Hemodynamic overload induces hypertrophy in cardiomyocytes that ultimately leads to cardiac decompensation and heart failure. The activation of several genes, termed the ‘fetal genes’, plays a key role in the process. However, the mechanisms that control the coordinated activation of hypertrophic genes are not completely understood. We hypothesize that a shift in chromatin folding during hypertrophy allows transcription factors, enhancers and differentially activated fetal genes promoters to physically interact in the nucleus. To explore the three dimensional organization of the chromatin in the heart we used primary cultures of both hypertrophied and quiescent neonatal rat ventricular myocytes, incubated with or without phenylephrine, respectively. The three dimensional organization of the chromatin around the ‘fetal genes’ promoters was resolved using multiplexed 4C-sequencing. This approach allowed us to detect the long-range chromatin-chromatin interactions of the fetal gene (Nppb, Acta1, Myh6, Myh7 and Cacna1g) promoters. We identified multiple in-cis, on the same chromosome as the promoter, and in-trans, on a different chromosome than the promoter, interactions and characterized them. Most of the genome-genome interactions between the ‘fetal gene’ promoters and distal sites were present both during quiescence and hypertrophy. Nevertheless, about 35%-40% of the interactions differed between the two states. The Unique chromatin-chromatin interactions during hypertrophy were highly and significantly enriched in differentially expressed genes and cardiac transcription factors binding sites. In conclusion, we have analyzed the three dimensional structure of the chromatin in both quiescent and hypertrophied cardiomyocytes. We show that chromatin in cardiomyocytes has a specific folding pattern that brings linearly distant loci together. We further show that cardiac hypertrophy is consistently associated with changes in both in-cis and in-trans three dimensional associations that brings transcription factors, enhancers and differentially activated fetal genes promoters to physically interact. These changes may allow the hypertrophic gene response.
