Abstract Body (Do not enter title and authors here): Background: Alternative splicing of SCN5A mRNA reduces Nav1.5 channel density in cardiomyopathy, impairing myocardial excitability. The long noncoding RNA LINC00667 has been linked to RNA regulation, but its role in SCN5A processing remains unclear.
Hypothesis: We hypothesized that downregulation of LINC00667 modifies SCN5A splicing, leading to diminished sodium current in cardiomyocytes.
Methods: LINC00667 expression was quantified in public datasets, human ventricular tissues, and iPSC-derived cardiomyocytes (iPSC-CMs). Nav1.5 protein levels were measured by Western blot, and SCN5A isoforms were assessed via exon-specific PCR. Computational predictions identified RBM25, RBM5, and TIA1 as potential LINC00667 binders, which were validated by RNA immunoprecipitation. LINC00667 was silenced in iPSC-CMs with siRNA, and peak I_Na was recorded by whole-cell patch-clamp. Two-tailed Student’s t-tests were used for statistics (P < 0.05).
Results: In failing human myocardium, LINC00667 was downregulated by 29% versus controls (0.207 ± 0.019 vs. 0.292 ± 0.022; P < 0.05), and similarly decreased under hypoxia in vitro. ASO-mediated reduction of LINC00667 decreased SCN5A mRNA levels and was accompanied by an approximately 36% reduction in peak sodium current at -20 mV (P < 0.01), without altering resting membrane potential. Silencing LINC00667 in iPSC-CM generated SCN5A splice variants lacking exons 4-7, 22-26, or 27-28, confirmed by PCR band shifts. In silico analysis nominated RBM25, RBM5, and TIA1 as candidate LINC00667 binders, and RNA immunoprecipitation demonstrated a mean 11.28 ± 0.47-fold enrichment with RBM25, 6.58 ± 0.18-fold with TIA1, and 2.20 ± 0.08-fold with RBM5 over IgG controls (P < 0.05).
Conclusions: The loss of LINC00667 can induce aberrant, pathogenic alternative splicing of the SCN5A transcript, which is associated with a reduction in peak sodium current (I_Na) in human cardiomyocytes. These findings identify LINC00667 as a critical regulator of SCN5A integrity and suggest that restoring its expression may preserve myocardial excitability in cardiomyopathy.