Cellular Dynamics in Engineered Human Myocardium

Introduction: Engineered human myocardium (EHM) is presently tested in patients with advanced heart failure and in parallel applied as a tool in drug discovery and modelling of cardiac pathophysiology. EHM are prepared from highly enriched induced pluripotent stem cell (iPSC)-derived cardiomyocytes and stromal cells. A key feature of EHM is sustainable spontaneous rhythmic contractility, which is similar to contractility in ventricular myocardium under nodal control. To gain a better understanding of the cellular and molecular underpinnings of contractility in EHM, we are building an EHM-Atlas comprised of functional, cellular and molecular data obtained from cultures maintained for up to 6 months. In a first step, we focus on cellular dynamics in EHM.

Methods and Results: EHM tissues were generated in a 48-well myrPlate format using iPSC-derived cardiomyocytes and stromal cells at an input ratio of 70:30 in a collagen hydrogel. After initial tissue formation, 100% of tissues were spontaneously beating after five weeks of culture, reaching maximum contraction forces at week six. Spontaneous beating rate (41±9 bpm; n=165) and contraction force (0.38±0.17 mN; n=165) under auxotonic conditions were stable over a period of three months. After four months, spontaneous beating rate and force of contraction gradually declined, with 57% of the EHM retaining visible spontaneous contractions (0.12±0.09 mN; n=52) at six months. Force measurements under isometric conditions in randomly picked EHM at defined preload, extracellular Ca2+ (4 mM) and electrical pacing (1.5 Hz) showed decreased contraction forces (0.58±0.17 vs 0.98±0.35 mN, p<0.05; n=6/7) and increased tissue tension in 3 vs 1 months old EHM (0.58±0.20 vs 0.20±0.05 mN; p<0.001; n=6/7). Single nuclei RNA sequencing (snSeq) confirmed the cell composition at the time of EHM casting (70% cardiomyocytes and 30% stromal cells). At 1 month, EHM contained 58% cardiomyocytes, with an apparent (relative) increase of the non-cardiomyocyte population, characterized by an enhanced abundance of activated (myo)fibroblasts and first evidence for the presence of vascular smooth muscle cells. At 3 months, we found major changes in the cellular composition: cardiomyocyte population decreased to less than 20%, while non-cardiomyocytes abundance was further increased. Interestingly, 15% of nuclei were identified as endothelial cells (EC), while <1% of nuclei at baseline and 1 month EHM were positive for EC transcripts. Direct comparison of functional and snSeq data revealed that 3 months EHM generate 60% of active force with less than 30% of relative cardiomyocyte content of 1 month EHM. Considering 30% decrease of total cell number within that time, this suggests an over 4-fold increase of force contribution per cardiomyocyte. The resting tension was 3-fold increased with an around double amount of fibroblasts in 3 vs 1 month old EHM, pointing towards increased fibroblast activation and extracellular matrix deposition.

Conclusion: EHM retain stable spontaneous contractility over a culture period of three months. A reduced cardiomyocyte to non-myocyte ratio was observed, while the contribution of individual cardiomyocytes to contraction force increased with time. Furthermore, endothelialization was noted in 3 months EHM. The obtained data set will provide the ground to decipher cell and transcript contribution to EHM development, which appears to simulate important aspects of human heart development in vivo.