Hemoglobin inhibits fibroblast-to-cardiomyocyte reprogramming via TLR2/TLR4-dependent chromatin compaction

Fibroblast-to-cardiomyocyte reprogramming is influenced by signals present in the injured heart. However, the nature of those signals is unclear. The efficacy of fibroblast-to-cardiomyocyte reprogramming was found to be negatively impacted by extracts derived from the freeze-thawing of normal cardiac tissue. Mass spectrometry identified the adult minor beta chain of hemoglobin (Hbb-b2) as the most abundant protein in the extract, prompting investigation of hemoglobin (Hb) as a candidate inhibitory factor. Indeed, Hb was found to suppress fibroblast-to-cardiomyocyte reprogramming by inhibiting the expression of various cardiomyocyte-specific markers and cardiomyocyte formation. Hb is known to interact with TLR2 and TLR4. While inhibition of either receptor was unable to reverse the effects of Hb, inhibition of both TLR2 and TLR4 reversed the suppressive effects of Hb. To clarify the mechanism, assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed. ATAC-seq revealed that Hb induced widespread transcription factor network remodeling, whereby stress-associated factor networks were induced and latent CEBP/PAR-bZIP networks were suppressed. These effects were localized to a region 500-750 bp upstream of the transcription start site in cardiomyocyte-specific genes. These findings identify Hb as a potential negative regulator of fibroblast-to-cardiomyocyte reprogramming, with implications for cardiac regeneration strategies.