HDAC2 inactivation in CREM-IbΔC-X transgenic mice counterregulates the proteome associated with the structural remodelling of atrial fibrillation

Objective: Atrial fibrillation (AF) is the most common cardiac arrhythmia and is associated with functional and structural atrial remodeling. We have previously shown that genetic inactivation of histone deacetylase 2 (HDAC2) reduces thrombus formation, atrial fibrosis and alterations of sarcomeres and mitochondria ultrastructure in CREM-IbΔC-X transgenic (TG) mice, a well-characterized model with extensive atrial remodeling and spontaneous AF development. Here, we investigated the impact of cardiomyocyte-specific inactivation of HDAC2 (KO) in atrial cardiomyocytes from TG mice on the atrial proteome to identify regulated pathways.

Material & Methods: Proteins were extracted from atrial tissue samples of TG, KO, TGxKO and control (CTR) mice. The proteome was analyzed using an ESI-LC-MS/MS system. Data analysis was performed using R and Ingenuity® pathway analysis (IPA). ShinyGO 0.81 was used for Cellular Component Ontology (GO-CC) enrichment analysis. 

Results: We detected 3949 proteins, of which 2416 were significantly regulated in TG vs. CTR. IPA revealed increased mitochondrial dysfunction, increased fibrosis and decreased overall cardiac function in TG vs. CTR, which is in good agreement with the impaired phenotype in TG. In TGxKO 363 proteins were significantly regulated vs. TG. Of these, 342 proteins were counterregulated. In males, 198 proteins were regulated in both TG vs. CTR and TGxKO vs. TG, and 196 of them were counterregulated in TGxKO vs. TG. In females, only 48 proteins were regulated in both TG vs. CTR and TGxKO vs. TG, but consequently counterregulated in TGxKO vs. TG. Furthermore, 17 proteins were exclusively regulated in TGxKO vs. TG females (e.g. Vcl, Vcam1 or Tppp), whereas 178 proteins were exclusively regulated in TGxKO vs. TG males, such as various collagen or myosin isoforms. Proteins regulated in TGxKO vs. TG were enriched in parts of the contractile apparatus, especially in the CC-terms “actin cyto-skeleton” and “actin filament bundle”. IPA revealed that pathways related to cytoskeletal reorganization and fibroblast formation were decreased - 20 in males and 12 in females, indicating changes in structural remodelling in both sexes. In addition, inflammatory activation and response were reduced in both. Only 120 proteins were regulated in KO vs. CTR, indicating an enhanced effect of HDAC2 inactivation under disease conditions. In a sex-specific analysis, 29 proteins were found to be regulated in KO males and 56 in KO females vs. CTR. GO analysis revealed enriched CCO terms such as “mitochondria”, “sarcomeres” and “contractile muscle fibers” in KO vs. CTR (analyzed atria, male/female, CTR: 5/6, KO: 6/5, TG: 4/5, TGxKO: 5/4).

Conclusion: The inactivation of HDAC2 in TG mice resulted in a reduction and counterregulation of proteomic changes related to the structural remodelling of AF. Sex influences which and how many target proteins are regulated by HDAC2 inactivation. In remodelled TG atria, the number of proteins affected by HDAC2 inactivation in female mice is fewer than in male mice. However, both sexes exhibited enriched and downregulated pathways related to cytoskeletal, contractile filament reorganisation and fibrosis as well as inflammation. Our results emphasize the therapeutic potential of inactivating or inhibiting HDAC2 to slow down the structural remodelling associated with atrial fibrillation.