Abstract Body: Introduction: Engineered Human Myocardium (EHM) is presently used for drug discovery and modeling of monogenic cardiomyopathy. Notably, standard four- to six-week EHM cultures exhibit characteristics similar to juvenile ventricular myocardium. We hypothesize, that prolonged culture of EHM recapitulates pathological cardiac remodeling and aging processes.
Methods and Results: We generated EHM (n=240) from human iPSC-derived cardiomyocytes and stromal cells in a collagen hydrogel. To assess spontaneous tissue contractions, we employed a video-optic recording system and observed stable contractions (force of contraction [FOC] in mN/mm2: 0.34 ± 0.01; n=240) in all EHM at 5 weeks of culture. Spontaneous contractions remained intact until 4 months (FOC in mN/mm2: 0.91 ± 0.06; n=159), after which they declined until 50% of the cohort lost spontaneous contractility at 6 months (FOC in mN/mm2: 0.35 ± 0.02; n=112). By 12 months, only 2 EHM showed macroscopically visible contractions (FOC in mN/mm2: 0.16; n=2). Diastolic tissue tension (resting tension [RT]), as a marker of fibroblast activity, remained stable until 8 weeks in culture (RT in mN/mm2: 0.6 ± 0.05; n=238), but increased significantly between 3 and 6 months (RT in mN/mm2: 2.5 ± 0.1 vs 10.0 ± 0.2; p<0.0001). In predefined endpoint measurements including RNA and single-nucleus sequencing, we observed development of a “myofibroblast signature” at 3 and 6 months with enrichment of transcripts encoding for ACTA2, POSTN and COL1A1. At 12 months, transcriptome signals indicative of cellular senescence (CDKN1A, SERPINE1) and inflammation (LIF, NFKB2) became apparent. Furthermore, analysis of medium supernatant by proximity extension assays (OLink) revealed increased abundance of aging-related proteins in older EHM cultures.
Conclusion: EHM exhibit stable contractile function and tissue tension from 1 to 3 months, serving as a model for the healthy myocardium within this timespan. Between 3 and 6 months, EHM show increased tissue tension and expression of myofibroblast markers, accompanied by decreased spontaneous contractility. Further cultivation up to 12 months accumulates transcriptome markers of cardiac senescence and secretion of aging-related proteins. These findings suggest that EHM can serve as a model for cardiac fibrosis and aging.