Abstract Body (Do not enter title and authors here): Introduction:
Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome, and its pathophysiology is not fully understood. Histone serotonylation has recently emerged as a novel epigenetic mechanism, and transglutaminase 2 (TGM2) has been identified as a responsible molecule mediating this process through its gulutaminase activity.

Aim:
This study aimed to investigate the significance of histone serotonylation in cardiomyocytes during the development of HFpEF.

Methods:
HFpEF was induced in a mouse model using a combination of salty drinking water, unilateral nephrectomy, and chronic exposure to aldosterone, referred to as the SAUNA model (Figure 1A). To assess the role of TGM2, cardiomyocyte-specific TGM2-deficient (CKO) mice were generated.

Results:
In wild-type mice, histone serotonylation (H3K4me3Q5ser) as well as nuclear TGM2 expression were significantly increased in the hearts at 4 weeks after SAUNA induction. In TGM2-CKO hearts, H3K4me3Q5ser levels were decreased by 80% compared to wild-type littermates (Figure 1B). Following 4 weeks of SAUNA exposure, TGM2-CKO mice showed exacerbated diastolic dysfunction, evidenced by increased E/A ratio, E/e’ ratio, and left atrial volume, compared to wild-type littermates (Figure 2). Furthermore, SAUNA-exposed TGM2-CKO mice exhibited a significant reduction in running distance by treadmill exercise testing and elevated lung weight. Ex vivo analysis using isolated cardiomyocytes revealed that time-to-50% relaxation in response to 1 Hz pacing were significantly prolonged in cardiomyocytes from TGM2-CKO mice, indicating impaired cardiomyocyte relaxation at a single cardiomyocyte level. CUT&RUN sequencing of cardiac tissue using an anti-H3K4me3Q5ser antibody revealed significant enrichment of gene ontology terms related to microtubule-associated pathways in SAUNA hearts compared to sham controls (Figure 3). In vitro, aldosterone stimulation promoted nuclear translocation of TGM2 in cardiac myocytes transfected with GFP-tagged TGM2, whereas a Ca²⁺-binding-mutants failed to localize to the nucleus, indicating its role of Ca²⁺-dependent TGM2 activation in this process.

Conclusion:
Cardiomyocyte-specific TGM2 deletion exacerbates diastolic dysfunction, exercise intolerance, pulmonary congestion, and impaired cardiomyocyte stiffness in the SAUNA model. These findings suggest that TGM2 plays a cardio-protective role and could represent a novel therapeutic target for HFpEF.