Background and Hypothesis: Expression of the cardiomyocyte (CM) protein S100A1, which improves contractile performance of the heart, sharply increases during postnatal myocardial maturation but declines rapidly in failing hearts. We therefore hypothesized that CMs are wired with transcriptional factors (TFs) that positively and negatively regulate S100A1’s gene locus activity. Understanding these reciprocal circuits may be relevant for advanced therapeutic modulation of S100A1’s abundance in diseased hearts.

Methods and Results: H9C2 rat cardiomyoblasts, an animal-free in vitro tool, displayed a strong concordant rise in S100A1 mRNA and protein levels (8.1+/-1.1 vs. cont.; n=9, p<0.05) amongst other CM markers (i.e., SERCA2a) over a 5-day CM differentiation protocol. Overall TF activity during this process was computationally inferred from TF binding site (TFBS) assessments in promoters of all actively regulated transcripts provided by a time-resolved (undifferentiated, day 0, 2 and 5) transcriptome analysis. From these TFs, EGR1 and SP1 amongst eight others were chosen due to their abundance both in differentiated H9C2-CMs and adult rat hearts and TFBS in the -1000 to +500 bp rat S100A1 gene promoter region due to a 5’ RACE-PCR based transcription start site identification in our model. To capture the relevant TFs from this group, we next co-incubated nuclear extracts from H9C2-CMs with biotinylated fragments of the aforementioned S100A1 promoter region coupled to streptavidin beads. S100A1 promoter-bound TFs were identified by mass spectrometry and only factors with a >2-fold enrichment over control were selected delivering EGR1 and SP1 as top hits. Subsequent siRNA-mediated silencing of both TFs yielded an EGR1 and SP1 knock-down dose-dependent inhibition (80%; p<0.05 vs. contr., n=9) and amplification (126%; p<0.05 vs. contr., n=9) of the rise both in S100A1 mRNA and protein levels in the H9C2-CM model that validated our comp./exp. pipeline and biological relevance of our TF hits, respectively.

Conclusion: Our study identified the known cardioprotective EGR1 and maladaptive SP1 as novel positive and negative TF regulators of CM S100A1 expression and targets for therapeutic S100A1 gene locus modulation i.e., by SP1 TFBS gene-editing.