBM25 and LUC7L3 in hypoxia-treated (shaded bars) and Ang II-treated (black bars) vs. normal control Jurkat cells (white bars) are shown at 48 h. HIF-1α is an indicator of hypoxia. mRNA abundances are normalized by β-actin. * P < 0.05 when compared with control (N=6). (B) Western blots quantification confirms the upregulation of RBM25 and LUC7L3 in Jurkat cells. Expressions of RBM25 and LUC7L3 are analyzed by time course. * P < 0.05 when compared with control (N=6). (C) The expression changes of SCN5A and the variants E28C and E28D in hypoxia-treated (shaded bars) and Ang II-treated (black bars) vs. untreated Jurkat cells (white bars) are shown at 48 h. * P < 0.05 when compared with control (N=6). Western blots indicate the downregulation of SCN5A in Jurkat cells with Ang II or hypoxia. (D) qPCR demonstrates that RBM25 and LUC7L3 siRNAs could block the induction of hypoxia on variants E28C and E28D. The representative results at 24h are shown. * P < 0.05 when compared with control (N=6). (E) qPCR demonstrates that RBM25 and LUC7L3 siRNAs could block the induction of variants E28C and E28D by Ang II. Representative results at 48 h are shown. * P < 0.05 when compared with control (N=6). Scrambled siRNA had no effect on the induction by Ang II (data not shown). The knockdown efficiency of RBM25 and LUC7L3 siRNAs are evaluated by Western blots and compared to control and scrambled RNA. (F) qPCR demonstrates that RBM25 and LUC7L3 overexpressions decrease the full length SCN5A transcript and increase the variants E28C and E28D at 48h. * P < 0.05 when compared with control (N=6). Exogenously expressed RBM25-GFP and LUC7L3-GFP are detected by Western blot analysis with anti-GFP at 48 h after transfection in Jurkat cells. The representative Western blots show the downregulation of the full-length SCN5A transcript in Jurkat cells with exogenously expressed RBM25 and LUC7L3 as compared to the control group. Full-length SCN5A RNA is unchanged with GFP expression alone.
Figure 4
Figure 4
The effect of RBM25 shRNA on the expressions of SCN5A and the variants E28C and E28D in Ang II-treated hESC-CMs. (A) Ang II (200 nmol/L) treatment is given to all the experiment groups on infection day 3. RT-PCR measurements are done at 24 h after Ang II treatment and normalized by β-actin. The expression changes of SCN5A and the variants E28C and E28D in Ang II-treated cardiomyocytes pre-infected by RBM25 pLKO.1 shRNA (shaded bars) and pre-infected by scrambled shRNA (black bars) vs. normal Ang II-treated cardiomyocytes (white bars) are shown at 24 h. * P < 0.05 compared with normal Ang II-treated cardiomyocytes (N=6). (B) Confocal microscopy shows a hESC-CM. The GFP fluorescence indicates the infection by pGIPZ lentiviral shRNAmir. RBM25 knockdown efficiency is evaluated by qPCR and Western blot (N=6). Scrambled shRNA has no effect on RBM25.
Figure 5
Figure 5
The effect of Ang II (200 nmol/L) on Na+ currents in hESC-CMs at 24 h after treatment. The representative current traces are shown in the control (A), Ang II-treated (B), Ang II-treated with pre-infection of RBM25 shRNA (C), and TTX -treated groups (D). The peak current statistics of the three experiment groups are shown in I–V curves (E), where the control group (n=3) is represented by filled squares, the Ang II-treated group (n=3) by filled circles, and the Ang II-treated group with pre-infection of RBM25 shRNA (n=3) by open triangles. The data are represented as mean ± SEM. The peak current is significantly reduced from −40 to +30 mV in the Ang II-treated group compared to the control group (P < 0.05), the maximum reduction was 91.1 ± 9.3 % at −30 mV. No difference is observed between the Ang II-treated group with pre-infection of RBM25 shRNA and the control group (E). The specificity of sodium channel current is tested with TTX (a specific sodium channel blocker) in Group D. Scrambled shRNA had no effect on the I-V relationship compared to Ang II alone (data not shown).
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